The methyltrienolone binding protein of JEG-3 cells and human placenta is localized within the nucleus and is tightly associated with chromatin.

Human placenta contains the methyltrienolone binding protein (MTBP), an androgen binding protein which is distinct from the androgen receptor. This study demonstrates that the human choriocarcinoma cell line (JEG-3) also contains the MTBP and that in both human placenta and JEG-3 cells the MTBP is located exclusively in the nucleus and in particular is associated with DNase 1 resistant chromatin.     

Characterisation and covalent labeling of the human placental methyltrienolone binding protein

Human placental cytosol contains an androgen binding protein which binds the synthetic androgen methyltrienolone (R 1881) with high affinity (Kd 8.7 nM) and with an average binding capacity of 518 fmol/mg cytosol protein. This study provides further evidence that this protein is distinguishable from classical androgen receptors on the basis of steroid specificity and sulphydryl group sensitivity. Covalent labeling studies have shown this protein, which we have called "the methyltrienolone binding protein", to have a mol. wt of 67,000 daltons.     

Separation of D- and L-amino acids by ion exchange column chromatography in the form of alanyl dipeptides

Summary A method of ion exchange column chromatography was developed for the determination of D- and L-amino acids in the form of diastereomeric dipeptide. First the protein containing samples were hydrolyzed with 6 molar hydrochloric acid, then the single amino acids were separated in an LKB automated amino acid analyzer with the LKB fraction collector. Following lyophilization, the single amino acids were transformed into alanyl dipeptides with tertiary-butyloxycarbonil-L-alanine-N-hydroxy-succinimide (t-BOC-L-Ala-ONSu) active ester. The alanyl dipeptides were easily separated from one another and the initial amino acids. Determination of the D- and L-amino acids in this form is relatively accurate and reproducible but takes some time (33–38 min). Accuracy of the determination is satisfactory. The coefficient of variation amounts to 3–5%. The use of the method is suggested to laboratories having an amino acid analyzer and wish to determine D-and L-amino acids in synthetic-amino acids complements, peptides or natural materials.     

Molecular basis for hypertension in the "type II variant" of apparent mineralocorticoid excess.

The syndrome of apparent mineralocorticoid excess (AME) is a heritable form of hypertension in which cortisol acts as a potent mineralocorticoid. The type I variant results in a severe clinical and biochemical phenotype and arises because of mutations in the gene encoding the type 2 isozyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD2), an enzyme responsible for the peripheral inactivation of cortisol to cortisone. Only mild abnormalities of cortisol metabolism have been found in the type II variant of AME, suggesting that it may be a separate gene defect. In an extensive consanguineous Sardinian pedigree affected with "type II" AME, a novel homozygous point mutation (C945T) was found in the human 11beta-HSD2 gene in four affected individuals. Thirteen family members were heterozygous for the resultant R279C amino acid substitution. The LOD score of linkage of the mutation to the disease was 3.23. Expression of the 11beta-HSD2 mutant cDNA resulted in an enzyme with reduced maximum velocity, but similar substrate affinity, compared with activity of the wild-type cDNA. Affected individuals were >30 years of age and had both mineralocorticoid hypertension and evidence of impaired metabolism of cortisol to cortisone. The heterozygote state was phenotypically normal but was associated with subtle defects in cortisol metabolism. AME represents a spectrum of mineralocorticoid hypertension with severity reflecting the underlying genetic defect in the 11beta-HSD2 gene; classification into distinct subtypes is inappropriate. Hypertensive populations should be screened to identify the prevalence of milder defects in 11beta-HSD2 in patients currently labeled as having "essential" hypertension.     

A new fourier transform approach for protein coding measure based on the format of the Z curve

MOTIVATION: At the core of most protein gene-finding algorithms are the coding measures used to make a decision on coding/non-coding. Of the protein coding measures, the Fourier measure is one of the most important. However, due to the limited length of the windows usually used, the accuracy of the measure is not satisfactory. This paper is devoted to improving the accuracy by lengthening the sequence to amplify the periodicity of 3 in the coding regions. RESULTS: A new algorithm is presented called the lengthen-shuffle Fourier transform algorithm. For the same window length, the percentage accuracy of the new algorithm is 6-7% higher than that of the ordinary Fourier transform algorithm. The resulting percentage accuracy (average of specificity and sensitivity) of the new measure is 84.9% for the window length 162 bp. AVAILABILITY: The program is available on request fromC.- T. Zhang. Contact: ctzhang@tju.edu.cn      

Expression of caveolin-1 enhances cholesterol efflux in hepatic cells

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains (caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.     

