RESUMEN

NADH:quinone oxidoreductases (NQOs) act as the electron entry sites in bacterial respiration and oxidize intracellular NADH that is essential for the synthesis of numerous molecules. Klebsiella pneumoniae contains three NQOs (NDH-1, NDH-2, and NQR). The effects of inactivating these NQOs, separately and together, on cell metabolism were investigated under different culture conditions. Defective growth was evident in NDH-1-NDH-2 double and NDH-1-NDH-2-NQR triple deficient mutants, which was probably due to damage to the respiratory chain. The results also showed that K. pneumoniae can flexibly use NQOs to maintain normal growth in single NQO-deficient mutants. And more interestingly, under aerobic conditions, inactivating NDH-1 resulted in a high intracellular NADH:NAD+ ratio, which was proven to be beneficial for 2,3-butanediol production. Compared with the parent strain, 2,3-butanediol production by the NDH-1-deficient mutant was increased by 46% and 62% in glycerol- and glucose-based media, respectively. Thus, our findings provide a practical strategy for metabolic engineering of respiratory chains to promote the biosynthesis of 2,3-butanediol in K. pneumoniae.

Asunto(s)

Klebsiella pneumoniae/crecimiento & desarrollo , Klebsiella pneumoniae/metabolismo , Ingeniería Metabólica , Quinona Reductasas/antagonistas & inhibidores , Quinona Reductasas/deficiencia , Butileno Glicoles/química , Butileno Glicoles/metabolismo , Klebsiella pneumoniae/citología , Klebsiella pneumoniae/enzimología , Quinona Reductasas/genética , Quinona Reductasas/metabolismo

RESUMEN

The cytosolic quinone oxidoreductases NQO1 and NQO2 protect cells against oxidative stress by detoxifying quinones and preventing redox cycling. In this study, we used double knockout (DKO) mice deficient for NQO1 and NQO2 to investigate the role of these antioxidative enzymes in a two-stage model of inflammatory skin carcinogenesis. In this model, tumors are caused by exposure to topical carcinogen dimethylbenz(a)anthracene or benzo(a)pyrene (BP) followed by twice weekly application of proinflammatory phorbol 12-myristate 13-acetate. On this classic chemical carcinogenesis protocol, DKO mice showed a significantly higher skin tumor frequency and multiplicity compared with control wild-type or single knockout mice. Analysis of skin from wild-type and DKO mice exposed to BP for 6, 12, or 24 hours revealed a relative delay in the activation of p53, p63, p19ARF, and apoptosis in DKO mice, consistent with a negative modifier role for NQO1/NQO2 in carcinogenesis. Our findings offer genetic evidence of the significance of quinone oxidoreductases NQO1 and NQO2 in limiting chemical skin carcinogenesis.

Asunto(s)

NAD(P)H Deshidrogenasa (Quinona)/genética , Quinona Reductasas/genética , Neoplasias Cutáneas/genética , Animales , Apoptosis/efectos de los fármacos , Benzo(a)Antracenos , Benzo(a)pireno , Western Blotting , Caspasa 3/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NAD(P)H Deshidrogenasa (Quinona)/deficiencia , Ornitina Descarboxilasa/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Quinona Reductasas/deficiencia , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic flavoprotein that catalyzes the two-electron reduction of quinones and quinoid compounds to hydroquinones. Although the role of a homologue, NAD(P)H:quinone oxidoreductase 1 (NQO1), is well defined in oxidative stress, neoplasia, and carcinogenesis, little is known about the mechanism of actions of NQO2 in these cellular responses. Whether NQO2 has any role in tumor necrosis factor (TNF) signaling was investigated using keratinocytes derived from wild-type and NQO2 knockout (NQO2-/-) mice. Although exposure of wild-type cells to TNF led to activation of nuclear factor-kappaB (NF-kappaB) and IkappaBalpha kinase, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation, this cytokine had no effect on NQO2-/- cells. Deletion of NQO2 also abolished TNF-induced c-Jun NH2-terminal kinase, Akt, p38, and p44/p42 mitogen-activated protein kinase activation. The induction of various antiapoptotic gene products (MMP-9, cyclin D1, COX-2, IAP1, IAP2, Bcl-2, cFLIP, and XIAP) by TNF was also abolished in NQO2-/- cells. This correlated with potentiation of TNF-induced apoptosis as indicated by cell viability, Annexin V staining, and caspase activation. In agreement with this, we also found that TNF activated NQO2, and NQO2-specific small interfering RNA abrogated the TNF-induced NQO2 activity and NF-kappaB activation. Overall, our results indicate that deletion of NQO2 plays a differential role in TNF signaling pathway: by suppressing cell survival signals and potentiating TNF-induced apoptosis.

