Carlsberg Res. Commun. Vol. 49, p. 179-186, 1984

THE EARLY DAYS OF by CICILY CHAPMAN-ANDRESEN

Institute of Biology and Anatomy, Universitetsparken 15, DK-2100 Copenhagen O.

Keywords: Pinocytosis, amoebae

1. INTRODUCTION photographs and descriptions of this process, The earliest suggestion of "cell drinking" showing that during active movements at the cell appears to be the medically orientated theory surface, droplets of the fluid medium were put forward by the American physician engulfed; these droplets were at first of irregular MELTZER in 1904 (51). This hypothesis, for shape, but became spherical as they passed into which there was no experimental evidence, was the interior of the cell, sometimes fusing with clearly influenced by METCHNIKOF~S (52) de- other droplets, and rapidly decreasing in size. It scription and definition ofphagocytosis in 1883. was in this classical paper that the term pinocy- If cells can eat, why should they not also drink? tosis first appeared, at the suggestion of Lewis' MELTZER'S theory sought to explain the forma- colleague, DAVID M. ROBINSON; the designa- tion of oedema by the transport of fluid though tion was derived from the Greek, rltvetv to drink, ceils, which "sipped" fluid from the surrounding and rvxo~ cell. Later LEWIS (47) observed the medium, a process which this author called same phenomenon in cultured malignant cells. "'. Apart from an observation by Thus the term pinocytosis appeared in the litera- EDWARDS (27) in 1925 on food cup formation ture 50 years after METCHNIKOFFhad described induced by neutral inorganic salt solutions in and defined . amoebae, there were no further published data The observations of MAST and DOYLE (49) in or theories on fluid uptake until 1930. In this 1934 on the uptake of fluid by freshwater amoe- year, a short editorial in the Journal of the bae, a process for which these authors also used American Medical Association by MAIN- Lewis' term pinocytosis, differed in two respects WARING (,48) under the title "Hydrophago- from LEWIS' descriptions of the process in mac- " brought to the attention of physicians rophages; in the droplets were the cine films of the movements of rat macro- engulfed directly at the cell surface, while the phages in culture which LEWIS had demon- amoebae formed channels on rather immobile strated at meetings in the United States. pseudopodia, at the base of which were MAINWARING pointed out that "the Lewis phe- pinched off from the channels. The other differ- nomenon", during which the macrophages cap- ence between pinocytosis in these two cell types tured and swallowed plasma from the surround- was that while the macrophages continually ing medium, could be of importance in cellular formed droplets from the nutrient medium, the nutrition, and in "mechanical filtration of puri- amoebae required a "stimulus", in the form of fication of body fluids", as well as "disposal of diluted sea water or egg albumin, before pinocy- souble antigens and .. synthesis of specific anti- tosis could take place: it should be noted here bodies". Then in 1931 LEWIS (46) published that while the medium was a nu-

Springer-Verlag 0105-1938/84/0049/0179/$01.60 C. CHAPMAN-ANDRESEN: Early days of pinocytosis

Figure 1. a and b. Pinocytosis in rat omental macrophages, a. Redrawn from MAST (46), the first paper on pinocytosis, b. Phase contrast photograph (CHAPMAN-ANDRESEN).C and d. Pinocytosis in fresh water amoebae. c. Drawing from MAST and DOYLE (49), the first paper on this subject, d. Phase contrast photograph (CHAPMAN-ANDRESEN).

trient medium, the medium, which was details of the morphology of the process (see a low concentration of inorganic salts, could not Figure 1). be considered as nutrient. Higher concentra- Meanwhile, since METCHNIKOFF'S first stu- tions of salts and of albumin, a protein, could dies and his further exploration ofphagocytosis however be nutritional importance, hence the through many animal groups from amoeba to early emphasis on this aspect of the significance man, considerable data had been accumulated ofpinocytosis. These early observations on mac- from in vivo and in vitro observations on a wide rophages and amoebae were all made on living variety of cells, which phagocytosed living or cells without the aid of phase contrast or other dead bacteria, organic or inorganic particles of accessory optics, but they give clear and full different types. The significance of phagocy-

