Biomedical subjects

M A Gray

Publications and source records attributed to M A Gray.

At least 37 records · Page 2Linked to original sources

PubMed2000

The qualities of an effective mentor from the student nurse's perspective: findings from a longitudinal qualitative study.

Parker and Carlisle (Journal of Advanced Nursing 24, 771-778) argue that there is a scarcity of empirical research focusing on issues such as supernumerary status and mentorship in Project 2000 courses from the students' perspective. This paper presents the findings of a longitudinal cohort study using Grounded Theory to discover the effect(s) of mentorship on student nurses following the introduction of the 1992 programme of education leading to a Diploma of Higher Education in Nursing and registration with the United Kingdom Central Council (UKCC). The cohort consisted of 10 students from a large Scottish College of Nursing & Midwifery who were interviewed on five occasions during the three years of their course. Students also kept a diary to record their thoughts and experiences regarding mentorship during their practice placements. In addition, a further seven students volunteered to participate by diary only. Data were analysed with the aid of NUD.IST and subjected to the constant comparative method of analysis. Findings indicate that Diploma students quickly lose their idealistic view of their mentor and over time develop an insight into the qualities they perceive are required of an effective mentor. Students quickly become aware of the importance of choosing good role models and learning their own mentor's likes and dislikes as they realize this impinges on the outcome of their assessment. As students move into their Branch programme, a gradual distancing from their mentor is evident. This coincides with a development in their confidence, skills and a holistic perspective of care.

Education, Nursing↗

PubMed2000

Characterization of a recombinant outer surface protein A (OspA) vaccine against Lyme disease.

Lyme disease, the most prevalent tick-borne disease in the United States, results from infection with Borrelia burgdorferi sensu stricto. Early studies of Borrelia burgdorferi sensu stricto identified outer surface protein A (OspA), a lipoprotein on the surface of spirochetes that could be the target of protective antibodies to this agent. Pasteur Mérieux Connaught has developed a Lyme vaccine, ImuLyme, using recombinant OspA protein (rOspA). Methods were developed to routinely assess the identity, quantity, structure, purity, biological activity, heterogeneity, stability, and potency of rOspA. In addition, several methods were performed on a series of lots to support the routine testing methods and further our understanding of the physicochemical characteristics of rOspA. These tests were electrospray mass spectrometry, circular dichroism, two-dimensional gel electrophoresis, amino acid analysis, peptide mapping with peptide sequencing, and the application of proteomic methodology to identify trace contaminant host cell proteins. The results of these methods indicate that the rOspA lots are composed of highly purified and properly processed and folded rOspA with trace amounts of E. coli host cell proteins.

Amino Acid Sequence↗

PubMed1999

Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel.

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.

Animals↗

PubMed1998

Calcium-activated chloride conductance in a pancreatic adenocarcinoma cell line of ductal origin (HPAF) and in freshly isolated human pancreatic duct cells.

Using the whole-cell patch-clamp technique, a calcium-activated chloride conductance (CACC) could be elicited in HPAF cells by addition of 1 microM ionomycin to the bath solution (66 +/- 22 pA/pF;Vm + 60 mV) or by addition of 1 microM calcium to the pipette solution (136 +/- 17 pA/pF; Vm + 60 mV). Both conductances had similar biophysical characteristics, including time-dependent inactivation at hyperpolarising potentials and a linear/slightly outwardly rectifying current/voltage (I/V) curve with a reversal potential (Erev) close to the calculated chloride equilibrium potential. The anion permeability sequence obtained from shifts in Erev was I > Br >/= Cl. 4,4'-Diisothiocyanatostilbene disulphonic acid (DIDS, 500 microM) caused a 13% inhibition of the current (Vm + 60 mV) while 100 microM glibenclamide, 30 nM TS-TM-calix[4]arene and 10 microM tamoxifen, all chloride channel blockers, had no marked effects (8%, -6% and -2% inhibition respectively). Niflumic acid (100 microM) caused a voltage-dependent inhibition of the current of 48% and 17% (Vm +/- 60 mV, respectively). In freshly isolated human pancreatic duct cells (PDCs) a CACC was elicited with 1 microM calcium in the pipette solution (260 +/- 62 pA/pF; Vm + 60 mV). The presence of this CACC in human PDCs could provide a possible therapeutic pathway for treatment of pancreatic insufficiency of the human pancreas in cystic fibrosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

A novel type of internal barium block of a maxi-K+ channel from human vas deferens epithelial cells.

