In the present experiment, tissue cultures of human cervical carcinoma, human metastasized carcinoma in lymph node, human upper jaw carcinoma, Bashford's mouse carcinoma, and normal mouse liver tissue were performed by using roller-tube method. The tissue growth patterns, cell counts and relative growth rate, hydrogen ion concentration in culture media, and histological examination of these tissues in culture were observed. The following are the results. 1. Epidermoid carcinoma of human cervix, rquamous cell carcinoma of human upper jaw, carcinoma simplex in metastasized lymph node, Bashford's mouse carcinoma, and normal mouse liver tissue were cultivated in the media composed of desiccated human plasma solution, chick embryo extract, and Hanks' balanced salt solution by using the roller-tube method. Epidermoid carcinoma of human cervix was successively maintained for 4 months, squamous cell carcinoma of human upper jaw and carcinoma simplex in metastasized lymph node were maintained for 2 weeks and 2 months respectively. Bashford's mouse carcinoma and normal mouse liver tissue were maintained for 1 month and 1 week respectively. 2. The tissue growth patterns of human and animal tumors, and normal mouse liver tissue showed the membraneous sheet formation like a halo. However, 3-4 weeks after tissue culture many small cell groups appeared and they were connected with each other. And this tissue growth pattern showed no difference between malignant and normal tissues. 3. Cell counts were taken in the culture of epidermoid carcinoma of human cervix, Bashford's mouse carcinoma and normal mouse liver tissue. The number of cells increased rapidly in malignant tissues when compared with that of normal mouse liver tissue. Mauy mitosis could be seen in the tissue cultures of squamous cell carcinoma and carcinoma simplex, but their cell counts were not carried out. 4. The pH changes in tissue culture media was estimated. The rapid increases of hydrogen ion concentration in the course of tissue culture was proportional to the cell increase rate, and this is one of the characteristics of malignant tissues in vitro. 5. The histological examination of the cells derived from tissue culture and original tissues was carried out. Atypical large polynuclear cells generating in the tissue culture of carcinoma simplex could be considered as representing the malignant trasformation. And abundant mitosis, anaplastic change, and rapid cell increase were used as the criteria for the evaluation of malignancy in every tissue cells derived from both normal and malignant tissues in vitro.
The roller-tube tissue cultures of human cervical carcinoma, Bashford's mouse carcinoma, and normal mouse liver tissue were performed in order to inquire into carbohydrate metabolism in the culture media. Glucose consumption, lactic acid, pyruvic acid, and α-ketoglutaric acid productions in cutlure media by human and animal tumors and by normal mouse liver tissue were estimated, and the following results have been obtained. 1. Glucose consumption and lactic acid production by human cervical carcinoma and Bashford's mouse carcinoma were in higher level than by normal mouse liver tissue. This was considered as the fact showing the high glycolysis of tumor tissues in vitro. 2. The ratios of lactic acid production/glcose consumption, pyruvic acid production/glucose consumption, α-ketoglutaric acid production/glueose consumption, in each tissue, were compared with each other. The ratio of lactic acid production/glucose consumption in every tissue turned out to be quite similar in values. But the ratios of pyruvic acid production/glucose consumption and α-ketoglutaric acid production/glucose consumption in Bashford's mouse carcinoma, were very low, about one third and one tenth of normal mouse liver tissue, respectively. In human cervical carcinoma, the ratio of pyruvic acid production/glucose consumption showed almost the same value as normal mouse liver tissue, but the ratio of α-ketoglutaric acid production/glucose consumption in hnman cervical carcinoma was half the value of normal mouse liver tissue. Thus pyruvic acid and α-ketoglutaric acid production, the components of tricarboyylic acid cycle, were reduced in tumor tissuen in vitro inspite of the same glucose consumption and lactic acid production as in normal mouse liver tissue in vitro. Therefore, the carbohydrate metabolism, as for the tricaroxylic acid cvcle in tumor tissues, turned out to be different from normal mouse liver tissue. 3. The changes of hydrogen ion concentration in the culture media were in step with the course of tissue cultures. And the pH in tissue culture media had a correlation with the lactic acid production by tissues in vitro. But the increase in the pH was closely correlated with the lactic acid, pyruvic acid and α-ketoglutaric acid productions by human cervical carcinoma, Bashford's mouse carcinoma, and normal mouse liver tissue in culture media.
