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The Survival of Tissues At Ice-Box Temperatures1
THE SURVIVAL OF TISSUES AT ICE-BOX TEMPERATURES1 ELSIE M. WALTER, M.A., HERMAN SHARLIT, M.D., AND JOSEPH C. AMERSBACH, M.D.
From the Skin and Cancer Unit, New York Post-Graduate Medical School and Ho8pital, Columbia University, New York, and the Institutum Divi Thomae, Cincinnati
In the course of experiments on skin metabolism, it was decided to destroy
in part the primary respiratory mechanism by storing the tissue at icebox temperature overnight. However, the results indicated no diminution in respiration. A study of the literature reveals that very little work has been done on the metabolism of adult tissues over a prolonged period after excision. It is well known that tumor tissue can be kept for several days after excision with no change in its glycolytic action; and that, on transplantation, the resulting growth is just as great as in the case of fresh material taken from the body (1). Likewise embryonic tissue can be cultured and successfully grown in vitro (2). Frog muscle at room temperature has a maximum respiration immediately after excision and then declines to a constant level (3). At 0°C. the respiration of suspensions of muscle remains unaltered for one and a half hours and then diminishes to almost zero after twentyfour hours (4). In rat muscle the respiration reaches a maximum an hour after excision and then declines for five or six hours to zero when kept at room temperature (3). Muscle brei from dogs, cows, lambs and horses loses its respiratory activity several hours after excision, but the liver brei from the same animals retains its respiratory activity intact even after twenty hours or more (5). In determinations of 02 consumption after 20, 40, 60 and 80 minutes, muscle becomes most rapidly exhausted, liver less and spleen least (6). The respiration of liver, heart or diaphragm markedly decreases after a few hours on ice, whereas, the respiration of kidney cortex and medulla after one to three hours is either unchanged or even greater than in the fresh condition (7). If tumor tissue is actually frozen with liquid air, the glycolytic property disappears and the tissue cannot be transplanted (1). Likewise rat heart, liver, kidney, lung, spleen and testes lose the ordinary 02 utilization by freezing with liquid air, but the enzyme system converting succinic to fumeric acid remains intact (8).
This report covers a study of the respiration of guinea pig skin immediately after excision and after a day or more in the icebox, with comparable studies on the liver and the kidney. EXPERIMENTAL
The largest lobe of the liver, the kidneys, as well as the skin from the ears of a full grown female guinea pig, were used in all experiments. The animal was killed by a sharp blow on the base of the skull and the tissues were removed immediately and placed in Ringer phosphate-dextrose (0.2 per cent dextrose,
pH 7.3) solution. A control experiment was run on the fresh tissue and the
'This research was made possible partly through a grant from the Lillia Babbitt Hyde Foundation. Received for publication, November 15, 1944. 235
236
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
remaining tissues were stored in the icebox at 4°C. in the Ringer solution, until
used. Immediately before testing the 02 consumption of the tissue, the skin was carefully cleaned of all fat and cartilage and cut into small sections, approximately 1 mm. square and placed in the manometer flasks. The liver and kidneys were sliced into very thin sections and likewise placed in the manometer
flasks. The respiration was measured at 37.5°C. in air by Warburg-Barcroft manometers, with the method described for animal skin and liver by Cooke, Kreke and Nutini (9). Seven manometers were used in the experiments, two TABLE 1 TISSUES STORED SEPARATELY
Time
% Orig. Resp.
TISSUES STORED TOGETHER
No Expts.
Time
% Orig. Resp.
No Expts.
