Allergic airway inflammation induces a pro-secretory epithelial ion transport phenotype in mice.

The airway epithelium is a central effector tissue in allergic inflammation and T-helper cell (Th) type 2-driven epithelial responses, such as mucus hypersecretion contribute to airflow obstruction in allergic airway disease. Previous in vitro studies demonstrated that Th2 cytokines also act as potent modulators of epithelial ion transport and fluid secretion, but the in vivo effect of allergic inflammation on airway ion transport remains unknown. We, therefore, induced allergic inflammation by intratracheal instillation of Aspergillus fumigatus extract or interleukin-13 in mice and determined effects on ion transport in native tracheal and bronchial tissues. We demonstrate that allergic inflammation enhanced basal Cl(-) secretion in both airway regions and inhibited epithelial Na(+) channel (ENaC)-mediated Na(+) absorption and increased Ca²(+)-dependent Cl(-) secretion in bronchi. Allergen-induced alterations in bronchial ion transport were associated with reduced transcript levels of α-, ß- and γENaC, and were largely abrogated in signal transducer and activator of transcription (Stat)6(-/-) mice. Our studies demonstrate that Th2-dependent airway inflammation produced a pro-secretory ion transport phenotype in vivo, which was largely Stat6-dependent. These results suggest that Th2-mediated fluid secretion may improve airway surface hydration and clearance of mucus that is hypersecreted in allergic airway diseases such as asthma, and identify epithelial Stat6 signalling as a potential therapeutic target to promote mucus hydration and airway clearance.

The Caenorhabditis elegans unc-17 gene: a putative vesicular acetylcholine transporter.

Mutations in the unc-17 gene of the nematode Caenorhabditis elegans produce deficits in neuromuscular function. This gene was cloned and complementary DNAs were sequenced. On the basis of sequence similarity to mammalian vesicular transporters of biogenic amines and of localization to synaptic vesicles of cholinergic neurons in C. elegans, unc-17 likely encodes the vesicular transporter of acetylcholine. Mutations that eliminated all unc-17 gene function were lethal, suggesting that the acetylcholine transporter is essential. Molecular analysis of unc-17 mutations will allow the correlation of specific parts of the gene (and the protein) with observed functional defects. The mutants will also be useful for the isolation of extragenic suppressors, which could identify genes encoding proteins that interact with UNC-17.

Expression of multiple UNC-13 proteins in the Caenorhabditis elegans nervous system.

The Caenorhabditis elegans UNC-13 protein and its mammalian homologues are important for normal neurotransmitter release. We have identified a set of transcripts from the unc-13 locus in C. elegans resulting from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but that vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. We have analyzed the sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. We have also isolated a deletion allele that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products.

Dephasing and diffusion on the alveolar surface.

We propose a surface model of spin dephasing in lung tissue that includes both susceptibility and diffusion effects to provide a closed-form solution of the Bloch-Torrey equation on the alveolar surface. The nonlocal susceptibility effects of the model are validated against numerical simulations of spin dephasing in a realistic lung tissue geometry acquired from synchotron-based µCT data sets of mouse lung tissue, and against simulations in the well-known Wigner-Seitz model geometry. The free induction decay is obtained in dependence on microscopic tissue parameters and agrees very well with in vivo lung measurements at 1.5 Tesla to allow a quantification of the local mean alveolar radius. Our results are therefore potentially relevant for the clinical diagnosis and therapy of pulmonary diseases.

Regulation of neurotransmitter vesicles by the homeodomain protein UNC-4 and its transcriptional corepressor UNC-37/groucho in Caenorhabditis elegans cholinergic motor neurons.

