RESUMEN
Atmospheric pressure plasma jets generate reactive oxygen and nitrogen species (RONS) in liquids and biological media, which find application in the new area of plasma medicine. These plasma-treated liquids were demonstrated recently to possess selective properties on killing cancer cells and attracted attention toward new plasma-based cancer therapies. These allow for local delivery by injection in the tumor but can be quickly washed away by body fluids. By confining these RONS in a suitable biocompatible delivery system, great perspectives can be opened in the design of novel biomaterials aimed for cancer therapies. Gelatin solutions are evaluated here to store RONS generated by atmospheric pressure plasma jets, and their release properties are evaluated. The concentration of RONS was studied in 2% gelatin as a function of different plasma parameters (treatment time, nozzle distance, and gas flow) with two different plasma jets. Much higher production of reactive species (H2O2 and NO2-) was revealed in the polymer solution than in water after plasma treatment. The amount of RONS generated in gelatin is greatly improved with respect to water, with concentrations of H2O2 and NO2- between 2 and 12 times higher for the longest plasma treatments. Plasma-treated gelatin exhibited the release of these RONS to a liquid media, which induced an effective killing of bone cancer cells. Indeed, in vitro studies on the sarcoma osteogenic (SaOS-2) cell line exposed to plasma-treated gelatin led to time-dependent increasing cytotoxicity with the longer plasma treatment time of gelatin. While the SaOS-2 cell viability decreased to 12%-23% after 72 h for cells exposed to 3 min of treated gelatin, the viability of healthy cells (hMSC) was preserved (â¼90%), establishing the selectivity of the plasma-treated gelatin on cancer cells. This sets the basis for designing improved hydrogels with high capacity to deliver RONS locally to tumors.
Asunto(s)
Antineoplásicos/farmacología , Gases em Plasma/farmacología , Especies de Nitrógeno Reactivo/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Gelatina/farmacología , Humanos , Tamaño de la Partícula , Soluciones , Propiedades de SuperficieRESUMEN
The coffee diterpene kahweol (KW; C20H26O3) is a cytoprotective agent exhibiting potent antioxidant actions, as demonstrated in several experimental models. In spite of the efforts to elucidate exactly how KW promotes cytoprotection, it was not previously examined whether KW would be able to protect mitochondria of human cells undergoing redox stress. In the present work, we have treated the human neuroblastoma SH-SY5Y cell line with KW at 0.1-10 µM for 12 h prior to a challenge with methylglyoxal (MG), a reactive dicarbonyl that impairs mitochondrial function. We have found that KW at 10 µM suppressed the loss of mitochondrial membrane potential (MMP) and the bioenergetics decline (including decreased activity of the mitochondrial complexes I and V and reduced production of adenosine triphosphate, ATP) in the MG-treated SH-SY5Y cells. KW also prevented the MG-elicited generation of reactive oxygen and nitrogen species (ROS and RNS, respectively) in the SH-SY5Y cells. In this regard, KW exerted an antioxidant effect on the membranes of mitochondria obtained from the MG-treated cells. The mitochondria-related effects induced by KW were blocked by inhibition of the phosphoinositide 3-kinase (PI3K)/Akt or of the p38 mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, silencing of the transcription factor nuclear factor E2-related factor 2 (Nrf2) suppressed the mitochondrial protection promoted by KW in the MG-challenged cells. Therefore, KW protected mitochondria by a mechanism associated with the PI3K/Akt and p38 MAPK/Nrf2 signaling pathways.
