Doxycycline has anti-tumour effects in a range of tumour systems. The aims of this study were to define the role mitochondria play in this process and examine the potential of doxycycline in combination with gemcitabine. We studied the... more
Doxycycline has anti-tumour effects in a range of tumour systems. The aims of this study were to define the role mitochondria play in this process and examine the potential of doxycycline in combination with gemcitabine. We studied the adenocarcinoma cell line A549, its mitochondrial DNA-less derivative A549 ρ° and cultured fibroblasts. Treatment with doxycycline for 5 days resulted in a decrease of mitochondrial-encoded proteins, respiration and membrane potential, and an increase of reactive oxygen species in A549 cells and fibroblasts, but fibroblasts were less affected. Doxycycline slowed proliferation of A549 cells by 35%. Cellular ATP levels did not change. Doxycycline alone had no effect on apoptosis; however, in combination with gemcitabine given during the last 2 days of treatment, doxycycline increased caspase 9 and 3/7 activities, resulting in a further decrease of surviving A549 cells by 59% and of fibroblasts by 24% compared to gemcitabine treatment alone. A549 ρ° cells...
Since the online publication of the article, the authors have noted errors with Table 2; this has now been corrected in both the HTML and the PDF.
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Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin... more
Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol/chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence f...
Research Interests: Mitochondria, Multidisciplinary, Mitochondrial DNA, Humans, Cerebellum, and 15 moreFemale, Male, Polymerase Chain Reaction, Substantia nigra, PLoS one, Aged, Middle Aged, Autopsy, Cell nucleus, Microarray Analysis, Brain Chemistry, Frontal Lobe, Parkinson Disease, Comparative Genomic Hybridization, and Case Control Studies
Pathologic ribonuclease H1 (RNase H1) causes aberrant mitochondrial DNA (mtDNA) segregation and is associated with multiple mtDNA deletions. We aimed to determine the prevalence of RNase H1 gene (RNASEH1) mutations among patients with... more
Pathologic ribonuclease H1 (RNase H1) causes aberrant mitochondrial DNA (mtDNA) segregation and is associated with multiple mtDNA deletions. We aimed to determine the prevalence of RNase H1 gene (RNASEH1) mutations among patients with mitochondrial disease and establish clinically meaningful genotype-phenotype correlations. RNASEH1 was analyzed in patients with (1) multiple deletions/depletion of muscle mtDNA and (2) mendelian progressive external ophthalmoplegia (PEO) with neuropathologic evidence of mitochondrial dysfunction, but no detectable multiple deletions/depletion of muscle mtDNA. Clinicopathologic and molecular evaluation of the newly identified and previously reported patients harboring RNASEH1 mutations was subsequently undertaken. Pathogenic c.424G>A p.Val142Ile RNASEH1 mutations were detected in 3 pedigrees among the 74 probands screened. Given that all 3 families had Indian ancestry, RNASEH1 genetic analysis was undertaken in 50 additional Indian probands with var...
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TRNT1 (CCA-adding transfer RNA nucleotidyl transferase) enzyme deficiency is a new metabolic disease caused by defective post-transcriptional modification of mitochondrial and cytosolic transfer RNAs (tRNAs). We investigated four patients... more
TRNT1 (CCA-adding transfer RNA nucleotidyl transferase) enzyme deficiency is a new metabolic disease caused by defective post-transcriptional modification of mitochondrial and cytosolic transfer RNAs (tRNAs). We investigated four patients from two families with infantile-onset cyclical, aseptic febrile episodes with vomiting and diarrhoea, global electrolyte imbalance during these episodes, sideroblastic anaemia, B lymphocyte immunodeficiency, retinitis pigmentosa, hepatosplenomegaly, exocrine pancreatic insufficiency and renal tubulopathy. Other clinical features found in children include sensorineural deafness, cerebellar atrophy, brittle hair, partial villous atrophy and nephrocalcinosis. Whole exome sequencing and bioinformatic filtering were utilised to identify recessive compound heterozygous TRNT1 mutations (missense mutation c.668T>C, p.Ile223Thr and a novel splice mutation c.342+5G>T) segregating with disease in the first family. The second family was found to have a ...