We have recently shown that a maxi-K+ channel from vas deferens epithelial cells contains two Ba2+-binding sites accessible from the external side: a "flickering" site located deep in the channel pore and a "slow" site located close to the extracellular mouth of the channel. Using the patch-clamp technique, we have now studied the effect of internal Ba2+ on this channel. Cytoplasmic Ba2+ produced a voltage- and concentration-dependent "slow" type of block with a dissociation constant of approximately 100 microM. However, based on its voltage dependence and sensitivity to K+ concentration, this block was clearly different from the external "slow" Ba2+ block previously described. Kinetic analysis also revealed a novel "fast flickering" block restricted to channel bursts, with an unblocking rate of approximately 310 s(-1), some 10-fold faster than the external "flickering" block. Taken together, these results show that this channel contains multiple Ba2+-binding sites within the conduction pore. We have incorporated this information into a new model of Ba2+ block, a novel feature of which is that internal "slow" block results from the binding of at least two Ba2+ ions. Our results suggest that current models for Ba2+ block of maxi-K+ channels need to be revised.

Barium↗

PubMed1998

Basal chloride currents in murine airway epithelial cells: modulation by CFTR.

We have isolated ciliated respiratory cells from the nasal epithelium of wild-type and cystic fibrosis (CF) null mice and used the patch-clamp technique to investigate their basal conductances. Current-clamp experiments on unstimulated cells indicated the presence of K+ and Cl- conductances and, under certain conditions, a small Na+ conductance. Voltage-clamp experiments revealed three distinct Cl- conductances. Itv-indep was time and voltage independent with a linear current-voltage (I-V) plot; Iv-act exhibited activation at potentials greater than +/- 50 mV, giving an S-shaped I-V plot; and Ihyp-act was activated by hyperpolarizing potentials and had an inwardly rectified I-V plot. The current density sequence was Ihyp-act = Iv-act >> Itv-indep. These conductances had Cl(-)-to-N-methyl-D-glucamine cation permeability ratios of between 2.8 and 10.3 and were unaffected by tamoxifen, flufenamate, glibenclamide, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited by Zn2+ and Gd3+. Itv-indep and Iv-act were present in wild-type and CF cells at equal density and frequency. However, Ihyp-act was detected in only 3% of CF cells compared with 26% of wild-type cells, suggesting that this conductance may be modulated by cystic fibrosis transmembrane conductance regulator (CFTR).

Animals↗

PubMed1997

Cross-resistance associated with development of resistance to isometamidium in a clone of Trypanosoma congolense.

Resistance to isometamidium was increased 94-fold in a clone of Trypanosoma congolense (clone IL 1180) by repeated subcurative treatment of infected mice for 11 months. This was associated with 3.4-, 33-, and 4.2-fold increases in resistance to diminazene, homidium, and quinapyramine, respectively. Both T. congolense IL 1180 and the resistant derivative were able to undergo cyclical development in Glossina morsitans centralis tsetse flies, producing hypopharyngeal infection rates of 40.0 and 39.8%, respectively.

Animals↗

PubMed1996

Characterization of whole cell chloride conductances in a mouse inner medullary collecting duct cell line mIMCD-3.

The chloride conductance of inner medullary collecting duct cells (mIMCD-3 cell line) has been investigated using the whole cell configuration of the patch clamp technique. Seventy-seven percent of cells were chloride selective when measured with a NaCl-rich bathing solution and a TEACl-rich pipette solution. Seventy-five percent of chloride-selective cells (90/144) had whole cell currents which exhibited an outwardly-rectifying (OR) current-voltage (I/V) relationship, while the remaining cells exhibited a linear (L) I/V relationship. The properties of the OR and L chloride currents were distinct. OR currents (mean current densities at +/- 60 mV of 66 +/- 5 pA/pF and 44 +/- 3 pA/pF), were time- and voltage-independent with an anion selectivity (from calculated permeability ratios) of SCN- (2.3), NO3- (1.8), ClO4- (1.7), Br- (1.7), I- (1.6), Cl- (1.0), HCO3- (0.5), gluconate- (0.2). Bath additions of NPPB, flufenamate, glibenclamide (all 100 microM) and DIDS (500 microM) produced varying degrees of block of OR currents with NPPB being the most potent (IC50 of approximately 50 microM) while DIDS was the least effective. Linear chloride currents had similar current densities to the OR chloride currents and were also time- and voltage-independent. The anion selectivity sequence was SCN- (2.5), NO3- (1.9), Br- (1.4), I- (1.1), Cl- (1.0), ClO4- (0.5), HCO3- (0.5), gluconate- (0.3). In contrast to the OR conductance, glibenclamide was the most potent and DIDS the least potent blocker of L currents. An IC50 of > 100 microM was observed for NPPB block. Neither OR of L chloride currents were affected by acutely or chronically increased intracellular cAMP and were not affected when intracellular Ca2+ levels were increased or decreased. The molecular identity and physiological role of OR and linear currents in mIMCD-3 cells are discussed.