Asunto(s)
Citoprotección , Diterpenos/farmacología , Mitocondrias/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Línea Celular Tumoral , Diterpenos/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Complejo I de Transporte de Electrón/deficiencia , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/fisiología , Enfermedades Mitocondriales/inducido químicamente , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Piruvaldehído , Especies de Nitrógeno Reactivo/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Genetic deficiency of protein kinase R (PKR) in mice was reported to enhance macrophage activation in vitro in response to interferon-γ (IFNγ) and to reduce the burden of Mycobacterium tuberculosis (Mtb) in vivo (Wu et al. PloS One. 2012 7:e30512). Consistent with this, treatment of wild-type (WT) macrophages in vitro with a novel PKR inhibitor (Bryk et al., Bioorg. Med. Chem. Lett. 2011 21:4108-4114) also enhanced IFN-γ-dependent macrophage activation (Wu et al. PloS One. 2012 7:e30512). Here we show that co-treatment with IFN-γ and a new PKR inhibitor identified herein to be highly but not completely selective likewise induced macrophages to produce more reactive nitrogen intermediates (RNI) and tumor necrosis factor alpha (TNF-α) and less interleukin 10 (IL-10) than seen with IFN-γ alone. Unexpectedly, however, this new PKR inhibitor had a comparable effect on PKR-deficient macrophages. Retrospective investigation revealed that the PKR-deficient mice in (Wu et al. PloS One. 2012 7:e30512) had not been backcrossed. On comparing genetically matched PKR-deficient and WT mice, we saw no impact of PKR deficiency on macrophage activation in vitro or during the course of Mtb infection in vivo. In addition, although 129S1/SvImJ macrophage responses to IFN-γ were greater than those of C57BL/6J macrophages, PKR was not required to mediate the IFN-γ-dependent production of IL-10, RNI or TNF-α in either strain. Together the data cast doubt on PKR as a potential therapeutic target for tuberculosis.
Asunto(s)
Interferón gamma/farmacología , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , eIF-2 Quinasa/antagonistas & inhibidores , Animales , Células Cultivadas , Femenino , Interleucina-10/biosíntesis , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especies de Nitrógeno Reactivo/biosíntesis , Tuberculosis/microbiología , Factor de Necrosis Tumoral alfa/biosíntesis , eIF-2 Quinasa/genéticaRESUMEN
In chronic kidney disease patients, high phosphate (HP) levels are associated with cardiovascular disease, the major cause of morbidity and mortality. Since serum phosphate has been independently correlated with inflammation, the present study aimed to investigate an independent direct effect of HP as a pro-inflammatory factor in VSMCs. A possible modulatory effect of vitamin D (VitD) was also investigated. The study was performed in an in vitro model of human aortic smooth muscle cells (HASMCs). Incubation of cells in an HP (3.3 mM) medium caused an increased expression of the pro-inflammatory mediators intercellular adhesion molecule 1 (ICAM-1), interleukins (ILs) IL-1ß, IL-6, IL-8 and tumour necrosis factor α (TNF-α) (not corroborated at the protein levels for ICAM-1), as well as an increase in reactive oxygen/nitrogen species (ROS/RNS) production. This was accompanied by the activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signalling as demonstrated by the increase in the nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κΒ) assessed by Western blotting and confocal microscopy. Since all these events were attenuated by an antioxidant pre-incubation with the radical scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), it is suggested that the inflammatory response is upstream mediated by the ROS/RNS-induced activation of NF-κΒ. Addition of paricalcitol (PC) 3·10-8 M to cells in HP prevented the phosphate induced ROS/RNS increase, the activation of NF-κΒ and the cytokine up-regulation. A bimodal effect was observed, however, for different calcitriol (CTR) concentrations, 10-10 and 10-12 M attenuated but 10-8 M stimulated this phosphate induced pro-oxidative and pro-inflammatory response. Therefore, these findings provide novel mechanisms whereby HP may directly favour vascular dysfunctions and new insights into the protective effects exerted by VitD derivatives.
