Acta Medica Okayama volume24 issue1

1) It has been found that the peripheral blood of hybrid adult dogs contains about 29.4 % of lymphocytes in average. When such a blood is passed through the glass wool column the leucocytes containing 68-90 % (81.6% in average) lymphocytes are obtained. 2) In the single culture of such lymphocytes alone and mixed culture both live lymphocytes and sonicated lymphocytes in the presence of 1% (vIv) PHA, the peak of the blastformation of lymphocytes is observed at culture hour 72. 3) In the abscence of PHA both single culture and mixed culture of lymphocytes show hardly any blastformation. 4) In the mixed culture of live lymphocytes with homogenate of sonicated lymphocytes with addition of 1% (vIv) PHA the rate of blast. formation observable at culture hour 72 and the rejection of kidney trans· plant 7 days after its transplantation of hybrid adult dogs show a direct correlation, demonstrating that the mixed lymphocyte reaction reflects accurately the difference in donor histocompatibility antigens against the recipient.

Eine Salieyl-Chinin-Lithium-Kombination wurde im Siemens Unterriehtsreaktor SUR 100 BE des Instituts fur Kernteehnik der Teehnisehen UniversiUit Berlin mit Neutronen aktiviert. AnsehlieBend erfolgte die Messung der spektralen Verteilung der Gamma-Zahlrate zu zwei versehiedenen Zeitpunkten naeh der Aktivierung. Es wird festgestellt, daB diese Salieyl-Chinin-Lithium-Kombination nur sehwaeh im SUR-Reaktor aktivierbar ist, vergliehen mit einer Goldsonde etwa 200 mal sehwaeher. Trotzdem konnten mit Hilfe eines 400-Kanal-Analysators die spektralen Verteilungen der Gamma-Energien dieses Praparates noch sehr genau bestimmt werden. Ein ausgepragter Gamma-Peak tritt auf bei der Energie 0.53 MeV. Er klingt ab mit einer Halbwertszeit von 1.6 h und ist vermut· lich auf ein Element der Tablettierhilfsstoffe zurUckzufUhren. Vom strahlenphysikalischen Gesichtspunkt ist das Praparat fUr die Human. medizin gut geeignet.

The role of -SH groups in mitochondrial energy transfer reaction was studied by observing the reduction of a disulphide, 5, 5'-dithiobis (2-nitrobenzoic acid), DTNB, a specific analytical agent for the estimation of -SH groups in biological materials, by addition of it to the isolated rat liver mitochondria in various respiratory states, as defined by CHANCE and WILLIAMS. 1. In the various respiratory states, states 1 to 5, the reduction of DTNB proceeds most rapidly at state 5, and most slowly at state 3. DTNB reduction at state 5 is suppressed by the partial oxidation of respiratory carriers with oxygen (state 4) and the addition of respiratory substrate does not affect the DTNB reduction. 2. The retardation in the reduction rate at state 3 is relieved partially by a respiratory inhibitor, KCN, and is intensified markedly by oligomycin, an inhibitor of oxidative phosphorylation. An uncoupler for oxidative phosphorylation, DNP, does not affect the reduction rate at state 3. At state 4 the reduction is stimulated by DNP and KCN, but is unaffected by oligomycin. The results suggest that the alteration in the functions of the energy transfer reaction in mitochondria is accompanied by changes in the occurrence and the functioning of -SH groups which can be detected by the reactivity with DTNB. The data suggest also that there are at least two kinds of -SH groups reacting with DTNB: the one is the -SH group which reacts DTNB actively when the respiratory carriers are kept reduced, and the other is the one which reacts actively when the respiratory carriers are kept oxidized, participating in the phosphorylating system and its reactivity with DTNB diminishes in the actively phosphorylating states (states 2 and 3).

With the purpose to clarified the mode of localization and mechanisms of activation of ATPase in the mitochondrial membrane, analyses were made on the properties of mitochondrial ATPase from the structural and functional aspects. The activation of ATPase by DNP and Mg++ and the oligomycin sensitivity were investigated in a series of inner membrane fragment samples obtained by ultrasonic irradiation and those samples obtained in the processes of isolation and purification of ATPase from rat liver mitochondria and beef heart mitochondria in parallel with electron microscope observations. As a result it has been found that the membrane fragments obtained from rat liver and beef heart mitochondria by ultrasonication exhibited high respiratory activity and unmasked ATPase activity which was charac· terized by remarkable stimulation by Mg++ and inhibition by oligomycin and azide. Therefore, mitochondrial ATPase seems to be bound fairly closely to the inner mitochondrial membrane. In the membrane fragments prepared by ultrasonication of intact mitochondria, ATPase activity was stimulated by DNP, but in the supernatant fractions was not. On the other hand, the supernatant fraction obtained from BHM and inner membrane fragments by severe sonication exhibits a marked ATPase activity and the activity incresed in each step of the purification on the treatments with acid, protamine and heat. Especially in the case of membrane fragments the protamine treatment can be omitted. Electron microscope observation of the fractions in each step of the purification proved the head pieces to be ATPase. The ATPase activity of solubilized head pieces is insensitive to oligo. mycin and coincides with the soluble ATPase of PULLMAN etat. (8) in the points of its cold labile property and optimum pH, but it shown no accele. ration of ATPase activity by DNP.

