| Project/Area Number |
03454127
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurophysiology and muscle physiology
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| Research Institution | University of Tokyo |
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Principal Investigator |
DAVID Saffen Tokyo University, Faculty of Medicine Lecturer, 医学部(医), 講師 (50231329)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1992: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1991: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | zif268 / PC12 / transcription factor / target gene / protein kinase C / nerve growth factor / calcium / muscarinic receptor / ムスカリン受容体 / zif268 / PC12D / チロシンキナーゼ / ターゲット遺伝子 / 海馬スライス / アンチセンス / 神経細胞 / 遺伝子発現 / ムスカリン性アセチルコリン受容体 / PC12細胞 / Cーキナ-ゼ / zinc finger |
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| Research Abstract |
In the present study we have demonstrated that nerve growth factor and the muscarinic acetlycholine receptor agonists carbachol and oxotremorine rapidly and dramatically increase levels of zif268 mRNA in PC12D cells, a subline of PC12 cells that undergoes rapid differentiation in response to nerve growth factor. Induction by muscarinic receptor agonists is completely blocked by pretreating the cells for 18 hours with phorbol diacetate, which induces the down- regulation of protein kinase C. By contrast, induction of zif268 mRNA increases by NGF is only partially blocked by phorbol ester pre- treatment. Induction by NGF is very sensitive to inhibition by the protein kinase inhibitors K252a (100 nM) and staurosporin (10 nM), suggesting that induction requires activation of the tyrosine kinase of the p140-trkA NGF receptor. Induction by carbachol and oxotremorine, but not by NGF, requires the influx of extracellular calcium via channels that are resistent to inhibition by the L-type calcium channel inhibitor nifedipine. These data suggest that zif268 mRNA induction by NGF and by muscarinic agonists takes place via, at least partially, independent pathways. We have also expressed the DNA binding domain of zif268 in E. coli as a maltose binding protein/ zif268 fusion peptide. This fusion protein can be isolated by affinity chromatography and binds to the zif268 DNA recognition sequence with high specificity and affinity. We are now attempting to use this fusion protein to isolate genes that are regulated by the zif268 transcription factor.
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