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Defects of mitochondrial dynamics are emerging causes of neurological disease. In two children presenting with severe neurological deterioration following viral infection we identified a novel homozygous STAT2 mutation, c.1836 C>A... more
Defects of mitochondrial dynamics are emerging causes of neurological disease. In two children presenting with severe neurological deterioration following viral infection we identified a novel homozygous STAT2 mutation, c.1836 C>A (p.Cys612Ter), using whole exome sequencing. In muscle and fibroblasts from these patients, and a third unrelated STAT2-deficient patient, we observed extremely elongated mitochondria. Western blot analysis revealed absence of the STAT2 protein and that the mitochondrial fission protein DRP1 (encoded by DNM1L) is inactive, as shown by its phosphorylation state. All three patients harboured decreased levels of DRP1 phosphorylated at serine residue 616 (P-DRP1(S616)), a post-translational modification known to activate DRP1, and increased levels of DRP1 phosphorylated at serine 637 (P-DRP1(S637)), associated with the inactive state of the DRP1 GTPase. Knockdown of STAT2 in SHSY5Y cells recapitulated the fission defect, with elongated mitochondria and decr...
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As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation. Human cytochrome c oxidase is composed of 13 subunits. The three major subunits form the... more
As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation. Human cytochrome c oxidase is composed of 13 subunits. The three major subunits form the catalytic core and are encoded by mitochondrial DNA (mtDNA). The remaining subunits are nuclear-encoded. The primary sequence is known for all human subunits and the crystal structure of bovine heart cytochrome c oxidase has recently been reported. However, despite this wealth of structural information, the role of the nuclear encoded subunits is still poorly understood. Yeast cytochrome c oxidase is a close model of its human counterpart and provides a means of studying the effects of mutations on the assembly, structure, stability and function of the enzyme complex. Defects in cytochrome c oxidase function are found in a clinically heterogeneous group of disorders. The molecular defects that underlie these diseases may arise from mutations of either mitoc...
Research Interests: Mitochondria, Bioenergetics, Early Childhood, DNA, Crystal structure, and 15 moreClinical Practice, Mitochondrial DNA, Humans, Cell fusion, Assembly, Animals, Cytochrome c oxidase, Mitochondrial Respiratory Chain, Enzyme, Cattle, Cell nucleus, Molecular Characterization, Amino Acid Sequence, Biochemistry and cell biology, and Molecular Sequence Data
In Huntington's disease (HD) the striatum and cortex seem particularly vulnerable. Mitochondrial dysfunction can also cause neurodegeneration with prominent striatal involvement very similar to HD. We first examined if mitochondrial... more
In Huntington's disease (HD) the striatum and cortex seem particularly vulnerable. Mitochondrial dysfunction can also cause neurodegeneration with prominent striatal involvement very similar to HD. We first examined if mitochondrial biogenesis, mitochondrial DNA (mtDNA) transcription, and the implications for mitochondrial respiratory chain (MRC) assembly and function differ between the striatum and cortex compared with the whole brain average in the healthy mouse brain. We then examined the effects of the mutant huntingtin transgene in end-stage R6/2 mice. In wild-type mice, mitochondrial mass (citrate synthase levels, mtDNA copy number) was higher in the striatum than in the cortex or whole brain average. PGC-1α and TFAM mRNA levels were also higher in the striatum than the whole brain average and cortex. mRNA reserve for MRC Complex proteins was higher in the striatum and cortex. In addition, in the cortex a greater part of mitochondrial mass was dedicated to the generation o...
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Complex III (CIII; ubiquinol cytochrome c reductase of the mitochondrial respiratory chain) catalyzes electron transfer from succinate and nicotinamide adenine dinucleotide-linked dehydrogenases to cytochrome c. CIII is made up of 11... more
Complex III (CIII; ubiquinol cytochrome c reductase of the mitochondrial respiratory chain) catalyzes electron transfer from succinate and nicotinamide adenine dinucleotide-linked dehydrogenases to cytochrome c. CIII is made up of 11 subunits, of which all but one (cytochrome b) are encoded by nuclear DNA. CIII deficiencies are rare and manifest heterogeneous clinical presentations. Although pathogenic mutations in the gene encoding mitochondrial cytochrome b have been described, mutations in the nuclear-DNA-encoded subunits have not been reported. Involvement of various genes has been indicated in assembly of yeast CIII (refs. 8-11). So far only one such gene, BCS1L, has been identified in human. BCS1L represents, therefore, an obvious candidate gene in CIII deficiency. Here, we report BCS1L mutations in six patients, from four unrelated families and presenting neonatal proximal tubulopathy, hepatic involvement and encephalopathy. Complementation study in yeast confirmed the delete...
