The molecular mechanism involved in the liganded thyroid hormone receptor suppression of the TSHbeta (thyroid-stimulating hormone beta, or thyrotropin beta) gene transcription is undetermined. One of the main reasons is the limitation of useful cell lines for the experiments. We have developed an assay system using non-pituitary CV1 cells and studied the negative regulation of the TSHbeta gene. In CV1 cells, the TSHbeta-CAT (chloramphenicol acetyltransferase) reporter was stimulated by Pit1 and GATA2 and suppressed by T3 (3,3',5-tri-iodothyronine)-bound thyroid hormone receptor. The suppression was dependent on the amounts of T3 and the receptor. Unliganded receptor did not stimulate TSHbeta activity, suggesting that the receptor itself is not an activator. Analyses using various receptor mutants revealed that the intact DNA-binding domain is crucial to the TSHbeta gene suppression. Co-activators and co-repressors are not necessarily essential, but are required for the full suppression of the TSHbeta gene. Among the three receptor isoforms, beta2 exhibited the strongest inhibition and its protein level was the most predominant in a thyrotroph cell line, TalphaT1, in Western blotting. The dominant-negative effects of various receptor mutants measured on the TSHbeta-CAT reporter were not simple mirror images of those in the positive regulation under physiological T3 concentration.
The data show a remarkably high incidence of thyroid autoantibodies in patients of middle of advanced age with sarcoidosis, especially in males, and a higher prevalence of Hashimoto's thyroiditis than in previous reports.
Cloned cDNAs for human liver serine -pyruvate aminotransferase (Ser -PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser -PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43 039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHsptl2, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser -PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser -PyrAT revealed 79.3% identity. Although human Ser -PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser -PyrAT.Serine -pyruvate aminotransferase (Ser -PyrAT) is an enzyme whose organelle localization and hormone responsiveness differ with the animal species. In human liver, this enzyme is entirely peroxisomal [l -31, while in cat and dog liver, it is entirely mitochondrial [3]. In rat liver, there are two types of this enzyme in terms of organelle distribution and hormone responsiveness: one is a mitochondrial matrix enzyme which is markedly induced on the administration of glucagon [4, 51 or insulin [6], and the other a glucagon-insensitive peroxisomal enzyme [4, 5, 71. It is well known that in eukaryotic cells each organelle participates in different cellular processes, and enzymes involved in these processes are synthesized being destined to be localized in a relevant subcellular compartment. Therefore, the localization of Ser -PyrAT in mitochondria and/or peroxisomes suggests that the enzyme in the different organelles participates in different metabolic processes.
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