Biomedical subjects

J Ian Mason

Publications and source records attributed to J Ian Mason.

10 recordsLinked to original sources

PubMed2003

Ovine steroid 17alpha-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme.

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.

17-alpha-Hydroxypregnenolone↗

PubMed2003

Carriers for type II 3beta-hydroxysteroid dehydrogenase (HSD3B2) deficiency can only be identified by HSD3B2 genotype study and not by hormone test.

OBJECTIVE: We investigated adrenal steroidogenic function relevant to 3beta-hydroxysteroid dehydrogenase (HSD3B2) activity in vivo and HSD3B2 genotype in clinically normal family members of patients with HSD3B2 genotype-proven HSD3B2 deficiency congenital adrenal hyperplasia (CAH) to determine whether genotype-proven carriers for HSD3B2 deficiency exhibit decreased enzyme activity analogous to the mildly decreased adrenal 21-hydroxylase activity in the carriers of CYP21 gene mutation. DESIGN/PATIENTS: Nineteen adult family members (ages median/range: 37/19-56 years) including 13 females and six males of six unrelated patients with HSD3B2 genotype-proven HSD3B2 deficiency were studied. MEASUREMENTS: All family members had HSD3B2 DNA analysis and an ACTH stimulation test (Cortrosyn 0.25 mg IV bolus) for determination of adrenal HSD3B activity. RESULTS: Ten of 13 females and five of six males were carriers of a proven or predictably deleterious mutation in one allele of the HSD3B2 gene, which was identified in the probands. ACTH-stimulated levels of 17-hydroxypregnenolone (delta5-17P), 17-hydroxyprogesterone (17-OHP), cortisol (F), dehydroepiandrosterone (DHEA) and androstenedione (delta4-A) and ratios of delta5-17P to 17-OHP, delta5-17P to F and DHEA to delta4-A, as well as increments of delta5-17P and DHEA values (ACTH-stimulated - baseline) in the genotype-proven female carriers (age, mean +/- SD: 36 +/- 6.7 years) and male carriers (age, mean +/- SD: 37 +/- 6.7 years) did not differ significantly from age-matched normal females (35 +/- 5.4 years, n = 20) and normal males (35 +/- 6 years, n = 10), respectively. There were no significant differences in any of the ACTH-stimulated hormonal levels or ratios between the female carriers with a seriously deleterious genotype (n = 5) and the female carriers with mildly deleterious genotypes (n = 5). These hormonal levels and ratios in three genotype-normal females and one genotype-normal male overlapped with those of the carriers. CONCLUSION: These data suggest that normal adrenal HSD3B2 activity is maintained in the genotype-proven carriers because heterodimers of mutant and wild-type HSD3B2 enzymes may be stable and exhibit similar activity compared to homodimers of wild-type enzymes, possibly by a relatively rate-unlimited effect of haplo-wild-type enzyme activity. However, we cannot preclude entirely the possibility of a limited expression of another HSD3B activity under ACTH stimulation contributing to the normal adrenal HSD3B activity in vivo in the HSD3B2 genotype-proven heterozygotes. Which mechanism plays a role in maintaining normal enzyme activity in the heterozygotes remains to be elucidated. The hormone findings in the genotypic-proven carriers for HSD3B2 deficiency also indicate that carriers for this disorder cannot be detected by a hormone test and can only be detected by HSD3B2 genotype study.

3-Hydroxysteroid Dehydrogenases↗

PubMed2002

Structure/function relationships responsible for the kinetic differences between human type 1 and type 2 3beta-hydroxysteroid dehydrogenase and for the catalysis of the type 1 activity.

