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1. In order to obtain direct evidence for the enzymatic identification of the head-pieces of the elementary particles in the inner mitochondrial membrane, the head-pieces were detached by sonication from the isolated inner membrane of beef heart mitochondria, purified by pursuing the particles with the electron microscope, and analyzed for enzymatic properties. 2. Electron microscope examination revealed that the isolated headpieces are the spherical particles about 90À in diameter which are quite similar in appearance to the head-pieces of the elementary particles lining the inner mitochondrial membranes. 3. The head-pieces are identified as ATPase sensitive to oligomysin when attached by stalks to the membrane, and become insensitive when detached or purified from the membrane. 4. The head-piece is labile to cold with respect to ATPase activity and morphology.

When cultured cells are used in experiments, It is very important to know from what kinds of cells the cultured cells are originated, and what characteristics the cultured cells maintain continuously in vitro Some properties of rat liver cells in long-term cultivation were examined for the purpose of identifying the cultured cells with parenchymal liver cells by investigating their functions. The production of rat serum albumin and α-globulin which is regarded as specific functions of liver parenchymal cells was detected in these cultured rat liver cells with the method of immunoelectrophoresis. Histochemically, acid phosphatase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, lactic dehydrogenase, and adenosine triphosphatase were demonstrated in the cultured rat liver cells which were morphologically epithelial. Alkaline phosphatase showed little activity in these cells. Glycogen was recognized by the periodic acid-Schiff technique, when bovine serum concentration in the culture fluid was reduced to 5 per cent. These histochemical findings of cultured rat liver cells were identical with those of parenchymal liver cells in vivo. These facts suggest that there is a possibility of the continuous cultivation of liver cells by the present methods and of the identification of the cultured cells with the parenchymal liver cells from their functions.

Changes of basophil leucocyte counts of the peripheral blood in bronchial asthma were investigated mainly by our improved method of KOVACS (4). The results are as follows. I) The basophils in bronchial asthma generally show a higher count than in healthy controls. 2) During the interval of repeated asthmatic attacks the basophil count is higher than in the asymptomatic period. 3) Particularly in an episode of asthmatic attack, the basophils increase immediately before the attack and decrease to the normal level or lower after the attack has begun. 4) During asthmatic attacks it may be possible to expect another attack, if the basophil count again tends to increase from the previous low count. 5) This counting method of basophils is easy enough for the routine examination like calculation of peripheralleucocytes. 6) Observation of the changes of peripheral basophils appears to be a useful laboratory aid for the diagnosis and therapeutic evaluation of bronchial asthma, making possible an early detection of the next attack. A discussion is given of the behavior of the basophils in bronchial asthma.

Human thyroid cancer cells in the pleural effusion were serially cultivated in vitro. Three kinds of cell lines were established from the same primary culture and were designated as PS, TS and TR lines, respectively. These three have been cultured for 574 days up to May 1, 1968. The cells of PS and TR lines were epithelial-like, whereas those of TS line revealed fibroblastic character. The chromosome numbers of PS and TR lines exhibited the modes near the hypertetraploid region, while TS line showed the mode of hypo-triploid number. Eosinophilic particles which were stained metachromatically by toluidine blue were present in the cytoplasm of these cells. The histochemical findings of the cells of each line were identical with those of thyroid cancer cells in vivo. The cells aggregated by the gyratory culture showed epithelial characters under microscopic observation of the sectioned specimens. The tumors produced in conditoned hamsters demonstrated undifferentiated cancer, which resembled the metastatic thyroid cancer of the patient. Neither collagen nor argentaffine fibers were detected with Van Gieson staining or silver impregenation.

The changes in muscle fibers after the crush injuries of the nerve were studied with rat sciatic nerve, and the following results were obtained. 1. After a severe crushing, the tendency of grouping of a single muscle fiber type was observed, although this scarcely occurred after slight injuries. 2. The muscle function and structure recovered better after crush injuries of the nerve than after the nerve reunification.

