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M A Gray

Publications and source records attributed to M A Gray.

At least 55 records · Page 3Linked to original sources
PubMed1994

CFTR and calcium-activated chloride currents in pancreatic duct cells of a transgenic CF mouse.

We have studied the cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride currents in pancreatic duct cells isolated from a transgenic cf/cf mouse created by targeted insertional mutagenesis. Adenosine 3',5'-cyclic monophosphate (cAMP)-activated CFTR chloride currents were detected in 78% (29/37) of wild-type cells, in 81% (35/43) of heterozygote cells, and in 61% (29/47) of homozygous cf/cf duct cells (P > 0.05, cf/cf vs. wild-type and heterozygote). The CFTR current density measured at membrane potentials of +/- 60 mV averaged 22-26 pA/pF in wild-type and heterozygote groups but only 13 pA/pF in cells derived from cf/cf animals (P < 0.05, cf/cf vs. wild-type and cf/cf vs. heterozygotes). In contrast, duct cells from animals of all three genotypic groups exhibited calcium-activated chloride currents that were of similar magnitude and up to 11-fold larger than the CFTR currents. We speculate that these transgenic insertional null mice do not develop the pancreatic pathology that occurs in cystic fibrosis patients because their duct cells contain 1) some wild-type CFTR generated by exon skipping and aberrant splicing and 2) a separate anion secretory pathway mediated by calcium-activated chloride channels.

Animals↗
PubMed1993

Drug sensitivity screening in vitro of populations of Trypanosoma congolense originating from cattle and tsetse flies at Nguruman, Kenya.

An in vitro assay that utilises in vitro-derived metacyclic trypanosomes was used to determine the drug sensitivity of 7 populations of Trypanosoma congolense collected from cattle and tsetse flies at Nguruman; a trypanosomiasis-endemic area in southwest Kenya. The metacyclic trypanosomes used in the assay were obtained from cultures initiated directly from either the blood of cattle with low levels of parasitaemia or from guts of infected tsetse flies. Sensitivities to isometamidium chloride, diminazene aceturate and homidium salts were assessed at various drug concentrations (0.5 ng-50 micrograms/ml). The results were compared with those obtained with two characterised laboratory populations. In spite of the fact that isometamidium chloride had not been widely used at Nguruman, two of the stocks (KE 3302 and KE 3303) expressed high levels of resistance to this drug (resistant to 100 ng/ml and 1000 ng/ml, respectively). In contrast, all of the populations examined were as sensitive, or more so, to diminazene aceturate than the sensitive laboratory clone IL 1180; two stocks (KE 3305 and KE 3306) were more sensitive (resistant to 0.5 microgram/ml, sensitive to 1 microgram/ml) and three stocks expressed the same level of sensitivity as IL 1180 (resistant to 1 microgram/ml, sensitive to 5 micrograms/ml). Since the results of the in vitro assay correlated well with field observations it was concluded that the assay would be a useful tool in epidemiological studies to determine the resistance phenotypes of trypanosome populations in the field, thereby enabling development of appropriate control measures for particular areas.

Animals↗
PubMed1993

An in vitro assay for drug sensitivity of Trypanosoma congolense using in vitro-derived metacyclic trypanosomes.

The sensitivity of seven populations of T. congolense to the salts of three trypanocides, diminazene, isometamidium and homidium, were determined in vitro using in vitro-derived metacyclic trypanosomes. The trypanosomes were incubated at 35 degrees C for 48 h with various drug concentrations (0.5 ng-50 micrograms/ml) and then transferred to cultures containing bovine endothelial-cell monolayers, to assess their viability over the following 5 days as compared to control trypanosomes that had been incubated without drug. The sensitivity to each drug was expressed as the minimum effective drug concentration which killed 100% of the trypanosomes in a given population within the 5 days. Using this assay, population IL 1180, characterised as being highly sensitive to all three drugs in vivo, required 10 ng/ml isometamidium chloride, 50 ng/ml homidium bromide or chloride and 5000 ng/ml diminazene aceturate to kill the entire population in vitro. In contrast, two derivatives of IL 1180 in which resistance to isometamidium had been induced in mice, IL 3343 and IL 3344, required isometamidium chloride at a concentration of 1000 ng/ml and 2000 ng/ml, respectively, to eliminate the populations. The in vitro results showed that the increase in level of resistance to isometamidium in these populations was associated with at least a 200-fold increase in resistance in both populations to homidium, but no increase in resistance to diminazene. KE 2887 and CP 81, two isolates expressing high levels of resistance to both isometamidium and homidium in mice and cattle, were both resistant in vitro to isometamidium chloride and homidium salts at 100 ng/ml. Furthermore, while the former population was resistant to 10,000 ng/ml diminazene aceturate, the latter was sensitive to 5000 ng/ml. IL 3274 and IL 3330, characterised as expressing high levels of resistance to all three drugs in vivo, were shown to be resistant to isometamidium chloride and homidium salts at 1000 ng/ml, and to diminazene aceturate at 10,000 ng/ml. Finally, the in vitro IC100 (concentration of drug required to eliminate 100% of the population) results were consistent with the maximum amounts of each drug detected in vivo.