Motor neuron function depends on neurotransmitter release from synaptic vesicles (SVs). Here we show that the UNC-4 homeoprotein and its transcriptional corepressor protein UNC-37 regulate SV protein levels in specific Caenorhabditis elegans motor neurons. UNC-4 is expressed in four classes (DA, VA, VC, and SAB) of cholinergic motor neurons. Antibody staining reveals that five different vesicular proteins (UNC-17, choline acetyltransferase, Synaptotagmin, Synaptobrevin, and RAB-3) are substantially reduced in unc-4 and unc-37 mutants in these cells; nonvesicular neuronal proteins (Syntaxin, UNC-18, and UNC-11) are not affected, however. Ultrastructural analysis of VA motor neurons in the mutant unc-4(e120) confirms that SV number in the presynaptic zone is reduced ( approximately 40%) whereas axonal diameter and synaptic morphology are not visibly altered. Because the UNC-4-UNC-37 complex has been shown to mediate transcriptional repression, we propose that these effects are performed via an intermediate gene. Our results are consistent with a model in which this unc-4 target gene ("gene-x") functions at a post-transcriptional level as a negative regulator of SV biogenesis or stability. Experiments with a temperature-sensitive unc-4 mutant show that the adult level of SV proteins strictly depends on unc-4 function during a critical period of motor neuron differentiation. unc-4 activity during this sensitive larval stage is also required for the creation of proper synaptic inputs to VA motor neurons. The temporal correlation of these events may mean that a common unc-4-dependent mechanism controls both the specificity of synaptic inputs as well as the strength of synaptic outputs for these motor neurons.

Heparan sulfate proteoglycans. Essential co-factors in receptor-mediated processes with relevance to the biology of the vascular wall.

Heparan sulfate (HS), a mixed bag of complex, heterogeneous and highly charged polysaccharides, is an essential co-factor in a large number of receptor-ligand interactions and cellular pathways. These co-factor functions depend on the binding-interactions of the HS chains with the ligand or receptor, or both. These binding interactions and the ensuing functional effects often depend on defined carbohydrate sequences within the HS chains, whereby the required sequences are not always represented within all natural forms of the polysaccharide. The proteins that are substituted with HS resort from a limited number of protein families, with different cellular, subcellular and supramolecular associations, and show differential activities in functional assays. It is likely that the natural co-factor functions of the HS proteoglycans depend on glycan-protein and protein-protein interactions that are subject to modulation, both at the glycan and protein levels.

Characterization of a basolateral electroneutral Na+/H+ antiporter in Atlantic lobster (Homarus americanus) hepatopancreatic epithelial vesicles

Purified basolateral membrane vesicles (BLMVs) were prepared from Atlantic lobster (Homarus americanus) hepatopancreas using a Percoll density gradient technique. Enrichments of the Na+/K+-ATPase and alkaline phosphatase activities of these vesicles were 15.4- and 1.2-fold, respectively. The presence of amiloride-sensitive Na+/H+ exchange was demonstrated. Contrary to electrogenic 2Na+/1H+ exchange on apical membranes from the same tissue, kinetic studies of Na+ transport by these basolateral membranes indicate an electroneutral antiport with a Km of 28±1.7 mmol l-1 and a Jmax of 1.74±0.13 µmol mg-1 min-1. Amiloride interacted at a single binding site (Ki=39 µmol l-1) and external Li+ was shown to be an effective competitive inhibitor of the exchange process (Ki=493 µmol l-1). The presence of a membrane-potential-sensitive, Na+-accepting ion channel was also demonstrated. The basolateral Na+/H+ exchanger physiologically resembles members of the NHE family of Na+/H+ antiporters described in vertebrates and departs from the apical electrogenic system previously described in lobster. Whether or not the basolateral Na+/H+ antiporter is an NHE isoform remains to be determined.

Ca2+ and Zn2+ are transported by the electrogenic 2Na+/1H+ antiporter in echinoderm gastrointestinal epithelium

