| Project/Area Number |
14370239
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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 |
Pediatrics
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
MINEGISHI Masayoshi Tohoku University, Hospital, Associate Professor, 病院, 助教授 (20211592)
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| Co-Investigator(Kenkyū-buntansha) |
OHASHI Yoshiyuki Tohoku University, Hospital, Research Associate, 病院・助手 (60250825)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥13,400,000 (Direct Cost: ¥13,400,000)
Fiscal Year 2004: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2003: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2002: ¥12,100,000 (Direct Cost: ¥12,100,000)
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| Keywords | Wiskott-Aldrich syndrome / WAS protein / beta-1 integrin / Signal transduction / Cytoskeletal organization / 細胞骨格 / beta1-インテグリン |
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| Research Abstract |
WASP, the product of the gene mutated in Wiskott-Aldrich syndrome (WAS), is expressed only in lymphohematopoietic cells and is the first identified member of an expanding family of proteins involved in signaling and cytoskeletal reorganization. Recent reports have helped to understand b1-integrin activation as an important role in controlling the F-actin polymerization. In the present stgudy, we investgigated whether the interaction between the signaling through b1-integrin and that associated with WASP could be involved in the cytoskeletal organization and explored the possibility of gene therapy for WAS patients. The results obtained in the experiments were showed as follows : 1)a novel monoclonal antibody (mAb) against WASP by immunizing mice with the recombinant protein was produced. The mAb was highly specific to WASP and suitable for Western blot analysis and immunoprecipitation. 2)A flow cytometric assay using the mAb identified expression of intracytoplasmic WASP in lymphocytes
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from normal individuals. Double staining analysis with cell surface CD3, CD19, abd CD56, and intracytoplasmic molecules revealed WASP expression in each subpopulation. With regard to WASP expression in patients with WAS and X-linked thrombocytopenia (XLT), peripheral blood mononuclear cells (PBMCs) from nine patients and Epstein-Barr virus-transformed B-lymphoblastoid cell lines from seven patients examined did not show WASP expression by flow cytometric analysis. These results were confirmed by Western blot analysis. 3)WASP expression was first demonstrated in the subset of peripheral blood T cells and NK cells from two WAS patients by double staining analysis. 4)WASP was tyrosine phosphorylated by solid-phase fibronectin stimulation in leukemic T cell line, L-KAW. The same results was obtained by immobilized CD29 mAb stimulation experiments. Moreover, preincubation with cytochalasin B before fibronectin stimulation inhibited WASP tyrosine phosphorylation. The result indicated that the alteration of F-actin-based cytoskeleton was involved in WASP tyrosine phosphorylation via b1-integrin. These findings suggested the association of WASP in the b1-integrin-mediated signaling pathways. Less
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