Electron Microscopic Study of “Adenosine Triphosphatase” Activity in Malignant Melanoma Cells of the Syrian Golden Hamster*

Vol. 44, No. 4

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ELECTRON MICROSCOPIC STUDY OF "ADENOSINE TRIPHOSPHATASE" ACTIVITY IX MALIGNANT MELANOMA CELLS OF THE SYRIAN GOLDEN HAMSTER* TAKASHI NAKAI, M.D., PH.l).t

In spite of the fact that tyrosinase activity iii melanocytes under various conditions has been extensively studied by means of lusto— chemical and biochemical methods during the

bedded in Epon according to Luft's method (6). The glutaraldehyde-fixed tissues were washed in cold 0.05 M cacodylate buffer containing 7.5 per

rent sucrose at pH 7.4 for several changes (2 hours total). They were then stored in the same solution last two decades, other enzymes appear to have at 0—4°C overnight. After storage, the fixed tissues received relatively little attention in the study were quickly frozen in dry ice, and thin (15 ,z) and frozen sections were cut in a cryostat (40 of figment cell growth. Recently the presence thick and collected in the buffered sucrose plus 0.05 M of adenosine triphosphatase (ATPase) activity MgCL at 0—4°C. They were transferred into the in melanocytes was histochemically demon- incubation mixture modified by Otero-Yilardebó

strated by Mustakallio (1), Cormane and (t al. (7)

from the standard ATPase medium of Waehstein and Meisel (8). The mixture used was Kalsbeek (2), of 8.3 >< 10 M adenosine triphosphate human epidermis at the light-microscopy level. eomposed (ATP) disodium salt, 2.5 X 10 M Pb(No:)2 and In the present report the localization of extra— 0.05 M MgC12 in Tris-maleate buffer at pH 7.2. The sections were incubated at 37°C for periods mitoehondrial adenosine triphosphatase activity was histochemically studied in the malig- of from 30 to 60 minutes. At various times thin were picked up from the medium, rinsed nant melanoma cells of the Syrian golden sections in distilled water, treated for less than 1 minute hamster under the light— and electron—micro- in 0.005 M acetic acid and transferred to ammoniurn sulfide, (NH4)25, for developing a dark scope. precipitate of lead sulfide (PbS). When a satisfacMATE1tTALS AND METHODS tory result was observed in the thin sections under the light microscope, the thick sections were taken A pigmented type of the mlignant melanoma out from the incubation mixture and rinsed in (code No. M. Mel 3) discovered by Fortner distilled water followed by brief treatment with et al. (4) was transferred to and maintained in 0.005 M acetic acid. These thick sections were adult male Syrian golden hamsters of the colony again washed in several changes of distilled water. of this laboratory by routine subcutaneous trans- Most of them were then postfixed in Palade's plantation. Morphological and biological charac- osmium tetroxide fixative for 1 hour at 0—4°C, teristics of this tumor were described in great and embedded in Epon according to Luft's procedetail (4), and they were remarkably similar to (lure (6). Some sections were embedded in Epon the features 01 the corresponding tumor in man. without the osmium refixation. In order to evaluAbout 10 days after transplantation, the tumors ate the specificity of the ATPase reaction, control and

Bradshaw et al. (3) in the

actively growing tip to approximately 1 cm in experiments were carried out at the light- and diameter were surgically removed and cut into electron—microscopic level. Control tissues were small blocks (2—3 mm thickness). Most of them incubated for 60 minutes in the substrate-free were rapidly immersed in 6.5 per cent glutaralde- medium or in the medium where 3 >< 10 M sohyde (5) buffered to nH 7.2 with 0.1 M cacodylate.

Fixation was carried out in the cold (0—4°C) for 2 hours. Some fresh blocks were further sliced into

dium fl-glycerophosphate was substituted for ATP at the same pH.