45Ca2+ uptake by purified brush-border membrane vesicles of starfish (Pycnopodia helianthoides) pyloric ceca was stimulated by an outwardly directed H+ gradient and this stimulation was enhanced by the simultaneous presence of an induced membrane potential (inside negative; K+/valinomycin). External amiloride (competitive inhibitor; Ki=660 µmol l-1) and a monoclonal antibody raised against proteins associated with the lobster (Homarus americanus) electrogenic 2Na+/1H+ antiporter both inhibited approximately half of the proton-gradient-stimulated 45Ca2+ uptake. These results suggested that Ca2+ might be transported by the electrogenic antiporter and that the crustacean antibody was inhibitory to the exchange function in echinoderms, as was recently shown in crustacean epithelial brush-border membrane vesicles. Carrier-mediated 45Ca2+ influx by amiloride-sensitive and amiloride-insensitive systems displayed the following kinetic constants: (amiloride-sensitive) Kt=66±2 µmol l-1; Jmax=0.173±0.002 pmol µg-1 protein 8 s-1; (amiloride-insensitive) Kt=18±0.3 µmol l-1; Jmax=0.100±0.001 pmol µg-1 protein 8 s-1. Zn2+ was a mixed inhibitor of 45Ca2+ influx by carrier-mediated transport, displaying a Ki of 920 µmol l-1. Mn2+, Cu2+, Fe2+ and Mg2+ also inhibited 45Ca2+ uptake, but the mechanism(s) of inhibition by these other cations was not disclosed. An equilibrium shift experiment showed that both Na+ and Zn2+ were able to exchange with equilibrated 45Ca2+ in these vesicles, suggesting that both monovalent and divalent cations were able to enter pyloric cecal cells through a common carrier-mediated transport system. In addition, the echinoderm electrogenic system appeared to exhibit a molecular component recognized by the crustacean antibody that may imply a similar epitope in the two animals.

Effectiveness of the California 1990-1991 tobacco education media campaign.

The California Department of Health Services conducted a $28,600,000 tobacco education media campaign in 1990 and 1991. An independent evaluation of the media campaign featured four waves of data-gathering, one prior to the campaign's beginning and three at intervals thereafter. In all, 29,264 students in grades 4-12 and 6,785 adult smokers provided data for the evaluation. Through telephone interviews for adults and written questionnaires for students, these participants supplied information so that each person could be classified as exposed or unexposed to the media campaign's advertisements. Five criterion variables were used in the evaluation: campaign awareness, tobacco use, smokers' intention to quit, nonsmokers' intention to start, and attitudes toward smoking. Based chiefly on the differences between the results of waves 1 and 4, we believe the media campaign had a number of positive effects on California students. For adult smokers, the results were mixed.

Do anti-smoking media campaigns help smokers quit?

As part of an evaluation of the 1990-91 anti-tobacco media campaign carried out by the California Department of Health Services, a study was conducted among 417 regular smokers who had quit during the period of the media campaign. In brief telephone interviews, all respondents identified up to three events or experiences that had influenced them to quit. In response to uncued questions, 6.7 percent of those interviewed indicated that they had been influenced to quit by an advertisement they had seen or heard on radio, television, or billboards. In response to direct questions about the media campaign, 34.3 percent of the respondents indicated that the media campaign's advertisement had played a part in their decision to quit. Applying the 6.7 percentage to the number of Californians who quit smoking in 1990-91, it can be estimated that for 33,000 former smokers, the anti-tobacco media advertisements were an important stimulus in their quit decision. Multiplying the 34.3 percent by the number of former California smokers who quit in 1990-91, the estimate of former smokers for whom the media campaign's advertisements played at least some part in their decision to quit rises to 173,000 persons. While causal attributions from such investigations should be made with caution, the evidence suggests that the 1990-91 campaign did influence substantial number of smokers in California to quit.

Ion transport processes of crustacean epithelial cells.

Epithelial cells of the gut, antennal glands, integument, and gills of crustaceans regulate the movements of ions into and across these structures and thereby influence the concentrations of ions in the hemolymph. Specific transport proteins serving cations and anions are found on apical and basolateral cell membranes of epithelia in these tissues. In recent years, a considerable research effort has been directed at elucidating their physiological and molecular properties and relating these characteristics to the overall biology of the organisms. Efforts to describe ion transport in crustaceans have focused on the membrane transfer properties of Na+/H+ exchange, calcium uptake as it relates to the molt cycle, heavy metal sequestration and detoxification, and anion movements into and across epithelial cells. In addition to defining the properties and mechanisms of cation movements across specific cell borders, work over the past 5 yr has also centered on defining the molecular nature of certain transport proteins such as the Na+/H+ exchanger in gill and gut tissues. Monovalent anion transport proteins of the gills and gut have received attention as they relate to osmotic and ionic balance in euryhaline species. Divalent anion secretion events of the gut have been defined relative to potential roles they may have in hyporegulation of the blood and in hepatopancreatic detoxification events involving complexation with cationic metals.

An analysis of pH tolerance and substrate preference of isolated skeletal muscle mitochondria from Bufo marinus and Rana catesbeiana.