Asunto(s)

Regulación Enzimológica de la Expresión Génica , Queratinocitos/enzimología , Quinona Reductasas/deficiencia , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Núcleo Celular/metabolismo , Ciclina D1/biosíntesis , Ciclooxigenasa 2/biosíntesis , Activación Enzimática/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Queratinocitos/fisiología , MAP Quinasa Quinasa 4/metabolismo , Metaloproteinasa 9 de la Matriz/biosíntesis , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinona Reductasas/genética , Quinona Reductasas/metabolismo , ARN Interferente Pequeño/genética , Neoplasias Cutáneas/patología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

NAD(P)H/NRH:quinone oxidoreductases (NQO1 and NQO2) protect against oxidative stress and neoplasia. Cross-breeding of NQO1-/- with NQO2-/- mice generated double-knockout (DKO) mice. DKO mice were born normal yet showed myelogenous hyperplasia as observed in single-knockout mice. DKO mice also showed bronchial-associated lymphoid tissue (BALT) that increased in number and size with age. BALT was absent in wild-type and single-knockout mice. Further analysis demonstrated infiltration of neutrophils and macrophages in BALT and significant increases in the serum cytokines TNFalpha, IL-6, and IL-1beta and increased expression of iNOS and higher nitric oxide in lung macrophages. The development of BALT in DKO mice presumably led to the release of cytokines and higher lung macrophage activation, because histologically spleen, thymus, and blood cultures and urine analysis showed absence of infection. Additionally, the DKO mice upon exposure to hyperoxia demonstrated severe intra-alveolar edema and perivascular inflammation and massive infiltration with neutrophils, compared with wild-type mice. These results suggest that NQO1 and NQO2 combined protect mice against lung inflammation, BALT, and hyperoxic lung injury.

Asunto(s)

Hiperoxia/inmunología , Pulmón/inmunología , Tejido Linfoide/inmunología , Ratones Noqueados , NADPH Deshidrogenasa/deficiencia , Quinona Reductasas/deficiencia , Animales , Northern Blotting , Western Blotting , Citocinas/sangre , Electroforesis en Gel de Poliacrilamida , Femenino , Citometría de Flujo , Hiperoxia/genética , Inmunohistoquímica , Lesión Pulmonar , Macrófagos/inmunología , Masculino , Ratones , Trastornos Mieloproliferativos/genética , NAD(P)H Deshidrogenasa (Quinona) , Reacción en Cadena de la Polimerasa

RESUMEN

The ND4L subunit is the smallest mitochondrial DNA-encoded subunit of the proton-translocating NADH-quinone oxidoreductase (complex I). In an attempt to study the functional and structural roles of the NuoK subunit (the Escherichia coli homologue of ND4L) of the bacterial NADH-quinone oxidoreductase (NDH-1), we have performed a series of site-specific mutations on the nuoK gene of the NDH-1 operon by using the homologous recombination technique. The amino acid residues we targeted included two highly conserved glutamic acids that are presumably located in the middle of the membrane and several arginine residues that are predicted to be on the cytosolic side. All point mutants examined had fully assembled NDH-1 as detected by blue-native gel electrophoresis and immunostaining. Mutations of nearly perfectly conserved Glu-36 lead to almost null activities of coupled electron transfer with a concomitant loss of generation of electrochemical gradient. A significant diminution of the coupled activities was also observed with mutations of another highly conserved residue, Glu-72. These results may suggest that both membrane-embedded acidic residues are important for the coupling mechanism of NDH-1. Furthermore, a severe impairment of the coupled activities occurred when two vicinal arginine residues on a cytosolic loop were simultaneously mutated. Possible roles of these arginine residues and other conserved residues in the NuoK subunit for NDH-1 function were discussed.

