| Project/Area Number |
06672202
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Faculty of Pharmaceutical Sciences, Teikyo University |
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Principal Investigator |
HORIE Shuichi Teikyo University, Faculty of Pharmaceutical Sciences, Department of Clinical Biochemistry, Lecturer, 薬学部, 講師 (60157063)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1995: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Thrombomodulin / Retinoic Acid / Transcription / Nuclear Receptor / RARE / Spl / CAT Assay / BxPC-3 Cell / Nuclear reccptor / BxPC-3 cell / トロンボモジュリン / レチノイン酸 / 腫瘍壊死因子 / 核内レセプター / 遺伝子転写 / レチノイン酸レセプター / 特異的塩基配列 / CATアッセイ |
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| Research Abstract |
We reported that thrombomodulin (TM) , the anticoagulant cofactor for activation of protein C,was up-regulated by treatment of endothelial cells with all-trans retinoic acid (RA) throgh the transcriptional acceleration. In the present investigation, the transcriptional regulatory sequence of the 5'-untranslated region of TM gene studied by transient expression assay in human pancreas BxPC-3 cells. Deletion mutants of plasmid constructs containing the TM promoter and chloramphenicol acetyltransferase (CAT) reporter gene exhibited that presence of proximal (-202 to 207) and distal (-1515 to -1531) RA-responsive regions. The distal one contains RA response element and the proximal one contains Spl site in the DNA sequence. The both sequences were additive in the increase in promoter activity by RA and mutations introduced into each sequence confirmed the functional response with RA-treatment. Transactivation study showed that cotransfection of the plasmid containing both the sequences with expression plasmid for RA receptor, such as RAR-alpha, -beta, -gamma and RXR-alpha, did not enhance RA-dependent promoter activity, though RAR-beta mRNA increased in BxPC cells treated with RA.Gel retardation experiment indicated the Spl protein not AP2 protein interacted with the Spl sequence where not with the Spl-deleted sequence. By treatment of the cells with RA time-dependent increases in mRNA levels of Spl, SPR-l and SPR-2 were observed. We concluded that two sites of genomic DNA regions particpated in the RA-dependent augmentation of TM gene expression.
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