Glucocorticoid receptors are associated with particles containing DNA and RNA in vivo

Chemical crosslinking of glucocorticoid-receptor complexes to associated components in living cells was performed by the use of formaldehyde. Glucocorticoid binding sites were predominantly located in nuclei, and could not be efficiently extracted by 0.3 M NaCl. Sonication was found to cause the release of about 40% of nuclear receptor complexes. By sucrose density gradient centrifugation of soluble extracts from nuclear sonicates, crosslinked receptor complexes were found in oligomeric forms under high salt conditions. Treatment of these extracts with hydrolytic enzymes showed that DNA and RNA were associated with crosslinked receptor complexes.     

Persistence of the control of the fluctuations of heart rate in the anesthetized and pithed adult rat. Effect of enteraminergic drugs]

Spontaneous fluctuations in heart rate were analyzed by spectral analysis in the ether anesthetized and pithed adult rat. In order to investigate the changes in the spectral pattern of the fluctuations, the selective 5HT-2 and -3 isoreceptor blockers ritanserin and BRL 43694A were used. In both the experimental conditions, ritanserin blockade led to dose-dependent increased fluctuations in HR low and high frequencies. In both the anesthetized and pithed rats, the low frequency range HR fluctuations were drastically reduced by BRL 43694A.     

Two functions encoded by adenovirus early region 1A are responsible for the activation and repression of the DNA-binding protein gene.

Human adenovirus early region 1A (E1A) gene products differentially regulate the expression of early region 2A (E2A) encoding the DNA-binding protein (DBP). In a microinjection system, plasmids containing the DBP gene associated with both its early (map coordinate 75) and late (coordinate 72) promoters, or only with the early promoter, are inefficiently expressed, and the presence of E1A DNA is required for full expression. In contrast, the E2A plasmid in which the DBP gene is associated solely with its late promoter, efficiently produces DBP, the synthesis of which is significantly inhibited by an E1A gene product. To identify which of the E1A products is responsible for either activation or repression of DBP gene expression, two E1A mutants (Ad5hr1 and Ad2/5pm975) have been tested in the microinjection system in the presence of different DBP plasmids containing either one or both promoters. The results obtained indicate that the product encoded by the E1A 13S mRNA is responsible for the stimulation of DBP produced from the early promoter and that the 12S mRNA codes for the product which represses the synthesis of DBP from the late promoter. These results were confirmed using clones in which the E2A early or late promoter was associated to the chloramphenicol acetyltransferase (CAT) gene and assayed for CAT activity after cell transfection in the absence or in the presence of wild-type or mutant E1A plasmids, and we have also shown that this promoter-dependent regulation is reflected in the relative amount of specific DBP mRNA.     

The level of pancreatic PLA2 receptor is closely associated with the proliferative state of rat uterine stromal cells

Rat uterine stromal cells (U_III) express pancreatic type PLA₂ (PLA₂-I) receptor and internalize the enzyme bound to receptors. Here, we investigate the proliferating effect and alterations in binding of PLA₂-I. There is a dramatic decline in PLA₂-I binding in U_III cells as they progress from a nonconfluent proliferating state (40,000 sites/cell) to a confluent state (1300 sites/cell). Intracellular concentration of PLA₂-I changed with the alteration in binding, suggesting that regulation in the PLA₂ binding capacity may have important implications in growth control mechanisms.     

The subcellular distribution of glucocorticoid-receptor complexes as studied by chemical crosslinking of intact HTC cells

Treatment of intact HTC cells with glutaraldehyde results in redistribution of glucocorticoid binding sites between cytosolic and nuclear fractions. The decrease in cytosolic receptors and their accumulation at the nuclear level were found to be directly related to the glutaraldehyde concentrations employed in our procedure and inversely related to the cell density of samples. When the data from eleven separate experiments were combined, and analyzed by linear regression of cytosolic and nuclear levels of receptor complexes vs the ratios between the DNA and glutaraldehyde concentration of our samples, two lines were obtained whose intercepts on the ordinate yielded values of cytosolic and nuclear receptors corresponding to 37.5 and 62.5% of the total cellular pool, respectively. When we compared the subcellular redistribution of glucocorticoid receptor to that of the cytosolic enzyme lactate dehydrogenase upon HTC cell crosslinking with glutaraldehyde, we found that the cytosolic and nuclear levels of the enzyme were 53.2 and 46.8% of the total content, respectively. If the subcellular distribution of glucocorticoid receptor is corrected for the artefactual redistribution induced by crosslinking, using the values obtained for lactate dehydrogenase, it can be concluded that glucocorticoid receptors in HTC cells are distributed between cytosol and nuclei in a ratio which is about 2:1. Our findings lend further support to theconclusion that only a portion of glucocorticoid receptor is cytosolic in intact cells.