Publications by authors named "Hugo Monteiro"

55 Publications

Molecular Dynamics Studies of Therapeutic Liquid Mixtures and Their Binding to Mycobacteria.

Front Pharmacol 2021 20;12:626735. Epub 2021 Apr 20.

Red Glead Discovery AB, Lund, Sweden.

Tuberculosis is an highly contagious disease still considered by the WHO as one of most infectious diseases worldwide. The therapeutic approach, used to prevent and treat tuberculosis targets the complex, comprises a combination of drugs administrated for long periods of time, which, in many cases, could cause several adverse effects and, consequently, low compliance of the patient to the treatment and drug-resistance. Therefore, therapeutic liquid mixtures formulated with anti-tuberculosis drugs and/or adjuvants in tuberculosis therapy are an interesting approach to prevent toxic effects and resistance to anti-tuberculosis drugs. The herein formulated therapeutic liquid mixtures, including ethambutol, arginine, citric acid and water under different molar ratios, were studied through a molecular dynamics approach to understand how ethambutol and arginine could be stabilized by the presence of citric acid and/or water in the mixture. To gain insights on how the uptake of these mixtures into the mycobacteria cell may occur and how a mycobacterial ABC transporter could contribute to this transport, multiple simultaneous ligand docking was performed. Interactions between citric acid and ethambutol involving the carboxyl and hydroxyl groups of citric acid with the amines of ethambutol were identified as the most critical ones. Water molecules present in the mixture provides the necessary network of hydrogen bonds that stabilize the mixture. Molecular docking additionally provided an interesting hypothesis on how the different mixture components may favor binding of ethambutol to an ABC importer. The data presented in this work helps to better understand these mixtures as well as to provide cues on the mechanisms that allow them to cross the mycobacterial cell membrane.
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http://dx.doi.org/10.3389/fphar.2021.626735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096353PMC
April 2021

AF-React study: atrial fibrillation management strategies in clinical practice-retrospective longitudinal study from real-world data in Northern Portugal.

BMJ Open 2021 03 29;11(3):e040404. Epub 2021 Mar 29.

Department of Community Medicine, Information and Health Decision Sciences (MEDCIDS), Faculty of Medicine of the University of Porto, Porto, Portugal.

Objectives: To determine the prevalence of atrial fibrillation (AF) and to assess how these patients are being cared for: what anticoagulants are being prescribed and are they being prescribed as recommended?

Design: Retrospective longitudinal study.

Setting: This study was conducted in the Regional Health Administration of Northern Portugal.

Participants: This study used a database that included 63526 patients with code K78 of the International Classification of Primary Care between January 2016 and December 2018.

Results: The prevalence of AF among adults over 40 years in the northern region of Portugal was 2.3% in 2016, 2.8% in 2017 and 3% in 2018. From a total of 63 526 patients, 95.8% had an indication to receive anticoagulation therapy. Of these, 44 326 (72.9%) are being treated with anticoagulants: 17 936 (40.5%) were prescribed vitamin K antagonists (VKAs) and 26 390 (59.5%) were prescribed non-VKA anticoagulants. On the other hand, 2688 patients of the total (4.2%) had no indication to receive anticoagulation therapy. Of these 2688 patients, 1100 (40.9%) were receiving anticoagulants.

Conclusions: The prevalence of AF is 3%. Here, we report evidence of both undertreatment and overtreatment. Although having an indication, a considerable proportion of patients (27.1%) are not anticoagulated, and among patients with AF without an indication to receive anticoagulation therapy, a considerable proportion (40.9%) are receiving anticoagulants. The AF-React study brings extremely relevant conclusions to Portugal and follows real-world studies in patients with AF in Europe, presenting some data not yet studied.
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http://dx.doi.org/10.1136/bmjopen-2020-040404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009225PMC
March 2021

Proteome Responses to Spinosad Exposure.

Toxics 2020 Dec 11;8(4). Epub 2020 Dec 11.

MARE-Marine and Environmental Sciences Centre, ESTM, Polytechnic of Leiria, 2520-641 Peniche, Portugal.

The potential of proteome responses as early-warning indicators of insecticide exposure was evaluated using the non-biting midge (Meigen) as the model organism. larvae were exposed to environmentally relevant concentrations of the neurotoxic pesticide spinosad to uncover molecular events that may provide insights on the long-term individual and population level consequences. The iTRAQ labeling method was performed to quantify protein abundance changes between exposed and non-exposed organisms. Data analysis revealed a general dose-dependent decrease in the abundance of globin proteins as a result of spinosad exposure. Additionally, the downregulation of actin and a larval cuticle protein was also observed after spinosad exposure, which may be related to previously determined life-history traits impairment and biochemical responses. Present results suggest that protein profile changes can be used as early warning biomarkers of pesticide exposure and may provide a better mechanistic interpretation of the toxic response of organisms, aiding in the assessment of the ecological effects of environmental contamination. This work also contributes to the understanding of the sublethal effects of insecticides in invertebrates and their molecular targets.
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http://dx.doi.org/10.3390/toxics8040117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768432PMC
December 2020

Ruminal acidosis, bacterial changes, and lipopolysaccharides.

J Anim Sci 2020 Aug;98(8)

Department of Animal Sciences, University of Florida, Gainesville, FL.

Acute and subacute ruminal acidosis (SARA) are common nutritional problems in both beef and dairy cattle. Therefore, the objective of this review is to describe how ruminal Gram-negative bacteria could contribute to the pathogenesis of ruminal acidoses, by releasing lipopolysaccharides (LPS; a component of their cell wall) in the ruminal fluid. When cattle consume excessive amounts of highly fermentable carbohydrates without prior adaptation, normal fermentation become disrupted. The fermentation of these carbohydrates quickly decreases ruminal pH due to the accumulation of short-chain fatty acids and lactate in the rumen. As a consequence, ruminal epithelium may be damaged and tissue function could be impaired, leading to a possible translocation of pathogenic substances from the rumen into the bloodstream. Such changes in fermentation are followed by an increase in Gram-positive bacteria while Gram-negative bacteria decrease. The lyses of Gram-negative bacteria during ruminal acidosis increase LPS concentration in the ruminal fluid. Because LPS is a highly proinflammatory endotoxin in the circulatory system, past studies have raised concerns regarding ruminal LPS contribution to the pathogenesis of ruminal acidosis. Although animals that undergo these disorders do not always have an immune response, recent studies showed that different Gram-negative bacteria have different LPS composition and toxicity, which may explain the differences in immune response. Given the diversity of Gram-negative bacteria in the rumen, evaluating the changes in the bacterial community during ruminal acidosis could be used as a way to identify which Gram-negative bacteria are associated with LPS release in the rumen. By identifying and targeting ruminal bacteria with possible pathogenic LPS, nutritional strategies could be created to overcome, or at least minimize, ruminal acidosis.
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http://dx.doi.org/10.1093/jas/skaa248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455920PMC
August 2020

In vitro evaluation of as direct-fed microbials in high-producing dairy cows diets.

