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Biomedical subjects

C Bear

Publications and source records attributed to C Bear.

12 recordsLinked to original sources
PubMed2001

Expression of CFTR and Cl(-) conductances in cells of pulmonary neuroepithelial bodies.

The pulmonary neuroendocrine cell system comprises solitary neuroendocrine cells and clusters of innervated cells or neuroepithelial bodies (NEBs). NEBs figure prominently during the perinatal period when they are postulated to be involved in physiological adaptation to air breathing. Previous studies have documented hyperplasia of NEBs in cystic fibrosis (CF) lungs and increased neuropeptide (bombesin) content produced by these cells, possibly secondary to chronic hypoxia related to CF lung disease. However, little is known about the role of NEBs in the pathogenesis of CF lung disease. In the present study, using a panel of cystic fibrosis transmembrane conductance regulator (CFTR)-specific antibodies and confocal microscopy in combination with RT-PCR, we demonstrate expression of CFTR message and protein in NEB cells of rabbit neonatal lungs. NEB cells expressed CFTR along with neuroendocrine markers. Confocal microscopy established apical membrane localization of the CFTR protein in NEB cells. Cl(-) conductances corresponding to functional CFTR were demonstrated in NEB cells in a fresh lung slice preparation. Our findings suggest that NEBs, and related neuroendocrine mechanisms, likely play a role in the pathogenesis of CF lung disease, including the early stages before establishment of chronic infection and chronic lung disease.

Animals↗
PubMed2000

Preventing carpal tunnel syndrome and cumulative trauma disorder: effect of carpal tunnel decompression excercises: an Oklahoma experience.

An Oklahoma meat packing company with 286 production workers experienced a high incidence of cumulative trauma disorder (CTD), specifically carpal tunnel syndrome (CTS). The company learned about the Carpal Tunnel Decompression Exercises (exercise program) through the American Academy of Orthopedic Surgeons' official web page. The Orthopaedic & Reconstructive Center assisted the company with implementation of the exercise program. The company reported their one-year follow up findings to the Governor's Council on Safety Policy (State of Oklahoma). The exercise program was the only ergonomic change the company implemented that year. The company's workers' compensation loss ratio improved from 13.89% to 11.61%. The company reported a net financial gain after comparing injury reduction versus work time lost to exercise. The incidence of Cumulative Trauma Disorder, excluding Carpal Tunnel Syndrome, was reduced 37% and Carpal Tunnel Syndrome was reduced 45.4%. The results of the exercise program were significant enough that the company made the exercise program permanent.

Adolescent↗
PubMed1998

Recombinant pICln forms highly cation-selective channels when reconstituted into artificial and biological membranes.

pICln has been proposed to be the swelling-activated anion channel responsible for ICl, swell, or a channel regulator. We tested the anion channel hypothesis by reconstituting recombinant pICln into artificial and biological membranes. Single channels were observed when pICln was reconstituted into planar lipid bilayers. In the presence of symmetrical 300 mM KCl, the channels had a high open probability and a slope conductance of 48 pS, and were outwardly rectifying. Reduction of trans KCl to 50 mM shifted the reversal potential by -31.2 +/- 0.06 mV, demonstrating that the channel is at least seven times more selective for cations than for anions. Consistent with this finding, channel conductance was unaffected by substitution of Cl- with glutamate, but was undetectable when K+ was replaced by N-methyl-D-glucamine. Reconstitution of pICln into liposomes increased 86Rb+ uptake by three- to fourfold, but had no effect on 36Cl- uptake. Phosphorylation of pICln with casein kinase II or mutation of G54, G56, and G58 to alanine decreased channel open probability and 86Rb+ uptake. When added to the external medium bathing Sf9 cells, pICln inserted into the plasma membrane and increased cell cation permeability. Taken together, these observations demonstrate that channel activity is due to pICln and not minor contaminant proteins. However, these findings do not support the hypothesis that pICln is the anion-selective ICl, swell channel. The observed cation channel activity may reflect an as yet to be defined physiological function of pICln, or may be a consequence of in vitro reconstitution of purified, recombinant protein.

Animals↗
PubMed1996

Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor.

Mice that have been made deficient for the cystic fibrosis transmembrane conductance regulator (Cftr) usually die of intestinal obstruction. We have created Cftr-deficient mice and demonstrate prolonged survival among backcross and intercross progeny with different inbred strains, suggesting that modulation of disease severity is genetically determined. A genome scan showed that the major modifier locus maps near the centromere of mouse chromosome 7. Electrophysiological studies on mice with prolonged survival show that the partial rectification of Cl- and Na+ ion transport abnormalities can be explained in part by up-regulation of a calcium-activated Cl- conductance. Identification of modifier genes in our Cftr(m1HSC)/Cftr(m1HSC) mice should provide important insight into the heterogeneous disease presentation observed among CF patients.

Animals↗
PubMed1995

Purification and characterization of recombinant cystic fibrosis transmembrane conductance regulator from Chinese hamster ovary and insect cells.

