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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.

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.

<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.

For the purpose to clarify further the residual catalase in the blood cell ghost, the ghost has been applied to Cyanogum and starch block electrophoresis and the results are briefly summarized as follows. 1. It has been demonstrated that after Cyanogum electrophoresis of the ghost after several washings, bubbling due to enzymatic reaction of catalase occurs near the points of origin, when the plate is immersed in hydrogen peroxide solution and also it has been proved the presence of catalase so firmly bound to the ghost that it is hardly moved by the electro phoresis. Even with the ghost of hypocatlasemia there can be detected catalase which is likewise hardly eluted from the ghost. 2. In the estimation of catalase activities of each fraction from red cell ghost by starch block electrophoresis there can be detected catalase near the point of origin, that is not eluted by the electrophoresis, and the activity of which corresponds to about 0.1 % of the total red cell catalase activity.

Compound 48/80, sinomenine, tween 20 and polyvinylpyrrolidone (PVP) were injected intravenously to dogs, in doses producing similar degree of profound hypotension, and changes in the plasma histamine content and coagulation time were followed on the blood from the femoral artery. After the injection of 48/80 or sinomenine plasma histamine rose rapidly and markedly, attaining its maximum within 2 minutes, but the increase was rather of a short duration. In contrast, after the injection of tween 20 or PVP a less marked increase in plasma histamine developed more slowly, but lasted longer. The blood coagulation time was prolonged in all the cases injected with 48/80, and occasionally with sinomenine. Both beginning and recovery of the prolongation of blood coagulation time were sluggish as compared with the changes of plasma histamine. Tween 20 and PVP did not induce any detectable change of the blood coagulation time. These data were discussed with reference to the sites of action of different histamine releasers.

1. For the settlement of carbon origin of urinary isovalthine, acetic acid-2-C14, valine-U-C14 or leucine-U-C14 was administered to rats together with isovaleric acid as an isovalthinuria inducer, and urinary isovalthine excreted was tested by autoradiography. As the results of which, it was found that these isotopic compounds were not the precursor of urinary isovalthine. Although the isovalthinuria inducing effect of isovaleric acid was fairly diminished by these isotopic compounds, urinary isovalthine was detected by paper electrophoresis. 2. Some metabolic products of these isotopic compounds were also inquired in urine and some tissues. The results were as follows: a) Acetic acid incorporated into urea, aspartate, serine, glutamate, proline, glycine, alanine, ornithine, ethanolamine, r-amino-buthyric acid (brain only), cholesterol and fatty acids. b) Valine incorporated into urinary glutamate and glycine, and tissue cholesterol and fatty acids. Valine was rapidly excreted in urine and found in a very small amount in liver digest. c) Leucine incorporated into urinary aspartate, serine, glutamate and glycine, and tissue cholesterol and fatty acids. 3. Several important problems of isovalthine studies to be elucidated were discussed.

Cb strain female mice were exposed to 800 p.p.m. of carbon tetrachloride for 3 hours by the use of newly devised gas chamber via constant current of gas. Contents of ATP, triglyceride and total lipid in the liver were measured at appropriate intervals after inhalation of carbon tetrachloride and compared to non-treated controls. And P : 0 ratio of the liver mitochondria was measured by oxymeter and morphological changes of liver mitochondria were observed by electron microscopy. The following results were obtained. 1. ATP conten t in the liver decreased slightly immediately after inhalation, rapidly decreased until 4 hours after inhalation and gradually decreased until 20 hours after inhalation. 2. Contents of total lipids increased slightly immediately after the exposure and increased gradually until 20 hours later. Contents of triglyceride in the liver increased at almost constant rate during and after the exposure. 3. P : 0 ratio of liver mitochondria did not change immediately after the exposure and gradually increased after the exposure, keeping parallel relation to decrease in ATP content in the liver. Decrease in ATP content in the liver after inhalation of carbon tetrachloride seems to be mainly due to uncoupling of oxidative phosphorylation of liver mitochondria. 4. Morphological changes of liver mitochondria were observed at 4 hours after the exposure by electron microscopy. 5. Decrease in ATP levels of the liver suggested to have a close relation to accumulation of lipid in the liver after the inhalation of carbon tetrachloride.

