Biomedical subjects

S D Morley

Publications and source records attributed to S D Morley.

At least 19 recordsLinked to original sources

PubMed2006

Characterization of the canine 3beta-hydroxysteroid dehydrogenase and its expression in the corpus luteum during diestrus.

3beta-Hydroxysteroid dehydrogenase (3betaHSD) is a key enzyme in the synthesis of bioactive steroid hormones. Objectives of the present study were to clone canine 3betaHSD and to investigate its expression in dog corpora lutea (CL) covering the periods of their formation, early and late regression (days 5, 15, 25, 35, 45, 65 after ovulation). Complete complementary DNA sequence was amplified by RACE PCR. Subsequent cloning revealed that the canine ovarian 3betaHSD transcript was composed of a 5'-untranslated region (5'-UTR) of 126 nucleotides, an open reading frame (ORF) of 1122 nucleotides and a 3'-UTR of 441 nucleotides. The putative ORF encoded a 374 amino acid protein which remains highly conserved (79-85% identity) between species. The transient expression of the cloned canine 3betaHSD in a mammalian heterologous cell expression system (HEK293T cells) identified the 3betaHSD activity as the only activity of this canine enzyme (absence of any detectable 17-hydroxysteroid dehydrogenase activity). Qualitative RT-PCR revealed expression of 3betaHSD on all days investigated and the signals were strongest on days 5 and 15, with day 25 intensity tending to decrease. However, variability between individual animals was high. The significant decrease in the expression of 3betaHSD towards the end of diestrus as indicated by Real Time PCR (p<0.01) and immunhistochemistry may indicate that the provision of progesterone is controlled by availability of the enzyme rather than the substrate.

17-Hydroxysteroid Dehydrogenases↗

PubMed2004

Validity of the 21-OH/LacZ transgenic mouse as a model for studying adrenocortical cell lineage.

Mosaic beta-galactosidase reporter staining patterns in the adult adrenal cortex of 21-OH/LacZ transgenic mice were compared to those observed in mouse chimeras and X-inactivation mosaics, which are known to have a lineage basis. This revealed similar patterns of blue and white radial stripes in all three experimental groups. Each blue stripe may contain one or more blue coherent clones of cells but this was taken into account by correcting the observed stripe numbers for the effects of different proportions of LacZ-positive (blue) and LacZ-negative (unstained) cells between adrenals. The corrected stripe numbers were similar in all three experimental groups, which supports the hypothesis that the stripes in the adrenals of 21-OH/LacZ transgenic mice are formed in a similar way to those in chimeras and X-inactivation mosaics (i.e., they have a lineage basis). This suggests that the 21-OH/LacZ transgenic mouse is likely to be a valid model for studying steroidogenic cell lineage in the adrenal cortex, thereby providing additional support for the centripetal migration hypothesis of adrenocortical cytogenesis.

Adrenal Cortex↗

PubMed2004

Promiscuous 3beta-hydroxysteroid dehydrogenases: testosterone 17beta-hydroxysteroid dehydrogenase activities of mouse type I and VI 3beta-hydroxysteroid dehydrogenases.

3Beta-hydroxysteroid dehydrogenase (3beta-HSD) activity is essential for the synthesis of all classes of steroid hormones, converting various delta5-3beta-hydroxysteroids into hormonally active delta4-3-ketosteroids in NAD+ -dependent reactions. Certain 3beta-HSD isoforms have been reported to exhibit additional dehydrogenase character (e.g., 17-hydroxysteroid dehydrogenase/reductase). We have investigated whether mouse type I (adrenal/gonadal) and type VI 3beta-HSDs (uterine/embryonic) display significant 17beta-HSD-like activity. Nonsteroidogenic HEK 293T cells were transiently transfected with pCMV-based expression vectors containing mouse type I and type VI 3beta-HSDs. Transfected cells expressing either mouse type I or type VI 3beta-HSD converted testosterone to androstenedione, albeit at rates one-tenth of those of pregnenolone to progesterone in similarly transfected 293T cells. Our findings demonstrate that the mouse 3beta-HSD I and VI isoforms can inactivate testosterone within an intact cell milieu. These findings are important not only in establishment of structure-function relationships, but also whenever murine systems are used for developmental/reproductive paradigms associated with human disorders.

17-Hydroxysteroid Dehydrogenases↗

PubMed2004

Vasoactive intestinal peptide (VIP) stimulates cortisol secretion from the H295 human adrenocortical tumour cell line via VPAC1 receptors.

