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For the purpose to obtain the information of the mechanism of protein uptake by the tumor cells, some cytochemical and electron microscopic observations were carried out on Ehrlich ascites tumor cells incubated with horseradish peroxidase (basic hemoprotein, molecular weight approximately 40,000) in vitro. In the earlier periods of the incubation peroxidase was found to be adsorbed on some area of surface of the tumor cells forming a thin protein layer, where an active pseudopodia formation was observed. With the lapse of time, the protein was taken in the deep cytoplasm by the infoldings of the cell membrane and accumulated in the cytoplasmic vesicles having limiting membrane. Concerning the accumulation of the protein into the vesicles, small tubular structures in the cytoplasm connecting the cell surface and the vesicles, were considered to participate in the intracellular transportation of peroxidase taken up. In cold environment (2°C), the formation of pseudopodia and deep inward infoldings of the cell membrane was inhibited and simultaneously the uptake of peroxidase stopped. Iodoacetate and sodium fluoride also effected to suppress the pseudopodia and infoldings formation moderetely, as well as uptake of peroxidase, though they did not stop completely. These facts have indicated that horseradish peroxdase is taken up by Ehrlich ascites tuimor cells through pinocytosis which involves energy-requiring process dependent upon glycolytic metabolism of the tumor cells.

For the purpose to reveal the changes in the metabolism of erythroblast in varied specialization stages the author observed the Feulgen DNA level of rabbit erythroblasts by microspectrophotometry. Observations were made on normal and anemic animals, and those receiving a mass red cell transfusion at the recovery stage of anemia where the early denucleation is stimulated. Observations have revealed that in normal erythropoiesis the DNA contents are kept at n to 2 n level from the proerythroblast to late basophilic stage, but in later stages, polychromatic and orthochromatic, DNA level per cell decreases gradually with advance of the cell specialization reaching the minimum level, nearly haploid level, at orthochromatic stage where most cells are believed to be denucleated. In blood depleted animals nearly the same pattern of DNA level was observed in connection with erythroid specialization as that in normal animal, except a relatively high DNA level in the later specialization. In the cases of the hemolytic anemia a similar tendency has been observed but the minimum level of DNA remains at a higher level, hypo-diploid level, in poly- and orthochromatic stages. Twenty-four hours after the mass red cell transfusion by which severe anemia has been recovered to the original level within one hour, the pattern of the DNA level of the erythroblast returns to the normal one showing a very low DNA level at the polyand the orthochromatic stages. The data indicate that the DNA synthesis of erythroblast kept at n to 2 n levels until the late basophilic stage begins to decline at polychromatic stage and reaches nearly haploid level at orthochromatic stage, but in active hemopoiesis the DNA synthesis is stimulated and the DNA contents are kept at a high level even in the late specialization stages, showing no relation between the denucleation and the low DNA level.

1) Normal karyotype of Donryu strain rat was determined according to the classification of KURITA et al. (8). Namely, the number of chromosomes was 42 in diploid cells, and chromosomes were divided into 3 groups (A, B and C) according to the position of centromere. A.group was consisted of 7 pairs of metacentric chromosomes, B-group 4 pairs of sub· meta.subtelocentrics and C.group 10 pairs of telocentrics and Y. 2) Among all chromosome pairs a pair of the longest telocentric chromosomes (C.l), 4 pairs of all the B.group, and the Y chromosome were recognizable. 3) The presence of polymorphism was demonstrated in the smallest submetacentric chromosomes (BA),: namely, (I) a homologous submeta. centric pair, (II) a homologous subtelocentric pair and (III) a heteromor. phic submeta and subtelocentric pair which seemed to be a hybrid from (I) and (II). To distinguish the polymorphism in their genotype from phenotype was impossible. 4) Animals with type III B·4 chromosomes were produced by type I and type II animals. 5) By checking the chromosomes of the inbred Donryu strain rats maintained over 40 generations by brother.sister mating at Nihon Rat Co., polymorphism in BA chromosomes was also recognized.

