| Project/Area Number |
11672313
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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 |
Laboratory medicine
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| Research Institution | Osaka Medical College |
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Principal Investigator |
SHIMIZU Akira Osaka Medical College, Faculty of Medicine Professor, 医学部, 教授 (00028581)
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| Co-Investigator(Kenkyū-buntansha) |
NAKANISHI Toyofumi Osaka Medical College, Faculty of Medicine Assistant Professor, 医学部, 講師 (10247843)
NAKAGAWA Toshimasa Osaka Medical College, Faculty of Medicine Associate Professor, 医学部, 助教授 (30237226)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | complement / HCV / RF / immunoglobulin V regeon / mass spectrometry / cold complent activation / cold activation / proteomics / 補体cold activation / 体細胞高頻度突然変異 / 生殖細胞型遺伝子 / 慢性関節リウマチ / HCV感染 / Ga抗原エピトープ / ビアコア / エレクトロスプレーイオン化質量分析 / 補体価 / クリオグロブリン / HCV感染B細胞 / プロテオーム解析 / アルコール長期多飲者 |
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| Research Abstract |
In vitro activation of the classical pathway of complement at a low temperature is known as cold activation of complete and was first described in the 1970s by Kitamura et al. The former Professor, Shinaya Inai, of the Department of Clinical Pathology, Osaka Medical College, was the leading investigator for this project. We first described the relationship between this phenomenon and HCV infection. Further we found that rheumatoid factor in the serum of hepatitis C virus-infected patients showed an increase in the titer during cold storage. We hypothesized that in the serum of some HVC -infected patients, the affinity of RF appears to be weak, and the binding between the RF and IgG coasted on latex may be inhibited by the complement components of fresh serum.
We tried to analyze the V regeon repertoir of RF obtained from HCV infected and cole activation positive sera. Proteomics strategy was applied to the analysis. Immunogloburin was separated by 2-D electrophoresis on sodium dodecyl sulfate polyacrylamide gels (SDS Page) followed by enzymatic proteolysis of the isolated spot and peptide mapping by mass spectrometry. The germ line sequence with less mutation rate was anticipated. This case the low avidity of RF, which react with ligand (Fc of IgG, aggregated or denatured) only in low temperature.
By these analysis we obtained the sequence, MDMRVPAQLLGLLLLR (Ig k light chain 1-16) ASQSISSYLINWYQQPGKAPK (IG k light chain 47-67)@
DSTYSLSSTLTLSK (Ig k light chain 55-68), These sequence may represent low mutated sequences.
We need more sequence from infected and non-infected which ole activation and without cold activation. Quantitative analysis of protein in ultra mini scale is possible using mass spectrometry and isotope-coded affinity tags. Affinity capture-release electrospray ionization mass spectrometry is also powerful strategy to analyze complex samples in ultra-mini scale. This is also applicable to examine the clonality and mutation diversity.
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