Ultrathin sections were cut on an LKB Ultrotome with glass knives, mounted on formvar-

smaller pieces (1 mm in diameter) and fixed in Palade's 1 per cent osmium tetroxide fixative for

coated grids, and stained in a saturated solution of

1 hour in the cold. After fixation, osmium-fixed materials were em-

uranyl acetate in distilled water for 30 to 60

This investigation was supported by grants C2893 and C-5717 from the National Cancei Insti-

minutes. They were examined in an RCA EMIl3F electron microscope operated at 50 KY. RESULTS

tute, National Institutes of Health, U.S. Public Health Service. Received for publication May 28, 1964.

* From the Division of Oncology, The Chicago Medical School, Institute for Medical Research, Chicago, Illinois.

t Present address: 11 Kamifuji-mae-cho, Koma-

gome, Bunkyo-ku, Tokyo, Japan.

Light Microscopy, Incubation of melanoma tissues in the ATPase medium resulted in the formation of a visible reaction product in the blood vessels and in many tumor cells (Fig. 1). The reaction product appeared to be accumu-

264

E. M. STUDY OF "ATPASE" IN MELANOMA CELLS

lated in tile periphery of the tumor eeHs, and

265

In the giutaraidehyde-fixed materiais incu-

dendritie processes became discernibie in some bated with ATP, the final product of the encelis. ATPase activity seemed to vary from one zymatic reaction was iocahzed to the ceH ceH to the other. Some celis were stained much membranes and Goigi zones of meianoma celis more heavily, and their ceH borders and cyto— (Figs. 3 and 4). Sites of ATPase iocahzation piasm showed dense deposits. No reaction prod- were characterized as intenseiy eiectron opaque,

uct deveioped when sections of the identical tumor were incubated in the control medium without ATP or in the medium where sodium fl-giycerophosphate was substituted for ATP at the same pH.

Electron Microscopy. An electron micrograph of a portion of melanoma ceHs fixed in Paiade's osmium tetroxide fixative is shown in Fig. 2. These ceHs contained an indented nucieus, round or ovai mitochondria, numerous pigment granuies and weil deveioped Goigi zones. Rough-surfaced endopiasmic reticuium and free ribosomes, the iatter often grouped into rosettes, Were comparatively numerous. Pigment granuies were round or poiygonal in profile and measured 100 to 450 mi in diameter. They contained various amounts of eiec— tron opaque materiai interpreted as meianin. Some irreguiar substructure was observed in the pooriy fiHed granuies which usuaily had a singie membrane. Pinocytic vesicies were rareiy found aiong the cell borders.

granuiar deposits of lead phosphate, PbPO4, under the eiectron microscope. Most of the tumor cells had the dense deposits on their membrane, although in some instances the reaction product was discontinuousiy deposited and faiied to form a continuous layer (Figs. 3 and 4). Lead phosphate was quite frequentiy deposited in either a spotty or linear fashion on the Goigi zones (Figs. 3 and 4). Mitochondrial ATPase activity appeared to be stiH retained even after the glutaraidehyde fixation, although greatiy diminished, and numerous precipitates of fine electron opaque particies were seen virtuaHy in ail mitochondria (Figs. 4,5 and 6). When the materiais were fixed in giutaraidehyde and then postfixed in osmium tetroxide, some pigment granuies having a heavy accumuiation of melanin showed a fairly high electron

opacity, which often made an exact evaluation of the enzymatic reaction in these granules difficuit. However, the majority of the pigment

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Fio. 1. Light photoniicrograph of a frozen section of the hamster melanoma incubated for 60 minutes in ATPase medium. Dark deposits of reaction product in the cell borders can be seen in many tumor cells. Some cells are stained darker, and dendritic processes are often discernible. Note small blood vessels shown in lower and right upper portions of the illustration. X 450.

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FIG. 2. Electron micrograph of a portion of melanoma cells containing many pigment granules, well-developed Golgi zones (G) and mitochondria (M). A centriole (C) and a portion of an indented nucleus (N) are also illustrated. X 18,000.

