Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
1.
Amino Acids ; 54(2): 169-180, 2022 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-34837556

RESUMEN

The human Dietary Approaches to Stop Hypertension-Sodium Trial has shown that ß-aminoisobutyric acid (BAIBA) may prevent the development of salt-sensitive hypertension (SSHT). However, the specific antihypertensive mechanism remains unclear in the renal tissues of salt-sensitive (SS) rats. In this study, BAIBA (100 mg/kg/day) significantly attenuated SSHT via increased nitric oxide (NO) content in the renal medulla, and it induced a significant increase in NO synthesis substrates (L-arginine and malic acid) in the renal medulla. BAIBA enhanced the activity levels of total NO synthase (NOS), inducible NOS, and constitutive NOS. BAIBA resulted in increased fumarase activity and decreased fumaric acid content in the renal medulla. The high-salt diet (HSD) decreased fumarase expression in the renal cortex, and BAIBA increased fumarase expression in the renal medulla and renal cortex. Furthermore, in the renal medulla, BAIBA increased the levels of ATP, ADP, AMP, and ADP/ATP ratio, thus further activating AMPK phosphorylation. BAIBA prevented the decrease in renal medullary antioxidative defenses induced by the HSD. In conclusion, BAIBA's antihypertensive effect was underlined by the phosphorylation of AMPK, the prevention of fumarase's activity reduction caused by the HSD, and the enhancement of NO content, which in concert attenuated SSHT in SS rats.


Asunto(s)
Fumarato Hidratasa , Hipertensión , Ácidos Aminoisobutíricos , Animales , Presión Sanguínea , Suplementos Dietéticos , Fumarato Hidratasa/genética , Fumarato Hidratasa/metabolismo , Hipertensión/tratamiento farmacológico , Hipertensión/prevención & control , Ratas , Ratas Endogámicas Dahl
2.
Artículo en Inglés | MEDLINE | ID: mdl-31332064

RESUMEN

In the last decade, carbon monoxide-releasing molecules (CORMs) have been shown to act against several pathogens and to be promising antimicrobials. However, the understanding of the mode of action and reactivity of these compounds on bacterial cells is still deficient. In this work, we used a metabolomics approach to probe the toxicity of the ruthenium(II) complex Ru(CO)3Cl(glycinate) (CORM-3) on Escherichia coli By resorting to 1H nuclear magnetic resonance, mass spectrometry, and enzymatic activities, we show that CORM-3-treated E. coli accumulates larger amounts of glycolytic intermediates, independently of the oxygen growth conditions. The work provides several evidences that CORM-3 inhibits glutamate synthesis and the iron-sulfur enzymes of the tricarboxylic acid (TCA) cycle and that the glycolysis pathway is triggered in order to establish an energy and redox homeostasis balance. Accordingly, supplementation of the growth medium with fumarate, α-ketoglutarate, glutamate, and amino acids cancels the toxicity of CORM-3. Importantly, inhibition of the iron-sulfur enzymes glutamate synthase, aconitase, and fumarase is only observed for compounds that liberate carbon monoxide. Altogether, this work reveals that the antimicrobial action of CORM-3 results from intracellular glutamate deficiency and inhibition of nitrogen and TCA cycles.


Asunto(s)
Antibacterianos/farmacología , Monóxido de Carbono/farmacología , Ciclo del Ácido Cítrico/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , Compuestos Organometálicos/farmacología , Aconitato Hidratasa/antagonistas & inhibidores , Aconitato Hidratasa/genética , Aconitato Hidratasa/metabolismo , Antibacterianos/química , Monóxido de Carbono/química , Ciclo del Ácido Cítrico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fumarato Hidratasa/antagonistas & inhibidores , Fumarato Hidratasa/genética , Fumarato Hidratasa/metabolismo , Fumaratos/metabolismo , Glutamato Sintasa/antagonistas & inhibidores , Glutamato Sintasa/genética , Glutamato Sintasa/metabolismo , Ácido Glutámico/metabolismo , Glucólisis/efectos de los fármacos , Glucólisis/genética , Ácidos Cetoglutáricos/metabolismo , Espectroscopía de Resonancia Magnética , Metabolómica/métodos , Compuestos Organometálicos/química , Oxidación-Reducción
3.
Cell Rep ; 19(8): 1631-1639, 2017 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-28538181

