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Red blood cell substitutes offer a solution to the problem of blood shortage and side effects of blood transfusion. Hemoglobin-based oxygen carriers (HBOCs) are one of the promising substitutes for red blood cells. Vasoactivity, which refers to the side effect of HBOCs that causes vasoconstriction and subsequent hypertension, limits the clinical application of HBOCs. In this study, an ex vivo method for the evaluation of vasoactivity induced by HBOCs was established based on isolated rat mesenteric artery vessels and the DMT120CP system. The DMT120CP system, equipped with a flowmeter, permits the control of intravascular pressure, pressure gradient, and flow conditions with high accuracy, simulating the physiological conditions for isolated vessels. The concentration of noradrenaline was optimized to 1 × 10-6â¼3 × 10-6 M. PEGylated bovine hemoglobin (PEG-bHb) was synthesized and perfused into the vessel for vasoactivity evaluation, with bHb as the positive control and PSS buffer solution as the negative control. PEG-bHb showed a hydration diameter of 15.5 ± 1.4 nm and a P50 value of 6.99 mmHg. PEG-bHb exhibited a colloid osmotic pressure of 64.1 mmHg and a viscosity of 1.73 cp at 40 mg/mL. The established vasoactivity evaluation method showed significant differences in samples (bHb or PEG-bHb) with different vasoactivity properties. The vasoconstriction percentage induced by PEG-bHb samples synthesized in different batches showed coefficients of variation less than 5%, indicating good applicability and repeatability. The established evaluation method can be applied to study the vasoactivity induction and elimination strategies, promoting the clinical application of HBOCs.
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Vascular production of nitric oxide (NO) regulates vascular tone. However, highly permeable NO entering the cardiomyocyte would profoundly impact metabolism and signalling without scavenging mechanisms. The purpose of this study was to establish mechanisms of cardiac NO scavenging. Quantitative optical studies of normoxic working hearts demonstrated that micromolar NO concentrations did not alter mitochondria redox state or respiration despite detecting NO oxidation of oxymyoglobin to metmyoglobin. These data are consistent with proposals that the myoglobin/myoglobin reductase (Mb/MbR) system is the major NO scavenging site. However, kinetic studies in intact hearts reveal a minor role (â¼9%) for the Mb/MbR system in NO scavenging. In vitro, oxygenated mitochondria studies confirm that micromolar concentrations of NO bind cytochrome oxidase (COX) and inhibit respiration. Mitochondria had a very high capacity for NO scavenging, importantly, independent of NO binding to COX. NO is also known to quickly react with reactive oxygen species (ROS) in vitro. Stimulation of NO scavenging with antimycin and its inhibition by substrate depletion are consistent with NO interacting with ROS generated in Complex I or III under aerobic conditions. Extrapolating these in vitro data to the intact heart supports the hypothesis that mitochondria are a major site of cardiac NO scavenging. KEY POINTS: Cardiomyocyte scavenging of vascular nitric oxide (NO) is critical in maintaining normal cardiac function. Myoglobin redox cycling via myoglobin reductase has been proposed as a major NO scavenging site in the heart. Non-invasive optical spectroscopy was used to monitor the effect of NO on mitochondria and myoglobin redox state in intact beating heart and isolated mitochondria. These non-invasive studies reveal myoglobin/myoglobin reductase plays a minor role in cardiac NO scavenging. A high capacity for NO scavenging by heart mitochondria was demonstrated, independent of cytochrome oxidase binding but dependent on oxygen and high redox potentials consistent with generation of reactive oxygen species.
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Mioglobina , Óxido Nítrico , Mioglobina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Óxido Nítrico/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Cinética , Miocitos Cardíacos/metabolismo , Oxidación-Reducción , Mitocondrias Cardíacas/metabolismo , Consumo de OxígenoRESUMEN
Hemin and heme-peroxidases have been considered essential catalysts for the nitrite/hydrogen peroxide (H2O2)-mediated protein nitration in vitro, understood as one of the main pathways for protein modification in biological systems. However, the role of nitric oxide (âNO) in the heme/hemin-induced protein nitration has not been studied in-depth. This is despite its reductive nitrosylating effects following binding to hemin and the possible involvement of the reactive nitrogen species in the nitration of various functional proteins. Here, the âNO-binding affinity of hemin has been studied along with the influence of âNO on the internalization of hemin into MDA-MB-231 cells and the accompanying changes in the profile of intracellular nitrated proteins. Moreover, to further understand the mechanism involved, bovine serum albumin (BSA) nitration was studied after treatment with hemin and âNO, with an investigation of the effects of pH of the reaction medium, generation of H2O2, and the oxidation of the tyrosine residues as the primary sites for the nitration. We demonstrated that hemin nitrosylation enhanced its cellular uptake and induced the one-electron oxidation and nitration of different intracellular proteins along with its âNO-scavenging efficiency. Moreover, the hemin/NO-mediated BSA nitration was proved to be dependent on the concentration of âNO and the pH of the reaction medium, with a vital role being played by the scavenging effects of protein for the free hemin molecules. Collectively, our results reaffirm the involvement of hemin and âNO in the nitration mechanism, where the nitrosylation products can induce protein nitration while promoting the effects of the components of the nitrite/H2O2-mediated pathway.
