560 results on '"Turnbull, Douglass M."'
Search Results
552. Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR.
- Author
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He L, Chinnery PF, Durham SE, Blakely EL, Wardell TM, Borthwick GM, Taylor RW, and Turnbull DM
- Subjects
- Humans, Mitochondrial Myopathies genetics, Muscle Fibers, Skeletal cytology, Muscle, Skeletal metabolism, Sensitivity and Specificity, Time Factors, DNA, Mitochondrial genetics, Muscle Fibers, Skeletal metabolism, Polymerase Chain Reaction methods, Sequence Deletion
- Abstract
Defects of mitochondrial DNA (mtDNA) are an important cause of disease and play a role in the ageing process. There are multiple copies of the mitochondrial genome in a single cell. In many patients with acquired or inherited mtDNA mutations, there exists a mixture of mutated and wild type genomes (termed heteroplasmy) within individual cells. As a biochemical and clinical defect is only observed when there are high levels of mutated mtDNA, a crucial investigation is to determine the level of heteroplasmic mutations within tissues and individual cells. We have developed an assay to determine the relative amount of deleted mtDNA using real-time fluorescence PCR. This assay detects the vast majority of deleted molecules, thus eliminating the need to develop specific probes. We have demonstrated an excellent correlation with other techniques (Southern blotting and three- primer competitive PCR), and have shown this technique to be sensitive to quantify the level of deleted mtDNA molecules in individual cells. Finally, we have used this assay to investigate patients with mitochondrial disease and shown in individual skeletal muscle fibres that there exist different patterns of abnormalities between patients with single or multiple mtDNA deletions. We believe that this technique has significant advantages over other methods to quantify deleted mtDNA and, employed alongside our method to sequence the mitochondrial genome from single cells, will further our understanding of the role of mtDNA mutations in human disease and ageing.
- Published
- 2002
- Full Text
- View/download PDF
553. Analysis of mitochondrial fatty acid oxidation intermediates by tandem mass spectrometry from intact mitochondria prepared from homogenates of cultured fibroblasts, skeletal muscle cells, and fresh muscle.
- Author
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Tyni T, Pourfarzam M, and Turnbull DM
- Subjects
- 3-Hydroxyacyl CoA Dehydrogenases deficiency, Acetylcarnitine analysis, Acetylcarnitine metabolism, Acyl-CoA Dehydrogenase, Carnitine analysis, Carnitine metabolism, Carnitine O-Palmitoyltransferase deficiency, Cells, Cultured, Fatty Acid Desaturases deficiency, Fibroblasts cytology, Humans, Metabolic Diseases diagnosis, Metabolic Diseases metabolism, Muscle, Skeletal cytology, Myoblasts cytology, Oxidation-Reduction, Skin cytology, Carnitine analogs & derivatives, Fatty Acids metabolism, Fibroblasts metabolism, Mass Spectrometry, Mitochondria metabolism, Myoblasts metabolism
- Abstract
Defects of mitochondrial fatty acid beta-oxidation are an important group of inherited metabolic disorders in children. Despite improved screening opportunities, diagnosis of these disorders is not often straightforward and requires enzyme analyses. Because therapy is effective in many of these disorders, rapid diagnosis is essential. We report a technique that allows analysis of fatty acid oxidation not only in cultured cells (fibroblasts, myoblasts, and myotubes) but also in fresh muscle homogenate. Fatty acid oxidation analysis was performed by incubating fresh muscle homogenate or harvested cultured cells with stable isotopically labeled palmitate. The intermediates generated were analyzed by tandem mass spectrometry. Results of patients with seven different beta-oxidation disorders were compared with controls. Acylcarnitine intermediates in patient samples could be easily differentiated from the control samples. The acylcarnitine profile of each beta-oxidation defect was compatible with localization of the enzyme defect. Both in patient and control samples, the same pattern of intermediates could be detected in fibroblasts, muscle cells, and fresh muscle homogenate. The procedure described allowed correct diagnosis of all the beta-oxidation defects studied. Utilization of fresh muscle samples reduces the delay in diagnosis related to tissue culture and is useful in diagnostic of patients with neuromuscular phenotype. Measurement of fatty acid oxidation intermediates from myoblasts or myotubes is an additional tool in investigating pathogenetic mechanisms of myopathy in beta-oxidation defects.
