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Résumé Introduction. Une interprétation fiable de l'albuminémie est essentielle pour l'évaluation nutritionnelle biologique (ENB) de la personne âgée. L'objectif principal était d'évaluer si une formule de correction proposée en cas d'inflammation prolongée pouvait être extrapolée aux situations d'inflammation biologique aiguës : albuminémie corrigée = albuminémie dosée + protéine C réactive (CRP)/25. Méthode. Cette étude observationnelle prospective monocentrique incluait des patients âgés hospitalisés de manière non programmée au CHU Grenoble-Alpes. Un recueil clinique et biologique était réalisé à J1 et J8. Le critère de jugement principal était la comparaison de l'albuminémie corrigée par la formule à J1 avec l'albuminémie dosée à J8. Résultat. Parmi 175 patients analysés, la moyenne de la CRP était de 64 mg/L (3 ; 324) à J1 et 24 mg/L (3 ; 99) à J8. Entre l'albuminémie corrigée à J1 et l'albuminémie dosée à J8, la corrélation était de p = 0,58, [IC 95 % : 0,47 ; 0,67], p < 0,001, et la différence moyenne de 2,9 mg/L (-13,5 ; 18), IC 95 %[-3,68 ; -2,20], p < 0,001. Entre l'albuminémie dosée à J1 et l'albuminémie dosée à J8, la corrélation était de p = 0,74, IC 95 % [0,66 ; 0,80], p < 0,001, et la différence moyenne de 0,4 mg/L (-14 ; 11), IC 95 % [-0,24 ; 1,02], p = 0,23. DISCUSSION: La formule proposée ne peut pas être extrapolée aux situations d'inflammation biologique aiguës au cours desquelles l'albuminémie dosée semble être stable. L'albuminémie dosée pourrait servir telle quelle de référence à l'ENB, en s'intégrant dans une histoire clinique. Abstract Introduction. A reliable interpretation of albumin is essential for the elderly's biological nutritional assessment (BNA), but is complex because many parameters change it. The main objective was to evaluate the extrapolation of a correction formula proposed for prolonged inflammation to acute biological inflammation situations: corrected albuminemia = measured albuminemia + CRP/25. METHOD: This prospective, single-center observation study included patients over 65 years of age who were unexpectedly hospitalized in geriatrics and internal medicine at Grenoble University Hospital, excluding carriers of active neoplasia, hepatic insufficiency, nephrotic syndrome, and those for whom a ABI persisted on the eighth day. A clinical and biological collection was made on the first and eighth days. The primary endpoint was the comparison of albuminemia corrected by the formula on day 1 with measured albuminemia on day 8. RESULTS: One hundred and seventy-five patients were analyzed. The average CRP was 64 (3; 324) mg/L on day 1 and 24 (3; 99) mg/L on day 8. Between corrected albumin at day 1 and albumin at day 8, the correlation was ρ = 0.58, 95%CI [0.47; 0.67], P < 0.001, and the mean difference of 2.9 (-13,5; 18) mg/L, 95%CI [-3.68;-2.20], P < 0.001. Between D1 albumin and D8 albumin, the correlation was ρ = 0.74, 95%CI [0.66; 0.80], P < 0.001, and the mean difference of 0.4 (-14; 11) mg/L, 95%CI [-0.24; 1.02], P = 0.23. DISCUSSION: The proposed formula cannot be extrapolated to acute inflammatory situations. Measured albuminemia appears to be stable during acute biological inflammation situations. Measured albuminemia could be used as a reference for BNA, integrated into a clinical history.