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FIG. 3. Tumor cells of malignant melanoma fixed in cold buffered glutaraldehyde and incubated for 1 hour with ATPase medium. The tissue is re-fixed in Palade's osmium tetroxide fixative. The dense

reaction product, lead phosphate, covers around the cell membrane in almost continuous fashion.

Note that Golgi zones also exhibit dense deposits of ATPase reaction product. Pigment granules show a lower electron opacity than the lead phosphate, thus indicating the possible absence of ATPase reaction product from these granules (see also Figs. 4—7). X 10,000. 267

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FIG. 4. Similar material as in Fig. 3. The reaction product deposited around the cell membrane (CM) is somewhat discontinuous in the lower part of the illustration. The final dense product appears in association with several Golgi zones, often outlining the parallel lamellae (arrows). Note the fine lead phosphate precipitates in the rnitochondria. X 16,500.

E. M. STUDY OF "ATPASE" IN MELANOMA CELLS

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FIGS. 5 AND (1. Portion of melanoma cells. The dense reaction product is localized in the cell membrane and Golgi zone. Mitochondiia exhibit fine lead phosphate precipitates. Note that no significant reaction product is seen in pigment granules. A scattering of some fine non-specific deposits is visible.

X 22,000 arid 36,500.

FIG. 7. Similar material as in Fig. 3, but the refixation in Palade's osmium tetroxide fixative is omitted. Portion of a melanoma cell is shown with the dense reaction product localized in the cell membrane. Note the absence of lead precipitates in pigment granules which show a relatively low electron opacity because of the onussion of osmium fixation. X 31,500.

granules, particularly those with incomplete granules except occasional insignificant precipimelanization, had a lower electron opacity tates of a few lead particles. The difficulty of than lead deposits, so that the latter could be distinguishing lead deposits from the pigment (listinguisliecI from the former if present. Careful and close inspection of many electron micrographs including Figs. 3—6 revealed that practically no reaction product occurred in the pigment

granules with a high electron opacity was over-

come by the examination of non-osmificated materials. Although preservation of the fine

structure was not as good as the osmium-

270

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refixed tissues, pigment granules seen in the sis of epidermal melanocytes of normal, human non-osmificated tissues had a much lower electron opacity (Fig. 7). Under this condition almost no accumulation of the reaction product was detected in the pigment granules (Fig. 7), while the cell membrane, Golgi zone and, to a much lesser extent, mitochondria showed the enzyme activity. In the control tissues incubated in the sub-

skin incubated in the ATPase medium of Wachstein et at. (9) that the cell membranes, mitochondria and possibly melanosomes were the sites of the enzyme activity. In the hamster melanoma cells, however, deposits of the reaction product were not found in the melanosomes, while the Golgi zones in addition to the cell membranes showed almost constantly a

strate-free medium or in the mixture where positive "ATPase" activity. It is now of considerable importance to sodium /3-glycerophosphate was substituted for ATP at the same pH, no lead deposits were evaluate the specificity of this reaction. Acobserved in any of the organdIes of melanoma cording to Bradshaw et al. (3), melanin grancells. ules in Malpighian cells show non-specific staining when ATPase is demonstrated by methods which utilize the sulfide conversion of Recently it was described by Sabatini et al. sulfur-metal compounds. However, this did not (5) that glutaraldehyde-fixed tissues showed an apply to the present experiment, since the reexcellent preservation of ultrastructural detail action product was not deposited in the melaDISCUSSION

and an adequate amount of various enzyme activities survived for histochemical demonstration. The combination of this fixation and histochemical incubation in the ATPase medium has made it possible to correlate the localization of "ATPase" with the well preserved fine structure of melanoma cells. The

nin granules of our material. Moreover, the treatment with ammonium sulfide was omitted for electron microscopic cytochemistry. There is also the possibility that the hydroly-

sis of ATP is due to the action of the nonspecific alkaline phosphatase. However, the extramitochondrial as well as mitochondrial

