Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q7LGC8 (HSD)
 3,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously demonstrated that cyclosporine inhibits testosterone (T) biosynthesis in vivo. To better understand the mechanism by which CsA inhibits T synthesis, interstitial cells were isolated from rat testes and incubated in the standard medium 199 with or without CsA (0-10 micrograms/ml) in the presence or absence of human chorionic gonadotropin (hCG, 10(-7) M) and 8-bromo cyclic AMP (cAMP, 0.5 mM) for 3 hr at 32 degrees C. The levels of cAMP and T were determined by RIA. CsA did not inhibit the basal secretion of T, but inhibited hCG-stimulated T production in a dose-dependent manner (4 ng/10(6) cells vs. 10 ng/10(6) cells at a CsA dose of 5 micrograms/ml, P less than 0.05). Radioligand binding of 125I-hLH to testicular membranes was not affected by CsA, as CsA did not compete with hCG/LH for binding sites (25-28% binding with or without CsA). Similarly, the MIX-stimulated cAMP production was not affected by CsA (24.03 +/- 1.09 vs. 20.60 +/- 0.38 pmol/10(6) cells), suggesting that CsA does not inhibit the accumulation of the second messenger. However, when interstitial cells were incubated with CsA in the presence of cAMP, a significant dose-dependent decline in T secretion was observed (7 ng/10(6) cells vs. 20 ng/10(6) cells at a CsA dose of 5 micrograms/ml). To determine whether CsA inhibits the steps beyond cAMP stimulation of T secretion, the kinetic parameters (Km and Vmax) of steroidogenic enzymes, delta 4-3 keto-17 alpha hydroxylase (17 alpha-hydroxylase), and delta 4-3 keto-17 beta hydroxy steroid dehydrogenase (17B-HSD) were determined by using Michaelis Menten analysis. Results are shown in the presence of CsA vs. no CsA: Km and Vmax values for 17 alpha-hydroxylase were (2.32 vs. 7.98 microM) and (27.96 vs. 100.97 pmol/mg protein/min), respectively. For 17B-HSD the Km and Vmax were (2.14 vs. 1.52 microM) and (15 vs. 15 pmol/mg protein/min), respectively. These results indicate that CsA inhibits the activity of 17 alpha-hydroxylase uncompetitively and 17B-HSD activity competitively. In conclusion the primary site for CsA inhibition is the cAMP stimulation and, CsA inhibits T synthesis at multiple sites.
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PMID:The mechanism of cyclosporine's action in the inhibition of testosterone biosynthesis by rat Leydig cells in vitro. 131 Jan 71

Genomic 17 beta-hydroxysteroid-dehydrogenase (17-HSD) clones were isolated from a human leucocyte genomic library using cDNA encoding human placental 17-HSD as a probe. The overlapping fragments spanned more than 21 kbp containing the duplications, 6.2 kbp of each, as well as 7 kbp upstream and 1.6 kbp downstream from the duplicated sequences. 17 complete and eight partial Alu elements were clustered in this area, covering about 30% of the region, including the borders of the duplications. Each duplication contained a 17-HSD gene and a conserved region of 1.56 kbp with 98% intercopy similarity. The exon structure of the 17-HSD gene II corresponded to the known cDNA species, but both genes contained a possible promoter region with TATA, GC and inverse CAAT boxes. The 5' flanking regions contained sequences similar to the consensus sequences of cis-acting elements, defined as regulators of 17-HSD gene expression. These putative sequences included estrogen and progesterone/glucocorticoid-response elements and a cyclic-AMP regulatory element.
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PMID:Genomic organization and DNA sequences of human 17 beta-hydroxysteroid dehydrogenase genes and flanking regions. Localization of multiple Alu sequences and putative cis-acting elements. 132 79