180 Carlsberg Res. Commun. Vol. 49, p. 179-186, 1984 C. CHAPMAN-ANDRESEN:Early days of pinocytosis

tosis-enhancing factors from normal and sensi- After confirmation of the need for protein or tized serum was realized, and the influence of salt (8, 24) in the medium before pinocytosis many factors, such as temperature, salts, pH and could take place, it soon became clear that not all hormones was studied. Reviews such as those of salts or all proteins were equally effective MUDD et al. (53) and of BERRY and SPIES (1) "inducers", as the solutes which were responsi- summarize the extensive research in this field up ble for the initiation of the process were now to 1949. It can thus be seen that phagocytosis had called. Using a quantitative method for estimat- early proved to be a subject of interest to medical ing the intensity of pinocytosis, based on the physiologists, and was soon attributed a role in mean number of channels formed under stan- certain diseases and in normal body repair dard conditions, the inducing capacity of a range functions. However, the concept ofpinocytosis, of water soluble proteins of differing iso-ionic during the first two decades after its first descrip- point was explored. The data showed that under tion, was either ignored, rejected as pathological conditions where the predominant charge of the in mammalian cells e.g. by BESSlS (2), or consi- protein was positive, pinocytosis was induced dered as a normal cellular process only in cul- (9). In general, non-toxic inorganic salts were tured cells and in Protozoa. effective inclucers at approximately neutral pH, although calcium salts were an exception: non- toxic basic dyes were also good inducers. 2. STUDIES ON PINOCYTOSIS AT Further experiments with these different CARLSBERG LABORATORIUM types of inducers confirmed the importance of The above introduction shows briefly the the interaction between cell surface and inducer background against which studies on pinocy- for the initiation ofpinocytosis; e.g. the presence tosis were started at Carlsberg Laboratorium, of fluorescein-labelled protein or basic dyes first at the Cytochemical Department, and from could be visualised on the cell surface. This first 1956 at the Physiological Department under phase of surface binding was independent of HEINZ HOLTER, who had again spotted an temperature (9) but was influenced by condi- important field in , which could be tions in the medium. Changes in the pH of the developed with the material and techniques medium and in its ionic strength, however, available at Carlsberg. influenced different types of inducers in differ- Cultures of the fresh water amoebae Amoeba ent ways, indicating that surface binding was proteus and Chaos carolinense (then known as several types, showing varying degrees of labil- C. chaos or Pelomyxa carolinensis) had been ity. These observations led to the division of kept in the laboratory for some years, for bioche- inducers into three types, according to the fac- mical, physiological and cytological studies, of-. tors which led to the release of the inducer from ten using microtechniques, which permitted the cell surface, or in modem terminology various types of measurements to be made on "enhance the receptor-ligand dissociation". single amoebae; studies with radioactive tracers Inorganic salts, as well as neutral salts of amino were also in progress. HEINZ HOLTER, together acids were effective as inducers only when the with a newly arrived guest from U.S.A., JOHN M. active solutes were present in the medium: on MARSHALL JR., then commenced studies on transfer to a non-inducing medium pinocytosis pinocytosis in fresh water amoebae. In these first ceased immediately. Surface-bound protein was experiments, the uptake of a protein, fluorescein not released on transfer of the amoebae to a labelled gamma globulin, was explored by mea- non-inducing medium of the same pH as that of surements of fluorescence in single amoebae. the inducing solution, while immediate release This (43) was the first of a series of studies on occurred if the pH of the non-protein medium different aspects of pinocytosis which issued differed (19). For basic dyes, the conditions were from HOLTER'Slaboratory during the next two similar to those shown by protein inducers. decades, the results of his collaboration with Finally, in a small group of compounds, so far guests from Denmark and abroad and with the comprising the copper phthalocyanin dye alcian laboratory's own co-workers. blue, and rhuthenium red, the dye, once bound