Liver tissue: average original respiration 3.13 Cu. mm. Oa/hr./mg. %
lday
2days
3da
4days 7days
Frozen
54 30 31 15 13 11
8 3 2 3 1 1
days
%
1
52 44 14 15 10 4
2 3 4 6
7
7 2 1 3 1 1
Kidney tissue: average original respiration 11.68 cu. mm. 02/hr./mg.
iday
2days 3days 4days 7days
Frozen
90 85 74 53 11 3
8
1
3
2 3 4
2 3 1 1
6 7
81 83 46 55 40 20
10 3 1 3 1 1
Skin tissue: average original respiration 1.58 cu . mm. Oa/hr./mg.
iday
100
12
2days 3days 4days 7days
99 63
6 2 3
1 2 3 4
25
1
14
1
Frozen
114
64 57
10
3
21
1
3
6
22 18
7
11
1
1
each for the three organs and one for thermo-barometric control. The tissues were always prepared as quickly as possible before they were placed in the manometer bath. After an equilibrium period of fifteen minutes, the 02 consumption was measured during a two hour period; readings were taken at least once an hour and oftener if necessary. The tissue was then carefully removed from the flasks, washed in distilled water and dried to constant weight at 100°C. Approximately 20 mg. (dry weight) of skin, 15 mg. of liver and slightly less than 10 mg. of kidney tissue were used in each manometer flask. Less kidney tissue
was used than the other tissues because it has a very high rate of respiration.
SURVIVAL OF TISSUES AT ICE-BOX TEMPERATURES
237
The respirations are expressed in terms of Qo, i.e., cubic millimeters of oxygen absorbed per milligram of tissue (dry weight) per hour. The tissues were stored in glass jars with tightly fitting screw caps and well covered with the Ringer solution. The volume of the air space in the jars was kept equal to, if not larger than, the volume of the solution. No arrangement was made for absorbing the CO2 given off by the tissues during the storing, for as Loser (10) pointed out, the presence of CO2 stabilizes the rate of 02 uptake of tissues in the presence of substrate for several hours and safeguards their ability to oxidize substrate; and in the absence of CO2 the maximum activity of the tissue slices is maintained for only a comparatively short time. In some of the experiments the tissues were stored in separate containers. In others, as a check on the possibility of bacterial growth in the case of the skin, the three tissues were stored together. When the tissues were kept longer than one day, the Ringer solution was changed daily.
An experiment was carried out to determine the effects of freezing. Here the respiration of the organs was determined immediately after removal from the animal as in the other experiments; then the tissues were quickly frozen with carbon dioxide snow on a freezing microtome. The tissues were then thawed in Ringer solution and the respiration again determined. A summary of the results is given in table 1. DISCUSSION
An examination of the data in the table shows that the order of survival is skin, kidney, liver, when the organs are stored separately. The skin respiration remains practically undiminished for three days, while the liver respiration is
approximately one half of the original respiration after only one day. The kidney survives much better than the liver, but it too begins to decline after the second day. The results seem logical, for one would expect skin, which is di-
rectly subjected to changes in environmental temperature, to survive better than the internal organs. Similarly, the kidney, whose function is to eliminate toxic materials, should survive the toxic products formed during storage better than liver. The experiment in which the tissues were frozen showed that within experimental error the respiratory process was destroyed by freezing. It is interesting to note that when the organs are stored together skin does not
survive much better than liver; and that the kidney also shows less vitality, although not so marked as skin. It seems probable that either liver or kidney or the combination of the two, produces some material which is also toxic to skin. Experiments were performed in which one specimen of skin was stored with liver and another with kidney, in an effort to determine which of the viscera
produced the toxic material. The results were quite variable, however, and no definite conclusions could be drawn. Also, some of the solution in which all the organs were stored together was added to the manometer flask containing
skin which had been stored alone. The solution seemed to have no definite effect on the respiration of the skin over the short interval of the experiment.
