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Michael J Welsh

Publications and source records attributed to Michael J Welsh.

At least 19 recordsLinked to original sources
PubMed2006

Eukaryotic operon-like transcription of functionally related genes in Drosophila.

Complex biological processes require coordinated function of many genes. One evolutionary solution to the problem of coordinately expressing functionally related genes in bacteria and nematodes is organization of genes in operons. Surprisingly, eukaryotic operons are considered rare outside the nematode lineage. In Drosophila melanogaster, we found lounge lizard (llz), which encodes a degenerin/ENaC cation channel, cotranscribed with CheB42a, a nonhomologous gene of unknown function residing <100 bp upstream. These two genes were transcribed from a single promoter as one primary transcript and were processed posttranscriptionally to generate individual mRNAs. The mechanism did not involve alternative splicing, and it differed from the trans splicing used in nematode operons. Both genes were expressed in the same tissues, and previous work suggested that both may be involved in courtship behavior. A bioinformatic approach identified numerous additional loci as potential Drosophila operons. These data reveal eukaryotic operon-like transcription of functionally related genes in Drosophila. The results also suggest that operon-based transcription may be more common in eukaryotes than previously appreciated.

Animals↗
PubMed2006

Acid-sensing ion channel 1a is a postsynaptic proton receptor that affects the density of dendritic spines.

Extracellular proton concentrations in the brain may be an important signal for neuron function. Proton concentrations change both acutely when synaptic vesicles release their acidic contents into the synaptic cleft and chronically during ischemia and seizures. However, the brain receptors that detect protons and their physiologic importance remain uncertain. Using organotypic hippocampal slices and biolistic transfection, we found the acid-sensing ion channel 1a (ASIC1a), localized in dendritic spines where it functioned as a proton receptor. ASIC1a also affected the density of spines, the postsynaptic site of most excitatory synapses. Decreasing ASIC1a reduced the number of spines, whereas overexpressing ASIC1a had the opposite effect. Ca(2+)-mediated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) signaling was probably responsible, because acid evoked an ASIC1a-dependent elevation of spine intracellular Ca(2+) concentration, and reducing or increasing ASIC1a levels caused parallel changes in CaMKII phosphorylation in vivo. Moreover, inhibiting CaMKII prevented ASIC1a from increasing spine density. These data indicate that ASIC1a functions as a postsynaptic proton receptor that influences intracellular Ca(2+) concentration and CaMKII phosphorylation and thereby the density of dendritic spines. The results provide insight into how protons influence brain function and how they may contribute to pathophysiology.

Acid Sensing Ion Channels↗
PubMed2006

Abnormal small heat shock protein interactions involving neuropathy-associated HSP22 (HSPB8) mutants.

Two mutations (K141E, K141N) in the small heat shock protein (sHSP) HSP22 (HSPB8) are associated with the inherited peripheral motor neuron disorders distal hereditary motor neuropathy type II and axonal Charcot-Marie-Tooth disease type 2L. HSP22 is known to form homodimers, heterodimers with other sHSPs, and larger oligomers. In an effort to elucidate the cellular basis for these diseases, we have determined the ability of mutant HSP22 to interact with itself, with wild-type HSP22, and with other sHSPs that are abundant in neurons. Using the yeast two-hybrid method, quantitative fluorescence resonance energy transfer in live cells, and cross-linking, we found aberrantly increased interactions of mutant HSP22 forms with themselves, with wild-type HSP22, and with the other sHSPs, alphaB-crystallin, and HSP27. Interaction with HSP20 was not affected by the mutations. The data suggest that each mutant form of HSP22 has a characteristic pattern of abnormal interaction properties. A mutation (S135F) in HSP27 that is also associated with these disorders showed increased interaction with wild-type HSP22 also, suggesting linkage of these two etiologic factors, HSP22 and HSP27, into one common pathway. Increased interactions involving mutant sHSPs may be the molecular basis for their increased tendency to form cytoplasmic protein aggregates, and for the occurrence of the associated neuropathies.

