INTERNATIONAL REVIEW OF CYTOLOGY V1, Volume 1 (v. 1)

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUMEI

This Page Intentionally Left Blank

INTERNATIONAL

Review of Cytology EDITED BY G. H. BOURNE

J. F. DANIELLI

London Hospital Medical College London, England

Zoology Department King's College London, England

VOLUME I

Prepared Under the Auspices of The International Society for Cell Biology

ACADEMIC PRESS INC. PUBLISHERS NEW YORK

1952

Copyright 1952, by ACADEMIC PRESS INC. 125 East 23rd Street, New York 10, N. Y All Rights Reserved NO PART OF THIS BOOE MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOW WRITTEN PERMISSION FROM THE PUBLISHERS

Library of Congress Catalog Card Number : (52-5203)

PRINTED I N T H E UNITED STATES OF AMERICA

Contributors to Volume I L. G. E. BELL,King's College, University of London, England. K . A. BISSET,University of Birmningham, Birininghm, England. L. H . BRETSCHNEIDER, University of Utrecht, Holland. R. BROWN,University of Leeds, Leeds, England. G. FANKHAUSER, Princeton University, Princeton, New Jersey. R. J . GOLDACRE, Chester Beatty Research Institute, London, Elzgland.

G. GOMORI, University of Chicago, Clzicago, Illinois. A. D. HERSHEY, Carnegie Institution of Washington, Cold Spring Harbor, New York.

ARTHURHUGHES,Strangeways Research Laboratory, Cawbridge, England.

C. LEONARD HUSKINS,University of Wisconsin, Madison, Wisconsin. GEORGE W . KIDDER, Amherst ColEege, Amherst, Massachusetts. WILLIAMMONTAGNA, Brozrm University, Providence, Rhode Island. TH. ROSENBERG, Steno Mentorial Hospital and Nordisk Insulinlaboratorium, Gentofte, Denmark.

LORDROTHSCHILD, Cambridge University, Cambridge, England. MARCUS SINGER, Harvard Medical Sclzool, Boston, Massachusetts. M. M. SWANN,Cambridge University, Cawbridge, England. W . WILBRANDT, University of Berne, Berne, Switzerland.

This Page Intentionally Left Blank

Foreword Much has happened in the world of Cytology since Schleiden ant1 Schwann enunciated their “cell theory” in 1838 and 1839. Beginning with the study of cell structure cytology rapidly became functional and chemical with the development of organic and biochemistry, Then with the rise of the dye industry and with the application of dyestuffs to the cell, delineation of structure became fatally facile and cytological chemistry sank more or less into obscurity. Gradually during the present century, however, the new developments in chemistry and the development of new physical and chemical microtechniques have led to an outburst of activity in the fields of cytochemistry and fine structure and their relation to cell physiology-all of which might well be incorporated in the inclusive term of “Cell Biology.” In recent years there has been a remarkable increase in the volume of published research in this field, and like so many others, it has now become too large to be adequately covered by any individual without the assistance of regular review service. Some years ago we decided to provide such a periodical review and were gratified to find that the International Society for Cell Biology welcomed this proposal and agreed that the review should be issued under its auspices. It is aimed to produce this review annually and to make it truly international. Among the authors of the present volume are seven British, five American, one Dutch, one Danish and one Swiss and in later volumes we hope to obtain a greater proportion of continental authors. It is proposed to keep the scope of the “International Review of Cytology” as wide as possible-to deal with all aspects of Cell Biology, including morphological and chemical studies of both cells and tissues. Papers presenting new theories of general interest will be welcomed. It should be pointed out that the articles contained in the present and subsequent volumes are not intended to cover completely any particular aspect of Cell Biology. The various chapters are individual and unrelated reviews of specific subjects by experts in those fields who have contributed at the invitation of the Editors. Succeeding volumes will follow the same pattern, and over a period of years the whole field of cytology and cell physiology will be covered. The policy of the Editors has been to obtain reviews which are critical discussions of data already published elsewhere or of new theoretical contributions. A certain amount of new work has been and will be admitted in some articles where it is thought that it makes for completeness or otherwise embeIlishes the review. GEOFFREY H. BOURNE Januwy 1952 JAMES F. DANIELLI

This Page Intentionally Left Blank

CONTENTS Some Historical Features in Cell Biology BY ARTHURHUGHES. Strangeways Research Laboratory. Cambridge. England I. Introduction ........................................................ I1. Microscopy ........................................................ I11. Nucleic Acids ..................................................... IV. Study of Living Cells .............................................. V . References .........................................................

1 1 2 5 6

Nuclear Reproduction BY C. LFQNARD HUSKINS, University of Wisconsin. Madison. Wisconsin I . Introduction ........................................................ I1. Nuclear or Cell Division ........................................... I11. Chromosome Reproduction .......................................... IV. Variation in Nuclear Size .......................................... V. Reduction in Chromosome Number .................................. VI . Conclusions ........................................................ VII . References . !.......................................................

9 10 12 15 18 21 34

Enzymic Capacities and Their Relation to Cell Nutrition in Animals

I. I1. I11. IV . V.

BY GWRGEW . KIDDER,Amherst College, Amherst. Massachusetts Introduction ........................................................ Nutritional Requirements of Tetrahymena ........................... Nutritional Requirements of Higher Animal Cells .................... Conclusion ......................................................... References .........................................................

27 28 32 32 33

T h e Application of Freezing and Drying Techniques in Cytology

I. I1. I11. I v. V.

BY L. G . E. BELL.King’s College. University of London. England Introduction ........................................................ Method ............................................................ Comparison with Histological Fixation .............................. Advantages of Freezing and Drying Techniques ...................... References .........................................................

35 36 53 57 62

Enzymatic Processes in Cell Membrane Penetration

Steno Memorial Hospital and Nordisk Insliliitlaboratorium. BY TH. ROSENBERG. Gentofte. Denmark A N D W . WILBRANDT. University of Berne. Berne. Switzerland I . Introduction ........................................................ I1. Some General Considerations ....................................... I11. Enzymes with Non-Penetrating Substrates ........................... I V. The Enzymatically Controlled Transport ............................. V . Transport of Glucose ............................................... VI . Conclusion ......................................................... V I I . References .........................................................

65 66 68 70 70 85 89

Bacterial Cytology BY K . A . BISSET.University of Birmitiglsam. Binrririghan.. Etiglawi

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

.

.

Introduction ........................................................ The Bacterial Nucleus .............................................. Growth and Cell Division in Bacteria ................................ Granular Inclusions ................................................ Bacterial Flagella .................................................. Specialized and Reproductive Methods .............................. Cytology and Systematics .......................................... References .........................................................

9.3 96 98 100 102 102 103 104

Protoplast Surface Enzymes and Absorption of Sugar BY R . BROWN.Uiiiversity of Leeds. Leeds. Eiiglaiid

I . Introduction

. I1. I11.

I V.

V.

VI

.

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

Nature of Experimental Material ................................... Characteristics of Absorption Process ............................... Enzyme Systems in the External Surface ............................ Discussion ......................................................... References .........................................................

107 107 110 114 117 118

Reproduction of Bacteriophage BY A . D . HERSHEY. Carizegie Iiistitutiou of Washington. cold Spririg Harbor N e w York

I . Introduction

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

.

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

Ideas about Origin ................................................. Ideas about Growth ................................................ Program and Objectives ........................................... Facts about Growth ................................................ Conclusions ........................................................ References .........................................................

119 119 120 123 126 133 133

The Folding and Unfolding of Protein Molecules as a Basis of Osmotic W o r k BY R . J . GOLDACRE. Chester Beatty Research Institute. London. England I . Introduction ......................................................... I1. The Folding and Unfolding of Protein Molecules in Living Cells ...... I11. Osmotic Work-The Accumulation of Material against a Concentration Gradient in Amoeba .............................................. I V. Osmotic Work in Other Cells ...................................... V The Inversion Tube Analogy ....................................... V I . Osmotic Work in Metazoijn Cells ................................... VII . Fungi ............................................................. VIII . General Discussion on Osmotic Work ............................... IX. Concluding Remarks ............................................... X . References .........................................................

.

135 136 141 . 147 150 151 153 153 161 163

Nucleo-Cytoplasmic Relations in Amphibian Development BY G . FANKHAUSER, Princeton University. Princeton. New Jersey I . Introduction ......................................................... I1. Quantitative Changes in Cytoplasm of Egg ...........................

165 166 168 177 179 181

I11. Quantitative Changes in the Nucleus : Polyploidy and Haploidy ....... I V . Unbalanced Chromosome Combination (Aneuploidy) ................. V. Invisible Chromosome Changes (Gene Mutations?) .................. V I . Development without Chromosomes ................................. V I I Nucleo-Cytoplasmic Relations in the Early Development of Species 183 Hybrids ......................................................... 188 VIII . Summary and Conclusions .......................................... 192 I X . References .........................................................

.

Structural Agents in Mitosis BY M. M. SWANN, Canabridge University. Cambridge. England

I. I1 I11. IV. V

.

VI

. .

Introduction ........................................................ Birefringence Changes in the Sea Urchin Egg during Mitosis ......... Further Evidence on the Release of Chemical Agents in Mitosis ...... The Nature of Chemical Agents in Mitosis .......................... Conclusion ......................................................... References .........................................................

195 197 203 208 209 210

Factors Which Control the Staining of Tissue Sections with Acid and Basic Dyes BY MARCUSSINGER. Harvard Medical School. Boston. Massachusetts I . Introduction ........................................................ 211 I1. The Influence of pH of the Staining Solution on the Interaction of Dye and Protein

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

I11. The Nature of the Influence of pH on Staining

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

I V. The Site of Dye-Binding and the Nature of the Bond between Dye and Protein .......................................................... V The Relation between the Isoelectric Point and Staining .............. V I The Ionic Strength of the Dye Solution ............................. VII . The Influence of Dye Concentration ................................. VIII . The Affinity of Dyes ............................................... I X . The Influence of Fixation and Other Modifications of Tissues on Subsequent Staining ................................................. X . The Influence of Temperature of the Staining Solution ............... X I . Some Observations on the Kinetics of Staining ...................... XI1. The Reversibility of Staining Reactions ; Equilibrium of Staining. and Other Factors Which Influence Staining ........................... XI11. References .........................................................

. .

215 221 224 230 233 236 237 212 215 246 248 250

The Behavior of Spermatozoa in the Neighborhood of Eggs BY LORDROTHSCHILD. Cambridge Uwiversity. Cambridge. England

I. I1 I11. I V. V.

.

Introduction ........................................................ The Block to Polyspermy .......................................... Chemotaxis of Spermatozoa ........................................ Conclusion ......................................................... References .........................................................

257 258 260 263 263

The Cytology of Mammalian Epidermis and Sebaceous Glands BY WILLIAMMONTAGNA. Brown UvGiverSity. Proz&fe.iEe. Rhode Island

. .

I I1 I11. IV.

Introduction ........................................................ The Epidermis .................................................... The Sebaceous Glands ............................................. References .........................................................

263 266 290 299

The Electron-Microscopic Investigation of Tissue Sections

BY L. H. BRETSCHNEIDER. University of Utrecht. Holland I. I1. I11. IV. V. VI

.

Objectives ......................................................... The History of Ultramicrotomy .................................... The Influence of Fixation upon the Electron-Optical Image ........... Primary Nuclear and Plasmatic Ultrastructures ..................... Secondary Ultrastructure of Cells .................................. References .........................................................

305 308 313 311 316 321

The Histochemistry of Eeterases BY G. GOMORI.Uiiiversity of Chicago. Chicago. lllinois

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

335

AUTHOR INDEX............................................................ SUBJECT INDEX............................................................

337 349

References

Some Historical Features in Cell Biology ARTHUR HUGHES Strangeways Research Laboratories, Cambridge, England.

CONTENTS

I. 11. 111. IV. V.

Introduction ........................................................... Microscopy ............................................................ Nucleic Acids .......................................................... Study of Living Cells .................................................. References .............................................................

Page 1 1 2 5 6

I. INTRODUCTION One of the questions which confronts the historian of science is the problem of the relations between the progress of discovery and advances in technique. How fully at each stage are the available methods and apparatus utilized? In the history of microscopical observation, it is clear that the imperfections of contemporary instruments have not exclusively determined the pace of research. The era of discovery which began in the late seventeenth century associated with the great names of Hooke, Leeuwenhoek, Grew, and Malpighi was not maintained, and although the succeeding century was not barren of microscopical researches, these tended to become more scattered as the century advanced. It is certainly true that the scope of the eighteenth century microscope with uncorrected lenses was limited. However, Sachs (1890) says, with respect to botanical microscopy : There was in fact no original phytotomic research in the first fifty or sixty years This state of decline must not be ascribed to imperfect of the last century; no one saw and described clearly even what can be seen microscopes only; with the naked eye o r with very small magnifying power; I t is not easy to discover the causes of this decline in phytotomy in the first half of the 18th century, but one of the most important appears to lie in the circumstance that botanists did not make the knowledge of structure the sole aim in their anatomical investigations, but sought it chiefly for the purpose of explaining physiological processes. D 2461

...

...

...

...

11. MICROSCOPY

The fundamental improvement in the microscope which resulted from the development of the achromatic objective was made between 1815 and 1830 (Mayall, 1886; Carpenter, 1857; Chevalier, 1839), but this great technical advance did not take effect in the several branches of microscopy at the same time. The study of plant histology was resumed very 1

2

ARTHUR HUGHES

early in the nineteenth century (Sachs, 1890, p. 256), well before improved microscopes were available and at a time when the other branches of microscopy were still dormant, for Dobell (1932) says that “from the standpoint of protozoology and bacteriology the first quarter of the last century is a blank.” (p. 381) Carpenter (1857, p. 7) tells us the same with regard to animal histology. The elements of microscopic structure can be more readily apprehended by simple methods in plants than in animals, and thus some scope in the botanical field still remained in the early nineteenth century for the uncorrected objective. Fresnel ( 1824) found that a contemporary achromatic microscope had no advantage at magnifications greater than 200 X. Indeed, we are told that Meyen, as late as 1836, still preferred an English eighteenth century instrument (Sachs, 1890, p. 258) though this may to some extent illustrate the notorious conservatism of microscopists. It is difficult at this period to trace in detail the parallel course of optical development and the resulting enhanced powers of observation. One cannot tell, for instance, from Amici’s paper of 1824 what microscope he used in following the growth of the pollen tube, here described for the first time, though it was probably his reflecting instrument. This he had developed subsequent to some early efforts in constructing an achromatic lens. The principle of the two aplanatic foci, on which the construction of the achromatic objective has since been based was described by J. J. Lister in 1830. Three years previously, however, he had constructed an object glass sufficiently corrected to enable Hodgkin to make some observations on the structure of animal cells and tissues which mark the real beginning of vertebrate histology. In a short paper (Hodgkin and Lister, 1827) are described the shape of the human erthyrocyte, the construction of muscle fibers and the fibrous network of the arterial intima. The then current view that all animal tissues were composed of uniform globules did not survive this refutation. Baker (1948, 1949) is admirably tracing this, and other aspects of the development of cellular theory. 111. NUCLEIC ACIDS

It is probable that early in the nineteenth century the technical development o€ the microscope and its use in biological research were reacting one upon another, as is indeed true also of the present day. This is not the only feature of contemporary cell biology which is reminiscent of an earlier period ; the reawakening of interest during the last twenty years

SOME HISTORICAL FEATURES I N CELL BIOLOGY

3

in the problems of the distribution and functions of the nucleic acids within the cell provides a striking parallel with the cell biology of the 1870’s. In this renaissance again, advances in technique have both facilitated and have been evoked by progress in the study of the cell. It may not be without interest to trace some of these parallel features which extend in some instances almost to the details of research. As is well known, Friedrich Miescher in the late 1860’s prepared from pus cells a substance with stronger acidic properties than any organic cell constituent then known ; this he termed “nuclein” ; it was further distinguished by a high content of phosphorous (Miescher, 1871). The first stage of this investigation has a very modern sound, for he separated the pus leucocytes into nuclear and cytoplasmic fractions. The isolated nuclei were clearly recognizable as such; within them was a nucleolus, they were merely slightly smaller than those of intact cells. Nor was Miescher alone in preparing separated nuclei ; a method for bird erthyrocytes was described at about the same time by Brunton (1870). Among the properties of nuclein which Miescher described were its solubility in alkalis and its resistance to peptic digestion. These criteria were used some ten years later by Zacharias (1881) in a cytochemical study. The use of reagents under the microscope to identify the chemical constituents of biological material is much older than this, for as Dr. Baker (1943) has shown, the subject begins with Raspail in the late 1820’s. Zacharias’ work however was probably the first example of the use of enzymes in microscopical study. H e found that the nucleus of the frog erthyrocyte, and the macronuclei of Vorticella and Paramecium all remained behind when the rest of the cell was digested with pepsin, but dissolved if soda was then added. His clearest results on plant cells were obtained with pollen mother cells in division. H e found that the “Kernplatten-elemente” of Strasburger, which Waldeyer ( 1888) later called “chromosomes,” retained their affinity for stains after treatment with pepsin. On the other hand, the spindle was digested away. Sixty years later, Mazia ( 1941) described how larval dipteran salivary chromosomes shrink when treated with pepsin, but retain their positive reaction toward the Feulgen reagent. H e concluded that the digested chromosome had lost the globulin-like protein of the matrix, but had retained the histones of the chromosome skeleton, which he found also to be resistant to pepsin. Numerous cell constituents, of course, are not attacked by this enzyme ; Zacharias (1883) gave the name of “Plastin” to cytoplasmic material which remains after peptic digestion, and it is not surprising that properties

4

ARTHUR HUGHES

common to plastin and nuclein should have led in those days to statements that nucleic acids occur in the cytoplasm. However, the first observations which we can interpret as a demonstration of this are due to van Hewerden in 1913, who then showed that the basophilia of the sea urchin egg is diminished by digestion with a preparation of what we now t e r n ribonuclease. Van Hewerden’s work has been recognized by recent investigators in this field (Catcheside and Holmes, 1947) but there still remains another type of study in microenzymology which has yet to be resumed. I n 1908 Adolf Oes published a study on the autolysis of cells, mostly in bean roots, which were incubated under toluene for periods from an hour to a day at 30-40“, and were then fixed and sectioned. The cytoplasm was relatively little affected by this treatment, but in mitotic cells from metaphase to telophase the chromosomes were digested away. Nuclei in prophase were more resistant, and those in interphase still more so. The autolysis proceeded in the presence of weak alkalis, but was inhibited by acids and by ions such as copper and magnesium. The only comparable experiment in modern times which I have come across is also due .to Mazia (1941), who found that there was no digestion of chromosomes in Drosophila salivary glands kept under toluene for seven days. Again, permanent nuclear elements resist autolysis. Another reminiscent feature of modern studies on cell nuclei is in their staining reactions. For instance, methyl green, used in Unna’s wellknown method, has recently been shown by Kurnick (1950) to react quantitatively with highly polymerized desoxyribonucleic acid. This dye was first introduced into microscopical technique by Calberla ( 1874). The most interesting early example of its use is Balbiani’s famous account of the salivary gland nucleus of the Chironomus larva (Balbiani, 1881). He described the chromosomes as a “cordon cylindrique,” the arrangement of which reminded him of an intestine. He found that only the bands took up methyl green ; other stains colored the nucleolar material, and that mixtures of the two resulted in “jolis effects de double coloration.” Not often have the staining reactions of cell constituents led to results as clear as this distinction between the nucleoli and the chromosomes. Ehrlich in 1879 realized the difference in effect of acid and basic dyes, but in later years physical explanations of staining increased in prominence ; Fischer in 1899 held that all staining was due to adsorption. The attempt was made by double staining to distinguish between free nucleic acid and nucleoprotein in the nucleus ; Lilienfeld in 1893 maintained that mitotic chromosomes and the resting nucleus differed in this respect, but two years later Heine was unable to confirm this distinction.

SOME HISTORICAL FEATURES IN CELL BIOLOGY

5

IV. STUDY OF LIVING CELLS One of the features of the cell biology of the last century is the occasional prominence of the study of living cells. Here again at the present day there is a tendency in this direction, now encouraged by the Zernicke phase microscope. The stained preparation, which has long been nearly the exclusive means of approach to the problems of cell structure did not at once acquire this predominance when dyeing technique was introduced into biology. In the 1870’s it was still customary to observe both stained and unstained material in microscopical researches. I n the elucidation of the complex events by which cells divide, the study of the living cell in the process of division played a conclusive part in that annus nzirabilis, 1879, for then three separate authors described the course of nuclear and cell division in life. Strasburger (1879) followed the process in the stamina1 hair cell of Tmdescantia while larval Amphibia were chosen by Schleicher (1879) and by Flemming (1879), who respectively studied cells in cranial cartilage and the skin. These two authors published their papers in the same volume of one journal. The fundamental importance of these studies is illustrated by a remark of Nordenokiold in referring to the editions of Strasburger’s “Zellbildung and ZelltheiIung,” which respectively preceded and followed this year : Even in the first edition of his said work (1875), Strasburger makes the nucleus of the egg cell in the plants he investigated dissolve upon fertilization and its mass disperse into the plasm of the cell; in the latter are then formed a number of concretions] which give rise to fresh nuclei. In the third edition (1880) on the other hand, it is asserted that examples of independent cell-formation can no longer be cited from the vegetable kingdom ; fresh nuclei invariably arise through the division of older ones C1928, p. 5351.

It is of much interest to compare Strasburger’s drawings with Bilai-’s photographs of the Tradescantia hair cell in division, which were published in 1929. One can recognize the parallel arrangement of the chromosomes in late prophase in Strasburger’s drawings, the significance of which he was not then aware. Attention may be drawn to two other nineteenth century studies on living cells, which have yet to be resumed in this era. In 1875 Ranvier described in his textbook of Histology how leucocytes in Amphibian lymph within a moist chamber can be seen to undergo amitotic division and says the observation is simple enough for a class exercise. He also described how fragments of elder pith inserted in the dorsaI lymph sac of the frog become infiltrated by leucocytes. In 1887, Arnold repeated

6

ARTHUR HUGHES

this work, by combining the two observations. H e mounted this infiltrated pith on a coverslip under sterile conditions, sealed the preparation, and was then able to observe the behavior of the leucocytes which wandered out of the pith fragment for a period of four or five days. Arnold confirmed Ranvier’s account of this amitotic division, and as far as I know these are the only descriptions in living cells of direct nuclear fission being followed by cytoplasmic division. It might be claimed that Ranvier and Arnold were the first parents of tissue culture, and it would surely be worth someone’s while to try their experiments again. The second example goes back still farther into the last century and relates closely to a subject of present day interest. Balbiani in 1864 described movements which occur in the nucleoli of the oocytes of spiders. He said that under the microscope this activity could be watched for several hours in the larger oocytes of an excised ovary. There was an ameboid movement of the whole nucleolus and also a continuous change in size of vacuoles within. H e was convinced that these were normal events because inspection of an ovary immediately on incision showed that all phases of activity were to be seen in individual nucleoli. Bradfield (1949) has found that the oocyte nucleolus of the spider gives a very strong positive alkaline phosphatase reaction, and is rich in ribonucleic acid. If these facts mean that this body is actively concerned in synthetic activity, as Caspersson would claim, it is possible that the nucleolar movements are related thereto, and so we may here have the possibility of watching material being synthesized in a cell under the microscope. Thr scope for futher study of such a system needs no emphasis. In conclusion, one may say that since in some ways cell biologists are on old ground working with new implements it would be as well if they recognized more fully the antiquity of their sites and looked out for thc old forgotten tracks, along which something of value might still be found A few more ideas, however ancient, would still be useful in cell biology. V. REFERENCES Amici, G. B. (1824) Ann. Sci. nat., 4 41. Arnold, J. (1887) Arch. mikr. Anat., SO, 205. Baker, J. R. (1943) J. Quekett micr. C!., Ser. 4, 1, 256. Baker, J. R. (1948) Quart. 1. micr. Sci., 89, 103. Baker, J. R. (1949) Quart. J. micr. Sci., 90, 87. Balbiani, E. G. (1864) C. R. SOC.Riol., Ser. 4, 1, 64. Balbiani, E. G. (1881) Zoo!. AM., 4, 637. Bslaf, K. (1929) 2. Zellforsch., 10, 73. Bradfield, J. R. G. (1949) Exp. Cell Res., Suppl. 1, 338

SOME HISTORICAL FEATURES I N CELL BIOLOGY

7

Brunton, T. L. (1870) 1. A w t . Physiol., Ser. 2, 3, 91. Calberla, E. (1878) Morph. Jb., 3, 635. Carpenter, W. B. (1857) The Microscope and Its Revelations, 2nd ed. London. exp. Biol., 1, 2 5 . Catcheside, D. G., and Holmes, B. (1947) Synp. SOC. Chevalier, C. (1839) Des microscopes et de leur usage. Paris. Dobell, C. (1932) Antony van Leeuwenhoek and His “Little Animals.” London. Ehrlich, P. (1879) Arch. Anat. Physiol. (Physiol. Abt.), p. 571. Fischer, A. (1899) Fixierung, Farbung und Bau des Protoplasmas. Jena. Flemming, W. (1879) Arch. mikr. Anat., 16, 302. Fresnel, A. J. (1824) Ann. Sci. nat., 3, 345. Heine, E. (1895) 2. physiol. Chem., P,494. van Herwerden, M. A. (1913) Arch. Zellforsch., 10, 431. Hodgkin, T., and Lister, J. J. (1827) Phil. Mag., 2, 130. Kurnick, J. B. (1950) Ex#. Cell Res., 1, 151. Lilienfeld, L. (1893) Arch. Anat. Physiol. (Physiol. Abt.), p. 391. Lister, J. J. (1830) Phil. Trans., p. 187. Mayall, J. (1886) J . Sot. A r t s , 34, 1055. Mazia, D. (1941) CoZd Sprifig Harb. Symp. qiruiit. Biol., 9, 40. Miescher, F. (1871) Hoppe-Seyleu‘s Med-chem. Urrtersttchiricgen, 46, 441. Nordenskiold, E. (1928) The History of Biology. Translated by L. B. Eyre, New York. Tudor Publishing Co. Oes, A. (1908) Bot. Z., 16, 89. Ranvier, L. (1875) Trait4 technique h’histologie. Paris. Sachs, J. v. (1890) History of Botany. Translated by H. E. F. Garnsey, Oxford. Schleicher, W. (1879) Arch. mikr. Aptat., 16, 248. Strasburger, E. (1879) Jena. Z . 19, Sitzungsber, p. 93. Strasburger, E. (1879) J e w . 2. Nafurw., 19, Sitzungsber, p. 93. Waldeyer, W. (1888) Arch. mikr. Apia#., 32, 1. Zacharias, E. (1881) Bot. L.,39, 169. Zacharias, E. (1883) Bot. Z., 41, 209.

This Page Intentionally Left Blank

Nuclear Reproduction* C. LEONARD HUSKINS Department of Botany, University of Wiscoiisin, Madison, Wiscowin. CONTENTS Pllgc

I. 11. 111. IV. V. VI. VII.

Introduction ......................................................... Nuclear or Cell Division ............................................. Chromosome Reproductioii ............................................ Variation in Nuclear Size ............................................. Reduction in Chromosome Number ..................................... Conclusions .......................................................... References ...........................................................

9 10 12 15 18 21 24

I. INTRODUCTION I t is difficult to find anything new to say about morphological aspects of nuclear reproduction, but there are cytochemical and genetic data which should be correlated with the descriptive in any evaluation of the problem. It may also be useful to emphasize the distinct aspects of some of the processes that conlnlonly occur in association. Any correlated analysis or evaluation must at the present time contain many speculations, but I do not think that harmful, provided facts and ideas are plainly differentiated. Everyone knows that reproduction of the elements within the nucleus is not the same thing as reproduction of the nucleus itself, yet a number of geneticists, for example, have failed to make this distinction explicit in their consideration of problems of gene action. Usually this makes no difference to the argument, but it can do so when as in some recent papers on the possible role of heterochromatin in differentiation, conclusions as diverse as that of Caspersson (1939) and others, that heterochromatin is concerned with the division of the chromosomes is cited along, for instance, with Darlington and Thomas’ (1941) conclusion that it is responsible for supernumerary divisions of the pollen cell in Sorghum, and so on, without any indication being given that these are very different processes or that the evidence for the conclusions has been obtained from very different observational levels. Though chromosome and nuclear reproduction are both normally antecedent to reproduction of the cell, any one of the three processes can, of course, occur without either one of the others.

* Presented at the Seventh International Congress of Cell Eiology, Yal: University, September 4-8, 1950. 9

10

C. LEONARD H U S K I N S

If the geneticist sometimes errs by implying nuclear and cell division when his evidence relates only to gene or chromosome reproduction, the experimental cytologist in the past often paid too little attention to the chromosome and genes. Total disregard is not possible today, especially when once widely divergent disciplines are brought together in a congress such as this, but there is still evident need, even in the abstracts of our program, for all of us to make more extended use of each other’s data.

11. NUCLEAR OR CELLDIVISION Professor Heilbrunn will show later in this program that “cell division is not necessarily initiated by an increase in cell permeability, nor is it always accompanied by an overall increase in cell permeability,” and will present evidence on the gelatin-liquefaction cycle associated with mitosis. H e maintains that : “The colloid-chemical theory offers a logical explanation of all the known facts” of cell division. It is evident that in his argument he is including both karyokinesis and cytokinesis but not chromosome reproduction. I shall therefore concentrate chiefly on chromosomes, especially since it is the field with which our research group is predominantly concerned. As for the correlation between karyokinesis and cytokinesis, we may say that the concept of nucleoplasmic ratio determining nuclear and cell division, which was derived predominantly from morphological observations, can no longer be considered seriously in its original form, but that the data it subsumed must still be taken into consideration and that the concept itself is not wholly invalid. As for morphogenesis, while fully realizing that nothing should be regarded as unimportant in the present embryonic state of our knowledge, I shall for today also assume that we need pay little attention to nuclear or cell division or size as such in this connection. W e know, e.g., Weisz (1947), that cell mass may influence the course of differentiation and also that nuclear and cell size may have striking effects in some cases. The latter is evidenced by the differences, both morphological and physiological, sometimes found between diploids and their autopolyploids. But in some cases an increase of the chromosome number appreciably increases neither nuclear size nor any other characteristic. Correlatively, differences in the number of nuclei per cell do not appear to be causally related to differentiation. I n Acetabularia, for instance, Hammerling ( 1946) has found that nuclear divisions normally begin only after niorphogenesis is completed, However, a young nucleus will divide when transplanted into an old system, which, since the nucleus controls the differentiation, can be taken to indicate that nuclear division is ultimately regulated

NUCLEAR REPRODUCTION

11

through the mediation of its own products. Schulze (1939) dismissed the possibility that increase of chromosome material, either by polyteny or polyploidy, is involved in the increase of nuclear size which accompanies differentiation in this alga, but the material does not seem sufficiently favorable for this to be ruled out by a descriptive cytological analysis. DNA measurements should give a more decisive answer. In dikaryotic fungi a gene in one nucleus can dominate in its effect over a gene in the other, just as one allele over another in a diploid or polyploid nucleus. Heterosis also is exhibited in heterokaryonts. If we look for functional significance of the multinucleate condition it seems to be found not in anything connected with differentiation but in the occurrence of nuclear competition, whereby nuclei containing disadvantageous genes are apparently subject to adverse selection, as found in Neurospora. The reported stability of the polykaryotic Mucor species after an initial adaptive period when first placed on artificial media (Hesseltine, unpublished thesis, University of Wisconsin) could also have this explanation. Fankhauser (1948) has shown that while a polynucleate condition causes abnormal cleavage in frogs and toads, it does not do so in most Urodeles. I n these, fertilization is normally polyspermic but at the critical stage in development the principal sperm nucleus unites with the egg nucleus, and the accessory nuclei begin to degenerate. The latter may go through prophase, and hence doubtless through chromosome reproduction, but not through mitosis. Barber (1942) found that orchid pollen grains with sub-haploid nuclei could divide normally if separated by only a thin cell wall from grains with a full haploid chromosome complement. As shown by Clark (1942) even fragmentation of the nucleus has no necessary effect on development and germination of corn pollen so long as all the fragments remain in the cell. To conclude this section of our discussion on the limited significance of nuclear or cell division for differentiation and development, brief reference may be made to a few data on the time and scope of gene action relative to mitosis and cytokinesis. Berrill and Huskins (1936) stimulated discussion of this issue by proposing that “energic” replace the term “resting” nucleus. C. Stern (1938) showed that in a number of decisive cases specific genes interact with the cytoplasm during the energic state of the nucleus. He pointed out that in some cases it is possible, as in some examples of pollen dimorphism, that gene-controlled substances exert visible effects only after the breakdown of the nuclear membrane, but decisive evidence of this was, at that time lacking. I am not aware of any more definitive evidence having been advanced since then. H. Stern

12

C. LEONARD HUSKINS

(1946) showed that there is an increased permeability to sucrose of the plasma membrane during meiosis and postmeiotic mitosis in Trillium pollen and that it begins to rise before breakdown of the nuclear membrane. Data on permeability during endomitosis are lacking and should be sought, but sugar intake need, of course, bear little or no relation to diffusion of nuclear products, Jones (1947) shows that changes in the nucleus may have visible effects in the cytoplasm and that gene-determined pigments in corn may be either cell-limited or diffusible over a considerable area of tissue. Commoner (1949), from further analyses of somatic mutations at the A locus, suggests that genetic determination of the anthocyanin content occurs before cell enlargement and that the specific action of the A gene is based on initiation of production of a precursor. The radial pattern suggests to him distribution of the gene product during formation of constituent cells ; evidence on the issue of influence during mitosis might be found here. 111. CHROMOSOME REPRODUCTION Let us turn to the problem of chromosome reproduction without nuclear division. Almost without exception, biology textbooks teach that the chromosome number is constant in all somatic cells of a multicellular organism. A brief sketch of the development of this concept may be of interest and not without value for future work and concept formulation. Weismann (1893) wrote: “With certain exceptions . . , the number of chromosomes is constant for each species.” Wilson (1900) went further : “The remarkable fact has now been established with high probability that every species of plant or animal has a fixed and characteristic number of chromosomes which regularly recurs in the division of all its cells; and in all forms arising by sexual reproduction, the number is even.” I t remained for 0. Hertwig (1918) to formulate in detail the “law of constancy of chromosome numbers”: “This law tells us that the number of chromosomes in all cdls of a plant or animal species, with the occurrence of nuclear division, is always exactly the same whether we are dealing with epidermis, cartilage, muscle, or glandular cells, etc. However, . . the egg and sperm contain one half the number of chromosomes of the somatic cells. This also is a lawful phenomenon.” One embryologist later extrapolated the law to the extent of writing: “All cells whether they continue to divide or not ultimately contain the same genetic proteins in equal quantities.” There were many factors involved in the gradual consolidation of the law of constancy of chromosome numbers. Nemec in 1904 and 1910 had

.

NUCLEAR REPRODUCTION

13

discovered tetraploid nuclei in chloralized roots, but Strasburger ( 1907) was convinced that they could not persist and therefore were of no significance. H e even explained away as obviously due to some “disturbing influences, such as wounding by small animals, etc.,” his own discovery of rows of tetraploid cells in control roots. Of this Winkler (1916) wrote : [trans.] “This last remark of Strasburger is extraordinarily characteristic. I t shows that the conviction that in normal somatic tissue only diploid cells could occur has become a dogma under the influence of which the best plant cytologist comes at once to the opinion that the occurrence of tetraploid cells found therein must be pathological, without even considering any other possibilities.” Winkler himself, after establishing with certainty the occurrence of polyploid cells and tissue in Solanum species and of the production of polyploid plants from the callus of grafts, concludes : “since the germ cells always arise directly from embryonic tissues they will always have the typical chromosome number and hand it down to the next generation. The constancy of chromosome number is safeguarded even when there is vegetative reproduction, since plants grow with their growing points which, by definition, are always embryonal. . . . W e therefore come to the view that the regular occurrence of polyploid cells in the somatic tissue of higher plants by no means refutes the laws of constancy of chromosome number but must be expected in view of the importance of the chromosome number for cell size.” Except possibly for the last clause, with its teleological flavor, this is as clear and acceptable a statement on polysomaty as could be made today. Why has it so very generally been ignored, not only by textbook writers but also by most research workers ? It must be remembered that from the time the correlation of chromosome behavior in meiosis and of Mendelian factors in segregation and recombination was first clearly enunciated it took about a quarter of a century to establish the “chromosome theory of Keredity” to the satisfaction of the overwhelming majority of biologists. As late as the 1920’s there were some who still considered the chromosome complement as a variable characteristic which was no more and no less a part of the plant phenotype than, say, the number and shapes of the leaves. The important general concepts involved in the chromosome theory of heredity were most conclusively established by showing that in exceptional organisms with chromosome numbers or arrangements of their parts that deviated from the norm of the species, such as haplo-IV Drosophila, trisomic Oenothera, tetraploid Datura and translocation stocks of Drosophila, maize, etc., the genetic behavior was altered correlatively.

14

C. LEONARD HUSKINS

By 1937 it was realized that colchicine produces polyploid cells by inhibiting spindle action, and in view) of the importance of polyploidy in plant breeding there was immediately a wide search for other chemical agencies that would induce it. I t is not surprising, therefore, that when polyploid divisions were found in mature plant tissues that had been stimulated by “growth substances” it was assumed that the polyploidy was caused by the treatment. It seemed to me, however, that the effect of “auxins” was more probably stimulation of divisions in already polyploid nuclei. Geitler (1948) apparently reached this conclusion at about the same time and during the war he effectively established that “polysomaty” is common in the leaves and stems of a large series of plants, as he had earlier found it is in many insects. He had stimulated divisions by wounding. Unaware of his work, we had initiated complementary studies on roots with indole acetic acid in dosages that gave results quickly enough for us to determine that at least the higher polyploid nuclei had not been produced after initiation of the treatment (Huskins and Steinitz,

1948). Since polysomaty has not been considered to have any effect on genetic behavior, it was of no significance in that stage of the rapidly developing science of genetics where establishment of rules of transmission was the major goal. The question today is whether or not it is significant in developmental or physiological genetics. Let us first consider from this point of view some of the recent work, including that of members of our own group, on polysomaty. Incidentally, there is much confusion due to unsatisfactory terminology in many discussions of nuclear reproduction. I have been accused of confusing endomitosis, endodivision, polysomaty, and polyteny because I have not always in all contexts differentiated sharply between them. I risk this charge again and for the same reason as previously, namely, that I think the distinctions unimportant at the present time in discussions of possible functions. They are not unimportant in descriptive cytology, and I do not ignore them in that context. Further, since I have been so widely misunderstood on another point, despite two separate and specific warnings in my original speculative discussion on the possible significance of polysomaty ( Huskins, 1947), let me here emphasize that polysomaty as suck cannot possibly be of any great general significance in differentiation. It may be in special cases; in all cases it docs prove that chromosome, and therefore gene, reproduction continues after nuclear or cell division ceases and thereby opens the way to coilsideration of the possibility that gene action i s correlated zcuWa gene reproduction. The cyto-

NUCLEAR REPRODUCTION

15

logical evidence, now generally accepted, that the chromatid is not a transversely unitary structure, shows that Mendelian segregation involves units at a higher level of integration than those resolvable by even such a relatively crude analyzer as the light microscope. We may therefore justifiably consider the possibility that the ultimate units which may be effective in differentiation could be at a very much lower level. There is considerable evidence that, at the microscopic level, reproduction of the component strands of a chromosome is not uniform throughout its length. The possibility that reproduction of the materials making up a Mendelian gene may be differential for different genes in different tissues therefore becomes almost an a priori probability. The problem is to devise methods for testing i t ; such are, of course, appearing as soon as the problem is envisaged. IV. VARIATION IN NLTCLEAR SIZE To return to simpler levels of discussion: Huskins and Steinitz (1948) attempted to analyze the great variation in nuclear size in differentiated regions of Rhoeo roots. Evidence obtained by counting the number of heterochromatic bodies (which was the method devised by Geitler for insect tissues) and by treatment with indole acetic acid, coincided in showing the variation to be correlated with degree of polysomaty. Similar results were obtained with barley (Leonard-Bennett, unpublished). Duncan and Ross (1950) have shown that in nuclei of niaize endosperm undergoing mitosis the normal triploid number of chromosomes is usually present. However, in giant energic nuclei a high degree of polyteny is observed in regions of chromosomes marked, for observational purposes, by heterochroinatic “knobs.” They have, further, shown a different range in nuclear size in different areas of the endosperm. An apparent reduction in the size of endosperm nuclei adjacent to the embroyo as its development proceeds is of special interest and is currently being studied further. Nuclear and cell volume, chromosome and chromatid number in pith cells of Nicotium towentosu are being studied by Dr. Muriel Bradley. Her data show, iiiter ulia that in this material nuclear volume is related directly to chromatid and not to chromosome number. I t seems to make little or no difference whether 8n chromatids are present as the 4n number of ordinary two-chromatid chromosomes or as 2n chromosomes each having four chromatids. The data show also that following the halving of the total cell volume after telophase there is within each of the polyploid classes a relatively slight increase in nuclear volume which is followed by something like a doubling before the next prophase. These morphological data accord with cytochemical studies. They support indirectly the as-

16

C, LEONARD HUSKINS

sumption made from microchemical analysis by Boivin, Vendrely and Vendrely (1948), Mirsky and Ris (1949), and others that with certain exceptions the DNA content is the same in all somatic cells. Certain variations in DNA content have been related, obviously correctly, to reproduction of the chromosomes and to polyploidy. Relating the constancy, apart from polysomaty, to the concept that the genotype is the same in all cells, it is usually concluded that DNA is an essential, and probably constant, component of the gene. The most recent published work along these lines is that of Swift (1950) who has shown, by absorption spectrophotometry following Feulgen staining, that the nuclei of ten different somatic tissues of young and adult mice all show approximately the same amount of DNA except for some of those in the liver, pancreas, thymus, blood lymphocytes, and Sertoli cells which contained two or four times the common amount. Occasional rare intermediate values were found and presumed to be associated with mitosis. Mouse spermatid nuclei had half the DNA of the common somatic nuclei. Primary spermatocytes had four times and secondary spermatocytes twice the spermatid value. Some premeiotic sex cells had the somatic amount and some twice as much. The former predominate in testes of 1- to 10day old mice and the latter are commoner at maturity. Nuclei of six tissues of adult frogs all had approximately the same amount of D N A (excepting for a few liver nuclei which had twice as much), and this was slightly less than twice that characteristic of the mouse. From studies of embryonic mouse liver and Amblystoma larvae, it was shown that DNA content “builds up in the interphase nucleus before the visible onset of prophase” and that “during the visible stages of mitosis no DNA is synthesized.” I n the Malpighian tuhule nuclei of a grasshopper four classes of DNA content with the ratio 1 :2:4:8 were found. In our laboratory (Bloch and Patau, unpublished) the relative DNA content of mouse liver nuclei has been determined, following Feulgen staining, with both an electro photometric and a visual microphotometer, the latter instrument having been designed by Dr. K. Patau. The results with liver of adult mice of pure lines and their hybrids agree with those of Swift and confirm that the step from one class to the next higher is a very accurate doubling of the DNA content accompanied by a doubling of nuclear size. In embryonic liver the DNA content ranges from 1 :2. It doubtless is reflecting the synthesis of DNA in nuclei preparing to undergo mitosis. Intermediate values between higher classes may be reflecting chromosome reproduction preparatory to either mitosis or endomitosis. Schrader and Leuchtenberger ( 1949) have stressed the

N U CLEAR REPRODUCTION

17

variation “which may be due to different degrees of polyteny” in different tissues of Tradescantia. Since oral presentation of this article, Swift (1950) has reported findings in maize and Tradescantia. In our laboratory, measurements are being made on Allium and Tradescantia tissues (Nelson, unpublished). For the roots the results are similar to those of Swift, excepting that he reports none of the lowest class in the elongation region of maize roots but several higher multiples, whereas in the same region of Allium roots, Nelson finds large numbers of the lowest and second classes, very few of the third, and none higher. Interesting additional findings by Nelson are : (1) that the doubling which precedes mitosis occurs early in the interphase period (unpublished data of Dr. Alma Howard, Radiotherapeutic Research Unit, Hammersmith, on uptake of radioactive phosphorus, are in accord) ; ( 2 ) that the guard cells of stomata have constantly the lowest amount of DNA normal for diploid cells, while other cells of the epidermis have double this amount ; (3) immediately after the first division of the microspore nucleus the resulting “vegetative” and “generative” nuclei have the same (haploid) amount of DNA but as the pollen grain “ripens” the generative nucleus doubles its content - in readiness for its division - while the content of the vegetative remains constant, contrary to the opinion of the many descriptive cytologists who have noted its fainter staining. In all of the foregoing, the findings confirm the ideas of Jacobj (1925) and many subsequent workers that increase of nuclear size may be caused by geometric increase of chromosome number, i.e., by polysomaty. It has, however, also long been clear that nuclear size can increase greatly without change in chromosome number. In some cases it is now evident that this may be correlated with increase in degree of polyteny, as shown by Duncan and Ross in maize endosperm. In yet others it may have little or no relation to the “chromatin” content. Schrader and Leuchtenberger ( 1950) have shown cytochemically that in the very different-sized nuclei which characterize different lobes of the testes of Armelitis albopunctatus, a hemipteran insect, the DNA content is approximately the same; it is the total protein and the RNA content that are correlated with nuclear, nucleolar and cytoplasmic volume. Apart from internally regulated “permanent” changes in nuclear volume, there are, of course, changes which appear to be correlated with developmental or physiological factors. Metz and his students (Buck and Roche, 1938) have shown that osmotic and mechanical pressure changes may cause reversable increase or decrease in nuclear and chromosome size as great as 25 per cent. The diverse effects of various fixatives on nuclear size are also, of course, well recognized.

18

C. LEONARD HUSKINS

To summarize this discussion of polysomaty we may safely say: (1) The concept of genic and chromosome identity of all cells obviously needs both amplification and circumscription; (2) Many more data on its occurrence are needed before we can even decide whether or not it, as stich, is at all likely to be found of any general significance in differentiation and development. (Regular and characteristic differences in number of chromosomes in certain tissues such as Geitler and others have found in various insects and in DNA content such as Nelson is finding in plant epidermis seem to point affirmatively, whereas the difference in these regards between morphologically similar regions of maize and onion roots and many other data point negatively.) ; (3) The newer techniques if used on specially chosen materials, particularly if these permit a concomitant genetic analysis, are capable of giving us answers to problems that were unassailable by the methods of cytological study available until very recently; (4) Whatever the relationship of D N A to ultimate gene structure, it seems certain that it is precisely related to the reproduction of the chromatid, which is the subdivision of the chromosome that is significant for Mendelian heredity-so far there is no evidence of a chromosome’s being able to divide without first doubling its DNA content, but, of course, very few studies of this question have yet been made: ( 5 ) Though chromosome reproduction must “normally” precede nuclear division, either of the two processes can proceed independently of the other -the relationship is parallel to that of cytokinesis to karyokinesis.

v.

REDUCTION IN

CHROMOSOME

NUMBER

We may next consider the process of reduction of chromosome number. To the long-established account of the process in germ cells the only important item to be added from current studies with the newer techniques seems to be the discovery (Swift, Nelson, and probably many others unpublished) that the D N A content reaches the same heightened level by pachytene of the first meiotic division as that typical for early mitotic prophase. Therefore, two divisions without an intervening increase during their interkinesis are required to restore the normal relationship between number of chromosomes and nuclear D N A content since the former has been halved. W e have moved far from the concept held by many cytologists not so long ago that the first division was the reduction division, and the second an unexplained concomitant ; genetics has long shown that both divisions are essential to genetic reduction, the occurrence at first prophase of four chromatids in each bivalent indicated the same from the descriptive cytological aspect, and now we see that they are needed to restore cytochemical balance also.

NUCLEAR REPRODUCTION

19

Somatic reduction and segregation have long been known but little investigated until recently. Many textbooks list Winkler’s ( 1910) Solununz danoriniaiticm as having originated by somatic reduction, but none that I know of list his 1916 cases. I believe (see Huskins, 1948) the former to be an erroneous interpretation while the latter are clearly valid. Winkler’s 1916 remarks warrant quotation as a basis for discussion of current studies. [translation] To obtain such reversions to the diploid normal form from a tetraploid gigas type, the cells which gave rise to the atavistic tissue complex must have undergone a reduction-division. We may therefore not doubt the possibility that reductiondivisions occur in somatic cells. What stimulates them has to be left undecided. That the tetraploid condition of the nucleus by itself should have caused a tendency for the reduction of the increased chromosome number cannot be assumed, since tetraploid types exist and in general persist. It will also have to remain undecided for the time being whether the halving of the chromosome number occurs by typical reduction-divisions or otherwise. As a matter of fact it will be very difficult to find such a reduction-division since the reversions, at least so far, have occurred rarely and quite irregularly, that is in places which could not be predicted.

Two cases are reported in which somatic reduction appears to be a regular process: (1) prior to meiosis in the hermaphrodite gonad of a coccid (Hughes-Schrader, 1927) and (2) in the ileum of mosquito larvae (Berger, 1941 ; Grell, 1946). Somatic pairing and segregation are, of course, well known in the Diptera, but adequate data are lacking on reduction, though it was early reported by Bridges. Bateson (1926) insisted that there was much genetic evidence for somatic segregation in various tissues, but most such cases investigated in the past quarter century have been interpreted as somatic mutation, which he warned against as likely to obscure the issue. Recent evidence indicates that in any specific case both must be considered as possible explanations until the one is ruled out. The sporadic occurrence of chromosome pairing and/or reduction in somatic tissues has frequently been recorded. For example, Gates (1912) observed it in the nucellus of Oenotheru Zutu; Ludford (1935) and others have recorded it in tumors and tissue cultures of tumors; Metz (1942) found in a Sciara hybrid a salivary gland nucleus containing chromosomes from only one of the parents; Love (1936) found pollen mother cells that had undergone reduction prior to meiosis. East ( 1934), Nishiyama (1933), Kiellander (1941), Sparrow (1941), and Vaarama (1949) obtained plants with reduced, ancestral, chromosome numbers among the progeny of polyploid strawberries, oats, Poa, wheat, and Ribes, respectively. Brown (1947) found a reduced sector in an unbalanced poly-

20

C. LEONARD HUSKINS

ploid cotton plant. Upcott ( 1939) observed irregularly reduced chroniosome numbers in tetraploid Primnula kewensis; it is possible that by attributing these to “split spindles” she may have diverted attention from the problem as Strasburger, in Winkler’s opinion, quoted earlier lierein (1916), diverted attention from polysoniaty by assuming that it must be pathological. Following sporadic discovery of haploid cells in various plant roots, consideration of Caspersson’s ( 1939) suggestion that nucleic acid plays a role in synapsis and chromosome division led to a search for somatic reduction in preparations of Allium root tips treated by Dr. M. Kodani (1948) with sodium nucleate. Many cells with two reduced groups of chromosomes were found (Huskins, 1948). They were later found also in roots grown in solutions rich in phosphates (Galinsky, 1949). More recently it has been found (Huskins and Cheng, 1950, and unpublished) that prolonged low temperature treatment also increases the frequency with which “reductional grouping” occurs. There is also evidence that genetic factors affect the frequency with which reduced tissues or organs occur. In one strain of tetraploid Rhoeo we have obtained diploid and triploid roots and shoots with and without treatment. In another strain, treatments increase the frequency of reductional groupings, but no reduced tissues have yet been obtained. Allen, Wilson, and Powell (1950) have recently compared the sodium nucleate results with the chromosome groupings that occur after colchicine treatment. An extensive study has been made by Patau and Steinitz (1951) on the origin of reductional groupings and of reduced cells (see also, Patau, 1950). It is clear that somatic reduction is of not infrequent spontaneous occurrence, that its frequency can be increased by various treatments and “natural” conditions such as low temperatures, and therefore that polyploidy is a reversible evolutionary process. Battaglia ( 1948) has recently reported that somatic reduction occurs regularly in the basal portion of the style of Sambucus, and Christoff and Christoff (1948) report it in the integumental cells of Hieracium. If these and the cases in insects mentioned earlier are confirmed and extended, the “law of constancy of chromosome numbers” and our concepts of nuclear reproduction will have to be extended in this direction also. Besides polysomaty, polyteny, and reduction, which affect the total chromosome complement, there is also aneusomaty, i.e., the occurrence of cells with variable numbers of individual chromosonles, to be taken into account. It appears probable that aneusomaty (not to be confused with aneuploidy which refers to deviations between, not within, organisms)

NUCLEAR REPRODUCTION

21

most commonly involves chromosomes that are wholly or in large part heterochromatic. Too little carefully controlled work has yet been done on aneusomaty to warrant any conclusions on either the mechanism of its occurrence or its significance. Duncan (1945) concluded that in the root tips of an orchid the occurrence of variable numbers of chromosomes was due to a differential rate of reproduction of euchromatic and heterochromatic chromosomes. Darlington and Thomas ( 1941) attributed similar variable numbers in Sorghum to selective elimination in the roots but not in the shoots. Randolph (1941) presents numerous cytogenetic data which show that in maize the problem of B chromosome function and behavior is very complex. In Cimex, Darlington (1939) found from 0 to 12 extra X chromosomes in the males, with the average number higher in natural populations (9.0) than in mass cultures (4.3). H e correlates cycles of chromosome and centromere division with the “differential precocity” of autosomes, M chromosomes, and sex chromosomes and functionally relates the various changes observed to “adaptive balance” in sex-determining mechanisms. The mechanism of variability is related to “the state or precocity” of the centromere and the relative size of the chromosomes. These issues would carry us far beyond the scope of the present review, but they lead up (as Darlington points out in his Appendix 11) to the problem of preferential segregation, which is also beyond present scope except that it must be pointed out that special spindle mechanisms exist which provide for elimination of whole sets of chromosomes. The best analyzed of such cases is probably Sciara, for which both genetic and cytological data are available. It seems probable that such elimination is functionally related to chromatin diminution that involves only parts of chromosomes and that some form of differential reproduction may be basic to all such. Investigation with the newer techniques and with wider concepts in mind than those which guided earlier descriptive studies may lead to the establishment of some of the generalizations which at present are almost entirely speculative.

VI. CONCLUSIONS To conclude we may summarize a few of the data which, though none alone may be conclusive, together suggest a need for revision and extension of some of our more orthodox concepts on various aspects of the reproduction of the nucleus and its components and of the role of the nucleus in differentiation and development. First, against the simplest unitary concept of the gene there is, to repeat, the cytological evidence that there are, frequently at least, more microscopically separable strands in both the

22

C. LEONARD HUSKINS

mitotic and meiotic chromosomes than there are chromatids, which are the unitary gene strings of Mendelian segregation. The concept of the Mendelian gene as made up of identical “lamellae” would fit this. (The term “lamella” must not be taken to connote an undue simplicity in either the concept or the writer ; it may at our present stage of concept-forming serve as the equivalent of the beads-on-a-string model which was useful when the linear order of the genes was the issue.) Against it are the onehit radiation data, together with the occurrence of reverse mutations. It would be easy to imagine a change in one “lamella” being transmitted to all the others if the process went only one way, but not if it goes either way with anything like the same frequency, as some few gene mutations do. However, the one-hit hypothesis is not unassailable. For recent discussions of it see Muller (1950) and Opatowski (1950). Secondly, against the concept of gene identity of all cells (leaving aside whole chromosome changes for the moment) there are observations which are taken to indicate : (1) that heterochromatic and euchromatic parts of chromosomes may reproduce at different rates (Schultz, 1941) ; (2) that the banding pattern and length and breadth is visibly different in the same giant chromosomes from different tissues (see Kosswig, 1948) ; (3) that “specific chromosome loci [produce] lateral loops” (Duryee, 1950) ; and (4) that different parts of the salivary gland nuclei give strikingly unequal phosphatase reaction (see Brachet and Jeener, 1948), which suggests the possibility of variation from one gene to another in speed of renewal of phosphorus in the DNA. This would leave open the alternatives of phosphorus renewal in DNA playing a role in synthesis of proteins concerned with growth or of differential reproduction of chromosome regions. Differential reproduction of gene lamellae would provide a mechanism for differentiation not envisaged by Goldschmidt and probably not compatible with his present ideas on the gene, but it can be related conceptually to his early theory of timed, sequential physiological activity of the genes. Thirdly, it now seems fairly certain that the DNA content of nuclei from different tissues is constant, or, more strictly speaking, that it is constant relative to the total number of chromatids per nucleus, whatever the tissue. However, the R N A and protein content are both variable in different tissues. Chargaff (1950) reports that the D N A from different species differs in chemical composition and puts out the interesting suggestion: “It would be gratifying if one could say-but this is for the moment no more than an unfounded speculation-that just as the desoxy-pentose nucleic acids of the nucleus are species-specific and con-

NUCLEAR REPRODUCTION

23

cerned with the maintenance of the species, the pentose nucleic acids of the cytoplasm are organ-specific and involved in the important task of differentiation.” Daly, Allfrey, and Mirsky (1950), however, dispute his findings. As for the possible role of the nucleus in differentiation, it is pointed out by Dunn ( 1949), Gluecksohn-Schoenheimer (1949), and others that the developmental reactions controlled by mutated genes are in some cases very like those occurring in normal differentiation, and that they are possibly “more fundamental and perhaps much closer to gene action than we suspect now.” Weiss (1950) emphasizes that the term differentiation has been very loosely used and that morphological criteria have played too large a part in the classification of cellular changes. H e classes as “modulations” those changes which are reversible and stresses the fact that many specialized cells cannot “dedifferentiate.” It is, of course, generally agreed that though permanent changes take place in many cells, the early crude concepts of differentiation always being determined by segregation of particles during early segmentation of animal eggs, or of its determination by any simple type of regulated gene segregation or mutation, are quite untenable. Further, the concept of genic identity of all cells was an essential step in the development of our understanding of Mendelian heredity and of the essential differences between asexual and sexual reproduction for which the basic mechanisms are mitosis and meiosis. Have we now reached the stage when we can profitably consider the possibility that our concept of the gene of hereditary transmission subsumes the “gene of differentiation and development” ( 1947) and that the two must now be distinguished? So long as we assume the chromosomal genes to be the same in all cells we are forced either to consider the cytoplasm as the seat of the primary differentiating materials on which the genes act or as (see Schultz, 1950) the variable member of the reciprocally interacting units-these are analytically the same, though the latter is conceptually more satisfying. The assumption that there must be units in the cytoplasm that determine differentiation has led to the very fruitful discoveries of the entities that currently are most frequently referred to as plasmagenes though the concepts this term implies cause its rejection by many (see Schultz, 1950). Plasmagenes that are found to be dependent on chromosome genes in their function, even though autonomous in reproduction (Ephrussi, 1950) do not of course conflict with the orthodox concept of gene identity of all cells. If, however, we should be forced to the conclusion maintained by Darlington (1949) that the only difference between nuclear genes and plasmagenes is that the latter “have been denied . . . the gift . . . of coordinated seg-

24

C. LEONARD HUSKINS

regation at meiosis,” we run into logical, or at least semantic difficulties. Though nucleus and cytoplasm are probably always interacting, the Acetabularia and other evidence seem to me to indicate that the nucleus primarily controls differentiation. But if cellular differentiation should in other cases be determined primarily by fully autonomous cytogenes which arise during ontogeny, as Sonneborn (1949) and others have suggested, then such cells are by definition genetically different even though we call their new “genes” plasmagenes. These cells should show different hereditary capacities not only in vegetative reproduction but also in the sexual reproduction of plants, if such differentiated cells can ever give rise, however remotely and indirectly, to female ,gametes. This latter would demonstrate definitely that the plasmagenes really are genes in the accepted meaning of the term, but it is the concept of chromosome identity, not of genic identity, of all cells that can be saved by the concept of two sorts of genes differing only in their location. This with the data on constant DNA content appeals to those of us who picture the chromosomes, but not necessarily the genes, as characterized by DNA at least during their period of reproduction and also of “division.” W e have, however, seen the limitations of “the law of constancy of chromosome numbers” and the constancy of chromosome parts seems very likely to prove even more limited. Which brings us back to nuclear reproduction: it is a very complex process which normally comprises many subsidiary processes which for the present we can safely classify into only two, viz., chromosome reproduction and chromosome separation. Various types of separation are accomplished by mitosis, endomitosis, and meiosis, each with many variants, but in each there is an essential basic uniformity. To these processes we must add the very incompletely known mechanisms of somatic reduction and of aneusomaty and differential elimination of parts of chromosomes, whole chromosomes of special types, and of genomes or sets of chromosomes. W e do not have to consider amitosis, for most of the descriptive work on it has long ago been shown to include errors of interpretation and the evidence of genetics shows clearly that it cannot be a normal process of nuclear reproduction, if by normal we mean the production of nuclei continuing to have potentially unlimited capacity for further reproduction. VII. REFERENCES Allen, N. S., Wilson, G. B., and Powell, S. (1950) J. Hered. 41, 159. Barber, H. N. (1942) J. Getwt., 48, 97. Bateson, W. (1926) 1. Genet., 16, 201: Battaglia, Emilio (1948) Nuovo G. bot. it&., 65, I.

NUCLEAR REPRODUCTION

25

Berger, C. A. (1941) Cold Spr. Harb. Symp. quant. Biol., 9, 191. Berrill, N. J., and Huskins, C. L. (1936) Amer. Nut., 70, 258. Boivin, A., Vendrely, R., and Vandrely, C. (1948) C. R. Acad. Sci., 226, 1061. Brachet, J., and Jeener, R. (1948) Biochim. Biophys. Acta, 2, 423. Bridges, C. B. (1930) Science, 72, 405. Brown, Meta S. (1947) Amer. J . Bot., !M, 384. Buck, John B., and Boche, Robert D. (1938) Coll. Net., 19. Caspersson, T. (1939) Arch. exp. Zellforsch., 22, 655. Chargaff, E. (1950) Experentia, 6, 201. Christoff, M., and Christoff, M. A., (1948) Genetics, 53, 36. Clark, F. J. (1942) Genetics, 27, 137. Commoner, B. (1949) Abst. Amer. J. Bot., 86, 822. Daly, M. M., Allfrey, V. G., and Mirsky, A. E. (1950) J . yen. Phjsiol., SS, 497. Darlington, C. D. (1939) J. Genet., 39, 101. Darlington, C. D. (1949) Hereditas Suppl., 1949, 189. Darlington, C. D., and Thomas, P. T. (1941) Proc. roy. Soc., B190, 127. Duncan, R. E. (1945) Amer. J. Bot., 32, 506. Duncan, R. E., and Ross, J. G. (1950) J . Hered., 41, 259. Dunn, L. C. (1949) Anniversary Symp., Jackson Laboratory, Ear Harbor, Maine. Duryee, William R. (1950) Ann. N . Y. Acad. Sci., 60, 920. East, E. M. (1934) Genetics, 19, 167. Ephrussi, Boris (1950) VIIth Int. Congr. Cell Biology, Yale University, p. 24. Fankhauser, G. (1948) Ann. N. Y . Acad. Sci., 49, 684. Galinsky, Irving (1949) J. Hered., 40, 289. Gates, R. R. (1912) Ann. Bot., 26, 993. Geitler, Lothar 1948) Ost. bot. Z., 9, 277. Gluecksohn-Schoenheimer, S. (1949) Growth, 9, 163. Grell, Sister Mary (1946) Gewtics, 31, 60-76, 77-94. Hammerling, J. (1946) Natzwwissenschaften, SS, 337. Heilbrunn, L. V. (1950) VIIth Int. Congr. Cell Biology, Yale University, p. 36. Hertwig, 0. (1918) Das Werden der Organismen. Fischer, Jena. Hughes-Schrader, S. (1927) 2. Zellforsch., 6, 509. Huskins, C. L. (1947) Amer. Nut., 81, 401. Huskins, C. L. (1948) J . Hered., 99, 311. Huskins, C. L. (1950) I. Hered., 41, 13. Huskins, C. L., and Cheng, K. C. (1950) J . Hered., 41, 13-18. Huskins, C. L., and Steinitz, L. M. (1948a) J. Hered., 99, 34. Huskins, C. L., and Steinitz, L. M. (1948b) J. Hered., 99, 66. Jacobj, W. (1925) Arch. EnfzuMech. Org., lU6, 124. Jones, D. F. (1947) Proc. eat. Acad. Sci., Wash., 99, 363. Kiellander, C. I. (1941) Svemk bot. Tidskr., 96, 321. Kodani, Masuo (1948) I. Hered., 83, 115. Kosswig, C. (1948) Proc. 8th Int. Congr. Genet., Stockholm. Love, R. M. (1936) Nature, Lond.,1S8, 589. Ludford, R. J. (1935) Arch. exp. Zellforsch., 17, 411. Metz, G. W. (1942) Amer. Nut., 88, 623. Mirsky, A. E., and Ris, Hans (1949) Nature, Lond., la,666. Muller, H. J. (1950) I . cell. comp. PhySioJ., 35, 9.

26

C. LEONARD HUSKINS

Nemec, B. (1904) Jb. z k s . Bot., 89, 645. Nemec, B. (1910) Das Problem der Befruchtungsfrage. Berlin. Nishiyama, I. (1933) Jap. 1. Genet., 8, 107. Opatowski, I. (1950) Genetics, I, 56. Patau, Klaus (1950) Genetics, MI 128. Patau, Klaus, and Steinitz, L. M. (1951) (In press.) Randolph, L. F. (1941) Genetics M, 608. Schrader, F., and Leuchtenberger, Cecilie (1949) Proc. nat. Acad. Sci., Wash, 86, 464. Schrader, F., and Leuchtenberger, Cecilie (1950) Ezp. Cell Res., 1, 421. Schultz, Jack (1941) Cold Spr. Harb. Symp. qzcant. Biol., 9, 55. Schultz, Jack (1950) Science, 111, 403. Schulze, K. L. (1939) Arch. Protistertk., 92, 167. Sonneborn, T. M. (1949) AMY. Scient., 87, 33. Stern, C. (1938) Amer. Nat., 7!2, 350. Stern, Herbert (1946) Philos. Tram., 40, 141. Strasburger, E. (1907) Jb. w'ss. Bot., 44, 482. Sparrow, A. H., Huskins, C. L., and Wilson, G. B. (1941) Canud. J. Res., 19, 323. Swift, H. H, (1950a) Proc. nut. Acad. Sci., Wash., SS, 643. Swift, H. H. (1950b) Physiol. ZoGI., 2S, 169. Upcott, M. (1939) J. Genet., 89, 79. Vaarama, A. (1949) Hereditas I,136. Weismann, August (1893) The Germ Plasm. W. Scott Ltd. London. Weiss, P. A. (1950) Quart. Rev. Biol., I,177. Weisz, P. B. (1947) J . Morph., 81, 45. Wilson, E. B. (1900) The Cell. Macmillan Co., N. Y. Winkler, Hans (1910) Ber. dfsch. bot. Ges., 28, 116. Winkler, Hans (1916) 2. Bot., 8, 417.

Enzymic Capacities and Their Relation to Cell Nutrition in Animals" GEORGE W. KIDDER Biological Laboratory, Amherst College, Amherst, Massachusetts

CONTENTS Introduction .......................................................... Nutritional Requirements of Tetrahymena .............................. Nutritional Requirements of Higher Animal Cells ....................... Conclusion ............................................................ V. References ............................................................

I. 11. 111. IV.

Page 27 28 32 32 33

I. INTRODUCTION Intensive studies of mammalian and avian nutrition have been stimulated by the practical importance of these higher animals in man's economy. It is well recognized that our newer knowledge of animal nutrition has led to the production and maintenance of more and better food animals, and this knowledge has been used to tremendous advantage in bettering man's own nutritional status. In studies of animal nutrition one is almost invariably studying, however, the nutritional conditions of a community of heterogeneous organisms. Nutritional requirements of the laboratory rat, for instance, have been determined largely for the rat plus his associated microflora and microfauna. Evaluation of enzyme systems capable of synthesizing biologically important compounds are often impossible when working with the whole animal. Techniques involving the use of sulfonamids and antibiotics to reduce the population of intestinal bacteria have frequently been successful in demonstrating enzyme lacks which otherwise were obscured. There appear to be two sets of results which can be expected when dealing with a higher animal, together with the intestinal bacteria, yeasts, and molds. First, a requirement for an organic compound may not manifest itself on account of the ability of highly synthetic organisms, within the gut, to produce sufficient amounts of the compound to supply the needs of the host tissues. This certainly has been demonstrated in the case of folic acid and vitamin BIZ, where deficiences in laboratory mammals can rarely be demonstrated without antibiotics. The second possibility to be kept in mind is that gut microorganisms may rob the host *Given before the Cell Nutrition session of the Seventh International Congress of Cell Biology.

27

28

GEORGE W . KIDDER

of important nutritional materials, thereby cutting down on the efficiency of the host tissues. This may well be the explanation for the marked growth-promoting powers of aureomycin and streptomycin, as first reported by Stokstad and his associates (1950). If one is interested in the basic enzyme patterns of whole animals and wishes to study them by standard nutritional methods, then one must meet two conditions if definitive results are to be obtained. The animal in question must be freed of all associated organisms (be rendered bacteria- and fungus-free). During this process it is often necessary to use chemically crude materials to support growth and maintenance. The next step is to develop a diet composed of chemically known constituents. When these two conditions are met, one is then in a position to study the natural enzymatic capacities and limitations which are the result of gene mutations accumulated through the evolutionary history of the organism. 11. NUTRITIONAL REQUIREMENTS OF TETRAHYMENA Up to the present time, techniques have been developed for only one animal genus to meet both these conditions: free of associated organisms and maintained on a chemically defined medium. This is the animal microorganism Tetrahynuena. The discussion of enzymatic capacities and limitations in animal cells whicli follows, therefore, will deal mainly with this unicellular animal. In making biochemical and enzymatic comparisons, however, between Tetrahymena and higher animals, one must remember that the comparisons are to be made between the protozoan and whole organisms and not between the protozoan and isolated cells or tissues from the multicellular types. I would like to review briefly the nutritional requirements of Tetrah p e m and point out some similarities and differences which are to be noted between this organism’s enzyme capacities and those which have been deduced for higher forms. The basic enzyme pattern for the synthesis of specific amino acids has been modified, in animals, by loss, from that posssessed by many bacteria. Synthesizing bacteria can and do construct all the amino acids from simple N-containing compounds such as nitrates and ammonium salts. Loss of many of the enzymes responsible for these syntheses has gradually resulted in what we can call the “animal pattern.” Tetrahymena, like the rat, has lost enzymatic capacities for the synthesis of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophane, and valine (Kidder and Dewey, 1945b, c, 1947b). Arginine can be synthesized with difficulty, indicating defective enzyme systems in regard to at

ENZYMIC CAPACITIES AND CELL NUTRITION I N ANIMALS

29

least some portion of this configuration. This corresponds also to the rat, where mature rats can synthesize arginine at a rate which allows maintenance, but growing rats must have an exogenous supply to supplement the arginine which they make. Serine appears to be much more important in the metabolism of Tetrahymen0 than it does in the higher forms. Growth fails in Tetrahynzena in the absence of serine when relatively high levels of certain other amino acids are present in the medium (Kidder and Dewey, 1947b). For practical purposes, therefore, serine may be considered essential, but only because its rate of synthesis is low, not absent. Certain interrelationships among the amino acids may be demonstrated here, as they can be in higher forms. Thus tyrosine is synthesized by the oxidation of the p-position of the phenylalanine but reductases for this position are lacking (Kidder, 1947). Similarly cystine is formed from methionine, but methionine cannot be synthesized from cystine (Kidder, 1947). On the other hand, Tetruhynzena has lost the ability to deacetylate tryptophane (Dewey, Kidder, and Parks, 1951). N-acetyl-tryptophane is without activity, whereas it has been reported to replace tryptophane for mammals. This does not mean that Tetrahymena has lost its deacetylating enzymes, however, as N-acetyl-leucine and N-acetylmethionine were able to replace leucine and methionine respectively. In addition to the essential amino acids, which must be balanced correctly for most efficient utilization by the cell (Kidder and Dewey, 1947b ; Dewey, Parks, and Kidder, 1951) , certain non-essential amino acids prove to be somewhat stimulatory. Some growth stimulation can be demonstrated in Tetrahymenu by alanine, aspartic acid, glutamic acid, glycine, and proline (Dewey, Parks, and Kidder, 1951 ; Kidder and Dewey, 1 9 4 9 ~ ) . It would appear that this simply reflects the ability of the organism to incorporate amino acids from the medium and thereby save energy otherwise required for reactions which it is perfectly capable of performing. Investigations of the well-known relationship between arginine, citrulline, and ornithine in the urea cycle have indicated that ornithine and/or citrulline do play a role in the metabolism of Tetrulaywna, and both are synthesized via arginine (Dewey, Kidder, and Parks, 1951). Just what these roles are, however, has not been worked out, although it does not appear to have anything to do with the urea cycle. Ammonia is the nitrogen end product of amino acid metabolism here, and neither urea nor uric acid is formed. Dextrose spares amino acids in the nutrition of Tetrahymenu but no

30

GEORGE W. KIDDER

carbohydrate source is essential to metabolism. Likewise acetate is stimulatory and spares the vitamin protogen, to be mentioned later (Kidder, Dewey, and Parks, 1950). Inorganic requirements are difficult to determine due to traces of various minerals found in amino acids and in other supposedly pure constituents of the medium. Where high requirements exist, the detection of essential ions becomes possible. It has been shown, for instance (Kidder, Dewey, and Parks, 1951), that phosphate, magnesium, copper, iron, and potassium are required. Likewise it can be demonstrated that calcium is non-essential. In the case of calcium, removal by oxalate or citrate can be accomplished. Citrate and oxalate inhibition can be completely overcome by additions of magnesium but not by calcium. Unlike the mammal, Tetrahymena is dependent upon an exogenous source of pyrimidine and purine (Kidder and Dewey, 1945a; 1948; 1949a, b ; Kidder, Dewey, Parks, and Heinrich, 1950). Of the naturally occurring pyrimidine bases only uracil will fulfill the requirements of this organism. Cytosine deaminase is lacking as are also enzymes for the decarboxylation of orotic acid. Cytidine deaminase is present, however, as cytidine is as active, on a molar basis, as is uracil or uridine. There is good evidence to show that thymine is synthesized from non-pyrimidine sources, as neither thymine or thymidine will spare uracil. Guanine is an absolute requirement for Tetruhymena, and no other naturally occurring base can act as a substitute. Adenine and hypoxanthine spare guanine, while xanthine and uric acid are both inert. I n the purine system the sugar-base linkage does not appear to be the limiting factor, for adenosine cannot be converted to guanosine. Xanthine oxidase appears to be lacking in Tetrahymena as evidenced by two observations: first, 2-aminopurine is inert and it has been found that xanthine oxidase converts this compound to guanine (Lorz and Hitchings, 1950) ; second, hypoxanthine is as active, on a molar basis, as is adenine in sparing guanine. This indicates that all the hypoxanthine is being aminated to adenine and none oxidized to the inert xanthine. Advantage has been taken of the knowledge of the enzyme patterns of Tetruhymena in purine metabolism to pilot experiments which have revealed a qualitative biochemical difference between certain neoplastic cells and normal cells of mice (Kidder, Parks, and Woodside, 1949). When one considers the vitamins, it becomes apparent that Tetrahymenu, like higher animals, has lost enzymes for the synthesis of the major B-group (Kidder and Dewey, 1951). An exception is biotin. No requirement for biotin exists and, what is more conclusive, raw

ENZYMIC CAPACITIES AND CELL NUTRITION I N ANIMALS

31

egg white and crystalline avidin have no inhibitory effect. Another exception is vitamin BIZ. It can be shown by appropriate assay techniques that vitamin BIZ activity increases in a culture of Tletrahymena, originally devoid of BIZ. Like all animals so far studied, Tetrahymena is dependent upon an exogenous source of PGA (Kidder and Dewey, 1947d, 1949d). It lacks enzymes for coupling the pteridine portion of the molecule to the p-aminobenzoyl-glutamic acid portion, and also lacks the necessary enzymes for the peptide linkage between the carboxyl of pteroic acid and the amino group of glutamic acid. Like higher animals, and distinctly in contrast to bacteria, Tetrahymenu possesses conjugases which enable it to utilize conjugated PGA such as the tri- and heptaglutamates. Recently it has been shown that what has been called the citrovorum factor (Sauberlich and Baumann, 1948) is a substituted PGA. This substance was shown by Broquist, Stokstad, and Jukes (1950) and others to be far more effective in releasing the inhibitory action of 4-amino PGA or aminopterin in chicks than was PGA itself. This and many other observations make it seem probable that PGA may not be the active molecule after all but must be changed to citrovorum factor (CF), which in turn becomes a part of the coenzyme-enzyme complex leading to a vital reaction. I n as much as PGA can be utilized by animals, they must pbssess the enzyme necessary for changing it to CF. The recent work of Shive, Bardos, Bond, and Rogers (1950) has indicated that C F (which they call folinic acid) may be a formylated PGA, unsaturated beyond the condition of PGA itself. W e can say, therefore, that animals including Tetrahymenu have retained the enzymes necessary for this formylation and dehydrogenation while these enzymes have been lost in the bacterium, Leuconostoc citroaorum. Tetrahymena is dependent upon a new member of the B vitamin complex which has been called protogen (Stokstad, Hoffmann, Regan, Fordham, and Jukes, 1949). This factor appears to be identical with the socalled acetate factor and the pyruvate oxidase for certain lactic acid bacteria (Snell and Broquist, 1949). That it functions during enzymatic oxidation of pyruvate to acetate can be shown manimetrically, and minute amounts of protogen dispel the, acetate requirement in Lactobacilli I n our animal system, however, protogen is active in one or more additional vital reactions since the addition of acetate only spares, never replaces protogen (Kidder, Dewey, and Parks, 1950). It may be that protogen is more active than even vitamin BIZ, and so far conditions of protogen depletion have not been attained in higher animals. Assays of various

32

tiEORGE W. KIDDER

plants and animals show it to be very widespread in nature (Keevil, 19.50), and it can be safely predicted that this factor will be found to function in more organisms than Tetralaymena and the lactic acid bacteria.

REQUIREMENTS OF HIGHER ANIMAL CELLS 111. NUTRITIONAL I would like to consider briefly the implications of what I have discussed on the nutrition of higher animal cells. I believe we should draw a sharp distinction between nutrition in a higher organism and nutrition in any of its isolated tissue cells on the basis of enzymatic capacities. W e know, for example; that the enzyme complex of cells of the liver are far more elaborate than those of most other tissues. There is, in fact, a segregation of enzymatic capacities during histogenesis so that the cells of one set of tissues develop certain enzymes while other cells develop other sets. In the intact animal, therefore, the nutritional requirements reflect the lack of synthetic ability after all the synthetic enzyme systems of the different tissues have been added together. W e know, for instance, that cells in tissue culture appear to require heat labile fractions from serum and/or embryo juice. If the embryo juice is heated, certain necessary configurations appear to be destroyed. And yet we know there is no such requirement for heat labile compounds in the intact aseptic animal. The group at Notre Dame (Reyniers, Trexler, and Ervin, 1946; Reyniers, Trexler, Wagner, Luckey, and Gordon, 1947) have shown that rats, and other animals, can be grown in the absence of other organisms on heat-sterilized foods. The biochemical and metabolic differences exhibited between cells of a higher organism when in Vivo and iiz vitro may be accounted for by the segregation of synthetic enzyme systems. Thus tissue 1 may synthesize compound A for itself and other tissues. Tissue 2 may synthesize compound B and utilize performed compound A. So in the whole economy of the organism this segregation of enzyme systems has resulted in a strict symbiosis. If this is so, then, with our present limited knowledge of the complex biologically important compounds, successful tissue cultures may be expected on chemically defined media, only when a sufficiently large number of representative tissue types are allowed to contribute one to the other. IV. CONCLUSION In spite of the apparent pessimism about the growth of animal tissues in chemically defined media, I believe that serious attempts should be continued. Drawing on our experience of many years with Tetrahymena and our experience of only a year with attempts at tissue culture, I would

ENZYMIC CAPACITIES AND CELL NUTRITION I N ANIMALS

33

like to suggest what I believe to be of prime importance for future trials. It would seem logical that an attempt should be made to supply the tissues with all known active compounds. In looking over the various formulas for tissue culture media, three types of compounds are most frequently missing : vitamin BIZ, protogen, and pyrimidine and purine bases. Moreover I would suggest the substitution of citrovorum factor for PGA, on the chance that your type of tissue lacks the specific formylating and dehydrogenating enzymes necessary for the conversion. I believe it would be wise to substitute pyridoxal and pyridoxamine for pyridoxine for the same reason. In addition to purine and pyrimidine bases I would suggest the use of both ribose and desoxyribose nucleosides, again to guard against the lack of enzymes for the sugar-base linkages. Finally I would suggest the use of the natural configurations of the amino acids used. It has been found for Tetrahynzena (Dewey, Kidder, and Parks, 19Sl), as has been found in other systems, that a number of the D-amino acids are inhibitory, and that this inhibition occurs even at the fifty-fifty ratio of the racemic mixture. D-histidine, D-leucine, and D-serine are strong growth inhibitors. The stakes are high, for if the various normal and abnormal tissues can be handled quantitatively in a chemically defined medium, many of the problems of animal biochemistry and metabolism will be simplified. V. REFERENCES Broquist, H. P., Stokstad, E. L. R., and Jukes, T. H. (1950) Fed. Proc., 9, 18. Dewey, V. C., Kidder, G. W., and Parks, R. E. (1951) J . biol. Chew. (In press.) Dewey, V. C., Parks, R. E., and Kidder, G. W. (1950) Arch. Biochem., 29, 281. Keevil, C. S., Jr. (1950) Unpublished Thesis, Amherst College, Massachusetts. Kidder, G. W. (1947) Ann. N. Y. Acad. Sci., 49, 99. Kidder, G. W., and Dewey, V. C. (1945a) Arch. Biochem., 8, 293. Kidder, G. W., and Dewey, V. C. (1945b) Arch. Biochem., 6, 425. Kidder, G. W., and Dewey, V. C. (1945~) PhySioZ. Zodl., 18, 137. Kidder, G. W., and Dewey, V. C. (1947a) Proc. nut. Acad. Sci., Wash., S, 95. Kidder, G. W., and Dewey, V. C. (1947b) Proc. nut. Acad. Sci., Wash., 59, 347. Kidder, G. W., and Dewey, V. C. (1948) Proc. nat. Acad. Sci., Wash., 34, 566. Kidder, G. W., and Dewey, V. C. (1949a) J. biol. Chem., 178, 383. Kidder, G. W., and Dewey, V. C. (1949b) J. biol. Chem., 179, 181. Kidder, G. W., and Dewey, V. C. (1949~) Arch. Biochem., 20, 433. Kidder, G. W., and Dewey, V. C. (1949d) Arch. Biochem., 21, 66. Kidder, G. W., and Dewey, V. C. in Biochemistry of Protozoa (1951) A. Lwoff, ed. Academic Press, N. Y. Kidder, G. W., Dewey, V. C., and Parks, R. E. (1950) Arch. Biochem., 27, 463. Kidder, G. W., Dewey, V. C., and Parks, R. E. (1951) Physiol. Zoiil., 24, 69. Kidder, G. W., Dewey, V. C., Parks, R. E., and Heinrich, M. H. (1950) Proc. nut. Acad. Sci., Wash., 86, 431.

34

GEORGE W. KIDDER

Kidder, G. W., Dewey, V. C., Parks, R. E., and Woodside, G. L. (1949) Scictice, log, 511. Kidder, G. W., and Fuller, R. C. (1946) Science, 104, 160. Lorz, D. C., and Hitchings, G. H. (1950) Fed. Proc., 9, 1Y7. Reyniers, J. A., Trexler, P. C., and Ervin, R. F. (1946) Lobund Repts., No. 1, Notre Dame, Indiana. Reyniers, J. A., Trexler, P. C., Ervin, R. F.,Wagner, M., Luckey, T. D., and Gordon, H. A. (1947) Lobund Repfs., No. 2, Notre Dame, Indiana. Sauberlich, H. E., and Baumann, C. A. (1948) J . b i d . Chem., 176, 165. Shive, W., Bardos, T. J., Bond, T. J., and Rogers, L. L. (1950) J. Amer. Chem. SOC.,72, 2817 Snell, E. E., and Broquist, H. P. (1949) Arch. Biochem., 23, 326. Stokstad, E. L. R., Hoffmann, C. E., Regan, M., Fordham, D., and Jukes, T. H. (1949) Arch. Biochcm., 20, 75. Stokstad, E. L. R., and Jukes, T. H. (1950) Proc. SOC.exp. B i d . Med., 73, 523.

The Application of Freezing and Drying Techniques in Cytology L. G. E. BELL Department of Zoology, King’s College, University of London

CONTENTS Page I. Introduction .......................................................... 35 11. Method .............................................................. 36 1. Quenching ...................................................... 36 2. Drying ............................ ......... ...... 41 3. Microtomy . . . . . . . . . ............... 50 111. Comparison with Histologica ............... 53 IV. Advantages of Freezing and Drying Techniques .......................... 57 V. References ...............................................

I. INTRODUCTION The method of dehydrating a material by a process of vacuum desiccation at a low temperature is of great scientific and industrial importance. The biochemist and pharmacist use the method on a large scale in the preparation of proteins, hormones, and other labile products. The bacteriologist uses the method extensively in the preservation of cultures. These applications are discussed by Flosdorf (1949). More recently physiologists have been using the method in an attempt to preserve life in animal cells (Smith and Polge, 1950). The cytological use of the method seems to have been among the earliest applications and to have developed independently of the other and later uses. Leeuwenhoek was among the first workers to dry tissues for microscopical examination, but it was Altmann (1890) who originated the method in use today. His procedure was to freeze small pieces of tissue and to keep them over sulfuric acid in vacuo at a temperature of -20°C for some days. The tissue was then infiltrated with xylene or paraffin wax in wacuo. The sections from such material were then floated on to fixative solutions to study the action of fixation. Altmann recommends as low a drying temperature as possible. H e remarks that the method is difficult and time consuming. Early workers in cytochemistry were well aware of the criteria for accurate, work and the artifacts of chemical fixation. Among these was Mann (1902) who used Altmann’s method and recommends it for cytochemical work. H e introduced cooling the tissue in alcohol cooled by a mixture of solid carbon dioxide and alcohol, but largely negatived this

35

36

L. G. E. BELL

improvement by quenching whole frogs and afterwards breaking up the animal into small pieces for drying. H e used a drying temperature of -30°C. Bayliss (1915) dried tissue at -35°C to be below the eutectic temperature of the tissue salts. H e was discouraged from using the method as his sections disintegrated when brought into contact with water. Other authors mention the method (Romeis, 1932) , but little was done until Bensley and his co-workers elaborated and improved the method (Gersh, 1932). Since the publication of Gersh’s paper the method has been used extensively in the United States (Scott, 1933; Goodspeed and Uber, 1934; Hoerr, 1936 ; Simpson, 1941a. I n other countries development has been slower. Lison (1936) mentions the method but says it is too specialized for general use. Scandinavian workers have used the technique (HydCn, 1943 ; Sjostrand, 1944). Recently, Reed and Udall (19SO), quoting unpublished experiments, refer to the results of freeze drying as disappointing, but from the few details given in their paper, it is clear that they are not acquainted with recent work. The increasing availability of the necessary physical equipment is an important factor in the wider use of the method. 11. METHOD The procedure of cytological freezing and drying is conveniently divided into three stages: The initial sampling and cooling of the tissue, the vacuum desiccation, and the preparation of the dried tissue for microscopical examination. These steps will be considered separately.

1. Quenching This involves taking a sample of tissue and immersing it in a cold bath. By this means, all chemical reactions and diffusion of substances in the tissue are slowed down to a minimum. Material such as protein and salt which is dissolved in the water of the tissue is precipitated from solution at the crystal boundaries of the ice crystals formed from the free water of the tissue. This precipitation of material means that the more minute the ice crystals formed in the tissue, the more faithfully will the structure of the frozen tissue reflect that of the original sample. The larger the ice crystals, the larger the distance dissolved material will be moved from its original site to be precipitated at the crystal boundaries. In a fixed volume of a solidifying liquid the number of crystals formed is directly proportional to the number of nuclei of crystallization and inversely proportional to the rate of growth in size of the crystals. The larger the number of crystals in a given volume, the smaller will each

FREEZING AND DRYING TECHNIQUES I N CYTOLOGY

37

crystal be. This means that a high rate of nucleation and a low rate of growth is desirable to obtain small crystals. Luyet (1938) has shown that the rate of growth of ice crystals in gelatin-water mixtures is reduced by twice for 1 per cent gelatin to three hundred and fifty times slower than pure water for 3 per cent gelatin. It is to be expected that the protein material in tissues will be able to slow the rate of growth of ice crystals in a similar way. The growth-retarding action of material in solution depends on its concentration and its structure. At certain concentrations, many substances have such a retarding action that crystal growth is entirely inhibited, and a solid with a subcrystalline or vitrified structure is obtained on cooling. The mechanism of retardation involves the binding of water by hydration of the dissolved molecules (Luyet, 1938), the adsorption of the precipitated material at the growing crystal boundaries (Adam, 1941) and the dissolved molecules hindering diffusion of water from the liquid phase to the growing crystal nuclei. Though the growth-retarding action of tissue substances is not sufficient to prevent the formation of ice crystals by itself, it is probable that it influences their final size and, as will be discussed later, the growth involved in recrystallization. The most important factor under the control of the experimenter is the rate of nucleation. It is first necessary to consider the extent to which water can be cooled without crystallization. Kistler (1936) has shown that small droplets of water can be cooled to about -35°C before crystallization begins. Similar critical temperatures are found by Cwilong (1945) and Schaeffer (1946). Using this value of -35"C, Fisher, Holloman, and Turnbull (1949) have shown that the rate of nucleation of water is very small up to the critical temperature and then increases very rapidly. Between -33°C and -43°C the rate of formation of new crystal nuclei is increased by a factor of lo1*. These considerations show that the more quickly the tissue can be cooled to well below the critical temperature, the finer the ice crystal structure. If a system can be cooled quickly enough to a temperature where the rate of nucleation is very large or infinite, a non-crystalline or vitrified structure is formed, even in the absence of dissolved material. If the concentration of dissolved substances is insufficient by itself to inhibit crystallization entirely, it is clear that it may slow crystal growth enough to enable the temperature to be reached where the rate of nucleation is very great, before the solution has solidified. This is confirmed by the work of Luyet, who shows that it is very difficult to vitrify pure water, but much easier to vitrify solutions of sugars and glycerine.

38

L. G. E. BELL

Factors which govern the rate of cooling and hence the rate of nucleation are the thermal conductivity of the cooling bath and the temperature difference between the tissue and the bath. A high conductivity and a large temperature difference are desirable. These criteria have been emphasized by Scott (1933), Hoerr (1936), and Simpson (1941a). The earlier work by Gersh and others used liquid air directly as the cooling bath. This was satisfactory in establishing a large temperature gradient, but had a very poor conductivity because of the formation of a film of vaporized air around the specimen. Gersh (1932) used large pieces of tissue, 0.5 by 0.5 by 1 cm., in which case the conductivity of the tissue becomes a limiting factor. The results obtained with such pieces do not seem to have been very good as Bensley and Gersh (1933) say “The quality of fixation by this method is much better than might be expected and can be compared favourably in some cases with that obtained by ordinary histological fixatives.” Hoerr ( 1936) mentions that he obtained improved results in quenching by wrapping the piece of tissue in liver or spinal cord. Simpson (1941a) confirms this and further shows that if a large enough piece of tissue is used, the crystal artifacts form three zones, an outer zone with a well-preserved structure, an intermediate zone very badly preserved, and an indifferently preserved inner zone. Simpson is inclined to ascribe the very bad intermediate zone to the effect of the pressure exerted by the outer hard shell of frozen tissue, It is possible, however, that this intermediate zone reflects the conditions where the solidification temperature is too high for rapid nucleation, but the rate of crystal growth is great, while the temperature in the inner zone is too high for rapid nucleation and also too high for the rate of crystal growth which gives the large artifacts of the intermediate zone. These circumstances would give the observed large ice crystal artifacts of the intermediate zone and the smaller artifacts of the inner zone. Evidence for this interpretation comes from the demonstration by Simpson that the extent of the three zones could be varied by altering the temperature of the cooling bath. With selected temperatures, both the outer and intermediate zones could be eliminated. Using a bath at -90°C only the inner zone was obtained. As Simpson points out, for cytological work it is best to take such a size of sample and to use such a cooling bath as will give only the outer zone type of structure with artifacts of minimal size. In order to improve the conductivity of the cooling bath, various liquids have been used. Scott (1933) used ethyl alcohol at -100°C. H e says better results were obtained than with liquid air alone. The disadvantages of this liquid are that it solidifies near -115°C and that near its melting

FREEZING AND DRYING TECHNIQUES I N CYTOLOGY

39

point is a viscous liquid, which means that stirring is inefficient. Hoerr (1936) uses pentane (m.p. -131"C), and impure isopentane, which remains liquid until -190"C, cooled with liquid nitrogen. He says that the best results were obtained with a bath at -190°C. Simpson (1941a) confirms this. Emmel (1946) lowers the melting point of pure isopentane by dissolving dry butane in it. The author has found that the addition of three volumes of propane to one volume of isopentane gives a bath that is still very fluid at -190°C; even better is propane by itself, with a melting point near -185°C. By quenching a small piece of tissue in these low temperature baths it is possible to obtain a structure which does not show ice crystal artifacts with the light microscope. Simpson (1941a) recommends that the bath temperature should be at least -165"C, the melting point of pure isopentane, and preferably as near -190°C as possible. H e says that above these temperatures the next best result was obtained by using a bath a t --6o"C when the quality of fixation was that of his inner zone. Pease and Baker (1949), quenching small pieces of muscle in liquid air, found no advantage in using isopentane. Hoerr (1936) emphasized that the cooling of such inflammable liquids as isopentane by liquid air was attended by great hazard and said it was much safer to use liquid nitrogen. If liquid nitrogen is not obtainable, by using only metal Dewar flasks and metal tubes, with extreme care it is possible that liquid air could be safely used with these liquids. The arrangement for a cooling bath can be very simple, consisting of a wide test tube containing the isopentane supported in a Dewar flask containing liquid nitrogen. A simple stirrer is needed for the isopentane. %hen using pure isopentane an indication of the bath temperature is given by the presence of solid isopentane at the bottom of the tube. With mixed baths it is necessary to use a thermo-couple to measure the bath temperature, if it needs to be known accurately. The piece of tissue can be dropped in the bath or preferably plunged in on a piece of metal gauze or strip (Gersh, 1932) or cardboard (Simpson, 1941a). Long narrow forceps make a convenient tool for submersing the specimens. The different thermal contractions of metal and tissue release the specimen once it is in the bath. Some authors use metal baskets for the specimens (Pease and Baker, 1949; Sjostrand, 1944), but the use of a container must hinder the rapidity of the quenching. Once the specimens are quenched they are allowed to stay in the bath until transference to the drying apparatus, or stored a t solid carbon dioxide and alcohol temperatures (Mendelow and Hamilton, 1950). It is essential that the specimens do not warm up beyond the selected drying temperature.

40

L. G . E. BELL

The most perfect quenching can only preserve the structures present in the tissue the instant before it enters the bath. I t is therefore necessary to obtain the sample as rapidly as possible to minimize post-sampling artifacts. Bartelmez ( 1940), in an investigation using both chemical and freezing drying fixation on uterine epithelium demonstrated that cellular material shows aggregation and vacuolation within thirty seconds after removal from the animal. This emphasizes the need for rapid sampling of fresh material if the advantages of the freeze drying method are not to be wasted. Some authors (Hoerr, 1936; Simpson, 1941a) have sought to improve the quality of the fixation by withholding water from the experimental animal, supposing that the partially dehydrated tissues, having a lesser amount of water than usual, would quench more satisfactorily. This may be so, but it raises the problem of evaluating changes in the material induced by this procedure, and the method cannot be recommended as a general procedure. Most animal tissues can be quenched so as to give negligible ice crystal artifacts. The least satisfactory results are obtained with loose connective tissue, nervous tissue, testis, and bone marrow (Simpson 1941a). With such tissues it is important to use as small a sample as possible and to use a cooling bath near -190°C. The author has obtained the best results with testis by making a thick smear, about one tubule thick, on thin celloidin and quenching strips of this material in propane at -185°C. Goodspeed and Uber (1934) quenched anthers and root tips in liquid air. They reported a coarse reticulate structure at the exterior of the specimens with a finer structure inside. It seems that their material had the characteristics of the intermediate zone and inner zone of Simpson. It is probable that the cellulose cell walls of plant material act as thermal insulators and make efficient quenching more difficult. It is possible to obtain good results, using small samples and an isopentane bath. Quenched specimens are transferred to the drying apparatus in gauze baskets, on small trays or by a spatula, all of which must first be cooled to the temperature of the quenching bath. It is not necessary to drain off all the adhering isopentane from the specimens, as most pumping systems can deal with a small amount, but it is better not to carry over large amounts on the specimens. The transference must be done rapidly into an apparatus at or below the selected drying temperature. Hoerr (1936) recommends that the specimens should be warmed up rather slowly to the drying temperature and says this gives better results. Simpson (1941a) used a procedure of freezing and substitution to dis-

FREEZING AND DRYING TECHNIQUES I N CYTOLOGY

41

tinguish artifacts of quenching from those of later stages. This procedure consists of placing the quenched specimen in water-soluble organic solvents at temperatures of from -40°C to -78°C. Methyl cellusolve and ethyl alcohol were among those used. These solvents dissolve the ice out of the specimen, and on warming up to room temperature the specimen retains those distortions originally caused by the quenching. It is not certain at what stage this substitution of alcohol for water occurs. It may well take place during the warming up stage. This means diffusion artifacts are possible and that the fixation is essentially chemical. For morphological studies it can be used as a second best to complete freeze drying. The author has found it useful as a quick method of checking the quality of quenching of difficult material. 2. Drying

A critical factor in drying is the temperature. Altmann dried his material at -20°C and suggests -30°C would be better. Mann (1902) dried at -30°C; Bayliss (1915) selected a temperature of -35°C with the intention of operating below the eutectic temperature of the tissue salts. Gersh (1932) used -2O"C, Goodspeed and Uber (1934) -3O"C, as have many later workers. Scott and Hoerr (1950) recommend temperatures below -30°C as giving better results. There are not sufficient quantitative physical data concerning the various factors which influence drying temperature to enable a drying temperature to be selected theoretically. It is, however, instructive to consider the qualitative aspects of this problem as they must be taken into account in evaluating the results of this method. The structure of the specimen immediately after efficient quenching will be that of a vitrified or a microcrystalline solid. It is well known, and the fact is used. extensively in metallurgy, that such a quenched structure is very unstable. The changes that tend to take place are: devitrification, recrystallization to relieve the mechanical stresses of the sudden contraction in quenching, and crystal growth. The magnitude of such changes depends on the increase in temperature of the specimen and also on the amount of material present other than the ice. Luyet (1938) says that a vitrified 1 molar solution of sucrose devitrifies in 10 seconds at -26"C, 60 seconds at -3O"C, and is stable at -35°C. H e further says that with ten or less molecules of water to one of sucrose there is no devitrification, but with higher concentrations of water devitrification starts at -50°C. The same type of result is obtained with other solutions. The action of the dissolved substances is the same as in quench-

42

L. G. E. BELL

ing, namely to hinder the growth of ice crystals. It seems likely that recrystallization and increase in size of ice crystals will not become appreciable until the specimen warms to the devitrification temperature. The extent of such recrystallization which may involve the movement of material at the boundaries of the old crystals will depend therefore on the temperature of the specimen and the length of time it is kept at that temperature. The devitrifying temperature of protoplasm is not accurately known. Burton and Oliver (1935) give the devitrifying temperature of pure water as -8O"C, so it may be taken that that of protoplasm is above this temperature. Recent work which has a bearing on this problem is that of Hazel, Parker, and Schipper (1949). This work concerns the behavior of quenched silica sols. When allowed to thaw rapidly from the quenched state to room temperature, the colloidal system remains stable, but holding the quenched specimen at any temperature above -55°C produces a coagulation of the sol. This is explained by the authors as being due to a recrystallization to a more stable crystal lattice at -55°C. It seems likely that some of the coagulation results from the concentration of the colloidal particles at the crystal boundaries. The authors say that the extent of coagulation is modified by the presence of salts in the solution. It is certain that the structure of quenched protoplasm will be influenced by similar changes. It is not yet possible to evaluate these changes quantitatively, but a drying temperature near -55°C seems to be indicated by these considerations. Another factor influencing the drying temperature first mentioned by Bayliss is the melting point of the eutectic mixtures in the tissue. Scott and Hoerr (1950) point out that the eutectic temperatures for such a complex mixture as protoplasm are very difficult to arrive at. The binary eutectic which may be present in the tissue, with the lowest melting point, is that of calcium chloride and water, with a melting point of -54.9"C. The tissue eutectics will be more complex tertiary and quaternary systems, which generally have lower eutectic temperatures than simple binary systems. Workers studying salt distribution in tissues have generally dried below -55°C in order to minimize diffusion of material which might occur if liquid eutectic was formed. Scott has shown that tissue dehydrated below -30°C shows a different ash distribution from that dehydrated a t -2WC. Why -30°C should be a critical temperature is not clear, if only euctectic mixtures are considered, but if another factor-recrystallization-becomes appreciable above this temperature, this would help to explain the result. Scott (1943) uses a drying temperature of -63°Cin his micro-incineration studies. It is not likely that

FREEZING AND DRYING TECHNIQUES IN CYTOLOGY

43

eutectics will be present in quenched tissue as they represent an equilibrium condition, and the constituents of the tissue do not have time to reach equilibrium during the very rapid quenching process. Much of the salt content of protoplasm is likely to be closely associated with protein, and the amount of free salt available for eutectic formation may be quite small. This consideration may explain why Pease (1947) was unable to find free salt on electron microscope examination of frozen and dried myosin from potassium chloride solutions. His explanation of the lack of free salt deposits is that during the dehydration at -72°C free ions become exposed and evaporate. It is true that at low temperatures the ionization of substances is depressed and the attractive forces may be sufficiently modified to enable evaporation to take place, but further experimental evidence is needed before the explanation of Pease can be accepted. H e suggests that experiments with radioactive salts should settle the problem. Most workers who are concerned with the larger molecules in tissues and the preservation of cytological structure seem to agree that drying at or below 4 ° C gives superior results to drying at higher temperatures, but that still lower temperatures prolong the drying time with no great improvement in the quality of the preparation. Much published work is based on material dried at -30°C. The work previously cited of Hoerr, Simpson, and others shows that the results obtained on material dried at -20°C or above are not of good quality. It is important to remember that due to the poor conductivity of the tissue and the use of high vacua in drying that it will be difficult to maintain a specimen accurately at any drying temperature. Evaporation of the ice during drying will cause the surface of the specimen to be cooler than the bulk of the tissue. However, it is possible to ensure that a specimen is not exposed to a higher temperature than the selected drying temperature during dehydration, even if, in fact, its actual temperature is lower. Details of the drying apparatus used by earlier workers are not available. The task of the drying apparatus is to remove the water from a previously quenched specimen held at the selected drying temperatiure. A drying apparatus must have provision for maintaining the drying temperature and for removing water vapor from the gas space of the apparatus. The most useful range of drying temperatures is from -30°C to -70°C. Temperatures down to -40°C can be attained by mechanical refrigerators, but the refrigerator needs to be of exceptionally good design and workmanship to give trouble-free operation. Goodspeed and Uber (1934) use a refrigerator at -30°C, Sylvdn (1950) has a re-

44

L. G. E. BELL

frigerated dryer running at - 4 7 " C , a commercial dryer, Aloe Co. (1947), uses a refrigerating unit. Gersh (1932) uses a method of boiling a volatile liquid under a controlled pressure. H e uses liquid ammonia boiling at 1304 mm. Hg, which gives -20°C. Harris, Sloane, and King (1950) use methyl chloride boiling at 350 mm. Hg, to give a temperature of -50°C. These liquids are contained in a jacket around the drying chamber. The disadvantages of this method seem to be mainly those of ventilation and the complication of the ancillary apparatus. A method first used in freezing drying by Mann (1902) is the use of a mixture of ethyl alcohol and solid carbon dioxide. This mixture gives a temperature of approximately -78°C. It is a stable mixture when contained in a Dewar flask and will remain cold as long as there is solid carbon dioxide present. While there is still carbon dioxide in the mixture it is non-inflammable. The temperature of the mixture is too low for most freeze drying, and the apparatus must include a method of warming the drying chamber or specimens to the drying temperature. Packer and Scott (1942) use a vacuum jacket around the drying chamber which reduces the heat conduction between the bath and the chamber. With a pressure in the 0.5-inch gas space of 1 mm. Hg, they obtain a temperature at - W C in the drying chamber. Their apparatus is so designed that an electric heater in the vacuum jacket could be used to raise the drying chamber temperature if necessary. An apparatus manufactured by W. Edwards and Co. uses an electric heater inside the drying chamber controlled by a bimetallic strip thermostat mounted on the heater support. An ingenious method of controlling the temperature of the drying chamber is to use the melting point of a low melting point liquid. Mendelow and Hamilton (1950) use a paste of solid and liquid ethyl oxalate which maintains a temperature of 4 C so long as there is solid present. These authors say that an occasional addition of a small quantity of solid carbon dioxide is sufficient to maintain some solid ethyl oxalate in the mixture. The method is obviously applicable to other temperatures with suitable liquids, e.g., nonane m.p. --53"C, isopropyl ether m.p. -60°C. This method is certainly the simplest and most economical to apply. Solid carbon dioxide is a fairly cheap and easily available material in all countries where ice cream is manufactured. As mentioned previously, the most that can be expected of a temperature control under the conditions in a freeze dryer is that the specimen is not allowed to warm up above the drying temperature. Its actual temperature may well be lower than the temperature of the heater.