Since skin survives for several days after removal from the body, Doctor
238
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
0. D. Meeker has suggested that further studies should be made regarding the availability for graft of skin removed at operation or immediately following death, and subsequently stored. SUMMARY
A study of the primary respiratory process of skin, kidney and liver, kept separately in the icebox for several days indicates that the skin respiration remains approximately normal for three days before showing much change; the kidney respiration declines slowly; but the liver respiration falls to about 50 per
cent of its initial respiration after one day. However, when the organs are stored in the same container, the skin respiration declines very much like that of liver. Freezing with carbon dioxide snow, as for sectioning, destroyed the primary process of respiration in all three tissues. 1. WARBURG, OTTO. The Metabolism of Tumors, London Constable and Co. 2. CARREL, ALEXIS. Jour. Exper. Med., 15: 516—528, 1912, ibid 18: 287—299, 1913, Pozzi,
F. Bul. de l'Acad. de Med., 67:475, 1913, Ebeling, A. A. Jour. Exper. Med., 17:273— 285, 1913.
3. FLETCHER, W. M. Lactic Acid Formation, Survival Respiration and Rigor Mortis in Mammalian Muscle, Jour. Physiol., 47: 360—380, 1913—14.
4. RABBENE, A. Respiratory Exchange of Tissues, Physiol. Abst., 10: 330, 1925. 5. BATTELLI, F., AND STERN, C. Research on the Primary Respiration of Tissue, Jour. Physiol. and Path. Gen., 9: 1—16, 1907. 6. DOMINI, GIOVIANNI, AND PERUZZI, PIETRO. The Exhaustion of the Respiratory Capacity in Vitro of Certain Tissues, Boll. Soc. Ital. Biol. Sper., 10: 493—6, 1935. 7. KiSci, BRUNO. Respiration Values of Fresh Mammalian Tissues, Biochem. Z., 280: 55—7, 1935.
8. LYNEN, FEODOR. Respiration of Animal Tissues after Freezing in Liquid Air, Z. Physiol. Chem., 264: 146—152, 1940.
9. Cooi, E. S., KEEKE, C. W., AND NUTINI, L. G. Fractions from Yeast Which Stimulate the Respiration of Yeast and Animal Tissues; Studies Inst. Divi Thomae, 2: 23—37, 1938.
10. LOSER, H. A Critical Analysis of the Tissue Slice Method in Manometric Experiments, Biochem. Jour., 36: 319—335, 1942.
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.
From the Skin and Cancer Unit, New York Post-Graduate Medical School and Ho8pital, Columbia University, New York, and the Institutum Divi Thomae, Cincinnati
In the course of experiments on skin metabolism, it was decided to destroy
in part the primary respiratory mechanism by storing the tissue at icebox temperature overnight. However, the results indicated no diminution in respiration. A study of the literature reveals that very little work has been done on the metabolism of adult tissues over a prolonged period after excision. It is well known that tumor tissue can be kept for several days after excision with no change in its glycolytic action; and that, on transplantation, the resulting growth is just as great as in the case of fresh material taken from the body (1). Likewise embryonic tissue can be cultured and successfully grown in vitro (2). Frog muscle at room temperature has a maximum respiration immediately after excision and then declines to a constant level (3). At 0°C. the respiration of suspensions of muscle remains unaltered for one and a half hours and then diminishes to almost zero after twentyfour hours (4). In rat muscle the respiration reaches a maximum an hour after excision and then declines for five or six hours to zero when kept at room temperature (3). Muscle brei from dogs, cows, lambs and horses loses its respiratory activity several hours after excision, but the liver brei from the same animals retains its respiratory activity intact even after twenty hours or more (5). In determinations of 02 consumption after 20, 40, 60 and 80 minutes, muscle becomes most rapidly exhausted, liver less and spleen least (6). The respiration of liver, heart or diaphragm markedly decreases after a few hours on ice, whereas, the respiration of kidney cortex and medulla after one to three hours is either unchanged or even greater than in the fresh condition (7). If tumor tissue is actually frozen with liquid air, the glycolytic property disappears and the tissue cannot be transplanted (1). Likewise rat heart, liver, kidney, lung, spleen and testes lose the ordinary 02 utilization by freezing with liquid air, but the enzyme system converting succinic to fumeric acid remains intact (8).
