Concerted action of ENaC, Nedd4-2, and Sgk1 in transepithelial Na(+) transport.

The epithelial Na(+) channel (ENaC), located in the apical membrane of renal aldosterone-responsive epithelia, plays an essential role in controlling the Na(+) balance of extracellular fluids and hence blood pressure. As of now, ENaC is the only Na(+) transport protein for which genetic evidence exists for its involvement in the genesis of both hypertension (Liddle's syndrome) and hypotension (pseudohypoaldosteronism type 1). The regulation of ENaC involves a variety of hormonal signals (aldosterone, vasopressin, insulin), but the molecular mechanisms behind this regulation are mostly unknown. Two regulatory proteins have gained interest in recent years: the ubiquitin-protein ligase neural precursor cell-expressed, developmentally downregulated gene 4 isoform Nedd4-2, which negatively controls ENaC cell surface expression, and serum glucocorticoid-inducible kinase 1 (Sgk1), which is an aldosterone- and insulin-dependent, positive regulator of ENaC density at the plasma membrane. Here, we summarize present ideas about Sgk1 and Nedd4-2 and the lines of experimental evidence, suggesting that they act sequentially in the regulatory pathways governed by aldosterone and insulin and regulate ENaC number at the plasma membrane.

Analysis of the expression of toll-like receptors 2 and 4 and cytokine production during experimental Leishmania chagasi infection

Toll-like receptors (TLRs) recognise pathogen-derived molecules and influence immunity to control parasite infections. This study aimed to evaluate the mRNA expression of TLRs 2 and 4, the expression and production of the cytokines interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17, IL-10 and transforming growth factor (TGF)-β and the production of nitric oxide (NO) in the spleen of mice infected with Leishmania chagasi. It also aimed to evaluate any correlations between mRNA expression TLR2 and 4 and cytokines and NO production. Infection resulted in increased TLR2-4, IL-17, TNF-α and TGF-β mRNA expression during early infection, with decreased expression during late infection correlating with parasite load. IFN-γ and IL-12 mRNA expression decreased at the peak of parasitism. IL-10 mRNA expression increased throughout the entire time period analysed. Although TGF-β, TNF-α and IL-17 were highly produced during the initial phase of infection, IFN-γ and IL-12 exhibited high production during the final phase of infection. IL-10 and NO showed increased production throughout the evaluated time period. In the acute phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17, NO, IL-10 and TGF-β expression and parasite load. During the chronic phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17 and TGF-β expression and parasite load. Our data suggest that infection by L. chagasi resulted in modulation of TLRs 2 and 4 and cytokines.

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

Interaction of Leukocyte Elastase Inhibitor/L-DNase II with BCL-2 and BAX

Leukocyte Elastase Inhibitor (LEI, also called serpin B1) is a protein involved in apoptosis among other physiological processes. We have previously shown that upon cleavage by its cognate protease, LEI is transformed into L-DNase II, a protein with a pro-apoptotic activity. The caspase independent apoptotic pathway, in which L-DNase II is the final effector, interacts with other pro-apoptotic molecules like Poly-ADP-Ribose polymerase (PARP) or Apoptosis Inducing Factor (AIF). The screening of LEI/L-DNase II interactions showed a possible interaction with several members of the BCL-2 family of proteins which are known to have a central role in the regulation of caspase dependent cell death. In this study, we investigated the regulation of LEI/L-DNase II pathway by two members of this family of proteins: BAX and BCL-2, which have opposite effects on cell survival. We show that, in both BHK and HeLa cells, LEI/L-DNase II can interact with BCL-2 and BAX in apoptotic and non-apoptotic conditions. These proteins which are usually thought to be anti-apoptotic and pro-apoptotic respectively, both inhibit the L-DNase II pro-apoptotic activity. These results give further insight in the regulation of caspase-independent pathways and highlight the involvement of the intracellular environment of a given protein in the determinism of its function. They also add a link between caspase-dependent and independent pathways of apoptosis.

An autoregulatory loop controlling CYP1A1 gene expression: role of H(2)O(2) and NFI.

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H(2)O(2) catabolism), thus implying that H(2)O(2) is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H(2)O(2), a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H(2)O(2) production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell.

New electrocardiographic criteria for discriminating between Brugada types 2 and 3 patterns and incomplete right bundle branch block.