Animals↗
PubMed1993

cAMP-regulated whole cell chloride currents in pancreatic duct cells.

Using the whole cell configuration of the patch-clamp technique, we have identified an adenosine 3',5'-cyclic monophosphate (cAMP)-regulated chloride conductance in pancreatic duct cells. Basal whole cell currents in single isolated cells were very low (approximately 5 pA/pF) but could be stimulated 17-fold by elevation of intracellular cAMP. The cAMP-activated currents exhibited 1) a high chloride selectivity, 2) a near linear current-voltage relationship, 3) time and voltage independence, 4) block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 5) an anion selectivity sequence based on permeability ratios of SCN > NO3 > Br > Cl > I > HCO3 > F > ClO4 > gluconate. Currents in single cells ran down within a few minutes; however, stable chloride currents could be recorded from duct cell clusters in which four or five cells were in electrical communication. We present evidence suggesting that these cAMP-regulated currents are carried by cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Physiologically, these CFTR channels act in parallel with chloride-bicarbonate exchangers to facilitate bicarbonate secretion across the apical plasma membrane of the duct cell.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Response of Trypanosoma congolense in goats to single and double treatment with diminazene aceturate.

Diminazene aceturate is one of a limited number of compounds currently marketed for treatment of trypanosomiasis in cattle, sheep and goats. The pharmacokinetics of the compound in goats suggest that double treatment with diminazene aceturate might enhance the compound's therapeutic activity. A study was therefore conducted in goats using two clones of Trypanosoma congolense, IL 3274 and IL 1180, which were previously shown to be resistant and sensitive, respectively, to single treatment with diminazene aceturate. The results indicated that, as compared to single treatment, double treatment with diminazene aceturate at a dose of 7.2 mg kg-1 bodyweight, at either eight or 24 hour intervals, did not greatly enhance the therapeutic activity of the drug. Furthermore, treatment with the same drug dose eliminated infections with T congolense IL 3274 when treatment was administered 24 hours after infected Glossina morsitans centralis had fed, but failed to do so if treatment was delayed until after goats were detected to be parasitaemic. This suggests that failure of T congolense IL 3274 to respond to treatment with diminazene may not be due to drug resistance per se.

Animals↗
PubMed1991

Axenic in vitro cultivation of Trypanosoma vivax trypomastigote forms.

Cultures of Trypanosoma vivax stocks were initiated with tsetse-derived metacyclics and with organisms isolated from mouse blood. They were propagated as trypomastigote forms at 35 degrees C initially in the presence of bovine endothelial cells. All stocks were subsequently adapted to axenic culture conditions. The standard medium consisted of HEPES-buffered MEM with 1% MEM non-essential amino acids, containing 2 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, 100 micrograms/ml streptomycin, 0.2 mM hypoxanthine, 0.01 mM bathocuproine sulphonate, 0.1 mM L-cysteine, 0.1 mM 2-mercaptoethanol and 20% caprine or bovine serum. Cultured trypanosomes were morphologically similar to bloodstream forms found in vivo and they retained infectivity after in vitro cultivation.

Animals↗
PubMed1990

Non-selective cation channel on pancreatic duct cells.

