Research Center Biomedica En Red En El Area Tematica Of Enfermedades Hepaticas gestivas

Barcelona, Spain

Research Center Biomedica En Red En El Area Tematica Of Enfermedades Hepaticas gestivas

Barcelona, Spain
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Donato M.T.,Hospital la Fe | Donato M.T.,Research Center Biomedica En Red En El Area Tematica Of Enfermedades Hepaticas gestivas | Donato M.T.,University of Valencia | Hallifax D.,University of Manchester | And 7 more authors.
Drug Metabolism and Disposition | Year: 2010

Cryopreserved human hepatocytes and other in vitro systems often underpredict in vivo intrinsic clearance (CLint). The aim of this study was to explore the potential utility of HepG2 cells transduced with adenovirus vectors expressing a single cytochrome P450 enzyme (Ad-CYP1A2, Ad-CYP2C9, or Ad-CYP3A4) for metabolic clearance predictions. The kinetics of metabolite formation from phenacetin, tolbutamide, and alprazolam and midazolam, selected as substrates probes for CYP1A2, CYP2C9, and CYP3A4, respectively, were characterized in this in vitro system. The magnitude of the Km or S50 values observed in Ad-P450 cells was similar to those found in the literature for other human liver-derived systems. For each substrate, CLint (or CLmax), values from Ad-P450 systems were scaled to human hepatocytes in primary culture using the relative activity factor (RAF) approach. Scaled Ad-P450 CLint values were approximately 3- to 6-fold higher (for phenacetin O-deethylation, tolbutamide 4-hydroxylation, and alprazolam 4-hydroxyaltion) or lower (midazolam 1′-hydroxylation) than those reported for human cryopreserved hepatocytes in suspension. Comparison with the in vivo data reveals that Ad-P450 cells provide a favorable prediction of CLint for the substrates studied (in a range of 20-200% in vivo observed CLint). This is an improvement compared with the consistent underpredictions (<10-50% in in vivo observed CLint) found in cryopreserved hepatocyte studies with the same substrates. These results suggest that the Ad-P450 cell is a promising in vitro system for clearance predictions of P450-metabolized drugs. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics.

Panes J.,Hospital Clinic IDIBAPS | Panes J.,Research Center Biomedica En Red En El Area Tematica Of Enfermedades Hepaticas gestivas | Lopez-Sanroman A.,Hospital Ramon y Cajal | Bermejo F.,Hospital Of Fuenlabrada | And 14 more authors.
Gastroenterology | Year: 2013

Background & Aims A small placebo-controlled trial reported the efficacy of mercaptopurine therapy for children newly diagnosed with Crohn's disease, yet little is known about the efficacy of early thiopurine therapy in adults. Methods We performed a prospective double-blind trial of adult patients with a recent (<8 weeks) diagnosis of Crohn's disease. Patients were randomly assigned to groups given azathioprine (2.5 mg · kg-1 · day-1, n = 68) or placebo (n = 63) at 31 hospitals from February 2006 to September 2009. Corticosteroids but no other concomitant medications were allowed for control of disease activity. The primary measure of efficacy was sustained corticosteroid-free remission. Results After 76 weeks of treatment, 30 patients treated with azathioprine (44.1%) and 23 given placebo (36.5%) were in sustained corticosteroid-free remission (difference of 7.6%; 95% confidence interval, -9.2 to 24.4%; P =.48). The rates of relapse (defined as Crohn's Disease Activity Index score >175) and corticosteroid requirements were similar between groups. A post hoc analysis of relapse, defined as a Crohn's Disease Activity Index score >220, showed lower relapse rates in the azathioprine group than in the placebo group (11.8% vs 30.2%; P =.01). Serious adverse events occurred in 14 patients in the azathioprine group (20.6%) and 7 in the placebo group (11.1%) (P =.16). A larger percentage of patients in the azathioprine group had adverse events that led to study drug discontinuation (20.6%) than in the placebo group (6.35%) (P =.02). Conclusions In a study of adults with Crohn's disease, early azathioprine therapy was no more effective than placebo to achieve sustained corticosteroid-free remission but was more effective in preventing moderate to severe relapse in a post hoc analysis. EudraCT 2005-001186-34. © 2013 by the AGA Institute.

