|Budget Amount *help
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
1)Based on the results of the protein primary structures of the newly born baby/infant type pepsinogen (PGF) and chymosin (CHY) identified by our cDNA cloning, synthetic peptides were prepared to immunize rabbits and 2 kinds of antibodies were obtained against PGF Immunohistochemistry revealed that PGF appeared on Day 16 of rat fetuses and the producing cells would, at least in part, constitute a gastric gland in the future.
2)We have been performing genomic cloning of these genes with the rat genomic library previously established using the PGF cDNA we cloned. To date, we have examined 41 positive clones, but all of them were clones for PGC and we have not obtained genomic clones for PGF.
3)We extracted RNA and protein soluble fractions from gastric mucosal epithelial tissue of rat fetuses using the separation method of the gastrointestinal epithelium that we developed. In the later fetal period, from the morphology formation period of the stomach to birth, we investigated the expressio
n of PGF CHY, and adult type pepsinogen (PGA) at the gene levels (by quantitative PCR) and protein levels (by zymography). As a result, the PGF gene expression first appeared on Day 15 in the fetal stomach, and later the expression of the CHY gene appeared. The expression of the PGF gene was mainly observed in the period of morphological formation of the stomach and a trace of PGA expression could be found. Just before birth, the CHY gene was upregulated to exceed the expression of PGF, and in the weaning period, in turn, the PGA expression exceeded the CHY expression.
4)Using the in vivo experiment system on newly born rats, we found that the expression of the pepsinogen genes switched from PGF to PGA by stimulating the rats with differentiation-inducing factors on the gastric mucosa, such as adrenal corticosteroids. Using the primary culture system of fetal gastric epithelium that we developed, we confirmed that the gene expression switched from PGF or CHY to PGA by administering differentiation-inducing factors on gastric epithelial cells, such as steroids and dibutyl-cAMP. Less