The protein metabolism of human cervical carcinoma, Bashford's mouse carcinoma, and normal mouse liver tissue have been studied with special reference to the culture media, and the following results were obtained. 1. The whole protein in the culture media of human cervical carcinoma, Bashford's mouse carcinoma, and normal mouse liver tissue was estimated. And as the results it was found that with lapse of time of the culture, human cervical carcinoma and Bashford's mouse carcinoma showed a higher protein consumption than that by normal mouse liver tissue. 2. In every tissue culture albumin was increased as compared with that in the control, but there was no difference between tumor tissues and normal tissue. 3. α-globulin in the media of human cervical carcinoma had no deviation from the control. α-globulin in the media of Bashford's mouse carcinoma showed a derease when compared with that of the control on one day after tissue cnlture, but with lapse of time in tissue culture it was increased. In the culture media of normal mouse liver tissue, α-globulin showed an increase as compared with the control on one day after tissue culture, but a remarkable decrease was found on 3 days afterward. As for normal mouse liver tissue, α-globulin showed the lower value than human cervical carcinoma and Bashford's mouse carcinoma during the course of tissue cultures. 4. The remarkable increase of β-globulin in the culture media of human cervical carcinoma could be seen. In the media of Bashford's mouse carcinoma, β-globulin showed a decrease on 3 days after tissue culture. β-globulin in the culture media of normal mouse liver tissue revealed an increase on one day after tissue culture, but on third day Bashford's mouse carcinoma and normal mouse liver tissue presented almost the same value. And both tissues showed an increase on five days cfter the tissue culture. 5. γ-globulin in every tissue culture was found to show a decrease as compared with the control along with the lapse of culture time. And in this respect no difference was found between tumor tissues and normal tissue.
When high hydrostatic pressure (50 to 500 kg/cm2) was applied to isolated longitudinal muscle strip (10 to 15 mm long, 2 to 3 mm wide) of frog intestine hanging in Ringer's solution, following changes of its length and rhythmical movement were observed. 1) Under high hydrostatic pressure up to 100 kg/cm2 the rhythmical movement of the strip is accelerated in frequency and tonus effectively, depending on neurogenic nature, and at 300 to 500 kg/cm2 it weakens or disappears. 2) When the pressure of about 300 to 500 kg/cm2 is applied to the muscle strip, it lengthens quickly at first and then conversely shortens gradually after a certain limit of extension, and again the strip contracts rapidly but temporarily and then relaxes slowly. 3) These lengthening and subsequent shortening of the strip under high hydrostatic pressure of 300-500 kg/cm2 seem to be of myogenic nature and not of neurogenic nature.
High hydrostatic pressure (50 to 500 kg/cm2) was applied to isolated longitudinal muscle strip (10 to 15 mm long, 2 to 3 mm wide) of frog intestine immersed in Ringer's solution containing some drugs, and from the observation of changes of the tonus and rhythmical movement of the preparation following results were obtained. 1) When a drug acts upon the strip during compression, such as magnesium sulfate, barium chloride, acetylcholine or atropine sulfate, the changes on the strip result in algebraic summation of the pressure- and drug-effects. But the action of adrenaline chloride on the strip is reversed under high hydrostatic pressure. Namely, the movement of the strip inhibited by adrenaline or pressure respectively, but it is augumented when the pressure is applied on the strip immersed in the adrenaline-Ringer's solution. 2) The stimulative effect on the muscle strip under high hydrostatic pressure up to 100 kg/cm2 is of neurogenic nature. But under pressure from 300 to 500 kg/cm2 the first instaneous lengthening of the strip and successive slow shortening are observed and this effect is of myogenic nature. 3) The temporary contraction of the strip immediately after decompression may be considered as an adaptive process in biological phenomenon.
High hydrostatic pressure (500 kg/cm2 and 1, 000 kg/cm2) was applied to an enucleated eyeball and eyeballs of the decapitated frog head, which were suspended in Ringer's solution. Next, some autonomic drugs were added to the solution to make them act on the eyeballs before, during or after compession. By observing the pupillary changes, the following results were obtained. 1) After decompression the pupil becomes wide at first and then narrow. But the miosis after decompression does not occur in the same manner in the enucleated eye as with those of the decapitated head. 2) The effects of autonomic drugs also vary according to the difference whether the eye has connection with the central nervous system or not. 3) These facts appear to prove that high hydrostatic pressure has marked influence on the central nervous system. Its sensitivity to autonomic drugs is remarkably raised by high pressure.
Hemolytic and precipitin reaction of nucleic acid and nucloprotein fractions from Sh. flexneri 2a and St. aureus were studied and the results as follow are obtained. 1 In these microorganisms, hemolytic reactions of nucleic acid fractions were negative, and precipitin reactions were positive. 2 Nucleoprotein fraction from Sh. flexneri 2a grown on glucose added medea showed a stronger positive hemolytic reaction, whereas that from St. aureus tended to negative. 3 Nucleic acid fractions from these microorganisms grown on Fe++ deficient medea by adding α, α'-dipyridyl showed a negative hemolytic reactions. 4 From these results it could be seen that the immunological properties of nucleic acid or nucleoprotein fractions were affected by the composition of culturing medea.
