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1.
BJOG ; 119(13): 1564-71, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23121125

ABSTRACT

OBJECTIVE: To study gene expression profiles in human endothelial cells incubated with plasma from women who developed pre-eclampsia and women with normotensive pregnancies. DESIGN: A case-control study. SETTING: A longitudinal nested case-control study within three maternity units. POPULATION: A mixed obstetric population attending maternity hospitals in Glasgow. METHODS: Plasma was obtained at both 16 and 28 weeks of gestation from 12 women: six women subsequently developed pre-eclampsia (cases) and six women, matched for age, body mass index (BMI) and parity, remained normotensive (controls). Human umbilical vein endothelial cells (HUVECs) were incubated with plasma for 24 hour before RNA isolation. MAIN OUTCOME MEASURES: Gene expression profiles were compared between the two gestational time points using Illumina(®) HumanHT-12 v4 Expression BeadChips. Differential mRNA expression observed in microarray experiments were validated using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and gene networks were analysed using Ingenuity(®) pathway analysis. RESULTS: There was a significant difference in the expression of 25 genes following incubation with plasma from controls, and an increase in the expression of 11 genes following incubation with plasma from cases, with no overlap between the two groups (false discovery rate, FDR < 0.05). There was a 3.74-fold (FDR < 0.001) increase in the expression of the c-Fos gene (FOS) when HUVECs were incubated with control plasma from 16 and 28 weeks of gestation, with no significant difference between the two time points with plasma from cases. Similar findings for FOS were obtained by qRT-PCR. CONCLUSIONS: Plasma from women who subsequently develop pre-eclampsia appears to contain factors that lead to the dysregulation of FOS in endothelial cells during pregnancy. Reduced expression of c-Fos may lead to impaired vasculogenesis, and thereby contribute to the development of pre-eclampsia.


Subject(s)
Gene Expression Regulation , Genes, fos , Human Umbilical Vein Endothelial Cells , Pre-Eclampsia/genetics , Transcriptome , Case-Control Studies , Cells, Cultured , Female , Gene Expression Profiling , Gene Regulatory Networks , Genetic Markers , Humans , Longitudinal Studies , Oligonucleotide Array Sequence Analysis , Plasma , Pre-Eclampsia/blood , Pregnancy , Pregnancy Trimester, Second , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
2.
Endocrinology ; 146(12): 5287-93, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16179417

ABSTRACT

Aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) catalyze the production of aldosterone and corticosterone, respectively, in the rat adrenal cortex. Recently, there has been some debate as to whether these corticosteroids are also produced in the hearts of rodents and humans, possibly contributing to the development of hypertrophy and myocardial fibrosis. To investigate this, we have used our established, highly sensitive real-time quantitative RT-PCR method to measure CYP11B1 and CYP11B2 mRNA levels in adrenal and cardiac tissue from several rat models of cardiovascular pathology. We have also studied isolated adult rat ventricular myocytes treated with angiotensin II and ACTH. Total RNA was isolated from the adrenal and cardiac tissue of 1) male Wistar rats with heart failure induced by coronary artery ligation and sham-operated controls; 2) stroke-prone spontaneously hypertensive rats and Wistar Kyoto rats as controls; 3) cyp1a1Ren-2 transgenic rats and Fischer controls; 4) isolated adult Sprague-Dawley ventricular myocytes incubated with 11-deoxycorticosterone (DOC), DOC plus angiotensin II, or DOC plus ACTH. Adrenal CYP11B2 expression was significantly increased in transgenic rats compared with Fischer controls (1.3 x 10(9)+/- 1.2 x 10(9) vs. 2.1 x 10(7) +/- 7.0 x 10(6) copies/microg RNA; P < 0.05). There were no other significant differences in adrenal CYP11B2 or CYP11B1 expression between the model animals and their respective controls. Cardiac CYP11B1 and CYP11B2 mRNA transcript levels from all in vivo and in vitro groups were never greater than 100 copies per microgram total RNA and therefore too low to be detected reproducibly. This suggests that cardiac corticosteroid production is unlikely to be of any physiological or pathological significance.