Animals↗
PubMed2006

Acid-sensing ion channels: advances, questions and therapeutic opportunities.

Extracellular acid can have important effects on neuron function. In central and peripheral neurons, acid-sensing ion channels (ASICs) have emerged as key receptors for extracellular protons, and recent studies suggest diverse roles for these channels in the pathophysiology of pain, ischemic stroke and psychiatric disease. ASICs have also been implicated in mechanosensation in the peripheral nervous system and in neurotransmission in the central nervous system. Here, we briefly review advances in our understanding of ASICs, their potential contributions to disease, and the possibility for their therapeutic modification.

Acid Sensing Ion Channels↗
PubMed2006

Access to modern contraception.

Access to modern contraception has become a recognized human right, improving the health and well-being of women, families and societies worldwide. However, contraceptive access remains uneven. Irregular contraceptive supply, limited numbers of service delivery points and specific geographic, economic, informational, psychosocial and administrative barriers (including medical barriers) undermine access in many settings. Widening the range of providers enabled to offer contraception can improve contraceptive access, particularly where resources are most scarce. International efforts to remove medical barriers include the World Health Organization's Medical Eligibility Criteria. Based on the best available evidence, these criteria provide guidance for weighing the risks and benefits of contraceptive choice among women with specific clinical conditions. Clinical job aids can also improve access. More research is needed to further elucidate the pathways for expanding contraceptive access. Further progress in removing medical barriers will depend on systems for improving provider education and promoting evidence-based contraceptive service delivery.

Contraception↗
PubMed2006

Conformational changes resulting from pseudophosphorylation of mammalian small heat shock proteins--a two-hybrid study.

The human genome codes for 10 so-called mammalian small heat shock or stress proteins (sHsp) with the various tissues expressing characteristic sets of sHsps. Most sHsps interact with each other and form homo- and heterooligomeric complexes. Some of the sHsps are phosphoproteins in vivo, and phosphorylation has been implicated in the regulation of complex size and composition. In this study, we analyze, by the 2-hybrid method, the reporter gene activation pattern of several sHsp pairs that previously have been demonstrated to interact. We show that pseudophosphorylation (mimicry of phosphorylation) of the homologous phosphorylation sites Ser15 and Ser16 in Hsp27 and Hsp20, respectively, modulates characteristics of these sHsps that can be detected by their ability to activate reporter genes in suitable 2-hybrid assays. Pseudophosphorylation of the separated N-terminus of Hsp27 alone is not sufficient for the activation of the reporter genes, whereas the separated C-terminus is sufficient. We conclude that pseudophosphorylation of Hsp27 and Hsp20 at their N-termini results in conformational changes that can be detected by their interaction with other sHsps. Pseudophosphorylation of alphaB-crystallin at Ser19, in contrast, had no detectable consequences.

Amino Acid Sequence↗
PubMed2005

erbB1 functions as a sensor of airway epithelial integrity by regulation of protein phosphatase 2A activity.

Two enzymes, protein phosphatase 2A and atypical protein kinase C, are associated with the tight junction and regulate its function. For example, phosphorylation of the tight junction protein occludin is required for its incorporation into the junction. The association of a kinase and phosphatase with the tight junction suggests that a balance between their activities exists and is required for normal tight junction function. This hypothesis predicts that loss of epithelial integrity may disrupt this balance in such a way as to facilitate restoration of epithelial integrity. Our previous data have shown that apically localized growth factors segregate from their basolaterally localized erbB receptors. Loss of epithelial integrity allows ligand access to the basolateral membrane where it immediately binds to and activates erbB receptors. We found that activation of erbB1 leads to phosphorylation of protein phosphatase 2A, inhibiting its activity. Importantly, this phosphorylation event was dependent on factors in the overlying airway surface liquid; washing away this liquid prevented phosphorylation. erbB1-mediated inhibition of phosphatase activity would shift the balance in favor of the kinase such that tight junction proteins would regain their phosphorylation, allowing for their incorporation into the junction complex. This mechanism provides a rapid means of sensing the loss of epithelial integrity and subsequently restoring barrier function.

