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D J Wilkinson

Publications and source records attributed to D J Wilkinson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Missense mutation (G480C) in the CFTR gene associated with protein mislocalization but normal chloride channel activity.

We have identified a novel CFTR missense mutation associated with a protein trafficking defect in mammalian cells but normal chloride channel properties in a Xenopus oocyte assay. The mutation, a cysteine for glycine substitution at residue 480 (G480C), was detected in a pancreatic insufficient, African-American, cystic fibrosis (CF) patient. G480C was found on one additional CF chromosome and on none of 220 normal chromosomes, including 160 chromosomes from normal African-American individuals. Western blot analysis and immunofluorescence studies revealed that, in 293T cells, the encoded mutant protein was not fully glycosylated and failed to reach the plasma membrane, suggesting that the G480C protein was subject to defective intracellular processing. However, in Xenopus oocytes, a system in which mutant CFTR proteins are less likely to experience an intracellular processing/trafficking deficit, expression of G480C CFTR was associated with a chloride conductance that exhibited a sensitivity to activation by forskolin and 3-isobutyl-1-methylxanthine (IBMX) that was similar to that of wild-type CFTR. This appears to be the first identification of a CFTR mutant with a single amino acid substitution in which the sole basis for disease is mislocalization of the protein.

Animals↗
PubMed1993

Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the traffic ATPase superfamily, possesses two putative nucleotide-binding folds (NBFs). The NBFs are sufficiently similar that sequence alignment of highly conserved regions can be used to identify analogous residues in the two domains. To determine whether this structural homology is paralleled in function, we compared the activation of chloride conductance by forskolin and 3-isobutyl-1-methylxanthine in Xenopus oocytes expressing CFTRs bearing mutations in NBF1 or NBF2. Mutation of a conserved glycine in the putative linker domain in either NBF produced virtually identical changes in the sensitivity of chloride conductance to activating conditions, and mutation of this site in both NBFs produced additive effects, suggesting that in the two NBFs this region plays a similar and critical role in the activation process. In contrast, amino acid substitutions in the Walker A and B motifs, thought to form an integral part of the nucleotide-binding pockets, produced strikingly different effects in NBF1 and NBF2. Substitutions for the conserved lysine (Walker A) or aspartate (Walker B) in NBF1 resulted in a marked decrease in sensitivity to activation, whereas the same changes in NBF2 produced an increase in sensitivity. These results are consistent with a model for the activation of CFTR in which both NBF1 and NBF2 are required for normal function but in which either the nature or the exact consequences of nucleotide binding differ for the two domains.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Mercury blockade of thiazide-sensitive NaCl cotransport in flounder urinary bladder.

The effects of HgCl2 on ion transport were investigated using isolated sheets of flounder urinary bladder, a model epithelium that is capable of electrically silent NaCl absorption and electrogenic K secretion. Exposure of the mucosal surface of the bladder to submicromolar doses of HgCl2 reduced K secretion, but the effect was not due to blockade of apical K channels. Rather, the effects of HgCl2 were virtually identical to those seen with experimental maneuvers that blocked the thiazide-sensitive NaCl cotransporter in the apical membrane, e.g., hydrochlorothiazide, Cl-free solutions, and Na-free solutions. Mucosal HgCl2 also blocked 22Na absorption, suggesting that the effect of the metal was mediated by blockade of NaCl entry. The effects of HgCl2 had a rapid onset and were readily reversed by washing, suggesting a noncovalent binding reaction. The abundance of polyanionic Hg complexes in salt solutions prompts the speculation that one of these may bind to the Cl-binding site on the cotransporter, thereby blocking it. The results provide the first evidence that the thiazide-sensitive NaCl cotransporter is a specific site of action for inorganic mercury.

Animals↗
PubMed1993

Expression of an abundant alternatively spliced form of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is not associated with a cAMP-activated chloride conductance.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP-activated chloride (Cl-) channel, and expression of the full length gene in vitro is sufficient to correct the Cl- conductance defect that is characteristic of cystic fibrosis (CF) epithelial cells. Alternatively spliced forms of CFTR mRNA have been identified in several tissues from normal individuals. One of the alternative transcripts, often present at high levels, results in the in-frame deletion of exon 9. Translation of this transcript would result in a CFTR protein missing the amino terminal portion of the first nucleotide binding fold (NBF). To evaluate the possible function of this form of CFTR, a cDNA representing this transcript (CFTR delta 9) was transduced into CFPAC cells, which are derived from a CF patient. CFTR delta 9 RNA was expressed in the transduced cell lines, but only immature, incompletely glycosylated protein was detectable by Western blot analysis. No increase in cAMP-activated anion permeability was detectable by 125I efflux assay or by means of the halide sensitive dye 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ). In a second assay system, in vitro synthesized mRNA representing CFTR delta D9 was injected into Xenopus oocytes, but expression of this alternatively spliced form of CFTR was not associated with the appearance of Cl- conductance. These results suggest that the protein produced by the CFTR delta 9 transcript is not properly processed and is not capable of generating Cl- conductance in response to cAMP. Whether this alternative transcript has some other function or represents 'noise' in the mRNA splicing mechanism remains unresolved.

Alternative Splicing↗
PubMed1993

Tetraethylammonium-sensitive apical K+ channels mediating K+ secretion by turtle colon.

