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S M Wilson

Publications and source records attributed to S M Wilson.

At least 55 records · Page 3Linked to original sources
PubMed1999

The simultaneous measurement of epithelial ion transport and intracellular free Ca2+ in cultured equine sweat gland secretory epithelium.

We explored the relationship between nucleotide-evoked changes in intracellular free calcium ([Ca2+]i) and anion secretion by measuring [Ca2+]i and I(SC) simultaneously in Fura-2-loaded, cultured equine sweat gland epithelia. Apical ATP, UTP or UDP elicited sustained increases in [Ca2+]i that were initiated by the mobilization of cytoplasmic Ca2+ but maintained by Ca2+ influx. However, although these nucleotides also increased I(SC), this response was transient whereas the [Ca2+]i signals were sustained. Experiments in which external Ca2+ was removed/replaced showed that Ca2+ entering nucleotide-stimulated cells elicited very little change in ISC. Cross desensitization experiments showed that UTP-stimulated epithelia became insensitive to ATP but that UTP could increase both [Ca2+]i and ISC in ATP-stimulated cells by activating 'pyrimidinoceptors' essentially insensitive to ATP. Thapsigargin evoked a sustained rise in [Ca2+]i that was accompanied by a maintained increase in ISC. However, this increase in ISC was dependent upon external Ca2+ and so the responses to nucleotides and thapsigargin have different properties. ATP increased ISC in thapsigargin-treated cells without causing any rise in [Ca2+]i while ionomycin increased both parameters. The data therefore show that apical P2Y receptors allow nucleotides to increase ISC via two mechanisms, one of which appears to be [Ca2+]i-independent control of anion channels.

Animals↗
PubMed1999

Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells.

Osteopontin (OPN) has been associated with enhanced malignancy in breast cancer, but its functional role in this disease is poorly understood. To study the effect of OPN on cellular invasiveness, basal OPN expression was first assessed in members of a progression series of human mammary epithelial cell lines (21PT: immortalized, non-tumorigenic; 21NT: weakly tumorigenic; 21MT-1: tumorigenic, weakly metastatic; MDA-MB-435 cells: tumorigenic, highly metastatic). The two lines which expressed lowest basal levels of OPN (21PT, 21NT) were then examined for up-regulation of invasive behavior in response to exogenous or transfected (endogenous) OPN. Both 21PT and 21NT showed increased invasiveness through Matrigel when human recombinant (hr)OPN was added to the lower chamber of transwells. Both also showed a cell migration response to hrOPN. Populations of 21PT and 21NT cells stably transfected with an OPN-expression vector showed higher levels of cell invasiness than control vector transfectants. Examination of transfectants for mRNA of a number of secreted proteases showed that only urokinase-type plasminogen activator (uPA) expression was closely associated with OPN expression and cellular invasiveness. Treatment of the parental 21PT and 21NT cells with exogenous hrOPN resulted in increased uPA mRNA expression and increased urokinase activity of the conditioned media. Both increased cell migration and induction of uPA expression are thus potential mechanisms of increased invasiness of breast epithelial cells in response to OPN.

Breast↗
PubMed1999

Effect of luminal nucleotides on Cl- secretion and Na+ absorption in distal bronchi.

P2Y receptor agonists stimulate Cl- secretion across both normal and cystic fibrosis (CF) airway epithelia, and therefore have potential for use in the treatment of CF. Although CF pathology is manifest primarily in the distal airways, most studies of P2Y-receptor-mediated airway epithelial ion transport have used cells cultured from proximal regions. Here we report the results of studies of P2Y-receptor-mediated ion transport in distal bronchi isolated from porcine lungs, cannulated and perfused. A luminal microelectrode was used to record transepithelial potential difference (PD) and cable analysis was applied to determine resistance (Rt) and equivalent short-circuit current (I(SC)). Luminal UTP (100 micromol/l) transiently hyperpolarized PD (from -5.8+/-0.3 to -6.5+/-0.4 mV) and increased I(SC) (from 47+/-6 to 55+/-8 microA cm(-2)) before inhibiting PD to below the pre-UTP level (-5.0+/-0.4 mV). The decline was attenuated by pretreatment with amiloride, and additional treatment with bumetanide inhibited the initial hyperpolarization, suggesting that UTP stimulates Cl- secretion and inhibits Na+ absorption across distal bronchi. Luminal addition of P2Y1 [ADP, 2-methylthio-ATP (2MeSATP)] and P2Y6 (UDP) receptor agonists had no effect on ion transport. Pretreatment with thapsigargin (0.3 micromol/l) did not prevent the UTP-induced increase in PD and I(SC) but attenuated the secondary fall in PD. Pretreatment with BAPTA/AM (50 micromol/l), however, had no effect on the response to UTP. Additional studies of isolated bronchial epithelial cells using Fura-2 showed that UTP increases [Ca2+]in, and this increase is blocked by pretreatment with thapsigargin. These results suggest that in intact distal bronchi luminal UTP stimulates Cl- secretion by a Ca2+-independent mechanism and inhibits Na+ absorption by a Ca2+-dependent mechanism. Both effects are likely to favour increased hydration of the airway surface, and may therefore be beneficial in CF.