As a step towards the elimination of Japanese encephalitis virus in natural surroundings, we inoculated pigs, rabbits and chicks with inactivated Japanese encephalitis vaccine supplemented with complete or incomplete Freund's adjuvant twice at one-week interval. Subsequently, we compared HI antibody titers of the groups inoculated with vaccine containing complete Freund's adjuvant (pigs, rabbits, chicks), of the group inoculated with vaccine containing incomplete adjuvant (rabbits), ar;d of the groups inoculated with vaccine containing no adjuvant (pigs, rabbits, chicks), and also observations on changes in the antibody titers due to natural infection. In a certain portion of these animals neutralizing antibody titers were also determined. The results of this study are briefly summarized as follows. 1. In the groups of pigs and rabbits inoculated with vaccine containing complete Freund's adjuvant, titers of HI antibody and neutralizing antibody were higher than those inoculated with vaccine containing no adjuvant and their high titers persisted. Further, in the group of chicks inoculated with inactivated Japanese encephalitis vaccine containing complete Freund's adjuvant, HI antibody titers were higher and persistent as compared with the antibody titers in the chicks inoculated with inactivated Japanese encephalitis vaccine alone. 2. In the rabbits inoculated with inactivated Japanese encephalitis vaccine contammg incomplete adjuvant, HI antibody titers were lower than in those receiving the vaccine with complete adjuvant, but it has been demonstrated clearly that vaccination of inactivated Japanese encephalitis vaccine supplemented with incomplete adjuvant brings about less sideeffects. Hence such a method of vaccination can be applied as the vaccination with least side-effects. 3. With respect to natural infection of swine, on August 27 when the pigs were thought to have been infected, there was observed a rise in antibody titers. And on being infected with Japanese encephalitis, the antibodies formed in those pigs inoculated with inactivated Japanese ence- phalitis vaccine with or without complete adjuvant proved to be all 2-ME resistant type, whereas the antibodies produced in the control groups not receiving such a vaccination were 2-ME sensitive antibody.

The amygdalofugal fibers were studied III the cat with the silver method of NAUTA-GYGAX. 1. The amygdalofugal fibers are distributed by way of the stria terminalis, the longitudinal association bundle, the inferior thalamic peduncle, and the medial forebrain bundle. 2. The amygdalofugal fibers running through the longitudinal association bundle arise in the lateral principal, intermediate principal nuclei and the lateral and possibly intermediate parts of the periamygdaloid cortex, and terminate in the lateral preoptic nucleus, the bed nucleus of the anterior commissure, the olfactory tubercle, the nucleus of the diagonal band of Broca, the nucleus accumbens, the medial and posterior septal nuclei and the basal part of the head of the caudate nucleus. In addition, there are scattered fibers coursing along the longitudinal association bundle proper. These fibers may have a widespread origin from the amygdaloid complex. The longitudinal association bundle contributes no fibers to the medial forebrain bundle. 3. The fibers, originating from the lateral principal, intermediate principal and medial principal nuclei, join the medial forebrain bundle to distribute widely in the lateral hypothalamic nucleus. A few fibers are seen to reach the ventromedial hypothalamic nucleus, and are considered to arise in the medial principal nucleus. 4. By way of the inferior thalamic peduncle some fibers from the amygdaloid complex course dorsally into the medial part of the dorsomedial thalamic nucleus at its caudal levels. They may arise widely from the amygdaloid complex. A few of them extend farther dorsally to reach the lateral habenular nucleus and the parataenial nucleus. They probably originate from the lateral principal nucleus. 5. The fibers forming the stria terminalis originate from the medial principal nucleus, the medial nucleus, the periamygdaloid cortex and the cortical nucleus, and are distributed in the bed nucleus of the stria terminalis and the lateral preoptic nucleus (preoptic component), as well as the medial preoptic nucleus, the anterior hypothalamic nucleus and the ventromedial hypothalamic nucleus (supracommissural component). The cortical nucleus, particularly its caudal part, and possibly the medial part of the periamygdaloid cortex are regarded as the main sources of the stria terminalis fibers ending in the hypothalamic region. The intermediate principal and lateral principal nuclei do not appear to contribute fibers to the stria terminalis. 6. The ventromedial hypothalamic nucleus receives amygdalofugal fibers both from the medial principal nucleus by way of the medial forebrain bundle, and from the cortical nucleus via the stria terminalis. 7. In addition to intrinsic internuclear fibers within the amygdaloid complex, some of the fibers from the complex are distributed to the ventralmost part of the putamen, the medial part of the claustrum, the periamygdaloid cortex, the prepiriform area and the anterior amygdaloid area, but do not reach the hippocampus.