RESUMEN

Fumarase catalyzes the interconversion of fumarate and L-malate in the tricarboxylic acid cycle. The Dahl salt-sensitive (SS) rat, a model of salt-sensitive hypertension, exhibits fumarase insufficiencies. To investigate the mechanism mediating the effect of fumarase-related metabolites on hypertension, we considered the pathway in which L-malate can be converted to oxaloacetate, aspartate, argininosuccinate, and L-arginine, the substrate of nitric oxide (NO) synthase. The levels of aspartate, citrulline, L-arginine, and NO were significantly decreased in the kidneys of SS rats compared to salt-insensitive consomic SS.13BN rats. Knockdown of fumarase in human kidney cells and vascular endothelial cells resulted in decreased levels of malate, aspartate, L-arginine, and NO. Supplementation of aspartate or malate increased renal levels of L-arginine and NO and attenuated hypertension in SS rats. These findings reveal a multi-step metabolic pathway important for hypertension in which malate and aspartate may modulate blood pressure by altering levels of L-arginine and NO.


Asunto(s)
Arginina/metabolismo , Ácido Aspártico/metabolismo , Hipertensión/metabolismo , Malatos/metabolismo , Óxido Nítrico/metabolismo , Animales , Regulación hacia Abajo , Fumarato Hidratasa/metabolismo , Técnicas de Silenciamiento del Gen , Riñón/metabolismo , Ratas Endogámicas Dahl
4.
Anticancer Res ; 35(12): 6639-53, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26637880

RESUMEN

BACKGROUND/AIM: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare autosomal dominant disorder characterized by fumarate hydratase (FH) gene mutation. It is associated with the development of very aggressive kidney tumors, characterized by early onset and high metastatic potential, and has no effective therapy. The aim of the study was to establish a new preclinical platform for investigating morphogenetic and metabolic features, and alternative therapy of metastatic hereditary papillary renal cell carcinoma type 2 (PRCC2). MATERIALS AND METHODS: Fresh cells were collected from pleural fluid of a patient with metastatic hereditary PRCC2. Morphogenetic and functional characteristics were evaluated via microscopy, FH gene sequencing analysis, real-time polymerase chaine reaction and enzymatic activity measurement. We performed bioenergetic analysis, gene-expression profiling, and cell viability assay with 19 anti-neoplastic drugs. RESULTS: We established a new in vitro model of hereditary PRCC2 - the NCCFH1 cell line. The cell line possesses a c.1162 delA - p.Thr375fs frameshift mutation in the FH gene. Our findings indicate severe attenuation of oxidative phosphorylation and glucose-dependent growth of NCCFH1 cells that is consistent with the Warburg effect. Furthermore, gene-expression profiling identified that the most prominent molecular features reflected a high level of apoptosis, cell adhesion, and cell signaling. Drug screening revealed a marked sensitivity of FH(-/-) cells to mitoxantrone, epirubicin, topotecan and a high sensitivity to bortezomib. CONCLUSION: We demonstrated that the NCCFH1 cell line is a very interesting preclinical model for studying the metabolic features and testing new therapies for hereditary PRCC2, while bortezomib may be a potential efficient therapeutic option.


Asunto(s)
Carcinoma de Células Renales/genética , Fumarato Hidratasa/metabolismo , Adolescente , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Humanos , Técnicas In Vitro , Masculino
5.
J Am Chem Soc ; 137(2): 564-7, 2015 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-25469852

