RESUMEN

Lignin, a major plant cell wall component, has an important role in plant-defense mechanisms against pathogens and is a promising renewable carbon source to produce bio-based chemicals. However, our understanding of microbial metabolism is incomplete regarding certain lignin-related compounds like p-coumaryl and sinapyl alcohols. Here, we reveal peripheral pathways for the catabolism of the three main lignin precursors (p-coumaryl, coniferyl, and sinapyl alcohols) in the plant pathogen Xanthomonas citri. Our study demonstrates all the necessary enzymatic steps for funneling these monolignols into the tricarboxylic acid cycle, concurrently uncovering aryl aldehyde reductases that likely protect the pathogen from aldehydes toxicity. It also shows that lignin-related aromatic compounds activate transcriptional responses related to chemotaxis and flagellar-dependent motility, which might play an important role during plant infection. Together our findings provide foundational knowledge to support biotechnological advances for both plant diseases treatments and conversion of lignin-derived compounds into bio-based chemicals.

Asunto(s)

Lignina , Xanthomonas , Xanthomonas/metabolismo , Xanthomonas/genética , Lignina/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Ciclo del Ácido Cítrico , Quimiotaxis , Aldehído Oxidorreductasas/metabolismo , Aldehído Oxidorreductasas/genética

RESUMEN

Formaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert cytotoxicity through DNA and DNA-protein crosslinking, likely contributing to the onset of the human DNA repair condition Fanconi Anaemia. Mutations in the genes coding for FA detoxifying enzymes underlie a human inherited bone marrow failure syndrome (IBMFS), even in the presence of functional DNA repair, raising the question of whether FA causes relevant cellular damage beyond genotoxicity. Here, we report that FA triggers cellular redox imbalance in human cells and in Caenorhabditis elegans. Mechanistically, FA reacts with the redox-active thiol group of glutathione (GSH), altering the GSH:GSSG ratio and causing oxidative stress. FA cytotoxicity is prevented by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which metabolizes FA-GSH products, lastly yielding reduced GSH. Furthermore, we show that GSH synthesis protects human cells from FA, indicating an active role of GSH in preventing FA toxicity. These findings might be relevant for patients carrying mutations in FA-detoxification systems and could suggest therapeutic benefits from thiol-rich antioxidants like N-acetyl-L-cysteine.

Asunto(s)

Aldehído Oxidorreductasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Anemia de Fanconi/metabolismo , Formaldehído/toxicidad , Glutatión/metabolismo , Aldehído Oxidorreductasas/genética , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Daño del ADN , Modelos Animales de Enfermedad , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Formaldehído/metabolismo , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Oxidación-Reducción , Estrés Oxidativo

RESUMEN

OBJECTIVES: An altered retinol metabolism might play a role in the development of nonalcoholic fatty liver disease (NAFLD). Tocopherols (TF) modulate metabolic pathways and have been proposed as a complementary treatment of obesity-induced metabolic alterations. Moreover, there is evidence suggesting that TF may modulate retinol metabolism. The aim of this study was to evaluate whether the dietary supplementation of α- and γ-TF modulates the expression of hepatic retinaldehyde dehydrogenases, RALDH1, RALDH2, and RALDH3 (involved in retinol metabolism) and, lipogenic factors sterol regulatory element binding protein-1c (SREBP-1c) and cluster differentiation 36 (CD36) in an animal model of diet-induced NAFLD. METHODS: Male C57BL/6J mice were divided into four groups: a control diet (CD) group (10% fat, 20% protein, 70% carbohydrates); a CD + TF group (α-tocopherol: 0.7 mg·kg·d-1, γ-tocopherol: 3.5 mg·kg·d-1); a high-fat diet (HFD) group (60% fat, 20% protein, 20% carbohydrates); and a HFD + TF group (0.01 mL·g body weight·d-1), for 12 wk. General parameters (body-adipose tissue weight, glucose-triacylglyceride serum levels), liver steatosis (histology, liver triacylglycerides content), and hepatic RALDH1, RALDH2, RALDH3, SREBP-1c and CD36 (qPCR, quantitative polymerase chain reaction; IHQ, immunohistochemistry) were measured. RESULTS: TF supplementation in HFD-fed mice decreased the presence of lipid vesicles (90%) and total lipid content (75%) and downregulated the expression of RALDH1, RALDH3, SREBP-1c, and CD36. CONCLUSIONS: The present study demonstrated that α- and γ-TF (1:5 ratio) might play a role in modulating retinol metabolism in the prevention of NAFLD induced by a HFD.

