International Review of Cytology: A Survey of Cell Biology, Volume 8

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUMEVIII

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INTERNATIONAL

Review of Cytology EDITED BY

G. H. BOURNE

J. F. DANIELLI

Department of Anatomy Emory University Emory University, Georgia

Department of Zoology King’s College London, England

VOLUME

VIII

Prepared Under the Auspices of The International Society for Cell Biology

ACADEMIC PRESS, New York and London 1959

Copyright 0, 1959, by

ACADEMIC PRESS INC. All Rights Reserved NO PART OF T H I S BOOK M AY BE REPRODUCED I N A N Y FORM, BY PHOTOSTAT, MICROFILM, OR A N Y OTHER MEANS, W I T H O U T WRITTEN PERMISSION FROM T H E PUBLISHERS.

ACADEMIC PRESS INC. 111 FIFTHAVENUE NEW YORK 3, N. Y. United Kingdom Edition Published by ACADEMIC PRESS INC. (LONDON) LTD. 40 PALLMALL, LONDON SW 1

Library of Congress Catalog Card Number 52-5203

PRINTED I N T H E UNI TED STATES OF AMERICA

Contributors to Volume VIII J . B. BRIDGES, Department of Anatomy, Queen’s University, Belfast, Ireland

EDUARDO DE ROBERTIS, Director of the Instituto de Anatomia General y Embrwlogia, Facultad de Ciencias Mddicas, Buenos Aires, Argentina D. A. T . D I C K , Department of Human Anatomy, Oxford University, Oxford, England1

C. F. EHRET,Diviswn of Biological and Medical Research, Argonne National Laboratory, Lemont, Illinois MILTONFINGERMAN, Department of Zoology, Newcomb College, Tulane University, N e w Orleans, Louisiana I. M . GLYNN, Cambridge University, Cambridge, England

DAVIDA. HALL,Nufield Gerontological Research Unit, Department of Medicine, School of Medicine, Leeds, England FREDERIC L. HOCH,Biophysics Research Laboratory of the Department of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Massachusetts H. HOLTER, Department of Physiology, Carlsberg Laboratory, Copenhagen, Denmark LEAH MIRIAMLOWENSTEIN; Department of Human Anatomy, Oxford University, Oxford, England2 CHARLESOBERLING, Institut de Recherches sur le Cancer, Villejuif (Seine), France E. L. POWERS, Diviswn of Biological and Medical Research, Argonne National Laboratory, Lcmont, Illinois R. D. PRESTON, Department of Botany, University of Leeds, Leeds, England

D. B. ROODYN, Medical Research Council, Radwbwlogical Research Unit, Hamiell, Engla*td3 BERTL. VALLEE,Biophysics Research Laboratory of the Departlnent of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Marsachusetts 1 2

3

Present address : Carlsberg Laboratory, Copenhagen, Denmark. Present address : Beth Israel Hospital, Boston, Massachusetts. Present address : George Washington University School of Medicine, Wash., D. C .

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CONTENTS

CONTRIBUTORS TO VOLUME VIII

............................................

V

The Structure of Cytoplasm CHARLES OBERLINC

I. I1 I11. I V. V. VI . VII . VIII . IX X. XI . XI1

.

.

.

Introduction ...................................................... Definition of Cytoplasmic Structures ................................ Mitochondria ..................................................... Mitochondria1 Regeneration ........................................ Pathological Aspects of Mitochondria .............................. Ergastoplasm ..................................................... Microsomes ...................................................... Origin of Ergastoplasm ........................................... Golgi Apparatus .................................................. Centriole ......................................................... Conclusion ........................................................ References ........................................................

1

2 4 5 8 15 18 18 21 26 28 28

Wall Organization in Plant Cells R . D . PRESTON Introduction ...................................................... The X-Ray Diagram and Electron-Microscopic Appearance of Cellulose The Chemical Composition of Microfibrils .......................... The Biosynthesis of Microfibrils .................................... The Synthesis and Orientation of Microfibrils at a New Cytoplasmic Surface ......................................................... V I . References ........................................................

I. I1. I11. I V. V.

33 36 40 51 53 58

Submicroscopic Morphology of the Synapse EDUARDO DE ROBERTIS I. I1 I11. I V. V.

.

Introduction ...................................................... Morphology of the Synaptic Region ................................ Submicroscopic Morphology and Function of the Synapse ............ Summary ......................................................... References ........................................................

61 63 76 93 94

The Cell Surface of Paramecium C. F. EHRET A N D E. L. POWERS I. I1. I11 I V. V.

.

The Problem ..................................................... The Evidence ..................................................... Synthesis and Outlook ............................................ Acknowledgments ................................................. . References ........................................................

97 99 128 132 132

The Mammalian Reticulocyte LEAHMIRIAMLOWENSTEIN

I. I1. I11. I v. V. V I. VII . V I I I. IX .

Introduction ...................................................... Techniques in the Examination of Reticulocytes ...................... Morphology ...................................................... Physical Properties ................................................ Biochemistry ...................................................... Physiology ....................................................... Reticulocytes in Disease ........................................... Acknowledgments ................................................. References ........................................................

136 136 141 143 149 154 163 165 166

The Physiology of Chromatophores MILTONFINGERMAN

I. I1. I11. IV . V. V I. VII. V I I I.

Introduction ...................................................... Classification of Chromatophore Responses .......................... Functional Significance of Color Changes .......................... Chromatophores of Arthropods .................................... Chromatophores of Fishes ......................................... Chromatophores of Amphibians .................................... Chemical Nature of Chromatophorotropins ........................ References ........................................................

175 176 177 181 202 204 205 206

The Fibrous Components of Connective Tissue with Special Reference to the Elastic Fiber DAVIDA . HALL

I. I1. I11. I V. V. VI . V I I.

Introduction ...................................................... Morphological Studies on Collagen and Elastic Fibers .............. Biochemical Studies on Collagen and Elastic Fibers .................. The Physiology of Connective-Tissue Fibers ........................ The Production of Elastic Material from Collagen .................. Conclusions ....................................................... References ........................................................

212 213 226 234 239 246 247

Experimental Heterotopic Ossification

J . B . BRIDGES I. I1. I11. I V. V. V I. VII . VIII .

Introduction ...................................................... Heterotopic Ossification and the Urinary Tract ...................... Injection of Extracts of Skeletal Tissues ............................ Injections of Irritants and Other Traumatic Experiments ............ Implants of Devitalized Skeletal Tissues ............................ Conclusions ....................................................... Acknowledgments ................................................. References ........................................................

253 254 262 267 269 276 276 276

A Survey of Metabolic Studies on Isolated Mammalian Nuclei D . B. ROODYN I. I1. I11. I V. V. V I. VII .

Introduction ...................................................... Methods for Isolating Nuclei ...................................... Biochemical Studies on Isolated Nuclear Fractions .................. Validity of Studies on Isolated Nuclei .............................. Conclusions ....................................................... Acknowledgments ................................................. References ........................................................

279 280 285 316 335 337 337

Trace Elements in Cellular Function BERTL . VALLEEA N D FREDERIC L . HOCH

I. I1. I11. I V. V. VI . VII .

Introduction ...................................................... Emission Spectrography ........................................... Metalloenzymes and Metalloproteins ................................ Metal-Enzyme Complexes ......................................... Metals in Subcellular Fractions ..................................... Summary ......................................................... References ........................................................

345 347 350 367 375 380 381

Osmotic Properties of Living Cells

. .

D . A T DICK

.

General Introduction .............................................. Theory of Osmotic Pressure ...................................... Osmotic Properties of Protein Solutions ............................ The Relationship between Volume and Osmotic Pressure a t Equilibrium in Living Cells .................................................. V . Kinetics of Osmotic Volume Changes in Living Cells ................ VI . Acknowledgments ................................................. VII . References ........................................................

I I1. I11. I V.

388 388 395 404 427 443 443

Sodium and Potassium Movements in Nerve. Muscle. and Red Cells

I . M . GLYNN

.

I I1. I11. I V.

Introduction ...................................................... Outline .......................................................... Evidence ......................................................... References ........................................................

449 450 451 477

Pinocytosis H . HOLTER Introduction ...................................................... Morphological Aspects of Pinocytosis .............................. Induction of Pinocytosis ........................................... Attempts to Measure the Uptake of Fluid .......................... Evidence for Adsorption on the Cell Surface ........................ Dehydration of Pinocytosis Vacuoles ................................ Permeability of Pinocytosis Vacuoles ............................... Concluding Remarks .............................................. References ........................................................

481 482 488 490 492 494 498 502 503

AUTHORINDEX............................................................

505

SUBJECT INDEX............................................................

529

I. I1. I11. IV . V. VI . VII . VIII . IX .

The Structure of Cytoplasm’ CHARLES OBERLING Institut de Recherches I. 11. 111. IV.

V VI. VII. VIII. IX. X. XI. XII.

stir

le Cancer, Villejuif (Seine), France

Introduction ...................................................... Definition of Cytoplasmic Structures ............................. Mitochondria ..................................................... Mitochondria] Regeneration ....................................... Pathological Aspects of Mitochondria ............................. Ergastoplasm .................................................... Microsomes ...................................................... Origin of Ergastoplasm ......................................... Golgi Apparatus ................................................. Centriole ........................................................ Conclusion ....................................................... References .......................................................

Page

1 2 4

5 8 15 18 18 21 26 28 28

I. INTRODUCTION Perhaps nothing indicates more clearly the recent progress of cytology than the very title of this report. Thirty years ago such a subject would have been unthinkable, and Frey-Wyssling ( 1955), in his article “Die submikroskopische Struktur des Cytoplasmas,” was right when he quoted the following passage by Guillermond, Mangenot, and Plantefol, dated 1933, which vividly sums up, the opinion of that time: “Le cytoplasme proprement dit se prksente, sur le vivant comme une substance colloidale homoghne, translucide, optiquement vide i l’ultramicroscope.” This “optical emptiness,” indeed, obsessed cytologists, and this had as logical consequences : ( 1 ) the dismissal as artifacts of all the granular, reticular, alveolar, and spongy structures attributed to cytoplasm, and an extraordinary distrust, beneficial to some degree, of all the procedures of fixation; (2) the decline of morphology as a method of cell investigation for, in spite of its ever-improving techniques, it finally led only to the discovery of an “emptiness.” Thus the necessity arose of introducing new methods, better qualified, as it seemed, to improve our knowledge in the field of cytology: biochemistry and physical chemistry. My generation has lived through this period, and those who, in spite of their disappointments, had remained morphologists at heart suffered a good deal. For human nature will always move between extremes. Having 1 Report presented by the IXth International Congress for Cell Biology, St. Andrews, August-September, 1957. This work was supported by La Mutuelle ghnhrale de 1’Education Nationale.

1

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CHARLES OBERLING

been everything, cytomorphology suddenly dwindled down to nothing, and hardly any cell structure remained unquestioned. Chromatin, membranes, mitochondria, brush borders, spindle fibers, basophilic filaments, etc., were considered as polyphasic colloidal systems, precipitates, interfaces, coacervates, or simply artifacts of fixation. Perhaps we are too much inclined nowadays to smile at the excesses of those physicochemists. W e should not underestimate the value of their investigations to which we owe a number of data that modern cytology can but confirm: the discontinuity of the cytoplasmic gel, the existence of different phases separated by membranes, with enormous surfaces, ideally conceived to obtain maximum chemical efficiency in minimum space. On the whole, cytologists before World War I1 had come to the conclusion that cytoplasm was organized, but on a purely chemical, or physicochemical basis. Later, phase-contrast and electron microscopy were to show that the organization of cytoplasm is really structural in the sense of the morphologists. Indeed, morphology does not end at the micron but reaches to the angstrom. W e no longer have a no man’s land between molecular arrangements and the cytological structures actually studied, where we have to speak another language and admit our ignorance. For this had been the final conclusion reached by the physicochemists. No one perhaps has worded this better than Duclaux (1934) in his “Introduction au Traite de Chimie-Physique.” His conclusion was that in the present state of our knowledge it is impossible for us to explain the physicochemical conditions existing in the cell and, consequently, thh vital functions. “It is,” he says, “just as absurd to make man understand life as it would be to make a motorcar describe the factory where it had been built.” His conclusion, I fear, has been satisfactory neither to morphologists nor to chemists.

11. DEFINITIONOF CYTOPLASMIC STRUCTURES The outlook appears less gloomy today, above all because morphology has given us once more a firm basis, showing that all chemical and physicochemical reactions characteristic of life are realized in a structural context within reach of our methods of investigation. Oddly enough, these structures were by no means unknown. They were discovered toward the end of the nineteenth century, and classical cytologists had recognized their functional importance, often with surprising accuracy. These structures were too delicate to be adequately described with the optical microscope ; they belong essentially to the range of ultramicroscopy, and only some of their outlines emerge from that domain. But, as we have said above, they have not actually been discovered by

T H E STRUCTURE O F CYTOPLASM

3

modern cytologists. Therefore, it would be supremely unjust, as well as misleading, to give them new names under the pretext that they are better described now that we see them with bigger lenses. Among these fundamental structures of cytoplasm, mitochondria, ergastoplasm, and the Golgi apparatus are at present well recognized. They should be thought of as essential cytoplasmic components, and, in my opinion, cytoplasm should no longer be considered as “what remains of protoplasm when deprived of all clearly characterized structures” (Frey-Wyssling, 1955). Such a definition, though well in line with the general sense in which the term of cytoplasm has been employed since it was coined by Strasburger in 1882, is no longer in conformity with our present state of knowledge. How could we speak of cytoplasmic structures if we consider as cytoplasm only the portion located between the structures ? Having recognized that cytoplasm always possesses structure and that new structures are likely to be added to the existing ones, w e have to consider as being part of the cytoplasm all morphological components which are found there in a constmt and ubiquitous fashion. The abovementioned organelles are then in accordance with this definition, and they will therefore be our main concern when speaking of the structure of cytoplasm. One question still remains to be settled before we start. Are we entitled to consider as real the pictures we see in the elctron microscope? Our answer is “yes,” and our arguments are the following: 1. The types of structures as shown by the electron microscope have been seen in living cells and photographed or filmed by the phase-contrast microscope.2 2. Whenever it has been possible to compare the same cells-alive under the phase-contrast microscope, or fixed under the electron microscope-a perfect agreement in the pictures has been observed. 3. The same methods of fixation and observation reveal all the structures side by side, showing their existence in all cells, even in those which are very different both phylogenetically and functionally. 4. The appearance of these structures depends to a large extent on the perfect preservation of the cells. In order to obtain excellent pictures, cells must be fixed from the living state. This proves both the great sensitivity of the observed structures and the reliability of our technical 2 Chondrioma: motion pictures by Fell and Hughes, LettrC, Gey, FrCdCric and Chevremont ; see articles : FrCderic and Chevremont (1952). Ergastoplasma : Palay and Wissig (1953) ; Thiery (1955) ; Deitch and Murray (1956) ; E. Shelton, in Haguenau (1958). Golgi apparatus : Gatenby and Moussa (1950) ; Beams and Tahmisian (1953) ; Dalton and Felix (1953, 1954a, b, c, 1957) ; Lacy (1954).

4

CHARLES ORERLING

procedures in detecting such structural changes as take place immediately after the vital functions have ceased. 5. The technique of fractionated ultracentrifugation, the use of which has become general in modern cytological research, thanks to the pioneer work of Claude (1941-1948), has enabled us to isolate these structures physically, as it were, and obtain them in a more or less pure state, and in sufficient quantity to permit biochemical investigations, thus furthering close collaboration between morphologists and biochemists in the field of cytology. 6. These structures, as they appear to us in the electron microscope, do not always present the same aspect, but vary according to the evolutionary phases and the pathological conditions to which the cell may have been submitted. These pictures make sense and strengthen our faith in our methods of morphological investigation. Furthermore, they enable us to examine these structures not only from a static point of view but also from a more dynamic angle. It is my intention to emphasize the latter aspect of the problem. 111. MITOCHONDRIA It is unnecessary to review the purely morphological features of these components described in detail by Palade ( 1953a, b ) , Sjostrand (1953), Sjostrand and Rhodin (1953a, b), and Sjostrand and Hanzon (1954a, b, c). In the discussion between Palade and the Swedish authors on the meaning of the inner partitions (cristae) or septae, we agree with the former author: they are probably always connected at one point at least with the outer membranes, an opinion for which, however, it may be difficult to give absolute evidence. The study of embryonic chondrioconts carried out by Ferreira (unpublished observations) in our institute also supports this view. They show initial states of partitions formed by an inward projection of the inner membrane. These pictures, by the way, are liable to produce the illusion of tubules. All cytologists are aware nowadays of the various aspects of these inner partitions : they are usually transverse, sometimes longitudinal, especially in muscle cells ; they may be tubular within steroid-secreting cells (Palade, 1953a, b ; Lever, 1955; Belt and Pease, 1956) and also in Protozoa (Rudzinska and Porter, 1953 ; Sedar and Porter, 1955 ; FaurC-FrCmiet and Rouiller, 1955 ; Tahmisian et al., 1956). One of the points of mitochondria1 morphology still under discussion concerns the open forms, namely, the existence of direct communications between the content of the chondrioconts and the surrounding cytoplasm through openings in the membranes. Powers et al. (1955) admit a direct

T H E STRUCTURE OF CYTOPLASM

5

continuity between the mitochondrial tubes and the cytoplasm in Pnramecia. Lever ( 1956), investigating the behavior of mitochondria in the adrenal cortex of hamsters, was led to suppose the existence of pores in the mitochondrial membranes. H e described them as “openings which may seal up or become exceedingly small.” Mitochondria would then behave as “osmometers with a leak.” Wohlfarth-Bottermann ( 1957) in Paramecium and Weissenfels (1957) in mouse tumor cells noted what they thought to be an expulsion of the mitochondrial content into the ambient cytoplasm. It is extremely difficult to form an opinion on the reality of the images put forward on this subject by the various authors. In Protozoa, where the limiting membranes often show little contrast, intercommunication of the inner tubules and cytoplasm seems possible. In Metazoa, however, where the double-layered membranes always appear very distinctly, figures showing breaks allowing subsequent communication of mitochondria with other structures need still further substantiation. The transformation of mitochondria into secretion granules often asserted by classical cytologists has not been confirmed by electron microscopy except in a few cases such as, for instance, in the adrenal (Lever, 1956; Belt and Pease, 1956). The production of the granulomere in thrombocytes, and possibly also the formation of certain leukocyte granules and of the “osmiophilic bodies” (corps osmiophiles) in alveolar cells (Fig. l ) , may be illustrations of a similar process.

IV. MITOCHONDRIAL REGENERATION The much-discussed problem of mitochrmdrial regeneration is on the way to its final solution. Detailed observations of Chevremont and FrCdCric ( 1952) resulting from the analysis of their films have revealed that, as a rule, new mitochondria are formed by already existing mitochondria during the end phase of mitosis and in the early postmitotic growing period of the cytoplasm. The chondrioconts involved are partitioned by longitudinal splitting or transverse scission, processes duly authenticated by a wealth of convincing images obtained by the cine camera and the electron microscope (Fawcett, 1955). The possibility of mitochondria appearing de novo has been much discussed, and in this case microsomes especially have been considered as the matricial elements (Chantrenne, 1947 ; Brachet, 1949, 1952 ; Zollinger, 1950; Eichenberger, 1951, 1953). But in this field technical procedures play an all-important part, and we are aware nowadays that all conclusions based on the examination of granules obtained by ultracentrifugation without a study of ultrathin sections of those same pellets in the electron

6

CHARLES OBERLING

FIG.1. Human lung. “Osmiophilic body” in an alveolar cell. ~137,000.

T H E STRUCTURE OF CYTOPLASM

i

7

microscope should not be relied on. Such duly established and indispensable checks would no doubt have demonstrated that among the alleged “microsomes” there also existed very small-sized mitochondria (ultrachondrioma) and microbodies. These latter formations have been described by Rhodin (1954) in the convoluted tubules of the mouse kidney as corpuscules measuring 0.1 to 0.5 p, of oval or elongated shape, with a single membrane surrounding a homogenous or finely granulated central mass void of any inner structure. Rouiller and Bernhard (1956) found similar formations with an osmiophilic central nucleus in liver cells where they played very obviously the part of precursors in the generation of mitochondria. These structures, by the way, should not be confused with the granules described by Novikoff et al. (1956) and considered by these authors as counterparts of the lysosomes of de Duve et al. (1955). In cases of intense mitochondrial regeneration after partial hepatectomy or carbon tetrachloride intoxication there is an increase of the above-mentioned bodies both in number and in volume. Their central core spreads, acquires an inner structure in the shape of parallel lamellae, and all transitions appear, leading toward more and more perfect mitochondrial forms. Of course the morphological figures alone do not in themselves convey enough evidence for one to ascertain in which direction the process develops; it might equally well be a case of gradually degenerating mitochondria changing into microbodies. According to Rouiller and Bernhard ( 1956), however, the possibility of microbodies evolving toward mitochondria is the only likely one. Indeed, these structures appear after periods of wide-spread mitochondria destruction, and their appearance coincides with the period of regeneration and not of destruction. Identical facts may be observed during embryonic development. In the liver, as well as in the pancreas, young forms of mitochondria are represented by microbodies, as shown by Ferreira (1957). The same is the case with tumors (Weissenfels, 1957, and personal observations), I n conclusion we would say that the formation of mitochondria i s similar to that of the plasts which grow at the expense of the pro-blastids (Strugger, 1950). The pro-mitochondria, the existence of which had been supposed by Frey-Wyssling (1955), would then be represented by the microbodies and would have no connection whatever with microsomes. W e are thus brought to the supposition that, besides the conventionalsized mitochondria (measuring more than 270 p) , smaller bodies must exist which do not appear in the optical microscope but are, nevertheless, clearly individualized mitochondria, even if their organization is imperfect.

8

CHARL E S OBERLING

This leads us to a problem discussed some years ago when Porter and Thompson ( 1947) described elongated, wavy, or spirochete-like filamentous formations in the cytoplasm of the sarcoma cells of rats. The authors named these formation “growth granules,’’ believing that they had an important part to play in growth and probably also in the formation of nucleic acid. Oberling et al. (1950) found in human leukemic cells and, later on, in other neoplastic and nonneoplastic cells as well, particularly in exudate cells (Bernhard et al., 1950; Hare1 and Oberling, 1954), similar structures which were more polymorphous. They considered them as a very small-sized chondrioma, a sort of “ultrachondrioma.” The same formations were described later by Selby and Berger (1952) and by Selby et al. (1956) in tumor cells. In ultrathin sections they are less conspicuous than in “spread” cells. Now the existence of ultramicroscopic chondrioconts first suggested by Dalton et al. (1949) is a frequent occurrence in blood tumor cells where the ultrachondrioma seems to be especially developed. Therefore, in our opinion, there is no need to homologize the “growth granules” with other types of structures (Porter, 1955-1956).

V. PATHOLOGICAL ASPECTSOF MITOCHONDRIA Knowledge is progressing at an extremely swift pace, and mitochondrial pathology already represents a chapter of such importance that it would be beyond the scope of this report to attempt even a broad outline of the main data. Modifications of mitochondria according to age, nutrition, and various functional conditions, as well as lesions brought about by all kinds of substances, such as detergents, narcotics, and enzymatic poisons, have been extensively studied (Weiss, 1955 ; FrkdCric, 1954 ; Dempsey, 1956, and others). Abnormal storage of certain substances and the retention of abnormal substances are important characteristics of mitochondria1 pathology. In order to understand these phenomena, we must remember that there is a constant interchange of substances between the chondrioconts and the surrounding cytoplasm. In other words, there exists a transmitochondrial membrane. It is commonplace to say that mitochondria function as osmometers, but this statement, though faithfully reprinted in all the textbooks of cytology, is incorrect if expressed in this general form. It may perhaps be applied to isolated mitochondria, severed from their natural surroundings and consequently already altered. Zn vivo, there is no visible relationship between the state of hydration of the mitochondria and the state of hydration of the cytoplasm. On the other hand, we know

T H E STRUCTURE O F CYTOPLASM

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that mitochondria retain electively certain cations such as potassium (Stanbury and Mudge, 1953) ; that they may be permeable to large molecules and impermeable to much smaller ones (Hogeboom et al. 1953) ; and that certain substances such as thyroxine have an effect on the permeability of liver mitochondria (Emmelot and Bos, 1956) but not on other ones. These and numerous other observations lead to the conclusion that the mitochondrial membranes are endowed with a selective permeability, the laws of which are as yet unknown. Certain substances, after having penetrated into the interior of the mitochondria, may then, for some reason, be retained there. I n fact, these phenomena of retention play an important role in the pathology of these organelles. The simplest example is the retention of water, which results in a more or less important swelling of the mitochondria with subsequent cell lesions, known by the name of “cloudy swelling” since the time of Virchow (1858). This modification, a detailed description of which was given by Gansler and Rouiller (1956), may be studied in a number of pathological states : fasting (Fig. 2), disturbances in cell hydration, various poisonings, neoplastic transformation, etc. The mitochondria grow larger and larger, the matrix lightens, and the membranes of the inner zone grow smaller and finally disappear or are replaced by a sort of reticulum. In many places, the outer membranes disappear too, and this leads to a fusion of mitochondria. Very likely in this last phase the lesions are irreversible, whereas it has been proved that in the first stage they are perfectly curable. Other examples of storage are provided by the retention of iron pigments (Zingg and Zollinger, 1951), of ferritin (Kuff and Dalton, 1957; Bessis and Breton-Gorius, 1957a, b) of silver granules (Dempsey and Wislocki, 1955), of gall pigments (Gansler and Rouiller, 1956), of prekeratinous substances (keratohyalin bodies of Sheldon and Zetterqvist, 1955), of melanin (Woods et al., 1949), and of carcinogenic hydrocarbons (Graffi, 1939, 1940, 1941). The presence of a substance inside a mitochondrion may be purely accidental, and it does not mean, of course, that this substance has been produced there; it does not even display an influence of mitochondrial activity on the intermediate metabolism of that substance. Under various circumstances, storage can also appear as a degenerative phenomenon. The stored substance itself may become an obstacle to normal functioning, or its appearance may be the manifestation of a degenerative process taking place outside the chondriocont. The abnormal substance may also be the result of the disintegration of the mitochondrion itself. This applies more particularly to the appearance

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CHARLES OBERLING

FIG.2. Cloudy swelling in the liver cells of a rat after fasting (4 days). M,mitochondria ; Er, ergastoplasm ; m, microbodies ; c m . , cell membrane. x23,800.

of neutral fats or intensely osniiophilic lipids inside the mitochondria (Fig. 3 ) . It is interesting to note that at the level of mitochondria the same difficulties are now experienced as at the cellular level in classical pathology. We need only remember the delicate and often impossible differentiation between fatty degeneration, lipophanerosis, and fatty infiltration. Since we now have to deal with structures whose chemical composition may be determined quantitatively, there is no doubt that the study of these FIG. 3. Fatty degeneration of mitochondria in the rat liver 18 hours after partial hepatectomy. M , mitochondria ; N , nucleus ; n.m., nuclear membrane ; c.m., cell membrane. x34,OOO.

T H E STRUCTURE OF CYTOPLASM

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CHARLES OBERLING

processes, pursued simultaneously among morphological and biochemical lines, will yield more satisfactory results than those on the cellular scale. This observation also applies to the study of protein storage as a consequence of the absorption of alien proteins, by the kidney cells, a problem which has been studied by Zollinger (1950), Riittimann ( 1951), Rhodin (1954), Gansler and Rouiller (1956), and Miller and Sitte (1956). Mitochondria of convoluted tubules swell and become filled with a dense substance, more or less homogeneous, which blurrs the characteristic pattern ; they divide into fragments and are transformed into hyalin granules (Fig. 4). Here again, the study of the processes involved would benefit greatly from modern cytochemical techniques. Despite the repeated assertions of various investigators, it is not even clearly established that the hyalin masses stored in the chondrioconts actually correspond to the injected protein. The degenerative processes of mitochondria can appear under various images which, at the present state of our knowledge, have no more than a purely descriptive value : fatty, floccular, vacuolar, filamentous, ribbonlike degeneration (Fig. 5 ) (Schulz, 1956). Sometimes concentric arrangement of the inner partitions accompanies these degenerative processes. The behavior of mitochondria in cancer cells is very important, since these organites play such a fundamental role in cellular respiration, and, according to the well-known theory of Warburg, cancer has been linked to a respiratory deficiency of the cell. This question is complex, and those interested in it should refer to the articles of Selby (1953), Howatson and Ham ( 1956), Dalton et al. (1949), Lindberg and Ernster (1954), Bernhard and Oberling (1956), and Selby et al. (1956). There are cancers which show a well-developed and scarcely modified chondrioma. Sometimes we even see an extraordinary increase of mitochondria, so that the cancer cells seem to be literally crowded with them. In most cases, however, the chondrioma is severely injured, and the number of chondrioconts is obviously reduced in comparison with corresponding normal cells in hepatomas, for instance. The mitochondria are of irregular size, and their structure is more or less undifferentiated as in embryonic cells. In most cases degenerative phenomena are patent, appearing as flocular degeneration, or cloudy swelling with disappearance of the inner partitions (Fig. 6 ) . On the whole, mitochondria1 lesions in cancer cells are frequent but neither constant nor specific. It may be that they are merely the result of the circulatory troubles so common in cancerous tissues. On the other hand, their extraordinary frequency does not eliminate the possibility

T H E STRUCTURE O F CYTOPLASM

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FIG.4. Protein storage in the mitochondria of the rat kidney (convoluted tubule) after repeated injections of ovalbumin. M , mitochondria ; gh, hyalin granulation, bm, basal membrane. ~28,000. (Gander and Rouiller, 1956.)

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CHARLES OBERLING

T H E STRUCTURE OF CYTOPLASM

15

that a primary lesion may be directly responsible for the altered metabolism of the neoplastic cell.

FIG.6. Very pronounced swellink of mitochondria in a human hepatoma. N, nucleus ; wn., nuclear membrane. x31,OOO. (Courtesy of Camain and Bernhard, 1956.)

VI. ERGASTOPLASM (Fig. 7)

I shall outline only some of the main problems arising in the consideration of this fundamental structural component of cytoplasm, the history, ultrastructure, and biochemistry of which have been ably reviewed by Haguenau ( 1958). The first problem concerns nomenclature. The difficulti& here arise from our still incomplete knowledge, especially as far as cytochemistry is concerned. Furthermore, the study of ergastoplasm has been initiated almost simultaneously by different study groups with a completely difFIG.5. Ribbonlike degeneration of the mitochondria in the alveolar cells of a rat maintained in an atmosphere with increased CO, pressure. (R.Z.), ribbonlike degeneration ; M, mitochondria ; N , nucleus ; n, nucleolus. x63,OOO. (Courtesy of Dr. Schulz.)

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CHARLES OBERLING

FIG.7. Organized ergastoplasm in the exocrine pancreas. Er, ergastoplasm; mitochondria ; n.m., nuclear membrane. ~31,000.

M,

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ferent background and proceeding from different starting points. When, simultaneously with Dalton et al. ( 1950), our Villejuif team (Bernhard et d.,1951; Bernhard et d.,1952) found in the liver cells and later in other cells, (especially pancreas and salivary glands) very osmiophilic filaments and membranes, we immediately thought of the basophilic filaments of classical cytology, in other words, ergastoplasm. This soon proved to be fully justified. Weiss (1953) published a comprehensive study of the ergastoplasm in the pancreas, and Palade (1953b) demonstrated that the roughness of the membranes described by us was actually due to granules rich in ribonucleic acid. There is, therefore, no doubt that the basophilic filaments or the ergastoplasm of Garnier and Bouin are made up by the amalgamation of granules and membranes forming a complicated system of sacks, clefts, vesicles, cysternae, or tubular cavities. This typical arrangement has appropriately been called “organized” ergastoplasm by Howatson and Ham (1955, 1956). But the same structural components are not always associated. They may be found separated in the cytoplasm, and we have then to consider ribonucleoprotein granules, on the one hand, and membranes or vesicles on the other. From a quite different angle, the investigations of the Rockefeller school of Porter and Palade came across the same components. Their work grew out of the first electron-microscope investigations of cell structures performed on spread cells, cultivated on Formvar and not on tissue sections. In those early preparations of connective tissue cells, a reticular pattern of the cytoplasmic ultrastructure was quite apparent and led to the conception of an “endoplasmic reticulum.” I do not intend at this point to criticize this term. On the contrary, I should like to emphasize the importance of the principle underlying a concept which, as a whole, has been amply confirmed. It stresses the separation of cytoplasm into two main phases by a more or less elaborate membranous system similar to the pulp and the sinuses of a reticular tissue, like the spleen. The cavities of this system are connected with one another and with the exterior, possibly through the pathways of the Golgi system, as we shall see later. In this general sense, the endoplasmic reticulum or canalicular system retains its significance. But it must be remembered that the ergastoplasm and the Golgi apparatus, though they are in some way parts of this canalicular system, are highly specialized structures, which should retain their individuality. The physiological significance of the various aspects under which ergastoplasm may reveal itself is probably connected with its functional state. If the ribonucleoprotein granules are the main support of the synthetic activity and especially of the protein synthesis, the scattered or diffuse type of ergastoplasm, often predominant in embryonic

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or tumor cells, may be connected with the elementary building-up process of cellular protein and other fundamental components going on in the growing cell. The oriented, canalicular type of the organized ergastoplasm would then be the appropriate structure for the highly specialized and polarized activity of any cell engaged in secretory activities. VII.

MICROSOMES

It should be stressed here that the so-called microsome fraction of cellular ultracentrifugates is composed almost exclusively of ergastoplastic components (Fig. S), all the membranes being transformed into small vesicles during the grinding of the cytoplasmic structures which always precedes the centrifugation (Bernhard et ul., 1954; Chauveau et ul., 1955; Palade and Siekevitz, 1956a, b ; Lindberg and Ernster, 1954; Novikoff, 1956). The great amount of knowledge which has been accumulated since Claude (1941) first characterized microsomes as a definite and constant cytoplasmic component (Brachet, 1952 ; Hirsch, 1955) thus becomes immediately available for the ergastoplasm. In fact, it is the study of “microsomes” which has disclosed the fundamental significance of ergastoplasm in protein synthesis. VIII. ORIGINOF ERGASTOPLASM The formation of ergastoplasmic structures is still quite obscure. In the embryonic pancreas, Ferreira ( 1957) has noticed ribonucleoprotein granules, often arranged in a very definite geometric pattern, such as rosettes or spirals, within a denser osmiophilic texture, corresponding probably to the future membrane (Fig. 9 ) . I n regenerating liver cells after partial hepactectomy, starving, or intoxication with CCL, membranes seem to appear prior to the granules (Fawcett, 1955; Bernhard and Rouiller, 1956). But the reverse too may happen. In some instances, granules may appear anew on remaining membranes (Oberling and Rouiller, 1956). The formation of ergastoplasmic membranes by invagination of the cell membrane (Palade, 1955) probably occurs but is not, in our opinion, a phenomenon of general significance. The close relationship between organized ergastoplasm and basophilic structures being established beyond doubt, the question arises whether basophilia, as such, is related to the granules alone, as claimed by Palade and Siekevitz (1956a). I t is well known that in some types of cells, such as the lymphocytes or the silk glands of the silkworms, where basophilia is intense, no membranes are formed and only granules are present.

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FIG.8. Microsome pellet of the liver (rat) obtained by ultracentrifugation (40,000

x

g, 20 minutes). Numerous ribonucleoprotein granules and vesicles (ergastoplasmic membranes). x45,OOO.

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CHARLES OBERLING

I t has also been shown that the granules, when isolated by ultracentrifugation, with or without deoxycholate, contain the major part of R N A present in the microsome fraction. It is therefore reasonable to link basophilia with the small granular component. But this does not mean that all cytoplasmic RNA is concentrated solely in the granules. This was suggested by Kuff et al. (1956) and was again brought to light through investigations carried out in our institute by Chauveau et al. (1957). It

FIG.9. Ergastoplasm in the embryonic pancreas (rat). The ribonucleoprotein granules are arranged in a regular geometric pattern within a dense osmiophilic ground substance corresponding probably to the future membrane (arrows). ~ 5 4 , 0 0 0 . (Courtesy of Dr. Ferreira.)

appears from their work that meinbranes too may have a high R N A content, and treatment with deoxycholate may unmask it in a granular form. But we do not know whether these membranes, in contrast to those of the Golgi apparatus, have basophilic staining properties. The same uncertainties are met with when we investigate the origin of the RNA present in the cytoplasmic structure. Since the pioneer work of Caspersson (1950) and Brachet (1952) we have been brought to think of the nucleus as the origin of the cytoplasmic RNA, and very convincing pictures have been published to show the passage of basophilic material

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from the nucleus through the nuclear membrane, its accumulation on the surface of the nucleus, and its final diffusion into the cytoplasm. (Caspersson, 1950; Vogt and Vogt, 1947; Altmann, 1949-1952, Hirsch, 1955 litt.). This view seemed in perfect accordance with the close morphological similarity between the nucleolar granules and the RNA granules of the cytoplasm. Moreover, the nucleolus is often conspicuously close to the nuclear membrane. This tendency is strikingly apparent in neoplastic cells, where deep invaginations of the membrane afford a direct and extensive contact with the nucleolar substance and seem to promote a possible extrusion of nucleolar material into the cytoplasm. Such a migration, however, with few exceptions so far (Anderson and Beams, 19.56), has not been convincingly demonstrated with the electron microscope, and we have to admit that if it occurs, which is very likely, it takes place in a form not detectable by our methods. The study of the pathology of ergastoplasm is still in its infancy. W e know about quantitative variation in relation to different functional states, about the scattering of ergastoplastic structures in cancer cells (Fig. l o ) , and the dilatation of cysternae in liver cells which is responsible for one type of the “vacuolar degeneration” in classical cytopathology . Oberling and Rouiller (1956) have described this lesion in CC1, intoxication of the liver (Fig. 11). It is interesting that Bassi and Bernelli-Zazzera (1957) observed the same in liver cells from hypoxic rats, especially since liver lesions resulting from CCll poisoning have been linked with circulatory troubles due to the obstruction of the intralobular capillaries by the swollen liver cells (Glynn and kimsworth 1948). IX.

GOLGIAPPARATUS

The Golgi apparatus provides a striking example of the superiority of modern electron-microscope technique in cytological investigations. With a simple gesture, like the Hexenmister in the well-known poem, it has brought to an end one of the most vehement scientific controversies in modern times. I n an emotionally cleared atmosphere, our concept of the Golgi complex has evolved into the idea of Golgi structures; the authenticity of which are no longer under discussion. The credit for this goes to Dalton and Felix ( 1953-1957). Their description of unmistakable sheets of paired membranes and vesicles devoid of ribonuclein granules embedded in a slightly osmiophilic ground substance has been confirmed by Sjostrand and Hanzon (1954b), Haguenau and Bernhard (1955), and others. The striking point is the uniformity of this structure in the most different species and in cells functionally as distinct as nervous, muscular, and glandular cells. There is, therefore, no doubt that this structure is a

22

CHARLES OEERLING

FIG.10. Scattered ergastoplasm in the cytoplasm of a tumor cell (Ehrlich's ascites carcinoma). N , nucleus ; rt.m., nuclear membrane. x53,OOO.

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23

FIG.11. Dilatation of the ergastoplasmic cysternae in the liver cell of a rat 60 minutes after injection of carbon tetrachloride. M , mitochondria; b.c., bile canaliculus. x 36,000.

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CHARLES OBERLING

very fundamental one, its nature unfortunately being still rather conjectural. The part played by the Golgi apparatus in secretion seems obvious. Whereas the formetion of secretion products probably takes place within the ergastoplasm, the appearance of the products in the shape of secretion granules generally occurs in the Golgi apparatus. Convincing examples may be found in the hypophysis (Haguenau and Bernhard, 1955), exocrine pancreas ( Sjostrand and Hanzon, 1954b), endocrine pancreas ( Wissig, 1956 ; Ferreira, 1957), and biliary pigments (personal observations). It is also known that the Golgi apparatus (idiosome) plays an important part in the secretion of the acrosome of spermatids (Clermont and Haguenau, 1955 ; Burgos and Fawcett, 1955 ; Grass6 et al., 1956). The secretion products in Golgi structures are probably condensed. It may also be that some of them are wrapped in membranes and rejected in this form. It appears from the work of Weiss (1955) and Palay (1958) that after having crossed the striated membranes the fats absorbed by the intestinal cells collect in the Golgi apparatus. It seems, therefore, that the Golgi apparatus is a sort of gate or lock between the living matter itself and the exterior of the cell. Certain signs, moreover, lead to the belief that the Golgi apparatus, in regulating the passage of and in concentrating and conditioning the substances leaving or entering the cytoplasm, is showing only one of the aspects of a much more important functional assignment. Gatenby et d. (1955) have reported a certain similarity between the Golgi apparatus and the contractile vacuoles of Protozoa. Electron-microscope examination has revealed the existence of parallel membrane systems, similar to the Golgi structures, in contact with the walls of these vacuoles. On the other hand, it becomes more and more obvious (Bartley et al., 1954) that there exists a constant circulation of water through the cell. This circulation, on account of the hypertonicity of the intracellular media (Robinson, 1950) requires an active rejection of excess liquid entering through the cellular membrane. Because of the biphasic arrangement of the cytoplasm, this exchange cannot possibly take place at random through the cellular membrane but requires within the cell a device to secure a constant circulation in a given direction. This same structure would have to maintain the osmotic pressure gradient and control the ionic exchange between the intra- and extracytoplasmic media. Now the Golgi apparatus, considered in respect to all we know of its functions and its structure, seems ideally suited to fulfill these circulatory, secretory, and osmoregu-

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latory functions. This role, which is of primary importance for cellular functions, would account perfectly for the universality of this structure (Fig. 12). The pathology of the Golgi apparatus is still largely unknown, but it is certainly important. The hypertrophy of these structures under the influence of certain hormones produces giant Golgi structures, as may be seen in tumors of the hypophysis (Fig. 13) (Severinghaus, 1937; Wolfe and Wright, 1938; Haguenau and Lacour, 1954) and in less apparent, but still

FIG. 12. Golgi apparatus in a flagellate (Chromulirtu psummobiu). M, mitochondria ; C, chromoplast; N , nucleus. x39,OOO. (Rouiller and FaurC-FrCmiet, 1958.)

very important hypertrophies of certain prostatic tumors (Bothe et al., 1950). In view of the location of the Golgi apparatus, it must be admitted that all the cellular modifications that occur in the paranuclear region, such as the formation of some inclusions and the “plastinreaction” described by Lipschutz (1931), must be in some way connected with these structures. W e should mention as a contribution to this subject the part played by the Golgi apparatus in the formation of virus corpuscules corresponding probably to the milk factor of Bittner (Fig. 14) (Bernhard et al., 1956). In fact, there exists a close topographic relationship between the Golgi area and these virus corpuscules: the Golgi components seem to play a direct part in the formation of virus membranes.

26

CHARLES OBERLING

X. CENTRIOLE The centriole, though conspicuous especially during mitosis, is probably a constant feature even during the interphase and logically belongs to the cytoplasmic structures. It has been observed by Bessis and Locquin (1950) and by Policard and Bessis (1952) in the living cell. Its ultrastructure was first described by de Harven and Bernhard (1956). When studying numerous normal and abnormal cells, especially cancer cells, as

FIG.13. Hypertrophy of the Golgi apparatus in a pituitary tumor. x27,OOO.

well as thymus and spleen cells with colchicine-blocked mitosis, these authors on numerous occasions observed organelles undoubtedly corresponding to centrosomes, They were of a peculiar cylindrical shape with strongly osmiophilic walls and contained a certain number of tubules lying parallel to each other and to the long axis of the cylinder. The appearance of rings very similar to the basal bodies of cilia undoubtedly represents, therefore, cross sections of these structures. This similarity between centrosomes and basal bodies of cilia is interesting, since, morphologically speaking, it assimilates two structures which are physiologically related to movement as was first stressed by Henneguy (1898), thus justifying

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FIG.14. Golgi apparatus in the cell of a mouse mammary carcinoma. Arrows, virus particles ; v, Golgi vesicles ; c.m., cell membrane ; e.c., excretory canaliculus ; N, nucleus ; a.m., nuclear membrane. x41,OOO.

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CHARLES OBERLING

the old concept of the “kinetosoms” (a term coined by Allen, 1912) and strongly advocated by Chatton (1930). XI. CONCLUSION Much remains to be said. I should speak of the ground substance in which no constant structure has so far been discovered and which, for this reason, is often called “hyaloplasm.” But, like Frey-Wyssling ( 1955), I am convinced that here, too, the protein or lipoprotein molecules are arranged in a certain pattern, more plastic perhaps than that of other structures, but nevertheless clearly definite. I n certain cells, distinct fibrillar or reticular arrangements of the hyaloplasm have already been found. It is not clear for the moment, however, whether these structures are peculiar to special cells or ubiquitous, like the structures discussed so far. New methods of fixation and other developments of present-day techniques will, no doubt, reveal yet unknown structures of cytoplasm. Nevertheless, it must be borne in mind that our present fixation techniques are adapted to the macromolecular level and that higher magnifications, which would bring cytomorphology into the realm of molecular chemistry, would find us utterly unprepared as far as the preservation of structures is concerned. The ribonucleoprotein granules or the nucleotide chains may then well appear as being miserably fixed according to our present procedures, as, for instance, the ergastoplasm or the mitochondria in Carnoy’s fluid. Each degree of magnification needs its mode of preservation, and I am afraid that it is this prerequisite and not the magnifying power of electron or proton microscopy which limits our scope and will prevent us, for the time being, from filling completely the gap between morphology and chemistry. But these problems are the concern of the future. For the present, let us enjoy the tremendous opportunities which are immediately within our reach and which warrant rich harvests and exciting discoveries for generations of workers. XII. REFERENCES Allen, C. E. (1912) Arch. Zellforsch. 8, 12. Altmann, H. W. (1949) 2. Naturforsch. 4, 138. Altmann, H. W. (1952) 2. Krebsforsch. MI, 632. Anderson, E., and Beams, H. W. (1956) J. Biophys. Biochem. Cytol. 2, 439. Bartley, W., Davies, R. E., and Krebs, H. A. (1954) Proc. Roy. Soc. B1&, 187. Bassi, M., and Bernelli-Zazzera, A. (1957) Nature 179, 256. Beams, H. W., and Tahmisian, T. N. (1953) Cytologia (Tokyo) 18, 157. Belt, W. D., and Pease, D. C. (1956) J. Biophys. Biochem. Cytol. 2, 369. Bernhard, W., and Oberling, Ch. (1956) Can. Cancer Conf. 2, 59. Bernhard, W., and Rouiller, Ch. (1956) J . Biophys. Biochem. Cytol. 2, 73. Bernhard, W., Braunsteiner, H., Febvre, H. L., Harel, J., Klein, R., and Oberling, Ch. (1950) Rev. hintatol. 6, 746.

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29

Bernhard, W., Gautier, A., and Oberling, Ch. (1951) Compt. rend. soc. biol. 146,

566. Bernhard, W., Haguenau, Fr., Gautier, A., and Oberling, Ch. (1952) 2. Zellforsch. u. mikroskop. Anat. 37, 281. Bernhard, W., Gautier, A., and Rouiller, Ch. (1954) Arch. anat. microscop. 43, 236. Bernhard, W., Guirin, M., and Oberling, Ch. (1956) Acta Unio Intern. contra Cancrum 12, 544. Bessis, M., and Breton-Gorius, J. (1957a) J . Biophys. Biochem. Cytol. S, 503. Bessis, M., and Breton-Gorius, J. (1957b) Compt. rend. 244, 2846. Bessis, M.,and Locquin, M. (1950) Compt. rend. soc. biol. 144, 483. Bothe, A. E.,Dalton, A. J., Hastings, W. S., and Zillesen, F. 0. (1950) J . Natl. Cancer Inst. 11, 239. Brachet, J. (1949) Bull. soc. chim. biol. 91, 724. Brachet, J. (1952) “Le r6le des acides nucliiques dans la vie de la cellule et de I’embryon.” Masson, Paris. Burgos, M. H., and Fawcett, D. W. (1955) J . Biophys. Biochem. Cytol. 1, 287. Camain, R., and Bernhard, W. (1956) Personal communication. Chantrenne, H. (1947) Biochim. et Biophys. Acta 1, 437. Caspersson, T. (1950) “Cell Growth and Cell Function.” Norton, New York. Chatton, E. (1930) Arch. zool. ital. 16, 169. Chauveau, J., Gautier, A., MOUE,Y., and Rouiller, Ch. (1955) Compt. rend. 241,

337. Chauveau, J., Mouli, Y., and Rouiller, Ch. (1957) Exptl. Cell Research lS, 398. Chhremont, M., and FrekIdCric, J. (1952) Arch. biol. ( L i i g e ) Bs, 259. Claude, A. (1941) Cold Spring Harbor Symposia Quant. Biol. 9, 263. Claude, A. (1944) A.A.A.S. Research Conf. Cancer 223. Claude, A. (1946) J. Exptl. Med. 84, 51. Claude, A. (194748) Harvey Lectures Ser. 4S, 121. Clermont, Y.,and Haguenau, Fr. (1955) Compt. rend. M, 708. Dalton, A. J., and Felix, M. D. (1953) Am. J . Anat. 92, 277. Dalton, A. J., and Felix, M. D. (1954a) A m . I. Anat. 94, 171. Dalton, A. J., and Felix, M. D. (1954b) “Fine structure of cells.” 7th Congr. Cell Biol. (Leiden) 274. Dalton, A. J., and Felix, M. D. (1954~) Am. J . Anat. 94, 171. Dalton, A. J., and Felix, M. D. (1956) 1. Biophys. Biochem. Cytol. 2, 79. Dalton, A. J., and Felix, M. D. (1957) Symposia SOC.Exptl. Biol. No. 10, 148. Dalton, A. J., Kahler, H., Kelly, M. G., Lloyd, B. J., and Striebich, M. J. (1949) J . Natl. Cancer Znst. 9, 439. Dalton, A. J., Kahler, H., Striebich, M. J., and Lloyd, B. (1950) J . Natl. Cancer Znst. 11, 439. Deitch, A. D., and Murray, M. R. (1956) J . Biophys. Biochem. Cytol. 2, 433. Dempsey, E. W. (1956) J. Biophys. Biochem. Cytol. 2, 305. Dempsey, E. W., and Wislocki, G. B. (1955) J . Biophys. Biochem. Cytol. 1, 99. Duclaux, J. (1934) “Introduction au Trait6 de Chimie-Physique.” Paris. Duve, C. de, Pressman, B. C.,Gianetto, R., Wattiaux, R., and Appelmans, F. (1955) Biochem. J . 80, 604. Eichenberger, M. (1951) Elektronenmikroskopische Beobachtungen iiber die Entstehung der Mitochondrien aus Mikrosomen. Inaugural Dissertation, Zurich. Eichenberger, M. (1953) Exptl. Cell Research 4, 275.

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Emmelot, P., and Bos, C. J. (1956) Esptl. Cell Research 12, 191. FaurC-FrCmiet, E., and Rouiller, Ch. (1955) Compt. rend. 241, 678. Fawcett, D. W. (1955) J. Natl. Cancer Znst. 16, 1475. Ferreira, D. (1957) J. Ultrastructural Research 1, 14. FrMCric, J. (1954) Ann. N.Y. Acad. Sci. 68, 1246. FrCdCric, J., and Chevremont, M. (1952) Arch. biol. ( L i i g e ) 63, 109. Frey-Wyssling, A. (1955) Protoplasmatologia, Hdb. d. Protoplasmaforsch. IIA2. “Die submikroskopische Struktur des Cytoplasmas.” Springer-Verlag, Vienna. Gander, H., and Rouiller, Ch. (1956) Schweiz. 2. allgem. Pathol. u. Bakteriol. 19,217. Gatenby, J. B., and Moussa, T. A. A. (1950) J. Roy. Microscop. SOC.70, 342. Gatenby, J. B., Dalton, A. J., and Felix, M. D. (1955) Nature 176, 301. Glynn, L. E., and Himsworth, H. P. (1948) Clin. Sci. 6, 235. Graffi, A. (1939) 2. Krebsforsch. 49, 177. Graffi, A. (1940) 2. Krebsforsch. 50, 196. Graffi, A. (1941) 2. Krebsforsch. 62, 165. GrassC, P.-P., Carasso, N., and Favard, P. (1956) Ann. sci. nat. 2001.et biol. animale 18, 339. Haguenau, Fr. (1958) Intern. Rev. Cytol. 7 , 425. Haguenau, Fr., and Bernhard, W. (1955) Arch. anat. microscop. 44, 27. Haguenau, Fr., and Lacour, F. (1954) “Fine structure of cells.” 7th Congr. Cell Biol. (Leiden) 317. Harel, J., and Oberling, Ch. (1954) Brit. I. Cancer 8, 353. Harven, E. de, and Bernhard, W. (1956) 2. Zellforsch. u. mikroskop. Attat. 45, 378. Henneguy, L. F. (1898) Arch. anat. microscop. 1, 481. Hirsch, G. C. (1955) Allgemeine Stoffwechselmorphologie des Cytoplasmas. “Hdb. d. allg. Pathol.” Vol. 11, Part 1, p. 92. Springer, Berlin. Hogeboom, G. H., Schneider, W. C., and Striebich, M. J. (1953) Cancer Research 13, 617. Howatson, A. F., and Ham, A. W. (1955) Cancer Research 16, 62. Howatson, A. F., and Ham, A. W. (1956) Can. Cancer Conf. 2, 17. Kuff, E. L., and Dalton, A. J. (1957) J. Ultrastructural Research 1, 62. Kuff, E. L., Hogeboom, G. H., and Dalton, A. J. (1956) J. Biophys. Biochem. Cytol. a, 33. Lacy, D. (1954) J. Roy. Microscop. SOC.74, 1. Lever, J. D. (1955) Am. I . Anat. 97, 409. Lever, J. D. (1956) J. Biophys. Biochem. Cytol. 2, 313. Lindberg, O., and Ernster, L. (1954) Chemistry and physiology of mitochondria and microsomes. “Plasmatologia” IIIA4, 1. Springer, Vienna. Lipschiitz, B. (1931) 2. Krebsforsch. 34, 299. Miller, F., and Sitte, H. (1956) Verhandl. deut. Ges. Pathol. 39, 183. Novikoff, A. B. (1956) Science l24, 969. Novikoff, A. B., Beaufay, H., and Duve, C. de (1956) J. Biophys. Biochem. Cytol. 2, 179. Oberling, Ch., and Rouiller, Ch. (1956) Ann. anat. pathol. 1, 401. Oberling, Ch., Bernhard, W., Braunsteiner, H., and Febvre, H. L. (1950) Bull. assoc. franc. itude cancer 37, 1. Palade, G. E. (1953a) J. Histochem. and Cytochem. 1, 188. Palade, G. E. (1953b) J. Appl. Phys. 24, 1419.

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31

Palade, G. E. (1955) J . Biophys. Biochem. Cytol. 1, 59. Palade, G. E., and Siekevitz, P. (1956a) J . Biophys. Biochem. Cytol. 2, 171. Palade, G. E., and Siekevitz, P. (1956b) J . Biophys. Biochem. Cytol. 2, 671. Palay, S. L. (1958) “Morphology of Secretion.” Yale Univ. Press, New Haven, Connecticut. Palay, S. L., and Wissig, S. L. (1953) Anat. Record 116, 301. Policard, A., and Bessis, M. (1952) Compt. rend. 284, 913. Porter, K. R. (1955-56) Harvey Lectures Ser. 61, 175. Porter, K. R., and Thompson, H. P. (1947) Cancer Research 7, 431. Powers, E. L., Ehret, C. F., and Roth, L. E. (1955) Biol. Bull. 108, 182. Rhodin, J. (1954) “Correlation of ultrastructural organization and function in normal and experimentally changed proximal convoluted tubule cells of the mouse kidney.” Karolinska Institute, Stockholm. Robinson, J. R. (1950) Proc. Roy. SOC.B197, 378. Rouiller, Ch., and Bernhard, W. (1956) J . Biophys. Biochem. Cytol. 2, 355. Rouiller, Ch., and Faur6-FrCmiet, E. (1958) Exptl. Cell Research 14, 47. Rudzinska, M. A., and Porter, K. R. (1953) Anat. Record 116, 363. Riittimann, A. (1951) Schweiz. Z . allgem. Pathol. u. Bakteriol. 14, 373. Schulz, H. (1956) Naturwiss. 43, 205. Sedar, A. W., and Porter, K. R. (1955) J . Biochem. Biophys. Cytol. 1, 583. Selby, C. C. (1953) Cancer Research 19, 753. Selby, C. C., and Berger, R. E. (1952) Cancer 6, 770. Selby, C. C., Biesele, J. J., and Grey, C. E. (1956) Ann. N . Y . Acad. Sci. 68, 748. Severinghaus, A. E. (1937) Physiol. Revs. 17, 556. Sheldon, H., and Zetterqvist, H. (1955) Exptl. Cell Research 10, 225. Sjostrand, F. S. (1953) Nature 171, 30. Sjostrand, F. S., and Hanzon, V. (1954a) Experientia 10, 367. Sjostrand, F. S., and Hanzon, V. (1954b) Exptl. Cell Research 7, 393. Sjostrand, F. S., and Hanzon, V. (1954~) Exptl. Cell Research 7, 415. Sjostrand, F. S., and Rhodin, J. (1953a) J . Appl. Phys. 24, 116. Sjostrand, F. S., and Rhodin, J. (1953b) Exptl. Cell Research 4, 426. Stanbury, S. W., and Mudge, G. H. (1953) Proc. SOC.Exptl. Biol. Med. 82, 675. Strugger, R. (1950) Naturwiss. 37, 166. Tahmisian, T. N., Powers, E. L., and Devine, R. L. (1956) J. Biochem. Biophys. Cytol. Suppl. 325. ThiCry, J. P. (1955) Rev. hbmnutol. 10, 745. Virchow, R. (1858) “Die Cellularpathologie,” p. 267. Berlin. Vogt, C., and Vogt, 0. (1947) Aerztl. Forsch. 1, 1, 43. Weiss, J. M. (1953) J . Exptl. Med. 98, 607. Weiss, J. M. (1955) J. Exptl. Med. 102, 775. Weissenfels, N. (1957) 2. Naturforsch. 12b, 168. Wissig, S. L. (1956) Thesis, Yale Univ., New Haven, Connecticut. Wohlfarth-Bottermann, K. E. (1957) 2. Naturforsch. lab, 164. Wolfe, J. W., and Wright, A, W. (1938) Endocrinology 29, 200. Woods, M. W., du Buy, H. G., Dean, Burk, and Hesselbach, M. L. (1949) J . Natl. Cancer Znst. 9, 311. Zingg, W., and Zollinger, H. U. (1951) Mikroskopie 6, 72. Zollinger, H. U. (1950) Rev. htmatol. 6, 696.

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Wall Organization in Plant Cells R. D. PRESTON Department of Botany, University of Leeds, Leeds, England

I. Introduction

......................................................

11. The X-Ray Diagram and Electron-Microscopic Appearance of Cellulose ............................................................. 111. The Chemical Composition of Microfibrils ......................... IV. The Biosynthesis of Microfibrils .................................. V. The Synthesis and Orientation of Microfibrils at a New Cytoplasmic Surface .......................................................... VI. References .......................................................

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36 40 51

53 58

I. INTRODUCTION The fibrous nature of plant cell walls has forced itself on the notice of investigators ever since the first detailed observations by Grew published in 1682. It is true that for about 150 years after these first statements doubt was repeatedly expressed concerning their validity, but these were invariably themselves based on faulty observation. As the optical system of the light microscope was improved during the nineteenth century, cell walls appeared progressively fibrous down to the limits of resolution, and the postulated units of structure became smaller and smaller, until finally studies under the polarizing microscope were found to demand the presence of rodlets smaller in diameter than the wavelength of visible light. The evidence relating to observations under the light microscope was based both on intact cell walls and on walls which had been mechanically treated or chemically swollen. With intact cell walls, the major evidence concerned the frequent appearance of striations. Thus Dippel ( 1879), Schmitz ( 1880), Krabbe ( 1887), and Strasburger ( 1898) explained the appearance of these striations in terms of contact faces between adjacent “screw bands” in intimate contact. Again Wiesner (1892) was led to the interesting speculation that the wall is composed of “dermatosomes” separated by layers of some protein or its derivative, a residue of the original protoplast. Although subsequent chemical determinations seem to indicate that the protein content of cell walls is in general too low to allow the presence of the kind of structure figured by Wiesner, this suggestion still retains some interest in view of the current belief that in primary walls at least the protoplasm and the wall interpenetrate. Wiesner further considered these minute bodies to be aggregated into fibrils, which produce the appearance of striations, and finally into wall layers. With swollen or mechanically distorted walls, attention has been repeatedly called in the literature to the development of particles of a somewhat 33

34

R. D. PRESTON

higher order of magnitude generally in the form of long threadlike bodies termed fibrils. These have been described, for instance, by Criiger (1854) for wood fibers, by Reimers (1922) for phloem fibers of several plants (see also Steinbrinck, 1927; Herzog, 1910; Herzog and Jancke, 1928), by Balls (1922) and Dischendorfer (1925) for cotton hairs, and also by several investigators for algae (van Iterson, 1933 ; Preston and Astbury, 1937; Nicolai and Frey-Wyssling, 1938). The diameter of these fibrils appears to be of the order of 0.4 p in the unswollen condition (Balls, 1922), and they were throughout considered to be built of subunits which themselves are elongated. It was shown by Ritter and by Farr and her co-workers (e.g., Farr and Sisson, 1934) that partial hydrolysis causes a falling apart into fusiform bodies and finally into minute ellipsoidal particles. Although all these observations were taken collectively as proof that the walls of plant cells are fundamentally fibrillar in nature, a word of warning was occasionally expressed that the appearance of fibrils consequent on swelling or disintegration of walls is no guarantee of the presence of fibrils in untreated material-a point of view which was emphasized, for instance, by the work of Bailey and Kerr (1935), who showed that in the walls of xylem elements the cellulose and lignin consist of interpenetrating matrices grading down to the limits of microscopic visibility. These authors suggested that the so-called fibrils are merely dissected fragments of such a network. The application of the method of X-ray diffraction analysis to cell walls had already, however, by that time placed the fundamentally fibrillar nature of cell walls beyond any possibility of doubt. It was clear that, particularly in elongated cells, the cellulose component consists of long molecular chains which, in certain regions at least of the wall, are arranged parallel to each other and spaced regularly the same distance apart in a space lattice ( Sponsler and Dore, 1926 ; Meyer and Mark, 1928 ; Mark and Meyer, 1929). It was further known that one possible interpretation of the X-ray diagram of the cellulose occurring in cell walls involved the presence in the cell wall of rodlets called micelles, to use the term proposed much earlier by Nageli, of the order of 50 A. in diameter and at least 600 A. long (Hengstenberg and Mark, 1929). It was further realized that cjther phenomena exhibited by cell walls demanded the presence of larger units than this, and indeed a synthesis of all the evidence at that time available led Frey-Wyssling (1937) to propose a model of wall structure closely resembling the modern view. The first observations of intact cell walls in the electron microscope by Preston et al. (1948a) and a few weeks later by Frey-Wyssling et al. (1948) placed the existence of fibrillar units of submicroscopic dimen-

WALL ORGANIZATION I N PLANT CELLS

35

sions beyond any doubt. It has now become progressively clearer that native cellulose, wherever it is found, occurs in the form of microfibrils and that the fibrils of the older workers are therefore aggregates of these units. Electron diffraction analysis (Preston and Ripley, 1954b) has shown conclusively that the microfibrils in VaZonia consist of parallel molecular chains of cellulose, and it has become the custom to identify microfibrils seen in cell walls as cellulosic. Later work, however, has shown that this cannot always be substantiated, and there is now no doubt that other molecular species found in walls are incorporated in the microfibrils. Roelofsen and Kreger (1954) were the first to show, using collenchymatous cells of Petasites vulgaris, that the pectic compounds so common in walls may also occur in the form of microfibrils. It has been known for some considerable time that microfibrils visible in cell walls generally cannot in any case be identical physically. It is now quite certain also that the microfibrils visible in the wall do not always correspond to the same molecular species. Even when the X-ray and electron diffraction diagrams suggest that the microfibrils may be referred to cellulose, it is not invariably true that the only sugar involved in the structure is glucose. It is the purpose of the present article to review the more recent evidence concerning the constitution of microfibrils in plant cell walls. This topic is of some considerable importance, not only for structural reasons alone, but because the microfibrils are undoubtedly produced by enzyme complexes situated on or near the surface of the cytoplasm, and the detailed delineation of the products of reaction of these enzymes is one way, perhaps at present the only way, of defining these complexes. Although attention here will for this reason be confined largely to the microfibrils, it must be emphasized that these form by no means the only component of wall structure. Indeed, it is already clear in the literature, and will be emphasized again below, that the cellulose microfibrillar fraction of a wall is sometimes so small as to be quantitatively negligible. This is particularly true of the walls of growing cells in higher plants (Bonner, 1936; Allsopp and Misra, 1940) and of some algae (Cronshaw et aZ., 1958). On the basis of the relative quantity alone of the “encrusting” components of cell walls it seems inevitable that the behavior of cell walls, particularly as affecting the growth processes of cells, may often be determined to a large extent by the properties of these amorphous materials.

36

R. D. PRESTON

11. THE X-RAY DIAGRAMA N D ELECTRON-MICROSCOPIC APPEARANCE OF CELLULOSE It will be shown later in this review that there is now considerable doubt as to the precise meaning to be put on the term “cellulose,” but for the moment we shall accept the general criteria that, if a cell wall material is optically anisotropic, can be made even if with difficulty to stain with iodine and sulfuric acid or chlorozinc iodide, and gives spacings in the X-ray diagram of the order of 3.9, 5.4, and 6.1 A., then the substance may be called cellulose. On the basis of these criteria, then the microfibrils of this substance we call cellulose range in diameter, over a wide variety of plant species, from something less than 100 A. to something rather over 200 A. (Ranby and Ribi, 1950 ; Preston et al., 1948a ; Preston, 1951) in the form of ribbons which are flatter than they are wide (Preston, 1951). They can be either straight or very much contorted. This dissimilarity between microfibrils from species to species both in diameter and in flexibility itself suggests some difference in architecture. W e may compare two extreme cases, the microfibrils of Valonk cellulose on the one hand with those of the cellulose of conifer cambium on the other. In the former the microfibrils are large in diameter, are approximately straight over considerable distances extending to much more than 10 p, and the X-ray diagram (Plate I, Fig. 1) is so sharp that it suggests the microfibrils to be wholly crystalline (Balashov and Preston, 1955 ; Preston and Cronshaw, 1958). In the primary cellulose of conifer cambium cell walls, however, the microfibrils are much thinner, follow a tortuous path, and the X-ray diagram (Plate I, Fig. 2) suggests that the crystallites of which they are composed are no more than 25 A. in diameter, about onefourth or one-fifth the size of the microfibril diameter. In these two cases the microfibrils can hardly be identical physically. Further, it is found PLATEI FIG.1. X-Ray diagram of a single piece of Vulonio wall, beam normal to wall surface. CuK, radiation, flat film, specimen-film distance ca. 3 cm. Note that all the arcs are sharp in the radial direction. This implies large crystalline size. FIG.2. X-Ray diagram of a flattened strip of conifer cambium, beam parallel to flattened faces and perpendicular to the length of the fusiform initials. Radiation details as in Fig. 1. Note that the arcs are wide radially, indicating a small crystalline size. FIG.3. Electron micrograph of isolated Vololziu microfibrils treated with Ag. Unshadowed ; X 30,000. Note that the microfibrils appear uniform. FIG.4. Electron micrograph of isolated microfibrils of conifer wood treated with Ag. Unshadowed; X 30,000. Note that the microfibrils have taken up Ag heterogeneously as evidenced by the variation in opacity along their lengths.

WALL ORGANIZATION I N PLANT CELLS

37

chemically that some celluloses, for instance the celluloses of wood and of jute, can be broken down by acid treatment to small fusiform bodies of the order of 50 to 100 A. in diameter (Ranby, 1949, 1951; Mukherjee and Woods, 1953), whereas Valonia cellulose must be treated by much stronger acid in order to break down the fibrils, and even then the microfibrils become transposed into cellulose I1 and apparently fail to break down into these small fusiform bodies (Preston, 1951). Again, it has been

38

R. D. PRESTON

concluded by Jorgenson ( 1949) that the noncrystalline regions are much more extensive in wood cellulose than they are in cotton cellulose. The indications are clearly that among the different celluloses in nature there is a marked variability in the degree of crystallinity. It is particularly to be stressed at this point that, although in any one species the elementary microfibrils may perhaps aggregate together to give fibrils of greater diameter, these findings show the larger microfibrils of, for instance, Valonia not to be multiples of microfibrils of the size of those found in, for instance, wood. This variability can also be demonstrated, perhaps more directly, by observation of the so-called “small-angle scattering” of X-ray diagrams (Kratky and Porod, 1954; Heyn, 1950, 1955). In X-ray diagrams of fibrous substances, in addition to the wide-angle scattering shown for Valonia cellulose in Plate I, Fig. 1, there occurs, close to the center of the diagram and resolved only when the specimen-to-film distance is increased, say, to 10 ,or 20 cm., a streak along the line .corresponding to the equator. This is interpreted as scatter from individual particles with the possibility of some interparticle interference. Its presence alone, however, demonstrates that the crystallites involved in the scattering are long narrow rods. In passing from the center of the diagram along this equatorial scatter there occurs a sudden minimum in the intensity of the scattered radiation, followed by a much weaker scatter ranging up to the position of the arcs in the wide-angle diagram. The position of minimum intensity is related to the particle size, and the fact therefore that the angular position of this minimum from the center varies with celluloses of different origin suggests that the constituent particles are of different diameters. Heyn (1955) has shown that the average particle diameters in, for instance, jute and cotton cellulose are 25 A. and 55 A., respectively. If we remember that the microfibrils of these two celluloses are not markedly different in diameter, it is clear that the microfibrils must differ in over-all crystallinity. Phenomena which may be related to this difference can under suitable conditions be observed in the electron microscope. If, for instance, widely separated microfibrils of Valonia are treated with silver nitrate, exposed to light, washed with distilled water and mounted in the electron microscope, the appearance is that presented in Plate I, Fig. 3. The microfibrils appear to be completely uniform, and there is no evidence that silver has been taken into them. If it has, then the distribution of silver is uniform. When, however, microfibrils of wood cellulose are similarly treated, they present the entirely different appearance represented in Plate I, Fig. 4. Here the microfibrils are by no means uniform in density and, traveling

WALL ORGANIZATION I N PLANT CELLS

39

along any one of them, one passes through regions of higher and regions of lower density. In parts, small electron-opaque particles can be seen either to be incorporated in the microfibrils or to be attached to them. One interpretation of such a phenomenon would be that in wood cellulose there are crystalline regions within which silver ions cannot penetrate, and other less crystalline or noncrystalline regions in which the structure as a whole is more porous and into which silver ions can migrate and become attached. On somewhat similar lines it has been known for some considerable time that, when celluloses of commercial interest are treated with copper salts under suitable conditions, then the copper can be fixed in the material, and it is on this basis that some wood preservatives are designed. One such method consists in treating a cellulosic product with aqueous copper formate and heating, when a considerable proportion of the copper fails subsequently to become washed out in water. W e have recently shown in this laboratory (Belford et al., 1957) that, under certain conditions, the copper forms a complex with some constituent of the microfibrils, giving an electron diffraction diagram which resembles neither any known copper salt nor cellulose itself. This is apparently the basis on which the wood preservatives have their effect. No such phenomenon occurs, however, with Valonia cellulose. Since it is clear that copper ions are unlikely to penetrate into the crystalline regions of cellulose, the copper-microfibril complex must occur in the noncrystalline regions, and this observation therefore reinforces the idea that cellulose microfibrils vary widely in degree of crystallinity. It is now known (Belford et al., 1958) that this adsorption of metal ions is, under the conditions used, confined to the surfaces of the microfibrils, so that the microfibrils become clothed in a single layer of copper ions. The difference revealed between Valonia and most other celluloses is confined to these surfaces. Further evidence that microfibrils are not uniform along their lengths is furnished by frequent observations-and this applies to Valoniu cellulose equally with other celluloses-of a type illustrated in Plate 11, Fig. 6. When microfibrils have been treated in some way (mere boiling in water is often sufficient) and are shadowcast before observation, their appearance suggests that small pieces have been removed. Since the shadow is nevertheless continuous, this removal must have occurred either in the last stage of shadowing or in the electron microscope. An alternative explanation would be that small localized regions had developed to which metal particles fail to adhere. I n either case the suggestion is clearly of discontinuities in structure periodically along the microfibrils.

40

R. D. PRESTON

111. THE CHEMICAL COMPOSITION OF MICROFIBRILS In nature, cellulose is invariably associated with other polysaccharides or polysaccharide derivatives including hexosans, pentosans, and polyglycuronic acids. Among these, xylan, mannan, araban, galactan, and polygalacturonic acid are prominent. Many of these can be removed by mild treatments such as treatment with dilute alkali, but it has been known for a long time that it is difficult to remove xylan or mannan completely from a cell wall without simultaneously degrading the cellulose (Norman, 1937; Brims, 1947; Schmidt et al., 1930). Schmidt in particular has provided evidence that some of the xylan is very closely associated with cellulose. H e has shown for instance that “skeletal substances” may be obtained from wood, seed husks, and other material by careful purification of the originally lignified material with chlorine dioxide in several stages. When this material is treated with dilute caustic soda, then some of the xylan is dissolved. The remaining insoluble material, however, is shown to ,consist of cellulose and “insoluble” xylan in approximately simple integral proportions. For instance, in red beech wood of any age and from any locality the proportion of cellulose to “insoluble” xylan is very nearly 3 to 1 (Schmidt et al., 1930, 1931), and a similar figure is found for the stones of cherries and California plums. On the other hand, a figure of 2 to 1 is quoted for Silesian plum stones at various stages of development. Schmidt interprets this as implying esterlike linkage between the two components. It has similarly been shown by Ritter and Kurth (1933) that even the celluloses of wood obtained by the most careful purification processes including treatment with 17.5% caustic soda may still contain very considerable quantities of xylan resistant to treatment. These observations have to a large extent been neglected during the past twenty years or so, but more recent observations using more critical methods have brought their importance to the fore. Jermyn and Isherwood (1956) have devised a method for the complete chemical analysis of cell walls using paper partition chromatography. Working largely on the cell walls of pear fruits, but also on other materials, they have shown PLATEI1

FIG.5. Inset. X-Ray diagram of a bundle of parallel filaments of a fresh-water Cladobhoru. Radiation details as in Fig. 1. Beam perpendicular to filament length, filament length parallel to longer edge of page.

FIG.6. Electron micrograph of Valonia microfibrils treated with dilute H,SO,. Shadowed Pd-Au; X 45,000. Note that pieces are missing here and there from the microfibrils, but the shadows are continuous.

WALL ORGANIZATION I N P L A N T CELLS

41

42

R. D. PRESTON

that the cellulose which can be extracted from cell walls normally contains a high proportion of xylan, so that when this cellulose is finally hydrolyzed both glucose and xylose can be detected on the chromatogram. This method has since been modified in this laboratory to apply to algal cell walls (Cronshaw et al., 1958) and has been worked in combination with the methods of X-ray analysis and electron microscopy. This combination has the advantage that, on the one hand, the sugars and sugar derivatives present in wall extracts and in the final residue after extraction are well documented and, on the other, the structural changes in the wall at each stage are watched so that it becomes possible to locate spatially within the wall the materials present in the various extracts and in the final residue. The results have proved so illuminating that it is proposed now to carry on a survey of higher plant celluloses, but for the moment attention must be confined to the algae. In all, fifteen species have been examined, four from the Chlorophyceae, seven from the Phaeophyceae, and four from the Rhodophyceae. All the algae were collected on the northeast coast of England in the first week of January, 1957, at a time when the storage products are at a minimum (Fogg, 1953). The results therefore refer almost exclusively to cell wall materials. Prior to observation the algae were frozen in liquid air, ground to a flour, boiled in ethanol for half an hour, washed, and dried in. vacuo from acetone. This formed the starting material. The subsequent extraction process gave four fractions : the water-soluble material, the alkali-soluble material, the residue from the alkali treatment, and the residue from the alkali treatment after chlorination. At each stage the solid material was examined by X-ray analysis and by electron microscopy. In all cases the treatment progressively removed the amorphous material visible in the natural untreated cell wall. The final material after complete extraction consists entirely of microfibrils except for sporadic globular inclusions which are interpreted as impurities deposited during the long extraction process. With one exception, which will be referred to again below, this proved to be the general picture. The percentage composition of these cell walls as judged by this fractionation method is given in Table I, where the residue after complete extraction is referred to as a-cellulose, although the meaning of this will need to be examined later on. Two striking features emerge, namely the high, occasionally very high, percentage of water-soluble material in the cell wall, and the comparatively low, sometimes extremely low, content of cellulose. The sugars and sugar acids present in each of the extracts are given in Table 11. Attention may be drawn immediately to two prominent features in Table 11. Firstly, uronic acids are present in the water-soluble fraction of all species except Chaeto-

43

WALL ORGANIZATION I N PLANT CELLS

morpha, but do not appear in any of the subsequent fractions except in Lawinaria, where they are present in the cellulose both before and after chlorite treatment. Secondly, and of more importance, the so-called cellulose, even after complete extraction, nevertheless commonly contains sugars other than glucose. The sole exception is Cladophora rupestris, although only small amounts of arabinose are in addition present in Chaetumorpha melagonium. It is interesting to note that these are the only two species in the tables in which it was already known, before these experiments, that cellulose I in the form also present in higher plants is the skeletal substance. One notable and quite unexpected result is that the species of TABLE I CELLWALLCOMPOSITION IN SOMEALGAE Watersoluble Alga fraction (%) Cladophora rupestris 31.5 Chaetomorpha melagonium 41.5 Enteromorpha sp. 30 Ulva lactuca 52

Alkalisoluble fraction (%) 2 8 39 25

Chloritesoluble fraction (%) 38 9.5 9 4

a-Cellulose

(%I 28.5 41 21 19

Halidrys siliquosa Fucus serratus Himanthalia lorea Ascophyllum nodosum Pelvetia canaliculata Laminaria sacchorina Laminaria digitata

62 44.5 67 68.5 70 59 49

14 29 14 16 16 17.5 25

10 13.5 11 8.5 12.5 5.5 6

14 13.5 8 7 1.5 18 20

Ptilota plumosa Grifithsia jlosculosa Rhodymenia palmuta Porphyra sp.

36 41.5 50 49

17.5 14 36.5 47.5

23 22.5 6.5 0

24 22 7 3.5

the red algae, Pmphyra, contains as its skeletal substance a polysaccharide which yields only mannose, not glucose, on hydrolysis. It is especially to be noticed that, since the residue from the complete extraction contains little other than microfibrils, then both the glucose and the other sugars which result on hydrolysis of this material, particularly when the other sugars are present in concentrations similar to that of glucose, must be derived from the microfibrils themselves and not from any encrusting amorphous material. The X-ray diagrams of the material after the various extractions presented observations of great interest. With two of the green algae, Cladophora and Chuetomorpha, the X-ray diagram is already well known (Plate 11, Fig. S), and this diagram remains unchanged throughout the

TABLE I1 SUGARS PRESENT I N VARIOUSWALL FRACTIONS (The letters S, M, W refer to the intensity on the chromatogram: S = Strong; M = Medium; W = Weak.)

Wall fraction Water-soluble fraction

Cladophora rupestris

S Uronic acid S Galactose S Glucose M Arabinose W Xylose

Hemicellulose

Enteromorpha

S Arabinose M Galactose

S Glucose M Uronic acid M Galactose W Xylose

S Uronic acid S Glucose S Xylose W Rhamnose

S Uronic acid M Galactose M Glucose M Xylose M Fucose

S Arabinose

S Xylose S Rhamnose M Glucose

S Galactose S Arabinose S Rhamnose W Xylose

S Xylose S Fucose

S Glucose M Xylose M Fucose

SP.

Ulva lactuca

Halidrys siliquosa and all Fucales

Chaetomorpha melagonium

a-Cellulose before chlorite

S Glucose M Galactose M Arabinose W Xylose

S Glucose M Arabinose

S Glucose M Xylose M Rhamnose

S Glucose S Xylose

a-Cellulose after chlorite

S Glucose

S Glucose W Arabinose

S Glucose M Xylose M Rhamnose

S Glucose S Xylose

S Glucose

W Xylose W Fucose

TABLE I1 (continued)

L. digitata Wall fraction Water-soluble fraction

Hemicellulose

a-Cellulose before chlorite

a-Cellulose after chlorite

and L. saccharina S Uronic acid M Galactose W Xylose W Fucose

Ptilota plumosa

Grifithsia flosculosa

S Galactose

S Galactose

M M M M W

M Uronic acid M Xylose

Uronic acid Glucose Xylose Ribose Arabinose

Rhodymenia palmata

Porphyra

S Xylose M Galactose W Uronic acid W Glucose W Ribose

S Galactose M Uronic acid M Fucose W Ribose W Mannose

S Xylose

S Xylose M Galactose M Mannose S Mannose W Xylose

S Xylose S Fucose

S Xylose

M Uronic acid S Glucose W Xylose

S Glucose S Galactose M Xylose

S Glucose S Galactose M Xylose

S Glucose S Xylose

S Glucose M Uronic acid

S Glucose W Galactose W Xylose

S Glucose W Galactose W Xylose

S Glucose S Xylose

S Xylose

SP.

: r r 0

$

*z * 2 ; c1

N

Y

z

v

*

r

1:

+I

n M

r S Mannose

t:

46

R. D. PRESTON

treatment. In both cases it is apparent that the “cellulose” is largely a glucan, and it is clear that when cellulose is present in this form then the treatment given has no effect on the crystalline structure, as evidenced by the absence of change in the X-ray diagram. With all the other species, however, the extraction progressively caused a most profound change in 4

5

6

7

E

9

10

A. Natural

8. After extraction with hat water

C. After extraction with 4N alkali

D. After treatmeni with chlorite

PLATEI11 FIG.7. Intermolecular spacings of a series of algae, before and after treatment, as determined by X-ray diffraction analysis. The numbers refer to algae, as follows: 1. Enteromorpha. 2. Ulva. 3. Halidrys. 4. Fucus. 5 . Himanthulia. 6. Ascophyllum. 7. Pelvetia. 8. Laminaria saccharina. 9. Laminaria digitata. 10. Ptilota. 11. Grifithsia. 12. Rhodymenia. 13. Porphyra. The spacings are indicated by the positions of solid circles the diameter of which .gives a rough measure of the intensity of the corresponding diffraction arcs.

the diagrams and therefore in the crystalline component remaining after extraction. These changes are illustrated in Plate 111, Fig. 7, in which the intermolecular spacings present in each of the species are represented by the position of a circle whose diameter represents roughly the intensity of the reflection from the corresponding planes. It will be observed that,

WALL ORGANIZATION I N PLANT CELLS

47

both in the untreated wall and in the wall after extraction with hot water, the diagrams are very variable, and none of them corresponds to the main spacings to be expected from cellulose, namely 2.65 A., 3.9 A., 5.4 A., and 6.1 A. After extraction with alkali, however, the X-ray diagrams of the residues are now much more uniform, and the spacings around 2.5, 3.9, and 4.3 A. are beginning to assume uniform prominence. After further treatment with chlorite the diagrams of all species become almost

(B) PLATE I11 (continued) FIG.8A. Diagrammatic representation of the appearance in transverse section of a microfibril. The solid diagonal lines represent the trace in this plane of chains of glucose units; the broken lines represent other sugar derivatives. In the central lattice the chains are regularly spaced in a crystalline array. Outside this the packing is disturbed by the presence of other molecular species. FIG.8B. Diagrammatic representation of a microfibril in longitudinal view. Each line represents a single molecular chain. The central core of chains in crystalline array is interrupted by a region of less perfect crystallinity, figured as being caused by the interpolation of other molecular species.

exactly alike, with prominent spacings at 2.5, 3.9, and about 4.4 A. This change from rich X-ray diagrams that are very variable among the species to X-ray diagrams comparatively poor in diffraction arcs and uniform among the species is most spectacular. With all species except Ctadophora and C h t o m o r p h a the microfibrils are randomly arranged, and in all cases, with the exception of Pmphyra, they remain apparently untouched during the extraction process as judged by observation in the electron microscope. With Porphyra, however, we

48

R. D. PRESTON

have the anomalous condition that microfibrils are no longer visible after the alkali extraction. The residue remaining presents the appearance of an accumulation of particles the dimensions of which are comparable to the diameter of the original microfibrils. The conclusion is inescapable, therefore, that the alkali extract contains polysaccharides from the microfibrils. As is clear from Table 11, these polysaccharides hydrolize to give a mixture of xylose, galactose, and mannose, whereas the remaining particulate fraction after the final treatment hydrolyses to give only mannose. The microfibrils in this instance, therefore, presumably contain xylan, galactan, and mannan, but glucose was not identified in any of the four fractions. It is clear then that the microfibrils after the complete extraction usually contain polysaccharides derived from other sugars as well as from glucose. It remains to determine whether or not any of the sugars and sugar derivatives extracted during the treatment from the walls of algae other than Porphyra are also derived from the microfibrils. Here the results of X-ray analysis presenfed in Plate 111, Fig. 7, are of paramount importance. Two interpretations of the change in the X-ray diagrams during extraction are possible. 1. The extractions may progressively be removing crystalline components other than those in the microfibrils which remain after the extraction, and the change in the X-ray diagram is due to the removal of these other crystalline components. 2. The crystalline components are confined to the microfibrils, and the progressive extraction is removing material from the microfibrils, leaving the residue within the microfibrils in a different crystallographic form. Comparison of the diagrams given by the untreated walls with those of the microfibrils after the full treatment shows that the diffracted arcs at about 3.9 A. and at about 4.4 A. are much more variable in the former than in the latter. This final concentration of the arcs closely around 3.9 A. and 4.4 A. clearly suggests that we are dealing here with a change in crystallinity and not merely with a removal of crystalline components. It seems, therefore, quite certain that the internal structure of the microfibrils is much more complex than had hitherto been supposed. Whether the sugars other than glucose occur in the same molecular chain as glucose, or whether whole chains of glucose and whole chains of the other sugars lie side by side in the microfibril, is not yet clear. It is known that the complete extraction of xylan from xylan-rich celluloses has a chemical effect on the cellulose remaining (Norman, 1937 ; Brims, 1947), and either increases the degree of crystallinity of the cellulose (Astbury et al., 1935) or reduces it (Preston and Allsopp, 1939), depending pre-

WALL ORGANIZATION I N PLANT CELLS

49

sumably on the details of the extraction procedure. Although this lends the fullest support to the conclusion that nonglucose sugars are present in microfibrils, it still does not enable us to determine precisely how this inclusion occurs. Evidence of a more indirect nature than that discussed in this article has led several authors to a somewhat similar suggestion. Several investigators (Preston et al., 1948b; Howsmon, 1949; Wardrop, 1949; Foster and Wardrop, 1951) have pointed to the fact that, when cell walls are treated with dilute hydrochloric acid, the size of the cellulose crystallites increases. This can arise only if cellulose chains in the amorphous regions on the outside of the micelles crystallize on the surface of the micelle. This in turn occurs presumably because the acid hydrolysis is removing, from between the cellulose chains in the noncrystalline regions, interfering molecular species of some other kind. The general picture, therefore, of cellulose architecture presented here is not out of harmony with current opinion. The experimental verification of the presence of cellulose in the cell wall is thus becoming progressively more difficult. It was shown some time ago that the generally accepted staining reaction with iodine and sulfuric acid or chlorozinc iodide is not sufficient (Nicolai and Preston, 1952). It is now clear that even if this staining reaction is combined with the appearance of microfibrils in the electron microscope this is still not sufficient. If in addition the material gives a sharp X-ray diagram containing the arcs characteristic of cellulose in their correct relative positions and intensities, then these three criteria together are often sufficient. If, however, the X-ray diagram is diffuse, with arcs only reminiscent of those to be attributed to cellulose, then this clearly falls short of proving the presence of cellulose. This is on the assumption that cellulose shall be defined as a compound containing long molecular chains of which each unit is @-glucoseand @-glucoseonly. This has not hitherto been strictly adhered to, since it has been stated on many occasions that celluloses in general contain carboxyl groups. Now that it is realized that, closely associated with cellulose and perhaps inseparable from it, there are several other sugars as well as sugar acids, it becomes difficult to allow the presence of a small quantity of any of them in a substance which shall be given the same name. It seems better in principle to adhere to a rigid definition such as that enunciated here.* The microfibrils, the crystalline regions within them, and the fusiform rodlets into which they can be chemically dissected are all known to be

* The nomenclature of celluloses has now been examined elsewhere (A. Myers and R. D. Preston, Proc. Roy. SOC.In press).

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flattened bodies (Preston, 1951 ; Kratky and Schossberger, 1938 ; Mukherjee and Woods, 1953). The flattened “faces” of the crystalline regions (the micelles) and of the fusiform bodies are known to be parallel to particular planes in the cellulose structures spaced 6.1 A. apart, and the same is true for the whole microfibrils of Valonia (Preston and Astbury, 1937; Preston, 1952; Cronshaw and Preston, 1958) and possibly for all cellulose microfibrils. This, together with their diameter and the constitution outlined above, enables a reasonably specific picture to be given of the internal architecture of the microfibrils. The earlier model presented by Frey-Wyssling (1954) depicts a microfibril some 200 A. by 100 A. in transversal view within which are found four micelles each some 70 A. by 30 A., separated and surrounded by randomly disposed cellulose chains. This is evidently conceived along the correct lines, but it can hardly be accepted as strictly accurate on geometrical grounds alone. For the dimensions of the microfibril correspond to those in Valonia in which the micelles are certainly broader than 70 A., whereas the dimensions of the “micelles” correspond to those of, for instance, wood cellulose, the microfibrils of which are only about 100 A. wide. A model more closely in accord with the present evidence is presented in Plate 111, Fig. 8. The microfibrils consist of a central core in which the molecular chains of cellulose are arranged in a regular space lattice (Plate 111, Fig. 8A). This is surrounded by a “skin” of two or three molecular chains which are not packed neatly into a lattice on account of the intermixture with molecular chains which are not, or not entirely, polyglucose. Along the length of the microfibril the proportion of “foreign” chains becomes in places so large, or lattice distortions of other kinds occur so frequently, that sections of the microfibril become entirely paracrystalline (Plate 111, Fig. 8B). It is not clear whether in those microfibrils containing tightly bound xylan and other polymers of sugars other than glucose these “foreign” components also take part in the building of the central core. As far as the evidence goes at present, it appears that those microfibrils which are sensibly straight yield glucose only on hydrolysis, and it may be that this linear arrangement is maintained by hydrogen bonding. Equally, with one or two possible exceptions, those microfibrils which are tortuous hydrolyze to yield other sugars in addition to glucose. It could be suggested, therefore, that within microfibrils which, in undisturbed walls, take a tortuous path there occurs a high proportion of noncellulosic chain molecules. This may be of some importance in the question of the mechanism of orientation.

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IV. THEBIOSYNTHESIS OF MICROFIBRILS Some of the modern evidence relating to the biochemistry of cellulose synthesis has already been reviewed elsewhere (Preston, 1958). We are concerned here largely, however, with the biosynthesis of the whole microfibril rather than of any one component of it. The major question to be decided is whether microfibrils are produced by lateral association of the long molecular chains which compose them, or whether the synthesis occurs as an end synthesis so that microfibrils, once formed, increase by the addition of units at the ends-whether microfibrils in fact grow in width or in length. The general consensus seems to be that it is the latter form of synthesis and not the former. The evidence for this comes from several sources. When the innermost layer of the cell wall of Yalonk is observed in the electron microscope with the cytoplasm still attached, there can be observed, in the cytoplasm and in close contact with the wall, aggregates of corpuscular bodies from which microfibrils appear to radiate. These corpuscular bodies are rather larger in diameter than are the microfibrils, and the suggestion has been made that these are “islands of synthesis” at which the microfibrils are made (Preston and Kuyper, 1951 ; Preston et al., 1953). Similar observations have also been made with the walls of conifer cambium (Preston and Ripley, 1954a) and in the walls of parenchyma in etiolated internodes of broad beans (Williams et d., 1955). This is clear evidence of end synthesis. Wardrop and Dadswell (1952) have made a critical examination of the development of the secondary wall in the tracheids of several species of conifer and have reached the conclusion that here again the microfibrils are synthesized at their ends. Perhaps the most conclusive evidence concerns the development of extracellular microfibrils in the cellulose-producing bacterium Acetobacter xylinum. This has been the object of study for many years, and it has been repeatedly claimed that the appearance of microfibrils is preceded by the appearance of amorphous material with no observable intermediates. It has recently been stated, however (Colvin et al., 1957), that, if cellulose-free Acetobmter xylinum is incubated with glucose and observations are made during the first few minutes, then short rodlets can be observed which progressively increase in length. If this can be substantiated, it provides the clearest possible evidence for the synthesis of cellulose by end synthesis. Although this would be in harmony with the known syntheses of other polysaccharides (with, for instance, the known necessity for the presence of small amounts of starch before starch synthesis can occur from glucose), it nevertheless poses a number of problems concerning the structure particularly of celluloses as at present envisaged, and perhaps even more particularly of the now-suspected complexes in the microfibrils. I n the

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structure of cellulose, the currently accepted model of Meyer and Mark involves the conception that neighboring molecular chains of cellulose are oriented in opposing directions. If the microfibrils are produced by end synthesis from an enzyme complex, then it seems unlikely that the enzyme complex could spin out cellulose chains oriented in opposite directions. It seems much more probable that all the cellulose chains would be pointing in the same direction. Synthesis would then presumably proceed at one end only, and the observation both in conifer cambium (Preston and Ripley, 1954a) and in the primary wall of sporangiophores of Phycomyces (Roelofsen, 1949) that one end of a microfibril lies free would harmonize with this point of view. It is therefore necessary to examine the reasons why the currently accepted unit cell of cellulose has this particular configuration. The chief argument is derived from the observation that, when cellulose is dissolved and reprecipitated, it nevertheless produces a crystalline complex. The argument is that, since in this crystalline complex, of which it is supposed that chain molecules have been separated and then reunited, the chances are that half the chains will be pointing one way and half the other way, then this must have been the case in the original cellulose. It does not follow that this alternation in direction is as regular as the currently accepted model demands. As a second and secondary piece of evidence, it is true that in making scale models of cellulose it is difficult to build the unit cell even with the current conception of the run of cellulose chains. With all the cellulose chains lying parallel to each other and pointing in the same direction, it is virtually impossible to construct a scale model. Neither piece of evidence seems, however, in the least convincing, and if end synthesis is substantiated it would seem inevitable that the unit cell must be called into question. It is therefore significant that Honjo and Watanabe ( 1958) have, on the basis of low-temperature electron diffraction, questioned the accepted structure of cellulose I. It appears now, moreover, that we have to allow for the incorporation within the microfibril of sugars other than glucose, and of sugar derivatives such as glycuronic acid. Therefore, either the enzyme complex responsible for the production of microfibrils must be such as to be able to handle sugars and sugar derivatives indiscriminately, or it must from time to time change in character so that different sugars or sugar derivatives can be incorporated periodically. Only the determination of the spatial relationships, within the microfibril, of the various components now known to be there can decide on this point. One further and equally important problem arises concerning the spatial relationships between the enzyme complex and the microfibril produced.

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It is a question whether the enzyme complex spins out the microfibril from a precursor, the enzyme complex remaining fixed and the microfibril progressively moving out of it, or whether the microfibril, once produced, is itself fixed and there is then a necessity for the enzyme complex to move as the microfibril grows. It may be that the particular process at work varies with the species concerned and the condition of growth. With Vulonh, for example, it seems inconceivable that microfibrils could be spun out of a stationary complex and pushed over the surface of the existing wall while remaining straight and parallel to its neighbors. Wardrop and Dadswell ( 1952), however, envisage this last phenomenon as occurring and go so far as to suggest that the twisting of microfibrils round each other observed in Valonia (Preston and Kuyper, 1951) is due indeed to the pushing of microfibrils away from the center of synthesis. It seems quite incredible that such a process can occur with this particular species. Until a great deal more is known about the organization of the microfibrils in a cell wall laid down at a new cytoplasm-environmental interface, no decision can be made on this point. It is for this reason particularly that observations on new wall formation may prove of the utmost value.

V. THE SYNTHESIS A N D ORIENTATION OF MICROFIBRILS AT NEW CYTOPLASMIC SURFACE

A

During the past few years considerable attention has been paid to the organization of a cell wall laid down at a new cytoplasmic surface. For observations of this kind the algae lend themselves very well, and the definitive observations have been made solely with organisms of this type. The algae belonging to the Cladophorales are particularly suitable both because the cell wall microfibrils apparently consist entirely of cellulose in the strict sense defined above, and because the production of swarmers is at times prolific and these can be made to develop in the laboratory. Nicolai (1957) has made an intensive study of this material, and her results may be summarized briefly as follows. The first formed wall, developed within 2 hours of the settling of a swarmer, consists of rather sparse microfibrils widely separated and in random arrangement. The X-ray diagram, however, has shown most conclusively that these microfibrils do not consist of cellulose in the strict sense referred to above. Within a few days, the new layers of wall formed within this outer envelope show all the features of the wall of the adult filaments, consisting of two parallel sets of microfibrils arranged in two separate layers, the orientation changing through 90" from one layer to the next. The X-ray diagram at this stage is typical of cellulose I. Both

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sets of microfibrils are, however, preceded by broad bands apparently within the cytoplasm, within which the microfibrils subsequently develop. The general appearance illustrated in Plate IV, Fig. 9, suggests that these

-

PLATE IV FIG.9. Electron micrograph of a sporeling of Chaetomorpha melagoniwm, 24 hours after settling, contents removed. Shadowed Pd-Au; X 15,000. Note the coarse transverse bands, parts of a slow spiral closing into two poles, one at each end of the spqreling. These coarse bands appear to be in the cytoplasm and contain already occasional microfibrils. (Photo by E. Nicolai.)

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opaque coarse bands constitute cytoplasmic organs within which the cellulose microfibrils subsequently form. This would appear to be at first sight inconsistent with the idea of end synthesis. In attempting to give even a tentative interpretation of this phenomenon, it is profitable to consider first the recent observations made on the structure of the mature vesicles and developing aplanospores of Valonia. This species takes the form of individual vesicles which are globular in form and arise from spores very much as do the filaments of the Cladophorales. In this case, however, the cell remains globular. The pioneer work of Preston and Astbury (1937) showed that in the mature vesicle the wall contains two sets of cellulose chains lying almost at right angles to each other, subsequently shown to correspond to two sets of microfibrils again approximately at right angles (Preston et al., 1948a). A complete investigation of the structure of a whole vesicle showed that these two sets are organized in a very specific way (Plate V, Fig. 10). One set of microfibrils, as we may now call them, runs as meridians joining two “poles” on the globular vesicle. The other set forms a series of slow left-hand spirals which open out from one pole and close in toward the other. These observations were made by the method of X-ray diffraction, and evidence was presented that occasionally a third direction of orientation was present. With the onset of electron microscopy it soon became clear that this third direction of orientation is more frequent than might have been expected (Wilson, 1951; Steward and Miihlethaler, 1953), an observation which led Cronshaw and Preston (1958) to a reinvestigation of the whole wall of the vesicle. The model which these latter workers present is illustrated in Plate V, Fig. 11. I t will be seen that the model is essentially that of Preston and Astbury (Plate V, Fig. l o ) , apart from the presence of the third orientation. The microfibrils lying in this third orientation are, however, relatively much more infrequent than are those of the other two sets, both because the lamellae in the wall which carry them are more infrequent, and because within any one lamella the microfibrils are much more loosely arranged in this third set than they are in the other two. I n the main, therefore, the adult vesicle resembles rather closely in wall structure the swarmers of the Cladophorales soon after settling. The Valonia vesicles can in fact be induced to produce aplanospores rather easily, and a few days after settling these aplanospores show essentially the structure of the adult vesicle. It seems therefore quite probable that the structure of the sporelings in the Cladophmales represents rather closely that of the adult vesicle in Valonia. Now in announcing this model of the structure of the mature Valonia vesicle, Cronshaw and Preston ( 1958) have made speculations concerning

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the possible mode of development of the particular structure observed, and the necessity is pointed out of knowing which set of microfibrils comes first among the major sets, the meridional or the spiral. If, for instance,

PLATE V FIG.10. Model of wall structure of a whole vesicle of Valolzia ventricosa proposed by Preston and Astbury in 1937. The broad tape shows the run of the cellulose chains. One set of chains forms a slow left-hand spiral, and the others form a steep left-hand spiral.

PLATE V FIG.11. A similar model proposed by Cronshaw and Preston in 1958. This is identical with that of Fig. 10 except for the presence of a third spiral, right-handed in sign. The microfibrils of this third set are much less numerous than are those of the other two.

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the meridional comes first, then this might be envisaged as starting at a pole. If cellulose microfibrils are produced in random orientation at any one localized region of the cytoplasmic surface of a spore, and if the microfibrils growing at their ends pass round the spore with a tendency to remain straight (as cellulose microfibrils would in fact do on account of the intense hydrogen bonding), then these microfibrils would form great circles around the spore and converge to a second pole at the antipodes. This is a possible, even if rather unlikely, method whereby the set of meridional microfibrils might be initiated. In order to produce the spiral set which would next be deposited, it is then necessary to suppose that this particular orientation of microfibrils imposes, on the cytoplasm which has produced them, a second orientation at right angles, so that the second set of microfibrils is laid down almost at right angles to the first. This would then impose a switch through approximately a right angle again for the third set and so forth. If, on the other hand, the cellulose microfibrils produced at the localized region all begin strictly parallel to each other, then this parallel band of microfibrils passing around a spore which is actually ellipsoidal in shape would naturally pass round as a spiral converging to “poles” at the two ends. of the spore. It would then be necessary to invoke the kind of mechanism envisaged above for the successive switch through a right angle in subsequent layers. Returning now to the observations of Nicolai (1957) illustrated in Plate IV, Fig. 9, it seems that in Chaetonzorpha, apart from a few sparsely arranged microfibrils which lie more or less at random over the surface of the young sporeling with a tendency, however, to form meridians, the first organized wall lamella is that containing microfibrils arranged in a slow spiral, and the appearance of these microfibrils is preceded by coarse bands foreshadowing the orientation of the microfibrils and appearing apparently in or just below the cytoplasmic surface. The implication is clearly that here we have an orienting mechanism in the cytoplasm which ensures that the cellulose microfibrils shall be laid down parallel to each other and take a spiral path around the cell. Taking this together with the probability that the microfibrils grow at least at one end, it could be supposed that the enzyme system responsible for cellulose synthesis is to be found in these coarse cytoplasmic bands. It could be, for instance, that the whole band throughout its length is, in a broad sense, an enzyme complex which can produce cellulose; and whether or not it does so depends on the presence or absence of microfibril ends. In that case, once the microfibril is initiated, growth will proceed from at least one end and, as the microfibril grows, successive neighboring regions of the coarse bands will take over the duty of synthesis. The microfibrils would then be deposited in the cytoplasm and oriented by the cytoplasm. This would

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be in harmony with the fact that the microfibrils both in Valonia and Cladophora are often twisted around each other and with the conclusion, which is now becoming general, that the cell wall and cytoplasm are not completely separable. It would also be in harmony with the observation of Myers et d.(1956) (which refers, however, to a red alga) that any new lamella is produced not at the actual apparent cytoplasm-wall interface itself but just within this, so that as the new lamella becomes part of the wall there is trapped, between it and the wall already laid down, a layer of cytoplasm. In the two species of plant-Valonia and Cladophora-for which these particular speculations have been made, the microfibrils in the wall hydrolyze to give glucose only among the hydrolytic products. The condition in other cells in which the microfibrils contain sugars other than glucose may well be much more complicated. These, however, could be fitted into this same general picture if we assume that, in the coarse bands which precede the microfibrils, there exists a number of enzyme complexes capable of producing glucans, arabans, galactans, xylans, etc., and whether or not these materials are produced might then depend on the presence or absence of the constituent sugars. I n those plants which produced, for instance, glucose and xylose in the correct place at the correct time, these two sugars could incorporate into the microfibrils. Differences in the constitution of the microfibril between different species would in that case depend rather on variations in the metabolic machinery within the cytoplasm responsible for the production and interconversion of saccharides and not necessarily on the presence or absence of any specific enzyme system. VI. REFERENCES Allsopp, A., and Misra P. (1940) Biochem. J . 34, 1078. Astbury, W. T., Preston, R. D., and Norman, A. G. (1935) Nature 136, 391. Bailey, I. W., and Kerr, T. (1935) J . Arnold Arboretum (Harvard Univ.) 16, 273. Balashov, V., and Preston, R. D. (1955) Nature 176, 64. Balls, W.L. ( 1 9 2 ) Proc. Roy. SOC.B93, 426. Belford, D. S., Preston, R. D., Cook, C. D., and Nevard, E. H. (1957) Nature 180, 1081. Belford, D. S., Myers, A., and Preston, R. D. (1958) Nature 181, 1518. Bonner, J. (1936) Jahrb. wiss. Botan. 82, 377. Brims, B. M. (1947) J . Council Sci. Ind. Research 20, 276. Colvin, J. R., Bayley, S. T., and Beer, M. (1957) Biochim. et Biophys. Acta aS, 652. Cronshaw, J., and Preston, R. D. (1958) Proc. Roy. SOC.B148, 137. Cronshaw, J., Myers, A., and Preston, R. D. (1958) Biochim. et Biophys. Acto 27, 89. Criiger, H. (1854) Botan. Z t g . 12, 57. Dippel, L. (1879) Abhandl. senckenberg. nuturforsch. Ges. 2, 154.

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Dischendorfer, H. (1925) Angew. Botan. 7, 57. Farr, W. K., and Sisson, W. A. (1934) Contribs. Boyce Thompson. Znst. 6, 309. Fogg, G. E. (1953) “The Metabolism of the Algae.” Wiley, New York. Foster, D. H., and Wardrop, A. B. (1951) Australian J . Sci. Research Ser. A 4,

412. Frey-Wyssling, A. (1937) Protoplasma 27, 372. Frey-Wyssling, A. (1954) Science 119, 80. Frey-Wyssling, A., Miihlethaler, K., and Wyckoff, R. W. G. (1948) Experientia 6, 12, 475. Hengstenberg, J., and Mark, H. (1929) Z . Krist. 69, 271. Herzog, A. ( 1910) “Untersuchungen d. natiirlichen und kiinstlichen Seiden.” Dresden. Herzog, R. O., and Jancke, W. (1928) Z . physik. Chem. (Leijzig) Al39, 235. Heyn, A. N. J. (1950) J . Am. Chem. SOC.72, 2284, 5768. Heyn, A. N. J. (1955) J . Appl. Phys. !26, 519. Honjo, G.,and Watanabe, M. (1958) Nature 181, 326. Howsmon, J. A. (1949) Textile Research J . 19, 153. Iterson, G. van, Jr. (1933) Chem. Weeklblad 30, 6. Jermyn, M. A., and Isherwood, F. A. (1956) Biochem. J. 64, 123. Jorgenson, L. (1949) Acta. Chem. Scand. 3, 786. Krabbe, G. (1887) Jahrb. cviss. Botan. 18, 346. Kratky, O.,and Porod, G. (1954) 2. Elektrochem. MI, 918. Kratky, O.,and Schossberger, F. (1938) Z . physik. Chem. (Leipzig) B39, 145. Mark, H., and Meyer, K. H. (1929) Z . physik. Chem. (Leibzig) B2, 115. Meyer, K. H., and Mark, H. (1928) Ber. 61B, 593. Mukherjee, S. M., and Woods, J. H. (1953) Biochim. et Biophys. Acta 10, 499. Myers, A., Preston, R. D., and Ripley, G. W. (1956) Proc. Roy. SOC.B144, 450. Nicolai, E. (1957) Nature 180, 491. Nicolai, E.,and Frey-Wyssling, A. (1938) Protoplasma 30, 403. Nicolai, E.,and Preston, R. D. (1952) Proc. Roy. SOC.B140, 244. Norman, A. G. ( 1937) “Biochemistry of Cellulose, Polyuronides, Lignin, etc.” Oxford Univ. Press, London and New York. Preston, R. D. (1951) Discussions Faruday SOC.No. 11, 165. Preston, R. D. (1952) “Molecular Architecture of Plant Cell Walls.” Chapman and Hall, London. Preston, R. D. (1958) “Handbuch der Pflanzenphysiologie” Vol. VI, pp. 323. Springer, Berlin. Preston, R. D., and Allsopp, A. (1939) Biodynamica 63, 1. Preston, R. D., and Astbury, W. T. (1937) Proc. Roy. SOC.Bl22, 76. Preston, R. D., and Cronshaw, J. (1958) Nature 181, 248. Preston, R. D., and Kuyper, B. (1951) J . Exptl. Botany 2, 247. Preston, R. D., and Ripley, G. W. (1954a) J . Exptl. Botany 6, 410. Preston, R. D., and Ripley, G. W. (195413) Nature 174, 76. Preston, R. D., Nicolai, E., Reed, R., and Millard, A. (1948a) Nature 162, 665. Preston, R. D., Wardrop, A. B., and Nicolai, E. (1948b) Nature 162, 957. Preston, R. D., Nicolai, E., and Kuyper, B. (1953) J . Exptl. Botany 4, 40. Ranby, B. G. (1949) Acta. Chem. Scand. 3, 649, Ranby, B. G. (1951) Discussion Faraday SOC.No. 11, 158. Ranby, B. G.,and Ribi, B. (1950) Experientia 6, 12. Reimers, H. (1922) Mitt. Forsch. Znst. TextilstoffeKarlsruhe 109.

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Ritter, G. J., and Kurth, E. F. (1933) Znd. Eng. Chem. 26, 1250. Roelofsen, P. A. (1949) Biochim. et Biophys. Acta 3, 518. Roelofsen, P.A., and Kreger, D. R. (1954) J . Exgtl. Botany 6, 24. Schmidt, E., Meihel, K., Nevros, K., and Jandebeur, W. (1930) Cellulosechemie 11, 49, 73. Schmidt, E., Yuan-Chi Tang, and Jandebeur, W. (1931) Cellzilosechemie 12, 185. Schmitz, F. R. (1880) S . B . Niederhein Ges. Nut.-u. Heilk. 37, 200. Sponsler, 0. L., and Dore, W. H. (1926) Colloid Symposium Monograph 4, 174. Steinbrinck, C. (1927) Naturwiss. 16, 978. Steward, F. C., and Miihlethaler, K. (1953) Ann. Botany (London) 17, 295. Strasburger, E. (1898) Jahrb. m‘ss. Botan. 31, 511. Wardrop, A. B. (1949) Nature 164, 366. Wardrop, A. B., and Dadswell, H. (1952) Australian J. Sci. Research 6,385. Wiesner, J. (1892) “Die Elementarstr. usw.” Vienna. Williams, W. T., Preston, R. D., and Ripley, G. W. (1955) J. Exptl. Botany 6, 451. Wilson, K. (1951) Ann. Botany (London) 16, 279.

Submicroscopic Morphology of the Synapse’ EDUARDO D E ROBERTIS Director of the Instituto de Anatomia General y Embriologia, Facultad de Ciencias Mfdicas, Buenos Aires, Argentina Page I. Introduction . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 61 11. Morphology of the Synaptic Region ......................... . ..... 63 A. Preliminary Observations with the Electron Microscope . . . . . . . . 66 B. Ultrastructure of the Synaptic Region : General Description . . 66 C. Ultrastructure of Typical Terminal Synapses .. . . . .. . . . . . . . . .. . . 68 1. Synaptic Membrane ... ................ ................ .... 68 2. Mitochondria .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. .. . . . . . . . . . . 69 3. Synaptic Vesicles . .. . . . ... . . . . .. .... .. . . .. . . .. . . . . . . . . . ... 69 D. Submicroscopic Structure of Some Special Synapses . . . . . . . . . . . . 70 1. Invertebrate Synapses . . . . . . . .. . . . . .. .. . . . . . . . . . . . . . . . . . . .. 70 2. Ultrastructure of the Neuromuscular Junction . .. . . . . . . . . . . . . 72 3. Innervation of the Electric Organ . .. . . . . . . . . . . .. . . . . . . . . . . . . 74 4. Synapses in Sympathetic Ganglia . .... . .... . . . ... . . . .. . . . . . . 74 5. Ultrastructure of Some Peripheral Nerve Endings. . . . . . . . . . . . . 74 6. Microvesicles in Regenerating Nerves ... . . .. .. . . . .. . . . . . . . 76 111. Submicroscopic Morphology and Function of the Synapse . . . . . . . . . . . . . 76 A. Degenerative Changes of the Synapse .. . . . ... . . . .. . . . . . . . . .. ... 78 B. Physiological Changes in Synapses of the Retinal Rods and Cones 79 C. Changes of the Synapse after Nerve Stimulation . .. . . . . . . . . . . . . . 80 D. Dimensions and Physiology of Synapses .................... .. 85 E. Functional Role of Synaptic Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . 90 IV. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . ... . .. . . . ... . ..... 93 V. References . . . . . .. . . . .. . . . . .. . . . . . .. . ... . .. .. .. .. .. . . . . .. .. .. .. .. . 94

I. INTRODUCTION The concept of the synapse, or synaptic junction, although first elaborated by physiologists to explain how nerve elements may exert excitatory or inhibitory actions on other nerve cells (Sherrington, 1897), had from its very beginning a definite morphological basis. The so-called “neuron doctrine,” masterly developed by Cajal (see Cajal, 1934), established that the individual nerve cells are not in continuity but in close contact at certain points, where the functional connections may be effected. The synaptic junction may be considered as a specialized locus of contact, at which synaptic excitatory or inhibitory influences are transmitted and act on other cells (see Eccles, 1957). As synaptic regions, in a strict sense, we shall consider the special zones 1 Some of the latest part of this work was helped by the grant B-1549 of the National Institute of Neurological Diseases and Blindness.

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of contact between two neurons, between a receptor and a neuron, and the neuromuscular junction, thus embodying all the regions “anatomically differentiated and functionally specialized for the transmission of the liminal excitations from one element to the following in an irreciprocal direction” (Arvanitaki, 1942). These typical polarized synapses are the great majority in the nervous system of both vertebrates and invertebrates? At the synaptic junction the two cellular elements-one presynuptic and another postsynuptic-are intimately apposed, and both of them have specialized functions that can be differentiated from the signal reaching the junction or the all-or-nothing impulse originating in the postsynaptic element and conducted by the following axon (see Eccles, 1957; Luco and Davidovich, 1957). Modern physiological and pharmacological investigations have demonstrated that synaptic junctions have indeed electrophysiological and chemical properties which can be differentiated from the rest of the neuron (see Fatt, 1954; Feldberg, 1954). These physiological advances for many years were not paralleled by progresses in morphological and structural studies of the synapse. In his review published in 1942, Bodian stressed the importance of learning more about the structure of the synapse, pointing out that, since the classical works of Cajal, Retzius, Ehrlich, and others, little but technical refinement has been contributed to the methodology of study of synapses. The generally used silver staining techniques gave a considerable body of information about the size, shape, and position of the nerve ending on the postsynaptic element, but not about the intimate structure of the terminal or the interface between the ending and the postsynaptic surface. Although the morphological aspects revealed by the optical microscope gave little background for a satisfactory explanation of synaptic function, it was hoped that the enormous resolving capabilities of the electron microscope would provide more fundamental details of structure. In fact, within the range of resolution that can be now achieved in tissue sections, the macromolecular structures revealed are better related to the chemical morphology of molecular complexes and to the intimate physicochemical mechanisms of cell physiology. This review will be concerned with studies of the synaptic junction carried out during the last five years with the electron microscope. As these investigations are still fragmentary and concerned only with a few types of synapses, it is difficult at this point to establish any kind of 2 This definition would exclude the natural or artificially produced contacts (generally axo-axonic) which were designated ebltases by Arvanitaki and also the contacts with reciprocal transmission found in some giant axons of invertebrates which have been named quasiartificial synapses or contacts by Bullock (1953).

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generalization. The results so far obtained, however, have settled some of the controversies which in the past derived from the limited resolving power of the light microscope and the vagaries of the silver staining techniques and have established the existence of a submicroscopic component, “the synaptic vesicles” (De Robertis and Bennett, 1954, 1955), which seem to have some relationship to the physiology of the synaptic junction (see De Robertis, 1957).

11. MORPHOLOGY OF T H E SYNAPTIC REGION The light microscope shows the extreme variation in shape, position, and dimensions which occurs at synaptic contacts. In his general review published in 1934, Cajal described eleven types of synapses divided into two groups of axosomatic and axodendritic junctions, according to the point of contact between the axon and the postsynaptic element. Although not recognized by Cajal, axo-axonic synapses are also found particularly in the neighborhood of the axon hillock. These axo-axonic synapses are the most commonly observed in invertebrates in which neurons are usually monopolar. One should not exclude the possibility of dendrodendritic junctions as proposed by Estable (1953). The existence of polarized synapses between two homologous elements (axon-axon, dendritedendrite), as we shall see later, can be easily explained on the basis of the submicroscopic organization of the synaptic region. The junction may be of the terminal type, in which it is the axon terminus or ending that establishes contact with the postsynaptic surface. In this case the ending may be of different shapes and classified as bud or foot ending, if there is a widening of the terminal; club ending, if the axon is wider and there is no enlargement of the terminal; or c a l k or c u p ending, if the ending covers a large zone of the cell surface (Bodian, 1942, 1952). The complex variety of terminals found in the ventral acoustic nucleus of mammals has been described by Estable et al. (1953). The foot endings or boutons observed on the soma and dendrites of motoneurons may be considered as prototype of endings. Barr (1939) and Haggar and Barr (1950) have calculated that over a thousand endings may cover the surface of a motoneuron, and up to 38% of it may be occupied by synaptic contacts. The close packing of terminals is confirmed in isolated motoneurons (Chu, 1954), in preparations with modifications of silver staining (Armstrong et al. 1956; Rasmussen, 1957), and in lowpower electromicrographs. According to Wyckoff and Young ( 1956j , the Since . the number of end feet can be estimated as 15 to 20 per 1 0 0 ~ ~ total surface area of a motoneuron is about 10,000p2,there are not less than 2000 end feet per cell (see Fig. 1A and B ) .

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The complex morphology of other types of synaptic junction has been recently reviewed by Bodian (1952) and von Horstmann (1957). Bodian emphasized the difficulties of studying in detail the morphology of synapses in which the contact is not terminal but along the axon, such as the synapses “en passant,” the climbing fibers or spiral synapses, and the glomerular synapses of the cerebellum. In all these cases it has been suggested that the contact may be effected by means of an intervening layer of material such as the ground substance with special conductance properties, postulated by Cajal (1934), or by a layer of glial cytoplasm, as held by de Castro (1942, 1950). The concept of the gliotheca was generalized by de Castro for all types of synapses and even for the neuromuscular junction (Noel, 1950). Very few cytological details of the structure of the synaptic region may be observed with the optical microscope. In synapses of the giant Mauthner cells stained with cytological methods the presence of a synuptk membrane or synaptolemma between the terminal and the postsynaptic cytoplasm has been revealed (Bartelmez and Hoerr, 1933 ; Bodian, 1940). Since the true synaptic membrane is of submicroscopic dimensions, the synaptolemma probably corresponds to the limit between the two contacting elements. In some cases neurofibrils have been observed within the terminal. Also the presence of mitochondria1 granules has been detected preferably on the proximal side of the synaptic junction. These granules probably correspond to the so-called “neurosomes” observed by Held (1897) within the glomeruli of the cerebellum. Mitochondria are also concentrated in integrative regions of the brain, forming vast synaptic FIG. 1. Diagram showing bouton-like synaptic junctions a t different magnifications with the optical and electron microscope. (A) Illustrates a motoneuron as seen a t medium power of the optical microscope. The nucleus (N),the axon ( A ) , and the dendrites (d) are indicated. Numerous bouton-like endings make synaptic contact with the surface of the pericaryon (axosomatic junctions) and of the dendrites (axodendritic junctions). Enclosure B is magnified ten times in B. ( B ) End feet ( e ) , as seen at high magnification with the optical microscope. The afferent axons are enlarged at the endings. The presence of mitochondria is indicated. Enclosure C is magnified about six times with the electron microscope in C. (C) Diagram of an end foot as observed with the electron microscope. Mitochondria ( m ) , neuroprotofibrils (nf), and synaptic vesicles (m) are shown within the ending. Three clusters of synaptic vesicles become attached to the presynaptic membrane ( p s n t ) ; these are probably active points (ap) of the synapse. Both the psm and the subsynaptic membrane (ssm) show higher electron density. The glial membrane is shown in dotted lines (gm). Enclosure D is magnified about twenty times in D. ( D ) Diagram of the synaptic membrane as observed with high-resolution electron microscopy (see description in the text). Some synaptic vesicles (m) are seen attached to the p s m and opening into the synaptic cleft (sc)

.

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fields or neuropiles (Scharrer, 1945), and in the terminal axoplasm of neuromuscular junctions (Noel, 1950).

A . Preliminary Observations with the Electron Microscope The early electron-microscope observations published in 1953 settled some of the above-mentioned controversies on the morphology of the synapse. Pease (1953) pointed out that end feet come in direct contact with the surface of nerve cells. In the axo-axonic synapses of the giant fibers of the squid and of the abdominal ganglia of the crayfish, Robertson (1953) found that the distance between the synaptic membranes was of the order of 600 A. I n his work, however, the distortion introduced by the extraction of the plastic may have altered the relationship between the membranes. Estable et ul. (1953) found in synapses of the ventral acoustic ganglia of the cat and dog that the minimal distance between the pre- and postsynaptic cytoplasm was 320 A, which corresponds approximately to the thickness of a double membrane. In the synapse between retinal rods and bipolar cells, Sjostrand (1953) observed that there is an intimate contact with considerable digitation of the postsynaptic ending into the adjacent region of the rod cell. These early observations and all the recent ones indicate that at the level of the junction there is a direct contact of membrane surfaces without interposed cellular material alien to the two pre- and postsynaptic components. This invalidates the supposition that the synaptic terminal is surrounded by a glial sheath or by any kind of ground substance. Furthermore, the observation of a neat delimitation of both the preand postsynaptic cytoplasm confirms and extends to a submicroscopic level the concept of the individuality of the nerve element which is implicit in the neuron doctrine of Cajal. The reticularist hypothesis, which still has its followers, cannot be maintained, even in those regions of the central nervous system called neuropiles, where most of the elements are of submicroscopic dimensions. The reticular appearance is the result of technical artifacts, plus the limited resolving power of the optical microscope to detect those structures and their boundaries. These facts indicate that for an exact interpretation all structures below 1 to 0.5 p should be studied with the electron microscope.

B.

Ultrastructure of the Synaptic Region: General Description

In spite of the obvious differences existing between synapses of the peripheral and the central nervous system, between the axosomatic, the axodendritic, and the axo-axonic, between the different types of synaptic endings and the synapses “en passant,” and so forth, from the submicro-

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scopic point of view there are details that are common to all of them. In the diagrams of Figs. 1 and 2 some of the most common types of synapse are indicated. In all of them there is a presynaptic element which has a

FIG.2. Diagram of different types of synaptic junction. ( A ) A Synapse between a rod and a bipolar cell (see description in the text) : p , a blind projection of the presynaptic membrane (psm) ; d, dendrites of the bipolar cell ; er, endoplasmic reticulum; gm, glial membrane. The main characteristic of this junction is the invagination of the psm and penetration of the dendrite into the ending. (B) Ending of a neuromuscular junction. Several active points on the p s m are indicated. The main difference from other synapses is the folding of the ssm, forming the subsynaptic or postjunctional folds (ssf) (see description in the text). (C) Type of lateral junction between an axon ( A ) and an electroplaque of the electric organ of the eel. Synaptic vesicles are present along the axon at synaptic contacts. Continuity of neuroprotofibrils (nf) is observed. Sc represents Schwann cell (Diagram based on an electron micrograph of Luft ; see also Fig. 11).

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different relationship with the postsynaptic one. In Fig 1C the bouton ending of the axon is applied to the postsynaptic surface of a motoneuron, which is flat or may have a small depression. Figure 2C is a synapse “en passant,” as observed by Luft on the plates of electric organ and probably similar to other synapses of this type in the central nervous tissue. In Fig. 2A the postsynaptic element penetrates deeply into the presynaptic one, as in the case of the retinal rod (Sjostrand, 1953; DeRobertis, 1955a; De Robertis and Franchi, 1956) and cone synapses with the bipolar cells. A similar relationship of membranes is probably found in synapses of the stellate ganglion of Loligo (Young, 1939) and in the crayfish abdominal ganglia (Robertson, 1953) and is probably most frequent in invertebrate neuropile (De Robertis and Bennett, 1954, 1955). Figure 2B indicates the case observed in the neuromuscular junction, in which the nerve endings are deeply embedded into grooves of the postsynaptic element (Couteaux, 1947, 1955) and the postsynaptic membrane is extensively folded ( Palade, 1954 ; Reger, 1954 ; Robertson, 1956), forming the so-called subneural apparatus of Couteaux. C.

Ultrastructure of Typical Terminal Synapses

1. Synaptic Membrane. Detailed descriptions of a bouton-like synapse as found in motoneurons and endings of the ventral acoustic ganglion have been published by De Robertis (1955a, b, 1956) and by Palay (1956, 1957b). In the nerve terminal, a surface membrane, an amorphous matrix, mitochondria, synaptic vesicles, neuroprotofibrils, and a few tubules or vesicles of the endoplasmic reticulum may be found. The surface membrane, usually of 50 to 70 A thick, is continuous with that of the axon membrane and with the presynaptic membrane which comes into direct contact with the postsynaptic surface membrane to form the synaptic junction proper. Eccles (1957) has propounded the term subsynaptic to this juxtaposed region of the postsynaptic membrane (Fig. lC, D ) . The surface membrane of the terminal is usually covered by glial processes in central synapses or by the Schwann cell in peripheral synapses (indicated by a broken line in Fig. 2C). ‘At this junction, however, its presynaptic part becomes entirely free and comes into direct contact with the subsynaptic membrane. Palay (1957a, b) has described in some cases small glial processes interposed between the terminal and the postsynaptic surface, but these do not obstruct direct contact. At the junction, both the pre- and subsynaptic membranes may show differentiated regions, which appear as spots or patches of higher electron density. These regions were first described by De Robertis (1955a, b, 1956) in the acoustic ganglion and more recently by Palay (1957b), who finds them to be

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150 to 200mp in length. At these patches, which are probably active points of the synapse (see below), the synaptic vesicles make a closer contact with the presynaptic membrane (see Figs. 2C and 4 ) . According to Palay, this complex formed by a cluster of synaptic vesicles associated with an area of the synaptic membrane and the subjacent synaptic cleft may be considered a morphological subunit of the synaptic membrane. Both the presynaptic and subsynaptic membranes are about 60 A thick and are separated by an intervening space-the synaptic cleft-of about 120 to 200 A. The synaptic cleft represents the real discontinuity of cell cytoplasm at the level of the junction. The continuity of this cleft with the extracellular spaces may be traced particularly in peripheral synapses. In central ones the cleft is continuous only with narrow interstitial clefts, since open extracellular spaces are not found in the central nervous tissue. High-resolution observations in retinal synapses indicate that both the pre- and subsynaptic membranes may be even more complex (De Robertis, 1957). Two dense lateral layers and a central one of lower density have been observed within the 60-A thickness of both membranes (Fig. 1D). 2. Mitochondria. Mitochondria are frequently observed within the terminal among the synaptic vesicles, but their number varies considerably from one type of synapse to another. Thus they are very abundant in the glomeruli of the cerebellum within the expanded terminals of the mossy fibers (Palade, 1954; De Robertis, 1955a; Palay, 1956) (Fig. 3 ) . In sections of the ventral acoustic ganglia there are only a few per terminal, and in synapses between the retinal rods and bipolar cells of the rabbit there are generally no mitochondria in the neighborhood of the synapse (De Robertis, 1955a; De Robertis and Franchi, 1956) (Fig. 5 ) . The mitochondria show the typical structure with the double lamellar crests, described by Palade ( 1952), which are frequently oriented longitudinally. This variability in concentration, the location of mitochondria generally far from the membrane, and their function in the oxidative cycle make its direct intervention in synaptic transmission, as suggested by Bodian ( 1942), very improbable. 3. Synaptic Vesicles. Under the name of “synaptic vesicles” De Robertis and Bennett ( 1954) described a special vesicular component present in the synapse. I n their early report on synapses of the frog sympathetic ganglia and the neuropile of the earthworm, they described the presynaptic location and the intimate relationship of some of these vesicles with the synaptic membrane. Almost simultaneously Palade (1954) and Palay (1954) reported an agglomeration of small vesicles in the axon endings of several synapses of the central nervous system and in the neuromuscular junction. The full paper of De Robertis and Bennett

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(1955), submitted for publication in May 1954, described in greater detail the relationship of the membranes with the synaptic vesicles. It was suggested that they may flow toward the synaptic membrane, perforate it, and discharge their content into the intermembranal space, and even go across the postsynaptic membrane to be destroyed at the postsynaptic cytoplasm. It was speculated at that time that acetylcholine or other chemical synaptic mediators could be associated with the synaptic vesicles. A t least from the quantitative point of view, the synaptic vesicles represent the most important, constant, and specific component of the synaptic terminal. Being confined almost exclusively to the proximal side of the synaptic region, the synaptic vesicles are the only elements which may confer to the synaptic region the necessary asymmetry for a polarized functional activity. The amount and disposition of the synaptic vesicles vary in different synapses, but in all cases one may observe their close association with the synaptic membrane (Figs. 3, 4, and 8). The profiles of synaptic vesicles are spherical or oval in shape with a dense limiting membrane 40 to 50 A thick and a content that is slightly denser than the matrix. The long diameter varies between 200 and 650 A. So far, extensive measurements have been made only in the retinal synapses, showing histograms with a high peak between 350 to 400 A and a mean diameter of 386 A (De Robertis and Franchi, 1956) (Fig. 9 ) . In the frog sympathetic ganglia the vesiculous material is very compact and fills the extreme distal part of the terminal. I n the ventral acoustic ganglion and in the glomeruli of the cerebellum the synaptic vesicles occupy the entire terminal with a rather homogeneous distribution (Fig. 5 ) ; in the rod-bipolar cell junction they are accumulated at an enlargement or expansion of the rod cell in which the postsynaptic element digitates and penetrates very deeply (Figs. 6, 7, and 8).

D. Submicroscopic Structure of Some Special Synapses 1. Invertebrate Synapses. In invertebrates the most commonly observed synapses are of the axo-axonic type. Neurons are usually monoFIG. 3. Electron micrograph of a bouton-like ending of the olfactory bulb of the rat showing three mitochondria ( m ) and numerous synaptic vesicles (m). G corresponds to glial processes ; d, dendrite. ( ~ 8 9 , 0 0 0 . ) FIG.4. Axodendritic synapse of the olfactory bulb. The zone of contact is indicated between the arrows. Three active points ( u p ) are indicated in the synaptic membrane. At these points the synaptic cleft is wider than in the rest of the junction, and dense material is present on both sides. See the intimate relationship of synaptic vesicles with the active points. My, myelin sheath ; psm, presynaptic membrane; ssm, subsynaptic membrane ; G, glia. ( ~ 8 9 , 0 0 0 . )

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polar, and the cell body is generally apart from the synaptic junction. The lack of vessels and the scanty number of glial elements favors the constitution of vast synaptic fields or dense neuropiles, where there is a great number of synaptic contacts per volume unit (Bullock, 1952). In the neuropile of the nerve cord of the earthworm, De Robertis and Bennett (1954, 1955) observed a complex tangle of unmyelinated nerve fibers in contact with no interposed glial elements. The fibers interdigitate extensively, forming complex and ramifying profiles. Mitochondria and endoplasmic reticulum are observed, but not typical neuroprotofibrils. Scattered in the neuropile there are regions containing large concentrations of synaptic vesicles of 200 to 400 A. Specialized areas of synaptic contact were recognized in which the postsynaptic membrane invaginates into the presynaptic one. Some synaptic vesicles were closely related to the presynaptic membrane, and a few of them were found in the interspace or synaptic cleft. Furthermore, faint ghostlike vesicular objects were observed in some postsynaptic fibers. These observations were interpreted as suggestive that vesicles may move toward the presynaptic membrane, perforate it, and discharge their contents into the interspace, and some of them may even enter and be destroyed in the postsynaptic cell. In arthropod neuropile, De Robertis and Franchi (1954) made similar observations of synaptic fields with synaptic vesicles. Recently Edwards (1957a, b) described the presence of numerous mitochondria and synaptic vesicles within the axon near or at the neuromuscular junction of annelid muscle and in the flight leg and abdominal muscles of higher insects. 2. Ultrastructure of the Neuromuscular Junction. The study of the fine structure of the neuromuscular junction is of considerable interest in view of remarkable advances made by physiologists by means of the microelectrode technique (see Tiegs, 1953 ; Fatt, 1954 ; del Castillo and Katz, 1956a, b ) . The complex structure revealed by the light microscope and the important studies on the histochemical location of cholinesterases have been reviewed by Couteaux (1955). In mammalian (Palade, 1954; Reger, 1954), amphibian (Reger, 1957), and reptilian synapses (Robertson, 1954, 1956), a close relationship was found between the branches of the innervating axons and the synaptic trough or grooves formed,by the sarcolemma. No interposed glia (teloglia) could be observed between the two contacting elements (see Couteaux, 1955). The main difference between the neuromuscular junction FIG. 5. Electron micrograph of a synapse of the cerebellum of a rat. The enlarged irregularly shaped endings correspond to mossy fibers that establish several contacts with dendrites ( d ) ; ap indicates active points. (x72,OOO.)

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and other types of synapse is found in the fact that at the subsynaptic membrane the sarcolemma differentiates in a very special manner. Folds approximately 800 A thick and 0 . 7 ~ long are formed which run transversely across the elongated axons, so providing channels between the interstitial spaces and the synaptic clefts. These folds, called “postjunctional” by Robertson ( 1956), were recognized earlier with the optical microscope and named subneural apparatus by Couteaux (1947) and Couteaux and Taxi (1952). Another characteristic of the neuromuscular synapse is the fact that the cleft is apparently wider (about 500 A ) and more complex than in other synapses. Within the axon, terminal mitochondria and numerous synaptic vesicles of 200 to 600 A may be observed (Palade, 1954; Robertson, 1956; Reger, 1957) (see Fig. 10). The relationship of these synaptic vesicles with the presynaptic and subsynaptic membranes and the synaptic cleft must be studied in normal and different physiological conditions. 3. Innervation of the Electric Organ. Electroplaques of the electric organ of different families of fishes were studied by Luft (1956). The plates are supplied with numerous nerve endings on one surface. The nerve fibers make lateral contacts upon papillae of the electroplate. At the junction the axon becomes closely approximated to the plate surface and is separated by a synaptic cleft of about 500 A. Beyond the synapse the axon is covered by Schwann cell cytoplasm. Synaptic vesicles accumulate at the site of contact, but they are less numerous in other parts of the axon (see Figs. 2C and 11). Neuroprotofibrils are present within the axon. 4. Synapses in Sympathetic Ganglia. In the abdominal sympathetic ganglia of the bullfrog, synaptic junctions upon the cell body and the emerging axon were recognized by De Robertis and Bennett ( 1954, 1955). I t was found that the Schwann cell covering of the fiber does not extend over the enlarged presynaptic ending, and the direct contact is frequently made in a depression of the postsynaptic neuron. Numerous densely packed synaptic vesicles were observed at the ending near the synaptic membrane. In Fig. 12 an axon-dendritic synapse in sympathetic ganglion of the cat is shown. It is interesting that in this synapse there is an accumulation of mitochondria in the postsynaptic cytoplasm. 5. Ultrastructure of Some Peripheral Nerve Endings. Vesicles similar to those found in typical synapses have been found in terminals about FIG. 6. Electron micrograph of a rod-bipolar cell synapse (see diagram of Fig. 2A) : d indicates the dendrites penetrating into the rod spherule which is totally filled with synaptic vesicles. A lateral synapse between the spherule and a dendrite is marked with arrows ( d ) ; gc, glial cell ; gin, glial membrane. ( ~70,000.)

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the hair cells of the cochlea (Engstrom and Sjostrand, 1954; Smith, 1957) and certain hair cells of the vesticular organ of the guinea pig (Wersall, 1956). If they were contained in real primary afferent fibers, the vesicles would occur in these cases on the postsynaptic side of the junction. This would be an exception to the rule of the presynaptic location of synaptic vesicles. The possibility exists, however, that these endings belong to tips of efferent fibers ending on the receptor (Rasmussen, 1953). It has been suggested that stimulation of these efferent fibers may produce a depolarization and reduction of impedance at the receptor (Engstrom and Sjostrand, 1954). Synaptic granules (vesicles) have also been described in clublike endings in the taste buds (Engstrom and Rytzner, 1956; Trujillo-CCnoz, 1957). Endings of the splanchnic nerve in the adrenal medulla of rabbits show a large concentration of typical synaptic vesicles that can be modified under nerve stimulation (see below, De Robertis and Vaz Ferreira, 1957b). 6. Microvesicles in Regenerating Nerves. The first observation of a vesicular material in regenerating nerve fibers was made in tissue cultures of the nervous system of the chick embryo (De Robertis and Sotelo, 1952). The growing endings of the fibers showed an enlarged mass with fingerlike processes, filled with tightly packed microvesicular material. Recently Estable et al. (1957) found in the growing tips of regenerating adult nerve fibers, after severance of the sciatic nerve, the appearance of numerous densely packed microvesicles 200 to 700 A in diameter. In regenerating limbs of Amblystomu, Hay (1957) found bulbous nerve endings containing numerous synaptic vesicles and small mitochondria. According to the author, each ending applied to two or more epithelial cells and resembled a synapse.

MORPHOLOGY AND FUNCTION OF T H E SYNAPSE 111. SUBMICROSCOPIC Since a general review of the subject under this title was presented at the Symposium on Submicroscopic Organization and Function of Nerve Cells (De Robertis, 1957), only some data will be summarized and discussed here. FIG.7. Rod-bipolar cell synapse showing the penetrating dendrites ( d ) and in p a process or blind infolding of the presynaptic membrane. Numerous synaptic vesicles become attached to this process. Gc, glial cell. ( x 114,000.)

FIG.8. Similar to Fig. 7. The relationship of the presynaptic membrane (psm) with the subsynaptic (ssm) and the synaptic cleft (sc) is shown. Synaptic vesicles attached to the psm are indicated with arrows ; em, surface membrane of the ending ; Gm, glial membrane. (x114,OOO.)

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A. Degenerative Changes of the Synapse The optical microscope has revealed that the alterations of the synaptic junction after section of the afferent axon consist in swelling and subsequent fragmentation and disintegration of the endings (Hoff, 1932 ;

x

x

I

40.

20-

Rod Synapses

Cone Synapses

FIG.9. Histogram showing the distribution of sizes (in percentage) of the synaptic vesicles. Rod synapses: ( A) rabbit exposed for 4 hours to sunlight; (B) in darkness for 24 hours; (C) in darkness for 46 hours; ( D ) in darkness for 9 days. Cone synapses : ( A ) Rabbit under sunlight for 4 hours ; (B) in darkness for 9 days. (Taken from De Robertis and Franchi, 1956.) Foerster et al., 1933; Hoff and Hoff, 1934; Gibson, 1937; Glees et d., 1946). I n the central nervous system, swelling of nerve endings has been seen as early as 24 hours after section. In peripheral synapses and particularly in the neuromuscular junction, nerve transmission fails before the

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axon has ceased to conduct (Titeca, 1935; Lissak et al., 1939; Eyzaguirre et ul., 1952). Similar changes in sympathetic ganglia have been correlated with decrease in acetylcholine content (Coppee and Bacq, 1938 ; McIntosh, 1938). Electron-microscope observations in normal ventral acoustic ganglion (Fig. 13A) and after destruction of the cochlea have revealed a sequence of degenerative changes in the synaptic endings (De Robertis, 1956). These involve swelling of the matrix, agglutination and lysis of synaptic vesicles, lysis and disintegration of mitochondria, and finally detachment and breakdown of the membrane at the synaptic junction (Fig. 13B, C, D ) . The first (after 22 hours) and more marked changes are those of the synaptic vesicles, and it has been suggested that they may be related to the early physiological deterioration of synaptic transmission (De Robertis, 1956, 1957).

B. Physiological Changes in Synapses of the Retinal Rods and Cones A detailed description of the submicroscopic organization of rod and cone synapses with bipolar cells was published by De Robertis and Franchi (1956). The observations extend the finding of synaptic vesicles to synapses between two receptors and the corresponding neurons. One of the striking characteristics of these synapses, as first observed by Sjostrand (1953), is the fact that the dendrites of the bipolar cells penetrate and digitate into the enlarged terminal endings of the rod and cone cells (Figs. 2A, 6, 7, 8 ) . This intimate and complex junction appears in the section showing the very bizarre profiles of a folded synaptic membrane. The presynaptic membrane shows blind infolds projecting into the terminals around which synaptic vesicles tend to accumulate (Figs. 2A and 8 ) . In Fig. 6, in addition to the most common rod synapse, there is a lateral junction of the rod spherule with a dendrite of the bipolar cell. This type of junction has been described by Polyak (1941). I n order to search for physiological changes, rabbits were maintained in complete darkness for periods of 24 hours to 9 days. Others, after dark adaptation, were submitted to intense light stimulation. I n dark-adapted animals the most significant fact is the accumulation of a large number of synaptic vesicles around the presynaptic membrane and processes (Fig. 8 ) . After 46 hours in darkness, and particularly after 9 days, there is a definite and striking reduction in size of the synaptic vesicles, both in rod and in cone synapses (Fig. 9) (see De Robertis and Franchi, 1956). In darkadapted animals, after stimulation by intense light the opening of synaptic vesicles into the synaptic cleft is frequently observed and also the passage of some of them into the cleft and even beyond the subsynaptic membrane. In the postsynaptic cytoplasm there are ill-defined ghostlike vesicles

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and filamentous material, apparently related to the disintegration of vesicles. Illustrations of these findings are shown in Figs. 5, 6, and 7 cf De Robertis (1957). These results were interpreted as indicative of an active flow of vesicles under the stimulation by light. Eccles and Jaeger (1958) have made the interesting suggestion that the liberation of the synaptic vesicles at the retinal synapses is effected by the hyperpolarization induced in the receptor cell through the photochemical reaction. In fact, according to Svaetichin (1953, 1957), there appears to be no impulse mechanism in the cone but only an increase in polarization from -45 to about -70 mv. This effect is much smaller than the depolarization occurring in the neuromuscular junction and presumably in other synapses. According to Eccles and Jaeger, “the invaginated synaptic membranes of the rod and cones may be a device for slowing down loss by diffusion and ensuing a cumulative action of small quantities of transmitter liberated over many milliseconds by the relatively small hyperpolarization.” This could be also in line with the long delays that are involved in photochemical mechanisms.

C . Changes of the Synapse after Nerve Stimulation Since the discovery of the synaptic vesicles in 1953 (De Robertis and Bennett, 1954, 1955), attempts were made by the authors to induce visible changes in the synapse by electrical stimulation. Several of these attempts failed because of technical difficulties in preparing the material for the electron microscope, but interesting observations were made about the fixation of some peripheral synapses with osmium tetroxide. It was observed with Professor Amassian at the University of Washington in December, 1953, that complete stopping of synaptic transmission in the FIG.10. Electron micrograph of a neuromuscular junction of the intercostal muscle of the mouse. ( A ) At the bottom, miofibrils ( m f ) , showing the Z lines and other structural details. On top, large amounts of sarcoplasm containing numerous mitochondria (m) (sarcosomes) and three sarcosomic nuclei (sn). The sarcoplasm is limited by the sarcolemmata (s) and by a differentiation of the sarcolemmata a t the level of the neuromuscular junction. This differentiation consists in infoldings of the subsynaptic membrane, the so-called postjunctional folds or subsynaptic folds (ssf), and constitute as a whole the subneural apparatus of Couteaux. Above this are endings ( e ) of an afferent axon containing a few mitochondria (m) of smaller size than the sarcosomes. T o the top left is a Schwann cell covering the ending. (~10,000.) ( B ) Enclosure B of Fig. 10A seen a t higher magnification. The ending with a smooth presynaptic membrane (psm) is in contact with the subsynaptic folds (ssf). The interspace is the synaptic cleft (sc) . Within the ending, mitochondria (nc) and numerous synaptic vesicles ( s v ) are seen. T o interpret this electron micrograph see the diagram of Fig. 2B. (X40,OOO.)

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celiac ganglion of Rana catesbiamz took place in about 10 seconds (Fig. 14). In Bufo arenurum Hensel the postsynaptic response disappeared in 5 to 10 seconds (De Robertis and Luco, 1954, unpublished observations). The results, although far from permitting the study of single nerve impulses, opened the possibility of detecting changes induced by coarser charges, such as overstimulation and fatigue of the synapse. For this purpose the nerve endings at the adrenal medulla of the rabbit were found to be better suited than the sympathetic ganglia. In this material nerve supply is abundant and belongs almost entirely to the homolateral splanchnic nerve which can be easily stimulated (see Teitelbaum, 1942). The preganglionic nerve fibers are cholinergic (Feldberg et al., 1933, 1934) and innervate the chromaffin cells directly without intercalated neurons. This type of junction is generally considered to be of synaptic nature (Rosenblueth, 1950). The postsynaptic signal that can be recorded in this system is the amount of adrenaline, noradrenaline, or total catechol secreted into the adrenal vein under electrical stimulation (Rapela and Coviin, 1954) or the analysis of the histochemical and submicroscopic changes of the stimulated adrenal cells with the electron microscope (De Robertis and Vaz Ferreira, 1957a). Preliminary accounts of the findings have been published (De Robertis and Vaz Ferreira, 1957b ; De Robertis, 1957). In the normal nerve ending, synaptic vesicles and other components of the synapse are found (Fig. 15). In the postsynaptic cell the large catechol droplets, surrounded by a thin membrane, and the content of reduced osmium are observed. Prolonged electrical stimulation of the splanchnic nerve induces striking changes in the synaptic vesicles. With a stimulus of 400 supramaximal pulses per second, known to produce fatigue of the ending and diminished output of catechol (Rapela and Covian, 1954), considerable depletion of synaptic vesicles occurs together with less significant alterations of the matrix and mitochondria (Fig. 16). On the other hand, with a stimulus of 100 pulses per second, known to FIG.11. Electron micrograph of a synaptic junction a t the surface of an electroplaque of the eel. This is a lateral synapse similar to that shown in the Diagram of Fig. 20 in a section along the axon. On top the cross section of the axon covered by the Schwann cell (Sc) is shown. At the junction the axon is free and contains synaptic vesicles ( s v ) . A synaptic vesicle apparently opening into the synaptic cleft (sc) is marked with an arrow ; ssm, subsynaptic membrane ; Ep, electroplaque. Note that the synaptic cleft is rather wide in this synapse (about 450 A ) . (x44,OOO.) (Courtesy of J. Luft.) FIG.12. Axon-dendritic synapse in a sympathetic ganglion of the cat. Between arrows is the synaptic junction. The endings contain synaptic vesicles and mitochondria. Observe that the dendrite contains neuroprotofibrils along the axis and sev--q1 mitochondria near the junction. ( x45,OOO.)