Subject(s)
Cardiovascular Diseases/enzymology , Cytochrome P-450 CYP11B2/metabolism , Myocardium/enzymology , Rats/metabolism , Steroid 11-beta-Hydroxylase/metabolism , Adrenal Glands/enzymology , Animals , Animals, Genetically Modified , Cells, Cultured , Cytochrome P-450 CYP11B2/genetics , Disease Models, Animal , Gene Expression , Heart Ventricles , Male , Myocytes, Cardiac/enzymology , RNA, Messenger/metabolism , Rats, Inbred Strains , Steroid 11-beta-Hydroxylase/genetics
3.
Scott Med J ; 47(1): 7-9, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11980293

ABSTRACT

The localisation of quantitative trait loci (QTL) is the first step towards gene identification. This is then verified by the construction of reciprocal congenic strains. The hypertensive SHRSP and normotensive WKY strains were used in a speed congenic approach to confirm the existence of a QTL on rat chromosome 2. Systolic baseline and salt loaded blood pressures were measured by radiotelemetry. Transfer of the chromosome 2 blood pressure QTL region from WKY into an SHRSP background significantly reduced blood pressure, with the increased significance at the salt loaded period, compared to the SHRSP. The reciprocal congenic blood pressure showed a significantly increased baseline systolic pressure compared to the WKY, with no change in significance at the salt loaded period. Thus we have successfully captured a gene(s) which contribute to blood pressure regulation in both congenic strains. This will facilitate further positional cloning of the causative genes first in this model and then in human essential hypertension.


Subject(s)
Blood Pressure/genetics , Hypertension/genetics , Quantitative Trait, Heritable , Analysis of Variance , Animals , Animals, Congenic , Chromosome Mapping , Male , Rats , Rats, Inbred SHR , Rats, Inbred WKY
4.
Physiol Genomics ; 6(2): 99-104, 2001 Jul 17.
Article in English | MEDLINE | ID: mdl-11459925

ABSTRACT

A genetic variant of the gene for the alpha(1)-isoform of Na(+)-K(+)-ATPase (Atp1a1) was suggested to be involved in the pathogenesis of salt hypertension in Dahl rats through altered Na(+):K(+) coupling ratio. We studied Na(+)-K(+) pump activity in erythrocytes of Dahl salt-sensitive (SS/Jr) rats in relation to plasma lipids and blood pressure (BP) and the linkage of polymorphic microsatellite marker D2Arb18 (located within intron 1 and exon 2 of Atp1a1 gene) with various phenotypes in 130 SS/Jr x SR/Jr F(2) rats. Salt-hypertensive SS/Jr rats had higher erythrocyte Na(+) content, enhanced ouabain-sensitive (OS) Na(+) and Rb(+) transport, and higher Na(+):Rb(+) coupling ratio of the Na(+)-K(+) pump. BP of F(2) hybrids correlated with erythrocyte Na(+) content, OS Na(+) extrusion, and OS Na(+):Rb(+) coupling ratio, but not with OS Rb(+) uptake. In F(2) hybrids there was a significant association indicating suggestive linkage (P < 0.005, LOD score 2.5) of an intragenic marker D2Arb18 with pulse pressure but not with mean arterial pressure or any parameter of Na(+)-K(+) pump activity (including its Na(+):Rb(+) coupling ratio). In contrast, plasma cholesterol, which was elevated in salt-hypertensive Dahl rats and which correlated with BP in F(2) hybrids, was also positively associated with OS Na(+) extrusion. The abnormal Na(+):K(+) stoichiometry of the Na(+)-K(+) pump is a consequence of elevated erythrocyte Na(+) content and suppressed OS Rb(+):K(+) exchange. In conclusion, abnormal cholesterol metabolism but not the Atp1a1 gene locus might represent an important factor for both high BP and altered Na(+)-K(+) pump function in salt-hypertensive Dahl rats.