In order to study the influence of pressure effects on the cell division of sea urchin eggs we have performed a series of experiments to enable us to understand the meaning and the importance of morphogenesis of the embryo. Sea urchin eggs used are of Temnopleurus tereumatics, in the stages of unfertilization to early blastulae. The experimental results can be summarized as follows. 1) In unfertilized eggs the elevation of fertilization membrane is delayed by high pressure of 300 kg/cm2 applied for 15 minutes. The delay is in proportion to the pressure intensity and duration. Neverthless, furrowing process of the egg does proceed after the pressure is withdrawn. 2) Likewise, in fertilized eggs, the furrowing are retarded under high pressure, in fertilized eggs, the furrowing are retarded under high pressure, in proportion to the pressure intensity and duoration. In most cases, however, the rate of the cell division catches up with that of the control before reaching the stages of bastulae. 3) The deformities of pluteus can be observed in applying the pressure neither to unfertilized, nor to fertilized eggs in all stages.
In this experiment, the authors obserbed the effect of Pretiron (TSH) on the transplantation and growth of three kinds of trnsplantable tumors of animals. 1) Ehrlich Ascites Tumor Mice weighing approximately 20 g were used. All mice were transplanted with Ehrlich Ascites Tumor intraabdominally. Pretiron (TSH) was intramuscularly injected daily for the period of 23 days, beginning 3 days prior to the transplantation of tumor. In the Pretiron (TSH) treated group, the growth of free ascites tumor cells was markedly inhibited and the survival days of these mice were prolonged (20-50 days). In the ascites of this group of mice, the infiltration of numerous lymphocyte-like cells was recognized. On the other hand, the survival days of the control group of mice were less than 20 days and the marked growth of free ascites tumor cells was obserbed. 2) Fibrosarcoma 58. The growth of transplanted tumor in the Pretiron (TSH) treated group is moderately inhibited. In the tumor tissue of Pretiron treated group, the more maked infiltration of lymphocyte-like cells was seen than iu the control group. 3) Mammary Carcinoma in R Strin of Mice The growh of transplanted tumors of Pretiron (TSH) treated group was markedly inhibited and the majority of these tumoss disappeared. Histologicaly, numerous lymphocyte-like cells was found to infiltrating in the stroma of the tumor tissues. On the contrary, in the control group, the transplanted tumors grew well in all mice. From these findings it is concluded that Pretiron (TSH) has an inhibitory action on these tumors. Although the mechanism of antitumor action of Pretiron (TSH) is unknown, it is interestinh that Pretiron (TSH) promotes the infiltration of lymphocyte-like cells in tumor tissues as obserbed in this experiment.
It has been suggested, since many years, that endocrine function of the pancreas may be related to the peptic ulceration of the stomach. There has been, however, no unanimous opinion on this item. To clarify this problem, the author investigated, from several points of view, the changes in gastric mucosa following administration of NPH-Insulin to the rat. By the intramuscular administration of NPH-Insulin hemorrhagic erosion of the gastric mucosa, instead of ulceration, was observed especially on the corpus of the glandular stomach. The occurrence of erosion was related to the severity of hypoglycemia produced by the Insulin and the rats showing erosion of the gastric mucosa were always associated with hypoglycemic shock. In the rats with gastric erosion weight of their adrenal glands was increased, concentration of K in sera was decreased, and concentration of Na in sera was increased. The occurrence of erosion in the gastric mucosa following administration of NPH-Insulin was completely hindered by vagotomy and was slightly depressed by administration of Chlorpromazin and of Dietazin. In the adrenaletomized rat, the erosion was more marked even after administration of small dosis of NPH-Insulin, and was not hindered by vagotomy. Gastric secretory function in rat with erosion of the gastric mucosa was depressed by administration of the Insulin. In the histological study of the stomach, contraction of the arteries in the submucosa, dilatation and stagnation of the veins in the submucosa and the mucosa were observed. The erosion did not develop to chronic ulcer even after repeated administration of the Insulin. It was, therefore, confirmed that circulatory disturbance in the gastric mucosa caused by vascular change which was produced by means of vagal stimulation played a main role on the occurrence of erosion in the administration of the Insulin, and was also surmized that the reduced resistance of the gastric mucosa caused by hypoglycemia promoted formation of erosion.
It was discussed, in part I, that endocrine function of the pancreas may be related to peptic ulceration of the stomach, though many problems on this item remain unknown. To clarify the influence of hyperglycemia induced by damage of the pancreas on the ulceration of the stomach, the anthor studied, in this part, changes in gastric mucosa and the secretory function of the stomach in Alloxan-diabetic rat. The incidence of the gastric erosion in Alloxan-diabetic rat following administration of NPH Insulin was equal to that in the non·diabetic. Gastric secretory function in Heidenhain's pouch of dog was elevated in all cases on the day of administration of Alloxan, and in half of the cases the function remained elevated during the course of diabetic state. In Alloxan-diabetic rat, however, gastric secretory function was depressed and the depression was much more remarkable in the cases in which adrenalectomy was added. No ulcer was observed in gastric mucosa of Alloxan-diabetic rat. It was, therefore, surmized that the hyperglycemia induced by Alloxan was not related to the ulceration of the stomach.