FREEZING AND DRYING TECHNIQUES I N CYTOLOGY

45

The desiccation of the specimen depends on the fact that solids have a vapor pressure of their own molecules in equilibrium with the solid. If the vapor pressure of a substance in the atmosphere over a solid of that substance is greater than the equilibrium pressure at that temperature, then material will pass from the vapor to the solid. This process is condensation. If the vapor pressure is less than the equilibrium vapor pressure, then some solid will pass to the vapor. This is evaporation. Material is actually always evaporating, but at equilibrium the same amount is also condensing, so the net change is zero. If the vapor pressure above a solid is maintained by some means below the. equilibrium pressure for any temperature, then the solid will evaporate completely. Packer and Scott ( 1942), using simple kinetic theory for evaporation rates, work on the assumption that the desiccating action involves pumping water vapor from the specimen. This leads them to conclude that high capacity oil diffusion pumps are necessary. The diffusion pump on their analysis is needed to reduce the pressure in the system below the vapor pressure of the specimen so that negligible recondensation of water takes place. The high capacity is needed because they consider pumping the water vapor from the specimens expanded to the pressure of the system. This gives very large volumes of water vapor to be handled by the pumps. They calculate the volume of water vapor evaporating from a square centimeter of ice at 4 0 ° C and at the pressure in their apparatus would be 1600 liters per second. This is more than a hundred times the capacity of the diffusion pumps used, as the authors point out. They ascribe the ability of their apparatus to handle the water vapor to unknown factors slowing the diffusion of water vapor from the specimen. They regard the traps of phosphorus pentoxide and of carbon dioxide alcohol as protection for the pumps. It seems, however, that it is these traps which actually do the drying by removing the water vapor from the system. It appears to the author that the above analysis neglects several factors, the main one being that the essential point in drying specimens is to have in the system a trap which continuously removes water from the gas space of the system. This trap may be either chemical, such as phosphorus pentoxide, which combines chemically with the water, or a surface held at such a low temperature that water condensing on it has an extremely low vapor pressure so that re-evaporation is negligible. Such temperatures are obtained by using liquid air or carbon dioxide alcohol mixtures. The other main factor to consider is that at the low pressures employed the distance that a molecule in the gas space can travel before hitting another molecule, known as the mean free path, becomes com-

L. G . E. BELL

46

parable with the dimensions of the apparatus. If the trap is farther away from the specimens than the mean free path, the rate of drying will be governed by diffusion of water vapor from the specimens to the trap. The greater this distance the longer the process will take, and in some dryers the distance is very great. It is clear that if the trap is brought within the distance of the mean free path from the specimen, most water molecules evaporating from the specimen at the vapor pressure of the specimen will pass directly to the trap without suffering any deflecting collisions. There can be no quicker process of drying than this, in fact it is molecular distillation. The limiting factor then becomes the vapor pressure of the specimens, and this is controlled by the temperature of the specimens. Many workers have designed dryers on the basis of the work of Packer and Scott, using oil diffusion pumps to give very low pressures. The traps are often placed a long distance from the drying chamber and though possibly within the mean free path at the pressures used, the connecting tubing usually has at least two bends in it. This means that a molecule will suffer at least two collisions before reaching the trap, and the drying time will be correspondingly increased. The basic principles of a freeze drier can be best appreciated by considering Fig. 1, which is a cross section of an idea1 dryer. The bottom Top plate

Trap a t -78.C.

G=

-

Bottom plate

To vacuum pump

Specimen at -4O'C.

FIG.1.

is assumed to be kept at the drying temperature and the top at -78°C to act as the trap. As the distance between top and bottom is increased, so must the vacuum be raised to increase the mean free path of the molecules. If the distance between top and bottom plates is 0.5 cm. the vacuum need only be 1W2mm. H g for the water molecules to.leave the specimen and pass directly to the trap. If the distance is increased to 4.5 cm. the vacuum must be lowered to 10-* mm. H g for water molecules to pass directly to the trap. Lowering the vacuum further will not increase the rate of drying which is governed by the vapor pressure of the specimen. If the drying temperature is lowered to --6O"C, the drying time will be correspondingly longer, and it can be calculated from the kinetic equa-

FREEZING AND DRYING TECHNIQUES I N CYTOLOGY

47

tions used by Packer and Scott that ice will evaporate ten times more slowly at -60°C than at -40°C. It is necessary that the trap should have such a small vapor pressure of water that the evaporation from it is negligible compared with the evaporation from the specimen. A trap at -78°C has a vapor pressure of water approximately two hundred times lower than a specimen of ice at -40°C. Details of dryers designed as pumps have been published by Gersh (1932), Packer and Scott (1942), Scott and Hoerr (1950), Harris, Sloane, and King (1950), Sylven (1950), and Pease and Baker (1949). These all employ diffusion pumps. Goodspeed and Uber (1934) and Sjostrand (1944) have built dryers using only rotary oil pumps. The dimensions of their apparatus are much larger than the mean free paths at the operating pressures. Sjostrand dries at a pressure of from to mm. Hg, giving a mean free path of from 0.5 to 5 cm. The trap is at least 50 cm. away from the specimen and is connected at right angles to the drying chamber by a narrower tube. It is not surprising that this apparatus takes from one to two weeks to dry at 4 ° C . The apparatus of Goodspeed and Uber suffers from similar disadvantages and takes two weeks to dry at -32°C. The author has found that an apparatus manufactured commercially (Edwards and Co., 1950) can be used as a short path condensation drier. The quenched specimens are held on trays inside an annular electric heater. The heater is suspended in a glass tube immersed in a carbon dioxide alcohol bath. Using a two-stage rotary pump operating at approximately 5 X lWS mm. Hg, the water vapor leaves the specimens at, say, -40°C and condenses at the bottom of the tube at -78°C opposite the end of the heater, or on a charge of phosphorus pentoxide placed in the bottom. The water does not react with the phosphorus pentoxide at such low temperatures, but when the dryer is allowed to warm up to room temperature when drying is finished the water is absorbed and cannot pass back to the specimens. The drying times with this apparatus are of the order of two or three days at -40°C. This apparatus operates on a short condensation path, but the design does not take into account the full possibilities of molecular distillation. The author feels that evidence supporting the molecular distillation analysis of freeze drying is provided by the recent paper of Mendelow and Hamilton (1950). These authors, using an all metal apparatus, maintain the specimens at -40°C a short distance from a liquid nitrogen cold trap inserted directly into the gas space above the specimens. Using

48

L. G. E. BELL

an oil diffusion pump to give a vacuum of 5 X lCP4 mni. H g they are able to dry their specimens in seven hours, which is a very great decrease in drying time compared with any other published data. The authors, however, do not seem to have appreciated the reasons why their design is so efficient. They follow the reasoning of Packer and Scott and assume it is essential to lower the pressure in the system below the vapor pressure of ice at the temperature of the specimen. They state that in their opinion the short drying time is due to the use of liquid nitrogen as a trap. The author suggests that the reason for the great efficiency of Mendelow and Hamilton’s apparatus is that the vacuum they use of 5 X 1 V mm. H g is more than sufficient to increase the mean free path of the water molecules to the dimensions of the apparatus. This means that most of the water molecules will pass directly from the specimens to the trap without suffering collisions with any other molecule. Further, a surface at the temperature of liquid nitrogen will hold a molecule of water at the first impact without a rebound. The drying times given in the literature vary greatly. Scott (1937) gives three days at -32°C; Emmel (1946) gives one to two weeks at - W C to -78°C; Scott and Hoerr (1950) say several days to weeks at 4 C ; Harris et al. (1950) says two days at -50°C. Factors apart from the apparatus which will influence the drying time are the size and shape of the specimen, and whether many specimens are loaded in one chamber in such a way as to obstruct each other. Hoerr (1936) points out that a limiting factor may be the diffusion of water vapor out of the specimen but says that it is quite rapid. Packer and Scott (1942) emphasize that little is known about this process and suggest that it slows evaporation considerably. They also point out that evaporation produces surface cooling and so slows the process. Gaseous diffusion may not be the controlling factor; as is well known, molecules migrate easily along solid surfaces. Volmer and Estermann (1921) give data I t seems concerning the phenomena with mercury crystals at -63°C. possible that such a migration of water molecules could occur in the solidified tissue giving the result that evaporation occurred mainly from the surface of the block. However, there is as yet no experimental evidence which can help to evaluate the extent to which either process controls the rate of drying. To obtain the best quenching for cytological purposes it is necessary to have a thin specimen. This kind of specimen will also have the best dirhensions for rapid drying. Hoerr (1936) points out that normally all the water is not removed from frozen dried tissue even by running the dryer for four or five days

FREEZING A N D DRYING TECHNIQUES I N CYTOLOGY

49

after completion. H e finds that frozen dried tissue loses from to 1 per cent of its original weight after drying to constant weight in an incubator. Mendelow and Hamilton (1950) have investigated the dryness of a variety of frozen dried tissues. They obtain the loss in weight of the dried specimens after heating to constant weight at 50°C in a vacuum oven. They find that most frozen dried tissues lose between 0.2 and 1 per cent of their original wet weight after heating to constant weight. The most probable source of error in their method is the loss of volatile constituents other than water from the tissue during the heating at 50°C. This error, if present, would make the results obtained for residual water content maximum figures. The completeness of drying will depend finally on the attainment of equilibrium of vapor pressures between the trap and the specimen. When the vapor pressure of the specimen equals the vapor pressure of the trap, no more water will be removed from the tissue by the trap. It is probable that the water left in the dried tissue represents water bound by protein or by salts and will have a very low vapor pressure. For this reason it is best to use a trap having the lowest possible vapor pressure. This consideration means that liquid nitrogen and liquid air traps will dry more completely than any others. However, as the majority of freeze dryers use either phosphorus pentoxide or carbon dioxide alcohol mixtures, it seems that for most practical purposes the degree of desiccation obtained by their use as traps is adequate. The problem of deciding when tissue is dried has been mentioned by numerous workers. The normal vacuum gauges such as the McCleod and Pirani gauges are accurate instruments for true gases but cannot be used to measure water vapor pressures. For this reason they are not usefully employed on freeze driers except as leak detectors. Packer and Scott (1942) applied the method of measuring the pressure differences between two ionization gauges sealed into the apparatus. They assume that when both gauges register the same pressure no more water vapor is being pumped along the apparatus, as there is no pressure gradient. It appears to the author that for this method to be useful the electrodes of the two gauges should be sealed into the gas space of a straight tube for accurate measurements of a pressure gradient. The diagram given by Packer and Scott shows one gauge connected to a wide tube containing the phosphorus pentoxide trap, while the other is connected to a narrower tube leading off the wider tube at right angles. Packer and Scott say that the results of this method are sometimes erratic. In an apparatus with the optimum short distance between the trap and

50

L. G. E. BELL

the specimens, gauges are of little use for determining the dryness of the specimens. Gersh (1947) sums up the situation very well in saying that the time of drying varies considerably with temperature, size, and shape of specimen and that the operator learns empirically when drying should be complete. Most dryers have been constructed of glass. Scott and Hoerr (1950) list the advantages of this material as cheapness, ease of finding and repairing leaks, and reduced adsorption of gases compared with metal tubing. The main disadvantage of this material is fragility, and where an apparatus is to be used by many workers, perhaps inexperienced in handling glass, there is no doubt this outweighs the advantages. The author has found a drier constructed almost entirely of metal very satisfactory in use. Glass is very useful where one end of a tube needs to be kept colder than the other, as metal tubing has too high a thermal conductivity for such a task. The apparatus of Mendelow and Hamilton is constructed entirely of metal except for a glass liquid nitrogen trap.

3. Microtomy The dried material can be treated in several ways. The most widely used method is to inflltrate with wax in vacuo. Many driers can be loaded (Scott, SylvCn, Harris) with degassed wax which is melted when the sample is dry. The sample is then infiltrated directly. There is little doubt that this is the most desirable method. It avoids exposure of the sample to the atmosphere and removes the risk of absorption of moisture or of oxidation. However, it is quite practicable to allow the sample to warm up to room temperature when dry and then to remove it quickly from the dryer to an embedding apparatus, which need consist only of a tube containing degassed wax connected to a vacuum line. The apparatus is evacuated, and the wax is melted by heating the tube in hot water. If the sample is dry, it will sink in the melted wax, and any moisture present will cause vigorous bubbling. Some authors transfer the dried tissues directly to melted paraffin, with or without first warming to room temperature. (Gersh, 1947). This seems more drastic than is desirable, as dropping cold tissue directly into hot paraffin will cause a very rapid rise in temperature. Simpson (1941a) infiltrates the tissue first with ethyl alcohol for a week or more to remove the last traces of moisture not removed by the drying, and to reduce shrinkage in paraffin embedding, Emmel (196) uses four hours in alcohol. Simpson recommends celloidin embedding without the use of heat for the best preservation of general histological detail. The author has found in-

FREEZING AND DRYING TECHNIQUES IN CYTOLOGY

51

filtration with methyl benzoate or methyl benzoate celloidin very satisfactory as a preliminary to the use of some of the ester wax mixtures available. Goodspeed and Uber (1934) remark on the difficulty of infiltrating frozen and dried plant tissue with wax directly. This has also been found by the author who has confirmed that the pretreatment of the dried tissue with butyl alcohol recommended by Goodspeed and Uber is beneficial. I t is not clear why infiltration with wax should be difficult in this case. The surfaces left exposed by the removal of water may be hydrophilic and not wetted by the non-polar wax, or the cellulose cell walls may offer mechanical obstruction to the entry of liquids. Harris et ad. (1950) caution against the use of a high vacuum in degassing the embedding wax. They suggest volatile constituents may be lost, giving a brittle wax which cuts badly. Hoerr (1936) cuts freehand sections of unembedded material. H e shows that his sections cleared in liquid paraffin contain many irregular vacuoles, probably due to ice crystals. Similar sections after thirty seconds exposure to aqueous toluidine blue show no vacuoles. This experiment illustrates the hygroscopic nature of dried tissue and the ability of frozen dried protein to become rehydrated. The rehydration could be prevented by first exposing the material to alcohol. In frozen and dried tissue unaltered by subsequent treatment, the tissue proteins retain a large part of their original solubility. Hoerr noted that this solubility might vary and suggested that the variation was due to the action of the hot paraffin in embedding or the use of petroleum ether to remove the wax from the section. H e therefore used' freehand sections for solubility studies and showed that the solubility decreased after standing at room temperature or paraffin embedding, and more slowly in the refrigerator. (Bensley and Hoerr, 1934). Hoerr has also stated that it is possible to reduce the solubility of tissue protein by the action of sunlight on frozen dried tissue in a vacuum desiccator. It is not certain how far this is an oxidizing or a thermal action, and it does not seem desirable to use it for cytochemical or enzyme studies. Heat also makes frozen dried tissue insoluble, and it is likely that the heat generated at the cutting edge of the microtome knife will have a similar action. Although such treatment may be undesirable for critical solubility studies, the tissue obtained in paraffin sections is still extremely soluble and able to absorb water. Hoerr (1936) shows that when freehand sections of nervous tissue are placed in aqueous fixatives, myelin takes up water rapidly with the formation of myelin figures. Most cytochemical or cytological techniques require the use of aqueous solutions, and the tissue substances must be made in-

52

L. G. E. BELL

soluble before expasure to such solutions. Frozen dried sections disintegrate when flattened on warm water in the usual way. Such sections are best flattened on a warm clean or albumenized slide by gentle pressure of a clean finger or a fine brush. If the ribbon of sections is attached first at one end to the slide and the ribbon is supported at the other end by forceps, it can be lowered on the slide a small portion at a time, starting from the attached end. The ribbon expands just as it comes down on the warm slide and gentle stretching by a fine brush will remove most of the creases from the section. The sections are then consolidated on the slide by gentle pressure. It is possible to flatten paraffin wax sections on a clean warm mercury surface and to remove the sections on a warm glass slide lowered onto the mercury surface, being careful not to trap air between the slide and the sections. Alcohol and diacetin can also be used, but are not so effective. After sections have been flattened by any of these methods, it is best to use gentle finger pressure and a few hours in an incubator to fix the sections to the slide, especially after mercury flattening, and if albumen has not been used. Mendelow and Hamilton (1950) overcome the difficulty of cutting thin sections without creases by cutting single sections and coating the block with paraffin before cutting the next. This gives a greater total thickness of wax which is not easily crumpled. Sections thus mounted are treated in the usual way by removal of wax in a non-polar solvent and passing through ethyl alcohol to water. It is usual to let the sections remain in absolute ethyl alcohol at least fifteen minutes to make the sections reBistant to water. Some authors (Gersh and Catchpole, 1949) keep sections overnight in absolute alcohol. If the tissue has been kept in alcohol before embedding, as in Simpson’s method, the sections may be floated on water. It is not clear what is the action of absolute alcohol on the dried tissue, and it is not known what materials may be removed from the section by this solvent. The action is not the same as that of an alcoholic fixative which involves precipitation and aggregation of material and the movement of substances by diffusion currents. Mancini (1948) avoids the action of alcohol by treating his sections after the removal of wax by the vapor of iodine or a non-polar solution of iodine to reveal glycogen. Unfortunately there are few cytochemical reactions which can be used in this way. It is possible to float the sections onto the fixatives usually used in cytology or cytochemistry, as was originally done by Altmann. The author has used 80 per cent alcohol in this procedure and finds the results with this fixative not so satisfactory as the dry flattening methods. Sec-

FREEZING AND DRYING TECHNIQUES I N CYTOLOGY

53

tions in the water-soluble waxes with the lipoid preserved can be floated on aqueous fixatives such as neutral formalin, which flattens the sections and dissolves the wax at the same time. The use of ultraviolet absorption and phase contrast microscopy enables observations to be made on sections that have had the wax removed by liquid paraffin and are mounted in the same liquid. This material will be freer than most sections from artifacts caused by the action of solvents. This kind of technique should be useful in studying cell structures such as Golgi complexes, which have been criticized as being artifacts, due to the action of some solvents (Palade and Claude, 1949). For ultraviolet absorption microscopy, in order to diminish absorption of ultraviolet light due to scatter at retractive index boundaries, it has been usual to mount frozen dried sections in glycerine. The protein imbibes glycerine and the sharp boundaries of different refractive indices are diminished (Caspersson, 1950). Fulham and Gessler (1946) use a eutectic mixture of camphor and naphthalene as an embedding medium for use in a highspeed microtome and sublime off the embedding medium without the use of solvents. Harris et al. (1950) mount frozen dried sections onto photographic emulsions without the use of solvents and emphasize that only in this way can it be possible to preserve all the radioactive material in the tissue. 111. COMPARISON WITH HISTOLOGICAL FIXATION

Frozen dried tissue, though free from most of the artifacts of chemical fixation, has a structure which reflects the treatment it has undergone. In the initial quenching there may be up to a 2 per cent volume contraction due to the cooling. This contraction can give rise to cracks, especially in large pieces of tissue. These cracks are always easily recognizable and are not likely to be confused with natural spaces in the tissue. The solid elements in the tissue will have been deposited around the boundaries of ice crystals. This means that structure will only be resolvable down to the size of the ice crystals. Any resolution below this size will only reveal the ice crystal artifact. I t is possible to prepare most tissues by freezing drying so that they do not show ice crystal artifacts in the light microscope. It is not known how much orientation the precipitated material will undergo a t the growing crystal face, but it is well known that crystal faces influence the orientation of material deposited on them. In this connection it is interesting to recall that Pease and Baker (1949) state that the birefringence of froien dried striped muscle was much greater

54

L. G. E. BELL

than that of chemically fixed muscle. Luyet (1938) showed that gelatinwater mixtures when quenched in liquid air showed a laminated structure. It is often possible to detect a similar laminated structure in frozen dried tissue with the higher powers of the microscope. It is especially obvious in fluid filled tissue spaces and in the ground substance of connective tissue. The better the quenching, the less obvious is this artifact. It is probably always present, but may be below the size resolved by the microscope. This laminated structure probably owes its origin to the same mechanism which gives a solid eutectic a laminated structure. This involves alternate supercooling and precipitation of material over short distances. For many cytochemical techniques the best cytological preservation is not essential. As long as the artifacts are not resolved at the power of the microscope used, they will not influence the interpretation. The chemical fixation of tissues has always been the object of investigation by histologists who have tried to determine what happens when a piece of living tissue is placed in solutions of protein denaturants and precipitants. Altmann developed the freeze drying technique to investigate this problem. Baker (1945, 1950) gives a review of the classical work of Fisher, Hardy, and others on this problem. Baker emphasizes that to elucidate the action of complex mixed fixatives is very difficult and for this reason advocates the use of simple fixatives where there is a possibility of understanding the mode of action. Cytochemists need fixatives that do not react with the chemical groups or substances they wish to identify. Fixatives such as 80 per cent alcohol and neutral formalin are popular for this reason. Unfortunately cytochemical fixatives often do not preserve structure very well, apart from this disadvantage it is important to realize the existence of others. The object of cytochemistry is to estimate qualitatively and quantitatively, in their natural cytological positions, the various chemical substances occurring in cells. Most cytochemistry is done on dead fixed cells. Ultraviolet microscopy of surviving cells has its own special problems and will not be considered here (Faraday Society Symposium, 1950). Cytochemistry does not require the use of large pieces of tissue. Pieces 0.5 mm. thick or less are usually adequate. Even so, as fixatives penetrate slowly (Bolles Lee, 1950) there is ample time for post mortem changes to occur before fixation is complete. That these post mortem changes may be serious has been shown by Bartelmez (1940). The penetration of fixatives sets up diffusion currents in the tissue. These sweep cell substances into fictitious localizations and concentrations. The

FREEZING AND DRYING TECHNIQUES IN CYTOLOGY

55

case of glycogen is a well-known example. One of the first applications of freeze drying (Gersh, 1932) was to confirm Fisher’s work on the even distribution of glycogen in the liver cell. Mancini (1948)in a comprehensive investigation of glycogen in animal tissues, clearly showed, by comparing frozen dried material with Zenker or Bouin fixed material, that glycogen may even be displaced extracellularly. He showed that frozen dried muscle contains glycogen only inside the fibers, while workers using chemical fixatives (Dempsey, Wislocki, and Singer, 1946) have described intra- and extra- fibrillar glycogen. Mancini further shows the presence of glycogen in skin and fatty tissue, while it could not be detected in similar tissue after chemical fixation. An important aspect of Mancini’s work is his use of non-polar solvents to carry out his glycogen reaction. He is thus able to reduce diffusion artifacts in the test to a minimum. Reactions which can be carried out in non-polar solvents or in strong alcoholic solutions are especially useful in reducing the possibility of diffusion, or removal of water-soluble materials from the specimen during a cytochemical reaction. The reaction of Hotchkiss for carbohydrate can be modified so that all the reagents are in 60 per cent alcohol (Staple, 1949). This reduces the possible diffusion of glycogen and muco protein in a section. Several workers have shown by comparing frozen dried tissue with chemically fixed tissues that materials may be seriously translocated in fixation. Gersh (1933-34)in an investigation of rabbit kidney describes differences in the distribution of uric acid between frozen dried and alcohol fixed tissue. H e finds that frozen dried material shows uric acid in Bowman’s space and only in the lumen of the tubules which all contain it. Alcohol fixed material shows no uric acid in Bowman’s space, little in the lumen of the tubules, and some in the cytoplasm and nuclei of the tubular cells. This work clearly shows the very bad diffusion which may occur with molecules the size of uric acid. With small inorganic ions, diffusion may be even more serious. By dehydrating at a low temperature (-6O’C.) it is possible to prevent the diffusion of inorganic ions up to the time that sections are cut. Scott and his co-workers have worked extensively on the distribution of calcium and magnesium in frozen dried tissue. Scott (1943) has reviewed this work. H e uses the method of micro-incineration and by taking a motion picture of the process shows that the picture remains static during the incineration. H e points out that this means any distortion which occurs is not observable at a magnification of 800x and therefore will not influence the interpretation of the results. Efforts by Gersh to establish unequivocally the site of chloride

56

L. G . E. BELL

and phosphate have been criticized by Scott and Packer (1939) on the grounds that although diffusion is prevented up to mounting the section on the slide, diffusion very likely occurs during the cytochemical reaction. The problem of localizing chloride does not yet seem to have been solved. The non-destructive nature of freeze drying means that enzymes retain their activity very much better after this process than by chemical fixation. Hoerr (1936) showed that ground up frozen dried liver suspended in saline retains its glycolytic activity, glycogen disappearing from the tissue with the fqrmation of glucose. Several workers have published comparisons of alkaline phosphatase activity and distribution in frozen dried tissue. Emmel (1946), comparing frozen dried and cold acetone fixed kidney and intestine, uses Gomori's (1939) phosphatase method with an incubation time of three hours. He finds substantially the same distribution with the two methods of fixation. H e remarks on the excellent preservation of cytological structure. It appears, however, that using such long incubation times may introduce diffusion artifacts and that a more critical comparison, both of activity and distribution, would be obtained by the use of very much shorter incubation times. Bevelander and Johnson (1950) make extensive use of frozen dried embryo pig heads in an investigation of the development of membrane bone by histochemical methods. Frozen dried material has been used for cytological investigations. Mitochondria were studied by Bensley and Gersh (1933). They found them insoluble in alkalis, acetic acid, and organic solvents. Using Bensley's Millon reaction for tyrosine it is shown that a large part is protein. The results of this work and that of Bensley and Hoerr (1934) on the solubility of frozen dried liver protein led to the work of Bensley and others in isolating cellular constituents from fresh tissue. This work has been reviewed by Hoerr (1943).Nuclear structures have been studied by Bensley (1933) and Goodspeed, Uber, and Avery (1935). Bensley, using Amblystoma and rodent tissues, demonstrates that the material staining with hematoxylin and giving a positive Feulgen reaction in interkinetic nuclei is concentrated on the inner surface of the nuclear membrane and also as an investment around the nucleolus. H e also reports that nuclear ground substance gave a slight positive Feulgen reaction. Goodspeed et d. investigated the dividing nuclei of root tips of Lilium, which had been quenched in liquid air and dried at -32°C. The cytological preservation does not seem to have been of the highest quality, but they were able to demonstrate the multi-strand nature of chromosomes. They report four chromonemata at anaphase and eight at metaphase.

FREEZIXG A N D DRYING TECHNIQUES I N CYTOLOGY

57

One of the most striking differences seen between frozen dried and cheniically fixed material is the almost complete absence of shrinkage in the former. This is well demonstrated in kidney. Chemically fixed kidney shows the usual capsular space between the glomerulus and Bowman’s capsule, frozen dried kidney shows the glomerulus filling Bowman’s capsule completely, leaving no capsular space. This absence of shrinkage may be a disadvantage to the cytologist making chromosome analyses, for which purpose the shrunken chromosomes obtained by chemical fixation are easier to observe. Simpson ( 1941b) investigated the Golgi apparatus in various frozen dried tissues of Cavia. He used freehand sections of unstained as well as stained material. He was not successful in obtaining an osmium impregnation of the Golgi apparatus, but obtained an even impregnation through the tissue. This was possibly due to the preservation of reducing groups such as sulfhydryl, normally destroyed to a large extent especially by fixatives containing oxidizing agents. Gersh (1949) has studied a component in the Golgi apparatus which gives a positive reaction with the periodate and leuco-fuchsin test for vicinal hydroxyl groups (Hotchkiss, 1948).

IV. ADVANTAGES OF FREEZING AND DRYINGTECHNIQUES The advantages of frozen dried material for quantitative cytochemistry have been emphasized by Caspersson and his school. This is now the standard method adopted by these workers for preparing material for ultraviolet absorption measurements. HydCn ( 1943) compares carnoy fixed mrve cells with frozen dried material and points out the clumped and precipitated appearance of the carnoy nuclei compared with the frozen dried material. He states that chemical fixatives such as acetic alcohol and formalin precipitate protein in clumps and cause shrinkage and that these artifacts will cause false absorption of ultraviolet light due to diffuse reflection and scattering of light at the surfaces. His photographs of frozen dried ganglion cells photographed at 2570A show diffuse dark masses of absorbing material in the cytoplasm, while carnoy fixed cells show sharply defined, more deeply absorbing areas. The author has compared the action of some cytochemical fixatives with frozen dried material. In rat kidney, frozen and dried, phase contrast and ultraviolet photographs show large evenly absorbing nuclei with no thick nuclear membrane. The cytoplasm is homogenous and contains well-preserved mitochondria. Absorbing material is preserved in the lumen of the proximal tubules. Intertubular structures are preserved.

58

L. G . E. BELL

FREEZING AND DRYING TECHNIQUES IN CYTOLOGY

59

FIG.2. Rat kidney, frozen dried, 4-micron section mounted in nonane. Visual phase contrast. FIG.3. Rat kidney, carnoy fixation, 4-micron section mounted in nonane. Visual phase contrast. FIG.4. Same section as Fig. 2, 2570A, Quartz Objective. NA 1.25. Condenser NA 0.4. FIG. 5. Rat kidney, carnoy fixation, photographed as Fig. 4. FIG.6. Rat walker tumor frozen dried, 4-micron section mounted in glycerine, photographed as Fig. 4. FIG.7. Rat walker tumor, carnoy fixation, 4-micron section mounted in glycerine, photographed as Fig. 4. FIGS.2-7 are from original photographs by R. J. King and E. M. F. Roe. FIG.8. Rat kidney, fixed by freeze drying, 5~ section reacted with tetrazo dianisidine and K acid. FIG.9. Rat kidney, carnoy fixed, rest as Fig. 8. FIG.10. Rat kidney, acetic formol fixed, rest as Fig. 8.

60

L. G. E. BELL

FREEZING AND DRYING TECHNIQUES I N CYTOI.OGY

61

Rat kidney fixed in carnoy shows smaller nuclei with a clumped appearance and heavy nuclear membrane. The nucleolus appears larger than in frozen dried sections. The cytoplasm shows clear spaces and a clumped appearance. Mitochondria are not preserved. There is no absorbing material in the lumen of the tubules. These points are illustrated in Figs. 2-5. I n similarly treated material from the Walker rat tumor, frozen dried sections show evenly absorbing nuclei and cytoplasm with a fine nuclear membrane. Carnoy material shows shrunken nuclei with a clumped appearance and a heavy nuclear membrane (Figs. 6-7). In the frozen dried material it is seen that there is even absorption in the cytoplasm of the metaphase cell, while the carnoy fixed metaphase cell shows a transparent area around the metaphase plate. This means the chemical fixation has removed absorbing material from its original site, either completely from the cell or possibly onto the chromosomes. Using the tetrazo-benzidine reagent for tyrosine, histidine, and tryptophan end groups (Danielli, 1947) the author has found that in rat kidney, frozen dried nuclei are large and evenly staining with a more densely staining nucleolus. Nuclei from acid fixatives such as acetic alcohol or 10 per cent formol plus 5 per cent acetic acid, show a more clumped structure with a heavy nuclear membrane. These points are illustrated by Figs. 8-10. Nuclei from neutral formalin material show an even absorption which is apparently much heavier than in the other fixations, but this may be due to the greater shrinkage with this fixative after paraffin embedding. Only in the frozen dried material are the mitochondria preserved, and they stain more deeply than the cytoplasm. The cytoplasm with the other fixatives mentioned shows a heterogenous appearance, the brush borders in acetic formol material appearing darker than the cytoplasm. Frozen dried material shows staining material in the lumen of the proximal tubules. There is little left in the lumen of the chemically fixed tubules. Sjostrand (1944) shows photographs of frozen dried kidney stained with iron hematoxylin with material in the lumen of the proximal tubules. What the chemical nature is of the substances translocated by chemical fixation is not yet known. It is clear, due to their content of protein end groups, that they are of a protein nature, and therefore presumably of a molecular weight, approaching that of at least the smaller protein molecules. This underlines the necessity of interpreting the cytochemical results on chemically fixed tissue with extreme caution. Even in frozen dried material, though the rapid quenching reduces diffusion artifacts to a minimum, the action of the various solvents used to treat the sections is not completely understood. The extent to which material is removed from

62

L. G. E. BELL

sections by alcohols and other reagents needs investigation. When quantitative measurements are to be made, especially on protein end groups the degree of denaturation or unfolding of the protein molecules will influence the results. It is not known to what extent the end groups of frozen dried protein are available and how their availability may be influenced by various reagents and the length of time elapsing between drying and carrying out the test. The observations of Hoerr (1936) on the solubility of frozen dried tissues has a bearing on this point. It seems clear, however, that even allowing for these uncertainties frozen dried material should be used wherever possible for cytochemical work in preference to chemical fixation. The rapid-quenching of freezing drying should be useful in studying rapidly changing structures such as cilia, or the state of capillaries. Such studies have not yet been very extensive. V. REFERENCES Adam, N. K. (1941) The Physics and Chemistry of Surfaces, 3rd ed., Oxford. Oxford University Press. Aloe Co. (1947) Catalogue U. S. A. Altmann, R. (1890) Die elementarorganism und ihre Beziehungen zu den Zellen. Leipzig. Baker, J. R. (1945 and 1950) Cytological Technique, 1st and 2nd ed. London. Bartelmez, G. W. (1940) Anat. Rec., 77, 509. Bayliss, W. M. (1915) Principles of General Physiology, p. 17. Longmans, Green & Co. London. Bensley, R. R. (1933) Anat. Rec., 58, 1. Bensley, R. R., and Gersh, I. (1933) Anat. Rec., 57, 205. Bensley, R. R., and Hoerr, N. L. (1934) Anat. Rec., SO, 251. Bevelander, G., and Johnson, P. L. (1950) Anat. Rec., 108, 1950. Bolles Lee, A. (1950) Microtomists Vade Mecum, 11th ed. J. & A. Churchill. London. Burton, E. F., and Oliver, W. F. (1935) Proc. TOY.Soc., 158, 166. Caspersson, T. (1950) Cell Growth and Cell Function. Norton & Co. New York. Cwilong, B. M. (1945) Nature, Lond., Iw,361. Danielli, J. F. (1947) S. E. B. Symfiosia, 1, 101. Dempsey, E. W., Wislockie, C. B., and Singer, N. (1916) Atid. Rec., 96, 221. Edwards & Co. (1950) Catalogue, London. Emmel, V. M. (1946) Anat. Rec., 06, 159. Faraday Society ( 1950) Symposium on Microspectography. Fisher, J. C., Holloman, J. H., and Turnbull, D. (1949) Scierrce, 109, 168. Flosdorf, E. W. (1949) Freeze Drying. Rheinhold Publishing Co., N. Y. Fullam, E. F., and Gessler, A. E. (1946) Rev. sci. Insfriwieir., 17, 23. Gersh, I. (1932) h u t . Rec., 88, 309. Gersh, I. (1933-4) Anat. Rec., 68, 369. Gersh, I. (1947) The Aloe Co. Cutalogwc.

FREEZING AND DRYING TECHXIQUES I N CYTOLOGY

-

63

Gersh, I. (1949) Arch. Path., 47, 99. 457. Gersh, I., and Catchpole, H. R. (1949) Ailat. Rec., I, Gomori, G. (1939) Proc. SOC.exp. Biol. Med., 9, 23. Goodspeed, T. H., and Uber, F. M. (1934) Proc. mt. Acad. Sci., Wash., a0, 495. Goodspeed, T. H., Uber, F. M., and Avery, P. (1935) Univ. Calif. Pub. Bot., 18, 33-44. Harris, J. E., Sloane, J. F., King, D. T. (1950) ‘Nature, Lond., 166, 25. Hazel, F., Parker, J. A,, and Schipper, E. (1949) Scielzce, 110, 160. Hoerr, N. L. (1936) Anat. Rec., 63, 293. Hoerr, N. L. (1943) Bid. Symposia, 10, 185. Hotchkiss, R. D. (1948) Arch. Biochem., 16, 131. HydCn, H. (1943) Acta physiol. scad., Suppl. VII, 6. Kistler, S. S. (1936) J. Amer. C k m . Soc., 68, 901. Lison, L. (1936) Histochemie animale. Paris. Gauthier-Villars. Luyet, (1938) Biodynumica, 2. Mancini, R E. (1948) Anat. Rec., 101,149. Mann, G. (1902) Physiological Histology. Oxford, pp. 139, 142. Oxford University Press. Mendelow, H., and Hamilton, J. €3. (1950) Anat. Rec., 101, 443. Packer, 0. M., and Scott, S. H. (1942) J. Tech. Methods No. 22, 85. Palade, G. E., and Claude, A. (1949) I . Morph., 88, 35. Pease, D. C. (1947) Science, 108, 543. Pease, D .C., and Baker, R. F. (1949) Amer. J. Afraf.,84. Reed, R., and Udall, K. M. (1950) J . roy. micr. SOC.London, 70, 92. Romeis, B. (1932) Taschenbuch der mikroscopischen Technik. Berlin, p. 93. Schaeffer, V. J. (1946) Science, 104, 457. Scott, G. H. (1933) Protoplamia, a0, 133. Scott, G. H. (1937) McClung’s Microscopical Technique, 2nd ed. Hoeber, New York. 643. Scott, G. H. (1943) B i d . Symjosia, 10, 277. Scott, G. H., and Hoerr, N. L. (1950) Medical Physics, 2, 293. Chicago. Year Book Publishers. Scott, G. H., and Packer, D. M. (1939) Awat. Rec., 74, 31. Simpson, W. L. (1941a) Anat. Rec., 80, 173. Simpson, W. L. (1941b) d4nat. Rrc., 80, 329. Sjostrand, F. (1944) Acta. Anat., Suppl. 1. Smith, A. V., and Polge, C. (1950) Naftrre, Lorrd.. 166, 668. Staple, P. H. (1949) Private communication. SylvCn, B (1950) Private communication. Voliner, M . and Estermann, I. (1921) Z. Pltys., I, 1. 13.

This Page Intentionally Left Blank

Enzymatic Processes in Cell Membrane Penetration TH. ROSENBERG Biochemistry Deportment, Strno Memorial Hospital and Nordisk Insdinlab., Denmark

W. WILBRANDT Department of Pharmacology, University of Beme, Berrce, Switserlarrd CONTENTS Page 65 66

I. Introduction ........................ ..................... 11. Some General Considerations . . . . . . . . . . . . . . ...................... 111. Enzymes with Non-Penetrating Substrat:s . . . ............. 1. Invertase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

IV. V.

VI. VII.

3. Trehalase . . . . . . . . . . . . . . . . . . . . .................... 4. Amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5. Phosphatases .................................................... 6. Phosphorylating enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Enzymatically Controll ............................... Transport of Glucose . . . . . ............................... 1. Muscle . . . . . . . . . . . . . ............................... 2. Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Erythrocyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Kidney and Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion ......................................................... References . . . . . . . . . . . . . . . . . . . ................

68 68 68

68 69 69 69 70 70 71 73 75 79

85 89

I. INTRODUCTION

Es ist anzunehmen, dass der physiologische Import und Export ein komplizierter, unanalysierter, an die Lebenstatigkeit der Zelle gebundener Vorgang in der Zelloberflache, der Plasmahaut ist. Dieser Vorgang setzt meist unter bestimmten Bedingungen ein; diese sind uns noch nicht geniigend bekannt. Es ist nicht anders denkbar, als dass fur solche Aktion der Plasmahaut eine komplizierte Organisation erforderlich ist. [R, HOBER,Physikalische Chemie der Zellen und Gewebe, 1911.1 The cell membrane has been widely considered as a passive structure allowing solutes to penetrate according to the properties of their molecules such as size, membrane solubility, and electric charge. More recently evidence has begun to accumulate indicating that, in addition, the membrane is a part of the cell machinery which contains special enzymes participating both in control of cell metabolism and transport of substances. In the present review an attempt is made to show some general principles and possible mechanisms underlying this activity rather than to give a complete representation of the pertinent published data. The reviewers are aware that this implies the introduction of concepts and 65

66

TH. ROSENBERG AND W. W I L B R A N M

suggestions some of which may appear rather hypothetical. They feel, however, that at the present stage an outline and discussion of some possible fundamental elements may be more useful than a compilation of observed facts. It was decided therefore to focus the interest on the role of surface enzymes in glucose transport which so far appears to have been studied most extensively,

11. SOMEGENERALCONSIDERATIONS Considering the submicroscopic dimension of the cell membrane, the evidence for the membrane localization of enzymes is necessarily more indirect than direct. The methods of histochemistry introduced the possibility of localizing enzyme activity with surprising accuracy. Their resolution power remains insufficient so far to enable decision to be made as to whether the enzyme is attached to the cell membrane. Therefore, for this purpose, histochemical results can be used only in combination with evidence derived from other observations. So far this has been possible mainly with regard to alkaline phosphatase, which in kidney, intestine, and other organs has given very clear and interesting pictures. (For a review see Bradfield, 1950.) In the indirect evidence available the most conclusive is based on impermeability of the membrane for substrates or enzyme effectors (inhibitors or activators). If a cell shows enzymatic action on a non-penetrating substrate or if the activity of a cellular enzyme is affected directly by non-penetrating substances, the site of the enzyme involved must be the outer surface of the cell membrane. The localization of an enzyme on the inner surface of the membrane (which should by no means be regarded as less frequent or less important), cannot, of course, be similarly demonstrated with equal ease and conclusiveness. A special group of enzymes with non-penetrating substrates are those whose action resembles those involved in digestion in the alimentary canal. These act by splitting larger, non-penetrating molecules of a substrate into smaller units which can be metabolized in the cell interior. They have adequately been termed “digestive enzymes” by Rothstein (1950). Wider biological importance appears to be attached to those processes which in the present review will be termed “enzymatically controlled transport.” Some introductory remarks concerning the meaning of this notion seem adequate. The essential feature of an enzymatically controlled transport is the

ENZYMATIC PROCESSES IN CELL MEMBRANE PENETRATION

67

combined action of two surface enzymes separated by the cell membrane whereby the substrate is temporarily changed to a membrane-soluble transport form capable of penetrating the membrane. The existence of this transport form will thus be confined essentially to the membrane proper. It may be assumed to consist of the penetrating substrate and some other molecule or molecules which may be regarded as “membrane carriers.” The transport direction of this substrate-carrier complex will be determined by the natural diffusion tendencies of both substrate and carrier. If these tendencies are opposite and if the resulting transport follows the tendency of the carrier rather than of the substrate, the latter will move from lower to higher chemical or electrochemical potential. This case will be termed “thermodynamically active transport.” It should be pointed out that the thermodynamically active transport is a possible, but by no means a necessary consequence of the participation of enzymes in membrane penetration; in other words, in general it represents a special case of enzymatically controlled transport. The essential point for a continuous thermodynamically active transport appears to be the maintenance of the carrier gradient which usually will require both enzymatic formation and elimination of the carrier on the two sides of the membrane. A more detailed discussion has been given by Rosenberg (1948). The identification of a transport as enzymatically controlled meets with greater difficulties if it is not thermodynamically active. Indirect criteria then have to be used. They include: 1. Non-conformity with the laws of diffusion, particularly a constant penetration rate independent of the concentration difference. Minor deviations may possibly be ascribed to variations of activity coefficients in the membrane or to a change of the membrane structure by high concentrations of the penetrating solute in the membrane. 2. Competition of simultaneously penetrating substrates. The limitations of this criterion coincide to a certain degree with those of the foregoing in so far as the principle of independent diffusion streams holds strictly only for dilute solutions. 3. High structural specificity, particularly stereospecificity. Although the possible existence of stereospecific pore structures cannot be denied in principle such “keyholes” have never been demonstrated and would appear to have no reasonable degree of probability. 4. Action of enzyme effectors (inhibitors or activators) on the rate of transport, provided that their effect cannot be ascribed to a changed concentration difference of the penetrating substances.

68

TH. ROSENBERG A N D W. WJFBRANDT

As to other criteria, occasionally an unusually high temperature coefficient of the penetration rate has been taken as an indication for chemical transport mechanisms. Danielli and Davson (1934) have shown, however, that high energy barriers in the, path of diffusion may lead to temperature coefficients of considerable magnitude (“activated diffusion”), without participation of chemical reactions. None of the above criteria alone will be conclusive and it will depend on the case under consideration, how much weight can be attributed to any of them. 111. ENZYMES WITH NON-PENETRATING SUBSTRATES

Disregarding the possibility of an enzyme action over long distances (through the cell membrane), the outside localization of enzymes acting on non-penetrating substrates seems to be conclusive. A number of the enzymes belonging to this group clearly are of the digestive type, particularly carbohydrases of microorganisms. Also certain phosphatases have been assigned to this group. 1. Invertase. Wilkes and Palmer (1933) studied the pH-activity curves of yeast invertase in free solution and in the living cells and found them practically identical. They concluded that the locus of the enzyme in the cell must have free exchange with the external medium as regards [HI+ ions and that the enzyme would be supposed to function on the surface of the cells. 2. Lactuse. Essentially the same behavior was later found concerning lactase in yeast by Myrback and Vasseur (1943), who arrived at the same conclusion as regards the localization of the enzyme. 3. Trehulase. Through a different approach the conclusion of outside localization was reached in the case of trehalase. Myrback and Oertenblad (1936) found that yeast cells contain trehalose which is not metabolized although the cells contain an enzyme trehalase which splits trehalose added to the suspension. Thus apparently the naturally occurring enzyme and substrate are separated by the cell membrane, and the enzyme must be sited on the outer surface of the membrane. Accordingly the pH-fermentation curve for added trehalose was found by Myrback and Vasseur (1943) to differ substantially from that for glucose, showing a narrow optimum about pH 5 , whereas glucose fermentation has a wide range of practically constant activity without a sharp maximum, extending from about pH 2-3 to beyond p H 7. The pH optimum of trehalose fermentation agreed well with that found earlier by Myrback and Oertenblad (1937) for yeast trehalase in solution ( p H 5-6), indicating that the rate of hydrolysis on the outside of the cell was the limiting factor.

69

ENZYMATIC PROCESSES 1.N CELL MEMBRANE PENETRATION

4. Amylace. According to Rahn and Leet ( 1949) Streptococcus bovis forms adaptive amylase in the presence of insoluble starch in the medium. In this case both the adaptive enzyme and the enzyme involved in the process of adaptation appear to be situated on the outside of the membrane. 5 . Phosphatases. Rothstein and Meier ( 1948) described surface phosphatases in yeast cells acting among others on adenosinetriphosphate. The outside localization in this case was established by quantitative recovery of the added nitrogen, pentose ester, phosphate, and total phosphate in the outside medium by experiments with ATP containing PS2in which no P3*entered the cells. I n later experiments Rothstein showed that yeast cells utilize phosphorylated sugars only after enzymatic dephosphorylation, the inorganic phosphate remaining outside. Inhibition of the surface phosphatases by molybdate deprived the cells of the ability to metabolize hexose phosphates. Vishniac ( 1950) described hydrolysis of tripolyphosphate, which also may be considered as non-penetrating into yeast cells. 6, Phosphorylating enzymes. Synthetic enzymes likewise must in some cases be localized on the surface of cells. Lindberg (1948, 1950) working with 32Pphosphate on sea urchin eggs studied the equilibration of the external inorganic phosphate with both inorganic phosphate and energy-rich phosphate of the cell. In the case of the unfertilized egg his experiments showed an increase of specific activity first in the inorganic phosphate and somewhat later in the energy-rich phosphate of the cell which, however, did not rise above 0.03 per cent of the specific activity of the external inorganic phosphate and remained constant at this level. His conclusion that on the outer surface of the eggs energy-rich phosphate was formed from inorganic phosphate, both fractions being unable to penetrate the membrane, appears to be reasonable although the striking fact that the equilibrium specific activities of the two fractions were found identical does not clearly follow from this assumption. Since according to prevailing views formation of energy-rich phosphate from inorganic phosphate involves coupling to other processes, a further consequence would appear to be that the total reaction on the surface is still more complex. In the case of the fertilized eggs equilibrium was not reached within the time of the experiment (135 minutes), indicating slow penetratibn. The continuous rise of the activity, however, was considerably steeper and reached much higher values in the energy-rich fraction, indicating that the formation of the energy-rich bonds in this case likewise must have taken place on the outer surface of the cell. It seems possible that r

r

70

TH. ROSENBERG AND W. WILBRANDT

the presence of energy-rich phosphate bonds on the outer surface of a cell as shown in these experiments may be of a more general significance for enzymatically controlled transports. IV. THE ENZYMATICALLY CONTROLLED TRANSPORT The number of enzymatically controlled transports used by the organism is probably greater than we know at present. In many cases no compelling criteria are available, and sometimes the enzymatic nature of a transport can only be supposed. There is mainly the alternative possibility of a << trapping mechanism,” which has frequently been assumed and which it may be necessary to consider. If a molecule after penetrating the membrane by diffusion is transformed (enzymatically or not) into a form unable to diffuse back, it may be said to be trapped. Trapping will result in a higher rate of penetration by maintaining a steeper gradient and may lead to an apparent accumulation if the transformed and the unchanged molecules are not considered separately. Thus thermodynamically active transport may be simulated. If two stages of trapping are arranged in series and if the second trapped form is the same as the original unchanged molecule, true active transport may result. This case will be discussed later in some detail. In higher organisms enzymatically controlled transports may be used as partial mechanisms in different cell functions, among which excitation and recovery mainly in muscle and nerve, secretory activity through epithelial cells, and regulation of cell metabolism seem most conspicuous at present. Whether an enzymatically controlled transport is thermodynamically active o r not will depend not only on the nature and localization of the enzymes involved, but also on the more or less fortuitous substrate concentrations on the two sides of the membrane. A separate treatment of active transport will therefore not be attempted in the following discussion. V. TRANSPORT OF GLUCOSE The problem of glucose penetration into living cells has attracted attention as early as cell permeability itself. Many authors have been struck by the fact that cell membranes seemed to be impermeable to this main fuel of the living cell when tested with current permeability methods. As early as 1902 Overton postulated a lipoid-soluble penetration form of glucose. Hober ( 1911) in his well-known textbook, Physikalische Chemie dm Zellen und der Gavebe, introduced the term “physiologische Permeabilitit,” which was supposed to involve “active” processes.

ENZYMATIC PROCESSES I N CELL MEMIsRANE PENETRATION

71

1. Muscle

Overton in his classical studies using osmotic methods arrived at the conclusion that the membrane of muscle fibers is impermeable to glucose. Later experiments by Eggleton (1935) in which the distribution volume of glucose in frog muscles was found to agree with the extracellular space seemed to confirm this conclusion. If the conditions in these experiments may be considered rather remote from physiological, this would apply at least to a much lesser degree to experiments by Lundsgaard (1939) on artificially perfused hind-limb preparations of cats. Three important results emerged from these experiments : (1 ) Glucose disappears from the perfusion fluid and consequently must be taken up by the muscle cells. (2) The concentration of free glucose inside the muscle fiber as determined by reducing methods is, however, practically zero, independent of the outside concentration and of the rate of uptake. (3) Insulin increases the rate of uptake by as much as 200 per cent. Lundsgaard concluded that glucose cannot enter the cell by simple diffusion and that the observed penetration must involve some “active” process. This process must be the point of attack of insulin. Considering the zero concentration of inside glucose, the insulin-sensitive process must be located on the surface of the muscle cell. This would be in harmony with the known size of the insulin molecule. A further result of Lundsgaard’s experiments is also important in considering the question of whether the “active” process consists of a trapping mechanism or of an enzymatically controlled transport. Confirming earlier workers he found that the rate of glucose uptake is not proportional to the glucose concentration but shows a clear tendency to become constant at higher glucose concentrations. As no free glucose was found in the cell, this again would appear to be a strong indication against a trapping mechanism. Concerning the nature of the ‘“active” process in the case of the muscle cell as well as in several others, certain observations point to phosphorylation reactions being involved. Sacks (1944, 1948) studied the uptake of Psa into the muscles of cats. Among the fractions of adenosinetriphosphate, creatine-phosphate, fructose-6-phosphate, and glucose-6-phosphate two and four hours after the injection into fasting animals he found that glucose-6-phosphate showed a two to three times higher specific activity than the others. H e concluded a primary formation of hexose nionophosphate on the outer surface of the fiber and made the somewhat surprising assumption that the ester splits

72

TH. ROSENBERC AND W. WILBRANDT

during penetration through the membrane, glucose entering the cell and phosphate remaining outside. Feeding of glucose further increased the specific activity of glucose&phosphate by more than 100 per cent which would not seem to support the interpretation given later by Kalckar et al. (1944) in a similar study that the high activity of glucose-&phosphate might be due to phosphorylysis of preformed glycogen in the membrane. A decisive extension of our knowledge concerning the action of insulin was brought about by the well-known discovery of Colowick, Cori, and Slein (1947) that insulin in the presence of pituitary and adrenocortical hormones in vitro is able to accelerate the hexokinase reaction, thus reproducing in vitro the physiological hormonal balance of glucose uptake. These findings in combination with the increase of glucose uptake in the presence of insulin may be regarded as further support of the hypothesis that phosphorylations are involved in glucose uptake. For an attempt to picture a detailed mechanism by which glucose uptake might depend on phosphorylation processes, the main difficulty appears to be that hexose phosphate esters according to general experience are unable to penetrate cell membranes. In the case of the muscle fiber this appears clearly from the retention of hexose phosphates in the cell. Their poor membrane penetration certainly may be attributed to their ionic charge and to the resulting low lipoid or membrane solubility. It may safely be concluded that none of the known orthophosphate esters of hexoses can be the transport form. I n attempting to combine the postulates of the enzymatically controlled transport as pictured above with a phosphorylating mechanism as suggested by the observations just mentioned, we thus have to search for an uncharged and diffusible compound arising from an outside reaction between glucose and ATP and, after penetration of the membrane, being transformed into glucosed-phosphate. Discussing the physico-chemical basis of the biological functions of energy-rich phosphates Rosenberg ( 1950) has given chemical arguments in favor of a primary formation of monometaphosphate compounds which would have properties in harmony with essential features of various ATP functions. For the problem of glucose uptake a glucose metaphosphate ester would offer the required electroneutrality and possibly a certain lipoid solubility. Furthermore, the intracellular formation of glucose orthophosphate would occur readily by mere hydration. One of the consequences of such a mechanism would be the presence

E N Z Y M A T I C PROCESSES I N CELL M E M B R A N E PENETRATION

73

of ATP outside the membrane. The work of Lindberg on the sea urchin egg has shown the possibility of such a localization. According to this interpretation the hexokinase reaction would consist of several steps which in the living cell would be spatially separated by the cell membrane. Insulin then would act on one of the steps located outside. 2. Yeast There are rather strong indications to support the assumption that the uptake of glucose by the yeast cell is controlled by enzymes. For the rate of uptake under anaerobic conditions Runnstrom, Sperber, and Feller (1938) found a saturation level at rather low concentrations. No data concerning the internal glucose concentration were given. Under aerobic conditions the glucose uptake seemed to be a rather complicated process. Within a single experiment it would appear from the kinetics of glucose disappearance outside that the internal glucose concentration was practically zero. But, in different experiments the rate seemed to depend also on how much glucose had already been taken up by the cells, decreasing as this amount increased. Numerous other reports on the relationship between outer glucose concentration and rate of fermentation or respiration do not permit a conclusion as to whether this relationship is based on enzymatic reactions on the cell surface or in the interior of the cell. Wertheimer ( 1934) concluded from experiments with galactose that this sugar is not taken up by the cell. Recently much new evidence concerning the uptake of hexoses by yeast was obtained by the use of non-penetrating enzyme inhibitors, particularly uranyl salts. Barron, Muntz and Gasvoda (1948/1949) found inhibition of fermentation and oxidation of glucose by.smal1 amounts of uranyl salts. No inhibition, however, was found in the case of other substrates like acetic acid, ethanol, malic and citric acid. They inferred the surface localization of the inhibition from the fact that it was easily reversed by the addition of non-penetrating compounds forming complexes with uranyl, particularly A T P and hexose diphosphate. The conclusion drawn was that the inhibition was due to a change in permeability, the membrane becoming impermeable to glucose, but not to other substrates. The possibility of a surface enzyme inhibition was considered, but was dismissed for various reasons, among others because uranyl salts had not been found to inhibit hexokinase. This point will be discussed later. Rothstein and co-workers (1948, 1949, 1951), continuing and extending

74

TH. ROSENBERG AND W. WILBRANDT

this work, introduced the assumption of surface enzymes being involved in glucose uptake. Rothstein et al. (1951) found that the uptake of other hexoses likewise is inhibited by the uranyl ion, and Rothstein and Larrabee (1948) were able to show that uranyl ions penetrate into the cell only to a negligible extent. The uptake of glucose under aerobic conditions was considerably less sensitive than under anaerobic mnditions. By comparing the thermodynamic stability of the complex between uranyl and the assumed cell surface compound with a great number of organic and inorganic uranyl complexes, Rothstein and Meier (1951) and Rothstein et al. (1951) came to the conclusion that the uranyl ions exert the inhibition by combining reversibly with surface groups similar to polymetaphosphates. Some critical remarks with regard to this conclusion may be justified. It is based on the observation that the dissociation tendency of the yeast-uranyl complex is about the same as that of polymetaphosphateuranyl complexes, and much higher than the dissociation tendencies of a great number of other complexes. These dissociation tendencies are derived from the mass law and are thus correct for the complexes in solution. In the case of the cell, however, the formation of the complex is not a homogeneous reaction. The mass law therefore cannot be used here. The use of an adsorption isotherm which will be adequate may lead to an equation formally identical with the mass law, but the constants will not have exactly the same meaning. In other words, the dissociation tendency of a complex may largely depend on whether both the complex and dissociation products are in aqueous solution or whether the complex and one of the dissociation products are fixed on a surface, only one of the dissociation products being in the solution. Thus in the opinion of the reviewers, from the figures of Rothstein and Meier it is not possible to decide.whether the complexing groups on the yeast cell belong to polymeric metaphosphates, nucleic acids, pyrophosphate, tripolyphosphate, A T P or others. A few experiments by Rothstein and Meier at different pH values, however, support the view that these groups belong to the class of phosphates. Experiments by Vishniac ( 1950) showing inhibition by tripolyphosphate of yeast hexokinase and of glucose fermentation would suggest that, as discussed above for the case of muscle, hexokinase and A T P on the outer surface may be involved in the process of glucose uptake. The inhibition by uranyl ions then may be due to complex formation with ATP. The observation of Barron et d. (1948-49) mentioned above that uranyl does not inhibit hexokinase would then most likely be attributable tq the

ENZYMATIC PROCESSES I N CELL MEMBRANE PENETRATIOS

75

comparatively large amounts of ATP used in such experiments. The findings of Lindberg concerning ATP on the surface of the sea urchin egg may be recalled in this connection. 3. ERYTHROCYTE

It was early recognized that the glucose permeability of red cells presents striking features. I n the first investigations of Grijns (1896) and Hedin (1897, 1898) the red cells seemed to be impermeable to glucose. Later experiments by Rona and Michaelis ( l W ) , Rotia and Doblin ( 1911), Kozawa (1914) Wilbrandt ( 1938) , and others showed that a marked species specificity exists, the red cells of some species appearing glucose permeable, others not. Only those of man and of the higher apes were shown to allow a rapid penetration of glucose. Much discussion has arisen with respect to the question whether glucose occurring naturally in the blood or added to blood is equally distributed between plasma and cells. After recognition of various sources of error, the general conclusion was reached that an equilibration of glucose concentration occurs at least in human blood. From the evidence available Peters and Van Slyke (1946) did not hesitate to conclude “that the membrane of the human red blood cell permits the free passage of glucose by diffusion.J’ Experiments of Wilbrandt and Rosenberg (1951) with the use of osmotic methods on human red cells have so far confirmed the equality of inside and outside concentrations in equilibrium over a wide range of concentrations. As to the finding of Klinghoffer (1940) that from extremely concentrated solutions only a limited amount of glucose penetrates the cells, it should be pointed out that the rate of penetration in high glucose concentrations is exceedingly low as discussed later. Nevertheless the glucose penetration into human red cells is beyond doubt an enzymatically controlled transport for which the criteria discussed above could be shown to be positive without exception. One point of special interest in this membrane transport is its close relation to the active transcellular transport of glucose in kidney and intestine. As regards the criterion of concentration dependence, the rate of glucose uptake in human red cells is not proportional to the concentration difference. This was indicated early by a striking difference between the very slow penetration in osmotic experiments involving high concentrations and the rapid uptake in experiments with physiological concentrations using chemical methods. (Ege, 1920, 1921 ; Guensberg, 1947; Kozawa, 1914; Masing, 1913, 1914; Rona and Michaelis, 1909; Rona and Doblin, 1911).

76

TH. ROSENBERG A N D

W. WILBRANDT

Ege (1921) inferred from such experiments a primary rapid adsorption to the outer surface of the cells followed by a much slower penetration. Recently in osmotic experiments concentrations between 0.25 and 20 to 30 per cent could be compared (Guensberg, 1947; Wilbrandt et ul., 1947; Wilbrandt and Rosenberg, 1950, 1951). From the lowest concentrations which are still within the range of physiological blood sugar levels the equilibration time was a matter of seconds, from the highest a matter of hours, with a continuous transition. These experiments show the inadequacy of the adsorption interpretation, because in osmotic experiments adsorbed glucose could not be mistaken as having penetrated. The rapid disappearance of glucose from low concentrations must actually be attributed to glucose uptake into the interior of the cells. A closer examination of the time course of penetration revealed that the kinetics must be basically different from those of a simple physical diffusion process (Guensberg, 1947). An attempt was made to find the underlying rate-concentration dependence by graphical evaluation of penetration rates from experiments on inward as well as outward passage, using a wide range of concentrations on both sides of the membrane (Wilbrandt & Rosenberg, 1951). For the sake of greater clarity it was found convenient to introduce the term cis for the side where glucose enters and trum where it leaves the membrane. In experiments on outward passage of glucose the surprising observation was made that in this case the penetration even from high glucose concentrations on the cis side is extremely rapid. This indicates that the slowing effect of high glucose concentrations is chiefly exerted on the trans rather than on the cis side of the membrane. Plotting the rate of penetration against the glucose concentration on the cis side at constant trans concentrations, instead of the straight line expected in the case of a simple diffusion, a saturation curve was obtained with constant rate of penetration (saturation level) at higher cis concentrations. This saturation level decreases rapidly with rising trans concentrations. These results resemble to a certain degree the above mentioned type of the rate-concentration curve of glucose uptake in muscle as well as the respective curves of the transcellular glucose transports through kidney and intestinal epithelia, which will be discussed later. The criterion of competition was tested with glucose, arabinose, and xylose (Wilbrandt, 1947). From a mixture of these three sugars the

ENZYMATIC PROCESSES I N CELL MEMBRANE PENETRATION

77

total rate of penetration was practically the same as from solutions of the components alone, although the total sugar concentration was three times higher. Thus the three sugars appear to compete for a common transport mechanism. As to structural specificity two observations may be mentioned. Among the hexoses the following relative rates of penetration in human red cells were found (Wilbrandt, 1938) : D-glucose 44, D-galactose 66,D-mannose 183, D-fructose 5.2, L-sorbose 34. Even more surprising was the finding that L-arabinose penetrates readily, D-arabinose practically not at all, while for xylose the reverse holds (Wilbrandt, 1947). Thus the penetrating forms of glucose, arabinose, and xylose agree in the configuration at carbon atoms 1, 2 and 3. As to the fourth criterion (the change of penetration rate under the action of enzyme effectors) in certain respects the erythrocyte as a single cell offers special working conditions in comparison with epithelial layers. The possibility of following the membrane penetration in both directions and of restricting the action of non-penetrating inhibitors to one side of the membrane allows a differentiation of the action on the cis and the trans enzyme. Inhibitors affecting the rate of glucose penetration so far investigated are phloridzin (Wilbrandt, 1947; LeFevre, 1947, 1948) and related substances [phloretin and a phosphorylated phloretin, the latter an extremely strong inhibitor of alkaline phosphatase, Diczfalusy et al., 19501, heavy metals and metallo-organic compounds : mercury, gold, and p-chloromercuribenzoate (LeFevre, 1947 ; Wilbrandt, 1950), narcotics in high concentrations : chloral hydrate, ethyl urethane, and phenobarbital (Wilbrandt and Haemmerli, 195l ) and lachrymators : chlorpicrine, allylisotliiocyanate, and bromacetophenone (Wilbrandt, 1950). With most of them the inhibition of penetration reaches 100 per cent in higher concentrations, indicating that the membrane is virtually impermeable to the unchanged glucose molecule. As far as has been tested, the inhibition is restricted to monosaccharides. The inhibitory action of the phloridzin group can be readily released by washing out or by dilution, that by ally1 isothiocyanate cannot. None of these inhibitors is specific enough to allow conclusions as to the nature of the transport enzymes involved. The fact that several enzymes known to be inhibited by phloridzin in some way are related to reactions involving phosphate again points to a possible role of phosphorylations. Some of the others have been described as hexokinase inhibitors (Dixon, 1948) and in fact have been chosen for this reason.

78

TH. ROSENBERG AND W. WILBRANDT

Our knowledge of the specificity of the inhibitions involved and the properties of hexokinase in different cells is so incomplete, however, that definite assumptions concerning the enzymes and enzyme reactions involved should be postponed. Due to the above mentioned special conditions in experiments with single cells the results obtained with the phloridzin group allow certain conclusions concerning minimum number, spatial distribution, and mode of cooperation of the enzymes involved, independent of their nature. T h e phosphorylated phloretin which is polymeric and ionized, will not penetrate the membrane. Its action will therefore be restricted to the outer surface of the cell. In concentrations up to 0.2 per cent it never showed any inhibition of glucose penetration at 37°C from outside to inside. The outward passage was markedly inhibited, practically 100 per cent in high concentrations and partially in concentrations down to 0.002 per cent. This startling result indicates that glucose entrance and exit must, on the outside of the cell, occur through different enzymatic pathways. Of the outer enzymes involved the phosphorylated phloretin inhibits only the trans enzyme of glucose exit and not the cis enzyme of entrance. Less definite results have so far been obtained with regard to the inner enzymes. Non-penetrating inhibitors of course cannot be used here. Phloretin and phloridzin in concentrations inhibiting alkaline phosphatase show strong inhibition of glucose exit immediately after the addition of the inhibitors, whereas entrance is inhibited less strongly. In the case of phloridzin, however, the inhibition was shown to increase with time, presumably reflecting inward penetration of phloridzin. Thus, as in the case of exit here likewise the trans enzyme seems to be inhibited by the phloridzin group. In other words, the inhibitory action of the phloridzin group seems to affect the dissociation of glucose and membrane carrier on both sides of the membrane. A further postulate required by the exclusive action of phosphorylated phloretin on outward penetration appears to be a rectifier effect of the enzyme system. If the trans enzyme of exit could be used by the cell as cis enzyme for entrance, the phosphorylated phloretin would necessarily have to show partial inhibition of entrance too. Furthermore if the cis enzyme of entrance could serve as trans enzyme for exit, the inhibition of exit could not be complete. The interpretation of this rectifier effect is an interesting problem which will not be discussed here. Considering the specialized function and relatively low metabolism of the red cell, the complexity of the mechanisms involved in glucose pene-

ENZYMATIC PROCESSES IN CELL MEMBRANE PENETRATION

79

tration may seem surprising. Apparently it has to be considered as an inherited remnant, indicating the general biological occurrence of the phenomena discussed here. The similarities to the conditions in kidney and intestine and their bearing on the mechanisms involved in the active transports in these organs will be evident from the following discussion.

4. KIDNEYAND INTESTINE Both kidney and intestine have the common function of providing or saving substances of vital importance to the organism, particularly foodstuffs. In the intestine they are taken from the stream of ingesta, in the kidney from the stream of glomerular filtrate, which due to the strange mode of action of the kidney contains both waste products and essential nutrient material without discrimination. I n both organs the glucoseabsorbing epithelia are characterized histologically by the special structure of brush borders on the lumen side. In both cases histochemical methods reveal a particularly high activity of alkaline phosphatase. Finally from the evidence available at present both may be assumed to work with closely related mechanisms, since the functional characteristics show a striking agreement. Thus the glucose-absorbing epithelia of the small intestine and of the proximal tubules in the kidney show so many features in common both morphologically and functionally that it seems justified to treat them jointly. The first point to be mentioned is that, in contrast to muscle and red cell, the glucose transport here clearly is (or can be) thermodynamically active. I n the case of the kidney this is immediately evident from the fact that the normal final urine leaving the tubules is practically glucose free. The admirable microanalyses of A. N. Richards and his group (reviewed by Richards, 1938) have shown that in the glomerular filtrate the glucose concentration equals that of blood and that during the passage through the proximal tubule it is continuously lowered by reabsorption. The reabsorption thus occurs from lower to higher chemical potential. I n the case of the intestine, because of the variable fluid volume in the lumen, special experimental conditions had to be realized to render the active nature of the transport clearly evident. This was accomplished by Barany and Sperber (1939) who followed glucose absorption from an isotonic sodium sulfate solution. On account of the low absorbability of sulfate, the fluid volume under these circumstances becomes practically constant. The concentration of glucose then can be shown to fall considerably below the blood sugar level, indicating thermodynamically active transport.

80

T H . ROSEKBERG A N D W. WILBRANDT

As regards the indirect criteria, the relationship between rate of transport and concentration in both cases is not linear. In the kidney it shows a clear saturation level, as was first demonstrated by Shannon and Fisher ( 1938) in experiments involving determination of glucose excretion in the urine and of glomerular filtration rate. Plotting glucose concentration in the glomerular filtrate (== blood plasma concentration) against the rate of reabsorption (rate of glucose filtration in the glomeruli, as given by glomerular filtration rate times plasma concentration, minus rate of excretion in the urine) a curve was obtained which indicated a constant rate of absorption at concentration values in the glomerular filtrate above 200 mg. per cent. In the intestine the relationship between sugar concentration and rate of absorption has been investigated repeatedly (Cori, 1925 ; Verzir, 1935 ; Donhoffer, 1935 ; Westenbrink, 1936; Barany und Sperber, 1942; Vidal-Sivilla, 1950). The conclusion to be drawn from the various results obtained would be that the relation is clearly not linear, the rate increasing with concentration more slowly at higher than at lower concentration values, but that a level of constant rate is not reached even at the highest concentrations tested. The interpretation suggested from inhibition experiments discussed later (Donhoffer, 1935) that intestinal glucose absorption consists of two parallel streams, one of which is an “active” transport, the other a simple diffusion, might account for this seeming difference between intestine and kidney. This assumption, however, requires further investigation. On the other hand, it should be pointed out, that the conditions as regards glucose concentration in kidney and intestine experiments differ in three points, each of which may possibly be involved. First, the absolute lumen concentrations in the intestine experiments were much higher than in the kidney ; second, the absorption in the intestine, as opposed to the kidney, mainly occurred from higher to lower concentrations; and third, the experimental variation of the concentration in the kidney involved both lumen and plasma concentrations, in the intestine the lumen concentration only. Competition in absorption experiments with mixtures of different monosaccharides has been found in the intestine as well as in the kidney (Cori, 1925 ; Shannon, 1938). Likewise high structural specificity was repeatedly found in both organs (Nagano, 1902; Hamburger, 1924; Cori, 1925; Wilbrandt and Laszt, 1933 ; Hober, 1933). In the intestine hexoses were found to penetrate faster than pentoses, and among the hexoses considerable differences exist. According to experiments of Hamburger (1924) the kidney even differentiates between optical stereoisomers like the

ENZYMATIC PROCESSES I N CELL MEMBRANE PENETRATION

a-

81

and 8-form of galactose. To a rather surprising degree the series of increasing absorbability of monosaccharides was found to agree in both organs, although both mammals and amphibia were investigated. Among the inhibitors most interest was aroused by phloridzin whose effect on intestinal absorption was first described by Nakazawa (1922), later by Lundsgaard (1933), by Wertheimer (1933), and Oehnell and Hober (1939). The most direct evidence that phloridzin glycosuria is due to inhibition of glucose reabsorption appears to be the microanalysis of tubular fluid obtained by micropuncture and microperfusion in the experiments of Walker and Hudson ( 1937). In recent experiments Bogdanov and Barker (1950) found that in the intestine phloridzin does not inhibit the absorption of fructose in contrast with that of glucose and galactose. With the exception of the thermodynamically active character of the transport in kidney and intestine the similarity between the two organs and the red cells in general and even in details appears sufficiently remarkable to assume at least partial mechanisms in common. Accepting this inference the first conclusion to be discussed concerns the general features of the mechanism. Before entering this discussion a short note on the mechanisms suggested hitherto seems appropriate. Hober as early as 1899 assumed a chemical reaction of sugar in the epithelial cells of the intestine which would maintain a steep concentration gradient. The same suggestion was later made by Verzir (1931). Wilbrandt and Laszt (1933) assumed that the reaction is a phosphorylation. Lundsgaard (1933) and Wertheimer ( 1933) showing the inhibition of glucose absorption from the intestine by phloridzin, linked the problem of intestinal absorption to that in the kidney. Lundsgaard (1935) and Kalckar (1937) assumed for both organs phosphorylation at one end of the cell interior and dephosphorylation at the other. Recently Drabkin ( 1948) ascribed the phosphorylation to hexokinase and the dephosphorylation to phosphatase. These views differ in the number and details of the transporting elements rather than in their essential mechanism. Although the effect of the postulated reactions on the rate of diffusion through the membrane by way of the establishment of a steep gradient is stressed, this effect on the membrane passage is quantitative rather than qualitative. I n other words, diffusion occurs in the form of the unchanged free substrate molecule through a membrane which is virtually permeable to this molecule. A carrier function appears only in the interior of the cell where diffusion occurs in the form of the substrate-carrier complex, supposedly glucose phosphate.

82

TH. ROSENBERC A N D W. WILBRANDT

In the case of the erythrocyte no transcellular transport is involved ; the only structure traversed by glucose is the cell membrane and the transport mechanism therefore clearly is a membrane phenomenon. Considering the far-reaching similarity of the mechanisms shown above, it would seem highly improbable that the red cell uses a membrane carrier system, and the epithelial cell in kidney or intestine a cytoplasmic carrier system. Because of the different solubilities in cytoplasm and membrane, this would most certainly mean fundamentally different reactions which would hardly be in harmony with the close parallelism actually found. A second remark as to the alternative membrane carrier or cytoplasm carrier concerns the efficiency of the system. I n the cytoplasmic carrier system (essentially a special case of the double trapping mechanism mentioned earlier) the membranes are freely permeable to the substrate, although possibly not to the substrate carrier complex. Furthermore the operating space for the carrier, the cytoplasm, must necessarily be freely permeable both to the diffusing substrate-carrier complex and to the substrate itself. Back diffusion therefore would be inhibited only as long as the machine runs, and even if it runs at high speed a considerable energetic loss by back diffusion of substrate in the cytoplasm is unavoidable. The efficiency thus will be poor as compared with that of the membrane carrier system which operates with a membrane permeable only to the carrier and the carrier-substrate complex. It may be added that in cases of active ion transport (also occurring in epithelial cells), the carrier must necessarily operate in the membrane. This is particularly convincing in the case of hydrogen ion transport through the gastric epithelium. To apply here the mechanism of a cytoplasmic carrier would necessitate the assumption of coexisting p H values between about 1 and 7 within the same cell. A third point related to the question of membrane carrier or cytoplasmic carrier, the localization of phosphatase in the cell, will be discussed later. Concerning the details of the mechanism, in particular the nature of the participating enzymes, the action of phloridzin appears to be of central importance. I n the erythrocyte experiments there is evidence that phloridzin inhibits a trans enzyme catalyzing the dissociation of a glucose-carrier complex. On account of the general parallelism this type of action would a priori appear likely for the epithelial cells too. There is, however, additional indirect evidence. Ellinger and Lambrechts (1937) studied the action of colored azo derivatives of phloridzin. Among three such compounds two produced

ENZYMATIC PROCESSES I N CELL MEMBRANE PENETRATION

83

glycosuria; while a third, containing a sulfonate group, was inactive. The active compounds could be shown to enter the tubule cells, the inactive one not, which points to an intracellular action. If phloridzin acts intracellularly and, as concluded above, affects the dissociation of the substrate-carrier complex after passage through the luminal cell membrane and if furthermore the assumption of some sort of phosphorylation is to be upheld, the point of attack must be dephosphorylation rather than phosphorylation, in contrast with the original suggestion. This would be consistent with more recent findings on the enzymes inhibited by phloridzin, which include phosphorylase and phosphatase, whereas no inhibition of hexokinase has been reported. Phosphorylase, catalyzing the phosphorylysis of glycogen, a reaction which does not involve free glucose, would not appear to have any relation to the membrane transport of glucose. It may, however, have been involved in the experiments of Lundsgaard and others in which an inhibition of phosphate uptake in muscle brei and other systems by phloridzin was found. With regard to phosphatase a synthetic action of this enzyme appears to be excluded on account of the free energy of hydrolysis of glucose-6phosphate (3000 cal.) determined recently by Meyerhof and Green (1949). Thus an action of phloridzin on phosphatase would rather affect dephosphorylation or possibly transphosphorylations such as have been shown by Meyerhof and Green (1950). The assumption that phosphatase is in some manner involved in the transport of glucose is supported not only by the action of phloridzin but in a rather impressive way also by histochemical investigations on the distribution of alkaline phosphatase. In numerous studies (Gomori, 1939 ; Bourne, 1943/44; Dempsey and Deane, 1945/46; Ross and Ely, 1949) the brush borders of both intestinal and tubular epithelia have been shown to be extremely rich in alkaline phosphatase activity. In particular Bourne (1943/44) in a comprehensive study showed that the distribution of the enzyme in the intestinal tract coincides strictly with the regions of sugar absorption : positive reactions in duodenum, jejunum and rectum, negative in stomach and colon. In the kidney likewise the localization of alkaline phosphatase as shown histochemically and that of glucose reabsorption as known from micropuncture experiments coincide, both being found in the proximal tubules, In addition it was found (Wilmer, 1944) that the epithelium of the aglomerular kidney of Opsanus tau is devoid of alkaline phosphatase. Furthermore Marsh and Drabkin ( 1947) found considerable inhibition

84

T H . ROSENBERG A N D W. WILBRANDT

of alkaline phosphatase in undiluted kidney homogenates of phloridzin poisoned animals. They supposed that negative results of earlier authors in this respect had been due to higher dilutions used. Negative evidence based on histochemical methods as presented by Kritzler and Gutman (1941) is open to the criticism that loss of the inhibitor during the histochemical procedure appears possible. In erythrocytes the occurrence of alkaline phosphatase has been reported both on the basis of histochemical results (Bourne, 1943/44) and of enzymatic studies (Ranganathan and Patwardhan, 1949). Concerning the cell localization of the enzyme it should be emphasized that the histochemical pictures clearly show the bulk of the activity concentrated on the lumen side of the epithelial cells. This again would not be consistent with the assumption of the hexokinase-phosphatasesystem in the cytoplasm as pictured by Drabkin and discussed above. The principal difficulty arising from the fact that orthophosphate esters do not penetrate through cell membranes has been mentioned above in the discussion concerning the muscle. It was pointed out that various considerations favor the possibility that the penetrating ester is a metaphosphate, considerations which likewise might apply to the cases in question now. On the basis of such view penetration in the form of a metaphosphoric ester might be suggested as a primary step common to all five transports discussed : muscle, yeast cell, erythrocyte, kidney, and intestine cell. The further reactions then would differ, depending on the enzymatic conditions in the different cells, which would vary according to the use the cells have to make of the sugar. In muscle which is devoid of alkaline phosphatase no inhibitory action of phloridzin on glucose transport has been described. Here the carriersubstrate complex would not be dissociated, the ester being hydrated to glucose-&phosphate and taken up into the metabolic chain of reactions. The biological role of the enzymatically controlled transport then would be the quantitative regulation of the rate of metabolism, partly under the influence of insulin and other hormones. Possibly the situation in yeast cells (with the exception of the hormonal control) would be similar. In kidney and intestine, the substrate-carrier complex would be dissociated, and the metaphosphate, (at least in part) would be finally hydrated. The free energy of this reaction would enable the transport to become thermodynamically active. In red cells the carrier would remain in the membrane, possibly again as a metaphosphate ester of some membrane constituent such as a sterol, continuing to operate cyclically via ATP. The transport in this case would show no tendency to beconie thermodynamically active.

ENZYMATIC PROCESSES I N CELL MEMBRANE PENETRATION

85

Since phloridzin inhibits the transport both in erythrocyte and epithelial cells the common inhibited function of phosphatase might be a trans esterification rather than a hydrolysis, by analogy with the transphosphorylations by phosphatase described by Meyerhof and Green (1950).

VI. CONCLUSION Some concluding remarks may be made concerning the proposed elements of the transport mechanisms, their possible bearing outside the field of transport, the transport of substrates other than sugar, and the outlook for future developments. First it should be pointed out that the “enzymatically controlled transport” as defined here, does not appear to contain any elements that would be considered essentially new. The immediate driving force for the transport is the natural diffusion tendency of a compound due to a concentration difference. The movement of this compound, the substrate-carrier complex, from one enzyme protein to another has its counterpart in wellknown coupled enzyme systems. For instance, as Warburg (1948) has pointed out, in the hydrogen transfer by pyridine nucleotides reduced nucleotide (the hydrogen nucleotide complex) moves from one enzyme protein to the other. The same is true for adenosinetriphosphate (the phosphate adenosinediphosphate complex) in the transphosphorylation of glucose from phosphopyruvic acid. The assumption finally that such a compound is restricted to the membrane due to its solubility properties appears natural on the basis of Overton’s work. The carrier thus might with equal justification be considered as a lipoid-soluble or membranesoluble co-enzyme. The particular combination of these elements, however, may have implications that reach beyond the problems of membrane transport and which may be briefly touched upon. They concern the properties and actions of enzymes in vivo and in vitro, the possible regulatory function of membrane enzymes on the rate of metabolism and the possible role in isotope experiments. Enzyme pairs (or groups) of enzymatically controlled transports, due to their close vicinity and presumably strong attachment to the membrane, easily may appear as enzyme units. The properties of such “coupled enzymes” then are liable to show differences in vitro and in vivo, as not infrequently observed. To mention only two possibilities: if the cis and the trans enzyme have widely different p H activity characteristics, the in vitro action of the coupled enzyme may appear extremely low or it may show quite unexpected dependence on the pH.

86

TH. ROSENBERG AND W. WILBRANDT

Also the possibility may be mentioned that for some reason, of the two enzyme reactions I and I1 involved, I limits the overall rate in vivo, whereas in vitro the conditions are such that I1 becomes limiting. The striking fact for instance, that insulin activates hexokinase in solution only in the presence of antagonistic hormones, whereas in the living muscle cell such hormones are not necessary for the demonstration of the activation (Villee and Hastings 1949), may be due to such a situation. If hexokinase is a coupled enzyme in which reaction I (outside the muscle fiber) is limiting in viwo, and reaction I1 in solution, the addition of antagonistic hormones might be necessary in solution to slow reaction I sufficiently to make it limiting again so that its activation by insulin can be demonstrated. Finally the observations on autolysis should be mentioned. For the assumed separation of enzyme and substrate in Vivo and.the onset of enzymatic reactions under the conditions of autolysis membrane enzymes would provide a most natural explanation. Also autolysis as a necessary step in the preparation of certain enzymes may possibly be referred to their membrane enzyme character. The possible regulatory role of membrane enzymes with respect to the rate of metabolism was discussed in connection with the action of insulin, It may be added that it is probably not restricted to the initial steps in the sequence of reactions. The fact that A T P in cases like that of Lindberg and probably in others is partly located on the outer surface of the cells where it presumably also will be used and have to be regenerated points to the probable existence of more surface enzymes. If in a chain or a cycle of metabolic reactions membrane enzymes are involved at any point and if the overall rate is limited at this point, it may become subject to the influence of external factors, two of which may be briefly mentioned. First, such a factor may be a protein hormone. Considering the fact that these hormones (including insulin as discussed above) can hardly be assumed to penetrate cell membranes, their action remains quite obscure as long as intracellular processes are assumed as the point of attack. A second possibility of special interest is that of an enzymatically transported substance S, whose carrier C is a metabolite of the membrane enzyme reaction referred to. The diffusion tendency of S may then speed up the reaction by the establishment of a diffusion stream of SC. I n this case the rate relationship between metabolism and transport is mutual. Steward (1937) described the stimulation of metabolism in potato disks by addition of salts which are actively taken up. Fenn and Cobb (1934/35) similarly stimulated the metabolism in muscle by addition of potassium. Both cases may be pertinent here.

ENZYMATIC PROCESSES I N CELL MEMBRANE PENETRATION

87

The last point concerns the interpretations of results obtained in tracer experiments. The existence of enzymes and of coenzymes on the outer surface of cells may occasionally mislead one in the evaluation of changes in the specific activities. If for instance, as in Lindberg’s experiments, ATP exists in two fractions outside and inside the cell which are not in equilibrium and if this situation is not recognized, conclusions drawn as to the sequence of events may be quite erroneous. The example of glucose transport was chosen for discussion in this review because the number of pertinent observations and of cell types investigated as well as the conclusiveness of the results obtained appeared greater than in other transports. In no way was this selection meant to imply that the principles involved in other cases should be fundamentally different. In the opinion of the reviewers the basic elements will be common in some form to most of the “active” transports, but will necessarily differ in details concerning the nature of the enzymes and of the carriers involved. Particularly the presence of ionic charges will introduce special conditions. Only a few brief remarks on some of the existing observations will be made. Of other substrates phosphate, amino acids, and cations, particularly hydrogen ions have been the most studied so far. With all of them thermodynamically active transport is apparently possible. To the most impressive extent this holds for hydrogen ions in the stomach, where the concentration ratio is more than lo6, the free energy of transport being of the order of 10,OOO calories per mol HCl secreted. This enormous concentration ratio together with the observed fact (Davies and Ogston, 1950) of a high efficiency (as judged by the ratio of chemical work done to the surplus oxygen uptake) characterizes the requirements for an adequate interpretation. They are met in a quantitatively satisfactory way by a theory of Davies and co-workers (Crane, Davies, and Longniuir 1948; Davies, 1950/51; Davies and Ogston, 1950) which involves enzymatic reactions on both sides of the membrane and membrane carriers, and thus has essential features in common with the “enzymatically controlled transport” as defined above. A certain weakness would appear to lie in the assumption of two coexistent different mechanisms, one transporting hydrogen taken from hydrogen donors, the other hydrogen ions from the water. Also the more recent views of Conway (Conway and Brady, 1950; Conway et d., 1950) on acid secretion by yeast cells and gastric epithelium, although less detailed stoichiometrically and energetically, contain the elements of membrane carriers (with somewhat universal complex affinities).

88

T H . ROSENBERG A N D W. WILBRANDT

Cation penetration in nerve has become a probleni of carrier transport through the discoveries of Hodgkin and his group concerning the ion permeability changes during excitation and recovery (Hodgkin and Huxley, 1950). The movements of sodium and potassium in excitation must be ascribed to independent permeability changes for which special carriers are assumed. During recovery thermodynamically active transport of sodium occurs. I n marine algae Osterhout (1933) has early suggested a carrier mechanism for cation penetration. Phosphate penetration into yeast cells has been shown to be almost completely inhibited by azide (Spiegelman and Kamen, 1947). Considering the manifold affinities of phosphate its transport may be assumed to be linked with that of various other substances. Sperber and Renvall (1941) , for instance, offered evidence for such a linkage with thiazine transport. Amino acids resemble sugar in some ways. They are absorbed from the intestine at rates not proportional to the concentration difference (Hober and Hober, 1937) and reabsorbed in the kidney from lower to higher chemical potential (Robbins and Wilhelm, 1933), and show competition phenomena in tubular reabsorption (Pitts, 1943a, b) . In other respects they show special features. According to Christensen and co-workers (Christensen and Streicher, 1948, 1949 ; Christensen et al., 1948) and to Krebs and co-workers (Stern et al., 1949) various amino acids are taken up from lower to higher chemical potential in mammalian cells. The work of Gale and co-workers (Gale, 1947a, b, 1949; Gale and Taylor, 1947a, b ; Gale and Mitchell, 1947; Gale and Rodwell, 1949; Taylor, 1947, 1949) has revealed similar .phenomena in bacteria and yeast cells. This accumulation appears to depend on the metabolism of the cells, in a manner differing considerably in the various cell types investigated. Nothing is known about the enzymes directly involved in these cases, and in fact an enzymatically controlled transport in the meaning of the term used here has been assumed so far only with respect to hydrogen ions as mentioned and of alkali ions (Ussing, 1947, 1949). A few remarks concerning the possibilities of future development may finally be added. To obtain more information on membrane enzymes the systematic search for non-penetrating inhibitors which proved so useful in the case of red cells may be a hopeful approach. Paradoxical as it may seem, cases with thermodynamically active transport, although they are more easy. to recognize as enzymatically controlled, may offer greater difficulties than others in the elucidation of the

ENZYMATIC PROCESSES I N CELL MEMBRANE PENETRATION

89

enzymes directly involved. This arises from the fact that in these cases, due to the necessity of a continuous carrier flow across the membrane, possibly including generation and decomposition of the carrier on the two sides, reactions in the cytoplasm are bound to become involved. Thus inhibitors acting on the transport may well affect enzymes other than those on the membrane. I n some cases the indirect nature of such an inhibitory action can be made evident, if the addition of metabolites whose formation was inhibited reverses the inhibition of the transport. The reversal in this case shows that the cycle can be “bridged” at the point of interruption, which would not be possible if this point were directly concerned with the membrane transport. Examples published are numerous, for instance the inhibition of phenol red secretion in the mesonephros of the chicken by iodoacetate (Beck and Chambers, 1935), which can be reversed by the ad8tion of lactate, pyruvate, or succinate. One point which, in suggestions of particular transport mechanisms, has been widely neglected and on which progress will depend to a large degree, is the necessary direct and, in the case of equilibrium reactions, stoichiometrically quantitative connection between the transport reactions and the energy-providing reactions assumed. The chemical and energetic requirements of a transport cannot be considered separately. They are but different aspects of the same process. We wish to thank Dr. A. Goldstein for reading the manuscript and making valuable suggestions.

VII. REFERENCES Barany, E. H., and Sperber, E. (1939) Skattd. Arch. Physiol., 81, 290. Barany, E. H.,and Sperber, E. (1942) Ark. Zool., %A, No. 1. Barron, E. S. G., Muntz, J. A,, and Gasvoda, B. (1948/49) J . gen. Physiol., 82, 163. Beck, L. V., and Chambers, R. (1935) J. cell. comp. Physiol., 6, 441. Bogdanove, E. M.,and Barker, S. B. (1950) Proc. SOC.exp. Biol. Med., 76, 77. Bourne, G. (1943/44) Quart. 1. exp. Physiol., 32, 1. Bradfield, J, R. G. (1950) Biol Rex, 26, 113. Christensen, H.N.,and Streicher, J. A. (1948) J . Biol. Chem., 175, 95. Christensen, H.N.,and Streicher, J. A. (1949) Arch. Biochem., 23, 96. Christensen, H.N.,Streicher, J. A,, and Elbinger, R. L. (1948) I . biol. Chem., I=, 515. Colowick, S. P., Cori, G . T., and Slein, M.W . (1947) J . biol. Chem., 166, 583. Conway, E. J, and Brady, T. G. (1950) Biochem. J., 47, 360. Conway, E.J., Brady, T. G., and Carton, E. (1950) Biochem J., 47,369. Cori, C. F. (1925) J. hiol. Chem., 86, 691.

90

TH. ROSENBERG A N D W. WILBRANDT

Crane, E. E., Davies, R. E., and Longmuir, N. M. (1948) Biochem. J., IS, 321. Danielli, J. F., (1945) 1. exp. Biol., 22, 110. Danielli, J. F.,and Davson, H. (1934) J . cell. comp. Physiol., 6, 495. Davies, R. E. (1950/51) Gastroenterologio, 76, 78. Davies, R. E., and Ogston, A. G. (1950) Biochem. I., 46, 324. Dempsey, E. W.,and Deane, H. W. (1945/46) J . cell. comb. PhySiol., 27, 159. Diczfalusy, E., Ferno, O., Hogberg, B., Linderoth, T., and Rosenberg, Th. (1950) Abstr. XVIII Int. Physiol. Congr., p. 177. Dixon, M. (1948) Biochem. SOC. Symposia No. 2, Cambridge University Press, Cambridge, England. Donhoffer, S. (1935) Arch. cxp. Path. Pharmakol., 177, 689. Drabkin, D. L. (1948) Proc. Amer. Diabetes Asso., 8, 171. Ege, R. (1920) Biochem. Z.,111, 189. Ege, R. (1921) Biochem. Z.,114, 88. Eggleton, M. G. (1935) J . PhyySiol., 84, 59P. Ellinger, P., and Lambrechts, A. (1937) C. R. SOC.Bwl., M,261. Fenn, W.O.,and Cobb, D. M. (1934) J. gen. Physiol. 17, 629, (1935) Amer. J . Physiol., ll2, 41. Gale, E. F. (1947a) 1. gen. Microbiol., 1, 53. Gale, E. F.(1947b) J. gen. Microbiol., 1, 327. Gale, E. F. (1949) 1. gem. Microbiol., 8, 369. Gale, E. F., and Mitchell, P. D. (1947) J. gen. Microbiol., 1, 299. Gale, E. F.,and Rodwell, A. W. (1949) J. gen. Microbiol., 8, 127. Gale, E. F.,and Taylor, E. S. (1947a) J . gen. Microbiol., 1, 77. Gale,’E. F., and Taylor, E. S. (1947b) 1. gen. Microbiol., 1, 314. Gomori, G. (1939) Proc. SOC.exp. Biol. Med., 42, 23. Grijns, G. (1896) Arch. ges. Physiol., 69, 86. Guensberg, E. (1947) Inaugural Dissertation, Berne. Hamburger, H. S. (1924) Ergebn. Physiol., !AS, 77. Hedin, S. G. (1897) Arch. ges. Physiol., 68, 229. Hedin, S. G. (1898) Arch. ges. Physiol., 70, 525. Hober, R: (1899) Arch. ges. PhySiol., 74, 246. Hober, R. (1911) Physikalische Chemie der Zellen und der Gewebe, Leipaig, p. 264. Hober, R. (1933) Arch. ges. Physiol., !BB, 181. Hober, R., and Hober, J. (1937) 1. cell. corn). Physiol., 10, 401. Hodgkin, A. L., and Huxley, A. F. (1950) Abstr. XVIII Int. Physiol. Congr. p. 36. Hodgkin, A. L., and Huxley, A. F. (1950) Personal communication. Kalckar, H. M. (1937) Ewymologia, 2, 47. Kalckar, H. M.,Dehlinger, J., and Mehler, A. (1944) J . Biol. Chem., 154, 275. Klinghoffer, K. A. (1940) A m . I. Physiol., 180, 89. Kozawa, S. (1914) Biochem. Z.,SO, 231. Kritzler, R. A.,and Gutman, A. B. (1941) Am. 1. Physiol., 184, 94. LeFevre, P. G. (1947) Biol. Bull., 98, 224. LeFevre, P. G. (1948) I. gen. Physiol., W , 505. Lindberg, 0. (1948) Ark. Kemi Min. Geol., 26B, 13. Lindberg, 0. (1950) Exp. Cell. Res., 1, 105. Lundsgaard, E, (1933) Biochem. Z.,264, 209. Lundsgaard, E. (1933) Biochem. Z.,264, 221. Lundsgaard, E. (1935) Skond. Arch. Physiol., 72, 265.

