| Project/Area Number |
11470006
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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 |
General anatomy (including Histology/Embryology)
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| Research Institution | KYOTO PREFECTURAL UNIVERSITY OF MEDICINE |
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Principal Investigator |
KAWATA Mitsuhiro KYOTO PREFECTURAL UNIVERSITY OF MEDICINE, PROFESSOR, 医学部, 教授 (60112512)
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| Co-Investigator(Kenkyū-buntansha) |
MORITA Noriyuki KYOTO PREFECTURAL UNIVERSITY OF MEDICINE, ASSISTANT PROFESSOR, 医学部, 講師 (50239662)
NISHI Mayumi KYOTO PREFECTURAL UNIVERSITY OF MEDICINE, ASSISTANT PROFESSOR, 医学部, 講師 (40295639)
OZAWA Hitoshi KYOTO PREFECTURAL UNIVERSITY OF MEDICINE, ASSOCIATE PROFESSOR, 医学部, 助教授 (60169290)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2000: ¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1999: ¥8,400,000 (Direct Cost: ¥8,400,000)
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| Keywords | GFP / CULTURED CELLS / GLUCOCORTICOID RECEPTOR / MINERALOCORTICOID RECEPTOR / ESTROGEN RECEPTOR / ANDROGEN RECEPTOR / REALTIME IMAGING / CYTOPLASM-NUCLEUS TRAFFICKING / リガンド |
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| Research Abstract |
GFP-GR chimera construct was transfected into the cultured cells. By using luciferase assay GFP-GR chimera system was confirmed to be functional. DEX-inducible mouse mammary tumor virus promoter (MMTV)-Luc reporter was induced by DEX.Real-time imaging study has shown that in the absence of ligand GFP-GR was present in the cytoplasm of cells. Exposure of DEX to cultured cells induced the nuclear accumulation of GFP-GR and its translocation from cytoplasm-to-nucleus was time-dependent manner. Thirty min after DEX treatment GFP-GR was completely translocated into the nucleus. No green fluorescence was observed in the nucleolus. The same results were observed in the primary cultured hippocampal neurons and cortical glia. GFP-GR from the small cytoplasm of the perikarya to the nucleus was observed after 15 to 30 min after the DEX as well as corticosterone treatment, indicating that there was no conspicuous difference of trafficking manner of GR in the presence of the ligands among cell types. GFP-MR chimera construct was also transfected into COS-1 cells, hippocampal neurons, and cortical glia. As in the case of GFP-GR, GFP-MR chimera construct should be verified to be transcriptionally active before observing the real-time imaging. GFP-MR was observed in the cytoplasm of most of the COS-1 cells without the ligand. Aldosterone, an agonist for MR, induced the translocation of GFP-MR from the cytoplasm to the nucleus in a time-dependent manner. Within 30 min after the treatment green fluorescence was completely present in the nucleus and nucleolus was devoid of GFP-MR.Corticosterone treatment caused the similar accumulation of GFP-MR in the nucleus of the COS-1 cells where no fluorescence was observed at the nucleolus. Cells that were transfected by GFP-ERa construct and were not treated by estrogen showed the GFP-ERa in the nucleus. Nucleolus was devoid of GFP-ERa.
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