Animals↗

PubMed1996

A mathematical model of the pancreatic ductal epithelium.

A mathematical model of the HCO-3-secreting pancreatic ductal epithelium was developed using network thermodynamics. With a minimal set of assumptions, the model accurately reproduced the experimentally measured membrane potentials, voltage divider ratio, transepithelial resistance and short-circuit current of nonstimulated ducts that were microperfused and bathed with a CO2/HCO-3-free, HEPES-buffered solution, and also the intracellular pH of duct cells bathed in a CO2/HCO-3-buffered solution. The model also accurately simulated: (i) the effect of step changes in basolateral K+ concentration, and the effect of K+ channel blockers on basolateral membrane potential; (ii) the intracellular acidification caused by a Na+-free extracellular solution and the effect of amiloride on this acidification; and (iii) the intracellular alkalinization caused by a Cl--free extracellular solution and the effect of DIDS on this alkalinization. In addition, the model predicted that the luminal Cl- conductance plays a key role in controlling both the HCO-3 secretory rate and intracellular pH during HCO-3 secretion. We believe that the model will be helpful in the analysis of experimental data and improve our understanding of HCO-3-transporting mechanisms in pancreatic duct cells.

Animals↗

PubMed1996

Volume-sensitive chloride currents in primary cultures of human fetal vas deferens epithelial cells.

Using the patch-clamp technique, we have identified a large, outwardly rectifying, Cl--selective whole-cell current in primary cultures of human vas deferens epithelial cells. Whole-cell currents were time- and voltage-dependent and displayed inactivation following depolarising pulses >/= 60 mV. Currents were equally permeable to bromide (PBr/PCl = 1.05 +/- 0.04), iodide (PI/PCl = 1. 06 +/- 0.07) and Cl-, but significantly less permeable to gluconate (PGluc /PCl = 0.23 +/- 0.03). Currents spontaneously increased with time after establishing a whole-cell recording, but could be inhibited by exposure to a hypertonic bath solution which reduced inward currents by 68 +/- 4%. Subsequent exposure of the cells to a hypotonic bath solution led to a 418 +/- 110% increase in inward current, indicating that these currents are regulated by osmolarity. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic acid (100 microM) produced a rapid and reversible voltage-dependent block (60 +/- 5% and 10 +/- 7% inhibition of current, measured at +/- 60 mV, respectively). Dideoxyforskolin (50 microM) also reduced the volume-sensitive Cl- current, but with a much slower time course, by 41 +/- 13% and 32 +/- 16% (measured at +/- 60 mV, respectively). Tamoxifen (10 microM) had no effect on the whole-cell Cl- current. These results suggest that vas deferens epithelial cells possess a volume-sensitive Cl- conductance which has biophysical and pharmacological properties broadly similar to volume-sensitive Cl- currents previously described in a variety of cell types.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1996

PspA, a protection-eliciting pneumococcal protein: immunogenicity of isolated native PspA in mice.

PspA is a surface exposed virulence factor of S. pneumoniae that can elicit protective immunity to pneumococcal sepsis in mice. It can be released from pneumococci by washing them with a solution containing 2% choline chloride, by growing pneumococci in media containing 1.2% choline chloride, or by growing pneumococci in media in which the choline has been replaced by ethanolamine. Our results indicate that PspA is the major protection-eliciting antigen in each of these preparations. Two injections of < or = 1 microgram of native PspA purified by use of a choline-Sepharose column are highly immunogenic in BALB/c and CBA/N mice, and even in the absence of adjuvant can elicit protection against otherwise fatal sepsis with 100 times the LD50 of S. pneumoniae. Fragments comprising the N-terminal 115 and 245 amino acids of PspA were able to elicit protection but only in the presence of complete Freund's adjuvant (CFA). In the absence of CFA the 245 amino acid fragment was less than 1/100 as immunogenic as native PspA.

