eNOS T-786C polymorphism affects atorvastatin-induced changes in erythrocyte membrane fluidity

Statins have pleiotropic effects, including endothelial nitric oxide synthase (eNOS) upregulation and increased nitric oxide formation, which can be modulated by a genetic polymorphism in the promoter region of the eNOS gene (T-786C). Here, we report our investigation of whether this polymorphism modulates the effects of atorvastatin on the fluidity of erythrocyte membranes. We genotyped 200 healthy subjects (males, 18-60 years of age) and then randomly selected 15 of these with the TT genotype and 15 with the CC genotype to receive placebo or atorvastatin (10 mg/day oral administration) for 14 days. Cell membrane fluidity was evaluated by electron paramagnetic resonance (EPR) and spin-labeling method. The EPR spectra were registered on a VARIAN-E4 spectrometer. Thiobarbituric acid-reactive species (TBA-RS) and plasma membrane cholesterol were determined in the erythrocytes. Atorvastatin reduced membrane fluidity in CC subjects (P < 0.05) but not in those with the TT genotype (P > 0.05). While no significant differences were found in plasma membrane cholesterol concentrations, higher TBA-RS concentrations were found in the CC subjects than in the TT subjects (P < 0.05). These findings suggest that a short treatment with atorvastatin is disadvantageous to subjects with the CC genotype for the T-786C polymorphism compared to those with TT genotype, at least in terms of the hemorheological properties of erythrocytes.

Simvastatin therapy in cyclosporine A-induced alveolar bone loss in rats

Background and Objective: Cyclosporine A treatment is important in the therapy of a number of medical conditions; however, alveolar bone loss is an important negative side-effect of this drug. As such, we evaluated whether concomitant administration of simvastatin would minimize cyclosporine A-associated alveolar bone loss in rats subjected, or not, to experimental periodontal disease. Material and Methods: Groups of 10 rats each were treated with cyclosporine A (10 mg/kg/day), simvastatin (20 mg/kg/day), cyclosporine A and simvastatin concurrently (cyclosporine A/simvastatin) or vehicle for 30 days. Four other groups of 10 rats each received a cotton ligature around the lower first molar and were treated similarly with cyclosporine A, simvastatin, cyclosporine A/simvastatin or vehicle. Calcium (Ca(2+)), phosphorus and alkaline phosphatase levels were evaluated in serum. Expression levels of interleukin-1 beta, prostaglandin E(2) and inducible nitric oxide synthase were evaluated in the gingivomucosal tissues. Bone volume and numbers of osteoblasts and osteoclasts were also analyzed. Results: Treatment with cyclosporine A in rats, with or without ligature, was associated with bone loss, represented by a lower bone volume and an increase in the number of osteoclasts. Treatment with cyclosporine A was associated with bone resorption, whereas simvastatin treatment improved cyclosporine A-associated alveolar bone loss in all parameters studied. In addition, simvastatin, in the presence of inflammation, can act as an anti-inflammatory agent. Conclusion: This study shows that simvastatin therapy leads to a reversal of the cyclosporine A-induced bone loss, which may be mediated by downregulation of interleukin-1 beta and prostaglandin E(2) production.

Anti-inflammatory effect of atorvastatin ameliorates insulin resistance in monosodium glutamate-treated obese mice