Carlsberg Res. Commun. Vol. 49, p. 179-186, 1984 181 C. CHAPMAN-ANDRESEN: Early days of pinocytosis

the uptake and fate of material ingested by pinocytosis. Further methods available at that time for measuring uptake was the use of radio- active compounds as tracers; rather few were then available compared with the vast range now commercially produced. In the first experi- t] b c ments, glucose, labelled with C-14, was used, as carbohydrates had been shown to be non-induc- ing (17), and hence might give a measure of the volume of fluid ingested. Non-radioactive albu- min was used to induce pinocytosis, and the fate ?& of the ingested material was followed by direct measurements of radio activity and by autora- diography. The glucose was metabolised by the d e amoebae, thus showing that it penetrated the Figure 2. Diagrams of different, microscopically visible pinocytic vacuoles, and that in addition to water, modes of ingestion in amoebae, a. Typical pinocytic significant amounts of solute entered during channel in salt solution, b. Pinocytic channel in solu- pinocytosis. In further studies (18) an attempt tion of tobacco mosaic virus, c. "Bottle-shaped" cavity was made to differentiate between the uptake of in solution of tobacco mosiac virus, d. Cavity formed fluid and that of surface-bound solute. C-14 in optically clear methionine solution, e. Formation glucose was again chosen as the label for fluid of food containing small ciliate. From uptake, while serum albumin, labelled with CHAPMAN-ANDRESEN and PRESCOTT (24). 1-131 was used to indicate uptake of solute. Comparison of the ratios of isotopes in the inducing solution and within the amoebae after to the surface, remained firmly attatched despite pinocytosis showed that the albumin was con- all possible changes in pH or ionic strength of the centrated during pinocytosis, and that the entry medium, that living amoebae could tolerate (9, of fluid was low. Adsorption of the albumin to 10). the filaments of the mucous coat could be seen The second phase of pinocytosis, the active by electron microscopy; the glycocalyx had be- phase of channel formation, which followed come extended to about three times its usual rapidly after surface binding, was dependent of length of ca. 100 nm. The weight of protein temperature, and sensitive to inhibitors ofglyco- entering by pinocytosis could amount to ca. 40% lysis and oxidative phosphorylation (11). Dur- of the dry mass of the amoeba. Although the use ing intense pinocytosis active locomotion of glucose, which is metabolized by the amoe- ceased, and the contractile vacuole ceased to bae, as the marker for fluid uptake was by no function. The appearance of the channels at the means ideal, yet these data showed the very large tips of non-locomotory pseudopodia was rapid, amounts of solute which could enter the cell and the channels exhibited peristaltic move- during pinocytosis. In later experiments using ments as they disappeared into the organelle- I- 131 labelled albumin as inducer, it was shown containing cytoplasm at their base. The width of that the pinocytosed albumin was degraded the channels depended on the type of inducer, within the cell, indicating that pinocytosed pro- being very narrow, ca. l I.tm, in acid protein tein followed the normal digestive path in the solutions, and to 5 Ixm in some basic dye amoebae (6). Radioactive tracers were also used solutions. In fact a whole spectrum of channels to measure pinocytic uptake of inorganic salts types could be observed, the largest being the (16). bottle-shaped vacuoles formed in virus solutions Using a different type of protein marker, the (24) (see Figure 2). iron core of ferritin, the uptake and fate of this After these morphological observations, in- protein was studied, using the modern method terest returned to quantitative measurements on of atomic adsorption for measuring the iron