The gonadotrophic regulation of progesterone production by rat granulosa cells was examined in a chemically-defined medium containing FSH, dibutyryl cyclic AMP [Bu)2cAMP) and the calcium ionophore, A23187. FSH and A23187 alone significantly enhanced the production of pregnenolone, progesterone and its metabolite, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) from endogenous substrate(s). Stimulation of progesterone production by A23187 was accompanied by an increase in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) but not 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity, as attested by enhancement of the metabolism of exogenous pregnenolone to progesterone but not of progesterone to 20 alpha-OH-P. In contrast, although (Bu)2cAMP increased pregnenolone and progesterone production and the metabolism of exogenous progesterone to 20 alpha-OH-P, it failed to stimulate the conversion of exogenous pregnenolone to progesterone. The increase in progesterone production and in the conversion of exogenous pregnenolone to progesterone by FSH and A23187 was concentration- and time-dependent. Whereas maximal stimulation of de-novo progesterone synthesis by FSH was evident by 6 h (earliest time examined), a significant increase in the conversion of exogenous pregnenolone to progesterone in the presence of FSH or the ionophore was not noted until 12 h of incubation. Although a small but significant increase in progesterone production was also noted as early as 6 h of incubation in the presence of the calcium ionophore, this was markedly smaller than that elicited by FSH. We conclude that the calcium ionophore A23187 and (Bu)2cAMP have similar as well as distinct effects on progesterone production in rat granulosa cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Influence of A23187 and dibutyryl cyclic AMP on progestagen production by rat granulosa cells in vitro. 254 59

The inhibitory (relaxation) effects of five purine derivatives (ATP, ADP, AMP, Adenosine and Guanosine) on guinea pig tracheal and lung parenchymal smooth muscle were investigated. The tracheal spirals and parenchymal strips were bisected longitudinally and all twins submaximally precontracted with histamine. Isoproterenol was applied to one set of tracheal and parenchymal strips, and one of the purine derivatives to the other to produce cumulative concentration-effect relationships. For tracheal tissues, the isoproterenol curves did not differ significantly and could be pooled for comparison. Analysis of the EC50 values by Tukey's Studentized (HSD) test of mean isoproterenol and purine curves from tracheal tissue showed that isoproterenol values differed significantly from those of all the purines; there were no significant differences between the purine values. In parenchymal strips, isoproterenol values could not be pooled. Comparison of EC50 values for each purine derivative with its' isoproterenol group showed that ATP and adenosine did not cause significantly different values from isoproterenol, AMP did cause significant differences, and that ADP and guanosine could not be compared for failure to cause 50% relaxation. ATP responses were not significantly different from those of beta, gamma-methylene ATP, which only slowly degrades, suggesting that ATP has specific receptors in the airways of guinea pigs. In this study purines produced better relaxation in trachea than in parenchyma. A difference in purine receptor distribution between trachea and parenchyma is suggested.
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PMID:Inhibitory effects of selected purine derivatives on guinea pig pulmonary tissues. 370 10

The aim of this study was to determine whether steroidogenesis occurs in human immature oocytes aspirated from follicles during gynecologic laparotomy. delta 5-3 beta-Hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activities were detected by Dickmann and Dey's reaction medium consisting of 1.8 mg substrate (pregnenolone or 17 beta-estradiol [E2]), 4 mg nicotinamide-adenine dinucleotide, 2 mg nitro-blue tetrazolium, 10 ml 0.1 M phosphate buffer. The activity of adenylate cyclase was examined by ultrastructural-cytochemical study using 5'-adenylyl-imidodiphosphate (AMP-PNP) as a substrate in Vorbrodt's medium of 0.005 M AMP-PNP, 0.001 M MgCl2, 0.02 M SrCl2, 0.01 M NaF, 0.002 M theophylline, 0.01 M Tris-HCl. Furthermore, in indirect immunofluorescence study, the presence of endogenous progesterone and E2 and the activity of delta 5-3 beta-HSD were demonstrated. The results suggest that steroidogenesis, through the adenylate cyclase-cyclic adenosine 3':5'-monophosphate system, in human oocytes may play some important role in oocyte maturation, fertilization, and early embryonic development. The implication of steroid-producing activities of the human oocytes for cytoplasmic maturation is discussed.
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PMID:Cytochemical study of steroid-producing activities of human oocytes. 630 92