Absorption↗
PubMed1999

P2Y receptor-mediated Ca2+ signalling in cultured rat aortic smooth muscle cells.

1. ATP, UTP, ADP and ADP-beta-S elicited Ca2+ -signals in cultured aortic smooth muscle cells although ADP, UDP and ADP-beta-S gave approximately 40% of the maximal response seen with ATP and UTP. Adenosine, AMP or alpha,beta-methylene-ATP had no effect. These responses were attributed to P2Y2/4 and P2Y1 receptors, which we assumed could be selectively activated by UTP and ADP-beta-S respectively. 2. The response to UTP was reduced (approximately 50%) by pertussis toxin, whilst this toxin had no effect upon the response to ADP-beta-S. This suggests P2Y2/4 receptors simultaneously couple to pertussis toxin-sensitive and -resistant G proteins whilst P2Y1 receptors couple to only the toxin-resistant proteins. 3. Repeated stimulation with UTP or ADP-beta-S caused desensitization which was potentiated by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and attenuated by staurosporine. 4. TPA completely abolished sensitivity to ADP-beta-S but the response to UTP had a TPA-resistant component. In pertussis toxin-treated cells, however, TPA could completely abolish sensitivity to UTP and so the TPA-resistant part of this response seems to be mediated by pertussis toxin-sensitive G proteins. 5. Loss of sensitivity to UTP did not occur when pertussis toxin-treated cells were repeatedly stimulated with this nucleotide, suggesting that pertussis toxin-sensitive G proteins mediate this effect. The toxin did not, however affect desensitization to ADP-beta-S.

Adenosine Diphosphate↗
PubMed1999

P2Y2 receptor-mediated inhibition of ion transport in distal lung epithelial cells.

1 Rat foetal distal lung epithelial cells were plated onto permeable supports where they became integrated into epithelial sheets that spontaneously generated short circuit current (ISC). 2 Apical ATP (100 microM) evoked a transient fall in ISC that was followed by a rise to a clear peak which, in turn, was succeeded by a slowly developing decline to a value below control. Apical UTP evoked an essentially identical response. 3 UDP and ADP were ineffective whilst ATP had no effect when added to the basolateral solution. These effects thus appear to be mediated by apical P2Y2 receptors. 4 The rising phase of the responses to ATP/UTP was selectively inhibited by anion transport inhibitors but persisted in the presence of amiloride, which abolished the inhibitory effects of both nucleotides. Thus, apical nucleotides appear to evoke a transient stimulation of anion secretion and sustained inhibition of Na+ absorption. 5 Basolateral isoprenaline (10 microM) elicited a rise in ISC but subsequent addition of apical ATP reversed this effect. Conversely, isoprenaline restored ISC to its basal level following stimulation with ATP. Apical P2Y2 receptors and basolateral beta-adrenoceptors thus allow their respective agonists to exert mutually opposing effects on ISC.

Adenosine Triphosphate↗
PubMed1999

P2 receptor modulation of voltage-gated potassium currents in Brown adipocytes.

