|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1994 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1993 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1992 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Results obtained from this project are summerized as follws :
(1) Retinoic acid-binding activity of nuclear extracts from rat liver and testis was determined by using a sucrose-density-gradient assay. From the sedimentation analysis, and the comparison with cloned RARs, it is likely that these binding activities represent endogeneous RARs. Futhermore we showed that these binding activities were constant irrespective of the retinoid status in the rat.
(2) Gene expression of three nuclear retinoid X receptors (RXRalpha, beta, gamma) was examined by Northern blot anlysis in various rat tissues. The RXRalpha mRNA was detected in most tissues and paticulaly expressed at a high level in the liver. The RXRbeta transcripts were expressed ubiquitously, and particulary at high level in the brain and testis. In the liver, heart, kidoney and lung, the RXRgamma mRNA was specifically detected. The effect of retinoid, vitamin D and thyroid hormone status on the gene expression of RXRalpha, beta and gam
ma. Though retinoid and vitamin D did not affect the mRNA level of three RXRs, the mRNA levels of two genes were controlled by thyroid hormone. Namely, positive (RXRbeta) and negative (RXRgamma) regulations by thyroid hormone were observed with no effect on the gene expression of RXRalpha.
(3) We have tried to elucidate the level at which thyroid hormone (TH) regulate the gene expression of RXRbeta and RXRgamma in the rat. A RNA synthesis inhibitor (actinomycin D), but not a protein synthesis (cycloheximide), blocked the induction of RXRbeta mRNA by TH.On the other hand, none of these drugs inhibited the decrease by TH.Nuclear run-on assays showed that the transcription rate of the RXRbeta gene was positively regulated by TH,whereas, the transcription of RXRgamma gene was not controlled by TH.
(4) The in vitro isomerization of all-trans-and 9-cis-retinoic acids (RAs) was evaluated by high performance liquid chromatography after oral administration to rat. All-trans-and 13-cis-RAs, but not 9-cis-RA,were detected in the serum of normal rat. When an excess of either all-trans-RA or 9-cis-RA was intragastrically administrated to the retinoid-depleted rats, a rapid isomer exchange between 9-cis-and all-trans-RAs along with appearans of the administered RA occurred shortly after the dose (30 min). RA rapidly isomerized when an excess of either all-trans-or 9-cis-RA was administered to normal rats. Furtheremore, the degrees of induction of cellular retinol-binding protein type II (CRBP II,target gene for 9-cis-RA) and RARbeta (for all-trans-and 9-cis-RA) did not differ 4 h after administration of either 9-cis-RA or all-trans-RA.However, unlike all-trans-RA,the RAR-specific synthetic retinoid did not induce the CRBP II gene.
(5) Functional domains of retinol-binding protein (RBP) and transthyretin (TTR) were determined by means of fusion proteins which were generated as protein fusing on the N-terminal domain of RBP and TTR respectively. The results may lead us to a conclusion that the binding domain of RBP molecule to the cell membrane receptor may be located on the C-terminal, whereas, the retinol and TTR binding domeins on the other side, the N-terminal. Moreover, the thyroxin and RBP binding sites on TTR molecule may locate near by on the N-terminal, which differ from domain involved in the tetramer formation function which may be located on the C-terminal. Less