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.

For the purpose to reveal the changes in the metabolism of erythroblast in varied specialization stages the author observed the Feulgen DNA level of rabbit erythroblasts by microspectrophotometry. Observations were made on normal and anemic animals, and those receiving a mass red cell transfusion at the recovery stage of anemia where the early denucleation is stimulated. Observations have revealed that in normal erythropoiesis the DNA contents are kept at n to 2 n level from the proerythroblast to late basophilic stage, but in later stages, polychromatic and orthochromatic, DNA level per cell decreases gradually with advance of the cell specialization reaching the minimum level, nearly haploid level, at orthochromatic stage where most cells are believed to be denucleated. In blood depleted animals nearly the same pattern of DNA level was observed in connection with erythroid specialization as that in normal animal, except a relatively high DNA level in the later specialization. In the cases of the hemolytic anemia a similar tendency has been observed but the minimum level of DNA remains at a higher level, hypo-diploid level, in poly- and orthochromatic stages. Twenty-four hours after the mass red cell transfusion by which severe anemia has been recovered to the original level within one hour, the pattern of the DNA level of the erythroblast returns to the normal one showing a very low DNA level at the polyand the orthochromatic stages. The data indicate that the DNA synthesis of erythroblast kept at n to 2 n levels until the late basophilic stage begins to decline at polychromatic stage and reaches nearly haploid level at orthochromatic stage, but in active hemopoiesis the DNA synthesis is stimulated and the DNA contents are kept at a high level even in the late specialization stages, showing no relation between the denucleation and the low DNA level.

Localization of IgM and IgG sypylitic antibodies in the sera of patients and the experimental syphylitic rabbits was examined by the gel filtration on Sephadex G.200 column. I) In the case of late syphylitic patients; OGATA test-reactive antibodies were contained in IgM and IgG fractions. On the other hand, RPCF test-reactive antibody was contained only in IgG fraction. This discrepancy may be due to the difference in antigens; Cardiolipin.resicin and T. P. Reiter protein. 2) In the case of the experimental syphylitic rabbits; The results were as follows. a) Variation in the level of the titer. The peaks of the titer were seen 3-4 weeks after inoculation of T. P. Nichols by OGATA test, VDRL test and RPCF test, thereafter titers decreased. On the other hand, the titer kept on rising up to 2 months and maintained high level during the periods of 3, 4 and 5 months by TPHA test. b)Transformation of antibody from IgM to IgG. Transformation of antibody from IgM to IgG was seen 3-4 weeks after inoculation by all four tests; OGATA test, VDRL test, RPCF test and TPHA test, and such a transformation was completed 3 months after inoculation.

1) Normal karyotype of Donryu strain rat was determined according to the classification of KURITA et al. (8). Namely, the number of chromosomes was 42 in diploid cells, and chromosomes were divided into 3 groups (A, B and C) according to the position of centromere. A.group was consisted of 7 pairs of metacentric chromosomes, B-group 4 pairs of sub· meta.subtelocentrics and C.group 10 pairs of telocentrics and Y. 2) Among all chromosome pairs a pair of the longest telocentric chromosomes (C.l), 4 pairs of all the B.group, and the Y chromosome were recognizable. 3) The presence of polymorphism was demonstrated in the smallest submetacentric chromosomes (BA),: namely, (I) a homologous submeta. centric pair, (II) a homologous subtelocentric pair and (III) a heteromor. phic submeta and subtelocentric pair which seemed to be a hybrid from (I) and (II). To distinguish the polymorphism in their genotype from phenotype was impossible. 4) Animals with type III B·4 chromosomes were produced by type I and type II animals. 5) By checking the chromosomes of the inbred Donryu strain rats maintained over 40 generations by brother.sister mating at Nihon Rat Co., polymorphism in BA chromosomes was also recognized.