Transl Anim Sci 2020 Jan 21;4(1):214-228. Epub 2019 Dec 21.

Department of Animal Sciences, University of Florida, Gainesville, FL.

The objectives of this study were: 1) to compare the effects of live yeast (LY), yeast fermentation product (YFP), a mix of (MLP), and included as additives in dairy cows' diets on in vitro ruminal fermentation and gas production (GP); and 2) to evaluate the effects of as direct-fed microbials (DFM) in dairy cows' diets on in vitro ruminal fermentation, GP, nutrient digestibility, and N metabolism. Three experiments were carried out: Exp. 1 had the objective to compare all additives regarding ruminal fermentation parameters: an Ankom GP system was used in a completely randomized design, consisting of four 48 h incubations, and eight replications per treatment. There were eight treatments: a basal diet without additive (CTRL) or with one of the following additives: LY, YFP, MLP, or at four levels (% of diet Dry Matter (DM)): 0.05% (L1), 0.10% (L2), 0.15% (L3), and 0.20% (L4). In Exp. 2, a batch culture was used to evaluate ruminal fermentation, and CO and CH production using the same treatments and a similar experimental design, except for having 16 replications per treatment. Based on Exp. 1 and 2 results, Exp. 3 aimed at evaluating the effects of the on ruminal true nutrient digestibility and N utilization in order to evaluate the use of as DFM. The treatments CTRL, MLP, L1, and L2 were used in a replicated 4 × 4 Latin square design using a dual-flow continuous culture system. Data were analyzed using linear and nonlinear regression; treatment means were compared through contrasts, and L treatments in Exp. 1 and 2 were tested for linear, quadratic, and cubic effects. In Exp. 1, all treatments containing additives tended to reduce OM digestibility as well as reduced total volatile fatty acids (VFA) concentration and total GP. The YFP had greater OM digestibility than LY, and MLP treatment had greater total VFA concentration compared to treatments. In Exp. 2, additives reduced CO production, and there were no major differences in CH. In Exp. 3, all additives reduced NH-N concentration. In conclusion, pH and lactate concentration were not affected in all three experiments regardless of additive tested, suggesting that these additives may not improve ruminal fermentation by pH modulation; and may improve ruminal N metabolism when used as DFM in high-producing dairy cows' diets, mainly by reducing NH-N concentration.
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http://dx.doi.org/10.1093/tas/txz187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994042PMC
January 2020

Assessment of fipronil toxicity to the freshwater midge Chironomus riparius: Molecular, biochemical, and organismal responses.

Aquat Toxicol 2019 Nov 4;216:105292. Epub 2019 Sep 4.

MARE - Marine and Environmental Sciences Centre, ESTM, Polytechnic Institute of Leiria, Peniche, Portugal.

Fipronil is a phenylpyrazole insecticide that entered the market to replace organochlorides and organophosphates. Fipronil impairs the regular inhibition of nerve impulses that ultimately result in paralysis and death of insects. Because of its use as a pest control, and due to runoff events, fipronil has been detected in freshwater systems near agricultural areas, and therefore might represent a threat to non-target aquatic organisms. In this study, the toxicity of fipronil to the freshwater midge Chironomus riparius was investigated at biochemical, molecular, and whole organism (e.g. growth, emergence, and behavior) levels. At the individual level, chronic (28 days) exposure to fipronil resulted in reduced larval growth and emergence with a lowest observed effect concentration (LOEC) of 0.081 μg L. Adult weight, which is directly linked to the flying performance and fecundity of midges, was also affected (LOEC = 0.040 μg L). Additionally, behavioral changes such as irregular burrowing behavior of C. riparius larvae (EC = 0.084 μg L) and impairment of adult flying performance were observed. At a biochemical level, acute (48 h) exposure to fipronil increased cellular oxygen consumption (as indicated by the increase of electron transport system (ETS) activity) and decreased antioxidant and detoxification defenses (as suggested by the decrease in catalase (CAT) and glutathione S-transferase (GST) activities). Exposure to fipronil also caused alterations in the fatty acid profile of C. riparius, since high levels of stearidonic acid (SDA) were observed. A comparison between exposed and non-exposed larvae also revealed alterations in the expression of globins, cytoskeleton and motor proteins, and proteins involved in protein biosynthesis. These alterations may aid in the interpretation of potential mechanisms of action that lead to the effects observed at the organism level. Present results show that environmentally relevant concentrations of fipronil are toxic to chironomid populations which call for monitoring of phenylpyrazole insecticides and of their ecological effects in freshwaters. Present results also emphasize the importance of complementing ecotoxicological data with molecular approaches such as proteomics, for a better interpretation of the mode of action of insecticides in aquatic invertebrates.
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http://dx.doi.org/10.1016/j.aquatox.2019.105292DOI Listing
November 2019

Nitric oxide stimulates a PKC-Src-Akt signaling axis which increases human immunodeficiency virus type 1 replication in human T lymphocytes.

Nitric Oxide 2019 12 17;93:78-89. Epub 2019 Sep 17.

Department of Medicine/Infectious Diseases, Universidade Federal de São Paulo, São Paulo, Brazil; Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, São Paulo, Brazil.