ENZYMATIC PROCESSES I N CELL MEMBRANE PENETRATION

91

Lundsgaard, E. (1939) Upsala Lakare forenings forhandlinger XLV, p. 141. Marsh, J. B., and Drabkin, D. L. (1947) J . bwl. Chem., 168, 61. Masing, E. (1913) Arch. ges. PhySol., 149, 227. Masing, E. (1914) Arch. ges, Physiol., l60,401. Meyerhof, O., and Green, H. (1949) J . biol. Chem., 178, 655. Meyerhof, O., and Green, H. (1950) J . biol. Chem., 189, 377. Myrback, K., and Oertenblad, B. (1936) Biochem. Z., 288, 329. Myrback, K., and Oertenblad, B. (1937) 2. Biockm., Bl, 61. Myrback, K., and Vasseur, E. (1943) Z. physiol. Chem., 477, 171. Nagano, J. (1902) Arch. ges. Physiol., 90, 389. Nakazawa, F. (1922) Tohoku J . Exp. Med., 3, 288. Oehnell, R., and Hijber, R. (1939) J . cell. comp. Physiol., 15, 161. Osterhout, W. J. V. (1933) Erg. Physiol., 98, 967. Overton, H. (1902) Arch. ges. Physiol., a,115. Peters, J. R., and Van Slyke, D. D. (1946) Quantitative Clinical Chemistry: Vol. I, Interpretations. Williams and Wilkins, Baltimore. P. 155. Pitts, R. F. (1943a) Am. 1. Physiol., 140, 156. Pitts, R. F. (1943b) Am. J . Physiol., 140, 535. Rahn, O., and Leet, M. (1949) J . Back, 68, 714. Ranganathan, S., and Patwardhan, V. N. (1949) Indium J. med. Res., 37, 233. Richards, A. N. (1938) Proc. roy. SOC.,Bl26,398. Robbins, S., and Wilhelm, M. L. (1933) Arch. ges. Physiol., aSa, 66. Rona, A., and Michaelis, L. (1909) Biochem. Z., 1660; 10, 375. Rona, A., and Doblin, H. (1911) Biochem. Z., 31, 215. Rosenberg, Th. (1948) Acfa. chem. scand., 2, 14. Rosenberg, Th. (1950) Rep. Steno Mem. Hosp. 6. Nord. Insulin Lab., 4, 59. Ross, M. H., and Ely, J. 0. (1949) 1. cell. comp. Physio?., S4, 71. Rothstein, A. (1950) Abstr. XVIII Int. Physiol. Congr., p. 421. Rothstein, A., Frenkel, A., and Larrabee, C. (1948) 1. cell. comp. Physiol., 83, 261. Rothstein, A., and Larrabee, C. (1948) J . cell. comj. P h y ~ o l . ,32, 247. Rothstein, A., and Meier, R. (1948) J . cell. comp. Phyysiol., Sa, 77. Rothstein, A., and Meier, R. (1949) J. cell. comp. Physiol., 34,97. Rothstein, A., and Meier, R. (1951) J . cell. comp. Physiol. (In press,) Rothstein, A., Meier, R., and Hurwitz, L. (1951) J . cell, comp. Physiol. (In press.) Runnstrom, J., Sperber, E., and Feller, W. (1938) Natumkenschaften, 26, 547. Sacks, J. (1944) Amer. J. Physiol., la, 145. Sacks, J. (1948) Cold Spr. Harb. Symp. quant. Biol., 13, 180. Shannon, J. A. (1938) Amer. J . Physiol., l22, 775. Shannon, J. A., and Fisher, S. (1938) A m r . J . Physiol., 222, 765. Sperber, E., and Renvall, S. (1941) Biochem. Z., SlO, 160. Spiegelman, S., and Kamen, M. D. (1947) Cold Spr. Harb. Symg. quant. Biol., l2, 211. Stern, J. R., Eggleston, Hems R., and Krebs, H. A. (1949) Biochem. J . , 44, 410. Steward, F. C. (1937) Trans. Faraday SOC., 33, 1006. Taylor, E. S. (1947) J . gelz. Microbiol., 1, 86. Taylor, E. S. (1949) J . gem. Microbiol., 3, 211. Ussing, H. H. (1947) Nature, 160, 262. Ussing, H. H. (1949) Physiol. Rev., 29, 127. Ussing, H. H. (1950) Abstr. XVIII Int. Physiol. Congr., p. 58.

92

TH. ROSENBERG AND W. WILBRANDT

Verzlr, F. (1931) Ergebn. Physiol., 83, 391. Verzir, F. (1935) Biochem. Z., a76, 17. Vidal-Sivilla S. (1950) Rev. esp. Fidol., IV, 131. Villee, C. A., and Hastings, A. B. (1949) I . biol. Chem., 179, 673. Vishniac, W. (1950) Arch. Biochem., 20, 167. Walker, A. M., and Hudson, C. L. (1937) Amer. I . Phydol., ll8, 130. Warburg, 0. (1948) Wasserstoff iibertragende Fermente. Berlin. P. 36. Wertheimer, E. (1932) Kolloid-Z., 81, 181. Wertheimer, E. (1933) Arch. ges. Physiol., aQ8, 514. Wertheimer, E. (1934) Protoplasma, 21, 522. Westenbrink, H. G. (1936) Nature, Lord., 138, 203. Wilbrandt, W. (1938) Arch. ges. Phydol., !M, 302. Wilbrandt, W. (1947) Helv. physiol. Acto, S, C64. Wilbrandt, W. (1950) Arch. exp. Path. Pharmkol., 2l2, 9. Wilbrandt, W., Guensberg, E., and Lauener, H. (1947) Helv. ghysiol. Actu, 6, C20. Wilbrandt, W., and Haemmerli, A. (1951). HeIv. physiol. Acta, (In press.) Wilbrandt, W., and Laszt, L. (1933) Biochem. Z., 269, 398. Wilbrandt, W., and Rosenberg, Th. (1950) Helv. physiol. Acta, 8, C82. Wilbrandt, W., and Rosenberg, Th. (1951) ESP. Cell. Res., (In press.) Wilkes, B. G., and Palmer, E. T. (1933) I . gen. Physol., ls, 233. Wilmer, H. A. (1944) Arch. Path., 87, '227.

Bacterial Cytology K. A. BISSET Department of Bacteriology, University of Birmingham, England

CONTENTS I. 11. 111. IV. V. VI. VII. VIII.

Introduction ....................................................... The Bacterial Nucleus ............................................. Growth and Cell Division in Bacteria ................................ Granular Inclusions ................................................ Bacterial Flagella .................................................. Specialized Reproductive Methods ................................... Cytology and Systematics ........................................... References .........................................................

Page 93

96 98 100 102 102 103 104

I. INTRODUCTION This review is not intended to be an exhaustive survey of the literatureupon the subject, but rather to bring to the attention of cytologists, familiar with other types of cell, some of the more fundamental studies upon bacterial cytology, and to give, at the same time, a general picture of the present state of our knowledge and some Indication of the trend of research. A fuller account of available methods of study and some attempt at correlation of the information upon various groups of bacteria is given in the author’s recent monograph (Bisset, 19504. Until recently the progress of bacterial cytology has been considerably retarded by several factors. The first of these is obvious; bacteria are so small that the entire cell of many species is no greater in size than the smallest structures comprising animal or plant cells. The second and third factors might, upon casual consideration, appear to be advantages. Bacteria are of such great practical importance as pathological and biochemical agents that the number of persons engaged upon their study has for long been very considerable, in comparison with those who have been similarly engaged upon most other natural groups, and microscopical examination, in the routine methods of these studies, is facilitated by the resistance of the bacterial cell to the simple techniques of drying and heat fixation, by means of which they can be examined and even identified microscopically within rather broad limits. A useful and widely employed adjunct to these techniques in routine bacteriology is Gram’s stain. Bacteria stained by one of the pararosaniline violet dyes and “mordanted” with iodine solution may or may not be capable of resisting decolorization

93

94

K . A. BISSET

with organic solvents. Those which retain the blackish Gram-complex are termed Gram-positive. This character is of considerable assistance in the practical identification of bacterial species, but serves to obscure cytological detail. I t has been suggested from time to time that a wide variety of different dyes may be employed and that almost any mild oxidizing agent can replace the iodine in this reaction. Recently, however, Bartholomew and Mittwer (1950) and Mittwer, Bartholomew, and Burton (1950) have brought evidence to show that the specificity of both the violet dye and of the iodine in this reaction is greater than had been supposed. The practical result of these various factors has been that attention has been focussed upon practical problems to the almost complete exclusion of fundamental, biological studies. At the same time, the simple, “routine” methods of bacteriological microscopy have resulted in neglect of the more complex techniques required for true cytological study. It would nevertheless be misleading to suggest that nothing was known of bacterial cytology until recently. The work of Schaudinn (1902, 1903) upon sporulation and cell division has been proved correct in general outlines, giving due allowance for the difficulty, by the techniques then available, of demonstrating the vegetative nucleus. Gutstein ( 1924) stained the nuclei of several bacterial species by methods fundamentally similar to those which are now generally employed. An even more striking demonstration of the bacterial nucleus was given by Paillot (1919) in Giemsa-stained preparations, the clarity of which was not exceeded for twenty years. One of the first convincing descriptions of the bacterial life cycle was that of Stoughton (1929, 1932), who showed the formation of the resting cell and some of the associated cytological processes. All these early researches, although they covered a period of approximately thirty years, suffered from a number of very similar defects. They were isolated studies of single bacterial species (or of a very small number of species) and seldom of those with which the majority of bacteriologists were most familiar. Schaudinn and Paillot worked with bacteria isolated from insects, Stoughton with a plant pathogen. It was thus tempting for bacteriologists to discount these observations as inapplicable to the bacteria which they commonly encountered. For this reason also, although modern bacterial cytology may be considered to have received considerable stimulus from the work of Badian (1930, 1933a) and Krzemieniewska (1930) upon myxobacteria and cytophagas, it was the demonstration by Stille (1937) and Piekarski (1937) of the Feulgen-positive nuclear granules of the typhoid bacterium and other well-known species which first attracted real attention.

BACTERIAL CYTOLOGY

95

Piekarski’s subsequent demonstrations by ultraviolet light and by the HC1-Giemsa technique ( 1938-1939) were followed by the magnificent photomicrographs of Robinow (1942, 1944) , which clearly showed the nucleus, stained by the latter method, as a rod-shaped structure, lying transversely to the long axis of the cell. The details of the nuclear cycle were known for myxobacteria (Badian, 1930, 1933a ; Krzemieniewska, 1930 ; Klieneberger-Nobel, 1947a) and for sporing bacilli (Badian, 1933b ; Klieneberger-Nobel, 1945) several years before the similar condition of non-sporing eubacteria was recognized (Bisset, 1949a, 1950a), although the work of Stoughton (1932), albeit upon a rather exceptional type of bacterium, clearly foreshadowed this knowledge. The study of the cell envelopes and surface structures of bacteria has followed a similar course to that of the study of the nucleus. The accurate, if rather incomplete, observations of Schaudinn (1902, 1903) have been confirmed, at least partially, by many later workers. Bisset (1939, 1947) extended Schaudinn’s demonstration of the two major methods of cell division, and Robinow (1945), Pringsheim and Robinow (1947), and Bisset (1947, 1948a, 194913) showed that many bacteria were composed of numerous small cells, and were not, as had usually been assumed, unicellular. Investigations of the nucleus, where inadequate attention has been given to the problem of the cell envelopes, has led to the description of multinucleate cells which were, in fact, multicellular bacteria. This applies, to some extent, to the descriptions of Curyophanon (a very large bacterium from cow dung) by Peshkoff (1940), of Mycobucterium tuberculosis by Brieger and Robinow (1947), although these workers apparently suspected the existence of cross walls which they were unable to demonstrate, and of Bacillzls mthracis, by Flewett (1948). A very extensive study of M . tuberculosis by Knaysi, Hillier, and Fabricant ( 1950), employing both electron and photomicroscopy, is rendered much less valuable than it should have been by the failure of these workers to appreciate the multicellularity of this organism. The same criticism applies to the recent studies of DeLamater (1951) upon supposed “mitotic spindles” and “fusion tubes” in Bacillus megatherium. Cross walls have been demonstrated by many different techniques : Burdon ( 1946) stained the lipoid components, and Pennington (1949) and Jarvi and Levanto (1950) stained similar polysaccharide structures by the use of periodic acid and silver nitrate. Lamanna and Mallette (1950) investigated the nucleic acids of the cell envelopes. The variety of these cytochemical reactions serves to emphasize the complexity of these surface

96

K. A. BISSET

structures. The nature of the surface materials which obscure the bacterial nucleus is discussed in the next section. As the majority of recent cytological studies have been published in English, detailed reviews have been published in other languages (Piekarski, 1949; Leonardi, 1949; Parvis, 1949).

11. THE BACTERIAL NUCLEUS In many bacteria the nucleus can readily be demonstrated by classical cytological methods, and this has been done by many workers in the past. Failure to recognize the nucleus has been due most frequently to the practice of examining heat-fixed preparations, which are both shrunken and increased in opacity, and to the strong affinity for basic dyes of the bacterial cytoplasm, especially the surface layers and cell membrane. This stainable material, which is composed largely of ribose nucleic acids (Henry and Stacey, 1943, 1946) , masks the underlying nuclear structures. According to Henry and Stacey, these nucleic acids are responsible for the phenomenon of Gram-positivity, where this occurs. This, however, has been contradicted by Mitchell and Moyle (1950), who attribute Grampositivity to the presence of a phosphoric ester, which is not correlated with the proportion of ribose nucleic acids present. Whichever of these materials is responsible for the masking effect of the surface material, the practical difficulty can be overcome by means of a mild hydrolysis in dilute hydrochloric acid, which has the effect of reducing the.affinity of the surface structures for dyes while at the same time rendering the deoxyribose nucleic acids of the nucleus more readily stainable. This is the basis of the acid-Giemsa technique, which is now widely used for bacterial cytology. It is, of course, obviously derived from the Feulgen reaction, but produces much clearer preparations. By this means Robinow (1942, 1944, 1945), extending the observations of Piekarski (1937) and others recorded in the previous section, demonstrated that the bacterial nucleus, in very young cultures, consisted of dumb-bell shaped rods, lying transversely to the long axis of the bacterium and dividing longitudinally (Figs. la, 4). Confirmation of this mode of division was made, in living material, by phase-contrast microscopy (Tulasne, 1949 ; Stempen, 1950). Haselmann and Kappel (1949) demonstrated the nucleus of cocci by the same technique. The definition obtained by this method, however, still compares unfavorably with that of classical microscopy. It was subsequently shown (Bisset, 1948b, 1948~)that the nuclear structures are paired, and that they divide, even in young cultures, not

BACTERIAL ,.CYTOLOGY

97

FIG.1. The Nucleus of the typhoid bacterium. a. Vegetative cell with paired chromosome complexes. b. Resting cell with vesicular nucleus of markedly yeastlike character.

by simple fission alone, but also by a method analogous to syngamy in fungi. I t appears (Bisset, 1948d) that certain bacterial species do not possess this chromosome-like nucleus but a vesicular structure of more typical appearance, and also (Bisset, 1949a, 195Oa, 1950b) that this vesicular nucleus was found in the resting cells of bacteria in which the vegetative nucleus was chromosomal and, presumably, semipermanently mitotic (Fig, lb). Similar cycles are well known in the case of myxobacteria (Badian, 1930, 1933a ; Beebe, 1941 ; Klieneberger-Nobel, 1947a), in cytophagas (Krzemieniewska, 1930 ; Grace, 1949), streptomyces and actinomyces (Klieneberger-Nobel, 1947b ; Morris, 1951) and sporing bacilli (Badian, 1933b; Klieneberger-Nobel, 1945 ; Bisset, 195Oc), mainly because these bacteria possess well-marked resting stages, the formation of which could be studied. The parallel condition in eubacteria escaped notice for a long time because the resting cell is often inconspicuous and appears only in the senescent stages of culture in many strains. The process of maturation of the even more inconspicuous resting cell of the tubercle bacillus was, however, fully described at a much earlier date (Lindegren and Mellon, 1933), although this interesting work was almost completely ignored. Despite superficial differences between the resting cells of the various bacteria, the cytological processes accompanying their maturation are fundamentally similar (Fig. 2). The chromosome-like vegetative nucleoids fuse to form a longitudinal rod-shaped fusion nucleus, which divides and

98

K . A. BISSET

born@@@@ e

f

9

abed

FIG. 2. Maturation of resting cell. A general scheme, typical of nearly all bacteria: a, vegetative cell; b, rod-shaped fusion nucleus; c, d, division and reconjugation of nucleus ; e, f, reduction ; g, mature resting cell.

subsequently recombines, either sexually or autogamously, usually the latter. This fusion is followed by a reduction division of the nucleus, either accompanied by cell division (Lindegren and Mellon, 1933) or within the undivided cell (Bisset, Grace, and Morris, 1951). One daughter nucleus degenerates or is rejected, the other becomes the nucleus of the resting cell, spore or microcyst, as the case may be. In addition to the papers quoted, which are mainly interpretative and based upon stained preparations, the reduction process in the sporulation of a large Bacillus has been observed throughout by phase-contrast microscopy (Pulvertaft, 1950). By this technique the rejected nucleoid could actually be observed to be extruded from the sporangium. As well as the numerous studies of the bacterial nucleus performed by methods which are now classical, modifications of these techniques have been invented. These do not require detailed comment; Cassel (1950) employed cold perchloric acid for the hydrolysis procedure ; Smith (1950) recommended fixation in formaldehyde after hydrolysis ; and the effect of formaldehyde upon the stainability of bacteria was also investigated by Pennington (1950). None of these refinements of technique has been productive of any notably original observation.

111. GROWTH AND CELLDIVISION IN BACTERIA Although, as stated above, Schaudinn (1902, 1903) recorded a number of fundamental and reasonably accurate observations upon the mode of

division of the bacterial cell, showing that the rigid cell wall may form new cross walls, which split when the cell divides (Fig. 3), or, in other morphological types, may divide by a constrictive ingrowth of the cell wall (Fig. 4), more attention appears to have been attracted by the description by Graham-Smith ( 1910) of the “post-fission movements.” These are the “snapping,” “slipping,” or “whipping” movements of daughter bacilli upon division, and they depend upon observation of growing bacteria in the restricted conditions between a cover-slip and a block of solid medium, in which Graham-Smith and several later workers (Nutt,

BACTERIAL CYTOLOGY

FIG.3. Multicellular bacilli and cocci. This type of bacillus is divided by cross walls and subdivided by septa derived from the cell-membrane, by which new cross-walls are secreted. The two isolated bacilli are at an earlier stage of division.

FIG.4. Cell division in bacteria. New cell wall material is secreted at cell tips and septa. 1927; Seal, 1936) examined them. When growth is not thus mechanically restricted the “post-fission movements” do not occur (Bisset, 1939) ; nevertheless this unimportant artifact has been quoted in innumerable textbooks of bacteriology, in default of more valuable information upon cell division. It was shown by Bisset (1939, 1947, 194%) that the septum-formation and constriction modes of division occur in many different bacterial groups, and that the former results in the production of multicellular bacilli, even cocci. The cells are divided by septa in various stages of formation, and characteristically a bacillus of this type is divided in the center by a complete cross wall, by the splitting of which the bacillus divides, and each half is subdivided by septa in process of formation, including a component derived from the cell membrane (Fig. 3). In growth and cell division the new cell walls are secreted by specialized areas of the underlying cell membrane, which stain strongly with basic dyes, because of the concentration of secretory nucleic acids. In cell division of either type, a secretory septum is first formed across the cell,

100

K. A. BISSET

and by it the new cross wall or the constrictive ingrowth of the cell wall is secreted (Fig. 4) (Robinow, 1945; Bisset, 1947). The growth of the cell wall occurs at the tip of the bacterium, usually at only one tip, where a similar concentration of nucleic acids underlies the thin, newly formed wall (Figs. la, 4) (Bisset, 1951). Certain bacteria, notably corynebacteria and mycobacteria are subdivided into as many as twelve or more small cells, by means of cross walls (Bisset, 1949b ; Burdon, 1946), but these are formed in a rather less regular pattern (Fig. 5).

Fig. 5. Typical corynebacteria. In dried preparations of C. diphtheria the shrunken contents of the enlarged terminal cells are the “metachromatic granules.”

The various structures concerned in cell division can be demonstrated with varying degrees of difficulty. The cell wall normally resists staining, but can be rendered stainable by mordanting with tannic acid (Robinow, 1945). By this means the complex, multicellular structure of many different bacterial species can be demonstrated (Bisset, 1948a, 1949b; Bisset and Moore, 1949). Cell membranes and septa derived from them can be stained by simple dyes and by the acid-Giemsa technique (Robinow, 1945; Bisset, 1947). The existence and thickness of this structure can be calculated from physical considerations (Mitchell, 1949). The secretory septa and “growing points” are clearly seen in preparations fixed by classical cytological methods and may stain so strongly, in contrast with the remainder of the cell, as to be confused with nuclear structures ( Imsenecki, 1948; Bondarenko-Zozulina, 1949; DeLamater, 1951).

IV. GRANULAR INCLUSIONS The routine methods of drying and heat fixation of bacterial material have led to the description of several different types of granular inclusion in the cell, some of which are genuine, but most of which are unquestionably artifacts, or other structures so distorted as to be unrecognizable. Much attention has been given to the “metachromatic granules” of

BACTERIAL CYTOLOGY

101

Corynebucterium diphtheriae, which appear as darkly stained terminal granules in a dumb-bell-shaped bacterium, when stained by one of the appropriate diagnostic methods such as Neisser’s stain. Recently Wachstein and Pisano (1950) have devised a new method for staining these and similar granules with lead salts, and Oeding (1949) has reinvestigated their constitution, and states (in agreement with a long-established doctrine) that they are composed of volutin, although, like most users of this term, he fails to define it. It has been shown (Bisset, 1949b) that these granules are the shrunken contents of the enlarged, terminal cells of C. diphtheriae, and that their appearance in dried preparations is an artifact (Fig. 5 ) . The granular appearance of Mycobacterium tuberculosis can be explained in the same way. Granules of glycogen-like substance do, in fact, appear in certain bacterial species and may be reserve food material (Lewis, 1941). Globules of elementary sulfur appear in the cells of photosynthetic sulfur bacteria, in some cases (Molisch, 1912). Lipoid granules and sulfur granules may be present simultaneously (Vahl, 1910). The so-called bipolar staining is, at least partially, an optical illusion, because of the concentration of stainable material in the surface layers of the bacterial protoplasm, across which a longer path is traversed by light passing tangentially through the tips of the cell (Fig. 6). Wei, Tchan,

a

b

FIG.6. “Bipolar staining.” The passage of light by paths of different length through the chromophilic cortex of the bacterial protoplasm (a) gives the effect of a concentration of stainable material at the poles of the image (b) in lightly stained preparations.

and Pochon ( 1948), however, investigated certain “polar granules” in Pasteuvella and Pfeiferella and concluded that they were nuclear bodies. From their figures it is impossible to say with what they were, in fact, dealing. Many bacteria stain strongly at one pole, because of the concentration of nucleic acids in the growing point (Figs. la, 4) (Bisset, 1951).

102

K. A. BISSET

V.

BACTERIAL FLAGELLA

Except in the case of myxobacteria, which can move upon surfaces by the muscular contraction of the cell wall, all bacterial motility is by means of flagella. These flagella are very fine structures, spirally coiled, and composed, at least in the case of Protezcs, of a myosin-like, fibrous protein, (Astbury and Weibull, 1949). The hagella point away from the direction of motion and may become twisted together at the pole of the cell. Mainly upon this appearance Pijper (1946) propounded his theory that the apparent flagella are “twirls” of polysaccharide mucus, drawn off from the slime layer of the cell by virtue of its motility, which is due to some other (rather ill-explained) cause. This is contrary to all the available evidence upon bacterial flagella. By electron microscopy it can be seen that the flagella pass through the cell wall (van Iterson, 1947) and are attached to the blepharoplasts (Fig. l a ) (Houwink and van Iterson, 1950; Bisset and Hale, 1951). The microcyst or resting cell of flagellated bacteria is devoid of flagella, which may be observed to grow out through the cell wall as the microcyst germinates, usually commencing at one pole of the cell (Bisset and Hale, 1951). The arrangement of the flagella, whether singly or in tufts at the poles of the cell, or in larger or smaller numbers, all over its surface, is probably indicative of varying degrees of adaptation to a terrestrial existence (Bisset, 195Od) ; polar flagellation is most efficient for swimming in a dilute medium ; peritrichous flagellation bestows some degree of ability to move in viscous materials, and even upon moist surfaces.

VI. SPECIALIZED REPRODUCTIVE METHODS Many bacteria appear habitually to reproduce by means of very small bodies, gonidia, which may be so small as to be capable of passing an antibacterial filter. Reports of this phenomenon in mycobacteria (Wyckoff and Smithburn, 1933; Wyckoff, 1934) are possibly based upon the ability of this multicellular organism to fragment into tiny, individual cells (Bisset, 1949b), an ability which is shared by corynebacteria and similar multicellular bacteria. In other genera, however, notably Azotobacter (Jones, 1920; Lohnis, 1921) and Bacillus (Allen, Appleby, and Wolf, 1939), the evidence of specialized, reproductive gonidia is very convincing. The swarmers of Rhizobium are well known. Closely associated with this problem is that of the L-organisms, resembling the infective agent of bovine pleuropneumonia, which were first described by Klieneberger (1935, 1936) as symbionts or parasites in

BACTERIAL CYTOLOGY

103

cultures of Streptobacillus monilifomis, but which were shown by Dienes (1939, 1946) to be stages in the life cycle of this and other bacteria. Dienes claimed that the minute colonies, which so closely resembled those of the pleuropneumonia organism, were released from swellings upon the bacterial threads, and that the tiny bacilli of which they were composed grew up into a new generation of typical bacteria (Fig. 7). This view was long

FIG.7. L-type reproduction in bacteria. Diagrammatic scheme of the condition in Streptobacillus moniliformis. Fusion of nuclear granules in a bacillary filament gives rise to a swelling from which an L-colony is released. After reproduction in this phase the small elements grow into a new generation of bacilli.

contested, but eventually accepted by Klieneberger ( Klieneberger-Nobel, 1949). Similar L-forms have been observed to occur in cultures of Neisseria (Brown and Hales, 1942), of Fusifornzis ( Klieneberger-Nobel, 1947c), of Salmonella and Shigella (Dienes, Weinburger, and Madoff, 1950 ; Weinburger, Madoff, and Dienes, 1950), and of Proteus (Dienes, 1946; Freundt, 1950; and others). Spirochaetes may also reproduce both by transverse fission and by the production of cysts, which may release numerous, tiny gonidia (Hampp, Scott and Wyckoff, 1948), or germinate into single spirochaetes (DeLamater, Wiggall, and Haanes, 1950).

VII. CYTOLOGY AND SYSTEMATICS The author has discussed, at some length, in a recent monograph (Bisset, 195Oa) the problem of the application of recent advances in our knowledge of morphology and cytology to the study of bacterial syste-

104

K. A. BISSET

matics. Very few papers have been published upon the subject, but it has been shown that the supposed typical appearance of many bacteria gives a misleading notion of their actual structure (Bisset, 1948a, 1949b), that such taxonomic groups as Coccaceae are morphologically heterogeneous (Bisset, 1949c) and that some degree of order can be established within groups in which morphological criteria are, apparently, of little value, by comparison of such characters as the morphology of the microcyst in Bacteriaceae (Bisset, 1 9 5 0 ~ )or of the type of branching in Actinomycetales (Bisset and Moore, 1949). It is even possible to discern some kind of evolutionary system among bacteria, from spiral, aquatic types, with polar flagella, to those capable of progress upon solid surfaces and of producing airborne spores or microcysts for the distribution of the species (Bisset, 195Od). Recently Cowan (1950) has produced an outline classification of bacteria, based to some extent upon the author’s suggestions. VIII. REFERENCES Allen, L. A., Appleby, J. C., and Wolf, J. (1939) Zbl. Bakt., Abt. II, 100, 3. Astbury, W. T.,and Weibull, C. (1949) Nature, Lond., la, 280. Badian, J. (1930) Acta. SOC.Bot. Polon. 7 , 55. Badian, J. (1933a) Acfa. SOC.Bot. Polon., 10, 361. Badian, J. (1933b) Arch. Mikobiol., 4, 409. Bartholomew, J. W., and Mittwer, T. (1950) Stain Tech., I,103. .2 , , 193. Beebe, J. M. (1941) 1. B Q C ~ 4 Bisset, K. A. (1939) 1. Path. Bact., 48, 427. Bisset, K. A. (1947) I . gen. Microbiol., 2, 83. Bisset, K. A. (1948a) 1. gen. Microbiol., 2, 126. Bisset, K. A. (1948b) 1. Hyg., Comb., 46, 173. Bisset, K. A. (1948~) J . gen. ddicrobiol., 2, 248. Bisset, K. A. (1948d) 1. Hyg., Camb., 46, 264. Bisset, K. A. (1949a) J. Hyg., Camb., 47, 182. Bisset, K. A. (1949b) 1. gen. Microbiol., 8, 93. Bisset, K. A. (1949~) 1. gen. Microbiol., 8, ii. Bisset, K. A. /1950a) The Cytology and Life History of Bacteria. Livingstone. Edinburgh. Bisset, K. A. (1950b) Exp. Cell. Res., 1, 473. Bisset, K. A. (1950~) 1. gen. Microbiol, 4, 1 . Bisset, K. A. (1950d) Nature, Lond., 166, 431. Bisset, K. A. (1950e) J. gen. Microbiol., 4, 413. Bisset, K. A. (1951) J. gen. Microbwf., 5, 161. Bisset, K. A., Grace, J. B., and Morris, 0. E. (1951) Exp. Cell. Res. 8, 388. Bisset, K. A., and Hale, C. M. F. (1951) J . gen. Microbiol., 8, 156. Bisset, K. A., and Moore, F. W . (1949) 1. gen. Microbiol., 8, 387. Bondarenko-Zozulina, M. 1. (1949) Microbiologia (Moscow), l8, 346. Rrieger, E. M., and Rohinow, C. F. (1947) J. Hyg., Camb., 4S, 413.

BACTERIAL CYTOLOGY

105

Brown, T. McP., and Hales, G. S. (1942) J . Bact., 4$, 82. Burdon, K. L. (1946) J . Bact., 63, 665. Cassel, W.A. (1950) J . Bact., 1,185. Cowan, S. T. (1950) An Outline Classification of Bacteria. National Collection of Type Cultures. DeLamater, E. D., Wiggall, R. H., and Haanes, M. (1950) J . exp. Med., a,239. DeLamater, E. D. (1951) Cold Spr. Harb. Symp. quant. Biol., 16. (In press.) Dienes, L. (1939) J. Infect. Dis., 66, 24. Dienes, L. (1946) Cold Spr. Harb. Symp. quant. Biol., 11, 51. Flewett, T. H. (1948) J . gen. Microbiol., 2, 325. Freundt, E. A. (1950) Acta path. microbiol. scand., W ,159. Grace, J. B. (1949) Personal communication. Graham-Smith, G. S. (1910) Parasitology, 3, 17. Gutstein, M. (1924) Zbl. Bakt., Abt. I. I, 393. Hampp, E. G., Scott, D. B., and Wyckoff, R. W. G. (1948) J. Bact., 66, 755. Haselmann, H., and Kappel, W. (1949) Arch Derm. Syph., N . Y., 187, 501. Henry, H. E., and Stacey, M. (1943) Nature, Lond., 151, 671. Henry, H. E., and Stacey, M. (1946) Proc. roy. Soc., BUS, 391. Houwink, A. L., and van Iterson, W. (1950) Biochim. Biophys. Acta., 6, 10. Imsenecki, A. A. (1948) Microbiologia, (Moscow), 17, 218. van Iterson, W. (1947) Eiochim. Biophys. Acta, 1, 527. Jarvi, O., and Levanto, A. (1950) Act. path. microbiol. scand., 27, 473. Jones, D. H. (1920) J . Bact., 6, 325. Klieneberger, E. (1935) J . Path. Bact., SO, 93. Klieneberger, E. (1936) J , Path. Bact., 43, 587. Klieneberger-Nobel, E. (1945) J. Hyg., Carnb., 44, 99. Klieneberger-Nobel, E. (1947a) ' 1. gen. Microbiot., 1, 33. Klieneberger-Nobel, E. (1947b) J . gen. Microbiot., 1, 22. Klieneberger-Nobel, E. (1947~) 1. Hyg., Camb., 45, 407. Klieneberger-Nobel, E. (1949) J . gen. Microbiol., S, 434. Knaysi, G., Hillier, J., and Fabricant, C. (1950) J. Bacf., So, 423. Krzemieniewska, H. (1930) Acta SOC.Bot. Polon., 7, 507. Lamanna, C., and Mallette, M. F. (1950) J. Bact., So, 499. Leonardi, G. (1949) Boll. Inrt. sierotor. Milano, 28, 188. Lewis, I. M. (1941) Bact. Rev., 6, 181. Lindgren, C. C., and Mellon, R. R. (1933) Proc. Soc. exp. Biol. Med., SO, 110. Lohnis, F. (1921) Mem. nuf. Acad. Sci., 16, 1. Mitchell, P. (1949) Symposium: The Bacterial Surface. SOC.gen. Microbiol., p. 55. Mitchell, P., and Moyle, J. (1950) Nature, Lond., 166, 218. Mittwer, T., Bartholomew, J. W., and Burton, J. K. (1950) Stain Tech., I,103. Mofisch, H. (1912) Zhl. Bakt., Abt. II, 33, 55. Morris, 0. E. (1951) J . H y g . Camb., 49, 46. Nutt, M. M. (1927) J . H y g . Camb., a8, 44. Oeding, P. (1949) Acta path. microbiol. scand., 26, 886. Paillot, A. (1919) Ann. Inst. Pasteur, 33, 403. Parvis, D. (1949) Boll. Ist. Sieroter. Milano, 28, 61. Pennington, D. (1949) J. Bact., 67, 163. Pennington, D. (1950) J. Bact., 69, 617. Peshkoff, M. A. (1940) J. gen. Biol. (Moscow), 1, 598.

106

K. A. BISSET

Piekarski, G . (1937) Arch. Mikrobiol., 8, 428. Piekarski, G. (1938) Zbl. Ba&t., Abt. I, 142, 69. Piekarski, G. (1939) Zbl. Bakt., Abt. I , 144, 140. Piekarski, G. (1949) Ergebn. H y g . Bakt. I, S,333. Pijper, A. (1946) 1. Path, Bact., MI 325. Pringsheim, E. G., and Robinow, C. F. (1947) J . gen. Microbiol., 1, 267. Pulvertaft, R. J. V. (1950) 1. gem Microbiol., 4, XIV. Robinow, C. F. (1942) Proc. roy. SOC.,Bilao, 299. Robinow, C. F. (1944) J . Hyg., Camb., 4, 413. Robinow, C. F. (1945) Addendum t o : The Bacterial Cell. Dubos. Schaudinn, F. (1902) Arch. Profistenk., 1, 306. Schaudinn, F. (1903) Arch. Protistmk, 2, 421. Seal, S. C. (1936) Indian J . Med. Res., W , 991. Smith, A, G. (1950) I. Bacf., 09, 575. Stempen, H. (1950) I. Bacf., So, 81. Stille, B. (1937) Arch. Mikrobiol., 8, 125. Stoughton, R. H. (1929) Proc. roy. Soc., BlM,469. Stoughton, R. H. (1932) Proc. roy. SOC.,B111, 46. Tulasne, R. (1949) C. A . Acud. Sci., B9, 561. Vahl, C. (1910) Zbl. Bakt., Abt. 11, S, 178. Wyckoff, R. W. G. (1934) Amer. Rev. Ticberc., aS, 389. Wyckoff, R. W. G., and Smithburn, K. C. (1933) I. infect. DiS., a,201. Wachstein, W., and Pisano, M. (1950) I . Bact., 69, 357. Wei, W. P., Tchan, Y. T., and Pochon, J. (1948) Ann*. Inst. Pmteur, 76, 87.

Protoplast Surface Enzymes and Absorption of Sugar R. BROWN Botany Department, University of Leeds, Ensland

CONTENTS I. Introduction ......................................................... 11. Nature of Experimental Material ..................................... 111. Characteristics of Absorption Process .................................. IV. Enzyme Systems in the External Surface .............................. V. Discussion ........................................................... VI. References ..........................................................

Page 107 107 110 114 117 118

I. INTRODUCTION Sugar absorption by plant tissues, it has been suggested, (Dormer and Street, 1949) depends on phosphorylation mechanisms operating in the external surface of the protoplast. The data that are reviewed here, while they do not’provide any further evidence in favor of this hypothesis, do suggest that the rate of sugar absorption may be determined by the rates of certain enzyme reactions proceeding in the external surface. This conclusion is reached from a consideration of the observation that with excised extending root tissue throughout the period of extension the rate of absorption remains constant. The interpretation of this observation in the present connection requires some reference to (1) the nature of the experimental material, (2) the characteristics of the absorption process, and (3) reactions that are probably due to surface enzymes. These topics are considered below in this order, and the treatment of these is followed by (4) a general discussion of the main topic of this paper. 11. NATURE OF EXPERIMENTAL MATERIAL The data that are considered here have been obtained with cells excised from the extending zone of the root. In the normally growing root, cells are formed by division in the meristematic zone at the tip, and certain of these cells then subsequently enlarge by a process of extension which probably involves the growth of all components of the system. While extension is proceeding, division continues in the tip, and thus at increaslng distances from the tip the tissue consists of cells in progressively advanced stages of extension. This situation provides the opportunity for studying the process of extension as it occurs in a normally growing organ. Sections are taken at increasing distances from the tip, observations are made on the sections, and the results are then related to the number of cells each

107

108

R. BROWN

section contains. With this technique it has been shown that enlargement of the cell in the root is accompanied by increase per cell in the mass of wall material, of protein, and respiration (Brown and Broadbent, 1950). Further it has been shown that certain enzyme activities and the rate of sugar absorption per cell increase as the tissue is composed of progressively more fully extended cells (Brown and Cartwright, 1951). It may be emphasized that these observations refer to different zones of the growing region of the intact root in which extension is accompanied by an increase in protein content. The zone of the root through which extension proceeds in many species extends from the tip to about 5 mm. behind it, and a fragment cut out of the root in the zone 1.5 to 3.0 mm. from the tip is therefore composed of cells that have not completed their development. Such a fragment, however, when transferred to a medium containing sugar increases considerably in length. The increase in length may be greater than fourfold and it has been shown that the increase is due entirely to cell extension. The observations that are considered here have been obtained with such excised fragments. The techniques used in excising and culturing the segments have been described elsewhere (Brown and Sutcliffe, 1950). The segments when they are excised consist of normal parenchymatous cells with a cellulose wall, a peripheral layer of cytoplasm, and a central vacuole. The data of Fig. 1 show the growth that is made by Czlcurbita fragments when provided with 2 per cent fructose and exposed to air and an atmosphere containing 5 per cent oxygen; also the growth made in 5 per cent fructose and exposed to air. Clearly the effect of aeration on growth is considerable. Whereas with air the fragment increases from 1.5 to 5.6 mm. in 2 per cent fructose, with 5 per cent oxygen it increases only to 2.7 mm. Further it is significant that the growth in 5 per cent fructose is considerably lower than it is in 2 per cent. When the fragments are excised, the tissue is of course separated from nutrient supplies from the apex and from the mature parts of the root. It is therefore not surprising that extension when it occurs in a medium containing sugar only has different characteristics from the process that occurs in the intact root. In particular it has been found that during the growth of the excised fragments provided with sugar only there is little or no change in protein content. The interpretation of absorption data obtained with growing tissue must depend of course on the nature of the relationship between absorption and growth. When cell division is involved in the growth, the solute whose absorption is being studied may stimulate division and thus increase

PROTOPLAST SURFACE ENZYMES AND ABSORPTION OF SUGAR

109

TIME, hours

FIG.1. Growth of Cuczlrbita segments in 5 per cent (5) and in 2 per cent fructose (2) solutions. Continuous lines with air, broken line with 5 per cent oxygen.

the number of absorbing units. This complication is not involved with the tissue of this investigation. On the other hand, the possibility still remains with extending tissue that absorption is determined simply by utilization of the solute in the synthesis of structural cellular components. In that event absorption is likely to be determined simply by the growth of the cells. It is evident, however, from a comparison of Figs. 1 and 2 that with respect to sugar absorption such is not the case. Thus while the rate of growth decreases with time, the rate of absorption does not, and secondly while the rate of absorption increases with concentration of the medium, the rate of growth does not. The rate of absorption is greater from a 5 than it is from a 2 per cent solution of sugar, whereas the rate of growth is much greater in the 2 than in the 5 per cent solution. On the other hand, since absorption is not determined by growth, it is evident that the fact that growth occurs implies the incidence of additional

110

R. BROWN

18 16 -

i4

-

u)

e

t

?

12-

z

2 0 + a

lo8-

a

sm

6-

a

4-

2-

TIME, hours

FIG.2. Absorption of fructose

by Cwzcrbito segments. Symbols as in Fig. 1.

variables in the experimental situation through which the process of absorption can be further analyzed. In the present connection it is of some importance that since there is a fourfold increase in the length of the fragment this implies about the same increase in the surface area of the cells. Secondly since this increase occurs in circumstances in which there is little increase in protein content, it is evident that there must be a corresponding change in the thickness of the protoplast in the sense of a decrease in the quantity of protein between corresponding areas of the vacuolar and external surfaces. The significance of these changes is discussed below. 111. CHARACTERISTICS OF ABSORPTION PROCESS The data of Fig. 2 show the course of absorption with time when Cucurbita fragments are suspended in 2 and 5 per cent fructose and exposed to air and when immersed in 2 per cent fructose and exposed to an atmo-

PROTOPLAST SURFbCE ENZYMES A N D ABSORPTION OF SUGAR

---_

0

FIG.3. Respiration of

I

I

I

I

(2

24

36

40

111

2.0

TIME, hours Cucurbito segments in fructose solutions.

Symbols as in

Fig. 1.

sphere containing 5 per cent oxygen. It is significant that with full aeration the quantity absorbed increases linearly with time and the rate of absorption therefore remains constant. Clearly absorption is independent of surface area. Further the rate of absorption decreases when the partial pressure of oxygen is reduced. In this connection the data of Fig. 3 which show the respiration rates observed with the experimental treatments of Fig. 2 are of some significance. I t is evident that in the lower partial pressure of oxygen the respiration rate is considerably lower than it is in air. At the same time it may be emphasized that the lower rate of absorption with 5 per cent oxygen is not a result of the lower growth rate that the data of Fig. 1 show occurs in this medium. The converse is probably the case

112

R. BROWN

since, as shown by the curves of Fig. 4, the internal concentration of reducing sugar is greater when the absorption occurs in air than when it occurs with 5 per cent oxygen. The values of Fig. 4* further show that as a result of absorption in air the 5.0

2.0

2.0 I-

E I

I

I

I

FIG.4. Internal concentration of reducing sugar in Cucurbita segments. Symbols as in Fig. 1. internal concentration of reducing sugar may reach a higher level than the external. These values based on total reducing sugar do not, of course, show that toward the end of the experimental period there is a gradient for fructose increasing from without inwards. This may be the case, or the difference may be due to the internal accumulation of other reducing sugars formed from fructose. Whatever the position however the results of Fig. 4 suggest than in the cell there is a barrier to the free diffusion of sugar. This inference is further supported by the fact that sugar does not leak out of the tissue at a detectable rate when it is transferred to water.

* A comparison of Figs. 2 and 4 shows that while absorption is proceeding the internal concentration of reducing sugar may not change. This is due to the fact that solute absorption may be accompanied by a considerable increase in water content.

PROTOPLAST SURFACE ENZYMES AND ABSORPTION OF SUGAR

113

Clearly sugar absorption in this tissue is an active process which requires the consumption of respiratory energy and which involves the operation of a transport mechanism across a diffusion barrier. The further elucidation of the mechanism requires a consideration of the position of the diffusion barrier. Most of the sugar absorbed by the cell undoubtedly accumulates in the vacuole, and absorption therefore involves movement of the solute from the external medium across the cytoplasm and into the vacuole. Thus the resistance to diffusion may be considered in terms of three possibilities: that it is either over the whole thickness of the cytoplasm, that it is at the external surface, or at the internal surface between the cytoplasm and the vacuole. The first possibility may be disregarded since the rate of absorption does not change as the thickness of the cytoplasm decreases during growth. I n relation to the second and third possibilities, the respiration data are of some significance. When sugar is not provided in the medium, some growth occurs but respiration decreases. Thus growth as such does not involve an increase in respiration. When sugar is provided, respiration increases, and this increase is clearly related to the concentration of sugar in the cytoplasm. Further, in sugar media the rate of respiration increases with time (Fig. 3), suggesting that the concentration in the cytoplasm increases

TIME, hours

FIG.5. Sucrose cleavage in 5 per cent sucrose solution by maize segments.

114

R. BROWN

with time. Now this could not occur with a condition of free diffusion between the medium and the internal surface of the cytoplasm. I n that event the concentration in the cytoplasm would follow that in the medium, and since this does not increase, respiration would either remain constant or decrease with time. In fact respiration increases and follows the internal concentration of sugar. Clearly the data suggest that free diffusion may occur between the cytoplasm and the vacuole, but not between the cytoplasm and the external medium. Thus it is probable that the barrier to free diffusion is on the external surface of the protoplast and that the transport mechanism operates across this barrier. A further aspect of the absorption mechanism is suggested by the stability of the rate of absorption. The constant rate is maintained while there is a fourfold increase in the surface of the protoplast. This suggests that transport across the external barrier is being controlled by a comparatively small fraction of that surface. It is evident that during extension material is being deposited in the surface layer which does not affect the rate of absorption. I n the fully extended cell at least 75 per cent of the surface is clearly inert with respect to the mechanism of absorption. The proportion of inert surface may of course be greater than this, since the whole of the surface in the unextended cell may not be involved in absorption. The evidence suggests that transport across the external surface is controlled by a series of reaction points scaftered over it, these points being immediately accessible to the sugar in the external medium. The rate of absorption remains constant, since these reactive islands become separated during the course of extension by the interposition between them of non-reactive material. In the next section evidence is assembled which suggests that the reactive points may be enzymes.

SYSTEMS IN THE EXTERNAL SURFACE IV. ENZYME Evidence for the operation of enzymes in the limiting external protoplast layer of other plant cells and tissues has been obtained by earlier workers (Wilkes and Palmer, 1933 ; Rothstein and Meier, 1948 ; Said and Fawzy, 1949; Street and Lowe, 1950). Some evidence is also available for the tissue with which this series of observations has been made. It has been found that when extending tissue is suspended in water there is a marked decrease in the weight of probable wall components (Fig. 6 ) . Since the wall is over the external surface of the cell, this necessarily implies an effect due to enzymes over that surface. Again the position of the cellulose wall indicates that at least part of the enzyme mechanism involved in its formation must be localized over the external surface (Brown and Sutcliffe, 1950).

PROTOPLAST SURFACE ENZYMES AND ABSORPTION OF SUGAR

FIG.6. Cellulose contents of Cucurbita segments in 2 per in water (B).

cent fructose

115

(A) and

Further evidence of surface enzyme systems has been obtained from studies on the medium in circumstances in which there is little or no solute absorption. This condition is secured when the tissue is exposed to anaerobic conditions. With prolonged exposure to such conditions, however, there is a danger of autolysis with a consequent dissolution of the barriers between the solute and the enzymes in the body of the cell. This danger has been avoided in these studies by using low partial pressures of oxygen instead of completely anaerobic conditions. With 5 per cent oxygen while the absorption is limited the little that occurs continues throughout the experimental period, indicating that the position is not being affected by the death of the cells (Fig. 2). With this technique it has been found that when extending root fragments of maize are suspended in sucrose solution there is a rapid accumulation of reducing sugar in the medium (Burstrom, 1941 ; Dormer and Street, 1949; Brown and Sutcliffe, 1950).

116

R. BROWN

Chromatographic examination of the culture fluid has also shown that 2-ketogluconic acid is also probably formed. (Brown, Johnson, and Robinson, 1951). The formation of this acid has also been found when the tissue is suspended in glucose. Thus these observations suggest that over the external surface of the protoplast of cells in extending root tissues enzyme systems are present which promote the cleavage of sucrose, and the conversion of glucose to 2-ketogluconic acid. Of the systems which have been identified as being probably in the surface none has been studied quantitatively in any detail. Nevertheless data are available which suggest that during growth the rates of sucrose cleavage and of wall formation remain more or less constant. The data of Fig. 5 have been obtained by determining the decrease in the content of sucrose at different times in an aerated medium. In such a medium reducing sugar accumulates, but not as rapidly as it does under relatively anaerobic conditions. If the assumption is valid that the sugar is absorbed as hexose after an independent cleavage of the sucrose, then the rate of sucrose elimination from the medium is a measure of the cleavage enzyme activity. The data of Fig. 5 suggest that, although during growth the area of the surface increases, the rate of cleavage remains constant with time. The rate of wall formation is shown by the data of Fig. 6. These data represent the weight of material in the tissue after different times which is not dispersed by heating in 2 per cent sulfuric acid. The material is probably predominantly cellulose, and the increase in weight therefore probably represents an increase in cellulose content. It is clear that weight increases more or less linearly with time and that the rate of formation is therefore again approximately constant with time. It may be suggested that both with sucrose cleavage and with wall formation the constant rate while surface area is increasing is due to a constant number of enzyme islands which become separated by the interposition of inert material during growth. Said and Fawzy (1949) have already suggested that in certain mature cells invertase islands occur on the outer surface of the protoplast. It is significant that the constant rate of sucrose cleavage is maintained in a system in which there is little change in protein content during the period of growth. On the other hand, in the intact root it has been shown that as the cell expands, the rate of cleavage increases. In the intact root, however, the protein content also increases. Clearly in the intact root net increase in protein provides for the deposition in the surface of the specific enzyme protein.

PROTOPLAST SURFACE ENZYMES AND ABSORPTION OF SUGAR

117

V. DISCUSSION It has been suggested above that the rate of absorption of sugar is determined by a series of reaction points scattered over the external surface of the protoplast. This conclusion is based on the observation that with excised extending root tissue the rate of absorption remains constant during growth. A similar relation operates with enzyme systems that are probably located in the surface. The similarity suggests that the reaction points involved in absorption may themselves be enzymes. Clearly all the evidence available is compatible with this interpretation. At the same time it must be emphasized that the similarity does not demonstrate that enzymes are in fact involved. It is possible that the reaction points are of another character altogether, and the hypothesis must be taken as a tentative suggestion. It has been shown that in the intact root, with absorption as with sucrose cleavage, as extension proceeds the rate of absorption increases. In terms of the hypothesis proposed here this implies that the increase in protein that occurs in the root provides the condition for the deposition of additional protein enzyme units in the surface. This does not occur with excised tissue, since the change in protein content during growth is only slight. The interpretation proposed here requires that the enzymes that control absorption shall be on the external surface of the protoplast in the sense that they a m immediately accessible to solutes in an external fluid. If that is the case, however, it has been argued that the rates of the appropriate reactions should vary intimately with the hydrogen ion concentration of the medium. On the other hand, certain preliminary data obtained with root fragments suggest that absorption may not vary intimately with changes in pH. It may be emphasized, however, that where two or more enzymes are involved the rate of the overall reaction may not change with pH, although that of each of the individual enzymes may do so. Thus in the following systemEl

E,

A+B+C

where a product C is derived from a substance A through an intermediate B, the A to B reaction being catalyzed by an enzyme El,and the B to C reaction by an enzyme Ez, then, if El and EZ have different p H optima, the rate of production of C may not be affected by pH. If El has an optimum at p H 5.0, and Ez one at p H 7.0, then at p H 5.0 the reaction catalyzed by EZis rate limiting and at pH 7.0 El is rate limiting for the

118

R. BROWN

rate of production of C. The net effect may be no change in the overall rate of production of C when the p H is changed from 5.0 to 7.0. ACKNOWLEDGEMENT

I have to thank Dr. A. W. Johnson for permission to quote certain unpublished results which are discussed in the text. VI. REFERENCES

Brown, R., and Broadbent, D. (1950) J . exp. Bot., 1, 249. Brown, R., and Cartwright, P. (1951) Unpublished data. Brown, R., Johnson, A. W., and Robinson, E. (1951) Unpublished data. Brown, R., and Sutcliffe, J. F. (1950) J. en#. Bot., 1, 88. Burstrom, H. (1941) Ann. agric, CON. S~e&n,9, 264. Dormer, K. J., and Street, H. E. (1949) Arcre. Bot., l8,199. Rothstein, A., and Meier, R. (1948) J. cell. comp. PhySiol., a,77. Said, H. and Fawzy, H. (1949) Nafure, US, 605. Street, H. E., and Lowe, J. S. (1950) Ann. Bot., 14, 307. Wilkes, B. G., and Palmer, E. T. (1933) J. Cen. Physiol., 16, 233.

Reproduction of Bacteriophage A. D. HERSHEY* Department of Genetics, Carnegie Institution of Warltirtgton, Cold Spring Harbor, New York CONTENTS

I. 11. 111. IV. V. VI. VII.

Introduction ........................................................ Ideas about Origin .................................................. Ideas about Growth ................................................. Program and Objectives ............................................. Facts about Growth ................................................. Conclusion .......................................................... References ..........................................................

Page 119 119 120 123 126 133 133

I. INTRODUCTION Anyone who works with bacteriophage feels rather complacent about the experimental familiarity with the growth of phage which present techniques make possible. The deeper questions of mechanism, on the other hand, have to be approached with what might be called absolute shyness. As a result, conversations about reproduction of phage tend either to revert to technological problems or to remain at the level of small talk. I shall try to avoid technological problems. In this paper I propose to discuss ideas, objectives, and facts, in that order. 11. IDEAS ABOUTORIGIN The question of what viruses are, which arose naturally enough during the period of their discovery, is not a pressing one at the moment since it is a question to which further discussion is not likely to contribute, and the study of the properties of viruses does not require the investigator to form any opinion about their nature. In so far as this problem can be said to exist, it can be stated in terms of two or three general hypotheses concerning origin. The development of these hypotheses, which I shall not attempt to trace here, has been reviewed by Doerr (1938). Viruses (or certain viruses) may represent descendants of prototypes which evolved from still simpler forms, and which existed before the appearance of the larger cellular forms on earth. Viruses (or certain other viruses) may have arisen from cells, in either of two ways. One

* The work of the author and his collaborators is being supported by a grant from the National Institutes of Health, U. S. Public Health Service. 119

120

A. D. HERSHEY

possibility requires a long series of loss variations, incidental to the adaptation to a parasitic mode of existence, to the point where only a few minimal structures essential to reproductive and genetic function remain (Green, 1935). The other possibility envisions the abrupt emergence from a previously virus-free cell, by mutation or other accident, of a fragment of its hereditary apparatus henceforth possessing the properties of the full-blown virus (Darlington, 1944). The two variations of the hypothesis of cellular origin differ only with respect to the number of intermediate steps by which the emergence of the virus from the cell is supposed to have occurred. The hypothesis of cellular origin is attractive chiefly because of the possibility of its eventual experimental proof. On the other hand, the exclusion of the first hypothesis would further complicate our notions concerning the origin of cells. No experimental evidence bearing on these questions exists, nor is there any in immediate prospect. I n particular, the historical method of tracing origins appears to be permanently closed to us. To this extent the various hypotheses are virtually equivalent. It is surely meaningless to ask whether the virus is a chemical substance produced by the host, or an autonomous living parasite, or an autocatalytic protein, or a naked nucleus, without reference to some theory of origin, or to some experimental criterion by which the terms used can be defined. At the present time, the various theories of the nature of the virus lead to identical predictions concerning the outcome of any experiment which it is feasible to perform. This view of the virus problem (which is only a special case of the problem of biology as a whole) is evidently shared by most experimenters today, who have largely ceased to discuss it in favor of a systematic approach to the problems of constitution and behavior (Pirie, 1937, 1946).

ABOUTGROWTH The current ideas about growth of phage likewise fall into three classes. I. Bacterial precursors may be converted into phage by a reaction catalyzed by phage, the analogue being the formation of trypsin from trypsinogen (Krueger and Scribner, 1939; Northrop, 1939). If the analogy is to mean anything, it must be assumed that the conversion of precursor into phage does not require energy from other reactions. 11. Phage may grow as an obligate intracellular parasite, requiring both nutrients and metabolic apparatus to be supplied by the host. 111. When a phage particle infects a bacterium both lose their identifying characteristics and merge to form a unit specifically organized for the production of a particular kind of phage. 111.

IDEAS

REPRODUCTION OF BACTERIOPHAGE

121

From one point of view, these three statements merely emphasize one or another aspect of the facts of phage growth. For example, phage is certainly formed from bacterial constituents. But nutritional and other studies show that the conditions necessary for phage formation are essentially those necessary for biosynthesis in general. The bulk of the carbon, nitrogen, and phosphorus incorporated into phage are assimilated by the bacterium after infection (Kozloff, Putnam, and Evans, 1950). Furthermore, immunological tests reveal no structural relation between characteristic bacterial constituents on the one hand and characteristic phage constituents on the other. The first hypothesis is not, therefore, subject to test at present. The intracellular-parasite analogy is adequate in every respect, as has been recognized by d’Herelle and many other virologists. In particular, it accounts in biologically understandable terms for the fact that infection of a cell by phage may result. in death of the host and growth of the phage, in death of the phage and growth of the host, or in growth of both simultaneously. It also accounts for the unlimited number of distinct viruses that can multiply in any one kind of cell. The weakness of the analogy is that it suggests no particular line of inquiry into the mechanism of growth, unless it be the attempt to cultivate the virus in artificial media. The third class of ideas is closely related to the second, with practical rather than logical differences. For instance, thinking of the infected cell as a fully integrated unit does not encourage attempts to cultivate the virus in artificial media. Such thinking is basically optimistic, however, in that it leaves open the possibility that the intracellular form of the virus may be of molecular dimensions. This possibility revives the biologist’s dream of a model system for the study of growth in which the fundamental replicating structures possess biospecific markers completely unrelated to those contained in the accessory metabolic apparatus on which replication depends. In fact, if there is any fundamental characteristic peculiar to ideas of class I11 it must be precisely this: that the phage is supposed to supply to the bacterium patterns to be copied, and the bacterium to supply everything else. Such a notion is the natural successor to some of the earliest ideas about phage (Troland, 1917; Muller, 1922; Wollman, 1925). It is a favorable trend, therefore, that several lines of attack on the mechanism of viral growth are being guided by ideas of this kind (Luria, 1950; Cohen, 1949; Hershey and Rotman, 1949; Kozloff et d.,1950; Benzer et al., 1950). A specific idea of unusual interest is that of Luria (1947), summarized in his 1950 paper. According to this idea the intracellular virus is a

122

A. D. HERSHEY

collection of independently replicating parts, which have to be reassembled to form the virus particles that finally emerge from the cell. Some of the facts, to explain which this idea was brought forth, will be referred to in another part of this paper. Luria’s (1950) paper should be consulted, however, for a full account of the theory. Luria’s idea has met with considerable scepticism. Part of this arises from experimental findings to be considered below. Part of the criticism, however, is based on the difficulty of visualizing the process called for. The following discussion is intended to show that criticisms of the second kind do not apply. Suppose one tries to form the simplest possible picture of growth compatible with ideas of class I11 and with the facts that replication and genetic recombination occur. The required assumptions are that the phage contributes nothing but the pattern for its replication, and that genetic recombination is somehow connected with the process of replication. Following the ideas of Wright (1941) and Delbriick (1941)’ one supposes that the phage particle after entering the cell has to break down into a two-dimensional fabric of monomolecular thickness before replication can begin. This fabric is copied not as a whole, but as partial replicas, which detach from the primary pattern and are completed only after repeated attachments to the same or homologous patterns. Recombination is thus explained as the result of the copying in a single fabric‘of the marked regions of patterns coming from two different parental lines. If it is assumed further that not only completed fabrics, but also the partial replicas, can serve as patterns for replication, we have a special case of Luria’s hypothesis. This assumption is not necessary, but neither can it be considered a complication of mechanism. The only alternatives to this general picture are: that the ideas of class I11 as here defined are wrong; that replication occurs in a manner no one has yet imagined; or that genetic recombination has nothing to do with replication. In the latter case partial replicas are not needed. The ideas just presented have no authorship. They developed from suggestions made to the writer by A. H. Sturtevant and M. Delbriick in January, 1948. One version of the general notion is referred to by Hershey and Rotman (1949). Doermann (1950) is thinking along the same lines. As we have shown, the main points are equivalent to Luria’s (1947) hypothesis, and it is chiefly this fact we wish to point out here, For convenience we shall refer to the specific ideas outlined above as the partial replica hypothesis, and to the third general class of ideas as the hypothesis of total integration.

REPRODUCTION OF BACTERIOPHAGE

123

The question arises how genetic recombination may be accounted for if ideas of class I11 are incorrect. One alternative is evidently zygote formation by intracellular phage particles. A second possibility belonging to class I1 is the following. Partial replica formation might occur within organized, parasitic particles and be transferred passively from particle to particle in functional form. This is a possible analogy to what happens in pneumococcus transformations. We shall refer to this as the geneticleakage hypothesis. T o summarize, the available ideas about growth of phage can be stated in potentially distinguishable form as follows : I. Phage is formed from an organized precursor present in the bacterium before infection. During phage growth the bacterium synthesizes more precursor, and the phage catalyzes the conversion of precursor to phage by spontaneous reactions. In my opinion this hypothesis is untenable, as I have stated it, and is untenable or meaningless if stated otherwise. 11. The phage multiplies intracellularly in the form of an organized parasite possessing differentiated genetic and physiologic structures. Genetic recombination occurs either by matings between particles, or by genetic leakage from particle to particle. 111. Phage multiplies intracellularly in the form of a structure possessing at least one dimension of molecular magnitude. This structure must comprise the full gene complement of the resting stage, but little else. Genetic recombination is explained by either ( a ) or ( b ) below. ( a ) Luria’s hypothesis of independent replication of subunits. I assume that both the number and the dimensions of these units are fixed, or that they are determined in a given bacterium by events occurring before the onset of multiplication. Since the hypothesis originally put forward by Luria implied restrictions of this kind, it can be distinguished from the following, which has no such restrictions. ( b ) The hypothesis of partial replica formation. I believe that this hypothesis is formally equivalent to Luria’s, except that it has been freed from the restrictions mentioned above. It does not require independent replication of subunits, but this difference is trivial. IV. PROGRAM AND OBJECTIVES The most numerous class of coliphages, brought together on the basis of antigenic relatedness and morphologic identity, is the one containing T2 and T4. These are among the largest of the viruses. They are tadpole-shaped particles measuring about 0.1 micron in diameter. The

124

A.

D. HERSHEY

particles are subject to osmotic shock, and presumably possess some sort of membrane (Anderson, 1949). Recent electron micrographs by T. F. Anderson (personal communication) show that the particles attach to bacteria by the ends of their tails. When T 2 or T4 attacks strain B of Esclzericlzia coli, the growth of the phage is terminated by lysis of the infected cell. Nothing further is known about the visible features of the infection. The beautiful new microscopic techniques of Anderson promise to repair this defect. The growth of T2 or T 4 on B may be taken as an example of a relatively well-studied type of virus-bacterium relationship, and this paper will be limited principally to the discussion of this example. There are, however, many coliphages quite different from T2, and virus-bacterium relationships exist which are quite different from that of T 2 to B. The study of these systems is just beginning, and it is not yet clear how much generality our information about the growth of T 2 possesses. gram, of which the dry matter The particle of T2 weighs about consists chiefly of protein (60 per cent) and desoxypentose nucleic acid (40 per cent), The antigenic specificity shows that a large part of this material is characteristic of the virus. The facts outlined above already suggest the main types of attack on the problem of viral growth. I would list these as follows: (a) Study of the connection between bacterial metabolism and viral growth, the immediate goal being a new chemistry of parasitism. ( b ) Study of protein synthesis in a system in which an appreciable part of the synthetic capacity of the cell can be directed at the will of the investigator to the formation of proteins foreign to the cell. ( c ) Study of desoxypentose nucleic acid synthesis, which changes from a minor to a major activity of the cell after infection. It is worth recalling here that one of the big questions of biology is whether this is a qualitative as well as a quantitative change. ( d ) A search for specific intermediates in the development of the virus, with the aim of learning what degree of physiologic complexity the intracellular phage possesses. With reference to the major hypotheses I1 and 111, perhaps the initial question could be formulated as follows. Does or does not the replication of phage-specific substances occur within a phage-specific membrane ? It would be out of place here to discuss specific methods for these several lines of attack, each of which is being vigorously pursued in more than one laboratory. My purpose is rather to express a particular logical and historical bias pertinent to the problem of choice of program.

REPRODUCTION OF BACTERIOPHAGE

125

One remarkable historical fact is that the discovery of the structural complexity of T 2 did not at all improve the status of the intracellular parasite idea at the expense of the notion of total integration-at any rate not among phage workers; it did among some bystanders. Since there is no experimental basis for this favoritism with respect to ideas, it must be put down to scientific optimism. A second remarkable historical fact is the following. Repeated attempts have been made to break open infected bacteria in order to count the number of phage particles inside. These attempts have uniformly resulted in failure to obtain infective phage. The old-fashioned interpretation of this result was that the method had failed. The current interpretation is that infected bacteria do not contain infective phage. Needless to say, this change has been authorized in part by improvements of method. My contention is that the optimistic intellectual environment to which phage workers are exposed has also had its influence. Perhaps the ripening fruits of physical science, which have caused some indigestion in other quarters, are providing the vitamins biology has sometimes lacked. It may be recalled also that the first fruitful attempt to break open infected bacteria was likewise a failure with respect to its immediate objective. Delbruck and Luria (1942) tried to lyse bacteria infected with T 2 by superinfecting with the more rapidly lysing phage T1. This experiment led into a number of still half-explored byways, and eventually to the discovery of genetic recombination of viruses. This line of development has been reviewed several times (Delbriick and Bailey, 1946; Hershey, 1946). It has had a good deal to do with the genesis (or revival) of the total integration hypothesis. The point of my digressions is this: if it were not for the hopeful promise contained in the total integration hypothesis, it is doubtful whether the search for organized intermediates in viral growth would now seem urgently necessary. That is to say, if it were simply a matter of working out the life cycle of another intracellular parasite, or of identifying a specific by-product of the parasitic process, items ( a ) , ( b ) , and (c) in my list of problems might take precedence over ( d ) . I can document this opinion. Burnet discovered a virus-specific small-particle substance in phage lysates in 1933, which I think he interpreted, perhaps correctly, as a specific secretion of an intracellular parasite. Until recently this observation had not even been confirmed (Luria and Bertani, personal communication). Now, owing I believe to the prevailing optimism I have tried to define, Burnet’s observation raises questions of fundamental import.

126

A. D. HERSHEY

I should not leave the impression that phage workers are excessively confident ; although Cohen (1949), Luria ( 1950), and Delbriick (195Ob) show the same optimism, Delbriick (1949) in a philosophical lecture adds a candid, somber warning. If we define the optimism of phage workers as the expectation that something is really going to be learned from phage about growth, it is the reviewer’s opinion that neither optimism nor pessimism can logically be justified at this time. If an error is to be made, it is well to err on the side of the former. Two excellent reviews on the subject of phage growth have appeared during the current year (Benzer et al., 1950; Luria, 1950). For this reason I have felt free in the following discussion to foreshorten facts somewhat, in the hope of discovering a tendency toward convergence on one or another of the general hypotheses stated earlier in this paper. In this I have mostly failed, but record the attempt for what it is worth. V. FACTSABOUT GROWTH When a particle of phage T2 or T 4 attaches to a bacterium, there follows a latent period of some twenty-five minutes before the infected cell lyses to liberate a hundred or more phage progeny. The latent period thus defines a maximal generation time of about three minutes. The various ambitions of the experimenter range from measurement of the generation time to elucidation of the basic process of replication. No one of these ambitions has yet been accomplished, but several leading questions have been answered. Doermann (1948) succeeded in lysing infected bacteria at various times before the end of the latent period and counting the partial yields of infective phage particles. H e found that during the first half of the latent period no phage particles can be obtained. Beginning with the second half of the latent period, phage particles reappear and there is a linear rise in number with a maximum at the end of the normal latent period. The striking fact is that the infecting phage seems to disappear completely, soon after entering the cell, so that we may speak of a dark period, occupying the first half of the latent period, during which no viable phage particles can be recovered by artificially induced lysis. Clues to what is going on during the dark period of viral growth come from several sources. Experiments on genetic recombination in mixed viral infections form the starting point for this discussion. If one infects bacterial cells with one particle each of a host-range mutant and a plaque type mutant of phage T2, one finds that nearly every bacterium liberates a crop of phage containing both parental types together with a larger or

REPRODUCTION OF BACTERIOPHAGE

127

smaller number of double-mutant and wild type recombinants. Now if recombination (by factorial substitution) occurred before multiplication started, a given bacterium infected with two different phage particles could liberate parental types or recombinant types, but not both. The results exclude this possibility, and also go much farther. It is found, rather unexpectedly, that the distribution of numbers of recombinants among the single-cell yields is nearly random, as if each particle of recombinant virus arose independently of others in the same bacterium. Recombinant virus does not, therefore, multiply to form clones of its kind (Hershey and Rotman, 1949). This shows that multiplication ceases before or as recombination occurs. Doermann and Dissosway (1949) studied genetic recombination in prematurely lysed bacteria. They found that the first crop of phage, obtained by lysing cells in the middle of the latent period, already contains nearly the same proportion of recombinant types as is found at the end of the latent period. In their experiments, also, the distribution of numbers of recombinants was not clonal. These results show that both multiplication and genetic recombination occur during the dark period. W e are thus led to postulate a noninfectious, vegetative form of the virus, as opposed to the resting, infectious form characteristic of its extracellular existence. T o summarize our conclusions up to this point, we say that immediately after infection the phage is transformed from its familiar resting form into a vegetative form about which we know little except that it multiplies and is noninfectious. I n this form it multiplies considerably during the first half of the latent period. During the second half of the latent period, multiplication of vegetative phage may continue, but the characteristic process is the transformation of vegetative into resting phage. Genetic recombination may be a separate process, occurring after multiplication has ceased and before transformation into resting phage has begun, or it may be incidental to a process in which groups of phage particles collaborate in bringing about their transformation from the vegetative to the resting condition. The fact that phage transformed early is just about as likely as phage transformed late to contain genetic markers coming from the progeny of two different phage particles makes the first alternative a little more plausible than the second. Somewhat more information was gained from experiments in which bacteria were infected with three different mutant phages (Hershey, unpublished). It was found that recombinants carrying markers derived from three different parental lines are formed, and that the efficiency of triparental recombination is remarkably high. Triparental recombination,

128

A. D. HERSHEY

as expected, is subject to the same linkage restrictions that are observed in biparental crosses. For either linked or unlinked factors, the quantitative results may be stated as follows with reference to some alternative mechanisms of interaction. If genetic recombination occurs during random matings between pairs of phage particles, each particle would have to mate in succession with three other particles to explain the observed yields of triparental recombinants. If the interaction involves groups of several phage particles, or if it is brought about by “genetic leakage,” the yields are easily explained. Finally, the observed yields of triparental recombinants accord very precisely with either statement (IIIa or IIIb) of the hypothesis of total integration. A choice is possible on other grounds, however. Luria’s hypothesis, in a form that postulates a fixed number of subunits, fails to account for linkage. A second form, in which to account for linkage it is assumed that a phage particle breaks up into subunits along cleavage lines determined by accident in a given bacterium and that the subunits multiply, incorrectly predicts a nonrandom distribution of numbers of recombinants among single-cell yields of virus. The partial-replica hypothesis (IIIb) , to which the notion of self-reproducing subunits is superfluous, accounts for all the known facts concerning genetic recombination. The very limited theoretical gains made by these relatively penetrating types of observation point to only one conclusion: that still more powerful experimental methods are needed. A second type of information about the dark period of viral growth is obtained by focusing attention on the behavior of the bacterial host during infection. It is found that the energy metabolism continues without gross change during the whole of the latent period, but that typical processes of bacterial growth, such as elaboration of enzymes and synthesis of pentose nucleic acid, come to a standstill (Cohen, 1949). The synthesis of desoxypentose nucleic acid is of particular interest, because about 40 per cent of the weight of resting phage is composed of this substance. During the first third of the latent period, there is a net synthesis of nuclein bases (Cohen, 1950), a rapid assimilation of phosphorus that is eventually incorporated into phage (Hershey, unpublished), but no net accumulation of polymerized nucleic acid (Cohen, 1949). There is a suggestion that bacterial DNA may be degraded during this period (Kozloff, et al., 1950). Our knowledge of the overall economy of DNA metabolism is inadequate to answer two key questions; we do not know how much DNA vegetative phage contains, nor do we know when during the dark period vegetative phage starts multiplying.

REPRODUCTION OF BACTERIOPHAGE

129

During the second quarter of the latent period, DNA begins to accumulate in infected bacteria and continues to do so at an undiminished rate throughout the remainder of the latent period. It seems reasonable to suppose that this nucleic acid is drawn on for the needs of the virus. According to Cohen (1949), practically all the DNA synthesized is incorporated into virus (or destroyed). We are thus led to conclude either that multiplication of vegetative phage continues throughout the latent period or that vegetative phage contains little DNA. During the dark period marked cytological changes occur in the infected cell. These changes . are specific for different phages (Luria and Human, 1950). During the first five minutes T2 and T4 cause rapid transformation of the chromatin bodies into peripheral masses. After seven to nine minutes the cells begin to fill with granular chromatin. From the chemical and cytochemical investigations, we can conclude at present only that after infection the activities of the cell are profoundly modified, and that the bacterium plays an active role in the propagation of the virus, but one scarcely defined as yet. The third line of evidence concerning the sequence of events during the dark period comes from an analysis of the effects of ultraviolet irradiation (Luria and Latarjet, 1947) or x-irradiation (Latarjet, 1948) on infected cells at various times during the latent period. In brief, these experiments consist in determining the survivor curves of infected bacteria, with respect to their subsequent ability to generate resting phage, as a function of dose of radiation. In principle the survivor curves can yield information about both the number per bacterium and the individual radiation sensitivity of the vegetative phage, provided the population is homogeneous in both respects. Unfortunately, the proviso regarding homogeneity is not satisfied in practice. Conclusions from these experiments have, therefore, to be stated with extreme caution. The experiments with ultraviolet radiation reveal a rapid decrease in apparent radiation sensitivity of individual phage, without apparent change in number per bacterium, during the first nine minutes. This effect may be due to the accumulation of ultraviolet-absorbing material in the cell, to changes in the phage particles themselves, to an increased number of phage per bacterium, or to more than one cause. Unmistakable multiplication of phage is not detected before the twelfth or thirteenth minute. The data show clearly that the population does not remain sufficiently uniform to permit further interpretation of results. The experiments with x-radiation yield qualitatively similar results. They show that considerable multiplication occurs before the tenth minute

130

A.

D. HERSHEY

and, less certainly, that none occurs in most of the bacteria before the sixth or eighth minute. This result leads to the remarkable conclusion that multiplication of vegetative phage may be confined principally to the second quarter of the latent period. These results call for a reappraisal of the data on nucleic acid accumulation, which occurs chiefly during the second half of the latent period. If multiplication is really limited to the second quarter of the latent period, we have to conclude that the vegetative phage does not contain much nucleic acid. Luria (1950) has suggested that the bulk of the nucleic acid may be synthesized and incorporated into phage during the conversion of vegetative into resting phage, rather than during the period of multiplication proper. This suggestion implies that the genetic specificity of the phage is independent of its major DNA content. The apparent decrease in x-ray sensitivity occurs later and is much smaller in magnitude than the decrease in sensitivity to ultraviolet light, suggesting that the latter may be due to the accumulation of UV-absorbing materials that shield the phage (Latarjet, 1948). However, several recent lines of evidence (Luria, Watson, Dulbecco, personal communications) point to fundamental differences between U V damage and x-ray damage to phage. The possibility now seems good that vegetative phage is considerably less sensitive to U V damage than resting phage. This change in apparent sensitivity does not occur in bacterial cells deprived of food (Benzer et d.,1950). Studies of the effects of radiation on resting phage have yielded an extensive and rapidly growing body of information, only a small part of which will be referred to here. Luria has described a very remarkable phenomenon called multiplicity reactivation, by which two or more ultraviolet-inactivated, individually non-infective, resting phage particles are able to infect a single bacterium and to pool resources to produce a more or less normal yield of phage. To explain multiplicity reactivation he has proposed a mechanism of unit substitution analogous to genetic recombination, and has postulated the independent multiplication of the units involved. According to his view, recently summarized in a beautiful essay (Luria, 1950), what we have called the vegetative form of the phage particle consists of a number of independently replicating subunit structures. I have already pointed out certain shortcomings of this hypothesis and have outlined a modified version, the partial replica hypothesis, more consistent with the facts of genetic recombination. Dulbecco has found that ultraviolet-inactivated phage can be revived by

REPRODUCTION OF BACTERIOPHAGE

131

visible light (Dulbecco, 1950) and has reinvestigated multiplicity reactivation both in the dark and after maximal photoreactivation (personal communication). He concludes that at least part of the UV damage is of a generalized physiological nature (cf. Luria, 1944) and that multiplicity reactivation may be explained in terms of the mutual repair of physiological damage. Dulbecco’s interpretation is evidently very different from Luria’s, and it must be stated that no decisive test of either type of hypothesis has yet been possible. At the moment it appears that the principal clue to the nature of vegetative phage provided by radiobiological experiments is the apparent reduction in sensitivity to U V irradiation, which might be interpreted in terms of the plausible hypothesis that intracellular phage undergoing replication can dispense with some of the UV-sensitive structures possessed by resting phage (Dulbecco and Delbruck, personal communication). The hypothetical UV-sensitive structures cannot be concerned merely with penetration into the bacterium, since UV-inactivated phage kills bacteria (Delbriick and Luria, 1942) and causes the same initial cytological changes that are seen after infection with active phage (Luria and Human, 1950). Cohen (1950) finds that nucleic acid synthesis fails, but that protein synthesis proceeds in bacteria infected with UV-inactivated phage. These findings are compatible with, but do not prove, the idea that the inactivated phage starts off something like the normal process of infection, which continues through the first quarter of the latent period and then stops. The fact that the major decrease in UVsensitivity does not occur until the second quarter of the latent period, on the other hand, implies that the sensitive structures are essential during the first quarter. The following comparison may be noted. Genetics furnishes a powerful tool, whose initial applications fail to discriminate among the obvious alternative hypotheses. Radiation techniques furnish a potentially more powerful tool, whose initial applications yield results that cannot be simply explained by any of the obvious alternative hypotheses. Perhaps a tool of intermediate power is needed, or some new hypotheses. Luria (1950), applying a technique he proposed some years ago, has obtained the first and only information about phage replication proper. This comes from an analysis of the distribution of numbers of spontaneous phage mutants arising in small populations of infected bacteria. H e finds that the distribution is clonal, with a large number of two-particle clones. This indicates that the replication of the genetically marked units is binary and geometric, analogous to the multiplication of bacteria rather than to

132

A. D. HERSHEY

replication by a fixed number of master patterns. This finding also strengthens the conclusion previously drawn, that multiplication precedes genetic recombination. By way of calling attention to a new technique, I should like to point out how this result might be confirmed. We have recently found (Hershey et d.,1951) that it is possible to assay the specific radioactivity of phage containing PS2 by measuring the rate of death of the phage. Suppose now that PS2is added to the culture after multiplication has already started. Evidently the phosphorus assimilated early will be nonradioactive, and that assimilated late will be radioactive. If phage particles are being formed by a linear process, the first formed will be nonradioactive, the last formed will be radioactive. If growth is geometric, however, all phage particles of a given generation will be formed (ideally) at the same time, and all members of the final generation will be equally radioactive. Since phage die at a rate proportional to the specific radioactivity of their constituent phosphorus, the two alternatives can be easily distinguished from the form of the curve of survival. For several reasons this method may fail to substantiate or refute Luria’s finding. One reason, as pointed out above, is that during the latter part of the latent period there may be two populations of phage in each bacterium, one multiplying and one resting. If this is the case, one would expect to get radioactive and nonradioactive phage, whatever the mechanism of growth. This discussion has been confined to the behavior of phages resembling T2. As other phages begin to be studied in similar detail, the similarities and differences in behavior are bound to assist in the interpretation of results. Some of the least-studied examples of viral behavior are worth mentioning here, in the hope that more than one phenomenon of general biological interest can soon be brought under one label. A variety of phage-bacterium relationships have been described in which the virus attaches to and kills susceptible bacterial cells but no viral progeny can be recovered (Hershey and Bronfenbrenner, 1948). In such cases it is worth inquiring whether the failure of the virus may be not a failure to multiply, but a failure of the transformation from the vegetative to the resting condition. The discovery of a system of this kind might provide the means for an analysis of the nature of the transformation. The evidence for cell-to-cell transmission of virus in lysogenic bacteria which rarely yield infective virus (Lwoff and Gutmann, 1950) already suggests a failure of this kind. A similar example among the animal viruses is that of the benign papilloma of the cottontail rabbit, which produces sterile lesions when introduced into the skin of the domes-

REPRODUCTION OF BACTERIOPHAGE

133

tic rabbit (Shope, 1950). The term masked virus used to describe situations of this kind would be equally appropriate for what I have called vegetative T2. Other analogies will doubtless occur to the reader. It should be mentioned that in some respects the study of influenza virus infection in the fertile hen’s egg has progressed farther than the study of phage infection. A characteristic product of infection, which appears before the crop of new virus has formed, has been identified by immunological methods (Hoyle, 1948; Henle and Henle, 1949). Schlesinger (1950) has shown that similar substances are formed in tissues which do not support viral growth. The inference is strong that these substances are precursors of the virus.

VI. CONCLUSIONS The sequence of events during the intracellular existence of phages like T2 can be summarized as follows. Soon after entering the cell, resting phage is transformed into vegetative phage. These two forms of virus have no known attribute in common except genetic continuity. Each has one known capacity that is unique: resting phage can infect bacteria, vegetative phage can multiply intracellularly. There seems to be a marked difference between the two forms in sensitivity to ultraviolet light. Multiplication begins some time before the midpoint of the latent period and may be confined to the second quarter. The multiplication itself is geometric by twos. Beginning at the midpoint of the latent period, the vegetative progeny are converted to the resting form at a linear rate. Genetic interaction immediately precedes or is part of this process. VII. REPERENCES Anderson, T. F. (1949) Bot. Rev.,15, 464. Benzer, S., et al. (1950) In Delbriick (1950a). Burnet, F. M. (1933) Brit. J . exp. Path., 14, 100. Cohen, S. S. (1949) Bact. Rev., lB, 1. Cohen, S. S. (1950) Personal communication. Darlington, C. D. (1944) Nature, Lond., lJj4, 164. Delbriick, M. (1941) Cold SPY. Harb. Symp. quanf. Biol., 9, 122. Delbriick, M. (1949) Tvans. Cow. Acud. Sci., 88, 173. Delbriick, M. (1950a) Viruses 1950. California Institute of Technology, Pasadena. Delbriick, M. (1950b) In Benzer et al. (1950.) Delbriick, M., and Bailey, W. T. (1946) Cold Spr. Harb. S p j . quafit. Biol., 11, 33. Delbriick, M., and Luria, S. E. (1942) Arch. Biochem., 1, 111. Doermann, A. H. (1948) Came& Znst. Wash. YY. Bk., 47, 176. Doermann, A. H. (1950) Personal communication. Doermann, A. H., and Dissosway, C. (1949) Cumegie In&. Wwh. Yr. Bk.,48, 170.

134

A. D. HEBSHEY

Doerr, Von R. (1938) In Doerr and Hollauer, Handbuch der Vitusforschung, Julius Springer, Berlin. Dulbecco, R. (1950) J. Bact., 69, 329. Green, R. G. (1935) S c k e , 82, 443. Henle, W., and Henle, G. (1949) J. exp. Med., SO, 23. Hershey, A. D. (1946) Cold SPY.H w b . Symp. quont. Biol., 11, 67. Hershey, A. D., and Bronfenbrenner, J. (1948) I n Rivers, T. M., Viral and Rickettsial Infections of Man, Lippincott, Philadelphia. Hershey,'A. D., and Rotman, R. (1949) Genetics, 84, 44. Hershey, A. D., Kamen, M., Kennedy, J., and Gest, H. (1951) J. gen. Physiol., S4, 305.

Hoyle, L. (1948) Brit. I. exp. Path., 29, 390. Kozloff, L., Putnam, F. W., Evans, E. A., Jr. (1950) In Delbriick (1950a). Krueger, A. P., and Scribner, E. J. (1939) I. gem Physiol., a,699. Latarjet, R. (1948) I. gen. Physiol., 31, 529. Luria, S. E. (1944) Proc. nat. Acad. ScL, Wash., W, 393. Luria, S. E. (1947) Proc. nat. Acad. Sci., Wash., S8, 253. Luria, S. E. (1950) Sciewe, 111, 507. Luria, S. E., and Human, M. L. (1950) J . Bwt., 6B, 551. Luria, S. E., and Latarjet, R (1947) 1. Bact., a,149. Lwoff, A., and Gutmann, A. (1950) Ann. Znst. Pastew, 78, 1. Muller, H. J. (1922) Amer. Nat., 68, 32. Northrop, J. H. (1939) Crystalline Enzymes. Columbia University Press, New York. Pirie, N. W. (1937) In Needham, J., and Green, D. E., Perspectives in Biochemistry, Cambridge University Press, Cambridge. Pirie, N. W. (1946) A m . Rev. Biochem., lii, 573. Schlesinger, R. W. (1950) Proc. SOC.exp. Biol. Med., 74 541. Shope, R. E. (1950) I n Delbriick (19%). Troland, L. T. (1917) Amer. Nat., 61, 321. Wollman, E. (1925) Ann. Znst. Pasfew, S9, 789. Wright, Sewall (1941) Physiol. Rev.,a,487.

The Folding and Unfolding of Protein Molecules as a Basis of Osmotic Work R. J. GOLDACRE Chester Beatty Research Institute, London. CONTENTS

I. Introduction ................................ .................... 11. The Folding and Unfolding of Protein Molecules in Living Cells . . . . . . 111. Osmotic Work-The Accumulation of Material against a Concentration Gradient-in Amoeba ....................... IV. Osmotic Work in Other Cells ............................. V. The Inversion Tube Analogy .... .............................. VI. Osmotic Work in Metazoon Cells . .......... VII. Fungi .......................... .. VIII. General Discussion on Osmotic Work ........................ .. IX. Concluding Remarks ............................................... X. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Page 135 136

150

151

153 153 161 163

I. INTRODUCTION Osmotic work, that is, the accumulation of substances against a concentration gradient, is an essential activity of all living cells. The body cells concentrate material from the blood, freshwater plants are always richer in salts’than the medium in which they grow, and in general the life of a cell depends on its being able to take up substances from a low concentration in the surrounding medium and concentrate them within itself. How this is done has been, until recently, a complete mystery. I n 1943 it was possible for Bull to write “One of the important unsolved problems of physiology is how the cell does work-work of any kind. The problem of heat production has been extensively investigated and is understood; but how the body transforms chemical into mechanical or into osmotic work is a complete blank.” Since then some light has been thrown on the details of muscular contraction; but the position in regard to osmotic work is much the same. The theories which have been put forward have been severely criticized. Dean, reviewing the position in 1947, points out that while osmotic work is a universal phenomenon in cells, ignorance of its mechanism is general. Ussing (1949) reviews the position in regard to active transport of potassium, sodium, and hydrogen ions. It is of little interest to enumerate the theories that have been proposed; some do not stand up

135

136

R. J . GOLDACRE

well to a logical analysis, others conflict with experimental facts. One or two examples may suffice. Brooks (1937) and Lundegardh (1940) put forward an electrochemical theory to explain the accumulation of inorganic ions by plant cells.. Electrical forces caused the diffusion of potassium ions into the cell to replace hydrogen ions diffusing out, the latter being produced in abundance from metabolic carbonic acid. However, cells accumulate cations and anions simultaneously, and the attempt to combine cation and anion accumulatory mechanisms has led to the postulation of a membrane with a mosaic structure, composed partly of cation-permeable and partly of anion-permeable areas. Such a membrane, however, would act as no barrier to any ion but would rather hasten the attainment of equilibrium (Hober, 1946). It has been suggested that glucose is accumulated through its phosphorylation inside the cell, so that the concentration inside the cell is kept lower than outside (Verzir, 1936). However, the secretion of glucose right through a sheet of cells, as in the kidney tubules, would require a distribution of phosphatase within the cells which is the opposite of that found (Dixon, 1949). If the cell had a special mechanism for every substance it accumulated, there would be thousands of different accumulatory mechanisms in it. Yet when placed in a solution of a new dye it coulp not have met before, a cell starts accumulating it into the vacuoles instantly. It seems much more likely that the cell has a general accumulatory mechanism with which it can accumulate practically all substances to some extent, and that it can if necessary be specialized to deal with a particular substance, and that this specificity is of the order of that met with in proteins. In this paper evidence is presented that, in the Amoeba, protein molecules fold up at one end of the cell and unfold a t the other, and that this process results in osmotic work. The corresponding process in types of cells other than the Amoeba is also discussed.

11. THEFOLDING AND UNFOLDING OF PROTEIN MOLECULES IN LIVINGCELLS Many processes in living cells suggest that protein molecules are reversibly folding and unfolding there. The most familiar example is the muscle cell, in which K. H. Meyer (1928, 1929) and later Astbury and his co-workers (Astbury, 1937, 1938, 1940, 1946; Astbury and Dickinson, 1935, 1940) extensively showed by x-ray diffraction that the protein chains were oriented parallel to the muscle fiber axis and became folded when the muscle contracted. Banga and Szent-Gyorgyi (1942) have ex-

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

137

tracted niyosin-like proteins from a wide variety of different cells, such as kidney (“renosin”), liver, brain, and lung; these resemble myosin in the property of existing both in the globular and fibrous condition, being reversibly interconvertible from one to the other, of having a large capacity for adsorbing potassium ions and of having a solubility greatly dependent on the bound phosphate. (Szent-Gyorgyi, 1947). Protoplasm, wherever it occurs, has an innate rhythmic contractility (Seifriz, 1942) ; Astbury (1940) has gone so far as to suggest that wherever contractility occurs in nature, whether in the most elementary forin of protoplasm or in highly specialized tissues, it is due to the shortening of protein fibers by molecular folding. Protein-unfolding in vitro is usually irreversible (“denaturation”), though there are well-known exceptions to this, e.g., insulin, pepsin, trypsin, serum albumin (Neurath et aE., 1944), and the cell must have some means of refolding the protein chains into their original configuration (it did it in the first place, at least), otherwise the cell would soon become full of denatured insoluble protein. That the cell can fold up protein monolayers was shown by the microinjection experiments of Chambers and Kopac (1940) who, however, did not give an explanation for their results. Chambers injected an oil drop into an ameba and caused the drop to expand and contract by moving the plunger of the syringe backwards and forwards. The contracting oil drop wrinkled if the ameba was dead or cytolyzed, but did not wrinkle as long as the ameba was alive. These experiments have been repeated by the present author and interpreted as follows. Oil drops in cells have an interfacial tension of practically zero (Harvey and Shapiro, 1934; E. N. Harvey, 1937). This is due to a monolayer of protein at the oil-water interface (Danielli and Harvey, 1934 ; Danielli, 1938). Now protein monolayers would collapse as the area available on the surface of the contracting oil drop became reduced, and do in fact do so, as shown by the wrinkles on the contracted oil in dead amebae. Since this does not happen in living amebae and the protein monolayers are undoubtedly there, they must be going into solution. But protein monolayers are “denatured” and insoluble. The cell evidently has some means of folding them up into the soluble globular form. How it does this is unknown, but possibly some ATP-like substance is involved (which would diffuse out of dead amebae) in a mechanism similar to that operating in the cell membrane of the ameba’s tail [which contracts and dissolves when A T P is injected intracellularly (Goldacre and Lorch, 1950) 1.

138

R. J. GOLDACRE

That unfolding of proteins may be required in certain cases of enzyme activity, where presumably groups buried within the molecule are brought to the surface, has been postulated by Eyring, Johnson, and Gender (1946), who found that the unfolding of proteins is accompanied by an unusually large change in molecular volume (about 1 per cent per 100 residues), and that unfolding can be prevented by the application of as little as 400 atmospheres pressure. This pressure was shown to inhibit the action of invertase, to influence the sol-gel change in myosin, the luminescence of bacteria, and to cause the reversible disappearance of the spindle in mitosis. It also causes the cortical gel in amebae to liquefy, so that extended pseudopodia break up into a chain of spheres (Marsland and Brown, 1936). It is hard to imagine the synthesis of proteins in a cell from a protein template, unless the template is completely unfolded at some stage (with the forming molecule adsorbed on it), otherwise a part of the pattern would be left out (Eyring et d., 1946; see also Goldacre, Loveless, and Ross, 1949). In any case, the living cell does make globular proteins, and since these contain folded chains buried within the molecule well outside the range of ordinary valence forces from outside, these molecules must have been folded up by the cell. Evidence of the cell’s ability rapidly to fold up protein monolayers comes from a study of ameboid movement (Goldacre and Lorch, 1950). Locomotion of the ameba is due to the contraction of the cortical gel at the rear end of the cell. After contracting, the gel liquefies and is squeezed forward up the central channel of the cell to form more gel on the walls of the advancing pseudopod. The cell membrane also contracts at the tail, as can be seen by the moving together of lateral pseudopodia on both sides of the cell as the tail approaches them (Fig. 1). This membrane contraction at the tail continues indefinitely, and it is evident that it must be dissolving, otherwise the cell would soon become full of membrane. The tail is wrinkled like a collapsed monolayer. This is probably due to the two-dimensional compression arising from the contraction of the cortical gel attached to it. The cell completely renews its membrane during the time it moves through its own length, i.e., about 2 minutes. The membrane forms at the advancing end of the cell in much the same way as a protein solution spreads at an oil-water interface. This spreading occurs spontaneously, and since there is plenty of protein in the protoplasm (and always a trace in the medium, otherwise the amebae cytolyze) and lipoid in the plasma membrane (Davson and Danielli, 1943), new cell membrane would form spontaneously if the membrane were blown out.

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

139

FIG.1. Atrocba discoides, photographed in three successive positions, showing the contraction of the cortical gel, and of the membrane attached to it, at the rear of the cell ; and the wrinkling of the membrane. (From Goldacre and Lorch, Nature, 166, 497, 1950.)

The more interesting process is the refolding of the protein monolayers in the double lipoprotein film of the plasma membrane, since refolding of the protein monolayers is difficult to bring about in z d r o and almost always results in collapse of the film into insoluble fibers. The low surface tension of the plasma membrane which is less than 1 dyne per centimeter (Davson and Danielli, 1943) shows that the protein monolayers in it must be near their collapse pressure. The lipoid itself in the membrane would exert a surface tension against water of about 20 dynes per centimeter, so that the surface pressure of the adsorbed protein monolayer, equal to the surface tension lowering, must be also 20 dynes per centimeter. This is close to the collapse pressure of most protein monolayers (Langniuir, 1939).

140

R. J. GOLDACBE

That it is in fact at the collapse pressure is shown by the data of Cole (1932), who flattened cells mechanically so as to increase their surface area by about 50 per cent. In spite of this the surface tension of the cells remained practically zero (0.1-0.2 dynes/cm.) . Now normally, a change of surface pressure of about 20 to 25 dynes per centimeter is needed to change the area of protein monolayers by 50 per cent (Langmuir, 1939) unless the film is at its collapse pressure, when a change in area is accompanied by no change in surface pressure. The membrane evidently expands by drawing on globular protein and lipoid in the protoplasm adjacent to it, and contracts by folding up and becoming globular protein in the plasmasol. This must certainly be so in the ameba, for it completely renews its membrane about every two minutes (the time it takes to move through its own length) otherwise the cell would soon become visibly full of membrane. The changes occurring in the ameba’s tail are paralleled by the action of A T P on the actomyosin gel. Thus, addition of A T P causes liquefaction of the gel, and a contraction of an actomyosin fiber (Szent-Gyorgyi, 1947). Goldacre and Lorch (1950) injected A T P into the ameba and found that it did cause contraction and liquefaction of the cortical gel. The ameba is like a muscle cell in which the contraction is slow and confined to the rear end. Unlike in muscle, the contracted protein is soluble and is squeezed away to relax again at the opposite end of the cell. This suggests a picture of the ameba in terms of protein molecules as in Fig. 2. The protein molecules in the liquid part of the cytoplasm must be folded up, since almost all known proteins gel when unfolded, e.g., by heating, when in the high concentration that exists in cells (1040 per cent). The protein molecules in the gel would be unfolded in order to form an interlocking network of fibers. The change from gel into sol as a result of the folding up of the molecules would thus cause a contraction during the first part of this process before the extended molecules had completely disengaged from one another. The size of protein molecules makes it feasible to handle them diagrammatically as shown in Fig. 2 ; for the length of an ameba (0.3 mni. for A . discoides) could be spanned by the unfolded polypeptide chain of a protein molecule of molecular weight lOO,OOO,ooO (or 10oO molecules of mol. wt. 100,OOO) and the cortical gel (10 microns thick) would correspond to the thickness of about 10,OOO wet protein monomolecular layers. In this model, the events in the membrane are paralleled by the events in the cytoplasm next to it. In the front of the cell, unfolding of protein to form new membrane is accompanied by unfolding of cytoplasn~icpro-

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

141

FIG. 2. Diagrammatic representation of an ameba, showing the suggested degree of folding of the protein molecules at various parts of the cell. (From Goldacre and Lorch, Nature, 166, 497, 1950.)

tein to form cortical gel; and in the back, folding of membrane monolayers during the progressive reduction of area of the tail is accompanied by folding of the cytoplasmic extended protein chains to form plasmasol. The dissolved membrane contributes to this plasmasol.

111. OSMOTIC WORK-THEACCUMULATION OF MATERIAL AGAINST A CONCENTRATION GRADIENT-IN AMOEBA When protein molecules are unfolded they have more surface area available for the adsorption of other molecules than when they are folded up. The side chains and other groups which were used to hold one part of the polypeptide chain to another part in the folded globular molecule become free and turn toward the solution and can then adsorb other substances. This was demonstrated chemically. For example, ovalbumin adsorbed eight times as much dye when unfolded as a monolayer than when in a globular form in solution (Goldacre, 1951). Similarly, Oster and Grimsson (1949) found that tobacco mosaic virus increased its uptake of dye 300 times when denatured. This difference between ovaibumen and the virus is to be expected because the larger molecules have a higher proportion of the side chains buried within the molecule. The process is represented diagrammatically in Fig. 3.

142

R. J. GOLDACRE

0

0

O

0

0 O0 0

0

m

0

0 0

0

0

000 0 0 0

o o o o o o o o c 1

b C FIG.3. Diagram showing the change in adsorption when protein molecules unfold, a

and desorption on refolding. (a) folded protein in dye solution; ( b ) unfolded protein with adsorbed dye molecules; (c) refolded protein, with shed dye.

In the ameba the unfolded molecules in the cortical gel and plasma membrane should adsorb the material from the environment, and when they fold up in the tail, they should desorb it. Thus, in time a large amount of material should be released into the tail. This is exactly what was found by experiment. A short account has been published (Goldacre and Lorch, 1950). Amebae placed in a dilute solution of neutral red (the optimum was found to be 0.003 per cent) accumulated the dye in their tails, but only when they were actively streaming (Figs. 4 and 5 ) . When the ameba had moved about half its length, the staining of the tail became conspicuous. The advancing pseudopod was relatively colorless. On the other hand, amebae which were still became stained uniformly all around the periphery. This shows that the color in the‘tail is not due to a stainable body in it but is a result of the movement of the cell. If the ameba died or cytolyzed, the whole of the cytoplasm instantly became stained, much more intensely than when alive. This shows that the whole of the cytoplasm is stainable, and that it has a mechanism for releasing itself from the dye it has taken up. Figure 5 is a diagram of three types of uptake. The dead cell stains so rapidly because the membrane is much more permeable than when alive (Davson and Danielli, 1943). The protein which is released from the dye streams forward and takes part in further accumulatory cycles. Why the neutral red does not stream forward with it is not clear. It does not appear to be trapped in a vacuole as in plant cells (see below), but when in high enough concentration causes some local precipitation of protoplasm, which can be felt as a hard lump with the microdissecting needle. When the cell is in a solution of much lower concentration, the dye (not being concentrated enough to cause precipitation) does stream forward with the cytoplasm and eventually appears distributed in what have been called “neutral red vacuoles” (Carrel and Ebeling, 1926 ; Hopkins, 1938).

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

143

FIG.4. Alrcoeba discoides after streaming for 15 minutes in 0.003 per cent neutral red solution, showing accumulated dye in tail (see top of figure). (From Goldacre and Lorch, Nature, 166, 497, 1950).

To test whether the precipitating power of the dye was a factor, various other dyes were tried. With methylene blue and brilliant green, a much higher proportion of the dye streamed forward, though it still accumulated in the tail. The advancing pseudopod was markedly colored, especially with methylene blue, and a greater concentration of it in the external medium was required for the accumulation in the tail to be apparent at all. Various other cationic dyes accumulated in the tail also, e.g., proflavine (2 :8-diaminoacridine) and crystal violet, though they were toxic and tended to prevent streaming, which of course did not allow the effect to develop, except in a rather narrow critical range of concentration. Anionic dyes such as Lissamine green, rose Bengal, Chicago

144

R. J. GOLDACRE

(0)

(C)

FIG.5. Diagram showing in 0.003 per cent neutral red solution, ( a ) successive stages of an active ameba, ( b ) an inactive ameba, (c) a dead ameba. Intensity of dye shown by closeness of shading.

blue and the coninion indicators, e.g., bromthymol blue, did not enter the cell and were not toxic in high concentration. The behavior of the amebae in solutions of various concentrations of neutral red in Chalkley’s medium at pH 6.5 is given in Table I. TABLE 1 Conc. of Dye (per cent)

Behavior

0.02

Cell becomes rounded, and red all over, very little streaming. After 15 min. dye is in granules in center of cell.

0.01

Cell stains heavily all around periphery, very little streaming ; after 15 min. tendency to blister-membrane lifts away from cytoplasm as with anesthetics. When rescued at 30 min. and placed in culture medium, cell recovers, starts streaming and the red goes into tail.

0.005

At 15 min. heavily stained around periphery, sporadically puts out clear pseudopod, which becomes red after min. Streaming is sluggish.

0.0025

At 14 rnin., cell very active, much accumulation in tail.

0.0012

At 5 min., cell active but no appreciable uptake. A t 17 min., active, faint red tails. A t 53 rnin., active, each cell has a conspicuous red blob in tail. 3 hr., deep red crystals in the cells.