This report covers a study of the respiration of guinea pig skin immediately after excision and after a day or more in the icebox, with comparable studies on the liver and the kidney. EXPERIMENTAL
The largest lobe of the liver, the kidneys, as well as the skin from the ears of a full grown female guinea pig, were used in all experiments. The animal was killed by a sharp blow on the base of the skull and the tissues were removed immediately and placed in Ringer phosphate-dextrose (0.2 per cent dextrose,
pH 7.3) solution. A control experiment was run on the fresh tissue and the
'This research was made possible partly through a grant from the Lillia Babbitt Hyde Foundation. Received for publication, November 15, 1944. 235
236
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
remaining tissues were stored in the icebox at 4°C. in the Ringer solution, until
used. Immediately before testing the 02 consumption of the tissue, the skin was carefully cleaned of all fat and cartilage and cut into small sections, approximately 1 mm. square and placed in the manometer flasks. The liver and kidneys were sliced into very thin sections and likewise placed in the manometer
flasks. The respiration was measured at 37.5°C. in air by Warburg-Barcroft manometers, with the method described for animal skin and liver by Cooke, Kreke and Nutini (9). Seven manometers were used in the experiments, two TABLE 1 TISSUES STORED SEPARATELY
Time
% Orig. Resp.
TISSUES STORED TOGETHER
No Expts.
Time
% Orig. Resp.
No Expts.
Liver tissue: average original respiration 3.13 Cu. mm. Oa/hr./mg. %
lday
2days
3da
4days 7days
Frozen
54 30 31 15 13 11
8 3 2 3 1 1
days
%
1
52 44 14 15 10 4
2 3 4 6
7
7 2 1 3 1 1
Kidney tissue: average original respiration 11.68 cu. mm. 02/hr./mg.
iday
2days 3days 4days 7days
Frozen
90 85 74 53 11 3
8
1
3
2 3 4
2 3 1 1
6 7
81 83 46 55 40 20
10 3 1 3 1 1
Skin tissue: average original respiration 1.58 cu . mm. Oa/hr./mg.
iday
100
12
2days 3days 4days 7days
99 63
6 2 3
1 2 3 4
25
1
14
1
Frozen
114
64 57
10
3
21
1
3
6
22 18
7
11
1
1
each for the three organs and one for thermo-barometric control. The tissues were always prepared as quickly as possible before they were placed in the manometer bath. After an equilibrium period of fifteen minutes, the 02 consumption was measured during a two hour period; readings were taken at least once an hour and oftener if necessary. The tissue was then carefully removed from the flasks, washed in distilled water and dried to constant weight at 100°C. Approximately 20 mg. (dry weight) of skin, 15 mg. of liver and slightly less than 10 mg. of kidney tissue were used in each manometer flask. Less kidney tissue
was used than the other tissues because it has a very high rate of respiration.
SURVIVAL OF TISSUES AT ICE-BOX TEMPERATURES
237
The respirations are expressed in terms of Qo, i.e., cubic millimeters of oxygen absorbed per milligram of tissue (dry weight) per hour. The tissues were stored in glass jars with tightly fitting screw caps and well covered with the Ringer solution. The volume of the air space in the jars was kept equal to, if not larger than, the volume of the solution. No arrangement was made for absorbing the CO2 given off by the tissues during the storing, for as Loser (10) pointed out, the presence of CO2 stabilizes the rate of 02 uptake of tissues in the presence of substrate for several hours and safeguards their ability to oxidize substrate; and in the absence of CO2 the maximum activity of the tissue slices is maintained for only a comparatively short time. In some of the experiments the tissues were stored in separate containers. In others, as a check on the possibility of bacterial growth in the case of the skin, the three tissues were stored together. When the tissues were kept longer than one day, the Ringer solution was changed daily.