Considering that inflammation contributes to obesity-induced insulin resistance and that statins have been reported to have other effects beyond cholesterol lowering, the present study aimed to it whether atorvastatin treatment has anti-inflammatory action in white adipose tissue of obese mice, consequently improving insulin sensitivity. Insulin sensitivity in vivo (by insulin tolerance test); metabolic-hormonal profile; plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and adiponectin; adipose tissue immunohistochemistry; glucose transporter (GLUT) 4; adiponectin; INF-alpha; IL-1 beta; and IL-6 gene expression; and I kappa B kinase (IKK)-alpha/beta activity were assessed in 23-week-old monosodium glutamate induced obese mice untreated or treated with atorvastatin for 4 weeks. Insulin-resistant obese mice had increased plasma triglyceride, insulin, TNF-alpha, and IL-6 plasma levels. Adipose tissue of obese animals showed increased macrophage infiltration, IKK-alpha (42%, P < .05) and IKK-beta (73%, P < .05) phosphorylation, and INF-alpha and IL-6 messenger RNA (mRNA) (similar to 15%, P < .05) levels, and decreased GLUT4 mRNA and protein (30%, P < .05) levels. Atorvastatin treatment lowered cholesterol, triglyceride, insulin, INF-alpha, and IL-6 plasma levels, and restored whole-body insulin sensitivity. In adipose tissue, atorvastatin decreased macrophage in and normalized IKK-alpha/beta phosphorylation; INF-alpha, IL-6, and GLUT4 mRNA; and GLUT4 protein to control levels. The present findings demonstrate that atorvastatin has anti-inflammatory effects on adipose tissue of obese mice, which may be important to its local and whole-body insulin-sensitization effects. (C) 2010 Published by Elsevier Inc.

Three-Dimensional Quantitative Structure-Activity Relationship Study of Antitumor 2-Formylpyridine Thiosemicarbazones Derivatives as Inhibitors of Ribonucleotide Reductase

In order to extend previous SAR and QSAR studies, 3D-QSAR analysis has been performed using CoMFA and CoMSIA approaches applied to a set of 39 alpha-(N)-heterocyclic carboxaldehydes thiosemicarbazones with their inhibitory activity values (IC(50)) evaluated against ribonucleotide reductase (RNR) of H.Ep.-2 cells (human epidermoid carcinoma), taken from selected literature. Both rigid and field alignment methods, taking the unsubstituted 2-formylpyridine thiosemicarbazone in its syn conformation as template, have been used to generate multiple predictive CoMFA and CoMSIA models derived from training sets and validated with the corresponding test sets. Acceptable predictive correlation coefficients (Q(cv)(2) from 0.360 to 0.609 for CoMFA and Q(cv)(2) from 0.394 to 0.580 for CoMSIA models) with high fitted correlation coefficients (r` from 0.881 to 0.981 for CoMFA and r(2) from 0.938 to 0.993 for CoMSIA models) and low standard errors (s from 0.135 to 0.383 for CoMFA and s from 0.098 to 0.240 for CoMSIA models) were obtained. More precise CoMFA and CoMSIA models have been derived considering the subset of thiosemicarbazones (TSC) substituted only at 5-position of the pyridine ring (n=22). Reasonable predictive correlation coefficients (Q(cv)(2) from 0.486 to 0.683 for CoMFA and Q(cv)(2) from 0.565 to 0.791 for CoMSIA models) with high fitted correlation coefficients (r(2) from 0.896 to 0.997 for CoMFA and r(2) from 0.991 to 0.998 for CoMSIA models) and very low standard errors (s from 0.040 to 0.179 for CoMFA and s from 0.029 to 0.068 for CoMSIA models) were obtained. The stability of each CoMFA and CoMSIA models was further assessed by performing bootstrapping analysis. For the two sets the generated CoMSIA models showed, in general, better statistics than the corresponding CoMFA models. The analysis of CoMFA and CoMSIA contour maps suggest that a hydrogen bond acceptor near the nitrogen of the pyridine ring can enhance inhibitory activity values. This observation agrees with literature data, which suggests that the nitrogen pyridine lone pairs can complex with the iron ion leading to species that inhibits RNR. The derived CoMFA and CoMSIA models contribute to understand the structural features of this class of TSC as antitumor agents in terms of steric, electrostatic, hydrophobic and hydrogen bond donor and hydrogen bond acceptor fields as well as to the rational design of this key enzyme inhibitors.

Rapid and slow modulation of nitrate reductase activity in the red macroalga Gracilaria chilensis (Gracilariales, Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.

