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Hydroxysteroid Dehydrogenase (hydroxysteroid + dehydrogenase)
Terms modified by Hydroxysteroid Dehydrogenase Selected AbstractsCinnamic Acid Esters as Potent Inhibitors of Fungal 17,-Hydroxysteroid Dehydrogenase , A Model Enzyme of the Short-Chain Dehydrogenase/Reductase SuperfamilyCHEMINFORM, Issue 46 2004Stanislav Gobec Abstract For Abstract see ChemInform Abstract in Full Text. [source] Structure of the G225P/G226P mutant of mouse 3(17),-hydroxysteroid dehydrogenase (AKR1C21) ternary complex: implications for the binding of inhibitor and substrateACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009Urmi Dhagat 3(17),-Hydroxysteroid dehydrogenase (AKR1C21) is a unique member of the aldo-keto reductase (AKR) superfamily owing to its ability to reduce 17-ketosteroids to 17,-hydroxysteroids, as opposed to other members of the AKR family, which can only produce 17,-hydroxysteroids. In this paper, the crystal structure of a double mutant (G225P/G226P) of AKR1C21 in complex with the coenzyme NADP+ and the inhibitor hexoestrol refined at 2.1,Å resolution is presented. Kinetic analysis and molecular-modelling studies of 17,- and 17,-hydroxysteroid substrates in the active site of AKR1C21 suggested that Gly225 and Gly226 play an important role in determining the substrate stereospecificity of the enzyme. Additionally, the G225P/G226P mutation of the enzyme reduced the affinity (Km) for both 3,- and 17,-hydroxysteroid substrates by up to 160-fold, indicating that these residues are critical for the binding of substrates. [source] Crystallization, X-ray diffraction analysis and phasing of 17,-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2005Alberto Cassetta 17,-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17,-HSDcl) is an NADP(H)-dependent enzyme that preferentially catalyses the oxidoreduction of oestrogens and androgens. The enzyme belongs to the short-chain dehydrogenase/reductase superfamily and is the only fungal hydroxysteroid dehydrogenase known to date. 17,-HSDcl has recently been characterized and cloned and has been the subject of several functional studies. Although several hypotheses on the physiological role of 17,-HSDcl in fungal metabolism have been formulated, its function is still unclear. An X-ray crystallographic study has been undertaken and the optimal conditions for crystallization of 17,-HSDcl (apo form) were established, resulting in well shaped crystals that diffracted to 1.7,Å resolution. The space group was identified as I4122, with unit-cell parameters a = b = 67.14, c = 266.77,Å. Phasing was successfully performed by Patterson search techniques. A catalytic inactive mutant Tyr167Phe was also engineered, expressed, purified and crystallized for functional and structural studies. [source] Discovery of Adamantyl Ethanone Derivatives as Potent 11,-Hydroxysteroid Dehydrogenase Type,1 (11,-HSD1) InhibitorsCHEMMEDCHEM, Issue 7 2010Xiangdong Su Dr. Abstract 11,-Hydroxysteroid dehydrogenases (11,-HSDs) are key enzymes regulating the pre-receptor metabolism of glucocorticoid hormones. The modulation of 11,-HSD type,1 activity with selective inhibitors has beneficial effects on various conditions including insulin resistance, dyslipidemia and obesity. Inhibition of tissue-specific glucocorticoid action by regulating 11,-HSD1 constitutes a promising treatment for metabolic and cardiovascular diseases. A series of novel adamantyl ethanone compounds was identified as potent inhibitors of human 11,-HSD1. The most active compounds identified (52, 62, 72, 92, 103 and 104) display potent inhibition of 11,-HSD1 with IC50 values in the 50,70,nM range. Compound 72 also proved to be metabolically stable when incubated with human liver microsomes. Furthermore, compound 72 showed very weak inhibitory activity for human cytochrome P450 enzymes and is therefore a candidate for in,vivo studies. Comparison of the publicly available X-ray crystal structures of human 11,-HSD1 led to docking studies of the potent compounds, revealing how these molecules may interact with the enzyme and cofactor. [source] Regulation of oocyte maturation in fishDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2008Yoshitaka Nagahama A period of oocyte growth is followed by a process called oocyte maturation (the resumption of meiosis) which occurs prior to ovulation and is a prerequisite for successful fertilization. Our studies using fish models have revealed that oocyte maturation is a three-step induction process involving gonadotropin (LH), maturation-inducing hormone (MIH), and maturation-promoting factor (MPF). LH acts on the ovarian follicle layer to produce MIH (17,, 20,-dihydroxy-4-pregnen-3-one, 17,, 20,-DP, in most fishes). The interaction of ovarian thecal and granulosa cell layers (two-cell type model), is required for the synthesis of 17,,20,-DP. The dramatic increase in the capacity of postvitellogenic follicles to produce 17,,20,-DP in response to LH is correlated with decreases in P450c17 (P450c17-I) and P450 aromatase (oP450arom) mRNA and increases in the novel form of P450c17 (P450c17-II) and 20,-hydroxysteroid dehydrogenase (20,-HSD) mRNA. Transcription factors such as Ad4BP/SF-1, Foxl2, and CREB may be involved in the regulation of expression of these steroidogenic enzymes. A distinct family of G-protein-coupled membrane-bound MIH receptors has been shown to mediate non-genomic actions of 17,, 20,-DP. The MIH signal induces the de novo synthesis of cyclin B from the stored mRNA, which activates a preexisting 35 kDa cdc2 kinase via phosphorylation of its threonine 161 by cyclin-dependent kinase activating kinase, thus producing the 34 kDa active cdc2 (active MPF). Upon egg activation, MPF is inactivated by degradation of cyclin B. This process is initiated by the 26S proteasome through the first cut in its NH2 terminus at lysine 57. [source] Reduced metabolites mediate neuroprotective effects of progesterone in the adult rat hippocampus.DEVELOPMENTAL NEUROBIOLOGY, Issue 9 2006The synthetic progestin medroxyprogesterone acetate (Provera) is not neuroprotective Abstract The ovarian hormone progesterone is