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R J Turner

Publications and source records attributed to R J Turner.

At least 37 records · Page 2Linked to original sources
PubMed2000

The role of cysteine residues in tellurite resistance mediated by the TehAB determinant.

TehATehB is a tellurite (TeO(2-)(3)) resistance determinant found on the Escherichia coli chromosome. Normally silent, it specifies a minimal inhibitory concentration (MIC) of 2 microg K(2)TeO(3)/ml unless upregulated or present on a multicopy plasmid which results in an MIC of 128 microg/ml. Both TehA and TehB have three cysteine residues. Oligonucleotide site-directed mutagenesis was carried out to systematically replace all six cysteine residues by alaninies. The results showed that cysteine residues in both TehA and TehB play a role in tellurite resistance: A single cysteine change had no effect, however increasing combinations of two or three cysteine substitutions demonstrated strong phenotypic effects with minimal inhibitory concentrations ranging from 16-64 microg K(2)TeO(3)/ml. A cysteine-free mutant in which all six cysteine residues were replaced by alanines maintained a MIC of 16 microg/ml. Further investigations on the role of cysteines in resistance were studied using thiol reactive reagents on the soluble subunit TehB. These studies confirmed that TehB is a dimer and undergoes a conformational change with tellurite and S-adenosyl-l-methionine binding. Studies using native and SDS denaturing PAGE under reducing and oxidizing conditions suggested that a cysteine in TehB is involved in binding tellurite.

Alanine↗
PubMed2000

One of two genes encoding glycyl-tRNA synthetase in Saccharomyces cerevisiae provides mitochondrial and cytoplasmic functions.

In the yeast Saccharomyces cerevisiae, two genes (GRS1 and GRS2) encode glycyl-tRNA synthetase (GlyRS1 and GlyRS2, respectively). 59% of the sequence of GlyRS2 is identical to that of GlyRS1. Others have proposed that GRS1 and GRS2 encode the cytoplasmic and mitochondrial enzymes, respectively. In this work, we show that GRS1 encodes both functions, whereas GRS2 is dispensable. In addition, both cytoplasmic and mitochondrial phenotypes of the knockout allele of GRS1 in S. cerevisiae are complemented by the expression of the only known gene for glycyl-tRNA synthetase in Schizosaccharomyces pombe. Thus, a single gene for glycyl-tRNA synthetase likely encodes both cytoplasmic and mitochondrial activities in most or all yeast. Phylogenetic analysis shows that GlyRS2 is a predecessor of all yeast GlyRS homologues. Thus, GRS1 appears to be the result of a duplication of GRS2, which itself is pseudogene-like.

Amino Acid Sequence↗
PubMed2000

Severe impairment of salivation in Na+/K+/2Cl- cotransporter (NKCC1)-deficient mice.

The salivary fluid secretory mechanism is thought to require Na(+)/K(+)/2Cl(-) cotransporter-mediated Cl(-) uptake. To directly test this possibility we studied the in vivo and in vitro functioning of acinar cells from the parotid glands of mice with targeted disruption of Na(+)/K(+)/2Cl(-) cotransporter isoform 1 (Nkcc1), the gene encoding the salivary Na(+)/K(+)/2Cl(-) cotransporter. In wild-type mice NKCC1 was localized to the basolateral membranes of parotid acinar cells, whereas expression was not detected in duct cells. The lack of functional NKCC1 resulted in a dramatic reduction (>60%) in the volume of saliva secreted in response to a muscarinic agonist, the primary in situ salivation signal. Consistent with defective Cl(-) uptake, a loss of bumetanide-sensitive Cl(-) influx was observed in parotid acinar cells from mice lacking NKCC1. Cl(-)/ HCO(3)(-) exchanger activity was increased in parotid acinar cells isolated from knockout mice suggesting that the residual saliva secreted by mice lacking NKCC1 is associated with anion exchanger-dependent Cl(-) uptake. Indeed, expression of the Cl(-)/ HCO(3)(-) exchanger AE2 was enhanced suggesting that this transporter compensates for the loss of functional Na(+)/K(+)/2Cl(-) cotransporter. Furthermore, the ability of the parotid gland to conserve NaCl was abolished in NKCC1-deficient mice. This deficit was not associated with changes in the morphology of the ducts, but transcript levels for the alpha-, beta-, and gamma-subunits of the epithelial Na(+) channel were reduced. These data directly demonstrate that NKCC1 is the major Cl(-) uptake mechanism across the basolateral membrane of acinar cells and is critical for driving saliva secretion in vivo.

