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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.

An attempt was made to find out the nature of catalase coritained in the red cell, especially in the ghost, For this the red cell ghost isolated were washed several times with CO2-saturated water or deionized water and the catalase activity per gram protein of the ghost was estimated. It was found that despite several washings, the catalase activity/gram protein of the ghost do not decrease as compared with the activity of the original red cell solution, indicating the presence of catalase in the ghost. In the case of hypocatalasemic blood the catalase activity in the ghost shows similar behaviors as with normal blood cells. It is assumed theoretically that there are two kinds of catalase having different affinity to the red cell ghost. Namely, one that is readily released from the ghost and the other that has a strong affinity. The affinity of hypocatalasemic blood to the ghost seems to be somewhat weaker.

The following conclusions were drawn from the above data concerning the RES function to sequestrate 51Cr-labelled heat-damaged iso-erythrocytes in mice. (l) When the hematological disorders of mice were induced, the RES of the liver and spleen reacted in the same manner. (2) The RES function of the bone marrow and liver were observed to react reversely except in the case of splenectomized mice. (3) Human gamma globulin hypersensitization and chloramphenicol administrations suppressed RES function of the bone marrow and augmented that of the liver and spleen. (4) The RES function of the bone marrow was activated after splenectomy. (5) The massive human gumma globulin administration was followed by the increased RES function of the bone marrow and by the suppressed one of the liver and spleen.

1. A cytochrome oxidase-rich submitochondrial membrane (green membrane) was obtained from beef heart mitochondria after extraction of flavoproteins, cytochrome b, Cll C, etc. by treating with deoxycholate and potassium chloride. 2. The green membrane was formed by self assembly from the membrane fragments (flat sheets), which derived from the cristae membrane of mitochondria and had essentially the same particulate structure as the green membrane. 3. The green membrane exhibited regular arrays of small particles on the surface, measuring approximately 50 to 60 A in diameter with center to center distance of about 70 A. These particles sometime were arranged in a woven structure on the surface. 4. Both the configuration of the particles and the regularity of the arrangement were influenced by detergents and temperature. 5. Green membranes as well as beef heart mitochondria and electron transfer particles commonly retained membrane-structure after sonication and exhibited higher specific activity of cytochrome oxidase than that of purified cytochrome oxidase, if the activity is calculated on the basis of heme a concentration (sec1 / 10 m,lJ.moles of heme a/3 ml). The results suggest that the active sites of cytochrome oxidase are arranged on the surface of these membranes. 6. From these results and other experimental findings, an intimate correlation between cytochrome oxidase and the particles observed on the green membranes is suggested.

<P>The mitochondrial, the microsomal, and the supernatant fractions were prepared from the cell homogenate of tumors induced by viruses, such as adenovirus type 12, SV 40, and Rous sarcoma virus, etc. and the antigenicities of these fractions were investigated. In the virus-induced tumors, there existed no antigenicity common to the mitochondrial and the microsomal fractions as in the tumors induced by chemical carcinogens, and the highest antigenicity was recognized in the mitochondrial fraction. Therefore, the properties of the tumor cell mitochondria were precisely investigated with virus-induced tumor mitochondria. 1. The mitochondria of tumors induced by viruses have clearly the specific antigenicity. 2. This specific antigenicity of virus.induced tumor mitochondria IS common to all the virus-induced tumors used in the present study. 3. This tumor mitochondria.specific antigenicity is found commonly in all the tumor mitochondria in the present experiments. 4. The specific cancer antigenicity of tumor cell mitochondria does not exist in normal organ mitochondria, but the regenerating organ mitochondria exhibit a slight antigenicity common to cancer cell mitochondria.

The regional lymph node cells of the mice sensitized with Ehrlich ascites tumor cells is known to possess a substance that shows antitumor activity on target cells (JTC-II cells). For the purpose to clarify the localization of this substance the regional lymph node cells from such sensitized mice were treated with trypsin solution of different concentrations (1.0 %, 0.2 %, and 0.01 %), and the tissue culture was carried out with JTC.II cells. As a result it was found that these lymph node cells lost antitumor activity. Next, by the differential contrifugation of these sensitized lymphocytes we obtained F1 fraction (700 g, sediment), F2 (8,500 g sediment), F3 (100,000 g sediment) and F4 (100,000 g supernatant). In the presence of each of these fractions tissue culture was conducted with JTC-II cells as target cells, and it was found that the substance with antitumor activity is contained abundantly in F2 fraction (8,500 g sediment) and F4 fraction (100,000 g supernatant). After giving due consideration to the results of these two experiments and also to the available data in the literature, we assume that the substance with antitumor activity is contained in the cell membrane component.

In the mixed tissue culture of mouse lymphocytes with addition of PHA the rate of the appearance of large and intermediate cells increases markedly, but which side of the two cell groups have reacted stronger remains obscure. In order to solve this problem, mixed cultures were conducted in such a way that only one cell group of the two would react. Namely, one cell group was exposed to C0 6).irradiation (Table 2) prior to the culture and cultured with another viable cell group (FJ test group, Table 2) to see the percentage of the appearance of large and intermediate cells. Simultaneously, the skin homograft from respective donor mouse was transplanted to each other and the survival days of each skin graft were compared. As a result it has been shown that the percentage of blastformation and the survival time of the skin transplant in each group prove to be in an inverse relation. The results of these mixed cultures indicate that the extent of blast. formation reflects significantly the difference in B-2 histocompatibility antigens.

In this review of the chemistry, biochemistry and chemical pathology of the bile pigments I am well aware that I shall ask many more questions than I am able to answer. It seems appropriate that the subject should be considered under the five headings: -Chemistry; Formation from Haem Proteins; Transport in the Blood; Conjugation and Transport to the Bile; and Changes in the Gut. I shall conclude with a brief account of the early labelled bilirubin. Because investigation of the pathological significance of the chemical changes undergone in the body is possible only if the chemical structures of the bile pigments are accurately known, my department in London has been very much concerned during the last 15 years with the chemistry of the bile pigments. The work I shall describe has been carried out in collaboration with Dr. NICHOLSON, Dr. KULCZYCKA, Dr. COLE, Dr. PETRYKA and more recently by Mr. STOLL and Miss LEMMON.