Oxidative activity of the type 2 isozyme of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) predominates in human sebaceous glands

Interactions between infiltrating T cells and keratinocytes via the secretion of the TH1 cytokines interleukin (IL) 2 and interferon gamma (INF-gamma), the keratinocyte growth factor transforming growth factor alpha (TGF-alpha) and the cytokines IL-6 and IL-8 are thought to be the predominant mechanisms inducing skin lesions in psoriatic patients. Systemic treatment of psoriasis with fumaric acid derivatives (FAEs) has been reported to be effective in the treatment of psoriasis, but the mode of action is still unknown. To clarify this phenomenon, keratinocytes from psoriatic patients as well as from healthy volunteers were mono- and cocultured with HUT 78 T cells with/without the addition of FAEs; the cytokine concentrations were then measured in the culture supernatants. Furthermore, mRNA expression was determined in epidermal growth factor (EGF) -activated keratinocytes as well as in phytohaemagglutinin (PHA)-activated HUT 78 T cells. Only dimethylfumarate (DMF) diminished IL-6 and TGF-alpha secretion in the psoriatic cocultures. However, it did not have this effect on cocultures from control subjects or on monocultures. DMF suppresses EGF-induced TGF-alpha mRNA induction in psoriatic keratinocytes. DMF inhibited INF-gamma secretion in all cultures but stimulated the IL-10 secretion. This immunomodulation away from the TH1 cytokine IFN-gamma to the TH2 cytokine IL-10 was confirmed in HUT 78 T cells by Northern blot analysis. An increased number of eosinophils is a known side-effect in patients treated with this drug, suggesting a clinical relevance of this immunomodulation in vivo. This immunomodulation and the suppression of cytokines from the psoriatic cytokine network could be responsible for the beneficial effect of DMF in the treatment of a hyperproliferative and TH1 cytokine-mediated skin disease.     

Deleterious mutations at the mitochondrial ND3 gene in South American marsh rats (Holochilus)

Statistical analyses of DNA sequences have revealed patterns of nonneutral evolution in mitochondrial DNA of mice, humans, and Drosophila. Here we report patterns of mitochondrial sequence evolution in South American marsh rats (genus Holochilus). We sequenced the complete mitochondrial ND3 gene in 82 Holochilus brasiliensis and 21 H. vulpinus to test the neutral prediction that the ratio of nonsynonymous to synonymous nucleotide changes is the same within and between species. Within H. brasiliensis we observed a greater number of amino acid polymorphisms than expected based on interspecific comparisons. This contingency table analysis suggests that many amino acid polymorphisms are mildly deleterious. Several tests of the frequency distribution also revealed departures from a neutral, equilibrium model, and these departures were observed for both nonsynonymous and synonymous sites. In general, an excess of rare sites was observed, consistent with either a recent selective sweep or with populations not at mutation-drift equilibrium.     

Steroid sulphotransferase and 17beta-hydroxysteroid dehydrogenase activities in Ishikawa human endometrial adenocarcinoma cells

In 20 rats bronchitis was evoked by permanent 12-week long inhalation of SO2. For this purpose a special chamber with gas supply was prepared. In all rats exposed to SO2 changes in cellularity of BALF were found (a statistically significant increase of the percent of macrophages and neutrophils). Histologic examination revealed bronchial changes especially in epithelium. The method applied, based on an original technology is cheap and effective and can be recommended in developing experimental bronchitis.     

Five novel missense mutations of the Lewis gene (FUT3) in African (Xhosa) and Caucasian populations in South Africa

A new bivalent ligand of formula 3 which results from the covalent coupling of two sumatriptan molecules with a p-xylyl spacer at the level of the sulfonamide nitrogen has been prepared and evaluated as a 5-HT1B/1D receptors agonist. In vitro experiments at 5-HT1B human cloned receptors (Ki = 0.64 nM; EC50 = 0.58 nM) and at the level of the contraction of the New Zealand white rabbit saphenous vein (pD2 = 6.6) demonstrate the superior potency of dimer 3 as a 5-HT1B receptor agonist when compared to sumatriptan or zolmitriptan. Interestingly enough, the new bivalent agonist 3 was found to induce hypothermia in the guineapig upon oral administration suggesting good oral activity and access to the brain.     

