| Project/Area Number |
03557034
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
内科学一般
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| Research Institution | THE UNIVERSITY OF THE AIR |
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Principal Investigator |
KITO Shuzo The University of the Air Div.of Health Sciences Professor, 教養学部, 教授 (00010140)
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| Co-Investigator(Kenkyū-buntansha) |
MIYOSHI Rie Tokyo Women's Sch.of Medicine Dept.of Pharm.Instructor, 助手 (80209965)
SEMBA Jun'ichi The University of the Air Div.of Health Sciences Associate Professor, 教養学部, 助教授 (30183429)
野本 照子 東京女子医科大学, 教授 (20075154)
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| Project Period (FY) |
1991 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥17,000,000 (Direct Cost: ¥17,000,000)
Fiscal Year 1993: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1992: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1991: ¥13,300,000 (Direct Cost: ¥13,300,000)
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| Keywords | calcium ion / fluorometry / cyclic AMP / A kinase / regulatory subunit / catalytic subunit / isothiocyanate / D1 and D2 receptors / A Kinase / holoenzyme / dopamine / D2受容体 / D1受容体 / 培養神経細胞 / Cyclic AMP / A-kina se / 蛍光標識 / tetramethylrhodamin / fluorescein / catalytic unit / regulatory unit / rhodamine |
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| Research Abstract |
The Method of intracellular calcium ion assay in which fura 2 and other calcium chelating reagents are used for fluorometry, has been well established. As for an qually important bioactive substance, cyclic AMP assay in living cell has not been successful.
In this study, we tried to assay cyclic AMP in living cells by fluorometry and to observe three dementionally by means of motor-driven focus controller in its image analysis.
cDNA of regulatory subunit of cyclic AMP dependent protein kinase (A kinase) was inserted into escherichiacoli and the protein was prepared.
For fluorescence labelling, tetramethylrhodamine was used for regulatory subunit and fluorescein isothyanate for catalytic subunit. These labelled proteins were microinjected into cells.
When 2 molecules of each of regulatory and catalytic subunit are in tetramer, fluorescence light from catalytic subunit can be the excitation light to regulatory subunit. When these subunit molecules are separated, fluorescence light from catalytic subunit itself is detected. This is the principle of fluorescence assay of cyclic AMP.We used mixed culture of the rat striatum in which D1 and D2 receptors coexisted. As widely known, D1 and D2 receptors act in opposite directions as regards to cyclic AMP level within cells. Activated D1 receptor decrease cyclic AMP, while D2 receptor increase it. For these opposite directions of action to cyclic AMP, it was not always possible to assay cyclic AMP at detectable level.
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