The hepatomas of the Donryu rats induced by feeding 4.dimethyl. aminoazobenzene for more than 191 days were transplanted into the brain of newborn rats of the same strain and the formed tumors were transplanted into the peritoneal cavity of adult rat of the same strain for the purpose to obtain transplantable strain of ascites hepatoma. As the result 4 lines of transplantable ascites hepatomas have been establised. The cells of these 4 hepatomas resembled their original liver tumor cells, respectively, showing the similar morphologic appearance to their mother cell. They showed less differentiated or more malignant characteristics in those taken from the tumor at the more advanced stages of DAB feeding. The liver tissues from the rat fed on DAB for 191 days had no tumor inducing activity when they were inoculated into the brains of the newborn rats (C 74). The liver tumors of the rats fed for more than 236 days produced the tumors in brain, which was serially transplantable (C 82), and kept the original morphologic pattern through serial transplantation and even in those growing in ascites. The tumor cells of the C 82 line showed the least malignancy among the 4 lines of ascites hepatoma established. Those of the C 83 line, which originated from the rat fed on DAB for 264 days, demonstrated the type of well.differentiated liver cell carcinoma with the trabecular arrangement of the tumor cells, but in ascites form they grew more rapidly than those of C 82. Those having most malignant characteristics were the cells of C 84.A which were derived from the rat fed on DAB for 312 days, and they were of the type of undifferentiated liver cell carcinoma. The island forming capacity of the C 84·A cells was the weakest among those of the 4 lines. C 84·B cells were also those derived from the same rat as that from which C 84.A originated and also showed the type of poorly differentiated liver cell carcinoma, but less malignant than those of C 84.A.

The disappearance of nucleolus has been traced in the rat erythroid cells in relation with the cell specialization under varying conditions, i. e. in anemia with or without treatment by bromouracil and aminopterin. To make the findings more reliable the observations have been made on tissue section as well as on the smeared samples as the nucleolus becomes often indistinct in smeared cell. The results indicate that under anemic condition nucleolus is lost by the late basoplilic stage. Treatment with bromouracil retained the nucleoli and cytoplasmic basophilicity till later stage of cell specialization suggesting some similar mechanism of RNA disintegration both in nucleolus and cytoplasm.

1. Dogs anesthetized with pentobarbital sodium were mainly used and effects of the distention of the small intestine on the movements of the gall bladder and the sphincter of Oddi were investigated. 2. The distention of the small intestine (jejunum or ileum) inhibited the rhythmic contraction of the gall bladder and duodenal movements, and relaxed the tone of the sphincter of Oddi, resulting in an increase of the outflow of fluid through the orifice of the common bile duct. 3. After cutting the bilateral thoracic splanchnic nerves together with extirpation of the bilateral upper lumbar sympathetic trunks, the inhibitory response on the movements of the gall bladder and the tone of the sphincter of Oddi was completely abolished. The vagus nerve did not take part in the reflex response described above. The transection of the spinal cord at the level between Thl and Th2 produced no change in the reflex responses. 4. Fwm the results described above it may be supposed that effects of the distention of the small intestine on the movements of the gall bladder and the sphincter of Oddi are produced via the thoracic and lumbar splanchnic nerves through the reflex center which is located in the spinal cord.

Adl2-induced tumors were homogenized and fractionated by the SCHNEIDER'S method. The CF test with the serum from tumor-bearing hamsters revealed the predominant presence of T-antigen in the mitochondrial and microsomal fractions. Hence, the rabbit was immunized with the microsomal fraction of tumors, and its serum was used to prepare the fluorescent antibody to T-antigen. The direct staining of the tumor cells with so prepared fluorescent antibody gave a staining pattern similar to the indirect staining with the serum of tumor-bearing hamsters. It thus appeares possible to stain T-antigen by the direct immunofluorescent method using the serum of rabbits hyperimmunized with the microsomal fraction of Ad12-induced tumors.

It is said blastformation can hardly be observed in the tissue culture of mouse lymphocytes. However, in our experiments of mouse lymphocytes (obtained either from axillary or cervical lymph nodes) mixed with various cells in combination of other cells as A+C3H, A+C57BL, or C3H+C57BL, it has been verified that these lymphocytes readily undergo blastformation in the presence of PHA (phytohemagglutinin M) as adjuvant. In the single tissue culture of these lymphocytes without PHA, the blastformation is observable in 6 per cent of the cells, while in the presence of PHA it is seen in 13. 7 per cent of the cells. In the cases of mixed cultures blastformation is observable in 14 per cent in the absence of PHA, whereas it is seen in 35.4 per cent in the presence of PHA. There is obviously a significant difference (p=O.OOI) in the blast. formation when cultured in the presence of PHA, and its reproducibility also proves to be quite high.

As the results of investigating the antigenicities of various fractions from the membrane systems of cancer cells, it has been found that the remarkable cancer-specific antigenicity exists in cancer cell mitochondria. With a particular reference to this antigenicity of cancer cell mitochondria, the antigenicities of the mitochondria of various kinds of rat ascites tumors and those of tumor-bearing rat liver mitochondria have been compared with those of normal rat liver mitochondria. In addition, it has been demonstrated that a strong tumor antitransplantability is induced when the recipient rat is immunized with the tumor cell mitochondria. In order to support these experimental facts, enzymatic activities of cancer cell mitochondria have been investigated also biochemically after treating the mitochondria with the antiserum to these mitochondria. 1. The most remarkable cancer specific antigenicity exists in mitochondria among the membrane systems of cancer cells. This cancer mitochondria-specific cancer antigenicity is common to all the ascites tumor mitochondria used here. 2. The original tissue- or organ-specific antigenicities diminish or disappear at the carcinogenic transformation of cells. 3. The tumor-bearing-organ specific antigenicity appears in the organs of animals bearing tumor. 4. The tumor antitransplantability is acquired when rats are immunized with these tumor cell mitochondria. 5. The inhibition of mitochondrial ATP·ase and respiratory activities is observed when the cancer cell mitochondria are treated with the anti. serum to the mitochondria.