Expression and characterization of recombinant type 2 3 alpha-hydroxysteroid dehydrogenase (HSD) from human prostate: demonstration of bifunctional 3 alpha/17 beta-HSD activity and cellular distribution

In androgen target tissues, 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) may regulate occupancy of the androgen receptor (AR) by catalyzing the interconversion of 5alpha-dihydrotestosterone (5alpha-DHT) (a potent androgen) and 3alpha-androstanediol (a weak androgen). In this study, a 3alpha-HSD cDNA (1170 bp) was isolated from a human prostate cDNA library. The human prostatic 3alpha-HSD cDNA encodes a 323-amino acid protein with 69.9%, 84.1%, 99.4%, and 87.9% sequence identity to rat liver 3alpha-HSD and human type 1, type 2, and type 3 3alpha-HSDs, respectively, and is a member of the aldo-keto reductase superfamily. The close homology with human type 2 3alpha-HSD suggests that it is either identical to this enzyme or a structural allele. Surprisingly, when the recombinant protein was expressed and purified from Escherichia coli, the enzyme did not oxidize androsterone when measured spectrophotometrically, an activity previously assigned to recombinant type 2 3alpha-HSD using this assay. Complete kinetic characterization of the purified protein using spectrophotometric, fluorometric, and radiometric assays showed that the catalytic efficiency favored 3alpha-androstanediol oxidation over 5alpha-DHT reduction. Using [14C]-5alpha-DHT as substrate, TLC analysis confirmed that the reaction product was [14C]-3alpha-androstanediol. However, in the reverse reaction, [3H]-3alpha-androstanediol was oxidized first to [3H]-androsterone and then to [3H]-androstanedione, revealing that the expressed protein possessed both 3alpha- and 17beta-HSD activities. The 17beta-HSD activity accounted for the higher catalytic efficiency observed with 3alpha-androstanediol. These findings indicate that, in the prostate, type 2 3alpha-HSD does not interconvert 5alpha-DHT and 3alpha-androstanediol but inactivates 5alpha-DHT through its 3-ketosteroid reductase activity. Levels of 3alpha-HSD mRNA were measured in primary cultures of human prostatic cells and were higher in epithelial cells than stromal cells. In addition, elevated levels of 3alpha-HSD mRNA were observed in epithelial cells derived from benign prostatic hyperplasia and prostate carcinoma tissues. Expression of 3alpha-HSD was not prostate specific, since high levels of mRNA were also found in liver, small intestine, colon, lung, and kidney. This study is the first complete characterization of recombinant type 2 3alpha-HSD demonstrating dual activity and cellular distribution in the human prostate.     

Cell type-specific expression of 17 beta-hydroxysteroid dehydrogenase type 2 in human placenta and fetal liver

The enzymatic actions of the 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isozymes are crucial in steroid hormone metabolism/physiology. The type 1 isozyme catalyzes the conversion of the biologically inactive C18 steroid, estrone, to the active estrogen, 17 beta-estradiol, and the enzyme is predominantly expressed in the syncytiotrophoblast of the placenta and the granulosa cells of the ovary. 17 beta HSD type 2 is highly expressed in placenta, liver, and secretory endometrium and catalyzes the conversion of bioactive estrogens and androgens to biologically inactive 17-ketosteroid counterparts. The expression pattern of 17 beta HSD type 2 protein was determined in human term placenta and fetal liver by immunohistochemical analysis using monoclonal antibodies directed against distinct epitopes of the 17 beta HSD type 2 protein. In placenta, the protein was detected in the endothelial cells of fetal capillaries, but not in cytotrophoblasts or syncytiotrophoblast. There was dichotomous immunostaining seen among pairs of cotyledonary vessels and chorionic vessels. In the liver, on the other hand, staining was detected in the hepatocytes, but not in the cells lining blood vessels. We conclude that the cell type-specific localization of 17 beta HSD type 2 is in accord with the proposed physiological role of the enzyme, namely to protect tissues, in this case the fetus, from bioactive estrogen and androgen.     