Human immunodeficiency virus (HIV) infections are typically accompanied by high levels of secreted inflammatory cytokines and generation of high levels of reactive oxygen species (ROS). To elucidate how HIV-1 alters the cellular redox environment during viral replication, we used human HIV-1 infected CD4T lymphocytes and uninfected cells as controls. ROS and nitric oxide (NO) generation, antioxidant enzyme activity, protein phosphorylation, and viral and proviral loads were measured at different times (2-36 h post-infection) in the presence and absence of the NO donor S-nitroso-N-acetylpenicillamine (SNAP). HIV-1 infection increased ROS generation and decreased intracellular NO content. Upon infection, we observed increases in copper/zinc superoxide dismutase (SOD1) and glutathione peroxidase (GPx) activities, and a marked decrease in glutathione (GSH) concentration. Exposure of HIV-1 infected CD4T lymphocytes to SNAP resulted in an increasingly oxidizing intracellular environment, associated with tyrosine nitration and SOD1 inhibition. In addition, SNAP treatment promoted phosphorylation and activation of the host's signaling proteins, PKC, Src kinase and Akt. Inhibition of PKC leads to inhibition of Src kinase strongly suggesting that PKC is the upstream element in this signaling cascade. Changes in the intracellular redox environment after SNAP treatment had an effect on HIV-1 replication as reflected by increases in proviral and viral loads. In the absence or presence of SNAP, we observed a decrease in viral load in infected CD4T lymphocytes pre-incubated with the PKC inhibitor GF109203X. In conclusion, oxidative/nitrosative stress conditions derived from exposure of HIV-1-infected CD4T lymphocytes to an exogenous NO source trigger a signaling cascade involving PKC, Src kinase and Akt. Activation of this signaling cascade appears to be critical to the establishment of HIV-1 infection.
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http://dx.doi.org/10.1016/j.niox.2019.09.004DOI Listing
December 2019

Double Monoubiquitination Modifies the Molecular Recognition Properties of p15 Promoting Binding to the Reader Module of Dnmt1.

ACS Chem Biol 2019 10 18;14(10):2315-2326. Epub 2019 Sep 18.

CIC bioGUNE , 48160 Derio , Spain.

The proliferating cell nuclear antigen (PCNA)-associated factor p15 is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15 gene is overexpressed in several types of human cancer, and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15 is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its N-terminal tail. Here we present an NMR conformational characterization of p15 monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15 is monomeric, intrinsically disordered, and binds to PCNA as nonubiquitinated p15 does but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15 shows that the ubiquitin moieties, separated by eight disordered residues, form transient dimers because of the high local effective ubiquitin concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15 is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15 directly interacts with PCNA and recruits Dnmt1 for maintenance of DNA methylation during replication.
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http://dx.doi.org/10.1021/acschembio.9b00679DOI Listing
October 2019

Nitric oxide and interactions with reactive oxygen species in the development of melanoma, breast, and colon cancer: A redox signaling perspective.

Nitric Oxide 2019 08 19;89:1-13. Epub 2019 Apr 19.

New York University School of Medicine, New York, NY, USA.

Cancer development is closely related to chronic inflammation, which is associated with identifiable markers of tumor progression, such as uncontrolled cell proliferation, angiogenesis, genomic instability, chemotherapeutic resistance, and metastases. Redox processes mediated by reactive oxygen species (ROS) and nitric oxide (NO) within the inflammatory tumor microenvironment play an essential role in directly influencing intercellular and intracellular signaling. These reactive species originating in the cancer cell or its microenvironment, mediate the epithelial-mesenchymal transition (EMT) and the mesenchymal-epithelial transition (MET). However, intracellular interactions between NO and ROS must be controlled to prevent cell death. Melanoma, breast, and colon cancer cells have developed a mechanism to survive and adapt to oxidative and nitrosative stress. The mechanism involves a spatial-temporal fine adjustment of the intracellular concentrations of NO and ROS, thereby guaranteeing the successful development of cancer cells. Physiological concentrations of NO and supra physiological concentrations of ROS are prevalent in cancer cells at the primary site. The situation reverses in cancer cells undergoing the EMT prior to being released into the blood stream. Intracellular supra physiological concentrations of NO found in circulating cancer cells endow them with anoikis resistance. When the anoikis-resistant cancer cells arrive at a metastatic site they undergo the MET. Endogenous supra physiological concentrations of ROS and physiological NO concentrations are prevalent in these cells. Understanding tumor progression from the perspective of redox signaling permits the characterization of new markers and approaches to therapy. The synthesis and use of compounds with the capacity of modifying intracellular concentrations of NO and ROS may prove effective in disrupting a redox homeostasis operative in cancer cells.
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http://dx.doi.org/10.1016/j.niox.2019.04.009DOI Listing
August 2019

S-nitrosothiols and HS donors: Potential chemo-therapeutic agents in cancer.

Redox Biol 2019 10 5;27:101190. Epub 2019 Apr 5.

Department of Biochemistry, Center for Cellular and Molecular Therapy - Universidade Federal de São Paulo - Campus São Paulo, São Paulo, Brazil. Electronic address:

Nitric Oxide (NO) and Hydrogen Sulfide (HS) are components of an "interactome", which is defined as a redox system involving the interactions of RSS, RNS and ROS. Chemical interaction by these species is common and is characterized by one and two electron oxidation, nitrosylation, nitration and sulfuration/polysulfidation reactions. NO and HS are gases that penetrate cell membranes, are synthesized by specific enzymes, are ubiquitous, regulate protein activities through post-translational modifications and participate in cell signaling. The two molecules at high concentrations compared to physiological concentrations may result in cellular damage particularly through their interaction with other reactive species. NO and HS can interact with each other and form a variety of molecular species which may have constructive or destructive behavior depending on the cell type, the cellular environment (ex. oxygen tension, pH, redox state), where the products are produced and in what concentrations. Cross talk exists between NO and HS, whereby they can influence the generation and signaling behavior of each other. Given the above mentioned properties of NO and HS and studies in cancer cells and animal models employing NO and HS donors that generate higher than physiological concentrations of NO and HS and are effective in killing cancer cells but not normal cells, lend credence to the possibility of the utility of these donors in an approach to the treatment of cancer.
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http://dx.doi.org/10.1016/j.redox.2019.101190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859576PMC
October 2019

Toxicity of the insecticides spinosad and indoxacarb to the non-target aquatic midge Chironomus riparius.