Asunto(s)
Mediadores de Inflamación/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Fosfatos/farmacología , Aorta/citología , Aorta/metabolismo , Calcitriol/administración & dosificación , Calcitriol/farmacología , Núcleo Celular/metabolismo , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Ergocalciferoles/farmacología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Especies de Nitrógeno Reactivo/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción ReIA/metabolismoRESUMEN
BACKGROUND/AIMS: NO and reactive nitrogen species (RNS) are thought to be physiologically important effectors of mitochondrial calcium transport, but this issue was not studied in a living organism. According to literature, the modulation of Ca2+ uptake could influence RNS production via the action on mitochondrial NO synthase (mtNOS). The aim of this work was to study the effect of in vivo administration of NO donor nitroglycerine (NG) on matrix Ca2+ accumulation, RNS production and mtNOS activity. METHODS: Ca2+ uptake was studied spectrophotometrically with arsenazo-III. The amounts of stable RNS (nitrite, nitrate and nitrosothiols) and L-citrulline, the product of enzymatic NOS activity, were determined analytically. RESULTS: NG administration resulted in dose-dependent short-term increase in Ca2+-uptake accompanied by essential rise in L-citrulline and RNS content in mitochondria. In parallel, dose-dependent elevation of hydroperoxide production was detected. Ca2+-uniporter activity was not affected, but mitochondrial permeability transition pore (MPTP) was effectively blocked by NO. CONCLUSION: Our results indicate that MPTP blockage by NO was the primary cause for the increase in calcium uptake which eventually resulted in the activation of mtNOS and RNS production. Improved Ca2+ accumulation in mitochondria, together with MPTP blockage, may contribute to well-known cardioprotective effects of pharmacological donors of nitric oxide.
Asunto(s)
Calcio/metabolismo , Mitocondrias/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Nitroglicerina/farmacología , Especies de Nitrógeno Reactivo/biosíntesis , Animales , Transporte Biológico/efectos de los fármacos , Calcio/farmacocinética , Citrulina/metabolismo , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/fisiología , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/fisiología , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/fisiología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Nitratos/metabolismo , Óxido Nítrico Sintasa/metabolismo , Nitritos/metabolismo , Ratas Wistar , S-Nitrosotioles/metabolismoRESUMEN
In the current study, we investigated the effects of carp Il10 on phagocytes and lymphocytes. Carp Il10 shares several prototypical inhibitory activities on phagocytes with mammalian IL-10, including deactivation of neutrophils and macrophages, as shown by inhibition of oxygen and nitrogen radical production, as well as reduced expression of proinflammatory genes and mhc genes involved in Ag presentation. Similar to mammalian IL-10, carp Il10 acts through a signaling pathway involving phosphorylation of Stat3, ultimately leading to the early upregulation of socs3 expression. To our knowledge, this is the first study of the effects of Il10 on lymphocytes in fish. Although Il10 did not affect survival and proliferation of T cells from naive animals, it greatly promoted survival and proliferation of T cells in cultures from immunized animals, but only when used in combination with the immunizing Ag. Preliminary gene expression analysis suggests that, under these circumstances, carp Il10 stimulates a subset of CD8+ memory T cells while downregulating CD4+ memory Th1 and Th2 responses. In addition to the regulatory effect on T cells, carp Il10 stimulates proliferation, differentiation, and Ab secretion by IgM+ B cells. Overall, carp Il10 shares several prototypical activities with mammalian IL-10, including downregulation of the inflammatory response of phagocytes, stimulation of proliferation of subsets of memory T lymphocytes, and proliferation, differentiation, and Ab secretion by IgM+ B lymphocytes. To our knowledge, this is the first comprehensive analysis of biological activities of fish Il10 on both phagocytes and lymphocytes showing functional conservation of several properties of Il10.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Carpas/inmunología , Inmunoglobulina M/biosíntesis , Interleucina-10/inmunología , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Inmunoglobulina M/inmunología , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/inmunología , Inflamación/inmunología , Interleucina-10/farmacología , Macrófagos/inmunología , Fagocitos , Especies de Nitrógeno Reactivo/biosíntesis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Nickel sulfate stimulates inducible nitric oxide synthase (i-NOS) and increases serum nitric oxide concentration by overproduction of reactive nitrogen species due to nitrosative stress. The present study was undertaken to assess possible protective role of L-ascorbic acid as an antioxidant against nickel induced pulmonary nitrosative stress in male albino rats. We studied the effect of the simultaneous treatment with L-ascorbic acid (50 mg/100 g b. wt.; orally) and nickel sulfate (2.0 mg/100 g b. wt.; i.p.) on nitric oxide synthesis by quantitative evaluation of serum i-NOS activities, serum and lung nitric oxide, L-ascorbic acid and protein concentrations of Wistar strain male albino rats. We have further studied histopathological changes in lung tissue after nickel sulfate treatment along with simultaneous exposure of L-ascorbic acid. Nickel sulfate treatment significantly increased the serum i-NOS activity, serum and pulmonary nitric oxide concentration and decreased body weight, pulmonary somatic index, serum and lung L-ascorbic acid and protein concentration as compared to their respective controls. Histopathological changes induced by nickel sulfate showed loss of normal alveolar architecture, inflammation of bronchioles, infiltration of inflammatory cells and patchy congestion of alveolar blood vessels. The simultaneous administration of L-ascorbic acid and nickel sulfate significantly improved all the above biochemical parameters along with histopathology of lung tissues of rats receiving nickel sulfate alone. The study clearly showed a protective role of L-ascorbic acid against nickel induced nitrosative stress in lung tissues.