182 Carlsberg Res. Commun. Vol. 49, p. 179-186, 1984 C. CHAPMAN-ANDRESEN: Early days of pinocytosis content of the protein, and the old method of in was explored in a wide rage of vertebrate cells vivo centrifugation for following the association (42, 44), and indicated that while cells of the of the protein to different types of cell organelles, reticulo-endothelial system, of kidney tubules, and for measuring the turn over of and of young intestinal epithelium, and also (15). In addition to the light microscopic studies cultured malignant cells and various types of on living amoebae, fine structural studies were leucocytes ingested the protein, other tissue cells begun; the first electronmicrograph ofa pinocy- appeared to be inactive in this respect. Other tic channel, induced in Amoeba proteus by a studies included the uptake of fluid droplets solution of sodium glutamate, perhaps not very from a nutrient medium by the ciliate Tetrahy- elegant by modern standards, but clear, was mena (23), uptake by plant cells (3), and by cells published in 1960 (21). Further fine structural from mammalian peritoneal exudates (7). studies reflected differences in binding lability of Lectures and reviews by HOLTER (30 - 41) the inducing solutes with the appearance of the brought the experimental studies of the labota- mucout coat, or glycocalyx, and the details of the tory, many of which were published in Carlsberg first phases of pinocytosis of alcian blue (29). Laboratorium's own Journal, to a wider audi- This useful copper phathalocyanine dye, visible ence. by both light and electron microscopy, was also The excellent working conditions and facili- a marker in studies aiming to relate the path of ties of the laboratory, the free atomsphere, where pinocytosed material through the cell, with that one could do "one's own thing", but have access of the path taken by the normal food of the to the support which discussions, suggestions amoeba, living ciliates. Here again, a double and criticisms from HOLTER gave, all contri- labelling technique, with Stentor polymorphus, buted to the progress of these studies, which were which contains symbiontic green algae, was the only one of the many fields of research in the marker for normal digestion (phagocytosis) and laboratory. alcian blue the marker for pinocytosed material, With HOLTER'S retirement in 1971, and the both markers visible by light and electron mi- ensuing changes and reorganization of the Phy- croscopy (12, 22). By following a number of siological Department, the study of pinocytosis carefully timed sequences, it was found that the passed from Carlsberg Laboratorium. However, alcian blue labelled secondary lysomomes, some of these studies continue in Copenhagen, which then fused with the Stentor-containing at the University's Institute of Cell Biology and food vacuoles, as well as with each other, so that Anatomy, where the first observations on finally both labels were found before egestion in vacuole formation in Tetrahymena (23) have led the same vacuoles. Lysosomal , as indi- to a series of papers by NILSSON (55, 56, 57, 61), cated by acid phosphatase, were also found in on various aspects of uptake, combined with these vacuoles up to egestion (20). physiological and fine structural studies. Fresh Further studies on pinocytosis of inorganic water amoebae are still cultured at the same salts, and the inhibiting action of calcium early Institute, at which studies on pinocytosis still found to differ in its inducing capacity (5, 25) proceed (13, 14). These studies at the University refocussed attention on the cell surface, on the of Copenhagen have been made possible by the mechanism of the first phase of induction, on the generous support of the Carlsberg Foundation. idea of the glycocalyx as a type of -exchange resin, containing weakly acid groups, and on the significance of the permeability of the plasma 3. CONCLUSION , with its changes in transmembrane This account of studies on pinocytosis at conductance and impedance (4). Carlsberg does not intend to imply that pinocy- In all the studies so far mentioned in this tosis was not studied, and studied extensively as section, fresh water amoebae were used as the time passed, in other institutions. However, it is experimental animals. However, other or- not appropriate here to mention more than a few ganisms and cell types were also included. The of the many developments which took place uptake of fluorescein-labelled plasma proteins during this period. Following LEWIS' classical

Carlsberg Res. Commun. Vol. 49, p. 179-186, 1984 183 C. CHAPMAN-ANDRESEN:Early days of pinocytosis