In the preceding report, the author studied the changes of tissue mast cells (TMC) in the specimens of liver biopsy specimens of viral hepatitis. The present study was attempted to confirm, by animal experiments, the data described in the previous report. Animals used were male dogs, mice and rabbits. Chloroform and carbon tetrachloride were administrated for experimental damage of the liver tissue. The experiments gave the following results: 1. TMC were found both in intralobular and periportal region of the liver. Destructive processes such as dispersion, degranulation, destruction and condensation of the granule in TMC were found in an early stage of the liver damage. A marked increase in number and morphological changes of TMC as the remarkable proliferation of the connective tissue in periportal and intralobular regions were ncted in such chronic stage. 2. A marked decrease in number and remarkable morphological changes of TMC were observed in the cases on an adrenocorticosteroid hormon therapy. 3. TMC were chiefly found in the periportal region of rat's liver, and a increase in number and morphological changes were found when the connective tissue increased in the periportal field after long-term administration of hepato-toxic drugs. 4. The asministration of the adrenal cortical hormones induced a marked decrease in number and destractive and condensed chages of TMC granules at this stage. 5. Administrations of the remedies for liver damage such as glucronic acid, thioctic acid, multi-vitamines and communin induced a slight increase in mumber and slight changes in the morphological findings comparing with control groups. The only remarkable change were the condensation of TMC granules. 6. Only a few TMC were found in rabbit's liver, and there were minimal or no changes in TMC, even in the stage of marked damage of the liver tissue. 7. These results lead to the following conculsions: There is a close relation between the proliferation of the connective tissue in dog's and rat's liver and the changes of TMC.
The method of micro-quantitative analysis of bile pigment precursors in erythrocytes was studied and various experimental observations were made for the elucidation of its physiological significance. The following results were obtained: 1. Studies of the fluorescent micro-quantitative analysis made it clear that bile pigment precursors in erythrocytes can be determined in the 0.1-5.0 μg/ml biliverdin chloroform solution. Observing its absorption spectrum and fluorescent spectral characteristics, it was also made clear that all the bile pigment precursors were determined by the fluorescence of choleterin zincic complex salt. 2. Gardikas' method of extracting intracorpuscular bile pigment precursors under an anaerobic condition without adding ascorbic acid proved to be better than that of R. Lemberg of extracting by adding ascorbic acid under an aerobic condition. 3. Among normal erythrocytes of various animals were found no significant specific differences, the amount of bile pigment precursors being about 15 μg/Hb. 4. When dog's erythrocytes were iucubated at 37°C. after the addition of ascorbic acid, bile pigment precursors in them attained to a remarkably great amount within several hours. This is considered to have resulted from the action of ascorbic acid in choleglobin formation in erythrocytes. 5. 4 hours after phenylhydrazine HCl was administered to a rabbit, the amount of bile pigment precursors in erythrocytes in circulating blood began to increase and attained to its maximum in 4-48 hours. The amount of easily split off blood iron also increased in parallel with it. 6. When canine blood was taken in A. C. D. solution and kept at 4°C., the amount of bile pigment precursors in erythrocytes gradually increased with the course of time, showing a sudden increase after 4 weeks. The amount of easily split off blood iron showed a similar variations. 7. In the erythrocytes of a phlebotomized anemic rabbit, the amount of bile pigment precursors decreased at the same time as reticulocytes showed a remarkable increase. The amount of easily split off blood iron showed a similar tendency of decrease. 8. From the above results the following conclusions were drawn. Bile pigment precursors which are usually observed in normal erythrocytes are increased by chemicals such as ascorbic acid and phenylhydrazine HCl, or by preservation, while in the case of anemia by phlebotomies where immature erythrocytes increase, the amount of bile pigment precursors decreases. Consequently, bile pigment precursors in erythrocytes physiologically increase with deterioration.
In order to study the possibility of porphyrin to become bile pigment precursors, the relations between free porphyrin in erythrocytes, especially protoporphyrin, and intracorpuscular bile pigment precursors were experimentally observed. The results were as follows: 1. A part of protoporphyrin which increased was observed to change quickly into bile pigment precursors in the erythrocytes of a phlebotomized anemic or phenylhydrazine HCl administered rabbits where reticulocytosis occurred. 2. This transformation of protoporphyrin into bile pigment precursors were also observed in preserving canine erythrocytes where reticulocytosis was caused by phlebotomies. 3. Judging from the variations of intracorpuscular easily split off blood iron, it seemed appropriate to consider that this transformation occurred after the formation of heme or hemoglobin. In the above experiment, however, of preserving erythrocytes of a phlebotomized anemic dog, the plainly direct transformation of protoporphyrin into bile pigment precursors were also observed. 4. In preserving canine erythrocytes with or without the addition of ascorbic acid, the increase of protoporphyrin and of bile pigment precursors were slso observed. However, it is difficult to account for the relations between these two; the increase of bile pigment precursors may be ascribable to the breakdown of hemoglobin. 5. Intracorpuscular free coproporphyrin was observed to increase in those erythrooytes after phlebotomies. phenylhydrazine HCl administration, or preservation with the addition of ascorbic acid. This increase may more properly be ascribed to protoporphyrin in the formation process of hemoglobin than to bile pigment precursors. On the other hand, free uroporphyrin in erythrocytes did not show any significant variations. 6. Protoporphyrin mixed with chloroform extract did not hinder the microquantitative analysis of bile pigment precursors by fluorescence.