With the purpose to elucidate further the properties of the supernatant F4 obtained by centrifugation at 100, 000 g from the regional lymph node cells of the Cb mice sensitized with EHRLICH ascites tumor cells, the supernatant (cf. Report 13) was subjected to the following treatments:. The supernatant (F4) was first diluted variously with Hanks solution. 2. F4 was passed through Seitz filter. 3. Heated at 56°C for 30 minutes. 4. It was frozen and thawed. 5. Treated with O. 01 96 trypsin solution. Each of F4 frations so treated was used in the tissue culture of JTC-II cells (derived from EHRLICH cancer cells) as target cells. As a result we found that the antitumor factor passes th rough Seitz filter, and it loses its antitumor activity by 4-fold dilution or over. Likewise F4 loses its activity by freezing-thawing treatment as well as by trypsin treatment, while by heat treatment at 56°C for 30 minutes, it still retains its activity. From these finding, it is assumed that the antitumor factor contained in F4, fraction is not serum antibody but is a protein associated with the cell membrane.

For the purpose of revealing whether the sensitivity of the erythropoiesis to actinomycin D (AMD) differs among different animal species, and to see the acting site of AMD on erythroid cell specialization stage, the author observed the hourly change of the blood cell counts and bone marrow cells after AMD administration to mice, rats and rabbits, and obtained the following results: 1. The data indicated that the erythropoiesis of ra bbit is sensitive to AMD, as much as that of mice, while the rat is resistant to AMD, and its erythropoiesis is not affected by the similar dose of AMD as in the case of mouse and rabbit. 2. The morphologic observations on the eradication process of erythroblasts in the bone marrow of mice and rabbits indicates that AMD acts as to inhibit the transformation of the stem cell to the proerythroblast but not on the erythroblast in the course of specialization. The time required for the eradication coincided with the time of the proerythroblast to the mature red cell. 3. Discussion has been made on the possibility of the common stem cell to erythroid and granulocytic cells in relation to the lymphoid cells in bone marrow and their blast form.

The following conclusions were drawn from the ferrokinetic studies using 59Fe in mice, whose hematological disorders were induced by various treatments. 1. The ferrokinetics in the normal mice were studied. 2. Chloramphenicol (CP) administration in mice first induced ferrokinetics disturbances and then suppressed erythropoiesis. 3. Splenectomy induced hyper-erythropoiesis in the bone marrow, and CP administration after splenectomy suppressed this hyper-erythropoiesis. 4. Human gamma-globulin (H.G.G.) caused hypersplenism and a marked suppression of erythropoiesis in the bone marrow, and Chlorabulin administration suppresed erythropoiesis. Finally, the author has summarized the relationship of the RES function and hematopoiesis in mice as follows. 1. The spleen and liver reacted in the same manner with respect to the RES function to sequestrate 51Cr-labelled heat-damaged erythrocytes when hematological failures were induced. 2. The spleen and bone marrow reacted reversely with regard to the RES function. 3. When the RES function, especially that of the spleen was accentuated, the suppression of hematopoiesis was observed. 4. Chloramphenicol administration was followed by the suppressed hematopoiesis and the accentuated RES function. 5. Splenectomy accentuated the RES function in the bone marrow and liver, and also increased hematopoiesis in the bone marrow. 6. Human γ-globulin hypersensitization induced hyperfunction of the RES, especially of the spleen and suppression of the hematopoiesis.

It has been demonstrated that by the mixed cultures in the presence of PHA the combination of those cells whose H-2 antigens differ from each other shows a higher rate and more significant difference of blastformation than in the combination where non-H-2 antigens differ (Table 1). The blastformation observable in the combinations where non-H-2 histocompatibility antigens or sex.linked antigens are weaker, is not, so marked as the difference seen of the blastformation in the case with H-2 isoantigens. This in vitro lymphocyte stimulation test can be applied to the histocompatibility test in the combinations of strong H-2 antigens.