OBJECTIVES: The aim of this study was to evaluate new electrocardiographic (ECG) criteria for discriminating between incomplete right bundle branch block (RBBB) and the Brugada types 2 and 3 ECG patterns. BACKGROUND: Brugada syndrome can manifest as either type 2 or type 3 pattern. The latter should be distinguished from incomplete RBBB, present in 3% of the population. METHODS: Thirty-eight patients with either type 2 or type 3 Brugada pattern that were referred for an antiarrhythmic drug challenge (AAD) were included. Before AAD, 2 angles were measured from ECG leads V(1) and/or V(2) showing incomplete RBBB: 1) α, the angle between a vertical line and the downslope of the r'-wave, and 2) β, the angle between the upslope of the S-wave and the downslope of the r'-wave. Baseline angle values, alone or combined with QRS duration, were compared between patients with negative and positive results on AAD. Receiver-operating characteristic curves were constructed to identify optimal discriminative cutoff values. RESULTS: The mean β angle was significantly smaller in the 14 patients with negative results on AAD compared to the 24 patients with positive results on AAD (36 ± 20° vs. 62 ± 20°, p < 0.01). Its optimal cutoff value was 58°, which yielded a positive predictive value of 73% and a negative predictive value of 87% for conversion to type 1 pattern on AAD; α was slightly less sensitive and specific compared with β. When the angles were combined with QRS duration, it tended to improve discrimination. CONCLUSIONS: In patients with suspected Brugada syndrome, simple ECG criteria can enable discrimination between incomplete RBBB and types 2 and 3 Brugada patterns.

Role for inducible cAMP early repressor in promoting pancreatic beta cell dysfunction evoked by oxidative stress in human and rat islets.

AIMS/HYPOTHESIS: Pro-atherogenic and pro-oxidant, oxidised LDL trigger adverse effects on pancreatic beta cells, possibly contributing to diabetes progression. Because oxidised LDL diminish the expression of genes regulated by the inducible cAMP early repressor (ICER), we investigated the involvement of this transcription factor and of oxidative stress in beta cell failure elicited by oxidised LDL. METHODS: Isolated human and rat islets, and insulin-secreting cells were cultured with human native or oxidised LDL or with hydrogen peroxide. The expression of genes was determined by quantitative real-time PCR and western blotting. Insulin secretion was monitored by EIA kit. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Exposure of beta cell lines and islets to oxidised LDL, but not to native LDL raised the abundance of ICER. Induction of this repressor by the modified LDL compromised the expression of important beta cell genes, including insulin and anti-apoptotic islet brain 1, as well as of genes coding for key components of the secretory machinery. This led to hampering of insulin production and secretion, and of cell survival. Silencing of this transcription factor by RNA interference restored the expression of its target genes and alleviated beta cell dysfunction and death triggered by oxidised LDL. Induction of ICER was stimulated by oxidative stress, whereas antioxidant treatment with N-acetylcysteine or HDL prevented the rise of ICER elicited by oxidised LDL and restored beta cell functions. CONCLUSIONS/INTERPRETATION: Induction of ICER links oxidative stress to beta cell failure caused by oxidised LDL and can be effectively abrogated by antioxidant treatment.

Mitofusins 1/2 and ERRalpha expression are increased in human skeletal muscle after physical exercise

Mitochondrial impairment is hypothesized to contribute to the pathogenesis of insulin resistance. Mitofusin (Mfn) proteins regulate the biogenesis and maintenance of the mitochondrial network, and when inactivated, cause a failure in the mitochondrial architecture and decreases in oxidative capacity and glucose oxidation. Exercise increases muscle mitochondrial content, size, oxidative capacity and aerobic glucose oxidation. To address if Mfn proteins are implicated in these exercise-induced responses, we measured Mfn1 and Mfn2 mRNA levels, pre-, post-, 2 and 24 h post-exercise. Additionally, we measured the expression levels of transcriptional regulators that control mitochondrial biogenesis and functions, including PGC-1alpha, NRF-1, NRF-2 and the recently implicated ERRalpha. We show that Mfn1, Mfn2, NRF-2 and COX IV mRNA were increased 24 h post-exercise, while PGC-1alpha and ERRalpha mRNA increased 2 h post-exercise. Finally, using in vitro cellular assays, we demonstrate that Mfn2 gene expression is driven by a PGC-1alpha programme dependent on ERRalpha. The PGC-1alpha/ERRalpha-mediated induction of Mfn2 suggests a role of these two factors in mitochondrial fusion. Our results provide evidence that PGC-1alpha not only mediates the increased expression of oxidative phosphorylation genes but also mediates alterations in mitochondrial architecture in response to aerobic exercise in humans

Effectiveness Of 2% And 4% Papain Gels In The Healing Of Venous Ulcers

OBJECTIVE To analyze the effectiveness of 2% and 4% papain gels in tissue repair of venous ulcers. METHOD Quasi-experimental study with consecutive sample of 16 patients with 30 venous ulcers treated at the outpatient clinic of a teaching hospital, from April to November in 2011, using a form for clinical assessment of the patient and its lesion. Variables were analyzed by Wilcoxon and McNemar test (p < 0.05). RESULTS Most participants were female; aged between 51 and 59 years; obese; with hypertension. Regarding ulcers, there was an average decrease of 7.9 cm2 (50% of its original size) in 90 days; 20% of the ulcers completely healed within 56.67 days. There was an increase in epithelialization, significant reduction in the slough and edema, improved depth, in the type and amount of exudate (p < 0.0001). CONCLUSION 2% and 4% papain gels were effective in healing venous ulcers.

Similarities and dissimilarities between the Movement ABC-2 and the Zurich Neuromotor Assessment in children with suspected developmental coordination disorder.

