Navegando por Palavras-chave "Mitochondrial diseases"

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    Complex I spectrophotometric assay in cultured cells: Detailed analysis of key factors
    (Elsevier B.V., 2013-04-01) Oliveira, Katia Klug [UNIFESP]; Kiyomoto, Beatriz Hitomi [UNIFESP]; Rodrigues, Andresa De Santi [UNIFESP]; Tengan, Celia Harumi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The diagnosis of mitochondrial encephalomyopathies caused by complex I (C-I) deficiency relies mainly on the spectrophotometric C-I assay. Considered difficult, this assay lacks reliability and has high nonspecific activity. We studied the key factors of this assay in cultured cells (cybrid and fibroblast): ubiquinone analogues, rotenone inhibition to determine specific activity, and mode of permeabilization of mitochondria] membranes. We showed that ubiquinone 1 allows a more stable and reliable assay, better results were obtained with rotenone inhibition done in the same assay, and mitochondria] permeability was improved just by freeze/thawing the sample prior to the assay. (C) 2012 Elsevier Inc. All rights reserved.
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    Metabolic studies of a patient harbouring a novel S487L mutation in the catalytic subunit of AMPK
    (Elsevier Science Bv, 2018) Arita, Juliana Harumi [UNIFESP]; Barros, Mario H.; Ravagnani, Felipe Gustavo; Ziosi, Marcello; Sanches, Livia Rentas; Picosse, Fabíola Rosa [UNIFESP]; Lopes, Tania Oliveira; Aguiar, Patricia de Carvalho [UNIFESP]; Macabelli, Carolina Habermann; Chiaratti, Marcos R.; Pedroso, Jose Luiz [UNIFESP]; Quinzii, Catarina M.; Barsottini, Orlando Graziani Povoas [UNIFESP]; Ferreiro-Barros, Claudia Cristina [UNIFESP]
    AMP-activated protein kinase (AMPK) regulates many different metabolic pathways in eukaryote cells including mitochondria biogenesis and energy homeostasis. Here we identify a patient with hypotonia, weakness, delayed milestones and neurological impairment since birth harbouring a novel homozygous mutation in the AMPK catalytic alpha-subunit 1, encoded by the PRKAA1 gene. The homozygous mutation p.S487L in isoform 1 present in the patient is in a cryptic residue for AMPK activity. In the present study, we performed the characterization of mitochondrial respiratory properties of the patient, in comparison to healthy controls, through the culture of skin fibroblasts in order to understand some of the cellular consequences of the PRKAA1 mutation. In these assays, mitochondrial respiratory complex I showed lower activity, which was followed by a decrement in the mtDNA copy number, which is a probable consequence of the lower expression of PGC-1 alpha and PRKAA1 itself as measured in our quantitative PCRs experiments. Confirming the effect of the patient mutation in respiration, transfection of patient fibroblasts with wild type PRKAA1 partially restore complex I level. The preliminary clinic evaluations of the patient suggested a metabolic defect related to the mitochondrial respiratory function, therefore treatment with CoQ10 supplementation dose started four years ago and a clear improvement in motor skills and strength has been achieved with this treatment.
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    The mutations m.5628T > C and m18348A > G in single muscle fibers of a patient with chronic progressive external ophthalmoplegia
    (Elsevier B.V., 2012-09-15) Gamba, Juliana [UNIFESP]; Kiyomoto, Beatriz Hitomi [UNIFESP]; Oliveira, Acary Souza Bulle [UNIFESP]; Gabbai, Alberto Alain [UNIFESP]; Schmidt, Beny [UNIFESP]; Tengan, Celia Harumi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    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. (c) 2012 Elsevier B.V. All rights reserved.