Mitochondrial activities in human cultured skin fibroblasts contaminated by Mycoplasma hyorhinis

BACKGROUND: Mycoplasma contaminations are a recurrent problem in the use of cultured cells, including human cells, especially as it has been shown to impede cell cycle, triggering cell death under various conditions. More specific consequences on cell metabolism are poorly known. RESULTS: Here we report the lack of significant consequence of a heavy contamination by the frequently encountered mycoplasma strain, M. hyorhinis, on the determination of respiratory chain activities, but the potential interference when assaying citrate synthase. Contamination by M. hyorhinis was detected by fluorescent imaging and further quantified by the determination of the mycoplasma-specific phosphate acetyltransferase activity. Noticeably, this latter activity was not found equally distributed in various mycoplasma types, being exceptionally high in M. hyorhinis. CONCLUSION: While we observed a trend for respiration reduction in heavily contaminated cells, no significant and specific targeting of any respiratory chain components could be identified. This suggested a potential interference with cell metabolism rather than direct interaction with respiratory chain components

ACO2 mutations: A novel phenotype associating severe optic atrophy and spastic paraplegia

International audienc

Mitochondria are physiologically maintained at close to 50 degrees C

In endothermic species, heat released as a product of metabolism ensures stable internal temperature throughout the organism, despite varying environmental conditions. Mitochondria are major actors in this thermogenic process. Part of the energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport, but a substantial proportion is released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured mitochondrial temperature in situ under different physiological conditions. At a constant external temperature of 38 degrees C, mitochondria were more than 10 degrees C warmer when the respiratory chain (RC) was fully functional, both in human embryonic kidney (HEK) 293 cells and primary skin fibroblasts. This differential was abolished in cells depleted of mitochondrial DNA or treated with respiratory inhibitors but preserved or enhanced by expressing thermogenic enzymes, such as the alternative oxidase or the uncoupling protein 1. The activity of various RC enzymes was maximal at or slightly above 50 degrees C. In view of their potential consequences, these observations need to be further validated and explored by independent methods. Our study prompts a critical re-examination of the literature on mitochondria.Peer reviewe

Conduite de projets dans un département de développment médical en entreprise

En mars 2009, les laboratoires pharmaceutiques Abbott France ont confié aux équipes du Développement Médical deux grands projets en parallèle. Nous avons donc eu pour première mission la préparation d'une étude épidémiologique observationnelle de phase IV (post AMM) nommée Ob'tension , au sein de l'unité médicale de Cardiologie. Pour la réalisation de cette étude, nous avons entre autre crée les documents essentiels (protocole, cahiers d'observation...), effectué les soumissions réglementaires pour l'autorisation de la mise en place de l'étude, tout en respectant le planning des dates prévisionnelles, et le budget attribué. En second projet, nous avons été chargées d'élaborer un cahier des charges dans l'objectif de développer un nouvel outil informatique au sein du service d'Information Médicale, avec pour pré requis de créer un outil plus performant que l'outil actuel, tout en répondant au mieux aux besoins des utilisateurs.ROUEN-BU Médecine-Pharmacie (765402102) / SudocSudocFranceF

Mutation in the mitochondrial translation elongation factor EFTs results in severe infantile liver failure

Background & AimsMultiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and often result in hepatic failure. A significant fraction of patients present mitochondrial DNA depletion but a number of cases remain unexplained. The aim of our study was to identify the disease causing gene in a kindred with intrauterine growth retardation, neonatal lactic acidosis, liver dysfunction and multiple respiratory chain deficiency in muscle.MethodsHomozygosity mapping was performed by 50K SNP genotyping and candidate genes were successively analyzed by direct sequencing on genomic DNA of the family members.ResultsSNP genotyping detected several regions of homozygosity in which we focused our attention to genes involved in mitochondrial translation. We sequenced the TSFM gene, encoding the mitochondrial translation factor EFTs and identified a homozygous mutation changing a highly conserved arginine into a tryptophan (R312W).ConclusionsThis mutation has been previously reported in two unrelated kindred presenting two distinct syndromes (fatal mitochondrial encephalomyopathy and hypertrophic cardiomyopathy respectively). The description of a third syndrome associated with a same TSFM mutation gives support to the broad clinical and genetic heterogeneity of mitochondrial translation deficiencies in human. It suggests that mitochondrial translation deficiency represents a growing cause of hepatic failure of mitochondrial origin in infants