For the purpose to reveal the correlation between molecular structure and biochemical functions of cytochrome oxidase the author studied purified cytochrome oxidase by using high resolution electron microscope and biochemical methods. 1. Cytochrome oxidase was purified from the cytochrome oxidase-rich submitochondrial membrane (green membrane), obtained from beef heart mitochondria, by three different methods; modification of the method of OKUNUKI et ai., method of FOWLER et ai. and modification of the method ofJACOBS et ai. All the preparations showed a high specific activity under appropriate conditions and consisted mainly of small particles measuring approximately 80 to 90 A. in diameter. 2. The particle, measuring approximately 80 to 90 A. in diameter, took a cylindrical form measuring about 70 A. in diameter at the base and 95 A. in height in an appropriate condition. Many experimental results indicate that the particle is the smallest, fundamental unit of the active cytochrome oxidase. For this reason it was designated as the unit particle of cytochrome oxidase (abbreviated as UPCO). 3. The molecular weight of the unit particle, calculated from its volume and average density (1.24) of lipoproteins (3: 7), was about 270,000. The value was roughly twice the minimum molecular weight of 128, 000 calculated from the heme a content. Accordingly, it is considered that the unit particle contains two heme a molecule and two copper atoms. 4. It was suggested electron microscopically that the particle collected in the 22.6 S position by sucrose gradient ultracentrifugal analysis was a dimer of the unit particle of cytochrome oxidase and also that the particle collected in the 5. 7 S position was a half of the unit particle of cytochrome oxidase. 5. It was also suggested that the particle observed on the green membrane was a subunit of cytochrome oxidase, containing one heme a and one copper atom, and the unit particle of cytochrome oxidase was constituted of two of the particles observed on the green membrane. Namely, the results indicate that the molecular state of cytochrome oxidase on the green membrane apparently differs from that of the purified cytochrome oxidase.

In order to elucidate the molecular organization of mitochondrial inner membrane, biochemical and electron microscope observations were made on the formation of membrane structure and function by the purified complexes of the electron transfer chain of beef heart mitochondria. Purified complex III (CoQ-cytochrome c reductase) and complex IV (cytochrome oxidase) were soluble in the presence of bile salts. They were, however, aggregated to form membrane by washing out the bile salts. When the membranous complexes III and IV were mixed, both membranes were separate by density gradient centrifugation and the vesicle which contained both complexes could not be formed and CoQH2-oxidase activity was hardly re;tored. When the mixture of the solubilized complexes III and IV were diluted to remove the bile salts, a membranous vesicle in which both complexes were assembled was formed. CoQH2-oxidase activity was restored in accordance with the formation of the membrane. The membrane which contained any desired propotion of each complexes could be obtained. These facts indicate that the complexes of the electron transfer chain conjugate two-dimentionally each other and form the membrane to carry electrons from substrate to oxygen most efficiently.

Effect of ATP and substrates on 2,4-dinitrophenol-induced adenosine triphcsphatase (E. C. 3.6. 1. 4.) activity and respiration of isolated rat liver mitochondria has been investigated. 1. The oxidation of sodium succinate inhibited the action of 2, 4-DNP on the induction of adenosine triphosphatase activity in the mitochondria. 2. A moderately large amount of sodium succinate restored the suppressed mitochondrial respiration due to 2, 4-DNP. 3. Adenosine-5'-triphosphate (ATP) restored quantitatively the released and inhibited mitochondrial respiration due to 2,4-DNP, and its prior addition prevented also quantitatively the action of 2,4-DNP on the mitochondrial oxygen up-take. These ATP effects were oligomycin sensitive, and they were considered to manifest their actions through the phosphorylation system.

1. The cells used in the present experiments were lymph-node cells from inbred mice, and over 98 % cells were proven to be small lympho-cytes. Therefore, those cells that have undergone blastformation are all those derived from small lymphocytes. 2. When homogenate of one cell group is cultured with live cells of the other pairing group, there occurs blastformation. In the presence of PHA, such a blastformation becomes more marked. 3. The optimal concentration of PHA (phytohemagglutinin)-M added to the mixed culture is found to be 1% (v/v). 4. The maximum rate of blastformation in the mixed culture is observed at the culture hour 48, being much faster than in the mixed culture between two live cell groups. 5. In the mixed cultures between subcellular fractions prepared from cell homogenate by centrifugation and live cells, the transplantation antigenic potential (histocompatibility antigenic potential) is seen in the mitochondrial and the microsomal fractions, especially marked in the latter. 6. In the observations carried out by various combinations of these inbred mice, it has been demonstrated that the rate of blastformation induced by the addition of cell homogenate or sediment fractions prepared from the homogenate reflects quite accurately the differences in H-2 antigens.

The author has described modern thrombolytic therapy of arterial and venous thrombosis and emboli by therapeutic fibrinolysis and other drugs also methods and effects of local and parenteral application of fibrinolysin preparations, dosage, control, indications. Contraindications, side-effects and their treatment with fibrinolysin antagonists and therapy with fibrinolysin combined with anticoagulants and antibiotics are discussed.