RESUMEN

Development of cell-permeable small molecules that target enzymes involved in energy metabolism remains important yet challenging. We describe here the discovery of a new class of compounds with a nutrient-dependent cytotoxicity profile that arises from pharmacological inhibition of fumarate hydratase (also known as fumarase). This finding was enabled by a high-throughput screen of a diverse chemical library in a panel of human cancer cell lines cultured under different growth conditions, followed by subsequent structure-activity optimization and target identification. While the highest cytotoxicity was observed under low glucose concentrations, the antiproliferative activities and inhibition of oxygen consumption rates in cells were distinctly different from those displayed by typical inhibitors of mitochondrial oxidative phosphorylation. The use of a photoaffinity labeling strategy identified fumarate hydratase as the principal pharmacological target. Final biochemical studies confirmed dose-dependent, competitive inhibition of this enzyme in vitro, which was fully consistent with the initially observed growth inhibitory activity. Our work demonstrates how the phenotypic observations combined with a successful target identification strategy can yield a useful class of pharmacological inhibitors of an enzyme involved in the operation of tricarboxylic acid cycle.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Fumarato Hidratasa/antagonistas & inhibidores , Línea Celular Tumoral , Ciclo del Ácido Cítrico/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Metabolismo Energético/efectos de los fármacos , Inhibidores Enzimáticos/toxicidad , Fumarato Hidratasa/metabolismo , Glucosa/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos
6.
Mol Med Rep ; 6(2): 429-33, 2012 08.
Artículo en Inglés | MEDLINE | ID: mdl-22580600

RESUMEN

Artemisinin, the active ingredient of the Chinese medicinal herb Artemisia annua L., and its derivatives (ARTs) are currently widely used as anti-malarial drugs around the world. In this study, we found that dihydroartemisinin (DHA), one of the main active metabolites of ARTs, inhibited the proliferation of human hepatocarcinoma BEL-7402 cells in a concentration-dependent manner. To interpret the mechanisms involved, an analysis of the mitochondrial proteome was performed employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Seven mitochondrial proteins including fumarate hydratase, 60 kDa heat shock protein, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, two subunits of ATP synthase and NADPH:adrenodoxin oxidoreductase were identified to be differentially expressed between the control and DHA-treated groups. Our results indicate that the imbalance of energy metabolism induced by DHA may contribute, at least in part, to its anti-cancer potential in BEL-7402 cells.


Asunto(s)
Artemisininas/farmacología , Proliferación Celular/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/análisis , Proteoma/análisis , 3-Hidroxiacil-CoA Deshidrogenasas/análisis , Antineoplásicos Fitogénicos/farmacología , Artemisia annua/química , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Electroforesis en Gel Bidimensional , Metabolismo Energético , Enoil-CoA Hidratasa/análisis , Fumarato Hidratasa/análisis , Humanos , Mitocondrias/enzimología , ATPasas de Translocación de Protón Mitocondriales/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
7.
Integr Biol (Camb) ; 3(11): 1135-42, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22005712

RESUMEN

The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.


Asunto(s)
Metabolismo Energético/fisiología , Técnicas de Inactivación de Genes , Interferencia de ARN/fisiología , Adenosina Trifosfato/metabolismo , Animales , Dióxido de Carbono/metabolismo , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Respiración de la Célula/efectos de los fármacos , Respiración de la Célula/fisiología , Ciclo del Ácido Cítrico/fisiología , Embrión de Mamíferos/citología , Metabolismo Energético/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Fibroblastos/metabolismo , Fumarato Hidratasa/deficiencia , Fumarato Hidratasa/genética , Eliminación de Gen , Glucólisis/fisiología , Células HeLa , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Ácido Láctico/metabolismo , Ratones , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Proteínas de Transporte de Nucleótidos/genética , Oligomicinas/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/fisiología , ARN Interferente Pequeño/genética
8.
J Plant Physiol ; 168(7): 730-3, 2011 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-21194788