3 beta-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5 alpha-androstane-3 beta,17 beta-diol and dehydroepiandrosterone as substrates

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-->4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3 beta-HSD/delta 5-->4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3 beta-HSD activity. For this purpose, we compared the efficiencies of a 3 beta-hydroxy-5-ene steroid (DHEA) and a 3 beta-hydroxy-5 alpha-reduced steroid (5 alpha-androstane-3 beta,17 beta-diol, 5 alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (Km) for 5 alpha-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent Km of placental 3 beta-HSD for 5 alpha-A-diol was in the range of 18 to 40 mumol/l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme, 17 mumol/l for 5 alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5 alpha-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to Kms and rates of product formation was obtained by use of purified placental 3 beta-HSD with DHEA, pregnenolone, and 3 beta-hydroxy-5 alpha-androstan-17-one (epiandrosterone) as substrates: the Km of 3 beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mumol/l, and for epiandrosterone 25 mumol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein.min and, with epiandrosterone, 127 nmol/mg protein.min. With placental homogenate as the source of 3 beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [3H]5 alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [3H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5 alpha-A-diol (40 mumol/l).(ABSTRACT TRUNCATED AT 400 WORDS)     

A new polymorphic restriction site in the human 11 beta-hydroxysteroid dehydrogenase type 2 gene

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) inactivates glucocorticoids in the kidney and thus prevents glucocorticoids from occupying the non-selective mineralocorticoid receptor in epithelial tissues. Mutations in the HSD11B2 gene have been found to cause the syndrome of apparent mineralocorticoid excess, a rare autosomal recessive disease characterized by severe hypertension. Thus, this locus could also be an ideal candidate involved in the etiology of primary hypertension. We identified a polymorphism in exon 3 characterized by a GAG to GAA transition at codon 178, with the loss of an Alu I restriction site and analysed it in an association study using end-stage renal disease patients, diabetic or essential hypertensive patients and control subjects. Two-hundred and eighty nine subjects and patients were analysed; the genotype was determined by amplification of genomic DNA and subsequent digestion with Alu I restriction enzyme. The prevalence of the Alu I allele was 8.6% in healthy control subjects (n = 116). This prevalence was lower (chi 2 P = 0.035 vs. controls) than the 18.0% in a group of renal transplant patients (n = 61). The corresponding values for patients with diabetes mellitus (n = 25), hypertension (n = 41) and patients on dialysis (n = 46) were 4.0%, 4.8% and 4.3%, respectively. There was no correlation between blood pressure and the marker in non-ESRD subjects. These data indicate the presence of a polymorphic marker in exon 3 of the HSD11B2 gene; this marker is associated with end-stage renal disease but not with essential hypertension in humans.     

Effect of nomegestrol acetate on estrone-sulfatase and 17beta-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E1S: 5 x 10(-9)M), nomegestrol acetate blocked very significantly the conversion of E1S to E2. In the MCF-7 cells, using concentrations of 5 x 10(-6)M and 5 x 10(-5) M of nomegestrol acetate, the decrease of E1S to E2 was, respectively, -43% and -77%. The values were, respectively, -60% and -71% for the T-47D cells. Using E1S at 2 x 10(-6) M and nomegestrol acetate at 10(-5) M, a direct inhibitory effect on the enzyme of -36% and -18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E1: 5 x 10(-9)M) this estrogen is converted in a great proportion to E2. Nomegestrol acetate inhibits this transformation by -35% and -85% at 5 x 10(-7)M and 5 x 10(-5)M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, -48%, at 5 x 10(-5)M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17beta-HSD activities which converts E1S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E2 can open new clinical possibilities in breast cancer therapy.     