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Hemina , Nitritos , Hemina/química , Hemina/metabolismo , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico , Nitritos/metabolismo , Albúmina Sérica Bovina/química , Tirosina/químicaRESUMEN
Blood transfusion is the key to life in case of traumatic emergencies, surgeries and in several pathological conditions. An important goal of whole blood or red blood cell transfusion is the fast delivery of oxygen to vital organs and restoration of circulation volume. Whole blood or red blood cell transfusion has several limitations. Free haemoglobin not only loses its tetrameric configuration and extracts via the kidney leading to nephrotoxicity but also scavenges nitric oxide (NO), leading to vasoconstriction and hypertension. PFC based formulations transport oxygen in vivo, the contribution in terms of clinical outcome is challenging. The oxygen-carrying capacity is not the only criterion for the successful development of haemoglobin-based oxygen carriers (HBOCs). This review is a bird's eye view on the present state of the PFCs and HBOCs in which we analyzed the current modifications made or which are underway in development, their promises, and hurdles in clinical implementation.
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Sustitutos Sanguíneos , Fluorocarburos , Sustitutos Sanguíneos/uso terapéutico , Hemoglobinas/uso terapéutico , Humanos , Óxido Nítrico/uso terapéutico , Oxígeno/uso terapéuticoRESUMEN
INTRODUCTION: Muntingia calabura from the Muntingiaceae family has been documented for several medicinal uses. The combinations of drying treatment and extracting solvents for a plant species need to be determined and optimised to ensure that the extracts contain adequate amounts of the bioactive metabolites. OBJECTIVE: Evaluate the metabolite variations and antioxidant activity among M. calabura leaves subjected to different drying methods and extracted with different ethanol ratios using proton nuclear magnetic resonance (1 H-NMR)-based metabolomics. Methodology The antioxidant activity of M. calabura leaves dried with three different drying methods and extracted with three different ethanol ratios was determined by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) scavenging assays. The metabolites variation among the extracts and correlation with antioxidant activity were analysed by 1 H-NMR-based metabolomics. RESULTS: Muntingia calabura leaves extracted with 50% and 100% ethanol from air-drying and freeze-drying methods had the highest total phenolic content and the lowest IC50 value for the DPPH scavenging activity. Meanwhile, oven-dried leaves extracted with 100% ethanol had the lowest IC50 value for the NO scavenging activity. A total of 43 metabolites, including sugars, organic acids, amino acids, phytosterols, phenolics and terpene glycoside were tentatively identified. A noticeable discrimination was observed in the different ethanol ratios by the principal component analysis. The partial least-squares analysis suggested that 32 compounds out of 43 compounds identified were the contributors to the bioactivities. CONCLUSION: The results established set the preliminary steps towards developing this plant into a high value product for phytomedicinal preparations.
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Antioxidantes , Etanol , Espectroscopía de Resonancia Magnética , Metabolómica , Extractos Vegetales/farmacología , Hojas de la PlantaRESUMEN
Nitric oxide (NO), a radical gas molecule produced by nitric oxide synthase, plays a key role in the human body. However, when endogenous NO is overproduced by physiological disorders, severe inflammatory diseases such as rheumatoid arthritis (RA) can occur. Therefore, scavenging NO may be an alternative strategy for treating inflammatory disorders. In our previous study, we developed a NO-responsive macrosized hydrogel by incorporating a NO-cleavable cross-linker (NOCCL); here, we further evaluate the effectiveness of the NO-scavenging nanosized hydrogel (NO-Scv gel) for treating RA. NO-Scv gel is simply prepared by solution polymerization between acrylamide and NOCCL. When the NO-Scv gel is exposed to NO, NOCCL is readily cleaved by consuming the NO molecule, as demonstrated in a Griess assay. As expected, the NO-Scv gel reduces inflammation levels by scavenging NO in vitro and shows excellent biocompatibility. Furthermore, the more promising therapeutic effect of the NO-Scv gel in suppressing the onset of RA is observed in vivo in a mouse RA model when compared to the effects of dexamethasone, a commercial drug. Therefore, our findings suggest the potential of the NO-Scv gel for biomedical applications and further clinical translation.