Asunto(s)

Enfermedad del Hígado Graso no Alcohólico , Retinaldehído , Aldehído Oxidorreductasas/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Suplementos Dietéticos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Retinaldehído/metabolismo , Tocoferoles/metabolismo

RESUMEN

Rhodococcus opacus PD630 accumulates significant amounts of triacylglycerols (TAG), but is not able to de novo synthesize wax esters (WE) from structural unrelated carbon sources, such as gluconate. In this study, strain PD630 was engineered to produce WE by heterologous expression of maqu_2220 gene, which encodes a fatty acyl-CoA reductase for the production of fatty alcohols in Marinobacter hydrocarbonoclasticus. Recombinant cells produced ca. 46% of WE and 54% of TAG (of total WE+TAG) from gluconate compared with the wild type, which produced 100% of TAG. Cell growth was not affected by the heterologous expression of MAQU_2220. Several saturated and monounsaturated WE species were produced by cells, with C18:C16, C16:C16 and C16:C18 as main species. The fatty acid composition of WE fraction in PD630maqu_2220 was enriched with C16:0, C18:0, whereas C16:0, C18:0 and C18:1 predominated in the TAG fraction. Significant amounts of WE and TAG were accumulated by PD630maqu_2220 from whey, an inexpensive waste material from dairy industries, without affecting cell biomass production. This is the first report on WE synthesis by R. opacus from gluconate, which demonstrates that lipid metabolism of this bacterium is flexible enough to assimilate heterologous components for the production of new lipid derivatives with industrial interest.

Asunto(s)

Ésteres/metabolismo , Rhodococcus/genética , Rhodococcus/metabolismo , Ceras/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Industria Lechera , Escherichia coli/genética , Residuos Industriales , Ingeniería Metabólica , Triglicéridos/metabolismo , Suero Lácteo/química , Suero Lácteo/metabolismo

RESUMEN

Exogenous supply of nitric oxide (NO) increases drought tolerance in sugarcane plants. However, little is known about the role of NO produced by plants under water deficit. The aim of this study was to test the hypothesis that drought-tolerance in sugarcane is associated with NO production and metabolism, with the more drought-tolerant genotype presenting higher NO accumulation in plant tissues. The sugarcane genotypes IACSP95-5000 (drought-tolerant) and IACSP97-7065 (drought-sensitive) were submitted to water deficit by adding polyethylene glycol (PEG-8000) in nutrient solution to reduce the osmotic potential to -0.4 MPa. To evaluate short-time responses to water deficit, leaf and root samples were taken after 24 h under water deficit. The drought-tolerant genotype presented higher root extracellular NO content, which was accompanied by higher root nitrate reductase (NR) activity as compared to the drought-sensitive genotype under water deficit. In addition, the drought-tolerant genotype had higher leaf intracellular NO content than the drought-sensitive one. IACSP95-5000 exhibited decreases in root S-nitrosoglutathione reductase (GSNOR) activity under water deficit, suggesting that S-nitrosoglutathione (GSNO) is less degraded and that the drought-tolerant genotype has a higher natural reservoir of NO than the drought-sensitive one. Those differences in intracellular and extracellular NO contents and enzymatic activities were associated with higher leaf hydration in the drought-tolerant genotype as compared to the sensitive one under water deficit.