Vasoactive intestinal peptide (VIP) shows a wide tissue distribution and exerts numerous physiological actions. VIP was shown in a dose-dependent manner to increase cortisol secretion in the NCI-H295R human adrenocortical carcinoma (H295) cell line (threshold dose 3.3x10(-10) M, maximal dose 10(-7) M), coupled with a parallel increase in cAMP accumulation. Receptor-specific agonists were employed to determine which of the two known VIP receptor subtypes was involved in cortisol secretion. Treatment with the VPAC1 receptor agonist, [K(15), R(16), L(27)]VIP(1-7)/GRF(8-27), produced a dose-dependent increase in H295 cell cortisol secretion (threshold dose 10(-11) M, maximal dose 10(-7) M) similar to that seen with VIP. Meanwhile, the high-affinity VPAC2 receptor agonist, RO-25-1553, failed to stimulate significantly cortisol or cAMP production from H295 cells. Inhibition of VIP-mediated H295 cell cortisol secretion by PG97-269, a competitive VPAC1-specific antagonist, produced parallel shifts of the dose-response curve and a Schild regression slope of 0.99, indicating competitive inhibition at a single receptor subtype. VIP is known also to interact with the PAC1 receptor, albeit with lower affinity (EC(50) of approximately 200 nM) than the homologous ligand, PACAP (EC(50) of approximately 0.5 nM). PACAP stimulated cortisol secretion from H295 cells (EC(50) of 0.3 nM), suggesting the presence of functional PAC1 receptors. However, stimulation of cortisol secretion by nanomolar concentrations of VIP (EC(50) of 5 nM), coupled with real-time PCR estimation that VPAC1 receptor transcripts appear 1000-fold more abundant than PAC1 transcripts in H295 cells, makes it unlikely that VIP signals via PAC1 receptors. Together, these data suggest that VIP directly stimulates cortisol secretion from H295 cells via activation of the VPAC1 receptor subtype.

Animals↗

PubMed2004

Virtual screening in structure-based drug discovery.

Recent advances in structure determination and computational methods have encouraged the development of structure-based virtual screening. Here we survey progress in the field and review the most recent methods, validation experiments and real applications, including an in-house example of hit identification for the oncology target Hsp90. These results provide a basis for discussing the current state of structure-based virtual screening and to outline the developments that are expected to have a major impact in the near future.

Combinatorial Chemistry Techniques↗

PubMed2000

Acute regulation of the bovine gene for the steroidogenic acute regulatory protein in ovarian theca and adrenocortical cells.

Upregulation of the steroidogenic acute regulatory protein (StAR) is implicated in the rapid synthesis and secretion of steroidogenic cells to produce steroids in response to stimulation by trophic hormones of the gonadal and stress axes. In the present study, we have assessed the kinetics of both StAR gene transcription and protein biosynthesis in primary cell cultures of bovine adrenocortical and ovarian theca cells, under conditions of acute stimulation by corticotrophin (ACTH) and luteinizing hormone (LH), respectively. In both cell systems, detectable upregulation of StAR gene transcription occurred within 1-2 h, reaching maxima at 4 h (theca cells) or 6 h (adrenocortical cells). mRNA levels returned rapidly to baseline, by 12 h or 24 h, respectively. Specific StAR protein levels were assessed by western blotting using a novel antibody raised against a bovine StAR peptide, and showed a similar fast upregulation, albeit delayed by 1-2 h compared with the mRNA. The response of the cultured theca cells was more acute than that of the adrenocortical cells, possibly reflecting the propensity of the LH receptor to desensitize rapidly, unlike the ACTH receptor. The primary bovine theca cell cultures were also used for fully homologous transfection studies using various deletion promoter-reporter constructs of the bovine StAR gene. Kinetic analysis of the results indicated that the acute transcriptional response resides within the proximal (-315 bp) promoter region, which includes two putative responsive elements for the steroidogenic factor-1. More distal promoter regions may be involved in modulating the specificity of expression by combining enhancer and inhibitory functions.

Adrenal Cortex↗

PubMed2000

Effects of cellular mediator agonists on cortisol and steroid acute regulatory (StAR) protein in bovine zona fasciculata (ZF) cells.

