| Research Abstract |
1) The identification of phosphorylated histones preceding apoptotic cell death It is reported that the apoptosis is induced by calyculin A and okadaic acid, which are the protein phosphatase inhibitors in various cell lines. We had already found that the histone phosphorylation precedes thymocyte apoptosis induced by these drugs. In this study, the identification of phosphorylated histones was tried. The phosphorylation of histone HI, H2A and H3 increased prior to calyculin A-induced DNA fragmentation in thymocytes. On the other hand, each histone contents did not change by the treatment of the cells with calyculin A. To clear whether the chemical modification of histones is important as a common mechanism of apoptosis, the chemical modification of histones undergoing apoptosis of human astrocytes was also examined. When the human astrocytes were cultured by growth medium after exposure to saline, the cells were injured. This cell damage showed features of the apoptosis. The phosphory
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lation of histones H2A preceded in apoptotic cell deatn.
2) The effect of chemical modification of histones for the chromatin structure The DNA fragmentation is the biochemical hallmark of the apoptosis, and the DNase concerning apoptotic DNA fragmentation has also been clarified. However, the detailed molecular mechanism of DNA fragmentation has not sufficiently been clarified. It is known that chemical modification of histones is related to the transcriptional control by the change of the structure of chromatin DNA in the nucleus. We tried to verify the chromatin structural change in apoptotic process. CD spectrum of the soluble chromatin prepared from the calyculin A treated cell nucleus was measured. Though the calyculin A treatment did not affect for the structure of DNA, cc-helical content in the calyculin A treated chromatin was decreased. The chromatin structural change was also observed in dexamethasone induced thymocyte apoptosis. The sensitivity for the DNase increased prepared chromatin from the calyculin A treated cell from control. These results suggest that the phosphorylation of histone H2A was important as a common mechanism of the apoptotic process. In addition, the histone phosphorylation made the chromatin structure relax by decreasing ahelical content of the chromatin and the sensitivity for DNase increased by change of chromatin structure.
3) The search of a novel apoptosis inducer. A novel apoptosis inducer was searched in order to clear whether the chromatin structural change is a common mechanism of apoptosis. YO-2 synthesized by Okada et.al induced apoptosis in rat thymocytes. Therefore, this compound seemed to be useful as an inducer of apoptosis, which examined the importance of the chromatin structural change in the apoptotic cell death.
4) The study on the mechanism of apoptotic cell death in cultured rat astrocytes. Reperfusion of cultured rat astrocytes with Ca^<2+>-containing medium after exposure to Ca^<2+>-free medium caused an increase in intracellular Ca^<2+> concentration followed by delayed cell death, including apoptosis. Some compounds such as T-588 and CV-2619 protected cultured rat astrocytes against reperfusion injury. In addition, CGMP inhibited the mitochondrial permeable transition pore via the activation of PKG and prevented apoptotic cell death. Less
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