The correlation of infectivity to the length of SV 40 DNA by dilute and undiluted passage was described. DNA was extracted from purified virions by Marmur's method, and propagation of virus was done using VERO cells. The infectivity of SV 40 (simian virus 40) ran parallel with the length of its DNA. Undiluted passage caused shortening of DNA and decrease in infectivity, but when these undiluted group was passaged dilutely, length of DNA approached the original length and the infectivity recovered. In undiluted passage group small circular DNAs under 1.0,11_, so far not reported in the SV 40 DNA, were found in low frequency. Replicating form and dimers were also noted from virions and the nuclei of SV 40 infected VERO cells.

1. The mixed lymphocyte culture test where live lymphocytes of female mice are cultured with supersonicated cell homogenate of isogeneic male mice does not reflect the difference in H-Y histocompatibility antigen alone. 2. When non-H-2 antigens are cumulated in various combinations, the rate of blastformation becomes greater than the combination of C3H ♀ themselves. 3. When non-H-2 antigens and H-Y antigens are added in various combinations, the rate of blastformation becomes significantly higher than in the combination of C3H ♀ themselves.

For the purpose to clarify the distribution of DNA in mouse ascites sarcoma cells (SR-C3H) induced by Rous sarcoma virus (Schmidt-Ruppin strain), quantitative assays were carried out by SCHMIDT-THANNHAUSERSCHNEIDER'S method using subcellular fractions isolated from SR-C3H cells and C3H mouse liver as a control tissue, and simultaneously electron microscopic observations were conducted with the rotary shadowed preparations of the SDS-phenol extracted nucleic acids by the protein monolayer technique. The results are briefly summarized as follows. 1. The RNA/DNA ratios in SR-C3H cells and liver cells were 2.3 and 3.7, while those in nuclear fraction of SR-C3H cells and liver cells were 0.34 and O. 56, respectively. The electron micrographs of nuclear nucleic acids revealed a DNA-RNA complex-like structure. 2. DNA and RNA contents of SR-C3H mitochondria were found to be 3.1 and 24 fl-g per mg of protein, respectiVely, which proved to be greater than those of liver mitochondria. The mean values of the contour length of circular DNA molecules in highest frequency group observed in the electron micrographs were 4.88 μ. in SR-C3H mitochondria and 5.08 μ. in mouse liver mitochondria. There could be observed circular molecules of duplicated-length in both mitochondrial DNA's and small circular molecules in SR-C3H mitochondrial DNA. 3. In the microsomal and supernatant fractions of SR-C3H cells and mouse liver cells, the ratios of DNA to RNA gave several percent by chemical analysis and this percentage was particularly high in the supernatant of SR-C3H cells. On the other hand, in the electron micrographs, the fibrous structure was significantly recovered in the supernatant nucleic acids of SR-C3H cells, but with difficulty in the other three fractions. This fibrous structure measured 1.13 μ in the mean value of the length and was considered to be DNA as it readily disappeared after the treat· ment with DNase.

For the purpose to reveal the relation between cell death and nuclear stainability by supravital staining with basic dyes observations have been made on the cells of bone marrow, peripheral blood and lymph node from anemic and non-anemic rabbit, rat, mouse and chicken, and thymus from young mouse. The cells were stained supravitally in blood serum, isotonic saline, calcium chloride and sucrose solutions with the dyes; brilliant cresyl blue (B. C. B), Nile blue (N. B.), neutral red (N. R.), Janus green (J.G.) and eosin (E.). The following results were obtained: 1. In the presence of blood serum all the living cell nuclei observed were not stained supravitally, except some mature erythroblasts and nucleated red cells. 2. In isotonic saline, CaCl2 and MgCl2 solutions all the erythroid cell nuclei were stained deep by B. C. B., N. B., N. R., slightly by J. G. but not by E. In stainability the younger the cell is the deeper in its nuclear staining. The nuclei of other cell strains were not stained. 3. In isotonic sucrose the nuclei of mature granulocyte were also stained by B. C. B. and N. B. but not by other dyes. The nuclei of lymphoid cells and myeloid cells appeared pale without being stained by any dyes. The nuclei of erythroblasts in sucrose solution were stained deeper with B. C. B. and N. B. than those in isotonic saline. The differences between supravital stainability of the nuclei among the cells belonging to different strain and among those of the same strain but in different maturation stage and the nuclear staining after cell death have been discussed from the possible dissociation of DNA from histone.