Structure-activity relationships of hypervalent organochalcogenanes as inhibitors of cysteine cathepsins V and S

A new series of organotelluranes were synthesized and investigated, and the structure-activity relationships in cysteine proteases inhibition were determinated. It was possible to identify the relevance of structural components linked to the reactivity of these compounds as inhibitors. For example, dibromo-organotelluranes showed to be more reactive than dichloro-organotelluranes towards cysteine cathepsins V and S. Besides, no remarkable enantio-selectivity was verified. In general the achiral organotelluranes were more reactive than the chiral congeners against cysteine cathepsins V and S. A reactivity order for organochalcogenanes and cysteine cathepsins was proposed after the comparison of the inhibitory potencies of organotelluranes with the related organoselenanes. (C) 2011 Elsevier Ltd. All rights reserved.

Caffeic and chlorogenic acids in Ilex paraguariensis extracts are the main inhibitors of AGE generation by methylglyoxal in model proteins

The present study concentrates on the evaluation of the anti-glycation effect of some bioactive substances present in yerba mate (Ilex paraguariensis): 5-caffeoylquinic acid, caffeic acid and a sapogenin (oleanolic acid). Bovine serum albumin and histones were incubated in the presence of methylglyoxal with or without the addition of 5-caffeoylquinic acid, caffeic acid and oleanolic acid. After the incubation period, advanced glycation end product (AGE) fluorescence spectra were performed and protein structural changes were evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. Chlorogenic acid, caffeic acid are the main substances responsible for the anti-glycation effect of mate tea. (C) 2009 Elsevier B.V. All rights reserved.

Insights into the Specificity of Thioredoxin Reductase-Thioredoxin Interactions. A Structural and Functional Investigation of the Yeast Thioredoxin System

The enzymatic activity of thioredoxin reductase enzymes is endowed by at least two redox centers: a flavin and a dithiol/disulfide CXXC motif. The interaction between thioredoxin reductase and thioredoxin is generally species-specific, but the molecular aspects related to this phenomenon remain elusive. Here, we investigated the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductase (ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2). We showed that ScTrxR1 was able to efficiently reduce yeast thioredoxins (mitochondrial and cytosolic) but failed to reduce the human and Escherichia coli thioredoxin counterparts. To gain insights into this specificity, the crystallographic structure of oxidized ScTrxR1 was solved at 2.4 angstrom resolution. The protein topology of the redox centers indicated the necessity of a large structural rearrangement for FAD and thioredoxin reduction using NADPH. Therefore, we modeled a large structural rotation between the two ScTrxR1 domains (based on the previously described crystal structure, PDB code 1F6M). Employing diverse approaches including enzymatic assays, site-directed mutagenesis, amino acid sequence alignment, and structure comparisons, insights were obtained about the features involved in the species-specificity phenomenon, such as complementary electronic parameters between the surfaces of ScTrxR1 and yeast thioredoxin enzymes and loops and residues (such as Ser(72) in ScTrx2). Finally, structural comparisons and amino acid alignments led us to propose a new classification that includes a larger number of enzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification.

NAD(P)H Oxidase Participates in the Palmitate-Induced Superoxide Production and Insulin Secretion by Rat Pancreatic Islets

Nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase complex has been shown to be involved in the process of glucose-stimulated insulin secretion (GSIS). In this study, we examined the effect of palmitic acid on superoxide production and insulin secretion by rat pancreatic islets and the mechanism involved. Rat pancreatic islets were incubated during 1 h with 1 mM palmitate, 1% fatty acid free-albumin, 5.6 or 10 mM glucose and in the presence of inhibitors of NAD(P)H oxidase (DPI-diphenyleneiodonium), PKC (calphostin C) and carnitine palmitoyl transferase-I (CPT-I) (etomoxir). Superoxide content was determined by hydroethidine assays. Palmitate increased superoxide production in the presence of 5.6 and 10 mM glucose. This effect was dependent on activation of PKC and NAD(P)H oxidase. Palmitic acid oxidation was demonstrated to contribute for the fatty acid induction of superoxide production in the presence of 5.6 mM glucose. In fact, palmitate caused p47(PHOX) translocation to plasma membrane, as shown by immunohistochemistry. Exposure to palmitate for 1 h up-regulated the protein content of p47(PHOX) and the mRNA levels of p22(PHOX), gp91(PHOX), p47(PHOX), proinsulin and the G protein-coupled receptor 40 (GPR40). Fatty acid stimulation of insulin secretion in the presence of high glucose concentration was reduced by inhibition of NAD(P)H oxidase activity. In conclusion, NAD(P)H oxidase is an important source of superoxide in pancreatic islets and the activity of NAD(P)H oxidase is involved in the control of insulin secretion by palmitate. J. Cell. Physiol. 226: 1110-1117, 2011. (C) 2010 Wiley-Liss, Inc.

Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic beta-cell line

Chronic exposure of pancreatic beta-cells to saturated non-esterified fatty acids can lead to inhibition of insulin secretion and apoptosis. Several previous studies have demonstrated that saturated fatty acids such as PA (palmitic acid) are detrimental to beta-cell function compared with unsaturated fatty acids. In the present study, we describe the effect of the polyunsaturated AA (arachidonic acid) on the function of the clonal pancreatic beta-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects. When added to beta-cell incubations at 100 mu M, AA can stimulate cell proliferation and chronic (24 h) basal insulin secretion. Microarray analysis and/or real-time PCR indicated significant AA-dependent up-regulation of genes involved in proliferation and fatty acid metabolism [e.g. Angptl (angiopoietin-like protein 4), Ech1 (peroxisomal Delta(3.5),Delta(2.4)-dienoyl-CoA isomerase), Cox-1 (cyclo-oxygenase-1) and Cox-2, P < 0.05]. Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects. Moreover, concomitant incubation of AA with PA dose-dependently attenuated the detrimental effects of the saturated fatty acid, so reducing apoptosis and decreasing parameters of oxidative stress [ROS (reactive oxygen species) and NO levels] while improving the GSH/GSSG ratio. AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-kappa B (nuclear factor kappa B) and the p47 subunit of NADPH oxidase in PA-treated cells. These findings indicate that AA has an important regulatory and protective beta-cell action, which may be beneficial to function and survival in the `lipotoxic` environment commonly associated with Type 2 diabetes mellitus.

Effects of cyclooxygenase inhibitors on parasite burden, anemia and oxidative stress in murine Trypanosoma cruzi infection

Prostaglandins are known to be produced by macrophages when challenged with Trypanosoma cruzi, the etiological agent of Chagas` disease. It is not known whether these lipid mediators play a role in oxidative stress in host defenses against this important protozoan parasite. In this study, we demonstrated that inducible cyclooxygenase-mediated prostaglandin production is a key chemical mediator in the control of parasite burden and erythrocyte oxidative stress during T. cruzi infection in C57BL/6 and BALB/c mice, prototype hosts for the study of resistance and susceptibility in murine Chagas` disease. The results suggested the existence of at least two mechanisms of oxidative stress, dependent or independent with regard to the nitric oxide and cyclooxygenase pathway, where one or the other is more evident depending on the mouse strain.

Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells

Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in many experimental models using phagocytic and non-phagocytic cells. Currently, there is some controversy about the efficacy of apocynin in non-phagocytic cells, but in phagocytes the reported results are consistent, which could be due to the presence of myeloperoxidase in these cells. This enzyme has been proposed as responsible for activating apocynin by generating its dimer, diapocynin, which is supposed to be the active compound that prevents NADPH oxidase complex assembly and activation. Here, we synthesized diapocynin and studied its effect on inhibition of gp91(phox) RNA expression. We found that diapocynin strongly inhibited the expression of gp91(phox)mRNA in peripheral blood mononuclear cells (PBMC). Only at a higher concentration, apocynin was able to exert the same effect. We also compared the apocynin and diapocynin efficacy as inhibitors of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production in response to lipopolysaccharide (LPS)-activated PBMC. Although apocynin did inhibit TNF-alpha production, diapocynin had a much more pronounced effect, on both TNF-alpha and IL-10 production. In conclusion, these findings suggest that the bioconversion of apocynin to diapocynin is an important issue not limited to enzymatic activity inhibition, but also for other biological effects as gp91(phox) mRNA expression and cytokine production. Hence, as diapocynin can be easily prepared from apocynin, a one-step synthesis, we recommend its use in studies where the biological effects of apocynin are searched. (C) 2010 Elsevier Inc. All rights reserved.