ErbB Receptors↗
PubMed2005

Interactions of HSP22 (HSPB8) with HSP20, alphaB-crystallin, and HSPB3.

Seven of the 10 mammalian small heat shock proteins (sHSP) are expressed in muscle where they constitute 3% or more of total protein. sHSPs interact with one another, and these interactions are believed to be important for their functions. In cell types expressing multiple sHSPs, it is of interest to know which sHSPs interact with one another. We have previously shown that HSP22 interacts with itself as well as with HSP27, MKBP, and cvHSP. Using yeast two-hybrid assays and Förster resonance energy transfer microscopy, we now show that HSP22 also can interact with two additional members of the sHSP family, alphaB-crystallin and HSP20. We also show that HSP22 is found in HPLC fractions of primate cardiac muscle containing high molecular weight complexes that include alphaB-crystallin and HSP20. Our results suggest that a variety of oligomers composed of different proportions of different sHSPs may form in cell types expressing multiple sHSPs.

Binding Sites↗
PubMed2005

Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR.

Gene transfer of CFTR cDNA to airway epithelia is a promising approach to treat cystic fibrosis (CF). Most gene transfer vectors use strong viral promoters even though the endogenous CFTR promoter is very weak. To learn whether expressing CFTR at a low level in a fraction of cells would correct Cl(-) transport, we mixed freshly isolated wild-type and CF airway epithelial cells in varying proportions and generated differentiated epithelia. Epithelia with approximately 20% wild-type cells generated approximately 70% the transepithelial Cl(-) current of epithelia containing 100% wild-type cells. These data were nearly identical to those previously obtained with CFTR expressed under control of a strong promoter in a CF epithelial cell line. We also tested high level CFTR expression using the very strong cytomegalovirus (CMV) promoter as well as the cytokeratin-18 (K18) promoter. In differentiated airway epithelia, the CMV promoter generated 50-fold more transgene expression than the K18 promoter, but the K18 promoter generated more transepithelial Cl(-) current at high vector doses. Using functional studies, we found that with marked overexpression, some CFTR channels were present in the basolateral membrane where they shunted Cl(-) flow, thereby reducing net transepithelial Cl(-) transport. These results suggest that very little CFTR is required in a fraction of CF epithelial cells to complement Cl(-) transport because transepithelial Cl(-) flow is limited at the basolateral membrane. Thus they suggest a broad leeway in promoter strength for correcting the CF gene transfer, although at very high expression levels CFTR may be mislocalized to the basolateral membrane.

Bronchi↗
PubMed2005

Lysozyme secretion by submucosal glands protects the airway from bacterial infection.

Submucosal glands are abundant (approximately 1 gland/mm2) secretory structures in the tracheobronchial airways of the human lung. Because submucosal glands express antibacterial proteins, it has been proposed that they contribute to lung defense. However, this concept is challenged by the fact that mice do not have submucosal glands in their bronchial airways, yet are quite resistant to bacterial lung infection. The contribution of airway submucosal glands to host defense is also debated as a pathophysiologic component of cystic fibrosis lung disease. Here, we asked whether submucosal glands protect airways against bacterial infection. By comparing tracheal xenograft airways with and without glands, we found that the presence of glands enhanced bacterial killing in vivo and by airway secretions in vitro. Moreover, immunodepletion studies suggested that lysozyme is a major antibacterial component secreted by submucosal glands. These studies provide evidence that submucosal glands are a major source of antibacterials critical for maintaining sterile airways.

Animals↗
PubMed2005

A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia.