Bovine liver NADP(+)-dependent dihydrodiol dehydrogenase (DD3) is extremely sensitive to SH reagents such as N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid). NEM produced time- and concentration-dependent inactivation of DD3 in a pseudo-first-order reaction manner. This inactivation was prevented by NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, 2',5'-ADP and 2'-AMP but not by substrates, NAD+, nicotinamide mononucleotide or 5'-ADP.DD3 was absorbed by an affinity column of thiopropyl-Sepharose 6B, but enzyme incubated with both NEM and NADP+ was not. Moreover, one [14C]NEM molecule was incorporated into a cysteine of DD3 in the presence, and two cysteines of DD3 in the absence, of NADP+. These results suggested that two cysteine residues were modified per enzyme molecule by NEM, one was protected by NADP+ and the other had no significant function for the enzyme activity. Two radiolabelled peptides (P1 and P2) produced by the digestion with lysyl endopeptidase of [14C]NEM-modified DD3 could be separated by reverse-phase HPLC. P1, which was radiolabelled by [14C]NEM only in the absence of NADP+, showed the following sequence; H2N-Tyr-Lys-Pro-Val-Xaa-Asn-Gln-Val-Glu- NEM.Cys-His-Pro-Tyr-Phe-Asn-Gln-Ser-Lys-COOH (Xaa indicates a possible cysteine residue). This sequence was very similar to that of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD/DD) (residues 184 to 201) and was also highly conserved in the aldo-keto reductase superfamily. The sequence of P2, which had radioactivity in both the absence and presence of NADP+, also contained an NEM-modified cysteine and was similar in sequence to the regions located in loop A of rat 3 alpha-HSD/DD. The present study suggests that P1, which may have a cysteine residue corresponding to Cys-193 of rat 3 alpha-HSD/DD, functions in the alteration of DD3 activity depending on the modulation of NADP(+)-binding ability through a thiol/disulphide exchange reaction similar to that of rat 3 alpha-HSD/DD shown in our previous results; while P2, which may have a cysteine residue corresponding to Cys-145 of rat 3 alpha-HSD/DD, may be located near the surface of the enzyme molecule.
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PMID:The role of cysteine in the alteration of bovine liver dihydrodiol dehydrogenase 3 activity. 764 30

Excess glucocorticoids impair fetal growth and cause teratogenesis. Placental 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) catalyzes the inactivation of cortisol to cortisone, preventing the high maternal cortisol levels from reaching the fetal circulation and thus preserving the low cortisol fetal environment. In previous work, an NADP-dependent isoform of 11 beta HSD has been purified from rat liver, a cDNA isolated, and the human homolog cloned. However, much evidence suggests tissue-specific 11 beta HSD activities that cannot be explained by the liver-type isoform. Therefore, we have partially purified human placental 11 beta HSD and compared it to the enzyme in rat liver. Human placental subcellular fractions exhibited NAD-dependent 11 beta HSD activity, but showed little activity with NADP. The enzyme had a pH optimum of 7-8.5 (peak, 7.7), was only sparingly soluble in detergents (solubility with Triton X-100 was very poor), and exhibited little latency or change in pH profile in detergent solution. By contrast, rat liver 11 beta HSD was exclusively NADP dependent and was easily solubilized by a wide range of detergents (including Triton X-100), revealing substantial latency and altered pH profile [optimum of 10, becoming 7-10 (peak, 9.5) in detergent]. These data do not merely reflect species differences, as rat placental 11 beta HSD was similar to the human placental isoform. AMP affinity chromatography, which was completely without affinity for rat liver 11 beta HSD, achieved a 1000-fold purification of human placental 11 beta HSD. This had Km values for corticosterone (mean +/- SE, 14 +/- 1 nM) and cortisol (approximately 55 nM) that were over 100 times lower than that for liver 11 beta HSD. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis allowed identification of a band (apparent mol wt, 40,000) that correlated consistently with human placental 11 beta HSD activity (contrasting with a mol wt of 34,000 for rat liver 11 beta HSD). Thus, the NAD-dependent human placental 11 beta HSD is distinct from the previously characterized rat liver isoform and may be the product of a separate gene.
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PMID:Human placental 11 beta-hydroxysteroid dehydrogenase: evidence for and partial purification of a distinct NAD-dependent isoform. 850 62

The type 2 isoform of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD2), which catalyzes the conversion of cortisol to hormonally inactive cortisone in man, is principally expressed in the placenta and mineralocorticoid target tissues, kidney and colon. To date, few studies have addressed the regulation of this novel 11 beta-HSD2 isoform. We have characterized the nature and regulation of the 11 beta-HSD activity expressed in a human cytotrophoblastic cell line, the JEG-3 choriocarcinoma cell. The 11 beta-HSD activity in JEG-3 cell homogenates required NAD+ as cofactor with NADP+ ineffective and demonstrated a high affinity for cortisol (apparent Km 31 nM). Incubation of JEG-3 cells with forskolin and dibutyryl cyclic AMP increased 11 beta-HSD2 activity several-fold in a time-dependent manner, while treatment with phorbol ester had little, if any, effect on 11 beta-HSD2 activity. Northern blot analysis of RNA isolated from JEG-3 cells after these treatments demonstrated a marked increase in a 1.9 kb 11 beta-HSD2 mRNA species in cells treated with forskolin for 24 h. We conclude that 11 beta-HSD2 is regulated by activation of the protein kinase A pathway, but not the protein kinase C pathway in human choriocarcinoma cells, and that this regulation occurs at a pretranslational level. JEG-3 cells provide an excellent model for further studies on the regulation of 11 beta-HSD2 gene expression in human trophoblast tissue.
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PMID:Regulation of 11 beta-hydroxysteroid dehydrogenase type 2 activity and mRNA in human choriocarcinoma cells. 878 85