neuroprotective in different experimental models of neurodegeneration. In the nervous system, progesterone is metabolized to 5,-dihydroprogesterone (DHP) by the enzyme 5,-reductase. DHP is subsequently reduced to 3,,5,-tetrahydroprogesterone (THP) by a reversible reaction catalyzed by the enzyme 3,-hydroxysteroid dehydrogenase. In this study we have analyzed whether progesterone metabolism is involved in the neuroprotective effect of the hormone in the hilus of the hippocampus of ovariectomized rats injected with kainic acid, an experimental model of excitotoxic cell death. Progesterone increased the levels of DHP and THP in plasma and hippocampus and prevented kainic-acid-induced neuronal loss. In contrast to progesterone, the synthetic progestin medroxyprogesterone acetate (MPA, Provera) did not increase DHP and THP levels and did not prevent kainic-acid-induced neuronal loss. The administration of the 5,-reductase inhibitor finasteride prevented the increase in the levels of DHP and THP in plasma and hippocampus as a result of progesterone administration and abolished the neuroprotective effect of progesterone. Both DHP and THP were neuroprotective against kainic acid. However, the administration of indomethacin, a 3,-hydroxysteroid dehydrogenase inhibitor, blocked the neuroprotective effect of both DHP and THP, suggesting that both metabolites are necessary for the neuroprotective effect of progesterone. In conclusion, our findings indicate that progesterone is neuroprotective against kainic acid excitotoxicity in vivo while the synthetic progestin MPA is not and suggest that progesterone metabolism to its reduced derivatives DHP and THP is necessary for the neuroprotective effect of the hormone. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Effects of brominated flame retardants and brominated dioxins on steroidogenesis in H295R human adrenocortical carcinoma cell lineENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2007Ling Ding Abstract Brominated flame retardants (BFRs) and brominated dioxins are emerging persistent organic pollutants that are ubiquitous in the environment and can be accumulated by wildlife and humans. These chemicals can disturb endocrine function. Recent studies have demonstrated that one of the mechanisms of endocrine disruption by chemicals is modulation of steroidogenic gene expression or enzyme activities. In this study, an in vitro assay based on the H295R human adrenocortical carcinoma cell line, which possesses most key genes or enzymes involved in steroidogenesis, was used to examine the effects of five bromophenols, two polybrominated biphenyls (PBBs 77 and 169), 2,3,7,8-tetrabromodibenzo- p -dioxin, and 2,3,7,8-tetrabromodibenzofuran on the expression of 10 key steroidogenic genes. The H295R cells were exposed to various BFR concentrations for 48 h, and the expression of specific genes,cytochrome P450 (CYP11A, CYP11B2, CYP17, CYP19, and CYP21), 3,-hydroxysteroid dehydrogenase (3,HSD2), 17,-hydroxysteroid dehydrogenase (17,HSD1 and 17,HSD4), steroidogenic acute regulatory protein (StAR), and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR),was quantitatively measured using real-time polymerase chain reaction. Cell viability was not affected at the doses tested. Most of the genes were either up- or down-regulated, to some extent, by BFR exposure. Among the genes tested, 3,HSD2 was the most markedly up-regulated, with a range of magnitude from 1.6- to 20-fold. The results demonstrate that bromophenol, bromobiphenyls, and bromodibenzo- p -dioxin/furan are able to modulate steroidogenic gene expression, which may lead to endocrine disruption. [source] Short-term cortisol infusion in the brachial artery, with and without inhibiting 11,-hydroxysteroid dehydrogenase, does not alter forearm vascular resistance in normotensive and hypertensive subjectsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 12 2002S. H. M. Van Uum Abstract Background Vascular tone is increased in primary hypertension, and glucocorticoids affect vascular tone. Local cortisol availability is modulated by activity of 11,-hydroxysteroid dehydrogenase (11,-HSD). As this activity may be decreased in patients with primary hypertension, vascular sensitivity to cortisol may be increased in these patients. We studied the acute effect of cortisol on forearm vascular resistance (FVR) by infusing cortisol directly into the brachial artery, both with and without inhibition of 11,-HSD, in normotensive and hypertensive subjects. Design Twenty normotensive volunteers and 20 patients with primary hypertension participated in the study. After a 10-min infusion of vehicle (glucose 5%), cortisol was infused into the brachial artery in three stepwise increasing doses (3·5, 10·5 and 35 µg per 100 mL of forearm volume), each for 10 min. Next, the participants received placebo or 500 mg glycyrrhetinic acid (GA) orally, and 150 min later the same infusion schedule was repeated. Forearm vascular resistance was measured during the last 5 min of the infused vehicle and of each dose. Arterial and forearm venous plasma samples for measurement of cortisol and cortisone were taken at the end of the infusions of glucose 5% and the highest cortisol dose. Results In both normotensive and hypertensive subjects, neither the infusion of cortisol nor the administration of GA changed FVR. Also 2 h after the cortisol infusion there remained no change in FVR in both the normotensive and hypertensive groups who received placebo. Following the infusion of the highest cortisol dose, total plasma cortisone levels in the venous plasma were decreased compared with levels in the arterial plasma (36 ± 3 and 49 ± 4 nmol L,1, respectively, P < 0·05). The protein-bound venous cortisone was 37·1 ± 4·8 nmol L,1 during the vehicle compared with 23·9 ± 3·7 nmol L,1 during the cortisol infusion (P < 0·01), whereas the free cortisone level was not altered by the cortisol infusion. Conclusions In both normotensive and hypertensive subjects, high-dose cortisol infusion both with and without 11,-HSD inhibition did not change FVR either immediately or after 2 h. We could not demonstrate in vivo 11,-HSD activity