Errasti-Murugarren E.,University of Barcelona | Errasti-Murugarren E.,Research Center Biomedica En Red En El Area Tematica Of Enfermedades Hepaticas gestivas | Errasti-Murugarren E.,Institute Of Recerca Biomedica | Diaz P.,University of Chile | And 6 more authors.
Molecular Pharmacology | Year: 2011

The plasma membrane distribution and related biological activity of nucleoside transporter proteins (NTs) were investigated in human syncytiotrophoblast from term placenta using a variety of approaches, including nucleoside uptake measurements into vesicles from selected plasma membrane domains, NT immunohistochemistry, and subcellular localization (basal, heavy, and light apical membranes as well as raft-enriched membranes from the apical domain). In contrast with other epithelia, in this epithelium, we have identified the high-affinity pyrimidine-preferring human concentrative nucleoside transporter (hCNT) 1 as the only hCNT-type protein expressed at both the basal and apical membranes. hCNT1 localization in lipid rafts is also dependent on its subcellular localization in the apical plasma membrane, suggesting a complex cellular and regional expression. Overall, this result favors the view that the placenta is a pyrimidine-preferring nucleoside sink from both maternal and fetal sides, and hCNT1 plays a major role in promoting pyrimidine salvage and placental growth. This finding may be of pharmacological relevance, because hCNT1 is known to interact with anticancer nucleoside-derived drugs and other molecules, such as nicotine and caffeine, for which a great variety of harmful effects on placental and fetal development, including intrauterine growth retardation, have been reported. Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics.

Amezaga N.,Health science Research Institute Germans Trias i Pujol | Sanjurjo L.,Health science Research Institute Germans Trias i Pujol | Julve J.,Research Center Biomedica en Redde Diabetes y Enfermedades Metabolicas Asociadas | Julve J.,Biomedical Research Institute Sant Pau | And 17 more authors.
Journal of Leukocyte Biology | Year: 2014

AIM is expressed by macrophages in response to agonists of the nuclear receptors LXR/RXR. In mice, it acts as an atherogenic factor by protecting macrophages from the apoptotic effects of oxidized lipids. In humans, it is detected in atherosclerotic lesions, but no role related to atherosclerosis has been reported. This study aimed to investigate whether the role of hAIM extends beyond inhibiting oxidized lipid-induced apoptosis. To accomplish this goal, functional analysis with human monocytic THP1 cells and macrophages differentiated from peripheral blood monocytes were performed. It was found that hAIM reduced oxLDL-induced macrophage apoptosis and increased macrophage adhesion to endothelial ICAM-1 by enhancing LFA-1 expression. Furthermore, hAIM increased foam cell formation, as shown by Oil Red O and Nile Red staining, as well as quantification of cholesterol content. This was not a result of decreased reverse cholesterol transport, as hAIM did not affect the efflux significantly from [3H] Cholesterol-laden macrophages driven by plasma, apoA-I, or HDL2 acceptors. Rather, flow cytometry studies indicated that hAIM increased macrophage endocytosis of fluorescent oxLDL, which correlated with an increase in the expression of the oxLDLR CD36. Moreover, hAIM bound to oxLDL in ELISA and enhanced the capacity of HEK-293 cells expressing CD36 to endocytose oxLDL, as studied using immunofluorescence microscopy, suggesting that hAIM serves to facilitate CD36-mediated uptake of oxLDL. Our data represent the first evidence that hAIM is involved in macrophage survival, adhesion, and foam cell formation and suggest a significant contribution to atherosclerosis-related mechanisms in the macrophage. © Society for Leukocyte Biology.

Ayuso-Tejedor S.,University of Zaragoza | Abian O.,University of Zaragoza | Abian O.,CIBER ISCIII | Abian O.,Research Center Biomedica En Red En El Area Tematica Of Enfermedades Hepaticas gestivas | Sancho J.,University of Zaragoza
Protein Engineering, Design and Selection | Year: 2011

Increasing protein stability is interesting for practical reasons and because it tests our understanding of protein energetics. We explore here the feasibility of stabilizing proteins by replacing underexposed polar residues by apolar ones of similar size and shape. We have compared the stability of wild-type apoflavodoxin with that of a few carefully selected mutants carrying Y → F, Q → L, T → V or K → M replacements. Although a clear inverse correlation between native solvent exposures of replaced polar residues and stability of mutants is observed, most mutations fail to stabilize the protein. The promising exceptions are the two Q → L mutations tested, which characteristically combine the greatest reduction in polar burial with the greatest increase in apolar burial relative to wild type. Analysis of published stability data corresponding to a variety of mutant proteins confirms that, unlike Y → F or T → V replacements, Q → L mutations tend to be stabilizing, and it suggests that N → L mutations might be stabilizing as well. On the other hand, we show that the stability changes associated to the apoflavodoxin mutations can be rationalized in terms of differential polar and apolar burials upon folding plus a generic destabilizing penalty term. Simple equations combining these contributions predict stability changes in a large data set of 113 mutants (Y → F, Q → L or T → V) similarly well as more complex algorithms available on the Internet. © The Author 2010. Published by Oxford University Press. All rights reserved.

