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M J Welsh

Publications and source records attributed to M J Welsh.

At least 127 records · Page 7Linked to original sources
PubMed1996

Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia.

Adenoviridae↗
PubMed1996

Expression of inositol 1,4,5-trisphosphate receptors changes the Ca2+ signal of Xenopus oocytes.

The receptors for the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] form a family of closely related proteins that play an important role in regulating the free intracellular Ca2+ concentration. To test the hypothesis that changing the expression level of Ins(1,4,5)P3 receptors could alter the Ins(1,4,5)P3-mediated Ca2+ signal, we overexpressed Ins(1,4,5)P3 receptor type 1 (InsP3R-1) or type 3 (InsP3R-3) in Xenopus laevis oocytes. Expression of InsP3R-1 increased the velocity of the propagating waves of intracellular Ca2+ release but did not affect the Ins(1,4,5)P3-induced entry of extracellular Ca2+ across the plasma membrane. In contrast, expression of intracellular Ca2+ but markedly increased the magnitude and duration of Ca2+ influx. Immunolocalization studied revealed InsP3R-3 at the endoplasmic reticulum, with a relatively stronger signal at or near the plasma membrane. The results suggest that changing the expression level of an InsP3R can alter the Ins(1,4,5)P3-mediated Ca2+ signal and that InsP3R-1 and InsP3R-3 may have different biological functions.

Animals↗
PubMed1996

Localization of epithelial sodium channel subunit mRNAs in adult rat lung by in situ hybridization.

The transport of Na+ through amiloride-sensitive sodium channels (ENaC) plays a major role in the absorption of fluid across the pulmonary epithelium. The proteins forming the ENaC channel are encoded by three genes in the rat (alpha-, beta-, and gamma-rENaC). According to Northern blot, all three subunit mRNAs were expressed in adult rat lung. Each subunit was expressed as a single transcript of approximately 3.7, 2.2, and 3.2 kb for alpha-, beta-, and gamma-rENaC, respectively. To localize the alpha-, beta-, and gamma-rENaC subunit mRNAs, we used in situ hybridization. Frozen and paraffin-embedded tissues were hybridized with sense and antisense 35S-labeled riboprobes. The alpha-rENaC mRNA was most abundant and was expressed diffusely in epithelia of the trachea, bronchi, bronchioles, and alveoli. At the alveolar level, alpha-rENaC was expressed in type II cells. The beta- and gamma-rENaC mRNAs were most abundant in the bronchial and bronchiolar epithelia. All three subunits were expressed in the renal cortical collecting duct in a pattern similar to that previously reported by other investigators. Thus the rENaC subunit mRNAs are expressed in regions of the lung where functional Na+ absorption is found. These results are consistent with an important role for ENaC in the absorption of Na+ and fluid across the pulmonary epithelium in all regions of the lung.

Animals↗
PubMed1996

Mutation of R555 in CFTR-delta F508 enhances function and partially corrects defective processing.

The most common cystic fibrosis mutation deletes phenylalanine 508 in CFTR (CFTR-F508). This mutation causes the loss of CFTR Cl- channel activity by disrupting biosynthetic processing so that mutant protein does not reach the plasma membrane. It also decreases the rate at which mutant channels open. To identify second-site mutations that could reverse the effects of delta F508, we used a chimeric yeast STE6/CFTR system bearing the delta F508 mutation. This chimera manifests defective mating. After mutagenesis of the first nucleotide-binding domain, we found that mutation R555K partially restored mating. However, it also increased mating in the chimera lacking the delta F508 mutation. When we introduced the R555K mutation into human CFTR-delta F508, we found that the loss of apical Cl- current caused by delta F508 was partially restored, predominantly due to a partial correction of the delta F508 processing defect. Analysis of single CFTR Cl- channels showed that the R555K mutation did not correct the prolonged closed time associated with delta F508, rather it increased activity of both wild-type and delta F508 channels by prolonging the duration of bursts of activity. These data suggest that the region around residue R555 in the first nucleotide-binding domain is important both in determining the ability of the delta F508 protein to be properly processed and in determining channel function.

ATP-Binding Cassette Transporters↗
PubMed1996

Immunofluorescence localization of the 90-kDa heat-shock protein to cytoskeleton.

