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Biomedical subjects

K M O'Shaughnessy

Publications and source records attributed to K M O'Shaughnessy.

At least 19 recordsLinked to original sources
PubMed2005

The functional significance of genetic variation within the beta-adrenoceptor.

The beta-1 adrenoceptor is an archetypal G-coupled protein receptor that controls sympathetic responses in the heart, kidney and adipocytes. It has been widely exploited as a drug target with the development of antagonists to treat cardiovascular diseases such as hypertension, angina and heart failure. Signalling through the receptor is modulated by desensitization and beta1- adrenoceptor down-regulation. It is also affected by in vitro substitution of specific amino acid residues within the beta-1 adrenoceptor. Amino acid substitutions also occur naturally due to polymorphic variation within the human beta-1 adrenoceptor gene itself. Since these variants are common (typically being present in > 5% of the population), the pharmacogenetic implications are enormous. A number of these variants have been identified, although two have been the particular focus of recent publications: a serine to glycine substitution at position 49 (49S > G) and an arginine to glycine at position 389 (389R > G). The data on the in vitro behaviour of these two receptor variants is reviewed here, along with the evidence that they may affect both the risk of cardiovascular disease and the therapeutic response to beta-1 adrenoceptor antagonists.

Animals↗
PubMed2003

Greater inotropic and cyclic AMP responses evoked by noradrenaline through Arg389 beta 1-adrenoceptors versus Gly389 beta 1-adrenoceptors in isolated human atrial myocardium.

1. We studied the biochemical and contractile responses of isolated human myocardial tissue expressing native receptor variants of the 389G>R beta(1)-adrenoceptor polymorphism. 2. Right atrial appendage was obtained from homozygous RR patients (n=37) and homozygous GG patients (n=17) undergoing elective cardiac surgery. The positive inotropic effect of noradrenaline in these tissues, mediated through beta(1)-adrenoceptors, was studied using electrically stimulated (1 Hz) atrial strips, as well as the effects of noradrenaline on cyclic AMP levels and cyclic AMP-dependent protein kinase. 3. Tissue from RR homozygotes (n=14) showed significantly increased inotropic potency to noradrenaline (-log EC(50), M=6.92+/-0.12) compared to GG homozygotes (n=8, -log EC(50), M=6.36+/-0.11, P<0.005). This difference was not dependent on tissue basal force. 4. Tissue cyclic AMP levels (pmol mg(-1)) were also greater in RR homozygotes (basal 34.8+/-3.7 n=12, 300 nM noradrenaline 41.4+/-7.6 n=9, 30 micro M noradrenaline 45.2+/-3.2 n=22, 0.2 mM isoprenaline 48.3+/-4.2 n=16) compared to GG homozygotes (basal 30.7+/-4.4 n=5, 300 nM noradrenaline 32.6+/-6.92 n=5, 30 micro M noradrenaline 38.1+/-3.1 n=8, 0.2 mM isoprenaline 42.6+/-5.2 n=6, P=0.007). There were no differences between the variants in terms of cyclic AMP-dependent protein kinase activity. 5. These data provide the first evidence that enhanced G-protein coupling of the R389 beta(1)-adrenoceptor variant reported in rodent fibroblast expression systems is also present in native human receptors. The functional consequence of this is to significantly alter the inotropic potency of beta(1)-adrenoceptor activation depending on its genotype at the 389 position.

Adrenergic beta-1 Receptor Agonists↗
PubMed2001

A polymorphism of the human matrix gamma-carboxyglutamic acid protein promoter alters binding of an activating protein-1 complex and is associated with altered transcription and serum levels.