The properties of so-called ether extractable bilirubin were studied, and the following results were obtained: 1. In comparing the pigment extracted by adding ether to jaundiced serum with crystalline bilirubin, some remarkable differences were observed between the ether solutions of them both, that is, differences in an absorption curve, a diazo reaction, and the reactions to acid and alkali. 2. The ether solution was found to contain both direct and indirect reacting bilirubins, the former taking the greater part in quantity. 3. This direct reacting bilirubin was considered to be of ester in type which we call ester-type bilirubin as it was presumed to have a phosphate ester, showing its absorption maximum at 415 mμ. 4. In the same ether solution was also found a lipid stain positive substance, changing the properties of both direct and indirect reacting bilirubins to some extent. 5. This lipid stain positive substance was supposed to combine, in a certain form, with both direct and indirect reacting bilirubins and to play an important role as a carrier in the process of ether extraction of both direct and indirect reacting bilirubins from the serum.
According to the results of studying E. E. P. positive phenomena from the viowpoint of the properties of the serum, some E. E. P. positive cases were examined in order to consider the clinical significance of E. E. P. The results were follows: 1. E. E. P. could be extracted from the E. E. P. positive serum by ether but not by chloroform. After it was extracted by ether, however, E. E. P. became chloroform-soluble. 2. When the serum was acidified after the ether extraction of E. E. P., a yellow pigment could be extracted again by ether, which was found to be the indirect reacting bilirubin. 3. The E. E. P. positive jaundiced serum was observed to contain much direct reacting bilirubin even after the ether extraction of E. E. P. 4. The lipid stain positive substance could be extracted by ether in a great quantity at a time, which it could not be extracted by chloroform. This substance was considered to play an important role in E. E. P. positive phenomena. 5. E. E. P. was presumed to combine with β-globulin through the combination of the lipid stain positive substance with β-globulin in the serum. 6. By comparing proteins in E. E. P. positive serum by paper electrophoresis before and after the ether extraction of E. E. P., it was found that β-globulin decreased considerably after this extraction. This fact also confirmed that the lipid stain positive substance could be extracted by ether, because this substance was combined with β-globulin. 7. For examining the presence of E. E. P., it was found to be better to observe the diazo layer after the direct diazo reaction than to examine the ether extract. 8. The E. E. P. positive jaundiced serum did not necessarily result from carcinomatous biliary obstruction but from a severe intrahepatic or extrahepatic biliary obstruction independently of its cause.
It is well known that in malignant tumors, their glycolysis are marked both under aerobic and anaerobic conditions, and this is the most important biochemical property common to all malignant tumors. Using the New Yoshida Tumor, the author investigated the quantitative relation in its glycolysis and the effect of thionin on its respiration in order to observe some aspects of its terminal respiration system and besides, the effect of some inhibitors on its glycolysis was studied. The results were as follows: 1) It was found that as reported up to this time, both under aerobic and anaerobic conditions, the glycolysis by this tumor was as marked as by other sorts of tumors and lactic acid was formed in a great quantity. 2) When glucose was added as the substrate, its endogenous respiration was inhibited and the higher the concentration of glucose was, the more marked the inhibitory effect was and yet, in any low concentration of glucose, its respiration was not accelerated. 3) The respiration inhibited by addition of glucose was recovered by addition of thionin: In this case, however, lactic acid formation was decreased. 4) When T. C. A. cycle intermediates were added as substrates, its respiration was little accelerated, but by addition of thionin at the same time as the intermediates, its respiration was accelerated to sme degree. Further, the respiration accelerated by addition of thionin was reduced after a long time reaction and conversely inhibited slightly. 5) KCN inhibited its respiration, but had no effect on glycolysis itself at all, whereas monoiodacetic acid, 2, 4-dinitrophenol, aureomycin and NaF inhibibited not only its respiration but its glycolysis at the same time.