RESUMEN

The effects of Zn excess on carboxylate metabolism were investigated in sugar beet (Beta vulgaris L.) plants grown hydroponically in a growth chamber. Root extracts of plants grown with 50 or 100µM Zn in the nutrient solution showed increases in several enzymatic activities related to organic acid metabolism, including citrate synthase and phosphoenolpyruvate carboxylase, when compared to activities in control root extracts. Root citric and malic acid concentrations increased in plants grown with 100µM Zn, but not in plants grown with 50µM Zn. In the xylem sap, plants grown with 50 and 100µM Zn showed increases in the concentrations of citrate and malate compared to the controls. Leaves of plants grown with 50 or 100µM Zn showed increases in the concentrations of citric and malic acid and in the activities of citrate synthase and fumarase. Leaf isocitrate dehydrogenase increased only in plants grown with 50µM Zn when compared to the controls. In plants grown with 300µM Zn, the only enzyme showing activity increases in root extracts was citrate synthase, whereas the activities of other enzymes decreased compared to the controls, and root citrate concentrations increased. In the 300µM Zn-grown plants, the xylem concentrations of citric and malic acids were higher than those of controls, whereas in leaf extracts the activity of fumarase increased markedly, and the leaf citric acid concentration was higher than in the controls. Based on our data, a metabolic model of the carboxylate metabolism in sugar beet plants grown under Zn excess is proposed.


Asunto(s)
Beta vulgaris/efectos de los fármacos , Beta vulgaris/metabolismo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Zinc/toxicidad , Citrato (si)-Sintasa/metabolismo , Ácido Cítrico/metabolismo , Activación Enzimática/efectos de los fármacos , Fumarato Hidratasa/metabolismo , Malatos/metabolismo
9.
Cancer Genet Cytogenet ; 196(1): 45-55, 2010 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-19963135

RESUMEN

Energy deregulation and abnormalities of tumor cell metabolism are critical issues in understanding cancer. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is an aggressive form of RCC characterized by germline mutation of the Krebs cycle enzyme fumarate hydratase (FH), and one known to be highly metastatic and unusually lethal. There is considerable utility in establishing preclinical cell and xenograft models for study of disorders of energy metabolism, as well as in development of new therapeutic approaches targeting of tricarboxylic acid (TCA) cycle enzyme-deficient human cancers. Here we describe a new immortalized cell line, UOK 262, derived from a patient having aggressive HLRCC-associated recurring kidney cancer. We investigated gene expression, chromosome profiles, efflux bioenergetic analysis, mitochondrial ultrastructure, FH catabolic activity, invasiveness, and optimal glucose requirements for in vitro growth. UOK 262 cells have an isochromosome 1q recurring chromosome abnormality, i(1)(q10), and exhibit compromised oxidative phosphorylation and in vitro dependence on anaerobic glycolysis consistent with the clinical manifestation of HLRCC. The cells also display glucose-dependent growth, an elevated rate of lactate efflux, and overexpression of the glucose transporter GLUT1 and of lactate dehydrogenase A (LDHA). Mutant FH protein was present primarily in edematous mitochondria, but with catalytic activity nearly undetectable. UOK 262 xenografts retain the characteristics of HLRCC histopathology. Our findings indicate that the severe compromise of oxidative phosphorylation and rapid glycolytic flux in UOK 262 are an essential feature of this TCA cycle enzyme-deficient form of kidney cancer. This tumor model is the embodiment of the Warburg effect. UOK 262 provides a unique in vitro and in vivo preclinical model for studying the bioenergetics of the Warburg effect in human cancer.


Asunto(s)
Carcinoma de Células Renales/genética , Fumarato Hidratasa/genética , Neoplasias Renales/genética , Leiomiomatosis/genética , Modelos Biológicos , Animales , Western Blotting , Carcinoma de Células Renales/enzimología , Carcinoma de Células Renales/metabolismo , Línea Celular , Metabolismo Energético , Fumarato Hidratasa/metabolismo , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Técnicas In Vitro , Neoplasias Renales/enzimología , Neoplasias Renales/metabolismo , Leiomiomatosis/enzimología , Leiomiomatosis/metabolismo , Ratones , Ratones Desnudos
10.
Hypertension ; 54(2): 255-60, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19546378