1. For the purpose to obtain parabiotic rats having well maintained humoral circulation, the author observed parabionts having coerio-anastomosis and vascular anastomosis. 2. In the parabiotic rats having coerio-auastomosis when one of the parabionts was prevented from taking food and water by mouth sealing, the animals died within 5 to 6 days just as the control animals subjected to complete starvation, indicating that in coerio-anastomosis no appreciable humoral exchange was established between the two parabionts. 3. In vascular parabiosis having cross anastomosis of the aortas with polyethylene tubules, the animals died about 24 hours after the operation because of blocking thrombosis formed in the polyethylene tubules. 4. In the vascular parabiosis having cross anastomosis of the aortas by the homologous thoracic aorta animals did not survive through the operation but those having parallel anastomosis of the aortas survived after the operation for 3 weeks at largest and they seem to serve as useful tool for the parabiosis experiment.

In vitro and in vivo experiments were conducted for the purpose to determine whether or not the antitumor factor found in the regional lymph node cells of the mouse sensitized with Ehrlich tumor cells would transfer its antitumor activity to normal lymph node cells. In in vivo experiments normal lymph node cells incubated at 5°C for 60 minutes in the supernatant containing the antitumor activity have shown the antitumor activity against JTC-11 cells in mixed culture. Namely, it hs been demonstrated that the antitumor activity in the supernatant can be transferred directly to normal lymph node cells in vitro. In the in vitro experiments, the same results as in in vivo experiments were obtained. The antitumor activity against JTC-11 cells has been detected in the lymph node cells obtained on 4, 7 and 9 days after subcutaneous and intraperitoneal injections of the supernatant having antitumor activity. Next, we tried DNase and RNase treatments of the sensitized supernant to observe the transfer factor-like snbstance. The results indicate that, while the passive transfer is possible with the supernatant treated with DNase, it is not with the RNase-treated supernatant. From these findings it is assumed that the factor (in the sensitized supernatant) capable of conferring the antitumor activity is an RNA-dependent substance (or a substance closely associated with RNA) and is probably different from the antitumor factor reported in Parts 1 and 2.

For the purpose to clarify the mechanism of phagocytosis or pinocytosis, the observations on the tumor ascites, including the macrophages as well as the tumor cells, were carried out by incubating with the iron colloid with or without pretreatment by several inhibibitors of glycolysis, oxidative phosphorylation and respiration, or under hypotonic or cold environments. The results have demonstrated that there are three steps in the phagocytosis. The first step is the adhesion of the substance to the cell surface, which is not an energy-requiring process. The second step is the engulfing which proceeds by using the energy supplied by glycolysis. The third is the accumulation of the substance into the vesicles through the canaliculi connecting the cell surface with the vesicles. The discussion was made on the existence of the active site on the cell surface to which the substance can be adhered, and the accumulation mechanism of the material into the phagocytic vesicles by the membrane flow, the flowing movement of the outer lipid layer of a unit membrane through the canaliculi which connect the cell surface to the phagocytic vesicles.

The liver cells obtained from a calf have been cultured continuously for 257 days in total at present (May 31, 1967). The primary culture was maintained in rotatory culture for about 2 months with gradual and continuous cell proliferation. The two original strains, LD-BS20 and LD-CS20, have been maintained in static culture since 4th subcultivation. Three substrains, LD-BS10, YLE-BS20 and LD-CS10, were derived from the original strains. Two kinds of appropriate media, in which the cells could be subcultured with trypsin without severe damages and maintained with some characteristic functions of liver cells, were reported. The one consisted of 20 per cent bovine serum, 0.4 per cent lactalbumin hydrolysate and saline D, and the other was added with 0.08 per cent yeast extract to the above mentioned medium. Calf serum examined was not so effective as bovine serum for cell proliferation. Morphologically, the cultured cells resembled parenchymal liver cells quite well. The cells spread wide with abundant pale staining cytoplasm and their large nuclei, oval or round, generally contained one to several nucleoli. The cells as well as the nuclei varied considerably in size, some being two to four times larger than others. Binuclear, trinuclear or polynuclear cells were also observed. No silver impregnated fiber was detected among the epithelial cells. Two attempts to characterize cell types in culture were made. First, the presence of glycogen was tested with PAS reaction and saliva digestion procedure. Secondly, the albumin formation in cultured liver cells was examined with the fluorescent antibody technique. The fact that both albumin and glycogen were observed in the cells suggests strongly that there is a possibility of the continuous cultivation of liver cells by the present method, and by these procedures it seems possible to identify functionally the cultured cells with the parenchymal liver cells.