Primary cultures of bovine ZF cells were incubated for 1 h or 6h with the agonists 8-bromo-cyclic AMP (8-Br-cAMP) an activator of protein kinase A (PKA), the phorbol ester phorbol 12-myristate 13-acetate (PMA) a protein kinase C (PKC) activator, the Ca2+ ionophore, A23187, or the L-type Ca2+ channel agonist Bay K8644. Both cortisol secretion (determined by radioimmunoassay of cell medium) and cellular StAR protein levels (quantified by western blotting) were significantly increased at 6h, by all agonists. However, while all agonists stimulated cortisol secretion at 1h, StAR protein levels remained unchanged by these treatments. We conclude that in bovine ZF cells, StAR protein synthesis can be regulated by mechanisms involving activation of PKA, PKC and Ca2+ influx. However, a net increase in cellular StAR protein does not appear to be essential for the initiation for the first stage of acute steroidogenesis.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2000

Role of arachidonic acid metabolism in ACTH-stimulated cortisol secretion by bovine adrenocortical cells.

We have studied the effects of inhibitors of arachidonic acid (AA) metabolism, nordihydroguaiaretic acid (NDGA), a lipoxygenase (LPX) inhibitor, and indomethacin (INDO), a cyclooxygenase (COX) inhibitor, on cortisol secretion and StAR protein in primary cultures of bovine adrenal zona fasciculata (ZF) cells. NDGA inhibited cortisol secretion in response to both 10(-12) M and 10(-8) M ACTH. AA (10(-4) M) partially reversed the inhibition of cortisol secretion by NDGA at 10(-12) M ACTH but not at 10(-8) M ACTH. On the other hand, INDO potentiated the cortisol response to 10(-12) M ACTH. Neither NDGA nor INDO significantly affected StAR protein levels. These results suggest a StAR protein-independent role for the LPX and COX pathways in acute cortisol secretion, and support the hypothesis that LPX products of AA metabolism are key cellular signals when bovine ZF cells are acutely stimulated by physiological concentrations of ACTH (10(-12) M).

Adrenocorticotropic Hormone↗

PubMed2000

Properties of an adrenal medullary protein immunorelated to steroid acute regulatory (StAR) protein.

Immunohistochemistry using a StAR peptide antiserum had previously revealed strong staining in rat and bovine adrenal medulla, suggesting the presence of a protein immunogenically related to StAR. Western blots of bovine medulla tissue homogenates showed the principal adrenal medullary immuno-reactive species to have a higher molecular weight (50 kDa) compared to StAR protein (30 kDa). Subcellular fractionation localised the 50 kDa species principally to the medulla cytosol. StAR peptide antiserum binding to both the 30 kDa and 50 kDa species could be specifically competed by the peptide antigen. These data suggest that the adrenal medullary immuno-reactive species and StAR protein are distinct entities, which share some features in common.

Adrenal Medulla↗

PubMed1997

Renin-1 is essential for normal renal juxtaglomerular cell granulation and macula densa morphology.

The secretion of renin from granules stored in renal juxtaglomerular cells plays a key role in blood pressure homeostasis. The synthesis and release of renin and the extent of granulation is regulated by several mechanisms including signaling from the macula densa, neuronal input, and blood pressure. Through the use of a gene-targeting vector containing homology arms generated using the polymerase chain reaction, we have inactivated the Ren-1(d) gene, one of two mouse genes encoding renin, and report that lack of renin-1(d) results in altered morphology of the macula densa of the kidney distal tubule and complete absence of juxtaglomerular cell granulation. Furthermore, Ren-1(d-/-) mice exhibit sexually dimorphic hypotension. The altered growth morphology of the macula densa in Ren-1(d)-null mice should provide a tool for the investigation of the JG cell-macula densa signaling. Furthermore, the current data indicate that expression of the Ren-1(d) gene is a prerequisite for the formation of storage granules, even though the related protein renin-2 is present in these mice, suggesting that renin-1(d) and renin-2 are secreted by distinct pathways in vivo.

Animals↗

PubMed1996

Comparison of the activity of carp and rat beta-actin gene regulatory sequences in tilapia and rainbow trout embryos.

A comparative study on the level of expression of lacZ reporter constructs driven by equivalent carp and rat beta-actin regulatory sequences was carried out in embryos of tilapia and rainbow trout. DNA was microinjected into fertilised tilapia and rainbow trout eggs and the embryos/fry were assayed at various developmental stages for beta-galactosidase expression. We provide evidence to demonstrate that the carp beta-actin promoter/ lacZ reporter gene is expressed at higher levels than the equivalent rat beta-actin construct in both species.

Actins↗

PubMed1996

Multiple signal transduction systems regulate angiotensin II type 1 (AT1) receptor mRNA expression in bovine adrenocortical cells.

