| Project/Area Number |
12557231
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
応用薬理学・医療系薬学
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| Research Institution | The University of Tokushima |
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Principal Investigator |
FUKUI Hiroyuki The University of Tokushima Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (90112052)
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| Co-Investigator(Kenkyū-buntansha) |
TAIRA Kazunari The University of Tokyo, Graduate School of Engineering, Faculty of Engineering, Department of Chemistry and Biotechnology, Professor, 大学院・工学研究科, 教授 (10261778)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥13,400,000 (Direct Cost: ¥13,400,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2000: ¥9,100,000 (Direct Cost: ¥9,100,000)
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| Keywords | ribozyme / protein kinase C / isozyme / G protein-coupled receptor / histamine H1 receptor / receptor desensitization / gene expression / signal transduction / ヒスタミンH_1受容体 / 受容体アップ調節 / cGMP依存性蛋白キナーゼ / アップレギュレーション |
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| Research Abstract |
Histamine H1 receptors mediate type-1 allergy in peripheral tissues and histaminergic neurotransmission in CNS. Activation of the protein kinase C (PKC) induced histamine H1 receptor desensitization, leading to the suppression of the receptor signaling. PKC activation also induced histamine H1 receptor up-regulation through gene expression, enhancing the receptor signaling. Different protein kinase C isozymes were considered to be involved in the two regulatory mechanisms of histamine H1 receptor signaling. In order to identify PKC isozymes in the two mechanisms, specific ribozyme to knock out each PKC isozyme was designed using a vector containing cDNA for ribozyme, pcDNA3. 1 Zeo+CMV(-). Initially, five ribozyme inserts selected from PKC-α sequences at 5'-site, Ribo-PKC-α1 〜 Ribo-PKCα5 were made. Ribozyme generating plasmids, pcDNA3. 1 Zeo+CMV(-)-PKC-α1 〜 pcDNA3. 1 Zeo+CMV(-)-PKC-α5, were constructed using pcDNA3. 1 Zeo+CMV(-). The plasmid was transfected with histamine H1 receptor expressing U373 astrocytoma cells. High transfection rate was essential for the purpose. The transfection rate to U373 cells, however, was not high. Stable transfection was achieved to obtain the PKCα knock out cell line expressing the ribozyme. U373 cells expressing ribozyme, however, failed in knocking out PKCα. Effective transfection reagent with low toxicity, PolyFect Transfection Reagent, showed very high transfection rate (70-80%) to HeLa cells. Transfection of the plasmid and expression of the ribozyme could not knock out PKCα in HeLa cells. Then the plasmid was transfected to HeLa cells with very low level of PKC by the treatment of PKC-activating phorbol ester. The effect of the ribozyme on PKCα generation was very limited in the PMA-treated HeLa cells. In conclusion, establishment of the PKC knock out was not successful due to the difficulties of condition settings and several unknown reasons.
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