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Biomedical subjects

R J Turner

Publications and source records attributed to R J Turner.

At least 91 records · Page 5Linked to original sources
PubMed1996

Evidence that quantal Ca2+ release in HSG cells is not due to 'all-or-none' release from discrete Ca2+ stores with differing sensitivities to IP3.

We demonstrate that the application of the muscarinic agonist carbachol to the human salivary epithelial cell line HSG elicits the now well-known phenomenon of 'quantal' Ca2+ release; namely, that the application of a submaximal concentration of agonist results in the release of only a portion of the agonist-sensitive intracellular Ca2+ pool. One explanation that has been proposed to account for this effect is that there are multiple intracellular Ca2+ stores, each with a different agonist sensitivity, which release Ca2+ in an 'all-or-none' fashion. We test this hypothesis in intact HSG cells with an experimental protocol designed to preferentially load less-agonist-sensitive stores with 40Ca2+ and more-agonist-sensitive stores with 45Ca2+. However, contrary to the expectations of the above explanation, these cells do not preferentially release 45Ca2+ in response to low concentrations of agonist. Thus our data suggest that quantal Ca2+ release must arise from some other property of the stores or their Ca2+ release channels.

Calcium↗
PubMed1996

Polarization and chemokinesis of ovine and human neutrophils in response to Fasciola hepatica excretory-secretory products.

The effect of Fasciola hepatica excretory-secretory products (ES products) on the polarization and chemokinesis of sheep and human neutrophils was investigated. Flukes were cultured overnight in RPMI-1640 medium, and the resulting ES products concentrated by dialysis (cut-off 1.2 kDa) before use. At the concentrations used toxicity tests showed no effect on neutrophil viability, but ES products were capable of causing morphological changes and chemokinesis in both sheep and human neutrophils. The polarizing effect of the ES products was found to be greater in the case of sheep neutrophils with 81% of cells polarizing at 80 micrograms total ES protein/ml as opposed to 36% of human neutrophils. Chemokinesis in response to 80 micrograms total ES protein/ml was significant (P < 0.01) in both species with sheep producing the stronger response. Delipidation of the ES products with Lipidex caused no loss of polarizing activity, nor did the purified lipid fraction show any polarizing effect. ES products lost polarizing activity after heat treatment or trypsin digestion. Preliminary fractionation on an S-300 Sephacryl column suggested the presence of at least four fractions capable of causing polarization in sheep neutrophils.

Analysis of Variance↗
PubMed1996

Effects of selenium and vitamin E on the immune responses of domestic animals.

The gross effects of selenium and vitamin E deficiency on animal production and the potential benefits of supplementation are recognised, and the influence of these micronutrients on the resistance of animals to a variety of infections has also been reported. As a result, deficiencies could compromise the immune system and result in a decline in production and performance before gross effects become apparent. Data obtained by laboratories studying different animal species indicate that the responses of domestic animals, particularly ruminants, differ from those of human beings and laboratory animals as well as differing among themselves. This paper provides a comprehensive review of the effects of selenium and vitamin E on the immune responses of domestic animals and discusses their effects with respect to the differences in the basal nutritional status of the animals concerned, the type of supplements used, the route and timing of their administration and the different agents which have been used to stimulate an immune response.

Animals↗
PubMed1996

Unravelling the familial tendency to aneurysmal disease: popliteal aneurysm, hypertension and fibrillin genotype.

