Three related orphan nuclear receptors that are expressed in the brain, NGFI-B, Nurr1, and NOR-1, were studied to compare their function as transcriptional activators. NGFI-B was able to activate (in the absence of added hormone) in CV1 cells both an NGFI-B-responsive luciferase reporter gene (containing eight copies of a response element for NGFI-B upstream of a basal prolactin promoter driving the luciferase gene, NBRE(8)-LUC), a similar thyroid hormone-receptor-responsive reporter gene (TRE(3)-LUC), and a reporter gene with an authentic promoter from a Xenopus vitellogenin gene containing two binding sites for the estrogen receptor (vit-LUC). NGFI-B activated NBRE(8)-LUC and TRE(3)-LUC (but not the vitLUC) with an amino-terminal activation domain. Nurr1 was less promiscuous as a transcriptional activator, activating.the NBRE(8)-LUC better than NGFI-B, but less than NGFI-B at the other reporter genes. NOR-1 activated only the NBRE(8)-LUC reporter gene. These results indicate that closely related nuclear receptors may differentiate between response elements or promoters and that different activation mechanisms exist depending on the promoter. This may contribute to regulation of specificity of target gene expression in the brain.
Organic anions of particular importance to biochemistry such as Krebs cycle intermediates, glycolysis intermediates, simple fatty acids, adenine nucleotides and CoA derivatives can be quantitatively extracted from a buffered solution by high-molecular-weight ammonium salts in an organic solvent. Phosphate salts of tertiary amines in chloroform were the most efficient extractants. The isolation procedure was found to be an example of amine neutralization. The effect of pH, different inorganic anions, volume ratios between the two phases, concentration of the isolated anions and concentration of the ammonium salts have been investigated. The extraction technique has been applied to rapid and sensitive radiochemical methods for the determination of acetylcholinesterase and 4-aminobutyrate aminotransferase activities.Previously, we have demonstrated that sodium tetraphenylboron (Kalignost) in heptanone or alkylnitriles constitutes a powerful liquid cation exchanger [I]. The complex has successfully been used in the isolation of several cations such as acetylcholine or aromatic amines from aqueous solutions [l] and has made it possible to develop highly sensitive radiochemical enzyme assays for measurements of choline acetyltransferase
The activities of five enzymes involved in acetyl-CoA synthesis, pyruvate dehydrogenase complex, ATP citrate lyase, carnitine acetyltransferase, acetyl-CoA synthetase, and citrate synthase, were determined in normal nucleus interpeduncularis and nucleus interpeduncularis in which cholinergic terminals were removed following lesion of the habenulointerpeduncular tract. The activities of aspartate transaminase, fumarase, and GABA transaminase also were determined to compare the effect of lesion on other mitochondrial enzymes which are not linked to the biosynthesis of ACh. In normal nucleus interpeduncularis the activities of carnitine acetyltransferase and pyruvate dehydrogenase complex were higher than the activity of ChAT (choline acetyltransferase), whereas the activities of acetyl-CoA synthetase and citrate synthase were considerably lower than that of ChAT. The effect of the lesion separated the enzymes into two groups: the activities of pyruvate dehydrogenase complex, carnitine acetyltransferase, fumarase and aspartate transaminase decreased by 30--40%, whereas the activities of the other enzymes descreased 5--15%. ChAT activity was in all cases less than 15% of normal. It could be concluded that none of the acetyl-CoA synthesizing enzymes decreased to the degree that ChAT did. Only pyruvate dehydrogenase complex and carnitine acetyltransferase seem to be localized in cholinergic terminals to a significant degree. ATP citrate lyase as well as acetyl-CoA synthetase seem to have less significance in supporting acetyl-CoA formation in cholinergic nerve terminals.
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