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It is well known that eosinophils are involved in tyrosine nitration. In this study, we evaluated tyrosine nitration by rat eosinophils isolated from peritoneal fl uid and constituent eosinophils in the stomach. Rat peritoneal eosinophils activated with 1 μM phorbol myristate acetate (PMA) and 50 μM NO2 ﾝ showed immunostaining for nitrotyrosine only in smaller cells, despite the fact that eosinophils are capable of producing superoxide (O2·ﾝ). Free tyrosine nitrating capacity after incubation with PMA and NO2 ﾝ was 4-fold higher in eosinophils than in neutrophils. Catalase and ｸ- and ｺ -tocopherol inhibited free tyrosine nitration by reactive nitrogen species from eosinophils but not that by peroxynitrite. Superoxide dismutase augmented free tyrosine nitration by activated eosinophils and peroxynitrite. The concentration of nitric oxide released from eosinophils was relatively low (0.32 μM/106 cells/h) and did not contribute to the formation of nitrotyrosine. On the other hand, most constituent eosinophils constituent in the rat stomach stimulated by PMA and NO2 ﾝ showed tyrosine nitration capacity. These results suggest that intact cells other than apoptotic-like eosinophils eluted in the intraperitoneal cavity could not generate reactive species responsible for nitration by a peroxidase-dependent mechanism. In contrast, normal eosinophils in the stomach were capable of nitration, suggesting that the characteristics of eosinophils in gastric mucosa are diff erent from those eluted in the peritoneal cavity.

Anthrax is essentially a disease of grazing herbivorous animals. The most common form of the disease is cutaneous anthrax, which accounts for 95% of all cases. We report here 39 cutaneous anthrax cases in humans that were seen in Eastern Anatolia over a six-year period. The clinical presentation was malignant edema in 16 of the cases (41%) and malignant pustule in 23 (59%). A secondary bacterial infection was present in 13 patients (33.3%) in the vicinity of the lesions. The agent was observed using Gram-stained smears in 25 patients (64%), and Bacillus anthracis was isolated from 15 patients (38.5%). All of the patients were treated with penicillin G or penicillin procaine, except one patient who had a penicillin allergy. One patient with cervical edema (2.5%) died as a result of laryngeal edema and sepsis syndrome. In conclusion, we found that the appearance of the skin lesion of cutaneous anthrax may vary, and this fact, combined with the rarity of this disease, which contributes to a general lack of experience among medical personnel, may make diagnosis difficult in nonagricultural settings

Healthy subjects 40 years old were used as controls in a study of stellate cells (S-100 protein-containing cells, or S-100 cells) in subjects with chronic alcoholism and fatty liver or fatty cirrhosis. S-100 cells were sparsely found in the adenohypophysis of control subjects, and these cells sometimes formed small clusters. However, in chronic alcoholics with fatty liver or fatty cirrhosis, the number of stellate cells in the anterior pituitary tended to be 17 times higher than it was in the control group. No increase in the number of S-100 positive cells that constitute the large and small follicles in the intermediate pituitary. The physiological function of the S-100 protein has not yet been identified. The fact that an increase in prolactin-secreting and growth hormone-secreting cells, as well as a decrease in gonadotrophs were observed in the hypophysis of alcoholics suggests that the function of stellate cells may be closely related to these phenomena. Our results also imply that the stellate cells found in the anterior and intermediate pituitary differ in function although they both produce S-100 proteins.

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

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

By injecting 131I-Lipiodol into lymphatics of the dorsum of dog feet, the distribution of 13JI in the lymph nodes and other principal organs as well as its histological effect were studied periodically after the injection for the period of two months. The characteristic feature of J3JI distribution was the fact that J31I was accumulated into lymph nodes markedly higher than in any other organs and it was retained there over a long period of time. Histological examinations of the lymph nodes revealed a marked lymphocytopenia, the loss of germinal center, practically complete loss of lymphoid elements already 5 days after injection, and marked fibrosis. In the lung a considerable J3JI·distribution could be seen in early stage:, but with lapse of time it decreased rapidly. The distribution in other organs such as liver, spleen, bone marrow, kidney, ureter, bladder, thyroid gland, pancreas, testicles and small and large intestines was negligible in amount, and any specific histologic effect of irradiation could not be recognized in these organs including the lung. From these results, the authors concluded that 131I-Lipiodol has a selective activity on lymph nodes by injecting it via lymphatics and it is a safe method in clinical application to treat the patients bearing malignant lymphoma or metastatic lymph nodes.

Constitutional lipid peroxidation in randomly selected 32 cases of clinically advanced carcinoma from human gastrointestinal tract (20 cases), breast (8 cases) and kidney (4 cases) was examined histochemically in frozen sections using cold Schiff's reagent. Only two cases of gastrointestinal carcinoma were positive by the reagent. Non-cancerous parenchymal cells were negative. These findings suggest that detectable constitutional lipid peroxidation seldom occurs in either cancerous or normal tissues. The capacity for normal and neoplastic tissues to undergo lipid peroxidation was also studied by incubation with an iron-NADPH pro-oxidant system. Normal parenchymal cells showed, to various degrees, a positive reactivity. In gastrointestinal carcinoma, 6 out of 7 cases of well differentiated adenocarcinoma reacted positively, whereas 2 out of 8 cases of moderately to poorly differentiated adenocarcinoma disclosed weakly positive reactions. Mucinous adenocarcinomas (4 cases) were all negative. Signet-ring cell carcinoma (1 case) was positive. One out of 8 cases of breast cancer also showed positive reaction. Four renal cell carcinomas were all negative. Cancer cells have lower capacity to undergo lipid peroxidation than normal cells, when the iron-NADPH pro-oxidant system was employed. In gastrointestinal carcinoma, the ability to undergo lipid peroxidation by the iron-NADPH pro-oxidant seems to be correlated with their histological differentiation. This fact may suggest that differences in lipid composition or the NADPH enzyme system exist between well differentiated and poorly differentiated gastrointestinal malignancies.