PURPOSE: To screen patients with abdominal aortic aneurysm for popliteal aneurysm and investigate cardiovascular and genetic risk factors associated with aneurysmal disease at more than one site (generalised aneurysmal disease). SUBJECTS, DESIGN AND SETTING: All patients referred to the Regional Vascular Surgical Service at Charing Cross Hospital with unruptured abdominal aortic aneurysm between 1989 and 1993 were screened for popliteal aneurysms, using ultrasonography. MAIN OUTCOME MEASURES: Palpation of a popliteal aneurysm or ultrasonographic detection of popliteal dilatation, where the ratio maximum popliteal fossa diameter/suprageniculate popliteal diameter was > or = 1.5, in relation to cardiovascular and genetic risk factors. RESULTS: Clinical examination detected popliteal aneurysms in only 11/232 patients (5%), but ultrasonography demonstrated the presence of popliteal aneurysm in a further 13 patients, 24/232 in total (10%). Multivariate regression identified four independent factors associated with popliteal dilatation disease: age (p = 0.046), height (p = 0.006), systolic hypertension (p = 0.037) and triglyceride concentration (p = 0.009). Generalised aneurysmal disease and systolic blood pressure were associated with polymorphic variation in the fibrillin-1 gene, but not with variations in the apolipoprotein B and type III collagen genes. CONCLUSIONS: Few patients with abdominal aortic aneurysm (10%) also have popliteal aneurysms: the risk of popliteal dilatation increases with age, height, systolic blood pressure, triglyceride concentration and fibrillin genotype. The strong interaction between fibrillin genotype and blood pressure may contribute to the familial tendency to aortic aneurysm.

Age Factors↗
PubMed1996

Calcium release in HSY cells conforms to a steady-state mechanism involving regulation of the inositol 1,4,5-trisphosphate receptor Ca2+ channel by luminal [Ca2+].

In many cell types, low concentrations of inositol 1,4,5-trisphosphate (IP3) release only a portion of the intracellular IP3-sensitive Ca2+ store, a phenomenon known as "quantal" Ca2+ release. It has been suggested that this effect is a result of reduced activity of the IP3-dependent Ca2+ channel with decreasing calcium concentration within the IP3-sensitive store ([Ca2+]s). To test this hypothesis, the properties of IP3-dependent Ca2+ release in single saponin-permeabilized HSY cells were studied by monitoring [Ca2+]s using the Ca(2+)-sensitive fluorescent dye mag-fura-2. In permeabilized cells, blockade of the sarco/ER Ca(2+)-ATPase pump in stores partially depleted by IP3 induced further Ca2+ release via an IP3-dependent route, indicating that Ca2+ entry via the sarco/ER Ca(2+)-ATPase pump had been balanced by Ca2+ loss via the IP3-sensitive channel before pump inhibition. IP3-dependent Mn2+ entry, monitored via quenching of luminal mag-fura-2 fluorescence, was readily apparent in filled stores but undetectable in Ca(2+)-depleted stores, indicating markedly reduced IP3-sensitive channel activity in the latter. Also consistent with reduced responsiveness of Ca(2+)-depleted stores to IP3, the initial rate of refilling of these stores was unaffected by the presence of 0.3 microM IP3, a concentration that was clearly effective in eliciting Ca2+ release from filled stores. Analysis of the rate of Ca2+ release at various IP3 concentrations indicated a significant shift of the IP3 dose response toward higher [IP3] with decreasing [Ca2+]s. We conclude that IP3-dependent Ca2+ release in HSY cells is a steady-state process wherein Ca2+ efflux via the IP3 receptor Ca2+ channel is regulated by [Ca2+]s, apparently via changes in the sensitivity of the channel to IP3.

Calcium↗
PubMed1996

The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs.

The sequenced members of a novel family of small, hydrophobic, bacterial multidrug-resistance efflux proteins, which we have designated the small multidrug resistance (SMR) protein family, are identified and analysed. Two distinct clusters of proteins were identified within this family: (i) small multidrug efflux systems; and (ii) Sug proteins, potentially involved in the suppression of groEL mutations. Hydropathy and residue distribution analyses of this family suggest a structural model in which the polypeptide chain spans the membrane four times as mildly amphipathic alpha-helices. The roles of specific residues, a possible mechanistic model of drug efflux, and the primary physiological role(s) of the SMR proteins are discussed.

Amino Acid Sequence↗
PubMed1996

Ion transport systems in human labial acinar cells.