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EDUARDO D E ROBERTIS

8

A

C

D

FIG.13. Diagram showing: ( A ) Some details of the submicroscopic structure of a synaptic ending in the normal acoustic ganglion. Description similar to that of Fig. 1C: st, stalk of the synaptic ending; nj, neuroprotofibrils; g, glia; SyE,synaptic ending ; sm, synaptic membrane ; Psy, postsynaptic cytoplasm. Three active points are indicated with arrows. ( B ) Twenty-two hours after destruction of the cochlea. (C and D ) After 44 hours. The sequence B-C-D corresponds to the most common and progressive degenerative changes observed in the endings (see description in the text). (Taken from De Robertis, 1956.)

FIG.14. Tracing of the postsynaptic response of the celiac ganglion of Rana catesbinno stimulated with supramaximal pulses a t a frequency of 2 per second. In the upper line the time a t which the osmium tetroxide is dropped on the ganglion is indicated. After 10 seconds practically all synapses have ceased transmission. (Experiment made by Prof. Amassian at the University of Washington in December, 1953.)

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induce near maximal output of catechol (Rapela and Covian, 1954), there is a definite increase in the number of synaptic vesicles and in the liberation of them at the synaptic cleft (Fig. 17 ; see also Fig. 10 of De Robertis, 1957). In Fig. 18 the results of measurements of synaptic vesicles per square micron of the surface of nerve endings are indicated. In the control the mean number is 82.6 vesicles per square micron. With 100 pulse; per second the mean increases to 132.7 vesicles per square micron, and with stimulation of 400 pulses per second it decreases considerably with a mean of 29.2 per square micron. These striking changes of the synaptic vesicles under electrical stimulation with different frequencies confirm the presumption that they play a physiological role in synaptic transmission, as first postulated by De Robertis and Bennett (1954, 1955). These experiments suggest that a balance exists between the formation of synaptic vesicles and release of the transmitter. The equilibrium may be altered in one sense or the other, according to the frequency of the stimulus (De Robertis and Vaz Ferreira, 1957b). D. Dimensions and Physiology of Synapses One of the most general conclusions that can be drawn from the submicroscopic analysis of synapses is that, in spite of the differences in morphology, distribution, and geometry of synaptic regions, they offer basic similarities. These are essentially: 1. The discontinuity between the cytoplasm of the two apposed elements of the synapse. 2. The direct contact of the presynaptic and subsynaptic surface membranes, separated only by an interspace of 100 to 500 A. 3. The presence of synaptic vesicles on the presynaptic side of the synapse. All these characteristics have been observed in synapses of vertebrates and invertebrates ; in peripheral and central synapses ; in terminal or lateral synapses ; in some synapses between receptors and neurons ; in the neuromuscular junction ; and in some neuroeffectors (see above). These factors suggest that an essentially analogous physiological mechanism may be involved in all synaptic junctions. Similar conclusions have recently been reached by physiologists, especially by the use of intracellular recording (see del Castillo and Katz, 1956a, b ; Eccles, 1957). The two essential types of synaptic actions, the excitatory and the inhibitory, are produced by an ionic flux across the synaptic cleft into the subsynaptic membrane. I n excitatory synapses a depolarization of the adjacent postsynaptic membrane occurs ( Fatt and Katz, 1951; Fatt, 1954; del Castillo and Katz, 1954, 1956a, b ; Eccles,

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EDUARDO DE ROBERTIS

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1957), whereas in inhibitory synaptic action the ion flux leads either to hyperpolarization (Coombs et al., 1955a, b) or to antagonization of the depolarization induced by excitation (Fatt and Katz, 1953a, b ; Kuffler and Eyzaguirre, 1955) (Fig. 19B).

normal

1001s

4001 s

FIG.17. Diagram showing nerve endings of the adrenal medulla of the normal rabbit and after stimulation for 10 minutes with supramaximal pulses of 100 and 400 per second.

According to Eccles and Jaeger (1958) the functional operation of the synapse may involve the following processes : 1. The action potential causes the liberation of a transmitter substance from the presynaptic terminal into the synaptic cleft. 2. The liberated transmitter substance diffuses across the synaptic cleft to the subsynaptic membrane. FIG.15. Electron micrograph of a nerve ending of the adrenal medulla of the normal rabbit interposed between adrenal cells. The ending contains mitochondria and numerous synaptic vesicles: sm, synaptic membrane. In the adrenal cell large catechol-containing droplets ( c d ) and mitochondria are seen. ( x 57,000.) FIG.16. Electron micrograph of a nerve ending of an adrenal gland whose splanchnic nerve was stimulated a t 400 pulses per second for 10 minutes. The most significant change is the great depletion of synaptic vesicles (m) ; cd, catechol-containing droplets. ( ~ 3 3 , 0 0 0 . )

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3. The molecules of the transmitter become attached to specific sites of the subsynaptic membrane, and profound alterations of ionic permeability occur. 4. The consequent ionic flux alters the polarization of adjacent areas of the postsynaptic membrane, and thus a current flow (synaptic potential) is induced (Fig. 19A).

Control

Mean

Kx)

p u l a ~ ~ / ~ Mean

400 pulses/¶ Mean

FIG.18. Diagram showing results of measurements of synaptic vesicles per square micron of synaptic ending in control specimens and in rabbits with stimulation of the splanchnic nerve at 100 and 400 pulses per second for 10 minutes (see description in the text).

5. The effect of the transmitter substance on the subsynaptic membrane is ended by its removal by enzymatic destruction and diffusion into the interstitial spaces. When acetylcholine is the transmitter, its destruction may be partially effected by cholinesterase. “In summary we may state that the synapse is a device for applying minute amounts of a specific chemical substance to the specialized receptor area of the subsynaptic membrane, which in turn becomes highly permeable to some or all ions. The resulting ionic current through the subsynaptic membrane becomes effective by passing through the synaptic cleft and so to the remainder of the postsynaptic membrane” (Eccles and Jaeger, 1958). Eccles (1957) and Eccles and Jaeger ( 1958) have discussed the dimensional requirements of the synaptic cleft which will permit the efficient and

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rapid application of the transmitter and the relatively wide areas needed for the free flow of current. They have calculated the diffusion of a transmitter such as acetylcholine through a cleft of 200 A to occur in a few microseconds. Even with a distance of 500 A an effective distribution of acetylcholine could take place in about 10 psec. Therefore the dimensions

A

Inhibitory

I

Excitatory

FIG. 19. ( A ) Diagram of a synaptic ending similar to the type illustrated in Fig. lC, showing the lines of postsynaptic current flow when the subsynaptic membrane is influenced by the liberation of the transmitter substance (T) into the synaptic cleft. (B) Schematic representation of the functional operation of inhibitory and excitatory synapses. The resting potential is in both cases -70 mv. Under the action of the liberated inhibitory substance (Is) the potential is raised to -80 mv, and by the action of the excitatory substance (Es) it is diminished to 0 mv. The voltages driving the inhibitory and excitatory currents are thus -10 mv and +70 mv, respectively. (Taken from Eccles, 1957, and slightly modified.)

of the synaptic cleft shown by the electron microscope are of the kind needed for an efficient action of the transmitter. The areas needed for the passage of the postsynaptic currents must be relatively large-of the order of a few square microns-in view of the specific resistance offered by the cleft and the subsynaptic membrane. ( F o r an interesting discussion, see Eccles, 1957 ; Eccles and Jaeger, 1958 ; and also Palay, 1957b).

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E. Functional Role of Synaptic Vesicles An interpretation of the possible role of synaptic vesicles in the physiology of synaptic transmission should take into consideration the important findings made with microelectrodes in neuromuscular junctions. Fatt and Katz (1952) found that, in amphibian muscle, end plates are the seat of spontaneous subthreshold activity. This is manifested by miniature endplate potentials of the order of 1/100 of the synaptic potential in response to a nerve impulse (Fig. 20).

(A)

(B)

FIG.20. ( A ) Spontaneous miniature end-plate potentials recorded by intracellular electrodes at the end plate. ( B ) At a distance of 2 mm, in the same muscle fiber, the end-plate potentials are not recorded. In the lower part, taken at higher speed and lower amplification, the response to a nerve stimulus is shown. (Reproduced from Fatt and Katz, 1952.)

Different pharmacological properties of the miniature end-plate potentials led the authors to postulate that they must be due to the release of acetylcholine by the endings. Feldberg (1945) had already suggested that cholinergic nerve endings, even at rest, continually discharge small amounts of acetylcholine and replace it by chemical synthesis. The miniature end-plate potentials, however, could not be produced by simple molecular diffusion of acetylcholine, and Fatt and Katz (1952, 1953a, b) suggested that the release of the chemical mediator must be in multimolecular or quanta1 units (del Castillo and Katz, 1955, 1956a, b ; Boyd

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and Martin, 1956 ; Liley, 1956) arising from the synchronous discharge of a large number of acetylcholine ions. Since the amplitude of the spontaneous potential is only about onehundredth of the functional response to a nerve impulse, "it may be concluded that the apparatus for the release of acetylcholine at a junction is subdivided into large number of units (at least loo), each of which is able to operate independently of the rest" (Fatt, 1954). The authors believe that under the action of driving forces, such as their own thermal agitation catelect rotonus Presynaptic impulses

Excess K' ions

POST-ACTIVAT ION

\

'\ I

/

Activation depressed by Mg" excess ca+* deficiency

QUANTAS of ACH

(synaptic vesicles)

-1

Blocked by botulinum toxin

I DEPOLARIZATION of ENDPLATE (endplate potential) '

FIG.21. Diagram of the factors influencing the mechanism by which quantas of ACH (acetylcholine or synaptic vesicles) are ejected into the synaptic cleft. (Reproduced from Eccles, 1957.)

and the electric fields across the membrane, these quanta1 units of acetylcholine are suddenly discharged at localized points of the endings. The depolarization occurring at the arrival of the nerve impulse would produce a large synchronized action and thus the simultaneous discharge of many units which determine the end-plate potential. In Fig. 21 are illustrated some of the factors believed to be operative in the ejection of acetylcholine from nerve endings (Eccles, 1957). These physiological findings and theoretical considerations find extraordinary support in the submicroscopic organization of the synapse. The

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observations made on the structure and relationship of the synaptic vesicles with the membranes and their behavior in different physiological and pathological conditions (De Robertis, 1955a, b, 1956 ; De Robertis and Franchi, 1956; De Robertis and Vaz Ferreira, 1957b) are all consistent with the concept that the synaptic vesicle may represent the quuntal unit of acetylcholine postulated by Fatt, K a t z , and del Castillo (Fig. 21). In our first papers (De Robertis and Bennett, 1954, 1955), we suggested that acetylcholine and other chemical mediators could be associated with particles or vesicles of submicroscopic size. W e also postulated that the synaptic vesicles may move toward the presynaptic membrane and discharge their contents at the junction. The opening of synaptic vesicles and even their passage through the synaptic cleft and postsynaptic cytoplasm was postulated on the basis of observations in the earthworm neuropile (De Robertis and Bennett, 1954, 1955). Luft (1956) observed the opening of vesicles into the synaptic clefts of the electric organ; this process was most evident in the retinal synapses of dark-adapted animals after intense illumination (De Robertis, 1957). It seems possible that acetylcholine or other chemical mediators may be synthesized at the ending and segregated into packets surrounded by a membrane. The synaptic vesicles may then flow toward a position adjacent to the synaptic membrane. These points of attachment of the vesicles with the presynaptic membrane will constitute the active spots of the synapse observed by De Robertis (1955a, b) and Palay (195713) (Figs. 1C and 4). (For a biophysical consideration of these active spots see del Castillo and Katz, 1956a). One may postulate that in the resting condition single vesicles may spontaneously and randomly burst and discharge their content at localized spots of the junction, originating the miniature end-plate potentials of Fatt and Katz (1952). If the resting condition is prolonged, as in the case of dark-adapted animals, an accumulation of vesicles at the presynaptic membrane would occur (De Robertis and Franchi, 1956). When a propagated electrical disturbance in the form of an action potential reaches the presynaptic membrane, including a depolarization or a hyperpolarization of the nerve terminal, many vesicles will synchronously open at the synaptic interspace and liberate their contents of acetylcholine or other transmitter substances (Fig, 21). This process may in some cases, such as in the retinal synapses, involve the passage and rapid destruction of the vesicles into the postsynaptic cytoplasm. These concepts of flow and discharge of the synaptic vesicles are in agreement with those postulated by Bennett (1956) of membrane vesiculation as a mechanism for active transport and ion pumping. They also involve a dynamic structure for the membrane, with the possibility of local

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breakdown and restoration during synaptic transmission. This dynamic structural concept of the synapse is in agreement with physiological experiments suggesting that the chemical transmitter short-circuits the synaptic membrane (Fatt and Katz, 1953a, b ) . This results in a reduction of the resistance as well as the potential of the membrane and would be indicative of a large increase of permeability to all ions (del Castillo and Katz, 1954). According to Palay (1957b) the synaptic junction must be considered dynamic not only in its physiology but in its morphology as well. “After all, this is not a soldered junction of two hot wires, but a living system. . . . The processes of nerve cells may well be in constant play, flowing and shifting in position and in shape, as they do in tissue culture preparations. The contact points may shift from one position to another by gliding over the postsynaptic surface. At least, we may easily imagine a dynamic “scintillation” of the clustered synaptic vesicles, discharging now at one point, now at another. Such speculations are not fantastic, but are merely extensions of current knowledge concerning the dynamic life of the cell” (Palay, 1957b). IV.

SUMMARY

The electron-microscope study of synaptic regions has revealed a highly differentiated and specific submicroscopic organization, which seems to be specially fitted to carry out the transmission of the nerve impulse. In spite of differences in morphology, distribution, and geometry, synaptic regions have the following basic similarities : 1. A definite discontinuity between the cytoplasm of the two apposed cellular elements of the synapse, confirming that the individuality of the neuron applies to the finest submicroscopic expansions. 2. A direct contact of the presynaptic and subsynaptic surface membranes separated only by an interspace of 100 to 500 A, indicating that at the junction no other cellular material alien to the two synaptic elements is interposed. 3. The presence of a special microvesicular material-the synaptic vesicles (De Robertis and Bennett, 1954)-on the presynaptic side of the synapse. These structural similarities suggest that an essentially analogous physiological mechanism may be involved in all synaptic junctions. The intimate relationship of the synaptic vesicles with the junction ; their early lysis in degeneration of the synapse ; the fact that under physiological stimulation the actual flow of vesicles into the synaptic cleft and even into the near postsynaptic cytoplasm may be observed in some synapses ; the changes in size that can be found by disuse of the junction ; and finally the intense modifications of the number of synaptic vesicles

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after nerve stiniulation-all are indicative of the direct intervention of this submicroscopic component in synaptic transmission. Furthermore the presence of clusters of synaptic vesicles in contact with certain zones of the presynaptic membrane is probably an indication that there may be active “scintillating” points in the functional operation of the synapse which is in agreement with recent electrophysiological studies. The most appealing possibility is that synaptic vesicles may represent quanta1 units of a chemical transmitter, such as acetylcholine, as has been postulated by physiologists. The spontaneous discharge of single synaptic vesicles may give rise to the miniature end-plate potentials recorded by microelectrodes in the neuromuscular junction (Fatt and Katz, 1952). When a propagated electrical disturbance in the form of a nerve impulse reaches the junction, inducing a depolarization (excitatory synapses) or a hyperpolarization (inhibitory synapses), many synaptic vesicles may liberate the transmitter and determine a large end-plate potential, which in turn gives rise to the depolarization of the postsynaptic element. It seems possible that acetylcholine or other chemical mediators may be synthesized at the ending, segregated into packets by a limiting membrane, and then flow toward a position adjacent to the synaptic membrane ready for instantaneous discharge at the arrival of the nerve impulse. These concepts of flow and discharge of synaptic vesicles are in agreement with similar mechanisms observed in the synthesis and excretion of other neurohormones such as adrenaline and noradrenaline (De Robertis and Vas Ferreira, 1957a). They involve a dynamic structure for the membrane, with the possibility of local breakdown and restoration during synaptic transmission. These morphological and physiological correlations at a submicroscopic level should be continued by a closer collaboration of physiologists working with microelectrodes and electron microscopists. Furthermore they should be integrated with the study of the patterns of chemical and enzymatic organization which are operative at the different synapses.

V. REFERENCES Armstrong, J., Richardson, K. C., and Young, J. Z. (1956) Stain Technol. 31,263. Arvanitaki, A. (1942) 1. Neurophysiol. 5, 108. Barr, L. M. (1939) I . Anat. 74, 1. Bartelmez, G. W., and Hoerr, N. L. (1933) J . Comp. Neurol. 67,401. Bennett, H. S. (1956) J . Biophys. Biochem. Cytol. 2, Suppl. 99. Bodian, D. (1940) J . Comp. Ncurol. 73, 323. Bodian, D. (1942) Physiol. Revs. 22, 146. Bodian, D. (1952) Cold Spring Harbor Symposia Quant. Biol.17,1. Boyd, I. A., and Martin, A. R. (1956) I . Physiol. (London) 13a,61. Bullock, T. H. (1952) Cold Spring Harbor Symposia Quant. Biol.17,267. Bullock, T.H. (1953) J . Comp. Neurol. 28, 1.

SUBMICROSCOPIC MORPHOLOGY OF T H E S Y N A P S E

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Cajal, S. R. y (1934) Trab. inst. Cajal invest. biol. (Madrid) 24, 1. Castillo, J. del, and Katz, B. (1954) J. Physiol. (London) 126,546. Castillo, J. del, and Katz, B. (1955) J. Physiol. (London) 128,3%. Castillo, J. del, and Katz, B. (1956a) Prog. Biophys. and Biophys. Chew. 6, 121. Castillo, J. del, and Katz, B. (195613) J. Physiol. (London) 132,630. Castro, F. de (1942) Trab. inst. Caial invest. biol. (Madrid) 34,217. Castro, F. de (1950) Verhandl. deut. Ges. Pathol. 34 Tgg. Chu, L. W. (1954) J. Comp. Xeurol. 100,381. Coombs, J. S., Eccles, J. C., and Fatt, P. (1955a) J. Physiol. (London) 130, 374. Coornbs, J. S., Eccles, J. C., and Fatt, P. (1955b) J. Physiol. (London) l30,326. CoppCe, G., and Bacq, Z. M. (1938) Arch. intern. physiol. 47, 312. Couteaux, R. (1947) Rev. can. biol. 6, 563. Couteaux, R. (1955) Intern. Rev. Cytol. 4, 335. Couteaux, R., and Taxi, J. (1952) Arch. anat. microscop. morphol. exptl. 41, 352. De Robertis, E. (1955a) Ac fa Neurol. Latinoam. 1, 1. De Robertis, E. (1955b) Anat. Record 121,284. De Robertis, E. (1956) J. Biophys. Biochem. Cytol. 2, 503. De Robertis, E. (1957) “Submicroscopic Morphology and Function of the Synapse.” Exptl. Cell Research Suppl. 6, 347 (1958). De Robertis, E., and Bennett, H. S. (1954) Federation Proc. l3,35. De Robertis, E., and Bennett, H. S. (1955) J. Biophys. Biochem. Cytol. 1, 47. De Robertis, E., and Franchi, C. M. (1954) J. Appl. Phys. 26, 1162. De Robertis, E., and Franchi, C. M. (1956) J. Biophys. Biochem. Cytol. 2, 307. De Robertis, E., and Sotelo, J. R. (1952) Exptl. Cell Research 3, 433. De Robertis, E., and Vaz Ferreira, A. (1957a) Exptl. Cell Research 12, 568. De Robertis, E., and Vaz Ferreira, A. (1957b) J . Biophgs. Biochem. Cytol. 3, 611. Eccles, J. C. (1957) “Physiology of Nerve Cells.” Johns Hopkins Press, Baltimore, Maryland. Eccles, J. C., and Jaeger, J. C. (1958) Proc. Roy. SOC.Bl48, 38. Edwards, G. A. (1957a) Anat. Record 528, 542. Edwards, G. A. (1957b) Anat. Record 128, 543. Engstrom, H., and Rytzner, C. (1956) Ann. Otol. Rhinol. & Laryngol. 65,361. Engstrom, H., and Sjostrand, F. S. (1954) Acta @to-laryngol. 44, 490. Estable, C. (1953) “Symposium on the Synapses, Montevideo” in press. Estable, C., Reissig, M., and De Robertis, E. (1953) J. Appl. Phys. 24, 1421. Estable, C., Acosta-Ferreira, W., and Sotelo, J. R. (1957) 2. Zellforsch. U. mikroskop. Anat. 46, 387. Eyzaguirre, C., Espindola, J., and Luco, J. (1952) A cfa Physiol. Latinoam. 2, 213. Fatt, P. (1954) Physiol. Revs. 34, 674. Fatt, P., and Katz, B. (1951) J. Physiol. (London) 116, 320. Fatt, P., and Katz, B. (1952) J. Physiol. (London) 117, 109. Fatt, P., and Katz, B. (1953a) Acta Physiol. S c a d . aS, 117. Fatt, P., and Katz, B. (1953b) J. Physiol. (London) 121, 374. Feldberg, W. (1945) Physiol. Revs. 26, 596. Feldberg, W. (1954) Pharmacol. Revs. 6, 85. Feldberg, W., and Minz, B. (1933) Arch. ges. Physiol. PfEiiger’s a83, 657. Feldberg, W., Minz, B., and Tsudrnizura, H. (1934) J. Physiol. (London) 80, 15; 81, 286. Foerster, O., Gagel, O., and Sheenan, D. (1933) 2. Anat. u. Ent.wicklungsgeschichte 101, 553.

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Gibson, N. C. (1937) Arch. Neurol. and Psychiat. 38,1145. Glees, P., Meyer, A., and Meyer, M. (1946) J. Anat. 80, 101. Haggar, R. A., and Barr, M. L. (1950) 1. Comp. Neurol. 93, 17. Hay, E. D. (1957) Anat. Record 128,562. Held, H. (1897) Arch. Anat. u. Physiol. Anut. Abt., Suppl. 273. Hoff, E. C. (1932) Proc. Roy. SOC.B111, 175. Hoff, E. C., and Hoff, H. E. (1934) Brain 67, 175. Horstmann, E. von (1957) Deut. med. Wochschr. 82, 731. Kuffler, S. W., and Eyzaguirre, C. (1955) J. Gen. Physiol. 39, 155. Liley, A. W. (1956) J. Physiol. (London) 132,650. Lissak, K.,Dempsey, E. W., and Rosenblueth, A. (1939) A m . 1. Physiol. 128,45. Luco, J. V., and Davidovich, A. (1956) Rev. medicinu (Argentina) 16, 295. Luft, J. (1956) J. Biophys. Biochem. Cytol. Suppl. 2,229. McIntosh, F.C. (1938) Arch. intern. Physiol. 47,312. Noel, R. (1950) Biol. mid. (Paris) 39,319. Palade, G. E. (1952) 1. Ezptl. Med. 95,285. Palade, G. E. (1954) Anat. Record 118,335. Palay, S.L. (1954) Anat. Record 118,336. Palay, S. L. (1956) J. Biophys. Biochem. Cytol. 2,193. Palay, S. L. (1957a) Progr. in Neurobiol. II. Ultrastructure and Cellular Chem. Neural Tissues, p. 31. New York, Hoeber. Palay, S. L. (195%) “The Morphology of Synapses in the Central Nervous System.” Exptl. Cell Research Suppl. 6,275 (1958). Pease, D. C. (1953) Anat. Record 115,359. Polyak, S.L. (1941) “The Retina.” Univ. Chicago Press, Chicago, Illinois. Rapela, C. E., and Coviin, M. R. (1954) Rev. soc. arg. biol. SO, 157. Kasmussen, G. L. (1953) J. Comp. Neurol. 99, 61. Rasmussen, G. L. (1957) “New Research Techniques of Neuroanatomy,” p. 27. C. C Thomas, Springfield, Illinois. Reger, J. F. (1954) Anat. Record 118,344. Reger, J. F. (1957) Exptl. Cell Research 12,662. Robertson, D. (1953) Proc. SOC.Exptl. Biol. Med. 82,219. Robertson, D. (1954) Federation Proc. 13, 119. Robertson, D. (1956) 3. Biophys. Biochem. Cytol. 2, 381. Rosenblueth, A. (1950) “The Transmission of Nerve Impulses.” Wiley, New York. Scharrer, E. (1945) J. Comp. Neurol. 83,237. Sherrington, C. S. (1897) The central nervous system. In Sir Michael Foster’s “A Textbook of Physiology,” 7th ed. Macmillan, London. Sjostrand, F. S. (1953) 1. Appl. Phys. 24, 1422. Smith, C. A. (1957) Anat. Record 127,483. Svaetichin G. (1953) Acta Physiol. Scand. 29,Suppl. 106,565. Svaetichin, G. (1957) Acta Physiol. Scand. 99, Suppl. 154, 17. Teitelbaum, H. A. (1942) Quart. Rev. Biol. 17, 135. Tiegs, 0. W. (1953) Physiol. Revs. 33,90. Titeca, J. (1935) Arch. intern. physiol. 41, 1. Trujillo-CCnoz, 0. (1957) 2. Zellforsch. u. mikroskop. Anat. 46, 272. Wersall, J. (1956) Acta Oto-Laryngol. Suppl. No. ls, 1. Wyckoff, R. W. G., and Young, J. Z. (1956) Proc. Roy. Soc. B144,440. Young, J. Z. (1939) Phil. Trans. Roy. SOC.B229, 465.

The Cell Surface of Paramecium’’2

c. F. EHRET

AND

E. L. POWERS

Division of Biological and hfedical Research, Argonne Nationul Laboratory, Lemont, Illinois

I. The Problem ..................................................... 11. The Evidence .................................................... A. Generalized Surface (or Pellicle) System ..................... 1. Introduction .............................................. 2. Historical Perspectives ................................... 3. Contemporary Views ..................................... B. Specialized Food-Intake (or Gullet) System ................... 1. Introduction .............................................. 2. Historical Perspectives ................................... 3. Contemporary Views ..................................... C. Replication Mechanisms ...................................... 111. Synthesis and Outlook ........................................... IV. Acknowledgments ................................................ V. References .......................................................

I.

Page 97 99 99 99 99 104 114 114 122 124 125 128 132 132

THEPROBLEM

When it is viewed from a sufficiently large distance-or at a sufficiently small magnification-a cell is simply a cell. On closer inspection it is difficult to find any “ordinary” cell, and one’s own favorite material, whatever it happens to be, generally appears as quite an oddity alongside some hypothetical type. Quite apart from any considerations of its cellularity or acellularity, Paramecium has appeared as such an oddity. It has been endowed by its investigators with not one but two layers of a chicken-wirelike silver-line system ; with a torpedolike defense system that underlies the outer pellicle in the form of the explosive and cytologically unique trichocysts; and with a food-intake system that is almost yeastlike in its supposed capacity to “bud-off” a new gullet whenever the cell chooses to divide. If true, these are indeed unique characteristics for an organism to possess without losing its right to the term “cell.” In recent years phase contrast and electron microscopy have allowed a closer third look at our object, and the present paper will show how the results of these investigations affect the cellular status of Paramecium and some of its distant relatives. W e include in this review all papers that have contributed significantly Work performed under the auspices of the U. S. Atomic Energy Commission. We presented much of the original material in this review at the Round Table on Ultrastructure o f the Protozoa sponsored jointly by the Society of Protozoologists and American Society of Zoologists at Stanford University, August 26-29, 1957. 1

2

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to an understanding of the surface structure of Paramecium published since the last comprehensive review of the subject by Taylor (1941). For purposes of clarity we have avoided discussing again the confusing synononiy of archaic terms not strictly pertinent to contemporary understanding (of historical and etymological interest are the comparisons of terms by Taylor, 1941, and Parducz, 1958a). The general form of Paramecium reminded Thompson (1952) of a “partial realization of the nodoid” ; to generations of less imaginative textbook writers after Joblot (1718), it was like a slipper. W e have considered this cell as an organization of its composite systems and have shown that the surface elements are physically dissectible into at least two distinct systems ; ( 1 ) the generalized surface or pellicle system, and (2) the specialized food-intake or gullet system (Ehret and Powers, 1956). The pellicle system covers the cytoplasm and has cilia more or less uniformly distributed on its outside ; the food-intake system is an elaboration of the cell surface that has ciliated as well as nonciliated surfaces on its tubelike inside. These two systems will be considered separately. But first a brief survey of the methods of analysis that have been employed seems desirable. The light microscope has been used extensively on fixed and stained cells but rarely on unstained specimens (Worley, 1933). Conventional staining methods have been employed, including especially Loeffler’s stain and bichromic osmic with which Schuberg (1905) made the first extensive descriptions of pellicular and granular patterns. The “dry” silver method was used by Klein (1958, summary) in an extensive series of papers that described “silver-line” or “neuroformative” systems in many ciliates. Alternative “wet” silver methods have been employed by most other workers including J. and G. von Gelei (1932, 1937, 1939), Chatton and Lwoff (1930), and Corliss ( 1953). Phase-contrast microscopy has been used to advantage on unfixed animals, either whole and intact or compression-dissected (Ehret and Powers, 1955, 1956), and on digitonin-fixed cells (Child and Mazia, 1956; Pitelka, 1956). These methods have been extremely valuable in recognizing some of the fixation artifacts that were present in presumably well-prepared silver-impregnated specimens (Ehret and Powers, 1957a ; Yusa, 1957). Electron microscopy has been employed on formalin-fixed ultrasonically disintegrated cells (Metz et al., 1953) and on thick- or thin-sectioned osmium-fixed cells (Powers et al., 1956; Ehret and Powers, 1956, 1957a; Sedar and Porter, 1955; Roth, 1957; Watanabe, 1957). W e believe that none of these methods singly can lead to a comprehensive understanding of the structure of any cell. Since all methods result

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to some extent in the production of artifacts, it is essential that the final generalized description provide rational explanations for the existence of each artifact, and relate it to the undisturbed living cell.

11. THEEVIDENCE A . Generalized Surface (or Pellicle) System

1. Introduction. According to some earlier views, certain line patterns that show up in the pellicle after silver impregnation and certain other treatments seemed to represent a hierarchy of “fibrillar” systems encasing the cell. A combination of the views of Schuberg (19O5), Klein (1928, 195S), J. and G. von Gelei (1932, 1937), and the Chatton school would include at least three strata of “fibrillar” systems ; these are an outer and an inner lattice, each chicken-wirelike, and sandwiched in between them a system of longitudinal fibrils from which the cilia were thought to originate, extending through holes in the outer lattice. Later views have included the same three strata, minus one. Using the electron microscope, Metz et al. (1953) failed to see the inner lattice but did identify an outer one and the underlying cilium-base interconnecting (kinetodesmal) fibrils. Sedar and Porter (1955), like Lund ( 1933) before them, assert that the outer lattice is mostly a pattern of cytoplasmic ridges and not fibers. In contrast also to Metz et al. (1953), however, they do claim to identify the inner lattice of G. von Gelei. Our concept of the structure of the cell surface, based chiefly on recent evidence, is new, although it contains some of the elements of notions of earlier authors. This new view eliminates both of the lattices as independent fibrillar systems, substituting a corpuscular organelle-packing concept for the “layered-wire” models of cell-border organization. The remaining bona fide fibrils that we recognize in the old patterns (the kinetodesmal fibrils) are directly comparable with those observed in the ground substance of other ciliated cells (Fawcett and Porter, 1954; Fawcett, 1958a) and are more specifically identifiable by us as the individual elements of the fibers of the infraciliature of other ciliates as described by Chatton and Lwoff ( 1930) and by Metz et al. (1953). In addition to these there may be certain fiberlike structures such as the so-called motorium, which appears in the gullet region to consist of an aggregation of 2O-mp filamentous components as described by Roth (1958). 2. Historuul Perspectives. Although most of the interpretations of surface structure through the time of Taylor’s review are incorrect, the well-presented evidence of some of the earlier workers, particularly in the publications of J. and G. von Gelei, must be taken into account and explained. Figure 1 shows a photomicrograph of the silver-impregnated

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surface of Paramecium caudatum prepared by G. von Gelei (1937). This represents the classical outer silver-line system. The loci of cilia and trichocysts are evident between and upon the hexagonal and rhomboidal patterns. These important constituents of the silver-line system were thought to be structural in nature and fibrillar in substance. Practically

FIG.1. Silver-line patterns on the surface of Parameciuin caudatum (Bielschowsky’s ammoniacal silver nitrate, reduced in sunlight: from G. von Gelei, 1937). A, B. Dorsal and ventral views of the so-called “outer lattice.” Starting with a cilium base in the center of any polygon, one can read up or down along the Schuberg patterns “cilium-trichocyst-cilium . C. The so-called “infraciliary lattice system” of an entire cell; the excretory pores of the contractile vacuoles are marked

. .”

be.

all preparations have been,interpreted so as to take these patterns into account (Schuberg, 1905; Klein, 1928; von Gelei, 1932; Parducz, 1957, 1958a, b ) . Some longitudinal line patterns between the cilium and trichocyst loci are also evident. These were mistakenly thought to represent the actual cilium-base interconnectives (now termed kinetodesma) and were called “longitudinal connecting fibers.” It is important to note that these vertical

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lines appear neither so straight nor so regular in thickness on the photographs as they do in the diagrammatic renderings of this surface: In some cases they even appear double and have been separately termed Nebenfibrillen (G. von Gelei, in Parducz, 1957). Because the best early descriptions of these longitudinal line patterns were presented by Schuberg ( 1905), we shall refer to them as “Schuberg patterns.” These patterns are to us exactly what they were to Schuberg; however, their structural interpretation is quite different, as we shall discuss below. They constitute one of the greatest sources of confusion in the literature to this date. Figure 1C represents the classical “infraciliary lattice system” of G. von Gelei. Note especially the irregularities of the inner pattern-including starlike configurations like those that Schuberg had described as feine Linien in the hexagonal fields of the pellicula thirty years earlier. Other aspects of this characteristically variable “infraciliary lattice” include a triangular pattern and a foamlike pattern (described as schumurtig), each unit of which is highly irregular in its shape and period. For an appreciation of the enormous diversity of patterns found in both the outer and the inner lattice, but especially the latter, the original paper of G. von Gelei should be consulted. Worley’s (1933) observations were similar in some respects. Using light microscopy and freshly crushed cells, he observed connecting fibers between the bases of cilia in the unstained cell. In his Fig. 2 he shows a series of bodies (imperfectly resolved, but in some and possibly in all cases double) in the vestibular region of the pellicle. These bodies occur at a linear frequency of about one per micron. From the lower left of each body (or pair) a fiber about 3 to 4 p long passes slightly downward and sharply to the left in the figure ; because of its length it underlies about three neighboring bodies and laps the fibers of those neighbors. Worley’s interpretation of these as the fibers that interconnect the bases of the cilia was almost correct. It is our view, however, that Worley was in error in associating these kinetodesma with the Schuberg patterns observed after silver impregnation; he referred to them in a hybrid terminology as “specialized longitudinal fibers of the silver line system,” implying a connection. Worley failed to note that one can see on his photographs the Schuberg patterns, and at the same time the kinetodesmal fibrils. The Schuberg patterns are faintly evident in some areas of his figure as parallel to and midway between the kinetodesma, but more nearly overlying the paired bodies than are the kinetodesma. Because of the curved coursing of the kinetodesmal fibrils, these fibrils come to lie nearly midmy between the r m s of paired bodies-i.e., lateral to the cilia and the longitudinal lines that appear to connect them. Thus, although Worley’s studies of

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impulse transmission emphasized the effects of severing the cilium-base interconnections (or kinetodesma), he described them as effects on the silver-line system. This error in identity led him to conclude that the silver-line system is of significance in the conduction of metachronal impulses (Worley, 1934), a conclusion accepted on the same grounds by Wichterman ( 1953). The ultrastructural basis for these Schuberg patterns and their real relation to surface structure we describe later (Figs. 3-9). The latest period in these investigations began with the convincing evidence of Metz et al. (1953) of the compound structure of the kinetodesmos. The method involved formalin fixation, sonic “dissection,” and electron-microscopic viewing of the resulting fragments. The kinetodesmos is seen to be “a bundle of tapering fibrils, each arising independently from a kinetosome, and extending for a modest distance along the bundle” (Pitelka and Metz, 1952; Metz et al., 1953). These authors (correctly in our view) regarded the kinetodesmal unit as a portion of a subpellicular k i n e t ~ or , ~ inner fiber system. Each unit consists of three parts: (1) the cilium which ends internally at (2) the kinetosome. From each kinetosome originates ( 3 ) the tapering fibril that parallels the body surface. The “fibrils from a longitudinal row of kinetosomes overlap in shingle-like fashion to form a tight bundle. This bundle is the kinetodesma ,(sic) of the light microscopist.” In addition to the kinety system, Metz et al. (1953) also describe an outer fibrillar lattice system intimately associated with the pellicular membrane. In the center of each polygon of the outer lattice is a ring-shaped structure through which the cilium passes ; near this ringshaped structure a second smaller “accessory” ring-shaped thickening is found. Between the outer lattice system and the kinety system “no obvious connection exists.” These authors found no evidence for the inner lattice system of G. von Gelei (1937) but agreed that if it exists it might have been destroyed by the sonic treatment. Unfortunately Metz and Westfall ( 1954) treat kinetodesma synonymously with “silver-line fibers of the light microscopists,” although several clues to their separability were visible in the photographs described by Metz et al. (1953) ; “one other structure associated with the pellicle 3 The terminology of the Ghatton-Lwoff school (Lwoff, 1950) is so well entrenched in the literature and so internally self-consistent (kinetosome, kinety, kinetodesmal fibril, kinetodesma, desmodexy) that it merits adoption on the operationally descriptive (nondeductive) level. In this sense, the implication of genetic continuity attached to the definition of kinetosome (Chatton and Skguela, 1940; Lwoff, 1950) should be regarded merely as an unproved hypothesis. The ultrastructure of the kinetosome as a cilium base, and as distinguished from the blepharoplast of a flagellum, has been treated by Roth in the modern context (1957, 1959).

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is evident in some preparations. This is a strand or fiber extending across the polygon from one trichocyst attachment point to the ciliary ring and on to the next trichocyst attachment point. . . The nature of these is not clear. They appear to be fibers of the same sort and continuous with the lattice fibers. . . . However, they are not present in all preparations, particularly those subjected to prolonged sonic treatment, and their form is somewhat variable. If this material should take a silver stain, it could well have been confused with the primary (kinety) fibrillar system.” In our interpretation, developed below, this is the silver-stainable material, whereas the kineties are not, and with silver impregnation this material is identified as the Schuberg patterns of the surface. Another clue came from the apparently displaced positioning of kinetodesmal fibrils with reference to the kinetosomes. “Inspection of the figures shows that the kinetodesmal bundles lie to one side of the kinetosomes and that the individual fibrils curve laterally from their kinetosomal origins to join the main bundle. This condition is probably exaggerated somewhat by the action of surface tension when the preparation is dried. In this connection it should be noted that the kinetodesma (sic) appears as a line directly connecting adjacent kinetosomes in most light microscope studies.” The electron micrographs of osmium-fixed sections demonstrate that the kinetodesmal bundles are lateral to the bases of the cilia. The probable basis for the misinterpretation is the failure, longstanding in the literature, to distinguish between the “directly connecting” patterns (Schuberg patterns) and the true kinetodesmal bundles lying between the rows of adjacent kinetosomes. The usefulness of these two papers for their photographic data and correct observations (like the earlier papers of Klein, Schuberg, the von Geleis and Lund) should not be lost sight of in the light of the less important nomenclatural inconsistencies we have tended to emphasize above. Unfortunately, slight inconsistencies have a way of becoming amplified at the interpretive level, and it is essential that they be thoroughly comprehended. I n an electron-microscope study of thin sections of Paramecium multimicronucleaturn, Sedar and Porter (1955) concur with Metz et al. that the kinetodesma are composed of fibrils that “overlap in a shingle-like fashion” and agree to the existence of a kinety or inner fibrillar system. They deny, however, the existence of an outer fibrillar system and attribute it to the cytoplasmic ridges of “a polygonal ridgework with depressed centers” that underlies both a plasma membrane and “two closely opposed membranes (together 250 A thick) constituting the pellicle.” They conclude that a gelled layer of ectoplasm may maintain the form of the

.

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ridges ; that this may be the material made visible by silver impregnation ; and that the thick fragments of the sonically dissected cells could mislead an observer into interpreting the ectoplasmic ridges as fibers. On the other hand, Sedar and Porter “confirm” the existence of an infraciliary lattice system like that of G. von Gelei (1937), separate and distinct from the kinetodesmal system. They assert that ( 1 ) it is well separated from the ridges in the plasma membrane, (2) it is located at the level of the kinetosomes, and ( 3 ) it is difficult to confuse with the outer polygonal system. Thus, Sedar and Porter recognized as fibrillar systems both the kinety system and an infraciliary lattice system but explained the outer lattice of the light microscopist as a misinterpretation of the pattern of cortical ridges. W e believe that “cortical ridges” do not explain the outer lattice pattern, nor do they account for the Schuberg patterns of the old silver-line system which the authors make no attempt to explain. A complete summary of recent work and ideas is presented by Parducz (1957, 1958a, b), who arrived at a most inclusive concept of surface structure in Paramecium. Unlike Grell’s reconstruction ( 1956), which almost entirely ignores the classical evidence, Parducz’s considerations are based on all the pertinent data, both classical and contemporary. H e concluded that no less than four “clearly discernible fibrillar systems can be distinguished in the peripheral-about 2p thick-zone of the ectoplasma if we proceed inward; ( 1 ) external lattice, (2) network of interciliary fibrils, ( 3 ) subpellicular bundles of ciliary root-processes, (4) G. von Gelei’s infraciliary or internal fibrillous network.” It is regrettable that Parducz failed to attempt a three-dimensional construction of the surface structure despite his essentially correct analysis of the sum of necessary and sufficient patterns present in two dimensions at various levels of optical sectioning. Parducz should also be credited with discriminating between kinetodesma and “interciliary fibrils” for the first time in any extensive account ; we had independently pointed out this important distinction on two previous occasions (Ehret and Powers, 1956, 195713) but had further indicated the nonfibrillar character of the “interciliary fibrils” (longitudinal connecting fibers) and had then referred to them as Schuberg patterns. 3. Contemporary Views. A disarmingly simple view of the generalized surface structures or pellicle system was obtained by us using the phase-contrast microscope and unfixed or living Paramecium bursaria (Ehret and Powers, 1956). After dissection by rapid compression and decompression between slide and cover slip, the pellicle system appears as sheetlike fragments, each composed of close-packed polyhedral organelles (Fig. 2 A ) . Some of these organelles float entirely free of a fragment

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and then appear as spheroids, with kinetodesmal and ciliary fibrils still intact (Fig. 2C). Such phase-contrast images compare favorably with the light microscope images observed by Worley (1933) and substantiate the general correctness of his views, once the terminology is made internally consistent. (The pattern of pellicle close packing is generally hexagonal ; one exception appears in the region of the food-intake system, or

FIG.2. Phase-contrast micrographs of compression-dissected fragments of untixed pellicle system of P. bursoria showing the ciliary corpuscles that compose this surface. A. Close (hexagonal) packing of the corpuscles; kinetodesmal fibers a t upper right. B. Frayed edge of pellicle sheet. C. Isolated clusters of corpuscles, and individual corpuscles with cilia and kinetodesmal fibrils still attached. One trichocyst tip with a discharged shaft in its wake is seen in the lower right quadrant. gullet, where it is rhomboidal.) This was indeed a surprise: if the pellicle surface is entirely composed of corpuscular organelles packed together like a single layer of soft peas packed in the bottom of a bowl, then actual or optical sections of these would indeed yield hexagonal patterns. What then of the three possible layers of fibers? Certainly the “middle” layer was no artifact-the kinetodesmal fibers had been evident in unfixed preparations even in the light microscope, and ultrastructural characteristics

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had been described. The possibility arose that the other “fibrillar systems” did not exist, a possibility that required electron microscopy for its full resolution. The geometrical solution to the problem came after a study of hundreds of sections. Its principal key was in the recognition of the physical basis for the Schuberg patterns (Ehret and Powers, 1956, 1957b). These patterns are clearly evident on superficial tangential sections such as at the periphery of the thick section in Fig. 3 A ; in some regions double patterns are seen about the cilia (opm, Fig. 3 B ) , whereas elsewhere the patterns, though continuous throughout the spaces peripheral to the cytoplasm, are linear, or of free and variable form. The patterns are those to be expected of sectioned sheets rather than of sections of wirelike fibers. Other electron micrographs of thin sections repeat pattern variations on this same theme; on Figs. 3B and 4, for example, note the irregular and star patterns, as well as the double lines representing the Schuberg patterns (see also Fig. 3 in Sedar and Porter, 1955). In sections such as these, all the G. von Gelei “infraciliary lattice” patterns are represented, and in every case its substance is continuous with the sheetlike substance of the Schuberg patterns. It should be realized that the epithet “infraciliary” has no observational basis without an accurate tridimensional visualization of the pellicle, and this von Gelei apparently did not have. In cross sections of these elements their interrelationships were seen most clearly (Fig. 5A and B ) . Figure 5B is a nearly diagrammatic view of a unit of surface structure entirely consistent with the phase-contrast view. With phasecontrast microscopy, however, resolution of even so gross a pattern as the ~

~~

~

FIG.3. Electron micrographs of tangential sections at the pellicle surface through the plane A-A, Fig. 8. A. Thick section near tip of cell. The gray lines extending anteroposteriorly across the polygons are sections of membranes that we interpret to be equivalent to the Schuberg patterns. Cilium and trichocyst loci read up and down as in Fig. 1. B. A thin superficial section of the pellicle. Note that the cilium lies within a boat-shaped circumciliary space;4 the wall of this space is composed of a membrane the texture of which is coarsely filamentous and beady. This outer peribasal membrane in turn encloses a peribasal space that half surrounds each cilium in the surface plane. The outer peribasal membrane is the physical representative of the historical Schuberg patterns. 4 Key to notations on figures. c = cilium; cs = circumciliary space; em = endoral membrane; fc = filamentous component; g = gullet; ipm = inner peribasal membrane ; j = junction between adjacent ciliary corpuscles at their peribasal membranes; k = kinetosome; kf = kinetodesmal fibril; m = mitochondrion; og = old gullet; opm = outer peribasal membrane; o m = old ribbed wall; p = peniculus; pbs = peribasal space; ps = parasomal sac; q = quadrulus; rw = ribbed wall; t = trichocyst; to = trichocyst opening.

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circumcilkry space (cs) had been quite difficult. The principal components of the nearly apple-shaped organelle that we call the ciliary corpuscle are as follows: an outer peribasal membrane ( o p m ) entirely surrounds the centrally located one or two cilia ; its outer portion is continuous with the ciliary membrane ; its outer surface is deeply depressed centrally about the cilia, forming a boat-shaped depression that we call the circumciliary space. Below this is a peribasal space, separated from the cytoplasm by an inner peribasal membrane ( i p m ; the plasma membrane of Sedar and Porter, 1955). An outer surface of this inner peribasal membrane is continuous with the inner surface of the outer peribasal membrane. The junction points between adjacent ciliary corpuscles are along the junctions of these two. The integrity of corpuscles and intercorpuscular junctions is shown in Fig. 6A, and these figures also show the bundles of kinetodesmal fibrils coursing through cytoplasmic bays to the right of their kinetosome row of origin (Fig. 6A, looking posteriorly; Fig. 6B looking anteriorly). In Fig. 5A, the shinglelike overlap of kinetodesma is demonstrated (as reported by Metz et &., 1953, and Sedar and Porter, 1955) ; in Fig. 6A and B evidence of clockwise spiralization is shown (viz., clockwise decrease in size of wedge-shaped X-sections of the kinetodesmal fibrils). In our material, spiralization of kinetodesma is more commonly observed than shinglelike overlap of the fibrils of the bundles (see also Fig. 7 A and B ) ; and evidence of spiralization of similar magnitude (about 60”per micron) can be seen in some of the figures of Metz et al. (1953, their Figs. 6 and 8). (This is in addition to their observations of the periodic structure of an individual fibril that takes the form of a spiral with a period of about 400 A.) In Fig. 6B, although the outer peribasal membrane has been separated into two surfaces, some of the membranes separating adjacent peribasal spaces have remained intact. Further evidence of the integrity of the ciliary corpuscle is given by its persistence during conjugation, even in the region of contact between cells of complementary mating type. In Fig. 6C the conjugants are shown to be joined at the junction points of adjacent ciliary corpuscles ; the kinetosomes, the outer and inner peribasal membranes, and peribasal spaces are each seen to persist during conjugation, although the fate of the cilia is unknown (they appear fragmented) ; cytoplasmic continuity between conjugants is FIG.4. A thin superficial section of the pellicle of P. bursark that continues from Fig. 3B. Note the alternating patterns of cilium-trichocyst-cilium . , the coarsetextured appearance of the outer peribasal membrane and the diversity of its patterns in section, and the junction point between membranous components of adjacent circumciliary corpuscles. Such junctions are the physical representatives of the historical “outer lattices.”

..

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FIG.5. Sections vertical to the pellicle surface of P. aurelia through the plane C-C of Fig. 8. In A the kinetodesmal fibrils in the cytoplasmic bays between cilia are prominent. In B , the relationships among all the membranes are demonstrated.

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seen at some of the intercorpuscular junctions. In Fig. 6F, kinetodesmal fibrils are seen in the sometimes pronounced ectoplasmic ridges that exist below the inner peribasal membrane and between rather than below the junction points of adjacent ciliary corpuscles. Such ridges appear as cytoplasmic bays below the junction points (Figs. 5A, 6A and B ) and are the loci of the kinetodesma; they therefore cannot be responsible for the polygonal “outer-lattice” pattern that overlies the kinetodesmal bundles in a zigzag (see especially section A-A, Fig. 8, in which the position of the ridge is that of the kinetodesma). In Fig. 6 0 and E another component adjacent to the kinetosomes, called the parasomd sac, is shown. This is a short, cone-shaped depression that probably corresponds to the Nebenkiirn of von Gelei (1932) and the accessory ring of Metz et ul. (1953). It is present but unidentified in the figures of Sedar and Porter ( 1955). (Their “accessory kinetosomes” are like the anterior kinetosomes of the paired-cilia in our Fig. 7A and B ) . This parasomal sac opens into the boat-shaped circumciliary space (and the fluid environment of the cell !) by passing from the level of the kinetosome through the inner peribasal membrane, the peribasal space, and the outer peribasal membrane (Figs. 6F, 7A and B ) . The sac opening is next to the base of the membrane of the posterior cilium (see immediately below). The common occurrence of two cilia in certain areas of the pellicle, particularly anteroventrally and circumorally, has been noted by numerous investigators. Sedar and Porter interpret such pairs as evidence of cilium duplication. This view is open to question and not held by us. W e note that the kinetodesmal fibril arises from the anterior filaments of the posterior kinetosome when two of these occur. It then advances anteriorly and laterally (to the right). A diagrammatic representation of several views of “typical” ciliary corpuscles packed to form the pellicle surface is given in Fig. 8. Guide lines are provided to facilitate “sectioning” with a straight-edge (to simulate optical or actual sectioning). References to these levels are made in the legends of some of the photographs of sections. Note that through X-X the outer peribasal membrane viewed from above would appear as the Nebenfibrillen ; through Y-Y as inter.zi1iarfasern ; and through 2-2 of the inner peribasal membrane as components of the infraciliary lattice system. Some of the names that have been applied through the years to the resulting patterns are listed in Table I, together with the actual region of the corpuscle that has been observed. Some of the famous controversies of the older literature become meaningless when the structural basis of the patterns is realized [such as whether that system called direkt ver-

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bindendes System does (Klein) or does not (von Gelei) connect with that called indirekt verbindendes System. Obviously Klein was more nearly correct, since the patterns arise from surfaces continuous with one another]. If the ciliary corpuscles composing the cell surface are like a layer of packed peas, then the trichocysts underlying the surface in orderly array are somewhat like a second packing layer. If the analogy were complete, however, instead of lying directly along the ciliary rows as they do (ciliumtrichocyst-cilium . . .) they ought to pack regularly at the corners of the hexagons and between the ciliary rows. (Actually, such corner placements of trichocysts are the most frequent exceptional displacements that are seen! See Fig. 3A and Schuberg, 1905.) Instead, the kinetodesma occupy these positions, with the trichocysts being found usually midway between. A remarkable ultrastructural similarity between the cilium and the trichocyst has been observed in certain cross sections of tips of these unusual organelles. The heavy-walled bull’s-eye appearance in cross sections of the tips of trichocysts in nondividing cells (Fig. 7) is in contrast to the delicate fibrillar constitution of similar tips (from new trichocysts?) in dividing cells (Fig. 6 0 , E ) . The dimensions of these fibrils, about 20 to 30 mp, are similar to those seen in the paired filaments of the cilium. Although the resemblance to the cilium is striking, no clear-cut counts of nine or eighteen peripheral fibrils have been observed to date. Nevertheless, beFIG.6. Various sections through the surface of Paramecium. A . Vertical section in the plane defined by points C-C of Fig. 8 looking toward the rear of the cell. The progressive decrease in the size of the kinetodesmul fibril in the apparent counterclockwise (really clockwise) direction is proof of the spiralization of the elements of the kinetodesma. B and C are reproduced to the same scale. B. A section through points C-C of P. bursaria looking anteriorly. The clockwise spiral arrangement is again obvious. The outer peribasal membrane has been separated into two component membranes ; the continuity between the inner component of the outer peribasal membrane and the outer component of the inner peribasal membrane is clearly demonstrated. C. A section vertically through the adjacent surfaces of two conjugating cells of P. bursaria demonstrating the maintenance of integrity of the ciliary corpuscle during conjugation ; the persistence of kinetosomes and the cytoplasmic continuity between the two conjugants are evident at the junction points ( j ) . D. Section through plane defined approximately by A-A in Fig. 8. The two kinetosomes and the associated parasomal sac together with one portion of the kinetodesmal fibril. This section demonstrates the almost filamentous composition of the cap of the tip of the trichocyst. In E (a section at a slightly deeper level ‘than D ) the same components are observed with the difference that the “filamentous elements” appear more densely organized. In this section there appear to be seven groups (some filamentous pairs?). F. A section through the plane D-D of Fig. 8 at a magnification equivalent to that of D. The relationship of the parasomal sac to the ciliary corpuscle is shown.

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cause of the submicroscopic fibrillar composition and other structural similarities, especially in young trichocysts and cilia, we regard the trichocyst as an organelle homologous with the cilium.

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The most serious challenge to our view that the infraciliary lattice system of G. von Gelei represents patterns arising from various sections through the ciliary corpuscle is contained in the papers by Roth (1958) and by Sedar and Porter (1955). Roth (1958) has shown that filaments measuring about 21 mp in diameter occur very generally near flagellar bases and beneath pellicular membranes in protozoa. H e interpreted these as fundamental filaments of fibrillar systems that form bundles and interconnect ciliary bases. Although we are in general agreement with his interpretations, we note a possible exception to the particular instance cited for Paramecium. Analysis of the patterns of the bases of the ciliary corpuscle in tangential sections reveals that the stratum in the cytoplasm at which the filamentous components appear is the one at which slices through the inner peribasal membrane should occur. The periodic rather than continuous appearance of these patterns is also consistent with their intimate association with the ciliary corpuscle. W e therefore interpret these “filamentous” components as constituting either the warp and woof of the inner peribasal membrane proper (as in the opm, Figs. 3A and 4 ) , or a filamentous fraying immediately contiguous to that lower membrane surface. Roth correctly relates these patterns to some of those also observed by Sedar and Porter (1955). In either case, since all the gross patterns of G. von Gelei’s infraciliary lattice system can be seen in sections of what is unquestionably a ciliary corpuscle, its structural basis must reside therein. A diagrammatic reconstruction of the pellicle system, with its surface of packed ciliary corpuscles and its second packing layer of trichocysts, is shown in Fig. 9.

B. Specialized Food-Intake (or Gullet) System

1. Introductwn. I n 1957 we described the gullet as a second system of packed organelles. This tubelike system is clearly and entirely dissectible by compression from the pellicle system of organelles, and, despite certain bizarre aspects, it is remarkably similar in a fundamental way to its counterpart, the pellicle (Ehret and Powers, 1956, 1957a). By presenting the essential gross aspects of our contemporary view of gullet structure first, the historical perspective will be more comprehensible to the reader. The edge of the open tube at its point of attachment to the pellicle system FIG.7. Two oblique sections of P. aurelia through the surface in the plane defined approximately by A-A of Fig. 8. These demonstrate chiefly the relationship of the parasomal sac ( p s ) to the circuinciliary space ( c s ) and the kinetosomes ( k ) . The filamentous components, because of their positions, are interpreted as frontal sections of the inner peribasal membranes of the ciliary corpuscle.

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FIG.8. Interpretive diagrams of a ciliary corpuscle and of idealized sections of it. The guide lines adjacent to the'diagrams define planes of sections described in earlier figures. A-A. A thin tangential section, through portions of three kineties. Kinetodesmal fibrils are shown leaving the anterior region of the posterior cilium in each corpuscle; this point of departure is below the level of the inner peribasal membrane and would not be visible in an actual thin section of so superficial a slice. The line around the two cross-sectioned cilia and the parasomal sac represents one contour on the outer peribasal membrane delineating the boat-shaped circumciliary space. The junction

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FIG.9. A reconstruction of the pellicle system as it would appear in perspective view. The relationships of the kinetodesma and trichocysts to the hexagonally packed ciliary corpuscles that form the outer surface of Paramecium are also shown.

points between the membranes of the adjacent ciliary corpuscles are represented diagrammatically. The actual appearance of those multiple-membrane junctions is seen in Fig. 4. Cross sections of trichocyst tips appear sandwiched between adjacent ciliary corpuscles and in a line parallel to and between the kinetodesmal bundles. B-B. A long section vertical to the pellicle and along a column of trichocysts and cilia. Kinetodesmal fibrils are seen leaving the anterior filaments of the posterior kinetosome in each corpuscle; a kinetodesmal bundle has been included to show its relationship to the other surface elements. C-C. A diagonal section vertical to the pellicle and through the posterior cilium of each corpuscle. As in B-B, the central filaments of the cilia are seen in longitudinal sections to terminate before reaching the kinetosome. The intercorpuscular junctions are shown again by a line representing membrane interspaces. D-D. A cross section vertical to the pellicle and through the anterior edge of the posterior cilium of a ciliary corpuscle. A kinetodesmal fibril is shown leaving a peripheral ciliary filament; it ascends to the right of the kinetosome to join five other spirally arranged kinetodesmal fibrils. The section includes a longitudinal section of a trichocyst and some of the membranous fabric of the sliced junction regions of contiguous corpuscles. E. A reconstruction of the ciliary corpuscle as it would appear in perspective view. The relationship of trichocysts to ciliary corpuscles is shown, a s well as the opening of the parasomal sac into the circumciliary space near the base of the posterior cilium. For purposes of clarity, the kinetodesma have been omitted, but see Fig. 9.

TABLE I TERMS THATHAVEBEEN INTERPRETIVE A COMPARISON OF THE ULTRASTRUCTURAL ANATOMY OF THE CILIARY CORPUSCLE WITH CERTAIN PATTERNS BY VARIOUSAUTHORS APPLIEDTO SURFACE Section of ciliary corpuscles represented by observed pattern

1. Cilium base;

Schuberg (1905) -

Basalkorperchen

Klein (1928-1958)

J. and G. von Gelei ( 1932-1939)

Metz et al. (1953)

3. Kinetodesmal fibril

4. Intercorpuscular junction

Kinetosome

Kinetosome ; accessory kinetosome

Basalkorperchen

Basalkorn ( ?)

Accessory ring ; cone with pitted center

Not identified

Nebenkorn

Not identified ; or Kinetodesmal fibril Terminulaste; or Neuronem( 2 )

Kinetodesmal fibril ; infraciliary lattice system ( ? )

Einzelne Zilienwurzeln

Polygonules Gitter; aiisseres Stiitzgeriistsystem

Pellicular Aiissere ridge pattern ; Gittersystem infraciliary lattice system

~

Pellicula ; hezagonelen Feldchen

Parducz (1957, 1958)

Nebenkorn (Z?) Basalkorn

kinetosome

2. Parasomal sac

Sedar and Porter (1955)

Zndirekt verbindendes System

Nebenkorn

Outer lattice system

M

TABLE I (continued) Section of ciliary corpuscles represented by observed pattern

Schuberg (1905)

5. Outer peribasal membrane At a high or inter- Feine Linien mediate point of the circumciliary space At a low point of Langslirtien the circumciliary space 6. Inner peribasal membrane At a high or intermediate point At a low point

Klein (1928-1958)

J. and G. von Gelei (1932-1939)

Metz et al. (1953)

Sedar and Porter (1955)

Parducz (1957, 1958)

* Direkt ver bindendes System; Zirkularfibrille Direkt verbindendes System; Interziliarfasern

Nebenjibrillen

Neuroneme System ; inf raziliare Gittersystem

Pellicle membrane Trichocyst-totrichocyst strand or fiber

Nebenfibrillen

n M

r r

Interziliare Fasernsystem

$ n M

0

q +d

> >

m Schuumartiger, inf raziliare Gittersystem Infraziliare , Gittersystem

Not seen Not seen

Infraciliary lattice system

Innere Gittersystem

Infraciliary Innere lattice system ; Gittersystem endoplasmic reticulum ( ?) ; plasma membrane

i5

z

2

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L. POWERS

FIG.10. Phase-contrast micrographs of an unfixed preparation of the gullet region of P. bursaria. In A the suture line between the rhomboidal and hexagonal regions (the ciliary suture of Rees, 1922) leads to and across the vestibular region of the pellicle system. The observer is looking directly into the buccal cavity, the anterior of the cell being at the top. B is a focal plane just below that of A. In the center of the picture some ends of the cilia of the gullet are seen; to the right the basal regions of these cilia and their organization into precise arrays is seen; the vestibular region of the pellicle is to the left in the picture. C. At a deeper focal level the gross shape of the gullet is seen.

is called the buccal overture, and the “empty” (actually cilium-filled) space within the tube is the buccal cavity (we adopt the terminology of .Corliss, 1955). The slightly conical or funnel shape assumed by the pellicle near the buccal overture is an indistinctly defined region that has been called the vestibulum by some authors ; it should be regarded merely as a region of the pellicle and not as a distinct anatomical structure. The gross relationships of the pellicle system to the gullet system are shown FIG.11. A transverse section through a specimen of P. bursaria at the level of the buccal overture looking anteriorly (from Ehret and Powers, 1957a). A11 the organelle complexes of the gullet system are demonstrated. Directly above the macronucleus are eight columns of cilia constituting the ventral and dorsal peniculi ; clockwise from those are four columns of cilia making up the quadrulus: above that is the ribbed wall.

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in Figs. 10 and 11. If we view the cell with anthropomorphic sympathy, we can imagine it to have a right and left side and can define “head” as anterior (the usual direction of swimming), and vestibulum as ventral. The gullet is seen to consist of three long columns of cilia and one nonciliated ribbed wall region (Fig. 12A, B ) . Each column consists of four cilia abreast, and is about 85 to 90 cilia long. The ciliary columns are, from left to right, ventral peniculus, dorsal peniculus, and quadrulus ; the quadrulus is dorsally situated, and to its right and below is the nonciliated ribbed wall that terminates in the endoral membrane (Fig. 12B). 2. Historical Perspectives. Both Maupas (1883) and Hertwig (1889) recognized the presence of numerous cilia within the gullet, and their nearly synchronous beating led them to consider these ciliary complexes as “membranelles.” J. von Gelei ( 1934a) identified the “membranelles” as ventral and dorsal peniculus (brush) and Vierermembran, later retermed quudrulus (Hyman, 1940). H e also recognized the Rippenfusern (retermed ribbed zmll, Ehret and Powers, 1957a) and, at its anterior, near the right edge of the buccal overture, a row of paired cilia, the endoral “membrane.” Von Gelei also recognized the correct topographical relationship of these complexes to one another ; the quadrulus and peniculus spiral ventrally (clockwise looking posteriorly) into the gullet cavity. Anteriorly the quadrulus is somewhat separated from the peniculi, and the four columns of cilia are disposed 1, 1, 2 from left to right. Posteriorly these become as close together as the four cilia of each peniculus ; in the region in which the quadrulus comes to lie directly to the right of the dorsal peniculus, the ventral peniculus ends, and for several rows only two quadriciliated columns remain. The general correctness of these observations of von Gelei is demonstrated in Figs. 11 to 14. Note the remarkable resemblance between these electron micrographs of the peniculus and quadrulus and those presented by Mannweiler and Bernhard (1958) of renal tumors of the hamster. Lund’s (1933) diagrams of the gullet have been widely reproduced in FIG.12. Various views of the gullet of P. bursaria photographed with phasecontrast optics. A and B are’to the scale represented by D. A and B, digitoninfixed preparations, showing the peniculus and quadrulus in A lying beneath the packed ciliary corpuscles; in B the endoral membrane is seen as one or two lines of dots at the right (ventral) end of the ribbed wall (the lines running toward the right of the picture). C. Compressed unfixed preparation showing all the complexes of the gullet except the endoral membrane (from Ehret, 1958). D and E. Unfixed preparations demonstrating the geometry of the ribbed wall. ( D , from Ehret and Powers, 1957a).

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texts (Wichterman, 1953) and even in a later research paper (Parducz, 1958b). In this interpretive reconstruction, Lund contended that the peniculi and quadrulus form a network that traverses like railroad tracks across another network of “pharyngeal fibers” continuous externally with the pellicle “fiber network.” H e also represented the cilia within each column as if they were rectangularly packed ; i.e., lines connecting adjacent cilia form angles of either 45”, 90”,135”, or 180” on his models. Yusa (1955), following the lead of Opton (1942), who had reported peniculi consisting of other than eight columns of cilia, presented a phylogenetically oriented study of the gullet of Paramecium. Yusa concluded that penicular counts constituted criteria of taxonomic utility in distinguishing between aurelia and bursaria groupings of species within the genus. W e challenged this view, since we had photographic evidence for only eight (the aurelia number) columns of penicular cilia in our own stocks of P . bursaria; our photographs of Yusa’s material revealed also only eight columns, and Yusa credited us with directing his attention “to the variation in the number of rows constituting peniculi of P . bursaria, strains C and D” (first italics ours). Although it is entirely conceivable that numerical variations may occur in the peniculus, no evidence in proof of this idea has been presented to date. One of the great difficulties in the analysis of gullet organelles by means of silver methods is the frequent and marked distortions of the closely spaced gullet kinetosomes (-0.46 p between centers of gullet cilia versus > 1 p between centers of pellicle cilia). Another difficulty is in the attempt to interpret from the low resolution of the light microscope the intricate patterns of such small structures. Some of the observations of J. von Gelei (1934b) even in this difficult region stand up with remarkable precision, however. Thus, he reported a row of large serrated granules separating the normal space between ventral and dorsal peniculus-a pattern suggestive of this, though possibly an optical illusion, is seen between quadriciliated peniculi in the phase micrographs in Fig. 12A, C. The physical basis for the serrated gap pattern between the rows is seen in Fig. 13 as a column of parasoma1 sacs that lies between ‘the quadriciliated units of the quadrulus and dorsal peniculus at the junction of those complexes, and also between dorsal and ventral peniculus (Ehret and Powers, 1957a; Fig. 2). It is interesting to note that von Gelei carefully termed these nonciliated sacs Nebenkiirnern rather than Basulkiirnern consistent with the terms applied by him to pellicle parts (Table I ) . These sacs are evident also in one of the figures presented by Watanabe (1957). 3. Contemporary Views. Our comparison of electron micrographic with phase-contrast evidence ( 1957a) confirmed the gross anatomical rela-

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tionships and added the necessary resolution to make precise the penicular counts. The periodic patterns of peniculus, quadrulus, and especially of the ribbed wall are strikingly reminiscent of the longitudinally viewed pellicle patterns. Thus, the ciliated complexes of the gullet as well as the nonciliated ribbed wall have periodic patterns resembling those of packed ciliary corpuscles of the pellicle, but with a frequency of repetition about three to four times as great; parasomal sacs are present, as in the pellicle, but more frequently abreast four rather than abreast one or two kineto-

FIG.13. Electron micrograph of a thick section of P. bursaria posterior to the buccal overture. Note the conspicuous column of parasomal sacs between the dorsal peniculus and the quadrulus at the junction point of these two complexes. somes ; peribasal spaces around individual cilia are evident as well as peribasal membranes (Fig. 14) ; kinetodesmal fibrils are also evident (Fig. 14), but the kinetodesma run well below the surface, rather than in the roomy bays of cytoplasm available for such fiber bundles at the less crowded pellicle system ; the nonciliated ribbed wall “units” are also provided with “kinetosomes” (Fig. 120, E ) that appear single in one orientation (Fig. 15A) and double (Fig. 15B) in another.

C. Replication Mechanisms Because of the extensive commitments that various authors (viz. Lwoff,

1950;Weisz, 1954) have made regarding the mechanism of surface replication during cell division, brief mention is made here. It is, however,

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FIG. 14. Electron micrograph of a section through one ciliary complex of the gullet of P. bursariu showing particularly the disposition of the kinetodesma in relationship to the kinetosomes. The spaces between the cilia are interpreted to be the peribasal space of this region.

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not only premature but beyond the scope of this present review to catalog extensively all the contradictory views on replication itself ; the state of progress is such that the general account by Tartar (1941) as well as the specific one on Paramecium by von Gelei ( 1 9 3 4 ~ )are remarkably up-todate. Since our best information from the time of Hertwig to the present is on the gullet, we shall confine our attention to that system. There is general agreement that a new gullet is formed during division which passes to the posterior daughter cell ; the anterior daughter retains the old gullet. Hertwig described this as a budding process and supposed that the new ciliary “membranelles” divided from the old. This view was essentially shared by von Gelei, and numerous Paramecium experimentalists have agreed to it (e.g., Sonneborn, 1947; Hanson, 1955). FaurC-Fremiet (1949) reported that the new gullet formed from the old one at the expense of the old quadrulus. Yusa (1957) reported de novo formation of peniculus, quadrulus, and bud from the right posterior wall of the buccal cavity, posterior to the endoral membrane. H e described the anlage as initially tubular, with six rows of granules; two of these are supposed to give rise to the peniculi, and he states that “only some time after the complex separation of the proter and opisthe do the two peniculi gain their full complement of rows of basal granules.” H e correctly observes that the integrity of the old organelles is maintained throughout division. An essentially similar view is presented by Roque for P.aramecium (1956a, b) and for the similarly constructed holotrich Disematostoma ( 1957a, b) . Roque contends, however, that the kinetosomes of the endoral membrane multiply to provide those of the new gullet; in this sense she considers the endoral membrane of Disematostoma as a “stomatogenic kinety.” The concept of stomatogenic kinety in distantly related S t a t o r has been convincingly challenged by Tartar (1956, 1957), who observes that the first appearance of the buccal anlage “clearly cuts across several of the body stripes” in the region of contrast between narrow and wide stripes. It is interesting to note that the gullet and gullet anlage in Paramecium lie in a similar region of pattern contrast (between hexagonal and rhomboidal patterns, Fig. IOA) . More recently, Ehret and Powers (1958) have observed the following sequence of events in gullet replication. The new gullet develops de novo from nonciliated progenitor organelles, microsomal in size, that appear in the pellicle surface between the vestibular kineties and the buccal overture. The randomly oriented organelles become linearly ordered (Fig. 16A) into three sets of about 90 organelles each. Each organelle gives rise to four cilia. The resulting three ribbonlike sets of quadriciliated organelles (Fig. 16B) develop later into quadrulus, dorsal peniculus, and ventral peniculus,

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respectively. At the time of micronuclear anaphase, the three ribbons are composed of rectangularly packed, laterally linked, actively beating cilia. Contrary to Yusa’s claim, at this stage the full complement of organelles needed for the ciliated complexes of the new gullet system are located on the pellicle surface. By early telophase, old and new gullets have separated, presenting the superficial appearance of budding ; at this time invagination at the cell surface occurs, and the new endoral membrane and pellicular organelles of the new vestibular region appear. Hexagonal repacking of the subsets of cilia, and ribbed-wall development are final stages in the development of this cell organelle system to functional maturity (Fig. 16A, B ) .

111. SYNTHESIS A N D OUTLOOK W e have traced the conceptual evolution of the surface structure of Paramecium from the early ideas of stratified fibrillar systems to our most recent one of systems of close-packed organelles. Despite superficial appearances, almost all the structural evidence in the literature is consistent, regardless of its sources and of the various methods by which it was obtained. Understandably, interpretations have differed, and, although some of the early interpretations have to be rejected, the elements of many appear to be correct and can be retained. With the advantage given by the higher resolution in electron-microscope preparations, we have been able to suggest a general description of the structure of the cell surface of Puruwcium that takes all the observations of other authors into consideration, that allows reinterpretation of their interpretations, and that reconciles some apparent inconsistencies. In one aspect, the cell surface is simple. The basic unit of surface structure is the ciliary corpuscle, an organelle between 0.25 and 2 p in diameter, ciliated or not externally, and with intracytoplasmic connecting fibrils. The two-dimensional (cross-sectional) patterns that arise from the closepacking of these three-dimensional corpuscular organelles give rise to the appearance of wire patterns in the old silver-line methods. The trichocysts are arranged beneath, in line between the kinetodesmal fibrils that course beneath the layer of corpuscles, and lateral to their centers. At their tips, trichocysts are homologous with cilia. The packing patterns of the foodFIG. 15. Electron micrograph of a transverse section of the ribbed-wall region of the gullet of P. bursaria. In A the “kinetosomes” associated with the individual elements of the ribbed wall are evident. Lateral to this region, the ribbed wall in section takes on a different aspect (upper left and left). The spaces of the elements may be homologous to the prri6asal spaces of the ciliary corpuscle. In B the sometimes-double appearance of the kinetosomes of the ribbed wall is shown.

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C.’F.EHRET AND E. L. POWERS

intake system resemble those of the general surface (or pellicle) system of organelles with two exceptions: (1) the frequency of cilia three to four times that of the pellicle and (2) a nonciliated ribbed wall region, otherwise similar (possessing kinetosomes) , exists. This food-intake system is replicated at cell division not by budding a new gullet from an old one, but by the laying down of three rows of nonciliated “microsomes” in the pellicle surface between the ribbed wall and the pellicle organelles. From

FIG.16. Phase-contrast micrographs of unfixed preparations of P. bursaria demonstrating the origin of the new gullet. In A, between A and og three or four lines of “microsomes” are apparent. This is in the region of the pellicle just to the right of the ribbed wall in Fig. 11. B. A preparation after differentiation of the microsomes into three columns of quadriciliated units lying parallel to the old gullet, on the surface of the animal. These are the qziadrulus and peniculus of the as yet uninvaginated new gullet.

each “microsome” four cilia arise, excluding the possibility that these cilia originate from previously existing cilia or kinetosomes in that immediate vicinity. Thus Paramecium is viewed as a cell whose surface is composed of at least two systems of corpuscular organelles : the generalized pellicle system and the gullet system. In both systems patterns associated with close packing are evident.

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We suppose that gross cytological patterns may be established in many cells by the elaboration and packing of such basic structural elements as those of Paramecium. A possibly similar case of the resolution of packed organelles as the basis of superficially bizarre cellular complexity is seen in the work of Fawcett with other freely dispersed cells: human spermatozoa. Fawcett (1958b) shows, for example, that the remarkable springshaped spiral filament of the spermatozoon is composed ultrastructurally of mitochondria packed like a string of sausages about a cilium-containing axial sheath. The implication of organelles in the generalized cell structure has been treated extensively elsewhere (Ehret, 1958). Of immediate practical interest will be the search for homologies of the ciliary corpuscle in related Protozoa, since many of the preparations in the literature of the ciliates reveal evidence of close-packed structures (e.g., the work on Ezlplotes by Turner, 1933, and that on Colpoda by Taylor and Garnjobst, 1939). In retrospect it occurs to us that it was perfectly reasonable for Schuberg to speculate that the coordinated cilia of the protozoan surface were interconnected in a nerve network represented by the very patterns that he observed. It was hardly more difficult in 1905 to cite an already flourishing related literature in support of such logic than it is today. With such a precedent, it followed naturally for Klein and von Gelei to invent complex terminologies that developed, almost competitively, multiple variations on the same neuronematous-neuroformative theme. With these established it then became difficult for subsequent workers to see through the quantity of terms anything but nerve and structural fibers in the beautiful geometrical patterns revealed by the reduced silver. The solution to the silver-line problem was preceded by the realization of new substantive elements within the patterns. Thus Tartar (1941) had considered the “atomistic” character of the units of pattern differentiation, “each type being of constant size after its brief development.” Characterization followed, and Metz et al. (1953) gave evidence of the structure of such units in the infraciliature ; finally the relationship of ciliary corpuscles to these units and to surface packing was realized. In conclusion it seems clear that over the past 50 years, as a consequence of optical sectioning, selective deposition of stains, and disruption of elements, portions of actual sheets and surfaces have been termed “fibrils.” This is not to say that the ultrastructural warp and woof of these surfaces may not involve fibrous proteins but simply that to call the edge of a sheet a “fibril” is a misinterpretation. With the “fibrillar lattices” eliminated, the old questions regarding their interconnectives seem pointless : the cilium and kinetodesmal fibril appear to be integral components of a cor-

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puscular organelle that constitutes a primary packing unit for the complexes and systems of the free cell border.

IV. ACKNOWLEDGMENTS We wish to express our gratitude to our colleagues at Argonne National Laboratory for their help in several matters, and especially to Dr. Friedrich Wassermann for his advice and encouragement on numerous occasions. The electron-microscope studies were made with the assistance of Dr. L. E. Roth; Mrs. Eleonore Larsen made the drawings for Figs. 8. and 9. V. REFERENCES Chatton, 8., and Lwoff, A. (1930) Compt. rend. soc. biol. 104, 834. Chatton, 8., and SCguCla, J. (1940) Bull. biol. France Belg. 74, 349. Child, F. M., and Mazia, D. (1956) Experientia l2,4. Corliss, J. 0. (1953) Stain Technol. 28, 97. Corliss, J. 0. (1955) J. Protozool. 2, Suppl. 12. Ehret, C. F. (1958) In “Symposium on Information Theory in Biology” (H. Yockey, ed.), pp. 218-229. Pergamon Press, London. Ehret, C. F., and Powers, E. L. (1955) Exptl. Cell Research 9, 241. Ehret, C. F., and Powers, E. L. (1956) J . Protozool. 3, Suppl. 5. Ehret, C. F., and Powers, E. L. (1957a) J. Protozool. 4, 55. Ehret, C. F., and Powers, E. L. (1957b) J . Protozool. 4, Suppl. 9. Ehret, C. F., and Powers, E. L. (1958) J . Protozool. 6, Suppl. 11. FaurC-Fremiet, E. (1949) Compt. rend. 13th congr. intern. zool., Paris 215. Fawcett, D. W. (1958a) I n “Frontiers in Cytology” (S. L. Palay, ed.), pp. 19-41. Yale Univ. Press, New Haven, Connecticut. Fawcett, D. W. (1958b) Intern. Rev. Cytol. 7, 195. Fawcett, D. W., and Porter, K. (1954) J. Morphol. 94, 221. Gelei, G. von (1937) Arch. Protistenk. 82, 331. Gelei, J. von (1932) Arch. Protistenk. 77, 152. Gelei, J. von (1934a) Arch. Protistenk. 82, 331. Gelei, J. von (1934b) Matemat. Termtszettud. Brtesito Magyar Tudomcinyos Akad. 61, 717. Gelei, J. von (1934~)Zool. Anz. 107, 161. Gelei, J. von (1939) Arch. Protistenk. 92, 245. Grell, K. G. (1956) “Protozoologie.” Springer, Berlin. Hanson, E. D. (1955) Proc. Natl. Acad. Sci. US.41, 783. Hertwig, R. (1889) Abhandl. bayer. Akad. Wiss. Miinchen, 17, 150. Hyman, L. (1940) “The Invertebrates : Protozoa through Ctenophora.” McGrawHill, New York. Joblot, L. (1718) “Descriptions et usages de plusieurs nouveaux microscopes.” In Wichterman (1953). Klein, B. (1926) Arch. Protistenk. 66, 243. Klein, B. (1928) Arch. Protistenk. 62, 177. Klein, B. (1958) J. Protozool. 6, 99. Lund, E. E. (1933) Univ. Calif. (Berkeley) Publs. Zool. 39, 35. Lwoff, A. (1950) “Problems of Morphogenesis in Ciliates.” Wiley, New York.

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Mannweiler, KI., and Bernhard, W. (1958) J. Ultrastructural Research 1, 158. Maupas, E. (1883) Arch. 2001. exptl. et gin. 1, 427. Metz, C. B., Pitelka, D. R., and Westfall, J. A. (1953) Biol. Bull. 104, 408. Metz, C. B., and Westfall, J. A. (1954) Biol. Bull. 107, 106. Opton, E. M. (1942) Anat. Record 84, 485. Parducz, B. (1957) Ann. Hist.-Nat. Musei Natl. Hung. 8, 231. Parducz, B. (1958a) Acta Biol. Acad. Sci. Hung. 8, 191. Parducz, B. (1958b) Acta Biol. Acad. Sci. Hung. 8, 219. Pitelka, D. R. (1956) J. Biophys. Biochem. Cytol. 2, 423. Pitelka, D. R., and Metz, C. B. (1952) Biol. Bull. 103, 282. Powers, E. L., Ehret, C. F., Roth, L. E., and Minick, 0. T. (1956) 1. Biophys. Biochem. Cytol. 2, Suppl. 341. Rees, C. W. (1922) Univ. Calif. (Berkeley) Publs. Zool. 20, 333. Roque, M. (19%) Compt. rend. 242, 2592. Roque, M. (1956b) Compt. rend. 243, 1564. Roque, M. (1957a) Compt. rend. 244, 2657. Roque, M. (1957b) Compt. rend. 244, 2849. Roth, L. E. (1957) J . Biophys. Biochein. Cytol. 3, 985. Roth, L. E. (1958) J . Ultrastructzcral Research 1, 223. Roth, L. E. (1959) I . Protozool. in press. Schuberg, A. (1905) Arch. Protistenk. 6, 61. Sedar, A., and Porter, K. R. (1955) J . Biophys. Biochem. Cytol. 1, 583. Sonneborn, T. M. (1947) Advances in Genet. 1, 263. Tartar, V. (1941) Growth (Third Growth Symposium) 6, 21. Tartar, V. (1956) In “Cellular Mechanisms in Development and Growth” (D. Rudnick, ed.), pp. 73-100. Princeton Univ. Press, Princeton, New Jersey. Tartar, V. (1957) J . Exptl. Zool. 136, 53. Taylor, C. V. (1941) I n “Protozoa in Biological Research” (Calkins and Summers, eds.), pp. 191-270. Columbia Univ. Press, New York. Taylor, C. V., and Garnjobst, L. (1939) Arch. Protistenk. 92, 73. Thompson, D’Arcy W. (1952) “Growth and Form,” 2nd ed. Cambridge Univ. Press, London. Turner, J. P. (1933) Biol. Bull. 64, 53. Watanabe, K. (1957) Ochanomizu lgaku Zasshi 6, 455. Weisz, P. B. (1954) Quart. Rev. Biol. 29, 207. Wichterman, R. (1953) “The Biology of Paramecium.” Blakiston, New York. Worley, L. (1933) Proc. Natl. Acad. Sci. US. 19, 323. Worley, L. (1934) J. Cellular Comp. Physiol. 6, 53. Yusa, A. (1955) J. Protozool. 2, Suppl. 6. Yusa, A. (1957) I . Protozool. 4, 128.

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The Mammalian Reticulocyte LEAH MIRIAM LOWENSTEIN

Department o f Human Anatomy. Oxford University. Oxford. England1 I . Introduction ...................................................... I1. Techniques in the Examination of Reticulocytes ..................... A . Methods of Obtaining Reticulocytes ........................... B Staining Methods ............................................. c. Examination of Unstained Reticulocytes ....................... D . Examination of Electron-Microscope Preparations ............... E. Counting Reticulocytes ....................................... 111 Morphology ...................................................... A . Inner Structure .............................................. B. Vacuoles ..................................................... I V Physical Properties ............................................... A . Shape ....................................................... B. Size ......................................................... C. Density and Refractive Index ................................. D. Osmotic Resistance ........................................... E. Resistance to Hemolytic Agents ............................... F. Adhesiveness ................................................. G. Charge ...................................................... V . Biochemistry ..................................................... A Ions and Water ............................................. B . Stroma ....................................................... C. Hemoglobin ................................................. 1. Iron ..................................................... 2. Porphyrin ............................................... 3. Amino Acids ............................................. D . Nucleic Acids ............................................... E. Enzymes ..................................................... V1. Physiology ....................................................... A . Occurrence in the Bone Marrow ............................... B. Age of Cells When Released from the Bone Marrow ............. C. Factors Which Influence the Release of Reticulocytes from the Bone Marrow ................................................ 1. Adhesiveness ............................................. 2. Neural Factors ............................................ 3. Humoral Factors .......................................... 4. Oxygen Tension ........................................... D . The Occurrence of Reticulocytes in the Peripheral Blood ...... E. The Maturation of Reticulocytes ............................... F. Cell Division ................ .............................. VII . Reticulocytes in Disease ........................................... A . Sickle Cell Anemia ...........................................

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B. Malaria ...................................................... C. Heinz Body Anemia ........................................... VIII. Acknowledgments ................................................. IX. References .......................................................

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I. INTRODUCTION The reticulocyte is a red blood cell in the last stages of development. In appearance it is irregular, ovoid, and devoid of inner detail when viewed unstained under the ordinary light microscope. A variety of granules and filaments may be observed in the unstained cell under the phase contrast and electron microscopes. The mammalian reticulocyte is formed in the bone marrow by the loss of the nucleus from the normoblast. It begins to mature in the bone marrow and finishes its development into an erythrocyte in the peripheral circulation. Under normal conditions, reticulocytes in mammals comprise 0.2 to 5% of the total circulating red blood cells. Reticulocytes of other vertebrates retain their nuclei and will not be discussed in this review. The reticulocyte is identified by the appearance of a reticulum on staining with a supravital dye (Ehrlich, 1881). As the reticulocyte matures, the amount of reticulum diminishes. When the reticulum disappears entirely, the cell is called an erythrocyte, although for several days the young erythrocyte retains some of the characteristics of the reticulocyte. Research on the reticulocyte prior to 1920 centered mainly on the morphology of the cell. After the percentage of reticulocytes in the blood was recognized as an important index of blood regeneration, its role in erythropoiesis and its maturation were investigated. I n recent years the structure of the reticulocyte has been examined with the phase contrast and electron microscopes, and studies of its metabolism have been initiated. Literature on reticulocytes has been reviewed previously by Key (1921), Davidson (1930), Orten (1934), Ninni (1949), Plum ( 1949), and Seip ( 1953). I N THE EXAMINATION OF RETICULOCYTES 11. TECHNIQUES A . Methods of 0btaining Reticulocytes Normally less than 5% of the circulating red blood cells of man and the common laboratory animal are reticulocytes. Larger percentages may be obtained from the blood of patients with increased red blood cell regeneration such as occurs in many of the hemolytic anemias or in the treatment of pernicious anemia. In certain anemias the blood may contain more than 30% reticulocytes (Minot et al., 1928a ; Valentine, 1928 ; Riddle, 1930; Young and Lawrence, 1945; Haenel, 1949; Plum, 1949; Dacie, 1954 ; Crosby and Rappaport, 1956 ; Wintrobe, 1956). Reticulocyte per-

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centages of about 30 to 40 can be induced in experimental animals by withdrawal of 10 to 20% of their blood volume daily for 1 week (for examples see Key, 1921 ; Pearse, 1926; Heath and Daland, 1930; Sjovall, 1936; Plum, 1942, 1949; Burt et al. 1951). A reticulocyte count of over 90% can be induced by subcutaneous injections of neutralized phenylhydrazine hydrochloride or acetylphenylhydrazine. Injections of 30 mg./kg. body weight daily or every other day for four doses is usually sufficient to produce an intensive reticulocytosis in rabbits, dogs, and mice (for examples, see Heinz, 1901; Paton and Goodall, 1903; Price-Jones, 1911 ; Duesberg, 1931 ; Henriques and Prskov, 1939; Cruz, 1941 ; Rapoport et al., 1944; Borsook et al., 1952; Jones et al., 1953; Rubinstein et al., 1956). Reticulocytes obtained from anemic subjects may be abnormal, and caution should be exercised in attributing characteristics of these cells to normal reticulocytes. B. Staining Methods The reticulum of the reticulocyte can be stained supravitally with many dyes, including methylene blue, azure I and 11, methyl violet, gentian violet, Janus green B, and neutral red (for reviews see Key, 1921 ; Davidson, 1930 ; Fiessinger and Laur, 1930). The stain commonly used is brilliant cresyl blue, which stains the reticulum a deep blue (Cesaris-Demel, 1907). The general techniques of staining reticulocytes today are essentially the same as those of earlier workers (see Fiessinger and Laur, 1930). Solutions of 0.5 to 1% of the dye in normal saline or in absolute ethyl alcohol are used, and either wet or dry preparations are made. I n the “wet” technique, equal amounts of blood and a saline solution of the dye are mixed and placed on a slide or counting chamber. Alternatively, a drop of blood is put on a slide on which an alcoholic solution of the dye has previously been dried. The preparation is covered with a cover slip ringed with Vaseline or placed in a moist chamber, and incubated for several minutes. I n the “dry” technique, blood is either smeared directly onto slides covered with dry stain, or it is first incubated with a saline solution of the dye and then smeared onto slides and air-dried. The “dry” preparation can be examined directly under oil immersion. Permanent smears can be made by fixing the supravitally stained smears in methyl alcohol for several minutes and counterstaining with a Romanowsky dye. During fixation the alcohol washes out the vital stain, and the reticulum absorbs the basic dye of the Romanowsky stain (Nittis, 1938). The permanent smears are useful in distinguishing reticulocytes from erythrocytes with Heinz bodies, since Heinz bodies are no longer visible after alcohol fixation and counterstaining (Dustin, 1942). On comparing the different methods of preparing reticulocyte smears, Osgood and Wilhelm (1934) found that the most satisfactory technique

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is to incubate equal amounts of blood and saline solution of the dye and to prepare dry smears of the mixture. Incubation at 37°C. for 10 to 15 minutes gives the reticulocytes adequate time to absorb the dye (Seip, 1953; Dacie, 1956). Reticulocytes may have a tendency to adhere to glass (Key, 1921) ; hence, for accuracy in counting, silicone-treated tubes may be used to incubate the blood (Seip, 1953). Increasing the concentration of dye results in a denser reticulum (Davidson, 1930), and different supravital dyes produce reticula of slightly altered appearance (Key, 1921). When blood smears are fixed by alcohol, formaldehyde, acid, osmium tetroxide, or heat, the reticulum is not visible on subsequent staining. Instead a diffuse or granular basophilia appears. The basophilia is also produced by Romanowsky stains where alcohol fixation is used, and by the addition of solutions of certain substances such as liver extracts to the blood (Key, 1921; Bruckner, 1927; Gawrilow, 1929; Heath and Daland, 1931 ; Davidson, 1930). It has been claimed that reticulocytes do not take up supravital stains readily in aqueous solutions of liver extracts, amino acids, dextrose, calcium chloride, and certain other substances (Heath and Daland, 1931). A number of the solutions used by these authors were hypertonic. This produces crenation of reticulocytes, and crenation decreases the ability of the cells to absorb the supravital dyes (Key, 1921 ; Davidson, 1930). Dilute isotonic solutions of amino acids were found not to affect the staining of reticulocytes (Nizet and Robscheit-Robbins, 1950).

C . Examination of Unstained Reticulocytes No inner structure is visible in the intact unstained reticulocyte viewed through the light microscope with conventional or with dark-field illumination. Inner detail becomes evident, however, when the cells are hemolyzed (Erb, 1865 ; Key, 1921 ; Brecher, 1948) or if they are examined by phasecontrast microscopy. The cells are most readily examined under phase contrast when placed in a solution of bovine plasma albumin with a refractive index slightly greater than that of the cell (Barer and Joseph, 1955). With this method the optical halo around the cell is negligible, the interior of the cell becomes light, and inner structural bodies show up clearly. Other suspension media which have been used are plasma (Discombe, 1950), saline, or 5% gelatin (Bessis, 1949). In these media the hemoglobin tends to obscure inner detail (Feissly and Ludin, 1949).

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D . Examination of Electron-Microscope Preparations I n examining unstained reticulocytes under the electron microscope, the method of Palade (1952) is generally used. The cells are first fixed in 1 to 2% isotonic osmic tetroxide buffered at p H 7.0 for 15 to 30 minutes,

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dehydrated with alcohol, and washed several times with the embedding plastic. The mixture of cells and plastic is placed in a gelatin capsule, allowed to set, and thin sections are cut (Braunsteiner et al., 1956 ; Chalfin, 1956 ; Pease, 1956). Such sections are not very satisfactory, however, because the cells do not fix well and the density of the hemoglobin obscures the inner structure. Placing the cells in hypotonic saline before fixing decreases the cell density and improves the microscopic appearance of the cell (Wolpers, 1956). Hemolysis of the reticulocytes produces better visualization of the cell membrane but results in the distortion of inner structures. The cells are hemolyzed in distilled water, and the resulting ghosts are centrifuged and fixed in 1 to 2% osmic acid. A drop of this suspension is placed on a collodion membrane and dried (Bernhard et al., 1949; Bessis, 1950; Braunsteiner and Bernhard, 1950; Peters and Wigand, 1950). Intact cells may also be spread on the membrane before hemolysis (Brunner and Vallejo-Freire, 1956).

E . Counting Reticulocytes The reticulocyte count is most frequently expressed as the percentage of reticulocytes in a number of consecutively counted red blood cells. It has been claimed that reticulocytes are randomly distributed only in “wet” but not in “dry” preparations (Ramsey and Warren, 1932; Marcussen, 1939; Nizet and Govaerts, 1947) ; but a random distribution of the cells can be seen in well-spread dry smears ( Seip, 1953; Dacie, 1956; Lowenstein, 1958). In blood smears of normal animals with reticulocyte counts of less than about 276, the reticulocyte distribution approximates to a Poisson distribution (Ramsey and Warren, 1932 ; Nizet and Govaerts, 1947; Schneiderman and Brecher, 1950) ; with higher reticulocyte counts the cell distribution is binomial (Biggs, 1948). In order to make reticulocyte counts meaningful, the standard error of the proportion of reticulocytes should be determined. The following formula may be used:

where S.E. is the standard error of p ; p is the proportion of reticulocytes ; and m is the number of reticulocytes counted (Haldane, 1945; Dacie, 1956). With reticulocyte counts under 2747, the formula may be simplified A to : Y S.E. = dtn -2 The number of reticulocytes that must be counted for a desired accuracy can be determined by letting S.E. equal the desired standard error and ~

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solving the formula for m (Dacie, 1956). Reticulocyte counts can be compared with each other for significance by calculating the standard error of p and consulting the appropriate statistical tables for the limits of expectation for p (see, for example, Fisher and Yates, 1943). The total number of cells required to be counted for a given degree of accuracy decreases as the reticulocyte percentage increases. When less than 1% reticulocytes are present, a total of 5000 to 10,OOO cells must be counted for a standard error of under 10% (Krumbhaar, 1930; Marcussen, 1939; Seip, 1953 ; Dacie, 1956) ; when the reticulocyte percentage is lo%, a total of only 900 cells need be counted for the same accuracy. In the example with 10,OOO cells, about 9900 erythrocytes and 100 reticulocytes would have been counted; in the example with 900 cells, the numbers would have been about 810 and 90, respectively. Methods of counting have been devised, however, in which fewer erythrocytes need be counted, since, for a given accuracy, no more erythrocytes than reticulocytes need be counted (Woolf, 1950). The technique of counting approximately equal numbers of reticulocytes and erythrocytes is performed by counting a given number of reticulocytes in a series of microscopic fields and approximately the same number of red blood cells in a series of microscopic fields. The number of red blood cells in the microscopic fields used for counting the reticulocytes is then calculated (Woolf, 1950; Dacie, 1956). Another method is to use an optical disc consisting of a small square drawn inside a larger square, with a known area ratio of the two squares. The reticulocytes in the larger square and the erythrocytes in the smaller square are counted, and the number of erythrocytes in the larger square is calculated (Brecher and Schneiderman, 1950; Schneiderman and Brecher, 1950; Woolf, 1950). It has been claimed that reticulocytes are arranged on smears in a distribution more regular than chance, and that therefore small deviations in reticulocyte counts are significant (Jacobsen et al., 1947) ; but this somewhat startling claim could not be verified (Biggs, 1948). Estimates of the number of reticulocytes per cubic millimeter of blood are occasionally made in order to determine the extent of erythropoiesis. This value can most easily be found by counting reticulocytes in a hemocytometer ( Friedlander and Wiedemer, 1929 ; Franke, 1931), although this method of counting is less accurate than the percentage count, since the reticulocytes with little reticulum are often hard to distinguish from erythrocytes (see Orten, 1934).

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111. MORPHOLOGY A . Inner Structure Different methods of examining the reticulocyte reveal different and sometimes conflicting details of its inner structure. When reticulocytes are stained with brilliant cresyl blue, a deep blue reticulum appears. The reticulum varies in amount according to the age of the cell. It may consist of one small granule or of a dense clump of interwoven strands which, in dry smears, occupies one third of the cell area (Rosin and Bibergeil, 1904 ; Cesaris-Demel, 1907 ; Lee et al., 1916 ; Seyfarth, 1927 ; Moldawsky, 1928; Davidson, 1930; Eaton and Damren, 1930; Riddle, 1930; Heilmeyer, 1931 ; Trachtenberg, 1932 ; Nicolle, 1936). In blood smears stained with a Romanowsky dye, no reticulum is apparent in any of the red blood cells. Instead, a diffuse or granular basophilia appears. These basophilic cells were claimed to be reticulocytes, because the percentage of reticulocytes in supravitally stained blood smears was similar to the percentage of basophilic cells in Romanowsky-stained smears of the blood (Biondi, 1908; Hawes, 1909; Schilling-Torgau, 1911 ; Pepper and Peet, 1913; Key, 1921; Briickner, 1927; Brookfield, 1928; Davidson et ol., 1928; Gawrilow, 1929; Davidson, 1930; Heath and Daland, 1930). More recently this claim has been supported chemically. The application of ribonuclease to smears stained with either type of dye resulted in the disappearance of both the reticulum and the basophilia (Dustin, 1944; Thoma, 1950). Because the reticulum of the reticulocyte can be demonstrated by supravital stains and not by Romanowsky stains, it was suggested that the reticulum is an artifact of staining and is formed from the diffuse basophilic substance of the cell by the action of the supravital dyes. This view was corroborated by the study of reticulocytes stained with acridine-orange and seen under the fluorescence microscope. As the concentration of dye was raised, filaments began to form, and a reticulum appeared (Kosenow, 1952). Nevertheless, certain structures have been seen inside the cell. When reticulocytes are hemolyzed, granules become visible in the unstained cell. This phenomenon was first described by Erb (1865), wha discovered the reticulocyte in hemolyzed blood samples. Under the phase-contrast microscope, up to 50 small granules have been seen in one reticulocyte (Bessis, 1949). The granules of hemolyzed cells have been observed in greater detail under the electron microscope. They measure from 0.1 to 0.7 p in diameter. The larger granules appear to have been deflated, whereas the smaller granules are regular and spherical. These observations were first described by Bernhard et al. (1949) and have been confirmed by Bessis (1950), Braunsteiner and Bernhard (1950), H u g et al. (1950), Peters

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and Wigand ( 1950), Jung ( 1956), and Wolpers ( 1956). The appearance of the granules under the electron microscope varies according to the technique used for hemolysis. When reticulocytes were hemolyzed in over 10 volumes of distilled water, only circular granules were seen. When thin blood smears were placed in solutions of formaldehyde or sucrose or partially dried before hemolysis, however, long filaments similar to those produced by vital dyes were seen (Brunner and Vallejo-Freire, 1956). The relation between the granules and the reticulum produced by supravital stains is not yet clear. Key (1921) claimed that these granules were fragments of reticulum, and Brecher (1948) found that the granules in hemolyzed cells coalesced into an intact reticulum on the addition of supravital stains and that frequently a reticulum appeared independently of the granules. Feissly and Ludin ( 1949), however, believed the granules to be identical with the reticulum. From these observations and from electron microphotographs some authors have concluded that the reticulum is not an artifact but a pre-existing structure. The granules seen in hemolyzed cells have been considered to arise from the filaments of the reticulum by swelling of the filaments during osmotic hemolysis (Bernhard et al., 1949). Partial drying of the reticulocyte prevented the filaments from swelling into granules during hemolysis (Brunner and VallejoFreire, 1956). The electron microphotographs of reticulocytes have also been interpreted to mean that granules and not filaments compose the inner structure in intact cells, and that the granules line up to form a reticulum on staining (Bernhard et al., 1949 ; Braunsteiner and Bernhard, 1950; Wolpers, 1956). H u g et al. (1950) and Schilling (1951) believe the granules to be artifacts of hemolysis. Observations on the unstained intact reticulocytes reveal a different picture of the cell morphology. The granules are not numerous in the intact cell, nor do they resemble the reticulum. Under the phase-contrast microscope a few granules and small filaments are visible when the cells are suspended in plasma or saline (Bessis, 1949; Rind and Stobbe, 1957). No inner detail at all was found by Moeschlin (1949a, b). When reticulocytes are placed in albumin solutions of a refractive index close to that of the cells, up to five delicate filaments are seen in some of the reticulocytes, and small granules are found in others. These structures are not visible in all reticulocytes and probably occur only in the younger cells. They are smaller and thinner than the reticulum produced by supravital staining and by hemolysis (Lowenstein, 1958). Granules were observed to coalesce into a reticulum in the presence of supravital dye (Ralph, 1947). These granules stained with Janus Green B and therefore were thought to be mitochondria. In sections of reticulocytes examined under

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the electron microscope only an occasional dark granule or structure resembling a mitochondrion has been found (Braunsteiner et al., 1956; Brunner et al., 1956 ; Chalfin, 1956 ; Pease, 1956 ; Wolpers, 1956; Lowenstein, 1958). In conclusion, observations on reticulocyte morphology do not present a clear picture of cellular detail. Since a reticulum is not visible in the living reticulocyte viewed under the phase-contrast microscope, the reticulum is most probably an artifact of staining. It consists of a combination of dye, ribonucleic acid, and possibly other materials, such as lipoprotein (Laur, 1932; Ma, 1932; Sen0 et al., 1953a). Mitochondria have also been invoked as constituents of the reticulum, but there is no experimental evidence for this. The heavy granular structure of the hemolyzed reticulocyte seen under the electron microscope is also an artifact of fixation, probably identical in nature to the punctate basophilia produced on Romanowsky staining. The ribonucleic acid of cells other than reticulocytes is associated chiefly with the endoplasmic reticulum which is distributed throughout the cytoplasm (see Holter, 1952). On centrifugation of broken cells under well-defined conditions the endoplasmic reticulum gives rise to a high-speed sediment referred to as microsomes. In the reticulocyte no endoplasmic reticulum is visible under the electron microscope. In centrifuged samples of hemolyzed reticulocytes, however, the centrifugal fraction which contains most of the ribonucleic acid corresponds to the “microsomal” fraction obtained with other cells. The only structures visible in living reticulocytes are a few mitochondria, which disappear as the cell matures. B. V u u o l e s When reticulocytes are allowed to stand for over 15 minutes or are stained with neutral red, they acquire vacuoles (Key, 1921 ; Dustin, 1944; Bessis, 1950; Discombe, 1950; Schwind, 1950). The vacuoles are not present in the intact living cell. IV.

PHYSICAL PROPERTIES

A . Shape The reticulocyte is usually an irregular ovoid cell, as seen in wet preparations. The cells have also been observed to have an irregular form similar to that of a clover leaf (Gripwall, 1938; Moeschlin, 194%, b). The shape may be changeable, since the cells have been observed to possess contractile motion. This motility may be a factor aiding the entry of reticulocytes into the blood stream (Ralph, 1947; Bessis, 1949; Rind and Stobbe, 1957). I t is not known how the shape of a reticulocyte is transformed from an irregular ovoid into a biconcave disc during matura-

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tion. It has been suggested that this conversion in shape is mediated by the spleen (Miller et al., 1942).

B. Size The red blood cell gradually diminishes in size during its development (Thorell, 1947a, b) . The normoblast is a larger cell than the reticulocyte ; the reticulocyte is in turn larger than the erythrocyte (Hawes, 1909; Key, 1921 ; Dameshek and Schwartz, 1940). The macrocytosis encountered in some anemias or in the recovery from some anemias is due to the increased percentage of reticulocytes in the blood (Cruz, 1934 ; Wintrobe, 1934; Lawrason et ul., 1949). Reticulocyte size has usually been determined from measurements of cell diameters in dry blood smears (Davidson and McCrie, 1928; Persons, 1929; Hegner, 1938; Paolino, 1949) and from calculations of the mean cell volume of a given number of reticulocytes (Wintrobe, 1934; Rapoport et al., 1944; Lawrason et al., 1949; Burt et al., 1951; Betke and Rodig, 1955; Kiinzer et ul., 1955; Weicker and Fichsel, 1955 ; Weicker et al., 1955 ; Chalfin, 1956; Weicker, 1956). Cell diameters have been measured on dry blood smears stained with a supravital dye. I n normal blood the diameter of reticulocytes was found to be about 8.5 p, 1 p greater than that of the erythrocyte (Davidson and McCrie, 1928; Hegner, 1938). In pernicious anemia the reticulocytes were also found to be greater in diameter than the megalocytes (Davidson and McCrie, 1928). In secondary anemia the reticulocyte diameter was only slightly larger than the diameter of normal erythrocytes, but reticulocytes in secondary anemia develop into small hypochromic erythrocytes (Persons, 1929), and hence the developmental size ratio is similar. The decrease in the mean diameter of reticulocytes during maturation was measured by Paolino (1949). Although it was estimated that reticulocytes first increased and then decreased in diameter, the data were not analyzed for statistical significance. The method of measuring reticulocyte diameters in dry smears is not very reliable. Reticulocytes become rounder and smaller when they absorb the supravital dye (Di Gugliemo and La Manna, 1948). In addition, the reticulocyte is said to be a thinner cell than the erythrocyte (Smith, 1891; Moeschlin, 1949a; Chalfin, 1956) and in dry smears may be more flattened and distorted than the erythrocyte. Estimations of the reticulocyte volume vary markedly, and no adequate comparison between the size of a normal reticulocyte and a normal erythrocyte has yet been recorded. Measurements of the mean corpuscular volume have been made by estimations of the packed cell volume and the cell count of a blood sample. The samples may contain 100% reticulocytes (Burt et el., 1951), or the percentages of reticulocytes in a cell sample

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may be accounted for by a formula such as that used by Weicker (1956). In chronic anemia of rabbits produced by repeated bleeding, the reticulocyte was found to be 1.2 to 1.5 times the volume of the erythrocyte (Burt et ul., 1951; Chalfin, 1956). In phenylhydrazine anemia of rabbits, dogs, or swine, the volume of a reticulocyte has been estimated as 2 to 3.5 times the volume of a normal erythrocyte (Cruz, 1941 ; Rapoport et al., 1944; Lawrason et al., 1949; Kunzer et al., 1955 ; Weicker et al., 1955 ; Weicker and Fichsel, 1955). In human hemolytic anemia the reticulocyte volume has also been estimated at 2 to 3 times that of the normal erythrocyte (Betke and Rodig, 1955; Weicker and Fichsel, 1955). The decrease in volume during reticulocyte maturation is probably due to loss of water from the cell, although it has been suggested that the volume is halved by the division of reticulocytes (Weicker et al., 1955; Weicker, 1956). The decrease in volume is gradual, and after transformation into a young erythrocyte the cell still has not diminished to its final volume (Cruz, 1941). Although reticulocytes are larger than erythrocytes, the exact difference in volume is hard to estimate. Measurements of the mean corpuscular volume involve errors in cell count, reticulocyte count, and packed cell volume; and when these are subjected to mathematical manipulation to calculate reticulocyte volume, such as was done by Weicker et d. (1956), the errors are increased. The amount of plasma trapped in the hematocrit tube is another possible source of error: it may vary with the percentage of reticulocytes in the sample, since reticulocytes have an ovoid shape and may pack differently from erythrocytes. Moreover, reticulocytes produced by anemias cannot properly be compared with normal erythrocytes, since the abnormally sized reticulocytes mature into erythrocytes that may be larger or smaller than normal.

C . Density and Refractive Index The reticulocyte is more dense than the normoblast but less dense than the erythrocyte (Handovsky, 1912 ; Key, 1921 ; Davidson, 1930). Reticulocytes from anemic rabbits were estimated to have a density of 1.105 g./ml. cells, compared with a density of 1.122 g./ml. cells for the mature anemic cells (Chalfin, 1956). The density of normal human erythrocytes containing 33% hemoglobin is about 1.094 g./ml. (Reznikoff, 1923). Anemic rabbit reticulocytes and erythrocytes usually contain less than 30% hemoglobin, and their density is therefore less than 1.094 g./ml. The values for cell density obtained by Chalfin indicate a hemoglobin concentration of over 44% in the cells, which is most unlikely. When a sample of blood is centrifuged or allowed to stand, the reticulocytes tend to remain

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in the top layer of cells. This behavior permits the separation of reticulocytes from erythrocytes (Handovsky, 1912 ; Heath and Daland, 1930; Stephens, 1938; Pritchard, 1949; Keitel et al., 1955; Allison and Burn, 1955; Chalfin, 1956). The separation is facilitated by the centrifugation of the blood in a solution of albumin and saline with a density between that of the erythrocyte and the reticulocyte (Ferrebee et al., 1946; Vallee et al., 1947; Allison and Burn, 1955). Measurements of individual cells have indicated that the refractive index of the reticulocyte is less than that of the erythrocyte (Gaffney, 1957 ; Lowenstein, 1958).

D.

Osmotic Resistance

The osmotic resistance of the red blood cell can be defined as its ability to remain intact in hypotonic solutions. When a red blood cell is placed in solutions of increasingly lower osmotic pressure, water diffuses into the cell, which results in an increase in cell volume. The surface area remains unaltered until, at a critical hypotonicity, the cell membrane becomes injured and the cell hemolyzes. Hemolysis tests on reticulocytes have been performed by putting the cells in hypotonic sodium chloride solutions and determining the extent of hemolysis, either by counting the remaining unhemolyzed cells or by estimating the amount of hemoglobin released. The tests are the most reliable when the blood is well oxygenated, the released hemoglobin is measured colorimetrically, and the osmotic pressure, pH, and temperature of the solutions are carefully controlled (Jacobs and Parpart, 1931; Dacie and Vaughan, 1938; Parpart et al., 1947; Hendry, 1948, 1949). The concentration of sodium chloride in which normal red blood cells hemolyze varies from 0.44 to 0.30%. The osmotic resistance of reticulocytes is usually measured in comparison with that of erythrocytes. Results of measurements by different authors are listed in Table I. It is evident from this table that there exists a wide variation in results. This variation is partly due to errors of technique. Early authors who counted remaining unhemolyzed cells in their tests may have confused hemolyzed reticulocytes with unhemolyzed reticulocytes, since the hemolyzed cells contain granules and may look unhemolyzed under the light microscope. I n addition, a large counting and mixing error is associated with this counting technique (Daland and Zetzel, 1936). Some of the experiments can also be criticized as to the interpretation of results. Normal reticulocytes are not easily obtainable in large enough amounts for testing ; therefore, reticulocytes have been used which were obtained from anemic animals. These anemic reticulocytes may have a different resistance from normal erythrocytes and reticulocytes. Variations in the resistance of reticulocytes occur in different diseases, and

TABLE I RESISTANCE OF RETICULOCYTES, COMPARED TO THE OSMOTIC RESISTANCE OF ERYTHROCYTES” THEOSMOTIC Osmotic Type of erythrocyte comDisease Species resistanceb pared with reticulocytec Author (s) Rabbit Greater Same Simrnel (1919) Normal Greater Minot and Buckman (1923) Same Man Same Same Whitby and Hynes (1935) Man Less Normal Cathala and Daunay (1908) Man Icterus neonatorum Greater Same Sabrazes and Leurat (1908) Man Less Same Goldbloom and Gottlieb (1929) Man Same Normal Smith and Brown (1906) Horse Chronic secondary anemia Greater Same Simmel (1919) Man Same Same Rabbit Key (1921) Varies Same Man Buckman and MacNaugher (1923) Varies Same Daland and Zetzel (1936) Man Same Same Rabbit Daland and Zetzel (1936) Same Less Stewart et al. (1950) Dog Greater Same Rabbit Chalfin (1956) Normal Greater Man Bauer and Aschner (1919) Recovery from acute anemia Greater Same Man Simmel (1919) Normal Greater Man Miiller-Neff (1936) Normal Less Cruz et 01. (1941) Dog Same Simmel (1919) Same Man Hemolytic anemia Same Same Man Valentine (1928) Varies Same Man Daland and Zetzel (1936) Less Same Man Buckman and MacNaugher (1923) Pernicious anemia Varies Same Man Daland and Zetzel (1936) Rabbit Same Same Phenylhydrazine anemia Pepper and Peet (1913) Greater Same Exteriorized spleen Stephens (1940) Dog . . The early literature on this subject is not included in this table. In this column the osmotic resistance of reticulocytes is compared with that of erythrocytes. 0 “Same” indicates that reticulocytes were compared with erythrocytes of the same disease ; “normal” indicates that reticulocytes were compared with normal erythrocytes. 0

i~

148

LEAH MIRIAM LOWENSTEIN

reticulocytes from different patients with the same disease have been found to vary in resistance (Daland and Zetzel, 1936). It is not possible from the conflicting evidence to state whether the normal reticulocyte is more or less resistant than the normal erythrocyte. The osmotic resistance of the red blood cell varies with the shape of the cell. In general, the greater the difference between the volume of a cell in isotonic solutions and its volume when spherical, the greater is the degree of osmotic resistance. Therefore, a small volume/thickness ratio of the cells indicates an increased osmotic resistance (Haden, 1934; Castle and Daland, 1937). Neither the thickness nor the volume of reticulocytes has been satisfactorily measured; and it is not known if the surface area of the reticulocyte and the erythrocyte are the same. Until these measurements are accurately determined, the behavior of the reticulocyte in hypotonic solutions cannot even be satisfactorily predicted.

E. Resistance to Hemolytic Agents Reticulocytes may behave differently from erythrocytes in resistance to certain hemolytic agents. Reticulocytes have been claimed to be more resistant than erythrocytes to hemolysis induced by heat (Isaacs et al., 1925), lead ( P a r s e , 1926), ammonium propionate and salicylate (Dziemian, 1942), alkali (Stephens, 1940), and specific circulating hemolysins (Cruz, 1941). They were found to be less resistant than erythrocytes to hemolysis induced by storage in vitro (Gabrio et al., 1954). They show a varied resistance to hemolysis by saponin (Zucker and Kesten, 1928; Mermod and Dock, 1935). I t is difficult to draw conclusions from these experiments. They have not been repeated, and it is not known whether results vary as much from experiment to experiment as they do in measurements of the osmotic resistance of reticulocytes.

F. Adhesiveness Reticulocytes may be more adhesive than erythrocytes, as is manifest by their tendency to stick together and to stick to glass (Key, 1921 ; Seyfarth, 1927 ; Davidson et al., 1928 ; Davidson, 1930). The adhesiveness may be a factor in holding the young reticulocyte in the bone marrow (Davidson, 1930).

G . Charge Reticulocytes were shown to move in a different manner from erythrocytes in an electrophoretic field, which may indicate that they adsorb less ions than do erythrocytes (Stephens, 1 9 4 ) .

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149

V. BIOCHEMISTRY A . Ions and Water Measurements indicate that reticulocytes contain more sodium, potassium, magnesium, chloride, and phosphorus than erythrocytes, but the calculated ratios of the cations are not in agreement (Kay, 1930; Henriques and Prskov, 1939; Guest and Rapoport, 1941; Rapoport, et al., 1944 ; Keitel et al., 1955 ; Kruszynski, 1955 ; Chalfin, 1956). Reticulocytes and young erythrocytes also have a larger water content than mature erythrocytes (Bodansky and Dressler, 1927 ; Keitel et d.,1955 ; Chalfin, 1956). Measurements on individual reticulocytes by the technique of immersion refractometry indicate that the water concentration of the reticulocyte is about 5% greater than that of the erythrocyte (Gaffney, 1957; Lowenstein, 1958). This higher water content during maturation of the reticulocyte helps to maintain the osmotic equilibrium of the cell with the plasma, since the reticulocyte contains a greater number of solute molecules than the erythrocyte. As the reticulocyte matures, the solutes diminish in amount or disappear entirely from the cell, which results in a decrease of cellular osmotic pressure. Osmotic equilibrium with the plasma is maintained by the loss of water from the cell.

B. Stroma Since the reticulocyte is a larger cell than the erythrocyte, it may be expected to contain more stroma. The amount of stroma in rabbit reticulocytes produced by phenylhydrazine was found to be 2 to 4.5 times as great as that of normal erythrocytes, but the measurements included the denatured hemoglobin produced in the cells by the drug (Ponder and Velick, 1939). Moreover, anemic erythrocytes may contain twice as much stroma as normal erythrocytes (Tishkoff et al., 1953). Reticulocytes contain more lipid phosphorus than do erythrocytes (Rapoport et al., 1944; Ruhenstroth-Bauer and Hermann, 1950; Burt et al., 1951). This indicates an increased amount of stroma; however, the amount of lipid per unit of cell surface in the reticulocyte may be less than in the erythrocyte (Dziemian, 1942). C. Hemoglobin The hemoglobin content of the developing red blood cell increases as the cell matures, and when the reticulocyte stage is reached, nearly all the hemoglobin of the cell has been synthesized (Thorell, 1947a, b). The reticulocyte is still capable of forming small amounts of hemoglobin. One of the first claims that reticulocytes synthesize hemoglobin was made by Reimann (1942), who noted a rise in the hemoglobin content of reticulocytes during incubation in vitro. The synthesis has been demonstrated by

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LEAH MIRIAM LOWENSTEIN

the incorporation of iron (Walsh et al., 1949; Kruh et al., 1953) and of amino acids into hemoglobin (London et al., 1950; Borsook et al., 1952; Nizet and Lambert, 1953a, b ; Kruh et al., 1956; Rabinovitz and Olson, 1956). 1. Iron. On incubating reticulocytes with radioactive iron in vitro, more of the assimilated iron was found attached to the stroma than was incorporated into heme. I t was postulated from these results that iron is attached to a stroma-acceptor before entering into the formation of hemoglobin (Walsh et al., 1949; Borsook et al., 1957). Iron uptake was increased if the cells were washed in saline several times and copper ions added to the incubation mixture. Under optimum experimental conditions reticulocytes have been found to take up more iron than can be used for hemoglobin synthesis (Ellis et al., 1954). 2. Porphyrin. Blood containing large numbers of young red cells was found to exhibit a marked fluorescence under ultraviolet light (Keller and Seggel, 1934; Seggel, 1934; Lageder, 1936; de Langen and Grotepass, 1938a, b). The fluorescence is due to the presence of protoporphyrin in reticulocytes and young erythrocytes (Watson and Clarke, 1937 ; Stasney and McCord, 1942; Watson et al., 1944; Watson, 1946, 1950). Coproporphyrin also occurs in the reticulocytes (Watson, 1950). 3. Amino Acids. The incorporation of amino acids into hemoglobin was demonstrated by experiments in which reticulocytes were incubated with radioactive amino acids in Vitro, the hemoglobin was isolated, and its radioactivity measured. The amino acids tested were glycine, histidine, leucine, lysine, phenylalanine, serine, tryptophan, tyrosine, and glutamic acid (London et al., 1950; Neuberger and Niven, 1951; Borsook et d., 1952; Nizet and Lambert, 1953a, b ; Kruh et al., 1956; Rabinovitz and Olson, 1956; Borsook et al., 1957). Di- and tripeptides were also utilized in hemoglobin synthesis after they were hydrolyzed into their constituent amino acids in the incubation mixture (Nizet and Lambert, 1954b). Glycine was incorporated mainly into heme but also into globin (London et al., 1950; Nizet and Lambert, 1953b; Kruh et al., 1956) ; and phenylalanine was incorporated into globin (Nizet and Lambert, 1953b). The incorporation of the amino acids was accelerated by the addition to the incubation mixture of iodothyronines or choline or extracts of spleen, reticulocytes, erythrocytes, yeast, or liver (Borsook et al., 1952 ; Lambert, 1953a; Lybeck et al., 1954). An accelerating factor in liver extracts was identified as a fructose amine (Borsook et al., 1955; Lowy and Borsook, 1956). Amino acid incorporation into proteins was also increased by the addition of preincubated plasma or by large doses of X-irradiation of blood in witro or in vivo before incubation (Nizet et al., 1954; Nizet and Lambert, 1954a). Retardation or inhibition of amino

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151

acid incorporation was observed when cyanide, sulfur mustards, or 2mercaptoethylamine was added to the incubation mixture (Lambert, 1953b ; Nizet and Lambert, 1953b ; Paoletti et d., 1956). Retardation of synthesis also occurred when all the sodium in the saline of the incubation mixture was replaced by potassium, or when lead, mercury, or aureomycin was added (Borsook et al., 1957). Glycine uptake was inhibited by arsenate and dinitrophenol (Riggs et d.,1952). During 24 hours 0.3 to 0.9% of the total heme of red blood cells was formed in blood samples containing varying percentages of reticulocytes in different stages of maturity (London et al., 1950). From this data it can be roughly calculated that over 5% of the heme of the erythrocyte is synthesized during the reticulocyte stage. The uptake of glycine by the reticulocyte diminishes in amount as the cell matures (Gavosto and Rechenman, 1954), and most of the heme synthesis occurs while the reticulocyte is still in the bone marrow.

D . Nucleic Acids Ribonucleic acid ( R N A ) , which is associated with protein synthesis (see Caspersson, 1951), is present in the cytoplasm of the developing red blood cell and declines in amount as the cell matures (Caspersson and Thorell, 1941 ; Thorell, 1947a). Small amounts of R N A exist in reticulocytes (Masing, 1911; Kay, 1930; Caspersson and Thorell, 1941 ; Ruhenstroth-Bauer and Hermann, 1950; Burt et al., 1951). The R N A phosphorus content of the average reticulocyte was found to be 1.3 to 5X pg. per cell, as compared with 6.9 X lo-' pg. per average bone marrow cell (white blood cells included) (Davidson et al., 1951). Values of a similar order were obtained by Ruhenstroth-Bauer and Hermann (1950). On supravital staining, R N A is precipitated to form part of the reticulum (Dustin, 1944; Thoma, 1950; Burt et al., 1951). Reticulocyte maturation is defined by the gradual loss of reticulum; and the decrease of ribonucleic acid in the cell is thus used as a measure of maturation. Its disappearance marks the transformation of the reticulocyte into an erythrocyte. The synthesis of ribonucleic acid in the reticulocyte has been demonstrated by the in vitro incorporation of radioactive glycine into RNA (Kruh and Borsook, 1955). Radioactive phosphorus is incorporated more quickly into reticulocytes than erythrocytes, but it is not known if the phosphorus is incorporated into R N A (Lovegrove et al., 1952). Reticulocyte maturation in vitro was retarded by the addition of thiourea and thiouracil, substances which are antagonistic to the incorporation of uracil into R N A (Nizet, 1946b, 1948, 1952). Reticulocytes possess little or no DNA (Davidson et al., 1951 ; Burt et al., 1951; Holloway and Ripley, 1952; Goetzke et al., 1954). Several

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LEAH M I R I A M LOWENSTEIN

studies have been made on the role of R N A in protein synthesis using reticulocytes (Holloway and Ripley, 1952 ; Koritz and Chantrenne, 1954 ; Rabinovitz and Olson, 1956; Schweiger et d.,1956; Eriksen, 1958). A protein in association with R N A was shown to be formed as an intermediate during hemoglobin synthesis. Reticulocytes were incubated with radioactive leucine and hemolyzed, and the radioactivity of cell fractions separated by centrifugation was measured. It was found that the radioactive amino acid was first incorporated into the cell fraction which contains R N A and then into the soluble protein fraction, which contains hemoglobin (Rabinovitz and Olson, 1956). This work was confirmed by Eriksen ( 1958). Thorell (1947a) suggested that, since the amount of R N A declines in the developing red blood cell before hemoglobin is formed, R N A is therefore responsible for protein synthesis but not protein differentiation. However, the mass of globin is synthesized before the heme is attached to it (Lagerlof et al., 1956). R N A may therefore be involved in globin differentiation but take no part in the attachment of heme to the globin molecule in the formation of hemoglobin (Hammarsten et al., 1953). Other experiments have indicated that R N A may play a role in protein synthesis. A correlation has been demonstrated between the reticulocyte count of a sample, the amount of R N A in the sample, and the amount of amino acid incorporation into protein (Holloway and Ripley, 1952). No direct relationship between R N A and protein synthesis can be deduced from these experiments, since the amounts of substances other than RNA, e.g., enzymes and water, are also correlated with the number of reticulocytes. Koritz and Chantrenne ( 1954) have suggested that R N A is responsible for protein differentiation rather than synthesis. Measurements of the amounts of R N A and of certain proteins and of amino acid incorporation into protein were made with blood samples containing different numbers of reticulocytes in various stages of maturity. Their results therefore cannot be applied directly to an interpretation of the time relationship between the R N A concentration and the rate of protein synthesis in single reticulocytes.

E . Enzymes The reticulocyte represents a metabolic transition between the active normoblast and the erythrocyte. Respiratory quotients of reticulocytes have been frequently measured since Morawitz (1909) and Warburg (1909) found that anemic blood cells consume more oxygen than normal erythrocytes. The oxygen consumption in reticulocytes has been estimated to be up to 30 times as high as in erythrocytes. The early literature on this subject is reviewed by Orten (1934) ; see also Jacobsen and Plum, 1944 ; Jones et al., 1953; Goetzke et d.,1954; Rapoport, 1956. The rate of

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glycolysis is also much higher in reticulocytes than in erythrocytes (Barer et al., 1929; Engelhardt and Lyubimova, 1936; Jones et al., 1953; Rubinstein et al., 1956), and reticulocytes will not mature unless adequate amounts of glucose are present (Nizet, 1943, 1946b ; Rapoport and Strassner, 1955 ; Strassner, 1956). The citric acid cycle has been shown to exist in reticulocytes (Jones et al., 1953 ; Rapoport and Hofmann, 1955 ; Rubinstein et al., 1956). Enzymes present in the reticulocyte can be divided into two groups, those present in equal activity in both reticulocyte and erythrocyte, and those present in increased concentrations in the reticulocyte. Enzymes in the first group consist of the following: lactic and isocitric dehydrogenases, and diphosphopyridine nucleotidase (Hofmann et al., 1956 ; Rubinstein et al., 1956). Enzymes in the second group include hexokinase, aconitase, fumarase, cytochrome oxidase, pyrophosphatase (Rubinstein et al., 1956), succinic dehydrogenase and succinic dehydrogenase inhibitor (Rapoport and Gerischer-Mothes, 1955 ; Rapoport and Hofmann, 1955 ; Rapoport and Neiradt, 1955 ; Rapoport and Gerischer-Mothes, 1956 ; Rapoport et al., 1956 ; Rubinstein et al., 1956 ; Rapoport, 1956) ; cholinesterase (Pritchard, 1949; Sabine, 1951 ; Scudamore et at., 1951 ; Allison and Burn, 1955; Ellis et al., 1956), glyoxylase and catalase (Allison and Burn, 1955), cathepsin (Goetzke and Rapoport, 1954; Goetzke et d.,1954; Ellis et al., 1956), glycylglycine dipeptidase, leucine aminopeptidase, alkaline organic pyrophosphatase, and acid inorganic pyrophosphatase (Ellis et d.,1956), alkaline phosphatase (Kerppola, 1951), carbonic anhydrase (Koritz and Chantrenne, 1954), and nuclease (Lindigkeit, 1956). Hemolyzates of reticulocytes have a lower respiratory activity than whole cell suspensions. Nicotinamide, methylene blue, or diphosphopyridine nucleotide, when added separately or together, stimulated the respiration of the reticulocyte hemolyzates (Hofmann and Rapoport, 1955 ; Rapoport and Hofmann, 1955). Greater activity of some enzymes is claimed for hemolyzates than for whole cell suspensions (Rubinstein et at., 1956). Hemolyzates of reticulocytes have been found to cleave adenosine triphosphate and diphosphopyridine nucleotide rapidly, triphosphopyridine nucleotide and thiamine pyrophosphate slowly, and flavine adenine dinucleotide not at all. The hydrolysis of triphosphopyridine nucleotide by the hemolyzates is inhibited by nicotinamide, similar to the inhibition by nicotinamide of the hydrolysis of diphosphopyridine nucleotide by diphosphopyridine nucleotidases (Hofmann et d.,1956). The activity of many of the enzymes declines during maturation at a different rate from the decline of R N A (Rubinstein et al., 1956). The disappearance of RNA marks the transformation of the reticulocyte to an erythrocyte, but certain

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LEAH MIRIAM LOWENSTEIN

enzymes persist in the young erythrocyte for a few days (Sabine, 1951 ; Kubinstein et al., 1956).

PHYSIOLOGY The reticulocyte is formed in the bone marrow and completes its development into an erythrocyte in the peripheral blood. Young reticulocytes contain more reticulum than old reticulocytes ; and reticulocyte maturation is most conveniently measured by the gradual disappearance of the reticulum from the cell (Gawrilow, 1929; Heilmeyer, 1931 ; Trachtenberg, 1932; Nicolle, 1936). The classification most commonly used is that of Heilmeyer, in which the reticulocytes are divided into four groups. In group O M , the reticulum appears in the form of a dense clump; in group two, in a wreath ; in group three, the wreath has disintegrated ; and in group four, only a few scattered granules of the reticulum remain. Each reticulocyte passes through all four stages in succession. VI.

A.

Occurrence in the Bone Marrow

In spite of many studies on the enumeration of bone marrow cells, the proportion of reticulocytes in the bone marrow has been little investigated ; Romanowsky dyes, which are commonly used to stain the marrow, do not produce the reticulum, and the reticulocytes on the smears are often ignored. It has been estimated that the ratio of reticulocytes to erythrocytes is 3 to 4 times as high in the bone marrow as in the peripheral blood (Pokrowsky, 1929; Dameshek et al., 1937; Ungricht, 1938; Forssell, 1939; Plum, 1942). Marrow punctures are often diluted, however, with up to an equal amount of peripheral blood (Berlin et al., 1950), and some observers hold that the bone marrow contains no erythrocytes at all (Istomanova, 1926; Nizet, 1947 ; Seip, 1953). It is of interest to compare the percentage of reticulocytes with that of other young blood cells in the bone marrow. Steele (1933) calculated that 52% of the erythroid cells in the normal human bone marrow are reticulocytes, and it can be estimated from this that approximately 25% of all the young marrow cells are reticulocytes. Other calculations agree with this percentage; Seip (1953) found a reticulocyte count of 27%, and de Vries et al. (1956), 18 to 26%. I n hyperplastic anemias this may rise to over 70% (de Vries et al., 1956). The normal ratio of myeloid to nucleated red cells averages about 4 to 1, with a range between 8 to 1 and 2 to 1 (Young and Osgood, 1935 ; Osgood and Seaman, 1944; Vaughan and Brockmyre, 1947; Dacie and White, 1949; Leitner et ad., 1949; Wintrobe, 1956). If reticulocytes are included with the erythroid cells, the ratio becomes about 1 to 1, and this gives a better picture of the proportions of developing white and red blood cells in the marrow.

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THE MAMMALIAN RETICULOCYTE

The proportions of reticulocytes of different age groups in the bone marrow, determined by various authors, are listed in Table 11. Seip’s figures are probably the most accurate, since his estimates allow for the presence in the bone marrow smears of reticulocytes from the peripheral blood. His calculations indicate that there are no reticulocytes of group 4 in the marrow. TABLE I1 AGEOF RETICULWYTES I N THE BONEMARROW AND BLOOD Bone marrow (% of total reticulocytes)

Blood

(% of total reticulocytes)

Age group@

Age group

Author

1

2

3

4

Author

1

Forssell (1939) Plum (1941 ) Seip (1953)

14

14

28

44

1.3

14

15

28

43

24

42

34

0

Heilmeyer (1931) Trachtenberg (1932) Nizet (1946a) Seip (1953)

2

3

4

10.0 18.7 70

0.6

7.5 18.7 73.2

0

9.0 20.4 70.6

0.1

7.0 32.0 60.9

5 Classified according to Heilmeyer (1931). The youngest reticulocytes are placed in group 1, and the oldest in group 4.

B . A g e of Cells W h e n Released from the Bone Marrow It is not certain at what stage of maturation the developing red blood cell leaves the bone marrow. It has been held that under normal conditions a proportion of the red blood cells mature into erythrocytes before leaving the bone marrow (Minot et al., 1928a ; Minot et d.,1928b ; Heath and Daland, 1930; Young and Lawrence, 1945; Wintrobe, 1956). Minot et 02. and Wintrobe arrived at this conclusion because they noted that the increase in the number of red blood cells in patients treated for pernicious anemia was greater than the increase in the number of reticulocytes. The continual entry and maturation of reticulocytes in the blood stream were not taken into account, however. Young and Lawrence (1945) also concluded that some red blood cells must enter the circulation as erythrocytes. They took into account the reticulocyte maturation time and estimated that it is 140 hours. The erythrocyte survival time calculated from this figure was much greater than the accepted value of 120 days, unless it was assumed that erythrocytes as well as reticulocytes entered the circulation. The maturation time of 140 hours is longer, however, than that obtained by other authors (see Table 111). Moreover, Young and Lawrence (1945) used the total maturation time of the reticulocyte in their

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LEAH MIRIAM LOWENSTEIN

calculations, whereas the average length of time the reticulocyte spends in the blood should have been used. The balance of the evidence favors the theory that red blood cells leave the bone marrow while still reticulocytes. The increase in the number of circulating red blood cells was calculated to equal the rise in circulating reticulocytes in patients with pernicious anemia treated with liver extracts (Riddle, 1930; Koller, 1939) and in rabbits recovering from TABLE I11 THEMATURATION TIMEOF RETICULOCYTES Percentage reticulocytes Maturation before time maturation (hours)

Animal

Disease

Rabbit Man Man Man Rabbit Man Man

Secondary Normal Pernicious Pernicious Secondary Pernicious Anemia

Rat

Secondary anemia

anemia

6

anemia anemia anemia anemia

1-16 245 18-33 10-42 7-20

Guinea pig Hypoxia Dog Rabbit Man

Man

Pyrodine anemia Secondary anemia Hereditary spherocytosis Phenylhydrazine anemia Hemolytic anemia

Man Dog

Rabbit

48-72 48 120-240 48 96-144 96144 24-48 96

9

96-144

12-90 10-20 5-14

48-72 44 8

47-70,'

8

73

140

Normal Normal

1-4 1-12

47 31

Dog

Secondary anemia

2-17

Man

Normal

Man Man

Normal Neonate Normal

1-2

,

,

29

60-80

48 48

Author (s) Pepper (1922) Denecke (1923) Cohn rt al. (1928) Riddle (1930) Heath and Daland (1930) Heath and Daland (1930) Heilmeyer and Westhauser (1932) Creskoff and Fitz-Hugh (1937) Gordon and Kleinberg (1938) Cruz (1941) Plum (1942) Baar and Lloyd (1943) Baar and Lloyd (1943) Young and Lawrence (1945) Nizet (1947) Nizet and RobscheitRobbins (1950) Nizet and RobscheitRobbins (1950) Baldini and Pannacciulli (1953) Seip (1953) Kiinzer (1955) Kiinzer (1955)

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hemorrhage (Gordon, 1934). The age at which reticulocytes leave the bone marrow was estimated from cell counts by the following authors : Heilmeyer (1931), group 4 ; Ungricht (1938), groups 1 and 2 ; Koller ( 1939), group 2 ; Nizet ( 1946a), usually group 4 ; and Seip ( 1953), groups 3 and 4. Nizet (1946a) established a cross circulation between two dogs, of which one had cells labeled with Heinz bodies. The ratio of cells with Heinz bodies to unaffected cells was the same in the blood and in the bone marrow of the recipient dog, and it was concluded that the erythrocytes present in the bone marrow were due to the admixture of peripheral blood. In other experiments, when hypoxia was produced in man, an immediate increase in reticulocytes occurred in the peripheral blood. The increase consisted largely of reticulocytes in age group 3. It was postulated that blood cells leave the bone marrow as reticulocytes of this age group (Seip, 1953). During periods of intensified blood production, the reticulocytes leave the bone marrow at an earlier age than normal. C. Factors Which Influence the Release of Reticulocytes from the Bone Marrow

The factors that influence blood regeneration have been studied extensively in recent years ; however, the factors responsible for the direct liberation of reticulocytes from the bone marrow have been little investigated. Experiments to test the release of reticulocytes into the blood usually consist in determining the rise in reticulocytes in the peripheral blood after applying the desired stimulus to the experimental animal. The increase in reticulocytes must be measured within several hours, preferably within 5 hour. If the reticulocyte response occurs later than this, it is probably due to an increase in blood production. Reticulocyte counting must be carefully done and the counting error determined to ascertain if the change in the count is statistically significant. Four general factors have been considered to influence the release of reticulocytes from the bone marrow : diminishing adhesiveness of the reticulocyte, neural stimulation, the action of humoral substances, and the oxygen tension of the blood. 1. Adhesiveness. The theory of adhesiveness has already been mentioned. It was suggested that the cellular adhesiveness of normoblasts is a factor in holding the cells in the bone marrow. This adhesiveness diminishes during maturation of the reticulocyte, leading to the liberation of the reticulocyte from the bone marrow (Davidson, 1930). This theory has not been confirmed, and reticulocytes already in the peripheral blood retain some of their adhesiveness (Key, 1921). 2. Neural Factors. Reticulocytes in the blood have been shown to increase in response to stimulation of various parts of the nervous system.

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Although irritation of the whole nervous system by electric shock does not produce a rise in reticulocytes (Clare et d.,1944 ; Hortling, 1947), irritation of the nervous system by ventricular or lumbar puncture was shown to produce a rise in reticulocytes in normal man and animals (du Bois, 1934; Beer, 1942; Seip, 1953) and in human subjects with an increased spinal fluid pressure (Ginzberg and Heilmeyer, 1932). Stimulation of the brain by diathermy evoked a reticulocyte response in patients with hyperplastic bone marrow (Denecke, 1935 ; Dockhorn, 1936). An attempt to define the brain center that regulates the release of reticulocytes was made by Heilmeyer (1933), who found that puncture of the diencephalon causes a rise in reticulocytes within 24 hours. Seip (1953) found that vertebral but not carotid angiography results in an increase of reticulocytes in the blood, which suggests that the center is located in the basal part of the brain. It has not yet been demonstrated whether the erythropoiesis center and the reticulocytosis center in the brain are identical. Stimulation of the sympathetic nervous system or the giving of sympathomimetic drugs has a variable effect on reticulocytosis. Direct stimulation of the sympathetic lumbar trunk, or the sympathetic plexuses around the liver and esophagus, or the great splanchnic nerve, was found to produce a reticulocyte response in 15 minutes which subsided after 100 minutes (Linke, 1953, 1955a, b) . A disappearance of the reticulocytosis after 100 minutes seems unlikely, however, since reticulocytes usually remain in the blood for 25 hours (Nizet, 1947 ; Seip, 1953 ; Kiinzer, 1955). Denervation of the carotid sinus produced a fall in reticulocytes (Durinyan, 1956). Increases in the reticulocyte count lasting only about 1 to 2 hours after the injection of epinephrine have been noted (Dazzi, 1921; Istomanowa and Chudoroscowa, 1930; Benhamou and Nouchy, 1931 ; Paschkis and Schwoner, 1934; Chatterjea et al., 1953). Capillary blood was used in these experiments, and it has been suggested that the transient rise in the reticulocyte count was due not to the release of reticulocytes from the bone marrow but to the increased number of the larger, more adhesive reticulocytes in the constricted peripheral vessels from which the blood was withdrawn (Seip, 1953). Kinkel and Diercks (1936) found a drop in the reticulocyte count after the injection of epinephrine, and Hortling (1947) and Seip (1953) found that epinephrine had no effect on the reticulocyte count. The bone marrow is extensively supplied with myelinated and unmyelinated nerve fibers that form plexuses around the blood vessels and penetrate into the marrow pulp (see Grant and Root, 1952). Stimulation of the parasympathetic nerves to the bone marrow resulted in a rise in reticulocytes in the majority of animals tested, as did cutting the sympa-

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thetic nerves to the bone marrow (Okinaka et al., 1938). Injections of carbachol (Seip, 1953) and pilocarpine (Kinkel and Diercks, 1936), parasympathomimetic drugs, also produced a rise in reticulocytes. Parasympathetic stimulation may therefore be a factor responsible for the liberation of reticulocytes into the peripheral blood. It cannot be stated with certainty, however, from the experiments on neural stimulation that the release of reticulocytes into the blood is produced on direct stimulation of the bone marrow nerves. 3. Humoral Factors. The existence of humoral factors which regulate erythropoiesis has been established (Grant and Root, 1952). Humoral factors may also influence the release of reticulocytes. Plasma from patients with high reticulocyte counts was found to raise the reticulocyte count when injected into normal men (Oliva et al., 1949) but this work could not be confirmed (Seip, 1953). Plasma from erythroblastotic infants injected into man produced a rise in reticulocytes within 30 minutes after injection (Seip, 1955b). This humoral factor has not been identified. X-irradiation of young rats produced a rise in reticulocytes within 30 minutes, although the total number of circulating red blood cells did not change (Hajdukovic and Stosic, 1957). This reticulocytosis may be mediated by a humoral factor. The humoral and neural mechanisms of reticulocyte release may be interrelated, as indicated by studies on parabiotic rats connected by their abdominal cavities with no nerve and few blood vessel communications. When air was injected into the cerebral ventricles of one rat, a rise in reticulocytes in the blood of both animals was observed 4 hours later. Extirpation of the spleen, adrenals, or pancreas had no effect on this phenomenon, and it was concluded that the substance responsible for the reticulocytosis was produced by the liver (Beer, 1942). It is possible, however, that the circulations of both rats were so intermingled after 4 hours that the circulation of the unstimulated rat received reticulocytes from the stimulated rat and no humoral factor was involved. In addition, the kidney, bone marrow, or nervous system were not excluded as possible sites of production of a humoral factor. This factor may arise from the central nervous system, since the injection into normal rabbits of cerebrospinal fluid from rabbits with anemia or with electric stimulation applied to the diencephalon produced a rise in reticulocytes within 2 to 4 hours (Agostini, 1950). The factor may enter the cerebrospinal fluid from the blood stream, however. It has been suggested that the bloodregulating center in the brain acts on the bone marrow through parasympathetic nerves and also regulates the production of a reticulocytosis factor in the liver (Seip, 1953). There is no evidence at present that the liver produces this factor.

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4 . Oxygen Tension. A decrease in oxygen tension in the atmosphere has long been known to increase blood production (Bert, 1882). This decrease does not have a direct effect on the bone marrow, since a lowered oxygen tension in bone marrow cultures retards the rate of maturation of the developing red blood cells (Rosin and Rachmilewitz, 1948; Magnussen, 1949). In addition, the oxygen tension of the bone marrow does not usually differ from normal in states of increased or decreased erythropoiesis (Grant and Root, 1947; Berk et al., 1948; Schwartz and Stats, 1949) ; and erythropoiesis proceeds at the same rate in different parts of the skeleton that have different oxygen tensions (Eranko and Karvonen, 1955). Hypoxia in man, produced by severe muscular effort, pneumothorax, or breathing air with a low oxygen tension, led to an immediate rise in reticulocytes in the blood (Riska, 1950a, b ; Seip, 1953). Cerebral ischemia due to syncope also raised the reticulocyte count (Bgie and Benestad, 1954). The hypoxia may result in the production of a reticulocytosis humoral factor in the same manner as it does in the production of an erythropoietic factor (see Grant and Root, 1952). Further work is needed to establish whether the two factors are identical.

D. The Occurrence of Reticulocytes in the Peripheral Blood During the first 3 months of gestation in the human being, the blood contains 90 to 100% reticulocytes; this percentage falls to 15 to 20 by the sixth month ( Seyfarth and Jiirgens, 1928 ; Windle, 1941 ; Wintrobe, 1956) and continues to decrease until birth, when there is an average of 2 to 670 reticulocytes in the blood (see Orten, 1934 ; Josephs, 1936; Wollstein, 1938; Waugh et al., 1939; Windle, 1941 ; Shapiro and Bassen, 1941 ; Wegelius, 1948; Gairdner et al., 1952; Seip, 1955a). The level of reticulocytes rises for the first 3 days after birth (Wegelius, 1948; Nuss, 1952; Seip, 1955a) and falls to a normal or low level by the eighth day (FaxCn, 1937; Wegelius, 1948). The percentage then remains approximately the same throughout adolescent and adult life (Nizet, 1946a ; Leichsenring et al., 1955; Seip, 1953), except for a possible rise in the third month of life when the blood production also rises slightly ( FaxCn, 1937 ; Wegelius, 1948; Glaser et al., 1950; Seip, 1955a). There are few data on the possible change in the reticulocyte count during old age. Reticulocyte counts in healthy people over 60 years of age have been listed as 0.6% (Ventura, 1941 ; Newman and Gitlow, 1943) and 2% (Shapleigh et al., 1952). This variation in results is probably due to a difference in technique. Newman and Gitlow counted only 500 cells per sample, using “dry” preparations, whereas Shapleigh et al. used “wet” preparations. Animals also have raised reticulocyte counts at birth (see Plum, 1949).