Matrix gamma-carboxyglutamic acid protein (MGP) is a mineral-binding extracellular matrix protein synthesized by vascular smooth muscle cells (VSMCs) and chondrocytes that is thought to be a key regulator of tissue calcification. In this study, we identified four polymorphisms in the promoter region of the human MGP gene. Transfection studies showed that the G-7A and T-138C polymorphisms have an important impact on in vitro promoter activity when transiently transfected into VSMCs. We found that one of these polymorphisms (T-138C) is significantly correlated with serum MGP levels in human subjects. Promoter deletion analysis showed that this polymorphism lies in a region of the promoter critical for transcription in VSMCs. This region contains a potential activating protein-1 (AP-1) binding element located between -142 and -136. We have demonstrated that the T-138C polymorphism results in altered binding of an AP-1 complex to this region. The -138T allelic variant binds AP-1 complexes consisting primarily of c-Jun, JunB and its partners Fra-1 and Fra-2 in rat VSMC. Furthermore, the -138T variant form of the promoter was induced following phorbol 12-myristate 13-acetate treatment, while the -138C variant was refractive to phorbol 12-myristate 13-acetate treatment, confirming that AP-1 factors preferentially bind to the -138T variant. This study therefore suggests that a common polymorphism of the MGP promoter influences binding of the AP-1 complex, which may lead to altered transcription and serum levels. This could have important implications for diseases such as atherosclerosis and aortic valve stenosis, since it strongly suggests a genetic basis for regulation of tissue calcification.

Animals↗
PubMed2001

Glycerol-enhanced mini-polyacrylamide gel electrophoresis for the separation of differentially expressed DNA fragments in cDNA representational difference analysis.

Representational difference analysis (RDA) is a widely used technique in molecular biology. However, in practice, its efficiency depends on a rapid and reliable separation of the RDA fragments prior to cloning. To achieve this, we have compared and combined the separation efficiencies of conventional and MetaPhor agarose gel electrophoresis (MAGE) with a glycerol-enhanced mini-polyacrylamide gel electrophoresis (PAGE) system (Gem-PAGE). As anticipated, MetaPhor agarose provided significantly improved resolution over conventional agarose electrophoresis, but the latter remains useful to rapidly confirm the presence of RDA-enriched difference products and direct the concentration of MetaPhor agarose subsequently used for further fragment separation. Additional improvements in resolution were possible by using the Gem-PAGE system. The effect of glycerol on band definition of PAGE was most noticeable as the acrylamide to glycerol ratio (A:G) approached 1:2. Gels in which the A:G ratio was significantly above or below this resulted in both poor morphology and impaired resolution of the bands. By exploiting sequentially agarose-based and Gem-PAGE electrophoresis, the goal in RDA of "one band one product" is now realizable.

Acrylic Resins↗
PubMed2001

The genetics of essential hypertension.

Essential hypertension is an escalating problem for industrialized populations. It is currently seen as a 'complex' genetic trait caused by multiple susceptibility genes the effects of which are modulated by gene-environment and gene-gene interactions. Nevertheless, the success to date in identifying these susceptibility genes has been very limited. A number of candidates has been proposed, but demonstrating consistently the linkage or association with hypertension has been problematic. The data for angiotensinogen is undoubtedly the most extensive and meta-analysis has confirmed a significant association overall, although the risk contributed by this gene appears to be modest (odds ratio of 1.2). Identifying further genes - probably conferring even smaller attributable risks - represents a major challenge for future developments in this area. This contrasts markedly with the success that has been achieved in the past 5 years in solving the molecular genetics of a number of rare familial hypertension syndromes. The true incidences of some of these disorders may be higher than first appreciated, but it is still unclear if the genes for these syndromes also play a part in essential hypertension. A more complete understanding of the genetic basis of essential hypertension should be possible in the coming years using new strategies that take advantage of the information provided by the human genome project. This knowledge will irrevocably change the way we approach this disease in terms of its diagnosis, risk assessment for end-points such as stroke and heart disease, and the customised treatment that might be offered in the future.

Animals↗
PubMed2000

The gain-of-function G389R variant of the beta1-adrenoceptor does not influence blood pressure or heart rate response to beta-blockade in hypertensive subjects.