Human labial acini were assayed for the presence of ion transport systems associated with salivary fluid secretion using microfluorometric methods. Na(+)-K(+)-Cl- contransport and Na+/H+ exchange activities (determined by their bumetanide and amiloride sensitivities, respectively) were found at levels approximately 50% of those seen in similarly assayed rat parotid acini, but little, if any, C1-/HCO3-exchange activity was observed. Also, when human labial acini were stimulated with the muscarinic agonist carbachol, little evidence of the intracellular acidification associated with HCO3- secretion by other salivary glands was found. Na+/H+ exchange activity in human labial acini was downregulated (approximately 40%) by beta-adrenergic stimulation and upregulated (approximately threefold) by muscarinic stimulation. In contrast, beta-adrenergic stimulation produced only a marginally significant increase in Na(+)-K(+)-Cl- cotransport activity, and muscarinic stimulation was without effect. We include that basolateral Na(+)-K(+)-Cl- cotransport appears to be the dominant mechanism driving Cl- secretion and thereby fluid secretion in this tissue.

Adrenergic beta-Agonists↗
PubMed1996

Immunomodulation of sheep and human lymphocytes by Fasciola hepatica excretory-secretory products.

Fasciola hepatica excretory-secretory (ES) products (30 micrograms ml-1 total ES protein upwards) significantly decreased tritiated thymidine (3HTh) uptake in phytolectin-stimulated sheep lymphocytes. Lower doses of ES products, however, caused an increase in 3HTh uptake in sheep cells stimulated with phytohaemagglutinin (PHA) and Concanavalin A (Con A) and, indeed, ES products were mitogenic on their own. In contrast, 3HTh uptake by human lymphocyte cultures in the presence of phytolectins was progressively inhibited by increasing doses of ES products, and ES products were not mitogenic on their own. The observed inhibitory effects on 3HTh incorporation were not due to toxicity of ES products.

Animals↗
PubMed1995

Involvement of direct phosphorylation in the regulation of the rat parotid Na(+)-K(+)-2Cl- cotransporter.

We identify a 175-kDa membrane phosphoprotein (pp175) in rat parotid acini whose properties correlate well with the Na(+)-K(+)-2Cl- cotransporter previously characterized functionally and biochemically in this tissue. pp175 was the only phosphoprotein immunoprecipitated by an anti-Na(+)-K(+)-2Cl- cotransporter antibody and the only membrane protein whose phosphorylation state was conspicuously altered after a brief (45-s) exposure of acini to the beta-adrenergic agonist isoproterenol. Phosphopeptide mapping provided evidence for three phosphorylation sites on pp175, only one of which was labeled in response to isoproterenol treatment. The half-maximal effect of isoproterenol on phosphorylation of pp175 (approximately 20 nM) was in excellent agreement with its previously demonstrated up-regulatory effect on cotransport activity. Increased phosphorylation of pp175 was also seen following acinar treatment with a permeant cAMP analogue and with forskolin, conditions that have likewise been shown to up-regulate the cotransporter. Combined with earlier results from our laboratory, these data provide strong evidence that the up-regulation of the cotransporter by these agents is due to direct phosphorylation mediated by protein kinase A. AlF(-)4 treatment, which results in an up-regulation of cotransport activity comparable with that observed with isoproterenol (approximately 6-fold), caused a similar increase in phosphorylation of pp175. However, hypertonic shrinkage and treatment with the protein phosphatase inhibitor calyculin A, which also up-regulate the cotransporter (approximately 3-fold and approximately 6-fold, respectively) caused no change in the phosphorylation level. Furthermore, although acinar treatment with the muscarinic agonist carbachol results in a dramatic up-regulation of cotransport activity and a concomitant phosphorylation of pp175, no phosphorylation of pp175 was seen with the Ca(2+)-mobilizing agent thapsigargin, which is able to fully mimic the up-regulatory effect of carbachol on transport activity. Taken together, these results indicate that direct phosphorylation is only one of the mechanisms involved in secretagogue-induced regulation of the rat parotid Na(+)-K(+)-2Cl- cotransporter.

Animals↗
PubMed1995

A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine selectively activates chloride efflux from human epithelial and mouse fibroblast cell lines expressing the cystic fibrosis transmembrane regulator delta F508 mutation.

Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) stimulates 36Cl- efflux from pancreatic CFPAC-1 cells which bear the delta F508 genotype common to most cases of cystic fibrosis [Eidelman et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5562-5566]. By contrast, correction of the cystic fibrosis defect by retrovirus-mediated gene transfer renders the resulting CFPAC-PLJ-CFTR cells insensitive to CPX. We now report that CPX also activates chloride efflux from the CF tracheal epithelial cell line IB3-1 bearing a delta F508 allele, but not if the IB3-1 cells have been repaired by transfection of the wild-type CFTR gene. Similar results were obtained with recombinant NIH 3T3 cells, in which CPX activates 36Cl- efflux from cells expressing the CFTR (delta F508) gene product but not from 3T3 cells expressing the wild-type CFTR. In all three cell types expressing CFTR (delta F508), CPX was found to activate 36Cl- efflux in a dose-dependent manner over the concentration range of 1-30 nM and then gradually lose potency at higher CPX concentrations. Six CPX analogues, A1 receptor antagonists of affinity similar to that of CPX, were found to be much less effective than CPX at activating 36Cl- efflux from CFPAC-1 cells. These included 2-thio-CPX. CPT (8-cyclopentyl-1,3-dimethylxanthine),3,4-dehydro-CPX,3-F-CPX,3-1-CPX, and KW-3902 (8-noradamantyl-1,3-dipropylxanthine).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Anion dependence of bumetanide binding and ion transport by the rabbit parotid Na(+)-K(+)-2Cl- co-transporter: evidence for an intracellular anion modifier site.

The anion dependence of [3H]bumetanide binding and 22Na+ transport by the rabbit parotid Na(+)-K(+)-2Cl- co-transporter was studied in acinar basolateral membrane vesicles (BLMVs). Cl-, Br- and NO3- have a biphasic effect on binding consistent with the presence of two anion sites associated with the bumetanide binding event, a high-affinity stimulatory site and a lower-affinity inhibitory site. We show that formate shares only the stimulatory site and SO4(2-) only the inhibitory site. The initial rate of [3H]bumetanide binding was stimulated by formate or low [Cl-] and inhibited by SO4(2-) or high [Cl-], but the rate of [3H]bumetanide dissociation was not affected by the presence of these anions in the dissociation medium. However, when [3H]bumetanide was bound to BLMVs in the presence of formate its rate of dissociation was more than four times faster than when binding took place in the presence of Cl-. These observations indicate that the binding of bumetanide and the stimulatory anion are ordered such that the anion must necessarily bind first and subsequently cannot dissociate until after bumetanide dissociates. In zero-trans-flux experiments, extravesicular SO4(2-) and formate had no effect on 22Na+ transport via the co-transporter [Turner and George (1988) J. Membr. Biol. 102, 71-77]. Thus neither of the anion sites associated with bumetanide binding is a Cl- transport site. However, we show here that SO4(2-) inhibits transport when present in the intravesicular space. Since the BLMV preparation is predominantly oriented cytosolic-side-in, this observation indicates the existence of an inhibitory cytosolic anion modifier site. Our data suggest that this site is identical to the inhibitory anion site associated with bumetanide binding.

Animals↗
PubMed1995

Mutation of a type II keratin gene (K6a) in pachyonychia congenita.

Pachyonychia congenita (PC) is a rare autosomal dominant condition characterized by multiple ectodermal abnormalities. Patients with Jadassohn-Lewandowsky Syndrome (MIM #167200; PC-1) have nail defects (onchyogryposis), palmoplantar hyperkeratosis, follicular hyperkeratosis and oral leukokeratosis. Those with the rarer Jackson-Lawler Syndrome (MIM #167210; PC-2) lack oral involvement but have natal teeth and cutaneous cysts. Ultra-structural studies have identified abnormal keratin tonofilaments and linkage to the keratin gene cluster on chromosome 17 has been found in PC families. Keratins are the major structural proteins of the epidermis and associated appendages and the nail, hair follicle, palm, sole and tongue are the main sites of constitutive K6, K16 and K17 expression. Furthermore, mutations in K16 and K17 have recently been identified in some PC patients. Although we did not detect K16 or K17 mutations in PC families from Slovenia, we have found a heterozygous deletion in a K6 isoform (K6a) in the affected members of one family. This 3 bp deletion (AAC) in exon 1 of K6a removes a highly conserved asparagine residue (delta N170) from position 8 of the 1A helical domain (delta N8). This is the first K6a mutation to be described and this heterozygous K6a deletion is sufficient to explain the pathology observed in this PC-1 family.