RESUMEN

In a previous proteomic study, we found dramatic differences in fumarase in the kidney between Dahl salt-sensitive rats and salt-insensitive consomic SS-13(BN) rats. Fumarase catalyzes the conversion between fumarate and l-malate in the tricarboxylic acid cycle. Little is known about the pathophysiological significance of fumarate metabolism in cardiovascular and renal functions, including salt-induced hypertension. The fumarase gene is located on the chromosome substituted in the SS-13(BN) rat. Sequencing of fumarase cDNA indicated the presence of lysine at amino acid position 481 in Dahl salt-sensitive rats and glutamic acid in Brown Norway and SS-13(BN) rats. Total fumarase activity was significantly lower in the kidneys of Dahl salt-sensitive rats compared with SS-13(BN) rats, despite an apparent compensatory increase in fumarase abundance in Dahl salt-sensitive rats. Intravenous infusion of a fumarate precursor in SS-13(BN) rats resulted in a fumarate excess in the renal medulla comparable to that seen in Dahl salt-sensitive rats. The infusion significantly exacerbated salt-induced hypertension in SS-13(BN) rats (140+/-3 vs125+/-2 mm Hg in vehicle control at day 5 on a 4% NaCl diet; P<0.05). In addition, the fumarate infusion increased renal medullary tissue levels of H2O2. Treatment of cultured human renal epithelial cells with the fumarate precursor also increased cellular levels of H2O2. These data suggest a novel role for fumarate metabolism in salt-induced hypertension and renal medullary oxidative stress.


Asunto(s)
Fumarato Hidratasa/metabolismo , Hipertensión/enzimología , Hipertensión/genética , Malatos/metabolismo , Ácido Succínico/metabolismo , Análisis de Varianza , Animales , Biomarcadores/metabolismo , Western Blotting , ADN Complementario/análisis , Modelos Animales de Enfermedad , Hipertensión/fisiopatología , Médula Renal/efectos de los fármacos , Médula Renal/metabolismo , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Masculino , Estrés Oxidativo/fisiología , Distribución Aleatoria , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Dahl , Cloruro de Sodio Dietético/farmacología
11.
Plant Cell Environ ; 29(4): 710-9, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17080620

RESUMEN

Low N availability induces carbohydrate accumulation in leaf cells, which often causes suppression of photosynthesis. Under low N supply, excess carbohydrates would be preferentially respired by the non-phosphorylating pathways, such as the alternative oxidase (AOX) and uncoupling protein (UCP), which would suppress the excessive increase in the ratio of C to N (C/N ratio). In leaves, however, responses of these pathways to the low N stress are still unknown. We examined the mitochondrial respiratory pathways in spinach leaves grown at three different N availabilities to clarify whether the respiratory pathways change depending on the N availabilities. With the decrease in N availability, leaf respiratory rates per leaf area decreased, but the rates on the leaf N basis were comparable. Using fumarase activities of whole leaf extracts and isolated mitochondria, we estimated mitochondrial protein contents per leaf N. The contents increased with the decrease in the N availability, that is, at the low N availability, N was preferentially invested into mitochondria. On the mitochondrial protein basis, capacities of cytochrome pathway (CP) and cytochrome c oxidase (COX) were comparable regardless of the N availabilities, whereas both AOX capacity and the amounts of AOX protein increased with the decrease in the N availability. Some enzymes of tricarboxylic acid (TCA) cycle, especially NAD-dependent malic enzyme (NAD-ME), showed higher capacities under lower N. On the other hand, amounts of UCP did not differ amongst the N availabilities. These results indicated that, under low N stress, AOX will be preferentially up-regulated and will efficiently consume excess carbohydrates, which leads to suppressing the rise in the C/N ratio to a moderate level.


Asunto(s)
Mitocondrias/metabolismo , Nitrógeno/metabolismo , Spinacia oleracea/metabolismo , Metabolismo de los Hidratos de Carbono , Carbono/metabolismo , Ciclo del Ácido Cítrico/fisiología , Transporte de Electrón/fisiología , Complejo IV de Transporte de Electrones/metabolismo , Fumarato Hidratasa/metabolismo , Inmunohistoquímica , Canales Iónicos/análisis , Canales Iónicos/metabolismo , Mitocondrias/fisiología , Proteínas Mitocondriales/análisis , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/análisis , Oxidorreductasas/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Extractos Vegetales/química , Hojas de la Planta/anatomía & histología , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Proteínas de Plantas/metabolismo , Spinacia oleracea/anatomía & histología , Spinacia oleracea/fisiología , Proteína Desacopladora 1
12.
J Inherit Metab Dis ; 28(5): 799-800, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16151915

RESUMEN

A fumarase-deficient patient expressed a novel phenotype of congenital cerebral ventricular dilatation and periventricular cysts. The patient was a compound heterozygote for two mutations that are the only ones among the 12 published mutations that have been found in multiple, unrelated, fumarase-deficient patients.