Regulation of AT1 receptor mRNA expression is an important determinant of angiotensin II-induced steroidogenesis. We have PCR-amplified the bovine adrenal AT1 receptor coding region using primers designed from the published bovine AT1 receptor sequence. This has been used as a probe on Northern blots to detect changes in the levels of AT1 receptor mRNA in primary cultures of bovine zona fasciculata cells in response to activation of several different signal transduction mechanisms in addition to two major adrenal steroid products, cortisol and aldosterone. AT1 receptor mRNA decreased in response to 6hr AII (10 nM) treatment, but returned to basal levels following 48h AII treatment. This effect was mimicked by the phorbol ester PMA (1 microM) and the calcium ionophore A23187 (1 microM), both singly and in combination. Activation of the cAMP pathway by ACTH (1 nM) and 8-bromo-cAMP (0.1 microM) also decreased AT1 receptor mRNA levels. In contrast, both IGF-1 (10 ng/ml) and potassium ions (12 mM) increased the levels of AT1 receptor mRNA. Finally, cortisol (10 microM) but not aldosterone (100 nM) decreased AT1 receptor mRNA. We conclude that the regulation of AT1 receptor mRNA in bovine zona fasciculata cells could involve several different signal transduction systems in addition to adrenocortical steroids themselves.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Adrenocortical-specific transgene expression directed by steroid hydroxylase gene promoters.

The 5'-flanking regions of genes for three mouse adrenal steroid hydroxylases were analyzed for their ability to direct adrenal cortex-specific beta-galactosidase (beta-gal) reporter expression both in cell culture and transgenic mice. The 5'-flanking regions chosen were from the genes for steroid 21-hydroxylase (21-OHase), expressed throughout the adrenal cortex and mediating both glucocorticoid and mineralocorticoid synthesis, and aldosterone synthetase (AS) and steroid 11 beta-hydroxylase (11 beta-OHase), which catalyze respectively the terminal steps of mineralocorticoid synthesis in the zona glomerulosa and glucocorticoid synthesis in the zona fasciculata/reticularis. While 5.0 kb of 11 beta-OHase gene 5'-flanking region and 5.4 kb of the AS gene 5'-flanking region mediated respectively moderate and low levels of beta-gal reporter expression in Y1 adrenocortical tumor cells, neither of these 5'-flanking regions was able to direct reporter expression to the appropriate adrenocortical zone of transgenic mice. This suggests that additional regulatory elements, lying outside these 5'-flanking regions, are required for 11 beta-OHase and AS gene expression in the intact mouse. In contrast, 6.4 kb of the mouse 21-OHase A gene 5' flanking region was able to direct specific beta-galactosidase reporter expression, in both Y1 cells and transgenic mice, indicating that elements directing adrenal cortex-specific gene expression in vivo are located not more than 6.4 kb 5' of the 21-OHase gene transcription start site.

Adrenal Cortex↗

PubMed1996

Variegated expression of a mouse steroid 21-hydroxylase/beta- galactosidase transgene suggests centripetal migration of adrenocortical cells.

5'-Flanking sequences (6.4 kb) of the mouse steroid 21-hyrodxylase (21-OHase) A gene linked to a LacZ reporter gene directed appropriate cell-specific expression in cultured Y1 adrenocortical tumor cells and in the adrenal cortex of transgenic mice. The transgene expression initiated at the same stage of adrenal development as the endogenous 21-OHase gene (embryonic day 11.5). Although the endogenous 21-OHase gene is expressed throughout the adrenal cortex, the 21-OHase/beta-gal transgene showed a strikingly variegated pattern of adrenocortical expression in all 10 transgene-expressing mouse lines examined. This presents as radial stripes of beta-gal staining transcending the classical zonal structure of the adrenal cortex but paralleling the columnar arrangement of cells of the zona fasciculata on the centripetal organization of the adrenocortical blood supply. To the extent that the variegated pattern of 21-OHase/beta-gal transgene expression depicts adrenocortical cell lineage, these results suggest that all cells within an individual stripe have a common clonal origin; the radial pattern of clonally derived cells argues that cellular migration maintains the adult adrenocortical cell population. Adrenal glands of developing embryos also exhibited a variegated pattern of 21-OHase/beta-gal transgene expression. However, this presented as islands of beta-gal reporter staining within the developing gland, suggesting that the rapid embryonic adrenal growth phase, which precedes the establishment of the classic adrenocortical zonal structure, may be governed by cellular mechanisms distinct from those responsible for maintenance of the adult adrenocortical cell population.