Adeno-associated viruses (AAVs) such as AAV5 that transduce airway epithelia from the apical surface are attractive vectors for gene transfer in cystic fibrosis (CF). However, their utility in CF has been limited because packaging of the insert becomes inefficient when its length exceeds approximately 4,900-5,000 bp. To partially circumvent this size constraint, we previously developed a CF transmembrane conductance regulator (CFTR) transgene that deleted a portion of the R domain (CFTRDeltaR). In this study, we focused on shortening the other elements in the AAV expression cassette. We found that portions of the CMV immediate/early (CMVie) enhancer/promoter could be deleted without abolishing activity. We also tested various intervening sequences, poly(A) signals, and an intron to develop an expression cassette that meets the size restrictions imposed by AAV. We then packaged these shortened elements with the CFTRDeltaR transgene into AAV5 and applied them to the apical surface of differentiated CF airway epithelia. Two to 4 weeks later, the AAV5 vectors partially corrected the CF Cl(-) transport defect. These results demonstrate that a single AAV vector can complement the CF defect in differentiated airway epithelia and thereby further the development of effective CF gene transfer.

Cells, Cultured↗
PubMed2005

p38 MAPK/HSP25 signaling mediates cadmium-induced contraction of mesangial cells and renal glomeruli.

The environmental pollutant cadmium affects human health, with the kidney being a primary target. In addition to proximal tubules, glomeruli and their contractile mesangial cells have also been identified as targets of cadmium nephrotoxicity. Glomerular contraction is thought to contribute to reduced glomerular filtration, a characteristic of cadmium nephrotoxicity. Because p38 MAPK/HSP25 signaling has been implicated in smooth muscle contraction, we examined its role in cadmium-induced contraction of mesangial cells. We report that exposure of mesangial cells to cadmium resulted in 1) cell contraction, 2) activation of MAP kinases, 3) increased HSP25 phosphorylation coincident with p38 MAP kinase activation, 4) sequential phosphorylation of the two phosphorylation sites of mouse HSP25 with Ser15 being phosphorylated before Ser86, 5) reduction of oligomeric size of HSP25, and 6) association of HSP25 with microfilaments. Exposure of isolated rat glomeruli to cadmium also resulted in contraction and increased HSP25 phosphorylation. The cadmium-induced responses were inhibited by the specific p38 MAP kinase inhibitor SB-203580, and cadmium-induced phosphorylation of HSP25 was inhibited by expression of a dominant-negative p38 MAP kinase mutant. These findings tentatively suggest that cadmium-induced nephrotoxicity results, in part, from glomerular contraction due to p38 MAP kinase/HSP25 signaling-dependent contraction of mesangial cells. With regard to the cellular action of HSP25, these data support a change in paradigm: in addition to its well-established cytoprotective function, HSP25 may also be involved in processes that ultimately lead to adverse effects, as is observed in the response of mesangial cells to cadmium.

Actin Cytoskeleton↗
PubMed2005

ADP inhibits function of the ABC transporter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity.

ADP interacts with the nucleotide-binding domains (NBDs) of the cystic fibrosis transmembrane conductance regulator (CFTR) to inhibit its Cl- channel activity. Because CFTR NBD2 has reversible adenylate kinase activity (ATP + AMP<==> ADP + ADP) that gates the channel, we asked whether ADP might inhibit current through this enzymatic activity. In adenylate kinases, binding of the two ADP molecules is cooperative. Consistent with this hypothesis, CFTR current inhibition showed positive cooperativity for ADP. We also found that ADP inhibition of current was attenuated when we prevented adenylate kinase activity with P1,P5-di(adenosine-5') pentaphosphate. Additional studies suggested that adenylate kinase-dependent inhibition involved phosphotransfer between two nucleotide diphosphates. These data indicate that the adenylate kinase reaction at NBD2 contributed to the inhibitory effect of ADP. Finding that ADP inhibits function via an adenylate kinase activity also helps explain the earlier observation that mutations that disrupt adenylate kinase activity also disrupt ADP inhibition. Thus, the results reveal a previously unrecognized mechanism by which ADP inhibits an ABC transporter.