The 90-kDa heat-shock protein, hsp90, is an abundant and conserved protein located predominantly in the cytoplasm. Previous reports have localized a portion of the hsp90 to microtubules and to microfilaments. Here, we show that colocalization of hsp90 with microtubules is seen after long-term but not short-term fixation of rat pulmonary endothelial cells with formaldehyde. Under conditions of methanol fixation, a significant portion of both hsp90 and the hsp90-associated protein p23 is present on fibrillar structures extending throughout the cytoplasm of endothelial cells, however, when cells are treated with colcemid under conditions that eliminate microtubules, the fibrils condense into bright rope-like bundles located in the immediate perinuclear area and extending toward the cell periphery. Identical images are observed with an antibody against vimentin and the pattern seen after colcemid treatment is classical for intermediate filaments. Preabsorption of the anti-hsp90 antibody with purified hsp90 prevents the immunofluorescence but preabsorption with purified p23 does not, and the reverse is the case for immunofluorescence produced by the anti-p23 antibody. These results suggest that hsp90 is able, either directly or indirectly via other proteins, to associate with both microtubules and microfilaments.

Animals↗
PubMed1996

Adenovirus-mediated generation of cAMP-stimulated Cl- transport in cystic fibrosis airway epithelia in vitro: effect of promoter and administration method.

Cystic fibrosis (CF) is a common autosomal recesses disease in which loss of CFTR-Cl- channel function of defective cAMP-stimulated Cl- transport transfer across airway epithelia. Recombinant adenoviruses have shown progress as vectors with which to transfer CFTR cDNA to CF airway epithelia. Here we investigated variables involved in adenovirus-mediated transfer of CFTR by measuring cAMP- stimulated Cl- transport in CF airway epithelia grown as monolayers on permeable filter supports. When we compared the effects of different promoters, we found that persistent correction of Cl- transport was obtained when the vector contained the E1a promoter, or to a lesser extent the PGK promoter. Vector containing the CMV promoter produced a greater initial cAMP-stimulated Cl- current, but the duration of correction was shorter and the infection procedure itself increased CFTR expression, suggesting that high input doses of virus stimulate expression. We compared the level of expression, measured with a beta-galactosidase reporter of CFTR mRNA, with CFTR-mediated Cl- transport. Even low levels of expression generated significant Cl- current and marked increases in expression produced only modest increments in Cl- current. Correction of the CF Cl- transport defect was also improved when the concentration of adenovirus vector was high and when the duration of contact with the epithelium was prolonged. These findings may help optimize the ability of adenovirus vectors encoding CFTR to correct the CF Cl- transport defect.

Adenoviridae↗
PubMed1995

Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel.

Liddle's syndrome is an inherited form of hypertension caused by mutations that truncate the C-terminus of human epithelial Na+ channel (hENaC) subunits. Expression of truncated beta and gamma hENaC subunits increased Na+ current. However, truncation did not alter single-channel conductance or open state probability, suggesting there were more channels in the plasma membrane. Moreover, truncation of the C-terminus of the beta subunit increased apical cell-surface expression of hENaC in a renal epithelium. We identified a conserved motif in the C-terminus of all three subunits that, when mutated, reproduced the effect of Liddle's truncations. Further, both truncation of the C-terminus and mutation of the conserved C-terminal motif increased surface expression of chimeric proteins containing the C-terminus of beta hENaC. Thus, by deleting a conserved motif, Liddle's mutations increase the number of Na+ channels in the apical membrane, which increases renal Na+ absorption and creates a predisposition to hypertension.

Amiloride↗
PubMed1995

Binding of the proline-rich region of the epithelial Na+ channel to SH3 domains and its association with specific cellular proteins.

The amiloride-sensitive epithelial Na+ channel (ENaC) is an important component of the Na(+)-reabsorption pathway in many epithelia. The identification of three subunits of ENaC (alpha, beta and gamma), as well as results from a number of functional and biochemical studies, suggests that functional Na+ channels are composed of a complex of proteins. To learn about possible interactions of the channel with other proteins, we studied the alpha-subunit of rat and human ENaC. We found that the proline-rich C-terminal domains of both rat and human alpha-ENaC, expressed as glutathione S-transferase fusion proteins, bound to SH3 domains in vitro. A 116 kDa protein from a human lung adenocarcinoma cell line (H441) was specifically bound by the human alpha-ENaC C-terminal fusion protein and by a shorter 18-amino acid proline-rich peptide derived from the larger fusion protein. The 116 kDa protein was not glycosylated and was not phosphorylated on tyrosine or by cyclic AMP-dependent protein kinase (PKA). A 134 kDa protein which was also bound by the human alpha-ENaC C-terminal fusion protein was a substrate for phosphorylation by PKA. These data suggest that the proline-rich C-terminal tail of alpha-ENaC may interact with other proteins that control its function, regulation or localization.

Adenocarcinoma↗
PubMed1995

Pyrophosphate stimulates wild-type and mutant cystic fibrosis transmembrane conductance regulator Cl- channels.

