Validation of endogenous controls for gene expression studies in peripheral lymphocytes from war veterans with and without PTSD.

BACKGROUND: Selection of appropriate endogenous control is a critical step in gene expression analysis. The aim of this study was to evaluate expression stability of four frequently used endogenous controls: beta-actin, glyceraldehyde-3-phosphate dehydrogenase, beta2-microglobulin and RNA polymerase II polypeptide A in peripheral blood mononuclear cells from war veterans with and without posttraumatic stress disorder (PTSD). The study was designed as to identify suitable reference gene(s) for normalization of gene expression in peripheral blood mononuclear cells in response to war trauma and/or PTSD. RESULTS: The variability in expression of the four endogenous controls was assessed by TaqMan Real-time RT-PCR in peripheral blood mononuclear cells from: war veterans with current PTSD, those with lifetime PTSD, trauma controls and healthy subjects. Expression stability was analyzed by GeNorm and NormFinder software packages, and by direct comparison of Ct values. Both, GeNorm and NormFinder identified beta-actin and glyceraldehyde-3-phosphate dehydrogenase as a pair of genes with the lowest stability value. CONCLUSIONS: The combination of beta-actin and glyceraldehyde-3-phosphate dehydrogenase appeared to be the most suitable reference for studying alterations in gene expression in peripheral blood mononuclear cells related to vulnerability and resilience to PTSD, as well as to trauma-provoked developing of this disorder and recovery from it. Using glyceraldehyde-3-phosphate dehydrogenase, beta-actin and beta2-microglobulin as individual endogenous controls would provide satisfactory data, while RNA polymerase II polypeptide A could not be recommended.

Interaction of rat renal glucocorticoid receptor with Hsp90 and Hsp70 upon stress provoked by mercury.

The influence of mercury on the association of rat kidney glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70 was investigated. The GR heterocomplexes with Hsp90 and Hsp70 were immunopurified from the renal cytosol of rats administered different doses of mercury (1, 2 and 3 mg Hg kg(-1) b.w.). A quantitative immunoblotting procedure was applied to determine the levels of GR, Hsp90 and two nucleocytoplasmic Hsp70 isoforms (constitutive Hsp73 and inducible Hsp72) in the renal cytosol, as well as the amounts of these proteins within GR heterocomplexes immunoprecipitated by anti-GR antibody. Mercury was found to stimulate GR association with all the examined Hsps. The most prominent effect of the metal was stimulation of Hsp72 interaction with GR. On the other hand, the metal administration led to an increase of Hsp90 level in the cytosol, while the cytosolic levels of Hsp70 isoforms remained unaltered. These findings suggest that association of Hsps, at least Hsp70, with the GR might be ascribed to changes in the affinity of their interaction rather than to changes in the Hsp availability in the cytosol. Therefore, GR heterocomplex assembly seems to be a controlled process enabling chaperoning and functioning of the GR to be in concert with physiological demands.

Mercury influences rat liver tyrosine aminotransferase activity and induction by dexamethasone.

The effects of mercury (Hg) on basal and dexamethasone-induced tyrosine aminotransferase (TAT) activity in rat liver were studied. Comparison of TAT activity after in vitro and in vivo mercury application revealed the influence of the metal only when applied in vivo, suggesting that the effects are expressed at the level of TAT gene transcription. Intraperitoneal administration of mercury at 1, 2 or 3 mg Hg kg(-1) b.w. 4 h before decapitation was shown to stimulate the basal activity of TAT. The most prominent increase was observed 4 h after the metal administration. When applied at 1 and 2 mg Hg kg(-1) b.w. mercury was also shown to reduce partially the extent of the enzyme induction by dexamethasone, which was injected intraperitoneally at 5 mg kg(-1) b.w. 5 h before death. The highest dose of mercury (3 mg Hg kg(-1) b.w.) almost completely abolished the dexamethasone effect. The finding that mercury increases basal activity of the enzyme while decreasing its induction by dexamethasone suggests that stimulatory effects of this metal on TAT activity are probably mediated by factors other than glucocorticoids.

Mercury stimulates rat liver glucocorticoid receptor association with Hsp90 and Hsp70.

The subject of the present study is the influence of mercury on association of rat liver glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70. The glucocorticoid receptor heterocomplexes with Hsp90 and Hsp70 were immunopurified from the liver cytosol of rats administered with different doses of mercury. The amounts of co-immunopurified apo-receptor, Hsp90 and Hsp70 were then determined by quantitative Western blotting. The ratio between the amount of heat shock protein Hsp90 or Hsp70 and the amount of apo-receptor within immunopurified heterocomplexes was found to increase in response to mercury administration. On the other hand, the levels of Hsp90 and Hsp70 in hepatic cytosol remained unaltered. The finding that mercury stimulates association of the two heat shock proteins with the glucocorticoid receptor, rendering the cytosolic heat shock protein levels unchanged, suggests that mercury affects the mechanisms controlling the assembly of the receptor heterocomplexes.