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Literature on the reticulocyte counts in the peripheral blood of animals, including man, has been reviewed by Orten (1934), who gives an average value of about 0.8% for man. Later work, in which the counts were carefully performed, indicates that the value is somewhat higher, between 1.5 and 1.7%, or 67,000 to 95,000 reticulocytes per cubic millimeter (Osgood, 1935; Nizet, 1946a; Seip, 1953). The age of reticulocytes in the blood, according to the method of Heilmeyer, have been determined by several authors (see Table 11). These figures agree closely, considering the individual variations involved in classifying the age of the cells. Variations in the reticulocyte count have been claimed to occur diurnally (Goldeck and Heinrich, 1949; Goldeck and Stoffregen, 1952; Goldeck, 1953), daily (Ederle, 1933; Langendorff and Reisner, 1936; Barbier. 1939), or seasonally (Friedlander and Wiedemer, 1929; Grunke and Diesing, 1936; Pintor and Grassini, 1957), but these deviations are probably not significant (Nizet, 1946a ; Engelbreth-Holm and Videbaek, 1948 ; Seip, 1953). Although no reticulocytosis accompanies postprandial leukocytosis, it was claimed that reticulocytes in the blood increased 1 hour after the ingestion of certain amino acids (Kohl, 1951). The reticulocyte count in aminals depends on a number of variables, among them the rate of blood production, the length of life of the erythrocyte, and the rate of maturation of the reticulocyte. From a knowledge of the reticulocyte count and the length of time the reticulocytes spend in the blood, it is possible to calculate the life of the erythrocyte in man (Heilmeyer and Westhauser, 1932; Baar and Lloyd, 1943; Young and Lawrence, 1945 ; Seip, 1953).

E. The Maturation of Reticulocytes The reticulocyte is formed in the bone marrow and finishes its development in the peripheral circulation. Under conditions of increased blood regeneration, reticulocytes are released into the blood at an earlier age than normal. Assuming that reticulocyte maturation in the blood proceeds at the same rate as it does in the bone marrow, these young reticulocytes can be used to determine the total maturation time of the reticulocyte. Such experiments have been conducted in vitro and in vivo. In vitro methods consist of incubating whole blood with or without added substances at 37"C., whereas in vivo experiments usually consist of injecting reticulocyte-rich blood into patients with aplastic anemia or into animals. Maturation is determined by the gradual disappearance of the reticulocytes and of the stainable reticulum. On plotting a graph of time as the abscissa and the percentage of reticulocytes in samples incubated in witro as the ordinate, a curve asymptotic to the abscissa is obtained. It was thought

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that the rate of reticulocyte maturation could be expressed by a first-order equation (Plum, 1942) ; but experimental maturation curves deviate from such a relationship (Nizet, 1947 ; Baldini and Pannacciulli, 1953 ; Seip, 1953). The average life of the reticulocyte appears to be between 40 and 80 hours, of which the last 25 hours are spent in the peripheral blood. Values as divergent as 8 to 140 hours have been obtained by different authors (Table 111). This wide variation is due to several factors: (1) In many of the calculations the length of life of the reticulocyte in the bone marrow was ignored. (2) In some experiments the disappearance of reticulocytes from incubated samples was estimated only once every 24 hours. (3) The maturation time varies with the composition of the incubation medium. Reticulocyte maturation is retarded if sufficient glucose or certain amino acids are not present (Nizet, 1943 ; Plum, 1944 ; Nizet and Robscheit-Robbins, 1950 ; Rubinstein et al., 1956; Strassner, 1956). The maturation time is lengthened in cultures incubated in low oxygen tension, at temperatures under 37"C., or if cyanide, urethan, thiouracil, or thiourea is added (Pepper, 1922 ; Heath and Daland, 1930 ; Heilmeyer and Westhauser, 1932; Jacobsen and Plum, 1944; Nizet, 1946b, 1948, 1952). Cells incubated in sera of pernicious anemia patients have been found to have a shorter maturation time than normal (Baar and Lloyd, 1943) ; cells incubated in the serum of malarial patients show a decreased maturation time (Fabiani and Orfila, 1954). The maturation of reticulocytes may be accelerated by extracts of liver, spleen, and stomach, and fumarate and oxaloacetate (Plum, 1942, 1943a, b ; 1944; Jacobsen and Plum, 1942a, b, 1943, 1944; Plum and Plum, 1943; Seno et al., 1953b). Plum has postulated the existence of a specific reticulocyte-ripening factor normally present in the blood, which contains tyrosine and which varies in potency in different species of animals. However, some of Plum's results are based on deviations in the cell count which may not be statistically significant (Biggs, 1948). Heath and Daland ( 1930) and Nizet (1946b) have failed to confirm some of these results. Liver extracts injected into fetal rats with 100% reticulocytes did not mature the cells (Fitz-Hugh et al., 1936). Serum with erythropoietic activity from anemic rabbits had no influence on the rate of reticulocyte maturation (Strassner, 1956).

F . Cell Division From measurements of the mean corpuscular hemoglobin content of rabbit reticulocytes, it has been estimated that reticulocytes contain up to twice as much hemoglobin as erythrocytes (Rapoport et al., 1944; Weicker and Fichsel, 1955 ; Weicker et al., 1955). Moreover, the reticulocyte volume has been estimated at 2 to 3 times the volume of the erythrocytes. A consideration of these factors has led to the postulate that reticulo-

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cytes divide (Weicker, 1955; Weicker et al., 1955). This division would necessarily be amitotic, since the cells possess no nucleus and very little or no deoxyribonucleic acid (Davidson et al., 1951; Burt et al., 1951). Amitotic division has been described rarely for the erythropoietic series (Sabin et al., 1924; Doan et al., 1925) ; but it has been suggested on the basis of indirect experimental exidence that more than one reticulocyte arises from a normoblast (Emmel, 1914 ; Plum, 1947 ; Bostrom, 1948 ; Duran-Jorda, 1950). T o test the possible amitotic division of reticulocytes, radioactive reticulocytes, produced by the injection of radioactive iron into anemic rabbits, were injected into normal rabbits, and cell maturation was followed by means of autoradiographs. If a reticulocyte divides, its radioactivity would be diminished by half. The radioactivity per cell did not significantly change during maturation, and it was concluded that division of the reticulocyte does not occur (Lowenstein, 1959). The postulate of Weicker et al. (1955) that reticulocytes divide is therefore incorrect. The work that led to this postulate is open to the criticism that the mean hemoglobin content of anemic reticulocytes was compared with that of normal erythrocytes. The comparison may not be justified because reticulocytes produced by phenylhydrazine may contain more hemoglobin than is normal and may mature into erythrocytes containing more hemoglobin than is normal. VII.

RETICULOCYTES IN DISEASE

The percentage of reticulocytes in the blood has been used as an index of erythropoiesis in experimental work and in clinical medicine. ,Conditions which influence blood production, such as anoxic anoxia, anemic anoxia, hormones, and humoral substances, have been studied by noting changes in the percentage of reticulocytes in the blood before and after the application of various stimuli. The subject was reviewed by Grant and Root (1952). Because of the clinical significance of the reticulocyte level in the blood, reticulocyte counts have become a routine laboratory procedure. An increase of reticulocytes occurs in diseases of red blood cell destruction, such as acquired hemolytic anemia, hereditary spherocytosis, uremia, toxic anemia, malaria, and sickle cell anemia. A decrease in reticulocytes occurs in apIastic crises of hereditary spherocytosis and sickle cell anemia, in aplastic anemia, and in certain hormone deficiencies. The reticulocyte count is also used as an indication of blood regeneration during the treatment of hemorrhage, and of pernicious and iron deficiency anemia. The variations in reticulocyte count in specific diseases and under experimental conditions of erythropoiesis have been reviewed by Orten ( 1934), Ninni (1949), Plum (1949), and Wintrobe (1956) and will not be considered

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here. The reticulocyte differs from the erythrocyte, however, in its behavior in several diseases.

A . Sickle Cell Anemia In moist preparations of blood from patients with sickle cell anemia, reticulocytes were observed to sickle less readily than erythrocytes ( Scriver and Waugh, 1930; Diggs and Bibb, 1939). It has been demonstrated that, although the general rate of sickling is the same for older reticulocytes as for erythrocytes, the younger reticulocytes assume the sickled form slowly. In addition, the reticulocytes have not been found in the irreversibly sickled state (Watson, 1948). This decreased response of the young reticulocyte to low oxygen tension may partly be due to the lower hemoglobin concentration in reticulocytes than erythrocytes. Reticulocytes are rarely seen in the sickled form in dry blood smears from patients with sickle cell anemia (Sydenstricker et al., 1923 ; Murphy and Shapiro, 1945 ; Watson, 1948) ; and it has been suggested that sickled reticulocytes tend to resume their original shape when smeared onto a glass slide because they are more “elastic” than erythrocytes (Watson, 1948). Reticulocyte sickling does not interfere with the uptake of supravital stains (Watson, 1948) ; conversely, the stain may actually accelerate sickling (HansenPruss, 1936). B. Malaria Reticulocytes and erythrocytes appear to be infected with malarial parasites at different rates. I n man, Plasmodium Vivax generally shows a predilection for reticulocytes (Craik, 1920 ; Eaton, 1934 ; Jacobsthal, 1936; Shushan et al., 1937; Hegner, 1938; Kitchen, 1938, 1939; Vryonis, 1939; Ferrebee et al., 1946; Chwatt, 1948; Jones, 1951), whereas Plasmodium mahriae preferentially invades erythrocytes (Kitchen, 1939 ; Chwatt, 1948) . Reticulocytes and erythrocytes are equally infected with Plasmodium falciparum (Jacobsthal, 1936 ; Shushan et al., 1937 ; Malamos, 1937 ; Kitchen, 1939 ; Chwatt, 1948). Malarial parasites were also found to infect reticulocytes preferentially in birds, monkeys, and mice (Ben-Harel, 1923; Hegner and Hewitt, 1937; Hegner, 1938; Jones, 1951 ; Fabiani et al., 1952a; Singer, 1954). Divergent views exist on this selectivity, however (Eaton, 1934 ; Baserga, 1937; Malamos, 1937 ; Hegner, 1938 ; Hingst, 1938). The reason for this predilection is not known. Reticulocytes contain enzymes and other substances not present in erythrocytes, and these may be necessary for the growth of some types of parasite. I t has been suggested that all blood cells are invaded in the reticulocyte stage and that the apparent selectivity for reticulocyte or erythrocyte is due to the

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severity of the anemia (Eaton, 1934; Hegner, 1938). Since the anemia produced by P. && is not severe, reticulocytes enter the blood in age groups 3 or 4, become infected, and mature into erythrocytes quickly ; but in the more severe anemia produced by P. vivux, a large number of young reticulocytes enter in the blood. The young reticulocyte, after infection, remains in the reticulocyte stage longer than the older reticulocyte, and this increases the proportion of parasitized reticulocytes compared with the proportion of parasitized erythrocytes (Kitchen, 1938). The severity of the malaria caused by P. berghei and P. cathemerium can be increased if a reticulocytosis is produced in the animal before it is infected (Fabiani et ul., 1952b; Fabiani et al., 1952c; Singer, 1954). It has been suggested that relapses in human malaria are correlated with an increase of reticulocytes in the blood, for example at high altitudes (Hegner, 1938). The malarial parasites were also thought to be attracted to the reticulocyte because of the adhesiveness of reticulocyte (Vryonis, 1939). These theories remain unproved.

C . Heinz Body Anemia Large refractile granules, called Heinz bodies, are formed in red blood cells exposed to certain chemicals in vivo and in vitro. Substances that produce these bodies are mainly benzene derivatives with nitro, amino, hydrazino, and sulfonamido groups, such as phenylhydrazine, sulfa drugs, nitroanaline, and nitrotoluenes. The composition of the Heinz body is not fully known, but various forms of denatured hemoglobin are among its constituents (Dustin, 1942 ; Beavan and White, 1954). The literature on Heinz bodies has been reviewed by Fertman and Fertman (1955). Although Heinz bodies may be formed in up to 100% of the erythrocytes exposed to one of the provoking chemicals, they are very rarely found in reticulocytes (Bratley et al., 1931 ; Cruz, 1941 ; Dustin, 1942; Gajdos and Tiprez, 1945; Lawrason et d.,1949; Webster, 1949; Wolpers, 1956), an observation which awaits explanation. I t has been suggested that Heinz body formation is enzymic (Moeschlin, 1942 ; Gajdos and Tiprez, 1947 ; Brenner and Allison, 1953); and the different enzyme content of the reticulocyte may be a factor in preventing the formation of Heinz bodies. VIII. ACKNOWLEDGMENTS I am grateful to R. Barer, J. Lowenstein, and W. Bartley for reading and criticizing the manuscript, and to Misses J. Ellingham and M. Venables for assisting in the translation of some of the German articles. This review was written while the author was in receipt of a grant from the Medical Research Council, England.

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IX. REFERENCES Agostini, L. (1950) Riv. biol. (Perugia) 42,339. Allison, A. C., and Burn, G. P. (1955) Brit. J. Haematol. 1,291. Baar, H. S.,and Lloyd, T. W. (1943) Arch. Disease Childhood 18, 1. Baldini, M., and Pannacciulli, I. (1953) Boll. soc. ital. biol. sper. 29, 1291. Barbier, H. (1939) Klin. Wochschr. 18, 1496. Barer, A. P.,Needles, R. J., and 'Baldridge, C. W. (1929) Proc. SOC.Exptl. Biol. Med. 27, 176. Barer, R., and Joseph, S. (1955) Quart. J. Microscop. Sci. 96,1. Baserga, A. (1937) Riv. malariol. 16,38. Bauer, J., and Aschner, B. (1919) Deut. Arch. klin. Med. 190, 172. Beavan, G. H., and White, J. C. (1954) Nature 173,389. Beer, A. G. (1942) Folia Haematol. 66,222. Benhamou, E.,and Nouchy, A. (1931) Compt. rend. SOC. biol. 107,827. Ben-Harel, S. (1923) A m . J. H y g . 3, 652. Exptl. Berk, L., Burchenal, J. H., Wood, T., and Castle, W. B. (1948) Proc. SOC. Biol. Med. 69, 316. Berlin, N. I., Hennessy, T. G., and Gartland, J. (1950) J . Lab. Clin. Med. 36, 23. Bernhard, W., Braunsteiner, H., and Mangini, H. (1949) Compt. rend. soc. biol. 1p3, 1513. Bert, P. (1882) Compt. rend. 94, 805. Bessis, M. (1949) Rev. hfmatol. 4, 294. Bessis, M. (1950) Blood 6, 1083. Betke, K.,and Rodig, I. (1955) Klin. Wochschr. 33, 911. Biggs, R. (1948) Nature 162, 457. Biondi, C. (1908) Folia Haemutol. 6,205. Bodansky, M., and Dressler, 0. G. (1927) Quart. J . Physiol. 17, 157. B$e, J., and Benestad, A. M. (1954) Acta Med. Scand. 149, 345. Borsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighley, G., and Lowy, P. H. (1952) J. Biol. Chem. 196,669. Borsook, H., Abrams, A., and Lowy, P. H. (1955) J. Biol. Chem. 215, 111. Borsook, H., Fischer, E. H., and Keighley, G. (1957) J. Biol. Chem. 239, 1059. Bostrom, L. (1948) Acta Med. Scand. 131, 303. Bratley, F. G., Burroughs, H. H., Hamilton, D. M., and Kern, C. (1931) A m . J. Med. Sci. 182, 597. Braunsteiner, H., and Bernhard, W. (1950) Acta Haematol. 3, 167. Braunsteiner, H., Fellinger, K., and Pakesch, F. (1956) Acta Haematol. 16, 322. Brecher, G. (1948) Proc. SOC.Exptl. Biol. Med. 69, 89. Brecher, G., and Schneiderman, M. (1950) A m . J . Clin. Pathol. 20, 1079. Brenner, S., and Allison, A. C. (1953) Experientia 9, 381. Brookfield, R. W. (1928) I . Pathol. Bacteriol. 31, 277. Briickner, H. (1927) Arch. 'Hyg. 98,95. Brunner, A., Jr., and Vallejo-Freire, A. (1956) Exptl. Cell Research 10,55. Brunner, A., Jr., Vallejo-Freire, A., and Souza Santos, P. (1956) Experientia

lz, 255. Buckman, T. E., and MacNaugher, E. (1923) J. Med. Research 44,61. Burt, N. S., Murray, R. G. E., and Rossiter, R. J. (1951) Blood 6,906. Caspersson, T. (1951) Symposia SOC.Exptl. Biol. N o . 1, 115. Caspersson, T.,and Thorell, B. (1941) Chromasoma 2, 132.

T H E MAMMALIAN RETICULOCYTE

167

Castle, W. B., and Daland, G. A. (1937) Arch. Internal Med. 60,949. Cathala, V., and Daunay, R. (1908) Compt. rend. soc. biol. 64,801. Cesaris-Demel, A. (1907) Folia Haematol. 4, Suppl. 1. Chalfin, D. (1956) J. Cellular Comp. Physiol. 42,215. Chatterjea, J. B., Dameshek, W., and Stefanini, M. (1953) Blood 8,211. Chwatt, L. J. (1948) Ann. Trop. Med. Parasitol. 42, 101. Clare, F. B., Cress, C. H., and Gellhorn, E. (1944) Ann. Internal Med. 21. 653. Cohn, E. J., Minot, G. R., Alles, G. A., and Salter, W. T. (1928) J . k o l . Chem.

77. 325. Craik,.R. (1920) Lancet 1, 1110. Creskoff, A. J., and Fitz-Hugh, T. (1937) I. Clin. Invest. 16,680. Crosby, W. H.,and Rappaport, H. (1956) Blood 11, 929. Cruz, W. 0. (1934) Mem. inst. Omaldo Cruz aS, 427. Cruz, W. 0. (1941) Am. J. Med. Sci. 203, 781. Cruz, W.O.,Hahn, P. F., Bale, W. F., and Balfour, W. M. (1941) Am. J. Med. Sci. 202, 157. Dacie, J. V. (1954) “The Haemolytic Anaemias,” p. 171. Churchill, London. Dacie, J. V. (1956) “Practical Haematology,” p. 24. Churchill, London. Dacie, J. V., and Vaughan, J. M. (1938) J . Pathol. Bacteriol. 46,341.. Dacie, J. V., and White, J. C. (1949) J. Clin. Pathol. 2, 1. Daland, G.A., and Zetzel, L. (1936) Am. J. Med. Sci. 191,467. Dameshek, W., and Schwartz, S. 0. (1940) Medicine 19, 231. Dameshek, W., Henstell, H. H., and Valentine, E. H. (1937) Ann. Internal Med.

11, 801. Davidson, J. N., Leslie, I., and White, J. C. (1951) Lancet 1, 1287. Davidson, L. S. P. (1930) Edinburgh Med. J. 37, 425. Davidson, L.S. P., and McCrie, J. G. (1928) Lancet 2, 1014. Davidson, S., McCrie, J. G., and Gulland, G. L. (1928) Lancet 1, 847. Dazzi, A. (1921) Morg. Arch. 63,93. Denecke, G. (1923) Z.ges. exptl. Med. 36, 179. Denecke, G. (1935) Verhandl. deut. Ges. inn. Med. 47,243. Diggs, L. W., and Bibb, J. (1939) J. Am. Med. Assoc. 112, 695. Discombe, G. (1950) Acta Haematol. 3, 151. Doan, C. A., Cunningham, R. S., and Sabin, F. R. (1925) Contribs. Embryol.

16, 163. Dockhorn, E. (1936) Folia Haenzatol. 64,248. du Bois, A. H. (1934) Sang 8,343. Duesberg, R. (1931) Arch. exptl. Pathol. Phannakol. Naunyn-Schmiedeberg’s la,

249. Duran-Jorda, F. (1950) Acta Med. Scand. 136,275. Durinyan, R. A. (1956) Byull. Eksptl. Biol. Med. 42, 15 (Abstr. Sov. Med.

1967, 284). Dustin, P. (1942) Rev. belge sci. mtd. 14,48. Dustin, P. (1944) Arch. biol. (Paris) 66, 285. Dziemian, A. J. (1942) 1. Cellular Comp. Physiol. a0, 135. Eaton, P. (1934) Am. J. Trop. Med. 14,431. Eaton, P., and Damren, F. L. (1930) South. Med. J . 23, 311. Ederle, W. (1933) Med. Welt 7, 1095. Ehrlich, P. (1881) Klin. Wochschr. 17,405. Ellis, D., Sewell, C. E., and Skinner, L. G. (1956) Nature 177, 190.

168

LEAH MIRIAM LOWENSTEIN

Ellis, G. S., Brandt, C. S., and Thacker, E. J. (1954) Science 119,94. Emmel, V. E. (1914) Am. J . Anat. 16,128. Engelbreth-Holm, J., and Videbaek, A. (1948) Blood 3, 612. Engelhardt, V. H., and Lyubimova, M. N. (1936) Compt. rend. akad. sci. U.R.S.S. 2, 329 (Chem. Abstr. SO, 7661). Eranko, O., and Karvonen, M. J. (1955) Ann. Paed. Fenn. 1, 179. Erb, W. (1865) Arch. pathol. Anat. u. Physiol. Virchow’s 34, 138. Eriksen, L. (1958) Exptl. Cell Research 13,624. Fabiani, G., and Orfila, J. (1954) Compt. rend. soc. biol. 148,670. Fabiani, G., Clausse, J., and Fulchiron, C. (1952a) Compt. rend. soc. biol. 146,1580. Fabiani, G., Clausse, J., and Fulchiron, C. (1952b) Compt. rend. soc. biol. 146,1587. Fabiani, G., Fulchiron, C., and Clausse, J. (1952~)Compt. rend. soc. biol. 146,1584. FaxCn, N. (1937) Acta Paed. 19 Suppl. 1, 1. Feissly, R., and Ludin, H. (1949) Rev. htmatol. 4, 481. Ferrebee, J. W., Gibson, J. G., and Peacock, W. C. (1946) 1. Infectious Diseases

78, 180. Fertman, M. H., and Fertman, M. B. (1955) Medicine 34, 131. Fiessinger, N.,and b u r , C.-M. (1930) Sang 4, 257. Fisher, R. A.,and Yates, F. (1943) “Statistical Tables for Biological, Agricultural, and Medical Research,” p. 44. Oliver & Boyd, London. Fitz-Hugh, T., Creskoff, A. J., and Taylor, H. B. (1936) J. Clin. Invest. 16,468. Forssell, J. (1939) Acta Med. Scand. Suppl. 101, 1. Franke, K. (1931) Med. Klin. ( M u n i c h ) 27, 431. Friedlander, A.,and Wiedemer, C. (1929) A.M.A. Arch. Internal Med. 44,209. Gabrio, B. W., Finch, C. A., Linde, W., and Rupen, A. (1954) J. Clin. Invest.

33, 242. Gaffney, F. M. (1957) Brit. J. Haemutol. 3,311. Gairdner, D., Marks, J., and Roscoe, J. D. (1952) Arch. Disease Childhood 27, 214. Gajdos, A.,and Tiprez, G. (1945) Compt. rend. SOC. biol. 139,545. Gajdos, A.,and Tiprez, G. (1947) Sang 18, 35. Gavosto, F., and Rechenman, R. (1954) Biochim. et Biophys. Acta 13,583. Gawrilow, R. (1929) Folio Haematol. 98, 246. Ginzberg, R., and Heilmeyer, L. (1932) Arch. Psych. 97,719. Glaser, K., Limarze, L. R., and Poncher, H. G. (1950) Pediatrics 6, 789. Goetzke, E., and Rapoport, S. (1954) Biochem. 2. 326, 53. Goetzke, E., Lindigkeit, R., and Rapoport, S. (1954) Biochem. 2. 326, 48. Goldbloom, A.,and Gottlieb, R. (1929) A.M.A. J. Diseases Children 38, 57. Goldeck, H. (1953) Acta Med. Scand. Suppl. 278, 83. Goldeck, H., and Heinrich, W. D. (1949) Acta Haematol. 2, 167. Goldeck, H., and Stoffregen, J. (1952) 2.vergleich. Physiol. 34,41. Gordon, A. S. (1934) Proc. SOC.Exptl. Biol. Med. 31, 563. Exptl. Biol. Med. 38, 360. Gordon, A. S.,and Kleinberg, W. (1938) Proc. SOC. Grant, W.C.,and Root, W. S. ‘(1947) Am. J . Physiol. l60,618. Grant, W . C., and Root, W. S. (1952) Physiol. Revs. 32,449. Gripwall, E. (1938) Acta Mrd. Scand. Suppl. 96,1. Grunke, W., and Diesing, J. (1936) Klin. Wochschr. 16, 1190. Guest, G. M., and Rapoport, S. (1941) Physiol. Revs. 21, 410. Di Gugliemo, L., and La Manna, C. (1948) Boll. SOC. ital. biol. sper. 24, 108. Haden, R. L. (1934) Am. J. Med. Sci. 188, 441. Haenel, U. (1949) Acta Haematol. 2, 298.

THE MAMMALIAN RETICULOCYTE

169

Hajdukovic, S., and Stosic, L. (1957) Nature 179, 821. Haldane, J. B. S. (1945) Nature 166, 49. Hammarsten, E., Thorell, B., Aqvist, S., Eliasson, N., and Akerman, L. (1953) Exptl. Cell Research 6, 404. Handovsky, H. (1912) Arch. exptl. Pathol. Pharmakol. Naunyn-Schmiedeberg’s 69, 412. Hansen-Pruss, 0. C. (1936) J. Lab. Clin. Med. a,311. Hawes, J. B. (1909) Boston Med. Surg. J. 161, 493. Heath, C. W., and Daland, G. A. (1930) A.M.A. Arch. Internal Med. 46, 533. Heath, C. W., and Daland, G. A. (1931) A.M.A. Arch. Internal Med. 48, 133. Hegner, R. (1938) Am. J. Hyg. 27, 690. Hegner, R., and Hewitt, R. (1937) J. Pararitol. aS, 557. Heilmeyer, L. (1931) Deut. Arch. klin. Med. 171, 123. Heilmeyer, L. (1933) Verhandl. deut. Ges. inn. Med. 4S,113. Heilmeyer, L., and Westhauser, R. (1932) Z. klin. Med. 121, 361. Heinz, R. (1901) Beitr. pathol. Anat. 29, 299. Hendry, E. B. (1948) Edinburgh Med. J. 66,427. Hendry, E. B. (1949) Edinburgh Med. J. 66,320. Henriques, V., and Qrskov, S. L. (1939) Skand. Arch. Physiol. 82, 86. Hingst, H. E. (1938) Am. J. Trop. Med. 18, 361. Hofmann, E. C. G., and Rapoport, S. (1955) Biochem. Z. 326, 499. Hofmann, E. C. G., Rapoport, S., and Kesselring, K. (1956) 2. physiol. Chem. 304, 157. Holloway, B. W., and Ripley, S. H. (1952) J. Biol. Chem. 196, 695. Holter, H. (1952) Advances in Enzymol. 19, 1. Hortling, H. (1947) Acta Med. Scand. Suppl. 201, 1. Hug, O., Lippert, W., and Moser, P. (1950) Compt. rend. 1st congr. intern. microscop. electron. 672. Isaacs, R., Brock, B., and Minot, G. R. (1925) J. Clin. Invest. 1,425. Istomanova, T. S. (1926) Z. exptl. Med. 62, 140. Istomanova, T. S., and Chudoroscowa. (1930) 2. exptl. Med. 71, 212. Jacobs, M. H., and Parpart, A. K. (1931) Biol. Bull. 60,95. Jacobsen, E., and Plum, C. M. (1942a) Acta Physiol. Scand. 4, 272. Jacobsen, E., and Plum, C. M. (194213) Acta Phydol. Scand. 4,278. Jacobsen, E., and Plum, C. M. (1943) Acta Physiol. Scand. 6, 1. Jacobsen, E., and Plum, C. M. (1944) Acta Physiol. Scand. 7, 168. Jacobsen, E., Plum, C. M., and Rasch, G. (1947) Acta Pathol. Microbiol. Scand. 24, 554. Jacobsthal, E. (1936) Klin. Wochschr. 16, 942. Jones, E. S. (1951) Trans. Roy. SOC.Trop. Med. Hyg. 46, 19. Jones, E. S., Maegraith, B. G., and Gibson, Q. H. (1953) Ann. Trop. Med. Parasitol. 47, 431. Josephs, H. W . (1936) Medicine 16, 307. Jung, F. (1956) Folk Haematol. 74, 258. Kay, H. D. (1930) Brit. 1. Exptl. Pathol. 11, 148. Keitel, H. G., Berman, H., Jones, H., and MacLachlan, E. (1955) Blood 10, 370. Keller, C . J., and Seggel, K. A. (1934) Folia Haemotol. 62, 241. Kerppola, W. (1951) Blood 6, 454. Key, J. A. (1921) A.M.A. Arch. Internal Med. a8, 511. Kinkel, H., and Diercks, C. (1936) Monatsschr. Kinderheilk. 87, 229.

170

LEAH MIRIAM LOWENSTEIN

Kitchen, S. F. (1938) A m . J. Trop. Med. 18, 347. Kitchen, S. F. (1939) South. Med. J. 32, 679. Kohl, H. (1951) Deut. 2. Verdauungs.- u. Stofwechselkrankh. 11, 274. Koller, F. (1939) Deut. Arch. klin. Med. 184, 568. Koritz, S.B., and Chantrenne, H. (1954) Biochim. et Biophys. Acta lS,209. Kosenow, W. (1952) Acta Haematol. 7, 360. Kruh, J., and Borsook, H. (1955) Nature 176, 386. Kruh, J., Dreyfus, J. C., and Schapira, G. (1953) Bull. soc. chim. biol. 36, 1181. Kruh, J., Dreyfus, J. C., Schapira, G., and Padieu, P. (1956) Compt. rend. soc. biol. 160, 1119. Krumbhaar, E. B. (1930) Sang 4,267. Kruszynski, J. (1955) Acta Anat. !24, 164. Kiinzer, W. (1955) 2. Kinderheilk. 77, 249. Kiinzer, W., Ambs, E., and Schneider, D. (1955) 2. Kinderheilk. 77, 90. Lageder, K. (1936) Klin. Wochschr. 16,296. Lagerlof, B., Thorell, B., and Akerman, L. (1956) Exptl. Cell Research 10,752. Lambert, S. (1953a) Arch. intern. physiol. 61,462. Lambert, S . (1953b) Arch. intern. physiol. 62, 132. Langen, C. D.de, and Grotepass, W. (1938a) Acta Med. S c a d . 94,245. Langen, C. D. de, and Grotepass, W. (1938b) Acta Med. S c a d . 97,29. Langendorff, H., and Reisner, A. (1936) Folia Haematol. 66, 88. Laur, C. M. (1932) “Les reticulocytes et les reticulocytoses.” Doin, Paris. Lawrason, F. D., Eltzholtz, D. C., Sipe, C. R., and Schork, P. K. (1949) Blood

4, 1256. Lee, R. I., Minot, G. R., and Vincent, B. (1916) I. A m . &fed. Assoc. 67,719. Leichsenring, J. M., Norris, L. M., Lamison, S. A., and Halbert, M. L. (1955) A . M . A . J . Diseases Children 90, 159. Leitner, S. J., Britton, C. J. C., and Neumark, E. (1949) “Bone Marrow Biopsy,” p. 20. ,Churchill, London. Lindigkeit, R. (1956) Folia Haematol. 74,251. Linke, P. G. (1953) 2. Biol. 106,292. Linke, P. G. (1955a) 2. Biol. 101,429. Linke, P. G. (1955b) 2. Biol. 108, 1. London, I. M., Shemin, D., and Rittenberg, D. (1950) J. Biol. Chem. 183,749. Lovegrove, T. D.,De Luca, H. A., and Rossiter, R. J. (1952) Can. J. Med. Sci.

30, 143. Lowenstein, L. M. (1958) D.Phi1. Thesis, Oxon. Lowenstein, L. M. (1959) Exptl. Cell Research. In press. Lowy, P. H., and Borsook, H. (1956) J. A m . Chem. SOC.78, 3175. Lybeck, H., Lambert, S., Nizet, A., and Barac, G. (1954) Arch. intern. physiol. 62, 304. Ma, W.-C. (1932) Chinese J: Physiol. 6,271. Magnussen, J. D. (1949) Acta Pharmacol. Toxicol. 6,153. Malamos, B. (1937) Klin. Wochschr. 16, 885. Marcussen, P. V. (1939) F o l k Haemutol. 61, 49. Masing, E. (1911) Arch. exptl. Pathol. Pharmukol. Naunyn-Schmiedeberg’s 68, 71. Mermod, C.,and Dock, W. (1935) A . M . A . Arch. Internal Med. 66,52. Miller, E. B., Singer, K., and Dameshek, W. (1942) Proc. SOC.exptl. Biol. Med.

49, 42. Minot, G. R., and Buckman, T. E. (1923) A m . J. Med. Sci. 166,469.

THE MAMMALIAN RETICULOCYTE

171

Minot, G. R., Murphy, W. P., and Stetson, R. P. (1928a) Am. J. Med. Sci.176, 581. Minot, G. R., Cohn, E. J., Murphy, W. P., and Lawson, H. A. (1928b) Am. J. Med. Sci. 176, 599. Moeschlin, S. (1942) Folia Haematol. 66, 345. Moeschlin, S. (1949a) Acta Haematol. 2, 399. Moeschlin, S. (1949b) Schwciz. med. Wochschr. 79, 842. Moldawsky, J. W. (1928) F o l k Haematol. 36,145. Morawitz, P. (1909) Arch. exptl. Pathol. Phrmakol. Naunyn-Schmiedeberg’s 80, 298. Miiller-Neff, H. (1936) Folia Haematol. 66, 18. Murphy, R. C. Jr., and Shapiro, S. (1945) Ann. Internal Med. 23, 376. Neuberger, A., and Niven, J. S. F. (1951) J. Physiol. (London) 1l2, 292. Newman, B., and Gitlow, S. (1943) Am. J. Med. Sci.aOa, 677. Nicolle, P. (1936) Arch. inst. Pasteur Tunis 26, 437. Ninni, M. (1949) “I Reticolociti.” Typografio de Libro, Pavia. Nittis, S. (1938) A m . J. Mcd. Sci. 196, 177. Nizet, A. (1943) Acta Biol. Bclge S, 314. Nizet, A. (1946a) Acta Med. Scand. 124, 590. Nizet, A. (1946b) Compt. rend. soc. biol. 140, 1076. Nizet, A. (1947) Acta Med. Scand. 127, 424. Nizet, A. (1948) Arch. intern. pharmacodynamie 77, 141. Nizet, A. (1952) Science 116,290. Nizet, A., and Govaerts, J. (1947) Sang 18,466. Nizet, A., and Lambert, S. (1953a) Arch. intern. physiol. 61, 120. Nizet, A., and Lambert, S. (1953b) Bull. soc. chim. biol. S6,771. Nizet, A., and Lambert, S. (1954a) Arch. intern. physiol. 62, 540. Nizet, A., and Lambert, S. (1954b) Bull. soc. chim. biol. S6, 307. Nizet, A., and Robscheit-Robbins, F. S. (1950) Blood 6, 648. Nizet, A., Lambert, S., Herve, A., and Bacq, Z. M. (1954) Arch. intern. physiol. 62, 129. Nuss, M. A. (1952) Voprosy Pediat. i Okhrang Materinstva i Detstva 20,47. Okinaka, S., Asai, I., Morikawa, K., and In6, S. (1938) Klin. Wochschr. 17, 1752. Oliva, G., Chiuini, F., and Tramontana, C. (1949) Acta Med. Scand. lSS, 27. Orten, J. M. (1934) Yale J. Biol.Med. 6, 519. Osgood, E. E. (1935) A.M.A. Arch. Internal Med. 66, 849. Osgood, E. E., and Seaman, A. J. (1944) Physiol. Revs. 24, 46. Osgood, E. E., and Wilhelm, M. M. (1934) J. Lab. Clin. Med. 19, 1129. Palade, G. E. (1952) J. Exptl. Med. 96, 285. Paoletti, C., Boiron, M., Truhaut, R., and Tubiana, M. (1956) Compt. rend. soc. biol. 160, 1084. Paolino, W. (1949) Acta Med. Scand. 136, 141. Parpart, A. K., Lorenz, P. B., Parpart, E. R., Gregg, J. R., and Chase, A. M. (1947) J. Clin. Invest. 26, 636. Paschkis, I., and Schwoner, A. (1934) Med. Klin. (Munich) SO, 900. Paton, D. N., and Goodall, A. (1903) J . Physiol. (London) 29, 411. P a r s e , H. E. (1926) A.M.A. Arch. Internal Med. 57, 715. Pease, D. C. (1956) Blood 11, 501. Pepper, 0. H. P. (1922) A.M.A. Arch. Internal Med. SO, 801. Pepper, 0. H. P., and Peet, M. M. (1913) A.M.A. Arch. Internal Med. l2,81. Persons, E. L. (1929) J. Clin. Invest. 7, 615.

172

LEAH MIRIAM LOWENSTEIN

Peters, D., and Wigand, R. (1950) Klin. Wochschr. 28, 649. Pintor, P. P., and Grassini, V. (1957) Acta Haematol. 17, 122. Plum, C. M. (1941) A c f a Med. Scand. 107, 11. Plum, C. M. (1942) Ac fa Physiol. Scand. 4, 259. Plum, C. M. (1943a) A c f a Physiol. Scand. 6, 165. Plum, C. M. (1943b) Acta Physiol. Scand. 6, 175. Plum, C. M. (1944) Acta Med. Scand. 117, 437. Plum, C. M. (1947) Blood Special Issue 1, 42. Plum, C. M. (1949) Ac fa Haematol. 2, 317. Plum, C. M., and Plum, R. (1943) Acta Physiol. Scand. 6, 380. Pokrowsky, W. I. (1929) Folio Haematol. 39,265. Ponder, E., and Velick, S. (1939) J. Physiol. (London) 97, 5P. Price-Jones, C. (1911) 1. Pathol. Bacteriol. 16, 48. Pritchard, J. A. (1949) Am. J . Physiol. 168, 72. Rabinovitz, M., and Olson, M. E. (1956) Exptl. Cell Research 10, 747. Ralph, P. H. (1947) Anaf. Record 98, 489. Ramsey, R., and Warren, C. 0. (1932) Quart. J . Exptl. Physiol. !22, 49. Rapoport, S. (1956) F o l k Haematol. 74, 276. Rapoport, S., and Gerischer-Mothes, W. (1955) Z. physiol. Chem. 303, 167. Rapoport, S., and Gerischer-Mothes, W. (1956) Z. physiol. Chem. 904, 213. Rapoport, S., and Hofmann, E. C. G. (1955) Biochern. 2.326, 493. Rapoport, S., and Neiradt, C. (1955) Z. physiol. Chem. 302, 179. Rapoport, S., and Strassner, W. L. (1955) Biochim. et Biophys. Acta 17,276. Rapoport, S., Guest, G. M., and Wing, M. (1944) Proc. SOC.Exptl. Biol. Med. 67, 344. Rapoport, S., Gerischer-Mothes, W., and Neiradt-Hiebsch, C. (1956) Z. physiol. Chem. 304, 207. Reimann, F. (1942) Schweiz. Z. Pathol. u. Bakteriol. 6, 343. Reznikoff, P. (1923) J . Exptl. Med. 38,441. Riddle, M. C. (1930) A.M.A. Arch. Internal Med. 46, 417. Riggs, T. R., Christensen, H. N., and Palatine, I. M. (1952) J . Biol. Chern. 194, 53. Rind, H., and Stobbe, H. (1957) Folia Haematol. 1, 219. Riska, N. (19504 A c f a Med. Scand. Suppl. 237, 1. Riska, N. (1950b) Experienfia 6, 345. Rosin, A., and Rachmilewitz, M. (1948) Blood 3, 165. Rosin, H., and Bibergeil, E. (1904) Z. klin. Med. 64, 197. Rubinstein, D., Ottolenghi, P., and Denstedt, 0. F. (1956) Can. J . Biochem. and Physiol. 34, 2 2 . Ruhenstroth-Bauer, G., and Hermann, G. (1950) Z. Naturforsch. 6, 416. Sabin, F. R., Austrian, C. R., Cunningham, R. S., and Doan, C. A. (1924) J . Exptl. Med. 40, 845. Sabine, J. C. (1951) Blood 6, 151. Sabrazes, J., and Leurat, E. (1908) Coinpt. rend. SOC. biol. 64, 423. Schilling, V. (1951) Deut. Gesulzdheitsw. 6, 1037. Schilling-Torgau, V. (1911) Folia Haematol. 11, 327. Schneiderman, M., and Brecher, G. (1950) Biometrics 6, 390. Schwartz, B. M., and Stats, D. (1949) J . Clin. Invest. 28, 736. Schweiger, H. G., Rapoport, S., and Scholzel, E. (1956) Nature 178, 141. Schwind, J. L. (1950) Blood 6, 597.

T H E MAMMALIAN RETICULOCYTE

173

Scriver, J. B., and Waugh, T. R. (1930) Can. med. Assoc. J. 23, 375. Scudamore, H. H., Vorhaus, L. J., and Kark, R. M. (1951) Blood 6, 1260. Seggel, K. A. (1934) Folia Haematol. 62, 250. Seip, M. (1953) Acta Med. Scand. Suppl. 2.82. Seip, M . (1955a) Acta Paediat. 44, 355. Seip, M. (1955b) Acta Paediat. 44, 507. Seno, S., Kanda, K., Matsuba, K., Haba, K., and Saito, M. (1953a) M e i Med. J . 4, suppz. 1, 43, Seno, S., Kawai, K., Sakabe, S., and Nishikawa, K. (1953b) M e i Med. J. 4, Suppl. 1, 11. Seyfarth, C. (1927) Folia Haematol. 94, 7. Seyfarth, C., and Jiirgens, R. (1928) Arch. pathol. Anat. u. Physiol. Virchow's 266, 676. Shapiro, L. M., and Bassen, F. A. (1941) Am. J . Med. Sci. 202, 341. Shapleigh, J. B., Mayes, S., and Moore, C. V. (1952) In "Cowdry's Problems of Ageing" (A. I. Lansing, ed.), p. 290. Williams and Wilkins, Baltimore, Maryland. Shushan, M., Blitz, O., and Adams, C. G. (1937) J. Lab. Clin. Med. 22, 364. Simmel, H. (1919) Verhandl. Congr. inn. Med. S6, 144. Simmel, H. (1926) Folia Haematol. S2, 97. Singer, I. (1954) J. Infect. Diseases 94, 159. Sjovall, H. (1936) Arch. pathol. mirrobiol. Srand. Suppl. 27. Smith, T. (1891) Trans. Assoc. Am. Physicians 6, 263. Smith, T., and Brown, H. R. (1906) J. Med. Research 16, 425. Stasney, J., and McCord, W. M. (1942) Proc. SOC.Exptl. Biol. Med. 61,340. Steele, B. F. (1933) 1. Exptl. Med. 67, 881. Stephens, J. G. (1938) Nature 141, 1058. Stephens, J. G. (1940) J. Physiol. ( L o n d o n ) 99, 30. Stewart, W. B., Stewart, J. M., Izzo, M. J., and Young, L. E. (1950) J. Exptl. Med. 91, 147. Strassner, W. (1956) F o l k Haematol. 74, 271. Sydenstricker, V. P., Mulherin, W. A., and Houseal, R. W. (1923) A.M.A. J. Diseases Children 26, 132. Thoma, K. (1950) Klin. Wochschr. 2.8, 215. Thorell, B. (1947a) Acta Med. Scand. Suppl. 200. Thorell, B. (1947b) Cold Spring Harbor Symposia Quant. Biol. 12,247. Tishkoff, G. H., Robscheit-Robbins, F. S., and Whipple, G. H. (1953) Blood 8, 459. Trachtenberg, F. (1932) Folia Haematol. 46, 1. Ungricht, M. (1938) Folia Haematol. 60, 145. Valentine, F. C. 0. (1928) J. Pathol. Bacteriol. 31, 473. Vallee, B. L., Hughes, W. L., Jr., and Gibson, J. G., 11. (1947) Blood Special Issue 1, 82. Vaughan, S. L., and Brockmyre, F. (1947) Blood Special Issue 1, 54. Ventura, L. (1941) G i o ~ n .clin. Med. 22, 43. de Vries, A., Izak, G., and Bruderman, M. (1956) Rev. himatol. 11,486. Vryonis, G. (1939) Am. 1. H y g . SO, C41. Walsh, R. J., Thomas, E. D., Chow, S. K., Fluharty, R. G., and Finch, C. A. (1949) Science 110, 396.

174

LEAH MIRIAM LOWENSTEIN

Warburg, 0. (1909) Z . physiol. Chem. 69, 112. Watson, C. J. (1946) Blood 1, 99. Watson, C. J. (1950) Trans. Assoc. Am. Physicians 6!J, 219. Watson, C. J., and Clarke, W. 0. (1937) Proc. SOC.Exptl. Biol. Med. S6, 65. Watson, C. J., Grinstein, M., and Hawkinson, V. (1944) J. Clin. Invest. a3, 69. Watson, J. (1948) Blood 3, 465. Waugh, T. R., Merchant, F. T., and Maughan, G. B. (1939) Am. J . Med. Sci. 198, 646. Webster, S. H. (1949) Blood 4, 479. Wegelius, R. (1948) Acta Paediat. S6, Suppl. 4. Weicker, H. (1955) Schweiz. med. Wochschr. 86, 947. Weicker, H. (1956) 2. Kinderheilk. 77, 544. Weicker, H., and Fichsel, H. (1955) Klin. Wochschr. SS, 1074. Weicker, H., Erbsen, H., and Wild, M. (1955) Klin. Wochschr. SS, 962. Whitby, L. E. H., and Hynes, M. (1935) 1. Pathol. Bacteriol. 40,219. Windle, W. F. (1941) J . Pediat. 18, 538. Wintrobe, M. M. (1934) J. Clin. Invest. 13, 669. Wintrobe, M. M. (1956) “Clinical Hematology,” p. 33. Kimpton, London. Wollstein, M. (1938) “Handbook of Hematology” (H. Downey, ed.), Vol. 2, p. 931. Kimpton, London. Wolpers, C. (1956) Klin. Wochschr. S4, 61. Woolf, B. (1950) Edinburgh Med. J . 67, 536. Young, L. E., and Lawrence, J. S. (1945) J . Clin. Invest. 24, 554. Young, R. H., and Osgood, E. E. (1935) A.M.A. Arch. Intern. Med. 66, 186. Zucker, T. F., and Kesten, H. D. (1928) Am. J. Physiol. 87, 280.

The Physiology of Chromatophores MILTON FINGERMAN Department of Zoology, Newcomb College, Tulane University, New Orleans, Louisiana I. 11. 111. IV.

V. VI. VII. VIII.

Page . 175 Introduction ................................................. . 176 Classification of Chromatophore Responses .................... . 177 Functional Significance of Color Changes .................... . 181 Chroniatophores of Arthropods .............................. . 181 A. Sources of Chromatophorotropins ........................ . 183 B. Crustaceans ............................................. 183 1. Isopods ................................................... 185 2. Brachyurans .............................................. 193 3. Natantians ................................................ 196 4. Astacurans ................................................ 201 5. Stomatopods .............................................. 201 C. Insects ...................................................... 202 Chrornatophores of Fishes ........................................ 204 Chromatophores of Amphibians .................................... 205 Chemical Nature of Chromatophorotropins ......................... 206 References .......................................................

I. INTRODUCTION Rapid strides have been made within the past fifteen years in several aspects of the physiology of chromatophores. The number of investigators interested in color changes appears to be greater now than at any time in the past. Since 1944 several reviews concerning the physiology of color changes in vertebrates and invertebrates have appeared (Brown, 1944, 1948a, 1952 ; Panouse, 1947 ; Parker, 1948 ; Waring and Landgrebe, 1950; Scharrer, 1952a; Knowles and Carlisle, 1956; Pickford and Atz, 1957). The most comprehensive review (Parker, 1948) covered the period from 1910 through 1943. The emphasis herein, therefore, will be on a critical evaluation of the literature published since 1943. T o minimize repetition of information in earlier reviews, older material will be mentioned only when necessary for a lucid explanation of recent work. Although this review deals with the chromatophores of vertebrates and invertebrates, emphasis will be placed on the crustaceans, the class most intensively investigated in recent years. Previously, investigations of chromatophores of vertebrates far outnumbered those of invertebrates. The distribution of space in Parker’s book (1948) testifies to this disproportionate interest; 237 pages were devoted to the vertebrates, and only 56 to the invertebrates. The pendulum is now swinging in the opposite direction, and the disparity is disappearing. 175

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11. CLASSIFICATION OF CHROMATOPHORE RESPONSES

Color changes have been divided into two categories, morphological and physiological. The former involves increases or decreases in the quantity of pigment in an organism and does not fall within the domain of this review. Fox (1953) wrote an excellent volume on the chemical nature of pigments in animals and discussed this type of alteration of body pigmentation. Physiological color changes involve alterations on appropriate stimulation in the degree of dispersion of pigment granules in chroinatophores. The generally accepted view is that the chromatophores of all groups, with the exception of the cephalopods, have a fixed cell outline and that the pigment migrates into and out of the processes (Matthews, 1931 ; Brown, 1935a). The older, alternative view which states that chromatophores are ameboid is still held by a few investigators, however. The shade of an organism depends on the number of chromatophores and the nature of the pigment or pigments each cell contains in addition to the degree of dispersion of the contained pigment. Early workers simply described the shade of an organism in macroscopic terms such as dark, intermediate, or light. Obviously for quantitative work another system had to be found. Several have been tried. The most successful and most popular is that of Hogben and Slome (1931) , who divided the entire range from maximum concentration of pigment to maximum dispersion into five stages. According to their scheme stage 1 represents the most concentrated condition of the pigment, stage 5 the most dispersed, and stages 2, 3, and 4 the intermediate conditions. The obvious advantages of this system are that (1) it allows direct, accurate observation of individual chromatophores and (2) it facilitates graphic representation of the changes of the degree of pigment dispersion. The photoelectric method of Hill et ul. (1935), whereby the fraction of incident light reflected from a unit area of skin surface is measured, was devised to eliminate the subjective aspect of the method of Hogben and Slome. The obvious defect in the photoelectric method is that the amount of reflected light depends on both the degree of pigment dispersion and the number of chromatophores. Furthermore, in the same piece of skin one pigment may disperse and another concentrate, but the photoelectric method reveals only the net change in light absorption by the pigmented surface and not the changes in the individual chromatophores. Physiological color changes may be evoked by a number of factors ; the most important are light and temperature. Responses of chromatophores to light may be divided into two categories, primary and secondary. Primary color changes typically occur through routes other than the eyes, i.e. by direct action of light on the chromatophores, the more common

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type, or through an extraocular reflex. Primary responses, although typically associated with larval or embryonic pigment cells that become fully functional before the eyes are operative, may be elicited by chromatophores of adults. Secondary responses depend on the nature of the background and not on the quantity of light impinging on the eye; the degree of pigment dispersion is determined by the ratio of the amount of light directly incident on the eye to the quantity of light impinging on the eye after reflection from the background. German investigators called this ratio “the albedo.” In most adults the primary response is dominated by the secondary one. Waring and Landgrebe (1950) revised the classification of responses of chromatophores to light as follows : (1) an uncoordinated nonvisual or dermal response which is independent of the eyes, central nervous system, and the pituitary, so that the chromatophores almost certainly behave as independent effectors ; (2) a coordinated nonvisual response which is independent of the eyes but involves either nervous or pituitary coordination between a stimulus received by some receptor other than the pigment cells and the chromatophores themselves ; ( 3 ) a secondary ocular response which results in melanin dispersion in specimens on a black background; and (4) a tertiary ocular response which results in melanin concentration when specimens are on a white background. The first two categories are merely subdivisions of the classical primary response, and the last two are subdivisions of the classical secondary response. In the opinion of this reviewer, division of the classical secondary response is unwarranted, and, furthermore, the restricted definition of the term “secondary response” proposed by Waring and Landgrebe can lead only to confusion in the literature.

111. FUNCTIONAL SIGNIFICANCE OF COLORCHANGES The three common functions of color changes are protective coloration, therrnoregulation, and displays associated with mating. Dispersion of dark pigments when an organism is put on a dark background obviously better adapts an organism for both aggression and survival. For example, Sumner (1935) demonstrated the value of protective coloration in the mosquito fish, Gumbusia patruelis. H e placed pale and dark fish into black and into white tanks and found that predatory birds captured a smaller percentage of the light fish in a white container than the dark ones and fewer dark fish in a black container than light ones. Concomitant with the ability to change coloration for protection of the individual should be the ability to select the background that would assure maximum protection. Evidence along this line was presented by Brown and Thompson (1937), who showed that in eight species of fresh-water

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fishes those adapted to a black background tended to select black more frequently than the fishes adapted to a white background. The rate of change of choice after a change of background in the silver-mouthed minnow, Ericymba buccata, was approximately the same as the rate of change in skin coloration. Brown (1939) showed that the crayfish, Orconectes immunis, also has the ability to select between black and white backgrounds. Primary responses of chromatophores to light have been noticed in a wide variety of invertebrates and vertebrates. Quite often these responses are intimately associated with responses to temperature interpreted as thermoregulatory. Brown and Sandeen ( 1948) determined the responses of the black and white chromatophores of the fiddler crab, Uca pugilator, to light and temperature. Both pigments dispersed with increased illumination and showed a temperature response. The black pigment tended to concentrate as the temperature was raised above or lowered below about 15°C. The white pigment, on the other hand, tended to disperse as the temperature increased above or decreased below about 20°C. The responses to high illumination and temperature may be thermoregulatory for protection of the protoplasm of the crabs. The optimal condition of the chromatophore system to produce dissipation of light and heat when the temperature is high would be, as is the actual case, dispersion of white pigment and concentration of black. Concentration of the black pigment tends to diminish the surface area that absorbs light, but dispersion of the white tends to increase the area that reflects it. Pautsch (1952) demonstrated that the chromatophores of the zoea of the shrimp, Crangm crangm, exhibit only a primary response; the pigment disperses with increased illumination. The responses of the chromatophores of the grasshopper, Kosciuscola tristis, to temperature were described by Key and Day (1954). Near 15°C. the grasshopper is a dull black, and above 25°C. a bright green with intermediate tints at the intermediate temperatures. O n clear days the insects become pale 2 to 3 hours after sunrise and then begin to turn dark again in the late afternoon. This color change is probably thermoregulatory, permitting the grasshopper to minimize the heating effects of the midday sun. Fingerman and Tinkle (1956) studied the responses of the white chromatophores of two species of prawns, Palumonetes pugio and Palaemonetes paladosus, to light and temperature. The responses of both species were qualitatively similar. The white pigment dispersed with increase in total illumination but concentrated with increased temperature. If the temperature responses of the white pigment were thermoregulatory, then with increase in temperature the white pigment should disperse rather

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than concentrate in order to increase the area of the body surface that is able to reflect light and heat efficiently. Since heat and bright light are usually concomitant in nature, as in sunlight, the antagonistic responses to light and temperature may be a mechanism to maintain a steady state of the white chromatophores. Melanin of the blue crab, Cdlinectes sapidus, also shows a response to total illumination. No response to light between 1 and 120 foot-candles was apparent, but between 120 and 3000 foot-candles the melanin dispersed (Fingerman, 1956a). As the temperature increased from 10" to 28"C., the melanin of Cdlinectes concentrated. These responses may be thermoregulatory. The tendency of melanin to concentrate with increased temperature, thereby reducing the light-absorbing area, may be a primitive attempt at homoiothermism. Furthermore, the antagonistic responses to light and temperature tend to maintain the pigment dispersion at a steady state, which may be important in protective coloration. I n spite of fluctuations in light and temperature the degree of pigment dispersion in animals on a particular background would remain nearly constant. The older literature on thermoregulatory use of melanophores by vertebrates is well known. At low temperatures melanin disperses so that more radiant energy is absorbed by the dark skin and then the pigment concentrates as the body temperature rises, the same situation as described above for the grasshopper. Further support of the thermoregulatory concept of chromatophores was offered by Deanin and Steggerda (1948), who demonstrated spectrophotometrically that more light is reflected from the skin of a frog with concentrated black pigment as a result of adaptation to a white background than from skin with dispersed melanin as a result of having kept the donor on a black background. These investigators also showed that more light whose wavelengths are at the red end of the spectrum than at the violet end is reflected from the skin of frogs on black and on white backgrounds. This seems significant in view of the greater heating capacity of the red rays than of the violet rays. Edgren (1954) showed that the tree frog, Hyla versicolor, was dark at low temperatures, 3" to 5 "C., and lightened with increased temperature. Secondary responses to light have been described among a wide variety of animals. Some species have the ability to mimic colored backgrounds as well as shades of gray, thereby demonstrating a concomitant ability to discriminate colors independent of intensity. A demonstration of this ability was shown by Kuhn (1950) for the cephalopods Sepia oficinalis and Octopus vulgaris. Both species possess black, yellow, and orange chromatophores in addition to iridophores. Brown and Sandeen (1948) showed that in the fiddler crab, Uca pug&

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lator, a specific background (albedo) response operates so that when specimens are on a white background the black pigment is more concentrated and the white pigment more dispersed than the corresponding pigments in crabs on a black background. This species also shows a 24-hour rhythm of color change, however, manifested by concentration of black and white pigments at night and dispersion during the day. This rhythm is the primary determinant of the coloration of the fiddler crab. The albedo response itself does not produce enough background adaptation to produce sufficient obliterative coloration that could have survival value. Brown (1950) found that the red pigment of this same crab exhibited extensive responses to background and is, therefore, better adapted for alteration of the shade of body in accordance with the background than are the black and white pigments. The red pigment dispersed in specimens on a black background and concentrated in those on a white background. The black and the red chromatophores of the blue crab, Callinectes sapidus, also show a specific background response (Fingerman, 1956a). Both pigments were more concentrated in crabs in a white pan than in specimens in a black container. The blue crab also shows a 24-hour rhythm of color change; both pigments are more dispersed by day than by night. In contrast to the situation observed in the fiddler crab, in the blue crab the albedo response and 24-hour rhythm contribute equally to the coloration of the organism. Furthermore, in the blue crab the changes in degree of dispersion of the black pigment observed when crabs were changed from black to white backgrounds and back again were greater than the changes of the red pigment, the reverse of the situation in Uca pugilator. The pigment in the white chromatophores of Palaemonetes pugw and Palaemonetes paladosus also responds to background. Dispersion occurs when specimens are placed on a white background and concentration on a black one (Fingerman and Tinkle, 1956). Fingerman ( 1957a) showed that in the dwarf crayfish, Cambarellus shufeldti, the red pigment dispersed maximally and the white pigment concentrated maximally when the specimens were placed on a black background at 22" to 28°C. The pigmentary states reversed themselves when specimens were on a white background. The white pigment of the crayfish, Orconectes clypeatus, showed the same background responses as that of Cambarellus, but the red pigment behaved differently (Fingerman, 1957b). The red pigment of Orconectes would disperse maximally when specimens were put in a black container but only concentrated to an intermediate state (stage 3 ) when Orconectes were on a white background. Maximal red pigment concentration did not occur in specimens kept on a white background for 32 days. In vertebrates secondary color changes characterize the later larval

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stages and the entire adult period. The organisms are typically dark on a dark background and light on a white one. The most striking secondary responses of all the vertebrates are shown by fishes. For example, in Ericymba buccuta the average diameter of the melanin masses varies in a directly proportional fashion with the ratio of incident to reflected light (Brown, 1936). Strong background responses have also been observed in the elasmobranch, Scyllium canicula, by Waring (1938). Adaptation to background in both vertebrates and invertebrates depends on a spatial separation within the retina of receptor elements, stimulation of which produces either a lightening or a darkening response (Smith, 1938 ; Hogben and Landgrebe, 1940). Smith ( 1938) showed, on the basis of experiments in which different portions of the eyes of the isopod Ligia oceu-nica were covered with an opaque material or stimulated differentially, that stimulation of the dorsal portion of the retina resulted in melanin dispersion and stimulation of the ventral and lateral portions resulted in blanching. Essentially the same conclusions were arrived at by Hogben and Landgrebe ( 1940) for the stickleback, Gmterosteus muleatus. Photoreceptors concerned with black background responses were in the floor of the retina below the optic nerve ; those associated with the white background response were in the center of the retina above and below the blind spot. Presumably, since the upper portion of the retina of specimens on a white background is stimulated by reflected light much more than it is when specimens are on a black background, the lightening response is called forth because the upper portion then receives adequate stimulation from the background. Color changes associated with mating have not received much attention. Hadley (1929) showed that male lizards, Anolis porcatus, show a striking change from green to brown while pairing with a female. The significance of this color change is unknown. One possibility is that this color change is used to drive off other males.

IV. CHROMATOPHORES OF ARTHROPODS Color changes in arthropods, among the more striking in the animal kingdom, are due to chromatophores which lie among or proximal to the hypodermal cells as well as among the deeper organs. Migration of pigment within these unicellular chromatophores in response to background is controlled by hormones alone. A . Sources of Chromatophorotropins The information concerning the origin of chromatophorotropins in insects is meager when compared with that for the crustaceans. Early workers were able to localize the source to the head but to no specific structure

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(Janda, 1934, 1936). Teissier ( 1947), Kopenec ( 1949), and DupontRaabe (1950) showed that the supraesophageal ganglia of several insects produced a chromatophorotropin. A slight chromatophorotropic effect was shown by extracts of the corpora cardiaca, an observation that might now be expected, since Scharrer later (1952b) showed that in the cockroach, Leucophaea maderue, the pars intercerebralis of the brain and the corpora cardiaca form a functionally related group of neuroglandular organs in which the corpus cardiacum serves as a reservoir for neurosecretory material from the brain. In crustaceans the situation is much more complicated than in the insects, since chromatophorotropins are found in the sinus gland, the optic ganglia, and the remaining central nervous organs. Much interest has been centered recently on the histology and cytology of these structures, and, as a result, considerable information has accumulated. In spite of the abundant literature, however, little information correlating morphology with physiology is available. The structures in the eyestalks that have received considerable attention are the sinus gland and the X organ of the medulla terminalis. The former structure was first described by Hanstrom (1933) and since then has been the subject of considerable interest. Originally the sinus gland was considered the source of the chromatophorotropins in the eyestalk (Hanstrom, 1937). The portion of the eyestalk that contained the sinus gland always contained most of the chromatophorotropic activity ; eyestalks from forms whose sinus glands were located in the head near the supraesophageal ganglia (e.g., Emerita tulpoidu) were chromatophorotropically inactive. Brown (1940) also found that virtually all the activity of the eyestalks of the prawn, Palaemonetes vulgaris, was in the sinus gland. Several investigators in the early 1950's began working on several phases of crustacean endocrinology with specimens whose sinus glands alone had been removed from the eyestalks (Knowles, 1950, 1952; Bliss, 1951; Frost et al., 1951 ; Have1 and Kleinholz, 1951 ; Passano, 1951a, b ; Travis, 1951 ; Welsh, 1951). The effects of sinus gland ablation were not the same as those after eyestalk removal. The evidence suggested that the sinus gland is merely a storage and release center for neurosecretory material produced elsewhere in the eyestalk. This concept is the only tenable explanation of studies of molting in eyestalkless and sinus glandless fiddler crabs (Passano, 1953). Likewise, what is known about chromatophorotropins in the eyestalks falls into line with this hypothesis. The sinus gland receives many axons from the X organ of the medulla terminalis (Bliss, 1951; Bliss and Welsh, 1952 ; Enami, 195l a ; Passano, 1951a, b). The material found in the sinus gland is considered by these

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authors to originate in the neurosecretory cells of the medulla terminalis X organ and to migrate along axons to the sinus gland where it is stored. The staining properties of the materials change as they move along the axon from the point of origin to the point of storage. So far the problem has not been solved as to whether the staining properties of the hormonal materials change or those of a carrier material. No histological evidence has appeared to show beyond doubt that the sinus gland itself produces a hormone. Chromatophorotropins, in addition to being found in the eyestalk, can be extracted from the supraesophageal ganglia, circumesophageal connectives, and thoracic and abdominal nerve cords. Brown (1933) supplied the first clear-cut evidence of this when he found that extracts of central nervous organs of the prawn, Palmmowtes vulgaris, concentrated the dispersed red pigment of eyestalkless specimens. Since then the nervous systems of numerous crustaceans have been shown to be sources of chromatophorotropins. Enami (1951a, b) described the histology and cytology of the sinus gland, optic ganglia, and supraesophageal ganglia of three species of the crab, Sesarma. Knowles (1953) described the structure of the tritocerebral commissure and postcommissure organs in the shrimp, Penaeus brasiliensis, and the prawn, Lea d er serratus. Matsumoto (1954a, b) described the cell types in the thoracic ganglion of the fresh-water grapsoid crab, Eriocheir japonicus. Durand ( 1956) described the neurosecretory cell types in the eyestalks and supraesophageal ganglia of the crayfish, Orconectes wirilis. Enami, Matsumoto, and Knowles correlated the distribution of neurosecretory cells with the presence of chromatophorotropins.

B . Crustaceans

1. Isopods. The fact that isopods have functional melanophores has been known for many years. In several species background responses and 24-hour rhythms of pigment migration evidenced by melanin dispersion by day and concentration by night have been observed. Kleinholz (1937) found that Ligia baudinima showed a background response when kept in black and in white containers and, when the isopods were in darkness, a 24-hour rhythm of color change. Injection of aqueous extracts of heads into dark specimens brought about melanin concentration. Smith (1938), by an ingenious series of experiments involving differential rates of background adaptation, showed that Ligia oceunica must have hormones that disperse and concentrate melanin. Enami (1941a) found that Ligia exotica shows striking background changes as well as a 24-hour rhythm of color change. The rhythm is exhibited by specimens in constant light and in darkness. The animals

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were palest about midnight and then rapidly darkened to a maximum about 6 A.M. The melanin then gradually concentrated throughout the day. Enami (1941b) found that an extract of the head of Ligia exotica caused definite melanin dispersion when injected into light specimens. The same extract injected into specimens on a black background caused a slight transitory concentration of melanin. Nagano (1949) also studied the pigmentary system of Ligia exotica and found, in contrast to the results of Enami (1941b), that the response to extracts of heads was pigment concentration alone ; no dispersion was apparent. Fingerman ( 1956b) reinvestigated the chromatophore system of Ligia exotica because of the conflicting results of Enami (1941a) and Nagano (1949). The results of Fingerman agreed with those of Enami. Ligia exotica appears to be an exception among isopods that have been investigated ; in all other species of isopods head extracts primarily concentrate rather than disperse melanin. Extracts of sinus glands and central nervous organs dispersed melanin (Fingerman, 1956b). In addition, the rapid background responses and 24-hour rhythm of color change described by Enami were confirmed. In a series of papers Okay (1943, 1945a, b, 1946) reported the results of his observations of the chromatophore system of Sphueromu serratwm, Idothea baltica, Ligia italic&, Tylos latreilli, and Armadillidium granulatum. Extracts of their heads induced melanin concentration. Heads of the latter two species, however, were not capable of causing pigment concentration until they were boiled. Carstam and Suneson (1949) found that extracts of heads of Idothea neglecta concentrated melanin in Idothea. In addition these extracts injected into specimens of L e a d e r adsterslls on a white background dispersed the pigment in red chromatophores but dispersed only the yellow pigment in the red-yellow ones. Furthermore, when these extracts were injected into eyestalkless Leander the red pigment in the yellow-red chromatophores all over the body concentrated and the pigment in the red chromatophores in the carapace, but not in the abdomen, concentrated. Trachelipus rathkei, a terrestrial isopod, showed weak physiological color changes in response to changes in background ( McWhinnie and Sweeney, 1955). As is typical of isopods the melanophores of Trachelipus showed a primary response to light ; the melanin dispersed further with increased illumination. Extracts of the sinus glands of Trachelipus which are located at the distal end of each optic tract dispersed the red pigment of a crayfish, Cambarus sp. Extracts of the optic tracts and the cerebral ganglia induced weak dispersion of the red pigment of Cambarus, whereas extracts of the circumesophageal connectives or any segment of the thoracic cord induced strong concentration of the same pigment. Responses of Trachelipus to

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injected extracts were inconclusive but did suggest that the reactions of the pigmentary system of Truchelipus are opposite to those of Cambarus. The best possible explanation of all the results summarized above is that isopods contain two chromatophorotropically active substances, pigment-dispersing and pigment-concentrating, and that the relative amount of each varies from organ to organ and from species to species so that in some instances the obvious effect of extracts is melanin concentration and in others melanin dispersion. The same situation appears to be true among the higher crustaceans. 2. Bruchyurans. Crustaceans were divided by Brown (1948a) into three groups on the basis of the response of their dark chromatophoral pigments to eyestalk removal. One group, exemplified by Pahmonetes, included the majority of the Mysidacea, Natantia, and Astacura. The dominant dark pigments of these animals disperse widely, resulting in a darkened body. The second type is characterized by Crago. Eyestalkless specimens show an intermediate mottled coloration. Some of the dark chromatophores have their pigment broadly dispersed, others are in an intermediate condition, and still others have theirs fully concentrated. The third type of response, exemplified by the crab Uca, was exhibited by all the Brachyura (true crabs) that had been investigated at that time. Eyestalk removal in these forms yielded a permanently pale condition of the body due to maximum concentration of the dominant dark pigment. The only reported exception to the last category was reported by Enami (1951b), who found that removal of both eyestalks from specimens of three species of crab, S e s a m intermedia, S. h m a t o c h e i r , and S. d e b n i , resulted in permanent dispersion of their dark pigments. Enami’s work will be discussed further below, but first some of the work with crabs that are in accord with Brown’s scheme will be considered. One of the early investigators, Carlson (1935), showed that removal of both eyestalks from the fiddler crabs Uca pugnux and Uca pugilator resulted in blanching, and injection of extracts of the eyestalk resulted in melanin dispersion, darkening. Sandeen ( 1950) determined the actions and distribution of chromatophorotropins in the fiddler crab, Uca pqihtor. She found two chromatophorotropins, one that disperses black pigment and another that concentrates white pigment, but no evidence of a black pigment-concentrating substance. These substances appeared to be mutually antagonistic to each other such that the presence of a large amount of one decreased the expression of the other. Enami (1943) studied the pigmentary system of the fiddler crab, Uca dubia, and reported that eyestalk removal resulted in blanching just as in other species of Uca. Surprisingly, he also reported that the sinus gland and the central nervous

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system contained a black pigment-concentrating hormone but no black pigment disperser. The results of Enami are subject to criticism because he did not show the results of injections of saline as a control. The reviewer has observed (unpublished) that injection of saline into Uca pugilator will have as great a pigment-concentrating effect as the extracts used by Enami. Fingerman ( 1 9 5 6 ~ ) considered the problem of the existence of a melanin-concentrating substance in fiddler crabs, since ( 1) the results of Enami (1943) are subject to criticism, and (2) Sandeen (1950) had found no evidence for such a substance in Uca pugihtor. Injection of extracts of known sources of chromatophorotropins of Uca pugihtor into specimens with maximally dispersed melanin causes no greater pigment-concentrating effect than does saline. By perfusion of isolated legs with blood, Fingerman was able to demonstrate the presence of (1) a black pigment-dispersing hormone in the blood of Uca pugiiator that had maximally dispersed melanin and (2) a black pigment-concentrating hormone in the blood of crabs with maximally concentrated melanin. Maximally dispersed melanin in the chromatophores of a leg of Uca will gradually concentrate when the leg is removed from the body. The rate of melanin concentration was slowed by perfusion of blood from dark specimens and increased by blood from specimens whose black pigment was maximally concentrated. Control legs were perfused with sea water. These results could not have been obtained unless a lightening factor were in the blood of the pale Uca and a darkening factor in the blood of the dark crabs. As is becoming evident with more investigation, the chromatophores of all crustaceans appear to be controlled by pigment-dispersing and pigment-concentrating substances. Brown (1950) found that eyestalk removal resulted in maximal concentration of red pigment in Uca pugihtor. Extracts of the sinus glands and all the major parts of the central nervous system contained two materials, one that dispersed red pigment and another that concentrated it. The red pigment-concentrating principle dominated the response when high concentrations of the antagonists were present concurrently. The red pigment-concentrating activity of each extract was of much shorter duration than the red pigment-dispersing activity ; the latter often lasted three to four times as long as the ,former. Brown and Fingerman (1951) showed by extraction of supraesophageal ganglia with absolute isopropyl alcohol that the black and red pigment-dispersing substances in Uca pugilat,or were not identical. The alcohol-soluble fraction had much black-dispersing but little red-dispersing activity. The reverse was true of the alcohol-insoluble fraction. Fingerman and Fitzpatrick (1956) showed that the pigment in the

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melanophores of female specimens of Uca pugilutor was more dispersed than the melanin in males. Removal of the large chela from the male resulted in approximately equal coloration of the males and females. Furthermore, the greater the number of appendages removed from both sexes, the darker were the crabs. Presumably, removal of the appendages resulted in a decreased circulatory space so that the melanin-dispersing hormone could not become as diluted as in intact crabs. Perez-Gonzilez ( 1957) presented evidence that the black pigmentdispersing hormone in the sinus gland of Uca pugilator is contained within granules that possess a semipermeable membrane. The release of hormone is facilitated when the granules are placed in hypotonic media, boiled, exposed to detergents, or frozen and then thawed. Bowman (1949) found that the sinus gland of the grapsoid shore crab, Hemigrapsus oregonensis, was a more potent source of a material that dispersed melanin than were the optic ganglia. H e also stated that this substance is “possibly in the brain and thoracic mass of ganglia.” The fact that Bowman did not show this conclusively is surprising, since the supraesophageal and the thoracic ganglia of all other brachyurans that have been investigated contain chromatophorotropins. The probable explanation for this deficiency is the small quantity of supraesophageal and thoracic tissue assayed. The volume was equal to one-half a sinus gland of Hemigrapsus. This minute amount of nervous tissue obviously could not evoke a strong chromatophorotropic response, since the hormones are much less concentrated in these tissues than in the sinus gland. Matsumoto ( 1954b) studied the endocrine control of the chromatophores of the fresh-water crab, Erwcheir japonicus. Melanin of eyestalkless crabs was maximally concentrated, but the red pigment was maximally dispersed, the reverse of the condition of red pigment in eyestalkless fiddler crabs. The supraesophageal ganglia, thoracic ganglia, and eyestalks of Eriocheir contained a material that dispersed black pigment. Extracts of the thoracic ganglia and eyestalks concentrated red pigment. Matsumoto ( 1954b) had determined the types and distribution of neurosecretory cells in the thoracic ganglion of Erwcheir. The distribution of black pigment-dispersing and red pigment-concentrating hormones correlated with the distribution and frequency of one cell type that he called “B-cells.” The physiology of the black and the red chromatophores of the blue crab, Cdlinectes sopidus, was determined by Fingerman ( 1956a). His results agree essentially with those of Matsumoto (1954b) with Eriocheir. After removal of the eyestalks from blue crabs the black pigment concentrates maximally and the red pigment disperses maximally. The sinus glands, optic ganglia, supraesophageal ganglia, circumesophageal connec-

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tives, and thoracic ganglia contained materials that dispersed black pigment and concentrated red pigment. An antagonism was apparent between these two substances such that a large amount of one principle decreased the expression of the other. Comparison of the order of decreasing potency of the sinus gland, optic ganglia, supraesophageal ganglia, circumesophageal connectives, and the thoracic ganglia of Callinectes on both black and red chromatophores with the activities of similar tissues of crabs reported in the literature showed no clearly defined trends. Determination of the alcohol solubility of the chromatophorotropins in the sinus glands and circumesophageal connectives of Callinectes revealed that the black pigment-dispersing hormone from the two sources had to be different in some way, whereas the red pigment-concentrating hormone from the two sources had the same alcohol solubility. The black pigment-dispersing hormone of the circumesophageal connectives was much less soluble in alcohol than the hormone in the sinus gland with the same function. Enami (1951b) described the chromatophore system of three species of Sesarma with special emphasis on S. hamaatockir. Removal of the eyestalks resulted in a blanching of short duration followed by permanent darkening ; the black pigment was maximally dispersed, the white nearly maximally concentrated, and the red and vermilion pigments were in an intermediate condition. The behavior of these pigments is obviously quite different from those in Uca. Extracts of the sinus glands and central nervous organs, including the optic ganglia, concentrated the red and vermilion pigments. Extracts of the supraesophageal ganglia and medulla terminalis of adults concentrated the black pigment and dispersed the white. Evidently, at least two chromatophorotropins are present. Knowles and Carlisle (1956) stated in their review that Enami (1951b) extracted the medulla terminalis ganglionic X organ separately and found that it contained one chromatophorotropin only. This reviewer can find no support for this statement in the original paper of Enami. Rhythms of color change have been described in more detail for crabs than for any other group of animals. These cycles are probably due to rhythmical release of chromatophorotropins from the sinus glands and central nervous organs and for this reason will be considered in some detail in this review. The mechanism whereby these cycles are maintained is still far from understood (Fingerman, 1957c ; Stephens, 1957a). Abramowitz (1937) first described the 24-hour rhythm of color change in Uca. The crab was dark by day and pale by night. Brown and Webb (1948) found that the rhythm persisted for as long as 30 days in constant darkness and that the frequency was unaffected by temperatures from 6" to 26"C., but the amplitude decreased with decrease in temperature. When

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the rate of metabolic processes was greatly reduced by temperatures at 0" to 3"C., the 24-hour rhythm of Uca pugnax was delayed by an interval closely approximating the time of exposure to the low temperature. The same authors (Brown and Webb, 1949) analyzed the 24-hour rhythm of Uca pugnux further and found that (1) the phases of the chromatophore rhythm could be reversed by illuminating the animals at night and keeping them dark by day, (2) exposure to 6-hour periods of illumination alternating with 6 hours of darkness resulted in a 24-hour rhythm that was 6 hours out of phase with solar day-night, and (3) such a rhythm, 6 hours out of phase, may persist for several days in constant darkness and then gradually return to the previously established rhythm if the last period of illumination occurred when the animals were entering the night phase, but the shifted rhythm showed no persistence when the last period of illumination occurred when the animals were entering the day phase of the rhythm. Further analysis of the results led to the adoption of an hypothesis involving two centers of rhythmicity in the animal, each one capable of having its rhythm altered independently of the other, and with one of the centers influencing the second, which is in turn responsible for the rhythmical release of chromatophorotropins. Webb (1950) showed that the basic 24-hour frequency of the rhythm of color change in Uca pugnax could not be altered by exposure to alternating light and dark periods of 16 hours each. She also clarified further the mechanism underlying phase shifts by light and dark changes. Brown and Stephens (1951) found that changes in the length of photoperiod induced persistent changes in the amplitude of the 24-hour cycle of Uca pugnax. The greater the photoperiod, the greater is the amplitude of the rhythm. These authors also postulated two centers of rhythmicity controlling the melanophores, quite similar to the hypothesis of Brown and Webb (1949). Brown and Stephens (1951) postulated that one of the two centers would respond to darkness by calling for the secretion of a black pigment-concentrating substance and to light by evoking release of a black pigment-dispersing hormone. Brown and Hines (1952) showed that the amplitude of the 24-hour rhythm of Uca pugnux exposed to constant illumination varies inversely with the intensity of illumination. The reduction in amplitude at low intensities was primarily the result of a decrease in the amount of melanin concentration that occurred during the night phase of the rhythm. Brown et al. (1954a) studied further the mechanism involved in shifting the phases of the persistent 24-hour rhythm of color change in Uca pugnux by subjecting crabs to a series of combinations of brighter illumination by night and dimmer illumination by day. A graded series in the amount

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of shift of the phase of the rhythm with respect to solar day-night was obtained that was capable of being interpreted in terms of two operating factors: (1) the strength of the stimulus in the form of the dark to light change and (2) the absolute brightness of the higher illumination. Webb et al. (1954) found a 24-hour rhythm in eyestalkless Uca pugilator which they suggested might be due to cyclically varying quantities of black pigment-concentrating hormone in the blood. Extracts of eyestalks were less effective in dispersing black pigment at night than during the daytime, presumably because of rhythmical release of more black pigment-concentrating hormone at night than by day, since during the nighttime melanin of intact specimens is concentrated. Stephens ( 1957b) showed that the 24-hour melanophore rhythm of Uca pugnax can be shifted out of phase with solar day-night cycles by exposing animals maintained in darkness to temperatures between 9.5" and 18°C. during the summer. The amount of the shift appeared to depend on the time of day the crabs were first exposed to the lower temperature and also on the time of day they were warmed. Persistent tidal and semilunar rhythms of color change have been described in three species of fiddler crabs: in Uca pugnux by Brown et al. (1953), in Uca pugilator and Uca specwsa by Fingerman (1956d), and in the blue crab, Cdlinectes sapidus, by Fingerman (1955). These crabs darkened by day and lightened by night in accordance with their 24-hour rhythm of color change. The tidal rhythm of color change in Uca pugnax collected near Woods Hole, Massachusetts, was evidenced by a supplementary dispersion of melanin near the time of low tide, which was superimposed on the 24-hour rhythm and progressed across the latter rhythm at the tidal rate of 48.8 minutes per day. The frequency of the tidal cycle was 12.4 hours. By virtue of possessing rhythms with both 12.4- and 24.0-hour frequencies, the crabs also possessed a 14.8-day cycle, the interval between days on which these two rhythms repeated similar time relations to one another. No loss of synchrony of the tidal rhythm of color change of fiddler crabs in the laboratory was evident when compared with animals on the beach still subject to the rhythmic tidal changes. To determine whether the two rhythmic mechanisms, 24-hour and tidal, were completely independent of each other or were in some way associated, the phases of the 24-hour rhythm were shifted abruptly backward by three consecutive midnight-to-6 A.M. periods of illumination. Analysis of the 24-hour and tidal rhythms of the specimens of Uca pugnux revealed that, with reference to a control group kept in constant darkness, the 24-hour rhythm had been shifted backward 4.9 hours and the tidal rhythm 4.6 hours. The tidal rhythm, therefore, appeared to be functionally associated

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with the 24-hour rhythm, because shifting the latter produced corresponding shifts in the phases of the tidal rhythm. The melanophores on the legs of Uca p u g w collected near Woods Hole tend to concentrate their pigment after autotomy during the daytime (Hines, 1954). The degree of melanin dispersion determined 30 minutes after autotomy reflected both a 24-hour and a tidal rhythm. Relatively little melanin concentration occurred after autotomy near the time of low tide. More concentration was observed when legs were isolated near the time of high tide during the daytime. Just as is true of the 24-hour rhythm, the endogenous tidal rhythm of color change of Uca pugnax is temperature-independent between 13" and 30°C. (Brown et al., 1954b). Brown et al. (1955a) presented evidence for a reversible influence of cosmic ray showers on the chromatophore system of Uca pugnax. Increased concentrations of cosmic ray showers resulted in increased pigment concentration during the initiation of transition into the day phase of the 24-hour cycle and increased melanin dispersion during at least most of the remaining hours of the day. No evidence has been presented as yet that the capacity to exhibit a response to alterations in intensity of cosmic ray showers is in any way normally operative in the maintenance of the precise 24-hour cycles of color change. Evidence for an endogenous component of a lunar rhythm of color change has been presented for the fiddler crab, Uca pugnax, by Brown et ul. (1955b). Fiddler crabs were transported from Woods Hole to Berkeley, California, within a 24-hour period. When data from Uca in California were compared with the data for Uca still in Woods Hole, there appeared to be no tendency for the cycles of the crabs in California to drift away from the controls in Woods Hole. The crabs were able to mark off quite accurately periods of solar and lunar day-lengths. The persistent tidal and semilunar rhythms of color change in the blue crab, Callinectes sapidus, were similar to those of Uca pugnax. The Callinectes, however, were collected in a region of diurnal tides (Lake Pontchartrain, Louisiana), whereas the Uca pugnax were collected in a region of semidiurnal tides (Woods Hole). The tidal rhythm of Callinectes had a 12.4-hour frequency just as the tidal rhythm of Uca pugnax. The time between successive low tides in a region with diurnal tides is 24.8 hours, however. Evidently the center of tidal rhythmicity in Callinectes operates solely on the basis of tides spaced 12.4 hours apart, independent of the nature of the tides, high or low. The tidal rhythm of Uca pugnax is set to exert its maximal effect near the times of low tide, whereas the blue crabs showed no difference between their rhythmical responses at times of high and low tides.

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The tidal rhythms of color change of Uca pugilator and Uca specwsa were observed in specimens collected on different portions of the beach at Ocean Springs, Mississippi, where the tides are diurnal (Fingerman, 1956d). Analysis of the tidal rhythms of both species revealed that the Uca speciosa behaved as if low tide occurred for them 7.5 hours earlier in the day than low tide for the Uca pugilator. The phase difference between the two species was explained according to the following hypothesis. As the water begins to recede after a high tide, in effect low tide occurs earlier for the Uca speciosa in their burrows among the marsh grass than for the Uca pugilator in their burrows closer to the actual low-tide mark. The Uca speciosa would, therefore, be free to leave their burrows and feed earlier than the species living in the sand, thus accounting for their tidal rhythm’s being set 7.5 hours earlier. Actual measurement of the beach revealed that the water should require 4.9 hours to move from one set of burrows to the next. Recently the hypothesis of Fingerman (1956d) that the phase difference between the tidal rhythms of Uca pugilator and Uca specwsa at Ocean Springs was due to the time for the receding water to pass from one set of burrows to the next was tested (Fingerman, 1957d). Specimens of Uca pugilator were collected at Ocean Springs from two isolated groups of burrows that were different distances from the high-tide mark. Measurements of the beach revealed that the receding water should reach the lower burrows 1.6 hours after the set of burrows closer to the high-tide mark began to uncover. Observation of the color changes of both groups of Uca pugilator in the laboratory revealed that the crabs from the burrows closer to the low-tide mark behaved as if low tide occurred for them 1.6 hours later in the day than for the fiddler crabs from the burrows higher on the beach. The phase difference observed by Fingerman (1956d) was evidently, therefore, not merely a species difference. Fingerman et al. (1958) continued the analysis of the tidal rhythm of color change in Uca pugilator and showed that the phase differences were additive. The phase differences found previously in the laboratory with crabs from Ocean Springs were 7.5 and 1.6 hours, the sum being 9.1 hours. Crabs collected from the two extreme sets of burrows showed in the laboratory a phase difference of 9.8 hours. Actual observation of the movement of the water on the beach showed that the set of burrows closer to the low-tide mark began to uncover 7.3 hours later each day than the set near the high-tide mark, and crabs from the low-tide burrows came to the surface 10.1 hours later than crabs from the high-tide burrows. Interestingly, the value observed in the laboratory was between those found in the field. These investigators also recorded the daily pigmentary excur-

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sion of the melanophores of a group of Ucu pugilator living above the high-tide mark where they were not exposed to the rise and fall of the tides. A 24-hour rhythm of color change was apparent, but the overt expression of the tidal rhythm was suppressed, apparently because these crabs were free to leave their burrows at any time, whereas crabs living in the intertidal zone were free to leave only when the burrowing area was uncovered by the receding water. Statistical analysis of the data, however, revealed a semilunar rhythm of color change. 3. Nutantiam. Historically the Natantians are the most important group of crustaceans as far as color changes are concerned, because evidence that background adaptation in crustaceans is under hormonal rather than nervous control was presented first for this suborder (Koller, 1925, 1927, 1928; Perkins, 1928). Koller (1925, 1927) showed that bloodborne substances are involved. H e found that blood from a shrimp, Crago vulgaris, dark as a result of having been maintained on a black background, darkened a light animal kept on a white background. Perkins (1928) then found that eyestalk extracts from the prawn, Pahemmetes vulgaris, would concentrate the dispersed red pigments of eyestalkless specimens. In the same year, Koller (1928) showed that the eyestalks of Crago contained a substance that would blanch this shrimp. Since then the volume of information about the control of the chromatophores of this group has increased tremendously. The physiology of the chromatophores of Crago septemspinosus was described in a series of papers by Brown and Ederstrom (1940), Brown and Wulff (1941), Brown (1946), Brown and Saigh (1946), and Brown and Klotz (1947). Briefly, the sinus gland of Crago contains a taillightening hormone. The tritocerebral commissure of Crago contains two mutually antagonistic hormones, a general darkening one and a bodylightening one. No additional information about the endocrine control of the chromatophores of this genus has appeared recently. Nagano (1943) found that the red pigment of eyestalkless specimens of the shrimp, Paratyu compressa, remained permanently dispersed. Concentration of this pigment was effected by eyestalk extract. Knowles (1952) found that the sinus gland is ineffective on the white pigment of the prawn, L ead er adspersus, whereas extracts of the entire eyestalk concentrated this pigment. Permanent dispersion of the white pigment was apparent in eyestalkless specimens, but this pigment would concentrate and disperse when sinus glandless specimens were placed on black and on white backgrounds, respectively. Knowles (1953, 1954) found that the nervous system and eyestalks of the shrimp, P e w u s brasiliensis, contained two substances, one that concentrated red pigment and another that concentrated white pigment.

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Brown et al. ( 1952) found two antagonistically functioning chroniatophorotropins in the nervous system of P h m o n e t e s vulguris; one concentrated the red pigment and the other dispersed it. The duration of the effect of the concentrating hormone was shorter than that of the dispersing hormone. The sinus gland and tritocerebral commissure contained approximately equal quantities of the pigment concentrating hormone, but the sinus gland contained little or none of the dispersing factor found in extracts of the entire eyestalk. Thus at least one animal from each of the three categories of crustaceans devised by Brown (1948a) on the basis of the condition of the pigment of eyestalkless specimens has been shown to possess a hormone that disperses the dark pigment and one that concentrates it. Panouse (1946) published a paper describing the chromatophore system of Le,mder serratus in which he reported that the sinus gland and central nervous organs each contained a substance that would concentrate red pigment. H e was also able to disperse the red pigment with extracts of the supraesophageal ganglia, but he thought the latter effect was a nonspecific response and not due to a pigment-dispersing hormone. In view of the results of Brown et al. (1952) described above it would appear that Panouse was unduly cautious. He thought the dispersing effect was nonspecific because (1) the degree of dispersion was not as great as the concentrating effect, and (2) the concentrating effect was completed sooner than the dispersing effect. Both statements apply equally well to the red pigment-dispersing hormone of Palaemolzetes. Furthermore, these are the same characteristics shown by the red pigment-dispersing hormone of the dwarf crayfish, Cumbarellus shufeldti, that were reported by Fingerman (1957a). Panouse (1946) also stated that the blue pigment of Leonder sewatus at Roscoff, France, appeared in the chromatophoral branches when the red pigment concentrated and that the blue pigment appeared to be formed from the red pigment. This observation is the same as that of Brown (1934, 1935a, b) concerning the origin of the blue pigment in Pulaemonetes vu2guri.s. In contrast to the observations of Panouse (1946), Scheer and Scheer (1954) reported that specimens of the same species, Leunder serratus, from Naples, Italy, possessed red and blue pigments that were not in the same chromatophores. Both types of chromatophores underwent small but significant cyclical changes in degree of pigment dispersion during the intermolt cycle. Four groups of chromatophores, two red and two blue, were distinguished on the basis of the independence of migration of their contained pigment during the intermolt cycle. The degree of pigment dispersion in each group could be correlated with the duration of

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one or more stages of the intermolt cycle, thereby suggesting that the hormonal factors which control chromatophore pigment dispersion are also concerned in the metabolic processes of the intermolt cycle. The eyestalks and supraesophageal ganglia of Leander serratus contained substances that concentrated the pigments in the four groups of chromatophores. In addition, the eyestalks contained a factor that dispersed the pigment in one group of blue chromatophores. The supraesophageal ganglia contained the latter hormone and one that dispersed the pigment in one type of red chromatophore. The authors postulated that at least five different chromatophorotropins must be present to explain the cyclical color changes. Interestingly enough, eyestalk removal did not completely abolish the cyclical color changes during the intermolt period ; presumably chromatophorotropins from the central nervous system were responsible for these changes. The authors found no response when extracts of eyestalks and supraesophageal ganglia of Lysmuta seticaudata were injected into this species as well as Lea de r serratus. This apparent lack of chromatophorotropins should certainly be verified. The difference between the origin and location of the blue pigment in the specimens from the Roscoff and Naples populations may have arisen as a result of geographical isolation of the populations. A similar problem of geographical diversity has arisen with respect to molting. Carlisle (1954) found that removal of the eyestalks from specimens of L e a d e r serrutus at Plymouth, England, lengthened the intermolt period, whereas Drach (1944) had clearly shown that the intermolt period of specimens of this species collected at Roscoff decreased after eyestalk ablation. With specimens from Naples, Scheer and Scheer (1954) noted that molting was less frequent in eyestalkless than in intact specimens. In an attempt to clear up this conflict Carlisle (1955) showed that populations of Leader serratus from Plymouth, Roscoff, and Concarneau show readily distinguishable, characteristic differences in color pattern. H e does not feel that these populations are subspecies, nor did he find signs of sterility in crossbreeding experiments with members of the three populations. Carstam (1951) showed that the hypodermis of the lateral portions of the carapace of Lea d er adspersus, Cancer pagurus, Momarus prulgaris, and Idothea neglectu contains an enzyme that inactivates the red pigmentconcentrating hormone in the sinus gland of L e a d e r adspersus. This enzyme, which is also present in the hepatopancreas, is destroyed by boiling for 5 minutes. Knowles et ul. ( 1955) investigated the chromatophorotropins of Leander serratus with filter paper electrophoresis. They found in the sinus gland and postcommissure organs a relatively immobile substance that they called the A-substance. It is electropositive at p H 7.8 and concen-

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trates the pigment in the large and small red chromatophores. Another molecule with low mobility, called the B-substance, is electronegative at p H 7.8. It concentrates the pigment in the large red chromatophores but disperses the pigment in the small red chromatophores. The B-substance is found only in the postcommissure organs. Knowles and Carlisle (1956) attempted to identify the A- and B-substances with chromatophorotropins found in other organisms. Such an attempt may be somewhat premature, since we have so little information concerning the number of chromatophorotropins involved in the color-change process in any one form. In experiments of this sort we must face the possibility that two different substances with the same or very similar mobilities and the same charge are present on the filter paper strip and might, therefore, be considered one substance. Such an occurrence may account for the opposite effect of the B-substance on the large and small red chromatophores, whereas the A-substance has one effect on these chromatophores. Knowles (1956) set forth some criteria which he recommends that all investigators use to decide whether a color change is due to the presence of a chromatophorotropin or is a nonspecific response : ( 1 ) Some response should occur within 5 minutes; ( 2 ) the response should last at least 30 minutes; ( 3 ) the response should be one that can be found in normal specimens under normal conditions ; and (4)the injected substance should not be toxic. 4 . Astmuruns. Until recently the Astacurans were among the most neglected crustaceans as far as studies of the physiology of chromatophores are concerned. This fact is quite surprising in view of their relatively large size and world-wide distribution. Brown and Meglitsch (1940) showed that the sinus gland of the crayfish, Orcmectes immunis, contains two chromatophorotropins, white pigment-dispersing and red pigment-concentrating. McVay ( 1942), also working with Orconectes immunis, demonstrated that the central nervous organs were a potent source of red and white pigment-concentrating substances. When the eyestalks were removed, both pigments dispersed maximally. The eyestalks, supraesophageal ganglia, circumesophageal connectives, and thoracic and abdominal nerve cords of the dwarf crayfish, Cambarellus shufeldti, contain at least' four chromatophorotropins ; red pigment-dispersing, red pigment-concentrating, white pigment-dispersing, and white pigment-concentrating (Fingerman, 1957a). Pigment-dispersing and pigment-concentrating hormones can be shown to be present in the same extract by injecting it into two groups of crayfish, one with the pigment under consideration maximally dispersed and the other with this pigment maximally concentrated. The red and white pigments in this crayfish also

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disperse maximally when both eyestalks are removed, which is in agreement with the classification of Brown (1948a), who grouped the Astacurans with the majority of the Natantians. Contrary to his description of the chromatic groups of crustaceans, however, the major effect of injection of eyestalk extract of dwarf crayfish on its red pigment is not concentration but dispersion. The eyestalks contain only a very small quantity of red pigment-concentrating hormone relative to the amount of dispersing hormone. The circumesophageal connectives contained more red pigment-concentrating hormone than any other nervous tissue examined, which was also true in Palaemonetes, as shown by Brown et al. ( 1952). In order to learn something of the “resting states” of the red and white pigments, portions of the carapace with the associated chromatophores were removed from crayfish and placed in physiological saline, thereby removing the chromatophores from the hormonal influence of the blood. The red pigment then concentrated and the white pigment dispersed (Fingerman, 1957a). Evidently the “resting states” for these two pigments are opposite each other. Chromatophores in the lateral portions of the carapace were slower to arrive at the appropriate “resting state” than those in the dorsal portion. Reciprocal blood transfusions between dwarf crayfish that had been on black and on white backgrounds for 2 hours revealed that the blood contained at all times hormones that dispersed and concentrated the red and white pigments. The degree of dispersion of these pigments at any time appeared to be determined by the relative quantity of each hormone in the blood. These results differ from those of Koller (1925, 1927), who observed that blood from a black shrimp, Crago vulgaris, darkened a light animal, whereas blood from a white donor had neither a lightening nor a darkening effect when injected into -either light or dark individuals. Responses of chromatophores on the body were compared with the responses of chromatophores on pieces of carapace in saline to tissue extracts that contained pigment-concentrating and pigmenkdispersing hormone. Pigment in isolated chromatophores showed little, if any, tendency to disperse when extracts were applied, whereas appreciable dispersion of the red and the white pigments was apparent when extracts were injected into crayfish and allowed to circulate throughout the body. This may be the reason McVay (1942) did not find red and white pigment-dispersing hormones in the central nervous organs of Orconectes immunis ; she used isolated chromatophores in her experiments. The amount of pigment migration in dwarf crayfish in response to a chromatophorotropin was in part a function of the initial stage of the pigment. For example, more concentration was evident when the same

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extract was applied to red pigment that was maximally dispersed at the time of removal of the carapace than to pigment in an intermediate state, even though maximal pigment concentration did not occur in either case. The effects of long-term background adaptation on the chromatophore system of Cambarellus shufeldti were determined by Fingerman and Lowe (1957a), who showed for the first time that the ability to change color in an invertebrate is facilitated through active use of the chromatophore system and becomes sluggish after a period of disuse. These investigators also demonstrated the changes that occur in the endocrine sources, blood titers, and target organs during a long-term stabilization of the chromatophores on a specific background. Specimens of Cambarellus were collected and maintained on black and on white backgrounds for 3 weeks. During this time the rates of red and white pigment concentration and dispersion after appropriate background changes progressively diminished. Likewise, the inherent tendencies of red pigment on isolated pieces of carapace to concentrate and of white pigment to disperse gradually disappeared. In addition to this physiological change the red chromatophores of the crayfish kept on a black background changed morphologically ; the number of processes in the chromatophores increased, the central body disappeared, and the processes of adjoining chromatophores intermingled to such an extent that the chromatophores appeared to have lost their individuality when observed under the microscope. To understand better the progressive decrease in ability to change color, the quantities of chromatophorotropins in the circumesophageal connectives and in the blood of specimens kept on black and on white backgrounds for 2 hours and for 2 weeks were compared. The results showed that the quantity of the hormone not needed to maintain the appropriate degree of red pigment dispersion or concentration increased in the circumesophageal connectives. For example, the amount of red pigment-dispersing hormone increased in the circumesophageal connectives of specimens with maximally concentrated red pigment as a result of having been kept on a white background for 2 weeks. The titers of red pigment activators in the blood also changed during the 2 weeks the crayfish were maintained on the black and the white backgrounds. Less of the hormone whose concentration in the circumesophageal connectives had increased was found in the blood, whereas the' red pigment activator necessary to maintain the appropriate degree of background adaptation had increased. Kleinholz (1957) had stated that all results obtained in studies of background adaptation in crustaceans could be explained with the chromatophorotropins in the eyestalk without invoking the secretory products of the supraesophageal ganglia or the circumesophageal connectives. The experiments described above provided the first evidence that the secretory

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products of the circumesophageal connectives play a role in the normal physiology of color change. In still another study of the chromatophore system of Cambarellus, alterations in the titer of red pigment-dispersing hormone in the blood after transfer of specimens from black to white and from white to black backgrounds were determined ( Fingerman and Lowe, 1957b). The titer of red pigment-dispersing hormone decreased in the blood of crayfish placed on a white background and increased in the blood of Cambarellus put on a black background. However, more red pigment-dispersing hormone was present in the blood of crayfish that had been on the black background for 30 minutes than in the blood of Cambarellus that had been on the black background for 15, 60, or 120 minutes. Obviously more red pigment-dispersing hormone was secreted just after the transfer from a white to a black background than was necessary to maintain the red pigment maximally dispersed. Although several plausible explanations for this phenomenon may be suggested, one of the more likely ones is that an overabundance of red pigment-dispersing hormone was secreted to disperse the pigment rapidly, and once the pigment was dispersed the excess was excreted or inactivated. Fingerman and Lowe (1957b) also determined the electrophoretic behavior at p H 7.4 and 7.8 of the chromatophorotropins in the supraesophageal ganglia with the circumesophageal connectives attached of dwarf crayfish. The hormone that concentrated the pigment in the large dark-red chromatophores was electropositive, whereas the one that dispersed this pigment was oppositely charged. The rates of disappearance of chromatophorotropins from extracts of the eyestalks and the circumesophageal connectives of dwarf crayfish on standing at room temperature also were determined by Fingerman and Lowe ( 1957b). Red and white pigment-concentrating substances of the circumesophageal connectives disappeared at a much faster rate than the red and white pigment-dispersing hormones. The red pigment-dispersing and white pigment-concentrating hormones were the first to disappear from eyestalk extracts. The predominant hormones ( Fingerman, 1957a) are the first to disappear from extracts of both the eyestalks and the circumesophageal connectives, whereas those which are in small quantity are relatively persistent molecules. Fingerman and Lowe (1957b) have tried to explain why in some crustaceans the dark pigment concentrates maximally and in other it disperses maximally after eyestalk removal. The ultimate condition of the pigment in chromatophores of crustaceans after eyestalk removal has been thought for a long time to depend on the chromatophorotropins produced by the eyestalks. For example, after removal of the eyestalks of the fiddler

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crab, Uca pugilator, black pigment becomes maximally concentrated ( Sandeen, 1950). This condition was thought to be due to removal of the source of black pigment-dispersing hormone. Results obtained with Cambarellus do not support this concept, however. Red pigment of eyestalkless Canzbarellus is maximally dispersed, yet the predominant chromatophorotropin produced by the eyestalks that affects red pigment has a dispersing action ( Fingerman, 1957a). The ultimate stage of the chromatophores of eyestalkless individuals is probably determined by hormones released from central nervous organs remaining after eyestalk removal and not primarily to the absence of chromatophorotropins from the eyestalk. The hormones that are not predominant quantitatively in the remaining central nervous organs, but are relatively stable molecules, probably determine the final stage of the chromatophores of eyestalkless crayfish and perhaps of all crustaceans. Fingerman (1957b) found that the eyestalks and central nervous organs of Orconectes clypeatzls are sources of red pigment-dispersing and pigmentconcentrating hormones. Direct injection into the body of extracts of central nervous organs as well as of entire eyestalks immediately after preparation did not produce red pigment dispersion. If the extracts were left at room temperature for 2 hours prior to injection, however, the red pigment-concentrating hormone became inactivated and the red pigmentdispersing hormone could then express itself. Reciprocal blood transfusions between specimens on black and on white backgrounds revealed that the state of the pigment appeared to depend on the relative concentrations of dispersing and concentrating hormones in the blood, just as was found for Carnbarellus shufeldti by Fingerman ( 1957a). Since direct injection of fresh extracts of tissues of Orconectes did not produce red pigment dispersion in Orconectes, it was of interest to determine whether these fresh extracts would disperse red pigment in Cambarellus, and vice versa ( Fingerman, 1957f). Extracts of central nervous organs as well as eyestalks of Orconectes caused concentration and dispersion of red pigment in Cambarellus. Extracts of eyestalks of Cambarellus caused dispersion as well as concentration of red pigment of Orcomectes. No dispersion of red pigment in Orconectes was caused by central nervous organs of Cambarellus. A possible explanation of these data is that Orconectes has an excellent feedback mechanism associated with its chromatophore system so that any displacement qf red pigment in crayfish on a white background to a more dispersed state is rapidly met with release of red pigment-concentrating hormone which antagonizes the added dispersing hormone. Extracts of the eyestalks of Cambarellus alone were able to overcome this mechanism because of the large amount of dispersing hormone in the eyestalk relative to the concentrating substance.

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Fingerman and Lowe (1958) showed that boiling extracts of the supraesophageal ganglia with the circumesophageal connectives attached of Cambarellus resulted in an increased titer of red pigment-dispersing hormone. Maintenance of extracts at room temperature for 2 hours had the same effect. These results were interpreted on the basis of release of additional hormone from the interior of neurosecretory granules, a mechanism similar to that described by Perez-Gonzilez (1957) for the black pigmentdispersing hormone in the sinus gland of Uca pugilator. Obviously, the chromatophore systems of Cambarellus shufeldti and Orconectes clypeatus, the only crayfishes examined in any detail recently, differ from each other considerably. W e should, therefore, proceed with caution in transferring information from one species of crayfish to another. 5. Stomatopods. The Stomatopods are another group that has been neglected. Brown (1948b) found that the mantid shrimp, Chloridella empusa, shows secondary color changes in response to background. This shrimp is a dark slate color when on a black background and a pale yellow when on white. Specimens from which both eyestalks were removed blanched permanently. However, extracts of these eyestalks had no perceptible effect on eyestalkless Chloridella but did darken eyestalkless Uca and when injected into eyestalkless Crag0 strongly darkened the telson and uropods and lightened the remainder of the body. Knowles (1954) reported that the postcommissure organs of the central nervous system in Squilla mantis contain large quantities of a substance that disperses the dark pigments of eyestalkless specimens. Obviously much work remains to be done before the chromatophore system of a single species of Stomatopod is well understood. C. Insects The most recent review of color changes in insects is that of Scharrer (1952a). The fact that color changes in insects are controlled by at least one hormone originating in the head (Janda, 1934) has been known for a long time. Only recently, however, has further detailed information been gathered about the process. Dupont-Raabe (1949) found that the brain of Carausius morosus was the major source of a hormone that darkened this stick insect; the corpora allata had no effect, and the corpora cardiaca only a slight one. Brainless specimens of C a r a w k become pale gray because of migration of the melanin granules in the hypodermal cells to the innermost depths of these cells. Injection of chromatophorotropin provokes the migration of the melanin toward the surface so that the animals become darker in color. Carlisle et d.(1955) reported in preliminary form the results of their studies of filter paper electrophoresis of chromatophorotropins in CarazGsius

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morosus. Their data were reported in final form in the paper by Knowles et al. (1955). Filter paper electrophoresis of extracts of the brain and the corpora cardiaca revealed that each contained a substance capable of dispersing melanin in Carausius. At p H 7.8 the chromatophorotropin in the corpora cardiaca was electropositive. At the same p H the substance in the brain had an extremely low mobility and was found on both sides of the origin; the authors called this the C-substance. Scharrer (1952b) had shown that in the cockroach, L e u c o p h a ma$erm, the pars intercerebralis of the brain and the corpora cardiaca form a functionally related group of neuroglandular organs, similar to the medulla terminalis X organ-sinus gland complex of crustaceans, in which the corpus cardiacum serves as a reservoir for neurosecretory material produced in the brain. If such a relationship existed between the corpora cardiaca and brain of Cm-aasiw, then the C-substance might be formed from the brain substance as it moves along the axons to the corpora cardiaca.

V. CHROMATOPHORES OF FISHES Color changes of fishes have been studied more intensively than those of amphibians or reptiles, the other vertebrates with functional chromatophores. The reviews of Waring and Landgrebe (1950) and of Pickford and Atz (1957) were restricted to the vertebrates. Secondary color changes in fishes require, first of all, eyes and afferent pathways from the eyes to the central nervous system. Nerves may then either run ( 1 ) to endocrine organs where chromatophorotropins are released or ( 2 ) directly to the pigment cells where chemical mediators are liberated at the nerve endings; or ( 3 ) in some instances mechanisms 1 and 2 may cooperate. If the chromatophores are not innervated, they are referred to as aneuronic. The skate, Raja erinucea, has this type of melanophore (Parker, 1937). If a single nerve fiber is present (niononeuronic chromatophores) , its activity is always pigment-concentrating, as was found in the dogfish, Mustelus canis, by Parker and Porter (1934). In most teleosts such as the killifish, Fundulus heteroclitus, pignientdispersing and pigment-concentrating fibers (dineuronic chromatophores) are present (Parker, 1934): In some species, evidence has been obtained for lightening and darkening hormones that are produced by the pituitary (Hogben and Slome, 1931) , whereas in others evidence for only a melanindispersing hormone is available. The pituitary of some fishes does not appear to have a melanin activator. Later publications have not changed the basic concepts of the control of color changes presented in the review by Parker (1948). Progress

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would naturally be slower with the vertebrates, since so much information had already been accumulated by Parker, Hogben, and their associates. Weisel (1950) tested the effects of pituitary extracts from four fishes on the melanophores of a variety of other fishes. Pituitaries were removed from the sheepshead, Pimelometopon pulchrum ; the barracuda, Sphyraenu argetea ; the yellow fin tuna, Neothmnus mmropterus ; and the white sea bass, Cynoscim nobilis. The extracts darkened the round stingray, Urobatis MZeri, and the black bullhead, Ameiurzls melus, and lightened the mosquito fish, G,ambusia afinis, the California killifish, Fundulus parvipinnus, the opaleye, Girella nigricam, the mudsucker, Gillichthys mirabilis, the green sunfish, Lepomis cyanellus, and the grunion, Leuresthes tenuis. Robertson (1951) determined the responses of the melanophores of the rainbow trout, Salmo gairdneri, to a variety of materials. H e found that melanin concentration was induced by asphyxia, bright light, high temperature, potassium chloride, and adrenaline. Dispersion was produced by acetylcholine and extracts of the posterior lobe of the pituitary. Cortisone was ineffective. Breder and Rasquin (1950) found that when adrenalin was injected into an angel fish, Chaetodipterus fuber, the pigment in the dermal melanophores dispersed and the melanin in the iris and meninges concentrated. These same authors reported in 1955 that this fish showed an albedo response and that melanin dispersion occurred in response to increased illumination. Umrath and Walcher ( 1951) found that the chromatophores of the teleost, Macropodus opercuhris, are completely independent of pituitary control. Pituitary extracts had no effect even after spinal section. Kohler ( 1952) found that adrenocorticotropic hormone (ACTH) dispersed the pigment in the melanophores of intact, adrenalectomized, and hypophysectomized specimens of the minnow, Phoxinus b v i s , as well as in isolated portions of the skin. Abolin long ago (1925) had shown that adrenaline concentrated melanin in specimens of this same species. Pickford (1956), on the other hand, found no response to ACTH by hypophysectomized Fundulus heteroclitus. Healey ( 1951, 1954) found that the chromatophore responses of Phoxinus lamis were not interfered with by spinal section. He concluded that the color changes of this species were strictly hormonal. Briseno Castrejon and Stevens Flores ( 1955) found that pituitary extracts of Cau-assius auratus, a goldfish, caused melanin dispersion in this species. Enami (1955) found two antagonistic responses when extracts of the hypothalamus and the pituitary were injected into a Japanese catfish, Pararilurus asotus, whose melanin was in an intermediate degree of dispersion as a result of hypophysectomy. A marked localized pallor appeared

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at the site of injection while the rest of the body darkened. H e believes the pigment-concentrating hormone is of hypothalamic origin. Brantner (1956) showed that extracts of the posterior lobe of the pituitary of preand postspawning European bitterlings, Rhodeus amarus, caused maximal pigment dispersion. VI.

CHROMATOPHORES OF AMPHIBIANS

Color changes in amphibians appear to be mediated by hormones alone. No conclusive evidence has been uncovered to show that the chroniatophores of amphibians are directly innervated. In some amphibians evidence for only a darkening hormone, intermedin, is available, whereas in others, such as the South African clawed toad, Xenopus laevis, two chromatophorotropins are found, intermedin and a pigment-concentrating substance in the pars tuberalis of the pituitary (Hogben and Slome, 1936). Recent investigations have involved a wide variety of species. Mussbichler and Umrath (1950) found that the tree frog, Hyla arborea, is darkened by intermedin and lightened by adrenaline. Rowlands (1950, 1952) found that dampness induced melanin dispersion in the frog, R a m temporurh, and that dehydration caused lightening. Skin receptors appeared to be involved because blinding by removal of the eyes or cautery of the optic chiasma obliterated background responses but not the responses to moisture. In intact frogs the humidity response dominated the background one. Sieglitz (1951) showed that extracts of the skin of R a m temporuriu contained a melanin-concentrating substance which was not adrenaline. Wright ( 1955) studied with photoelectric recording equipment the behavior of the melanophores in excised portions of the skin of the frogs R a m pipiens and R a m chmitans. Their melanin dispersed in response to intermedin and concentrated in the presence of adrenaline. The rates of concentration and dispersion of melanin increased with increase in temperature. Sodium iodoacetate, malonate, and fluoride at least partially inhibited melanin concentration. Wright postulated that his results showed that the energy of glycolysis was necessary for blanching. Triphenyltetrazolium chloride inhibited the responsiveness of the melanophores to intermedin ; energy exchange may have been blocked by this chemical. Amphibians usually respond to excitement stimuli and adrenaline by blanching. Two exceptions to this rule have been reported. I n 1909 Siedlecki reported that the Javanese “flying frog,” Polypedatus reinwarti, darkened on excitement. More recently Burgers et d. (1953) found that Xenopus laevis darkened in response to excitement stimuli or injection of adrenaline. Ketterer and Remilton ( 1954) confirmed the observations

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of Burgers et al. that excitement results in darkening of Xenopus. Hudson and Bentley (1955) also found that adrenaline darkened specimens of Xenopus. Burgers (1956) provided further information about the control of melanophores in Xenopus laevis. H e demonstrated that pigment dispersion after excitement stimuli was not due to intermedin. These stimuli cause secretion by the skin glands of a pigment-dispersing substance which acts directly on the melanophores but which is not intermedin. The excitement darkening reaction was probably due to the action of adrenaline coupled with the pigment-dispersing substance in the skin secretion. Burgers also studied the relationship between the chemical structure and melanophore activity of several analogs of adrenaline. A hydroxyl group must be in the three and the four position of the phenyl nucleus for a positive effect on melanophores. A hydroxyl group at the 1’-C atom of the side chain is important. Substitution of a hydrogen atom for this hydroxyl group decreased the activity; a methoxy group eliminated it. Chang (1957) found that thyroxine induced indirect melanin concentration in X m o p u s laevis. This hormone appeared to stimulate the release of a neurohumor of cholinergic nature, which in turn controlled the release of melanophore-stimulating hormone from the pituitary. Triiodothyronine had no chromatophorotropic effect.

VII. CHEMICALNATUREOF CHROMATOPHOROTROPINS Several groups of investigators have attempted to determine the cheniical structure of chromatophorotropins in both vertebrates and invertebrates. So far only the structure of the melanocyte-stimulating hormone from porcine pituitary glands has been elucidated. Two groups of investigators, working independently, have arrived at the same conclusion concerning the structure of this molecule. They are ( 1 ) Harris and Roos (1956) and ( 2 ) Geschwind et al. (1956, 1957); Geschwind and Li (1957). The molecule is an octadecapeptide, a chain of 18 amino acids : H-asp * glu gly * pro * tyr * lys met glu * his * phe * arg * try * gly * ser pro * pro * lys asp.-OH. The molecular weight is 2177. Current interest in the nature of the chroniatophorotropins in crustaceans is also very high. These substances appear to be small polypeptides, just as is intermedin. The A-substance of Knowles et al. (1956) is destroyed by trypsin and by acid hydrolysis, indicative of peptide bonds. This substance is not inactivated by amine oxidase or by orthodiphenoloxidase, thereby suggesting that the A-substance is not a catechol amine. Ostlund and Fange (1956) increased by means of column chromatography the titer of a substance in the eyestalks of the shrimp, Pandalus

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borealis, that concentrated the pigment in the small and the large red chromatophores of Leunder udspersus. The authors stated that “it is probable that the hormone is a simple aromatic amine”. They now feel, however, (personal communication) that the hormone is not a lowmolecular-weight simple aromatic amine. Finally, PCrez-GonzLlez ( 1957) found that the hormone in the sinus gland of Uca pugilator that dispersed black pigment was inactivated by chymotrypsin and, therefore, may be a polypeptide. The chromatophorotropic substances of vertebrates and invertebrates ultimately may be shown to be closely related molecules (Florey, 1952). VIII. REFERENCES Abolin, L. (1925) Arch. mikroskop. Anut. u. Entwicklungsmech. 104, 667. Abramowitz, A. A. (1937) Biol. Bull. 72, 344. Bliss, D. E. (1951) Anat. Record 111, 502. Bliss, D. E., and Welsh, J. H. (1952) Biol. Bull. 103, 157. Bowman, T. E. (1949) Biol. Bull. 96, 238. Brantner, G. (1956) Z . vergleich. Physiol. 33, 324. Breder, C. M., Jr., and Rasquin, P. (1950) Science 111, 10. Breder, C. M., Jr., and Rasquin, P. (1955) Zoologica 40,85. Briseno Castrejon B., and Stevens Flores, I. (1955) Anales escuela nacl. rienc. biol. ( M e x . ) 8, 203. Brown, F. A., Jr. (1933) Proc. Natl. Acad. Sci. U.S . 19, 327. Brown, F. A., Jr. (1934) Biol. Bull. 67, 365. Brown, F. A., Jr. (1935a) I. Exptl. 2001.71, 1. Brown, F. A., Jr. (1935b) 1. Morphol. 67, 317. Brown, F. A., Jr. (1936) Biol. Bull. 70, 8. Brown, F. A., Jr. (1939) Ecology 20, 507. Brown, F. A., Jr. (1940) Physiol. Zool. El, 343. Brown, F. A., Jr. (1944) Quart. Rev. Biol. 19, 32-46, 118-143. Brown, F. A., Jr. (1946) Physiol. 2001.19, 215. Brown, F. A., Jr. (1948a) Zn “The Hormones” ( G . Pincus and K. V. Thimann, eds.), Vol. I. Academic Press, New York. Brown, F. A., Jr. (194813) Anat. Record 101, 732. Brown, F. A., Jr. (1950) Biol. Bull. 98, 218. Brown, F. A., Jr. (1952) “Action of Hormones in Plants and Invertebrates.” Academic Press, New York. Brown, F. A., Jr., and Ederstrom, H. E. (1940) J . Exptl. Zool. 86, 53. Brown, F. A., Jr., and Fingerman, M. (1951) Federation Proc. 10, 20. Brown, F. A., Jr., and Hines, M. N. (1952) Physiol. Zool. 26, 56. Brown, F. A., Jr., and Klotz, I. M. (1947) Proc. SOC.Exptl. Biol. Med. 64, 310. Brown, F. A., Jr., and Meglitsch, A. (1940) Biol. Bull. 79, 409. Brown, F. A., Jr., and Saigh, L. M. (1946) Biol. Bull. 91, 170. Brown, F. A., Jr., and Sandeen, M. I. (1948) Physiol. Zool. 21, 361. Brown, F. A., Jr., and Stephens, G. C. (1951) Biol. Bull. 101, 71. Brown, F. A., Jr., and Thompson, D. H. (1937) Cope& p. 172. Brown, F. A., Jr., and Webb, H. M. (1948) Physiol. Zool. 21, 371. Brown, F. A., Jr., and Webb, H. M. (1949) Physiol. Zool. !H,i 136.

T H E PHYSIOLOGY OF CHROMATOPHORES

'

207

Brown, F. A., Jr., and Wulff, V. J. (1941) J. Cellular Comp. Physiol. 18, 339. Brown, F. A,, Jr., Webb, H. M., and Sandeen, M. I. (1952) J. Exptl. 2001.120, 391. Brown, F. A,, Jr., Fingerman, M., Sandeen, M. I., and Webb, H. M. (1953) J. Exptl. 2001.la,29. Brown, F. A., Jr., Fingerman, M., and Hines, M. N. (1954a) Biol. Bull. 106, 308. Brown, F. A., Jr., Webb, H. M., Bennett, M. F., and Sandeen, M. I. (1954b) Physiol. Zool. 27, 345. Brown, F. A., Jr., Bennett, M. F., and Ralph, C. L. (1955a) Proc. SOC.Exptl. Biol. Med. 89, 332. Brown, F. A., Jr., Webb, H. M., and Bennett, M. F. (195513) Proc. Natl. dcad. Sci. U . S. 41, 93. Burgers, A. C. J. (1956) Doctorate Thesis, G. W. van Der Wiel and Co., Arnhem, Netherlands. Burgers, A. C. J., Boschman, Th. A. C., and van de Kamer, J. C. (1953) Acta Endocrinol. 14, 72. Carlisle, D. B. (1954) J . Marine Biol. Assoc. United Kingdom SS, 61. Carlisle, D. B. (1955) J. Marine Biol. Assoc. United Kingdom S4, 559. Carlisle, D. B., Dupont-Raabe, M., and Knowles, F. G. W. (1955) Compt. rend. M ,665. Carlson, S. Ph. (1935) Proc. Natl. Acad. Sci. U. S. 21, 549. Carstam, S. Ph. (1951) Nature 167, 321. Carstam, S. Ph., and Suneson, S. (1949) Kgl. Fysiograf. Sallskap. Lund Forh. 19, 1. Chang, C. Y . (1957) Science, l26, 121. Deanin, G. G., and Steggerda, F. R. (1948) Proc. SOC.Exptl. Biol. Med. 67, 101. Drach, P. (1944) Bull. Biol. 78, 40. Dupont-Raabe, M. (1949) Compt. rend. aa8, 130. Durand, J. B. (1956) Biol. Bull. 111, 62. Edgren, R. A. (1954) Proc. SOC.Exptl. Biol. Med. 87, 20. Enami, M. (1941a) Japan. J. Zool. 9,497. Enami, M. (1941b) Japan. J. Zool. 9, 515. Enami, M. (1943) Proc. Imp. Acad. ( T o k y o ) 19, 693. Enami, M. (1951a) Biol. Bull. 101,241. Enami, M. (1951b) Biol. Bull. 100, 28. Enami, M. (1955) Science l21, 36. Fingerman, M. (1955) Biol. Bull. 109,255. Fingerman, M. (1956a) J. Exptl. 2001.199, 87. Fingerman, M. (195613) Tulane Studies Zool. 5, 137. Fingerman, M. (1956~) Science la, 585. Fingerman, M. (1956d) Biol. Bull. 110, 274. Fingerman, M. (1957a) Tulane Studies 2001.I, 137. Fingerman, M. (1957b) Anat. Record 128, 548. Fingerman, M. (1957~) A m . Naturalist 91, 167. Fingerman, M. (1957d) Biol. Bull. 112, 7. Fingerman, M. (1957e) Physiol. Zool. SO, 142. Fingerman, M. (1957f) Anat. Record 128,549. Fingerman, M., and Fitzpatrick, C. (1956) Biol. Bull, 110, 138. Fingerman, M., and Lowe, M. E. (1957a) Physiol. Zool. 90, 216.

208

MILTON FINGERMAN

Fingerman, M., and Lowe, M. E. (1957b) Tulane Studies 2001.6, 149. Fingerman, M., and Lowe, M. E. (1958) Biol. Bull. 114,317. Fingerman, M., and Tinkle, D. W. (1956) Biol. Bull. 110, 144. Fingerman, M., Lowe, M. E., and Mobberly, W. C., Jr. (1958) Limnol. and Oceanog. 5, 271. Florey, E. (1952) Biol. Zentr. 71, 499. Fox, D. L. (1953) “Animal Biochromes and Structural Colours.” Cambridge Univ. Press, New York. Frost, R., Saloum, R., and Kleinholz, L. H. (1951) Anat. Record, 111, 572. Geschwind, I. I., and Li, C. H. (1957) J . Am. Chem. SOC.79, 615. Geschwind, I. I., Li, C. H., and Barnafi, L. (1956) J. A m . Chem. SOC.78, 4491. Geschwind, I. I., Li, C. H., and Barnafi, L. (1957) J . Am. Chem. SOC.79, 620. Hadley, C. E. (1929) Bull. Museum Comp. Zool. Harvard 69, 10s. Hanstrom, B. (1933) 2001.Jahr. Abt. Anat. u. Ontog. Tiere 66, 367. Hanstrom, B. (1937) Kgl. Svenska Vetenskapsakud. Handl. 16, 1. Harris, J. I., and Roos, P. (1956) Nature 178, 90. Havel, V. J., and Kleinholz, L. H. (1951) Anat. Record 111, 571. Healey, E. G. (1951) J. Exptl. Biol. 28, 297. Healey, E. G. (1954) J. Exptl. Biol. 91, 473. Hill, A. V., Parkinson, J. L., and Solandt, D. Y. (1935) J . Exptl. Biol. 12, 397. Hines, M. N. (1954) Biol. Bull. 107, 386. Hogben, L.T., and Landgrebe, F. W. (1940) Proc. Roy. SOC.Bl28, 317. Hogben, L. T., and Slome, D. (1931) Proc. Roy. SOC.B108, 10. Hogben, L. T., and Slome, D. (1936) Proc. R o y . SOC.B120, 158. Hudson, B., and Bentley, G. A. (1955) Lancet i, 775. Janda, V. (1934) Mkm. soc. sci. BohPme C1. sci. 1-31. Janda, V . (1936) 2001.Anz. 116, 177. Ketterer, B.,and Remilton, E. (1954) J. Endocrinol. 11, 7. Key, K. H. L., and Day, M. F. (1954) Australian J. 2001.2, 309. Kleinholz, L. H. (1937) Biol. Bull. 72, 24. Kleinholz, L. H. (1957) “Recent Advances in Invertebrate Physiology.” Univ. Oregon Press, Eugene, Oregon. Knowles, F. G. W. (1950) Biol. Bull. 98, 66. Knowles, F. G. W. (1952) Physiol. Comparata et Oecol. 2, 289. Knowles, F. G. W. (1953) Proc. R o y . SOC.B141, 248. Knowles, F. G. W. (1954) Pubbl. stao. zool. Napoli 24, Suppl. 74. Knowles, F. G. W. (1956) Ann. sci. nut. 2001.et biol. animale 18, 325. Knowles, F. G. W., and Carlisle, D. B. (1956) Biol. Revs. Cambridge Phil. SOC. s1, 3%. Knowles, F. G. W., Carlisle, D. B., and Dupont-Raabe, M. (1955) J. Marine Biol. Assoc. United Kingdom 34, 611. Knowles, F. G. W., Carlisle, D. B., and Dupont-Raabe, M. (1956) Compt. rend. 242, 825. Kohler, V . (1952) Natarrwiss. S9, 554. Koller, G. (1925) Verhandl. deutsch. zool. Ges. SO, 128. Koller, G. (1927) Z. vergleich. Physiol. 6, 191. Koller, G. (1928) Z . vergleich. Physiol. 8, 601. Kopenec, A. (1949) Z . vergleich. Physiol. S1, 490. Kiihn, A. (1950) Z . vergleich. Physiol. 92, 572.

THE PHYSIOLOGY OF CHROMATOPHORES

209

McVay, J. A. (1942) Doctorate Thesis, Northwestern University, Evanston, Illinois. McWhinnie, M. A,, and Sweeney, H. M. (1955) Biol. Bull. 108, 160. Matsumoto, K. (1954a) Biol. Bull. 106,60. Matsumoto, K. (1954b) Biol. J. Okayama Univ. 1, 234. Matthews, S. A. (1931) J. Exptl. Zool. 68, 471. Miissbichler, H., and Umrath, K. (1950) 2. vergleich. Physiol. 32, 311. Nagano, T. (1943) Sci. Repts. TBhoku Imp. Univ., Fourth Ser. 17, 223. Nagano, T. (1949) Sci. Repts. TBhoku Imp. Univ., Fourth Ser. 18, 167. Okay, S. (1943) RPU.fac. sci. univ. Istanbul, Sir. B . Fasc. 3, 9, 204. Okay, S. (1945a) Rev. fac. sci. univ. Istanbul, Sir. B. Fasc. 5, 9, 1. Okay, S. (1945b) Rev. fac. sci. univ. Istanbul, Sir. B. Fasc. 2, 10, 116. Okay, S. (1946) Compt. rend. ann. et arch. soc. turq. sci. phys. et nat. 12, 101. Ostlund, E., and Finge, R. (1956) Ann. sci. nut. Zool. et biol. animale 18, 325. Panouse, J. B. (1946) Ann. inst. Ocianog. (Monaco) 23, 65. Panouse, J. B. (1947) Annie Biol. 2S, 33. Parker, G. H. (1934) Proc. Natl. Acad. Sci. U. S. 20, 306. Parker, G. H. (1937) Proc. Am. Phil. SOC.77, 223. Parker, G. H. (1948) “Animal Colour Changes and their Neurohumours.” Cambridge Univ. Press, London. Parker, G. H., and Porter, H. (1934) Biol. Bd1. 66, 30. Passano, L. M. (1951a) Anat. Record 111, 502. Passano, L. M. (1951b) Anat. Record 111, 559. Passano, L. M. (1953) Physiol. Comparata et Oecol. 3, 155. Pautsch, F. (1952) Bull. intern. acad. polon. sci. Classe sci. math. nat. Skr. B 1961 (7/10) 511. PCrez-Gonzilez, M. D. (1957) Biol. Bull. 113, 426. Perkins, E.B. (1925) 1. Exptl. Zool. 60, 71. Pickford, G. E. (1956) Anat. Record 126, 603. Pickford, G. E., and Atz, J. W. (1957) “The Physiology of the Pituitary Gland of Fishes.” New York Zoological Society, New York. Robertson, 0. H. (1951) Physiol. 2061. 24, 309. Rowlands, A. (1950) J. Exptl. Biol. W ,446. Rowlands, A, (1952) I . Exptl. Biol. 29, 127. Sandeen, M. I. (1950) Physiol. 2061. 2S, 337. Scharrer, B. (1952a) “Action of Hormones in Plants and Invertebrates.” Academic Press, New York. Scharrer, B. (1952b) Biol. Bull. 102, 261. Scheer, B. T., and Scheer, M. A. R. (1954) Pubbl. staz. zool. Napoli 26, 397. Siedlecki, M. (1909) Biol. Centr. 26, 704. Sieglitz, G. (1951) 2. vergleich. Physiol. SS, 99. Smith, H. G. (1938) Proc. Roy. SOC.BUS, 250. Stephens, G. C. (1957a) A m . Naturalist 91, 135. Stephens, G. C. (1957b) Physiol. 2001. SO, 55. Sumeer, F. B. (1935) Am. Naturalist 69, 245. Teissier, G. (1947) Conzpt. rend. 235, 204. Travis, D. F. (1951) Anat. Record 111, 503. Umrath, K.,and Walcher, H. (1951) 2. vergleich. Physiol. SS, 129. Waring, H. (1938) Proc. Roy. SOC.Bl26, 264.

210

MILTON FINGERMAN

Waring, H., and Landgrebe, F. W. (1950) In “The Hormones” (G. Pincus and K. V. Thimann, eds.), Vol. 11. Academic Press, New York. Webb, H. M. (1950) Physiol. 2001. 2S, 316. Webb, H. M., Bennett, M. F., and Brown, F. A.; Jr. (1954) Biol. Bull. 106, 371. Weisel, G. F. (1950) Biol. Bull. 99, 487. Welsh, J. H. (1951) Anat. Record 111, 442. Wright, P. A. (1955) Physiol. 2001. 28, 204.

The Fibrous Components of Connective Tissue with Special Reference to the Elastic Fiber DAVID A . HALL Nufield Gerontological Research Unit. Department of Medicine. School of Medicine. Leeds. England Page

I. Introduction ..................................................... I1. Morphological Studies on Collagen and Elastic Fibers ............... A . Introduction ................................................. B Histology .................................................... 1. Methods .................................................. 2. Theories of Staining ...................................... C. Morphological Studies on the Pathological Involvement of Elastic Tissues ...................................................... 1. Senile Elastosis .......................................... 2. Ehler's Danlos Syndrome-Rubber Skin ................... 3. Pseudoxanthoma Elasticum ............................... 4. Colloid Millium .......................................... 5 . Obliterative Diseases of Elastic Fibers ..................... D . Structural and Physical Aspects of Connective-Tissue Fibers ... 1. Morphology under the Light Microscope ................... 2. Electron-Microscope Appearance .......................... 3. X-Ray Diffraction Studies on Connective Tissue ............. 4. Physical Properties of Collagen and Elastin ............... 5 . Model Structures for Elastic Fibers ....................... I11. Biochemical Studies on Collagen and Elastic Fibers ................. A . Chemical Composition of Collagen and Elastin ................ 1. Amino Acid Analyses .................................... 2. Polysaccharide as a Component of Connective Tissue ........ 3. Lipid ..................................................... B. Distribution of Collagen and Elastic Fibers ................... 1. Methods of Determination ................................. 2. The Relative Distribution of Collagen and Elastin .......... C. The Enzymatic Susceptibility of Elastin and Collagen ........... 1. The Elastase Complex and Its Physiological Significance ... 2. Specificity of the Enzymes ................................. I V. The Physiology of Connective-Tissue Fibers ....................... A . Fibrogenesis ................................................. 1. Embryonic Tissue ........................................ 2. Wound Healing ......................................... B. The Aging of Connective-Tissue Fibers ....................... 1. Changes in Collagen ..................................... 2. Changes in Elastic Fibers ................................. V . The Production of Elastic Material from Collagen ................. A . Structural Evidence ......................................... 1. Histochemical Evidence .................................. 2. Electron-Microscope Studies ............................... 3. X-Ray Diffraction ........................................

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B. Chemical Evidence ........................................... 1. Alkali Treatment ......................................... C. Evidence for the Heterogeneity of Collagen ................... 1. Sequence Studies ......................................... 2. The Action of Collagenase ................................ 3. The Effect of Phthalate Buffer on Collagen ................. VI. Conclusions ...................................................... VII. References .......................................................

I. INTRODUCTION The year 1955 saw the reintroduction of a concept (Burton et al., 1955; Hall et al., 1955a) that had engaged the interest of histologists since the days of Unna (18%). It had, however, been relegated to the limbo reserved for those hypotheses that have failed to withstand the test of subsequent research. This particular concept concerned the relationship between collagen and elastic fibers, especially with regard to the possibility of direct conversion of the former into elastic fibers in the animal body. Although such a concept could explain much of the histological evidence for the existence of material with tinctorial properties intermediate between those of collagen and elastica, it appeared to receive its death blow with the appearance of the first relatively complete and accurate analyses for the protein constituents of these connective-tissue fibers (Bowes and Kenten, 1948; Stein and Miller, 1938). In view of the reawakened interest in connective tissue, this review should serve a twofold purpose. Not only will it provide one of the original authors with a medium for discussing the present status of the hypothesis, but it will enable him to attempt to fill a gap long apparent in the studies of elastic tissue, namely the examination of the properties of elastic fibers as they fit into the general physiology of connective tissue, especially in comparison with the properties of collagen fibers. In the first three major sections to follow, therefore, evidence is presented to justify the consideration of fibrous components of connective tissue as a group, and not as individuals. In the fourth section is gathered together the not inconsiderable mass of evidence for the consideration of two members of the group-collagen and elastic fibers-as being more intimately related than has hitherto been assumed likely. The literature relating to connective-tissue fibers has been reviewed from the point of view of the elastic fiber, and hence references to collagen are by no means complete. Selection has been made solely to provide an adequate background against which to compare the properties of the elastic fiber.

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11. MORPHOLOGICAL STUDIES ON COLLAGEN A N D ELASTIC FIBERS

A . Introduction Connective tissue contains three major groups of components : cellular structures, fibrous elements, and the amorphous semisolid gel in which they are embedded. A complete coverage of all aspects of connective tissue can be attempted in a review of this nature only at the expense of detail, and in view of the author’s own interests it was decided to restrict the review to a consideration of the fibers alone, and to consider these from the point of view of the elastic fibers and their relationship to the other fibrous element, collagen. Such a narrow division is not completely practicable, and at various stages in the discussion nonfibrous components will be introduced. The origin of the fibers is, for instance, dependent on specific cellular activity, and such activity will have to be considered when dealing with the general question of fibrogenesis. Similarly, quantitative relationships between the fibers and the amorphous component appear to be of considerable importance in considerations of pathological and aging changes (Sobel and Marmorston, 1956). At this point it may be appropriate to introduce the question of nomenclature. Semantics, probably more than any other factor, has obscured the results of connective-tissue research, and it would appear that the time has come for stricter definition, especially in the elastin field. The definitions suggested below are the sole responsibility of the author, but they are essentially similar to those agreed on by a number of workers in the elastin field who met during a symposium on connective tissue in 1956 and whose individual contributions to the subject are recorded in Connective Tissue (Tunbridge, 1957). Elastin: The name given to a derived protein, obtained from elastic tissue by techniques aimed at the removal of as much extraneous material-ther protein, polysaccharide, etc.-as possible, without causing undue degradation of the protein (cf. Partridge and Davies, 1955). The term is also used loosely in general phrases which do not presuppose the precise structural level under consideration. Elastic fibers: A morphological term which can be employed in either physicochemical or histological context to define the intact fibrous element. I t may or may not contain material in addition to the protein elastin (cf. Hall, 1957c, and Partridge et al., 1957). Elastica-staining : An adjectival phrase with purely histological connotations, which presupposes neither a specific fibrous structure nor a given chemical composition. Elastic tissue: A tissue rich in elastic fibers or other elastic-staining material, which exhibits as a, whole the property typical of individual elastic fibers-namely elasticity. Elastomucin: A considerable amount of tautology can be avoided if the mucopolysaccharide associated with the elastic fiber be given the name elustomucin. The

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term was introduced by Hall et al. (1952) to describe a protein-polysaccharide complex, the existence of which could explain a number of facts concerning the structure, enzymatic resistance, and degradation products of elastic fibers. Partridge and Davies (1955) have, however, doubted whether such a complex is a fundamental component of the fiber. Whether such is the case or not, a certain amount of polysaccharide, either by loose bonding or because it is an integral part of the fiber, can be regarded as being more closely associated with the elastic elements of tissue than are the polysaccharides of the ground substance. T o this material the name elastomucin can most usefully be applied. Collagen: The position with regard to collagen is just as confusing, since in this case a single term is used to describe everything from the molecular level to the fiber bundle, whether observed under the electron microscope, the light microscope, or the naked eye. Also there is the added confusion of soluble collagens, both natural and derived. It would appear imperative that the nomenclature of collagen should be rationalized as soon as possible, but in view of the fact that the present review deals mainly with the relationship between elastic fibers and collagen as a fiber species, which it will not in general be necessary to define more rigorously, the author will delegate this task to others. It would appear to be a formidable one, since no general agreement on nomenclature was reached at the symposium mentioned above. Ground substance : The amorphous matrix enveloping the fibrous components will be given this general term. I t is not assumed that it has the same composition throughout the body, and indeed qualitative differences between the polysaccharides present in various sites may have a profound bearing on the physiology of the fibrous components. Our knowledge of the comparative chemistry of connective-tissue ground substances is, despite the activities of Meyer and his co-workers (Meyer and Rapport, 1951), still very fragmentary.

Characterization of connective-tissue components was originally the outcome of extensive histological observation, and it is, therefore, not surprising that in the majority of the earlier reviews and monographs the problem was approached almost entirely from this point of view (e.g., Fleming, 1876 ; Popa, 1936). More recently, with the advent of improved chemical and physical methods, the spectrum of techniques available for the examination of connective-tissue fibers has been broadened, and certain differences in properties between the various fibers have been observed. The characterization of fibers by histological techniques, by its very nature, permitted the identification, in pathological tissue, of intermediate structures with staining properties midway between those of individual fiber species. Chemical analysis,. confined in the first place to nonpathological tissue, tended on the other hand toward more rigorous definitions and consequently resulted in the specialized consideration of the individual fibers rather than the fiber complex as a whole. Hence later reviews have dealt specifically with one particular fiber species (Dempsey and Lansing, 1954 ; Kendrew, 1953), and little comparative work has been reported. Notable exceptions have been the series of conferences organized by the Josiah

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Macy Foundation (Ragan, 1950), the book “Connective Tissue in Health and Disease” ( Asboe-Hansen, 1954), and shorter reviews on the physiology of connective tissue (Baker and Abrams, 1955). Even in these, however, comparison of the properties of the fibers themselves is limited, and in the main collagen has received far more attention than elastin.

B. Histology 1. Methods. The fibers of connective tissue can be divided by histological methods into three main groups : collagen, elastic, and reticular fibers. Although the identity of these fibers in a number of sites has been associated with the appearance of a particular staining reaction, the names by which they are known have been derived from a consideration of their physical, chemical, or morphological properties as they appear en masse in those tissues in which each particular species predominates. One of the main difficulties has been the correlation of chemical and histological observations on those tissues that contain mixtures of fibers, or in which a particular fiber is present only in small amounts (Hall, 1951). Until recently it had been assumed, for instance, that the presence of orceinpositive material in a tissue meant of necessity that elastic fibers were present, or that argyrophilic fibers from all sites consisted of the same type of reticulin. I n spite of these weaknesses, histological methods have been of considerable importance in studies of connective tissues, in providing the basis on which, in many instances, other disciplines have built. Many collagen stains are taken up to a limited extent by elastic fibers. For instance, both fibers are slightly acidophilic and hence stain with eosin. On the other hand, certain collagen stains do differentiate between collagen and elastic fibers. Mallory’s aniline blue stain (1900, 1936), for example, stains collagen blue and elastic tissue red, and similar differentiation occurs under ideal conditions with Masson’s trichrome stain ( 1929). The variability of the staining properties of elastic fibers with these stains, however, rendered it necessary for specific elastic stains to be devised. Two such are Unna’s acid orcein method (1896) and any one of a number of modifications of Weigert’s method (1898). With most of these stqins a small degree of generalized staining of collagen fibers occurs, and the amount of elastica-staining material in sections containing both collagen and elastica from, for example, human aorta, depends on the stain employed, indicating that not all the areas taking up one elastica stain are capable of being stained by another. This implies either that a proportion of the collagen in elastic tissue has different properties from the rest, or that the elastica-staining material is itself heterogeneous. The latter may well be the case, since even with a single stain, e.g., Hart’s (1908) modi-

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fication of Weigert’s stain, not only do elastic fibers in different tissues stain to different shades, but elastic lamellae and elastic fibrils in a single section of aorta are quite different in their stained appearance. Polysaccharide can be demonstrated in elastic tissue by the periodic acid-Schiff ( P A S ) reaction ( McManus, 1956), although in native fibers this effect is slight. This is as would be expected, in view of the fact that, even after exhaustive purification (Partridge and Davies, 1955) under conditions that bring about the partial removal of any elastomucin sheath (Hall, 1957c), not all the polysaccharide is removed (Wood, 1958). Fibers that have been subjected to mild digestion with elastase, however, show the presence of considerable amounts of metachromatic polysaccharide (Balo et ul., 1954; Saxl, 1957a). It would appear that in the intact fiber the polysaccharide in the elastomucin layer (Hall ct aJ., 1952) must be combined so firmly and in such a fashion to the protein that it is incapable either of reacting with periodic acid or of aligning the dye molecules to produce metachromasia. Rinehart and Abul-Haj ( 1951), however, using a modification of Hale’s (1946) method, showed the presence of an outer layer in the intact elastic fiber which was rich in acidic polysaccharide. The P A S reaction on collagen, on the other hand, gives very variable results (positive-Wislocki, 1952 ; negative-Leblond, 1950), but it has been pointed out that slight variations in technique could affect the intensity of staining to such an extent as to account for these two extremes. This makes the histological picture even more confusing, and one must compare these results with those obtained by other disciplines to be able to reach a decision concerning their validity. The observations by Jackson (1954) and Wood (1953) on the physical properties of collagen fibers treated with reagents specific for polysaccharides indicate that, at the intact-fiber level, polysaccharide is associated with the collagen in such an intimate fashion as to effect its physical stabilization. It would appear, therefore, that collagen and elastic fibers are similar in that they both contain polysaccharide. The identity of the polysaccharide is indeterminable by histological techniques, and chemical methods are still incapable of differentiating between polysaccharides derived from two or more components of such a complex tissue. If histological differentiation of intact collagen and elastin is difficult, the separate identification of these two fibers in the presence of partially degraded material derived from either or both is even more unsatisfactory. Unna (1890) classified the structures observable in connective tissue as : collagen, collacin, collastin, elastin, and elacin. H e regarded them as a series of products with tinctorial properties which merged imperceptibly into one another. The same stain was used for all these elements, and it

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was difficult to identify any particular structure unless a full spectrum from collagen to elacin were present for comparison. Fullmer and Lillie (1957) have shown that variations in p H can affect the differential staining properties of collagen elastin. Staining with resorcinol-fuchsin depends on the adsorption of the dye onto the fibers by forces that are antagonized by polar groupings. Collagen and elastin differ chemically, among other things, in the amounts of glutamic and aspartic acid present in each, and at p H values in the range 2 to 3 all the acid groupings on the side chains of elastin are back-titrated, whereas an appreciable number of those of collagen remain ionized. This permits differentiation between elastin and collagen, since the latter repels the dye while the former adsorbs it. At lower p H values, however, all acid side chains are backtitrated on both fibers, and both take up the stain. Variations in pH, however, may occur locally owing to the proximity of acidic polysaccharide, and stains based on phenomena that are susceptible to such changes cannot be universally applicable. Gillman et al. (1954) studied the possibilities of a number of elastica stains as a means of differentiating, not only between collagen and elastin, but also between normal and degenerate elastica-staining material. They discarded many of’ the usual elastica stains-orcein (Unna, 18%), Weigert’s ( 1898), Verhoeff’s ( 1908), Gomori’s aldehyde-fuchsin ( 1950), etc.-but demonstrated that a number of other stains could differentiate between true elastic fibers and other elastica-staining material which they suggested should be called “elastotically degenerate collagen.” Positive reactions with a number of these stains could be correlated with the liberation of polysaccharide from the fibers, during the induction of general elastica-staining properties or the adsorption of polysaccharide onto the surface of degenerate fibers. Gillman’s stains have not been employed to their full as yet, as will be seen from the fragmentary evidence presented in Section 11. C concerning those pathological conditions in which elastoses appear to occur, but their use may facilitate the differentiation of many structures hitherto thought to be similar. 2. Theories of Staining. Braun-Falco (1956) examined the way in which the two stains aldehyde-fuchsin (Gomori, 1950) and resorcinolfuchsin (Weigert, 1898) react with elastic tissue. By blocking reactive groups in both collagen and elastin he demonstrated that the specificity of the staining reaction depends on the availability of polar groups. This is in agreement with the observations of Fullmer and Lillie mentioned above (1957), in which alterations in p H produce a similar effect. BraunFalco concluded that the adsorption of the dye molecule onto the fiber is brought about by the reaction of basic centers in the dye through a dipole with the main chains of the fiber.

