Regulation of inducible nitric oxide synthase expression in rat mesangial cells and isolated glomeruli

Regulation of inducible nitric oxide synthase expression in rat mesangial cells and isolated glomeruli. The presence of the inducible isoform of nitric oxide synthase (iNOS) in glomerular mesangial cells facilitates the synthesis of nitric oxide (NO) after stimulation with cytokines or lipopolysaccharide (LPS). As the role of NO within the glomerulus may be important in conditions such as glomerulonephritis, we have studied the effect of dexamethasone (DX) and pirrolidine dithiocarbamate (PDTC), an inhibitor of the nuclear transcription factor, NF-κB activation on the induced synthesis of NO in rat mesangial cells (RMC). LPS, tumor necrosis factor-alpha (TNF-α) and the combination of both were able to induce NO synthesis in a dose-dependent manner as measured with the determination of NO2- levels. Treatment with LPS (10 µg/ml) + TNF-α (100 ng/ml) for eight hours was the most potent stimulus for iNOS activity. DX (1 µM) had an inhibitory effect on LPS-, TNF-α- and LPS + TNF-α-induced NO synthesis (51.2, 42.5 and 68% of inhibition, respectively). The inhibitory effect of DX was confirmed using a reporter cell bioassay, whereas cGMP was measured as a reflection of bioactive NO. DX inhibited induced NO synthesis when RMC were exposed to this agent before (16hr of pretreatment, 75.7% inhibition) or at the same time (8hr of cotreatment, 61.2% inhibition) as TNF-α + LPS but not four hours after the stimuli. Northern blot analysis showed marked blunting of mRNA expression in RMC treated with DX, in concordance with functional studies. Both actinomycin D and cycloheximide significantly inhibited NO synthesis and iNOS mRNA expression. PDTC (100 µM) was able to inhibit the iNOS activity induced by LPS and TNF-α independently (56.8 and 49.9% inhibition, respectively), and in combination (79.1% inhibition). PDTC (1 to 100 µM) inhibited LPS + TNF-α-induced NO synthesis and iNOS mRNA expression in a concentration-dependent fashion (69 to 86% inhibition of NO synthesis and 50 to 100% inhibition of mRNA expression). Addition of PDTC four hours after exposure to TNF-α + LPS was still able to markedly inhibit NO synthesis. The effects of DX and PDTC were also demonstrated in isolated glomeruli, where two different combinations of inductive stimuli for NO synthesis were employed. Our results establish DX and PDTC as useful tools to study the regulation of NO synthesis in the mesangial cell and glomerulus, and suggest that NF-κB is involved in the transcriptional regulation of iNOS in RMC

Regulation of endothelin synthesis by extracellular matrix in human endothelial cells

Regulation of endothelin synthesis by extracellular matrix in human endothelial cells.BackgroundVascular diseases are characterized by the presence of structural changes and the progressive loss of endothelial function. Although the biochemical basis of these structural changes have started to be outlined, it seems that accumulation of normal extracellular matrix proteins as well as the appearance of interstitial collagens, mainly collagen type I, characterize this process. On the other hand, a role for endothelial vasoactive factors has been proposed in the genesis of endothelial dysfunction, and it is generally accepted that changes in extracellular matrix composition may modify cell behavior.MethodsExperiments were designed to test the influence of the supporting matrix on endothelin-1 (ET-1) synthesis by endothelial cells. Northern blot experiments were performed to analyze the prepro-endothelin-1 (prepro-ET-1) mRNA expression. ET-1 production was measured by ELISA.ResultsCells grown on collagen type I (Col I) showed an increase of prepro-ET-1 mRNA level when compared with cells cultured on collagen type IV (Col IV). According to these results, the release of ET-1 to culture medium was also higher in Col I-grown cells than in those cultured on Col IV. Treatment of cells with a peptide that interferes with Col I integrins (D6Y), or with protein tyrosine kinase inhibitors such as genistein and herbimycin, completely abolished the effect of Col I. Moreover, experiments with antibodies against integrins suggest that these cell surface receptors could be involved in the modulation of ET-1 system by extracellular matrix.ConclusionsThese results suggest that the presence of an abnormal extracellular matrix could stimulate endothelin synthesis by human endothelial cells, through integrin activation

Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: studies on TGF-ß1 regulation

This work was supported by grants from Ministerio de Ciencia y Tecnología (MCYT-SAF2007-62471, MCYT-SAF2010-16198; PI070695) and Redes Temáticas de Investigación Cooperativa en Salud from Instituto de Salud Carlos III (ISCIII-RETIC RD06/00160002

A Computer-Driven Scaffold-Hopping Approach Generating New PTP1B Inhibitors from the Pyrrolo[1,2-a]quinoxaline Core

Protein tyrosine phosphatase 1B (PTP1B) is a very promising target for the treatment of metabolic disorders such as type II diabetes mellitus. Although it was validated as a promising target for this disease more than 30 years ago, as yet there is no drug in advanced clinical trials, and its biochemical mechanism and functions are still being studied. In the present study, based on our experience generating PTP1B inhibitors, we have developed and implemented a scaffold-hopping approach to vary the pyrrole ring of the pyrrolo[1,2-a]quinoxaline core, supported by extensive computational techniques aimed to explain the molecular interaction with PTP1B. Using a combination of docking, molecular dynamics and end-point free-energy calculations, we have rationally designed a hypothesis for new PTP1B inhibitors, supporting their recognition mechanism at a molecular level. After the design phase, we were able to easily synthesize proposed candidates and their evaluation against PTP1B was found to be in good concordance with our predictions. Moreover, the best candidates exhibited glucose uptake increments in cellulo model, thus confirming their utility for PTP1B inhibition and validating this approach for inhibitors design and molecules thus obtained

Recursos humanos. Breve mirada en el sector de seguros

Màster de Direcció d'Entitats Asseguradores i Financeres, Universitat de Barcelona, Facultat d'Economia i Empresa, Curs: 2003-2004, Tutor: José Luis Pérez TorresEste trabajo intenta ser una aproximación a la situación actual del mercado laboral nacional, y en particular al sector de las empresas aseguradoras, a sus tamaños por diferentes segmentos de asalariados, en provincias y comunidades españolas. También a la evolución del 69 % de entidades de este sector: la composición de su plantilla, jornada laboral, promoción, etc., durante los años 2000 al 2004. Además, se introducen comentarios sobre management y problemas del entorno social y empresarial. Los datos obtenidos para hacer el análisis los he conseguido con la información recogida del INE y de ICEA. Las lecturas especializadas en Economía, Derecho Laboral, Recursos Humanos, Sociología y Psicología, han ayudado para ampliar la visión de la gestión empresarial. El mercado laboral nacional sirven como introducción para después poder valorar mejor la situación actual de las empresas de seguros, sin olvidar la gestión de los recursos humanos y de su importancia en la dirección empresarial y sobre todo de las personas de sus necesidades y de sus inquietudes

Insight into the mechanism of molecular recognition between human Integrin-Linked Kinase and Cpd22 and its implication at atomic level

Pseudokinases have received increasing attention over the past decade because of their role in different physiological phenomena. Although pseudokinases lack several active-site residues, thereby hindering their catalytic activity, recent discoveries have shown that these proteins can play a role in intracellular signaling thanks to their non-catalytic functions. Integrin-linked kinase (ILK) was discovered more than two decades ago and was subsequently validated as a promising target for neoplastic diseases. Since then, only a few small-molecule inhibitors have been described, with the V-shaped pyrazole Cpd22 being the most interesting and characterized. However, little is known about its detailed mechanism of action at atomic level. In this study, using a combination of computational chemistry methods including PELE calculations, docking, molecular dynamics and experimental surface plasmon resonance, we were able to prove the direct binding of this molecule to ILK, thus providing the basis of its molecular recognition by the protein and the effect over its architecture. Our breakthroughs show that Cpd22 binding stabilizes the ILK domain by binding to the pseudo-active site in a similar way to the ATP, possibly modulating its scaffolding properties as pseudokinase. Moreover, our results explain the experimental observations obtained during Cpd22 development, thus paving the way to the development of new chemical probes and potential drugs

Tripeptides as Integrin-Linked Kinase Modulating Agents Based on a Protein-Protein Interaction with alfa-Parvin

