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Biomedical subjects

M J Welsh

Publications and source records attributed to M J Welsh.

At least 163 records · Page 9Linked to original sources
PubMed1994

dT diaphorase: increased enzyme activity and mRNA expression in oxidant stress of skin.

DT diaphorase is a flavoprotein that enzymatically transfers two electrons from quinones as intermediate substrates and has been reported to increase its activity in the liver after exposure to toxicants. In this series of experiments, we tested the hypothesis that DT diaphorase also increases its activity after exposure to oxidants following gradient ischemia in skin. Using dorsal rat flaps, oxidant stress was induced immediately or during a 7-day period of preconditioning as a bipedicle flap before the distal attachment was divided. DT diaphorase activity (delta Abs/min/100 g) or expression of message was measured during the period of preconditioning to determine the relationship between skin survival, enzyme activity, and expression of message. There was 4.7 +/- 0.8 cm of skin necrosis in the distal end of acute flaps while the preconditioned flaps had no skin necrosis after the distal attachment was divided. In the acute flaps, the DT diaphorase activity was equal throughout the flap for the first 6 hr. After 24 hr of ischemia, the DT diaphorase activity was significantly higher in the proximal end of the flap (1.83 +/- 0.21 delta Abs/min/100 g) than that in the distal end (0.005 +/- 0.01 delta Abs/min/100 g), which was significant (P < 0.05). In the preconditioned flaps, enzyme activity did not increase but there was as 50-fold increase in DT diaphorase activity at the distal end 24 hr after they were divided (P < 0.05). Maximal enzyme induction of DT diaphorase activity occurred after 4 days of preconditioning and correlated with the maximal expression of mRNA. These studies provide the first evidence that DT diaphorase enzyme activity is inducible after oxidant stress. The data also suggests that DT activity remains elevated for at least 6 hr of ischemia and may be a potential source of anti-oxidant activity in ischemic skin.

Animals↗
PubMed1994

Chaperone functions of the heat shock proteins associated with steroid receptors.

Mammalian steroid receptors exist in hormone-free cells in a heterocomplex that contains the three heat shock proteins hsp90, hsp70 and hsp56. Some protein kinases, including pp60v-src and v-Raf, exist in similar cytosolic heterocomplexes containing hsp90 and a 50 kDa protein of unknown function, pp50. The four proteins--hsp90, hsp70, hsp56 and pp50--exist together in a heterocomplex independent of the presence of steroid receptors and protein kinases. Both the receptor and the protein kinase heterocomplexes can be formed by a protein folding-heterocomplex assembly system in reticulocyte lysate that carries out an hsp70-dependent attachment of the proteins to the preformed heat shock protein complex. Association of receptors with this structure occurs at the termination of receptor translation and is critical for maintenance of the receptors in a transcriptionally inactive state in the absence of hormone. We discuss how this preformed protein folding structure may be involved in the subsequent targeted trafficking of steroid receptors through the cytoplasmic space to the nucleus.

Animals↗
PubMed1994

Nucleotide and deduced amino acid sequences of the matrix (M) and fusion (F) protein genes of cetacean morbilliviruses isolated from a porpoise and a dolphin.

Morbilliviruses have been isolated from stranded dolphins and porpoises. The present paper describes the cloning and sequencing of the porpoise morbillivirus (PMV) F gene and of the dolphin morbillivirus (DMV) M and F genes and their flanking regions. The gene order of the DMV genome appeared to be identical to that of other morbilliviruses. A genomic untranslated region of 837 nucleotides was found between the translated DMV M and F gene regions. The predicted DMV M protein were highly conserved with those of other morbilliviruses. Both the deduced PMV and DMV F0 proteins exhibited three major hydrophobic regions as well as a cysteine rich region, a leucine zipper motif and a cleavage motif allowing cleavage of the F0 protein into F1 and F2 subunits. Apparently the DMV F0 cleavage motif was not modified by adaptation of DMV to Vero cells. The predicted PMV and DMV F proteins were 94% identical. Comparisons with the corresponding sequences of other morbilliviruses demonstrated that the cetacean morbillivirus does not derive from any known morbillivirus but represents an independent morbillivirus lineage.