Metallopeptidase inhibitors arrest vital biological processes in the fungal pathogen Scedosporium apiospermum

P>Scedosporium apiospermum is an emerging agent of opportunistic mycoses in humans. Previously, we showed that mycelia of S. apiospermum secreted metallopeptidases which were directly linked to the destruction of key host proteins. In this study, we analysed the effect of metallopeptidase inhibitors on S. apiospermum development. As germination of inhaled conidia is a crucial event in the infectious process of S. apiospermum, we studied the morphological transformation induced by the incubation of conidia in Sabouraud-dextrose medium at 37 degrees C. After 6 h, some conidia presented a small projection resembling a germ-tube. A significant increase, around sixfold, in the germ-tube length was found after 12 h, and hyphae were exclusively observed after 24 h. Three distinct metallopeptidase inhibitors were able to arrest the transformation of conidia into hyphae in different ways; for instance, 1,10-phenanthroline (PHEN) completely blocked this process at 10 mu mol l-1, while ethylenediamine tetraacetic acid (EDTA) and ethylene glycol-bis (beta-aminoethyl ether; EGTA) only partially inhibited the differentiation at up to 10 mmol l-1. EGTA did not promote any significant reduction in the conidial growth, while PHEN and EDTA, both at 10 mmol l-1, inhibited the proliferation around 100% and 65%, respectively. The secretion of polypeptides into the extracellular environment and the metallopeptidase activity secreted by mycelia were completely inhibited by PHEN. These findings suggest that metallo-type enzymes could be potential targets for future therapeutic interventions against S. apiospermum.

Quantitative structure-activity relationships for a series of inhibitors of cruzain from Trypanosoma cruzi: Molecular modeling, CoMFA and CoMSIA studies

Human parasitic diseases are the foremost threat to human health and welfare around the world. Trypanosomiasis is a very serious infectious disease against which the currently available drugs are limited and not effective. Therefore, there is an urgent need for new chemotherapeutic agents. One attractive drug target is the major cysteine protease from Trypanosoma cruzi, cruzain. In the present work, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) studies were conducted on a series of thiosemicarbazone and semicarbazone derivatives as inhibitors of cruzain. Molecular modeling studies were performed in order to identify the preferred binding mode of the inhibitors into the enzyme active site, and to generate structural alignments for the three-dimensional quantitative structure-activity relationship (3D QSAR) investigations. Statistically significant models were obtained (CoMFA. r(2) = 0.96 and q(2) = 0.78; CoMSIA, r(2) = 0.91 and q(2) = 0.73), indicating their predictive ability for untested compounds. The models were externally validated employing a test set, and the predicted values were in good agreement with the experimental results. The final QSAR models and the information gathered from the 3D CoMFA and CoMSIA contour maps provided important insights into the chemical and structural basis involved in the molecular recognition process of this family of cruzain inhibitors, and should be useful for the design of new structurally related analogs with improved potency. (C) 2009 Elsevier Inc. All rights reserved.

Discovery of novel Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase inhibitors

Based on its essential role in the life cycle of Trypanosoma cruzi, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been considered a promising target for the development of novel chemotherapeutic agents for the treatment of Chagas` disease. In the course of our research program to discover novel inhibitors of this trypanosomatid enzyme, we have explored a combination of structure and ligand-based virtual screening techniques as a complementary approach to a biochemical screening of natural products using a standard biochemical assay. Seven natural products, including anacardic acids,. avonoid derivatives, and one glucosylxanthone were identified as novel inhibitors of T. cruzi GAPDH. Promiscuous inhibition induced by nonspecific aggregation has been discarded as specific inhibition was not reversed or affected in all cases in the presence of Triton X-100, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. The structural diversity of this series of promising natural products is of special interest in drug design, and should therefore be useful in future medicinal chemistry efforts aimed at the development of new GAPDH inhibitors having increased potency. (C) 2009 Elsevier Ltd. All rights reserved.