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Hospitalización , Estado Nutricional , Anciano , Albúminas , Atención a la Salud , Humanos , Estudios ProspectivosRESUMEN
A reliable interpretation of albumin levels is essential when assessing nutrition in elderly people, but this is complex as it is affected by a number of parameters. The main objective of this study was to evaluate whether a correction formula proposed for prolonged inflammation could be extrapolated to acute biological inflammation situations: corrected albuminemia = measured albuminemia + CRP/25. This prospective, single-centre observation study included patients over 65 years of age who were the subject of an unscheduled hospitalisation in the geriatrics and internal medicine departments of Grenoble University Hospital, excluding carriers of active neoplasia, hepatic insufficiency, nephrotic syndrome and those who continued to present with an acute biological inflammation on the eighth day. Clinical and biological samples were taken on the first and eighth days. The primary objective was the comparison of albumin levels, corrected using the formula on day (d) 1, with albumin levels measured on d8. One hundred and seventy-five patients were analysed. Average CRP was 64 (3; 324) mg/L on d1 and 24 (3; 99) mg/L on d8. Between the corrected albumin levels on d1 and albumin levels measured on d8, the correlation was ρ = 0.58, 95% CI [0.47; 0.67], P < 0.001, with a mean difference of 2.9 (-13.5; 18) mg/L, 95% CI [-3.68-2.20], P < 0.001. Between the albumin levels measured on d1 and d8, the correlation was ρ = 0.74, 95%CI [0.66; 0.80], P < 0.001, with a mean difference of 0.4 (-14; 11) mg/L, 95%CI [-0.24; 1.02], P = 0.23. The proposed formula cannot be extrapolated to acute inflammatory situations. Albumin levels appear to be stable during acute biological inflammation. Albumin levels could be used as a reference for biological nutritional assessments and be integrated into the patient's clinical history.
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Osteoarthritis (OA) is a multidimensional health problem and a common chronic disease. It has a substantial impact on patient quality of life and is a common cause of pain and mobility issues in older adults. The functional limitations, lack of curative treatments, and cost to society all demonstrate the need for translational and clinical research. The use of OA models in mice is important for achieving a better understanding of the disease. Models with clinical relevance are needed to achieve 2 main goals: to assess the impact of the OA disease (pain and function) and to study the efficacy of potential treatments. However, few OA models include practical strategies for functional assessment of the mice. OA signs in mice incorporate complex interrelations between pain and dysfunction. The current review provides a comprehensive compilation of mouse models of OA and animal evaluations that include static and dynamic clinical assessment of the mice, merging evaluation of pain and function by using automatic and noninvasive techniques. These new techniques allow simultaneous recording of spontaneous activity from thousands of home cages and also monitor environment conditions. Technologies such as videography and computational approaches can also be used to improve pain assessment in rodents but these new tools must first be validated experimentally. An example of a new tool is the digital ventilated cage, which is an automated home-cage monitor that records spontaneous activity in the cages.
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Osteoartritis de la Rodilla , Anciano , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Osteoartritis de la Rodilla/terapia , Evaluación de Resultado en la Atención de Salud , Dolor , Dimensión del Dolor , Calidad de VidaRESUMEN
Objective: X-ray Phase Contrast Imaging (PCI) is an emerging modality that will be in the next few years available in a wider range of preclinical set-ups. In this study, we compare this imaging technique with conventional preclinical modalities in an osteoarthritis mouse model. Method: Phase contrast technique was performed on 6 post-mortem, monoiodoacetate-induced osteoarthritis knees and 6 control knees. The mice knees were then imaged using magnetic resonance imaging and conventional micro computed tomography. Examples of imaging surrogate markers are reported: local distances within the articular space, cartilage surface roughness, calcified cartilage thickness, number, volume and locations of osteophytes. Results: Thanks to PCI, we can show in 3D calcified cartilage without contrast agent by a non-invasive technique. The phase contrast images reveal more details than conventional imaging techniques, especially at smaller scales, with for instance a higher number of micro-calcifications detected (57, 314 and 329 for MRI, conventional micro-CT and phase contrast imaging respectively). Calcified cartilage thickness was measured with a significant difference (p â< â0.01) between the control (23.4 â± â17.2 âµm) and the osteoarthritis induced animal (46.9 â± â19.0 âµm). Conclusions: X-ray phase contrast imaging outperforms the conventional imaging modalities for assessing the different tissue types (soft and hard). This new imaging modality seems to bring new relevant surrogate markers for following-up small animal models even for low-grade osteoarthritis.