Two distinct genes encode the 93% homologous type 1 (placenta, peripheral tissues) and type 2 (adrenals, gonads) 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) in humans. Mutagenesis studies using the type 1 enzyme have produced the Y154F and K158Q mutant enzymes in the Y(154)-P-H(156)-S-K(158) motif as well as the Y269S and K273Q mutants from a second motif, Y(269)-T-L-S-K(273), both of which are present in the primary structure of the human type 1 3beta-HSD/isomerase. In addition, the H156Y mutant of the type 1 enzyme has created a chimera of the type 2 enzyme motif (Y(154)-P-Y(156)-S-K(158)) in the type 1 enzyme. The mutant and wild-type enzymes have been expressed and purified. The K(m) value of dehydroepiandrosterone is 13-fold greater, and the maximal turnover rate (K(cat)) is 2-fold greater for wild-type 2 3beta-HSD compared with the wild-type 1 3beta-HSD activity. The H156Y mutant of the type 1 enzyme has substrate kinetic constants for 3beta-HSD activity that are very similar to those of the wild-type 2 enzyme. Dixon analysis shows that epostane inhibits the 3beta-HSD activity of the wild-type 1 enzyme with 14-17-fold greater affinity compared with the wild-type 2 and H156Y enzymes. The Y154F and K158Q mutants exhibit no 3beta-HSD activity, have substantial isomerase activity, and utilize substrate with K(m) values similar to those of wild-type 1 isomerase. The Y269S and K273Q mutants have low, pH-dependent 3beta-HSD activity, exhibit only 5% of the maximal isomerase activity, and utilize the isomerase substrate very poorly. From these studies, a structural basis for the profound differences in the substrate and inhibition kinetics of the wild-type 1 and 2 3beta-HSD, plus a catalytic role for the Tyr(154) and Lys(158) residues in the 3beta-HSD reaction have been identified. These advances in our understanding of the structure/function of human type 1 and 2 3beta-HSD/isomerase may lead to the design of selective inhibitors of the type 1 enzyme not only in placenta to control the onset of labor but also in hormone-sensitive breast, prostate, and choriocarcinoma tumors to slow their growth.

3-Hydroxysteroid Dehydrogenases↗

PubMed2002

Dissecting human adrenal androgen production.

The human adrenal cortex produces aldosterone, cortisol and the so-called adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). Within the adult adrenal, the zona glomerulosa produces aldosterone, the zona fasciculata cortisol and the zona reticularis both DHEA and DHEAS. The processes regulating aldosterone and cortisol synthesis are well defined; however, the mechanisms regulating the production of DHEA(S) remain elusive. The emphasis of this review is based on increasing evidence that cytochrome b(5), DHEA sulfotransferase and 3 beta-hydroxysteroid dehydrogenase play crucial roles in regulating production of DHEA(S). Insight into the mechanisms that regulate the synthesis of these key components of DHEA(S) synthesis should provide important clues to the regulation of adrenal androgen biosynthesis.

3-Hydroxysteroid Dehydrogenases↗

PubMed2002

Baboon cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17).

Human cytochrome P450 17alpha-hydroxylase (CYP17) catalyses not only the 17alpha-hydroxlation of pregnenolone and progesterone and the C17,20-side chain cleavage (lyase) of 17alpha-hydroxypregnenolone, necessary for the biosynthesis of C21-glucocorticoids and C19-androgens, but also catalyses the 16alpha-hydroxylation of progesterone. In efforts to understand the complex enzymology of CYP17, structure/function relationships have been reported previously after expressing recombinant DNAs, encoding CYP17 from various species, in nonsteroidogenic mammalian or yeast cells. A major difference between species resides in the lyase activity towards the hydroxylated intermediates and in the fact that the secretion of C19-steroids take place, in some species, principally in the gonads. Because human and higher primate adrenals secrete steroids, CYP17 has been characterized in the Cape baboon, a species more closely related to humans, in an effort to gain a further understanding of the reactions catalysed by CYP17. Baboon and human CYP17 cDNA share 96% homology. Baboon CYP17 has apparent Km and V values for pregnenolone and progesterone of 0.9 micro m and 0.4 nmol.h-1.mg protein-1 and 6.5 micro m and 3.9 nmol.h-1.mg protein-1, respectively. Baboon CYP17 had a significantly higher activity for progesterone hydroxylation relative to pregnenolone. No 16alpha-hydroxylase and no lyase activity for 17alpha-hydroxyprogesterone. Sequence analyses showed that there are 28 different amino acid residues between human and baboon CYP17, primarily in helices F and G and the F-G loop.

Adrenal Glands↗

PubMed2002

Differences in substrate and inhibitor kinetics of human type 1 and type 2 3beta-hydroxysteroid dehydrogenase are explained by the type 1 mutant, H156Y.