24 hr. small red vacuoles and deep red crystals distributed throughout the cytoplasm. Red vacuoles often have a deep red crystal in the center.

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

145

The best concentration showing rapid accumulation is thus 0.003 per cent. Above this streaming is inhibited. In neutral red the lateral pseudopodia were usually suppressed, and the cell proceeded in a monopodal manner (as in Fig. 4). It is interesting to compare the amount of dye accumulating in the tail during the folding up of a known area of cell membrane with the amount of dye that is adsorbed by an equal area of ovalbumin monolayer. \IThen an ameba of 300 microns length and 60 microns diameter has moved through ten times its own length (taking about 20 minutes), the amount of membrane folded up is: 10 X 2~ X 30 X 300 pz

=

6 X

cm.2

and the concentration of dye in the tail is about 1 to 10 per cent (estimated by visually comparing the depth of color with that of test tubes of known concentration and thickness). il.1 The ovalbumin monolayer adsorbs at pH 7 from a 1.8 X solution (O.OOO6 per cent) of neutral red 0.3 equiv. dye/equiv. protein (Goldacre, 1951), i e . , lo00 grams monolayer adsorbs 60 grams dye,

6 X

cm.2 monolayer adsorbs 36 X

Volume of ameba's tail is about =

1/20

X

T

X 30

g. dye

+&,volume of cell X 300 p 3

=

5 X 10-8 CIII.

therefore concentration of'neutral red in tail is

36 5

x x

10'8

=

7

x

10-4

=

0.07%

ie., a 100-fold enrichment. But the observed amount is about 100 times as much as this. It is likely, therefore, that some of the cortical gel is participating also. Since it can be seen stained a faint pink all along the cell, this is not unexpected. The capacity of the cortical gel is 10,OOO times that of the plasma membrane, since there is room for about 10,OOO protein monolayers in a 20 per cent protein gel 10 microns thick. Now the amount accumulated is only 100 times the amount that would be accounted for by the protein in the membrane, assuming its adsorptive power is the same as that of ovalbumin. This suggests that the cortical gel is participating in the accumulation to the extent of about 1 per cent of its full adsorbing capacity. This may be due to two causes:

146

R. J. GOLDACRE

1. The time is not sufficient for equilibrium-only two minutes is available after the gel is formed before it liquefies. 2. Other substances from the medium are competing for adsorption e.g., food and salts, all of which are concentrated by the accumulatory mechanism. This calculation, though rough, is adduced to show the feasibility of the protein-folding mechanism of accumulation and to show that it is quantitatively adequate to account for ( a ) the amount of accumulation that takes place and ( b ) the concentrations that can be dealt with in the external and internal media. The accumulation of neutral red in the tail of actively streaming amebae seems to have been overlooked by other workers. This is probably because they used too low a concentration of dye and observed after a much longer time, so that only the secondary effects of the accumulation were noticed (the so-called “neutral red vacuoles” and “neutral red granules”, which appear to be coacervation or precipitation effects in the cytoplasm resulting from its chronic exposure to low concentrations of neutral red.) For example, Koehring (1930) reported that a 1 :1O,OOO,OOO solution of neutral red stained the food vacuoles and granules of amebae after 24 hours, but only in life. The present author’s interpretation of this is that the living ameba’s accumulatory mechanism is operating as it does in higher concentrations of neutral red, but the concentration in the tail is too low to be visible; and it is taken up by the granules as fast as it is accumulated (as happens also after several hours of exposure in the higher concentrations used by the author). Thus the medium bathing the granules, etc., after a few hours is much richer in neutral red than when the accumulatory mechanism is inactive in death. For 100 to loo0 times the concentration of 1 :1O,~,OOOwas found necessary in order to stain the granules of dead amebae appreciably. This enrichment could be reached in a few hours of streaming. This is consistent with ivi vitro adsorption experiments (Goldacre, t951). Ovalbumin is about half-saturated with neutral red when the dye concentration is lob4M , (0.002 per cent) whereas in a 1 :1O,OoO,OOO solution the proteins are only about %OO saturated which, in the thickness of the ameba, gives a depth of color too little to be detected by the eye.*

* The = 0,001.

optical density would be only 0.003 X 104 X 1/10 X 1/200 X 1/10

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

147

IV. OSMOTIC WORKIN OTHERCELLS Turning now to other kinds of cells the question arises whether proteinfolding is the basis of the accumulatory mechanisms in them also and whether accumulation is correlated with cytoplasmic streaming as in the ameba. The streaming in many plant cells and in some animal cells (preferably observed by time-lapse cinematography) often occurs in many directions at once and has a complexity so bewildering that it is difficult to discern what is happening. Fortunately, the streaming in the root hairs of plants, which are single elongated cells, has a simplicity comparable with that in the ameba. The hair is much narrower than the ameba (20 microns cf. 60 microns), but may be several times as long. A typical root hair has a vacuole near the tip with the nucleus on the proximal side (Fig. 6 ) .

FIG.6. Diagram of a typical root hair showing manner of accumulation of neutral red.

The cytoplasm streams back and forth over the vacuole, reversing bodily about every 5 minutes. This reversal occurs over the whole of the surface of the vacuole, i.e., there is not in general a stream going in the opposite direction over part of the surface. The distance of the vacuole from the tip of the cell varies rhythmically with time, as shown in Fig. 7. The volume of the vacuole does not change during this rhythm, as was proved by measuring simultaneously the distance from the tip of the cell to both ends of the vacuole, by means of the optical micrometer. This was best done when the cell was immersed in neutral red solution

148

R. J. GOLDACRE

and actually accumulating the dye, which went into the vacuole and increased the contrast between the end of the vacuole and the cytoplasm, the latter not being perceptibly colored. This shows that the cytoplasm has a true bodily motion over the vacuole, and in this respect the cell resembles the inversion tube described below. Cytoplasm and vacuolar membrane move practically together, so that vacuolar membrane forms at one end of the vacuole and dissolves at the other, where presumably the protein molecules in it are folding up. It was found that the dye accumulated at the end of the vacuole toward which the cytoplasm was streaming. It could be seen as a diffusing cloud, tumbling toward the center of the vacuole (Fig. 6). Then, when the streaming reversed, the dye accumulated at the other end. Usually, after two or three reversals the vacuole had such a high concentration of dye in it that streaming ceased. Figure 8 shows photographs of root hairs accumulating neutral red.

I

t:

10

TIME

15

MINUTES

20

FIG.7. Showing rhythmic reversal of streaming of cytoplasm in root hair, in terms of distance of vacuole from end of cell.

Here again it is possible to correlate the dye accumulated with the adsorption of neutral red on protein monolayers in witro. The area of nionolayer passing over the vacuole was determined by the optical micrometer, from the change of volume of the terminal cap of cytoplasm (OA,Fig. 6) and the thickness of the cytoplasm streaming over the vacuole. The concentration of neutral red was measured by comparing the depth of color in the vacuole with neutral red solutions in test tubes.

PROTEIK MOLECULES AS A BASIS O F OSMOTIC WORK

149

FIG. 8. Photographs showing ( a ) accumulation of neutral red in vacuoles of root hairs, (b) the same under higher power. Calculation: Diameter of vacuole = 20 p, length = 50 p. Thickness of protoplasm passing over vacuole = 1 p. (20 X lO-4)2 X w X 50 X 10-4 = 1 6 X 1O-Q nil. Volume of vacuole =

Surface area induced in 20-p cube of protoplasm as it squeezes over vacuole = (2 X lO-a)3 X VlO-4 = 8 X 10.5 cm.2. Maximum amount of dye that can be

adsorbed on this area ( 1 mg./sq. meter) is:

8 X 10-6 X

10-3 X

So concentration of dye in vacuole will be 8 X 10-12 16 x 10-9

10-4 = 8 X 10-12 g.

=

after vacuole has moved 20 p in one direction.

0.5 g/l

150

R. J. GOLDACRE

If the gel in the 1 micron thick layer of cytoplasm between the vacuole and the cell wall is involved also (and, in contrast to the ameba, this is likely since the dye must traverse the gel before it can get to the membrane folding up) then loo0 times this amount could be adsorbed, i.e., concentration after one cycle would be 500 grams per liter. The actual amount lies closer to the higher figure since solid dye is eventually observed in the vacuole, appearing to form from a supersaturated solution, as the vacuolar liquid becomes lighter when red crystals appear in it. If more accurate experiments show a general agreement between calculated and observed concentrations, then there would be no room for any other mechanism of dye accumulation and protein-folding would be the mechanism of osmotic work.

V. THEINVERSION TUBEANALOGY A working model of an accumulatory mechanism, similar to that in the root hair, can be made from a long glass tube closed at both ends and about three-fourths filled with protein solution. When the tube is inverted, the bubble of air runs from one end to the other. Protein monolayer forms spontaneously on the surface of the bubble and is carried by the solution running down the walls and collapsed against the ascending meniscus. With 0.1 per cent egg albumin, which is irreversibly denaturable, a permanent fiber of collapsed protein about 15 cm. long forms on each inversion of the 50-cm.tube. With a reversibly denaturable protein, such as serum albumin, the fiber becomes only a few millimeters long, since it dissolves at the end as the molecules fold up and go into solution when compressed in two dimensions. If dye is added to such a solution, it is adsorbed on the protein monolayer and desorbed when it folds up, and is released from the trailing end of the bubble as an intensely colored streamer, and becomes distributed by convection throughout the solution again. The process can be repeated indefinitely. A suitable solution for showing this effect is a 0.1 per cent solution of serum albumin and a 0.001 per cent solution of crystal violet in a tube about 50 a n . long and 7 mni. internal diameter. Thus, in inversion tube, ameba and root hair, concentrated dye is released from the trailing edge of the air bubble, plasma membrane and vacuole respectively, and the degree of concentration over that in the bulk of the solution is about the same in each, ie., even solid particles of concentrate can be obtained from a surrounding medium containing, say 1 :100,ooO. The depth of color of granules appearing in amebae and root hairs was foiund to be as great as that of solid particles of neutral red of the same di-

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

151

mensions. Likewise, the concentration effected by the inversion tube is very great. With surface-active dyes (in the absence of protein which adsorbs non-surface active substances also), such as brilliant green, solid particles of dye were formed at the trailing edge of the bubble, and when the bubble had stopped, could be seen darting over the water surface like camphor, leaving a colored trail behind, until they were dissolved completely. There is no limit to the degree of concentration attainable except the final volume in which the collapsed monolayers are dissolved. I n the root hair this is a small vacuole, about 20 microns in diameter, and concentrations near saturation are reached in a few minutes in an active cell. It is surprising that the cell continues to function until the vacuolar concentration is as high as this. This is probably because the cytoplasm itself remains free of the dye which is imprisoned in the vacuole whose membrane is relatively impermeable to it. I n fact, it only appears to be permeable where the vacuolar membrane is folding up, which might be expected to cause some disorganization in any barrier in it. For if the vacuolar membrane were not impermeable, the dye which had already been accumulated in the vacuole would be taken u p when the streaming is reversed and accumulated at the other end of the vacuole. This is not so, and the membrane takes the dye up from the cytoplasm instead, which has a much lower concentration-too little to detect by eye. But the accumulation of dye would tend to drive the folding-unfolding cycle in the opposite direction (for the adsorption of dye tends to unfold the protein molecule), and the protein molecule would refold with difficulty in a strong dye solution. It was indeed found that streaming ceased after about 15 minutes in 0.003 per cent neutral red, by which time the vacuole was so dense that it was difficult to see the opposite side of the cell through it (Fig. 8b).

VI. OSMOTIC WORKIN METAZOON CELLS Fibroblasts of chick and rat move in tissue culture like amebae, with a well-defined persistent tail and lateral pseudopodia. The pseudopodia are often extremely thin, suggesting that the cortical gel is much thinner relative to the cell’s width than in the ameba, which is also suggested by cinematographic observation of streaming in the cells. Speeded-up cinematography of these cells [e.g., the films of Canti (1920), Pomerat (1949), Hughes (1949), and Waymouth (1950)l show that the contractions in the cytoplasm are not confined to the tail region and retracting pseudopodia but exist in the bulk of the cell also. It is as if the various contractile elements in the cell have gained a measure of

152

R. J. GOLDACRE

independence-a fibroblast is like an ameba in which the contractile elements have been “liberated” from the cortical gel and not forced to contract all at once in a given region. This dissociation of cellular movement as a whole from chemical activity might be an advantage for a cell which is normally stationary in a tissue. Movement in the ameba may be a by-product of the chemical activity of the foldable proteins, as heat is a by-product in all cells. It would be expected that each contractile element in the cell would act as an independent accumulatory mechanism. In support of this it was found that fibroblasts in neutral red solution in about half an hour accumulated the dye in three or four diffuse clouds at various parts of the cell. In general the major site of accumulation was not the tail, although in a few cases accumulation in the tail was observed. This is probably because the amount of cytoplasmic streaming contributing to the ameboid motion is small compared with the total in the cell. Accumulation was particularly conspicuous as a cap on a rapidly growing oil drop in Rous sarcoma cells undergoing fatty degeneration (drops grow to the width of the cell in a few hours) suggesting that perhaps the oil is shed from a fibrous lipoprotein as it folds up. Another frequent site of accumulation was as a cap on one side of the nucleus. This was especially marked in snail embryonic cells (100-cell embryo) suspended in Ringer. Tissue cells of the rat freshly suspended in Ringer behaved similarly. As with amebae, it was noticeable that dead cells took up the dye instantly, whereas living cells still had large clear regions after half an hour. After a few hours to a day, the concentrated patches of neutral red often induced a vacuolization of the cytoplasm and the dye appeared in the vacuoles and not in the cytoplasm. This secondary change, after the primary rapid accumulation as a diffuse cloud, has been noted by other workers, e.g., Carrel and Ebeling (1926) who used much more dilute dye solutions, of the order of 1 :l,OOo,OOO and observed after 24 hours. The rapid accumulation of dyes in the diffuse patches, which is a conspicuous and interesting feature both in amebae and other cells, seems to have escaped attention. One or two passing references to it are made, the authors briefly dismissing the phenomenon by saying “the dye diffused in unevenly,” as if the experiment were a failure. But uneven diffusion could not explain the existence of regions of high concentration completely surrounded by regions of low concentration.

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

153

VII. FUNGI The mold Neurospora, which has septate hyphae, was found to take up the dye at the end of each cell, as in amebae (Fig. 9). The dye first appeared at the septum and gradually spread toward the center of the cell.

FIG.9. Photograph of Nezcrospora after 5 to 10 minutes in neutral red, showing accumulation of dye at one end of each cell. In some of the cells there were vacuoles already, and in these cases the dye went straight into the vacuoles. Neurospora therefore, in its accumulatory mechanism, occupies a position intermediate between the ameba and the plant root hair cells. O N OSMOTIC WORK VIII. GENERALDISCUSSION

Let us consider the adsorption-desorption mechanism of osmotic work more closely. What determines whether adsorbed molecules will be desorbed by the purely mechanical'effect of folding up the polypeptide chain? That they can be desorbed in this member is shown by the inversion tube experiment described above. In many proteins (e.g., keratin, myosin, egg albumin) the number of basic side chains is approximately equal to the number of acid side chains (Speakman and Hirst, 1933). At the physiological pH these are almost entirely ionized and some form salt links which help to hold the polypeptide chains in a definite folded configuration. When the molecule is unfolded, the salt links are pulled apart and an equal number of sites become available for the adsorption of positive and negative ions. Potassium ions, or neutral red cations, for example, will be adsorbed from the surrounding medium onto the negatively charged carboxyl groups. When the protein molecule is tending to refold, there will be competition for adsorption on the carboxyl group between the adsorbed cation and the positively charged side chains of the proteins. Since the latter are present in very high effective concentration, and, especially when the protein is nearly refolded, in the neighborhood of the carboxyl

154

R. J. GOLDACRE

group of the salt link to which they were formerly attached, competition begins to favor the side chain and the adsorbed cation is displaced. The desorption of a monolayer by reduction of the area on which it is adsorbed can be well demonstrated at the oil-water interface. If a dilute (about 20 mg./l.) aqueous solution of a surface active dye, such as Lissamine green (a sulfonated triphenylmethane dye) is shaken with about half its volume of chloroform (in which the dye is insoluble), the emulsion of chloroform in water so formed settles in a few minutes. Each drop of chloroform has an adsorbed monolayer of dye on it,* and the color is carried down by the chloroform as it settles, leaving the aqueous phase practically colorless. As the drops of chloroform coalesce, the total surface area on which the dye is adsorbed is decreased, and the dye is squeezed off the surface back into the water. Thus between the chloroform and the clear water there appears a very thin layer, about Ifi0-5 mm. thick, of highly concentrated aqueous dye solution ; its concentration, measured colorimetrically after running it out of a separating funnel, was between 100 and lo00 times that of the clearer solution above it, depending on the amount of convection during the separation. The dye was quite free in the solution and could be stirred back into the aqueous layer so as to bring the system back to its original unshaken condition. The cycle could be repeated indefinitely. The author has had for four years a sealed demonstration tube which has been taken through the cycIe hundreds of times. Accumulation of .the dye could be prevented by a trace of detergent of the same charge. Various dyes which were not surface active and not concentrated in this system (e.g., bromcresyl green) could be made to do so by the addition of a trace of oppositely charged detergent (e.g., cetyl pyridinium bromide) which apparently made a surface-active salt. The mechanism is thus adaptable and versatile and could concentrate, for example, potassium ions. Thus it is no longer necessary to imagine that some substances diffuse into a cell against a concentration gradient. They would diffuse into a region of lower concentration in the cell because it has been lowered by adsorption (except in those regions where the substance has been desorbed, such as vacuoles, where it is isolated from the rest of the cell). If the displaced ions are shed into a vacuole, the vacuolar membrane will to a large extent prevent the accumulated product from taking further part in the accumulatory cycle. That penetration of the vacuolar mem*Calculation shows that an area produced by converting all the chloroform to l/lO-mm. drops is sufficient to adsorb all the dye as a monolayer.

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

155

brane occurs during accumulation is an experimental fact. The vacuole is the first site of accumulation of free ions (Guilliermond, 1941). How this entry occurs, mainly in one direction, is unknown, but if the accuniulatory protein is, at one stage of the cycle, a part of the vacuolar membrane (which is probably lipoprotein in nature) penetration would naturally occur along with the protein. The plasma membrane of the ameba dissolves continually into the cytoplasm at the tail. In this way adsorbed molecules to which the membrane was impermeable (such as glucose) could be taken into the cell through a membrane normally impermeable to them. The question arises, is an adsorbed monolayer of glucose, potassium ion, or any other substance a significant contribution to the amount in the cell ? The protein in a cell the size of Amoeba proteus or A . discoides is sufficient to form only about 10,OOO plasma membranes. An ameba renewing its membrane every two minutes, would, in the period from one mitosis to the next (about two days) expose an area equal to about 1500 times its own surface area. It could thus adsorb an amount comparable with that adsorbable by the whole of the protein in the cell, an amount which, if free, could have a concentration in the cell of up to 15 per cent. Let us consider to what extent the osmotic work mechanism postulated is consistent with the available data on accumulatory mechanisms in living cells. I n general it appears that substances being accumulated are first taken up by the protoplasm and later shed into a depot such as a vacuole. For example, Brooks (1939) found in Valonia that the protoplasm took up rubidium ions from the surrounding medium, becoming eventually 39 times richer than the surrounding medium. Then rubidium passed into the cell sap and into the external medium, so that the accumulation ratio for the protoplasm fell rapidly. Similar results were found when the process was followed by radioactive potassium. This is just what one would expect if the protein molecules which had adsorbed the ions suddenly folded up and shed them. (It is to be noted that potassium, though not usually regarded as a strongly adsorbed ion, is nevertheless no exception to the fact that all ions are adsorbed to some extent on all surfaces, depending on the relative charges, polarizability, van der Waals attraction, etc. Szent-Gyorgyi ( 1947) has reported a considerable adsorption of potassium ions by myosin: from half to seven equivalents of potassium ion per equivalent of myosin (17,600 grams), within the range 0.01-0.1 M KC1 and p H 6.5-7.5). Guilliermond (1941) has shown that many dyes, such as methylene

156

R. J. GOLDACBE

blue, Janus green, and neutral red penetrate the cells of various plants and “stain” the vacuoles. This is essentially a vital phenomenon, possibly only during the life of the plant. High concentrations of dye may cause the death of the cell. At this stage the vacuole suddenly becomes colorless, and the entire cytoplasm takes on the color of the dye. This suggests that at death the proteins suddenly become “denatured,” i.e., unfold and so take up the dye. Yeast cells in a 0.005 per cent solution of neutral red accumulated the dye in their vacuoles, the concentration in the external medium falling in half an hour to 0.0015 per cent (Guilliennond, 1941). After another half an hour the vacuoles began to lose their dye and the concentration in the medium rose until at the end of an hour all the cells were de-stained and the concentration in the medium had risen almost to its original value: the yeast, after accumulating the dye in its vacuoles, excreted it into the medium. It is obvious that if proteins are unfolding and folding continually, then many substances will be accumulated which are of no use to the cell. In this category are rubidium and the dyes mentioned above. The cell cannot help accumulating them if they are present in the external medium. The tubules of the kidneys have the power of concentrating some dyes, but not others (Hober and Woolley, 1940). On analysis, it is found that only those dyes with a marked amphipathic structure are concentrated, and if a markedly polar group is inserted in the non-polar end, the dye is concentrated no longer. Some examples of dyes accumulated, with the corresponding non-accumulated analogue, are given in Table 11. Hober (1940) has suggested that the difference between the two classes of dyes is due to their adsorption on a surface at some stage of the accumulatory process. How the dye becomes released from the surface is developed in this paper. When frog kidneys are perfused with a phenol red solution, the dye is concentrated by the tubules; the concentration of the dye could be varied 100-fold above a critical concentration in the perfusion fluid without influencing the rate of secretion (Scheminsky, 1929). This is consistent with there being a limiting factor set by the rate at which the protein of the accumulatory mechanism can fold and unfold. Once the dye is adsorbed to near the saturation point of the protein, no increase in concentration will increase the rate of turnover. The number of cycles of folding and unfolding of a protein should be proportional to the amount of material accumulated and to the chemical energy expended. Lundegardh and Burstrom (1933, 1935), and Robert-

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

157

TABLE I1 Dye Accumulated

Dye not Accumulated

-8

SO&a

HO

N = N o

SOSNa

SOaNa (Cloth red 2R)

son and Turner (1945) have shown that when root tissue was placed in solutions of various salts, the cells accumulated the salt and consumed more oxygen than before. The extra salt-induced respiration (measured as COZ) is proportional to the amount of salt accumulated. (Both salt accumulation and the extra salt-induced respiration were inhibited by cyanide, whereas the basic “ground” respiration was not.) One of the most interesting examples of an accumulatory mechanisiii occurs in the stomach. The oxyntic cells in the gastric mucosa secrete a solution. which is about a million times richer in hydrogen ions than the blood. Various attempts have been made to explain this secretion of decinornial hydrochloric acid by juggling with metabolic carbonic acid and sodium chloride (Conway and Brady, 1947; Bull and Gray, 1945; etc.) or with electrical potentials across membranes, permeable only to the hydrogen ion, etc. (Davies, Longmuir, and Crane, 1947; Hollander, 1943; Rehm, 1943, 1945; etc.). But the alkaline tide, i.e., the production of an exactly equivalent amount of alkali in the blood (Hanke, Johannesen and Hanke, 1931), shows that metabolic carbonic acid, or any other organic acid synthesized from a neutral precursor such as glucose; cannot be responsible for

158

R. J. GOLDACRE

the hydrochloric acid; there is merely a separation of acid from alkali. Again, membranes tend to be permeable to all cations or to all anions, and selective permeability to a single ion is not established (Davson and Danielli, 1943). The simplest explanation is that hydrogen ion accumulation is a particular function of the general cation-accumulatory mechanism which all cells possess. The amount of adsorption of hydrogen ions on folded and unfolded protein molecules is different. Thus, the isoelectric point of egg albumin increases by half a unit on surface denaturation (Monger, 1938). Conversely, a pH increase occurs when the egg albumin molecule is unfolded by the action of heat, ultraviolet light, urea or surface forces (Neurath et al., 1944). Hence it is evident that the refolding of this molecule would liberate hydrogen ions to the solution. The way in which a protein molecule might be specialized so as to produce much greater p H changes is shown in Fig. 10. The specialized

I

+H

-P+

0

2

FIG.10. The folding of a protein so as to release hydrogen ions to the solution.

protein would have a configuration such that positively charged side chains (e.g., as in lysine and arginine) often come opposite to weakly acid side chains (in the adjacent protein lamina), of pK, greater than 7 (such as tyrosine p& 9.8-10.5, cysteine pK0 9.1-10.8) so that at the physiological p H of 7 the hydrogen ion is attached to most of the acid side chains in the unfolded molecule. When the molecule is refolding, competitive adsorption from the positively charged side chain forces the hydrogen ion off into the solution (into a vacuole). The process is then repeated. The pH to be expected from a single cycle may be calculated. If it is assumed that the cross link shown in Fig. 10 occurs at every fifth residue along the chain (which is the frequency of salt links in myosin) and that the concentration of this protein is 10 per cent (as is myosin in muscle), of average residue weight 100, then the molarity of the cross

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

-

159

link is 100/(100 X 5) 0.2. Thus, if one cycle were 100 per cent efficient, it would produce a p H of 0.7. This is ample, for the p H of the gastric secretion is about 1-2. If the vacuoles of hydrochloric acid produced in this way are excreted bodily by the oxyntic cells, so that they travel down the lumen of the gland and burst in the stomach, the protoplasm of the oxyntic cells would be left alkaline. This would equilibrate by dialysis with the blood across the anion-permeable cell membrane, resulting in the alkaline tide which eventually appears in the urine. The unfolded protein molecule is versatile because of the variety of side chains and of the number of ways of arranging them along the backbone ; the possible use of certain kinds of side chains in ion accumulation has been indicated. Examples of possible ways in which protein molecules might be specialized to take up particular un-ionized molecules are as follows : 1. Non-ionized substances would be adsorbed by virtue of their attraction for different side chains of the protein, e.g., alcohols for the -OH of serine, threonine, tyrosine, and perhaps CONHz of citrulline ; and fatty substances by virtue of their attraction for the non-polar groups. 2. Urea, which is concentrated by the kidney tubules of the frog and some fish (Marshall and Crane, 1924) has the power to unfold proteins by breaking the hydrogen bonds between adjacent polypeptide chains ; but the concentration required is high and about 3-6 molar. The forces which oppose the unfolding, apart from the relative stability of -CONHbonded to urea compared with -CONH- bonded to its counterpart in an adjacent polypeptide chain (Huggins, 1943), are those due to the mutual attraction of the side chains which are roughly in contact with one another and pointing at right angles to the protein laminae. The forces due to hydrogen bonds between amido groups act in the plane of the laminae. Thus, anything increasing the repulsive forces between the side chains would weaken the hydrogen bonds and lessen the resistance to unfolding by urea. This property would be possessed by a protein in which the distribution of side chains was such that, when the chain was folded, similarly ionized side chains occurred next to one another in adjacent chains. Urea would act as its own unfolding agent in such a weakened protein. Refolding might be achieved by esterification of the (charged) acid groups, converting a repulsion into a van der Waals attraction. The original condition of the protein would next be regained by its hydrolysis by an esterase, thus completing the cycle and leading chemical energy from another source into the process (coupling).

160

R. J. GOLDACRE

3. Water, which is accuniulated, e.g., by the salivary gland [the secretion of which contains much less salt than the blood (Hober, 1946)], could be withdrawn from a solution by the hydration of an unfolded protein which then refolds dong the planes in which water is adsorbed, shedding it ultimately into the lumen of the gland. It is not suggested that these are actual mechanisms operating in the kidney and salivary gland respectively, but they are merely put forward to illustrate the adaptability of the protein molecule t o particular ends. I n all the accumulatory cycles postulated it is necessary that the protein complete a cycle and return to its original state. This involves the intervention of both unfolding and refolding agents, though at different parts of the cell. The state of folding of globular proteins is influenced by a great variety of agents, including mechanical stresses, heat, light, pressure, pH, surface-active agents, high concentrations of alcohols, amides, and other relatively inert organic substances, and of inorganic salts (Neurath, et al., 1944). In general, any substance which is strongly adsorbed will unfold the protein by splitting the laminae (through competing with one lamina for adsorption on the surface of another). A change in the surface activity of a compound at the protein-water interface would bring about a change in the extent of folding of the protein. (This change in surface activity may be brought about by an extraneous chemical reaction, occurring in a discrete part of the cell. Thus the release of energy in one reaction could be used to cause an accumulatory mechanism to proceed.) Conversely a change in the charge of a strongly bound molecule, such as by hydrolysis of an ester group, would change the condition of the protein. Whether the changes brought about in actomyosin by adenosine triphosphate are due to this kind of change or not remains to be shown. This sort of change may extend beyond actomyosin to protoplasm in general. Goldacre and Lorch (1950) have shown that the effect of A T P on the ameba’s cortical gel (the basis of the accumulatory mechanism) is similar to its effect on actomyosin. Osmotic work with neutral red, in ameba and in plant root hairs, occurs only during cytoplasmic streaming. Since streaming and osmotic work both appear to result from the reversible folding of protein molecules, it seems likely that they would occur together in other cells. This appears to be so in two instances of accumulation followed by means of radioactive phosphorus. An egg cell is quiescent and neither streams nor takes up phosphate. A few minutes after fertilization however, stream-

PROTEIN MOLECULES AS A BASIS OF OSMOTIC WORK

161

ing begins and phosphate is steadily accumulated, (Whitely, 1949). Conversely, amebae which have been enucleated, or enucleate fragments, stream only feebly and intermittently, in contrast to the vigorous and continuous streaming beforehand, and when the nucleus is put back again (Clark, 1942; Commandon and de Fonbrune, 1939; Radir, 1931; Lorch and Danielli, 1950) , and the feebly streaming enucleated fragments take up less than one third as much phosphate as the nucleated controls (Mazia and Hirshfield, 1950). Moreover, the post-fertilization respiration rate in sea urchin eggs is four to five times that before fertilization, and KCN strongly inhibits the respiration of the fertilized eggs but has no effect on unfertilized ones (Runnstrom, 1930). IX. CONCLUDING REMARKS Osmotic work and cytoplasmic streaming are interrelated. Both processes, however, appear to be merely offshoots of a much more important process. The energy required for osmotic work is only a small fraction of that actually used in the extra respiration that salt accumulation induces, and the work involved in ameboid movement itself is small compared with the oxygen consumed. Robertson ( 1941) has calculated that the salt-induced respiration results from an expenditure of energy of about 200 times the osmotic work involved in the concentration of potassium occurring in the same time. Similarly, Heilbrunn ( 1937) has shown that ameboid movement required only a surface tension difference of 0.01 dyne per centimeter between the front and the back of the cell. Protein folding and unfolding appear therefore to be required for something much more fundamental in the cell. This probably involves enzyme reactions which are assisted by this rhythmic change in form of the molecule. The products of the reaction would be squeezed off the surface of the enzyme when it folded up, but while adsorbed it would be kept out of the back reaction. This may also be concerned with the folding up of the newly formed protein molecules themselves. I n fact, Steward and Preston (1941) have shown that accumulation does not occur in potato discs unless protein synthesis is taking place. The foldingunfolding cycle in the cell would be analogous to the driving wheel in a workshop, which can be harnessed to drive various machines by letting in a clutch. The machines driven would be the other enzymes which change their state of folding rhythmically (in changing each molecule of substrate) but which are unable to fold up of their own accord. Their adsorption (clutch mechanism) on to the contractile myosin fibril would do this for them.

162

R . J. GOLDACRE

I n this way ATP, which works the folding-unfolding cycle, could be harnessed to drive any other reaction which involves the folding-unf olding cycle (coupling). A muscle cell for example is.not something apart but an ordinary cell in which the already contractile protein chains are aligned so that contraction of the cell all at once becomes a conspicuous feature. The same contractile chains can do other things as well, e.g., osmotic work and secretion. Nervous impulse, the electrical cells of fish, and secretion may all turn out to be different aspects of the same phenomenon, the cell’s geometry being altered so that some particular aspect of protein folding is brought to the fore. It might be fitting to conclude with some stimulating remarks made by Astbury and Szent-Gyorgyi on protein folding and living processes. Astbury (1946) speaking on the structure of the protein molecule, says: “Such a plan is sufficient to explain the observed striking physical unity amid chemical diversity . . . the keratin-myosin-fibrinogen group represents the power of the cell to synthesise elastic fibroproteins of a standard molecular form, the details of which can be adapted to as many ends as the process of differentiation demands.” Szent-Gyorgyi ( 1947), having shown that the actomyosin-ATP system is extraordinarily sensitive to changes in the ionic balance of the medium, points out that the same ionic balance is repeated in different organs for normal activity. If systems other than the actomyosin-ATP systems are sensitive to metallic ions (as, for example, the cell membrane undoubtedly is), then the o p timum for each system is the same. This suggests that the same basic mechanism operates in a wide variety of cellular functions. Summing up more cautiously, he states : “Another most fascinating problem is to find out how far the relationships found in muscle represent general principles in living matter, or how far they are specific cases only. Some observations suggest that the different functions of different organs are closely related, with the same basic mechanism adapted to specific purposes. “Caffeine, which produces contraction in muscle, produces increased renal and nervous activity. Veratrine, which provokes protracted contraction in muscle, produces prolonged flow of saliva. If the same key opens different slots, then the mechanism of these slots cannot be any too different.”

163

PROTEIN MOLECULES AS A BASIS O F OSMOTIC WORK

X. REFERENCES Astbury, W. T. (1940) Proc. Roy. SOC.Lond., Bl29, 307. Astbury, W. T. (1937) Tram. Faraday Soc., 84, 378. Astbury, W. T. (1946) Nature, Lond., 167, 121. Astbury, W. T., and Bell, F. 0. (1941) Nature, Lond., 147, 696. Astbury, W. T., and Dickinson, S. (1935) Nature, Lond., 136, 95. Astbury, W. T., and Dickinson, S. (1940) Proc. Roy. SOC.Lond., Bl29, 307. Astbury, W. T., Dickinson, S., and Bailey, K. H. (1935) Nature, Lond., 136, 95. Astbury, W. T., and Lomax, R. (1935) I. Chem. Soc., 846. Banga, I., and Szent-Gyorgyi, A. (1942) Ewymologia, 9, 111. Brooks, S. C. (1937) Trans. Faraday SOC.,SS, 1006. Brooks, S. C. (1939) J. cell. comp. Physiol., M ,649. Bull, H. B. (1943) Physical Biochemistry, Wiley, New York, 324. Bull, H. B., and Gray, S. C. (1945) Gastroent., 4, 175. Canti, R. G. (1920) Film available at Roy. Micr. SOC.Library. Carrel, A., and Ebeling, A. H. (1926) I. exp. Med., 44, 261. Chambers, R., and Kopac, M. J. (1940) private communication to J. Needham, quoted in Biochemistry and Morphogenesis (1950) Cambridge, p. 657. Clark, A. M. (1942) Aust. I . exp. Biol. med. Sci., 20, 241. Cole, K. S. (1932) J . cell. comp. Physiol., 1, 1. Commandon, J., and de Fonbrune, P. (1939) C. R. SOC.Biol., Paris, 1S0, 740. Conway, E. J., and Brady, T. (1947) Nature, Lond., 169, 137. Danielli, J. F. (1938) Cold Spr. Harb. Symp. quant. Biol., 6, 190. Danielli, J. F., and Harvey, E. N. (1934 ) I . cell. comp. Physiol., 6, 483. Davies, R. E., Longmuir, N. M., and Crane, E. E. (1947) Nature, Lond., 159, 468. Davson, H., and Danielli, J. F. (1943) Permeability of Natural Membranes. Cambridge. Dean, R. B. (1947) Chem. Rev., 41, 503. Dixon, M. (1949) Multi-Enzyme Systems. Cambridge, pp, 49, 54. Eyring, H., Johnson, F., and Gender, R. (1946) I. phys. Chem., 60, 453. Goldacre, R. J. (1951) In preparation. Goldacre, R. J., and Lorch, I. J. (1950) Nature, Lond., 166, 497. Goldacre, R. J., Loveless, A., and Ross, W. C. J. (1949) Nature, Lond., 163, 667. Guilliermond, A. (1941) The Cytoplasm of the Plant Cell. Chronica Botanica Co., Waltham, Mass., pp. 129-145, 189. Hanke, M. E., Johannesen R. E., and Hanke, M. M. (1931) Proc. SOC.exp. Biol. Med., aS, 698. Harvey, E. N. (1937) Trans. Faraday SOC., SS, 946. Harvey, E. N., and Danielli, J. F. (1938) Biol. Rev., 13, 319. Harvey, E. N., and Shapiro, E. (1934) J. cell. comp. Physiol., 6, 255. Heilbrunn, L. V. (1937) Outline of General Physiology, London. 247. Hober, R. (1940) Cold Spr. Harb. Molzogr., 8. Hober, R. (1941) Schweiz. med. Wschr. 71, 241. Hober, R. (1946) The Physical Chemistry of Cells and Tissues, p. 595. Hober, R., and Woolley, P. M. B. (1940) J . cell. comp. Physiol., 16, 34. Hober, R., and Woolley, P. M. B (1940) J. cell. comb. Physiol., 16, 63. Hollander, F. (1943) Gastroent., 1, 40. Hopkins, D. L. (1938) Biodynamica, 2, 1. 1

164

R. J. GOLDACRE

Huggins, M. L. (1943) Chcm. Rev., 82, 195. Hughes, A. (1949) Film shown to Roy. Micr. SOC.and various other societies. Koehring, V. (1930) J. Morph., 49, 45. Langmuir, I. (1939) Proc. Roy. SOC.8170, 1. Lorch, I. J., and Danielli, J. F. (1950) Nature, Lond., 168, 329. Lundegardh, H. (1939) Nature, Lond., 148, 203. Lundegardh, H. (1940) A". agric. Coll. Sweden, 8, 234. Lundegardh, H. (1940) Nature, Lo&., 146, 114. Lundegardh, H., and Burstrom, H. (1933) Biochem. Zeitschr., 201, 235. Lundegardh, H., and Burstrom, H. (1935) Biochem. Zeitschr., 277, 723. Marsland, D. H., and Brown, D. E. S. (1936) J. cell. comb. Physiol., 8, 136. Marshall, E. K., and Crane, M. M. (1924) A m y . J. Physiol., 70, 465. Mazia, D., and Hirshfield, H. I. (1950) Science, 111, 297. Meyer, K. H. (1928) Ber. dtsch. chew. Ces, a,1932. Meyer, K. H. (1929) Biochem. Zeitschr., 214, 253. Mongar, L. S. (1938) I . phys. Chew., a,71. Neurath, J., Greenstein, J., Putnam, F., and Erikson, J. (1944) Chem. Rac., 36, 157. Oster, G., and Grimsson, H. (1949) Arch. Biocbm., M, 119. Pomerat, C. M. (1949) Film produced at Texas Univ. shown to Chester Beatty Research Institute, 1949. Radir, P. L. (1931) Profofilasma, l2, 42. Rehm, W. (1943) A w r . I, Physiol., l88, 1. Rehm, W. (1945) Amer. J. Physwl., 144, 701. Robertson, R. N. (1941) Aust. J. ex). Biol. Med., l9, 265 Robertson, R. N. (1944) Aust. 1. exp. Biol. Med., a,237. Robertson, R. N., and Turner, J. S. (1945) AM#.J exp. Biol. Med., 23, 63. Runnstrom, J. (1930) Protoplasma, 10, 106. Seifriz, W. (1942) Symposium on Structure of Protoplasm, Iowa, p. 264. Scheminsky, F. (1929) Pfiiigers Arch., Bl, 641. Speakman, J. B., and Hirst, M. (1933) Tram. Faruuhy Soc., 29, 148. Steward, F. C., and Preston, C. (1941) Pluttt Phydol., 16, 85. Szent-Gyorgyi, A. (1947) Chemistry of Muscular Contraction, Academic, New York. See also 2nd ed., 1951. Ussing, H. H. (1949) Physiol. Rev., 29, 127. Verzk, F. (1936) Absorption from the Intestine. London. Waymouth, C. (1950) Film produced in the Chester Beatty Institute, private communication. Whitely, A. H. (1949) Amer. Nat., (w, 249.

Nucleo-Cytoplasmic Relations in Amphibian Development G. FANKHAUSER Dcpartrrreiii qf Biology, Prirtcctori Utrivtmsity, Princetort, New Jerscy

CONTENTS

I. Introducti~ii ......................................... 11. Quantitative Changes in Cytoplasm of Egg ................... 111. Quantitative Changes in the Nucleus : Polyploidy and Haploidy . . . . . . . . 1. Chromosome Number, Nuclear and Cell Size ..................... 2. Cell Size, Cell Number, and Body Size .......................... 3. Chromosome Number, Cell Size, and Differentiation : Regulation of Cell Number and Cell Shape ...................... 4. General Effects of Total Quantity of Chromosome Material versus Special Effects of Gene Dosage . . . . . . . . . . . . . . . . . . ... IV. Unbalanced Chromosome Combinations 1. Uniformly Aneuploid Embryos . ... 2. Complex Mosaics with Irregular me Numbers . . . . . . . . . V. Invisible Chromosomal Changes (Gene Mutations?) . . . . . . . . . . . . . . . . . . VI. Development without Chromosomes .................................. 1. Non-Nucleated Cells in the Cleavage of Androgenetic Eggs . . . . . . 2. Cleavage of Eggs without Functional Chromosomes .............. VII. Nucleo-Cytoplasmic Relations in the Early Development of Species Hybrids .................................................... 1. Nucleo-Cytoplasmic Interactions during Fertilization ... 3. Analysis of Nucleo-Cytoplasmic Incompatibility in Diploi and Androgenetic Haploid Hybrids ............................. 3. Nucleo-Cytoplasmic Relatio Species Characteristics . . . . . . ....................... .................. .... VIII. Summary and Conclusions . . . . IX. References .........................................................

Page

168 170 170 173 173 177 177 179 179 181 181 181 183

184 187 188 192

I. INTRODUCTION Interactions between nucleus and cytoplasm obviously are of the greatest importance in all stages of amphibian development. Long before fertilization, preparations begin in the slowly growing oocyte for the creation of a definite organization of the egg cytoplasm which is revealed as soon as the egg becomes available for experimentation, at the time of fertilization. These preparations are made under the control of the oocyte nucleus which manifests its participation in these activities in structural changes involving both the nucleoli and the chromosomes. The latter interrupt meiosis in the diplotene stage and transform into the diffuse “lampbrush” chromosomes whose structure is still only partly understood. I n the cytoplasm, histochemical tests uncover a pattern of a variety of substances

165

166

ti. FANKHAUSER

and the existence of “microsomes” which operate as centers of enzymatic activity (cf. Brachet, 1944). Once the egg is mature and fertilization is initiated, again numerous problems of nucleo-cytoplasmic relations arise, such as the nature of the interactions between the sperm head and the egg cytoplasm, between the sperm and egg nuclei, the mechanism of the inhibition of accessory sperm nuclei in the normally polyspermic eggs of urodeles, and the nature of the factors that control the proper timing of the nuclear and centrosomal cycles during mitosis. Since these interactions in the preparatory phases of development were discussed briefly in an earlier publication (Fankhauser, 1948) this review will be limited primarily to experimental studies of nucleo-cytoplasmic relations in embryonic and later development.

CHANGESI N CYTOPLASM OF EGG 11. QUANTITATIVE It is relatively simple to study the effects of changes in the initial amount of cytoplasm and yolk present in the egg at the time of fertilization. In newts, the blastomeres of the two-cell stage may be separated by means of a loop of fine hair. The operation frequently produces normal twirl embryos of half size if the division takes place in the plane of bilateral symmetry. The size of the cells in the various organs is the same as in controls developed from whole eggs, but their number is reduced (Penners, 1935, Tritoja taeniutus and T. alpestris). However, if small twin larvae from isolated blastomeres of the two-cell stage are raised on an unlimited food supply, they will gradually catch up in growth with the controls and at metamorphosis reach approximately normal size (Spemann and Falkenberg, 1919, Fig. l, Plate l , Triton tacninttu; Fankhauser, 1945a, 11. 48, Triturus wiridescens) , Recently, the influence of egg volume on growth was investigated more thoroughly by Briggs (1949), who took advantage of the great range of variation in the size of the eggs produced by a single female Rana pipiens. The large and small eggs selected for the experiment had a volume ratio of 1.73 to 1. At the end of embryonic development (age ten days), before feeding began, the ratio of the weights of the large and small animals was still 1.68 to 1. After two or three weeks of maximal feeding, the small tadpoles began to grow faster, and at 63 days the weight ratio of the two groups had diminished to 1.08 to 1. It has not been possible so far to determine the mechanism that makes this compensatory growth possible. It may be either a relatively greater food intake of the small larvae, or a better utilization of the ingested food. It is more difficult to increase the amount of cytoplasm in an egg.

NUCLEO-CYTOPLASMIC RELATIONS IN AMPHIBIAN DEVELOPMENT

167

PLATE I Left. Sections through hindbrain of three larvae of Triticrus vividcscerts in early hindlimb-bud stage, with various chromosome numbers, taken a t level of entrance of eighth cranial nerve. Magnification approximately 93 X. From top t o bottom : Haploid, diploid, tetraploid. The area of the cross section of the brain is approximately the same in the three larvae because of the compensation of the changes in cell size by regulation of cell number. Right. Enlargement of parts of Same or adjacent sections to show giant neurones (Mauthner’s cells). Magnification approximately 329 X. From top to bottom : haploid (left side), diploid (right side), tetraploid (left side). The haploid has formed two Mauthner’s cells on both sides; the tetraploid possesses a pair of enlarged Mauthner’s cells.

168

G . FANKHAUSER

Fusion of two eggs in the two-cell stage is possible and sometimes produces a single, normal embryo of giant size (Mangold and Seidel, 1927) ; such embryos have not been raised to feeding stages so far. O n the other hand, eggs that are considerably larger than normal may also be obtained from polyploid females in the axolotl; these develop into large embryos but, eventually, will grow into animals of about normal size, regardless of the number of chromosomes they possess. The available evidence clearly shows that the body size typical for the species is independent of the initial mass of cytoplasm present in the egg. I t would be interesting to determine the minimum amount of cytoplasm in an egg fragment required to produce an animal of normal body size. However, the situation is complicated by the fact that the mere quantity of cytoplasm is not alone deciding the outcome. Invisible, qualitative differences exist between diff erent regions of the egg at fertilization. There is a simple pattern of cytoplasmic differentiation which includes an important area on the dorsal side of the egg, the future center of organization in the dorsal lip of the blastopore. Without a sufficient share of this region, gastrulation and the establishment of a normal embryonic axis are not possible. I

111. QUANTITATIVE CHANGESIN

THE

NUCLEUS

POLYPLOIDY AND HAPLOIDY In the amphibian egg it is relatively easy to obtain quantitative changes in the nucleus. On the one hand, we can let nature perform this delicate operation for us. Among embryos of the American newt, Triturus viridescens, raised in the laboratory from untreated eggs, between 1 and 2 per cent are triploid, i.e., they possess an additional, third set of chromosomes. A much smaller proportion have four or even five sets (tetraploids and pentaploids) , Such spontaneous polyploids probably arise from the accidental formation of unreduced gametes, usually eggs. Besides polyploids, haploid embryos also occur spontaneously ; probably they owe their origin to a process of partial fertilization, with only one set of chromosomes participating in development. Similar observations have been made in several other species of amphibians, notably in the axolotl (cf. the review by Fankhauser, 1945a). The chromosome number of living amphibian larvae may be easily determined in whole-mounts of the amputated tip of the tail, since the epidermis of the thin, transparent tailfin usually contains mitotic figures. Introduction of this method also made it possible to find simple and effective methods for the experimental induction of polyploidy. In amphibian

NUCLEO-CYTOPLASMIC RELATIONS I N A M P H I B I A N DEVELOPMENT

169

eggs, the second maturation division is normally arrested at metaphase until the sperm contacts the egg surface; the second polar body is given off from half an hour to an hour later. Refrigeration of freshly fertilized eggs for several hours (Fankhauser and Griffiths, 1939 ; Griffiths, 1941), or treatment at 36 to 37°C for ten minutes (Fankhauser and Watson, 1942), frequently suppresses the formation of the second polar body and thus produces a high percentage of triploid embryos. Cytological studies on heat-treated eggs showed that the effect of the abnormal temperature is not a complete suppression of the anaphase separation of the chromosomes in the maturation spindle, with consequent formation of a diploid egg nucleus, as had been anticipated on the basis of earlier observations on the effects of abnormal temperatures on meiosis in plants. Instead, the temperature shock produces a change in the surface film of the egg and releases the maturation spindle from its anchorage ; the division continues below the surface and produces two haploid egg nuclei both of which fuse with the haploid sperm nucleus to form a triploid zygote (Fankhauser and Godwin, 1948). ' In the axolotl, the only amphibian species in which polyploid individuals have been raised to sexual maturity,l the breeding of polyploids produces numerous off spring with a variety of abnormal chromosome complements. Matings between triploid females and diploid males, in addition to a majority of embryos with chromosome numbers ranging from the diploid to the triploid, give rise to several per cent tetraploid offspring, because of the not infrequent formation of unreduced, triploid eggs. Very rarely, heptaploid embryos, with seven sets of chromosomes, also appear among the offspring of such niatings (Fankhauser and Humphrey, 1950). Tetraploid females, in turn, when mated to diploid males, produce large numbers of triploid and near-triploid off spring. Haploid embryos have been produced for many years by various methods all of which aim to prevent the formation of a diploid cleavage nucleus (cf. Fankhauser, 1937, 1945a). Haploid development may proceed either with the maternal set of chromosomes, following artificial stimulation of the egg (parthenogenesis) or activation by a spermatozoon (gynogenesis), or with the paternal set (androgenesis) . 1 Kawamura (1951, 1. Sci. Hiroshima Uwizi., Ser. B, Div. 1, l2, 1) reports successful artificial insemination of eggs of diploid female Triturw flyrrhogaster with sperm of a triploid male, and of eggs of three triploid females with sperm of diploid males. The number of eggs produced by the triploid females was very small, and the great majority of triploid females proved to be completely sterile. Kawamura (1951), ibid., l2, 11 also obtained a small number of offspring from triploid males of the frog, Rana nigromoculata, by artificial insemination of eggs of diploid females.

170

ti. FANKHAUSER

1. Chroinosonze Nuinber, Nuclear and Cell Size

Haploid and polyploid embryos may be recognized in life as soon as the pigment cells (melanophores) appear since the size of these cells varies with the number of chromosome sets present in the nucleus. The nuclei themselves increase their size approximately in direct proportion to the number of chromosome sets, as is most clearly seen in the epidermis cells of the tailfin because these cells and their nuclei are very flat discs of quite uniform thickness. The area of outline drawings of the nuclei as seen from the surface thus represents a good index of their volume. In Tritwrus viridescens, measurements have shown that the combined areas of twenty epidermis nuclei have the following values : haploid = 1.0, diploid 2.32, triploid 3.5, pentaploid 5.0. With higher chromosome numbers the increase is less than expected, perhaps because pentaploid epidermis nuclei are somewhat less flattened than the smaller nuclei of the other chromosome classes. Because of the indistinctness of the cell boundaries in ordinary preparations of salamander tailtips, measurements of the cell size have not been made, except in unicellular glands of the epidermis which increase in size in proportion to the chromosome number. In certain tissues, such as the mesenchyme of the tailfin, the number of nuclei per unit area and their spacing are a convenient index of differences in chromosome number (Fig. 1). In the frog, Rana pipiens, the boundaries of the epidermis cells are clearly visible in tailtip preparations. The triploid/diploid ratio for the area of the nuclei was found to be 1.50, that for the area of the cells 1.51 (Briggs, 1947). In this species, the proportional increase in cell size is evident as eariy as the tailbud stage, because the ectoderm at this time consists of a mixture of lightly and darkly pigmented cells (Fig. 2). Recently, Gallien and Muguard (1950) have described a silver staining technique for the boundaries of epidermis cells in tailtips of the newt, Pleurodeles waltlii; this procedure should prove very useful in establishing the actual nucleo-cytoplasmic ratio in different tissues at various levels of heteroploidy.

-

-

-

2. Cell Size, Cell Number, aitd Body Size In spite of the increase in the size of the individual cells in polyploids, the body size of the embryos and larvae remains remarkably constant. In Triturus z&idesceiw, at the beginning of feeding, triploid, tetraploid, and pentaploid larvae are of approximately the same size as the diploid. The increase in cell size is compensated by a corresponding decrease in cell number in various organs and tissues. In the living animals this be-

NUCLEO-CYTOPLASMIC RELATIONS IN A M P H I B I A N DEVELOPMENT

f

6 8

8

N

I

171

., 8

C

1 t *a-

??

I)

FIG.1. Size and spacing of nuclei of mesenchyme in tailfin of five young axolotl larvae with different numbers of chromosomes (haploid, diploid, triploid, tetraploid, and heptaploid) , Magnification approximately 255 X .

FIG.2. Size and spacing of lightly pigmented cells in head ectoderm of intact embryos of Rana pipiens at tailbud stage (haploid, diploid, and triploid). The lightly pigxhented cells have moved between the deeply pigmented surface cells from the deeper layers of the ectoderm following gastrulation. Magnification approximately l l O X . After Briggs (1947, Fig. 1).

172

G. FANKHAUSER

comes clearly expressed as soon as the melanophores develop : the greater the chromosome number, the smaller is the number of individual pigment cells in any given area of the body. The balance between cell size and cell number that keeps the size of the organ constant is easily seen in the cartilages of the carpus and tarsus, in the brain (Plate I ) and in small sense organs such as the lateral line organs (Fankhauser, 1941). In haploids, on the other hand, the smaller cell size is partly compensated by an increase in cell number. The best haploid newt larvae are very nearly normal size and show almost twice the normal number of cells in different organs. I n the most advanced haploid obtained so far, which died at the completion of metamorphosis (Baltzer, 1922 ; Fankhauser, 1938), almost complete regulation in the size of some glandular organs was obtained in one of two ways: either by increase in the size of the individual tubules, as in Harder’s gland in the lower eyelid, or by multiplication of the number of units which individually remained smaller than in the diploid, as in the follicles of the thyroid. The adjustment of cell number reaches a critical point in the special case of Mauthner’s cells, a single pair of giant ganglion cells located in the medulla at the entrance of the eighth cranial nerve. The tetraploid cannot reduce the cell number; it still has a pair of super-giant neurones. A haploid larva, on the other hand, may compensate and develop two Mauthner’s cells on either side (Plate I). That polyploid, particularly triploid, amphibian larvae possess normal body size has been amply confirmed by observations on several other species, viz., Tm’turus pyrrhugaster, Eurycea bislineato, axolotl (cf. Fankhauser, 1945a), Triton alpestris (Fischberg, 1947), Rana pipiens (Briggs, 1947). Evidence on later growth and on the size reached by adult polyploids is derived from the observations of Humphrey on axolotls (Fankhauser and Humphrey, 1950, and unpublished observations). Triploids vary considerably in size; more often they are normal; some are as large as the largest diploids. I n spite of their general vigor, the life span appears to be reduced; in their third and fourth years they eat less and become emaciated; usually they die before four years, while diploids in the same colony may live to six or seven years. Some adult tetraploid axolotls are as large as the best diploids; all these so far have been of mixed axolotl and Ainblystowaa tigrinurn ancestry. Others, including all those of pure axolotl stock, are inferior in size and sometimes runty. Pentaploids have been raised to ages over a year and a half, but not to maturity. They are, as a rule, much retarded

NUCLEO-CYTOPLASMIC RELATIONS IN AMPHIBIAN DEVELOPMENT

173

in growth and tend to develop a more or less pronounced axial curvature. Again there is considerable individual variation ; one pentaploid was almost normal in body length until death occurred at the age of seven months.

3. Chromosome N u d e r , Cell Size, and Diferentiatioiz: Regulation of Cell Number and Cell Shape During the embryonic period, the general course of differentiation as well as its rate of progress appear normal in triploid, tetraploid, and pentaploid embryos, as far as external inspection shows. Detailed microscopical examination may yet reveal some minor differences in the differentiation of certain organs or tissues. The haploids are usually retarded and abnormal, although the degree of abnormality varies considerably with the species, with embryos of newts being in general less affected than those of axolotls and of frogs. Occasional haploid newt embryos differentiate normally and with only slight retardation. In general, all the processes of morphogenesis are highly adaptable to differences in cell size which may range from one-half normal in the haploid, to two and one-half normal in the pentaploid. The two heptaploid axolotl embryos obtained so far were both abnormal; still they developed to an advanced embryonic stage and formed all essential organs, in spite of the large size of their cells. The adjustments to the alterations in cell size of heteroploid embryos, however, are not expressed in cell numbers alone. In some organs they involve changes in the shape of the individual cells as well. This is particularly striking in structures that consist essentially of single layers of cells, such as the epidermis of the young larva, the tubules of the pronephros, the pronephric ducts, and the epithelium of the lens of the eye. The thickness of these layers remains practically constant and normal from the haploid to the pentaploid. In the haploid, the cells are more nearly cuboidal. I n the polyploids, the large cells are greatly flattened to form a layer of normal diameter. In this way, the normal structure of various organs can be maintained (Fankhauser, 1945b).

4. General Efects of Total Quantity of Chromosome Muterial versau Special Effects of Gene Dosage The primary effects of changes in the number of sets of chromosomes are quantitative and expressed in the increase in size of the nuclei and cells. The latter is, presumably, a consequence of the increase in total mass of the chromosome material, although observations on plants have demonstrated that the cell size may be affected differently in different

174

C . FANKHAUSER

strains, with different genetic backgrounds. I n other words, there is evidence for genetic control of this “mass effect” of the chromosome material. So far, no significant differences have been discovered in the changes in cell size in polyploids of different species of amphibia, although sufficiently accurate studies have not been made to preclude the possibility. Whether or not the genetically “inert” heterochromatin plays a special role in the relations of chromosome number to nuclear and cell size remains an open question. The normal course of development of polyploid einbryos up to the pentaploid level shows that whole sets of genes may be multiplied without disturbing the harmonious activity of the gene complex, as long as all genes are present in the same number. Theoretically, certain qualitative effects of polyploidy may still be expected for the following reasons : (1) The mere increase in nuclear and cell size may affect the activity of genes differentially by changing the dimensions of the stage on which they act (cf. Goldschmidt, 1937). At the same time, this may be an important factor in determining the limit of tolerance for polyploidy, which is clearly surpassed at the heptaploid level. (2) Most, but not all, of the genes may produce their maximum effect in double dose in the diploid ; addition of one or more genes would not enhance their expression. Some genes, however, may have a cuniulative effect in multiple dose, as in the classical case of the gene for yellow endosperm in corn (Randolph and Hand, 1940). This differential effect of polyploidy on gene expression, in some cases at least, might be connected with the availability of cytoplasmic substrates with which the gene products finally interact. The substrates may be present in limited amount for some genes, in excess for others. (3) If the diploid is heterozygous for several or inany factor pairs, addition of one or more sets of chromosomes may change the dominance relations of different genes to different degrees. Incomplete dominance in the triploid condition has recently been established for the gene affecting the pigment pattern in the axolotl. In the diploid, gene D (for “dark”) is dominant over d (for “white”) so that D D and Dd animals are both dark and indistinguishable. Recessive white larvae (dd) have few melanophores with very restricted distribution. Triploids of the constitution Ddd show fewer melanophores than Dd diploids or DDD triploids, although the cells still have a normal distribution (Dalton, 1 9 5 0 ~ ) . The question whether changes in cell size or special gene effects are involved is raised in the interpretation of the usually abnormal development and poor viability of haploids (for a discussion of the older litera-

NUCLEO-CYTOPLASMIC RELATIONS I S AMP H I B I A N DEVELOPMEKT

175

ture see Fankhauser, 1937, 194Sa). The haploid cells, because of their small size, may have a lowered metabolic efficiency as compared with the diploid. Such a view seems to be supported by the fact that in the Hymenoptera, where haploidy of the males is a constant phenomenon, the cell size of the haploid males is essentially the same as in diploid females, a t least in those species (Apis, Habrobrucorr) that have been most thoroughly investigated. The reduced cell size of the “original” haploid males may have been gradually restored to normal through a slow process of selection of genetic factors causing an enlargement of the haploid cells. On the other hand, the low viability of haploids may be a simple consequence of the unmasking of recessive lethal or at least detrimental genes with a wide distribution in the population. This would account for the appearance of occasional more vigorous haploid larvae. This interpretation is also supported by the discovery of a recessive lethal factor in the axolotl to be discussed in a later section (Humphrey, 1948). The fluid imbalance created by this factor is similar to, but not identical with, the edema common in haploids. Furthermore, at the haploid level as at the polyploid, one may also have to consider the possibility of a disturbance of gene balance, if some genes produce the same effect in single dose as in double, while others have a more or less reduced activity. The fact that the “best” haploid newt obtained so far, which completed metamorphosis, developed from an egg fragment of Triton tueniutins and not from a whole egg seemed to give support to the view that a reduction in the initial mass of cytoplasm and yolk present in a haploid egg has a beneficial effect since it returns the nucleo-cytoplasmic ratio to the normal value. More recently, however, Fischberg ( 1947) has raised a haploid from a refrigerated whole egg of Tritou alpestris to the age of 289 days, and at least to the beginning of metan~orphosis. Similarly, a whole egg of the axolotl, not exposed to any special treatment, developed into an unusually normal and vigorous larva that lived for 116 days, while the many other spontaneous haploids found in this species all showed the usual haploid syndrome and died early, before feeding (Humphrey and Fankhauser, unpublished). On the other hand, the importance of the quantitative relations between the nucleus and the total mass of cytoplastn and yolk present in the egg has again been demonstrated by Briggs (1949) for haploid embryos of Rnnn pipicns. Following neurulation, the androgenetic haploids developing from eggs of normal size (“large haploids”) acquired the characteristics of the haploid syndrome typical for frog embryos, viz., small head, small and abnormal eyes, short gills, frequent dorsal curvature of

176

G. FANKHAUSER

the posterior part of the axis, incomplete fusion of the opercular fold; later, the animals developed ascites and edema, and all died between twelve and eighteen days from insemination without feeding. In “small” haploids, developing from eggs of slightly more than one-half normal size, most of these symptoms appeared but were less severe. Two-thirds of the animals were able to feed ; they did not display edema or ascites and grew as tadpoles for periods up to nine months. The interpretation of this striking difference is not clear. In the small haploid eggs, the nucleo-cytoplasmic ratio was almost normal from the beginning of development, as compared with the abnormally small ratio in the large haploid eggs. This difference did not express itself until after the neurula stage, although the nucleo-cytoplasmic ratio in the large egg returned to normal at the end of cleavage or by the time of the beginning of gastrulation. Measurements on sections showed that the reduction in egg size led to a larger reduction in the size of the gut mass than of most other organs, which would facilitate normal differentiation of the gut. At the same time, in the small haploids the blood circulation was much improved over that of the large haploids, so that the process of yolkbreakdown would occur more normally. The reason for the better development of circulation is not obvious, unless it be again correlated with the relatively smaller mass of yolk present. A further approach to the analysis of the effects of liaploidy is indicated by the occurrence (either spontaneous or as a result of refrigeration of eggs) of chromosomal mosaics, i.e., of larvae that are haploid in large areas of the body and diploid (or triploid) in the remainder. Haploid portions of such mosaics usually develop better than the corresponding portions of completely haploid embryos. This may be caused by a beneficial, “vitalizing” effect of normal, diploid tissues on adjacent haploid ones, through the contribution of some important substances not produced in sufficient quantity by the haploid cells themselves. Signs of a quantitative deficiency in the activity of the haploid nucleus have actually been found. In the retarded haploid portions of the haploid-diploid mosaics developed from dispermic eggs of the frog, Brachet (1944) discovered a reduced amount of ribose nucleo-protein. On the other hand, Briggs (1946) found that haploid frog embryos are more sensitive to the inhibiting effects of hexenolactone, possibly because of the production of subnormal amounts of various -SH enzymes. Another possible interpretation of the improved performance of some haploid-diploid mosaics rests on the assumption that certain organs or tissues are more adversely affected by the haploid constitution than others

NUCLEO-CYTOPLASMIC RELATIONS IN AMPHIBIAN DEVELOPMENT

177

and would thus become the most important factor limiting the development of haploids. A systematic study of the many possible combinations of haploid and diploid territories in mosaics may decide the question whether good viability of the mosaic is always correlated with the diploid or predominantly diploid constitution of certain tissues. The problem could also be approached in a more direct way by grafting primordia of various organs from haploid gastrulae or young embryos into diploid hosts and following the development of such artificial mosaics.

IV. UNBALANCED CHROMOSOME COMBINATIONS (ANEUPLOIDY) 1. Uniformly Aneuploid Embryos While the amphibian embryo tolerates the addition of whole sets of chromosomes very well, it is easily upset by quantitative alterations in the nucleus of a much smaller scope. If, instead of complete sets, one or more single chromosomes are added to the diploid complement, development becomes almost always abnormal. A large number and variety of such “aneuploid” embryos with unbalanced chromosome complements may be obtained by mating triploid axolotl females with diploid males (Fankhauser and Humphrey, 1950). At meiosis in triploid eggs, the third set of chromosomes is distributed more or less at random between the two poles of the spindle and eggs result that vary in chromosome number from haploid to diploid. Following fertilization by a normal haploid sperm, the eggs begin development with chromosome numbers ranging from diploid (28) to triploid (42). The actual distribution of chromosome numbers, as determined in tailtip preparations of the embryos surviving to the stage of tail-clipping, follows closely the expected normal curve, with a shift of the peak from the 35- to the 33-chromosome class, probably because of the lagging and loss at meiosis of an average of two chromosomes of the third set. Thirteen hundred and fifteen embryos, about two-thirds of the total of 2027 obtained, completed their embryonic development, but only twelve aneuploid larvae survived beyond the third month. The few animals surviving past the first year were much retarded in growth and never reached adult size or sexual maturity. The great majority of the aneuploid embryos became abnormal at an early stage. One of the most frequent syndromes combined a complete failure of the circulation to become established with small gills, and edema (Fig. 3). In other cases delayed and subnormal circulation was accompanied by stasis and hemorrhages in various organs and by ascites. These or similar features are observed occasionally in diploid embryos ;

178

G. FANKHAUSER

b FIG. 3. Three hyperdiploid axolotl embryos from spawning of a triploid female mated with a diploid male, showing typical features. Magnification approximately 6 X . After Fankhauser and Humphrey (1950). a. 29 chromosomes ( 2 N f l ) ; no circulation, extreme microcephaly and ascites. b. 34 chromosomes (2N-I-6) ; no circulation, microcephaly, ascites, and large blister under epidermis on right. c. 33 chromosomes (2N-t-5) ; good circulation, some stasis in liver, slight fluid accumulation in gills and pronephros. Ten days later, this animal showed defective circulation, pronounced edema of head, and ascites.

however, their combined and frequent occurrence in aneuploid embryos is typical. Unfortunately, the morphological individuality of the chromosomes of the haploid set of the axolotl is not sufficiently striking to allow identification of the extra chromosonies present in aneuploid embryos, as can be done in some plants, e.g., Datura, and to assign definite effects to the presence of single chromosomes. Even the expected correlation between the number of extra chromosomes and the severity of the symptoms is frequently broken, since embryos with a single extra chromosome may be more severely affected than others with several (Fig. 3 ) . In spite of this general unspecificity of the effects the fact remains that the establishment and maintenance of the circulation are particularly sensitive to chromosomal imbalance. It is hardly a coincidence that the development of the circulatory system is also affected by other disturbances of the nucleo-cytoplasmic relations, as in haploid frog embryos ( Briggs, 1949) and in hybrid embryos derived from crosses between southern and northern races of Ratla pipiens (Moore, 1946).

NUCLEO-CYTOPLASMIC RELATIONS I N A M P H I B I A N DEVELOPMENT

2.

179

CoPnplen Mosaics with Irregular Clironzosome Nuwibers

Embryos of this type show a complicated pattern of various abnorinal and mostly unbalanced chromosome numbers in different cells. They appear with high frequency in experiments on androgenesis in newts which produce a relatively small percentage of uniformly haploid embryos because of the normally polyspermic insemination of the urodele egg. In the course of normal fertilization, a single sperm nucleus fuses with the egg nucleus. The independent division of the accessory sperm nuclei is normally arrested at the time of the first cleavage mitosis through an inhibiting influence originating in the diploid mitotic system. When the egg nucleus is removed, some of the accessory sperm nuclei divide ; abnormal mitoses are frequent, particularly multipolar figures in which the chromosomes are distributed irregularly between the three or four poles. Such eggs segment abnormally and form more or less irregular blastulae that show a complex mosaic of abnormal chromosomes numbers in most or all of their cells. None of these eggs survives through gastrulation (Tritoii palmatus, Fankhauser, 1934; Triturus viridescens, Fankhauser and Moore, 1941; Kaylor, 1941). Close cytological analysis, which made full use of the pronounced morphological individuality of the chromosomes of the haploid set of Tritoii palmatus, showed that the composition of many chromosome complements in such androgenetic blastulae is much more abnormal and unbalanced than the chromosome number alone would indicate. Even in a complement of eighteen chromosomes, some members of the haploid set of twelve may be completely absent while others are represented two or three times. Furthermore, occasional groups with the normal haploid number were discovered that contained only two of the twelve individual chromosomes, each of these being represented six times. The origin of such extreme combinations could be explained by the observation that two or more abnormal mitoses of various types may occur in succession during cleavage, producing increasingly abnormal chromosome complements. The fact of the early death of such eggs with multiple irregular chromosome numbers confirms Boveri’s conclusion reached in his studies on the effects of multipolar mitosis in dispermic sea urchin eggs, viz., that a complete, haploid set of chromosomes is essential for normal development from the gastrula stage on.

V. INVISIBLE CHROMOSOME CHANGES(GENE MUTATIONS ?) Besides the recessive factor for white in the axolotl, which expresses itself in the embryo as soon as the pigment pattern develops, only one

180

G. FANKHAUSER

other Mendelian factor with early expression in the embryo is known at present. The trait, known as “fluid imbalance,” was discovered by Humphrey (1948) in his axolotl colony and was shown to be caused by a single recessive factor. Matings between two heterozygous animals (Ff) of normal appearance produced almost exactly 25 per cent affected embryos among their offspring (2107 of a total of 8441). The trait first appears in tailbud stages as a marked enlargement of the head and suprabranchial region by excess fluid; later on, fluid may also accumulate caudal to the gills (Fig. 4). The gills are defective, gill circulation often fails to develop and the larvae die within a short time after hatching. The factor “fluid imbalance,” which may be either a gene mutation or a deficiency for a short segment, is a most welcome demonstration of the existence of lethal factors in amphibians which, prior to this discovery, had already played an important role in theoretical discussions of the causes of the poor development of amphibian haploids. It is important to note, however, that the condition is clearly distinguishable from the edema of

9 b

FIG.4. Recessive lethal factor “fluid imbalance” in axolotl embryo. a. Embryo in tailbud stage showing lethal trait, viewed from ventral surface; accumulation of fluid in head and suprabranchial region. b. Normal control in same stage. c. Larva with lethal trait at hatching; fluid accumulation in branchial region and pronephros ; gills poorly developed, with deficient circulation. d. Normal control in same stage. Magnification approximately 6 X . Modified after Humphrey (1948, Fig. 4).

NUCLEO-CYTOPLASMIC RELATIONS

IN AMPHIBIAN DEVELOPMENT

181

the haploid syndrome, because of the location of the swelling in the head and suprabranchial region, and can be recognized in haploid embryos that occur spontaneously in Ff x Ff crosses where all diploid offspring are normal. VI. DEVELOPMENT WITHOUT CHROMOSOMES Uoveri’s conclusion that nuclear factors begin to play an important part at the time of gastrulation is also confirmed in a striking manner by observations on the development of partially or Completely non-nucleated eggs which invariably fail to gastrulate normally. Conversely, these cases are also of interest since they demonstrate that development to the blastula stage may proceed in the absence of chromosomes.

1. Non-Nucleated Cells in the Cleavage of Androgenetic Eggs

As has been mentioned in a preceding section, the development of antlrogenetic eggs or egg fragments of newts is frequently disturbed by the independent division of some of the accessory sperm nuclei. Abnormal, multipolar mitosis of such nuclei may produce asters not associated with any chromosomes; in addition, new asters (cytasters) may appear in the egg apart from any nuclei. Such asters may divide repeatedly and induce the formation of complete cleavage furrows ; blastulae result with more or less extensive areas of non-nucleated cells containing single or dividing asters. Cleavage, in some cases, may also take place in the complete absence of an achromatic apparatus. Non-nucleated cells have been found in eggs developing androgenetically of Triton palmatus (Fankhauser, 1929, 1934b), Triturus zkridescens (Fankhauser and Moore, 1941) , and the axolotl (Stauffer, 1945). The formation of cleavage furrows in nonnucleated cells provided with asters has also been observed by Tchou-Su ( 1931) in an egg of Hyla fertilized by sperm of Rana. 2. Cleavage of Eggs without Functional Chro.omosomes Jollos and Peterfi (1923) removed the second maturation spindle from fertilized eggs of the axolotl and found that a delayed and abnormal cleavage may follow without participation of the sperm nucleus or sperm centrosome. However, the cleavage shown in their figure is so irregular that it resembles fragmentation observed in eggs on the verge of cytolysis. On the other hand, among the androgenetic eggs of the axolotl mentioned above, Stauffer (1945) found one completely and regularly cleaved blastula which on sections showed no trace of chromosome material in any of the cells, while asters were present in some. Cleavage of this egg had

182

G . FANKHAUSER

been normal but delayed; presumably, the sperm nucleus failed to function following removal of the maturation spindle of the egg and was resorbed at an early stage, while the sperm centrosome functioned as an active division center. More recently, Briggs, Green, and King (1951) have made a detailed study of the development of frog’s eggs without functional chromosomes. If eggs of Rana pipiens are inseminated with heavily x-irradiated sperm of R. pipiens or R. c a t e s b i w , they develop as gynogenetic haploids, with the maternal set of chromosomes alone. The sperm does not contribute its haploid set of chromosomes but activates the egg and also contributes its centrosome which becomes associated with the egg nucleus. If now the latter is also eliminated immediately after laying by simple puncture of the egg at or near the animal pole, the egg is left without functional chromosomes. It shows regular but delayed cleavage ; the cleavage furrows usually do not extend to the vegetal pole. Later cleavages are restricted mainly to the animal hemisphere and produce a partial blastula in which from one-third to three-fourths of the animal hemisphere are divided into small cells. Although cleavage ceases after about 33 hours, the cellular parts of the blastula survive for one to four additional days without any signs of differentiation. Cytological studies showed that the irradiated sperm chromatin is not completely inert but distributed during cleavage to a minority of cells on one side of the egg. I n these it appears in the form of small nuclear vesicIes or chromosomal fragments. Chromatin-containing and chromatinfree cells continue to divide at about the same rate, but the former seem to survive longer in old blastulae than the latter. The heavily damaged sperm chromatin, which is still capable of increasing in amount during cleavage, may play some role in maintaining the life of the cell for a short time. Further studies revealed striking differences in the behavior of the irradiated sperm chronlosomes depending on their origin. Pipiens sperm chromatin is more widely distributed during cleavage than catesbiurtu sperm chromatin, and is present in approximately 200 to 300 out of lo00 cells. Moreover, the irradiated sperm chromatin has a different fate in these enucleated eggs and in eggs containing a normal egg nucleus and developing into haploid, gynogenetic embryos. I n the latter, much of the irradiated chromatin degenerates within the first twenty-four hours and is eliminated from the blastomeres in the early blastula. In order to test the capacity of “achromosomal” cells for further development, groups of such cells from day-old, arrested blastulae were grafted

NUCLEO-CYTOPLASMIC RELATIONS IN A M P H I B I A N DEVELOPMENT

183

to various sites in normal hosts. The grafts healed normally but were eliminated by the hosts after periods ranging from a few hours to four days, i.e., the grafted cells, with or without irradiated cliromatin, failed to resume cleavage, to differentiate, or to survive longer than in their original position. Whatever essential substances were lacking in the achromosomal cells could not be supplied by diffusion from adjacent normal cells. VII.

NUCLEO-CYTOPLASMIC RELATIONSI N THE EARLYDEVELOPMENT OF SPECIES HYBRIDS

While the changes in the nucleus of the egg described so far were predominantly of a quantitative character, the combination of eggs and sperms belonging to different species introduces also qualitative differences that have proved extremely useful in the analysis of nucleo-cytoplasmic relations in development from at least three different viewpoints. 1. Nucleo-Cytoplasmic Interactioizs during Fertilization

Cross-fertilization between numerous species of anurans has provided interesting material for the analysis of the processes of fertilization, in the form of a whole series of modifications of fertilization brought about by the failure of normal interactions between the sperm and the egg cytoplasm. The cytological studies of Tchou-Su (1931) have revealed the following series of phenomena of “partial” fertilization. (1) The spermatozoon fails to penetrate but activates the egg by contact; the egg gives off the second polar body, the egg nucleus enters a nionocentric mitosis ; there is no cleavage because of the absence of a functional centrosome (e.g., Rana 0 x HyZu 8). (2) Following penetration there is no interaction between the sperm head and the egg cytoplasm; the sperm head remains condensed and is eventually resorbed, while the sperm centrosome associates itself with the egg nucleus to produce a normal mitotic figure; the egg develops by gynogenesis into a haploid tadpole (“false hybrid” of G. Hertwig; e.g., Hyla 0 x Pelobates 3). ( 3 ) The interaction between the sperm head and the egg cytoplasm is subnormal and produces a small sperm nucleus which fuses with the egg nucleus ; however, during telophase of the first cleavage mitosis the sperm chromosomes are eliminated and development continues by haploid gynogenesis’ (e.g., HyZa 0 x ~ u f o (4) Fertilization and cleavage are normal without elimination of sperm chromosomes. The incompatibility between the latter and the egg cyto9

“>.

184

G . FANKHAUSEB

plasm is demonstrated by the arrest of development at the gastrula stage (e.g., Bufo 9 x Rana 3 ) . An early cessation of development of the hybrid, following normal fertilization phenomena, as in the last example, has been found in a number of hybrid combinations and subjected to detailed analysis.

2. Analysis of Nucleo-Cytoplasmic Incompatibility in Diploid and Androgenetic Haploid Hybrids A detailed study of the factors involved in the early arrest of development in such cases is of obvious importance since the block to the progress of development must be caused by the incompatibility between the foreign chromosomes and the cytoplasm (and chromosomes) sf the egg. The situation is especially favorable for experimental treatment since the analysis can be extended by combining hybridization with androgenesis and testing directly the developmental possibilities of the combination of cytoplasm of species A with a haploid set of chromosomes of species B, following removal of the chromosomes of A. Since the problems of early lethality are similar in both types of experiments, the evidence gained from diploid hybrids and from androgenetic, haploid hybrids will be reviewed together, reserving the application of hybrid androgenesis to the study of the development of species characteristics for the following section. For detailed references to the literature the reader is referred to the recent reviews by Baltzer ( 1940, 1949, 1950). For convenience, androgenetic hybrid combinations will be designated by placing the name of the species furnishing the egg cytoplasm but no chromosomes in parentheses. The final stage to which development proceeds varies considerably with different hybrid combinations. In the androgenetic hybrid Triton (pdinatus) 9 x Salamandra inaculosa d development is arrested before gastrulation ; in the corresponding diploid hybrid gastrulation is not completed. In several androgenetic hybrids, such as Triton (palmatus) 9 x T. cristatus 3, Tritoi, (dpestris) 9 x T . palmatus 8 , Rana palzcstris 9 x R. pipiens 3, development proceeds through neurulation or to the formation of the eye vesicles ; in the combination Triton (taeniatus) x T . palinatus the lethal effect is not expressed until an advanced embryonic stage with branching gills and pigment. The lethal effect is more often general, affecting all regions of the embryo. In the androgenetic hybrid Triton palinatus or taeniutus x T . crisfatus, however, the effect is strictly localized in the head mesodet-1x1. Pycnosis and degeneration of the cells are not preceded by visible mitotic disturbances, with the exception of the diploid and androgenetic hybrids

N UCLEO-CYTOPLASMIC RELATIONS I N A M P H I B I A N DEVELOPMENT

185

Triton palmatus x Salunaandra (and, possibly, Rana esculenta x R. fusca), where failure of the chromosomes to separate normally during anaphase is a striking phenomenon in the middle blastula stage. The causes of the early arrest of development have been investigated in a number of ways. Valuable information has been obtained by physiological and biochemical methods which define more accurately the nature of the block to development. In the arrested hybrid gastrulae Rana pipiens x sylvatica there is a marked reduction in respiration and glycolysis (Barth and Jaeger, 1947 ; Gregg, 1948). In the lethal combination Rana esculenta x R. fusca the synthesis of ribosenucleoprotein is reduced (Brachet, 1944). The foreign chromosomes are not only unable to take part in basic metabolic processes but also seem to interfere with the activity of the maternal chromosomes. Moore (1947) has suggested that the foreign genes compete with the maternal ones for a substrate present in limited amount and form an analog that cannot take the place of the substance formed by the normal cells. More important still was the discovery that the development of hybrid tissue may be resumed when parts of hybrid gastrulae are grafted to normal embryos where they survive and may differentiate normally. In one combination only has transplantation failed to improve the performance of the cells ; in the androgenetic hybrid (Triton palmatus) ? x Salamandra d, all grafts so far have failed to survive. In some cases transplants from all regions of the hybrid gastrula differentiate normally in their new site and survive until the hosts have developed into free-swimming larvae ; such full viability is shown by grafts from the diploid hybrid Triton palmatus x Salanzandra, and from the androgenetic hybrid Triton (dpestris) x T . palwtatus. In the hybrid Ram pipiens x R. sylvatica, differentiation of the grafts is limited ; ectudermal grafts show a reduced competence in response to inductive stimuli of normal embryos, while grafts from the dorsal lip have a limited power to induce secondary structures in competent ectoderm of a normal host (Moore, 1947, 1948). In the most thoroughly analyzed combination, the androgenetic hybrid Triton. (palmatus) or (tueniatits) x T.cristatzts, the behavior of the grafts depends both on their prospective significance and on their relations to the host tissues. If a series of relatively small grafts are made to normal embryos, the different organ territories of the androgenetic hybrid gastrula show a clear-cut gradation in their response: (1) head mesoderm, which is the focus of degeneration in the intact androgenetic hybrid, degenerates ; (2) muscle segments and notochord both contain some pycnotic nuclei ;

186

G . FANKHAUSER

but muscle fibrils differentiate normally while the notochord cells begin to differentiate but do not become vacuolated; (3) neural tube and epidermis differentiate normally to tlie condition reached in young larval stages (Hadorn, 1932). When similar pieces of androgenetic hybrid gastrulae are explanted in vitro in salt solution, the epidermis and notochord alone show normal differentiation ; neural tissue and somites survive but fail to differentiate, demonstrating the importance of the host environment in furthering their differentiation when grafted (Hadorn, 1934). This dependence is more clearly shown by the development of chimaeras, consisting of the anterior half of a normal palnzatus gastrula and tlie posterior half of an androgenetic hybrid gastrula. Epidermis and notochord differentiate well, but the neural tissue develops poorly, except in the zone of contact with normal host tissues where histological differentiation is normal ; this stimulating effect decreases rapidly with increasing distance from the boundary (“histogenetic stimulation,” Hadorn, 1935, 1937). I n his most recent theoretical discussions of the beneficial effects of transplantation Baltzer ( 1949, 1950) offers three possible interpretations. (1) The grafts are able to survive and differentiate because they have been removed from the harmful or lethal influence of the disintegrating head mesoderm ; the differentiation of tlie graft cells is autonomous, i.e., independent of any stimulation by the surrounding host tissues. This would mean that the palmatus nucleus may be effectively replaced by the cristatus nucleus in its functions during embryonic development and differentiation of the tissues under consideration. (2) The differentiation of the grafts is made possible entirely by the proximity of normal host tissues and is dependent on the transfer of substances from the host to the hybrid tissues which the latter are unable to elaborate. (3) The cells of the graft differentiate autonomously, not under the “guidance” of the cristatus chromosomes cooperating with the palmatus cytoplasm, but because of a much earlier “predetermination” of the characteristics of these tissues in the cytoplasm of the oocyte, before fertilization, under the control of the maternal gene complex. Which of these interpretations, alone or in combination, is the correct one cannot be decided without additional evidence, although the “histogenetic stimulation” of hybrid cells by host tissue seems to be well established by the observations on chimaeras which ‘emphasize the complications introduced by the graft-host relationships.

NUCLEO-CYTOPLASMIC RELATIONS IN A M P H I B I A N DEVELOPMENT

187

3. Nztclco-Cytoplasnaic Relatiom in the Developnzetit of Species Characteristics Experiments on hybrid androgenesis, combining cytoplasm and nucleus belonging to two different species, were undertaken originally to determine the relative role of these two components in the development of species characteristics. Unfortunately, androgenetic hybrids of any of the combinations tried could not be raised to a stage where species characteristics first appear. I t was only through the use of transplantation that this critical period could be approached. So far, three experiments of this type have been at least partially successful. (1) A large piece of ectoderm from a gastrula of the combination Triton (palmatus) x T. cristatus was grafted to a normal embryo of T . alpestris where it differentiated into normal skin and survived metamorphosis of the host animal (Hadorn, 1936). At metamorphosis, a specific difference between T . palmatus and T. cristatus becomes apparent in the surface structure of the epidermis; in T . cristatus the surface is smooth; in T. palmatus the epidermis forms small protuberances consisting of short columns of cornified cells. The epidermis developed froin the graft, with cytoplasm of T. palmatiis and nuclei of T . cristatzls, showed the typical palmatus protuberances. The cristatus chromosomes apparently had no influence on this characteristic which followed the maternal species entirely ; probably, this trait, although it appears late in development, is “determined” very early in the cytoplasm of the egg before fertilization. However, the interpretation of the result is complicated by the fact that the epidermis of alpestris, serving as host, also forms protuberances. (2) The species of California newts can be recognized in early larval stages by distinctive pigment patterns. In Triturus torosus, the melanophores form two compact, dorsal bands, while in T . rivularis the individual pigment cells are scattered. Hybrids between the two species show an intermediate condition which also develops when neural crest from hybrid embryos is grafted to embryos of either one of the pure species (Twitty, 1936). The basic difference determined by the genetic constitution of the two species appears to be a difference in the rate of differentiation of the melanophores and in the ultimate degree of differentiation and melanization attained (Twitty, 1945, 1949) . Androgenetic hybrids with rivzrlaris cytoplasm and torosus chromosomes do not live to the stage of pigment formation, but their neural crest may be transplanted to normal toroszis hosts where it produces a pigment pattern that is essentially like torosus. However, an increase in the number of pigment cells on the flank and the degree of dispersion in this region show a definite influence of the rizrziluris cytoplasm (Dalton, 1946).

188

G. FANRHAUSER

(3) The third experiment involves androgenetic hybrids between two races of the axolotl, rather than between two species. The recessive white race shows a limited number of melanophores in a very restricted distribution, compared with the black race. It seems that this difference is caused by the absence of a substance in the “white” epidermis which is necessary for melanin formation in the propigment cells below ; a graft of skin from a white donor prevents the melanization of the pigment cells of black hosts in the region under the graft (Dushane, 1935). Baltzer (1947) produced androgenetic hybrids with cytoplasm of the black race and a haploid nucleus of the white. A piece of ectoderm of this hybrid grafted to a black neurula formed a large area of epidermis and prevented melanization of the pigment cells of the black host. If the mechanism of melanin formation is correctly interpreted (cf. the recent work of Dalton, 195Oa, b) , the result of this experiment demonstrates that the peculiarity of the white epidermis is determined by a chromosomal factor acting early in development and not by “predetermination” by maternal genes in the cytoplasm of the oocyte before fertilization. Altogether, the limited evidence available points to the importance of both nucleus and cytoplasm in the development of early species characteristics. The constitution of the cytoplasm of the egg emerges as more important than had been anticipated, since it may control characteristics that appear relatively late in development. Such long-range cytoplasmic effects are no longer too difficult to visualize if particles with various degrees of genetic continuity and dependence on nuclear genes exist in the cytoplasm of the egg. A N D CONCLUSIONS VIII. SUMMARY The experimental evidence reviewed in this paper, though incomplete in many respects, demonstrates clearly the existence during development of nucleo-cytoplasmic relations at various levels. ( 1 ) Within the nucleus, a delicate balance exists between the genes of the whole complex. Whole sets of genes may be added without disturbing development; addition of one or more single chromosomes to the diploid complement, however, almost invariably results in abnormal development. Not alone viability and growth, but differentiation as well are affected, particularly the development of circulation. The fact that certain groups of genes are present in triplicate, while the others are in the normal duplicate condition, disturbs the activity of the gene complex. The mechanism of this effect of a numerical imbalance among the genes remains obscure ; it may involve competition for various raw materials in the cytoplasm. (2) The study of unviable hybrid embryos deals more directly with

NUCLEO-CYTOPLASMIC RELATIONS I N AMPHIBIAN DEVELOPMENT

189

the relations between the chromosomes and their cytoplastnic environment. In the diploid hybrid which fails to develop beyond the gastrula or neurula stage, there remains the possibility of a conflict between the two sets of chromosomes within the nucleus. Even here, however, it is perhaps itlore logical to transfer the site of conflict into the cytoplasm of the cells if one follows Moore’s (1947) suggestion that the foreign genes compete with the maternal ones for cytoplasmic substrates and form related analogs that cannot be used properly by the cells of the developing embryo. The depression in respiratory activity and in the synthesis of ribonucleic acid, which was demonstrated in the arrested hybrid gastrulae of frogs, shows that the foreign genes fail to operate properly in the control of basic biochemical processes, possibly because of specific differences in the structure of proteins. To what extent the genetically “inert,” heterochromatic sections of the chromosomes, rather than the euchromatic gene-carrying portions, may be involved in such biochemical activity cannot yet be discussed profitably because of the lack of critical evidence. In androgenetic hybrids, the incompatibility exists obviously between the foreign chromosomes and the maternal cytoplasm. In those combinations where the corresponding diploid hybrid is completely viable, we meet the problem of the nature of the relations between the two chromosome sets within the diploid nucleus which are not antagonistic as in the lethal combinations. With the maternal chromosomes present and taking the lead, the paternal ones are able to cooperate properly, although they are incapable of doing so when left alone in the strange cytoplasm. One of the most interesting and at the same time puzzling features of the analysis of hybrid lethality is the beneficial effect of transplantation which has been observed in the majority of combinations. I n a normal host environment, all or most of the grafted tissues remain alive and differentiate normally. It has not been possible as yet to decide between two interpretations of this improved performance : (a) The hybrid cells may be able to develop normally, under their own power, once they have passed the critical stage and have been removed from the lethal environment of the degenerating hybrid embryo ; this would in turn presuppose that the nuclei of the two species are interchangeable as far as their activity in the differentiation of these particular tissues is concerned, unless we assume a “predetermination” of these characteristics before fertilization, under the effect of the maternal gene complex. ( b ) The hybrid cells are “vitalized” by the intimate relations with the normal host tissues from which substances essential for differentiation

190

’C. FANICHAUSER

diffuse into the graft cells that are themselves still unable to manufacture them. That such “histogenetic stimulation” (Hadorn, 1937) by the host exists is shown negatively by the generally poor differentiation of explants in zdro and, positively, by the normal differentiation of hybrid tissues in the boundary zone of chimaeras, where they are in contact with the tissues of the other coniponent derived from a normal embryo. The possibility that faulty gene-cytoplasm relations may be corrected by bringing in substances (gene products ?) from neighboring healthy cells opens a very interesting perspective of gene action in development. (3) Further light is thrown on the nucleo-cytoplasmic relations during cleavage of the egg by the observation that cells without chromosomes can survive and divide repeatedly until the blastula stage is reached. During this period of rapid cell division there seems to be little time for synthetic activities that require nuclear control. The fact that non-nucleated cells fail to survive through gastrulation and to differentiate when grafted to normal embryos demonstrates participation of the nucleus at this stage and can be correlated with biochemical evidence of increased enzymatic activity beginning at the end of cleavage. The longer survival of cells with remnants of sperm chromatin that had been heavily damaged by x-irradiation, as compared with completely achroniosomal cells, may indicate that some components of the damaged chromosomes (heterochromatin?) are still active in a limited way. (4) The graded increase in nuclear and cell size in amphibian embryos with various degrees of polyploidy reaffirms the existence of a definite nucleo-cytoplasmic ratio. Just how and why the regulation of cell size is brought about remains unknown. In haploid embryos developing from whole eggs, an additional mitosis at the end of cleavage would, theoretically, reduce the cell size to one-half of the diploid value and thus restore the normal nucleo-plasmic ratio. Conversely, omission of the last cleavage mitosis in a tetraploid blastula would produce large tetraploid cells with the same nucleo-cytoplasmic ratio as exists in the diploid cells. This interpretation becomes inadequate when one considers the fact that the same ratio is obtained in diploid and haploid embryos regardless of the size of the original egg cell, whether normal, tliree-fourths, or onehalf normal. There remains also to be explained the regulation in triploid embryos which involves an increase by 50 per cent over the diploid cell size. Briggs (1947, pp. 257-58) has suggested that the adjustment takes place, at least in part, when cell growth begins at the end of the period of rapid cell division during cleavage. In triploid frog embryos the cells are definitely larger than in diploids in the gastrula stage which suggests

NUCLEO-CYTOPLASMIC RELATIONS I N A M P H I B I A N DEVELOPMENT

191

that a regulation in triploid mitotic rate and cell size occurs during gastrulation or late blastula stages. At this time the cells acquire new synthetic activities, in which nucleo-cytoplasmic interactions are important. If the nucleus becomes active in protein synthesis at this stage, it might also acquire the ability to regulate cell size, if the synthesis of cytoplasmic substances were proportional to the chromosome number. A suitable prolongation of the first “growth interphase” would regulate cell size and cell number in one step; from this time on, the mitotic rate in both diploid and triploid embryos would again be equal, as actual observations show it to be. (5) One of the outstanding features of polyploid development in amphibians is the normal character of morphogenesis at the triploid, tetraploid, and pentaploid levels. Embryonic differentiation is to a high degree independent of cell size. The upper limit of polyploidy compatible with normal morphogenesis has not been established ‘with certainty. The fact that the two heptaploid embryos discovered so far were both abnormal may well indicate that, with cells of approximately three and one-half times the normal size, some of the morphogenetic processes are meeting with serious obstacles. While early development of tetraploid and pentaploid embryos is normal, their later growth and viability are often adversely affected, although with considerable individual variations. The most plausible explanation would seem to be that the large polyploid cells do not function at maximum efficiency. The interactions between the nucleus and the cytoplasm, and between the latter and the environment, must be influenced by the increase in cellular dimensions. The distance between the surface of the cell and that of the nucleus may be a critical factor, as well as the relations between cell surface and cell volume. Until more definite information is available, another more strictly genetical interpretation must also be considered. The multiplication of identical sets of genes beyond a certain point may have a harmful effect, perhaps because some genes when present in multiple dose increase their activity more than others and thus disrupt the harmony of the genetic system. (6) In spite of these limitations the adaptability of embryonic development to differences in cell size remains truly remarkable. Increases in the dimensions of the individual cells are balanced by a regulation of cell number and, in thin-layered organs, also by an adjustment of cell shape, so that the size and structure of the organs and the size of the body remain essentially normal. One could not wish for a more forceful reminder that

192

G . FANKHAUSER

the subdivision of the embryo into cells is in some respects incidental and subject to the regulating influences of the embryo as a unit. The mechanisms that determine the normal limits of growth and thus the final body size remain unknown. All we have learned is that growth, within wide limits, is independent of the initial mass of cytoplasm of the egg and of the size and number of the individual cells, since changes in cell size invariably induce compensatory changes in cell number. It appears as if the total mass of living material that is produced during developnient were fixed by the genetic constitution of the species. It does not matter how much or how little the embryo starts with, or how the material is divided up into individual units. This subordination of the cells to the developing embryo as a whole, since it in turn must be under the control of the genes within the nucleus, emphasizes the complexity of nucleo-cytoplasmic relations in amphibian development. IX. REFERENCES Baltzer, F. (1922) Verh. schweie. nalurf. Ges., 248. Baltzer, F. (1940) Naturwissenschaften, S,177, 1%. Baltzer, F. (1947) Rev. mime Zool., 54, 260. Baltzer, F. (1949) XI11 Congr Int. Zool.,234. Baltzer, F. (1950) Rev. suisse Zool.,57, suppl. No. 1, 93. Barth, L. G., and Jaeger, L. (1947) Physiol. Zool., 20, 117. Brachet, J. (1944) Embryologie Chimiqw. Masson, Paris. Briggs, R. (1946) Growth, 10, 45. Briggs, R. (1947) J. exp. Zool., 106, 237. Briggs, R. (1949) J . exp. Zool., 111, 255. Briggs, R.,Green, E. U., and King, T. J. (1951) J . exp. 2001.. 116, 455. Dalton, H. C. (1946) J . ezp. Zool., 103, 169. Dalton, H. C. (195Oa) J. exp. Zool., 116, 17. Dalton, H. C. (1950b) J. exp. Zool., 116, 157. Dalton, H. C. (1950~) Auat. Rec., 108, No. 3, 30. Dushane, G. P. (1935) J. exp. Zool., 72, 1. Fankhauser, G. (1929) Rev. suisse Zool., 86, 179. Fankhauser, G. (1934a) J . ex$. Zool., 67, 349. Fankhauser, G. (1934b) J. exp. Zool., 08, 1. Fankhauser, G. (1937) J. Hered., 28, 1. Fankhauser, G. (1938) J. Morph., Ba, 393. Fankhauser, G. (1941) J. Morph., 66, 161. Fankhauser, G. (1945a) Quart. Rev. Biol., 20, 20. Fankhauser, G. (1945b) J. exp. Zool., lw, 445. Fankhauser, G. (1948) A m N. Y. Acad. Sci., 48, 684. Fankhauser, G., and Godwin, D. (1948) Proc. nat. Acad. Sci.,LVuslt.. 34, 544. Fankhauser, G., and Griffiths, R. B. (1939) Proc. nat. Acad. Sci., Wash., 26, 233. Fankhauser. G., and Humphrey, R. R. (1950) J. cxp. Zool., 116,207.

NUCLEO-CYTOPLASMIC RELATIONS I N AMPHIBIAN DEVELOPMENT

193

Fankhauser, G., and Moore, C. (1941) J. Morph., 68, 387. Fankhauser, G., and Watson, R. C. (1942) Proc. nat. Acad. Sci., Wash., 28, 436. Fischberg, M. (1947) Genetica, 24, 1. Gallien, L.,and Muguard, H. (1950) C. R. SOC.B i d , Paris, 144, 657. Goldschmidt, R. (1937) Amer. Not., 71, 83. Gregg, J. R. (1948) J . exp. Zool., 109, 119. Griffiths, R. B. (1941) Getietics, 26, 69. Hadorn, E. (1932) Arch. EiifwMCch. Org., l26, 495. Hadorn, E. (1934) Arch. EiztwMech. Org., 131, 238. Hadorn, E. (1935) Rev. d s s e Zool., 49, 417. Hadorn, E. (1936) Verh. dtsclz. zool. Ges., 97. Hadorn, E. (1937) Arch. ElttwMecR. Org., 136, 400. Humphrey, R. R. (1948) J. Hered., 89, 255. Jollos, V., and Peterfi, T. (1923) Biol. Zbl., 43, 286. Kaylor, C. T. (1941) Biol. Bull., 81, 402. Mangold, O., and Seidel, F. (1927) Arch. EntwAlech. Org., 111, 593. Moore, J. A. (1946) Genetics, 31, 304. Moore, J. A. (1947) J . exp. Zool., lM, 349. Moore, J. A. (1948) J. exp. Zool., 108, 127. Penners, A. (1935) 2. w'ss. Zool., 146, 463. Randolph, L. F., and Hand, D. B. (1940) J: agric. Res., 60, 51. Spemann, H., and Falkenberg, H. (1919) Arch. EnfwMrrh. Org., 45, 371. Stauffer, E. (1945) Rev. suisse Zool., 62, 231. Tchou-Su (1931) Archs. Attat. uiicr., 27, 1. Twitty, V. C. (1936) J. ezp. Zool., 74, 239. Twitty, V. C. (1945) J. exp. Zool., 100, 141. Twitty. V. C. (1949) Growth Symfiosiiini, 9, 133.

