Analyses of responsible domain inducing functional difference of polymorphic murine osteopontin protein
| Project/Area Number |
15590346
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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 |
Experimental pathology
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| Research Institution | Ehime University |
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Principal Investigator |
MIYAZAKI Tatsuhiko Ehime University, School of Medicine, Instructor, 医学部, 助手 (80239384)
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| Co-Investigator(Kenkyū-buntansha) |
NOSE Masato Ehime University, School of Medicine, Professor, 医学部, 教授 (70030913)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | Osteopontin / Allelic polymorhism / Cell Free protein synthesis / Structural analysis protein / Cytokine / Functional analysis / Congenic inbred mice / Glomerulonephritis / IL-1β / 1prマウス / 蛋白多型 / 無細胞蛋白合成システム / 構造解析 / サイトカイン / 蛋白機能解析 / コンジェニックインブレッド |
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| Research Abstract |
We previously identified Opn as a candidate gene susceptible to glomerulonephritis in MRL/lpr mice by genome wide screening using (MRL/lpr x C3H/lpr)F_2 mice, followed by a functional assay of synthetic polymorphic OPN peptides of MRL and C3H type. To identify the responsible domain to the functional difference between these alleles, we generated mutant Opn peptides which were modified in substituted amino acid between two alleles followed by structural and functional analyses. Also, to determine the role of the allelic difference in vivo, we made selective congenic mice of Opn gene locus which have C3H allele Opn in MRL background, and then, carried out in vivo and histopathological analyses on the congenic mice.
1)We synthesized MRL/Mp type (allele a) and C3H/HeJ type (allele b) OPN peptide using Cell Free protein synthesis system, then analyzed these peptides by SDS-PAGE and Western blotting.
2)Also, using PCR based mutagenesis technique, we generated mutated Opn peptides which were m
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odified a substitution site between allele a and allele b, then analyzed as same as above.
3)To identify the responsible polymorphic domain, we carried out bioassays using modified polymorphic Opn peptides on splenic cells and macrophages derived from MRL-Fas^<+/+> mice. As a result of these analyses, we manifested a certain substitution site dramatically modified the functional difference in cytokine inducing in this system. That polymorphic site might seem to be involved in the functional difference of these alleles.
4)We established Opn locus specific congenic inbred mice strain MRL/lpr-Opn^<C3H/C3H> in MRL/lpr x (MRL/lpr x C3H/lpr)N_<13>F_2 mice. By using MRL/lpr-Opn^<C3H/C3H>, MRL/lpr-Opn^<MRL/C3H>, and MRL/lpr- Opn^<MRL/MRL> mice in N_<13>F_2 mice, we confirmed that MRL/lpr-Opn^<C3H/C3H> mice had marked reduction in incidence of glomerulonephritis.
These results suggest that an amino acid substitution in allelic polymorphism of murine Opn may play a critical role for the development of glomerulonephritis in MRL/lpr mice. Less
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Report
(3 results)
Research Products
(24 results)