Abian O.,Hospital Universitario Miguel Servet | Abian O.,Aragon Health science Institute ICS | Abian O.,University of Zaragoza | Abian O.,Research Center Biomedica En Red En El Area Tematica Of Enfermedades Hepaticas gestivas | And 12 more authors.
Molecular Pharmaceutics | Year: 2011

Gaucher disease (GD) is a disorder of glycosphingolipid metabolism caused by deficiency of lysosomal glucocerebrosidase (GlcCerase) activity, due to conformationally or functionally defective variants, resulting in progressive deposition of glycosylceramide in macrophages. The glucose analogue, N-butyldeoxynojirimycin (NB-DNJ, miglustat), is an inhibitor of the ceramide-specific glycosyltransferase, which catalyzes the first step of glycosphingolipid biosynthesis and is currently approved for the oral treatment of type 1 GD. In a previous work, we found a GlcCerase activity increase in cell cultures in the presence of NB-DNJ, which could imply that this compound is not only a substrate reducer but also a pharmacological chaperone or inhibitor for GlcCerase degradation. In this work we compare imiglucerase (the enzyme currently used for replacement therapy) and velaglucerase alfa (a novel therapeutic enzyme form) in terms of conformational stability and enzymatic activity, as well as the effect of NB-DNJ on them. The interaction between these enzymes and NB-DNJ was studied by isothermal titration calorimetry. Our results reveal that, although velaglucerase alfa and imiglucerase exhibit very similar activity profiles, velaglucerase alfa shows higher in vitro thermal stability and is less prone to aggregation/precipitation, which could be advantageous for storage and clinical administration. In addition, we show that at neutral pH NB-DNJ binds to and enhances the stability of both enzymes, while at mildly acidic lysosomal conditions it does not bind to them. These results support the potential role of NB-DNJ as a pharmacological chaperone, susceptible of being part of pharmaceutical formulation or combination therapy for GD in the future. © 2011 American Chemical Society.

Abian O.,University of Zaragoza | Abian O.,Research Center Biomedica En Red En El Area Tematica Of Enfermedades Hepaticas gestivas | Vega S.,University of Zaragoza | Sancho J.,University of Zaragoza | Velazquez-Campoy A.,University of Zaragoza
PLoS ONE | Year: 2013

The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn+2-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn+2, the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn+2-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease. © 2013 Abian et al.

Vega S.,University of Zaragoza | Abian O.,University of Zaragoza | Abian O.,Instituto Aragones Of Ciencias Of La Salud Iacs | Abian O.,Research Center Biomedica En Red En El Area Tematica Of Enfermedades Hepaticas gestivas | Velazquez-Campoy A.,University of Zaragoza
Biochimica et Biophysica Acta - General Subjects | Year: 2016

Background Conformational changes coupled to ligand binding constitute the structural and energetics basis underlying cooperativity, allostery and, in general, protein regulation. These conformational rearrangements are associated with heat capacity changes. ITC is a unique technique for studying binding interactions because of the simultaneous determination of the binding affinity and enthalpy, and for providing the best estimates of binding heat capacity changes. Scope of review Still controversial issues in ligand binding are the discrimination between the "conformational selection model" and the "induced fit model", and whether or not conformational changes lead to temperature dependent apparent binding heat capacities. The assessment of conformational changes associated with ligand binding by ITC is discussed. In addition, the "conformational selection" and "induced fit" models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins. Major conclusions Conformational equilibrium is a major contribution to binding heat capacity changes. A simple model may explain both conformational selection and induced fit scenarios. A temperature-independent binding heat capacity does not necessarily indicate absence of conformational changes upon ligand binding. ITC provides information on the energetics of conformational changes associated with ligand binding (and other possible additional coupled equilibria). General significance Preferential ligand binding to certain protein states leads to an equilibrium shift that is reflected in the coupling between ligand binding and additional equilibria. This represents the structural/energetic basis of the widespread dependence of ligand binding parameters on temperature, as well as pH, ionic strength and the concentration of other chemical species. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences - Principles and Applications, edited by Fadi Bou-Abdallah. © 2015 Elsevier B.V. All rights reserved.