Research Interests: Mitochondria, Biological Sciences, Humans, Mutation, Female, and 14 moreAnimals, Male, Cytochrome c oxidase, Mitochondrial Respiratory Chain, Expression, Proteins, Electron Transport, Newborn Infant, Liver Failure, Amino Acid Sequence, Base Sequence, Brain Diseases, Molecular Sequence Data, and Medical and Health Sciences
... including Leigh syndrome, chronic progressive ophthalmoplegia, Kearns Sayre syndrome, and fatal and benign infantile mitochondrial myopathy) ~ In addition ... DNA sequencing data predict as N terminus: QQK [AM Maarse, APGM van Loon,... more
... including Leigh syndrome, chronic progressive ophthalmoplegia, Kearns Sayre syndrome, and fatal and benign infantile mitochondrial myopathy) ~ In addition ... DNA sequencing data predict as N terminus: QQK [AM Maarse, APGM van Loon, H. Riezman, I. Gregor, G. Schatz ...
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To characterize the spectrum of mutations in the OPA1 gene in a large international panel of patients with autosomal dominant optic atrophy (adOA), to improve understanding of the range of functional deficits attributable to sequence... more
To characterize the spectrum of mutations in the OPA1 gene in a large international panel of patients with autosomal dominant optic atrophy (adOA), to improve understanding of the range of functional deficits attributable to sequence variants in this gene, and to assess any genotype-phenotype correlations. All 28 coding exons of OPA1, intron-exon splice sites, 273 bp 5' to exon 1, and two intronic regions with putative function were screened in 94 apparently unrelated white patients of European origin with adOA by single-strand conformational polymorphism (SSCP)-heteroduplex analysis and direct sequencing. Clinical data were collated, and putative mutations were tested for segregation in the respective families by SSCP analysis or direct sequencing and in 100 control chromosomes. Further characterization of selected splice site mutations was performed by RT-PCR of patient leukocyte RNA. Staining of mitochondria in leukocytes of patients and control subjects was undertaken to ass...
Research Interests: Adolescent, Biological Sciences, Humans, Child, Mutation, and 15 moreData Collection, Pedigree, Phenotype, Aged, Middle Aged, Genetically Modified, Genotype, Adult, Clinical Data, Large Scale, Base Sequence, Molecular Sequence Data, DNA mutational analysis, Child preschool, and Medical and Health Sciences
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The assembly of cytochrome-c oxidase was studied in human cells cultured in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using two-dimensional PAGE (blue native PAGE and... more
The assembly of cytochrome-c oxidase was studied in human cells cultured in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using two-dimensional PAGE (blue native PAGE and tricine/SDS/PAGE) and subsequent western blots were developed with monoclonal antibodies against specific subunits of cytochrome-c oxidase. Proteins were also visualized using metabolic labeling followed by two-dimensional electrophoresis and fluorography. These techniques allowed identification of two assembly intermediates of cytochrome-c oxidase. Assembly of the 13 subunits of cytochrome-c oxidase starts with the association of subunit I with subunit IV. Then a larger subcomplex is formed, lacking only subunits VIa and either VIIa or VIIb.
