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R G Sutcliffe

Publications and source records attributed to R G Sutcliffe.

At least 37 records · Page 2Linked to original sources
PubMed1995

New members of the 3 beta-hydroxysteroid dehydrogenase gene family.

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.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Blood pressure in genetically hypertensive rats. Influence of the Y chromosome.

We used a cross between the stroke-prone spontaneously hypertensive rat (SHRSP) strain and the Wistar-Kyoto (WKY) normotensive strain to elucidate the genetic basis of hypertension. Previous studies have reported conflicting evidence for the contribution of the Y chromosome to hypertension in these models. To investigate further the role of the Y chromosome in hypertension, we performed two large reciprocal crosses: one with the SHRSP as a male progenitor of the cross, yielding 60 F2 rats, and another with the WKY as a male progenitor, yielding 83 F2 rats. The resulting F2 hybrids were phenotyped with the use of a radiotelemetry system (Data Sciences) for measurement of systolic, diastolic, and mean arterial pressures as well as heart rate and motor activity continuously for 96 hours at baseline and after 1% NaCl was added to the rats' drinking water for 12 days. Male F2 hybrids with the SHRSP grandfather had significantly higher average systolic, diastolic, and mean arterial pressures at baseline compared with male F2 hybrids with the WKY grandfather (188.7 +/- 18.1 versus 168.9 +/- 11.5, 130.3 +/- 14 versus 115.7 +/- 7.3, and 159.1 +/- 15.8 versus 141.5 +/- 9.4 mm Hg, respectively). These differences were also observed after salt loading (197.9 +/- 22.1 versus 176.8 +/- 11.7, 136.5 +/- 17.3 versus 120.7 +/- 7.6, and 166.7 +/- 19.5 versus 148 +/- 9.7 mm Hg, respectively; P < .0001 for each comparison). These results suggest that the SHRSP Y chromosome contains a locus or loci that contribute to hypertension in SHRSP/WKY F2 hybrids.

Animals↗
PubMed1995

Microsatellite polymorphism analysis allows the individual assignment of the rat 11 beta-hydroxylase gene (Cyp11b1) and the rat aldosterone synthase gene (Cyp11b2) to chromosome 7 using rat x mouse somatic cell hybrids and identifies differences between and within various rat strains.

Mouse hepatoma x rat hepatocyte hybrids that segregate rat chromosomes were used to determine the chromosomal location of the rat genes encoding 11 beta-hydroxylase and aldosterone synthase (Cyp11b1 and Cyp11b2 respectively). By means of species-specific restriction fragments and microsatellite markers both genes were mapped to rat chromosome 7. The Cyp11b1 microsatellite marker was subsequently found to vary in length between and within rat strains. Furthermore, we compared the sequences of Cyp11b1 markers in two genetically hypertensive strains of rat with their normotensive counterparts. Previous studies have indicated that 11 beta-hydroxylase activities in Milan and Lyon hypertensive strains are different from their respective genetic controls. The Cyp11b1 microsatellite regions from Lyon hypotensive and normotensive strains of rat were similar and were both shorter by 15 bases than that of the Lyon hypertensive strain. The Cyp11b1 marker in Milan hypertensive (MHS) and normotensive (MNS) strains differ from all the Lyon strains and from each other. The MHS marker is 12 bases shorter than that of MNS rats. These differences in microsatellite length may provide useful polymorphic markers in cosegregation studies of genetic hypertension in rats.

Animals↗
PubMed1995

The human 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) gene cluster on chromosome 1p13 contains a presumptive pseudogene; 3 beta-HSD and CYP17 do not segregate with dominantly inherited hirsutism.

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).

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Mutation in 3 beta-hydroxysteroid dehydrogenase type II associated with pseudohermaphroditism in males and premature pubarche or cryptic expression in females.

A mutation (A82T) is described in the coding sequence of the gene for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type II that is associated with variable clinical consequences. Four homozygotes are described, all of which showed elevated levels of delta 5 steroids consistent with 3 beta-HSD deficiency. Two males from a consanguineous family were found to be homozygous for A82T and were affected with pseudohermaphroditism. They differed in their degree of mild salt loss. In the same family a female was found to be homozygous for A82T, but was clinically normal and had no history of premature pubarche or of abnormal menstrual cycles. However, in an apparently unrelated family, the A82T mutation was found in a female affected with premature pubarche. This is the first report of a proven mutation in 3 beta-HSD type II associated with premature pubarche.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Mutation in the human gene for 3 beta-hydroxysteroid dehydrogenase type II leading to male pseudohermaphroditism without salt loss.

