| Project/Area Number |
12670436
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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 |
内科学一般
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| Research Institution | Saitama Medical School |
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Principal Investigator |
TSUZAKA Kensei Saitama Medical School, Medicine, Assistant Prof., 医学部, 講師 (00245490)
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| Co-Investigator(Kenkyū-buntansha) |
TAKEUCHI Tsutomu Saitama Medical School, Medicine, Prof., 医学部, 教授 (50179610)
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| Project Period (FY) |
2000 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2003: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | Systemic lupus erythematosus / TCRζ / mRNA3'UTR / alternative splicing / MA5.8 / signal transduction / mRNA3'UTR / In vitro translation / dimerization / 半定量PCR法 |
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| Research Abstract |
We recently reported that expression of the T-cell receptor ζchain (TCRζ) was significantly decreased or absent in systemic lupus erythematosus (SLE) patients due to the TCRζ mRNA with alternatively spliced short 3'UTR (TCRζ mRNA/as-3'UTR) (Int. Immunol 1998, J.Autoimmunity 1998, Arthritis Rheum, 1999). Here we investigated the mechanisms of the decreased expression of TCRζ in SLE T cells. 1)By the semi-quantitative PCR, TCRζ mRNA/as-3'UTR was predominantly detected in the SLE patients compared with the normal controls. 2)By using the in vitro translation assay, TCRζ mRNA/w-3'UTR consistently coded much more 16-kD TCRζ monomer and homodimer than TCRζ mRNA/as-3'UTR. 3) TCRζ cDNA/w-3'UTR and TCRζ cDNA/as-3'UTR was amplified and ligated into pDON-AI vector. Then the MA5.8 mutants were constructed by transferring the recombinant retrovirus. Western blot analysis using an anti-TCRζ antibody showed decreased expression of the TCRζ monomer and homodimer in AS3'UTR cells. FACS analysis and the IP using the cell surface paroteins using TIA-2 and anti-CD3ε antibody demonstrated downregulation of the cell surface expression of TCRζ and TCR/CD3 complex on the AS3'UTR cells. The expression of IL-2 and CD28 in the AS3'UTR cells was up-regulated after stimulation with anti-CD3 antibody. Also the TCRζ mRNA stability of AS3'UTR cells was significantly lower than that in WT cells. From these observations, predominant expression of TCRζ mRNA/as-3'UTR in SLE T cells is responsible for the decreased expression of TCRζ because of the reduced stability of TCRζ mRNA.
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