Asunto(s)

Proteínas de Escherichia coli/química , Proteínas de la Membrana/química , NADH Deshidrogenasa/química , Subunidades de Proteína/química , Quinona Reductasas/química , Secuencia de Aminoácidos , Secuencia Conservada , Complejo I de Transporte de Electrón/química , Proteínas de Escherichia coli/genética , Ácido Glutámico/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADH Deshidrogenasa/deficiencia , NADH Deshidrogenasa/genética , Mutación Puntual , Potenciometría , Procesamiento Proteico-Postraduccional/genética , Estructura Terciaria de Proteína/genética , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , Quinona Reductasas/deficiencia , Quinona Reductasas/genética , Análisis de Secuencia de Proteína

RESUMEN

Quinone oxidoreductases (NQO1 and NQO2) are cytosolic proteins that catalyze metabolic reduction of quinones and its derivatives to protect cells against redox cycling and oxidative stress. In humans, a high percentage of individuals with myeloid and other types of leukemia are homo- and heterozygous for a null mutant allele of NQO1. The NQO2 locus is also highly polymorphic in humans. Recently, we generated NQO1-/- and NQO2-/- mice deficient in NQO1 and NQO2 protein and activity, respectively. These mice showed no detectable developmental abnormalities and were indistinguishable from wild type mice. Interestingly, all the mice lacking expression of NQO1 and NQO2 protein demonstrated myelogenous hyperplasia of the bone marrow and increased granulocytes in the peripheral blood. Decreased apoptosis contributed to myelogenous hyperplasia. The studies on short-term exposure of NQO1-/- mice to benzene demonstrated substantially greater benzene-induced toxicity, as compared to wild type mice.

Asunto(s)

Benceno/toxicidad , Médula Ósea/patología , Trastornos Mieloproliferativos/enzimología , NAD(P)H Deshidrogenasa (Quinona)/genética , Quinona Reductasas/genética , Animales , Médula Ósea/enzimología , Predisposición Genética a la Enfermedad , Humanos , Hiperplasia/inducido químicamente , Hiperplasia/enzimología , Hiperplasia/genética , Leucemia/inducido químicamente , Leucemia/enzimología , Leucemia/genética , Ratones , Ratones Noqueados , Trastornos Mieloproliferativos/inducido químicamente , Trastornos Mieloproliferativos/genética , NAD(P)H Deshidrogenasa (Quinona)/deficiencia , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Polimorfismo Genético , Quinona Reductasas/deficiencia , Quinona Reductasas/metabolismo

RESUMEN

NRH:Quinone oxidoreductase 2 (NQO2) is an enzyme that catalyzes the reductive metabolism of quinones. C57BL/6 NQO2-/- mice lacking NQO2 gene expression were generated in our laboratory. The dorsal skin of NQO2-deficient mice was exposed to 7,12-dimethylbenz(a)anthracene (DMBA) or benzo(a)pyrene alone (complete carcinogen) or with 12-O-tetradecanoylphorbol-13-acetate (TPA) (initiation/promotion model) to determine the in vivo role of NQO2 in chemical carcinogenesis. The NQO2-/- mice showed significantly increased tumor frequency with DMBA + TPA when compared with their wild-type littermates. The benzo(a)pyrene + TPA also showed increase in tumor incidence in NQO2-/- mice but to a less extent than DMBA. DMBA alone resulted in low frequency of tumor development with no difference in susceptibility between wild-type and NQO2-/- mice. Benzo(a)pyrene alone failed to induce tumors in either wild-type or NQO2-/- mice. Histologic analysis of the NQO2-/- mice tumors demonstrated proliferative activity. The treatment of NQO2-/- mice skin with benzo(a)pyrene failed to significantly increase tumor suppressor protein p53 and p53-regulated growth-related protein p21 and proapoptotic protein Bax as observed in case of wild-type mice. These results demonstrate that NQO2 protects against DMBA- and benzo(a)pyrene-induced skin carcinogenesis and suggest that NQO2 protection might be against tumor promotion. The results also suggest that lack of induction of p53, p21, and Bax proteins might contribute to increased sensitivity of NQO2-/- mice skin to benzo(a)pyrene carcinogenicity.