Asunto(s)
Ácido Ascórbico/administración & dosificación , Pulmón/metabolismo , Óxido Nítrico Sintasa de Tipo II/sangre , Óxido Nítrico/biosíntesis , Animales , Masculino , Níquel/toxicidad , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Ratas , Especies de Nitrógeno Reactivo/biosíntesis , Estrés FisiológicoRESUMEN
A single intraperitoneal injection of a gram-positive pathogen Clostridium perfringens (Cp) causes a remarkable down-regulation the constitutive nitric oxide synthase (cNOS) with a simultaneous increase in the activity of inducible NOS (iNOS) and the level of reactive nitrogen species in the rat brain major regions (cortex, striatum, hippocampus and hypothalamus) at 48 h post-administration of Cp. Treatment by both a semiconductor laser (SCL) and/or a light-emitting diode (LED) with same wavelength, energy density and time exposure (continuous wave, λ=654 nm, fluence=1.27 J/cm(2), time exposure=600 s) could modulate brain nitrergic response following Cp-infection. Besides, unlike the LED, the SCL-irradiation prevents the cNOS inhibition in all the studied brain regions and might be useful in restoring its function in neurotransmission and cerebral blood flow, along with providing a protective effect against nitrosative stress-induced iNOS-mediated injury in the brain regions.
Asunto(s)
Infecciones por Clostridium/radioterapia , Clostridium perfringens/efectos de la radiación , Hipotálamo/efectos de la radiación , Láseres de Semiconductores/uso terapéutico , Neuronas Nitrérgicas/efectos de la radiación , Animales , Infecciones por Clostridium/enzimología , Hipotálamo/enzimología , Hipotálamo/microbiología , Masculino , Neuronas Nitrérgicas/metabolismo , Óxido Nítrico Sintasa de Tipo I/efectos adversos , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Distribución Aleatoria , Ratas , Especies de Nitrógeno Reactivo/biosíntesis , Especies de Nitrógeno Reactivo/efectos de la radiación , Resultado del TratamientoRESUMEN
In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (Mφ) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient Mφ revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected Mφ. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli- or S. aureus-infected wild-type Mφ, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX(-/-) NOS2(-/-) Mφ further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX(-/-) NOS2(-/-) Mφ cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.
Asunto(s)
Hipoxia de la Célula , Escherichia coli/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Mitocondrias/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Staphylococcus aureus/inmunología , Animales , Transporte de Electrón , Escherichia coli/crecimiento & desarrollo , Macrófagos/microbiología , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Especies de Nitrógeno Reactivo/biosíntesis , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
Beta-glucans participate in the processes of repair, metabolism and detoxification, and affect the overall health of the body counteract the pathological conditions of reactive oxygen and nitrogen and the processes in which they participate. Reactive oxygen species (ROS) and nitrogen (RNS) play an important role in the pathogenesis of many diseases. Production of ROS is an integral part of aerobic metabolism of cells. Physiological concentrations of ROS play an important role in proper functioning of many cellular processes, and their overproduction occurs during induced oxidative stress. Very closely associated with oxidative stress is nitrosative stress. Nitric oxide (NO) reacts with molecular oxygen, superoxide anion radical and metal cations to give more reactive oxygen species. Reactive oxygen and nitrogen react with proteins to cause impairment of their function by oxidation or nitrosylation of amino acid residues, which can direct the path of apoptotic cells. In addition, nitric oxide enhances the effect induced by cyclooxygenase and becomes a mediator of inflammation.