Prenyldiphosphate synthase, subunit 1 (PDSS1) and OH-benzoate polyprenyltransferase (COQ2) mutations in ubiquinone deficiency and oxidative phosphorylation disorders

Coenzyme Q(10) (CoQ(10)) plays a pivotal role in oxidative phosphorylation (OXPHOS), as it distributes electrons among the various dehydrogenases and the cytochrome segments of the respiratory chain. We have identified 2 novel inborn errors of CoQ(10) biosynthesis in 2 distinct families. In both cases, enzymologic studies showed that quinone-dependent OXPHOS activities were in the range of the lowest control values, while OXPHOS enzyme activities were normal. CoQ(10) deficiency was confirmed by restoration of normal OXPHOS activities after addition of quinone. A genome-wide search for homozygosity in family 1 identified a region of chromosome 10 encompassing the gene prenyldiphosphate synthase, subunit 1 (PDSS1), which encodes the human ortholog of the yeast COQ1 gene, a key enzyme of CoQ(10) synthesis. Sequencing of PDSS1 identified a homozygous nucleotide substitution modifying a conserved amino acid of the protein (D308E). In the second family, direct sequencing of OH-benzoate polyprenyltransferase (COQ2), the human ortholog of the yeast COQ2 gene, identified a single base pair frameshift deletion resulting in a premature stop codon (c.1198delT, N401fsX415). Transformation of yeast Δcoq1 and Δcoq2 strains by mutant yeast COQ1 and mutant human COQ2 genes, respectively, resulted in defective growth on respiratory medium, indicating that these mutations are indeed the cause of OXPHOS deficiency

Large-Scale Deletion and Point Mutations of the Nuclear NDUFV1 and NDUFS1 Genes in Mitochondrial Complex I Deficiency

Reduced nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest complex of the mitochondrial respiratory chain and complex I deficiency accounts for ∼30% cases of respiratory-chain deficiency in humans. Only seven mitochondrial DNA genes, but >35 nuclear genes encode complex I subunits. In an attempt to elucidate the molecular bases of complex I deficiency, we studied the six most-conserved complex I nuclear genes (NDUFV1, NDUFS8, NDUFS7, NDUFS1, NDUFA8, and NDUFB6) in a series of 36 patients with isolated complex I deficiency by denaturing high-performance liquid chromatography and by direct sequencing of the corresponding cDNA from cultured skin fibroblasts. In 3/36 patients, we identified, for the first time, five point mutations (del222, D252G, M707V, R241W, and R557X) and one large-scale deletion in the NDUFS1 gene. In addition, we found six novel NDUFV1 mutations (Y204C, C206G, E214K, IVS 8+41, A432P, and del nt 989–990) in three other patients. The six unrelated patients presented with hypotonia, ataxia, psychomotor retardation, or Leigh syndrome. These results suggest that screening for complex I nuclear gene mutations is of particular interest in patients with complex I deficiency, even when normal respiratory-chain–enzyme activities in cultured fibroblasts are observed

Pitfalls in Monitoring Mitochondrial Temperature Using Charged Thermosensitive Fluorophores

