| Project/Area Number |
09660085
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
NAGAO Masaya KYOTO UNIVERSITY, GRADUATE SCHOOL OF BIOSTUDIES, ASSOSIATE PROFESSOR, 生命科学研究科, 助教授 (10237498)
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| Co-Investigator(Kenkyū-buntansha) |
MASUDA Seiji KYOTO UNIVERSITY, GRADUATE SCHOOL OF BIOSTUDIES, ASSOSIATE PROFESSOR, 生命科学研究科, 助教授 (20260614)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | oxygen sensor / hypoxia / erythropoietin / transcription / cell technology / 酵素センサー / 低酵素 |
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| Research Abstract |
Erythropoietin (Epo) is a glycoprotein, and its sugar chains are important for its in vivo biological activity. So, this year we examined the biological activities of recombinant Epo (rEpo) produced by cultured Chinese hamster ovary (CHO) cells in normoxia or hypoxia and analyzed the structure of its sugar chains. For this purpose we used the promoter of lactate dehydrogenase A gene, which is active in CHO cells and its vicinal hypoxia-response enhancer (HRE) stimulates the promoteractivity efficiently in hypoxia. We have prepared a number of permanent CHO cell lines producing rEPO under control of this promoter/HRE. There was little difference in the in vitro and in vivo activities, and glycosylation between Epo produced by the cells in 21% and 2% oxygen. Further more, forced expression of hypoxia-inducible factor-1α (HIF-1α) enhanced Epo production in all oxygen concentrations. These results indicate that a biological strategy based on the hypoxic induction of gene transcription provides a novel system which guarantees a high productivity even under low oxygen concentration.
HIF-1 is involved in hypoxia response. AKT kinase thought to be involved in the signal transudation of hypoxia response. So we co-transfected a constitutive active AKT kinase expression plasmid and a luciferase reporter plasmid under control of LDH A promoter/HRE into Cos1 cells. Expression of constitutive active AKT kinase alone could not stimulate the expression of the reporter in normoxic condition, but it could potentiate the expression of the reporter in hypoxia. It was reported that dominant-negative AKT could depress the hypoxic induction. These results suggest that AKT kinase is necessary but not sufficient for hypoxic induction.
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