Antigenicity of the peptide of ribosomal digest in Ehrlich ascites tumor was studied. The peptide was purified by DEAE Sephadex A-50 column chromatography. The peptide was electrophoretically basic, single, and 1.32 S20w sedimentation coefficient with poor content of tyrosine and phenylalanine. The maximum absorbancy was at 267 mμ. Mice and rabbits were immunized with the mixture of the purified peptide with Freund's complete adjuvant. The inhibitory effect of immune γ-globulin on the tumor growth was demonstrated in vitro cultured JTC-11 cells. A single precipitin line was observed between rabbit antiserum and tumor cell extract of Ehrlich ascites cells in ouchterlony double diffusion chamber and immunoelectrophoresis. The sedimentation coefficient of the effective fraction in immune-serum was 17 S20w. The precipitin line was observed at β2-γ region in immunoelectrophoresis.

A unique low density lipoprotein was obtained from the tumor transplanted with a cultured cell line of Ehrlich ascites tumor, JTC-ll cell. The tumor low density lipoprotein electrophoretically migrated as a single band, and the mobility was different from that of other organs. The chemical composition of lipid, cholesterol and phospholipids in tumor low density lipoprotein were characteristic. The flotation rate was Sf 5.9, and thus the molecular weight was estimated to be about 130 x 104. The inhibitory effect on tumor growth of the immune serum was most elevated at 25th day after the intraperitoneal administration of tumor low density lipoprotein. The main fraction effective for inhibition of tumor growth existed in γ-globulin.

1. Both EACA and AMCHA clearly showed an anti-inflammatory effect, by intravenous, intramuscular, or oral route, against inflammatory edema produced in rats by intracutaneous injection of rabbit's anti-rat serum, carrageenin, histamine, serotonin, or bradykinin, as tested by the punch method. 2. The two compounds also showed inhibitory action against the cotton pellet granuloma when used in a larger dose. 3. There was virtually no difference between the two compounds in their anti-inflammatory activity, in spite of the fact that antiplasmin activity of AMCHA is evidently greater than that of EACA. In addition, there was no increase in fibrinolysis at the site of antiserum inflammation in rats. Therefore, it would be difficult to presume that the anti-inflammatory action of these compounds is due to their antiplasmin activity. 4. Salicylates, pyrazolidine derivatives, and non-steroidal antiinflammatory agents like flufenamic acid inhibited degranulation of isolated rat mast cells induced by compound 48/80 and also inhibited ATP-32Pi exchange reaction in rat liver mitochondria, but such actions were not observed in EACA or AMCHA. 5. Anti-inflammatory effect of EACA and AMCHA did not decrease after adrenalectomy but did become weak in hypophysectomized rats. EACA did not increase blood sugar level in normal rats so that its antiinflammatory action is not due to hyperglycemia, and the effect of hypophysectomy may not be correlated to carbohydrate metabolism. 6. Anti- inflammatory effect of EACA and AMCHA appeared more rapidly after intramuscular or oral administration than by intravenous injection but the effect was not fortified after their in vitro incubation with tissues of stomach, intestine, or liver.

Exfoliative cytologic studies on normal oral cavity and on the postexodontic wound healing of infants were carried out and the following results were obtained: 1) The keratinization of cells was found marked in such regions as buccal mucosa, mucobuccal fold, gingiva and palate in that order. 2) As for changes in the distribution of cells and leucocytes, the cell distribution in the period of 15-21 postexodontic days proved to be identical with that of normal exfoliated cells. Namely, the wound healing from exfoliative cytologic aspects takes place during the period of 15-21 postexodontic days.