Animals↗

PubMed1996

Two barium binding sites on a maxi K+ channel from human vas deferens epithelial cells.

Using the patch clamp technique, we have investigated the blockade of maxi-K+ channels present on vas deferens epithelial cells by extracellular Ba2+. With symmetrical 140 mM K+ solutions, Ba2+ produced discrete blocking events consisting of both long closings of seconds duration (slow block) and fast closings of milliseconds duration (flickering block). Kinetic analysis showed that flickering block occurred according to an "open channel blocking" scheme and was eliminated by reducing external K+ to 4.5 mM. Slow block showed a complex voltage-dependence. At potentials between -20 mV and 20 mV, blockade was voltage-dependent; at potentials greater than 20 mV, blockade was voltage-independent, but markedly sensitive to the extracellular K+ concentration. These data reveal that the vas deferens maxi-K+ channel has two Ba2+ binding sites accessible from the extracellular side. Site one is located at the cytoplasmic side of the gating region and binding to this site causes flickering block. Site two is located close to the extracellular mouth of the channel and binding to this site causes slow block.

Barium↗

PubMed1995

cAMP-activated chloride channels in a CFTR-transfected pancreatic adenocarcinoma-derived cell line, pANS6.

Pancreatic adenocarcinoma cell lines rarely express the CFTR gene, despite the high levels of CFTR protein that are present in primary pancreatic duct cells. We have attempted to generate a non-CF pancreatic adenocarcinoma cell line that stably produces high levels of CFTR mRNA and protein by transfecting a vector containing the CFTR cDNA, driven by a strong mammalian promoter, into the poorly differentiated pancreatic adenocarcinoma cell line, Panc-1. The pANS6 pancreatic duct cell line expresses substantial levels of CFTR mRNA, but little CFTR protein. Despite this we were able to detect low conductance chloride channels in 40% of patches, stimulated with cAMP, that have similar biophysical properties to CFTR.

Adenocarcinoma↗

PubMed1995

Volume-activated chloride currents in pancreatic duct cells.

We have used the patch clamp technique to study volume-activated Cl- currents in the bicarbonate-secreting pancreatic duct cell. These currents could be elicited by a hypertonic pipette solution (osmotic gradient 20 mOsm/l), developed over about 8 min to a peak value of 91 +/- 5.8 pA/pF at 60 mV (n = 123), and were inhibited by a hypertonic bath solution. The proportion of cells which developed currents increased from 15% in freshly isolated ducts to 93% if the ducts were cultured for 2 days. The currents were ATP-dependent, had an outwardly rectifying current/voltage (I-V) plot, and displayed time-dependent inactivation at depolarizing potentials. The anion selectivity sequence was: ClO4 = I = SCN > Br = NO3 > Cl > F > HCO3 > gluconate, and the currents were inhibited to a variable extent by DIDS, NPPB, dideoxyforskolin, tamoxifen, verapamil and quinine. Increasing the intracellular Ca2+ buffering capacity, or lowering the extracellular Ca2+ concentration, reduced the proportion of duct cells which developed currents. However, removal of extracellular Ca2+ once the currents had developed was without effect. Inhibiting protein kinase C (PKC) with either the pseudosubstrate PKC (19-36), calphostin C or staurosporine completely blocked development of the currents. We speculate that cell swelling causes Ca2+ influx which activates PKC which in turn either phosphorylates the Cl- channel or a regulatory protein leading to channel activation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Calcium-activated chloride conductance is not increased in pancreatic duct cells of CF mice.