Two distinct genes encode the human type 1 (placenta, mammary gland) and type 2 (adrenal, gonad) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD). We have produced the Y154F, H156Y, and K158Q mutant enzymes in the Y154-P-H156-S-K158 motif of the human type 1 3beta-HSD/isomerase. The H156Y mutant was created to produce a chimera of the type 2 enzyme motif (Y154-P-Y156-S-K158) in the type 1 enzyme. The wild-type (WT) 1 and 2 plus the mutant enzymes were expressed and purified. The Km for dehydroepiandrosterone and Ki for epostane measured with both the H156Y mutant and WT 2 are 13-fold to 17-fold greater than those values obtained with the WT 1 3beta-HSD. The Y154F and K158Q mutants exhibit no 3beta-HSD but have significant isomerase activity. Thus, H156 in WT 1 vs. Y156 in WT 2 accounts for the substantially higher affinity of WT 1 3beta-HSD activity for these substrate and inhibitor steroids relative to the WT 2 enzyme.

Androstenols↗

PubMed2002

A novel nonstop mutation in the stop codon and a novel missense mutation in the type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene causing, respectively, nonclassic and classic 3beta-HSD deficiency congenital adrenal hyperplasia.

We investigated two novel point mutations in the human type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene causing a mild and a severe form of 3beta-HSD deficiency congenital adrenal hyperplasia. The first is a nonstop mutation in the normal stop codon 373 of the gene in exon IV [TGA (Stop) --> TGC (Cys) = Stop373C) identified from one allele of a female child with premature pubarche whose second allele had an E142K mutation. The Stop373C mutation predictably results in an open reading frame and a mutant-type (MT) II 3beta-HSD protein containing 467 amino acid residues, compared with the 372 amino acid residues of wild-type (WT) protein. The second is a homozygous missense mutation in codon 222 [CCA (Pro) --> ACT (Thr) = P222T] in the gene identified from a female neonate with salt-wasting disorder. The pcDNA vectors containing the constructs of WT II 3beta-HSD cDNA, WT cDNA with the open reading frame (WT cDNA(+)), MT Stop373C with the open reading frame (Stop373C(+)) and MT P222T cDNA were transfected in COS-I and 293T cells and expressed a similar amount of 3beta-HSD mRNA. The enzyme activity in intact cells using pregnenolone and dehydroepiandrosterone as substrate in the medium (1 micromol/liter) was identical between the WT cDNA and the WT cDNA(+), but was decreased to 27% of the WT enzymes at 6 h by MT Stop373C(+) enzyme, and was undetectable by P222T enzyme. In the homogenates of the cells, both MT Stop373C(+) and P222T enzyme activities and enzymes were undetectable despite clear detection of WT enzyme activities and WT enzymes. LH response to an LHRH analog stimulation in the pubertal female with the Stop373C/E142K genotypes and in a pubertal female with compound 273/318 frameshift genotypes were comparable to and higher than control females, respectively. In conclusion, a structurally lengthy MT II 3beta-HSD enzyme due to a nonstop mutation was relatively detrimental in intact cells causing the nonclassic phenotype of 3beta-HSD deficiency. A missense P222T mutation was seriously detrimental, causing the classic phenotype of 3beta-HSD deficiency. The undetectable Stop373C and P222T enzymes on Western blottings, together with the respective in vivo and in vitro data, suggest that a relative instability of Stop373C enzyme and a profound instability of the P222T enzyme are likely the detrimental molecular mechanisms. The increased LH in the female with the frameshift genotype and the appropriate LH response in the female with the nonstop genotype correlated with predictably severe and mild ovarian type II 3beta-HSD deficiency, respectively.

3-Hydroxysteroid Dehydrogenases↗

PubMed2002

Newly proposed hormonal criteria via genotypic proof for type II 3beta-hydroxysteroid dehydrogenase deficiency.

