1998 Fiscal Year Final Research Report Summary
Role of myosin-binding poteins in myofibrillogenesis
| Project/Area Number |
08044191
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Field |
動物生理・代謝
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| Research Institution | DEPARTMENT OF BIOLOGY,CHIBA UNIVERSITY |
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Principal Investigator |
OBINATA Takashi Chiba Univ.Dept.of Biology Professor, 理学部, 教授 (40012413)
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| Co-Investigator(Kenkyū-buntansha) |
STOCKDALE Fr スタンフォード大学, 医学部, 教授
WELIKSON Rob コーネル大学, 医学部, 研究員
FISCHMAN Don コーネル大学, 医学部, 教授
SATO Naruki Chiba Univ.Dept.of Biology Research Associate, 理学部, 助手 (40261896)
WELIKSON Robert E. Cornell Univ. Medical School Research Associate
STOCKDALE Frank E. Stanford Univ.Medical School Professor
FISCHMAN Donald A. Cornell Univ Medical School Professor
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| Project Period (FY) |
1996 – 1998
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| Keywords | C-protein / H-protein / Myosin-binding protein / Myofibril / Myofibrillogenesis / Muscle cells / Myosin / Actin |
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| Research Abstract |
C-protein is a major myosin-binding protein characteristic of striated muscles. In order to clarity the roles of C-protein in myofibrillogenesis, we carried out the following experiments and obtained the results as described.
1) Mouse C-protein cDNA sequences and C-protein isoform expression
The cDNAs encoding mouse fast skeletal (F) and slow skeletal (S) C-proteins were cloned and their entire sequences were determined. Cardiac (C)-isoform is known to be expressed first and transiently during development of chicken skeletal muscles, but in mouse skeletal muscle, C-isoform was not detected through developmental stages. S-isoform became detectable first, followed by appearance of F-isoform, In the degenerating mouse skeletal muscles, expression of S-isoform is increased.
2) Two isoforms in chicken cardiac C-protein and their expression
We detected two variants (type I and type II) in chicken cardiac C-protein and determined their entire sequences ; they differ only by the inclusion or exclu
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sion of a sequence of 15 amino acid residues (P-seq) which includes a phosphorylation site for A-kinase. We prepared the antibody specific for P-seq. By immunocytochemical methods with this and the other monoclonal antibody to cardiac C-protein (C-315), we found that two isoforms were coexpressed and colocalized in both atrial and ventricular muscles through development, but type I was scarcely expressed in embryonic skeletal muscle. These observations were confirmed by a RT-PCR method.
3) Competition of C-protein (MyBP-C) with H-protein (MyBP-H) for binding to myosin in cardiac muscle cells
C-Protein and H-protein are myosin-binding proteins which localize in A-band regions of sarcomeres. To clarify functional relationship of these two myosin binding proteins, we introduced cDNAs encoding these proteins into primary cultures of chicken myocytes and examined their assembly into myofibrils. The results obtained indicate that overexpression of MyBP-C or MyBP-H in cultured cardiac myocytes can reduce the content of MyBP-H or MyBP-C, respectively. These data indicate that MyBP-H can compete with MyBP-C for binding to myosin in the cells. Less
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[Publications] Nagaoka, R., Minami, N., Hayakawa, K., Abe, H.and Obinata, T.: "Quantitative analysis of low MW-actin-binding proteins, cofilin, ADF and profilin, expressed in developing and degenerating chicken skeletal muscles." J.Muscle Res Cell Motil.17. 463-473 (1996)
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[Publications] Endo, T., Matsumoto, K., Hama, T., Ohtsuka, Y., Katsura, G.and Obinata, T.: "Distinct troponin T genes are expressed in embryonic/larvel tail striated muscle and adult body wall smooth muscle in ascidian." J.Biol.Chem.271. 27855-27862 (1996)
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[Publications] Obinata, T., Nagaoka-Yasuda, R., Ono, S., Kusano, K., Mohri, K., Ohtaka, Y., Yamashiro, S., Okada, K., and Abe, H.: "Low molecular-weight G-actin binding proteins involved in the regulation of actin assembly during myofibrillogenesis." Cell Str.Func.22 (1). 181-189 (1997)
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[Publications] Begum, S., Komiyama, M., Toyota, N., Obinata, T., Maruyama, K.and Shimada, Y.: "Differentiation of muscle-specific proteins in chicken somites as studied by immuno-fluorescence microscopy." Cell Tiss.Res.293. 305-311 (1998)
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[Publications] Kurasawa, M., Sato, N., Matsuda, A., Koshida, S., Totsuda, T.and Obinata, T.: "Differentiation expression of C-protein isoforms in developing and degenerating mouse striated muscles." Muscle & Nerve. 22. 196-207 (1999)
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[Publications] Soeno, Y., Yajima, H., Kawamura, Y., Kimura, S., Maruyama, K.and Obinata, T.: "Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells." Mol.Cell Biochem.(in press). (1999)
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