Asunto(s)

Cocarcinogénesis , Proteínas Proto-Oncogénicas c-bcl-2 , Quinona Reductasas/deficiencia , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/enzimología , Animales , Benzo(a)Antracenos , Benzo(a)pireno , Carcinógenos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Susceptibilidad a Enfermedades , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/biosíntesis , Quinona Reductasas/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis , Proteína X Asociada a bcl-2

RESUMEN

BACKGROUND: The phase II enzyme NAD(P)H :quinone oxidoreductase 1 (NQO1) catalyzes quinone detoxification, protecting cells from redox cycling, oxidative stress, mutagenicity, and cytotoxicity induced by quinones and its precursors. We have used NQO1(-/-) C57BL/6 mice to show that NQO1 protects them from skin cancer induced by the polycyclic aromatic hydrocarbon benzo[a]pyrene. Herein, we used NQO1(-/-) mice to investigate whether NQO1 also protects them against 7,12-dimethylbenz[a]anthracene (DMBA), where methyl substituents diminish primary quinone formation. METHODS: Dorsal skin of NQO1(-/-) or wild-type C57BL/6 mice was shaved. When tested as a complete carcinogen, DMBA (500 or 750 microg in 100 microL of acetone) alone was applied to the shaved area. When tested as a tumor initiator, DMBA (200 or 400 nmol in 100 microL of acetone) was applied to the shaved area; 1 week later, twice-weekly applications of phorbol 12-myristate 13-acetate (PMA)-10 microg dissolved in 200 microL of acetone-to the same area began and were continued for 20 weeks. Tumor development was monitored in all mice (12-15 per group). All statistical tests were two-sided. RESULTS: When DMBA (750 microg) was tested as a complete carcinogen, about 50% of the DMBA-treated NQO1(-/-) mice but no DMBA-treated wild-type mouse developed skin tumors. When DMBA (both concentrations) was used as a tumor initiator, NQO1(-/-) mice developed larger tumors at a greater frequency than their wild-type littermates. Twenty-three weeks after the first PMA treatment in the tumor initiator test, all 30 NQO1(-/-) mice given 400 nmol of DMBA had developed skin tumors, compared with 33% (10 of 30) of treated wild-type mice (P<.001). CONCLUSIONS: NQO1(-/-) mice are more susceptible to DMBA-induced skin cancer than are their wild-type littermates, suggesting that NQO1 may protect cells from DMBA carcinogenesis.

Asunto(s)

Quinona Reductasas/deficiencia , Neoplasias Cutáneas/enzimología , 9,10-Dimetil-1,2-benzantraceno , Animales , Carcinógenos , Susceptibilidad a Enfermedades/enzimología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Cutáneas/inducido químicamente

RESUMEN

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme that detoxifies quinones and reduces oxidative stress. A cysteine-to-threonine (C --> T) substitution polymorphism at nucleotide 609 of the NQO1 complementary DNA (NQO1 C609T) results in a lowering of NQO1 activity. Individuals homozygous for this mutation have no NQO1 activity, and heterozygotes have low to intermediate activity compared with people with wild type. DNA samples from 493 adult de novo acute leukemia patients and 838 matched controls were genotyped for NQO1 C609T. The majority of cases were diagnosed as acute myeloid leukemia (AML) (n = 420); 67 as acute lymphoblastic leukemia (ALL); and 6 as other forms of acute leukemia. The frequency of cases with low or null NQO1 activity (heterozygote + homozygous mutant) was significantly higher among total acute leukemia case subjects compared with their matched controls (odds ratio [OR] = 1.49; 95% confidence interval [CI], 1.17-1.89). Both ALL (OR = 1.93; 95% CI, 0.96-3.87) and AML case subjects (OR = 1.47; 95% CI, 1.13-1.90) exhibited a higher frequency of low or null NQO1 genotypes than controls. For de novo AML, the most significant effect of low or null NQO1 activity was observed among the 88 cases harboring translocations and inversions (OR = 2.39; 95% CI, 1.34-4.27) and was especially high for those harboring inv(16) (OR = 8.13; 95% CI, 1.43-46.42). These findings were confirmed in a second group of 217 de novo AML cases with known cytogenetics. Thus, inheritance of NQO1 C609T confers an increased risk of de novo acute leukemia in adults, implicating quinones and related compounds that generate oxidative stress in producing acute leukemia.