Animals↗
PubMed2000

Evidence that type I, II, and III inositol 1,4,5-trisphosphate receptors can occur as integral plasma membrane proteins.

A number of previous reports have suggested that inositol 1,4, 5-trisphosphate receptors (IP(3)Rs) are present in the plasma membranes of cells. We confirm this directly in the present study by demonstrating that a significant proportion of the IP(3)Rs found in A431 cells, Jurkat cells, and rat parotid acini can be biotinylated by the extracellular application of sulfo-N-hydroxysuccinimide-biotin to intact cells. This labeling cannot be accounted for by the reaction of sulfo-N-hydroxysuccinimide-biotin with intracellular IP(3)Rs since calnexin and the SERCA2 ATPase, both integral membrane proteins of the endoplasmic reticulum, are not labeled under the same experimental conditions. Individual IP(3)R subtypes were detected using subtype-specific antibodies. A431 cells expressed only the type-3 IP(3)R, and 23% of this protein was in the biotinylated (plasma membrane) fraction. Jurkat cells and rat parotid cells expressed all three IP(3)R subtypes. Contrary to earlier results suggesting that only the type-3 IP(3)R might localize to the plasma membrane, we found that significant amounts (5-14%) of all three subtypes could be identified in the biotinylated fractions of Jurkat and rat parotid cells. Our results suggest a role for IP(3)Rs in plasma membrane as well as intracellular membrane function.

Amino Acid Sequence↗
PubMed2000

Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli.

Dimethyl sulfoxide (Me(2)SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under anaerobic growth conditions and is required for anaerobic growth with Me(2)SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support growth on glycerol-Me(2)SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membrane-bound DmsABC with greatly reduced enzyme activity and inefficient anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.

Amino Acid Sequence↗
PubMed2000

The structural unit of the secretory Na+-K+-2Cl- cotransporter (NKCC1) is a homodimer.

The oligomeric state of the secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in rat parotid plasma membranes was studied using the reversible chemical cross-linker DTSSP [3, 3'-dithiobis(sulfosuccinimidyl propionate)]. The monomeric apparent molecular mass of NKCC1 is approximately 170 kDa. However, we show here that this protein migrates as a approximately 355 kDa complex on SDS-PAGE gels after membrane treatment with DTSSP, indicating that NKCC1 exists as an oligomer in the plasma membrane. The stability of this oligomer is such that it is not disrupted by solubilization of the membrane by low concentrations of the nonionic detergent Triton X-100 (0.3%) or the mild ionic detergent deoxycholate (20 mM); however, higher concentrations of Triton X-100 or treatment with the denaturing detergent SDS do result in destabilization of the NKCC1 complex. In additional experiments, we immunoprecipitated the 355 kDa cross-linked complex from biotinylated membranes, then cleaved the cross-linking bonds and analyzed the resulting components of the NKCC1 oligomer by avidin blotting, silver staining, and 2D electrophoresis. In these studies, we were unable to detect the presence of any proteins other than NKCC1 itself in the 355 kDa oligomer, suggesting that this complex is an NKCC1 dimer. Strong evidence for this conclusion was provided by a quantitative analysis of the molecular sizes of oligomers formed by full-length NKCC1 and an N-terminally truncated version of NKCC1 expressed in HEK293 cells. Taken together, our data provide convincing evidence that the dominant structural unit of NKCC1 in the plasma membrane is a homodimer.

Animals↗
PubMed2000

PUVA-related punctate keratoses of the hands and feet.

BACKGROUND: We have observed, in patients undergoing high-dose PUVA treatment, a type of keratosis not previously described. The lesions usually occur on the sides of the palms or soles and are clinically distinct. They are generally painless and often go unnoticed by patients. OBJECTIVE: We sought to further characterize these lesions both clinically and histologically. METHODS: Patients attending a PUVA clinic were screened for these keratoses. Other PUVA-related complications were recorded. Representative lesions were photographed, and biopsy specimens were taken. RESULTS: Biopsy specimens were taken from lesions in 10 patients. All had plaque psoriasis and had received high UVA doses (>1000 J/cm(2)) through PUVA therapy. All patients had PUVA-induced keratoses elsewhere, but the number varied greatly between patients. The hand and foot keratoses were well defined and circular and had a characteristic histologic appearance, with a sharp demarcation between normal and abnormal markedly pale-staining epidermis. CONCLUSION: These lesions are a further cutaneous manifestation of prolonged PUVA therapy.

Adult↗
PubMed2000

A retrospective study of outcome of Mohs' micrographic surgery for cutaneous squamous cell carcinoma using formalin fixed sections.

