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1.
Clin Neuropathol ; 27(6): 414-23, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19130740

RESUMEN

UNLABELLED: Leber's hereditary optic neuropathy (LHON) is a maternally inherited mitochondrial disorder, leading to a selective loss of retinal ganglion cells (RGC) and degeneration of the optic nerve, which results in severe visual impairment or even blindness. The primary causes are point mutations of the mitochondrial DNA (mtDNA), associated with aminoacid exchanges in complex I of the electron transport chain (ETC), which are thought to disturb oxidative ATP generation in the mitochondria. The major side effect of the antibiotic ethambutol, commonly used in tuberculosis therapy, is a retinopathy, which may lead to selective RGC loss, if not detected in an early stage. Moreover, LHON was reported to be elicited by ethambutol in some mutation carriers. OBJECTIVE: The present study intended to measure a possible synergism between mitochondrial dysfunction, caused by the most common LHON mutation (G11778A) and caused by ethambutol, which may lead to a higher cytotoxicity of the drug in LHON cells. MATERIAL: An NT2/D1 teratoma-derived LHON cybrid line and the parental cells. METHOD: Determination of ethambutol toxicity in both lines, using a microtiter tetrazolium assay, luminometric measurement of ATP/ADP ratios and determination of mtDNA copy numbers by Real-time PCR. RESULTS: Short-term ethambutol toxicity occurred only at micromolar concentrations, far beyond the estimated plasma peak concentrations of patients under antibiotic therapy. No significant difference occurred between both cell lines. The ATP/ADP ratios in the cybrids were surprisingly low, but showed no correlation with the mutational status of drug-treated cells. The mtDNA copy number of treated LHON and parental cells did not differ significantly. CONCLUSIONS: Ethambutol shows no synergism with the most common primary LHON mutation with respect to mitochondrial energy production or mtDNA replication in cybrid cells, although the issue of ATP decline should be further addressed in neuronally differentiated cybrids with complete OXPHOS dependency.


Asunto(s)
Antituberculosos/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Etambutol/administración & dosificación , Mutación/genética , Atrofia Óptica Hereditaria de Leber/tratamiento farmacológico , Atrofia Óptica Hereditaria de Leber/genética , Antituberculosos/efectos adversos , Técnicas de Cultivo de Célula , Línea Celular Tumoral , ADN Mitocondrial/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Etambutol/efectos adversos , Humanos , Modelos Neurológicos , Atrofia Óptica Hereditaria de Leber/patología , Fosforilación Oxidativa/efectos de los fármacos , Teratoma
2.
Clin Neuropathol ; 26(4): 164-8, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17702497

RESUMEN

UNLABELLED: The pathogenesis of the selective loss of dopaminergic neurons in idiopathic Parkinson's disease (PD) has not been understood up to now. Respiratory chain dysfunction and accumulation of mitochondrial DNA deletions to biochemically relevant levels have been observed in the dopaminergic neurons. However, respiratory chain defects have also been reported in other tissues, pointing to a generalized component of oxidative stress in PD. Recently, somatic point mutations in a narrow region of the complex I polypeptide ND5 (codons 120 - 150) were suggested to separate PD patients from age-matched controls, using frontal cortex homogenates. OBJECTIVE: The present study intended to analyze whether those recently described ND5 mutations may also generally occur in skeletal muscle tissue of PD patients, in which complex I dysfunction had been measured earlier with biochemical approaches. MATERIAL: Skeletal muscle biopsy samples of 5 PD individuals with a previously characterized biochemical complex I defect and of 5 age-matched controls were used. METHOD: DNA was extracted from the muscle samples. The relevant ND5 region was PCR-cloned using a high fidelity Pfu polymerase and a low number of PCR cycles (15). Amean number of 96 clones were randomly selected from the ampicillin plates and sequenced by the dye terminator method to allow the detection of low abundance mutations with a sensitivity around 1%. RESULTS: Mutations between codons 120 and 150 were only slightly more frequent in PD versus controls (60 versus 40% of samples affected), while this ratio had been 100 versus 12.5% in frontal cortex. CONCLUSIONS: In contrast to results reported for PD frontal cortex, low-level ND5 mutations between codons 120 and 150 do not accumulate severely in biochemically affected skeletal muscle samples of PD patients.