Mutation scanning of the beta1-adrenoceptor gene has identified a polymorphism, G389R, that markedly affects G-protein coupling of the receptor and resulting cAMP production. We have investigated the effect of this functionally active polymorphism on clinical response to beta-adrenoceptor blockade. Two cohorts of untreated hypertensive patients randomly assigned to a beta1-selective beta-blocker at the start of antihypertensive therapy were studied retrospectively to see if the G389R polymorphism influenced the response in terms of blood pressure and heart rate. The blood pressure and heart rate responses to treatment were assessed 4 weeks later and compared with the G389R genotype, ascertained by PCR/restriction fragment length polymorphism. The falls in blood pressure and heart rate for the first group (n = 92) by genotype were: GG, 20.1 +/- 3.5/13.9 +/- 2.7 mmHg (systolic/diastolic blood pressure), 18.4 +/- 2.2 beats/min; GR, 20.0 +/- 2.2/15.0 +/- 1.3 mmHg, 16.5 +/- 1.5 beats/min; RR, 20.8 +/- 2.3/13.4 +/- 1.1 mmHg, 16.0 +/- 1.4 beats/min. For the second group (n = 55) the corresponding falls were: GG, 17.0 +/- 4.3/11.2 +/- 3.4 mmHg, 12.0 +/- 3.5 beats/min; GR, 16.6 +/- 1.8/14.4 +/- 1.1 mmHg, 13.1 +/- 2.1 beats/min; RR, 18.0 +/- 1.6/13.0 +/- 1.4 mmHg, 14.4 +/- 1.4 beats/min. The G389R genotype also failed to have a significant effect on pretreatment blood pressure or heart rate in either group. These data suggest that, despite clear functional differences between the G389R receptor variants expressed in vitro, the polymorphism does not affect the haemodynamic response of hypertensive subjects to chronic beta1-adrenoceptor blockade.

Adrenergic beta-Antagonists↗
PubMed1999

A common variant of the endothelial nitric oxide synthase (Glu298-->Asp) is a major risk factor for coronary artery disease in the UK.

BACKGROUND: Endothelium-derived nitric oxide (NO) is synthesised from L-arginine by endothelial nitric oxide synthase (eNOS) encoded by the NOS 3 gene on chromosome 7. Because reduced NO synthesis has been implicated in the development of coronary atherosclerosis, which has a heritable component, we hypothesised that polymorphisms of NOS 3 might be associated with increased susceptibility to this disorder. METHODS AND RESULTS: Single-strand conformation polymorphism analysis of NOS 3 identified a G-->T polymorphism in exon 7 of the gene which encodes a Glu-->Asp amino acid substitution at residue 298 of eNOS. We investigated the relationship between this Glu(298)-->Asp variant and atherosclerotic coronary artery disease (CAD) using 2 independent case-controlled studies. In the first study (CHAOS), cases consisted of 298 unrelated patients with positive coronary angiograms and controls were 138 unrelated healthy individuals ascertained through a population health screen. In the second study (CHAOS II), the cases were 249 patients with recent myocardial infarction (MI), and a further 183 unrelated controls. There was an excess of homozygotes for the Asp298 variant among patients with angiographic CAD, and among patients with recent MI when compared with their respective controls (35.9% versus 10.2%, P<0.0001 in CHAOS, and 18.1% versus 8.7%, P<0.02 in CHAOS II). In comparison to Glu(298) homozygotes, homozygosity for Asp(298) was associated with an odds ratio of 4.2 (95% CI, 2.3 to 7.9) for angiographic CAD and 2.5 (95% CI, 1.3 to 4.2) for MI. CONCLUSIONS: Homozygosity for a common NOS 3 polymorphism (894 G-->T) which encodes a Glu298-->Asp amino acid substitution in eNOS is a risk factor for angiographic CAD and recent MI in this population.

Aged↗
PubMed1999

Factor V Leiden and thermolabile methylenetetrahydrofolate reductase gene variants in an East Anglian preeclampsia cohort.

Preeclampsia is a heritable condition that develops as a result of widespread vascular endothelial dysfunction. The thrombotic tendency in this condition has suggested a number of candidate genes, and there have been recent reports of positive association with the Leiden variant of factor V and the thermolabile variant of methylenetetrahydrofolate reductase. We attempted to reproduce these results in a large cohort of well-characterized women with preeclampsia, recruited prospectively within the East Anglian region of the United Kingdom. Women in the preeclampsia cohort (n=283) were genotyped for both the Leiden variant (G1691A) of factor V and the thermolabile variant (C677T) of methylenetetrahydrofolate reductase. Genotype and allele frequencies were compared with those of 2 control groups, one consisting of women recruited prospectively (n=100) from the same maternity hospital as the subjects and another consisting of normotensive women (n=100) from East Anglia. No significant differences were detected. Specifically, the carrier rate for the Leiden variant was 5.3% in the preeclampsia group and 5. 5% in the combined control group. T677 homozygotes for methylenetetrahydrofolate reductase were 11% and 11.5% in the 2 groups, respectively. We conclude that there is no evidence of association of preeclampsia with either of these 2 polymorphisms in our study population.

