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R J Turner

Publications and source records attributed to R J Turner.

At least 73 records · Page 4Linked to original sources
PubMed1998

Alpha-periodicity analysis of small multidrug resistance (SMR) efflux transporters.

Proteins in the small multidrug resistance (SMR) family of transport proteins are about 110 amino acids in length and are predicted to have four transmembrane helices. This family is divided into a two groups, one of which we have referred to as small multidrug pumps (Smp) and confer resistance to a wide variety of quaternary ammonium compounds through a proton-drug efflux antiport mechanism. Members of the second group within this family have, as yet, not had their substrate profile characterized and are referred to as Sug proteins. Alpha-periodicity analysis was conducted on a set of six homologous proteins of the SMR family consisting of three established Smp and three Sug proteins. Several amino acid properties were used in the analysis including hydropathy, variability, and a substitution matrix for lipid exposed amino acids. The scanning window was varied between 8 and 14 residues and the alpha-periodicity was calculated from the peaks in the Fourier transform power spectra in the region between 3.0 and 4.3 residues/turn. This analysis adds to the hydropathy analysis to give a more confident prediction of which residues are within the lipid bilayer for each of the four transmembrane helices. Information was also obtained that allowed for the identification of zones within each transmembrane helix that face the interior of the helical bundle on one side and are lipid exposed on the other face.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of the cation-chloride cotransporter family.

Na-K-2Cl cotransporters, Na-Cl cotransporters and K-Cl cotransporters have recently been shown to constitute a new gene family of membrane transport proteins. At least five distinct members of this family of cation-chloride cotransporters have been found in vertebrates and several as yet functionally uncharacterized sequences have been identified in insects, worms and yeast. The relationships among the various known members of this gene family is briefly reviewed along with our current knowledge about the topological structure of these proteins in the plasma membrane.

Animals↗
PubMed1998

New insights into the upregulation and function of the salivary Na+-K+-2Cl- cotransporter.

In many exocrine epithelia, the Na+-K+-2Cl- cotransporter is the main provider of cellular chloride entry during transepithelial salt and water secretion. Because of its accessibility and hormonal responsiveness, the salivary gland has recently emerged as a convenient preparation in which to study the regulation and characteristics of this transport protein. In this review, we summarize recent findings from our laboratory which demonstrate that muscarinic, alpha1-adrenergic and peptidergic stimulation of rat parotid acinar cells induce a dramatic (up to twenty-fold) upregulation of Na+-K+-2Cl- cotransporter activity. Our results indicate that this effect is dependent on the rise in intracellular calcium concentration ([Ca2+]i) that accompanies stimulation, and is not a consequence of the KCl loss and the concomitant cell shrinkage associated with fluid secretion. In addition, we show that the effect of muscarinic stimulation on the cotransporter can be blocked by inhibitors of phospholipase A2, by a general inhibitor of arachidonic acid metabolism, and by specific inhibitors of the cytochrome P450 pathway. These data argue strongly for the involvement of a product of the cytochrome P450 pathway of arachidonic acid metabolism in upregulation of the salivary Na+-K+-2Cl- cotransporter.

Animals↗
PubMed1997

Circumferential deformation and shear stress induce differential responses in saphenous vein endothelium exposed to arterial flow.