Asunto(s)

Sequías , Óxido Nítrico/metabolismo , Saccharum/metabolismo , Saccharum/fisiología , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Genotipo , Nitrato-Reductasa/genética , Nitrato-Reductasa/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/fisiología , S-Nitrosoglutatión/metabolismo

RESUMEN

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.

Asunto(s)

Aldehído Oxidorreductasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos/genética , Mioblastos/metabolismo , Óxido Nítrico/metabolismo , Aldehído Oxidorreductasas/antagonistas & inhibidores , Aldehído Oxidorreductasas/genética , Animales , Diferenciación Celular , Fusión Celular , Embrión de Pollo , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Cisteína/análogos & derivados , Cisteína/metabolismo , Cisteína/farmacología , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Desarrollo de Músculos/efectos de los fármacos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citología , Mioblastos/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , S-Nitrosoglutatión/metabolismo , S-Nitrosotioles/metabolismo , S-Nitrosotioles/farmacología , Transducción de Señal , Guanilil Ciclasa Soluble/genética , Guanilil Ciclasa Soluble/metabolismo , Guanilil Ciclasa Soluble/farmacología , Tionucleótidos/farmacología , Triazenos/farmacología

RESUMEN

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.

Asunto(s)

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 & desarrollo

RESUMEN

Mycobacterium tuberculosis (M. tuberculosis) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant systems could facilitate novel drug developments. In this work, we characterized the product of the gene Rv1608c from M. tuberculosis, which according to sequence homology had been annotated as a putative peroxiredoxin of the peroxiredoxin Q subfamily (PrxQ B from M. tuberculosis or MtPrxQ B). The protein has been reported to be essential for M. tuberculosis growth in cholesterol-rich medium. We demonstrated the M. tuberculosis thioredoxin B/C-dependent peroxidase activity of MtPrxQ B, which acted as a two-cysteine peroxiredoxin that could function, although less efficiently, using a one-cysteine mechanism. Through steady-state and competition kinetic analysis, we proved that the net forward rate constant of MtPrxQ B reaction was 3 orders of magnitude faster for fatty acid hydroperoxides than for hydrogen peroxide (3×106vs 6×103M-1s-1, respectively), while the rate constant of peroxynitrite reduction was (0.6-1.4) ×106M-1s-1 at pH 7.4. The enzyme lacked activity towards cholesterol hydroperoxides solubilized in sodium deoxycholate. Both thioredoxin B and C rapidly reduced the oxidized form of MtPrxQ B, with rates constants of 0.5×106 and 1×106M-1s-1, respectively. Our data indicated that MtPrxQ B is monomeric in solution both under reduced and oxidized states. In spite of the similar hydrodynamic behavior the reduced and oxidized forms of the protein showed important structural differences that were reflected in the protein circular dichroism spectra.

Asunto(s)

Aldehído Oxidorreductasas/química , Proteínas Bacterianas/química , Ácidos Grasos/química , Mycobacterium tuberculosis/química , Peroxirredoxinas/química , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Grasos/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Cinética , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/enzimología , Oxidación-Reducción , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tiorredoxinas/genética , Tiorredoxinas/metabolismo

RESUMEN

Autism spectrum disorders (ASDs) are a range of complex neurodevelopmental conditions principally characterized by dysfunctions linked to mental development. Previous studies have shown that there are more than 1000 genes likely involved in ASD, expressed mainly in brain and highly interconnected among them. We applied whole exome sequencing in Colombian-South American trios. Two missense novel SNVs were found in the same child: ALDH1A3 (RefSeq NM_000693: c.1514T>C (p.I505T)) and FOXN1 (RefSeq NM_003593: c.146C>T (p.S49L)). Gene expression studies reveal that Aldh1a3 and Foxn1 are expressed in ~E13.5 mouse embryonic brain, as well as in adult piriform cortex (PC; ~P30). Conserved Retinoic Acid Response Elements (RAREs) upstream of human ALDH1A3 and FOXN1 and in mouse Aldh1a3 and Foxn1 genes were revealed using bioinformatic approximation. Chromatin immunoprecipitation (ChIP) assay using Retinoid Acid Receptor B (Rarb) as the immunoprecipitation target suggests RA regulation of Aldh1a3 and Foxn1 in mice. Our results frame a possible link of RA regulation in brain to ASD etiology, and a feasible non-additive effect of two apparently unrelated variants in ALDH1A3 and FOXN1 recognizing that every result given by next generation sequencing should be cautiously analyzed, as it might be an incidental finding.

