Molecular Biological Studies on mechanisms of Vitamin A sigunal transduction

Research Project

Project/Area Number	07660174
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	食品科学・栄養科学
Research Institution	Tokyo University of Agriculture
Principal Investigator	MASUSHIGE Shoichi Tokyo University of Agriculture, Faculty of Agriculture, Department of Agricultural Chemistry, Professor, 農学部, 教授 (70078153)
Co-Investigator(Kenkyū-buntansha)	YAMAMOTO Yuji Tokyo University of Agriculture, Faculty of Agriculture, Department of Agricultu (50240130)
Project Period (FY)	1995 – 1996
Project Status	Completed (Fiscal Year 1996)
Budget Amount *help	¥2,600,000 (Direct Cost: ¥2,600,000) Fiscal Year 1996: ¥1,300,000 (Direct Cost: ¥1,300,000) Fiscal Year 1995: ¥1,300,000 (Direct Cost: ¥1,300,000)
Keywords	Retinol / Retinoic acid / Retinoic acid receptor (RAR) / Rtinoid X receptor (RXR) / Vitamin A / Trnsthyretin (TTR) / Rtinol binding-protein (RBP) / レチノイン酸 / 分子生物学
Research Abstract	Results obtained from this project are summerized as follows : 1 NEW PATHWAYS OF VITAMIN A METABOLISM AND FUNCTIONS OF NEW METABOLITES (1) Functions of Bacterlally Expressed Rat Retinol-binding Protein Retinol-binding protein (RBP) was expressed in Eshericia coli using the cDNA for rat RBP,and characterized. The expressed RBP was fused to maltose-binding protein (MBP) at the N-terminal end (MBP-RBP), and MBP was enzymatically removed frome the MBP-RBP from proteinase factor Xa. The binding of retinol and transthyretin (TTR) to the recombinant RBP was monitored by mean of gel filtration. The recombinant RBP specifically bound to retinol with an affinity similar to that of purified RBP from rat serum. Furhtermore, the retinol-bound recombinant RBP formed hetero-complexes with TTR similar to RBP.Thus, the results showed that the recombinant RBP expressed in E.coli is as functional as serum RBP in terms of retinol and TTR bindings. (2) New Metabolic Pathways of Vitamin A New metabolic pathway o … More f vitmin A especially retinoic acid was studied. The results obtained were detailedly described in the form 12. 2 SEACH FOR A NEW VITAMIN A DEPENDENT GENE AND ITS FUNCTIONS (1) Retinoic Acid Differentially Up-Regulates the Gene Expression of Retinoic Acid Receptor alpha and gamma Isoforms in Embryo and Adult Rats The divers biological effects of retinoic acid (RA) are excerted by its nuclear receptor-mediated gene expression. One of the two nuclear retinoic acid receptor subfamilies is composed of three subtypes of the all-trans RA receptor (RAR-alpha, RAR-beta, and RAR-gamma). Futhermore, several isoforms are generated from each of three RARs by differential promorter usage and/or altanative splicing. It is thus thought that the developmental stage-specific actions of RA are modulated through the spatio-temporal expression of the subtype and isoforms of RARs. In this project, the auto-regulation of the RAR subtypes (RAR-alpha total, RAR-beta total, and RAR-gamma total) and their major isoforms (RAR-alpha1, alpha2, RAR-beta1, beta2 and RAR-gamma1, gamma2) by RA was examined by mean of Northern blotting in the 11.5 day embryo and maternal tissues by administrating pregnant rats with an excess of all-trans RA.The expression of RAR-beta isoform as well as the RAR-beta total was auto-regulated by RA in all maternal tissues and embryos ixamined. The gene expression of RAR-alpha2, which was not affected by RA in the maternal tissues, was up-regulated in embryos, though there were no significant effects of RA on the levels of RAR-alpha1 and the alpha total. Like-weise, RA did not affect the levels of RAR gamma1 and gamma total. However, unlike RAR-alpha2, RAR-gamma2 expression was up-regulated by RA only in the Maternal tissues. Thus, these results indicates that two retinoic acid receptor isoforms (RAR-alpha2 and RAR-gamma2) are differentially auto-regulated in embryo and adult rats. (2) Characters of A Retionoic Acid Induced Gene, KT-1 Expression of vitamin A induced gene designated KT-1 was examined. The detail of results was described in form 12. Less