Subject(s)
Hypertension/genetics , Hypertension/metabolism , Lipids/blood , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Blood Pressure , Cholesterol/blood , Enzyme Inhibitors/pharmacology , Erythrocytes/drug effects , Erythrocytes/enzymology , Hypertension/physiopathology , Ion Transport , Male , Ouabain/pharmacology , Polymorphism, Genetic , Rats , Rats, Inbred Dahl , Rubidium/metabolism , Sodium/metabolism , Sodium Chloride/administration & dosage , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors
5.
Hypertension ; 37(2 Pt 2): 391-7, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11230306

ABSTRACT

We have previously demonstrated that the SHRSP Y chromosome contains a locus that contributes to hypertension in SHRSP/WKY F2 hybrids and that SHRSP exhibit an increased vulnerability to focal cerebral ischemia after permanent middle cerebral artery occlusion (MCAO). This increased vulnerability is inherited as a codominant trait, and a putative role for the Y chromosome has been suggested in F1 hybrids. The objective of this study was to investigate further the role of Y chromosome in blood pressure (BP) regulation and in the vulnerability to cerebral ischemia. We have constructed consomic strains by selectively replacing the Y chromosome from WKY rats with that of SHRSP, and vice versa, by using a marker-assisted breeding strategy. Permanent MCAO was carried out by electrocoagulation, with infarct volume expressed as a percentage of the ipsilateral hemisphere. Systolic blood pressure was measured by radiotelemetry during a baseline period of 5 weeks followed by a 3-week period of salt loading. We observed that the transfer of the Y chromosome from WKY onto SHRSP background significantly reduced systolic BP in consomic strains, SP.WKYGlaY(w) (n=6) versus SHRSP (n=6) (209.2+/-10.4 mm Hg versus 241.7+/-7.7 mm Hg, F=5.88, P=0.038) during the salt-loading period. In the reciprocal consomic strain, WKY.SPGlaY(s) (n=5), systolic BP was increased compared with WKY parental strain (n=6) (147.6+/-2.4 mm Hg versus 132.6+/-5.1 mm Hg, F=6.11, P=0.035) during baseline. Infarct volumes in consomic strains were not significantly different from their respective parental strain: WKY.SPGlaY(s) (n=7) versus WKY (n=7), 22.8+/-3.7% versus 22.2+/-8.0%, 95% CI=-12.7, 4.2, P=0.3; SP.WKYGlaY(w) (n=7) versus SHRSP (n=6), 37.7+/-4.4% versus 33.6+/-7.6%, 95% CI=-20.3, 12.1, P=0.5. We conclude that the SHRSP Y chromosome harbors a locus contributing to systolic BP, whereas no contribution to vulnerability to cerebral ischemia can be detected.


Subject(s)
Infarction, Middle Cerebral Artery/physiopathology , Y Chromosome/physiology , Animals , Blood Pressure/physiology , Body Weight , Crosses, Genetic , Electrocoagulation , Genetic Markers , Infarction, Middle Cerebral Artery/etiology , Infarction, Middle Cerebral Artery/genetics , Male , Myocardium/pathology , Organ Size , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Y Chromosome/genetics
6.
J Reprod Fertil Suppl ; 57: 3-10, 2001.
Article in English | MEDLINE | ID: mdl-11787165

ABSTRACT

Ovaries of bitches are relatively inactive during anoestrus despite apparently adequate circulating FSH concentrations. Alternative FSH receptor (FSH-R) transcripts in bitches might hinder the follicular response to gonadotrophins, which may account for anoestrus. The expression of the full length FSH-R and novel isoforms in bitches was examined using in situ hybridization and RT-PCR analysis. Various PCR primers to the FSH-R were used and its expression was assessed in ovarian tissue at different stages of the oestrous cycle. RT-PCR amplification of the extracellular domain (exon 1-10) was generally successful, indicating that cFSH-R expression (> 90%) occurs throughout the oestrous cycle. Two FSH-R isoforms were sequenced, but there were no clear differences in the pattern of expression between anoestrus and other stages of the oestrous cycle, except that isoform expression was less frequent (30% occurrence) in prepubertal bitches. Data from in situ hybridization showed clear expression of the FSH-R in secondary and antral follicles, and corpora lutea. It was concluded that there is no evidence of a change in the expression of the FSH-R specific to anoestrus.