Adaptation of saphenous vein to the hemodynamic stresses of the arterial circulation is critical to the maturation of vein bypass grafts. We have investigated early adaptive responses of venous endothelium by placing excised human saphenous vein in a bypass circuit with either venous or arterial flow conditions, using external stenting to resolve the effects of longitudinal (shear) from circumferential stress. Endothelial protein concentrations were assessed by immunostaining area (ratio of protein/CD31) and Western blotting of endothelial cell lysates (staining ratio protein/vWf). In both unstented and stented veins nitric oxide synthase increased after 90 min of arterial flow: twofold increase of immunostaining area (P = 0.001), four- to fivefold increase by Western blotting (P = 0.02), and increased A23187mediated maximum endothelium-dependent relaxation of vein rings (P = 0.01). In unstented veins, ICAM-1 concentration was increased after 45 min of arterial flow: twofold increase by immunostaining (P = 0.001) and Western blotting (P = 0.038), with maximum fibrinogen-mediated endothelium-dependent relaxation increasing from 55.9+/-4.9 to 97+/-2.1% (P = 0.01). In contrast, in unstented veins there was a threefold decrease of VCAM-1 and no change in P-selectin after arterial flow for 45 and 90 min, respectively. However, no changes in ICAM-1 and VCAM-1 were observed in stented veins. The flow-induced alterations in nitric oxide synthase, ICAM-1, and VCAM-1 were abolished when 3 mM tetraethylammonium ion (K+ channel blocker) was included in the vein perfusate. The very rapid changes in ICAM-1 and VCAM-1 expression are a response to circumferential stress, whereas the slower upregulation of nitric oxide synthase is a response to longitudinal (shear) stress. Similar changes could influence the adhesiveness of endothelium in newly implanted saphenous vein bypass grafts.

Arteries↗
PubMed1997

Upregulation of Na(+)-K(+)-2Cl- cotransporter activity in rat parotid acinar cells by muscarinic stimulation.

1. The effects of fluid secretory stimuli on the Na(+)-K(+)-2Cl- cotransporter in rat parotid acini were investigated. Cotransporter activity was measured using NH4+ as a K+ surrogate and following cotransporter-mediated NH4+ fluxes by monitoring intracellular pH. 2. A dramatic upregulation (15- to 20-fold) of acinar Na(+)-K(+)-2Cl- cotransporter activity was induced by muscarinic, alpha 1-adrenergic and peptidergic stimuli. A half-maximal effect of the muscarinic agonist carbachol was observed at approximately 0.5 microM. 3. Our results indicate that the rise in intracellular calcium concentration ([Ca2+]i) which accompanies these stimuli is both a necessary and a sufficient condition for this effect; but it is not a consequence of the KCl loss and concomitant isotonic shrinkage caused by increased [Ca2+]i as it persists when these effects are prevented. 4. The effect of muscarinic stimulation on the cotransporter can, however, be blocked by inhibitors of phospholipase A2 (4-bromophenacylbromide and manoalide), by a general inhibitor of arachidonic acid metabolism (5,8,11,14-eicosatetraynoic acid) and by specific inhibitors of the cytochrome P450 pathway (methoxsalen and ketoconazole). 5. These latter results argue strongly for the involvement of a product of the cytochrome P450 pathway of arachidonic acid metabolism in upregulation of the salivary Na(+)-K(+)-2Cl- cotransporter. 6. Owing to the complexity of the arachidonic acid cascade a wide variety of agents could potentially interfere with this upregulation of the cotransporter, and thereby result in decreased salivary fluid production. We suggest that such an effect could underlie the dry mouth (xerostomia) that occurs as an unexplained side-effect of many commonly prescribed medications.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1997

Effect of Fasciola hepatica excretory-secretory products on the metabolic burst of sheep and human neutrophils.

F. hepatica excretory-secretory (ES) products were found to inhibit superoxide output in phorbol myristate acetate (PMA)-stimulated sheep and human neutrophils as measured by spectrophotometry. Luminol-enhanced chemiluminescence by PMA-stimulated neutrophils from both species was again inhibited. However, nitric oxide output by PMA-stimulated human neutrophils was significantly increased in the presence of ES products, whilst in sheep it was inhibited. No major effects were noted using resting neutrophils. The results are discussed in relation to the evolution of parasite defence mechanisms.

Animals↗
PubMed1997

Evidence that up-regulation of the parotid Na(+)-K(+)-2Cl- cotransporter by hypertonic shrinkage is not duplicated by isotonic shrinkage.