Amino Acid Sequence↗
PubMed1994

Phosphate stimulates CFTR Cl- channels.

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.

3T3 Cells↗
PubMed1994

Effect of ATP concentration on CFTR Cl- channels: a kinetic analysis of channel regulation.

Phosphorylated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels require nucleoside triphosphates, such as ATP, to open. As the concentration of intracellular ATP increases, the probability of the channel being open (Po) increases. To better understand how ATP regulates the channel, we studied excised inside-out membrane patches that contained single, phosphorylated CFTR Cl- channels and examined the kinetics of gating at different concentrations of ATP. As the ATP concentration increased from 0.1 to 3 mM the mean closed time decreased, but mean open time did not change. Analysis of the data using histograms of open- and closed-state durations, the maximum likelihood method, and the log-likelihood ratio test suggested that channel behavior could be described by a model containing one open and two closed states (C1<==>C2<==>O). ATP regulated phosphorylated channels at the transition between the closed states C1 and C2: as the concentration of ATP increased, the rate of transition from C1 to C2 (C1-->C2) increased. In contrast, transitions from C2 to C1 and between C2 and the open state (O) were not significantly altered by ATP. Addition of ADP in the presence of ATP decreased the transition rate from C1 to C2 without affecting other transition rates. These data suggest that ATP regulates CFTR Cl- channels through an interaction that increases the rate of transition from the closed state to a bursting state in which the channel flickers back and forth between an open and a closed state (C2). This transition may reflect ATP binding or perhaps a step subsequent to binding.

3T3 Cells↗
PubMed1994

Correction of cAMP-stimulated fluid secretion in cystic fibrosis airway epithelia: efficiency of adenovirus-mediated gene transfer in vitro.

Adenovirus vectors are a promising vehicle to deliver cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia. However, the value of adenovirus vectors will depend on the efficiency with which the vector can correct the defective fluid transport that is though to underlie the pathogenesis of the disease. To address the efficiency of gene transfer, we applied adenovirus vectors expressing CFTR (Ad2/CFTR-1) or beta-galactosidase to the mucosal surface of primary cultures of airway epithelial cells grown as polarized epithelial monolayers on permeable filter supports. These conditions provide a model that reproduces the physiology of the airways in vivo. We found that after adding 1 moi Ad2/CFTR-1 to the mucosal surface, cAMP agonists stimulated fluid secretion that was within the range observed in epithelia from normal subjects. When we measured electrolyte transport, we found that as little as 0.1 moi partially restored cAMP-stimulated Cl- secretion, and at 10 moi Cl- secretion was in the normal range. A related vector encoding beta-galactosidase generated activity in approximately 20% of cells at an moi of 1 and 90% of cells at an moi of 10. These data suggest that Ad2/CFTR-1 is very efficient at restoring normal fluid and electrolyte transport to CF airway epithelia. Thus, they suggest that relatively low input doses could be used for gene transfer to CF airway epithelia.

Adenoviridae↗
PubMed1994

Second messenger specificity of the inositol trisphosphate receptor: reappraisal based on novel inositol phosphates.

To further understand how the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] interacts with its intracellular receptor, we injected 47 highly purified inositol phosphate (InsP) positional isomers in Xenopus oocytes and compared their potency in releasing intracellular Ca2+. The potency of the Ca(2+)-releasing InsPs spanned four orders of magnitude. Seven compounds, including the novel inositol 1,2,4,5-tetrakisphosphate [D/L-Ins (1,2,4,5)P4] and D/L-Ins(1,4,6)P3, had a very high potency. All of these highly active InsPs shared the following structure: two D-trans-equatorial phosphates (eq-P) and one equatorial hydroxyl (eq-OH) attached to ring carbons D-4, D-5, and D-6 (or to the structurally equivalent D-1, D-6, and D-5 carbons). This permissive structure was not sufficient for Ca2+ release, because it was also found in two inactive compounds, Ins(1,6)P2 and Ins(1,3,6)P3. To be active, InsPs also required the structural equivalent of a D-3 eq-OH and/or a D-1 eq-P. Together, our data reveal how the structure of the InsP molecule affects its ability to release Ca2+.