We have identified a non-selective cation channel on pancreatic duct cells. These epithelial cells secrete the bicarbonate ions found in pancreatic juice; a process controlled by the hormone secretin, which uses cyclic AMP as an intracellular messenger. The non-selective channel is located on both apical and basolateral plasma membranes of the duct cell, is equally permeable to sodium and potassium, and has a linear I/V relationship with a single-channel conductance of about 25 pS. Channel opening requires the presence of 1 microM Ca2+ on the cytoplasmic face of the membrane, and is also increased by depolarization. Intracellular ATP, ADP, magnesium, and a rise in pH all decreased channel activity. The channel was not affected by 10 mM TEA, 1 mM Ba2+ or 0.5 mM decamethonium applied to the cytoplasmic face of the membrane, but 0.5 mM quinine caused a flickering block which was more pronounced at depolarizing potentials. We observed the channel only rarely in cell-attached patches on unstimulated duct cells, and acute exposure to stimulants did not cause channel activation. However, after prolonged stimulation, the proportion of cell-attached patches containing active channels was increased 9-fold. The role of this channel in pancreatic duct cell function remains to be elucidated.

Adenosine Diphosphate↗
PubMed1990

Regulation of maxi-K+ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation.

Using the patch-clamp technique we have identified a Ca2(+)-sensitive, voltage-dependent, maxi-K+ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of approximately 200 pS in excised patches bathed in symmetrical 150 mM K+, and was blocked by 1 mM Ba2+. Channel open-state probability (Po) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted the Po/voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration, Po remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca2+ concentration from 2 mM to 0.1 microM. However, activated channels were still blocked by 1 mM Ba2+. Channel Po was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase. We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K+ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca2+. Physiologically, these K+ channels may contribute to the basolateral K+ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Anion selectivity and block of the small-conductance chloride channel on pancreatic duct cells.

Rat and human pancreatic duct cells have small-conductance Cl- channels in their apical plasma membranes. These channels are regulated by secretin and adenosine 3',5'-cyclic monophosphate and may function in parallel with Cl(-)-HCO3- exchangers to allow HCO3- secretion from the duct cell. Using the patch-clamp technique, we have now determined the anion permeability sequence of the channel as NO3- greater than Br- approximately I- approximately Cl- much greater than HCO3- much greater than gluconate. From this we conclude 1) that anion permeation involves a weak interaction with charged sites inside the channel pore, 2) that because of the low HCO3-/Cl- permeability ratio it is unlikely that significant amounts of HCO3- could be secreted directly via the channel, and 3) that channel permeability may determine the anion selectivity of secretion. We also show that 5-nitro-2-(3-phenylpropylamino)benzoic acid blocks the small-conductance Cl- channel, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid has no effect.

Animals↗
PubMed1989

New observations on cyclical development of Trypanosoma vivax in Glossina.

It is widely held that cyclical development of Trypanosoma vivax in Glossina is confined to the proboscis. This view has been re-examined in a series of experiments. Teneral G. morsitans centralis were fed on a goat infected with T. vivax IL 1392 and dissected 1-2 h after feeding. The infection rates in the labrum and hypopharynx were 40% and 0%, in contrast to 82% and 58%, respectively, observed in a control group dissected on day 25. This suggested that in a significant number of tsetse, cyclical development of T. vivax was initiated at sites other than the proboscis. Subsequent experiments revealed the presence of trypomastigotes, pre-epimastigotes and epimastigotes in the cibarium/oesophageal region of tsetse dissected 1-48 h after an infected feed. To investigate this further, tsetse proboscides were excised at intervals beginning 1 h after an infected feed, and transferred to in vitro culture conditions. Parasite multiplication and full cyclical development were only observed in proboscides excised 4 h or later after the infected bloodmeal. Thus, it would appear that at least in a number of tsetse, T. vivax cyclical development initially occurs in the cibarium/oesophageal region from where parasites migrate to the food canal of the proboscis and development is completed to infective metatrypanosomes in the hypopharynx.

Animals↗
PubMed1989

In vitro development of metacyclic Trypanosoma simiae derived from bloodstream trypomastigotes.

A stock of Trypanosoma simiae was transformed into procyclic forms at 26 C in a semi-defined maintenance medium. After transformation, the trypanosomes were maintained in a modified Eagle's MEM medium. On day 35 of cultivation, epimastigotes attached to the bottom of the culture flask. From day 44 onwards, metacyclic-like trypanosomes were observed. Subcutaneous injections into pigs of trypanosome suspensions obtained from cultures on day 10 were not infective, whereas culture-derived metacyclics (days 44, 63 and 69) were highly pathogenic.