Adenosine Diphosphate↗
PubMed2004

Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain.

ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.

Adenosine Triphosphate↗
PubMed2004

Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity.

Compounds that enhance either the function or biosynthetic processing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel may be of value in developing new treatments for cystic fibrosis (CF). Previous studies suggested that the herbal extract curcumin might affect the processing of a common CF mutant, CFTR-DeltaF508. Here, we tested the hypothesis that curcumin influences channel function. Curcumin increased CFTR channel activity in excised, inside-out membrane patches by reducing channel closed time and prolonging the time channels remained open. Stimulation was dose-dependent, reversible, and greater than that observed with genistein, another compound that stimulates CFTR. Curcumin-dependent stimulation required phosphorylated channels and the presence of ATP. We found that curcumin increased the activity of both wild-type and DeltaF508 channels. Adding curcumin also increased Cl(-) transport in differentiated non-CF airway epithelia but not in CF epithelia. These results suggest that curcumin may directly stimulate CFTR Cl(-) channels.

Animals↗
PubMed2004

Sodium arsenite exposure alters cell migration, focal adhesion localization and decreases tyrosine phosphorylation of focal adhesion kinase in H9C2 myoblasts.

Exposure to the environmental toxicant arsenic is reported to produce a variety of effects including disruption of signal transduction pathways, cell proliferation, and apoptosis. This suggests that arsenite may not have specific targets but rather extremely broad effects. The present study was designed to test the hypothesis that arsenite alters signaling involved in focal adhesion structure and function in cultured myoblasts. H9C2 cells were exposed to 1, 2.5, 5, or 10 microM sodium arsenite for 48 h. MTT metabolism and staining by neutral red, trypan blue, and propidium iodide showed that sodium arsenite treatments of 5 microM or less were not overtly cytotoxic. At these doses, sodium arsenite did not affect the amount of polymerized actin in cells, rate of protein synthesis, or amounts of vinculin, talin, paxillin, and focal adhesion kinase (FAK) in cells. However, sodium arsenite-treated cells contained fewer focal adhesions with an altered distribution pattern. Sodium arsenite exposure caused a dose-dependent reduction in cell migration and cell attachment rates. The average area of substrate covered by a cell was also reduced, although the average volume of cells was not significantly affected. Sodium arsenite exposure resulted in reduced tyrosine phosphorylation of FAK, its substrate paxillin and the FAK auto- phosphorylation site, Tyr397. Our results indicate that sodium arsenite can alter focal adhesion structure and function, thus affecting cell attachment and migration and possibly other aspects of focal adhesion function such as integrin signaling. These diverse consequences may be mediated by a relatively specific inhibition of FAK tyrosine phosphorylation, modifying scaffolding proteins.

Animals↗
PubMed2004

Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels.

Ca2+ toxicity remains the central focus of ischemic brain injury. The mechanism by which toxic Ca2+ loading of cells occurs in the ischemic brain has become less clear as multiple human trials of glutamate antagonists have failed to show effective neuroprotection in stroke. Acidosis is a common feature of ischemia and is assumed to play a critical role in brain injury; however, the mechanism(s) remain ill defined. Here, we show that acidosis activates Ca2+ -permeable acid-sensing ion channels (ASICs), inducing glutamate receptor-independent, Ca2+ -dependent, neuronal injury inhibited by ASIC blockers. Cells lacking endogenous ASICs are resistant to acid injury, while transfection of Ca2+ -permeable ASIC1a establishes sensitivity. In focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene protects the brain from ischemic injury and does so more potently than glutamate antagonism. Thus, acidosis injures the brain via membrane receptor-based mechanisms with resultant toxicity of [Ca2+]i, disclosing new potential therapeutic targets for stroke.

Acid Sensing Ion Channels↗