Using patch voltage-clamp techniques, we find there are two components to the voltage-gated potassium current (IKv) in rat brown adipocytes. The components differ in their gating and responses to purinergic stimulation, but not their pharmacology. IKv-A recovers from inactivation at physiological membrane potentials, while IKv-B inactivation recovers at more negative potentials. Both currents are >90% blocked by similar concentrations of quinine and tetraethylammonium, but not by beta-dendrotoxin, charybdotoxin, or apamin. The two current components are differentially modulated by extracellular ATP. ATP shifts the voltage dependence of IKv-A inactivation negative by 38 +/- 5 mV (n = 35, +/-SEM) and shifts activation by -14 +/- 2 mV in whole-cell experiments. ATP did not affect the steady state inactivation voltage dependence of IKv-B, but did apparently convert IKv-A into IKv-B. The pharmacology of the inactivation shift is consistent with mediation by a P2 purinergic receptor. Purinergic stimulation of perforated-patch clamped cells causes hyperpolarizing shifts in the window current of IKv-A by shifting inactivation -18 +/- 4 mV and activation -7 +/- 2 mV (n = 16). Since perforated-patch recordings will most closely resemble in vivo cell responses, this ATP-induced shift in the window current may facilitate IKv activation when the cell depolarizes. IKv activity is necessary for the proliferation and differentiation of brown adipocytes in culture (Pappone, P.A., and S.I. Ortiz-Miranda. 1993. Am. J. Physiol. 264:C1014-C1019) so purinergic modulation of IKv may be important in altering adipocyte growth and development.

Adipose Tissue, Brown↗
PubMed1999

Pyrimidine nucleotide-evoked inhibition of cyclic AMP accumulation in equine epithelial cells.

Uridine triphosphate (UTP) evoked inhibition of adrenaline-evoked cAMP accumulation in cultured equine epithelial cells (EC50, 1.8 +/- 0.2 microM) and this effect was mimicked by 5-Br-UTP (EC50, 6.6 +/- 1.8 microM) and uridine diphosphate (UDP; EC50, 96 +/- 26 microM). This inhibitory action of UTP was abolished by pre-treating cells with pertussis toxin (10 ng ml-1, 24 h). UTP (EC50, 2.3 +/- 0.3 microM) and 5-Br-UTP (EC50, 29.4 +/- 9.4 microM) also increased intracellular free calcium ([Ca2+]i) whilst UDP did not; the two effects are thus differentially sensitive to these pyrimidine nucleotides. ATP evoked cAMP accumulation in control cells and this response was unaffected by pertussis toxin. There is, therefore, no indication that ATP activates the pertussis toxin-sensitive inhibitory pathway. The UTP-evoked inhibition of cAMP accumulation was abolished by isobutylmethylxanthine (IBMX, 5 mM) and so the negative control over cAMP levels appears to be mediated by receptors that are selectively activated by pyrimidine nucleotides and permit control over phosphodiesterase activity.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Evaluation of reverse transcription-PCR and a bacteriophage-based assay for rapid phenotypic detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis.

New rapid phenotypic assays for the detection of rifampin resistance in Mycobacterium tuberculosis have recently been described, but most of these require liquid cultures, which reduces the utility of many tests in terms of turnaround times. In the United Kingdom, over 90% of rifampin-resistant isolates are also resistant to isoniazid, so rifampin resistance can be used as a sensitive marker for multidrug-resistant tuberculosis. In this study, two new rapid phenotypic assays were compared to the standard resistance ratio method on 91 clinical isolates of M. tuberculosis. One, the phage amplified biologically (PhaB) assay, has been described previously and is based on the inability of susceptible isolates of M. tuberculosis to support the replication of bacteriophage D29 in the presence of inhibitory doses of rifampin. The other employed reverse transcription (RT)-PCR to demonstrate a reduction in inducible dnaK mRNA levels in susceptible isolates treated with rifampin. After incubation for 18 h with 4 microg of rifampin per ml, the PhaB assay showed concordance with the resistance ratio method for 46 of 46 (100%) susceptible and 31 of 31 (100%) resistant isolates, while RT-PCR showed concordance for 46 of 48 (96%) susceptible and 35 of 36 (97%) resistant isolates. We believe these assays provide a reliable rapid means of susceptibility testing with a total turnaround time of only 48 h, although the PhaB assay is better in terms of its lower technical demand and cost and its applicability to tuberculosis susceptibility testing in developing countries.

Antibiotics, Antitubercular↗
PubMed1999

Evaluation of a bacteriophage-based assay (phage amplified biologically assay) as a rapid screen for resistance to isoniazid, ethambutol, streptomycin, pyrazinamide, and ciprofloxacin among clinical isolates of Mycobacterium tuberculosis.

