Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

PGH2, TxA2 and PGI2 have potent and differentiated actions on human uterine contractility

The contractile response to three different prostanoids of the isolated human myometrium and the different layers of the utero-tubal junction (UTJ) was studied $\text{in vitro}$. The prostaglandin endoperoxide, PGH₂, stimulated contractility of both the myometrium and the outer and inner muscle layers of the UTJ, whereas the intermediate layer of the UTJ was inhibited. Thromboxane A₂ generated from PGH₂ and a thromboxane synthase preparation caused a stimulation of both the myometrium and all three layers of the UTJ. The stimulatory response to TxA₂ occurred at concentrations as low as 50–70 pg/ml. The sodium salt of PGI₂ was found to relax both the myometrium and all the layers of the UTJ. Intravenous administration of PGI₂ in repeated doses between 2–8 μg induced facial flushing and headache but had little if any effect on $\text{in vivo}$ uterine contractility. At least under $\text{in vitro}$ conditions, these short-lived prostanoids and/or their metabolites apparently have a specific action on uterine contractility, an action which is manifested at comparatively low concentrations.     

Activation of the dioxin and glucocorticoid receptors to a DNA binding state under cell-free conditions

The activation in vitro of dioxin and glucocorticoid receptors from a non-DNA binding to a DNA binding state was characterized. Ligand-free dioxin and glucocorticoid receptors were partially co-purified from rat liver cytosol, and both receptors sedimented at 9 S following labeling with the respective ligand. The 9 S forms of the dioxin and glucocorticoid receptors have previously been shown to represent heteromeric complexes containing the Mr approximately equal to 90,000 heat shock protein. The 9 S ligand-free or ligand-bound glucocorticoid receptor was converted to the monomeric 4-5 S form upon exposure to 0.4 M NaCl even in the presence of the stabilizing agent molybdate. Under identical conditions, the 9 S ligand-free and ligand-bound dioxin receptor forms remained essentially intact. However, in the absence of molybdate, the dioxin receptor could be converted to a 4-5 S form upon exposure to high concentrations of salt. These results indicate that the glucocorticoid receptor readily dissociates from the 9 S to the 4-5 S form even in the absence of hormone, whereas both the ligand-free and ligand-occupied 9 S dioxin receptor forms represent more stable species. Gel mobility shift experiments revealed that the 4-5 S glucocorticoid receptor interacted with a glucocorticoid response element both in the absence and presence of ligand. On the other hand, occupation of the dioxin receptor by ligand greatly enhanced the ability of the receptor to be activated to a form that binds to its target enhancer element. Once dissociated, the monomeric form of the dioxin receptor was also able to interact with its DNA target sequences even in the absence of ligand. Thus, ligand binding efficiently facilitates subunit dissociation of the dioxin receptor but is not a prerequisite for DNA binding per se. Given the apparent stability of its non-DNA binding 9 S form, the dioxin receptor system might be a useful model for the investigation of the mechanism of activation of soluble receptor proteins.     

3-(3-Aryloxyaryl)imidazo[1,2-a]pyridine sulfones as liver X receptor agonists

Replacement of a quinoline with an imidazo[1,2-a]pyridine in a series of liver X receptor (LXR) agonists incorporating a [3-(sulfonyl)aryloxyphenyl] side chain provided high affinity LXR ligands 7. In functional assays of LXR activity, good agonist potency and efficacy were found for several analogs.