Sci Total Environ 2019 May 20;666:1283-1291. Epub 2019 Feb 20.

MARE - Marine and Environmental Sciences Centre, ESTM, Instituto Politécnico de Leiria, Peniche, Portugal.

Spinosad and indoxacarb are two relatively new insecticides mainly used in agriculture to control insect pests. However, at their current application rates, non-target aquatic insect species may also be impacted. In this study, larvae of the non-biting midge Chironomus riparius were exposed in the laboratory to both insecticides and their effects evaluated at the organismal level, using standard ecotoxicological tests, and at the biochemical level, by monitoring specific oxidative stress, neuronal, and energy metabolism biomarkers. Chronic exposure to both insecticides compromised growth and emergence of C. riparius. Short-term exposures revealed alterations at biochemical level that might be related to the toxicological targets of both insecticides. Growth and development time were the most sensitive endpoints at individual level for both pesticides, while at the biochemical level, the electron transport system activity was the most sensitive biomarker for spinosad exposure, suggesting an increase in energy demands associated with the activation of defense mechanisms. Glutathione-S-transferase was the most sensitive biomarker for indoxacarb exposure, underlining the role of this enzyme in the detoxification of indoxacarb. Additionally, changes in lactate dehydrogenase and glutathione peroxidase activities were observed for both insecticides, and evidences of oxidative damage were found for spinosad. This study contributes to the growing knowledge on sublethal effects of novel insecticides on non-target aquatic invertebrates and strengthens the usefulness of biochemical biomarkers to support the interpretation of their potentially deleterious effects on aquatic insects near agricultural fields.
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http://dx.doi.org/10.1016/j.scitotenv.2019.02.303DOI Listing
May 2019

HIV-1 infection modulates IL-24 expression which contributes to cell apoptosis in vitro.

Cell Biol Int 2019 May 12;43(5):574-579. Epub 2019 Mar 12.

Disciplina de Infectologia, Laboratório de Retrovirologia, Universidade Federal de São Paulo, São Paulo, Brazil.

Although interleukin-24 (IL-24) has been extensively explored in the immunopathologies of autoimmune diseases, neoplasms, and infections, its role in HIV-1 infection has not been thoroughly elucidated to date. Therefore, the objective of this study was to evaluate the gene and protein expressions of IL-24 at the initial moments of HIV infection in PBMCs. Due to the pro-apoptotic role of IL-24, we evaluated the protein expression of caspase-3, as well as Annexin V/Propidium Iodide flow cytometry and phosphorylation of ERK, which may induce an apoptotic signal block when phosphorylated. The results of this study demonstrated that HIV-1 infection had an impact on the gene and protein expressions of IL-24 and ERK. Annexin V/Propidium Iodide assay demonstrated decrease in the mechanisms of apoptosis in infected cells after incubation of IL-24 neutralizing antibody. Studies on how HIV-1 regulates IL-24 expression may play a role in characterizing viral persistence mechanisms and designing antiretroviral strategies.
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http://dx.doi.org/10.1002/cbin.11111DOI Listing
May 2019

Amitraz toxicity to the midge Chironomus riparius: Life-history and biochemical responses.

Chemosphere 2019 Apr 4;221:324-332. Epub 2019 Jan 4.

Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal.

Acute and chronic toxicity of the formamidine pesticide amitraz to the midge Chironomus riparius was assessed using conventional ecotoxicological tests and biochemical approaches (biomarkers). Amitraz is mainly used as an ectoparasiticide in veterinary medicine, but also in agriculture and apiculture. However, information of amitraz toxicity to non-target invertebrates is limited. Besides the impairment of developmental and emergence rates (reduced larval growth, emergence, and delayed development time) caused by chronic exposure to amitraz, acute exposures induced alterations in the antioxidant enzymes glutathione peroxidase (GPx) and catalase (CAT), and in energetic metabolism biomarkers, lactate dehydrogenase (LDH) and electron transport system (ETS) activities. Moreover, lipid peroxidation (LPO) increased by amitraz exposure. Our results reveal potential secondary effects of amitraz to invertebrates and biomarkers that may aid in the interpretation of sub-lethal toxic responses to amitraz. These results add information concerning the potential outcomes of amitraz exposure to freshwater invertebrates underlining the importance of risk assessment studies of formamidine pesticides.
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http://dx.doi.org/10.1016/j.chemosphere.2019.01.018DOI Listing
April 2019

Conformational study of the electronic interactions and nitric oxide release potential of new S‑nitrosothiols esters derivatives of ibuprofen, naproxen and phenyl acids substituted (SNO-ESTERS): Synthesis, infrared spectroscopy analysis and theoretical calculations.

Spectrochim Acta A Mol Biomol Spectrosc 2019 Jan 11;207:132-142. Epub 2018 Sep 11.

Department of Chemistry, Institute of Environmental, Chemical and Pharmaceutical Sciences, Universidade Federal de São Paulo - Campus Diadema, Brazil. Electronic address:

The conformational study on the new S‑nitrosothiols esters (SNO-ESTERS): para-substituted (X = H, OMe, Cl and NO) S‑nitrosothiol derivatives 2‑methyl‑2‑(sulfanyl)propyl phenylacetates (R1), 2‑(4‑isobutylphenyl)propanoate (ibuprofen, R2), and 2‑(4‑isobutylphenyl)propanoate of 2‑methyl‑2‑(nitrososulfanyl)propyl (naproxen, R3) was performed using infrared spectroscopy (IR) in solvents with increasing polarity (CCl, CHCl, and CHCN), and theoretical calculations, to determine the preferential conformer and the potential of these compounds to release nitric oxide (NO). S‑Nitrosothiols were synthesized by esterification reactions, using chlorides of the corresponding carboxylic acids, with good yields (~60%). IR results showed that these compounds presented only one conformation, and the experimental data were supported by the theoretical results obtained by density functional theory (DFT) calculations using the 6311+G (2df, 2p) basis set. The calculations revealed that all S‑nitrosothiols presented one preferential anticlinal (ac) geometric conformation, which agrees with the data obtained experimentally in CCl. These conformers are stabilized by intramolecular hydrogen bonds. Examination of the geometry with regard to the RSNO group revealed that these compounds are preferentially in the trans (anti) conformation. The calculation of the orbital interactions using the Natural Bond Orbital (NBO) method showed that the n → σ hyper-conjugative interaction increases the SN bond length. The strong n → π interaction and electronic delocalization induces a partial π character to the SN bond. The weak σ bond indicates strong delocalization of the electron pair in O (NO) by the n → σ interaction, thereby increasing the capacity of NO release from SNO-ESTERS.
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http://dx.doi.org/10.1016/j.saa.2018.09.020DOI Listing
January 2019

Src kinase activation by nitric oxide promotes resistance to anoikis in tumour cell lines.