- Published
- 2002
- Full Text
- View/download PDF
554. Mitochondrial dysfunction in a cell culture model of familial amyotrophic lateral sclerosis.
- Author
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Menzies FM, Cookson MR, Taylor RW, Turnbull DM, Chrzanowska-Lightowlers ZM, Dong L, Figlewicz DA, and Shaw PJ
- Subjects
- Amyotrophic Lateral Sclerosis pathology, Animals, Cell Line, Cell Survival drug effects, Cell Survival genetics, Culture Media, Serum-Free pharmacology, Electron Transport Complex I, Electron Transport Complex II, Electron Transport Complex III metabolism, Electron Transport Complex IV metabolism, Energy Metabolism, Enzyme Activation drug effects, Enzyme Activation genetics, Enzyme Inhibitors pharmacology, Glycolysis drug effects, Humans, Iodoacetates pharmacology, Mice, Mitochondria metabolism, Mitochondria pathology, Mitochondria ultrastructure, Mitochondrial Diseases pathology, Motor Neurons drug effects, Motor Neurons pathology, Multienzyme Complexes metabolism, Mutagenesis, Site-Directed, NADH, NADPH Oxidoreductases metabolism, Oxidoreductases metabolism, Succinate Dehydrogenase metabolism, Superoxide Dismutase-1, Transfection, Amyotrophic Lateral Sclerosis metabolism, Mitochondrial Diseases metabolism, Motor Neurons metabolism, Superoxide Dismutase genetics, Superoxide Dismutase metabolism
- Abstract
The molecular mechanisms by which mutations in the gene for Cu/Zn superoxide dismutase (SOD1) lead to the selective death of motor neurones in familial amyotrophic lateral sclerosis (FALS) remain incompletely understood. Previous evidence has indicated that mitochondrial abnormalities may develop during motor neurone injury, but several important questions remain unanswered. We have developed a cell culture model of FALS in which a motor neurone cell line (NSC34) has been stably transfected to express normal or mutant human SOD1 at levels approximating to those seen in the human disease. The aims of the study were to: (i) investigate whether morphological mitochondrial abnormalities occur at expression levels of mutant SOD1 close to physiological levels; and (ii) determine whether the presence of mutant SOD1 causes abnormalities of mitochondrial respiratory chain function and changes in cellular bioenergetic parameters in motor neuronal cells. Using this cellular model, we demonstrate that the presence of mutant SOD1 results in the development of abnormally swollen and pale staining mitochondria. These morphological changes are accompanied by biochemical abnormalities with specific decreases in the activities of complexes II and IV of the mitochondrial electron transfer chain. These same complexes are inhibited when control NSC34 cells are subjected to oxidative stress induced by serum withdrawal. The decrease in respiratory chain complex activity in the presence of mutant SOD1 was not accompanied by decreased expression of representative proteins present in these complexes. Motor neuronal cells expressing mutant SOD1 showed increased cell death when exposed to oxidative stress by serum withdrawal, whereas the presence of normal human SOD1 exerted a protective effect. Under basal, unstressed culture conditions, no change in the ATP : ADP ratio was observed in the presence of mutant SOD1. However, the mitochondrial changes associated with the presence of mutant SOD1 clearly had adverse cellular bioenergetic consequences as shown by increased cell death in the presence of pharmacological inhibition of the glycolytic pathway. We conclude that one important mechanism by which mutant SOD1 causes motor neurone injury involves inhibition of specific components of the mitochondrial electron transfer chain. Therapeutic measures aimed at protecting mitochondrial respiratory chain function may be useful in SOD1 related familial and possibly other forms of amyotrophic lateral sclerosis.