Asunto(s)
Quistes/diagnóstico , Fumarato Hidratasa/deficiencia , Errores Innatos del Metabolismo/diagnóstico , Encéfalo/patología , Análisis Mutacional de ADN , ADN Complementario/metabolismo , Electroencefalografía , Resultado Fatal , Femenino , Fumarato Hidratasa/genética , Fumaratos/sangre , Fumaratos/orina , Heterocigoto , Histidina/química , Humanos , Recién Nacido , Ácidos Cetoglutáricos/química , Leucomalacia Periventricular/metabolismo , Lisina/química , Errores Innatos del Metabolismo/complicaciones , Ácido Metilmalónico/química , Mutación , Fenotipo , Ácido Pirúvico/química , Ultrasonografía
13.
Proc Soc Exp Biol Med ; 221(2): 147-52, 1999 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10352126

RESUMEN

Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I.


Asunto(s)
Cobre/deficiencia , Estrés Oxidativo , Cobre/farmacología , Deficiencia de Citocromo-c Oxidasa , Complejo II de Transporte de Electrones , Complejo III de Transporte de Electrones/metabolismo , Fumarato Hidratasa/metabolismo , Células HL-60 , Humanos , Mitocondrias/metabolismo , Complejos Multienzimáticos/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Oxidorreductasas/metabolismo , Fenilhidrazinas , Proteínas/análisis , Succinato Deshidrogenasa/metabolismo , Superóxido Dismutasa/análisis
14.
Arch Biochem Biophys ; 348(1): 65-74, 1997 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-9390175

RESUMEN

A cDNA EST clone encoding the C-terminal portion of Arabidopsis thaliana fumarase was identified by homology analysis. A fragment of cDNA encoding the N-terminal region of fumarase was amplified from a cDNA library using PCR and cloned. Genomic DNA corresponding to the coding region of fumarase was amplified and cloned. Arabidopsis fumarase was expressed as a chimeric fusion protein and polyclonal antibodies were generated. Fumarase was purified to near-homogeneity (over 600-fold) from etiolated Pisum sativum mitochondria. The identification of fumarase was confirmed by a combination of immunoblot and N-terminal amino acid sequencing. Kinetic analysis of highly purified fumarase yielded a KM(malate) of 0.45 mM and a Vmax(malate) of 650 mumol of fumarate/min/ mg. The pea fumarase was inhibited by the alpha-keto acids pyruvate and alpha-ketoglutarate at low millimolar concentrations. Adenylates were highly inhibitory; the degree of this inhibition was reduced in the presence of Mg2+, suggesting that uncomplexed adenylates are the inhibitory species.


Asunto(s)
Arabidopsis/enzimología , Fumarato Hidratasa/metabolismo , Genes de Plantas , Pisum sativum/enzimología , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Secuencia de Bases , Clonación Molecular , Escherichia coli/enzimología , Fumarato Hidratasa/química , Fumarato Hidratasa/aislamiento & purificación , Humanos , Cinética , Mitocondrias/enzimología , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Solanum tuberosum , Porcinos
15.
Lipids ; 32(1): 7-12, 1997 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9075187

RESUMEN

The temporal distribution of ATP/citrate lyase (ACL) activity in developing seeds of Brassica napus L. closely paralleled both that of acetyl-CoA carboxylase and the overall rate of lipid biosynthesis. Maximum ACL activities (250 nmol acetyl-CoA formed min-1.g fresh seed) were recorded between 35 to 42 d after pollination and, if the in vitro data could be extrapolated to the situation in vivo, could account for half of the acetyl-CoA required for the measured rate of fatty acid biosynthesis during seed development. The enzyme appeared to be localized in a subcellular compartment, which was clearly separated from mitochondria on a sucrose gradient and by differential centrifugation, and which corresponded to the chloroplast organelle.


