Nitric oxide reduces Cl- absorption in the mouse cortical collecting duct through an ENaC-dependent mechanism.

Since nitric oxide (NO) participates in the renal regulation of blood pressure, in part, by modulating transport of Na(+) and Cl(-) in the kidney, we asked whether NO regulates net Cl(-) flux (JCl) in the cortical collecting duct (CCD) and determined the transporter(s) that mediate NO-sensitive Cl(-) absorption. Cl(-) absorption was measured in CCDs perfused in vitro that were taken from aldosterone-treated mice. Administration of an NO donor (10 μM MAHMA NONOate) reduced JCl and transepithelial voltage (VT) both in the presence or absence of angiotensin II. However, reducing endogenous NO production by inhibiting NO synthase (100 μM N(G)-nitro-l-arginine methyl ester) increased JCl only in the presence of angiotensin II, suggesting that angiotensin II stimulates NO synthase activity. To determine the transport process that mediates NO-sensitive changes in JCl, we examined the effect of NO on JCl following either genetic ablation or chemical inhibition of transporters in the CCD. Since the application of hydrochlorothiazide (100 μM) or bafilomycin (5 nM) to the perfusate or ablation of the gene encoding pendrin did not alter NO-sensitive JCl, NO modulates JCl independent of the Na(+)-dependent Cl(-)/HCO3(-) exchanger (NDCBE, Slc4a8), the A cell apical plasma membrane H(+)-ATPase and pendrin. In contrast, both total and NO-sensitive JCl and VT were abolished with application of an epithelial Na(+) channel (ENaC) inhibitor (3 μM benzamil) to the perfusate. We conclude that NO reduces Cl(-) absorption in the CCD through a mechanism that is ENaC-dependent.

Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta.

Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

MALT1 auto-proteolysis is essential for NF-κB-dependent gene transcription in activated lymphocytes.

Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.

Application of real-time PCR for total airborne bacterial assessment : comparison with epifluorescence microscopy and culture-dependent methods

Traditional culture-dependent methods to quantify and identify airborne microorganisms are limited by factors such as short-duration sampling times and inability to count nonculturableor non-viable bacteria. Consequently, the quantitative assessment of bioaerosols is often underestimated. Use of the real-time quantitative polymerase chain reaction (Q-PCR) to quantify bacteria in environmental samples presents an alternative method, which should overcome this problem. The aim of this study was to evaluate the performance of a real-time Q-PCR assay as a simple and reliable way to quantify the airborne bacterial load within poultry houses and sewage treatment plants, in comparison with epifluorescencemicroscopy and culture-dependent methods. The estimates of bacterial load that we obtained from real-time PCR and epifluorescence methods, are comparable, however, our analysis of sewage treatment plants indicate these methods give values 270-290 fold greater than those obtained by the ''impaction on nutrient agar'' method. The culture-dependent method of air impaction on nutrient agar was also inadequate in poultry houses, as was the impinger-culture method, which gave a bacterial load estimate 32-fold lower than obtained by Q-PCR. Real-time quantitative PCR thus proves to be a reliable, discerning, and simple method that could be used to estimate airborne bacterial load in a broad variety of other environments expected to carry high numbers of airborne bacteria. [Authors]

Corticosterone mediates the condition-dependent component of melanin-based coloration

The handicap principle of sexual selection theory states that colourful phenotypic traits signal aspects of individual quality because only individuals in prime condition can afford to produce and bear conspicuous traits. Melanin-based pigments participate in the elaboration of many secondary sexual characters and, given their role in sexual selection, melanin-based coloration may therefore honestly reject individual quality. Although the expression of melanism is usually under genetic control, in some species it is condition dependent. However, the underlying physiological mechanism is yet unknown. Based on the negative feedback link between corticosterone and melanogenesis ( melanocortins, tyrosinase) in response to stressful environmental factors, we hypothesize that corticosterone mediates the condition-dependent component of melanism. This hypothesis predicts that stressful factors induce a rise in circulating corticosterone which inhibits the secretion of melanocortins and tyrosinase and in turn melanin production. We tested this prediction by manipulating the level of corticosterone at the time of melanin production in nestling barn owls, Tyto alba, a species showing heritable variation in the degree of phaeomelanism from reddish-brown to white. The finding that corticosterone-implanted nestlings produced feathers with less phaeomelanic coloration than placebo-implanted nestlings is consistent with the hypothesis that the environment-mediated reduction in the degree of melanism is, at least in part, caused by a rise in corticosterone. In species in which the expression of melanin-based coloration is condition dependent, we now need a test showing that individuals with less corticosterone and more melanin-based signals are individuals in better condition.

Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors controlling the expression of genes involved in lipid homeostasis. PPARs activate gene transcription in response to a variety of compounds including hypolipidemic drugs as well as natural fatty acids. From the plethora of PPAR activators, Scatchard analysis of receptor-ligand interactions has thus far identified only four ligands. These are the chemotactic agent leukotriene B4 and the hypolipidemic drug Wy 14,643 for the alpha-subtype and a prostaglandin J2 metabolite and synthetic antidiabetic thiazolidinediones for the gamma-subtype. Based on the hypothesis that ligand binding to PPAR would induce interactions of the receptor with transcriptional coactivators, we have developed a novel ligand sensor assay, termed coactivator-dependent receptor ligand assay (CARLA). With CARLA we have screened several natural and synthetic candidate ligands and have identified naturally occurring fatty acids and metabolites as well as hypolipidemic drugs as bona fide ligands of the three PPAR subtypes from Xenopus laevis. Our results suggest that PPARs, by their ability to interact with a number of structurally diverse compounds, have acquired unique ligand-binding properties among the superfamily of nuclear receptors that are compatible with their biological activity.

The Visual Dysphagia Questionnaire: Evaluating the first Tool for Assessing Food Consistency-Dependent Dysphagia in Eosinophilic Esophagitis

Background and Aims: The international EEsAI study group iscurrently developing the first a ctivity index specific forEosinophilic Esophagitis (EoE). None of the existing dysphagiaquestionnaires take into account the consistency of theingested food t hat considerably impacts the symptompresentation. Goal: To d evelop and evaluate an E oE-specificquestionnaire assessing dysphagia caused by foods of differentconsistencies.Methods: B ased on patient interviews and chart reviews, a nexpert panel ( EEsAI study g roup) identified internationallystandardizedfood prototypes t ypically a ssociated with EoErelateddysphagia. Food consistencies were c orrelated withEoE-related d ysphagia, t aking into account p otential f oodavoidance and f ood processing. This V isual D ysphagiaQuestionnaire (VDQ) was piloted in 20 patients and is currentlyevaluated in a cohort of 150 adult EoE patients.Results: T he following 8 food c onsistency prototypes w ereidentified: soft foods (pudding, jelly), grits, toast bread, Frenchfries, dry rice, ground meat, raw fibrous f oods (eg. apple,carrot), s olid m eat. Dysphagia was r anked o n a 4-point Likertscale (0=no difficulties; 3= severe difficulties, food will not pass).First analysis demonstrated that severity of dysphagia is relatedto the eosinophil load and presence of esophageal strictures.Conclusions: T he VDQ i s the first EoE-specific tool f orassessing dysphagia caused by i nternationally-standardizedfoods of different consistencies. This instrument also addressesfood avoidance behaviour and food processing habits. This toolperformed well in a p ilot study a nd is currently evaluated in acohort of 150 adult EoE patients.

Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.

New multicomponent capsules for immunoisolation.

A new generation of microcapsules based on the use of oligomers which participate in polyelectrolyte complexation reactions has been developed. These freeze-thaw stable capsules have been applied as a bioartificial pancreas and have resulted in normoglycemia for periods of six months in concordant xenotransplantations. The new chemistry permits the control of permeability and mechanical properties over a wide range and can be adapted both to microcapsule and hollow fiber geometries rendering it a robust tool for encapsulation in general. Methods, and metrics, for the characterization of the mechanical properties and permeability of microcapsules are presented.

Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.

Regulation of glucagon secretion by glucose transporter type 2 (glut2) and astrocyte-dependent glucose sensors.

