| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1999: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Protein kinase C (PKC) plays a crucial role a receptor-mediated signal transduction affecting diverse range of cellular responses such as cell proliferation, differentiation, and tumor promotion. PKC is a unique enzyme, which is activated by 1,2-diacylglycerol (DAG) produced from receptor-mediated hydrolysis of inositol phosphate. On the other hand, active oxygen species such as superoxide, hydrogen peroxide, and hydroxy radical have been suggested to play important roles in many pathological events such as inflammation, autoimmune diseases, ischemia-reperfusion injury, atherosclerosis and cardiovascular diseases, and carcinogenesis. This suggests a close relationship between the action of PKC and active oxygen species-induced dysfunction and cellular damage, however, this possibility has not been thoroughly examined. In the present study, we observed that 1-stearoyl-2-linoleoylglycerol hydroperoxide (SLG-OOH) and SLG Hydroxide (SLG-OH) stimulated the PKC activity isolated from rat bra
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in more efficiently than that of unoxidized SLG in the presences of phosphatidylserin and calcium ion. Then, the PKC activation effect of oxidized DAG has also been tried on 7 repsentative PKC isoforms, which were most pronouncedly expressed in rat brains, and alpha and delta isoforms were found to be intensely activated by the oxidized DAGs. Furthermore, we attempts to prove the neurodegenerative effects of DAG-OOH on cultured neurons overexpressing alpha and delta isoforms. As the cultured cells, neuronal cells established from 18-day rat fetus cerebral cortex (PN cells) were employed for the experiments. We used an adenovirus vector system in PN cells, which allows expression of PKC alpha and delta gene at a high level. These cultured neurons treated by streptolysin O to let the penetration of natural DAG and DAG-OOH into cells. Those treated cells were observed by 1) phase contrast microscopy, 2) cell viability, 3) electron microscopy, 4) immunoblot analysis of phoshorylated tau, 5) immunohistochemistry to localize phosphorylated tau. Within 6h of exposure to DAG-OOH, PN cells overexpressing PKC delta isoform, exhibited neuritic thinning and characteristic beading, while PN cells overexpressing alpha isoform did not shows such changes. The MEK inhibitor PD98059 prevented its neurodegeneration caused by DAG-OOH. Those lesions were observed routine EM which revealed the microtubule (MT) disassembly in the lesions. Immunoblot analysis showed that tau was partially phosphorylated on Thr 181, Ser 202 and Thr 205 in those DAG-OOH-treated cells. Phosphorylation of tau may have caused in MT disassembly and accumulated in cell body and beading lesions. Those findings indicate that the pronounced activation of PKC delta by DAG-OOH may specifically be responsible to elicit the neurodegeneration. Less
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