An established tool for the assessment of motor performance in children with developmental coordination disorder (DCD) is the Movement-ABC-2 (M-ABC-2). The Zurich Neuromotor Assessment (ZNA) is also widely used for the evaluation of children's motor performance, but has not been compared with the M-ABC-2. Fifty-one children (39 males) between 5 and 7 years of age with suspected DCD were assessed using the M-ABC-2 and the ZNA. Rank correlations between scores of different test components were calculated. The structure of the tests was explored using canonical-correlation analysis. The correlation between total scores of the two motor tests was reasonable (0.66; p<0.001). However, ZNA scores were generally lower than those of M-ABC-2, due to poor performance in the fine motor adaptive component and increased contralateral associated movements (CAM). The canonical-correlation analysis revealed that ZNA measures components like pure motor skills and CAM that are not represented in the M-ABC-2. Furthermore, there was also no equivalent for the aiming and catching items of the M-ABC-2 in ZNA. The two tests measure different motor characteristics in children with suspected DCD and, thus, can be used complementary for the diagnosis of the disorder.

Prostaglandin E2 promotes integrin alpha Vbeta 3-dependent endothelial cell adhesion, rac-activation, and spreading through cAMP/PKA-dependent signaling.

We have recently reported that the inhibition of endothelial cell COX-2 by non-steroidal anti-inflammatory drugs suppresses alpha(V)beta(3)- (but not alpha(5)beta(1)-) dependent Rac activation, endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047). Here we investigated the role of the COX-2 metabolites PGE(2) and TXA2 in regulating human umbilical vein endothelial cell (HUVEC) adhesion and spreading. We report that PGE(2) accelerated alpha(V)beta(3)-mediated HUVEC adhesion and promoted Rac activation and cell spreading, whereas the TXA2 agonist retarded adhesion and inhibited spreading. We show that the cAMP level and the cAMP-regulated protein kinase A (PKA) activity are critical mediators of these PGE(2) effects. alpha(V)beta(3)-mediated adhesion induced a transient COX-2-dependent rise in cAMP levels, whereas the cell-permeable cAMP analogue 8-brcAMP accelerated adhesion, promoted Rac activation, and cell spreading in the presence of the COX-2 inhibitor NS-398. Pharmacological inhibition of PKA completely blocked alpha(V)beta(3)-mediated adhesion. A constitutively active Rac mutant (L61Rac) rescued alpha(V)beta(3)-dependent spreading in the presence of NS398 or, but did not accelerate adhesion, whereas a dominant negative Rac mutant (N17Rac) suppressed spreading without affecting adhesion. alpha(5)beta(1)-mediated HUVEC adhesion, Rac activation, and spreading were not affected by PGE(2), 8-brcAMP, or the inhibition of PKA. In conclusion, these results demonstrate that PGE(2) accelerates alpha(V)beta(3)-mediated endothelial cell adhesion through cAMP-dependent PKA activation and induces alpha(V)beta(3)-dependent spreading via cAMP- and PKA-dependent Rac activation and may contribute to the further understanding of the regulation of vascular integrins alpha(V)beta(3) by COX-2/PGE(2) during tumor angiogenesis and inflammation.

Importance of IL28B gene polymorphisms in hepatitis C virus genotype 2 and 3 infected patients.

BACKGROUND & AIMS: Genetic variation in the interleukin 28B (IL28B) gene has been associated with the response to interferon-alfa/ribavirin therapy in hepatitis C virus (HCV) genotype 1-infected patients. The importance of three IL28B single nucleotide polymorphisms (rs8099917, rs12980275 and rs12979860) for HCV genotype 2/3-infected patients is unknown. METHODS: In patients with chronic hepatitis C genotype 2/3 (n=267), IL28B host genotypes (rs8099917, rs12980275 and rs12979860) were analyzed for associations with sustained virologic response (SVR) to antiviral therapy with (pegylated) interferon-alfa and ribavirin and with respect to epidemiological, biochemical, and virological parameters. For comparison, hepatitis C genotype 1 patients (n=378) and healthy controls (n=200) were included. RESULTS: The rs12979860 CC genotype, lower age, and genotype 2 were significantly associated with SVR in HCV genotype 2/3-infected patients (p=0.01, p=0.03 and p=0.03, respectively). No association was observed for rs8099917 and rs12980275. In addition, an SVR in patients with rapid virologic response (RVR) was associated with the rs12979860 CC genotype (p=0.05), while for non-RVR no association was found. Furthermore, a significant association with a higher baseline viral load was observed for all three IL28B genotypes in genotype 1/2/3-infected patients. Finally, increasing frequencies of the rs12979860 CC genotypes were observed in genotype 1- (33.9%), genotype 3- (38.9%), and genotype 2-infected (51.9%) patients in comparison with healthy controls (49.0%) (p<0.01). CONCLUSIONS: In genotype 2/3-infected patients, rs12979860 was significantly associated with SVR. The frequency of the rs12979860 CC genotype is lower in HCV genotype 1 vs. genotype 2/3 patients. All major IL28B genotypes are associated with HCV-RNA concentration.