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C J Wardle

Publications and source records attributed to C J Wardle.

12 recordsLinked to original sources
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

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

Characterization of DNASE I hypersensitive sites in the 120kb 5' to the CFTR gene.

The chromatin structure of 120kb of genomic DNA 5' to the CFTR gene has been analysed in a number of CFTR expressing and non-expressing cell types, including primary genital duct epithelial cells. Novel DNAse I hypersensitive sites have been observed at -79.5kb and -20.5kb 5' to the ATG translation start codon of the CFTR coding sequence. Neither of these sites appears to show strong correlation with CFTR expression in the cell types investigated, hence they are unlikely to reflect the sites of binding of the major CFTR tissue specific regulator(s). However, they may still play an important part in the complex series of events involved in the regulation of CFTR transcription.

Base Sequence↗
PubMed1995

Down-regulation of CFTR expression in an HT29-derived cell line by stable antisense RNA production.

In order to examine the interactions of the cAMP activated CFTR chloride ion channel with other chloride ion channels present in epithelial cells we have generated a model based on antisense mRNA down regulation of CFTR expression. The HT29-derived epithelial cell line CJC4-1 has integrated a plasmid that is constitutively expressing CFTR exons 1-6 in the antisense orientation, from the MoMULV LTR. This cell line shows a reduction in cAMP-activated chloride efflux, as measured by iodide efflux assay, in comparison to the parental HT29 cell line and a sense transfected control.

Cell Line↗
PubMed1995

Molecular analysis of the ovine cystic fibrosis transmembrane conductance regulator gene.

There is a need for a large-animal model to investigate the etiology and biology of cystic fibrosis (CF) lung disease and to study potential therapies. The development and electrophysiology of the sheep airway have been shown to exhibit close functional parallels with the human airway, particularly with respect to the respiratory epithelium. We have cloned and sequenced the ovine cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. It shows a high degree of conservation at the DNA coding and predicted polypeptide levels with human CFTR: at the nucleic acid level there is a 90% conservation (compared with 80% between human and mouse CFTR cDNA); at the polypeptide level, the degree of similarity is 95% (compared with 88% between human and mouse). Northern blot analysis and reverse transcription-PCR have shown that the patterns of expression of the ovine CFTR gene are very similar to those seen in humans. Further, the developmental expression of CFTR in the sheep is equivalent to that observed in humans. Thus, overall a CF sheep should show lung pathology similar to that of humans with CF.

Amino Acid Sequence↗
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↗
PubMed1993

Expression of the cystic fibrosis gene in human foetal tissues.

In order to examine the onset of the cystic fibrosis (CF) disease process, the expression of the cystic fibrosis gene (CFTR) has been examined in mid-trimester human foetal tissues by in situ hybridization. CFTR mRNA was detected in the epithelia of pancreatic ducts, small intestine, colon, genital ducts, lung and trachea. The majority of these sites of CFTR expression in the foetus are similar to those seen in adult tissues. However, epithelia of the lung, that contain very little CFTR mRNA in the adult, express high levels of CFTR mRNA in the foetus. Since the lung is the major site of pathology and morbidity in CF these findings have implications for treatment.

Cystic Fibrosis↗
PubMed1991

Twentieth-century influences on the development in Britain of services for child and adolescent psychiatry.

Modern comprehensive multidisciplinary mental-health services for children and adolescents have four origins: psychology from 1890, psychoanalysis from 1906, the child-guidance movement from 1920, and the children's departments of psychiatric teaching hospitals from 1930. Post-war changes in society and reform, especially the NHS Act 1946, contributed to rapid development of services and an increasingly wide range of sophisticated therapeutic interventions; professional and interdisciplinary associations and trans-Atlantic exchange were also influential. In the last three decades a succession of official inquiries, reports, legislation and reorganisations have had a damaging effect. Children and their services have been prey to causes célèbres, fashion and the exaggerated fads and foibles of the media and politicians; they have thrived best when society and their carers were tolerant, and loving, sought good qualities to augment, not evil to exorcise, and succeeded in balancing structure and control with flexibility and freedom to grow. Planners should review the past before acting.

Adolescent↗
PubMed1990

Mass sociogenic illness by proxy: parentally reported epidemic in an elementary school.

"In a cluster of illness reported among students at an elementary school parents mentioned many signs and symptoms including headache, pallor, dark circles under the eyes, nausea, and vomiting--which they attributed to exposure to recurrent leaks of natural gas at the school. It is likely that the parents spread among themselves the notion of toxic exposure at the school. A questionnaire revealed no spatial clustering, but increased reports of symptoms were related to intense media coverage. A thorough environmental and epidemiological investigation was negative, there being no evidence of a continuing gas leak or other potential causes. At a strictly biological level, the complaints in this reported 'cluster' apparently represented the sporadic occurrence of common childhood illnesses. The possibility of an epidemic from toxic exposure at the school caused intense parental concern and led to a major public health problem. The established term 'mass sociogenic illness' seems inapplicable here because complaints did not come principally from the students and the apparent epidemic illness was not transmitted among them. The term 'mass sociogenic illness by proxy' is proposed to describe this incident, in which transmission in one group (the parents) resulted in reports of an epidemic in another group (students)."

Child↗
PubMed1989

High efficiency expression of transfected genes in a Drosophila melanogaster haploid (1182) cell line.

Drosophila tissue culture cells have been important in the study of homologous promoters and more recently in the study of mammalian transcriptional factors such as CTF and SP1 which bind and stimulate transcription from transfected genes. In this paper we show that a Drosophila melanogaster haploid cell line (1182-4), not previously used for transfection studies, is capable of taking up and expressing DNA without the use of a facilitating agent such as calcium phosphate. Furthermore expression from a variety of Drosophila promoters such as copia, heatshock and rudimentary as well as a mammalian promoter RSV-LTR, show between 20 and over 100 times more activity in 1182-4 cells than in D.hydei DH33 or D.melanogaster S3, or D1 cell lines. This cell line should prove to be particularly useful for the analysis of weak promoters and heterologous transcription factors.

Animals↗