Estudo do papel da proteína multifuncional APE1/Ref-1 sobre a resposta inflamatória na meningite bacteriana

Despite advances in antibiotic therapy, bacterial meningitis (BM) remains with high mortality and morbidity rates in worldwide. One important mechanism associated to sequels during disease is the intense inflammatory response which promotes an oxidative burst and release of reactive oxygen species, consequently leading to cell death. Activation of DNA repair enzymes during oxidative stress has been demonstrated in several neurological disorders. APE1/Ref-1 is a multifunctional protein involved in DNA repair and plays a redox function on transcription factors such as NFkB and AP-1.The aim of this study was assess the role of APE1/Ref-1 on inflammatory response and the possibility of its modulation to reduce the sequels of the disease. Firstly it was performed an assay to measure cytokine in cerebrospinal fluid of patients with BM due to Streptococcus pneumoniae and Neisseriae meningitides. Further, a cellular model of inflammation was used to observe the effect of the inhibition of the endonuclease and redox activity of APE1/Ref-1 on cytokine levels. Additionally, APE1/Ref-1 expression in cortex and hippocampus of rat with MB after vitamin B6 treatment was evaluated. Altogether, results showed a similar profile of cytokines in the cerebrospinal fluid of patients from both pathogens, although IFNy showed higher expression in patients with BM caused by S. pneumoniae. On the other hand, inhibitors of APE1/Ref-1 reduced cytokine levels, mainly TNF-α. Reduction of oxidative stress markers was also observed after introduction of inhibitors in the LPS-stimulated cell. In the animal model, BM increased the expression of the protein APE1/Ref-1, while vitamin B6 promoted reduction. Thereby, this data rise important factors to be considered in pathogenesis of BM, e.g., IFNy can be used as prognostic factor during corticosteroid therapy, APE1/Ref-1 can be an important target to modulate the level of inflammation and VIII oxidative stress, and vitamin B6 seems modulates several proteins related to cell death. So, this study highlights a new understanding on the role of APE1/Ref-1 on the inflammation and the oxidative stress during inflammation condition

Increased pancreatic islet mass is accompanied by activation of the insulin receptor substrate-2/serine-threonine kinase pathway and augmented cyclin D(2) protein levels in insulin-resistant rats

It is well known that glucocorticoids induce peripheral insulin resistance in rodents and humans. Here, we investigated the structural and ultrastructural modifications, as well as the proteins involved in beta-cell function and proliferation, in islets from insulin-resistant rats. Adult male Wistar rats were made insulin resistant by daily administration of dexamethasone (DEX; 1mg/kg, i.p.) for five consecutive days, whilst control (CTL) rats received saline alone. Structure analyses showed a marked hypertrophy of DEX islets with an increase of 1.7-fold in islet mass and of 1.6-fold in islet density compared with CTL islets (P < 0.05). Ultrastructural evaluation of islets revealed an increased amount of secreting organelles, such as endoplasmic reticulum and Golgi apparatus in DEX islets. Mitotic figures were observed in DEX islets at structural and ultrastructural levels. Beta-cell proliferation, evaluated at the immunohistochemical level using anti-PCNA (proliferating cell nuclear antigen), showed an increase in pancreatic beta-cell proliferation of 6.4-fold in DEX islets compared with CTL islets (P < 0.0001). Increases in insulin receptor substrate-2 (IRS-2), phosphorylated-serine-threonine kinase AKT (p-AKT), cyclin D(2) and a decrease in retinoblastoma protein (pRb) levels were observed in DEX islets compared with CTL islets (P < 0.05). Therefore, during the development of insulin resistance, the endocrine pancreas adapts itself increasing beta-cell mass and proliferation, resulting in an amelioration of the functions. The potential mechanisms that underlie these events involve the activation of the IRS-2/AKT pathway and activation of the cell cycle, mediated by cyclin D(2). These adaptations permit the maintenance of glycaemia at near-physiological ranges.

Tobacco smoke-induced left ventricular remodelling is not associated with metalloproteinase-2 or -9 activation

Aim: To investigate the role of MMP-2 and MMP-9 in cardiac remodelling induced by tobacco smoke exposure in rats.Methods: Rats were allocated into two groups: C (n = 9): control animals; ETS (n = 9): exposed to tobacco smoke. After 4months, the animals underwent echocardiography, morphometric study and determination of MMP-2 and MMP-9 activity.Results: ETS rats had larger diastolic (C= 15.6 +/- 1.2 mm/kg, ETS = 18.0 +/- 0.9 mm/kg; p < 0.001) and systolic (C= 7.3 +/- 1.2 mm/kg, ETS = 9.2 0.9 mm/kg; p = 0.001) ventricular diameters adjusted for body weight. Fractional shortening (C= 53 +/- 4.8%, ETS = 48 +/- 3.3%; p = 0.031) and ejection fraction (C= 0. 89 +/- 0.03 5 ETS = 0. 86 +/- 0.02; p = 0.03 0) were smaller in the ETS group. Myocyte cross-sectional area (C= 245 8 mu m(2), ETS=253 8 mu m(2); p = 0.028) was higher in ETS rats. There were no differences in MNtP-2 (C=50 +/- 14%; ETS 43 +/- 11%, p 0.22 +/- 8) or MMP-9 (C=0.36 +/- 0.3%; ETS=0.62 +/- 0.3%, p=0.630) activity between the groups.Conclusion: MMP-2 and MMP-9 did not participate in the remodelling process induced by tobacco smoke exposure. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Platelet activation: ultrastructure and morphometry in platelet-rich plasma of horses