1. The effects of varying pH and substrate on isolated skeletal muscle mitochondria from Bufo marinus and Rana catesbeiana were investigated. 2. For both species, VO2 max significantly decreased at all pH < 7.3 (P < 0.05), while maximum values were observed at a pH range of 7.3-7.6 with B. marinus maintaining a greater VO2 max than R. catesbeiana. 3. Respiratory control values (RCR) decreased significantly at all pH < 6.9 for both species (P < 0.05). 4. Isolated mitochondria from both species were maintained at pH = 7.2 and O2 consumption measured under five separate substrate conditions. 5. A rank preference was established based upon state 3 and RCR values. 6. Substrate preference was identical for both species and interspecific comparisons revealed differences in state 3 respiration and coupling.

Three Drosophila mutations that block associative learning also affect habituation and sensitization.

Drosophila melanogaster has been cultured with shock to avoid various odors. Mutants that failed to learn this task have been isolated. Here we report tests on these mutants for more elementary types of behavioral plasticity--habituation and sensitization of a reflex. Fruit flies have taste receptors on their feet. When a starved, water-satiated fly has sucrose applied to one foot, it usually responds by extending its proboscis. In normal flies this feeding reflex shows habituation: application of sugar to one foot depresses responsiveness through the contralateral leg for at least 10 min. The reflex also shows brief sensitization application of concentrated sucrose solution to the proboscis increases subsequent responsiveness to tarsal stimulation for 2-5 min. In three associative learning mutants , the proboscis-extension reflex is present with a normal threshold but behavioral modulation of the response is altered. The dunce, turnip, and rutabaga mutants all habituate less than normal flies. In addition, sensitization wanes unusually rapidly in dunce and rutabaga flies, lasting less than a minute in the case of dunce.

Role of the invertebrate electrogenic 2Na+/1H+ antiporter in monovalent and divalent cation transport.

In recent years, an electrogenic 2Na+/1H+ antiporter has been identified in a variety of invertebrate epithelial brush-border membranes of gut, kidney and gill tissues. The antiporter differs significantly in its physiological properties from the electroneutral 1Na+/1H+ antiporter proposed for vertebrate cells. In all invertebrate cells examined, the antiporter displayed a 2:1 transport stoichiometry, responded to an induced transmembrane potential and exhibited a high binding affinity for the divalent cation Ca2+, which acted as a competitive inhibitor of Na+ transport. A monoclonal antibody specific for the crustacean electrogenic antiporter inhibited 2Na+/1H+ exchange, but was without effect on Na(+)-dependent D-glucose transport. Immunoreactivity was localized at hepatopancreatic brush-border and vacuolar membranes, antennal gland coelomosac podocytes and posterior gill epithelial cells-all locations were published reports described unique cation exchange kinetics. Significant fractions of Ca2+ transport into invertebrate cells across brush-border membranes occurred by an electrogenic, amiloride-sensitive exchange process, probably by the 2Na+/1H+ antiporter, and this transport was markedly inhibited by exogenous zinc and cadmium. A recently identified electroneutral, amiloride-sensitive, hepatopancreatic epithelial basolateral Na+/H+ antiporter was uninfluenced by the brush-border monoclonal antibody, exhibited an apparent 1:1 transport stoichiometry and possessed a minimal divalent cation specificity. Calcium transport at this epithelial pole occurred by the combination of a Ca2+/Na+ antiporter, an ATP-dependent Ca(2+)-ATPase and a verapamil-sensitive calcium channel. These crustacean brush-border and basolateral transporters may play significant roles in calcification and heavy metal detoxification.

Identified neurons in C. elegans coexpress vesicular transporters for acetylcholine and monoamines.

We have identified four neurons (VC4, VC5, HSNL, HSNR) in Caenorhabditis elegans adult hermaphrodites that express both the vesicular acetylcholine transporter and the vesicular monoamine transporter. All four of these cells are motor neurons that innervate the egg-laying muscles of the vulva. In addition, they all express choline acetyltransferase, the synthetic enzyme for acetylcholine. The distributions of the vesicular acetylcholine transporter and the vesicular monoamine transporter are not identical within the individual cells. In mutants deficient for either of these transporters, there is no apparent compensatory change in the expression of the remaining transporter. This is the first report of neurons that express two different vesicular neurotransmitter transporters in vivo.