Rapid molecular assays for the detection of mutations associated with rifampin resistance in Mycobacterium tuberculosis are commercially available. However, they are complex and expensive and have predictive values of 90 to 95%. Molecular assays for other drugs are less predictive of resistance. Ideally, assays based on phenotypic markers should be used for susceptibility testing, but these can take weeks to complete. We previously described a rapid phenotypic assay, the phage amplified biologically (PhaB) assay, for the rapid determination of rifampin and isoniazid susceptibility in clinical isolates of M. tuberculosis. In this study, we extended the assay to the study of ethambutol, pyrazinamide, streptomycin, and ciprofloxacin. After the optimization of antibiotic concentrations and incubation conditions, the assay was applied to each drug for a total of 157 isolates. The correlations between the results of the PhaB assay and the resistance ratio method were 94% for isoniazid, 96% for streptomycin, 100% for ciprofloxacin, 88% for ethambutol, and 87% for pyrazinamide. For ciprofloxacin, ethambutol, and pyrazinamide, significantly better correlations were found when a 90% reduction in plaque count was used as the cutoff. Turnaround times for the PhaB assay were 2 to 3 days, compared with 10 days for the resistance ratio method. We believe that this low-cost assay may have widespread applicability for the rapid screening of drug resistance in M. tuberculosis isolates, especially in developing countries.

Antitubercular Agents↗
PubMed1999

Specificity of IS6110-based DNA fingerprinting and diagnostic techniques for Mycobacterium tuberculosis complex.

Restriction fragment length polymorphism and hybridization of DNA extracted from Mycobacterium tuberculosis, nontuberculous mycobacteria, and nonmycobacterial species with a probe derived from IS6110 confirmed that IS6110 was specific to M. tuberculosis complex. In addition, DNA amplification with IS6110-specific primers yielded a 181-bp fragment only in DNA from M. tuberculosis complex isolates.

Bacteriological Techniques↗
PubMed1999

Intranet technology in Scottish NHS Trusts.

Intranet technology has the potential to turn everything we've taken for granted in internal communication upside down. This paper looks at the uptake and use of Intranet technology in NHS Trusts in Scotland and discusses some of the uses of an Intranet system.

Attitude to Computers↗
PubMed1999

Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis.

High resolution intravital videomicroscopy has provided a powerful tool for directly observing steps in the metastatic process, and for clarifying molecular mechanisms of metastasis and modes of action of anti-metastasis therapeutics. Cells previously have been identified in vivo using exogenously added fluorescent labels, limiting observations to a few cell divisions, or by natural markers (e.g. melanin) expressed only by specific cell types. Here we tested the utility of stable green fluorescent protein (GFP)-transfected cells for monitoring and quantifying sequential steps in the metastatic process. Using CHO-K1 cells that stably express GFP, we document the visualization and quantification by intravital videomicroscopy of sequential steps in metastasis within mouse liver, from initial arrest of cells in the microvasculature to the growth and angiogenesis of metastases. Individual, non-dividing cells, as well as micro- and macrometastases could clearly be detected and quantified, as could fine cellular details such as pseudopodial projections, even after extended periods of in vivo growth. We quantified the size distribution of micrometastases and their locations relative to the liver surface using 50 micrometer thick formalin-fixed tissue sections. The data suggest preferential growth and survival of micrometastases near the liver surface. Furthermore, we observed a small population of single cells that persisted over the 11 day observation period, which may represent dormant cells with potential for subsequent proliferation. This study demonstrates the advantages of GFP-expressing cells, coupled with real-time high resolution videomicroscopy, for long-term in vivo studies to visualize and quantify sequential steps of the metastatic process.

Animals↗
PubMed1999

African American mothers' responses to hospitalization of an infant with serious health problems.

PURPOSE: To describe African American mothers' experiences related to the hospitalization of an infant with serious health problems. DESIGN: Retrospective descriptive. SAMPLE: 19 African American mothers with premature and term infants who were hospitalized at birth for serious health problems related to sequelae of prematurity or birth defect. MAIN OUTCOME VARIABLE: African American mothers' recollections about the hospitalization of their seriously ill infant. RESULTS: The mothers worried primarily about when the baby could go home. Their greatest source of stress was separation from the infant. Seeing their sick infant was also stressful and evoked shock, fear, denial, guilt, and helplessness. Mothers sought hope by seeking information and cues from the infant and by praying to God. Mothers established a relationship with their infant by visiting regularly and by learning how to care for him. Some mothers feared getting attached to an infant who might die. Mothers' highest source of satisfaction was support from the health care team.

Adaptation, Psychological↗
PubMed1999

Drug extrusion, 125I- efflux and the control of intracellular [Ca2+] in drug-resistant ovarian epithelial cells.