Free Radic Res 2018 May 13;52(5):592-604. Epub 2018 Apr 13.

a Department of Biochemistry- Center for Cellular and Molecular Therapy (CTCMol) , Escola Paulista de Medicina - Universidade Federal de São Paulo , Campus São Paulo, São Paulo , Brazil.

Tumour progression involves the establishment of tumour metastases at distant sites. Resistance to anoikis, a form of cell death that occurs when cells lose contact with the extracellular matrix and with neighbouring cells, is essential for metastases. NO has been associated with anoikis. NO treated HeLa cells and murine melanoma cells in suspension triggered a nitric oxide (NO)-Src kinase signalling circuitry that enabled resistance to anoikis. Two NO donors, sodium nitroprusside (SNP) (500 µM) and DETANO (125 µM), protected against cell death derived from detachment of a growth permissive surface (experimental anoikis). Under conditions of NO-mediated Src activation the following were observed: (a) down-regulation of the pro-apoptotic proteins Bim and cleaved caspase-3 and the cell surface protein, E-cadherin, (b) up-regulation of caveolin-1, and (c) the dissociation of cell aggregates formed when cells are detached from a growth permissive surface. Efficiency of reattachment of tumour cells in suspension and treated with different concentrations of an NO donor, was dependent on the NO concentration. These findings indicate that NO-activated Src kinase triggers a signalling circuitry that provides resistance to anoikis, and allows for metastases.
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http://dx.doi.org/10.1080/10715762.2018.1455095DOI Listing
May 2018

The expression of endothelial and inducible nitric oxide synthase and apoptosis in intestinal ischemia and reperfusion injury under the action of ischemic preconditioning and pentoxifylline.

Acta Cir Bras 2017 Nov;32(11):935-948

PhD, Associate Professor, Division of Surgical Techniques and Experimental Surgery, Department of Surgery, UNIFESP, Sao Paulo-SP, Brazil. Conception and design of the study, critical revision, final approval.

Purpose: To investigate the expression of nitric oxide synthase (NOS) and apoptosis associated with ischemic preconditioning (IPC) and pentoxifylline (PTX) in intestinal ischemia (I) and reperfusion (R) injury.

Methods: Thirty male rats were assigned to 5 groups: (CG), no clamping of the superior mesenteric artery (90 minutes); (IR-SS) saline + ischemia (30 minutes) + reperfusion (60 minutes); (IR-PTX) PTX + ischemia (30 minutes) + reperfusion (60 minutes); (IPC-IR-SS) 5 minutes of ischemia + 5 minutes of reperfusion (IPC) + saline + I(30 minutes)+R(60 minutes); and (IPC-IR-PTX) IPC + PTX + I(30 minutes)+ R(60 minutes).

Results: The application of IPC and PTX showed a significantly lower immunohistochemistry reaction for active caspase-3 (P<0.05) compared to IR+SS. The number of cells immunoreactive to BCL-2 was higher in the IR-PTX group (P>0.05). The NOS-2 expression (qRTPCR) in the IR-PTX group (P<0.05) was higher than the values for the IPC+IR-SS and IPC-IR-PTX groups. The NOS-3 expression was significantly upper in the IPC-IR-PTX group than in the CG (P<0.05), the IR-SS (P<0.05) and the IR-PTX (P<0.05) groups.

Conclusions: The BCL-2 and active caspase-3 showed beneficial effects on PTX and IPC. The expression of NOS-2 and NOS-3 in the IPC and IPC-PTX groups showed no synergistic effect.
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http://dx.doi.org/10.1590/s0102-865020170110000005DOI Listing
November 2017

Thioredoxin promotes survival signaling events under nitrosative/oxidative stress associated with cancer development.

Biomed J 2017 Aug 29;40(4):189-199. Epub 2017 Jul 29.

New York University School of Medicine, New York, NY, USA. Electronic address:

Accumulating mutations may drive cells into the acquisition of abnormal phenotypes that are characteristic of cancer cells. Cancer cells feature profound alterations in proliferation programs that result in a new population of cells that overrides normal tissue construction and maintenance programs. To achieve this goal, cancer cells are endowed with up regulated survival signaling pathways. They also must counteract the cytotoxic effects of high levels of nitric oxide (NO) and of reactive oxygen species (ROS), which are by products of cancer cell growth. Accumulating experimental evidence associates cancer cell survival with their capacity to up-regulate antioxidant systems. Elevated expression of the antioxidant protein thioredoxin-1 (Trx1) has been correlated with cancer development. Trx1 has been characterized as a multifunctional protein, playing different roles in different cell compartments. Trx1 migrates to the nucleus in cells exposed to nitrosative/oxidative stress conditions. Trx1 nuclear migration has been related to the activation of transcription factors associated with cell survival and cell proliferation. There is a direct association between the p21Ras-ERK1/2 MAP Kinases survival signaling pathway and Trx1 nuclear migration under nitrosative stress. The expression of the cytoplasmic protein, the thioredoxin-interacting protein (Txnip), determines the change in Trx1 cellular compartmentalization. The anti-apoptotic actions of Trx1 and its denitrosylase activity occur in the cytoplasm and serve as important regulators of cell survival. Within this context, this review focuses on the participation of Trx1 in cells under nitrosative/oxidative stress in survival signaling pathways associated with cancer development.
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http://dx.doi.org/10.1016/j.bj.2017.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136292PMC
August 2017

Heparan sulfate proteoglycan deficiency up-regulates the intracellular production of nitric oxide in Chinese hamster ovary cell lines.