Base Sequence↗
PubMed1998

Linkage of Gordon's syndrome to the long arm of chromosome 17 in a region recently linked to familial essential hypertension.

Gordon's syndrome (GS) is a salt-sensitive, hyperkalaemic, familial hypertension syndrome which may masquerade in milder forms as essential hypertension. The response of hyperakalaemia to dietary salt restriction and to mineralocorticoids is heterogeneous, suggesting genetic heterogeneity. In a recently published study using small pedigrees, possible linkage of GS to chromosomes 1 and 17 was described. Studying the largest pedigree so far reported with GS, and using fluorescent-labelled microsatellite markers, we sought evidence of linkage to chromosomes 1 and 17. In this family there was no segregation of GS with any of the markers for chromosome 1. On chromosome 17, however, evidence of linkage was found with a maximum multipoint LOD score of 2.4 (the maximum LOD possible from the pedigree) over markers D17S250 and D17S934. This represents strong evidence in this pedigree for a responsible gene mutation on the long arm of chromosome 17. Recent reports of linkage for essential hypertension and, especially, familial essential hypertension within the same area of chromosome 17 containing the D17S934 marker raise the possibility that the same gene may be responsible for familial essential hypertension and for Gordon's syndrome.

Chromosomes, Human, Pair 1↗
PubMed1996

Imaging allergen-invoked airway inflammation in atopic asthma with [18F]-fluorodeoxyglucose and positron emission tomography.

BACKGROUND: Airway inflammation is a feature of asthma and can be quantified invasively with bronchial lavage and endobronchial histology. Inflammatory foci can be imaged non-invasively with positron emission tomography (PET) and [18F]-fluorodeoxyglucose (18FDG) to quantify glucose uptake in activated granulocytes. We used this technique to study airway inflammation in asthma. METHODS: Nine men with mild atopic asthma were studied. In five, we studied the effect of bronchoscopic segmental allergen challenge on 18FDG uptake. Allergen was instilled into the posterior segment of the right upper lobe; a similar volume (20 mL) of isotonic saline was instilled into the posterior segment of the left upper lobe. At 1-32 h after instillation, PET with 18FDG was done. In the other four patients, we administered aerosolised allergen. FINDINGS: 18FDG uptake was increased four-fold in the right compared with the left upper lobe (geometric mean of ratios 4.30, 95% Cl 2.39-7.72, p=0.002). Aerosolised administration of allergen did not significantly increase 18FDG uptake. INTERPRETATION: These data show that local allergen-invoked airway inflammation can be visualised with 18FDG and PET in asthma. The cellular localisation of the 18FDG signal remains to be determined.

Adult↗
PubMed1994

Inhibition of nitric oxide synthesis in vascular smooth muscle by retinoids.