The application of Ca2+ mobilizing secretagogues to rat parotid acini results in a significant decrease in cell volume (15-30%) due to isotonic salt loss. It is often assumed that the effects of such an isotonic volume decrease can be mimicked by anisotonic cell shrinkage. We demonstrate that the Na(+)-K(+)-2Cl- cotransporter in these cells is up-regulated by Ca2+ mobilizing secretagogues as well as by cell shrinkage in hypertonic media. However, we find that although the protein kinase inhibitors staurosporine (0.3 microM) and K252a (0.6 microM) significantly blunt the latter up-regulation, they are without effect on the former. These observations suggest that hypertonic and isotonic shrinkage do not result in the activation of the same intracellular signalling pathways, and indicate that anisotonic volume perturbations may not provide good experimental models of physiologic isotonic volume changes.

Ammonium Chloride↗
PubMed1997

Expression and epitope tagging of the membrane anchor subunit (DmsC) of Escherichia coli dimethyl sulfoxide reductase.

Escherichia coli dimethyl sulfoxide reductase is a heterotrimer comprising a catalytic subunit (DmsA), an electron transfer subunit (DmsB) and an integral membrane anchor subunit (DmsC). DmsC is not antigenic and the production of antibodies to this subunit has not been successful. We have tagged DmsC at the C-terminus with a dystrophin-specific amino acid sequence (dysp) to which antibodies are readily available. We were able to use this tagging technique to monitor expression and localization of DmsC in E. coli and non-muscle eukaryotic cells. Growth properties of wild-type E. coli, strain HB101, overexpressing DmsC:dysp suggest that the expression of DmsC is lethal to E. coli. The lethality could be overcome by utilizing an E. coli F0F1 ATPase mutant as the host. Growth conditions of culture density, duration of induction, temperature of incubation after induction and media conditions were investigated to optimize DmsC:dysp accumulation levels. In order to alleviate the problem arising from the toxicity of DmsC, expression in eukaryotic tissue culture was also explored. A plasmid expressing DmsC:dysp was transfected into COS-1 or McA-RH777 cells. The presence of expressed DmsC:dysp was confirmed using specific anti-dysp antibodies and immunofluorescence microscopy analysis revealed that the DmsC:dysp was localized to the endoplasmic reticulum. Expression of DmsC:dysp did not appear to be toxic to the eukaryotic cells. These data suggest methodologies to overcome lethality problems associated with the overexpression of integral membrane proteins like DmsC.

Amino Acid Sequence↗
PubMed1997

Expression of Escherichia coli TehA gives resistance to antiseptics and disinfectants similar to that conferred by multidrug resistance efflux pumps.

The genes tehAB located at 32.3 min on the Escherichia coli chromosome were initially identified by their ability to mediate resistance to potassium tellurite (128 micrograms of K2TeO3 per ml) when overexpressed with a high-copy-number plasmid. The genes encode an integral membrane protein (TehA) of 36 kDa with 10 putative transmembrane segments and a second protein (TehB) of 23 kDa. Overexpression of TehAB results in hypersensitivity to dequalinium CI and methyl viologen (paraquat). Expression of TehA alone gives similar hypersensitivity. Overexpression of TehA gave resistance to tetraphenylarsonium CI, ethidium bromide, crystal violet and proflavin. The efflux of ethidium, measured by fluorescence quenching, revealed that TehA transported ethidium at twice the control rate and 10% of the rate of the highly resistant efflux transporter Emr Eco. Addition of tellurite had no effect on ethidium transport. In addition to the ethidium transport assay, a proflavin fluorescence assay which was approximately 200-fold more sensitive was also used. TehA was also found to have proflavin efflux activity. The addition of TeO32- to the proflavin transport assay on TehA caused a 20% increase in transport rate. Both ethidium and proflavin transport were found to be energy dependent.

Amino Acid Sequence↗
PubMed1997

An association between arterial pulse pressure and variation in the fibrillin-1 gene.