Asunto(s)

Aldehído Oxidorreductasas/genética , Trastorno del Espectro Autista/genética , Exoma , Factores de Transcripción Forkhead/genética , Receptores de Ácido Retinoico/genética , Tretinoina/metabolismo , Adulto , Aldehído Oxidorreductasas/metabolismo , Animales , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/patología , Secuencia de Bases , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Encéfalo/patología , Niño , Estudios de Cohortes , Colombia , Embrión de Mamíferos , Femenino , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Linaje , Pruebas Psicológicas , Receptores de Ácido Retinoico/metabolismo , Elementos de Respuesta , Transducción de Señal

RESUMEN

Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a homodimeric molybdenum-containing protein that catalyzes the hydroxylation of aldehydes to carboxylic acids and contains a Mo-pyranopterin active site and two FeS centers called FeS 1 and FeS 2. The electron transfer reaction inside DgAOR is proposed to be performed through a chemical pathway linking Mo and the two FeS clusters involving the pyranopterin ligand. EPR studies performed on reduced as-prepared DgAOR showed that this pathway is able to transmit very weak exchange interactions between Mo(V) and reduced FeS 1. Similar EPR studies but performed on DgAOR samples inhibited with glycerol and ethylene glycol showed that the value of the exchange coupling constant J increases ~2 times upon alcohol inhibition. Structural studies in these DgAOR samples have demonstrated that the Mo-FeS 1 bridging pathway does not show significant differences, confirming that the changes in J observed upon inhibition cannot be ascribed to structural changes associated neither with pyranopterin and FeS 1 nor with changes in the electronic structure of FeS 1, as its EPR properties remain unchanged. Theoretical calculations indicate that the changes in J detected by EPR are related to changes in the electronic structure of Mo(V) determined by the replacement of the OHx labile ligand for an alcohol molecule. Since the relationship between electron transfer rate and isotropic exchange interaction, the present results suggest that the intraenzyme electron transfer process mediated by the pyranopterin moiety is governed by a Mo ligand-based regulatory mechanism.

Asunto(s)

Aldehído Oxidorreductasas/química , Desulfovibrio gigas/enzimología , Molibdeno/química , Conformación Proteica , Aldehído Oxidorreductasas/antagonistas & inhibidores , Aldehído Oxidorreductasas/metabolismo , Dominio Catalítico , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/química , Cinética , Ligandos , Oxidación-Reducción , Especificidad por Sustrato

RESUMEN

Objective. To conduct a health impact assessment (HIA) to quantify health benefits for several PM and O3 air pollution reduction scenarios in the Mexico City Metropolitan Area (MCMA). Results from this HIA will contribute to the scientific support of the MCMA air quality management plan (PROAIRE) for the period 2011-2020. Materials and methods. The HIA methodology consisted of four steps: 1) selection of the air pollution reduction scenarios, 2) identification of the at-risk population and health outcomes for the 2005 baseline scenario, 3) selection of concentration-response functions and 4) estimation of health impacts. Results. Reductions of PM10 levels to 20 μg/m³ and O3 levels to 0.050ppm (98 µg/m³) would prevent 2300 and 400 annual deaths respectively. The greatest health impact was seen in the over-65 age group and in mortality due to cardiopulmonary and cardiovascular disease. Conclusion. Improved air quality in the MCMA could provide significant health benefits through focusing interventions by exposure zones.

