| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Based on the published data about 3-D configuration of CGRP, we have attempted to produce some CGRP fragments and performed bioassay with those new peptides. However, we could not particularly find any bioactive peptides.
1) Production of CGRP fragments and screening - In addition to commercially available human CGRP_<1-37>, CGRP_<8-37>, CGRP_<19-37>, CGRP_<22-37> and [Cys(Acm)^<2,7>]-CGRP, we have produced CGRP_<1-12>, reverse-CGRP_<1-8> and reverse-CGRP_<1-14>. On screening with cAMP production and other indeces, we have found that CGRP_<8-37>, CGRP_<22-37> and [Cys(Acm)^<2,7>]-CGRP have substantial agonistic or antagonistic action. 2) Effects of bioactive CGRP fragments on intracelllar signaling pathways - In osteoblastic MG63 cells, CGRP_<8-37> potently anta gonized CGRP_<1-37> action in cAMP production. CGRP_<22-37>, also had a weak antagonistic action. [Cys(Acm)^<2,7>]-CGRP showed both a medium antagonistic and a weak agonistic action. Similar results were obtained in either gingival fibroblastic Gin-1 or oral epithelial SCC25 cells. 3) Binding affinity of bioactive CGRP fragments - Fluoro-CGRP bound to its receptors in a concentration-dependent fashion, and its binding was specifically replaced with CGRP_<8-37>, but not clearly with [Cys(Acm)^<2,7>]-CGRP. 4) Effects of CGRP fragments on cell proliferation - Any CGRP fragments produced here failed to reproducibly influence the proliferation, but only CGRP_<1-37> weakly stimulated Gin-1 proliferation. 5) Effects of CGRP fragments on cell apoptosis - Any CGRP fragments did not apparently induce apoptosis in any cell types used here. 6) Expression of CGRP receptor subtypes in periodontal tissues - Expression of both CRLR and RAMP-I proteins were detected using those specific antibodies either in periodontal tissues or in any cell types used here.
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