J Cell Physiol 2018 04 9;233(4):3176-3194. Epub 2017 Oct 9.

Departamento de Bioquímica-UNIFESP, São Paulo, São Paulo, Brazil.

We investigated the role of glycosaminoglycans (GAGs) in the regulation of endothelial nitric oxide synthase (eNOS) activity in wild-type CHO-K1 cells and in xylosyltransferase-deficient CHO-745 cells. GAGs inhibit the integrin/FAK/PI3K/AKT signaling pathway in CHO-K1 cells, decreasing the phosphorylation of eNOS at Ser1177. Furthermore, in CHO-K1 cells, eNOS and PKCα are localized at sphingolipid- and cholesterol-rich domains in the plasma membrane called caveolae. At caveolae, PKCα activation stimulates the phosphorylation of eNOS on Thr495, resulting in further inhibition of NO production in these cells. In our data, CHO-745 cells generate approximately 12-fold more NO than CHO-K1 cells. Increased NO production in CHO-745 cells promotes higher rates of protein S-nitrosylation and protein tyrosine nitration. Regarding reactive oxygen species (ROS) production, CHO-745 cells show lower basal levels of superoxide (O ) than CHO-K1 cells. In addition, CHO-745 cells express higher levels of GPx, Trx1, and catalase than CHO-K1 cells, suggesting that CHO-745 cells are in a constitutive nitrosative/oxidative stress condition. Accordingly, we showed that CHO-745 cells are more sensitive to oxidant-induced cell death than CHO-K1 cells. The high concentration of NO and reactive oxygen species generated by CHO-745 cells can induce simultaneous mitochondrial biogenesis and antioxidant gene expression. These observations led us to propose that GAGs are part of a regulatory mechanism that participates in eNOS activation and consequently regulates nitrosative/oxidative stress in CHO cells.
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http://dx.doi.org/10.1002/jcp.26160DOI Listing
April 2018

The role of ischemic preconditioning and pentoxifylline in intestinal ischemia/reperfusion injury of rats.

Acta Cir Bras 2017 Jul;32(7):559-567

PhD, Associate Professor, Division of Surgical Techniques and Experimental Surgery, Department of Surgery, UNIFESP, Sao Paulo-SP, Brazil. Conception and design of the study, critical revision, final approval.

Purpose:: To investigate the role of ischemic preconditioning (IPC) and pentoxifylline (PTX) in intestinal mucosa ischemia/reperfusion injury (IR).

Methods: : Thirty rats were assigned to 5 groups (N=6): (CG): no clamping of the superior mesenteric artery (90 min.); (IR-SS): saline + ischemia (30 min.) + reperfusion (60 min.); (IR-PTX): PTX + ischemia (30min.) + reperfusion (60 min.); (IPC-IR-SS): 5 min. of ischemia + 5 minutes of reperfusion (IPC) + saline + ischemia (30 min.) + reperfusion (60 min.); (IPC-IR-PTX ): 5 min. of ischemia + 5 min. of reperfusion (IPC) + PTX + 30 min. of I + 60 minutes of R.

Results: : The IR-PTX, IPC-IR-SS and IPC-IR-PTX groups had significantly lower scores of mucosa damage than the IR-SS group. IR-PTX group showed higher scores than the IPC-IR-PTX group, in accordance with the hypothesis of a favorable effect of IPC alone or in association with PTX. Additionally, IPC-IR-SS had a higher damage score than the IPC-IR-PTX. The villi height and crypt depth were similar in all groups. The villi height in the IR-SS was significantly lower.

Conclusion:: Ischemic preconditioning or pentoxifylline alone protect the intestinal mucosa from ischemia/reperfusion injury. However, they do not have a synergistic effect when applied together.
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http://dx.doi.org/10.1590/s0102-865020170070000007DOI Listing
July 2017

. Focus on "Balance between -nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation".

Am J Physiol Cell Physiol 2017 08 21;313(2):C131-C133. Epub 2017 Jun 21.

Division of Biochemistry, Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

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http://dx.doi.org/10.1152/ajpcell.00127.2017DOI Listing
August 2017

Effects of Static or Oscillating Dietary Crude Protein Levels on Fermentation Dynamics of Beef Cattle Diets Using a Dual-Flow Continuous Culture System.

PLoS One 2016 30;11(12):e0169170. Epub 2016 Dec 30.

Department of Agriculture, Nutrition, and Veterinary Sciences, University of Nevada, Reno, Nevada, United States of America.

The objective of this study was to evaluate the effects of increasing dietary crude protein (CP) levels and also comparing the effects of static versus oscillating dietary CP on ruminal nutrient digestibility, ruminal fermentation, nitrogen (N) metabolism, and microbial efficiency in beef cattle diets using a dual-flow continuous culture system. Eight fermenters (1,223 ± 21 mL) were used in a replicated 4 x 4 Latin square design with periods lasting 12 d each (8 d for adaptation and 4 d for sampling). Dietary treatments were: 1) 10% CP, 2) 12% CP, 3) 14% CP, and 4) 10 and 14% CP diets oscillating at 48-h intervals. Experimental diets consisted of 50% orchard hay and 50% concentrate. Fermenters were fed 72 g/d and solid and liquid dilution rates were adjusted to 5.5 and 11%/h, respectively. Data were analyzed using the MIXED procedure in SAS with α = 0.05. Apparent and true ruminal digestibilities of dry matter and organic matter were not affected (P > 0.05) by increasing dietary CP, nor by oscillating dietary CP. Total volatile fatty acids concentration and molar proportions of acetate, propionate, butyrate, valerate, iso-butyrate and iso-valerate were not affected (P > 0.05) by increasing or oscillating dietary CP. Ruminal NH3-N concentration increased linearly (P < 0.01) in response to increasing dietary CP. Total N, non-ammonia N, and rumen undegraded protein flows did not differ among treatments or between oscillating dietary CP and static 12% CP. Microbial N and NH3-N flows and microbial efficiency did not differ when comparing oscillating versus static CP (P > 0.05). However, there was a quadratic effect (P < 0.05) for these variables when dietary CP was increased. These results indicate that either ruminal microorganisms do not respond to oscillating CP levels or are capable of coping with 48-h periods of undernourishment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169170PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201265PMC
July 2017

Inducible Nitric Oxide Synthase in the Carcinogenesis of Gastrointestinal Cancers.