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Schwarz and Dettmer (1953) and Dettmer (1952) have shown by electron-microscope examination that the dye is bound to the amorphous matrix of the fiber. They suggest that if this material is destroyed by elastase the fiber no longer stains with Weigert’s stain (see also Lansing, 1951), since the fibrils that lie in the interior of complete fibers do not stain with elastica stains. If this is true, it would be expected that treatment such as that employed by Partridge and Davies (1955), which, according to Hall (1957c), may remove the larger part of the elastomucin coat, would result in the destruction of elastica-staining properties. This is, however, not so (Partridge and Davies, 1955 ; Wood, 1958), but much of the elastica-staining material does appear to be associated with those portions of the fiber that dissolve most rapidly. Sax1 (1957a) showed that elastic fibers still retained their structure after they ceased to take up elastic stains. Sachar et al. (1955) utilized the release of stain from stained elastic tissue as a measure of elastolytic activity, but Findlay (1954) claimed that the staining of elastic tissue with resorcinal-fuchsin produced inhibition of elastolysis. H e drew attention, however, to the facc that this was true only of intact tissue, whereas Sachar et al. used pow. dered elastin. Similarly the observations by Partridge and Wood regarding the retention of staining properties by purified elastin may be due to the presence of the small amount of polysaccharide and p-protein (cf. Section 111. A ) which is still present. These results are not necessarily at variance but may appear to differ on account of the varying degrees of disorganization or partial purification to which the starting material is subjected. Both orcein and resorcinol-fuchsin contain phenolic compounds, and their mode of attachment to elastin would appear to be similar (Michaelis, 1901). Fullmer and Lillie (1956), for instance, have shown that staining with orcein is independent of p H and is not affected by blocking the hydroxyl, amino, carboxyl, or aldehyde groups. No studies similar to those of Braun-Falco for resorcinol-fuchsin regarding the staining of altered collagen have been attempted for orcein, but Tunbridge et al. (1952) pointed out that the partial degradation of collagen by proteolytic enzymes such as pepsin induced in the early stages of attack a high degree of affinity for orcein, and it may well be that exactly similar reactions account for the specificity of both dyes. The main conclusion to be deduced from histological observation, therefore, is that many of the older techniques for differentiation between collagen and elastic fibers, and more especially “elastotically degenerate collagen,” are inadequate. Although recent studies on the mode of action of the stains, when taken in conjunction with chemical studies on the

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connective tissue fibers, may enable more specific stains to be devised, the results so far obtained have been meager. C.

Morphological Studies on the Pathological Involvement of Elastic Tissues

Increases in material having elastica-staining properties have been reported in a number of pathological conditions, especially those in which involvement of the dermis occurs. 1. Senile Elastosis. Kissmeyer and With (1922) found an increase in elastica-staining material in dermis from subjects between the ages of 26 and 40 but pointed out that the changes are most marked in the exposed sites from fair-skinned individuals. This was confirmed by Dick (1947) and by Tattersall and Seville (1950), and, although Ejiri (1936, 1937) did not differentiate between exposed and unexposed skin surfaces, his general findings were in agreement and were supported by the negative observation of Hill and Montgomery (1940) that no changes occur in skin from covered regions of the body. In general there is an increase in elastica-staining material in exposed tissue of elderly subjects, at the expense of collagen-staining fibers. It has been reported (Findlay, 1954) that in the deeper layers of the dermis the finer elastic fibers degenerate with the production of (‘elacin,” a lessening of Weigert-positiveness, and the appearance of islands of PASpositive material. Nearer the surface, masses of PAS-positive material unite to form the elastin-like colloid to which the name (‘collacin” has been given. There is no evidence from histological studies alone, that the elasticastaining material present in these pathological tissues differs from that present in normal tissue except that the material that appears in the upper layers of senile dermis is composed of broad ribbonlike structures as opposed to the fine fibers originally present. An explanation of the different appearance of the fibers and evidence for their origin was first given by Tunbridge et al. ( 1952), who by electron-microscope studies showed that there is no increase in true elastic fibers in senile skin, but that exposed areas contain large quantities of bent and broken collagen fibers coated loosely with amorphous particulate material. They also showed that short periods of treatment of native collagen with pepsin produce material having similar staining properties and a similar appearance under the electron microscope. Lansing ( 1951) and Findlay (1954), however, studying the solubility of these senile fibers under the action of elastase, showed that senile elastica is very susceptible to elastase and hence assumed that normal elastic fibers occur in abundance in senile tissue.

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The validity of such an assumption is, however, dependent on proof of the absolute specificity of elastase. There was no evidence against such a concept at that time, but subsequent studies have shown that elastase is capable of attacking thermally denatured collagen (Banga, 1953 ; Hall et al., 1953). More recently Hall (1957b) has shown that a soluble fragment of high molecular weight which can act as a substrate for elastase can be obtained from collagen by the action of collagenase. It would appear that certain elastase-resistant polysaccharides and procollagen must be removed from collagen by either thermal or chemical denaturation (Balo et al., 1956) before it can serve as a substrate for elastase. The specific groupings attacked may be similar to those attacked by chymotrypsin, since both enzymes hold certain synthetic peptides in common as substrate (Grant and Robbins, 1957). 2. Ehler’s Danlos Syndrome-Rubber Skin. As the trivial name for this condition implies, it is associated with hyperelasticity of the dermis. In normal skin the dermis consists of bundles of collagen fibrils, which are in themselves virtually inextensible. The mobility and tone of the skin is maintained by the fact that the bundles lie at an angle to one another, so that stresses may be taken up by distortion of the normal “weave” of the collagen bundles, before force is exerted on any individual bundle or fiber. Jensen (1955) has suggested that to account for hyperelasticity one must assume that the network of collagen bundles is abnormal and that the bundles lie roughly parallel to one another. An applied force is thus capable of separating the bundles, with consequent distention of the dermis. This theory, however, fails to take account of two salient factors. The model proposed for the dermis cannot account for the strength of the tissue, on the one hand, and, on the other, the dermis contains unusually large amounts of elastin. If the collagen fibers do not form an interwoven pattern, the strength of the skin can be dependent only on the ground substance, and this could not account for the appreciable, although markedly reduced, stability of the tissues. Histologically, dermis from subjects with Ehler’s Danlos syndrome is characterized by the presence of a high concentration of elastica-staining material, differing from that observed in senile elastosis, however, in that the fibers more nearly resemble those of normal dermis, differing only in number, and Tunbridge et al. (1952) showed by electron-microscope examination that this was in fact a true’elastosis. Sax1 and Graham (in Bourne, 1956), searching for a systemic cause for the condition, showed that elastic tissue from a bulla, excised from the anterior surface of a hypermobile knee, was highly susceptible to the action of the enzyme

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elastase, whereas apparently normal elastic tissue from adjacent regions was almost completely resistant to the enzyme. Such resistance could be imparted to tissue by the adsorption during life of an inhibitory substance from the serum, via the tissue fluids. An increased serum inhibitor level was observed, but the full implications of this have not yet been discussed. 3. Pseudoxanthoma Elasticum. This condition, which is also associated with marked cutis iaxa, has been described as an elastosis on the basis of increased elastica-staining material which shows signs of degeneration. Thomas and Rook (1949) and Hannay (1951) have stated, however, that the “elacin” which is present is too abundant to have originated solely from the elastic fibers originally present, and ascribe the elastica staining to degenerate collagen. Here again, as in senile elastosis, the elastica-staining material after which the condition is named has been shown by electron-microscope studies to be due to degraded collagen (Tunbridge et al., 1952). 4 . Colloid Millium. I n this condition, the elastic fibers originally present in the skin swell, and when stained with Mallory’s phosphotungstic acid hematoxylin stain demonstrate orange masses of swollen material coalescing where the fibers cross (Findlay, 1954). The ultimate stage is an amorphous, nonfibrous mass of PAS-positive material. This dissolves rapidly under the action of elastase, revealing the remains of fibers which have not progressed so far in degradation and which are more slowly dissolved by the enzyme. Findlay suggests that total elastolysis is assisted in the case of colloid millium by the fact that the elastomucin has been separated from the fiber. Since he employed crude elastase preparations which most probably contained both proteolytic and mucolytic components (Hall, 1957a), these observations are in complete accord with present views on elastase action. 5. Obliterative Diseases of Elastic Fibers. In the fibrous tumors of the skin of patients suffering from infection by the helminthic parasite Onchocerca volvulus the elastic fibers of the affected dermis are completely destroyed. I n the burnt-out stage of the disease, although the damaged collagen bundles may re-form perfectly, there is evidence that elastic fibers are not re-formed (Jamison and Kershaw, 1956). In this the condition differs from lathyrism which arises from feeding the seeds of the sweet pea (Lathyrus odoratus), in which case there is evidence that elastic fibers, if already formed, are unaffected, although their initial formation is prevented if the causative agent is administered early enough.

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D. Structural and Physical Aspects of Connective-Tissue Fibers 1. Morphology under the Light Microscope. Examination of unstained preparations of connective tissue under the light microscope demonstrates collagen and elastic fibers as two distinct species. Collagen occurs in bundles of straight or slightly wavy, white fibers which form networks but which show no signs of branching. Elastic fibers, on the other hand, are fine individual elements which follow an irregular course through the tissue lying on and around the bundles of collagen fibrils. They branch and anastomose with one another freely. This description of elastic material refers to the fibers as they occur in dermis and in the interlamellar areas of arterial media, but not to the lamellar structures of the media and the broad fibers of ligament which differ from the fibrils in their staining and physical properties as well as by morphological criteria. The internal organization of these various structures cannot be seen at the level of the light microscope, but certain broad assumptions regarding the arrangement of the various subfibrous components can be made from a consideration of their appearance in polarized light. Collagen fibers from all sources show an appreciable degree of birefringence, but this is not true of elastic components. Thus it may be deduced that the internal structure of the collagen fiber is more highly oriented than that of the elastic fiber. 2. Electron-Microscope Appearance. The deduction referred to above is borne out by an examination of connective tissue under the electron microscope. Collagen fibers characterized by a regularly repeating system of cross-striations at 640-A. intervals (Wolpers, 1944 ; Gross and Schmitt, 1948; Gross, 1949; Wyckoff, 1949) and by finer interband structures (Hoffmann et al., 1952) differ considerably from elastin (Wolpers, 1944 ; Gross, 1949). Hall et al. (1955b) described the gross structure of elastic material from aorta and from ligainentum nuchae and showed that the amorphous structure ascribed to the elastic fiber as a result of observations with polarized light was borne out by electron-microscope studies. In the aorta at least two major components were apparent, one frankly fibrous, the other appearing to consist of a network of fibers covered with an electron-dense, formless coating ; Keech et al. ( 1956) have since reported the existence of many apparently different forms of elastic fiber in dermal preparations. The electron opacity of the outer layer of both the elastic fiber and the lamella has rendered it impossible to obtain more than fragmentary evidence concerning its inner structure from an examination of intact material.

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During fragmentation, however, small areas of the outer sheath are stripped away, revealing a finer fibrillar structure within. This disturbance of the architecture of the elastic structures, which is an essential concomitant of the teasing process, makes it difficult to differentiate between true substructures and those that arise as artifacts in the preparation procedure. The advent of the thin-section technique (Neuman et al., 1949) should enable more detailed localization of smaller elements to be made. Rhodin and Dalhamn (1955), however, examining the lamina propria of the trachea of the rat, showed that little structural detail could be observed within this fiber in the intact state. Hall et al. (1952, 1955b) and Lansing et al. (1952) utilized crude elastase preparations to examine the finer structure of elastic elements. Both groups showed that a finer fibrillar structure was revealed after the removal of the outer electron-opaque coating. The collapse of the structures also appeared to present evidence for the penetration of this amorphous phase in between the individual fibrils. It would, therefore, be more correct to refer to it as an amorphous matrix. Owing to the crude nature of the elastase, it was not possible to stop the reaction after the removal of the matrix, and hence details of the underlying fibrils could not be obtained, since they were degraded simultaneously. Sax1 (unpublished work), using purified enzyme preparations, has been able to demonstrate fine detail in the inner fibrillar structures. 3. X - R a y Diffraction Studies on Connective Tissue. The X-ray diffraction pattern of elastin was first investigated by Kolpak (1935), who found unstretched fibers to give diffuse rings, whereas stretched samples gave a meridional arc corresponding to a spacing of 3 A. and equatorial spots characteristic of spacings of 11.5, 5.9, and 4.6 A. Astbury (1938, 1940) attributed these findings to the presence of small quantities of collagen fibers, which, although themselves fully oriented, were arranged haphazardly in the elastic fiber. At such low concentrations it was impossible to identify a collagen powder diagram superimposed on that of an elastic fiber, but when the collagen fragments were aligned by stretching the elastic fibers the more discrete collagen fiber diagram became visible. Astbury drew attention to the fact that prolonged boiling prevented the observation of a collagen pattern even after stretching. H e felt, however, that elastin should be included in the collagen group of fibers, as opposed to the keratin-myosin-epidermin-fibrinogengroup, and suggested that it might represent a member that was permanently in a contracted state on account of its thermal transition point’s being below that of the animal body. Bear (1944) recorded small-angle X-ray patterns of collagen, as faint arcs on a photograph of beef ligament, but again ascribed these to the

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presence of collagen as an impurity, and in a review of the collagen fiber (1956) suggested that elastin should not be included in the collagen group. When reviewing the position in 1953 Kendrew assessed the evidence as adequate to justify the exclusion of elastin from the collagen group, but electron-microscope studies may necessitate a reconsideration of this point of view. Collagen fibrils have been observed in elastic tissue by a variety of workers, but there is still no direct evidence as to whether structures having the characteristic spacings of collagen at either electron-microscope (640-A.) or X-ray diffraction (2.86-A.) levels constitute an integral part of the elastic fiber structure. 4 . Physical Properties of Collagen and Elastin. The tensile properties of collagen and elastin differ considerably, the Young’s modulus of the elastic fibers being smaller than that of collagen by a factor between 400 and lO,oOO, whereas the extension at break is 20 to 30 times as great (Buck, unpublished results quoted in Burton, 1954 ; Krafka, 1937). Much of the early work on the thermoelasticity of elastic fibers was carried out on whole ox ligament. Meyer and Ferri (1936) and Wohlisch et al. (1943) showed that ligament behaved as a rubberlike solid up to 100% extension and that extensibility was independent of time up to 50% extension. Lloyd and Garrod (1946) later showed that similar elastic properties, which could be represented by a model consisting of a steel spiral spring maintained in a partially compressed state by a rubber band, could also be demonstrated in elastic fibers freed from collagen and ground substance. Wood (1954) examined the effect of various reagents which might remove or destroy polysaccharide present in both collagen and elastic fibers. H e showed that mucopolysaccharides appeared to be of greater importance in stabilizing the collagen component of the tissue than the elastic fibers. H e also reported that the collagen associated with the elastic fibers appeared abnormal in that it could be extended by 70 to 75% and pointed out that Banga (1949) had reported an abnormal collagen in association with elastic fibers on the basis of determination of flow birefringence on the protein extracted from aorta with urea solution. Wood’s recent studies on the tensile properties of reconstituted elastic material (1958) have confirmed that as far as stretching phenomenon are concerned elastic fibers are not dependent on a polysaccharide component for the type of load-extension relationship observed. The dependence of collagen on polysaccharide for its thermal stability and resistance to extension has been studied by Jackson (1954). It would appear that at the fiber level extensibility can be increased, as also can solubility, with a concomitant lowering of the thermal transition tempera-

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ture, by procedures that remove polysaccharide. Hall and Reed (1957) ascribed the variations in thermal stability which could be associated with differences in collagenase susceptibility to changes in the polysaccharide content of the fibers rather than to fundamental changes in amino acid analysis. 5. Model Structures for Elastic Fibers. The nature of the internal fibrillar structure of elastin has been in doubt for some time. Hall et al. (1952, 1955b) could not determine the fine detail of fibrils released by elastase because of the simultaneous degradation of fibrils and amorphous matrix. Gross (1949) claimed to have revealed helical structures under the action of trypsin, although these were later shown to be contaminants derived from trypsinogen (Franchi and De Robertis, 1951 ; Gross, 1951). Lansing et al. (1952) also identified helical fibrils in the elastase degradation products of the elastic fibers of ligament. Another possibility was introduced by the observations of Schwarz and Dettmer (1953), who claimed to have revealed striated fibrils akin to collagen by treatment of aortic tissue with elastase. These observations led Banga (1953) to suggest that collagen fibrils constituted the core of all elastin fibers. She drew attention to the fact that workers other than Schwarz and Dettmer had invariably employed elastic fiber preparations obtained by heat treatment of elastic tissue. She showed that thermally denatured collagen is susceptible to elastase action, and hence any collagen at the center of heated elastic fibers would be altered to an elastase-susceptible form. This suggestion was questioned by Hall et al. (1953), who pointed out that Schwarz and Dettmer did not present adequate evidence that the collagen fibers surrounding the elastica had been removed prior to attack by elastase, although they agreed with the premise that thermally denatured collagen could act as substrate for the enzyme. Keech and Reed (1957a) showed that elastic fiber preparations obtained from aorta or ligament by treatment with boiling 2% acetic acid (Gross, 1949; Hall et al., 1952, 1955b) were complex structures from which collagen fibers could be liberated by short periods of treatment with collagenase. Based on the foregoing morphological evidence, and supported by biochemical studies, a model structure for elastic fibers was proposed by Hall ( 1 9 5 7 ~ ) . H e suggested that elastic fibers are essentially biphasic-an outer layer which contains polysaccharide and protein surrounding an inner layer which consists solely of protein. Romhanyi (1955), on the other hand, suggested that elastin might consist of at least three concentric cylindrical structures. H e based his argument on the apparent changes in diameter of elastic fibers after treatment with either aniline or phenol or after staining with resorcinol-fuchsin.

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Romhanyi’s experiments indicate that it is difficult by purely optical means to define the exact boundary of the fiber, since different reagents reveal “cylinders” of varying diameters. Hall ( 1 9 5 7 ~ )drew attention to this phenomenon when comparing the results of enzymatic studies on elastic fibers with those of Partridge and co-workers (1955) using purely chemical methods. The main difference between the two sets of results could be ascribed to the fact that the amount of tissue included within the hypothetical outer surface of the fiber could be varied in such a way as to include or exclude the majority of the polysaccharide-rich material. Since fibers from which the majority of the polysaccharide had been removed still retained their structure, Partridge suggested that polysaccharide was not a necessary component. A similar conclusion has been advanced by Wood (1958) on the basis of physical determinations on reconstituted elastic material. Many of the properties of reconstituted elastin differ quantitatively, however, from those of the native fibers, and, although this may be an indication of the degree of main chain hydrolysis which occurs during solution with oxalic acid, it might also represent the effect of the removal of an outer structure rich in polysaccharide. Morphological and enzymatic studies, therefore, indicate that the line of demarcation between the native elastin fiber and the surrounding ground substance should be drawn in such a position that an appreciable amount of mucopolysaccharide is included, and that the presence of this material may have a considerable part to play in the stabilization of the fiber. Chemical and physical examination of the fibers, on the other hand, dealing with properties which appear to be those of the protein alone, indicate that the fiber is delineated by a cylindrical surface enclosing only fibrous protein. These two sets of views are not necessarily in conflict, since the same properties are not selected for comparison.

111. BIOCHEMICAL STUDIES ON COLLAGEN AND ELASTIC FIBERS A . Chemical Composition of Collagen and Elastin Prior to the last ten or twenty years, analyses of protein- and polysaccharide-containing tissue preparations have not been dependable. This has been especially true of insoluble structures such as collagen and elastin. Amino acid analyses of the entities which purport to be the pure proteins from these tissues have become available only recently, as methods of analysis have improved, but even now results must be considered in relationship to the methods of preparation employed. The position with respect to polysaccharide is even more complex. Carbohydrate may be present in connective tissue either free or combined in the amorphous

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phase, or combined either loosely or by firmer linkages with the fibrous structures themselves. Methods of preparation may well bring about only partial fractionation of these different species of polysaccharide, especially if two or more are combined in any one site. 1. Amino Acid Analyses. Studies of the amino acid analysis of collagen preceded those on elastin because of the greater ease with which analyses could be performed on the derived protein, gelatin. Recent analyses for collagen (Bowes et al., 1955) have justified the assumption that the conversion from collagen to gelatin is accomplished without the removal of amino acids or small peptides. Hence it must be assumed that the recent discovery of between 20 and 30% of a protein with an entirely foreign analysis in gelatin (Russell, 1957) implies the presence of a similar degree of heterogeneity in collagen itself. The analysis of elastin has until recently been accomplished only by the removal of all other extraneous material by methods depending on the relative inertness of elastin to chemical attack. Thus Stein and Miller (1938) stated that elastic tissue could be boiled for prolonged periods in water, dilute acids, or alkalies or strong urea solutions without any variation in the amino acid analysis of the residue. Hall (1951, 1955) showed, however, that this was only partially true. Analyses such as those obtained by Lansing et al. (1951) for old aortic elastin are dependent on the reagent employed for purification. The material by which the amino acid composition of such preparations differed from the classical elastin analysis is resistant to boiling 2% acetic acid but dissolves after prolonged treatment with boiling 40% urea solution. Even the resistance of young elastin to boiling urea solutions is dependent on the tissue-solvent ratio. It would appear, therefore, that with the exception of the classic amino acid analysis for ox ligamentum elastin, young aortic elastin, etc., which appears constant, all variant analyses for elastin are a function of the method of purification. The analysis of native elastin can be compared and shown to agree substantially with that of soluble elastin preparations obtained by the action of oxalic acid on purified elastic fibers (Adair et al., 1951). Partridge et al. (1955 ; 1957) showed that elastin dissolves rapidly in boiling 0.25 M oxalic acid with the release of acidic acids, and the production of two species of soluble protein (a- and p-elastins). Chemical studies indicated that the two derived proteins, although they differ considerably in molecular weight ( 6 7 , O and 5500) , do not show marked differences in the number of N-terminal residues. Partridge et al. suggested that the p-protein consists, on the average, of two chains containing 27 residues, and the a-protein of seventeen chains with 35 residues each. These chains appear to be linked laterally to one another by acid-resistant cross linkages,

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the nature of which is still obscure. The rates of production of these two fragments preclude the formation of the smaller 0-fraction from the larger a-component, although Bowen (1953) provided evidence that such a relationship might occur in the case of fractions obtained from urea-soluble elastin. In general these findings may be taken as indicating that the gross structure of elastic fiber is heterogeneous. If this is so, however, the analytical evidence would indicate that both species of protein, although differing in size and chain organization, are chemically similar. Hall ( 1 9 5 7 ~ )suggested that the 0-protein is present in the amorphous coat and matrix of the fiber, and the more highly polymerized a-material constitutes the internal fibrillar element. No evidence has as yet been advanced which suggests that elastin is chemically heterogeneous such as would appear to be the case with collagen and gelatin (Russell, 1957), but there is increasing evidence for structural heterogeneity (cf. also Section 11. D ) . Even doubts as to the unitary nature of both collagen and elastin, however, do not invalidate comparisons of the amino acid composition of the two proteins if adequate specifications of the methods of preparation are provided. Figures are now available which account for 96% of the amino acids of collagen and 106% of those of elastin (Bowes et al., 1955; Partridge and Davies, 1955), and it appears unlikely that significant changes in these values will be introduced by subsequent work. Collagen and elastin differ from most other proteins in that the sum of their glycine and proline residues together amount to 33 and 41%, respectively, of the total number of residues. Another qualitative similarity is the presence of hydroxyproline in both proteins. The values are, however, quantitatively different. Collagen with 11.07 g. of residues per 100 g. has seven times as much hydroxyproline as elastin. This again may be due to heterogeneity in the elastic fiber, since the small amount of hydroxyproline present in elastin could be accounted for by the retention of collagen in the elastin preparation (cf. Section 11. D ) . No method of preparation which has so far been employed, however, has resulted in the complete removal of hydroxyproline. Harkness et al. (1957) showed that in dog artery the amount of hydroxyproline in elastin may vary from 2 to 1% from animal to animal, but lower values were not obtained. Neither protein contains tryptophan, tyrosine, or cystine in significant amounts, but there the similarity ceases. Elastin is predominantly nonpolar, only 0.1% of the amino nitrogen being provided by polar amino acids, as opposed to the 28.9% of collagen. Their place is taken by monoaminomonocarboxylic acids, especially those of larger residue weight. The net result of these differences on the titration curves of collagen and elastin has been discussed by Bowes and Kenten (1948) and Bendall (1955).

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The latter, using rigorously purified material, showed that the number of polar groups which could be calculated from the titration curve for elastin were in close agreement with the analytical figure for these amino acids, thus indicating that in the preparations employed (Partridge and Davies, 1955) no other polar groupings were present. 2. Polysaccharide as a Component of Connective Tissue. The evidence for the presence of polysaccharide as an integral part of collagen or elastic fibers is as yet mainly inferential and not analytical, the main reason being the difficulties experienced in determining whether the bonds holding the polysaccharide in close association with the fibers are sufficiently strong to justify the classification of the polysaccharide as a structural component. Part of the polysaccharide can, however, be so classified. Both collagen and elastin can withstand a considerable degree of chemical treatment without losing their fundamental fibrous structure. After such treatment a small amount of polysaccharide can still be identified in the protein preparations (collagen: 0.42%, Grassmann et ul., 195713; elastin: 0.3%, Partridge and Davies, 1955; 0.1, Wood, 1958). The identity of the sugars obtained by hydrolysis of this polysaceharide has been determined in the case of collagen, glucose and galactose (Grassmann and Schleich, 1935 ; Gross et al., 1952), fucose (Glegg et al., 1953), and glucosamine (Schneider, 1940, 1949). In elastin Lloyd (in Bourne, 1956) has shown that a small amount of polysaccharide remains inseparable from the protein even after prolonged extraction with hot sodium chloride and cold calcium chloride solutions. A further fraction remains attached to protein; but the complex can be extracted by neutral calcium chloride solution. This material is insoluble in buffer solutions after removal of the calcium chloride by dialysis but can be made to pass into solution under the action of elastase without the liberation of aldehyde groups. Among polysaccharides more easily, extractable from connective tissue, Meyer has identified hyaluronic acid, chondroitin sulfates A, B, and C, heparatin sulfate, and kerotosulfate. Since both collagen and elastin are present in the ligament, and the interfibrous spaces are filled with polysaccharide-rich ground substance, it is difficult to assess the exact site of attachment of any one polysaccharide. This can, however, be attempted by histochemical and electron-microscope methods. Rinehart and AbulH a j (1952) have shown the existence of acid polysaccharide on the surface of the elastic fibers, and Hall et al. (1952, 1955b) have shown that elastase can remove acid polysaccharide from the surface and interfibrillar regions of elastic lamellae in aorta before destroying the underlying protein structure.

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3. Lipid. The involvement of lipid in the structure of connectivetissue fibers has been suggested for some time. Reticulin contains, in addition to the collagen protein, a mixture of lipid and polysaccharide (Kramer and Little, 1953), and Little and Windrum ( 1954) have reported that a relatively high proportion of the native collagen fiber consists of myristic acid. Lansing et al. (1952) suggested that elastase was, in fact, a lipase on the grounds that under the action of the enzyme Sudanophilic droplets of low density were liberated from elastic tissue. Sax1 (1957a) recognized the presence of both neutral and acid lipid in whole elastic tissue, but pnly the neutral lipid was liberated by elastase, the acidic lipid being destroyed. The addition of serum protein resulted in the hydrolytic fission of the neutral fat. B. Distribution of Collagen and Elastic Fibers 1. Methods of Determination. Early studies on the distribution of the various connective-tissue fibers were performed by histologists employing differential staining methods. The results obtained were sufficiently accurate to justify classification of tissues as predominantly collagenous, elastic, or of mixed composition containing appreciable concentrations of both fibers. Quantitative values for the relative proportions of elastic fibers were difficult to obtain, however. The advent of chemical analysis permitted numerical values to be ascribed to the relative concentrations of the separate components, and, based on these figures, many theories relating tissue architecture to function were propounded. The indiscriminate use of these values may be no more justifiable than the use of visual assessment of stained areas of a section. Employed with circumspection, however, chemical analyses of tissue can present evidence of considerable importance in studies of the changes occurring in tissue during differentiation, growth, and senescence or with the onset of pathological conditions. The apparent inertia of elastic fibers to chemical attack proved an important property in devising methods of analysis. Most procedures have consisted in treatment with boiling water, acetic acid, or alkali, or autoclaving with these reagents (Lowry et al., 1941; Neuman and Logan, 1950). I n choosing appropriate conditions, the period of treatment was determined to ensure the removal of all extractable material. Gross (1949) and Hall et al. (1952), for instance, stated that treatment with acetic acid brought about the removal of all morphologically discernible collagen from elastic tissue. This method has been criticized by Partridge and Davies (1955) as being too drastic. I t is, however, possible that their apparently milder reagents bring about preferential removal of polysaccharide-rich fragments from the elastic fiber itself. Retention of collagen or ground

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substance, on the one hand, or removal of part of the elastic fiber, on the other, tends to produce extremes of analytical values, which differ considerably. Moreover, the existence in aorta, in addition to collagen and elastin, of a third component which could not be dissolved by acetic acid but which was more soluble in boiling 40% urea solution than true elastin indicated the fallacy of applying any one analytical procedure to a tissue (Hall, 1951, 1955). Since the material is retained with elastin after extraction with acid or water, all methods based on this type of procedure give erroneous values for elastin content in aorta. The amount of elastin in the tissue can be calculated either from the dry weight of the residue or from the hydroxyproline content of a hydrolyzate of the residue (Harkness et al., 1957), the collagen content being calculated from hydroxyproline determinations on the extracted protein. Harkness et al. showed that hydroxyproline content of the elastin varied by over 100% in twelve dogs, thus making it difficult to determine by this method the amounts of elastin present. Similarly, figures for collagen extracted by p H 5 phthalate buffer (Hall, 1957b) calculated on the hydroxyproline content, when compared with the actual amount of protein extracted, present the paradoxical situation that the collagen content of the extracted protein is 175%. Hence all numerical values for collagen/ elastin ratios in tissue must be considered in relationship to the method of analysis. 2. The Relative Distribution of Collagen and Elastin. Despite these drawbacks to the assessment of numerical values for collagen and elastin content, interesting observations have been made, notably the contribution of Harkness et al. (1957). These workers, studying the collagen and elastin content of the aorta of dogs, employed autoclaving with water followed by boiling with decinormal alkali to remove first the collagen, and second a fraction--"dry material other than collagen and elastin"-which they did not analyze. They reported that in adult dogs, the ratio elastin/ elastin plus collagen remains roughly constant at 50 to 60% from the aortic valves to within some 5 cm. of the diaphragm. The proportion of elastin then decreases rapidly and remains at a value between 25 and 30% throughout the abdominal aorta and the iliac, femoral, and saphenous arteries. A similar drop occurs at the point of departure of the arteries at the upper confines of the thorax. In young animals the drop is from 70% to 60% (newborn), 50% (3 weeks), and 35% (6 weeks).

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C. The Enzymatic Stcsceptibility of Elastin and Collagen

1. The Elastase Complex and Its Physiological Significance. The earliest positive observations on the action of mammalian enzymes on elastic tissue were reported by Ewald (1890), but he employed crude enzyme preparations, and it was not until 1949 that Balo and Banga isolated an enzyme in a partially purified form from pancreas (1949a). This appeared to be specific for elastic fibers. Their studies arose from a search for the causative agent for the degradation of elastic fibers and lamellae such as occurs in aortic media during arteriosclerosis. The activity of the enzyme can be controlled by a component of serum which has positive inhibitory activity, and Balo and Banga (1949b) suggested that the decrease in the inhibitor which could be correlated with the onset of arteriosclerosis could account for an apparently unchecked activity of the enzyme resulting in medial degeneration. Lansing (1955) pointed out that the decrease in inhibitor content could also be correlated with increased age of the patient and might have no direct connection with the onset of degeneration, a point of view which was also accepted by Balo and Banga themselves (1953) and which constrained them to suggest that elastase might be concerned with the synthesis of elastic fibers as well as their disruption, and that it might be a failure of synthesis that accounted for the changed appearance of the elastic fibers in arteriosclerosis. The whole question of the physiological significance of the enzyme is bound up with the ultimate results of as yet unsuccessful attempts to prove that it is present in the circulating blood. If we assume that elastic fibers are synthesized within the body, some elastoclastic mechanism must also be operative if generalized elastosis is not to occur. In the adult animal (Slack, 1954) the turnover of glycine in the elastic tissues is very small, and it may well be that the amounts of enzyme necessary for this low rate of catabolism may lie below the threshold of analysis. There may, however, be positive removal of elastase from the circulation by the formation of a triple complex between enzyme, substrate, and inhibitor (Saxl, 1957a), and this may be of significance in those pathological conditions in which resistance to elastase attack is apparent. A considerable amount of contradictory circumstantial evidence regarding the systemic or digestive role of elastase has been reported recently. Lansing et al. (1953), studying the teleost fish Lophius piscatorius, in which the pancreas is located in two anatomically separate sites, showed that elastase is secreted only by islet cells. Further evidence for this was obtained by Carter (1956), who reduced the elastase content of dog pancreas by the administration of cobalt. Hall et al. (1952) also reported

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their inability to identify elastase in the pancreas of the human subjects who were diabetic. Balo and Banga (1950), however, stated that in the pancreas elastase occurred in conjunction with an inhibitor which could be removed by dialysis or by acid treatment. Grant and Robbins (1955) also identified an inactive elastase in pancreas and suggested that the removal of the “inhibitor” represented the conversion of a zymogen to the free enzyme. Kokas et al. (1951) demonstrated the presence of the zymogen in pancreatic juice and hence assumed that elastase was a product of acinar tissue. From these conflicting reports it is evident that the exact location of the cells that synthesize and secrete elastase remains in doubt. 2. Specificity of the Enzymes. Balo and Banga (1949b) reported that the elastin-elastase system was specific in so far as native elastin fibers cannot be degraded by any other of a considerable variety of enzymes. The selective removal of elastin and failure to attack collagen suggest that elastase may be specific; this is, however, not the case. Elastase activity is not restricted to the solubilization of a single substrate. This more general activity was first recorded by Banga (1953) and Hall et al. (1953) , who simultaneously reported that thermally denatured collagen could also act as substrate for the enzyme. The latter group of workers also claimed that elastase could degrade the insoluble proteins of the lens. These observations were confined to relatively crude enzyme preparations which might also contain other proteolytic enzymes. It has been pointed out that similar differences exist between the susceptibility to trypsin of collagen fibers before and after thermal denaturation, and the effects observed may not be typical of elastase. Other evidence is, however, available for the interaction between elastase and partially degraded collagen. Both Findlay (1954) and Dempsey and Lansing (1954) quoted the elastase susceptibility of the elastica of senile elastosis as indicating that true elastic fibers were synthesized during the onset of the senile changes. Tunbridge et al. ( 1952), however, showed by electronmicroscope studies that this material was in fact denatured and partially degraded collagen. Balo et d. (1956) showed that the metacollagen (cf. Section V. A) was also susceptible to elastase, and Hall has demonstrated ( 195713) that certain high-molecular-weight material obtained from collagen after its dissolution by collagenase (CZ. wekhii) is broken down into smaller molecules by e1astase.l Treatment of whole elastic tissue with elastase at the lower optimum pH of 7.8 (Hall, 1957a) results in the liberation of metachromatic 1 The general question of the importance of the polysaccharide in determining the susceptibility of elastic fibers in aorta to elastase attack has recently been discussed by Yu and Blumenthal (1958).

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material from the fibers, and also from the surrounding ground substance. A similar effect has been observed by Rinaldini (1958), who has employed elastase preparations rich in mucolytic activity to separate aggregated cells for tissue culture. It would appear that the linkage between polysaccharide and protein is attacked and that either the same polysaccharide is present in ground substance as in the fiber, or the mucolytic enzyme is no more specific than the proteolytic component. Grant and Robbins (1957) advanced the hypothesis that the role of elastase in metabolism might be that of an endopeptidase on the basis of a comparison between the activity of various elastase preparations, trypsin, and chymotrypsin on a variety of substrates. Elastase in a relatively high degree of purity showed appreciable activity with acetyl-L-tyrosine ethyl ester ( A T E E ) as substrate, thus demonstrating its similarity to chymotrypsin. Under similar conditions, however, elastase also digested casein and hemoglobin, and thus its A T E E activity might be due to the presence of chymotrypsin in the elastase preparations. On the other hand, the most active elastase preparation was one precipitated from solution by dialysis, a phenomenon not observed with chymotrypsin. This possible relationship has not yet been fully elucidated.

IV. THE PHYSIOLOGY OF CONNECTIVE-TISSUE FIBERS A . Fibrogenesis The question of elastic-fiber formation in vivo is not difficult to review, since little if any concrete evidence exists from which the mode of fibrogenesis can be determined. Much of the evidence discussed below is negative and can be assessed only by comparison with the positive findings available for collagen production. Two main lines of approach have been examined : (1) the production of fibers in actively growing and differentiating tissue, and (2) the replacement of lost tissue in wound healing. These aspects have been studied by reference to fibrogenesis in whole organisms and in tissue culture. 1. Embryonic Tissue. The fibroblast has been identified as the instigator of fibrogenesis, although the actual site of fiber formation has long been in doubt. It was originally suggested that fibrin fibers originating as extrusions of fibroblast cytoplasm were converted into collagen fibers extracellularly. This hypothesis was based on early histological studies and has 'now been completely discounted, although Buck (1953) has suggested that fibrin may, by its contraction, play a part in stretching and orienting the collagen fibers during their production. In an actively differentiating tissue such as embryonic mesenchyme, the production of fibers is far in excess of the cellular content, and it would appear unlikely

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that all the fibers could originate within the cells themselves in a fibrous state. Although Doljanski and Romlet (1933) claimed that in tissue culture formation of collagen fibers could be initiated in areas far removed from the cells, the work of Porter (1951) and Fitton-Jackson (1956) has shown that the fibroblasts do have a primary role to play in fibrogenesis, and fiber formation starts in the immediate vicinity of the cells. Fitton- Jackson ( 1954) showed that certain cytoplasmic granules within the fibroblast, which contain both protein and mucopolysaccharide, are associated with the formation of intercellular material. It is suggested (Fitton-Jackson, 1956) that the fibroblasts evolve globular proteins similar in composition to collagen, and polysaccharides from which the ground substance is derived. In the region outside the cell, the initial stages in fiber formation take place with the production of a primitive collagen fiber with a band periodicity of about 210 A. At this stage each fibril is surrounded by a region of relatively low electron density, but as the fibers increase in diameter the size of this region decreases. The increasing diameter of the fibers during growth appears to be due to an uptake of material from the ground substance, and this latter thus assumes a fundamental role in fibrogenesis. It would appear proved that the formation of the initial fibrils requires the interaction of collagen precursors and some component of the environment. The exact nature of the precursors evolved by the fibroblast is in doubt, but Harkness et al. (1954) have shown that material extracted from tissues by neutral salts and having many properties of collagen, including that of being able to act as starting material for the regeneration of collagen fibrils, demonstrates a far higher rate of turnover for glycine than do other collagenous fractions of the tissue. One is left with the hypothesis that callagen fibers are formed from a soluble precursor evolved by the fibroblast, by combination with some component of the extracellular mass, but the way in which these phenomena are controlled is as yet unknown. If the mode of formation of collagen fibers is little understood, that of elastic fibers is shrouded in even deeper mystery. First, although there have been numerous claims, no one has conclusively proved the existence of an elastoblast. Robb-Smith (1954) states that elastic fiber production always follows the appearance of collagen in the embryo and in healing wounds. It would appear likely that a similar extracellular process to that which brings about the formation of collagen fibers initiates the fibrogenesis of elastic fibers. Either the fibroblast is induced to produce a variant of its normal precursor, or, as was suggested by Hall et d. (1955a),

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elastic fibers may be evolved by the interaction of a selection of the precursor fragments secreted by the elastoblast, with certain components of the ground substance. This presupposes that the precursor material secreted by the fibroblast is not a single protein species but a mixture of polypeptides of differing amino acid composition, such that the selection of a certain number affords the necessary building bricks for the production of elastic fibers instead of collagen. It is interesting in this context to recall that Schultz (1922) suggested that new elastica-staining material was derived from a similar source to collagen but was saturated with the polysaccharides of ground substance. Hass (1939) thought that elastic fibers might be formed from fibroblastic products as a fibrillary membrane at lipid interfaces in the surrounding tissue. Lipid is always closely associated with elastic elements, and such a mode of fibrogenesis might explain the deposition of elastic lamellae in vascular walls. Elastin fibers cannot be obtained in tissue culture from undifferentiated mesenchyme, but they can be caused to proliferate in differentiated tissue in which they already exist (Maximow, 1929). Even adult tissue, however, may not always be capable of supporting the regeneration of elastic fibers. For instance, keloid or even normally regenerating scar tissue contains little or no elastica. 2. Wound Healing. In early studies of tissue regeneration in the healing wound, as in the case of embryonic differentiation, suggestions were made that plasma clots or fibrin fibrils (Baitsell, 1946; Nageotte and Guyon, 1930) were converted into collagen fibrils. Here again, however, the only role the fibrin is likely to play would appear to be that of a matrix or template in association with which fibrogenesis may occur, and which by its physical properties may exert orienting forces on the fibers during synthesis. The most important observations on wound healing have been studies on the retention of granuloma tissue in wounds in animals with vitamin C deficiency. Wolbach and Howe (1926) first showed that wounds in ascorbic acid-deficient animals failed to heal. Danielli et al. (1945) showed that, although at low levels of ascorbic acid intake large amounts of reticulin were formed, the appearance of the wound was abnormal. The metachromatic material which is present in the granuloma decreases as the wound heals, owing to fiber formation, but in scorbutic subjects the metachromatic granulomatous tissue remains. Elster ( 1950), Robertson (1952), and Perrone and Slack (1951) showed that ascorbic acid was not necessary for the maintenance of already formed collagen fibers but appeared to be required for fibrogenesis. The properties and constitution

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of newly formed tissue have been studied by Robertson and Schwartz (1952) and by Jackson ( 1957), making use of the considerable amount of granuloma tissue which can be induced by injection of carrageenin solutions. Jackson showed by tracer studies that during the development of the tissue neutral salt-soluble collagen was produced first, and this was then consolidated into insoluble collagen, probably by combination with a tyrosine-rich mucopolysaccharide (Bowes et al., 1956). The amount of soluble collagen extracted from the fibers by acid increases with the progress of the granuloma, showing that the initial stages of the consolidation of the neutral salt-soluble material into the fibers are not complete. Jackson also reported the presence of a water-soluble hydroxyproline-containing fraction, whose function in fibrogenesis he was unable to determine. Since Robertson and Schwarz (1952) have shown that in scorbutic granuloma (which is a permanent phenomenon as opposed to the transient nature of the condition induced by carrageenin) the main protein constituent is devoid of hydroxyproline, it may be that the fragment rich in hydroxyproline combines with the products of fibroblastic activity at a late; stage in the production of the neutral salt-soluble collagen. Elastic fibers often do not occur in scar tissue at all, and never occw early, a@ an explanation of this might be that the presence of the hydroxyproline-cantaining fraction in granuloma tissue produces a situation in which the whole of the products of fibroblastic activity go to the production first of neutral salt-soluble collagen, and then insoluble collagen fibrils, whereas: in normal tissue in the absence of excessive amounts of the hydroxyqroline-containing material part of the fibroblastic products will be avaitable for the synthesis of elastic fibers.

B. The Aging of Connective-Tissue Fibers 1. Changes in Collagen. Electron-microscope studies have shown that the perifidic striations of collagen fibers at 640 A. are universally apparent in tissues at all ages from 1 hour to 89 years (Gross and Schmitt, 1948). I n embryonic tissue, however, Porter ( 1951) and Fitton-Jackson (1954) have shown that fibrils with cross striations of 210-A. periodicity are visible. The exact nature of the structural patterns which bring about these changes in electron opacity at regular intervals along the fiber axis is as ye$ unknown, but it would appear that there is no change in this structure after the fiber has become fully mature. The change observed in the transition from embryonic to infant tissue may represent the process of stabilization, which Bowes et al. (1956) have suggested may be due to the association of the collagen precursor, or its immediate solid successor with a mucopolysaccharide. The presence of mucopolysaccharide sur-

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rounding infant fibers is well substantiated, and there is evidence that at least in certain tissues this material decreases with age (Happey et al., 1953 ; Sobel and Marmorston, 1956). Chemically, little evidence has been advanced to indicate changes in amino acid analysis with age, although Hall and Reed (1957), examining a small population of normals, showed evidence for a trend in the hydroxyproline content toward lower values with increasing age. By studying the argyrophilia which appears to be associated with the polysaccharide content of collagen fibers, Schwarz (1957) has been able to obtain evidence for the lateral aggregation of collagen molecules and fibrils to form larger fibers resulting in an apparent “crystallization” in the adult fibers. This “consolidation” or “crystallization” is accompanied by a change in the reactivity of the fibers toward disruptive reagents. Orekhovitch et al. (1948) showed that a far smaller amount of acid-soluble collagen could be obtained from adult dermis than from young tissue, and Banfield (1952) has rendered these observations more precise by studying the effect of dilute acetic acid on pure collagen preparations. H e was thus able to show that, not only is there less extractable material in old tissue, but the fibers themselves are more resistant to extraction. The earliest stage in this series of structures of increasing stability is represented by the granuloma tissue induced by carrageenin (Jackson, 1957) in which an increasing amount of material can be extracted with acid throughout the whole period of fibrogenesis and resorption. 2. Changes in Elastic Fibers. The structure of the elastic fiber is so variable that it is not easy to determine whether degradation is associated with age purely on morphological grounds. Much of the apparently degenerate elastin, Unna’s so-called elacin, which can be seen in a variety of elastoses of the dermis has been shown (Tunbridge et al., 1952) to consist of degraded collagen. Where the conditions are not so advanced as to warrant the term “elastosis,” little change is observed (Hill and Montgomery, 1940). It is mainly in the arteries that age changes have been observed in elastic tissue. In the media of arteriosclerotic vessels, marked changes are observed in the morphology and tinctorial properties of the elastic fibers (Carlson, 1949) and in the amounts of polysaccharide associated with them. Sax1 (1957b) has shown that the area of aortic media most susceptible to elastase attack varies with age. I n middle age groups, the region most markedly attacked by the mucolytic fraction of elastase is the upper third of the media in which lipid changes occur in atheroma. Lansing et al. (1951) have shown that changes begin to be apparent in aortic elastin between the ages of 15 and 25 years and hence may be taken as being dependent on the maturity of the subject and as

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antecedent to the more gross changes associated with atherosclerosis. The fact, however, that the changes appear in the main to be restricted to those arteries in which atherosclerotic changes ultimately occur may indicate that they are in effect the early stages of a pathological process, and not solely aging phenomena. Biochemically, one of the characteristic changes in elastic fibers is their increased affinity for calcium, which may rise from 0.6% in the first decade to an average value of 6.8% by the seventh to eighth decade, and in extreme cases even to values in excess of 13%. Running parallel with these changes are apparent variations in the amino acid composition of the elastin. Lansing et al. report that there is an increase in the concentration of aspartic and glutamic acids, which they suggest provides the site for the binding of increasing concentrations of calcium. Their amino acid analyses indicate a rise in certain amino acids, and a decrease in others, and, in addition to those shown to increase by Lansing, the hydroxyproline content is also raised (Hall, 1951, 1955). Hall also showed that treatment with boiling urea solution was capable of separating from a preparation of aortic elastin a fraction rich in hydroxyproline, aspartic acid, glutamic acid, arginine, lysine, and histidine, leaving behind a substance which had the classic amino acid analysis of young elastin or ox ligamentum nuchae elastin. These observations led to the suggestion that the preparation of “old elastin” described and analyzed by Lansing et al. is, in fact, a mixture of two proteins, one of which has a far higher dicarboxylic amino acid content than either elastin or Lansing’s “old elastin.” This component is, however, closely attached to the true elastin, and hence the whole complex separates together. Small numbers of anisotropic fibers can be observed in dermal or vascular tissues from elderly subjects (Hall et al., 1958). These fibers appear to consist of a protein core enveloped in cellulose, with the complex fibresis similar to the cellulose fibers occurring in the tunic of the ascidians (Meyer et al., 1951). O n the basis of this it is suggested that with aging the organism may revert to a more primitive metabolic level, and degraded collagen fibrils become coated with cellulose.

V. THE PRODUCTION OF ELASTIC MATERIALFROM COLLAGEN A. Structural Evidence 1. Histochemical Evidence. The original observations of Burton et d. (1955) which led to the promulgation of the conversion hypothesis were made on dermal preparations purified according to the method of Neuman ( 1949).

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After treatment with buffer solutions of p H range 7 to 10.4 for prolonged periods of time, pancreatic enzyme concentrates, and solutions of phthalate buffer ( p H 5) with or without the addition of sodium metaperiodate, significant changes occurred in the tissue powder. Masses of amorphous material and high concentrations of anisotropic lamellae were observed in close association. When the reagents were perfused under pressure through thin discs of dermis (Hall, 1956), the spatial relationship of the amorphous material and the anisotropic structures was more easily seen. A certain proportion was free, some loosely adhering to the lamellae, and other lamellae were completely coated with it. This amorphous material stained deeply with Hart’s modification of Weigert’s stain. I n addition to these masses of amorphous Weigert-positive material, large numbers of structures histologically indistinguishable from elastin, in the form of wavy black-staining fibrils, were observed. 2. Electron-Microscope Studies. At the electron-microscope level, the transformation of the collagen fibrils was equally obvious. Collagen fibrils appeared in all stages of degeneration, from the condition in which only the edges of the fiber appeared to have lost their sharp outline, to one of complete gelatinization. Some, however, never reached this stage of total degeneration but became coated with amorphous material. It has been suggested (Smith, 1957) that the structures observed, to which the term “elastin-like” was applied on account of their marked similarity to this material, are in fact collagen fibers coated with gelatin. A comparison of the appearance of these fibers with the frankly gelatinized structures illustrated in Keech and Reed (1957a) indicates the significant difference between the two types of collagen degradation. Gelatinization is accompanied by the appearance of diffuse ill-defined masses hardly distinguishable from the background. The production of elastica is also characterized by the appearance of masses of amorphous material, but these are sharply defined, are electron-opaque, and stand out clearly from the background. Fibers exist (Burton et al., 1955, Fig. 2) in which part is converted to elastica, the remainder retaining its collagenous characteristics. Keech and Reed (1957a) compared the effect of boiling water on the appearance of collagen that has not been subjected to alkaline treatment with that of elastica produced in this way. Untreated collagen gelatinizes, whereas the synthetic elastica merely consolidates and assumes an appearance even more nearly like that of true elastic fibers. Attention was drawn to the similarity of these structures to the products of heat treatment on the irregular electron-opaque structures obtained from young collagen after treatment with collagenase. Disruption of the opaque outer layer reveals an essentially elastic struc-

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ture. Keech et al. (1957a) suggest that these “moth-eaten fibers” represent an intermediate stage in the conversion of collagen to elastin. Keech and Reed ( 1957b) also show that collagenase, elastase, hyaluronidase, and ultrasonic radiation all have the same effect in producing elastin-like material from the “moth-eaten fibers.” Balo et al. (1956) suggest that the residue from collagen fibers, after the removal of polysaccharide and procollagen, either by thermal or chemical denaturation, has a similar appearance to the synthetic elastica produced by alkaline treatment of collagenous tissue. They do not, however, feel justified in calling the material elastin, but give it the name metacollagen. Since the starting material employed by these workers consisted of rat-tail tendons, essentially free of ground substance, it is possible that the material to which they give the name metacollagen is, in fact, not identical to synthetic elastica. The production of elastica-staining areas and material identifiable as elastic fibers under the electron microscope is a more efficient process in whole tissue than in purified collagen. Hence, the ground substance may play an important role in the production of synthetic elastica-staining material, and it may be that metacollagen differs from elastica in the absence of this component. 3. X - R a y Diffraction. As mentioned in Section 11. D, it appears that collagen and elastin differ in that collagen has a highly oriented structure producing a complex array of arcs on an X-ray diffraction photograph, whereas elastin in the unstretched state gives a picture which is representative of an amorphous structure. Ramachandran and Santhanan ( 1957), however, have reinvestigated the X-ray diffraction pictures obtained from collagen, chemically altered collagen, and elastin and have suggested not only that elastin should be classed with collagen but that its molecular structure may well be built up of a triple-chain structure similar to that of collagen (Ramachandran and Kartha, 1954; 1955a, b ; Ramachandran, 1956). They showed that two diffuse rings of 4.4-A. and 2.2-A. spacings and a sharper ring at 11 to 12 A. are typical of elastin. The 2.2-A. spacing is reported for the first time and appears to be similar to rings that occur in pictures obtained from native and thermally shrunk collagen and gelatin. They claim even greater similarity between elastin and collagen fibers denatured by treatment with nickel nitrate and calcium chloride. Since certain of these reactions are reversible, it would not appear likely that the identity of the two sets of X-ray pictures is indicative of conversion from collagen into elastin such as is implied by Burton et al. (1955) and Hall et al. (1955a). If conversion does occur, the material studied by Ramachandran and Santhanan may represent a state in which the correct structural align-

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ment is attained, without the removal of those portions of the molecule which are redundant to the elastin structure.

B. Chemical Evidence

1. Alkali Treatment. Alkaline buffers, especially borate, p H 8.7, have been employed (Burton et al., 1955; Hall, 1956) to bring about an apparent conversion of collagen into elastin-like structures. The early studies were concerned mainly with an assessment of the hydroxyproline relationships of the tissues and the extracts. Hydroxyproline-rich protein fractions were obtained by the exhaustive extraction of tissue preparations for periods of up to 3 days. Division of the extracts according to time of extraction produced protein fractions of varying hydroxyproline content. The period during which the protein with the highest hydroxyproline content was extracted varied with the age of the subject, being early in the young tissue and appearing only after protracted extraction in elderly subjects. Continuous fractionation of the extract by perfusing tissue with alkaline buffer under pressure permitted the collection of five protein fractions. Of these, three had appreciable hydroxyproline content. One had a hydroxyproline content of between 30 and 40%, and the other two were similar to collagen. Hall (1956) showed that a similar amount of material was extracted from whole calf skin, and purified tissue with borate buffer and similar amounts of elastin were obtained in the residue, but the physical properties of the residue from the former was more nearly like the native elastic fiber than that obtained from purified tissue. C. Evidence for the Heterogeneity of Collagen One of the major criticisms against the acceptance of the hypothesis that collagen may be converted in vivo into elastic fibers arises from a consideration of their respective amino acid compositions. Collagen is characterized (Bowes et al., 1956) by the presence of high concentrations, of glycine, proline, and hydroxyproline which together account for 437’0 of the residues in the molecule. Elastin, on the other hand, although containing roughly similar amounts of glycine and proline, is relatively free of hydroxyproline (1.270). It has also a far lower concentration of polar amino acids. Their place is .taken by the monoaminomonocarboxylic acids all of which are increased in amount, especially valine, which is present in seven times as high a concentration in elastin as in collagen (Partridge and Davies, 1955). Thus any conversion of collagen into elastin would have to be associated with the removal of a large proportion of the hydroxyproline and polar amino acids, and all the valine would have to remain in the residue. In view of the relative valine concentrations, this

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cannot represent more than one-seventh of the original collagen, and Harkness et al. (1957) have pointed out that the efficiency of the reaction in terms of turnover of one protein species to the other can only be of a similar order. Tristram (1957) and Smith (1957) have also doubted whether such conversion could proceed without complete hydrolysis to the amino acid level followed by resynthesis. Such a sequence of reactions could hardly be accepted as explaining the phenomenon reported by Burton et al. (1955) in which, after perfusion with borate buffer at p H values between 8 and 9, collagen fibers were directly converted to elastinlike material. Not only would complete hydrolysis followed by resynthesis be unlikely to effect the production of the elastin-like material at the same site as the original collagen fibers, since the released amino acids would be leached from the tissue by the perfusing fluid, but also it is inconceivable that sufficient energy could be available in such an in vitro system for the resynthesis of the necessary peptide bonds. Similarly, if such a series of reactions were accepted as an explanation of a possible in vivo conversion (Hall et al., 1955a), the elastin could not be said to be derived directly from the collagen fibers, since the released amino acids would join the amino acid pool, and those employed in the resynthesis could have come from the catabolism of any tissue, or from some exogenous source. Such profound degradation was indeed never envisaged by the authors of the hypothesis, and it is of interest to consider how such a concept arose. 1. Sequence Studies. Bergmann and Niemann (1936), basing their hypothesis on the fragmentary analyses then available, suggested that collagen consisted of a repeating tripeptide containing glycine, proline, or hydroxyproline, with another amino acid as the third component. An assessment of this suggestion in association with wide-angle X-ray diffraction data enabled Astbury (1940) to devise the first structural model for collagen. Since then, small-angle X-ray diffraction studies and polarized infrared observations, both on collagen and on synthetic peptides rich in either glycine or proline, have permitted the evolution of more-complex structures for collagen (Pauling and Corey, 1953; Ramachandran and Kartha, 1955a, b ; Rich and Crick, 1955 ; Cowan et al., 1955) which ascribe a triple helical structure to collagen. As a relic of the Bergmann-Niemann hypothesis, however, the initial assumption was made that a repeating triad of amino acids occurred in the molecular chain. Schroeder et al. (1953, 1954) and Kroner et al. (1953, 1955) suggested that the original gly-pro or (hypro)-R structure was untenable, since they were able to isolate, from among the peptides obtained by partial hydrolysis of collagen, a tetrapeptide gly-pro-hypro-gly. They also suggested on the basis of their

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evidence that there might be regions that were devoid of pyrrolidine residues. Even the possibility of regions of varying analysis did not prevent these authors from adhering to the spirit of the Bergmann-Niemann hypothesis, if not to its letter. They replaced the triad by a tetrad, however, on very flimsy evidence. The hydroxyproline content of the tetrapeptide units was only 1.17% of the total hydroxyproline content of the collagen, but Kroner et al. (1955) report the existence, in their partial hydrolyzates, of three tripeptides in which hydroxyproline is flanked by other nonpyrrolidine amino acids. The hydroxyproline content of these tripeptides-ala-hypro-gly, gly-hypro-gly, and ser-hypro-gly-together represents 2.28% of the whole hydroxyproline content of the molecule. Their decision to ascribe one particular structure to the whole of the collagen molecule on the basis of an analysis of just over 1% of the total and in the face of evidence in support of another structure representing over 2% of the molecule would appear to be unjustifiable. Evidence from larger molecules derived by partial enzymatic degradation of collagen has been presented by Grassmann et al. (1957a). They obtained five or six peptides of considerable chain length (ranging from 20 to 79 amino acid residues). These showed marked variations in amino acid analysis, and, as a general rule, it was possible to show that areas rich in proline and hydroxyproline were devoid of polar amino acids, and vice versa. Even greater variations could be observed between the individual peptides. For instance, one with a chain length of 79 residues contained 11 proline and 12 alanine residues, whereas another polypeptide (43 residues long) contained only 3 alanine residues to 10 prolines. Even if the larger peptide contained the one with fewer residues, the alanine content of the 35 extra residues would have to be 25% of the total, as opposed to the 7% in the smaller peptide, or the 8% of the whole molecule. Similarly, a decapeptide containing 4 arginine and 5 glycine residues was identified. There would appear to be evidence in favor of marked heterogeneity in the collagen molecule, yet here again the fractions considered by Grassmann et al. only amount to 4.2% of the whole protein, and hence strict extrapolation to an analysis for the whole molecule is not justifiable. Grassmann points out, however, that areas of some 30 amino acid residues, completely devoid of polar groups, such as appear to exist, could represent structurally repeating elements, whose length would be roughly similar to one turn of the triple helix of Ramachandran and Kartha and of the same order of magnitude as a single light interperiod band in an electron micrograph. On the whole, it would appear that until more extensive portions of the collagen molecule have been analyzed, there is little reason for assuming

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more than a marked degree of heterogeneity in the molecule. One fact does appear to emerge. Rigidly repeating units of triad or tetrad nature cannot account for the whole of the chain structure of collagen. 2. The Action of Collagenase. The hydrolysis procedures utilized for the preparation of the peptide fractions reported above have of necessity broken down the proteins to a considerable degree. Much of the material obtained is useless for sequence studies, being in the form of free amino acids. Other workers, employing collagenase, have performed digestions which have resulted in the production of polypeptides of much longer chain length. Although it is impossible at this stage to obtain sequence data for these compounds, their gross analyses have been sufficient to demonstrate even further the existence of a considerable degree of heterogeneity in collagen. Mandl (private communication) has isolated a polypeptide with a molecular weight of some 7000 which contains neither hydroxyproline nor proline. This peptide was derived from collagen by treatment with a collagenase preparation from CZ. histolyticum. I n similar studies employing the enzyme from Cl. weZchii, Hall (1957b) reported the preparation from collagen of a polypeptide of high molecular weight (retained by a dialysis membrane) which was relatively devoid of hydroxyproline. Apparently the enzyme acted preferentially on those regions of the molecule that were rich in hydroxyproline. 3. The Effect of Phthulate Buffer on Collagen. When it was first suggested that elastin-like material could be produced from collagen (Burton et al., 1955 ; Hall et al., 1955a), it was reported that alkaline extraction of collagen resulted in the degradation of the fibers with the release of protein fragments rich in hydroxyproline. These results were amplified by Hall (1957b), who showed that perfusion of tissue with borate buffer brought about the release of a number of polypeptide units one at least of which contained a content high in hydroxyproline. Veis and Cohen ( 1956), however, suggested that collagen should be considered as a collection of “segments of varying length and cross section due to differences in cross-link distribution and the lateral ordering of side chains. The segments are chain networks held together by sets of acid stable bonds, while the segments contain and are held in the gross structure by acid labile bonds and physical forces. All bonds are, however, alkali stable.” It would appear, therefore, that alkali treatment of collagen would be the most likely to produce partial degradation products of sufficiently high a degree of complexity for the evolution of another structure to be accomplished without recourse to peptide bond formation. This indeed is what was observed. Electron micrographs of collagen treated with buffers of

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varying p H showed the presence of elastic fiber masses only within a circumscribed p H range. Since this was in the region of low alkalinity, a necessary factor in the conversion might be the availability of polysaccharide, rendered soluble by the mildly alkaline conditions. At lower p H little polysaccharide would be extracted; at higher p H it would be destroyed. It was of significance, therefore, that phthalate buffer was found to have a specific effect. Hall (1957b) reported that extraction of collagen with phthalate results in the removal of material rich in hydroxyproline. The extracted material differs from that isolated from collagenase-digested collagen, however, in that the majority of the hydroxyproline remains with the high-molecular-weight material.

VI. CONCLUSIONS In the earlier sections of this review evidence was presented indicating the desirability of considering the elastic fiber and the elastic lamellae as members of a group of fibrous components of connective tissue. Collagen appears to be relatively constant in composition throughout the tissues, although even here the differences discovered between collagenous tissue at various ages may be mirrored in variations from tissue to tissue. As yet the major studies on chemical composition and physical properties of collagen have been carried out on dermis and tendon, in view of the ease with which collagen from these sites can be obtained in a pure state. For elastin, evidence for variability already exists. True elastic fibers from dermis or aorta may be similar in composition to the classical elastin of ligament, but the aorta also contains components that appear to be similar to elastin in many of their properties but have markedly dissimilar amino acid analysis. I n the last part, evidence is reviewed for the concept that, as well as being members of the same group of fibrous proteins, collagen and elastic fibers may be even more closely related. The points of similarity are as follows : ( 1) They have a common source in the same fibroblasts ; (2) they contain certain amino acid sequences in common which act as specific foci of attack for elastase ; (3) collagen on degradation picks up material from the ground substance, which may be polysaccharide alone, or may include polypeptides to give a protein with many of the physical properties of elastin. The evidence against this concept is based mainly on amino acid sequence studies for collagen. These are still, however, so incomplete as to preclude their use as a true basis for criticism. The only fact that has emerged from them is that areas of heterogeneity do exist in collagen, and these may be sufficiently extensive to permit conversion of the type sug-

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gested to occur without recourse to “cataclysmic degradation” (Tristram,

1957). The evidence from conditions such as lathyrism or onchocerciasis are more difficult to discount. If collagen can be converted into old elastin, then even the prevention of elastic fiber formation in lathyrism or the complete destruction of the elastic network in onchorcerciasis should not prevent the ultimate replacement of the lost elastic fibers by the body, with collagen fibers as source. Two possibilities present themselves : either the collagen in these conditions becomes highly resistant to attack, and hence cannot take part in elastin production ; or true elastic fibers are required as a matrix before the old elastin can be laid down. Full elucidation of the problem will no doubt have to await intensive sequence studies on collagen fibers, collagen degradation products, elastic fibers, and “old elastin.”

VII. REFERENCES Adair, G. S., Davies, H. F., and Partridge, S. M. (1951) Nature 167, 605. Asboe-Hansen, G. (ed.) (1954) “Connective Tissue in Health and Disease.” Munksgaard, Copenhagen. Astbury, W. T. (1938). Trans. Faraday SOC.34, 378. Astbury, W.T. (1940). 1. Intern. SOC.Leather Trades’ Chem. 24, 69. Baitsell, G. (1946) 1. Exptl. Med. 29, 736. Baker, B. L.,and Abrams, G. D. (1955) Ann. Rev. Physiol. 17, 61. Balo, J., and Banga, I. (1949a) Schweiz. 2. Pathol. u. Bakteriol. 12, 350. Balo, J., and Banga, I. (194913) Nature 164, 491. Balo, J., and Banga, I. (1950) Biochem. I. 46, 384. Balo, J., and Banga, I. (1953) Arch. Physiol. Acad. Sci. Hung. 4, 187. Balo, J., Banga, I., and Schuler, D. (1954) Acta Morphol. Acad. Sci. Hung. 4, 141. Balo, J., Banga, I., and Szabo, D. (1956) J . Gerontol. 11, 242. Banfield, W. (1952) Anat. Record 114, 157. Banga, I. (1949) 2. Vitamin-, Hormon-, u. Fermentforsch. 2, 408. Banga, I. (1953) Nature 172, 1099. Bear, R. S. (1944) J. Am. Chem. SOC.68, 1297. Bear, R. S. (1956) J . Biophys. Biochem. Cytol. 2, 363. Bendall, J. R. (1955) Biochem J . 61, 31. Bergmann, M., and Niemann, C. (1936) J. Biol. Chem. 115, 77. Bourne, G. H. (1956) Nature 177, 467. Bowen, T.J. (1953) Biochem. J . 66,766. Bowes, J. H., and Kenten, R. H. (1948) Biochem. J. 4, 358. Bowes, J. H., Elliott, R. G., and Moss, J. A. (1955) Biochem. J . 61, 143. Bowes, J. H., Elliott, R. G., and Moss, J. A. (1956) Biochem. J . 63, 1. Braun-Falco, 0. (1956) Arch. klin. u. ezptl. Dermatol. 203, 256. Buck, R. C. (1953) J. Pathol. Bacteriol. 66, 1. Burton, A. C. (1954) Physiol. Revs. 34, 619. Burton, D., Hall, D. A., Keech, M. K., Reed, R., Saxl, H., Tunbridge, R. E., atid Wood, M. J. (1955) Nature 176, 966.

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Carlson, A. J. (1949) In “Geriatric Medicine” (E. J. Steiglitz, ed.), p. 47. Saunders, Philadelphia, Pennsylvania. Carter, H. E. (1956) Science 123, 669. Cowan, P. M., McGavin, J., and North, A. C. T. (1955) Nature 176, 1062. Danielli, J. F., Fell, H. B., and Kodicek, E. (1945) Brit. J. Exptl. Pathol. 26, 367. Dempsey, E. W., and Lansing, A. I. (1954) Intern. Rev. Cytol. 3, 437. Dettmer, N. (1952) 2. Zellforsrh. u. mikroskop. Anat. 37, 89. Dick, J. (1947) J. Anat. 81, 201. Doljanski, L., and Romlet, F. C. (1933) Arch. Pathol. Anat. u. Physiol. Virchow’s 291, 260. Ejiri, I. (1936) Japan. J . Dermatol. Urol. 40, 46, 173. Ejiri, I. (1937) Japan. J. Dermatol. Urol. 41, 8, 64, 95. Elster, S. K. (1950) J. Biol. Chem. 186, 105. Ewald, A. (1890) Z.Biol. 26, 1. Findlay, V. H. (1954) Brit. J. Dermatol. 66, 16. Fitton-Jackson, S. (1954) Proc. Roy. SOC.B142, 536. Fitton-Jackson, S. (1956) Proc. Roy. S O C .B144, 556. Fleming, W. (1876) Arch. mikroskop. Anat. u. Entwicklungsnaech. 12, 391. Franchi, C. M., and De Robertis, E. (1951) Proc. SOC.Exptl. Biol. Med. 76, 515. Fullmer, H. M., and Lillie, R. D. (1956) J . Histochem. and Cytochenz. 4, 64. Fullmer, H. M., and Lillie, R. D. (1957) J. Histochem. and Cytochem. I, 11. Gillman, T., Penn, J., Bronx, D., and Roux, M. (1954) Nature 174, 789. Glegg, R. E., Eidinger, D., and Leblond, C. P. (1953) Science 118, 614. Gomori, G. (1950) A m . J. Clin. Pathol. 20, 665. Grant, N. H., and Robbins, K. C. (1955) Proc. SOC.Exptl. B i d . Med. 90,264. Grant, N. H., and Robbins, K. C. (1957) Arch. Biochem. Biophys. 66, 396. Grassmann, W., and Schleich, H. (1935) Biochem. 2. 277, 230. Grassmann, W., Hanig, K., Endres, H., and Reidel, A. (1957a) In “Connective Tissue” (R. E. Tunbridge, ed.), p. 308. Blackwell, London. Grassmann, W., Hoffmann, U., Kiihn, K., Horman, H., Endres, H. and Wolf, K. (1957b) In “Connective Tissue” (R. E. Tunbridge, ed.), p. 157. Blackwell, London. Gross, J. (1949) J. Ezptl. Med. 89, 699. Gross, J. (1951) Proc. SOC.Exptl. Biol. Med. 78, 241. Gross, J., and Schmitt, F. 0. (1948) J . Exptl. Med. 88, 555. Gross, J., Highberger, J. H., and Schmitt, F. 0. (1952) Proc. SOC.Exptl. Biol. Med. 80, 462. Hale, C. W. (1916) Nature 167, 802. Hall, D. A. (1951) Nature 168, 513. Hall, D. A. (1955) Biochem. J . 59, 459, 465. Hall, D. A. (1956) Ezperientia Suppl. 4, 19. Hall, D. A. (1957a) Arch. Biochem. Biophys. 67, 366. Hall, D. A. (1957b) Gerontologia 1, 347. Hall, D. A. (1957~) In “Connective Tissue” (R. E. Tunbridge, ed.), p. 238. Blackwell, London. Hall, D. A., and Reed, R. (1957) Nature 180, 243. Hall, D. A., Reed, R., and Tunbridge, R. E. (1952) Nature 170, 264. Hall, D. A., Tunbridge, R. E., and Wood, G. C. (1953) Nature 172, 1099. Hall, D. A., Keech, M. K., Reed, R., Saxl, H., Tunbridge, R. E., and Wood, M. J. (1955a) J . Gerontol. 10, 388.

CONNECTIVE TISSUE FIBERS

249

Hall, D. A., Reed, R., and Tunbridge, R. E. (1955b) Exptl. Cell Research 8, 35. Hall, D. A., Lloyd, P. F., Saxl, H., and Happey, F. (1958) Nature 181, 470. Hannay, P. W. (1951) Brit. J. Dermatol. 63, 92. Happey, F., MacRae, T. P., and Naylor, A. (1953) In “Nature and Structure of Collagen” (J. T. Randall, ed.), p. 65. Butterworths, London. Harkness, R. D., Marko, A. M., Muir, H. M., and Neuberger, A. (1954) Biochem. J . W,558. Harkness, M. L. R., Harkness, R. D., and McDonald, D. A. (1957) Proc. Roy SOC. B146, 541. Hart, C. (1908) Zentr. Pathol. 19, 1. Hass, G. M. (1939) A.M.A. Arch. Pathol. 27, 15. Hill, R., and Montgomery, H. (1940) J. Invest. Dermatol. 3, 231. Hoffmann, U. von, Nemetschek, T., and Grassmann, W. (1952) Z . Naturforsch. 7b, 509. Jackson, D. S. (1954) Biochem. I . 66, 639, 699. Jackson, D. S. (1957) Biochem. J . 66, 277. Jamison, D. G., and Kershaw, W. E. (1956) Ann. Trop. Med. Parasitol. 60, 514. Jensen, L. H. (1955) Dermatologica 110, 108. Keech, M. K., and Reed, R. (1957a) Ann. Rheumatic Diseases 16, 35. Keech, M. K., and Reed, R. (1957b) Ann. Rheumatic Diseases 10, 198. Keech, M. K., Reed, R., and Wood, M. J. (1956) I. Pathol. Bacteriol. 71, 477. Kendrew, J. C. (1953) In “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. 2, Part B, p. 848. Academic Press, New York. Kissmeyer, A., and With, C. (1922) Brit. J. Dermatol. 34, 175. Kokas, E., Foldes, I., and Banga, I. (1951) Acta Physiol. Acad. Sci. Hung. 2, 323. Kolpak, H. (1935) Kolloid-2. 73, 129. Krafka, J. Jr. (1937) A.M.A. Arch. Pathol. 23, 1. Kramer, H., and Little, K. (1953) In “Nature and Structure of Collagen” (J. T. Randall, ed.), p. 33. Butterworths, London. Kroner, T. D., Tabroff, W., and McGarr, J. (1953) J. Am. Chem. SOC.76, 4084. Kroner, T. D.,Tabroff, W., and McGarr, J. (1955) J. A m . Chem. SOC.77, 3356. Lansing, A. I. (1951) In “Chemical Morphology of Elastin Fibres in Connective Tissues” (C. Ragan, ed.), p. 45. Josiah Macy Jr. Foundation, New York. Lansing, A. I. (1955) Ciba Foundation Symposium, Ageing, 1954 p. 88. Lansing, A. I., Roberts, E., Ramasarma, G. B., Rosenthal, T. B., and Alex, M. (1951) Proc. SOC.Exptl. Biol. Med. 76, 714. Lansing, A. I., Rosenthal, T. B., Alex, M., and Dempsey, E. W. (1952) Anat. Record 114, 555. Lansing, A. I., Rosenthal, T. B., and Alex, M. (1953) Proc. SOC.Exptl. Biol. Med. 84, 689. Leblond, C. P. (1950) Am. J. Anat. 86, 1. Little, K., and Windrum, G. M. (1954) Nature 174, 789. Lloyd, D.J., and Garrod, M. (1916) SOC.Dyers Colourists Symposium on Fibrous Proteins p. 24. Lowry, 0. H., Gilligan, D. R., and Katersky, E. M. (1941) J. Biol. Chem. 189, 795. McManus, J. F. H. (1956) Nature 168, 202. Mallory, F. B. (1900) J. Exptl. Med. 6, 15. Mallory, F. B. (1936) Stain Technol. 11, 101. Masson, P. (1929) 1. Tech. Methods 12, 75.

250

DAVID A . H A L L

Maximow, A. (1929) 2. mikroskop.-anat. Forsch. 17, 625. Meyer, K. H., and Ferri, E. (1936) Arch. ges. physiol. Pfliiger’s 238, 78. Meyer, K., and Rapport, M. M. (1951) Science 113, 596. Meyer, K. H., Huber, L., and Kellenberger, E. (1951) Experientia 7, 216. Michaelis, L. (1901) Deut. med. Wochschr. 27, 219. Nageotte, J., and Guyon, L. (1930) Arch. biol. (Libge) 41, 1. Neuman, R. E. (1949) Ph.D. Thesis, University of Cincinnati, Cincinnati, Ohio. Neuman, R. E., and Logan, M. A. (1950) I. Biol. Chem. 139, 794. Neuman, S. B., Borysko, E., and Swerdlow, M. (1949) J. Research Natl. Bur. Standards 43, 183. Orekhovitch, V. N., Tustanovskii, A. A., Orekhovitch, K. D., and Plotnikova, N. E. (1948) Biokhimiya l3, 55. Partridge, S. M., and Davies, H. F. (1955) Biochem. J. 61, 21. Partridge, S. M., Davies, H. F., and Adair, G. S. (1957) In “Connective Tissue” (R. E. Tunbridge, ed.), p. 222. Blackwell, London. Pauling, L., and Corey, R. B. (1953) Proc. Roy. SOC.Bl41, 31. Perrone, J. C., and Slack, H. G. B. (1951) Biochem. J. 48, iv; 49, lxxii. Popa, G. T. (1936) Morphol. Jahrb. 78, 79. Porter, K. R. (1951) I n “Connective Tissues” (C. Ragan, ed.), p. 126. Josiah Macy Jr. Foundation, New York. Ragan, C. (ed.) (1950 et seq.) “Connective Tissues.” Josiah Macy Jr. Foundation, New York. Ramachandran, G. N. (1956) Nature 177, 710. Ramachandran, G. N., and Kartha, G. (1954) Nature 174, 269. Ramachandran, G. N., and Kartha, G. (1955a) Nature 176, 593. Ramachandran, G. N., and Kartha, G. (1955b) Proc. Indian Acad. Sci. 4aA, 216. Ramachandran, G. N., and Santhanam, M. S. (1957) Proc. Indian Acad. Sci. UA, 124. Rhodin, J., and Dalhamn, T. (1955) Exptl. Cell Research 9, 371. Rich, A., and Crick, F. H. C. (1955) Nature 176, 915. Rinaldini, L. M. (1958) in press. Rinehart, J. F., and Abul-Haj, S. K. (1951) A.M.A. Arch. Pathol. 62, 189. Rinehart, J. F., and Abul-Haj, S. K. (1952) J. Natl. Cancer Inst. 13, 232. Robb-Smith, A. H. T. (1954) In “Connective Tissue in Health and Disease” (G. Asboe Hansen, ed.) , p. 15. Munksgaard, Copenhagen. Robertson, W. von B. (1952) J. Biol. Chem. 196, 403. Robertson, W.von B., and Schwarz, B. (1952) J. Biol. Chem. 201, 689. Romhanyi, G. (1955) Acta Morphol. Acad. Sci. Hung. 5, 311. Russell, G. (1957) Nature 181, 102. Sachar, L. A., Winter, K. K., Sicher, N., and Frankel, S. (1955) Proc. SOC.Exptl. Biol. Med. 90, 323. Saxl, H. (1957a) Gerontologip 1, 142. Saxl, H. (1957b) Proc. 4th Conf.of Gerontol. Merano. In press. Schneider, F. (1940) Collegium 97. Schneider, F. (1949) Angew. Chem. 61, 259. Schroeder, W. A., Honnen, L., and Green, F. C. (1953) Proc. Natl. Acad. Sci. U.S. 39, 23. Schroeder, W. A., Kay, L. M., LeGette, J., Honnen, L., and Green, F. C. (1954) J. Am. Chem. SOC.76, 3556.

CONNECTIVE TISSUE FIBERS

25 1

Schultz, A. (1922) Arch. pathol. Anat. u. Physiol. Virchow’s 289, 415. Schwarz, W. (1957) In “Connective Tissue” (R. E. Tunbridge, ed.), p. 144. Blackwell, London. Schwarz, W., and Dettmer, N. (1953) Arch. pathol. Anat. u. Physiol. Virchow’s 339, 243. Slack, H. G. B. (1954) Nature 174, 512. Smith, R. H. (1957) Progr. in Biophys. and Biophys. Chem. 8, 217. Sobel, H., and Marmorston, J. (1956) I. Gerontol. 11, 1. Stein, W. H., and Miller, E. G. (1938) J. Biol. Chem. V25, 599. Tattersall, R. N., and Seville, R. (1950) Quart. J. Med. 19, 151. Thomas, E. W. P., and Rook, A. J. (1949) Proc. Roy. SOC.Med. 42, 142. Tristram, G. R. (1957) Nature 180, 690. Tunbridge, R. E. (ed.) (1957) “Connective Tissue.” Blackwell, London. Tunbridge, R. E., Tattersall, R. N., Hall, D. A., Astbury, W. T., and Reed, R. (1952) Clin. Sci. 11, 315. Unna, P. G. (1890) Monatsch. grakt. Dermutol. 11, 365. Unna, P. G. (1896) “Histopathology of the Diseases of the Skin” (translated by N. Walker), p. 984. Macmillan, New York. Veis, A., and Cohen, J. (1956) J. A m . Chem. SOC.18, 6244. Verhoeff, F. H. (1908) 1. Am. Med. Assoc. 60, 876. Weigert, C. (1898) Centr. allgem. Pathol. u. pathol. Anat. 9, 290. Wislocki, G. B. (1952) Am. J. Anat. 91, 233. Wohlbach, S. B. and Howe, P. R. (1926) A.M.A. Arch. Pathol. 1, 1. Wohlisch, E., Weitnauer, H., Griinig, W., and Rohrbach, R. (1943) Kolloid-Z. 104, 14. Wolpers, C. (1944) Klin. Wochschr. 4S, 169. Wood, G. C. (1953) Biochem. J. 56, xxxiv. Wood, G. C. (1954) Biochim. et Biophys. Actu 16, 311. Wood, G. C. (1958) Biochem. 1. 69, 539. Wyckoff, R. W. G. (1949) “Electron Microscopy Techniques and Applications.” Interscience, New York. Yu, S. Y., and Blumenthal, H. T. (1958) J . Gerontol. 13, 366.

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Experimental Heterotopic Ossification J. B. BRIDGES Department of Anatomy, Queen’s University, Belfast, Ireland Page I. Introduction ...................................................... 253 11. Heterotopic Ossification and the Urinary Tract ..................... 254 A. Ligation of the Renal Vessels ................................. 254 B. Transplants to the Urinary Tract ............................. 258 C. Damage to the Urinary Tract ................................. 259 D. Transplants of Urinary Tract ................................. 260 111. Injection of Extracts of Skeletal Tissues ........................... 262 IV. Injections of Irritants and Other Traumatic Experiments ............. 267 A. Irritants into Muscle ......................................... 267 B. Irritants into the Eye ........................................ 268 C. Application of Sclerosing Solutions to Blood Vessels ............. 268 D. Trauma by Irradiation ....................................... 269 V. Implants of Devitalized Skeletal Tissues ........................... 269 VI. Conclusions ....................................................... 276 VII. Acknowledgments ................................................. 276 VIII. References ........................................................ 276

I. INTRODUCTION Pathologists have long been accustomed to finding bone in human tissues which has no direct connection with the skeleton, and there are many reports in the literature of bone having formed in the soft tissues of animals after various experimental procedures. It is also generally accepted that bone formation requires the active participation of specialized cells, the osteoblasts, and that such cells are present during, and responsible for, heterotopic ossification. In normal circumstances osteoblasts are only found in association with bone formation inside the natural periosteal boundaries of the skeleton, and it is a reasonable hypothesis that they are a specialized race of cells descended from those that originally differentiated from the mesenchyme at genetically determined centers of ossification in the developmental period. The fact that cells apparently not of this lineage can differentiate into osteoblasts under certain conditions raises questions of great theoretical importance for the understanding of bone formation generally, whether it be in the embryonic centers of ossification, during normal growth, in the course of fracture repair, or after the transplantation of bony tissue. For, either these processes are to be interpreted as the result of the proliferation, migration, and functional differentiation of specialized cells uniquely situated in respect to the bony tissues involved, or they are to be 253

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regarded as phenomena within the capacity of any connective tissue, and emphasis is to be placed on the factors that determine their bone-forming activities, rather than on the morphology or situation of the cells taking part. Of course, in the second eventuality it is not necessary to assume that every kind of connective tissue cell is capable of conversion to an osteoblast, but only that some of the cells in all connective tissues possess this property. Unfortunately many observers have been uncritical in their claims to have observed heterotopic formation ; in particular, claims based on the histological examination of transplants of living bone, periosteum, and bone marrow placed in the soft tissues cannot be accepted because of the possibility that viable osteoblasts had been carried over in the transplant. Nevertheless there is a considerable amount of acceptable evidence derived from experiments in which there is no possibility that skeletal osteoblasts were involved, except on the unlikely assumption that they were transported via the blood stream. It is the purpose of this review to examine such evidence and to try to unravel the course of events and the factors responsible for them.

11. HETEROTOPIC OSSIFICATION AND

THE

URINARY TRACT

Several lines of experimental evidence point to the conclusion that the transitional epithelium of the urinary tract is able, under certain conditions, to induce bone formation in adjacent connective tissues. Thus bone has been found in the kidney after ligation of the renal vessels, around transplants of urinary bladder epithelium to the soft tissues, in association with fascia1 transplants in, and fistulae of, the urinary bladder, and as an accidental finding in association with other pathological or experimental disturbances of the urinary tract.

A . Ligation of the Renal Vessels Sacerdotti and Frattin ( 1902) reported heterotopic ossification in the kidney after ligation of the renal vessels: they found bone in 3 out of 4 rabbits 3 to 4 months after the operation. Pocharissky (1905), Liek ( 1908), Maximow (1906), and Asami and Dock ( 1920) have since confirmed these findings. Pocharissky (1905) found bone in the kidneys in 3 of 5 rabbits after simple ligation of the renal vessels. Liek (1908) modified the experimental technique by wrapping omentum around the ischemic kidney to establish a more abundant collateral circulation and found bone histologically as early as 17 days after ligation. H e compared the results of ligating the renal vessels with and without ligation of the ureter and found that, whereas inclusion of the ureter failed to give hetero-

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topic ossification in any of the 4 rabbits used, bone appeared in all 12 rabbits in which the ureter was not included. In Sacerdotti and Frattin’s study, however, bone did form in one case where the ureter had been ligated. Maximow ( 1906), after confirming the original report, studied the origin of the marrow cells found in association with the heterotopic bone but came to no definite conclusions. Asami and Dock (1920), in a comprehensive paper, investigated the histological changes occurring in 13 such ischemic kidneys. Bone was first seen after 28 days, just under the transitional epithelium of the ureteric pelvis, and they described its subsequent extension and maturation. Harvey ( 1907) reported that intermittent occlusion of the renal vessels in the rabbit over a period of 35 days, produced by daily manual pressure through the abdominal wall, resulted in bone formation in the kidney. It is noteworthy that all the workers mentioned employed the rabbit; Liek also ligated the renal vessels in the cat but found no ossification subsequently in the ischemic kidney. Sacerdotti and Frattin stated that bone first appeared as a thin plate in the connective tissue underlying the transitional epithelium of the ureteric pelvis in the neighborhood of the renal papilla. I t was coarsely fibered and appeared to be formed by direct “calcification” of the connective tissue. Later this bone was resorbed and replaced by mature lamellar bone. Liek believed that bone formation began in close proximity to deposits of calcium salts in the ischemic kidney. Asami and Dock stated that there was initially an “accumulation of fibroblasts immediately under the transitional epithelium of the pelvis to form a membrane which lays down bone.” Direct transformation of hyaline connective tissue to bone was found only in the vicinity of pre-existing bone, and they interpreted this as a secondary mode of ossification differing from, and following on, the primary ossification under the epithelium of the ureteric pelvis. Yet another mode of ossification was found in one of the 13 kidneys studied, namely the deposition of bone on the walls of vascularized channels eroded in plaques of calcified necrotic kidney tissue, associated with which there seemed to be a conversion of fibroblasts into osteoblasts. These reports of experimentally produced heterotopic ossification in ischemic kidneys did much to discount the earlier explanations of natural heterotopic bone which claimed that all such bone arose from displaced skeletal elements (Busch, 1878; MacEwen, 1912), at the same time lending support to the concept that bone could arise by a metaplasia of connective tissue cells. Sacerdotti and Frattin interpreted bone formation in ischemic kidneys as a direct metaplasia of connective tissue and likened it to the normal process of ossification in developing cranial bones. These authors, however, made no suggestion as to the stimulus responsible for

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the initiation of such metaplasia. As to the origin of the bone marrow found within the ectopic bone, they could not decide between local metaplasia and colonization by blood-borne cells. Liek thought that calcium deposits in the ischemic kidney initiated bony metaplasia. H e believed that the necessary requirements for bone formation were young connective tissue and a neighboring deposit of calcium salts, and that both these conditions developed after renal pedicle ligation. In support of this view Liek referred to the work of Litten (1881) and von Kossa (1901), who had described calcification in the kidney after arrest of the renal circulation. Such calcification has been confirmed by Wells et al. (1911), who showed that even a temporary arrest in the renal circulation for 1 hour was followed by calcification demonstrable by histological and analytical means 36 hours later. I n further support of his thesis, Liek claimed that the cat’s blood is relatively deficient in calcium as compared with the rabbit’s, and that this explained the failure of ischemia to promote heterotopic ossification in the kidney of the cat. This view that calcium deposits are instrumental in the initiation of bone formation in ischemic kidneys was contested by Asami and Dock, however, on the grounds that calcification was apparently confined to the cortex, but the first-formed bone lay in the medulla just below the persisting ureteric transitional epithelium. They did not, however, imply that the transitional epithelium was the stimulus to bone formation. Recently we (Bridges, 1958) have found that heterotopic ossification regularly follows ligation of the renal vessels in the rat and guinea pig (Figs. 1 4 ) as well as in the rabbit. Heterotopic ossification in all three

FIG.1. Bone formation in the kidney of a rat 140 days after ligation of the renal (X15)

vessels.

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species began in the medulla either just under the surviving transitional epithelium of the ureteric pelvis or in close association with cords of transitional epithelial cells apparently migrating from the ureteric pelvis into the fibrotic medulla. Once established, bone formation extended throughout the medulla and into the cortex. In some areas the appearances

FIG.2. Same specimen as above, showing the closeness of the bone to the persisting transitional epithelium of the ureteric pelvis. (X100).

FIG.3. Invading cords of transitional epithelial cells are seen in the Haversian spaces of the induced bone scarcely distinguishable from the osteoblasts lining the surfaces of the bony trabeculae. (X450)

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were superficially similar to endochondral ossification, the hyalinized tubular remains of the cortex being eroded like the calcified cartilage of an epiphyseal plate. In other areas the bone spread into and incorporated the dense collagen fibers of the fibrotic medulla. Between the bony trabeculae fully hemopoietic marrow eventually formed. Cartilage was never found in the kidneys prior to the commencement of ossification, or associated with ossification, but in one rabbit kidney recovered after 195 days

FIG.4. Bone formation in the kidney of a guinea pig 100 days after ligation of the renal vessels. (X100)

discrete nodules of cartilage were found close to bony trabeculae among the hyalinized tubular remains which were undergoing resorption.

B. Transplants to the Urinary Tract Strauss (1914) repaired artificial defects in dogs’ ureters with pedunculated flaps of fascia from the abdominal wall and found that such introduced fascia became converted into a rigid bony tube. Neuhof (1917) found, in 14 dogs, that fascia transplanted into a defect in the urinary bladder wall became ossified, although this did not occur after similar transplants to several other hollow organs, except in one case where bone formed in the stomach after gastroenterostomy. The invariable association

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of bone with fascial grafts to the urinary tract led Strauss and Neuhof to the hypothesis that the fascia imbibed from the urine calcium salts which stimulated bony metaplasia. Neuhof also believed this bony metaplasia was in part an adaptive response to mechanical stress, leading to a strengthening of the weakened bladder wall. Phemister and Dabbs (cited in Phemister, 1923) likewise found that fascial grafts to the bladder always became ossified in the dog, but in 4 rabbits and a sheep no bone was found. They also believed that calcium salts imbibed from the urine stimulated bone formation in the fascial grafts and argued that the relative alkalinity of the urine in the rabbit and sheep hindered calcium uptake by the graft. In spite of an attempt to render the dogs’ urine less acid by dietary means, however, heterotopic bone formation still occurred. C.

Damage to the Urinary Tract

Simple injury to, or irritation of, the urinary tract without transplantation of connective tissue has also been followed by bone formation. Thus, Leuckart (1876) and Weinland (1859) reported ossification around the ureteric pelvis of the coatimundi and mink infested with the parasitic worm Eustrongylus gigans. This parasite causes damage to the renal tissue, and hydronephrosis and ossification occurred in the thickened fibrosed wall of the ureteric pelvis. Phemister ( 1923) described spontaneous heterotopic ossification in three cases of renal calculi attached to the renal pelvis in man, and Kretschmer ( 1928), Chauvin and Rouslacroix (1929), and Abbott and Goodwin ( 1932) reported ossification in infra-umbilical scars after suprapubic prostatectomy : bone was especially liable to form in the walls of bladder fistulae. While studying the repair process after excision of the lower pole and part of the pelvis of the ureter of the dog’s kidney, Pearce (1909) reported ossification in the fibrous tissue of the scar. The bone was present either immediately under the persisting transitional epithelium of the ureteric pelvis, or else apposed to invading cords of transitional epithelial cells which had spread into the scar tissue of the medulla “like a carcinoma.” Huggins (1931) reported ossification in the granulation tissue under the transitional epithelium after fulguration of the ureteric pelvis of dogs with a high-frequency alternating current, and after it was painted with 95% phenol. Bone has also been described in association with nests of transitional epithelial cells arising during the repair of the ureteric wall in dogs after part of it had been excised (Boyarsky and Duque, 1955).

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D . Transplants of Urinary Tract Huggins ( 1931) began his classic studies of experimental heterotopic bone formation by verifying the previous reports of bone formation in fascial grafts to the dog’s urinary bladder. He then systematically analyzed the various factors that could have played a part in such ossification. H e excluded the urine as a factor on the grounds that bone formed in fascial transplants to the urinary bladder even after the urine had been by-passed through the transplantation of the ureters to the anterior abdominal wall. H e noted the spread of transitional epithelium from the edges of the bladder wall over the fascial graft and the close association of the bony tissue with this epithelium. To test the possibility that transitional epithelium might be the factor responsible for inducing new bone formation, he transplanted part of the bladder wall autogenously to the rectus sheath in dogs. H e found such a graft was quickly transformed into a cyst lined with transitional epithelium, and bone formed in the connective tissue of the rectus sheath just beneath the proliferating epithelium. Bone formed round every one of nine such epithelial cysts. Transplants of bladder wall without mucosa, however, were uniformly unsuccessful. I n the rabbit, however, heterotopic bone was found only once in 6 similar experiments. From this work it was clear that proliferating transitional epithelium was able to ind.uce bone formation in the connective tissues of the dog’s rectus sheath. The initial report by Huggins of osteogenesis after bladder mucosal grafts excited wide interest. His observations have been confirmed in dogs by Abbott and Goodwin (1932), Regen and Wilkins (1934), Gomori ( 1943), Abbott and Stephenson ( 1945) , and Marshall and Spellman (1954). Similar results have been obtained in the guinea pig by Huggins et al. (1936), Gomori (1943), Abbott and Stephenson (1945), and Loewi (1954), and in the cat by Abbott et al. (1938) and Johnson and McMinn (1955). Starting from this genuine and repeatable example of experimental bone induction, Huggins and others modified the conditions of the experiment in an attempt to discover the causal factors. Thus, Huggins et al. (1936) found that bladder grafts to the spleen and liver failed to induce bone unless fascia from the abdominal wall was grafted with it. It was also found that gall bladder epithelium transplanted to the rectus sheath regularly formed bone in the guinea pig and sometimes in the dog (Huggins and Sammett, 1933). I n a study of the repair of fractures in bladder-induced bone in dogs, it was found that healing took place along normal lines, indicating that the induced bone behaved like a normal element of the skeleton. In other

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words, the transitional epithelium had behaved like an embryonic organizer and produced a permanent alteration in cellular behavior and potentialities (Huggins et al., 1934). Huggins et al. (1936) investigated the capacity of different connective tissues throughout the body to form bone in response to the presence of transitional epithelium. They found that only some connective tissues were capable of becoming osteogenic. Thus, the normal connective tissue lying adjacent to the epithelial lining of the urinary tract very rarely ossified, but a bladder mucosal graft placed on the peritoneal surface of the bladder readily induced bone. Huggins interpreted these results to mean that there were various races of fibroblasts in the body, which, though morphologically indistinguishable, were functionally diverse in their response to proliferating epithelium. This concept is supported by Parker’s ( 1933) observation that fibroblasts from different organs of the developing embryo display specific rates of cell division in vitro, which persist throughout development. Johnson and McMinn (1955) studied the behavior of auto- and honiografts of bladder mucosa placed in the rectus sheath in the cat. After 2 days both types of graft formed epithelial-lined cysts, but whereas in the autografts the epithelium persisted in a healthy state, in homografts infiltration by inflammatory cells began after 5 days and the graft was eventually destroyed. Johnson and McMinn ( 1956) further reported that both auto- and homografts of urinary bladder mucosa can induce bone formation as early as 10 days after transplantation to the rectus sheath in the cat. Gomori ( 1943) studied the histochemical changes around bladder grafts placed in the rectus sheath and found scattered phosphatase-rich fibroblasts in the host connective tissues in the vicinity of the epithelial cyst. The number of these phosphatase-rich cells gradually increased, and then around them coarsely fibrillar osteoid was laid down near the epithelium although separated from active contact with it by a narrow phosphatasefree zone. After 13 days calcium salts were deposited in the osteoid to 4 give true bone. In an attempt to expldt the bone-inducing power of transitional epithelium, extracts of bladder mucma have been injected around healing fractures by Copher et al. (1932) and Eskelund and Plum (1950), but without success. Copher and Key ( 1934), however, placed urinary bladder mucosa in l-cm. defects of the dog’s ulna and obtained bony union, but similar fractures without bladder mucosa did not unite. They also claimed that the presence of the mucosa somehow prevented atrophy of the fractured ends of the ulna. Copher (1935) further found that bladder mucosa

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placed in contact with hyaline costal cartilage in dogs induced hypertrophic changes and then bony replacement ; Loewi ( 1954), however, was unable to confirm these changes in the cartilage of the guinea pig, although ossification occurred in the connective tissue surrounding the bladder mucosal graft. Roome and McMaster (1934) reported that the amount of heterotopic bone induced by bladder mucosal grafts placed in the leg fascia of the dog was increased by the venous stasis produced by tying off the main leg veins. Working along different lines, Abbott et ul. ( 1938), Dragstedt (1931), and Dragstedt and Kearns (1932) found that parathyroidectomy retarded the induction of bone by bladder epithelium in the soft tissues of dogs. The general conclusion from all these various lines of evidence is that, in certain animals at least, the transitional epithelium of the urinary tract, when stimulated by a diversity of means, becomes capable of inducing bone formation in a number of connective tissues placed in contact with it. As yet, however, we know little of the nature of the inducing stimulus in the epithelium, or of its mode of action. The heterotopic bone induced by transitional epithelium is histologically and histochemically indistinguishable from normal bone, and its reactions to injury and hormones are orthodox. OF EXTRACTS OF SKELETAL TISSUES 111. INJECTION

Levander (1938), from a study of the fate of autogenous bone grafts in the soft tissues of the rabbit, came to the conclusion that the bone cells of such grafts died within 4 days and that new bone found later was metaplastic in origin. H e argued from this that an osteogenetic substance must have diffused out from the bone graft and induced bony metaplasia in the surrounding connective tissue. Accordingly, he attempted to extract the hypothetical osteogenetic inductor from bone and fracture callus, testing the potency of his extracts by injections into the thigh muscles of rabbits. Although aqueous extracts failed to induce ossification, injections of alcoholic extracts resulted in cartilage or bone formation in 15 out of 70 animals. Injections of alcohol alone (60 animals), or extracts of muscle and connective tissue (20 animals), however, failed completely to induce bone or cartilage. Levander believed these results proved his hypothesis that a specific osteogenetic substance was present in bone and fracture callus; and he suggested that it might enter the blood stream in certain circumstances and initiate heterotopic bone formation in the soft tissues. Annersten (1940) confirmed Levander’s results. He found that alcoholic extracts of homogenous bone in 50% of cases and of heterogenous bone in 25% of cases were successful in inducing cartilage or bone in the

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muscles of rabbits. Finding that one of 10 injections of extracts of homogenous kidney, and one of 6 injections of alcoholic-HC1-dialyzed urine, induced bone or cartilage, he considered there was substance in Levander’s argument that a bone inductor might enter the blood stream and be excreted by the kidney. Annersten attempted to elicit some of the properties of the bone inductor substance by fractionating procedures with various solvents and claimed that the inductor was a lipoid or a steroid but not a phospholipid. Bertelsen (1944) tried to identify the component of growing bone which produced the inductor. H e tested separately extracts of growing bones, periosteum, cortical bone, bone marrow, and epiphyseal cartilage and found that bone marrow gave the highest number (10 of 12 experiments) of positive results. Extracts of whole bone gave inductions in 6 out of 12 cases, periosteum in 4 out of 12, cortical bone in 5 out of 12, and epiphyseal cartilage in 4 out of 12 animals. In all these experiments, however, a preliminary injection of 40% alcohol was made into the rectus femoris a few days before the extract was injected. H e found that none of 10 injections of alcohol alone, but one of 10 injections of HC1-alcohol, induced bone formation ; he ascribed this single positive result to periosteal stimulation by the injection. I n 41 control injections only one positive result was obtained, viz., with an alcoholic extract of liver. Bertelsen concluded : “The osteogenetic substance was chiefly concerned with bone marrow.” Levander, Annersten, and Bertelsen had all used a preliminary injection of alcohol to stimulate granulation tissue formation, for it was thought that proliferating connective tissue cells were the most likely to respond to any inductor which might be present in these extracts. Bertelsen attempted to stimulate granulation tissue formation mechanically by pinching the rectus femoris with forceps, but in none of 24 animals did subsequent injections of skeletal tissue extracts induce bone or cartilage. He also found that his extracts were without effect on heart fibroblasts in vitro. H e suspected that the preliminary alcohol injection used in the previous experiments may have had some effect on the muscles which disposed them to bone induction a.fter the alcoholic extract injections. Lacroix (1945, 1947) produced cartilage, bone, and bone marrow in the thigh muscles of growing rabbits after injection of alcoholic extracts of epiphyseal cartilage from newborn rabbits. H e described the induced cartilage and bone as being well organized, the cartilage in some specimens closely resembling a normal epiphyseal plate even to the ossification groove (encoche de Ranvier) and perichondral ring of bone. The induced bony tissue, on the other hand, resembled part of a growing diaphysis and even showed the normal type of metaphyseal remodeling. H e concluded that

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alcoholic extracts of very young epiphyseal cartilage induced a more highly organized form of bone and cartilage than did extracts of the bone and callus from older animals. H e suggested the name “osteogenin” for the inducing substance apparently present in skeletal tissues and extractable with alcohol. Levander and Willstaedt ( 1946), in a brief communication, claimed that the highest osteogenetic activity was to be found in the crude fatty acid fraction of the alcoholic extracts of bone marrow. Rendano (1942) obtained cartilage in one out of 22 injections of bone extracts into the thigh muscles of rabbits. Martin-Lagos and Romero (1946) were very sceptical of the “osteogenin” hypothesis, for they found that injection of 40% alcohol alone produced cartilage and/or bone in 7 of 87 rabbits. Nevertheless, Hartley et al. (1949) found that the injection of alcoholic extracts of the ends of long bones of young rabbits into the thigh muscles of older animals resulted in bone and/or cartilage in 3 out of 11 animals, whereas all 11 control injections of 95% alcohol alone were unsuccessful. Heinen et al. (1949) gave a good review of the “osteogenin” literature, and they repeated the experiments of Annersten. In 7 of 24 injections of alcoholic extracts of bone they found bone or cartilage formation, but they obtained positive results also in 35 of 77 injections of alcohol alone into muscles. In other words, they found a higher incidence of bone and cartilage production after alcohol alone than after alcoholic extracts. Moreover, they claimed that the bone and cartilage resulting from alcohol injections alone were just as well organized as the tissues described by Lacroix. Wachsmuth (1950), however, found in 8 cases that alcohol injected into the thigh muscles of rabbits did not produce bone or cartilage, although positive results were obtained in 12 of 13 extracts of whole bone, 8 of 17 extracts of periosteum, 11 or 18 extracts of cortical bone, and 11 of 14 extracts of bone marrow. Roth (1950) likewise found that all his 8 injections of alcohol alone gave negative results, but alcoholic extracts of fresh bone gave 5 positive results out of 8. Willstaedt et al. (1950) found that alcoholic extracts of heterogenous bone marrow (calf) were as successful as homogenous ones in stimulating osteogenesis in rabbits’ muscles. They considered the inductor responsible for bone formation to have some of the properties of a phosphatide. Schreiber (1950) found that alcoholic extracts of growing rabbit bones were osteogenetic in the newt. Pfeiffer (1950) found that alcoholic extracts of bone failed to produce

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cartilage or bone after injection into the testis of the mouse in 33 cases. Mondolfo (1950) and Fogliati (1950) failed to obtain bone or cartilage after injection of alcoholic extracts of bone into rabbit muscles, as did Constance (1954) after injections of cell-free aqueous extracts of bone into the thigh muscles of guinea pigs. Lacroix (1951a) exactly repeated the experiments of Heinen et al. (1949) but found no bone or cartilage after injecting alcohol alone into the leg muscles of 9 young rabbits. Hartley and Tanz (1951) reported that alcoholic extracts of autogenous bone injected into the thigh muscles of rabbits gave cartilage or bone in 3 of 11 cases, whereas 11 control injections of the extracting agent (95% alcohol) did not. Of 16 injections of concentrated alcoholic extract of bone marrow, 5 resulted in bone or cartilage formation, but none of 36 injections of extracts of bone from which the periosteum and marrow had been removed was successful. No bone or cartilage was obtained with aqueous extracts, but alcoholic extracts of calf bone marrow resulted in bone or cartilage formation in 5 of 32 intramuscular injections into rabbits. Hartley and Tanz discuss their results very cautiously. They think an osteogenetic substance might be present in the skeleton but admit that the results could be due to the nonspecific irritant properties of the extracting agent. Lindahl and Orell (1951) tested various bone extracts by repeated injection into the leg muscles of mice and guinea pigs and subcutaneously in man but found neither bone nor cartilage at the site of any of the injections. Stephenson (1952) reported that injections of alcoholic extracts of sternal and ear cartilage, of benzene extracts of cartilage, of phosphatase solution, and of acidified alcohol were uniformly unsuccessful in the formation of cartilage or bone in the leg muscles of rats. Heinen (personal communication, 1956) thinks that the existence of a specific osteogenetic substance is undecided. H e emphasizes the close topographical relationship of the ectopic bone and cartilage in his experiments to the dense connective tissue found in the posterior part of the rabbit’s rectus femoris muscle and feels that variations in the results obtained by other workers might be due to differences in the site of injections into the rectus femoris muscle. Recently we (Bridges and Pritchard, 1958) completely failed to induce bone or cartilage formation in the leg muscles 30 days after the injection of 40% alcohol into 27 rabbits, 12 rats, 18 guinea pigs, 16 mice, and 6 fowl muscles. Connective tissue had replaced muscle fibers in some localized areas in the muscle, but elsewhere the muscle fibers stained normally. In

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one of 4 rabbit leg muscles injected with undiluted methylated spirits, however, cancellous bone formation was noted in the midst of a mass of fibrous tissue deep in the muscle (Fig. 5 ) . Numerous osteoblasts lined the trabeculae, and primitive marrow filled the spaces between them. No bone or cartilage was found in 4 rats or in 4 guinea pig muscles after similar injections.

FIG.5. Bone formation in the rectus femoris muscle of a rabbit 30 days after the injection of methylated spirits. (XIOO)

Thus, of the 91 intramuscular injections of alcohol in rabbits, rats, guinea pigs, and hens, bone was ouly found in a single instance, namely, in a rabbit whose rectus femoris had been injected with undiluted methylated spirits 30 days previously. At first sight the evidence that alcohol alone might induce bone or cartilage in rabbits’ muscles seeiiis fatal to the “osteogenin” hypothesis. It must be realized, however, that the majority of workers have failed to get positive results with alcohol alone. Thus, in no one of 123 injections of alcohol alone into rabbit muscle was bone or cartilage produced in the experiments of Levander and seven other workers. Nevertheless the high percentage of positive results of Heinen et al. must be accounted for. Lacroix suggests, without evidence, that the alcohol diffused through the thigh muscles to the femur and extracted osteogenin from the bone, and that the osteogenin then initiated bone or cartilage formation in the muscle.

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A different interpretation may be put on the results both with alcohol injections alone, and with alcoholic and other extracts of skeletal tissues, arising out of the finding (Bridges and Pritchard, 1958) that muscle of all kinds (skeletal, cardiac, and smoothj in the rabbit, when devitalized by alcohol and other means, induces cartilage and bone when transplanted beneath the kidney capsule. It can thus be argued that the muscle itself liberates the inductor, and the alcohol and extracts may act as nonspecific irritants. I t should be stressed, however, that alcohol and extracts of skeletal tissues have only been shown to cause bone and cartilage formation in the muscles of the rabbit.

Iv.

INJECTIONS OF IRRITANTS A N D O T H E R

TRAUMATIC EXPERIMENTS

There are a number of reports in the literature of heterotopic ossification subsequent to mechanical damage to muscles and the injection of irritant substances into muscles or into the eye. Such ossification has also followed the painting of blood vessels with sclerosing solutions and the application of large doses of X-rays to the lungs. It is difficult to see any common denominator in these experiments apart from tissue damage at the time of operation. Haga and Fujimura (1903) reported ossification in rabbit muscles after hammer blows to the animals’ legs. Masadu (1929) confirmed these results. Bertelsen (1944), however, was unable to demonstrate bone formation in the rectus femoris muscles of 24 rabbits after pinching with forceps and then injecting alcoholic extracts of bone. Nevertheless von Seemen (1929) reported bone or cartilage after injection of bone autolyzates into previously traumatized muscles of rabbits. Neither trauma nor the autolyzate was effective alone, so it would appear that trauma may be a necessary, though not a sufficient, condition for heterotopic ossification. Trauma most likely acts by stimulating the production of a granulation tissue on which an inductor (probably liberated from the muscle) can exert its metaplastic stimulus.

A . Irritants into Muscle Calcium salts injected into muscles have occasionally given rise to new bone formation. Wiirm (1930) found that artificial bone salt (calcium phosphate-calcium carbonate mixture) was effective in young rabbits but not in older ones, and other workers have employed the much more irritant calcium chloride solutions. Asami and Dock (1920) found no bone formation 50 days after injecting calcium salts (calcium chloride, carbonate, and sodium phosphate mixture) into the leg muscles or under the skin of 5 rabbits. On the other hand, Heineii et al. (1949) found that intramuscular

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injections of 1 or 2% calcium chloride solutions resulted in bone and/or cartilage in 9 of 56 animals. Previous to this Annersten (1940) had obtained bone with a mixture of alcohol, calcium chloride, and disodium phosphate injected into the leg muscles of rabbits in 2 out of 33 experiments. Stephenson (1952) found cartilage formation in the leg muscles of a single rat 10 days after the injection of a saturated solution of monobasic calcium phosphate. Blum (1944) injected a mixture of alkaline phosphatase, calcium chloride, and sodium alginate into rabbit muscle and found bone formation in a few animals. In view of the results of Heinen et al. with calcium chloride solutions alone, it is likely that the calcium chloride was the effective agent in these last two experiments, though Blum thought he had demonstrated the bone-inducing power of phosphatase. Slessor and Wyburn ( 1948) repeated Blum’s experiments, however, and found no bone formation in any of 6 animals. Nor did we (Bridges and Pritchard, 1958) find that pellets containing calf intestinal alkaline phosphatase induced any bone or cartilage when placed under the kidney capsule or subcutaneously iir rabbits. The irritant properties of quinine have also been used to provoke osteogenesis in muscle. Severi (1933), noting the reports of occasional heterotopic ossification in man after repeated injections of quinine hydrochloride in the treatment of malaria, found that similar repeated injections into the thigh muscles of rabbits resulted in bone formation in 3, and cartilage in 2, of 15 such experiments.

B. Irritants into the Eye Calcium salts have not been effective in producing heterotopic ossification in the eye. Bisgard (1936) studied the effect of injecting calcium salts into the anterior chamber of rabbits’ eyes in which living bone had previously been grafted and found that the presence of these salts did not stimulate osteogenesis. In a series of 6 rabbits he injected a mixture of calcium carbonate ( 14%), calcium phosphate (85%), and magnesium phosphate ( 1 % ) into the eye but found no bone formation after 126 days. Using a similar experimental technique, Ray et al. (1952) injected a synthetic hydroxyl apatite containing calcium carbonate into the eyes of 6 guinea pigs but found no. bone formation in the damaged eye. Formic acid, however, injected into the vitreous body of the eyes of 26 rabbits, resulted in bone or cartilage formation in 12 cases (Imai, 1930). C. Application of Sclerosing Solutions to Blood Vessels

Harvey (1907) found that application of 3% silver nitrate or 2% cupric sulfate to the abdominal aorta of 10 mature rabbits resulted in bone or

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osteoid tissue forming in the calcified media of the vessels in 8 cases. H e thought the bone arose by nietaplasia of the connective tissues replacing the necrotic media whose blood supply from the vasa vasorum had been destroyed by the sclerosing solution.

D. Trauitia by Irradiation Engelstad (1934) found that irradiation of the lungs of rabbits with X-rays caused degenerative changes and fibrosis. In 15 of 28 rabbits so treated, he found bone or cartilage in the fibrous tissue. It should be noted that workers who placed calcium salts in soft tissues did so to test the hypothesis that calcium was a specific inductor of heterotopic bone. It is clear, however, from the variety of traumatic and irritant stimuli which have resulted in bone formation, that the calcium salts even when successful probably acted only as nonspecific irritants. This is borne out by the fact that of the calcium salts tried only the soluble and highly irritant chloride led to bone forma tion, whereas other relatively insoluble calcium salts were unsuccessful. The most likely explanation for all these results is that mechanical trauma liberates inductors from the cells it damages or destroys, and that these inductors stimulate bone formation in the young granulation tissue which develops around the site of injury.

V. IMPLANTS OF DEVITALIZED SKELETAL TISSUES It is evident that new bone appearing in or around an implant of dead bone or cartilage in the soft tissues remote from the skeleton must be a genuine example of bone induction. The appearance of new bone in association with a transplant of living bone, however, or a piece of dead bone transplanted into the living skeleton, cannot be taken as evidence of induction because the osteogenetic cells might have come from the graft, or host, or both. Nevertheless a great many authors have assumed induction on no stronger grounds than doubtful histological evidence from living bone grafts. There is no point in pursuing here the endless controversy as to whether bone grafts live or die in whole or in part, whether they act as inductors, as reservoirs of calcium salts, or simply as a medium for conducting ossification from the host : the problem of induction by skeletal tissues will not be solved by a study of bone grafted to bone ; unequivocal evidence can be obtained only from devitalized skeletal tissues implanted into soft tissues where direct spread of osteogenetic cells from the living skeleton can be ruled out. Thus, only evidence of bone or cartilage induction in soft tissues around skeletal implants devoid of living cells need be considered.

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Barth (189.5) found bone 6 weeks after he had introduced calcined bone into the peritoneal cavity of the cat. Osteoblasts appeared and laid down bone on the walls of channels excavated in the dead bone by vascular granulation tissue. H e considered that local calcium deposits in the region of granulation tissue were the necessary and sufficient conditions for bone formation. Nageotte (1918) found new bone in 75% of a series of over 20 cases where alcohol-devitalized homogenous cartilage was implanted subcutaneously into the ear of the rabbit. Later, he reported new bone formation around alcohol-devitalized bone implants in the ear ( 1920). Asami and Dock (1920) compared the fate of living and boiled hyaline and elastic cartilage placed in the subcutaneous tissues. In no instance did living or boiled hyaline cartilage grafts induce bone formation, but in one experiment with boiled elastic cartilage they found bone and marrow formation in the dense connective tissue around the implant. Polletini ( 1922) repeated Nageotte’s experiments using subcutaneous and subfascial sites for implantation. H e reported new bone or cartilage formation in the connective tissue around 2 of 12 alcohol-fixed cartilage implants, and 4 of 19 similarly devitalized bone implants. As he could find no direct continuation between the implants and the induced bone or cartilage, he concluded that these tissues were produced by a metaplasia of the connective tissues under the influence of a chemical substance diffusing out of the original implant. Didier and Guyon (1928) found that alcohol-fixed cartilage implants in the shoulder region and linea alba of rabbits were replaced by bone and fatty marrow. They thought bone formation was due to metaplasia of the connective tissue around the implant. Wiirm (1930) described new bone formation in 6 of 10 animals when he transplanted boiled bone into the connective tissue between the abdominal muscles of the rabbit. Orell (1934) reported new bone formation around bone implants devitalized either by chemical means or by cooking and placed in the subcutaneous tissues of man. H e thought the only difference between the reactions to living and dead implants was the longer time taken for the vascular connective tissue of.the host to penetrate the devitalized implants. Bisgard (1936) compared the fate of living and boiled bone autografts placed in the anterior chamber of the rabbit’s eyes. H e found new bone formation in 3 of the implants of boiled bone and associated it with vascular invasion of the graft. Kimball (1949) found bone in one of 5 transplants of boiled homogenous bone placed in the anterior chamber of the guinea pig’s eye.

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Dupertuis (1941) noted early bone formation in one of 6 pieces of alcohol-fixed rabbit’s ear cartilage examined after 216 days in the subcutaneous tissues of another rabbit. Rohlich ( 1941) described bone around alcohol-devitalized homogenous bone implanted in the muscles of rabbits. H e thought that an inductor present in the implants diffused into the connective tissue and initiated bony metaplasia. Engstrom and Orell (1943) compared the fate of implants of fresh bone and fresh bone devitalized b,y repeated freezing and thawing, in the subcutaneous tissues of man. As both types of implant produced new bone in their vicinity, they concluded that the bone implants contained a substance capable of inducing the surrounding connective tissues to form bone. Lacroix ( 1951b) reported that slivers of alcohol-devitalized cortical bone placed beneath the kidney capsule of rabbits were replaced after 4 to 7 months by a lentiform ossicle filled with hemopoietic marrow. Boiled bone, however, did not result in bone formation. Lacroix claimed that the new bone which replaced the implanted bone was initiated by “osteogenin” released from the graft and that this inductor was destroyed by boiling but not by immersion in alcohol. Urist and McLean (1952) reported new bone formation in 2 of 3 boiled, and one of 3 frozen, implants of fracture callus placed in the anterior chamber of the rat’s eye. New bone formation developed from ingrowing perivascular connective tissue and was laid down both around and within the devitalized cartilage of the implant. De Bruyn and Kabisch (1955) found the incidence of new bone formation after autogenous implants of bone to the thigh muscles of rabbits to be reduced from 18 out of 21 to 2 out of 26 by prior immersion of the implant in liquid nitrogen. The incidence of bone formation around similar homogenous implants was likewise decreased after freezing. De Bruyn and Kabisch accounted for these results by suggesting that a bone inductor present in the implants was destroyed by freezing. Lacroix (1953) reported bone induction by living fracture callus placed under the kidney capsule of the rabbit, but later (1956) he stated that alcohol-killed callus and epiphyseal cartilage also showed bone induction in these circumstances. Peer (1955) observed new bone formation in grafts of alcohol-preserved rib and nasal septal cartilage which had been buried in human subcutaneous tissues for periods up to 12 years. Alcohol-preserved human septal cartilage implants began to be eroded and replaced by connective tissue after about 12 months, and bone was laid down on the walls of the eroded

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cartilage. H e described the process as being very similar to normal endochondral ossification. We (Bridges and Pritchard, 1955, 1958) have recently shown that autogenous and homogenous tissues of the rabbit that contain hypertrophic cartilage, but not those that contain nonhypertrophic hyaline or elastic cartilage, when devitalized in certain ways and placed beneath the kidney capsule or in the ear, were able to induce bone formation in the host tissues. Thus, devitalized fracture callus recovered from a healing tibia1 fracture, and devitalized epiphyseal plate cartilage (Fig. 6) regularly induced bone formation after 25 days under the kidney capsule, whereas xiphisternal and ear cartilage, cortical bone, skin, heterogenous hyper-

FIG.6. Bone and marrow formation in an implant of alcohol-fixed homogenous epiphyseal cartilage after 40 days under the kidney capsule of a rabbit. (X45)

trophic cartilage, and callus implants that contained no hypertrophic cartilage were unsuccessful. Implants of alcohol-devitalized costal cartilage showed bony replacement at their hypertrophic but not at their hyaline ends. Devitalized auto- and homogenous skeletal, cardiac, and visceral muscle, on the other hand, induced cartilage, which in turn was replaced by bone, when placed beneath the kidney capsule. Hypertrophic cartilage retained its bone-inducing properties after devitalization with alcohol, acetone, 0.01 N HCl, and heating to 55°C. (Fig. 7), but not after treatment with 1% NaOH or heating to 100°C. Cardiac, skeletal, and smooth muscle induced cartilage after devitalization in alcohol and acetone (Figs. 8-10). Although it was extremely unlikely that any cells could survive these devitalizing procedures, attempts were made to cultivate alcoholtreated callus fragments in vitro, but without success, whereas pieces of living callus produced a flourishing outgrowth of cells.

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Direct effects of the devitalizing agents on the connective tissue as an explanation for induction were ruled out by the fact that implants were well washed of residual alcohol before implantation, that heating without.

FIG. 7. Bone formation in an implant of autogenous fracture callus (heated to 55°C. for 3 minutes before implantation) after 35 days under the kidney capsule of a rabbit. (X34)

FIG.8. Nodules of cartilage in and around an implant of alcohol-fixed homogenous skeletal muscle after 35 days under the kidney capsule of a rabbit. (X100)

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chemical treatment gave similar results, that induction was limited to hypertrophic cartilage and muscle implants, and that alcohol painted on the kidney capsule or implants of gelatin sponge immersed in alcohol produced only an increase of connective tissue in the kidney capsule with-

FIG.9. Cartilage formation in an implant of acetone-fixed autogenous skeletal muscle after 35 days under the kidney capsule of a rabbit. (X34)

FIG.10. Cartilage formation in an implant of alcohol-fixed homogenous ileum after 35 days under the kidney capsule of a rabbit. (X34) out bone or cartilage formation. ‘The site of implantation did not seem a significant factor, as similar results were obtained in the ear and under the kidney capsule. Callus implants recovered from 9 to 95 days after implantation were studied. Bone formation began on the walls of vascularized channels eroded in the cartilage by multinucleated giant cells and proceeded until

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the implant had been replaced by an ossicle with cortex, medulla, and periosteum (Fig. 11j . With the subcapsular implants the medulla contained hemopoietic marrow, but subcutaneous implants developed nonhemopoietic marrow, possibly owiiig to the lower temperature of the ear site. Cartilage formation began between the swollen fibers of the muscle implants but was sometimes in the form of isolated nodules in the adjacent connective tissue of the kidney capsule. The cartilage was of the hypertrophic type with large cells and relatively scant matrix; it was later invaded by blood vessels and replaced by bone, resulting in a lentiform marrow-filled ossicle after 65 days.

FIG. 11. Lens-shaped marrow-filled ossicle replacing alcohol-fixed autogenous callus after 65 days under the kidney capsule of a rabbit. (X45) W e concluded that these results represented genuine bone and cartilage inductions, for the implants contained no living skeletal cells, and the kidney capsule site used was remote from the skeleton. W e consider that the inductor or inductors responsible were most likely protein in nature and arose from the devitalized implants whence they diffused out to stimulate osteogenesis or chondrogenesis in the connective tissue of the host's kidney capsule. New bone formation has been found on the surface of the cartilage component of alcohol-fixed homogenous fracture callus 100 days' after it was placed in the brain of a rat, whereas alcohol-fixed autogenous or homogenous callus failed to excite any reaction when placed under the rat kidney capsule after 40 or 60 days. These results indicate that bone induction by hypertrophic cartilage may also occur in the rat but that a

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much longer period of adaptation is necessary (Thompson and Bridges, unpublished results, 1958).

VI.

CONCLUSIONS

Spontaneous heterotopic ossificalion may occur in almost any connective tissues of the body and has been extensively described, but with few definite etiological factors emerging. Bone and cartilage have also been induced experimentally in many different ways, but general principles are not easily adduced from these results. The association of both experimentally and spontaneously induced heterotopic bone with proliferating transitional epithelium, however, is well established in several species. There also seems little doubt that hypertrophic cartilage and muscle of all kinds possesses bone- and cartilage-inducing powers in the rabbit. The presence of inductors for bone and cartilage in other tissues and other species has not been conclusively demonstrated. Much hitherto promising work is suspect because of the ability of extracting and devitalizing agents to produce heterotopic bone and cartilage of themselves after the manner of certain nonspecific irritants. Until the presence of inducing agents in bone has been unequivocally established in a number of species, it would be wise to defer judgment on the many claims that induction plays an important role in bone repair and transplantation. Perhaps “once fibroblastic, sonietimes osteoblastic ; once osteoblastic, always osteoblastic” is as near to a summarizing aphorism as one can go at the present in this field.

VII. ACKNOWLEDGMENTS The author acknowledges gratefully the helpful and constructive criticisms of Professor J. J. Pritchard, who read the manuscript, and also the Editors of the Journal of Anatomy and the Journal of Urology for permission to reproduce Figs. 6-11 and Figs. 1-4, respectively.

VIII. REFERENCES Abbott, A, C., and Goodwin, A. M. (1932) Can. Mcd. Assoc. 1. 26, 393. Abbott, A. C.,and Stephenson, E. (1945) Can. Med. Assoc. J . 62, 358. Abbott, A. C., Goodwin, A. M., and Stephenson, E. (1938) J. Urol. 40, 294. Annersten, S. (1940) Acta Chip. Scand. 84, Suppl. NO.60, 1. Asami, G., and Dock, W. (1920) J . Exptl. Med. 32, 745. Barth, A. (1895) Beitr. pathol. Anat. u. allgcm. Pathol. 17, 65. Bettelsen, A. (1944) Acta Orthopaed. Scand. 16, 139. Bisgard, J. D. (1936) A . M . A . Arch. Surg. 33, 926. Blum, G. (1944) Lancet 2, 75. Boyarsky, S., and Duque, 0. (1955) J. Urol. 73, 53. Bridges, J. B. (1958) I. Urol. 79, 903.

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Bridges, J. B., and Pritchard, J. J. (1955) J. Anat. 89, 580. Bridges, J. B., and Pritchard, J. J. (1958) J. Anat. 91, 28. Busch, F. (1878) Deut. Z . Chir. 10, 59. Chauvin, E.,and Rouslacroix, A. (1929) 1. 8rol. m i d et chir. 27, 465. Constance, J. J. (1954) J. Pathol. Bacteriol. 68, 381. Copher, G. H. (1935) Ann. Surg. 102, 927. Copher, G. H., and Key, J. A. (1934) A.M.A. Arch. Surg. 29, 64. Copher, G. H., Key, J. A., and West, E. S. (1932) Proc. Soc. Exptl. Biol. Med. 29, 640. De Bruyn, P. P. H., and Kabisch, W. T. (1955) A m . I. Anat. 96, 375. Didier, R., and Guyon, L. (1928) Compt. rend. soc. biol. 98, 443. Dragstedt, C. A. (1931) A m . J. Physiol. 97, 517. Dragstedt, C. A., and Kearns, J. E. (1932) A m . J. Physiol. 100, 262. Dupertuis, S. M. (1941) A . M . A . Arch. Surg. 43, 32. Engelstad, R. B. (1934) Act a Radiol. Suppl. 19, 1. Engstrom, H., and Orell, S. (1943) 2. mikroskop.-anat. Forsch. 63, 283. Eskelund, V., and Plum, C. M. (1950) Act a Orthopaed. Scand. 19, 433. Fogliati, E. (1950) Chir. org. movimento 94, 129. Gomori, G. (1943) A m . J. Pathol. 19, 197. Haga, and Fujimura, (1903) Arch. klin. Chir. Langenbecks 72, 64. Hartley, J., and Tanz, S. S. (1951) A . M . A . Arch. Surg. 65, 845. Hartley, J., Tanz, S. S., and Schneider, M. (1949) J. M t . Sinai Hosp. N . Y . 16, 383. Harvey, W. H. (1907) J. Med. Research 12, 25. Heinen, J. H., Jr., Dabbs, G. H., and Mason, H. A. (1949) J. Bone and Joinf Surg. 31A, 765. Huggins, C. B. (1931) A . M . A . Arch. Surg. 22, 377. Huggins, C. B., and Sammett, J. F. (1933) J . Exptl. Med. 66, 393. Huggins, C. B., McCarroll, H. R., and Dahlberg, A. A. (1934) J. Exptl. Med. SO, 199. Huggins, C. B., McCarroll, H. R., and Blocksom, B. H. (1936) A.M.A. Arch. Surg. 32, 915. Imai, N. (1930) Arch. Ophthalmol. Graefe’s l26, 267. Johnson, F. R., and McMinn, R. M. H. (1955) J. Anat. 89, 450. Johnson, F. R., and McMinn, R. M. H. (1956) J. Anat. 90, 106. Kimball, R. M. (1949) M. S. Thesis, Tulane University, New Orleans, Louisiana. Kossa, J. von (1901) Beitr. pathol. Anat. u. allgem. Pathol. 29, 163. Kretschmer, H. L. (1928) J. Urol. 20, 477. Lacroix, P. (1945) Nature 166, 576. Lacroix, P. (1947) I. Bone and Joint Surg. 29, 292. Lacroix, P. (1951a) “The Organization of Bones” (translated by S. Gilder), p. 217. Churchill, London. Lacroix, P. (1951b) “The Organization of Bones” (translated by S. Gilder), p. 194. Churchill, London. Lacroix, P. (1953) Acta Chir. Belg., 62, 877. Lacroix, P. (1956) Ciba Foundation Symposium, Bone Structure and Metabolism p. 144. Leuckart, R. (1876) “Die Parasiten des Menschen,” Vol. 2, p. 396. Levander, G. (1938) Surg. Gynecol. Obstet. 67, 705. Levander, G., and Willstaedt, H. (1946) Nature 167, 587.

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Liek, E. (1908) Arch. klin. Chir. Langenbecks 86, 118. Lindahl, O., and Orell, S. (1951) Acta Chir. Scand. 101, 136. Litten, M. (1881) Arch. pathol. Anat. u. Physiol. Virchow’s 83, 508. Loewi, G. (1954) J . Pathol. Bacteriol. 68, 419. MacEwen, W. (1912) “The Growth of Bone,” Vol. 1, p. 116. Maclehose, Glasgow. Marshall, V. F., and Spellman, R. M. (1954) Transplant. Bull. 1, 150. Martin-Lagos, F., and Zarapico Romero, M. (1946) Trabajos inst. nacl. cienc. mid. (Madrid) 6, 173. Masadu, R. (1929) Japan. J . Dermatol. Urol. 29, 423; Abstract, p. 33. Maximow, A. (1906) Anat. Anz. 28, 609. Mondolfo, S. (1950) Rev. ortop. traum. 19, 47. Nageotte, J. (1918) Compl. rend. soc. biol. 81, 113. Nageotte, J. (1920) Compt. rend. 171, 280. Neuhof, H. (1917) Surg. Gynecol. Obstct. 24, 383. Orell, S. (1934) Acta Chir. Scand. 74, 1, Suppl. No. 31. Parker, R. C. (1933) J . Exptl. Med. 108, 393. Pearce, R. M. (1909) J. Med. Research 20, 53. Peer, L. A. (1955) “Transplantation of Tissues,” p. 102. Williams & Wilkins, Baltimore, Maryland. Pfeiffer, C. A. (1950) Proc. SOC.Exptl. Biol. Med. 71, 388. Phemister, D. B. (1923) Ann. Surg. 78, 239. Pocharissky, J. F. (1905) Beitr. pathol. Anat. u. allgem. Pathol. 38, 135. Polettini, B. (1922) Arch. ital. chir. 6, 179 [Abstract, J. Am. Med. Assoc. 80, 360 (1923) 1. Ray, R. D., Degge, J., Gloyd, P., and Mooney, G. (1952) J . Bone and Joint Surg. MA, 638. Regen, E. M., and Wilkins, W. E. (1934) J. Lab. Clin. Med. 20, 250. Rendano, C. (1942) Ann. ital. chir. 21, 249. Rohlich, K. (1941) 2. mikroskop.-anat. Forsch. 60, 132. Roome, N. W., and McMaster, P. E. (1934) A.M.A. Arch. Surg. 29, 54. Roth, H. (1950) Schweiz. med. Wochschr. 80, 1051. Sacerdotti, C., and Frattin, G. (1902) Arch. pathol. Anat. u. Physiol. Virchozv’s 168, 431. Schreiber, B. (1950) Boll. soc. ital. biol. sper. 26, fasc. 4, 526. Seemen, H. von (1929) Deuf. 2. Chir. 217, 60. Severi, R. (1933) Pathologica 26, 611. Slesser, A., and Wyburn, G. M. (1948) Lancet, 1, 212. Stephenson, K. L. (1952) Plastic Reconstr. Surg. 9, 302. Strauss, A. A. (1914) Surg. Gynecol. Obstet. 18, 78. Urist, M. R., and McLean, F. C. (1952) J . Bone and Joint Surg. MA, 443. Wachsmuth, G. (1950) Arch. klin. Chir. Langenbecks 266, 58. Weinland, D. F. (1859) Arch. Naturgeschichte 1, 283. Wells, H. G., Holmes, H. F., and Henry, G. R. (1911) J . Med. Research 26, 373. Willstaedt, H., Levander, G., and Hult, L. (1950) Acta Orthopaed. Scand. 19, 419. Wiirm (1930) Verhandl. deut. pathol. Ges. 26, 191.

A Survey of Metabolic Studies on Isolated Mammalian Nuclei D . B . ROODYN Medical Research Council Radiobiological Research Unit. Harwell. England1 Page I . Introduction ...................................................... 279 I1. Methods for Isolating Nuclei ....................................... 280 A . List of Methods Used ....................................... 280 B. Homogenizers ................................................ 280 C. Methods of Separating Nuclei from Homogenate ............... 284 I11. Biochemical Studies on Isolated Nuclear Fractions ................. 285 A . In Vitro Isotope Incorporation Studies ......................... 285 B . Enzyme and Nitrogen Determinations ....................... 287 C. DNA and RNA ............................................. 316 I V. Validity of Studies on Isolated Nuclei ............................. 316 A . Contamination with Nonnuclear Material ..................... 316 1. Adsorption of Material onto Nuclei ....................... 316 2. Endoplasmic Reticulum (a-Cytomembranes) and “Microsomes” 321 322 3 . Mitochondria .............................................. 324 4. Red Blood Cells ........................................... 325 5. Unbroken (Whole) Cells .................................. 6. Bile Duct Canaliculi, Cell Walls, and Cell Debris ............. 326 B . Possible Loss of Protein by Isolation in Aqueous Media ........ 327 1. Structure and Permeability of Nuclear Membrane .......... 328 329 2. Effect of Washing Nuclei ................................... 3. Comparative Experiments with Aqueous and Nonaqueous Methods .................................................. 331 C. Damage to Nuclei ............................................. 331 1. Appearance under the Microscope ........................... 331 2. Enzymatic Criteria of Damage ............................. 332 333 3. Effect of Homogenate on Nuclei ........................... 4. Yield of Nuclei and Appearance of DNA in Nonnuclear Fractions ...................................................... 334 V . Conclusions ....................................................... 335 V I . Acknowledgments ................................................. 337 VII . References ....................................................... 337

I. INTRODUCTION The aim of this review is to present a survey of the results of biochemical investigations on nuclear fractions isolated from homogenates of mammalian tissues and, also, of the attempts made to determine whether the activities observed are true properties of the nuclei or are due to artifacts arising from the technique. The reader is referred to the following authors for general reviews on the technique of “differential centrifugation” : Bradfield ( 1950), Schneider 1 Present address : George Washington University School of Medicine, Washington, D . C.

279

280

D. B. ROODYN

and Hogeboom ( 1951, 1956), Dounce (1952a, b), Holter (1952), Hogeboom et al. (1953), Duve and Berthet (1954), Claude (1954), Hogeboom and Schneider (1955), Davidson (1957), and Novikoff and Podber (1957). For reviews on the isolation and enzymatic properties of nuclei he is referred to Dounce ( 1948, 1950, 1952a, b, 1954, 1955), Mazia (1952), Allfrey et al. ( 1955b), Brachet (1957), and Siebert and Smellie (1957).

11. METHODSFOR ISOLATING NUCLEI A . List of Methods Used The reviews given above and the individual references in the text provide details of the many methods that have been used for nuclear isolation. Although the subject has already been well reviewed and discussed, it is possibly worth while to present a list of the methods used, in historical order (Table I). B. Homogenizers The most commonly used homogenizer is that described by Potter and Elvehjem (1936), which is similar in some respects to the apparatus of Hagan (1922). Corper and Cohn (1936) described an apparatus very similar to that of Potter and Elvehjem, except that there is a smaller surface area for grinding the tissue and a glass hood is fitted over the grinding rod in order to prevent bacterial contamination. Various modifications have been described of the Potter-Elvehjem apparatus. One of the problems is ground-glass formation, and it is the common practice nowadays to make the head of the apparatus from some durable form of plastic (e.g., Kamphausen and Morton, 1956). Dounce ( 1948) described a Potter-Elvehjem apparatus with a tapered conical pestle and mortar. Wilbur and Skeen (1950) use a simple rubber plunger in a test tube, operated by hand and not by motor. Harris and Mehl (1954) have a modification that permits continuous passage of material through the homogenizer which is thus useful for the preparation of large volumes of homogenate. Philpot and Stanier ( 1956) describe a conical hand-operated plunger type of homogenizer, and Dounce (1955) used a ball-and-socket type of apparatus. Lang and Siebert (1955a) describe a tapered motordriven adaptation of the Potter-Elvehjem device, the tissue having been previously ground in a hand-driven homogenizer. The high-speed Waring blendor was commonly used in early work on isolation of cell nuclei (Dounce, 1943a) but is not used a great deal now. Allfrey et al. (1957), however, have recently used a low-speed blendor for the isolation of thymus nuclei. More-complex types of homogenizer have been reported, usually precision worked from stainless steel. Thus Lang and Siebert (1952) describe a stainless-steel “kern mill” that has

TABLE I LIST OF METHODS USEDFOR ISOLATION OF MAMMALIAN NUCLEI Reference

Method of cell breakage

Isolation medium ; comments

Behrens (1932)

Freeze-dry tissue and grind in ball mill

Flotation, using various mixtures of benzene and carbon tetrachloride

Stoneburg (1939)

Grind in fine-toothed meat chopper

5% citric acid, followed by pepsin plus HCI

Marshak (1941)

5% citric acid

Haven and Levy (1942)

2% citric acid

Mayer and Gulick (1942)

Grind frozen tissue in power mill

Adaptation of Behrens (1932) method

Dounce (1943a)

Waring blendor

0.002 M citric acid, pH 6.0 to 6.2

Dounce (1943b)

Waring blendor

Citric acid added to pH 3.8 to 4.0

Claude (1946)

Mortar

0.85% NaCl

Dounce (1948)

Waring blendor or modified Potter-Elvehjem

Homogenized in distilled H,O, citric acid then added to p H 6.0

Schneider (1947)

Potter-Elvehjem

Alkaline water

Hogeboom et al. (1948)

Potter-Elvehjem

0.88 M sucrose

Schneider (1948)

Potter-Elvehjem

0.25 M sucrose

Price et al. (1948)

Potter-Elvehj em

0.88 M sucrose/O.Ol M phosphate, pH 7.2 or 0.14 M NaCI/O.Ol M phosphate, pH 7.2 0.25 M sucrose/0.008 M citric acid

Arnesen et al. (1949) Schneider and Petermann (1950)

Glass homogenizer

0.88 M sucrose/0.0018 M CaCl,

Dounce et al. (1950)

Freeze-dry and grind in ball mill

Various mixtures of petroleum ether, benzene, and carbon tetrachloride ; modification of Behrens (1932) method

TABLE I (Continued) Reference Wilbur and Anderson (1951)

Method of cell breakage Hand-plunger type of homogenizer

N

Isolation medium ; comments

w

Layering procedure, using three solutions to separate nuclei, whole cells, and mitochondria : 0.0094 M KH,P0,/0.0125 d!f K,HP0,/0.0015 M NaHCO, plus 0.145 M ,0.218 M , or 0.272 M sucrose, pH 7.1 Dilute acetic acid

Stedman and Stedman (1951) Potter-Elvehjem followed by Shearing blades

Sucrose/phosphate/CaCl, mixture

Behrens and Taubert (1952)

Organic solvents ; freeze-drying omitted

Hogeboom et al. (1952)

Potter-Elvehjem

0.25 h4 sucrose/0.0018 M CaCI, for homogenate, layered over 0.34 M sucrose/0.00018 M CaCI,, sediment resuspended in 0.25 M sucrose/ 0.00018 M CaC12

,, 3Z

Maver et al. (1952)

Allfrey et al. (1952)

Tissue freeze-dried and ground in a ball mill

Various mixture of organic solvents (cf. Behrens, 1932)

Lang and Siebert (1952)

Precision homogenizer of stainless steel, with controlled gap

40% sucrose

Dounce and Litt (1952)

Waring blendor

1% gum arabic solution, pH of homogenate adjusted with citric acid, to pH 6.0 Glycerol or sucrose, specific gravity 1.194, for homogenization, diluted afterward

Glass mortar

0.0009 M CaCI,

Dallam and Thomas (1953) Johnson, Albert, and Wagshall (see Weiss, 1953) Dounce (1954) Schneider (1955a)

2% gum arabic

Potter-Elveh jem

70% glycerol

*

56

0 0

TABLE I (Continued) Reference

Method of cell breakage

Anderson (1955b) Emery and Dounce (1955a)

Isolation medium ; comments 2.2 M sucrose, speficic gravity 1.4, prolonged spin, only nuclei sediment

Chauveau (1952) Plunger homogenizer Ball-and-socket homogenizer

Medium of graded sucrose concentration, centrifuging in sector-shaped tubes 0.44 M sucrose or 0.25 M sucrose/0.004 M CaCI,, or 0.44 M sucrose adjusted to various pH values (pH 5.8 to 6.3) with dilute citric acid 10% glucose

Chalazonitis and Otsuka (1956) Philpot and Stanier (1956)

Hand-plunger homogenizer of conical shape

0.3 M sucrose/40% glycerol/O.l2 M potassium glycerophosphate

Roodyn (19%)

Potter-Elvehjem

Graded sucrose medium, containing 0.00018 M CaCl,

Allfrey et al. (1957)

Low-speed Waring blendor Precision homogenizer

Sucrose-CaCl, media

Emanuel and Chaikoff (1957)

z 2 0 m

5 r n

2 U

8 P

5 a"

2

r

E

284

D. B. ROODYN

a known and controlled gap. Emanuel and Chaikoff (1957) describe a hydraulic homogenizer in which the gap through which the tissue is forced can be accurately controlled. The relation between gap and cell breakage is given, as well as the effect of different methods of homogenizing on the final yield of nuclei (as measured by D N A ) . Poort (1957) has designed a precision homogenizer that is suitable for cell breakage in highly viscous media (i.e., 70% glycerol). Since these accurately made devices are still fairly recent and are, as yet, not in general use, there has not been a great deal of material published on the systematic control of the method of cell breakage, and it is hoped that they will be very useful in giving much-needed information about the optimal gaps, shearing forces, tissue concentration, and time of homogenization required to produce maximal cell breakage with minimum damage to the cell constituents. In this connection, it may be noted that Anderson (1956) has pointed out that the shearing forces in the PotterElvehjem apparatus can operate over a much wider range if the head is moved up and down, rather than rotated. C. Methods of Separating Nuclei from Homogenate Behrens (1932) separated nuclei from cytoplasmic material by flotation in media of carefully controlled specific gravity. Later (Behrens, 1938) he used gradient density systems of organic solvents in order to find the correct specific-gravity level for plant nuclei. Apart from the adaptations of the Behrens method (Mayer and Gulick, 1942; Dounce et al., 1950; Allfrey et al., 1952) an example of separation by spinning to specific-gravity equilibrium is the method of Chauveau ( 1952), in which homogenates are spun for a long time at high speed in 2.2 M sucrose, specific gravity 1.4, and it is found that only the nuclei sediment to the bottom of the tube. A second approach is to rely on differences in sedimentation rates. A great improvement in the separation between nuclei and mitochondria came with the introduction of simple layering techniques (Wilbur and Anderson, 1951) as distinct from simply centrifuging the whole homogenate. One can layer over a simple medium (Hogeboom et al., 1952) or over one of graded specific gravity (Anderson, 1955b; and Roodyn, 1956a). The technique of gradient density centrifuging (Brakke 1951, 1953) has been applied to the fractionation of cytoplasmic particles as well (for example, Holter et al., 1953; Kuff and Schneider, 1954; Thomson and Mikuta, 1954), and one of its difficulties is in the clean separation of fractions at the end of the experiment. Anderson (1955b), Randolph and Ryan (1950), Hogeboom and Kuff (1954), and Phelpstead and

METABOLIC STUDIES O N ISOLATED NUCLEI

285

Roodyn (1957) describe devices for doing this, the latter containing an enclosed Perspex shutter for cutting fractions and thus eliminating fluid loss around the edge of the cutting surface. The behavior of layered liver homogenates during centrifuging has been examined in detail by Anderson (1955b), and he noted several causes of cross-contamination that can occur. Apart from this, new types of centrifuge have been developed. Lindhal (1948, 1956) has described a counter-streaming centrifuge, and it has been used for the fractionation of nuclei (Bonnichsen et al., 1957). Philpot and Stock (1955) described an air-driven drum centrifuge in which the homogenate is introduced over a layering fluid flowing over the inside surface of the drum. The sediment collects in grooves on the inner surface of the drum. It may be mentioned that simple sedimentation without centrifuging is sufficient for some purposes. For example, Dounce and Beyer (194813) separated nuclei from fiber by using a simple sedimentation apparatus consisting of a measuring cylinder with a movable transverse shutter. Also, Arnesen et al. (1949) separated whole cells from nuclei by simple sedimentation in a measuring cylinder. Finally it may be said that there seems little hope in using electrophoretic methods for nuclear isolation, since Philpot and Stanier (1954) observed that applied electrical forces did not cause appreciable movement of nuclei, although they did cause shifting of the nuclear contents.

111. BIOCHEMICAL STUDIES ON ISOLATED NUCLEAR FRACTIONS

A . I n Vitro Isotope Incorporation Studies It is not in the scope of this review to consider the results obtained from injecting radioactive tracers into the whole animal, isolating the nuclei, and then measuring the uptake (for example, Smellie et al., 1955; Daly et al., 1952 ; Allfrey et al., 1954) , since such experiments study the in vivo metabolism of the nucleus, while it is still in its natural environment. The reader is referred to Brown and Roll (1955), Smellie (1955), and Siebert and Smellie (1957) for comprehensive reviews on this very interesting subject. Similarly, the author will not discuss experiments in which tissue slices were incubated in the presence of label and subsequently fractionated (e.g., Weinman et al., 1956). An interesting type of experiment in between in vitro and in vivo work is described by Korner and Tarver (1957) in which rats were fed radioisotope and the release of activity from the isolated fractions on incubation in vitro was studied. With labeled amino acids it was possible to find conditions in which the isolated radioactive nuclear fraction did not liberate isotope into the medium but actually incorporated it.