Asunto(s)

Leucemia/enzimología , Leucemia/epidemiología , Quinona Reductasas/genética , Enfermedad Aguda , Adulto , Anciano , Sustitución de Aminoácidos , Estudios de Casos y Controles , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Citogenética , Femenino , Frecuencia de los Genes , Pruebas Genéticas , Genotipo , Humanos , Leucemia/etiología , Masculino , Persona de Mediana Edad , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Quinona Reductasas/deficiencia , Factores de Riesgo

RESUMEN

The first component of the mitochondrial electron-transport chain is especially complex, consisting of 19 nuclear and seven mitochondrion-encoded subunits. Accordingly, a wide range of clinical manifestations are produced by the various mutations occurring in human populations. In this study, we analyze the subunit structure of complex I in fibroblasts from two patients who have distinct clinical phenotypes associated with complex I deficiency. The first patient died in the second week of life from overwhelming lactic acidosis. Severe complex I deficiency was evident in her fibroblasts, since alanine oxidation was markedly reduced whereas succinate oxidation was normal. Absence of a 20-kDa subunit was demonstrable when newly synthesized proteins were immunoprecipitated from pulse-labeled fibroblasts by anti-complex I antibody. Disordered assembly or decreased stability of the complex was suggested by deficiency of multiple subunits on Western immunoblots. The second patient exhibited a milder clinical phenotype, characterized by moderate lactic acidosis and developmental delay in childhood and by onset of seizures at 8 years of age. Oxidation studies demonstrated expression of the complex I deficiency in fibroblasts, but no subunit abnormalities were detected by immunoprecipitation or Western immunoblotting. This report demonstrates the utility of cultured fibroblasts in studying mutations affecting synthesis and assembly of complex I.

Asunto(s)

Mitocondrias/química , Quinona Reductasas/deficiencia , Alanina/química , Southern Blotting , Niño , ADN/análisis , Fibroblastos/química , Humanos , Recién Nacido , Peso Molecular , NAD(P)H Deshidrogenasa (Quinona) , Oxidación-Reducción , Fenotipo , Pruebas de Precipitina , Succinatos/química , Ácido Succínico

RESUMEN

A 24-year-old male had a deficiency of the complex I (NADH coenzyme-Q-reductase) of the mitochondrial respiratory chain, which clinically presented as a mitochondrial encephalomyopathy, with lactic acidosis and stroke-like episodes (MELAS syndrome). The encephalopathic episodes were preceded by migraine and were characterized by focal deficit signs, motor partial seizures and hypodense areas in the CT scan. An echocardiographic diagnosis of hypertrophic cardiomyopathy without intracavitary thrombi was made. It is suggested that hypertrophic cardiomyopathy is caused by the mitochondrial abnormalities that have been reported in the myocardium, and that migraine and cerebral infarctions are associated with abnormalities in the mitochondria from the endothelium and smooth muscle fibres of the cerebral small arteries and arterioles.

