Acta Medica Okayama volume23 issue4

l) The submitochondrial particle system can synthesize ATP in the early phase (220 seconds after the accition of ADP) in the presence of sodium succinate and Pi, in spite of the absence of the hexokinase-glucose system, and this phosphorylation is inhibited by oligomycin. 2) The submitochondrial particle system can synthesize ATP by the base-acid transition (proton pulse) only in the presence of ADP and Pi, in spite of the absence of oxidizing substrates and the hexokinase-glucose system, and this phosphorylation is dependent on the span of pH change, and is inhibited by oligomycin and 2, 4-dinitrophenol. 3) The role of the proton vector in the oxidative phosphorylation and the proton ejection was discussed from the stand point of a new hypothesis.

Among various photosensitizing dyes, 4, 4'-dimethyl 3, 3'-di-n-heptyl-8- {2-(4-methyl-3-n-heptylthiazole) }-2, 2'-dicarbocyanin diiodide (abb. NK19), even in an extremely low concentration, is known to inhibit the proliferation of bacteria and tissue culture cells (1, 2, 3). With respect to the mechanism of such inhibitory action no other property of this NKl9 is known except that it has a marked adsorptive property to protein (4). As a step toward the elucidation of the mode of biological effect, the author studied the effect of NK19 on the energy transfer reaction of Irat liver mitochondria, followed by comparison with the mode of actions of various other inhibitors of the oxidative phosphorylation (5). NK19. NKl9 can be prepared by letting 2, 4-dimethylthiazole heptyliodide react with ethylorthoformate in anhydrous acetic acid. We used NKI9, a product of Nihon Kanko Shikiso Research Laboratories. The molecular structure is as in the following and in its MeOH state it has maximum absorbancy at 590 m,a. For the use in experiment it was made into 1 mg/ml of MeOH and was stored in the dark until used.

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.

With a constant gas-exposure chamber newly devised, the author had Cb mice (females weighing 16.0 ± 1.5 g) inhale 600 ppm (in average) of 1, 1, 2, 2-tetrachloroethane for 3 hours. Then, the total Iipid, triglyceride and ATP levels in the liver were estimated before, immediately after, 4 hours and 8 hours after the exposure. The results of the observations are briefly summarized as follows: 1. It has been demonstrated by the chemical quantitative analyses of total lipid and others that the exposure to 1, 1,2, 2-tetrachloroethane induces fatty liver in mice. 2. Both total lipid and triglyceride levels increased almost linearly from the time of exposure up to 8 hours later. The ratio, triglyceride: total lipid, increased with lapse of time after the exposure, and of the lipid components, the increase of triglyceride was marked. 3. The hepatic ATP level decreased almost linearly from the time of exposure to 8 hours later. The value, total lipid × ATP, hardly differed from that of the control even after the exposure, and there was observed a parallel relationship between the rate of increase in total lipid level and the rate of decrease in the hepatic ATP level. 4. The intensity of hepatotoxicity of 1, 1, 2, 2-tetrachloroethane proved to be practically the same at that of carbon tetrachloride.

For the purpose of revealing whether AMD inhibits the RNA synthesis of erythroblasts in an effective dose in vivo to eradicate erythroid cells in rabbit bone marrow, the author observed the RNA synthesis by H3-uridine incorporation in vitro and RNA level on the cells from the anemic animals taken at a certain period after a single injection of AMD in a small dose of 50 and 100μg/kg body weight. The data revealed that by such a small dose of injection of AMD the RNA synthesis of erythroid precursors, early basophilic and proerythroblast stages, was successfully suppressed without any suppressing effect on the RNA synthesis of erythroblasts in the later stages of specialization, indicating that there are at least two kinds of RNA synthesis: one seen mainly in the earlier stages of specialization and the other one seen mainly in the later stages, and they can be distinguished from each other by the AMD sensitivity.

1. After the centrifugation of sonicated heavy beef heart mitochondria at 75, 000 × g for 10 minutes, the supernatant was centrifuged at 144, 000 × g for 30 minutes. The residue was revealed being composed of vesicular inner membrane fragments (ETPH), about 600 to 1000 Å. in diameter, showing a morphological homogeneity and a high capacity of oxidative phosphorylation. 2. The Pia ratio of the ETPH in the presence of succinate and of NADH2 was 1.68 and 2.54, respectively, and the corrected Pia value for O2 gas equilibrium was 1. 01 and 1.40, respectively. 3. The capacity of oxidative phosphorylation in ETPH fraction was parallel to the activity of the oligomycin. sensitive ATPase in these fractions. 4. The P/0 ratio of ETPH was decreased to about 50 % by hypotonic treatment. The decrease of P/0 ratio was restored to the level of about 90 % by incubating the ETPH with ATP and BSA. In the instance where the P/0 ratio was low level in the hypotonic medium, the surface structure of ETPH was observed as a swollen form and the head pieces of the elementary particles were clearly observed in contrast to the solid surface structure of ETPH in the isotonic medium. 5. The P/0 ratio of ETPH was decreased to about 60 % by relatively severe sonication, and after separating the residue from the supernatant, that of the residue decreased further to about 40 %. The P/0 ratio of the residue was restored to the level before the separation on the addition of the supernatant containing oligomycin-insensitive ATPase. 6. A discussion was made on the correlation between the surface structure and the activities at terminal phosphorylation step of ETPH after the simple physico-chemical treatment.

1) In order to study the molecular structure and electron transfer activities of mitochondrial inner membrane, dissolution and reconstitution of membranous structure and function of the inner membrane of beef heart mitochondria were carried out. 2) The inner membrane of mitochondria could be dissolved into some unit of particles 70-140 Å in diameter by the treatment with bile salts at the concentration 0.5 mg of deoxycholate per mg of protein, 0.5 mg of cholate per mg of protein and 74.5 mg of crystalline potassium chloride per ml of the suspension. 3) The dissolved unit particles readily reaggregated into a vesicular membrane simultaneously restoring over-all electron transfer activities by the removal of bile salts with dilution of the suspension.4) Isolated electron transfer unit particle fraction contammg all components of the electron transfer chain but no structural protein were soluble in aqueous solution due to some residual bile salts used in the preparation. The removal of bile salts by dilution led the dispersed particles to aggregate into membrane and restore their over-all enzymatic activities. 5) From these results and the results of the reconstitution of membrane from purified complexes as described in the previous paper, it may be concluded as follows: The mitochondrial inner membrane may consist of several kinds of repeating unit particles conjugating each other with adjacent particles. It is necessary for over·all enzymatic activities that some unit components aggregate into a single vesicular membrane. Structural proteins may play an important role in the constitution of the membranous structure and in the over-all enzymatic activities.