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Antiinflamatorios/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Nanogeles/uso terapéutico , Óxido Nítrico/antagonistas & inhibidores , Animales , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/patología , Ratones , Óxido Nítrico/inmunologíaRESUMEN
This study aimed to improve the antioxidant and nitric oxide scavenging activities of ginseng marc fermented by Pediococcus acidilactici, thereby creating a biofunctional resource with improved anti-inflammatory capability. P. acidilactici was inoculated in 1% ginseng marc extract; cell viability, pH, and total titratable acidity were measured. Total phenolic and flavonoid contents were measured using Folin-Ciocalteu reagent and colorimetric method. Ferric reducing antioxidant power (FRAP), ß-carotene, and sodium nitroprusside (SNP) assays were used to evaluate functionality. Polyphenols and flavonoids totaled 33.7 ± 0.4 and 10.0 ± 0.4 mg/g of solid, respectively, at 24 h fermentation. P. acidilactici had 40 nM ß-galactosidase and 20 nM ß-glucosidase activities. Antioxidative activities increased up to 34.5 and 10.2%, respectively, as measured via FRAP and ß-carotene assays. Anti-inflammatory activity of the fermented extract-as measured via SNP assay-increased 342%, suggesting that ginseng marc fermented by P. acidilactici could be used in food or pharmaceutical industries.
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BACKGROUND: Skin forms an important part of human innate immune system. Wrinkles, thinning and roughening of skin are some of the symptoms that affect the skin as it ages. Reactive oxygen species induced oxidative stress plays a major role in skin aging by modulating the elastase enzyme level in the skin. Extrinsic factors that affect skin aging such as UV radiation can also cause malignant melanoma. Here we selected four medicinal plant materials, namely, leaves of Nyctanthes arbor-tristis, unripe and ripe Aegle marmelos fruit pulp and the terminal meristem of Musa paradisiaca flower and investigated their anti-aging properties and cytotoxicity in vitro individually as well as in a poly herbal formulation containing the four plant extracts in different ratios. METHODS: The phytochemical contents of the plant extracts were investigated for radical scavenging activity and total reducing power. Based upon its anti-oxidant properties, a poly herbal formulation containing leaves of Nyctanthes arbor-tristis, unripe and ripe fruit pulp of Aegle marmelos, and the terminal meristem of Musa paradisiaca flower in the ratio 6:2:1:1 (Poly Herbal Formulation 1) and 1:1:1:1 (Poly Herbal Formulation 2), respectively were formulated. RESULT: It has been observed that the Poly Herbal Formulation 1 was more potent than Poly Herbal Formulation 2 due to better anti-oxidant and anti-elastase activities in NIH3T3 fibroblast cells. In addition Poly Herbal formulation 1 also had better anti-cancer activity in human malignant melanoma cells. CONCLUSION: Based on these results these beneficial plant extracts were identified for its potential application as an anti-aging agent in skin creams as well as an anti-proliferation compound against cancer cells.