286

D. B. ROODYN

The main studies with direct in vitro methods have been concerned with the uptake of labeled amino acids and P32-labeled inorganic phosphate. Dealing first with amino acids, Lang et al., (1953a) found that isolated pig kidney nuclei incorporated CI4-labeled glycine into protein, lipids, and nucleic acids. A most extensive study of amino acid incorporation has been carried out by Allfrey and co-workers (Allfrey et al., 1955a, 1957; Allfrey and Mirsky 1957a, b ) . The work was done on calf thymus nuclei isolated in sucrose-calcium chloride media. Alanine-l-C14, glycine-l-C14, methionine S35, and lysine-2-C14 are all taken up into the nuclear protein. The uptake is inhibited by anaerobic conditions, by dinitrophenol, NaCN, NaN3, antimycin A, Dicumarol, and Janus green B, and, what is of the greatest interest, by the removal of DNA (by treatment with DNAase). The activity is restored to DNAase-treated nuclei by adding DNA from several sources, RNA, synthetic polyadenylic acid ( Grunberg-Manago and Ochoa, 1955) and by certain degradation products of DNA. It is not restored by nucleotides, but there is a slight restoration by dinucleotides. The incorporation depends on a preliminary activation of the nucleus and possibly on R N A synthesis. The uptake is greatest into a protein fraction closely associated with DNA and into a fraction easily soluble in phosphate buffer at p H 7.1. These workers also demonstrated the uptake of orotic a ~ i d - 6 - Cand ~ ~ adenosine-8-C14 into the R N A of thymus nuclei, the relatively insoluble R N A (nuclear R N A ) being the most active. This biochemical heterogeneity of nuclear R N A has been confirmed by Logan and Davidson (1957) from in vivo studies. Also Friedkin and Wood (1956) have shown that isolated rabbit thymus nuclei can incorporate thymidine-C14 into their DNA. Siekevitz (1952) studied the uptake of radioactive alanine into proteins from various liver fractions and found that the rate of uptake per milligram of protein was about the same in the nuclear fraction and the homogenate. The nuclear fraction used was contaminated with whole cells, and it was difficult to determine whether the uptake was truly nuclear. The uptake of labeled amino acids by isolated liver nuclei has been reported by Grant and Rees (1958), who observed incorporation of (2-C14) glycine into proteins of nuclei isolated in sucrose-CaClz media. Uptake of P32 into nuclear R N A was also observed. Ficq and Errera ( 1956) have confirmed, by the use of autoradiographic techniques, that isolated thymus nuclei incorporate C14-phenylalanine, the incorporation being inhibited by 50 to 70% after DNAase treatment and restored on addition of DNA. The incorporation is not inhibited by ribonuclease. It is of great interest that it was found that X-radiation (950 r ) or ultraviolet light (2200 ergs/mm2) produced a 50% inhibition of the incorpora-

METABOLIC STUDIES ON ISOLATED NUCLEI

287

tion. In a more detailed report Ficq and Errera (1958) have suggested that there might be some heterogeneity in the nuclear population as regards uptake of labeled amino acid. Apart from using labeled amino acids there have also been in vitro studies with P32-labeled orthophosphate. Siebert et al. ( 1953b) showed incorporation with isolated pig kidney nuclei into lipids, RNA, and protein. The labeled lipids formed were found not to be nucleic acid precursors (Lang et al., 1953b). Logan and Smellie ( 1956) found that direct uptakes in witro by nuclei of inorganic P32was not much higher than the controls, and they therefore did the following kind of experiments: Nuclei from radioactive rats were incubated with the cytoplasmic fraction from nonradioactive rats (and vice versa) so as to prepare (‘composite” homogenates. The nuclei were then reisolated after incubation. It was found by these means that the nuclear fraction does not contribute label to the cytoplasmic RNA, but that, on the contrary, the cytoplasmic fraction (essentially the soluble component) transfers P32to nuclear DNA and RNA. (The action of the soluble component is stimulated by the cell particles.) Finally it may be said that Weiss (1953) has shown that nuclear fractions obtained from beef thyroid homogenates can incorporate 1131 into diiodotyrosine and thyroxine.

B. Enzyme and Nitrogen Determinations The determinations we shall first consider are those which have been done with no reference to the homogenate from which the nuclei are isolated and which are therefore expressed in absolute enzyme units. Such assays are difficult to tabulate, although some are briefly noted in Table I1 by the comments (‘present’’ or “trace.” Similarly on some occasions activities have been reported “absent,” although no test has been made on the homogenate. Accurate comparisons with the homogenate have not been made because the results are still preliminary, or the activity is of a complex nature which does not give satisfactory balance sheets, or the particular worker was satisfied that the results from nuclei alone had a validity in themselves. Some results have been obtained about the role of nuclei in oxidative phosphorylation. Siekevitz ( 1952) could not detect oxidative phosphorylation in a crude nuclear fraction isolated from rat liver, and, similarly, Brody and Bain (1952) attributed the activity in brain nuclear fractions to mitochondrial contamination. The nuclear fraction influences mitochondrial phosphorylation, however. Potter et al. (1951) found that added nuclei stimulated mitochondrial oxygen uptake and caused an output of inorganic phosphate, probably because of phosphatase activity. In contrast to this,

288

D. 3. RBODYN

however, Johnson and Ackermann ( 1953) found that phosphate esterification by isolated chick liver mitochondria was greatly enhanced by the addition of the nuclear fraction, which itself did not carry out phosphorylation. Subsequently it has been shown, however, that this effect is probably nonspecific, since heated nuclei and serum albumin both enhance mitochondria phosphorylation (Stern and Timonen, 1955). Osawa et al. (1957) have reported that isolated thymus nuclei can oxidatively phosphorylate endogenous adenylic acid to A T P . Unlike mitochondria1 phosphorylation (Lehninger, 1949) the activity is not inhibited by calcium chloride. The P: 0 ratios for this nuclear phosphorylation are not given. It is inhibited by antimycin, cyanide, azide, and dinitrophenol but not by Dicumarol, Janus green B, or methylene blue. Another complex activity that has been studied in glycolysis. It was found by LePage and Schneider ( 1948) that complex interactions occurred during the fractionation of glycolytic activity. For example the (low) glycolysis observed in the nuclear fraction from a Flexner- Jobling carcinoma was greatly enhanced by adding the microsome fraction, which itself was inactive. Lang and Siebert (1951) have discussed the glycolytic activity of isolated nuclei in relation to possible energy sources, oxidative enzymes being very deficient. Several very important reactions in the field of nucleotide metabolism have been reported either in isolated nuclei or in nuclear extracts. Smith and Mills (1954) reported that guinea pig liver nuclei can split, by pyrophosphorolysis, either uridine diphosphate glucose ( U D P G ) or uridine diphosphate acetyl glucosamine ( U D P A G ) , giving uridine triphosphate ( U T P ) as one of the products. There are also degradative enzymes present which convert UTP to U D P and UDPAG to U D P , uridylic acid, and uridine. The pyrophosphorylase has been freed from these, however, by later work (Mills et al., 1954). Although D P N synthesis is localized solely in the nucleus (Hogeboom and Schneider, 1952b; see also Branster and Morton, 1956), F A D synthesis, which occurs by a very similar type of reaction (Schrecker and Kornberg, 1950), has been reported to be localized exclusively in the soluble fraction of liver (Schneider, 1955b). As far as the author is aware, no study has yet been reported of the site of T P N synthesis (Kornberg, 1950) Shonk and Boxer (1957) have made the interesting observation that in normal liver the synthesis of deoxyribose5-phosphate occurs mainly in the nuclear fraction, although in malignant hepatoma the synthetic activity is demonstrated in all cell fractions. As regards polynucleotide metabolism, Heppel et al. (1956) isolated an enzyme from guinea pig liver nuclei that degraded synthetic polyadenylic acid to small poly- or oligonucleotides containing 5’-phosphomonoester end groups.

METABOLIC STUDIES ON ISOLATED NUCLEI

289

Adenosine and adenylic acid were also liberated. Also, Hilmoe and Heppel (1957) have demonstrated by a tracer technique the presence of polynucleotide phosphorylase in guinea pig liver nuclei. This observation is of the greatest interest, even though net synthesis of polynucleotide could not be demonstrated because of the presence of contaminant nucleases. Apart from these observations, which are rather difficult to tabulate, the rest of the enzyme assays carried out on isolated nuclei have been brought together in Table 11, together with the nitrogen assays on the fractions used. The method of presentation requires some explanation : First, only assays that have been carried out directly on nuclear fractions are reported. For example, assays on combinations of mitochondria and nuclei (e.g., some experiments reported by Hers et al., 1951) are not included, nor is it assumed, if most of the enzyme is found in the soluble fraction, that there is none in the nuclear fraction (for example, see Hogeboom and Schneider, 1952a; Friedkin and Roberts, 1954; Bremer and Gloor, 1955). Some of the results presented have been calculated indirectly or from diagrams and are liable to error. The reader is therefore strongly recommended to refer to the original paper for exact data. Because of space, the comments and desciption of isolation medium have necessarily been compressed and hence might have lost a certain accuracy. The results are expressed as follows: % T : Percentage of the total enzyme activity in the homogenate that is found in the nuclear fraction. R.C. : ‘(Relative concentration” is defined as Enzvme activitv/me. N of nuclear fraction Enzyme activity/mg. N of homogenate % R : Percentage recovery (i.e., sum of all fractions) of activity originally present in the homogenate. % N: Percentage of the total nitrogen of the homogenate that is found in the nuclear fraction. ( I t may be noted that the term “relative concentration” is slightly less cumbersome than “per cent specific activity” and is also equal to % T divided by % N ) . The following symbols have been used in the tables. ( a ) and ( b ) : These symbols are used when more than one result has been obtained by the same fractionation technique. This is usually due to the author’s using two methods of enzyme assay. (c) : In some cases values for the homogenate are not given but the activity per milligram of N of the nucleus-free cytoplasmic fraction is given. The value for

TABLE I1 ENZYME A N D NITRIXENDETERMINATIONS

3

(For explanation of symbols, see text) Reference* Method Acid phosphatase 1 2 2 104 3 3 4 73 5 73 6 6 1 7 8 8 9 9 3 3 10 6 3 3

5 11 12 13 14 16

73 111 126 13 14 23

Tissue

0.25 M SU 0.25 M SU 0.25 M SU 0.25 1ci SU 0.25 M SU 0.25 M SU/O.OOl M Versene 0.25 M SU/O.OOl M Versene 0.88iM SU 0.88M su HZO Rat liver parenchyma cells 0.25 M SU/O.OOl M Versene Rat liver (fatty) 0.25 M SU HZO Mouse liver 0.25 M su 0.88M su Rat kidney 0.88M su: Nuclei contaminated with “droplets” Nuclear fraction washed 0.25 M SU Dog cerebrum Human cerebral cortex Citric acid, p H 6.0, mean of 15 biopsies Bull prostate Rat liver

Acetyl coenzyme A deacylase 16 104 Rat liver

*

Isolation medium and comments

For references, see p. 313.

0.25 M SU/0.005 M Versene

%T

R.C.

%R

%N

7.3 6.1 12.4 12.0 6.1 3.6 3.6 7.0 4.8 6.4 2.4 9.4 8.6 1.o 7

0.46

98.6 99.8

15.8

0.47 0.49

101 102.8 101.8 94.5 95.0 98.4

0.35

103.5

0.6 (p) 0.27

21.0 (PI 13.3 15.0 9.6 6.9

m !a 0

2

1:

0.44

104 93.6

15.5

1.02 0.75

92.4 101.7

17.9 15.0

0.7

1.01 0.2

99.2

3.5

6.2

0.31

89.6

18.3 11.2 -10

P

TABLE I1 (Continued) Reference Method

Tissue

Isolation medium and comments

"/o T

R.C.

%R

DJoN

Aconitase

17 18

73 104

Adenosine deaminase 19 20 20 20 19 19 20 20 20 20 19 20 19 19 20 20

Rat liver

Rabbit cerebral cortex

Calf liver

Fetal calf liver Calf thymus

Calf intestinal mucosa Calf kidney Calf kidney cortex Fetal calf kidney Calf pancreas Fetal calf pancreas Calf heart

0.25 M SU ; assay a t : p H 7.3 p H 5.8 0.25 M S U ; as substrate: Citrate nL-Isocitrate Organic solvents Organic solvents 0.25 M SU/0.0018 M CaCI, Organic solvents Organic solvents Organic solvents 0.25 M SU/0.0018 M CaCI, Organic solvents Organic solvents Organic solvents Organic solvents Organic solvents Organic solvents Organic solvents

8.0 14.0 5.7 2.2

130.4 92.0 0.98 0.58 2.08 2.08 (c) 0.20 2.08 (c) 0.66 (c) 1.oo 0.90 0.19 (c) Trace Trace Trace 1.63 (c) 1.63 (c) 6.00 (c)

Adenosine-J'-Phosp hatase

20

20

Calf Calf Calf Calf

intestinal mucosa thymus heart liver

Organic Organic Organic Organic

solvents solvents solvents solvents

0.04 (c) 0.11 (c) 0 0.10 (c)

100.7 101.3

5.8 4.0

TABLE I1 (Continued)

n, ~

~~

Reference Method

Tissue

Adenosine-5’-Phosphatase 8 8 Rat liver 20 20 Calf liver Calf intestinal mucosa Ca!f thymus Calf heart Adenosine Triphosphatase (ATPase) 3 3 . Rat liver 21 104

22

111

8 23 24 3 3

8 23 127 3 3

25 26 23 27

127 69 23

Isolation medium and comments

0.88M

40

solvents solvents solvents solvents

0.25 M SU 0.25 M S U ; in assay: No M g + + , no D N P No Mg+ +, D N P present M g + + present, no D N P Mg+ + and D N P present 0.88 M SU ; in assay : No metals C a + + present: ( a ) (b) M g + + present 0.88 M SU ; M g + + present in assay 40% SU ; Ci+ + present in assay Alkaline H,O

HZO

Rat fatty liver

0.25 M SU

Rat hepatoma Embryonic chick liver Pig liver Calf liver

Alkaline H,O 0.25 M SU ; M g + + present in assay 40% SU ; Ca+ + present in assay Organic solvents

HZO

20

su

Organic Organic Organic Organic

%T

R.C.

%R

2.66 100 0.18 (c) 0.06 (c) 0.09 (c) 0

%N

N

15

20 3.0 0.28 0.68 0.38 18.0 34.0 18.0 20.0 10.0 26.8 19.7 25.3 25.3 12.4 29.5

P

fd 5f1 0 0

90.0 1.18 1.90 101 1.33 74 1.25 105 0.67 95.0 1.28 2.58 (d) 104

10.4 (d)

99.0

24.8 (d)

0.5 (d) 1.42 0.12 (c)

17 17 17 17 15.0

31:

TABLE I1 (Continued) Reference Method

28 28

128 128

23 23 27

23 23 20

Aldolase 29 30 30 32

111 77 77 32

31 32

31 32

33 34 35 36

Tissue Rat infant cerebrum Rat adult cerebrum Pig kidney Rat kidney Calf kidney Calf intestinal mucosa Calf thymus Calf heart Rat liver

33 34

35 36

Dog kidney Sheep pancreas

Isolation medium and comments

0.25 M SU ; in assay : M g + + present C a + + present 0.25 M S U ; in assay: Ca+ + present M g + + present 40% SU ; Ca+ + present in assay 40% SU ; Ca+ + present in assay Organic solvents Organic solvents Organic solvents Organic solvents 0.88M su 0.25 M SU/0.0018 M CaCI,: (a) (b) 0.25 M SU/0.0018 M CaCI,, Waring Blendor used to homogenize 0.00018 M CaCI, 0.25 M SU/0.0018 M CaCl,/maleate : pH 5.75 pH 6.00 pH 6.42 pH 6.75 Citric acid, pH 6.0 Distilled H,O plus CaCI, Organic solvents Dilute citric acid Dilute citric acid

%T

R.C.

%R

%N

15.6 15.*5

1.08 1.08

101.2 101

15 15

25.3 20.5

1.42 98.7 96.8 1.34 0.72 0.40 0 0 0.09 (c) 0

16.0 16.0

3.0 12.5 27.6

1.44 2.90

100 %.O 98.5

9.0 9.5

8.5 16.3

1.33 1.92

100.4 97.6

6.4 8.5

31.1 19.7 10.0 5.2

0.78 1.81 1.22 0.74 0.4 Trace Trace 0.2 Absent

82.5 98.8 99.5 98.0

39.8 10.9 8.2 7.0

TABLE I1 (Continued) Reference Method

Tissue

Isolation medium and comments

N

%T

%R

%N

2.75

100.4

14.6

0.74 1.0

98.9 103.8 98.0 110

15

R.C.

p"

Alkaline phosphatase

37 3 38

5

39 8 40

73 3 104 73

111 8 40

41 3 3

41 3 3

37

73

11

Rat liver

111

0.25 M su 0.25 64 su 0.25 M su Conditions of assay varied (e.g., M g + + concentration, pH, and type of buffer used) ; two extreme results given 0.25 M SU 0.88M SU 0.88M SU 0.44M su 0.25 M SU/O.O04 M CaCl, 0.44M SU, pH 5.8: ( a ) (b) 0.44M SU, pH 6.0: ( a ) (b) 0.44M SU, pH 6.2 0.44M SU, pH 6.3: ( a ) (b) Dilute citric acid

HZO Rat fatty liver Regenerating rat liver: 1 day 72 hr. Mouse liver

0.25 M HZO

su

0.25 M su 0.88M SU 0.88M SU

0.6 10.6 40.1

6.6 37.4 10.6 15.0 -15

Present 4 14 2.9 23.5 6.6 3.12 32.5 1.92

-95

W

0

*z

U

8.4 8.6 9.0

31.8 10.4

3.0 1.61 0.66

p

96.0 101.8

15.5

Reference Method

11

111

27 20 27

20 20 20

20 27 19 19 27 13 14

20 20 20 19 20 13 14

Tissue Regenerating mouse liver 65 hr. Horse liver Fetal horse liver Calf liver Calf intestinal mucosa Fetal intestinal mucosa Calf thymus Calf kidney Dog cerebrum Human cerebral cortex

TABLE I1 (Continued) Isolation medium and comments

0.88M SU Organic solvents Organic solvents Organic solvents Organic solvents Organic solvents Organic solvents Organic solvents 0.25 M SU/0.0018M CaCl, Organic solvents 0.25 M S U Citric acid, pH 6.0,mean of 15 biopsies

%T

R.C.

%R

%N

20.7

1.10

98.8

18.8

0.28 (c) 0.11 (c) 0.12 (c) 0.035 (c) 0.035 (c) 0.005 (c) 0.06 (c)

0.18 0.06 (c)

40.0 3.3

Amine Oxidase

42 42 43 43

104 111 111

Rat liver

0.25 M SU 0.88M S U 0.88M SU Dilute citric acid

1.04 (p) 101.5 0.66 (p) 98.0 0.98 99.5 Absent

Rat liver, pig kidney

40% sucrose

Absent

Rat liver

Dilute citric acid: Osborne-Mendel rats Wistar rats Dilute citric acid : Hepatoma 31 Walker carcinosarcoma

57

31.5 22.0 21.9

30.2 (p) 33.0 (p) 23.1

U

cn

0

L-Amino acid oxidase

44

125

D-Amino acid oxidase

45 46

41 41

Rat liver from animal bearing transplantable tumors

0.78 0.50 1.2 0

N

$

TABLE I1 (Continued) Reference Method

Tissue

p-Aminobenzoate acetylase 47 47 Pigeon liver p-Aminohippuric acid synthesis 48 104 Mouse liver

Isolation medium and comments

%T

0.25 M SU or Ringer 0.25 M SU; in assay: O.OOO5 M A T P + fumarate 0.0005 M A T P + glutamate 0.001 M A T P + fumarate 0.001 M A T P glutamate

+

R.C.

%R

%N

40.5 77.0 73.0 93.0

14.9 14.9 12.6 12.6

Absent

0.02 3.0 1.2 1.4

0.013 0.20 0.10 0.11

Amylase

50

50

51 52

-

27

20

Arginase 53 38 54 55 54

53 104 73

51

77

Rat pancreas

0.88 M SU : Controls : unfiltered homogenate filtered homogenate Pilocarpine-injected : unfiltered homogenate filtered homogenate

Dog pancreas Pig pancreas Pigeon pancreas Rat pancreas Horse pancreas Beef pancreas

0.25 M su 40% su 40% su 40% SU Organic solvents Organic solvents

Rat liver

0.25 M SU 0.25 M SU 0.25 M SU 40% SU 0.25 M SU/0.0018 M CaCl,

P 13.0 2.0

74.0 66.0

9.0 2.0

63.0 51

m

d

Present 1.31 0.17 1.14 0.025 (c) 0.071 (c)

33.6 35.0 9.3

12.0 2.3 1.96 1.17 2.10

83.5 84.3

14.6 17.9 4.5

TABLE I1 (Continued) ~

Reference Method 56 41 57 35

111 41 33 35

102 27 20 27

102 20 20 20

57

33

Tissue

Rat liver Regenerating rat liver Dog liver Horse liver Fetal horse liver Calf liver Calf kidney Rat kidney

Isolation medium and comments Distilled H,O Dilute citric acid Dilute citric acid Organic solvents Dilute citric acid Organic solvents Organic solvents Organic solvents Organic solvents Organic solvents Distilled H,O followed by dilute citric acid

%T 36.0

Carbonic anhydrare 14 14 Catalase 38 41

14

0

z

U

0.25 A4 SU ; in assay: No D P N D P N present

17.0 12.0

F 8

80.0 92.0

H

z

Human cerebral cortex

Dilute citric acid, mean of 15 biopsies

4r

0.37

E

104 41

34

34

35 20

35

27

%N

Absent Absent Absent

Lamb kidney Hen kidney Betaine aldehyde oxidase 58 104 Rat liver

R.C. %R 2.50 93.0 0.4-0.5 1.17 Present 2.75 0.95 0.57 (c) 0.16 (c) 0.54 (c) 1.10 (c)

20 20

Rat liver

Horse liver (fetal) Horse liver

0.25 M SU Dilute citric acid Distilled H,O/CaCl, Organic solvents Organic solvents

4.5

0.31 Trace Trace 0.54.6 Absent 0.71

104.5

14.6

N

3

Reference Method

Tissue

TABLE I1 (Continued) Isolation medium and comments

%T

73

60 52 61

125

10

6

61

61

Rat liver

61

0.25 M SU 0.25 M SU/O.OOl M Versene 40% SU 40% SU ; cysteine present

31.2 4.0

0.25 M SU/O.OOl A4 Versene

4.8

SU/phosphate/CaClz :

72 hr.

5 days 59 61

59 59

73 61

73 73

100.4 1.58 106.5 0.30 1.11 1.08 (c) 0 0.053

22.1 13.3

Absent

SU/phosphate/CaClz : (a) (b) Rat liver parenchyma cells Regenerating rat liver : 48 hr.

%N

0.32

Calf liver Calf kidney

Cathegsin 59

DJoR

R.C.

N u3 00

Rat hepatoma Rat (DAB) hepatoma

0.25M SU

Rat adenocarcinoma

SU/phosphate/CaClz :

Rat lymphosarcoma

SU/phosphate/CaCl, :

Rat spleen

SU/phosphate/CaC12 :

Rat spleen Rat kidney

0.25 M S U 0.25 M S U

16.0

SU/phosphate/CaClz :

26.4 25.9

0.69 0 0.44 0 0.58 0 0.24 1.00 0 0.23 0.63 0.47 0 0.2 0.45 0.32 0.69 1.06

83.9

6.9

97.9

30.2

99.8 99.0

44.5 22.9

p

TABLE I1 (Continued) Reference Method

60 52 60 52

125

-

Tissue Pig kidney

125

-

Pig pancreas

Isolation medium and comments

40% 40% 40% 40%

%T

SU

R.C.

%R

%N

0.65 0.53 (c) 1.32

SU,cysteine present SU,neutral proteinase SU, great activation of nuclei by

cysteine

6.10 (c)

z

s

Choline acetylase

62 62

128 62

Rabbit brain

0.25 M SU 0.25 A4 SU

6.9 2.7

k0 cc1

87.0 90.0

rn

Cholinesterase 1. Acetylcholine as substrate 63 6 Rat liver

64 14

111 14

Human cerebral cortex

2. Acetyl-$-methylcholine as substrate Rat liver 78 111

64

111

64

104

Rabbit liver

0.25 M SU 0.88M su Dilute citric acid, mean of 15 biopsies

18.6 17.5

1.14 0.73

108.7 86.5

16.4 23.9

0.94

2s rn 8 M

U

v1

0

0.88M su 0.88M su 0.88M su 0.25M SU 0.25M SU

r

(a) (b) (a) (b)

7 0 13 13.2 4.8

0.38

22.0 18.0 18.6 30.0 13.3

0.75 0.73 0.87

5

75 60.3 86.8 94.5 92.8

25.5 34.5

85.0 83.2 92.6 84.3 102.7

23.9 25.5 34.5

Z

C

c1 P

E!

3. Benzoylcholine as substrate

65 64

111

64

104

in

Rat liver

0.88M su

Rabbit liver

0.88M 0.88M 0.8844 0.25 A4

su su su su

(a> (b) (c)

3

TABLE I1 (Continued) Reference Method

64

111

64

64 66 66

66 66

Tissue

Isolation medium and comments

%T

Guinea pig liver

0.25 M SU 0.88M SU 0.15M NaCl

Dog pancreas

0.25 M SU 0.15M NaCl

10.0 5.2 52.0 17.0 29.0

Rat liver

0.25 M SU 0.88M su

16.0 9.9

R.C.

%R 87.0 79.2 93.3 87.5 98.0

%N

s”

Choline oxidase

67 68 45 46

104 111 41 41

Rat liver from animals bearing transplantable tumors

Citrate dehydrogenase 23 Pig kidney Citrate oxidase 29 111 Rat liver Zsocitric Dehydrogenase 69 73 Mouse liver

70

73 18

Rabbit cerebral cortex

Dilute citric acid

0.80 Absent

Hepatoma Walker Carcinosarcoma

Absent Absent

40% SU

Trace

0.88M su

Trace

94.0 97.0

12.4

P m 56

0.25 M SU 0.88M su 0.25 M su : Tissue not frozen Tissue frozen and thawed

0 0

3.0 2.0

0.19 0.14

97.9 104.5

12.9 14.1

3.18 3.47

143 103

4.05 4.05

Cysteine desulfhydrase

71

71

Rat liver

0.25M SU

104

Rat liver

0.25 M SU: (a)

2.55

Cytochronte c

72

(b)

26.3 24.9

1.15 1.24

85.5 95.1

20.1 20.1

TABLE I1 (Continued) Reference Method

73 74 72

73 73 41 104

Tissue

Isolation medium and comments

0.25 M S U 0.88M su Rat liver Liver from rats given carbon tetrachloride

Dilute citric acid

0.25 M S U : (a) (b)

75

20

%T 10.0 5.6

Liver from rats given phosphorus

0.25 M S U : (a)

Calf liver Calf heart Calf kidney cortex

(b) Organic solvents Organic solvents Organic solvents

R.C.

%R

0.68 0.42

63.0 68.0

%N 13.8 13.2

Trace

24.0 28.8

1.07 1.31

95.0 93.5

21.2 21.2

27.2 23.7

1.06 1.24

94.6 104.9

20.7 20.7

99.0

15.8

94.7 92.8

20.3 (p) 10.4 (d)

88.9 %.O

13.3 12.6 12.6 10.05

Absent 0.08 0.08

CJitochrome oxidme

1 76 110 24 41 6 77

2 73 110 127 41 6 77

78 10

25 75 79 14

6 127 73 79 14

Rat liver

0.25 M S U 0.25 M su 0.25 M SU Alkaline H,O Dilute citric acid

0.25 M SU/O.OOl M Versene 0.25 M SU/0.0018M CaCI, : (a) (b) Homogenate filtered 2% gum arabic Rat liver parenchyma cells 0.25 M SU/O.Ool M Versene Rat hepatoma Alkaline H,O Calf thymus 0.25 M S U Bull spermatozoa Human cerebral cortex Citric acid, p H 6.0,mean from 15 biopsies

16.5 10.5 5.4 10.1 1.01 1.40 0.23 0.6 9.1

1.04 1.17 0.54 (p) 0.53 (d) 0.5-0.6 0.76 0.08 0.11 0.018 0.10 0.087 0.36 (d)

91.5 73.3 104

E

P r

E

6.9 24.8 (d)

Absent 0.48

0.89

w

0,

TABLE I1 (Continued) Reference Method

Tissue

Deoxyribonuclease (DNAase) 80 129 Rat liver 6 6 80 77 81 77 82 127 80 129 Rat liver tumor (from DAB) 77 10 6 Rat liver parenchyma cells 109 73 Mouse liver 109 77 12 126 Rat kidney

83

125

Pig kidney

84

84

Calf thymus

82

41 82 111 130 111 127 82 41

Mouse leukemic tissue

Isolation medium and comments

0.25 M 0.25 M 0.25 M 0.25 M 0.14M

su SU/O.OOl M Versene SU/0.0018 M CaClz SU/0.0018 M CaCI, NaCl

0.25 M SU 0.25 M SU/O.OOl M CaC1, 0.25 M SU/O.001 M Versene 0.25 M su 0.25 M SU/0.0018 M CaCI, 0.8844 su: Nuclear sediment not washed Nuclei washed 40% sucrose; activity depends on pH and Mg+ + concentration 0.85% NaCV0.002M CaCI, Citric acid, pH 4.8 0.2 M K phosphate/magnesium phosphate 0.88M SU 0.88 M SU/0.0018 M CaCl, 0.88 M SU 0.14M NaCl 0.2 M K phosphate/magnesium phosphate Dilute citric acid

%T

R.C.

%R

%N

6.6 5.3 0.55 6.9

0.51

100.2 95.3

13.3

0.69 Present

96.1

9.9

0.40

18.1 14.5 4.8 10.3 <1

0.98

100.3

0.69 1.00

90.0 104.2

6.9 10.5

24.4 15.0

1.36 1.00

95.9 90.9

17.9 15.0

37.0

Present 0.68 Absent

76.0

54.0

Absent Present Trace Trace Trace Trace Absent

s

P td 0

*1: U

TABLE I1 (Continued)

Reference Method

Tissue

DPN-cytochrome c redwtase Rat liver 110 110 76 73 6 6 76 128 Rat cerebral cortex Calf thymus 75 73

Isolation medium and comments

0.25M 0.25 M 0.25 M 0.25 M 0.25 M

SU SU SU/O.OOl M Versene SU SU

%T

R.C.

%R

%N

12.0

0.60 (p) 97.4 1.04 0.63 104.2 1.12 0.23

20.3

8.4

13.3

D P N nucleosidase

Rat liver 85

104

D P N synthesis 86 73 77

Mouse liver

86

0 2 5 M su Low substrate concentration High substrate concentration 0.25 M SU 0.25 M SU/0.0018M CaC1, ; liver forced through metal disk 0.25 M SU layered over 0.34 M SU ; disk not used before homogenization

Enolase 33

41

Rat liver

Dilute citric acid

Esterase 63

6

Rat liver

0.25 M SU ; as substrate :

38 87 52 8 88

104 87

8 88

Indoxylacetate a-Naphthyl acetate 0.25 M SU 0.25 M SU ; cholesterol esterase 40% su

0.88M

su

0.88 M SU/O.Ol M phosphate buffer

37 34

136.0 132.0

71.0

4.66

104

15.6

69.0

6.12

99

11.2

92.0

5.5

99

16.9

91.8 87.7 101.0 127.1 92.7 105 90.9

16.4 16.4 14.6

0.50

6.3 5.5 6.5 14.9 0.5 10.0 4.3

0.38 0.33 0.44 0.1 1 0.66 0.35

15

(PI

TABLE I1 (Continued) Reference Method

41 35 89 20

41 35 127 20

Tissue

Dilute citric acid Organic solvents Mouse liver 0.14M NaCl Horse liver Organic solvents Fetal horse liver Organic solvents Calf liver Organic solvents Calf kidney Organic solvents Calf kidney cortex Organic solvents Calf thymus Organic solvents Calf heart Organic solvents Calf pancreas Organic solvents Calf intestinal mucosa Organic solvents Fetal calf intestinal mucosa Organic solvents

Fructose-6-phosphatare 90 90 Rat liver Fumarase 6 6 Rat liver 91 73 Mouse liver

77 104

Rabbit cerebral cortex Glucose-1-phosphatase 90 90 Rat liver Gb~ose-6-phosphatase 1 2 Rat liver

18

6 63

7

6 6 1

Isolation medium and comments

%T

%N

0.62 93.0 0.80 (c) 0.17 (c) 0.076 (c) 0.10 (c) 0.023 (c) 0.035 (c) 1.27 (c) 0.32 (c) 0.09 (c) 0.085 (c)

27.5

i?

0.5 Present

17.0

0.88M su

2.0

0.25M 0.25M 0.25M 0.25M

SU/O.001 M Versene SU SU/0.0018M CaCI, SU

8.0 9.3 3.9 2.5

0.88M

su

0

0.25 M su 0.25 M SU/O.001 M Versene 0.25 M SU/O.001M Versene 0.25 M SU!O.001 M Versene

%R

R.C.

10.7 6.8 6.3 9.7

P

m

70 0.60 0.62 0.39 0.36

91.7 100.1 101 100.8

13.3 15.2 9.8 7.3

38 0.67 0.51 0.38

99.7 92.8 93.9 100.4

15.8 13.3 16.4

TABLE I1 (Continued) Reference Method

92

92

90 93 10

90 77 6 fl-Glucuronidase 6 6 6 10 94 73

20

12

20

126

Tissue

Isolation medium and comments

0.25 M 0.25 M 0.88M 0.25 M Rat liver parenchyma cells 0.25 M

su

SU/0.0018 M CaCI, SU/O.001 M Versene

Rat liver 0.25 M SU/O.OOl M Versene Rat liver parenchyma cells 0.25 M SU/O.OOl M Versene Mouse liver: adult 0.25 M S U 0.25 M su 4 days old Regenerating mouse liver, 4 days 0.25 M SU Horse liver Organic solvents Fetal horse liver Organic solvents Calf liver Organic solvents Calf thymus Organic solvents Calf heart Organic solvents Calf pancreas Organic solvents Calf intestinal mucosa Organic solvents Fetal calf intestinal mucosa Organic solvents Calf kidney Organic solvents Rat kidney 0.88M su

Glucuronide synthesis Rat liver 95 73 Glutamic dehydrogenuse Mouse liver % 73

96 77

SU/mixed buffer SU/KHCO,

0.25 M

"/o T

R.C.

"/o R

%N

25.0 6.0 2.9 6.1 2.7

0.61 0.39

93.0 92.0 79.5 95.1 95.0

6.9

4.3 2.8 14.0 15.0

0.30 0.40 0.74 0.71

96.1 93.1 98.0 99.0

13.3 6.9 19.0 21.0

12.0

0.62 101 0.17 (c) 0.13 (c) 0.17 (c) 0.05 (c)

16.1

su

0.25M S U : (a) (b) 0.25 M SU, layered over 0.34M SU 0.25 M SU/O.O018 M CaCI,

Trace Trace 0.25 (c) 0.25 (c) 0.12 (c) 0.90 89.9

20.0

17.9

Absent

29.0 27.0 8.9 1.6

1.31 1.15 0.78 0.18

106 103 97.0 101

22.4 23.5 11.0 9.1

Isolation medium and comments

TABLE I1 (Continued) Reference Method Tissue

%T

R.C.

%R

%N

90.7

14.0

E

Glutathione hydrolysis

97

125

Pig kidney

40% SU

Rat liver

0.25 M SU

Rat liver

0.9% NaCl, pH 8.0

Present

Glutathione reductase

98

73

0.3

0.02

Glutaminuse I Glyceraldehyde dehydrogenase 23 125 . Pig kidney a-Glycerophosphate dehydrogenase 49 49 Rat liver Rat liver ; animals given carbon tetrachloride Glycolysis 29 111 Rat liver 99 104 Rabbit liver Flexner- Jobling rat carcinoma Hexose diphosphutase 90 90 Rat liver

14.5

1.08 (d) 99.4

14.0 (d)

0.32

40% SU

P m

0.88M SU

14.3

88.5

0.88M SU

22.3

115.3

0.88M su 0.25 M SU

5.2 12.5

90 69

22.2

0.25 M

su

18.0

55

37.2

0.88M

su

0

6.4

Histanzinase

100 101

43 69

Rabbit liver Pig kidney

a-Ketoglutarate oxidase 29 111 Rat liver

0.88M su 0.88M su 0.25M SU

0.88M

su

42.0 27.7 23.0

1.23

Trace

99.5 101.2 100.7

35.0

TABLE I1 (Continued) Reference Method Leiccine amidase 59 73

60

125

Tissue Rat Rat Rat Rat Pig

liver primary hepatoma spleen kidney kidney

Lactic dehydrogenare 33 41 Rat liver Pig kidney 23 125 Lipme 52 102 27

-

102 20 20 Malic dehydrogenaase 23 125 Myokinase 22 103

111 104

Isolation medium and comments

0.25 M SU 0.25M SU 0.25 M su 0.25 M SU 40% sucrose; assay: At pH 4.6 At pH 8.3

%T

R.C.

%R

%N

3.6 5.94 14.2 6.2

0.66 0.23 0.33 0.54

104.3 98.9 99.8 99.9

22.1 30.2 44.5 22.9

Present 0.19 (c)

Pig pancreas Guinea pig liver Horse pancreas Beef pancreas

40% SU Organic solvents Organic solvents Organic solvents

Pig kidney

40% SU

Rat liver Mouse liver

0.88M su 0.25 M SU

z

rn

0.40 0.28 0.12

Dilute citric acid 40% SU: (a) (b)

1.2

iml U

rn

1.30 106.7 0.048 0.015 (c) 0.08 (c)

t:

5 Z

Nucleoside phosphorylase 19 19 Calf liver Calf liver 19 20 20 20 Calf liver

0.25 M SU/O.0018 M CaCl, Organic solvents Organic solvents

C

<0.08

P E?

4.5 15.4

66.0 93.6 0.04 1.oo 1.00 (c)

2v3

Reference Method

19

20 19

Tissue

TABLE I1 (Continued) Isolation medium and comments

G,

%T

Fetal calf liver Organic solvents Horse liver Organic solvents Fetal horse liver Organic solvents Calf kidney Organic solvents Calf kidney cortex Organic solvents Calf heart Organic solvents Calf intestinal mucosa Organic solvents Fetal calf intestinal mucosa Organic solvents Calf pancreas Organic solvents Fetal calf pancreas Organic solvents Calf thymus Organic solvents Calf thymus Organic solvents Calf thymus 0.25M SU/0.0018M CaCl,

R.C.

%R

%N

2.08 (c) 2.74 (c) 1.36 (c) 1.2 (c) 1.0 (c) 4.4 (c) 0.24 (c) 0.24 (c) 0.75 (c) 1.63 (c) 0.37 (c) 0.47 0.60

P W %

0 0

Octanoate oxidase

104 29

Rat liver Rat liver

0.25 M su 0.88M SU

2.8

104 111 104

Rat liver Rat liver Rat kidney

0.25 M SU

10.5

115

Rat liver

0.25 M SU layered over 0.34M SU;

Pig kidney

as substrate : GlycyIglycine Triglycine Carbobenzoxyglutamyl-tyrosine 40% SU ; as substrate :

104 111

0.21

13.3

Trace

Oxaloacetate oxidase

105 29

105

0.88M 0.25 M

su su

0.73 0.37

Peptidase

115

60

125

101

14.4

Trace

8.6

Absent Absent Present

a 00

82.8

23.1

s2

Reference Method

Tissue

TABLE I1 (Continued) Isolation medium and comments

%T

carbobenzoxyglutamyl-glutaminyltyrosine, at 2 pH values : (a) carbobenzoxyglycyl-phenylalanine, at 2 pH values : (a)

Phosphotransferase 4 73 Proline oxidase 44 44 44

Dilute citric acid

Rat liver

Dilute citric acid Dilute citric acid, grind tissue

Rat liver

0.25 M

su

Rat liver

Dog cerebrum

0.25 M SU H2O 0.25 M su H2O 0.25 M SU

Pgruvate oxidase 29 111

Rat liver

0.88M

Rhodanese 38 6

Rat liver Rat liver

0.25 M SU 0.25 M SU/O.OOl M Versene

104 6

Z

Absent Absent

Y

v1

0

r ? I

Fatty rat liver

13

10.0

su su

40% 40%

13

0.26 0.66

0

Rat liver Pig kidney

Pyrophosphatase 3 3

%N

10.5 3.75 Present

(b) Rat liver

%R

20.6 0.37

(b)

106 106 Phosphory h e 33 41

R.C.

8

28.1 12.9 9.4 8.6 2.0

su

Z

4r E?

Trace

15.4 15.1

1.06 1.14

84.2 106.7

14.6 13.3

G,

Reference Method

Tissue

Rihonuclease (RNAme) 107 73 Rat liver

108

108

6 10 109

6 6 73

12

126

84

84

TABLE I1 (Continued) Isolation medium and comments

R.C.

%R

%N

94.0 96.0

11.3 13.8 Present

5.5 4.6 10.2 <1

0.41 0.66 1.oo

93.4 87.6 85.4

13.3 6.9 10.5

23.6 13.9 49.0

1.32 0.93 0.91

98.0 97.3 101.0

17.9 15.0 54.0

P

110

0.25 M SU/O.OOl M Versene

10.1

0.76

Rat liver

0.25 M SU

10.1

0.50 (p) 90.6

20.3 (PI

Rat liver

0.25M s u : 13.6

0.68 0.90

13.8 13.8

9.8 10.5

87.0 0.53 (p) 85.1

20.3 (PI 20.3 (PI

0.47

13.2

72.7

13.3

Succinic oxidase

73 110 73

73 110 73

W

56

0 0

z

Z

Succinic dehydrogeme

110

0

0.25 M SU; assay:

At acid pH At alkaline pH 0.25 M S U ; studied over wide pH range 0.25M SU/O.OOlM Versene Rat liver parenchyma cells 0.25M SU/O.OOl M Versene Mouse liver 0.25 M S U 0.25M SU/0.0018 M CaCl, Rat kidney 0.88M su: Nuclei not washed Nuclei washed Calf thymus 0.85% NaC1/0.002 M CaCl,

Succinate-cytochrome c reductase 6 6 Rat liver

2i

%T

No cytochome c Plus cytochrome c 0.25 M s u : No cytochrome c Plus cytochrome c 0.88M su: No cytochrome c

81.0

TABLE I1 (Continued) Reference Method

8 68 111 112 113 114 112 24 30 25 24

8 111 111 111 113 114 111 127 77 127 127

Tissue

Rat (DAB) hepatoma Rat kidney

Isolation medium and comments Plus cytochrome c 0.88M su 0.88 M S U 0.88M su 0.88M su 0.88M su 0.9% NaCl H,O Alkaline H,O 0.25M SU/0.0018 M CaCI, Alkaline H,O Alkaline H,O

%T

R.C.

%R

%N

8.4 9.0 6.4 18.4 14.0 20.0 11.1 4.0 6.6 1.48 11.5 7.6

0.63 0.60 0.52 0.82 0.82 0.79 0.86 (d) 0.33 0.64 (d) 0.17 0.46 (d) 0.46 (d)

76.0 82.0 85.0 97.0 90.0 95.0 90.4 92.0 94.0 97.5 91.0 96.5

13.2 15.0 12.4 22.5 17.0 13.0 (d) 12.0 10.4 (d) 9.0 24.8 (d) 16.4 (d)

104

116

104

117 118

73 104

Rat liver Rat liver

111

7

8

m

2

m

Sulfatase

116

K

+3

1

131 118 T P N cytochrome c reductase 6 6 Rat liver Mouse liver 69 73

2

0.25 M SU ; as substrate, p-nitrophenylsulfate 0.25 M SU ; as substrate, p-acetylphenylsulfate 0.25 M S U 0.25M SU : Male rats Female rats 0.88 M SU : Male rats Female rats 0.25 M SU/O.OOlM Versene : (a) (b) Sucrose/phosphate

0.25 M SU/O.OOlM Versene 0.25 M S U

9.1

109.2

12.5 -12 12.3 15.3 22.1 22.6 6.2 9.3

82.8 -80 83.7 94.1 88.7 91.2 102.2 96.7

U

8

1:

c:

0

r

E

Absent

10.8 12.0

0.81 0.68

105.2 104

13.3 G,

c

Reference Method

Tissue

TABLE I1 (Confinued) Isolation medium and comments

%T

R.C.

%R

19.5 18.5

0.84 -

104 108

%N

zN

~

Triacetic lactonase 119 73 127 Trypsin 52

-

Uracil decarboxylase 120 111

Rat kidney

0.25 M su -4lkaline H,O

Pig pancreas

40% SU: No activation Activated by enterokinase

Rat liver

Ureidosuccinate synthesis 121 121 Rat liver Uricase 122 6 112 8 112 45

77 10 46 122

73 6 111 8 111 41 77 6 41 73

0.25 M SU

P 0.25 M SU/O.Ol M Versene

0.25 M SU 0.25 M SU/O.OOl M Versene 0.88M su 0.88M su Rat liver Distilled H,O Dilute citric acid: Osborne Mendel rats Wistar rats 0.25 M SU/0.0018 M CaCI, Rat liver parenchyma cells 0.25 M SU/O.OOl M Versene Rat livers from animals Dilute citric acid: with transplantable tuHepatoma 31 mors Walker carcinosarcoma 0.25 M su Mouse liver

Rat liver

1.02 (c) 5.05 (c)

m

16.0

0.48

64

32.6

6.3 8.2 7.0 7.0 5.0

0.38 0.62 0.41 0.47 0.41

106.2 94.9 92.0 96.0 85.0

16.5 13.3 17.0 15.0 12.0

1.01 2.7

1.46 1.30 0.097 0.39

99.0 95.0

10.05 6.9

2.9

1.32 1.48 0.27

96.5

10.8

TI 8* 1:

Reference Method

Tissue

TABLE I1 (Continued) Isolation medium and comments

%T

Horse liver Fetal horse liver Calf liver Calf kidney

Organic Organic Organic Organic

Rat liver

0.25M SU

Xanthine dehydrogenase 124 111 Rat liver

0.25M SU

0

0.25 M SU 40% SU 40% SU

0

27 20 27

20 20 20

solvents solvents solvents solvents

R.C.

%R

%N

104.3

14.6

64.0

14.6

0.05 (c) 0.02 (c) Absent Absent

Vitamin A esterasc

123

73

Xanthine oxidase 124 111

44

1. 2. 3. 4.

5. 6. 7 8. 9. 10. 11. 12. 13. 14.

44 44

Rat liver Rat liver Pig kidney

Appelmans et al. (1955). Berthet and Duve (1951). Dianzani (1954). Brawerman and Chargaff (1955). Allard et al. (1954). Duve et al. (1955). Gianetto and Viala (1955). Novikoff et 02. (1953). Palade (1951). Wattiaux et 02. (1956). Tsuboi (1952). Straus (1956). Waked and Kerr (1955). Richter and Hullin (1951).

Absent

Absent Absent

REFERENCES TO TABLE I1 15. Siebert et a2. (1955). 16. Szekely (1955). 17. Dickman and Speyer (1954). 18. . Shepherd and Kalnitsky (1954). 19. Stern and Mirsky (1953). 20. Allfrey et al. (1952). 21. Lardy and Wellman (1953). 22. Novikoff et 01. (1952). 23. Lang and Siebert (1951). 24. Schneider (1946a). 25. Schneider (194613). 26. Johnson and Xckermann (1953). 27. Stern et al. (1952). 28. Jordan and March (1956).

0 1:

2

w

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

Kennedy and Lehninger (1949). Roodyn (1956a). Roodyn (1957). Roodyn (1956h). Dounce (1948). Dounce and Barnett (1952). Dounce et al. (1950). Dounce and Beyer (19484. Rosenthal et al. (1952). Ludewig and Chanutin (1950). Novikoff (1952). Emery and Dounce (1955a). Dounce (1943a). Hawkins (1952). Cotzias and Dole (1951). Lang and Siebert (1950). Lan (1943). Lan (1944). Chauveau and Hung (1952). Kielley and Schneider (1950). Dianzani (1951). Laird (1957). Hokin (1955). Siebert and Smellie (1957). Rosenthal (1953). Rosenthal et al. (1956). Lang et al. (1951). Schein and Young (1952). Dounce and Beyer (1%8b). Williams (1952b). Maver and Greco (1951)

REFERENCES TO TABLE I1 (Continued) 60. Lang and Siebert (1955a, b) . 61. Maver et al. (1952). 62. Hebb and Smallman (1956). 63. Underhay et al. (1956). 64. Goutier and Goutier-Pirotte (1955a). 65. Goutier and Goutier-Pirotte (1954). 66. Goutier and Goutier-Pirotte (1955b). 67. Williams (1952a). 68. Kensler and Langermann (1951). 69. Hogeboom and Schneider ( 1950). 70. Shepherd (1956). 71. Jackson et al. (1955). 72. Dianzani and Viti (1955). 73. Schneider and Hogeboom (1950). 74. Dounce (1943b). 75. Stern and Timonen (1954). 76. Brody et al. (1952). 77. Hogeboom et al. (1952). 78. Dounce (1954). 79. Zittle and Zitin (1942). 80. De Lamirande et al. (1954). 81. Rotherham et al. (1956). 82. Webb (1953). 83. Siebert et al. (1953). 84. Brown et al. (1952). 85. Sung and Williams (1952). 86. Hogeboom and Schneider (1952b). 87. Schotz et al. (1954). 88. Heller and Bargoni (1950). 89. Omachi et al. (1948). 90. Swanson (1950).

2 P

91. 92. 93. 94. 95.

96. 97. 98.

99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111.

REFERENCES TO TABLE I1 (Contitwed) Schein et al. (1951). 112. Kuff (1954). 113. Schneider (1947). Hers et al. (1951). 114. Shepherd and Kalnitsky (1951). Roodyn (unpublished observations). 115. Rademaker and Soons (1957). Walker (1952). 116. Dodgson et al. (1955). Dutton and Storey (1954). Hogeboorn and Schneider (1953a). 117. Roy (1954). Lang et al. (1951). 118. Dodgson et al. (1954). Rall and Lehninger (1952). 119. Meister (1952). LePage and Schneider (1948). 120. Rutman et al. (1954). Cotzias and Dole (1952). 121. Reichard (1954). Vallette et al. (1954). 122. Hogeboorn and Schneider (1952a). Behrens (1939). 123. Ganguly (1954). Kielley and Kielley (1951) . 124. Villela et al. (1955). Schneider (1948). 125. Lang and Siebert (1952). Schneider and Potter (1949). 126. Straus (1954). Miller and Dounce (reported by Dounce, 1952a). 127. Claude (1946). De Larnirande et al. (1954). 128. Brody and Bain (1952). Roth (1954). 129. Allard et al. (1952). Schneider and Hogeboorn (1952). 130. Schneider and Peterrnann (1950). Strittmatter and Ball (1954). Hogeboorn et al. (1948). ’ 131. Wilbur and Anderson (1951).

316

D. B. ROODYN

Enzyme activity/mg. N of nuclear fraction Enzyme activity/mg. N of cytoplasmic fraction is then recorded under the R.C. column, but followed by the letter (c). ( d ) : When the amount of tissue in the various fractions is measured by dry weights, the values for R.C. and % N are followed by the letter ( d ) ( p ) : Similarly, when the amount of tissue is based on protein determinations, the values for R.C. and % N are followed by the letter (p). SU: Sucrose.

.

The table is restricted to experiments on nuclei obtained from mammalian tissues. C. D N A and R N A The distribution of these nucleic acids is of interest as an aid to the assessment of the recovery of nuclei after fractionation (DNA) and as an indication of the amount of cytoplasmic contamination (RNA/DNA ratios). The results of some nucleic acid determinations are presented in Tables 111 and IV, although the data are by no means comprehensive.

IV. VALIDITY OF STUDIES ON ISOLATED NUCLEI I n the section above a survey has been made of the results of biochemical determinations on isolated nuclear fractions from a variety of tissues, the nuclei having been isolated by a number of methods. Unfortunately in many cases (inspection of Table I1 will soon reveal this) there is serious lack of agreement between the results obtained by different methods. Quite clearly the methods used are exposed to several sources of error, and therefore until some idea has been obtained as to how serious is this error it is not possible to understand the results given above. The attempts reported in the literature to study these errors will now be given. A . Contamination with Nonnuclear Material

Unless the nuclei are completely free of contaminant material the possibility must always be considered that all the activity observed in it is due to contamination. It is to be hoped that future research on the nucleus will provide more accurate methods for estimating contamination than are at present available. 1. Adsorption of Material onto Nuclei. It is possible that fine cytoplasmic debris or soluble proteins become adsorbed onto the surface of the nuclei during isolation and hence follow the nuclei through the purifica-

TABLE I11 DNA ANALYSES Reference" Method

1 2 3 4 5 6

7 8 9 10 12 6 13

1 1 21 5 5 5 7

Tissue Rat liver

0.88M

9 10 20 5 13 13 1

15 16 22 14 12 2 17

20 11 13 1 20 1

ii

su

0.88M su 0.88M su 0.88M su 0.88M su 2.2M su 2.2M su Alkaline H,O Distilled H,O 0.25 M SU/O.O018M CaCI, : (a)

8

3 14

Isolation medium and comments

0.25 M su 0.25 M SU 0.25 M su

Mouse liver Rabbit liver Rat kidney Rat brain

0.25 M

su

Alkaline H,O 0.88M SU/O.OlM phosphate 0.25 M SU/O.0018M CaCI,

0.25M

su

Alkaline H,O

0.25 M su 0.25 M SU,two fractions used : (a) fb) \

0

References for Table I11 are listed at the end of Table IV.

,

R.C.

103 89 93 84.5 91.0 57.3

7.40 7.00 4.86 5.30 4.41 5.35 4.05 5.32 11.2 11.2 10.0 (d) 7.40 7.35 8.40 8.80 12.8

85.4 95.0 87.0 93.2 99.5 100 105 69 11

2.3 3.9 (d) 2.6 (d) 8.32 4.46 6.1 (d) 4.5 1.68 0.82

80.0

(b) (C) Flexner- Jobling rat carcinoma Rat (DAB) hepatoma

%T 99.0 100.5 87.4 90.0 99.0 91.0 107

%R

%N

13.3 10.3 17.9 17.0 22.5 17.0 90.0

15.0

10.4 (d) 12.0 104 94.2 9.50 4.5

98.0 102

93.0 93.0

37.2 24.8 (d) 33.6 (d) 11.2 22.2 16.4 (d) 23.1

TABLE IV RNA ANALYSES Reference Method

Tissue

1 2 3 4 5 6 7 8 9 10 11 12 6 11 13

1 1 21 5 5 5 7 8 9 10 11 20 5 11 13

3 14

13 1

15 16 14 12 2 17

20 11 1 20 1 17

Rabbit liver Rat kidney

18

19

Pig thyroid

Rat liver

Flexner-Jobling rat carcinoma Rat (DAB) hepatoma

Rat brain

Isolation medium and comments

0.25 M su 0.25 M S U 0.25 M su 0.8844 su 0.88M su 0.88M su 0.88 M SU 0.88 M SU 2.2 M SU 2.2M su 0.88 -44 SU/O.Ol 21.1 phosphate Alkaline H,O Distilled H,O 0.14 M NaCI/O.Ol d l phosphate 0.25 M SU/0.0018 M CaCl, : (a) (b) 0.25 M SU Alkaline H,O 0.88 M SU/O.Ol M phosphate 0.25 M su Alkaline H,O 0.25 M SU 0.25 M SU, two fractions used : (a) (b) Dilute citric acid

%T

R.C.

%R

%N

13.8 11.8 9.1 15.0 18.9 16.0 24.8 10.0

1.04 96.8 0.82 101 0.53 93.2 0.88 94.0 0.84 96.5 0.94 92.0 0.94 91.2 0.66 90.0 0.73 0.73 0.7 (p) 88.0 0.72 (d) 98.5 0.66 101 0.7 (p) 86.0 1.05 1.10 0.6

13.3 14.4 17.9 17.0 22.5 17.0

9.0 7.5 8.0 29.0 13.6 11.3 3.2 37.8 11.9 25.0 28.0 10.7 15.6 39.0 12.0 7.5

1.02 0.49 (d) 0.74 (p) 1.25 0.65 (d) 0.68 1.1 0.9

97.5 103 98.0 101.9 98.5 108 98 98

15.0

13 (PI 10.4 (d) 12.0 40 (PI 12.6 9.5 4.5 37.2 24.8 (d) 33.6 (PI 22.2 16.4 (d) 23.1

P

E

0

3

1:

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Schneider (1948). Schneider and Potter (1949). Rosenthal et al. (1956). Novikoff et al. (1952). Hogeboom et al. (1948). Schein et al. (1951). Schneider (1947). Novikoff et al. (1953). Chauveau et al. (1956). Chauveau et al. (1957). Price et al. (1948).

TO TABLES I11 AND IV REFERENCES 12. Schneider (1%). 13. Hogeboom et al. (1952). 14. LePage and Schneider (1948). 15. Schneider ( 1 W b ) . 16. Price et al. (1949). 17. Brody and Bain (1952). 18. Rerabek (1952). 19. Dounce and Beyer (1948b). 20. Claude (1946). 21. Schneider and Hogeboom (1950). 22. Hogeboom and Schneider (1952%).

rn cr

320

D. B. ROODYN

tion process. Attachment of cytoplasmic debris to the nucleus or the presence of a thin cytoplasmic film are usefully shown up by methyl greenpyronine or aceto-orcein fast green (Kurnick and Ris, 1948), the latter stain giving good contrast between nucleoplasm, nucleoli, and cytoplasm. Observation under the light microscope may be deceptive, however, and electron microscopy has revealed debris attached to apparently clean nuclei (Davison and Mercer, 1956). There have been many investigations into the possibility that nuclear enzymes have been adsorbed from the cytoplasm. Dounce and Beyer (1948b) carried out some interesting experiments with arginase. It was found that rat liver nuclei has arginase activity but that lamb kidney and chicken liver nuclei were very deficient in the enzyme, because the whole homogenates themselves had very weak arginase activity. Addition of liver arginase either to suspended nuclei or to the homogenates from which the nuclei were isolated only produced a small increase in the activity of chicken liver and lamb kidney nuclei, the final activity being much less than that of isolated rat liver nuclei. It was therefore concluded that the activity of the rat liver nuclei was not due to adsorption. Ohlmeyer (1949) made a detailed study of the binding of added acid. prostatic phosphatase to isolated nuclei and to nucleic acid. The adsorption of enzyme onto nuclei is related to their nucleic acid content and is optimal at p H 4.5. Beinert (1951) used isotopically labeled cytochrome c and hence was able to determine with accuracy how much activity in a given cell fraction was derived from the original homogenate and how much from added cytochrome c. By these means he was able to show that the entire cytochrome c content of nuclear fractions, isolated in 0.25 M sucrose, was due to artificial readsorption. Novikoff (1952) suspended nuclei in purified alkaline phosphatase and found an increase in nuclear activity even after six washings, the increase being proportional to the amount of added enzyme. Similarly Roodyn (unpublished observations) has found that addition of liver soluble fraction to homogenates results in an increase in the aldolase activity recovered in the nuclear fraction, the increase being proportional to the amount of soluble enzyme added. Klose et al. (1952) mixed isolated nuclei with the supernatants obtained from the livers of rats fed radioactive amino acids (cf. Logan and Smellie, 1956) and measured the radioactivity of the reisolated nuclei. It was concluded that exchange of soluble protein did occur between nuclei and the supernatants, the effect being greatest at high p H values. Allfrey et al. (1952) concluded that hemoglobin is not adsorbed onto nuclei isolated in organic solvents, since mixing a thymus homogenate with red blood cell debris from fowl erythrocytes gave colorless thymus nuclei on isolation. Finally Dounce

METABOLIC STUDIES O N ISOLATED NUCLEI

321

et al. (1953) concluded that the cytochrome oxidase activity of nuclei isolated in distilled water at p H 6.0 was due to the adsorption of fine mitochondria1 fragments onto the nuclei. The effect was apparently prevented by the addition of gum arabic to the isolation medium. The mitochondrial fragments could, in some instances, be made visible by immersing the nuclei in 2,3,5-triphenyltetrazolium chloride solutions. 2. Endoflusmic Reticulum (a-Cytomembranes) md “Microsomes.” There are indications from electron microscopy that the nuclear membrane has an intimate connection with the cytoplasmic membrane system (“endoplasmic reticulum,” “ergastoplasm,” or the a-cytomembranes of Sjostrand, 1956). Pollister et ul. ( 1954) reported threadlike material running continuously from nucleus to cytoplasm at certain parts of the nucleocytoplasmic boundary of Frog oijcytes. Gay (1955, 1956) has studied the relationship between the endoplasmic reticulum and the nuclear membrane in the giant salivary glands of Drosophila mehogaster. It was observed under certain conditions that pockets of the nuclear membrane evaginated, broke away from the nucleus, and contributed to the formation of secretion granules and endoplasmic reticulum. The nuclear outpushings were identified by their characteristic annulate structure. Dr. J. D. Lever has very kindly sent the author an electron micrograph showing perfect connection between the outer nuclear membrane and the endoplasmic reticulum in the embryonic adrenal gland of the rat (J. D. Lever, Department of Anatomy, University of Cambridge, England, private communication). Watson (1955) reported continuity at some points between the outer nuclear membrane and the endoplasmic reticulum in rat spleen reticular cells and suggested that the perinuclear cisterna (i.e., both nuclear membranes plus the perinuclear space included between them) is really a modified part of the endoplasmic reticulum. Swift (1956) reports the presence, in the cytoplasm of a number of tissues, of annulate lamellae that closely resemble the nuclear membrane in structure and often lie near the nucleus. Similarly De Robertis (1954) has observed loose material resembling endoplasmic reticulum (ergastoplasm) lying on both sides of the nuclear membrane in nerve cells and suggests that at least part of the dense basophilic substance of the ergastoplasm has a nuclear origin. It is very likely, therefore, that the nuclear membrane might retain some of its cytoplasmic attachments after cell breakage. This could result in two effects. First, the densely osmophilic particles that line the endoplasmic reticulum and contribute to the “microsome” fraction obtained by differential centrifuging probably continue over part of the surface of the nucleus. One might therefore expect some similarities between the enzymatic properties of the nuclear and microsomal fractions. It has been noted that the

322

D. B. ROODYN

“microsomal” contamination of the nuclear fraction is often greater than the mitochondrial contamination. For example, Roodyn (unpublished observations) observed 6.1 % of the total glucose-6-phosphatase activity of a rat liver homogenate in the nuclear fraction isolated by the method of Hogeboom et d. (1952) (glucose-6-phosphatase is a characteristic microsomal enzyme; Hers et al., 1951). The succinoxidase and cytochrome oxidase activities in nuclear fractions obtained by this method are considerably less than this (Hogeboom et ul., 1952; Roodyn, 1956a), these two enzymes giving a measure of mitochondrial contamination. It is interesting in this connection that Chiquoine ( 1953) found by histochemical techniques that glucose-6-phosphatase is concentrated near the nuclear membrane in mouse liver cells. A second effect resulting from the connection between nuclear and cytoplasmic membranes might be that elements of the latter remain attached to the isolated nuclei as fine strands which would not be visible under the ordinary microscope. Indeed Davison and Mercer (1956) have revealed attached debris by electron microscopy. Chauveau et al. ( 1957), however, report that nuclei isolated in 2.2 M sucrose are completely free of microsomes or elements of the endoplasmic reticulum. 3. Mitochondria. The results of the direct estimation of mitochondrial contaminations by counting procedures (Allard et al., 1952 ; Shelton et d.. 1952, 1953) are presented in Table V. Many workers have suggested that the activity they observed in crude nuclear fractions was partly due to mitochondrial contamination (e.g., Cotzias and Dole, 1951, for amine oxidase), and it has frequently been found that techniques that produce less mitochondrial contamination also result in lower activities recovered in the nuclear fraction-for example, with cathepsin (Maver et ul., 1952), glutamic acid dehydrogenase (Hogeboom and Schneider, 1953a), and fumarase (Kuff, 1954). Allard et al. ( 1954) have calculated the contribution of mitochondrial contamination to the acid phosphatase activity observed in nuclear fractions from mitochondrial counts. I n rat liver, for example, 6.1% of the total activity was found in the nuclear fraction, but after correction for mitochondrial contamination this value fell to only 1.0%. Hogeboom et al. (1952) reduced the cytochrome oxidase content to very low levels and were able to obtain a clear correlation in several fractionations between the mitochondrial Contamination in the nuclear fraction and the cytochrome oxidase activity. It was possible to say quite definitely that d l the enzyme activity in the nuclear fraction was due to mitochondria present. (This was not the case with uricase, however.) For some time there had been considerable confusion as regards the status of cytochrome oxidase, since Dounce (1948)

TABLE V MITOCHONDRIAL CONTAMINATION OF NUCLEAR FRACTIONS Reference Method 1 7 2 3 3 3

4 5 6 4 1. 2. 3. 4.

7 7 6 3 3

Tissue Rat liver

Mouse liver

Allard et al. (1952). Rotherham et nl. (1956). Hogeboom et nl. (1952). Hogeboom and Schneider (1952b).

Isolation medium and comments 0.25 M SU 0.25 M SU/0.0018 M CaCI, 0.25 M SU/0.0018 M CaCI, : Unfiltered homogenate Filtered homogenate : (a) (b) 0.25 M SU 0.25 M SU 0.25 M SU layered over 0.34 M S U 0.25 M SU/0.0018 M CaCI, 0.25 M SU/0.0018 M CaCI,

%T

R.C.

13.2 5.0

%N

9.9

1.07 0.28 0.31 15.0 8.8 8.3 1.3 1.8

REFERENCES TO TABLE V 5. Schneider and Hogeboom 6. Hogeboom and Schneider 7. Schneider and Honeboom -

%R 98.3

0.085 0.028 0.032

88.0 100.3

0.87 0.73 0.14 0.19

99.0

(1952). (1953a). (1950). . .

12.6 10.05 9.5 15.6 10.5 11.0 9.1 11.2

324

D. B. ROODYN

had claimed to have found significant amounts of this enzyme in nuclei isolated in dilute citric acid. I n later work (Dounce et al., 1953), however, it was stated that the activity was due to contamination with mitochondrial fragments. The contribution of mitochondrial contamination to activity observed in the nuclear fraction has also been studied by Roodyn (1956a). In the case of aldolase, mitochondrial contribution was excluded because of the low activity of mitochondria (Kennedy and Lehninger, 1949) and because there was no correlation between the aldolase and succinoxidase activity in several fractionations. I n the case of succinoxidase activity, the mitochondrial contribution was clearly shown up, however, by sedimenting the (crude) nuclear fraction for a short time over a medium of graded sucrose concentration. There was no correlation between nuclei and succinoxidase distribution along the length of the tube. Part of the succinoxidase was found in fractions lying above the nuclei and containing the contaminant mitochondria, and the remainder was recovered below the bulk of the nuclei, in a fraction rich in unbroken cells. I n one fractionation, approximately 95% of the succinoxidase activity of the crude nuclear fraction was associated with the contaminating mitochondria. Chauveau et al. (1957) have claimed that isolation of nuclei in very concentrated sucrose solutions effects complete separation from contaminant mitochondria. (If enzymatic checks are made of this observation, it should not be forgotten that very high sucrose concentrations could possibly inhibit certain mitochondrial enzymes). 4 . Red Blood Cells. The blood cell contamination in nuclear fractions can be estimated by direct counts. I n addition, Gordon and Nurnberger (1955) have described a method for the determination of the amount of whole blood in tissue homogenates, which is of use as a check of the efficiency of perfusion. It is clearly most convenient to remove blood by perfusion of the organ before homogenization. I n liver this is relatively simple, perfusion with isotonic saline proceeding quite easily through the portal vein, by back-perfusion through the inferior vena cava or via the left heart through the whole animal. There have been some estimates of the contribution of blood cells to nuclear activity (Dodgson et al., 1954, for arylsulfatase ; Cotzias and Dole, 1951, for amine oxidase; and Roodyn, 1956a, for aldolase), Kuff (1954) has suggested that the low fumarase activity present in nuclear fractions might be due to red blood cells. Related to the question of contamination with red blood cells is the subject of nuclear hemoglobin. Clearly if the tissue used is not perfused before homogenization and hypotonic media are used, the hemoglobin liberated from the lysed erythrocytes could very easily become adsorbed onto the

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nuclei. As this hemoglobin would presumably be easily washed off the nuclei again, it is of interest to observe that Ehrenstein and Hevesy (1956) report a residual water-insoluble component in the hemoglobin found in liver nuclei after isolation in aqueous media. This nonextractable hemoglobin is one hundred times greater in amount in fetal as compared to maternal liver and incorporates Fe69 very actively (Bonnichsen et al., 1957). It is of interest in this connection that the nucleus plays an important part in the iron accumulation observed after in vitro incubation of rat liver cell suspensions (Bass et al., 1957). 5. Unbroken (Whole) Cells. This represents a very serious form of contamination because the intact cell might be able to carry out complex reactions which would be falsely ascribed to the nuclei. The hazard is particularly great because separation of nuclei from unbroken cells is difficult and whole cells might be found only in the nuclear fraction. I n other words a complex process might be found to be localized only in the nuclear fraction, and absent from all other fractions. In this respect the custom of some authors to refer to the “nuclear and whole-cell fraction” is to be highly recommended. As with mitochondria, several authors have suggested that the low activities observed in the nuclear fraction were due to whole cells (e.g., LePage and Schneider, 1948, with glycolysis ; De Lamirande et al., 1954a, with deoxyribonuclease ; Dodgson et al., 1954, with arylsulfatase) . Hogeboom et al. (1952) found slightly more uricase (1.0% of the total) than cytochrome oxidase (0.3% of the total) in nuclear fractions prepared in sucrose-CaC12 media. They assumed that this was due to whole cells. As there was perfect correlation between the mitochondria1 contamination and the cytochrome oxidase activity, it must therefore be assumed that whole cells do not estimate for cytochrome oxidase, but that they do for uricase. Uricase probably behaves similarly to succinoxidase in this respect. It was observed (Roodyn, 1956a, using the method of Hogeboom et al., 1952) that 1.3% of the whole cells and 1.5% of the succinoxidase were recovered in the nuclear fraction. I n several parallel fractionations, the succinoxidase was always slightly greater than the whole-cell contamination. Also, repeated washing of the nuclear fraction failed to remove residual succinoxidase, although all the mitochondria had been washed out. It is clear that both mitochondria and whole cells therefore contribute to the succinoxidase observed in the nuclear fraction. The fact that whole cells estimate for succinoxidase, but not for cytochrome oxidase, appears a little strange, but it is suggested that it is because succinate can enter the cell, but added ascorbic acid (used in the cytochrome oxidase assay) cannot (Schneider and Potter, 1943).

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In expressing whole-cell contamination as a percentage of the number of whole cells present in the liver before homogenization, the percentage of binucleate cells must be taken into account, if the results are calculated from nuclear counts on the homogenate. Harrison (1953) obtained a value of 30% by counting binucleate cells in suspensions of separated cells (St. Aubin and Bucher, 1952). Similar results were obtained by Bucher and Glinos (1950), who also observed that the percentage of binucleate cells is considerably greater in young than in adult rats, so that calculations of percentage whole-cell contaminations might be affected by the age of the animals. Perhaps the most important case to consider is whether the amino acid incorporation observed by Allfrey and co-workers in isolated thymus nuclei is due to contaminant thymocytes. On the basis of certain tonicity experiments in various media, Brown (1955) suggested that a large proportion of the nuclei used by Allfrey et al. were actually small intact thymocytes. Allfrey and Mirsky ( 1955), however, concluded from electron-microscope studies that there were only about 30 small thymocytes per 1000 nuclei. Similarly, using the same preparations, Ficq and Errera (1956) found 3% whole cells and 8% nuclei surrounded by a thin layer of cytoplasm. Using the refractometric method of Barer et al. (1956), they observed, however, that 24% of the structures present were intact cells, so that there seemed to be some confusion as to the actual contamination present. Fortunately, Ficq and Errera (1956, 1958) were able to confirm the findings of Allfrey et al. directly by using autoradiographic techniques and observed continuous uptake of label into nuclei that were free of attached cytoplasm. This independent confirmation of results obtained from bulk methods by autoradiography is of the greatest interest and holds out great promise for the future as a valuable measure of the true activity of nuclei. Another example, this time of a negative nature, of the use of other techniques to confirm a finding from bulk methods is the observation of Sacks et ul. (1957) that the small amount of glycogen present in isolated nuclear fractions is found only in the unbroken cells contaminating the preparation. This result was obtained by histochemical study of nuclear smears. 6. Bile Duct Canuliculi, Cell Walls, and Cell Debris. This kind of contamination is rather difficult to define and certainly very hard to measure quantitatively. Under its general heading are included small pieces of cell debris, pieces of fiber, partially broken cells, and “cell walls.” As regards the latter, Palade ( 1951 ) described contamination in nuclear fractions by “lp thick sheets of amorphous refringent material that probably represents the membranes of two adjacent cells together with the lamina of interposed cement.” Similarly, Hogeboom et ul. ( 1.952) describe

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“irregularly shaped, very thin structures that appear to be collapsed cell membranes.” Anderson ( 1955a) reports that the contamination with cell walls, fragments of broken cells, and whole cells in liver homogenates is pH-dependent, being much greater at p H 6.0 than at p H 7.88. Roodyn (1956a) made an approximate estimate of the aldolase activity of crude liver fibrous tissue and concluded that it could not contribute greatly to the activity observed in the nuclear fraction. There is little doubt that there is a relation between the amount of fiber contamination and the efficiency of removal of the blood vessel and connective-tissue framework of the tissue before homogenization. Also, in liver, the bile duct canaliculi can easily contaminate the nuclear fraction. It is therefore common practice to force the tissue through a suitable grid or mesh (e.g., Dounce, 1955) before cell breakage and then to filter the homogenate through flannelet or lint. (Vallette et al., 1954, use glass wool to eliminate connective tissue.) It is interesting that the material so removed is not altogether devoid of enzymatic activity. For example, Ludewig and Chanutin ( 1950) observed significant concentrations of esterase and alkaline phosphatase in the material remaining on the mesh. Cotzias and Dole (1951), however, found little amine oxidase in connective tissue and concluded that it did not contribute to nuclear activity. In connection with these problems, the question arises in liver as to how much of the bile duct system is removed before preparation of the homogenate and, hence, what proportion of the nuclei studied are derived from parenchyma cells and what proportion from the cells that make up the bile duct system. The relative proportion of bile duct to parenchyma has been determined in whole liver (Striebich et al., 1953) but it is of course very difficult to know this proportion in the nuclear fraction. Fortunately it has been shown by Wattiaux et al. (1956) that the enzyme distribution pattern in cell fractions of purified parenchyma cells is not very different from that in whole liver, although of course this may not be true for all enzymes. For example, Novikoff (1952) observed a great increase in the alkaline phosphatase activity of the nuclear fraction during liver regeneration. H e hesitated to ascribe this increase to nuclear activity, however, since during regeneration there was a great increase in the alkaline phosphatase activity of the bile duct caniculi. Also there was an increase in the activity of the sinusoidal (enlarged Kupffer), cells so that possibly only a small proportion of the parenchyma nuclei in regenerating liver contributed to the increase in activity.

B. Possible Loss of Protein by Isolation in Aqueous Media It is possible that the nucleus contains an easily soluble protein component which can be washed out during isolation. For this to be so it is

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clear that the nuclear membrane must be permeable to protein, so that the study of the structure and properties of the nuclear membrane is relevant to the problem. 1. Structure and Permeability of the Nuclear Membrane. Although many inferences have been made about the structure of the nuclear membrane as a result of studies with the ordinary light microscope or the polarizing microscope (see Baud, 1949), the most striking information has come from electron-microscope studies, in particular after the introduction of ultrathin sectioning techniques (cf. Watson, 1953). As a result of the study of a wide range of biological material it is now possible to generalize by stating that the nuclear membrane is a double structure, there really being inner and outer laminae. It is also found that these membranes either meet at a number of places in order to form “plates” or are completely pierced by holes or “pores.” This fenestrate or annulate structure of the nuclear membrane has been observed in plant cells (De, 1957), Amoeba proteus (Bairati and Lehmann, 1952 ; Harris and James, 1952 ; Pappas, 1956; and Greider et al., 1956), a protozoan parasite, Gregarinu melanopli (Beams et al., 1957), Chironomus larvae (Bahr and Beermann, 1954), the larval insect Heliothus obsoletu (Beams et al., 1956), snail, surf clam, and grasshopper oocytes (Rebhun, 1956), Triturus and Xenopus oocytes (Callan et al., 1949; Callan and Tomlin, 1950; Gall, 1954), sea urchin and starfish oocytes (Afzelius, 1955), nerve ganglia from Hirudo medicinalis (Bretschneider, 1952), chick embryo cells (Kautz and De Marsh, 1955), spinal ganglion cells of the rabbit (Dawson et d.,1955), neurons from ganglia, medulla oblongata, and cerebellar cortex of the rat (Palay and Palade, 1955), and finally a wide range of endodermal, mesodermal, and ectodermal tissues of the rat (Watson, 1955). The diameters of the pores range from 250 to 1000 A. and would clearly allow the passage of the largest molecules if they represented true openings. Unfortunately, it is not completely established that the nuclear membrane is actually pierced by “holes.” First, not all studies have revealed these structures. For example, Sjostrand and Hanzon (1954) reported the appearance of the nuclear membrane in the exocrine cells of mouse pancreas as a single uninterrupted thin line (see, however, Watson, 1954). Also Hartmann (1955) could fipd little evidence for fenestration of the nuclear membrane in cells from the rat central nervous system. Second, it is not certain that the pores or annulae actually represent gaps, since Kautz and De Marsh (1955), using chick embryo cells, have found that the pores in the membrane are as electron-dense, if not denser, than the surrounding nuclear membrane. Sjostrand (1956) expresses the view that there is not yet sufficient evidence to regard these pores as true holes and mentions

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the possibility that they might be caused by shrinking of the nuclear membrane. Apart from these structural studies, which suggest, but do not prove, that the nuclear membrane is freely permeable to protein, there have been some more direct experiments. Holtfreter (1954) observed the passage of hemoglobin into frog oocyte nuclei, which readily lost it when suspended in hemoglobin-free solutions. The nuclei were also permeable to sucrose and gum arabic. In contradiction to this, however, Goldstein and Harding (1950) found that egg albumin did not penetrate the nuclear membrane of such nuclei, although they were freely permeable to salts and sugars. Anderson (1953b) has shown that isolated liver nuclei are permeable to DNAase and RNAase which pass readily into the interior of the nucleus and affect its microscopic appearance. Philpot and Stanier (1957) have also shown that added protamine and histone can pass easily into the interior of isolated rat liver nuclei and so affect the type of granularity that they show. Because the soluble DPN-synthesizing enzyme is still present in nuclei after isolation in aqueous media but is completely released when the nuclei are disrupted by sonic vibrations (Hogeboom and Schneider, 1952b), it has been suggested (Hogeboom and Schneider 195313) that possibly, in this case at least, a protein is retained within the nucleus by an impermeable membrane. The use of sonic vibrations, however, might have caused depolymerization of nucleic acid and hence release of protein that was normally bound to the nucleic acid (Anderson, 1 9 5 3 ~ ) .Using ultrasonic vibrations, Roodyn (1957) was able to disrupt all the nuclear membranes without causing significant release of soluble material (aldolase) from the nuclei, in agreement with the observations of Dounce (1952a). It would be interesting to see the effect of short (l-minute) treatment of nuclei with ultrasonic vibrations on the DPN-synthesizing enzyme. The nucleus does not seem to swell and burst in hypotonic media, and it has been shown by Anderson and Wilbur (1952) and Philpot and Stanier (1956) that many of the optical and size changes in nuclei placed in various media are due to alterations in the state of the nucieohistone gel and not to swelling or shrinkage because of tonicity effects. For example, nuclei isolated in a hypotonic medium still retained significant amounts of the normally soluble enzyme aldolase (Roodyn, 1957). 2. Eflect of Washing Nuclei. If nuclei contain a component that is easily washed out during isolation in some media, washing nuclei prepared by better media should release this component. In addition, there is the feeling that an enzyme that can be readily washed out of the nuclear fraction is not a true “nuclear” enzyme but has probably been adsorbed onto

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the nuclei. As a result there have been many experiments on the effects of washing isolated nuclei. Dounce (1943a) found that washing nuclei isolated in dilute citric acid with Ringer-phosphate removed hemoglobin in two washings. Lactic acid dehydrogenase was diminished by washing, but the esterase and arginase activities were relatively unchanged. Brown et al. (1952) observed a fall in the deoxyribonuclease activity of isolated thymus nuclei after repeated washing, but it was not clear whether this was due to release or to inactivation of enzyme. Neff (1948), reported by Schneider and Hogeboom (1952), found that this enzyme could be completely washed off isolated liver nuclei with 0.013 M M g S 0 4 and concluded that all the activity initially found was due to adsorption. Arginase has been studied in detail by Rosenthal (1953) and Rosenthal et al. (1956). It was found that washing with solutions containing bivalent cations caused a great release of enzyme. Thus 0.01 M C a + + , M g + + , o r Mn+ + salts extracted 80 to 90% of the arginase activity from crude nuclear fractions, but only 10 to 20% of the nitrogen. The effect of the cations was independent of the type of anion present. Also with arginase, Dounce and Barnett (1952) found that three washings with 0.9% NaCl extracted 30 to 40% of the activity of nuclei isolated in dilute citric acid. These workers also found that this washing released 60 to 70% of the aldolase activity of the nuclei. Washing with 0.25 M or 0.88 M sucrose released little aldolase. Similar results were obtained by Roodyn (1956a, 1957), who showed that repeated washing of isolated nuclei with 0.25 M sucrose/0.00018 M CaC12 did not result in release of aldolase, the fall in activity observed being due to inactivation of the enzyme. Suspension of the nuclei in media of ionic strength 0.17, however, resulted in the release of 95 to 100% of their aldolase activity. Finally, Emery and Dounce (1955a) found that 0.9% NaCl released about 25% of the (magnesiumactivated) alkaline phosphatase of nuclei isolated in 0.44 M sucrose. These workers have also studied the extraction of nuclei prepared by the Behrens technique (Emery and Dounce, 1955b). It can be seen from the above that it might be possible to generalize by saying that nuclei contain a component that is easily washed out by salt solutions that are approximately isotonic. It is interesting to note, therefore, that Pollister and Leuchtenberger (1949) found that immersing tissue sections in physiological saline at 5°C. for 3 hours caused the loss of about 75% of the protein in guinea pig liver nuclei. The nucleus seems to be able to lose protein quite easily without altering the appearance of the membrane. Thus Wang et al. (1953) found that even after exhaustive extraction of rat liver nuclei with M NaCl some of the nuclei retained their original shape and had intact membranes.

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3. Comparative Experiments With Aqueous and Nonuqueous Methods. Kay et al. (1956) have made a detailed comparison of the protein and nucleic acid content of nuclei prepared in dilute citric acid and in organic solvents, but they did not study enzymes. Stern and Mirsky (1953) compared the adenosine deaminase, nucleoside phosphorylase, and glucose6-phophate dehydrogenase activity of calf liver nuclei prepared in aqueous and nonaqueous media, and it was concluded that a considerable loss of enzyme occurred after isolation in aqueous media. It was also found, however, that treatment of nuclei isolated in sucrose media with organic solvents affected their activity and sometimes produced an activation of the nuclear enzyme. Also, in some cases assays were not carried out directly on the nuclei but on extracts from them, so that differences in activity could possibly have been due to differences in extractibility of protein from the two kinds of nuclei. It is unfortunate that there have been no other studies, apart from those of Stern and Mirsky, on the enzyme activity of nuclei isolated in aqueous media with respect to the three enzymes in question. Thus Hogeboom and Schneider (1952a) only showed that 90% of the nucleoside phosphorylase and adenosine deaminase of liver was recovered in the soluble fraction and did not report any direct assays on the nuclear fraction. Similarly Mueller and Miller (1949) did not carry out direct assays on isolated nuclei but merely found that the bulk of the glucose-6-phosphate dehydrogenase activity was in the soluble fraction. Also, Glock and McLean (1953) found that all this enzyme was soluble (i.e., not sedimented by 20,0009 for 60 minutes) ; however, 0.15 M KCl was used, and protein might have been extracted from the nuclei. As Stern and Mirsky (1953) used a Waring blendor in the preparation of the aqueous nuclei, it is also possible that their method favored loss of nuclear protein (see Section IV.c.3 below). It cannot therefore be said definitely at the moment that nuclei isolated in aqueous media are deficient in the enzymes studied and hence that the use of organic solvents is essential for the study of their distribution. C . Damage to Nuclei Even though the nuclei isolated might be quite free of contaminant material and still retain a soluble protein component, they might nevertheless be damaged in some other way by the isolation procedure. The difficulty, however, is to establish reliable criteria of nuclear damage. 1. Appearance under the Microscope. Philpot and Stanier (1956) have made a detailed study of the effect of various suspension media on the appearance of rat liver nuclei under the phase-contrast microscope. Many

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of the optical changes observed were freely reversible and hence could not have resulted from permanent nuclear damage. Anderson and Wilbur (1952) indicated that many of the effects of various solutions on the appearance of the nuclei were due to changes in the state of nucleohistone gel (particularly its degree of hydration) and not to irreversible changes in nuclear structure. Thus Ris and Mirsky (1949) showed that isolated chromosomes are capable of reversible extension and condensation on being taken from sucrose to saline and that the changes in appearance of the nucleus are due to the alterations in the chromosomes. It must be concluded, therefore, that the state of granularity of the nucleoplasm is not an accurate measure of permanent nuclear damage. Apart from the texture of the nucleoplasm, however, other effects can be observed under the microscope that indicate nuclear damage. Anderson (1953a) demonstrated the appearance of “blebs” on the nuclear surface when liver nuclei were placed in dilute solutions of MgC12 or CaClz in the absence of sucrose. The blebs are impermeable to protein and probably arise from phospholipid material present in localized parts of the nucleus (Anderson, 1 9 5 3 ~ ) . Also, nuclei can become distorted in shape and elongated (Hogeboom et ul., 1952). Severe damage can pull the nuclei out into threadlike structures (Lamb, 1949). Torn membranes are shown up by the nuclear contents partially extruding into the suspension medium. Electron-microscope studies have been of great use (Davison and Mercer, 1956; Chauveau et aZ., 1956, 1957) in revealing torn nuclear membranes and attached cytoplasmic debris, and in studying the state of the nucleoplasm. It is to be hoped that future work with the electron microscope will provide useful information about nuclear damage in the various isolation methods. 2. Enzymatic Criteria of Damage. There are few clear cases of damage affecting the enzymatic properties of the nucleus. Hogeboom and Schneider (1952b) showed that forcing the liver through a perforated metal disk, in order to remove connective tissue, caused damage to the nuclei and also resulted in a lower yield of DPN-synthesizing enzyme in the nuclear fraction. ( A similar effect was observed by Roodyn, 1956b, with aldolase.) It was pointed out by Duve and Berthet (1954) that in the results of Hogeboom and Schneider (1952b) invariably a greater percentage of DNA than DPN-synthesiz’ing enzyme was found in the nuclear fraction. This indicates that it is possible to lose enzyme from the nuclei without losing DNA. It must be concluded that loss of enzyme from the nuclear fraction is not due to the complete fragmentation of nuclei with a resultant failure to sediment. As mentioned above, however (Section IV.B.l) loss of DPN-synthesizing enzyme from nuclei is not necessarily an index of damage to the nuclear membrane.

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Allfrey et al. (1957) mention that mechanical damage to nuclei destroys their ability to incorporate labeled amino acids, and it is to be hoped that this will provide a useful enzymatic index of damage, although as yet few details have been given. 3. E f f e c t of Homogenute on Nuclei. There have been several reports that suggest that nuclear damage (probably by the action of nucleolytic enzymes) can occur while the nuclei are in the homogenate prior to isolation, and that the ideal nuclear isolation medium must protect not only the nucleus but also cell particles. Anderson (1953b) has reported that liver nuclei rapidly empty if incubated at 37°C while still in the homogenate. Similarly, Philpot and Stanier (1956) observed that emptying of nuclei occurred much more rapidly in nuclear fractions that were heavily contaminated with cell particles than in purified nuclear fractions. If potassium glycerophosphate was added to the suspension medium, however, the nuclei did not empty, even though they were left in the presence of cell particles. It was concluded that the glycerophosphate was acting by preventing the lysis of “lysosomes,” cell particles described by Duve et al. (1955) as containing several degradative enzymes (including deoxyribonuclease) . It has been shown by Appelmans and Duve ( 1955) that glycerophosphate can provide partial osmotic protection to these granules. It may be noted that under certain circumstances the DNA of rat liver homogenates can remain unattacked after incubation at 37°C for as long as one hour (Roodyn, unpublished observations). These interactions between nuclei and cell particles can certainly produce changes in the activity finally recovered in the nuclear fraction. Roodyn (1956b, 1957) has studied the effect of the homogenate on nuclear aldolase and observed that delay in fractionation can sometimes result in a marked redistribution of enzyme between nuclei and supernatant. In addition, treatment of the homogenate for 30 seconds with the Waring blendor resulted in a fall in nuclear activity, although complete disruption of the nuclei by ultrasonic vibrations, once they hed been isolated, produced no release of enzyme. It is likely, therefore, that the effect of the Waring blendor is indirect (i.e., by fragmenting cell particles). Closely related to this are the observations of Dounce and co-workers on gel formation in nuclei. Dounce (1955) has suggested that a good index of nuclear integrity is their ability to form gels when placed in alkali. ( I t may be noted that Philpot and Stanier, 1956, give as. a measure of integrity the ability of isolated nuclei to form a “halo” when placed in M NaCl, the effect being due to DNA swelling through the nuclear membrane.) Dounce and Monty (1955) have studied this gel formation in

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detail and report that 300 r or 900 r of X-irradiation greatly reduces the ability of nuclei to form gels. In relation to the influence of the homogenate on the nucleus, Dounce et al. (1955) have shown the presence of an enzyme in liver mitochondria that inhibits gel formation in nuclei. Dounce et ul. (1957) have stated that DNAase I is the important factor in this inhibition, although proteases may also be involved. Mitochondria1 DNAase I1 is apparently not involved in this “degelling” action. This subject has also been studied by Goutier-Pirotte (1956). The influence of cytoplasmic enzymes on the nucleus during isolation seems well established, therefore, and constitutes yet another hazard in nuclear isolation. 4. Yield of Nuclei and Appearance of D N A in Nonnuclear Fractions. If not all the nuclei are sedimented, some DNA will be found in the fraction lighter than the nuclei (i.e., the heavy mitochondria). If, however, the nuclei are completely fragmented during the homogenization then DNA will be found in fractions depending on the size and sedimentation properties of the nuclear fragment. That such fragmentation can occur has been shown very clearly by Bass et al. (1956). Excessive homogenization of liver resulted in a great fall in the number of nuclei that could be counted in the homogenate. Sedimentation of the nuclear fraction showed that there was an increase in the DNA content of the supernatant corresponding with the amount of nuclear destruction. Fifteen minutes of treatment in the Potter-Elvehjem apparatus resulted in the destruction of half the nuclei. Reference to Table I11 will show that most determinations of the distribution of DNA have been carried out on nuclear fraction and homogenate only, so that the data for total recovery of D N A and distribution in other cell fractions is rather limited. Price et al. (1948) found 95% of the D N A in the nuclear fraction, 5% in the large granules, and none in other fractions in rat liver. Using liver tumor tissue, however, they found about 4% of the total D N A in the soluble fraction and 1.3% in the microsome fraction. They ascribed the appearance of D N A in these fractions to the presence of small (0.5- to 5-p) Feulgen-positive particles derived from infiltrating polymorphonuclear leukocytes and from disintegrating tumor cells. Keller (1951) observed high concentrations of DNA in the microsome fraction after treatment of rat liver homogenates with the Waring blendor. Similarly, Rerabek ( 1952) found considerable amounts of DNA in cytoplasmic fractions of swine thyroid fractionated in dilute citric acid and suggested that part of this was due to an artifact. It was suggested, however, that the chondriomes (cell particles) might really have DNA, not due to nuclear contamination. Brody and Bain (1952) report that the mitochondrial, microsomal, and soluble fractions of rat brain homogenates contain 576, 576, and 376, respectiveIy, of the total DNA.

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On the assumption that DNA is localized only in the nucleus, the data in Table I11 indicate the yield of nuclei in the various methods used. Nuclear yield can also be estimated from counts, however. Wilbur and Anderson (1951) mention a yield of about 5% by their method, and Chauveau et al. (1956) mention a yield of 45 to 50%. The author has not been able to find a report of the nuclear yield in preparations obtained in dilute citric acid or in organic solvents.

V. CONCLUSIONS It is hoped that this survey will be of use in revealing obvious gaps in our knowledge of the enzymatic properties of mammalian nuclei. Perhaps certain of these could be pointed out here. First, it is strikingly obvious that although our knowledge of rat liver nuclear fractions is quite large the experiments carried out on other tissues (even mouse, rabbit, or guinea pig liver) are relatively few in number. Although it is obviously important to have a clear understanding of at least one mammalian tissue, there are dangers that the results obtained might have only special significance and that some of the effects with rat liver might not apply to other animals. Second, a great deal of quantitative study has been made with a relatively small number of enzymes (e.g., cytochrome oxidase, succinoxidase, acid phosphatase, alkaline phosphatase, ATPase, and arginase) , but other enzymes of interest (eg., enolase) have not been studied in detail. Also it is unfortunate that, at the time of writing, some of the enzymes that have been studied with the greatest detail (e.g., cytochrome oxidase, succinoxidase, and uricase) are absent from the nucleus, whereas several enzymes that are probably present have not yet been quantitatively fractionated (e.g., U D P G synthesis and polynucleotide phosphorylase) . Third, it is anachronistic that we have rather complete information about the degree of contamination and the nuclear recovery, together with detailed comparative balance sheets with the simultaneous study of cytoplasmic fractions from the same homogenate, in nuclear fractions prepared by methods that quite obviously give serious contamination (e.g., the methods of Hogeboom et al., 1948, Schneider and Hogeboom, 1950), whereas the information available on nuclear recovery and cytoplasmic contamination in methods that obviously yield better nuclear fractions is fragmentary. Finally, there is an obvious need for experiments on the same tissue, studying the same enzyme, with nuclear fractions obtained by the several methods available. Thus there is extremely fragmentary information about the enzymatic properties of rat liver nuclei isolated in organic solvents,

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although these are the nuclei most commonly used for studies with aqueous media. Although it is apparently difficult to obtain satisfactory preparations with rat liver using organic solvents (Dounce et al., 19501, even comparative results with crude nuclear fractions would be of the greatest interest. Apart from these indications of certain deficiences in our knowledge, it might, perhaps, be useful to attempt some sort of classification of the results in Tables 11, 111, and I V into the following groups: P1:

P2: P3: A3: A2: Al:

Enzymatic activity in the nuclear fraction shown to be associated with nuclei present, and not with the contaminant material. Enzymatic activity localized predominantly in the nuclear fraction and absent from cytoplasmic fractions. Relative concentration greater than 1.0. Relative concentration less than or equal to 1.0. No enzymatic activity detected in the nuclear fraction. All enzymatic activity in the nuclear fraction quantitatively accounted for by contaminant material present.

Applying these classifications to the results for mouse and rat liver nuclear fractions isolated in aqueous media, we obtain the results shown in the accompaning groupings (the assigning of some enzymes to the group shown has been somewhat arbitrary because of conflicting data, and the reader is referred to the tables and original references for the details). Provided the method of enzyme assay is valid and suitable recoveries of both enzyme and nuclei are obtained after fractionation, it seems to the author that enzymes falling into groups A1 and A2 are absent from nuclei, whereas enzymes falling into groups P1 and P 2 are present. This clearly should not be taken as a rigid rule, but only as a guide to assess the significance of results in any particular case. The results that fall into groups A3 and P3 are felt by the author to be indicative of absence and presence, respectively, but requiring further study before a definite answer can be given. It is interesting to observe in this connection that ribonucleic acid ( R N A ) falls into group A3, and the exact contribution of contamination to nuclear R N A is yet to be established (see Mauritzen et al. 1952). P1 Aldolase DNA

P2 DPN synthesis DNA

P3 Adenosine-5'-phosphatase Aldolase Alkaline phosphatase Arginase ATPase (Ca+ +-activated) D P N synthesis

METABOLIC STUDIES ON ISOLATED NUCLEI

A1 Cytochrome c (Beinert, 1950) Cytochrome oxidase Glutamic dehydrogenase Succinoxidase Uricase A2 L-Amino acid oxidase p-Aminobenzoic acid acetylase Cathepsin Glucuronide synthesis a-Ketoglutarate oxidase Peptidase Vitamin A esterase Xanthine dehydrogenase Xanthine oxidase A3 Acid phosphatase Acetyl CoA deacylase Amine oxidase ATPase (Mg+ +-activated) p-Aminohippuric synthesis Catalase

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A3 (cont.) Choline esterase Choline oxidase DNAase DPN-cytochrome c reductase Enolase Esterase Fumarase Glucose-6-phosphatase B-Glucuronidase Glutaminase Isocitric dehydrogenase Leucine amidase Lactic dehydrogenase Octanoate oxidase Phosphorylase Phosphotransferase Pyruvate oxidase Rhodanese RNA Succinic dehydrogenase SulfataseTPN-cytochrome c reductase Uracil decarboxylase

Because of the multiplicity of errors and artifacts possible in the study of the enzymatic properties of isolated nuclei, the author has refrained from drawing too definite conclusions about the metabolic significance of the findings reported. It is hoped that future work will not only reduce or eliminate these errors, but also reveal new enzymatic pathways that are the exclusive property of the nucleus.

VI. ACKNOWLEDGMENTS The author would like to thank Dr. P. Hagen, Dr. E. Kennedy, Dr. N. Madsen, and Dr. F. Isherwood, for useful correspondence and Mr. J. W. P. Phelpstead for help with proofreading. H e is also most grateful to Mr. J. St. L. Philpot and Dr. Jean E. Stanier for many useful and stimulating discussions on the subject of the nucleus.

VII. REFERENCES Afzelius, B. A. (1955) Exptl. Cell Research 8, 147. Allard, C., Mathieu, R., de Lamirande, G., and Cantero, A. (1952) Cancer Research 12, 407. Allard, C., de Lamirande, G., Faria, H., and Cantero, A. (1954) Can. J . Biochem. Physiol. 32, 383. Allfrey, V. G., and Mirsky, A. E. (1955) Science l.21,879.

338

D. B. ROODYN

Allfrey, V. G., and Mirsky, A. E. (1957a) Proc. Natl. Acad. Sci. US.43, 589. Allfrey, V. G., and Mirsky, A. E. (1957b) Proc. Natl. Acad. Sci. US.43, 821. Allfrey, V. G., Stern, H., Mirsky, A. E., and Saetren, H. (1952) 1. Gen. Physiol. 36, 529. Allfrey, V. G., Daly, M. M., and Mirsky, A. E. (1954) J. Gen. Physiol. 38, 415. Allfrey, V. G., Mirsky, A. E., and Osawa, S. (1955a) Nature 176, 1042. Allfrey, V. G., Mirsky, A. E., and Stern, H. (1955b) Advances in Enzymol. 16, 411. Allfrey, V. G., Mirsky, A. E., and Osawa, S. (1957) J. Gen. Physiol. 40, 451. Anderson, N. G. (1953a) Exptl. Cell Research 4, 306. Anderson, N. G. (1953b) Exptl. Cell Research 6, 361. Anderson, N. G. (1953~) Science 117, 517. Anderson, N. G. (1955a) Exptl. Cell Research 8, 91. Anderson, N. G. (1955b) Exptl. Cell Research 9,446. Anderson, N. G. (1956) J. Biophys. Biochem. Cytol. 2, 219. Anderson, N. G., and Wilbur, K. M. (1952) J. 8Gen. Physiol. 36, 781. Appelmans, F., and Duve, C. de (1955) Biochem. I. 69,426. Appelmans, F., Wattiaux, R., and Duve, C. de (1955) Biochem. J. 69,438. Arnesen, K., Goldsmith, Y., and Dulaney, A. D. (1949) Cancer Research 9, 669. Bahr, G. F., and Beermann, W. (1954) Exptl. Cell. Research 6, 519. Bairati, A., and Lehmann, F. E. (1952) Experientia 8, 60. Barer, R., Joseph, S., and Esnouf, M. P. (1956) Science 123, 24. Bass, A. D., McArdle, A. H., and Grisham, J. W. (1956) Science 123,372. Bass, R. L., Bernick, S., and Saltmann, P. (1957) Exptl. Cell Research 13, 395. Baud, C. A. (1949) Bull. histol. appl. et tech. microscop. 26, 99. Beams, H. W., Tahmisian, T. N., Anderson, E., and Devine, R. L. (1956) Proc. S O C Exptl. . Biol. Med. 91, 473. Beams, H. W., Tahmisian, T. N., Devine, R. L., and Anderson, E. (1957) Exptl. Cell Research 13, 200. Behrens, M. (1932) 2.physiol. Chem. 209, 59. Behrens, M. (1938) 2.physiol. Chem. 263, 185. Behrens, M. (1939) 2.physiol. Chem. 268, 27. Behrens, M., and Taubert, M. (1952) 2.physiol. Chem. 291, 213. Beinert, H. (1951) J. hiol. Chem. 190, 287. Berthet, J., and Duve, C. de (1951) Biochem. J. 50, 174. Bonnichsen, R., von Ehrenstein, G., Hevesy, G., and Schliack, J. (1957) Acta Chem. Scand. 11, 120. Brachet, J. (1957) “Biochemical Cytology.” Academic Press, New York. . 113. Bradfield, J. R. G. (1950) Biol. Revs. Canzb. Phil. S O C26, Brakke, M. K. (1951) J. A m . Chem. SOC.73, 1847. Brakke, M. K. (1953) Arch. Biochem. Biophys. 46,275. Branster, M. V., and Morton, R. K. (1956) Biochem. J. 63,640. Brawerman, G., and Chargaff, E. (1955) Biochim. et Biophys. Acta 16, 524. Bremer, J., and Gloor, V. (1955) Acta Chem. Scand. 9,689. Bretschneider, L. H. (1952) Intern. Rev. Cytol. 1, 305. Brody, T. M., and Bain, J. A. (1952) J. B i d . Chem. 196, 685. Brody, T. M., Wang, R. I. H., and Bain, J. A. (1952) J . Biol. Chem. 198, 821. Brown, G. B., and Roll, P. M. (1955) In “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 11, p. 341. Academic Press, New York. Brown, J. R. C. (1955) Science 121, 511.

METABOLIC STUDIES O N ISOLATED NUCLEI

339

Brown, K. D., Jacobs, G., and Laskowski, M. (1952) J. Biol. Chem. 194, 445. Bucher, N. L. R., and Glinos, A. D. (1950) Cancer Research 10, 324. Callan, H. G., and Tomlin, S. G. (1950) Proc. Roy. SOC.B197,367. Callan, H. G., Randall, J. T., and Tomlin, S. G. (1949) Nature 169, 280. Chalazonitis, N., and Otsuka, M. (1956) Compt. rend. a49, 978. Chauveau, J. (1952) Compt. rend. 2S6, 902. Chauveau, J., and Hung, L. V. (1952) Compt. rend. 2S6, 1248. Chauveau, J., MOUE,Y., and Rouiller, Ch. (1956) Exptl. Cell Research 11, 317. Chauveau, J., Mouli, Y., and Rouiller, Ch. (1957) Bull. SOC. chim. biol. 39, 1521. Chiquoine, D. (1953) J. Histochem. and Cytochem. 1, 429. Claude, A. (1946) J. Exptl. Med. 84, 51. Claude, A. (1954) Proc. Roy. Soc. Bl42, 177. Corper, H. J., and Cohn, M. L. (1936) I. Lab. Clin. Med. 21,428. Cotzias, G. C., and Dole, V. P. (1951) Proc. SOC.Exptl. Biol. Med. 78, 157. Cotzias, G. C., and Dole, V. P. (1952) J. Biol. Chem. 196,235. Daly, M. M., Allfrey, V. G., and Mirsky, A. E. (1952) J . Gen. Physiol. 36, 173. Dallam, R. D., and Thomas, L. (1953) Federation Proc. 12, 193. Davidson, J. N. (1957) Brit. Med. Bull. 13, 77. Davison, F. P., and Mercer, E. H. (1956) Exptl. Cell Research 11, 237. Dawson, I. M., Hossack, J., and Wyburn, G. M. (1955) Proc. Roy. SOC.B144, 132. De, D. N. (1957) Exptl. Cell Research 12, 181. De Lamirande, G., Allard, C., and Cantero, A. (1954a) Can. I. Biochem. and Physiol. 32, 35. De Lamirande, G., Allard, C., Costa, H. C. da, and Cantero, A. (1954b) Science 119, 351. De Robertis, E. (1954) J. Histochem. and Cytochem. 2, 341. Dianzani, M. U. (1951) Arch. Fisiol. 60, 187. Dianzani, M. U. (1954) Biochim. et Biophys. Acto 14, 514. Dianzani, M. U., and Viti, I. (1955) Biochem. J. 69, 141. Dickman, S. R., and Speyer, J. F. (1954) J . Biol. Chem. W , 67. Dodgson, K. S., Spencer, B., and Thomas, J. (1954) Biochem. J. 66,177. Dodgson, K. S., Spencer, B., and Thomas, J. (1955) Biochem. J. 69,29. Dounce, A. L. (1943a) 1.Biol. Chem. 147, 685. Dounce, A. L. (194313) J. Biol. Chem. 161,221. Dounce, A. L. (1948) Ann. N . Y . Acad. Sci. 60, 982. Dounce, A. L. (1950) Zn “The Enzymes” (J. B. Sumner and K. Myrback, eds.), Vol. I, Part A, p. 187. Academic Press, New York. Dounce, A. L. (1952a) Exptl. Cell Research Siippl. 2, 103. Dounce, A. L. (1952b) J. Cellular Comp. Physiol. 39 Suppl. 2, 43. Dounce, A. L. (1954) Intern. Rev. Cytol. 3, 199. Dounce, A. L. (1955) In “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 2, p. 93. Academic Press, New York. Dounce, A. L,and Barnett, S. R. (1952) J. Cellular Comp. Physiol. 39 Suppl. 2, 50. Dounce, A. L., and Beyer, G. T. (1948a) J. Biol. Chem. 173, 159. Dounce, A. L., and Beyer, G. T. (1948bj J. Biol. Chem. 174,859. Dounce, A. L., and Litt, M. (1952) Federation Proc. 11, 203. Dounce, A. L., and Monty, K. J. (1955) J. Biophys. Biochem. Cytol. 1, 155. Dounce, A. L., and Tishkoff, G. H., Barnett, S. R., and Freer, R. M. (1950) J. Gen. Physiol. SS, 629.

340

D. B. ROODYN

Dounce, A. L., Kaye, E. R. M., and Pate, S. (1953) Federation Proc. 12, 198. Dounce, A. L., Witter, R. F., Monty, K. J., Pate, S., and Cotton, M. A. (1955) I. Biophys. Biochem. Cytol. 1, 139. Dounce, A. L., O’Connell, M. P., and Monty, K. J. (1957) J. Biophys. Biochem. Cytol. 3, 649. Dutton, G. J., and Storey, I. D. E. (1954) Biochem. J. 67,275. Duve, C. de, and Berthet, J. (1954) Intern. Rev. Cytol. 3, 225. Duve, C. de, Pressman, B. C., Gianetto, R., Wattiaux, R., and Appelmans, F. (1955) Biochem. J . 60, 604. Ehrenstein, G. von, and Hevesy, G. (1956) Acta Physiol. Scand. 38, 184. Emanuel, C. F., and Chaikoff, I. L. (1957) Biochim.et Biophys. Acta 24, 254. Emery, A. J., and Dounce, A. L. (1955a) J. Uiophys. Biochem. Cytol. 1, 315. Emery, A. J., and Dounce, A. L. (1955b) I. Biophys. Biochem. Cytol. 1, 331. Ficq, A., and Errera, M. (1956) Arch. intern. physiol. et biochem. 64, 533. Ficq, A., and Errera, M. (1958) Exptl. Cell Research 14, 182. Friedkin, M., and Roberts, D. (1954) J. Biol. Chem. 207, 245. Friedkin, M., and Wood, H. (1956) J. Biol. Chem. 220, 639. Gall, J. F. (1954) Exptl. Cell Research 7, 197. Ganguly, J. (1954) Arch. Biochem. Biophys. 62, 186. Gay, H. (1955) Proc. Natl. Acad. Sci. U S . 41, 370. Gay, H. (1956) 1. Biophys. Biochem. Cytol. 2 Suppl. 407. Gianetto, R., and Viala, R. (1955) Science l!2l, 801. Glock, G. E., and McLean, P. (1953) Biochem. I. 66, 400. Goldstein, L., and Harding, C. V. (1950) Federation Proc. 9, 48. Gordon, M. W., and Nurnberger, J. I. (1955) J. Histochem. and Cytochem. 3, 130. Goutier, R., and Goutier-Pirotte, M. (1954) Arch. intern. physiol. 62, 151. Goutier, R., and Goutier-Pirotte, M. (1955a) Biochim. et Biophys. Acta 16, 361. Goutier, R., and Goutier-Pirotte, M. (1955b) Biochim. et Biophys. Acta 16, 558. Goutier-Pirotte, M. (1956) Biochim. et Biophys. Acta %, 394. Grant, H. C., and Rees, K. R. (1958) Proc. Roy. SOC.B148, 117. Greider, M. H., Kostir, W. J., and Frajola, W. J. (1956) I. Biophys. Biochem. Cytol. 2 Suppl. 445. Grunberg-Manago, M., and Ochoa, S. (1955) I. A m . Chem. SOC.77, 3165. Hagan, W. A. (1922.) J. Exptl. Med. 36, 711. Harris, E. S., and Mehl, J. W. (1954) Science 120, 663. Harris, P., and James, T. W. (1952) Experientia 8, 384. Harrison, M. F. (1953) Nature 171, 611. Hartmann, J. F. (1955) J. Comp. Neurol. 99, 201. Haven, F. L., and Levy, S. R. (1942) Cancer Research 2, 797. Hawkins, J. (1952) Biochem. I. 60, 577. Hebb, C. O., and Smallman, B. N. (1956) Nature 178, 365. Heller, L., and Bargoni, N. (1950) Arkiv Kemi 1, 447. Heppel, L. A., Ortiz, P. J., and Ochoa, S. (1956) Science 123, 415. Hers, H. G., Berthet, J., Berthet, L., and Duve, C. de (1951) Bull. soc. chim. biol. 33, 21. Hilmoe, R. J., and Heppel, L. A. (1957) I. Am. Chem. SOC.79, 4810. Hogeboom, G. H., and Kuff, E. L. (1954) J. Biol. Chem. 210, 733. Hogeboom, G. H., and Schneider, W. C. (1950) J. Biol. Chem. 186, 417. Hogeboom, G. H., and Schneider, W. C. (1952a) I. Biol. Chem. 196, 161.

METABOLIC STUDIES ON ISOLATED NUCLEI

34 1

Hogeboom, G. H., and Schneider, W. C. (1952b) J. Biol. Chem. 19’7, 611. Hogeboom, G. H., and Schneider, W. C. (1953a) J. Biol. Chem. 204, 233. Hogeboom, G. H., and Schneider, W. C. (1953b) Science 118, 419. Hogeboom, G. H., and Schneider, W. C. (1955) In “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 2, p. 199. Academic Press, New York. Hogeboom, G. H., Schneider, W. C., and Palade, G. E. (1948) J. Biol. Chem. 172, 619. Hogeboom, G. H., Schneider, W. C., and Striebich, M. J. (1952) J. Biol. Chem. 196, 111. Hogeboom, G. H., Schneider, W. C., and Striebich, M. J. (1953) Cancer Research 19, 617. Hokin, L. E. (1955) Biochim. et Biophys. Acta 18, 379. Holter, H. (1952) Advances in Enzymol. 13, 1. Holter, H., Ottesen, M., and Weber, R. (1953) Experientia 9, 346. Holtfreter, J. F. (1954) Exptl. Cell Research 7 , 95. Jackson, R. M., Albert, S., and Reeves, A. (1955) Proc. SOC.Exptl. Biol. Med. 88, 594. Johnson, R. B., and Ackermann, W. W. (1953) J. Biol. Chem. 200, 263. Jordan, W. K., and March, R. (1956) J. Histochem. and Cytochem. 4, 301. Kamphausen, H., and Morton, R. K. (1956) Biochem. J. 69, 647. Kautz, J., and De Marsh, Q. B. (1955) Exptl. Cell Research 8, 394. Kay, E. R. M., Smellie, R. M. S., Humphrey, G. F., and Davidson, J. N. (1956) Biochem. J. 62, 160. Keller, E. R. (1951) Federation Proc. 10, 206. Kennedy, E. P., and Lehninger, A. L. (1949) J. Biol. Chem. 179, 957. Kensler, C. J., and Langermann, H. (1951) 1. Biol. Chem. 192, 551. Kielley, R. K., and Schneider, W. C. (1950) J. Biol. Chem. 186, 869. Kielley, W. W., and Kielley, R. K. (1951) J. Biol. Chem. 191, 485. Klose, S., Frankel, D., Miller, L. L., and Dounce, A. L. (1952) Exptl. Cell Research Suppl. 2, 113. Kornberg, A. (1950) J. Biol. Chem. 182, 805. Korner, A., and Tarver, H. (1957) J. Gen. Physiol. 41, 219. Kuff, E. L. (1954) I. Biol. Chem. m,361. Kuff, E. L., and Schneider, W. C. (1954) J. Biol. Chem. 206, 677. Kurnick, N. B., and Ris, H. (1948) Stain Technol. 2S, 17. Laird, A. K. (1957) Biochim. et Biophys. Acta I,56. Lamb, W. G. P. (1949) Nature 164, 109. Lan, T. H. (1943) J. Biol. Chem. 161, 171. Lan, T. H. (1944) Cancer Research 4, 37. Lang, K., and Siebert, G. (1950) Biochem. 2. Sao, 492. Lang, K., and Siebert, G. (1951) Biochem. 2. S22, 196. Lang, K., and Siebert, G. (1952) Biochem. 2. S22, 360. Lang, K., and Siebert, G. (1955a) In “Handbuch der physiologisch und pathologisch chemischen Analyse,” 10th ed., Vol. 2, p. 537. Springer, Heidelberg. Lang, K., and Siebert, G. (1955b) Ann. Acad. Sci. Fennicae Ser. A , 11, No. 80, 73. Lang, K., Siebert, G., Lucius, S., and Lang, H. (1951a) Biochem. 2. 321, 538. Lang, K., Siebert, G., and Miiller, L. (1951b) Nafurwiss. 38,528. Lang, K., Lang, H., Siebert, G., and Lucius, S. (1953a) Biochem. 2. SM, 217. Ling, K., Siebert, G., and Rossmiiller, G. (1953b) Natumiss. 40, 293.

342

D. B. ROODYN

Lardy, H. A., and Wellman, H. (1953) J. Biol. Chem. a01, 357. Lehninger, A. L. (1949) J. Biol. Chem. 178, 625. LePage, G. A., and Schneider, W. C. (1948) J. Biol. Chem. 176, 1021. Lindhal, P. E. (1948) Nature 161, 648. Lindhal, P. E. (1956) Biochim. et Biophys. Acta 21, 411. Logan, R., and Davidson, J. N. (1957) Biochim. et Biophys. Acta 24, 196. Logan, R., and Smellie, R. M. S. (1956) Biochim. et Biophys. Acta 21, 92. Ludewig, S. L., and Chanutin, A. (1950) Arch. Biochem. aS, 441. Marshak, A. (1941) J. Gen. Physiol. 26, 275. Mauritzen, C. M., Roy, A. B., and Stedman, E. (1952) Proc. Roy. SOC.Bl40, 18. Maver, M. E., and Greco, E. (1951) J. Natl. Cancer Znst. 12, 37. Maver, M. E., Greco, E. A., Lgivtrup, E., and Dalton, A. J. (1952) 1. Natl. Cancer Znst. 13, 687. Mayer, D. T., and Gulick, A. (1942) J. Biol. Chem. 146, 433. Mazia, D. (1952) In “Modern Trends in Physiology and Biochemistry” (E. S. G. Barron, ed.), p. 77. Academic Press, New York. Meister, A. (1952) Science 116, 521. Mills, G. T., Ondarza, R., and Smith, E. E. B. (1954) Biochim. et Biophys. Acfa 14, 159. Mueller, G. C., and Miller, J. A. (1949) I. Biol. Chem. 180, 1125. Neff, R. J. (1948) Thesis, University of Missouri, Columbia, Missouri. Novikoff, A. B. (1952) Exptl. Cell Research Suppl. 2, 123. Novikoff, A. B., and Podber, E. (1957) J. Histochem. and Cytochem. 6, 552. Novikoff, A. B., Hecht, L., Podber, E., and Ryan, J. (1952) J. Biol. Chem. 194, 153. Novikoff, A. B., Podber, E., Ryan, J., and Noe, E. (1953) J. Histochem. and Cytochem. 1, 27. Ohlmeyer, P. (1949) 2. Naturforsch. 4b, 263. Omachi, A., Barnum, C. P., and Glick, D. (1948) Proc. SOC.Exptl. Biol. Med. 67, 133. Osawa, S., Allfrey, V. G., and Mirsky, A. E. (1957) J. Gen. Physiol. 40, 491. Palade, G. E. (1951) Arch. Biochem. SO, 144. Palay, S. L., and Palade, G. E. (1955) I. Biophys. Biochem. Cytol. 1, 69. Pappas, G. D. (1956) J . Biophys. Biochem. Cytol. 2 Suppl. 431. Phelpstead, J. W. P., and Roodyn, D. B. (1957) Exptl. Cell Research B,673. Philpot, J. St. L., and Stanier, J. E. (1954) Nature 174, 651. Philpot, J. St. L., and Stanier, J. E. (1956) Biochem. I . 63, 214. Philpot, J. St. L., and Stanier, J. E. (1957) Nature 179, 102. Philpot, J. St. L., and Stock, D. A. (1955) Biochem. J. 60 Proc. xlii. Pollister, A. W., and Leuchtenberger, C. (1949) Proc. Natl. Acad. Sci. U.S.1, 66. Pollister, A. W., Gettner, M., and Ward, R. (1954) Science 120, 789. Poort, C. (1957) Biochim. et Biophys. Acta 26, 32. Potter, V. R., and Elvehjem,, C. A. (1936) 1. Biol. Chem. 114, 495. Potter, V. R., Lyle, G. G., and Schneider, W. C. (1951) J. Biol. Chem. 190, 293. Price, J. M., Miller, E. C., and Miller, J. A. (1948) J. Biol. Chem. 173, 345. Price, J. M., Miller, J. A., Miller, E. C., and Weber, G. M. (1949) Cancer Research 9, 96.

Rademaker, W., and Soons, J. B. J. (1957) Biochim. et Biophys. Acta 24, 451. Rall, T. W., and Lehninger, A. L. (1952) J. Biol. Chem. 194, 119. Randolph, M. L., and Ryan, R. R. (1950) Science ll2, 528.

METABOLIC STUDIES ON ISOLATED NUCLEI

343

Rebhun, L. I. (1956) I. Biophys. Biochem. Cytol. 2, 93. Reichard, P. (1954) Acta Chem. Scand. 8, 795. Rerabek, J. (1952) Biochim. et Biophys. Acta 8, 389. Richter, D.,and Hullin, R. P. (1951) Biochem. I. 48, 406. Ris, H., and Mirsky, A. E. (1949) J. Gen. Physiol. Sa, 489. Roodyn, D.B. (1956a) Biochem. 1. 64,361. Roodyn, D. B. (1956b) Biochem. J. 64, 368. Roodyn, D. B. (1957) Biochim. et Biophys. Acta 26, 129. Rosenthal, 0. (1953) Federation Proc. 12, 260. Rosenthal, O.,Novack, B. G., and Brodie, J. W. (1952) Federation Proc. 11, 276. Rosenthal, O.,Gottlieb, B., Gorry, J. O., and Vars, H. M. (1956) J. Biol. Chem.

223,469. Roth, J. S. (1954) J. Biol. Chem. 208, 181. Rotherham, J., Schottelius, D. D., Irvin, J. L., and Irvin, J. M. (1956) J. Biol. Chem. aa3, 817. Roy, A. B. (1954) Biochim. et Biophys. Acta 14, 149. Rutman, R. J., Canterow, A., and Paschkis, K. E. (1954) J. Biol. Chem. 210, 321. Sacks, J., Johnston, P. M., Morton, J. H., and Harvey, J. A. N. (1957) Exptl. Cell Research 12, 537. St. Aubin, P. M. G., and Bucher, N. L. R. (1952) Anat. Record 1l2, 797. Schein, A. H., and Young, E. (1952) Exptl. Cell Research 3, 383. Schein, A. H.,Podber, E., and Novikoff, A. B. (1951) J. Biol. Chem. 190, 331. Schneider, W.C. (1946a) J. Biol. Chem. 166, 585. Schneider, W.C. (1946b) Cancer Research 6, 685. Schneider, W.C. (1917) Cold Spring Harbor Symposia Quant. Biol. 12, 169. Schneider, W.C. (1948) I. Biol. Chem. 176, 259. Schneider, R. M. (1955a) Exptl. Cell Research 8, 24. Schneider, W. C. (1955b) 3rd Intern. Congr. Biochem., Brussels, 1955, Conf. and Repts. p. 305. Schneider, W.C., and Hogeboom, G. H. (1950) J. Biol. Chem. 183, 123. Schneider, W.C., and Hogeboom, G. H. (1951) Cancer Research 11, 1. Schneider, W.C., and Hogeboom, G. H. (1952) J. Biol. Chem. 198, 155. Schneider, W.C., and Hogeboom, G. H. (1956) Ann. Rev. Biochem. 26, 201. Schneider, R. M., and Petermann, M. L. (1950) Cancer Research 10, 751. Schneider, W.C., and Potter, V. R. (1943) J. Biol. Chem. 149, 217. Schneider, W.C., and Potter, V. R. (1949) 1. B i d . Chem. 177, 893. Schotz, M. C., Rice, L. I., and Alfin-Slater, R. B. (1954) J. Biol. Chem. 207, 665. Schrecker, A. W.,and Kornberg, A. (1950) J. Biol. Chem. la,795. Shelton, E.,Schneider, W. C., and Striebich, M. J. (1952) Anat. Record ll2, 388. Shelton, E., Schneider, W. C., and Striebich, M. J. (1953) Exgtl. Cell Research 4, 32. Shepherd, J. A. (1956) J. Histochem. and Cytochem. 4, 47. Shepherd, J. A., and Kalnitsky, G. (1951) I. Biol. Chem. 192, 1. Shepherd, J. A., and Kalnitsky, G. (1954) J. Biol. Chem. 207, 605. Shonk, C. E.,and Boxer, G. E. (1957) Federation Proc. 16, 247. Siebert, G.,and Smellie, R. M. S. (1957) Intern. Rev. Cytol. 6, 383. Siebert, G.,Lang, K., Lucius-Lang, S., Herkert, L., Stark, G., Rossmiiller, G., and Jockel, H. (1953a) 2. physiol. Chem. 296, 229. Siebert, G., Lang, K., Lucius, S., and Rossmiiller, G. (1953b) Biochem. 2. 324,311.

344

D. B. ROODYN

Siebert, G., Jung, G., and Lang, K. (1955) Biochem. 2. Sas, 464. Siekevitz, P. (1952) 1. Biol. Chem. 196, 549. Sjostrand, F. S. (1956) Intern. Rev. Cytol. 6, 456. Sjostrand, F. S., and Hanzon, V. (1954) Exptl. Cell Research 7, 393. Smellie, R. M. S. (1955) In “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 2, p. 393. Academic Press, New York. Smellie, R. M. S., Humphrey, G. F., Kay, E. R. M., and Davidson, J. N. (1955) Biochem. I. 60, 177. Smith, E. E. B., and Mills, G. T. (1954) Biochim. et Biophys. Acta 13, 386. Stedman, E.,and Stedman, E. (1951) Phil. Trans. Roy. SOC.London B236, 565. Stern, H., and Mirsky, A. E. (1953) J . Gen. Physiol. 37, 177. Stern, H., and Timonen, S. (1954) J . Gen. Physiol. 38, 41. Stern, H., and Timonen, S. (1955) Exptl. Cell Research 9, 101. Stern, H., Allfrey, V., Mirsky, A. E., and Saetren, H. (1952) J. Gen. Physiol. 36, 559. Stoneburg, C. A. (1939) J. Biol. Chem. 129, 189. Straus, W. (1954) I . Biol. Chem. 207, 745. Straus, W. (1956) J. Biophys. Biochem. Cytol. 2, 513. Striebich, M. J., Shelton, E., and Schneider, W. C. (1953) Cancer Research 13, 279. Strittmatter, C. F., and Ball, E. G. (1954) J . Cellular Comp. Physiol. 43, 57. Sung, S-C., and Williams, J. N. (1952) I. Biol. Chem. 197, 175. Swanson, M. (1950) J. Biol. Chem. 184, 647. Swift, H. (1956) J . Biophys. Biochem. Cytol. 2 Suppl. 415. Szekely, M. (1955) Acta Physiol. Acad. Sci. Hung. 8, 291. Thomson, J. F., and Mikuta, E. T. (1954) Arch. Biochem. Biophys. 61, 487. Tsuboi, K. K. (1952) Biochim. et Biophys. Acta 8, 173. Underhay, E., Holt, S. J., Beaufay, H., and Duve, C. de (1956) I. Biophys. Biochem. Cytol. 2, 635. Vallette, G., Cohen, Y., and Burkard, W. (1954) Compt. rend. soc. biol. 148, 1762. Villela, G. G., Mitidieri, E., and Affonso, 0. R. (1955) Nature 176, 1087. Waked, N., and Kerr, S. E. (1955) J . Histochem. and Cytochem. 3, 75. Walker, P. G. (1952) Biochem. J. 61, 223. Wang, T. Y., Mayer, D. T., and Thomas, L. E. (1953) Exptl. Cell Research 4, 102. Watson, M. L. (1953) Biochim. et Biophys. Acta 10, 1. Watson, M. L. (1954) Biochim. et Biophys. Acta 16, 475. Watson, M. L. (1955) J. Biophys. Biochem. Cytol. 1, 257. Wattiaux, R.,Baudhuin, P., Berleur, A. M., and Duve, C. de (1956) Biochem. 1. 63, 608. Webb, M. (1953) Exptl. Cell Research 6, 16. Weinman, E. O., Lerner, S. R., and Entenman, C. (1956) Arch. Biochem. Biophys. 64, 164. Weiss, B. (1953) I. Biol. Chem. Ml,31. Wilbur, K. M., and Anderson, N. G. (1951) Exptl. Cell Research 2, 47. Wilbur, K. M., and Skeen, M. V. (1950) Science 111, 304. Williams, J. N. (1952a) I. Biol. Chem. 194, 139. Williams, J. N. (1952b) J . Biol. Chem. 196, 37. Zittle, C. A., and Zitin, B. (1942) J . Biol. Chem. 144, 99.

Trace Elements in Cellular Function' BERT L. VALLEE

AND

FREDERIC L. HOCH

Biophysics Research Laboratory of the Department of Medicine, Harvard Medical School and The Peter Bent Brigham Hospital, Boston, Massachusetts I. Introduction ...................................................... 11. Emission Spectrography .......................................... 111. Metalloenzymes and Metalloproteins ............................... A. Definitions ................................................... B. Zinc Metalloenzymes .......................................... 1. Carbonic Anhydrase ...................................... 2. Bovine Pancreas Carboxypeptidase ......................... 3. Pyridine Nucleotide-Dependent Zinc Metallodehydrogenases . . C. Cadmium Protein from Equine Kidney Cortex ................. IV. Metal-Enzyme Complexes ........................................ A. Definitions ................................................... B. Metal Ion-ATP Interactions ................................. V. Metals in Subcellular Fractions ................................... A. Fractionations in Isotonic Sucrose ........................... B. Other Media and Dietary Variations ......................... C. Effects of Carbon Tetrachloride ............................. VI. Summary ........................................................ VII. References .......................................................

Page 345 347 350 350 353 353 354 355 365 367 367 371 375 375 377 378 380 381

I. INTRODUCTION Many possible approaches have been suggested and pursued toward elucidating the functions of metallic elements in biochemistry and physiology. The present review will comment on some hypotheses and on some experimental principles which seem pertinent to the problem but must be regarded as a partisan's view of a larger arena of effort. Collectively, the elements to be discussed have variously and inappropriately been named trace elements, micronutrients, rare elements, or minor elements (Vallee, 1952), terms which by implication refer either to their concentrations or to their supposed functions in tissues. These appellations themselves focus on the reasons for grouping these elements together arbitrarily. Common factors are the difficulties encountered in their measurement because of the low concentrations in which they are found and a 1 Based on a lecture given at the International Congress for Cell Biology, St. Andrews, Scotland, August to September, 1957. The original investigations reported in this chapter were supported by grants-in-aid by the Office of Naval Research, the National Institutes of Health of the Department of Health and Welfare, and the Howard Hughes Medical Institute.

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distinct resistance to definitive appraisal of their physiological function. This phenomenology is a rather negative basis for taxonomy, and, understandably, it has not easily led to constructive hypotheses. The a priori assumption, reached by many, that there might be analogies or identities in the functions of these elements solely because of analogies in their concentrations does not necessarily seem justifiable, a deduction strengthened by the available evidence. The further conclusion that the criteria for appraisal of the biological effectiveness of metals have been ill-defined also appears unavoidable. An establishment of such criteria is essential before the vagaries incident to investigative efforts are to be removed, so that their scope may be extended. Much effort has centered on phenomenological gross or microscopic observations of animals, plants, or microorganisms in which a deficiency, imbalance, or intoxication was induced by nutritional manipulations of metals. Although a great deal of knowledge has accumulated in this manner (Underwood, 1956), comprehension of the mode of action of metals in biology has not grown at a similarly rapid rate. The complexities of experimental design are reflected in many instances by the various interpretations which are made of similar data. An explanation for the function of some of the so-called trace metals has been sought (Kubowitz, 1937; Keilin and Mann, 1938, 1940; Warburg, 1949; Dawson, 1950; Mallette, 1950; Nelson, 1950; Vallee, 1951, 1955) in their association with proteins, particularly with those exhibiting enzymatic activity. Although these elements may be associated with other molecules, e.g., cobalt and vitamin Blz (Smith, 1948; Rickes et ul., 1948), the study of metal-enzyme interactions seems to offer special opportunities in an operational sense. The investigations of the metabolic roles of the trace elements, which will be described here, have been based, on the one hand, on the concept of their association with enzymes. Once the nature of such an association is defined, the metabolic function of the metal may be identified with the significance that the enzyme-catalyzed chemical reaction plays in the cellular economy. The other basis of these investigations has been the conclusion that it is necessary to identify and to measure the metals of the biological system under study. The low concentrations and the large numbers of the metals intrinsic to biological systems, even partially purified ones, impose stringent demands on the analytical method to be used. The intellectual demands of accurate, precise, sensitive, and complete metal analyses cannot be avoided, and, at the same time, the operational needs for economy of effort and expense must also be met. In the experiments to be cited,

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emission spectrography, which fulfills these postulates, has been employed as an analytical tool for metal analyses.

11. EMISSION SPECTROGRAPHY In view of the above considerations, the analytical detection of metals becomes a significant aspect of work with metalloproteins and metalloenzymes. Many of the sensitive and highly precise procedures common to analytical chemistry and instrumental analysis may be applicable. Sensitivity of the technique is of great importance, since the total material available for analysis is usually severely limited. Spectrophotometry, spectrography, polarography, and other electrochemical techniques, X-ray and electron optics, ion-exchange and chromatographic separations, and isotope methods including neutron activation analysis (Tobias and Dunn, 1949) are examples of methods commonly employed. Combinations of these methods often become useful and are sometimes indispensable (Harrison et al., 1948; Tobias and Dunn, 1949; Snell and Snell, 1949; Sandell, 1950; Ahrens, 1950; Nachtrieb, 1950; Mellon, 1950; Muller, 1951 ; Kolthoff and Lingane, 1952 ; Hall, 1953 ; Lederer and Lederer, 1953; Samuelson, 1953; Smith, 1953 ; Lingane, 1953 ; Delahay, 1954 ; Weissberger, 1949-1954). The principles, applications, and promise of emission spectroscopy as a procedure in protein chemistry have received little attention. Elements excited by sufficient energy emit light of characteristic wavelengths. The accepted mechanism of this effect is the excitation of atoms to higher electronic energy states, followed by the emission of radiation when the excited atoms return to lower energy states, or when ionization takes place by ejection of an electron from the atom. The various transitions from higher to lower states are accompanied by the emission of radiation of specific wavelengths, characteristic of the element excited. The appearance of different lines of the same element, and of lines of different elements, is a function of the source employed for the excitation of the sample (Nachtrieb, 1950). Two essentially independent processes condition this phenomenon : ( 1) volatilization ,of the inorganic salts which constitute the sample ; ( 2 ) excitation of the atom to a higher energy state. When the direct-current arc is employed as a source, its essentially thermal characteristics first cause volatilization of the elements into the arc column. The rate of diffusion into the arc column is a function of the anode temperature, which in turn is a function of the applied wattage, the mass of the atoms, and-for ions-the field of electric and magnetic force. Since the direct-current arc operates at temperatures varying from 3000" to 8000°K (Seminova, 1946), elements present as components of compounds with low boiling points may volatilize very rapidly and be lost

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from the arc stream prior to excitation (Vallee and Peattie, 1952). The excitation of atoms and ions, once present in the direct-current arc, is a function of the electrode material, the shape and separation of the electrodes, the voltage drop across the system, and the amperage applied. When the radiation emitted from one or several elements is dispersed by a prism or a diffraction grating, its component wavelengths may be recorded on a photographic film or plate, where the light at each wavelength will bring about blackening in the form of a line. The light-gathering power of the spectrograph, the range of the spectrum recorded, and the characteristics of the photographic plates employed are all additional variables to be considered in the evaluation of both the qualitative and quantitative data obtained (Ahrens, 1950). The technique of emission spectrography has the unique advantage of allowing the simultaneous qualitative and quantitative analysis of most of the biologically important elements quickly and accurately. This is true for elements present in concentrations as low, in some instances, as 1x to 1 x g. per gram of sample. Under appropriately controlled conditions, the light emitted, and hence the blackening of the photographic plates, is proportional to the amount of the element present in the sample. The density of lines is therefore an index of the amount of the element present, and the blackening can be measured accurately with a microdensitometer (Fig. 1) . Recently developed emission spectrographic techniques (Vallee, in preparation) allow the simultaneous measurement of some twenty elements with high sensitivity, accuracy, and precision, and with samples no larger than a few milligrams. Whereas quantitative emission spectrographic analysis yields accurate identification and quantification of elements present, attempts at semiquantitative interpretation frequently lead to erroneous conclusions. This is an unfortunate circumstance and has often led to avoidance of this method of analysis in spite of its obvious and desirable features. Although spectroscopy is simple in principle, the achievement of accurate, quantitative data is time-consuming and tedious, mostly owing to the problems of quantitative photometry. With the advent of photoelectric devices for measurement of line intensities (Margoshes and Vallee, 1956), these drawbacks should be, greatly diminished. Emission spectrography has been used in biology almost exclusively for the analysis of whole tissues. The metal content of an organ having been obtained, how is the finding to be translated in terms of the function of the living organism? The problem is similar to that which confronts an organic chemist who tries to establish the structural and functional characteristics of a new compound. It is not profound to state that carbon,

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hydrogen, oxygen, nitrogen, sulfur, and metal analyses would be of relatively little use. If a mixture of such compounds were analyzed, it is dubious that much meaningful information would be discerned by such a procedure. Yet, this is the situation in which the biological chemist and spectroscopist finds himself when dealing with tissues. A given metal in a biological material exists as a constituent of many different compounds, serving totally different functions. Since most of these compounds and their func-

FIG.1. Spectrochemical working curve for calcium. Portions of the spectrum obtained with 1, 3, 10, and 50 parts per million of calcium are shown a t the right. The density of the Ca 3968.5-A. line, indicated by the arrows, is measured with a microdensitometer and is plotted, on the left, as an intensity relative to that of an internal standard, the V 4395.2-A. line. Relative intensity is a linear function of the logarithm of the concentration of the metal.

tions have not as yet been discerned, and only the sum of their constituent metal atoms can be measured, the biological interpretation of the analytical results is, at the least, problematic. The approaches to be discussed may constitute a partial answer. A further comment is deserving of emphasis. The elements under discussion have been designated collectively as “trace” elements, because they are encountered in relatively low concentrations in tissues. The data to be presented are a further reminder that tissues constitute a heterogeneous mixture of natural products, the majority of which do not contain metals. In effect, they constitute a diluent for such metal-containing compounds.

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Individual metalloproteins can be purified and isolated by removal of this inert, extraneous material, resulting in marked aggregation of the metal into the milligram range, as will be demonstrated. Although a metal may, therefore, be a minor “trace” constituent with reference to the whole organ, it may be a major component with reference to one or several proteins of that organ. No conclusion concerning the function of a metal can, therefore, readily be drawn from data of metal concentrations in whole tissues. It may not require emphasis that the amount of biological material available for analysis is often severely limited, particularly if highly purified products such as enzymes must be analyzed, when only milligram quantities are usually available. At this juncture, simultaneous qualitative and quantitative spectrochemical analysis becomes unique. The limitation of total sample precludes analysis for so many elements by almost any other procedure. The contamination hazards, however, are analogous to those encountered in any other method for trace analysis. The role of metals in systems of varying biological complexity may be studied spectroscopically. I t is of advantage, and for this purpose often necessary, to choose for examination biological systems which are most susceptible to a joint study of a given metal, of the related biological activity or function, and of the organic component. As a result, this approach leads to the perusal of tissues or tissue components from species covering a considerable range of the biological spectrum. It also demands the examination of systems of widely varying complexity themselves. These may be classified, albeit somewhat arbitrarily, as ( 1 ) isolated molecular species, (2) organized particles of living cells, and ( 3 ) living organisms. A series of studies on highly purified enzyme systems serves as an example of results obtained from such an approach at the molecular level.

111. METALLOENZYMES AND METALLOPROTEINS A. Definitions Emission spectrography is peculiarly suited to the exploration and the investigation of enzymes containing a fixed amount of a specific metal per protein molecule, both in the “natural” state and in the purified state. This group of substances has been termed wtalloenzywes, and this appellation is a purely operational one in the sense of Bridgman (1946) (Ogden and Richards, 1947). The use of the term does not denote any judgment on the presumed differences in function, composition, or structure between these and other enzymes which react differently with metals (metal-enzyme complexes, Section I V ) . It does single out those proteins and enzymes

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35 1

which are associated with metals in such a way as to permit certain general experimental approaches to be detailed. This definition is made in the terms of the operational methods employed to examine metalloenzymes, to assist in clarifying the distinction between abstract constructs and the more concrete results of experimental experience. The criteria to be given, therefore, simply derive from current experimental considerations and are not exclusive of others yet to be found. Nor does the term imply that metals may not participate in catalysis under circumstances not encompassed by these criteria, although different interpretations of this definition have been made ( Malmstrom, 1956). The large number of enzymes which involve metals in their action, but do not fall within the scope of this operational system, continues to present a challenge. In metalloenzymes, at least three primary parameters can be measured independently to examine their interdependence: (1) the protein, (2) the metal, and (3) the activity. Thus, these enzymes present a particularly suitable group of substances both for physical-chemical definition of metalprotein interactions and also for extrapolation of information from such simple systems toward the understanding of metabolic mechanisms. The unique chemical specificity and reactivity of metals provide a powerful basis for such studies. The characteristics of metalloenzymes have been reviewed in detail elsewhere (Vallee, 1955). Some features will be summarized briefly here to serve as a basis for the subsequent illustrations of the principles involved in the work on specific metalloenzymes. When metalloenzymes are isolated in completely purified form, the ratio (R1) of metal (Me) to protein (P) reaches a limiting value: Me R1 = P The ratio ( R 2 ) of activity ( A ) to protein reaches a limiting value : A R2 = P Therefore, the ratio (R3) of metal to activity also reaches a limiting value: R 3 -- -Rl = - Me (3) R2 A Simultaneously all metals present, but neither intrinsically associated with nor functionally related to the enzyme, and therefore extrinsic to it, are removed without adversely affecting the activity : 2(extraneous metals) Activity +o (4)

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With complete purification the number of gram atoms of the intrinsic metal per mole of apoenzyme reaches an integral value, attesting to the specificity of their association and implying stoichiometry. The metal seems to be unique, and its removal, which is accomplished with difficulty, results in concomitant loss of activity. The metal is not easily restored to its original site, and restoration of activity is similarly difficult. These considerations all imply specific and chemically distinct binding sites. When a metalloenzyme interacts with a coenzyme or a prosthetic group, the numbers of moles of coenzyme and the numbers of gram atoms of metal contained in the apoenzyme (R4)usually show a numerical correspondence expressed by an integral number :

where a, b = 1, 2, 3 . . . . a / b = 1, 2, 3 . . . . Operationally, equation 5 is obtained in the form

The functional implications of compositional findings have been examined with metal-binding inhibitors. The considerations governing their use have been detailed elsewhere (Vallee, 1955). These inhibitors have been postulated to diminish catalytic activity by binding to the metal in situ on the apoenzyme, forming a mixed complex, as shown in equation 7: [EMe]

+ nI P

[EMe] I,

(7)

indicating that the active metalloenzyme [ EMe] reversibly combines with n molecules of inhibitor, I, to yield the enzymatically inactive complex [EMe] I,,. The brackets indicate the great strength of association between enzyme and metal. As an alternative, it might be expected that the inhibitor, I, would inactivate the enzyme by removing the metal. The reversibility of this reaction would be governed by the reassociation of the metal with the protein, in addition to the ,dissociation of the metal-inhibitor complex. [EMe]

+ n I # E + Me-I,

(8)

The zinc-containing metalloenzymes and metalloproteins have been particularly characterized and studied from these points of view (Vallee, 1958), by both emission spectrography and chemical methods (Vallee and Gibson, 1948; Hoch and Vallee, 1949) for measurements of zinc.

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B . Zinc Metalloenzymes According to the operational definitions for a metalloprotein and a metalloenzyme (Vallee, 1955), seven zinc metalloproteins have been characterized thus far : the carbonic anhydrase of bovine erythrocytes (Keilin and Mann, 1940) ; a zinc-containing protein of human leukocytes (Hoch and Vallee, 1952; Vallee et al., 1954) ; the carboxypeptidase of bovine pancreas (Vallee and Neurath, 1954, 1955) ; the alcohol dehydrogenase of yeast (Vallee and Hoch, 1955a, b) and of equine liver (Theorell et al., 1955; Vallee and Hoch, 1956, 1957) ; the glutamic dehydrogenase of bovine liver (Vallee et al., 1955) ; and the lactic dehydrogenase of rabbit skeletal muscle (Vallee and Wacker, 1956). Later findings are consistent with the existence of yet additional pyridine nucleotide-dependent metallodehydrogenases (Vallee et al., 1956a), although the possibility of their final inclusion in this group of zinc enzymes awaits their purification from extraneous proteins and metals, and their characterization in this homogeneous state. Until 1953, the only conclusively proved role of zinc in metabolism was as a component of carbonic anhydrase. 1. Carbonic Anhydrase. Carbonic anhydrase was first described as a protein catalyst in 1932 (Brinkman et a l . ) and was purified from erythrocytes shortly thereafter (Meldrum and Roughton, 1932a, b, 1933). The most frequently used preparative method extracts the enzyme from bovine erythrocytes (Keilin and Mann, 1940). Carbonic anhydrase has never been obtained in ultracentrifugally or electrophoretically pure form ; the claims of its crystallization (Scott and Fisher, 1942) have been questioned (Keilin and Mann, 1944). The molecular weight of carbonic anhydrase from bovine erythrocytes is approximately 30,000 (Eirich and Rideal, 1940; Smith, 1940; Petermann and Hakala, 1942). Zinc was first identified as a constituent of bovine erythrocyte carbonic anhydrase (Keilin and Mann, 1939, 194O), and this was quickly confirmed (Leiner and Leiner, 1940; Hove et al., 1940). Plant carbonic anhydrase also contains zinc (Day and Franklin, 1946). The exact molar stoichiometry of zinc in carbonic anhydrase is not known, possibly because adequately purified samples have not been analyzed simultaneously for zinc, extraneous metals, and protein dispersity. Based on a molecular weight of 30,000, the reported zinc contents of bovine erythrocyte carbonic anhydrase vary from 0.92 and 1.04 (Scott and Mendive, 1941) to 1.52 (Keilin and Mann, 1940) and 1.46 (Hove et al., 1940) gram atoms per mole. The zinc contents of carbonic anhydrase from erythrocytes of the ox, sheep, and man may differ (Keilin and Mann, 1940). Although the manner of zinc-protein binding is obscure, the bond is firm, as shown by electrodialysis (Scott and Mendive, 1941) and exchange

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experiments with Zns5 (Tupper et al., 1951) ; ZnS5 ions did not exchange with enzyme zinc over a period of 32 days. This enzyme is inhibited by a number of agents known to form complexes with metal ions, and which are thus thought to inhibit by combining with zinc: cyanide, sulfide, and azide (Meldrum and Roughton, 1933), 2,3-dimercaptopropanol (Webb and Van Heyningen, 1947) and thiocyanate (Davenport, 1939). The inhibition by sulfide is disputed (Leiner and Leiner, 1940; Van Goor, 1948). Cyanide is a potent reversible inhibitor, 85% inactivation occurring at 4 X M (Keilin and Mann, 1940). The inactivations caused by these complexing agents indicate that zinc may be an active enzymatic site (Keilin and Mann, 1940) ; no data either on the kinetics of these inhibitions or on the effects of chelating agents have been published. No information is extant to indicate that the inhibitory action of the sulfonamides (Keilin and Mann, 1940) is exerted through the zinc atoms of the enzyme. 2. Bovine Pancre.as Carboxypeptidae. The carboxypeptidase first crystallized in 1937 (Anson, 1937) and purified from bovine pancreatic juice (Green and Neurath, 1954) has a molecular weight of 34,300 (Putnam and Neurath, 1946). Recent quantitative spectrographic analyses and chemical measurements demonstrate that this carboxypeptidase is a zinc metalloenzyme (Vallee and Neurath, 1954, 1955 ; Vallee, 1955). Each enzyme molecule contains 1 atom of zinc, and all other metals detected, including magnesium, are present in stoichiometrically insignificant amounts. The active role of zinc in the carboxypeptidase molecule is illustrated by the concomitant rise of the zinc: protein (equation 1 ) and the activity : protein (equation 2 ) ratios in the course of enzyme purification (Vallee and Neurath, 1955). Other extraneous metals decrease in concentration as zinc and specific activity increase (equation 4 ) . With the first crystallization, the zinc content becomes 0.98 gram atom per mole of carboxypeptidase ; repeated crystallizations do not change this ratio, but there is an increase of specific activity, concomitant with the further removal of extraneous metals. The zinc atom is firmly bound to the protein of carboxypeptidase, as is indeed the case with all the known zinc metalloenzymes. Prolonged dialysis against water or ammoniacal solutions does not alter the zinc content of the crystalline enzyme, but 1,lO-phenanthroline removes zinc. On the other hand, some zinc not firmly associated with the enzyme is removed during the purification from pancreatic juice ; this zinc may derive from contamination introduced in reagents, water, or glassware, or may represent other zinc-containing organic moieties, or ionic zinc in pancreatic juice.