Calcium-activated anion secretion is elevated in the pancreatic ductal epithelium of transgenic cf/cf mice which lack the cystic fibrosis transmembrane conductance regulator (CFTR). To elucidate whether this effect is due to increased activity of calcium-activated chloride channels, we have studied the relationship between CFTR and calcium-activated chloride currents in pancreatic duct cells isolated from Cambridge cf/cf mice. CFTR chloride currents activated by cAMP were detected in 59% (29/49) of wild-type cells and in 50% (20/40) of heterozygous cells. However, we could not detect any CFTR currents in the homozygous cf/cf cells (0/25). The maximum CFTR current density measured at a membrane potential of 60 mV was 23.5 +/- 2.8 pA/pF (n = 29) in wild-type cells, and about half that value, i.e. 12.4 +/- 1.6 pA/pF (n = 20) in heterozygotes (P = 0.004). Calcium-activated chloride currents were detected in 73% (24/33) of wild-type, 75% (21/28) of heterozygous and in 58% (7/12) of homozygous cf/cf cells. There was no significant difference between the steady-state calcium-activated current densities in the three genotypic groups; the current measured at 60 mV being 527 +/- 162 pA/pF (n = 24) from wild-type, 316 +/- 35 pA/pF (n = 21) from heterozygote and 419 +/- 83 pA/pF (n = 7) from homozygous cells. Our data suggest that lack of CFTR does not enhance the calcium-activated chloride conductance in murine pancreatic duct cells.

Animals↗

PubMed1995

Chloride channels and cystic fibrosis of the pancreas.

Cystic fibrosis (CF) affects approximately 1 in 2000 people making it one of the commonest fatal, inherited diseases in the Caucasian population. CF is caused by mutations in a cyclic AMP-regulated chloride channel known as CFTR, which is found on the apical plasma membrane of many exocrine epithelial cells. In the CF pancreas, dysfunction of the CFTR reduces the secretory activity of the tubular duct cells, which leads to blockage of the ductal system and eventual fibrosis of the whole gland. One possible approach to treating the disease would be to activate an alternative chloride channel capable of bypassing defective CFTR. A strong candidate for this is a chloride channel regulated by intracellular calcium, which has recently been shown to protect the pancreas in transgenic CF mice. Pharmacological intervention directed at activating this calcium-activated Cl- conductance might provide a possible therapy to treat the problems of pancreatic dysfunction in CF.

Animals↗

PubMed1995

Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells.

Activation of protein kinase C (PKC) inhibits adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated fluid secretion in rat pancreatic ducts (N. Ashton, R. L. Evans, and B. E. Argent. J. Physiol. Lond. 452: 99P, 1992). Using the patch-clamp technique, we have investigated whether this inhibition of fluid secretion results from an effect of PKC on cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels. Exposure to 100 nM 4 beta-phorbol 12,13-dibutyrate (PDBu) had no effect on CFTR current density in unstimulated duct cells, but caused a 31% increase in the magnitude of CFTR currents recorded from cells stimulated with cAMP. Furthermore, prolonged (2-4 h) exposure of stimulated duct cells to 100 nM PDBu (a condition that should downregulate PKC) significantly slowed the rate at which CFTR currents run down after establishing a whole cell recording. A similar effect was observed with calphostin C (500 nM), a specific inhibitor of PKC. Thus, although inhibition of ductal fluid secretion by PDBu is unlikely to be explained by an effect on CFTR, modulation of PKC activity can affect both the magnitude and stability of CFTR currents in pancreatic duct cells.

Animals↗

PubMed1994

Maxi K+ channels on human vas deferens epithelial cells.

The vas deferens forms part of the male reproductive tract and extends from the cauda epididymis to the prostate. Using the patch clamp technique, we have identified a Ca(2+)-activated, voltage-dependent, maxi K+ channel on the apical membrane of epithelial cells cultured from human fetal vas deferens. The channel had a conductance of approximately 250 pS in symmetrical 140 mM K+ solutions, and was highly selective for K+ over Na+. Channel activity was increased by depolarization and by an elevation of bath (cytoplasmic) Ca2+ concentration, and reduced by cytoplasmic Ba2+ (5 mM) but not by cytoplasmic TEA (10 mM). Channel activity was also dependent on the cation bathing the cytoplasmic face of the membrane, being higher in a Na(+)-rich compared to a K(+)-rich solution. We estimated that up to 600 maxi K+ channels were present on the apical membrane of a vas cell, and that their density was 1-2 per mu 2 of membrane. Activity of the channel was low on intact cells, suggesting that it does not contribute to a resting K+ conductance. However, fluid in the lumen of the human vas deferens has a high K+ concentration and we speculate that the maxi K+ channel could play a role in transepithelial K+ secretion.

Calcium↗