| Project/Area Number |
16390532
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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 |
Functional basic dentistry
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| Research Institution | Health Sciences University of Hokkaido |
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Principal Investigator |
TANIMURA Akihiko Health Sciences University of Hokkaido, SCHOOL OF DENTISTRY, ASSOCIATE PROFESSOR (70217149)
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| Co-Investigator(Kenkyū-buntansha) |
TOJYO Yosuke HEALTH SCIENCES UNIVERSITY OF HOKKAIDO, SCHOOL OF DENTISTRY, PROFESSOR (90111731)
MORITA Takao HEALTH SCIENCES UNIVERSITY OF HOKKAIDO, SCHOOL OF DENTISTRY, ASSISTANT PROFESSOR (20326549)
NEZU Akihiro HEALTH SCIENCES UNIVERSITY OF HOKKAIDO, SCHOOL OF DENTISTRY, ASSISTANT PROFESSOR (00305913)
東城 庸介 北海道医療大学, 歯学部, 教授 (90111749)
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| Project Period (FY) |
2004 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥14,170,000 (Direct Cost: ¥13,900,000、Indirect Cost: ¥270,000)
Fiscal Year 2007: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 2004: ¥4,900,000 (Direct Cost: ¥4,900,000)
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| Keywords | inositol 1,4,5-trisphosphate / fluorescent resonance energy transfer / fluorescent probe / fluorescent proteins / intracellular calcium ion / screening / imaging / inositol 1,4,5-trisphosphate receptor / イノシトール三リン酸 / イノシトール三燐酸 / カルシウム / FRET / IP_3受容体 / calcium / IP3受容体 |
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| Research Abstract |
LIBRA is a fluorescent biosensor for inositol 1,4,5-trisphosphate (IP_3), which is composed of the ligand-binding domain of the rat type III IP_3 receptor (IP_3R) and cyan and yellow fluorescent proteins. In the present study, we developed a series of IP_3 biosensors that exhibit strong pH stability and varying affinities for IP_3, as well as a method for the quantitative measurement of cytosolic concentrations of IP_3([IP_3]_i] in single living cells. Improved biosensors and the method for the quantitative measurement were used to elucidate IP_3dynamics during agonist-induced Ca2+ oscillations. Our result demonstrated cell type-dependent differences in IP_3dynamics; a non-fluctuating rise in [IP_3]_i and repetitive IP_3spikes during Ca^2+ oscillations in COS-7cells and HSY-EA1cells, respectively. The size of IP_3spikes in HSY-EA1cells varied from10to100nM, and the second and later spikes were initiated before the decline of [IP_3]_i to resting levels. While [IP_3]_i showed clear fluctuations at the beginning of Ca^2+ oscillations, repetitive spikes of [IP_3]_i gradually obscured during Ca^2+ oscillations. In addition, [IP_3]_i spike peaks were preceded by Ca^2+ spike peaks. These observations do not support the requirement of IP_3fluctuations in Ca^2+ oscillations.
It is also found that LIBRA and its IP_3-insensitive variant should facilitate the detection of specific agonists and antagonists of the ligand-binding site of IP_3Rs. We applied this method for the screening of novel ligand for IP_3Rs. On the basis of computational docking of the ligands to the binding domain of the type I IP_3R, we synthesis of four novel metabolically stabilized analogues of IP_3. Of these analogues, two compounds were novel IP_3 ligands using LIBRA. We further examined the subtype specificity of adenophostin derivatives using series of LIBRA with ligand-binding domains of IP_3Rs from type I, II, or III.
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