electron micrographs revealed that the electron opaque reaction product, lead phosphate, pro-

lead deposits were not present in the melanoma tissues incubated in the medium without ATP

localized in the cell membranes and Golgi zones

phosphate was substituted for ATP. Novikoff

duced by the hydrolysis of ATP was clearly

or in the medium where sodium fl-glycero-

of hamster melanoma cells. A light accumula- et al. (10) strongly suggested, according to tion of the fine lead precipitates also occurred their results of histochemical and cell frac-

in the mitochondria. It is our interpretation, tionation studies of ATPase activity in the however, that no significant ATPase reaction liver, that the enzyme activity shown by the Wachstein and Meisel method (8) with ATP product was present on the pigment granules. Several recent papers (1—3) have dealt with

is a specific ATPase rather than a non-specific alkaline phosphatase. Novikoff et al. (10) and

the presence of ATPase activity in normal epidermal melanocytes observed under the Novikoff (11) emphasized that pH-activity light microscope. In an attempt to demonstrate curves are of considerable value for distinncural elements of human epidermis by means of histochernical reactions, Mustakallio (1)

guishing substrate-specific phosphatases in tis-

interpreted that the cell membrane is a possible

his co-workers (12—16). In sections of kidney,

sues, particularly in the liver and spleen,

found that the dendritic network of Langer- where there is relatively low alkaline phoshans' cells and probably of melanocytes as well phatase activity present. They felt that the specific ATPase can be histochemically demonas the papillary nerves and their intraepider- strable at pH 7.2, which is used in the Wachmal extensions showed a positive ATPase ac- .stein-Meisel method, and that the activity of tivity. Subsequently, Cormane and Kalsbeek non-specific alkaline phosphatase can be neg(2) also reported the presence of ATPase ac- lected. Moreover, this view has received further tivity in dendritic cells, presumably Langer- supports from a serics of electron microscopic hans' cells, of the normal human skin. They studies of various tissues done by Novikoff and

site of the enzyme activity (2). Bradshaw

liver and hepatomas as well as some other

et al. revealed in their light-microscopic analy- tissues incubated with various nucleosidephos-

E. M. STUDY OF "ATPASE" IN MELANOMA CELLS

271

phates, the reaction product is always accumu- within mitochondria of rat colonic mucosa lated to specific membranes (12—16). Since the fixed overnight in formalin when frozen seccytoplasm of melanoma cells is known to pos- tions, prepared from the tissue which was once sess a relatively low level of alkaline phos- chilled at —70°C, were incubated with ATP. phatase activity (17, 18), the enzyme activity The mitochondria of rat kidney treated in the demonstrated under our experimental condi- identical manner failed to give a positive re-

tions is most likely the specific magnesium- action (7). Our finding that some degree of activated "ATPase". the "ATPase" activity survived under our exIt is of great interest that the "ATPase" perimental conditions may add another exactivity is localized in the malignant melanoma cells which show a rapid multiplication, high

ample to that of Otero-Vilardebó et al. (7).

invasiveness and active melanin synthesis, since ATPase is one of the nucleosidephos-

SUMMARY

Malignant melanoma of Syrian golden hamphatases which are known to mediate the high ster was fixed in cold 6.5 per cent glutaraldelevel energy necessary for a variety of synthetic hyde. Frozen sections were incubated in a modand functional activities of the cell (19). It is ified Wachstein-Meisel ATPase medium and also believed that ATPase appears to play a examined under the light- and electron-micro-

role in the transport of inorganic phosphate scope. The magnesium-activated "ATPase" (20, 21) and Na and K (22, 23) across cell was localized in the cell membrane and Golgi membranes. The ultrastructural localization of "ATPase" demonstrated in the Golgi zone and cell membrane of the melanoma cells appears to be relevant to the functions of these organdIes. Possible roles and functional significance of nucleosidephosphatases in the membrane system of cells are well documented and discussed in great detail by Novikoff et al. (14). They suggested that the split of ATP or other energy-rich nucleosidephosphate might lead to alteration of a contractile protein in the cytomembrane, as in the mechanism of cell transport. In our material, however, more studies on various nucleosidephosphatases will be required to evaluate the functional significance in our present observation. Cytochemical studies of ATPase as well as other phosphatases in

zone of the melanoma cells. Mitochondria ap-

the melanocyte system of Syrian hamsters

G. R.: Transplantable tumors of the Syrian

under various experimental conditions are in progress.

