Biomedical subjects
Publications and source records attributed to S Ikegami.
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Single-stranded circular DNA of phage fd was microinjected into Xenopus laevis eggs and DNA synthesis was analyzed after incubation. An efficient DNA synthesis occurred in the eggs: 2.7 ng of DNA was newly formed in an egg whose volume is approximately 900 nl during a 5-h incubation at 20 degrees C. The DNA synthesis was sensitive to aphidicolin, suggesting that DNA polymerase alpha participates in the synthesis. Agarose gel electrophoresis showed that full size of fd DNA is synthesized and that replicative form I of fd DNA is the major form of the newly produced DNAs. These results suggest that priming of DNA synthesis, elongation of DNA chain and ligation of DNA strands take place co-operatively in the egg with single-stranded DNA as a template.
In fertilized echinoderm eggs, the male and female pronuclei fuse to form the diploid nucleus even in the presence of aphidicolin, a specific inhibitor of eukaryotic DNA polymerase-alpha. The subsequent first cleavage is independent of chromosomes but dependent on spindle and amphiaster. The fate of DNA originally existing in the fused nucleus during achromosomal cleavage of fertilized sea urchin and starfish eggs induced by aphidicolin was determined using antidenatured DNA antibody. The nucleus is not formed in the divided daughter cells at the two-cell stage but the nuclear-envelope-free chromatin mass which is unassociated with mitotic apparatus remains in the center region of embryos, especially near the first cleavage furrow. These results indicate that the condensed and nonreplicated chromatin can not be associated with spindle and asters in the presence of aphidicolin.
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The presumptive muscle cells (B5.1 blastomeres) were isolated from 16-cell-stage embryos of the ascidian, Ciona intestinalis. The isolated cells were allowed to divide either twice or three times thereafter. Then further divisions of the cells were continuously inhibited by a simultaneous treatment with aphidicolin (a specific inhibitor of DNA synthesis) and cytochalasin B (an inhibitor of cytokinesis). When development of muscle-specific acetylcholinesterase in these division-arrested progeny cells of B5.1 blastomeres was examined histochemically, the B5.1 blastomeres which had been allowed two further divisions did not produce any detectable acetylcholinesterase activity. Whereas those which had been allowed three further divisions showed the tissue-specific enzyme activity. These results provide further evidence for the presence of a quantal DNA replication cycle for the tissue-specific enzyme development, which is qualitatively different from the other DNA replication cycles.
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Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.
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The present study was designed to determine the effect of purified viscous polysaccharides on pancreatic exocrine secretion and biliary output. Male Sprague-Dawley rats aged 5 weeks were fed either on the control diet which contained no fiber or on the experimental diets that contained 5% indigestible polysaccharides (agar, kappa-carrageenan, Na-alginate and konjac mannan). After pretreatment with each diet for 2 weeks, the diet was discontinued overnight, and the animals were then allowed to eat 4 g of the test diet in 1 h. Immediately after this, a polyethylene tube was placed in the common bile duct, under anesthesia. Combined, free drained pancreatic-bile juice was collected for the following 1 h. Long-term (2 weeks) administration of Na-alginate and konjac mannan gave rise to increases in the volume of secreted juice and in the output of total bile acids, amylase and protease, whereas that of agar and kappa-carrageenan had no affect. Since a single dose of Na-alginate and konjac mannan was ineffective as regards pancreatic-bile secretion, the elevated secretion observed over prolonged periods may result from processes of adaptation against highly viscous polysaccharides.
dNTP pools are quite low in immature oocytes of the starfish, expand during the 1-methyladenine-induced maturational process and thereafter reach a maximal level (approx. 35, 20, 15 and 5 fmoles/egg for dTTP, dCTP, dATP and dGTP, respectively) which is maintained in overmatured eggs. Maturing oocytes were inseminated at the stage just before extrusion of the first polar body and determination of dNTP pools during early embryogenesis showed the same expansion pattern as that of the 1-methyladenine-treated oocytes. Therefore, the increase in dNTP pools during early embryogenesis is dependent on 1-methyladenine (1-MA) but independent of fertilization. Aphidicolin, a specific inhibitor of eukaryotic DNA polymerase alpha, has no effect on dNTP pool size in 1-methyladenine-treated oocytes, but causes considerable expansion of dNTP pools in fertilized eggs which cleave achromosomally in the presence of the drug.
DNA synthesis was studied during the overmaturational stage of starfish eggs. Some DNA synthesis took place in the overmature eggs of the starfish. The DNA synthesis was resistant to aphidicolin. Judging from its circularity and small molecular size, the DNA synthesized is of mitochondrial origin.
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The present studies were conducted to characterize the relevance of a microsomal mixed function oxidase system to the polychlorinated biphenyls (PCB)-induced vitamin A reduction and endogenous lipid peroxide formation in the liver of rats. And also, this study dealt with an influence of scavengers on the hepatic lipid peroxide formation stimulated by PCB. Rats were given a 0.01% PCB diet supplemented with adequate nutrients, for 14 days. In an experiment, secobarbital was injected subcutaneously for the degradation of hepatic microsomal cytochrome P-450 heme. A marked liver enlargement and a significant increase of total liver lipid content were observed in the PCB group. The secobarbital enhanced the PCB-induced liver enlargement but no effect of secobarbital on the lipid content was recognized. PCB significantly induced hepatic microsomal cytochrome P-450, but not both cytochrome b5 and NADPH-cytochrome c reductase. The secobarbital suppressed the induction of cytochrome P-450 caused by PCB to approximately one-half. The hepatic vitamin A content significantly decreased on PCB administration and the secobarbital slightly enhanced the PCB-induced vitamin A reduction. However, the vitamin A content in the secobarbital-injected control group decreased to nearly the same levels as in the PCB groups. Therefore, it was presumed that the hepatic microsomal mixed function oxidase system, especially the cytochrome P-450, was possibly not directly involved in the PCB-induced hepatic vitamin A reduction or that a metabolic system related to mixed function oxidase system was involved in the reduction. On the other hand, the hepatic lipid peroxide content tended to increase on PCB administration though no significant difference was recognized. In contrast, the hepatic lipid peroxide content significantly increased in the secobarbital-injected PCB group as compared with the secobarbital-injected control group. However, there was no difference in the lipid peroxide contents between the control groups with and without the injection of secobarbital, and also between the PCB groups. The hepatic vitamin E contents lowered in the secobarbital-injected groups but no effect was observed on PCB administration. The glutathione peroxidase activity decreased significantly on PCB administration and the secobarbital further decreased the activity. Therefore, it was suggested that the significant increase and tendency of increase in hepatic lipid peroxide contents in the PCB groups with and without injection of secobarbital were ascribed to an insufficiency of lipid peroxide scavengers in the liver.(ABSTRACT TRUNCATED AT 400 WORDS)