We have developed procedures to purify highly functional recombinant cystic fibrosis transmembrane conductance regulator (CFTR) from Chinese hamster ovary (CHO) cells to high homogeneity. Purification of CHO-CFTR was achieved using a combination of alkali stripping, alpha-lysophosphatidylcholine extraction, DEAE ion-exchange, and immunoaffinity chromatography. Insect CFTR from Sf9 cells was purified using a modification of the method of Bear et al. (Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M. and Riordan, J. R. (1992) Cell 68, 809-818), which included extraction with sodium dodecyl sulfate, hydroxyapatite, and gel filtration chromatography. Characterization of the properties of purified CFTR from both cell sources using a variety of electrophysiological and biochemical assays indicated that they were very similar. Both the purified CHO-CFTR and Sf9-CFTR when reconstituted into planar lipid bilayers exhibited a low pS, chloride-selective ion channel activity that was protein kinase A- and ATP-dependent. Both the purified CHO-CFTR and Sf9-CFTR were able to interact specifically with the nucleotide photoanalogue 8-N3-[alpha-32P]ATP with half-maximal binding at 25 and 50 microM, respectively. These values compare well with those reported for 8-N3-[alpha-32P]ATP binding to CFTR in its native membrane form. Thus CFTR from either insect or CHO cells can be purified to high homogeneity with retention of many of the biochemical and electrophysiological characteristics of the protein associated in its native plasma membrane form. The availability of these reagents will facilitate further investigation and study of the structure and function of CFTR and its interactions with cellular proteins.

Adenosine Triphosphate↗
PubMed1993

The delta F508 mutation decreases the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. Determination of functional half-lives on transfected cells.

Deletion of the phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most prevalent mutation in cystic fibrosis (CF). This mutation (delta F508CFTR) leads to a reduced cAMP-sensitive Cl- conductance in epithelial cells. While the mutant protein can function as a Cl- channel, it seems to be misprocessed and unable to accumulate at normal levels in the plasma membrane. Under conditions where the biosynthetic block of delta F508CFTR is not complete, the residence time of delta F508CFTR in the plasma membrane is a critical determinant of the cAMP-sensitive Cl- conductance. To assess the stability of the mutant and wild-type CFTR, we compared their functional half-lives at the plasma membrane of transfected Chinese hamster ovary cells. The plasma membrane Cl- conductance was assessed by patch-clamp recordings and/or by fluorimetric determinations of the membrane potential. Accumulation of delta F508CFTR in the plasma membrane was promoted by growing the transfected cells at reduced temperature (24-28 degrees C), and was verified by immunoblotting and by detecting the appearance of a plasmalemmal cAMP-activated Cl- conductance. Subsequently increasing the temperature to 37 degrees C inhibited further delivery of newly synthesized delta F508CFTR to the surface membrane. By studying the time dependence of the disappearance of the Cl- conductance, the functional half-life of the mutant protein at the plasma membrane was determined to be < 4 h, which is considerably shorter than the half-life of wild-type CFTR (> 24 h). The latter was estimated by terminating protein synthesis or secretion with cycloheximide or brefeldin A, respectively. Inhibition of protein synthesis did not alter the rate of disappearance of delta F508CFTR at 37 degrees C, validating the difference in turnover between mutant and wild-type CFTR. These results indicate that the structural abnormality of delta F508CFTR affects not only the delivery of the protein to the plasma membrane, but also its stability therein. Moreover, they suggest that overcoming the processing block at the endoplasmic reticulum may not suffice to restore normal Cl- conductance in CF.

Animals↗
PubMed1993

Novobiocin forms cation-permeable ion channels in rat fetal distal lung epithelium.

The antibiotic novobiocin has been previously reported to increase Na+ transport in frog skin, presumably by attenuation of Na+ self-inhibition of Na+ channels. To determine whether novobiocin had similar effects and utilized a similar mechanism in mammalian Na(+)-transporting tissues, we studied its effect on ion transport by primary cultures of fetal distal lung epithelium (FDLE) cultured from 20-day gestationally aged rats (term = 22 days). Novobiocin (10 mM) increased short-circuit current and markedly decreased the resistance in FDLE monolayers mounted in Ussing chambers. Fura-2 single-cell studies showed that novobiocin increased intracellular Ca2+ concentration and that this resulted from extracellular sources. Nystatin-perforated patch-clamp techniques demonstrated that novobiocin increased nonrectifying cation whole cell currents without inducing detectable anion currents. Novobiocin created nonrectifying monovalent cation-selective channels in lipid bilayers. These studies demonstrated that novobiocin affects the bioelectric properties of Na+ transporting lung epithelium and that this likely occurs by the formation of ion-permeant channels in their lipid membranes.

Animals↗
PubMed1992

Regulation of CFTR expression and function during differentiation of intestinal epithelial cells.

CFTR, the protein defective in cystic fibrosis is regulated during differentiation of intestinal epithelial cells. The undifferentiated cells (Caco-2 and HT-29) show a lower level of CFTR mRNA, while a 10-fold increase is seen in differentiated cells. These differences correlate well with those of other intestinal-specific genes, including sucrase-isomaltase, villin and alpha 1-antitrypsin, indicating that the regulation is cell specific. In Caco-2 cells the increase in CFTR mRNA cannot be accounted for by increased transcription of the gene. These data indicate that CFTR mRNA stabilizing factor(s) might be present in differentiated cells. The higher levels of CFTR mRNA in differentiated cells are accompanied by decreased protein levels, indicating, as well, involvement of translational control in the regulation of CFTR in these cells. Finally, fully differentiated cells show lowered levels of cyclic AMP-activated C1- transport, the characteristic function of CFTR. Thus, CFTR function in differentiated cells is modulated by a complex interaction of regulatory elements. Caco-2 and HT-29 cells provide a suitable in vitro system in which to study the mechanism of regulation of CFTR.

Blotting, Western↗