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Dialysis at p H below 5.5, or against 1,lO-phenanthroline, removes zinc with concomitant loss of activity (Vallee and Neurath, 1954). Restoration of one gram atom of zinc per mole of enzyme fully restores activity (Vallee et al., 1 9 5 8 ~ ) . Carboxypeptidase is inhibited by a number of agents which can form complexes with metals. The apparent inhibitions by sulfide, cyanide, citrate, oxalate, pyrophosphate, and cysteine were taken to corroborate earlier conclusions that carboxypeptidase was a metalloenzyme (Smith and Hanson, 1948, 1949). Some of these substances (pyrophosphate, oxalate, citrate) did not, however, affect initial rates of hydrolyses (Neurath and DeMaria, 1950). On the other hand, metal-chelating agents such as 1,lO-phenanthroline, a,a’-dipyridyl, and others (Vallee and Neurath, 1955), which form strong complexes with zinc ions, inhibit initial rates completely. These agents do not inhibit when first exposed to equimolar amounts of zinc, cupric, or ferrous ions, indicating that their chelating sites are the effective ones in inhibition. Addition of zinc ions to carboxypeptidase already inhibited by 1,lo-phenanthroline restores enzymatic activity, demonstrating the reversibility of this reaction. The sulfonamides, which inhibit carbonic anhydrase strongly, are without effect on carboxypeptidase. 3. Pyridine Nucleotide-Dependent Zinc Metallodehydrogelulses. a. Yeast and equine liver alcohol dehydrogenuses. An alcohol dehydrogenase was crystallized from brewer’s yeast in 1937 (Negelein and Wulff) and is usually now prepared from baker’s yeast (Racker, 1950). The enzyme, prepared according to Racker, has a molecular weight of 150,000 and binds 3.6 diphosphopyridine nucleotide ( D P N ) molecules (Hayes and Velick, 1954) ; it usually contains an inactive component comprising up to 20% of the mass of the crystalline enzyme. A preliminary report (Keleti, 1956) indicates that the enzyme prepared from baker’s yeast and that from brewer’s yeast are not identical. Although they have certain properties in common, their isoelectric points and ultraviolet extinction coefficients differ, suggesting differences in their structure and composition. Immunological studies ( Antoni and Keleti, 1957) further indicate a dissimilarity of these enzymes ; both enzymes completely absorb the homologous as well as the heterologous rabbit antiserum, but the quantitative interactions are not identical. They appear to be zinc metalloenzymes, both being inhibited by chelating agents, but their zinc contents are not reported (Keleti, 1956). This raises questions concerning the identity of the enzymes which different investigators have studied. The DPN-dependent alcohol dehydrogenase crystallized from horse liver (Bonnichsen and Wassen, 1948; Bonnichsen and Brink, 1955) differs in

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many respects from the yeast enzyme, although it catalyzes the same reaction. Liver alcohol dehydrogenase is less specific enzymatically than is yeast alcohol dehydrogenase, the former also oxidizing vitamin A alcohol (Bliss, 1949, 1951) and glycerol (Holzer and Schneider, 1955). This liver alcohol dehydrogenase has a molecular weight of 84,000 (Ehrenberg, 1957) and reacts with two D P N molecules at neutral p H values (Theorell and Bonnichsen, 1951) . The crystalline yeast alcohol dehydrogenase, prepared from baker’s yeast according to Racker, contains four atoms of zinc per molecule (Vallee and Hoch, 1955a, b) as measured by spectrographic and chemical analyses. We have termed this zinc, which is enzymatically active, intrinsic. The identification of zinc in this enzyme followed the principles implied by equations 1 to 4. The enzyme was isolated from yeast according to now standard methods, and the activity, protein, and all metals were measured in all fractions. The concomitant rise in activity and zinc was evident as purification progressed from the crude material to the crystals. Simultaneously all other metals initially present are removed, R1, Rz,and Rs become constant, and R4 approaches 0. These relationships are observed reproducibly and are maintained in preparations which are homogeneous by electrophoresis and ultracentrifugation. The early studies with yeast alcohol dehydrogenase ( Y A D H ) led to the prediction, based on equations 5 and 6, that liver alcohol dehydrogenase ( L A D H ) would be found to contain two atoms of zinc per molecule (Vallee and Hoch, 1955a, b). This was rapidly confirmed qualitatively (Theorell et al., 1955) and quantitatively (Vallee and Hoch, 1956, 1957). Examination of liver enzyme according to the principles of equations 1 to 4 demonstrated that zinc is firmly bound, reaching a constant value of 2 gram atoms per mole of enzyme. The concomitant rise of zinc and activity during purification, the accompanying elimination of extraneous metals therewith, and the equality of zinc and D P N stoichiometry (Theore11 and Bonnichsen, 1951) all confirm that liver alcohol dehydrogenase, like yeast alcohol dehydrogenase, is a zinc metalloenzyme (Vallee and Hoch, 1957). Occasionally, yeast or liver enzyme preparations are obtained which, although homogeneous by physical-chemical criteria, contain zinc in excess of 4 or 2 gram atoms per mole, respectively. W e have termed this excess zinc extrinsic to the enzyme, since this zinc is not involved in enzyme action, and the activity is generally lower in its presence. Zinc in excess of the four atoms in YADH-extrinsic zinc-can be removed by dialysis against 0.1 M phosphate buffer at p H 6, together, presumably, with other contaminating metals. Increases in enzymatic activity

TRACE ELEMENTS IN CELLULAR FUNCTION

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are effected by removal of these extraneous metals. Like yeast alcohol dehydrogenase, liver alcohol dehydrogenase loses intrinsic zinc and activity par; passu on dialysis at p H values below 5.5 (Vallee and Hoch, unpublished data). The critical sensitivity of the enzyme to pH values below 6 had been observed in the preparation of the enzyme (Bonnichsen and Brink, 1955). Zinc is firmly incorporated into the yeast alcohol dehydrogenase molecule, as attested to by the constancy of R1 and Rs. The correspondence of the zinc content of the enzyme and the numbers of moles of D P N bound is in conformity with equations 5 and 6. Recent studies have demonstrated not only the firm incorporation but also the failure of such intrinsic zinc to exchange with adventitious, extrinsic zinc, and experiments with ZnS5 have differentiated between them (Vallee et al., 1958b). The incorporation of Zna5 by biosynthesis permits the unequivocal differentiation of the intrinsic, enzymatically active zinc atoms of [ (YADH)Zn4] from those present adventitiously. It also allows the precise determination of the number of intrinsic gram atoms of zinc per mole of YADH apoenzyme in its state in nature, contrary to other assumptions which have been made (Wallenfels et al., 1957). The measurement of protein, activity, zinc, and Zna5 allows the measurement of the numbers of gram atoms of zinc per mole of protein, which we have previously found to be 4, on the basis of spectrographic and microchemical measurements. I n addition, radioactivity measurements can be used now. By employing all these, the numbers of gram atoms can be calculated accurately by using the averages of different purified fractions : the values are between 3.96 and 4.06. These data seem to confirm the previous spectrographic findings, The presence of zinc in yeast alcohol dehydrogenase has been confirmed. It was reported that the enzyme contains five atoms of zinc (Wallenfels et ul., 1957), binds five diphosphopyridine nucleotide molecules (Wallenfels and Sund, 1957a, b), and can utilize added zinc ions to form up to thirty-five active enzymatic sites (Wallenfels et al., 1957). It is possible that the differences observed may be characteristic of the enzymes from brewer’s and baker’s yeasts (Keleti, 1956). I t should be pointed out, however, that the protein purity of these preparations is not stated. The average number of gram atoms of zinc per mole of alcohol dehydrogenase was 5.9 in the first, 5.22 in the second, and 4.7 in the third crystals. Fourth crystals were not reported. The number 5 was apparently deduced from the average of the second and third crystallizations, but, since the zinc content was falling with progressive crystallization, further

358

BERT L. VALLEE A N D FREDERIC L. H O C H

crystallizations might well have lowered the zinc content further to reconcile it with the 4 gram atoms previously and subsequently reported. The isotope experiments cited, as well as further experiments in our laboratory, have failed to confirm the suggestion (Wallenfels et al., 1957) that added extrinsic zinc up to 35 gram atoms becomes enzymatically active. An inhibition of enzymatic activity is observed when compared to the control activity, in conformity with previous observations (Vallee and Hoch, 1955b). The differences in the characteristics of Y A D H preparations isolated in different laboratories may be related to current findings in our laboratory. Yeast alcohol dehydrogenase is an unstable molecule. Under certain conditions, an active fraction with a turnover number of over 45,000can be isolated from the crystalline material. 1,lO-phenanthroline can split this enzyme into smaller components (Vallee, 1958). The apparent disparities discussed here may be resolved through studies of the inhomogeneity of enzyme preparations. Exposure of yeast alcohol dehydrogenase to p H levels below 6 also results in rapid loss of activity; the loss of activity is directly proportional to the removal of intrinsic zinc from the enzyme. Thus, on dialysis, 50% of the intrinsic zinc and the activity are lost at p H 5 , and all zinc and activity at p H 4.5. The crucial operational differences between metalloenzymes and metalenzyme complexes are emphasized by the fact that efforts to restore activity to this zinc-depleted enzyme by additions of zinc ions alone, together with coenzyme, substrates, or sulfhydryl-containing compounds, or by variations of p H or anions have thus far been unsuccessful (Vallee and Hoch, unpublished data). Sulfhydryl groups, apparently involved in the enzymatic action of YADH, may maintain the structural integrity of the protein, or possibly may bind the zinc atoms (Hoch and Zotos, 1957; Hoch and Vallee, 1958b). Whatever the details of the molecular configuration and stoichiometry may be, all findings reported so far are consistent with an active role for the zinc atoms in the mechanism of action of yeast alcohol dehydrogenase. This enzyme can therefore be employed to good advantage to explore further the detailed chemical structure of the zinc site and its effect on activity. Studies with metal-binding inhibitors further demonstrate the enzymatic role of zinc. A large number of chelating and complexing agents capable of combining with zinc inhibit the enzyme under various conditions (Vallee and Hoch, 1955a, b ; Hoch and Vallee 1956a). The presumable mode of interaction of such agents is generalized in equations 7 and 8. The features of the inhibition by one of these chelating agents, 1,lO-phenanthroline,

I

TRACE ELEMENTS I N CELLULAR FUNCTION

3 59

have been studied intensively in an effort to elucidate the role of the zinc atoms in yeast and liver alcohol dehydrogenase (Hoch and Vallee, 1956a, b ; Vallee and Hoch, 1956, 1957; Hoch et d.,1958b; Williams et al., 1958). In the yeast enzyme, two types of interaction between enzyme zinc and 1,lO-phenanthroline can be distinguished, both leading to characteristic inactivations (Williams et ul., 1958; Hoch et al., 1958b). The first is an instantaneous reversible combination between each zinc atom on the enzyme and one 1,lO-phenanthroline ( O P ) molecule (Hoch et at., 1958b) ; YADH.Zn

+ O P pKI Y A D H - Z n - O P

(9) The formula YADH .Zn represents each zinc atom of [ (YADH) Znr] as an independent active site. The existence of this reaction is substantiated by spectrophotometric studies which characterize the inactive enzyme-zincinhibitor complex, YADH-ZneOP (Vallee et al., 1957a, 1958a) and is of the type shown in equation 7. The inhibition proceeding through this interaction is competitive between 1,lO-phenanthroline and diphosphopyridine nucleotide or reduced diphosphopyridine nucleotide, but noncompetitive between 1,lo-phenanthroline and ethanol or acetaldehyde. This is consistent with the following mechanism for yeast alcohol dehydrogenase action :

+ D P N p YADH-ZneDPN YADHaZneDPN + C 2 H 5 0 H & YADHaZn-DPNH + CHsCHO + H +

YADH.Zn

YADH-Zn-DPNHP YADH-Zn

+ DPNH

(10) (11) (12)

Zinc is here depicted as a site for binding of D P N or D P N H to form the active enzyme-coenzyme complex. The stoichiometric correspondence between the four atoms of zinc and the four molecules of coenzyme known to be capable of binding to yeast alcohol dehydrogenase, together with the observation that D P N and D P N H are attached at the same enzymatic site (Hayes and Velick, 1954), strengthens this conclusion. The evidence from the OP-inhibition kinetics indicates that the substrates, ethanol and acetaldehyde, do not bind to yeast alcohol dehydrogenase at its zinc atoms to form a ternary complex, although such a complex at this site has been postulated (Wallenfels and Sund, 1957b; Mahler and Douglas, 1957). This instantaneous interaction, however, is not the only type of reactivity of yeast alcohol dehydrogenase zinc atoms toward 1,lO-phenanthroline. If the enzyme is exposed to 1,lO-phenanthroline, and samples of this mixture

360

BERT L. VALLEE AND FREDERIC L. HOCH

are assayed for activity after standing for various times, a progressive inhibition occurs in addition to the immediate one (Vallee and Hoch, 1955b; Hoch and Vallee, 1956b). The kinetics of this inhibition at pH 7.5 in 0.1 M phosphate buffer at 0.2”C are consistent with the further binding of a second molecule of 1,lO-phenanthroline to each zinc atom in the inactive complex Y A D H - Z n - O P (Williams et al., 1958) : k YADH-Zn-OP O P + YADH.Zn-OP2 (13)

+

This interaction is irreversible in an enzymatic sense and proceeds at a pseudo-first-order rate dependent on the velocity constant, k. The irreversibility of equation 13 denotes that the “undissociable” YADH -Zn OP2 complex represents a pool of irretrievably inactivated enzyme derived from the dissociable YADH.Zn.OP complex. With time, the size of this inactivated pool of enzyme increases, more dissociable complex is immediately formed in accord with equation 9, and the net result is a slow loss of free, active enzyme. It is hypothesized that the zinc atoms become slowly available to combine with the additional 1,lO-phenanthroline molecule through irreversible changes in the protein. This slow type of inhibition is observed with many other metal-binding agents and yeast alcohol dehydrogenase and other pyridine nucleotidedependent metallodehydrogenases (Vallee and Hoch, 1955b ; Vallee et al., 1956a) and may have its counterpart in the large number of enzymatic inhibitions reported to depend on preincubations. The details of the enzymatic role of zinc in yeast alcohol dehydrogenase and the structure of this enzymatic site are being pursued through such studies. The active role of the zinc atoms of liver alcohol dehydrogenase is also demonstrated by the inhibiting effects of metal-binding agents (Vallee and Hoch, 1957), but a difference exists in the interaction of 1,lO-phenanthroline with [ (LADH)Zn2] and with [ (YADH) Zn4]. Whereas the yeast alcohol dehydrogenase exhibits both the immediate and the timedependent inhibition with 1,lO-phenanthroline, liver alcohol dehydrogenase exhibits, under identical conditions, only the immediate reversible inhibition (Vallee and Hoch, 1957). Either dilution of the inhibited enzyme or addition of zinc or cupric ions can reverse the OP-inhibition of liver alcohol dehydrogenase ; preincubation of liver alcohol dehydrogenase and 1,lO-phenanthroline produces no further time-dependent inactivation at 23°C. Thus the zinc atoms of [ (LADH)Zn2] react differently from those of [ (YADH) Zn4]. The kinetics of the immediate l,10-phenanthroline inhibition of liver alcohol dehydrogenase are similar in some respects to those with yeast alcohol dehydrogenase. One molecule of 1,lO-phenanthroline reacts with

TRACE ELEMENTS I N CELLULAR FUNCTION

361

each zinc atom to produce the inactivation through the formation of an LADH Zn OP complex (Vallee et al., 1957a, 1958a). Both D P N and D P N H compete with 1,lO-phenanthroline (Vallee et ul., in preparation), indicating that the coenzyme is bound at or near the zinc atoms, as in yeast alcohol dehydrogenase, equations 9 and 12. Ethanol and acetaldehyde both deviate, however, from pure noncompetition with 1,lO-phenanthroline, so that the reaction shown for yeast alcohol dehydrogenase in equation 11 may not be followed by liver alcohol dehydrogenase. This difference may be related to the ability of liver alcohol dehydrogenase to reduce D P N without the addition of ethanol (Kaplan and Ciotti, 1954). It is important to point out that such examination of activity with a metal-binding agent only illuminates the participation of zinc in catalysis but does not permit any conclusions concerning that of other groups, such as sulfhydryls, for instance. 4,7-Dihydroxy-l,lO-phenanthroline,although inhibiting enzymatic activity, does not alter the number of titratable -SH groups, thus eliminating a dual locus of action of this compound as an explanation of the inhibitory phenomena, and further affirming the specificity of the inhibition for zinc (Hoch and Zotos, 1957; Hoch and Vallee, 195813). Nor do these studies bear on the manner in which zinc plays its role in the molecular events of enzymatic dehydrogenation. Although preliminary p H titrations indicated a complex between D P N and zinc ions in solution (Vallee and Hoch, 1955b), these observations and subsequent reports (Wallenfels and Sund, 1957c) are not borne out by additional experiments with Z ~ I ~ ~ ( O in Hour ) ~laboratory, showing the absence of complexation (Vallee et ul., 1956a). Similar evidence has been obtained independently (Kaye, 1955). b. Bovine liver glutamic d e h y d r o g e w e . The DPN-dependent glutamic dehydrogenase crystallized after purification from beef liver (Olson and Anfinsen, 1952) differs from the other enzymes of this group in some important respects. The zinc metallodehydrogenases described in this section and other enzymes depending on pyridine nucleotides all act on primary or secondary alcoholic groups, whereas the glutamic dehydrogenase is the only known enzyme of this group which catalyzes the reversible oxidative deamination of an amino acid. Having a molecular weight of 1,OOO,OOO, this glutamic dehydrogenase is much larger a molecule than the other pyridine nucleotide-dependent dehydrogenases that have been studied. The glutamic dehydrogenase of beef liver is a zinc metallodehydrogenase (Vallee et al., 1955 ; Adelstein and Vallee, 1956, 1958). All crystalline preparations contain zinc in stoichiometrically significant amounts and do not consistently contain significant amounts of other metals. The average

362

BERT L. VALLEE A N D FREDERIC L. HOCH

zinc content of eight different crystalline preparations was 3.4 1.0 atoms per molecule. This is a rather high coefficient of variation, 30%, which derives both directly and indirectly from the large molecular weight of the protein. Until the resolution of this difficulty, the exact number of gram atoms of zinc in bovine glutamic dehydrogenase cannot be specified better than stating that there are between 2 and 4 atoms per molecule. A recent report (Ito, 1957) confirms the metalloenzyme nature of the glutamic dehydrogenases isolated from human, pig, and ox livers. They are said to contain 5 to 6 gram atoms of zinc per mole, which is removed by dialysis at p H 4.5. Because of the low zinc : protein mass ratio of glutamic dehydrogenase, and the relatively large amounts of other zinc metalloproteins in liver, the correlation between zinc content and glutamic dehydrogenase activity is not striking until successive crystallizations are performed. At that time, the greatest elimination of other metals occurs, and a concomitant rise of zinc content and activity is observed. The zinc metalloenzyme nature of glutamic dehydrogenase is further shown by the inhibiting effects of metal-binding agents. 1,lO-Phenanthroline inhibition of this enzyme, as with the yeast and liver alcohol dehydrogenases, is competitive with DPN, indicating that the coenzyme may be attached to glutamic dehydrogenase at or near its zinc atoms (Vallee et ol., 1955 ; Ito, 1957). The large molecule of bovine liver glutamic dehydrogenase is depolymerized in the presence of 1,lO-phenanthroline or D P N H , forming four enzyme subunits (Frieden, 1958a, b) . This further indicates the importance of zinc in the binding of DPNH, and possibly in maintaining the structure of the large glutamic dehydrogenase monomer. c. Rabbit skeletal muscle lactic dehydrogenase. The redox interconversion of lactic and pyruvic acids is catalyzed by a DPN-dependent enzyme isolated and crystallized from rabbit skeletal muscle (Beisenherz et al., 1953). This enzyme has not yet been completely characterized, and its molecular weight is not known. The crystalline enzyme is not homogeneous, showing at least three components of different mass on ultracentrifugation (unpublished data). The heterogeneity of the lactic dehydrogenases isolated from the same organ of an animal, from different organs of the same animal, and from different animals has been demonstrated recently (Wieland and Pfleiderer, 1957). There is zinc in all analyzed preparations of the crystalline rabbit muscle lactic dehydrogenase or, perhaps more properly, dehydrogenases (Vallee and Wacker, 1956; Vallee et al., 1956a), the average being 637 pg. of zinc per gram of protein. These preparations have all contained metals other

TRACE ELEMENTS I N CELLULAR F U N C T I O N

363

than zinc as well, but in very variable amounts. With purification, the zinc content alone rises with increasing activity ; the concentration of the other metals does not follow any consistent course. The inhomogeneity of the enzyme and the existence of many different lactic dehydrogenases in one organ, however, make it difficult to interpret the variable zinc content. Like the zinc metallodehydrogenases discussed above, this enzyme is inhibited either immediately or after preincubation with a number of metalbinding reagents ; some of these inhibitions can be prevented and reversed by the addition of Zn++ ions. The inhibition by 1,lO-phenanthroline is competitive with DPN, indicating that this coenzyme is bound at or near a zinc atom. Apparently, zinc plays a functional role in the activity of skeletal muscle lactic dehydrogenase. Skeletal muscle lactic dehydrogenase thus resembles yeast alcohol dehydrogenase, liver alcohol dehydrogenase, and glutamic dehydrogenase in all major respects. The final establishment of the molar stoichiometry of zinc awaits further purification of this enzyme. d . Other pyridine nucleotide-dependent metallodehydrogelwlses. The hypothesis that a metal is a component of many, if not all, of the pyridine nucleotide-dependent dehydrogenases is a natural extension of the studies on yeast alcohol dehydrogenase, liver alcohol dehydrogenase, glutamic dehydrogenase, and skeletal muscle lactic dehydrogenase discussed in Section 111. B. 3. Absolute proof of the hypothesis awaits their examination when the remaining enzymes have been obtained in completely pure form, with respect both to metals and to protein, a not inconsiderable task. The hypothesis is strengthened, however, if further consistencies are found, and it can be discarded if major inconsistencies appear. The presence of metals in highly active, though not necessarily pure, enzyme preparations, together with the demonstration of inhibition by metalbinding agents, would validate this suggestion. These considerations apply equally to completely purified and to partially purified proteins, and it follows that the metalloprotein nature of the pyridine nucleotide-dependent dehydrogenases can be examined by investigation of readily available enzyme preparations. Accordingly, a series of active but not completely purified dehydrogenases were examined. Zinc, copper, and iron were the only metals found (Vallee et al., 1958a) consistently in significant concentrations in the glyceraldehyde-3-phosphatedehydrogenase of yeast (Cori et ul., 1948) and rabbit muscle (Beisenherz et al., 1953) ; the a-glycerophosphate dehydrogenase of rabbit muscle (Beisenherz et al., 1953) ; the malic dehydrogenase of pig heart (Straub, 1942) ; and the glucose-6-phosphate dehydrogenase of yeast, a TPN-dependent enzyme (Kornberg, 1950). These

364

BERT L. VALLEE A N D FREDERIC L. HOCIT

three elements all form strong complexes with 1,lO-phenanthroline, 8-hydroxyquinoline, and sodium diethyldithiocarbamate, and these reagents all inhibit these enzymes under appropriate conditions (Vallee et al., 1956a). Magnesium, calcium, barium, and aluminum were also present in all enzyme preparations, but these form complexes only with 8-hydroxyquinoline. Thus, the inhibitions observed are best explained by an interaction of the complexing agents with zinc, copper, or iron. Neither a metal-free active pyridine nucleotide-dependent enzyme was found, nor a pyridine nucleotide-dependent enzyme that was not inhibited under the appropriate constellation of experimental conditions. A recent report (Langer and Engel, 1958) shows that human placental estradiol-l7fl-dehydrogenase,a DPN- or TPN-dependent enzyme, is inhibited by 1,lO-phenanthroline, 8-hydroxyquinoline, and S-hydroxyquinoline-5-sulfonic acid. Activity of this enzyme is increased up to 34% by low concentrations of Zn++. No metal contents were reported, but this dehydrogenase was not obtained in a homogeneous form. These findings are consistent with the hypothesis of metallodehydrogenases. In addition to these enzymes, it has been reported that the DPNdependent formic dehydrogenase of peas is inhibited by azide, cyanide, and 8-hydroxyquinoline (Davidson, 1951) , and the DPN-dependent isocitric dehydrogenase of yeast by azide and cyanide (Kornberg and Pricer, 1951). The inhibitions of these and other dehydrogenases with cyanide and semicarbazide are ambiguous because of the presumed interaction of these reagents with either the pyridine nucleotide or a substrate. Interpretation of these findings must now include a possibility that these inhibitors may combine with a functional metal atom of the apoenzyme. I n the absence of stated conditions of exposure of enzyme to inhibitor, reported absences of inhibitions of dehydrogenases with metal-binding agents cannot be interpreted.* The inhibition studies with purified and less pure zinc metallodehydrogenases indicate that zinc may serve as an organizer of reversible attachments between apoenzyme and coenzyme, a role for which it seems well suited by virtue of the stability of its valence state and its complexes. The detection of zinc in the dehydrogenases joins these enzymes with others of the respiratory metalloenzyme group : hemes (Lemberg and Legge, 1949), metalloflavoproteins (Mahler, 1956), and copper oxidases (Kubowitz, 1937; Keilin and Mann, 1938; Dawson, 1950; Nelson, 1950; Mal-

* Alkaline phosphatase of swine kidney has recently been shown to be a zinc enzyme (Mathies, 1958). Zinc content and activity are linearly related as purification progresses. The best preparation contained 0.15% of firmly bound zinc, but exhibited three components by ultracentrifugal analysis, so that no molecular stoichiometry is yet established. The enzyme is inhibited by Versene, KCN, a,a’-dipyridyl.

TRACE ELEMENTS I N CELLULAR FUNCTION

365

lette, 1950), emphasizing the salient role of metals in oxidative catalysis. Apparently, these oxidative enzymes all capitalize on the properties of metals : iron, copper, zinc, molybdenum. The process of photosynthesis involves magnesium as a component of chlorophyll. The phosphorylative pathways, common to both oxidation and photosynthesis, which transform energy to biologically utilizable forms also seem to involve metal ions. The maintenance of biological structure against the second law of thermodynamics thus does seem to be founded on an inorganic core. It might be well to point out that the participation of a metal in coenzyme-dependent dehydrogenation reactions was completely unsuspected. The discovery of zinc enzymes may have been delayed for SO long because zinc proteins, as well as zinc salts, have no visible color, whereas the red iron and the blue copper enzymes attract attention. The identification of zinc metalloenzymes obtained from mammalian livers, and involved in alcohol oxidation and in ammonia metabolism, has led to the study of zinc metabolism in a human disease involving the liver and both of these functional loci: post-alcoholic hepatic cirrhosis (Vallee et al., 1956b, 1957b). A disturbance in zinc metabolism was found in patients with this disease consisting of abnormally low serum and liver zinc concentrations, and abnormally high urinary excretion of the metal. It may be suspected that the cellular and enzymatic function of zinc may hold significant clues to the solution of this problem of pathology. Schwarz and Foltz (1957) have found, by chemical analysis, that 1 part in 10 million of selenium prevents dietary necrosis of the liver in rats, defining an essential role for this element in physiology. The relation of this discovery to the findings on zinc is uncertain, but the unexpected demonstration that two elements heretofore considered to be accidental contaminants are of significance in pathology is at least impressive, and would seem to promise more surprises.

C.

Cadmium Protein from Equine Kidney Cortex

The discussion of zinc metalloenzymes illustrates systems in which a metal and an enzymatic activity are studied jointly. The operational concept of a metalloprotein further allows a “transperiodic” approach to the function of metals in biology : metals with similar atomic structure might also be predicted to be capable of associating with proteins or enzymes functionally. Traces of many metals have been found in various biological systems, although these metals are not presently known to have any distinct or precisely defined relationship to specific biological events. If hemovanadin, the vanadium-containing protein of ascidians, is included, it is apparent that metalloproteins and metalloenzymes incorporate transi-

366

BERT L. VALLEE AND FREDERIC L. HOCH

tion elements of atomic numbers 23 to 30. Macromolecules containing the elements 24 (chromium), 27 (cobalt), and 28 (nickel) have not been identified so far, although cobalt, of course, is found in vitamin BIZ. The similarity of their atomic structure to that of the other transition elements has suggested the possibility that they will be found in metalloproteins or other organometallic compounds of biological importance (Vallee, 1954). The similarity between the atomic structure and the chemical behavior of cadmium and zinc suggests that cadmium might also occur as an integral part of a natural substance. Such a complex has never previously been demonstrated, although cadmium has been found in the organs of various species (Maliuga, 1941 ; Klein and Wichmann, 1945 ; Voinar, 1952). Colorimetric analyses of the kidneys of different animals led to the choice of the cortex of the horse kidney for fractionation, because of its exceptionally high cadmium content (Table I ) . Successive fractionations of TABLE I CADMIUM IN MAMMALIAN KIDNEYS@ (Micrograms per gram wet weight) Species Horse Man Cattle Lamb Dog @

Cortex

Medulla

94 36 0.6 3.1

1.7 12 0.2 0.6

0.4

0.1

From Kagi and Vallee (unpublished data, 1958).

horse kidney cortex with ethanol and ammonium sulfate result in products containing 20 to 25 mg. of cadmium per gram dry weight of trichloroacetic acid-precipitable material ( Margoshes and Vallee, 1957). More recently fractions containing up to 43 mg. of cadmium per gram of protein have been obtained (Kagi and Vallee, unpublished data, 1958). Ultracentrifugation in a synthetic boundary cell shows the final products of seven successive fractionations to be monodisperse, with sedimentation constants (uncorrected for viscosity and diffusion) varying from 1.10 to 1.98 Svedbergs (Kagi and Vallee, unpublished data, 1958). Electrophoresis of the final material of a preparation in acetate, tris, and veronal buffers, p0.05,reveals one component at p H 4.5,two at p H 5.5, and three at p H 6.5 and 8.6. While the main component, forming 70% of the total material at p H 6.5 and 8.6,moves toward the anode, the two minor ones move toward the cathode. At lower pH, cathodic migration of all components is observed. Cadmium is bound to the main and one of the minor components, both giving a positive ninhydrin reaction on paper. Staining with dithizone specifically for zinc further indicates that

TRACE ELEMENTS I N CELLULAR FUNCTION

367

zinc is also bound to the main component (Kagi and Vallee, unpublished data, 1958). The fractions were analyzed colorinietrically and by emission spectrography. The cadmium content rises throughout the purification, a thirtyfold increase from the first extract to the last fraction. Cadmium is not removed by dialysis at p H 7 but is removed by treatment with hot trichloroacetic acid. With the exception of zinc, the other metals, present initially and introduced during fractionation, are removed and are low in concentration in the final material (Table 11). Isomorphism cannot be excluded as an explanation of the substantial, although lesser, increase of zinc content as fractionation proceeds, nor can the possibility be ruled out that cadmium is associated with one, and zinc with another, of the three electrophoretically separable fractions. The product contains 14% nitrogen, measured on the material precipitated by trichloroacetic acid. It reacts positively with biuret and ninhydrin. Hydrolysis and paper chromatography show serine, glycine, aspartic, and glutamic acids, among other amino acids not identified. The last fraction contains about 1% of hexosamine. A carbazole test for uronides is negative (Margoshes and Vallee, 1957). Titration with AgN03 (Benesch et d.,1955) and p-CMB (Boyer, 1954) reveals a high content of reactive sulfhydryl groups (Kagi and Vallee, unpublished data, 1958). The ultraviolet absorption spectra of purified preparations at p H 7 or p H 12 do not exhibit maxima, but distinct shoulders at 290 and 282.5 mp are present (Kagi and Vallee, unpublished data, 1958), indicating a low content of aromatic groups. Absorption bands have not been found in the visible region. The infrared spectrum of a potassium bromide pellet of the lyophilized product closely resembles those obtained for several proteins. The low sedimentation constant and the high metal content of this material indicate a protein of low molecular weight, probably containing a small number of cadmium atoms per molecule (Margoshes and Vallee, 1957). Thus far, no biological activity has been ascribed to this protein. The remarkable aggregation of cadmium would lead to the inference that it has a significant biological function. At present, however, it remains a metalloprotein in search of a biological activity. IV.

METAL-ENZYME COMPLEXES

A . Definitions The foregoing discussion of metalloenzymes has focused on but one aspect of the problem of the function of metals in the cell. Metals are not always firmly incorporated into proteins, and not all enzymes involving a

TABLE I1 EMISSION SPECTROGRAPHIC AND COLORIMETRIC ANALYSES OF HORSE KIDNEYCORTEX FRACTIONS (Cadmium was determined by colorimetry (Color.) and, along with all other metals, spectrographically (Spec.). Protein was measured by dry weight of material precipitated by trichloroacetic acid. Data are expressed as micrograms per gram wet weight of cortex, and as micrograms per gram of protein for the fractions.) Margoshes and Vallee (1957) Preparation C Preparation E Cadmium (Color.) Fraction Cortex

82

Cadmium

Kagi and Vallee (unpublished data, 1958) Preparation C Cadmium Zinc Othermetals"

Zinc

Other metals"

( Spec.1

(Spec. ) 829

(Color.) (b)

(Spec.) 117

(Color.)

(Spec.)

83.7

137

91

(Spec.) 66

W

E+I r <

*

( Spec. 1 5,740

r r

3,630

E

(c)

(c)

* 3

11,900

5,560

4,900

2m

25,100

30,900

7,310

29,700

37,000

13,500

23,00O(f)

VII 24,500 22,400 24,200 5,880 2,470 43,200 Mg, Ca, Ba, Sr, Al, Fe, Mn, Fe, Cr, P b ; Na, K, Cu not determined. b Sample lost. c Step omitted in modified fractionation scheme. d Sample size insufficient. e Protein concentration too small to measure by trichloroacetic acid precipitation. f Contamination with Mg and Ca introduced with ammonium sulfate.

40,100

7,600

3,740

I I1

1,130 3,050

754 3,440

687 2,630

340 1,440

2,410 2,200

1,100

1,140

(C)

(C)

I11

17,100

8,900

9,730

3,600

4,910

16,100

V

(e)

17,100

(d)

(6)

45,2OO(f)

VI

(b)

21,600

4,910

15,400(1)

20,700

400

r 0 c1

w

TRACE ELEMENTS I N CELLULAR FUNCTION

369

metal in their action can be isolated with the metal in situ. There is also the large body of information, as yet incompletely synthesized, on the group of biological catalysts referred to and operationally defined as metalenzyme-complexes (Vallee, 1955). These are activated or inactivated when various metal ions are added to the apoenzyme in Vitro. Magnesium, for example, activates the numerous and important enzymes which split and transfer phosphate groups, among them the phosphatases and the many enzymes concerned in the reactions involving adenosine triphosphate ( A T P ) . Adenosine triphosphate is required in such diverse functions as muscle contraction ; protein, nucleic acid, fat, and coenzyme synthesis ; glucose utilization ; methyl group transfer ; sulfate, acetate, and formate activation ; and oxidative phosphorylation, to name but a few. By inference, therefore, the activating action of magnesium extends to all these functions. I n addition to thiamine pyrophosphate, magnesium is a cofactor in enzymatic decarboxylations. Certain peptidases also are said to require it for their activity. Because of its effects in so large a number of diverse enzyme systems, magnesium enzyme complexes will be discussed here briefly as examples of these systems. The subject of magnesium metabolism has been reviewed recently (Wacker and Vallee, 1958). Alkaline phosphatase from mammalian kidney was the first enzyme found to be activated by magnesium ions (Erdtman, 1927 ; Tauber, 1949). The alkaline phosphatases from red cells and bone, which have not been purified, are also activated by magnesium (Lehninger, 1950), although several other divalent metals may substitute for magnesium in bringing about activation (Jenner and Kay, 1931; Abul-Fad1 et al., 1949; AbulFad1 and King, 1949a, b ; Neuman, 1949). A phosphatase isolated from yeast, and homogeneous by physical and chemical criteria, is activated by magnesium and is inhibited competitively by calcium (Bailey and Webb, 1944; Tsuboi and Hudson, 1956). The alkaline phosphatases are inhibited by M beryllium ions. This inhibition is reversed by nickel, manganese, and cobalt, but not by magnesium (Dubois et ul., 1949; Klemperer et ul., 1949; Aldridge, 1950). Like many similar enzymes, the acid phosphatase from prostate is activated by magnesium and inhibited by fluoride (Ohlmyer, 1945 ; Sadasivan, 1952). Magnesium seems to activate all the enzymes which catalyze the transfer of phosphate from adenosine triphosphate to a phosphate receptor, or from a phosphorylated compound to adenosine diphosphate. When these systems are isolated, however, magnesium can usually be replaced by manganese ; the requirement is therefore not absolute. It is not known whether these findings are of physiological significance. A complete discussion of all the enzymes which comprise this group is beyond the scope of this paper.

370

BERT L. VALLEE A N D FREDERIC L. H O C H

Lardy has reviewed the subject in a comprehensive fashion, and the reader is referred to his paper for complete details (Lardy, 1951). The activating effect of magnesium in the hexokinase reaction was recognized in the earliest studies of this system (Lohmann, 1930). Examples of the most important magnesium-activated transphosphorylating enzymes include the purified hexokinases obtained from various sources (Colowick et al., 1947 ; Kunitz and McDonald, 1946), fructokinase (Cori and Slein, 1947), creatinine transphosphorylase (Boyer et al., 1942), phosphopyruvic transphosphorylase (Lehmann, 1935) , acetyltransphosphorylase (Kaplan and Lipmann, 1948), the enzyme forming T P N from D P N (Kornberg, 1950), the enzymatic phosphorylation of riboflavine (Kearney and Englard, 1951a, b), and the phosphorylation of flavine mononucleotide ( Schrecker and Kornberg, 1950). Other systems which utilize adenosine triphosphate as a substrate are also activated by magnesium : the formation of “active sulfate” (Hilz and Lipmann, 1955) : Mg+ + ATP sulfate AMP-sulfate pyrophosphate

+

-

+

and the synthesis of diphosphopyridine nucleotide from nicotinamide mononucleotide and adenosine triphosphate. Phosphoglucomutase, the enzyme which monitors the conversion of glucose-l-phosphate to glucosed-phosphate, is activated by magnesium (Cori et al., 1938; Jagannathan and Luck, 1949). Several of these enzymes require potassium as an additional cofactor. The entire case of the function of the metals, as defined on this basis, rests on the activity of an enzyme. Some activity is usually present without addition of a metal ions, but this activity is either increased or decreased on its addition. In general, it is thought that metal ions exert their effects on enzyme activity in these systems through the formation of easily dissociable catalytic complexes, either with the enzyme:

E

+

Me # E - M e E-Me S s E-Me-S

or with the substrate:

+

+ S e Me.S + Me-S p E-MeaS

Me E

(15)

The final result in both cases is the active enzyme-substrate complex, E s M e - S , and for this reason the mechanisms of action shown in equations 14 and 15 often cannot be distinguished from each other by measurements of activity alone. Since such observations are made in vitro, and since

TRACE ELEMENTS I N CELLULAR F U N C T I O N

37 1

the metal is not attached firmly enough to the protein to allow their joint isolation in their natural state, it is difficult, on the basis of present knowledge, to ascertain whether or not these in vitro findings have significance for events as they occur in the cell. The interpretation of data on metal-enzyme complexes is further complicated by the fact that different metal ions often substitute for one another in bringing about enzymatic activation. Some have felt that physiological significance should be attributed to that ion giving the highest activity. There is little evidence to justify this presumption, although it may yet be proved to be correct in some cases. Others have felt that most importance should be entailed to the activating metal which is most abundant in the cell (Edlbacher and Baur, 1938), and the latter type of interpretation has been prevalent in spite of the fact that adequate data on abundance have not been available. The complexity of the problem is emphasized by the fact that neither physical nor chemical parameters have been found which might permit the systematic arrangement of these loosely bound metals in a manner analogous to that proposed in chelate model systems, for instance. It is reasonable to assume that metals may interact with a large number of basic and acidic side chains of proteins which are suitable for the coordination of cations. In fact, they will compete with the simplest cation, H+, for these sites. I t is to be expected, therefore, that enzyme activity will readily be affected by such interactions ; since the H+ concentration markedly influences the activity, it does not seem reasonable to expect otherwise of the other cations. This subject has been reviewed thoroughly (Williams, 1953). Thus, the problem of the metal-enzyme complexes is a difficult one, and one not easily solved with existent approaches, although no value judgment is implied as to the importance or significance of these systems. These interactions of metal ions with enzyme systems are most easily followed by measurements of enzyme activity, and the degree of activation or inhibition is taken as a measure of the degree of interaction. Such studies are easily performed and are, perhaps because of this, widely practiced. The following investigations may serve as an example both of the type of data obtainable on metal-enzyme complexes and of the large proportion of complexity which underlies the deceptive simplicity of the data themselves.

B. Metal Ion-ATP Interactions Various experiments (Hoch and Vallee, 1955; Hoch et al., 1958a) designed to illuminate these problems point to the need for the continued correlation of composition with structure and activity.

372

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L.

VALLEE AND FREDERIC L. H O C H

The succinic dehydrogenase-cytochrome c reductase system of rat liver mitochondria was assayed by using triphenyltetrazolium as an electron acceptor (Kun and Abood, 1949; Brodie and Gots, 1951). The activity measurements are reproducible, with a 5% coefficient of variation. The addition of A T P increases this enzymatic activity. Activity is normally about 27 millimicromoles of formazan produced per hour per milligram dry weight of mitochondria. Concentrations of A T P between 5 X lo-* and 2 X 10-3M double this activity, and lesser or greater concentrations of A T P produce smaller degrees of activation (Hoch and Vallee, 1955). Since metal ions have usually been found to be active components of systems involving A T P , the effects of their addition to this system were assayed. The addition of either Mg++, M n + + , or N i + + chlorides to a system containing 5 X M A T P increases activity further. Increasing concentrations of metal ions produce increased activation, up to fourfold over the inactivated system. Maximal activation occurs at about 5 x 10-3M metal-that is, when metal ions are isomolar with the ATP. Over a range of A T P and metal concentrations, this relation of optimal activation at isomolar concentrations holds. These are the only three activating cations found; others, like Ca++, inhibit the effect of A T P (Hoch and Vallee, 1955). This is of some interest, because A T P is not known to be required in this redox system, and it might be acting at a site common to both the oxidative and the phosphorylating mitochondrial apparatuses. Since Mg+ + or Mn+ + are activators in many systems involving A T P (McElroy, 1953 ; Section IV. A ) , the maximum observed might reflect an interaction of A T P with the intrinsic metals of the mitochondria themselves. Manometric measurements reveal that A T P and Mg+ + produce little or no change in succinoxidase activity. The observed increase in succinic dehydrogenase activity confirms the results of the triphenyltetrazolium assay, and cytochrome oxidase activity is actually decreased. These observations seem to minimize the probability that the activity changes in the triphenyltetrazolium assay are due to mitochondrial structural alterations, and to demonstrate an effect of A T P and cations on an oxidative enzyme site. The interpretation of such data appears relatively direct : such maximal activations at isomolar metal and cofactor concentrations may be taken to indicate that the metal forms an active complex either with the enzyme or with ATP, as in equations 14 and 15. This interpretation takes into account the fact that mitochondria contain metals, but assumes that when Mg++, for instance, is added to the system it acts only additively with the Mg+ + contained in mitochondria.

THACE ELEMENTS I N CELLULAR FUNCTION

373

Spectrographic measurements demonstrate, however, that mitochondria contain significant concentrations of metals (Table 111). These data were recalculated for molar concentrations from the analytical values obtained by Thiers and Vallee (1957). Magnesium and calcium are present, respectively, in the concentrations of 3 x mole per liter of and 8 x mitochondria, concentrations similar to those activating or inhibiting the system when added. Manganese is present at 3 X 10-6M, and iron, zinc, and copper are also found in comparable amounts. Since these are significant concentrations, it may be justly questioned whether the single added cations actually do act independently of other metals already present. TABLE I11 METALSIN MITOCHONDRIA [Moles of metal (XlO4) per liter of mitochondria] Metal

Moles

Metal

Moles

Metal

Moles

Mg Mn Ca

30 0.3 8

Na K Fe

9 40 4

Zn cu

1 0.5

Therefore, activity was examined as a function of the concentration of more than one added metal ion, necessitating a factorial experimental design ; the functions chosen were f l and fz :

At each A T P concentration, the sum of the two metal ions added, M g + + and M n + + , for instance, was constrained in a set of definite ratios to the A T P concentration, the function fl. Within this sum, the proportion of Mg+ + to Mn+ + was also varied, the function f2. Activity was expressed as a resultant of these two functions. T o present such data, a three-dimensional plot is used, with fl and fz as the base lines, and activity as a surface at various heights above them. If the assumption that one metal acts independently of another is tenable, the surface should exhibit a tentlike appearance, with each cross section corresponding to a line of activation, i.e., with maximal activity at isomolarity. Figure 2 shows the results when the concentration of A T P is 1x M , and Mg+ + and M n + + are added as chlorides. The level of activity in the presence of A T P alone, without added metals, is considered to be the base line for metal activation. The surface above it represents activation, that below it, inhibition. When M n + + is present in concentrations ten times that of Mg concentration, maximal activity occurs, but at about one-half the concentration, rather than at the isomolar point between

374

BERT L. VALLEE A N D FREDERIC L. HOCH

Mn+ + and A T P (Hoch and Vallee, 1955). The experimental data therefore differ from those predicted on the basis of a single univariate theory. When Mg+ + is ten times the Mn+ + concentration, the maximal activation occurs between isomolarity and four times that value, again a different result from that occurring when only M g + + is added, and the peak occurs at equal A T P and metal concentrations. I n fact, when the total metals are high, the presence of more M n + + produces inhibition. These results would indicate that the effect of one of these cations is not independent of the presence of the other.

Mg

+ Mn

ATP FIG.2. Effects of ATP, M g + + , and Mn++ on the rate of reduction of triphenyltetrazolium by mitochondria. ATP concentration is 1 X lO-3M, and the activity (the height above the base line) in the presence of A T P alone is indicated by the shaded horizontal plane. Activity is shown as a surface and as a function of both the sum of the metal ion concentrations relative to ATP (fl, equation 16), and the ratio of Mg+ + to Mn+ + (f2), the receding horizontal abscissa.

When A T P concentration is lowered to 5 x 10-4M, there is significant activation over a very wide range of metaI concentrations and ratios. When M g + + is present in high proportions, there is activation at almost all metal concentrations. No inhibition occurs here. This differs markedly from the simple form of the activation curves when just one metal is varied and further indicates the cation effects are dependent on each other. Figure 3 shows the results when, instead of M g + + and Mn++, both of these being activating ions, Mg++ and Ca++ are varied, C a + + being an ion that inhibits. A T P concentration is 5 x 1 0 - 3 M . The inhibition of activity as a function of only calcium concentration is apparent, as is the previously shown isomolar maximal activation by Mg++. In the presence of mixtures of added Mg++ and Ca++, however, both the inhibition and the activation become lesser in degree, and they are spread over a wider range of total metal concentration. Here the two cations seem to antag-

TRACE ELEMENTS I N CELLULAR FUNCTION

375

onize each other’s action, and the resultant activity is very much a function of their interrelationship, not of their individual concentrations. These data do not fit the hypothesis that the changes of activity observed when a metal ion is added to an enzyme system bear no relation to the presence of other metal ions. The activity observed is a complex function of the metal and ligand concentrations, and different degrees of activation or inhibition can be obtained under appropriate conditions. The data thus caution against oversimplifications of interpretation of activation experiments in which only one or two variables are examined. It is clearly diffi-

FIG.3. Effect of ATP, Mg++, and Ca++ on the rate of reduction of triphenyltetrazolium by mitochondria. A T P concentration is 5 X 10-3 M. Representation is as in Fig. 2. cult to take a stand in any controversy concerning the functional consequences of metal ion additions in vitro unless the composition of the enzyme system under study is known. This applies not only to mitochondria, but to more purified systems, which can also be shown to contain metals. In view of the known multivariable influences on the function of biological systems, these conclusions need not be startling.

V. METALSIN SUBCELLULAR FRACTIONS A . Fractionations in Isotonic Sucrose Quantitative knowledge of metal content is necessary information for the interpretation of similar functional results with subcellular fractions. Such studies also demonstrate the association of metals with cell constituents, quite apart from functional considerations and the ultimate correlation with enzyme systems. Differential centrifugation to 0.25 M sucrose (Hogeboom et al., 1948)

376

BERT L. VALLEE A N D FREDERIC L. HOCH

has been employed together with emission spectroscopy (Thiers and Vallee, 1956, 1957) to determine the normal distribution of metals. Tissues were obtained from 25 normal rats and used in a statistically designed experiment. Connective tissue, nuclei (and whole-cell residue), mitochondria, microsomes, and clear supernatant were analyzed. All contained copper, zinc, manganese, iron, calcium, magnesium, sodium, and potassium. The metal contents of each fraction, expressed as per cent of total metal relative to per cent of total liver nitrogen, are shown in Fig. 4. The height of each bar represents the enrichment of a particular metal in a fraction relative to its concentration in the liver as a whole. The bar widths repre-

FIG.4. Content of Na, K, Mg, Ca, Zn, Fe, Mn, and Cu in the cellular fractions of rat liver, obtained by differential centrifugation in 0.25M sucrose. The height of each bar represents the metal content of each fraction, expressed as a percentage of the total content of that metal in liver divided by the same parameter for nitrogen. This normalization allows direct comparison between different fractions and metals. The width of each bar represents the relative nitrogen content of the fraction. sent relative amounts of nitrogen per fraction. The distribution patterns are precise, and they are characteristic for each metal and fraction. Repeated determinations on tissues from the same rat population over a period of two and a half years show no significant change. The concentrational functions and the graphical form of Fig. 4 are specifically chosen to allow all the data to be presented simultaneously, a significant feature of this approach, and of emission spectrography. An area of resolved information is thus produced, with which function may be correlated when such information becomes available. Several points worthy of further investigation appear in Fig. 4. In the connective tissue,

TRACE ELEMENTS I N CELLULAR FUNCTION

377

all the metals are low and significantly uniform, except for calcium. Calcium, magnesium, and manganese, the elements already implicated in mitochondrial metabolism, are at very high relative levels in the mitochondria. The supernatant fluid has a high zinc level, consistent with its high concentration of some zinc enzymes. I n all of the fractions; iron and manganese seem to vary inversely to each other. Actually, the sum of the heights of the iron and manganese bars is exactly two units, within the error of measurement, in all fractions except connective tissue.

B. Other Media and Dietary Varktions These patterns are stable, but they depend on the medium used for fractionation. The original method of Hogeboom et al. ( 1948), employing

FIG.5. Content of Na, Mg, Ca, Zn, Fe, Mn, and Cu in the cellular fractions of rat liver, obtained by differential centrifugation in 0.25 M sucrose and 0.1 M potassium ethylenediaminetetraacetate (Versene) . Representation is as in Fig. 4.

0.25 M sucrose, was used for the experiments in Section V. A. Figure 5 shows the pattern obtained when 0.01 M potassium ethylenediaminetetraacetate (Versene) is added to the medium (Thiers and Vallee, unpublished data, 1958). The differences observed due to the presence of Versene are not only those which would be predicted on the basis of solubilization of metals by chelation. For example, the relative calcium and zinc contents of connective tissue are elevated. Iron is high in the microsonies, copper in the nuclei and cell residue, and manganese and magnesium in the supernatant fluid. Most metals show markedly lowered relative levels in mitochondria. Thus Versene, which has been of interest to biochemists because

378

BERT L. VALLEE AND FREDERIC L. H O C H

of its ability to bind alkaline earths, actually affects the distribution patterns of all the metals except sodium, and possibly potassium. A dietary metal deficiency has also been found to affect the distribution patterns of metals (Thiers and Vallee, unpublished data, 1958). Rats fed a diet which produces low serum magnesium levels showed the character1957). Data were istic pathology of magnesium deficiency (Vitale et ,d., obtained from normal rats and from those fed such diets containing two different low magnesium levels. Changes in metal content are observed only in mitochondria and connective tissue, and there only in the concentrations of magnesium, calcium, and iron. Calcium and magnesium in connective tissue rise in order of decreasing serum magnesium level, a result consistent with pathological findings. I n mitochondria, calcium, magnesium, and iron levels all decrease markedly with increasing severity of the deficiency symptoms. Thus the diet produces changes which are selective in terms both of relative metal content and of cellular fraction affected. With increasing magnesium deficiency, the differences in magnesium content of the mitochondria are much greater than those in the serum, which is generally considered the barometer of body chemistry. In the average of all fractions (that is, the whole tissue), no trend in data is discernible, however, and all the concentrations are in the normal range, the numbers actually being 6.0, 5.5, and 6.6 pg. of magnesium per milligram of nitrogen. These data show clearly how multidirectional spreading of the information contained in the whole cell can disclose striking relationships which may not be obvious and may, indeed, be completely obscured, when whole tissues are analyzed directly, while they may be resolved into distinctly abnormal tissue components.

C . Effects of Carbon Tetrachloride The distribution patterns of metals also may change, owing to factors other than dietary metal intake. Findings in our laboratory (Thiers and Reynolds, 1958) indicate that the functional and anatomical changes in the livers of rats poisoned orally with carbon tetrachloride also include reproducible major changes in the metal contents of the cell fractions, which depend on the temporal progression of the metabolic lesion. Carbon tetrachloride, 0.25 ml. per 100 g., was administered to rats by stomach tube, and the animals were sacrificed sequentially. The metal content of the mitochondria specifically changes within a few hours, the calcium content rising rapidly and reaching a maximum of more than ten times the normal value in 16 to 20 hours, then falling again. The potassium

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379

content decreases concomitantly, but slightly later, to values about onetenth of the normal, and subsequently rises again. Mitochondria1 magnesium and sodium also show significant but lesser degrees of change, but the sum of these four major cations remains relatively constant. Figure 6 shows the changes in concentration of these metals 16 hours after administration of carbon tetrachloride. After 40 hours, the changes in calcium are no longer confined to the mitochondria. All fractions reflect the increase then.

FIG.6. Content of Na, K, Mg, and Ca in the mitochondria of rat liver 16 hours after administration of carbon tetrachloride. The ordinate represents the concentrations of these metals, in the mitochondria of treated rats, relative to those in untreated rats, on a logarithmic scale. Relative concentrations plotted above the line showing the normal levels represent increases, those below, decreases, after carbon tetrachloride feeding. The mitochondria1 metal changes appear to be accompanied by enzymatic alterations ; uncoupling of oxidative phosphorylation and decrease in the rate of oxidation of octanoic acid have been observed (Reynolds, Thiers, and Vallee, unpublished data, 1958). These findings offer an opportunity for the correlation of the composition of cellular components with functional changes brought about by a variety of agents. Such studies may provide some common chemical or composi-

380

BERT L. VALLEE AND FREDERIC L. HOCH

tional denominators for the understanding of the role of metals in cellular physiology. VI. SUMMARY The function of metals in living systems may be viewed as a problem of general biological importance, affecting the interpretation of phenomena in all phyla and species. Although many of the implications of the presence of metals have been recognized through specific problems in different fields of biological specialization, few investigations have approached the function of metals in cellular physiology as one problem, starting with the known chemical characteristics of metal ions. This latter approach rests heavily, on the one hand, on the qualitative and quantitative memurement of the metals present in a system and, on the other hand, on the judicious choice of the biological system to be analyzed, so that the metal measurements can be interpreted in terms of function and strwture. The experimental approach exemplified in this review, for instance, places emphasis on examination of highly purified enzyme systems, or of the organized cellular organelles derived from certain species susceptible to nutritional manipulation. Thus, a horizontal and vertical examination of the comparative biochemistry of metals has produced some answers as to the function of metals that cut across the conventional boundaries between the specific experimental subjects. In this manner, many of the pyridine nucleotide-dependent dehydrogenases have been shown to be metalloenzymes. This generalization apparently transcends marked species differences. The identification of the alcohol dehydrogenases as zinc enzymes exemplifies this. Nutritional experiments with Neurosporu crassu by Nason et al. (1951, 1953) suggested that zinc participates in the dehydrogenation of ethanol. The metal was identified as a functional component in the purified crystals of the alcohol dehydrogenase of baker’s yeast (Vallee and Hoch, 1955a, b) and the prediction of the presence and, indeed, of the stoichiometry, of zinc in the horse liver alcohol dehydrogenase (Vallee and Hoch, 1955b) was borne out by experiment (Theorell et al., 1955; Vallee and Hoch, 1956, 1957). Thus, by intellectual extrapolation, the observation was extended from unicellular organisms to the vertebrates, and zinc was shown to play similar roles in this basic metabolic process-similar, but not identical, since the enzymes in the vertebrates have apparently become modified through evolution, and the zinc atoms there show subtle differences in their reactivity (Vallee et &., 1957b). Such studies on metalloenzymes reveal directly a function of a metal in a tissue, and reduce phenomenological observations to a common chemical denominator. The function of the metal is represented by the metabolic

TRACE ELEMENTS I N CELLULAR FUNCTION

38 1

significance of the catalyzed chemical reaction. Thus, the known high zinc content of the vertebrate retina (Leiner and Leiner, 1940) becomes functionally meaningful when it is correlated with the presence of vitamin A1 alcohol dehydrogenase (Bliss, 1951), which is similar to the liver alcohol dehydrogenase (Bliss, 1949; Theorell and Bonnichsen, 1951). The identification of zinc in retina, and its measurement, are necessary but not sufficient criteria of the importance and biological role of zinc in that organ. The specific role of zinc can be defined by cmrebted analytical, functional, and structural studies, and the results on isolated biochemical components can serve as a valid basis for the assessment of the function of elements in Vivo. The investigations of hepatic cirrhosis (Vallee et d.,1956b, 1957b) translate such biochemical mechanisms to the pathological physiology of man. This approach has just begun to bear fruit. The bases for the “transphylar” comparisons of one metal have been mainly ones of similar biochemical or biological function. An equally promising area is that of comparison of metal ions with similar chemical and physical reactivity: a “trans-periodic” approach. This has resulted in the first identification of cadmium as a component of a naturally occurring protein, and further studies in this area are indicated. Such “inorganic” biochemistry serves to reduce biological complexity to common chemical denominators, allowing a measure of predictability in even complex systems. All these studies have benefited greatly, both experimentally and intellectually, from the development of emission spectrography as an analytical device for biological work. In fact, this technique has shown itself to be of unexpectedly high value in protein characterization. By continued and joint study of composition, structure, and function, much may be expected in the exploration of the role of metals in cellular physiology. VII.

REFERENCES

Abul-Fadl, M. A. M., and King, E. J. (1949a) Biochem. J . 44, 431. Abul-Fadl, M. A. M., and King, E. J. (1949b) Biochem. J. 44, 435. Abul-Fadl, M. A. M., King, E. J., Roche, J., and Thoai, N. V. (1949) Biochem. J. 44, 428. Adelstein, S. J., and Vallee, B. L. (1956) Federation Proc. 15, 505. Adelstein, S. J., and Vallee, B. L. (1958) J . Biol. Chem. in press. Aldridge, W. M. (1950) Nature 166, 772. Ahrens, L. H. ( 1950) “Spectrochemical Analysis.” Addison-Wesley, Cambridge, Massachusetts. Anson, M. L. (1937) J . Gen. Physiol. a0, 663. Antoni, F., and Keleti, T. (1957) Nature 179, 1020. Bailey, K., and Webb, E. C. (1944) Biochem. J . 38, 394. Beisenherz, G., Boltze, H. J., Biicher, T., Czok, R., Garbade, K. H., Meyer-Arendt, E., and Pfleiderer, G. (1953) 2. Naturforsch. 8b, 555.

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BERT L. VALLEE A N D FREDERIC L. HOCH

Benesch, R. E., Lardy, H. A., and Benesch, R. (1955) I. Biol. Chem. 216, 663. Bliss, A. F. (1949) Biol. Bull. 97, 221. Bliss, A. F. (1951) Arch. Biochem. 31, 197. Bonnichsen, R. K., and Brink, N. G. (1955) Biochem. Preparations 4, 29. Bonnichsen, R. K., and Wassen, A. M. (1948) Arch. Biochem. 18, 361. Boyer, P. D. (1954) J. Am. Chem. SOC.76, 4331. Boyer, P. D., Lardy, H. A., and Phillips, P. H. (1942) I. Biol. Chem. 146, 673. Bridgman, P. W. (1946) “The Logic of Modern Physics.” Macmillan, New York. Brinkman, R., Margaria, R., Meldrum, N. U., and Roughton, F. J. W. (1932) I . Physiol. (London) 75, 3. Brodie, A. F., and Gots, J. S. (1951) Science 114, 40. Colowick, S. P., Cori, G. T., and Slein, M. W. (1947) I. Biol. Chem. 168, 583. Cori, G. T., and Slein, M. W. (1947) Federation Proc. 6, 245. Cori, G. T., Colowick, S. P., and Cori, C. F. (1938) I. Biol. Chem. 124, 543. Cori, G. T., Slein, N. W., and Cori, C. F. (1948) J. Biol. Chem. 173, 605. Davenport, H. W. (1939) J. Physiol. (London) 97, 32. Davidson, D. C. (1951) Biochem. J. 49, 520. Dawson, C. R. (1950) In “Copper Metabolism” (W. D. McElroy and B. Glass, eds.), p. 18. Johns Hopkins Press, Baltimore, Maryland. Day, R., and Franklin, J. (1946) Science 104, 363. Delahay, P. (1954) “New Instrumental Methods in Electrochemistry.” Interscience, New York. Dubois, K. P., Cochran, K. W., and Mazur, M. (1949) Science 110, 420. Edlbacher, S., and Baur, H. (1938) 2. physiol. Chem. 265, 275. Ehrenberg, A. (1957) Acta Chem. Scand. 11, 1257. Eirich, F. R., and Rideal, E. K. (1940) Nature 146, 541. Erdtman, H. (1927) 2. physiol. Chem. 172, 182. Frieden, C. (1958a) Biochim. et Biophys. Acta 27, 431. Frieden, C. (195813) Federation Proc. 17, 223. Green, N. M., and Neurath, H. (1954) In “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. 2, Part B, Chapter 25. Academic Press, New York. Hall, C. (1953) “Introduction to Electron Microscopy.” McGraw-Hill, New York. Harrison, G. R., Lord, R. C., and Loofbourow, J. R. (1948) “Practical Spectroscopy.” Prentice-Hall, New York. Hayes, J. E., Jr., and Velick, S. F. (1954) I . Biol. Chem. 207, 2 5 . Hilz, H., and Lipmann, F. (1955) Proc. Natl. Acad. Sci. US. 41, 885. Hoch, F. L., and Vallee, B. L. (1949) J . Biol. Chem. 181, 295. Hoch, F. L., and Vallee, B. L. (1952) J . Biol. Chem. 195, 531. Hoch, F. L., and Vallee, B. L. (1955) Nature 176, 256. Hoch, F. L., and Vallee, B. L. (1956a) Federation Proc. 16, 93. Hoch, F. L., and Vallee, B. L. (1956b) J . Biol. Chem. 221, 491. Hoch, F. L., and Vallee, B. L. (1958a) In “Trace Elements” (C. A. Lamb, 0. Bentley, and J. M. Beattie, eds.), p. 337. Academic Press, New York. Hoch, F. L., and Vallee, B. L. (1958b) In “Symposium on Sulfur in Proteins” (R. Benesch, ed.). Academic Press, New York, in press. Hoch, F. L., and Zotos, B. (1957) Federation Proc. 16, 359. Hoch, F. L., Tyler, H. R., and Vallee, B. L. (1958a) Federation Proc. 17, 243. Hoch, F. L., Williams, R. J. P., and Vallee, B. L. (1958b) J . Biol. Chem. a32, 453.

TRACE ELEMENTS IN CELLULAR FUNCTION

383

(1948) J . Biol. Chem. 172, 619. Holzer, H., and Schneider, S. (1955) Angew. Chem. 67, 276. Hove, E., Elvehjem, C. A., and Hart, E. B. (1940) J. Biol. Chem. 136, 425. Ito, K. (1957) Osaka Daigaku Zgaku Zasshi 9, 1059 [Chem. Abstr. 62, 4726b ( 1958)1. Jaggannathan, V., and Luck, J. M. (1949) J . Biol. Chem. 179, 561. Jenner, H. O., and Kay, R. (1931) J . Biol. Chem. 93, 733. Kaplan, N. O., and Ciotti, M. M. (1954) J. Biol. Chem. 221, 431. Kaplan, N. O., and Lipmann, F. (1948) J . Biol. Chem. 176, 459. Kaye, M. A. G. (1955) Biochim. et Biophys. Acta 18, 456. Kearney, E. B., and Englard, S. (1951a) Arch. Biochem. Biophys. 32, 223. Kearney, E. B., and Englard, S. (1951b) Federation Proc. 10, 205. Keilin, D., and Mann, T. (1938) Proc. Roy. SOC.Bl%, 187. Keilin, D., and Mann, T. (1939) Nature 144, 442. Keilin, D., and Mann, T. (1940) Biochem. J. 34, 1163. Keilin, D.,and Mann, T. (1944) Nature 189, 107. Keleti, T. (1956) Acta Physiol. Acad. Sci. Hung. 9, 415. Klein, A. K., and Wichmann, H. J. (1945) J. Assoc. Off. Agr. Chemists 28, 257. Klemperer, F. W., Miller, G. H., and Hill, C. (1949) J. Biol. Chem. 180, 281. Kolthoff, I. M., and Lingane, J. J. (1952) “Polarography,” Vols. 1 and 2. InterHogeboom, G. H., Schneider, W. C., and Palade, G. E.

science, New York. Kornberg, A. (1950) J . Biol. Chem. la,805. Kornberg, A., and Pricer, W. E., Jr. (1951) J . Biol. Chem. 189, 123. Kubowitz, F. (1937) Biochem. 2. aSa, 221. Kun, E., and Abood, L. G. (1949) Science 109, 144. Kunitz, M., and McDonald, M. R. (1946) J . Gen. Physiol. 29, 393. Langer, L. J., and Engel, L. L. (1958) J. Biol. Chem. 2SS, 583. Lardy, H. A. (1951) In “Phosphorus Metabolism” (W. D. McElroy and B. Glass, eds.), Vol. I, p. 477. Johns Hopkins Press, Baltimore, Maryland. Lederer, E., and Lederer, M. (1953) “Chromatography, A Review of Principles and Applications.” Elsevier, New York. Lehmann, H. (1935) Biochem. 2. 281, 271. Lehninger, A. L. (1950) Physiol. Revs. 30, 393. Leiner, M., and Leiner, G. (1940) Biol. Zentr. 60, 449. Lemberg, R., and Legge, J. W. (1949) “Hematin Compounds and Bile Pigments.” Interscience, New York. Lingane, J. J. (1953) “Electroanalytical Chemistry.” Interscience, New York. Lohmann, K. (1930) Natumiss. 19, 180. McElroy, W. D. (1953) In “Symposium on Nutrition” (R. M. Herriott, ed.), p. 262. Johns Hopkins Press, Baltimore, Maryland. Mahler, H. R. (1956) Advances in Enzymol. 17, 233. Mahler, H. R., and Douglas, J. (1957) J . A m . Chem. SOC.79, 1159. Maliuga, D. P. (1941) Compt. rend. acad. sci. U.R.S.S.31, 145. Mallette, M. F. (1950) In “Copper Metabolism” (W. D. McElroy and B. Glass, eds.), p. 48. Johns Hopkins Press, Baltimore, Maryland. Malmstrom, B. G. (1956) “The Mechanism of Metal-Ion Activation of Enzymes. Studies on Enolase.” Almqvist and Wiksells Boktryckeri AB, Uppsala. Margoshes, M., and Vallee, B. L. (1956) Zn “Methods of Biochemical Analysis” (D. Glick, ed.), p. 353. Interscience, New York.

384

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Margoshes, M., and Vallee, B. L. (1957) J . Am. Chem. SOC.79, 4813. Meldrum, N. U., and Roughton, F. J. W. (1932a) J . Physiol. (London) 76, 3 p. Meldrum, N. U., and Roughton, F. J. W. (1932b) J . Physiol. (London) 76, 15 p. Meldrum, N. U., and Roughton, F. J. W. (1933) J . Physiol. (London) 80, 113. Mellon, M. G., ed. ( 1950) “Analytical Absorption Spectroscopy.” Wiley, New York. Muller, 0. ( 1951) “The Polarographic Method of Analysis.” Chemical Education Publishing Co., Easton, Pennsylvania. Nachtrieb, N. (1950) “Principles and Practice of Spectrochemical Analysis.” McGraw-Hill, New York. Nason, A., Kaplan, N. O., and Colowick, S. P. (1951) 1. Biol. Chem. 188, 397. Nason, A., Kaplan, N. O., and Oldewurtel, H. A. (1953) J . Biol. Chem. 201, 435. Negelein, E., and Wulff, H.-J. (1937) Biochem. 2. 293, 351. Nelson, J. M. (1950) In “Copper Metabolism” (W. D. McElroy and B. Glass, eds.) , p. 76. Johns Hopkins Press, Baltimore, Maryland. Neuman, H. (1949) Biochim. et Biophys. Acta S, 117. Neurath, H., and DeMaria, G. (1950) J. Biol. Chem. 188, 653. Ogden, C. K., and Richards, I. A. (1947) “The Meaning of Meaning.” Harcourt, Brace, New York. Ohlmeyer, P. (1945) 2. physiol. Chem. 383, 1. Olson, J. A., and Anfinsen, C. B. (1952) J . Biol. Chem. 197, 67. Petermann, M. N., and Hakala, N. V. (1942) I. Biol. Chem. 146, 701. Putnam, F. W., and Neurath, H. (1946) J . Biol. Chem. 166, 603. Racker, E. (1950) J . Biol. Chem. 184, 313. Rickes, E. L., Brink, N. G., Koniuszy, F. R., Wood, T. R., and Folkers, K. (1948) Science 108, 134. Sadasivan, V. (1952) Nature 170, 421. Samuelson, 0. (1953) “Ion Exchangers in Analytical Chemistry.” Wiley, New York. Sandell, E. B. (1950) “Colorimetric Determination of Traces of Metals,” 2nd ed. Interscience, New York. Schrecker, A. W., and Kornberg, A. (1950) J. Biol. Chem. 182, 295. Schwarz, K., and Foltz, C. M. (1957) J. Am. Chem. SOC.79, 3292. Scott, D. A., and Fisher, A. M. (1942) 1. Biol. Chem. 144, 371. Scott, D. A., and Mendive, J. R. (1941) J . Biol. Chem. 140, 445. Seminova, D. P. (1946) Compt. rend. mad. sci. U.K.S.S. 51, 683. Smith, E. C. B. (1940) Biochem. I . 34, 1176. Smith, E. Lester (1948) Nature 162, 144. Smith, E. L., and Hanson, H. T. (1948) J . Biol. Chem. 176, 997. Smith, E. L., and Hanson, H. T. (1949) I. Biol. Chem. 179, 803 Smith, 0. C. (1953) “Inorganic Chromatography.” Van Nostrand, New York. Snell, F., and Snell, C. T. f1949) “Colorimetric Methods of Analysis,” Vols. 1 and 2. Van Nostrand, New York. Straub, F. B. (1942) 2. physiol. Chem. a16, 63. Tauber, H. (1949) “Chemistry and Technology of Enzymes.” Wiley, New York. Theorell, H., and Bonnichsen, R. (1951) Acta Chem. Scand. 5, 1105. Theorell, H., Nygaard, A. P., and Bonnichsen, R. (1955) Acta Chem. Scond. 9, 1148. Thiers, R. E., and Reynolds, E. S. (1958) Federation Proc. 17, 537.

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Thiers, R. E., and Vallee, B. L. (1956) Federation Proc. 16, 371. Thiers, R. E., and Vallee, B. L. (1957) J . Biol. Chem. B6, 911. Tobias, C. A., and Dunn, R. A. (1949) Science 109, 109. Tsuboi, K. K., and Hudson, P. B. (1956) Arch. Biochem. Biophys. 61, 197. Tupper, R., Watts, A., and Wormall, R. W. E. (1951) Biochem. J . 60, 429. Underwood, E. J. (1956) “Trace Elements in Human and Animal Nutrition.” Academic Press, New York. Vallee, B. L. (1951) Sci. Monthly 62, 368. Vallee, B. L. (1952) Nutrition Revs. 10, 65. Vallee, B. L. (1954) In “Principles of Internal Medicine” (T. Harrison, ed.), p. 437. Blakiston, New York. Vallee, B. L. (1955) Advances in Proteifi Chem. 10, 317. Vallee, B. L. (1958) 4th Intern. Congr. Biochem., Vienna, in press. Vallee, B. L., and Gibson, J. G., 11. (1948) J . Biol. Chem. 176, 435. Vallee, B. L., and Hoch, F. L. (1955a) J . Am. Chem. SOC.77, 821. Vallee, B. L., and Hoch, F. L. (1955b) Proc. Natl. Acud. Sci. US.41, 327. Vallee, B. L., and Hoch, F. L. (1956) Federation Proc. 16, 619. Vallee, B. L., and Hoch, F. L. (1957) I. Biol. Chem. 226, 185. Vallee, B. L., and Neurath, H. (1954) J. Am. Chem. SOC.76, 5006. Vallee, B. L., and Neurath, H. (1955) J. Biol. Chem. 217, 253. Vallee, B. L., and Peattie, R. W. (1952) Anal. Chem. !&, 434. Vallee, B. L., and Wacker, W. E. C. (1956) I. A m . Chem. SOC.78, 1771. Vallee, B. L., Hoch, F. L., and Hughes, W. L., Jr. (1954) Arch. Biochem. Biophys. 48, 347. Vallee, B. L., Adelstein, S. J., and Olson, J. A. (1955) J . Am. Chem. SOC.77, 5196. Vallee, B. L., Hoch, F. L., Adelstein, S. J., and Wacker, W. E. C. (1956a) J. .4m. Chem. SOC.78, 5879. Vallee, B. L., Wacker, W. E. C., Bartholomay, A. F., and Robin, E. D. (1956b) N . Engl. J. Med. 266, 403. Vallee, B. L., Coombs, T. L., and Williams, R. J. P. (1957a) Federation Proc. 16, 264. Vallee, B. L., Wacker, W. E. C., Bartholomay, A. F., and Hoch, F. L. (1957b) New Engl. J . Med. 1 7 , 1055. Vallee, B. L., Coombs, T. L., and Williams, R. J. P. (1958a) J . Am. Chem. SOC. 80, 397. Vallee, B. L., Kagi, J. H. R., and Hoch, F. L. (1958b) Federation Proc. 17, 326. Vallee, B. L., Rupley, J., Coombs, T. L., and Neurath, H. (1958~) J . Am. Chem. SOC.80, 4750. Van Goor, H. (1948) Enzymologia 19, 73. Vitale, J. J., Nakamura, M., and Hegsted, D. M. (1957) J. Biol. Chem. aa8, 573. Voinar, A. 0. (1952) Trudy Konf. Mikroelement 1950, 580 (Akad. Nauk S.S.S.R., Translation R- J-296, of Associated Technical Services, East Orange, New Jersey). Wacker, W. E. C., and Vallee, B. L. (1958) New Engl. J . Med. !d69,431, 475. Wallenfels, K., and Sund, H. (1957a) Biochem. 2.339, 17. Wallenfels, K., and Sund, H. (1957b) Biochem. Z . 333, 59. Wallenfels, K., and Sund, H. (1957~) Biochem. Z . 333, 41. Wallenfels, K., Sund, H., Faessler, A., and Burchard, W. (1957) Biochem. 2.Sas, 31.

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Warburg, 0. (1949) “Heavy Metal Prosthetic Groups and Enzyme Action.” Oxford Univ. Press, New York. Webb, E. C., and Van Heyningen, R. (1947) Biochem. J . 41, 74. Weissberger, A., ed. (1949-1954) “Physical Methods of Organic Chemistry,” 2nd ed., Parts 1 and 2 (1949), Part 3 (1954). Interscience, New York. Wieland, T., and Pfleiderer, G. (1957) Biochem. 2. 329, 112. Williams, R. J. P. (1953) Biol. Revs. a8, 381. Williams, R. J. P., Hoch, F. L., and Vallee, B. L. (1958) J . Biol. Chem. B 2 , 465.

Osmotic Properties of Living Cells D . A . T. DICK Department of Hunznn Anatomy. Oxford University. Oxford. England1 Page

I. General Introduction .............................................. I1. Theory of Osmotic Pressure ....................................... A Introduction ................................................. B. Thermodynamic Derivation of Osmotic Pressure Laws ........ C. The Osmotic Coefficient ....................................... D . Units of Osmotic Pressure .................................... E. Summary ..................................................... I11. Osmotic Properties of Protein Solutions ........................... X . The Donnan Effect ........................................... B . Ion Binding by Proteins ...................................... C. Hydration of Proteins in Solution ............................. D . Entropy of Mixing of Protein Solutions ....................... E Summary .................................................... I V . The Relationship between Volume and Osmotic Pressure at Equilibrium in Living Cells ............................................ A . Theory ...................................................... B. Assumptions Underlying Theoretical Treatment ................. C. Techniques of Volume Measurement ............................ 1. Direct Measurement of Diameter of Spherical Cells .......... 2. Hematocrit Methods ...................................... 3 Measurement of Concentration of Nonpenetrating Solute ...... 4. Angular Diffraction of Light by Cell Suspensions (Halometry) 5 . Measurement of Conductivity of Cell Suspensions ............ 6 Measurement of Opacity of Cell Suspensions ................ 7. Measurement of Changes in the Solid and Water Contents of the Cell .................................................. D . Volume-Osmotic Pressure Relationships in Erythrocytes ........ E Volume-Osmotic Pressure Relationships in Cells Other than Erythrocytes ................................................. F. Osmotic Behavior of the Nucleus ............................... G. Conclusion ................................................... V . Kinetics of Osmotic Volume Changes in Living Cells ................. A Introduction .................................................. B. Theory of Osmotic Water Permeability ......................... C Methods of Measuring Osmotic Water Permeability ............. D Osmotic Permeability Coefficients of Cells ....................... E. Difference between Osmotic and Diffusion Methods of Measuring Water Permeability ........................................... F. The Rate of Diffusion of Water through the Cell Protoplasm .... G. Conclusion ................................................... VI . Acknowledgments ................................................. VII . References .......................................................

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388 388 388 389 392 393 395 395 396 398 399 399 403 404 404 407 413 414 414 415 415 416 416 417 418 422 426 427 427 427 428 431 432 436 438 442 443 443

1 Present address : Physiological Department, Carlsberg Laboratory, Copenhagen, Denmark .

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I. GENERAL INTRODUCTION The relation of the water content of cells to the osmotic pressure of the immersion medium has been much studied. Nevertheless the physicochemical principles underlying the osmotic properties of cells are still not well understood. It is the object of this review not so much to attempt a wide survey as to assemble information for the examination of some fundamental aspects of the subject from a limited point of view. Discussion has been confined to isolated cells maintained under normal or nearly normal physiological conditions. The review has further been limited to animal cells; the osmotic properties of plant cells are in general modified, except in hypertonic solutions, by their possession of a cell wall capable of withstanding considerable hydrostatic pressures. Discussion of the effect on the osmotic properties of the cell of various factors in the external environment-e.g., temperature, variation in ionic composition, damage to the cell membrane-has been omitted for reasons of space. The great advantages of a thermodynamic treatment of the osmotic properties of the cell have only recently been appreciated. Since so little is known with certainty of the structure and functioning of the cell membrane, it has seemed appropriate to avoid discussion of mechanism altogether and to take an empirical approach, using thermodynamic methods to do no more than reconcile the known properties of the cell with the properties of the cell membrane and the physicochemical properties of the cell constituents so far as these are known. OF OSMOTIC PRESSURE 11. THEORY

A . Introduction When a solution is separated from a quantity of the pure solvent by a membrane which is permeable to the solvent and not to the solute, then solvent tends to be drawn through the membrane into the solution so as to dilute it. The movement of solvent can be prevented by applying a certain hydrostatic pressure to the solution. This pressure is called the osmotic pressure. The osmotic pressure is thus defined in terms of certain experimental conditions ; it is an expression of a specific physical property of the solution, but it comes into existence only when the defined conditions are fulfilled. It does not refer to any particular theory of the mechanism of the phenomenon, such as the bombardment of solute or solvent molecules on the membrane. In the present state of knowledge discussion of the mechanism involved is in fact of no practical importance and is best avoided. It must be noted that the component whose movements are observed is the solvent and not the solute. I n the modern theoretical treatment

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of osmotic pressure, attention has therefore come to be directed to the solvent and not to the solute as in the classical treatment of van’t Hoff. Movement of solvent is described in terms of its chemical potential. The chemical potential is a thermodynamic quantity which is used to measure the tendency to flow. It is defined in such a way that a fluid always flows from a situation of higher to one of lower chemical potential; when no movement of a fluid takes place across a permeable membrane, then its chemical potential is the same on both sides and the system is said to be in equilibrium. The chemical potential of a solvent is commonly altered in two different ways (although other ways are possible) : by the admixture of a solute with the solvent, which lowers its chemical potential, and by the application of pressure to the solvent, which raises its chemical potential. Thus the flow of solvent across a semipermeable membrane (i.e., permeable only to the solvent) is associated with the lowering of the chemical potential of the solvent in the solution due to the presence of the solute ; and the flow is prevented by raising this chemical potential by the application of pressure until it is again equal to the chemical potential of solvent in the pure solvent, so that the system comes into equilibrium. The osmotic pressure may thus be accurately defined as the excess pressure which must be applied to a solution to bring it into equilibrium with the pure solvent when they are separated by a perfectly semipermeable membrane. Attention has been drawn by Hildebrand ( 1955), Babbitt ( 1955), and Chinard and Enns (1956) to the limitations of the original van’t Hoff law of osmotic pressure and to the great advances made by applying to osmotic pressure theory thermodynamic concepts such as the chemical potential in place of kinetic concepts based on analogy with the gas laws. The following is a simplified account of the main results of this approach. Thermodynamic treatments can be found in standard works on the subject (Glasstone, 1948 ; Butler, 1946 ; Guggenheim, 1950; Lewis and Randall, 1923). B. Thermodynamic Derivation of Osmotic Pressure Laws It has been seen that the chemical potential of a solvent can be altered in two principal ways, by addition of solute and by application of pressure. The mathematical relations expressing these facts are as follows. The relation between the change of chemical potential and the amount of solute added (if we assume that the solution is ideal, and that the temperature, pressure, and amount of solvent are kept constant) is given by the equation

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where p1 and plo are the chemical potentials of the solvent in the solution and in the pure solvent, respectively, and nl and n2 are the numbers of gram-molecules of solvent and solute in the solution. (The derivation of this and other equations used here is given in the works quoted above.) The quantity nl/(nl nz) is called the mole fraction and is a measure of concentration. Since its value is necessarily less than 1.0, its logarithm is negative, and thus pl is lower than pl0. The relation between the change of chemical potential and the applied pressure, when the temperature and composition of the solution are kept constant, is given by the equation

+

where P is the total applied pressure, and V1 is a quantity known as the partial molal volume of the solvent in the solution, which is approximately equal to the volume occupied by one gram-molecule of solvent in solution. The raising of the chemical potential of the solvent by increasing the applied pressure by an amount, TI, which equals the osmotic pressure, is obtained by integrating equation 2 thus :

If it is assumed that Yl remains constant with change of pressure, i.e., that the solvent is incompressible (this assumption is almost exactly true), then Apl

= (Po

+ TI)8,

- Po 81 = TIPI

(4)

Thus the increase of chemical potential is proportional to the increase of pressure. But since at osmotic equilibrium the reduction of the chemical potential of the solvent due to the presence of solute is exactly balanced by the increase of chemical potential produced by the osmotic pressure, then the net change of chemical potential of the solvent in the solution is zero ; the chemical potential of solvent in the solution is equal to that in the pure solvent. In mathematical terms, that is,

+ Apl

(due to pressure) = 0 On substituting equations 1 and 4 in equation 5, there results Apl (due to solute)

or

(5)

39 1

OSMOTIC PROPERTIES OF LIVING CELLS

This equation relates the osmotic pressure to the mole fraction of solvent in the solution. If it is assumed that nl is much greater than n2, i.e., that the solution is very dilute, the equation may be simplified as follows : Since

n2 (when nl

*

> n2)

then

or

But nlPl = V l , whic and thus

is the partial volume of solvent in the solution,

or

where m is the molal concentration of solvent in the solution (for definition, see below). Equations 9 and 10 are modified expressions of the Boyle-van’t Hoff law. By deriving them in this way and not as originally by analogy with the gas laws, which is now known to be invalid, it is seen first that the law is an approximation which is entirely dependent on the assumptions that the solution is both ideal and extremely dilute. (An ideal solution is one which obeys Raoult’s law.) Second, the quantity Vl in equation 9 is the volume of solvent in the solution and not the volume of the solution itself, Correspondingly m in equation 10 is a molal concentration [weight of solute/weight (* volume) of solvent] and not a molar concentration (weight of solute/volume of solution). This difference makes clear the significance of the quantity known as the “osmotically inactive volume” as it occurs in the simplified form of the Boyle-van? Hoff law conventionally applied to living cells :

II (Y- b ) = K ( a constant)

(11) where V is the total cell volume, and b is the “osmotically inactive volume.” I t can be seen that ( V - b ) corresponds to Vl in equation 9 and thus expresses the amount of solvent water in the cell ; b is merely that quantity which has to be subtracted from the total cell volume to obtain it and

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consists of the volume of the cell solute plus that of any solid in the cell which is not in solution. The term “osmotically inactive volume” as applied to this is meaningless and may even be misleading, since b includes the crystalloid fraction of the cell solute which is responsible for the major part of the intracellular osmotic pressure. The term “nonsolvent volume” has already been used for b, and its use is clearly preferable. It must be noted that b as used in the van der Waals gas equation is the co-volume, which is not the same as the actual volume of the gas molecules (it is approximately four times the volume of the molecules). The use of b in osmotic equations must therefore be clearly distinguished from the van der Waals b. Teorell (1952) has stated that b “clearly has nothing to do with the eigenvolume of the solutes.” This statement appears inconsistent with the thermodynamic derivation of the osmotic pressure law.

C . The Osmotic Coeficient

A further modification must be introduced into the Boyle-van’t Hoff law before it can be applied in practice. It has been seen that two basic assumptions underlie the law-first that the solution concerned is extremely dilute, and second that the solution shows ideal behavior. In order to overcome these limitations, a correction factor, the osmotic coefficient (+), is introduced into the equation so as to reconcile the predictions of theory with the behavior of real solutions. The modified forms of equations 9 and 10 are IIVl = + R T n 2 (12) and II = + R T m (13) As a consequence of this modification an assumption underlying the simple Boyle-van’t Hoff law of equation 11 is revealed. It is assumed that RT% = K ( a constant)

+

Here R is the gas constant; T, the absolute temperature, is usually kept constant during osmotic experiments ; n2 is constant, provided that there is no net passage of solute across the cell membrane during the experiment. The assumption of the constancy of here the average molal osmotic coefficient of the intracellular solute, is, however, not generally valid. In spite of the effects of electrostatic attraction between ions and of ionic hydration ( a good account of the treatment of these effects by means of the Debye-Huckel theory is given by Bull, 1951)’ the osmotic coefficients of the crystalloid fractions of the cell solute are indeed practically constant over the range of concentrations which occur in living cells (see Robinson and Stokes, 1955). But the osmotic coefficients of many

+,

OSMOTIC PROPERTIES OF LIVING CELLS

393

proteins have been shown to increase sharply with concentration, even when this is expressed in molal units (see Fig. 1 ) . This is partly due to the binding of water of solvation by protein molecules, thus diminishing the amount capable of acting as solvent to the protein molecules themselves or to other solutes. Ogston (1956) has, however, pointed out that water of solvation is in fact a purely theoretical concept used to interpret certain experimental results, and so the amount of its contribution to the observed osmotic coefficients remains uncertain. Another possible cause of the anomalous osmotic pressures of protein solutions is the large partial molar entropy of mixing of protein solutions (see Section 111.D). This has been explained and at least partly accounted for as a consequence of the great difference in the size of solute and solvent molecules in protein solutions. The osmotic properties of protein solutions and the theories used to account for them will be examined in greater detail in Section 111. Whatever the cause of the large osmotic coefficients of proteins, it is clear that they are likely to produce anomalies in the osmotic behavior of all cells which contain significant amounts of intracellular protein. One important result is that the contribution of the protein component to the total intracellular osmotic pressure may be unexpectedly high. For example, in the human erythrocyte, although the molar concentration of hemoglobin is only 0.005 (if we assume 33.5 g. of H b per 100 ml. of corpuscles and a molecular weight of 67,000), the corresponding molar osmotic coefficient is 3.55 (calculated from the data of Adair, 1929), so that the partial osmotic pressure of the hemoglobin in the cell is 17.5 m-osm. and not 5 m-osm. as expected. This is not an insignificant contribution to a total intracellular osmotic pressure of 300 m-osm. Second, when the concentration of the intracellular protein falls, as during osmotic swelling, the osmotic coefficient drops sharply. Thus the simple form of the Boylevan’t Hoff law will not be obeyed, since in the equation

n(V-b) = K K , which is equivalent to 4 RTn2, is no longer constant. The result is to make b appear to be larger than it actually is or, conversely, to make the apparent water content of the cell, as determined by measurements of osmotic equilibria, smaller than the water content determined by direct methods. D . Units of Osnzotk Pressure There is much confusion as to the use of the terms “osmole” and “milliosmole.” Milliosmoles have been frequently used merely as units of mass of solute; they have been defined as “the sum of the millimoles of undissociated solutes and the milliequivalents of dissociated solutes” (Deyrup, 1953) and used in concentration units such as milliosmoles per liter.

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D. A. T. DICK

2.

2.

I-

: z w I. Y

0.5

t

LIMITS OF CONCENTRATIONS PRESENT IN ERYTHROCYTE.

SOL%; I

0

I

I

0

I

CHLORID~qms{100ml. :[tolvent I

20

I

I

30 40 HAEMOCLOBIN qms/100mt.of solvent 10

I.

I 50

FIG.1. Variation of the molal osmotic coefficients of hemoglobin and sodium chloride with concentration. These must be carefully distinguished from molar osmotic coefficients (see Section 11. D ) . Dotted lines represent approximately the limits of concentration produced during osmotic swelling of the erythrocyte, sodium chloride being taken as typical ,in its osmotic behavior of the crystalloid fraction of the erythrocyte solute.

OSMOTIC PROPERTIES O F L IVI N G CELLS

395

Such a unit is tacitly assumed to express the osmotic pressure of the solution. This assumption is not correct, however, because no account is taken of the osmotic coefficients of the solutes. Since the osmotic coefficient relates a concentration to an osmotic pressure (see equation 13), two osmotic coefficients exist corresponding to the two methods of expressing concentration. If molal concentrations are used, then the molal osmotic coefficient must be applied to obtain the osmotic pressure; for molar concentrations the molar osmotic coefficient must be used. For dissociated electrolytes the osmotic coefficients must be applied to the total gram-ion concentration present. It is thus greatly preferable to use the milliosmole directly as a unit of osmotic pressure. One osmole is defined as the osmotic pressure of a 1.0 molal solution of an ideal nonelectrolyte, i.e., 22.4 atmospheres at 0°C. An osmolar solution so defined has a freezing-point depression of 1.86”C. The osmolarity of a real solution can thus be determined directly from freezing-point measurements, but to calculate it from the molality (or molarity) of the solute the appropriate molal (or molar) osmotic coefficient must be used. This unit has the advantage of being independent of the temperature, apart from very small variations due to the effect of temperature on the osmotic coefficient. The unit milliosmole, used in this review, is based on the above definition unless otherwise stated.

E. Summary An outline of the thermodynamic derivation of the laws of osmotic pressure is given. It is shown that the simple Boyle-van? Hoff law is based on a number of approximations which are not generally valid. The chief assumption is that the average osmotic coefficient of the intracellular solutes is constant. The osmotic coefficient of proteins in solution is usually large, however, and increases rapidly with concentration. This is due in part to the great difference in size of protein and water molecules and also to binding of water of solvation to the protein. In consequence, living cells which contain a significant amount of protein cannot be expected to obey the simple Boyle-van’t Hoff law. I t is seen that the so-called “osmotically inactive volume” is simply the volume of solute plus the volume of any solid in the cell which is not in solution. The unit “milliosmole” is used not as a unit of mass but as a unit of osmotic pressure, and a definition is given.

PROPERTIES OF PROTEIN SOLUTIONS 111. OSMOTIC Protein forms an important component of the solute in every living cell. It has been known since the classical work of Sgkensen (1917) that concentrated protein solutions show great departures from ideal

396

D. A. T. DICK

behavior. For this reason a discussion of the osmotic properties of protein solutions is essential for an understanding of the osmotic behavior of living cells. The nonideality of protein solutions is due to a variety of causes of which the most important are ( 1 ) the Donnan effect, (2) ion binding by the protein, ( 3 ) hydration, and (4) nonideal entropy of mixing.

A . The Donmn Eflect When proteins are not at their isoelectric point, they discharge or combine with protons to form charged ions, usually of multiple valency. Since the p H of the body fluids and of the cell interior probably lies in the region 6.5 to 7.5 (Caldwell, 1956) and thus on the alkaline side of the isoelectric point of most proteins, the latter normally exist in the body fluids as multivalent anions. If a membrane is impermeable to one species of ion such as these protein ions, then an unequal distribution of the remaining permeable ions is set up across the membrane of a type first described and accounted for on a thermodynamic basis by Donnan (191 1 ) . It has not been clearly appreciated, however, that there are two kinds of Donnan equilibrium in physiological systems: (1) with a difference in osmotic pressure across the membrane, and (2) without a difference of osmotic pressure across the membrane. The distinction between these two kinds depends on the nature of the ions to which the membrane remains permeable. It follows from the nature of water as an ionizing solvent that H + and OH- ions can effectively permeate any water-permeable membrane provided only that one other ion, either anion or cation, is also permeable, since changes in H+ and OH- concentrations can be produced by combination of the permeable ion with the solvent. Hydrogen and hydroxyl ions are, however, never present in sufficient concentration at physiological pH to affect the chemical potential of the solvent and hence the osmotic pressure. Osmotically significant Donnan distributions are therefore always due to the other permeable ions present. In the first type of Donnan equilibrium the membrane is impermeable to protein ions but the remaining permeable ions include both cations (in addition to the hydrogen ion) and anions (in addition to the hydroxyl ion). When the protein is, confined to one side of the membrane, there is a difference of osmotic pressure across the membrane partly due to the impermeable protein ions and partly due to the unequal distribution of the permeable ions. There is also a difference of electrical potential and of pH. Such a system can be maintained in equilibrium only if a hydrostatic pressure is applied to the solution containing the impermeable ion. Such conditions obtain in the capillary membrane as was demonstrated by the

OSMOTIC PROPERTIES O F L I V I N G CELLS

397

observations of Landis (1927), who confirmed the theory of Starling ( 18%). In the second type of Donnan equilibrium the membrane is effectively impermeable to protein and to either anions or cations. Thus the remaining permeable ions consist only of either anions or cations (in addition to hydrogen or hydroxyl ions), so that in order to preserve electrical neutrality no net passage of electrolytes across the membrane is permitted. Even if the protein is confined to one side of the membrane, provided there is initially some impermeable ion on both sides, an equilibrium can be attained in which there is no difference of osmotic pressure across the membrane. There is therefore no difference of hydrostatic pressure. Nevertheless a distribution of the permeable ions (anions or cations) takes place which obeys the Donnan rule. Thus with a membrane permeable only to anions and hydrogen and hydroxyl ions

[XI1 where

[x,],

[y;],

[ x c ] , [ Y c ] , and

and

[z;]

[ZB]

are the external concentrations;

are the internal concentrations of the an-

and [Hz] are the external and internal hydrogen ions; and [H:] ion concentrations. There is therefore a difference of p H and of electrical potential across the membrane. These conditions have been shown to obtain in several types of cells (see Table 11). Although fluxes of cations across the cell membrane can occur, efflux always equals influx, so that no net transfer of cations occurs ; i.e., there is a net or effective impermeability to cations and permeability only to water and anions (evidence reviewed by Caldwell, 1956; see particularly Van Slyke et al., 1923; also recent work by Swan et al., 1956). Indeed, as shown in the erythrocyte by Davson (1936), effective impermeability to electrolytes (caused by the effective impermeability of either cation or anion) is an essential condition for survival of all cells, since in its absence the unbalanced internal colloid osmotic pressure will cause swelling and disruption of the cell (see also Wilbrandt, 1948). In these circumstances the internal osmotic pressure of the cell may be considered as made up of the partial osmotic pressures due to the protein and electrolyte components separately (allowance has to be made, however, for the effects of ion binding by the protein), and no Donnan term appears in considering the effect of the protein, account being taken only

398

D. A. T. DICK

of the presence in the cell of an equivalent amount of ion of the opposite charge (gegenion) to maintain electrical neutrality. This applies particularly to the calculation of the osmotic coefficient of the cell protein. The osmotic coefficient used in this case is that derived from the partial osmotic pressure of the protein after calculating and subtracting the Donnan element from the total osmotic pressure observed in an osmometer which normally employs a collodion membrane, freely permeable to electrolytes. The Donnan effect as applied to biological phenomena has been reviewed by Henderson ( 1928), Bolam ( 1932), Overbeek ( 1956), and Harris (1956), and in some aspects affecting intracellular p H by Caldwell (1956).

B. Ion Binding by Proteins It is well known that proteins are capable of binding small ions, particularly organic anions and cations and transition metal and alkaline earth cations (for review see Klotz, 1953). These ions are not, however, present in sufficient concentration in biological fluids to make the binding effect of osmotic significance. I t is only recently that binding of any of the ions commonly present in high concentration, e.g., C1-, Na+, and K + , has been demonstrated to an extent sufficient to affect the total osmotic pressure of the protein-salt mixture. Evidence for the binding of C1- ions to bovine plasma albumin was first obtained during osmotic pressure studies by Scatchard et al. (1946). They concluded that each albumin molecule bound about six ions within a certain range of conditions-albumin concentration 1 to 676, to sodium chloride concentration 0.05 to 0.2 M , and p H 4.2 to 8.2. More recent estimates of the ion binding of isoionic serum albumin are those of Scatchard et al. (1950), who found 11 C1- ions bound per molecule of human serum albumin, and of Scatchard et al. (1957), who found an average of 9.16 C1- ions bound per molecule of bovine serum mercaptalbumin in 0.1 M sodium chloride solution. Lindenbaum and Schubert (1956) concluded from investigations of the binding of a variety of organic anions to serum albumin that important causes of binding were low reactivity of the bound ion with the solvent water, attraction of the anion by cationic centers in the protein, and van der Waals forces between the protein and alkyl side chains and aromatic rings in the organic anion. Lewis and Saroff ( 1957), using electrodes which were selectively permeable to cations, claim to have found evidence of binding of Na and K to the muscle proteins myosin A and myosin B. The effect of ion binding is greatly to reduce the net osmotic effect of the pF6tein in a multi-component solution.

OSMOTIC PROPERTIES OF L IVI N G CELLS

399

C. Hydration of Proteins in Solution Hydration as it affects the osmotic properties of proteins denotes no more than an association of the protein with the water fraction of a multicomponent solvent which is closer than its association with any of the other solvent components such as electrolyte ions. This kind of hydration, which has been recognized for a long time, has been named selective solvation by Ogston (1956) ; the definition is similar to that used for “free” and “bound” water by Hill (1930). It may be measured by comparing the concentrations of a solvent component in a protein solution and in an ultrafiltrate or diffusate in equilibrium with it. Although hydration has an influence on the osmotic pressure of the protein, it cannot easily be measured by osmotic methods, since it is extremely difficult to distinguish its effects from those of other factors such as the anomalous entropy of mixing. Selective solvation must be clearly distinguished from total solvation which is calculated from hydrodynamic measurements on the basis of various assumptions as to the shape and structure of the protein molecule (Ogston, 1956). Comparison of the water and salt contents of protein crystals formed from saline solutions with the composition of the solution leads to a further value for the hydration, but this is based on an analogy between the state of the protein in the crystal and in the solution which may be unjustified. This estimate is therefore of doubtful relevance to dissolved proteins. McMeekin et al. (1954) give values for the selective solvation of various proteins in ammonium sulfate solution ranging from 0.24 to 0.31 g. of H 2 0 per gram of protein. Drabkin (1950), in a study of hemoglobin crystals, gives the hydration of hemoglobin as 0.339 g. of H20per gram of protein. Edsall (1953) indicates that the most probable values for hydration of many proteins in solution lie in the range 0.1 to 0.3 g. of H 2 0 per gram of protein, but these estimates are based for the most part on hydrodynamic measurements. Since hydration is no more than a theoretical concept used to account for experimental results in witro, the use of the concept to account for experimental results in vivo results in a circular argument. All that is being done is to use experimental data obtained in vitro to account for observations on cells in vivo, and the concept of hydration appears only as an unnecessary intermediary. I n this comparison it is sometimes best to use the osmotic coefficient, which is an empirical quantity that does not depend on any particular theory of the mechanism involved.

D. Entropy of Mixing of Protein Solutions The expression (given in equation 1 ) for the change in chemical potential of the solvent on mixing with a solute is derived from a theoretical con-

400

D. A. T. DICK

sideration of the entropy change on mixing. That a change of energy (or, strictly, of free energy) takes place during the process of mixing, even when there is no change of temperature, hydrostatic pressure, or volume of the mixture, is due to a change in the entropy or heat capacity of the system. The entropy is related to the degree of randomness or thermodynamic probability of the system. By the methods of statistical mechanics based on the consideration of the number of possible ways of arranging the solute and solvent molecules in the rnixture4.e.) on the probability of any one arrangement-the total change of entropy or heat capacity of the system on mixing solvent and solute may be calculated (for a simple derivation see Butler, 1946), and the result for an ideal solution is

The partial molal entropy of mixing of the solvent alone is AS1

= -R

In

nl

nl

+ nz

But the partial entropy of mixing of the solvent is related to the chemical potential or free energy change thus : Ap1

=

AH1 -

ThSl

( 16)

whereH1 is the change of heat content of the system. If there is no volume or temperature change on mixing, then the heat content remains unchanged, and therefore

=0

(17) On substituting in equation 16 by means of equations 15 and 17, equation 1 results : AH1

Apl

= RT In

nl

nl

+

n2

It is, however, a basic assumption of the derivation of equation 14 that the molecules of solvent and solute should be interchangeable in the solution, i.e., should be of the same order of size. When this condition is not fulfilled, a very different estimate of the partial molar entropy of mixing of the solvent results. Theoretical expressions for the partial molar entropy of mixing of solvent with a macromolecular solute were first obtained by Huggins ( 1942) and Flory ( 1942). Of expressions suitable for application to solutions with a nonuniform distribution of macromolecular segments and solvent molecules such as protein solutions in the range of concentrations present in living cells, the two simplest are those of Flory (1945) and Schulz (1947). Flory used a lattice model for the solution and considered

401

OSMOTIC PROPERTIES OF LIVING CELLS

the possible distributions on the lattice points of solvent molecules or of segments of the macromolecule equivalent in size to solvent molecules, the macromolecule being supposed to consist of a long chain of such segments. The resulting expression was

+

Here vz is the volume fraction of the solute [Let v2 = V 2 / ( V 1 V 2 ) , where V l and V 2 are the volumes of the solvent and solute, respectively, in the solution] ; x is the number of segments in the macromolecule (i.e., 8 2 / P 1 , where P 1 and 8, are the volumes of the molecules of solvent and solute) ; s is the swelling factor

(

i.e, the ratio

gross hydrated volume of solute molecule volume of solid in molecule

)

and f = f(x/s), which equals 1.0 when x / s is very large, as is normally the case with protein solutions. When the swelling factor is taken as unity (i.e., the molecule is considered unhydrated) , Flory’s expres-:on reduces to Rv~ AS1 = -(X 1 4v2 21.3~2~) (19)

+

+

Schulz (1947) employed the model of a rigid spherical solute molecule and in estimating the number of possible configurations took account of the excluded volume surrounding the actual molecule itself into which the center of another solute molecule cannot penetrate ; his equation was

This is equivalent to

since V1 = nlV1 and V 2 = nzVz and therefore v2

=

n2 8

+

2

%Vl n2v2 Thus if terms in vZ2are neglected, Schulz’ and Flory’s expressions give the same result: Rv, AS1 = - ( 1 4~2) (22) X

+

The applicability of these theoretical expressions for the entropy of mixing to real solutions of proteins may be judged by using them to calculate

402

D. A. T. DICK

predicted values of the osmotic coefficient. When equations 18 and 21 are used to calculate the osmotic pressure in a protein solution of given concentration, the values of the entropy of mixing which they give are inserted into an equation relating the osmotic pressure to the entropy of mixing of the solvent, which is obtained by combining equations 4, 5, 16, and 17, thus: -IIB1 = -TAS1 (23) When the substitution is made, we obtain from equation 18 II = RTm' (1 4sk2 2 1 . 3 3 f 2 ~ ~ ~ ) (24)

+

+

where m' is the molar concentration ; i.e., m'

=

-

n2

nlBl

+

n2P2

v2 -x

. -1

PI

By comparison with equation 13, it is seen that the value of the molar osmotic coefficient, is 4' = 1 4sU2 Z1.3S2~2~ (25) (taking f = 1.0)

+',

+

+'

+

since is defined by the equation - n = 4' RTm'. Similarly from equation 21 we obtain 4' = 1 4 ~ 2- 8.06~2~ (26) I n a solution containing 10 g. of protein per 100 ml. of solution, if the partial specific volume of the protein is 0.75, then v2 = 0.075. Thus the predicted molar osmotic coefficient is 1.42 from equation 25, if the swelling factor, s, is taken as 1.0 (i.e., the solute molecule is considered to be unhydrated). If s = 1.3 (if we assume a hydration of 0.3 g. of H 2 0 per gram of protein), then the predicted molar osmotic coefficient is 1.59. The prediction from equation 26 (derived from Schulz' formula) is 1.25, and that from equation 22 is 1.30. These predictions may be compared with molar osmotic coefficients calculated from available experimental data for various proteins and shown in Table I. These values have been calculated from the experimental data by correcting molal to molar concentrations when necessary and by interpolation or short extrapolation from the experimental points. It must be emphasized that the possible effects of the heat of mixing, hydration, and ion binding by the protein have been ignored in these calculations, although allowance has been made for the influence of the Donnan effect where the protein solution observed was not at isoelectric point. Nevertheless it may be concluded that a considerable part of the nonideality of the behavior of proteins in aqueous solutions may be accounted for by the nonideal entropy of mixing which results from the great difference in the size of the solvent and solute molecules.

+

403

OSMOTIC PROPERTIES OF LIVING CELLS

Other possible expressions for the entropy of mixing of macromolecular solutions are contained in a review by Flory and Krigbaum (1951) and are discussed by Hildebrand and Scott ( 1950), Mark and Tobolsky ( 1950), Guggenheim ( 1952), and Flory ( 1953). TABLE I OSMOTICCOEFFICIENTS OF VARIOUSPROTEINS

Protein

Human Human Human Human Human Human Human

al-lipoprotein a,-globulin &-globulin y-globulin y-globulin y-globulin y-globulin

Observed molar osmotic coefficient (in soln. of conc. Molecular 10 g./100 ml. weight soln.)

78,000 600,000 93,000 140,000 200,000 155,000 156,000

0.76 8.64 1.71 1.08 1.37 1.18 1.12

References

. Oncley et al.

(1947)

A correction was made in this case for the effect of ion binding to the isoionic protein.

E. S u m w y Although the Donnan distribution of electrolyte increases the osmotic pressure exerted by a protein across a membrane permeable to electrolyte, no such effect occurs when the membrane is effectively impermeable to electrolyte as is the cell membrane; the osmotic pressure exerted in this case is the partial osmotic pressure of the protein ion alone. The net osmotic pressure of the protein may, however, be affected by ion binding. Hydration of proteins in solution is not to be regarded necessarily as a mechanical reality but as a theoretical concept used t o account for experimental

404

D. A.

T.

DICK

results in vitro ; use of the concept to account for observations on cells in vivo results in a circular argument, since all that is being done is to compare physicochemical observations in vivo and in vitro. By statistical mechanical methods it is shown that, when there is a great difference between the size of solute and solvent molecules, an abnormally large entropy of mixing is to be expected, which in turn produces a large osmotic coefficient. It is shown that this theory is capable of accounting at least in part for the osmotic coefficients of proteins which are actually observed. IV.

THERELATIONSHIP BETWEEN VOLUME A N D OSMOTIC PRESSURE AT EQUILIBRIUM I N LIVING CELLS A . Theory

Volume-osmotic pressure relationships in living cells have conventionally been analyzed by means of the simple Boyle-van’t Hoff law given by Luck6 and McCutcheon (1932) in their classic review:

I I ( Y - b ) = IIo(V0 - b ) = K (27) where and Vo are the original or isotonic osmotic pressure and total cell volume. Ponder ( 1948) expressed volume-osmotic pressure relations in the erythrocyte in terms of the equation

+

Y = W(l/T - 1) 100 (28) where V = total cell volume, W = isotonic water content, and T = relative tonicity. This equation is another expression of the simple Boyle-van’t Hoff law and may be easily derived from equation 27 if W is equated to ( V O- b ) , T to IT/&, and it is remembered that Ponder expressed water and solid concentrations not as fractions in accordance with conventional practice but as percentages ( V o = 100%). Ponder’s equation has two nonessential but inconvenient disadvantages : ( 1) Its form obscures the fact that it is derived from the simple Boyle-van’t Hoff law, and (2) the unorthodox symbols employed lead to confusion with those of standard thermodynamic terminology ; e.g., T is conventionally used for absolute temperature. The original expression used by Luck6 and McCutcheon (1932), or a more accurate modification of it, is thus to be preferred to Ponder’s equation. The fact that the erythrocyte does not obey the simple Boyle-van’t Hoff law was first clearly demonstrated by Ponder (see Table 111) ; swelling in hypotonic and shrinkage in hypertonic solutions is always less than that predicted by the law. Thus if equation 28 is used in calculations from experimental data, the value of W obtained is always less than the true isotonic water content of the cell as measured by direct methods. Ponder

OSMOTIC PROPERTIES OF L IVI N G CELLS

405

(1933a, b) expressed the discrepancy between theory and observation by multiplying W by an empirical factor R (normally less than 1.0) before insertion in the equation, thus obtaining the apparent isotonic water content, R W ( R has been represented here by a bold-faced R to distinguish it from the gas constant). Equation 28 thus becomes

+

V = R W ( l / T - 1) 100 (29) For the reasons given above, further description of the theory of the osmotic behavior of living cells will be based on Luck6 and McCutcheon’s (1932) equation. Thus if equation 27 is differentiated and the osmotic pressure is expressed in units of the isotonic osmotic pressure SO that no= 1.0, the result is dV = Vo - b = K = Wo ( 30) d ( l/W where W Ois the apparent isotonic water content of the cell. But it has been shown (Dick and Lowenstein, 1958; see also below) that equation 27 is an approximation, so that the equalities expressed in it are not exact. In consequence, Wo # Vo - b as is otherwise suggested by equations 27 and 30; i.e., W o is only the apparent and not the true isotonic water content of the cell; it is actually only an empirical expression of dV/d( l/lT), i.e. dV W (31) - d ( l / n ) ( = Ponder’s R W ) If W,, the true isotonic water content of the cell, is defined as W , = Vo - b ( = Ponder’s W ) (32) then Ponder’s R in this terminology is WO dV . -1 R== Wm d(l/n) W, The correct value of W oand hence a theoretical expression for Ponder’s R has been obtained by using a more accurate form of the Boyle-van’t Hoff law (Dick, 1958b). It has already been shown (from equations 11 and 12 above) that this is lT(V - b ) = 9 RTn2 (33)

+

Equation 27 is true only if remains constant throughout the range of concentrations produced in osmotic experiments. Since this condition is not fulfilled, owing to the large and concentration-dependent osmotic coefficients of the proteins, a more accurate value of Wo is obtained by differentiating equation 33 correctly, i.e, treating 9 as a function of n (since

406

D. A. T. DICK

the molar concentration of intracellular solute and hence external osmotic pressure, n) ; thus

+ depends on the

O n inserting values at the isotonic osmotic pressure, there results

W o = +oRTn2-

(2)

n = 1

-

RTn2

(35)

since IIo = 1.0, and with (p = +o at isotonic osmotic pressure. But from equations 32 and 33, on inserting values at the isotonic osmotic pressure,

= doRTn2

(36)

-

(37)

since IIo = 1.0. From equations 35 and 36,

wo = w, -

(g)

n = 1

RTn2

but d+/dn is in general positive, owing to the increase of the osmotic coefficient of the protein fraction of the cell solute with concentration (and hence osmotic pressure), and therefore

wo < w m and

<

1.0

Since this equation is derived from basic thermodynamic principles, it is of general validity for all cells, provided the assumptions on which it is based (see Dick, 1958b) are fulfilled. It has been used to calculate a predicted value of R for the erythrocyte which is in agreement with the experimental results (Dick, 1958b) and also with the results of previous numerical calculations (Dick and Lowenstein, 1958). This prediction of course applies only to physiologically normal erythrocytes, e.g., in defibrinated or heparinized blood samples. Other values of R are found in abnormal states of the erythrocyte or after the use of certain anticoagulants, and it has been suggested that these are due to an alteration in the physical state of the internal protoplasmic solution (Ponder, 1948). In accordance with equations 27 and 30, volume-osmotic pressure data

OSMOTIC PROPERTIES OF LIVING CELLS

407

are best interpreted by plotting cell volume against the reciprocal of osmotic pressure (Ponder, 1955) or by calculating the resulting relationship by regression analysis (Dick and Lowenstein, 1958). Methods employing the direct relationship between volume and osmotic pressure involve unnecessarily cumbersome mathematics which necessitate crude approximations (eg., Cristol and Benezech, 1946a; Hamburger and MathC, 1952, P. 46).

B. Assumptions Underlying Theoretical Treatment At least three assumptions underly the present theoretical treatment of volume-osmotic pressure relationships described in the preceding section : 1. That the intracellular osmotic pressure with which the equation deals can be taken to be equal to the osmotic pressure of the external medium. 2. That there is no net passage of solute across the cell membrane during the attainment of osmotic equilibrium between the cell and its environment. 3. That the protein component of the cell is in true solution or at least is capable of osmotic behavior as if it were in solution.

1. Although the assumption of osmotic equality between the cell interior and the environment was long tacitly accepted, it has recently been challenged. It has been observed that mammalian tissue slices immersed in isotonic saline solutions swell by imbibition of fluid when their metabolism is impaired by low temperatures, anoxia, or metabolic inhibitors, and that this swelling can be prevented by immersion in strongly hypertonic solutions (Stern et al., 1949; Opie, 1949; Opie and Rothbard, 1953 ; Robinson, 1950, 1953; Aebi, 1952; Adolph and Richmond, 1956; Riecker et al., 1957). Similar swelling of tissues in vivo by hypothermia or anoxemia has been shown by D’Amato (1954) and Hamburger and MathC (1951). If the tissue is reincubated aerobically at 37°C. in the presence of a suitable substrate, swelling is much less and fluid may be re-expelled from tissue which has already swelled (Stern, et al., 1949; Robinson, 1950, 1952a; Aebi, 1952). These observations have been interpreted by Robinson (1953), Opie (1954), and Bartley et al. ( 1954) as evidence that the cell interior is normally hypertonic to its environment and that a steady state is maintained only by continuous active expulsion of water. It has, however, been clearly shown that the fluid which enters the cells in conditions of impaired metabolism is not pure water but water plus sodium and chloride ions in a mixture approximately isotonic with the immersion medium (Mudge, 1951 ; Conway and Geoghegan, 1955 ; Leaf, 1956; Whittam, 1956; Gaudino, 1956) (although Schwartz and Opie, 1954, claimed

408

D. A. T. DICK

that sodium entered only the extracellular space during equilibration of tissue slices in saline) ; thus, whatever other evidence may be produced in favor of the theory of normal intracellular hypertonicity, the interpretation of tissue swelling by impaired metabolism as evidence for the theory is certainly not valid. Conway and McCormack (1953) attempted to settle the question by direct measurement of the intracellular osmotic pressure. They measured the freezing-point depression of tissues frozen and pulverized in liquid oxygen and then mixed with 0.95% saline at 0°C. It was found that the freezing-point depression increased rapidly with time even at 0°C.) and this was shown to be due to autolytic changes (Conway et al., 1955). To obtain the true osmotic pressure of the tissue in vivo, freezing-point depressions were measured at different times and extrapolated to zero time. It was concluded that the osmotic pressures of the tissues examined did not differ significantly from that of homologous serum. Brodsky et al. (1956) have criticized the method of Conway and his colleagues on the grounds that delay in equilibrating the tissue-saline mixture may occur and that the osmotic pressure measured is therefore that of the isotonic saline and not that of the tissue. Brodsky et al. obtained evidence of delay in mixing of about 10 minutes in liver samples mixed with hypertonic saline but not in those mixed with distilled water. After this interval tissue-saline mixtures showed a steady increase in freezing-point depression similar to that found by Conway and McCormack (1953). Brodsky et al., however, have extrapolated osmotic pressure to zero time only from data obtained in the first 10 minutes of the experiment, when mixing is probably incomplete ; and statistical methods of regression analysis were apparently not used. But after the first 10 minutes diffusion equilibrium has almost certainly been attained whether or not the tissue has been completely disrupted, and thereafter the variation in osmotic pressure is almost certainly due to autolysis. Thus, extrapolation to zero time of data obtained during the first 10 minutes is not equivalent to Conway and McCormack’s procedure ; significant comparisons can be made only by extrapolating from data obtained after the first 10 minutes. Such extrapolations have been performed on the data of Brodsky et al. by the present author using regression analysis. The results for the tissue osmotic pressure of liver so obtained are different from those given by Brodsky et al. in their paper-i.e., from isotonic saline mixtures 378 m-osm. (S.E. 17.4) instead of 317 to 340 m-osm. ; from hypertonic saline mixtures 324 m-osm. (S.E. 20.0) instead of zero ; and from distilled water mixtures 31 1 m-osm. (S.E. 10.1) instead of 300 to 305 m-osm. Thus in contrast with Conway and McCormack’s results hypertonic saline and distilled water mixtures

OSMOTIC PROPERTlES OF L I VI N G CELLS

409

gave a tissue osmotic pressure similar to that of plasma, and only isotonic saline mixtures gave an extrapolated osmotic pressure significantly different from the plasma osmotic pressure of 300 m-osm. Brodsky et al. have also given estimates of the osmotic pressure of many different undiluted tissue samples ; values ranged from 286 m-osm. to 827 m-osm., but it was suggested that the high values may be due to keeping the tissue at 0°C. for several hours, which from the findings of Conway et al. (1955) would be expected to result in a considerable degree of autolysis. Data of Brodsky et al. on the variation with time of tissue osmotic pressure measured without admixture of saline further suggest a hypertonic tissue ; however, the difficulties of measurement of accurate freezing points in viscous tissue homogenates must be very great as, unless equilibrium is rapidly attained, too low values (and thus high osmotic pressure estimates) result (see Dick and Lowenstein, 1958). Values of freezing-point determinations obtained on undiluted homogenates must therefore be treated with reserve. The definite conclusion that “the freezing point depression of freshly excised frozen tissues . . . is greater than that of plasma” does not appear to be justified by the rather inconsistent results presented in the paper of Brodsky et al. (1956). Appelboom ( 1957) has recently found from freezing-point determinations on boiled tissues that the intracellular osmotic pressure is not significantly different from that of plasma. Maffly and Leaf (1959) have reached a similar conclusion from melting-point determinations on frozen tissues. The finding of Robinson (1952b) that the base content of kidney cortex cells is higher than that of the extracellular phase does not necessarily imply a higher intracellular osmotic pressure, since some of the base may be present in an osmotically inactive state, e.g., bound to protein molecules (see Section 111. B above), or, as suggested by Mudge (1953), the excess of base may be needed to offset the osmotic deficiency due to polyvalent anions in the cell. Nichols and Nichols (1953) calculated from the concentrations of the known constituents of the erythrocyte and plasma that the total osmotic pressure within the erythrocytes of patients recovering from acidosis is only 274 m-osm. as compared with 302 m-osm. in the plasma. They concluded that the discrepancy was only apparent and was probably due to the presence of unknown solutes in the cell which had not been taken into account. However, the authors calculated the osmotic contribution of the hemoglobin without taking account of its large osmotic coefficient, so that the calculated value was only 4.5 m-osm. instead of the correct value of 16 m-osm. When this correction is made, a large part of the observed discrepancy is accounted for.

410

D. A. T. DICK

The assumption of osmotic equality does not seem to be unequivocally contradicted by any evidence so far available; as suggested by Darrow and Hellerstein (1958), it appears to be at least permissible to continue to make it. As indicated by Manery (1954),the success of theories based on this assumption in accounting for the osmotic behavior of living cells is itself a point in its favor. Theoretical treatments of cellular osmotic phenomena may be considered as dependent on proof of this assumption. This is not yet available. It must be noted, however, that most osmotic theories will be disturbed only when it is shown not only that osmotic equality does not occur between cells and environment but that there is not even a linear relationship between the intracellular and extracellular osmotic pressures, since even this modified condition would satisfy the requirements of the theories. 2. In a discussion of the relation between cell volume at osmotic equilibrium and electrolyte movements across the cell membrane, two points must be noted. First, the attainment of osmotic equilibrium is rapid, owing to the high permeability of the cell to water ; therefore since osmotic experiments, for the determination both of equilibrium and of kinetics, are of short duration-usually less than one hour-only electrolyte movements occurring within a period of a few hours need be considered. Second, it is necessary to discuss only the actual distribution of solute which occurs in cells and determines osmotic phenomena ; discussion of the mechanism which produces that distribution is not directly relevant to the problem. Also, since obviously only net transfers of solute and not actual effluxes or influxes determine over-all solute distributions, only net transfers are significant from the osmotic point of view. Available data on net cation transfers in various cells and tissues under physiological conditions are given in Table 11. It is seen that losses of potassium from individual cells and portions of undamaged rat diaphragm into media containing mostly sodium with small quantities of potassium are very small within the first few hours. There is some indication that they may be balanced (or even outweighed) by gains of sodium. On the other hand, in tissue slices of kidney cortex and guinea pig brain, the movements of ions are different; in the first there is a gain instead of a loss of potassium, and in the second the loss of potassium is much more rapid than in isolated cells. These differences may be associated with tissue damage during the preparation of the slice or with uptake of an actively transported anion, e.g., a-ketoglutarate. The evidence demonstrates that net cation transfer across the membranes of erythrocytes, leukocytes, and muscle cells is negligible within the first few hours of immersion in saline solutions. Since net anion transfer is dictated by cation

411

OSMOTIC PROPERTIES OF L I V I N G CELLS

transfer, owing to the necessity for preserving electrostatic neutrality, the assumption that no net transfer of solute across the cell membrane occurs during the attainment of osmotic equilibrium appears to be justified by TABLE I1 NET CATIONMOVEMENT I N CELLSIMMERSED I N PLASMA OR PHYSIOL~CICAL SALINE

Cell

Temperature (C.1

Human erythrocyte

25

K loss Time (% of interval initial (hours) conc.)

1 3

5 20 Human erythrocyte

Human erythrocyte Human erythrocyte Chicken erythrocyte Duck erythrocyte Rabbit leukocyte Rat muscle Rat muscle Guinea pig brain Kidney cortex

Na gain (% of initial conc.)

1.1 2.3 3.3 6.0

Ponder (1947a)

2 3 5 12

0.8 1.3

1.3 2.1

3.2 5.4

4.7

5 40

1 1

1.5 4.5

-

-1

37

6

1.3

0.4 (loss)

25

7.9

,

37

3

37

Several

Nil

Nil

37 38 37

6 12 4

Nil 2.0 Nil

Nil

40

1

5.2(gain)

-

37

10min.

0.5 5.0 (approx.) (approx.)

6.5

References

1

Na

1

+ K)

Ponder (1947b) (immersed in 0.1 M NaCl) Davson (1937)

[

Maizels (1954)

(% of total initial

+

Na K) Hunter et a/. (1956) Tosteson and Robertson (1956) Hempling (1954) Creese (1954) Calkins et al. (1954)

-

21.0

(% of total initial

Terner et al. (1950)

{

(% of total initial

+

Na K) Whittam and Davies

the limited evidence available. Transfers of certain nonelectrolytes such as glucose or urea may of course occur, but these substances are not present in sufficient concentration to be of osmotic significance. It may be noted that, since the cell membrane appears to present no

412

D. A . T. DICK

distinct barrier to the passage of anions as it does to cations, an exchange of anions may readily occur. Thus if erythrocytes are placed in a solution containing a divalent anion, a two-for-one exchange with intracellular univalent anions may occur, resulting in diminution of the intracellular osmolarity and hence shrinkage of the cell (Parpart, 1940). A similar phenomenon has recently been demonstrated in Ehrlich mouse ascites tumor cells (Hempling, 1958). Conway ( 1957) has reviewed evidence that, contrary to previous opinion, the muscle cell is permeable to C1- ions. Ponder (1953) found that the concentrations of solutions of the alkaline earth chlorides needed to maintain erythrocytes at the same volume as in 1.0% sodium chloride were 0.105 M BaC12, 0.1 10 M MgC12, 0.130 M SrC12, and 0.10M CaC12. Since the respective osmotic coefficients are NaCl 0.928, BaC12 0.843, MgC12 0.861, SrC12 0.850, and CaCl2 0.854 (Robinson and Stokes, 1955), and thus the corresponding osmotic pressures are NaCl 317 m-osm., BaC12 266 m-osm., MgCl2 284 m-osm., SrC12 332 m-osm., and CaC12 256 m-osm., it is seen that, with the exception of SrC12, a lower concentration of divalent ion is needed to maintain the isotonic volume in saline solution ; a possible interpretation of the phenomenon might be that a limited two-for-one exchange of divalent for univalent cations takes place similar to that found for anions. Heilbrunn (1952, p. 127) reported similar findings in sea urchin eggs immersed in calcium chloride solutions. Grim (1953) has provided a theoretical treatment for predicting the osmotic properties of a membrane which is permeable to both water and solutes. This treatment is applicable, however, only when the permeability of the solutes is comparable with that of the solvent. 3. The evidence bearing on the physicochemical organization of solute and solvent in the cell interior is very unsatisfactory. Ponder (1948, 1949, 1955) has brought forward some indirect evidence for the conclusion that the erythrocyte possesses an internal structure formed either by an unidentified nonhemoglobin component or by interaction or orientation between the hemoglobin molecules themselves. Ponder has also suggested that the osmotic behavior of abnormal erythrocytes, e.g., crenated forms in oxalated blood or the paracrystalline rat red cell, is due to gelation in the cell interior. Dervichian et al. (1947) have concluded from comparative X-ray diffraction studies of erythrocytes, hemoglobin solutions, and hemoglobin crystals that the arrangement of the hemoglobin molecules in the erythrocyte is intermediate between the order present in a crystal and the disorder of a dilute solution. Frey-Wyssling (1953) has suggested that the protein molecules in the cell interior are connected by various types of junction or chemical bond. These junctions are considered to be highly labile, and

OSMOTIC PROPERTIES OF L IVI N G CELLS

413

changes in them produce gel-sol transitions in the protoplasm which are responsible for protoplasmic flow. Further evidence on the viscosity and elasticity of the protoplasm is interpreted as demonstrating a state intermediate between that of sol and gel. The bearing of electron-microscope evidence on the physicochemical state of the living protoplasm, though apparently direct, depends largely on assumptions about the relation of the fixed tissue to the living state. Frey-Wyssling ( 1955, 1957) has described four types of organization in the ground structure of the cytoplasm-reticular, granular, fibrillar, and lamellar. Palade ( 1955, 1956) has described an endoplasmic reticulum in the cytoplasm of a great variety of cells (Frey-Wyssling suggests that this may be equated to his lamellar organization). The reticulum has been found to be highly labile in character and is to this extent consistent with Frey-Wyssling’s views. The only cell type in which the endoplasmic reticulum has not so far been found is the mature erythrocyte, but this may be due to the extreme difficulty of preparing adequate thin sections of the erythrocyte. Thus it appears to be equally unlikely either that the protein component of the cell interior is in simple solution or that it is merely an insoluble structural framework. It must be remembered that so far as the osmotic properties of the cell are concerned this is a quantitative and not a qualitative question. What matters is not the mere fact that a structural component influences the osmotic behavior of the cell but the degree of that influence. It is equally possible that an apparently solid morphological structure may have a comparatively small quantitative effect on the solute properties of the protein composing it or, on the other hand, that the solute properties of the intracellular protein may be affected to a large extent by ultramicroscopic intermolecular chemical bonds which are revealed only by indirect evidence. It will be seen later that volume-osmotic pressure relationships in living cells may be interpreted so as to have a bearing on this question.

C . Techniques of Volume Measurement The difficulty of investigating volume-osmotic pressure relationships in living cells lies mainly in the measurement of cell volume. It is necessary, however, to measure only the relative and not the absolute cell volume. A great number of techniques have been used for this purpose, and these may be listed as follows.

1. Direct measurement of diameter of spherical cells. 2. Hematocrit methods. 3. Measurement of concentration of nonpenetrating solute.

414

D. A. T. DICK

4. Angular diffraction of light by cell suspensions (halometry). 5. Measurement of conductivity of cell suspensions. 6. Measurement of opacity of cell suspensions. 7. Measurement of changes in the solid and water contents of the cell. In a restricted compass it is possible only to outline the methods and to indicate, where appropriate, the scope and accuracy of the technique. 1. Direct Mearurement of Diameter of Spherical Cells. This method has been used extensively in measurements on marine invertebrate eggs (see Table IV). I t suffers only from the disadvantage that, since the calculated volume is proportional to the cube of the diameter, the percentage error of volume is three times the percentage error of the linear measurement. The absolute size of the cell is thus an important factor in the application of this technique, since as the diameter of the cell increases the percentage error falls ; the technique is thus more suitable for large cells such as sea urchin eggs rather than small ones such as leukocytes or sphered red cells. 2. Hematocrit Methods. The hematocrit technique involves centrifuging suspended cells at high speed and measuring the volume of packed cells. The application of the method to measurements of erythrocyte volume has been extensively reviewed by Ponder (1948). Recent determinations of the amount of trapped plasma in the packed cell column after centrifugation have shown that considerable errors can arise from this source (Leeson and Reeve, 1951 ; Chaplin and Mollison, 1952 ; Bernstein, 1955 ; Ebaugh et al., 1955). The extent of this effect in cells at other than isotonic osmotic pressures has been described by grskov (1946). Parpart and Ballantine (1943) used very high speeds of centrifugation and suggested that packing was practically complete after centrifugation at 30,000g for 5 minutes. Workers using the high-speed hematocrit at 10,OOO to 12,000 r.p.m. (grskov, 1946 ; Ponder, 1950) have obtained relative volurnes which give R values comparable with those resulting from methods of entirely different kinds (see Table 111). The modified van Allen dilution method, centrifuging at 2500 to 3000 r.p.m. (Guest and Wing, 1942), has given similar results (Guest, 1948). Side1 and Solomon ( 1957), however, who centrifuged at 7800s for 30 minutes obtained an R value of 0.64, and Ponder and Barreto (1957), who centrifuged at 20,OOOg for 90 minutes, an R value of 0.78. Hendry ( 1954) has criticized the hematocrit technique for measuring the relative volume of swollen erythrocytes in hypotonic solutions. H e claimed that such cells are more sensitive to compression during centrifugation than are cells in isotonic solution. This criticism, if valid, implies that the hematocrit is unreliable even if the duration and force of centrif-

OSMOTIC PROPERTIES OF LIVING CELLS

415

ugation are carefully standardized ; it cannot be expected to estimate accurately even relative volume changes. The hematocrit method has also been applied to measuring the volume of sea urchin eggs (Shapiro, 1935). The values obtained were on an average 2.5% greater than those given by the combination of hemocytometer count and measurement of diameter, but the range of the discrepancy was high (from +28.2% to -15.5%). Using the same material Clowes and Krahl (1936) found that the hematocrit results were on average 8% higher than the hemocytometer values. The discrepancy appears to be due to the presence of jelly adhering to the egg surface. Since the amount of jelly is very variable (Shapiro, 1935), the hematocrit appears to be unreliable for this purpose. 3. Measurement of Concentration of Nonpenetrating Solute. This method has been applied to the erythrocyte. The concentration of some substance in the plasma or suspending fluid either added artificially or naturally present is measured before and after osmotic volume changes in the erythrocytes. From the concentrations the volumes of the plasma and hence the volumes of the cells are easily measured. Substances measured have been artificially added hemoglobin (Ponder and Saslow, 1930), the dye Evans’ blue (Shohl and Hunter, 1941; Ponder, 1944), and the plasma proteins (Hendry, 1954). Provided the basic assumptions-that the measured substance is truly nonpenetrating and is not adsorbed on the erythrocyte surface-are satisfied, the method appears to be reliable, since it gives results consistent with values of R obtained by other methods (see Table 111). 4. Angular Diffraction of Light by Cell Suspensions (Halometry). The history and technique of this method have been fully reviewed by Ponder (1948, 1950). When a narrow beam of light is passed through a suspension of fine spherical articles, it becomes surrounded by bright and dark rings due to scattering of the original straight beam of light. The angular distance of the rings of scattered light from the source beam depends on the diameter of the particles which cause the scattering. From measurements of the angle the diameter of the particles can be calculated. This method has chiefly been applied to erythrocytes. Thus use of it involves two difficulties: (e) Before the method is applied to measuring the volume of erythrocytes, they must first be turned into spheres ; Ponder ( 1929) claimed that disc-sphere transformation can be produced without change of cell volume, but unpublished observations of Lowenstein and Dick (1958) show that, although in the first stage of the transformation a crenated sphere is produced which has a diminished diameter but the same volume as the original erythrocytes, this crenated sphere rapidly

416

D. A. T. DICK

increases in diameter and volume with disappearance of the original crenations. ( b ) Since the method involves the calculation of volume from diameter, it necessitates the inevitable trebling of the percentage error which has already been mentioned. 5. Measurement of Conductivity of Cell Suspensions. From measurements of the conductivity of a suspension of cells and of the suspending medium, the volume fraction of cells in the suspension can be calculated. Although the theoretical foundation of the method is sound, the equation used in the calculation contains a “form factor” which depends on the shape of the cell used. The “form factor” can be accurately calculated for a sphere or a spheroid, but for an irregular cell such as the erythrocyte it must be determined empirically by means of suspensions of known concentration calibrated by another method (eg., the hematocrit) . In addition to this source of uncertainty the conductivity of a suspension is liable to wide variations if it is stirred, due to formation of eddy currents. The method is therefore only of limited value. The application of the method to erythrocytes has been reviewed by Ponder (1948). 6. Measurement of Opacity of Cell Suspensions. The use of this technique for the measurement of red cell volume has been reviewed by Ponder (1948). The method has been applied to leukocytes by Shapiro and Parpart ( 1937), by Luck6 and Parpart (1954), by Luck6 et al. (1956), and by LeFevre and LeFevre ( 1952). The opacity of a suspension is expressed by the fraction Intensity of transmitted light Intensity of incident light The opacity of a suspension of cells depends on the amount of light lost by absorption and scattering. Neither of these quantities is amenable to accurate theoretical treatment. Besides the number and size of cells, absorption depends on the concentration of absorbing material in the cells, and the distribution of the cells. Scattering depends on the difference of refractive index between cell and immersion medium. Thus changes in the opacity of a cell suspension depend on a number of variables. Nevertheless it has been found experimentally in many cases that an approximately linear relationship exists between the opacity of the suspension and the total volume fraction of cells in the suspension. I t must be emphasized, however, that this relationship is purely empirical and has no satisfactory theoretical foundation. Although the method therefore cannot be used for measuring absolute cell volumes, it provides a useful method of measuring relative volume changes, particularly if these are rapid. The accuracy of the method is limited, however, to the accuracy of the volume-

OSMOTIC PROPERTIES O F L I VI N G CELLS

417

measuring method, usually the hematocrit, which is used in the calibration and the accuracy of the calibration technique itself. Relative cell volume has recently been measured by a variation of the opacimetric technique (Side1 and Solomon, 1957). Instead of measuring the transmitted light, the light scattered by a red cell suspension at right angles to the incident beam is measured. An empirical linear relation is found between the scattered light and the volume fraction of cells in the suspension. The method is subject to the same limitations as the opacimetric technique. 7. Measurement of Changes in the Solid and Water Contents of the Cell. From changes in the solid and water contents of the cell, relative changes in volume due to osmotic conditions can be calculated, provided it is assumed that volume changes are due solely to water transfer ; as seen in Section IV. B. 2, this assumption appears to be substantially justified by the evidence available. Methods of measuring the solid and water contents of the erythrocyte are reviewed by Ponder (1948). The density of the erythrocyte can be measured by various methods ; the hemoglobin content of cells is measured by centrifuging, lysing the packed cells, and estimating the hemoglobin present colorimetrically ; the erythrocyte water content is determined directly by centrifuging the cells at high speed and drying a weighed sample to constant weight. The estimations of hemoglobin and water content are of course dependent on the efficiency of centrifuging. The most recent technique applied for this purpose is that of immersion refractometry (Barer and Joseph, 1954, 1955a, b ; Barer, 1956). The refractive index of the cell cytoplasm is determined by immersing the cell in solutions of bovine plasma albumin of different refractive index until a condition of zero contrast is obtained between the cell and the background as seen in the phase-contrast microscope. The refractive index of the cell is then the same as that of the immersing medium, and from it the solid concentration of the cytoplasm may be directly calculated from the equation n

=

n,

+ aC

where n = the refractive index of the cytoplasmic colloidal solution.

nzo = the refractive index of the pure solvent, i.e., water. a = the average specific refraction increment of the cell solute. C = the solid concentration of the cell expressed as weight/volume of solution. For cells other than erythrocytes, a is taken as 0.0018, a value slightly less than the average for most proteins, to allow for the presence of lipoprotein in the cell (it is considered that the amount of the nonprotein

418

D. A. T. DICK

constituents of the cell is so small that it does not affect the value significantly). For the erythrocyte a is taken as 0.0019, which is again slightly less than the accepted value for hemoglobin. This method has been applied to erythrocytes (Dick and Lowenstein, 1958; Gaffney, 1958) and to chick heart fibroblasts in tissue culture (Dick, 1958a). The results obtained are consistent with recent values obtained by other methods (see Tables I11 and I V ) . Particular advantages of the refractometric technique for the measurement of volume-osmotic pressure relationships are its applicability to irregular cells and the fact that besides relative volume there is obtained at the same time a determination of the isotonic water content of cell which is needed in the calculation of Ponder’s R.

D. Volume-Osmotic Pressure Relatwnships in Erythrocytes Owing to its easy availability in quantity, the osmotic properties of the erythrocyte have been more extensively studied than those of any other kind of cell. The subject has been reviewed by Luck6 and McCutcheon (1932) and by Ponder (1933a, b, 1940, 1948, 1955). It is now well established that when the total cell volume is plotted against the reciprocal of osmotic pressure a linear relationship is obtained within certain limits of hypotonicity and hypertonicity (e.g., Dick and Lowenstein, 1958). Beyond the hypotonic limit the cell fails to increase in volume as expected, probably owing to the prehemolytic loss of ions from the cell. That the linearity found is more apparent than real is demonstrated by equation 34, but by means of the treatment described in Section IV. A it may be used to calculate the value of R for the erythrocyte. Data available from the literature are shown in Table 111. Observations made on cells from defibrinated or heparinized blood are shown first; these cells may be presumed to have been in a physiologically normal condition. I t is seen that the values of Ponder’s R are highly variable, especially in the earlier measurements. With improved techniques, however, values obtained from cells in hypotonic saline appear to lie for the most part in the range of 0.90 to 1.0. On the other hand, values from cells in hypertonic saline are much lower. This is in accordance with the theory presented in Section IV. A, since the osmotic coefficient of hemoglobin changes ever more rapidly with concentration at the high concentrations produced by hypertonic osmotic pressures, i.e., the value of d+/dII is greatly increased and a low value of R results. Part A of Table I11 shows R values obtained from cells in oxalated blood; it is seen that the majority of values lie in the range 0.5 to 0.8 and are thus much lower than those of cells from defibrinated or heparinized blood. This difference was first described by Ponder and Robinson (1934a). Since the enzyme mechanisms of cells in oxalated

OSMOTIC PROPERTIES OF LIVING CELLS

419

blood samples are grossly dislocated, some alteration in the osmotic properties of the cell is to be expected. Table 111, part C, shows one low R value for citrated blood, but the method used was unreliable. Several theories have been put forward to account for the value of R. T o avoid confusion these must be divided into two groups:

1. Theories applicable to the physiologically normal erythrocyte. a. Binding of water of solvation to the erythrocyte proteins (Jdrskov, 1946; Cristol and Benezech, 1946a; Ponder, 1948). It has been shown in Section 111. C that the status of water of solvation is rather nebulous and that it is in many cases merely a hypothetical concept used to interpret observed results. Cristol and Benezech (1948) have used the binding of water of solvation as an explanation of the fact that the chloride content of the erythrocyte is less than that of the plasma; such an interpretation ignores the effect of the electrical charge on the hemoglobin in producing an unequal Donnan distribution of the electrolyte. b. Increase of osmotic activity of salts in concentrated hemoglobin solutions (e)rskov, 1946). This possibility is very improbable, as protein-salt interactions in concentrated hemoglobin solutions are more likely to diminish than to increase the osmotic activity of the salt. c. Elasticity and resistance to shrinkage of hemoglobin molecules in concentrated solutions (Jdrskov, 1946). This theory is open to criticism on the ground that the elasticity of a true aqueous solution is so small as to be negligible. It is, however, possible that the partially gel-like properties of the intracellular solution of the erythrocyte (see Section IV. B. 3) confer some elasticity on it. d. An internal nonhemoglobin framework in the cell resists shrinking (Jdrskov, 1946; Ponder, 1940). Evidence for such an internal framework at least in the region close to the erythrocyte membrane has been put forward by Mitchison (1953) and Ponder (1949, 1951b), although the electron microscope has not so far confirmed it. e. Tension in the cell membrane (Cristol and Benezech, 1946b). A similar mechanism was proposed for the erythrocyte ghost by Teorell (1952). There is no evidence that the intact red cell membrane is capable of withstanding pressures of significant degree in relation to the large osmotic pressures involved (the isotonic osmotic pressure is 7 atmospheres). The prehemolytic failure of the erythrocyte to increase its volume proportionately to the fall of osmotic pressure is more likely to be due to leakage of solute than

n.

420

A. T. nrcK

TABLE I11 VALUESOF PONDER'S R I N ERYTHROCYTES Range of relative osmotic Ponder's Technique pressure R A . Defibrinated or Heparinked Blood Hematocrit (dilution) 1 - 1.5 0.66 Colorimetric Hypotonic 0.5 (approx. 1 Vapor pressure measurement 1-2.0 0.97 0.85-0.98 Hematocrit 1 - 0.65 Density measurement 1 - 0.65 Diffraction 1- 0.5 0.65-1.00

Animal Horse Rabbit Ox

Rabbit

Man Rabbit

ox Sheep Rabbit Rabbit Man

zL

Rabbit Dog Rabbit

1

Diffraction Diffraction Diffraction Diffraction Measurement of diameter

1- 0.41 1- 0.47 1 - 0.50 1 - 0.58 1-0.5

0.79

Conductivity Conductivity

1- 1.5 1- 1.5

0.72-0.95 0.73-0.96

0.69 - 2.0

0.58-0.81 0.58-0.91 0.6 -0.8

Hematocrit

Man

Water concentration 1- 2.56 1- 0.68 measurement Hematocrit (van Allen) 1-0.425

Man Man

Colorimetric Not stated

Man Man Man Man Man Man Chicken

Hematocrit 1 - 0.62 Hematocrit 1 2.39 Hematocrit (van Allen) 1 - 0.425 Not stated Hypertonic Hematocrit (dilution) 1 - 0.2 1- 0.45 Diff ractometry Hematocrit 1 - 2.0 1-0.7 Opacimetry 1- 2.0

1- 0.5 1 - 4.0

-

0.64-0.79

0'7(M'84 0.57-0.90 0.63-0.86

0.6 -0.73 0.5 o.74

I I

1}

Hill (1930) Ponder and Robinson (1934a)

Ponder (1935a)

Ponder (1935b) Parpart and Shull (1935)

Davson (1936) Guest and Wing ( 1942) Ponder (1944) Cristol and Benezech (1946a)

0.93-0.97 0.70

0.55-0.85

Hamburger (1898) Ponder and Saslow (1930)

Ponder (193%)

0.96-1.05

0.79 0.98 0.79 0.9 0.75-0.85

References

}

grskov (1946) Guest (1948) Giraud ef nl. (1950) Ponder (1950) Ponder (1951a)

42 1

OSMOTIC PROPERTIES O F LIVING CELLS

TABLE I11 (continued) Range of relative osmotic pressure

Ponder’s R

Animal Man

Technique Plasma concentration measurement

Man

Hematocrit

Man

Immersion refractometry 1 - 0.62 0.95 (S.E. 0.018)

References

1 - 0.58 0.97 (S.E. 0.011) 0.5- 1.7 0.78

Hendry (1954) Ponder and Barreto

(1957) Dick and Lowenstein

(1958) Man Immersion refractometry 1 - 0.56 0.90 (S.E. 0.035) Chicken Immersion refractometry 1-0.56 1.00 (S.E. 0.051)

Gaffney (1958)

B. Oxalated Blood Rabbit

Hematocrit (dilution)

1 - 1.5 1 - 0.5

1.06 0.83

}

Ege (1922)

Man

Hematocrit

1 - 0.75 1 - 2.9

0.65 0.54

Gough (1924)

Man

Hematocrit (dilution)

1-0.6 1 - 1.25

0.76 0.68

1

1-3.0 Sheep Rabbit

Man Rabbit

Diffraction Hematocrit (dilution) Hematocrit (dilution) Density measurement H b concentration measurement Water concentration measurement

1 - 0.46 1-2.0 1 - 0.56 1-0.5 1-0.65

0.6

Christensen and Warburg (1929) Krevisky (1930)

(approx. 1

0.47-0.59 0.15 0.58-0.73 0.63 0.55-0.72

1 - 0.65

0.53-0.69

1 - 0.65

0.6

Ponder and Saslow

(1931) Schi@dt (1932)

Macleod and Ponder

(1933) (approx.)