A unique feature of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is regulation by ATP through the two cytoplasmic nucleotide-binding domains (NBDs). To better understand this process, we asked how channel activity is affected by inorganic pyrophosphate (PPi), a compound that binds to NBDs in other proteins. PPi and three nonhydrolyzable PPi analogs reversibly stimulated the activity of phosphorylated channels. Kinetic modeling of single channel data demonstrated that PPi affected two distinct steps in channel regulation. First, PPi increased the rate at which channels opened. Second, once channels were open, PPi delayed their closure. PPi could only stimulate channels when it was applied in the presence of ATP. PPi also increased the photolabeling of CFTR by an ATP analog. These two findings suggest that PPi modifies the activity of ATP-dependent CFTR channel gating. Based on these and previous data, we speculate that the effects of PPi are mediated by binding of PPi to NBD2 where it regulates channel opening by NBD1, and then, because it is not hydrolyzed, it slows the rate of NBD2-mediated channel closing. Because PPi stimulated wild-type channels, we tested its effect on CFTR containing the cystic fibrosis mutations: delta F508, R117H, and G551S. PPi stimulated all three. PPi also stimulated endogenous CFTR in the apical membrane of permeabilized T-84 epithelia. These results suggest that PPi or an analog might be of value in the development of new approaches to the treatment of cystic fibrosis.

Adenosine Triphosphate↗
PubMed1995

Cellular and molecular barriers to gene transfer by a cationic lipid.

Cationic lipids are widely used for gene transfer in vitro and show promise as a vector for in vivo gene therapy applications. However, there is limited understanding of the cellular and molecular mechanisms involved. We investigated the individual steps in cationic lipid-mediated gene transfer to cultured cell lines. We used DMRIE/DOPE (a 1:1 mixture of N-[1-(2,3-dimyristyloxy) propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) as a model lipid because of its efficacy and because it is being used for clinical trials in humans. The data show that cationic lipid-mediated gene transfer is an inefficient process. Part of the inefficiency may result from the fact that the population of lipid-DNA complexes was very heterogeneous, even under conditions that have been optimized to produce the best transfection. Inefficiency was not due to inability of the complex to enter the cells because most cells took up the DNA. However, in contrast to previous speculation, the results indicate that endocytosis was the major mechanism of entry. After endocytosis, the lipid-DNA aggregated into large perinuclear complexes, which often showed a highly ordered tubular structure. Although much of the DNA remained aggregated in a vesicular compartment, there was at least a small amount of DNA in the cytoplasm of most cells. That observation plus results from direct injection of DNA and lipid-DNA into the nucleus and cytoplasm indicate that movement of DNA from the cytoplasm to the nucleus may be one of the most important limitations to successful gene transfer. Finally, before transcription can occur, the data show that lipid and DNA must dissociate. These results provide new insights into the physical limitations to cationic lipid-mediated gene transfer and suggest that attention to specific steps in the cellular process may further improve the efficiency of transfection and increase its use in a number of applications.

Cell Nucleus↗
PubMed1995

Changes in either cytosolic or nucleoplasmic inositol 1,4,5-trisphosphate levels can control nuclear Ca2+ concentration.

The free nucleoplasmic Ca2+ concentration ([Ca2+]n) may regulate many nuclear events, such as gene transcription. Since the nucleus may possess the enzymes necessary to generate the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), and because the nuclear envelope may enclose an Ins(1,4,5)P3-releasable Ca2+ store, we tested the hypothesis that nuclear and/or cytosolic levels of Ins(1,4,5)P3 can control [Ca2+]n. To assay [Ca2+]n, we measured the fluorescence of the Ca2+ indicator fluo 3 in the nucleus of Xenopus oocytes by confocal microscopy. When we injected Ins(1,4,5)P3 into the cytosol, [Ca2+]n increased. This increase in [Ca2]n still occurred when heparin was present in the nucleus, but was abolished when heparin was present in the cytosol, indicating that cytosolic Ins(1,4,5)P3 levels could control [Ca2+]n. When we injected Ins(1,4,5)P3 directly into the nucleus, [Ca2+]n increased, even when heparin was present in the cytosol, indicating that Ins(1,4,5)P3 could control [Ca2+]n from within the nucleus. These results provide functional evidence for Ins(1,4,5)P3 receptors facing the nucleoplasm and raise the possibility that a phosphoinositide cycle situated at the nuclear membranes can control Ca(2+)-dependent nuclear functions.

Aniline Compounds↗
PubMed1995

Mechanism of dysfunction of two nucleotide binding domain mutations in cystic fibrosis transmembrane conductance regulator that are associated with pancreatic sufficiency.

