| Project/Area Number |
12670117
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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 medical chemistry
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| Research Institution | Shimane Medical University |
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Principal Investigator |
TANIGAWA Yoshinori School of Medicine, Shimane Medical University, Professor, 医学部, 教授 (60084860)
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| Co-Investigator(Kenkyū-buntansha) |
MITANI Toshifumi School of Medicine, Shimane Medical University, Instructor, 医学部, 助手 (80335554)
並河 東明 島根医科大学, 医学部, 助手 (40304274)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2000: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Keywords | RAW 264.7 cell / iNOS / ubiquitin / proteasome / ユビキチン / 免疫抑制 |
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| Research Abstract |
1) In RAW 264.7 cells, PMA synergistically increased IFN-γ-induced iNOS activity, but PMA alone failed to increase iNOS activity. PMA might modulate iNOS induction as a cosignal with IFN-γ in RAW 264.7 cells because the synergistic effect of PMA was mediated through IRF-1. PMA together with IFN-γ increased iNOS mRNA without affecting the iNOS mRNA degradation, suggesting that the synergistic effect of PMA on IFN-γ-induced iNOS mRNA production may be depend on the elevation of the transcription rate.
2) The lysates from RAW 264.7 cells treated with IFN-γ and PMA were subjected to SDS/PAGE followed by immunoblotting with an anti-iNOS antibody. In addition to markedly augmented iNOS protein level in the density of 130 KDa band compared with the IFN-γ or PMA alone, the tailing to higher molecular complexes (>130 KDa) of iNOS were observed by immunoblots, particularly after the treatment of proteasomal inhibitors, such as lactacyctin, MG132 and N-acetyl-L-leucinyl-L-leucinyl-norleucinal.
3) Treatment with the proteasomal inhibitors resulted in the accumulation of iNOS, in contrast, calpastatin inhibitor and trypsn-like protease inhibitor did not lead to the accumulation of iNOS, and similar results were obtained using the lysosomal protease inhibitors lupeptin and pepstatin-A. These results suggested that the accumulated iNOS after proteasomal inhibition is ubiquitinated and the ubiquitination of iNOS is required for its degradation.
4) Partially purified iNOS and conjugase was incubated in the presence of ^<125>I-GST-ubiqutin and ATP. After the immunoprecipitation using anti-iNOS antibody, samples were subjected to SDS/PAGE ^<125>I-conjugation of >130 KDa was observed by autoradiography.
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