- Published
- 2002
- Full Text
- View/download PDF
555. Defining the importance of mitochondrial gene defects in maternally inherited diabetes by sequencing the entire mitochondrial genome.
- Author
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Choo-Kang AT, Lynn S, Taylor GA, Daly ME, Sihota SS, Wardell TM, Chinnery PF, Turnbull DM, and Walker M
- Subjects
- Aged, Female, Hearing Loss, Sensorineural genetics, Humans, Male, Middle Aged, Mitochondria, Muscle genetics, Muscle, Skeletal, Mutation, Neuromuscular Diseases genetics, RNA, Transfer, Leu genetics, Restriction Mapping, DNA, Mitochondrial genetics, Diabetes Mellitus genetics, Genome, Genomic Imprinting, Mitochondrial Diseases genetics
- Abstract
For any mitochondrial DNA (mtDNA) mutation, the ratio of mutant to wild-type mtDNA (% heteroplasmy) varies across tissues, with low levels in leukocytes and high levels in postmitotic tissues (e.g., skeletal muscle). Direct sequencing is the gold-standard method used to detect novel mutations, but can only reliably detect % heteroplasmy >25%, which is rare in leukocytes. Therefore, we investigated the role of mtDNA defects in maternally inherited diabetes by first screening for the A3243G tRNA(Leu(UUR)) mutation by restriction assay, followed by sequencing of the entire mitochondrial genome using skeletal muscle derived mtDNA. A total of 28 patients had maternally inherited diabetes either alone (group 1, n = 17) or with one or more additional features of mitochondrial disease, including bilateral sensori-neural deafness and neuromuscular disease (group 2, n = 11). Three patients (all from group 2) carried the A3243G mutation. Skeletal muscle mtDNA from eight group 1 patients and six more group 2 patients was sequenced. No pathogenic mutations were found in the group 1 patients, while two patients from group 2 had mutations at positions 12258 and 14709 in the tRNA serine and glutamic acid genes, respectively. We conclude, therefore, that screening for mtDNA mutations should be considered in patients with maternally inherited diabetes, but only when additional features of mitochondrial disease are present.
- Published
- 2002
- Full Text
- View/download PDF
556. Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups.
- Author
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Herrnstadt C, Elson JL, Fahy E, Preston G, Turnbull DM, Anderson C, Ghosh SS, Olefsky JM, Beal MF, Davis RE, and Howell N
- Subjects
- Africa, Asia, Europe, Evolution, Molecular, Genetic Variation genetics, Humans, Linkage Disequilibrium genetics, Phylogeny, Polymorphism, Genetic genetics, Recombination, Genetic genetics, DNA, Mitochondrial genetics, Ethnicity genetics, Haplotypes genetics, Racial Groups genetics
- Abstract
The evolution of the human mitochondrial genome is characterized by the emergence of ethnically distinct lineages or haplogroups. Nine European, seven Asian (including Native American), and three African mitochondrial DNA (mtDNA) haplogroups have been identified previously on the basis of the presence or absence of a relatively small number of restriction-enzyme recognition sites or on the basis of nucleotide sequences of the D-loop region. We have used reduced-median-network approaches to analyze 560 complete European, Asian, and African mtDNA coding-region sequences from unrelated individuals to develop a more complete understanding of sequence diversity both within and between haplogroups. A total of 497 haplogroup-associated polymorphisms were identified, 323 (65%) of which were associated with one haplogroup and 174 (35%) of which were associated with two or more haplogroups. Approximately one-half of these polymorphisms are reported for the first time here. Our results confirm and substantially extend the phylogenetic relationships among mitochondrial genomes described elsewhere from the major human ethnic groups. Another important result is that there were numerous instances both of parallel mutations at the same site and of reversion (i.e., homoplasy). It is likely that homoplasy in the coding region will confound evolutionary analysis of small sequence sets. By a linkage-disequilibrium approach, additional evidence for the absence of human mtDNA recombination is presented here.