Variability in the severity of cystic fibrosis (CF) is in part due to specific mutations in the CF transmembrane conductance regulator (CFTR) gene. To understand better how mutations in CFTR disrupt Cl- channel function and to learn about the relationship between genotype and phenotype, we studied two CF mutants, A455E and P574H, that are associated with pancreatic sufficiency. A455E and P574H are located close to conserved ATP binding motifs in CFTR. Both mutants generated cAMP-stimulated apical membrane Cl- currents in heterologous epithelial cells, but current magnitudes were reduced compared with wild-type. Patch-clamp analysis revealed that both mutants had normal conductive properties and regulation by phosphorylation and nucleotides. These mutants had normal or increased Cl- channel activity: A455E had an open-state probability (Po) similar to wild-type, and P574H had an increased Po because bursts of activity were prolonged. However, both mutants produced less mature glycosylated protein, although levels were greater than observed with the delta F508 mutant. These changes in channel activity and processing provide a quantitative explanation for the reduced apical Cl- current. These data also dissociate structural requirements for channel function from features that determine processing. Finally, the results suggest that the residual function associated with these two mutants is sufficient to confer a milder clinical phenotype and infer approaches to developing treatments.

Adenosine Triphosphate↗
PubMed1995

The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel contains two cytoplasmic nucleotide-binding domains (NBDs). After phosphorylation of the R domain, ATP interacts with the NBDs to regulate channel activity. To learn how the NBDs regulate channel function, we used the patch-clamp technique to study CFTR and variants which contained site-directed mutations in the conserved Walker A motif lysine residues in either NBD1 (K464A), NBD2 (K1250A and K1250M), or both NBDs simultaneously (K464A/K1250A). Studies in related proteins suggest that such mutations slow the rate of ATP hydrolysis. These mutations did not alter the conductive properties of the channel or the requirement for phosphorylation and ATP to open the channel. However, all mutations decreased open state probability. Mutations in NBD1 decreased the frequency of bursts of activity, whereas mutations in NBD2 and mutations in both NBDs simultaneously prolonged bursts of activity, as well as decreased the frequency of bursts. These results could not be attributed to altered binding of nucleotide because none of the mutants studied had reduced 8-N3ATP binding. These data suggest that the two NBDs have distinct functions in channel gating; ATP hydrolysis at NBD1 initiates a burst of activity, and hydrolysis at NBD2 terminates a burst.

3T3 Cells↗
PubMed1995

The evolution of morbilliviruses: a comparison of nucleocapsid gene sequences including a porpoise morbillivirus.

Sequence data for the nucleocapsid protein (N) gene of the porpoise morbillivirus including the very conserved middle section of the protein and the hypervariable C terminus are reported. Analysis of dissimilarity indices based on an alignment of the N proteins of various morbilliviruses identifies a variable region of the N protein from amino acids residues 121 to 145 and a hypervariable part from amino acids 400 to 517. This type of analysis can be usefully applied when protein sequences of five or more morbillivirus species are available. Regions of variability between species identified by this index also represent regions of variation within one species e.g. measles virus (MV). Hence, comparative analysis of different morbilliviruses provides an insight into the potentially variable parts of viral proteins. From the great and unexplained nucleotide sequence conservation observed within MV, it would appear that the various morbilliviruses have diverged from each other a very long time ago. However, the data do not yet allow us to estimate the time span of these divergences. The relatedness and the number of different morbillivirus species provides a unique database for study of the evolution of RNA viruses.

Amino Acid Sequence↗
PubMed1995

Structural and functional similarities between the nucleotide-binding domains of CFTR and GTP-binding proteins.

The opening and closing of the CFTR Cl- channel are regulated by ATP hydrolysis at its two nucleotide binding domains (NBDs). However, the mechanism and functional significance of ATP hydrolysis are unknown. Sequence similarity between the NBDs of CFTR and GTP-binding proteins suggested the NBDs might have a structure and perhaps a function like that of GTP-binding proteins. Based on this similarity, we predicted that the terminal residue of the LSGGQ motif in the NBDs of CFTR corresponds to a highly conserved glutamine residue in GTP-binding proteins that directly catalyzes the GTPase reaction. Mutations of this residue in NBD1 or NBD2, which were predicted to increase or decrease the rate of hydrolysis, altered the duration of channel closed and open times in a specific manner without altering ion conduction properties or ADP-dependent inhibition. These results suggest that the NBDs of CFTR, and consequently other ABC transporters, may have a structure and a function analogous to those of GTP-binding proteins. We conclude that the rates of ATP hydrolysis at NBD1 and at NBD2 determine the duration of the two states of the channel, closed and open, much as the rate of GTP hydrolysis by GTP-binding proteins determines the duration of their active state.

ATP-Binding Cassette Transporters↗