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The NADPH oxidase Nox4 is a multi-pass membrane protein responsible for the generation of reactive oxygen species that are implicated in cellular signaling but may also cause pathological situations when dysregulated. Although topological organization of integral membrane protein dictates its function, only limited experimental data describing Nox4's topology are available. To provide deeper insight on Nox4 structural organization, we developed a novel method to determinate membrane protein topology in their cellular environment, named Topological Determination by Ubiquitin Fusion Assay (ToDUFA). It is based on the proteolytic capacity of the deubiquitinase enzymes to process ubiquitin fusion proteins. This straightforward method, validated on two well-known protein's topologies (IL1RI and Nox2), allowed us to discriminate rapidly the topological orientation of protein's domains facing either the nucleocytosolic or the exterior/luminal compartments. Using this method, we were able for the first time to determine experimentally the topology of Nox4 which consists of 6 transmembrane domains with its N- and C-terminus moieties facing the cytosol. While the first, third and fifth loops of Nox4 protein are extracellular; the second and fourth loops are located in the cytosolic side. This approach can be easily extended to characterize the topology of all others members of the NADPH oxidase family or any multi-pass membrane proteins. Considering the importance of protein topology knowledge in cell biology research and pharmacological development, we believe that this novel method will represent a widely useful technique to easily uncover complex membrane protein's topology.
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Proteínas de la Membrana/química , NADPH Oxidasa 4/química , Animales , Membrana Celular/metabolismo , Citosol , Enzimas Desubicuitinizantes/metabolismo , Humanos , Métodos , Dominios Proteicos , Estructura Terciaria de Proteína , Proteolisis , Ubiquitina/metabolismoRESUMEN
NADPH oxidases, Nox, are a family of isoenzymes, composed of seven members, whose sole function is to produce reactive oxygen species (ROS). Although Nox catalyze the same enzymatic reaction, they acquired from a common ancestor during evolution, specificities related to their tissue expression, subcellular localization, activation mechanisms and regulation. Their functions could vary depending on the pathophysiological state of the tissues. Indeed, ROS are not only bactericidal weapons in phagocytes but also essential cellular signaling molecules and their overproduction is involved in chronic diseases and diseases of aging. The understanding of the mechanisms involved in the function of Nox and the emergence of Nox inhibitors, require a thorough knowledge of their nature and structure. The objectives of this review are to highlight, in a structure/function approach, the main similar and differentiated properties shared by the human Nox isoenzymes.
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NADPH Oxidasas , Animales , Evolución Molecular , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Terapia Molecular Dirigida/tendencias , Familia de Multigenes , NADPH Oxidasas/química , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Conformación ProteicaRESUMEN
The NADPH oxidases, Nox, are transmembrane hemoproteins, whose exclusive function is to reduce molecular oxygen to produce superoxide anion O2°- and consequently highly reactive oxidant and toxic oxygen species, ROS. Among the 7 NADPH oxidases expressed in humans, Nox4 is the sole Nox isoform present in human primary chondrocytes. Nox4 was suggested as one of the main actors involved in cartilage degradation in osteoarthritis. The stimulation of chondrocytes, the only cell present in cartilage, by IL-1ß results in the activation of Nox4. This leads to an increase of ROS production which in turn could regulate signaling pathways sensitive to oxidative stress such as gene-encoding matrix metalloproteases MMP1, MMP13 and Adamalysin ADAMTS4. A deep understanding of Nox4 structure/function and mechanisms of regulation could lead both to the identification of new therapeutic targets and to the development of innovative strategies for appropriate osteoarthritis treatment.
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Terapia Molecular Dirigida , NADPH Oxidasas/fisiología , Osteoartritis/tratamiento farmacológico , Humanos , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendencias , NADPH Oxidasa 4 , NADPH Oxidasas/química , Osteoartritis/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Interleukin-1ß (IL-1ß) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1ß stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1ß signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22(phox) heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1ß. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis.
