| Budget Amount *help |
¥3,950,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
In order to understand the phylogenetic characterization of amelogenin protein in lower vertebrates, cDNA sequences of amelogenin in reptile (iguana) and amphibia (newt) were determined, then immunohistochemical and western blot analyses were performed in them, using antibodies against the peptides consisting the N-terminus, C-terminus and central domain of their amelogenin. These antibodies were termed as follows by the recognizing portions of amelogenin in iguana and newt : IN (iguana N-terminus), IM (iguana central domain), IC (iguana C-terminus), NN (newt N-terminus), NM (newt central domain) and NC (newt C-terminus). ELISA analysis indicated that each antibody had a high specificity for the corresponding peptide. Immunohistochemical analysis was examined by the protein-A-gold method, for the iguana using IN,IM and IC, and for the newt using NN, NM and NC. Consequently, all antibodies, except NC, showed intense and uniform immunoreactivities over the enamel matrix and the secretory granules of the ameloblasts in iguana and newt, respectively. Only NC showed ununiform distribution of reactivities over the enamel matrix in newt. The intense reactions with NC were concentrated on the surface layer of an enamel matrix and decreased gradually toward the middle layer to deep layer. Similar phenomenon was reported by Uchida, et. al.(1991) by immunohistochemical demonstration of amelogenin in the porcine tooth enamel formation using the antibody against the C-terminus of the porcine amelogenin. The present study, therefore, may indicates that the newt has the degraded mechanism of the amelogenin similar to mammalian amelogenesis. Western blot analysis, however, showed the presence of 22 kDa amelogenin in both newt and iguana but the absence of low molecular weight of amelogenins under 20 kDa in them. These data suggest that the amelogenesis in reptile and amphibia were incomplete for the degradation of amelogenin proteins compared with that in mammals.
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