The surgical management of recurrent or large squamous cell carcinoma (SCC) can be challenging as tumours often extend beyond visible margins. Micrographic surgery is a potentially effective method of ensuring complete clearance of tumour. A retrospective study of all cases of SCC treated by micrographic surgery in this department between 1986 and 1996 has been done. Sixty-one patients were treated using a formalin-fixed paraffin-embedded tissue technique with a median follow-up of 4 years. In two cases there was local recurrence and in three others metastasis to local lymph nodes. The overall cure rate was 92% (56 of 61), which compares favourably with published series using chemosurgery and frozen tissue techniques. The results show that this technique of micrographic surgery is a satisfactory and cost-effective alternative to conventional frozen section techniques in the treatment of SCC. The formalin-fixed tissue method has the advantage of providing high-quality permanent histological sections using existing conventional pathology services.

Adult↗
PubMed2000

A controlled study of ultraviolet A sunbed treatment of psoriasis.

BACKGROUND: Ultraviolet (UV) A sunbeds are widely used by patients with psoriasis in an attempt to treat their skin disease. However, there is little evidence that UVA therapy improves psoriasis, and the long-term risks of sunbed exposure are not known. OBJECTIVES: To perform a randomized, placebo-controlled study of UVA sunbed therapy for psoriasis. METHODS: A sunbed and canopy unit was modified to allow UVA exposure on one side of the body (front and back), and 'placebo' visible light exposure on the other side of the body. We treated 38 patients with psoriasis, giving 12 exposures over a period of 4 weeks. Assessment was made using a modified Psoriasis Area and Severity Index (PASI) score, individual plaque assessment and patient questionnaire. RESULTS: In 17 patients (47%) the PASI score showed a greater reduction on the UVA side compared with placebo, in 11 patients (31%) no difference was recorded between the two sides, and in eight (22%) the improvement was greater on the placebo-treated side. Overall, the median pretreatment half-body modified PASI score was 4.4 units, reducing to 3.9 units on the UVA-treated side and 4.2 units on the placebo-treated side (P = 0. 044 for difference in response). Breakdown of the plaque score into the individual components of erythema, scale and thickness revealed significant improvement only with the score for erythema. Although the degree of improvement was small, 64% of patients felt that the response was sufficiently good that they would use a sunbed again to treat their psoriasis. CONCLUSIONS: Our results show that a short course of sunbed treatment does improve psoriasis in some patients, but that the degree of improvement is small.

Adult↗
PubMed2000

Arterial flow induces changes in saphenous vein endothelium proteins transduced by cation channels.

OBJECTIVES: expression of leukocyte adhesins and proteins controlling thrombosis is likely to be an important determinant of graft patency early following vein bypass. We have previously demonstrated rapid increase in endothelial ICAM-1 and nitric oxide synthase (eNOS) concentrations in human saphenous vein exposed to arterial flow. The aim of this study was to investigate whether ion-channel-blocking drugs could alter these flow-induced changes. METHODS: human saphenous vein segments, freshly excised from patients, were placed in a validated in vitro circuit using flow conditions shown to simulate arterial or venous circulations for 90 min, in the presence or absence of ion-channel blockers. The concentrations of ICAM-1, VCAM-1, eNOS and tissue factor (TF) were assessed by quantitative immunohistochemistry in vein exposed to flow and compared with that in freshly excised vein from the same patient. The endothelial protein concentration was calculated as the mean area of staining as percentage of that for the control protein CD31, using computer-aided image analysis. RESULTS: after arterial flow conditions the area ratio of ICAM-1 increased from 21.4+/-1.4 to 44.6+/-2.0%, of eNOS increased from 50.0+/-5.6 to 70.1+/-5.0%, of VCAM-1 decreased from 16.6+/-3.4 to 3.6+/-1.0%, whereas TF staining area ratio was unchanged. Inclusion of the non-selective K(+)channel blocker, tetraethylammonium, in the arterial perfusion solution abolished all these arterial flow-induced changes. Inclusion of the K(+)ATP channel blocker, glibenclamide, selectively abolished the arterial flow-induced changes in ICAM-1 and VCAM-1. Inclusion of the calcium channel blocker, nifedipine, abolished the arterial flow-induced changes in eNOS and VCAM-1 but increased the TF staining area ratio from 3.0+/-0.4 to 8.5+/-0.7%, p=0.01. Inclusion of the stretch-activated cation-channel blocker, gadolinium, enhanced the arterial flow-induced increase in eNOS, but prevented the arterial flow-induced increase in ICAM-1. CONCLUSIONS: perfusion of veins under arterial flow conditions with gadolinium was associated with low endothelial concentrations of ICAM-1, VCAM-1 and TF, but high levels of eNOS. Such a concentration of endothelial proteins may be advantageous in newly implanted vein grafts. In contrast, nifedipine could have adverse effects by promoting increase in TF concentration.