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Apoptosis , Condrocitos/enzimología , Hemo-Oxigenasa 1/fisiología , Metaloproteinasa 1 de la Matriz/metabolismo , NADPH Oxidasas/metabolismo , Monóxido de Carbono/farmacología , Condrocitos/fisiología , Fragmentación del ADN , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Hemo/biosíntesis , Humanos , Interleucina-1beta/fisiología , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , Osteoartritis/enzimología , Multimerización de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
NADPH oxidase Nox4 is expressed in a wide range of tissues and plays a role in cellular signaling by providing reactive oxygen species (ROS) as intracellular messengers. Nox4 oxidase activity is thought to be constitutive and regulated at the transcriptional level; however, we challenge this point of view and suggest that specific quinone derivatives could modulate this activity. In fact, we demonstrated a significant stimulation of Nox4 activity by 4 quinone derivatives (AA-861, tBuBHQ, tBuBQ, and duroquinone) observed in 3 different cellular models, HEK293E, T-REx™, and chondrocyte cell lines. Our results indicate that the effect is specific toward Nox4 versus Nox2. Furthermore, we showed that NAD(P)H:quinone oxidoreductase (NQO1) may participate in this stimulation. Interestingly, Nox4 activity is also stimulated by reducing agents that possibly act by reducing the disulfide bridge (Cys226, Cys270) located in the extracellular E-loop of Nox4. Such model of Nox4 activity regulation could provide new insight into the understanding of the molecular mechanism of the electron transfer through the enzyme, i.e., its potential redox regulation, and could also define new therapeutic targets in diseases in which quinones and Nox4 are implicated.
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Benzoquinonas/farmacología , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Calcio/metabolismo , Línea Celular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Humanos , L-Lactato Deshidrogenasa/metabolismo , Luminiscencia , Datos de Secuencia Molecular , NADPH Oxidasa 4 , NADPH Oxidasas/química , Oxidación-Reducción , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción GenéticaRESUMEN
The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 ± 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 ± 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 ± 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 ± 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the importance of this domain.
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NADH Deshidrogenasa/química , NADPH Oxidasas/química , Sistema Libre de Células , Citosol/enzimología , Escherichia coli/enzimología , Escherichia coli/genética , Células HEK293 , Humanos , NADH Deshidrogenasa/genética , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H(2)O(2) versus L-012 and cytochrome c for O(2)(·-). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H(2)O(2) to O(2)(·-) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O(2)()-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O(2)(·-). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H(2)O(2) but increased O(2)(·-) formation by Nox4 without affecting Nox1-dependent O(2)(·-) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H(2)O(2). Mutation of this but not of four other conserved histidines also switched Nox4 from H(2)O(2) to O(2)(·-) formation. Thus, H(2)O(2) formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O(2)(·-) egress and/or provide a source for protons, allowing dismutation to form H(2)O(2).
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Peróxido de Hidrógeno/metabolismo , NADPH Oxidasas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Mutación , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Donantes de Óxido Nítrico/farmacología , Ácido Peroxinitroso/genética , Ácido Peroxinitroso/metabolismo , Estructura Secundaria de Proteína , Superóxidos/metabolismoRESUMEN
Nox4, a member of Nox family of NADPH oxidase expressed in nonphagocytic cells, is a major source of reactive oxygen species in many cell types. But understanding of the role of Nox4 in the production of ROS and of regulation mechanism of oxidase activity is largely unknown. This study reports for the first time the generation and characterization of 5 mAbs against a recombinant Nox4 protein (AA: 206-578). Among 5 novel mAbs, 3 mAbs (8E9, 5F9, 6B11) specifically recognized Nox4 protein in HEK293 transfected cells or human kidney cortex by western blot analysis; mAb 8E9 reacted with intact tet-induced T-REx™ Nox4 cells in FACS studies. The other 2 mAbs 10B4 and 7C9 were shown to have a very weak reactivity after purification. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4, while mAbs 6B11 and 5F9 are directed to its cytosolic tail. Contrary to mAb 6B11, mAb 5F9 failed to detect Nox4 at the plasma membrane. Cell-free oxidase assays demonstrated a moderate but significant inhibition of constitutive Nox4 activity by mAbs 5F9 and 6B11. In conclusion, 5 mAbs raised against Nox4 were generated for the first time. 3 of them will provide powerful tools for a structure/function relationship of Nox4 and for physiopathological investigations in humans.
