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

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

Comparison of bioaugmented EGSB and GAC–FBB reactors and their combination with aerobic SBR for the abatement of chlorophenols

The biological abatement of 2,4,6-trichlorophenol (246TCP) and its chlorinated degradation byproducts using anaerobic and aerobic biological reactors coupled in series has been studied. The performance of an anaerobic fluidized bed biofilm reactor (FBBR) and expanded granular sludge bed (EGSB) reactors bioaugmented with Desulfitobacterium strains was compared within a wide range of 246TCP loading rates. The bioaugmentation of an EGSB reactor with Desulfitobacterium strains enhanced the chlorophenols removal efficiency and the stability against high toxic shocks. The FBBR showed an even higher stability, but also improved the dechlorination efficiency and required a shorter start-up period than the bioaugmented EGSB reactor. Thus, it was selected as the preferred anaerobic system. The subsequent treatment of the effluents from the anaerobic reactors in an aerobic sequencing batch reactor allowed complete dechlorination and improved mineralization up to 85% TOC reduction with a substantial abatement of the ecotoxicity, which was diminished in more than 80%Spanish MCI through the project CTM 2013-43803-

La Universidad Española 1889-1939. Repertorio de legislación

El libro recoge un repertorio de la legislación universitaria española entre los años 1889 y 1939 ordenado cronológicamente y acompañado de un índice de materias. En un sustancioso estudio preliminar se reflexiona sobre la publicidad de estas normas durante la edad contemporánea y se rastrean las distintas compilaciones existentes, intentando poner orden en un periodo complejo. El objetivo de la publicación es ofrecer a los investigadores un instrumento que enriquezca el cuadro de referencias utilizado y facilite nuevas incursiones, siempre dentro del marco de la regulación estatal

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

Somatostatin binding capacity, guanylate cyclase and tyrosine phosphatase activities during pancreatic proliferation in the rat induced by gastrectomy

Gastrectomy increased pancreatic growth and this effect was associated with an increase in the number of somatostatin-14 (SS) receptors (146% of control) without altering their affinity. SS increased guanylate cyclase activity twofold in pancreatic acinar membranes from gastrectomized rats. The gastrectomy decreased pancreatic SS-like immunoreactivity (SS-LI) content (55% of control levels) and tyrosine phosphatase activity (74% of control levels). Administration of proglumide (20 mg/kg, IF), a gastrin/cholecystokinin (CCK) receptor antagonist, suppressed the inhibitory effect of gastrectomy on basal tyrosine phosphatase activity and SS-LI content, which returned to control levels. Furthermore, proglumide suppressed the increase of the number of SS receptors and of SS-stimulated guanylate cyclase activity induced by gastrectomy. All this suggests that pancreatic acinar cell growth is associated with upregulation of SS receptors, which could represent a mechanism promoted by the cell to negatively regulate the mitogenic activity of pancreatic growth factors such as CCK. In addition, the results also suggest that the negative regulation of tyrosine phosphatase activity may be important in the events involved in the pancreatic hyperplasia observed after gastrectomy