Rodriguez-Ortigosa C.M.,University of Navarra | Rodriguez-Ortigosa C.M.,Research Center Medica Aplicada | Celay J.,University of Navarra | Olivas I.,University of Navarra | And 12 more authors.
Gastroenterology | Year: 2014

Background & Aims Bile salts inhibit their own production by inducing the nuclear receptor small heterodimer partner (SHP) (encoded by NR0B2), which contributes to repression of the gene encoding cholesterol 7α-hydroxylase (CYP7A1), a key enzyme for the control of bile salt synthesis. On the other hand, bile salts stimulate hepatic synthesis of nitric oxide. We investigated the role of nitric oxide signaling in the control of CYP7A1 expression and the involvement in this process of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which participates in intracellular propagation of nitric oxide signals.Methods We studied the effects of inhibitors of nitric oxide synthesis (L-NG-nitroarginine methyl ester [L-NAME]) or protein nitrosylation (via dithiothreitol) on bile salt homeostasis in male Wistar rats placed on a cholate-rich diet for 5 days and in cultured primary hepatocytes. S-nitrosylation of GAPDH was assessed using a biotin-switch assay. Interacions of SHP with other proteins and with the Cyp7a1 promoter sequence were studied using immunoprecipitation and chromatin immunoprecipitation (ChIP) assays. We reduced the GAPDH levels in H35 cells with small interfering RNAs. GAPDH nitrosylation was assessed in normal and cholestatic rat and human livers.Results Rats placed on cholate-rich diets and given L-NAME had increased intrahepatic and biliary levels of bile salts, and deficiency in repression of CYP7A1 (at the messenger RNA and protein levels) in liver tissue, despite preserved induction of SHP. In cultured hepatocytes, L-NAME or dithiothreitol blocked cholate-induced down-regulation of CYP7A1 without impairing SHP up-regulation. In hepatocytes, cholate promoted S-nitrosylation of GAPDH and its translocation to the nucleus, accompanied by S-nitrosylation of histone deacetylase 2 (HDAC2) and Sirtuin 1 (SIRT1), deacetylases that participate, respectively, in the formation of Cyp7a1 and Shp repressor complexes. Knockdown of GAPDH prevented repression of CYP7A1 by cholate, and blocking nuclear transport of nitrosylated GAPDH reduced cholate-induced nitrosylation of HDAC2 and SIRT1; this effect was accompanied by abrogation of Cyp7a1 repression. Cholate induced binding of SHP to HDAC2 and its recruitment to the Cyp7a1 promoter; these processes were inhibited by blocking nitric oxide synthesis. Levels of nitrosylated GAPDH and nitrosylated HDAC2 were increased in cholestatic human and rat livers reflecting increased concentrations of bile salts in these conditions.Conclusions In rat liver, excess levels of bile salts activate a GAPDH-mediated transnitrosylation cascade that provides feedback inhibition of bile salt synthesis. © 2014 by the AGA Institute.

Martinez-Clemente M.,University of Barcelona | Ferre N.,University of Barcelona | Titos E.,University of Barcelona | Titos E.,Research Center Biomedica En Red En El Area Tematica Of Enfermedades Hepaticas gestivas | And 11 more authors.
Hepatology | Year: 2010

We have shown that Alox15, the gene encoding for 12/15-lipoxygenase (12/15-LO), is markedly up-regulated in livers from apolipoprotein E-deficient (ApoE-/-) mice, which spontaneously develop nonalcoholic fatty liver disease secondary to hyperlipidemia. In the current study, we used ApoE-/- mice with a targeted disruption of the Alox15 gene to assess the role of 12/15-LO in the development and progression of hepatic steatosis and inflammation. Compared with ApoE-/- mice, which exhibited extensive hepatic lipid accumulation and exacerbated inflammatory injury, ApoE/12/15-LO double-knockout (ApoE-/-/12/15-LO-/-) mice showed reduced serum alanine aminotransferase levels; decreased hepatic steatosis, inflammation, and macrophage infiltration; and decreased fatty acid synthase, tumor necrosis factor α (TNFα), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-18, and IL-6 expression. Remarkably, disruption of Alox15 attenuated glucose intolerance and high-fat diet-induced insulin resistance, up-regulated insulin receptor substrate-2, and exerted opposite effects on hepatic c-Jun amino-terminal kinase and adenosine monophosphate-activated protein kinase phosphorylation, known negative and positive regulators of insulin signaling, respectively. In adipose tissue, the absence of Alox15 induced significant reductions in the expression of the proinflammatory and insulin-resistant adipokines MCP-1, TNFα, and resistin while increasing the expression of glucose transporter-4. Interestingly, compared with ApoE-/- mice, which exhibited increased hepatic caspase-3 staining, ApoE-/-/12/15-LO-/- mice showed attenuated hepatocellular injury. Consistent with this finding, hepatocytes isolated from ApoE-/- mice were more vulnerable to TNFα-induced programmed cell death, an effect that was not observed in hepatocytes carrying a targeted disruption of the Alox15 gene. Conclusion: Collectively, our data suggest a potentially relevant mechanism linking 12/15-LO to the promotion of hepatic steatosis, insulin resistance, and inflammation in experimental liver disease of metabolic origin. Copyright © 2010 American Association for the Study of Liver Diseases.

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