To define the hormonal criteria via genotypic proof for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) deficiency in the adrenals and gonads, we investigated the type II 3beta-HSD genotype in 55 patients with clinical and/or hormonal presentation suggesting compromised adrenal with or without gonadal 3beta-HSD activity. Fourteen patients (11 males and 3 females) had ambiguous genitalia with or without salt wasting and with or without premature pubarche. One female neonate had salt wasting only. Twenty-five children (4 males and 21 females) had premature pubarche only. Fifteen adolescent and adult females had hirsutism with or without menstrual disorder. The type II 3beta-HSD gene, including the promoter region up to -1053 base, all exons I, II, III, IV, and exon and intron boundaries, was sequenced in all subjects. Eight patients had a proven or predictably deleterious mutation in both alleles of the type II 3beta-HSD gene, and 47 patients had no apparent mutation in the gene. ACTH-stimulated (1 h post iv bolus of 250 microg Cortrosyn) serum 17-hydroxypregnenolone (Delta5-17P) levels and basal and ACTH-stimulated ratios of Delta5-17P to cortisol (F) in the genotypic proven patients were unequivocally higher than those of age-matched or pubic hair stage matched genotype-normal patients or control subjects (n = 7-30 for each group). All other baseline and ACTH-stimulated hormone parameters, including dehydroepiandrosterone (DHEA) levels, ratios of Delta5-17P to 17-OHP and DHEA to androstenedione in the genotype-proven patients, overlapped with the genotype-normal patients or control subjects. The hormonal findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3beta-HSD deficiency congenital adrenal hyperplasia (numeric and graphic reference standards from infancy to adulthood are provided): ACTH-stimulated Delta5-17P levels in 1) neonatal infants with ambiguous genitalia at or greater than 378 nmol/liter equivalent to or greater than 5.3 SD above the control mean level [95 +/- 53 (SD) nmol/liter]; 2) Tanner I children with ambiguous genitalia at or greater than 165 nmol/liter equivalent to or greater than 35 SD above the control mean level [12 +/- 4.3 (SD) nmol/liter]; 3) children with premature pubarche at or greater than 294 nmol/liter equivalent to or greater than 54 SD above Tanner II pubic hair stage matched control mean level [17 +/- 5 (SD) nmol/liter]; and 4) adults with at or greater than 289 nmol/liter equivalent to or greater than 21 SD above the normal mean level [25 +/- 12 (SD) nmol/liter]. ACTH-stimulated ratio of Delta5-17P to F in 1) neonatal infants at or greater than 434 equivalent to or greater than 6.4 SD above the control mean ratio [88 +/- 54 (SD)]; 2) Tanner I children at or greater than 216 equivalent to or greater than 23 SD above the control mean ratio [12 +/- 9 (SD)]; 3) children with premature pubarche at or greater than 363 equivalent to or greater than 38 SD above the control mean ratio [20 +/- 9 (SD)]; and 4) adults at or greater than 4010 equivalent to or greater than 221 SD above the normal mean ratio [29 +/- 18 (SD)]. Conversely, the hormonal data in the genotype-normal patients suggest the following hormonal criteria are not consistent with 3beta-HSD deficiency congenital adrenal hyperplasia: ACTH-stimulated Delta5-17P levels in children with premature pubarche up to 72 nmol/liter equivalent to up to 11 SD above the control mean level, and in hirsute females up to 150 nmol/liter equivalent to up to 12 SD above the normal female mean level [28 +/- 10 (SD) nmol/liter]; and ACTH-stimulated Delta5-17P to F ratio in children with premature pubarche up to 67 equivalent to up to 5 SD above the control mean ratio, and in hirsute females up to 151 equivalent to up to 10 SD above the normal mean ratio [32 +/- 12 (SD)]. These findings help define newly proposed hormonal criteria to accurately predict inherited 3beta-HSD deficiency.

3-Hydroxysteroid Dehydrogenases↗

PubMed2000

Studies on the onset of Leydig precursor cell differentiation in the prepubertal rat testis.