Asunto(s)
Neoplasias/metabolismo , Neoplasias/prevención & control , Especies de Nitrógeno Reactivo/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , beta-Glucanos/metabolismo , Animales , Humanos , Óxido Nítrico/metabolismo , Oxidación-Reducción , Estrés Oxidativo , SuperóxidosRESUMEN
BACKGROUND: The purpose of the present study was to evaluate the effect of carotenoid astaxanthin (ASTA) on human neutrophils treated with high glucose and free fatty acids (FFA) on the phagocytic capacity and reactive oxygen/nitrogen species production. METHODS: The following parameters were evaluated: phagocytic capacity of neutrophils by using zymosan particles, intracellular and extracellular superoxide anion (lucigenin and DHE probes), hydrogen peroxide (H(2)O(2) - phenol red), nitric oxide (Griess reagent) production, and maximal activity of G6PDH. RESULTS: There was a decreased phagocytic capacity of human neutrophils treated with high glucose (30 mM) or FFA (0.1mM) and a partial restoring of the phagocytic capacity after ASTA-treatment was observed. ROS and RNS production was increased in neutrophils due to both high glucose and FFA. This increase in ROS/RNS production was also partially prevented by ASTA treatment. Both glucose and FFA increased the G6PDH activity. We show that ASTA provides a modest improvement of cellular functions after cells have been treated with high glucose and FFA. CONCLUSIONS: In summary, this study showed that both high glucose and a mixture of FFA are potent inducers of ROS/RNS production on neutrophils as observed by higher levels of superoxide anion, hydrogen peroxide and NO production. Also, these metabolites decrease the phagocytic capacity of neutrophils and increase the G6PDH activity. Overall, ASTA-treatment was able to reduce partially ROS/RNS production by reducing the availability of NADPH and recover phagocytic capacity of neutrophils.
Asunto(s)
Ácidos Grasos no Esterificados/farmacología , Glucosa/farmacología , Neutrófilos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Especies de Nitrógeno Reactivo/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Adulto , Femenino , Glucosafosfato Deshidrogenasa/metabolismo , Humanos , Masculino , Xantófilas/farmacología , Adulto JovenRESUMEN
Peroxynitrite (ONOO(-)) is a reactive nitrogen species formed when nitric oxide (NO) reacts with the superoxide anion (O(2)(-)). It was first identified as a mediator of cell death in animals but was later shown to act as a positive regulator of cell signaling, mainly through the posttranslational modification of proteins by tyrosine nitration. In plants, peroxynitrite is not involved in NO-mediated cell death and its physiological function is poorly understood. However, it is emerging as a potential signaling molecule during the induction of defense responses against pathogens and this could be mediated by the selective nitration of tyrosine residues in a small number of proteins. In this review we discuss the general role of tyrosine nitration in plants and evaluate recent evidence suggesting that peroxynitrite is an effector of NO-mediated signaling following pathogen infection.
Asunto(s)
Ácido Peroxinitroso/biosíntesis , Plantas/metabolismo , Especies de Nitrógeno Reactivo/biosíntesis , Modelos Biológicos , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Transducción de Señal , Estrés FisiológicoRESUMEN
Oxidative and nitrosative stress induced by ROS/RNS play crucial roles in a wide range of physiological processes and are also implicated in various diseases, including cancer and neurodegenerative disorders. Sensitive and selective methods for the detection of ROS/RNS based on fluorescent and luminescent probes are of great use in monitoring the in vivo production of these species and elucidating their biological functions. This critical review highlights recent advances that have been made in the development of fluorescent and luminescent probes employed to monitor various ROS/RNS (132 references).