1. Apical membrane K+ channels in turtle colon were identified and characterized using current fluctuation analysis. 2. Under short-circuit conditions in NaCl-Ringer solution, the power density spectrum (PDS) of the short-circuit current (Isc) sometimes exhibited a clearly discernible Lorentzian component, indicating spontaneous fluctuations produced by a population of apical ion channels. The Lorentzian component had a characteristic corner frequency (fc) which averaged 10.2 +/- 0.9 Hz (mean +/- S.E.M., n = 20). 3. The power of the spontaneous fluctuations was enhanced (So increased) by manoeuvres that depolarize the apical membrane electrical potential (Va). Discernible fluctuations were enhanced or induced by raising the serosal K+ concentration ([K+]s = 50-115 mM, Na+ replacement), by clamping the transepithelial potential (Vt) to serosa-positive values, or by blocking basolateral K+ channels with Ba2+. 4. Mucosal amiloride (100 microM) attenuated the spontaneous fluctuations observed in NaCl-Ringer solution but had no effect in the presence of serosal high K+, indicating that amiloride did not block the K(+)-permeable channels but these channels resided in the same cells as the amiloride-sensitive Na+ channels. 5. Raising the mucosal K+ concentration attenuated spontaneous fluctuations. 6. In the presence of serosal high K+ and mucosal amiloride, the spontaneous fluctuations were often accompanied by a reversed Isc consistent with K+ secretion. These conditions were used to test the effects of putative channel blockers. 7. Mucosal Ba2+ and tetraethylammonium (TEA+) were effective inhibitors of the spontaneous fluctuations and the reversed Isc. At a concentration of 10 mM, TEA+ was maximally effective but the TEA+ analogues tetramethylammonium (TMA+) and tetrapropylammonium (TPrA+) were much less effective. Mucosal Rb+ or Cs+ did not inhibit at a concentration of 10 mM. 8. Mucosal lidocaine (200 microM), quinidine (200 microM), or diphenylamine-2-carboxylate (DPC, 1 mM) had little or no effect on the spontaneous fluctuations and reversed Isc. Quinine (100 microM), 4-aminopyridine (1 mM), and apamin (100 nM) were also without effect. 9. Mucosal TEA+ (10 mM) abolished the active secretory K+ flux measured in the presence of serosa-positive transepithelial potentials. 10. These experiments identified a population of TEA(+)-sensitive, apical K+ channels which mediate active K+ secretion in turtle colon. Sensitivity to external TEA+ distinguishes these channels from basolateral K+ channels in turtle colon and demonstrates similarity to apical K+ channels in mammalian colon.

Amiloride↗
PubMed1992

The occurrence of bacteraemia associated with the use of oral and nasopharyngeal airways.

In order to determine whether placement of oral or nasopharyngeal airways during anaesthesia induces a significant bacteraemia, 36 ASA grade 1 or 2 patients undergoing body surface surgery, in whom it was anticipated that spontaneous respiration would be maintained, were randomly allocated to one of two groups. One group was given oral airways and the other, nasopharyngeal. A series of blood samples, which were taken before and after airway insertion, were cultured aerobically and anaerobically. Nasal and oral swabs were taken at the same time. Single isolates of Corynebacterium species and Acinetobacter species, together with two isolates of Staphylococcus epidermidis were grown from one culture bottle from 4 of the 36 patients studied. None of the oral or nasal swabs, or any of the subsequent or previous blood samples produced positive cultures for these organisms in these four patients. We believe that these results represent skin commensal contamination rather than bacteraemia in these four patients, and that bacteriological considerations should not influence decisions about the type of airway used during anaesthesia. However, prophylactic antibiotic therapy should still be provided for all high risk cases.

Adult↗
PubMed1991

Chloride conductance expressed by delta F508 and other mutant CFTRs in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is associated with expression of a chloride conductance that is defective in cystic fibrosis (CF). Xenopus oocytes injected with RNA coding for CFTR that contained mutations in the first nucleotide binding fold (NBF1) expressed chloride currents in response to raising adenosine 3',5'-monophosphate (cAMP) with forskolin and 3-isobutyl-1-methylxanthine (IBMX). The mutant CFTRs were less sensitive than wild-type CFTR to this activating stimulus, and the reduction in sensitivity correlated with the severity of cystic fibrosis in patients carrying the corresponding mutations. This demonstration provides the basis for detailed analyses of NBF1 function and suggests potential pharmacologic treatments for cystic fibrosis.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Dr F.P. de Caux--the first user of curare for anesthesia in England.

Curare was used in the 19th century in England by a wide variety of scientists, physicians and veterinarians. Their experiments indicated many of the properties of the drug, but its clinical usage remained very limited and was reserved for cases of tetanus, hydrophobia and strychnine poisoning. Griffith and Johnson are usually credited with the introduction of curare into clinical anaesthesia in 1942, but a Dr F.P. de Caux working at the North Middlesex Hospital, London, in 1928 utilised curare in a series of seven patients. His work was not widely publicized and this contribution to anaesthetic history has been overlooked by subsequent authors.

Adjuvants, Anesthesia↗
PubMed1991

Francis Percival de Caux (1892-1965). An anaesthetist at odds with social convention and the law.

All doctors practice medicine within the confines of what is termed 'acceptable practice'. This acceptable practice is delineated by medical ethics, the actions of one's colleagues, social custom, and the laws of the country. Failure to conform to any or all of these constraints may result in professional ostracism or even loss of liberty. The life and work of Frances Percival de Caux clearly shows these effects in their most damaging manner.

Abortion, Illegal↗
PubMed1990

Ginger root--a new antiemetic. The effect of ginger root on postoperative nausea and vomiting after major gynaecological surgery.

The effectiveness of ginger (Zingiber officinale) as an antiemetic agent was compared with placebo and metoclopramide in 60 women who had major gynaecological surgery in a double-blind, randomised study. There were statistically significantly fewer recorded incidences of nausea in the group that received ginger root compared with placebo (p less than 0.05). The number of incidences of nausea in the groups that received either ginger root or metoclopramide were similar. The administration of antiemetic after operation was significantly greater in the placebo group compared to the other two groups (p less than 0.05).

Adolescent↗