OBJECTIVE: To investigate whether variation in the fibrillin-1 gene was associated with blood pressure in healthy middle aged men, as had been observed in patients with abdominal aortic aneurysm. DESIGN, SETTING, AND PATIENTS: Middle aged men (n = 245), aged 50 to 61 years, were recruited from one of the nine general practices participating in the second Northwick Park heart study. Blood samples were obtained for the preparation of genomic DNA and analysis of plasma variables. MAIN OUTCOME MEASURES: Systolic, diastolic, and pulse pressures; fibrillin-1 genotype characterised with a four allele variable tandem nucleotide repeat polymorphism in intron 28. RESULTS: In healthy middle aged men only three common genotypes were observed: 2-2 (frequency 54.1%), 2-3 (16%) and 2-4 (15%). The mean arterial systolic (and pulse) pressure varied according to fibrillin-1 genotype: 2-4 genotype, 126-3 (47.6) mm Hg; 2-2 genotype, 131.0 (51.3) mm Hg; and 2-3 genotype, 135.5 (54.2) mm Hg. The median pulse pressure was 50 mm Hg. Distribution of men around the median pulse pressure, according to genotype, showed a significant trend for patients of 2-4 genotype to have the lowest pulse pressures, those of 2-2 genotype to have intermediate pressures, and those of 2-3 genotype to have the highest pulse pressures (p = 0.003 for healthy men). CONCLUSIONS: There appears to be a significant association between fibrillin-1 genotype and arterial pulse pressure in men aged 50 to 61 years.

Alleles↗
PubMed1997

Influence of hypercholesterolemia and adventitial inflammation on the development of aortic aneurysm in rabbits.

Abdominal aortic aneurysms are characterized by intimal atherosclerosis, disruption and attenuation of the elastic media, and a variable adventitial inflammatory infiltrate. We have developed an animal model of this disorder to evaluate the contribution of hypercholesterolemia, medial injury, and adventitial inflammation to aneurysmal dilatation. To accomplish this, we used periaortic application of calcium chloride, which induced both medial injury with calcification and endothelial injury. Ultrasonography was used to demonstrate the dilatation and thickening of the aortic wall. Over the first 3 weeks after periaortic application of 0.25 mol/L CaCl2, the external aortic diameter increased from 3.5 +/- 0.5 to 4.2 +/- 0.8 mm, but the ID remained unchanged. This apparent wall thickening was accompanied by vascular remodeling, and biochemical changes included approximately 50% reduction in tissue hydroxyproline concentration and increased activity of gelatinases (matrix metalloproteinase [MMP]-2 and MMP-9). Independently, cholesterol feeding to induce hypercholesterolemia or the concomitant periaortic application of thioglycollate had little effect on the histological, biochemical, or diameter changes. Together, hypercholesterolemia and thioglycollate were associated with rapid aortic dilatation in CaCl2, treated animals but not controls: after 3 weeks, the ID and OD had doubled, the OD increasing from 3.5 +/- 0.4 to 7.1 +/- 0.4 mm, P = .005. The remarkable feature that accompanied this dilatation was the infiltration of cells, mostly foamy macrophages, into the adventitia, with a further reduction in hydroxyproline concentration. Adventitial inflammation may provide the critical stimulus to dilatation of an aorta with preexisting intimal and medial injury.

Animals↗
PubMed1997

An immunological assessment of group-housed rabbits.

Laboratory rabbits kept in barren 'traditional' cages tend to develop stereotypic behaviours and bone deformities. We have used an alternative regime, housing adult does as groups of four or five in floor pens (2.5-3 m2) supplied with hiding places and bedding. High- and low-ranking members of each group were identified, and their immunological status compared in terms of blood leucocyte function (chemiluminescence and mitogen tests), complement activity, and antibody production to soluble and cellular antigens. We found no evidence of immunosuppression, either in groups of a 'docile' breed (New Zealand White) or Dutch crosses. These results, together with the animals' general health and ease of handling, lead us to conclude that group-housed does are suitable for raising antisera and other purposes, provided that they are adequately monitored.