Antioxid Redox Signal 2017 06 31;26(18):1059-1077. Epub 2016 Oct 31.

1 Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Frederick, Maryland.

Significance: Gastrointestinal (GI) cancer taken together constitutes one of the most common cancers worldwide with a broad range of etiological mechanisms. In this review, we have examined the impact of nitric oxide (NO) on the etiology of colon, colorectal, gastric, esophageal, and liver cancers. Recent Advances: Despite differences in etiology, initiation, and progression, chronic inflammation has been shown to be a common element within these cancers showing interactions of numerous pathways. NO generated at the inflammatory site contributes to the initiation and progression of disease. The amount of NO generated, time, and site vary and are an important determinant of the biological effects initiated. Among the nitric oxide synthase enzymes, the inducible isoform has the most diverse range, participating in numerous carcinogenic processes. There is emerging evidence showing that inducible nitric oxide synthase (NOS2) plays a central role in the process of tumor initiation and/or development.

Critical Issues: Redox inflammation through NOS2 and cyclooxygenase-2 participates in driving the mechanisms of initiation and progression in GI cancers.

Future Directions: Understanding the underlying mechanism involved in NOS2 activation can provide new insights into important prevention and treatment strategies. Antioxid. Redox Signal. 26, 1059-1077.
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http://dx.doi.org/10.1089/ars.2016.6850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488308PMC
June 2017

The Ig V complementarity-determining region 3-containing Rb9 peptide, inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor.

Peptides 2016 11 26;85:1-15. Epub 2016 Aug 26.

Department of Microbiology, Immunology and Parasitology, Cell Biology Division and Experimental Oncology Unit (UNONEX), Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil; Recepta Biopharma, São Paulo, SP, Brazil. Electronic address:

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the VCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.
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http://dx.doi.org/10.1016/j.peptides.2016.08.006DOI Listing
November 2016

Basal neutrophil function in human aging: Implications in endothelial cell adhesion.

Cell Biol Int 2016 Jul 11;40(7):796-802. Epub 2016 May 11.

Department of Biological Sciences, Federal University of São Paulo, Diadema, SP, Brazil.

Much attention has been drawn to the pro-inflammatory condition that accompanies aging. This study compared parameters from non-stimulated neutrophils, obtained from young (18-30 years old [y.o.]) and elderly (65-80 y.o.) human volunteers. Measured as an inflammatory marker, plasmatic concentration of hs-CRP was found higher in elderly individuals. Non-stimulated neutrophil production of ROS and NO was, respectively, 38 and 29% higher for the aged group. From the adhesion molecules evaluated, only CD11b expression was elevated in neutrophils from the aged group, whereas no differences were found for CD11a, CD18, or CD62. A 69% higher non-stimulated in vitro neutrophil/endothelial cell adhesion was observed for neutrophils isolated from elderly donors. Our results suggest that with aging, neutrophils may be constitutively producing more reactive species in closer proximity to endothelial cells of vessel walls, which may both contribute to vascular damage and reflect a neutrophil intracellular disrupted redox balance, altering neutrophil function in aging.
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http://dx.doi.org/10.1002/cbin.10618DOI Listing
July 2016

Effects of Partial Replacement of Corn with Glycerin on Ruminal Fermentation in a Dual-Flow Continuous Culture System.

PLoS One 2015 23;10(11):e0143201. Epub 2015 Nov 23.

Department of Agriculture, Nutrition, and Veterinary Sciences, University of Nevada, Reno, Nevada, United States of America.

The objective of this study was to evaluate the effects of partially replacing dry ground corn with glycerin on ruminal fermentation using a dual-flow continuous culture system. Six fermenters (1,223 ± 21 ml) were used in a replicated 3x3 Latin square arrangement with three periods of 10 d each, with 7 d for diet adaptation and 3 d for sample collections. All diets contained 75% concentrate and three dietary glycerin levels (0, 15, and 30% on dry matter basis), totaling six replicates per treatment. Fermenters were fed 72 g of dry matter/d equally divided in two meals/d, at 0800 and 2000 h. Solid and liquid dilution rates were adjusted daily to 5.5 and 11%/h, respectively. On d 8, 9, and 10, samples of 500 ml of solid and liquid digesta effluent were mixed, homogenized, and stored at -20°C. Subsamples of 10 ml were collected and preserved with 0.2 mL of a 50% H2SO4 solution for later determination of NH3-N and volatile fatty acids. Microbial biomass was isolated from fermenters for chemical analysis at the end of each experimental period. Data were analyzed using the MIXED procedure in SAS with α = 0.05. Glycerin levels did not affect apparent digestibility of DM (PLin. = 0.13; PQuad. = 0.40), OM (PLin. = 0.72; PQuad. = 0.15), NDF (PLin. = 0.38; PQuad. = 0.50) and ADF (PLin. = 0.91; PQuad. = 0.18). Also, glycerin inclusion did not affect true digestibility of DM (PLin. = 0.35; PQuad. = 0.48), and OM (PLin. = 0.08; PQuad. = 0.19). Concentrations of propionate (P < 0.01) and total volatile fatty acids (P < 0.01) increased linearly and concentrations of acetate (P < 0.01), butyrate (P = 0.01), iso-valerate (P < 0.01), and total branched-chain volatile fatty acids, as well as the acetate: propionate ratio (P < 0.01) decreased with glycerin inclusion. Linear increases on NH3-N concentration in digesta effluent (P < 0.01) and on NH3-N flow (P < 0.01) were observed due to glycerin inclusion in the diets. Crude protein digestibility (P = 0.04) and microbial N flow (P = 0.04) were greater in the control treatment compared with the other treatments and responded quadratically with glycerin inclusion. Furthermore, the inclusion of glycerin linearly decreased (P = 0.02) non-ammonia N flow. Glycerin levels did not affect the flows of total N (PLin. = 0.79; PQuad. = 0.35), and dietary N (PLin. = 0.99; PQuad. = 0.07), as well as microbial efficiency (PLin. = 0.09; PQuad. = 0.07). These results suggest that partially replacing dry ground corn with glycerin may change ruminal fermentation, by increasing total volatile fatty acids, and propionate concentration without affecting microbial efficiency, which may improve glucogenic potential of beef cattle diets.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143201PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4657883PMC
June 2016

Nitric oxide: Protein tyrosine phosphorylation and protein S-nitrosylation in cancer.

