| Project/Area Number |
05670896
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Hematology
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| Research Institution | 1st Department of Internal Medicine, Faculy of Medicine, University of Tkyo |
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Principal Investigator |
HIGASHIHARA Masaaki Tokyo University Medicine Assistant, 医学部(病), 助手 (80165084)
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| Co-Investigator(Kenkyū-buntansha) |
WATABE Shugo Tokyo University Agriculture Associated P., 農学部, 助教授 (40111489)
YONEYAMA Akiko Tokyo University Medicine Assistant, 医学部(病), 医員 (50175684)
MIURA Noboru Tokyo University Medicine Medical Staff, 医学部(病), 医員
SUNAGA Shinji Tokyo University Medicine Assistant, 医学部(病), 助手
小山 真理子 東京大学, 医学部(病), 医員
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Stroma cells / myosin / phosphorylation / phosphatase / monoclonal antibody / モノクロナル抗体 / 骨髄幹細胞 / 細胞骨格蛋白 / サイトカイン / フォスファターゼ / 血小板 |
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| Research Abstract |
We made Monoclonal antibodies (12 clones) specifi for myosin heavy chain of non-muscle cells, although we could not detect the transformation of myosin heavy chain in several stroma cells. We also made DNA probes for these myosin isoforms. However, RT-PCR was unsuccessful using mRNA of HEL cells. Using cDNA of myosin heavy and light chain, we tried the cloning of myosin heavy and light chain of human hematopoietic cells. The problem is that cDNA library of human bone marrow cells we made, was not good. We tried the tranfection of cytokine genes into stroma cell lines (HFL,HUC-Fm) using retrovirus vetors. However, data were variable and non-reprodusible.
We Made success in making ten monoclonal antibodies agaist myosin-associated protein phosphatase (MAPP), which was gift of Dr.M.Ikebe, CWRU.However all antibodies had lgM isotype and unfavorable for immunoblotting. Thus we made now clones (lgG) against non-catalytic subunit of phosphatase. We are now working on the characterization of th
… More
ese antibodies. In the clarifying the role of myosin phosphorylation on chilling -induced platelet shape change, four types of shape change were observed and these changed in a time-dependent fashion. The atractive finding is that these shape change were correlated with the extent of phosphorylation of LC20, and these changes were reversible. Contribution of myosin on the shape changes was further confirmed by two findings. First, association of myosin into the triton X-insoluble fractions were proceded in a time-ddependent fashion. Secondly, electron microscopic observation shwed that myosins were associated in the bundles of fibers in the filopodias. We previously reported the purifiation of myosin-associated protein phosphatase and Q10 of this phosphatase is very high.i.e.about 5.0 in contrast to MLCK 2.0. The suppression of this phosphatase activity was much more than that of MLCK during the change in temperature from 37C to 0-4ﾟC.This unbalace can induced the phosphorylation of LC20 even if [Ca^<2+>] i does not increase by chilling. Less
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