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Objetivo. Realizar una evaluación de impacto en salud (EIS) que documente los beneficios en salud ante diversos escenarios de reducción de PM10 y O3 en el aire de la Zona Metropolitana del Valle de México (ZMVM). Los resultados contribuyen al sustento científico del plan de gestión de calidad del aire (PROAIRE 2011-2020). Material y métodos. La metodología de EIS comprende cuatro pasos: 1) selección de los escenarios de reducción, 2) identificación de la población en riesgo y de los eventos en salud para el año basal 2005, 3) selección de las funciones de concentración-respuesta y 4) estimación del impacto en la salud. Resultados. Reducciones de PM10 a 20μg/m³ y de O3 a 0.050ppm (98 µg/m³) evitarían, respectivamente, cerca de 2 300 y 400 muertes por año. El mayor impacto se observa en el grupo de más de 65 años y en la mortalidad por causas cardiopulmonares y cardiovasculares. Conclusiones. Mejorar la calidad del aire en la ZMVM podría reflejar importantes beneficios para la salud focalizados por zonas o áreas de exposición.

Asunto(s)

Pseudomonas putida/metabolismo , Estirenos/metabolismo , Aldehído Oxidorreductasas/metabolismo , Biodegradación Ambiental , Compuestos Epoxi/metabolismo , Proteínas de Escherichia coli , Ácido Glutámico/metabolismo , Isomerasas/metabolismo , Oxidación-Reducción , Consumo de Oxígeno , Fenilacetatos/metabolismo , Pseudomonas putida/enzimología , Pseudomonas putida/crecimiento & desarrollo , Estireno , Succinatos/metabolismo , Ácido Succínico

RESUMEN

Water stress is one of the most severe problems for plant growth and productivity. Using the legume Lotus japonicus exposed to water stress, a comparative analysis of key components in metabolism of reactive nitrogen and oxygen species (RNS and ROS, respectively) were made. After water stress treatment plants accumulated proline 23 and 10-fold in roots and leaves respectively, compared with well-watered plants. Significant changes in metabolism of RNS and ROS were observed, with an increase in both protein tyrosine nitration and lipid peroxidation, which indicate that water stress induces a nitro-oxidative stress. In roots, ·NO content was increased and S-nitrosoglutathione reductase activity was reduced by 23%, wherein a specific protein nitration pattern was observed. As part of this response, activity of NADPH-generating dehydrogenases was also affected in roots resulting in an increase of the NADPH/NADP(+) ratio. Our results suggest that in comparison with leaves, roots are significantly affected by water stress inducing an increase in proline and NO content which could highlight multiple functions for these metabolites in water stress adaptation, recovery and signaling. Thus, it is proposed that water stress generates a spatial distribution of nitro-oxidative stress with the oxidative stress component being higher in leaves whereas the nitrosative stress component is higher in roots.

Asunto(s)

Lotus/metabolismo , Estrés Oxidativo , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Agua/metabolismo , Aldehído Oxidorreductasas/metabolismo , Western Blotting , Deshidratación , Activación Enzimática , Pruebas de Enzimas , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido , Lotus/enzimología , Lotus/fisiología , NADP/metabolismo , NADPH Deshidrogenasa/metabolismo , Óxido Nítrico/metabolismo , Fenotipo , Extractos Vegetales/análisis , Extractos Vegetales/metabolismo , Hojas de la Planta/enzimología , Hojas de la Planta/fisiología , Raíces de Plantas/enzimología , Raíces de Plantas/fisiología , Prolina/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

By applying metabolic control analysis and inhibitor titration we determined the degree of control (flux control coefficient) of pyruvate:ferredoxin oxidoreductase (PFOR) and bifunctional aldehyde-alcohol dehydrogenase (ADHE) over the fluxes of fermentative glycolysis of Entamoeba histolytica subjected to aerobic conditions. The flux-control coefficients towards ethanol and acetate formation determined for PFOR titrated with diphenyleneiodonium were 0.07 and 0.09, whereas for ADHE titrated with disulfiram were 0.33 and -0.19, respectively. ADHE inhibition induced significant accumulation of glycolytic intermediates and lower ATP content. These results indicate that ADHE exerts significant flux-control on the carbon end-product formation of amoebas subjected to aerobic conditions.