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Antioxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Elastasa Pancreática/antagonistas & inhibidores , Extractos Vegetales/farmacología , Aegle/química , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , Células 3T3 NIH , Óxido Nítrico/metabolismo , Oleaceae/química , Plantas Medicinales/química , Especies Reactivas de Oxígeno/metabolismo , Envejecimiento de la PielRESUMEN
The aim of the present study was to evaluate the total phenolic and lipid content, fatty acids profiles and in vitro antioxidant activities of aqueous and solvent extracts of the red seaweed Asparagopsis taxiformis, through six different investigations. The present study demonstrated that phenol contents (mg gallic acid/g dry weight) were highest in the aqueous and methanolic extracts, followed by the ethanolic, hydroethanolic and hydromethanolic extracts. The lowest phenol contents were identified in the three remaining extracts: Butanolic, petroleum ether and acetone extracts. Furthermore, the total lipid content of the algae powder amounted to 2.85% of dry weight. The fatty acid methyl ester profiles analysed by gas-liquid chromatography represented indicated that fatty acids comprised 91.0±0.3% of total algae lipids. The saturated to unsaturated fatty acid contents amounted to 23.2±0.1 and 67.9±0.4% respectively. C13:0 (tridecanoate), C15:0 (pentadecanoate) and C17:0 (heptadecanoate) represented 47.4% of the total saturated fatty acids. Notably, the two most abundant unsaturated fatty acids, C15:1 (pentadecenoate) and C18:2 (octadecadienoate) represented 13.4 and 11.4% respectively, of the total unsaturated fatty acid content. Furthermore, the results of the antioxidant screening performed at 1.0 mg/ml, revealed that aqueous and methanolic extracts exhibited higher inhibition against superoxide and nitric oxide radicals and excellent radical scavenging activity [with half maximal inhibitory concentration (IC50) values 5.1 and 15.0 µg/ml, respectively], demonstrating improved antioxidant behavior when compared with standard ascorbic acid (which has an IC50 value of 3.7 µg/ml). Scavenging activity of the aqueous and methanolic extracts exhibited a strong peroxidation inhibition against linoleic acid emulsion system at a concentration of 300 µg/ml in comparison to the butylated hydroxyltoluene. Although all the studied extracts exhibited ferric reducing power, the aqueous and methanolic extracts had greater hydrogen donating ability. By contrast, hydromethanolic, ethanolic, hydroethanolic, butanolic, acetone and petroleum ether extracts exhibited weak antioxidant behavior. The antioxidant activity of potent seaweed species identified in the current study means that as well as being used as a functional food, they may be developed as novel pharmaceutical compounds and may be used as anti-ageing agents.
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Despite the significant impacts of light on nitric oxide (NO) levels in plants, the mechanism underlying the influence of this environmental factor on NO metabolism remains poorly understood. A critical mechanism controlling NO levels in plant cells relies on the S-nitrosylation of glutathione (GSH), giving rise to S-nitrosoglutathione (GSNO), which can be either stored or degraded depending on the cellular context. Here, we demonstrate that a strict balance is maintained between NO generation and scavenging during tomato (Solanum lycopersicum) seedling deetiolation. Given the absence of accurate methods in the literature to estimate NO scavenging in planta, we first developed a simple, robust system to continuously monitor the global in vivo NO scavenging by plant tissues. Then, using photomorphogenic tomato mutants, we demonstrated that the light-evoked de-etiolation is associated with a dramatic rise in NO content followed by a progressive increment in NO scavenging capacity of the tissues. Light-driven increments in NO scavenging rates coincided with pronounced rises in S-nitrosothiol content and GSNO reductase (GSNOR) activity, thereby suggesting that GSNO formation and subsequent removal via GSNOR might be key for controlling NO levels during seedling deetiolation. Accordingly, treatments with thiol-blocking compounds further indicated that thiol nitrosylation might be critically involved in the NO scavenging mechanism responsible for maintaining NO homeostasis during deetiolation. The impacts of both light and NO on the transcriptional profile of glutathione metabolic genes also revealed an independent but coordinated action of these signals on the regulation of key components of glutathione and GSNO metabolisms. Altogether, these data indicated that GSNO formation and subsequent removal might facilitate maintaining NO homeostasis during light-driven seedling deetiolation.
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Etiolado , Homeostasis/efectos de la radiación , Luz , Óxido Nítrico/metabolismo , Plantones/metabolismo , Plantones/efectos de la radiación , Aldehído Oxidorreductasas/metabolismo , Depuradores de Radicales Libres , Glutatión/química , Glutatión/metabolismo , Óxido Nítrico/química , Reacción en Cadena de la Polimerasa , Plantones/crecimiento & desarrolloRESUMEN
A new series of N-(6-arylbenzo[d]thiazol-2-yl)acetamides were synthesized by C-C coupling methodology in the presence of Pd(0) using various aryl boronic pinacol ester/acids. The newly synthesized compounds were evaluated for various biological activities like antioxidant, haemolytic, antibacterial and urease inhibition. In bioassays these compounds were found to have moderate to good activities. Among the tested biological activities screened these compounds displayed the most significant activity for urease inhibition. In urease inhibition, all compounds were found more active than the standard used. The compound N-(6-(p-tolyl)benzo[d]thiazol-2-yl)acetamide was found to be the most active. To understand this urease inhibition, molecular docking studies were performed. The in silico studies showed that these acetamide derivatives bind to the non-metallic active site of the urease enzyme. Structure-activity studies revealed that H-bonding of compounds with the enzyme is important for its inhibition.