Integrin-linked kinase (ILK) has emerged as a controversial pseudokinase protein that plays a crucial role in the signaling process initiated by integrin-mediated signaling. However, ILK also exhibits a scaffolding protein function inside cells, controlling cytoskeletal dynamics, and has been related to non-neoplastic diseases such as chronic kidney disease (CKD). Although this protein always acts as a heterotrimeric complex bound to PINCH and parvin adaptor proteins, the role of parvin proteins is currently not well understood. Using in silico approaches for the design, we have generated and prepared a set of new tripeptides mimicking an alpha-parvin segment. These derivatives exhibit activity in phenotypic assays in an ILK-dependent manner without altering kinase activity, thus allowing the generation of new chemical probes and drug candidates with interesting ILK-modulating activities

Integrin Linked Kinase (ILK) Downregulation as an Early Event During the Development of Metabolic Alterations in a Short-Term High Fat Diet Mice Model.

Background/Aims: Diabetes type 2, metabolic syndrome or non-alcoholic fatty liver disease are insulin resistance-related metabolic disorders, which lack a better prognosis before their full establishment. We studied the importance of the intracellular scaffold protein integrin linked kinaes (ILK) as a key modulator in the initial pathogenesis and the early progression of those insulin resistance- related disorders. Methods: Adult mice with a global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. Weights, blood glucose and other systemic biochemical parameters were determined in animals under fasting conditions and after glucose or pyruvate intraperitoneal injections to test their tolerance. In RNA or proteins extracted from insulin-sensitive tissues, we determined by reverse transcription?quantitative PCR and western blot the expression of ILK, metabolites transporters and other metabolism and inflammatory markers. Glucose uptake capacity was studied in freshly isolated tissues. Results: HFD feeding was able to early and progressively increase glycaemia, insulinemia, circulating glycerol, body weight gain, liver-mediated gluconeogenesis along this time lapse, but cKD-ILK have all these systemic misbalances exacerbated compared to CT in the same HFD time lapse. Interestingly, the tisular expression of ILK in HFD-fed CT was dramatically downregulated in white adipose tissue (WAT), skeletal muscle and liver at the same extent of the original ILK downregulation of cKD-ILK. We previously published that basal STD-fed cKD-ILK compared to basal STD-CT have different expression of glucose transporters GLUT4 in WAT and skeletal muscle. In the same STD-fed cKD-ILK, we observed here the increased expressions of hepatic GLUT2 and WAT pro-inflammatory cytokines TNF-? and MCP-1. The administration of HFD exacerbated the expression changes in cKD-ILK of these and other markers related to the imbalanced metabolism observed, such as WAT lipolysis (HSL), hepatic gluconeogenesis (PCK-1) and glycerol transport (AQP9). Conclusion: ILK expression may be taken as a predictive determinant of metabolic disorders establishment, because its downregulation seems to correlate with the early imbalance of glucose and glycerol transport and the subsequent loss of systemic homeostasis of these metabolites.Instituto de Salud Carlos III-ISCIIIComunidad de MadridFondo Europeo de Desarrollo Regional-FEDERInstituto Ramon y Cajal de Investigación Sanitária-IRYCISFundación Renal Iñigo Álvarez de Toledo-FRIA

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Ibeta pathway activation

15 p.Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs.We have demonstrated that H-rasgene deletion produces mice hypotensionviaa soluble guanylate cyclase-proteinkinase G (PKG)–dependent mechanism. In this study, we analyzed the consequences of H-rasdeletiononcardiacremodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Leftventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H-ras2/2) and control wild-type (H-ras+/+) mice, as were extracellular matrix proteinexpression. Increased cardiac PKG-Ibprotein expression in H-ras2/2mice suggests the involvement of this proteinin heart protection.Ex vivoexperiments on cardiac explants could support this mechanism, as PKG blockadeblunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H-ras2/2mice. Geneticmodulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3b-dependent activation ofthe transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iboverexpressionin H-ras2/2mouse embryonic fibroblasts. This study demonstrates that H-rasdeletion protects against AngII-induced cardiac remodeling, possiblyviaa mechanism in which PKG-Iboverexpression could play a partial role, andpoints to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.Instituto de Salud Carlos IIIUniversidad de AlcaláFundación SenefroFEDE