This Page Intentionally Left Blank

Structural Agents in Mitosis* M. M. SWANN D<par-tiiient of Zoology, Cambridge Uaiversity, Cambridge, Eikglatkd

CONTENTS

Page

I. Introduction ......................................................... 195 11. Birefringence Changes in the Sea Urchin Egg during Mitosis . . . . . . . . . . . 197

111. Further Evidence on the Release of Chemical Agents in Mitosis . . . . . . . . . . 203

Iv. The Nature of Chemical Agents in Mitosis ............................ V. Conclusion ........................................................... VI. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

208 209 210

I. INTRODUCTION In the course of mitosis, the physical properties of cells undergo a series of changes. Cytoplasmic viscosity, membrane tension and permeability, birefringence and light scattering all rise and fall in a definite pattern. Most of these changes are concerned in some way with the formation and disappearance of the mitotic figure or with the surface activity involved in cleavage; but so little is known of the mechanisms involved, that the significance of the various changes is far from clear. The multiplicity of theories about mitosis and cleavage is evidence enough of the state of confusion that exists. A full understanding of some of these physical changes would probably lead to the solution of a great many cytological problems. If, for instance, the decrease in birefringence in the spindle at anaphase could be interpreted for certain in molecular terms, the cause of chromosome movement might become clear. As it is, the physical data to be obtained from studies of living cells are too scanty for drawing any but the most tentative conclusions. Sometimes, however, it is possible to reach beyond difficulties of this kind, by investigating phenomena at a different level. The study of nervous conduction is an interesting case in point. Many aspects of conduction, such as its self-propagating nature and the rise and fall of the action potential were understood in outline many years ago; the physico-chemical mechanisms responsible for the action potential, on the other hand, are only now beginning to be understood. To reach beyond the difficulties inherent in interpreting scanty physical data implies, in the case of mitosis, a study of what may be called the behavior of the physical changes. Much work has, of course, already been

* A ,short version of this paper was delivered at the Seventh International Cytological Congress, held at Yale University in September, 1950. 195

196

M . M . SWANN

done on these lines, particularly with regard to the timing of the various changes in the mitotic cycle. There are, for example, the observations of Heilbrunn (1943) on cytoplasmic viscosity, VlCs (1921) on refractive index, Herlant (1920) on permeability, and Raven (1948) on membrane tension, to mention only a few. But curiously enough, not much attention has been paid to the spatial patterns of these changes. No doubt the limitations of the experiniental methods are responsible for this ; it is scarcely possible for instance, to measure refractive index, membrane tension, or permeability in different regions of the cell. Viscosity can be measured at different points, though with difficulty, and the observations of Carlson (1946) are a valuable beginning in this direction. But for some time to come, practical difficulties are likely to limit the study of spatial and temporal variations in the physical properties of dividing cells, to methods involving direct microscopical observation. Since there is so little work on the variation of physical properties in space as well as time, it is not surprising that we know little of what causes such changes. This is not a deficiency peculiar to mitosis; it is true of almost all the activities of the cell that involve extensive changes of protoplasmic structure : muscular contraction, ameboid and ciliary movement, protoplasmic streaming and rnorphogenetic movements, to mention only the more important ones. All structural changes involve, ultimately, changes in the forces acting between molecules and molecular aggregates. A considerable amount is known about the effect of various physical and chemical factors on such forces in protein and other model systems (Frey-Wyssling, 1948). Temperature, for instance, affects the cohesion of lipophilic groups ; the presence of ions affects the amount of bound water, and hence the amount of hydrogen bonding; p H affects salt linkages] and r H probably affects various other forms of valency bond. But the extent to which the living cell employs any of these mechanisms is uncertain. Though Heilbrunn (1943) attaches great importance to changes in calcium concentration, the evidence on the whole suggests that the internal environment of the cell is surprisingly constant. On the other hand, it is possible that some of these factors, including no doubt calcium ions, may be employed locally as intermediary mechanisms ; but if so, they must presumably be controlled by other more specific means. There are, in any case, certain general reasons for supposing that the structure of the living cell is controlled by highly specific substances with an essentially catalytic effect. Hormones, for example, are usually effective in very great dilution; so are various inhibitors such as colchicine which appear to act on the

197

STRUCTURAL AGENTS I N MITOSIS

mechanisms controlling structure. But whether the ultimate controlling agents act directly, or whether they operate through some of the mechanisms discussed above, is not known. If we look at the specific mechanisms that exert an effect on protoplasmic structure in living systems, we find essentially two different types : selfpropagating action potentials, and hormones or comparable chemical agents. The self-propagating type of mechanism is most highly developed in nerve, but is found also in muscle and a few other cells. Being primarily electrical it appears to be limited to the surface of cylindrical cells.* Whether this form of transmission can affect the internal structure of cells directly is uncertain. The fact that nerve endings liberate various types of hormone, which can themselves stimulate muscles and other organs, suggests that chemical intermediaries are usually necessary. The chemical type of control is widely distributed in living systems. Acetyl choline, adrenaline, and histamine are particularly well known for their effects on muscle, though it is not clear whether they act directly on the contractile mechanism or on the conduction mechanism of the muscle fiber. The effect of these and other hormones on the expansion and contraction of chromatophores, however, is presumably a direct one. Auxins appear to have a direct effect on protoplasmic streaming in plants, and in muscle it seems that adenosine triphosphate is directly concerned in altering protoplasmic structure. It is possible that self-propagating mechanisms are important at some stage in the control of the more rapid cell functions, such as ciliary and flagellar movement. Ameboid movement, mitosis, and protoplasmic streaming, on the other hand, are so slow that action potentials must be ruled out. Only further research, however, will show whether the control in these cases is by chemical agents. CHANCES 11. BIREFRINGENCE

I N THE SEA URCHIN

EGGDURING

MITOSIS

The first detailed study of birefringence in mitotic figures was that of Schmidt (1937, 1939) ; references to other work are given by Swann (1951a). Schmidt found that the spindle of the sea urchin egg was positively birefringent with respect to its length, and the asters positive with respect to their radii. H e also found that the birefringence, which was strongest at metaphase, disappeared almost completely during anaphase. Since protein fibers are positively birefringent with respect to their length, he supposed the mitotic figure to consist of such fibers, and ~

* Though the only self-propagating mechanisms so far demonstrated are primarily electrical, it is possible that there may also be purely chemical ones.

198

M. M. SWANN

since muscle and protozoan myonemes lose their birefringence to a greater or lesser extent when they contract, he supposed further that the decrease in birefringence during anapliase was due to contraction. His ideas have lent considerable support to the traction fiber hypothesis of chromosome separation. Beyond measuring the maximum birefringence of the mitotic figure, Schmidt did not make a quantitative study, nor did he examine the changes during anaphase in any detail. Using ordinary methods of observation, indeed, this is scarcely possible. Recently, however, Swann and Mitchison (1950) have made certain refinements in polarised light technique, which open the way to a more detailed examination of weakly birefringent structures in living cells. They describe various means of obtaining increased sensitivity, and show how, by taking photographs and measuring them with a densitometer, it is possible to construct a series of curves of retardation for structures whose birefringence varies from point to point and moment to moment. The various problems involved in applying this technique to sea urchin eggs are dealt with by Swann (1951a). In the same paper he examines in some detail the birefringence of the living spindle and asters of Psamwchinus miliaris at metaphase (Plate I ) . From curves of retardation it is possible to calculate curves for coefficient of birefringence, exactly in the case of the asters, and approximately in*the case of the spindle. Both spindle and aster curves show essentially the same features (Figs. 1 and 2) ; coefficient of birefringence is nil in the centrosomes, rises to a maximum at about 5 microns from the center, and then falls again to a minimum at the periphery of the asters, and at th'e equator of the spindle. The rise in coefficient of birefringence to a maximum at 5 microns represents, of course, the build-up of orientation round the centrosomes. The fall beyond 5 microns is not so easily accounted for. Since the total amount of material in the mitotic figure does not vary significantly, it must be due either to changes in molecular and micellar structure, or to a decreasing proportion of the material of the mitotic figure being oriented. A structure of submicroscopic fibrils radiating from the centrosomes like the spokes of a wheel, for instance, would give an inverse square law fall in coefficient of birefringence, if the material between the fibrils were not oriented. In fact, the observed fall in coefficient of birefringence corresponds fairly closely to an inverse square, from which it is tentatively assumed that the structure of the spindle and asters may be one in which submicroscopic fibrils radiate from the centrosomes. A certain amount of other evidence points in the same direction. On the

STRUCTURAL AGENTS IN MITOSIS

199

PLATEI. Photographs from a time-lapse film, taken in polarized light, of an egg of the sea urchin Psammechinus miliaris. 450 X. Compensated. Fertilization membrane removed. (1) 52 minutes after fertilization, prophase ; (2) 56 minutes, metaphase; (3) 58 minutes, early anaphase; (4) 60 minutes, late anaphase; (5) 62 minutes, telophase; (6) 64 minutes, starting to cleave.

200

M. M . SWANN

fiistance along the spindle axis,* microns from the equator

FIG.1. Coefficient of birefringence of a spindle at metaphase.

i j

Distance from the aster center, microns

FIG.2. Coefficient of birefringence of an aster at metaphase.

other hand it is always possible that the mitotic figure is really a homogeneous body, in which all the material present is oriented; if so, the fall in coefficient of birefringence with distance from the centers, must be due to molecular and micellar changes. Further analysis is not possible without evidence about form and intrinsic birefringence. A detailed interpreta-

STRUCTURAL AGENTS I N MITOSIS

201

tion of structure is not necessary, however, for an examination of the changes in the spindle and asters during anaphase. These are described by Swann (1951b). By using time-lapse photography and constructing curves for retardation and coefficient of birefringence, it is possible to compare the birefringence of living spindles or asters at successive stages in anaphase. The results show that the decrease in birefringence noticed by Schmidt is not uniform, but proceeds on a definite, though somewhat complicated, pattern. The birefringence of the spindle is at a maximum in nietaphase; it first decreases in anaphase at the equator, though without any change elsewhere. In the course of a minute or two, quite a large region in the center of the spindle is affected. But as time goes on, it becomes apparent that there are in reality two separate regions in which the birefringence is decreasing more rapidly than elsewhere ; these are centered somewhere between the equator and either pole. Later still, the birefringence decreases mainly in the region close to either pole, the equator remaining more or less constant. In short, the decrease of birefringence starts at the equator and then moves towards either pole. The region affected by the decrease at any particular moment extends over a number of microns and is by no means sharply defined. There is an interesting correspondence between the movements of these regions of decreasing birefringence and the familiar pattern of movement of the chromosomes. They also start at the equator in metaphase and move apart in two groups towards either pole. I n the sea urchin egg, unfortunately, the chromosomes are very small and invisible in the living cell. But by following single eggs under polarized light and then fixing and staining them, it is possible to show that there is not merely a general similarity between the patterns of movement of the chromosomes and the regions of decreasing birefringence, but that there is a precise correspondence. The chromosome groups actually lie within the region of maximum birefringence decrease. Since the chromosome groups are small and compact, and since the chromosomes themselves appear not to be birefringent, it seems likely that the decrease must be due, not to any opposite sign of birefringence in the chromosomes, but to an indirect effect exerted by them on the structure of the spindle. The changes in the asters during cleavage lend further weight to this idea. For the first few minutes of anaphase they show no decrease in birefringence at all; but at the time when the chromosomes reach the poles of the spindle, the asters also begin to show a decrease. This begins near the centrosomes, and, in the course of the next few minutes, moves steadily outwards at the rate of about 5 or 6 microns per minute.

202

M. M. SWANN

Returning to the two mechanisms for producing structural changes, it will be seen that we have here to decide between a self-propagating process, initiated by the chromosomes, and the release of a chemical agent from the chromosomes. Fortunately, there is one clear-cut difference between mechanisms of these two types which enables us to make a decision with some certainty. Once started, self-propagating mechanisms are necessarily independent of the subsequent behavior of the initiating stimulus. The nerve action potential for instance, is not affected by the removal or repetition of the original stimulus. A diffusion mechanism, on the other hand, is inevitably affected by the subsequent behavior of the source. In the case of the birefringence changes it is evident that the movements of the chromosomes do affect the pattern of change. Provisionally therefore, we may accept the hypothesis that the changes in spindle and aster structure are brought about by the release of a chemical agent from the chromosomes, rather than by any self-propggating mechanism. The decrease of birefringence is not the only change in the mitotic figure that takes place in anaphase. From the curves of spindle and aster birefringence, it is evident that there is a marked growth of the whole mitotic figure during this period (see Plate I). The spindle lengthens, and the asters increase in radius. I t might be supposed that such growth is simply part of a general growth going on throughout mitosis; but this does not appear to be the case. Curves from birefringence data and from fixed material, show that both spindle and asters reach a certain size quite early in prophase and remain more or less constant for the rest of prophase and metaphase. In anaphase, however, there is a sharp increase in size. The same effect was noticed in the chick spindle by Hughes and Swann (1948) and is discussed further by Swann ( 1 9 5 1 ~ ) . It is evident that, once formed, the whole mitotic figure remains in a more or less static condition until the beginning of anaphase, when it suddenly beconies able to incorporate and orient fresh material. In the case of the asters this leads simply to an increase in radius, but in the spindle it leads mainly to an increase in length. The cause of this sudden change in the power of aggregation of the cytoplasm is not clear. Since it appears to affect the whole mitotic figure more or less simultaneously it is not possible to decide between a self-propagating and a diffusion mechanism; nor is there any reason to connect the change with the chromosomes. But since the growth clearly represents a second type of structural change, which starts at the same moment as the first type discussed earlier, it is perhaps not unreasonable to regard it as being due to a similar mechanism, that is to say, to the release of a second type of chemical agent.

STRUCTURAL AGENTS I N MITOSIS

203

There is considerable evidence that outside the mitotic figure, the cytoplasm of the sea urchin egg at metaphase is in a fluid condition (Heilbrunn, 1943; Chambers, 1924). Seen in ordinary light it is not fibrous, nor is it birefringent; there is, in fact, every reason for supposing it to be unoriented and structureless. At this stage therefore, it is possible to distinguish two different protoplasmic configurations : one, in the spindle and asters, which is fibrous in ordinary light, and strongly birefringent ; the other, outside the spindle and asters, which is structureless. At anaphase however, both these configurations undergo a change. The spindle and asters show a marked fall in birefringence, to between a third and a sixth of their metaphase value. The structureless outer cytoplasm on the other hand, having previously been isotropic, shows a slight rise in birefringence, since it becomes incorporated into the growing mitotic figure. But although it becomes fibrous in appearance, it never becomes strongly birefringent like the original metaphase spindle and asters. The final result is two large asters, which virtually fill the cell, but are only weakly birefringent . Let the structureless configuration in the cytoplasm be called A, the weakly birefringent one B, and the strongly birefringent one C. The changes that occur in anaphase are then A to B and C to B. The meaning of these changes is not yet clear. The decrease in coefficient of birefringence involved in the change from C to B, however, may well be the result of randomization or folding up of protein chains, which is usually associated with contraction. This is the interpretation originally put forward by Schmidt, and there is no reason to think that it is not substantially correct. If so, it would seem that the chromosomes must be responsible for initiating their own movement. This may be of some significance in view of the instances cited by Schrader (1944) where chromosomes appear to behave autonomously.

111. FURTHER EVIDENCE ON

RELEASEOF CHEMICALAGENTSI N MITOSIS

THE

Further evidence on the release of chemical agents from the chromosomes comes from a study of the cell surface using various optical methods, (Mitchison and Swann, 1952). A number of changes are visible, which fall into two groups, one at the beginning of anaphase, the other at cleavage. From shortly after fertilization until the end of metaphase, the cortex of the sea urchin egg scatters little or no light under a particular form of dark-ground microscopy known as vertical illumination. At the beginning of anaphase, however, at the same time as the structureless

204

M. M . S W A N N

regions of the cytoplasm begin to be incorporated into the mitotic figure, the cortex suddenly develops a marked color. At the same time, the hyaline layer outside the cortex, which is only faintly birefringent from fertilization onwards, becomes more strongly birefringent. These two changes are clearly correlated in time with the A to B structural change in the cytoplasm, and may therefore be caused by the same chemical agent. More striking changes are apparent at the surface during cleavage. During anaphase, the light-scattering visible under vertical illumination is apparent all round the egg. Just before cleavage begins, this scatter decreases at either pole of the aell. As cleavage progresses, the subpolar and later the equatorial regions of the surface also decrease (Plate 11, 5-8.) Corresponding changes can be seen in the cortex by polarized light, though there appear to be no marked changes of birefringence in the hyaline layer, which must be removed with calcium free sea water if the changes in the cortex proper are to be seen. Before cleavage the cortex is slightly birefringent, being negative in a tangential direction. As cleavage begins, there is a decrease in birefringence, which starts at either pole of the cell, and later spreads round to the subpolar and equatorial regions. Similar changes in birefringence are mentioned by Monroy ( 1945). Two points about the changes at cleavage are important. The first is their general timing. It was mentioned earlier that the decrease of birefringence in the aster ( C to B), spreads outwards at about 5 or 6 microns per minute. Since, at the end of anaphase, the chromosome groups lie at the poles of the spindle, which are then about 30 microns from the surface, the change should take some 5 or 6 minutes to reach the cortex. It is significant that cleavage normally begins at about this time after the decrease of birefringence first becomes visible in the asters. Secondly, since the chromosome groups lie at either pole of the spindle, they are considerably nearer to the poles of the cell than the equator. A chemical agent released from the chromosomes will therefore reach the poles of the cell before it reaches other parts of the surface. The observed pattern of decrease in birefringence and light scattering in the cortex in fact, is exactly what would be expected, if the changes were to be caused by the same chemical agent as causes the decrease in birefringence in the spindle and asters. It is interesting that this pattern of optical changes corresponds precisely to the pattern of cortical expansion found in another species of sea urchin egg by Dan, Yanagita, and Sugiyama (1937). The significance of these various changes from the point of view of the mechanism of cleavage, is discussed by Mitchison (1952) and Swann (1952). Evidence of a different kind in favor of the release of chemical agents

STRUCTURAL AGENTS I N MITOSIS

205

PLATE11. Fertilization and cleavage in the egg of the sea urchin. Figs. 1-1 ?dia?%. Figs. 5-8 PU?-aCf?ntrOtUslividus. 14. Photographs from a time-lapse film, taken by ordinary dark-ground illumination. 300 X . (1) unfertilized egg; (2) sperm has just entered at about 8 o'clock; (3) a few seconds after 2, the brightening of the cortex has spread about halfway round the egg; ( 4 ) 20 seconds after the entry of the sperm, the brightening has spread all round the egg. 5-8. Photographs of an egg in cleavage, taken by vertical illumination. 300 X. The effect of four quadrants is the result of using a polarizing system to cut out light reflected from the coverslip; the whole egg surface does in fact scatter light. (5) 60 minutes after fertilization, egg in telophase; (6) 61.5 minutes; (7) 62.5 minutes ; (8) 64.5 minutes. PSOJJlJJlCChinUS

206

M . M. SWANN

comes from a study of the fertilization reaction in the sea urchin egg, using ordinary dark-ground illumination. From time to time, various authors have noticed that a wave spreads round the surface of the egg at fertilization, without, however, describing it in detail. Recently Rothschild and Swann (1949) have taken time-lapse films of the phenomenon. They find that the surface of the unfertilized egg scatters very little light but that shortly after the attachment of the spermatozoon, a bright patch appears round the point of entry. This brightening gradually spreads round the egg, taking about 20 seconds in all (Plate 11, 1-4). They believe the change to be associated with the block to polyspermy (Rothschild and Swann, 1950, 1951a, b). They also plotted the time course of the wave moving round the egg. Initially it spreads at a more or less constant rate ; it then slows down slightly at the equator, and finally speeds up considerably. For a number of reasons they conclude that the change is not likely to be due to a self-propagating mechanism of an action potential type, and they suggest that the time relations are of the right order for diffusion. Rothschild (1949) has calculated the type of curve to be expected, assuming the change to be due to the release by the sperm head, of an active substance which then diffuses through the body of the egg. The theoretical and observed curves bear a fairly close resemblance to each other. The particular shape of the observed curve suggests a molecular weight for the diffusing substance of some tens of thousands, but this conclusion must be treated with considerable reserve. Corresponding changes in the cell surface at fertilization can be observed by vertical illumination. Here the position is the reverse of that under ordinary dark ground ; the unfertilized egg shows a strong scatter which disappears on fertilization (Mitchison and Swann, 1952). The cortex of the unfertilized egg, like the cortex before cleavage, shows a slight birefringence, which also disappears at fertilization ( Monroy, 1935). Both vertical illumination and polarized light appear to show the same spread of a change round the surface of the egg as does ordinary dark ground, but for practical reasons the time course is more difficult to follow. Not only do the changes at fertilization suggest that a chemical agent is released from the sperm head, but it will be noticed that the changes visible by vertical illumination and polarized light are almost identical with those to be seen at cleavage. Since the sperm head consists largely of nuclear material, it would not be surprising if it were to release the same chemical agent as the chromosomes. There is yet another interesting parallel between events at fertilization and at anaphase. Immediately after fertilization, the sperm aster starts

STRUCTURAL AGENTS I N MITOSIS

207

to form round the sperm head. The cytoplasm of the unfertilized egg has a low viscosity, and appears to be structureless in ordinary and polarized light. But unlike the spindle and asters of prophase and metaphase, the sperm aster built up from this structureless cytoplasm, though strongly fibrillar, is only weakly birefringent. In fact, it closely resembles the asters of late anaphase, which, it will be remembered, are built up in their outer regions from the structureless cytoplasm of metaphase. It is interesting too, that the sperm aster grows in radius at about the same rate as the asters in anaphase, namely about 2 to 3 microns per minute. It was suggested earlier that the growth of the asters in anaphase may be the result of the release of a second chemical agent. I t seems possible at least, that this same agent is also released by the sperm head and causes the growth of the sperm aster. Beyond these observations on the sea urchin egg, there is little evidence for the release of chemical agents from the chromosomes or the sperm head, though there are a number of points in the literature which may be relevant to the idea. One of the most interesting suggestions is that of Metz (1933) on the curious type of anaphase found in the first spermatocyte division of Sciara. H e argues that the movement of the chromosomes must be due to some activity of their own, and that “this activity operates by bringing about a progressive alteration in the physical state of the protoplasm adjacent to the chromosome.” Somewhat similar observations were made by Chalkley (1935) on mitosis in the ameba. He claimed that protoplasmic viscosity was lowered in the neighborhood of the telophase nucleus and put forward a theory of cell division based on the idea that the presence of daughter nuclei at either side of the cell caused ameboid movement in opposite directions, with the result that the cell pulled itself in two. Similarly, Chambers (1938) noticed that the well-known “bubbling” of tissue culture cells began near the daughter nuclei at either pole of the dividing cell, and only later reached the equator. The changes involved in the maturation of the sea urchin egg are another interesting problem. Whereas the cytoplasm of the mature egg is capable of forming asters, that of the immature egg is not. Fry (1925) reviewing the evidence, attributes this to the release of substances from the germinal vesicle when it breaks down, There seems to be little direct evidence on whether nuclear substances can affect protoplasmic structure, though the existence of proteolytic enzymes in spermatozoa has been reported by a number of workers (e.g., Lundblad, 1949).

208

M. M. S W A N N

IV. THE NATUREOF

THE

CHEMICAL AGENTSI N MITOSIS

The evidence for the existence of chemical agents which are released from the chromosomes o r the sperm head is only indirect. The isolation of nuclear substances with an effect on protoplasmic configurations in the living cell is clearly necessary to establish the hypothesis, and it is as well to make it clear that, as yet, there is no direct evidence of this sort whatsoever. There is, however, a certain amount of more indirect evidence of some interest. The ratio of total chromosome volume to cell volume in the sea urchin egg is only about 1:104. The ratio of the volume of the sperm head to the whole cell is even less than this. It follows that any chemical agents in the chromosomes or the sperm head can only be present in very small quantities, and must be catalytic in nature. The non-committal term “structural agent” would perhaps be suitable for them. Cytochemical tests on sea urchin eggs (Swann, unpublished) have not shown that any of the recognizable substances are released from the chromosomes into the cytoplasm during anaphase. The only evidence on the nature of the structural agents comes from the work described earlier on the change in light scattering at fertilization. Diffusion considerations suggest a molecular weight of some tens of thousands, though this figure may well be seriously in error. But whereas the cortical changes at fertilization take only about 20 seconds, the changes in the mitotic figure at anaphase take several minutes. The rate of movement of the decrease of birefringence in the spindle is so slow in fact, that it would seem to imply a molecule of at least virus-like dimensions. This seems unlikely, though it is not, presumably, out of the question. There are two other possible explanations. In the case of the unfertilized egg, the structural agents released from the sperm head diffuse through a structureless medium of low viscosity, whereas in mitosis, they have to diffuse through the oriented structure of the mitotic figure. This might possibly account for the slowing down, since it is known that when the size of a molecule approaches the pore size of the gel in which it is moving, the rate of diffusion is greatly reduced. An alternative explanation is that the structural agents do not move freely, but are subject to trapped diffusion; that is to say, they are bound by the protoplasmic structure, so that the diffusion front does not move on until all the traps in its way are filled. Under these circumstances the apparent rate of diffusion will be governed in reality by the rate of release of the substance in question. If this is the case, it becomes necessary to suppose that at fertilization the structural agents are released very rapidly, whereas at

STRUCTURAL AGENTS I N MITOSIS

209

anaphase one of them is only released slowly. It will be remembered that the asters start growing at the very beginning of anaphase, so that the second structural agent, associated with this growth, must either be released rapidly, or must diffuse in the normal way. The release of substances from the chromosomes during anaphase has a certain parallel in the apparent disappearance of nucleic acids from the chromosomes in the later stages of mitosis. It has been supposed by Brachet (1947) that nucleic acids are released directly into the cytoplasni at the end of mitosis, after conversion to the ribose form, and reabsorbed again on to the chromosomes in prophase. Such a scheme would explain why the cytoplasm of many cells is basophilic in interphase, but loses its staining capacity at metaphase, when the nucleic acid charge on the chromosomes is at a maximum. But it is now thought unlikely that DNA and R N A are directly interconvertible (Villee et d.,1949). The extensive studies of the Caspersson school, however, seem to support the view that some nucleic acid, at any rate, is lost from the nucleus in the later stages of mitosis (Caspersson, 1950). Ris (1947) finds that the DNA content of grasshopper chromosomes decreases in anaphase and telophase, while Jacobson and Webb (1950) claim that nucleic acids are discharged onto the spindle at anaphase in chick tissue culture cells. On the other hand, Lison and Pasteels (1951) find that the DNA content of sea urchin nuclei is somewhat lower in metaphase than interphase. Although there is a possible parallel between the behavior of nucleic acids and the postulated behavior of the structural agents, there is no direct evidence to connect the two. The idea is nevertheless an interesting one that is not altogether out of keeping with what is known of nucleic acids. It is well established that they are connected in some way with protein synthesis, both in the nucleus and the cytoplasm, and it is known that nucleotides are constituents of certain coenzymes, while one particular nucleotide, adenosine triphosphate has an effect on the structure of muscle proteins.

V. CONCLUSION The idea that chemical agents are responsible for the structural changes of the cell, has a number of points to commend i t : it fits in with what is known of biological controlling mechanisms in general ; it links together mitosis and cleavage and relates them to fertilization; and it allows of the possibility that individual chromosomes may behave to some extent independently. It is easy to see also, how it might give rise to a cyclical controlling mechanism, for if chemical agents are released from the chro-

210

M . M. S W A N N

mosomes at one stage, they must presumably recondense there at some other stage, or else be broken down in the cytoplasm, so that a freshly synthesized batch may be released. But there may be other explanations for the observed patterns of structural change during mitosis, a i d the idea of structural agents must stand or fall in the end on whether it is possible to identify or isolate nuclear substances with a catalytic effect on the structure of living protoplasm. This is not likely to be an easy problem. VI. REFFXENCES Brachet, J. (1947) Embryologie Chimique. Masson, Paris. Carlson, J. G. (1946) Biol. Bull., 90, 109. Caspersson, T. (1950) Cell Growth and Cell Function. New York. Chalkley, H. W . (1935) Protoplasma, 24, 607. Chambers, R. (1925) Chapter in Cowdry’s General Cytology. Chicago. Dan, K., Yanagita, T., and Sugiyama, M. (1937) Protoplasma, !28, 66. Frey-Wyssling, A. (1948) Submicrascopic Morphology of Protoplasm and Its Derivatives. Elsevier, New York. Fry, H. J. (1925) J. exp. Zool., 43, 49. Heilbrunn, L. V. (1943) Outline of General Physiology. Philadelphia. Herlant, M. (1920) Arch. Biol., Paris, So, 517. Hughes, A. F., and Swann, M. M. (1948) I . exp. Riol., 26, 45. Jacobson, W., and Webb, M. (1950) J . Physiol., ll2, 2 P. Lison, L., and Pasteels, J. (1951) Arch. Uiol., Paris, 6!2, 1. Lundblad, G. (1949) Nature, Lord., 169, 643. Metz, C. W. (1933) Biol. Bull., 64, 333. Mitchison, J. M. (1952) Symp. SOC.ex+. Biol., 6. ( I n press.) Mitchison, J. M., and Swann, M. M. (1952) ( I n preparation). Monroy, A. (1945) Experientia, 1, 335. Raven, Chr. P. (1948) Biol. Rev., aS, 333. Ris, H . (1947) Cold. Spr. Harb. Symp. q w n t . Biol., 12, 158. Rothschild, Lord (1949) J. exp. Biol., 26, 177. Rothschild, Lord, and Swann, M. M. (1949) J . exp. Biol., 26, 164. Rothschild, Lord, and Swann, M. M. (1950) J . exp. Biol., !27, 40. Rothschild, Lord, and Swann, M. M. (1951a) Exp. Cell Rcs., 2, 137. Rothschild, Lord, and Swann, M. M. (1951b) J. exp. Biol., 28, 403. Schmidt, W. J. (1937) Die Doppelbrechung von Karyoplasma, Zytoplasnia und Metaplasma. Berlin. Schmidt, W. J. (1939) Chronzosoma, 1, 253. Schrader, F. (1944) Mitosis. New York. Swann, M. M. (1951a) J. exp. Biol., 28, 417. Swann, M. M. (1951b) J . exp. Biol., 28, 434. Swann, M. M. (19.51~) Chapter in The Mitotic Cycle, by A. F. Hughes. London. Swann, M. M. (1952) Symp. SOC.exp. Biol., 6. (In press.) Swann, M. M., and Mitchison, J. M. (1950) J . ex). Biol., 27, 226. Villee, C. A., Lowens, M., Gordon, M., Leonard, E., and Rich, A. (1949) J. cell. comb. Physiol., 83, 93. VKs, F. (1921) C. R. SOC.Biol., 86, 494.

Factors Which Control the Staining of Tissue Sections with Acid and Basic Dyes* MARCUS SINGER? Department of Anatomy, Harvard Mcdical School, Bostotc, Massachusetts CONTENTS I. Introduction ....................................................... 11. The Influence of pH of the Staining Solution on the Interaction of Dye and Protein ................................................. 111. The Nature of the Influence of pH on Staining ....................... IV. The Site of Dye Binding and the Nature of the Bond between Dye and Protein ................................. V. The Relation between the Isoelectric Point and Staining . . . . VI. The Ionic Strength of the Dye Solution ............................. VII. The Influence of Dye Concentration ................ VIII. The Afinity of Dyes ............................................. IX. The Influence of Fixation and Other Modifications of Tissues on Subsequent Staining ............................................ X. The Influence of Temperature of the Staining Solution . . . . . . . . . . . . . . . XI. Some Observations on the Kinetics of Staining ....................... XII. The Reversibility of Staining Reactions ; Equilibrium of Staining and Other Factors which Influence Staining .................. ... .............................. ... XIII. References .............

Page 211 215 221

233 236

242 245 246

248 250

I. INTRODUCTION Acid and basic dyes are employed in histology mainly to reveal the morphology of cells and tissues. Yet, these stains may also be used in the histochemical study of cellular and tissue proteins, an application which was appreciated in a number of early works on the staining reaction (for example, Miescher, 1874 ; Ehrlich, 1879a, b ; Lilienfeld, 1893 ; Michaelis, 1900, 1901, 1911 ; Pappenheim, 1901, 1917; Heidenhain, 1902, 1903; Mann, 1902; Magnus, 1903; Bethe, 1905). Mann (1902) in his remarkable book on histology noted: “It is not enough to regard dyes as simply acids or bases, as oxidizers or reducers, but we must aim at microchemical methods, and endeavour to know the composition of the dye and the tissue, to apply tests in a purposive way. Not till then will progress be made in the most difficult of all branches of Physiology, namely inicrochemistry.’’ But relative to the total literature on staining, interest in the

* This work was supported in part by funds received from the Eugene Higgins Trust. t Present address: Cornell University, Ithaca, N. Y . 21 1

212

M A R C U S SINGER

use of acid and basic dyes as histochemical tools has always been a limited one. A major reason for this has been the purely morphological demands which such growing fields as genetics, embryology, experimental inorphology, and endocrinology have hitherto made on the use of dyes and staining techniques. Another and perhaps more important reason for the limited histochemical application of staining procedures has been the absence of adequate information on the nature of the staining reaction. A prerequisite of a histochemical analysis of tissue sections with dye is, of course, a working knowledge of the mechanism of the staining reaction. The present review hopes to define the more important factors which govern interactions of acid and basic dyes with proteins and is preliminary to a second article, now in preparation, which applies this information to histochemical study of proteins of cells and tissues. Many facts of interest have appeared in the literature on staining of proteins, and it is one of the purposes of the present work to review the most salient of these and, whenever possible and necessary, to reinterpret them in the light of modern concepts. The review of literature is not confined to histological sources but also considers pertinent works in the field of protein, dye and textile chemistry. Much work has been done in the latter field on the protein-dye interaction, but these works have been largely ignored by histologists except for the detailed references and the comparison of textile and tissue staining of Pappenheim (1901; see also Baker, 1945) and for casual notations by other workers. The textile literature is particularly instructive since, as in the case of tissues, the substrate is also in a solid, insoluble state. Indeed, even studies of dyeing of cellulose derivatives and polyamide fibers are instructive for tissue staining. In contrast to that of textile materials, however, the stainable substance of cells and tissues is much more complex and heterogeneous and shows considerable variation from one region to the next in the kind, the interrelations, and the arrangement of substances. Much of the early literature on staining was devoted to the controversy, then extant, on whether the staining process is physical or chemical in nature. These speculations are adequately covered in one sense or another in many works such as those of Hofmeister (1891), Fischer (1899), Pappenheim (1901), Heidenhain (1902, 1903), Mann (1902), Dreaper (1906), Schwalbe (19O7), Zacharias ( 1908), Michaelis ( 1911), Gee and Harrison (1910), Pelet-Jolivet (1910), Harrison (1911), Bancroft (1914a, b, c, 1915 a, b), Briggs and Bull (1922), von Mollendorff and Krebs (1923), Unna (1928), Conn and Holmes (1928), Holmes

STAINING OF

TISSUE SECTIONS

213

(1929), Stearn and Stearn (1929, 1930), Zeiger (1938), Conn (1940), Baker (1945), Bourne (1951), and therefore no attempt will be made to review and evaluate these works systematically. However, a few comments taken from the literature may be pertinent in placing this controversy in proper perspective for the ensuing discussion of the forces which influence staining. In the light of modern information on intermolecular forces “the different theories are merely different ways of looking at the same phenomenon” (Vickerstaff, 1950). Mann (1902) wrote in similar vein fifty years ago when, in describing his views on the nature of staining, he noted : “Before discussing whether chemical or physical factors are at work in staining, the question must be asked, Is there any difference between chemistry and physics? There is not. Frankland used to teach that chemistry is but a branch of physics, and the whole recent development of chemistry points to the importance of studying chemical interaction apart from those conditions where substances join in definite molecular proportions. Even adsorption, which is perhaps the best example of physical action, has certain resemblances to chemical action. This matter has been gone into to make the reader realize that hard and fast lines cannot be drawn between chemical and physical action.” These sentiments have since been reemphasized in views of others. For example, the working assumption of Rawlins and Schmidt (1930) in analyzing the mode of combination between certain dyes and gelatin granules (see also Goldstein, 1949) was the fundamental statement of Langmuir ( 1916, 1917) that “there is no present justification for dividing interatomic (or intermolecular) forces into physical and chemical forces. It is much more profitable to consider all such forces as strictly chemical in nature. Evaporation, condensation, solution, crystallization, adsorption, surface tension, etc., should all be regarded as typical chemical phenomena.” Michaelis (1920) in a theoretical work on staining pointed out that practically all phenomena of staining with the exception, perhaps, of dye uptake of charcoal, could be described in chemical terms. He later noted ( 1926) that processes of adsorption, generally considered by physical theorists to be the mechanism of interaction between dye and substrate, “do not represent a fundamental contrast to chemical union.” This view was stressed by Stearn and Stearn (1930). Similar thoughts and sentiments concerning the significance of this controversy have been expressed by Dubos (1945), Baker (1945), Dempsey and Wislocki (1946), Rideal (1950), Vickerstaff (1950), Bourne (1951), and others. Studies of the interaction of dyes and proteins, although originally directed toward elucidating the physical or chemical nature of staining, un-

214

MARCUS SINGER

covered many facts which are of greater interest here than the controversy which originally stimulated them. These facts will be focused upon in some detail. The experiments were varied and considered one or another aspect of the staining process. Some were concerned especially with the structure of dyes ; others with the nature of the dye reaction ; and still others dealt primarily with the physical-chemical nature of the protein interactant. In analyzing the staining of protein, many substances were employed as “models,” and along with tissue sections, subjected to a variety of dyes and conditions of staining. The model substances included, among others, gelatin, glue, wool, charcoal, celloidin, fibrin film, filter paper, and soluble proteins (e.g., casein, albumin). From these many and varied investigations the obvious fact emerges that there are numerous and diverse factors which profoundly influence the dye uptake of proteins of cells and tissues. The more salient of these factors are considered in detail in this review since they throw light upon the nature of the staining mechanism. The variable which affects dyeing most profoundly is pH, and consequently most extensive treatment is given it. Other factors of importance are ionic strength of the dye medium, concentration of dye, nature and affinity of the dye, fixation and other chemical modification of the protein, temperature, diffusion, rate of staining, and the chemical and physical characteristics of the protein. With few exceptions, the discussion is confined to aqueous solutions of molecularly dispersed acid and basic dyes. Staining under conditions other than these is frequently done in the histological laboratory, and it is appreciated here that the forces which operate under such conditions cannot be equated with those described here and are worthy of separate consideration at another time. Finally, as for the chemistry and constitution of acid and basic dyes, no attempt will be made here to discuss this subject in detail. Instead, the reader is referred to appropriate works. A brief summary of features of these dyes important for the following discussion is, however, pertinent here. Works which treat this subject in proper detail are the concise and illuminating descriptions by Conn ( 1940), Baker ( 1945), and Bourne (1951), and the more detailed ones by Rowe (Colour Index, 1924), Mayer ( 1934), Pratt ( 1947), Fierz-David and Blangey ( 1949). Acid (anionic) and basic (cationic) dyes are aromatic compounds of various complexities containing a water-solubilizing group, which in the case of acid dyes is frequently a sulfonic radical (--SOsH) but which also may be a carboxyl or hydroxyl group. In the latter event a nitroso, nitro, or another hydroxyl group is present. The solubilizing group of the basic

215

STAINING O F TISSUE SECTIONS

dye is an amino group or one of its derivatives. These acid and basic groups are also in large part the groups responsible for the reaction of the dye with the protein, for which activity they have been termed auxochronzes. The acid dyes are generally used as the sodium salt of the dye acid ; basic dyes are prepared as chlorides, sometimes as acetates, sulfates, or as still other salts. There may be both acid and basic groups in a dye molecule, in which instance the overall acid or basic tendency is determined by the relative strength of these groups. Certain unsaturated groups of the dyes are chiefly responsible for their color, although the auxochrome may influence the quality and intensity of the color. Groups which impart the color are called chronzophorcs and include among others the carboxyl group >C = 0 ; the azo group -N = N- ; the nitroso group -N = 0 ; the nitro group -N Go ; the quinoid group 0 = <=> 0 = <=> = N ; and the ethylene group -CH = CH -. 1 0

=

OF p H OF THE STAINING SOLUTION ON 11. THE INFLUENCE INTERACTION OF DYE A N D PROTEIN

0 or

THE

Control of pH, although reached on a highly empirical basis, is implicit in many staining techniques where acid or alkali is used to enhance or depress staining. Instances of such control are abundant among the methods listed in books of histological technique (e.g., see Baker, 1945, Bourne, 1951). For example, among the various methylene blue solutions recommended by Mallory (1944) for staining of basophilic substances are Loeffler’s solution which includes sodium hydroxide, Kiinne’s with phenol, Gabbett’s with sulfuric acid, Unna’s with potassium carbonate, Goodpasture’s with potassium carbonate and acetic acid, and Sahli’s with borax. Examples of substances which are used to control staining with acid dye may be found in the work of Maneval (1941) on bacteria. References in early histological literature to acidic or basic substances which enhance dye uptake (“accentuators”) or which otherwise control the intensity of staining were frequent. The mechanism of action of these substances was not agreed upon, but it was evident to many (see Mann, 1902) that alkaline substances accentuate basic dye uptake and acid substances favor the acid one. Examples of basic accentuators listed by Mann (1902) are bicarbonate, soap, pyridine, sodium borate, aniline, and potassium or sodium hydroxide. Some acid ones are phenol and sulfuric, acetic, and oxalic acids. There are many other examples in the literattire where the effective mechanism of enhancing or otherwise controlling staining is the use of acid or alkali. The influence of acid

216

MARCUS SINGER

and base on staining has also been appreciated by wool and silk dyers who have employed various acids as well as salts to enhance and control dyeing of textiles (see e.g., Brown, 1901b), an effect which today is still the subject of considerable interest and research. A number of early studies were devoted to the experimental analysis of the effect of acid and base on staining. Mathews (1898) observed that proteins could be precipitated from solution as the salt of acid dyes such as acid fuchsin, erythrosin, orange red, and methyl blue if a few drops of dilute acetic acid were added to the dye-albumin mixture. Basic stains did not give this reaction under such conditions, but when brought into protein solutions made alkaline with sodium carbonate a precipitate was formed of the protein in combination with the dye. H e stated, in summary, “The acid stains will combine with albumoses only in acid solutions and the basic stains will combine with the albumoses only in alkaline solution, when they form insoluble colored compounds.” That these reactions held true for solid, coagulated protein as well as protein in solution was also demonstrated by Mathews. “If two pieces of coagulated egg albumin be brought the one into slightly acid and the other into alkaline solutions of thionin, the stain poured off after a few seconds, and the albumin washed in water, the piece that has been in the alkaline solution will be an intense purple, the other barely tinged with color.” H e observed similar results when he extended his observations to tissue sections of liver, kidney, and muscle of the frog. “I find that sections of the above-mentioned tissue, if immersed for an instant in one-tenth per cent sodium carbonate solution before staining or if stained in solutions of the basic stains made slightly alkaline with sodium carhomte show the cytoplasm deeply stained, as well as the chromatin. These reactions, which are identical with those of the albumoses, show that in alkaline solution many of the basic dyes will combine with the albumin molecule whether in cytoplasm or nucleus.” This work of Mathews which was written at a time when little was known about the dissociation and reaction of proteins was one of the earliest important contributions to our understanding of acid-base relations in staining ; yet, except for the detailed treatment which Mann (1902) gave it, little reference has been made to it. Mann (1902) was also impressed by the effect of acidity or alkalinity of the staining solution on the degree of dye-binding. H e believed that in cases where staining was favored the acid or alkali prepared the protein to receive the dye radical. At that time proteins were described as pseudoacids and pseudobases whose acidic or basic nature could be brought out by various substances or conditions. An alternative explanation ad-

STAINING OF TISSUE SECTIONS

217

vanced by some textile chemists at this time was that the accentuators acted upon the dye ; thus, in the case of the accentuating action of acid in dyeing of wool with acid dyes, the acid accentuator caused the liberation of the dye acid which then reacted with the basic groups of the protein to form a stable salt. Bethe (1905) observed that the degree of staining of tissue sections varied with the acidity of the staining solution. He stained sections of spinal cord, sublingual gland, mammary gland, and kidney in toluidine blue solutions of constant dye concentration but containing different amounts of acid or alkali. Alkali favored and acid inhibited the uptake of the basic dye. This was true for all structures of cells and tissues, but the degree of staining at any given level of acid or base varied from one structure to another. A e described the staining of given structures by curves which depicted the intensity of staining as a function of the amount of acid or alkali. The curves showed that in highly alkaline solution all structures stained with the basic dye ; with decreasing alkalinity the tissue substances gradually lost their affinity for toluidine blue. The order of loss varied, however, with the tissue structure. For example, in weakly alkaline solution tracts and glia of the central nervous system no longer stained, whereas solutions of approximate neutrality were required before fibrous connective tissue and colostrum of mammary gland failed to stain. Cartilage and mucus continued to stain even in very acid solutions. Other early workers who stressed the importance of acid and alkali in dyeing were Spiro (1897), Brown (1901a), Heidenhain (1902, 1903), Halphen and Riche ( 1904, 1905), Pelet- Jolivet ( 1910), and Harrison (1911). As information was accumulated on the dissociation of protein in aqueous solution interest in the effect of acid and alkali increased greatly. Michaelis (1920) in a theoretical work on the nature of the staining reaction was among the first to emphasize the relation between the binding of acid and basic dye at different concentrations of hydrogen ion and the amphoteric nature of the protein. Loeb (1922, 1924) showed that the binding of acid or basic dye by gelatin followed the pH of the dye bath, high p H favored basic dye uptake, and low p H enhanced staining with acid dye. Staining with either dye appeared to be minimal at the isoelectric point, but some interaction with acid dye occurred above and with basic dye below the isoelectric point. Staining of collagen powder also varied with p H (Thomas and Kelly, 1922). Briggs and Bull (1922) concluded from their studies of dyeing of wool that the “hydrogen ion concentration of the dye-bath is the most important single factor affecting the process of dyeing” and that “many of the assistants or re-

218

MARCUS SINGER

strainers, used in dyeing, produce an appreciable and often a great change in the hydrogen ion concentration of the dye-bath and their action in many cases is due more to this change than to any other specific action.” They observed that high pH favored binding of the basic dye and low pH that of the acid one. Hobbins (1923, 1924, 1926) studied the influence of acidity on the staining of plant tissues and reported that potato tuber tissue, Elodea leaves, and the mycelium of fungi responded as amphoteric colloids being stained with either acid or basic dye according to the p H of the solution. A number of important works bearing upon the influence of p H in staining bacteria were published by Stearn and Stearn (1924, 1925, 1928a, b). In solutions of varying p H the bacteria stained in a fashion similar to the protein structures discussed above; at low pH, acid fuchsin (acid dye) was taken up, but at higher ,pH, gentian violet (basic dye) was bound. When the intensity of staining was plotted as a function of pH, curves decreased with increasing p H for the acid dye, but were the reverse for basic dyes. Furthermore, such staining curves were displaced along the pH axis for different bacterial species (see also McCalla and Clark, 1941). Stearn and Stearn (1928a, b, c) also investigated the action of various chemical decolorizers on the dye fastness of stained bacteria and observed that decolorizers which were especially effective were acidic and basic ones, The acidic ones, for example phenol aldehyde, selectively removed basic dye, and basic decolorizers removed acid dyes. Pischinger (1926) , a student of Bethe, reexamined the influence of pH on staining and applied the method of p H control for the histochemical analysis of tissue structure. He studied quantitatively the binding of acid and basic dye by gelatin, egg white, thymus, and cartilage over a range of pH. The amount of bound dye was determined colorimetrically after extraction from the stained product. In each instance dye uptake followed the p H relations described above, namely that basic dye binding was inhibited as the pH of the staining solution was lowered until finally, at a given pH and below, no dye was bound; and staining with acid dye occurred most readily at low p H but dropped progressively and eventually ceased with gradual elevation of pH. Differences were observed among these protein substances in the degree of staining at any given p H or in the critical pH which marked the boundary between loss of staining capacity and increasing dye uptake. The proteins of tissue sections when subjected to p H variations responded in a manner similar to that of the model substances. As in Bethe’s earlier work, Pischinger plotted somewhat quantitatively the intensity of staining of given tissue structures

STAINING OF TISSUE SECTIONS

219

against pH. The curves obtained thereby were different for each tissue structure, a fact which led Pischinger and others to introduce pH staining control as a method of histochemical characterization and study of tissue proteins. The importance of p H in staining of tissue sections and in histochemical study of tissue proteins was independently recognized and reported by other workers at about the time of Pischinger’s publications. A series of publications, in which dyes were used in the study of tissue proteins emanated from the laboratories of the University of Missouri (Robbins, 1923, 1924, 1926; Naylor, 1926; Stearn, 1931, 1933; Stearn and Stearn, 1924, 1925, 1928a, b, c, 1%9, 1930; and Levine, 1940). These studies sought to extend to the proteins of cells and tissues the information on protein reactions published by Loeb (1922). Robbins (1923, 1924) applied the staining reaction at different p H to study of the isoelectric point of the proteins of fungi. Naylor (1926) showed that the staining of plant tissues followed the p H relations reported by Loeb for gelatin staining. In addition to studying dye binding from solutions of an individual dye, he analyzed staining from solutions containing both an acid (eosin) and basic (methylene blue) dye. At high p H only methylene blue was bound, but as the acidity of the staining solution was increased, the sections at first became purplish because of the partial binding of eosin as well and, then, at a low p H the sections were stained exclusively with eosin. The use of an eosin-methylene blue combination over a p H range was also applied with similar results to staining of tissue, particularly blood cells, and of bacteria by Tolstoouhov (1927, 1928, 1929), who also evaluated independently the use of dyes in the study of proteins of cells and tissues. Other contributions at this time to the study of the relations between p H and dye uptake of tissue and cellular proteins were made by Pulcher (1927) and Haynes (1928). The control of acidity has a longer history in textile dyeing, and it is evident in reviewing the literature that some of the experiences of textile colorists have been drawn upon by histologists. The history of pH control in dyeing of textile fibers has been summarized recently by Seymour, Agnew, Crumley, and Kelly (1948). The influence of pH and other factors on the dyeing of wool and various textile fibers is reviewed in a number of articles, important among which are works by Rose (1942), Abbot, Crook, and Townend (1947), and a journal-sponsored review article in the American Dyestuff Reporter (1948). Wool is dyed with acid dyes which are generally applied in acid solution between p H 2 and 5,

220

MARCUS SINGER

depending on the type of dye employed. Sulfuric acid is used to establish the lower p H and acetic acid the higher. Finally, studies have been reported recently on the influence of pH and other factors on the binding of dye in a relatively pure protein system (Singer and Morrison, 1948). Films of fibrin of known protein content, purity, and thickness prepared from products of fractionation of human blood (Cohn, 1945; Ferry and Morrison, 1946, 1947) were used as a model substance. This model protein was of particular value because as initially prepared it was native and undenatured. Moreover, it was in a solid and insoluble form and thus was similar in condition to cellular and tissue proteins of histological sections. Some films were modified by chemical or physical denaturants such as are used in fixing and then subjected to progressive staining under a variety of conditions until equilibrium was reached. The dye uptake was measured quantitatively by photometric means and expressed in terms of the amount of fibrin. Many factors influenced staining of fibrin, but primary among these was pH. The relations observed with pH variation are exemplified in the curves of Fig. 1. The curves resemble those for the titration of protein with CAST GREEN

MmFNNE U E

vn

FIG.1. The influence of pH on the staining of fibrin film with acid (fast green) and basic dye (methylene blue). Film fixed in formaldehyde (10 per cent Tor 10 hours) dye concentration, 5 X 10-6 M.;ionic strength, 0.02. (Singer and Weiss, unpublished.)

acid and base. The importance of pH for staining is emphasized particularly by the fact that there are p H levels where the protein shows no affinity for a dye even though immersed for very long periods in a solution of high concentration. The results of these studies were applied to the histochemical characterization and identification of proteins of cells and tissues (Dempsey and Singer, 1946; Dempsey, Wislocki, aiid Singer, 1946; Dempsey, Bunting, Singer, and Wislocki, 1947 ; Wislocki, Weather-

22 1

STAINING O F TISSUE SECTIONS

ford, and Singer, 1947; Singer and Wislocki, 1948; Wislocki, Singer, and Waldo, 1948 ; Singer, 1949 ; Dempsey, Singer, and Wislocki, 1950).

111. THENATUREOF

THE

INFLUENCE OF pH

ON

STAINING

The profound influence which p H exerts on staining reflects in large part the sensitivity of the dissociation characteristics of proteins to alterations in the solution environment. Before proceeding to a description of the dissociation of proteins under various conditions of p H it is well to summarize at first some chemical information about proteins pertinent to the discussion. A characteristic feature of proteins in solution is that they are amphoteric, that is, they contain at the same time both basic and acidic groups which by their dissociation give rise respectively to positive and negative charges on the protein molecule. These acidic and basic groups comprise the free side groups of certain amino acids, the terminal amino and carboxyl groups of the protein molecule, and, finally, the charged substances which niay be conjugated to the protein. Free basic side groups (substituted ammonium : NH3+) are found in the amino acids-lysine, histidine, and arginine ; and acidic ones (carboxyl : COO-) in glutamic, hydroxyglutamic, and aspartic amino acids. Another acid group in addition to free carboxylic acid is the hydroxyl one of certain amino acids (e.g., of tyrosine, serine) . Many amino acids have no dissociating group other than their single amino and carboxyl ones which are used in the peptide linkage and, therefore, do not impart a charge to the protein unless they are located terminally in the polypeptide chain. Of special interest among the conjugated proteins are those which contain acid groups such as nucleoprotein (phosphoric acid) and mucoprotein (uronic and sulfuric acids). Proteins differ according to the nature and number of their constituent amino acids and their conjugated substances. For example, free basic groups may be relatively more abundant in one protein and acidic ones in another. -kcording to the relative number of acidic and basic groups and their degree of dissociation, the net or overall charge on the protein mole-’ cule at a particular time will be positive (excess basic dissociation), negative (excess acidic dissociation), or zero (isoelectric point). Positive and negative charges exist in the protein molecule even at the isoelectric point (defined as the p H of a solution in which the protein does not migrate in an electrical field and therefore in which it is electrically neutral), although the net charge is zero. Thus, the electrically neutral form of the protein molecule is a dipolar ion (Zwittem’on or amphion) and its where R structure may be represented in general as H,+N-R-COO-,

z 2

MARCUS SINGER

represents the polypeptide chain and NHs', COO- the ionized basic and acidic side and free terminal groups of the constituent amino acids. The degree of ionization of the free acidic or basic groups of proteins depends on the pH of the solution. When acid is added to the solution, the dissociation of the free carboxylic acid group (as well as any conjugated acid group, e.g., phosphoric acid) is decreased and that of the free amino is increased, and the protein becomes less negatively charged. The reverse tendency in ionization and charge follows upon addition of alkali to the solution. The responses of protein to changes in p H may be indicated according to the following formulations : Isoelectric condition

A

B

NHs+-R-COONH3+-R-COO-

acid alkali

NHs+-R-COOH NHs -R-COO-

If the pH of the solution is below the isoelectric point the protein tends to respond increasingly in the direction described by formula A and above the isoelectric point according to B. It is presumed that at extremes of p H (approximately 2 and 11) complete ionization of the substituted ammonium or carboxylic groups is attained, and in these regions the respective net positive or net negative charge on the protein is maximal. At p H levels intermediate between these extremes and the isoelectric point, the net charge on the protein falls somewhere between the zero of the isoelectric point and the maximum, being positive below the isoelectric point and negative above. The ability of proteins to take up acid or basic dye according to the acidity of the environment is an expression of these amphoteric properties and of the charge on the dye ion. Basic dyes are generally chloride salts of a dye base, whereas acid dyes are in general the sodium salts of a dye acid. In the following discussion these salts are assumed to be completely ionized. Actually a small amount of the dye exists in solutions of ordinary pH as the undissociated dye acid or base whose concentration is probably influenced by alterations in p H of the solution. In those cases where dye ion association is prevalent at the p H of staining it is important to stress that the degree of dissociation of the dye would play an important part in the extent of staining. But, with few exceptions, the dissociation constants of these dye acids or bases fall in the extremes of p H and consequently ordinary levels of pH, such as those considered here, will hardly influence the degree of dye dissociation. Indeed, the variations in dissociation of the dye with alteration of p H are evidently insignificant when compared with that of the protein. Consequently, variations in ioniza-

STAINING O F TISSUE SECTIONS

223

tion of the dye may be ignored in most cases in the influence of p H on staining. The uptake of dye over a range of p H (Fig. 1) follows fairly closely the alterations in electrostatic charge on the protein. Basic dye, being cationic, may be expected to be attracted to protein with an excess negative charge. And, the staining conditions which favor increase in the net negative charge, such as low acidity (high p H ) of the staining solution, should also favor basic dye uptake-a relation borne out by previously described studies of staining as a function of pH. On the other hand, increase in the net positive charge, as the p H is lowered, favors the increased attraction of acid (anionic) dye. As dissociation proceeds and the groups are uncovered, the amount of dye bound increases until at the extremes of p H complete dissociation occurs and the maximum dye binding is attained. The above description of the effect of electrostatic alterations of the protein molecule on staining is based on the experiences and ideas of many workers. The early works of Brown (1901a), Mann (1902), Gee and Harrison (1910), and Harrison ( 1911) explained staining as an electrostatic attraction between dye and protein. These early interpretations were further elaborated as more detailed information on the nature of the bond between organic substances was obtained (Michaelis, 1920 ; Loeb, 1922; Stearn and Stearn, 1928a; Tolstoouhov, 1928; Craig and Wilson, 1937; Kelley, 1939b; Zeiger, 1938; Conn, 1940; Levine, 1940; McCalla, 1941 ; Rose, 1942 ; Neale, 1947 ; Gerstner, 1949 ; Vickerstaff, 1950). It will be shown later that there are other forces besides electrostatic ones which operate in the interaction of dye and protein. Yet, the electrostatic ones explain most effectively the observed variation of staining with pH and cannot be ignored even if it is finally shown that other forces provide the bond which combines the dye and protein. In the latter event, the electrostatic forces would play the important role of attracting the dye and protein within the range of the binding forces (see below). Finally, it should also be noted here that alteration in p H of the dye liquor also affects staining in another, albeit less important way. Swelling of the protein framework may occur as the p H is shifted beyond the isoelectric point. The increased separation of the micellae attendant upon swelling facilitates penetration of dye ions, particularly aggregates, which otherwise might be excluded from more central binding sites (Gerstner, 1949). Further thoughts on swelling phenomena as they relate to dyeing are noted below.

224

MARCUS SINGER

IV. THESITEOF DYEBINDING AND BETWEEN

THE

NATURE OF

THE

BOND

DYEAND PROTEIN

Electrostatic forces operate over relatively long distances and, consequently, are particularly effective in attracting the dye ion to the protein. The actual site on the protein to which the dye is bound and the nature of the bond have been frequently speculated upon. The most prevalent and firmly established view holds that the site of binding is the charged group of opposite sign which has attracted the dye ion and that the bond is an electrostatic one (also called salt linkage or primary zdence, coulombic, or ionic bond). Accordingly, the actual combination may be described by ordinary laws of chemical combination as a salt formation between dye and protein (Loeb, 1922). The idea that the dye-protein reaction is a salt formation was advanced by textile chemists more than a hundred years ago and is also prevalent in early histological literature (see reviews of Pappenheim, 1901; Mann, 1902 ; Heidenhain, 1902; Pelet-Jolivet, 1910). Mann ( 1902) repeatedly described staining phenomena as salt linkages ; for example, he stated that “Proteid, changed in this manner, can readily interact with the ions derived from the salt which we employ as a dye, and in consequence chemical union between the kat-ions of the tissue and the an-ions of the dye (or the an-ions of the tissue with the kations of the dye) can readily take place, provided the tissue and the dye are brought into contact with one another in a common solvent, i e . , in a fluid which allows of electrical dissociation of both the proteid and the dye.” And elsewhere he said, “The conception I have formed of precipitated proteids is that each component molecule, in addition to adhering probably only physically to its neighbour, is still endowed with a number of side chains, which are unsatisfied after the removal of the fixing reagent, and which, under suitable conditions, may attract toward themselves color radicals of the opposite electrical sign. Thus, for example, a tissue side-chain may be a sufficiently strong kat-ion to combine with the an-ion radical of-picric acid, and the unsatisfied nucleic acid radical in nucleins may withdraw the kat-ion on methylene-blue from its an-ion chlorine. Thus dyes and tissue-molecules can adhere chemically to one another by their side-chains.” Salt-like combinations between dye and protein were also emphasized by Mathews (1898), and, Nietzke ( 1901) observed in wool dyeing that “Certain facts speak for the view that the unions of dyes with fibres are nothing but salt-like unions, in which the fibre, analogously to an amido acid, plays in the one case the part of an acid, in the other case that of a base.” Weber (1894) concluded that “the amido group of the wool combines with the sulphonic group of the dye, while the

STAINING OF TISSUE SECTIONS

225

carboxyl group of the wool remains unaffected.” H e believed, moreover, that basic dye reacted with the carboxyl group, leaving the amino group unaffected. In the early literature on the affinity of chromatin for basic dye a number of works appeared which described the interaction between nucleic acid and basic dye as salt formation (see for example Miescher, 1874; Lilienfeld, 1893 ; Mathews, 1898). Description of dye binding as a salt linkage involving primary valence forces has been repeatedly advanced for reaction between various dyes and soluble proteins such as casein and albumin, or solid ones like gelatin and wool (some references are : Michaelis, 1920, 1947; Loeb, 1922, 1924 ; Chapman, Greenburg, and Schmidt, 1927 ; Hewitt, 1927 ; Rakusin, 1928; Rawlins and Schmidt, 1929, 1930; Stearn and Stearn, 1929, 1930; Stearn, 1931 ; Craig and Wilson, 1937; Ender and Miiller, 1937; Fraenkcl-Conrat and Cooper, 1944; Peters, 1945; Schmidt, 1945; Abbot, Crook, and Townend, 1947 ; Sokolova, 1948; Veller, 1948). The charged groups which woul_d form salts with acid dye are the free basic groups of the amino acids lysine, histidine, and arginine. Those to which basic dye would be bound are the free carboxyl groups of aspartic, glutamic, and hydroxyglumatic acids, the hydroxy groups of certain other amino acids, and the free acidic groups of phosphoproteins and mucoproteins. If a salt linkage is formed between dye and protein, one would expect that the amount of dye bound corresponds to the number of free acid or basic groups on the protein molecule. Such stoichiometric proportions between the quantity of dye and the number of binding .sites in the protein molecule have been looked for by many workers to support the view of salt linkages by primary valence bonds. The total number of acidic or basic groups available for interaction with dye may be calculated from the amino acid content of the protein or may be determined by titration of the protein with acid or alkali. In determining the number of dyeing sites for comparison with the calculated number of available sites, staining is done at extremes of pH where there is presumably maximum dissociation of acidic and basic groups of the protein and, consequently, where maximum uncovering of the binding sites occurs. The extremes of p H at which these groups are maximally dissociated are respectively 2 and 11 for the free amino and carboxyl groups. If the reaction is a stoichiometric one, there should presumably be a one-to-one combination between dissociating groups of the protein and dye ion at these extremes of pH. Such a result obtained in many of the works cited above where it was demonstrated that the amount of dye bound was equivalent to the calculated number of basic or acid groups.

226

MARCUS SINGER

Other methods have been used for determining the stoichionietry of dye binding by protein. A unique one was employed by Stearn (1931) in his conductometric titrations of sodium gelatinate and sodium nucleinate with basic dye. H e observed alterations in electrical conductivity of the solution as the basic dye displaced hydrogen ion and calculated therefrom the amount of dye bound. His results supported those obtained by direct measurement of the dye taken up by protein. McCalla (1941) described the stoichiometry of the reaction of dye and protein in bacteria treated with MgS04 by observing the amount of Mg++displaced by the basic dye, methylene blue. The stoichiometry of staining was further studied in bacteria by observing the amount of hydrogen ion ( p H ) released in the course of staining with methylene blue (McCalla, 1941; McCalla and Clark, 1941). If combination of acid dye is determined by basic groups of the protein, then destruction of these groups should be followed by loss in capacity to bind acid dye. Experiments directed toward this end have been reported for wool in which the fiber was deaminated with nitrous acid (Speakman and Stott, 1934; Speakman and Elliott, 1943). But, deamination though extensive was never complete, and a small amount of acid dye was still taken up by the fiber. Further loss in acid dye binding was obtained by acetylation of the wool after deamination. Early experiments along these lines were done by Gelmo and Suida (1905), who looked for various groups in the fiber by first neutralizing them in various ways and then observing the alteration in dyeing. While a large body of information has been accumulated to support the view of dye binding in stoichiometric proportions by primary valence forces, there are many instances, however, where the amount of dye bound does not reflect the number of dissociating groups on the protein. Because of some of these results, as well as still other information to be presented below, the possibility has been advanced that forces other than coulombic ones may also operate in staining, though to various degrees depending on the substrate and the dye. In studies of dye binding of the solid protein, fibrin, much less dye appeared to be bound at extremes of pH than the number of groups available in the fibrin molecule (Ferry, Singer, et al., 1947; Singer and Morrison, 1948). Indeed, the quantity of dye bound at these levels of p H varied according to conditions of staining other than pH, such as ionic strength and dye concentration (see also studies with safranine 0 of Fraenkel-Conrat and Cooper, 1944). Another situation in which the number of dye equivalents probably differs from the number of binding sites obtains whenever an aggregate of dye ions rather than a single one is bound. Aggregation is quite common in solutions of dye,

STAINING O F TISSUE SECTIOXS

227

and dye is frequently taken up by the substrate in the form of aggregates ranging from dimers to colloidal particles. In the staining of agar and other sulfated substances the basic dye is probably bound as an aggregaterather than a molecular unit-to the charged side chains of the substrate, a mode of binding which would explain the metachromasia of the stained product (Michaelis and Granick, 1945 ; Michaelis, 1947). Another example, where stoichiometric proportions with the primary charged groups do not obtain, appeared in studies of the interaction of various inorganic and organic acids and wool at low p H where more acid was bound than expected from the basic amino acid content; and the conclusion was drawn that either feeble basic groups, such as the amide groups of glutamine and asparagine and of peptide nitrogen or an entirely different mechanism of binding was responsible (Steinhardt and Harris, 1940; Peters, 1945 ; Carlene, Fern, and Vickerstaff, 1947 ; Abbot, Crook, and Townend, 1947; Vickerstaff, 1950). The amide groups may become charged at very low pH and thus become sites for ionic interaction with acid dye, as is shown by a rise in binding of certain acid dyes between p H 1 and 2 (Abbot, Crook, and Townend, 1947). Strong support for binding by amide nitrogen has been obtained in the study of nylon dyeing. Vickerstaff (1950) described interaction of dye and amide groups by way of hydrogen bonding (see below) and believed that amide combination is the source of main affinity of acid dyes and protein. According to him, combination with the amide group would occur particularly in neutral solution. He described two sites of amide binding; one which is adjacent to positively charged basic sites and the other not. In solutions of low p H the former sites are more effective and have the higher affinity for acid dye by virtue of the electrostatic attraction of dye ions to these regions. In this way he explained the stoichiotnetric correspondence often reported between amount of dye bound and the number of basic sites. Interest in amide groups as sites of dye attachment has been emphasized in recent years since the advent of polyamide fibers. Nylon contains some free amino and carboxyl groups, but various lines of evidence suggest that the amide groups with which this fiber abounds are particularly active as dye-binding sites. Another possible exception to stoichiornetric proportions and salt linkages is the observation, frequently made, that protein can bind acid dye, albeit minimally, above the isoelectric region where dye and protein have a similar charge, and basic dye below the isoelectric region where the interactants are both positive (Atkin and Douglas, 1924; Loeb, 1924; Grollman, 1925 ; Robbins, 1926; Chapman, Greenherg, and Schmidt, 1927 ;

228

MARCUS SINGER

Kelley, 1939a; Schmidt, 1945 ; Skinner and Vickerstaff, 1945 ; Klotz, 1946; Neale, 1946, 1947; Singer and Morrison, 1948; and others). These relations are evident in Fig. 1 where both cationic and anionic dye are bound in the pH range above and below the point at which the curves cross. It can be argued that the limited binding of dye at a p H where staining is opposed by electrostatic repulsion may be due to interaction of dye ions with occasional and isolated protein groups of opposite charge. Yet other possibilities have been advanced to explain dye uptake under circumstances in which dye and substrate have the same electrical sign and, indeed, under ordinary conditions of staining, too. Dye ions which reach the protein surface may be anchored there by specific and powerful short range forces such as van der Waals (Neale, 1947; see discussion below of cellulose dyeing in section on dye affinity). If such bonds are important in binding of dye, then the electrostatic forces would operate in assisting or opposing diffusion of dye to the binding sites. In their analysis of the interaction of wool and dye, Gilbert and Rideal, (1944) also suggested that other forces besides primary valence bonds may be effective, such as resonance bonds, van der Waals forces, and coordinate links (covalent bonds), perhaps by way of the chromophore and auxochrome groups of the dye. The fact that large molecules have a greater affinity for wool fibers than small ones, would be expected if forces in addition to ionic ones, as for example van der Waals forces, obtain in the binding of the dye (Steinhardt, Fugitt, and-Harris, 1941a, 1942). Moreover, it is possible that forces operating between protein and dye vary according to the dyestuff, being a simple salt linkage in the case of some, such as small ions, but including additional forces in the case of others such as those mentioned above (Skinner and Vickerstaff, 1945). The important hydrogen bond, already mentioned above, may be particularly effective in the binding of certain dyes (Vickerstaff, 1950). In hydrogen bonding, hydrogen acts as a bond between two atoms, especially highly electronegative ones such as oxygen and nitrogen. Hydrogen bond formation has been postulated between the amide group of the protein and the hydroxyl, amine, or azo group of the dye (Vickerstaff, 1950). Additional discussion of this method of dye binding is given below (see section on affinity of dyes). Another possible method of linking dye and protein emerged from study of shifts in the spectral absorption curves of azo dyes during certain reactions (Gerstner, 1949) ; in addition to ordinary ionic reactions the carboxyl groups of protein may bind dye by linkage with the azo group (also refer to Gillet, 1889, 1890). There are, conceivably, still other ways by which dye and protein may be

STAINING OF TISSUE SECTIONS

229

combined than those listed above. And, indeed, the forces involved in dye binding may vary as staining progresses, since there is some evidence that dye ions which are first bound influence the binding of subsequent ones in various ways, for example by repelling oncoming ones electrostatically (Klotz, Walker, and Pivan, 1946). The nature of the binding site and the forces involved may vary according to the characteristics and state of the protein. In the study of the stoichiometry of staining, soluble proteins were mainly used. Yet, there is good evidence to show that binding in a solid system, such as is encountered in histological sections, is different, and consequently the mechanism of staining of soluble proteins cannot be equated precisely with that for solid ones. Major differences are evident between the titration curves of dissolved and solid proteins (Speakman and Hirst, 1933; Lloyd and Bidder 1934 ; Speakman and Stott, 1934, 1935 ; Steinhardt and Harris, 1940; Steinhardt, Fugitt, and Harris, 1941b). The p H levels over which the titration curve shows little change with added acid is much greater than for soluble proteins. Moreover, whereas the pK values of soluble proteins are very similar to the corresponding amino acids, they are shifted to higher and lower p H levels for the basic and acidic groups respectively of the solid protein (Vickerstaff, 1950). These differences between insoluble and soluble proteins have been attributed to structural differences, to the effect of a two-phase system on the reaction as explained by the Donnan equilibrium, or to other means. It is quite evident that the conditions under which dye is bound in a three-dimensional solid protein are quite different from those for protein free in solution. I n the former instance Donnan effects operate just as effectively as though the protein were in solution and separated from the staining solution by a semipermeable membrane (Eliid, 1933), and, consequently, the solution within the protein meshwork differs from that without in terms of p H as well as ionic distribution. These differences have been emphasized and quantitated by Speakman and Peters (1949), who applied the Donnan theory of membrane equilibrium to titration of wool with acids and observed that the internal p H of wool differed from that of the external solution. They were able to calculate the internal p H but could not measure it directly (see also Vickerstaff, 1950; Gilbert and Rideal, 1944; Kitchener and Alexander, 1949 ; Peters and Speakman, 1949). There is further evidence to complicate and question the view that salt linkages and stoichionietric proportions are alone characteristic of staining. This other evidence is best considered in relation to the nature of the affinity of dyes, which is discussed below. Particularly instructive is the

230

MARCUS SINGER

information on cellulose derivatives where forces other than primary valence bonds explain dyeing most logically.

BETWEEN T H E ISOELECTRIC POINTAND STAININC; V. THERELATION The dyeing of protein in the isoelectric condition was studied by Loeb (1922) who emphasized that in this condition gelatin was relatively inert ; but, he later reported (1924) that gelatin could bind acid dye, albeit minimally, above or basic dye below, the isoelectric point. Nevertheless, the close relation between the isoelectric point and the relative absence of protein reactivity appeared to offer a means of defining the isoelectric point by staining procedures. Many attempts were subsequently made to apply such determinations of isoelectric points to the protein complexes of cells and tissues. Robbins (1923, 1924, 1926) stained the mycelium of fungi and tissue of the potato tuber with acid or basic dye and then washed them in buffer solutions of various p H and observed the degree of dye loss in each solution. The p H region of minimum retention of dye was considered the apparent isoelectric point of the protein complex. Because of the relation between dissociation of the protein at dieerent pH and combination with dye Stearn and Stearn (1924) concluded that the isoelectric point of bacteria could be determined from curves of staining with acid and basic dye. They reported that the crossing point of staining curves of acid and basic dye reflects more accurately the isoelectric condition than the region of minimal anion or cation retention. Therefore, they defined the isoelectric point ( 1928c) as “the hydrogen-ion concentration at which there is equal retention of cation and of anion.” Tolstoouhov ( 1929) , using a mixture of eosin and methylene blue buffered to various pH, defined the isoelectric point of blood cells and other tissue proteins as that p H where staining was an approximately equal combination (purple color) of the red and blue dye components. The isoelectric point of structures of plant cells was described by Naylor (1926) and Robbins (1926) , following observations on the pH of minimal acid and basic dye uptake, as a p H range rather than a specific point. The isoelectric point of hemoglobin in hemoglobin-containing cells was observed in fixed and unfixed specimens with acid and basic dye (Kindred, 1932, 1935). The crossing point of p H curves of acid and basic dye binding, or the region of minimal basic or acidic dye uptake was used as the criterion of the isoelectric condition by Pischinger (1926, 1927a) in histochemical studies of the proteins of cells and tissues. A whole series of studies of ‘%oelectric points” on a variety of tissue and cellular components was initiated by Pischinger’s work (Mommsen, 1927; Pulcher, 1927a ;

STAINING OF TISSUE SECTIONS

231

Schwarz-Karsten, 1927 ; Ochs, 1928 ; Pfeiffer, 1929 ; Seki, 1933c, 1934 ; Yasuzumi, 1933a, 1933b, 1934 ; Nishimura, 1934 ; Achard, 1935 ; Ikeda, 1935, 1936a, b ; Sturm, 1935; Fautrez, 1936; Yasuzumi and Matsumoto, 1936; for review of these and other works see Zeiger, 1938 and Levine, 1940). More recently the isoelectric point of structures of skin were defined using fluorescent dyes over a pH staining range (Bejdl, 1950; Stockinger, 1950). The accuracy of isoelectric determinations of proteins by staining procedures has been questioned. The close relation between the isoelectric point and the minimum of acid and basic dye uptake observed by Loeb for gelatin did not obtain for powdered hide collagen (Thomas and Kelly, 1922). When isoelectric point determinations were attempted from titration data or from study of the interaction of the protein with various substances, a number of difficulties appeared (Speakman and Stott, 1934). Between p H 5 and 7 there was no significant binding of alkali or acid by wool, and consequently the isoelectric point of wool could not be exactly defined but rather the values 5 to 7 were considered an isoelectric range. Comparison of the staining of extracted nucleoprotein with that of tissue nuclei showed that the isoelectric point of the nucleoprotein of the cell could not be determined by staining with the basic dye, toluidine blue (Kelley, 1939a, b). According to Kelley, staining depended on the amount of nucleic acid in the nucleoprotein and not on the isoelectric point. H e observed that toluidine blue was bound to nucleoprotein below its cataphoretic isoelectric point. Levine ( 1940) reexamined the concept of isoelectric point‘ determination of tissue proteins with dyes and concluded that the relatively qualitative curves of staining with p H are inadequate sources of such information. H e found that the crossing points and other characteristics of acid and basic dye curves upon which “isoelectric point” determinations were based varied with such factors as the nature and concentration of dye and buffer salts of the staining solution. Isoelectric point determinations varied by as much as 2 p H units when different dye pairs were used. Pfeiffer (1929, 1931) also questioned the belief that the point of‘ intersection of the dye curves represents the isoelectric point. Some pertinent observations concerning isoelectric determinations by staining procedures may be drawn from studies of fibrin film, an isolated and relatively pure protein system. The isoelectric point of powdered samples of fibrin film was determined electrocataphoretically and the molar binding of orange G and methylene blue analysed quantitatively over a range of p H (Singer and Morrison, 1948). The cataphoretic iso-

232

MARCUS SINGER

electric point of freshly prepared fibrin was 6.0,but the p H of crossing of orange G and methylene blue curves was 6.5 (Fig. 5, see Fig. 3A of Singer and Morrison, 1948). When denatured by heating (20 minutes at lZO”C), the apparent isoelectric point was 5.5 but the crossing point of acid and basic dyes was 6.3;for films heated for lesser times (1 minute at 100°C)the values were 5.7and 6.4respectively (refer to same figures). The disparity between the two values, although thoroughly apparent, is not great so that the p H of crossing diverges between one-half and one p H unit from the cataphoretic isoelectric point. If such quantitative procedures could be applied as readily to the microscopic and, yet, highly heterogeneous protein systems of tissue sections, it would be possible to approximate the isoelectric conditions of tissue elements by determining the pH at which there is equivalent binding of acid and basic dye. It must be stressed that such determinations would be only approximations. The divergence between the isoelectric point and the region of crossing of the curves may be due to binding of dye by forces other than coulombic ones. These additional forces do not determine the cataphoretic isoelectric point and, consequently, binding by them should only increase the disparity between the values. The deviation between the cataphoretic isoelectric point of fibrin and the pH a t which the acid and basic dye curves intersect one another varies with the dye pairs used (unpublished results). The pH of equimolar dyeing of formalin-fixed fibrin (cataphoretic isoelectric point of 5.2) with methylene blue and the following acid dyes were: orange 11, 5.0;picric acid, 5.1; orange G, 5.2; ethyl orange, 5.3; fast green, 5.5 ; sodium 2, 4-dihydroxyazobenzene-4-sulfonate, 5.6; light green, 5.7. These results are in agreement with the more qualitative observations of Levine ( 1940), who demonstrated variation of the intersection point for dye pairs in tissue staining. There are other factors as well which influence the crossing point. It is evident, therefore, from these observations that caution must be exercised in defining the isoelectric condition of proteins by their staining characteristics, particularly in tissue sections where the conditions of binding are not easily controlled and where adequate procedures for determining the ’quantity of bound dye are not yet developed. At best, perhaps, a broad isoelectric range may be described in which the actual isoelectric point would probably fall (“isoelectric range” of Naylor, 1926;“isoelectric zone” of Stearn, 1933 ; Speakman and Stott, 1934;see also discussion by Dubos, 1945,p. 68). Perhaps too much emphasis has been placed upon the relation between the isoelectric point and dye uptake. There are characteristics about the pH curves of staining which can be drawn upon in analyzing the protein

STAINING OF TISSUE SECTIONS

233

without any particular reference to the isoelectric point. For example, the region of the curve showing greatest rate of change with alteration in pH reflects a most sensitive and characteristic response of the protein to alterations in its electrostatic environment. This region can be defined by that p H at which a tangent along the steep part of the curve intercepts the pH axis; or it can be described as the p H which bisects the steepest part of the curve. If dye pairs are used then the pH at which curves cross may be used as a characterization point of the protein structure. There are still other ways of characterizing proteins according to the dye curves which they yield. Although the isoelectric point of the protein cannot be precisely defined by p H staining characteristics, yet it is possible to compare curves of different proteins and to draw conclusions therefrom on the relative position of the isoelectric point of these substances. From previous considerations of dissociation of proteins in relation to staining, it would follow that staining curves displaced to regions of higher p H reflect higher isoelectric proteins and those which lie lower on the p H axis represent lower isoelectric proteins. It is also possible to determine the direction of shift of the isoelectric point of a protein or protein complex following a physiological modification by comparing staining curves of the substance before and after modification (Levine, 1940; Singer and Morrison, 1948 ; Singer and Wislocki, 1948; Singer, 1949). A point which requires some emphasis here is that studies with dyes of the “isoelectric point” or other characteristics of proteins invariably and by necessity have been made on fixed proteins. Such chemical modification undoubtedly affects the protein, and the characteristics of dye binding does not reflect those of the native undenaturecl system, as will be evident in the discussion of the effect of fixation on staining (see below). The isoelectric point may be influenced profoundly by such chemica1 modification. Pischinger ( 1926, 1927a) and many others (see e.g., Zeiger, 1930b ; Seki, 1933b) have assumed in their studies that the isoelectric point of tissue proteins is not altered by alcohol fixation, a conclusion which has been criticized by Yasuzumi (1933a). VI. THE IONICSTRENGTH OF

THE

DYE SOLUTION

The foregoing discussion of the influence of p H on staining has emphasized that the protein amphion is sensitive to alterations in the pH of its environment and reflects these alterations by a change in charge. This being the case, it may be expected that factors other than acid or alkali which alter the electrolytic environment should also affect the electro-

234

MARCUS SINGER

static condition of the ampholyte and, thereby, its interaction with dye. The amount of dissolved salt represented either as neutral or buffer salts is such a factor. The activity of salt ions in solution is best expressed by the ionic strength (see Cohn and Edsall, 1943) rather than by other concentration expressions. Salt solutions of equal concentration measured by the latter means may have different ionic activities by virtue of a difference in number and valence of dissociable ionic groups. The ionic strength ( u ) of a solution of electrolyte is defined according to Lewis and Randall (1923) as half the sum of the concentration (molality) of each ion multiplied by the square of its valence: u = 8 mi Zf Increasing ionic strength of the dye solution decreases staining with both acid and basic dye, a result which is exemplified in Fig. 2 for the stainORANGE G

0.4

k

g a3 W

n J

a

-0

oa 0.1

s a 10-5 W.

-.\.

+:, ,.'

o -\ . 0 . 0

0

3

9@Y

4

1.2

Ionic strength

' 0

\e

0

g

METHYLENE BLUE

I I 10-sY.

%,bc@ /O s

6

0 8 0

0.01 0.04 0.15

I.o

3.8 36

.d

0.4

/

01

I

7

E

9

P"

FIG.2. The influence of ionic strength on staining of fibrin film with acid (orange G ) and basic dye (methylene blue). Taken from Singer and Morrison, J. B i d . Chem., 176, 1948. ing of fibrin with methylene blue and orange G. Steinhardt, Fugitt, and Harris (1941a) described such a depressing effect on the titration of soluble and fibrous proteins with various anions. Levine (1940) observed that the staining of tissue sections decreased with increasing concentration of buffer salt. A number of reasons have been advanced for this effect of the electrolyte on staining, among which are the remarks of Singer and Morrison (1948) on the dye binding of fibrin and the more detailed discussion of Elod (1933) in his work on the effect of neutral salt on the binding of dye anions by wool at low pH. According to Donnan's theory of membrane equilibrium, salt ions may influence the staining of solid proteins by altering the distribution of dye ions between the external solution and the solution within the protein itself. For this reason, the effective

STAINING OF TISSUE SECTIONS

235

staining concentration, namely the concentration of dye in the solution between and within the fibrillae of the protein probably differs from that of the dye bath. In solutions of high ionic strength the internal dye concentration in equilibrium with the stained micellae is evidently much less than in solutions where little or no salt is present. A quantitative interpretation of the effect of different ionic srengths on the interaction of solid protein and various acids according to Donnan’s theory has been elaborated by Speakman and Peters (1949) in studies of the binding by wool of sulfuric acid and hydrochloric acid singly or in the presence of different concentrations of KCl. The analysis of dyeing equilibria according to the Donnan theory has been summarized and evaluated by Vickerstaff (1950). In addition to the Donnan effect it is also possible that the salt ion competes with the color ion for the binding site on the protein molecule and thereby limits the quantity of dye bound (Pelet-Jolivet, 1910; Briggs and Bull, 1922; Elod, 1933; Speakman and Clegg, 1934; Skinner and Vickerstaff, 1945). I n describing such competition Elod (1933) showed that in dyeing of wool the small salt ion penetrates the fiber most rapidly by virtue of its greater diffusibility and is bound, but then secondarily is replaced by the larger dye ion which shows less tendency to dissociate after binding (see discussion below on dye affinity). When the concentration of salt ion is increased, less of the dye ion in competition with it is bound. Neale (1946, 1947) concluded that salt ions serve to suppress the electrostatic forces on the protein, thus decreasing the attraction to acid dye below the isoelectric point and basic above. It is interesting, as Neale and others have emphasized, that higher ionic strengths have the reverse effect on dye binding when the sign of the charge on the dye and the protein is the same (above the isoelectric point for acid dyes and below for basic ones). In these instances the positive effect of the salt on staining results from suppression of forces which would normally repel the dye ion. Since he attributed dye binding, in part, to the more powerful but short-range forces, such as hydrogen bonds and covalent links, the effect of the decreased potential of the protein surface is to allow more dye ions to come within the range of action of these forces (see discussion on dye affinity). I n this way the activation energy of the dyeing process is reduced and dyeing increased with added salt (Vickerstaff, 1950). Electrolyte also plays an important role in the dyeing of cellulose fibers, but in a manner quite different from that for ordinary dyeing of wool. Cellulose derivatives are negatively charged and yet are generally stained

236

MARCUS SINGER

with acid dyes. The affinity of the color anion for cellulose is quite low in the absence of electrolytes. As electrolytes are added to the color bath, dyeing increases markedly despite the similarity in charge of the color anion and the plant fiber. The effect of the salt ions is to dampen the mutual repulsion and allow dye to approach the cellobiose chains closely enough to be bound by hydrogen linkages. The effect of electrolyte on the staining of cellulose resembles its effect on dyeing of proteins above the isoelectric point with acid dye and below this point with basic dye. In both instances salt serves to facilitate dyeing. Finally, it is also possible that salt influences staining by acting on the dye itself. Increased salt causes the dye to form colloidal aggregates and even eventually to precipitate from solution (Michaelis, 1947). Large aggregates of the dye by virtue of their size find less ready access to intermicellar regions of the protein than single ions or dimers. Consequently, the effective concentration of dye in solution is lowered thereby, and the staining decreased.

VII. THEINFLUENCE OF DYE CONCENTRATION Variation in the amount of dye taken up by tissue sections according to the concentration of the dye bath is a matter of common knowledge to histologists. Greater amounts of dye are bound with increasing concentration as shown in Fig. 3 for both acid and basic dye. The amount of dye which is bound with increasing dye concentration is limited by the number of available binding sites (Knecht, 1889, 1904; Hofmeister, 1891, p. 224; ORANGE G

44.

METHYLENE BLUE Dye concentration

2.

o

- 1.2 -

I r ~ ~ M.- 5 0 SXIO'~ M. 0 2.5 ~ 1 0 M. . ~

x

-

0.8

a6

-.0.4

- 02 2

3

4

5

6

P"

7

8

9

FIG.3. The influence of dye concentration on staining of fibrin film with acid (orange G ) and basic dye (methylene blue). Taken from Singer and Morrison, J. Biol. Chem., 176, 1948.

STAINING OF TISSUE SECTIONS

237

Steinhardt, Fugitt, Harris, 1941a; Skinner and Vickerstaff, 1945). Except for the activity of other forces which may bind dye such as hydrogen bonds and van der Waals forces, once coulombic forces are satisfied little or no additional dye is bound. Limitation in the amount of dye taken up as staining proceeds is due, however, not merely to occupation of the dyeing sites by dye molecules but also to the effect of bound dye on the oncoming dye molecules. Klotz, Walker, and Pivan (1946) have pointed out in their studies of the adsorption of dye by serum albumin that steric hindrance or electrical effects on approaching dye molecules may result from already bound ones. The change in the rate of increase of bound dye with increase in dye concentration has been explained by Elod (1933) in terms of the Donnan equilibrium. Other experiments on the influence of dye concentration on staining are those of Craig and Wilson (1937).

VIII. THEAFFINITYO F DYES By ufinity is generally meant the tendency of a dye to combine with a given tissue structure. However, the term is userl quite loosely by histologists and may have a variety of implications. Often it implies a specificity between the substrate and a particular dye not shared by other dyes. Yet, a given protein may be stained by any one of a number of acid or basic dyes provided conditions of staining, particularly such as pH, are adequate. And, consequently, from this viewpoint, specific affinities are not the rule and molecularly dispersed dyes of quite different character are taken up by the same protein. For example, the basic dye methyl green which is ordinarily considered very specific for desoxyribonucleoprotein will interact with proteins of cells and tissues in general in aqueous solution, provided the appropriate p H conditions are used (compare Michaelis, 1947). And other basic dyes will stain the same nucleoprotein quite well. The acid stain, aniline blue, will also stain cells and tissues widely when applied under conditions of low pH, although as employed in triple staining methods it is considered fairly specific for collagen. Mathews (1898) appreciated this similarity in staining capacity of dyes at a time when dfferences or “specific affinities” were especially highlighted. Although protein shows little tendency to bind a particular acid or basic dye exclusively and reject others, nevertheless the degree of binding varies from one dye to the next. Some dyes are bound in greater amount than others. If the equivalent of acid dye or of other acid substance taken up by the protein is plotted against pH, notable differences in the number of equivalents of each substance are apparent, even though the conditions of staining are the same. Steinhardt, Fugitt, and Harris (1941a) recorded

238

MARCUS SINGER

differences between a variety of substances which at the extremes reached 2 units on the p H coordinate between the curves for HC1 and flavianic acid when the p H values were compared a t which half the maximum amount of acid was bound. Such differences in the curves of staining of various dyes were also recorded by Levine (1940) and Elod (1933) and are exemplified in Fig. 4 for staining of fibrin film.

PH

FIG. 4. Differences in the affinity of two acid dyes (light green and picric acid) revealed by their pH staining curves. Note the differences in the crossing points of these curves with the curve of methylene blue (see text on discussion of isoelectric points). Formaldehyde-fixed film (10 per cent for 10 hours) ; dye concentration, 5 X 10-6 M . ; ionic strength, 0.02. (Singer and Weiss, unpublished.)

The differences in affinity of dyes for solid proteins evidently depends on a number of factors (see also review of the factors which govern the affinity of soluble proteins for dyes and other interactants by Goldstein, 1949). As mentioned previously, it is conceivable that other groups on the protein molecule than amino or carboxyl ones may be involved in the reaction with different dyes. Some forces may be more available for combination with one dye than with another. For example, combination of protein with simple acids may involve primary valence bonds with substituted ammonium groups, whereas with other substances forces such as covalent links or hydrogen bonds may be involved simultaneously or alternatively (Klotz, Walker, and Pivan, 1946; Neale, 1946, 1947; Klotz and Walker, 1947). Neale (1947) in his study of affinity and its meaning in terms of electrochemistry of staining (see also Harrison, 1948) concluded that short-range forces such as are present in covalent links or hydrogen bonds are responsible for the specific affinity of a dye for a particular fiber. The long-range electrostatic forces oppose or assist these more powerful short-range forces. But, even with electrostatic repulsion of the dye (e.g., below the isoelectric point with basic dye or above with

STAINING OF TISSUE SECTIONS

239

acid dye) some dye is bound, nevertheless, by the short-range forces, although work evidently must be done (thermal agitation) to bring the dye ion to the surface of the fiber. Another explanation of differences in affinity is based on studies of the degree of dissociation of various protein-anion combinations ( Steinhardt, 1940; Steinhardt and Harris, 1940; Steinhardt, Fugitt, and Harris, 194la, b, 1942). When the dissociation was great, as occurred after reaction with chloride ion, the affinity as recorded in the titration curve was much less than when the dissociation was slight as occurred with protein-dye combinations (compare Chapman, Greenberg, and Schmidt, 1927). The dissociation constants, calculated for various combinations of protein and anion, showed wide variations (also Steinhardt, 1942). Therefore, affinity of the dye was directly related to the degree of association of the color ion with the binding site of the protein. The differences in affinity were correlated with differences in size of the anion, and it was noted that with few exceptions increasing order of affinity followed increase in molecular weight and was higher in aromatic than in aliphatic ions of the same size (compare Klotz and Walker, 1947; Klotz, Triwush, and Walker, 1948). In the exceptions, considerations of shape of the molecule and its relation to steric hindrance were offered as an alternative possible explanation of differences in affinity (cf. Goldstein, 1949, p. 146). The difference in combining capacity of various acids with protein was great. When chloride ion was taken as unity, then the combining capacity of picric acid was 758 and that of Orange I1 over 23400 (Rose, 1942). It has also been stated by textile chemists as a general rule that affinity increases with molecular weight and, moreover, with complexity of the dye ion and with the introduction of additional polar groups (Abbot, Crook, and Townend, 1947; Lemin and Vickerstaff, 1947). In connection with size differences of dye molecules, Speakman and Clegg (1934) and Speakman and Smith (1936) believed that in the case of wool the cystine (-S-S-) and salt linkages (-COO-, -NH3+) offer resistance to the penetration of large dye molecules, but at low pH, salt linkages are broken since the carboxyl groups are displaced from combination and the freed amino groups combine with the added acid. As a result of lowered cohesion of the micelles the structure swells with water and is now accessible to large dye molecules which are then trapped in the protein fiber. The tendency of dye ions to aggregate in solution and to stain as aggregates will also affect the penetration and therewith the affinity of the dye for the protein. There are evidently other possibilities involving the number and kind

240

MARCUS SINGER

of reacting groups on the dye molecule (Speakman and Clegg, 1934; Townend and Simpson, 1946 ; Gerstner, 1949) and the physical structure of the protein as well as that of the dye (see also Seki, 1933a; Zeiger, 1938). For each dye with more than one binding site in its molecule it would be important to inquire how many of these sites are involved in the actual attachment of the dye molecule to the protein. For example, a dye molecule with two charges may be bound in equivalent or molecular fashion (Loeb, 1922; Elod, 1933; Speakman and Stott, 1935), and its mode of attachment may vary according to the protein. Abbot, Crook, and Townend (1947) have described this effect for the dyeing of nylon. When the number of acid groups of the dye molecule is increased from one to two, the affinity of the dye decreases since it is now spread over two sites rather than one. Or, dye with three or even four charged groups has been reported to occupy an equivalent number of oppositely charged sites. Because of the probable disparity in spatial arrangement of the charges on the dye and those on the protein, it is possible that an equivalent number of charged sites are neutralized rather than occupied by the dye ion (Vickerstaff, 1950). However, if only one binding group of the dye molecule is involved, the remaining groups may influence adversely the binding of oncoming ions by electrical effects or steric hindrance (Klotz and Walker, 1947; Vickerstaff, 1950). I n these instances the affinity of dye for protein must be weighed not merely in terms of its combining capacity but also, once bound, in relation to its effect on the approach of another dye ion to an adjacent site. Therefore, as staining proceeds, the available sites are no longer equivalent to the initial ones and the dyeing mechanism may be profoundly influenced and altered. In connection with these thoughts it may be well to note that there is no need to assume that all dyeing sites are equivalent, even at the onset of dyeing (Vickerstaff, 1950). Some may not be accessible to certain dye molecules, but readily available to others. Some correlation has been drawn between the shape of the dye molecule and the affinity for protein in that planar molecules are believed to have more affinity for wool than three-dimensional or linear ones (Steinhardt and Harris, 1940) ; but in the case of protein staining in general little information is available relating shape and affinity (Vickerstaff, 1950). The relation between the structure of the substrate and the shape and constitution of the dye has been studied in most detail in direct dyeing of cellulose fibers. Cellulose is dyed by color ions (anions) having the same charge (negative) as the fiber, and consequently, other forces than

STAINING OF TISSUE SECTIONS

241

electrostatic ones must be invoked to explain the uptake and fixing of the dye. Indeed, the electrostatic forces serve in this case to repel the dye ion rather than attract it, as in the case of ordinary protein staining. Dyes which combine directly with cellulose are generally sodium salts of sulfonated aromatic azo dyes. Once the dye anion is brought to the surface of the fiber (as described in preceding pages, factors of thermal agitation and addition of large quantities of electrolytes to the dye bath are important in this movement) and penetrates the water swollen regions between the cellobiose chains, the dye molecule is bound by short-range forces to the cellulose molecules (Neale, 1947 ; Vickerstaff, 1950). The nature of these forces and the problems involved in binding dyes of various structure and configuration have been speculated upon. Among the theories is that of hydrogen bonding (Rose, 1935) in which hydrogen of certain groupings of the fiber or of the dye acts as the electron acceptor. According to Rose at least two hydrogen links are required to bind a colored ion. Such linkages between the color ion and the linearly arranged cellobiose chains should be more readily formed with dye ions of certain shape and structure. Indeed, dye molecules used in direct dyeing of cellulose are in general long and chain-like and thus can contact the cellulose micellae more closely than non-linear ones (Meyer, 1928). Coplanarity of the various ring nuclei (benzene and naphthalene) also determines affinity of the dye (Hodgson, 1933) ; dyeing is favored when the rings lie in one plane. Finally, a high number of double bonds in the dye molecule seems also to be important (Schirm, 1935). This description of some of the theories of cellulose dyeing emphasizes that dye interaction depends to some degree upon the shape, size, and constitution of the dye molecule. The problem of the affinity of dye is undoubtedly complicated by still other factors than those described above. In non-aqueous solutions or under conditions of staining quite different from those considered in the present review, the extent of interaction may be still further influenced; and the reaction between a particular substrate and a dye may be specifically favored. Relatively little attention has been paid to the influence of special media, such as an alcoholic or phenolic one, as is employed, for example in methyl green-pyronin staining of nucleoprotein. Perhaps the function of the special media or other peculiar conditions of staining is specifically to enhance or to facilitate forces which may favor a particular dye and protein combination.

242

MARCUS SINGER

IX. THEINFLUENCE OF FIXATION AND OTHERMODIFICATIONS OF TISSUES ON SUBSEQUENT STAINING The obvious and important effect of fixation in histology is to denature and thereby render insoluble both the solid and dissolved proteins. In addition, fixation and other chemical or physical modification influence profoundly the subsequent binding of dye. In general, the living cell shows a selective affinity for some dyes which it concentrates and stores. This selective affinity is an expression of vital activities of living cells and bears little relation to forces which control the binding of dye in fixed and histologically prepared cells and tissues. After death, dyes of various character readily penetrate the cell, but the capacity to bind them is slight. However, when the cell is subjected to physical or chemical denaturing agents there is an immediate and pronounced increase in staining. The extracellular protein matrix of tissues also shows a limited affinity for dye until it is fixed. The precise nature of the effect of fixatives on proteins is not known. Elucidation of the alterations suffered by the protein during fixation is a problem of major importance in the study of the physical chemistry of tissue proteins since it is the modified and not the native protein which is studied under the microscope. Yet, relatively little work is being or has been done in recent years on fixation. Early workers devoted a considerable time to the elaboration of different fixatives designed to preserve the morphology of the cell and tissue with little change and yet to favor the staining of one or another morphological component. A considerable number of procedures was elaborated empirically or on the basis of certain chemical information. The procedures were reviewed admirably by Mann (1902), who also discussed, according to the information then available, the chemical and physical significance of the techniques. A more recent evaluation of fixation is given by Zeiger (193Oa, b, 1938). References to various methods of fixation and their general application may be obtained in technical works on histological procedures (for example Baker, 1945 ; McClung’s Handbook, Jones, 1950; Lee’s Vade-Mecum, Gatenby and Beams, 1950 ; Bourne, 1951). No attempt will be made to cover these works here. Instead, some general statements will be made about fixation based especially upon chemical information on denaturation of proteins and related information that is available in the histological literature, The manner in which fixation influences the subsequent staining is exemplified in experiments recently reported on films of fibrin (Singer and Morrison, 1948). These films are particularly suited for studies of

243

STAINING OF TISSUE SECTIONS

fixation, since the constituent fibrin of freshly prepared films may be considered native (Ferry, Singer, et al., 1947). Moreover, the protein is solid and insoluble having the three-dimensional relations typical of the structural proteins of cells and tissues. Consequently, the factors which obtain in fixation of fibrin film resemble somewhat those for cells and tissues. The interaction with dye of native fibrin and fibrin modified by various procedures is compared in Fig. 5. Freshly prepared fibrin has

i

I 2

10

08

06

0.4

0. I 09.

t

3

6

4

9

PH

FIG.5. The influence of different fixatives on subsequent staining of fibrin film. Note how the acid (orange G ) and basic (methylene blue) dye-binding curves shift according to the fixative (see text on discussion of isoelectric points and of fixation). Taken from Singer and Morrison, J. Biol. Chem., 176, 1948. little capacity to bind dye and resembles unfixed tissue protein in this way. In general, a notable increase in stainability with both acid and basic dye occurs after fixation, whether such fixation be p%sical (heat) or chemical in nature. However, the increase in acid and basic dye-binding capacity is not an equal one and in most cases varies according to the treatment. For example, there is a relatively greater increase in basic than in acid dye uptake following formaldehyde fixation. O n the other hand, the reverse is true after fixation with HgClz or some other salt of a heavy metal (cf. Kelley, 1939a). Fixatives appear to have two major effects on protein as reflected in the dye reaction. There is an initial effect of increase in affinity for both classes of dye attributable presumably to a physical reorganization of the protein, whereby charged and other groups to which the dye ion may attach are rendered more available to the dye (Singer and Morrison, 1918). This effect is in general shared by all fixatives. An increase in

244

MARCUS SINGER

availability of reacting groups resulting from denaturation of proteins has been frequently described in the chemical literature (see reviews of Neurath, Greenstein, Putnam, and Erickson, 1944 ; and Anson, 1946). Alteration in physical structure of the protein implies also an alteration in the permeability of the protein meshwork for dye molecules. The second effect is peculiar to the particular fixative and results in an alteration in the relative uptake of acid and basic dye. This effect may be attributed to a specific influence of the fixative on groups which bind the dye, or on other groups whose proximity or mere presence influences the binding of dye to adjacent sites. The fixative, depending on its nature, may introduce other ionizing and, therefore, dye-binding sites in the protein. It may cover specifically certain groups or in some other manner prevent dye interaction with these sites. The two effects of the fixative are well exemplified in heat denaturation of fibrin film (Ferry, Singer, et al., 1947). As a result of brief heating the acid and basic dye-binding capacity increased remarkably (see also Herrmann, Nicholas, and Boricious, 1950), an effect which corresponds to the first postulate. Upon prolonged heating a second effect appeared and gradually became pronounced ; the affinity for basic dye increased whereas that for acid dye declined. Associated with the second change there was a gradual drop in the isoelectric point of the protein (Singer and Morrison, 1948). Prolonged heating probably causes a gradual and progressive deamination of the protein and thereby a decrease in the number of positive groups available for acid dye. A similar drop in the isoelectric point and a similar alteration in staining is observed following denaturation with formaldehyde. But, in this instance, the secondary effect is accomplished by combination and therefore covering of the amino groups. A secondary effect in which metal ions combine with carboxyl groups presumably occurs with HgCl? and other heavy metal fixatives. In this way the basic dye affinity is relatively diminished and the acid one is increased. Other alterations, such as mordanting, which are secondarily imposed upon the primary fixation will further influence the staining of proteins by modifying the dyeing sites or so changing the physical characteristics of the protein structure (such as degree of swelling) as to alter the extent of penetration by the dye ion. Staining intensity varies with changes in the protein and, consequently, is a sensitive criterion of the modifications to which the protein was previously subjected. Indeed, slight differences in heat treatment of fibrin film were detected through alterations in dye uptake (Ferry, Singer, et al., 1947). Another study of staining of modifietl proteins was that of Fraenkel-Conrat (1944) ; and the effect of various chemical modifications

S T A I N I N G OF TISSUE SECTIONS

215

on the dye binding of wool was reviewed by Kienle, Royer and McCleary (1945), Lemin, Vickers and Vickerstaff (1946) ; Vickerstaff ( 1950). A number of interesting studies of the variation of staining with fixation have been reported in histological literature (see reviews by Zeiger, 193Oa, 1938). Tolstoouhov (1928) recorded the staining of blood cells in mixtures of eosin and methylene blue of various pH and following various fixations. The p H of approximately equal binding of these two dyes depended upon previous fixation. After fixation in solutions of salts of heavy metals, the cells had much less affinity for basic dye and the p H of equal binding rose. The reverse occurred after formalin fixation, whereas fixation with ethyl alcohol yielded stain affinities of an intermediate character. Zeiger (1930) studied alcohol and formalin fixation and believed that there was less shift in the isoelectric point of tissue proteins after alcohol fixation than with other common fixatives. Yasuzumi ( 1933) studied the effect of alcohol fixation on the isoelectric point of red blood cells. An extensive series of experiments has been done with various fixatives on egg albumen and tissue sections (Seki, 1933b) and on extracted nucleoprotein (Kelley and Miller, 1935 ; Kelley, 193913).