Abnormal regulation of the Ke 6 gene, a new 17beta-hydroxysteroid dehydrogenase in the cpk mouse kidney

The function encoded by the Ke 6 gene has been recently determined to be 17beta-hydroxysteroid dehydrogenase. Previously, the abnormal expression of the Ke 6 gene has been intimately associated with development of recessive polycystic kidney disease. The Ke 6 gene is normally expressed at very high levels in the kidney and liver and is severely down regulated in all recessive murine models of polycystic kidney disease that have been examined to date. Here, we report a detailed examination of the promoter region of the Ke 6 gene in normal mouse kidney cells (CTA) and in cells derived from mouse kidneys homozygous for the cpk (congenital polycystic kidney) mutation, using transfection analysis and DNA-protein gel shift assays. The minimal promoter region, P1 (+1 to -96), and a putative enhancer site, P3 (-165 to -256), within the Ke 6 gene 5' flanking sequence have been identified. We have also identified another region, P2 (-97 to -165), that may be responsible for the lower promoter activity of the Ke 6 gene in cpk cells. Furthermore, absence of binding of a 38 kDa nuclear protein to a 16 bp sequence element (P1A) within the minimal promoter of the Ke 6 gene suggests that the P1A element could be responsible for the overall reduction in promoter function in cpk cells.     

Leukocytes modulate 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity in human granulosa-lutein cell cultures

A randomised clinical trial was performed to investigate whether there is a need or advantage to splint two implants in the mandible retaining a hinging overdenture. Furthermore, patient satisfaction was evaluated for different attachment systems retaining the overdenture. Thirty-six edentulous patients were randomised into three groups of equal size and treated with either magnets, ball attachments or straight bars (reference group). After 3 years of observation no implants were lost in any of the groups. Besides, no statistically significant differences were noted for the peri-implant outcome. Although the bar group presented the highest retention force, the general satisfaction of the patients in the three groups did not differ. The patients with bar retentions showed less prosthetic complications of the retention elements but more at the level of the denture-supporting mucosa. Finally, the clinical outcome of all groups confirms that overdenture treatment demands regular controls with limited time intervals.     

11beta-hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex

Although oxidation of cortisol or corticosterone by 11beta-hydroxysteroid dehydrogenase (11beta-HSD) represents the physiological mechanism conferring specificity for aldosterone on the mineralocorticoid receptor in mineralocorticoid target tissues, little attention has been paid until now to the expression and activity of this enzyme in human adrenals. We have shown that human adrenal cortex expresses 11beta-HSD type 2 (11beta-HSD2) gene, and found a marked 11beta-HSD2 activity in microsomal preparations obtained from slices of decapsulated normal human adrenal cortices. Under basal conditions, adrenal slices secreted, in addition to cortisol and corticosterone (B), sizeable amounts of cortisone and 11-dehydrocorticosterone (DH-B), the inactive forms to which the former glucocorticoids are converted by 11beta-HSD. Addition of the 11beta-HSD inhibitor glycyrrhetinic acid elicited a moderate rise in the production of cortisol and B and suppressed that of cortisone and DH-B. ACTH and angiotensin II evoked a marked rise in the secretion of cortisol and B, but unexpectedly depressed the release of cortisone and DH-B. ACTH also lowered the capacity of adrenal slices to convert [3H]cortisol to [3H]cortisone. This last effect of ACTH was concentration-dependently abolished by both aminoglutethimide and cyanoketone, which blocks early steps of steroid synthesis, but not by metyrapone, an inhibitor of 11beta-hydroxylase. Collectively, these findings indicate that the human adrenal cortex possesses an active 11beta-HSD2 engaged in the inactivation of newly formed glucocorticoids. The activity of this enzyme is negatively modulated by the main agonists of glucocorticoid secretion through an indirect mechanism, probably involving the rise in the intra-adrenal concentration of non-11beta-hydroxylated steroid hormones.     