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Thrombomodulin-independent activation of protein C and specificity of hemostatically active snake venom serine proteinases - Crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator

Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 angstrom resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl1- instead of SO42-.

Measurements of inclusive W plus jets production rates as a function of jet transverse momentum in p(p)over-bar collisions root s=1.96 TeV

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Burnt sugarcane harvesting: Particulate matter exposure and the effects on lung function, oxidative stress, and urinary 1-hydroxypyrene

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Amifostine does not prevent activation of TGF beta 1 but induces smad 7 activation in megakaryocytes irradiated in vivo

Experiments were undertaken to assess the role of amifostine in the activation of latent TGFbeta1 and in the smad proteins cascade (smad 2/3, smad4, smad7), focusing on megakaryocytes, in the bone marrow irradiated in vivo. Non-irradiated megakaryocytes were negative for active TGFbeta1. Immunopositivity to active TGFbeta1 was detected in megakaryocytes 10 days after irradiation in amifostine- treated and untreated marrows. Smad 2/3 and smad 4 were strongly positive in the nucleus of megakaryocytes 10 days after irradiation. At the same time, a predominant hypocellular bone marrow with foci of hematopoiesis was observed with few megakaryocytes. An increase in the number of reticulin fibers was also seen. In amifostine-treated marrows, smad 2/3 and smad4 were not detected in the nucleus but were positive in the cytoplasm of megakaryocytes 10 days after irradiation. Coincidentally, bone marrows were cellular with megakaryocytes. Smad7 immunoexpression was detected in the cytoplasm of megakaryocytes in the non-irradiated, amifostine-treated and in the irradiated, amifostine-treated marrows. Data indicate that amifostine does not prevent latent TGFbeta1 activation in irradiated megakaryocytes. While TGFbeta1 signal transduction occurs in megakaryocytes in untreated bone marrows, it is inhibited in megakaryocytes in amifostine-treated marrows due to the induction of smad 7 activation. This is the first report showing smad 7 activation by amifostine. Our results also suggest a role for TGFbeta1 as an inhibitor of megakaryocytes in vivo. (C) 2002 Wiley-Liss, Inc.

GREEN-FUNCTION FOR A SPIN-1/2 PARTICLE IN AN EXTERNAL PLANE-WAVE ELECTROMAGNETIC-FIELD

The Green function for a spin-1/2 charged particle in the presence of an external plane wave electromagnetic field is calculated by algebraic techniques in terms of the free-particle Green function.

A novel biological activity for galectin-1: Inhibition of leukocyte-endothelial cell interactions in experimental inflammation

Galectin-1 (Gal-1), the prototype of a family of β -galactoside-binding proteins, has been shown to attenuate experimental acute and chronic inflammation. In view of the fact that endothelial cells (ECs), but not human polymorphonuclear leukocytes (PMNs), expressed Gal-1 we tested here the hypothesis that the protein could modulate leukocyte-EC interaction in inflammatory settings. In vitro, human recombinant (hr) Gal-1 inhibited PMN chemotaxis and trans-endothelial migration. These actions were specific as they were absent if Gal-1 was boiled or blocked by neutralizing antiserum. In vivo, hrGal-1 (optimum effect at 0.3 μg equivalent to 20 pmol) inhibited interleukin-1β-induced PMN recruitment into the mouse peritoneal cavity. Intravital microscopy analysis showed that leukocyte flux, but not their rolling velocity, was decreased by an anti-inflammatory dose of hrGal-1. Binding of biotinylated Gal-1 to resting and post-adherent human PMNs occurred at concentrations inhibitory in the chemotaxis and transmigration assays. In addition, the pattern of Gal-1 binding was differentially modulated by PMN or EC activation. In conclusion, these data suggest the existence of a previously unrecognized function of Gal-1, that is inhibition of leukocyte rolling and extravasation in experimental inflammation. It is possible that endogenous Gal-1 may be part of a novel anti-inflammatory loop in which the endothelium is the source of the protein and the migrating PMNs the target for its anti-inflammatory action.

Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counter-regulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages-cell types known to function in experimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS. Copyright © American Society for Investigative Pathology.

Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense effectors

An involvement of the transient receptor potential vanilloid (TRPV) 1 channel in the regulation of body temperature (T b) has not been established decisively. To provide decisive evidence for such an involvement and determine its mechanisms were the aims of the present study. We synthesized a new TRPV1 antagonist, AMG0347 [(E)-N-(7-hydroxy-5,6,7,8-tetrahydronaphthalen-1- yl)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)acrylamide], and characterized it in vitro. We then found that this drug is the most potent TRPV1 antagonist known to increase T b of rats and mice and showed (by using knock-out mice) that the entire hyperthermic effect of AMG0347 is TRPV1 dependent. AMG0347-induced hyperthermia was brought about by one or both of the two major autonomic cold-defense effector mechanisms (tail-skin vasoconstriction and/or thermogenesis), but it did not involve warmth-seeking behavior. The magnitude of the hyperthermic response depended on neither T b nor tail-skin temperature at the time of AMG0347 administration, thus indicating that AMG0347-induced hyperthermia results from blockade of tonic TRPV1 activation by nonthermal factors. AMG0347 was no more effective in causing hyperthermia when administered into the brain (intracerebroventricularly) or spinal cord (intrathecally) than when given systemically (intravenously), which indicates a peripheral site of action. We then established that localized intra-abdominal desensitization of TRPV1 channels with intraperitoneal resiniferatoxin blocks the T b response to systemic AMG0347; the extent of desensitization was determined by using a comprehensive battery of functional tests. We conclude that tonic activation of TRPV1 channels in the abdominal viscera by yet unidentified nonthermal factors inhibits skin vasoconstriction and thermogenesis, thus having a suppressive effect on T b. Copyright © 2007 Society for Neuroscience.

VEGF-C expression in oral cancer by neurotransmitter-induced activation of beta-adrenergic receptors

The aim of this study was to investigate the expression of vascular endothelial growth factor type C (VEGF-C) in oral squamous cell carcinoma (OSCC) cell lines through norepinephrine-induced activation of beta-adrenergic receptors. Human OSCC cell lines (SCC-9 and SCC-25) expressing beta-adrenergic receptors were stimulated with different concentrations of norepinephrine (0.1, 1, and 10 μM) and 1 μM of propranolol, and analyzed after 1, 6, and 24 h. VEGF-C gene expression and VEGF-C production in the cell supernatant were evaluated by real-time PCR and by ELISA, respectively. The results showed that beta-adrenergic receptor stimulation by different concentrations of norepinephrine or blocking by propranolol did not markedly alter VEGF-C expression by SCC-9 and SCC-25 cells. VEGF-C protein levels produced by oral malignant cell lines after stimulation with different norepinephrine concentrations or blocking with propranolol was statistically similar (p > 0.05) to those of the control group (nonstimulated OSCC cell lines). Our findings suggest that stimulation of beta-adrenergic receptors by means of norepinephrine does not seem to modulate the VEGF-C expression in OSCC cell lines. These findings reinforce the need for further studies in order to understand the responsiveness of oral cancer to beta-adrenergic receptor stimulation or blockage, especially with regard to VEGF-C production. © 2012 International Society of Oncology and BioMarkers (ISOBM).

Anxiogenic-like effect induced by TRPV1 receptor activation within the dorsal periaqueductal gray matter in mice

Pharmacological manipulation of TRPV1 (Transient Receptor Potential Vanilloid type-1) receptors has been emerging as a novel target in the investigation of anxiety states. Here, we attempt to show the role played by the TRPV1 receptors within the dorsal periaqueductal gray matter (dPAG), a midbrain structure strongly involved in the modulation of anxiety. Anxiety was assessed by recording spatiotemporal [percent open arm entries (%OE) and percent open arm time (%OT)] and ethological [e.g., head dipping (HD), stretched-attend postures (SAP)] measures in mice exposed to the elevated plus-maze (EPM). Mice received an intra-dPAG injection of the TRPV1 agonist capsaicin (0, 0.01, 0.1 or 1.0. nmol/0.2. μL; Experiment 1) or antagonist capsazepine (0, 10, 30 or 60. nmol/0.2. μL; Experiment 2), or combined injections of capsazepine (30. nmol) and capsaicin (1.0. nmol) (Experiment 3), and were exposed to the EPM to record spatiotemporal and ethological measures. While capsaicin produced an anxiogenic-like effect (it reduced %OE and %OT and frequency of SAP and HD in the open arms), capsazepine did not change any behavior in the EPM. However, when injected before capsaicin (1.0. nmol), intra-dPAG capsazepine (30. nmol-a dose devoid of intrinsic effects) antagonized completely the anxiogenic-like effect of the TRPV1 agonist. These results suggest that the anxiogenic-like effect produced by capsaicin is primarily due to TRPV1 activation within the dPAG in mice, but that dPAG TRPV1 receptors do not exert a tonic control over defensive behavior in mice exposed to the EPM. © 2013 Elsevier B.V.