Using the New Yoshida Tumor, the author investigated the energy metabolism by observing the inhibition of the respiration and the inorganic phosphorus intake of the cells by NaF, 2, 4-dinitrophenol (DNP), and aureomycin (AM). The following results were obtained. 1) By the addition of glucose into the reaction media, the respiration, the respiration of the tumor cells was inhibited to some extent compared to the control, though the inorganic phosphorus intake of that was retained almost same as the control. Whereas the addition of Mg into the media did not show remarkable effects on the respiration and on the phosphorus intake. 2) When glucose was not added into the media, the respiration was markedly inhibited by NaF in dilution of 10-1 M, but was rather accelerated by that in dilutions of 10-2 and 10-3 M: the phosphorus intake was also inhibited by NaF in diluticn of 10-1 M, and conversely accelerated by that to slight extent in dilutions of 10-2 and 10-3 M. However, as the addition of glucose into the media, it was also inhibited by NaF in dilution of 10-1 M; and conversely accelerated by that to slight extent in dilutions of 10-2 and 10-3 M. However, as the addition of glucose into the media, it was noticed that the respiration was inhibited by NaF in any dilutions and that the phosphorus intake was accelerated, especially in dilution of 10-2 M. 3) The endogeneous respiration and the phosphorus intake of the tumor cells were inhibited by DNP in any dilutions, 10-3, 10-4, and 10-5 M. The inhibitions of these were reduced as the addition of glucose into the media, respectively, especially on the phosphorus intake. 4) AM did not show inhibitory effect so remarkably on the phosphorus intake irrespective of the additicn of glucose; but showed considerable inhibition on the endogenueous repiration in dilutions of 10-3 and 10-4 M. The inhibited endogeneous respiration was not recovered by the addition of glucose in contrast with the inhibition by DNP. 5) The O2 uptake and the phosphorus intake were also studied on the cells. suspended in 0.85% NaCl, ascitic supernatant, and amniotic fluid of chick embryo, respectively, in all reaction media glucose was added as substrate. And both accelerations of the respiration and of the phosphorus intake were remarkable in order of 0.85% NaCl, amniotic fluid and ascitic supernatant. The inhibitory effects on the O2 uptake and the phosphorus intke by NaF, DNP, and AM were regarded to be similar to each other in respective cell suspensions mentioned above.
1. By means of microsphectrophotometry the author carried out quantitative analyses of DNA and protein in an individual cell, using Euplotes woodruffi and Ehrlich ascites tumor cells as the materials through Feulgen reaction and Naphthol yellow S stain, and studied the relation between the quantities of these substances and the mitotic cycle. The results are as follows. 2. The DNA content in the macronucleus of Euplotes is increased at two ends of the nucleus, and the part where DNA is increased and that which shows no DNA increase are demarcated by the formation of a reorganization band. 3. The DNA synthesis of Euplotes is most active in the latter stage of interphase; and the DNA content is completely doubled just before the cell division. 4. The protein synthesis of Euplotes cells is taking place at all phases of the mitotic cycle and there is a linear relation between the mitotic cycle and the protein contents of the cell. 5. Judging from the relationship between the DNA synthesis and the protein synthesis during the mitotic cycle of Euplotes cells, the DNA content and the protein content in an individual cell show a sigmoid curve between the two and also the similar curve can be observed in the case of Ehrlich ascites tumor cell. Therefore, it is assumed the protein synthesis is being likewise carried on all through the mitotic cycle of Ehrlich ascites tumor cells. 6. The author discussed about the relationship between the protein synthesis and the cell division.
Using sea-urchin eggs, Euplotes woodruffi, HeLa cells, and Ehrlich ascites tumor cells, the author studied the action of OX substance on the DNA and protein synthesis of these cells, and obtained the following results. 1. OX substance has been found to act as to impede the incorporation of 32P to DNA in Ehrlich ascites tumor cells. 2. OX substance leads the nuclei of Euplotes, HeLa and Ehrlich ascites tumor cells into a picnotic state. 3. OX substance is found to possess an action to diminish the DNA contents in the nuclei of HeLa and Ehrlich ascites tumor cells while it tends to increase transiently the DNA content in the nucleus of sea-urchin egg but later decreases it. 4. It has been found that OX substance brings about the disturbance on the mitotic apparatus or the functional disturbance in Ehrlich ascites tumor cells and sea-urchin eggs. 5. In addition, this substance acts as to inhibit the protein synthesis of Ehrlich ascites tumor cells. 6. From these findings, it seems that OX acts temporarily on the mitotic apparatus, thus interfering with the cell division as well as giving rise to the degeneration of the cells. accompanied by the secondary inhibition of the synthesis of DNA and RNA in the cells.