Subject(s)
Anestrus/metabolism , Dogs/physiology , Ovary/chemistry , RNA, Messenger/analysis , Receptors, FSH/genetics , Sexual Maturation/physiology , Amino Acid Sequence , Animals , Aromatase/genetics , Cholesterol Side-Chain Cleavage Enzyme/genetics , Corpus Luteum/chemistry , DNA Primers/genetics , Female , In Situ Hybridization , Molecular Sequence Data , Ovarian Follicle/chemistry , Protein Isoforms/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment
7.
Hypertension ; 35(1 Pt 2): 164-72, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10642293

ABSTRACT

Human essential hypertension is a complex, multifactorial, quantitative trait under a polygenic control. Several strategies have been developed over the last decade to dissect genetic determinants of hypertension. Of these, the most successful have been studies that identified rare mendelian syndromes in which a single gene mutation causes high blood pressure. The attempts to identify multiple genes, each with a small contribution to the common polygenic form of hypertension, have been less successful. Several laboratories focused their attention on rat models of genetic hypertension, which can be considered as a reductionist paradigm for human disease. Using numerous crosses between hypertensive and normotensive strains, investigators identified several quantitative trait loci (QTL) for blood pressure subphenotypes and for cardiovascular complications such as left ventricular hypertrophy, kidney failure, stroke, and insulin resistance. Furthermore, congenic strains have been produced to confirm the existence of some of these QTL and to narrow down the chromosomal regions of interest. A number of interesting strategies have been developed, including a "speed" congenic strategy perfected by our group in Glasgow. However, the limit of congenic strategy is estimated at 1 cM, which corresponds to 2x10(6) base pairs of DNA and approximately 50 candidate genes. It is envisaged that gene expression profiling with cDNA microarrays might allow a quick progression toward the gene identification within cardiovascular QTL. In parallel experimental effort, several laboratories have been developing gene transfer/therapy strategies with adenoviral or adeno-associated viral vectors used, for example, to overexpress protective vascular genes such as vascular endothelial growth factor or endothelial nitric oxide synthase. It is anticipated that further developments in positional cloning of susceptibility and severity genes in hypertension and its complications will lead to a direct transfer of these discoveries to essential hypertension in humans and will ultimately produce novel targets for local and systemic gene therapy in cardiovascular disease.


Subject(s)
Chromosome Mapping , Gene Transfer Techniques , Hypertension/genetics , Hypertension/therapy , Animals , Disease Models, Animal , Endothelium, Vascular/physiology , Humans
8.
Genomics ; 61(3): 277-84, 1999 Nov 01.
Article in English | MEDLINE | ID: mdl-10552929

ABSTRACT

Seven members of the human 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene family (HGMW-approved symbols HSD3BP1-HSD3BP5) have been cloned and physically mapped. HSD3B1 and 2 express 3beta-HSD enzymes; HSD3Bpsi1-5 are unprocessed pseudogenes that are closely related to HSD3B1 and 2 but contain no corresponding open reading frames. mRNA is expressed from psi4 and psi5 in several tissues, but with altered splice sites that disrupt reading frames. A 0.5-Mb contig of 3 yeast artificial chromosome and 32 bacterial artificial chromosome genomic clones contained no additional members of the gene family. The seven genes and pseudogenes mapped within 230 kb in the order HSD3Bpsi5-psi4-psi3-HSD3B1-psi1-psi2 -HSD3B2. HSD3B1 and 2 are in direct repeat, 100 kb apart. Six HSD3B2 mutations involve substitutions that are present in several of the pseudogenes. In four cases, mutations arose in CpG sites that are conserved within the gene cluster. The tendency for CpG sites to mutate by transition provides an adequate explanation for these HSD3B2 mutations, which are unlikely to be due to recombination or conversion within the gene family.