in the forearm vascular tissues. When binding of cortisone to CBG is changed, e.g. during cortisol infusion, arterio-venous changes in cortisone cannot reliably be used to assess (alterations in) local 11,-HSD activity. [source] Microglia express functional 11,-hydroxysteroid dehydrogenase type 1,GLIA, Issue 10 2010Andres Gottfried-Blackmore Abstract Glucocorticoids are potent regulators of inflammation exerting permissive, stimulatory, and suppressive effects. Glucocorticoid access to intracellular receptors is regulated by the activity of two distinct enzymes known as 11,-hydroxysteroid dehydrogenase (11,HSD) Type 1 and Type 2, which catalyze the activation or deactivation of glucocorticoids. Although expression of these enzymes in major organ systems and their roles in the metabolic effects of glucocorticoids have been described, their role in the inflammatory response has only recently started to be addressed. In this report, we have studied the expression and activity of 11,HSD Type 1 and Type 2 in microglia cells. Microglia, the brain's resident macrophages, initiate and orchestrate CNS inflammatory responses. Importantly, activated microglia are implicated in most neurodegenerative conditions, making them key subjects of study. We found that microglia expressed 11,HSD-1, but not 11,HSD-2, both in ex vivo FACS-sorted adult cells and in vitro primary cultures. 11,HSD-1 expression was increased in LPS-activated microglia. Moreover, 11,HSD-1 catalyzed the metabolic conversion of 11-dehydro-corticosterone into corticosterone (CORT), which potently reduced cytokine production in activated microglia. We propose that 11,HSD-1 may provide microglia with an intrinsic mechanism to autoregulate and inhibit proinflammatory mediator production through CORT formation. © 2010 Wiley-Liss, Inc. [source] Single nucleotide polymorphisms of 17,-hydroxysteroid dehydrogenase type 7 gene: Mechanism of estramustine-related adverse reactions?INTERNATIONAL JOURNAL OF UROLOGY, Issue 10 2009Takeshi Ozeki Objectives: To investigate the influence of single nucleotide polymorphisms (SNP) on transcription of the 17,-hydroxysteroid dehydrogenase (HSD17B7) gene. Methods: Luciferase reporter genes containing a 5,-flanking of the HSD17B7 gene, as well as the sequence around the SNP, were transfected into LNCaP and DU145 cells. Then, luciferase assays were carried out. Results: The presence of the G allele resulted in an increase of transcriptional activity derived from the 5,-flanking region of the HSD17B7 gene by 270% and 370% in LNCaP and DU145 cells, respectively. Transcriptional activity of the HSD17B7 gene containing the G allele was higher than that of the C allele. Conclusions: The transcriptional activity of the HSD17B7 gene containing the G allele is higher than that of the C allele. This difference in HSD17B7 expression may regulate the risk of peripheral edema as an adverse reaction induced by estramustine phosphate sodium. [source] The adrenal cortex and steroidogenesis as cellular and molecular targets for toxicity: critical omissions from regulatory endocrine disrupter screening strategies for human health?JOURNAL OF APPLIED TOXICOLOGY, Issue 2 2003Philip W. Harvey Abstract Current testing strategies to assess the endocrine disrupting properties of chemicals have omitted examination of the adrenal gland and do not adequately cover the process of steroidogenesis. Steroidogenesis is critical for adrenocortical function as well as that of the testes and ovaries, and presents multiple molecular targets for toxicity, ranging from general effects on all steroidogenic tissues (e.g. via StAR protein or CYP11A1 cholesterol side-chain cleavage) through to speci,c targets affecting only adrenocortical function (e.g. CYP11,/18 and glucocorticoid synthesis). Numerous chemicals of environmental relevance are now being shown to affect adrenocortical function both in vivo in aquatic species and in vitro in human cell lines, and given the vital role of the adrenal gland to human health and development, there is a strong case for including dedicated assessment techniques in screening batteries for endocrine-disrupting chemicals, not least to assist in general data interpretation (e.g. whether adrenal hypertrophy is due to stress or to a more sinister adrenocortical insuf,ciency). Cell lines such as H295R (derived from a human adrenocortical adenocarcinoma) currently exist that will allow assessment of cortisol production and most of the major enzymes and functional proteins in the steroidogenic pathway (e.g. StAR; CYP11A1/scc; CYP11,/18; CYP17; CYP19; CYP21; 3, -hydroxysteroid dehydrogenase). Adequate assessment of adrenocortical function, as with any component of the integrated endocrine system, probably also will require the development of speci,c in vivo methodology to include effects on hypothalamo-pituitary function. Finally, although there is currently no direct evidence that environmental exposure to endocrine-disrupting (oestrogenic) chemicals has actually caused adverse human health effects, lessons have been learned on their potential from the diethylstilboestrol case. Similar evidence exists from aminoglutethimide and etomidate on the lethal impact of unpredicted chemically induced adrenal insuf,ciency in sensitive human subgroups, and it would seem prudent to incorporate relevant tests for adrenal function and steroidogenesis into current regulatory validation programmes. Published in 2003 by John Wiley & Sons, Ltd. [source] Intracrine androgenic apparatus in human bone marrow stromal cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009Tarvo Sillat Abstract It was suggested that human mesenchymal stromal cells might contain an intracrine enzyme machinery potentially able to synthesize the cell's own supply of dihydrotestosterone (DHT) from dehydroepiandrosterone (DHEA) pro-hormone produced in the adrenal cortex in the reticular zone, which is unique to primates. Indeed, 3,-hydroxysteroid dehydrogenase (3,-HSD) and 5,-reductase enzyme proteins were expressed in resting mesenchymal stromal cells (MSCs) in vitro. However, the ,bridging' enzymes 17,-HSDs, catalysing interconversion between 