Rabbit

Hematocrit Density measurement Diffraction Hematocrit

1 - 0.65 1 - 0.65 1-0.75 1 - 0.68

0.71-0.85 0.54-0.72 0.65 0.77-0.89

Man

Hematocrit

1 - 0.6

0.71

Rabbit

Ponder and Robinson (1934a) Ponder and Robinson (193413) Castle and Daland

(1937) C. Citrated Blood

Horse

0.56

Slawinski (1936)

422

D. A. T. DICK

to the membrane tension of 0.75 atmosphere suggested by Cristol and Benezech. f. As discussed in Section IV. B. 3, it is possible that some degree of intermolecular linkage modifies the osmotic properties of the hemoglobin, e.g., makes the osmotic coefficient considerably greater than that of a corresponding solution in vitro. g . Anomalous molal osmotic coefficient of hemoglobin in solution. This was used to account for the difference in vapor pressure and freezing-point depression between whole and laked blood (Roepke and Baldes, 1942; Williams et al., 1955). It has been used to account quantitatively for R values (Dick and Lowenstein, 1958 ; Dick, 1958b) (see Section IV. A ) . It may be noted that this explanation merely interprets the osmotic behavior of the erythrocyte in terms of that of hemoglobin solutions in vitro. Discussion of the actual mechanism involved is avoided, although as has been rioted in Section 111. D the large partial molal entropy of dilution of hemoglobin in solution due to the large size of its molecule plays a most important part in causing the large osmotic coefficient. 2. Theories applicable to the abnormal erythrocyte. a. Gelatin of the corpuscle interior (Ponder, 1940, 1948). This is associated with crenation which is produced by the action of oxalate (Ponder, 1944) or of citrate at low temperature (Ponder, 1945). It is supposed that elastic forces resist swelling in the gelated corpuscle and produce a low R value. b. Leakage of solute from the cell (Ponder and Robinson, 1934b). As has been seen above (Section IV. B. 2 ) , this does not occur in cells kept under good physiological conditions but can be expected to occur whenever the conditions are disturbed. Although ion leakage may contribute to the effects, losses actually measured are insufficient to account for the entire depression of the R value which is found (Davson, 1936, 1937). Agna and Knowles (1955), Streeten and Thorn (1957), and Riecker (1957) have shown from studies of the effect of change of plasma osmotic pressure by water injection, drinking, or abnormal pathological states that the erythrocyte in vivo undergoes shrinking and swelling in response to external osmotic changes similar to that observed in vitro.

E.

Volume-Osmotic Pressure Relationships in Cells Other Than Erythrocytes As described in Section IV. A, a graphical plot of total cell volume against the reciprocal of the osmotic pressure may be used to find the

OSMOTIC PROPERTIES OF L I V I N G CELLS

423

value of Wo,the apparent isotonic water content of the cell. The apparent nonsolvent volume, b', may be defined by the equation

Wo = Vo - b'

(39) Emphasis in the interpretation of osmotic experiments in all cells except the erythrocyte has hitherto been placed on the calculation of b'. This emphasis appears, however, to be misplaced ; it is much more important to calculate the value of Wo, the apparent isotonic water content, and to compare it with W,, the true water content obtained by direct methods, and thus obtain the value of Ponder's R as in the erythrocyte. This aspect of experiments on marine invertebrate eggs and leukocytes has been neglected. Owing to this neglect, the actual water content of cells used in osmotic experiments has in many cases not been given. Table IV has been constructed from values for the actual water content, W,, which are calculated by applying to the dry weight the average specific volume of 0.75; but the dry weight employed in the calculation has often 'been obtained from a different source so that it need not necessarily apply to the cells actually used in the osmotic experiment. This is particularly the case with Arbacia eggs, whose dry weight may be increased by the presence of a layer of jelly on the surface of the egg (Shapiro, 1935). This may account for the high R values shown for Arbacia in' Table IV. Hamburger and Math6 (1952, p. 46) obtained volume-osmotic pressure data on leukocytes and claimed to find a direct linear relationship between the volume and the applied osmotic pressure; in view of the other evidence and the sound theoretical basis available which predicts a hyperbolic relationship in this case, Hamburger and MathC's interpretation of their results is improbable. Ross ( 1953), in experiments on snail spermatocytes, claimed that the cells suffered no change in volume between osmotic pressures corresponding to 0.5% and 0.8% NaCl; however, the scatter of his observations is so high that no conclusion regarding constancy or change of volume can safely be drawn, and the claim made seems unjustified. The interpretation of R values in other cells may be considered by using the hypotheses which have already been applied to the erythrocyte. Some modifications must be made, however, particularly to take account of the lower concentration of protein in such cells. Thus it has been shown that a n osmotic coefficient for the cytoplasmic proteins comparable with that of a similar protein solution in vitro is probably insufficient to account for the observed R value in chick heart fibroblasts (Dick, 1958a). The difference may be accounted for by a modification of the osmotic properties of the protein due to intermolecular bond formation.

TABLE IV NONSOLVENT VOLUME A N D PONDER’S R IN CELLSOTHER THANERYTHROCYTES

Species

Technique

Range of relative osmotic pressure

b

wo a

(see text)

W,

R0

References Lillie (1916) F a d - F r e m i e t (1924) Skowron and Skowron (1926) Ephrussi and Neukomm (1927)

A . Eggs of Marine and Aquatic Invertebrates (Unfertilized) Arbacia punctulata Sabellaria alveolata Sphuerechinus granularis

Measurement of diameter Measurement of diameter Measurement of diameter

1-0.4

0.370

0.63

0.80a

0.79

2.24-0.25

0.31~

0.69

0.750

0.92

1-0.43

0.330

0.67

-

-

Paracentrotus lividus

Measurement of diameter

1.16-0.36

0.46c

0.54

0.740

0.73

d r b a c k punctulata

Measurement of diameter

1-0.5

0.07 0.13 0.14 0.09 0.12 0.39 0.44 0.320 0.57

0.93 0.87 0.86 0.91

0.80b 0.80b 0.80b 0.80b 0.80b

%

0.61 0.56 0.68 0.43

0.08 0.21 0.21 0.28

0.92 0.79 0.79 0.72

Arbacia punctulata (fragments) ArbacM punctulata Chaetopterk pergamentaceous Ostrea virginica Arbacia punctulata Limnea stagnalis

Measurement Diffraction Measurement Diffraction Measurement Measurement

of diameter of diameter

1-0.6 1-0.6 1-0.4 1-0.4 1.134.5 1.5-0.5

Arbacia punctulata Paracentrotus lividus Strongylocentrotus interrnedius Strongylocentrotus nudus

Measurement Measurement Measurement Measurement

of of of of

1-0.6 Hypotonic 1-0.6 1-0.5

of diameter of diameter

diameter diameter diameter diameter

0.88

1.08 1.14 1.11

}

+I

McCutcheon et al. (1931)

-

-

0.80b 0.800 (approx. ) 0.80b 0.740

0.85 0.50 1.16 1.07

Shapiro (1948) Mettetal (1948)

-

-

-

}

:

U

Luck6 (1932) Luck6 et al. (1935) Shapiro (1941) Luck6 and Ricca (1941) Churney (1942) Raven and Klomp (1946)

-

p ?

Shinozaki (1951)

TABLE I V (continued)

Species

Technique

Range of relative osmotic pressure

WO a

b

(see text)

W,,,

Ra

-

-

References

B. Other Cells Egg of Murphysa gruvelyi Rat lymphocyte (Lewis strain) Rat lymphocyte (Wistar strain) Mouse lymphdcyte (C3H strain) Gardner mouse ascites tumor Lewis rat lymphoma Murphy-Sturm rat lymphosarcoma Mouse sarcoma cell Chick heart fibroblast (plasma clot culture) Chick heart fibroblast (fluid plasma culture) Chick heart fibroblast (saline culture)

Measurement of diameter Opacimetry Opacimetry Opacimetry Opacimetry Opacimetry

1-0.45 1-20 1-2.0 1-2.0 1-2.0 1-20

0.670 0.36 0.26 0.21 0.31 0.40

0.33 0.64 0.74 0.79 0.69 0.60

0.89 0.89a 0.89 0.86e

Opacimetr y

1-20

0.36

0.64

-

-1

0.74 0.78

-

-

0.87a

0.82

0.87

0.90 I 0.94 1

0.87

1.15

Measurement of linear dimensions Immersion refractometry Immersion refractometry

1.78-0.39 1-0.58

0.18

0.00

1.00

0.886

0.83 0.72 0.89 0.80 0.68

Krishnamoorthi (1951)

1 *

i Luck6 et a!. (1956)

30 $

T:u

M

-

v)

}

1

Brues and Masters (19364

Dick (1958a)

Calculated by the present author. Calculated by the present author from data of Hutchens et ul. (1942). 0 Calculated by Luck6 and McCutcheon (1932). d Calculated by the present author from data of Joseph (personal communication from the Department of Human Anatomy, Oxford). e Calculated by the present author from data of Hempling (personal communication from Department of Physiology, Cornell University Medical School, New York) .

% E:

s

Z c,

n M r

r

v)

a b

R

VI

426

D. A. T. DICK

The work of McDowell et al. (1955) on the apparent volume of distribution of solutes injected into nephrectomized animals and the clinical water and salt balance studies of Wynn and Houghton (1957) and Wynn (1957) have shown that cells in vivo shrink and swell in response to changes of external osmotic pressure less than would be expected on the basis of the simple Boyle-van? Hoff law. This finding is in accordance with the accumulated observations on individual cells. McDowell et d. considered that the effect was due to “an idiogenic increase of osmotic pressure in proportion to applied osmotic stresses tending to dehydrate cells”; but it is probably to be at least partly explained by the osmotic properties of the intracellular proteins. An exception to the normal osmotic behavior of cells is the egg of teleost fishes. There is evidence that the egg of Salmo does not change its volume with variation in the external osmotic pressure (Gray, 1932) and that this is due to an almost complete impermeability to water (Prescott, 1955). In the activated egg of Fundulus immersed in hypertonic solution or sea water a hydrostatic pressure develops within the chorion which is produced by colloids in the perivitelline space. This pressure in turn tends to compress the egg itself so that its volume diminishes (Kao et al., 1954; Kao, 1956). F. Osmotic Behavior of the Nucleus Most of the observations recorded in Table IV refer to volume changes in the whole cell. A few observations are available relating to the volume changes in the nucleus and other inclusions. Beck and Shapiro (1936) found that in starfish eggs the nucleus swells in hypotonic solutions as the whole cell does but proportionately to a slightly greater extent than the whole cell. Similar swelling was noted by Kamada (1936). Churney (1942) measured the volume of the nucleus and of, the whole cell at different osmotic pressures and found also slightly greater relative swelling in the nucleus. By the use of an inappropriate method of statistical analysis, however, he reached the erroneous conclusion that the nonsolvent volume of the cell and nucleus is zero at near isotonic osmotic pressures but becomes positive in anisotonic solutions. If a correct method of regression analysis is applied to Churney’s data and the apparent isotonic water content ( W o ) is calculated by equation 31, it is found that W Ofor the nucleus is higher than that for the whole cell; for Arbacla punctulata the values of W o are for the whole cell 0.68 (S.E. 0.02) and for the nucleus 0.84 (S.E. 0.07). This interpretation also provides a reasonable explanation for Beck and Shapiro’s results. The fact that, when the chick heart fibroblast is immersed in a solution of the same refractive index as the cytoplasm, the’ nucleus frequently appears (with positive phase con-

OSMOTIC PROPERTIES O F LIVING CELLS

427

trast) very slightly brighter than either cytoplasm or background also supports the suggestion that the solid concentration of the nucleus is slightly lower than that of the cytoplasm and the water content is correspondingly higher (Barer and Dick, 1957). In contrast with the above results Marshak (1957) found that the nuclei of starfish eggs did not change volume either in hypotonic or hypertonic solutions of polyhydric alcohols or sucrose. Since in these experiments the egg cytoplasm was found to swell in both hypotonic and hypertonic solutions and considerable morphological changes occurred (e.g., nuclear extrusion), it is probable that the physiological state of the cells was considerably altered by the solutions employed. Harris (1943) concluded that the pigment granules of the sea urchin egg swell and shrink along with the cytoplasm in the same way as the nucleus. G . Conclusion Deviation of the osmotic behavior of cells from the conventional Boylevan? Hoff law is expressed by Ponder’s empirical factor R. An equation connecting the average osmotic coefficient of the intracellular protein with the value of Ponder’s R has been given. In consequence R is a much more significant cellular parameter than the apparent nonsolvent volume, b‘, on which osmotic studies have been conventionally based. The value of R may be useful in drawing conclusions concerning the physical state of the intracellular proteins and the possible presence of intermolecular chemical bonds. In order that accurate values of R for different cells may become available, it is essential that in all future osmotic studies the true water content of the cell be determined independently for comparison with the apparent water content. The immersion refractometry technique offers considerable advantages for this purpose. OF OSMOTIC VOLUME CHANGES I N LIVING CELLS V. KINETICS A . Introduction

Because movement of solute across the cell membrane is negligible in physiological saline solutions, the volume changes in cells produced by changes of external osmotic pressure are due almost entirely to the entry or exit of water. A study of the kinetics of such changes is thus equivalent to a study of the permeability of the cell to water. It must be recognized, however, that this is not the only criterion of permeability to water. Permeability may also be studied by measuring the rate of diffusion of isotopically labeled water (HiO, HiO, or H20l8) into or out of the cell. The two methods are not equivalent because the mechanisms of water transport in the two cases are not the same. In the case of osmotic water

428

D. A. T. DICK

transfer a net flow of water occurs across the cell membrane, whereas in the diffusion method there is only an exchange of labeled for unlabeled water molecules without net transfer. The water permeability measured by the osmotic method is always higher than that obtained by the diffusion method (Prescott and Zeuthen, 1953). The reason for this difference will be discussed in Section V. E. It is essential to appreciate the distinction between the two water permeabilities before examining the theory of osmotic water transfer. The subject of water permeability of cells has been reviewed by Luck6 and McCutcheon ( 1932), Luck6 ( 1940), Brooks and Brooks ( 1941), Davson and Danielli (1952), Jacobs (1952), Ussing (1952), and Harris (1956). An extensive introductory account of theoretical aspects of diffusion and permeability processes has been given by Jacobs (1935).

B.

Theory of Osmotic Water Permeability It has been a basic assumption of all measurements of the water permeability of cells that resistance to the passage of water is effectively confined to the membrane of the cell. This assumption has been made more because it was considered essential to the mathematical treatment of the problem than because it was supported by evidence (this question is fully discussed in Section V. F), but it has nevertheless become implicit in the technical use of the term permeability and in the choice of units to measure it. The permeability or volume of water crossing the cell membrane is assumed to depend on ( 1) the area of the membrane, (2) the time of permeation, and (3) the difference of osmotic pressure between the two sides of the membrane. The conventional unit of water permeability is thus the number of cubic microns of water crossing one square micron of the cell membrane in one minute in response to a difference of osmotic pressure of one atmosphere, or p3/p2/min./atm. If the dimensions of this unit are reduced to the simplest form, it may be expressed with no ambiguity as p min.-' atm.-l. The osmotic water permeability unit described above may be converted into what may be called a concentration permeability unit by expressing the difference of osmotic pressure which appears in it as a difference of water concentration. The method of this calculation has been described by Frey-Wyssling (1946) and LZvtrup and Pigon (1951) and the transformation has been employed by Prescott and Zeuthen (1953) and Harris (1956). The resulting concentration permeability unit when reduced to its lowest dimensions is expressed as p/sec. The basic equation used in the interpretation of the kinetics of osmotic water flow is

OSMOTIC PROPERTIES O F LIVING CELLS

dV = kA(rI - n,)

429

(40)

dt where V is the volume of the cell, t the time of measurement, k the permeability coefficient, A the area of the cell membrane, and rI and He the internal and external osmotic pressures. Two assumptions underlying this equation are (1) that the mechanism of permeation of the cell membrane is one of simple diffusion, i.e. there are no energy barriers requiring activation energies to overcome them and (2) that the resistance of the membrane does not vary during osmotic volume changes. I n order to apply it in practice, equation 40 must be integrated ; several methods have been used for doing this. 1. I n the case of the erythrocyte which has a disc shape, the volume may be greatly increased without significant change in the area of the cell membrane. Jacobs ( 1932) therefore performed the integration by treating A as a constant. By substituting for V , using the simple equation IIV = rIOV0 (Jacobs’ symbols have been altered to conform to present usage), obtained , r10Vo drI - - - -- kA (rI - II,) r12 dt of which the integral form is drI kAt = -rIovo II2 (rI - rIe) or

he

where TIe is the osmotic pressure of the external medium. No allowance is made in this equation for the nonsolvent volume in the erythrocyte. Dick (1959a) used a similar expression in treating kinetics of osmosis in chick heart fibroblasts in a cover slip culture. These cells, being thinly spread out on the cover slip, alter their volume largely by changes of thickness, and the surface area available for water transfer remains practically constant. The substitution for V is performed by using an equation already obtained from equilibrium experiments (Dick, 1958a) : 0.817 I I o V o V = rI + b or dV 0.817rIoV0 (43)

so that the nonsolvent volume is allowed for.

430

D. A. T. DICK

2. I n spherical cells the area of the cell membrane may be expressed in terms of the volume of the cell by the equation A = (36.rr)%V ? S (44) and the integration is performed by treating the area as a function of the volume. The equation so obtained by LuckC et al. ( 1931 ) was

k ( 3 6 ~ ) ~ ~ (Yo f l o - b ) t = (Ye- b) X

where Vo = initial total cell volume. no = initial intracellular osmotic pressure. V, = total cell volume at final equilibrium with external medium. V = total cell volume at time t. This equation has been used in all subsequent studies of spherical cells. Historically, two equations which are now obsolete were used in permeability studies. Lillie ( 1916), in the first mathematical treatment of permeability, used a supposed analogy with monomolecular chemical reactions and obtained the equation

dV- K(II dt

- 11,)

where K is a constant; the surface area has been assumed constant and combined with the permeability coefficient, k, to form a different constant, K . The constant K could not of course be used to compare the permeabilities of cells of different area. The integral form of equation 46 was given by Lillie as 1 V , - Yo K=-ln (47) t - Vt

v,

This equation contains two errors, however : (1) it has not been integrated correctly, and (2) the assumption that the surface area does not alter during volume changes in spherical cells is obviously incorrect and leads to a dependence between the value of the external osmotic pressure and the permeability coefficient obtained by the use of Lillie's equation. LuckC and McCutcheon (1927), who used Lillie's equation, found just such a relationship in the eggs of Arbacia punctulata. Northrop (1927) corrected both of the errors in Lillie's equation. H e

OSMOTIC PROPERTIES OF L IVI N G CELLS

43 1

used a different approach, however, in estimating the effect of cell swelling or shrinking on the resistance of the membrane to the passage of water. His basic equation took account of the thickness as well as the area of the cell membrane (the symbols have been altered to conform) :

where k' is a constant, and h is the thickness of the cell membrane. If the volume of the membrane, m, is assumed to remain constant during changes of cell volume, then, since m is the product of the area and thickness of the membrane,

h =

m

A

and

If m is incorporated into the permeability coefficient, the resulting equation is

where k" is a new constant of permeability. A similar equation was also derived by Northrop on the different assumption that water transfer takes place through pores whose diameters change with change of area of the cell membrane. It was shown by .Luck6 et al. (1931) that the course of osmotic changes in the egg of Arbacia punctuhta is described equally well by equations 45 and 50, so that it is not possible to distinguish between them on empirical grounds ; nevertheless equation 45 has been preferred in subsequent studies, e.g., Luckk et al. (1956). C. Methods of Measuring Osmotic Water Permeability

As pointed out in the previous section, this is largely a question of determining the rate of change of cell volume in an anisotonic solution. Many of the methods of measurement of cell volume already described in Section IV. C have therefore been applied to the measurement of cell permeability, particularly diameter measurement, measurement of angular diffraction, measurement of opacity of cell suspensions, and recently immersion refractometry.

432

D. A. T. DICK

Two further methods of measuring cell water permeability of a different type are the hemolytic method of Jacobs applied to erythrocytes and the simultaneous measurement of water and solute permeability. When erythrocytes are hemolyzed in hypotonic solutions, the occurrence of hemolysis is always associated with a definite and constant increase in the volume of the erythrocytes. I n practice, since not all the cells of a given sample hemolyze at once, but the percentage of hemolysis increases with the degree of swelling of the erythrocytes, it is found that when cells are 75% hemolyzed the volume of the erythrocytes has always increased to 1.70 times their isotonic volume (Jacobs, 1934). The degree of hemolysis of the erythrocyte suspension is measured by a simple opacimetric method, and the time required to produce 75% hemolysis is noted; from the known increase in volume associated with this and the corresponding time the permeability coefficient of the erythrocyte is measured (Jacobs, 1932, 1934). I t has been shown, however (Jacobs, 1955), that the accuracy of the hemolytic method may be seriously affected by prehemolytic ion transfers across the cell membrane. When cells are suspended in isotonic saline which has been made hypertonic by the addition of a penetrating solute, they first shrink in response to the hypertonic environment, and then swell as the penetrating solute enters the cell, finally returning to their original volume when the penetrating solute has reached diffusion equilibrium across the cell membrane. I t has been shown by Jacobs and Stewart (1932) and by Jacobs (1933a, b) that, if the size and time of attainment of minimum volume are measured, then the permeability coefficients both of water and of the penetrating solute can be calculated. Methods for calculating the two permeability coefficients from the rate of swelling of cells in a pure solution of a penetrating solute have also been given by Jacobs (1933a, b) and applied to the erythrocyte (Jacobs, 1934). An equation for use when both penetrating and nonpenetrating solutes are present is given by Jacobs (1933~).

D. Osmotu Permeability Coefiicients of Cells Data collected from the literature have been summarized in Table V. Besides the permeability coefficients, there are also shown diffusion COefficients for the rate of water diffusion in the protoplasm. These have been calculated by neglecting the resistance of the cell membrane according to the method discussed in Section V. F. The ratio of surface area to volume is also shown for these cells for which sufficient data are available. It is seen that there is a large variation in the permeability coefficients and that this variation is related to the surface-volume ratio (this relationship is fully discussed in Section V. F).

TABLE V PERMEABILITY COEFFICIENT OF CELL. MEMBRANE AND DIFFUSIONCOEFFICIENT OF WATERIN CYTOPLASM (See Text)

1

Cell Amoeba proteus Amoeba Zebra fish egg Frog egg Xenopus egg Eggs of: Arbacia punctulata Arbacia punctulata Paracentrotus lividus Arbacia punctuluta

2

3

Direction of Technique water flow" Measurement in capillary' Ex tube End Diameter measurement Diameter measurement End End Diameter measurement End Diameter measurement

4

5

6

Permeability Diffusion coefficient coefficient Temperature (p min-1 (cm.Z/sec) ("C.) atm.-l) ( X 1010) 18-25 0.0268 -

-

-

0.017 0.020 0.059 0.072

-

7

8

Surfacevolume ratio (p2/p3)

0.039

0.009 0.003

1

References Mast and Fowler

(1935) Prescott and Zeuthen

(1953)

0.004

Diameter measurement Diffraction Diameter measurement Minimum volume method

End End End Ex

15 22 20-22 21-22

0.05 0.106 0.06-0.16 0.15

0.087

Diameter measurement Linear measurement

Ex End

20 14.5-16

0.133-0.187 0.125-0.25

0.086 0.234

Minimum volume method Diffraction (sea water) Diffraction (ethylene glycol solution) Diffraction (diethylene glycol solution) Diameter measurement

Ex End Ex

22-23

0.166 0.14-0.19 0.20

0.079

Ex

-

Luck6 et al. (1931) Luck6 et al. (1935) Mettetal (1948) Stewart and Jacobs

-

(1935) Fucus vesiculosis Peritrich Eggs of: Arbacia punctulata Arbacia punctulata Arbacia punctulata

Strongylocentrotus intermedius

Ex End

-

24

22.7

20.2-22.5

Resuhr (1935) Kitching (1938)

-

0.21

-- 1

0.212 0.197

-

Jacobs (1933a) Luck6 et al. (1951) Luck6 et al. (1951)

}

Shinozaki (1952)

J

End - endosmosis ; Ex -exosmosis. This value should probably be doubled since Mast and Fowler assumed the internal osmotic pressure of the Amoeba to be only Sm-osm. instead of l07m-osm. as measured by Lfivtrup and Pigon (1951). 0

b

TABLE V (continued)

1

Cell Strongylocentrotus Purpuratus Strongy locentrotus franciscanus Urechis caupo Patiria miniata Dendraster excentricus Pisaster ochraceous Strongy locentrotus intermedius Strongylocentrotus nudus Strongylocentrotus intermedius Strongy locentrotus nudus

2

6 Diffusion coefficient (cm.Z/sec)

7 Surfacevolume ratio

8

( X 1010)

(pz/p3)

References

-

0.072

Direction of water flowa End

Temperature

Technique Diameter measurement

("C.) 17-22

5 Permeability coefficient (p min-1 atm.-1) 0.093

Diameter measurement

End

17-22

0.128

0.047

Diameter measurement Diameter measurement Diameter measurement

End End End

17-22 17-22 17-22

0.138 0.206 0.207

0.049 0.033 0.047

Diameter measurement Diameter measurement in penetrating solvent Diameter measurement in penetrating solvent Diameter measurement

End End Ex End Ex End

17-22 22 19 22 22 20

0.409 0.27 0.25 0.45 0.38 0.2

0.035 -

End

Ex

24 23

0.25 0.38

-

End Ex End Ex End Ex End

22.5 22.5 22.5 22.5 22.5 22.5 21.3-25

0.12 0.17 0.38 0.41 0.44 0.46 0.49

0.078 0.078 0.092 0.092 0.057 0.057 0.060

Diameter measurement

Arbacia Punctulata

Diffraction

Cumingia tellenoides

Diffraction

Chaetopterus Pergamentaceous Chaetopterus Pergamentaceous

Diffraction Diameter measurement

3

4

Leitch (1931)

Shinozaki (1951)

LuckC et al. (1939)

LuckC et al. (1939) Shapiro (1941)

P P

C A

1

2

Cell

Technique

TABLE V (continued) 4 5 Permeability coefficient (p min-1 Direction of Temperature atm-1) ("C.) water flowa 0.410 Ex 3

6 Diffusion coefficient (cm.z/sec) ( X 1010)

7 Surfacevolume ratio

8

(pZ/p3)

References

Ex Ex

-

0.470 0.49c

2.8 13 15

0.86 0.90 0.62

Opacimetry

Ex

-

0.490

9.1

0.56

Opacimetry Opacimetry Diameter measurement

Ex Ex End

-

Room temp.

0.560 0.620 0.7

15 15 -

0.88 0.66 0.55

Opacimetry Opacimetry

End End

20-23 20-23

0.29 1.35

-

0.75

Diameter measurement Diameter measurement Hemolysis (penetrating solute) Hemolysis method Hemolysis method Immersion refractometry

End End End

21-26 21.5

2.2 0.7

0.02

Room temp.

2.5

-

End End End

Room temp. Room temp. 38

2.2 3.0

5.0 -

1'75

2.82

6.6

1.37

Dick (1958b)

End and E x

23-26

5.7

-

1.88

Side1 and Solomon (1957)

C3H mouse lymphocyte Wistar rat lymphocyte Murphy-Sturm lymphoma Gardner mouse ascites tumor Lewis rat lymphocyte Lewis rat lymphoma Chick heart fibroblast (plasma clot culture) Rabbit leukocyte Human leukocyte

Opacimetry Opacimetry Opacimetry

Giant axon of Loligo Giant axon of Sepia O x erythrocyte

Ox erythrocyte Human erythrocyte Chick heart fibroblast (fluid plasma culture) Diffraction Human erythrocyte

Brues and Masters (1936b) Shapiro and Parpart (1937)

Hill (1950) Jacobs (1931)

1.75

1.88

}

m 4

z

rn

8

c

5 z

0

cl

M F

t: Jacobs (1932)

Dr. H. G. Hempling of the Department of Physiology, Cornell University Medical College, New York, has corrected an error in the calculation of the published figures and the corrected values are given here by permission of Dr. Hempling. c

M

P

436

D. A. T. DICK

The relation of endosmosis to exosmosis may be seen from the table. Although the data of Luck6 et al. (1939) show that exosmosis is consistently slightly more rapid than endosmosis, the results of Shinozaki (1951) are inconsistent. The values given for the permeability of the erythrocyte are widely different. In view of the possibility of prehemolytic ion movements in the hemolytic method of Jacobs (Jacobs, 1955), the higher value of 5.7 p min.-' atm.-l given by Side1 and Solomon (1957) is to be preferred to those of Jacobs (1932, 1934). The rate of osmotic flow of HZ0 into erythrocytes was studied by Parpart (1935), who found that it was 44% slower than that of HzO. This finding was confirmed by Brooks (1935), but it was explained by the fact that the lower fugacity of Hi0 caused a lower osmotic pressure difference than expected. Luck6 and Harvey (1935), in a similar study on Arbacia eggs, equilibrated the cells first in an isotonic HZO solution before the beginning of the permeability experiment and under these conditions found no difference between the permeabilities of HZO and HzO.

E. Difference between Oslnotic and Diffusion Methods of Measuring Water Permeability It has already been indicated that two difficulties arise in comparing the value of water permeability obtained by measuring the net flow of water through the cell membrane in response to an osmotic gradient with that obtained by measuring the diffusion of labeled water through the membrane. The first difficulty arises from the different mechanisms in the two cases, the one involving net transfer and the other merely exchange of water molecules. The second arises from the impossibility of a strict comparison of the permeability coefficients used and this will be dealt with first. Jacobs (1935) claimed that Fick's law of diffusion is not applicable to substances in high concentration such as the water in biological systems (concentration approximately 55M) and that in consequence it is not possible to compare the permeability of water with that of other substances whose permeability is studied in low concentrations. The modern physico-chemical approach is, however, to regard Fick's law as applicable at all concentrations but to recognize that the value of the diffusion coefficient which is inserted into Fick's equation varies with the concentration. Thus although Jacobs' arguments are not now acceptable, his conclusion that permeability coefficients measured at widely different concentrations are not directly comparable is still valid. If this principle were applied to the comparison of osmotic permeability measurements

437

OSMOTIC PROPERTIES O F LIVING CELLS

with those obtained from the diffusion of isotopic water whose concentration is very low, then it might be expected to introduce into the comparison a further difficulty in addition to the basic difference of mechanism mentioned above In this case, however, since the molecules of normal and isotopic water are physically almost identical, the water concentration involved is the total concentration of normal and isotopic water which is of course similar in osmotic and isotopic experiments. A small difference in the permeability coefficients obtained may be expected analogous to the difference between self-diff usion and diffusion coefficients, but the discrepancy from this source is unlikely to be of practical importance. If the difficulty of comparison of permeability coefficients is ignored and the osmotic water permeability is expressed in terms of concentration difference-i.e., as grams of water passing through one square centimeter of cell membrane in one second with a difference of water concentration of one gram per cubic centimeter (this unit when reduced to its lowest dimensions is cm./sec., or, more conveniently, p/sec.)-then it is found that the osmotic is always greater than the diffusion permeability. This difference was demonstrated for several egg cell membranes and the amoeba by Prescott and Zeuthen (1953) and for the erythrocyte by Sidel and Solomon ( 1957). The available comparative data are summarized TABLE VI COMPARISON OF WATERPERMEABILITY COEFFICIENTS MEASURED BY AND OSMOTIC METHODS

Cell

Diffusion water Osmotic water permeability permeability coefficient coefficient ( d s e c .) (Wsec.1

Xenopus egg Zebra fish egg Amoeba

0.90 0.36 0.23

1.59 0.45 0.37

Human erythrocyte

53

125

THE

DIFFUSION

References

I J

Prescott and Zeuthen (1953)

{

Sidel and Solomon (1957) Paganelli and Solomon (1957)

in Table VI. It seems certain that a difference of this size has some fundamental cause. Koefoed-Johnsen and Ussing (1953) and Ussing and Andersen (1956) have suggested that this big difference is due to bulk flow of water through pores in the cell membrane and that the area and diameter of the pores may be calculated from it. Such calculations have been per-

438

D. A. T. DICK

formed by Paganelli and Solomon ( 1957) and by Nevis (1958). Chinard (1952) has claimed, however, that the conception of osmotic transfer of water through a membrane as a bulk flow is wholly mistaken. O n the other hand, Kuhn (1951), Pappenheinier (1953), and Garby (1955) claim that osmotic and hydrostatic pressures can exert exactly equivalent effects on a membrane, including a bulk flow of solvent through it. Pappenheimer (1953) has further produced evidence that if the radius of the postulated “pores“ in the biological membrane concerned exceeds 20 A. then a large part of any net solvent transfer either under a hydrostatic or under an osmotic pressure gradient consists of a bulk flow. Poiseuille’s law is applicable to such a flow, thus enabling the area and diameter of the pores to be calculated. I t must be noted, however, that none of the evidence produced by Pappenheimer is relevant to membranes having smaller pore radii than 20 A., although Renkin (1954) has produced a modified theory which appears to be applicable down to pore radii of 15 A. Although there is some evidence that Pappenheimer’s theory is applicable to a membrane similar to the capillary membrane (Mauro, 1957) and to frog gastric mucosa (Durbin et ul., 1956), there appears to be no experimental support for its application to the cell membrane. Harris (1956) has put forward an alternative explanation for the difference observed between the osmotic and diffusion permeability coefficients of the cell membrane; it is based on a solution of a similar problem in potassium transfer in nerve axons by Hodgkin and Keynes (1955). If the diffusing water molecules lie in long narrow pores in the cell membrane, then it is to be expected purely on grounds of statistical probability that the net transfer of water molecules, irrespective of label, will greatly exceed the flux of labeled water molecules under similar concentration gradients. Whatever the ultimate explanation may be, it may be concluded that Harris’ theory removes any compulsion to postulate bulk flow through the cell membrane merely on the grounds of the difference between the osmotic and diffusion permeability coefficients.

F.

T h e Rate of Diffusion of Water through the Cell Protoplasm

The almost universal assumption in permeability studies that water transfer under osmotic gradients is significantly resisted only at the cell membrane will now be examined. The diffusion of water both in the internal protoplasm of the cell and in the external medium is considered to take place with such rapidity that the time so occupied is negligible. This assumption has been introduced on grounds of mathematical convenience in order to obtain a numerical expression of the permeability of the cell, but no evidence has been produced to support it. O n the contrary, evidence is available which suggests that it is probably untrue.

OSMOTIC PROPERTIES O F L I V I N G CELLS

439

It has been shown by Kamada (1936) and by Beck and Shapiro ( 1936) that, when an invertebrate egg cell swells in hypotonic solution and the nucleus also swells, the time relations of nuclear swelling are not the same as those of the whole cell; both the onset and the termination of nuclear swelling are delayed. Kamada also noted that on transfer of an egg from tonicity A to a lower tonicity, B, the rate of swelling between the stages corresponding to an intermediate tonicity, C, and the final tonicity, B, was slower than that of an egg transferred to tonicity B after being first equilibrated at tonicity C. These facts point strongly to the conclusion that equilibration within the egg protoplasm is delayed because diffusion of water through the protoplasm requires a significant length of time. A further pointer to the same conclusion is seen by examining the permeability data presented in Table V. An inverse correlation exists between the size of the cell and its permeability coefficient. This may be expressed in the form of a direct correlation between the permeability coefficient and the surface-volume ratio. For spheres this ratio is inversely proportional to the radius, since Surface = 4x1.2

4 3

Volume = -4 and thus Surface - 3 Volume r The correlation is best examined by taking logarithms of both permeability coefficient and surface-volume ratio ; the resulting relationship is shown graphically in Fig. 2. The correlation is highly significant ( P < 0.001). (Only two values deviate considerably from the rest, those relating to the giant axons of Loligo and Sepia. It may be shown that the mean of these values deviates significantly ( P < 0.001) from the mean regression line, and thus it may be concluded that some other factor is operating in this case.) It is an obvious interpretation of this correlation that the apparent decrease in the permeability attributed to the cell membrane of the larger cells (which have small surface-volume ratios) is due to the length of time that water takes to diffuse through the proportionately larger volume of the internal protoplasm (see also Dick 1959b). In further investigation of this conclusion, it is useful to examine the consequences of making an opposite assumption regarding the cell, i.e., that the resistance to water entry is uniformly distributed through the protoplasm of the cell, including its membrane. This assumption makes possible an alternative mathematical treatment from which the average

440

D. A. T. DICK

diffusion coefficient of water in the cell protoplasm may be calculated. The equations used are given by Crank (1956) ; for spherical cells

for flat approximately planar cells such as the fibroblast in fluid culture (see Dick, 1959a).

M, = fractional volume increase at time t. Mm = fractional volume increase at equilibrium. a = radius of sphere at mean volume. 1 = thickness of cytoplasm at mean volume. D = diffusion coefficient. t = time of diffusion. ( I n order to simplify the mathematical treatment it is necessary to assume a volume-fixed boundary for diffusion equivalent to the position of the cell membrane when half of the total volume increase in time t has taken place.) The diffusion coefficients of water in the protoplasm calculated by this means are shown in column 6 of Table V. Two points may be noted about them : cm.2/sec. to 5 X 1. The range of values from 1.5 x cm?/sec. is much lower than the comparable diffusion coefficients for protein-water systems in vitro which lie for the most part between and 10-6 cm?/sec. (Edsall, 1953). 2. The diffusion coefficients are inversely correlated with the surfacevolume ratio as illustrated in Fig. 3 ; the correlation is statistically significant ( P < 0.001) . Both facts point to an important contribution by the membrane to the resistance of the cell to water entry. Since both the permeability of the cell membrane and the diffusion coefficient of water in the protoplasm are intrinsic properties of membrane and protoplasm, there is no obvious reason why they should be correlated with the absolute size or surface-volume ratio of the cell. By assuming that water entry into the cell is impeded by a nonuniform resistance, which is most intense in the cell membrane, but is also present in the protoplasm, the correlations which are found may be attributed to error in the calcula-

441

OSMOTIC PROPERTIES OF LIVING CELLS

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442

D. A. T. DICK

tions due to neglecting the role of either protoplasmic or membrane resistance. It is likely that in any given case the true permeability of the cell membrane is higher than the value calculated by neglecting the resistance of the protoplasm, and the true diffusion coefficient of water through the protoplasm is also higher than the value calculated by neglecting the resistance of the cell membrane. By making the further assumption that the permeability coefficient of the cell membrane and the diffusion coefficient of water through the protoplasm are practically uniform in all the cells studied, it would be possible to calculate the values of these quantities. Such a calculation is being attempted (Crank and Dick, 1959). It is of interest that Harris (1957) and Harris and Prankerd (1957) have concluded from a study of the kinetics of permeation that protoplasmic resistance as well as membrane resistance determines the rate of entry of cations into frog muscle and human and dog erythrocytes.

G. Conclusion There are a number of different methods of measuring and expressing the permeability of the cell to water which are based on various assumptions about the cell. The accuracy of these assumptions largely determines the significance of the different permeability values obtained. These methods and the units employed may be summarized as follows. A. Methods assuming instantaneous diffusion within the cell and giving an apparent permeability coefficient of the cell membrane. 1. Osmotic difference method. a. Expressed in osmotic units (p min.-' atm.-l). b. Expressed in concentration units (p sec.-l). 2. Diffusion method using isotopic water (p sec.-l). B. Method assuming uniform distribution of water resistance in both cell membrane and cytoplasm and giving an average diffusion coefficient of water in the protoplasm (cm.2 sec.-l). The simple conversion of osmotic permeability units into concentration permeability units involves theoretical difficulties which may or may not be of practical significance. The diffusion permeability measurement gives lower values than the osmotic one, probably owing to the statistical laws of molecular transfer by random movement in long narrow pores in the cell membrane. The concept of osmotic water movement as a bulk flow is possibly mistaken. Available evidence suggests that the basic assumptions underlying both methods A and B are erroneous. Water movement is probably most strongly impeded at the cell membrane, but the internal protoplasm of the cell also offers significant resistance. Consequently all permeability

OSMOTIC PROPERTIES OF LIVING CELLS

443

coefficients calculated by conventional methods are probably too low. In order to supply more evidence bearing on this conclusion, further studies of the osmotic permeability of isolated cells must include data of the cell dimensions for the calculation of the surface-volume ratio and sufficiently full time-volume data for the calculation of the diffusion coefficient of water in the protoplasm. VI.

ACKNOWLEDGMENTS

Many helpful suggestions and criticisms received from Drs. R. L. Baldwin, R . Barer, J. Crank, E. J. Harris, and A . G. Ogston, F.R.S. are gratefully acknowledged. VII. REFERENCES Adair, G. S. (1929) Proc. Roy. SOC. A M , 16. Adair, G. S., and Robinson, M. E. (1930) Biochem. J. 24, 1864. Adolph, E. F., and Richmond, J. (1956) A m . J. Physiol. 187, 437. Aebi, H. (1952) Helv. Physiol. Acta 10, 184. Agna, J. W., and Knowles, H. C. (1955) J. Clin. Invest. 34, 919. Appelboom, J. W. (1957) Federatiofi Proc. 16, 278. Babbitt, J. D. (1955) Science 122, 285. Barer, R. (1956) In “Physical Techniques in Biological Research” (G. Oster and A. W. Pollister, eds.), Vol. 111. Academic Press, New York. Barer, R., and Dick, D. A. T. (1957) Exptl. Cell Rcsearch Suppl. 4, 103. Barer, R., and Joseph, S. (1954) Quart. J . Microscop. Sci. 96, 399. Barer, R., and Joseph, S. (1955a) Quart. J. Microscop. Sci. 96, 1. Barer, R., and Joseph, S. (1955b) Quart. J . Microscop. Sci. 96, 423. Bartley, W., Davies, R. E., and Krebs, H. A. (1954) Proc. Roy. SOC.B l a , 187. Beck, L. V., and Shapiro, H. (1936) Proc. SOC.Exptl. Biol. Med. 34, 170. Bernstein, R. E. (1955) I. Clin. Puthol. 8, 225. Bolani, T. R. (1932) “The Donnan Equilibria.” G. Bell and Sons, London. Brodsky, W. A., Appelboom, J. W., Dennis, W. H., Rehm, W. S., Miley, J. F., and Diamond, I. (1956) J. Gen. Physiol. 40, 183. Brooks, S. C. (1935) I. Cellular Comp. Physiol. 7, 163. Brooks, S. C., and Brooks, M. M. (1911) “The Permeability of Living Cells.” Protoplasma-Monographien, Borntrager, Berlin. Brues, A. M., and Masters, C. M. (1936a) A m . J. Cancer 28, 314. Brues, A. M., and Masters, C. M. (1936b) A m . J. Cancer 28, 324. Bull, H. B. (1951) “Physical Biochemistry.” Wiley, New York. Burk, N. F. (1932) J. Biol. Chew. 98, 353. Butler, J. A. V. ( 1946) “Chemical Thermodynamics,” Macmillan, London. Caldwell, P. C. (1956) Intern. Rev. Cytol. 6, 229. Calkins, E., Taylor, I. M., and Hastings, A. B. (1954) A m . J. Physiol., 177, 211. Castle, W. B., and Daland, G. A. (1937) A . M . A . Arch. Internal Med. 60, 949. Chaplin, H., and Mollison, P. L. (1952) Blood 7, 1227. Chinard, F. P. (1952) Ant. J. Physiol. 171, 578. Chinard, F. P., and Ems, T. (1956) Science IN, 472. Christensen, I., and Warburg, E. J. (1929) Actu Med. Scand. 70, 286.

444

D. A. T. DICK

Churney, L. (1942) Biol. Bull. 83, 52. Clowes, G. H. A., and Krahl, M. E. (1936) J. Gen. Physiol. 20, 145. Conway, E. J. (1957) Physiol. Revs. 37, 84. Conway, E. J., and Geoghegan, H. (1955) 1. Physiol. (London) lSD, 438. Conway, E. J., and McCormack, J. I. (1953) 1. Physiol. (London) 120, 1. Conway, E. J., Geoghegan, H., and McCormack, J, I. (1955) J. Physiol. (London) 130, 427. Crank, J. (1956) “The Mathematics of Diffusion.” Clarendon Press, Oxford. Crank, J., and Dick, D. -4. T. (1959) In preparation. Creese, R. (1954) Proc. Roy. SOC.BlQ, 497. Cristol, P., and Benezech, C. (1946a) Arch. intern. physiol. 64, 148. Cristol, P., and Benezech, C. (1946b) Arch. intern. physiol. 54, 153. Cristol, P., and Benezech, C. (1948) 1. physiol. (Paris) 40, 160A. D’Amato, H. E. (1954) Am. J . Physiol. 178, 143. Darrow, D. C., and Hellerstein, S . (1958) Physiol. Revs. 98, 114. Davson, H. (1936) Biochem. J . SO, 391. Davson, H. (1937) J. Cellular Comp. Physiol. 10, 247. Davson, H., and Danielli, J. F. (1952) “The Permeability of Natural Membranes.’’ Cambridge Univ. Press, London. Dervichian, D. G., Fournet, G., and Guinier, A. (1947) Compt. rend. 224, 1848. Deyrup, I. (1953) J. Gen. Physiol. S6, 739. Dick, D. A. T. (1958a) Proc. Roy. SOC.Bl49, 130. Dick, D. A. T. (1958b) Exptl. Cell Research 14, 608. Dick, D. A. T. (1959a) Proc. Roy. SOC.BlM), 43. Dick, D. A. T. (1959b) Exptl. Cell Research in press. Dick, D. A. T., and Lowenstein, L. M. (1958) Proc. Roy. SOC.B148, 241. Donnan, F. G. (1911) 2. Elektrochem. 17, 572. Drabkin, D. L. (1950) J. B i d . Chem. 185, 231. Durbin, R. P., Frank, H., and Solomon, A. K. (1956) J. Gen. Physiol. 39, 535. Ebaugh, F. G., Levine, P., and Emerson, C. P. (1955) 3. Lab. Clin. Med. 46, 409. Edsall, J . T. (1953) In “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. I, Part B, pp. 549-726. Academic Press, New York. Ege, R. (1922) Biochem. 2. WO, 99. Ephrussi, B., and Neukomm, A. (1927) Compt. rend. SOC. biol. 96, 1291. FaurC-Fremiet, E. (1924) Arch. anat. microscop. 20, 212. Flory, P. J. (1942) 1. Chem. Phys. 10, 51. Flory, P. J. (1945) J . Chem. Phys. 13, 453. Flory, P. J. (1953) “Principles of Polymer Chemistry.” Cornell Univ. Press, Ithaca, New York. Flory, P. J., and Krigbaum, W. R. (1951) Ann. Rev. Phys. Chem. 2, 383. Frey-Wyssling, A. (1946) Ezperientia 2, 132. Frey-Wyssling, A. (1953) “Submicroscopic Morphology of Protoplasm.” Elsevier, Amsterdam. Frey-Wyssling, A. (1955) In “Protoplasmatologia” (L. V. Heilbrunn and F. Weber, eds.), Vol. 11, Part A2. Frey-Wyssling, A. (1957) “Macromolecules in Cell Structure.” Harvard Univ. Press, Cambridge, Massachusetts. Gaffney F. M. (1958) In preparation. Garby, L. (1955) Acta Physiol. Scand. 35, 88.

OSMOTIC PROPERTIES OF LIVING CELLS

445

Gaudino, M. (1956) Am. J. Physiol. 187, 75. Giraud, G., Latour, H., and Levy, A. (1950) Presse mtd. 66, 949. Glasstone, S. (1948) “Textbook of Physical Chemistry.” Macmillan, London. Gough, A. (1924) Biochem. J . 18, 202. Gray, J. (1932) J. Exptl. Biol. 9, 277. Grim, E. (1953) Proc. SOC.Exptl. Biol. Med. 83, 195. Guest, G. M. (1948) Blood 3, 541. Guest, G. M., and Wing, M. (1942) J . Clin. Invest. 21, 257. Guggenheim, E. A. (1950) “Thermodynamics.” North Holland Publ. Co., Amsterdam. Guggenheim, E. A. ( 1952) “Mixtures.” Clarendon Press, Oxford. Guntelberg, A. V., and Linderstr$m-Lang, K. (1949) Compt. rend. trav. lab. Carlsberg, str. chim. 27, 1. Hamburger, H. J. (1898) Arch. Anat. u. Physiol. Abt. Physiol. 317. Hamburger, J., and MathC, G. (1951) Presse mid. 69, 265. Hamburger, J., and Math&, G. (1952) “Metabolisme de l’eau.” Editions mkdicales, Flammarion, Paris. Harris, D. L. (1943) Biol. Bull. 86, 179. Harris, E. J. (1956) “Transport and Accumulation in Biological Systems.” Butterworths, London. Harris, E. J. (1957) J. Gen. Physiol. 41, 169. Harris, E. J., and Prankerd, T. A. J. (1957) J . Gen. Physiol. 41, 197. Heilbrunn, L. V. (1952) “Outline of General Physiology.” Saunders, Philadelphia. Hempling, H. G. (1954) J. Cellular Comp. Physiol. 44, 87. Hempling, H. G. (1958) J . Gen. Physiol. 41, 565. Henderson, L. J. (1928) “Blood. A Study in General Physiology.” Yale Univ. Press, New Haven, Connecticut. Hendry, E. B. (1954) Edinburgh Med. J . 61, 7. Hildebrand, J. H. (1955) Science, 121, 116. Hildebrand, J. H., and Scott, R. L. (1950) “The Solubility of Non-electrolytes.” Reinhold, New York. Hill, A. V. (1930) Proc. Roy. SOC.BlOS, 477. Hill, D. K. (1950) J. Physiol. (London) 111, 304. Hodgkin, A. L,and Keynes, R. D. (1955) J. Physiol. (London) 126, 61. Huggins, M. L. (1942) Ann. N.Y. Acad. Sci. 48, 1. Hunter, F. R. (1953) J. Cellular Comp. Physiol. U, 387. Hunter, F. R., Chalfin, D., Finamore, F. J., and Sweetland, M. L. (1956) J . Cellular Comp. Physiol. 47, 37. Hutchens, J. O., Keltch, A. K., Krahl, M. E., and Clowes, G. H. A. (1942) J. Gen. Physiol. %, 717. Jacobs, M. H. (1932) Biol. Bull. Ba, 178. Jacobs, M. H. (1933a) I. Cellular Comp. Physiol., 2, 427. Jacobs, M. H. (1933b) J. Cellular Comp. PhySiol. 3, 29. Jacobs, M. H. (1933~) J. Cellular Comp. Physiol. 3, 121. Jacobs, M. H. (1934) J. Cellular Comp. Physiol. 4, 161. Jacobs, M. H. (1935) Ergebn. Biol. 12, 1. Jacobs, M. H. (1952) In “Modern Trends in Physiology and Biochemistry” (E. S. G. Barron, ed.). Academic Press, New York. Jacobs, M. H. (1955) J. Cellular Comp. Physiol. IS, 356. Jacobs, M. H., and Stewart, D. R. (1932) I. Cellular Comp. Physiol. 1, 71.

446

D. A. T. DICK

Kamada, T. (1936) J . Fac. Sci. Univ. Tokyo Ser. I V 4, 215. Kao, C. Y. (1956) J . Gen. Physiol. 40, 91. Kao, C. Y., Chambers, R., and Chambers, E. L. (1954) J . Cellular Comp. Physiol.

44, 447. Kitching, J. A. (1938) J . Exptl. Biol. 16, 143. Klotz, I. M. (1953) In “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. I, Part B, pp. 727-806. Academic Press, New York. Koefoed-Johnsen, V., and Ussing, H. H. (1953) Acta Physiol. Scund. 28, 60. Krevisky, C. (1930) Biochem. J. 24, 815. Krishnamoorthi, B. (1951) Proc. Indian Acad. Sci. B34, 199. Kuhn, W. (1951) 2. Elecfrochem. 66, 207. Landis, E. M. (1927) A m . I. Physiol. 82, 217. Leaf, A. (1956) Biochem. J . 62, 241. Leeson, D., and Reeve, E. B. (1951) J . Physiol. (London) 116, 129. LeFevre, P. G., and LeFevre, M. E. (1952) J . Gen. Physiol. 36, 891. Leitch, J. L. (1931) Univ. Calif. (Berkeley) Publ. Zool. 38, 127. Lewis, G. N., and Randall, M. (1923) “Thermodynamics.” McGraw-Hill, New York. Lewis, M. S., and Saroff, H. A. (1957) J . A m . Chem. SOC.79, 2112. Lillie, R. S. (1916) Am. I. Physiol. 40, 249. Lindenbaum, A., and Schubert, J. (1956) I. Phys. Chem. 60, 1663. L@vtrup, S., and Pigon, A. (1951) Compt. rend. trav. lab. Carlsberg Sir. chim.

a,1.

LuckC, B. (1932) J . Cellular Comp. Physiol. 2, 193. LuckC, B. (1940) Cold Spring Harbor Symposia Quant. Biol. 8, 123. LuckC, B., and Harvey, E. N. (1935) J . Cellular Comp. Physiol. 6, 473. LuckC, B., and McCutcheon, M. (1927) J. Gen. Physiol. 10, 665. LuckC, B., and McCutcheon, M. (1932) Physiol. Revs. 12, 68. LuckC, B., and Parpart, A. K. (1954) Cancer Research 14, 75. LuckC, B., and Ricca, R. A. (1941) I. Gen. Physiol. 26, 215. LuckC, B., Hartline, H. K., and McCutcheon, M. (1931) I. Gen. Physiol. 14, 405. Luck&, B., Larrabee, M. G., and Hartline, H. K. (1935) J . Gen. Physiol. 19, 1. LuckC, B., Hartline, H. K., and Ricca, R. A. (1939) J . Cellular Comp. Physiol. 14, 237.

LuckC, B., Ricca, R. A., and Parpart, A. K. (1951) J . Natl. Cancer Inst. 11, 1007. LuckC, B., Hempling, H. G., and Makler, J. (1956) J . Cellular Comp. Physiol. 47, 107.

McCutcheon, M., LuckC, B., and Hartline, H. K. (1931) 1. Gen. Physiol. 14, 393. McDowell, M. E., Wolf, A. V., and Steer, A. (1955) Am. J . Physiol. 180, 545. Macleod, J., and Ponder, E. (1933) J . Physiol. (London) 77, 181. McMeekin, T. L., Groves, M. L., and Hipp, N. J. (1954) J . Am. Chem. SOC.76,

407. Maffly, L. H., and Leaf, A. (1959) Nature in press. Maizels, M. (1954) Symposiu SOC.Exptl. Biol. No. 8, 202. Manery, J. F. (1954) Physiol. Revs. 34, 334. Mark, H., and Tobolsky, A. V. (1950) “Physical Chemistry of High Polymeric Systems.” Interscience, New York. Marrack, J., and Hewitt, L. F. (1929) Biochent. J . 23, 1079. Marshak, A. (1957) Exptl. Cell Research 12, 599. Mast, S. O.,and Fowler, C. (1935) I. Cellular Comp. Physiol. 6, 151. Mauro, A. (1957) Science 126, 252.

OSMOTIC PROPERTIES OF LIVING CELLS

447

Mettetal, C. (1948) Bull. inst. ocianog. No. 926. Mitchison, J. M. (1953) J. Exptl. Biol. SO, 397. Mudge, G. H. (1951) A m . J. Physiol. 167, 206. Mudge, G. H. (1953) A m J. Physiol. 173, 511. Nevis, A. H. (1958) J. Gen. Physiol. 41, 927. Nichols, G., and Nichols, N. (1953) J. Clin. Invest. 32, 113. Northrop, J. H. (1927) J. Gen. Physiol. 11, 43. Ogston, A. G. (1956) Proc. Intern. Wool Textile Research Conf. 1955 Vol. B, p. 92. Commonwealth Scientific and Industrial Research Organisation, Melbourne. Oncley, J. L., Scatchard, G., and Brown, A. (1947) J. Phys. Chem. 61, 184. Opie, E. L. (1949) J. Exptl. Med. 89, 185. Opie, E. L. (1954) J. Exptl. Med. 99, 29. Opie, E. L., and Rothbard, M. B. (1953) J. Exptl. Med. 97, 483. Jdrskov, S. L. (1946) Acta Physiol. Scand. 12, 202. Overbeek, J. T. G. (1956) Progr. in Biophys. and Biophys. Chem. 6, 58. Paganelli, C. V.,and Solomon, A. K. (1957) J. Gen. Physiol. 41, 259. Palade, G. E. (1955) J. Biophys. Biochem. Cytology 1, 567. Palade, G. E. (1956) J . Biophys. Biochem. Cytology 2, Suppl. 85. Pappenheimer, J. R. (1953) Physiol. Revs. 33, 387. Parpart, A. K. (1935) J. Cellular Comp. Physiol. 7, 153. Parpart, A. K. (1940) Cold Spring Harbor Symposia Quant. Biol. 8, 25. Parpart, A. K., and Ballantine, R. (1943) Science 98, 545. Parpart, A. K., and Shull, J. C. (1935) J. Cellular Comp. Physiol. 6, 137. Ponder, E. (1929) I. Exptl. Biol. 6, 387. Ponder, E. (1933a) Cold Spring Harbor Symposia Quunt. Biol. 1, 170. Ponder, E. (1933b) Cold Spring Harbor Symposia Quunt. Biol. 1, 178. Ponder, E. (1935a) J. Physiol. (London) 83, 352. Ponder, E. (1935b) 1. Physiol. (London) I, 439. Ponder, E. (193%) Proc. SOC.Exptl. Biol. Med. 33, 156. Ponder, E. (1940) Cold Spring Harbor Symposia Quant. Biol. 8, 133. Ponder, E. (1944) J. Gen. Physiol. 27, 273. Ponder, E. (1945) J. Gen. Physiol. 29, 89. Ponder, E. (1947a) J. Gen. Physiol. 30, 235. Ponder, E. (1947b) J. Gen. Physiol. 30, 379. Ponder, E. ( 1948) “Hemolysis and Related Phenomena.” Churchill, London. Ponder, E. (1949) Discussion Faraday SOC.No. 6, 152. Ponder, E. (1950) J. Gen. Physiol. 33, 177. Ponder, E. (1951a) J. Gen. Physiol. 34, 567. Ponder, E. (1951b) J. Exptl. Biol. 28, 567. Ponder, E. (1953) J. Gen. Physiol. 36, 767. Ponder, E. (1955) In “Protoplasmatologia.” (L. V. Heilbrunn and F. Weber, eds.), Vol. X, Part 2. Ponder, E., and Barreto, D. (1957) Blood 12, 1016. Ponder, E.,and Robinson, E. J. (1934a) J . Physiol. (London) 83, 34. Ponder, E., and Robinson, E. J. (1934b) Biochem. J. B, 1940. Ponder, E.,and Saslow, G. (1930) I . Physiol. (London) 70, 18. Ponder, E., and Saslow, G. (1931) J. Physiol. (London) 73, 267. Prescott, D. M. (1955) J . Cellular Comp. Physiol. 46, 1. Prescott, D. M., and Zeuthen, E. (1953) Acta Physiol. Scand. 28, 77. Raven, C. P., and Klomp, H. (1946) Proc. Acad. Sci. Amsterdam c49, 101. Renkin, E. M. (1954) J. Gen. Physiol. 38, 225.

448

D. A. T. DICK

Resuhr, B. (1935) Protoplasma 24, 531. Riecker, G. (1957) Klin. Wochschr. p. 1158. Riecker, G., Zack, W., and Renschler, H. E. (1957) Arch. ges. Physiol. Pfliiger‘s 264, 245. Robinson, J . R. (1950) Proc. Roy. SOC.B131, 378. Robinson, J. R. (195%) Proc. Roy. SOC.B140, 135. Robinson, J. R. (1952b) Nature 169,713. Robinson, J. R. (1953) Biol. Rev. 28, 158. Robinson, R. A., and Stokes, R. H. (1955) “Electrolyte Solutions.” Butterworths, London. Roepke, R. R., and Baldes, E. J. (1942) I. Cellular Comp. Physiol. 20, 71. Ross, K. F. A. (1953) Quart. J. Microscop. Sci. 94, 125. Scatchard, G., Batchelder, A. C., and Brown, A. (1946) I. A m . Chem. SOC.68, 2320. Scatchard, G., Scheinberg, I. H., and Armstrong, S. H. (1950) J. Am. Chem. SOC. 72, 535. Scatchard, G., Coleman, J. S., and Shen, A. L. (1957) J. Am. Chem. SOC.79, 12. SchiZdt, E. (1932) I. Gen. Physiol. 16, 977. Schulz, G. V. (1947) 2. Naturforsch 2a, 27. Schwartz, I. L., and Opie, E. L. (1954) J. Clin. Invest. 38, 964. Shapiro, H. (1935) Biol. Bull. 66, 363. Shapiro, H. (1941) J . Cellular Comp. Physiol. 18, 143. Shapiro, H. (1948) J. Gen. Physiol. 32, 43. Shapiro, H., and Parpart, A. K. (1937) J. Cellular Comp. Physiol. 10, 147. Shinozaki, J. (1951) J. Fac. Sci. Hokkaido Univ. Ser. V I 10, 113. Shinozaki, J. (1952) 1. Fac. Sci. Hokkaido Univ. Ser. V I 11, 107. ShohI, A. T., and Hunter, T. H. (1941) J. Lab. Clin. Med. 26, 1829. Sidel, V. W., and Solomon, A. K. (1957) J. Gen. Physiol. 41, 243. Skowron, S. and Skowron, H. (1926) Bull. intern. acad. polon. sci. CIasse sci. math. nat. Sir. B 859. Slawinski, A. (1936) Bull. intern. mad. polon. sci. Classe mid. p. 951. SZrensen, S. P. L. (1917) Compt. rend. trav. lab. Carlsberg Sir. chim. 12, 1. Starling, E. H. (18%) J. Physiol. (London) 19, 312. Stern, J. R., Eggleston, L. V., Hems, R., and Krebs, H. A. (1949) Biochem I. 44, 410. Stewart, D. R., and Jacobs, M. H. (1935) J. Cellular Cmp. Physiol. 7, 333. Streeten, D. H. P., and Thorn, G. W. (1957) I. Lab. Clin. Med. 49, 661. Swan, R. C., Feinstein, H. M., and Madisso, H. (1956) I. Clin. Invest. 36, 607. Teorell, T. (1952) J. Gen. Physiol. !JS, 669. Terner, C., Eggleston, L. V., and Krebs, H. A. (1950) Biochem. I., 47, 139. Tosteson, D. C., and Johnson, J. (1956) J . Cellular Comp. Physiol. 47,147. Ussing, H. H. (1952) Advances in Enzymol. 13, 21. Ussing, H. H., and Andersen, B. (1956) Proc. 3rd Intern. Congr. Biochem. Brussels, 1955. Van Slyke, D. D., Wu, H., and McLean, F. C. (1923) J. Biol. Chem. 66, 765. Weber, H. H., and Stover, R. (1933) Biochem. 2. 269, 269. Whittam, R. (1956) I. Physiol. (London) 131, 542. Whittam, R., and Davies, R. E. (1953) Biochem. J. 66, 880. Wilbrandt, W. (1948) Helv. Physiol. Acta 6, 234. Williams, T. F., Fordham, C. C., and Hollander, W. (1955) J. Clin. Invest. s4, 971. Wynn, V. (1957) Lancet 2, 1212. Wynn, V., and Houghton, B. J. (1957) Quart. J. Med. 26, 375.

Sodium and Potassium Movements in Nerve, Muscle, and Red Cells I. M. GLYNN Cambridge University, Cambridge, England I. Introduction ...................................................... 11. Outline .......................................................... 111. Evidence ......................................................... A. Ion Distribution and the Resting Potential ..................... B. Ionic Equilibria in Red Cells ................................. C. Active Nerve and Muscle ..................................... D. Energy for Active Ion Movements ............................ 1. The Role of Adenosine Triphosphate ..................... E. The Link between Active Potassium Influx and Active Sodium Efflux ....................................................... F. The Passive Fluxes .......................................... G. Ion Transport Systems ....................................... IV. References .......................................................

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I. INTRODUCTION Living cells usually contain more potassium than sodium, and this is remarkable because in the fluid outside the cells-tissue fluid or the aquatic environment-sodium is nearly always more plentiful than potassium. Even in those few cells which do not contain more potassium than sodium, it is generally found that potassium is in higher concentration, and sodium in lower concentration, within the cell than in the external fluid. The origin of this unequal distribution is obscure. There are, of course, intracellular enzymes which are activated by potassium and inhibited by sodium, but such specificity is likely to be secondary to the ion distribution rather than its cause. Any cell which contains protein and which has a membrane permeable to water and salts has to face the problem of water entry, and it may be that cells early acquired the ability to balance the osmotic pressure of the cell protein by expelling sodium ions. If this were so, then the accumulation of potassium might originally have been the result merely of the excess of negative ions left within the cell; the smaller size and greater mobility of the hydrated potassium ion would help potassium accumulation, though a difference in the passive permeability to sodium and potassium is not essential to the theory. A third hypothesis is that the unequal distribution of sodium and potassium was, from the start, associated with cell excitability. The excitability of muscle and nerve cells, and of certain algal cells, is well known to depend on the unequal distribu449

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tion of sodium and potassium ions across the cell membrane, but here again it is difficult to know which is primary and which secondary. The purpose of this review is not, however, to consider the historical origin of the sodium and potassium distribution in living cells, but rather to discuss the way in which cells now control the movements of sodium and potassium across their membranes. About this much more is known. Discussion will be concerned mainly with muscle, nerve, and red cells, as it happens that these are the tissues that have been most studied-nerve and muscle largely with a view to understanding excitability, and red cells partly because of their convenience and partly because of a mistaken belief in their simplicity. During the past ten years it has become apparent that, in their behavior to sodium and potassium ions, the membranes of nerve, muscle, and red cells possess certain common features. It is too early to say whether these common features form a pattern which, occurring in nearly all living cells, is responsible for the characteristic sodium : potassium distribution, but this may prove to be so. There are, of course, other features of the permeability of nerve, muscle, and red cell membranes, which are peculiar to each tissue and which are related to the special functions of each type of cell. The procedure which will be adopted is first to describe in outline, and without evidence, what the common pattern of fluxes is believed to be, and how in nerve and muscle this pattern is altered during activity. The various lines of evidence that have led to the described view will then be considered. The three types of cell will be treated together, as it seems more useful to emphasize resemblances and differences than to give a connected historical account of the development of views about each. Nevertheless, some account of this development will be given.

11. OUTLINE The high potassium concentration and low sodium concentration found in nerve, muscle, and red cells is thought to depend on the activity of a pump in the membrane which uses metabolic energy to accumulate potassium and expel sodium. The active movements of potassium and sodium appear to be linked, though whether the ions move in a one-to-one ratio is uncertain ; the ratio may not be simple or even fixed. Unlimited increase in the potassium concentration, and fall in the sodium concentration, is prevented by passive leakage of sodium and potassium across the membrane. Leakage will occur in both directions, but the net movements will be outward for potassium and inward for sodium, and a steady state will be reached when the passive net movements of each ion just equal the active movements. The passive movements of sodium and potassium ions will carry charges across the membrane, so generating an electrical potential,

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and the pump may or may not contribute directly to this potential, depending on whether the ratio is one-to-one and whether the pumped cations are accompanied by anions. Ions, such as chloride, which can pass through the membrane but which, as far as is known, are not subject to active transport will distribute themselves in such a way that they are at the same electrochemical potential on the two sides of the membrane. It follows that in the steady state the equilibrium for chloride and similar ions is a true thermodynamic equilibrium, whereas that for sodium and potassium is not and indeed cannot be. If it were, the passive fluxes in the two directions would be equal, and active movements would cause departure from the steady state. If, however, the passive permeability to one of the pumped ions were high, so that the passive movements were very large compared with the active movements, the difference between the steady-state distribution and true thermodynamic equilibrium might be small. I t will be seen that this is probably true of potassium in resting nerve and muscle. During the spread of excitation in nerve and muscle it is believed that depolarization brought about by local circuits causes a rapid and very marked but transient rise in sodium permeability followed by a smaller but longer lasting rise in potassium permeability. While the membrane is highly permeable to sodium ions there is a net entry of sodium, and the inward movement of positive charge is responsible for the ascending part of the spike potential. At the top of the spike the potential is very far from the equilibrium potential for resting nerve, so that there is a net loss of potassium and gain of chloride, and when the sodium permeability is shut off these movements continue until equilibrium is restored. The delayed rise in potassium permeability, which coincides with the falling phase of the spike, facilitates the exit of potassium and so hastens the return to the resting potential. Each action potential results in a small gain of sodium and loss of potassium, but slightly increased activity of the exchange pump during the period following a burst of activity restores the original ion distribution. 111. EVIDENCE In what follows it will be assumed that the distribution of sodium and potassium depends on the permeability properties of the membrane and not on ion binding in the cell interior. The evidence against the view that bound ions play a large part in producing the ion distribution has been summarized by Keynes and Adrian ( 1956), Conway (1957), and Glynn (1957b) and appears to be overwhelming. Harris ( 1957) and Harris and Prankerd (1957) suggested that the kinetics of sodium and potassium exchange in muscle and red cells could be explained by supposing that the

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ions were impeded by slow intracellular diffusion as well as by the resistance of the cell membrane, and they postulated the existence within the cell of sodium- and potassium-specific sites able to bind a large proportion of the internal cations. But the deviation from first-order kinetics on which their theory is based can, as they mention, be explained alternatively by the existence of several compartments, and the possibility that their results arise from variation between individual cells is not excluded. In view of the known variation between cells in a population (Henriques and grskov, 1936; Conway, 1957) and the difficulties in the theory that a large proportion of the internal cations is bound, the alternative explanation seems preferable, unless it can be shown that the exchange of sodium or potassium in a single cell departs from first-order kinetics. This is a possibility that could be tested with single muscle fibers. The binding of a high proportion of intracellular cations has also been postulated by Shaw and Simon (1955a) and by Simon et al. (1957), but the grounds on which they argue that the distribution of sodium and potassium cannot be explained by the properties of the membrane are open to question. Their results are, however, more conveniently discussed later.

A . Ion Distribution and the Resting Potential Arrhenius’ theory of electrolytic dissociation was published in 1887, Nernst’s formula for the potential of a concentration cell in 1899, and Bernstein’s “membrane theory” of nerve conduction in 1902. Two years before Bernstein’s theory, Macdonald (1900) had shown that the potential between the surface and the cut end of a nerve was related logarithmically to the salt concentration in the bathing fluid, but, under the conditions he used, it is doubtful if the potential he measured bore much relationship to the normal resting potential. In its original form the “membrane theory” postulated that the nerve or muscle membrane was permeable to potassium, but not to sodium or anions; and supposed that the tendency of potassium ions to diffuse out of the fiber gave rise to the resting potential. In spite of a description of net chloride movement by Meigs and Atwood in 1916, the impermeability of the membrane to chloride was accepted fairly unquestioningly until about 1936, for reasons which are summarized by Fenn in a review of that year. Very briefly, impermeability to chloride accounted for the good agreement, in muscle, between the chloride space and histological estimates of extracellular space; it explained why the chloride content of a muscle was roughly proportional to the chloride concentration in the bathing fluid-a muscle in isotonic sucrose rapidly losing all its chloride-and it fitted in with the observation that the potential was very sensitive to the

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external potassium concentration but little affected when chloride was replaced by sulfate. In 1941 Boyle and Conway made a detailed study of the distribution of potassium and chloride in frog sartorius fibers placed in modified Ringer solutions containing different amounts of potassium. They found that, if the potassium content of the solution was increased by adding solid potassium chloride, then potassium and chloride entered the fiber, but the fiber volume at equilibrium was unchanged. If, however, the potassium concentration was increased by substituting potassium for sodium in the Ringer solution, then potassium and chloride entered the fiber accompanied by sufficient water to keep the internal potassium concentration constant. In both cases equilibrium was approached with a time constant of about one hour, and at equilibrium

where [ K]s, [ Cl] o, etc., represent the concentration of potassium chloride, etc., inside the fiber or in the bathing fluid. That is, the distribution of potassium and chloride appeared to be determined by a -Donnan relationship. Similar observations on crab nerve were made by Shanes in 1946. If the distribution of potassium and chloride represents a Donnan equilibrium, the potential across the membrane should be given by the Nernst equation ;

E

=

RT

log, F

P I* PI

0

where E is the membrane potential, and RT and F have their usual significance. Boyle and Conway verified this in two ways. They measured the potential between two points on the surface of a muscle in contact with pads of cotton wool soaked in Ringer solutions of different strengths, and they also measured the potential between the surface and the cut end of a muscle using as electrodes pads of cotton wool soaked in identical Ringer solutions. They found remarkably good agreement between the theoretical predictions and the observed values, although it now seems probable that the closeness of the agreement-at which they were “at first much surprised,” in view of the expected short-circuiting-was partly due to the setting up of diffusion potentials tending to offset the potential drop caused by the short circuit. Boyle and Conway found that at 4°C. the membrane potential and the distribution of potassium and chloride fitted a Donnan equation over

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a range of external potassium concentrations from 12 to 300 mM. Below 12 mM there was marked divergence from the theoretical behavior, and muscles showed a steady loss of potassium and gain in sodium. Since the muscle potassium remains constant in the intact animal with only 2.5 mM potassium in the plasma, it seems that the removal of the muscle from the body, or the use of artificial bathing fluids, alters the membrane in some way. Lack of phosphate in the bathing fluid is probably partly to blame, since Fenn and Cobb (1934) showed that, if phosphate at p H 7.2 was added to the Ringer solution, muscle potassium could be maintained constant at 4°C. with only 5 mM potassium outside. Similar divergence from simple Donnan behavior at physiological potassium concentrations was mentioned by Shanes (1946) in his experiments with crab nerves. Although Boyle and Conway were the first to regard the resting potential as a Donnan potential, a roughly logarithmic relationship between [ K l 0 and membrane potential had been described by Fischer in the frog sartorius in 1924, and by Cowan in Muiu nerve in 1934. Similar observations were made on two other sorts of crab nerve-Libiniu and Grapsus-by Shanes and Hopkins (1948), and on frog sciatic nerve by Shanes (1944) and by Feng and Liu (1949). All these authors used external electrodes, and their results are therefore subject to error because of short-circuiting, but all agree that the simple logarithmic relationship broke down when the external potassium concentration was less than about 10 mM. For further advance it was necessary to be able to measure membrane potentials without the short-circuiting error, and this was first done in squid giant axons by pushing fine electrodes longitudinally down the inside and measuring the potential across the membrane directly (Hodgkin and Huxley, 1939; Curtis and Cole, 1940). A method of wider application was developed in Gerard’s laboratory (Graham and Gerard, 1946; Ling and Gerard, 1949), where it was shown that KC1-filled micropipets with tip diameters of less than 0.5 p could be pushed through cell membranes without apparent damage and could therefore serve as intracellular electrodes. If the pipets are filled with 3 M KCl, the liquid junction potential between the electrode fluid and the cytoplasm can be greatly reduced (Nastuk and Hodgkin, 1950), although Adrian ( 1956) has shown that, unless the electrodes are carefully selected, junction potentials can still arise, due, he thinks, to blocking of the tip in such a way that the mobility of the Cl- ion is reduced and the difference in mobility of Na+ and K+ ions exaggerated. KC1-filled microelectrodes have been used to investigate the relationship between potassium distribution and membrane potential in frog muscle by Ling and Gerard ( 1950), Jenerick and Gerard ( 1953), Jenerick ( 1953), Harris and Martins-Ferreira ( 1955), Adrian ( 1956), and Conway (1957) ; and in squid axons by Hodgkin and Keynes

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(1955a, b) . A third method of avoiding short-circuiting errors, developed by Huxley and Stampfli (1951) for use on single myelinated nerve fibers, depends on balancing the potential to be measured with an externally applied potential, the fiber being so arranged that short-circuiting is the same for both. With a few exceptions, which will be discussed later, measurements made by the methods just outlined show that (1) with more than 10 mM potassium outside there is a logarithmic relationship between [ K l 0 and the membrane potential ; (2) even at high external potassium concentrations, the slope of the regression line of membrane potential on [ K l 0 is less than that predicted by the Nernst equation-i.e., 58 mv. for a tenfold change in [ K l 0 (Fig. 1 ) ; (3) at physiological external potassium concentrations the membrane potential of isolated nerve and muscle fibers is considerably less than the Nernst potential ; and ( 4 ) the discrepancy is much less for nerve and muscle fibers in situ in the living animal (Ling and Gerard, 1949; Moore and Cole, 1955 ; Hodgkin, 1958). The reason for the difference in behavior between isolated fibers and fibers in situ is not known, but it may be related to the inability of isolated fibers to maintain their internal potassium concentration when placed in artificial media containing potassium at the same concentration as in plasma. Conway’s theory (Boyle and Conway, 1941) does not allow for any contribution to the membrane potential from ions other than potassium and assumes that the fiber is in a steady state with potassium at the same electrochemical potential on the two sides of the membrane. If these assumptions are not made, to calculate the potential it is necessary to write down equations for the current carried by each sort of ion, and from these to find the potential for which the total current is zero. The first step involves assumptions about the way in which ions move in the membrane under the influence of osmotic and electrical forces; the second involves assumptions about the change of potential across the thickness of the membrane. A treatment of this sort was carried out by Goldmann ( 1943) and by Hodgkin and Katz ( 1949). If ions in the membrane move under the influence of diffusion and electrical forces in a manner similar to that of ions in free solution, then

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where I K , I N a , and Icl represent, respectively, the current carried by potassium, sodium, and chloride; uK, u N n , and uc1 are the mobilities of the ions in the membrane; CK, CNa,and Ccl are the concentrations of the ions at a distance x from the surface of the membrane; and $ is the electrical potential. Hodgkin and Katz showed that, if the potential gradient is assumed to be uniform across the whole thickness of the membrane, and the concen-

-

-$ 100 -

120

110

70 -

90

80-

60-

-

c

>

50

-

E 40-

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FIG.1. The effect of external potassium concentration on the membrane potential of the frog sartorius muscle. (From Adrian, 1956.) Abscissa : potassium concentration, millimolar (log scale). Ordinate : membrane potential, millivolts. 0,membrane potential in chloride solutions ; 0, membrane potential in sulfate solutions ; half-filled circles denote membrane potential in chloride solutions with half the normal concentration of NaCI. Where the number of muscles is not indicated by a figure in brackets, the point is the average value of four muscles. The lines are drawn according to the equation

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tration of each ion at the edge of the membrane is proportional to the concentration in the adjacent solution, then the membrane potential is given by

where p p , PNa, and pc1 are permeability constants for the individual ions and are themselves functions of the ionic mobilities in the membrane and partition coefficients. Because of the assumption of uniform potential gradient, this equation is generally referred to as the “constant field equation.” Since one of the premises on which it is based is that the ions move subject only to diffusion and the potential gradient, it does not allow for a direct contribution to the membrane potential from active ion movements. If p~ is large compared with f N a and pol, then at high external potassium concentrations the equation reduces to the familiar

RT

=log, F

P I

6

-

IK

L Hodgkin and Katz showed that the results of Curtis and Cole (1942) on squid axon could be fitted well in the physiological range of external potassium concentrations by taking p K : p N a : pol = 1 : 0.04 : 0.45 although at higher potassium concentrations a better fit was given by PK : PN. : PCI = 1 : 0.025 : 0.3. Since there is evidence that depolarization leads to an increase in potassium permeability (Hodgkin et al., 1949; Hodgkin and Keynes, 1955b), the need to change the ratio of the permeabilities to get a fit over the whole range of external potassium concentrations does not disprove the theory. In frog muscle Jenerick (1953), extending the work of Ling and Gerard (1950) and Jenerick and Gerard (1953), showed that the variation of membrane potential with [K], in the range 1 to 110mM could be described by the constant field equation, with p p : P N a : ~ C = I 1 :0.027: 0.23. Similar results were obtained by Harris and Martins-Ferreira (1955) with the South American frog Leptodactylus. Jenerick also measured the total membrane conductance by inserting two microelectrodes into a single fiber and observing the membrane potential during the application of a polarizing current. H e found that the increase in conductance as [K], was raised could be accounted for only if pg varied inversely with the membrane potential, but his reasoning involves the assumption that [ K]‘ is constant and has been criticized by Adrian (1956) on the grounds that the raising of [ K l 0 by the addition of solid potassium chloride would have led to an entry of potassium into the fiber.

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Adrian has made a careful study of the relationship between the resting potential of fibers of isolated frog sartorius muscles and the ratio of the potassium concentrations on the two sides of the fiber membranes. If muscles are placed in solutions of sodium and potassium sulfates containing different amounts of sucrose, they can be made to swell or shrink without taking up or losing potassium. With [K], constant at 75 mM, Adrian showed that the resting potential varied logarithmically with [ K ] 4. The slope of the regression line of potential on log [K]a corresponded to a shift of 50.2 mv. for a tenfold change in concentration, but the difference between this figure and the theoretical 58 mv. was probably largely due to changes in the internal activity coefficient resulting from changes in the internal ionic strength. The effect of [ K l 0 on the resting potential under conditions in which [K], could be assumed constant is shown in Fig. 1. The regression line of membrane potential on log [K], calculated for the muscles in solutions containing 25, 50, and 100 m M potassium has a slope which corresponds to a change of 52.3 mv. for a tenfold change in [K],, and to account for this on the constant field assumption Adrian calculated that the ratio p p : p N a must be about 30. (Chloride was assumed to be distributed passively according to the potential.) At physiological potassium concentrations a higher ratio was required (cf. the figures for squid axons), but it was difficult to test the equation at very low potassium concentrations because [ Cl] could not be assumed constant and p,, [ Cl]4 was no longer negligible compared with p K [ K ] ,. Adrian’s figure for the slope of the linear part of the graph of membrane potential against potassium distribution ratio is greater than those of Ling and Gerard ( 1950) and of Jenerick and Gerard ( 1953), but less than that found by Conway (1957). Ling and Gerard, and Jenerick and Gerard, varied the external potassium concentration by substituting potassium for sodium in isotonic Ringer solution and allowed the muscle to equilibrate for 10 minutes before the potential measurements were made. Conway (1957) has pointed out that this time may have been too short, and in experiments in which muscles were allowed to equilibrate overnight in solutions whose potassium concentrations had been raised by the addition of solid potassium chloride, he found a slope of 57 mv. for a tenfold change in ratio, i.e., not ,significantly different from the theoretical. In Adrian’s experiments, however, muscles were placed in sulfate solutions, in which equilibrium should be reached very rapidly, since sulfate is a nonpenetrating ion ; yet the slope was appreciably less than the theoretical. The difference between these results and those of Conway remains unexplained. Another way of investigating the relationship between membrane poten-

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tial and potassium distribution across the membrane is to deplete the tissue of potassium by storage at low temperature in a potassium-poor medium, and then to follow the change in potential as potassium is reaccumulated at a higher temperature in a medium richer in potassium. Stephenson (1958) soaked frog sartorii in potassium-free saline solution for 11 to 18 hours at 4" to 6"C., then transferred them to a saline solution containing 1 0 m M potassium at 25°C. for 50 minutes. During the second part of the experiment the muscles took up an average of 31 meq. of potassium per liter of fiber water and lost a similar amount of sodium, but, instead of the 9-mv. rise in potential predicted by the Nernst formula, Stephenson found that the average potential changed very little and there was a tendency for the potential of individual fibers to move toward 40mv. These observations are difficult to explain, but they may arise from the fact that potassium accumulation depends on all the fibers, whereas the potential measurements are made only on the surface fibers. Desmedt (1953) noticed that when frog sartorii were stored in potassium-free saline solution at 2" to 3°C. for more than 6 hours the surface fibers showed increased mechanical resistance to microelectrode puncture and did not show reversal of the overshoot when the muscles. were subsequently incubated in high-potassium solutions, even though analyses of the whole muscles showed satisfactory sodium extrusion. H e interpreted the changes in the surface fibers as signs of deterioration and found that they could be prevented by the presence of 0.2 mM potassium in the original storage solution. In the potassium-free saline solution the deeper fibers were thought to be protected by the small amount of potassium present in the interspaces as a result of continuous leakage. In a single experiment on a muscle soaked for 31 hours in 0.2 mM potassium saline solution and allowed to recover for 90 minutes in 1 0 m M potassium saline solution, the resting potential rose by 11 mv. during the recovery period (Desmedt, 1953). Another factor which may have influenced Stephenson's results is that both his storage and recovery solutions were free from calcium, and this may have increased the sodium permeability of the fibers (cf. Weidmann, 1955). Observations on the changes in resting potential produced by direct injection of salts have been made in squid axons by Grundfest et d.(1954) and by Hodgkin and Keynes (1956), and in frog sartorius muscle fibers by Falk and Gerard (1954). In interpreting the results of such experiments it is necessary to take into account the effects of both cations and anions and to consider the distribution of injected material along the fiber. Unless the change in concentration produced by the injection occurs over

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a length of fiber at least several times the space constant for passive potential spread ( 6 mm. for a squid axon in sea water-Cole and Hodgkin, 1939), the observed potential will not be given by the constant field equation even if the assumptions on which this equation is based are true. In squid axons, injection of potassium chloride sufficient to raise the internal potassium concentration by 30 mM had very little effect on the potential (Grundfest et al., 1954; Hodgkin and Keynes, 1956), but the chloride and potassium ions would have opposite effects and the expected change is only a few millivolts-the exact value depends on the ratio p K / p o l . More remarkable is the observation of Grundfest et al. that injections of potassium glutamate or aspartate of sufficient size to raise the internal potassium concentration three- or fourfold did not raise the resting potential. However, the onset of progressive decline in spike amplitude and of propagation block shortly after the injections, and irrespective of the internal ionic changes produced, suggests that the injection procedure may have damaged the membrane. I n frog sartorius fibers, Falk and Gerard found that the injection of very small volumes of 3 M KCl or 3 M NaCl had little effect on the resting potential. Since the intracellular chloride is so low, the effect of both salts should have been to cause a fall in resting potential, although exact predictions were not possible, as the distribution of the injected material within the fiber was not known. As a possible explanation of their results, Falk and Gerard suggest that the chloride conductance may be much less than has been generally assumed, but this seems unlikely both from the experiments of Levi and Ussing (1948) with C13* and from experiments of Hodgkin and Horowicz (Hodgkin, 1958) showing that for small displacements of potential the transport numbers are approximately 0.3 for potassium and 0.6 for chloride. Falk and Gerard point out that, if the injection caused a sudden increase in the resistance and capacitance of the fiber membrane, the recorded resting and action potentials might remain the same even if the equilibrium potential in the immediate neighborhood of the electrode was altered. Not enough is known about the effects of the injection procedure to say whether such changes in the electrical properties of the membrane are likely. Shaw et al. (1956a) have criticized the theory that the resting potential depends largely on the [K],/[K], ratio, since they were unable to find any correlation between the potassium content and resting potential of a large number of toad sartorius muscles both in situ and in Ringer solution. At the potassium concentrations at which their measurements were made, the constant field equation suggests that the potential would be sensitive to differences in the distribution of sodium and chloride and the

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permeabilities of the membrane to these ions, and it may be that variation in these factors was sufficient to swamp the effect of the differences in potassium concentration. It is difficult to assess the likelihood of this possibility, as the cause of the variation in potassium content is unknown, but it may be significant that the noncorrelation became apparent only in cells whose ionic concentration differed markedly from the average. How much the results were affected by differences between the ionic concentrations in the surface fibers and in the deeper fibers is not certain, though Shaw et al. point out that the constancy of the potential readings over 2 hours is evidence against deterioration of the surface fibers. In a later paper, Shaw et al. (1956b) describe experiments in which they measured the sodium, potassium, and chloride contents of toad muscles in vivo and in Ringer solution and found that the Donnan relationship [Kli[ClIi = [K],[CI], did not hold. But the intracellular concentrations are calculated on the assumption that the extracellular space is 15% of the muscle volume, and, though this is unlikely to lead to large errors in [K]( it could easily lead to extremely large errors in [Cl],. For example, if [Cl], is 100 mM, [ClIr is 2.5 mM, and the true extracellular space is 12.8%, then the assumption that it is 15% will lead to an estimate of [Cl], equal to zero. The large variation in the estimated figures for intracellular chloride (0 to 31.8 mM) can probably be accounted for in this way. In squid giant axons, Mauro (1954) has shown that a resting potential may still be recorded when both external and internal electrodes are Ag/AgCl and has argued that this result is incompatible with the passive distribution of chloride postulated by the Conway theory. But the argument is valid only if the Ag/AgCl electrode in the axoplasm behaves as a pure chloride electrode, and this is by no means certain. Conclusions. It may be helpful to summarize the conclusions that may be drawn from the evidence presented so far. They are: 1. The membrane potentials of nerve and muscle depend on the distribution of potassium across the membrane, both at high external potassium concentrations and at physiological concentrations. 2. At high external potassium concentrations the membrane potential and the distribution of potassium across the membrane can be described, to a first approximation, by a Donnan equilibrium. 3. At physiological potassium concentrations the membrane potential is less than that predicted from the potassium distribution by the Nernst formula, and it follows that, in the absence of ion binding in the cell interior, a steady state can be maintained only by the active uptake of potassium ions.

462

I. M. GLYNN

T o these three conclusions may be added a fourth. In the original Bernstein theory the membrane was regarded as impermeable to sodium, but it is clear that to preserve the low internal sodium indefinitely this impermeability must be complete and maintained under all reversible conditions. This view was made untenable, firstly, by observations of net sodium movements (Heppel, 1939 ; Steinbach, 1940) and, secondly and more clearly, by experiments with radioactive sodium (see, for example, Levi and Ussing, 1948; Heppel, 1939; Harris and Burn, 1949; Keynes, 1951a). It follows that the low sodium content of nerve and muscle fibers can be maintained only by the active extrusion of sodium ions.

B. Ionic Equilibria in Red Cells The red cell has long been known to be highly permeable to anions and to water, and, since the interior contains a high concentration of hemoglobin, it seemed that osmotic stability required that the membrane be impermeable to cations. For many years this impermeability was believed to be complete, but in the 1930’s it was noticed that when blood was stored in the cold the cells lost potassium and gained sodium, and that the movements were reversed if the cells were subsequently incubated with glucose or replaced in the circulation (Harris, 1941; Maizels and Patterson, 1940; Maizels, 1949). The leakage during cold storage might conceivably have been due to damage to the cell membrane, but it was unlikely that the cells suddenly acquired the ability to carry out active transport when they were incubated. It followed that there was probably a continuous exchange of sodium and potassium across the membrane even under normal conditions, and this has since been amply confirmed with tracers (Cohn and Cohn, 1939; Mullins et al., 1941). There have been no direct measurements of the potential across the red cell membrane, and no techniques are available to make them. The cell is so small that, even if it were possible to insert a KC1-filled microelectrode, within a very small fraction of a second sufficient KCl would have diffused from the tip to upset completely the potassium and chloride concentrations in the cell fluid. Nevertheless, the high permeability of the membrane to C1- and HC03- ions (Dirken and Mook, 1931 ; Luckner, 1939) and the fact that [HCOs-Io [Cl- l o - r W + I o [Cl-I4 [HC03-]i whatever the state of cell metabolism (Harris and Maizels, 1952), make it extremely probable that the distribution of these ions represents a Donnan equilibrium, and that the membrane potential is therefore given by [H+]c

-

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Since, under normal conditions, r is about 1.4, the potential will be about 9mv. with the outside positive-that is, much less than in resting nerve or muscle. The ratio [K],/[K], is about 35, so that the high internal potassium concentration cannot be explained on a Donnan basis, and, unless there is considerable ion binding, it follows that there must be active uptake of potassium. Similarly the ratio [Na]t/[Nalo = 1/15 points to the active extrusion of sodium. C. Active Nerve and Muscle

As long as the action potential was thought to be simply the disappearance of the resting potential, it was sufficient to suppose, with Bernstein, that during the spike the membrane broke down in the sense that it no longer served as a barrier to ions. The reversal of potential, demonstrated with intracellular electrodes, pointed to a more subtle change in the membrane. That this change involves a selective and very marked increase in sodium permeability is shown by observations of three kinds. First, it is found that most excitable tissues fail to conduct impulses in the absence of external sodium. Second, during activity nerve and muscle resemble a sodium concentration cell in the way in which the size of the reversed membrane potential varies with the external sodium concentration. Third, activation analysis shows a rise in sodium content during activity, and, with tracers, both influx and efflux can be shown to be increased. The dependence of excitability on external sodium has been tested in myelinated and unmyelinated nerve, striated and cardiac muscle, and Purkinje tissue, in experiments in which sodium has been replaced both by sugars and quaternary ammonium ions (Table I ) . Usually sodium is essential, though in crab muscle (Fatt and Katz, 1953) and in a group of small nerve fibers in the frog (Lorente de N6, 1949) excitability remained when sodium in the external medium was replaced by small quaternary ammonium ions. If a nerve or muscle fiber immersed in a solution resembling extracellular fluid suddenly becomes very permeable to sodium, there will be a net entry of sodium ions, and their positive charges will first reduce and then reverse the membrane potential. As the potential changes, the other ions, whose distribution was in a steady state, will show net movements, and the change of potential will therefore depend on the relative movements of all the different ions. If the permeability to sodium is much higher than to all the other ions, the initial net entry of sodium will con-

TABLE I OF SODIUM IONSON THE ACTIONPOTENTIALS OF DIFFERENT TISSUES EFFECT (Reprinted from Hodgkin, 1958)

Preparation

Substance used to replace Na or NaCl

Rapid reversible block in Na-free solution

Active membrane behaves like a sodium electrode, i.e. slope of 58 mV 5 ca. 5 mV for 10-fold change

& P

References

-

Hodgkin and Katz (1949) ; Cole (1955) Unpublished Katz (1947) ; Hodgkin and Katz (1949)

U

No

Fatt and Katz (1953)

5

Squid giant axon

Choline, dextrose

Yes

Yes

Sepia giant axon Crab (Carcinus) nerve fiber Crab muscle

Choline Choline, dextrose, sucrose Choline, and other quartenary ammonium salts Choline, dextrose

Yes Yes

No

Yes

Yes

Huxley 1951)

Choline, sucrose

Yes

Yes

Tetraethylammonium

Yes

No

Overton (1902); Nastuk and Hodgkin (1950) ; Hagiwara and Watanabe (1955) Hagiwara and Watanabe (1955)

Sucrose

Yes

Yes

Draper and Weidmann (1951)

Choline, sucrose Choline after atropine Choline

Yes Yes Yes

Yes

Brady and Wocdbury (1957) S. Weidmann (unpublished) Coraboeuf and Otsuka (1956)

Frog single myeh a t e d nerve fiber Frog sartorius muscle Frog sartorius muscle Dog Purkinje fibers Frog ventricle Turtle ventricle Guinea-pig ventricle

n

2

No No

and

Stampfli

(1949,

2

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tinue until the potential built up (“overshoot”) is sufficient to offset the concentration difference ; i.e. : RT “a16 E=loge F “a10 If the permeability to the other ions is not negligible compared with that to sodium, then the potential can be calculated only on the basis of further assumptions, including assumptions about the potential in the thickness of the membrane. The theory is therefore less easily tested. The methods available to test the relationship between the overshoot and the ratio [Na](/[Na], are the same as those used to test the similar relationship between the resting potential and [ K ] i./[ K ] 0 . [ Na], may be increased or decreased directly, and “a]{ may be altered by causing osmotic movements of water or by direct injection of sodium ions into the fiber. The results of a number of such investigations are summarized in Table I, which is taken from Hodgkin’s Croonian Lecture (1958). It is clear that in many tissues the simple equation holds-that is, the tissue behaves as though at the peak of the action potential the permeability to other ions is negligible compared with the permeability to sodium. A few experiments in which the simple relationship has not been observed are open to criticism on technical grounds. Thus the noncorrelation between sodium ratio and action potential found by Shaw et al. (1956a) is based on the assumption that the extracellular space in toad sartorius is 15%. But, because the internal sodium concentration is so low, small changes in extracellular space lead to very large changes in the calculated value of [Nali; indeed, strict application of the figure of 15% leads to negative internal sodium concentrations in some muscles. Shaw et d. (1956b) found no rise in overshoot when the external sodium concentration was increased, but Hodgkin ( 1958) suggests that the apparent contradiction between this result and the earlier results of Nastuk and Hodgkin (1950) may arise from the failure of Shaw et al. to allow for the osmotic shift of water when muscles were placed in hypertonic solutions. The difference between the results of experiments in which sodium was injected into squid axons by Hodgkin and Keynes (1956) and by Grundfest et al. (1954) probably arises from differences in technique. Hodgkin and Keynes injected into the fiber longitudinally by a method which ensured that the injected material was spread evenly over several millimeters of axon and found good agreement between the calculated change in “a]( and the observed action potential. Grundfest injected at a single point through a micropipet pushed transversely into the fiber, and the action potentials observed at the injected region may have been affected by elec-

466

I. M. GLYNN

trotonic spread from adjacent regions (see discussion by Hodgkin and Keynes, 1956). If the reversal of potential is brought about by the entry of sodium ions, then the charge on the quantity of sodium that enters during one impulse must be sufficient to reverse the potential across the membrane capacitance. In squid axons this capacitance has been shown not to change more than 2% during activity (Cole and Curtis, 1939), so that the minimal net sodium entry in pic0 moles per second is 1000*CVr/F, where C is the membrane capacitance in microfarads per square centimeter, V is the membrane potential in millivolts, and Y is the rate of stimulation in impulses per second. In fact the sodium entry should be greater than this to make up for the net loss of K + and the net gain of C1- ions which will occur as soon as the potential rises above the resting potential. Keynes and Lewis (1951), using the technique of activation analysis, showed that in Loligo and Sepia axons the net gain of sodium during activity was greater than the minimum predicted by the theory. From Na24 measurements, Keynes showed further that both influx and efflux of sodium were much increased so that the permeability must have been much increased. The difference between the influx and efflux determined with Na24 agreed well with the net entry determined by activation analysis. An increase in Na24 entry during activity has also been found by Rothenberg (1950) and by Grundfest and Nachmahnsohn ( 1950). If the repolarization of the membrane is brought about chiefly by the exit of potassium, then the net loss of potassium during each impulse should be roughly equal to the net gain of sodium. Potassium movements have been determined by methods similar to those used for measuring sodium entry (Keynes and Lewis, 1951; Keynes, 1951a, b) and also indirectly through the change in membrane conductance brought about by the accumulation of potassium ions in the film of saline adherent to a single nerve fiber immersed in oil (Hodgkin and Huxley, 1947; Weidmann, 1951) . Both direct and indirect methods point to fairly good agreement between potassium loss and sodium gain. By activation analysis, or the use of radioactive tracers, it is possible to measure the entry and exit of sodium and potassium associated with a period of activity, and hence to calculate the changes associated with a single impulse, but it is not possible to determine the time relations of the movements observed. On the other hand, impedance measurements such as those of Cole and Curtis (1939) allow the membrane current to be calculated at each moment during the action potential but do not permit identification of the ions carrying the current. To determine the changes in permeability to individual ion species during the passage of a single action

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potential, Hodgkin et al. (1952) developed the “voltage clamp” method. In this method, which was developed from the work of Cole (1949) and of Marmont (1949), the potential is suddenly displaced from its resting value and held at some predetermined level by a feedback amplifier. The current which the feedback mechanism has to pass through the membrane to keep the voltage constant gives a measure of the total ionic current at any instant, and the distinction between sodium and potassium currents is made partly by comparison of experiments carried out in the presence and absence of sodium (choline being used as a substitute), and partly with the help of parallel K42flux measurements. Figures 2 and 3 show some of the results obtained. A sustained depolarization leads to a transient increase in sodium permeability followed by a sustained rise in potassium permeability. The size of the increase in sodium permeability depends on the degree of depolarization, so that in an “unclamped” nerve there will be a critical level at which depolarization causes a sufficient degree of sodium permeability for the reaction to become explosive. This will be the threshold. Furthermore, in an “unclamped” nerve the rise in potassium permeability following depolarization will facilitate the exit of potassium ions and hence lead to repolarization and suppression of the potassium permeability to its resting value. From the “voltage clamp” data, Hodgkin and Huxley (1952a, b) developed empirical equations expressing changes in the permeability to sodium and potassium as functions of time and membrane potential and showed that such changes could account quantitatively for the form of the action potential.

D. Energy for Active Ion Movements The ion distribution which determines the resting potential, and which provides the driving force for the fluxes generating the action potential, is itself maintained by the active uptake of potassium and active elimination of sodium. In nerve, the energy for these active movements probably comes from aerobic metabolism, since they are inhibited by anoxia (Shanes, 1951) and by cyanide or azide (Hodgkin and Keynes, 1955). This appears to be true, too, of the nucleated red cells of reptiles and chicken (Maizels, 1954). On the other hand, ion movements in human red cells are unaffected by anoxia, cyanide, or azide, and also by the Krebs cycle inhibitors, malonate and fluoroacetate. The active fluxes do, however, require the presence of glucose and are inhibited by low concentrations of fluoride and iodoacetate (Harris, 1941 ; Danowski, 1941 ; Maizels, 1951) , presumably because they depend on energy from glycolysis. Similar sensitivity to inhibitors occurs in rabbit and in monkey red cells

468

I. M. GLYNK

(McKee et al., 1946). Duck red cells appear to occupy an intermediate position and can probably obtain energy for ion pumping from either aerobic or anaerobic metabolism (Tosteson and Robertson, 1956). The effects of anoxia are complicated, however, and can cause an increase or decrease in potassium influx, depending on the external potassium consodium conductance

potessium conductanca

.,

I

-

0

10mmho/cm2

I

.-.

26

I 0

I 2

I

L 4

0

2 time (msec)

4

6

8

FIG.2. Changes - in sodium and potassium conductance associated with different depolarizations at 6°C. (From Hodgkin, 1958, after Hodgkin and Huxley, 1952b.) The numbers attached to the curves give the depolarizations used. The circles are experimental estimates, and the smooth curves are solutions of the equations used to describe the changes in conductance. centration. Furthermore, after storage at 37°C. in the absence of glucose and in an atmosphere of nitrogen, duck red cells were unable to maintain their ionic composition without oxygen ; and Tosteson and Johnson ( 1957) suggest that the failure of chicken red cells to accumulate potassium anaerobically in Maizels’ (1954a) experiments may arise from .the use of cells stored overnight without oxygen rather than from a species difference. In muscle the evidence is conflicting. A combination of iodoacetate and

ION MOVEMENTS I N NERVE,

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cyanide does not lead to net loss of potassium from normal frog muscle (Ling, 1952), nor to a decrease in the extrusion of sodium from sodiumenriched frog muscle (Keynes and Maisel, 1954). Keynes and Maisel point out, however, that in their experiments muscles were still capable of contraction even after several hours of exposure to the inhibitors and so, presumably, still had energy available, perhaps from creatine phosphate. This explanation seems more acceptable than that of Ling, who supposes that the potassium distribution in muscle is maintained, not by active ion movements, but by ion binding. Conway (1957) has recently reported a considerable inhibition of net sodium extrusion in the presence of cyanide, mV m.mho/cd

FIG.3. The components of membrane conductance (9) during a propagated action potential (-V). (From Hodgkin and Huxley, 1952b.) The curves are derived from empirical equations developed by Hodgkin and Huxley to describe changes in the permeability to sodium and potassium as functions of time and membrane potential. but though his conditions were slightly different from those of Keynes and Maisel, and this difference may be significant, the position is unsatisfactory as, under conditions similar to those of Keynes and Maisel, Conway not only did not find inhibition but could not demonstrate net sodium extrusion. Shaw and Simon (1955b) describe an inhibitory effect of 1 mM iodoacetate on net sodium loss from toad skeletal muscle, but Conway (1957) has pointed out that under the conditions of their experiments it is not certain that the sodium loss represents an active extrusion. There is clearly need for further experiments on the effect of metabolic inhibitors on ion movements in amphibian muscle. In mammalian diaphragm muscle Creese (1954) showed that anoxia caused large downhill movements.

470

I. M. GLYNN

1. The Role of Adenosine Triphosphate. Whether the energy for active ion movements comes from glycolysis or respiration, the question arises : Is the energy made available as adenosine triphosphate ( A T P ) , or is the transport mechanism linked to a particular reaction in the glycolytic or respiratory sequence? The use of A T P as an energy carrier seems more likely on general grounds, and evidence for it is provided by the action of dinitrophenol. Dinitrophenol is thought to uncouple phosphorylation from oxidation, so that respiration is not accompanied by the synthesis of energy-rich phosphate bonds, and it has been shown to inhibit uphill ion movements in Loligo and Sepia axons (Hodgkin and Keynes, 1955a) and in chicken red cells (Maizels, 1954b). The action of dinitrophenol on ion movements might, of course, be unrelated to its uncoupling action, but it would then be necessary to explain why no inhibition occurs in red cells which depend on glycolysis for the production of energy-rich phosphate. In muscle, dinitrophenol has little or no effect on ion movements (Keynes and Maisel, 1954; Conway, 1957), but this may be because of the large store of creatine phosphate. If ion pumping depends on the presence of A T P , then under conditions in which A T P synthesis is stopped the decline in active movements should be related to the fall in A T P content. In squid axons subjected to cyanide or dinitrophenol, Caldwell (1956) found that there was a correlation between the concentration of A T P and arginine phosphate and the rate of sodium extrusion ; in a later paper ( 1957) he showed that at alkaline p H and with only 0.2 niM dinitrophenol the arginine phosphate fell without a corresponding fall in A T P , and under these conditions sodium extrusion continued. In somewhat analogous experiments on human red cells which had been depleted of potassium, Whittam (1958) showed that incubation in the absence of glucose led to a parallel decline in A T P content and in the rate of potassium accumulation, but there was also a fall in 2,3-diphosphoglyceric acid, and, except on general grounds, there is no reason to relate the potassium accumulation to the presence of one phosphate compound rather than the other. The most direct evidence that A T P is the energy source for active transport comes from experiments in which A T P has been added intracellularly. In squid axons poisoned with cyanide, Caldwell and Keynes (1957) found that sodium d u x was partially restored by the injection of A T P or of arginine phosphate. In the experiments with A T P the quantity of sodium extruded was roughly proportional to the amount of A T P injected ; control experiments with a solution of A T P which had been hydrolyzed by boiling showed no restoration of efflux. About four energyrich phosphate bonds were broken for each sodium ion extruded, but the

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47 1

injected A T P seemed to be less effective than the A T P already present in the nerve, since sodium d u x was not restored to normal even when sufficient A T P was injected to raise the intracellular concentration to the level found in a fresh axon. I n red cells, intracellular injection is not possible, but A T P may be made to enter the cells by taking advantage of the remarkable phenomenon of “reversible hemolysis” described by Szekely et al. (1952) and by Teorell (1952). If cells are lysed in a relatively small volume of water, and salt is then added to restore isotonicity, the permeability of the cell membrane returns toward normal, and, in the presence of suitable substrates, “reconstituted” cells can maintain concentration differences across the membrane (Teorell, 1952) and carry out active movements against concentration gradients (Straub, 1954; Gardos, 1954). Gardos has shown that, if the cells are lysed not in water but in an ice-cold 10% solution of the acid sodium salt of A T P , after “reversal” they contain about 4 mg of A T P per milliliter and are able to accumulate potassium actively without further addition of substrate. Unfortunately such cells hydrolyze A T P even in the absence of potassium, so it is not possible to relate A T P breakdown quantitatively to active transport. A feature of Gardos’ experiments that is not entirely satisfactory is that lysis was made to occur in relatively small volumes of fluid, so that the ghosts were probably heavily contaminated with intact cells; these may have been responsible for the small production of lactic acid that he observed. It is, however, rather unlikely that the intact cells contributed appreciably to the active potassium accumulation, since A T P was the only added substrate and the accumulation was not affected by the presence of arsenate.

E. The Link between Active Potassium Influx and Active Sodium Eflux A direct link between the active fluxes was first proposed by Steinbach (1940, 1952), who showed that in the absence of potassium frog muscle fibers lost potassium and gained sodium, and that if they were then transferred to a Ringer solution containing potassium they were able to expel some of the sodium and reaccumulate potassium. Working with NaZ4, Keynes (1954) demonstrated that the effect of potassium was on the sodium efflux, which was reduced in the absence of potassium and increased above normal at high external potassium concentrations. Similar observations were made on Sepia axons by Hodgkin and Keynes (1955a), and, since they found that sodium efflux was not increased by anodal polarization, they concluded that potassium did not act simply by changing the membrane potential. In the complete absence of potassium, Hodgkin

472

I. M. G L Y N N

and Keynes found that sodium efflux was reduced to about a third of its normal value, but since much greater reduction could be obtained with metabolic inhibitors it seemed that active extrusion was not completely abolished. They point out that, although this might appear to exclude the possibility of a tight linkage between sodium efflux and potassium influx, an alternative theory is that the net leakage of potassium occurring in potassium-free solutions prevents the concentration of potassium immediately outside the membrane from falling below 1 or 2 m M . Frankenhaeuser and Hodgkin (1956), from a study of the effects of potassium concentration on the size of the positive after-potential in Loligo and Sepia axons, have evidence that the Schwann cell acts as a diffusion barrier to potassium ions and therefore causes accumulation of potassium in the immediate vicinity of the membrane under conditions in which there is a net loss of potassium from the axon. In red cells, a reduction in sodium efflux at low external potassium concentrations was first noticed by Harris and Maizels in 1951. The effect was subsequently investigated by Shaw (1954) in horse red cells and by Glynn (1954, 1956) in human red cells. The reduction in sodium efflux resulting from removal of potassium from the outside medium is much smaller in red cells than in nerve or muscle, complete absence of potassium reducing sodium efflux by only about one-third. However, from measurements of sodium efflux over a wide range of external potassium concentrations and in the presence and absence of glucose, Glynn concluded that sodium efflux in human red cells was made up of separate active and passive components, and that the active component was completely abolished by the removal of potassium from the outside medium. Furthermore, the active components of both potassium influx and sodium efflux seemed to vary in the same way with the external potassium concentration, being half-maximal at a little more than 2 mM and reaching saturation at about 10 mM (Figs. 4 and 5 ) . Since the effect of the external potassium concentration occurred within a few minutes-that is, long before there could have been any appreciable change in the internal potassium concentration -a direct link between potassium influx and sodium efflux is indicated. This link may involve a 1 : 1 exchange of sodium for potassium, since removal of glucose led to a roughly similar drop in both fluxes. Although a linked potassium influx and sodium efflux may be a common feature of living cells, active movements of the two ions are not always obviously linked. Kidney tubules, for example, can transport sodium and potassium independently; frog skin and the lining of the rumen absorb sodium with an equivalent quantity of chloride (Dobson, 1955 ; Dobson and Phillipson, 1958) ; the isolated gill of Eriochsir pumps sodium and

ION M O V E M E N T S I N NERVE, MUSCLE, A N D RED CELLS

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potassium indiscriminately (Koch, 1954) ; and in Ulva lactuca, although potassium accumulation and sodium excretion normally occur together, they can be separated by poisoning the cells with metabolic inhibitors (Scott and Hayward, 1954). h

L

5 - 10-

i5

P

P

I

I

I

50

100 [KJ m-moldl.

150

FIG.4. The effect of external potassium concentration on the glucose-sensitive part of the potassium influx into human red cells. (From Glynn, 1956.) The continuous line represents the equation 0.96[K] , Y = 2.2+ WI, T O 4

-

----I--.

y when [KJ-m

c 3

-

'g

1

1

1

1

1

1

1

,

1

2

3

4

5

6

7

8

IKJ m-mole/l.

FIG.5. The effect of external potassium concentration on the glucose-sensitive part of the sodium efflux from human red cells. (From Glynn, 1956.). The continuous line represents the equation 0.8 [KI, Y = 1.8+ IKI,

When transport is across a cellular layer, however, two cell membranes are involved, and investigation of the over-all process alone may be deceptive. In frog skin, Koefoed-Johnson and Ussing (1956) suggest that, although only sodium is pumped across the membrane, the active process

474

I. M. G LYNN

may be an exchange of sodium for potassium at the inner surface of the cells. If the cells had outer membranes permeable to sodium but fairly impermeable to potassium, and inner membranes permeable to potassium but not to sodium (except through the pump), the over-all effect would be an inward transport of sodium. I t has been known for some years (Ussing, 1955) that potassium must be present at the inner surface of the skin for sodium transport to occur, and Koefoed-Johnson and Ussing (1956) have shown that if the chloride in the fluid bathing the skin is replaced by sulfate the outer surface of the skin behaves like a sodium electrode and the inner surface like a potassium electrode. There is no evidence that a mechanism similar to that postulated for frog skin is responsible for ion transport in other salt-regulating organs, but the possibility is interesting.

F . The Passive Fluxes Sodium and potassium will move passively across the cell membrane in both directions, but, because of the electrochemical-potential gradients, the movement of sodium will be predominantly inward and that of potassium predominantly outward. It might be expected that the passive flux of each ion would be proportional to the driving force for that flux, i.e., to the electrochemical-potential gradient down which the ions move, but quite often this seems not to be true. Thus when Sepia axons poisoned with dinitrophenol were placed in solutions containing K42-labeled potassium, the influx of K42 at constant membrane potential was not proportional to the external potassium concentration (Hodgkin and Keynes, 1955b). Similarly, when human red cells were placed in solutions containing different concentrations of sodium but the same concentration of chloride (so that the membrane potential was presumably always the same), the influx of sodium was not proportional to the external sodium concentration (Glynn, 1956). Ussing (1950) has shown that, when ions move through a membrane solely under the influence of their own kinetic energy, then, provided that the chance of penetration by any ion is unaffected by the presence of other ions of the same species, the ratio of the inward and outward fluxes is given by

where m, and denote the inward and outward fluxes, fi and ci, and fo and c,, denote the activity coefficient and concentration of the ion under consideration on the two sides of the membrane, z is the valency of the ion, E is the electrical potential, and R, T, and F have their usual mean-

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ings. I n Sepia axons poisoned with dinitrophenol, Hodgkin and Keynes (19SSb) found that the flux ratio for potassium was much greater than would be predicted from the Ussing formula ; and at constant membrane potential an increase in the external potassium concentration reduced the potassium efflux. They showed that this was the type of behavior to be expected if the potassium ions moved through the membrane in single file, along a narrow channel perhaps, or a series of special sites. There is no evidence to show whether similar behavior occurs in muscle; it is not found in red cells. A flux ratio less than that predicted by the Ussing formula has been found for passive sodium movements in human red cells (Glynn, 1956), and there is a little evidence suggesting that passive sodium efflux may be reduced at low external sodium concentrations. A similar, but much clearer effect has been found in frog muscle by Swan and Keynes (1956), who showed that the sodium efflux was reduced by a little more than half when choline or lithium was substituted for sodium in the bathing fluid. They suggest that the effect may be explained by a mechanism, first proposed by Ussing under the name of “exchange diffusion,” in which ions cross the membrane in combination with a carrier which in the uncombined state can cross less readily or not at all. In nerve, Hodgkin and Keynes (1955a) found that substitution of choline for sodium caused an increase in sodium efflux, but the experiments were done on unpoisoned axons, so it is not quite certain that the increase is in the passive efflux.

G. Ion Transport Systems The evidence that the cell membrane can take up potassium and expel sodium is extremely strong, but, except that the movements appear to be linked and that the energy probably comes from ATP, nothing is known of the mechanism. Various models have been suggested which involve hypothetical sodium- or potassium-specific carriers, but the selectivity must be very great, since the cell can take up potassium rather than sodium when the concentration of sodium in the bathing fluid is twenty to forty times the concentration of potassium. Of the few substances known to combine selectively with sodium or potassium, none shows a preference nearly big enough to account for the specific accumulation by the cell unless the mechanism were such that a penetrating ion underwent selection several times during its passage through the membrane. This does not seem very likely. Another possibility is that the selectivity depends on the existence in the membrane of enzymes specifically activated by potassium or sodium. Several enzymes activated by potassium and inhib-

TABLE I1 ENZYMES ACTIVATED BY UNIVALENT CATIONS (Reprinted from Glynn, 1956) Enzyme

Activated by

Source

Pyruvic-phosphoferase

Muscle and viscera of a variety of species

K, Rb, NH,

Fructokinase

Beef liver

+

Inhibited bY Na*, Li, Ca

-

Phospho-fructokinase

Yeast

Mg K or NH, weakly or Na very weakly K, NH4, Mg, Mn

Acetate activating system “Choline acetylase” Phospho-transacetylase Aldehyde dehydrogenase Galactosidase Apyrase

Pig heart Rat brain Clostridiiinz kliiyveri Yeast Bacterium coli Rat brain

K, Rb, NH, Na, Li K K, NH,, ? divalent ions Na, Li K, Rb, NH, Na*, Li, Cs K, Rb, NH, Na*, Li MK Na

*

In the absence of other ions sodium is weakly activating.

+

Na in high concentration

References Kackmar and Boyer (1953) !-

Hers (1952) Muntz (1947) von Korff (1953) Nachmansohn and John (1945) Stadtman (1952) Black (1951) Cohn and Monod (1951) Utter (1950)

F ti

r; 5 4

I O N M O V E M E N T S I N NERVE, M U S C L E , A N D RED CELLS

477

ited by sodium are known to exist (Table I I ) , but so far none of them has been implicated in active transport. Whatever the mechanism, there is evidence that, in the red cell at least, only a small fraction of the total surface is concerned. In 1953 Schatzmann found that one of the cardiac glycosides, strophanthin, inhibited the active uptake of potassium and elimination of sodium in red cells, and similar effects have been found with other cardiac glycosides, and on other tissues (Joyce and Weatherall, 1955 ; Glynn, 1955, 1957a ; Kahn and Acheson, 1955; Harris and Prankerd, 1955; Solomon et al., 1956; Johnson, 1956; Edwards and Harris, 1957), Schatzmann found that strophanthin did not affect respiration or glycolysis, and from the experiments of Glynn (1955, 1957a) and of Whittam (1958) it seems likely that the cardiac glycosides act directly on the pump, probably by displacing potassium from the carrier sites. Furthermore, since they act at extremely low concentrations, when relatively few molecules are present, they can be used to estimate the number of these sites. From a study of the effects of very low concentrations of scillaren A, Glynn calculated that the number of sites, on each red cell, responsible for that part of the potassium influx that is sensitive to scillaren A (about 80% of the total influx at physiological potassium concentrations) was, at most, of the order of 1003. This is a remarkably small number, but the turnover rate that it requires at each site is not impossibly high, and the diffusion of potassium ions up to the sites would not be limiting. The area of membrane occupied by a single site is, of course, unknown, but on any reasonable estimate it seems that only a minute fraction of the total area of membrane can be involved in active transport. IV. REFERENCES Adrian, R. H. (1956) J . Physiol. (London) 133, 631. Bernstein, J . (1902) Arch. ges. Physiol. PfEiiger’s 92, 521. Black, S . (1951) Arch. Biochem. 34, 86. Boyle, P. J., and Conway, E. J. (1941) J . Plzysiol. 100, 1. Brady, A. J., and Woodbury, J. W. (1957) Ann. N.Y. Acad. Sci. 65, 687. Caldwell, P. C. (1956) I. Physiol. (London) l32, 35P. Caldwell, P. C. (1957) Biochem. J . 67, 1P. Caldwell, P. C., and Keynes, R. D. (1957) J . Physiol. (London) 137, 12P. Cohn, M., and Monod, J. (1951) Biochem. Biophys. Acta 7, 153. Cohn, W. E., and Cohn, E. T. (1939) Proc. SOC.Exptl. Bi’ol. 41, 445-449. Cole, K . S . (1949) Arch. sci. physiol. 3, 253. Cole, K. S. (1955) In “Electrochemistry in Biology and Medicine,” p. 121. Wiley, New York. Cole, K. S., and Curtis, H. J. (1939) J. Gen. Physiol: 22, 649. Cole, K. S., and Hodgkin, A. L. (1939) 1. Gen. Physiol. a,671. Conway, E. J. (1957) Physiol. Revs. 37, 84. Coraboeuf, E., and Otsuka, M. (1956) Compt. rrizd. soc. biol. 243, 441.

478

I. M. G L Y N N

Cowan, S. L. (1934) Proc. Roy. SOC.B116, 216. Creese, R. (1954) Proc. Roy. SOC.B142, 497. Curtis, H. J., and Cole, K. S. (1940) 1. Cellular Comp. Physiol. 16, 147. Curtis, H. J., and Cole, K. S. (1942) J . Cellular Comb. Physiol. 19, 135. Danowski, T. S. (1941) J . Biol. Chem. 1S9, 693. Desmedt, J. E. (1953) J . Physiol. (London) 121, 191. Dirken, M. N. J., and Mook, H. W. (1931) J . Physiol. (London) 73, 349. Dobson, A. (1955) J . Physiol. (London) 128, 39P. Dobson, A., and Phillipson, A. T. (1958) 1. Physiol. (London) 140, 94. Draper, M. H., and Weidmann, S. (1951) J. Physiol. (London) 116, 74. Edwards, C., and Harris, E. J. (1957) J. Physiol. (London) lS6, 567. Falk, G., and Gerard, R. W. (1954) J . Cellular Comb. Physiol. 49, 393. Fatt, P., and Katz, B. (1953) 1. Physiol. (London) la,171. Feng, T.P., and Liu, Y. M. (1919) J . Cellular Comp. Physiol. S4, 33. Fenn, W. 0. (1936) Physiol. Revs. 16, 450. Fenn, W.O., and Cobb, D. M. (1934) J . Gen. Physiol. 17, 629. Fischer, E. (1924) Arch. ges. Physiol. Pfliiger's 203, 580. Frankenhaeuser, B., and Hodgkin, A. L. (1956) J . Physiol. (London) 131, 341. Gardos, G. (1954) Acto Physiol. Acad. Sci. Hung. 6, 191. Glynn, I. M. (1954) J . Phgsiol. (London) 126, 35P. Glynn, I. M. (1955) J. Physiol. (London) laa, 56P. Glynn, I. M. (1956) I . Physiol. (London) 134, 278. Glynn, I. M. (1957a) J . Physiol. (London) l!K, 148. Glynn, I. M. (1957b) Progr. in Biophys. and Biophys. Chem. 8, 241. Goldmann, D. E. (1943) J. Gerz. Physiol. 27, 37. Graham, J., and Gerard, R. W. (1946) J . Cellular Comp. Physiol. 28, 99. Grundfest, H., and Nachmansohn, D. (1950) Federation Proc. 9, 53. Grundfest, H., Kao, C. Y., and Altamirano, M. (1954) I . Gen. Physiol. 38, 245. Hagiwara, S., and Watanabe, A. (1955) J . Physiol. (London) 129, 513. Harris, E. J. (1957) J . Gen. Phgsiol. 41, 169. Harris, E. J., and Burn, G. P. (1949) Traits. Faradoy SOC.46, 508. Harris, E. J., and Maizels, M. (1951) 1. Physiol. (London) 113, 506. Harris, E. J., and Maizels, M. (1952) J. Physiol. (London) 118, 40. Harris, E. J., and Martins-Ferreira, H. (1955) J . Exptl. Biol. Sa, 539. Harris, E. J., and Prankerd, T. 4 . J. (1955) Biochem. J . 61, xix. Harris, E. J., and Prankerd, T. A. J. (1957) J . Gen. Physiol. 41, 197. Harris, J. E. (1941) J . Biol. Chem. 141, 579. Henriques, V., and grskov, S. L. (1936) Skarrd. Arch. Physiol. 74, 63. Heppel, L. A. (1939) Am. J . Physiol. 128, 440. Hers, H. G. (1952) Biochim. Biophys. Acto 8, 424. Hodgkin, A. L. (1958) Proc. Roy. SOC.B148, 1. Hodgkin, A. I.., and Huxley, A. F. (1939) Nature 144, 710. Hodgkin, A. L., and Huxley, A. F. (1947) J . Physiol. (London) 106, 341. Hodgkin, A. L., and Huxley, A. F. (1952a) J . Physiol. (London) 116, 449. Hodgkin, A. L., and Huxley, A. F. (1952b) J. Physiol. (London) 117, 500. Hodgkin, A. L., and Katz, B. (1949) J . Physiol. (London) 108, 37. Hodgkin, A. L., and Keynes, R. D. (1955a) J . Physiol. (London) 128, 28. Hodgkin, A. L., and Keynes, R. D. (1955b) J. Physiol. (London) 128, 61. Hodgkin, A. L., and Keynes, R. D. (1956) 1. Physiol. (London) 131, 592.

I O N MOVEMENTS IN NERVE, MUSCLE, A N D RED CELLS

Hodgkin, A. L., Huxley, A. F., and Katz, B.

479

(1952) I. Physiol. (London) 116,

424. Huxley, A. F., and Stampfli, R. (1949) J . Physiol. (London) 108, 315. Huxley, A. F., and Stampfli, R. (1951) J. Physiol. (London) 112, 496. Jenerick, H. P. (1953) J . Cellular Comp. Physiol. 42, 427. Jenerick, H. P.,and Gerard, R. W. (1953) J . Cellular Comp. Physiol. 42, 79. Johnson, J. A. (1956) Am. J. Physiol. 187, 328. Joyce, C. R. B., and Weatherall, M. (1955) J. Physiol. (London) l27, 33P. Kachmar, J. F., and Boyer, P. D. (1953). J . Biol. Chem. a00, 669. Kahn, J. B., Jr., and Acheson, G. H. (1955) J . Pharmacol. Exptl. Therap. 116, 305. Katz, B. (1947) J. Physiol. (Lmtdon) 106, 411. Keynes, R. D. (1951a) J. Physiol. (London) 114, 119. Keynes, R. D. (1951b) J . Physiol. (London) 113, 99. Keynes, R. D. (1954) Proc. Roy. SOC.Bl42, 359. Keynes, R. D.,and Adrian, R. H. (1956) Discussions Faruday SOC. No. 21, 265. Keynes, R. D., and Lewis, P. R. L. (1951) J . Physiol. (London) 114, 151. Keynes, R. D., and Maisel, G. W. (1954) Proc. Roy. SOC.B142, 383. Koch, H. J. (1954) In “Recent Developments in Cell Physiology” (J. A. Kitching, ed.), p. 15. Butterworths, London. Koefoed-Johnson, V., and Ussing, H. H. (1956) Abstr. Communs. 20th Intern. Physiol. Congr., p. 511. Levi, H., and Ussing, H. H. (1948) Acta Physiol. Scand. 16, 232. Ling, G. (1952) ZR “Phosphorus Metabolism” (W. D. McElroy and B. Glass, eds.), Vol. 2, p. 748. Johns Hopkins Press, Baltimore, Maryland. Ling, G., and Gerard, R. W. (1949) J . Cellular Comp. Physiol. S4, 383. Ling, G., and Gerard, R. W. (1950) Nature 166, 113. Lorente de N6, R. (1949) J . Cellular Comp. Physiol. s9 Suppl. Luckner, H. (1939) Arch. ges. Physiol. Pfliiger‘s 241, 753. Macdonald, J. C. (1900) Proc. Roy. SOC.67, 310. McKee, R. W., Ormsbee, R. A., Anfinsen, C. B., Geiman, Q. M., and Ball, E. G. (1946) J. Exptl. Med. 84, 569. Maizels, M. (1949) J . Physiol. (London) 108, 247. Maizels, M. (1951) J. Physiol. (London) 112, 59. Maizels, M. (1954a) J. Physiol. (London) 126, 263. Maizels, M. (1954b) Symposia SOC.Exptl. Biol. No. 8, 202. Maizels, M., and Patterson, J. H. (1940) Lancet 2, 417. Marmont, G. (1949) J . Cellular Comp. Physiol. sl, 351. Mauro, A. (1954) Federation Proc. 13, 96. Meigs, E. B., and Atwood, W. G. (1916) Am. J. Physiol. 40,30. Moore, J. W., and Cole, K. S. (1955) Federation Proc. 14, 103. Mullins, L. J., Fenn, W. O., Noonan, T. R., and Haege, L. (1941) Am. J. Physiol.

136, 93. Muntz, J. A. (1947) I. Biol. Chem. 171,653. Nachmansohn, D., and John, H. M. (1945) J . Biol. Chem. 168, 157. Nastuk, W. L., and Hodgkin, A. L. (1950) J . Cellular Comp. Physiol. 36, 39. Overton, E. (1902) Arch. ges. Physiol. Pfliiger’s 92, 346. Rothenberg, M. A. (1950) Biochim. et Biophys. Acta 4, 96. Schatzmann, H. J. (1953) Helv. Physiol. et Pharmacol. Acta 11, 346. Scott, G. T., and .flayward, H. R. (1954) J . Gen. Physiol. 37, 601.

480

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Shanes, A. M. (1944) J . Cellular Comp. Physiol. 29, 193. Shanes, A. M. (1946) J. Cellular Comp. Physiol. 27, 115. Shanes, A. M. (1951) J . Gcn. Physiol. 34, 795. Shanes, A. M., and Hopkins, H. S. (1948) J . Neurophysiol. 11, 381. Shaw, F. H., and Simon, S. E. (1955a) Australian J. Exptl. Biol. Med. Sci. 33, 153. Shaw, F. H., and Simon, S. E. (1955b) Nature 176, 1031. Shaw, F. H., Simon, S. E., and Johnstone, B. M. (1956a) J . Gen. Physiol. 40, 1. Shaw, F. H., Simon, S. E., Johnstone, B. M., and Holman, M. E. (1956b) J. Gen. Physiol. 40, 263. Shaw, T. I. (1954) Ph.D. Thesis, Cambridge University. Simon, S. E., Shaw, F. H., Bennett, S., and Muller, M. (1957) J . Gen. Physiol. 40, 753. Solomon, A. K., Gill, T. J., and Gold, G. L. (1956) J. Gen. Physiol. 40, 327. Stadtman, E. R. (1952) J. Biol. Chem. 196, 527. Steinbach, H. B. (1940) J. Biol. Chem. 1S3, 695. Steinbach, H. B. (1952) Proc. Natl. Acud. Sci. US.38, 451. Stephenson, W. K. (1958) J . Cellular Comp. Physiol. 60, 105. Straub, F. B. (1954) Acta Physiol. Acad. Sci. Hung. 4, 235. Swan, R. C., and Keynes, R. D. (1956) Abstr. Comnzuns. 20th Intern. Physiol. Congr., Brussels, p. 869. Szkkely, M., Minyai, S., and Straub, F. B. (1952) Acta Physiol. Acad. Sci. Hung. 3, 571. Teorell, T. (1952) 1. Gen. Physiol. 35, 669. Tosteson, D. C., and Johnson, J. (1957) 1. Cellular Comp. Physiol. SO, 169, 185. Tosteson, D. C., and Robertson, J. C. (1956) 1. Cellular Comp. Physiol. 47, 147. Ussing, H. H. (1950) Arta Physiol. Scand. 19, 43. Ussing, H. H. (1955) I n “Ion Transport across Membranes” ( H . T. Clarke and D. Nachmansohn, eds.), p. 3. Academic Press, New York. Utter, M. F. (1950) 1.Biol. Chem. 186, 499. von Korff, R. W. (1953) J . Biol. Chem. 203, 265. Weidmann, S. (1951) J . Physiol. (London) 114, 372. Weidmann, S. (1955) J . Physiol. (London) 129,.568. Whittam, R. (1958) J . Physiol. (London) 140, 479.

Pinocytosis H. HOLTER Defiartment of Pltysioloyy, Carlsbery Laboratory, Copenhagen, Denmark Page

I. Introduction ...................................................... 11. Morphological Aspects of Pinocytosis ............................. A. Light-Microscope Evidence .................................... B. Electron-Microscope Evidence ................................. 111. Induction of Pinocytosis ......................................... IV. Attempts To Measure the Uptake of Fluid ......................... V. Evidence for Adsorption on the Cell Surface ....................... VI. Dehydration of Pinocytosis Vacuoles ............................... VII. Permeability of Pinocytosis Vacuoles ............................. VIII. Concluding Remarks ............................................. IX. References .......................................................

I.

481 482 482 483 488 490 492 494 498 502 503

INTRODUCTION

The word “pinocytosis,” derived from the Greek “TIVCLV,” which means to drink, was coined as an analogy to “phagocytosis” by Warren Lewis and was intended to designate a process of active drinking by cells. In pinocytosis, the fluid is taken up discontinuously, in droplets that are engulfed or sucked in by the cell, and the process is thus morphologically quite different from the well-known uptake of fluid by diffusion through the cell surface. The phenomenon of pinocytosis was discovered by Warren Lewis in 1931 by means of time-lapse films of macrophages in tissue culture. Strangely enough, not much notice was taken of this important discovery for about twenty years, although Lewis himself in his truly classical papers (1931, 1937) described and analyzed the phenomenon very thoroughly and, in fact, anticipated many of the results and problems which more recent research has endowed with renewed actuality. Lewis’s own approach to the investigation of pinocytosis, the study of time-lapse films of cells grown in tissue culture, has been applied with greatly improved techniques by FrCdCric and ChPvremont ( 1952), Gey et al. (1954), Pomerat et al. (1954), Paul (1957), and probably several others whose work may have escaped the attention of the reviewer. In all these studies, Lewis’s original observations have been confirmed and extended, so ’that pinocytosis, as displayed by tissue culture cells, is today well known morphologically, within the limits of light microscopy. Another type of cell in which pinocytosis was discovered relatively early is the fresh-water amoeba. Mast and Doyle (1934) detected and correctly interpreted the phenomenon in Amoeba proteus and a number of related 481

482

H . HOLTER

organisms. Again, their discovery passed practically unnoticed for nearly twenty years. The amoebae, however, although not optically so favorable as tissue culture cells, offer special possibilities for an experimental and physiological study of pinocytosis. Such studies were therefore taken up by the reviewer and his collaborators, and they form the nucleus of the present review, which is based on a lecture delivered at the IXth International Congress for Cell Biology in St. Andrews, 1957. It is thus intended to give a presentation of certain aspects of pinocytosis, of problems, results, and conclusions that were mainly derived from our own work on amoebae (Amoeba proteus and Chaos chaos-Pelomyxa carolinensis), but which have been extended and modified in essential points by work done in other laboratories and with other objects. 11. MORPHOLOGICAL ASPECTSOF PINOCYTOSIS

A . Light-Microscope Evidence As described by Lewis, pinocytosis is entirely dependent on the presence of membranous ruffle pseudopodia. These membranes, by their undulating movements, enclose droplets of the fluid which surrounds the cells, thus forming vacuoles. The shape of these vacuoles is dependent initially on the shape of the membrane cavity, but as they enter the interior of the cell they soon become spherical. The average diameter of the primary pinocytosis vacuoles in macrophages is about 1 to 2p, but during their migration toward the perinuclear space they shrink in size unless they coalesce with other pinocytosis vacuoles. This type of pinocytosis by action of the folds of an undulating membrane is shown by a great variety of cells grown in tissue culture, and is described in detail in the time-lapse film studies mentioned in the Introduction. It has been demonstrated also to occur in leukocytes and ascites tumor cells (Bessis and Bricka, 1952 ; Easty et al., 1956; Chapman-Andresen, 1957, 1959), and in the elaiocytes of the coelomic fluid of echinoderms (Holter, unpublished observations, 1958). On closer inspection, it will probably be found in many other amoeboid cells which display membranous pseudopodia. The morphology of pinocytosis is somewhat different in the case of amoebae. The essential feature in these organisms is not the undulating function of a membranous pseudopod but the formation of a tubelike channel from the bottom of which the pinocytosis vacuoles are pinched off. This mechanism was seen and described by Mast and Doyle (1934), and to this day there exists no better schematic drawing of the phenomenon than that given by these authors, here reproduced in Fig. 1. The work of Mast and Doyle was done without phase-contrast or timelapse photography and is a remarkable example of acuity in microscopic

PINOCYTOSIS

483

observation. Later studies with modern technique have confirmed their description in all details, as demonstrated by Fig. 2, which is an unpublished phase-contrast photograph by David M. Prescott, who has kindly permitted its reproduction.

FIG. 1. Pinocytosis channels in Amoeba protews according to Mast and Doyle (1934). A . Channels in small pseudopods in various stages of. formation. B. Convoluted channels. C. Channel beginning to disintegrate at the inner end. D. Further disintegration showing that many drops of fluid are ingested in the formation of each channel.

The average diameter of a newly pinched-off pinocytosis vacuole in amoebae is 1 to 2 p ; the lifetime of an individual pinocytosis channel is of the order of a few minutes; and the number of channels in a cell during intense pinocytosis may be as high as 50 to 100, distributed all over its surface. The whole period of ingestion is usually about 30 minutes. The pinocytosis channels depicted in Figs. 1 and 2 represent the form most frequently observed, but it must be realized that channel formation in amoebae, although characteristic for these organisms, is a variable feature morphologically. Chapman-Andresen and Prescott ( 1956) have described a continuous series of channel forms, from food-cup-like cavities, strongly reminiscent of phagocytosis, to very narrow tubes yielding pinocytosis vacuoles of 1-p diameter or less (Fig. 3 ) .

B . Electron-Microscope Evidence All the instances of pinocytosis so far mentioned have been within the dimensions of microscopic visibility. If we turn to the scale that has been made accessible by the electron microscope, we find that pinocytosis through channels is not restricted to amoebae alone. Of other cells showing essentially the same type of pinocytosis, physiologically the most inter-

(2-4) FIG.2. Pinocytosis in Amoeba proteus in phase contrast. (Photographs by D. M. Prescott.) Amoebae starved for 24 hours, then immersed in 1% egg albumin in Prescott’s medium (balanced salt solution). 4 3 x phase objective, ocular 1OX. Pictures A and B were taken with 35-second interval from the same channels. The amoeba has moved in between, and the pictures do not match. The three most prominent channels in A, however, correspond to the three most prominent strings of vacuoles in B.

PINOCYTOSIS

485

(2B) (For legend, see facing page.)

486

H. HOLTER

esting ones are undoubtedly the cells of the brush-border epithelium which may well be regarded as a highly specialized organ for just this pinocytosis function. Several pictures are to be found in the literature which more or less clearly indicate pinocytosis to occur (the earliest one that I have found is in a paper by Sjostrand and Rhodin, 1953), but the most impressive is that shown in Fig. 4, for which I am much indebted to Drs. S. L. Clark, Jr., and D. Wochner (1958).

k.

a

b

C

e FIG.3. Various forms of pinocytosis channels in amoebae, according to ChapmanAndresen and Prescott ( 1956). a. Typical pinocytosis channel in salt solution. b. Pinocytosis channel in solution of tobacco mosaic virus. c. “Bottle-shaped” cavity in solution of tobacco mosaic virus. d . Cavity formed in optically clear methionine solution. e. For comparison : forniation of food vacuole containing small ciliate.

The work of the electron microscopists, referred to in the preceding paragraph, has resulted in an extremely interesting extension of the whole concept of pinocytosis. This is due mainly to brilliant observations made by Palade ( 1956), which have led Bennett (1956) to formulate a hypothesis of “membrane flow and membrane vesiculation.” Bennett’s hypothesis, schematically depicted in Fig. 5 (left), postulates that particles, molecules or ions, are in some way engaged by the cell surface membrane ( A , A ’ ) . If the membrane then flows or slides along the surface into a fold extending toward the cell interior, the engaged particles are transported into the recess ( B ) . Finally they are included in vesicles pinched off from the tip of the recess and may thence be moved off to some other portion of the cell. In addition, Bennett proposes a mechanism (Fig. 5 , right), by which vesicles are formed directly at the surface membrane. This mechanism consists again in the engagements of particles (a, b ) at the cell surface

PINOCYTOSIS

487

by appropriate forces, followed by an invagination of the surface at the loaded area (c) and a pinching off of the invaginated part (d, e). In both cases Bennett then assumes a breakdown of the isolated membrane, leading to a liberation of enclosed substances ( f ) .

FIG.4. Electron micrograph of jejunum of 3-day-old mouse (Clark and Wochner, 1958). RCA EMU 2E microscope, 40-p objective aperture. Original magnification X 6500.

It is obvious that this hypothetical mechanism very closely resembles what can actually be seen in amoebae, only on a much larger scale than that with which the hypotheses of Palade and Bennett are mainly concerned. Bennett, of course, was quite aware of the close connection between membrane vesiculation and pinocytosis, and Palade uses the expression “pinocytosis at the submicroscopic level.”

488

H. HOLTER

It will be seen from the following pages that, apart from Palade’s observations on mammalian tissue cells, the work with amoebae has furnished weighty reasons for accepting Bennett’s hypothesis. If we do so and regard it as an extension of the visible pinocytosis phenomenon toward molecular dimensions, we arrive at a definition of pinocytosis as a mechanism for the discontinuous uptake of solutions by invagination

1 FIG. 5. Membrane flow and membrane vesiculation, according to Bennett (1956). For explanation, see text.

and vesiculation of the cell surface. The quantities taken up at a single gulp cover a wide range from submicroscopic vesicles to large vacuoles, and there is no sharp demarcation between phagocytosis on one side, and molecular permeation o n ,the other. 111; INDUCTION OF PINOCYTOSIS In the definition given above, it was stated that pinocytosis is a discontinuous process. This is true in two senses: first, the fluid is taken

up not in a continuous stream but in sips or gulps, the size of which is determined by the size of the primary pinocytosis vacuoles. As we have

PINOCYTOSIS

489

seen, this size is extremely variable. Second, the process as such is discontinuous in the sense that the cells are not drinking all the time. In tissue cultures it has been observed by Lewis that pinocytosis is not going on continuously, but nothing is known about what makes the process start or stop. I n amoebae, the process can be initiated at will by the addition of certain substances, and this is perhaps the greatest advantage that amoebae offer in the study of pinocytosis. These organisms provide an opportunity of finding out which molecular properties are responsible for the induction. Mast and Doyle (1934) found evidence for the inducing action of certain substances, and at the Carlsberg Laboratory the question has been investigated for some time, but so far without much success. Broadly speaking, it can be said that the most efficient inducers of pinocytosis known at present are salts and proteins. Within these groups, however, there are specific differences. Chapman-Andresen ( 1958) has studied the induction of pinocytosis in Amoeba proteus by various simple inorganic salts and has found that different cations, such as sodium and potassium, induce slightly different morphological types of pinocytosis ,.with regard to the width of the channels and the way they are formed. She has also found that there exists, for every salt, an optimum range of concentration where pinocytosis is intense and where toxic or osmotic effects are still not serious enough to prevent the inducing effect. The optimum range also shows specific differences but with most salts investigated is in the neighborhood of 0.1 M . Strictly quantitative data are still lacking; but one thing is certain, namely, that there is no simple correlation between the osmotic conditions offered by various solutions and their pinocytosis-inducing efficiency. The same is true with regard to proteins and a number of other substances tested in our laboratory, mainly by Chapman-Andresen and Prescott ( 1956). Here again there are specific differences, remarkable especially within the protein group, but so far no simple correlation between chemical properties and inducing power has been found. The best inducers in our experience, besides inorganic salts, are rabbit y-globulin, gelatin, and Na glutamate (both D- and L-forms). Noninducers are all carbohydrates tested, whether low- or high-molecular, and also, surprisingly enough, the nucleic acids. All we can say at present is that for unknown reasons some substances seem to taste better than others. This is very unsatisfactory, since the problem is important. A better knowledge of the specificity of induction may furnish the key to our understanding of the mechanism of adsorption to the cell surface, which is supposed to initiate the process of pinocytosis. It is to be hoped that

490

II. HOLTER

further work with various inducing substances will yield more systematic evidence.

IV. ATTEMPTST o MEASURE THE UPTAKE OF FLUID Mast and Doyle (1934) detected pinocytosis in amoebae after immersing them in protein solutions, and, as mentioned before, they found that several other substances besides protein can act as inducers. They were well aware also of the potential physiological importance of pinocytosis, but they could not proceed very far in investigating the physiological implications of the process, because at that time there were no labeled substances available which would enable them to follow the fate of the pinocytosis vacuoles after ingestion.

FIG.6. Pinocytosis vacuoles in a section of the cytoplasm of Chaos chaos, 2 hours after the ingestion of fluorescein-labeled y-globulin. Fluorescence micrograph, X 600. (Holter and Marshall, 1954.)

The first tracer study of pinocytosis was carried out by Holter and Marshall ( 1954), who fed fluorescein-labeled y-globulin to the large multinuclear amoeba Chaos chaos (Pelomyxa carolinensis) (Fig. 6). By suitable micromethods the amount of fluorescent material present in an amoeba was determined and the corresponding uptake of fluid calculated, on the basis of the assumption that concentration of the protein solution would remain unchanged during the process of ingestion. Table I shows the uptake of fluorescent material for ten individual amoebae. From the values in the last column it is seen that the amount

491

PINOCYTOSIS

TABLE I DETERMINATION OF LABELED MATERIAL I N PROTEIN-FED AMOEBAE, AND CORRESPONDING CONTROL MEASUREMENTS Control amoebae

Reduced weight

No. 1 2 3 4

5 6

7 8 9 10 Mean -

(PLg. )

Volume in cuvette (PI.)

I525

I430

0.825 0.46 0.39 0.39 0.65 0.52 0.32 0.29 0.415 0.43

0.69 0.68 0.67 0.71 0.67 0.59 0.58 0.57 0.58 0.67

32.8 29.3 23.0 23.5 19.0 27.5 20.3 21.0 46.5 34.0

24.0 21.8 17.5 18.8 14.3 20.0 14.8 15.5 36.0 24.5

15zdLao

1.37 1.35 1.32 1.25 1.33 1.37 1.37 1.35 1.29' 1.39 1.34 f 0.01

0.47 ? 0.05 Amoebae fed on protein solution

Volume in cuvette

No. 1 2 3 4

5 6 7 8 9 10

( 4.)

I525

I430

0.69 0.69 0.69 0.60 0.57 0.60 0.64 0.61 0.60 0.60

32.5 49.0 40.5 50.0 53.5 51.5 46.0 48.0 48.5 53.5

12.3 17.8 16.3 20.5 22.3 19.5 15.3 19.0 17.5 18.5

Calculations: I,,, AIS25

= 1530 scale units, = 1525 - 1430 X 1.3.

(protein)

Volume ingested =

"525 X -

1530

"525

Volume of solution ingested (mpl.)

16 25 18.5 22.5 23.5 25.5 25.5 22.5 25.5 28.5

7.2 11.3 8.4 8.8 8.8 10.0 10.7 9.0 9.8 11.2

Mean

9.5 k 0.4

on this day.

(volume in cuvette).

All the amoebae were starved one day, isolated from the same culture at the same time, and the individuals from each group were analyzed alternately. Their treatment was thus exactly alike, except for the immersion in protein solution.