Contribution of protein kinase A and protein kinase C signalling pathways to the regulation of HSD11B2 expression and proliferation of MCF-7 cells

Contribution of the protein kinase A (PKA) and protein kinase C (PKC) signalling pathways to the regulation of 11beta-hydroxysteroid dehydrogenase type II (HSD11B2) gene expression was investigated in human breast cancer cell line MCF-7. Treatment of the cells with an adenylyl cyclase activator, forskolin, known to stimulate the PKA pathway, resulted in an increase in HSD11B2 mRNA content. Semi-quantitative RT-PCR revealed attenuation of the effect of forskolin by phorbol ester, tetradecanoyl phorbol acetate (TPA), an activator of the PKC pathway. It was also demonstrated that specific inhibitors significantly reduced the effect of activators of the two pathways. Stimulation of the PKA pathway did not affect, whereas stimulation of the PKC pathway significantly reduced MCF-7 cell proliferation in a time-dependent manner. A cell growth inhibitor, dexamethasone, at high concentrations, caused a 40% decrease in proliferation of MCF-7 cells and this effect was abolished under conditions of increased HSD11B2 expression. It was concluded that in MCF-7 cells, stimulation of the PKA signal transduction pathway results in the induction of HSD11B2 expression and that this effect is markedly reduced by activation of the PKC pathway. Activation of the PKC pathway also resulted in inhibition of cell proliferation, while activation of the PKA pathway abolished the antiproliferative effect of dexamethasone. These effects might be due to oxidation of dexamethasone by the PKA-inducible HSD11B2.     

11Beta-hydroxysteroid dehydrogenase type 2 and the regulation of surfactant protein A by dexamethasone metabolites

Glucocorticoid (GC) metabolism by the 11beta-hydroxysteroid dehydrogenase (HSD) system is an important prereceptor regulator of GC action. The HSD enzymes catalyze the interconversion of the endogenous, biologically active GC cortisol and its inactive 11-dehydro metabolite cortisone. The role of the HSD enzymes in the metabolism of synthetic GCs, such as dexamethasone (Dex), is more complex. The human lung is a classic GC-sensitive organ; however, the roles of the HSD enzymes (HSD1 and HSD2) in the human lung are poorly understood. In the present study, we examined the expression of the HSD enzymes in human adult and fetal lung tissues and the human lung epithelial cell line NCI-H441. We observed that human adult and fetal lung tissues, as well as H441 cells, express HSD2 protein and that it is upregulated by Dex (10(-7) M). By contrast, HSD1 protein was undetectable. We also show that the Dex-mediated regulation of surfactant protein A is attenuated by inhibition of HSD2 activity. Furthermore, we demonstrate that unlike the inactive, 11-dehydro metabolite of cortisol (i.e., cortisone), the 11-dehydro metabolite of Dex, 11-dehydro-Dex, competes for binding to the GC receptor (GR) in human lung epithelial cells and retains GR agonist activity. Together, these data suggest that differences exist in the biological activities of the metabolites of cortisol and Dex.     

Type I and type II corticosteroid receptor gene expression in the rat: effect of adrenalectomy and dexamethasone administration

We have used 32P-labeled cRNA probes directed against Type I (mineralocorticoid, high affinity glucocorticoid) and Type II (classical glucocorticoid) receptor mRNA to screen various tissues, and have investigated the effect of adrenalectomy (ADX) and dexamethasone (DM) administration on their levels in hippocampus. Both Northern blot and S1 nuclease analysis showed Type I mRNA to be high in hippocampus, colon, and heart; low in liver; and undetectable in thymus. Type II mRNA was high in liver, thymus, and brain; and low in testis and parotid. A transient increase in both hippocampal Type I and Type II mRNA was noted at 1-3 days post ADX. DM similarly elicited a rise in hippocampal Type I mRNA at 2-4 days after ADX, but prevented the ADX-induced increment in Type II mRNA. In contrast to the transient increase in Type I receptor mRNA levels, hippocampal levels of Type I receptors measured by [3H]aldosterone binding were constant 1-16 days post ADX. DM administration caused a doubling in Type I receptor levels over 4 days, with plateau levels at 4-16 days; previously, DM has been shown to lower Type II receptor levels in the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)