Human hypertension caused by mutations in the 11 beta-hydroxysteroid dehydrogenase gene: a molecular analysis of apparent mineralocorticoid excess

CONVERSION OF CORTISOL TO CORTISONE: 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is a microsomal enzyme complex which, in humans, catalyses the interconversion between biologically active cortisol and inactive cortisone. This prereceptor signalling mechanism is essential for maintaining the aldosterone selectivity of the intrinsically non-specific mineralocorticoid receptor and for modulating glucocorticoid access to the glucocorticoid receptor. Apparent mineralocorticoid excess (AME) is a syndrome of severe low-renin mineralocorticoid hypertension associated with marked hypokalaemia which arises from a congenital deficiency of 11 beta-HSD. In AME patients, therefore, it is cortisol and not aldosterone which behaves as a potent mineralocorticoid. ISOFORMS OF 11 BETA-HSD: Two isoforms of human 11 beta-HSD have now been characterized and cloned. The type 1 isoform (11 beta-HSD1) is a low-affinity reduced nicotinamide adenine dinucleotide phosphate (NADP) dependent dehydrogenase-oxoreductase which is expressed in predominantly glucocorticoid target tissues and the encoding sequence of which is normal in patients with AME. In contrast, the type 2 isoform (11 beta-HSD2) is a high-affinity NADP-dependent unidirectional dehydrogenase which is expressed in placenta and mineralocorticoid target tissues such as renal collecting ducts and distal colonic epithelia. Exon- and intron-specific polymerase chain reaction amplification of the 11 beta-HSD2 gene from genomic DNA from members of a consanguinous kindred with AME consistently revealed a single missense mutation (C1228T) in two affected sibs and twin stillbirths. This mutation in codon 374 of exon 5 of the 11 beta-HSD2 gene creates an inframe premature stop (TGA) and, as such, results in a truncated 11 beta-HSD2 protein lacking the carboxyl-terminal proline-rich 32 amino acids. In keeping with an autosomal recessive mode of inheritance, both parents were phenotypically and biochemically normal but were heterozygous for this mutation. Unique to this kindred were expression analyses of the native mutant 11 beta-HSD2 enzyme in the stillbirth-affected placenta, which was almost completely devoid of NADP-dependent 11 beta-dehydrogenase activity. Immunohistochemical and Western blot analyses revealed the absence of 11 beta-HSD2 protein using antisera raised against synthetic peptide sequences corresponding either to the carboxyl terminus or other domains of the enzyme. MISSENSE MUTATION: In this kindred with AME, congenital deficiency of 11 beta-HSD activity is due to a single missense mutation in exon 5 of the 11 beta-HSD2 gene. Simultaneous studies by two other groups have similarly revealed no gross deletions or rearrangements of the 11 beta-HSD2 gene, but have described a number of single point mutations and oligonucleotide deletions in exons 3, 4 and 5, and adjacent to a splice site in intron 3. Recombinant expression analysis of site-directed mutant 11 beta-HSD2 complementary DNA constructs suggests a correlation between the predicted severity of these mutations and the biochemical and clinical phenotype. AME AS A CAUSE OF HYPERTENSION: The mutations in the 11 beta-HSD2 gene, together with those currently being sought by us for other kindreds with AME, establishes AME as a monogenic cause of human hypertension and will provide insight into the structure-function relationships of this important enzyme.     