1. The aim of this study was to investigate the mechanism by which amphetamine exerts its inhibitory effect on testicular interstitial cells of male rats. 2. Administration of amphetamine (10(-12)-10(-6) M) in vitro resulted in a dose-dependent inhibition of both basal and human chorionic gonadotropin (hCG, 0.05 iu ml(-1))-stimulated release of testosterone. 3. Amphetamine (10(-9) M) enhanced the basal and hCG-increased levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in vitro (P<0.05) in rat testicular interstitial cells. 4. Administration of SQ22536, an adenylyl cyclase inhibitor, decreased the basal release (P<0.05) of testosterone in vitro and abolished the inhibitory effect of amphetamine. 5. Nifedipine (10(-6) M) alone decreased the secretion of testosterone (P<0.01) but it failed to modify the inhibitory action of amphetamine (10(-10)-10(-6) M). 6. Amphetamine (10(-10)-10(-6) M) significantly (P<0.05 or P<0.01) decreased the activities of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c17, and 17-ketosteroid reductase (17-KSR) as indicated by thin-layer chromatography. (t.l.c.). 7. These results suggest that increased cyclic AMP production, decreased Ca2+ channel activity and decreased activities of 3beta-HSD, P450c17, and 17-KSR are involved in the inhibition of testosterone production induced by the administration of amphetamine.
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PMID:The role of cyclic AMP production, calcium channel activation and enzyme activities in the inhibition of testosterone secretion by amphetamine. 938 14

3Alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) catalyze the interconversion between 5alpha-dihydrotestosterone (5alpha-DHT), the most potent androgen, and 3alpha-androstanediol (3alpha-diol), a weak androgen metabolite. To identify the rate-determining step in this physiologically important reaction, rat liver 3alpha-HSD (AKR1C9) was used as the protein model for the human homologues in fluorescence stopped-flow transient kinetic and kinetic isotope effect studies. Using single and multiple turnover experiments to monitor the NADPH-dependent reduction of 5alpha-DHT, it was found that k(lim) and k(max) values were identical to k(cat), indicating that chemistry is rate-limiting overall. Kinetic isotope effect measurements, which gave (D)k(cat) = 2.4 and (D)2(O)k(cat) = 3.0 at pL 6.0, suggest that the slow chemical transformation is significantly rate-limiting. When the NADP(+)-dependent oxidation of 3alpha-diol was monitored, single and multiple turnover experiments showed a k(lim) and burst kinetics consistent with product release as being rate-limiting overall. When NAD(+) was substituted for NADP(+), burst phase kinetics was eliminated, and k(max) was identical to k(cat). Thus with the physiologically relevant substrates 5alpha-DHT plus NADPH and 3alpha-diol plus NAD(+), the slowest event is chemistry. R276 forms a salt-linkage with the phosphate of 2'-AMP, and when it is mutated, tight binding of NAD(P)H is no longer observed [Ratnam, K., et al. (1999) Biochemistry 38, 7856-7864]. The R276M mutant also eliminated the burst phase kinetics observed for the NADP(+)-dependent oxidation of 3alpha-diol. The data with the R276M mutant confirms that the release of the NADPH product is the slow event; and in its absence, chemistry becomes rate-limiting. W227 is a critical hydrophobic residue at the steroid binding site, and when it is mutated to alanine, k(cat)/K(m) for oxidation is significantly depressed. Burst phase kinetics for the NADP(+)-dependent turnover of 3alpha-diol by W227A was also abolished. In the W227A mutant, the slow release of NADPH is no longer observed since the chemical transformation is now even slower. Thus, residues in the cofactor and steroid-binding site can alter the rate-determining step in the NADP(+)-dependent oxidation of 3alpha-diol to make chemistry rate-limiting overall.
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PMID:Dissection of the physiological interconversion of 5alpha-DHT and 3alpha-diol by rat 3alpha-HSD via transient kinetics shows that the chemical step is rate-determining: effect of mutating cofactor and substrate-binding pocket residues on catalysis. 1537 43


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