Amino Acid Sequence↗
PubMed1995

The tellurite-resistance determinants tehAtehB and klaAklaBtelB have different biochemical requirements.

The tehAtehB operon from the Escherichia coli chromosome (32.3 min) mediates resistance to potassium tellurite (K2TeO3) when expressed on a multicopy plasmid such as pUC8 (pTWT100). An MIC of 128 micrograms ml-1 is observed when tehAtehB is expressed in a wild-type host and grown on rich media. In this study, the tehAtehB determinant was transformed into mutants deficient in electron transport processes and/or thiol redox coupling within E. coli. These mutants included ubi, nad, cys, nar, trx, grx, gsh and sod. MICs of tehAtehB transformed into these mutants ranged from 1-16 micrograms K2TeO3 ml-1 compared to 0.03-2 micrograms ml-1 for strains transformed with a control plasmid. The tellurite-resistance determinant locus kilA cloned from the IncP alpha plasmid RK2Ter (pDT1558) was also investigated in these strains. This tellurite-resistance determinant showed little or no dependency on the host genotype. The ability of tehAtehB to mediate resistance in wild-type hosts is limited to rich medium. Rich medium may provide a key unidentified cofactor required by TehATehB that is not provided under minimal conditions. Again, the ability of the kilA determinant to mediate tellurite resistance was independent of medium conditions. These data suggest that either a reducing environment or electron-reducing equivalents are required for tehAtehB to mediate high levels of resistance to potassium tellurite. Therefore, the two resistance determinants studied here possess two very different biochemical mechanisms of resistance. Our data also suggest a mechanism for endogenous resistance to tellurite which involves nitrate reductase, superoxide dismutase, and thiol redox processes.

Culture Media↗
PubMed1995

Roles of the three transcriptional attenuators of the Bacillus subtilis pyrimidine biosynthetic operon in the regulation of its expression.

Expression of the Bacillus subtilis pyr operon is regulated by exogenous pyrimidines and the protein product of the first gene of the operon, PyrR. It has been proposed that PyrR mediates transcriptional attenuation at three untranslated segments of the operon (R.J. Turner, Y. Lu, and R.L. Switzer, J. Bacteriol., 176:3708-3722, 1994). In this study, transcriptional fusions of the pyr promoter followed by the pyr attenuation sequences, either individually or in tandem to a lacZ reporter gene, were used to examine the physiological functions of all three attenuators through their ability to affect beta-galactosidase expression. These fusions were studied as chromosomal integrants in various B. subtilis strains to examine the entire range of control by pyrimidines, PyrR dependence, amd developmental control of pyr gene expression. The nutritional regulation of each attenuator separately was roughly equivalent to that of the other two and was totally dependent upon PyrR, and that of tandem attenuators was cumulative. The regulation of a fusion of the spac promoter followed by the pyrP:pyrB intercistronic region to lacZ produced results similar to those obtained with the corresponding fusion containing the pyr promoter, demonstrating that attenuator-dependent regulation is independent of the promoter. Extreme pyrimidine starvation gave rise to two- to threefold-higher levels of expression of a pyr-lacZ fusion that lacked attenuators, independent of PyrR, than were obtained with cells that were not starved. Increased expression of a similar spac-lacZ fusion during pyrimidine starvation was also observed, however, indicating that attenuator-independent regulation is not a specific property of the pyr operon. Conversion of the initiator AUG codon in a small open reading frame in the pyrP:pyrB intercistronic region to UAG reduced expression by about half but did not alter regulation by pyrimidines, which excludes the possibility of a coupled transcription-translation attenuation mechanism. Developmental regulation of pyr expression during early stationary phase was found to be dependent upon the attenuators and PyrR, and the participation of SpoOA was excluded.