An experiment was carried out to determine the effects of freezing. Here the respiration of the organs was determined immediately after removal from the animal as in the other experiments; then the tissues were quickly frozen with carbon dioxide snow on a freezing microtome. The tissues were then thawed in Ringer solution and the respiration again determined. A summary of the results is given in table 1. DISCUSSION
An examination of the data in the table shows that the order of survival is skin, kidney, liver, when the organs are stored separately. The skin respiration remains practically undiminished for three days, while the liver respiration is
approximately one half of the original respiration after only one day. The kidney survives much better than the liver, but it too begins to decline after the second day. The results seem logical, for one would expect skin, which is di-
rectly subjected to changes in environmental temperature, to survive better than the internal organs. Similarly, the kidney, whose function is to eliminate toxic materials, should survive the toxic products formed during storage better than liver. The experiment in which the tissues were frozen showed that within experimental error the respiratory process was destroyed by freezing. It is interesting to note that when the organs are stored together skin does not
survive much better than liver; and that the kidney also shows less vitality, although not so marked as skin. It seems probable that either liver or kidney or the combination of the two, produces some material which is also toxic to skin. Experiments were performed in which one specimen of skin was stored with liver and another with kidney, in an effort to determine which of the viscera
produced the toxic material. The results were quite variable, however, and no definite conclusions could be drawn. Also, some of the solution in which all the organs were stored together was added to the manometer flask containing
skin which had been stored alone. The solution seemed to have no definite effect on the respiration of the skin over the short interval of the experiment.
Since skin survives for several days after removal from the body, Doctor
238
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
0. D. Meeker has suggested that further studies should be made regarding the availability for graft of skin removed at operation or immediately following death, and subsequently stored. SUMMARY
A study of the primary respiratory process of skin, kidney and liver, kept separately in the icebox for several days indicates that the skin respiration remains approximately normal for three days before showing much change; the kidney respiration declines slowly; but the liver respiration falls to about 50 per
cent of its initial respiration after one day. However, when the organs are stored in the same container, the skin respiration declines very much like that of liver. Freezing with carbon dioxide snow, as for sectioning, destroyed the primary process of respiration in all three tissues. 1. WARBURG, OTTO. The Metabolism of Tumors, London Constable and Co. 2. CARREL, ALEXIS. Jour. Exper. Med., 15: 516—528, 1912, ibid 18: 287—299, 1913, Pozzi,
F. Bul. de l'Acad. de Med., 67:475, 1913, Ebeling, A. A. Jour. Exper. Med., 17:273— 285, 1913.
3. FLETCHER, W. M. Lactic Acid Formation, Survival Respiration and Rigor Mortis in Mammalian Muscle, Jour. Physiol., 47: 360—380, 1913—14.
4. RABBENE, A. Respiratory Exchange of Tissues, Physiol. Abst., 10: 330, 1925. 5. BATTELLI, F., AND STERN, C. Research on the Primary Respiration of Tissue, Jour. Physiol. and Path. Gen., 9: 1—16, 1907. 6. DOMINI, GIOVIANNI, AND PERUZZI, PIETRO. The Exhaustion of the Respiratory Capacity in Vitro of Certain Tissues, Boll. Soc. Ital. Biol. Sper., 10: 493—6, 1935. 7. KiSci, BRUNO. Respiration Values of Fresh Mammalian Tissues, Biochem. Z., 280: 55—7, 1935.
8. LYNEN, FEODOR. Respiration of Animal Tissues after Freezing in Liquid Air, Z. Physiol. Chem., 264: 146—152, 1940.
9. Cooi, E. S., KEEKE, C. W., AND NUTINI, L. G. Fractions from Yeast Which Stimulate the Respiration of Yeast and Animal Tissues; Studies Inst. Divi Thomae, 2: 23—37, 1938.
10. LOSER, H. A Critical Analysis of the Tissue Slice Method in Manometric Experiments, Biochem. Jour., 36: 319—335, 1942.
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.