Leydig cells of the adult rat testis differentiate postnatally from spindle-shaped cells in the testis interstitium during the neonatal-prepubertal period. Which spindle-shaped cell types are the precursor for Leydig cells and the stimulus for initiation of their differentiation are, however, two unresolved issues. In the present study, our objectives were to identify unequivocally which spindle-shaped cells are the precursors to Leydig cells and to test whether the initiation of their differentiation into Leydig cells depends on LH. Testes from fifteen groups of Sprague-Dawley rats (n = 4 per group) from 7-21 days of age were fixed in Bouin solution and embedded in paraffin. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome P450 side-chain cleavage (P450(scc)), 17alpha-hydroxylase cytochrome P450 (P450(c17)), and LH receptors (LHR) in interstitial cells (other than fetal Leydig cells) was observed using the avidin biotin method. Of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for all three steroidogenic enzymes, beginning from the 11th postnatal day. All three enzymes were expressed simultaneously in these cells, and their numbers increased significantly thereafter. Immunoexpression of LHR in a few of these cells was just evident for the first time on postnatal Day 12 (i.e., after acquiring the steroidogenic enzyme activity). Their numbers gradually increased with time. The number of immunolabeled cells per 1000 interstitial cells (excluding fetal Leydig cells and capillary endothelial cells) was not significantly different for the three steroidogenic enzymes tested at all ages; however, a lower value was observed for LHR at each time-point. Based on these observations, we suggest that 1) the precursor cell type for the adult generation of Leydig cells in the postnatal rat testis is the peritubular mesenchymal cells, 2) precursor cells acquire 3beta-HSD, P450(scc), and P450(c17) enzyme activity simultaneously during Leydig cell differentiation, and 3) onset of precursor cell differentiation during Leydig cell development does not depend on LH.

3-Hydroxysteroid Dehydrogenases↗

PubMed2000

Effects of thyroid and luteinizing hormones on the onset of precursor cell differentiation into leydig progenitor cells in the prepubertal rat testis.

Leydig cells in the adult rat testis differentiate during the neonatal-prepubertal period. However, the stimulus for the initiation of their differentiation is still not clear. In the present study our objectives were to test the effects of thyroid hormone and LH on the initiation of precursor cell differentiation into Leydig cells in the prepubertal rat testis. Four groups of Sprague-Dawley rats were used. All treatments began at postnatal Day 1. Rats in groups I, II, and III received daily s.c. injections of saline (200 microl, controls), triiodothyronine (T(3), 50 microg/kg body weight, hyperthyroid), and LH (ovine LH 10 microg/rat/day), respectively. Rats in group IV were made hypothyroid from postnatal Day 1 by adding 0.1% propylthiouracil (PTU) to their mother's drinking water. Testes of rats were collected at 7, 8, 9, 10, 11, 12, 16, and 21 days of age, fixed in Bouin's solution, and embedded in paraffin for immunocytochemical studies. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and LH receptors (LHR) in testicular interstitial cells (other than the fetal Leydig cells) was observed using the avidin-biotin method. In control rats, out of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for 3beta-HSD, beginning from the postnatal Day 11. However, positive immunolabeling for LHR was first detected in these cells at Day 12, i.e., after acquiring the steroidogenic enzyme activity. In T(3)-treated rats 3beta-HSD positive spindle-shaped cells were first observed at Day 9 (i.e., 2 days earlier than controls), and LHR-positive cells were first observed on Day 11 (2 days later than obtaining 3beta-HSD immunoactivity); they were exclusively the peritubular mesenchymal cells. The 3beta-HSD- and LHR-positive spindle-shaped cells were absent in the testis interstitium of LH-injected rats from Days 7 through 12 but were present at postnatal Day 16. In addition, more fetal Leydig cell clusters and fetal Leydig cells in mitosis were present in LH-treated rats compared to rats in all other treatment groups. Following their first detection, the number of positive cells for each protein continued to increase at each subsequent age in controls, T(3)-, and LH-injected groups. In PTU rats, 3beta-HSD and LHR-positive spindle-shaped cells were absent throughout the experimental period. From these observations, it is possible to suggest the following regarding the developing rat testis interstitium. 1) The precursor cells for the adult generation of Leydig cells in the postnatal rat testis are the peritubular mesenchymal cells. 2) Luteinizing hormone does not initiate the onset of mesenchymal cell differentiation into Leydig cells, instead it delays this process. However, daily LH treatment causes mitosis in fetal Leydig cells and increase in fetal Leydig cell clusters. 3) Thyroid hormone is critical to initiate the onset of mesenchymal cell differentiation into adult Leydig cells.

3-Hydroxysteroid Dehydrogenases↗