| Project/Area Number |
13470140
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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 | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SEKO Yoshinori The University of Tokyo, Faculty of Medicine, Lecturer, 医学部附属病院, 講師 (30240708)
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| Co-Investigator(Kenkyū-buntansha) |
SAKURAI Takeshi University of Tsukuba, pharmacology, Associate Professor, 基礎医学系, 助教授 (60251055)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 2002: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2001: ¥10,000,000 (Direct Cost: ¥10,000,000)
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| Keywords | G protein / orphan receptor / myocardial tissue / cardiac function / intracellular calcium |
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| Research Abstract |
1. (A) To identify the specific ligand s for orphan G protein -coupled receptors (GPCRs) expressed in myocardial tissue, we analyzed 7 GPCRs known to be expressed in cardiac myocytes and 6 other GPCRs of which we confirmed their expression in myocardial tissue. We extracted peptide -rich fraction from bovine myocardial tisuue with ether and C18 column and separated it by reverse -phase chromatography. Then we analyzed calcium releasing activity of these peptide fractions in HEK293T cells transfected with GPCRs by fluorescent imaging plate reader (FLIPR) assay (1^<st> screening). We found that there was a significant calcium release in one of the GPCRs -transfected cells with a certain fraction. We thought that this fraction contained a specific ligand for the GPCR because we confirmed the reproducibility five times. We are further purifying the ligand by second screening. (B) We also analyzed calcium release by using melanophore cells transfected wit h GPCRs, and found positive reactions in the same GPCR -transfected cells, however, we could not reproduce the result. 2. Because hypothalamic ependymal cells seem to secrete some humoral factors which specifically stimulates cardiac myocytes and pituitary cells by enhancing calcium release, we started their purification by FLIPR assay and melanophore cells. We found that some peptide fractions from culture supernatant of hypothalamic ependymal cells separated by reverse-phase chromatography stimulated calcium release in rat cardiac myocytes and a quail myocardial cell line. We are currently proceeding the purification by cAMP assay and reverse-phase chromatography.
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