| Project/Area Number |
04670257
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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 |
細菌学
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| Research Institution | Fujita Health University |
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Principal Investigator |
MIYAMA Akio Fujita Health University Microbiol.Professor, 医学部, 教授 (20075047)
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| Co-Investigator(Kenkyū-buntansha) |
IMAMURA Seiji Fujita Health University Microbiol.Res.Assistant, 医学部, 助手 (20247654)
KAWAMOTO Yasuko Fujita Health University Microbiol.Res.Associate, 医学部, 講師 (50090676)
井上 孝 藤田保健衛生大学, 衛生学部, 講師 (90121433)
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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 |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1992: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | iIEL / Granzyme A / Peptide antibody / Hybridoma / Cholera toxin / Forskolin / IEL / グランザイム / コレラ毒素 / CD_8陽性細胞 / 細胞傷害活性 |
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| Research Abstract |
1. We studied serine proteases in the cytoplasmic granules of murine intestinal intraepithelial lymphocytes(iIEL). Nothern blot analysis demonstrated that total cellular RNA in iIEL hybridized with oligo-probe of granzyme A but did not with that of granzyme B.We detected a 50-56 KDa serine protease highly reactive to DFP and an additional 28 KDa protease with a low affinity to DFP in nucleus-free lysates of iIEL.The 55 KDa protease was characterized as homodimer consisting of 33 KDa monomer and resided in the granules of IIEL.Its esterase activity displayd only against BLT and was inhibited by PMSF, DFP and FUT-175. Antibody raised against a synthetic peptide designed from CTL-granzyme A recognized 55 KDa protease but did not 33 KDa monomer.
2. Peroral administration of cholera toxin(CTx) suppresses BLT-esterase activity in mouse intestinal intraepithelial lymphocytes. One of the major effector molecules that exhibits such proteolytic activity in iIEL is a serine protease (granzyme A). To elucidate how CTx affect iIEL, we prepared T cell hybridoma (49D4) between BW5147 and iIEL.The proteolytic activity of 49D4 was suppressed by CTx, but not by forskolin, although both reagents agitated cytoplasmic cAMP level. Slot blot hybridization showed that CTx reduced granzyme A transcripts. Similarly, actinomycine D reduced granzyme A transcript but did not inhibit the proteolytic activity. These findings suggest that granzyme A has a long life span in the cytoplasma, thus proteolytic activity in these lymphocytes does not always correlate with the amount of granzyme A transcripts. From these results, possible explanation would be that CTx suppresses the proteolytic activity via a more rapid pathway than suppression on granzyme A transcription, and this pathway is cAMP independent.
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