A 5-year-old XY pseudohermaphrodite was found to have a defect of steroid biosynthesis consistent with a partial deficiency of the enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). Circulating concentrations of delta 5 steroids and delta 5 urinary steroid metabolites were elevated and remained elevated after orchidectomy. There was no evidence of salt loss, plasma renin being within normal limits, and no detectable glucocorticoid abnormality. The coding sequences of the genes for 3 beta-HSD types I and II were amplified by PCR and screened for mutations by denaturing gradient gel electrophoresis (DGGE) and manual and automatic DNA sequencing. A mutation in the gene for 3 beta-HSD type II was observed at codon 173 (CTA-->CGA), leading in the affected patient to a homozygous substitution in which the leucine at residue 173 was altered to an arginine (L173R). The propositus's 2-year-old XX sister was also homozygous for L173R and showed the biochemical characteristics of partial 3 beta-HSD deficiency without clinical symptoms or signs. The mutation segregated as an autosomal recessive. Three related heterozygous adult females showed evidence of a small over-production of delta 5 steroids and steroid metabolites and a variable reduction in ovarian function. Concentrations of delta 5 steroids and steroid metabolites in the heterozygous father of the propositus were within the normal range. These data are discussed in relation to the endocrine causes of pseudohermaphroditism and hirsutism. Evidence for tight linkage between the genes for 3 beta-HSD types I and II was obtained using a microsatellite polymorphism in the third intron of the gene for 3 beta-HSD type II and synonymous and non-synonymous mutations and polymorphisms in the gene for 3 beta-HSD type I. The latter polymorphisms were located 88 bp apart at the 3' end of the type I coding sequence and could be physically resolved as haplotypes using DGGE. The application of DGGE to the analysis of mutations in members of a multigene family is discussed.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Epitopic heterogeneity of human 3 beta-hydroxysteroid dehydrogenase in villous and extravillous human trophoblast.

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.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3 beta-hydroxy-5-ene steroid dehydrogenase.

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.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

A search for antibodies in term maternal sera to solubilized syncytiotrophoblast surface components. A passive haemagglutination assay yields negative evidence.

Using a passive haemagglutination assay, agglutinins against trophoblast components were detected in term maternal sera at titres above those of non-pregnant control sera. These agglutinin titres were independent of the family relationship between individual sera and trophoblast samples. They were unaffected by adsorption with excess packed fetal or paternal red blood cells or paternal lymphocytes. The agglutinins of both normal control and term maternal sera were found to elute in the macroglobulin fraction of serum having a molecular weight of greater than 350 kDa and were shown to be a component of euglobulin precipitates. The titres of the agglutinins were unaffected by adsorption using solid phase antibodies against human IgM and IgG from the sera. It was concluded that these agglutinins were not a maternal antibody response against trophoblast surface components.

Antibodies↗
PubMed1986

Detection of the mRNA to placental alkaline phosphatase by in vitro translation and immunoprecipitation.

RNA prepared from human placental syncytiotrophoblast tissue was analysed by in vitro translation and immunoprecipitation. The major polypeptide synthesized from syncytiotrophoblast mRNA was human placental lactogen, accounting for 15-20 per cent of the translated polypeptides. Immunoprecipitation using antibodies to placental alkaline phosphatase (PLAP) specifically precipitated polypeptides of 56 kD and 58 kD. The possible relationship between these polypeptides and those of placental microvillus PLAP is discussed.

Cell-Free System↗
PubMed1982

A quantitative assay for the measurement of immune responses directed against the human placenta.

A quantitative in vitro immune assay based on the classical chromium release assay has been developed to detect immune responses directed against alien antigens expressed by the developing foetus and present on the maternal-facing surface of the human placenta. A plasma membrane fraction from the surface of the placenta was prepared and the vesicles thus formed were radiolabelled with 51 Cr. The 51Cr-labelled vesicles, by various criteria, were found to be suitable for use as targets in a 'release assay'. Further, by means of experimentally immunised animals, the target membranes were shown to be capable of detecting both cellular and humoral anti-placental activity.

Animals↗
PubMed1982

Measurement of plasma placental proteins and estriol in the detection of intrauterine growth retardation.

This study was designed as an assessment of the clinical value of total estriol, placental lactogen (hPL), pregnancy specific glycoprotein (SP1), pregnancy-associated plasma protein-A (PAPP-A) and placental protein 5 (PP5), measured in a single sample of maternal serum, in the detection of intrauterine growth retardation (IUGR) in a large series of high risk pregnancies. Our results show the highest detection rate with SP1 (51%) and second highest with hPL (35%); estriol was less useful while PAPP-A and PP5 were of no value. However, there was 31% of false positives with SP1 compared with 14% for hPL. The best combination of the five parameters for the diagnosis of IUGR was achieved by measurement of both SP1 and hPL, with a low concentration of either protein indicating IUGR (sensitivity 63%, specificity 63%).

Estriol↗
PubMed1982

Localization of alpha-uterine protein in human endometrium.

Immunoperoxidase staining was used to investigate the origin of human alpha-uterine protein (AUP). Specific staining was observed in the glandular epithelium of the endometrium during the secretory phase of the menstrual cycle and during pregnancy, and in a patient on an oestrogen-progestagen contraceptive pill. The pattern of staining strongly suggests that AUP is secreted into the uterine lumen. The location and concentration of AUP in the uterus may explain the relative concentrations of AUP in amniotic fluid and maternal serum.

Amnion↗