Asunto(s)
Sondas Moleculares , Especies de Nitrógeno Reactivo/análisis , Especies Reactivas de Oxígeno/análisis , Colorantes Fluorescentes , Luminiscencia , Técnicas de Diagnóstico Molecular , Especies de Nitrógeno Reactivo/biosíntesis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Prolonged depolarization of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. Skeletal muscle excitation-coupled calcium entry relies on the interaction between the 1,4-dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. In this study, we directly measured excitation-coupled calcium entry by total internal reflection fluorescence microscopy in human skeletal muscle myotubes harbouring mutations in the RYR1 gene linked to malignant hyperthermia (MH) and central core disease (CCD). We found that excitation-coupled calcium entry is strongly enhanced in cells from patients with CCD compared with individuals with MH and controls. Furthermore, excitation-coupled calcium entry induces generation of reactive nitrogen species and enhances nuclear localization of NFATc1, which in turn may be responsible for the increased IL-6 released by myotubes from patients with CCD.
Asunto(s)
Calcio/metabolismo , Interleucina-6/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miopatía del Núcleo Central/metabolismo , Factores de Transcripción NFATC/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Hipertermia Maligna/genética , Microscopía Fluorescente , Músculo Esquelético/metabolismo , Mutación , Miopatía del Núcleo Central/genética , Reacción en Cadena de la Polimerasa , Especies de Nitrógeno Reactivo/biosíntesis , Especies de Nitrógeno Reactivo/metabolismoRESUMEN
Sepsis-induced acute kidney injury (AKI) is a complex disease characterized by generation of inducible nitric oxide synthase (iNOS)-derived reactive nitrogen species (RNS) by the renal tubular epithelium. While most in vitro models of sepsis use combinations of lipopolysaccharide and cytokines to simulate exposure to inflammatory mediators thought to play a role in sepsis, the relevance of these models is limited. To address the need for a model that more closely mimics the tubular microenvironment during sepsis, we developed an in vitro model where mIMCD-3 (murine tubular epithelial) cells are treated with media containing 5% serum collected from mice at 4 h after cecal ligation and puncture (CLP) or sham surgery (no sepsis). After exposure to CLP serum, induction of iNOS messenger RNA occurred and NO generation was significantly increased compared to sham. This increase was accompanied by increased RNS as measured by oxidation of 5-(and-6)-carboxy-2,7'-dichlorodihydrofluorescein diacetate (carboxy-H(2)DCF-DA) and 2-(3,6-diamino-9H-xanthen-9-yl)-benzoic acid, methyl ester (dihydrorhodamine 123) and moderate cytotoxicity in cells treated with CLP serum, similar to what is observed in mice subjected to CLP. Since iNOS has been shown to play an important role in sepsis-induced AKI, the iNOS inhibitor L-N(6)-(1-iminoethyl)-lysine (L-NIL) was tested in this in vitro model. L-NIL completely blocked NO generation, RNS generation, and cytotoxicity, similar to its effects in vivo. Therefore, this new in vitro model exhibits many of the characteristics observed in vivo, suggesting that it is a relevant model for studying the mechanism of sepsis-induced renal epithelial RNS generation and injury.