Asunto(s)

Encefalopatías/enzimología , Cardiomiopatía Hipertrófica/enzimología , Enfermedades Musculares/enzimología , Quinona Reductasas/deficiencia , Acidosis Láctica/enzimología , Adulto , Trastornos Cerebrovasculares/etiología , Enfermedades Carenciales/complicaciones , Enfermedades Carenciales/diagnóstico , Humanos , Masculino , Mitocondrias Musculares/enzimología , Enfermedades Musculares/patología , NAD(P)H Deshidrogenasa (Quinona) , Síndrome

Asunto(s)

Mitocondrias/patología , Enfermedades Musculares/patología , Acidosis Láctica/genética , Acidosis Láctica/patología , Trastornos Cerebrovasculares/genética , Trastornos Cerebrovasculares/patología , Deficiencia de Citocromo-c Oxidasa , ADN Mitocondrial/genética , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/patología , Humanos , Mitocondrias/metabolismo , Enfermedades Musculares/genética , NAD(P)H Deshidrogenasa (Quinona) , Oftalmoplejía/genética , Oftalmoplejía/patología , Quinona Reductasas/deficiencia

RESUMEN

Two major lines of evidence support the hypothesis that an impairment of mitochondrial function may underlie neuronal death in Parkinson's disease. First, the neurotoxicity of the parkinsonism-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is due to the generation of its 1-methyl-4-phenylpyridinium (MPP+) metabolite in the central nervous system; the toxicity of MPP+ is likely to result from its ability to block mitochondrial electron flow at the level of complex I. Second, recent studies have revealed a deficiency of mitochondrial complex I activity in the brain as well as other tissues of parkinsonian patients. This enzyme activity reduction might be explained by a defect in one or more of the genes coding for the subunits of complex I. Since seven of these genes are localized in the mitochondrial genome, it is conceivable that abnormal mitochondrial DNA (mtDNA) might play a role in the pathogenesis of Parkinson's disease. The entire sequence of the human mitochondrial genome is known, and human mtDNA can be isolated and rapidly analyzed using techniques such as the polymerase chain reaction. Therefore, identification of an easily detectable mtDNA alteration might ultimately be used as a marker for the diagnosis and screening of Parkinson's disease.

Asunto(s)

ADN Mitocondrial/fisiología , Enfermedad de Parkinson/etiología , Daño del ADN , Reparación del ADN , Biblioteca Genómica , Humanos , Mitocondrias/fisiología , Enfermedades Musculares/etiología , NAD(P)H Deshidrogenasa (Quinona) , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/fisiopatología , Quinona Reductasas/deficiencia

RESUMEN

We have previously reported the isolation of CHO cell lines resistant to mitomycin C under aerobic conditions of drug exposure. Here it is reported that these cell lines have the same response to mitomycin C under hypoxic conditions as do controls. The cells are shown to have lower levels of DT-diaphorase activity than controls, but similar levels of activity of NADPH:cytochrome c reductase, another enzyme involved in the metabolism of mitomycin C. Evidence for molecular defects in the DT-diaphorase gene or gene transcript is presented for the deficient cell lines. The consequences of this DT-diaphorase deficiency is further explored by testing the toxicity of menadione, an established enzyme substrate. The isolation of CHO cell lines deficient in DT-diaphorase activity and resistant to mitomycin C under aerobic but not hypoxic conditions suggests that mitomycin C reduction by this enzyme has a significant impact on cytotoxicity under aerobic but not hypoxic conditions. Similarly, DT-diaphorase metabolism of menadione does not appear to have a significant impact on cytotoxicity in CHO cells.

Asunto(s)

Mitomicinas/metabolismo , Quinona Reductasas/deficiencia , Animales , Southern Blotting , Hipoxia de la Célula , Línea Celular , Cricetinae , Cricetulus , ADN/análisis , Resistencia a Medicamentos , Genes , Mitomicina , Mitomicinas/toxicidad , Quinona Reductasas/genética

RESUMEN

We describe a 6-year-old boy who presented with progressive muscle weakness. Additional investigations revealed the existence of a myopathy and a pure motor neuropathy. Biochemical studies in muscle tissue showed a defect of NADH dehydrogenase (complex I). The patient dramatically improved on treatment with riboflavin and L-carnitine. Seven months after the start of the treatment, complex I activity was determined again and appeared to be normalized. Normalization of the enzymatic defect at this level has not been reported before. We provide a survey of nine patients with pure myopathy, associated with complex I deficiency and onset of symptoms in childhood.