- Published
- 2002
- Full Text
- View/download PDF
557. A high frequency of mtDNA polymorphisms in HeLa cell sublines.
- Author
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Herrnstadt C, Preston G, Andrews R, Chinnery P, Lightowlers RN, Turnbull DM, Kubacka I, and Howell N
- Subjects
- Base Sequence, HeLa Cells, Humans, Molecular Sequence Data, DNA, Mitochondrial genetics, Polymorphism, Genetic
- Abstract
The complete mtDNA sequences from the uncloned "founder" HeLa cells and from five sublines have been determined. These sequences all carry a common "core" of 38 single basepair alterations relative to the revised Cambridge Reference Sequence (CRS). The HeLa mitochondrial genome is of African descent and it is a member of the African L3 haplogroup. The sequence of the HeLa mtDNA resolves the uncertainty surrounding the mosaic composition of the original CRS for human mtDNA. Most importantly, we detected a total of eight polymorphisms that have arisen in the mtDNA coding region of different HeLa sublines. These observations suggest that HeLa mtDNA has a high rate of sequence divergence, relative to the phylogenetically-derived divergence rate for mtDNAs in the human population, which results from a relaxation of negative selection against the fixation of deleterious mutations. Furthermore, this high frequency of polymorphisms in HeLa mtDNA may reflect a process similar to the accumulation of somatic mtDNA mutations in human cancers. Preliminary analysis of single-cell derived subclone lines revealed the occurrence of another polymorphism and provided evidence for a large number of mtDNA segregation units.
- Published
- 2002
- Full Text
- View/download PDF
558. A roundabout route to gene therapy.
- Author
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Turnbull DM and Lightowlers RN
- Subjects
- Adenosine Triphosphatases genetics, Humans, Mitochondrial Proton-Translocating ATPases genetics, Models, Genetic, Mutation, Mutation, Missense, Phosphorylation, Saccharomyces cerevisiae metabolism, DNA, Mitochondrial genetics, Genetic Therapy methods
- Published
- 2002
- Full Text
- View/download PDF
559. Multiple neonatal deaths due to a homoplasmic mitochondrial DNA mutation.
- Author
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McFarland R, Clark KM, Morris AA, Taylor RW, Macphail S, Lightowlers RN, and Turnbull DM
- Subjects
- Adult, DNA Mutational Analysis, Female, Humans, Infant, Newborn, Leigh Disease genetics, Male, Pedigree, Phenotype, DNA, Mitochondrial genetics, Mitochondrial Diseases genetics, Mutation
- Abstract
Mutations of mitochondrial DNA (mtDNA) are an important cause of genetic disease. We describe a family with an unusual homoplasmic mutation that resulted in six neonatal deaths and one surviving child with Leigh syndrome. The mother is clinically normal, but a severe biochemical and molecular genetic defect was present in both a fatally affected child and the mother. This family highlights the role of homoplasmic mt-tRNA mutations in genetic disease.
- Published
- 2002
- Full Text
- View/download PDF
560. Leigh disease associated with a novel mitochondrial DNA ND5 mutation.
- Author
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Taylor RW, Morris AA, Hutchinson M, and Turnbull DM
- Subjects
- Adult, Child, Humans, Leigh Disease etiology, Male, Sequence Analysis, DNA, DNA, Mitochondrial genetics, Leigh Disease genetics, Mutation, Missense
- Abstract
Leigh disease is a genetically heterogeneous, neurodegenerative disorder of childhood that is caused by defects of either the nuclear or mitochondrial genome. Here, we report the molecular genetic findings in a patient with neuropathological hallmarks of Leigh disease and complex I deficiency. Direct sequencing of the seven mitochondrial DNA (mtDNA)-encoded complex I (ND) genes revealed a novel missense mutation (T12706C) in the mitochondrial ND5 gene. The mutation is predicted to change an invariant amino acid in a highly conserved transmembrane helix of the mature polypeptide and was heteroplasmic in both skeletal muscle and cultured skin fibroblasts. The association of the T12706C ND5 mutation with a specific biochemical defect involving complex I is highly suggestive of a pathogenic role for this mutation.
- Published
- 2002
- Full Text
- View/download PDF
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