Rats were depleted of skin histamine by more than 80 % by intraperitoneal injections of sinomenine with daily increasing doses for 6 days. In these rats, egg-white edema induced in the hind paws was inhibited by 68 % of control. The weight of the wall of granuloma pouch made by croton oil was also evidently smaller in the rat treated similarly with sinomenine than that of control. This suggests an important role of histamine participating in the inflammation. It has been observed that a variety of non-steroidal anti-inflammatory drugs inhibited both degranulation and histamine release induced by compound 48/80 of mast cells isolated from rat peritoneal fluid. The degranulation inhibiting actions of anti-inflammatory drugs were markedly decreased in the presence of glucose as in cases of dinitrophenol, dicumarol and warfarin which are known uncouplers of oxidative phosphorylation. Also, prevention of edema provoked by anti-rat serum is roughly correlated to a potency of degranulation inhibiting effect of anti-inflammatory agents. These observations suggest that there is a common mechanism between these two phenomena, and the prevention of mast cell degranulation by the anti-inflammatory agents is, at least, partially due to their uncoupling effects. A working hypothesis explaining the process of edema formation at the inflammatory site has. been made based on the data of the present experiment and other ob3ervations: a leakage of plasma into the tissue space from the gap between two adjacent endothelial cells which are contracted by released histamine may activate a kinin-forming system in the plasma, and kinin(s) may further aggravate a leakage. The mechanism of action of anti-inflammatory agents, which interfere with the histamine effect in inflammation, should be understood in twofold: one is prevention of histamine release from the tissue, mainly by inhibiting mast-cell degranulation, and the other is prevention of the contraction of endothial cells by their uncoupling activities.

MC-induced sarcomas produced under the skin on the back between　scapulas of C3H mice were transplanted　successively to the mice of the　same strain. Using the first and the second generation tumors, viable　tumor cells were prepared and with these tumor cells C3H mice were　inoculated. From these sensitized mice regional lymph　nodes were taken　out at certain intervals and lymph-node cells were prepared. These tumor　cells were mixed with regional lymph-node cells in the ratio of 1 : 10, and the mixed cells were transplanted subcutaneously on the back of C3H　mice, and the development and growth of tumors were observed at intervals.　As a result it was found that the inhibitory effect of these regional　lymph-node cells on the tumor growth was strong one to two weeks after　the transplantation, but beyond 3, or 4 weeks no inhibition　was observable.　In connection with the present in vivo experiments, some comments　were made on the available literature, and it has been demonstrated that　even in the cancer-bearing animal destined to die of tumors, at certain stage of cancer there is seen an inhibitory effect of the host on the tumor　growth by way of the lymphoid system and that such a response of the　host in vivo seems to be　correlated well with in vitro reaction.

For the purpo3e to determine exactly what stage of cell specialization the DNA level of erythroid cell nuclei begins to decline, the author observed the DNA level of erythroblasts in mitosis by microspectrophotometry and the DNA synthesis by flash labeling with H3-thymidine. The cell samples were obtained from the bone marrow of normal, blood-depleted and phenylhydrazine-treated animals, and the anemic animals received a mass red cell transfusion, all the animals being injected with colchicine 4 hours before obtaining the bone marrow sample. DNA level was measured on the smeared cells stained by Feulgen reaction and DNA synthesis by autoradiography on the smeared cells. Besides these, chromosome number was observed on the anemic rat erythroblasts at metaphase by air dry method. The observations indicated that the DNA level begins to decrease at polychromatic stage being accompanied by a decrease in TDH3-incorporation into DNA, reaching minimum level at orthochromatic cell both in DNA contents and synthesis. Chromosome numbers of erythroblasts of rat were irregular being distri buted between 42 to 20. The data have suggested that the DNA level of erythroblasts decreases only in the later stages of cell specialization, and at polychromatic stage the chromosome number may also decrease in rabbit at polychromatic stage by the cell division with an incomplete DNA replication. The high DNA level of the erythroblasts of rabbit, in severe anemia where most of the cells are denucleated at polychromatic and late basophilic stages, has been discussed from the view point of the insufficient DNA replication at polychromatic stages.