studies on cultured cells, GEY and co-workers first references in a paper from 1983 "Phagocyte- (28) and ROSE (58) continued with cinematogra- pathogenic microbe interactions" (59), two re- phic methods and analyses to collect data on views on pinocytosis, the first by HOLTER from different types of normal and malignant verte- 1959 (32), the second by a former co-worker brate cells in culture, especially with respect to (14). the inter-relationship between the pinocytic vacuoles and other cellular organelles, and the fate of the vacuoles. REFERENCES Other interesting studies on amoebae include 1. BERRY.J.L a T.D. SPIES: Phagocytosis. Medicine RUSTADS observations (59) on orientation of 28, 239-300 (1949) basic dyes at the cell surface, during the first, 2. BESSIS.M: Trait4 de Cytologie sanguine. Masson adsorptive phase of induction; competition was et Cie, Paris (1954) found between basic dyes and basic proteins, 3. BRADFUTE, O.E.C. CHAPMAN-ANDRESEN & W. indicating acidic sites at the surface. Other JENSEN: Concerning morphological evidence for pinocytosis in higher plants. Expt. Cell Res. 207- approaches were those of JOSEFSSON and co- 210 (1964) workers (45) who tested the influence of drugs on 4. BRANDT,P.W. & K.B. HENDIL: Properties of the pinocytosis on inorganic salts, and the inter- plasma membrane of Amoeba. In: Surface Chemi- action between different cations. A series of stry of Biological Systems. Plenum Press, 323-335 papers exploring fine structure during pinocy- (1970) tosis was started by the BONN group (62), to- 5, BRANDT.P.W. & KB. HENDIL:Plasma membrane gether with the fate of different types of inducers. permeability and pinocytosis in Chaos chaos. Further studies on surface binding of proteins Compt. Rend. Lab. Carlsberg 38, 297-313 (1972) and their fate were made by MARSHALL and 6, BROWNSTONE.Y.S. & C. CHAPMAN-ANDRESEN: co-workers (50, 54), using ferritin and methyl- Degradation of endocytosed albumin by Chaos chaos. Compt. Rend. Lab. Carlsberg 38, 297-313 ated ferritin, two proteins of similar structure, (1971) but differing in solubility and iso-ionic point. 7. CHAPMAN-ANDRESEN, C.: Some observations on From these and other data, it became grad- pinocytosis in leucocytes. Expt. Cell Research 12, ually clear that the distinction between pinocy- 397-399 (1957) tosis and phagocytosis as originally difined, i.e. 8. CHAPMAN-ANDRESEN, C,: Pinocytosis of inorga- between uptake of fluid or of solid material, nic salts by Amoeba proteus. Compt. Rend. Lab. was tenuous, as in both cases solute and fluid Carlsberg, Ser. chim. 31, 77-92 (1958) entered the cell simultaneously, and in both 9. CHAPMAN-ANDRESEN, C.: Studies on pinocytosis cases initiation was dependent on cell surface in amoebae. Compt. Rend. Lab. Carlsberg 33, 73-264 (1962) interactions. Numerous other designations had 10. CHAPMAN-ANDRESEN, C." Measurement of mate- been given to uptake processes, which have rial uptake by cells: Pinocytosis. In: Methods in essential similarities with pinocytosis and pha- Cell Physiology Vol. I, 277-304 (1964) gocytosis, these are listed in (9). In 1963, a new I I. CHAPMAN-ANDRESEN, CS: The effect of matabo- designation "" was proposed by DE lic inhibitors on pinocytosis in amoebae. Proto- DUVE (26) to cover all uptake processes involv- plasma 43, 103-105 (1967) ing active participation of the plasmamembrane 12. CHAPMAN-ANDRESEN,C.: Studies on endocytosis in the formation of vacuoles or channels, which in amoebae. The distribution of pinocytically in- contain components of the extracellular me- gested dyes in relation to food vacuoles in Chaos dium, and of which the plasmalemma forms chaos. I. Light microscopic observations. Compt. the limiting membrane. This term is now often Rend. Lab. Cadsberg 36, 161-187 (1967) 13. CHAPMAN-ANDRESEN,C.: Endocytic processes. In: used in place of pinocytosis, although the latter The Biology of Amoeba. Acad. Press 319-348 is still used, especially when the vacuoles formed (1973) are large, and the fluid component considerable. 14. Cr~APMAN-ANDRESEN,C.: Endocytosis in fresh- Finally, as an echo from the early days of water amebas. Phys. Rev. 57, 371-385 (1977) pinocytosis and its sequels at Carlsberg Labora- 15. CHAPMAN-ANDRESEN, C. & S. CHRISTENSEN: torium, I was interested to notice, among the Pinocytic uptake of ferritin by the amoebae Chaos