Experiments were undertaken using an ovarian adenocarcinoma cell line (A2780) and a drug-resistant strain (A2780.ad) derived from this line. P-glycoprotein could not be detected in A2780 cells but was essentially ubiquitous in A2780.ad cells, although removing the selective pressure for drug resistance led to reduced expression. However, the amount of P-glycoprotein present was used to predict the capacity of these cells to extrude rhodamine-123 (R-123) and their resistance to adriamycin, a cytotoxic drug. This accords with the role of P-glycoprotein as a drug pump. Although hypotonic solutions increased anion efflux from A2780 and A2780.ad cells, larger responses occurred in the parental line. Moreover, R-123 extrusion and anion efflux appeared to be mutually independent processes and so these data do not support the view that P-glycoprotein is involved in the control of volume-sensitive anion channels. Hypotonic solutions increased intracellular free calcium ([Ca2+]i) in drug-resistant cells but not in the parental line, and so establishing a drug-resistant strain may affect the control of [Ca2+]i during osmotic swelling. This could account for effects that were previously attributed to P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Expression of genes that contribute to proliferative and metastatic ability in breast cancer resected during various menstrual phases.

BACKGROUND: Retrospective studies show significant improvements in survival among women who had breast cancer resected during the luteal phase of their menstrual cycle compared with the follicular phase. We hypothesised that tumour tissue would show cyclical changes in expression of genes whose products might contribute to metastatic potential. METHODS: We studied 32 premenopausal women with operable breast cancer. We assayed hormones to define more accurately the menstrual phase during which surgery was done. We used northern blot analysis of RNA from fresh-frozen tumour specimens to study the patterns of expression of genes for proteolytic enzymes (cysteine proteinase cathepsin L and aspartyl proteinase cathepsin D; matrix metalloproteinases MMP-9 and MMP-2), tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2, and TP53. RESULTS: There was a significantly higher level of expression of RNA for cathepsin L, MMP-9, and TP53 (p=0.005, 0.03, 0.03, respectively) in tumours that were resected during the follicular and periovulatory phases of the menstrual cycle than at other times in the cycle. A similar but non-significant trend was seen for MMP-2 and cathepsin D. A non-significant trend in the opposite direction was seen for TIMP-1 and TIMP-2. INTERPRETATION: We found that tumour expression of genes that may contribute to proliferative capacity and metastatic potential can change in breast cancer during the course of the menstrual cycle. The finding could provide a molecular explanation for the reports of improved survival in some breast-cancer patients whose tumours were removed during the luteal phase of the menstrual cycle. Larger studies are required to extend our study, assess mechanisms of gene regulation, and verify any relevant influence in long-term survival.

Adult↗
PubMed1998

Nucleotide-evoked calcium signals and anion secretion in equine cultured epithelia that express apical P2Y2 receptors and pyrimidine nucleotide receptors.

1. Experiments with a spontaneously transformed equine epithelial cell line showed that certain nucleotides increased intracellular free calcium ([Ca2+]i) in cells plated on glass coverslips. The rank order of potency was ATP UTP > 5-Br-UTP, whilst UDP and ADP were ineffective. The response thus appears to be mediated by P2Y2 receptors. 2. Nucleotides also increased short circuit current (Isc) in cells grown into epithelial monolayers and the rank order of potency was UDP> UTP > 5-Br-UTP > ATP > ADP. The increase in [Ca2+]i and the rise in ISC thus have different pharmacological properties. Cross-desensitization experiments indicated that, as well as P2Y2 receptors, the monolayer cultures express at least one additional receptor population that allowed nucleotides to increase ISC. 3. The UDP-evoked increase in ISC was essentially abolished in BAPTA-loaded epithelia suggesting that this response is dependent upon increased [Ca2+]i. Moreover, experiments in which ISC and [Ca2+]i were measured simultaneously showed that the UDP- and ADP-evoked increases in ISC were accompanied by increases in [Ca2+]i. 4. When grown under conditions which favour the development of a polarized phenotype, these epithelial cells thus appear to express [Ca2+]i-mobilizing receptors sensitive to UDP and ADP that are not present in non-polarized cells on coverslips.

Adenosine Diphosphate↗
PubMed1998

Culture substrate-specific expression of P2Y2 receptors in distal lung epithelial cells isolated from foetal rats.

ATP and UTP did not evoke [Ca2+]i signals in rat foetal lung epithelial cells grown on glass but elicited clear responses in cells grown into functionally polarised epithelia on permeable supports. Moreover, P2Y2 receptor mRNA could not be detected in cells on glass by the polymerase chain reaction but this mRNA species was clearly expressed by polarised cells. P2Y2 receptor expression thus appears to be a feature of the polarised phenotype.

Adenosine Triphosphate↗