Asunto(s)
ATP Citrato (pro-S)-Liasa/metabolismo , Brassica/enzimología , Metabolismo de los Lípidos , Acetatos/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Fraccionamiento Celular , Centrifugación por Gradiente de Densidad , Cloroplastos/enzimología , Cloroplastos/metabolismo , Ácido Cítrico/metabolismo , Citosol/enzimología , Citosol/metabolismo , Fumarato Hidratasa/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , Concentración de Iones de Hidrógeno , Lípidos/biosíntesis , Malato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Mitocondrias/metabolismo , Aceites de Plantas/metabolismo , Semillas/enzimología , Semillas/metabolismo
16.
Plant Physiol ; 112(3): 1219-27, 1996 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-8938419

RESUMEN

The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells.


Asunto(s)
Fumarato Hidratasa/biosíntesis , Fumarato Hidratasa/química , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Escherichia coli , Humanos , Hígado/enzimología , Mitocondrias/enzimología , Datos de Secuencia Molecular , Peso Molecular , Hojas de la Planta , Raíces de Plantas , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Transcripción Genética
17.
Arch Biochem Biophys ; 321(1): 153-9, 1995 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-7639515

RESUMEN

The potential of fumarate reductase as a therapeutic target against the human pathogen Helicobacter pylori was investigated by studying the cytotoxicity of morantel, oxantel, and thiabendazole, known to inhibit the enzyme in parasitic worms. Nuclear magnetic resonance spectroscopy was employed to investigate the effects of the inhibitors on the fumarate reductase activity of laboratory-adapted and wild-type bacterial strains. Production of succinate from fumarate in H. pylori cells was inhibited by morantel, oxantel, and thiabendazole. Cell growth and viability techniques were used to examine the bacteriostatic and bactericidal effects of the three anthelmintics. Each of the antiparasites arrested growth and produced cell death in liquid cultures, although the minimal inhibitory and bactericidal concentrations of these compounds are such that they would not be of therapeutic use. The strength of the effects as measured by minimal inhibitory and bactericidal concentrations was oxantel > thiabendazole > morantel. The findings suggested that fumarate reductase is an essential component of the metabolism of H. pylori and as such constitutes a possible target for therapeutic intervention in the treatment of the bacterium.


Asunto(s)
Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Morantel/toxicidad , Pirantel/análogos & derivados , Succinato Deshidrogenasa/antagonistas & inhibidores , Tiabendazol/toxicidad , Aconitato Hidratasa/metabolismo , Antibacterianos/toxicidad , Fumarato Hidratasa/metabolismo , Helicobacter pylori/enzimología , Helicobacter pylori/crecimiento & desarrollo , Humanos , Cinética , Malato Deshidrogenasa/metabolismo , Pruebas de Sensibilidad Microbiana , Morantel/uso terapéutico , Pirantel/uso terapéutico , Pirantel/toxicidad , Tiabendazol/uso terapéutico
18.
Ann Nutr Metab ; 34(2): 93-7, 1990.
Artículo en Inglés | MEDLINE | ID: mdl-2164346

RESUMEN

Two groups (n = 5) of male weanling Wistar rats were housed individually and fed copper (Cu)-deficient (0.5 mg Cu/kg) diets either with or without methionine supplementation (18 g/kg) for 49 days. Plasma caeruloplasmin (EC 1.16.3.1) and erythrocyte superoxide dismutase (EC 1.15.1.1, CuSOD) activities were measured in blood. Tissue Cu levels and the activities of cytochrome c oxidase (EC 1.9.3.1, CCO) and CuSOD were measured in the heart and liver. Hepatic activities of the sulfhydryl-sensitive enzymes, creatine kinase (EC 2.7.3.2), fumarase (EC 4.2.1.2) glutathione S-transferase (EC 2.5.1.18) and lipoamide dehydrogenase (EC 1.6.4.3) were also measured. Apart from cardiac CCO activity all of the measured indices of Cu status were found to be significantly (p less than 0.05) decreased in the methionine supplemented rats. Although fumarase activity was significantly (p less than 0.05) decreased in the methionine-supplemented animals compared with controls, the activities of the other sulfhydryl-sensitive enzymes were not significantly decreased. These results suggest that some of the toxic effects of excess dietary methionine may be mediated through interference with copper metabolism rather than through the previously postulated inhibition of sulfhydryl-sensitive enzymes by metabolites of methionine.