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Anticuerpos Monoclonales/inmunología , Espacio Intracelular/metabolismo , NADPH Oxidasas/inmunología , NADPH Oxidasas/metabolismo , Animales , Mapeo Epitopo , Células HEK293 , Humanos , Espacio Intracelular/enzimología , Ratones , Ratones Endogámicos BALB C , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Biblioteca de Péptidos , Transporte de Proteínas , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Eliminación de SecuenciaRESUMEN
Oxidative stress is a metabolic situation used by immune cells to provide protection against infection. Under activation by threatening elements, phagocytes produce chemically toxic molecules, namely the reactive oxygen species (ROS) and reactive nitrogen species (RNS). This mechanism involves two types of enzymes: NAPDH oxidases (NOX) and NO synthases (NOS), which activities are versatile and not fully understood yet. In this regard, we studied in real-time the release of bursts of ROS and RNS by single PLB-985 cells, originating from a myeloid cell line prone to differentiate into neutrophil or monocyte-like phagocytes. A selective electrochemical detection of each ROS or RNS was conducted at platinized carbon fiber microelectrodes positioned at micrometric distances from single cells. Our results show (1) the existence of a NO synthase activity in PLB-985 cells and (2) the ability of NO synthases to provide a NOX activity in cells where NADPH oxidase (NOX2) is knocked out.
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NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Amidinas/farmacología , Bencilaminas/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Estrés Oxidativo/efectos de los fármacos , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio/efectos de los fármacosRESUMEN
NOX4 is an enigmatic member of the NOX (NADPH oxidase) family of ROS (reactive oxygen species)-generating NADPH oxidases. NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology is poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1 h) followed closely (2 h) by a release of ROS. Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (<24 h). In membrane preparations, NOX4 activity was selective for NADPH over NADH and did not require the addition of cytosol. The pharmacological profile of NOX4 was distinct from other NOX isoforms: DPI (diphenyleneiodonium chloride) and thioridazine inhibited the enzyme efficiently, whereas apocynin and gliotoxin did not (IC(50)>100 muM). The pattern of NOX4-dependent ROS generation was unique: (i) ROS release upon NOX4 induction was spontaneous without need for a stimulus, and (ii) the type of ROS released from NOX4-expressing cells was H(2)O(2), whereas superoxide (O(2)(-)) was almost undetectable. Probes that allow detection of intracellular O(2)(-) generation yielded differential results: DHE (dihydroethidium) fluorescence and ACP (1-acetoxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine) ESR measurements did not detect any NOX4 signal, whereas a robust signal was observed with NBT. Thus NOX4 probably generates O(2)(-) within an intracellular compartment that is accessible to NBT (Nitro Blue Tetrazolium), but not to DHE or ACP. In conclusion, NOX4 has a distinct pharmacology and pattern of ROS generation. The close correlation between NOX4 mRNA and ROS generation might hint towards a function as an inducible NOX isoform.
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NADPH Oxidasas/genética , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Citosol/efectos de los fármacos , Citosol/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Etidio/análogos & derivados , Etidio/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Ratones , Mitocondrias/efectos de los fármacos , NAD/metabolismo , NADP/metabolismo , NADPH Oxidasa 4 , Nitroazul de Tetrazolio/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Superóxidos/metabolismo , Tetraciclina/farmacología , Factores de TiempoRESUMEN
Cytochrome b(558) is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b(558) is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b(558) conformation. Recently, we confirmed this hypothesis by isolating cytochrome b(558) in a constitutively active form. To characterize active and inactive cytochrome b(558) conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b(558) purified from both resting and stimulated neutrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b(558). Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13B6 to bind preferentially to the active form of cytochrome b(558). All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b(558) in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformationally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo.