Research Interests: Kinetics, Mitochondria, Cell line, Humans, Assembly, and 12 moreCytochrome c oxidase, Monoclonal Antibodies, Mitochondrial Respiratory Chain, Gene, European, Oxidative phosphorylation, Doxycycline, Protein Conformation, Two dimensional Gel Electrophoresis, Biochemistry and cell biology, Cycloheximide, and Medical biochemistry and metabolomics
Research Interests: Mitochondria, Apoptosis, Multidisciplinary, Gene Silencing, Free Radical, and 15 moreDopamine, Mitochondrial DNA, Humans, Protein Kinases, Cell Death, Glutathione, PLoS one, Neuroblastoma, Mitochondrial dysfunction, Oxidative phosphorylation, Autosomal Recessive, Mitochondrial Membrane Potential, Cell Survival, Adenosine Triphosphate, and reverse transcriptase polymerase chain reaction
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Research Interests: Genetics, Principal Component Analysis, Gene expression, Cell line, Metabolic Bone Disease, and 15 moreHumans, Metabolic diseases, Mutation, Inborn errors of metabolism, Polymerase Chain Reaction, mRna expression levels, Fatty Acid, Amino Acid Profile, Metabolic pathway, Metabolic Disorder, High Sensitivity, Clinical Presentation, Gene expression profiling, fibroblasts, and premature termination codon
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Research Interests: Membrane Proteins, Molecular Genetics, Energy Metabolism, Western blotting, Humans, and 14 moreMutation, Female, Polymerase Chain Reaction, Cytochrome c oxidase, Infant, Alternative splicing, Proteins, Clinical Sciences, Alternative Splicing, Base Sequence, Mitochondrial Proteins, fibroblasts, hypertrichosis, and Neurodegenerative disorder
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Research Interests: Genetics, Immunohistochemistry, Mitochondria, Humans, Mutation, and 15 moreFemale, Male, Infant, ATP synthase, Mitochondrial Respiratory Chain, Electron Transport, Laboratory, Enzyme, Clinical Sciences, Adult, Multienzyme complexes, Immunophenotyping, Monoclonal Antibody, Immunoblotting, and fibroblasts
Controversy exists as to the clinical importance, cause, and disease specificity of the cytochrome oxidase (CO) activity reduction observed in some patients with Alzheimer's disease (AD). Although it is assumed that the... more
Controversy exists as to the clinical importance, cause, and disease specificity of the cytochrome oxidase (CO) activity reduction observed in some patients with Alzheimer's disease (AD). Although it is assumed that the enzyme is present in normal amount in AD, no direct measurements of specific CO protein subunits have been conducted. We measured protein levels of CO subunits encoded by mitochondrial (COX I, COX II) and nuclear (COX IV, COX VIc) DNA in autopsied brain of patients with AD whom we previously reported had decreased cerebral cortical CO activity. To assess disease specificity, groups of patients with spinocerebellar ataxia type I and Friedreich's ataxia were also included. As compared with the controls, mean protein concentrations of all four CO subunits were significantly decreased (-19 to -47%) in temporal and parietal cortices in the AD group but were not significantly reduced (-12 to -17%) in occipital cortex. The magnitude of the reduction in protein levels of the CO subunits encoded by mitochondrial DNA (-42 to -47%) generally exceeded that encoded by nuclear DNA (-19 to -43%). In the spinocerebellar ataxia disorders, COX I and COX II levels were significantly decreased in cerebellar cortex (-22 to -32%) but were normal or close to normal in cerebral cortex, an area relatively unaffected by neurodegeneration. We conclude that protein levels of mitochondrial- and nuclear-encoded CO subunits are moderately reduced in degenerating but not in relatively spared brain areas in AD and that the decrease is not specific to this disorder. The simplest explanation for our findings is that CO is decreased in human brain disorders as a secondary event in brain areas having reduced neuronal activity or neuronal/synaptic elements consequent to the primary neurodegenerative process.
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VON KLEIST-RETZOW, JÜRGEN-CHRISTOPH; YAO, JIANBO; TAANMAN, JAN-WILLEM; CHANTREL, KARINE; CHRETIEN, DOMINIQUE; CORMIER-DAIRE, VALÉRIE; RÖTIG, AGNÈS; MUNNICH, ARNOLD; RUSTIN, PIERRE; SHOUBRIDGE, ERIC A.