The rats which received the repeated intra peritoneal or intravenous　injections of methyl palmitate showed a marked depressed phagocytic activity　of the RES as shown by the clearance test with radioactive iron as　well as by histological observations and a significantly suppressed antibody　formation against the challenge by BSA.　Differing from the cases of the blockade of the RES made by PVP or　radiogold, the injection of methyl palmitate did not result in any injurious　effect on the lymph follicles of lymph nodes and spleen and the plasma　cells proliferation as revealed by the histological observation. Histochemical observations of iron phagocytosis of the RES done by　Perls stain revealed that methyl palmitate　suppressed the phagocytic activity　of the Kupffer cells of the liver dramatically and also suppressed the　phagocytic activity of the sinus-lining cells in spleen to a lesser degree.　The result indicates that the injection of methyl palmitate attacks the phagocytic　function of the RES selectively and induces the reduced immune　response of the organism without giving any damages to the proliferation of immunologically com petent cells.　The fact suggests that the RES lowered in their phagocytic activity　fails to produce the informational substance for immune　response, showing　a lower level in the antibody formation even in the presence of antigen　and proliferating immunologically competent cells.

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.

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.

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.

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.

1. For the purpose to clarify the relationship between the structural change and lipid composition of isolated rat liver mitochondria, lipid composition and swelling rate of mitochondria obtained from the rat of 3'-Me-DAB feeding and raised in cold room are measured, and the following results were obtained. 2. The mitochondria obtained from the liver of 3'-Me-DAB-fed rat and of rat raised in cold room show a low rate of swelling by addition of Na-oleate accompanied by the decrease in highly unsaturated fatty acids (C18:3 and C20:3or 4) and with the increase in saturated fatty acids (C16 and C18). 3. Activation energy for the mitochondrial swelling is about 16.2 Kcal in the mitochondria obtained from normal rat liver, but requires 19.7 Kcal in the mitochondria that show a low rate of swelling. The fatty acid composition, especially in glycerophosphatides which occupy about 80 per cent of total lipids, is a structural component of mitochondrial membrane, undergoes the change from former to latter in the following fashion: C16:0 21.73→32.10, C16:1 3.37→2.96, C18:0 25.0→29.75, C18:1 13.75→17.40, C18:2 23.90→16.0 and C20:3 or 4 12.23→1.79. 4. At the time of low rate swelling of mitochondria isolated from 3'-MeDAB- fed rat liver, there could be observed a marked increase of the acetone soluble lipid (simple lipids) in the total liver lipids and in the fatty acid distribution of the acetone-soluble lipids, oleic acid was markedly increased (0.838→3.81%/dry liver), despite the fact that in the acetone-insoluble fractions or in the mitochondria there are no marked changes in the oleic acid contents (1.84→2.56% or 0.212→0.246%/dry liver).

A patient with an unresectable hepatocellular carcinoma (HCC) who survived without active treatment 3 years and 8 months after histological diagnosis is described. The size of the liver, which was already quite huge at the time of diagnosis, changed little during the entire clinical observation. However, 2 months before death, his condition deteriorated rapidly following gastrointestinal bleeding due to the direct invasion of the stomach by HCC. A critical reason for the unusually long-term survival of the patient may stem from the facts that a well-differentiated and bile-producing HCC was extent in most encapsulated-tumor tissues and that liver cirrhosis was not present.

Our experience with Brachet test on twenty-four leukemic patients has shown a high degree of reliability of the test for differentiating acute leukemias. A standard method has been described which is simple enough to be carried out routinely. The test, however, is not without pitfalls and need be interpreted with some caution. From the fact that urine hydrolysis can be closely simulated by the enzymatic action of pure DNase solution, it is suggested that the urine factor responsible for the nuclear lysis is DNase excreted in the human urine. The possible mechanism and implication of the test have been discussed in relation to the results obtained.

1. The threshold of a muscle poisoned with monoiodoacetic acid for galvanic and induction current increases as the poisoning proceeds, though it shows temporary decrease of the threshold at an earlier stage of the poisoning. 2. The height of the muscular contraction (isometric as well as isotonic) of a poisoned muscle decreases gradually as the poisoning proceeds. 3. A fully poisoned muscle has a longer chronaxie than that of a normal one. It is observed that there are two types of increasing chronaxie in the course of poisoning. The one is that the chronaxie remained practically unchanged though the poisoning progresses and suddenly increases at the moment when contracture sets in, while the other is that it increases gradually until at last the poisoned muscle goes into rigor. 4. The absolute refractory period of a poisoned muscle shows a marked increase as the poisoning proceeds. Sometimes, at an earlier stage of poisoning, a slight shortening of the absolute refractory period is observed. 5. The maximum work performed by a poisoned muscle shows a rapid diminution as the poisoning goes on. On the other hand, parallel with it, the total moment of inertia of the recording system must be increased in order to attain the maximum work. This fact suggests that the viscous property of a poisoned muscle increases and that the energy developed by activity is wasted in overcoming this resistance. 6. These characteristics displayed by a poisoned muscle may fairly be explained under the supposition that a poisoned muscle falls into some sort of fatigue. In conclusion I wish to thank Prof. S. Oinuma for his help and advice during this experiment.