184 Carlsberg Res. Commun. Vol. 49, p. 179-186, 1984 C. CHAPMAN-ANDRESEN: Early days of pinocytosis

chaos measured by atomic adsorption of iron. 30. HOLTER, H.: Om pinocytose hos amober. Natu- Compt Rend. Lab. Carlsberg 38, 19-57 (1970) rens Verden 38, 257-269 (1954) 16. CHAPMAN-ANDRESEN,C. & D.A.T. DICK: Sodium 31. HOLTER, H.: Studi citochimici sulla pinocitosi and bromine fluxes in the amoeba Chaos chaos nelle amebe. Rendiconti dellstituto Superiore de L. Compt. Rend. Lab. Carlsberg 32, 265-289 Sanita, Suppl. 19, 51-70 (1956) (1962) 32. HOLTER. H.: Pinocytosis. Intern. Rev. Cytol. 8, 17. CHAPMAN-ANDRESEN.C. & H. HOLTER: Studies 481-505 (1959a) on the ingestion of ~4C glucose by pinocytosis in 33. HOLTER,H.: Problems ofpinocytosis -with special the amoeba Chaos chaos and Amoeba proteus. regard to amoebae. Ann. N. Y. Acad. Sci. 78, Expt. Cell Research, Suppl. 3, 52-63 (1955) 524-537 (1959b) [8. CHAPMAN-ANDRESEN,C. & H. HOLTER: Differ- 34. HOLTER.H.: The induction ofpinocytosis. Sympo- ential uptake of protein and glucose by pinocytosis sium on biological approaches to cancer therapy. in Amoeba proteus. Compt. Rend. Lab. Carlsberg Louvain. Acad. Press Inc. (1960) 34, 211-226 (1964) 35. HOLTER. H.: Pinocytosis. IUB/IUBS Symposium 19. CHAPMAN-ANDRESEN.C. & H. HOLTZER: The up- biological structure and function I, 157-168 take of fluorescent albumin by pinocytosis in (1961a) Amoeba proteus. J. Biophys. Biochem. Cytol. 8, 36. HOLTER.H.: Pinocytosis. CIBA Foundation Sym- 288-291 (1960) posium on enzymes and drug action. Acad. Press, 20.CHAPMAN-ANDRESEN.C. & D. LAGUNOFF: The dis- London, 30-39 (1961b) tribution of acid phosphatase in the amoeba Chaos 37. HOLTER, H.: HOW things get into cells. Scientific chaos L. Compt. Rend. Lab. Carlsberg 35, 419-438 American 205, 167-180 (1961) (1966) 38. HOLTER, H.: Pinocytosis. Proc. V Intern. Congr. 21. CHAPMAN-ANDRESEN.C. & JR. NILSSON: Elec- Biochemestry. II, 248-256 (1963) tron micrographs of pinocytosis channels in 39. HOLTER, H." Membrane in colleration with pino- Amoeba proteus. Expt. Cell Research 19, 631-633 cytosis. "Intracellular Membraneous Structure" (1960) eds. S. Seno and E.V. Cowdry. Japan Soc. Cell 22. CHAPMAN-ANDRESEN,C. & JR. NILSSON: Studies Biol. Okayama, pp. 451-465 (1965) on endocytosis in amoebae. The distribution of 40. HOLTER, H.: Physiologie der Pinocytose bei pinocytically ingested dyes in relation to food Amoben. Funktionelle und morphologische Orga- vacuoles in Chaos chaos. II. Electron microscopic nisation der Zelle. Sekretion und Exkretion. 2. observations using alcian blue. Compt. Rend. Lab. Konf. Gesellschaft Deutscher Naturforscher und Carlsberg 36, 189-207 (1967) Arzte. Springer-Verlag, Berlin, 119-146 (1965) 23. CHAPMAN-ANDRESEN, C. & JR. NILSSON: On 41. HOLTER, H.: Passage of particles and macro- vacuole formation in TetrahymenapyriformisGL. molecules through cell . Function and Compt. Rend. Lab. Carlsberg 36, 405-432 (1968) structure in micro-organisms. Symp. Soc. Gen. 24. CHAPMAN-ANDRESEN,C. & D.M. PRESCOTT: Stu- Microbiol. 15, 89-114 (1965) dies on pinocytosis in the amoebae Chaos chaos 42. HOLTER. H. & H HOLTZER: Pinocytic uptake of and Amoeba proteus. Compt. Rend. Lab. Cads- fluorescein-labelled proteins by various tissue berg set. chim. 30, 57-78 (1956) cells. Exptl. Cell Research 18, 421-423 (1959) 25. COOPER, B.A.: Quantitative studies of pinocytosis 43. HOLTER, n. & JM. MARSHALLJR." Studies on induced in Amoeba proteus by simple cations. pinocytosis in the amoeba Chaos chaos. Compt. Compt: Rend. Lab. Carlsberg 36, 385-403 (1968) Rend. Lab. Carlsberg, S6r. chim. 29, 7-37 (1954) 26. DE DUVE, C.: In: Ciba Found. Symp. Lysosomes. 44. HOLTZER, H. & S. HOLTZER" The in vivo uptake Churchill, London 126 (1963) of fluorescein-labelled plasma proteins. I. Mature 27. EDWARDS, GJ.: Formation of food-cups in Cells. Compt. Rend. Lab. Carlsberg 31,737-408 Amoeba induced by chemicals. Biol. Bull. 48, (1960) 236-239 (1925) 45. JOSEFSSON,J.-O.: Studies on the mechanism of 28. GEY, G.O.P. SHAPRAS, F.B. BANG & M.K. GEY: induction of pinocytosis in Amoeba proteus. Acta Some relations of inclusion droplets (pinocytosis Physiol. Scand. suppl. 432, 1-65 (1975) - Lewis) and mitochondria behaviour in normal 46. LEWIS. W.H.: Pinocytosis. John Hopkins Hosp. and malignant cells. In: Fine structure of ceils. Bull. 49, 17-27 (1931) Groningen, Noordhoff ( 1955) 47. LEWIS.W.H.: Pinocytosis by malignant cells. Am. 29. HAYWARD,AF.: Electron microscopy of induced J. Cancer 29, 666-679 (1937) pinocytosis in Amoeba proteus. Compt. Rend. 48. MAINWARING, J.: Hydrophagocytosis. J. Am. Lab. Carlsberg 33, 535-558 (1963) Med. Ass. 95, 1509 (1930)