Animals↗
PubMed1997

A genomewide linkage study of preeclampsia/eclampsia reveals evidence for a candidate region on 4q.

Preeclampsia (PE) and eclampsia (E) are potentially life-threatening conditions that can occur during human pregnancy. Generally considered to be different degrees of severity of the same disease process, the PE/E syndrome is thought to be predominantly genetic in origin, although its exact etiology and genetics are not fully understood. Here we report results of a genomewide linkage search for the gene(s) responsible for susceptibility to PE/E, using 15 informative pedigrees and 90 polymorphic DNA markers from all autosomes. Because of uncertainties concerning inheritance and diagnosis, four different models that assume maternal gene expression have been used to carry out LOD-score analysis. The region between D4S450 and D4S610 (2.8 cM) on the long arm of chromosome 4 was identified as a strong candidate region for a PE/E-susceptibility locus. The maximum multipoint LOD score within this interval was 2.9. Analysis of markers in the region around D4S450 and D4S610 by the affected-pedigree-member method also supported the possibility of a susceptibility locus in this region. However, to verify or exclude definitively linkage to this region, other groups of PE/E pedigrees will be required.

Australia↗
PubMed1996

Function of RNA secondary structures in transcriptional attenuation of the Bacillus subtilis pyr operon.

The Bacillus subtilis pyr operon is regulated by exogenous pyrimidines by a transcriptional attenuation mechanism. Transcription in vitro from pyr DNA templates specifying attenuation regions yielded terminated and read-through transcripts of the expected lengths. Addition of the PyrR regulatory protein plus UMP led to greatly increased termination. Synthetic antisense deoxyoligonucleotides were used to probe possible secondary structures in the pyr mRNA that were proposed to play roles in controlling attenuation. Oligonucleotides predicted to disrupt terminator structures suppressed termination, whereas oligonucleotides predicted to disrupt the stem of antiterminator stem-loops strongly promoted termination at the usual termination site. Oligonucleotides that disrupt a previously unrecognized stem-loop structure, called the anti-antiterminator, the formation of which interferes with formation of the downstream antiterminator, suppressed termination. We propose that transcriptional attenuation of the pyr operon is governed by switching between alternative antiterminator versus anti-antiterminator plus terminator structures, and that PyrR acts by UMP-dependent binding to and stabilization of the anti-antiterminator.

Bacillus subtilis↗
PubMed1996

Inositol 1,4,5-trisphosphate-dependent oscillations of luminal [Ca2+] in permeabilized HSY cells.

Oscillations in intracellular Ca2+ concentration ([Ca2+]i) are thought to play an important role in phosphoinositide-linked Ca2+ signaling events. We demonstrate corresponding inositol 1,4, 5-trisphosphate (IP3)-dependent oscillations of Ca2+ concentration within the lumen of the IP3-sensitive stores ([Ca2+]L) of saponin-permeabilized HSY cells by monitoring [Ca2+]L with the fluorescent Ca2+ indicator Mag-fura-2. The associated openings and closings of the IP3-sensitive Ca2+ release channel were detected via quenching of Mag-fura-2 fluorescence due to the entry of Mn2+, a Ca2+ surrogate, into the stores. Evidence for complimentary Ca2+ oscillations at the external surface of the stores was provided by the membrane-bound Ca2+ probe Calcium Green C18. The permeabilization of saponin-treated HSY cells to macromolecules was confirmed by demonstrating that permeabilized cells readily took up and lost (t1/2 approximately 46 s) a fluorescently tagged 70-kDa dextran. Our results impose a number of constraints on possible mechanisms for [Ca2+]i oscillations. In addition, they support recent proposals that [Ca2+]i oscillations arise directly from the (biphasic) effects of Ca2+ itself on IP3-sensitive Ca2+ channel activity.

Calcium↗
PubMed1996

Interaction between fibrillin genotype and blood pressure and the development of aneurysmal disease.