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Acetamidas/síntesis química , Antibacterianos/síntesis química , Antioxidantes/síntesis química , Benzotiazoles/síntesis química , Inhibidores Enzimáticos/síntesis química , Ureasa/antagonistas & inhibidores , Acetamidas/farmacología , Antibacterianos/farmacología , Antioxidantes/farmacología , Benzotiazoles/farmacología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/crecimiento & desarrollo , Hemólisis/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Óxido Nítrico/antagonistas & inhibidores , Phaseolus/química , Phaseolus/enzimología , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/química , Ureasa/químicaRESUMEN
Scavenging of nitric oxide (NO) often interferes with studies on NO signaling in cell-free preparations. We observed that formation of cGMP by NO-stimulated purified soluble guanylate cyclase (sGC) was virtually abolished in the presence of cytosolic preparations of porcine coronary arteries, with the scavenging activity localized in the tunica media (smooth muscle layer). Electrochemical measurement of NO release from a donor compound and light absorbance spectroscopy showed that cytosolic preparations contained a reduced heme protein that scavenged NO. This protein, which reacted with anti-human hemoglobin antibodies, was efficiently removed from the preparations by haptoglobin affinity chromatography. The cleared cytosols showed only minor scavenging of NO according to electrochemical measurements and did not decrease cGMP formation by NO-stimulated sGC. In contrast, the column flow-through caused a nearly 2-fold increase of maximal sGC activity (from 33.1 ± 1.6 to 54.9 ± 2.2 µmol × min(-1) × mg(-1)). The proteins retained on the affinity column were identified as hemoglobin α and ß subunits. The results indicate that hemoglobin, presumably derived from vasa vasorum erythrocytes, is present and scavenges NO in preparations of porcine coronary artery smooth muscle. Selective removal of hemoglobin-mediated scavenging unmasked stimulation of maximal NO-stimulated sGC activity by a soluble factor expressed in vascular tissue.
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Vasos Coronarios/metabolismo , Hemoglobinas/metabolismo , Óxido Nítrico/metabolismo , Túnica Media/metabolismo , Animales , Bovinos , GMP Cíclico/metabolismo , Citoglobina , Globinas/metabolismo , Haptoglobinas/metabolismo , Humanos , Técnicas In Vitro , Guanilil Ciclasa Soluble/metabolismo , PorcinosRESUMEN
Secondary metabolite contents (total phenolic, flavonoid, tocopherol, and tocotrienol) and antioxidant activities of Hashemi rice bran extracts obtained by ultrasound-assisted and traditional solvent (ethanol and 50:50 (v/v) ethanol-water) extraction techniques were compared. Phenolic and, flavonoid compounds were identified using ultra-high performance liquid chromatography and method validation was performed. Significant differences (p < 0.05) were observed among the different extraction techniques upon comparison of phytochemical contents and antioxidant activities. The extracts obtained using the ethanol-water (50:50 v/v) ultrasonic technique showed the highest amounts of total phenolics (288.40 mg/100 g dry material (DM)), total flavonoids (156.20 mg/100 g DM), and total tocotrienols (56.23 mg/100 g DM), and the highest antioxidant activity (84.21% 1,1-diphenyl-2-picrylhydrazyl (DPPH), 65.27% ß-carotene-linoleic bleaching and 82.20% nitric oxide scavenging activity). Secondary metabolite contents and antioxidant activities of the rice bran extracts varied depending of the extraction method used, and according to their effectiveness, these were organized in a decreasing order as follows: ethanol-water (50:50 v/v) ultrasonic, ethanol-water (50:50 v/v) maceration, ethanol ultrasonic and ethanol maceration methods. Ferulic, gallic and chlorogenic acids were the most abundant phenolic compounds in rice bran extracts. The phytochemical constituents of Hashemi rice bran and its antioxidant properties provides insights into its potential application to promote health.