Using various anti-carcinogenic agents (nitrogen mustard N-oxyd as an alkylating agent, 8-azaguanine as an anti-metabolite, and sarcomycin-Na, carzinophilin, mitomycin C, chromycin, actinomycin and sanamycin as inhibitory anti-carcinogenic agents) the authors studied the actions of these agents on the spermatozoa and eggs of sea-urchin by observing the rates of the membrane formation at insemination and egg division and obtaind the following results. 1. When treated with the solution of various anti carcinogenic agents diluted ten-fold the optimal qunatity for the human body for ten minutes, the spermatozoa of sea-urchin (Heliocidaris crassistina) are hardly affected by these agents. 2. In the cases where sea-urchin eggs (Hemicentrotus Pulcherrimus) are treated for 30 minutes with different anti-carcinogenic agents at various concentrations, the higher the concentration, the greater is the decrease in the rates of the membrane formation and egg division at insemination of normal spermatozoa, and in the case treated with the solution diluted 50-fold of the optimal quantity for the human body the inhibitory action of the agents increases in the ascending order of: carzinophilin >Thio TEPA > Nitrogen mustard > mytomycin > Sanamycin > chromomycin > actinomycin. 3. When treated with the 50-fold solution these agents as compared with the proximal concentration for the human body, at the intervals of 20, 40, 60, 80, and 90 minutes after the fertilization, mytomycin and carzinophilin markedly inhibit the fusion of male and female nuclei while chromomycin, sarcomycin and nitrogen mustard suppress the cell division at metaphase, and chromomycin has especially strong inhibitory action on the cell. 4. When the effects of these agents are compared with those of OX-substance with respect to their concentration, it has been clarified that the action of these agents on sea-urchin eggs and spermatozoa are weaker than that of OX substance.
In 1958, SENO et al has successfully fixed the dye granules in the supravitally stained bone marrow cells and Yoshida tumor cells by the use of mercuric potassium iodide solution which is known as the reagent for the quantative determination of NH3. But, the fixation method is not perfect for electron microscopy, because the resulted precitants of mercuric potassium iodine-dye complex is easily soluble in pure alcohol, and there is a possibility that the complex is lost during dehydration. Hereupon, the author tried to substitute the dye complex with mercuric sulfide which is insoluble in water, alcohol and ether and by this substitution the granules should be retained safely through dehydration for electron microscopy. Observations revealed that the stable complex of the dye-HgS can be produced in the site of dye granules by exposing the cells, which were previously stained supravitally and treated with mercuric potassium iodide, to (NH4)2Sx solution. The fine structure of the protoplasma proved to be retained through the treatment with (NH4) 2Sx solution giving almost the same picture as those treated only with mercric potassium iodide solution added an equal volume of 2% osmic acid solution (pH. 7.4). Electronmicroscopic observation on the cells stained supravitally with Janus green B and fixed by the method just mentioned revealed that Janus green B granules appear as opaque masses of uniform in density or rings having thick opaque walls and scanty in contents. Morphologic observation proved granules are the mitochondria themselves, because some of them showed the inner structure like cristae. Treatment with (NH4)2Sx seems to increase the opacity the dye granules, and the prolonged treatment results in the formation of black masses irregulare in shape. The result obtained is almost the same as demonstrated by SENO et al, but in my experiment the possility of solving out of dyes from some cytoplasmic area has been complexely devoided. And now it can safely be said that Janus green stains mitochondria selectively. Dye, even in its leuco type, is not detected in the ground substance of cytoplasm. These observations refute the theory proposed by LAZAROW and COOPERSTEIN in which they claim that the Janus green B enters into the cytoplasma diffusely, but mitochondria only can be recognized as the stained granules by the oxidation of Janus green B. There are some of mitochondria remain without staining, but any other cytoplasmic elements are not stained by Janus green B as far as the cells the author observed.
The author's own divice, as mentioned in part I, a method of substitution of the dye potassium mercuric iodide complex formed in the supravitally stained cells with mercuric sulfide complex, gave a good result to retain the dye granules in situe, revealing the selective staining of mitochondria with Janus green B. But the granules appeared too opaque to see the detailed inner structure of mitochondria. Then, the author tried to retaine the potassium mercuric iodide dye complex through the dehydration process by using a specific dehydration media for electronmicroscopy. In vitro experiment on the precipitants, the mercuric potassium iodide dye complexes, proved that this complex is dissolved easily in pure ethanol, but is not dissolved in 50% ethanol. On the author hand, the complex is not dissolved in ethyl-ether, but dissolved ether-ethanol mixture (1.1). The test done by lowering the ethanol contents in ether proved that the dye does not dissolved in the ether containing 20% ethanol. On the basis of these observations the author tried to retain the complex through dehydration and succeeded in observing the mercuric-dye complex in the cells under electron microscope by making the sections from the cells dehydrated through 50% alcohol, ether containing 10% of pure ethanol and then pure ether, imbibed with ether-methacrylatemonomer and methacrylatemonomer, and embedded in methacrylate by the aid of benzoyl peroxide. The electronmicroscope observation on the cells stained supravitally with Janus green B, fixed by the method deviced by SENO, dehydrated and embedded by the method just mentioned, proved that the minnte structure of the cells are kept as well as in those fixed with 1% osmic acid and dehydrated by the routine method. In the series of observation on the cells treated with Janus green B in the different period of staining, the processes of the invasion of dye into mitochondria and the deformation of the inner structure have been traced. After a short period of staining, the dye has acted as scarecely to increase the opacity of mitochondria but a marked change in they structure occured by the ring formation at one end of mitochondria. More deeply stained ones the appear very dark but in the dark area some opaque droplets are recognized. In this case, too, the less opaque pole were seen at the end of mitochondria, in which the structure of cristae were observed. These observations show that by the action of dye element of mitochondria seems to be segregated into 3 parts, less affine, modercetely affine and strong affine elements to me dye, with the destruction of the inner structure.