Debate as to whether abdominal aortic aneurysms (AAA) are caused by atherosclerosis or whether they have a strong genetic etiology continues. We have investigated the hypothesis that risk factors are likely to be strongest in patients with generalized aneurysmal disease. We screened 232 consecutive AAA patients for popliteal aneurysm and investigated cardiovascular and genetic risk factors in these patients. Ultrasonography demonstrated the presence of a popliteal aneurysm in 24 of 232 (10%) patients. Multivariate analysis identified four independent factors associated with popliteal aneurysm: age (p = 0.013), height (p = 0.017), triglyceride concentration (p = 0.009), and systolic blood pressure (p = 0.037). In the AAA patients a significant association of fibrillin-1 genotype was present, determined by a tandem repeat polymorphism, with both systolic and pulse pressure. The genotypes associated with the highest pressures were significantly more common among the patients with popliteal aneurysm, p = 0.03. Following these findings we investigated whether there was an association between fibrillin-1 genotype and blood pressure in a healthy population, 245 men aged 50-61 years. Again we found a significant association between fibrillin genotype and pulse pressure, p = 0.003. We suggest that a strong interaction occurs between fibrillin genotype and blood pressure which contributes to the development of aneurysmal disease.

Aging↗
PubMed1996

Gastric chief cells possess NK1 receptors which mediate pepsinogen secretion and are regulated by agents that increase cAMP and phospholipase C.

In order to determine whether tachykinins alter the function of chief cells and to characterize the receptors mediating the effect, we investigated the abilities of various substance P (SP)-related peptides to inhibit the binding of 125I-Bolton-Hunter labeled substance P (125I-BH-SP) and their abilities to alter cell function in dispersed chief cells from guinea pig stomach. Binding of 125I-BH-SP was saturable, reversible, time- and temperature-dependent and was inhibited by several SP-related peptides with relative potencies of SP = physalaemin (IC50:0.19 nM) > SP methyl ester (SP-ME) (IC50:3.3 nM) > eledoisin (IC50:6.1 nM) > neurokinin A (NKA) (IC50: 65 nM) > neurokinin B (NKB) (IC50:80 nM). Analyses of these binding data demonstrated that chief cells possess a high and low affinity class of binding sites. Neither 125I-NKA nor [phenylalanyl-3,4,5-3H]senktide demonstrated saturable binding to chief cells. Acid stripping experiments demonstrated rapid ligand internalization with 55% of the bound radioligand internalized by 10 min. Phospholipase C activating agents (carbachol, CCK-8), adenylate cyclase activating agents (secretin, VIP), TPA and the calcium ionophore, A23187, all inhibited the binding of 125I-BH-SP and it was due to inhibition of ligand internalization with no change in surface bound parameters. SP (0.1 microM) stimulated pepsinogen secretion but was 4-times less efficacious than CCK-8 (10 nM) or carbachol (1 mM). 10 nM SP stimulated a rapid increase in cytoplasmic free calcium concentration ([Ca2+]i) followed by a sustained elevation lasting 2 min. Single cell spectroscopy demonstrated SP (10 pM to 1 microM) did not cause calcium oscillations. The NK1 receptor antagonist, CP96,345 specifically inhibited the SP-stimulated changes in [Ca2+]i and pepsinogen secretion. The relative potencies of SP-related peptides to stimulate pepsinogen secretion and [Ca2+]i demonstrated a close agreement with their abilities to inhibit the binding of 125I-BH-SP, and comparison of the dose-response curves suggests occupation of the low affinity sites mediate changes in biologic activity. In conclusion, the present study demonstrates that chief cells possess a NK1 subtype of tachykinin receptor, occupation of the low affinity sites of this receptor cause calcium mobilization and pepsinogen secretion, and that binding to this receptor is regulated by agents that activate phospholipase C, adenylate cyclase, protein kinase C and calcium mobilization.

Animals↗