17,-ketosteroids and 17,-hydroxysteroids, were not found in resting MSCs, but 17,-HSD enzyme protein was induced in a dose-dependent manner by DHEA. Quantitative real-time polymerase chain reactions disclosed that this was mainly due to induction of the isoform 5 catalysing this reaction in ,forward', androgen-bound direction (P < 0.01). This work demonstrates that the MSCs have an intracrine machinery to convert DHEA to DHT if and when challenged by DHEA. DHEA as substrate exerts a positive, feed-forward up-regulation on the 17,-hydroxy steroid dehydrogenase-5, which may imply that DHEA-DHT tailor-making in MSCs is subjected to chronobiological regulation. [source] Corticosterone induces steroidogenic lesion in cultured adult rat leydig cells by reducing the expression of star protein and steroidogenic enzymesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008Srinivasan Rengarajan Abstract The present study was designed to investigate the dose-dependent direct effect of corticosterone on adult rat Leydig cell steroidogenesis in vitro. Leydig cells were isolated from the testis of normal adult male albino rats, purified on discontinuous Percoll gradient and plated in culture plates/flasks overnight at 34°C in a CO2 incubator under 95% air and 5% CO2 using DME/F12 medium containing 1% fetal bovine serum. After the attachment of cells, serum-containing medium was removed and cells were exposed to different doses (0, 50, 100, 200, 400, and 800 nM) of corticosterone using serum-free fresh medium for 24 h at 34°C. At the end of exposure period, cells were utilized for assessment of the activities and mRNA expression of steroidogenic enzymes (cytochrome P450 side chain cleavage enzyme, 3,-hydroxysteroid dehydrogenase, 17,-hydroxysteroid dehydrogenase, and cytochrome P450 aromatase) and steroidogenic acute regulatory protein gene expression. Testosterone and estradiol production were also quantified. Activities of cytochrome P450 side chain cleavage enzyme, 3,- and 17,-hydroxysteroid dehydrogenases were declined significantly in a dose-dependent manner after corticosterone exposure, while their mRNA expression were significantly reduced at higher doses of corticosterone exposure. The activity and mRNA expression of cytochrome P450 aromatase registered a significant increase at 100 nM dose of corticosterone whereas at 200,800 nM doses both the activity as well as the mRNA levels was significantly reduced below the basal level. StAR protein gene expression was significantly inhibited by higher doses of corticosterone employed. At all doses employed, corticosterone significantly reduced the production of testosterone by Leydig cells, while estradiol level registered a significant increase at 50 and 100 nM doses but at higher doses, it registered a significant decrease when compared to basal level. It is concluded from the present in vitro study that the molecular mechanism by which corticosterone reduces the production of Leydig cell testosterone is by reducing the activities and mRNA expression of steroidogenic enzymes and steroidogenic acute regulatory protein. J. Cell. Biochem. 103: 1472,1487, 2008. © 2007 Wiley-Liss, Inc. [source] Endogenous glucocorticoids decrease skeletal angiogenesis, vascularity, hydration, and strength in aged miceAGING CELL, Issue 2 2010Robert S. Weinstein Summary Aging or glucocorticoid excess decrease bone strength more than bone mass in humans and mice, but an explanation for this mismatch remains elusive. We report that aging in C57BL/6 mice was associated with an increase in adrenal production of glucocorticoids as well as bone expression of 11,-hydroxysteroid dehydrogenase (11,-HSD) type 1, the enzyme that activates glucocorticoids. Aging also decreased the volume of the bone vasculature and solute transport from the peripheral circulation to the lacunar-canalicular system. The same changes were reproduced by pharmacologic hyperglucocorticoidism. Furthermore, mice in which osteoblasts and osteocytes were shielded from glucocorticoids via cell-specific transgenic expression of 11,-HSD type 2, the enzyme that inactivates glucocorticoids, were protected from the adverse effects of aging on osteoblast and osteocyte apoptosis, bone formation rate and microarchitecture, crystallinity, vasculature volume, interstitial fluid, and strength. In addition, glucocorticoids suppressed angiogenesis in fetal metatarsals and hypoxia inducible factor-1, transcription and vascular endothelial growth factor production in osteoblasts and osteocytes. These results, together with the evidence that dehydration of bone decreases strength, reveal that endogenous glucocorticoids increase skeletal fragility in old age as a result of cell autonomous effects on osteoblasts and osteocytes leading to interconnected decrements in bone angiogenesis, vasculature volume, and osteocyte-lacunar-canalicular fluid. [source] Protein identification by peptide mass fingerprinting and peptide sequence tagging with alternating scans of nano-liquid chromatography/infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2003Toshiyuki Kosaka Abstract We have developed a method for protein identification with peptide mass fingerprinting and sequence tagging using nano liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). To achieve greater sensitivity, a nanoelectrospray (nano-ES) needle packed with reversed-phase medium was used and connected to the nano-ES ion source of the FTICR mass spectrometer. To obtain peptide sequence tag information, infrared multiphoton dissociation (IRMPD) was carried out in nano-LC/FTICR-MS analysis. The analysis involves alternating nano-ES/FTICR-MS and nano-ES/IRMPD-FTICR-MS scans during a single LC run, which provides sets of parent and fragment ion masses of the proteolytic digest. The utility of this alternating-scan nano-LC/IRMPD-FTICR-MS approach was evaluated by using bovine serum albumin as a standard protein. We applied this approach to the protein identification of rat liver diacetyl-reducing enzyme. It was demonstrated that this enzyme was correctly identified as 3-,-hydroxysteroid dehydrogenase by the alternating-scan nano-LC/IRMPD-FTICR-MS