Subject(s)
3-Hydroxysteroid Dehydrogenases/genetics , 3-Hydroxysteroid Dehydrogenases/metabolism , Cloning, Molecular , Multigene Family , Physical Chromosome Mapping , Base Sequence , Blotting, Southern , Chromosomes, Artificial, Yeast , Chromosomes, Bacterial , DNA Primers , Genomic Library , Humans , Molecular Sequence Data , Mutation , Phylogeny , Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
9.
Eur J Biochem ; 260(3): 911-7, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10103023

ABSTRACT

Six isoforms of the enzyme 3beta-hydroxysteroid dehydrogenase (3betaHSD) have been identified in the mouse, each the product of a distinct gene. Two of these isoforms (type I and type VI) are detectable in the adult testis but changes in their expression during development are unknown. In this study we have examined changes in testicular expression and localization of mRNA encoding the type I and type VI isoforms of 3betaHSD. Total 3betaHSD (type I plus type VI) mRNA was measured by reverse transcription-polymerase chain reaction and showed a peak of expression at day 5 after birth followed by a decline and then a further rise after day 10 that continued up to adulthood. When each isoform was measured individually it was clear that the type I isoform was expressed at all ages from embryonic day 13 to adulthood. In contrast, the type VI isoform was only expressed at significant levels during fetal life on embryonic day 13 and then not again until after day 10 postnatally. Expression of the type VI isoform mRNA increased markedly after day 10 so that by adulthood it was the predominant 3betaHSD isoform present in the testis. Closer examination of the timing of type VI expression showed that the isoform mRNA was first detectable at a significant level on day 11. In-situ hybridization confirmed that the type I isoform is the only one expressed in the fetal/neonatal animal and showed that expression was limited to the interstitial tissue. In the adult, both type I and type VI expression was within the interstitial tissue. The timing of 3betaHSD type VI mRNA expression suggests, strongly, that this isoform is expressed only by adult-type Leydig cells in the mouse testis and that this development starts shortly before day 11. The limited expression of the type VI isoform means that it will be a useful marker in studies of adult Leydig cell development.


Subject(s)
3-Hydroxysteroid Dehydrogenases/biosynthesis , Isoenzymes/biosynthesis , Testis/enzymology , Testis/growth & development , 3-Hydroxysteroid Dehydrogenases/genetics , Animals , Female , In Situ Hybridization , In Vitro Techniques , Isoenzymes/genetics , Leydig Cells/enzymology , Male , Mice , RNA, Messenger/biosynthesis , Testis/cytology
10.
Biol Reprod ; 57(3): 602-8, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9282997

ABSTRACT

Normal gonadal development is dependent upon stimulation by the gonadotropic hormones, and it is likely that control of ovarian development is regulated by expression of gonadotropin receptors. In this study, quantitative changes in LH- and FSH-receptor mRNA levels were measured in the ovary during development in normal mice and in hypogonadal (hpg/hpg) mice, which lack circulating gonadotropins. The relative abundance of alternate transcripts encoding LH and FSH receptors was also determined. Results show that shortened transcripts of the receptors were abundant at all ages. Full-length transcripts of the LH receptor were not detectable until postnatal Day 5 although shortened transcripts encoding the extracellular domain of the receptor were present from birth. Between Days 5 and 7, LH-receptor transcript levels showed a marked increase in normal animals but no change in hpg/hpg animals. FSH-receptor transcripts encoding all domains of the receptor were detectable at low levels at birth, increased in concentration between Days 3 and 5, and peaked at Day 10. In hpg/hpg animals, FSH-receptor mRNA levels were normal up to Day 7 but failed to increase thereafter. These results show that early development of both LH- and FSH-receptor mRNA levels in the ovary is gonadotropin-independent. This coincides with early folliculogenesis to the primary stage. Further development of LH-receptor mRNA levels is gonadotropin-dependent although FSH-receptor mRNA levels continue to increase independently until early secondary follicles are present.