Asunto(s)
Cobre/farmacocinética , Complejo IV de Transporte de Electrones/metabolismo , Hígado/metabolismo , Metionina/farmacología , Miocardio/metabolismo , Animales , Cobre/análisis , Cobre/deficiencia , Creatina Quinasa/análisis , Creatina Quinasa/metabolismo , Dihidrolipoamida Deshidrogenasa/análisis , Dihidrolipoamida Deshidrogenasa/metabolismo , Complejo IV de Transporte de Electrones/análisis , Fumarato Hidratasa/análisis , Fumarato Hidratasa/metabolismo , Glutatión Transferasa/análisis , Glutatión Transferasa/metabolismo , Corazón/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Metionina/administración & dosificación , Metionina/toxicidad , Miocardio/enzimología , Estado Nutricional , Ratas , Ratas Endogámicas , Compuestos de Sulfhidrilo
19.
Ann Neurol ; 13(1): 72-8, 1983 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-6219611

RESUMEN

The activity of the pyruvate dehydrogenase complex (PDHC) was reduced in affected areas of brain from patients with Huntington disease (caudate, putamen) and Alzheimer disease (frontal cortex) where choline acetyltransferase (CAT) activity was low. PDHC was also deficient in an area (Huntington hippocampus) where CAT was not significantly reduced. The activity of fumarase, an inner mitochondrial marker, was normal in all areas examined. The activities of PDHC and CAT correlated well in caudate, putamen, and amygdala but not in hippocampus or frontal cortex. Both total activity and activation of PDHC were below normal in fibroblasts from 4 patients with C-21 trisomy Down syndrome, who are at very high risk to develop Alzheimer disease. However, no abnormality of PDHC was detected in Huntington or Alzheimer fibroblasts. Deficiency of PDHC may play a role in the pathophysiology of Huntington and Alzheimer diseases, although it does not appear to be a primary defect. Loss of tissue oxidative capacity may relate to the reduction in cerebral metabolic rate and blood flow which are characteristic of many dementing illnesses.


Asunto(s)
Enfermedad de Alzheimer/enzimología , Demencia/enzimología , Enfermedad de Huntington/enzimología , Complejo Piruvato Deshidrogenasa/metabolismo , Amígdala del Cerebelo/enzimología , Núcleo Caudado/enzimología , Colina O-Acetiltransferasa/metabolismo , Lóbulo Frontal/enzimología , Fumarato Hidratasa/metabolismo , Hipocampo/enzimología , Humanos , Putamen/enzimología
20.
Brain Res ; 101(1): 103-11, 1976 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-1104081

RESUMEN

A flotation method for preparing synaptosomes, previously developed for work with squid nervous tissue, has now been successfully applied to the ventral nerve cord of lobster. Perhaps due to the greater content of connective tissue, homogenization of the lobster nerve cord was more difficult than with squid optic lobes and the yield of synaptosomes was lower. The synaptosomes fraction showed a 3.8-fold enrichment of bound acetylcholine relative to the homogenate and was almost 10 times richer in acetylcholine than a guinea pig cerebral cortical synaptosome fraction. The lobster synaptosomes accumulated choline rapidly when incubated at room temperature in sea water, and showed a high degree of occlusion of lactate dehydrogenase, thus confirming that they are sealed structures. The lobster can thus be added to the wide range of species from whose nervous systems synaptosomes can be isolated, and merits further study as a possibly rich source of cholinergic synaptosomes.


Asunto(s)
Sistema Nervioso Central/metabolismo , Técnicas Histológicas , Nephropidae/anatomía & histología , Sinaptosomas/metabolismo , Acetilcolina/metabolismo , Animales , Colina/metabolismo , Fumarato Hidratasa/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Fracciones Subcelulares/metabolismo , Sinaptosomas/enzimología , Sinaptosomas/ultraestructura
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA