| Project/Area Number |
04807021
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | University of Occupational and Environmental Health, School of Medicine |
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Principal Investigator |
HIRANO Hideyasu Univ.Occu.Env.Health ; Dept.Biochemistry Assistant Prof., 医学部, 講師 (50040241)
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| Co-Investigator(Kenkyū-buntansha) |
GOTOH Sadao Univ.Occu.Env.Health ; Dept.Biochemistry Assistant Prof., 医学部, 助教授 (50131917)
HIGASHI Ken Univ.Occu.Env.Health ; Dept.Biochemistry Professor, 医学部, 教授 (30028386)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1993: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1992: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | Fibronectin / Profilin / ECM / Basement membrane / Collagen / Controlling Morphology Proteins / Morphological control / Nephritis / 肝硬変 / DNA / RNA / 線維化 |
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| Research Abstract |
Extracellular adhesive proteins and membrane associated proteins actively function during wound healing, cell migration, differentiation, signal transduction, and during changes in morphology and motility.
We attempted to obtain cDNA that is actively expressed during early stage of liver injury. We assayd for positive cDNAs by Northern blot hybridization, sequenced the cDNA we obtained, and scanned the nucleic acid sequence data of GenBank and EMBL for homology matching. The sequence we found was for profilin, and its localization was determined using antibody against a synthetic peptide derived from the protein sequence. The antibody specificity was analyzed using Western blots of cytoplasmic extracts from fibrotic liver and spleen.
By screening lgt11 cDNA libraries using antibodies against basement membrane, 32 positive clones were obtained. The 21st positive clone coded for profilin, and its mRNA levels increased gradually and reached a maximum at 12 hrs after the first subcutaneous injection of the fibrogenic compound CC14 into rats. Further analysis was performed after the 13th injection of CC14 treated rat livers, and it was found that the acute phase increases in levels of profilin mRNA were similar either with or without previous chronic CC14 administration. Increased mRNA levels of profilin at 72 hrs after the last injection were also detected using chronically injected rat livers at the 10th and 12th week. Our affinity purified rabbit polyclonal antibody againt a profilin peptide(anti-mpFTM) reacted with profilin specifically on Western blots. The localization of profilin at newly forming fibrotic septa was detected by immunofluorescence microscopy using anti-mpFTM antibody.
We found increased profilin gene expression during early stage of liver injury and profilin localization at newly formed fibrotic septa. Our experimental results suggest that profilin actively functioned in liver fibro sis induced by carbon tetrachloride.
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