2004 Fiscal Year Final Research Report Summary
Analysis of stabilization and degradation mechanism of muscle fiber of vascular smooth muscle cell upon transformatioon
| Project/Area Number |
15590224
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General pharmacology
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| Research Institution | Mie University |
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Principal Investigator |
OKAGAKI Tsuyoshi Mie University, Faculty of Bioresouces, assistant professor, 生物資源学部, 助教授 (80185412)
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| Co-Investigator(Kenkyū-buntansha) |
OOI Atsushi Mie University, Faculty of Bioresouces, research associate, 生物資源学部, 助手 (70203693)
NAKAMURA Akio Gunma University, Graduate School of Medicine, lecturer, 大学院・医学研究科, 講師 (30282388)
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| Project Period (FY) |
2003 – 2004
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| Keywords | protein / pharmacology / myosin / cell, tissue / atherosclerosis / hypertension / brain blood vessel |
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| Research Abstract |
Cultured vascular smooth muscle cell line AC01 become to be in transformed state in the presence of PDGF, whereas it to be in well differentiated state in the presence of sodium butyrate (NaB). mRNA was isolated from AC01 cells cultured under these two types of conditions to make cDNA. We examined expression of several marker protein such as α-actin, high molecular weight caldesmon. By RT-PCR analysis, expression level of these two proteins was down regulated upon addition of PDGF, and up regulated in the presence of NaB. We further made subtraction library from these two types of cDNA as described below.
To examine the nature of p32, a myosin regulatory protein, in living smooth muscle cell, we raised antibody against N-terminal signal sequence of the protein. By indirect immunofluorescence microscopy the antibody stained cytoskeletal filaments, suggesting the localization on myosin filaments. And western blotting using antibody against p32mat, that stain both p32mat and p32FL, showed that most of p32 existing in cytoskeleton of AC01 cell is p32FL (42 kDa) but not p32mat (38 kDa). These observations suggest that p32 is binding to myosin filament as p32FL containing signal sequence. Although p32mat bind to myosin and stimulate its assembly, p32FL expressed in E. coli did not interact with myosin. The discrepancy indicates that p32FL should be post-translationaly modified to interact with myosin in vivo.
We further tried to make subtraction library to screen specific gene that is up regulated upon transformation. Using two types of cDNA that are obtained from PDGF treated and NaB treated cells, PCR-select subtraction have been performed to hybridize common gene in these two groups of cDNA. Subtracted cDNA was subcloned to plasmid to construct library.
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Research Products
(11 results)
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[Journal Article] Biochemical properties of ordinary and dark muscle myosin from carp skeletal muscle
Author(s)
Okagaki, T., Takami, M., Hosokawa, K., Yano, M., Higashi-Fujime, S., Ooi, A.
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Journal Title
Description
「研究成果報告書概要(和文)」より
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[Journal Article] Biochemical properties of ordinary and dark muscle myosin from carp skeletal muscle.
Author(s)
Okagaki, T., Takami, M., Hosokawa, K., Yano, M., Higashi-Fujime, S., Ooi, A.
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Journal Title
J.Biochem., (on revision)
Description
「研究成果報告書概要(欧文)」より