Animals↗
PubMed1994

cAMP and inositol 1,4,5-trisphosphate increase Ca2+ in HT-29 cells by activating different Ca2+ influx pathways.

Ca2+ plays a central role in regulating transepithelial fluid and electrolyte transport in intestinal epithelial cells. To investigate the mechanisms regulating the cytosolic free Ca2+ concentration ([Ca2+]c), we examined the effect of secretory agonists on [Ca2+]c in the intestinal epithelial cell line HT-29 clone 19A cells. We found that [Ca2+]c increased after addition of either adenosine 3',5'-cyclic monophosphate (cAMP)-dependent agonists or a D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]dependent agonist carbachol. Several lines of evidence suggest that cAMP- and Ins(1,4,5)P3-dependent agonists act through separate pathways. First, isoproterenol and forskolin increased cellular levels of cAMP but not Ins(1,4,5)P3, whereas carbachol increased cellular levels of Ins(1,4,5)P3 and stimulated inositol phosphate turnover without increasing cAMP. Second, carbachol increased [Ca2+]c by stimulating the release of Ca2+ from intracellular stores and influx of extracellular Ca2+. In contrast, cAMP agonists increased [Ca2+]c by stimulating Ca2+ influx alone. Third, the responses to maximal concentrations of cAMP agonists and carbachol were approximately additive. Finally, Ins(1,4,5)P3- but not cAMP agonist-dependent Ca2+ influx was inhibited by inorganic Ca2+ channel blockers. Thus, in intestinal epithelial cells, [Ca2+]c is regulated by at least two different second-messenger pathways, involving Ins(1,4,5)P3 or cAMP. In addition, cAMP stimulates influx of extracellular Ca2+ through a pathway distinct from that mediated by Ins(1,4,5)P3.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation and by intracellular nucleotides. The function of CFTR, like other recombinant ion channels, has generally been studied in single cells using voltage-clamp techniques. However, because CFTR is normally located in the apical membrane of epithelia we wanted to develop a system to study the function of recombinant CFTR expressed in an epithelium. We chose Fischer rat thyroid (FRT) epithelia for two reasons. First, when grown on permeable filter supports, FRT cells form polarized epithelia with a high transepithelial resistance. Second, they have no endogenous cAMP-regulated Cl- channels in their apical membrane. We expressed CFTR in FRT epithelia either transiently, using recombinant vaccinia virus, or stably, using a retrovirus. To measure apical membrane Cl- currents, we permeabilized the basolateral membrane to monovalent ions with nystatin and imposed a large transepithelial Cl- concentration gradient. cAMP agonists stimulated apical membrane Cl- currents in FRT epithelia infected with wild-type CFTR (vTF-CFTR) but not in FRT epithelia infected with either control virus (vTF7-3) or CFTR containing the delta F508 mutation (vTF-delta F508). These Cl- currents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Cloning, expression, and tissue distribution of a human amiloride-sensitive Na+ channel.

Amiloride-sensitive Na+ channels control, in part, fluid and electrolyte transport across epithelia in many organs. In the lung, they control the quantity and composition of the respiratory tract fluid and play a key role in the transition from a fluid-filled lung at the time of birth. Their function may also be altered in a number of diseases. The recent identification of an epithelial Na+ channel from rat colon allowed us to use a probe from that sequence to clone an amiloride-sensitive Na+ channel from human kidney, alpha hENaC. The cDNA had an open reading frame of 2,007 nucleotides and encoded a protein predicted to contain 669 amino acids. The amino acid sequence of alpha hENaC was 83% identical to that of the rat. The gene was mapped to chromosome 12 by polymerase chain reaction (PCR) of somatic cell hybrids. Transcripts of alpha hENaC were detected in human kidney, lung, liver, and pancreas. No message was detected in first- and second-trimester human fetal lung, indicating that alpha hENaC expression is developmentally regulated. In vitro transcription and translation of alpha hENaC produced a 74-kDa protein and translation in the presence of microsomal membranes produced a glycosylated 87-kDa protein. Expression of alpha hENaC in Xenopus oocytes produced currents that were amiloride sensitive and Na+ selective, properties consistent with the function of epithelial Na+ channels in native tissues.