X. THEINFLUENCE OF TEMPERATURE OF THE STAINING SOLUTIOK The temperature of the staining solution may influence staining in a number of ways. Probably the most pronounced effect is on the rate of diffusion or movement of the dye within the protein and, therefore, the rate of staining. The rate of dyeing is increased progressively with increased temperature so that equilibrium staining is reached much faster at higher temperatures than at lower ones. The notable effect of temperature on equilibrium staining is illustrated well in the example which Vickerstaff (1950) gives for the dyeing of wool fiber. Five months at 20°C would be required for equilibrium dyeing of wool which can be dyed in 1 hour at 100°C. This effect of temperature on the diffusivity of the dye has been treated quantitatively by determining the activation energy of dyeing (Vickerstaff, 1950). Dyes of poor diffusivity are affected most by temperature changes (Abbot, Crook, and Townend, 1947). Temperature of the dye bath is believed to have other influences besides alteration in the rate of diffusion of the dye. It may influence the affinity of the dye-protein interactants since the amount of dye bound at equilibrium decreases with increasing temperature (Boulton, Delph, Fothergill, Morton, 1933 ; Neale, 1933 ; Vickerstaff, 1950). At low temperatures equilibrium may be so slow in attainment that the erroneous impression is thereby given that less dye is bound at low temperatures (Vickerstaff,

246

MARCUS SINGER

1950). Steinhardt ( 1940) and Steinhardt, Fugitt, and Harris ( 1940b, c, 1912) studied the effect of three temperatures (0,25, and 50°C) on the affinity of various anions and wool. They observed that the p H association curves of these anions showed different degrees of sensitivity to temperature change. Larger, more tightly bound anions have greater heats of dissociation, and thus their affinity was altered less by increase in temperature than smaller more readily dissociated anions. Altered temperature may be expected to produce essential responses and chemical changes in the protein itself (Elod, 1933). Heating may cause a swelling of the fiber due to loosening of the micellar structure as has been reported by Speakman and Smith (1936) for wool dyeing. Penetration of the protein by the color ion is thereby facilitated. The effect would be particularly pronounced for large dye ions or for colloidal aggregates (Gerstner, 1949). The separation of the micellar sheets can occur by loosening of the covalent bonds between them but also by destruction of the disulfide linkages. At elevated temperature there may also be a decomposition of protein (see previous discussion of heat modification of fibrin). In the case of colloidally dispersed dyes, the effect of temperature is very marked since swelling of the protein which results from increased temperature allows greater penetration of dye into the protein. Moreover, the dye is more finely dispersed at higher temperature and aggregation tendencies are diminished (Speakman and Smith, 1936; Goodall, 1938, 1947). For most successful dyeing, dyes of low dispersivity require higher temperatures. This information explains the importance in tissue staining of elevated temperatures with colloidal . solutions of dye (for example, azocarmine in triple acid staining techniques). Other works of interest for further references and description of temperature effects in dyeing are those of Brown (1901a, b), Sheppard, Houck, and Ditmar (1942) ; and Royer, Zimmerman, Walter and Robinson (1948). The latter workers described the effect of extremely elevated temperatures (200"and 300°F) on dye uptake of textiles. In the histological literature there is relatively little other than empirical studies on temperature effects on staining. Some references may be had in the work of Ochs (1928), who also described temperature alterations of the staining of blood cells and gelatin.

XI. SOMEOBSERVATIONS ON THE KINETICS OF STAINING In preceding pages attention was focused primarily on the forces which bind molecularly dispersed dye to solid protein and on the various factors

STAINING OF TISSUE SECTIONS

247

which influence these forces or otherwise alter the dyeing process. Another important aspect of the staining process concerns forces involved in the movement of dye ions to the surface of the protein and their distribution to the binding sites. Histological staining is invariably done in concentrated solutions with a great excess of dye so that there is essentially no decrease in concentration as staining proceeds. Consequently, diffusion of dye ions to the protein surfaces must be extremely rapid and the movement must continue at a high level particularly when the solution or tissue is agitated during staining. Moreover, coulombic forces acting between dye and protein would serve to hasten the movement. Under conditions where the dye bath is gradually exhausted during the course of dyeing as is done in textile coloring, the problem of diffusion of dye in solution to the surface of the fiber is of greater import. Diffusion of the dye ion from the surface of the protein into the internal meshwork and thence into the intermolecular spaces of tissue structures to more deeply placed dyeing sites, constitutes, however, an important factor which evidently exerts a profound control on the rate of staining. An early and particularly lucid discussion of diffusion factors is given by Pappenheim (1901 ) . The importance of factors of diffusion in staining was stressed particularly in early works which supported the physical mechanism of staining (Gierke, 1885 ; Fischer, 1899; Knoevenagel, 1911 ; Pappenheim, 1917; von Mollendorff, 1923; von Mollendorff and Krebs, 1923; von Mollendorff and von Mollendorff, 1924; excellently reviewed by Zeiger, 1938). The importance of dye particle size and pore size in the protein was speculated upon as a mechanism of staining (e.g., the “Durchtrankungsfarbung” of von Mollendorff, 1923). Problems of diffusion of dye to the binding sites are also of importance in textile dyeing since the fiber and fibril sizes to be penetrated by the dye ion are large and the micellar network dense (Vickerstaff, 1949, 1950). Once the dye reaches the surface of the tissue section it is free to react with sites available at that position. Penetration of other dye ions to deeper staining sites must occur through this layer in which staining forces are already at least partly satisfied. Since the interaction of the dye with the surface sites is probably immediate, the rate of staining is determined in large part by the time for the dye ions to reach more central regions of the protein (Vickerstaff, 1950). The movement of dye ion to deeply placed sites is much slower than to the surface because of mechanical obstruction of the protein micellae and because of other forces acting between dye and protein and, indeed, between bound and free dye. The protein structure may provide an effective barrier to some dye ions and not others. Large dye ions may

248

MARCUS SINGER

penetrate with difficulty or not at all and dye aggregates may penetrate the interstices of certain meshworks but be excluded from others. The speed of penetration may vary from one region of the tissue to another according to the density of the charge in these regions. It may change as dyeing proceeds because of the gradual alteration of charge on the protein with staining. The ease of diffusion may also be affected by dye ions which are already bound according to conditions described in a preceding section on the affinity of dye. For example, dye ions with residual unsatisfied charges would repel oncoming ions. Such a repulsion would be particularly effective in influencing the rate of staining if the first dye reaction occurs at or near the surface of the tissue section. The rate of dyeing may follow closely the rate of diffusion of the dye ion into the protein but also may depend upon the affinity of the dye for the protein and upon the various conditions of staining such as pH, temperature, ionic strength, and dye concentration of the dye bath. The physical state of the protein is important not only as it may interfere with movement of dye but also in other ways. Degree of orientation of the fiber has been shown to affect greatly the degree of dye binding by cotton (Preston and Pal, 1947). This effect has also been described for nylon since dyeing may be enhanced by “relaxing” the fiber with heat treatment (Fidell, Royer, and Millson, 1948). Thus, highly oriented fibers show less affinity for dye than less oriented ones. XII. THE REVERSIBILITY OF STAINING REACTIONS ; EQUILIBRIUM OF STAINING A N D OTHER FACTORS WHICH INFLUENCE STAINING It is important to stress that staining is a reversible reaction and that when the solution environment of tissue sections is changed, there is a corresponding alteration in the equilibrium concentration of dye within the tissue. Dye may then be lost to the solution or removed from it. It is possible to wash out the stain in a solution free of dye particularly if the p H is adjusted upward in the case of acid dyes or downward in the case of basic ones-pH regions which would favor dissociation of the dye-protein combination. Although the reaction may be reversed and dye washed from the stained protein structure, the extent and rate of “desorption” varies with the histological structure, with the dye and with the washing conditions. Washing from tissue in which the protein is densely packed is conceivably more difficult than where the protein is more dispersed particularly when the dye has aggregated upon binding. And, dye of great affinity for a particular protein will show greater fastness than another dye and is removed only

STAINING OF TISSUE SECTIONS

249

with difficulty (Gerstner, 1949). Conditions of p H which do not favor binding of acid or basic dye are most favorable for stripping the dye from the protein. As already mentioned, elevated p H is most conducive for decolorizing acid dyes and low pH basic ones. The effect of acidity or alkalinity of the washing medium on destaining is considered in the early review of Pappenheim (1901) and the more recent work of Stearn and Stearn (1928a, b). Reversibility of the staining reaction is important for another reason, namely the redistribution of dye so that final staining is relatively even in a given protein structure despite rapid staining or destaining procedures which might be expected to favor uneven localization of the dye. In the redistribution, dye ions are “desorbed” from one site and transferred to another more deeply placed one. The ease of desorption and transference determines the final uniformity of the distribution. I t is known in textile staining that the migrating or leveling power of dyes differs greatly. The leveling capacity of dyes influences the course of the reaction. Dye which satisfies surface sites without tending to shift to deeper regions delays or prevents the expression of full staining capacity of the protein. Such staining stands in contrast to that with dye which rapidly migrates and distributes itself uniformly as staining proceeds. I n the former case dyeing is uneven and slow in reaching equilibrium. Various procedures of gradual alteration in dyeing conditions are used in the textile industry to improve and hasten the leveling of dye. Of interest among these is that leveling is favored by p H regions at which the charge of the protein is not extreme. There are still other factors which operate in the staining reaction. Most of these are poorly understood and, therefore, are only briefly considered here. If the dyeing time is lengthy and the temperature is elevated, decomposition of the protein may set in. In some instances the dye has been said to have a catalytic effect on degradation of the protein (Lemin and Vickerstaff, 1947). Ionic exchange has been described for the staining of ligno-cellulose with methylene blue (Sarkar and Chatterjee, 1948) and may operate more widely. The problems of staining with dye aggregates or suspensions which are widely used in histology (for example, Congo red, azocarmine, and trypan blue) have barely been touched upon in this review. The profound influence of mordants, media other than water, the effect of specific ions and the competition between dye ions of mixtures for similar sites deserve special study. Finally, the influence of brief staining times on dyeing must be touched upon. In progressive staining low dye concentrations are employed and

250

MARCUS SINGER

staining is allowed to proceed to equilibrium. Staining is more easily controlled under such circumstances where the time of staining is not a variable. If staining is not carried to equilibrium, then the time of immersion in the dye solution is an important factor in dye uptake. Short staining times at elevated dye concentrations are quite popular in many histological techniques. Since the rate of staining is greatest during the first few minutes of reaction, and levels off slowly thereafter, tissues may be effectively stained during brief immersion. The tissue is removed when the desired intensity of staining is reached or the tissue is overstained and then secondarily destained. The amount of dye taken up in a given time will depend on a number of factors, including the mobility of the dye ion in the tissue substrate, the speed of interaction, the leveling capacity, the affinity of the dye, and the conditions of staining. Consequently, the time of dyeing must vary with each dye. In order to compensate for individual differences in dyes the concentration of the dye may be changed or other conditions of staining varied. XIII. REFERENCES Abbot, E. B., Crook, H., and Townend, F. (1947) J . SOC.D y . Col., Bradford, 63. 462. Achard, J. (1935) 2. Zellforsch., 23, 573. Arner. Dyestuff Reporter. Proc. of Airtrr. Asso. Textile Chem. Col. (1918) drncr. Dyestuff Rep., 37, 149. Anson, M. L. (1946) Protein denaturation and the properties of protein groups. Advances in Protein Chem., 2. Atkin, R. W., and Douglas, F. W. (1924) J. Airzer. Leather Chem. Asso., 19, 528. Baker, J. R. (1945) Cytological Technique. Methuen Monog., London. Bancroft, W. D. (1914a) J. phys. Chem., l8, 1. Bancroft, W.D. (1914b) J . phys. Chem., l8, 118. Bancroft, W.D. (1914~) J. phys. Chem., 18, 385. Bancroft, W.D. (1915a) 1. phys. Chem., 19, 50. Bancroft, W.D. (1915b) J . phys. Chem., 19, 145. Bejdl, W. (1950) Mikroskopie, 6, 83. Bethe, A. (1905) Beitr. Chem. Physiol. Pathol., 6, 399. Bonin, W.,Frappier, J., and LararnCe, A. (1944) Rev. Canad. BioJ., C. R., 3. 481. Boulton, J., Delph, A. E., Fothergill, F., and Morton, T. H. (1933) J. Textile Inst., 24, 113. Bourne, G. (1951) Cytology and Cell Physiology, 2nd ed. Clarendon Press, Oxford. Briggs, T. R., and Bull, A. W. (1922) J. phys. Chem., 26, 844. Brown, R. B. (1901a) J. SOC.Dy. Col., Bradford, 17, 92. Brown, R. B. (1901b) J . SOC.Dy. Col., Bradford, 17, 125. Carlene, P. W., Fern, A. S., and Vickerstaff, T. (1947) J . SOC.D y . Col., Bradford, 0S, 388. Chapman, L. M., Greenberg, D. M., and Schmidt, C. L. A. (1927) J . biol. Chem., 72, 707.

STAINING O F TISSUE SECTIONS

25 1

Cohn, E. J. (1945) Blood and Blood Derivatives. Smithsonian Rep., Smithsonian Inst., Wash., p. 413. Cohn, E. J., and Edsall, J. T. (1943) Proteins, Amino Acids and Peptides. Reinhold Co., New York. Conn, H. J. (1940) Biological Stains. Humphrey Press Inc., Geneva, New York. Conn, H. J., and Holmes, W. C. (1928) Stain Tech., S, 94. Craig, R., and Wilson, C. (1937) Stain Tech., Za, 99. Dempsey, E. W., Bunting, H., Singer, M., and Wislocki, G. B. (1947) Anat. Rec., 98, 417. Dempsey, E. W., and Singer, M. (1946) Endocrin., 38, 270. Dempsey, E. W., Singer, M., and Wislocki, G. B. (1950) Stain Tech., 26, 73. Dempsey, E. W., and Wislocki, G. B. (1946) Physiol. Rev., 26, 1. Dempsey, E. W., Wislocki, G. B., and Singer, M. (1946) Anat. Rec. 96, 221. Dreaper, W. P. (1906) The Chemistry and Physics of Dyeing. London. Dubos, R. J. (1945) The Bacterial Cell. Harvard University Press, Cambridge, Massachusetts. Ehrlich, P. (1879a) Arch. Physiot., 166. Ehrlich, P. (1879b) Arch. Physiol., 571. Elod, E. (1933) Trans. Faraday SOC.,29, 327. Ender, W., and Miller, A. (1937) Melliand TextilDer., 18, 633. Fautrez, J. (1936) Bull. Histol. Appl., l8, 202. Ferry, J. D., and Morrison, P. R. (1946) Znd. Eng. Chem., 38, 1217. Ferry, J. D., and Morrison, P. R. (1947) J. Amer. Chem. SOC.,69, 400. Ferry, J. D., Singer, M., Morrison, P. R., Porsche, J. D., and Kutz, R. L. (1947) 1. Amer. Chem. Soc., 69, 409 Fidell, L. I., Royer, G. L., and Millson, H. E. (1948) Amer. Dyestuf Rep., 37, 166. Fierz-David, H. E., and Blangey, L. (1949) Fundamental Processes of Dye Chemistry. Interscience, New York. Fischer, A. (1899) Fixierung, Farbung, Bau des Protoplasmas. Jena. Fraenkel-Conrat, H. (1944) J. biol. Chem., 164, 227. Fraenkel-Conrat, H., and Cooper, M. (1944) J. biol. Chenz., 164, 237. French, R. W. (1930) Stain Tech., 6, 87. Gatenby, J. B., and Beams, H. W. (1950) The Microtomist's Vade-Mecum (Bolles Lee), Blakiston, Philadelphia. Gee, W. W. H., and Harrison, W. (1910) Trans. Faraday SOC.,6, 42. Gelmo, P., and Suida, W. (1905) Sitz. Akad. Wiss. Wien. Jan. Math.-nat. Kt. 114, Quoted from Pelet-Jolivet, 1910. Gerstner, H. (1949) Melliand Textilber., 30, 253; 302. Gierke, H. (1885) 2. m'ss-Mikr., 2, 13, 164. Gilbert, G. A., and Rideal, E. K. (1944) Proc. roy. SOC.,8182, 335. Gillet, C. (1889) Rev. gen. mat. cot., pp. 15, 189. Gillet, C. (1890) Rev. gen. mat. col., p. 339. Goldstein, A. (1949) 1. Pherm. exp. Therap., Pt. 11, 96, 102. Goodall, F. L. (1938) J. SOC.Dy. Col., Bradford, 64, 45 Goodall, F. L. (1947) Am. Dyestuf Rep., 36, 380. Grollman, A. (1925) J. biol. Chem., 64, 141. Halphen, G., and Riche, A. (1904) C. R. SOC.Biol., 140, 1408. Halphen, G., and Riche, A. (1905) Rev. gen. mat. cot., p. 200.

252

MARCUS SINGER

Harrison, W. (1Fi1) J. SOC.Dy. Col., Bradford, 27, 279. Harrison, W. (1948) J. SOC.Dy. Col., Bradford, 64, 248. Haynes, R. (1928) S t a h Tech., 8, 131. Heidenhain, M. (1902) Arch. ges. Physiol., SO, 115. Heidenhain, M. (1903) In : Enzyklopiidie mikr. Technik, 1, 335. Herrmann, H., Nicholas, J. S., and Boricious, J. K. (1950) J . biol. Chew., 184, 321. Hewitt, L. F. (1927) Biochem. J., 21, 1305. Highman, B. (1945) Stain Tech., 20, 85. Hodgson, H. H. (1933) 1. SOC.Dy. Col., Bradford, 49, 213. Hofmeister, F. (1891) Arch. exp. Path. Pharm., 28, 210. Holmes, W. C. (1929) Stain Tech., 4, 75. Ikeda, S. (1935) Folia Anqt. lap., 13, 141. Ikeda, S. (1936a) Folia Anat. Jap., 14, 107. Ikeda, S. (193613) Folio Anat. lap., 14, 175. Jones, R. McClung (1950) McClung’s Handbook of Microscopical Technique. Hoeber, New York. Kelley, E. G. (1939a) J. biol. Chem., l27, 55. Kelley, E.G. (1939b) J. biol. Chem., ia7, 73. Kelley, E. G., and Miller, E. C., Jr. (1935) J. biol. Chew., 110, 113. Kienle, R. H.,Royer, G. L., and McCleary, H. R. (1945) Aster. Dyestuf Rep., S4, 42.

Kindred, J. E. (1932) Anat. Rec., 63, 43. Kindred, J. E. (1935) Sta& Tech., 10, 7. Kitchener, J. A,, and Alexander, P. (1949) J. SOC.Dy. Col., Bradford, 6S, 284. Klotz, I. M. (1946) J. Amer. Chem. SOC.,68, 2299. Klotz, I. M., Triwush, H., and Walker, F. M. (1948) J. Amer. Chefit. Soc., 70,

2935.

Klotz, I. M., Walker, F. M., and Pivan, R. B. (1946) J. Amer. Chefit. SOC.,68,

1486.

Klotz, I. M., and Walker, F. M. (1947) J. Amer. Chem. SOC.,69, 1609. Knecht, F. (1889) Ber. dtsch. c h m . Ges., a,1120. Knecht, F. (1904) Ber. dtsch. chem. Ges., 87, 3479. Knoevenagel, H. E. A. (1911) 2. angew. Chem., 106. Langmuir, I. (1916) J. Amer. Chem. SOC.,36, 2221. Langmuir, I. (1917) 1. Amer. Chem. SOC.,89, 1848. Lemin, D.R., Vickers, E. J., and Vickerstaff, T. (1946) J. SOC.Dy. Col., Bradford, 62, 132.

Lemin, D. R., and Vickerstaff, T. (1947) J. SOC.Dy. Col., Bradford, 68, 405. Levine, N. D. (1939) Stah Tech., 14, 29. Levine, N. D. (1940) Stain Tech., 16, 91. Lewis, G. N.,and Randall, M. (1923) Thermodynamics and the Free Energy of Chemical Substances. McGraw-Hill, New York. Lilienfeld, L. (1893) Arch. Anat. PhySiol., PhySiol. Abf., 391. Lloyd, D. J., and Bidder, P. B. (1934) Tram. Faraday Soc., 81, 864. Loeb, J. (1922) Proteins and the Theory of Colloidal Behavior. McGraw-Hill, New York. Loeb, J. (1924) Proteins and the Theory of Colloidal Behavior, 2nd ed. McGrawHill, New York.

STAINING O F TISSUE SECTIONS

253

Magnus, W. (1903) Zellchemie. In: Encyklopadb mikr. Technik., 2, 1365. (See especially p. 177, Farbunganalytische Methoden). Mallory, F. B. (1944) Pathological Technique. W. B. Saunders Co., Philadelphia. Maneval, W. E. (1941) Stain. Tech., 16, 13. Mann, G. (1902) Physiological Histology. Methods and Theory. Oxford University Press. hlathews, A. (1898) Amer. J . Physiol., 1, 445. hlayer, F. (1934) Chemie der organischen Farbstoffe. Springer, Berlin. McCalla, T. M. (1941) Stain Tech., 16, 27. McCalla, T. M., and Clark, F. E. (1941) Stain Tech., 16, 95. Meyer, K. M. (1928) Melliand Textilber., 9, 573. Michaelis, L. (1900) Arch. mikr. Anat., 66, 558. Michaelis, L. (1901) Dtsch. med. Wchsch., 27, 219. Michaelis, L. (1911) Theorie des Farbeprozesses. In : Oppenheimer : Handb. d. Biochemie, Bd. 2. Michaelis, L. (1920) Arch. mikr. Anat., 94, 580. hlichaelis, L. ( 1926) Hydrogen ion concentration. Williams and Wilkins, Baltimore. Michaelis, L. (1947) Cold Spr. Harb. Symp. quant. Biol., 12, 131. Michaelis, L., and Granick, S. (1945) J . Am. Chem. SOC.,67, 1212. Miescher, F. (1874) Verh. Naturf. Gesellschaft, Basel, 6, 138. v. Mollendorff, F., and v. Mollendorff, M. (1924) Ergebn. Anat. Entw., 26. v. Mollendorff, W. (1923) Derm. Wchsch., 1417. v. Mollendorff, W., and Krebs, H. A. (1923) Arch. Amt., 97, 554. v. Mollendorff, F., and v. Mollendorff, M. (1924) Ergebn. Anat. Entw., 26. Mommsen, H. (1927) Folb Haematol., 54, 50. Naylor, E. E. (1926) A w r . J. Bot., 13, 265. Neale, S. M. (1933) J . SOC.C h m . Znd., 62, 88. Neale, S. M. (1946) Trans. Faraday SOC.,42, 473. Neale, S. M. (1947) J . SOC.Dy. Cot., Bradford, 63, 368. Neale, S. M., and Stringfellow, W. R. (1933) J . Textile Znst., !U, 145. Neurath, H., Greenstein, J. P., Putnam, F. W., and Erickson, J. 0. (1944) Chem. Rev., 34, 157. Nietzke, R. (1901) Farbstoffe. J. Springer, Berlin. Quoted from Mann (1902). Nishimura, T. (1934) F o l k A m t . Jap., l2, 357. Noble, E. I. (1945) J . SOC.Dy. Col., Bradford, 81, 328. Ochs, G. W. (1928) Folia Haematol., 37, 241. Pappenheim, A. (1901) Grundrisz der Farbchemie zum Gebrauch bei mikroskopischen Arbeiten. A. Hirschwald, Berlin. Pappenheim, A. (1917) Folia Haemtol., 92, 18. Pelet- Jolivet, L. (1910) Theorie des Farbeprozesses. Th. Steinkopf, Dresden. Peters, L., and Speakman, J. B. (1949) J . SOC.Dy. Cot., Bradford, 65, 285. Peters, R. H. (1945) J . SOC.Dy. Col., Bradford, 61, 95. Pfeiffer, H. (1929) P r o t o p l a m , 8, 377. Pfeiffer, H. (1931) 2. miss. Mikr., 48, 88. Pischinger, A. (1926) 2. Zellforsch., 5, 169. Pischinger, A. (1927a) Pfliiger’s Arch. ges. Physiol., 217, 205. Pischinger, A. (1927b) 2. Zellforsch. mikr. A m t . , 6, 347. Pratt, L. S. (1947) The Chemistry and Physics of Organic Pigments. Wiley, New York.

254

MARCUS SINGER

Preston, J. M., and Pal, P. (1947) J. SOC.Dy. Col., Bradford, 63, 430. Pulcher, C. (1927) Boll. Sac. ital. Biol. Sperm., 1, 223. Rakusin, M. A. (1928) Biochm. Z.,la,167. Kawlins, L. M. C., and Schmidt, C. L. A. (1929) J. biol. Chem., a,709. Rawlins, L. M. C., and Schmidt, C. L. A. (1930) J. biol. Chcni., 88,271. Rideal, E. K. (1950) Foreword to the Physical Chemistry of Dyeing (Vickerstaff) Imperial Chem. Ind., London. Robbins, W. J. (1923) Anw. J . Bot., 10, 412. Robbins, W. J. (1924) J. Sen. Pkysiol., 6, 259. Robbins, W. J. (1926) Univ. of Missouri Stud., 1, 3. Columbia, Missouri. Rose, F. L. (1935) Private Communication to Vickerstaff, 1950. Rose, R. E. (1942) Amer. Dyestuff Rep., 31, 204. Rowe, F. M. (1924) Colour Index. Sac. Dy. Col., Bradford, England. Royer, G. L., Zimmerman, C. L., Walter, H. J., and Robinson, R. D. (1948) T0.rtile Res. J., 18, 598. Sarkar, P. B., and Chatterjee, H. (1948) J . SOC.Dy. Col., Bradford, 64, 218. Schirm, E. (1935) I. fir&. Chem., la, 69. Schmidt, C. L. A. (1945) The Chemistry of the Amino Acids and Proteins. Charles C. Thomas, Springfield, Illinois. Schwalbe, C. G. (1907) Die neueren Farbetheorien. Stuttgart. Schwarz-Karsten, H. (1927) Dtsch. med. Wchsch., 65, 1820. Seki, M. (1933a) Folia Anat. Iap., 11, 377. Seki, M. (1933b) 2. Zellforsch. mikr. Anat., 18, 21. Seki, M. (1933~)2. Zellforsck. mikr. Anat., 18, 1. Seki, M. (1934) 2. ges. exp. Med., 94, 655. Seymour, R. B., Agnew, W., Crumley, J. A., and Kelly, A. J. (1948) A m r . Dycstuff Rep., 37, 689. Sheppard, S. E., Houck, R. C., and Dittmar, C. (1942) J. Phys. Chem., 46, 158. Singer, M. (1949) Proc. N . Y . Path. SOC., 90. Singer, M., and Morrison, P. R. (1948) I , biol. Chem., 176, 133. Singer, M.,and Wislocki, G. B. (1948) Anat. Rec., lOa, 175. Skinner, B. G.,and Vickerstaff, T. (1945) J. SOC.Dy. Col., Bradford, 61, 193. Sokolova, N. V. (1948) I . app1. Chem. (U.S.S.R.), 21, 966. Speakman, J. B.,and Clegg, H. (1934) I . SOC.Dy. Col., Bradford, 60, 348. Speakman, J. B.,and Elliott, G. H. (1943) J. SOC.D y . Col., Bradford, 59, 185. Speakman, J. B.,and Hirst, M. C. (1933) Trans. Faraday Soc., 29, 148. Speakman, J. B., and Peters, L. (1949) J. SOC.Dy. Col., Bradford, 66, 63. Speakman, J. B.,and Smith, S. G. (1936) J. SOC.Dy. Col., Bradford, 63, 121. Speakman, J. B.,and Stott, E., (1934) Trans. Faraday SOC.,30, 539. Speakman, J. B., and Stott, E. (1935) Trans, Faraday SOC.,31, 1425. Spiro, C. (1897) Uber physikalische und physiologische Selektion. Habilitationschrift. Strassburg. Stearn, A. E. (1931) I. biol. Chem., 91, 325. Stearn, A. E. (1933) J. Bact., 26, 21. Stearn, A. E.,and Stearn, E. W. (1924) Amer. J. Pub. Health, 14, 409. Stearn, A. E.,and Stearn, E. W. (1929) Stain Tech., 4, 111. Stearn, A. E.,and Stearn, E. W. (1930) Stairz Tech., I,17. Stearn, E. W., and Stearn, A. E. (1925) I . Bact., 10, 13.

STAINING OF TISSUE SECTIONS

255

Stearn. E. W., and Stearn, A. E. (1928a) Amer. J. Pub. Health, 18, 174. Stearn, E. W., and Stearn, A. E. (1928b) Stain Tech., 3, 81. Stearn, E. W., and Stearn, A. E. (1928~) Stain Tech., 3, 87. Stearn, E. W., and Stearn, A. E. (1929) Stain Tech., 4, 10.5. Steinhardt, J. (1940) Ann. N . Y. dcad. Sci., 41, 287. Steinhardt, J. (1942) I. Res. nut. Bur. Stand., a8, 191. Steinhardt, J., Fugitt, C. H., and Harris, M. (1940a) J. Res. Ifat. Birr. S t a d . , 26, 519. Steinhardt, J., Fugitt, C. H., and Harris, M. (1940b) Textile Res., 11, 72. Steinhardt, J., Fugitt, C. H., and Harris, M. (1940~) Amer. Dyestiif Rep., 29, 607. Steinhardt, J., Fugitt, C. H., and Harris, M. (1941a) J. Res. nat. Btw. S t a i d , 26, 293. Steinhardt, J., Fugitt, C. H., and Harris, M. (1941b) Atner. Dyestuff Rep., SO, 223, 250, 288. Steinhardt, J., Fugitt, C. H., and Harris, M. (1942) Anter. Dyestuf Rep., 31, 77. Steinhardt, J., and Harris, M. (1940) J. Res. nat. Bur. Stand., 24,335. Stockinger, L. (1950) Mikuoskopie, 6, 79. Sturm. K. (1935) 2. mikr.-anat. Forsch., 57, 595. Thomas, A. W., and Kelly, M. W. (1922) J. Amer. Chon. SOC.,44, 195. Tolstoouhov, A. V. (1927) Proc. N. Y. Path. Sac., 26, 147. Tolstoouhov, A. V. (1928) Stain Tech., 3, 49. Tolstoouhov, A. V. (1929) Stain Tech., 4, 81. Townend, F., and Simpson, G. G. (1946) J. Soc. Dy. Col., Bradford, 62,2, 47. Unna, P. G. (1928) Chromolyse. Aberhalden’s Handb. der Biol. Arbeitsmeth., Abt. V, pp. 1-62. Veller, E. A. (1948) J. a j p l . Chem. (U.S.S.R.),21, 1147. Vickerstaff, T. (1948) Rate of Dyeing. Imp. Chem. Ind. Technol. Monograph, No. 1, Manchester, England. Vickerstaff, T. (1949) Amer. Dyesfuf Rep., 36, 305. Vickerstaff, T. (1950) The Physical Chemistry of Dyeing. Imp. Chem. Ind., London. Weber, C. 0. (1894) J. Soc. Chem. Znd., 13, 120. Wislocki, G. B., and Singer, M. (1950) J. camp. Neurol., 92, 71. Wislocki, G. B., Singer, M., and Waldo, C. M. (1948) Aaat. Rec., 101, 487. Wislocki, G. B., Weatherford, H. L., and Singer, M. (1947) Anat. Rec., 99, 265. Wood, J. K. (1913) The Chemistry of Dyeing. London. Yasuzumi, G. (1933a) Folia Anat. Jap., 11, 267. Yasuzumi, G. (1933b) Folia Anat. Jup., 11, 415. Yasuzumi, G. (1934) Folia Anat. Jap., 12, 1. Yasuzumi, G., and Matsumoto, S. (1936) Foliu Anat. Jap., 14, 101. Zacharias, P. D. (1908) Die Theorie der Farbevorgange, Berlin. Zeiger, K. (1930a) Z . Wiss. Mikr., 47, 273. Zeiger, K. (1930b) 2. Zellforsch., 10, 481. Zeiger, K. (1938) Physikochemische Grundlagen der Histologischen Methodik. Th. Steinkopf, Dresden und Leipzig.

This Page Intentionally Left Blank

The Behavior of Spermatozoa in the Neighborhood of Eggs LORD ROTHSCHILD Department of Zoology, University o f Cambridge, Cambridge, England

CONTENTS I. Introduction ......................................................... 11. The Block to Polyspermy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Chemotaxis of Spermatozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. References ...........................................................

Page 257 258 260 263

I. INTRODUCTION The fact that only one spermatozoon normally participates in the fertilization reaction is one of the most striking and characteristic features of reproduction in many animal phyla. Monospermy implies that the fertilizing spermatozoon initiates a change in the egg surface such that other spermatozoa cannot fertilize or refertilize the egg. The nature of this block to polyspermy has been the subject of speculation for many years; but before these speculations can have any value it is necessary to try and find out, as quantitatively as possible, what the egg has got to contend with to prevent di-, tri-, and polyspermy. When an egg is placed in a suspension of spermatozoa, it is reasonable to assume that sperm-egg collisions take place. Apart from the reasonableness of this assumption several workers have mentioned in their papers that they have observed such collisions. It is natural to ask the question how many sperm-egg collisions occur ? The following treatment of this question, though defective in a number of respects, some of which will be mentioned later, is the nearest to a quantitative approach that has so far been found possible. Suppose that a container has in it a number of particles moving about in random directions. The number of particles per milliliter is n and their mean speed is C. If now a sphere of radius a is introduced into the container, the number of collisions 2,sustained by the sphere per second, is

Z=?ra2n?

(1)

Translating this result into terms of sea urchin eggs in suspensions of spermatozoa, the radius of a sea urchin egg (Psammechinus miliaris) is 50 microns, the mean speed of a sperm suspension is of the order of 200 microns per second at 18”C, for dilute suspensions, while the density n of the sperm suspension is under the control of the experimenter. If sea urchin semen (formerly called “dry sperm”) is diluted with sea water so that the concentration of spermatozoa in an egg suspension is lo6, lo6, 257

258

LORD ROTHSCHILD

or lo7 per milliliter, the number of collisions an egg will sustain per second is 0.16, 1.6, or 16. 11. THE BLOCKTO POLYSPERMY Although suggestions have on several occasions been made that the block to polyspermy is propagated over the egg surface at a high speed, in a fraction of a second, and is similar to an action potential in stimulated muscle, nerve, or plant cells, no experimental evidence has been adduced in support of this suggestion while some evidence exists in the contrary sense (Rothschild and Swann, 1949). The fastest post-insemination change which has so far been observed takes about 20 seconds to be progagated over the surface of the egg of P. miliaris at 18°C. This change in surface structure is most easily observed and its time course measured with dark ground illumination, though it is possible to see it with normal illumination or polarized light. Under dark ground illumination, the cortex of the egg scatters more light after this change has passed over the egg surface than before; the time relationships of this change in cortical structure are not inconsistent with the hypothesis that it is caused by the diffusion of a substance, mol. wt. + 10,0-20,000, derived directly or indirectly from the surface of the adhering sperm head, through the cytoplasm, the cortex of the egg being affected by this substance from the inside (Rothschild, 1949). I t can be shown that during the passage of this change only about half the number of spermatozoa colliding with the egg will in fact hit unaffected parts of the egg surface. This means that at sperm densities of 106, lo8, or 10’ per milliliter, an egg will sustain 1.6, 16, or 160 potential polysperniy-producing collisions during the passage of this cortical change. At a sperm density of lo8 per milliliter, which corresponds to an initial semen dilution of about 1 in 25,000, the incidence of polyspermy is very low, though not perhaps quite so low as the casual observer, who does not do counts, may imagine. Assuming for the moment that this “kinetic theory” of fertilization is not too wide of the mark, it is obvious either that the time relationships of the block to polyspermy are not similar to those of the 20-second cortical change, or that the probability of a sperm-egg collision being successful, in the sense that fertilization follows, is low. There are therefore four possibilities regarding the block to polyspermy and sperm-egg collisions : (1) that there is a high-speed block to polyspermy (a fraction of a second) and a high probability of a successful collision; (2) that there is a high-speed block to polyspermy and a low probability of a successful collision; (3) that there is a lowspeed block to polyspermy (10-20 seconds) and a high probability of a successful collision ; (4) that there is a low-speed block to polyspermy

BEHAVIOR O F SPERMATOZOA I N THE NEIGHBORHOOD O F EGGS

259

and a low probability of a successful collision. W e can dismiss the third alternative at once as, if this were correct, eggs inseminated with sperm suspensions of density lo7 per milliliter would on the average be more than lOO-spermic, which is not the case. W e can dispose of the first and second possibilities by means of the following experiment, which was carried out in collaboration with Dr. M. M. Swann. Spermatozoa were added to a vessel containing unfertilized sea urchin eggs so that the final concentration of the sperm suspension was 3 x lo6 per milliliter. (The concentration of eggs was less than one hundredth of this figure.) After the eggs had been in contact with this sperm suspension for 25 'seconds, the spermatozoa were suddenly killed but the eggs allowed to continue developing. Counts of monospermic, unfertilized and polyspermic eggs were made at the first cleavage stage. The results were: monospermic eggs, 85 per cent; unfertilized eggs, 13 per cent; polyspermic eggs, 2 per cent. In a second vessel exactly the same procedure was carried out, except that instead of killing the spermatozoa after they had bombarded the eggs for 25 seconds, more spermatozoa were added, bringing the final sperm concentration up to 3 X lo8. Counts at the first cleavage in this experiment showed the following percentages : monospermic eggs, 54 per cent; unfertilized eggs, 2 per cent; polyspermic eggs, 44 per cent. Now there are more than three times as many polyspermic eggs in the second experiment as there were unfertilized eggs in the first experiment. rn other words nearly half of the eggs which were fertilized in 25 seconds had not finished propagating their block to polyspermy in that time and became polyspermic because of the new sperm bombardment they received after the 25-second period was terminated. It may therefore be concluded that the block to polyspermy is relatively slow, of the order of 20 seconds, and that the low incidence of polyspermy is due in part to a low probability of a successful sperm-egg collision. This type of experiment, the novel feature of which centers round subjecting eggs to a known number of sperm-egg collisions, may enable the probability of a successful collision to be estimated, possibly both for heterologous and homologous fertilizations. Calculations however depend on the validity of the assumption that a suspension of spermatozoa can be treated analytically as if it were an assemblage of gas molecules. Spermatozoa undoubtedly do not collide with each other or with eggs elastically, but these factors are unlikely to interfere seriously with the analysis.

260

LORD ROTHSCHILD

.m...m... FIG.1. a. Movements of bracken spermatozoa in tap water. The circles indicate

where each track begins. Numbers at the beginning and end of each track refer respectively to time of start and duration of track in seconds. b. Movements of

111. CHEMOTAXIS OF SPERMATOZOA

The most serious objection concerns the possibility that a substance called Fertilizin or Gynogamone I, which attracts spermatozoa chemotactically, diffuses out of eggs of the same species. If this were so, the “kinetic” analysis, which assumes random sperm movement, might be seriously in error. The claim that chemotaxis occurs has been made for many years, though it is noteworthy that in her article on Fertilization in the 1946 Edition of the Encyclopaedia Britannica, Dr. Ethel Browne Harvey states (p. 189) that “The meeting of the egg and sperm is generally believed in animals to be by chance, not by chemical attraction.” Most workers, with the exception of Loeb (1914), have assumed that a substance which makes spermatozoa swim more quickly will act as an attractive agent. The argument runs rather like this: if a spermatozoon happens to be swimming in the direction of increasing concentration of

BEHAVIOR OF SPERMATOZOA IN T H E NEIGHBORHOOD OF EGGS

261

J

bracken spermatozoa after insertion of pipette (diameter 30 microns), containing 1 per cent sodium L-malate in agar-tap water gel, into the same sperm suspension as in Fig. la. Numbers at the beginning of tracks indicate time in seconds after insertion of pipette.

the activating substance, it will swim more quickly and therefore get nearer the source of the substance. If a spermatozoon happens to be swininiing in the direction of descending concentration of Fertilizin, it will swim more slowly and therefore get less far from the Fertilizin source. This argument is fallacious as can be seen from the following rather childish example. Suppose that in a drop of water containing spermatozoa we suddenly create an area which is lethal to spermatozoa. For the purposes of the argument it is assumed that the poisonous substance does not diffuse out of the lethal area. At the beginning of the experiment the spermatozoa, swimming in random directions, are uniformly distributed in the drop of water. Whenever a spermatozoon enters the lethal region by chance, it is killed, that is to say its movement is slowed up a great deal, and it stays there. In due course, therefore, nearly all the spermatozoa accumulate in a region where they swim more slowly:

262

LORD ROTHSCHILD

conversely, they will in these circumstances be repelled from a region where they are stimulated to swim more quickly. One implication of this argument is that it might be better from the egg’s point of view to make spermatozoa swim more slowly, rather than more quickly. Although many workers have found that egg secretions increase the O2 uptake of sea urchin spermatozoa, Hayashi (1946) found exactly the reverse effect, that the addition of egg secretions decreased oxygen consumption. Dr. E. S. Guzman Barr6n (1949, p. 45) says that Hayashi’s “techniques of measurement of 0 2 uptake and of dilution were faulty,” but I ought to mention that I have confirmed Hayashi’s results this year, using the standard Warburg technique. A slowing-up action of Fertilizin, if it occurred, would hardly be a sensitive enough mechanism to account for the alleged cases of chemotaxis. An alternative mechanism, which contains the necessary ingredients to account for the variability of the phenomenon, i.e., some people have observed chemotaxis toward egg water while others have not, was suggested to me by some ,experiments of Vasseur (1950) on the effect of egg jelly and calcium ions on the 0 2 uptake of sea urchin spermatozoa. When, for example, calcium is suddenly added to a suspension, there is a sudden increase in 0 2 uptake followed by an exponential decline to about the original rate. If the 0 2 uptake of sea urchin spermatozoa is a reflection of their speeds of movement, this type of response,. with an exponential adaptation to a basal speed-Orthokinesis with Adaptation-may cause the spermatozoa to move up a gradient of the stimulating substance, while the intensity of the effect will depend, inter alia, on the rate of decline in response. If, for example, adaptation to the stimulus is very slow, the mechanism will be very feeble. Before investigating the alleged chemotaxis of sea urchin spermatozoa toward Fertilizin, I thought it advisable to examine the phenomenon in a case where it is known to occur, in fern spermatozoa. Pfeffer (1884) showed qualitatively that fern spermatozoa are positively chemotactic towards L-malic acid. This phenomenon has now been examined quantitatively (Fig. 1). I have noted that they are also attracted by D-malic acid and nialeic acid, but not by fumarate, or several of the other participants or near participants in the tricarboxylic acid cycle, such as succinate, acetate, oxalacetate, pyruvate, or lactate. None of these substances, including D- and L-malate and maleate, appear to have any effect on the velocities of fern spermatozoa with the possible exception of lactate, which may have a slight inhibitory action. Dihydroxymaleic acid, which is directly oxidized by peroxidase is, on the other hand, toxic ; but it is not a chemotactic agent. No such behavior is observed when sea urchin spermatozoa are subjected to a gradient of egg secretions.

BEHAVIOR OF SPERMATOZOA I N THE NEIGHBORHOOD OF EGGS

263

IV. CONCLUSION From what has been said, it seems doubtful whether chemotaxis, at any rate in the case of sea urchin gametes, is likely to make the “kinetic” approach to fertilization seriously in error. There remains the jelly round the egg in those cases where it has not been removed. This is a subject which we have been examining in recent months. The results have not yet been analyzed. V. REFERENCES Barrbn, E. S. G., Gasvoda, B., and Flood, V. (1949) Biol. Bull. Woods Hole, 97, 44. Harvey, E. B. (1946) Encyclobaedia Britannica, 9, 188. Hayashi, T. (1946) Biol. Bull. Woods Hole, 90, 177. Loeb, J. (1914) J. ex#. Zool., 17, 123. Pfeffer, W. (1884) Unter Bot. Inst. Tiibingen, 1, 363. Rothschild, Lord (1949) J. exp. Biol., 20, 177. Rothschild, Lord, and Swann, M. M. (1949) J . exp. Biol., 20, 164. Vasseur, E. (1950) Ark. Kcmi. Min. Geol., 1, 393.

This Page Intentionally Left Blank

The Cytology of Mammalian Epidermis and Sebaceous Glands WILLIAM MONTAGNA Departmerit of Biology, Brown University, Providence, Rhode Islarid CONTENTS

I. Introduction . . . . .. . .. . . . .. . . . . ... . . . .. . . . . . . . . . . ...... ....... . ... .. .. 11. The Epidermis .... . . ... .. . .. . . . . . .. ... .... ... .......... . ... . . .... . . . . 1. General Description . . . . . . . . . . . . . . . . . . . . . . . . ............. 2. Intercellular Bridges and Tonofibrils . . . . . . . . . . . . . . . . . . . . . 3. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Golgi Element . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . 5. Lipids . . . . . . . . . . . . . . . . . . . . . ....................................... 6. Keratinization . 7. Sulfhydryl Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Basophilia . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Glycogen ...................................................... .. . . . .. 11. Phosphatase and Lipase . . . . . . . . . ................................ 12. Mineral Substances . . . . . . ................................ 13. Pigment . .. . . . . . . . 14. Mitotic Activity . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. The Sebaceous Glands ... . . .... . . . . . . . . . .. . . . . . . ...

4. Lipids

. .. . . . . . . .

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

Page 265 266 266 268 270 272 274 275 278 280 280 281 282 283 284 287

291

. . . . . . . . . 296

.. . . . .. . . . . . . . . . . . . . . . . . . . 297 8. Peroxidase ................. 9. Phosphatases and Lipases . . . ...................... 10. Growth and Proliferation . ...... . .... . . ... . . . . . .. . .. ... . ... . . .. 298 ........ .. . ... ,....................... . . . ... 299 IV. References . . . . . . . . . .

I. INTRODUCTION During recent years, research in our laboratory has been directed toward mammalian skin. Attention was at first focused on the sebaceous glands, but it soon became evident that neither sebaceous glands nor other cutaneous appendages could be studied alone. It was found, for instance, that when the sebaceous glands of the mouse are eradicated with one percutaneous application of methylcholanthrene in benzene, they regenerate from the cells of the external root-sheath of hair follicles only if these

266

WILLIAM MONTAGNA

follicles contain actively growing hair bulbs (Montagna and Chase, 1950). In addition, the epidermis of mice is much more resistant to injury by x-ray and/or methylcholanthrene, when the skin contains growing hairs, than when the hairs are in the club, or resting stage (Chase and Montagna, 1951). These findings and others draw attention to the skin as an integrated functional system. Although the literature which deals with the cytology, chemistry, and physiology of skin and cutaneous appendages is voluminous, there is a lack of harmony among the different findings. The study of the cytology of skin has been burdened by excessive nomenclature, disharmony, and hasty conclusions. This review considers only the cytology of the epidermis and of the sebaceous glands. The cytology of sudoriparous glands, of hair follicles (which deserve a thorough review in their own right) and of the dermis will be omitted. 11. THEEPIDERMIS

1. General Description Mammalian epidermis is composed of two principal layers : an inner stratum germinativum, which rests upon the dermis, and a horny superficial layer, the stratum corneum. The stratum germinativum has been called the mucous layer, stratum Malpighii, rete or mucus Malpigliii, rete mucosum, etc., by various authors. I n agreement with Cowdry (1932) and Hoepke (1927), stratum germinativum is to be preferred, since it is the germinal layer of the epidermis. It is composed of a lowermost layer of cells above the dermis, the stratum basale (stratum cylindricum of Kolliker), or basal layer, and a layer of variable thickness above the latter, the stratum spinosum, or spinous layer. The upper cells of the spinous layer show a progressive accumulation of granules, readily stainable with ordinary histological methods ; these cells form a layer called the stratum granulosum or granular layer. In the epidermis of the palms and soles, there is a hyalin layer above the stratum granulosum ; this layer is seldom colored by histological stains and was named stratum lucidum by Oehl (1857, cited from Martinotti, 1924). In the thinner epidermis of the general body surface, the stratum lucidum is seldom present. The outer cornified layer of the epidermis is composed of dead, flattened cells which when dissociated, resemble squamae (Kolliker, 1853). In the “pressure areas” of the skin, palms, and soles, the stratum corneum is very thick. The thickness of the epidermis is variable in different mammals and in different parts of the body of the same animals. In man it is relatively

CYTOLOGY OF MAMMALI AN EPIDERMIS

267

thick. Among the laboratory mammals, the normal epidermis of the mouse and the hamster is very thin and consists of a stratum germinativum two or three cells in thickness ; the spinous and granular layers are not distinct, and the stratum mrneum varies in thickness from 5 to 10 layers of cells. In the rat and guinea pig the epidermis is thicker and one can distinguish well-defined basal, spinous, and granular layers in the stratum germinativum. In the epidermis of the paws and digital pads of the cat, rat, guinea pig, and others, all the layers described, including the stratum lucidum, are present. In human epidermis, the cells in the basal layer are usually cuboidal or fusiform, although they may be columnar. Those of the stratum spinosum are polyhedral and become increasingly flattened as they ascend to the stratum granulosum (Schafer, 1912), where the cells are elongated horizontally. In the stratum lucidum and corneum the cells are flattened squamae. In histological sections the dermoepidermal junction of human skin appears as an undulating line. Epidermal cones and ridges (rete pegs) project into the dermis, enclosing between them highly vascular “dermal papillae.’’ When the epidermis is separated from the dermis, it becomes apparent that the epidermal cones and ridges seen in sections are in reality a series of branching ridges. The architecture of these epidermal ridges is more complex in skin areas which constitute “pressure areas,” palms and soles, than in the skin elsewhere in the body (Hoepke, 1927 ; Odland, 1950). In the normal skin of most laboratory mammals, such as the mouse, rat, and rabbit, epidermal ridges are usually lacking, and the dermoepidermal junction is fairly straight. The cells of the basal layer in human epidermis send into the dermis a number of delicate protoplasmic processes which provide a close union between the dermis and the epidermis (Schafer, 1912). Favre (1950) demonstrated in human skin that the spiral filaments of Herxheimer (vide infro) in the basal cells form radicles which project into the surface of the corium. The union between the basal cells and the corium is very intimate, and Martinotti ( 1914a,b), believed that delicate collagenic fibers from the corium are insinuated between the cells of the basal layer, and that the condensation of these fibrils forms a basement membrane. This concept, as we shall see below, is not correct. The cells of the basal layer are separated from the corium by a poorly defined basement membrane (Cowdry, 1932). Studies on the nature of the basement membrane by Herxheimer ( 1916), Laguesse ( 1919a,b), Born (1921), Frieboes (1920, 1921, 1922), Busacca (1922), Hoepke

268

WILLIAM MONTAGNA

(1924), Martinotti (1924), and many others are not in agreement with one another. Hefxheimer (1916) believed that a translucent basement membrane is intimately associated with the protoplasmic processes of the basal cells which project into the dermis. Frieboes (1920, 1921, 1922) demonstrated that the basement membrane is actually a complex argyrophilic reticulum whose meshes contain the cytoplasmic processes of the basal epidermal cells. Studying this problem, Dick (1947) concluded that it is difficult to determine whether reticular fibers form an open meshwork with interstices into which the cytoplasmic processes of the basal cells fit, or whether the meshwork forms a series of fibrils which run up to the cells and fix them either by entering them, or by filling the spaces between their basal processes. Odland (1950), in an excellent reinvestigation of this problem, has demonstrated that the argyrophilic reticulum “forms a continuous meshwork of delicate fibrils. Where the meshwork is penetrated by basal epithelial processes, the constituent reticular fibrils are compacted to form a network of coarse strands compressed between adjacent cell processes.” Odland also observed that the dermo-epidermal junction is morphologically adapted to “variable shearing forces to which the skin is exposed.” In palms and soles, this adaptation is “reflected by the relatively long basal epidermal processes as well as by the extensive development of the reticular net between the cell processes.” Moreover, in such areas, “epidermal ridges and cones attain a greater depth.” In contrast, in the skin of thighs or abdomen, the epidermal ridges are shallower and the union of the epidermis with the dermal reticulum is less distinct. 2. Intercellular Bridges and Tonofibrils Schultze ( 1864) first described the characteristic cytoplasmic processes which appear to connect the cells in the stratum germinativum. These intercellular bridges give dissociated epidermal cells the appearance of small burrs, and for this reason they have been called “prickle cells” or %pinous cells.” Chambers and de RCnyi (1925) believe they have deinonstrated that the intercellular bridges are real protoplasmic exteiisions which connect adjacent cells, since in human epidermis the effect of injury to a single cell is quickly transmitted to other cells connected to it by protoplasmic bridges. In human skin intercellular bridges are best defined in the stratum spinosum, and they gradually become obliterated in the stratum granulosum (Shapiro, 1924). Roughly midway between two cells, each protoplasmic bridge possesses a spindle-shaped swelling, the node of Bizzozero (Bizzozero, 1871). Studies in other animals have revealed well-defined intercellular bridges and nodes of Bizzozero, particu-

CYTOLOGY O F M A M M A L I A N EPIDERMIS

269

larly in the lip of the horse and ox, and in the epidermal lamina in the hooves of ungulates. Although the normal epidermis of the mouse does not contain visible intercellular bridges and nodes, these become visible in the skin of mice with avitaminotic dermatosis (Montagna, 195Oa), or in skin treated with such irritants as podophyllin and methylcholanthrene (Paletta et al , 1941 ; King, 1949). Epidermal cells contain in their cytoplasm delicate fibrils (first described by Ranvier, 1879) which appear to sweep from cell to cell by way of the intercellular bridges (see Shapiro’s, 1924, review of the literature). These fibrils, which in fresh or formalin-fixed frozen sections are anisotropic (Schmidt, 1937; Litvac, 1939), and which in paraffin sections are stainable with Heidenhain’s hematoxylin or with Mallory’s acid phosphotungstic hematoxylin, are the so-called tonofibrils of the epidermis. In human epidermis, tonofibrils are most distinct in the cells of the stratum germinativum ; they are lesI: clear in the stratum granulosum, and appear to be absent from the stratum corneum. The older literature which deals with these elements is reviewed by Weidenreich (1900), Rosenstadt (1910), Shapiro (1924), and Patzelt (1926). There is confusion concerning the nature and origin of tonofibrils, and much of it has stemmed from the discovery by Herxheimer (1889) of thick, undulating or spiral filaments in the cells of the stratum basale. Branca (1899) and Argaud (1914) considered the spiral filaments of Herxheimer to be intracellular fibrils. Favre and Regaud (1910a,b) and Regaud and Favre (1912), on the other hand, have stated that these structures are actually mitochondria of the basal cells, and suggested that the tonofibrils might develop from them ; Favre (1950) now denies that tonofibrils are related to the spiral filaments. Firket (1911 ) objected to such an interpretation and assumed that since the basal cells contain Herxheimer filaments as well as mitochondria, the spiral filaments must be young tonofibrils just formed from mitochondria. A different concept of the origin of tonofibrils was presented by Martinotti (1914a,b) who believed that they arise from a coalescence of cytoplasmic granules, and that they multiply by splitting longitudinally. Neither Martinotti nor other authors (Branca, 1899 ; Firket, 191l ) , who also considered that a multiplication of tonofibrils occurs by longitudinal splitting, actually observed this process. Studying the effect of podophyllin and methylcholanthrene on the skin of mice, King (1949) found that tonofibrils, which are scanty in the normal animal, undergo rapid hypertrophy and proliferation in skin rendered hypertrophied by these agents. Furthermore, tonofibrils are produced in spite of severe morphologic disturbances in the cytoplasm and nucleus of

270

WILLIAM MONTAGNA

the epidermal cells. King suggested that tonofibrils are intimately associated with the process of keratinization, a concept shared by many authors (vide infra) . In summary, tonofibrils are structures which seem to extend from cell to cell by way of the intercellular bridges. Their origin, exact nature, morphology, and significance are unclear, although they seem to be associated with keratinization. Although often confused with tonofibrils, the spiral filaments of Herxheimer, as will be shown later, appear to be mitochondria. 3. Mitochowidria Before discussing the mitochondria in epidermal cells one must agree with Cowdry (1918) that “the terminology of mitochondria is unnecessarily complicated and confusing. The confusion has resulted from incoordination and from hasty individual action in elaborating new names, often only to discard them in a new paper in favor of some other.” In a discussion of mitochondria and their shapes it is profitable to recall the observations of Lewis and Lewis (1915) in living tissue culture cells “that every type of mitochondria is continually changing shape and may assume as many as fifteen or twenty shapes in ten minutes.” In this review the term mitochondria will be used to designate those intracytoplasmic elements which are revealed in fresh tissues by supravital staining with Janus green, and in paraffin sections of postchromed tissues stained with Regaud’s or Heidenhain’s hematoxylin. As implied above, confusion has arisen from the existence of recognizable mitochondria, spiral filaments of Herxheimer, and tonofibrils in the same epidermal cell. In the cells of the stratum basale of human epidermis, Herxheimer (1889) described thick, spiral filaments which he first considered to be intracellular lymphatic spaces. Later, however, he interpreted them as shrunken parts of the cell membrane resulting from fixation artifact (Herxheimer and Miiller, 18%) ; this concept was shared by Schultze ( 1896). Most subsequent authors, among them Kromayer ( 1892), Rabl (1897), Firket ( 1911), and Kollmann and Papin ( 1914), concluded that the filaments of Herxheimer are the forerunners of epidermal fibrils. Favre and Regaud (1910a,b and Regaud and Favre, 191’2) demonstrated that the filaments in the basal layer of human epidermis are revealed by Regaud‘s method for mitochondria and that Herxheimer filaments are coexistent with typical mitochondria. By lengthening the period of postchromation, Favre (1920a,b; 1924) demonstrated spiral filaments in the cells of the spinous layer as well as in the basal layer. In mitochondria preparations, Favre found that the nodes of

CYTOLOGY OF MARIMALIAN EPIDERMIS

27 1

Bizzozero are stained selectively together with mitochondria and spiral filaments, and concluded that the nodes of Bizzozero are a part of the mitochondria1 complex. This point he reemphasized in two recent papers (Favre, 1946, 1950). From a lifelong study of mitochondria in human epidermis, Favre reveals several significant facts. H e has convincingly demonstrated that Herxheimer spiral filaments are actually mitochondria, although not all mitochondria are in this form. While postchromation of skin for one month is sufficient to demonstrate the spiral filaments in the stratum basale, longer postchroming is necessary to reveal them in the upper strata. In palmar and plantar epidermis, the spiral filaments are larger and more numerous and show less polymorphism than in the general body skin. In both types of epidermis they are aligned parallel to the long axis of the cells. In cells of the basal layer, the spiral filaments form a bed of radicles which extends into the dermis by way of the basal processes. Spiral filaments are found throughout the stratum germinativum, but in the upper strata they become thicker and less numerous. In the stratum granulosum they show a progressive fragmentation, but even in the superficial cells of this layer, recognizable fragments are scattered among the keratohyalin granules. From these observations the author concluded that mitochondria are directly concerned with the process of keratinization, and that since this is so, a process of insensible keratinization begins in the basal layer. Mitochondria are very sensitive indices of cellular change and damage. In psoriasis, and in other inflammatory conditions, for example, the nuclei of the epidermal cells in the stratum germinativum are displaced distally and the fragmented and polymorphic mitochondria become subnuclear. In epidermal neoplasms, mitochondria become strikingly polymorphic, and the nodes of Bizzozero disappear. Favre ( 1950) illustrates basal-cell carcinoma in which the mitochondria resemble those of normal spinous cells while in squamous-cell carcinoma they resemble those in normal basal cells. Parat ( 1928) cautions that mitochondria1 preparations often give deceiving and unreliable results. In the skin of newborn rats, stained supravitally with Janus green, Parat found mitochondria whose appearance and distribution correspond to the spiral filaments described by Favre. Even in the stratum granulosum he found numerous flexuous mitochondria and batonettes. Fixed and stained preparations gave results which were comparable to those stained supravitally. Parat’s studies with supravitally stained material give greater credence to Favre’s belief that the spiral filaments are mitochondria.

272

WILLIAM MONTAGNA

In summary, mitochondria are large and numerous in the basal layers of the stratum germinativuni ; in the stratum granulosum they become fragmented and dispersed. They are polymorphic, appearing either in the form of spiral filaments or batonettes. The work of Favre and Regaud gives abundant evidence that Herxhejmer filaments are mitochondria and that the nodes of Bizzozero contain mitochondria1 elements.

4 . Golgi Element Concerning the study of the Golgi apparatus in epidermis, the papers of Deineka (1912) , Cajal (1915), DaFano (1921), Tello (1923a,b), Ludford (1924, 1925), Cowdry and Scott (1928), and Parat (1928) are instructive. This puzzling organelle has been demonstrated almost exclusively with silver or osmium tetroxide impregnation methods. Cowdry and Scott (1928) and Parat (1928) appear to be the only ones who have stained the epidermis supravitally with neutral red for the study of the Golgi element. Ludford (1925) demonstrated the Golgi element in the cells of the epidermis of mice under normal and pathologic conditions with osmium tetroxide. H e described the Golgi apparatus in cells of the basal layer as a juxta-nuclear network or group of rodlets in the distal end of the cell (the mitochondria being heaped up at the proximal end). This precise polarity is lost in the upper cells of the spinous layer where the Golgi element is dispersed irregularly as are also the mitochondria. Golgi elements become fragmented and scattered at the onset of keratinizalion, and in cells laden with keratohyalin granules, they are no longer demonstrable with osmium tetroxide. Parat ( 1928), studying fresh epidermis of the newborn rat, stained supravitally with neutral red, found in the cells of the basal layer a supranuclear, compact mass of neutral red vacuoles which often descends along the sides of the nucleus (this is the “vacuome” of Parat). In the cells immediately above the basal layer, the system of neutral red stained vacuoles is as dense as in the basal cells; in cells nearer the stratum granulosum, the mass of vacuoles tends to become subnuclear and somewhat dispersed, but it remains always in the proximity of the nucleus. The mitochondria do not have such intimacy with the nucleus. Although the smaller vacuoles readily become colored with neutral red, the larger ones do so less easily. In the stratum granulosum, the masses of vacuoles become dispersed among the keratohyalin granules, and oftentimes keratohyalin granules are encircled by neutral red stained material. In the stratum granulosum, the larger vacuoles are located basally and along the sides of the nucleus, while the smaller ones are found apically. With continued keratinization the “vacuome”

CYTOLOGY OF MAMMALIAN EPIDERMIS

273

becomes more “rarefied” and disappears. This is in contrast with supravitally stained flexuous mitochondria1 filaments which are still present among the keratohyalin granules. Parat’s silver impregnation preparations show in the lower cells of the stratum germinativum a classical Golgi element which he described as a mass of compact rodlets and tubules. These structures, which comprise the “dictyosome,” have the same orientation as the “vacuome” ; ascending toward the surface cells they become dispersed (as also described ’by Cajal, 1915, Tello, 1923a,b, and Ludford, 1925). I n the stratum granulosum, scattered Golgi elements appear as “appendages” of the keratohyalin granules. These observations strengthen Parat’s belief that the Golgi element (vacuome or dictyosome) is involved in the formation of keratohyalin granules. Although it is not possible to determine from his work whether or not keratohyalin granules arise from the Golgi element, Parat has made an important contribution to the understanding of the nature of this organelle. He was the first cytologist to look for a Golgi net in living cells, and found instead a series of vacuoles. The subsequent work of Hirsch (1939), Worley (1944 ; see also Worley’s excellent review, 1946), Baker (1944, 1949), Cain (1947, 1949), Thomas (1948), and Palade and Claude (1949a,b) on the Golgi element in other cells has substantiated Parat’s observations and added to them. The classical Golgi net is an artifact which develops at the site of a system of spherules. One fact brought out especially by the school of Baker, and by Palade and Claude (1949a,b), is that Golgi vesicles are lipoidal in nature. Parat (1928) using Sudan I11 and Scharlach R found lipid droplets in the basal layer of the epidermis of the newborn rat, but riot in the cells of the more superficial layers. These granules, however, did not correspond to the “vacuome”; on the contrary, they seemed to coincide with granules which were supravitally stained with the “nadi” reagent (Parat interprets granules stained by the “nadi” reagent as lipids; this is an odd interpretation since according to Keilin, 1925, they should represent sites of cytochrome oxidase) . Nicolau ( 1911) demonstrated lipid granules in the basal layers of human epidermis, and Kollmann and Papin (1914) demonstrated osmiophobic but sudanophilic granules in the basal cells in the lining of the esophagus of the guinea pig. None of these authors, however, observed whether these granules correspond to the Golgi element. Montagna (1950b) studied the distribution of lipids in the epidermis of several laboratory mammals by coloring frozen sections with Sudan black. H e observed in the cells of the stratum germinativum a series of perinuclear lipid spherules which lie close to the nuclear membrane and usually cluster at the distal pole of

274

WILLIAM MONTAGNA

the nucleus. The cells of the stratum granulosuni contain only barely visible lipid granules. In the epidermal cells of the monkey, phospholipid granules, demonstrated with Baker’s (1946) acid hematein test, are identical with the perinuclear sudanophil bodies. Cain ( 1949) demonstrated comparable phospholipid bodies in the skin of the guinea pig, an animal not studied by Montagna. In preparations impregnated with silver or with osmium tetroxide, rodlets or granules appear in the same general areas in the cells occupied by the lipid granules. The perinuclear lipid granules in the epidermal cells, then, are comparable to the “vacuome” of Parat ( 19289, and must, but not without caution, be considered either as the Golgi complex or a part of it. Since the name “Golgi apparatus” has come to denote a variety of things to cytologists, Baker (1950) proposed the name “lipochondria” for these lipoidal spherules which are stainable supravitally with neutral red and which apparently correspond to Parat’s “vacuome.” 5. Lipids In addition to the perinuclear lipids described above, the epidermis often shows lipid granules in the intercellular spaces. One is cautioned against too literal an interpretation of these intercellular lipids. In poorly fixed tissues, or in tissues kept in the fixative longer than one month, the number of intercellular lipid droplets increases pari passu with the decrease of intracellular lipids. The nodes of Bizzozero in frozen sections treated with Sudan black also show some lipoidal content. In some unpublished observations on the skin of mice simultaneously treated with methylcholanthrene and x-radiated with lo00 r, we find that four days after treatment, the perinuclear lipid bodies discussed above (Montagna, 1950a,b) become fragmented and diffuse. Furthermore, numerous lipid particles are found in the intercellular spaces, a situation which is not encountered in the normal skin of the mouse. On the other hand, from seven to twelve days after treatment most of the epidermal cells, which have become tremendously hypertrophied, contain so much lipid that they resemble sebaceous cells. These cells resemble those in the epidermis of mice treated with tar, described by Ludford (1925). Lipids in the stratum corneum were first described by Ranvier (1898). These lipids must come from three sources: (1) intrinsically from lipophanerosis in epidermal cells ; (2) from the secretion of sebaceous glands ; and (3) from the secretion of sweat (Levin e t d.,1940, and Mickelsen and Keys, 1943, have demonstrated lipids in sweat ; Hoepke, 1927, Bunting et al. 1948, and Bunting, 1948, have demonstrated lipids in the secretory cells of sweat glands). Unna and Golodetz (1909) have shown in human

CYTOLOGY OF MAMMALIAN EPIDERMIS

275

skin that the cells of the stratum spinosum contain a relative abundance of free cholesterol but a paucity of cholesterol esters. The stratum corneum, on the other hand, contains approximately equal amounts of free and ester cholesterol. These authors state that while the cholesterol secreted by sebaceous and sweat glands is easily oxidized, the intracellular cholesterol of the epidermis is not. Kvorning (1949), in an analysis of lipids secreted upon the face of normal subjects, finds only small amounts of cholesterol. The stratum corneum in skin areas which contain no sebaceous glands is also sudanophilic, and the lipids must be derived from lipophanerosis and from sweat, if sweat glands are present. I n the skin of biotin-deficient mice where the sebaceous glands are plugged (Montagna, 1950a), the stratum corneum is sudanophilic, as is also the case in the skin of mice where sebaceous glands have been completely eliminated by local applications of methylcholanthrene (Montagna and Chase, 1950). Since mice have no other skin glands, these lipids must come from the unmasking of bound lipids in the epidermal cells. However, the sudanophilia of the stratum corneum in skin deprived of sebaceous secretion is not homogeneous as in the skin of normal animals, but appears granular. When frozen sections of skin of man (Montagna et al., 1948), or hamster (Montagna and Hamilton, 1949), or of all the other mammals studied are treated with Nile blue sulfate, the stratum corneum is colored pink, indicating perhaps the presence of neutral lipids. The stratum corneum is always Schultz-positive (indicating the presence of cholesterol or its esters), it is colored with Baker’s acid hematein test for phospholipids, and it is brilliantly birefringent (after extraction with organic solvents the intense birefringence is partially lost, the residual birefringence being a property of keratin). I n summary, the demonstrable lipids in the stratum germinativum are principally the perinuclear sudanophil bodies. The nodes of Bizzozero are mildly sudanophilic. The stratum corneum contains histologically demonstrable cholesterol esters, and possibly also neutral fats and phospholipids.

6. Keratinization The process of keratinization in epidermis has interested cytologists since the early descriptions by Langerhans (1873) and by Ranvier ( 1879). In human epidermis Ranvier recognized a “keratogenic” layer which Unna later called the stratum granulosum. Ranvier called the granules of the stratum corneum “dlkidine en graine” and the content of the cells of the stratum lucidum “ilkidine. en flaques.” Waldeyer (1882) called the granules in the stratum granulosum “keratohyalin” granules. In the

276

WILLIAM MONTAGNA

stratum granulosum, keratohyalin granules are usually aggregated at the poles of the nucleus. The granules may be stained with most basic dyes as well as with acid dyes such as Congo red, acid fuchsin, and “wasserblau.” Several morphologically detectable changes in the cells of the stratum granulosum accompany the formation of keratohyalin granules. There is a decrease in the number of mitochondria and Golgi elements; an apparent decrease and fragmentation of tonofibrils ; an apparent extrusion of nucleolar material ; and an increase in the cell volume. It is not known with certainty which cell component or components are responsible for the elaboration of keratohyalin granules. It is remarkable that a study which has engaged the talents of so many cytologists should have borne so few tangible results. That the keratohyalin granules develop at the expense of mitochondria is a thesis which has been pursued by Regaud and Favre since 1910. In his latest paper, Favre (1950) presented a detailed account of the metamorphosis of mitochondria in the different layers of the epidermis and concluded that since mitochondria are responsible for the elaboration of keratohyalin granules, the process of keratinization must actually begin, although imperceptibly, in the basal layer of the stratum germinativum. Parat (1928), on the other hand, implicated the “vacuome” or Golgi apparatus. Kollmann and Papin (1914) believed that keratohyalin granules represent transformed nucleolar extrusions. Ludford (1924) agreed in part with Parat and in part with Kollmann and Papin but stated that although the Golgi apparatus and the nucleolus are partially involved, the process of keratinization is essentially a function of the ground cytoplasm of the cell. Martinotti (1914a,b, 1915, 1921), in a series of studies based upon a battery of different stains applied to human skin, concluded that keratohyalin is formed from several sources : (1) epidermal fibrils by a process of “fibrillorhexis”; (2) from the ground cytoplasm, probably the basophilic granules in the cells of the stratum germinativum ; (3) from the nucleus by “karyolysis” ; and ( 4 ) from the cell membrane. Branca (1907) and Firket (1911) considered that keratinization begins upon the tonofibrils and then proceeds to the interfibrillar cellular substance and to the cell membrane. More recent literature (King, 1949) tends to support this view. Keratin can be demonstrated histochemically by the application of the Millon reagent or by the xanthoproteic test. Presumably, these tests are specific for proteins which contain tyrosine in their molecule, but actually a color reaction is given by nearly all proteins and phenolic compounds (Serra, 1946). Unna and Golodetz (1909) identified two types of keratin in epidermis :

CYTOLOGY OF MAMMALIAN EPIDERMIS

277

keratin A, which is insoluble in nitric acid and in a mixture of sulfuric acid and hydrogen peroxide, and keratin B, which is soluble in these reagents. According to Hawk et al. (1%7), the keratin molecule consists of closely packed polypeptide chains which are held together by the disulfide bond of cystine, the resistance to solvents and enzymes being associated with the close packing of the chains. The major portion of hair, horn, hoof, feather, nails, and the stratum corneum of the skin is made up of albuminoid proteins. The keratin of hair, nails, and other appendages contains from 3 to 5 per cent sulfur, while that of skin contains from 1 to 3 per cent, nearly all of which is cystine (Hawk ,et al., 1947). Keratin, according to Block and Vickery (1931), “is insoluble in dilute alkalies, in water and in organic solvents, and . . . on acid hydrolysis, yields such quantities of histidine, lysine, and arginine that the molecular ratios of these amino acids are respectively approximately as 1 :4 :12.” Wilkerson (1934) found that these amino acids are in a molecular ratio of 1 :5 :15. Wilkerson (1935) has shown further that the isoelectric points for keratin, hair, and nails are 3.70; 3.67; 3.78. He suggested that since the isoelectric points are practically the same and the basic amino acids are present in approximately the same molecular ratios, possibly the amino acids responsible for the acid groups are also present in a definite molecular ratio in these three chemically, physically, and embryologically related structures. These figures show the remarkable unity which exists among the keratins from different cutaneous appendages. If they are correct, they throw some doubt upon the conclusions of Martinotti (1914a,b, 1915, 1921) and others, who, on the basis of painstaking cytological studies concluded that there are several “different” types of keratins. It is pertinent here to mention the findings of Litvac (1939) from observations on skin cultured in vitro ; she found that in very young cultures all the epidermis is digested by pepsin and trypsin. In 10-day cultures, the young keratin is digested only by pepsin, and the mature keratin in 3-week old cultures is resistant to both enzymes. Astbury (1933) demonstrated that most mammalian keratins, in the normal state, give approximately the same x-ray diffraction diagrams, indicating a periodicity of 5.15 A, characteristic of the a form. When keratin is stretched, changing from a- to p-keratin, there is an extension of the polypeptide chains of about 100 per cent (Astbury and Woods, 1930). Alpha- and @-keratins seem to be stereoisomers corresponding to two different structures having similar molecular configurations. Derksen and Heringa (1936), studying the lip of the ox, Derksen e t d. (1938) the human nail, and Giroud and Champetier (1936) the chestnut of the

278

WILLIAM MONTAGNA

horse and the hoof of the calf, all report the same x-ray diffraction pattern of a-keratin. Derksen and Heringa, in agreement with the findings of Astbury, report that these keratins when placed in warm water give the diffraction pattern of @keratin. These authors conclude that the polypeptide skeleton possessing the periodicity of 5.15 A is preexistent in the stratum germinativum even before keratinization begins, and that the tonofilks are probably the elements involved. Champetier and Litvac (1939) studying thick sections of hoofs from embryonic calves note that the birefringence of the thick stratum corneum (produced by a-keratin, or keratin B of Unna) is destroyed in sections placed in chilled 2 per cent potassium hydroxide. The keratinized cell membranes (keratin A of Unna), on the other hand, remain intact and are isotropic. After digestion with pepsin and trypsin the tonofibrils in the stratum germinativum are destroyed, but these enzymes do not digest a-keratin in the keratinized region. This is explained by the fact that the keratinized cell membranes (keratin A of Unna), which are resistant to proteolytic enzymes, protect the enclosed a-keratin. Studies of the x-ray diffraction patterns of untreated epidermis confirm the findings of the authors named above in that diagrams corresponding to a-keratin are obtained where the tonofibrils are present, either in the non-keratinized stratum germinativum or in the keratinized upper layers. They conclude that the tonofibrils are responsible for the characteristic x-ray diffraction pattern of a-keratin. Alpha keratin under pressure, or after exposure in the autoclave, is transformed to /3 keratin. X-ray diffraction diagrams of re!atively pure (extracted with KOH) membrane keratin (keratin A of Unna) are unlike those of either a or 3, keratin, and must represent something quite different. These papers present interesting data, but whether or not the tonofibrils represent stages in keratinization or a stage in the polymerization of keratin molecules, as stated by King (1949), has not been conclusively shown.

7. Sulfhydryl Groups The presence of sulhydryl groups in the epidermis is closely related to the process of keratinization. After treatment of tissues with sodium nitroprusside, there is a moderate coloration in the stratum germinativum, a strong reaction in stratum granulosum and lucidum, and no reaction in the stratum corneum. Kaye (1924) believes that this reaction in the skin is due to glutathione. Walker (1925) denies the presence of glutathione and suggests that the nitroprusside reaction is due to a substance similar to, or identical with the thermostable sulfhydryl constituent of muscle, since pretreatment of sections of skin with alkyl isothiocyanate abolishes

CYTOLOGY OF MAMMALIAN EPIDERMIS

279

the reaction. Giroud and Bulliard (1930, 1934, 1935), on the basis of their results with nitroprusside, conclude that there are two sulfhydryl substances involved in two types of keratinization : one soluble, which is probably glutathione, involved in soft keratinization in the skin ; the other, in much larger quantities, associated with hard keratinization. Chhremont and Frederic (1943), utilizing a new technique (modification of the prussian blue reaction, whereby the -SH groups reduce ferric ferricyanide to ferric ferrocyanide to give a blue color), repeated the observations on the distribution of -SH groups in the skin of guinea pigs and of man. In the stratum germinativum there is a delicate color reaction. I n the stratum granulosum the keratohyalin granules are always intensely colored as are also the “flaques d’C1Cidine.” The stratum corneum is completely negative. These authors deny the presence of two types of keratinization proposed by Giroud and Bulliard. Frederic (1949) using the prussian blue reaction, studied the distribution of -SH groups in x-radiated skin of guinea pigs. If the injury is not too grave and the stratum granulosuin is still present, it shows no diminution of histochemically reactive -SH substances, although the reaction in the stratum germinativum is much weaker. Control skin of non-irradiated areas in the same animal also shows a diminution of the reaction in the stratum germinativum. During regeneration and repair of the injury, the epidermis shows an augmentation in the intensity of the reaction. Bennett and Yphantis (1948) have recently synthesized a reagent, 1-4 (chloromercuriphenylazo) -naphthol -2, which is apparently specific for the demonstration of sulfhydryl groups. Mescon and Flesch ( MS ) have applied this reagent to thin sections of fresh and fixed human skin and obtained results essentially similar to those revealed by the nitroprusside method. They report good staining in the stratum basale, a less intense staining in the stratum spinosum and minimal or no staining in the stratum corneum. Staining in the latter is intensified by longer exposure of sections to the reagent. The nuclei of cells of the stratum germinativum stain quite well. The staining of the stratum corneum, however slight, is interesting since these authors have shown by direct chemical methods, in agreement with Rudall (1946) and Gustavson (1949), that human horny scales contain some free sulfhydryl groups. Two possible roles might be attributed to the sulfhydryl groups in epidermis: one, that they may be concerned with cell division and proliferation ; the other, that they may undergo a transformation to disulfide substances in the stratum corneum. Hammett (1931), by the application of benzyl mercaptan, a sulfhydryl containing compound, to the skin of mice,

280

WILLIAM MONTAGNA

induced hypertrophy of the epidermis, accompanied by abundant mitotic activity and differentiation of a distinct stratum spinosum (normally not apparent in the epidermis of the mouse). H e inferred that the percutaneously applied sulfhydryl compound was responsible both for the accelerated mitotic activity and for epidermal differentiation. Admitting these facts, one must remember that both of these conditions can be induced by the application of almost any irritant. Concerning the second point, that sulfhydryl compounds may be concerned with keratinization, Litvac (1939) found that addition of cysteine to the culture media caused no acceleration of keratinization in epidermis cultured in vitvo.

8. Ascorbic Acid In conjunction with the studies on keratinization, Giroud, Leblond, and Ratsimamanga ( 1935a,b) have also studied the presence of ascorbic acid in epidermis. By the use of chemical and cytochemical methods these authors have demonstrated in the chestnut of the horse and in the hoof of the pig, as well as in the epidermis of the guinea pig, a considerable amount of ascorbic acid in the stratum germinativum, but only traces in the cornified layer. It is not without interest to find this correspondence between the distribution of this substance and that of the -SH groups in epidermis. 9. Basophilia The cells in the stratum germinativum are intensely basophilic when sections of skin are stained with basic dyes. The ground cytoplasm of the cells in the stratum granulosum is much less basophilic but the keratohyalin granules usually stain intensely. The stratum lucidum and the stratum corneum stain very weakly or not at all. Treatment of sections of skin before staining with a solution of ribonuclease buffered to pH 6.7 abolishes all the cytoplasmic basophilia (the basophilia of the nucleolus is also abolished) but the intact nuclei and the keratohyalin granules still stain clearly. This is presumptive evidence that the basophilia which is eliminated by the enzyme is due to ribonucleic acid. These observations can be extended also to the cells of the outer root-sheath of hair follicles. Dempsey et d. (1950) have shown that oxidation of sections of skin (frontal skin of the deer) with periodic acid before staining with toluidin blue, greatly enhances the basophilia normally present in epidermis. This induced basophilia does not correspond to the basophilia presumably due to ribonucleic acid. When nucleoproteins and/or acid mucopolysaccharides were destroyed by treating sections with hydrochloric acid and the sections then oxidized with periodic acid, and stained with toluidin blue, the epi-

CYTOLOGY OF M A M M A L I A N EPIDERMIS

28 1

dermis showed no diminution of dye uptake when compared with sections oxidized with periodic acid without acid hydrolysis. Sections treated only with hydrochloric acid and stained without oxidation showed complete loss of cytoplasmic basophilia, but the keratinized areas were still basophilic. Since these facts indicate that acid groups other than nucleic acids or mucopolysaccharides exist in the epidermis, the authors concluded tentatively that the newly formed basophilia is related to the sulfur content of proteins. Oxidation of disulfide in keratin and of sulfhydryl groups in the stratum germinativum might conceivably lead to the formation of sulfonic acids, responsible for the induced basophilia. There are, then, in the stratum germinativum three biologically active substances : sulfhydryl groups, ascorbic acid, and ribonucleic acid. Ascorbic acid might mediate the transformation of -SH groups in the stratum germinativum to -S-S- groups in the keratinized area. Perhaps nucleic acid plays a role in protein synthesis (Caspersson, 1947), a role which might also be played by the -SH groupings.

10. Glycogen Whereas chemical analyses of the skin (Folin et al., 1927, and Calvery et a,!., 1946) show that it contains glycogen, this substance is not generally demonstrable by cytochemical methods in the normal epidermis of man and laboratory mammals. In human epidermis, occasional cells in the upper stratum spinosum and granulosum do have some cytochemically demonstrable glycogen (Montagna et al., 1948), but most of those epidermal granules which recolor the Schiff reagent are visible even after previous treatment of sections with saliva or diastase. These substances, then, may represent polysaccharides other than glycogen (this is in striking contrast with the epithelial lining of the oral and buccal cavities, and of the vagina, whose epithelial cells are usually laden with glycogen granules). It is possible that the high glycogen values obtained in chemical analyses of skin correspond not to the glycogen of the epidermis, but rather to the glycogen stored in the cells of the outer root-sheaths of hair follicles and the glycogen in the sudoriparous glands. In the skin of all mammals studied, the cells of the outer root-sheath, up to the level of the sebaceous glands, contain variable amounts of demonstrable glycogen (in man, Lombardo, 1907; Sasakawa, 1921 ; and Montagna et al., 1948; in the rat, Johnson and Bevelander, 1946; in the rabbit and other mammals, Bolliger and McDonald, 1949). Glycogen is particularly abundant in the heavy sheaths of thick hairs (in human skin), and it is absent from the sheaths of follicles which contain resting “club” hairs.

282

WILLIAM MONTAGNA

In the skin of embryonic rats, Parat (1928) found glycogen in all the layers of the epidermis, except in the most superficial cells of the stratum corneum. In the basal cells it is present as large plaques, either infra- or supranuclear. Glycogen granules are numerous and large in the lower cells of the spinous layer; in the upper cells of this layer the granules decrease in size but they increase in number. I n the cells of the stratum granulosum, the whole cytoplasm shows diffuse glycogen content. Glycogen diminishes in the stratum lucidum and disappears in the stratum corneum. I n human embryos (Lombardo, 1907 ; Sasakawa, 1921) , during the first six months of fetal life, there is abundant glycogen in the epidermis and in all of the cutaneous derivatives. After-the sixth fetal month, glycogen diminishes in the epidermis and becomes restricted to those structures where it is found in postnatal life, i.e., in external root-sheaths of hair follicles and in the secretory epithelium of sudoriparous glands. I n an analysis of glycogen in skin under different pathologic conditions, these authors find that a stimulus, of whatever nature, inducing excessive epidermal proliferation causes a reappearance of glycogen in variable quantities. Under such conditions, there is thus an apparent reversion to the state observed during the first part of fetal life.

11. Phosphatases a d Lipme Gomori (1941) in his first report on the distribution of alkaline phosphatase in tissues, stated that with the exception of its capillaries, skin contains no demonstrable alkaline phosphatase. Bourne ( 1943) found strong phosphatase activity only in the sebaceous glands and hair follicles. Fisher and Glick (1947) described a slight amount of alkaline phosphatase activity only in the stratum granulosum of human epidermis. I n the epidermis of the mouse, Biesele and Biesele (1944) found an increase in epidermal alkaline phosphatase after treating the skin with methylcholanthrene. At best, then, there is a scant amount of alkaline phosphatase in the epidermis of all mammals studied. Unpublished observations on the distribution of alkaline phosphatase in the epidermis of the cat and rabbit show that the enzyme is present in irregularly scattered foci, usually around the pilosebaceous orifices. Thompson and Whittaker (1944), studying the skin of man and rat manometrically, have demonstrated two esterases. One is a specific active cholinesterase, the other, a non-specific aliphatic esterase. Cytochemically, neither cholinesterase nor non-specific esterases (lipase) have been demonstrated in the epidermis. Although lipase activity has been demonstrated by the method of Gomori (1946) in the sebaceous glands of man (Mon-

CYTOLOGY OF MAMMALIAN EPIDERMIS

283

tagna et d.,1948), mouse (Kung, 1949), and the hamster (Montagna and Hamilton, 1949) only small amounts were reported in the epidermis of man and hamster where it was confined to the stratum corneum. I t should be pointed out that considerable lipase (esterase) activity can be demonstrated cytochemically in the subcutaneous fat. One wonders from these scant data if the esterases described in slices of skin by Thompson and Whittaker may not correspond to the esterases revealed in the sebaceous glands and in the subcutaneous fat. 12. Mineral Substances

Epidermis normally contains minerals. These minerals have been studied both by chemical methods and by examination with dark field illumination of incinerated tissue sections. Using chemical methods, Suntzeff and Carruthers (1945) found that the average amounts of K, Na, Mg, and Ca per 100 mg. of human epidermis are 0.322, 0.122, 0.018, and 0.015 mg. respectively. MacCardle et d. (1941) studied the mineral content of human skin under normal and pathologic conditions by means of spectrophotometric analysis, expressing their values for Ca, Cu, Mg, I, P, and Zn in relative intensities of spectral lines. Relatively large amounts of Mg were found in normal skin. Study of skin by microincineration has made possible a partial understanding of the localization of these minerals. Unfortunately, specific identification of the individual elements is .not practical since nearly all of them leave a whitish ash, with the exception of iron whose ash is yellow to red. Furthermore, it is likely that after such treatment the original topographic relationship of these elements is disturbed. Since Na and K are the principal metals found in epidermis, it is reasonable to assume that the white ash residue represents both of them. Scott (1933) found in normal epidermis of man and other mammals a concentration of mineral ash in the distal-most layers of the epidermis. Cowdry and Andrew (1950) found large amounts of mineral matter in human skin, especially in the basal layer of the epidermis. The spinous cells contain visibly less ash than the basal layer, and ash is again abundant in the superficial layer of the stratum germinativum, and in the stratum corneum. The mineral ash content of the epidermis of the mouse, under normal and pathologic conditions, has been discussed in a review by Cowdry (1943). MacCardle et al, (1943) in an excellent account of mineral ash in human skin summarize their findings in the following way: “In the skin of infants under 1 year old, the stratum corneum, stratum granulosum and superficial spinous layer leave a bluish ash indicative of the presence of sodium and

284

WILLIAM MONTAGNA

potassium, while the deeper spinous, basal, and dermal layers contain much white ash of calcium and magnesium. In persons from 10 to 50 years of age all layers of the skin are heavily mineralized with white ash except the basal layer, which leaves mostly bluish ash. The basal cells, as well as the spinous cells, in the skin of persons between the ages of 10 and 50 years all possess thick perinuclear mantles of calcium and magnesium, except the ‘clear cells’, whose ash is blue and whose nuclei contain large amounts of iron. In children between infancy and 10 years of age it is only the stratum corneum and stratum granulosum that are constant in their heavy deposits of white ash. The other layers may or may not be heavily mineralized. At all ages there are single clear cells more or less ashless scattered about in the spinous and basal layers. The nuclei of these clear cells contain an unusual amount of iron.”

13. Pigwent The five pigments responsible for the color of normal skin are melanin, in the deeper layers of the epidermis; melanoid, a substance allied, $0 melanin, allegedly found throughout the epidermis ; carotene, in the stratum corneum, as well as in the subcutaneous fat; and reduced hemoglobin and oxyhemoglobin in the blood vessels of the dermis. The relative abundance of these pigments in human skin has been studied spectrophotometrically by Edwards and Duntley (1939 ; see also brief review by Jeghers, 1950). Of the five, melanin is the only one which lends itself completely to cytological studies. Melanin is a yellow to black pigment which is related in some way to the metabolism of tyrosine. It is resistant to nearly all chemical agents and is not modified even by concentrated acids. It is, however, somewhat soluble in concentrated KOH, or NaOH, and is bleached by hydrogen peroxide, potassium permanganate, and other reducing agents. Melanin is an argentaffin substance, reducing ammoniacal silver nitrate without the intervention of a reducing agent, and it is easily impregnated by all histological silver methods (Fontana, Bielchowski, del Rio-Hortega, Achucarro, etc.) (see Lison, 1936, for a diagnostic characterization of melanin and other pigments). The problems of pigment and melanogenesis in skin have been reviewed by Becker (1927, 1948), Meirowsky (1940), Masson (1948), Rawles ( 1948), and Lerner and Fitzpatrick (1950). In normal human epidermis, melanin is present in varying amounts in the basal layers of the stratum germinativum, but it may be present also throughout the stratum spinosum, and rarely, in the stratum granulosum and corneum. When abundant, melanin granules are evenly and diffusely distributed in the cytoplasm

CYTOLOGY OF M A M M A L I A N EPIDERMIS

285

of epidermal cells, especially in those of the basal layer; when less abundant, the granules form supranuclear caps. Pigmented dendritic cells occur in the epidermis couched between the basal cells, and resting against the basement membrane. These cells have been studied critically by Masson ( 1948), Billingham ( 1949) , and Zimmermann and Cornbleet (1948) in human skin, by Billingham and Medawar (1950) in the skin of guinea pigs, and by Chase et al. (1951) in the skin of the mouse. Billingham and Medawar consider that these cells form a definite and specific system which they have called the “epidermal glial system.” Since both dendritic cells and epidermal cells contain melanin granules, the presence of dendritic cells is masked in histological preparations. In scantily pigmented epidermis, dendritic cells are readily seen in silvered paraffin sections or under the phase contrast microscope (Chase et al., 1951). Dendritic cells can be demonstrated selectively by the application of the “dopaJJ (3-4 dihydroxyphenylalanine) reaction of Bloch ( 1917). This reaction was perfected by Laidlow and Blackberg (1932) and later modified by Becker (1935). After the use of dopa, the cytoplasm of the epidermal dendritic cells is stained selectively gray, grayish brown, or black, whereas the epidermal cells remain uncolored. The specificity of this technique is based on the assumption that an enzyme (dopa oxidase) present in the dendritic cells, but lacking in the epidermal cells, converts the substrate, dopa, into a dark pigment, dopamelanin (for critical comments on the specificity of this reaction see Rawles, 1948, and Lerner and Fitzpatrick, 1950). In naturally occurring melanogenesis, this same dopa oxidase presumably transforms natural chromogens in the cytoplasm of dendritic cells into melanin, Thus, any cell which is dopa-positive is considered as a cell which is capable of elaborating melanin and should, therefore, be called a melanoblast, with the exception of the granular leucocytes which are also dopa-positive. Those cells which contain melanin but which are dopa-negative are called melanophores, the assumption being that they have received their melanin from nearby melanoblasts. Masson believes that there is a sequence of centripetal maturation of melanin in the dendritic melanoblasts of human skin, the most distally located dendritic granules being the most mature. In some melanoblasts, whose cell bodies are clear and presumably contain only “premelanin,” there is a progressive accumulation of demonstrable melanin granules toward the ends of the dendrites. If the epidermal melanoblasts are the only source of epidermal melanin, this substance must be transferred from the melanoblasts to the epidermal

286

WILLIAM MONTAGNA

cells (melanophores) . Masson’s view is that the melanoblasts are comparable to glandular cells, which secrete their product by virtually injecting it by way of their dendrites into epidermal cells, a process for which he has coined the term “cytocrine.” Billingham ( 1949), Billingham and Medawar ( 1950), and Zimmermann and Cornbleet ( 1948) also believe in the “cytocrine” concept of pigment distribution. Despite the plausibility of this, no one has actually observed the “cytocrine” process in mammalian epidermis. In our laboratory, when melanogenesis is induced by x-radiation or by carcinogenic agents in naturally non-pigmented epidermis of the mouse, fine melanin granules are found around the nucleus and coarse ones at the periphery of the epidermal cells. In addition, pigment granules are often found in the nuclei of epidermal cells. These observations suggest that the nucleus is implicated in melanogenesis. The deceptiveness of histological preparations and the fact that these cells have been injured, would not justify such a conclusion. Meirowsky et al. (1950) state that in epidermal cells “melanin first appears in the nucleolus and spreads to the linin frame work of the nucleus. It appears in intracellular vacuoles and finally involves the entire nucleus and the membrane. It leaves the nucleus by way of the chromidial derivatives.” These authors give a good summary of the main investigations which deal with melanin formation in the nucleus of epidermal cells. Danneel and Lubnow (1936) and Lubnow (1939) have an entirely different view of the formation of pigment. They deny altogether the existence of dendritic cells. They assert that pigment is formed within the epidermal cells in association with, or at the expense of, the Golgi element or “lipochondria,” since these elements disappear at the same pace as pigment granules appear and increase in the cells. It is difficult to evaluate the relative merits of these three concepts. The fact that most authors believe in the “cytocrine” method of melanin distribution does not make it valid. It is conceivable that all these proposed processes may play a role in melanization. Singularly significant contributions to the problem of melanogenesis, have been made by Rothman et d. (1946) and Flesch ( 1949). They have obtained from human epidermis and from homogenates of rabbit skin a water-extractable, heat-stable, dialyzable, non-protein-like sulfhydryl compound which inhibits melanin formation in the tyrosine-tyrosinase system. These authors suggested the possibility that this substance occurs normally in melanoblasts and inhibits the action of the enzyme on the substrate. Melanogenic stimuli such as sunshine, x-rays, heat, and inflammatory

CYTOLOGY OF MAMMALIAN EPIDERMIS

287

diseases would oxidize or destroy these sulfhydryl compounds of the epidermis, leaving the enzyme free to act upon melanin precursors. 14. Mitotic Activity

Epidermal cells possess one spherical nucleus (occasionally two). In flattened or fusiform basal cells the nuclei become ovoid or elongated. The basophilic nuclear membrane encloses a granular, or finely reticulated chromatin, one or more nucleoli, and a variable number of chromatin nucleoli. The nucleolus has a dense basophilic membrane, a homogeneous, delicately basophilic content, and one or more peripheral heterochromatic granules. In the basal layers of the stratum germinativum, nuclei are more basophilic than they are in the upper layers. The apparent scantiness of mitotic activity in epidermis has always puzzled histologists. It did not seem plausible that the few mitotic figures encountered in the stratum germinativum could provide an adequate mechanism for the replacement of cells lost from exfoliation of the stratum corneum. Andrew and Andrew (1949) believe that a transformation of lymphocytes into cells indistinguishable from those of the epidermis make up for the relatively low numbers of mitotic figures seen in normal epidermis. Although it is possible that such transformations may occur, it would seem unlikely that they are to be considered seriously as a major factor in epidermal growth and repair (vide infra) . Flemming (1884) observed mitotic division in the deepest three layers of the epidermis of the snout of the pig. In human skin, Patzelt (1926) found them principally in the basal layer, but occasionally also in the second layer, and Pincus (1927) asserts that division occurs only in the basal layer. Thuringer (1924)) studying the epidermis of the scalp, found mitotic activity in the basal layer and throughout the stratum germinativum. The number of mitotic figures is greater in the middle and outer one-third than in the deeper layers. In the epidermis of the human prepuce, Thuringer (1928) encountered the majority of mitotic figures in the stratum spinosum; only 10.75 per cent of all mitotic figures were found in the basal layer. Since the axes of mitotic figures in the basal layer appeared to be parallel to the basement membrane, Thuringer believed that these cells do not move into the stratum spinosum. In this study he found focal groupings of mitotic figures which suggested to him the presence of “growth waves.’’ Cowdry and Thompson ( 1944), studying the foot pads of mice, found maximal mitotic frequency in the proximal and middle third of the stratum spinosum. In the epidermis of the rabbit, stimulated topically with testosterone propionate, Montagna et al. ( 1949)

288

WILLIAM MONTAGNA

also found mitotic figures more frequently in the lower layers of the stratum spinosum than in the basal layer. In agreement with Cowdry and Thompson, they concluded that in the basal layer, daughter cells may remain upon the basement membrane, or move up into the stratum spinosum. Thus, not only the basal cells but also those of the lower twothirds of the spinous layer must be considered as the germinal elements of the epidermis. The riddle of the scantiness of mitotic activity in the epidermis began to be solved by the work of Ortiz-Picon (1933) who found more abundant mitotic activity in mice killed at noon than in those killed a t night. Carleton (1934), also in the epidermis of the mouse, discovered a rhythmic mitotic activity with a maximum from 8 o’clock in the evening to midnight, and a minimum about noon. On the contrary, Cooper and Franklin (1940) found that the period of greatest mitotic frequency in mouse epidermis occurs at 10 o’clock in the morning, and that of least frequency at 10 o’clock at night, the number of mitotic figures in the morning being more than twice that at night. (These data agree essentially with those of Blumenfeld, 1939, on the epidermis of the rat.) Mice and rats are nocturnal, and their diurnal period of rest and sleep coincides with tissue repair. In human epidermis there is also a rhythmic mitotic cycle which, unlike that of nocturnal animals, is higher in the night hours than in the morning hours (Cooper and Schiff, 1938; Cooper, 1939; Broders and Dublin, 1939). The most thoroughgoing studies on mitotic activity in the epidermis of the mouse are those of W. S. Bullough (1946, 1948a,b, 1949a,b,c,d,e, 1950a,b; Bullough and Green, 1949; and Bullough and Van Oordt, 1950). In this series of papers, Bullough has studied not only the rhythmicity of epidermal mitotic activity, but also the physiologic factors which are responsible for the rhythmicity. In the diurnal cycle he found that in males, as well as in females, mitotic frequency extends from 10 A.M. to 4 P.M. with a peak at approximately 1 P.M. ; thus confirming that during bodily activity, the mitotic rate is low, and during sleep and rest it is high. Excessive muscular exercise is followed by an abnormal depression of the mitotic rate ; the same effect can be induced by extreme cold. In mice placed on starvation rations, mitotic activity drops considerably below that of normally fed controls. Since in these situations there is a drain of the sugar reserves in the body, it was suspected that an abundance of sugar might be a critical factor in the development of a high mitotic rate. It was demonstrated that injections of starch solution subcutaneously causes a marked rise in mitotic rate. Injection of a substance which inter-

CYTOLOGY OF M A M M A L I A N EPIDERMIS

289

feres with phosphorylation, such as phloridzin or insulin, depresses mitotic activity, whereas injections of disodium hydrogen phosphate together with starch induce greater mitotic activity than starch alone. There is a remarkable drop in mitotic rate in animals which are subjected to ischemic shock, and it has been pointed out that although shock raises the blood-sugar level, there is a coincident fall in the total oxygen consumption, indicating that less sugar is being oxidized. These studies indicate that the number of resting cells entering division at any moment is in direct proportion to the amount of sugar being oxidized. In the studies of mitotic rate in normal males and females and in animals treated with androgenic and estrogenic hormones, it was shown that these hormones also have a profound effect upon cell division. I n a study of the diurnal mitotic rate of male mice during each of the first twenty months of life, Bullough found that the life of the animals can be divided into four periods. During infancy the animals are still growing and their epidermal mitotic rate is generally high. During mature age, the mitotic rate is lowered; during the middle age which follows, mitotic rate increases, and in senility, it is again reduced. Coincident with these changes, there are changes in spontaneous bodily activity. In female mice, H. F. Bullough (1943, 1947) and W . S. Bullough (1946, 1948a,b, 1949a,b,c,d,e, 1950a,b) have shown that the epidermis undergoes a cyclic growth similar to that of the reproductive organs, and that the epidermal mitotic rate can be stimulated by injections of oestrone. During the estrous cycle, peaks of mitotic activity are obtained in the third day of diestrous and again in early estrus. These peaks coincide with the normal diurnal peaks at approximately 1 P.M. W. S. Bullough, in comparing the effect of glycogen and oestrone, reported that while both substances are mitogenic, the maximum stimulation obtained by an increased glycogen concentration is small compared with the stimulation obtained with estrone. I n males and females, the duration of each mitotic division was established at 2% hours. While glycogen and androgen increase the mitotic rate, neither has an effect on the duration of each division, which remains constant at 2% hours. Estrogen, on the other hand, not only increases the number of divisions, but also reduces the duration of each to less than one hour. These points and others demonstrate the complexities encountered in a study of mitosis. Reports of mitotic indices which do not take into account diurnal periodicity, state of nutrition and activity, the sex, age, and general physiological status of the animal, as well as physical environment, are difficult to assess.