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Antioxidantes/química , Flavonoides/química , Oryza/química , Fitoquímicos/química , Etanol/química , Depuradores de Radicales Libres/química , Oxidación-Reducción , Fenoles , Extractos Vegetales/química , Solventes/químicaRESUMEN
Paroxysmal nocturnal haemoglobinuria (PNH) is a progressive and life-threatening disease that causes thrombosis, end organ damage and impaired quality of life. Chronic uncontrolled complement activation leads to chronic haemolysis, causing progressive morbidities and early mortality. Hence, early diagnosis is essential for improved patient management and prognosis. Eculizumab (SOLIRIS®) specifically inhibits chronic, uncontrolled complement activation and is the first-in-class, humanised, monoclonal antibody targeting C5 within the terminal complement pathway. Eculizumab is the first and only approved treatment for PNH in adults and children.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Activación de Complemento/efectos de los fármacos , Proteínas Inactivadoras de Complemento/uso terapéutico , Hemoglobinuria Paroxística/complicaciones , Hemoglobinuria Paroxística/tratamiento farmacológico , Trombosis/etiología , Animales , Proteínas del Sistema Complemento/inmunología , Hemoglobinuria Paroxística/inmunología , Hemólisis , Humanos , Terapia Molecular DirigidaRESUMEN
The genus Mycobacterium is comprised of Gram-positive bacteria occupying a wide range of natural habitats and includes species that range from severe intracellular pathogens to economically useful and harmless microbes. The recent upsurge in the availability of microbial genome data has shown that genes encoding haemoglobin-like proteins are ubiquitous among Mycobacteria and that multiple haemoglobins (Hbs) of different classes may be present in pathogenic and non-pathogenic species. The occurrence of truncated haemoglobins (trHbs) and flavohaemoglobins (flavoHbs) showing distinct haem active site structures and ligand-binding properties suggests that these Hbs may be playing diverse functions in the cellular metabolism of Mycobacteria. TrHbs and flavoHbs from some of the severe human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae have been studied recently and their roles in effective detoxification of reactive nitrogen and oxygen species, electron cycling, modulation of redox state of the cell and facilitation of aerobic respiration have been proposed. This multiplicity in the function of Hbs may aid these pathogens to cope with various environmental stresses and survive during their intracellular regime. This chapter provides recent updates on genomic, structural and functional aspects of Mycobacterial Hbs to address their role in Mycobacteria.
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Proteínas Bacterianas/metabolismo , Hemoproteínas/metabolismo , Mycobacterium/metabolismo , Hemoglobinas Truncadas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biotransformación , Hemoproteínas/química , Hemoproteínas/genética , Redes y Vías Metabólicas , Modelos Moleculares , Mycobacterium/química , Mycobacterium/genética , Óxido Nítrico/metabolismo , Óxido Nítrico/toxicidad , Oxidación-Reducción , Oxígeno/metabolismo , Oxígeno/toxicidad , Conformación Proteica , Especies de Nitrógeno Reactivo/metabolismo , Especies de Nitrógeno Reactivo/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/toxicidad , Hemoglobinas Truncadas/química , Hemoglobinas Truncadas/genéticaRESUMEN
The dependence of the structure and function of cytoplasmic organelles in endothelial cells on constitutively produced intracellular nitric oxide (NO) remains largely unexplored. We previously reported fragmentation of the Golgi apparatus in cells exposed to NO scavengers or after siRNA-mediated knockdown of eNOS. Others have reported increased mitochondrial fission in response to an NO donor. Functionally, we previously reported that bovine pulmonary arterial endothelial cells (PAECs) exposed to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) developed a prosecretory phenotype characterized by prolonged secretion of soluble proteins. In the present study, we investigated whether NO scavenging led to remodeling of the endoplasmic reticulum (ER). Live-cell DAF-2DA imaging confirmed the presence of intracellular NO in association with the BODIPY C5-ceramide-labeled Golgi apparatus. Untreated human PAECs displayed a pattern of peripheral tubulo-reticular ER with a juxtanuclear accumulation of ER sheets. Cells exposed to c-PTIO showed a dramatic increase in ER sheets as assayed using immunofluorescence for the ER structural protein reticulon-4b/Nogo-B and the ER-resident GTPase atlastin-3, live-cell fluorescence assays using RTN4-GFP and KDEL-mCherry, and electron microscopy methods. These ER changes were inhibited by the NO donor diethylamine NONOate, and also produced by L-NAME, but not D-NAME or 8-br-cGMP. This ER remodeling was accompanied by Golgi fragmentation and increased fibrillarity and function of mitochondria (uptake of tetramethyl-rhodamine, TMRE). Despite Golgi fragmentation the functional ER/Golgi trafficking unit was preserved as seen by the accumulation of Sec31A ER exit sites adjacent to the dispersed Golgi elements and a 1.8-fold increase in secretion of soluble cargo. Western blotting and immunopanning data showed that RTN4b was increasingly ubiquitinated following c-PTIO exposure, especially in the presence of the proteasomal inhibitor MG132. The present data complete the remarkable insight that the structural integrity of three closely juxtaposed cytoplasmic organelles - Golgi apparatus, endoplasmic reticulum and mitochondria - is dependent on nitric oxide.