approach with accurate peptide mass fingerprinting and peptide sequence tagging. Copyright © 2003 John Wiley & Sons, Ltd. [source] Inhibiting Biosynthesis and/or Metabolism of Progestins in the Ventral Tegmental Area Attenuates Lordosis of Rats in Behavioural OestrusJOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2005S. M. Petralia Abstract In the ventral tegmental area (VTA), lordosis of rats is facilitated by 5,-pregnan-3,-ol-20-one (3,,5,-THP). Central 3,,5,-THP results from metabolism of peripheral progesterone, from the ovaries and/or adrenals, by sequential enzymatic activity of 5,-reductase and 3,-hydroxysteroid oxidoreductase (3,-HSOR). In addition, in glial cells, cholesterol is converted into pregnenolone by the P450 side-chain cleavage enzyme (P450scc), which is then metabolized to progesterone by 3,-hydroxysteroid dehydrogenase, and subsequently reduced to 3,,5,-THP. We hypothesize that, in the VTA, formation of 3,,5,-THP by both metabolism and biosynthesis is necessary for facilitation of lordosis of female rats. In Experiment 1, naturally-receptive rats received bilateral VTA infusions of a P450scc inhibitor, digitoxin (1 µg/side); a 5,-reductase inhibitor, finasteride (10 µg/side); digitoxin (1 µg/side) + finasteride (10 µg/side); or vehicle and were tested 3 h later for lordosis. In Experiment 2, the effects of VTA infusions of digitoxin, finasteride, digitoxin + finasteride, or vehicle on lordosis and midbrain and plasma 3,,5,-THP levels were examined. In Experiment 3, we investigated whether infusions of 3,,5,-THP to the VTA reinstated lordosis and midbrain 3,,5,-THP levels following administration of inhibitors. VTA infusions of digitoxin, finasteride, or digitoxin + finasteride, significantly and similarly reduced lordosis and midbrain, but not plasma 3,,5,-THP levels, compared to vehicle. Following receipt of inhibitor infusions, 3,,5,-THP to the VTA restored lordosis and midbrain 3,,5,-THP levels. These data suggest that, in the VTA, both central biosynthesis of progesterone and metabolism of progesterone (from central and/or peripheral sources) to 3,,5,-THP are important for mediating lordosis of rats. [source] Reduction of the Potential Anticancer Drug Oracin in the Rat Liver In-vitroJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2000BARBORA SZOTÁKOVÁ Studies on the metabolism of the potential cytostatic drug oracin have shown that a principal metabolite of oracin is 11-dihydrooracin (DHO). We conducted in-vitro experiments to investigate the extent of oracin carbonyl reduction in microsomal or cytosolic fractions and to find out the enzymes involved under these conditions. Among several inducers of rat cytochrome P450 only 3-methylcholanthrene caused a significant (P < 0.01) stimulation (1.9 times) of DHO production in microsomal fraction and the specific P4501A inhibitor ,-naphthoflavone significantly (P < 0.01) decreased (twice) the induced reduction activity. Cytochrome P4501A participates in oracin reduction in microsomes. 18,-Glycyrrhetinic acid, a specific inhibitor of hydroxysteroid dehydrogenase, significantly (P < 0.01) inhibited the production of DHO in the microsomal fraction (>95% inhibition) in comparison with the non-inhibited reaction. Statistically significant (P < 0.01) inhibition (95%) of DHO formation was caused by metyrapone, which is also the substrate of 11,-hydroxysteroid dehydrogenase. The main microsomal enzyme which catalyses the carbonyl reduction of oracin is probably 11,-hydroxysteroid dehydrogenase. Important oracin reduction to DHO in the cytosolic fraction was found. According to its specific sensitivity towards quercitrin (inhibition by 99%, P < 0.01), the enzyme responsible for DHO formation in the rat liver cytosol is postulated to be carbonyl reductase. [source] Origin and evolution of somatic cell testicular tumours in transgenic mice,THE JOURNAL OF PATHOLOGY, Issue 4 2010Silvina Quintana Abstract Transgenic mice bearing a construct in which the expression of the SV40 oncogene is directed by the AMH promoter (AT mice) develop testicular tumours in adult life. We aimed to study early steps of tumour development and characterize tumours at different ages by histological, morphometric, and immunohistochemical techniques. One- to 3-month-old AT mice depicted multifocal Leydig cell hyperplasia. The testicular volume occupied by interstitial tissue was significantly higher in 3-month-old AT mice in comparison with littermate controls. Between 5 1/2 and 7 months, microscopic interstitial tumours developed that progressively evolved to form large confluent areas of high mitotic index in 7- to 14-month-old AT mice. Tumour cells had the characteristics and histoarchitecture of Leydig cells, or formed solid cord-like structures reminiscent of those seen in Sertoli cell tumours. Hyperplastic areas and tumours diffusely expressed 3,-hydroxysteroid dehydrogenase (3,-HSD) in Leydig cell areas. AMH expression was negative in Leydig cell conglomerates and tumours and variable in cord-like tumours. The SV40 T antigen and markers of cell proliferation (PCNA) were intensely positive in hyperplastic cells and tumours. Control mice of similar ages showed neither hyperplasia nor tumours, and SV40 T expression was always negative. In conclusion, transgenic mice develop large testicular tumours that are preceded by interstitial hyperplasia and microtumours. The histological and immunohistochemical phenotype of tumours (Leydig and Sertoli cell differentiation, positive 3,-HSD, and variable AMH) suggests a mixed differentiation of somatic cells of the specialized gonadal stroma. The finding that an oncogene directed by a promoter specifically active in fetal Sertoli cells has given rise to testicular tumours of mixed differentiation is compatible with a common origin of Leydig and Sertoli cells from the specific stroma of the gonadal ridge, as supported by double labelling experiments in fetal mice showing co-localization of the transgene with Sertoli and Leydig cell markers. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Actions of glucocorticoid and their regulatory mechanisms in the ovaryANIMAL SCIENCE JOURNAL, Issue 2 2007Masafumi TETSUKA ABSTRACT Glucocorticoid (G) directly modulates ovarian functions through binding to G receptor. The actions of G are both agonistic and antagonistic depending on the developmental stage of follicles and corpora lutea (CL). During follicular maturation, G suppresses follicular differentiation by downregulating expression of P450 aromatase and luteinizing hormone (LH) receptor in granulosa cells. During ovulation, G protects the ovulatory follicle from inflammatory damage and promotes luteinization, ensuring a smooth transition of the follicle to CL. Throughout life the ovary is exposed to periodic and sporadic waves of G. The Ovary appears to cope with this situation by locally modulating levels of active G. The primary regulatory mechanism consists of two isoforms of 11,-hydroxysteroid dehydrogenase (11,HSD) that catalyze conversion between active and inactive G. During follicular maturation the levels of active G are suppressed by the dehydrogenase activity of 11,HSD, whereas during the ovulatory process, levels of active G are further increased by the oxo-reductase activity of 11,HSD. The expression of these enzymes is under the control of gonadotrpins and local regulatory factors such as cytokines, allowing the mechanism to act in coordination with major reproductive events. Thus the G system is an integral part of ovarian physiology, which ensures that the ovary experiences only beneficial effects of G. [source] Studies on a Tyr residue critical for the binding of coenzyme and substrate in mouse 3(17),-hydroxysteroid dehydrogenase (AKR1C21): structure of the Y224D mutant enzymeACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2010Urmi Dhagat Mouse 3(17),-hydroxysteroid dehydrogenase (AKR1C21) is the only aldo,keto reductase that catalyzes the stereospecific reduction of 3- and 17-ketosteroids to the corresponding 3(17),-hydroxysteroids. The Y224D mutation of AKR1C21 reduced the Km value for NADP(H) by up to 80-fold and completely reversed the 17, stereospecificity of the enzyme. The crystal structure of the Y224D mutant at 2.3,Å resolution revealed that the mutation resulted in a change in the conformation of the flexible loop B, including the V-shaped groove, which is a unique feature of the active-site architecture of wild-type AKR1C21 and is formed by the side chains of Tyr224 and Trp227. Furthermore, mutations (Y224F and Q222N) of residues involved in forming the safety belt for binding of the coenzyme showed similar alterations in kinetic constants for 3,-hydroxy/3-ketosteroids and 17-hydroxy/ketosteroids compared with the wild type. [source] Structure of the G225P/G226P mutant of mouse 3(17),-hydroxysteroid dehydrogenase (AKR1C21) ternary complex: implications for the binding of inhibitor and substrateACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009Urmi Dhagat 3(17),-Hydroxysteroid dehydrogenase (AKR1C21) is a unique member of the aldo-keto reductase (AKR) superfamily owing to its ability to reduce 17-ketosteroids to 17,-hydroxysteroids, as opposed to other members of the AKR family, which can only produce 17,-hydroxysteroids. In this paper, the crystal structure of a double mutant (G225P/G226P) of AKR1C21 in complex with the coenzyme NADP+ and the inhibitor hexoestrol refined at 2.1,Å resolution is presented. Kinetic analysis and molecular-modelling studies of 17,- and 17,-hydroxysteroid substrates in the active site of AKR1C21 suggested that Gly225 and Gly226 play an important role in determining the substrate stereospecificity of the enzyme. Additionally, the G225P/G226P mutation of the enzyme reduced the affinity (Km) for both 3,- and 17,-hydroxysteroid substrates by up to 160-fold, indicating that these residues are critical for the binding of substrates. [source] Structure of 3(17),-hydroxysteroid dehydrogenase (AKR1C21) holoenzyme from an orthorhombic crystal form: an insight into the bifunctionality of the enzymeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2007Urmi Dhagat Mouse 3(17),-hydroxysteroid dehydrogenase (AKR1C21) is a bifunctional enzyme that catalyses the oxidoreduction of the 3- and 17-hydroxy/keto groups of steroid substrates such as oestrogens, androgens and neurosteroids. The structure of the AKR1C21,NADPH binary complex was determined from an orthorhombic crystal belonging to space group P212121 at a resolution of 1.8,Å. In order to identify the factors responsible for the bifunctionality of AKR1C21, three steroid substrates including a 17-keto steroid, a 3-keto steroid and a 3,-hydroxysteroid were docked into the substrate-binding cavity. Models of the enzyme,coenzyme,substrate complexes suggest that Lys31, Gly225 and Gly226 are important for ligand recognition and orientation in the active site. [source] Crystallization, X-ray diffraction analysis and phasing of 17,-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2005Alberto Cassetta 17,-Hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17,-HSDcl) is an NADP(H)-dependent enzyme that preferentially catalyses the oxidoreduction of oestrogens and androgens. The enzyme belongs to the short-chain dehydrogenase/reductase superfamily and is the only fungal hydroxysteroid dehydrogenase known to date. 17,-HSDcl has recently been characterized and cloned and has been the subject of several functional studies. Although several hypotheses on the physiological role of 17,-HSDcl in fungal metabolism have been formulated, its function is still unclear. An X-ray crystallographic study has been undertaken and the optimal conditions for crystallization of 17,-HSDcl (apo form) were established, resulting in well shaped crystals that diffracted to 1.7,Å resolution. The space group was identified as I4122, with unit-cell parameters a = b = 67.14, c = 266.77,Å. Phasing was successfully performed by Patterson search techniques. A catalytic inactive mutant Tyr167Phe was also engineered, expressed, purified and crystallized for functional and structural studies. [source] Therapeutic manipulation of glucocorticoid metabolism in cardiovascular diseaseBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2009Patrick W.F. Hadoke The therapeutic potential for