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Anticuerpos Monoclonales/inmunología , Glicoproteínas de Membrana/inmunología , NADPH Oxidasas/metabolismo , Fagocitos/enzimología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Sistema Libre de Células , Células Cultivadas , Grupo Citocromo b/genética , Grupo Citocromo b/inmunología , Grupo Citocromo b/metabolismo , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Citometría de Flujo , Humanos , Immunoblotting , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/inmunología , Neutrófilos/citología , Neutrófilos/enzimología , Neutrófilos/metabolismo , Biblioteca de Péptidos , Fagocitos/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
NOX family NADPH oxidases are enzymes whose biological function is electron transport and the generation of reactive oxygen species (ROS). NOX enzymes in mammalian organisms have received most attention. However, NOX enzymes are widely distributed in different kingdoms of life. While they are not found in prokaryotes and most unicellular eukaryotes, they are present in fungi, plants, and animals. The identity of the ancestral NOX is not known, but most likely it: (i) possessed the basic NOX structure consisting of 6 transmembrane domains (containing two assymmetrical hemes) and a long cytoplasmic C-terminal (containing the FAD and NADPH binding sites); and (ii) emerged before the divergence of life into fungi, plants, and animals. During evolution, acquisition of a Ca(2+)-binding EF hand domain by an ancestral NOX, led to NOX5-like isoforms. DUOX isoforms presumably developed from a NOX5-like isoform through the additional acquisition of a peroxidase homology domain. The expression pattern of NOX enzymes is specific to each kingdom of life. Fungi express only ancestral-type isoforms, and plants only NOX5-like isoforms. NOX expression patterns in animals are complex and ancestral NOXes, NOX5-like isoforms and DUOXes are generally found. But there are exceptions; for example rodents lack NOX5 and Caenorhabditis elegans expresses only DUOXes. Biological functions of NOX enzymes include, among others, host defense, post-translational modification of proteins, and regulation cell growth and differentiation. In summary, the invention of NOX enzymes early in the development of life was a success story: there is no evidence of multicellular life without NOX enzymes.
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Mamíferos/metabolismo , NADPH Oxidasas/metabolismo , Animales , Evolución Molecular , Hongos/enzimología , Hongos/genética , Mamíferos/genética , Modelos Biológicos , NADPH Oxidasas/genética , Filogenia , Plantas/enzimología , Plantas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Reactive oxygen species (ROS) are regulators of redox-sensitive cell signaling pathways. In osteoarthritis, human interleukin-1beta is implicated in cartilage destruction through an ROS-dependent matrix metalloproteinase production. To determine the molecular source of ROS production in the human IL-1beta (hIL-1beta)-sensitive chondrocyte immortalized cell line C-20/A4, transfected cells were constructed that overexpress NAD(P)H oxidases. First, RT-PCR analysis showed that the C-20/A4 cell line expressed Nox2, Nox4, p22( phox ), and p67( phox ), but not p47( phox ). It was found that ROS production by C-20/A4 chondrocytes does not depend on PMA and ionomycin activation. This indicates that Nox2 was not involved in the production of ROS. In C- 20/A4 cells that overexpress Nox4, hIL-1beta stimulated ROS production three times more than the normal production of C-20/A4 cells. Moreover, there was a fourfold increase in the production of collagenase (MMP-1) by chondrocytes that overexpress Nox4. Interestingly, MMP-1 production in cells that overexpress Nox2 was not sensitive to hIL-1beta. These data suggest that under hIL-1beta stimulation, C-20/A4 chondrocytes produce MMP-1 through a Nox4-mediated, ROS-dependent pathway.