1. These studies examine the effect of retinoids on interleukin 1 beta (IL-1 beta)-induced nitric oxide synthase (NOS) activity in cultured rat aortic vascular smooth muscle (VSM) cells and isolated rat aortic rings. 2. All-trans-retinoic acid (all-trans-RA, 0.1-10 microM) and its active analogues produced concentration-dependent inhibition of IL-1 beta (0.1-10 ng ml-1)-induced nitrite production in cultured VSM cells. In contrast, the inactive retinoid, Ro 14-6113 (0.1-10 microM), had no effect on IL-1 beta-induced nitrite production. 3. Since some of the actions of retinoids are mediated by induction of transforming growth factor beta (TGF-beta), its effect on inducible NOS activity in VSM cells was examined. TGF-beta produced concentration-dependent (0.1-10 ng ml-1) inhibition of IL-1 beta-induced nitrite production and the maximum effect (approximately 90% inhibition) was significantly greater than that seen with all-trans-RA (approximately 70% with 10 microM). However, an anti-TGF-beta antibody (50 micrograms ml-1) which blocked the effect of exogenous TGF-beta (5 ng ml-1) did not significantly reverse the inhibitory action of all-trans-RA (10 microM). 4. In addition to inhibiting IL-1 beta-induced nitrite production, all-trans-RA (10 microM) reduced substantially inducible NOS mRNA and protein levels in IL-1 beta-induced VSM cells (P < 0.01). 5. Incubation of isolated rat aortic rings with IL-1 beta (10 ng ml-1) caused a progressive resistance of the rings to the vasoconstrictor action of phenylephrine (10 nM to 10 microM). This effect was abolished by the addition of the nitric oxide synthase inhibitor L-NG-monomethyl-L-arginine (L-NMMA, 1 mM). All trans-RA (10 micro M) also markedly and significantly reversed this IL-1p-induced vascular hyporeactivity(P<0.01).6. These data show that all-trans-RA and other active retinoids are able to block cytokine-stimulated expression of inducible NOS in cultured VSM cells and isolated aortic rings.

Amino Acid Oxidoreductases↗
PubMed1993

Potent leukotriene D4 receptor antagonist ICI 204,219 given by the inhaled route inhibits the early but not the late phase of allergen-induced bronchoconstriction.

ICI 204,219 is a potent and specific leukotriene D4 receptor antagonist that blocks both the early and late responses to allergen challenge in humans when given orally at a dose of 40 mg. Here we report our findings with an inhaled formulation of ICI 204,219 against allergen-induced bronchoconstriction. A group of 10 atopic subjects (mean age 25.6 +/- 4.2; 6 females; FEV1 > 90% of predicted; on inhaled beta 2-agonists only) were studied on 2 separate days 2 to 3 wk apart. In a randomized placebo-controlled trial they inhaled eight puffs of a standard metered dose inhaler containing either ICI 204,219 (200 micrograms/puff, total dose 1,600 micrograms) or propellant alone. They underwent bronchial allergen challenge 30 min later using a single concentration of allergen previously shown to lower the FEV1 by > or = 15%. FEV1 was monitored hourly for 10 h, and urine was collected for LTE4 determination. Inhalation of ICI 204,219 was well tolerated, with no adverse clinical or biochemical effects. There was no significant effect of ICI 204,219 inhalation on basal airway caliber (change in FEV1 30 min after inhalation was -149 +/- 67 ml after placebo versus 3 +/- 38 ml after ICI 204,219; p = 0.08). The early response to allergen was significantly inhibited by ICI 204,219 (the maximum fall in FEV1 was -21.2 +/- 6.1% after ICI 204,219 compared with -38.8 +/- 6.5% on placebo; p = 0.007).(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1993

Differential effects of fluticasone propionate on allergen-evoked bronchoconstriction and increased urinary leukotriene E4 excretion.

Allergen challenge is associated with an increased excretion of urinary leukotriene E4. The source of this increase is unknown, although the lack of effect of inhaled beta-agonists and sodium cromoglycate suggests that airway mast cells may not be involved. We investigated this further using a new and topically potent inhaled glucocorticoid, fluticasone propionate (FP). A group of 10 mild atopic asthmatic subjects (6 males; FEV1 > 60% of predicted; PC20 histamine < or = 8 mg/ml; and on inhaled beta 2-agonists only) were studied before and after a 2-wk period of FP (1,000 micrograms/day) or placebo administered by metered-dose inhalers as two puffs twice per day through a large-volume spacer. Treatments were assigned in a double-blind crossover fashion separated by a 3-wk washout period. The PC20 histamine was measured at the start and end of each treatment when subjects also received a bronchial allergen challenge. Urine was collected for 4 h after allergen challenge for determination of LTE4 using HPLC-RIA, and 2 h later the PC20 histamine measurement was repeated. The 2-wk treatment with FP significantly inhibited both early and late responses to allergen: the maximum % fall in FEV1 during the early (0 to 2 h) and late response (2 to 6 h) was 32.6 +/- 3.4 and 19.6 +/- 5.2, respectively, following placebo versus 19.5 +/- 4.5 and 3.6 +/- 2.6 following FP (both p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