The growth of JTC-11 cell line which was established from Ehrlich ascites carcinoma in vitro was inhibited by the addition of 2 per cent guinea pig serum to the control medium composed of 10 per cent bovine serum, 0.4 per cent lactalbumin hydrolysate and saline D. The concentration of guinea pig serum could be reduced to 0.02 per cent (lOγ of guinea pig serum protein/ml) with positive result, but 0.002 per cent guinea pig serum did not inhibit the growth at all. The inhibitory effect was not abolished by heating at 56°C, 66°C, and 70°C for 30 min but it was completely lost by heating at 100°C for 30 min. The inhibitory factor was undialyzable, and was inactivated with the treatment of 1mM HgCl2- Morphologically, the cells exposed to guinea pig serum showed pycnotic changes of the nuclei, accompanied by the formation of fine vacuole-like particles in the cytoplasm. Electron microscopic study revealed poor development of endoplasmic reticulum. There were more multivesicular bodies and large vacuoles with amorphous content in the cytoplasm of the damaged cells. The DNA synthesis in these cells was remarkably disturbed by 2 per cent guinea pig serum.

A case of arteriosclerotic retinopathy associated with retinal venous thrombosis was treated with Anginin and the following results obtained: 1) Visual acuity was improved from 0.03 to 0.7. 2) Retinal hemorrhages were absorbed and pipe-stem sheathing of the branch of retinal artery decrease, with white sheathing remaining partially. 3) It was therefore considered that the pipe-stem sheathing was decreased because Anginin removed venous spasm and improved the blood stream of the branch of the artery, and that the organic changes already established on the arterial wall would remain as white sheathing. 4) Anginin could not prevent retinal veins from changing into white lines. 5) Consequently the authors considered that Anginin may be a drug effectively used for retinal arteriosclerosis and retinal venous thrombosis associated therewith.

1. An oligomycin -sensitive ATPase was isolated and partially purified from beef heart mitochondria. The specific activity of ATPase sensitive to oligomycin of the fraction was five to eight times that of aged mitochondrial or of DNP-induced mitochondrial ATPase assayed under the same condition. 2. Electron micrographs of the partially purified oligomycin- sensitive ATPase reveal a structure in which headpieces are regularly attached by way of stalks to a thread-like structure derived from a superficial portion of base pieces. 3. A high concentration of the structured material coincided with a high activity of oligomycin-sensitive ATPase. When the headpieces were detached from the structure, the ATPase became insensitive to oligomycin. 4. The fraction of oligomycin -sensitive ATPase was essentially free of membrane structure and was contaminated with a small amount of cytochromes b and Cl but no cyt. a. Cytochrome concentrations of the preparations were indifferent to the activity of oligomycin sensitive ATPase. It follows that ATPase does not require cytochromes or membrane structure for its oligomycin sensitivity. 5. From these results it seems that the factor rendering ATPase sensitive to oligomycin should be contained in the stalks and/or the thread-like portion of basepieces of the structure. The structure is the simplest unit of oligomycinsensitive ATPase as yet obtained. 6. The structure was called "oligomycin-sensitive ATPase particles" (abbreviated as OSA particles). A unit of OSA particles consists of a headpiece attached by a stalk to a portion of base piece.

The results of our study may briefly be summarized as follows: 1) The irradiation with microrays (20∼30 watts) similar as 2,000 R and 5,000 R Gamma radiation did not substantially affect the activity of fibrinolysin (SK+SD). 2) By the irradiation method so far mentioned it has been demonstrated that the fibrinolytic activity of anticoagulant of the SK+SD preparation is preserved in all the clotting systems which we used. 3) Our findings indicate that it is possible to irradiate patients for therapeutical purpose with Radarmed (electromagneticrays) provided that there is produced some enhancing influence of the same blood clotting factors or systems. Together with earlier works in this field it appears that this method of the microirradiation could provide us with an important evidence on which we can base our further in vitro and in vivo radiohematologic studies; investigations with various preparations, types of radiation that are still underway9∼16.

For the purpose to know whether the annual increase of leukemia incidence in Japan is due to some leukemogenic factors or due to the increased detection rate, the authors made some statistical survey of autopsy cases in which the diagnosis is reliable and not any type of leukemias escape the detection. The results showed that acute leukemias, which are found mostly in younger age, is actually increasing. In addition, it has been deduced that among the suspected factors the increase in ionizing radiation will be one of the most probable factors for the increase in leukemia incidence

1. In the absorption spectra of crude catalase solution (Stages 2, 3, and 5) of normal blood, three absorption bands characterizing catalase molecules are recognized. 2. The three absorption bands specific for catalase cannot be found in acatalasemic blood extracts (Stages 2 and 3). 3. It is inferred that catalase is not present in the crude catalase extract from acatalasemic red blood cells.