Arteries↗
PubMed2000

Escherichia coli TehB requires S-adenosylmethionine as a cofactor to mediate tellurite resistance.

The Escherichia coli chromosomal determinant for tellurite resistance consists of two genes (tehA and tehB) which, when expressed on a multicopy plasmid, confer resistance to K(2)TeO(3) at 128 microg/ml, compared to the MIC of 2 microg/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found in S-adenosyl-L-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a approximately 20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.

Amino Acid Sequence↗
PubMed1999

Arterial flow conditions downregulate thrombomodulin on saphenous vein endothelium.

BACKGROUND: The antithrombogenic properties of venous endothelium may be attenuated when vein is implanted in the arterial circulation. Such changes may facilitate thrombosis, which is the final common pathway for saphenous vein arterial bypass graft occlusion. METHODS AND RESULTS: Using human saphenous vein in a validated ex vivo flow circuit, we investigated (1) the possibility that arterial flow conditions (mean pressure, 100 mm Hg, 90 cpm, approximately 200 mL/min) alter the concentration of proteins involved in regulating thrombosis at the vessel wall and (2) the influence of ion channel blockade on such effects. Concentrations of thrombomodulin and tissue factor were quantified by Western blotting (ratio of von Willebrand factor staining) and immunohistochemistry (as a percentage of CD31-staining area). Thrombomodulin concentrations after 90 minutes of venous and arterial flow conditions were quantified by immunostaining (68.9+/-4.8% and 41.0+/-3.0% CD31, respectively; P<0.01) and by Western blotting (1.35+/-0.20 and 0. 15+/-0.03 ratio of von Willebrand factor, respectively; P<0.01). The ability of endothelial cells to generate activated protein C also decreased from 62+/-14 to 19+/-10 ng. min-1. 1000 cells-1 (P=0.01). The significant reduction in thrombomodulin was attenuated if calcium was removed from the perfusate but not by external vein stenting. Inclusion in the vein perfusate of drugs that reduce calcium entry (including Gd3+, to block stretch-activated ion channels, and nifedipine) abolished the reduction in thrombomodulin concentration observed after arterial flow conditions. In freshly excised vein, negligible concentrations of tissue factor were detected on the endothelium and concentrations did not increase after 90 minutes of arterial flow conditions, although the inclusion of nifedipine caused the immunostaining to increase from 3.0+/-0.4% to 8.5+/-0.7% CD31 (P<0.02). CONCLUSIONS: In saphenous vein endothelium exposed to arterial flow conditions, there is rapid downregulation of thrombomodulin, sufficient to limit protein C activation, by a calcium-dependent mechanism.

Calcimycin↗
PubMed1999

Ion transport and signalling in human labial glands.

Recent reports characterizing the physiological properties of normal human labial acini are reviewed with particular emphasis on mechanisms related to fluid secretion. In contrast to the salivary glands of several experimental animals, human labial acinar cells do not appear to have a1-adrenergic receptors, substance P peptidergic receptors, or significant levels of Cl-/HCO3- exchange. Nor do they appear to secrete HCO3- in response to Ca2+ mobilizing stimuli. The data presently available indicates that fluid secretion by these glands is mainly under muscarinic control and is due to acinar Cl- secretion driven by a basolateral Na+ -K+ -2Cl- cotansporter.

Animals↗
PubMed1999

Regulation of the Bacillus subtilis pyrimidine biosynthetic operon by transcriptional attenuation: control of gene expression by an mRNA-binding protein.

The pyrimidine nucleotide biosynthetic (pyr) operon of Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which termination of transcription at points upstream of the genes being regulated is promoted by the binding of a regulatory protein, PyrR, to specific sequences in the pyr mRNA. Binding of PyrR to pyr mRNA is stimulated by uridine nucleotides and causes changes in the mRNA secondary structure. This model is supported by extensive molecular genetic analysis. PyrR, which is encoded by the first gene of the pyr operon, is also a uracil phosphoribosyltransferase, although it has little amino acid sequence resemblance to other bacterial uracil phosphoribosyltransferases. Purified B. subtilis pyrR promotes attenuation of pyr transcription in vitro and binds specifically to pyr RNA sequences. The crystallographic structure of PyrR demonstrates the similarity of its tertiary structure to other phosphoribosyltransferases and suggests the surface to which RNA binds. PyrR is widely distributed among eubacteria and appears to regulate pyr genes not only by the attenuation mechanism found in B. subtilis, but also by a coupled transcription-translation attenuation mechanism and by acting as a translational repressor. PyrR illustrates the concept that transcriptional attenuation is a much more widespread and mechanistically versatile mechanism for the regulation of gene expression in bacteria than is generally recognized.