Research Interests: Medical Genetics, Membrane Proteins, Biological Sciences, DNA, Humans, and 13 moreMutation, Female, Infant, Proteins, Amino Acid Sequence, Base Sequence, Amino Acid Substitution Rates, Medical, Mitochondrial Proteins, DNA mutational analysis, Child preschool, hypertrichosis, and Medical and Health Sciences
Research Interests: Medical Genetics, Biological Sciences, Mitochondrial DNA, Humans, Mutation, and 15 moreCell Death, Genetic Association Studies, Differential Diagnosis, Steady state, Skeletal Muscle, Western blot, Base Sequence, Ribonucleotide Reductase, Polyacrylamide Gel Electrophoresis, Cell Cycle Proteins, Cohort Studies, Medical, DNA sequence, DNA mutational analysis, and Medical and Health Sciences
Research Interests: Membrane Proteins, Biological Chemistry, Biological Sciences, Humans, Mutation, and 10 moreCopper, Biological, Cytochrome c oxidase, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Heme, CHEMICAL SCIENCES, Oxidation-Reduction, Mitochondrial Proteins, fibroblasts, and Medical and Health Sciences
Research Interests: Sequence Analysis, Human, Mitochondrial DNA, Humans, Female, and 15 morePEO, Enzyme, Introns, Aged, Human reproduction, Skeletal Muscle, Adult, Large Scale, Reverse Transcriptase, Premature Ovarian Failure, Parkinson Disease, DNA Polymerase, Protein Biosynthesis, Medical and Health Sciences, and primary ovarian insufficiency
ABSTRACT We studied six unrelated children with depletion of mitochondrial DNA (mtDNA). They presented with Leigh syndrome, infantile hepatocerebral mtDNA depletion syndrome, or Alpers-Huttenlocher syndrome. Several genes have been... more
ABSTRACT We studied six unrelated children with depletion of mitochondrial DNA (mtDNA). They presented with Leigh syndrome, infantile hepatocerebral mtDNA depletion syndrome, or Alpers-Huttenlocher syndrome. Several genes have been implicated in mtDNA depletion. Screening of candidate genes indicated that all six patients were compound heterozygous for missense mutations in the gene for the catalytic subunit of DNA polymerase (POLG). Three of the identified mutations, c.3328C>T (p.H1110Y), c.3401A>G (p.H1134R), and c.3406G>A (p.E1136K), have not been reported earlier. To investigate the functional consequences of the mutations, we carried out a series of biochemical assays in cultured fibroblasts. These studies revealed that fibroblast cultures from the patients with infantile hepatocerebral mtDNA depletion syndrome progressively lost their mtDNA during culturing, whereas fibroblast cultures from patients presenting with Leigh syndrome or Alpers-Huttenlocher syndrome had reduced but stable levels of mtDNA. DNA polymerase activity was below the normal range in all patient cultures, except for one; however, this culture showed low levels of the heterodimeric enzyme and poor DNA polymerase processivity. Parental fibroblast cultures had normal catalytic efficiency of DNA polymerase , consistent with the observation that all carriers are asymptomatic. Thus, we report the first patient with Leigh syndrome caused by POLG mutations. The cell culture experiments established the pathogenicity of the identified POLG mutations and helped to define the molecular mechanisms responsible for mtDNA depletion in the patients' tissues. The assays may facilitate the identification of those patients in whom screening for POLG mutations would be most appropriate. Hum Mutat 0, 1–8, 2008. © 2008 Wiley-Liss, Inc.
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Research Interests: Membrane Proteins, Mitochondria, Biological Sciences, Humans, Mutation, and 15 moreFemale, Male, Human Molecular Genetics, Cytochrome c oxidase, Biogenesis, Gene, Pedigree, Gen, Consanguinity, Codon, Base Sequence, Genetic Mapping, DNA mutational analysis, Child preschool, and Medical and Health Sciences
A cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The... more
A cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The probe was labelled with Bio-11-dUTP and detected by fluorescence. Subsequent R-banding indicated that the cytochrome c oxidase subunit VIb gene is localized in band 19q13.1, extending the evidence that the human nuclear genes of cytochrome c oxidase are not clustered.
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... 7 November 1989; revised 15 March 1990; accepted 18 May 1990; Received by H. van Ormondt. Available online 27 January 2003. ... Aquila, 14., Misra, D., Eulitz, M. and Klingenber8, M.: Complete amino acid sequence ofthe ADP ATP carrier... more
... 7 November 1989; revised 15 March 1990; accepted 18 May 1990; Received by H. van Ormondt. Available online 27 January 2003. ... Aquila, 14., Misra, D., Eulitz, M. and Klingenber8, M.: Complete amino acid sequence ofthe ADP ATP carrier from beef heart mitochondria. ...