Subject(s)
Ovary/growth & development , Ovary/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, FSH/genetics , Receptors, LH/genetics , Animals , Animals, Newborn , Base Sequence , DNA Primers/genetics , Female , Gonadotropins, Pituitary/deficiency , Hypogonadism/genetics , Hypogonadism/metabolism , Mice , Mice, Mutant Strains , Polymerase Chain Reaction
11.
J Mol Endocrinol ; 15(2): 167-76, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8800641

ABSTRACT

Four hirsute females from a family exhibiting idiopathic dominant hirsutism were examined. Basal blood levels of delta 5 and delta 4 steroids were within the normal range, but ACTH stimulation led to increases in 17-hydroxypregnenolone and dehydroepiandrosterone that were significantly above control levels. Using polymorphic genetic markers, the genes for cytochrome P450c1717 encoded by CYP17, and the type I and II forms of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) were found not to segregate with hirsutism in this family, though a base substitution was detected in the 3' end of exon 1 of the gene for 3 beta-HSD type I in three of the four patients investigated. Analysis of PCR patients amplification products by denaturing gradient gel electrophoresis (DGGE) and sequencing revealed a novel homologue of exon 3 of 3 beta-HSD. DNA of one of the affected patients was used to create a genomic library in lambda gem 11 and clones containing the novel homologue were obtained and partially sequenced. The equivalent clone was obtained from a genomic library of an unrelated normal individual. The sequences of the clones from patient and control were identical and homologous to exons 2-4 of human 3 beta-HSD types I and II. No difference was found in the PCR primer sites that flanked the exons 3 homologue which led to its detection on DGGE gels. In both clones, stop codons and deletions were identified in the exon 4 homologue, leading to the deduction that the sequence comes from a pseudogene, which we call 3 beta-HSD psi 1. The pseudogene mapped to chromosome 1p13. It was concluded that dominantly inherited idiopathic hirsutism in this rare kindred was not due to deficiencies in 3 beta-HSD types I, II, or psi or of CYP17).


Subject(s)
3-Hydroxysteroid Dehydrogenases/genetics , Chromosomes, Human, Pair 1 , Hirsutism/genetics , Multigene Family , Steroid 17-alpha-Hydroxylase/genetics , Base Sequence , Chromosome Mapping , DNA Primers , Exons , Female , Genes, Dominant , Genetic Markers , Genomic Library , Hirsutism/blood , Humans , In Situ Hybridization, Fluorescence , Male , Molecular Sequence Data , Pedigree , Point Mutation , Polymerase Chain Reaction , Polymorphism, Genetic , Reference Values , Steroids/blood
12.
Mol Cell Probes ; 9(2): 121-8, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7603471

ABSTRACT

Several bands of hydridization are detected when southern blots of human genomic DNA are proved with cDNA of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type I. Two experimental approaches were adopted to estimate the size of the 3 beta-HSD gene family. Firstly, primer designed to amplify 3 beta-HSD type I and II genes were found on occasion to amplify DNA products of appropriate length but which were resolved as distinct sequences by denaturing gradient gel electrophoresis (DGGE). Five of these novel bands were cloned and their sequences were found to be closely related to 3 beta-HSD types I and II. Secondly, 57 genomic clones were selected from two lambda genomic libraries by hybridization with exonic probes of 3 beta -HSD type I. These were screened for novel members of the gene family by pcr amplification using various combinations of PCR primers to the type I and II genes, particularly those primers that previously amplified novel PCR products from genomic DNA. Amplification products from (lambda) clones were screened for novel sequences by DGGE. As a result of these approaches, at least five new members of the 3 beta-HSD gene family were found, one of which locates to the 3 beta -HSD type I and II gene cluster on 1p13. The existence of additional closely related but distinct members of the gene family should be recognized as a potential complication when screening PCR fragments for mutations in the type I and II genes. DGGE was found to be an exceedingly rapid means of screening amplification products from (lambda) clones to search for novel members of the gene family.


Subject(s)
3-Hydroxysteroid Dehydrogenases/genetics , Multigene Family , 3-Hydroxysteroid Dehydrogenases/biosynthesis , 3-Hydroxysteroid Dehydrogenases/chemistry , Amino Acid Sequence , Base Sequence , Blotting, Southern , Cloning, Molecular , DNA Primers , DNA Probes , Exons , Genomic Library , Humans , Isoenzymes/biosynthesis , Isoenzymes/chemistry , Isoenzymes/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid
13.
Endocr Res ; 21(1-2): 485-94, 1995.
Article in English | MEDLINE | ID: mdl-7588414