Asunto(s)

Carnitina/uso terapéutico , Enfermedades Musculares/etiología , Quinona Reductasas/deficiencia , Riboflavina/uso terapéutico , Biopsia , Niño , Quimioterapia Combinada , Humanos , Masculino , Microscopía Electrónica , Músculos/patología , Músculos/ultraestructura , Enfermedades Musculares/tratamiento farmacológico , Enfermedades Musculares/patología , NAD(P)H Deshidrogenasa (Quinona)

RESUMEN

The functional consequences of large heteroplasmic mtDNA deletions were investigated in a group of 6 patients with chronic progressive external ophthalmoplegia (CPEO) syndromes. State III respiration rates corrected for age were low with site I and II substrates in all cases and cytochrome oxidase activity was depressed. The severity of impairment varied and is consistent with inclusion of a variable percentage of non-functioning mitochondria (with deleted mtDNA) in the pellet. Western blot studies with a holocomplex antibody battery revealed no abnormalities in subunit content of complexes III and IV. A deficiency of several complex I subunits in 3 cases suggests that abnormal nuclear-mitochondrial regulation of complex I assembly may follow large mtDNA deletions.

Asunto(s)

Deficiencia de Citocromo-c Oxidasa , ADN Mitocondrial/genética , Complejo III de Transporte de Electrones/deficiencia , Oftalmoplejía/metabolismo , Quinona Reductasas/deficiencia , Adulto , Deleción Cromosómica , Transporte de Electrón , Complejo III de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/genética , Femenino , Glutamatos/metabolismo , Ácido Glutámico , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , NAD(P)H Deshidrogenasa (Quinona) , Oftalmoplejía/genética , Fosforilación Oxidativa , Consumo de Oxígeno , Piruvatos/metabolismo , Ácido Pirúvico , Quinona Reductasas/genética , ARN de Transferencia/genética , Succinatos/metabolismo , Ácido Succínico

RESUMEN

We describe a young man with a progressive neurological disorder including myoclonus, mental retardation, muscle weakness and a mitochondrial myopathy (myoclonus epilepsy and ragged red fibres--MERRF). Multiple abnormalities of the mitochondrial respiratory chain in skeletal muscle are shown by direct measurement of the flux through the individual complexes, low-temperature redox spectroscopy and decreased immunodetectable subunits of complexes I and IV by immunoblotting. No abnormality of mitochondrial DNA was found. This is the first report of combined defects of complexes I, III and IV as a cause of this clinical syndrome. However, we propose that the occurrence of multiple respiratory chain defects may be more common than previously recognised and that this particular combination of defects, involving complexes I, III and IV, may be the predominant biochemical abnormality in MERRF.

Asunto(s)

Ataxia Cerebelosa/genética , Deficiencia de Citocromo-c Oxidasa , Complejo III de Transporte de Electrones/deficiencia , Epilepsias Mioclónicas/genética , Pérdida Auditiva Sensorineural/genética , Discapacidad Intelectual/genética , Mitocondrias/enzimología , Complejos Multienzimáticos/deficiencia , Enfermedades Neuromusculares/genética , Oxidorreductasas/deficiencia , Quinona Reductasas/deficiencia , Succinato Deshidrogenasa/deficiencia , Adulto , Ataxia Cerebelosa/enzimología , Preescolar , ADN Mitocondrial/análisis , Complejo II de Transporte de Electrones , Epilepsias Mioclónicas/enzimología , Pérdida Auditiva Sensorineural/enzimología , Humanos , Discapacidad Intelectual/enzimología , Masculino , NAD(P)H Deshidrogenasa (Quinona) , Enfermedades Neuromusculares/enzimología , Enfermedades Neuromusculares/patología , Fosforilación Oxidativa , Síndrome