Amino Acid Sequence↗
PubMed1999

Effect of Fasciola gigantica somatic extracts and excretory/secretory products on superoxide production by activated neutrophils.

Somatic extracts and excretory/secretory (ES) products from Fasciola gigantica suppress the release of toxic oxygen intermediates by activated sheep neutrophils. At the concentrations used both the somatic extracts and ES products had no effect on neutrophil viability and in both cases the inhibitory effect was reversible. In the somatic extracts, heating or treatment with trypsin destroyed the inhibitory effect. However, the ES products contained at least two immunomodulatory factors. One was less than 10 kDa, the other greater than 50 kDa and both factors were heat and trypsin stable. By acting locally and only causing temporary inhibition of neutrophil function F. gigantica can protect itself from immune mediated oxidative damage without compromising the host's response elsewhere.

Animals↗
PubMed1999

Obstetric history in women with surgically corrected adult urinary incontinence or pelvic organ prolapse.

STUDY OBJECTIVE: To compare obstetric histories of women who had surgical correction of urinary incontinence or pelvic organ prolapse with a similar group who did not. DESIGN: Case control study (Canadian Task Force classification II-2). SETTING: Urban, community-based, private practice teaching hospital. PATIENTS: Four hundred eighty women (age 51.4 +/- 13.0 yrs) who underwent corrective surgery for urinary incontinence, pelvic organ prolapse, or both, and whose obstetric history was obtainable through chart review. The control group was composed of 150 women (age 50.7 +/- 9.6 yrs) having routine screening mammography who completed a questionnaire regarding obstetric, gynecologic, and urologic history. MEASUREMENTS AND MAIN RESULTS: Patients and controls did not differ significantly in terms of age, race, height, weight, body mass index, or smoking history. Women who underwent surgery were of greater parity (2.5 +/- 1.2 vs 2.0 +/- 1.2, p <0.001), less often nulliparous (3% vs 18%, p <0.001), less likely to have had a cesarean delivery (4% vs 15%, p <0.001), and more likely to have had a vaginal delivery (94% vs 77%, p <0.001) than those with no surgery. The odds ratio of patients who had a vaginal delivery compared with controls was 4.7 (2.3-8.3), and that for cesarean delivery was 0.22 (0.11-0.43). Analysis of specific delivery information found that, compared with controls, patients were older by 4 years at time of their first delivery (28.9 +/- 4.9 vs 24.9 +/- 4.9 yrs, p <0.001) and more commonly received epidural analgesia intrapartum (87% vs 40%, p = 0.004). Comparisons within the patient group, categorized by indication for surgery, revealed that women who had surgery for either prolapse alone or for both prolapse and incontinence were most likely to have had vaginal deliveries (85% incontinence alone vs 94% prolapse alone vs 97% both, p <0.001). CONCLUSION: Increased parity, vaginal childbirth, maternal age at time of delivery, and use of epidural analgesia are associated with need for operative correction of pelvic organ prolapse or adult urinary incontinence. Conversely, cesarean delivery is associated with less need for surgical correction of incontinence or pelvic organ prolapse.

Case-Control Studies↗
PubMed1999

Personal resources and the social distribution of depression.

This paper addresses the hypothesis that gender, age, marital status, and SES matter for depression partly because of associated differences in the availability and/or impact of the personal resources of mastery and self-esteem. It is argued that findings indicating that the social distributions of these resources complement those for depression would provide preliminary support for this hypothesis. Based on a large urban community sample (n = 1,390), our findings fail to support the availability hypothesis in relation to marital status, provide only modest support in reference to age and gender, but yield compelling support in relation to socioeconomic status (SES). Indeed, variations in the availability of these resources, especially mastery, provide a largely, if not entirely, adequate explanation for the SES-depressive symptoms relationship and accounts for nearly half of the SES-Major Depressive Disorder relationship. Although the significance of mastery was more pronounced among women and unmarried persons, such differences did not contribute to understanding observed gender or marital status variations in depression.

Adolescent↗