Pioneer growth cone steering decisions mediated by single filopodial contacts in situ.

In grasshopper embryo limb buds, the sibling Ti1 pioneers are the first neurons to initiate axonogenesis. The pioneer growth cones migrate from the limb tip to the CNS along a in direction comprising discrete steering events. Filopodial exploration of the cellular terrain in the vicinity of the advancing growth cone appears to be important for steering. Some information is available on the identity of cells and cell types, on cell-surface characteristics, and on the involvement of basal lamina in these steering decisions. In the work reported here, we have used computer-enhanced fluorescence video microscopy to examine filopodial behavior and the process of growth cone migration and reorientation resulting from interactions with the normal guidance cues on the in situ substrate. We observed several different kinds of migration and steering events, which appear to be related to the absolute and relative affinities of the contacted substrates. On a relatively homogeneous substrate of intrasegmental epithelium, growth cones advance by extending veils between filopodia, as is commonly observed on uniform substrates in vitro. Where growth cones confront an orthogonal border between substrates of dissimilar affinity, they remain on the higher-affinity substrate by extending new branches along it. Subsequently, reorientation in the preferred direction on the higher-affinity substrate is accomplished by regression of branches extended in the nonselected direction. By contrast, a single filopodial contact with a very high-affinity substrate, such as a guidepost neuron, can reorient a growth cone, even when it is migrating on a favorable substrate. In this situation, the filopodium that contacts the high-affinity substrate expands in diameter until it becomes the nascent axon.

Neurogenetics of vesicular transporters in C. elegans.

The nematode Caenorhabditis elegans has a number of advantages for the analysis of synaptic molecules. These include a simple nervous system in which all cells are identified and synaptic connectivity is known and reproducible, a large collection of mutants and powerful methods of genetic analysis, simple methods for the generation and analysis of transgenic animals, and a number of relatively simple quantifiable behaviors. Studies in C. elegans have made major contributions to our understanding of vesicular transmitter transporters. Two of the four classes of vesicular transporters so far identified (VAChT and VGAT) were first described and cloned in C. elegans; in both cases, the genes were first identified and cloned by means of mutations causing a suggestive phenotype (1, 2). The phenotypes of eat-4 mutants and the cell biology of the EAT-4 protein were critical in the identification of this protein as the vesicular glutamate transporter (3, 4). In addition, the unusual gene structure associated with the cholinergic locus was first described in C. elegans (5). The biochemical properties of the nematode transporters are surprisingly similar to their vertebrate counterparts, and they can be assayed under similar conditions using the same types of mammalian cells (6, 7). In addition, mild and severe mutants (including knockouts) are available for each of the four C. elegans vesicular transporters, which has permitted a careful evaluation of the role(s) of vesicular transport in transmitter-specific behaviors. Accordingly, it seems appropriate at this time to present the current status of the field. In this review, we will first discuss the properties of C. elegans vesicular transporters and transporter mutants, and then explore some of the lessons and insights C. elegans research has provided to the field of vesicular transport.

Thermo-controlled device for inducing deep coagulation in the liver with the Nd:YAG laser.

BACKGROUND AND OBJECTIVE: To increase the effectiveness of laser-induced interstitial thermotherapy (LITT), a new thermo-controlled application system for minimal invasive intervention was designed. Our system consists of a laser applicator of 2.5 mm in diameter, insertion equipment, and a Nd:YAG-laser source. STUDY DESIGN/MATERIALS AND METHODS: A cylindrical light emitting fiber (1-6 cm in length) was placed in the center of the applicator. The surrounding tissue was irradiated through a Duran window at the distal end of the applicator. The power of the laser source was controlled dynamically by thermosensors in a water-cooling system of the laser applicator. The temperature at the surface of the Duran window was kept constant at approximately 60 degrees C, without charring the surrounding tissue. RESULTS/CONCLUSION: We obtained homogeneous coagulation zones. In in vitro experiments with pig livers, we reached ellipsoid coagulation volumes of 3 and 5 cm in diameter within 10 minutes, corresponding to a volume of approximately 25 cm3.