Ripglut1;glut2-/- mice have no endogenous glucose transporter type 2 (glut2) gene expression but rescue glucose-regulated insulin secretion. Control of glucagon plasma levels is, however, abnormal, with fed hyperglucagonemia and insensitivity to physiological hypo- or hyperglycemia, indicating that GLUT2-dependent sensors control glucagon secretion. Here, we evaluated whether these sensors were located centrally and whether GLUT2 was expressed in glial cells or in neurons. We showed that ripglut1;glut2-/- mice failed to increase plasma glucagon levels following glucoprivation induced either by i.p. or intracerebroventricular 2-deoxy-D-glucose injections. This was accompanied by failure of 2-deoxy-D-glucose injections to activate c-Fos-like immunoreactivity in the nucleus of the tractus solitarius and the dorsal motor nucleus of the vagus. When glut2 was expressed by transgenesis in glial cells but not in neurons of ripglut1;glut2-/- mice, stimulated glucagon secretion was restored as was c-Fos-like immunoreactive labeling in the brainstem. When ripglut1;glut2-/- mice were backcrossed into the C57BL/6 genetic background, fed plasma glucagon levels were also elevated due to abnormal autonomic input to the alpha cells; glucagon secretion was, however, stimulated by hypoglycemic stimuli to levels similar to those in control mice. These studies identify the existence of central glucose sensors requiring glut2 expression in glial cells and therefore functional coupling between glial cells and neurons. These sensors may be activated at different glycemic levels depending on the genetic background.

Concentration-dependent half-lives of polychlorinated biphenyl in sera from an occupational cohort.

Polychlorinated biphenyls (PCBs) are carcinogenic. Estimating PCB half-life in the body based on levels in sera from exposed workers is complicated by the fact that occupational exposure to PCBs was to commercial PCB products (such as Aroclors 1242 and 1254) comprised of varying mixtures of PCB congeners. Half-lives were estimated using sera donated by 191 capacitor manufacturing plant workers in 1976 during PCB use (1946-1977), and post-exposure (1979, 1983, and 1988). Our aims were to: (1) determine the role of covariates such as gender on the half-life estimates, and (2) compare our results with other published half-life estimates based on exposed workers. All serum PCB levels were adjusted for PCB background levels. A linear spline model with a single knot was used to estimate two separate linear equations for the first two serum draws (Equation A) and the latter two (Equation B). Equation A gave half-life estimates of 1.74 years and 6.01 years for Aroclor 1242 and Aroclor 1254, respectively. Estimates were 21.83 years for Aroclor 1242 and 133.33 years for Aroclor 1254 using Equation B. High initial body burden was associated with rapid PCB elimination in workers at or shortly after the time they were occupationally exposed and slowed down considerably when the dose reached background PCB levels. These concentration-dependent half-life estimates had a transition point of 138.57 and 34.78 ppb for Aroclor 1242 and 1254, respectively. This result will help in understanding the toxicological and epidemiological impact of exposure to PCBs in humans.

ENaC inhibition stimulates Cl- secretion in the mouse cortical collecting duct through an NKCC1-dependent mechanism.

In cortical collecting ducts (CCDs) perfused in vitro, inhibiting the epithelial Na(+) channel (ENaC) reduces Cl(-) absorption. Since ENaC does not transport Cl(-), the purpose of this study was to determine how ENaC modulates Cl(-) absorption. Thus, Cl(-) absorption was measured in CCDs perfused in vitro that were taken from mice given aldosterone for 7 days. In wild-type mice, we observed no effect of luminal hydrochlorothiazide on either Cl(-) absorption or transepithelial voltage (V(T)). However, application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid or application of a Na(+)-K(+)-ATPase inhibitor to the bath reduced Cl(-) absorption by ∼66-75% and nearly obliterated lumen-negative V(T). In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1a(loxlox)). Whereas benzamil-sensitive Cl(-) absorption did not depend on CFTR, application of a Na(+)-K(+)-2Cl(-) cotransport inhibitor (bumetanide) to the bath or ablation of the gene encoding Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) blunted benzamil-sensitive Cl(-) absorption, although the benzamil-sensitive component of V(T) was unaffected. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl(-) absorption is benzamil sensitive, whereas thiazide-sensitive Cl(-) absorption is undetectable. Second, benzamil-sensitive Cl(-) absorption occurs by inhibition of ENaC, possibly due to elimination of lumen-negative V(T). Finally, benzamil-sensitive Cl(-) flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.