Browsing by Subject "Ethanol"
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- ItemOpen AccessImpact of glucocorticoid receptor density on ligand-independent dimerization, cooperative ligand-binding and basal priming of transactivation: a cell culture model(Public Library of Science, 2013) Robertson, Steven; Rohwer, Johann M; Hapgood, Janet P; Louw, AnnGlucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoter-reporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs.
- ItemRestrictedThe LEA-like protein HSP 12 in Saccharomyces cerevisiae has a plasma membrane location and protects membranes against desiccation and ethanol-induced stress.(Elsevier, 2000) Sales, Kurt; Brandt, Wolf; Rumbak, Elaine; Lindsey, GeorgeThe LEA-like protein HSP 12 was identified as having a plasma membrane location in yeast. Gold particles, indicative of the presence of HSP 12, were observed on the external side of the plasma membrane when yeast grown to stationary phase were subjected to immunocytochemical analysis. Growth of yeast in the osmolyte mannitol resulted in an increased number of gold particles that were now observed to be present on both sides of the plasma membrane. No gold particles were observed using a mutant strain of the same yeast that did not express HSP 12. A model liposome system encapsulating the fluorescent dye calcein was used to investigate the protection by HSP 12 of membranes during desiccation. HSP 12 was found to act in an analogous manner to trehalose and protect liposomal membrane integrity against desiccation. The interaction between HSP 12 and the liposomal membrane was judged to be electrostatic as membrane protection was only observed with positively charged liposomes and not with either neutral or negatively charged liposomes. The ability of the wild-type and mutant yeast to grow in media containing ethanol was compared. It was found that yeast not expressing the HSP 12 protein were less able to grow in media containing ethanol. HSP 12 was shown to confer increased integrity on the liposomal membrane in the presence of ethanol. Ethanol, like mannitol, was found to induce HSP 12 protein synthesis. However, yeast grown in both ethanol and mannitol showed a decreased HSP 12 response compared with yeast grown in the presence of either osmolyte alone.