Tamas Pazmany | PubMed Health

PubMed2005

The major histocompatibility complex class II transactivator is differentially regulated by interferon-gamma and transforming growth factor-beta in microglial cells.

We evaluated the regulation of the major histocompatibility complex class II (MHC II) transactivator (CIITA) gene expression in two microglial cell lines, EOC2 and EOC20. We demonstrate that interferon-gamma (IFN-gamma) activates type III- and IV-CIITA mRNA and high levels of MHC II in EOC20. However, in EOC2 cells only low levels of type IV-CIITA mRNA and MHC II are detectable following IFN-gamma treatment. Transforming growth factor-beta1 (TGF-beta1) inhibits both type III- and IV-CIITA expression in EOC20 cells while, in EOC2 cells TGF-beta1 enhances IFN-gamma induced pIV-CIITA expression. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, abrogates the TGF-beta1 mediated repression of the IFN-gamma induced CIITA in EOC20. Evidence is presented that the TG-interacting factor (TGIF), a co-repressor known to recruit HDACs, plays a role in determining the effects of TGF-beta1 on microglial cells.

Animals↗

PubMed2005

Novel mutation of the CYP17 gene in two unrelated patients with combined 17alpha-hydroxylase/17,20-lyase deficiency: demonstration of absent enzyme activity by expressing the mutant CYP17 gene and by three-dimensional modeling.

The CYP17 gene, located on chromosome 10q24-q25, encodes the cytochrome P450c17 enzyme. Mutations of this gene cause the 17alpha-hydroxylase/17,20-lyase deficiency, which is a rare, autosomal recessive form of congenital adrenal hyperplasia. Approximately 50 different mutations of the CYP17 gene have been described, of which some mutations have been identified in certain ethnic groups. In this study, we present the clinical history, hormonal findings and mutational analysis of two patients from unrelated families, who were evaluated for hypertension, hypokalemia and sexual infantilism. In the first patient, who was a 37-year-old female, additional studies showed a large myelolipoma in the left adrenal gland, and a smaller tumor in the right adrenal gland. In the second patient, who was a 31-year-old phenotypic female, clinical work-up revealed a 46,XY kariotype, absence of ovaries and presence of testes located in the inner opening of both inguinal canals. Analysis of the CYP17 gene by polymerase chain reaction amplification and direct sequencing demonstrated a novel homozygous mutation of codon 440 from CGC (Arg) to TGC (Cys) in both patients. The effect of this novel mutation on 17alpha-hydroxylase/17,20-lyase activity was assessed by in vitro studies on the mutant and wild-type P450c17 generated by site-directed mutagenesis and transfected in nonsteroidogenic COS-1 cells. These studies showed that the mutant P450c17 protein was produced in transfected COS-1 cells, but it had negligible 17alpha-hydroxylase and 17,20-lyase activities. In addition, three-dimensional computerized modeling of the heme-binding site of the P450c17 enzyme indicated that replacement of Arg by Cys at amino acid position 440 predicts a loss of the catalytic activity of the enzyme, as the mutant enzyme containing Cys440 fails to form a hydrogen bond with the propionate group of heme, which renders the mutant enzyme unable to stabilize the proper position of heme. Based on these findings we conclude that expressing the CYP17 gene with functional analysis, combined with three-dimensional computerized modeling of the heme-binding site of the protein provide feasible tools for molecular characterizing of functional consequences of the novel CYP17 mutation on enzyme function.

Adrenal Hyperplasia, Congenital↗