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Condrocitos/metabolismo , Colagenasas/metabolismo , NADPH Oxidasas/genética , Sal Disódica del Ácido 1,2-Dihidroxibenceno-3,5-Disulfónico/farmacología , Línea Celular Transformada , Condrocitos/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/metabolismo , Interleucina-1beta/farmacología , Metaloproteinasa 1 de la Matriz/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutación/genética , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , Fosfoproteínas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , TransfecciónRESUMEN
The amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes (PHG1 and KIL1) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae. Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.
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Dictyostelium/fisiología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/fisiología , Proteínas de la Membrana/fisiología , Fagocitosis , Animales , Células Cultivadas , Dictyostelium/crecimiento & desarrollo , Dictyostelium/microbiología , Modelos Animales de Enfermedad , Drosophila/crecimiento & desarrollo , Drosophila/microbiología , Drosophila/fisiología , Klebsiella pneumoniae/patogenicidad , Proteínas de la Membrana/genética , Ratones , Mutación , Neumonía Bacteriana/metabolismo , Neumonía Bacteriana/microbiología , VirulenciaRESUMEN
Membrane-associated NADPH oxidase complexes catalyse the production of the superoxide anion radical from oxygen and NADPH. In mammalian systems, NADPH oxidases form a family of at least seven isoforms that participate in host defence and signalling pathways. We report here the cloning and the characterisation of slime mould Dictyostelium discoideum homologs of the mammalian heme-containing subunit of flavocytochrome b (gp91(phox)) (NoxA, NoxB and NoxC), of the small subunit of flavocytochrome b (p22(phox)) and of the cytosolic factor p67(phox). Null-mutants of either noxA, noxB, noxC or p22(phox) show aberrant starvation-induced development and are unable to produce spores. The overexpression of NoxA(myc2) in noxA null strain restores spore formation. Remarkably, the gene alg-2B, coding for one of the two penta EF-hand proteins in Dictyostelium, acts as a suppressor in noxA, noxB, and p22(phox) null-mutant strains. Knockout of alg-2B allows noxA, noxB or p22(phox) null-mutants to return to normal development. However, the knockout of gene encoding NoxC, which contains two penta EF-hands, is not rescued by the invalidation of alg-2B. These data are consistent with a hypothesis connecting superoxide and calcium signalling during Dictyostelium development.
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Diferenciación Celular , Dictyostelium/enzimología , Morfogénesis , NADPH Oxidasas/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Membrana Celular/enzimología , Células Cultivadas , Clonación Molecular , Dictyostelium/citología , Dictyostelium/crecimiento & desarrollo , Motivos EF Hand , Regulación del Desarrollo de la Expresión Génica , Genes Protozoarios , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , NADPH Oxidasas/química , NADPH Oxidasas/genética , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Superóxidos/metabolismoRESUMEN
We report here two atypical cases of X-linked CGD patients (first cousins) in which cytochrome b(558) is present at a normal level but is not functional (X91+). The mutations were localized by single-strand conformational polymorphism of reverse transcriptase-polymerase chain reaction amplified fragments and then identified by sequence analysis. They consisted in two base substitutions (C919 to A and C923 to G), changing His303 to Asn and Pro304 to Arg in the cytosolic gp91phox C-terminal tail. Mismatched polymerase chain reaction and genomic DNA sequencing showed that mothers had both wild-type and mutated alleles, confirming that this case was transmitted in an X-linked fashion. A normal amount of FAD was found in neutrophil membranes, both in the X91+ patients and their parents. Epstein-Barr virus-transformed B lymphocytes from the X91+ patients acidified normally upon stimulation with arachidonic acid, indicating that the mutated gp91phox still functioned as a proton channel. A cell-free translocation assay demonstrated that the association of the cytosolic factors p47phox and p67phox with the membrane fraction was strongly disrupted. We concluded that residues 303 and 304 are crucial for the stable assembly of the NADPH oxidase complex and for electron transfer, but not for its proton channel activity.