manipulation of glucocorticoid metabolism in cardiovascular disease was revolutionized by the recognition that access of glucocorticoids to their receptors is regulated in a tissue-specific manner by the isozymes of 11,-hydroxysteroid dehydrogenase. Selective inhibitors of 11,-hydroxysteroid dehydrogenase type 1 have been shown recently to ameliorate cardiovascular risk factors and inhibit the development of atherosclerosis. This article addresses the possibility that inhibition of 11,-hydroxsteroid dehydrogenase type 1 activity in cells of the cardiovascular system contributes to this beneficial action. The link between glucocorticoids and cardiovascular disease is complex as glucocorticoid excess is linked with increased cardiovascular events but glucocorticoid administration can reduce atherogenesis and restenosis in animal models. There is considerable evidence that glucocorticoids can interact directly with cells of the cardiovascular system to alter their function and structure and the inflammatory response to injury. These actions may be regulated by glucocorticoid and/or mineralocorticoid receptors but are also dependent on the 11,-hydroxysteroid dehydrogenases which may be expressed in cardiac, vascular (endothelial, smooth muscle) and inflammatory (macrophages, neutrophils) cells. The activity of 11,-hydroxysteroid dehydrogenases in these cells is dependent upon differentiation state, the action of pro-inflammaotory cytokines and the influence of endogenous inhibitors (oxysterols, bile acids). Further investigations are required to clarify the link between glucocorticoid excess and cardiovascular events and to determine the mechanism through which glucocorticoid treatment inhibits atherosclerosis/restenosis. This will provide greater insights into the potential benefit of selective 11,-hydroxysteroid dehydrogenase inhibitors in treatment of cardiovascular disease. [source] Selection and optimization of MCF-7 cell line for screening selective inhibitors of 11,-hydroxysteroid dehydrogenase 2CELL BIOCHEMISTRY AND FUNCTION, Issue 6 2010Chi Hyun Kim Abstract An 11,-hydroxysteroid dehydrogenase type 1 (11,-HSD1) produces glucocorticoid (GC) from 11-keto metabolite, and its modulation has been suggested as a novel approach to treat metabolic diseases. In contrast, type 2 isozyme 11,-HSD2 is involved in the inactivation of glucocorticoids (GCs), protecting the non-selective mineralocorticoid receptor (MR) from GCs in kidney. Therefore, when 11,-HSD1 inhibitors are pursued to treat the metabolic syndrome, preferential selectivity of inhibitors for type 1 over type 2 isozyme is rather important than inhibitory potency. Primarily, to search for cell lines with 11,-HSD2 activity, we investigated the expression profiles of enzymes or receptors relevant to GC metabolism in breast, colon, and bone-derived cell lines. We demonstrated that MCF-7 cells had high expression for 11,-HSD2, but not for 11,-HSD1 with its cognate receptor. Next, for the determination of enzyme activity indirectly, we adopted homogeneous time resolved fluorescence (HTRF) cortisol assay. Obviously, the feasibility of HTRF to cellular 11,-HSD2 was corroborated by constructing inhibitory response to an 11b-HSD2 inhibitor glycyrrhetinic acid (GA). Taken together, MCF-7 that overexpresses type 2 but not type 1 enzyme is chosen for cellular 11,-HSD2 assay, and our results show that a nonradioactive HTRF assay is applicable for type 2 as well as type 1 isozyme. Copyright © 2010 John Wiley & Sons, Ltd. [source] Glucocorticoids contribute to the heritability of leptin in Scottish adult female twinsCLINICAL ENDOCRINOLOGY, Issue 1 2004A. M. Wallace Summary objective, The precise interactions between glucocorticoids and leptin are complex and poorly understood. The aim of the study was to investigate whether the glucocorticoid/leptin interaction is influenced by shared environmental or genetic factors. design, We investigated the heritability of body mass index (BMI), circulating leptin and urinary glucocorticoid metabolites [tetrahydrocortisol (THF), alloTHF and tetrahydrocortisone (THE)] in 54 monozygotic (MZ) and 39 dizygotic (DZ) female twins. Analysis was performed using a structural equation modelling package Mx, developed by Neale. results, Leptin and BMI showed substantial heritability (68·3% and 71·3%, respectively). Bivariate analysis indicated that the genetic determinants of BMI and leptin are partly shared. Total cortisol metabolites (THF + alloTHF + THE), the (THE + alloTHF)/THE ratio [a marker of 11,-hydroxysteroid dehydrogenase (11HSD) activity] and the alloTHF/THF ratio (marker for 5,-reductase activity) followed an environmental pattern. The heritability of leptin was significantly lowered to 63·8% (P = 0·012) when values were corrected for the influence of total cortisol metabolites but unaffected by markers of 11HSD and 5,-reductase activity. conclusions, We confirm that the genetic influence on both BMI and the circulating leptin concentration is substantial and show that these genetic determinants are highly correlated. These genetic factors, which are more likely to be dominant than additive, can be modestly but significantly modified by urinary total cortisol metabolites implying an adrenal influence. [source] Grapefruit juice inhibits 11,-hydroxysteroid dehydrogenase in vivo, in manCLINICAL ENDOCRINOLOGY, Issue 1 2003Mario Palermo No abstract is available for this article. [source] Corticosterone induces steroidogenic lesion in cultured adult rat leydig cells by reducing the expression of star protein and steroidogenic enzymesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008Srinivasan Rengarajan Abstract The present study was designed to investigate the dose-dependent direct effect of corticosterone on adult rat Leydig cell steroidogenesis in vitro. Leydig cells were isolated from the testis of normal adult male albino rats, purified on discontinuous Percoll gradient and plated in culture plates/flasks overnight at 34°C in a CO2 incubator under 95% air and 5% CO2 using DME/F12 medium containing 1% fetal bovine serum. After the attachment of cells, serum-containing medium was removed and