RESUMEN

Neuropathological studies were carried out in two patients with mitochondrial encephalomyopathies in whom the underlying lesions in muscle mitochondrial DNA (mtDNA) and respiratory enzyme complexes have been investigated. The first, a man with Kearns-Sayre syndrome, died at the age of 49 years. Autopsy showed an old parietal lobe infarct, diffuse spongiform leukoencephalopathy of cerebral and cerebellar white matter and mild spongiform change in deep grey matter and brainstem nuclei. Heteroplasmy of skeletal muscle mitochondrial DNA with a 3.5 kb mtDNA deletion in one of two mtDNA populations was found. The second case, a woman, suffering from myoclonic epilepsy, cerebellar ataxia, bilateral sensorineural deafness, several 'stroke-like' episodes died at age 52. At autopsy, an old infarct was seen in the L internal capsule. Severe loss of neurons and gliosis were found in the dentate nuclei, moderate changes in the red nuclei and inferior olivary nuclei and mild changes in the substantial nigra and locus coeruleus. In both patients, skeletal muscle biopsy showed numbers of ragged-red fibres and intramitochondrial paracrystalline inclusions at electron microscopy. A defect in the synthesis of the ND5 subunit of the respiratory complex I was suggested in the second patient in whom a diagnosis of MELAS was made.

Asunto(s)

Ataxia Cerebelosa/genética , ADN Mitocondrial/genética , Epilepsias Mioclónicas/genética , Pérdida Auditiva Sensorineural/genética , Síndrome de Kearns-Sayre/genética , Enfermedades Neuromusculares/genética , Quinona Reductasas/deficiencia , Adulto , Encéfalo/patología , Ataxia Cerebelosa/patología , Deleción Cromosómica , Enfermedades en Gemelos , Epilepsias Mioclónicas/patología , Pérdida Auditiva Sensorineural/patología , Humanos , Síndrome de Kearns-Sayre/patología , Masculino , Mitocondrias Musculares/patología , NAD(P)H Deshidrogenasa (Quinona) , Enfermedades Neuromusculares/patología , Quinona Reductasas/genética , Síndrome

RESUMEN

The total sequences of mitochondrial DNA were determined in two patients with juvenile-onset mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) due to Complex I deficiency. Patients 1 and 2 had three and two unique point mutations, respectively, causing replacement of phylogenically conserved amino acids. A transition from G to A was found at nucleotide position 5601 in the alanine tRNA gene of Patient 2, and a transition from A to G was found at 3243 in the leucine (UUR) tRNA gene of both patients. The latter mutation located at the phylogenically conserved 5' end of the dihydrouridine loop of the tRNA molecule, and was present in two patients with adult-onset MELAS and absent in controls. These results indicate that a mass of mtDNA mutations including the A-to-G transition in the tRNA(Leu) gene is a genetic cause of MELAS.

Asunto(s)

ADN Mitocondrial/genética , Errores Innatos del Metabolismo/genética , Mitocondrias Musculares/enzimología , Mutación , Quinona Reductasas/genética , Adolescente , Secuencia de Aminoácidos , Composición de Base , Secuencia de Bases , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , NAD(P)H Deshidrogenasa (Quinona) , Conformación de Ácido Nucleico , Sondas de Oligonucleótidos , Quinona Reductasas/deficiencia , ARN de Transferencia de Leucina/genética , Síndrome

RESUMEN

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is thought to produce parkinsonism in humans and other primates through its inhibition of complex I. The recent discovery of mitochondrial complex I deficiency in the substantia nigra of patients with Parkinson's disease has provided a remarkable link between the idiopathic disease and the action of the neurotoxin MPTP. This article shows that complex I deficiency in Parkinson's disease is anatomically specific for the substantia nigra, and is not present in another neurodegenerative disorder involving the substantia nigra. Evidence is also provided to show that there is no correlation between L-3,4-dihydroxyphenylalanine therapy and complex I deficiency. These results suggest that complex I deficiency may be the underlying cause of dopaminergic cell death in Parkinson's disease.

Asunto(s)

Enfermedad de Parkinson/enzimología , Quinona Reductasas/deficiencia , Citrato (si)-Sintasa/metabolismo , Humanos , NAD(P)H Deshidrogenasa (Quinona) , Sustancia Negra/enzimología