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49. MAST, S.O. & W.L. DOYLE: Ingestion of fluids by 57. NILSSON.J.R.: Phagotrophy in Tetrahymena. Bio- Amoeba. Protoplasma 20, 555-560 (1934) chemistry and Physiology of Protozoa, 2nd Ed. 50. MARSHALL.JM. & V.T. NACHMIAS; Cell surface 2, Acad. Press, pp. 339-379 (1979) and pinocytosis. J. Histochem. Cytochem. 13, 58. ROSE.GG.: Microkinetospheres and VP satellites 92-104 (1965) of pinocytic cells observed in tissue cultures of 51. MELTZER, S.J.: Edema. A consideration of the Gey's strain HeLa with phase contrast cinemato- physiologic and pathologic factors concerned in graphic techniques. J. Biophys. Biochem. Cytol. its formation. Am. Med. 8, 191-199 (1904) 3, 697-704 (1957) 52. METCHNIKOFF, E.: Untersuchungen iaber de 59. RUSTAO, R.C.: Molecular orientation at the sur- mesodermalen Phagocyten einigen Wirbeltiere. face of amoebae during pinocytosis. Nature 183, Biol. Zentr. 3, 560-565 (1883) 1058-1059 (1959) 53. MUDD, S, M. McCuTCHEON & B. LUCKE: Phago- 60. RYTER. A. & C DE CH~,STELLIER: Phagocyte- cytosis. Physiol. Rev. 14, 210-275 (1934) pathogenic microbe interactions. Int. Rev. Cytol. 54. NACHMIAS. V.T. & JM. MARSHALL JR.: Protein 85, 287-327 (1983) uptake by pinocytosis in amoebae. Studies on 61. SKRIVER, L. & JR NILSSON: The relationship be- ferritin and methylated ferritin. IUB/IUBS Sym. tween energy-dependent phagocytosis and the rate Biological structure and function II, 605-619 of oxygen consumption in Tetrahymena. J. Gen. (1962) Microbiol. 109, 359-366 (1978) 55. NILSSON,J R.: Physiological and structural studies 62. WOH LFARTH-BOTTER MANN, K.E & W. STOCKEM: on Tetrahymena pyriformis GL. Compt. Rend. Pinocytose und Bewegung von Amoben. II. Per- Lab. Carlsberg 40, 215-355 (1976) manente und induzierte Pinocytose bei Amoeba 56. NILSSON,JR.: On food vacuoles in Tetrahymena proteus. Zeitschr. Zellforsch. 73, 444-474 (1966) pyriformis GL. J. Protozool. 24, 502-507 (1977)

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