Asunto(s)
Células Epiteliales/enzimología , Túbulos Renales/enzimología , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Especies de Nitrógeno Reactivo/biosíntesis , Sepsis/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Túbulos Renales/efectos de los fármacos , Lisina/análogos & derivados , Lisina/farmacología , Ratones , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/genética , ARN Mensajero/metabolismoRESUMEN
Macrophages produce reactive oxygen and nitrogen species (ROS/RNS) in response to immunological challenges. We have previously reported the real-time detection and quantification of released ROS/RNS by immunostimulated macrophages using constant potential amperometry, at four different potentials, with platinized carbon microelectrodes. As a methodological extension to that work, we sought to develop an electroanalytical method that would allow for the simultaneous monitoring of several ROS/RNS. Triple potential-step chronoamperometry at platinized carbon microelectrodes was found to provide satisfactory sensitivity and signal/noise ratio for this purpose. The title method was applied to the detection of endogenously produced ROS/RNS by single IFN-gamma/LPS/PMA stimulated RAW 264.7 macrophages. Significantly higher fluxes of H(2)O(2), ONOO(-), and NO* responses were detected over stimulated macrophages as compared to unactivated macrophages, consistent with the endogenous production of primary NO* and O(2)(*-) by both the inducible isoform of nitric oxide synthase (iNOS) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymatic systems in stimulated cells. Crucially, significant temporal variations in the release of each of the aforementioned species was evidenced using this method, which would not have been achievable with the use of either constant potential amperometry or classical biochemical methods such as the Griess assay.
Asunto(s)
Macrófagos/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Capacidad Eléctrica , Electroquímica , Interferón gamma/inmunología , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Ratones , Microelectrodos , Especies de Nitrógeno Reactivo/biosíntesis , Acetato de Tetradecanoilforbol/inmunología , Factores de TiempoRESUMEN
The infiltration and persistence of hematopoietic immune cells within the rheumatoid arthritis (RA) joint results in elevated levels of pro-inflammatory cytokines, increased reactive oxygen (ROS) and -nitrogen (RNS) species generation, that feeds a continuous self-perpetuating cycle of inflammation and destruction. Meanwhile, the controlled production of ROS is required for signaling within the normal physiological reaction to perceived "foreign matter" and for effective apoptosis. This review focuses on the signaling pathways responsible for the induction of the normal immune response and the contribution of ROS to this process. Evidence for defects in the ability of immune cells in RA to regulate the generation of ROS and the consequence for their immune function and for RA progression is considered. As the hypercellularity of the rheumatoid joint and the associated persistence of hematopoietic cells within the rheumatoid joint are symptomatic of unresponsiveness to apoptotic stimuli, the role of apoptotic signaling proteins (specifically Bcl-2 family members and the tumor suppressor p53) as regulators of ROS generation and apoptosis are considered, evaluating evidence for their aberrant expression and function in RA. We postulate that ROS generation is required for effective therapeutic intervention.
Asunto(s)
Artritis Reumatoide/inmunología , Artritis Reumatoide/terapia , Especies de Nitrógeno Reactivo/biosíntesis , Especies de Nitrógeno Reactivo/inmunología , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Animales , Artritis Reumatoide/patología , Supervivencia Celular/inmunología , HumanosRESUMEN
Toll-like receptors have been implicated in the recognition of various pathogens, including bacteria, viruses, protozoa and fungi. However, no information is available about Toll-like receptor 4 (TLR4) participation in Sporothrix schenckii recognition and the consequent triggering of the immune response to this fungal pathogen. Following activation of TLRs by ligands of microbial origin, several responses are provoked, including reactions in immune cells that may lead them to produce signalling factors that trigger inflammation. The present study was designed to elucidate the role of TLR4 during the host response to S. schenckii. TLR4-deficient (C3H/HeJ) and control mice (C3H/HePas) were infected with S. schenckii yeast cells and immune response was assessed over 10 weeks by assaying production of pro-inflammatory mediator (nitric oxide and tumour necrosis factor-alpha) and anti-inflammatory cytokine (interleukin-10) by peritoneal macrophages and their correlation with apoptosis in peritoneal exudate cells. We found that both pro-inflammatory and anti-inflammatory mediators are reduced in TLR4-deficient mice, suggesting the involvement of this receptor in the recognition of this infectious agent. Translocation into the nucleus of nuclear transcription factor, nuclear factor-kappaB, was also evaluated and showed higher levels in TLR-4 normal mice, consistent with the results found for cytokine production. We are showing here, for the first time, the involvement of TLR4 in S. schenckii recognition. Taken together, our results demonstrate that the activation of peritoneal macrophages in response to S. schenckii lipid extracts has different responses in these two mouse strains which differ in TLR4 expression, suggesting an important role for TLR4 in governing the functions of macrophages in this fungal infection.