Asunto(s)

Ácido Acético/metabolismo , Alcohol Deshidrogenasa/metabolismo , Aldehído Oxidorreductasas/metabolismo , Entamoeba histolytica/metabolismo , Proteínas Protozoarias/metabolismo , Aerobiosis , Alcohol Deshidrogenasa/antagonistas & inhibidores , Aldehído Oxidorreductasas/antagonistas & inhibidores , Disulfiram/farmacología , Entamoeba histolytica/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Etanol/metabolismo , Glucólisis/efectos de los fármacos , Cinética , Redes y Vías Metabólicas , Compuestos Onio/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Piruvato-Sintasa/antagonistas & inhibidores , Piruvato-Sintasa/metabolismo , Trofozoítos/efectos de los fármacos , Trofozoítos/metabolismo

RESUMEN

The enzyme S-nitrosoglutathione reductase (GSNOR) has an important role in the metabolism of S-nitrosothiols (SNO) and, consequently, in the modulation of nitric oxide (NO)-mediated processes. Although the mitochondrial electron transport chain is an important target of NO, the role of GSNOR in the functionality of plant mitochondria has not been addressed. Here, we measured SNO content and NO emission in Arabidopsis thaliana cell suspension cultures of wild-type (WT) and GSNOR overexpressing (GSNOR(OE)) or antisense (GSNOR(AS)) transgenic lines, grown under optimal conditions and under nutritional stress. Respiratory activity of isolated mitochondria and expression of genes encoding for mitochondrial proteins were also analyzed. Under optimal growth conditions, GSNOR(OE) had the lowest SNO and NO levels and GSNOR(AS) the highest, as expected by the GSNO-consuming activity of GSNOR. Under stress, this pattern was reversed. Analysis of oxygen uptake by isolated mitochondria showed that complex I and external NADH dehydrogenase activities were inhibited in GSNOR(OE) cells grown under nutritional stress. Moreover, GSNOR(OE) could not increase alternative oxidase (AOX) activity under nutritional stress. GSNOR(AS) showed constitutively high activity of external NADH dehydrogenase, and maintained the activity of the uncoupling protein (UCP) under stress. The alterations observed in mitochondrial protein activities were not strictly correlated to changes in gene expression, but instead seemed to be related with the SNO/NO content, suggesting a post-transcriptional regulation. Overall, our results highlight the importance of GSNOR in modulating SNO and NO homeostasis as well mitochondrial functionality, both under normal and adverse conditions in A. thaliana cells.

Asunto(s)

Aldehído Oxidorreductasas/metabolismo , Arabidopsis/metabolismo , Mitocondrias/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Línea Celular , Complejo I de Transporte de Electrón/metabolismo , Proteínas Mitocondriales/metabolismo , NADH Deshidrogenasa/metabolismo , Óxido Nítrico/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , S-Nitrosotioles/análisis

RESUMEN

Pseudomonas putida G7 is one of the most studied naphthalene-degrading species. The nah operon in P. putida, which is present on the 83 kb metabolic plasmid NAH7, codes for enzymes involved in the conversion of naphthalene to salicylate. The enzyme NahF (salicylaldehyde dehydrogenase) catalyzes the last reaction in this pathway. The nahF gene was subcloned into the pET28a(TEV) vector and the recombinant protein was overexpressed in Escherichia coli Arctic Express at 285 K. The soluble protein was purified by affinity chromatography followed by gel filtration. Crystals of recombinant NahF (6×His-NahF) were obtained at 291 K and diffracted to 2.42 Å resolution. They belonged to the hexagonal space group P6(4)22, with unit-cell parameters a = b = 169.47, c = 157.94 Å. The asymmetric unit contained a monomer and a crystallographic twofold axis generated the dimeric biological unit.

