Integrated analysis of the involvement of nitric oxide synthesis in mitochondrial proliferation, mitochondrial deficiency and apoptosis in skeletal muscle fibres.

Nitric oxide (NO) is an important signaling messenger involved in different mitochondrial processes but only few studies explored the participation of NO in mitochondrial abnormalities found in patients with genetic mitochondrial deficiencies. In this study we verified whether NO synthase (NOS) activity was altered in different types of mitochondrial abnormalities and whether changes in mitochondrial function and NOS activity could be associated with the induction of apoptosis. We performed a quantitative and integrated analysis of NOS activity in individual muscle fibres of patients with mitochondrial diseases, considering mitochondrial function (cytochrome-c-oxidase activity), mitochondrial content, mitochondrial DNA mutation and presence of apoptotic nuclei. Our results indicated that sarcolemmal NOS activity was increased in muscle fibres with mitochondrial proliferation, supporting the relevance of neuronal NOS in the mitochondrial biogenesis process. Sarcoplasmic NOS activity was reduced in cytochrome-c-oxidase deficient fibres, probably as a consequence of the involvement of NO in the regulation of the respiratory chain. Alterations in NOS activity or mitochondrial abnormalities were not predisposing factors to apoptotic nuclei. Taken together, our results show that NO can be considered a potential molecular target for strategies to increase mitochondrial content and indicate that this approach may not be associated with increased apoptotic events.

Nitric oxide synthesis is increased in cybrid cells with m.3243A>G mutation.

Nitric oxide (NO) is a free radical and a signaling molecule in several pathways, produced by nitric oxide synthase (NOS) from the conversion of L-arginine to citrulline. Supplementation of L-arginine has been used to treat MELAS (mitochondrial encephalopathy with lactic acidosis and stroke like syndrome), a mitochondrial disease caused by the m.3243A>G mutation. Low levels of serum arginine and endothelium dysfunction have been reported in MELAS and this treatment may increase NO in endothelial cells and promote vasodilation, decreasing cerebral ischemia and strokes. Although clinical benefits have been reported, little is known about NO synthesis in MELAS. In this study we found that osteosarcoma derived cybrid cells with high levels of m.3243A>G had increased nitrite, an NO metabolite, and increased intracellular NO, demonstrated by an NO fluorescent probe (DAF-FM). Muscle vessels from patients with the same mutation had increased staining in NADPH diaphorase, suggestive of increased NOS. These results indicate increased production of NO in cells harboring the m.3243A>G, however no nitrated protein was detected by Western blotting. Further studies are necessary to clarify the exact mechanisms of L-arginine effect to determine the appropriate clinical use of this drug therapy.

The mutations m.5628T>C and m.8348A>G in single muscle fibers of a patient with chronic progressive external ophthalmoplegia.

We identified a double mutation in a patient with chronic progressive external ophthalmoplegia, located in the tRNA(Ala) (m.5628T>C) and tRNA(Lys) (m.8348A>G) genes. Both mutations were previously described separately and considered pathogenic, however the same mutations were also reported as polymorphisms or phenotype modulator. We analyzed the proportion of each mutation in isolated muscle fibers by single fiber-polymerase chain reaction to investigate the contribution of each mutation to mitochondrial deficiency. Our findings demonstrated that the mutations were heteroplasmic in skeletal muscle and both mutations were present in all single muscle fibers. The proportions of the m.5628T>C mutation were not significantly different between normal and cytochrome-c-oxidase (COX) deficient fibers. However, a significant higher proportion of the m.8348A>G mutation was observed in COX deficient fibers. Homoplasmic m.8348A>G was only observed in COX negative fibers. In conclusion, we provide a piece of evidence toward the pathogenicity of the m.8348A>G mutation and suggest that m.5628T>C is probably a neutral polymorphism.

Características morfológicas da casca do ovo de avestruz / Morphologicals characteristics of the ostrich eggshell

A análise morfológica da casca do ovo da avestruz é importante, pois diversos fatores morfológicos podem estar relacionados com a eclodibilidade do ovo desta espécie no Brasil. Para tanto foram analisadas 60 amostras de ovos aparentemente normais (ovos eclodidos e com morte embrionária). Foram efetuadas observações que permitiram estabelecer: a espessura média da casca do ovo, a porosidade da casca destes ovos, e correlacionar estas variáveis com a eclodibilidade. Os resultados foram analisados estatisticamente mediante o teste de Duncan, o que mostrou que ovos com menor porosidade tem também uma menor eclodibilidade. A porosidade média dos ovos eclodidos é de 19,87 poros/cm², e dos ovos com morte embrionária é de 16,78 poros/cm². Não houve diferença estatística entre a espessura da casca nas regiões estudada, nem interação entre viabilidade e as regiões.