Bacillus subtilis↗
PubMed1995

Neither reduced uptake nor increased efflux is encoded by tellurite resistance determinants expressed in Escherichia coli.

Rates of uptake of the TeO3(2-) oxyanion were investigated in Escherichia coli cells containing tellurite resistance determinants from both plasmid (RK2Ter, R478, pMER610, MIP233, pHH1508a, pMUR) and chromosomal (tehAB) sources. The uptake was investigated to determine whether or not reduced uptake or increased efflux is involved in the tellurite resistance mechanism. Reduced TeO3(2-) uptake generated by cultures harboring arsABC from the plasmid R773, which has been previously shown to be an oxyanion efflux transporter, was used as the standard. Uptake curves were found to be essentially identical among E. coli cultures harboring the tellurite resistance plasmids RK2Ter, pMER610, pHH1508a, and pMUR and cultures harboring tellurite-sensitive control plasmids. Cultures harboring clones of the tehAB operon from E. coli showed no change in the TeO3(2-) accumulation. Cultures harboring R478 demonstrated reduced uptake. However, a subclone containing only the tellurite resistance determinant displayed no reduced uptake. This suggests that there may be another determinant on R478 other than the primary tellurite resistance determinant that gives rise to TeO3(2-) efflux. These results demonstrate that neither reduced uptake nor increased efflux is responsible for the tellurite resistance in the resistance determinants investigated here.

Adenosine Triphosphatases↗
PubMed1995

Na+ channels in membrane vesicles from intralobular salivary ducts.

Electrical potential-driven 22Na+ fluxes were measured in membrane vesicles prepared from male and female rat submandibular intralobular ducts. A relatively temperature-independent (Q10 = 1.45 +/- 0.15), amiloride-inhibitable (mean affinity constant approximately 1 microM), rheogenic Na+ transport pathway was observed. The relative potency of amiloride analogues for inhibition of this pathway was amiloride > ethylisopropyl-amiloride > phenamil, similar to that of the "low-amiloride-affinity" Na+ channel recently observed in a number of other tissues. These results are consistent with the existence of the apical Na+ channel thought to be involved in intralobular ductal salt reabsorption. No significant difference was found in the magnitude or pharmacology of electrogenic Na+ fluxes in vesicles prepared from male and female rat intralobular ducts, suggesting that the sexual dimorphism observed in this tissue is not reflected at the level of the apical membrane Na+ channel. Amiloride-sensitive 22Na+ fluxes in intralobular ductal membranes were of the same magnitude as 22Na+ fluxes measured in similarly prepared and assayed vesicles from the toad bladder, a tissue thought to be a rich source of amiloride-sensitive Na+ channels.

Amiloride↗
PubMed1995

CPX, a selective A1-adenosine-receptor antagonist, regulates intracellular pH in cystic fibrosis cells.

The selective A1-adenosine-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), has been reported to activate Cl- efflux from cystic fibrosis cells, such as pancreatic CFPAC-1 and lung IB3 cells bearing the cystic fibrosis transmembrane regulator(delta F508) mutation, but has little effect on the same process in cells repaired by transfection with wild-type cystic fibrosis transmembrane regulator (O. Eidelman, C. Guay-Broder, P. J. M. van Galen, K. A. Jacobson, C. Fox, R. J. Turner, Z. I. Cabantchik, and H. B. Pollard. Proc. Natl. Acad. Sci. USA 89: 5562-5566, 1992). We report here that CPX downregulates Na+/H+ exchange activity in CFPAC-1 cells but has a much smaller effect on cells repaired with the wild-type gene. CPX also mildly decreases resting intracellular pH. In CFPAC-1 cells, this downregulation is dependent on the presence of adenosine, since pretreatment of the cells with adenosine deaminase blocks the CPX effect. We also show that, by contrast, CPX action on these cells does not lead to alterations in intracellular free Ca2+ concentration. We conclude that CPX affects pH regulation in CFPAC-1 cells, probably by antagonizing the tonic action of endogenous adenosine.

Calcium↗