| Co-Investigator(Kenkyū-buntansha) |
山下 俊英 大阪大学, 医学部, 助手 (10301269)
YONEDA Takunari Osaka University Faculty of Medicine, assistant professor, 医学部, 助手 (70271179)
HORI Osamu Osaka University Faculty of Medicine, assistant professor, 医学部, 助手 (60303947)
TAMATANI Michio Osaka University Faculty of Medicine, associate professor, 医学部, 助教授 (30294052)
OGAWA Satoshi Kanazawa University School of Medicine, Professor, 医学部, 教授 (90283746)
PINSKY David コロンビア大学, 医学部(米国), 助手
STERN David コロンビア大学, 医学部(米国), 正教授
島田 昌一 大阪大学, 医学部, 助教授 (20216063)
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| Budget Amount *help |
¥11,000,000 (Direct Cost: ¥11,000,000)
Fiscal Year 1998: ¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 1997: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Research Abstract |
The 150kDa Oxygen-regulated protein (ORP150) was initially identified as a novel stress protein in hypoxic astrocytes and its amino acid sequence suggest its possible participation in cellular protein transport. Northern blot analysis showed abundant expression of its transciipts the kidney, and immunohistochemical analysis suggested the enhanced expression of ORP150 antigen in renal tubular epitheliurm. The ORP150 immunointensity in epithelium was colocalized with that of Tamm-Horsfall protein, a marker of tubular epithelium in thick ascending limb of Henle, suggesting a possible role of ORP150 in protein transport in tublar epithellum. In MDCK cells, a cell line representing the renal epitheliurm, exposure to hypoxia resulted in the increase of ORP150 antigen, as well as the incresae of GP8O (8OkDa glycoprotein) antigen immunoprecipitated by anti-ORP150 antibody. In the ORP150 antisense transformant MOCK cells, where the expression of OPR150 is strongly supressed, GP-80 antigen retai
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ned in the ER after the exposure to hypoxia Consistently, metabolic labelling showed the delay of GP8O maturation in antisense transformant cells in hypoxia, whereas matured form of GP8O was defected in wild type cells under hypoxia, indicating the role of ORP150 in protein transport, especially in hypoxia. The affinity chromatographic analysis of ORP150 to immobilized ATP-agarose suggested a higher affinity of ORP150 to ATP than those of GRP78/Bip and GRP94, other molecular chaperons functioning in the MDCK cells. Further, the ATP-hydrolysis analysis. showed the ORP150 can release GP8O in a lower ATP concentration To determine the contribution of 0RP150 to cellular processes underlying adaptation to hypoxia, a cell line stably-transfected to overexpress ORPl50 antisense RNA was created. In human embryonic kidney (HEK) cells stably overexpressing ORPl50 antisense RNA, ORP150 antigen and transcripts were suppressed to low levels in normoxia and hypoxia, whereas wild-type cells showed induction of ORP150 with oxygen deprivation. Inhibition of ORP150 expression in antisense transfectants was selective, as hypoxia-mediated enhancement of glucose-regulated protein (GRP) 78 and GRP94 were maintained. However, antisense ORP150 transfectants displayed reduced viability when subjected to hypoxia, compared with wild-type and sense transfected HEK cells. In contrast, diminished levels of ORP150 had no effect on cytotoxicity induced by other stimuli, including oxygen-free radicals and sodium arsenate. Although cellular ATP content was similar in hypoxia, comparing ORP150 antisense transfectants and will-type HEK cells, suppression of ORP150 expression was associated with accelerated apoptosis, based on DNA fragmentation and changes in nuclear morphology. Hypoxia-mediated cell death in antisense HEK transfectants did not cause an increase in caspase activity or in cytoplasmic cytochrome c antigen. A well-recognized inducer of apoptosis in HEK cells, staurosporine, caused increas Less
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