Role of 17 beta-hydroxysteroid dehydrogenase type 1 in endocrine and intracrine estradiol biosynthesis

Enzymes with 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity catalyse reactions between the low-active female sex steroid, estrone, and the more potent estradiol, for example. 17 beta-HSD activity is essential for glandular (endocrine) sex hormone biosynthesis, but it is also present in several extra-gonadal tissues. Hence, 17 beta-HSD enzymes also take part in local (intracrine) estradiol production in the target tissues of estrogen action. Four distinct 17 beta-HSD isozymes have been characterized so far, and the data strongly suggests that different 17 beta-HSD isozymes have distinct roles in endocrine and intracrine metabolism of sex steroids. Current data suggest that 17 beta-HSD type 1 is the principal isoenzyme involved in glandular estradiol production both in humans and rodents. During ovarian follicular development and luteinization, rat 17 beta-HSD type 1 is regulated by gonadotropins, and the effects of gonadotropins are modulated by steroid hormones and paracrine growth factors. Human 17 beta-HSD type 1 favors the reduction reaction, thereby converting estrone to estradiol both in vitro and in cultured cells. Hence, the enzymatic properties of the enzyme are also in line with its suggested role in estradiol biosynthesis. Interestingly, 17 beta-HSD type 1 is also expressed in certain target tissues of estrogen action such as normal and malignant human breast and endometrium. Hence, 17 beta-HSD type 1 could be one of the factors leading to a relatively high tissue/plasma ratio of estradiol in breast cancer tissues of postmenopausal women. We conclude that 17 beta-HSD type 1 has a central role in regulating the circulating estradiol concentration as well as its local production in estrogen target cells.     

An autosomal glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) polymorphism in human saliva

Glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) from human saliva has been demonstrated by the zymogram technique. Three phenotypes were found. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles Sgd-1 and Sgd-2.     

The effects of insulin on 3 beta-hydroxysteroid dehydrogenase expression in human luteinized granulosa cells

OBJECTIVE: We determined the relative effects of insulin and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human luteinized granulosa cells. METHODS: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treated with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3 beta HSD were evaluated. RESULTS: Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/microL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3 beta HSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02). Insulin (greater than 100 nmol/L) alone increased 3 beta HSD protein content as measured by Western analysis 1.8-2-fold over basal levels, whereas FSH alone increased protein content 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3 beta HSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3 beta HSD 3.2-fold. In the presence of insulin plus FSH, 3 beta HSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3 beta HSD. CONCLUSION: Insulin is a regulator of both 3 beta HSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.     

The codon 213 of the 11beta-hydroxysteroid dehydrogenase type 2 gene is a hot spot for mutations in apparent mineralocorticoid excess

In the kidney, the 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11betaHSD2) inactivates glucocorticoids to their inactive ketoforms and thus prevents endogenous glucocorticoids from occupying the nonselective mineralocorticoid receptor in epithelial tissues. Several mutations have been described in the 11betaHSD2 gene in the congenital syndrome of apparent mineralocorticoid excess. These mutations generate partially or completely inactive 11betaHSD2 enzymes. In the present work, we describe an already known mutation in a new patient affected by apparent mineralocorticoid excess, which results in an arginine-to-cysteine mutation (R213C) in the 11betaHSD2 enzyme. This mutation has been found in two other independent families. In vitro expression studies of this mutant provide evidence that the mutant protein is normally expressed, but its activity is abolished. The CGC-to-TGC (C-toT) transition at codon 213 can be considered a typical CpG-consequence mutation. The present finding suggests that the codon R213 of 11betaHSD2 is a hot spot for mutations in this gene, as shown by the occurrence of an R213C point-mutation in several families unrelated to each other.     

Alterations in protein aggregation and degradation due to mild and severe missense mutations (A104D, R157N) in the human phenylalanine hydroxylase gene (PAH)

Although the importance of the father for the development of the child is well known and although an increased presence of the father in the family system is often demanded, processes of active shutting out the father occur. The deterioration of relation between father and child is not seen as a result of pathological male personality, but rather as a result of family or couple system. The special situation for counsellors of such cases is discussed.