Morphological analysis of the ostrich eggshell is important because several factors can be related to morphological hatchability of eggs of this species in Brazil. Therefore, 60 samples of apparently normal eggs were analyzed (eggs hatched and with embryonic death). The observations were allowed to establish that: the average thickness of the eggshell, the porosity of the shell of the eggs, and correlate these variables with hatchability. The results were statistically analyzed by Duncan's test, which showed that eggs with less porosity also have a lower hatchability. The average porosity of hatched eggs is 19.87 pores/cm², and of eggs with embryonic death is pores/cm2 16.78. There was no statistical difference between the thickness of the shell in the regions studied, no interaction between viability and the regions studied.

The role of nitric oxide in muscle fibers with oxidative phosphorylation defects.

NO has been pointed as an important player in the control of mitochondrial respiration, especially because of its inhibitory effect on cytochrome c oxidase (COX). However, all the events involved in this control are still not completely elucidated. We demonstrate compartmentalized abnormalities on nitric oxide synthase (NOS) activity on muscle biopsies of patients with mitochondrial diseases. NOS activity was reduced in the sarcoplasmic compartment in COX deficient fibers, whereas increased activity was found in the sarcolemma of fibers with mitochondrial proliferation. We observed increased expression of neuronal NOS (nNOS) in patients and a correlation between nNOS expression and mitochondrial content. Treatment of skeletal muscle culture with an NO donor induced an increase in mitochondrial content. Our results indicate specific roles of NO in compensatory mechanisms of muscle fibers with mitochondrial deficiency and suggest the participation of nNOS in the signaling process of mitochondrial proliferation in human skeletal muscle.

Estudio descriptivo sobre las caracteristicas particulares que presentan determinadas problemáticas adolescentes (redescubrimiento del esquema corporal, orientación vocacional, problema de identidad y relación con la familia y la sociedad), y su relación con el uso de prótesis auditivas y la atención fonoaudiológica en adolescentes sordos que concurren a la escuela de educación especial N§ 2061 de la ciudad de Avellaneda, provincia de Santa Fe, a noviembre de 1999 / Description study of the particular characteristics that present determines problematics adolescents (rediscover of the body schema, vocational orientation, problem of identity and relation with the family and the society), and his relation with the

Esta investigación se realizó con el propósito de conocer las caractísticas particulares que presentan determinados conflictos propios de la adolescencia en sordos y su relación con el uso de prótesis auditivas y la atención fonoaudiológica. La población estudiada estuvo compuesta por 17 jóvenes sordos que concurren a la escuela de esucación especial n§ 2061 de la ciudad de Avellaneda, provincia de Santa Fe. Se realizó una entrevista referida a determinados puntos de proceso adolescente. Las preguntas de la misma fueron oritneadas a obtener respuestas a las que el sujeto diera cuenta a nivel conciente. Posteriormente se estableció un plan de análisis de los datos obtenidos, lo que posibilitó generalizar las respuestas de los adolescentes discapacitados y formular conclusiones. A partir de esto, se pudo evidenciar lo siguiente: . el 88,2 por ciento de los adolescentes investigados usan protesis auditivas; el 23,5 por ciento en forma continua y el 64,7 por ciento de manera discontinua. . Solo el 23,5 por ciento de estos jóvenes sordos reibió atención fonoaudiológica. . El 52,9 por ciento (9/17) de los jóvenes sordos estudiados presentaría determinadas problemáticas adolescentes en proceso de superación ya que ha respondido afirmativamente a las preguntas sobre: conformadad con el aspecto corporal, "subordinación de las identificaciones infantiles",elección laboral y/o educativa y buena interacción con la familia y la sociedad. Por otro lado se evidenció que todos los jóvenes ordos investigados que presentarían estas determinadas problemáticas adolescentes en proceso de superación usen prótesis auditivas (8 de manera discontinua y 1 en forma continua). Además se pudo observar que de los 9 jóvenes sordos estudiados (el 52,9 por ciento) que presentarían estas determinadas problemáticas adolescentes en proceso de superación sólo 2 de ellos ha recibido atención fonoaudilógica