ABSTRACT

Polymorphic genetic variation shows that the genes for human 3 beta-hydroxysteroid dehydrogenases (3 beta-HSD) types I and II are closely linked. The type II mutations A82T, S100N and L173R are associated with male pseudohermaphroditism and A82T is associated, with variable penetrance, with female premature puberty. When expressed in vitro A82T showed less than 5% of normal activity and L173R showed a 30-50% reduction in activity. PCR experiments and direct genomic cloning show that there is a larger family of 3 beta-HSD sequences which require to be tested for expression. The phenomenon of epitopic heterogeneity of 3 beta-HSD is discussed and is now shown to apply to testicular Leydig cells as well as extrauterine trophoblast. RT-PCR analyses indicate that the phenomenon is most likely to be due to post-translational modification affecting the carboxytermini 3 beta-HSD types I and II. This phenomenon may reflect a further level at which enzyme activity is regulated.


Subject(s)
3-Hydroxysteroid Dehydrogenases/genetics , Gene Expression Regulation, Enzymologic/physiology , Genetic Variation , Multigene Family , Polymorphism, Genetic , Amino Acid Sequence , Antigenic Variation , Base Sequence , Epitope Mapping , Genetic Linkage , Humans , Molecular Sequence Data , Mutation
14.
J Mol Endocrinol ; 12(3): 273-81, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7522459

ABSTRACT

A new monoclonal antibody (FDO26G) is described which was raised against purified human 3 beta-hydroxysteroid dehydrogenase type I (3 beta-HSD type I). FDO26G reacted strongly with villous syncytiotrophoblast, weakly with some trophoblast cells in chorion laeve, and not at all with extravillous trophoblast in cytotrophoblast cell islands and decidual trophoblast. All these types of trophoblast reacted strongly with monoclonal antibody FDO161G, which has previously been shown to react with 3 beta-HSD type I and, like FDO26G, reacts strongly with adrenal cells. Mapping experiments using a combination of lacZ fusion polypeptides and synthetic peptides located the FDO26G epitope to residues 354-366 at the C-terminal end of the molecule, a sequence that is identical in the type I and type II forms of the enzyme. The epitope contains a consensus for a casein kinase-II site with serine 359 as the candidate phosphorylation site. This suggested that the lack of reactivity of FDO26G to 3 beta-HSD in extravillous trophoblast might be due to phosphorylation at serine 359. Peptide 354-366 was synthesized with phosphoserine at residue 359 and its binding to FDO26G was compared with that of the unphosphorylated peptide. FDO26G bound the phosphopeptide at least as strongly as the unphosphorylated peptide. It is concluded that the lack of staining of extravillous trophoblast by FDO26G is due to the presence of a different sequence at residues 354-366 and that a hitherto unidentified third isoform of human 3 beta-HSD is expressed in these cells.


Subject(s)
3-Hydroxysteroid Dehydrogenases/immunology , Antibodies, Monoclonal/immunology , Chorionic Villi/enzymology , Epitopes/immunology , Pregnancy Proteins/immunology , Trophoblasts/enzymology , 3-Hydroxysteroid Dehydrogenases/genetics , Amino Acid Sequence , Animals , Cattle , Chorionic Villi/immunology , Consensus Sequence , DNA, Complementary/genetics , Extraembryonic Membranes/enzymology , Extraembryonic Membranes/immunology , Female , Humans , Immune Sera , Mice , Molecular Sequence Data , Phosphorylation , Pregnancy , Pregnancy Proteins/genetics , Pregnancy Trimester, First , Rats , Recombinant Fusion Proteins/immunology , Sequence Alignment , Sequence Homology, Amino Acid , Sheep/blood , Trophoblasts/immunology
15.
J Reprod Fertil ; 93(1): 149-56, 1991 Sep.
Article in English | MEDLINE | ID: mdl-1920284

ABSTRACT

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.


Subject(s)
3-Hydroxysteroid Dehydrogenases/isolation & purification , Antigens/isolation & purification , Trophoblasts/immunology , 3-Hydroxysteroid Dehydrogenases/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular/methods , DNA, Circular/genetics , Female , Fluorescent Antibody Technique , Humans , Male , Molecular Sequence Data , Placenta/enzymology , Placenta/immunology , Trophoblasts/enzymology
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