Animals↗
PubMed1989

Two types of chloride channel on duct cells cultured from human fetal pancreas.

Using the patch-clamp technique, we have identified two types of chloride channel on duct cells cultured from human fetal pancreas. The channel we observed most frequently exhibited slight outward rectification, had a conductance of 4-7 pS in cell-attached patches, and was present on the apical plasma membrane where it usually occurred in clusters. Its open-state probability was not markedly voltage dependent but was increased up to threefold by exposing duct cells to secretin (10 nM), dibutyryl cyclic AMP (1 mM), or forskolin (1 microM). The other type of chloride channel was only rarely observed. Its current-voltage relationship exhibited marked outward rectification, giving chord conductances of 19 pS for inward currents and 53 pS for outward currents. Although this channel could be activated by sustained depolarization of excised patches, once activated the open state probability was not voltage dependent. While the physiological role of these channels is not firmly established, the small-conductance channel might function in parallel with a Cl- -HCO-3 exchanger to provide a mechanism for electrogenic bicarbonate secretion from the duct cell.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells.

Using the patch clamp technique we have identified a small conductance ion channel that typically occurs in clusters on the apical plasma membrane of pancreatic duct cells. The cell-attached current/voltage (I/V) relationship was linear and gave a single channel conductance of about 4 pS. Since the reversal potential was close to the resting membrane potential of the cell, and unaffected by changing from Na+-rich to K+-rich pipette solutions, the channel selects for anions over cations in cell-attached patches. The open state probability was not voltage-dependent. Adding 25 mM-bicarbonate to the bath solution caused a slight outward rectification of the I/V relationship, but otherwise, the characteristics of the channel were unaffected. In excised, inside-out, patches the I/V relationship was linear and gave a single channel conductance of about 4 pS. A threefold chloride concentration gradient across the patch (sulphate replacement) shifted the single channel current reversal potential by -26 mV, indicating that the channel is chloride selective. Stimulation of duct cells with secretin (10 nM), dibutyryl cyclic AMP (1 mM) and forskolin (1 microM) increased channel open state probability and also increased the number of channels, and/or caused disaggregation of channel clusters, in the apical plasma membrane. Coupling of this channel to a chloride/bicarbonate exchanger would provide a mechanism for electrogenic bicarbonate secretion by pancreatic duct cells.

Animals↗
PubMed1988

Structural aspects of the sarcoplasmic reticulum K+ channel revealed by gallamine block.

We have studied single-channel conductance fluctuations of K+ channels present in the sarcoplasmic reticulum (SR) membrane systems of rabbit cardiac and skeletal muscle. K+ conductance through the channels is reversibly blocked by gallamine. Conductance block occurs only from the trans side of the channel and is resolved as a smooth reduction in the open state conductance. At a fixed K+ concentration, conduction decreases with increasing gallamine concentration and the data can be fitted to a single-site inhibition scheme. The degree of block seen at a constant gallamine concentration decreases as K+ concentration is increased, indicating competition between gallamine and K+. Gallamine block is voltage dependent, the degree of block increasing with increasing negative holding potential. Quantitative analysis of block yields a zero voltage dissociation constant of 55.3 +/- 16 microM and an effective valence of block of 0.93 +/- 0.12. We conclude that gallamine blocks by interacting with a site or sites located at an electrical distance 30-35% into the voltage drop from the trans side of the channel. This site must have a cross-sectional area of at least 1.2 nm2. The results of this study have been used to modify and extend our view of the structure of the channel's conduction pathway.

Animals↗
PubMed1986

Increased sugar transport in BHK cells infected with Semliki Forest virus or with herpes simplex virus.

Infection of BHK cells by SFV increases the rate of uptake of [3H]MeGlc and of [3H]dGlc at approximately 2 hours p.i. Infection by HSV increases the uptake of [3H]MeGlc and [3H]dGlc at approximately 10 hours p.i.; the increased uptake is prevented by acyclovir. It is concluded that an increased sugar uptake by infected cells reflects an increased rate of transport across the plasma membrane and is the result of cellular changes caused by virus infection.

3-O-Methylglucose↗