The body wall of the cercaria of Schistosoma japonicum is covered with a thin integument which is connected to epithel cells located under the uscle layer. On the outer and the basal surfaces of the integument are seen thin limiting membranes. In the matrix of the integument are distributed numerous dense granules, vacuoles and spines. The rootlet of the spine is attached to the basement membrane of the integument. The circular and longitudinal muscle layers, both underlying the integument, have smooth muscle fibers composed of thick and thin myofilaments. The cercaria possesses five pairs of secretion gland cells which are divided into two groups of three anterior and two posterior pairs. Both gland cells are filled with secretion balls. The tail of cercaria is likewise covered with a thin integumen t, whose structure is identically the same as the body integument. Beneath the integument are located thin circular and longitudinal muscle layers. The circular muscle cells have smooth muscle fibers, but the longitudinal muscle cells have striated muscle fibers. These muscle cells contain many large mitochondria. On observing the cross-sections of the tail at the flame cell level the arrangement of these muscle can be divided into four muscle groups and each muscle group reveals four or five muscle cells. The excretory system is well developed and has flame cells, excretory canal and bladder.

Morphological comparison at colonial level was made on a series of established liver cell lines derived from rats fed 4-dimethylaminoazo-benzene (DAB) for various periods of days for the purpose of elucidating more accurately the differences in morphology and growth patterns among these cell lines. Colonies of each cell line produced by the single cell plating technique were compared with regard to colony size, density and piling-up of cells, atypism and pleomorphism of cells, and the migration of cells from colonies. Plating efficiency of each cell line was also compared. The cultured rat liver cells obtained from those rats fed DAB for a longer period of days showed higher plating efficiency, and increased the incidence of large-sized, dense, and piled-up colonies, of colonies consisted of cells having nuclear atypism and pleomorphism, and of irregularly margined colonies with migrating cells. The correlation between the present results and the process of DABcarcinogenesis is discussed.

A non.specific esterase activity was demonstrated III the jejunum of rats by an azoindoxyl method. 1) Microvilli of the jejunal epithelial cells were remarkably stained in non-frozen specimens and feebly in frozen specimens. 2) The other cytoplasmic structures, i. e. mitochondria, endoplasmic reticulum, nuclear envelope, plasma membrane and multivesicular body showed a positive reaction product in frozen sections but not in non-frozen blocks.

A human fetal fibroblast strain, belonging to a group resistant to SV40 transformation, was transformed by SV40 through a multiple inoculation procedure. Two independently transformed cells were described in comparison with each other. The proportions of cells with the nuclei possessing V antigen were 2.9% at the 5th passage in one strain and 1.1 % at the 4th passage in another, and they declined gradually as frequent passages were repeated. The percentages of the transformed cells with V antigen-positive nuclei were, in both strains, quantitatively compatible with those of the cells with the nuclei full of virus particles in crystalline arrays, which were demonstrated by immunofluorescent studies and electron microscopy.

An electron microscopic observation was made on the DNA's extracted from purified HeLa cell nuclei, mitochondria, and the whole cell, and fractionated by ethidium bromide-cesium chloride density gradient method or sucrose density gradient method. Nuclear DNA presents mainly long linear DNA derived from fragmented chromosomal DNA. In addition to this, the existence of small circular DNA molecules measuring 0.32 -1.78 μ, was confirmed. Mitochondrial DNA was mainly circular DNA, which measured 4.87 μ in the mean value of the contour lengths in the highest frequency group, and small circular DNA molecules, measuring 0.3-1.01 μ in contour length, were also found in an extremely low frequency.

As a step in the elucidation of immunity of human cancer from the standpoint of homotransplantation immunity, we conducted mixed cultures of regional lymph node cells from C3H mouse isotransplanted with methylcholanthrene-induced sarcoma (MC-tumor) together with the primary culture MC-tumor cells, and observed the behaviors of these lymph node cells to the MC-tumor cells and compared the effects of these lymph node cells with lhose of normal mouse lymph node cells by counting the growth number of tumor cells and also by cinematography. As a result, it has been demonstrated that the regional lymph node cells from the mouse isotransplanted with the MC-tumor (2mm3 in size) acquire a strong antitumor activity by 14 days after the transplantation, but such antitumor activity diminishes and disappears in the terminal stage of cancer. When the number of these lymphocytes is increased, there can be observed some dosage effect, but no complete inhibition of the tumor growth can be attained. The cinematographic observations of these regional lymph node cells in the mixed culture with tumor cells reveal that lymphocytes of small to intermediate size aggregate onto the tumor cells and inhibit the movement of the latter.