492

H. HOLTER

ingested is remarkably constant. On the assumption mentioned above, the uptake is expressed as volume of protein solution ingested. The average value, 9.5mpl., corresponds to about 30 to 40% of the amoeba’s average volume. Lewis (1931), judging the diameter and number of the vacuoles in his macrophages, arrived at a similar order of magnitude for the uptake of fluid and in a later paper (1937) stated that the cells may take up several times their own volume within a few hours. According to this evidence, therefore, pinocytosis is a very intense process, and the volumes of fluid taken up are surprisingly high. It must be remembered, however, that the figures given in Table I do not take into account the possibility that the concentration of protein may be changed in the very act of ingestion, by its adsorption to the cell surface, as postulated in Bennett’s hypothesis.

V. EVIDENCE FOR ADSORPTION O N THE CELL SURFACE Marshall et al. (1958) presented convincing evidence that surface adsorption of the dissolved substance actually takes place during pinocytosis. Brandt ( 1958) extended Holter’s and Marshall’s work with fluorescent proteins. H e used fluorescein-labeled rabbit y-globulin directly, and also unlabeled proteins, to which labeled antibodies had been prepared according to the method of Coons and Kaplan (1950) and Marshall (1951, 1954). The result was the same in both cases, and Brandt was able to show (Fig. 7) that the pinocytosis-inducing protein is indeed adsorbed by the plasmalemma of Chaos chaos, that this surface structure is then ingested, and that the adsorbed protein coats the wall of the pinocytosis vacuoles. This remarkable confirmation of Bennett’s hypothesis is further supported by the results of Schumaker (1958), who investigated a similar problem by an indirect method. H e measured the rate at which ribonuclease and cytochrome C labeled with radioactive iodine were taken up by Amoeba proteus. H e found that during the first 5 minutes after immersion there is a fast and reversible uptake of protein which he interprets as an adsorption on the cell surface. This initial stage seems to be insensitive to cooling and metabolic inhibitors. The later stage of uptake is slower and is thought to represent the process of ingestion of the loaded surface membrane. This is irreversible, but its rate is affected by temperature and metabolic inhibitors. Marshall et al. (1958) studied the binding of lysozyme in the first stages of pinocytosis of this substance, which Chapman-Andresen and Prescott (1956) had shown to be a pinocytosis inducer. By means of differential centrifugation of amoeba (Chaos chaos) homogenates, Marshall and his

PI NOCYTOSIS

493

co-workers isolated a fraction which among other particulates contained broken sheets or fragments of plasmalemma. This fraction was treated with lysozyme under the same conditions as those in which living amoebae were observed to bind lysozyme. From this mixture was isolated a complex of lysozyme with some component of the amoeba particulate. Its analysis was not completed, but as a working hypothesis the authors assume that the substance in the amoeba homogenate which binds the basic protein is a mucoid or lipoid component of the membranes, acidic in

FIG.7. Fluorescence photomicrograph of a part of the periphery of Chaos chaos after immersion in 1% solution of fluorescent y-globulin. The plasmalemma (also in the pinocytosis channels) is coated with a fluorescent layer. Numerous pinocytosis vacuoles can be identified by their fluorescent walls. (Brandt, 1958.)

nature. Further evidence is necessary to decide whether or not this complex formation corresponds to the initial binding of lysozyme to the amoeba surface. As a result of the work reviewed in this section, it must be assumed that an adsorption of pinocytosis-inducing proteins on the cell surface actually takes place. It remains to be shown whether the same is true of other pinocytosis-inducing substances, such as salts. Furthermore, the adsorption behavior of noninducers, like carbohydrates, will have to be studied. before definite conclusions can be drawn.

494

H . HOLTER

From our present knowledge of the adsorption of proteins, however, it is clear that protein uptake cannot without reservation be taken as a measure of the volume of fluid ingested in pinocytosis. O n the other hand, there is ample evidence that large amounts of fluid are taken up together with the adsorption film. But for reliable determinations of the quantities of solvent and of solute ingested, a double label will be required. SOfar, no such determinations appear to have been carried out. There exists one set of data, however, which bears some relation to the problem. In the course of another study, discussed below, Chapman-Andresen and Holter (1955) fed radioactive glucose to C h o s chaos. Glucose in itself does not induce pinocytosis, and to bring about the ingestion of the glucose solution protein (serum albumin) was used as a vehicle. On the as yet unproved assumption that glucose as a noninducer is not adsorbed by the cell surface, the amount of radioactivity in the amoebae should be a true measure of the volume of solution ingested. Table I1 shows the result. A comparison of Table I1 and Table I shows two main points of difference. First, the average “uptake of fluid” is considerably higher in Table I. This might be (and has been) interpreted to indicate that y-globulin is a specifically better inducer of pinocytosis than the serum albumin used in the experiment of Table 11. Second, the variation in the fluid uptake is much greater in the experiment of Table 11. In the first experiment, the total amount of labeled substance taken up is the sum of the fraction that had been adsorbed to the surface and the fraction dissolved in the fluid ingested. In the second experiment, the uptake of labeled substance is probably dependent only on the amount of fluid ingested. If these assumptions are correct, the fact that in the latter case the uptake was smaller and much more irregular might be significant. It would indicate, in agreement with Bennett’s hypothesis, that surface adsorption is the dominant factor in pinocytosis, and that the uptake of fluid may, at least in certain cases, play a secondary role. VI.

DEHYDRATION OF PINOCYTOSIS VACUOLES

I n tissue culture cells, where the vacuoles are comparatively stationary and can be followed individually in time-lapse films, the shrinkage of the vacuoles can be seen directly. In Chaos chaos, where the cytoplasmic currents prevent direct observation, the shrinkage of vacuoles and the corresponding concentration of their contents can be demonstrated by centrifugation (Holter and Marshall, 1954). At the moment of ingestion, the density of the vacuoles is smaller than the average density of amoeba cytoplasm. Correspondingly, on centri-

UPTAKE OF C14 GLUCOSEBY Chaos chaos

Group I I1 I11

TABLE I1 FROM GLUCOSEAND GLUCOSE-PROTEIN SOLUTIONS

Total no. of amoebae

Feeding solution

+

Pringsheim solution glucose (without protein) Pringsheim-proteinglucose Pringsheim-protein-glucose (new batch protein, prewash in protein)

50

No. of amoebae ingesting activity corresponding to following volumes of active solution

> 0.004 PI. 0

(0%) 109

116

12 (11%) 16 (14%)

Between

< 0.0001 PI.

3 (6%) 54 (50%) 72 (62%)

47 (94%) 43 (39%) 28 (24%)

496

H . HOLTFA

fugation of an amoeba shortly after pinocytosis of a fluorescent protein solution, one finds the pinocytosis vacuoles in the centripetal part of the amoeba, not in a sharply delimited band, but more or less randomly spread over the light half. A few hours later, however, the vacuoles accumulate on centrifugation in a stratum rather close to the heaviest components of the cytoplasm (Fig. 8 ) . This indicates that a considerable concentration of the vacuole contents has taken place.

FIG. 8. Fluorescence micrograph of the heavy pole of Chaos chaos, centrifuged 2 hours after pinocytosis of fluorescent y-globulin. Brightly fluorescent protein vacuoles occur throughout the stratum containing the mitochondria and nuclei. X600. (Holter and Marshall, 1954.)

The shrinking process continues, and finally, when the vacuoles have been reduced to the size of granules, on centrifugation they accumulate in the same zone as the mitochondria (Fig. 9). The fact that pinocytosis granules behave like mitochondria, at least in the centrifugal field, is interesting, since it has been claimed by Gey et al. (1954) that mitochondria are formed by the fusion of shrunken pinocytosis granules with pre-existing mitochondria, or are produced de novo by the formation of chains of pinocytosis granules. A further indication of a possible connection between mitochondria and pinocytosis is furnished by Brandt’s (.1958) beautiful pictures of pinocytosis vacuoles in amoebae (Fig. 10). As shown in Fig. 10, the pinocytosis vacuoles display many infoldings of the vacuolar membrane, and one is, of course, struck by the morphological similarity of these structures and the cristae which are such an essential feature of electron-microscope pictures of mitochondria. From the observations on amoebae, therefore, there may also be derived

PINOCYTOSIS

497

FIG. 9. Fluorescence micrograph of the heavy pole of Chaos chaos, centrifuged 3 days after pinocytosis of fluorescent y-globulin. Oblique section. Nuclei appear as large ovoid bodies, pinocytosis granules brightly fluorescent. X600. (Holter and Marshall, 1954.)

FIG. 10. Fluorescence micrograph of pinocytosis vacuoles of Chaos chaos after immersion of 1% fluorescent y-globulin. Numerous infoldings of vacuolar walls. (Brandt, 1958.)

498

H. HOLTER

some arguments in favor of the assumption that pinocytosis may be concerned with the genesis of mitochondria. O n the other hand, it must be remembered that morphological resemblance and similarity in density may simply represent a case of physiological convergence. Considering what we know about the fine structure of mitochondria and their biochemical properties, it is certainly difficult to imagine a transition from a simple vacuole to a mitochondrion. But if the essential feature of this vacuole really is a highly developed internal surface with the characteristics of the plasmalemma, such a transition becomes less improbable with regard to enzymatic properties. This is an exceedingly interesting problem and a challenging task for cytochemists and electron microscopists.

PERMEABILITY OF PINOCYTOSIS VACUOLES So far, next to nothing is known about the physiological significance of pinocytosis. I n the literature, one quite often finds scattered pieces of evidence which make it seem probable that, in certain types of cells, there exists a mechanism which enables such cells to take up and incorporate high-molecular substances without breakdown to smaller molecules. It is very tempting to suppose that pinocytosis is this badly wanted mechanism, but before we can make such an assumption we need to know more of the ultimate fate of the solutes in the pinocytosis vacuoles. If Lewis (1931) is right in assuming that a digestion occurs during the transformation of the pinocytosis vacuoles to cytoplasmic granules, the notion of incorporation of high-molecular substances without change has to be abandoned. O n the other hand, pinocytosis takes place by invagination of the cell surface which is notoriously impermeable to many, especially high-molecular substances. The vacuole formed by this invagination is enclosed by a membrane which originally was a part of the cell surface and which, according to the evidence presented by Brandt (1958) and Marshall et al. (1958) preserves the morphological characteristics of the plasmalemma, even as vacuolar lining. There is no obvious reason for assuming, and no microscopical evidence to indicate, that the permeability of this membrane should be changed during the act of invagination ; but as long as it is unchanged, and if no digestion takes place, the contents of the vacuole must be assumed to be just as inaccessible to the organism as if they were still in the surrounding medium. For all these reasons, investigation of the permeability properties of the wall of pinocytosis vacuoles seemed to be highly desirable, and such an investigation has been begun by Chapman-Andresen and Holter ( 1955), who have used radioactive glucose as a model substance and Chaos chaos as test organisms. VII.

PINOCYTOSIS

499

The cell membrane of these amoebae is exceedingly impermeable. If they are immersed in a solution of glucose, they do not take it up to any appreciable extent (Chapman-Andresen and Holter, 1955), and their permeability is very low even to water, as was shown by L$vtrup and Pigoh (1951) and Prescott and Zeuthen (1952). In order to find out if, and how soon, the vacuolar membrane becomes permeable, ChapmanAndresen and Holter immersed Chaos chaos in a solution of C14-labeled glucose and serum albumin. The protein was unlabeled and acted only as an inducer of pinocytosis, causing the labeled glucose to be ingested into

FIG.11. Autoradiogram of 10-p section of Chaos chaos, freeze-dried 3% hours after pinocytosis of glucose-protein solution (0.5 and 1%). (Chapman-Andresen and Holter, 1955.) pinocytosis vacuoles. The distribution of radioactivity in the cytoplasm of the amoebae was then followed by means of an autoradiographic technique (Andresen et al., 1953). One of the autoradiograms obtained is shown in Fig. 11. Figure 11 shows that there is a considerable amount of activity evenly distributed in the cytoplasm of the amoebae. Such autoradiograms have been obtained as early as 30 minutes after the end of feeding, the earliest time at which, for technical reasons, fixation was possible. It is therefore reasonable to assume that the process of glucose resorption begins soon after the formation of the vacuoles. In other words, the impermeability of the cell surface is not maintained by the vacuolar membrane. Whether

500

H. HOLTER

the mechanism of penetration is a diffusion of unchanged glucose or a metabolic absorption by means of enzymatic equipment newly formed at the membrane is not known. It is also unknown if the autoradiogram is produced entirely by glucose or partly by some of the products of its metabolism. At any rate, it is certain that pinocytosed glucose is incorporated into the normal metabolism of the amoeba, on or after entering the cytoplasm. This has been shown by transferring amoebae to Cartesian divers, following their respiration, and measuring the radioactivity of the various compounds of the diver charge, as well as that of the surviving amoebae. The measurements were repeated on the same amoebae with suitable intervals. In this way it has been found (Fig. 12) that only about 15% of the activity introduced as glucose is incorporated in a more permanent way in the cytoskeleton. Of the activity given off by the amoebae, about 75% is recovered in respiratory carbon dioxide, while the rest is excreted into the surrounding medium. Figure 12 shows the loss of radioactivity after ingestion of active glucose by pinocytosis (curve A ) and after ingestion of radioactive ciliates (the amoebae’s normal prey) by phagocytosis (curve B ) . It is seen that there is no essential difference between curves A and B, except that the latter is somewhat steeper in the initial stage, owing to loss of radioactivity by defecation of undigestible remnants of ciliates in the first 48 hours after phagocytosis (Andresen and Holter, 1944). Curve C, on the other hand, drawn for comparison after the data of Holter and Marshall (1954), shows that elimination of the unphysiological labeling substance (fluorescent protein) follows an entirely different course and is complete within 5 to 6 days. W e have no means of ascertaining directly whether curve C depicts the digestion of the marked protein within the pinocytosis vacuoles, or outside the vacuoles in the cytoplasm, or whether it depicts simply the subsequent process of elimination of the labeling substance. Besides glucose, the incorporation of an amino acid, S36methionine, has also been studied (Chapman-Andresen and Prescott, 1956), with essentially similar results, both in Chaos chaos and A m e b a proteus. There can therefore be little doubt that at least low-molecular food substances given by pinocytosis are being utilized. About the fate of high-molecular substances, especially proteins, we know very little as yet. So far it has not been possible to study the uptake of protein by autoradiographic methods, since no soluble proteins have been available that were sufficiently radioactive for the requirements of the autoradiographic technique on a cytological scale. But even if such proteins could be obtained, they could probably only give information

501

PINOCYTOSIS

about the integrated nutritive utilization of the substance involved. Autoradiography can hardly solve the questions of by what mechanism and in what state the protein molecules are being transferred from the vacuoles to the cytoplasm. For this, some other experimental approach will have to be found.

st c

X

I

0

200 hours

I

40 0

FIG.12. Disappearance of radioactivity from Chaos chaos. A . After ingestion of glucose by pinocytosis. B. After ingestion of radioactive ciliates by phagocytosis. C. The excretion of fluorescent label after pinocytosis as found by Holter and Marshall (1954). Symbols indicate individual measurements on five amoebae. (ChapmanAndresen and Holter, 1955.) C14

The question is of crucial importance for the evaluation of the physiological role of pinocytosis. If pinocytosis is to be considered as a mechanism for the uptake of high-molecular-weight substances without breakup of the molecules, we must be able to answer the question of how the membrane barrier is overcome. It could be supposed that the vacuolar membrane is itself digested by cytoplasmic enzymes, so that the contents of the vacuoles are released into the cytoplasm. This supposition would involve some rather interest-

502

H. HOLTER

ing assumptions about the instability of a cytoplasmic structure in relation to the cell’s own enzymes, a kind of local cytolysis, so to speak ; but so far no evidence of this has been found. The vacuolar membrane is certainly not rigid, since the vacuoles can be deformed, can shrink, and can both coalesce and divide, but it has never been seen to disappear. Further evidence on this point is thus highly desirable. VIII.

CONCLUDING REMARKS

It will be seen from the results mentioned in the preceding section that pinocytosis has practical aspects for the study of certain physiological problems, at least in amoebae. As a laboratory method it enables the investigator to study the metabolism of substances that would not be taken up normally. It has been mentioned that an amoeba can be persuaded to ingest and utilize carbohydrate if this is offered together with a pinocytosis-inducing protein. Another instance is the ingestion of ribonuclease, the intracellular effects of which have been studied for years in Brachet’s laboratory (1955, 1956). It has been shown by Chapman-Andresen and Prescott (1956) and by Schumaker (1958) that ribonuclease enters amoebae by pinocytosis. So far, little is known whether or not similar effects will be found in cells of higher organisms where environmental conditions are entirely different. In a time-lapse film of macrophages Paul (1957) has demonstrated that the addition of insulin to the medium (already containing a variety of proteins) caused an impressive intensification of pinocytosis, and similar effects would probably be known in other instances, if only they were properly recognized. I n view of the great potential importance of pinocytosis in the physiology of many types of cells, a systematic search for instances of its occurrence would probably be rewarding. Some studies on macrophages with colloidal gold as a tracer (Gosselin, 1956) and on HeLa cells (Harford et al., 1957) have already yielded extremely interesting results concerning the mechanism of pinocytosis and the role of surface adsorption in the process. This, I think, is true in spite of the fact that Gosselin does not regard pinocytosis as a probable explanation for the phenomena of which he has studied the kinetics. Finally, mention must be made of another problem of pinocytosis, which has not been treated in this review, namely, the transport of pinocytosis vacuoles from the periphery of the cell to the perinuclear space. In tissue culture cells, where every vacuole can be followed on its way, this process is very impressive. Its mechanism has already occupied the attention of Lewis (1931), and its explanation, as membrane flow, is an essential

PINOCYTOSIS

503

feature of Bennett’s (1956) hypothesis. This problem has not been touched on in this review because studies on amoebae can contribute very little to its elucidation on account of the constant and chaotic cytoplasmic currents which are characteristic of these organisms. In the Introduction it was stated that the problems of pinocytosis are discussed mainly as problems that have emerged from our own work with amoebae. No complete coverage of the literature has been possible, and, inevitably, the presentation has been one-sided. Therefore I should like, in conclusion, to emphasize the encouraging fact that during the last years work in various fields has tended to converge. Only a few years ago Pomerat et al. (1954) wrote about pinocytosis: “It seems incredible that this important phenomenon has been almost totally neglected by cell physiologists.” Today this statement is no longer true. The fundamental importance of pinocytosis in cell physiology is realized by many workers, and it is to be hoped that a better understanding of its problems will emerge from their converging results. The most interesting feature of the progress during the last few years has been the change that has been brought about in the concept of pinocytosis itself. Indeed, it seems that the definition of the phenomenon may have to be modified so that less emphasis is placed on the uptake of fluid and more on the uptake of dissolved substances. This modification endows pinocytosis with very interesting aspects indeed, since, as Bennett has stressed, it connects pinocytosis with the whole problem of the active uptake and transport of substances by cells. It may well be that some of the more puzzling aspects of active transport will become clearer when we know more about pinocytosis.

IX. REFERENCES Andresen, N., Chapman-Andresen, C., Holter, H., and Robinson, C. V. (1953) Compt. rend. trav. lab. Carlsberg Str. chim. 28, 499. Andresen, N., and Holter, H. (1944) Contpt. rend. trav. lab. Carlsberg Shr. chim. 26, 107. Bennett, H. S. (1956) J . Biophys. Biochem. Cytol. 2, Part 2 (Suppl.) 99. Bessis, M., and Bricka, M. (1952) Rev. himutol. 7, 407. Brachet, J. (1955) Nature 176, 581. Brachet, J. (1956) Exptl. Cell Research 10, 255. Brandt, P. W. (1958) Exptl. Cell Research 16, 300. Chapman-Andresen, C. (1957) Exptl. Cell Research U,397. Chapman-Andresen, C. (1958) Compt. rend. trav. lab. Carlsberg 51, 77. Chapman-Andresen, C. (1959) in press. Chapman-Andresen, C., and Holter, H. (1955) Exgtl. Cell Research Suppl. 3, 52. Chapman-Andresen, C., and Prescott, D. M. (1956) Compt. rend. trav. lab. Carlsberg Shr. chim. SO, 57. Clark, S. L., Jr., and Wochner, D. (1958) Anat. Record l80, 286.

504

H . HOLTER

Coons, A. H., and Kaplan, M. H. (1950) J . Exptl. Med. 91, 1. Easty, D. M., Ledoux, L., and Ambrose, E. J. (1956) Biochim. et Biophys. Acto 20, 528. Frederic, J., and Ch&remont, M. (1952) Arch. biol. ( L i i g e ) 85, 109. Gey, G. O., Shapras, P., and Borysko, E. (1954) Ann. N.Y. Acad. Sci. 88, 1089. Gorselin, R. E. (1956) 1. Gen. Physiol. 39, 625. Harford, C. G., Hamlin, A., and Parker, E. (1957) J. Biophys. Biochem. Cyfol. 3, 749. Holter, H., and Marshall, J. M., Jr. (1954) Compt. rend. trav. lab. Carlsberg Sir. chim. 29, 7. Lewis, W. H. (1931) Bull. Johns Hopkins Hosp. 49, 17. Lewis, W. H. (1937) Amer. J. Cancer 29, 666. L@vtrup, S., and Pigon, A. (1951) Compt. rend. trav. lab. Carlsberg Sir. chim. 28, 1. Marshall, J. M., Jr. (1951) J. Exptl. Med. 94, 21. Marshall, J. M., Jr. (1954) Exptl. Cell Research 6, 240. Marshall, J. M., Jr., Schumaker, V. N., and Brandt, P. W. (1958) Ann. N.Y. Acad. Sci. in press. Mast, S. O., and Doyle, W. L. (1934) Protoplasma 20, 555. Palade, G. E. (1956) J. Biophys. Biochem. Cytol. 2, Part 2 (Suppl.) 85. Paul, J . (1957) Demonstration at IXth International Congress for Cell Biology, St. Andrews, 28th August-3rd September 1957. Pomerat, C. M., Lefeber, C. G., and Smith, McD. (1954) Ann. N . Y . Acad. Sci. 88, 1311. Prescott, D. M., and Zeuthen, E. (1953) Acta Physiol. Scand. 28, 77. Schumaker, V. N. (1958) Exptl. Cell Research 15, 314. Sjostrand, F. S., and Rhodin, J. (1953) Exptl. Cell Research 4, 426.

Author Index Numbers in italics indicate the pages on which the references are listed at the end of the article.

A Abbott, A. C., 259, 260,262,276 Abolin, L., 203, 206 Abood, L. G., 372, 383 Abramowitz, A. A., 188, 206 Abrams, A., 150,166 Abrams, G. D., 215, 247 Abul-Fadl, M. A. M., 369, 381 Abul-Haj, S. K., 216,229, 250 Acheson, G. H., 477,479 Ackermann, W. W., 288,313,341 Acosta-Ferreira, W., 76, 95 Adair, G. S., 213, 226, 227, 247, 250, 393, 403, 443 Adams, C. G., 164,173 Adelstein, S. J., 353, 360, 361, 362, 364, 381, 385 Adolph, E. F., 407, 443 Adrian, R. H., 451, 454, 456, 457, 477, 479 Aebi, H., 407, 443 Afionso, 0. R., 315,344 Afzelius, B. A., 328, 337 Agna, J. W., 422,443 Agostini, L., 159, 166 Ahrens, L. H., 347, 348, 381 Akerman, L., 152,169,170 Albert, S., 314, 341 Aldridge, W. M., 369, 381 Alex, M., 223,225, 227,230, 232,238, 249 Alfin-Slater, R. B., 314, 343 Allard, C., 313, 314, 315, 322, 323, 325, 337, 339 Allen, C. E., 28, 28 Alles, G. A., 156, 167 Allfrey, V. G., 280, 282, 283, 284, 285, 286, 288, 313, 320, 326, 333, 337, 338, 339, 342,344 Allison, A. C., 146, 153, 165, 166 Allsopp, A., 35, 48, 58, 59 Altamirano, M., 459, 460, 465, 478 Altmann, H. W., 21, 28 Ambrose, E. J., 482, 504 Ambs, E., 144, 145,170 Andersen, B., 437, 448 Anderson, E., 21,28,328,338

505

Anderson, N. G., 282, 283, 284, 285, 315, 327, 329, 332, 333, 335, 338, 344 Andresen, N., 499, 500, 503 Anfinsen, C. B., 361,384,468,479 Annersten, S., 262, 268, 276 Anson, M. L., 354, 381 Antoni, F., 355, 381 Appelbloom, J. W., 408, 409, 443 Appelmans, F., 7,29,313,333,338,340 Aqvist, S., 152, 169 Armstrong, J., 63, 94 Armstrong, S. H., 398, 448 Arnesen, K., 281, 285, 338 Arvanitaki, A., 62, 94 Asai, I., 159, 171 Asami, G., 254,255,267, 270,276 Asboe-Hansen, G., 215, 247 Aschner, B., 147, 166 Astbury, W. T., 34, 48, 50, 55, 56, 58, 59, 218, 219, 220, 221, 223, 233, 238, 243, 247, 251 Atwood, W. G., 452, 479 Atz, J. W., 175, 202, 209 Austrian, C. R., 163, 172

B Baar, H. S., 156, 161, 162,166 Babbitt, J. D., 389, 443 Bacq, Z. M., 79, 95, 150,171 Bahr, G. F., 328,338 Bailey, I .W., 34, 58 Bailey, K., 369, 381 Bain, J. A., 287, 293 (128), 314, 319, 334, 338 Bairati, A., 328, 338 Baitsell, G., 236, 247 Baker, B. L,215,247 Balashov, V., 36, 58 Baldes, E. J., 422, 448 Baldini, M., 156, 162, 166 Baldridge, C. W., 153, 166 Bale, W. F., 147, 167 Balfour, W. M., 147, 167 Ball, E. G., 315, 344, 468,479 Ballantine, R., 414, 447 Balls, W. L., 34, 58

506

AUTHOR INDEX

Balo, J., 216, 220, 232, 233, 241, 247 Banfield, W., 238, 247 Banga, I., 216, 220, 224, 225, 232, 233, 241, 247, 249 Barac, G., 150, 170 Barbier, H., 161, 166 Barer, A. P., 153,166 Barer, R., 138, 166, 326, 338, 417, 427, 443 Bargoni, N., 314, 340 Barnafi, L., 205, 208 Barnett, S. R., 281, 284, 314, 330, 336, 339 Barnum, C. P., 314, 342 Barr, L. M., 63, 94 Barr, M. L., 63, 96 Barreto, D., 414, 421, 447 Bartelmez, G. W., 64, 94 Barth, A., 270, 276 Bartholomay, A. F., 365, 380, 381, 385 Bartley, W., 24, 28, 407, 443 Baserga, A., 164, 166 Bass, A. D., 334,338 Bass, R. L., 325, 338 Bassen, F. A., 160,173 Bassi, M., 21, 28 Batchelder, A. C., 398, 403, 448 Baud, C. A., 328, 338 Baudhuin, P., 313, 327, 344 Bauer, J., 147, 166 Baur, R., 371, 382 Bayley, S. T., 51, 58 Beams, H. W., 3, 21, 28,328,338 Bear, R. S., 223,224, 247 Beaufay, H., 7,30, 314,344 Beavan, G. H., 165,166 Beck, L. V., 426, 439, 443 Beer, A. G., 158, 159,166 Beer, M., 51, 58 Beermann, W., 328, 338 Behrens, M., 281,282, 284,315,338 Beinert, H., 320, 337, 338 Beisenherz, G., 362,363,381 Belford, D. S., 39, 58 Belt, W. D., 4, 5, 28 Bendall, J. R., 228, 247 Benesch, R., 367, 382 Benesch, R. E., 367,382 Benestad, A. M., 160,166 Benezech, C., 407, 419, 420,444

Benhamou, E., 158, 166 Ben-Harel, S., 164, 166 Bennett, H. S., 63, 68, 69, 70, 72, 74, 80, 85, 92, 93, 94, 95, 486, 488, 503, 503 Bennett, M. F., 190, 191, 207, 210 Bennett, S., 452, 480 Bentley, G. A., 205, 208 Berger, R. E., 8, 31 Bergmann, M., 243, 247 Berk, L., 160, 166 Berleur, A. M., 313, 327, 344 Berlin, N. I., 154, 166 Berman, H., 146, 149,169 Bernelli-Zazzera, A., 21, 28 Bernhard, W., 7, 8, 12, 15, 17, 18, 21, 24, 25, 26, 28, 29, 30, 31, 122, 133, 139, 141, 142, 143,166 Bernick, S., 325, 338 Bernstein, J., 452, 477 Bernstein, R. E., 414,443 Bert, P., 160, 166 Bertelsen, A., 263,267, 276 Berthet, J., 280, 289, 313, 315, 322, 332, 333, 338, 340 Berthet, L., 289, 315, 322, 340 Bessis, M., 9, 26, 29, 31, 138, 139, 141, 142, 143,166,482, 503 Betke, K., 144, 145,166 Beyer, G. T., 285, 314, 319, 320,339 Bibb, J., 164, 167 Bibergeil, E., 141, 172 Biesele, J. J., 8, 12, 31 Biggs, R., 139, 140, 162,166 Biondi, C., 141, 166 Bisgard, J. D., 268, 270, 276 Black, S., 476, 477 Bliss, A. F., 356, 381, 382 Bliss, D. E., 182, 206 Blitz, O., 164, 173 Blocksom, B. H., 260,261, 277 Blum, G., 268, 276 Blumenthal, H. T., 233,251 Bodansky, M., 149, 166 Bodian, D., 62, 63, 64, 69, 94 BZe, J., 160, 166 Boiron, M., 151, 171 Bolam, T. R., 398, 443 Boltze, H. J., 362, 363, 381 Bonner, J., 35, 58

AUTHOR INDEX

507

Bonnichsen, R., 285, 325, 338, 353, 355, Brody, T. M., 287, 293 (128), 314, 319, 334, 338 356, 357, 380, 381,382,384 Bronx, D., 217, 248 Borsook, H., 137, 150, 151,166,170 Brookfield, R. W., 141,166 Borysko, E., 223, 250, 481, 496, 504 Brooks, M. M., 428,443 Bos, C. J., 9, 30 Brooks, S. C., 428, 436, 443 Boschman, Th. A. C., 204,207 Brown, A., 398, 403,447,448 Bostrom, L., 163, 166 Brown, F. A., Jr., 175, 176, 177, 178, 179, Bothe, A. E., 25, 29 180, 181, 182, 183, 185, 186, 188, 189, Bourne, G. H., 220,229,247 190, 191, 193, 194, 196, 197, 201, 206, Bowen, T. J., 228, 247 207, 210 Bowes, J. H., 212, 227, 228, 237, 242, 247 Brown, G. B., 285,338 Bowman, T. E., 187,206 Brown, H. R., 147,173 Boyarsky, S., 259, 276 Brown, J. R. G., 326,338 Boyd, I. A., 90,91,94 Brown, K. D., 314,330,339 Boyer, P. D., 367,370,382,476,479 Bruderman, M., 154, 167 Boyle, P. J., 453, 455, 477 Bruckner, H., 138, 141, 166 Boxer, G. E., 288,343 Brachet, J., 5, 18, 20, 29, 280, 338, 502, 503 Brues, A. M., 425,435, 443 Brunner, A., Jr., 139, 142, 143,166 Bradfield, J. R. G., 279,338 Bucher, N. L. R., 326,339,343,344 Brady, A. J., 464, 477 Bucher, T., 362, 363, 381 Brakke, M. K., 284,338 Buck, R. C., 234, 247 Brandt, C. S., 168 Brandt, P. W., 492, 493, 496, 497, 498, Buckman, T. E., 147,'166,170 Bull, H. B., 392, 443 503, 504 Branster, M. V., 288, 338 Bullock, T. H., 62,72,94 Burchard, W., 357, 358,385 Brantner, G., 204, 206 Burchenal, J. H., 160,166 Bratley, F. G., 165,166 Burgers, A. C. J., 204, 205, 207 Braun-Falco, O., 217, 247 Braunsteiner, H., 8, 28, 30, 139, 141, 142, Burgos, M. H., 24,29 Burk, N. F., 403, 443 143, 166 Burkard, W., 315,327,344 Brawerman, G., 313, 338 Brecher, G., 138, 139, 140, 142, 166, 172 Burn, G. P., 146, 153,166,462, 478 Burroughs, H. H., 165,166 Breder, C. M., Jr., 203,206 Burt, N. S., 137, 144, 145, 149, 151, 163, Bremer, J., 289, 338 166 Brenner, S., 165, 166 Breton-Gorius, J., 9, 29 Burton, A. C., 224, 247 Bretschneider, L. H., 328, 338 Burton, D., 212, 239, 240, 241, 242, 243, Bricka, M., 482, 503 245, 247 Bridges, J. B., 256, 265, 267, 272, 276, 277 Busch, F., 255, 277 Bridgman, P. W., 350,382 Butler, J. A. V., 389, 400, 443 Brims, B. M., 40, 48, 58 C Brink, N. G., 346, 355, 357, 382, 384 Brinkman, R., 353, 382 Cajal, S. R. y, 61, 63, 64, 95 Briseno Castrejon, B., 203, 206 Caldwell, P. C., 396, 397, 398, 443, 470, Britton, C. J. C., 154, 170 477 Brock, B., 148, 169 Calkins, E., 411, 443 Brockmyre, F., 154, 173 Callan, H. G., 328, 339 Brodie, A. F., 372,382 Camain, R., 15, 29 Brodie, J. W., 314, 343 Cantero, A., 313, 314, 315, 322, 323, 325, Brodsky, W. A,, 408, 409, 443 337, 339, 343

508

AUTHOR INDEX

Carasso, N., 24, 30 Carlisle, D. B., 175, 188, 195, 196, 201, 202, 205, 207, 208 Carlson, A. J., 238, 248 Carlson, S. Ph., 185, 207 Carstam, S. Ph., 184, 195, 207 Carter, H. E., 232, 248 Caspersson, T., 20, 21, 29, 151,166 Castle, W. B., 148, 160, 166, 167, 421, 443 Cathala, V., 147, 167 Cesaris-Demel, A., 137, 141, 167 Chaikoff, I. L., 283,284,340 Chalazonitis, N., 283, 339 Chalfin, D., 139, 143, 144, 145, 146, 147, 149,167,411,445 Chambers, E. L., 426, 446 Chambers, R., 426, 446 Chang, C. Y., 205, 207 Chantrenne, H., $29, 152, 153,170 Chanutin, A., 314, 327, 342 Chaplin, H., 414, 443 Chapman-Andresen, C., 482, 483, 486, 489, 492, 494, 498, 499, 500, 501, 502, 503 Chargaff, E., 313, 338 Chase, A. M., 146, 171 Chatterjea, J. B., 158, 167 Chatton, 8., 28,29,98,99, 102,132 Chauveau, J., 18, 20, 29, 283, 284, 314, 319, 322, 324,332,335,339 Chauvin, E., 259, 277 Chevremont, M., 3, 5,29,30,481,504 Child, F. M., 98,132 Chinard, F. P., 389,438,443 Chiquoine, D., 322, 339 Chiuini, F., 159, 171 Chow, S. K., 150,173 Christensen, H. N., 151, 172 Christensen, I., 421, 443 Chu, L. W., 63, 95 Chudorosheva, 158, 169 Churney, L., 424, 426, 444 Chwatt, L. J., 164,167 Ciotti, M. M., 361, 383 Clare, F. B., 158,167 Clark, S. L., Jr., 486, 487, 503 Clarke, W. O., 150,174 Claude, A., 4, 18, 29, 280, 281, 319, 339 Clausse, J., 164, 165, 168 Clermont, Y.,24, 29 Clowes, G. H. A., 415,425, 444,445

Cobb, D. M., 454, 478 Cochran, K. W., 369,382 Cohen, J., 245, 251 Cohen, Y., 315, 327, 344 Cohn, E. J., 155, 156,167,171 Cohn, E. T., 462, 477 Cohn, M., 476, 477 Cohn, M. L., 280,339 Cohn, W. E., 462,477 Cole, K. S., 454, 455, 457, 460, 464, 466, 467, 477, 478, 479 Coleman, J. S., 398, 448 Colowick, S. P., 370, 380, 382,384 Colvin, J. R., 51, 58 Constance, J. J., 265, 277 Conway, E. J., 407, 408, 409, 412, 444, 451, 452, 454, 455, 458, 469, 470, 477 Cook, C. D., 39, 58 Coombs, J. S., 87, 95 Coombs, T. L., 355, 359, 361, 363, 385 Coons, A. H., 492,504 Copher, G. H., 261,277 CoppCe, G., 79, 95 Coraboeuf, E., 464,477 Corey, R. B., 243,250 Cori, C. F., 363, 370, 382 Cori, G. T., 363, 370,382 Corliss, J. O., 98, 120, 132 Corper, H. J., 280, 339 Cotton, M. A., 334, 340 Cotzias, G. C., 314, 315, 322, 324, 327, 339 Couteaux, R., 68,72, 74, 95 Coviln, M. R., 82, 85, 96 Cowan, P. M., 243,248 Cowan, S. L., 454, 477 Craik, R., 164, 167 Crank, J., 440,442,444 Creese, R., 411, 444,469, 477 Creskoff, A. J., 156, 162,167,168 Cress, C. H., 158, 167 Crick, F. H. C., 243, 250 Cristol, P., 407, 419, 420, 444 Cronshaw, J., 35, 36, 42, 50, 55, 56, 58, 59 Crosby, W. H., 136,167 Criiger, H., 34, 58 Cruz, W. O., 137, 144, 145, 147, 148, 156, 165, 167 Cunningham, R. S., 163,167,172

AUTHOR INDEX

Curtis, H. J., 454, 457, 466, 477, 478 Czok, R., 362, 363,381

D Dabbs, G. H., 264, 265,266,267,277 Dacie, J. V., 136, 138, 139, 140, 146, 154, 167 da Costa, H. C., 314,315,339 Dadswell, H., 51, 53, 60 Dahlberg, A. A., 261, 277 Daland, G. A., 137, 138, 141, 146, 147, 148, 155, 156, 162, 167, 169, 421, 443 Dalhamn, T., 223, 250 Dallam, R. D., 282, 339 Dalton, A. J., 3, 8, 9, 12, 17, 20, 21, 24, 25, 29, 30, 282, 314, 322, 342 Daly, M. M., 285, 338, 339 D’Amato, H. E., 407,444 Dameshek, W., 144, 154, 158,167,170 Damren, F. L., 141,167 Danielli, J. F., 236, 248, 428, 444 Danowski, T. S., 467, 478 Darrow, D. C., 410, 444 Daunay, R., 147, 167 Davenport, H. W., 354,382 Davidovich, A., 62, 96 Davidson, D. C., 364,382 Davidson, J. N., 151, 163, 167, 280, 285, 286, 331,339,341,342,344 Davidson, L. S. P., 136, 137, 138, 141, 144, 145, 148, 157, 167 Davies, H. F., 213, 214, 216, 218, 226, 227, 228, 229, 230,242,247,250 Davies, R. E., 24, 28, 407, 411, 443, 448 Davison, F. P., 320, 322, 332, 339 Davson, H., 397, 411, 420, 422, 428, 444 Dawson, C. R., 346,364,382 Dawson, I. M., 328,339 Day, M. F., 178, 208 Day, R., 353, 382 Dazzi, A,, 158, 167 De, D. N., 328,339 Dean, Burk, 9, 31 Deanin, G. G., 179, 207 Deasy, C. L., 137, 150,166 De Bruyn, P. P. H., 271,277 de Castro, F., 64, 95 de Duve, C., 7, 29, 30, 280, 289, 313, 314, 315,322,327,332,338,340,344 Degge, J., 268, 278

509

de Harven, E., 26,30 Deitch, A. D., 3, 29 Delahay, P., 347, 382 de Lamirande, G., 313, 314, 315, 322, 323, 325, 337, 339 de Langen, C. D., 150,170 del Castillo, J., 72, 85, 90,92, 93, 95 De Luca, H. A., 151,170 DeMaria, G., 355, 384 De Marsh, Q. B., 328,341 Dempsey, E. W., 8, 9, 29, 79, 96, 214, 223, 225, 230, 233, 248,249 Denecke, G., 156, 158,167 Dennis, W. H., 408,409,443 Denstedt, 0. F., 137,153,154,162,172 De Robertis, E., 63, 66, 68, 69, 70, 72, 74, 76, 78, 79, 80, 82, 84, 85, 92, 93, 94, 95,225,248,321,339 Dervichian, D. G., 412, 444 Desmedt, J. E., 459,478 Dettmer, N., 218, 225, 248, 251 Devine, R. L., 4, 31, 328, 338 de Vries, A., 154, 1?3 Deyrup, I., 393, 444 Diamond, I., 408, 409, 443 Dianzani, M. U., 313,314,339 Dick, D. A. T., 405, 406, 407, 409, 418, 421, 422, 423, 425, 427, 429, 435, 439, 440, 442, 443, 444 Dick, J., 219, 248 Dickman, S. R., 313,339 Didier, R., 270, 277 Diercks, C., 158, 159,169 Diesing, J., 161, 168 Diggs, L. W., 164,167 Di Gugliemo, L., 144, 168 Dippel, L,33, 58 Dirken, M. N. J., 462, 478 Dischendorfer, H., 34, 59 Discombe, G., 138, 143,167 Doan, C. A., 163,167,172 Dobson, A., 472, 478 Dock, W., 148, 170,254, 255, 267, 270, 276 Dockhorn, E., 158, 167 Dodgson, K. S., 315,324, 325,339 Dole, V. P., 314, 315, 322, 324, 327, 339 Doljanski, L., 235, 248 Donnan, F. G., 396, 444 Dore, W. H., 34, 60 Douglas, J., 359, 383

5 10

AUTHOR INDEX

Dounce, A. L., 280, 281, 282, 283, 284, 285, 314, 315, 319, 320, 321, 322, 324, 327, 329, 330, 333, 334, 336, 339, 340, 341 Doyle, W. L., 481, 482, 483, 489, 490, 504 Drabkin, D. L., 399,444 Drach, P., 195, 207 Dragstedt, C. A., 262, 277 Draper, M. H., 464,478 Dressler, 0. G., 149,166 Dreyfus, J. C., 150,170 du Bois, A. H., 158, 167 Dubois, K. P., 369, 382 du Buy, H. G., 9, 31 Duclaux, J., 2, 29 Duesberg, R., 137, 167 Dulaney, A. D., 281, 285,338 Dunn, R. A., 347, 385 Dupertius, S. M., 271, 277 Dupont-Raabe, M., 182, 195, 201, 202, 205, 207, 208 Duque, O., 259, 276 Duran-Jorda, F., 163, 167 Durand, J. B., 183,207 Durbin, R. P., 438, 444 Durinyan, R. A., 158,167 Dustin, P., 137, 141, 143, 151, 165, 167 Dutton, G. J., 315, 340 Dziemian, A. J., 148, 149, 167

E Easty, D. M., 482, 504 Eaton, P., 141, 164, 165,167 Ebaugh, F. G., 414, 444 Eccles, J. C., 61, 62, 68, 80, 85, 87, 88, 89, 91, 95 Ederle, W., 161, 167 Ederstrom, H. E., 193, 206 Edgren, R. A., 179, 207 Edlbacher, S., 371, 382 Edsall, J. T., 399, 440,444 Edwards, C., 477, 478 Edwards, G. A., 72, 95 Ege, R., 421, 444 Eggleston, L. V., 407, 411, 448 Ehrenberg, A., 356, 382 Ehret, C. F., 4, 31, 98, 104, 106, 114, 120, 122, 124, 127, 131,132,133 Ehrlich, P., 136, 167 Eichenberger, M., 5, 29 Eidinger, D., 229, 248

Eirich, F. R., 353, 382 Ejiri, I., 219, 248 Eliasson, N., 152, 169 Elliott, R. G., 227, 228, 237, 242, 247 Ellis, D., 150, 153, 168 Ellis, G. S., 168 Elster, S. K., 236, 248 Eltholtz, D. C., 144, 145, 165, 170 Elvehjem, C. A., 280,342,353, 383 Emanuel, C. F., 283, 284,340 Emerson, C. P., 414, 444 Emery, A. J., 283,314,330, 340 Emmel, V. E., 163,168 Emmelot, P., 9, 30 Enami, M., 182, 183, 184, 185, 186, 188, 203, 207 Endres, H., 229, 244, 248 Engel, L. L., 364,383 Engelbreth-Holm, J., 161, 168 Engelhardt, V. H., 153, 168 Engelstad, R. B., 269, 277 Englard, S., 370, 383 Engstrom, H., 76, 95,271,277 Enns, T., 389, 443 Entenman, C., 285, 344 Ephrussi, B., 424, 444 Eranko, O., 160, 168 Erb, W., 138, 141,168 Erbsen, H., 144, 145, 162, 163, 174 Erdtman, H., 369, 382 Eriksen, L., 152, 168 Ernster, L., 12, 18, 30 Errera, M., 286,287, 326,340 Eskelund, V., 261, 277 Esnouf, M. P., 326, 338 Espindola, J., 79, 95 Estable, C., 63, 66, 76, 95 Ewald, A., 232, 248 Eyzaguirre, C., 79, 87, 95, 96

F Fabiani, G., 162, 164, 165,168 Fange, R., 205, 209 Faessler, A., 357, 358, 385 Faira, H., 313, 322, 337 Falk, G., 459, 478 Farr, W. K., 34, 59 Fatt, P., 62, 72, 85, 87, 90, 91, 92, 93, 94, 95, 463, 464, 478

AUTHOR INDEX

Faure-FrCmiet, E., 4, 25, 30, 31, 127, 132, 424, 444 Favard, P., 24, 30 Fawcett, D. W., 5, 18, 24, 29, 30, 99, 131, 132 FaxCn, N., 160, 168 Febvre, H. L., 8,28,30 Feinstein, H. M., 397, 448 Feissly, R., 138, 142, 168 Feldberg, W., 62, 82,90, 95 Felix, M. D., 3, 21, 24, 29, 30 Fell, H. B., 236, 248 Fellinger, K., 139, 166 Feng, T. P., 454, 478 Fenn, W. O., 452,454, 462,478,479 Ferreira, D., 7, 18, 20, 24, 30 Ferri, E., 224, 250 Fertman, M. B., 165,168 Fertman, M. H., 165,168 Ferrebee, J. W., 146,168 Fichsel, H., 144, 145, 162, 163,174 Ficq, A., 286, 287, 326,340 Fiessinger, N., 137, 168 Finamore, F. J., 411, 445 Finch, C. A., 148,150,168,173 Findlay, V. H., 218, 219, 221, 233, 248 Fingerman, M., 178, 179, 180, 184, 186, 187, 188, 189, 190, 192, 194, 196, 198, 199,200,201, 206,207,208 Fischer, E., 454, 478 Fischer, E. H., 150, 151,166 Fisher, A. M., 353, 384 Fisher, R. A., 140,168 Fitton- Jackson, S., 235, 237,248 Fitz-Hugh, T., 156, 162, 167, 168 Fitzpatrick, C., 186, 207 Fleming, W., 214, 248 Florey, E., 206, 208 Flory, P. J., 400, 403, 444 Fluharty, R. G., 150,173 Foerster, O., 78, 95 Fogg, G. E., 42, 59 Fogliati, E., 265, 277 Foldes, I., 233, 249 Folkers, K., 346, 384 Foltz, C. M., 365, 384 Fordham, C. C., 422, 448 Forssell, J., 154, 155, 168 Foster, D. H., 49, 59 Fournet, G., 412, 444

511

Fowler, C., 433, 446 Fox, D. L., 176,208 Frajola, W. J., 328, 340 Franchi, C. M., 68, 69, 70, 78, 79, 92, 95, 225, 248 Frank, H., 438, 444 Franke, K., 140, 168 Frankel, D., 320, 341 Frankel, S., 218, 250 Frankenhaeuser, B., 472, 478 Franklin, J., 353, 382 Frattin, G., 254, 278 FrCdCric, J., 3, 5, 8, 29,30, 481, 504 Freer, R. M., 281,284,314,336, 339 Frey-Wyssling, A., 1, 3, 7, 28, 30, 34, 50, 59,412,413, 428,444 Frieden, C., 362, 382 Friedkin, M., 286, 289, 340 Friedlander, A., 140, 161,168 Frost, R., 182, 208 Fujimura, 267, 277 Fulchiron, C., 164, 165, 168 Fullmer, H. M., 217, 218,248 G Gabrio, B. W., 148,168 Gaffney, F. M., 146, 149, 168, 418, 421, 444 Gagel, O., 78, 95 Gairdner, D., 160, 168 Gajdos, A., 165,168 Gall, J. F., 328, 340 Ganguly, J., 315, 340 Gansler, H., 9, 12, 13, 30 Garbade, K. H., 362, 363,381 Garby, L., 438, 444 Gardos, G., 471, 478 Garnjobst, L., 131, 133 Garrod, M., 224, 249 Gartland, J., 154, 166 Gatenby, J. B., 3, 24, 30 Gaudino, M., 407, 445 Gautier, A., 17, 18, 20, 29 Gavosto, F., 151, 168 Gawrilow, R., 138, 141, 154, 168 Gay, H., 321, 340 Geiman, Q. M., 468,479 Gellhorn, E., 158, 167 Geoghegan, H., 407,408,409,444

512

AUTHOR INDEX

Gerard, R. W., 454, 455, 457, 458, 459, 478, 479 Gerischer-Mothes, W., 153, 172 Geschwind, I. I., 205, 208 Gettner, M., 321, 342 Gey, G. O., 481,496,504 Gianetto, R., 7, 29, 313, 333, 340 Gibson, J. G., II., 146, 168, 173, 352, 385 Gibson, N. C., 78, 96 Gibson, Q. H., 137, 152, 153,169 Gill, T. J., 477, 480 Gilligan, D. R., 230, 249 Gillman, T., 217, 248 Ginzberg, R., 158, 168 Giraud, G., 420, 445 Gitlow, S., 160, 171 Glaser, K., 160, 168 Glasstone, S., 389, 445 Glees, P., 78, 96 Glegg, R. E., 229,248 Glick, D., 314, 342 Glinos, A. D., 326,339 Glock, G. E., 331,340 Gloor, V., 289, 338 Gloyd, P., 268, 278 Glynn, 1. M., 451, 472, 473, 474, 475, 476, 477, 478 Glynn, L. E., 21, 30 Goetzke, E., 151, 152, 153, 168 Gold, G. L., 477, 480 Goldbloom, A., 147, 168 Goldeck, H., 161, 168 Goldmann, D. E., 455,478 Goldsmith, Y., 281, 285, 338 Goldstein, L., 329, 340 Gomori, G., 217,248,260,261,277 Goodall, A., 137, 171 Goodwin, A. M., 259,260,262,276 Gordon, A. S., 156, 157,168 Gordon, M. W., 324,340 Gorry, J. O., 314, 319, 330, 343 Gosselin, R. E., 502, 504 Gots, J. S., 372, 382 Gottlieb, B., 314, 319, 330, 343 Gottlieb, R., 147, 168 Gough, A., 421, 445 Goutier-Pirotte, M., 314, 334, 340 Goutier, R., 314, 340 Govaerts, J., 139, 171 Graffi, A., 9, 30

Graham, J., 454, 478 Grant, H. C., 286,340 Grant, N. H., 220,233, 234, 248 Grant, W. C., 158, 159, 160, 163,168 Grass&,P. P., 24, 30 Grassini, V., 161, 172 Grassmann, W., 222,229, 244, 248,249 Gray, J., 426, 445 Greco, E., 282,314,322, 342 Green, F. C., 243, 250 Green, N. M., 354,382 Gregg, J. R., 146,171 Greider, M. H., 328, 340 Grell, K. G., 104, 132 Grey, C. E., 8, 12,31 Grim, E., 412, 445 Grinstein, M., 150, 174 Gripwall, E., 143, 168 Grisham, J. W., 334,338 Gross, J., 222, 225, 229, 230, 237, 248 Grotepass, W., 150, 170 Groves, M. L., 399, 446 Griinig, W., 224, 251 Grunberg-Manago, M., 286, 340 Grundfest, H., 459, 460, 465, 466,478 Grunke, W., 161,168 GuCrin, M., 25, 29 Guest, G. M., 137, 144, 145, 149, 162, 168,172,414, 420, 445 Guggenheim, E. A., 389,403,445 Guinier, A., 412, 444 Gulick, A., 281, 284, 342 Gulland, G. L., 141, 148, 167 Guntelberg, A. V., 403, 445 Guyon, L., 236,250,270, 277

H Haagen-Smit, A. J., 137, 150,166 Haba, K., 143,173 Haden, R. L., 148,168 Hadley, C. E., 181, 208 Haege, L., 462,479 Haenel, U., 168 Haga, 267, 277 Hagan, W. A., 280,340 Haggar, R. A., 63, 96 Hagiwara, S., 464, 478 Haguenau, Fr., 3, 15, 17, 21, 24, 25, 29, 30 Hahn, P. F., 147,167

AUTHOR INDEX

Hajdukovic, S., 159, 169 Hakala, N. V., 353, 384 Halbert, M. L., 160,170 Haldane, J. B. S., 139,169 Hale, C. W., 216, 248 Hall, C., 347, 382 Hall, D. A., 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 225, 226, 227, 228, 229, 230, 231, 232, 233, 235, 238, 239, 240, 241, 242, 243, 245, 246, 247, 248, 249, 251 Ham, A. W., 12, 17, 30 Hamburger, H. J., 420,445 Hamburger, J., 407, 423, 445 Hamilton, D. M., 165,166 Hamlin, A., 502, 504 Hammarsten, E., 152, 269 Handovsky, H., 145, 146,169 Hanig, K., 244, 248 Hannay, P. W., 221, 249 Hansen-Pruss, 0. C., 164,169 Hanson, E. D., 127,132 Hanson, H. T., 355,384 Hanstrom, B., 182, 208 Hanzon, V., 4, 21, 24, 31, 328,344 Happey, F., 238,239, 249 Harding, C. V., 329,340 Harel, J., 8, 28, 30 Harford, C. G., 502, 504 Harkness, M. L. R., 228, 231, 243, 249 Harkness, R. D., 228, 231,235,243,249 Harris, D. L., 427, 445 Harris, E. J., 398, 428, 438, 442, 445, 451, 454,457,462,472,477,478 Harris, E. S., 280, 340 Harris, J. E., 462, 467, 478 Harris, J. I., 205, 208 Harris, P., 328, 340 Harrison, G. R., 347,382 Harrison, M. F., 326,340 Hart, C., 215, 249 Hart, E. B., 353, 383 Hartley, J., 264, 265, 277 Hartline, H. K., 424, 430, 431, 433, 434, 436, 446 Hartmann, J. F., 328, 340 Harvey, E. N., 436, 446 Harvey, J. A. N., 326,343 Harvey, W. H., 255, 268,277 Hass, G. M., 236, 249

513

Hastings, A. B., 411, 443 Hastings, W. S., 25, 29 Havel, V. J., 182, 208 Haven, F. L., 281,340 Hawes, J. B., 141, 144,169 Hawkins, J., 314, 340 Hawkinson, V., 150, 174 Hay, E. D., 76, 96 Hayes, J. E., Jr., 355, 359, 382 Hayward, H. R., 473, 479 Healey, E. G., 203, 208 Heath, C. W., 137, 138, 141, 146, 155, 156, 162, 169 Hebb, C. O., 314,340 Hecht, L., 313, 319,342 Hegner, R., 144, 164, 165,169 Hegsted, D. M., 378, 385 Heilbrunn, L. V., 412, 445 Heilmeyer, L., 141, 154, 155, 156, 157, 158, 161, 162,168,169 Heinen, J. H., Jr., 264, 265, 266, 267, 277 Heinrich, W. D., 161,168 Heinz, R., 137, 169 Held, H., 64, 96 Heller, L., 314, 340 Hellerstein, S., 410, 444 Hempling, H. G., 411, 412, 416, 425, 431, 435, 445, 446 Hems, R., 407, 448 Henderson, L. J., 398, 445 Hendry, E. B., 146,169,414,415, 421,445 Hengstenberg, J., 34, 59 Henneguy, L. F., 26,30 Hennessy, T. G., 154,166 Henriques, V., 137, 149,169,452,478 Henry, G. R., 256,278 Henstell, H. H., 154, 167 Heppel, L A., 288,289,340,462,478 Herkert, L., 314, 343 Hermann, G., 149, 151,172 Hers, H. G., 289, 315, 322, 340, 476, 478 Hertwig, R., 122, 132 Herve, A., 150, 171 Herzog, A., 34, 59 Herzog, R. O., 34, 59 Hesselbach, M. L., 9, 31 Hevesy, G., 285,325,338,340 Hewitt, L. F., 403, 446 Hewitt, R., 164, 169 Heyn, A. N. J., 38,59

514

AUTHOR INDEX

Highberger, J. H., 229,248 Hildebrand, J. H., 389, 403, 445 Hill, A. V., 176, 208, 399, 420, 445 Hill, C., 369, 383 Hill, D. K., 435, 445 Hill, R., 219, 238, 249 Hilmoe, R. J., 289, 340 Hilz, H., 370, 382 Himsworth, H. P., 21, 30 Hines, M. N., 189, 191, 206,207,208 Hingst, H. E., 164, 169 Hipp, N. J., 399, 446 Hirsch, G. C., 18, 21,30 Hoch, F. L., 352, 353, 356, 357, 358, 359, 365, 362, 364, 365, 371, 372, 374, 380, 381, 382, 385, 386 Hodgkin, A. L., 438, 445, 454, 455, 457, 459, 460, 464, 465, 466, 467, 468, 469, 470, 471, 472, 474, 475, 477, 478, 479 Horman, H., 229, 248 Hoerr, N. L., 64, 94 Hoff, E. C., 78,96 Hoff, H. E., 78, 96 Hoffmann, U., 229, 248 Hofmann, E. C. G., 153,169,172 Hogben, L. T., 176, 181, 202,204,208 Hogeboom, G. H., 9, 20, 30, 280, 281, 282, 284, 288, 289, 313, 314, 315, 319, 322, 323, 325, 326, 329, 330, 331, 332, 335, 340,341,343,375, 377,383 Hokin, L. E., 314,341 Hollander, W., 422, 448 Holloway, B. W., 151, 152,169 Holman, M. E., 461, 465,480 Holmes, H. F., 256, 278 Holt, S. J., 314, 344 Holter, H., 143, 169, 280, 284, 341, 490, 494, 4%, 497, 498, 499, 500, 501, 503, 504 Holtfreter, J. F., 329, 341 Holzer, H., 356, 383 Honjo, G., 52, 59 Honnen, L., 243, 250 Hopkins, H. S., 454, 480 Hortling, H., 158, 169 Hossack, J., 328, 339 Houghton, B. J., 426, 448 Houseal, R. W., 164, 173 Hove, E., 353, 383 Howatson, A. F., 12, 17,30

Howe, P. R., 236,251 Howsmon, J. A., 49, 59 Huber, L., 239, 250 Hudson, B., 205, 208 Hudson, P. B., 369,385 Hug, O., 141, 142,169 Huggins, C. B., 259, 260, 261, 277 Huggins, M. L., 400,445 Hughes, W. L., Jr., 146,173,353,385 Hullin, R. P., 313, 343 Hult, L., 264, 278 Humphrey, G. F., 285, 331,341,344 Hung, L. V., 314,339 Hunter, F. R., 411, 420, 445 Hunter, T. H., 415, 448 Hutchens, J. O., 425, 445 Huxley, A. F., 454, 455, 464, 466, 467, 468,469, 478, 479 Hyman, L., 122, 132 Hynes, M., 147, 174

I Imai, N., 268, 277 Ino, S., 159, 171 Irvin, J. M., 314, 323, 343 Irvin, J. L., 314, 323,343 Isaacs, R., 148, 169 Isherwood, F. A., 40,59 Istomanova, T. S., 154, 158,169 Ito, K., 362, 383 Izak, G., 154, 167 Izzo, M. J., 147,173

J Jackson, D. S., 216, 224,237,238, 249 Jackson, R. M., 314,341 Jacobs, G., 314, 330, 339 Jacobs, M. H., 146, 169, 428, 429, 432, 433, 435,436,445, 448 Jacobsen, E., 140, 152, 162,169 Jacobsthal, E., 164, 169 Jaeger, J. C., 80, 87,88, 95 Jaggannathan, V., 370, 383 James, T. W., 328,340 Jamison, D. G., 221,249 Jancke, W., 34, 59 Janda, V., 182,201,208 Jandebeur, W., 40, 60 Jenerick, H. P., 454,457, 458, 479 Jenner, H. O., 369,383

AUTHOR INDEX

Jensen, L. H., 220, 249 Jermyn, M. A., 40,59 Joblot, L., 98, 132 Jockel, H., 314, 343 John, H. M., 476, 479 Johnson, F. R., 260, 261, 277 Johnson, J., 468, 480 Johnson, J. A., 477,479 Johnson, R. B., 288,313,341 Johnston, P. M., 326,343 Johnstone, B. M., 460,461,465,480 Jones, E. S., 137, 152, 153, 164,169 Jones, H., 146,149,169 Jordan, W. K., 315,341 Jorgenson, L., 38, 59 Joseph, S., 138,166,326,338,417,443 Josephs, H. W., 160,169 Joyce, C. R. B., 477,479 Jiirgens, R., 148, 160,173 Jung, F., 142, 169 Jung, G., 313,344

515

Kearns, J. E., 262, 277 Keech, M. K., 212, 225, 235, 239, 240, 241, 242, 243,245,247,248,249 Keighley, G., 137, 150, 151, 166 Keilin, D., 346, 353, 354, 364, 383 Keitel, H. G., 146, 149, 169 Keleti, T., 355, 357,381,383 Kellenberger, E., 239, 250 Keller, C. J., 150, 169 Keller, E. R., 334, 341 Kelly, M. G., 8, 12, 29 Keltch, A. K., 425, 445 Kendrew, J. C., 214, 224, 249 Kennedy, E. P., 314, 324,341 Kensler, C. J., 314, 341 Kenten, R. H., 212, 228, 247 Kern, C., 165, 166 Kerppola, W., 153, 169 Kerr, S. E., 313, 344 Kerr, T., 34, 58 Kershaw, W. E., 221, 249 Kesselring, K., 153, 169 K Kesten, H. D., 148, 174 Kabisch, W. T., 271, 277 Ketterer, B., 204, 208 Kachmar, J. F., 476, 479 Key, J. A., 136, 137, 138, 141, 142, 143, Kagi, J. H. R., 357,366,367,368,385 144, 145, 147, 148, 157, 169, 261, 277 Kahler, H., 8, 12, 17, 29 Key, K. H. L., 178,208 Kahn, J. B., Jr., 477, 479 Keynes, R. D., 438, 445, 451, 454, 455, Kalnitsky, G., 313, 315, 343 457, 459, 460, 462, 465, 466, 467, 469, Kamada, T., 426, 439, 446 470, 471, 472, 474, 475, 477, 478, 479, Kamphausen, H., 280, 341 480 Kanda, K., 143, 173 Kielley, R. K., 314, 315, 341 Kao, C. Y., 426, 446, 459, 460, 465, 478 Kielley, W. W., 315, 341 Kaplan, M. H., 492, 504 Kimball, R. M., 270, 277 Kaplan, N. O., 361, 370, 380, 383,384 King, E. J., 369, 381 Kark, R. M., 153,173 Kinkel, H., 158, 159, 169 Kartha, G., 241, 243,250 Kissmeyer, A., 219, 249 Karvonen, M. J., 160,168 Kitchen, S. F., 164, 165,170 Katersky, E. M., 230, 249 Kitching, J. A,, 433, 446 Katz, B., 72, 85, 87, 90, 92, 93, 94, 95, Klein, A. K., 366, 383 455,457,463,464,467,478,479 Klein, B., 98, 99, 100, 118, 119, 132 Kautz, J., 328, 341 Klein, R., 8, 28 Kawai, K., 162, 173 Kleinberg, W., 156, 168 Kay, H. D., 149,169 Kleinholz, L. H., 182, 183, 198, 208 Kay, L. M., 243, 250 Klemperer, F. W., 369,383 Kay, R., 369, 383 Klomp, H., 424, 447 Kaye, E. R. M., 285, 321, 324, 331, 334, Klose, S., 320, 341 340, 341, 344 Klotz, I. M., 193, 206, 398, 446 Kaye, M. A. G., 361,383 Knowles, F. G. W., 175, 182, 183, 188, Kearney, E. B., 370,383 193, 195, 196, 201, 202, 205, 207, 208

516

AUTHOR INDEX

Knowles, H. C., 422, 443 Koch, H. J., 473, 479 Kodicek, E., 236, 248 Koefoed-Johnsen, V., 437, 446, 473, 474, 479 Kohler, V., 203, 208 Kohl, H., 161, 170 Kokas, E., 233, 249 Koller, F., 156, 157, 170 Koller, G., 193, 197, 208 Kolpak, H., 223, 249 Kolthoff, I. M., 347, 383 Koniuszy, F. R., 346, 384 Kopenec, A., 182, 208 Koritz, S. B., 152, 153,170 Kornberg, A., 288, 341, 343,363, 364, 370, 383, 384 Korner, A., 285, 341 Kosenow, W., 141, 170 Kostir, W. J., 328, 340 Krabbe, G., 33, 59 Krafka, J., Jr., 224, 249 Krahl, M. E., 415,425, 444,445 Kramer, H., 230, 249 Kratky, O., 38, 50,59 Krebs, H. A., 24, 28,407, 411, 443,448 Kreger, D. R., 3 5 6 0 Kretschmer, H. L., 259, 277 Krevisky, C., 421, 446 Krigbaum, W. R., 403,444 Krishnamoorthi, B., 425, 446 Kroner, T. D., 243, 244,249 Kruh, J., 150, 151,170 Krumbhaar, E. B., 140,170 Kruszynski, J., 149, 170 Kubowitz, F., 346, 383 Kiihn, A., 179, 208 Kiihn, K., 229, 248 Kiinzer, W., 144, 145, 156, 158,170 Kuff, E. L., 9, 20, 30, 284, 315, 322, 324, 340, 341 KuWer, S. W., 87, 96 Kuhn, W., 438, 446 Kun, E., 372, 383 Kunitz, M., 370, 383 Kurnick, N. B., 320, 341 Kurth, E. F., 40, 60 Kuyper, B., 51, 53, 59

L Lacour, F., 25, 30 Lacroix, P., 263,265,271,277 Lacy, D., 3, 30 Lageder, K., 150,170 Lagerlof, B., 152, 170 Laird, A. K., 314, 341 La Manna, C., 144,167 Lamb, W. G. P., 332,341 Lambert, S., 150, 151,170,171 Lamison, S. A., 160,170 Lan, T. H., 314,341 Landgrebe, F. W., 175, 177, 181, 202, 208, 210 Landis, E. M., 397, 446 Lang, H., 286,314, 315,341 Lang, K., 280, 282, 286, 287, 288, 313, 314, 315, 341, 343, 344 Langendorff, H., 161, 170 Langer, L. J., 364,383 Langermann, H., 314, 341 Lansing, A. I., 214, 218, 219, 223, 225, 230, 232, 233, 238, 248,249 Lardy, H. A., 313, 342, 367, 370, 382, 383 Larrabee, M. G., 424,433,446 Laskowski, M., 314,330,339 Latour, H., 420, 445 Laur, C.-M., 137, 143,168,170 Lawrason, F. D., 144, 145, 165,170 Lawrence, J. S., 136, 155, 156, 161,174 Lawson, H. A., 155,171 Leaf, A., 407, 409, 446 Leblond, C. P., 216,229,248,249 Lederer, E., 347, 383 Lederer M., 347, 383 Ledoux, L., 482, 504 Lee, R. I., 141, 170 Leeson, D., 414, 446 Lefeber, C. G., 481, 503, 504 LeFevre, M. E., 416, 446 LeFevre, P. G., 416, 446 LeGette, J., 243, 250 Legge, J. W., 364,383 Lehmann, F. E., 328, 338 Lehmann, H., 370, 383 Lehninger, A. L., 288, 314, 315, 324, 341, 342, 369, 383 Leichsenring, J. M., 160, 170 Leiner, G., 353,354,381,383

517

AUTHOR INDEX

Leiner, M., 353, 354, 381, 383 Leitch, J. L., 434, 446 Leitner, S. J., 154, 170 Lemberg, R., 364, 383 Le Page, G. A., 288,315, 319,325,342 Lerner, S. R., 285,344 Leslie, I., 151, 163, 167 Leuchtenberger, C., 330, 342 Leuckart, R., 259, 277 Leurat, E., 147, 172 Levander, G., 262,264,277,278 Lever, J. D., 4, 5, 30 Levi, H., 460, 462, 479 Levine, P., 414, 444 Levy, A., 420, 445 Levy, S. R., 281,340 Lewis, G. N., 389, 446 Lewis, M. S., 398, 446 Lewis, P. R. L., 466, 479 Lewis, W. H., 481,492, 498, 502,504 Li, C. H., 205,208 Liek, E., 254, 256, 278 Liley, A. W., 91, 96 Lillie, R. D., 217, 218, 248 Lillie, R. S., 424, 430, 446 Limarze, L. R., 160,168 Lindahl, O., 265, 278 Lindberg, O., 12, 18, 30 Linde, W., 148, 168 Lindenbaum, A., 398, 446 Linderstrfim-Lang, K., 403, 445 Lindhal, P. E., 285, 342 Lindigkeit, R., 151, 152, 153,168,170 Ling, G., 454,455,457,458, 469, 479 Lingane, J. J., 347, 383 Linke, P. G., 158,170 Lipmann, F., 370,382,383 Lippert, W., 141, 142,169 Lipschiitz, B., 25, 30 Lissak, K., 79, 96 Litt, M., 282, 339 Litten, M., 256, 278 Little, K., 230, 249 Liu, Y. M., 454, 478 Lloyd, B. J., 8, 17, 29 Lloyd, D. J., 224, 249 Lloyd, P. F., 239, 249 Lloyd, T. W., 156, 161, 162,166 Locquin, M., 26, 29 Lfivtrup, E., 282,314, 322,342

Lfivtrup, S., 428, 433, 446,499, 504 Loewi, G., 260, 262,278 Logan, M. A., 230,250 Logan, R., 286,287, 320,342 Lohmann, K., 370, 383 London, I. M., 150, 151,170 Loofbourow, J. R., 347,382 Lord, R. C., 347,382 Lorente de No, R., 463,479 Lorenz, P. B., 146,171 Lovegrove, T. D., 151,170 Lowe, M. E., 192, 198, 199, 201, 207, 208 Lowenstein, L. M., 139, 142, 143, 146, 149, 163, 170, 405, 406, 407, 409, 418, 421, 422, 444 Lowry, 0. H., 230, 249 Lowy, P. H., 137, 150,166,170 Lucius, S., 286, 287, 314, 315, 341, 343 Lucius-Lang, S., 314, 343 Luck, J. M., 370,383 LuckC, B., 404, 405, 416, 418, 424, 425, 428, 430, 431, 433, 434, 435, 436, 446 Luckner, H., 462, 479 Luco, J. V., 62, 79, 95, 96 Ludewig, S. L., 314,327,342 Ludin, H., 138, 142, 168 Luft, J., 74, 82, 92, 96 Lund, E. E., 99, 122,132 Lwoff, A., 98,99, 102, 125,132 Lybeck, H., 150, 170 Lyle, G. G., 287, 342 Lyubimova, M. N., 153,168

M Ma, W.-C., 143, 170 McArdle, A. H., 334,338 McCarroll, H. R., 260,261, 277 McCord, W. M., 150,173 McCormack, J. I., 408, 409,444 McCrie, J. G., 141, 144, 148,167 McCutcheon, M., 404, 405, 418, 425, 428, 430, 431, 433, 446 McDonald, D. A., 228, 231, 243, 249 Macdonald, J. C., 452, 479 McDonald, M. R., 370,383 McDowell, M. E., 426, 446 McElroy, W. D., 372,383 MacEwen, W., 255, 278 McGarr, J., 243, 244, 249 McGavin, J., 243, 248

518

AUTHOR INDEX

McIntosh, F. C., 79, 96 McKee, R. W., 468,479 MacLachlan, E., 146, 149,169 McLean, F. C., 271,278,397, 448 McLean, P., 331, 340 Macleod, J., 421, 446 McManus, J. F. H., 216, 249 McMaster, P. E., 262, 278 McMeekin, T. L., 399, 446 McMinn, R. M. H., 260,261,277 MacNaugher, E., 147, 166 MacRae, T. P., 238,249 McVay, J. A., 196, 197,209 McWhinnie, M. A., 184, 209 Madisso, H., 397, 448 Maegraith, B. G., 137, 152, 153,169 M a y , L. H., 409, 446 Magnussen, J. D., 160,170 Mahler, H. R., 359, 364, 383 Maisel, G. W., 469, 470, 479 Maizels, M., 411, 446, 462, 467, 468, 470, 472, 478, 479 Makler, J., 416, 425, 431, 435, 446 Malamos, B., 164, 170 Maliuga, D. P., 366, 383 Mallette, M. F., 346, 364, 365, 383 Mallory, F. B., 215, 249 Malmstrom, B. G., 351, 383 Manery, J. F., 410, 446 Mangini, H., 139, 141, 142, 143, 166 Mann, T., 346, 353, 354, 364,383 Mannweiler, Kl., 122, 133 Minyai, S., 471, 480 March, R., 315, 341 Marcussen, P. V., 139, 140,170 Margaria, R., 353, 382 Margoshes, M., 348, 366, 367, 368, 383, 384 Mark, H., 34, 59,403, 446 Marko, A. M., 235,249 Marks, J., 160, 168 Marmont, G., 467, 479 Marmorston, J., 213, 238, 251 Marshak, A., 281,342,427,446 Marshall, J. M., Jr., 490, 492, 494, 496, 497,498, 500, 501,504 Marshall, V. F., 260, 278 Marrack, J., 403, 446 Martin, A. R., 91, 94 Martin-Lagos, F., 264, 278

Martins-Ferreira, H., 454, 457, 478 Masadu, R., 267, 278 Masing, E., 151, 170 Mason, H. A., 264, 265, 266,267, 277 Masson, P., 215, 249 Mast, S. O., 433, 446, 481, 482, 483, 489, 490, 504 Master, C. M., 425, 435, 443 MathC, G., 407, 423,445 Mathieu, R., 315,322, 323,337 Matsuba, K., 143, 173 Matsumoto, K., 183, 187, 209 Matthews, S. A., 176, 209 Maughan, G. B., 160,174 Maupas, E., 122, 133 Mauritzen, C. M., 336, 342 Mauro, A., 438,446,461,479 Maver, M. E., 282,314, 322, 342 Maximow, A., 236,250,254, 255, 278 Mayer, D. T., 281,284, 330, 342,344 Mayes, S., 160, 173 Mazia, D., 98, 132, 280, 342 Mazur, M., 369, 382 Meglitsch, A., 196, 206 Mehl, J. W., 280, 340 Meigs, E. B., 452, 479 Meihel, K., 40, 60 Meister, A., 315, 342 Meldrum, N. U., 353, 354,382,384 Mellon, M. G., 347, 384 Mendive, J. R., 353, 384 Mercer, E. H., 320, 322,332,339 Merchant, F. T., 160,174 Mermod, C., 148, 170 Mettetal, C., 424, 433, 447 Metz, C. B., 98, 99, 102, 108, 111, 118, 119, 131, 133 Meyer, A., 78, 96 Meyer-Arendt, E., 362, 363, 381 Meyer, K., 214, 250 Meyer, K. H., 34,59,224, 239, 250 Meyer, M., 78, 96 Michaelis, L., 218, 250 Mikuta, E. T., 284,344 Miley, J. F., 408, 409, 443 Millard, A., 34, 36, 55, 59 Miller, E. B., 144,170 Miller, E. C., 281, 319, 334, 342 Miller, E. G., 212, 227, 251 Miller, F., 12, 30

519

AUTHOR INDEX

Miller, G. H., 369, 383 Miller, J. A., 281, 319, 331, 334, 342 Miller, L. L., 320, 341 Mills, G. T., 288, 342, 344 Minick, 0. T., 98,133 Minot, G. R., 136, 141, 147, 148, 155, 156, 167,169,170,171 Minz, B., 82, 95 Mirsky, A. E., 280, 282, 283, 284, 285, 286, 288, 313, 320, 326, 331, 332, 333, 337,338,339,342,344 Misra, P., 35, 58 Mitchison, J. M., 419, 447 Mitidieri, E., 315, 344 Mobberly, W. C., Jr., 192, 208 Moeschlin, S., 142, 143, 144, 165,171 Moldawsky, J. W., 141, 171 Mollison, P. L., 414, 443 Mondolfo, S., 265, 278 Monod, J., 476, 477 Montgomery, H., 219, 238,249 Monty, K. J., 333, 334,339, 340 Mook, H. W., 462, 478 Mooney, G., 268, 278 Moore, C. V., 160,173 Moore, J. W., 455,479 Morawitz, P., 152, 171 Morikawa, K., 159, 171 Morton, J. H., 326, 343 Morton, R. K., 280,288,338,341 Moser, P., 141, 142,169 Moss, J. A., 227,228, 237,242,247 Moulk, Y., 18, 20, 29, 319, 322, 324, 332, 335, 339 Moussa, T. A. A., 3,30 Mudge, G. H., 9,31,407,409,447 Miihlethaler, K., 34, 55, 59, 60 Mueller, G. C., 331, 342 Miiller-Neff, H., 147, 171 Miiller, L., 314, 315, 341 Miissbichler, H., 204, 209 Muir, H. M., 235, 249 Mukherjee, S. M., 37, 50, 59 Mulherin, W. A., 164,173 Muller, M., 452, 480 Muller, O., 347, 384 Mullins, L. J., 462, 479 Muntz, J. A., 476, 479 Murphy, R. C., Jr., 164,171 Murphy, W. P., 136, 155,171

Murray, M. R., 3, 29 Murray, R. G. E., 137, 144, 145, 149, 151, 163, 166 Myers, A., 35, 39, 42, 49, 58, 58, 59

N Nachmansohn, D., 466,476, 478,479 Nachtrieb, N., 347, 384 Nagano, T., 184, 193,209 Nageotte, J., 236, 250, 270, 278 Nakamura, M., 378, 385 Nason, A., 380, 384 Nastuk, W. L., 454, 464, 465, 479 Naylor, A., 238, 249 Needles, R. J., 153, 166 Neff, R. J., 330,342 Negelein, E., 355, 384 Neiradt, C., 153, 172 Neiradt-Hiebsch, C., 153, 172 Nelson, J. M., 346, 364, 384 Nemetschek, T., 222, 249 Neuberger, A., 150,171,235, 249 Neuhof, H., 258, 278 Neukomm, A., 424, 444 Neuman, H., 369, 384 Neuman, R. E., 230,239,250 Neuman, S. B., 223, 250 Neumark, E., 154, 170 Neurath, H., 353,354,355,382,384,385 Nevard, E. H., 39,58 Nevis, A. H., 438, 447 Nevros, K., 40, 60 Newman, B., 160,171 Nichols, G., 409, 447 Nichols, N., 409, 447 Nicolai, E., 34, 36, 49, 51, 53, 55, 57, 59 Nicolle, P., 141, 154,171 Niemann, C., 243, 247 Ninni, M., 136, 163, 171 Nishikawa, K., 162, 173 Nittis, S., 137, 171 Niven, J. S. F., 150,171 Nizet, A., 138, 139, 150, 151, 153, 154, 155, 156, 157, 158, 160, 161, 162, 170, 171 Noe, E., 313, 319,342 N d l , R., 64,66, 96 Noonan, T. R., 462, 479 Norman, A. G., 40,48,58,59 Norris, L. M., 160, 170

520

AUTHOR INDEX

Pakesch, F., 139, 166 Palade, G. E., 4, 17, 18, 30, 31, 68, 69, 72, 74, 96, 138, 171, 281, 313, 315, 319, 326, 328, 335, 341, 342, 375, 377, 383, 413, 447,486,504 Palatine, I. M., 151, 172 Palay, S. L., 3, 24, 31, 68, 69, 89, 92, 93, 96, 328, 342 Pannacciulli, I., 156, 162, 166 Panouse, J. B., 175, 194,209 0 Pappas, G. D., 328, 342 Oberling, Ch., 8, 12, 17, 18, 21, 25, 28, Pappenheimer, J. R., 438, 447 29, 30 Paoletti, C., 151, 171 Ochoa, S., 286, 288,340 Paolino, W., 144, 171 O’Connell, M. P., 334,340 Parducz, B., 98, 100, 101, 104, 118, 119, dstlund, E., 205, 209 124,133 Ogden, C. K., 350,384 Parker, E., 502, 504 Ogston, A. G., 393, 399,447 Parker, G. H., 175, 202, 209 Ohlmeyer, P., 320,342,369,384 Parker, R. C., 261, 278 Okay, S., 184, 209 Parkinson, J. L., 176, 208 Okinaka, S., 159, 171 Parpart, A. K., 146, 169, 171, 412, 414, Oldewurtel, H. A., 380, 384 416, 420, 433, 434, 435, 436, 446, 447, Oliva, G., 159, 171 448 Olson, J. A., 353, 361, 362, 384,385 Parpart, E. R., 146, 171 Olson, M. E., 150, 152,172 Partridge, S. M., 213, 214, 216, 218, 226, Omachi, A., 314, 342 227, 228, 229,230, 242, 247,250 Oncley, J. L., 403, 447 Paschkis, I., 158, 171 Ondarza, R., 288, 342 Paschkis, K. E., 315,343 Opie, E. L., 407, 447, 448 Passano, L. M., 182, 208 Opton, E. M., 124,133 Pate, S., 321, 324, 334, 340 Orekhovitch, K. D., 238, 250 Paton, D. N., 137,171 Orekhovitch, V. N., 238, 250 Patterson, J. H., 462, 479 Orell, S., 265, 270, 271, 277, 278 Paul, J., 481, 502, 504 Orfila, J., 162, 168 Pauling, L., 243, 250 Ormsbee, R. A., 468,479 Pautsch, F., 178, 208 grskov, S. L., 137, 149, 169, 414, 419, 420, Peacock, W. C., 146,168 447,452, 478 Pearce, R. M., 259, 278 Orten, J. M., 136, 140, 152, 160, 161, 163, Pearse, H. E., 137, 148,171 171 Pease, D. C., 4, 5, 28, 66, 96, 139, 143,171 Ortiz, P. J., 288, 340 Peattie, R. W., 348, 385 Osawa, S., 280, 283, 286, 288, 333,338,342 Peer, L. A., 271, 278 Osgood, E. E., 137, 154, 161,171,174 Peet, M. M., 141, 147,171 Otsuka, M., 283, 339,464, 477 Penn, J., 217, 248 Ottesen, M., 284, 341 Pepper, 0. H. P., 141, 147, 156, 162, 171 Ottolenghi, P., 137, 153, 154, 162,172 Perez-GonzAles, M. D., 187, 201, 206, 209 Overbeek, J. T. G., 398, 447 Perkins, E. B., 193, 209 Overton, E., 464, 479 Perrone, J. C., 236, 250 Persons, E. L., 144,171 P Petermann, M. L., 281, 315,343 Padieu, P., 150, 170 Petermann, M. N., 353,384 Paganelli, C. V., 437, 438, 447

North, A. C. T., 243,248 Northrop, J. H., 430, 447 Nouchy, A., 158, 166 Novack, B. G., 314,343 Novikoff, A. B., 7, 18, 30, 280, 313, 314, 315, 319, 320, 327,342,343 Nurnberger, J. I., 324,340 Nuss, M. A., 160,171 Nygaard, A. P., 353, 356,380,384

52 1

AUTHOR INDEX

Peters, D., 139, 141, 142, 172 Pfeiffer, C. A., 264, 278 Pfleiderer, G., 362, 363, 381,386 Phelpstead, J. W. P., 284, 342 Phemister, D. B., 259, 278 Phillips, P. H., 370, 382 Phillipson, A. T., 472, 478 Philpot, J. St. L., 280, 283, 285, 329, 331, 333, 342 Pickford, G. E., 175, 202, 203, 209 Pigoh, A,, 428,433,446,499, 504 Pintor, P. P., 161, 172 Pitelka, D. R., 98, 99, 102, 108, 111, 118, 119, 131, 133 Plotnikova, N. E., 238, 250 Plum, C. M., 136, 137, 140, 152, 154, 155, 156, 160, 162, 163, 169, 172, 261, 277 Plum, R., 162, 172 Pocharissky, J. F., 254, 278 Podber, E., 280, 313, 315, 319, 342, 343 Pokrowsky, W. I., 154,172 Polettini, B., 270, 278 Policard, A., 26, 31 Pollister, A. W., 321, 330, 342 Polyak, S. L., 79, 96 Pomerat, C. M., 481, 503, 504 Poncher, H. G., 160,168 Ponder, E., 149, 172, 404, 405, 406, 407, 411, 412, 414, 415, 416, 417, 418, 419, 420, 421, 422, 446,447 Poort, C., 284, 342 Popa, G. T., 214, 250 Porod, G., 38, 59 Porter, H., 202, 209 Porter, K. R., 4, 8, 31, 98, 99, 103, 105, 108, 111, 114, 118, 119, 132, 133, 235, 237, 250 Potter, V. R., 280, 287, 315, 319, 325, 342, 343 Powers, E. L., 4, 31, 98, 104, 106, 114, 120, 122, 124, 127,132,133 Prankerd, T. A. J., 442, 445, 451, 477, 478 Prescott, D. M., 426, 433, 437, 447, 483, 486, 489,492,499, 500,503,504 Pressmann, B. C., 7, 29, 313, 333, 340 Preston, R. D., 34, 35, 36, 37, 39, 42, 48, 49, 50, 51, 52, 53, 55, 56, 58, 58, 59, 60 Price-Jones, C., 137, 172 Price, J. M., 281, 319, 334, 342 Pricer, W. E., Jr., 364, 383

Pritchard, J. A., 146, 153, 172 Pritchard, J. J., 256, 265, 267, 272, 277 Putnam, F. W., 354,384

R Rabinovitz, M., 150, 152, 172 Rachmilewitz, M., 160, 172 Racker, E., 355, 384 Rademaker, W., 315, 342 Ragan, C., 215, 250 Rall, T. W., 315, 342 Ralph, C. L., 191, 207 Ralph, P. H., 142, 143,172 Kamachandran, G. N., 241, 243,250 Ramasarma, G. B., 227,238,249 Ramon y Cajal, S., see Cajal, S. R. y, Ramsey, R., 139, 172 Ranby, B. G., 36,37,59 Randall, J. T., 328, 339 Randall, M., 389, 446 Randolph, M. L., 284, 342 Rapela, C. E., 82, 85, 96 Rapoport, S., 137, 144, 145, 149, 151, 152, 153, 162,168,169,172 Rappaport, H., 136, 167 Rapport, M. M., 214, 250 Rasch, G., 140, 169 Rasmussen, G. L., 63,76,96 Rasquin, P., 203, 206 Raven, C. P., 424,447 Ray, R. D., 268, 278 Rebhun, L. I., 328,343 Rechenman, R., 151, 168 Reed, R., 34, 36, 55, 59, 212, 214, 218, 219, 220, 221, 222, 223, 225, 229, 230, 232, 233, 235, 238, 239, 240, 241, 242, 243,245,247,248,249 Rees, C. W., 120,133 Rees, K. R., 286,340 Reeve, E. B., 414, 446 Reeves, A., 314, 341 Regen, E. M., 260,278 Reger, J. F., 68, 72, 74, 96 Rehm, W. S., 408, 409, 443 Reichard, P., 315, 343 Reidel, A,, 244, 248 Reimann, F., 149, 172 Reimers, H., 34, 59 Reisner, A., 161, 170 Reissig, M., 63, 66, 95

522

AUTHOR INDEX

Remilton, E., 204, 208 Rendano, C., 264, 278 Renkin, E. M., 438, 447 Renschler, H. E., 407, 448 Rerabek, J., 319, 334, 343 Resuhr, B., 433, 448 Reynolds, E. S., 378,384 Reznikoff, P., 145, 172 Rhodin, J., 4, 7, 12, 31, 223, 250, 486, 504 Ribi, B., 36, 59 Ricca, R. A., 424,433,436, 446 Rice, L. I., 314, 343 Rich, A., 243, 250 Richards, I. A., 350,384 Richardson, K. C., 63, 94 Richmond, J., 407, 443 Richter, D., 313, 343 Rickes, E. L., 346,384 Riddle, M. C., 136, 141, 156,172 Rideal, E. K., 353,382 Riecker, G., 407, 422,448 Riggs, T. R., 151,172 Rinaldini, L. M., 234, 250 Rind, H., 142, 143, 172 Rinehart, J. F., 216, 229, 250 Ripley, G. W., 35, 51, 52, 58, 59, 60 Ripley, S. H., 151, 152, 169 Ris, H., 320, 332, 341, 343 Riska, N., 160, 172 Rittenberg, D., 150, 151, 170 Ritter, G. J., 40, 60 Robb-Smith, A. H. T., 235, 250 Robbins, K. C., 220,233, 234, 248 Roberts, D., 289, 340 Roberts, E., 227, 238, 249 Robertson, D., 66, 68, 72, 74, 96 Robertson, J. C., 468, 480 Robertson, J. S., 411, 448 Robertson, 0. H., 203, 209 Robin, E. D., 365, 385 Robinson, C. V., 499, 503 Robinson, E. J., 418, 420, 421, 422, 447 Robinson, M. E., 403, 443 Robinson, J. R., 24, 31, 392, 407, 409, 412, 448 Robscheit-Robbins, F. S., 138, 149, 156, 162, 171, 173 Roche, J., 369, 381 Rodig, I., 144, 145, 166 Rohlich, K., 271, 278

Roelofsen, P. A., 35, 52, 60 Roepke, R. R., 422, 448 Rohrbach, R., 224, 251 Roll, P. M., 285, 338 Romhanyi, G., 225, 250 Romlet, F. C., 235, 248 Roodyn, D. B., 283, 284, 285, 314, 315, 322, 324, 325, 327, 329, 330, 332, 333, 342, 343 Rook, A. J., 221, 251 Roome, N. W., 262, 278 Roos, P., 205, 208 Root, W. S., 158, 159, 160, 163,168 Roque, M., 127, 133 Roscoe, J. D., 160, 168 Rosenblueth, A., 79, 82, 96 Rosenthal, O., 314, 319, 330,343 Rosenthal, T. B., 223, 225, 227, 230, 232, 238, 249 Rosin, A., 160, 172 Rosin, H., 141, 172 Ross, K. F. A., 423, 448 Rossiter, R. J., 137, 144, 145, 149, 151, 163, 166, 170 Rossmuller, G., 287, 314, 341,343 Roth, H., 264, 278 Roth, J. S., 315, 343 Roth, L. E., 4, 31, 98, 99, 102, 114, 133 Rothbard, M. B., 407, 447 Rothenberg, M. A., 466, 479 Rotherham, J., 314, 323, 343 Roughton F. J. W., 353, 354,382,384 Rouiller, Ch., 4, 7, 9, 12, 13, 18, 20, 21, 25, 28, 29, 30, 31, 319, 322, 324, 332, 335, 339 Rouslacroix, A,, 259, 277 Roux, M., 217, 248 Rowlands, A., 204, 209 Roy, A. B., 315, 336,342,343 Rubinstein, D., 137, 153, 154, 162, 172 Rudzinska, M. A., 4, 31 Riittimann, A., 12, 31 Ruhenstroth-Bauer, G., 149, 151, 172 Rupen, A,, 148, 168 Rupley, J., 355, 385 Russell, G., 227, 228, 250 Rutman, R. J., 315, 343 Ryan, J., 313, 319, 342 Ryan, R. R., 284, 342 Rytzner, C., 76, 95

AUTHOR INDEX

S Sabin, F. R., 163,167,172 Sabine, J. C., 153, 154, 172 Sabrazes, J., 147, 172 Sacerdotti, C., 254, 278 Sachar, L. A., 218, 250 Sacks, J., 326, 343 Sadasivan, V., 369, 384 Saetren, H., 282, 284, 313, 320,338,344 Saigh, L. M., 193, 206 St. Aubin, P. M. G., 326, 343 Saito, M., 143, 173 Sakabe, S., 162, 173 Saloum, R., 182, 208 Salter, W. T., 156, 167 Saltmann, P., 325, 338 Sammett, J. F., 260, 277 Samuelson, O., 347, 384 Sandeen, M. I., 178, 179, 185, 186, 190, 191, 194, 197,200, 206,207,209 Sandell, E. B., 347, 384 Santhanam, M. S., 241, 250 Saroff, H. A., 398, 446 Saslow, G., 415, 420,421,447 Saxl, H., 212, 216, 218, 220, 230, 232, 235, 238, 239, 240, 241, 242, 243, 245, 247, 248, 249, 250 Scatchard, G., 398,403, 447,448 Schapira, G., 150, 170 Scharrer, B., 175, 182,201,202,209 Scharrer, E., 66, 96 Schatzmann, H. J., 477, 479 Scheer, B. T., 194, 195, 209 Scheer, M. A. R., 194, 195, 209 Schein, A. H., 314,315,343 Scheinberg, I. H., 398, 448 Schilling, V., 142, 172 Schilling-Torgau, V., 141, 172 Schi$dt, E., 421, 448 Schleich, H., 229, 248 Schliack, J., 285, 325, 338 Schmidt, E., 40, 60 Schmitt, F. O., 222, 229, 237, 248 Schmitz, F. R., 33, 60 Schneider, D., 144, 145,170 Schneider, F., 218,229, 250 Schneider, M., 264, 277 Schneider, S., 356, 383

523

Schneider, W. C., 9, 30, 279, 280, 281, 282, 284, 287, 288, 289, 313, 314, 315, 319, 322, 323, 325, 326, 327, 329, 330, 331, 332, 335, 340, 341, 342, 343, 344, 375, 377, 383 Schneiderman, M., 139, 140,166,172 Scholzel, E., 152, 172 Schork, P. K., 144, 145, 165,170 Schossberger, F., 50, 59 Schottelius, D. D., 314, 323, 343 Schotz, M. C., 314, 343 Schrecker, A. W., 288,343,370,384 Schreiber, B., 264, 278 Schroeder, W. A., 243, 250 Schuberg, A., 98, 99, 100, 101, 112, 118, 119, 131, 133 Schubert, J., 398, 446 Schuler, D., 216, 247 Schultz, A., 236, 251 Schulz, G. V., 400, 401,448 Schulz, H., 12, 15, 31 Schurnaker, V. N., 492, 498, 502,504 Schwartz, B. M., 160, 172 Schwartz, I. L., 407, 448 Schwartz, S. O., 144,167 Schwarz, B., 237, 250 Schwarz, K., 365, 384 Schwarz, W., 218, 225,238, 251 Schweiger, H. G., 152,172 Schwind, J. L., 143,172 Schwoner, A., 158, 171 Scott, D. A., 353,384 Scott, G. T., 473, 479 Scott, R. L., 403, 445 Scriver, J. B., 164,173 Scudamore, H. H., 153, 173 Seaman, A. J., 154,171 Sedar, A. W., 4, 31, 98, 99, 103, 105, 108, 111, 114, 118, 119, 133 Seggel, K. A., 150,169,173 SCguCla, J., 102, 132 Seip, M., 136, 138, 139, 140, 154, 155, 156, 157, 158, 159, 160, 161, 162,173 Selby, C. C., 8, 12, 31 Seminova, D. P., 347,384 Seno, S., 143, 162, 173 Severi, R., 268, 278 Severinghaus, A. E., 25, 31 Seville, R., 219, 251 Sewell, C. E., 150, 153, 168

524

AUTHOR INDEX

Seyfarth, C., 141, 148, 160, 173 Shanes, A. M., 453,454,467, 479,480 Shapiro, H., 415, 416, 423, 424, 426, 434, 435, 439, 443, 448 Shapiro, L. M., 160, 173 Shapiro, S., 164, 171 Shapleigh, J. B., 160, 173 Shapras, P., 481, 496, 504 Shaw, F. H., 452, 460, 461, 465, 469, 480 Shaw, T. I., 472, 480 Sheenan, D., 78, 95 Sheldon, H., 9, 31 Shelton, E., 3, 322, 327, 343,344 Shemin, D., 150, 151,170 Shen, A. L., 398, 448 Shepherd, J. A., 313, 314,315,343 Sherrington, C. S., 61, 96 Shinozaki, J., 424,433,434, 436,448 Shohl, A. T., 415,448 Shonk, C. E., 288, 343 Shull, J. C., 420, 447 Shushan, M., 164, 173 Sicher, N., 218, 250 Sidel, V. W., 414, 417, 435, 436, 437, 448 Siebert, G., 280, 282, 285, 286, 287, 288, 313, 314, 315, 341,343,344 Siedlecki, M., 204, 209 Sieglitz, G., 204, 209 Siekevitz, P., 18, 31, 286, 287, 344 Simmel, H., 147, 173 Simon, S. E., 452, 460, 461, 465, 469, 480 Singer, I., 164, 165, 173 Singer, K., 144, 170 Sipe, C. R., 144, 145, 165,170 Sisson, W. A., 34, 59 Sitte, H., 12, 30 Sjostrand, F. S., 4, 21, 24, 31, 66, 68, 76, 79, 95, 96, 321, 328, 344, 486, 504 Sjovall, H., 137, 173 Skeen, M. V., 280, 344 Skinner, L. G., 150, 153,168 Skowron, H., 424, 448 Skowron, S., 424, 448 Slack, H. G. B., 232,236, 250, 251 Slawinski, A., 421, 448 Skin, M. W., 363, 370, 382 Slesser, A., 268, 278 Slome, D., 176, 202, 204, 208 Smallman, B. N., 314, 340

Smellie, R. M. S., 280, 285, 287, 314, 320, 331,341,342,343,344 Smith, C. A., 76, 96 Smith, E. C. B., 353, 384 Smith, E. E. B., 288, 342,344 Smith, E. Lester, 346, 384 Smith, E. L., 355,384 Smith, H. G., 181, 183, 209 Smith, McD., 481, 503, 504 Smith, 0. C., 347, 384 Smith, R. H., 240,243, 251 Smith, T., 144, 147, 173 Snell, C. T., 347, 384 Snell, F., 347, 384 Sobel, H., 213, 238, 251 SZrensen, S. P. L., 395, 448 Solandt, D. Y., 176, 208 Solomon, A. K., 414, 417, 435, 436, 437, 438,444,447,448,477, 480 Sonneborn, T. M., 127, 133 Soons, J. B. J., 315, 342 Sotelo, J. R., 76, 95 Souza Santos, P., 143,166 Spellman, R. M., 260,278 Spencer, B., 315,324, 325,339 Speyer, J. F., 313, 339 Sponsler, 0. L., 34, 60 Stadtman, E. R., 476, 480 Stampfli, R., 455,464, 478,479 Stanbury, S. W., 9, 31 Stanier, J. E., 280, 283, 285, 329, 331, 333, 342 Stark, G., 314, 343 Starling, E. H., 397, 448 Stats, D., 160, 172 Stasney, J., 150, 173 Stedman, Edgar, 282, 336,342,343 Stedman, Evelyn, 282, 314, 336, 342, 343 Steele, B. F., 154,173 Steer, A., 426, 446 Stefanini, M., 158, 167 Steggerda, F. R., 179, 207 Stein, W. H., 212, 227, 251 Steinbach, H. B., 462,471, 480 Steinbrinck, C., 34, 60 Stephens, G. C., 188, 189, 190, 206, 209 Stephens, J. G., 146, 147, 148, 173 Stephenson, E., 260, 262, 276 Stephenson, K. L., 265,268,278 Stephenson, W. K., 459, 480

AUTHOR INDEX

Stern, H., 280, 282, 284, 288, 313, 320, 331, 338, 344 Stern, J. R., 407, 448 Stetson, R. P., 136, 155, 171 Stevens Flores, I., 203, 206 Steward, F. C., 55, 60 Stewart, D. R., 432, 433, 445, 448 Stewart, J. M., 147,173 Stewart, W. B., 147, 173 Stobbe, H., 142, 143,172 Stock, D. A., 285,342 Stokes, R. H., 392,412, 448 Stoffregen, J., 161, 168 Stoneburg, C. A., 281,344 Storey, I. D. E., 315, 340 Stosic, L., 159, 169 Stover, R., 403, 448 Strasburger, E., 33, 60 Strassner, W., 153, 162,173 Strassner, W. L., 153,172 Straub, F. B., 363, 384, 471, 480 Straus, W., 313, 315, 344 Strauss, A. A., 258,278 Streeten, D. H. P., 422, 448 Striebich, M. J., 8, 9, 12, 17, 29, 30, 280, 282, 284, 314, 319, 322, 323, 325, 326, 327, 332,341,343,344 Strittmatter, C. F., 315, 344 Strugger, R., 7, 31 Sumner, F. B., 177, 209 Sund, H., 357, 358,359,361, 385 Suneson, S., 184, 207 Sung, S.-C., 314, 344 Svaetichin, G., 80, 96 Swan, R. C., 397,448,475,480 Swanson, M., 314, 344 Sweeney, H. M., 184, 209 Sweetland, M. L., 411, 445 Swerdlow, M., 223, 250 Swift, H., 321, 344 Sydenstricker, V. P., 164,173 Szabo, D., 220,233, 241, 247 SzCkely, M., 313,344,471, 480

T Tabroff, W., 243, 244, 249 Tahmisian, T. N., 3, 4, 28, 31, 328, 338 Tanz, S. S., 264, 265, 277 Tartar, V., 127, 131, 133 Tarver, H., 285, 341

525

Tattersall, R. N., 218, 219, 220, 221, 233, 238, 251 Tauber, H., 369, 384 Taubert, M., 282, 338 Taylor, C. V., 98, 131,133 Taylor, H. B., 162, 168 Taylor, I. M., 411, 443 Taxi, J., 74, 95 Teissier, G., 182, 209 Teitelbaum, H. A., 82, 96 Teorell, T., 392, 419, 448,471, 480 Terner, C., 411, 448 Thacker, E. J., 168 Theorell, H., 353,356, 380, 381,384 Thiers, R. E., 373, 376, 378,384,385 ThiCry, J. P., 3, 31 Thoai, N. V., 369, 381 Thoma, K., 141, 151,173 Thomas, E. D., 150, 173 Thomas, E. W. P., 221,251 Thomas, J., 315, 324, 325,339 Thomas, L,282, 339 Thomas, L. E., 330,344 Thompson, D. H., 177,206 Thompson, D’Arcy W., 98, 133 Thompson, H. P., 8,31 Thomson, J. F., 284, 344 Thorell, B., 144, 149, 151, 152, 166, 169, 170, 173 Thorn, G. W., 422, 448 Tiegs, 0. W., 72, 96 Timonen, S., 288, 314,344 Tinkle, D. W., 178, 180, 208 Tiprez, G., 165, 168 Tishkoff, G. H., 149, 173, 281, 284, 314, 336, 339 Titeca, J., 79, 96 Tobias, C. A., 347, 385 Tobolsky, A. V., 403, 446, Tomlin, S. G., 328,339 Tosteson, D. C., 411, 448. 468, 480 Trachtenberg, F., 141, 154, 155,173 Tramontana, C., 159, 171 Travis, D. F., 182, 209 Tristram, G. R., 243, 247, 251 Truhaut, R., 151, 171 Trujillo-CCnoz, O., 76, 96 Tsuboi, K. K., 313,344,369,385 Tsudniizura, H., 82, 95 Tubiana, M., 151, 171

526

AUTHOR INDEX

Tunbridge, R. E., 212, 213, 214, 218, 219, 220, 221, 222, 223, 225, 229, 230, 232, 233, 235, 238, 239, 240, 241, 242, 243, 245,247,248,249,251 Tupper, R., 354, 385 Turner, J. P., 131,133 Tustanovskii, A. A., 238, 250 Tyler, H. R., 371, 382

U Umrath, K., 203, 204, 209 Underhay, E., 314, 344 Underwood, E. J., 346, 385 Ungricht, M., 154, 157, 173 Unna, P. G., 212, 215, 216, 217, 251 Urist, M. R., 271, 278 Ussing, H. H., 428, 437, 446, 448, 460, 462, 473, 474, 479,480 Utter, M. F., 476, 480

V Valentine, E. H., 154,167 Valentine, F. C. O., 136, 147, 173 Vallee, B. L., 146, 173, 345, 346, 348, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 371, 372, 373, 374, 376, 380, 381, 381,382,383,384,385,386 Vallejo-Freire, A., 139, 142, 143, 166 Vallette, G., 315, 327, 344 van de Kamer, J. C., 204,207 Van Goor, H., 354,385 Van Heyningen, R., 354,386 van Iterson, G., Jr., 34, 59 Van Slyke, D. D., 397,448 Vars, H. M., 314, 319, 330,343 Vaughan, J. M., 146,167 Vaughan, S. L., 154,173 Vaz Ferreira, A., 76, 82, 85, 92, 94, 95 Veis, A., 245, 251 Velick, S., 149, 172 Velick, S. F., 355, 359, 382 Ventura, L., 160, 173 Verhoeff, F. H., 217, 251 Viala, R., 313, 340 Videbaek, A., 161, 168 Villela, G. G., 315, 344 Vincent, B., 141, 170 Virchow, R., 9, 31 Vitale, J. J., 378, 385

Viti, I., 314, 339 Vogt, C., 21, 31 Vogt, O., 21, 31 Voinar, A. O., 366,385 von Ehrenstein, G., 285, 325, 338,340 von Gelei, G., 98, 99, 100, 101, 102, 104, 118, 119, 132 von Gelei, J., 98, 99, 100, 111, 118, 119, 122, 124, 127, 132 von Hoffmann, U., 222, 249 von Horstman, E., 64, 96 von Korff, R. W., 476,480 von Kossa, J., 256, 277 von Robertson, W., 236, 237, 250 yon Seemen, H., 267, 278 Vorhaus, L. J., 153,173 Vryonis, G., 164, 165, 173 W Wachsmuth, G., 264, 278 Wacker, W. E. C., 353, 360, 361, 362, 364, 365, 369, 380,381, 385 Waked, N., 313, 344 Walcher, H., 203, 209 Walker, P. G., 315,344 Wallenfels, K., 357, 358, 359, 361, 385 Walsh, R. J., 150, 173 Wang, R. 1. H., 314,338 Wang, T. Y., 330,344 Warburg, E. J., 421, 443 Warburg, O., 152,174,346,386 Ward, R., 321, 342 Wardrop, A. B., 49, 51, 53,59,60 Waring, H., 175, 177, 181, 202, 209, 210 Warren, C. O., 139,172 Wassen, A. M., 355, 382 Watanabe, A., 464, 478 Watanabe, K., 98, 124,133 Watanabe, M., 52, 59 Watson, C. J., 150, 174 Watson, J., 164, 174 Watson, M. L., 321, 328, 344 Wattiaux, R., 7, 29, 313, 327, 333, 338, 340 Watts, A., 354, 385 Waugh, T. R., 160, 164,173,174 Weatherall, M., 477, 479 Webb, E. C., 354, 369,381,386 Webb, H. M., 188, 189, 190, 191, 194, 197, 206, 207, 210 Webb, M., 314, 344

527

AUTHOR INDEX

Weber, G. M., 319, 342 Weber, H. H., 403, 448 Weber, R., 284, 341 Webster, S. H., 165,174 Wegelius, R., 160, 174 Weicker, H., 144, 145, 162, 163,174 Weidmann, S., 459,464, 466,478,480 Weigert, C., 215, 217, 251 Weinland, D. F., 259, 278 Weinman, E. O., 285, 344 Weisel, G. F., 203, 210 Weiss, B., 282, 287, 344 Weiss, J. M., 8, 17, 24, 31 Weissberger, A., 347, 386 Weissenfels, N., 5, 7, 31 Weisz, P. B., 125, 133 Weitnauer, H., 224, 251 Wellman, H., 313, 342 Wells, H. G., 256, 278 Welsh, J. H., 182, 206,210 Wersall, J., 76, 96 West, E. S., 261, 277 Westfall, J. A., 98, 99, 102, 108, 111, 118, 119, 131, 133 Westhauser, R., 156, 161, 162,169 Whipple, G. H., 149, 173 Whitby, L. E. H., 147, 174 White, J. C., 151, 154, 163, 165, 166, 167 Whittam, R., 407, 411, 448, 470, 477, 480 Wichmann, H. J., 366,383 Wichterman, R., 102, 124,133 Wiedemer, C., 140, 161,168 Wieland, T., 362, 386 Wiesner, J., 33, 60 Wigand, R., 139, 142,172 Wilbrandt, W., 397, 448 Wilbur, K. M., 280, 282, 284, 315, 329, 332, 335, 338, 344 Wild, M., 144, 162, 163, 174 Wilhelm, M. M., 137,171 Wilkins, W. E., 260,278 Williams, J. N., 314, 344 Williams, R. J. P., 359, 360, 361, 363, 371, 382, 385, 386 Williams, T. F., 422, 448 Williams, W. T., 51, 60 Willstaedt, H., 264, 277, 278 Wilson, K., 55, 60 Windle, W. F., 160, 174 Windrum, G. M., 230, 249

Wing, M., 137, 144, 145, 149, 162, 172, 414, 420, 445 Winter, K. K., 218, 250 Wintrobe, M. M., 136, 144, 154, 155, 160, 163, 174 Wislocki, G. B., 9, 29, 216, 251 Wissig, S. L., 3, 24, 31 With, C., 219, 249 Witter, R. F., 334, 340 Wochner, D., 486,487,503 Wohlbach, S. B., 236, 251 Wohlfarth-Bottermann, K. E., 5,31 Wohlisch, E., 224, 251 Wolf, A. V., 426, 446 Wolf, K., 229, 248 Wolfe, J. W., 25, 31 Wollstein, M., 160, 174 Wolpers, C., 139, 142, 143, 165, 174, 222, 251 Wood, G. C., 216, 218, 220, 223, 224, 225, 226, 229, 233, 248,251 Wood, H., 286, 340 Wood, M. J., 212, 222, 235, 239, 240, 241, 242, 243, 245,247, 248,249 Wood, T., 160,166 Wood, T. R., 346,384 Woodbury, J. W., 464,477 Woods, J. H., 37, 50, 59 Woods, M. W., 9, 31 Woolf, B., 140, 174 Worley, L., 98, 101, 102, 105, 133 Wormall, R. W. E., 354,385 Wright, A. W., 25, 31 Wright, P. A., 204, 210 Wu, H., 397, 448 Wiirm, 267, 270, 278 W ~ l f f H.-J., , 355, 384 Wulff, V. J., 193, 207 Wyburn, G. M., 268,278,328,339 Wyckoff, R. W. G., 34,59,63, 96,222,251 Wynn, V., 426, 448

Y Yates, F., 140, 168 Young, E., 314, 343 Young, J. Z., 63,68, 94, 96 Young, L. E., 136, 147, 155, 156, 161, 173, 174 Young, R. H., 154,174 Yu, S. Y., 233, 251

528 Yuan-Chi Tang, 40, 60 Yusa, A., 98, 124, 127,133 Z Zack, W., 407, 448 Zarapico Romero, M., 264, 278 Zetterqvist, H., 9, 31 Zetzel, L,146, 147, 148, 167

AUTHOR INDEX

Zeuthen, E., 428, 433, 437, 447, 499, 504 Zillesen, F. O., 25, 29 Zingg, W., 9, 31 Zitin, B., 314, 344 Zittle, C. A., 314, 344 Zollinger, H. U., 5, 9, 18, 31 Zotos, B., 358, 361,382 Zucker, T. F., 148,174

Subject Index A Acetate, activation, cations and, 476 Accfobacter zylinum, microfibril synthesis in, 51 Aceto-orcein fast green, isolated nuclei and, 320 Acetylcholine, degeneration and, 79 quanta of, 90-92 synapse and, 88-89 synaptic vesicles and, 70 Acetyl coenzyme A deacylase, isolated nuclei and, 290, 337 Acetylphenylhydrazine, reticulocytes and, 137 Aconitase, isolated nuclei and, 291 Acridine-orange, reticulocytes and, 141 Action potential, sodium and, 463-467 Adenosine deaminase, isolated nuclei and, 291, 331 Adenosine phosphatase, isolated nuclei and, 292, 336 Adenosine triphosphatase, isolated nuclei and, 292-293, 336, 337 Adenosine triphosphate, enzyme activation and, 372-375 ion movements and, 470-471 magnesium and, 369, 372 Adhesiveness, reticulocytes and, 157 Adrenal, nervous stimulation of, 82 synaptic vesicles and, 76 Adrenaline, adrenal stimulation and, 82 chromatophores and, 203 Adrenocorticotropic hormone, chromatophores and, 203, 204 Albedo, background adaptation and, 180 Albumin, cell refractive index and, 417 chloride ions and, 398 nuclear membrane and, 329 pinocytosis and, 494 Alcohol, bone induction and, 267 Alcohol dehydrogenase, inhomogeneity of, 358 phenanthroline and, 359-361 zinc and, 355-361, 380

Aldehyde dehydrogenase, cations and, 476 Aldehyde-fuchsin, connective tissue and, 217 Aldolase, isolated nuclei and, 293, 320, 324,327, 329, 330, 332,336 Algae, cell walls of, 42-45 Alkali, elastic fiber production and, 242 Amine oxidase, isolated nuclei and, 295, 322, 324, 327, 337 Amino acid oxidases, isolated nuclei and, 295, 337 Amino acids, connective tissue and, 227-229 incorporation, nuclei and, 286, 326, 333 reticulocytes and, 150-151 sequence, collagen and, 243-245 p-Aminobenzoate acetylase, isolated nuclei and, 2%, 337 p-Aminohippuric acid, synthesis, isolated nuclei and, 296, 337 Amitosis, reticulocytes and, 162-163 Amoeba *roteus, pinocytos~s and, 481482, 489 Amphibians, chromatophorotropins of, 204-205 Amylase, isolated nuclei and, 2% Anemia, reticulocytes and, 136, 137, 145, 147, 163 Aniline blue, connective tissue and, 215 Anions, net transfer of, 412 Anolis porcatus, color changes in, 181 Anoxia, potassium uptake and, 467 Aorta, elastidcollagen ratio in, 231 Apyrase, cations and, 476 Arbacia, eggs, water content of, 423, 424, 426 Arginase, isolated nuclei and, 296-297, 320, 330, 336 Arginine phosphate, ion movement and, 470 Arteriosclerosis, elastase and, 232 Ascorbic acid, deficiency, wound healing and, 236 Astacurans, chromatophorotropins and, 196-201 Autolysis, osmotic pressure and, 408-409 Azide, potassium uptake and, 467

529

530

SUBJECT INDEX

B Basophilia, ergastoplasm and, 18-20 Beans, microfibril synthesis in, 51 Beryllium, phosphatase and, 369 Betaine aldehyde oxidase, isolated nuclei and, 297 Bicarbonate, erythrocytes and, 462 Bipolar cells, synapses of, 79-80 Bladder, mucosa, ossification and, 260-262 Blood, reticulocytes in, 160-161 Blood vessels, heterotopic ossification and, 268-269 Bone, devitalized, osteogenesis and, 269-276 extracts, heterotopic ossification and, 262-267 Bone marrow, reticulocytes in, 154-155 Boutons, motoneurons and, 63 Brachyurans, chromatophorotropins in, 185-193 Brain, cation movement and, 410-411 Brilliant cresyl blue, reticulocytes and, 137-138, 141 Brush-border epithelium, pinocytosis and, 486-487 C Cadmium, metalloproteins and, 365-367, 368 Calcium, adenosine triphosphate and, 374-375 elastic fibers and, 239 heterotopic ossification and, 256, 259, 267-268 Callinectes sapidus, albedo response in, 180 chromatophorotropins in, 187-188 color rhythms in, 190, 191 melanin in, 179 Cambarellits shufeldti, chromatophorotropins in, 194, 196, 19820 1 pigment dispersion in, 180 Cancer, mitochondria and, 12-15 Carasdus auratzis, melanophores of, 203 Carausius morosus, chromatophorotropins of, 201-202 Carbonic anhydrase, isolated nuclei and, 297 zinc and, 353-354

Carbon tetrachloride, metal distribution and, 378-380 Carboxypeptidase, zinc and, 354-355 Carrageenin, fibrogenesis and, 237 Cartilage, heterotopic ossification and, 258 hypertrophic, bone induction and, 272 Catalase, isolated nuclei and, 297-298, 337 Cathepsin, isolated nuclei and, 298-299, 322, 337 Cations, net transfer of, 410-412 Cells, binucleate, isolated nuclei and, 326 membranes, water permeability and, 440-442 osmotic permeability coefficients of, 432-436 osmotic pressure of, 408-410, 422-426 potassium content of, 449-450 refractive index of, 417-418 unbroken, isolated nuclei and, 325-326 volume measurement and, 413-418 water diffusion through, 438-442 Cellulose, cell walls and, 33-35 copper complexes and, 39 elastic fibers and, 239 electron microscopy of, 36-39 microfibril size and, 36 other polysaccharides and, 40-42 X-ray diagrams of, 36-39 Cell walls, alkali soluble material in, 42-45 a-cellulose in, 42-45 chlorite soluble material in, 42-45 composition of, 33-35 synthesis of, 53-58 water soluble material in, 42-45 Centrifuges, nuclear separation and, 285 Centriole, structure of, 26-28 Cerebellum, synaptic vesicles and, 70 Chaetodipterus faber, melanophore responses of, 203 Chaetomorphu, microfibril orientation in, 57-58 Chelating agents, metalloenzymes and, 354, 355 Chemical potential, osmotic pressure and, 389

SUBJECT INDEX

Chloride, erythrocytes and, 462 ionic equilibrium and, 451, 452-453, 474 reticulocytes and, 149 Chloridella empusa, chromatophorotropins and, 201 Choline acetylase, cations and, 476 isolated nuclei and, 299 Cholinesterases, isolated nuclei and, 299300, 337 Choline oxidase, isolated nuclei and, 300, 337 Chromaffin cells, stimulation of, 82 Chromatophores, functional significance of, 177-181 red, hormones controlling, 186 responses, classification of, 176-177 thermoregulation and, 178-179 Chromatophorotropins, amphibia and, 204-205 astachurans and, 196-201 brachyurans and, 185-193 chemical nature of, 205-206 chromatography of, 195-196, 199,202 fishes and, 202-204 inactivation of, 195 insects and, 201-202 isopods and, 183-185 natantians and, 193-196 rhythmicity and, 188-193 sources of, 181-183 stability of, 199-200 stomatopods and, 201 Chondroitin sulfates, connective tissue and, 229 Cilia, gullet and, 124 kinety system and, 102-103 paired, 111 Circumciliary space, 108 Citrate dehydrogenase, isolated nuclei and, 300 Clostridium, collagenase and, 245 Cloudy swelling, mitochondria and, 9 Cochlea, synaptic vesicles and, 76 Coenzymes, metalloenzymes and, 352 Collacin, senile elastosis and, 219 Collagen, aging and, 237-238

53 1

chemical composition of, 226-230 definition of, 214 distribution of, 230-231 elastic fiber production and, 239-246 electron microscopy and, 240-241 enzymatic susceptibility of, 232-234 heterogeneity of, 242-246 microscopic structure of, 222-223 Collagenase, collagen and, 245 elastase and, 220 elastic fibers and, 240-241 Colloid millium, elastic tissue and, 221 Conductivity, cell volume and, 416 Cones, synapses of, 79-80 Conifers, microfibril synthesis in, 51, 52 microfibrils of, 36-37 Conjugation, ciliary corpuscles and, 108110 Connective tissue, aging and, 237-239 components of, 213-215 fiber morphology, electron microscopy and, 222-223 light microscopy and, 222 models and, 225-226 physical properties and, 224-225 X-ray diffraction and, 223 fibrogenesis and, 234237 heterotopic ossification and, 261 histology, methods and, 215-217 staining theories and, 217-219 metals in, 376-377 polysaccharide and, 229-230 Constant field equation, 457 Copper, reticulocytes and, 150 Copper formate, microfibrils and, 39 Copper sulfate, bone induction and, 268269 Corpora cardiaca, chromatophorotropins and, 182 Cosmic rays, chromatophores and, 191 Crangon crangon, chromatophores of, 178 Crago, chromatophores of, 185 chromatophorotropins in, 193, 197

532

SUBJECT INDEX

Creatine phosphate, potassium distribution and, 469, 470 Cristae, structure of, 4 Cyanide, potassium uptake and, 467, 469 Cysteine desulfhydrase, isolated nuclei and, 300 Cytochrome c, isolated nuclei and, 300-301, 320, 337 uptake of, 492 Cytochrome oxidase, adenosine triphosphate and, 372 isolated nuclei and, 301, 321, 322-324, 325, 337 D Damage, heterotopic ossification and, 259 Degeneration, mitochondria and, 9-15 Deoxyribonuclease, isolated nuclei and, 302, 325, 329, 330, 333, 334,337 Deoxyribonucleic acid, amino acid incorporation and, 286 distribution in homogenates, 334-335 isolated nuclei and, 316-319 reticulocytes and, 151 Dermis, elastic fiber production and, 239240 Deuterium oxide, osmosis and, 436 Diameter, cell volume measurements and, 414 Diencephalon, reticulocytes and, 158 Diet, metal distribution and, 378 Dinitrophenol, ion movement and, 470, 474, 475 Diphosphopyridine nucleotide, phenanthroline and, 361 synthesis, isolated nuclei and, 288, 303, 329, 332, 336 zinc metalloenzymes and, 355-365 Diphosphopyridine nucleotide-cytochrome c reductase, isolated nuclei and, 303, 337 Diphosphopyridine nucleotide nucleosidase, isolated nuclei and, 303 Disease, elastic tissue and, 219-221 reticulocytes and, 163-165 Disematostoma, gullet replication in, 127 Donnan effect, erythrocytes and, 462-463 osmotic pressure and, 396-398 potassium and, 453-454

E Eggs, sea urchin, hematocrit and, 415 Ehler’s Danlos syndrome, elastic tissue and, 220-221 Elacin, senile elastosis and, 219 Elastase, arteriosclerosis and, 232 Ehler’s Danlos syndrome and, 220-221 elastic fibers and, 241 lipid and, 230 physiological significance of, 232-233 specificity of, 220, 233-234 polysaccharide and, 229 staining and, 216, 218 Elastics-staining, definition of, 213 Elastic fibers, aging and, 238-239 definition of, 213 distribution of, 230-231 models of, 225-226 production from collagen, chemical evidence for, 242 histochemical evidence for, 239-242 heterogeneity and, 242-246 tensile properties of, 224 X-ray diffraction and, 241-242 Elastic tissue, definition of, 213 disease and, 219-221 staining of, 215-219 Elastin, chemical composition of, 226-230 definition of, 213 enzymatic susceptibility of, 232-234 purification of, 227 X-ray diffraction of, 223-224 Elastomucin, definition of, 213-214 Electric organ, innervation of, 74 Electron microscopy, cellulose and, 36-39 elastic fibers and, 222-223, 240-241 pinocytosis and, 483-488 reliability of, 3-4 reticulocytes and, 141-142 Electrophoresis, reticulocytes and, 148 Embryo, fibrogenesis in, 234-236 Emerita talpoida, sinus gland of, 182 Emission spectrography, trace elements and. 347-350

SUBJECT INDEX

Endoplasmic reticulum, isolated nuclei and, 321-322 reticulocytes and, 143 Enolase, isolated nuclei and, 303,337 Entropy, protein solutions and, 399-403 Enzymes, nuclei and, 287-316,336-337 reticulocytes and, 152-154 Eosin, connective tissue and, 215 Epinephrine, reticulocytes and, 158 Epithelium, heterotopic ossification and,

260 Ergastoplasm, origin of, 18-21 structure o#, 15-18 Ericymba buccata, background response of, 181 protective coloration and, 178 Eriocheir, 183, 472 chromatophorotropins in, 187 Erythrocytes, alkaline earth chlorides and, 412 cation movement and, 410,411 halometry and, 415-416 hematocrit and, 414 internal structure of, 412-413 ionic equilibria in, 462-463,467-468,471,

533

Fiber, isolated nuclei and, 327 Fibrin, collagen and, 234,236 Fibroblasts, fibrogenesis and, 234-236 heterotopic ossification and, 255, 261 refractive index and, 426-427 volume-osmotic pressure and, 423,425 Fishes, chromatophorotropins in, 202-204 Fixation, reticulocytes and, 138 Fluorescein, labeled globulin and, 490-493 Fluoride, potassium uptake and, 467 Fluoroacetate, potassium uptake and, 467 Formic acid, heterotopic ossification and,

268 Formic dehydrogenase, inhibitors and,

364 Freezing point, osmotic pressure and, 408-

409 Fructokinase, cations and, 476 Fructose-6-phosphatase, isolated nuclei and, 304 Fucose, connective tissue and, 229 Fumarase, isolated nuclei and, 304, 322,

324, 337 Fundulus, 203 eggs, volume of, 426 melanophores of, 202, 203

477 isolated nuclei and, 324-325 nonpenetrating solutes and, 415 osmotic pressure and, 393, 404-405,409,

G Galactose, connective tissue and, 229 Galactosidase, cations and, 476 418-422 Gambusia, 203 separation from reticulocytes, 145-146 protective coloration and, 177 water content of, 417 Ganglia, synaptic vesicles and, 69,70,74 Esterase, isolated nuclei and, 303-304, Gasterosteus aculeatus, melanin disper327, 330, 337 sion in, 181 Estradiol-l7~-dehydrogenase, metals and, Gelatin, 364 collagen and, 227 Ethylenediaminetetraacetate, subcellular elastic fiber production and, 240 fractions and, 377-378 pinocytosis and, 489 Eustrongylus gigans, heterotopic ossifica- Gills, ion transport and, 472-473 tion and, 259 Globulin, pinocytosis and, 489,490 Evans blue, cell volume and, 415 Glucosamine, connective tissue and, 229 Eye, heterotopic ossification and, 268 Glucose, Eyestalks, chromatophorotropins and, connective tissue and, 229 182 pinocytosis and, 494,495,498-500 F potassium uptake and, 467 Fascia, heterotopic ossification and, 258- Glucose phosphatases, isolated nuclei and,

259, 260

304-305,322, 337

534

SUBJECT INDEX

Glucose-6-phosphate dehydrogenase, isolated nuclei and, 331 metals and, 363, 364 B-Glucuronidase, isolated nuclei and, 305, 337 ' Glucuronide, synthesis, isolated nuclei and, 305, 337 Glutamate, pinocytosis and, 489 Glutamic dehydrogenase, isolated nuclei and, 305, 322, 337 zinc and, 361-362 Glutathione, hydrolysis, isolated nuclei and, 306 Glutathione reductase, isolated nuclei and,

306 Glutaminase, isolated nuclei and, 306, 337 Glyceraldehyde dehydrogenase, isolated nuclei and, 306 metals and, 363-364 Glycerol, alcohol dehydrogenase and, 356 Glycerophosphate, nuclear damage and, 333 Glycerophosphate dehydrogenase, isolated nuclei and, 306 metals and, 363-364 Glycine, connective tissue and, 228, 242 Glycogen, isolated nuclei and, 326 Glycolysis, isolated nuclei and, 288, 306, 325 reticulocytes and, 153 Golgi apparatus, ergastoplasm and, 21-25 Gradients, nuclear separation and, 284285 Grafts, bone, heterotopic ossification and, 269 Ground substance, definition of, 214 fibrogenesis and, 235 Gullet, fine structure of, 122-125 gross structure of, 114-122 replication mechanisms and, 125-128 Gum arabic, nuclear membrane and, 329

H Halometry, cell volume and, 415-416 Heinz bodies, reticulocytes and, 137, 165 Hematocrit, cell volume measurement and, 414-415

Hemigrapsus oregonensis, chromatophorotropins in, 187 Hemoglobin, erythrocytes and, 415 hydration of, 399 isolated nuclei and, 320, 324-325, 329, 330 reticulocytes and, 149-151 Hemolysis, reticulocytes and, 138, 139, 148 water permeability and, 432 Heparin sulfate, connective tissue and, 229 Hexose diphosphatase, isolated nuclei and, 306 Histaminase, isolated nuclei and, 306 Histone, nuclear membrane and, 329 Homogenizers, nuclei and, 280-284 Humoral factors, reticulocytes and, 159 Hyaloplasm, structure and, 28 Hyaluronic acid, connective tissue and, 229 Hyaluronidase, elastic fibers and, 241 Hydroxyproline, connective tissue and, 228, 231, 242, 243, 244 wound healing and, 237 Hyla arborea, chromatophorotropins in, 204 Hyla versicolor, chromatophores in, 179 Hyperpolarization, retinal synapses and, 80 synaptic inhibition and, 86-87 Hypoxia, reticulocytes and, 160

I Illumination, chromatophores and, 181 Inhibitors, metalloenzymes and, 352, 355,358-359, 362, 363, 364 Insects, chromatophorotropins in, 182, 201-202 Insulin, pinocytosis and, 502 Intermedin, chemical nature of, 205 chromatophores and, 204 Iodoacetate, potassium uptake and, 468-469 Iron, nuclei and. 325

178354,

181-

467,

535

SUBJECT INDEX

reticulocytes and, 150 subcellular fractions and, 377 Isocitric dehydrogenase, inhibitors and, 364 isolated nuclei and, 300, 337 Isopods, chromatophorotropins in, 183185

J Janus Green B, reticulocytes and, 142

K Keratosulfate, connective tissue and, 229 a-Ketoglutarate, cation transfer and, 410 Ketoglutarate oxidase, isolated nuclei and, 306, 337 Kidney, cation movement and, 410, 411 heterotopic ossification and, 254-258 ion transport and, 472 Kinetodesma, gullet and, 125 interciliary fibrils and, 104 live cells and, 101-102 location of, 111 silver impregnation and, 100-101 spiralization of, 108 Kinetosomes, centriole and, 26-28 kinetodesma and, 102, 103 ribbed wall and, 125 Kinety, stomatogenic, 127 Kosciuscola tristis, chromatophores of, 178

L Lactic dehydrogenase, isolated nuclei and, 307, 330, 337 zinc and, 362-363 Lathyrism, elastic tissue and, 221 L e a d e r , 183, 184, 206 chromatophorins and, 193, 194-196 Leucine amidase, isolated nuclei and. 307. 337 Leucophaea maderac, chromatophorotropins in, 182, 202 Leukocytes, cation movement and, 410, 411 volume-osmotic pressure and, 423, 425 Ligia oceanica, chromatophorotropins in, 183-184 melanin dispersion in, 181

Lignin, cell walls and, 34 Lipase, isolated nuclei and, 307 Lipid, connective tissue and, 230 fibrogenesis and, 236 heterotopic ossification and, 263, 264 reticulocytes and, 149 Lipoprotein, specific refraction increment and, 417 Liver, bile duct nuclei and, 327 osmotic pressure and, 408-409 Lophius piscatorius, elastase in, 232 Lysmata seticaudata, chromatophorotropins in, 195 Lysosomes, mitochondria and, 7 nuclear damage and, 333 Lysozyme, pinocytosis and, 492-493

M Macrocytosis, reticulocytes and, 144 Macrophages, pinocytosis in, 481, 502 Macropodus opercdaris, chromatophores of, 203 Magnesium, deficiency, mitochondria and, 378 enzyme activation and, 369-370, 373375 reticulocytes and, 149 Malaria, reticulocytes and, 164-165 Malic dehydrogenase, isolated nuclei and, 307 metals and, 363-364 Malonate, potassium uptake and, 467 Manganese, enzyme activation and, 369,373-375 subcellular fractions and, 377 Mannose, cell walls and, 43, 48 Mating, color changes and, 181 Maturation, reticulocytes and, 155-157, 161-162 Medulla terminalis, chromatophorotropins and, 182-183 Melanin, concentrating hormone and, 186 thermoregulation and, 179 Melanophores, aneuronic, 202 Membrane potential, calculation of, 455-457 short-circuiting errors and, 454-455

5 36

SUBJECT INDEX

Membranes, ergastoplasm and, 18 ion permeability and, 451-452 pinocytosis and, 501-502 synaptic transmission and, 92-93 Metacollagen, elastica and, 241 Metalloenzymes, definitions of, 350-352 Metals, distribution, carbon tetrachloride and, 378-380 diet and, 378 enzyme complexes and, 367-375 mitochondria and, 373 subcellular fractions and, 375-380 Methionine, pinocytosis and, 500 Methyl green-pyronine, isolated nuclei and, 320 Microdensitometer, emission spectrography and, 348 Microfibrils, biosynthesis of, 51-53 composition of, 40-50 crystallinity of, 38-39 orientation of, 53-58 Microsomes, ergastoplasm and, 18 gullet replication and, 127, 130 isolated nuclei and, 321-322 metals in, 376 mitochondria1 regeneration and, 6-7 Microvesicles, regenerating nerve and, 76 Mitochondria, carbon tetrachloride and, 378-379 isolated nuclei and, 322-324 metals and, 373, 376-377 pathological aspects of, 8-15 pinocytosis granules and, 4%-498 regeneration of, 5-8 reticulocytes and, 142-143 sperm structure and, 131 structure of, 4-5 synapses and, 64-65, 69,72,74 , Mitosis, mitochondria and, 5 Motility, reticulocytes and, 143 Motoneurons, synapses and, 63, 68 Mucopolysaccharides, collagen and, 224225 Muscle, cation movement and, 410-411 heterotopic ossification and, 267-268

ion permeability of, 451, 468-469 membrane potential of, 457-458 potassium content of, 460-461 potassium injection and, 460 Mustelus canis, melanophores of, 202 Myokinase, isolated nuclei and, 307 Myristic acid, connective tissue and, 230

N Natantians, chromatophorotropins in, 193-196 Nerve, ion permeability of, 451 potassium injection and, 460 potassium permeability and, 466-467 regenerating, microvesicles in, 76 sodium permeability and, 466-467 Nerve endings, ultrastructure of, 74-76 Nervous system, chromatophorotropins and, 183 reticulocytes and, 157-159 Neuromuscular junction, degeneration and, 78-79 synapse and, 68 ultrastructure of, 72-74 Neuroprotofibrils, electric organ and, 74 invertebrate synapses and, 72 Neutral red, reticulocytes and, 143 Nonsolvent volume, osmotic pressure and, 392 Nu c1eit cell debris and, 326-327 damage to, 331-335 electron microscopy of, 328 endoplasmic reticulum and, 321-322 enzyme adsorption and, 316-321 enzymes, classification of, 336-337 fragmentation of, 334-335 gel formation and, 333-334 homogenate and, 333-334 isolation, contamination and, 316-327 methods of, 280-285 protein loss and, 327-331 isotope incorporation in, 285-316 membranes, permeability and, 328-329 metals in, 376 mitochondria and, 322-324 nonaqueous media and, 331

SUBJECT INDEX

osmotic behavior of, 426-427 ribonucleic acid and, 20-21 unbroken cells and, 325-326 washing of, 329-330 Nucleolus, ribonucleic acid and, 21 Nucleoside phosphorylase, isolated nuclei a d , 307-308, 331

I

0 Octanoate oxidase, isolated nuclei and, 308, 337 Octopus vulgaris, color mimicry and, 179 Onchocerca volvulus, elastic tissue and, 221 Opacity, cell volume and, 416-417 Orcein, elastic fibers and, 215, 217, 218 Orconectes, 183 chromatophorotropins in, 196-198, 200 protective coloration and, 178 white pigment of, 180 Osmole, definition of, 395 Osmotic coefficient, 432-436 protein and, 393, 398, 403 Osmotic pressure, cell volume and, 404-427 Donnan effect and, 396-398 erythrocytes and, 418-422 kinetics and, 427-443 metabolic activity and, 407-408 proteins and, 395-404 theory of, 388-395 units of, 393-395 Osmotic resistance, reticulocytes and, 146148 Osmoregulation, Golgi apparatus and, 2425 Osteogenin, bone formation and, 264, 271 Oxalacetate oxidase, isolated nuclei and, 308 Oxalic acid, elastin and, 227-228 Oxygen tension, reticulocytes and, 160

P Palaemonrtes, chromatophores of, 178-179, 180, 185 chromatophorotropins in, 182, 183, 193, 194 Pandahs borealis, chromatophorotropin of, 205-206

537

Paramecium, circumciliary space and, 108 gullet replication in, 125-128 fibrillar systems, electron microscopy of, 99, 02-1 4, 106-111 nomenclature and, 111, 118-119 phase contrast and, 104 silver impregnation and, 99, 100-101 food-intake system of, 114-128 kinetodesmal fibrils of, 99 mitochondria of, 5 pellicular ridges in, 103-104 peribasal space and, 108 Parasilurus asotus, melanophores of, 203204 Parasomal sac, cilium and, 111 gullet and, 124, 125 Parathyroid, heterotopic ossification and, 262 Paratya compressa? chromatophorotropins in, 193 Parasympathetic nerves, reticulocytes and, 158-159 Pars intercerebralis, chromatophorotropins and, 182 Pathology, mitochondria and, 8-15 Pectin, cell walls and, 35 Patapus brasiliensis, 183 chromatophorotropins in, 193 Peniculi, gullet and, 122-125 Peptidase, isolated nuclei and, 308-309, 337 Petasites vulgaris, cell walls of, 35 Peribasal membranes, 108 fibrils and, 114 Periodic acid-Schiff reaction, elastic tissue and, 216 Phase contrast, reticulocytes and, 138, 141, 142 1,lO-Phenanthroline, alcohol dehydrogenase and, 359-361 Phenylhydrazine, reticulocytes and, 137 Phosphatase, acid, isolated nuclei and, 290, 320, 322, 337 magnesium and, 369

538

SUBJECT INDEX

alkaline, isolated nuclei and, 294-295, 320, 327, 330, 336 magnesium and, 369 zinc and, 364 heterotopic ossification and, 268 Phosphate, incorporation of, 287 potassium retention and, 454 Phospho-fructokinase, cations and, 476 Phosphorus, reticulocytes and, 149, 151 Phosphorylase, isolated nuclei and, 309, 337 Phosphorylation, magnesium and, 370 oxidative, nuclei and, 287-288 Phospho-transacetylase, cations and, 476 Phosphotransferase, isolated nuclei and, 309, 337 Phoxinus laevis, melanophore responses of, 203 Phthalate, collagen and, 245-246 Phycontyccs, microfibril synthesis in, 52 Pinocytosis, definition of, 481 induction of, 488-490 measurement of, 490-492 mitochondria and, 496-498 morphological aspects of, 482-488 surface adsorption and, 492-494 vacuole dehydration and, 494-498 vacuole permeability and, 498-502 Pituitary gland, chromatophores and, 202203, 204 Plasmalemma, pinocytosis and, 493 Polynucleotide phosphorylase, isolated nuclei and, 289 Polypedatus reinwarti, melanophore responses in, 204 Polysaccharide, connective tissue and, 229-230 elastic tissue and, 213-214, 216, 225-226 fibrogenesis and, 236 Pores, cell membranes and, 438 Porphyrin, reticulocytes and, 150 Potassium, depletion, membrane potential and, 459 Donnan effect and, 453-454 enzyme activation and, 475-477 erythrocytes and, 462-463

net transfer of, 410-412 passive flux of, 474-475 pinocytosis and, 489 pumping of, 450-451 reticulocytes and, 149 sodium efflux and, 471-474 Pyruvic phosphoferase, cations and, 476 Proline, connective tissue and, 228, 242 Proline oxidase, isolated nuclei and, 309 Protamine, nuclear membrane and, 329 Protein, bone induction and, 275 elastic fibers and, 225-226 entropy of mixing and, 399-403 hydration of, 399 ion binding and, 398 isolated nuclei and, 320, 329 osmotic coefficient and, 393, 398, 403 osmotic properties of, 395-404 pinocytosis and, 489, 500-501 trace elements and, 350 Pseudopodia, pinocytosis and, 482 Pseudoxanthoma elasticum, collagen and, 22 1 Pyrophosphorylase, isolated nuclei and, 288, 309 Pyruvate oxidase, isolated nuclei and, 309, 337

Q Quadrulus, gullet and, 122-125 Quinine, heterotopic ossification and, 268

R Raja erinacea, melanophores of, 202 Rana temporaria, melanophore responses in, 204 Refractometry, cell volume and, 417-418 Regeneration, mitochondria and, 5-8 Renal vessels, ligation, ossification and, 254-258 Resorcinol-fuchsin, connective tissue and, 217, 218 Respiration, reticulocytes and, 152, 153 Resting potential, ion distribution and, 452-462 Reticulin, lipid and, 230 Reticulocytes, adhesiveness of, 148 bone marrow and, 154-155 cell division and, 162-163

SUBJECT INDEX

charge of, 148 counting of, 139-140 definition of, 136 density of, 145-146 electron microscopy of, 138-139 enzymes and, 152-154 Heinz body anemia and, 165 hemoglobin and, 149-151 hemolytic agents and, 148 inner structure of, 141-143 ions and, 149 malaria and, 164-165 maturation of, 161-162 method of obtaining, 136-137 nucleic acids and, 151-152 osmotic resistance of, 146-148 peripheral blood and, 160-161 refractive index of, 146 release, age and, 155-157 factors affecting, 157-160 shape of, 143-144 sickle cell anemia and, 164 size of, 144-145 staining of, 137-138 stroma of, 149 unstained, 138 vacuoles in, 143 water and, 149 Retina, melanin dispersion and, 181 synaptic vesicles and, 70 Rhodanese, isolated nuclei and, 309, 337 Ribbed wall, gullet and, 122 Ribonuclease, isolated nuclei and, 310, 329 reticulocytes and, 141 uptake of, 492, 502 Ribonucleic acid, ergastoplasm and, 17-21 isolated nuclei and, 286, 316 reticulocytes and, 151-152 Rods, synapses of, 79-80 Romanowsky dye, reticulocytes and, 137, 141 Rumen, ion transport and, 472 S Salmo, eggs, volume of, 426 melanophore responses of, 203

539

Sarcolemma, synapse and, 74 Scyllium canicula, background response in, 181 Secretory granules, Golgi apparatus and, 24 mitochondria and, 5 Selenium, pathology and, 365 Senile elastosis, histology of, 219 Sepia officinalis,color mimicry in, 179 Sesarma, 183, 185 chromatophorotropins in, 188 Sickle cell anemia, reticulocytes and, 164 Silver nitrate, bone induction and, 268269 Sinus gland, chromatophorotropins and, 182-183 Skin, ion transport and, 472, 473-474 Sodium, action potential and, 463-467 enzyme inhibition and, 475-477 erythrocytes and, 462-463 membrane permeability and, 462 passive flux of, 474-475 pinocytosis and, 489 potassium influx and, 471-474 reticulocytes and, 149 Solutes, nonpenetrating, cell volume and, 415 penetrating, erythrocytes and, 432 Spermatozoon, ultrastructure of, 131 Spherocytosis, reticulocytes and, 163 Squilla mantis, chromatophorotropins in, 201 Staining, elastic tissue and, 217-219 Standard error, reticulocyte counts and, 139-140 Stentor, gullet replication in, 127 Stomatopods, chromatophorotropins in, 20 1 Storage, mitochondria and, 9-12 Stroma, reticulocytes and, 149 Strophanthin, ion transport and, 477 Succinic dehydrogenase-cytochrome c reductase, activation of, 372 isolated nuclei and, 310 Succinic dehydrogenase, isolated nuclei and, 310, 337 Succinoxidase, adenosine triphosphate and, 372 isolated nuclei and, 310-311, 322, 324, 325, 337

540

SUBJECT INDEX

Sucrose, nuclear membrane and, 329 Sulfatase, isolated nuclei and, 311, 324, 325, 337 Sulfhydryl groups, alcohol dehydrogenase and, 358, 361 Supernatant, metals in, 376-377 Supraesophageal ganglia, chromatophorotropins and, 182, 183 Sympathetic nerves, reticulocytes and, 158 Synapse, definition of, 61-62 degenerative changes in, 78-79 electric organ and, 68 electron microscopy of, 65-66 hyperpolarization and, 80 imertebrate, 70-72 membranes and, 68-69, 92-93 mitochondria and, 69, 74 morphology, 63-93 electron microscopy and, 66 function and, 76-93 submicroscopic, 70-76 ultrastructure and, 66-70 nerve stimulation and, 80-85 physiology of, 85-89 retinal rods and, 68 stellate ganglion and, 68 transmitter substance and, 87-89 Synaptic vesicles, asymmetry and, 70 cochlea and, 76 degeneration and, 79 electric organ and, 74 functional role of, 90-93 nerve stimulation and, 82-85 retinal synapses and, 79-80 Synaptolemma, 64

T Taste buds, synaptic vesicles and, 76 Temperature, chromatophores and, 178179 Thermodynamics, osmotic pressure and, 389-392 Thermoregulation, chromatophores and, 178-179 Thyroxine, melanophores and, 205 mitochondria and, 9

Tides, color rhythms and, 190-191, 192193 Trace elements, emission spectrography and, 347-350 Transplants, heterotopic ossification and, 258-259, 260-262 Transport systems, ions and, 475-477 Trauma, heterotopic ossification and, 267, 269 Trichocyst, kinety system and, 103 location of, 112 structure of, 112-113 Triacetic lactonase, isolated nuc!ei and, 312 Triphosphopyridine nucleotide-cytochrome c reductase, isolated nuclei and, 311, 337 Trypsin, isolated nuclei and, 312

U Uca, chromatophores of, 178, 180, 185 chromatophorotropins in, 185-187, 206 color rhythms, diurnal, 188-191 lunar, 191 tidal, 190-191, 192-193 Ultrachondrioma, mitochondria and, 8 Ultrasound, elastic fibers and, 241 Ulva lachcca, ion transport and, 473 Uracil decarboxylase, isolated nuclei and, 312, 337 Urea, elastin and, 227 Ureidosuccinate, synthesis, isolated nuclei and, 312 Ureter, heterotopic ossification and, 254255, 258 Uricase, isolated nuclei and, 312-313, 322, 325, 337 Urinary tract, heterotopic ossification and, 254-262

V Vacuoles, contractile, 24 reticulocytes and, 143 Valine, elastic fibers and, 242-243 Vnlonia, cell walls of, 35, 36-37 microfibril orientation in, 55, 58 microfibril synthesis in, 51-53

541

SUBJECT INDEX

Viruses, Golgi apparatus and, 25 Vitamin A, alcohol dehydrogenase and, 356, 381 Vitamin A esterase, isolated nuclei and, 313, 337 Voltage clamp, ion permeability and, 467, 468 Volume, cellular, measurement of, 413-418 osmotic pressure and, 404-427

W Water, cell permeability and, 427-428 cell volume and, 417-418 diffusion in cells, 438-442 reticulocytes and, 145, 149 Water permeability, diffusion and, 436-438 measurement of, 431-432 Wounds, fibrinogenesis and, 236-237

X Xanthine dehydrogenase, isolated nuclei and, 313, 337 Xanthine oxidase, isolated nuclei and, 313, 337 Xenopus laevis, chromatophorotropins in, 204-205 X-ray diffraction, connective tissue and, 223-224 elastic fibers and, 241-242 X-rays, cellulose and, 36-39 heterotopic ossification and, 269 Xylan, cellulose and, 40-42, 48, 50 Xylose, microfibril formation and, 58 Z Zinc, alcohol dehydrogenase and, 355-361, 380 coenzymes and, 364-365 extrinsic, 356-357 metalloenzymes and, 353-365 pathology and, 365

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