Although it was not the purpose of this ex-

periment to demonstrate the mitochondrial ATPase activity, the light deposits of reaction product in mitochondria appeared to indicate

that a small amount of the enzyme activity still remained. It was reported by many investigators (5, 13, 24—29) that significant deposits due to ATPase in the mitochondria cannot be demonstrated in the electron microscope when tissues are subjected to prolonged treatment in chemical fixatives including aldehydes preceding the incubation with ATP. Recently, however, Otero-Vilardebó et al. (7) observed

an extensive deposition of reaction product

peared to retain some degree of the enzyme activity after the aldehydè fixatipn. However, no such activity was histochemically demonstrable in the melanosomes under the experimental conditions employed.

1.

REFERENCES K.: Adenosine triphosphatase

MTJSTAKALLIO,

activity in neural elements of human epidermis. Exp. Cell Res., 28: 449, 1962.

2. CORMANE, H. H. AND KALSBEEK, 0. L.: ATP-

hydrolyzing enzymes in normal human skin.

Dermatologica, 127: 381, 1963. 3. BRADSUAw, B., WACHSTEIN, M., SPENCE, J. AND ELIAS, J. M.: Adenosine triphosphatase

activity in melanocytes and epidermal cells of human skin. J. Histochem. Cytochem., 11: 465, 1963. 4. FORTNER, J. G., MAHY, A. G. AND SCHRODT,

(golden) hamster. Part 1: Tumors of the alimentary tract, endocrine glands and

melanomas. Cancer lies., 21 (Suppl.): 161, 1961.

5. Sn'rnci, D. 1)., BENSCH, K. AND BARRNETT,

R. J.: Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17: 19, 1963. 6. LIJFT, J. H.: Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409, 1961.

7. OTERO-VILARDEBó, L. R., LANE, N. AND

GODMAN, G. C.: Demonstration of nfitochondrial ATPase activity in formalin-fixed colonic epithelial cells. J. Cell Biol., 19: 647,

1962. 8. WACHSTEIN, M. AN!) MEISEL, E.: Histochemis-

try of hepatic phosphatases at a physiologic

pH with special references to the demonstration of bile canaliculi. Amer. J. Clin. Path., 27: 13, 1957.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

272

9. WACHSTEIN, M., BRADSHAw, M. AN!) ORTIZ,

J. M.: ilistochemical demonstration of mitochondrial adenosine triphosphatase activity in tissue sections. J. Histochem.

Cytochem., 10: 65, 1962. 10. Noviicon, A. B., HAUSMAN, 1). II. AND POD-

BER, E.: The localization of adenosine tn-

phosphatase in the liver: in situ staining and cell fractionation studies. J. ilistochem.

20. GOURLEY, 1). 711,

specific phosphatases. J. Histochem. Cytochem., 6: 251, 1958.

12. NovIKoFF, A. B.: Biochemical and staining reactions of cytoplasmic constituents. In:

mammalian red cells. Biochem. J., 58: 622, 1954.

22. KEYNES, II. 1).: The energy source for active

transport in nerve and muscle. In: Membrane Transport and Metabolism, p. 131.

A. Kleinzeller and A. Kotyk, eds. New York, Academic Press, Inc., 1960. 23. POsT, R. L. ANt) ALBRIGIIT, C. P.: Membrane

adenosine triphosphatase system as a part

of a system for active sodium and potassium

Developing Cell Systems and Their Control, p. 167. 1). Rudnick, ed. New York, Ronald Press Co., 1960. 13. ESSNER, E., N0vIKOFF, A. B. ANT) MASEK,

B.: Adenosine triphosphatase and 5 '-nucleotidase activities in the plasma membrane of liver cells as revealed by electron

microscopy. J. Biophys. Biochem. Cvtol., 4: 711,

1958.

