| Project/Area Number |
17300128
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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 |
Neurophysiology and muscle physiology
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| Research Institution | Yamaguchi University |
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Principal Investigator |
KOBAYASHI Sei Yamaguchi University, Grad. School of Med., Dept. of Mol. Physiol, Professor (80225515)
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| Co-Investigator(Kenkyū-buntansha) |
KISHI Hiroko Yamaguchi Univ., Grad. School of Med., Dept. of Mol. Physiol, Associate Professor (40359899)
KAWAMICHI Hozumi Yamaguchi Univ., Grad. School of Med., Dept. of Mol. Physiol, Assistant Professor (80363042)
加治屋 勝子 山口大学, 医学部, 助手 (00379942)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥15,740,000 (Direct Cost: ¥14,900,000、Indirect Cost: ¥840,000)
Fiscal Year 2007: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2006: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2005: ¥9,300,000 (Direct Cost: ¥9,300,000)
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| Keywords | Vascular Smooth Muscle / Mass Spectrometry / Membrane Raft / Signal Transduction / Functional Proteomics |
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| Research Abstract |
Vascular diseases are life-threatening diseases with high mortality in Japan. Abnormal vascular contraction, such as vasospasm, is known as major cause of sudden death due to vascular diseases. We identified sphingosylphosphorylcholine (SPC) as a novel mediator which causes abnormal vascular contraction mediated by Rho-kinase. In this study, we attempted to identify downstream molecules of SPC and their regulatory mechanisms, and obtained the following results.
1. We found that SPC-induced abnormal contraction depends on cholesterol, and cholesterol-enriched membrane domain, membrane raft, plays a pivotal role in abnormal contraction of human vascular smooth muscle. These results were introduced as very high impact discovery in the Editorial Section of Circulation Research.
2. We obtained recombinant proteins of mutated Fyn, namely, wild type of Fyn, constitutively active Fyn, and dominant negative Fyn. Using cytosolic application of these recombinant mutated Fyn proteins by membrane permeabilization, we demonstrated the first direst evidence that Fyn tyrosine kinase plays an important role in abnormal contraction of vascular smooth muscle induced by SPC and Rho-kinase.
3. We showed for the first time that Ca^<2+>-independent sliding of actomyosin in an in vitro motility assay of highly purified contractile proteins.
4. We succeeded to make lipid raft model membrane as a hybrid liposome and found that SPC selectively binds to membrane raft.
5. We succeeded to purify membrane raft fraction from human vascular smooth muscle, and identified several novel proteins localized in membrane raft, using functional proteomics by tandem mass spectrometer.
6. We identified several molecules which can selectively inhibit the signal transduction of abnormal vascular contraction, which was clarified by this study.
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