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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.

1. A respiratory-deficient mutant strain of yeast was obtained from wild strain of Saccharomyces servisiae by treatment with 4-nitroquinoline N-oxide. Ultrastructure and function of the wild or mutant strains and the mitochondrial fractions isolated from these strains were examined by biochemical and electron microscopic analyses. 2. The frequency of the respiratory-deficient mutant strain in yeast induced with 10-6M 4-nitroquinoline N-oxide was about 40 %. 3. Respiratory-deficient mutant strain is incapable of reducing 2, 3, 5-triphenyltetrazolium chloride salt and to grow on lactate medium. In addition to this, the mutant has been found to have lost its ability to take up oxygen in sodium succinate and pyruvate. 4. 4.Nitroquinoline N-oxide in the concentration that induces a mutant of yeast cells or its kin inhibits the oxygen uptake in normal strain. 5. The normal strain of yeast is characterized by difference spectrum corresponding to cytochromes a+as, band c+Cll respectively, whereas, the mutant strain containes almost no cytochromes a+ as, band C1 but contains normal or increased amount of cytochrome c. 6. Mitochondrial fraction isolated from mutant strain has largely lost its ability to oxidize succinate. On the other hand, NADH-, lactate-and cytochrome c-oxidase activities are reduced by about 1/17, 1/7 and 1/8 of that of normal strain, respectively. 7. Succinate dehydrogenase activity of mutant strain is almost zero. Moreover, this activity is not affected on the addition of phenazine methosulfate. NADH dehydrogenase activity of mutant stran is about 1/2 of normal strain. 8. The variations in mitochondrial structure of normal and mutant strain in the stationary phase have been followed with the aid of electron microscopy. In contrast to the normal strain, the mutant strain revealed distinct morphological changes in mitochondria, especially, the lack of cristae in its interior. The results have been interpreted to indicate that the mutant induced by 4.nitroquinoline N.oxide has a character of cyto. plasmic mutant.

For the purpose of revealing whether or not hemoglobin synthesis is inhibited by the AMD, the author estimated the hemoglobin level of AMD treated anilmals by microspectrophotometer, and found that the hemoglobin levels of all the developmental stages of erythroid cells were not inhibited by the AMD. The data indicated that about one half of mRNA for hemoglobin is synthesized in the early stage of specialization with the supplementary synthesis at the later stages and all these mRNA is stable and insensitive to AMD.

For the purpose to confirm the existence of the stem cells of the myeloid and erythroid cells in the circulating blood and to have the information of its morphologic entity, the author conducted morphologic observations on the peripheral and bone marrow cells of the x-irradiated parabionts having non-irradiated partners joined by the vascular parabiosis devised by the author, and the following results were obtained. 1. The control rats exposed to 1000R x-ray did not show any sign of recovery of hemopoiesis in bone marrow even 8 days after irradiation. 2. In the bone marrow and spleen of the lethally irradiated animals having non-irradiated partner in parabionts, precursor cells of granulocyte and erythrocyte appeared first 4 days after conjugation irrespective of the days after irradiation, and the hemopoiesis was restored completely on the sixth to the seventh day. The results have indicated that the circulat. ing blood has the stem cells which can dedifferentiate and transform into the precursor cells of the myeloid and erythroid cells within 3 days under adequate conditions. 3. On the basis of the morphologic observations on the peripheral blood of the parabiosis and non-irradiated rats revealded non-specific cells, a discussion was made on the possibility that some atypical lymphoid cells can serve as the stem cells of the myeloid and erythroid cells.

The composition of total lipid extracted with chloroform-methanol from the AV12-induced tumor was investigated by thin-layer and paper chromatography. The content of lecitin and sphingomyelin was somewhat decreased and a cerebroside was characteristically detected in this tumor.

Correlation of molecular structure with biochemical functions of the plasma membrane of the microvilli of intestinal epithelial cells has been investigated by biochemical and electron microscopic procedures. Repeating particles, measuring approximately 60 Åin diameter, were found on the surface of the microvilli membrane which had been isolated or purified from rabbit intestinal epithelial cells and negatively stained with phosphotungstic acid. These particles were proved to be inherent components of the microvillus membrane, attached to the outer surface of its trilaminar structure, and were designated as the elementary particles of the microvilli of intestinal epithelial cells. Biochemical and electron microscopic identification of these elementary particles has been carried out by isolation of the elementary particles with papain from the isolated microvillus membrane, followed by purification of the particles by chromatographies on DEAE-cellulose and Sephadex columns. The partially purified particles containing invertase and leucine aminopeptidase are similar in size and structure to those of the elementary particles in the microvillus membrane. Evidence indicates that each of the elementary particles coincide with or include an enzyme molecule such as disaccharidase or peptidase, which carry out the terminal hydrolytic digestion of carbohydrates and proteins, respectively, on the surface of the microvillus membrane. Magnesium ionactivated adenosine triphosphatase and alkaline phosphatase cannot be solubilized with papain but remains in the smooth-surface membrane after the elementary particles have been removed. Cytochemical electron microscopic observation revealed that the active site of magnesium ion-activated adenosine triphosphatase is localized predominantly in the inner surface of the trilaminar structure of the microvillus membrane.

We fractionated the red cells of Japanese acatalasemic individuals (four individuals in two families) by SAss's method and counted the number of reticulocytes and determined the catalatic activity by manometric and perborate methods on each fraction containing reticulocytes in the descending order from the upper to the lower layers. We also incubated each of these fractions at 37°C for 4, 10,24 and 48 hours, to see the catalatic activity along with the maturation of reticulocytes. Heat stability of catalatic fraction separated by DEAE column was also examined. The results of the study may briefly be summarized as follows. 1. a. There was no parallel relationship between the number of reticulocytes and the catalatic activity in Japanese acatalasemic red cells. b. There could be seen no decrease in catalatic activity while the number of reticulocytes did decrease by the incubation. 2. Heat stability of crude catalatic fraction from acatalasemic blood is approximately the same as that of crude catalase fraction from normal blood.