In recent years there has been a great deal of interest in the relationship of adrenocortical and pituitary hormons and their influence on the development of malignant tumors. During the past 3 years I have been studing the effect of dried tissues of adrenal and pituitary glands, implanted into subcutaneous tisuse on the tumor induction in the rats fed with dimethyl-amino-azobenzol (DAB) The animal used were inbred ones cultured in our institute from a pair of hybrid rats which showed a high susceptibility for DAB. The liver tumors appeared after about 120 days feeding of DAB. Malignant adenocarcinoma was predominant in the beginning and then typical hepatoma appeared. Continued feeding with DAB resulted in the rapid growth of tumors and the development to the more malignant types of hepatocellular or cholangiocellular carcinoma. Pituitary tissue effected an aceelerated tumor induction and the earlier development of the malignant type, Adrenal tissue showed a slight inhibitory effect on tumor growth. Simultaneous inplantation of pituitary and adrenal tissue represented a marked inhibitory effect on the tumor induction by DAB.
The marginal capillary plexus of the cornea of 305 healthy persons was studied by a micro-photographic apparatus especially for capillaries of skin and mucous membrane. And the following results were obtained: 1. The width of arterial foot and venous foot of the majority of them ranged between 67 μ and 1115 μ respectively, the ratio between these twoups being 1: 2. There was a tendency of the width narrowing with the persons over 60 years. 2. As for the width and length of capillary loop there was no difference as to the age, the former ranging between 3160 μ and the lattes, between 100150 μ with the majority of them. 3. The capillaries were straight seldom winding or zigzagging. 4. The dilatation of veins was found in 20.5% of them, those under 19 developing none. The dilatation increased with age, and it was found in 50.0% of those over 60 years. The fusiform and trangular dilatations were very characteristic to the marginal capillary plexus, their percentage being 5.2 and 3.3 respectively. Their appearance incrased with age. 5. The partial stricture and hemorrhage of artery were rarely found. 6. The intermittence of blood flow and the formation of granules in blood flow were found in 1020% of them respectively, the percentage of occurrence rising with age. 7. No hyperemia, congestion, and ischemia were found. 8. The cold weather narrowed the width of arterial and venous feet, reduced the length of capillary loop, stimulated the formation of granules, caused the intermittence of blood flow and reduced the speed of blood flow but it almost had nothing to do with the fusiform dilatation and triangular dilatations.
The marginal capillary plexus of cornea of the hypertensives was studied and the following results were obtained: 1. The width of arterial foot was 3 μ or less for 38.7% and 45 μ for 49.7% while it was less than 5 μ for the majority. This width was narrower than that of healthy persons. There was a tendency that the width narrowed with the age and the progress of hypertension. 2. The width of venous foot was 7 μ or less for 19.3% and 810 μ for 52.0% while it was less than 10 μ for the majority. This width was also narrower than that of healthy persons. There was a tendency that the width increased with the age and the rise of systolic as well as diastolic blood pressure. 3. As for the width and length of capillary loop and the direction of blood vessels, they were not much different from healthy persons and these things had nothing to do with the age and blood pressure. 4. Hemorrhage was seen though in a very few cases, and it was regarded as an in important clue to diagnosing. 5. The partial dilatations of venous foot were found in 75.0% of them. Those dilatations were fusiform or spindle-shaped, the occurrence of the former increasing with the age and the rise of systolic as well as diastolic blood pressure. It seemed that the factors of hypertension were the cause of the partial dilatations. 6. The occurrence of the intermittence and stasis of blood flow was more frequent than in healthy persons, the former affecting 39.2% and the latter, 8.2% of them. The frequency of occurrence increased with the age and the rise of systolic as well as diastolic blood pressure. 7. The speed of blood flow was slower than normal for 80.7% of them, the abnormality and its ingravescence increasing with the age and the rise of systolic blood pressure. 8. The formation of granules in blood flow was found in 56.7% of them, the percentage of granules being much higher than that of healthy persons. The frequency of occurrence rose with the age and the rise of systolic as well diastolic blood pressure. 9. The abnormality in blood volume was not characteristic in this case. 10. The comparative study of the arteries of the eye ground and the marginal capillary plexus of cornea revealed the existence of a close inter-relationship between them. 11. The effect of the cold weather on them was not much different from that on healthy persons, but it increased the width of venous foot and raised the ocurrence and ingravescence of the fusiform and spindle-shaped dilatation.