Adult↗
PubMed1994

Defective fluid transport by cystic fibrosis airway epithelia.

Cystic fibrosis (CF) airway epithelia exhibit defective transepithelial electrolyte transport: cAMP-stimulated Cl- secretion is abolished because of the loss of apical membrane cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, and amiloride-sensitive Na+ absorption is increased two- to threefold because of increased amiloride-sensitive apical Na+ permeability. These abnormalities are thought to alter respiratory tract fluid, thereby contributing to airway disease, the major source of mortality in this genetic disease. However, the underlying hypothesis, that fluid transport is abnormal in CF airway epithelia, has not been tested. Most conjecture about fluid transport is based on measurements of Na+ and Cl- transport performed under short circuit conditions in Ussing chambers. But such studies differ from in vivo conditions in that transepithelial voltage and mucosal fluid composition are held constant. Therefore, we measured fluid transport and mucosal electrolyte composition in primary cultures of CF airway epithelia without holding transepithelial voltage and ion concentration gradients at zero. In normal epithelia, cAMP agonists plus amiloride stimulated NaCl and fluid secretion. In CF epithelia, cAMP agonists failed to stimulate fluid or electrolyte secretion, changes consistent with the loss of CFTR Cl- channels. But in striking contrast to predictions based on Ussing chamber studies, CF epithelia absorbed fluid at a rate no greater than normal epithelia. Moreover, amiloride, which inhibits Na+ channels, failed to inhibit fluid absorption by CF epithelia. These results have important implications for understanding the pathogenesis of CF airway disease and for the design and evaluation of therapy.

Amiloride↗
PubMed1994

The hsp56 immunophilin component of untransformed steroid receptor complexes is localized both to microtubules in the cytoplasm and to the same nonrandom regions within the nucleus as the steroid receptor.

In their unliganded state, mouse glucocorticoid receptors (GR) that are overexpressed in the WCL2 line of Chinese hamster ovary cells are distributed in a nonrandom manner throughout all planes of the nucleus. These untransformed nuclear receptors exist in a heterocomplex containing three heat shock proteins, hsp90, hsp70, and hsp56, the latter being an immunophilin of the FK506 binding type whose cellular function is unknown. Because a knowledge of the cellular distribution of hsp56 could provide important clues to its function in steroid-receptor heterocomplexes, we have examined hsp56 localization in intact cells by indirect immunofluorescence using the UPJ56 antibody. The majority of hsp56 is located in the nucleus, with substantial amounts also visualized in the cytoplasm of intact cells. The cytoplasmic hsp56 was examined in rat pulmonary endothelial cells where the protein was found to colocalize with microtubules. The nuclear hsp56 was examined in the WCL2 cells, where the protein was found by confocal imaging to colocalize throughout all planes of the nucleus in the same mottled pattern as the overexpressed GR. Like the GR, the nuclear hsp56 is recovered largely in the cytosolic fraction after hypotonic rupture of WCL2 cells. An observation potentially related to the microtubule-associated fraction of hsp56 is that immunoadsorption of hsp56 from WCL2 cytosol is accompanied by coadsorption of the microtubule-associated protein-1C complex. These observations are discussed with respect to the possible biological functions of hsp56 in the folding and/or cytoplasmic-nuclear trafficking of the receptor.

Animals↗
PubMed1993

Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis.

To evaluate the potential of direct transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA for the treatment of cystic fibrosis (CF), we administered an E1-deficient adenovirus, encoding CFTR, to a defined area of nasal airway epithelium of three individuals with CF. This treatment corrected the Cl- transport defect that is characteristic of CF-affected epithelia. After treatment, there was a decrease in the elevated basal transepithelial voltage, and the normal response to a cAMP agonist was restored. We found no evidence of viral replication or virus-associated adverse effects, even at the highest dose tested (25 MOI). These data represent a small step in achieving long-term improvement of CF lung function by gene therapy.

Adenoviridae↗
PubMed1993

Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.

Alanine↗