290

WILLIAM MONTAGNA

111. THE SEBACEOUS GLANDS 1. General Description The distribution of sebaceous glands in man has been studied by Benfenati and Brillanti (1939). They are most numerous in the scalp, forehead, face, and chin, where there may be 400 to 900glands to each square centimeter of skin. Over the rest of the body there are fewer than 100 glands per square centimeter. They tend to be larger and more numerous in the midline of the body, especially of the back. They are lacking in the palms and soles and in the dorsum of the foot. The glands are largest where they are most numerous. In other mammals, sebaceous glands are widespread over the body skin, and they are largest and most numerous in the external auditory meatus and in the perianal region. Most sebaceous glands are appendages of the external root-sheath of hair follicles and open to the surface of the skin together with hairs. The size of the glands often varies inversely with the diameter of the hairs with which they are associated, the largest glands being found where hairs are small or wanting. This, however, does not always apply, since large glands may be associated with coarse hairs and vice versa, In addition to hair-associated glands, there are free sebaceous glands with excretory ducts opening directly onto the surface of the skin. In man, free sebaceous glands are found in the palpebrae (Meibomian glands), occasionally in the buccal mucosa, in the nipples (Perkins and Miller, 1926), in the preputium (Tyson’s glands), occasionally in the glans penis (De Sousa, 1931) and in the labia minora. In other mammals, aggregates of free sebaceous glands form preputial glands (in the rat and mouse), inguinal glands (rabbit), scent glands (shrew), and anal and circumanal glands (rodents, carnivores). The morphology, physiology, and growth of sebaceous glands have been reviewed by Schaffer (1927), Hoepke (1927), Pincus (1927), and Clara (1929). Sebaceous glands are holocrine, multiple acinar glands ; their general configuration is determined by their relative abundance (crowding), and by the nature of the dermis in which they lie (Clara, 1929). Whether or not sebaceous glands are associated with hair follicles, their fine morphology is similar. The details which follow are based primarily upon hair-associated glands, but they apply also to free sebaceous glands. The acini of the glands converge toward a common excretory duct which opens into the upper part of hair follicles. The stratified squamous epithelium of the excretory duct is continuous with the external rootsheath, which in turn, is continuous with the surface epidermis. The sebaceous acini are composed of closely packed, large, misshaped and

CYTOLOGY O F MAMMALIAN EPIDERMIS

291

moribund central cells, and small peripheral cells which are non-sebaceous, usually flattened, and resemble those of the epidermis. Most acini contain a lumen which is filled with sebum, a substance composed of lipids and cell debris. Not all the acini in the same glandular unit are in the same state of sebaceous maturity; in some, only the central cells are laden with lipid droplets, and there may be no lumen; in others there is a lumen, and lipid accumulation may extend to the periphery of the acinus. Just as keratinization Characterizes the end point in epidermal cells, lipid accumulation and fragmentation characterizes that of sebaceous cells.

2. Mitochondria Although mitochondria are discernible in sebaceous cells, there is very little information concerning them. They were described briefly by Nicolas et al. (1914), who believed that the lipid droplets first appear within the mitochondria1 filaments. As the lipid droplets become larger and coalesce, the accompanying mitochondria decrease in number. Such an interpretation is probable since Murray (1916) observed the formation of lipid globules within the mitochondria in the cells of a transplantable sarcoma of the guinea pig. Ludford (1925), in normal and pathologic skin of the mouse, found that mitochondria increase in numbers at the beginning of sebaceous lipid storage. With continued sebaceous accumulation they become fewer, and in mature cells there are only scattered fragments between the lipid droplets. Ludford did not find transition stages between mitochondria and lipid globules and is inclined to doubt such a relationship. Observations made in our laboratory on the mitochondria of sebaceous glands are not very informative on this point, but they are in agreement with those of Ludford. 3. Golyi Element Observations on the Golgi element were made by Ludford (1925) in the glands of the mouse, Bowen (1926, 1929) in the white inguinal glands of the rabbit, and Melczer and Deme (1943) in human sebaceous glands. The non-sebaceous cells at the periphery of the acini, and in the excretory ducts, contain perinuclear osmiophilic Golgi bodies. As lipid accumulation begins, the osmiophilic bodies increase in numbers, and the stored lipid droplets appear to develop within them. Melczer and Deme (1943) believe that the early lipid globules correspond to the Golgi internuni. As the lipid globules increase in size, the osmiophilic bodies become reduced to curved or crescentic rods or shells around them (Ludford, 1925). When the sebaceous cells attain maturity, there is practically no Golgi material left. The early lipid droplets in the center of the Golgi bodies are osmio-

292

WILLIAM MONTAGNA

phobic, but with continued growth they show a progressive blackening with osmium tetroxide (Ludford, 1925). Short treatment of osmicated sections with turpentine or potassium permanganate bleaches the lipid droplets but not the accompanying Golgi elements (Ludford, 1925 ; Melczer and Deme, 1943). In the sebaceous glands of the cat (Montagna, 1949a), the non-sebaceous peripheral acinar cells contain perinuclear osmiophilic bodies which at the onset of lipid storage appear as osmiophilic ringlets with an osmiophobic center. In mature cells, the large, osmiophobic lipid globules are surrounded by a delicate osmiophilic ring. In the degenerating sebaceous cells, there are minute osmiophilic fragments among osmiophobic lipid masses. This sequence of events was also described in the sebaceous glands of the hamster (Montagna and Hamilton, 1949). The only difference between the results of Montagna and those of the authors named above, is that the lipid droplets in the sebaceous glands in his preparation were nearly always osmiophobic. This discrepancy might be explained by the fact that Montagna’s preparations were post-osmicated a shorter period of time. Although osmium is not a specific cytochemical reagent, these results are constant, and they are similar to those obtained with Baker’s acid hematein test for phospholipids. In the sebaceous glands of man (Montagna et al., 1949; Suskind, 1951), the hamster (Montagna and Hamilton, 1949), and the dog (Montagna and Parks, 1948), there are acid hematein positive elements whose shape and distribution resemble those of the osmiophilic Golgi bodies. Montagna and Chase (1950) observed a transformation of sebaceous cells from the cells of the external root-sheath in the skin of mice treated with methylcholanthrene. When the skin of the mouse is painted with one application of this carcinogen, its sebaceous glands become fragmented and disappear within four days. They will regrow within one week from the cells of the external root-sheath, if the follicles contain actively growing hairs. In frozen sections colored with Sudan black, the cells of the external root-sheath possess discrete perinuclear sudanophil bodies similar to those described in epidermal cells. An increase in the size and number of perinuclear sudanophil bodies in focal groups of cells in the external root-sheath indicates the first sebaceous transformation. Young sebaceous cells are distinguished from non-sebaceous cells only by their increased lipid content. Since the perinuclear sudanophil bodies seem to be identical with the osmiophilic granules and the acid hematein-positive elements, one must assume that they are the Golgi bodies or “lipochondria.” From these observations, it is apparent, in agreement with Ludford, Bowen, and Melczer and Deme, that the Golgi element is implicated in some way in the process of lipid storage in sebaceous cells.

CYTOLOGY OF MAMMALIAN EPIDERMIS

293

4 . Lipids Quantitative chemical analyses of sebum are few and contradictory. For example, while Butcher and Parnell ( 1947), studying the surface sebum of the scalp, found that cholesterol makes up as much as 10 per cent of the lipids, Kvorning (1949) in a much larger number of subjects found only very small amounts of this substance. Since it is not possible to obtain for chemical analyses sebum which is free of corneal exfoliation, chemical values must be viewed with caution. Melczer and Deme (1942) studied the distribution of lipids in human sebaceous glands employing histochemical methods of doubtful specificity. They demonstrated four concentric layers of lipids. At the periphery of the glands, they found fatty acids, probably oleic acid. The next layer inward contained eicosyl alcohol, allegedly formed by a reduction of fatty acids. I n the third layer, comprising the cells undergoing sebaceous breakdown, they found triglycerides, and in the sebum an admixture of fatty acids, neutral fats, and esters of arachyl alcohol. Neither Montagna et d. (1948) nor Suskind (1951) found such layering in normal human sebaceous glands. The progression of lipid accumulation in young sebaceous acini begins in the center, and proceeds toward the periphery as the acini become mature (Montagna and Noback, 1946b, 1947). I n moderately mature acini the central cells are laden with large lipid globules ; these diminish in size and in numbers toward the periphery, and the outermost cells may contain only small perinuclear sudanophilic bodies. In fully mature acini even the most peripheral cells may be inflated with large lipid spherules. These observations hold good for the sebaceous glands of all the animals studied. For an overall study of lipids, Sudan black is excellent since it colors lipids indiscriminately. Sudan I11 and IV, being less powerful lipid dyes, may occasionally be more instructive. For example, in the sebaceous glands from the costovertebral pigmented spot of the hamster, Sudan I V shows a concentric stratification of lipids, but Sudan black does not (Montagna and Hamilton, 1949). When frozen sections of skin of man, mouse, hamster, rabbit, cat, and rat are treated with secondary osmication, only the sebum in the excretory ducts of the sebaceous glands becomes blackened. The newly formed sebum in the center of the acini, and the lipid droplets in sebaceous cells, are osmiophobic. I n the glands of the rabbit, the lipids in the peripheral acinar cells are weakly osmiophilic (Montagna, 1949b). Thus, the old sebum, being osmiophilic, may differ chemically from the sebum just formed and from the lipid droplets in the sebaceous cells both of which are osmiophobic.

294

WILLIAM MONTAGNA

Nile blue sulfate colors the mature sebum pink or red; the newly formed sebum is usually colored purple, and the sebaceous lipid droplets, pink. The peripheral cells contain purplish or blue lipid droplets. Although Nile blue is not a specific cytochemical reagent, a rose color usually indicates the presence of neutral lipids. One might surmise, then, that sebum and sebaceous cells contain appreciable amounts of triglycerides. Since the Fischler method for the demonstration of fatty acids is not a specific test (Lison, 1936), the results obtained with its use are of little value. Only the fact that in sebaceous glands the new sebum shows a positive reaction (blue-black), while the old sebum does not, is of some interest (Montagna and Hamilton, 1949). I n addition, when sections treated with this method are subsequently colored with Nile blue, the previously uncolored old sebum, as well as the discrete lipid droplets in the sebaceous cells, becomes pink. I n the skin of the rat treated with the Smith-Dietrich method for phospholipids, Montagna and Noback (1947) described a positive reaction at the periphery of the sebaceous acini. The more specific acid hematein test of Baker (1946; see also Cain’s critiques, 1947, 1950), indicates that phospholipids are present in the spongy cytoplasm of the mature and degenerating sebaceous cells, and in the sebum, as well as in the granules and rodlets which are comparable to the Golgi elements. These observations were made by Montagna and his associates in the glands of man, dog, cat, and hamster, and by Suskind (1951) in a large number of human sebaceous glands. It is not surprising that phospholipids are revealed in the sebum, since Engman and Kooyman (1934) in an analysis of surface lipids found that they contain slightly more than 1 per cent phospholipids. The application of the Schultz method for the demonstration of cholesterol or cholesterol esters shows a positive blue-green reaction, principally in the sebum. I n man (Montagna et al., 1948; Suskind, 1951), in the rat (Montagna and Noback, 1947), the dog (Montagna and Parks, 1948), and the hamster (Montagna and Hamilton, 1949), only the sebum and degenerating sebaceous cells become colored ; in the cat and rabbit (Montagna, 1949a,b) and in the monkey (Morisuye, 1950) a positive reaction is found throughout the mature glands. In the preputial glands of the rat, Montagna and Noback (1946b) found that only the sebum in the excretory ducts reveals digitonide crystals after treatment with digitonin. Free cholesterol combines with digitonin to form acetone-insoluble birefringent crystals. Digitonide crystals were not found in the gland of the general skin in the rat or other animals studied. Normal human sebaceous glands contain no free cholesterol but

CYTOLOGY OF MAMMALIAN EPIDERMIS

295

the stagnant sebum in comedones and in early acne cysts contains free cholesterol in abundance (Suskind, 1951) . When viewed under polarized light, sebaceous glands reveal variable amounts of anisotropic lipids. With minor exceptions, the distribution of these lipids corresponds to the color reaction obtained with the Schultz test. In human sebaceous glands, only the sebum shows consistent birefringence (Montagna et al., 1948). Suskind (1951) observed birefringent lipids in the glands of 30 out of 45 samples of human skin. In most of the 45 specimens the sebum in the pilosebaceous exits and in the excretory ducts was anisotropic. Suskind observed that those specimens in which sebaceous acini exhibit marked anisotropy also contain glands which give the most intense Schultz reaction. In the preputial glands of the rat (Montagna and Noback, 1946b) and in the sebaceous glands of the hamster (Montagna and Hamilton, 1949), only the sebum is consistently birefringent. In the glands of the rat, dog, and cat (Montagna and Noback, 1947 ; Montagna and Parks, 1948 ; Montagna, 1949a), birefringence is more extensive, being present in the degenerating and mature sebaceous cells as well as in the sebum. In these glands the sebum appears as a birefringent homogeneous mass ; in sebaceous cells birefringent lipids are in the form of spherules and acicular crystals. In the glands of the rabbit (Montagna, 1949b), the sebum is weakly birefringent, but the peripheral sebaceous cells contain abundant anisotropic spherules and acicular crystals. These birefringent lipids are not colored with Sudan IV, Nile blue sulfate, or Baker's acid hematein test. After short treatment with Sudan black, the spherocrystals are colored pink, but the acicular crystals either remain colorless or they are a light blue. With longer treatments in Sudan black the birefringent lipids become deeply colored and isotropic. After secondary osmication there is no birefringence in the peripheral lipids, but the blackened sebum shows increased anisotropy. Molisch fluid does not alter the peripheral birefringence, but enhances that of the sebum. The spherocrystals, but not the acicular crystals, are dissolved by 90 per cent alcohol ; 95 per cent alcohol or acetone removes all birefringent lipids. Heating to 60" C. destroys birefringence, but it reappears virtually unchanged as the sections are cooled to room temperature. Cooling to -2" C. increases the anisotropy of all sebaceous lipids, but the induced anisotropy is lost when the sections are rewarmed to room temperature. Anisotropy is not a physical property which allows a specific identification of lipids, but the manipulations mentioned above give it some characterization. The parallelism between birefringence and the Schultz

296

WILLIAM MONTAGNA

test strongly suggests that the birefringent lipids in sebaceous glands represent esters of cholesterol. Sebaceous glands, under near-ultraviolet light (3600 A ) , emit a yellow to orange light which is usually comparable in distribution to the birefringence. Montagna and his associates (1946b, 1947, 1948, 1949a) have studied the autofluorescence in the sebaceous glands of several species of animals. The sebum in the exits of the sebaceous ducts emits a brilliant yellow light, the new sebum and the degenerating sebaceous cells emit yellow or white light of low intensity which fades toward the periphery of the glands. I t is possible that the anisotropic Schultz-positive cholesterol esters are responsible for the emission of the autofluorescent light. There is in sebaceous glands an orderly progression of events which leads to the formation of sebum. Sebum contains histologically demonstrable cholesterol esters, phospholipids, and possibly triglycerides. The blocked sebum in comedones and in sebaceous cysts contains, in addition, free cholesterol. Cytochemically demonstrable differences appear between sebum in the excretory ducts and sebum just formed in the center of the glands. 5 . Glycogen Adult sebaceous glands contain practically no glycogen. Lombard0 (1907) and Sasakawa (1921) found glycogen in the sebaceous glands of human fetuses up to six months; after this age it disappears. In the adult, under several pathologic conditions, sebaceous glands as well as the epidermis may reacquire glycogen. Montagna et al. (1949) found some glycogen in the center of young sebaceous acini, and only traces in the sebaceous cells of the human external auditory meatus. In the adult glands of all animals studied there was no glycogen.

6. Basophilia The cytoplasm of the non-sebaceous cells in sebaceous glands is strongly basophilic. This has been described by Montagna and associates in the glands of several animals. The cytoplasmic basophilia is abolished by ribonuclease and is apparently due to ribonucleoproteins. Young sebaceous acinar buds are intensely basophilic. In more mature acini the peripheral cells stain clearly with basic dyes ; the more central cells do not. There is a progressive centripetal loss of basic staining, and in cells laden with lipid droplets the spongy cytoplasm appears acidophilic.

CYTOLOGY OF MAMMALIAN EPIDERMIS

297

7 . Nadi Reaction Fresh or formalin-fixed sections of the preputial glands of the rat treated with the M-nadi reagent show numerous indophenol-blue granules, especially in the peripheral acinar cells (Montagna and Noback, 1946b). The cytoplasm of non-sebaceous acinar cells is virtually full of indophenolblue granules. In cells which contain lipid globules, indophenol-blue granules form a perinuclear zone. In mature cells engorged with lipids, only a few scattered granules can be seen in the cytoplasm. I n the sebaceous glands of the general-skin of the rat, there is only a trace of indophenol-blue reaction (Montagna and Noback, 1947). In the sebaceous glands of man and of laboratory animals the reaction is scant or absent. 8. Peroxidase Benzidine peroxidase, like the indophenol-blue reaction, was demonstrated in the preputial glands of the rat (Montagna and Noback, 1946b). It is particularly marked in the sebum-forming central acinar cells and in the sebum, the mature sebum being the most reactive. In all other sebaceous glands little or no peroxidase has been demonstrated.

9. Phosphatases and Lipass Alkaline phosphatase activity in sebaceous glands was first demonstrated in human skin by Bourne (1943). It was described in the glands of the rat by Johnson and Bevelander (1946), and by Montagna and associates in the glands of several animals. Although there are species differences, all the sebaceous glands observed contain some alkaline phosphatase. The enzyme is most abundant in the glands of the cat and rat and least abundant in those of the hamster. In the cat and rat enzyme activity is copious in the peripheral acinar cells and diminishes gradually toward the center, where the mature sebaceous cells show practically none. I n the hamster there are only traces of alkaline phosphatase activity in the outer peripheral cells. Some acid phosphatase activity has been demonstrated in the sebaceous glands of man and various animals. This enzyme is more labile than alkaline phosphatase, and much of it is probably inactivated during histological manipulations. The glands of the hamster show much acid phosphatase (Montagna and Hamilton, 1949). The reaction is strong throughout the acinus, but it is weak or absent at the periphery of the acini and in the center where the cells are undergoing degeneration. The newly formed sebum in the center of the acini indicates intense activity ; the old sebum shows less.

298

WlLLIAM MONTAGNA

In most sebaceous glands, including those of man, lipase activity, demonstrated by the method of Gomori, is present, but not abundant. It has been shown by Kung (1949) in the glands of the mouse, and by Montagna and Hamilton (1949) in the glands of the hamster. In the hamster, lipase activity, indicated by the presence of dark brown lead sulfide granules, is scattered sparsely throughout the acihar cells. I n those cells which are undergoing sebaceous degeneration, enzyme activity is maximal. The sebum, new and old, exhibits much lipase activity, being dark brown to black. In immature acini, which do not contain sebum, the central cells alone show copious lipase activity.

10. G r w t h and Proliferation Although holocrine glands must overcome the problem of replacing cells lost in secretion, mitotic activity appears scant in them. Some authors (Bizzozero and Vassale, 1887; Stamm, 1914; Kyrle, 1925 ; Schaffer, 1927; and others) described mitotic division in the peripheral acinar cells and believed that this provides for the replenishing of cells in the center. Others (Bab, 1904; Brinkman, 1912; Clara, 1929; and others) believed that mitotic activity occurs almost entirely in the epithelium of the sebaceous ducts at their junction with the acini, and they envision that the new cells formed there glide down into the body of the glands. Mitotic activity is, indeed, found in both places described above (Parnell, 1949), but the concept of cells gliding down into the fundus of the glands is unfounded. Sebaceous glands of mice injected with colchicine in the morning hours and sacrificed five hours later at approximately 1 P.M. show numerous mitotic figures in the peripheral cells. This not only demonstrates that in mice mitotic activity takes place in the peripheral cell, but also that it follows a cyclic rhythm similar to that of the epidermis. Bullough (1946) has shown that mitotic division in the peripheral sebaceous cells of female mice, as in the epidermis, is correlated with the ovarian cycle, being maximal in early pro-estrus and minimal in the first day of diestrus; injection of estrone induces a peak in mitotic activity. Androgens also stimulate sebaceous activity (Hamilton, 1941, 1947 ; Hooker and Pfeiffer, 1943 ; Ebling, 1948). Montagna and Kenyon (1919) induced mitotic activity in the sebaceous glands of the rabbit with topical application of testosterone propionate. Without the use of a mitotic block, they observed a sharp rise in sebaceous mitotic activity, regardless of the time of day or night the animals were killed. I n these animals, although mitotic divisions were also present in the cells of the excretory ducts, they were most numerous in the non-sebaceous peripheral cells

CYTOLOGY OF MAMMALIAN EPIDERMIS

299

of sebaceous acini. Mitotic division occurred also in sebaceous cells containing a moderate amount of stored lipids. Sebaceous glands of kittens treated similarly showed the same results. Mitotic division in the sebaceous glands of the mouse, rabbit, and cat, then, occurs principally in the periphery of the acini. Parnell (1949) confirmed this in the glands of the rat. The concept of cells gliding into old acini is also objectionable because it implies that sebaceous acini, once formed, remain unchanged. Montagna and his associates (1946a, 1947, 1949) described sebaceous acini in a constant state of change. Epithelial buds, which grow from the walls of the excretory ducts, develop sebaceous kernels in their centers and grow into new sebaceous units. As the new acini expand, they may encroach upon nearby acini, fuse with them, and become a part of larger units, In such sebaceous complexes, the periphery of the fused elements is still outlined by small epithelial cells and fibroblasts adhering to them, forming trabeculae which separate the sebaceous units into locules. Adventitial sebaceous acini also develop from tabs of non-sebaceous cells at the periphery of the acini, protruding into the dermis as appendages of the parent acini. Such lateral buds grow and engulf smaller adjacent ones. Sebaceous kernels may develop anywhere along the acini, outside or in, where there are accumulations of non-sebaceous cells. The fact that mitotic activity is abundant at the periphery of the acini, combined with the vicissitudes just described, militates against the static concepts usually described in textbooks of histology. Furthermore, the inherent growth dynamics of these glands is demonstrated by their quick regeneration from the cells of the external root-sheath of hair follicles, when the glands have been completely destroyed by chemical agents (Montagna and Chase, 1950). ACKNOWLEDGMENTS T h e work of Montagna on skin and cutaneous appendages was supported in part by the United States Public Health Service. T h e author expresses his gratitude to Miss Helen Melaragno for her invaluable help in the preparation of this review and t o his colleagues a t Brown University for their counsel and patience. IV. REFERENCES Andrew, W., and Andrew, N. V. (1949) A m t . Rcc., lO4, 217. Argaud, R. (1914) C. R. SOC.B i d , 77, 61. J - . Ceut., 110, 210. Astbury, W. T. (1933) Sci. P Y ~ J ~T7vcnl. , 126, 913. Astbury, W. T., and Wwds, H. J . (1930) N f l t ~ r cLolttf., Bab, H. (1904) Beiti-. l i l i i t . A/c,d., 1;rstschr. Seriator, 1. Baker, J. R. (1944) Quart. J . rrricr. Sci, 86, 1. Baker, J. R. (1946) Qztart. J . naicr. Sci., 87, 441. Baker, J. R. (1949) Quart. J . niicr. Sci., 90, 293.

300

WILLIAM MONTAGNA

Baker, J. R. (1950) Proc. Liiztz. Soc. Lond., 162, 67. Becker, S. W. (1927) Arch. Drrm. Syph., 16, 259. Becker, S. W. (1935) Arch. Derm. Syph., 31, 190. Becker, S. W. (1948) Sp. Pztb. N . Y . Acad. Sci., 4, 82. Benfenati, A., and Brillanti, F. (1939) Arch. ital. D e r m Sifilo!~.b'rncrcol., 16, 33. Bennett, H. S., and Yphantis, D. A. (1948) J. A n w . Chem. Soc., 70, 3522. Biesele, J. J., and Biesele, M. M. (1944) Cancer Res., 4, 751. Billingham, R. E. (1949) J . Anet., 83, 109. Billingham, R. E., and Medawar, P. B. (1950) Heredity, 4, 141. Bizzozero, G. (1871) Centralbl. med. Wissensch., 9, 482. Bizzozero, G., and Vassale, G. (1887) I'irchozws Arch., 110, 155. Bloch, N. (1917) Arch. Derm Syph., U4, 129. Block, R. J., and Vickery, H. B. (1931) J. Biol. Chenz., 99, 113. Blumenfeld, C. M. (1939) Science, 90, 446. Bolliger, A., and McDonald, N. D. (1949) Ailst. J. e x p . Biol. J J I C ~ .Sci.,27, 223. Born, S. (1921) Derm. Z.,94, 324. Bourne, G. (1943) Qtrart. J. exp, Physiol., a,1. Bowen, R. H. (1926) Quart. J . nricr. Sci., 70, 193. Bowen, R. H. (1929) Quart. Rev. Biol.,4, 484. Branca, A. (1899) J. Anat. Physiol., 36, 764. Branca, A. (1907) J. Avat. Physiol., 43, 341. Brinkman, A. (1912) 2. ges. Ailat., Abs. 3 : Ergebn. Amt. Et~t.ro-Gcsck.,20, 1173. Broders, A. C., and Dublin, W. B. (1939) Proc. Mayo Clin., 14, 423. Bullough, H. F. (1943) J . Endocrirrol., 3, 280. Bullough, H. F. (1947) Nature, Lond., 169, 101. Bullough, W. S. (1946) Philos. Tratrs., BN1, 453. Bullough, W. S. (1948a) Proc. roy. Soc., B186, 233. Bullough, W. S. (1948b) Proc. roy. SOC.,BlM, 233. Bullough, W. S. (1949a) Nature, Lotid., 169, 680. Bullough, W. S. (1949b) J. e x p . B i d , 26, 83. Bullough, W. S. (1949~) J. exp. Riol., 26, 261. Bullough, W. S. (1949d) J . exp. Biol., 26, 287. Bullough, W. S. (1949e) Brit. J . Cancer, 3, 275. Bullough, W. S. (1950a) J. Eiidocrinol., 6, 340. Bullough, W. S. (1950b) Acta Pltysiol. Phartn. tried., 1.2, 357. Bullough, W. S., and Green, H. N. (1949) Nature, Lot~d.,164, 795. Bullough, W. S., and Van Oordt, G. J. (1950) Acta Eirdocrino?., 4, 291. Bunting, H. (1948) Anat. Rec., 101, 5. Bunting, H., Wislocki, G. B., and Dempsey, E. W. (1948) A n a t . Xec., 100, 61. Busacca, A. (1922) Arch. Dcwii. Syph., 141, 88. Butcher, E. O., and Parnell, J. P. (1947) J. Invest. Derni., 9, 67. Cain, A. J. (1947) Quart. J . P J I ~ C ~Sci., . 88,151. Cain, A. J. (1949) Oa-jord Sci., II, No. 2, 30. . 73. Cain, A. J. ( 1950) Biol. K E ~ ' .a6, . tTkIi7!. dftrdr.. 12, 127. Cajal, R. y. (1915) 7'r(16. Lab. I ~ r w s t hiol. Calvery, H. O., IJraize, J. H., and Laug, E. P. (1916) 'Phvsiol. Kelp.. 4, 195. Carleton, A. (1931) J . L'IJlfJt., 68. 251. Caspersson, T. (1947) Synrp. Soc. exp. Biol., No. 1, 127.

CYTOLOGY OF MAZI MALIAN EPIDERMIS

30 1

Chambers, R., and de Kinyi, G. S. (1925) Airier. J. Ailat., S6, 385. Champetier, G., and Litvac, A. (1939) Arch. Anat. micr., 36, 65. Chase, H. B., and Montagna, W. (1951) Proc. SOC.enp. Biol. Med., 76, 35. Chase, H. B., Rauch, H., and Smith, V. W. (1951) Physiol. Zool., 24, 1. ChCvremont, M., and Frederic, J. (1943) Arch. Biol., 64, 589. Clara, M. (1929) Ric. Morfol., 9, 121. Cooper, 2. K. (1939) J . Invest. Derm., 2, 289. Cooper, Z. K., and Franklin, C. (1940) Anat. Rec., 78, 1. Cooper, Z. K., and Schiff, A. (1938) Proc. SOC.exp. Biol. Med., 39, 323. Cowdry, E. V. (1918) Contribs. Embryol., No. 25, 8, 108. Carnegie Inst. Wash. (Pub. 271). Cowdry, E. V. (1932) Special Cytology. Vol. 1. Paul B. Hoeber, New York. Cowdry, E. V. (1943) Biol. Symp., 10, 131. Cowdry, E. V., and Andrew, W. (1950) J. Gerontol., 6, 97. Cowdry, E. V., and Scott, G. H. (1928) Arch Irist. Pasteur Tunis, 17, 233. Cowdry, E. V., and Thompson, H. C., Jr. (1944) Anat. Rec., 86, 403. DaFano, C. (1921) 7th Rep. Imp. Cancer Res. Fund, p. 67. Danneel, R., and Lubnow, E. (1936) Biol. Zbl., 66, 572. Deineka, D. (1912) Anat. Anz., 41, 289. Dempsey, E. W., Singer, M., and Wislocki, G. B. (1950) Stain Tech., a6, 73. Derksen, J. C., and Heringa, G. C. (1936) Gaz. mid. polonaise, 28 and 29 (cited from Derksen et al., 1938). Derksen, J. C., Heringa, G. C., and Weidinger, A. (1938) Acta m h l . Morph. Norm. Path., 1, 31. De Sousa, M. (1931) C. R. des sburzces, 108, 894. Dick, J. C. (1947) 1. Anat., 81, 201. Ebling, F. J. (1948) J. Endocririol., 6, 297. Edwards, E. A., and Duntley, S. Q. (1939) Amer. J . Aucat., 66, 1. Engman, M. F., and Kooyman, D. J. (1934) Arch Dlerm. Syph., 29, 12. Biol., 83, 349. Favre, M. (1920a) C. R. SOC. Favre, M. (1920b) C. R. SOC.Biol.,89, 351. Favre, M. (1924) C. R. SOC.Bwl., 91, 1220. Favre, M. (1946) Anrt. Derm. Syph., Paris, 6, 537 Favre, M. (1950) Ann. Derm. Syph., Paris, 10, 241. Favre, M., and Regaud, C. (1910a) C. R. Acad. Sci., UO,560. Favre, M., and Regaud, C. (1910b) Lyon mid. 22, 1132. Firket, J. (1911) Anat. Anz., 38, 537. Fisher, I., and Glick, D. (1947) Proc. SOC.exp. Biol. Med., 66, 14. Flemming, W. (1884) Arch. mikr. Allat., 29, 148. Flesch, P. (1949) Proc. SOC.exp. Biol. Med., 70, 136. Folin, O., Trimble, H. C., and Newman, L. H. (1927) 1. Biol. Chew., 76, 263. Frederic, J. (1949) d r c h . Biol.,60, 79. Friboes, W. (1920) Derm. Z., 91, 57. Friboes, W. (1921) Derm. z., S2, 1. Friboes, W. (1922) Arch. Derm. Syph., 140, 201. Giroud, A., and Bulliard, H. (1930) d r c h . klorpli. g i r l . exp., 29, 1. Giroud, A., and Bulliard, H. (1934) BuZZ. Histol. Tech. micr., 11, 169. Giroud, A., and Bulliard, H. (1935) Arch. Airat. wicv., 91, 271.

302

WILLIAM MONTAGNA

Giroud, A,, and Champetier, G. (1936) Bull. Sac. C h i . biol., Paris, U,656. Giroud, A.,Leblond, C. P., and Ratsimamanga, R. (1935a) C. R . SOC.Biol., lla, 321. Giroud, A., Leblond, C. P., and Ratsimamanga, R. (1935b) C. R. Asso. Allat. Montpelier, 1. Gomori, G. (1941) J. cell. comp. PhyJiol., 17, 71. Gomori, G. (1946) Arch. Path., U, 121. Gustavson, I<. H . (1949) Advances Prof. Chew, 6, 353. Hamilton, J. B. (1941) 1. Clin. Endocrind., 1, 570. Hamilton, J. B. (1947) Anat. Rec., 97, 340. Hammett, F. S. (1931) Protoplusma, U,331. Hawk, P. B., Oser, B. L., and Summerson, W. H. (1947) Practical Physiological Chemistry, Blakiston, Philadelphia. Herxheimer, K. (1889) Arch. Derm. Syph., 21, 645. Herxheimer, K. (1916) Derm. Z.,28, 63. Herxheimer, K., and Miiller, H. (1896) Arch. Derm. Syph., 96, 93. Hirsch, G. C. (1939) Protoplasma Monographien, 18. Hoepke, H. (1924) Anat. Anz., 68, 147. Hoepke, H. (1927) Mollendorffs Hand. naikr. Anat. Mensch., 8.1, 1, 55. Hooker, C. W.,and Pfeiffer, C. A. (1943) Eirdocrinol., Ba, 69. Jeghers, H. (1950) Med. Phys., 2, 984. Johnson, P. L., and Bevelander, G. (1946) Anaf. Rec., 96, 193. Kaye, M. (1924) Biochem. J. l8, 1289. Keilin, D. (1925) Proc. roy. SOC.,BW, 312. King, L. S. (1949) J. m t . Comer Insf., 10, 689. Kolliker, A. (1853) Manual of Human Histology. Vol. 1. Sydenham Society, London. Kollman, M., and Papin, L. (1914) Arch. A n d . Micr., 18, 193. Kromayer, E. (1892) Arch. mikr. ditat., 39, 141. Kung, S. K. (1949) J. not. Cancer Inst., 9, 435. Kvorning, S. A. (1949) Acta Pharmacol. Toxicol., 6, 383. Kyrle, J. (1925) Vorlesungen iiber Histobiologie der menschlichen Haut und ihrer Erkrankungen. Springer, Vienna and Berlin. Laguesse, E. (1919a) C. R . SOC.Biol., 8?, 435. Laguesse, E. (1919b) C. R. Soc. Biol.,82, 438. Laidlaw, G. F., and Blackberg, S. N. (1932) Amer. I . Pathol., 8, 491. Langerhans, P. (1873) Arch. mikr. Anat., 9, 730. Lerner, A. B., and Fitzpatrick, T. B. (1950) Physiol. Re7,., So, 91. Levin, 0.L,Silvers, S., and Behrman, H. T. (1940) Urol. czlfun. Rezc, 44,307. Lewis, M. R., and Lewis, W. H. (1915) Amer. J. Anaf., 17, 339. Lison, L. (1936) Histochimie Animale. Gauthier-Villars, Paris. Litvac, A. (1939) Arch. Anat. micr. S6, 55. Lombardo, C. (1907) Giorn. itul. Mal. wetter., 40, 448. Lubnow, E. (1939) 2. indukt. Abstamm-u. Vererb. Lehre, 67, 516. Ludford, R. J. (1924) Quart. J . micr. Sci., 88, 27. Ludford, R. J. (1925) Proc. roj. Soc. BW, 557. MacCardle, R. C.,Engman, M. F., Jr., and Engman, M. F., Sr. (1941) Arch. Derm. Syph., 44, 429. MacCardle, R. C., Engman, M. F., Jr., and Engman, M. F., Sr. (1943) Arch. Derm. 47, 335.

CYTOLOGY OF MAMMALIAN EPIDERMIS

303

Martinotti, L (1914a) Arch. ZelZforsch., l2, 457. Martinotti, L. (1914b) Anat. Am., 46, 321. Martinotti, L. (1915) Arch. Zellforsch., 19, 446. Martinotti, L. (1921) Arch. Zellforsch., 16, 377. Martinotti, L. (1924) 2. &s. Mikr., 4l, 202. Masson, P. (1948) The Biology of Melanomas. Sp. Pub. N. Y. Acad. Sci., 4, 15. Meirowsky, E. (1940) Brit. J. Derm., 82, 205. Meirowsky, E., Freeman, L. W., and Fischer, R. B. (1950) ZooEogica, 36, 29. Melczer, N., and Deme, S. (1942) Lkrmufologica, 86, 24. Melczer, N., and Deme, S. (1943) Arch. Derm. Syph., 188, 388. Mescon, H., and Flesch, P. Modification of Bennett’s method for the histocheniical demonstration of free sulfhydryl groups in skin. (MS) Mickelsen, O., and Keys, A. (1943) J. Biol. Chem., 149, 479. Montagna, W. (1949a) 1. Morph., I ,42. Montagna, W. (1949b) Anat. Rec., u14, 243. Montagna, W. (1950a) Proc. SOC.exp. Biol. Med., 79, 127. Montagna, W. (1950b) Quart. J . micr. Sci., 81, 205. Montagna, W., and Chase, H. B. (1950) Anuf. Rec., 107, 83. Montagna, W., and Hamilton, J. B. (1949) Amer. J. Anat., M, 365. Montagna, W., and Kenyon, J. (1949) Anat. Rec., 108, 365. Montagna, W., Kenyon, P., and Hamilton, J. B. (1949) J. ex#. Zool., 110, 3i9. Montagna, W., and Noback, C. R. (1946a) Ami. Rec., 96, 41. Montagna, W., and Noback, C. R. (1946b) Anat. Rec., 96, 111. Montagna, W., and Noback, C. R. (1947) Amer. J . Anat., 81, 39. Montagna, W., and Noback, C. R., and Zak, F. G. (1948) Amer. J. Ailat., 89, 409. Montagna, W., and Parks, H. F. (1948) A n d . Rec., 100, 297. Morisuye, J. M. (1950) A cytochemical study of the glands of the external auditory meatus of the monkey. Brown University (Thesis). Murray, J. A. (1916) J. Path. Bacf., 20,260. Nicolas, J., Regaud, C., and Favre, M. (1914) X V I I t h Znf. Corigr. Med., Set. 13, Derm.and Syph., p. 101. Nicolau, S. (1911) Ann. Derm. Syph., 2, 641. Odland, G. F. (1950) Anat. Rec., 108, 399. Ortiz-Picon, J. M. (1933) 2. Zellforsch., 19, 488. Palade, G. E., and Claude, A. (1949a) J. Morph., 86, 35. Palade, G. E., and Claude, A. (1949b) 1. Morph., I ,71. Paletta, F. X., Cowdry, E. V., and Lischer, C. E. (1941) Cower Rrs., 1, 942. Parat, M. (1928) Arch. Anat. micr., 24, 73. P m e l l , J. P. (1949) Amer. J. Anat., 86, 41. Patzelt, V. (1926) 2. mikro.-anat. Forsch., 5, 371. Perkins, 0. C., and Miller, A. M. (1926) J. Obstet. Gynaecol., 11, 789. Pincus, F. (1927) Jadassohn’s Handbuch der Haut und Geschlechtskrankheiten I. Springer, Berlin. Rabl, H. (1897) Haut. In Merkel-Bonnets Erpbn., 8, 339. Ranvier, L. (1879) C. R . Acad. Sci., 89, 667. Ranvier, L. (1898) Arch. Anat. mkr., 2, 510. Rawles, M. E. (1948) Physiol. Rev.,28, 383. Regaud, C., and Favre, M. (1912) C. R . SOC.Biol., 73, 328.

304

WILLIAM MONTAGNA

Rosenstadt, B. (1910) Arch. mikr. Anat., 76, 659. Rothman, S., Krysa, H. F., and Smiljanic, A. M. (1946) Proc. Soc. ex#. B i d . Med., a,208. Rudall, K. M. (1946) The structure of epidermal protein. From: Fibrous Protein, p. 16, Leeds. Sasakawa, M. (1921) Arch. Dernb. Sypk., 184, 418. Schafer, E. A. (1912) Quain’s Anatomy, Vol. 2. Textbook of Microscopic Anatomy. Longmans, Green, London. Schaffer, J. (1927) Das Epithelgewebe. Die Talgriisen. In : Mb’tleiidorfs Hand. mikr. Anat. Mensch. Schmidt, W. J. (1937) Prototlama, 29, 300. Schultze, F. E. (1896) S. B. preitss. Akad. Wiss. (Cited from Hoepke, 1927.) Schultze, M. (1864) Virchow’s Arch., 30, 260. Scott, G. H. (1933) Amer. 1. Ailat., 63, 243. Serra, J. A. (1946) Stain Tech., 21, 5. Shapiro, B. (1924) Quart. J. micr. Sci., 68, 101. Stamm, (1914) Mindeschrift for Japetus Steenstrup, Kobenhaun, p. 1 (cited from Schaffer, 1927). Suntzeff, V., and Carruthers, C. (1945) J . Biol Clzenr., 160, 567. Suskind, R. R (1951) J . Invest. Derm. (In press.) Tello, F. (1923a) Trab. Lab. I w e s t . bio?. Univ. Madr. (cited from Tello, 1923b). Tello, F. (1923b) Trab. Lab. Znzvst. biol. Univ. Madr., 21, 255. Thomas, 0.L. (1948) Quart. J. mkr. Sci., 89, 333. Thompson, R. H.S., and Whittaker, V. P. (1944) Biochem I., 88, 295. Thuringer, J. M. (1924) Anat. Rec., !B, 31. Thuringer, J. M. (1928) Anat. Rec., 40, 1. Unna, P. G., and Golodetz, L. (1909) Biochem. Z.,20, 469. Waldeyer, W. (1882) Brifr. Anat. Embryol. Hcnle-Festgabe (cited from Hoepke,

1927).

Walker, ‘E. (1925) Biochem. J., 19, 1085. Weidenreich, F. (1900) Arch. mikr. Anat., 66, 169. Wilkerson, V. A. (1934) J . Biol. Chem.. 107, 377. Wilkerson, V. A. (1935) J. Biol. Chem., 1l2, 329. Worley, L. G. (1944) J. Morph., 76, 261. Worley, L. G. (1946) Ann. N . Y. A c d . Sci., 47, 1. Zimmermann, A. A., and Cornbleet, T. (1948) J. Invest. Derm., 11, 383.

The Electron-Microscopic Investigation of Tissue Sections L. H. BRETSCHNEIDER Department of Zoology, University of Utrecht, Hollatd CONTENTS

I. Objectives ................................... ....... 11. The History of Ultramicrotomy . . . . . . . . . . . . . . 1. The Wedge-Sectioning Method ........................... 2. Low-Speed Microtomy (Below 1 Micron) 3. High-speed Microtomy ......................................... 4. The Thermal Expansion Method ................................ 111. The Influence of Fixation upon the Electron-Optical Image . IV. Primary Nuclear and Plasmatic Ultrastructures . . . . . . . . . . . . V. Secondary Ultrastructures of Cells ........................ 1. Perforated Nuclear Membranes ................................ 2. Neuroglial Reticula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cross-Striated Muscles ......................................... 4. Myelin Axonal Sheaths ........ ........ 5. Cell-Surface Structures ........ ........ 6. The Ultrastructure of Cnidia . . . 7. Extracellular Structures ....................................... 8. Cytopathological Investigations ................................. VI. References ........................ .... ....

Page 305

310 312

316 316 316 318 318 319 320 321

I. OBJECTIVES To obtain an electron-optical image by diffusion of the electronic rays in the electrostatic fields of the atoms of the object requires relatively thin objects; and this has led directly to attempts to make ultra-thin sections; for the electronic discharge is the result of the thickness of the object and the atomic weight of its atoms. The composition of organic structures having a low atomic weight (HI - Fesa) allows of thicknesses up to 0.5 micron, a relatively favorable figure for cytological purposes, which has encouraged cytologists in their efforts to produce sections thinner than the generally valid lowest figure of 1 micron. Thanks to the success of these efforts cytologists were no longer restricted to the use of structures whose original thickness was already below 1 micron; and the removal of this limitation constituted a promising step forward both for electron microscopy and cytology. For, it is now possible, by the electron-microscopic examination of tissue sections, to penetrate into the structure of all parts of the cell and investigate their finer structure directly and on the spot. The new method provides us with every possibility to verify the presence of supposedly pure ultra-structures, or, alternatively,

305

306

L. H. BRETSCHNEIDER

to discover completely new ones. In addition we are now able to eliminate the formerly unpassable light-optical border, and follow the cellular texture without a break, from the light-optically visible right down to the macromolecular region, visibly and directly. Electron microscopy, however, should not content itself with describing the finer structures ; its investigations will have to include the development, the physiology, and the topochemistry of cells and tissues. In this, there still are considerable difficulties to be overcome, especially because the electron-microscopic image is produced in a way totally different from the light-optical one, with the result that, as a rule, no direct adoption of recent cytological techniques is possible. Although we are only at the beginning of the electron-optical investigation of tissue sections, we may safely predict that the study of the finer cellular structures of cytobiology and cytopathology will take a new turn because of the advent of electron microscopy. It is, however, also fairly certain that successes in this field cannot be expected from the examination of tissue sections alone, but that, on the other hand, many different and mutually complementary methods will be required. One of the primary demands to be made of electron microscopy will be that its results will exceed those hitherto obtained by the light microscope. Unfortunately many an investigation has failed, as yet, to come up to this expectation. It is not enough that an electron-optical image gives a greatly enIarged light-microscopic picture, if it gives us no new insights or fresh knowledge. No doubt it is permissible, and often even desirable, for an electron-microscopic image, to fit in immediately beyond the light-optical border, providing always that it also goes a step further than that. Moreover, the purpose of electron microscopy need not exclusively be the finding of hitherto completely unknown facts; it may also render great services by the verification of our-often very uncertain-knowledge this side of the light-optical border. Indeed, given satisfactory technical conditions-fixation, thickness of the object, emission current and sharpness of the image-each newly investigated object offers new aspects. In this way we find, in using proper enlargements side by side, additions to our light-optical knowledge and new electron-optical insights into the finer structure of our objects. On the basis of electronmicroscopic investigation, old conceptions are modifiecl ; long-standing controversies are settled. The brief history of the investigation of tissue sections has so far yielded only sparse results. For investigators who had to develop the technique themselves, the object of their research was, at first, more or less seconcl-

ELECTRON-MICROSCOPIC

INVESTIGATION

OF TISSUE SECTIONS

307

ary ; their greatest problem was how to achieve success in their technical efforts. Purposeful application of methods with the object as the main issue, has only just begun. Now that the apparatus and the technique have been designed, they are also at other investigators’ disposal for their own purposes, see Table I. TABLE I Objects

Authors

Bacteria

I

Fernhndez-Morh (1950)

Newman, Borysko, Swerdlow (1950) Bretschneider (1950) Bretschneider (1949, 1950) Ciliata : Paramecium, Opalina, Isotricha Bretschneider (1949, 1950) Oocytes : Ascaris, Cyanea Beams (1950) Oocytes : Coregonus Bretschneider (1949, 1950) Nematocysts of Corynactis Newman, Borysko, Swerdlow (1950) Mouse red blood cells Bretschneider (1949) Intestinal epithelial cells of Ascaris Newman, Borysko, Swerdlow (1950) Intestinal epithelial cells of frog Intestinal epithelial cells of rat Rhoades (1949) Bretschneider (1951) in press Pancreas cells of mouse Nenman, Borysko, Swerdlow (1950) Liver cells of frog and mouse Pease and Baker (1948) Liver cells of rat Claude and Fullam (1946) Liver cells of guinea pig Richards, Anderson (1942) Cuticles of insects : Periplaneta. Culex Way (1950) Cuticles of Diataraxia Bretschneider (1949) Smooth muscle of M y t h Richards and Anderson (1942) Striated muscle of Periplaneta Bretschneider (1949) Striated muscle of Aeschna Bretschneider (1949) Striated muscle of Triton Danon and Kellenberger (1950) Striated muscle of frog Bretschneider (1951) Nerve cells and axons of cat Latta and Hartmann (1950) ; Schmitt (1949,-1950) Nerve axon of rat Fernindez-MorLn (1950) ; Rosza, et al. (1950) Schmitt (1949, 1950) Nerve axon of frog, man, Loligo Bretschneider (1949) Bone of Balaenoptera Carcinoma, breast, mouse Bretschneider (1950) Gessler, Grey, Schuster, Kelsch, Richter (1949) Neoplastic tissues Cells infected with tobacco mosaic virus Black, Morgan, Wyckoff (1950) Onion, root tip, cells oi nieristem

I

To give a systematic summary of the, as yet very modest, literature on the subject is not easy, since investigations are still more or less in the experimental phase, without any noticeably definite system having so far been followed in the choice of the objects.

308

L. H. BRETSCHNEIDER

11. THEHISTORY OF ULTRAMICROTOMY

The endeavor to obtain tissue sections thinner than 1 micron has led to various solutions, the main problem being that of the construcion of suitable microtomes. Up to the present, the following four solutions have, in principle, been found. 1. The Wedgc-Sectioning Method v. Ardenne (1939) was the first investigator to produce ultrathin sections, by applying the principle of wedge-shaped sections. H e used a LeitzMinot microtome with a revolving object holder, which enabled him to shift the object block by a slight angle (1 :ZOO) with respect to the blade in cutting each separate section. By adjusting the microtome at 1 micron he obtained sections whose thin extremity was just usable for electronmicroscopic examination. v. Ardenne further studied all the physical phenomena arising between the blade and the object during the cutting process and leading to deformation. Apart from v. Ardenne, however, no other investigator has since used this first method.

2. Low-Speed Microtowny (Below 1 Micron) Starting from the idea that the conventional lowest limit of 1 micron of most microtomes was merely based on the fact that ordinary histology had no need for thinner sections, a beginning was made with the construction of microtomes with an object displacement less than 1 micron. As early as 1896, Apathy had argued that there was nothing in the way of producing tissue sections thinner than 1 micron; but the time for this was not ripe until the advent of the electron microscope. Richards, Anderson, and Hance (1942) used a rotary Minot microtome with a special transinission (1 :4) of the object-shifting mechanism, which brought the thickness of the sections down to 250 mp. Pease and Baker (1948) used a Spencer microtome “820” with a transmission of l:lO, bringing the thickness of the sections down to 100 mp. The latter type of microtome, now on the market as a commercial product, has been used in recent years, especially in America, in different investigations (Rhoades, 1949; Schmitt and Geren, 1950; Beams, Evans, Baker, and Breemen, 1950). Bretschneider ( 1949), starting from an already old principle in microtome construction, by which the displacement of the object toward the blade is reduced by means of a lever with arms of different length, furthered the construction of the “Rocking” microtome (Cambridge), by which sections of 200 irip can be cut. The efficiency of this

ELECTRON-MICROSCOPIC INVESTIGATION OF T I S S U E SECTIONS

309

FIG.1. EXAMPLE FORLOW-SPEEDMICNOTOMY. 1. Section of the mammary carcinoma of the mouse (Bretschneider, 1950). Fixed with Champys fluid. Sectioned with the Cambridge Rocking microtome at 300 mp. Embedded in beeswax paraffin. Investigation with the experimental electron microscope from Le Poole at Delft, Holland. Emission voltage 110 kv. Magnification 4000X. 2. Part of a cell nucleus in the same section photographed at an electron magnification of 60,OOOX.

3 10

L. H. BRETSCHNEIDER

type for electron-niicroscopic investigation was clearly shown when it was used on the most divergent tissues, cells, and protozoa (Bretschneider 1949, 1950a, b, 1951). This type was followed up by Danon and Kellenberger (1950), who constructed a Rocking microtome by which, because of the wedge-shaped construction of the part moving the lever, a further reduction of the distance between the blade and the object was obtained. With this microtome it is theoretically possible to obtain sections down to 40 mp. Various investigations have already yielded proof to the effect that it is possible to obtain sections of only 100 vnp thickness by means of a microtome with a normal mechanical object displacement ; for this, however, certain harder embedding materials such as paraffin combined with beeswax and carnauba wax, ester wax or “wax 1OOo” are required, as well as cutting at low temperatures.

3. High-speed Microtomy Whereas, with the preceding microtomes, the method of cutting does not differ much as regards tempo and manipulation, high-speed microtomy is based on a new principle. Starting from the factors which may lead to deformation, as formulated by von Ardenne (1939), O’Brien and MacKinley (1943) endeavored to reduce these factors by increasing the speed of the blade movement. While a micrometer screw of the microtome moves the object toward the blade at a certain rate of speed, sections are cut by a very rapidly rotating blade (about 4O.OOO r.p.in.). The thickness of the sections is determined by the rate at which the object moves and the speed of the rotating blade. Sections down to 100 mp can be obtained in this manner. Fullam and Gessler (1946) undertook the construction of two different high-speed microtomes on the basis of this principle. Success in obtaining serviceable thin sections was shown to depend not only upon a suitable microtome, but also upon a special embedding medium. Since paraffin was found to be unsuitable, Claude and Fullam (1946) introduced a eutectic mixture of camphor and naphthalin. A special improvement for the correct stretching of the thin sections was applied with the “liquid reservoir” which Claude (1947-48) has designed. It is a well in front of the blade which is filled with water so that each section can expand directly on the surface of the water (Fig. 2). Besides the above-mentioned investigators and Gessler and Fullam (1948), Gessler, Grey, Schuster, Kelsch, and Richter (1948) used highspeed microtomy for the investigation of pathological tissues.

ELECTRON-MICROSCOPIC

INVESTIGATION

OF TISSUE SECTIOKS

311

Fig. 2. Example of high-speed microtomy. Sections of guinea pig liver (Claude and Fullam, 1946). Fixed by perfusion of osmium tetroxide. Embedded in camphornaphthalene. Sectioned with the high-speed rotary microtome at 500 nzp. 1. Light-optical picture of a 1-micron section. 2. Electron micrograph taken at 4000X. 3. Micrograph taken at 10,OOOX. Shows the fine ground structure of the cell plasm.

312

L. H. BRETSCHNEIDER

4. The Thermal Expansion Method Unlike the first three methods, according to which ultra-thin sections are obtained by purely mechanical means, this method is based upon thermal expansion of the object block immediately prior to the cutting of the section, No specially designed microtome is required. The only special device is a freezing chamber for the object holder. The “Spencer Microtome 820’ is at present most commonly used for the purpose. This new principle was introduced by Newman, Borysko, and Swerdlow ( 1950). The fixed specimen is placed in the monomer of butyl methacrylate and this medium is polymerized. Carbon dioxide cooling of the block first produces shrinkage. At this stage a few sections of 2 to 3 microns are cut, and the microtome is put in the &position. After an interval of a few seconds, during which thermal expansion of the polymer takes place, a thin section is cut. Sections down to 100 mp can be obtained, in accordance with the duration of the interval (Fig. 3 ) .

FIG. 3. Example of thermal expansion microtomy and embedding with methacrylate (Newman, Borysko, and Swerdlow, 1950). Onion root tip cells. Fixed in medium chrom-acetic. Embedded in methacrylate. Sectioned with the Spencer microtome (820) at 100 nib. Shadowed with chromium. Electronic magnification 750X and enlarged to 2600X.

ELECTRON-MICROSCOPIC

INVESTIGATION OF TISSUE SECTIOSS

313

Various tissues have already been investigated with the aid of the method. Rosza et al. (1950) and Duncan and Antes (1950) used it for neurological investigations. The thermal expansion method was further perfected by Fernindez-Morin (1950), who froze objects (bacteria), unfixed or fixed and embedded in gelatin, on the Spencer microtome, whereupon thermal expansion of the ice block made it possible to cut sections of 100 nzp. With the aid of this technique he succeeded in defining the intricate structure of axonal sheaths by electron-optical means (Fig. 4 ) .

111. THEI N F L U E N C E OF

F I X A T I O N UPON T H E

ELECTRON-OPTICAL IMAGE

A comparison of electron-microscopic results so far obtained by various investigators using different methods of fixation shows that light-optical results are frequently based to a considerable extent upon artifact formation. At this still experimental stage of the investigation of ultra-thin sections, this may very well prove to be the most valuable conclusion. The problem of the influence of fixation upon the electron-optical image has become increasingly urgent ever since the first attempts at electronmicroscopic determination of the ultrastructure of cells were made, for an interpretation of the electron-optical image largely depends on the solution of this problem. Numerous conventional cytological views have become dubious since Gessler and Fullam (1946) and Bretschneider (1949, 1950a) pointed out the marked differences in the nuclear and plasmatic ultrastructure of identical cells, which become apparent after fixation by means of substances containing OsO4 or sublimates. Electron microscopy, with its stronger magnifications, requires a perfection in the preparation of biological objects even greater than that required for light microscopy. Artifact formation, though unavoidable, is restricted as much as possible. To determine the influence of fixation upon the ultrastructure of objects within the limits of electron-optical magnification, 18 fixative substances were tested on the radicular cells of a plant (Bretschneider, 195Oa). The highest degree of coagulation was found to be produced only by mixtures of formalin and osmium tetroxide, preferably in combination with chromic acid or potassium dichromate. When these substances are used, the diameter of the plasma and karyolymph filaments (elementary parts of the finer structure) reduced to the minimum. This condition is presumed to resemble the natural condition of plasma as closely as possible. All other fixation substances, especially acid, markedly flocculent and rapidly diffusing ones, were found to be unsuitable for electron-microscopic purposes. These four substances also successfully used by other authors (Grey and Kelsch, 1948) ; not, however, in mix-

311

L. €1. BRETSCHNEIDER

tures, but successively in the following order : formaldehyde +potassium dichromate +osniic acid +chromic acid. The satisfactory effect produced by means of these substances is based upon making proteins and lipoids insoluble with regard to subsequent treatment and upon the fact that valence and niolecular interrelationships are hardly affected.

IV. PRIMARY NUCLEAR AND PLASMATIC ULTRASTRUCTURES In sections of 100-300 wzp successfully treated by Chanipy, Regaud or formalin fixation, the structure of the basic cytoplasm of each cell is clearly discernible ( Bretschneider, 1950a). After the above-mentioned forms of fixation, light microscopy hitherto invariably revealed a “liomogeneous” basic cytoplasm, whereas the plasma picture observed other forms of fixation was fibrillar, vacuolar, or granular. The most markedly fibrillar form of plasma was observed in axons and perikaryons. These observations finally led to the neurofibril theory. Various authors siniultaneously tried to verify this theory by electron-microscopic means (Schmitt and Geren, 1950; Rosza ct al., 1950; Bretschneider, 1950b; Fernbndez-Moran, 1950 ; Latta and Hartniann, 1950). All investigators finally denied the existence of preformed neurofibrils, whose occurrence in light-microscopic specimens must be regarded as an artifact. No actual neurofibrils, in the sense of light-microscopic investigation, were found after fixation by means of forniol (Rosza, Fernbndez-Morin) , Apathy’s mixture ( Bretschneider )-two of the fixation substances most commonly used in neuroliistology-OsOr or substances containing OsOl ( Bretschneider) . All electron-optical results revealed that axons, both of invertebrates and vertebrates, consist exclusively of axoplasni. This consists of long leptones, about 100 A thick, of undefinable length. They run a parallel or slightly undulating course, occasionally touching and thus forming points of contact relatively far apart. Rosza et al. (1950) observed that axonal leptones run through Ranvier’s nodes unaltered, without passing a membrane. The only bodies we found embedded in the axons were chondrioconts. Unlike the more or less parallel course of the axoplasm leptones, the structure of the basic cytoplasm shows a certain regularity. Leptones, 80 to 250 A thick, constitute a regular three-dimensional reticulum, the meshes of which have an axial length of 300 to 800 A (Bretschneider, 1950a, 1951). After successful fixation, this leptone reticulum was observed in all cells so far examined. After most of the other forms of fixation it is absent, due to structural coarsening. The regularity in the thickness of the leptones and the length of the meshes is suggestive of

ELECTRON-MICROSCOPIC

INVESTIGATION

OF TISSUE SECTIONS

31 5

a preformed condition rather than artifact. Aggregation of the leptones, as elementary parts of the basic cytoplasm, results in the formation of fibrillar or membranous structures of variable proportions. The long leptones of cells characterized by rapid growth, fission or physiological activity show nodular distentions at regular intervals, corresponding to the chromidia described by MonnC ( 1946-1951). The distensions are interpreted as parts of these elementary structures containing enzymes and plasmanucleotides connected with the physiology and autocatalysis of plasma. MonnC published detailed reports on this matter in 1946 and 1951. Electron-optical investigation of sections does not reveal the wellknown light-microscopic image of a nucleus, a fluid-filled bubble wherein chromatin particles and nucleoles are fixed upon linins. After fixation the karyolymph is likely to coagulate to a fine, irregular reticulum filling the entire nucleus. The same picture may be observed upon light microscopy after forniol or OsO4 fixation, in which case it is usually called a “honiogenized” nucleus. In view of the minute size of the karyolyniph particles (about 80-300 A) there is a tendency to interpret this minute structure as a product of the originally already finely dispersed, protein molecules in the karyolymph (Gessler and Fullam, 1946 ; Bretschneider, 1950a, b) . The empty nuclei observed electron-optically after the use of unsuitable fixation substances are interpreted by some authors (Beams et al.. 1950; Bretschneider, 1950a) as artifacts. The teinporary plasma fibrils best known from light-microscopic investigation are the linear structures of mitotic asters and spindles. Two contradictory electron-optical studies have so far appeared in the literature on this subject. In the radicular cells of Allium, studied both during the metaphase and the anaphase, Bretschneider ( 1950a) observed no fibrillar structures, but only parallel arrangements of plasma leptones. Beams, Evans, Baker, and Breemen ( 1950), however, clearly observed fibrils, about loo0 A thick, in the dividing blastonieres of Coregonus egg. Since different fixation techniques were used, however, the results are not comparable. Bretschneider ( 195Oa) proved that both alcohol and acetic acid, combined by Beams et al. with Tellyesniczky’s mixture and used as fixation substance, are unsuitable for the purpose and produce coarse plasma coagulation. Bretschneider himself used form01 and Regaud’s mixture, both excellent fixation substances which permit of visualization of leptones in their most minute form.

316

L. 1%. BRETSCHNEIDER

V. SECONDARY ULTRASTRUCTURES OF CELLS All data on tissues, cells, and protozoa so far obtained by electronoptical investigation of sections are given in Table I. Technical difiiculties prevented the discovery of new facts with regard to some of the objects mentioned which will, therefore, not be discussed further. New points of view arose regarding the following objects.

1. Perforated Nuclear Menzbraites. Callan, Randall, and Tomlin (1949) discovered a regular perforation of the two-layer nuclear membrane of isolated pronuclei of Amphibia. Bretschneider verified this observation on neuronal nuclei in a ganglion section from a leech, Hirudo medicinalis. The nuclear membrane shows pores with a diameter of 70 ntp at regular intervals. This observation, made on two extremely diverse cells (oocyte and neuron) suggests that this structural principle may occur in various cells. In fact, it may very well be quite common. 2. Neuroglial Reticula Sections made by Bretschneider (1951)* in the neuropiles of the abdominal marrow of Hirudo revealed reticular glial structures. These structures result from agglutination of fibrils, 40 nzp thick, to thicker bars, which arrange themselves in a plane to form the fenestrated membranes. In numerous places these bars bifurcate again into thin fibrils protracted throughout the neuropile. In view of the fact that these supportive structures are situated in the syncytial neuropile plasma, and although they are not of extracellular origin like collagen, elastin, or reticulin, they may be presumed to have developed under the influence of directive plasmatic forces. 3. Cross-Striated Muscles The available information on the electron-microscopic ultrastructure of the fibrils of cross-striated muscles has been extended considerably by numerous electron-optical investigations. The thinness of the structure made it possible to carry out most investigations on isolated fibrils. The problem of the orientation of the myosin filaments within the fibril remained to be solved. Rosza, Morgan, Szent-Gyorgyi, and Wyckoff (1950) proved with the aid of replicas of cross sections that the filaments are compactly arranged in a crystalline and not, as was presumed, in a cylindrical formation.

* In press.

ELECTRON-MICROSCOPIC INVESTIGATION OF TISSUE SECTIONS

3 17

FIG. 3. Example of the frozen section technique from Fernindez-Morin, 1950. Section of frog sciatic nerve. Fixed in osmic acid. Frozen section with the Spencer rotary microtome (820X) by means of the thermal expansion method. Shadowed with platinum-palladium. Magnification : 160,000X. Shows the myelin sheath from a transverse section. More than 20 sharp parallel lines arranged concentrically in the sheath can be counted here.

-411 these investigations concern diff erentiated fibrils. The myogenesis of young Triton larvae was investigated by Bretschneider (1949) with the aid of myotome sections. In the young niyoblasts, myosin filaments develop, 25 w p thick and showing not a trace of cross striation, which bundle themselves to form tlie fibrils. Subsequently, thin membranes develop at regular intervals in the sarcoplasm, perpendicular to the course of tlie fibrils ; they are observed throughout the plasnia as telophragms and niesophragms and give rise to annular densities surrounding the fibrils. This primary indication of cross striation is followed in older niyoblasts, probably originating from granular structures in the plasma, by the Q-zones, denser cylinders surrounding the fibrils at various points. Apart from the vertebrate muscles, the flying muscles of Peripluizefu aiiierirarra was studied in sections by Richards, Anderson, and Hance

318

L. II. BRETSCHNEIDER

(1942) and those of Aeschna by Bretschneider. In this case, too, the latter found clearly discernible telophragms and observed that the fibrils consisted of myosin filaments, 25 nap thick and of a nodular structure. Diskshaped corpuscles of a reticular ultrastructure were found in the Q-zone.

4 . Myelin Axonal Sheaths Light-microscopic data on the ultrastructure of myelin nerve sheaths are contradictory. Chemically, lipoproteins are known to be the most important factor, but their arrangement could not be determined by light microscopy as the structural picture is highly variable according to the technique used. After various unsuccessful electron-microscopic investigations by Rosza et al. (1950) and Duncan and Antes (1950), FernindezMorin succeeded in visualizing the actual structure by means of electronoptical examination of very thin frozen sections. The myelin sheaths proved to consist of a concentric arrangement of, alternately, protein lamellae of 5 nap and lipoid lamellae of 3 mp. Fernindez-Morhn’s success was based on his efficient technique. He prevented dissociation of the structure by ensuring excellent fixation, while abolishing the alcohol-xylene series, Unsatisfactory fixation is known to affect the lipoids, resulting in the development of the well-known neurokeratin reticulum which was also observed by electron-optical means (Rosza et al., 1950). FernandezMorin succeeded, where these authors had failed. H e demonstrated the disputed presence of axilemmae, visible as a separate, structured membrane on the border between axoplasm and myelin sheath. The thin neurilemmae, provided with collagenous fibrils, were demonstrated electronoptically by Schmitt (1950), Rosza et al. (1950), and Fernindez-Morin (1950). The last mentioned has elucidated the structure of axons by means of a comprehensive diagram.

5. Cell-Sitrface Structures The structure of the free surface of a number of cells was investigated by electron-optical examination of sections. Richards and Anderson (1942), for instance, published a contribution to our knowledge of the cuticular structure of insects. They determined the structure of the chitin cuticle of Periplnneta, which consists of compact protein-chitin lamellae of 150 t a p alternated with equally thick but less compact laniellae. Perpendicular to these layers, the cuticle is perforated by numerous very narrow spiral ducts which do not conimunicate with tlie cellular plasma but terminate abruptly at tlie periphery. Bretschneider (1949) found a similar perforated structure in the egg shell of Ascuris inegalocephala. Light niicro-

ELECTRON-MICROSCOPIC IWESTIGATION OF TISSUE SECTIONS

319

scopy revealed the so-called prismatic cuticular border on the free surface of cover cells in the skin of Amphibia. Electron-optical investigation showed this cuticle to consist of separate olivary corpuscles originating in the plasma and arranged side by side on the surface. They may slightly protrude and are reminiscent of the rhabdites of worms of lower order (Bretschneider, 1951). The brush border of intestinal cells also shows some similarity to this cuticle. Bretschneider (1949) observed that the brush border of intestinal cells of Ascaris consists of fibrillar structures, 80 mp {hick, which remain anchored in the cellular ectoplasm and often penetrate farther than this. No deviating basal structures such as granula or distensions were found. The cilia of three holotrichous ciliates investigated by Bretschneider (1949, 1950) showed a much higher level of differentiation. In analogy with the flagella of flagellates and the tail of spermatozoa, the structure was found to consist of a central axial fibril, a plasmatic mantle, a ciliary sheath, and a spiral fibril. The ciliary sheath extends in the ectoplasm as a solid body forming the ciliary root. Topography, implantation, and intercommunication of the cilia (the so-called silver lines) were studied in detail. 6. The Ultrastructiirg of Cnidia The cnidia of coelenterates show a very high level of differentiation within each cell. The minute proportions of these intracellular structures induced Bretschneider ( 1949, 1950) to institute an electron-optical investigation of sections froin the epithelium of Coryrlactis vim'dis. Although the anlage of the cnidial capsule and the cnidial filament in the vacuole of the cnidoblasts, their final form and structure is very intricate and specific. The capsule membrane proved to consist of protein filaments of 90 A which clearly showed cross striation, with a period length of about 280A. This cross striation is probably correlated with the turgor function of the capsular wall which invaginates itself into the capsule to form a coiled tube, under considerable torsion due to indentations of the wall, arranged in a spiral. Whereas the contents of the penetrants are not electron-optically visible, the capsule of the glutinants reveals a deposit probably containing m u c h The so-called lasso is also demonstrated as a more compact fibril (Bretschneider, 1951*).

7. Extracellular Str iictures Bone, as an extracellular basic substance of the substantia spongiosa of the vertebral bodies, was investigated by Bretschneider ( 1949) in BarZen-

*

In press.

320

L. H. BRETSCHNEIDEB

optera. The decalcified basic substance consists of an intricate pattern of interlocked collagenous fibrils, 100 ncp thick, masked by a homogeneous mass. The minute crevices in the basic substance probably contained the salts dissolved by decalcification, Fern6ndez-Morh ( 1950) examined cross-striated collagenous fibrils of neurilemmae. He observed a certain regularity in their course and caliber. The problem of the ultrastructure of cytoplasm is correlated with that of organic colloids composed of polymers. Electron-optical investigations have already revealed numerous similarities between the structural properties of extracellular structures composed of polymers and related cytoplasmic structures. Electron-microscopic investigation of colloid substances parallel and analogous with the investigation of cells is essential, as the simpler and more familiar models facilitate the interpretation of the data on the much more complicated systems of cells. Sections of various extracellular and “model” colloids such as the mesogloea of a coelenterate, the lymph of Hirudo, the perivitelline fluid of L h n a e a egg and agar-agar examined by Bretschneider (1951) showed that these colloids are also characterized by a specific size of their ultraparticles and, as a colloid basic structure, by a definite arrangement. Certain properties of the ultrastructures, such as cross-striation, established period lengths, arrangement of periods at equal levels in adjacent elements, mutual orientation, etc., could probably often be traced back to the structural properties of macromolecules. An entirely new field, between the chemical physics of colloids and biology, awaits electron-microscopic exploration.

8. Cytopafhological Investigations One of the first electron-microscopic successes in the field of biology was the discovery of viruses (Kausche, Pfankuch, and Ruska, 1939) which was followed by numerous other events. With the exception of bacteriophages, whose host could also be observed by electron-optical means, only extracellulary isolated and purified specimen could hitherto be studied. At present, electron-microscopic investigations of cell sections make it possible to analyze the actual vital phases and phases of multiplication of cells. Black, Morgan, and Wyckoff (1950) succeeded in investigating tobacco mosaic viruses in infected leaf cell sections. They proved that the entire cell is filled with mosaic virus particles, 100 A thick and 2800 A long, lying parallel and close together. Only the nucleus is free from these particles. Bretschneider and Miihlbock ( 1951) observed in sections of mouse mammary carcinoma certain islets of closely packed virus-like particles. Size, compactness, and form are suggestive of hyper-

ELECTRON-MICROSCOPIC INVESTIGATION OF TISSUE SECTIONS

321

trophic mitochondria wherein the virus probably multiplies. Rupture of the nuclear membrane, marked hypertrophy of the nucleus of the tumor cell, the occurrence of chromidial plasma structures, and various signs of degeneration are described as typical pathological characteristics. Pathological processes and structures in various pathological and neoplastic tissue sections were investigated electron-microscopically by Gessler, Grey, Schuster, Kelsch and Richter (1948). They also observed partial disappearance of the cellular and nuclear membrane, atrophy of the nuclei, karyolysis and the occurrence of fibrillar structures in the plasma. Although the above-mentioned investigations leave many details and problems unsolved, they constitute the first attempt at the investigation of the ultrastructure of cells in pathological conditions, which is of undeniable importance. VI. REFERENCES Ardenne, M. v. (1939) 2. .wiss. Mikr., 66, 8. Ardenne, M. v. (1941) NatunuissEnschaften, A ! S, 521, Baker, R. F.,and Pease, D. C. (1949) 1. appl. Phys., 21, 480. Beams, H. W., Evans, T. C., Baker, W. W., and Breemen, V. v. (1950a) . h a t . Rec., 101, 329. Beams, H. W., Evans,T. C., Baker, W. W., and Breemen, V. v. (1950b) Proc. SOC. ex$. Biol. Med., 74, 717. Black, L. M., Morgan, C., and Wyckoff, W. G. (1950) Proc. SOC.exp. Biol. Med., 73, 65.

Bretschneider, L. H. (1949) Proc. Konin Kl. Nederlan. Akad. Amsterdam, 63, 654. Bretschneider, L. H. (1950a) Proc. Konin KI. Nederlan. Akad. Amsterdam, 634 B. 675, 1476.

Bretschneider, L. H. (1950b) Mikroscopie, Vienna 6, 15, 257. Bretschneider, L. H. (1951) Proc. Konin KI. Nederlan. Akad. Alirstcrdam, 64, 89. Bretschneider, L. H., and Miihlbock, 0. (1951) (In press.) Callan, H. G., Randall, J., and Tomlin, S, G. (1950) Proc. Roy. SOC.187, 32. Claude, A., and Fullam, E. F. (1946) J. exp. Med., 88, 499. Claude, A. (1947-48) Heruey Lect., 47, 121. Danon, D., and Kellenberger, E. (1950) Arch. Sci. Gedve, 3, 169. Duncan, D., and Antes, L. (1950) Tex. Rep. Biol. Med., 8, 329. Fernhdez-Morhn, H. (1949) Experientia, 6, 339. Ferdndez-Morh, H. (1950) Exp. Cell. Res., 1, 309. Fullam, E. F., and Gessler, A. E. (1946) Rev. sci. Zmtrurn., 17, 23. Gessler, A. E., and Fullam, E. F. (1946) Amer. J . Anat., 78, 245. Gessler, A. E., and Grey, C. E. (1947) Ex#. Med. Surg., 6, 307. Gessler, A. E., and Grey, C. E. (1947) Ex#. Med. Surg., 4, 329. Gessler, A. E., Grey, C. E., Schuster, M. C., Kelsch, J. J., and Richter, M. N. (1948) Cancer Res., 8, 534. Kausche, G. A., Pfankuch, E., and Ruska, H. (1941) Natzirwiss., 573. Latta, H., and Hartmann, J. F. (1950) Proc. SOC.exp. Biol. Med., 74. 124. MonnC, L. (1948) Advances in Enzymology, 8, 30. Miihlbock, O., and Bretschneider, L. H. (1952) (In press.)

322

L. H. BRETSCHNEIDER

Newman, S. B., Borysko, E., and Swerdlow, M. (1950) Bur. Starid., 4S, 183. O’Brien, H. C., and McKinley, G. M. (1943) Scieme, 98, 455. Pease, D. C., and Baker, R. F. (1948) Proc. SOC.exp. Bwl. N . Y., 67, 470. Rhoades, R. P. (1949) Proc SOC.exp. BioL, N . Y., 73, 93. Richards, G. A., and Anderson, Th. F. (1942) J. Morph., 71, 135. Richards, G. A., Anderson, Th. F., and Hance, R. T. (1942) Proc. SOC.exp. Biol. Med., 61, 148. Rosza, G., Morgan, C., Szent-Gyiirgyi, A., and Wyckoff, R. W. G. (1950) Srience, ll!d, 653. Schmitt, F. O., and Geren, B. B. (1950) J. exp. Med., 91, 499. . niicr. Sci., 91, 145. Way, M. J. (1950) Q ~ o r t J.

The Histochemistry of Esterases” G. GOMORI Drpartinmt of Medicine, The University of Chicago, Chicago, Zll~iiois

For the purposes of this presentation esterases will be defined as enzymes hydrolyzing carboxylic acid esters of alcohols and phenols. The classification of esterases on the basis of biochemical investigations is still a partly controversial issue, but can be said to have crystallized around certain well-established principles, such as substrate specificity and the effect of activators and inhibitors. The following scheme, which includes only such enzymes as are of histochemical interest, is an attempt to integrate known biochemical differences between esterases into a logical system. (Table I.) This classification requires a few words of explanation. First of all, the differences mentioned are relative rather than absolute. For instance, esterase has no real preference for esters of aromatic acids but will hydrolyze them much more readily than lipase; D I P F P is actually a more powerful inhibitor of both types of cholinesterase than nitrogen mustard ; however, the ratio (Inhibition by DIPFP/Inhibition by nitrogen mustard) is much higher in the case of pseudocholinesterase than in the case of true cholinesterase. The differences between aliesterase and cholinesterase are much less marked than was initially believed. Actually, both aliesterase and cholinesterase will hydrolyze choline as well as noncholine esters, (Stedman, Stedman, and White, 1933; Easson and Stedman, 1937; Mendel and Rudney, 1943; Whittaker, 1949), and not even necessarily at very different rates. The main difference between the two large groups appears to be in their sensitivity to eserine and similar alkaloids. While aliesterases are very little influenced even by M eserine, cholinesterases are comM or less (Easson and Stedman, 1937; Richter pletely inhibited by and Croft, 1942). Enzymes of various sources may exhibit strikingly different properties. For instance, liver esterase shows all the typical features enumerated under “esterase,” and pancreatic lipase all the features of “lipase.” On the other hand, the enzymes of other organs such as the kidney, intestine, and the lung are very hard to classify because in some respects they may *This work has been done under grants from the Douglas Smith Foundation for Medical Research of The University of Chicago, and from the Pathology Study Section of the United States Public Health Service.

323

TABLE I Biochemical Differences Between Esterases I. Aliesterases (Richter and Croft, 1942) - substrates: esters of N-free alcohols. A. Lipase 1. Substrate preferences a. Chain length of fatty acid (Terroine, 1920; Schghheyder and Volqvartz, Long (>W 1944) b. Branching of chain of fatty acid (TerStraight chain roine, 1920) c. Aliphatic or aromatic nature of fatty Aliphatic acid (Terroine, 1920) Glycerol d. Nature of alcoholic moiety e.

Rates of hydrolysis of nitrophenol esters of C, to C. fatty acids (Huggins and Moulton, 1948)

f. Optical isomers (Willstgtter and Memmen, 1924; Ammon, 1930; Rona and Ammon, 1933)

w

2

B. Esterase

Short ( 4 2 ) Is0 chain

Aromatic Monohydric alcohols

n n

0

2<3<4<5

2<3>4>5

g

0

m

U

The two types of enzymes often favor opposite o p t i d isomers in an unpredictable way.

2. Activators and inhibitors. a. Quinine (ROM and Pavlovir, 1922; 1923; Rona and Takata, 1923; ROM and Haas, 1923)

Inhibition

No effect

b. Arsanilic acid (Rona and PavlovZ, 1922, 1923; Rona and Haas,1923)

No effect

Inhibition

c. Fluoride (Loevenhart and Peirce, 1906)

Slight inhibition

Marked inhibition

d. Bile acids (Willstatter and Memmen, 1924b)

Activation

Inhibition

TABLE I (Coittinued) Biochemical Differences Between Esterases

4

z

m

11. Cholinesterases - substrates : esters of choline. A. So-called true or specific cholinesterase 1. Substrate preference (Nachmansohn and Rothenberg, 1944)

Acetylcholine

B. So-called pseudo- or nonspecific cholinesterase Other choline esters

ze

rn

x

l! CI

2. Optimal substrate conc. (Mendel, Mundel, and Rudney, 1943; Augustinsson, 1948) 3. Selective inhibitors (Mendel and Hawkins, 1947; Adams and Thompson, 1948; Adams, 1949)

m

e

Low (klO-s.5 M )

Nitrogen mustard

High (-110-2 M )

Di-isopropylfluorophosphate (DIPFP);percaine (Zeller and Bisegger, 1943)

0

<

E1 M

cn

m

v1

326

G . GOMORI

behave as an esterase and in others as a lipase. Whether or not we are dealing with mixtures or with a family of closely related enzymes which differ from each other in various minor respects cannot be decided at this time. Altogether, the problem of classification is a much more complex one than it would appear from the relatively simple scheme of Table 1. It is beyond the scope of this presentation to quote the very extensive literature in detail. Histochemical techniques have produced a number of new and unexpected results which have changed our ideas about the specificity of esterases. In the case of biochemical studies, tissue extracts contain the constituents of a large variety of cells and an unpredictable array of enzymes. These enzymes can be separated into reasonably pure fractions only if the solubility properties of the individual components are exactly known. In histochemistry, on the other hand, a number of different enzymatic behaviors can be observed directly, within one sample, with as much sharpness as the resolving power of the microscope permits. In many cases a relatively few individual cells, scattered among a vast majority of cells of a different type, are found to be enzymatically different from their environment. Before going into the details of the results obtained by the use of histochemical techniques, it is necessary to review the chemical background of the methods. The natural substrates of lipases would be true fats and oils but, being insoluble in water, they cannot be used for histochemical purposes. (It is obvious that even the finest suspensions cannot penetrate cells and produce pictures reflecting the true localization of enzymes.) However, by esterifying fatty acids with highly hydrophilic alcohols, it is possible to prepare substances fairly closely related to fats and yet soluble in water. Such substances are (1) the Tweens, which are long-chained ( CIZto CIS) fatty acid esters of sorbitan, the remaining hydroxyl groups of which are etherified with ethylene oxide chains of various lengths, and (2) similar synthetic compounds, usually esters of polyethylene glycols, marketed under various trade names. On hydrolysis by enzymes they yield fatty acids which are precipitated, in the presence of Ca salts, as highly insoluble Ca soaps. The latter can be subsequently transformed first into lead soaps and then into brown-black lead sulfide. This technique will henceforth be referred to as the Tween technique (Gomori, 1945). The simplest histochemical substrates for esterases are acetates of naphthols, especially of a- and P-naphthol and of naphthol A S (the anilide of 2-hydroxy-3-naphthoic acid). I n the presence of diazonium salts the

THE HISTOCHEMISTRY OF ESTERASES

327

naphthol liberated enzymatically yields intensely colored, insoluble azo dyes. The original naphthol method for esterase as first described by Nachlas and Seligman (1949) is not entirely satisfactory because of the diffusion artifacts it produces. The following modifications yield sharp localizations : The a-Nnplithol Method. Into about 40 to 45 nil. of distilled water blow about 1 nil. of a 1 per cent solution of a-naphthol acetate in 50 per cent acetone. Add a few milliliters of a 0.2 M solution of N a 2 H P 0 4 (pH slightly in excess of 8 ) . Add about 20 to 50 mg. of either diazotizetl 4-benzoylamin0-2,5-dimethoxyaniline (Diazo Fast Blue RR Salt) or diazotized 4-ch1oro-2-aminoanisole (Diazo Red RC Salt) to the mixture and stir it thoroughly. Incubate sections at room temperature for five to twenty minutes, depending on the intensity of shade desired (with Fast Blue RR Salt, black; with Red RC Salt, rusty red). For optimal results it is highly advisable to stir the solution vigorously during incubation in order to prevent diffusion artifacts. In the case of very active tissues, it may be necessary to cool the mixture. The Naphthol AS Method. Naphthol A S acetate (melting point, 161+0.5"C) is not available commercially; it must be prepared by esterifying naphthol AS with acetic anhydride in pyridine. To about 5 to 10 ml. of propylene glycol add 0.5 ml. of a 1 per cent solution of naphth61 AS acetate in 50 per cent acetone, and shake the mixture. Fill it up with water to about 50 ml. with contipuous stirring. Add a few milliliters of a 0.2 M phosphate buffer of pHk6.5. The solution may be slightly opalescent, Add 20 to 50 mg. of diazotized ortho-amino-azotoluene (Diazo Garnet GBC Salt) and stir the mixture. Incubate slides at room temperature for thirty to ninety minutes. Sites of esterase activity will appear in a carmine red shade. The yellowish background can be removed almost completely with acid alcohol (1 per cent HCI in 80 per cent alcohol). Counterstain with a suitable nuclear dye (hematoxylin or alum carmine, depending on the shade of the reaction). Mount sections in glycerol-jelly. For cholinesterases, two methods have been published, the long-chained fatty acid ester technique (Gomori, 1948a) and the thiocholine technique (Koelle and Friedenwald, 1949). The latter method requires the use o f unfixed frozen sections and, judging from the illustrations given, localization is less sharp than with the former technique, which utilizes paraffinembedded tissue.

328

G . GOMORI

RESULTS The Twecit Technique. The hydrolysis of Tweens is influenced by the presence of a number of substances which are known to be inhibitors or activators from biochemical experiments (Gomori, 1948a). Some of the histochemical results are in perfect agreement with biochemical findings ; others are indicative of overlapping between enzymes. Bile acids, for example, differentiate sharply between pancreatic lipase, which is greatly activated, and esterases of other organs, which are markedly inhibited. The only other lipolytic enzyme besides pancreatic lipase to be activated by bile acids is that of the chief cells in a limited area of the mouse stomach. This enzyme is not found in every mouse. As a rule the reaction is considerably more intense in females than in males. The gastric enzyiiie of man, the dog and the rabbit is inhibited by cholates. Quinine, described as a specific inactivator of lipase, inhibits pancreatic and gastric (mouse) enzymes strongly ; however, its effect on the intestinal enzyme, which is

FIG.1. Chief cells in the depths of the gastric glands of the mouse, intensely staining by the Tween method (substrate : Tween 80).

an esterase rather than a lipase, is even more marked. In the presence of arsanilate, a typical inhibitor of esterase, the enzymes of the liver, kidney, and intestine are completely inactive, whereas the lipases of the pancreas and of the mouse stomach are but slightly influenced. The esterase of whole mouse testis is resolved histochemically into two com-

T H E HISTOCHEMISTRY O F ESTERASES

329

poneiits, that of the spermatic elements which is insensitive to arsanilate and that of the interstitial cells which is markedly inhibited by it. M ) has practically no effect on the hydrolysis of Eserine (up to Tweens When Tween-like compounds containing unsaturated fatty acids (oleic, ricinoleic, linoleic, etc.) are used as substrates, only the pancreas and the chief cells of the mouse stomach (“true lipase”) show a good activity. All the other sites that are active with saturated substrates are almost entirely negative (Gomori, 1949). Whether the minimal reaction at these latter sites is due to a slight activity of the enzyme toward the unsaturated substrate or to the presence of a small amount of saturated esters (as impurities) in the unsaturated compound cannot be decided. With the thaphtholic substrates, the distribution of the reaction is quite similar to, but more widespread than that obtained with the Tweens. Many structures, completely negative by the Tween method, are more or less intensely positive by the naphthol methods (Brunner’s glands, macrophages, nervous elements in a number of species ; convoluted tubules of the human kidney, etc.). This is not merely an expression of the superior sensitivity of the azo dye technique since the two types of reactions do not run parallel. Some of the Tween-negative structures are intensely naphthol-positive while others which show a strong Tween reaction are only moderately stained by the azo dye technique. In fact, there are a few examples of strongly Tween-positive structures which show no naphthol reaction at all (chief cells of the mouse stomach; spermatic elements of the mouse testis). The topographic patterns of activity as shown by the two naphtholic substrates mentioned (acetates of a-naphthol and of napthol AS), although closely similar, are not identical. There are a large number of structures which will stain far more intensely with one of the substrates than with the other one. For instance, the villi in the duodenum of the rat, certain unidentified cells in the heart of the rat, the septa1 cells of the mouse lung, etc., are strongly positive with a-naphthol and only faintly positive or entirely negative with naphthol AS ; the opposite holds for Brunner’s glands of the rat, for human mast cells, and for large motor cells of the brain of man and the rat. The pictures obtained with P-naphthol appear to be identical with those given by a-naphthol. This is surprising since naphthol AS is a derivative of P-naphthol. The specific enzymes responsible for the hydrolysis of the two naphtholic substrates will be referred to as “a-esterase” and “AS esterase,” respectively. The henzoates of the naphthols mentioned were also tried as histo-

.

330

G . GOMORI

FIG.2. The distribution of a-esterase in the duodenum of the rat. Villi intensely stained ; no reaction in Brunner’s glands.

chemical substrates. They are hydrolyzed very much more slowly than the corresponding acetates, but the pictures obtained with the two groups of esters are indistinguishable from each other. The existence of a specific “acetylesterase” (Jansen and co-workers, 1947 and 1948) in animal tissues lacks histochemical support. The activators and inhibitors mentioned under the Tween techniques have no effect whatsoever on the hydrolysis of naphthol esters, except that eserine ( loe5M) appears to inhibit the activity of the muscle spindles of the mouse considerably. The results of the long-chained fatty acid ‘ester technique for cholinesterase show a number of highly interesting features. First of all, species differences are very marked. Some species (mouse, dog) hydrolyze these esters very readily and many of their organs inhibit activity. In others (man) the sites of activity are very limited (arborizations around synipathetic ganglion cells, adrenal medulla) ; still others (practically all species below birds, phylogenetically speaking) do not seem to possess

THE HISTOCHEMISTRY OF ESTERASES

33 1

FIG.3. The distribution of AS-esterase in the duodenum of the rat. Brutiner’s glands intensely stained; no reaction in the villi.

cholinesterases which will attack these substrates despite the fact that they are known to hydrolyze acetylcholine very vigorously. Especially interesting is the complete histochemical inactivity of the electric organ of the electric eel, one of the best sources of acetylcholine esterase. As a rule, the rate of hydrolysis rapidly drops with increasing chain length. Lauroyl choline is the fastest and palniitoyl choline the slowest of the three substrates (C12,C14, and Cle) extensively investigated. However, some species (rat, pigeon) hydrolyze the palmitoyl ester more rapidly than the shorter-chained ones. There are also examples of organ specificity. The spermatic elements of the mouse do not attack lauroyl choline at all, myristoyl choline sluggishly and palmitoyl choline quite vigorously although in other mouse organs the substrate preference runs in the opposite direction. In a few cases the fine localization of activity varies with the substrate. In the sympathetic ganglia of the rabbit, the arborizations around ganglion cells stain with palmitoyl choline, and the satellite cells, with lauroyl and myristoyl choline.

332

G. GOMORI

FIG.4. Heart of the rat: peculiar cells with tentacle-like processes, strongly positive for a-esterase.

In the case of lauroyl or myristoyl choline as substrates

to low6M

of eserine abolishes all reaction. With palmitoyl choline, the activity of the muscle spindles of the mouse is completely inhibited; in other

organs inhibition is only partial. The type of cholinesterase denionstrated by the long-chained fatty acid technique cannot be defined in terms of the biochemically accepted nomenclature. Some of the “true” cholinesterases hydrolyze the histochemical substrates very readily; others do not. The same applies to the “pseudo” type of enzyme. The ability or inability to hydrolyze these substrates bisects the class of cholinesterases in a plane entirely different from that which divides them into the two biochemical types, and no common denominator can be found for the two classifications. The fhiocholine technique utilizes the acetate and butyrate of thiocholine (Koelle, 1950). It should be remarked here that the specificity of thiochoIine esters for the two types of cholinesterases is less marked than that of the corresponding choline analogues. Whereas acetylcholine and mecholyl are specific substrates for true cholinesterase and are but sluggishly hydrolyzed by the “pseudo” enzyme, the corresponding thio compounds are readily attacked by both types of cholinesterase. Butyryl thiocholine seems to be attacked by the nonspecific (“pseudo”) enzyme only. The nonspecific enzyiiie, regardless of the substrate used, is almost

T H E HISTOCHEMISTRY OF ESTERASES

333

completely inhibited by thirty minutes preincubation with M DIPFP, whereas the true enzyme is inhibited only to the extent of 40 per cent. According to Koelle, Friedenwald, Koelle, and Wurzbacher (1950), any reaction obtained with butyryl thiocholine as a substrate is due to the “pseudo” enzyme only, while any residual activity towards acetyl thiocholine after pretreatment with DIPFP must be ascribed to the true enzyme. The authors conclude that in the cat, the brain, spinal cord, striated muscle, preganglionic fibers, the smooth muscle of bronchioles and urinary bladder contain almost exclusively the specific (true) type of enyzme; whereas the sensory ganglia, the longitudinal muscle of the ileum, and the liver contain almost exclusively the nonspecific type ; other ganglia and the adrenal medulla, both types. It is interesting to note that in the liver of the cat the hydrolysis of acetylthiocholine is inhibited by D I P F P only to the extent of 70 per cent; this is contrary to the general consensus and the authors’ own statement that the hepatic enzyme is of the nonspecific type. At first glance, the results of histochemical methods appear to be chaotic. However, this impression is created mainly by the sketchy and arbitrary enumeration of findings in the preceding passages. After careful comparison of hundreds of slides, representing many tissues of a fair number of species, one is bound to arrive at the conclusion that animal tissues contain three cardinal types of esterases with narrowly defined substrate specificities. In addition, there are a number of enzymes of an intermediate character, hydrolyzing the substrates of two or even of all three, of the main types. Whether the transitions actually represent individual enzymes with intermediate properties or rather mixtures of the pure types cannot be decided at this time. The scheme to be shown is based on the results of the Tween, azo dye, and long-chained fatty acid choline ester techniques only; those of the thiocholine method, with which the writer has had no personal experience, are not included. Needless to say, it does not by any means claim to be a classification of esterases in general but only of those esterases which are demonstrable in paraffin sections by the methods enumerated. It is readily admitted that the classification suggested is quite complicated and yet incomplete in that it does not even attempt to analyze the puzzling differences among cholinesterases. However, on the basis of the relatively meager body of findings at hand it is hardly possible to present anything better than this tentative effort. A more satisfactory classification of esterases will have to be based on the systematic investigation of the tissues of many species by all the methods available.

334

G. GOMORI

TABLE I1 The Histochemical Classification of Esterases Substrates

Examples

Enz ypes

I. Main types A. Lipase B. Esterase

++--+ + -

- -

B,.a-Esterase

- -

B,. AS esterase

-

-

-

+ to* to +

-

&

C. Cholinesterase

11. Intermediate types

.

A-B

-

- - -

+

+-+++*

A-BI

+ - + t o -

A-B2

t

Chief cells of mouse stomach. Chief cells of rabbit and dog stomach; human renal tubules ; sympath. ganglia in several species Unidentified cells in the rat heart; ampullary gland of the mouse. Brunner’s glands of the rat ; cells of pancreatic islets of rat; motor cells of man and rat; astroglia of cat. Human adrenal medulla ; certain cells and tracts of mouse brain; arborizations around sympath. ganglion cells in several species. Chief cells of human stomach; pancreas of rat and mouse (also hydrolyze unsat. Tweens) ; bronchial epithelium in several species ; interstit. cells of rat testis. Duodenal villi of the rat; septa1 cells of mouse lung.

+ - t o + -

+ - - f- + - - + + +

No examples known.

A-C B-C

Bi-C B3-C

- - + - - -

Spermatic elements of mouse testis. Muscle spindles of mouse ; conductive system of dog’s heart ; Bowman’s capsules in dog’s kidney. No examples known. No examples known. Pancreas of several species (also hydrolyzes unsat. Tweens) ; liver and intestine of several species.

.4-B-C

-+ + + + +*+ + +

T H E HISTOCHEMISTRY OF ESTERASES

335

REFERENCES

Adams, D. H. 1949) Biochem. et Biophys. A d a , 3, 1. Adams, D. H., and Thompson, H. S. (1948) Biochem. J., La, 170. Ammon, R. (1929-1930) Fermenfforschung, 11, 459. Augustinsson, K. B. (1948) Nature, 162, 194. Easson, L. H., and Stedman, E. (1937) Biochem. J., 31, 1723. Gomori, G. (1945) Proc. SOC.Exptl. Biol. Med., 68, 362. Gomori, G. (1948) Proc. SOC.Exptl. Biol. Med., 67, 4. Gomori, C . (1948b) Proc. SOC.Exptl. Biol. Med., 68, 354. Gomori, G. (1949) Proc. SOC.Exptl. Biol. Med., 72 :697. Gomori, G. (1949) Proc. SOC.Exptl. Biol. Med., 72, 697. Huggins, C., and Moulton, S. H. (1948) J. Exptl. Med., 88, 169. Jansen, E. F., Jang, R., and MacDonnell, L. R. (1947) Arch. Bbchem., 16, 415. Jansen, E. F., Nutting, M. D. F., and Balls, A. K. (1948) I. Biol. Chem., 175, 975. Koelle, G. B., and Friedenwald, J. S. (1949) Proc. SOC.Exptl. Biol. Med., 70, 617. Koelle, G. B. (1950) J. Pharmacol. Exptl. Therap., 100, 158. Koelle, G. B., Friedenwald, J. S., Koelle, E. S., and Wurzbacher, W. (1950) J. Natl. Caiccer Inst., 10, 1364. Loevenhart, A. S., and Peirce, G. (1906-1907) J. Biol. C k m . , 2, 397. Mendel, B., and Hawkins, R. D. (1947) Biochem. J., 41, xxii. Mendel, B., Mundell, D. B., and Rudney, H. (1943) Biochem. J., 97, 473. Mendel, B., and Rudney, H. (1943) Biochem. J., 37, 59. Nachlas, M. M., and Seligman, A. M. (1949) 1. Natl. Cancer Znst., 9, 415. Nachlas, M. M., and Seligman, A. M. (1949) Anat. Record., 106, 677. Nachmansohn, D., and Rothenberg, M. A. (1944) Science, 100, 454. Richter, D., and Croft, P. G. (1942) Biochem. J., SS, 746. Rona, P., and PavloviE, R. (1922) Biochem. Z., 190, 225. Rona, P., and PavloviE, R. (1923) Biochem. Z., l34, 108. Rona, P., and Takata, M. (1923) Biochem. Z., 194, 118, Rona, P., and Haas, H. E. (1923) Biochem. Z., 141, 222. Rona, P., and Ammon, R. (1933) Enzymforschwg, 2, 50. Schghheyder, F., and Volqvartz, K. (1944) Enzymologia, 11, 178. Stedman, E., Stedman, E., and White, A. C. (1933) Biochem. J., 27, 1055. Terroine, E. F. (1920) Antz. sci. $tot. ZOO^., [XI 4, 1. Whittaker, V. P. (1949) Biochenz. J., 44, xlvi. Willstatter, R.. and Memmen, F. (1924) 2. physiol. Chem., 198, 216. Zeller, E. A,, and Bisegger, A. (1943) Helv. Chim. Acta, 26, 1619.

