Glycine oxidation and conversion into amino acids in Saccharomyces cerevisiae and Candida albicans.

Humans are exposed much more often to exogenous Saccharomyces cerevisiae (a baker's yeast) than exogenous Candida albicans (a highly infectious yeast) but suffer no apparent complications from S. cerevisiae. We hypothesize that variations in characteristics between these two species may be due, in part, to differences in glycine metabolism. In this study, we examined differences in glycine oxidation between C. albicans and S. cerevisiae. Both C. albicans and S. cerevisiae were cultured in glycine enriched media, followed by determination of glycine oxidation and amino acid concentrations in cells. Glycine was degraded to a much greater extent in C. albicans than in S. cerevisiae. Threonine concentrations and glycine oxidation were also elevated in C. albicans. Almost all of the disappearance of glycine from incubation media was accounted for by the formation of serine, threonine, and CO(2) in S. cerevisiae, whereas these products represented only 50% of the metabolized glycine in C. albicans. The unidentified metabolites of glycine in C. albicans, presumably purines, could contribute to its infectious capacity and this warrants further study.

Transcriptional regulation of dehydroepiandrosterone sulfotransferase (SULT2A1) by estrogen-related receptor alpha.

The estrogen-related receptors (ERRalpha, -beta, and -gamma) are a subfamily of orphan nuclear receptors (designated NR3B1, NR3B2, and NR3B3) that are structurally and functionally related to estrogen receptors alpha and beta. Herein we test the hypothesis that ERRalpha regulates transcription of the genes encoding the enzymes involved in adrenal steroid production. Real-time RT-PCR was first used to determine the levels of ERRalpha mRNA in various human tissues. Adult adrenal levels of ERRalpha transcript were similar to that seen in heart, which is known to highly express ERRalpha. Expression of ERRalpha in the adult adrenal was then confirmed using Western blotting and immunohistochemistry. To examine the effects of ERRalpha on steroidogenic capacity we used reporter constructs with the 5'-flanking regions of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A), 3beta-hydroxysteroid dehydrogenase type II (HSD3B2), 17alpha-hydroxylase/17,20-lyase (CYP17), and dehydroepiandrosterone sulfotransferase (SULT2A1). Cotransfection of these reporter constructs with wild-type ERRalpha or VP16-ERRalpha expression vectors demonstrated ERRalpha enhanced reporter activity driven by flanking DNA from CYP17 and SULT2A1. SULT2A1 promoter activity was most responsive to the ERRalpha and VP16-ERRalpha, increasing activity 2.6- and 79.5-fold, respectively. ERRalpha effects on SULT2A1 were greater than the stimulation seen in response to steroidogenic factor 1 (SF1). Transfection of serial deletions of the 5'-flanking DNA of the SULT2A1 gene and EMSA experiments indicated the presence of three functional regulatory cis-elements which shared sequence similarity to binding sites for SF1. Taken together, the expression of ERRalpha in the adrenal and its regulation of SULT2A1 suggest an important role for this orphan receptor in the regulation of adrenal steroid production.

The NGFI-B family of transcription factors regulates expression of 3beta-hydroxysteroid dehydrogenase type 2 in the human ovary.

The nerve growth factor-induced clone B (NGFI-B) family of transcription factors are orphan members of the steroid hormone receptor superfamily. The NGFI-B expression was recently shown in the rat ovarian tissue and appears to be regulated by gonadotrophins. The purpose of our study was to investigate the role of the three members of this family [NGFI-B, Nur-related factor 1 (NURR1) and neuron derived orphan receptor 1 (NOR-1)] in the transcription of genes that encode key steroidogenic enzymes and examine expression in the human ovary. Real-time RT-PCR was used to quantify mRNA expression levels of the NGFI-B family members in human ovarian follicles, corpora lutea and in human granulosa cells after FSH, phorbol ester (TPA) and forskolin treatment. NGFI-B was expressed at higher levels than both NURR1 and NOR-1 in both ovarian follicles and corpora lutea. In human granulosa tumour (HGT) cells, the NGFI-B expression increased after TPA, and to a lesser extent, after forskolin treatment. Treatment of primary cultures of human granulosa cells with forskolin and FSH rapidly increased the NGFI-B mRNA levels followed by an increase in 3beta-hydroxysteroid dehydrogenase type 2 (HSD3B2). Transcription of HSD3B2 was studied by transfecting NGFI-B and steroidogenic factor 1 (SF1) expression vectors with reporter constructs prepared with human steroidogenic acute regulatory protein, cholesterol side-chain cleavage, and HSD3B2 genes. NGFI-B increased the transcription of HSD3B2 in HGT cells which is significantly more than SF1. Mutation or deletion of the NGFI-B response element in the HSD3B2 promoter significantly reduced the NGFI-B-mediated transcription of HSD3B2. Therefore, our data suggest that the NGFI-B may play a significant role in up-regulation of HSD3B2 that leads to the increase in progesterone production that is seen in granulosa cells at ovulation.