Mitochondria are the source of internal heat which influences all cellular processes. Hence, monitoring mitochondrial temperature provides a unique insight into cell physiology. Using a thermosensitive fluorescent probe MitoThermo Yellow (MTY), we have shown recently that mitochondria within human cells are maintained at close to 50 degrees C when active, increasing their temperature locally by about 10 degrees C. Initially reported in the HEK293 cell line, we confirmed this finding in the HeLa cell line. Delving deeper, using MTY and MTX (MitoThermo X), a modified version of MTY, we unraveled some caveats related to the nature of these charged fluorophores. While enabling the assessment of mitochondrial temperature in HEK and HeLa cell lines, the reactivity of MTY to membrane potential variations in human primary skin fibroblasts precluded local temperature monitoring in these cells. Chemical modification of MTY into MTX did not result in a temperature probe unresponsive to membrane potential variations that could be universally used in any cell type to determine mitochondrial temperature. Thus, the cell-type dependence of MTY in measuring mitochondrial temperature, which is likely due to the variable binding of this dye to specific internal mitochondrial components, should imply cautiousness while using these nanothermometers for mitochondrial temperature analysis.11Nsciescopu

Prévention du carcinome rénal : l’approche nutrigénétique

La fréquence du cancer du rein (RCC), 3 % des cancers humains, augmente dans les pays industrialisés, laissant supposer l’intervention de facteurs toxiques (xénobiotiques et/ou alimentation, trichloréthylène, tabagisme et obésité). Le RCC survient le plus souvent sous forme sporadique mais est également retrouvé dans un contexte familial : la maladie de von Hippel-Lindau (VHL). L’existence, d’une part, d’une grande hétérogénéité intra- et inter-familiale dans le contexte du VHL et, d’autre part, la susceptibilité variable à des carcinogènes chimiques dans les formes sporadiques, laisse supposer, en outre, la participation de gènes modificateurs conditionnels. Afin d’identifier des sous-groupes d’individus particulièrement exposés ou, au contraire, protégés du fait de certains génotypes, nous avons collecté une série de 460 tumeurs et de patients appartenant à 79 familles VHL et développé un outil informatique, l’« universal mutation database » (UMD) pour les mutations du gène VHL, permettant de rechercher des corrélations. Les mutations du gène VHL à l’origine à la fois des RCC sporadiques et de la maladie de VHL sont de nature différente : 1) dans les tumeurs sporadiques, 83 % des mutations du gène VHL sont des mutations aboutissant à un décalage du cadre de lecture (délétion, insertion, non-sens = « frameshift »). Les 17 % restant comprennent des transversions (3/4) et des transitions (1/4). Cette proportion élevée de transversions suggère fortement l’implication de substances carcinogènes (fumée de tabac) dont l’impact est largement conditionné par la variabilité génétique de l’activité des enzymes de biotransformation ; 2) pour les formes familiales, les mutations de type faux sens prédominent dans 65 % des cas. Cette différence permet de définir un facteur pronostique de développer un RCC pour les patients VHL en fonction de la nature de la mutation germinale dont ils sont porteurs. Afin de repérer les génotypes conférant un risque élevé en présence de substances potentiellement carcinogènes, nous avons établi le génotype des patients pour 8 gènes (une cinquantaine de génotypes) impliqués dans le métabolisme des xénobiotiques. Cette étude fait apparaître une relation significative entre le développement d’un RCC et des combinaisons d’allèles comprenant : CYPIA1 ("variant"), NAT2 et NAT1 (acétyleurs lents) et GSTM1 (allèle nul). D’éventuelles associations entre les génotypes "à risque" et le profil des mutations somatiques observées chez les patients, mais aussi à différents stades tumoraux, pourraient aider à 1) préciser la nature de certains profils de mutagenèse en relation avec l’activité ou la déficience de telle ou telle enzyme du métabolisme des xénobiotiques et sous l’effet de tel ou tel carcinogène ; 2) montrer que, dans le contexte du VHL, certaines combinaisons d’allèles de ces différents gènes confèrent un risque particulier de développer certains types de tumeur. Ainsi, suivre "à la trace" des substances potentiellement carcinogènes, à la fois par l’empreinte laissée au niveau de l’ADN, ainsi qu’à travers les allèles conditionnellement délétères de gènes participant à leur détoxication, devrait permettre une meilleur prévention grâce à une alimentation personnalisée pour les individus présentant ces génotypes