14. No\TIK0FF,

A. B., ESSNER, E., GOLDEISCriER,

S. AND MARGARETA, H.: Nucleosidephos-

phatase activities of cvtomernbranes. Symp. Internat. Soc. Cell Biol., 1: 149, 1962. 15. ESSNER, E. AND NovIxoFF, A. B.: Cytological

studies on two functional hepatomas. Interrelations on endoplasmic reticulum, Golgi apparatus, and lysosornes. J. Cell Biol., 15: 289,

1962.

16. NOvIKOFF, A. B. AND ESSNER, E.: Pathological changes in cytoplasmic organelles. Fed.

Proc., 21: 1130, 1962.

17. GREENBERG, S. S., KOPAC, M. J. AND GORDON,

M.: Cytology and cytochernistry of melanoma cells. Ann. N.Y. Acad. Sci., 67: 55, 1956.

18. RosE, 0. 0.: Observations on human melanoma cells in tissue culture. Ann. N.Y. Acad. Sci., 100: 92, 1963. 19. LINDBERG, 0. AND ERNSTEH, L.: Chemistry

1958.

21. PRANKERD, T. A. J. AND ALTMANN, K. I.: A study of the metabolism of phosphorus in

Cytochern., 6: 61, 1958.

11. Noviico, A. B.: Distinguishing substrate-

R. H.: The role of adenosine tn-

in the transport of phosphate in the human erythrocyte. Arch. Biochern. 4: phosphate

transport. In: Membrane Transport and Metabolism, p. 219. A. JKleinzeller and A. Kotylc, eds. New York, Academic Press, Inc. 1960.

24.

Asiiwowni, C. T., LU1BEL, F. J. AND STEWART,

S. C.: The fine structural localization of adenosine triphosphatase in the small intestine, kidney, and liver of the rat. J. Cell

Biol., 17: 1, 1963. 25. DEI3EYER, J. M., DEMAN, J. C. H. AND PER-

S1JN, J. P.: ATPase activity on the intercalated disc and C hands of mouse heart

muscle. 1. Cell J3iol., 13: 452, 1962. 26. KAPLAN, S. E. AND NOVKOFF, A. B.: The

localization of adenosine trihosphatase

activity in rat kidney: Electron microscopic examination of reaction product in formalin-calcium fixed frozen sections. J. Histochem. Cytochem., 7: 295, 1959.

27. LAZARUS, S. S. AND BARDEN, H.: Histochernis-

try and electron microscopy of mitochondrial aclenosinetriphosphatase. j. Histo-

chern. Cytochern., 10: 285, 1962. 28. SCARPELLI, 1). (1. AND CRAIG, E. L.: The fine

localization of nucleoside triphosphatase activity in the retina of frog. J. Cell Biol.,

and physiology of mitochondria and micro-

17: 279,1963. 29. TICE, L. AND BARRNETT, II. J.: Fine structural

lleilbrunn and F. Weber, eds. Wien, Springer-

tivity in heart muscle myofibrils. J. Cell

somes. In: Protoplasmatologia Handbuch der Protoplasrnaforschung, p. 136. L. V. Verlag, 1954.

localization of adenosinetriphosphatase acBiol., 15: 401, 1962.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

94

linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.

24. Wynn, C. H. and Iqbal, M.: Isolation of rat

skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.

human epidermis, p. 511, The Epidermis

Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-

No warranty is given about the accuracy of the copy.

Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.

Cell Biol., 27: 603, 1965.

27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-

C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.

30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.

31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-

lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.

32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.

33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.

34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:

Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.

43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.

44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.

45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.

46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,

Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by

Res., 33: 176, 1964.