Adrenal Cortex↗

PubMed1996

Transgenics and essential hypertension.

Primary or "essential' hypertension is generally perceived to be a multifactorial or complex genetic trait. An individual's susceptibility to high blood pressure (BP) is influenced not only by the many genetic factors, which effect control through biochemical and physiological mechanisms, but also by environmental determinants. In a small proportion of human hypertensives the cause is a single genetic defect, exhibiting Mendelian characteristics. The vast heterogeneous majority, however, result from a multitude of contributing factors, making identification of the underlying etiology very difficult. We will briefly review a number of strategies which have helped to identify genetic factors involved in hypertension. These include the search for genetic defects in Mendelian forms of hypertension, intensive study of classical animal models such as the spontaneously hypertensive rat, and linkage analyses in animal models and hypertensive patients. We will then discuss the role which transgenesis can play in complementing and extending such analyses.

Animals↗

PubMed1994

Denatonium bitter tasting among transgenic mice expressing rat von Ebner's gland protein.

Von Ebner's gland protein (VEGP) is a secretory protein, which is abundantly expressed in the small von Ebner's salivary glands of the tongue. VEGP as component of the perireceptor environment around taste papillae might function as transporter of hydrophobic molecules, for example bitter substances. Here we report a new approach to investigate the physiological role of VEGP by expression of the cloned rat VEGP gene in transgenic mice. Taste papillae of mice, in contrast to rats, do not contain VEGP. The founder mouse 4345 and three offspring carry the transgene as shown by PCR analysis and saliva of the transgenic mice contains high amounts of VEGP. In two-bottle preference tests, transgenic and nontransgenic siblings show significantly different capabilities to taste the bitter compound denatonium benzoate at 10 microM. The reduced sensitivity of transgenic mice to denatonium benzoate points to a clearance function of VEGP the specificity of which for taste compounds and other molecules remains to be seen.

Animals↗

PubMed1992

Structural and computational investigations of the conformation of antigenic peptide fragments of human polymorphic epithelial mucin.

Human polymorphic epithelial mucins (PEM) are complex glycoproteins that are associated with breast and ovarian carcinomas. The PEM core protein consists of variable numbers of a tandem repeat sequence which contains a short antigenic hydrophilic region (Pro1-Asp-Thr-Arg-Pro-Ala-Pro7). High-field n.m.r. studies undertaken on antigenic 20- and 11-amino acid fragments of the PEM core protein in dimethyl sulphoxide have identified a type-I beta-turn to be present in the region Pro1-Asp-Thr-Arg4. This region includes and overlaps the identified type-I (Asp2-Thr-Arg4) and type-II (Arg4-Pro-Ala6) epitopes of anti-PEM monoclonal antibodies. The studies indicate that the beta-turn is stabilized by the presence of a salt-bridge interaction between Asp-2 and Arg-4. In order to probe the conformations accessible to the PEM peptides a computational study was undertaken independently on the peptide Pro-Asp-Thr-Arg-Pro using a modified Metropolis Monte Carlo algorithm. This study identified the n.m.r.-observed salt-bridge type-I beta-turn as the major low-energy conformer. These results suggest that this structural motif may be involved in the immune recognition of PEM.

Amino Acid Sequence↗

PubMed1992

Solution structures of nisin A and its two major degradation products determined by n.m.r.

The conformations of nisin and two major degradation products, nisin-(1-32)-peptide (nisin1-32) and des-delta Ala5-nisin1-32 (where delta Ala is alpha beta-didehydroalanine), in aqueous solution have been determined from n.m.r. data. Sequential assignments of the peptides using correlation spectroscopy ('COSY'), homonuclear Hartmann-Hahn spectroscopy ('HOHAHA'), nuclear Overhauser enhancement spectroscopy (NOESY), relayed NOESY and rotating-frame nuclear Overhauser spectroscopy (ROESY) experiments are presented, including stereospecific assignments of beta-methylene protons of the lanthionine residues. ROESY experiments are also used to detect flexible regions in the polypeptide chain. A dynamic-stimulated-annealing approach is used for structural determination. It can be concluded that all these peptides are flexible in aqueous solution, with no experimental evidence of preferred overall conformations; the only defined conformational features are imposed by the presence of the lanthionine residues. Low-temperature studies also reveal that des-delta Ala5-nisin1-32 adopts conformations similar to those when the ring is intact, suggesting that the loss of activity of this degradation product is due to the absence of the delta Ala5 residue rather than to the conformational consequences of ring-opening.

Amino Acid Sequence↗