Asunto(s)

Aldehído Oxidorreductasas/química , Pseudomonas putida/enzimología , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/aislamiento & purificación , Aldehído Oxidorreductasas/metabolismo , Cristalografía por Rayos X , Expresión Génica , Naftalenos/metabolismo

RESUMEN

Retinoic acid, the active form of the nutrient vitamin A, regulates several facets of neuronal plasticity in the hippocampus, including neurogenesis and synaptic strength, acting via specific retinoic acid receptors (RARs). Essential for conversion of vitamin A to retinoic acid is the enzyme retinaldehyde dehydrogenase (RALDH) and in the rodent hippocampus this is only present in the adjacent meninges where it must act as a locally released paracrine hormone. Little is known though about the expression of RALDHs and RARs in the human hippocampus. This study confirms that RALDH levels are very low in mouse neurons but, surprisingly, strong expression of RALDH protein is detected by immunohistochemistry in hippocampal neurons. The receptors RARα, ß and γ were also detected, each receptor exhibiting differing subcellular locations implying their potential regulation of both transcription and non-genomic actions. These results imply an essential function of retinoic acid in the human hippocampus likely to include regulation of neuronal plasticity.

Asunto(s)

Aldehído Deshidrogenasa/metabolismo , Hipocampo/metabolismo , Receptores de Ácido Retinoico/metabolismo , Retinal-Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Aldehído Oxidorreductasas/metabolismo , Animales , Autopsia , Giro Dentado/metabolismo , Giro Dentado/patología , Hipocampo/patología , Humanos , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Modelos Animales , Neuronas/metabolismo , Neuronas/patología

RESUMEN

Glutamyl-tRNA reductase (GluTR) is the first enzyme committed to tetrapyrrole biosynthesis by the C(5)-pathway. This enzyme transforms glutamyl-tRNA into glutamate-1-semi-aldehyde, which is then transformed into 5-amino levulinic acid by the glutamate-1-semi-aldehyde 2,1-aminomutase. Binding of heme to GluTR seems to be relevant to regulate the enzyme function. Recombinant GluTR from Acidithiobacillus ferrooxidans an acidophilic bacterium that participates in bioleaching of minerals was expressed in Escherichia coli and purified as a soluble protein containing type b heme. Upon control of the cellular content of heme in E. coli, GluTR with different levels of bound heme was obtained. An inverse correlation between the activity of the enzyme and the level of bound heme to GluTR suggested a control of the enzyme activity by heme. Heme bound preferentially to dimeric GluTR. An intact dimerization domain was essential for the enzyme to be fully active. We propose that the cellular levels of heme might regulate the activity of GluTR and ultimately its own biosynthesis.

Asunto(s)

Acidithiobacillus/enzimología , Aldehído Oxidorreductasas/metabolismo , Hemo/metabolismo , Acidithiobacillus/genética , Aldehído Oxidorreductasas/química , Aldehído Oxidorreductasas/genética , Catálisis , Escherichia coli/genética , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Transferrin (Tf) is a host glycoprotein capable of binding two ferric-iron ions to become holotransferrin (holoTf), which transports iron in to all cells. Entamoeba histolytica is a parasitic protozoan able to use holoTf as a sole iron source in vitro. The mechanism by which this parasite scavenges iron from holoTf is unknown. An E. histolytica holoTf-binding protein (EhTfbp) was purified by using an anti-human transferrin receptor (TfR) monoclonal antibody. EhTfbp was identified by MS/MS analysis and database searches as E. histolytica acetaldehyde/alcohol dehydrogenase-2 (EhADH2), an iron-dependent enzyme. Both EhTfbp and EhADH2 bound holoTf and were recognized by the anti-human TfR antibody, indicating that they correspond to the same protein. It was found that the amoebae internalized holoTf through clathrin-coated pits, suggesting that holoTf endocytosis could be important for the parasite during colonization and invasion of the intestinal mucosa and liver.