This Page Intentionally Left Blank

Author Index’” Numbers in italics indicate p a p on which the references are liatrd a t the end of each article.

A Abbot, E. E., 219, 225, 227, 239, 240, 243, 250

Achard, J., 231, 2j0 Adam, N. K., 37, 62 Adanis, D. H., 325, 335 Agnew, W., 219, 254 Alexander, P., 229, 252 Allen, L. A., 102, 104 Allen, N. S., 20, 24 Allfrey, V. G., 23, 25 Altmann, R., 35, 52, 54, 62 Amici, G. B., 2, 6 Ammon, R., 324, 335 Anderson, T. F., 124, 133 Anderson, Th. F., 307, 308, 317, 318, 323 Andrew, N. V., 287, 299 Andrew, W., 283, 287, 299 Anson, M. L., 244, 250 Antes, L., 313, 318, 321 Apathy, 308, Appleby, J. C.,102, 104 Ardenne, M. J., 308, 310, 321 Argaud, R., 269, 299 Arnold, J., 5, 6, 6 Astbury, W. T., 102, 104, 136, 137, 162, 163, 277, 278, 299 Atkin, R. W., 227, 250 Augustinsson, R. B., 325, 335 Avery, P., 56, 63

B Bab, H., 298, 299 Badian, J., 94, 95, 97, 104 Bailey, K. H., 163 Bailey, W. T., 125, 133 Baker, J. R., 2, 3, 6, 54, 62, 212, 213, 214, 215, 242, 250, 273, 274, 294, 299, 300 Baker, R. F., 39, 47, 53, 63, 308, 321 Baker, W. W., 308, 315, 321 Balbiani, E. G., 4, 6 Balls, A. K., 330 (see Jansen), 335 Baltzer, F., 172, 184, 186, 188, 192 Bancroft, W. D., 212, 250 Banga, I., 136, 140, 163

337

Barany, E. H., 79, 80, 89 Barber, H. N., 11, 21 Bardos, T. J., 31, 34 Barker, S. B., 81, 89 Barron, E. S. G., 73, 74, 89, 262, 263 Bartelmez, G. W., 40, 54, 62 Barth, L. G., 185, 192 Bartholomew, J. W., 94, 104 Bateson, W., 19, 24 Battaglia, Emilio, 20, 24 Baumann, C. A., 31, 34 Bayliss, W. M., 36, 41, 42, 62 Beams, H. W., 242, 251, 307, 308, 315, 321

Beck, L. V., 89, 89 Becker, S. W., 284, 285, 300 Beebe, J. M., 97, 104 Behrman, H. T., 274, 302 Bejdl, W., 231, 250 BElar, K., 5, 6 Bell, F. O., 163 Benfenati, A., 290, 300 Bennett, H. S., 279, 300 Bensley, R. R., 36, 38, 51, 56, 62 Benzer, S., 121, 126, 130, 133 Berger, C. A., 19, 25 Berrill, N. J., 11, 25 Bethe, A,, 211, 217, 218, 250 Bevelander, G., 56, 62, 281, 297, 302 Bidder, P. B., 229, 252 Biesele, J. J., 282, j00 Biesele, 11. ?.‘I., 282, 300 Billingham, R. E., 285, 286, 300 Bisegger, A., 335 Bisset, K. A., 93, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 104 Bizzozero, G., 268, 271, 272, 274, 298, 300 Black, L. M., 307, 320, 321 Blackberg, S. N., 285, 302 Blangey, L., 214, 251 Bloch, N., 300 Block, R. J., 277, 300 Blumenfeld, C. &I.,288, 300 Boche, Robert D., 17, 25 Bogdanove, E. M., 81, 89

338

AUTHOR INDEX

Boivin, A., 16, 25 Bolles Lee, A., 54, 62 Bolliger, A., 281, 300 Bond, T. J., 31, 34 Bondarenko-Zozulina, M . I., 100, 104 Bonin, W., 250 Boricious, J. K., 244, 252 Born, S., 267, 300 Borysko, E., 307, 312, 321 Boulton, J., 245, 250 Bourne, G., 83, 84, 89, 213, 214, 215, 242, 250, 282, 297, 300

Bowen, R. H., 291, 292, 300 Brachet, J., 22, 25, 166, 176, 185, 192, 209, 210

Bradfield, J. R. G., 6, 6, 66, 89 Brady, T. G., 87 (see Conway), 89, 157, 163

Branca, A., 269, 276, 300 Breeman, V. v., 308, 315, 321 Bretschneider, L. H., 307, 308, 310, 313, 314, 315, 316, 317, 318, 319, 320, 321

Bridges, C. B., 19, 25 Briggs, R., 166, 170, 171, 172, 175, 176, 178, 182, 190, lQ2, 217, 235, 250

Brillanti, F., 290, 300 Brinkman, A., 298, 300 Broders, A. C., 288, 300 Brooks, S. C., 136, 155, 163 Broquist, H. P., 31, 33 Brieger, E. M., 9S, 104 Broadbent, D., 108, 118 Bronfenbrenner, J., 132, 134 Broquist, H. P., 34 * Brown, D. E. S., 138, 164 Brown, Meta, S., 19, 25 Brown, R., 108, 114, 115, 116, 118 Brown, R. B., 216, 217, 223, 246, 250 Brown, T. McP., 103, 105 Brunton, T. L., 3, 7 Buck, John B., 17, 25 Bull, A. W., 217, 235, 250 Bull, H. B., 135, 157, 163 Bulliard, H., 279, 301 Bullough, H. F., 289, 300 Bullough, W. S., 288, 289, 298, 300 Bunting, H., 220, 251, 2’14, 300 Burdon, K. L,95, 100, 104 Burnet, F. M., 125, 133

Burstrom, H., 115, 118, 156, 164 Burton, E. F., 42, 62 Burton, J. K., 94, 105 Busacca, A., 267, 300 Butcher, E. O., 293, 300 C Cain, A. J., 273, 274, 294, 300 Cajal, R. y., 272, 273, 300 Calberla, E., 4, 7 Callan, H. G., 316, 321 Calvery, H. O., 281, 300 Canti, R. G., 151, 163 Carlene, P. 227, 250 Carleton, A., 288, 300 Carpenter, W. B., 1, 2, 7 Carrel, A., 142, 152, 163 Carlson, J. G., 196, 210 Carruthers, C., 283, 304 Carton, E., 87 (see Conway), 89 Cartwright, P., 108, 118 Caspersson, T . , 9, 20, 25, 53, 57, 62, 209,

w.,

210, 281, 300

Cassel, W. A., 98, 105 Catcheside, D. G., 4, 7 Catchpole, H. R., 51, 63 Chalkley, H. W., 207, 210 Chambers, R., 89, 89, 137, 163, 203, 207. 210, 268, 301

Champetier, G., 277, 278, 301, 302 Chapman, L. M . , 225, 227, 239, 250 Chargaff, E., 22, 25 Chase, H. B., 266, 275, 285, 292, 299, 301, 303

Chatterjee, H., 249, 254 Cheng, K. C., 20, 25 Chevalier, C., 1, 7 ChCvremont, M., 301 Christensen, H. N., 88, 89 Christoff, M., 20, 25 Christoff, M. A., 20, 25 Civilong, B. M., 37, 62 Clara, M., 290, 298, 301 Clark, A. M., 161, 163 Clark, F. E., 218, 226, 253 Clark, F. J., 11, 25 Claude, A., 53, 63, 307, 310, 321 Claude, E., 273, 303 Clegg, H., 235, 239, 240, 254

339

AUTHOR INDEX

Cobb, D. M., 86, 90 Cohen, S. S., 121, 126, 128, 129, 133 Cohn, E. J., 220, 234, 251 Cole, K. S., 140, 163 Colowick, S. P., 72, 89 Commandon, J., 161, 163 Commoner, B., 12, 25 Conn, H. J., 212, 213, 214, 223, 251 Conway, E. J., 87, 89, 157, 163 Cooper, M., 225, 226, 251 Cooper, 2. K., 288, 301 Cori, G. T., 72, 80, 89 Cornbleet, T., 285, 286, 304 Cowan, S. T., 104, 105 Cowdry, E. V., 266, 267, 269, 270, 272, 283, 287, 288, 301, 303

Craig, R., 223, 225, 237, 251 Crane, E. E., 87, 9 4 157, 163 Crane, M. M., 159, 164 Croft, P. G., 323, 324, 335 Crook, H., 219, 225, 227, 239, 240, 245, 250

Crumley, J. A., 219, 254

D Da Fano, C., 272, 301 Dalton, H. C., 174, 187, 188, 192 Daly, M. M., 23, 25 Dan, K., 204, 210 Danielli, J. F., 61, 62, 68, 90, 137, 138, 139, 142, 158, 161, 163, 164 Danneel, R., 286, 301 Danon, D., 310, 321 Darlington, C. D., 9, 21, 23, 25, 120, 133 Davies, R. E., 87, 90, 157, 163 Davson, H., 68, 90, 138, 139, 142, 158, 14.3

Dean, R. B., 163 Deane, H. W., 83, 90 de Fonbrune, P., 161, 163 Dehlinger, J., 72 (see Kalckar), 90 Deineka, D., 272, 301 De Lamater, E. D., 95, 100, 103, 105 Delbriick, M., 122, 125, 126, 131, 133 Delph, A. E., 245, 250 Deme, S., 291, 292, 293, 303 Dempsey, E. W., 55, 62, 83, 90, 213, 220, 221, 251, 274, 280, 301

de Rknyi, G. S., 268, 301 Derksen, J. C., 277, 278, 301

De Sousa, M., 290, 301 Dewey, V. C., 28, 29, 30, 31, 33 Dick, J. C., 268, 301 Dickenson, S., 136, 163 Diczfalusy, E., 77, 90 Dienes, L., 103, 105 Dissosway, C., 127, 133 Dittmar, C., 246, 254 Dixon, M., 77, 90, 136, 163 Dobell, C., 2, 7 Doblin, H., 75, 91 Doermann, A. H., 122, 126, 127, 133 Doerr, Von R., 119, 134 Donhiiffer, S., 80, 90 Dormer, K. J., 107, 115, 118 Douglas, F. W., 227, 250 Drabkin, D. L., 81, 83, 84, 90 Draize, J. H., 281, 300 Dreaper, W. P.. 212, 251 Dublin, W. B., 288, 300 Dubos, R. J., 213, 232, 251 Dulbucco, R., 130, 131, 134 Duncan, D., 313, 318, 321 Duncan, R. E., 15, 21, 25 Dunn, L. C., 23, 25 Duntley, S. Q., 284, 301 Duryee, William R., 22, 25 Dushane, G. P., 188, 192

E Easson, L. H., 323, 335 East, E. M., 19, 25 Ebeling, A. H., 142, 152, 163 Ebling, F. J., 298, 301 Edsall, J. T., 234, 251 Edwards, E. A,, 284, 301 Ege, R., 75, 76, 90 Eggleston, Hems R., 88 (see Stern), 91 Eggleton, M. G., 71, 90 Ehrlich, P., 4, 7, 211, 251 Elbinger, R. L., 88, 89 Ellinger, P., 82, 90 Elliott, G. H., 226, 254 EM, E., 229, 234, 235, 237, 238, 240, 246, 251

Ely, J. O., 83, 91 Emmel, V. M., 39, 48, SO, 56, 62 Ender, W., 225, 251 Engman, M. F., Jr., 283, 302

340

AUTHOR INDEX

Engman, M. F., Sr., 283, 294, 301, 302 Ephrussi, Boris, 23, 25 Erickson, J. O., 244, 253 Erikson, J., 137 (see Neurath), 158 (see Neurath), 160 (see Neurath), 164 Ervin, R. F., 32, 34 Estermann, I., 48, 63 Evans, E. A., Jr., 121, 128 (see Kozloff), 134

Evans, T. C., 308, 315, 321 Eyring, H., 138, 163

F Fabricant, C., 95, 105 Falkenberg, H., 166, 193 Fankhauser, G., 11, 25, 166, 168, 169, 172, 1173, 175, 177, 178, 179, 181, 192, 193 Fautrez, J., 231, 251 Favre, M., 267, 269, 270, 271, 272, 276, 291, 301, 303 Fawzy, H., 114, 116, 118 Feller, W., 73, 91 Fenn, W. O., 86, 90 Fern, A. S., 227, 250 Ferngndez-Moran, H., 307, 313, 314, 318, 320, 321 Ferry, J. D., 220, 226, 243, 244, 251 Ferno, O., 77 (see Diczfalusy), 90 Fidell, I-. I., 48, 251 Fierz-David, H. E., 214, 251 Firkct, J., 269, 270, 276, 301 Fischberg, M., 172, 175, 193 Fischer, A., 4, 7, 212, 247, 251 Fischer, R. B., 286, 303 Fisher, I., 282, 301 Fisher, J. C., 37, 54, 62 Fisher, S., 80, 91 Fitzpatrick, T. B., 284, 285, 302 Flemming, W., 5, 7, 287, 301 Flesch, P., 286, 301, 303 Flewett, T . H., 95, 105 Flood, V., 262, 263 Flosdorf, E. W., 35, 62 Folin, O., 281, 301 Fordham, D., 31, 34 Fothergill, F., 245, 250 Fraenkel-Conrat, H., 225, 226, 244, 251 Franklin, C., 288, 301 Frappier, J., 250

Frederic, J., 279, 301 Freeman, L. W., 286, 303 French, R. W., 251 Frenkel, A., 73 (see Rothstein), 91 Fresnel, A. J., 2, 7 Freundt, E. A., 103, 105 Frey-Wyssling, A., 196, 210 Friboes, W., 267, 268, 301 Friedenwald, J. S., 327, 333, 335 Fry, H. J., 207, 210 Fugitt, C. H., 228, 229, 234, 237, 239, 246, 255

Fullam, E. F., 53, 62, 307, 310, 313, 315, 321

Fuller, R. C., 34 G Gale, E. F., 88, 90 Gallien, L., 170, 193 Galitisky, Irving, 20, 25 Gasvoda, B., 73, 74 (see Barron), 89. 262, 263 Gatenby, J. B., 242, 251 Gates, R. R., 19, 25 Gee, W. W. H., 212, 223, 251 Geitler, Lothar, 14, 15, 25 Gelmo, P., 226, 251 Genderi R., 138, 163 Geren, B. B., 308, 314, 322 Gersh, I., 36, 38, 39, 44, 47, 50, 51, 55, 56, 57, 62, 63 Gerstner, H., 223, 228, 240, 246, 249, 251 Gessler, A. E., 53, 62, 307, 310, 313, 315, 321, 321 Gest, H., 134 Gierke, H., 247, 251 Gilbert, G. A., 228, 229, 251 Gillet, C., 228, 251 Giroud, A., 277, 279, 280, 301, 302 Glick, D., 282, 301 Gluecksohn-Schoenheimer, S., 23, 25 Godwin, D., 169, 192 Goldacre, R. J., 137, 138, 139, 140, 141, 142, 145, 146, 160, 163 Goldschmidt, R., 174, 193 Goldstein, A., 213, 238, 239, 251 Golodetz, L., 274, 276, 304 Gomori, G., 56, 63, 83, 90, 282, 302. 326, 327, 328, 329, 335

AUTHOR IIiDES

Goodall, F. L., 246, 251 Goodspeed, T. H., 36, 40, 41, 43, 47, 51, 56, 63

Gordon, H. A., 32, 34 Gordon, M., 209 (see Villee), 210 Grace, J. B., 97, 98, 105 Graham-Smith, G. S., 98, 105 Granick, S., 227, 253 Gray, S. C., 157, 163 Green, E. U., 182, 192 Green, H., 83, 85, 91 Green, H. N., 288, 300 Green, R. G., 120, 134 Greenberg, D . M., 225, 227, 239, 250 Greenstein, J., 137 (see Neurath), 158 (see Neurath), 160 (see Neurath), 164

Greenstein, J. P., 244, 253 Gregg, J. R., 185, 193 Grell, Sister Mary, 19, 25 Grey, C. E., 307, 310, 313, 321, 321 Griffiths, R. B., 169, 192 Grijns, G., 75, 90 Grimsson, H., 141, 164 Grollman, A., 227, 251 Guensberg, E., 75, 76 (see Wilbrandt), 90, 92

Guilliermond, A., 155, 156, 163 Gustavson, K. H., 279, 302 Gutman, A. B., 84, 90 Gutmann, A., 132, 134 Gutstein, M., 94, 105

H Haanes, M., 103, 105 Haas, H. E., 324, 335 Hadorn, E., 186, 187, 190, 193 Haemmerli, A., 77, 92 Hammerling, J., 10, 25 Hale, C . M. F., 102, 104 Hales, G. S., 103, 105 Halphen, G., 217, 251 Hamburger, H. S., 80, 90 Hamilton, J. B., 39, 44, 47, 48, 49, 50, 52, 63, 275, 283, 287, 292, 293, 294, 295, 296, 297, 298, 299, 302, 303 Hammett, F. S., 279, 302 Hampp, E. G., 103, 105 Hance, R. T., 308, 317, 322

34 1

Hand, D. B., 174, 193 Hanke, M. E., 157, 163 Harris, J. E., 44, 47, 48, 50, 51, 53, 63 Harris, M., 227, 228, 229, 234, 237, 239, 240, 246, 255

Harrison, W., 212, 217, 223, 23S,

251,

252

Hartmann, J. F., 307, 314, 321 Harvey, E. B., 260, 263 Harvey, E. N., 137, 163 Haselmam, H., 96, 105 Hastings, A. B., 86, 92 Hawkins, R. D., 325, 335 Hayashi, T., 262, 263 Haynes, R., 219, 252 Hedin, S. G., 75, 90 Hazel, F., 42, 63 Hawk, P . B., 277, 302 Heilbrunn, L. V., 161, 163, 196, 203, 210 Heidenhain, M., 211, 212, 217, 224, 252 Heine, E., 4, 7 Heinrich, M. H., 30, 33 Henle, G., 133, 134 Henle, W., 133, 134 Henry, H. E., 96, 105 Heringa, G. C., 277, 278, 301 Herlant, M., 196, 210 Herrmann, H., 244, 252 Hershey, A. D., 121, 122, 127, 132, 134 Hertwig, O., 12, 25 yan Herwerden, M. A., 4, 7 Herxheimer, K., 267, 268, 269, 270, 271, 272, 302

Hewitt, L. F., 225, 252 Highman, B., 252 Hillier, J., 95, 105 Hirsch, G. C., 273, 302 Hirshfield, H. I., 161, 164 Hirst, M., 153, 164 Hirst, M. C., 229, 254 Hitchings, G. H., 30, 34 Hodgkin, A. L., 88, 90 Hodgkin, T., 2, 7 Hodgson, H. H., 241, 252 Hober, J., 88, 90 Hober, R., 70, 80, 81, 88, 90, 91, 136, 156, 160, 163

Hogberg, R., 77 (see Diczfalusy), 90

312

AUTHOR INDEX

Hoepke, H., 266, 267, 274, 290, 302 Hoerr, N. L., 36, 38, 39, 40, 41, 42, 43. 47, 48, 50, 51, 56, 62, 63

Hoffmann, C. E., 31, 34 Hofmeister, F., 212, 236, 252 Hollander, F., 157, 163 Holloman, J. H., 37, 54, 62 Holmes, B., 4, 7 Holmes, W. C., 212, 251, 252 Hooker, C. W., 298, 302 Hopkins, D. L., 142, 163 Hotchkiss, R. D., 5 5 , 57, 63 Houck, R. C., 246, 254 Houwink, A. L., 102, 105 Hoyle, L., 133, 134 Hudson, C. L., 81, 92 Huggins, C., 324, 335 Huggins, M. L., 159, 164 Hughes, A., 151, 164 Hughes, A. F., 202, 210 Hughes-Schrader, S., 19, 25 Human, M. L., 129, 134 Humphrey, R. R., 169, 172, 175, 177, 178, 180, 192

Hurwitz, L., 73, 74 (see Rothstein), 91 Huskins, C. L., 11, 14, 15, 19, 20, 25 Huxley, A. F., 88, 90 HydCn, H., 36. 57, 63

I Ikeda, S., 231, 252 Imsenecki, A. A., 100, 105 von Iterson, W., 102, 105

J Jacobj, W., 17, 25 Jacobson, W., 209, 210 Jaeger, L., 185, 192 Jang, R., 330 (see Jansen), 335 Jansen, E. F., 330, 335 Jarvi, O., 95, 105 Jeener, R., 22, 23 Jeghers, H., 284, 302 Johanneson, R. E., 157, 163 Johnson, A. W., 116, 118 Johnson, F., 138, 163 Johnson, P. L., 56, 62, 281, 297, 302 Jollos, V., 181, 193 Jones, D. F., 12, 25

Jones, D. H., 102, 105 Jones, R. McClung, 242, 252 Jukes, T. H., 28, 31, 33

K Kalckar, H. M., 72, 81, 90 Kamen, M. D., 88, 91, 134 Kappel, w., 96, 105 Kausche, G. A., 320, 321 Kawamura, 169 Kaye, M., 278, 302 Kaylor, C. T., 179, 193 Keevil, C. S., Jr., 32, 33 Keilin, D., 273, 302 Kellenberger, E., 310, 321 Kelley, E. G., 223, 228, 231, 243, 245, 252 Kelly, A. J., 219, 254 Kelly, M. W., 217, 231, 255 Kelsch, J. J., 307, 310, 313, 321, 321 Kenyon, J., 287, 292, 296, 298, 299, 303 Keys, A., 274, 303 Kidder, G. W., 28, 29, 30, 31, 33 Kiellander, C. I., 19, 25 Kennedy, J., 134 Kienle, R. H., 245, 252 Kindred, J. E., 230, 252 King, D. T., 44, 47, 53, 63 King, L. S., 269, 270, 276, 278, 302 King, T. J., 182, 192 Kistler, S. S., 37, 63 Kitchener, J. A., 229, 252 Klieneberger, E., 102, 103, 105 Klieneberger-Nobel, E., 95, 97, 103, 105 Klinghoffer, K. A., 75, 90 Klotz, I. M., 228, 229, 237, 238, 239, 240, 252

Knecht, F., 236, 252 Knaysi, G., 95, 105 Knoevenagel, H. E. A., 247, 252 Kodani, Masuo, 20, 25 Koehring, V., 146, 164 Koelle, E. S., 333, 335 Koelle, G. B., 327, 332, 333, 335 Kolliker, A., 266, 302 Kollman, M., 270, 273, 276, 302 Kooyman, D. J., 294, 301 Kopac, M. J., 137, 163 Kosswig, C., 22, 25 Kozloff, L., 121, 128, 134

343

AUTHOR INDEX

Kozawa, S., 75, 90 Krebs, H. A., 88 (see Stern), 91, 212, 247, 253

Kritzler, R. A., 84, 90 Kromayer, E., 270, 302 Krysa, H. F., 286, 304 Krueger, A. P., 120, 134 Krzemieniewska, H., 94, 95, 97, 105 Kung, S. K., 298, 302 Kurnick, J. B., 4, 7 Kutz, R. L., 226 (see Ferry), 243 (see Ferry), 244 (see Ferry), 251 Kvorning, S. A., 275, 293, 302 Kyrle, J., 298, 302

L Laguesse, E., 267, 302 Laidlaw, G. F., 285, 302 La Manna, C., 95, 105 Lambrechts, A., 82, 90 Langerhaus, P., 275, 302 Langmuir, I., 139, 140, 164, 213, 252 Larrabee, C., 73 (see Rothstein), 74, 91 Laramke, A., 250 Laszt, L., 80, 81, 92 Latarjet, R., 129, 130, 134 Latta, H., 307, 314, 321 Lauener, H., 76 (see Wilbrandt), 92 Laug, E. P., 281, 300 leblond, C. P., 280, 302 Leet, M., 69, 91 LeFevre, P. G., 77, 90 Lemin, D. R., 239, 245, 249, 252 Leonard, E., 209 (see Villee), 210 Leonardi, G., 96, 105 Lerner, A. B., 284, 285, 302 Leuchtenberger, Cede, 16, 17, 26 Levanto, A., 95, 105 Levin, 0. L., 274, 302 Levine, N. D., 219, 223, 231, 233, 238, 2-52 Lewis, G. N., 234, 252 Lewis, I. M., 101, 105 Lewis, M. R., 270, 302 Lewis, W. H., 270, 302 Lilienfeld, L., 4, 7, 211, 225, 252 Lindberg, O., 69, 73, 86, 87, 90 Lindegren, C. C., 97, 98, 105 Linderoth, T.. 77 (see Diczfalusy), 90 Lischer, C. E., 269, 303

Lison, L., 36, 63, 209, 210, 284, 294, 302 Lister, J. J., 2, 7 Litvac, A., 269, 277, 278, 280, 301, 302 Lloyd, D. J., 229, 252 Lohnis, F., 102, 105 Loeb, J., 217, 219, 223, 224, 225, 227, 230, 240, 252, 260, 263

Loevenhart, A. S., 324, 335 Lomax, R., 163 Lombardo, C., 281, 282, 296, 302 Longmuir, N. M., 87, 90, 157, 163 Lorch, I. J., 137, 138, 139, 140, 141, 142, 160, 161, 163, 164

Lorz, D. C., 30, 34 Lave, R. M., 19, 25 Loveless, A., 138, 163 Lowe, J. S., 114, 118 Lowens, M., 209 (see Villee), 210 Lubnow, E., 286, 301, 302 Luckey, T. D., 32, 34 Ludford, R. J., 19, 25, 272, 273, 274, 276, 291, 292, 302

Lundblad, G., 207, 210 Lundegardh, H., 136, 156, 164 Lundsgaard, E., 71, 81, 83, 90, 92 Luria, S. E., 121, 122, 123, 125, 126, 128, 129, 130, 131, 133, 134

Luyet, B. J., 37, 41, 54, 63 Lwoff, A., 132, 134

M McCalla, T. M.,218, 223, 226, 253 MacCardle, R. C., 283, 302 McCleary, H. R., 245, 252 McDonald, N. D., 281, 300 MacDonnell, L. R., 330 (see Jansen), 335 McKinley, G. M., 310, 321 Madoff, S., 103, 105 Magnus, W., 211, 253 Mallette, M. F., 95, 105 Mdlory, F. B., 215, 253 Mancini, R. E., 52, 55, 63 Maneval, W. E., 215, 253 Mangold, O., 168, 193 Mann, G., 35, 41, 44, 63, 211, 212, 213, 215, 216, 223, 224, 242, 253

Marsh, J. B., 83, 91 Marshall, E. K., 159, 164 Marslalid, D. A., 138, 164

344

AUTHOR INDEX

Martinotti, L., 266, 267, 268, 269, 276, 277, 303

Masing, 75, 91 Masson, P., 284, 285, 303 Mathews, A., 216, 224, 225, 237, 253 Matsumoto, S., 231, 255 Mayer, F., 214, 253 Mayall, J., 1, 7 Mazia, D., 3, 4, 7, 161, 164 Medawar, P. B., 285, 286, 300 Mehler, A., 72 (see Kalckar), 90 Meier, R., 69, 73, 74 (see Rothstein),

Moore, C., 179, 181, 193 Moore, F. W., 100, 104, 104 Moore, J. A., 178, 185, 189, 193 Morgan, C., 307, 313, 314, 316, 318, 320, 321, 322

Morisuye, J. M., 294, 303 Morris, 0. E., 97, 98, 105 Morrison, P. R., 220, 226 (see Ferry),

91,

114, 118

Meirowsky, E., 284, 286, 303 Melczer, N., 291, 292, 293, 303 Mellon, R. R., 97, 98, 105 Memmem, F., 324, 335 Mendel, B., 325, 335 Mendelow, H., 39, 44, 47, 48, 49, 50, 52, 63

Mescon, H., 303 Metz, C . W., 207, 210 Metz, G. W., 19, 25 Meyer, K. H., 136, 164 Meyer, K. M., 241, 253 Meyerhof, O., 83, 85, 91 Michaelis, L., 75, 91, 211, 212, 213, 217, 223, 225, 227, 236, 237, 253

Mickelsen, O., 274, 303 Miescher, F., 3, 7, 211, 225, 253 Miller, A. M., 290, 303 Miller, E. C., Jr., 245, 252 Millson, H. E., 248, 251 Mirsky, A. E., 16, 23, 25 Mitchell, P., 96, 100, 105 Mitchell, P. D., 88, 90 Mitchison, J. M., 198, ,203, 204, 206, 210 Mittwer, T., 94, 104 v. Mollendorff, F., 247, 253 v. Mollendorff, M., 247, 253 v. Mollendorff, W., 212, 247, 253 Molisch, H., 101, 105 Mommsen, H., 230, 253 Mongar, L. S., 158, 164 Monnb, L,315, 321 Monroy, A., 204, 206, 210 Montagna, W., 266, 269, 273, 274, 275, 281, 283, 285, 287, 292, 293, 294, 295, 296, 297, 298, 299, 301, 303

228, 231, 232, 233, 234, 236, 242, 243 (see Ferry), 244 (see Ferry), 251, 254 Morton, T. H., 245, 250 Moulton, S. H., 324, 335 Moyle, J., 96, 105 Miiller, A., 225, 251 Miiller, H., 270, 302 Muguard, H., 170, 193

Miihtbock, O., 320, 321 Muller, H. J., 22, 25, 121, 134 Mundell, D. B., 325, 335 Muntz, J. A., 73, 74 (see Barron), 89 Murray, J. A., 291, 303 Myrback, K., 68, 91

N Nachlas, M. M., 327, 335 Nachmansohn, D., 325, 3.75 Nagano, J., 80, 91 Nakazawa, F., 81, 91 Naylor, E. E., 219, 230, 232, 253 Neale, S. M., 223, 228, 235, 238, 241, 245, 253

Nemec, B., 12, 26 Neurath, H., 244, 253 Neurath, J., 137, 158, 160, 164 Newman, L. H., 281, 301 Newman, S. B., 307, 312, 321 Nicholas, J. S., 244, 252 Nicolas, J., 291, 303 Nicolau, S., 273, 303 Nietzke, R., 224, 253 Nishimura, T., 231, 253 Nishiyama, I., 19, 26 Noback, C. R., 275, 281, 283, 293, 294, 295, 296, 297, 299, 303

Noble, E. I., 253 Nordenskiold, E., 5, 7 Northrop, J. H., 120, 134 Nutt, M. M., 98, 105 Nutting, M. D. F., 330 (see Jansen), 335

345

AUTHOR INDEX

0 O’Rrien, H. C., 310, 321 Ochs, G. W., 231, 246, 253 Odland, G. F., 267, 268, 303 Oes, A., 4, 7 Oeding, P., 101, 105 Oehnell, R., 81, 91 Oertenblad, B., 68, 91 Ogston, A. G., 87, 90 Oliver, W. F., 42, 62 Opatowski, I., 22, 26 Ortiz-Picon, J. M., 288, 303 Oser, B. L., 277, 302 Oster, G., 141, 164 Osterhout, W. J. IT., 88, 91 Overton. H.. 70, 71, 85, 91

P Packer, D. M., 44, 45, 46, 47, 48, 49, 56, 63

Paillot, A,, 94, 105 Pal, P., 248, 254 Palade, G. E., 53, 63, 273, 303 Paletta, F. X., 269, 303 Palmer, E. T., 68, 92, 114, 118 Papin, L., 270, 273, 276, 302 Pappenheim, A., 211, 212, 224, 247, 249, 253

Parat, M., 271, 272, 273, 274, 276, 282, 303

Parker, J. A., 42, 63 Parks, H. F., 281, 283, 292, 293, 294, 295, 296, 303

Parks, R. E., 29, 30, 31, 33 Parnell, J. P., 293, 298, 299, 300, 303 Parvis, D., 96, 105 Pasteels, J., 209, 210 Patau, Klaus, 20, 26 Patwardhan, V. N., 84, 91 Patzelt, V., 269, 287, 303 Pavlovif, R., 324, 335 Pease, D. C., 39, 43, 47, 53, 63, 307, 308, 321

Peirce, G., 324, 335 Pelet-Jolivet, L., 212, 217, 224, 235, 253 Penners, A., 166, 193 Pennington, D., 95, 98, 105 Perkins, 0. C., 290, 303 Peshkoff, M. A., 95, 105

Peterfi, T., 181, 193 Peters, J. R., 75, 91 Peters, L., 229, 235, 253 Peters, R. H., 225, 227, 253 Pfankuch, E., 320, 321 Pfeffer, w., 262, 263 Pfeiffer, C. A., 298, 302 Pfeiffer, H., 231, 253 Piekarski, G., 94, 95, 96, 106 Pijper, A., 102, 106 Pincus, F., 287, 290, 303 Pirie, N. W., 120, 134 Pisano, M., 101, 106 Pischinger, A., 218, 219, 230, 233, 253 Pitts, R. F., 88, 91 Pivan, R. B., 229, 237, 238, 252 Pochon, J., 101, 106 Polge, c., 35, 6 3 Pomerat, C. M., 151, 164 Porsche, J. D., 226 (see Ferry), 243 (see Ferry), 244 (see Ferry), 251 Powell, s., 20, 24 Pratt, L. S., 214, 253 Preston, c., 161, 164 Preston, J. M., 248, 254 Pringsheim, E. G., 95, 106 Pulcher, C., 219, 230, 254 Pulvertaft, R. J. V., 98, 106 Putnam, F., 137 (see Neurath), 158 (see Neurath), 160 (see Neurath), 164 Putnam, F. W., 121, 128 (see Kozloff), 134, 244, 253

R Rabl, H., 270, 303 Radir, P. L., 161, 164 Rahn, O., 69, 91 Rakusin, M. A., 225, 254 Randall, J., 316, 321 Randall, M., 234, 252 Randolph, L. F., 21, 26, 174, 193 Ranganathan, S. 84, 91 Ranvier, L., 5, 6, 7, 269, 274, 275, 303 Ratsimamanga, R., 280, 302 Rauch, H., 285, 301 Raven, Chr. P., 196, 210 Rawles, M. E., 284, 285, 303 Rawlins, L. M. C., 213, 225, 254 Reed, R., 36, 63 Regan, M., 31, 34

346

AUTHOR INDEX

Regaud, C., 27Q, 272, 276, 291, 301, 303 Rehm, W., 157, 164 Renvall, S., 88, 91 Reyniers, J. A., 32, 34 Rhoades, R. P., 307, 308, 322 Rich, A., 209 (see Villee) , 210 Richards, A. N., 79, 91 Richards, G. A., 307, 308, 317, 318, 322 Riche, A., 217, 251 Richter, D., 323, 324, 335 Richter, M, N., 307, 310, 321, 321 Rideal, E. K., 213, 228, 229, 251 Ris, H., 16, 25, 209, 210 Robbins, S., 88, 91 Robbins, W. J,, 218, 219, 227, 230. 254 Robertson, R. N., 157, 161, 164 Robinow, C. F., 95, 96, 100, 106 Robinson, E., 116, 118 Robinson, R. D., 246, 254 Rodwell, A. W., 88, 90 Rogers, L. L., 31, 34 Romeis, B., 36, 63 Rona, A., 75, 91 Rona, P., 324, 335 Rose, F. L., 254 Rose, R. E., 219, 223, 239, 241, 254 Rosenberg, Th., 67, 72, 75, 76, 77 (see Diczfdusy), 91 Rosenstadt, B., 269, 304 Ross, J. G., 15, 25 Ross, M. H., 83, 91 Ross, W. J., 138, 163 Rosza, G., 307, 313, 314, 316, 318, 322 Rothenberg, M. A., 325, 335 Rothman, S., 286, 304 Rothschild, Lord, 206, 210, 258, 263 Rothstein, A., 66, 69, 73, 74, 91, 114, 118 Rotman, R., 121, 122, 127, 134 Rowe, F. M., 214, 254 Royer, G. L., 245, 246, 248, 251, 252, 254 Rudall, K. M., 279, 304 Rudney, H., 325, 335 Runnstrom, J., 73, 91, 161, 164 Ruska, H., 320, 321

s Sachs, J. v., 1, 2, 7 Sacks, J., 71, 91 Said, H., 114, 116, 118

Sarkar, P. B., 249, 254 Sasakawa, M., 281, 282, 296, 304 Sauberlich, H. E., 31, 34 Schaeffer, V. J., 37, 63 Schafer, E. A,, 267, 304 Schaffer, J., 290, 298, 304 Schaudinn, F., 94, 95, 98, 106 Scheminsky, F., 156, 164 Schiff, A., 288, 301 Schipper, E., 42, 63 Schirm, E., 241, 254 Schleicher, W., 5, 7 Schlesinger, R. W., 133, 134 Schmidt, C. L. A., 213, 225, 227, 228, 239, 250, 254

Schmidt, W. J., 197, 198, 201, 210, 269, 304

Schrnitt, F. O., 307, 308, 314, 318, 322 Schplnheyder, F., 324, 335 Schrader, F., 16, 17, 26, 203, 210 Schultz, Jack, 22, 23, 26 Schultze, F. E., 270, 304 Schultze, M., 268, 304 Schulze, K. L., 11, 26 Schuster, M. C., 307,'310, 321, 321 Schwalbe, C. G., 212, 254 Schwarz-Karsten, H., 231, 254 Scott, D. B., 10.3, 105 Scott, G. H., 36, 38, 41, 42, 47, 48, 50, 55, 56, 63, 272, 283, 301, 304

Scott, S. H., 44, 45, 46, 47, 48, 49, 50, 63 Scribner, E. J., 120, 134 Seal, S. C., 99, 106 Seidel, F., 168, 193 Seifrez, 137, 164 Seki, M., 231, 233, 240, 245, 254 Seligman, A. M., 327, 335 Serra, J. A., 276, 304 Seymour, R. B., 219, 254 Shannon, J. A., 80, 91 Shapiro, B., 268, 269, 304 Shapiro, E., 137, 163 Sheppard, S. E., 246, 254 Shive, W., 31, 34 Shope, R. E., 133, 134 Silvers, S., 274, 302 Simpson, G. G., 240, 255 Simpson, L, 36, 38, 39, 40, 43, 50,

w.,

w.

57, 63

347

AUTHOR INDEX

Singer, M., 220, 221, 226, 228, 231, 232, 233, 234, 236, 242, 243, 244, 251, 254, 280, 301 Singer, N., 55, 62

Sjostrand, F., 36, 39, 47, 61, 63 Skinner, B. G., 228, 235, 237, 254 Slein, M. W., 72, 89 Sloane, J. F., 44, 47, 53, 63 Smiljanic, A. M., 286, 304 Smith, A. G., 98, 106 Smith, A. V., 35, 63 Smith, S. G., 239, 246, 254 Smith, V. W., 285, 301 Smithburn, K. C., 102, 104 Snell, E. E., 31, 34 Sokolova, N. V., 225, 254 Sonneborn, T. M., 24, 26 Sparrow, A. H., 19, 26 Speakman, J. B., 153, 164, 226, 229, 231, 232, 235, 239, 240, 246, 253, 254

Spemann, H., 166, 193 Sperber, E., 73, 79, 80, 88, 89, 91 Spiegelman, s., 88, 91 Spiro, C., 217, 254 Stacey, M., 96, 105 Stamm, , 298, 304 Staple, P. H., 55, 63 Stauffer, E., 181, 193 Stearn, A. E., 213, 218, 219, 223, 225, 226, 230, 232, 249, 254, 255

Stearn, E. W., 213, 218, 219, 223, 225, 230, 249, 254, 255

Stringfellow, W. R., 253 Sturm, K., 231, 255 Sugiyama, M., 204, 210 Suida, W., 226, 251 Summerson, W. H., 277, 302 Suntzeff, v., 283, 304 Suskind, R. R., 292, 293, 294, 295, 304 Sutcliffe, J. F., 108, 114, 115, 118 Swann, M. M., 198, 201, 202, 203, 204, 206, 207, 208, 210, 258, 259, 263

Swerdlow, M., 307, 312, 321 Swift, H. H., 16, 17, 25 SylvCn, B., 43, 47, 63 Szent-Gyorgyi, A., 136, 137, 155.

162. 163.

307, 313, 314, 316, 318, 322

T Takata, M., 324, 335 Taylor, E. S., 88, 90 Tchan, Y. T., 101, 106 Tchou-Su, 181, 183, 193 Tello, F., 272, 273, 304 Terroine, E. F., 324, 335 Thomas, A. W., 217, 231, 255 Thomas, 0. L., 273, 304 Thomas, P. T., 9, 21, 25 Thompson, H. C., Jr., 287, 288, 301 Thompson, H. S., 325, 335 Thompson, R. H. S., 282, 283, 304 Thuringer, J. M., 287, 304 Tolstoouhov, A. V., 219, 223, 230, 245, 255

Stedman, E., 323, 335 Steinhardt, J., 227, 228, 229, 234, 237, 239,

Townend, F., 219, 225, 227, 239, 240, 245,

240, 246, 255 Steinitz, L. M., 14, 15, 20, 25, 26 Stempen, H., 96, 106 Stern, C., 11, 26 Stern, Herbert, 11, 26 Stern, J. R., 88, 91 Steward, F. C., 86, 91, 161, 164 Stille, B., 94, 106 Stockinger, L., 231, 255 Stokstad, E. L. R., 28, 31, 33 Stott, E., 226, 229, 231, 232, 240, 254 Stoughton, R. H., 94, 95, 106 Street, H. E., 107, 114, 115, 118 Strasburger, E., 5, 7, 13, 26 Streicher, J. A., 88, 89

Trexler, P. C., 32, 34 Trimble, H. C., 281, 301 Triwush, H., 239, 252 Troland, L. T., 121, 134 Tulasne, R., 96, 106 Turnbull, D., 37, 54, 62 Turner J. S., 157, 164 Twitty, V. C., 187, 193

250, 255

U Uber, F. M., 36, 40, 41, 43, 47, 51, 56, 63 Udall, K. M., 36, 63 Unna, P. G., 212, 255, 274, 276, 304 Upcott, M., 20, 26 Ussing, H. H., 88, 91, 135, 164

348

AUTHOR INDEX

V Vaarama, A., 19, 26 Vahl, C., 101, 106 Van Oordt, G. J., 288, 300 Van Slyke, D. D., 75, 91 Vassale, G., 298, 300 Vasseur, E., 68, 91, 262, 263 Veller, E. A., 225, 255 Vendrely, C., 16, 25 Vendrely, R., 16, 25 VerzAr, F., 80, 81, 92, 136, 164 Vickers, E. J., 245, 252 Vickerstaff, T., 213, 223, 227, 228, 229, 235, 237, 239, 240, 241, 245, 247, 249,

250, 252, 254, 255

Vickery, H. B., 277, 300 Vidal-Swilla, S., 80, 92 Villee, C. A., 86, 92, 209, 210 Vishniac, W., 69, 74, 90 VICs, F., 196, 210 Volmer, M., 48, 63 Volqvartz, K., 324, 335

W Wachstein, W., 101, 106 Wagner, M., 32, 34 Waldeyer, W., 3, 7, 275, 304 Waldo, C. M., 221, 255 Walker, A. M., 81, 92 Walker, E., 278, 304 Walker, F. M., 229, 237, 238, 239, 240,

Weisz, P. B., 10, 26 Wertheimer, E., 73, 81, 92 Westenbrink, H. G., 80, 92 White, A. C., 323, 335 Whitely, A. H., 161, 164 Whittaker, V. P., 282, 283, 304, 335 Wiggall, R. H., 103, 105 Wilbrandt, W., 75, 76, 77, 80, 81, 92 Wilhelm, M. L., 88, 91 Wilkerson, V. A., 277, 304 Wilkes, B. G., 68, 92, 114, 118 Willstatter, R., 324, 335 Wilmer, H. A., 83, 92 Wilson, c., 223, 225, 237, 251 Wilson, E. B., 12, 26 Wilson, G. B., 20, 24 Winkler, Hans, 13, 19, 20, 26 Wislockie, C. B., 55, 62 Wislocki, G. B., 213, 220, 221, 233, 251, 254, 274, 280, 301

Wolf, J., 102, 104 Wollman, E., 121, 134 Wood, J. K., 255 Woods, H. J., 277, 299 Woodside, G. L., 30, 34 Wooley, P. M. B., 156, 163 Worley, L. G., 273, 304 Wurzbacher, W., 333, 335 Wyckoff, R. W. G., 102, 103, 105, 307, 313, 314, 316, 318, 320, 321, 322

Wright, Sewall, 122, 134

252

Walter, H. J., 246, 254 Warburg, O., 85, 92 Watson, R. C., 169, 193 Way, M. J., 307, 322 Waymouth, C., 151, 164 Weatherford, H. L., 221, 255 Webb, M., 209, 210 Weber, C. O., 224, 255 Wei, w. P., 101, 106 Weibull, C., 102, 104 Weidenreich, F., 269, 304 Weidinger, A., 277, 301 Weinburger, H. J., 10.3, 105 Weismann, August, 12, 26 Weiss, P. A., 23, 26

Y Yanagita, T., 204, 210 Yasuzumi, G., 231, 233, 245, 255 Yphantes, D. A., 279, 300 Z Zacharias, E., 3, 7 Zacharias, P. D., 212, 255 Zak, F. G., 275, 281, 283, 293, 294, 295, 296, 303

Zeiger, K., 213, 223, 231, 233, 240, 242, 245, 247, 255

Zeller, E. A., 335 Zimmerman, c. L., 246, 254 Zimmermann, A. A., 285, 286, 304

Subject Index A Absorption, intestinal, inhibition of, 82 of glucose, 80 Absorption spectrophotometry, of cells, 16 Absorption, sugar and cell division, 108, 109 in plants anaerobic, 115 characteristics, 110-114 mechanism, 107, 117, 118 rate, 114 Accentuators, staining, 215, 216, 217 Accumulatory mechanism, and inversion tube analogy, 150, 151 cellular, 136 of amebae, 146 Acetabularia, 10 Acetic acid, 262 Acetylcholine, 197, 331, 332 Acetylcholine esterase (see also under cholinestcrase), 33 1 Acetylesterase, 330 Acetyl thiocholine, 333 Achromosomal cell and enzyme activity, 190 Acid, accentuator, 217 aromatic, ester of, 323 effect of on staining, 216 Acid dye, nature of, 222, 223 Acid fuchsin, in bacterial staining, 218 A&, sebaceous gland, 290, 291 and lipid accumulation, 293 changes in, 299 Acne, 295 Actinomyces, 97 Actinomycetales, 104 Activation, Action potential, 195 Activation, egg, by sperm contact, 183 energy, dye, 245 of dyeing process, 235 Activator, of esterase, 324 sperm, 260, 261

Active group, on dye molecule, 239, 240 Activity, enzyme on naphtholic substrates, 329 mitotic, in epidermis, 287-290 Activity, mitotic rhythmic, 288 Actomyosin, gel-sol transformation, 140 Adaptation, and viruses, 120 Adenine, 30 Adenosinediphosphate, 85 Adenosinetriphosphatase, 69 Adenosinetriphosphate, 71, 72, 197 action on actomyosin, 140 action on cortical gel, 140, 160 distribution at cell membrane, 87 in protein folding, 137, 162 uranyl complex 74, 75 A. discoides, 140 Adrenaline, 197 Adrenocortical hormone, in glucose metabolism, 72 Adsorption, dye by proteins, 141, 142, 159 in staining, 213 Aeration, and root tip growth, 108 a-Esterase, 329 Affinity, dye and molecular shape, 240 and protein structure, 239-241 Agar, staining of, 227 Agent, chemical from chromosomes, 202, 203, 204, 206, 210

from germinal vesicle, from sperm, 206, 207-210 Agents, structural, in mitosis, 208-210 Aggregate, dye, formation of, 236 Aggregation, of dyes, 226, 227 Manine, 29 Albumin, 137, 216, 225

349

350

SUBJECT INDEX

Alcohol, hydrophilic, 326 Aliesterase, 323 Alkaline phosphatase, distribution in tissue, 56 inhibition, 84 in epidermis, 282 in intestine, 79, 83 in kidney, 79, 83 occurrence, 66 of sebaceous gland, 297 of spider oocyte, 6 Alkaline tide, 157, 159 Alkyl isothiocyanate, effect of, on skin sections, 278, 279 Allium 16 Allylisothiocyanate, 77 Amblystoma, 16 Amblystoma tigrinum, 172 Amino acids, 87, 88 configuration and activity, 33 essential, for Tetrahymena, 28, 29 free side groups of, 221 in keratin,. 277 non essential, for Tetrahymena, 29 relationships between, 29 sythesis, by bacteria, 28 p-amino-benzoyl-glutamic acid, 31 Aminopterin, 31 o-amino-azotoluene, 327 Ammonia, formation by Tetrahymena, 29 Amoeba, and protein folding, 136, 137 discoides, 155 dye adsorption studies, 142-146 proteus, 155 Amphion, 2 2 1 protein, 233 Amphoteric, nature of proteins, 221-223 Amylase, 69 Anaphase, birefringence, 197, 198, 202, 203, 207 Androgenesis, 169 Aneuploidy, 20, 177-179 Aneusomaty, 20, 2 1 Aniline blue, specificity of, 237

Anion, in protein-dye affinity studies, 239 Anisotropic lipid, in sebaceous glands, 295 Anthocyamin, genetic determination of, 12 Antibiotics, 2 7 (See individual members) Apis, 175 Aplanatic foci, 2 Arahinose, 76, 77 Arachyl alcohol, 293 Area, pressure, of skin, 266 Arginine, 158, 277 basic side group of, 221, 225 in urea cycle, 29 Arsanilic acid, 324, 328 Arterial intima, 2 Artifact, 313 diffusion, in quenching, 36, 40, 41 Awclius albopunctatus, 17 Ascads megdoccphda, 3 18 Ascorbic acid, in epidermis, 280 AS Esterase, 329 Ash, content of epidermis, 283, 284 Aspartic Acid, 29 acidic side group of, 221, 225 Aster, birefringence of, 198, 201, 204 electron-optical study of, 315 non chromosomal, 181 sperm, 206, 207 Aureomycin, 28 Autogamy, 98 Autolysis, activation and inhibition of, 4 Autosome, 21 Auxin, 197 in cell division, 14 Auxochrome, 215 group, in wool-dye interaction, 228 Avidin, 31 Axilemmae, 318 Axopiasm, 314, 318 Axon, leptone, 314 Azide, 88 Azodye,

SUBJECT INDEX

Azodye, and hydrogen bonding, 228 spectral absorption study, 228 Azocannine, 249 Azotobacter, 102

B Bacillus anthracis, 95 Bacillus negatherium, 95 Bacteria, 93, 94 as phage precursors, 120, 121, 123 effect of pH, on staining of, 218 embedding of, 313 evolutionary system, 104 infected, phage studies on, 125 intestinal in nutrition experiments, 27, 28 isoelectric point of, 230 maturation and cytology, 97, 98 photosynthetic, 101 stained, effect of chemical decolorizers on, 218 Bacteriophage electron microscopy of, 320 formation, 120 growth, 120-123 mutants, distribution in bacteria, 131,132 non reproductive, 132 reactivation, by ultraviolet radiation, 130, 131

sensitivity to radiation, 129, 130 studies with radio-isotopes, 132 transformations, 127 Baelenoptera, 319 Baker’s acid hematein stain, 274, 275, 292, 2 95

Base, effect of, on staining, 216 Basic dye, nature of, 222, 223 Basophilia, in epidermal cells, 280, 281 in sebaceous glands, 29b Benzidine peroxidase, 297 4-Benzoylamino-2, 5, dimethoxyaniline, 327 Benzyi mercaptan, 279, 280 Bile acids, 324, 328 and enzyme activation, 328 Binding, amide, of dye, 227 dye, and dye concentration, 236

351

and forces involved, 226 and pH, 219 by pure protein system, 220 by various tissues, 218, 219 forces in, 232 site of, on protein, 224 gelatin, of acid dye, 230 ion, of proteins, 244 protein of dyes, 216, 217 site of dye, 240, 241 wool, of sulfuric and hydrochloric acids, 235

Biotin deficient mice, 275 requirement of Tetrahymena, 30, 3 1 Bipolar staining, 101 Birefringence, and protein fibres, 197 cell, and mitosis, 195 coefficient of, 198-203 of chlosterol digitonide, 294 of frozen dried tissue, 53, 54 of sebaceous glands, 295 of stratum corneum, 278 Blastomere, dividing of coregonous egg, 315

newt, cells of, 166 Blastopore, 168 Blastula, irregular, 179 Blepharoplast, 102 Body size and polyploidy, 172, 173, 175-177 Bond, Cwlombic, 224 covalent, in dyeing, 228 hydrogen, effectiveness of, in dyeing, 228 ionic, 224 primary valence, 224, 225 Bonding, hydrogen and cellulose dyeing, 241

Bone, 319, 320 Brilliant green, adsorption by amoeba, 143 in adsorption experiments, 151 Bromacetophenone, 77 Bromcresyl green, 154 Bromthymol blue, 144 Brunner’s glands, 329 Brush border of intestinal cells, 319 Butyl, methacrylate, and ultramicrotomy, 312

Butyryl thiocholine, 332, 333

352

SUBJECT INDEX

C Caffeine, 162 Calcium, and cell structure, 196 effect on spermatozoa, 262 in Tetrahymena nutrition, 30 Capsule, Cnidal, 319 Carbohydrate, and mitotic activity, of epidermis, 288, 289 metabolism, of tetrahymena, 29, 30 Carbon dioxide, and ultramicrotomy, 312 Carbonic acid, 157 Carcinogenic agent, and melanogenesis, 286 Carcinoma, basal cell, and mitochondria, 271 Carnauba wax, 310 Carnoy preparations, 57-61 Carotene, 284 Carrier, in glucose absorption, 84 Cartilage, staining, at low pH, 216 Caryophanon, 95 Casein, 225 Cations, transport of, 87, 88 Cell, achromosomal, 181-183 adjacent, connection of, 268-270 basal, of epidermis, 267, 268 damage, and mitochondria, 271 division, 10 bacterial, 98-102 epidermis, and mitochondria, 270-272 epithelial, of vagina, oral and buccal cavities, 281 membrane, bacterial, staining, 100 non-nucleated, in developing embryo, 181

nuclei, staining reactions, 4 permeability, and cell division, 10 physical properties and mitosis, 195-197 pigmented, of epidermis, 285 radicular, 315 size and cell number, 170, 172, 173 surface, structures of, 317, 318 ultrastructure, 313 wall, bacterial, 98-100 staining, 100 components, changes, 114 rate of formation, 116 with unbalanced chromosome number, 179

Cellobiose, 241

Cellulose, 116 dyeing of, 228 and ion concentration, 235, 236 by color ions, 240, 241 Central Nervous System, staining of, 216 Centromere, 21 Centrosome, birefringence, 198 sperm, 181, 182 Cetyl pyridinium bromide, 154 Champy fixation, 314 Charge, on protein molecule, 221, 222 Chemical sites, in fixed tissue, 54-56 Chemotoxis, egg-sperm, 260, 262 Chicago blue, 143 Chitin, 318 Chironomous larvae, 4 Chloral hydrate, 77 4-Chloro-2-Aminoanisole,327 1-4,Chloromercuriphenylazo-2-naphthol, 2 79 Chloropicrine, 77 Cholesterol, in cells of skin, 275 in sebaceous gland, 294 in sebum, 293 occurrence, in sebaceous tissue, 294, 295 Choline, 323 analogues of, 332 Cholinesterase, 323 activity, in different species, 330, 331 classification of, 332 histochemical determination, 327 inhibition of, 323 of skin, 282 Chondrioconts, 314 Chromatid, structzlre, 15 and desoxyribonucleic acid, 18 Chromatin, and basic dyes, 225 bacterial, during phage infection, 129 in epidermal cells, 287 sperm, irradiated, 182 Chromatography, 116 Chromic acid, as tissue fixative, 313, 314 Chromidia, 315 Chromophore, 215 group in wool-dye interaction, 228 Chromosome, 3 abnormal patterns, 12-14

SUBJECT INDEX

amphibian of arrested hybrids, 185 and melanophores, 170, 172 aneuploid, effects of, 177, 178 birefringence, 201 complements in multipolar mitosis, 179 functional, and egg development, 181-183 “lampbrush”, 165 mitotic, 4 numbers, 12, 13 number, determination of, 168, 169 variation, 21 reproduction, aspects of, 22 salivary, reaction with pepsin, 3 separation, traction fibre theory, 198 staining reaction, 4 structure, 21, 22 Cilia, 319 Cimex, 21 Citrovorum factor, 31 Citrulline, 159 in urea cycle, 29 Cleavage, 259 of androgenic eggs, 181 sea urchin egg, and birefringence, 204 without sperm nucleus, 181, 182, 183 Cnida, ultrastructure of, 319 Coacervation, 146 Colchicine, 196 action of, 14 in reduction division, 20 Collagen, 231, 316 staining of, 217, 237 Collision, sperm-egg, 257, 258, 259 Colloid, amphoteric behavior of tissues as, 218 a t low temperatures, 42 changes of amoeba protein, 140 polymeric, 320 Color anion, affinity for, by cellulose, 236 Color radical, 224 Colostrum, 2 17 Comedones, 295 Competition, gene, 189 ionic, in staining, 235 Complex, mitochondrial, in epidermis, 27 1 substrate-carrier, 67 dissociation of, 83 uranyl-polyphosphate, 73, 74

353

Concurbita, 108, 109, 110 Conductivity, of cooling agents, 38, 39 Conductometric titrations, and dye-protein stoichiometry, 226 Cones, epidermal, 267, 268 Congo red, 249 Conjugase, in Tetrahymena, 31 Coplananty, 241 Contraction, protein, 137 Cooling agents, 38, 39 bath, 39-44 Copper, in Tetrahymena nutrition, 30 Corium, 267 Corynactis viridis, 3 19 , Corynebacteria, mo$hology, during growth, 100 reproduction, 102 Corynebacterium diphtheriae, metachromatic granules in, 100, 101 Costovertebral pigmented spot, 293 Covalent link, in dye binding, 235 Creatine-phosphate, 71 Cross-fertilization, 183, 184 Cross wall, bacterial, 95 Crystal violet, 143, 150 Cysteine, 158, 280 Cysthe in wool, 239 keratin, 277 synthesis, 29 Cytidine deaminase, 30 Cytochemistry, 35, 51, 52, 54 Cytochrome oxidase, 273 Cytocrine, 286 Cytokinesis, 10 Cytology, bacterial, 93-104 during phage infection, 129 Cytophagas, 94, 97 Cytoplasm, aggregation, in mitosis, 202 bacterial, 96 Cytoplasm basic microscopy of, 314 basophilia of, 280, 281 carrier system, in glucose absorption, 82 egg, 165 sea urchin egg, in mitosis, 203 thickness, and sugar absorption, 113 ultrastructure of, 320 Cytosine deaminase, 30

354

SUBJECT INDEX

D Datura, 13, 178 Dark period, of bacteriaphage growth, 126, 128

Deamination, of wool, 226 protein, by heating, 244 Decolorizers, 2 18 Dendritic cells, 285 Denaturation, and cytoplasmic staining, 156 cell, and dye penetration, 242 protein, 137 Dephosphorylation, glucose, 81 Dermatosis, avitaminotic, in mice, 269 Dermis, 267, 268 Desorption, of dye, 248, 249 Desoxyribonucleic acid, bacterial, 96 during phage infection, 128, 129 chromosome, content in mitosis, 29 concentration of pachytene, 18 content of cells during mitosis, 16, 17 content, of different tissue, 22 during reduction division, 18 in chromosomes and genes, 24 prior to mitosis, 16, 17 reaction with dyes, 4 of TZ phage, 124 Desoxyribonucleoprotein, staining of; 237 Desiccation of frozen tissue, 45 Development, amphibian, arrest of, 185 embryonic and cell size, 191, 192 2-8,Diaminoacridine; 143 Diastase, treatment of epidermis sections, 281

Diazo Fast Blue RR Salt, 327 Diazo Garnet GBC Salt, 327 Diazo Red RC Salt, 327 Diazonium Salt, reaction with naphthols, 326

Dictyosome, 273 Differentiation, amphibian tissue in salt solution, 186 and cytoplasmic influence, 23 cellular, and nuclear and cell size, 10 embryonic, amphibian, 172 nuclear units in, 14 Diffusion, cellular internal barrier, 112, 113, 114

dye, and staining solution temperature, 245

glucose, across membrane, 81, 82 of mitotic chemical agent, 208 of substrate across membrane, 66, 67 pumps, 45, 47 uneven, 152 Digestion, proteolytic, of keratin, 277 Digital pad, cat, 267 Digitonin, 294 Dihydroxymaleic acid, 262 3-4,Dihydroxyphenylalanine, 285 Diisopropylfluorophosphate, 323, 325 inhibition of pseudocholinesterase, 333 Diploid, autopolyploid, characteristics, 10 heterozygons, 174 Diptera, 19 Dissociation, constant, of dyes, 222, 223 of protein-anion complex and dye affinity, 239 protein, in aqueous solution, 217 Disulfide, oxidation, in keratin, 281 Division, amitotic of leucocytes, 5, 6 cell, 5 mitotic, in sebaceous glands, 299 nuclear, 5 regulation of, 10, 11 of accessory sperm nuclei, 179, 181 Donnan equilibrium, 229 and ionic strength-staining relationship, 234, 235 Dopa oxidase, 285 Dopa reaction, 285 Dopamelanin, 285 Drosophila, 13 Drying, apparatus, 43, 44 efficiency, 48, 49 time, 46, 48 Dry sperm, 257 Duodenum, rat, 329 Dye, 4 acid, basic nature of, 214, 215 affinity, effect on staining, 237-241 bacterial, 93-96 concentration, effect on staining, 236,237 fluorescent, 231 in cell accumulation studies, 156 lipid, 293

SUBJECT INDEX

nuclear, 327 protein, interaction and temperature, 245, 246

sulfonated aromatic azo, 241 Dyes utilization of, 211, 212 Dyeing, rate and temperature, 245, 246

E Edema, in aneuploids, 177 in haploids, 175, 176, 180 Effectors, enzyme, 66, 67 Egg, cortex, 258 sea urchin, 203, 204 fertilized, changes in, 258 fusion, 168 haploid, 175, 176 jelly, 262 sea urchin, 197, 198, 203 volume, 166, 168 yolk, 166 Eicosyl alcohol, 293 Elastin, 316 Electrochemistry, of staining, 238-241 Electron, discharge, and section thickness, 305

microscopy, and fixatives, 313, 314 objectives of, 306 of axons, 314 of extracellular structures, 319, 320 Electrostatic charge, of protein, 223 repulsion, staining in the presence of, 228

“8Ridine en flaques,” 275, 279 ‘%ldidine en graine,” 275 Embedding material, for ultramicrotomy, 310, 312

Embryo, aneuploid, 177-179 giant, 168 haploid, 169, 170 human glycogen in, 282 juice, 32 polyploid, 168, 170 body size of, 170, 172, 173 differentiation of, 173-177 Endodivision, 14 Endomitosis, 14 “Energic” state, of nucleus, 11 Energy, and sugar absorption, 113

355

Enzyme, activity, and protein folding, 138 adaptive, 69 autolysis, of membrane, 86 at cell surface, 65-68 Cis-trans. . . . ., 85 of membrane, 76, 78 coupled, 85 digestion of nuclei, 4 digestive, 66, 68 histological location, in cell, 315 in cell membrane, 66 in microscopy, 3 islands, in plant cell, 116 patterns, of whole animals, 28 phosphorylating, 69 proteolytic, 278 reactions, cellular and protein folding, 161

similarity between, 323-326 sulfhydryl, 176 surface, plant, 107, 114-117 ”symbiosis”, 32, 33 Enzymic capacity, during histogenesis, 32 Eosin-methylene blue, staining, at various pH’s, 219 Epidermis, basal layer, and mitochondria, 270-272

glial system of, 285, 286 human, 269 tonofibril of, 269 mammalian, composition, 266-268 neoplasms of, and mitochondria, 271 palmar and plantar, 271 thickness of, 266, 267 Erythrocyte, alkaline phosphatase of, 84 avian, 3 glucose permeabiilty, 75-79 human, 2 and glucose penetration, 75, 76 isoelectric point, 230 staining, 245 Escherichia coli, infection by phage, 124 Eserine, 323 inhibition of esterase, in mouse, 330 Ester, 323 high energy phosphate, 71 (see also under names of individual members.)

356

SUBJECT INDEX

Esterase, aliphatic, in skin, 282 biochemical difference, 324, 325 classification of, 333 definition of, 323 histochemical classification of, 324 of mouse testis, 328, 329 substrates, 326, 327 Estrus, mouse and mitosis, 289 Ethyl alcohol, as tissue cooling agent, 38, 39, 41, 44

fixation, 245 treatment of dried tissue, 52, 53 Ethyl urethane, 77 Eurycea bislimeata, 172 Eutectic mixture, 310 in tissue, 42, 43 Evaporation, 45, 46 Exercise, effect on mitotic rate, 288

F Factor, lethal, 180, 181 Fat, 326 subcutaneous, 283 Fatty acid, 294 esterification of, 326 Fermentation, glucose, 73 Fertilization, 257 and nucleo-cytoplasmic interactions, 183, 184

heterologous-homologous, 259 kinetic theory of, 258 “partial”, 183, 184 polyspermic, in urodeles, 11 in sea urchin, 257-263 reaction, in sea urchin egg, 206 Fertilizin, 260, 261, 262 Feulgen reaction, 96 on frozen dried tissue, 56, 57 Feulgen stain, 16 Fibre, cellulose, dyeing of, 235, 236 collagen, of bone, 320 polyamide, 2 12 reticular, in epidermis, 268 spiral, of cilia, 319 wool, and molecular affinity, 228 swelling and temperature, 246 Fibrillorhexis, 276

Fibrin, 220 affinity of dyes dye binding by, dyeing of, with film, isoelectric

for, 238 226

various dyes, 232 point, determination of,

231

fixation, effect of, on staining, 243 Fibroblasts, movement in tissue culture, 151, 152

Filament, cnidal, 3 19 myosin, 316-318 spiral, of Herxheimer, 267, 269, 270 and mitochondria, 270, 272 Fixation, chemical, 54 by cooling agents, 38 effect of, on staining, 242-245 formalin, 244, 245 influence of, on electron-optical image, 313, 314

of frozen dried tissue, 53 Fixative, effect of, on protein, 243, 244 Flagella, bacterial, 102, 103 structure, 319 Flattening, of dried sections, 52 Fluorescence, of sebaceous glands, 296 Folk acid, 27 Folinic acid, 31 Follicle, hair external root sheath of, 265, 266, 290 glycogen in, 282 Force, coulombic, 236 electrostatic, 223, 224 protein suppression by salt ions, 235 intermolecular, 196 primary valence, 226 pbysico-chemical, in staining, 2 12 short range and dye affinity, 238, 239 Van der Waals, 228, 237 Formalin, fixation, 313, 314 and neurofibrils, 314 Freeze drying apparatus, 46, 47 Fructose, 77 absorption by root tip, 108, 110 Fructose-6-phosphate, 7 1 Fungi, dikaryatic, 11 Fusiformis, 102 Fusion tubes, bacteria, 95

SUBJECT INDEX

G Gabbett’s methylene blue, 215 Galactose, 73, 77 Gametes, sea urchins, 263 Ganglia, sympathetic, 331 Gauges, in tissue drying, 49, 50 Gel, cortical, amebae, 138 adsorption by, 145, 146 and adenosinetriphosphate, 140 Gelatin, 225, 230, 231 interaction with dyes, 217 liquefaction cycle in mitosis, 10 Gene, and desoxyribonucleic acid synthesis, 22 and development, 188, 189 dosage, 173-177 in differentiation, 11 in polyploidy, 174 interaction with cytoplasm, 11 mutation, 179-181 reproduction and cell division, 14 specialization, 23 substances, 11, 12 Genomes, 24 Gentian violet (See also crystal violet) in bacterial staining, 218 Giemsa stain, 94, 95, 96 Gland, anal and circumanal, 290 holocrine, 290, 298 inguinal, 290 Meibomian, 290 preputial, rat, 294, 295, 297 sebaceous (see under sebaceous gland). scent, 290 sudoriparous, 266 glycogen in, 281 white inguinal, 291 Glans penis, 290 Glomerulus, glucose in, 79, 80 Glucose, 77 accumulation in cells, 136 distribution in blood, 75 penetration, 70 uptake, 71-75 in red blood cells, 75-79 Glucose-6-phosphate, fate, during penetration into cell, 72 formation, 7 1

357

Glutamic acid, 29, 3 1 acidic side group of, 221, 225 Glutathione, in epidermis, 278, 279 Glycine, 29 Glycogen, in epidermis, 281, 282 in membrane, 72 in sebaceous glands, 296 localization in tissue, 54 phosphorylysis, 83 substances, bacterial, 101 Glycolysis, amphibian, 185 Glycosuria, phloridzin, 81, 83 Golgi element, in frozen dried tissue, 57 in skin, 272-274 melanin formation, 286 of sebaceous gland, 291- 293 Golgi internum and lipids, 291, 292 Golgi net, 273 Gonidia, bacterial, 102, 103 Goodpasture’s methylene blue, 215 Graft, cell, 182, 183 Grafting, and tissue response, 185, 186 tissue of amphibian hybrids, 185 Gram reaction, of bacteria, 96 Gram stain, 93, 94 Granular inclusions, bacterial, 100-102 Granule, bacterial, metachromatic, 100 polar, 101 colored in amebae, 150 cytoplasmic, coalescence and tonofibril formation, 269 Feulgen-positive, 94 glycogen, 281, 282 heterochromatic, in epidermis, 287 indolphenol-blue, of sebaceous gland, 297 keratohyaline, 271-273, 275, 276, 280 lipid, in epidermis, 273 melanin, 284, 285 phospholipid, in epidermis, 274 pigment, distribution of, 286 skin, 275, 276 sudanophilic, 273 Group, acidic and basic, of protein, 221 reactive, protein production by denaturation, 244 sulfhydryl, 278, 280

358

SUBJECT INDEX

Growth bacterial, 98-102 of sebaceous glands, 298, 299 plant, 108-110 virus, mechanisms, 120-123, 124, 128 waves, 287 Guinea pig, epidermis of, 267 Guanine, 30 Gynogamone I, 260 Gynogenesis, 169, 183

H Habobracon, 175 Hamster, epidermis of, 267 Haploid, amphibian, 168 gynogenetic, 182 syndrome, 175, 176, 181 viability of, 174, 175 Harder’s gland, 172 Hemaglobin, isoelectric point, 230 reduced, in epidermis, 284 Heterochromatic bodies, in differentiation, 15

Heterochromatin, 9, 174 Heterosis, 11 Hexenolactone, 176 Hexokinase, 72, 86 inhibition of, 74 Hexokinase phosphatase, 84 Hieracium, 20 HirzKto medicinalis, 3 16 Histamine, 197 Histidine, 28, 277 basic side group of, 221, 225 Histochemistry, and enzyme studies, 326 substrates, enzymatic, 329, 330 Histone, of chromosome, 3 Hormones, 72, 196, 197 and epidermial mitosis, 289 and transport, 86 Horny scale, human, 279 Hyaline layer, of epidermis, 266 sea urchin egg, and birefringence, 204 Hybrid, amphibian, 183-188 and species characteristics, 187, 188 androgenic haploid, 184-188 diploid, 184-188 lethality, 189, 190

Hydrochloric acid, binding by wool, 235 secretion by stomach, 157, 158, 158 Hydrogen bonds, 159, 196 ion, adsorption by protein, 158 Hydroxyglutamic acid, acidic side group of, 221, 225

Hypoxanthine, 30

I Ice crystals, growth, in tissue, 36-39 Image, electron-microscope, 306 Indole acetic acid, 15 Infection, by bacteriophage, 121, 124 Infiltration, agents, for dried tissue, 50, 51 wax, of dried tissue, 50 Inhibition, of crystal growth, 37 Inhibitor, cholinesterase, 323 of esterases, 324, 328 of glucose penetration, 77-79 of melanin formation, 286,287 Insemination, polyspermic, 179 Insulin, 71, 137 and epidermal mitosis, 289 in hexokinase reaction, 86 mode of action, 72, 73 Intercellular bridges, 268-270 human, of stratum spinosum, 268 Intercellular, space, epidermal, and lipids, 274-275

Intestine, and glucose uptake, 79-85 Invertase, inhibition, by pressure, 138 in plant cell, 116 yeast, 68 Iodoacetic acid, 89 Ion, accumulation in cells, 136, 159 balance and actomyosin, 162 effect on sperm respiration, 262 hydrogen, displacement by basic dye, 226

in stomach, 87 released during bacterial staining, 226 exchange in staining, 249 salt competition with color ion, in staining, 235 Ionic strength, dye solution in staining, 233-236

Ionization, at low temperatures, 43 of proteins, 222

SUBJECT INDEX

Iron, Tetrahymena nutrition, 30 Ischemic shock, 289 Isoelectric point and protein charge, 221 and staining, 217 bacterial, determination of 230 cataphoretic, of nucleoprotein, 231 determination of, by staining technique, 230 erythrocyte and alcohol fixation, 245 fibrin apparant and cataphoretic difference, 232 of fungal protein, of keratin, 277 variation, and denaturation, 244 Isoelectric, range of wool, 231 Isoleucine, 28 Isopentane, as cooling agent, 39

J Janus green, 156, 271 staining of mitochondria, 270 Junction, dermoepidermal, in laboratory animals, 267

K Karolymph, 315 filament, 313 Karyokinesis, 10 Karyolysis, 276, 321 Keratin A, 277 Keratin B, 277 Keratin, in skin, 276-278 stereoisomers of, 277, 278 Keratinization, in tonofibrils, I70 insensible, 2 7 1 skin, 276, 277 soft and hard, 279 Keratohyaline, granules, formation, 276 “Kern-platten-elemente”, 3 2-Ketogluconic acid, 116 Kidney, glucose uptake, 19-85 Kinetics, staining, 246-248 Kunne’s methylene blue, 2 15

L Lactase, 68 Lactic acid, 262 bacteria, 31

359

Latent period, of bacteriophage infection, 126 ’ Lauroyl choline, 331, 332 Lens, microscope, 1, 2 Leptone, 314, 315 Lethal effect, in hybrid development, 184, 187 Leucine, 28 Leucocytes, fractionation of, 3 Leuconostoc citrovorum, 31 Leveling power, dye, 249 Life cycle, bacteria, 94, 97 Light-optical border, 306 Light scattering, cell, and mitosis, 195 of f e a t i o n process, 206, 207 of fertilized egg, 258 of sea urchin egg, 203-207 Ligno-cellulose, 249 Lmoleic acid, 329 Lipase, 323 distribution in mouse tissue, 329 inactivation, 328 in epidermis, 282, 283 in sebaceous gland, 297, 298 Lipid, and Golgi apparatus 273 in epidermal tissue, 273-275 in membrane, 71, 72 in sebaceous gland, 293 mitochondria, 291 in sebum, 291 of plasma membrane, 139, 140 perinuclear, in epidermis, 273, 274 storage, 291 Lipochondria, 274, 294 and melanin formation, 286 Lipophanerods, 274, 275 Lipoprotein, of myelin nerve sheath, 317 of plasma membrane, 139 Liquid air, 38 Liquid nitrogen, 39 Lissamine green, 143 and monolayer studies, 154 Liver esterase, 323 Locomotion, sperm, 260-263 LoefEer’s methylene blue, 215 L-organisms, 102, 103 Low temperature, and reduction division, 20

SUBJECT INDEX

Mesoglea, 320 Mesophragms, 317 Metabolism, bacterial, 128 cell, 65 of growing root tip, 108 M of haploid cells, 175 stimulation, by salts, 86, 87 Macrophage, 329 Metachromasia, 227 Magnesium, 30 Metaphase, birefringence, 197, 198, 202 Maleic acid, 262 Metaphosphate, 84 d-Malic acid, 262 Methionine, 28, 29 I-Malic acid, 262 Mallory’s acid phosphotungstic hematoxy- Methyl cellusolve, 4 1 Methylcholanthrene, 269, 274, 292 lin, 269 effect of, on sebaceous gland, 265, 266 Manipulation, of quenched tissue samples, Methylene blue, 156, 215, 249 40, 41 adsorption, by amoeba, 143 Mannose, 77 binding, by fibrin, 231 Mauthner’s cells, 172 magnesium displacement, 226 Mecholyl, 332 Methyl green, 4 Meiosis, 23 specificity of, 237 abnormal, 169 Melanin, distribution, in epidermis, 284, Methyl green-pyronin, 241 Microcyst, bacterial, 98, 102 285 Microenzymology, 2-4 formation, 286 Microincineration, of skin, 283 inhibition of, 286 Microscope, phase contrast, and bacteria, 98 properties, 284 phase, 5 Melanoblast, 285 Microscopy, dark-ground, vertical illumiMelanogenesis, 285 nation, 203, 206 inducement of, 286 development of, 1, 2 Melanoid, 284 electron (see under electron microscopy) Melanophores, 170, 172, 174, 187, 285, 286 phase contrast, 53, 57 Membrane, basement, 285, 288 ultraviolet absorption, 53, 54, 57 in epidermis, 267, 268 Microsome, 166 carriers, 67 Microtome, 308, 310, 312 system in glucose absorption, 82 rocking, 310 cell, 65, 66 Microtomy, high speed, 310 changes in amoeba, 138, 139 low speed, 308-310 contraction and expansion, 139, 140 Millon reagent, 276 keratinized, 278 Mineral requirements of tetrahymena, 30 substances, in “trapped”, 70 Minerals, in epidermis, 283, 284 formation of amoeba, 140, 141 Mitochondria, 269, 272, 273, 276 nuclear of epidermal cells, 287 as indices of cell damage, 271 perforated, 316 changes, in epidermal carcinoma, 271 plasma, amoeba role in cell accumulacytochemistry, 56 tion, 155 flexous, 271 vacuole, role in cell accumulation, 155 in skin, 270-272 Mendelian gene, 22 of sebaceous gland, 291 segregation, 15 polymorphic, 271 Meristem zone, 107

Luminescence, bacterial, 138 Lysine, 28, 158, 277 basic side group of, 221, 225 Lysis, bacterial, 125

36 1

SUBJECT INDEX

Mitosis, and permeability changes, 12 and structural changes, 202, 203, 207 epidermal effect of age on, 289 indices, factors affecting, 289 in epidermis, 280, 287-290 in scalp epidermis, 287 mouse, in epidermis tissue, 288 multipolar, 179 of sebaceous gland, 298, 299 rhythm, of sebaceous gland, 298 Modulated cellular changes, 23 Molecular weight, dye, 239 Molisch fluid, 295 Monospermy, 257 Mordant, 244 bacterial, 93, 94 Mosaic, chromosomal, 176, 177 Mounting, of frozen dried tissue, 52, 53 Mouse, epidermis, 266, 267, 269 Movement, ameboid, 138 surface tension requirements of, 161 bacterial “post-fission”, 98, 99 chromosome, 207 and cell birefringence, 201, 202 dye, and staining efficiency, 247, 248 Mucopolysaccharide, in epidermal cell, 280, 281

Mucoprotein, 221, 225 Mucor, 11 Mucosa, buccal, 290 Mucus, staining, 217 Mucus Malpighii, 266 Multicellvlar bacteria, 99 Multiplication, virus, 121-123 Muscle, cross-striated, 316-318 electron microscopy of, 316-318 fibre, 2 glucose permeability, 71 glucose uptake, 71, 72 proteins, folding of, 136, 137 Mutation, and viruses, 120 gene, and enzymic makeup, 28 somatic, 12 Mycelium, staining, 230 Mycobacteria, morphology and growth, 100

reproduction, 102 Mycobacterium tuberculosis, 95, 101

Myelii nerve sheath, 317 Myoblast, 317 Myonemes, 198 Myosin, 161 and bacterial flagella, 102 orientation of, in muscle, 316-318 properties, 137 Myristoyl choline, 331, 332 Myxobacteria, 94, 97 locomotion, 102

N Nadi reaction, 297 Nadi reagent, 273 Naphthol AS method, 327 a-naphthol, 326 method for esterases, 327 Neisseria, 103 Neoplasm, 321, 322 epidermal, 27 1 Neoplastic cells, in mice, 30 Neurilemmae, 320 Neurofibril, theory of, 314 Neuropile, 316 Neurospora, 11 adsorption studies, 153 Neutral red, 156 accumulation by yeast, 156 adsorption by amoeba, -42, 144, 146 by fibroblasts, 152 by root hair, 147, 148 study of Golgi element, 272 vacuoles, 142, 146 Nicotiana tomentosa, 15 Nile bleue sulfate, 295 effect of, on skin sections, 275 reaction of, with sebaceous glands, 294 Nipples, 290 Nitrogen, amide, 227 bases in Tetrahymena nutrition, 30 mustard, 323 Nitroprusside reaction, 278, 279 Node of Bizzozero, 268, 274, 275 and mitochondria, 270-272 Nuclear competition, in neurospora, 11 reproduction phases of, 24 size, and chemical agents, 17 volume and chromatids, 15

362

SUBJECT INDEX

Nucleation, of ice crystals, 37, 38 Nuclei, cell free, 3 Nucleic acids, 2, 3 bacterial, during growth, 100 transformation, during phage infection, 129, 130 concentration, and staining relationship, 231

exchange, during mitosis, 209 in chromosome division, 2 0 Nuclein, 3 Nucleo-cytoplasmic incompatibility,

184-

188

in haploid eggs, 175, 176 ratio and cell size, 190, 191 Nucleoids, bacterial, 97, 98 Nucleolus, and melanin formation, 285 movement and synthesis, 6 Nucleoplasmic ratio, 10 Nucleoprotein, 221 extracted, staining, 231 in epidermal cell, 280 Nucleus, and differentiation, 22, 23 and production of chemical agents, 2082 10

bacterial, 94, 95 characteristics, 97 division, 96, 97, 98 electron-optical stpdy of, 315 in differentiation, 23 in frozen dried tissue, 57-61 neuronal, 316 oocyte, 165 size, and cell size, 170, 173, 174 Nutrition, animal, 27 bacteriophage, 121 Nylon, dyeing of, 227, 240

0 Oenothera, 13 Oil drop, studies of protein folding, 137 Oleic acid, 293, 329 Olivary corpuscle, 319 One-hit hypothesis, 22 Oocytes, movement, 6 Orange G, 231 Orchid pollen, 11 Orientation, during mitosis, 198-203

Ornithine, 29 Orotic acid, 3 0 Orthokinesis, 262 Osmic acid, 314 reaction with sebaceous glands, 293 Osmiophilic Golgi bodies, 291, 292 Osmium tetroxide, 272, 313, 314 Osmotic pressure, and nuclear size, 17 of blood glucose, 75, 76 Osmotic work, 135 and protein folding, 150 mechanism, 153 Ovalbumin, 150 adsorption studies, 141, 145 Oxalacetic acid, 262 Oxidation, disulfide, 281 glucose, 73 Oxygen, tension and sugar absorption, 111 uptake of spermatozoa, 262 Oxyhemoglobin, 284

P Palmitoyl choline, 331, 332 Palpebrae, 290 Papillae, dermal, 267 Paramecium, 3 Pararosaniline violet, 93 Parasite, intracellular, 123 126 Parasitism, by bacteriophage, 121 Parthenogenesis, 169 Pasteurella, 101 Paw, cat, epidermis of, 267 Penetration, dye, 247, 248 glucose, inhibition, 77 kinetics, 76, 77 of sugars and competition, 76, 77 Pentose nucle\ic acid, bacterial, 128 Pepsin, 137 digestion of cell cytoplasm, 3 Peptide linkage, 159, 221 Periodic acid, 95

-

PerdHaneta anten'cana, 317 Peritrichous flagella, 102 PerivitelIine fluid, 320

Permeability, cell, and mitosis, 195, 196 during inhibition of glucose utilization, 73

SUBJECT INDEX

during meosis, 12 of enzyme effectors, 66 Peroxidase, 262 in sebaceous gland, 297 Pfeifferella, 101 pH, and bacteria staining, 218 and decolorizing stained material, 249 and dye uptake, 223 and isoelectric point of proteins, 222, 223 and protein-dye affinity, 220 effect of, on textile dyeing, 219, 220 role in staining, 215-218, 232, 233 Phenobarbital, 77 Phenol red, absorption by kidney, 156 Phenylalanine, 28, 29 Phloretin, 77, 78 Phloridzin, 77 and epidermal mitosis, 289 inhibition of glucose absorption, 81, 82, 83

Phosphatase, 69, 136 inhibition of, 83 in epidermis, 282, 283 in sebaceous glands, 297 Phosphate, energy rich, 69 in Tetrahymena nutrition, 30 uptake, by egg cell, 160, 161 Phospholipid, in sebaceous glands, 294 test for, 294 Phosphorous, in nucleic acid synthesis, 22 Phosphorylation, in ghcose metabolism, 71, 72

inhibition of, 77, 78 Phytotomy, 1 Pigments, cell, during mitosis, 12 in epidermis, 284-287 Pilosebaceous exit, 295 orifice, 282 Pituitary hormone, 72 pK, value of soluble proteins, 229 Plasmagenes, 23, 24 Plasma Ieptones, 315 Plasmanucleatides, 315 Plasmasol, 141 Plastin, 3 Pleurodeles waltlii, 170 Pleuropneumonia, 102

Pneumococcus transformation, 123

363

Podophyllin, 269 Polar body, formation, suppression of, 169 Polar flagella, bacterial, 102 Polarized light, and mitosis, 198, 201 Pollen tube, 2 Polyethylene glycbl, 326 Polyglutamates, 3 1 Polykaryotic fungi, 11 Polymerization, of butyl methacrylate, 312 of keratin, 278 Polypeptide, skeleton of keratin, 278 Polyploidy, 11, 13, 14 amphibian, 168 stimulation of, 13, 14 Polysaccharides and bacterial flagella, 102 in epidermis, 281, 282 Polysomaty, 14, 15, 18 Polyspermy, 257, 258 block, 206, 258 time factor in, 259 Polyteny, 11, 14 in eneric nuclei, 15 Postchromation technique in study of mitochondria, 270, 271 Potassium, adsorption of, 155 in Tetrahymena nutrition, 3 Pressure, effect of, on a-keratin, 278 313, 314

Precipitation, tissue, by quenching, 36 Premelanin, 285 Preputium, 290 Pressure, effect of, on a-keratin, 278 Prickle cell, 268 Primwla kervensis, 20 Proflavine, 143 Proliferation, of sebaceous gland, 298, 299 Proline, 29 Pronuclei, amphibian, 314 Prophase birefringence, 202 Protein, acid dye interaction, 227 albuminoid, in skin, 277, 278 alteration during h a t i o n , 242 amoeba, and membrane formation, 155 unfolding of, 140 amphoteric nature of, 217 and hydrogen ion liberation, 158

364

SUBJECT INDEX

and specific stains, 237-241 cellular, template, 138 chitin lamellae, 318 conjugated, 221 content, variation in tissue, 22 decomposition, by dyes, 249 denaturation, in tissue fixation, 61, 62 dried, rehydration of, 51 dye interaction, 212, 213, 214 and pH, 215 folding, 136, 141, 142, 151 and accumulatory mechanisms, 147 and cell accumulation, 156, 157 globular, 138, 140 folding of, 160 monolayers, 137 and dye accumulation, 141-150 collapse pressure, 139, 140 of growing root tip, 108, 110, 117 of T2 phage, 124, 131 precipitation by acid dyes, 216 soluble, in staining studies, 229 staining, 214, 215 swelling and temperature, 246 synthesis, in epidermis, 281 terminal amino and carboxyl groups, 221 unfolding, 137, 138, 141, 142, 151 mechanism, 153, 154, 160 Proteus, Bagella of, 102 Protogen, 30 relationship to acetate, 31 Protoplasm, contractibility, 137 quenched, 42 precipitation of, 142 streaming, in amoeba, 141-145 Protoplast, and sugar absorption, 114, 117 Psammechinus miliaris, 198, 2 5 7 , 258 Pseudoacid, 216 Pseudobase, 216 Pseudo-cholinesterase, 332 Pseudopodia, amoebae, 138 Psoriasis, 271 Pteroic acid, 31 Pyridine nucleotide, 85 Pyruvic acid, 262 oxidation, 31

Quinine, 324, 328 Q-zones, 317

Q R

Radioisotope, and bacteriophage, 132 Rana esculenta, 185 R a m palustris, 184 Rana fipiens, 166, 170, 172, 175, 178, 182, 185 R a m sylvatica, 185 Ranvier node, 314 Rat, and amino acid, 28, 29 R. catebiana, 182 Reaction centers, plant, 117 metabolic, and membrane enzymes, 86 sugar absorption, 117, 118 Recombination, genetic, of phage particles, 126, 127, 128 triparental, in bacteriophage, 127, 128 Reductase, 29 Reduction-division, 18-2I bacterial, 98 in somatic cells, 19, 20 phases during, 18, 19 Refractive index, and mitosis, 196 Regaud fixation, 314, 315 Regaud’s method, for mitochondria staining, 270 Reproduction, bacterial, 102, 103 chromosome, 9, 10, 14 of bacteriophage, 126 of gene lamellae, 22 Respiration, amphibian, 185 induced by salt accumulation, 157 plant, and sugar concentration, 113, 114 Rete mucosum, 266 Rete pegs, 267 Reticulin, 316 Reticulum, argyrophilic, of basement rnernbrane, 268 formed by leptones, 314 neurokeratin, 318 Reverse mutation, 22 R . fusca, 185 Rhabdite, 319 Rhizobium, 102 Rhoeo, 15

SUBJECT INDEX

Ribonuclease, 4 action on sebaceous gland, 296 effect of, on skin sections, 280 Ribonucleic acid, and nuclear size, 17 bacterial, 96 in epidermal cell, 280 in spider nucleolus, 6 Ribonucleoprotein, amphibian, 185 of haploids, 176 of sebaceous gland, 296 Ricinoleic acid, 329 Ridge, epidermal, 267, 268 Ringer solution, 152 Root hair, 147 tip, excised, 108 growth, 107, 108 Rose Bengal, 143 Rous sarcoma cells, 152 Rubidium, adsorption of, 115

S Sahli’s methylene blue, 215 Salamandra maculosa, 184, 185 Salivary gland, 160 Salmonella, 102 Salt, formation, in dye-protein reaction, 224 linkage, 224 and dye-binding, 225 in frozen tissue, 42, 43 Sambucus, 20 Sarcoplasm, 3 17 Scharlach R, 273 Schiff reagent, 281 Sciara, 19, 2 1 Sea urchih egg, basophilia, 4 enzymes of, 69 Sebaceous cell, 274, 281 gland, effect of methylcholanthrene on, 292 human, distribution, 290 morphology, 290, 291 mouse, regeneration of, 265, 266 Sebaceous kernals, 299 Sebum. 291-298 Sections, ultra thin, 305-312 Semen, sea ,urchin, 257

365

Septum, bacterial, formation in division, 99 secretory, 99, 10 Serine, 159 acidic side group of, 2 2 1 in Tetrahymena metabolism, 29 Sex cells and desoxyribonucleic acid content, 16 Sheath, ciliary, 319 myelin, 318 Shigella, 102 Silver lines, of ciliates, 3 19 Site, dye binding, 226 Skin, amphibian, structure of, 319 human, dermoepidermal junction of, 267 mitosis in, 285 isoekctric point, 231 keratinization of, 275-278 spinous layer of, 266 Smith-Dietrich phospholipid method, 294 Soap-calcium complex, 326 Sodium gelatinate, 226 Sodium nucleinate, 226 Solonum darwinianum, 19 Sorbitan, 326 Sorbose, 77 Sorghum, 9 Species, characteristics and nucleo-cytoplasmic relationship, 187, 188 Specificity, between dye and substrate, 237 structural, of sugars and penetration, 77 Spectrophotometric analysis, of skin minerals, 283 Spermatozoa, 257, 258, 259 amphibian, 182 chemotoxis of, 260 density, 258, 259 egg interaction, 166 fern, 262 inducement of birefringence, in egg, 206 tail of, 319 Spindle, birefringence of, 195, 198, 201, 204 electron-optical study of, 315 function in reduction, 2 1 Spinous cell, 268 Spirochaete, 103 Sporangium, bacterial, 98

366

SUBJECT INDEX

Spore, bacterial, 94 Staining, and electrostatic charge, 223 and isoelectric point relationship, 230233

and protein dissociation,. 221-223 fibrin ionic strength relationship, 234 intensity, and protein changes, 244, 245 lipid, 293 of model protein system, 220 of skin granules, 276 reversibility, 248-250 stoichiometry studies, 229 theories of, 212, 213 time, 249, 250 Stains, basic, and protein precipitation, 216 Starch, and epidermal mitosis, 289 Starvation, effect on mitosis, 288 Stereospecificity, of cell membrane, 67 Steric hindrance, and dye adsorption, 237, 239, 240

Sterioisomers, and sugar absorption, 80, 81 Stimuli, melanogenk, 286, 287 Stoichiometry, of protein-dye interaction, 225-230

Stomata guard cells, 17 Stratum basale, 266, 269, 279 Stratum corneum, 266, 267, 269, 275, 278, 279, 283

Stratum cyliidricum, 266 Stratum germinativum, 266, 268-272, 276278, 280, 281, 287

intercellular bridges of, 268-270 Stratum granulosum, 266, 271, 272, 274,

genetic, of virus, 122, 123 surface, of bacteria, 95, 96 of insect cell, 317, 318 Substantia spongiosa, 319 Substrate, carrier complex 8 1 82 cellular non-penetrating, 66 cytoplasmic, and gene interaction, 174 dye structure and affinity, 240, 241 membrane-soluble, 67 of esterases, 324 Subunit, bacteriophage, 121-123, 128, 130 Sucdnic acid, 262 Sucrose, absorption, 115 cleavage during plant growth, 116 crystal, at low temperature, 41 Sudan black, 273, 274, 292, 295 Sudan 111, 273 Sudanophila, 275 Sugar, accumulation in cell vacuole, 113 Sulfhydryl groups, in epidermis, 278-280 role of, 279, 280 in human horny scales, 279 skin distribution of, 279 Sulfonamides, 27 Sulfonic acids, 281 Sulfur, content of keratin, 277 in bacteria, 101 Supravital staining, 270 Surface, area and absorption, 110, 111 protein, enzyme units, in plants, 116, 117 tension, of plasma membrane, 139, 140 Syngamy, 97 Systematics, bacterial, 103, 104

275, 278-284

Stratum lucidum, 266, 267, 275, 278 Stratum Malpighii, 266 Stratum spinosum, 266, 275, 279, 281, 283, 284, 287

Streaming, protoplasmic, in enucleated amoebae, 161 in root hairs, 147, 148 Streptobacillus monilijormis, 103 Streptococcus bowis, 69 Streptomyces, 97 Streptomycin, 28 Stretching, molecular, of keratin, 277 Structure, bacteriophage, ultraviolet sensitive, 131

T Tailtip, salamander, 170 T . dpestds, 166, 172, 175, 184, 187 T . cristatus, 184, 187 Tellyesniczky’s mixture, 315 Telophragms, 317, 318 Temperature coefficient, of penetration rate, 68 cooling, critical for different tissues, 39, 40

critical, for crystal formation, 37, 38 drying for tissue, 36, 41-43 eutectic for tissue, 36, 41, 42, 43 inducement of polyploidy, 169

SUBJECT INDEX

of cooling agents, 38, 39 of staining sokution, 245, 246 vitrification, 41, 42 Tension, membrane, and mitosis, 195, 196 Testosterone propionate, 287, 298 Tetrahymena, 28, 29 Tetraploid nuclei, 13 Tetrazo-benzidine reagent, 61 Textile, dyeing, 212, 216, 217, 249 Thermal expansion method, for ultramicrotomy, 312, 313 Thickness, of tissue section, 305, 308, 310 Thiocholine technique, 332 for cholinesterase activity, 327 Thionin, 216 Threonine, 28, 159 Thymidine, 30 Thymine, synthesis in Tetrahymena, 30 Tissue, amphibian, 185 binding of dyes, 218, 219 colloid behavior of, on staining, 218 culture, 6 development and chromosome patterns, 176, 177

dried, treatment of, 50-53 drying, 41-50 carbon dioxide, 35, 44 frozen dried, cytochemistry of, 54-57 quenching, 36-41 reactions in, retardation, 36 structure, on cooling, 38, 41 samples, artifacts on cooling, 40 various, electron microscopy of, 307 Titration, of wool, 229 Tobacco mosaic virus, 320 adsorption studies, 141 Toluidin blue, 280 and nucleic acid staining, 231 staining and pH, 217 Tonofibril, and keratin x-ray diffraction pattern, 278 in keratohyaline granules, 276 of epidermis, 269, 270 of human epidermis, 269 Topochemistry, 306 T1 phage, in lysis of bacteria, 125 T2 phage, composition, 124 morphology, 123, 124

367

mutants, 126, 127

T4 phage, morphology, 123, 124 Tradescantia, 5, 17 Translocation, of chemicals in tissue, 55, 61 Transphosphorylation Transplantation, of nucleus, 10, 11 tissue, 186 Transport, active, 67, 70, 82 control of, 65-68 controlled and cell function, 70 controlled enzymatically, 66, 67, 70 glucose, 66, 70 rate-concentration relationship, 76, 77, 80

of substrates, mechanism, 85-89 Traps, for diffusion pump, 45, 46, 47 Trehalose, fermentation, 68 Tricarboxylic acids, and sperm chemotaxis, 262 Triglycerides, 294 . Triploid chromosomes, 15 Triton palmatus, 179, 181, 184, 185, 187 Triton taeniatus, 166, 175, 184 Triturus pyrrhogaster, 172 Triturus torosus, 187 Triturus viridescens, 166, 168, 170, 172, 179, 181

T . rivularis, 187 Trypan blue, 249 Trypsin, 120, 137 Trypsinogen, 120 Tryptophan, 28, 29 deacetylation, by Tetrahymena, 29 Tubules, kidney, and dye accumulation, 156

Tumors, 19 Turgor function, of cnida capsule wall, 319 Tween, 326 hydrolysis of, 328, 329 technique, 326, 328, 330 Typhoid bacterhrn, 94, 97 Tyrosinase, 286 Tyrosine, 158, 159, 284 acidic side group of, 221 color reaction, 276 synthesis, 29 Tyson's gland, 290

365

SUBJECT INDEX

U Ultramicrotomy, 308-3 13 Ultra-structure, 305, 306 cell, primary, 314, 315 secondary, 316, 321 Ultraviolet irradiation, bacterial, 129 Ultraviolet light, reaction of sebaceous gland, 296 Ungulate, hoove, 269 Unna’s methylene blue, 215 Uracil, 30 Uranyl-adenosinetriphosphate complex, 73, 14

Uranyl ion, 73, 74 Urea, and protein denaturation, 159 cycle, 29 Uridine, 30 Urodeles, 11

V Vacuole, cell, and cell accumulation mechanism, 154, 155 role in accumulation, 155 neutral red, in epidermal cells, 272 root hair, 147, 148, 149 “Vacuome” of Parat, 272, 274 Valine, 28 Valonia, 155 Veratrine, 162 Virus, and electron microscopy, 320 animal, 132 in mouse mammary carcinoma tissue, 320 multiplication, 127 origin, 119, 120 vegetative form, 127 Viscosity, cytoplasmic, during mitosis, 195, 196

Vitamin, and Tetrahymena, 30 Be analogue, 33

Bn, 27 stimulation of Tetrahymena, 3 1

Volume, molecular, 138 Volutin, 101 Vorticella, 3

W Water, crystallbation, 37, 38 in protein folding, 160 Wedge-sectioning method, 308 Wool, 225, 239 acetylation of, 226 amido groups of, 224, 225 deaminated fibres of, 226 dye binding, 245 groups of, 227 dye interaction, 228 dyeing, and salt concentration, 235 dyeing of, 217, 218 equilibrium dyeing, 245 koelectric range, 231

X Xanthme oxidase, 30 Xanthoproteic test, 276 X-chromosome, 2 1 X-irradiation, and melanogenesis, 286 bacterial, during phage infection, 129, 130

of mouse skin, 274 X-ray, diffraction, of keratin, 277 Xylose, 76, 17

Y Yeast, glucose uptake, 13-75 Yolk, egg, 166, 175, 176

z Zwitterion, 221, 222 Zones, tissue of crystals, 38, 39 Zygote, virus, 123