cells were exposed to different doses (0, 50, 100, 200, 400, and 800 nM) of corticosterone using serum-free fresh medium for 24 h at 34°C. At the end of exposure period, cells were utilized for assessment of the activities and mRNA expression of steroidogenic enzymes (cytochrome P450 side chain cleavage enzyme, 3,-hydroxysteroid dehydrogenase, 17,-hydroxysteroid dehydrogenase, and cytochrome P450 aromatase) and steroidogenic acute regulatory protein gene expression. Testosterone and estradiol production were also quantified. Activities of cytochrome P450 side chain cleavage enzyme, 3,- and 17,-hydroxysteroid dehydrogenases were declined significantly in a dose-dependent manner after corticosterone exposure, while their mRNA expression were significantly reduced at higher doses of corticosterone exposure. The activity and mRNA expression of cytochrome P450 aromatase registered a significant increase at 100 nM dose of corticosterone whereas at 200,800 nM doses both the activity as well as the mRNA levels was significantly reduced below the basal level. StAR protein gene expression was significantly inhibited by higher doses of corticosterone employed. At all doses employed, corticosterone significantly reduced the production of testosterone by Leydig cells, while estradiol level registered a significant increase at 50 and 100 nM doses but at higher doses, it registered a significant decrease when compared to basal level. It is concluded from the present in vitro study that the molecular mechanism by which corticosterone reduces the production of Leydig cell testosterone is by reducing the activities and mRNA expression of steroidogenic enzymes and steroidogenic acute regulatory protein. J. Cell. Biochem. 103: 1472,1487, 2008. © 2007 Wiley-Liss, Inc. [source] Effects of glycyrrhetinic acid derivatives on hepatic and renal 11,-hydroxysteroid dehydrogenase activities in ratsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 6 2003Yoshihito Shimoyama The purpose of this study was to examine the structure and activity relationships of glycyrrhetinic acid derivatives on the inhibition of hepatic and renal 11,-hydroxysteroid dehydrogenases (HSDs) in rats. Furthermore, we explored whether inflammatory effect of the derivatives is involved in the inhibition of 11,-HSD activity. 18,-Glycyrrhetinic acid (Ia) potently inhibited 11,-HSD activity of hepatic (IC50 (concentration giving 50% inhibition of cortisone production) = 0.09 ,m) and renal (IC50 = 0.36 ,m) homogenate. The inhibitory effect of 18,-glycyrrhetol (Id) modified at the 30-position of glycyrrhetinic acid was weaker than that of glycyrrhetinic acid itself. 18,-24-Hydroxyglycyrrhetinic acid (Ie), oxidized at the 24-position, remarkably reduced the inhibitory activity for both enzymes. 18,-11-Deoxoglycyrrhetinic acid (IIc) showed the same inhibitory effect as glycyrrhetinic acid on hepatic 11,-HSD activity, but less effect on renal 11,-HSD activity. Furthermore, the inhibitory activity of 18,-deoxoglycyrrhetol (IIa), modified at the 11- and 30-position, was markedly decreased. Dihemiphthalate derivatives (IIb, IIIb and IVb) of deoxoglycyrrhetol (IIa), 18,-olean-9(11), 12-diene-3,, 30-diol (IIIa) and olean-11, 13(18)-diene-3,, 30-diol (IVa), which are anti-inflammatory agents, also showed weak inhibition against both hepatic and renal 11,-HSDs. While glycyrrhetinic acid (200 mg kg,1, p.o.) significantly inhibited 11,-HSD activity in rat liver and kidney at 3 h after administration, compound IVb (100 mg kg,1, p.o.) had no effect on either enzyme activity. In addition, the circulating corticosterone level was slightly increased by glycyrrhetinic acid but not by compound IVb. These results suggest that the anti-inflammatory effects of compound IVb, derived from glycyrrhetinic acid, are not due to accumulation of steroids induced by the inhibition of 11,-HSD activity. Our data also showed that the 11-, 24- and 30-positions of glycyrrhetinic acid may play important roles in the differential inhibitory effects on 11,-HSD isozyme activity. [source] Therapeutic manipulation of glucocorticoid metabolism in cardiovascular diseaseBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2009Patrick W.F. Hadoke The therapeutic potential for manipulation of glucocorticoid metabolism in cardiovascular disease was revolutionized by the recognition that access of glucocorticoids to their receptors is regulated in a tissue-specific manner by the isozymes of 11,-hydroxysteroid dehydrogenase. Selective inhibitors of 11,-hydroxysteroid dehydrogenase type 1 have been shown recently to ameliorate cardiovascular risk factors and inhibit the development of atherosclerosis. This article addresses the possibility that inhibition of 11,-hydroxsteroid dehydrogenase type 1 activity in cells of the cardiovascular system contributes to this beneficial action. The link between glucocorticoids and cardiovascular disease is complex as glucocorticoid excess is linked with increased cardiovascular events but glucocorticoid administration can reduce atherogenesis and restenosis in animal models. There is considerable evidence that glucocorticoids can interact directly with cells of the cardiovascular system to alter their function and structure and the inflammatory response to injury. These actions may be regulated by glucocorticoid and/or mineralocorticoid receptors but are also dependent on the 11,-hydroxysteroid dehydrogenases which may be expressed in cardiac, vascular (endothelial, smooth muscle) and inflammatory (macrophages, neutrophils) cells. The activity of 11,-hydroxysteroid dehydrogenases in these cells is dependent upon differentiation state, the action of pro-inflammaotory cytokines and the influence of endogenous inhibitors (oxysterols, bile acids). Further investigations are required to clarify the link between glucocorticoid excess and cardiovascular events and to determine the mechanism through which glucocorticoid treatment inhibits atherosclerosis/restenosis. This will provide greater insights into the potential benefit of selective 11,-hydroxysteroid dehydrogenase inhibitors in treatment of cardiovascular disease. [source] | ||||||||||||||||