Asunto(s)
ADN Mitocondrial/genética , Complejo I de Transporte de Electrón/genética , Mitocondrias Musculares/genética , Proteínas Mitocondriales/genética , Músculo Esquelético/enzimología , Enfermedad de Parkinson/genética , Anciano , Estudios de Casos y Controles , Humanos , Persona de Mediana Edad , Mitocondrias Musculares/enzimología , Mutación , Enfermedad de Parkinson/enzimología , Valores de Referencia
3.
Neurobiol Dis ; 25(3): 536-44, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17169568

RESUMEN

The mechanism of retinal ganglion cell loss in Leber's hereditary optic neuropathy (LHON) is still uncertain, and a role of enhanced superoxide production by the mutant mitochondrial complex I has been hypothesized. In the present study, it was shown that LHON cybrids, carrying the np11778 mutation, became selectively more H(2)O(2) sensitive compared with the parental cell line only following short-term retinoic acid differentiation. They contained a decreased cellular glutathione pool (49%, p< or =0.05), despite 1.5-fold enhanced expression of the regulatory subunit of gamma-glutamylcysteine synthetase (p< or =0.05). This points to a reduction of the capacity to detoxify H(2)O(2) and to changes in thiol redox potential. The activity of the H(2)O(2) degrading enzyme glutathione peroxidase (GPx) and the activities of glutathione reductase (GR) and superoxide dismutase (SOD) were unaffected.


Asunto(s)
Antioxidantes/metabolismo , Complejo I de Transporte de Electrón/genética , Glutatión/metabolismo , Atrofia Óptica Hereditaria de Leber/genética , Atrofia Óptica Hereditaria de Leber/metabolismo , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Complejo I de Transporte de Electrón/metabolismo , Genotipo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Mitocondrias/enzimología , Mitocondrias/genética , Atrofia Óptica Hereditaria de Leber/patología , Oxidantes/farmacología , Mutación Puntual , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Teratoma , Tretinoina/farmacología
4.
Clin Neuropathol ; 24(4): 175-83, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16033134

RESUMEN

We investigated the effects of 2-methoxyestradiol (2-ME), a promising new antitumor agent, on viable cell number and nuclear morphology of malignant glioma cells (three human and one rat glioma cell lines) and analyzed the controversial role of death recepor 5 (DR5) upregulation in 2-ME induced apoptosis. Microtiter-tetrazolium (MTT) assays showed a significant reduction of viable cells after incubation with 2 microM and 20 microM 2-ME for 48 and 72 hours in all cultures. In the 20 microM concentration, there were even significant effects in the majority of shorter incubation periods. Hoechst 33258 stains showed a substantial amount of cells with nuclear fragmentation indicating a late stage of apoptosis after 20 microM 2-ME treatments of 24 hours and more. The role of the DR5-mediated extrinsic apoptotic pathway was further studied in the three human glioma cell lines; 50 ng/ml of the DR5 ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and 2 microM 2-ME showed no synergism, as determined by MTT assays. Real-time PCR revealed no significantly increased amount of DR5 mRNA, suggesting that receptor upregulation does not play a major role for 2-ME-induced apoptosis in glioma cells, in contrast to data for a breast cancer cell line in the literature.


Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Estradiol/análogos & derivados , Glioma/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral/metabolismo , 2-Metoxiestradiol , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Fragmentación del ADN/efectos de los fármacos , Sinergismo Farmacológico , Estradiol/uso terapéutico , Glioma/metabolismo , Humanos , Glicoproteínas de Membrana/uso terapéutico , ARN Mensajero/análisis , Ratas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/genética , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/uso terapéutico , Regulación hacia Arriba
5.
Urologe A ; 44(7): 780-4, 2005 Jul.
Artículo en Alemán | MEDLINE | ID: mdl-15952015

RESUMEN

Iatrogenic cavernous nerve lesions occurring during radical pelvic surgery often lead to irreversible erectile dysfunction. The nerve defects after excision of the neurovascular bundles must be reconstructed by interposition grafting to supply a permissive scaffold for oriented axonal regrowth. The use of autologous nerve grafts for the repair of human cavernous nerves during radical prostatectomy has been controversial regarding the limited success achieved with bilateral nerve grafting. Artificial nerve guides consisting of natural or synthetic materials have been successfully used for bridging peripheral nerve defects. The combination with Schwann cells, neurotrophic factors and extracellular matrix components has been shown to promote cavernous nerve regeneration.


Asunto(s)
Disfunción Eréctil/cirugía , Regeneración Tisular Dirigida/métodos , Regeneración Nerviosa , Procedimientos Neuroquirúrgicos/métodos , Pene/inervación , Células de Schwann/trasplante , Procedimientos Quirúrgicos Urológicos Masculinos/efectos adversos , Disfunción Eréctil/etiología , Humanos , Masculino , Guías de Práctica Clínica como Asunto , Pautas de la Práctica en Medicina , Recuperación de la Función , Resultado del Tratamiento
6.
Eur Urol ; 48(3): 372-8, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15964126

RESUMEN

OBJECTIVE: This article reviews novel restorative therapies for cavernous nerves that may be used to replace resected cavernous nerves at the time of pelvic surgery. METHODS: A literature-based presentation (Medline search) on current nerve replacement strategies was conducted with emphasis on neurobiological factors contributing to the restoration of erectile function after cavernous nerve injuries. RESULTS: A promising alternative to autologous nerve grafts for extending the length of successful nerve regeneration are artificial nerve guides. The addition of neurotrophic factors, extracellular matrix components and Schwann cells has been shown to promote cavernous nerve regeneration. Neurotrophic factors can be incorporated in the scaffold or can be supplied by cells seeded into the stroma. The regenerative capacity of these cells can be further enhanced by genetic modification with neurotrophic factor encoding genes. CONCLUSIONS: Artificial nerve guides, especially biodegradable ones containing growth-promoting factors or cells, are a promising option for the repair of cavernous nerve lesions.


Asunto(s)
Disfunción Eréctil/cirugía , Regeneración Tisular Dirigida/métodos , Regeneración Nerviosa/fisiología , Erección Peniana/fisiología , Pene/inervación , Células de Schwann/trasplante , Disfunción Eréctil/etiología , Humanos , Masculino , Recuperación de la Función
7.
Biochem Biophys Res Commun ; 332(1): 43-9, 2005 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-15896297

RESUMEN

A heterogeneous group of multisystem disorders affecting various tissues and often including neuromuscular symptoms is caused by mutations of the mitochondrial genome, which codes 13 polypeptides of oxidative phosphorylation (OXPHOS) complexes and 22 tRNA genes needed for their translation. Since the link between OXPHOS dysfunction and clinical phenotype remains enigmatic in many diseases, a possible role of enhanced apoptosis is discussed besides bioenergetic crisis of affected cells. We analyzed the proapoptotic impact of the mitochondrial 5kb common deletion (CD), affecting five tRNA genes, in transmitochondrial cybrid cell lines and found a slightly enhanced sensitivity to exogenous oxidative stress (H2O2) and a pronounced sensitization against death receptor stimulation (TRAIL) at a rather low CD heteroplasmy level of 22%. Mitochondrial deletions confer enhanced susceptibility against proapoptotic signals to proliferating cells, which might explain the elimination of deletions from hematopoietic stem cells.


Asunto(s)
Apoptosis/genética , ADN Mitocondrial/genética , Eliminación de Gen , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Osteosarcoma/genética , Osteosarcoma/patología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Humanos , Células Híbridas/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF
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