Asunto(s)
Antígenos Fúngicos/inmunología , Lípidos/inmunología , Macrófagos Peritoneales/inmunología , Sporothrix/inmunología , Esporotricosis/inmunología , Receptor Toll-Like 4/inmunología , Animales , Apoptosis/inmunología , Tolerancia Inmunológica/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-10/biosíntesis , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Endogámicos C3H , Necrosis/inmunología , Especies de Nitrógeno Reactivo/biosíntesis , Factor de Transcripción ReIA/metabolismo , Translocación Genética , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The purpose of this study is to compare the oxidative stress response to aerobic and anaerobic power testing, and to determine the impact of exercise training with or without glycine propionyl-L-carnitine (GPLC) in attenuating the oxidative stress response. Thirty-two subjects were assigned (double blind) to placebo, GPLC-1 (1g PLC/d), GPLC-3 (3g PLC/d) for 8 weeks, plus aerobic exercise. Aerobic (graded exercise test: GXT) and anaerobic (Wingate cycle) power tests were performed before and following the intervention. Blood was taken before and immediately following exercise tests and analyzed for malondialdehyde (MDA), hydrogen peroxide (H2O2), and xanthine oxidase activity (XO). No interaction effects were noted. MDA was minimally effected by exercise but lower at rest for both GPLC groups following the intervention (p = 0.044). A time main effect was noted for H2O2 (p = 0.05) and XO (p = 0.003), with values increasing from pre- to postexercise. Both aerobic and anaerobic power testing increase oxidative stress to a similar extent. Exercise training plus GPLC can decrease resting MDA, but it has little impact on exercise-induced oxidative stress biomarkers.
Asunto(s)
Carnitina/uso terapéutico , Ejercicio Físico/fisiología , Estrés Oxidativo/efectos de los fármacos , Complejo Vitamínico B/uso terapéutico , Adulto , Biomarcadores/sangre , Carnitina/administración & dosificación , Carnitina/farmacología , Método Doble Ciego , Prueba de Esfuerzo , Femenino , Humanos , Masculino , Estrés Oxidativo/fisiología , Especies de Nitrógeno Reactivo/biosíntesis , Especies de Nitrógeno Reactivo/sangre , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/sangre , Especies Reactivas de Oxígeno/metabolismo , Complejo Vitamínico B/administración & dosificación , Complejo Vitamínico B/farmacología , Adulto JovenRESUMEN
During the initial growth infection stage of Mycobacterium tuberculosis (Mtb), (*)NO produced by host macrophages inhibits heme-containing terminal cytochrome oxidases, inactivates iron/sulfur proteins, and promotes entry into latency. Here we evaluate the potential of (*)NO as an inhibitor of Mtb cytochrome P450 enzymes, as represented by CYP130, CYP51, and the two previously uncharacterized enzymes CYP125 and CYP142. Using UV-visible absorption, resonance Raman, and stopped-flow spectroscopy, we investigated the reactions of (*)NO with these heme proteins in their ferric resting form. (*)NO coordinates tightly to CYP125 and CYP142 (submicromolar) and with a lower affinity (micromolar) to CYP130 and CYP51. Anaerobic reduction of the ferric-NO species with sodium dithionite led to the formation of two spectrally distinct classes of five-coordinate ferrous-NO complexes. Exposure of these species to O(2) revealed that the ferrous-NO forms of CYP125 and CYP142 are labile and convert back to the ferric state within a few minutes, whereas ferrous CYP130 and CYP51 bind (*)NO almost irreversibly. This work clearly indicates that, at physiological concentrations (approximately 1 microM), (*)NO would impair the activity of CYP130 and CYP51, whereas CYP125 and CYP142 are more resistant. Selective P450 inhibition may contribute to the inhibitory effects of (*)NO on Mtb growth.