Asunto(s)

Alcohol Deshidrogenasa/metabolismo , Aldehído Oxidorreductasas/metabolismo , Clatrina/metabolismo , Endocitosis , Entamoeba histolytica/metabolismo , Interacciones Huésped-Patógeno , Transferrina/metabolismo , Alcohol Deshidrogenasa/aislamiento & purificación , Aldehído Oxidorreductasas/aislamiento & purificación , Vesículas Cubiertas por Clatrina/metabolismo , Humanos , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Espectrometría de Masas en Tándem

RESUMEN

BACKGROUND: Carbonic anhydrase (CA) is a ubiquitous enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction underlying diverse biochemical and physiological processes. Gamma class carbonic anhydrases (gamma-CAs) are widespread in prokaryotes but their physiological roles remain elusive. At present, only gamma-CA of Methanosarcina thermophila (Cam) has been shown to have CA activity. Genome analysis of a rhizobacterium Azospirillum brasilense, revealed occurrence of ORFs encoding one beta-CA and two gamma-CAs. RESULTS: One of the putative gamma-CA encoding genes of A. brasilense was cloned and overexpressed in E. coli. Electrometric assays for CA activity of the whole cell extracts overexpressing recombinant GCA1 did not show CO2 hydration activity. Reverse transcription-PCR analysis indicated that gca1 in A. brasilense is co-transcribed with its upstream gene annotated as argC, which encodes a putative N-acetyl-gamma-glutamate-phosphate reductase. 5'-RACE also demonstrated that there was no transcription start site between argC and gca1, and the transcription start site located upstream of argC transcribed both the genes (argC-gca1). Using transcriptional fusions of argC-gca1 upstream region with promoterless lacZ, we further demonstrated that gca1 upstream region did not have any promoter and its transcription occurred from a promoter located in the argC upstream region. The transcription of argC-gca1 operon was upregulated in stationary phase and at elevated CO2 atmosphere. CONCLUSIONS: This study shows lack of CO2 hydration activity in a recombinant protein expressed from a gene predicted to encode a gamma-carbonic anhydrase in A. brasilense although it cross reacts with anti-Cam antibody raised against a well characterized gamma-CA. The organization and regulation of this gene along with the putative argC gene suggests its involvement in arginine biosynthetic pathway instead of the predicted CO2 hydration.

Asunto(s)

Aldehído Oxidorreductasas/genética , Azospirillum brasilense/crecimiento & desarrollo , Proteínas Bacterianas/genética , Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/genética , Regulación Bacteriana de la Expresión Génica , Transcripción Genética , Aldehído Oxidorreductasas/metabolismo , Azospirillum brasilense/clasificación , Azospirillum brasilense/enzimología , Azospirillum brasilense/genética , Proteínas Bacterianas/metabolismo , Anhidrasas Carbónicas/metabolismo , Filogenia

RESUMEN

RATIONALE: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. OBJECTIVE: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. METHODS AND RESULTS: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. CONCLUSION: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels.

Asunto(s)

Diferenciación Celular , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Miocitos del Músculo Liso/metabolismo , Pericardio/metabolismo , Transducción de Señal , Tretinoina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Animales , Apoptosis , Comunicación Autocrina , Diferenciación Celular/genética , Células Cultivadas , Embrión de Pollo , Técnicas de Cocultivo , Vasos Coronarios/embriología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Morfogénesis , Miocitos Cardíacos/metabolismo , Comunicación Paracrina , Pericardio/embriología , Codorniz , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Técnicas de Cultivo de Tejidos , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/genética