Biomed J 2015 Sep-Oct;38(5):380-8

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, USA.

Cancer is a worldwide health problem leading to a high incidence of morbidity and mortality. Malignant transformation can occur by expression of oncogenes, over-expression and deregulated activation of proto-oncogenes, and inactivation of tumor suppressor genes. These cellular actions occur through stimulation of oncogenic signaling pathways. Nitric oxide (NO) can induce genetic changes in cells and its intracellular generation can lead to tumor formation and progression. It can also promote anti-tumor activities. The pro- and anti-tumor activities of NO are dependent on its intracellular concentration, cell compartmentalization, and cell sensitivity. NO affects a number of oncogenic signaling pathways. This review focuses on two oncogenic signaling pathways: NO-EGFR-Src-FAK and NO-Ras-EGFR-ERK1/2 MAP kinases. In these pathways, low to intermediate concentrations of NO/S-nitrosothiols (RSNOs) stimulate oncogenic signaling, while high concentrations of NO/RSNO stimulate anti-oncogenic signaling. Increasing knowledge on pro- and anti-tumorigenic activities of NO and related reactive species such as RSNOs has fostered the research and synthesis of novel NO-based chemotherapeutic agents. RSNOs, effective as NO donors and trans-nitrosylating agents under appropriate conditions, may operate as potential chemotherapeutic agents.
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http://dx.doi.org/10.4103/2319-4170.158624DOI Listing
December 2016

Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration.

Nitric Oxide 2015 May 24;47:40-51. Epub 2015 Mar 24.

Department of Biochemistry, Center for Cellular and Molecular Therapy-CTCMOL, Escola Paulista de Medicina /Universidade Federal de São Paulo, SP, Brazil. Electronic address:

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.
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http://dx.doi.org/10.1016/j.niox.2015.03.004DOI Listing
May 2015

Asparagopsis armata and Sphaerococcus coronopifolius as a natural source of antimicrobial compounds.

World J Microbiol Biotechnol 2015 Mar 15;31(3):445-51. Epub 2015 Jan 15.

Marine Resources Research Group (GIRM), ESTM, Polytechnic Institute of Leiria, 2520-641, Peniche, Portugal.

Methanol, n-hexane and dichloromethane extracts of twelve marine macro-algae (Rhodophyta, Chlorophyta and Heterokontophyta divisions) from Peniche coast (Portugal) were evaluated for their antibacterial and antifungal activity. The antibacterial activity was evaluated by disc diffusion method against Bacillus subtilis (gram positive bacteria) and Escherichia coli (gram negative bacteria). Saccharomyces cerevisiae was used as a model for the antifungal activity by evaluating the growth inhibitory activity of the extracts. The high antibacterial activity was obtained by the Asparagopsis armata methanolic extract (10 mm-0.1 mg/disc), followed by the Sphaerococcus coronopifolius n-hexane extract (8 mm-0.1 mg/disc), and the Asparagopsis armata dichloromethane extract (12 mm-0.3 mg/disc) against Bacillus subtilis. There were no positive results against Escherichia coli. Sphaerococcus coronopifolius revealed high antifungal potential for n-hexane (IC50 = 40.2 µg/ml), dichloromethane (IC50 = 78.9 µg/ml) and methanolic (IC50 = 55.18 µg/ml) extracts against Saccharomyces cerevisiae growth. The antifungal potency of the Sphaerococcus coronopifolius extracts was similar with the standard amphotericin B. Asparagopsis armata and Sphaerococcus coronopifolius reveal to be interesting sources of natural compounds with antimicrobial properties.
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http://dx.doi.org/10.1007/s11274-015-1797-2DOI Listing
March 2015

Heparin modulates the expression of genes encoding pro and anti-apoptotic proteins in endothelial cells exposed to intestinal ischemia and reperfusion in rats.

Acta Cir Bras 2014 Jul;29(7):445-9

Department of Surgery, UNIFESP, Sao Paulo, SP, Brazil.

Purpose: To investigate if expression of genes encoding pro and anti-apoptotic proteins in the rat enteric endothelial cells stimulated by intestinal ischemia followed by reperfusion (IR) can be modified by treatment with heparin (HP).

Methods: Eighteen adult Wistar rats were divided in three groups: sham group submitted to laparotomy only (SG), ischemia followed by reperfusion group (IRG); ischemia followed by reperfusion plus pretreatment with HP 100 mg.kg-1 (IRG+HP). Ischemia was performed by clamping of the superior mesenteric artery. After 60 min of ischemia, metal clamps were removed for reperfusion for 120 min. Gene expression of encoding pro (Casp1, Casp6, Casp3, Cflar, Fas and Pgl) and anti-apoptotic (Bcl2, Bcl2l1 and Naip2) proteins in rat enteric endothelial cells was evaluated by PCR microarray method.

Results: Compared to rat endothelial cells of SG, the expression of pro-apoptotic genes was up-regulated in IRG while anti-apoptotic genes were down-regulated. In contrast, the expression of anti-apoptotic genes in IRG+HP was up-regulated while pro-apoptotic genes was down-regulated compared to SG.

Conclusion: The attenuation by heparin of intestinal ischemia-reperfusion previously demonstrated in rodents could be related with ability of this drug to stimulate and reduce gene expression of encoding anti and pro-apoptotic proteins, respectively.
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http://dx.doi.org/10.1590/s0102-86502014000700006DOI Listing
July 2014

Endothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis.

Arch Biochem Biophys 2014 Sep 21;558:14-27. Epub 2014 Jun 21.

Department of Biochemistry, Center for Cellular and Molecular Therapy - CTCMol, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Electronic address:

Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.
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http://dx.doi.org/10.1016/j.abb.2014.06.011DOI Listing
September 2014