| Project/Area Number |
14580660
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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 |
Functional biochemistry
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| Research Institution | Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare |
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Principal Investigator |
HAYASHI Masami Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare, Tokyo Metropolitan Institute of Gerontology, Biomembrane Research Group, Research Associate, 東京都老人総合研究所・生体膜機能研究グループ, 研究助手 (00192279)
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| Co-Investigator(Kenkyū-buntansha) |
IWASHITA Yoshiko Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare, Tokyo Metropolitan Institute of Gerontology, Biomembrane Research Group, Senior Research Scientist, 福祉振興財団・東京都老人総合研究所・生体膜機能研究グループ, 副参事研究員 (50111498)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | CHOLESTEROL / FIBROBLAST / LIPID RAFTS / PERERINGOLYSIN O / VISUALIZATION / GREEN FLUORESCENT PROTEIN / MICRODOMAIN / ラフト / 線維芽細胞 / 脂質マイクロドメイン / 脂質ミクロドメイン / MDCK細胞 |
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| Research Abstract |
We prepared a non-cytolytic derivative of perfringolysin O (θ-toxin), a cholesterol-binding cytolysin, as a probe for cholesterol. It is already shown that the probe (BCθ; protease-nicked and biotinylated θ-toxin) binds selectively to cholesterol in cholesterol-rich membrane domains (lipid rafts) of intact cells, and is useful for visualizing cholesterol-rich membrane domains on the cell surface. To visualize further intracellular cholesterol-rich membrane domains of living cells, we established a cellular system. We have shown that the specificity and high affinity of BCθ for cholesterol and its property of targeting to cholesterol-rich membrane domains can all be ascribed to the C-terminal domain (D4) of θ-toxin. Based on this finding, we constructed a fused protein of D4 and enhanced green fluorescent protein (EGFP). First, the fused protein, EGFP-D4,was overexpressed in E. coli and purified for characterization. We confirmed that EGFP-D4 is a fluorescent protein targeting to cholesterol-rich membrane domains. Then, an EGFP-D4 DNA fragment was inserted into a vector for mammalian gene expression and introduced into mouse fibroblasts. We isolated several stable cell lines expressing EGFP-D4 at variable levels and showed that the expressed EGFP-D4 was partially associated with intracellular vesicular structures by fluorescence microscopy. Characterization of the vesicles is now under way.
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