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Refractory convulsive status epilepticus in infancy and childhood is a rare emergency situation. Metabolic disorders frequently underlie this condition, in particular... more
Refractory convulsive status epilepticus in infancy and childhood is a rare emergency situation. Metabolic disorders frequently underlie this condition, in particular Alpers' disease caused by POLG1 mutations. Status epilepticus may be the first symptom. A pathognomonic electroencephalography (EEG) signature may facilitate diagnosis of Alpers' disease and allow timely avoidance of valproic acid, which is contraindicated in this disorder because it may trigger fatal liver failure. We present five patients with Alpers' disease caused by mutations in POLG1. Age of onset ranged from 7 months to 10 years. Three of the five children died after 3 to 12 months after onset of status epilepticus. Two of these had liver failure associated with use of valproic acid; liver transplantation in one child did not prevent a fatal neurologic outcome. Convulsive status epilepticus was the first obvious sign of Alpers' disease in all children. All had focal clonic and complex-focal seizures; four of them developed epilepsia partialis continua. In four children, initial EEG showed unilateral occipital rhythmic high-amplitude delta with superimposed (poly)spikes (RHADS). Magnetic resonance imaging (MRI) revealed cortical and thalamic involvement in all, although there were only discrete abnormalities in one child. Metabolic investigations remained normal in three children. Alpers' disease is an important differential diagnosis in childhood refractory convulsive status epilepticus. Its EEG hallmark of RHADS is important for timely diagnosis, management, and counseling.
Research Interests: Electrophysiology, Magnetic Resonance Imaging, MRI, Electroencephalography, Magnetic Resonance Spectroscopy, and 14 moreHumans, Child, Mutation, Female, Male, Infant, Epilepsia, Clinical Sciences, Tritium, Retrospective Studies, Status Epilepticus, Nuclear Magnetic Resonance Imaging, Neurosciences, and Nino
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Cytochrome c oxidase isolated from a wild-type yeast strain and a mutant in which the gene for subunit VIa had been disrupted were used to study the interaction of adenine nucleotides with the enzyme complex. At low ionic strength (25 mM... more
Cytochrome c oxidase isolated from a wild-type yeast strain and a mutant in which the gene for subunit VIa had been disrupted were used to study the interaction of adenine nucleotides with the enzyme complex. At low ionic strength (25 mM potassium phosphate), in the absence of nucleotides, the cytochrome c oxidase activity of the mutant enzyme lacking subunit VIa was higher than that of the wild-type enzyme. Increasing concentrations of ATP, in the physiological range, enhanced the cytochrome c oxidase activity of the mutant much more than the activity of the wild-type strain, whereas ADP, in the same concentration range, had no significant effect on the activity of the cytochrome c oxidase of either strain. These results indicate an interaction of ATP with subunit VIa in the wild-type enzyme that prevents the stimulation of the activity observed in the mutant enzyme. The stimulation of the mutant enzyme implies the presence of a second ATP binding site on the enzyme. Quantitative titrations with the fluorescent adenine nucleotide analogues 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) and 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) confirmed the presence of two binding sites for adenine nucleotides per monomer of wild-type cytochrome c oxidase and one binding site per monomer of mutant enzyme. Covalent photolabeling of yeast cytochrome c oxidase with radioactive 2-azido-ATP further confirmed the presence of an ATP binding site on subunit VIa.(ABSTRACT TRUNCATED AT 250 WORDS)
Research Interests: Biochemistry, Saccharomyces cerevisiae, Kidney, Mutation, Animals, and 15 moreCytochrome c oxidase, Spectrophotometry, Electron Transport, Cattle, Myocardium, Isoenzymes, Protein Conformation, Species Specificity, Amino Acid Sequence, Binding Site, Biochemistry and cell biology, Adenosine Triphosphate, Adenosine Diphosphate, Molecular Sequence Data, and Medical biochemistry and metabolomics
Cytochrome oxidase catalyses the reduction of oxygen to water. The mitochondrial enzyme contains up to 13 subunits, 11 in yeast, of which three, Cox1p, Cox2p and Cox3p, are mitochondrially encoded. The assembly pathway of this complex is... more
Cytochrome oxidase catalyses the reduction of oxygen to water. The mitochondrial enzyme contains up to 13 subunits, 11 in yeast, of which three, Cox1p, Cox2p and Cox3p, are mitochondrially encoded. The assembly pathway of this complex is still poorly understood. Its study in yeast has been so far impeded by the rapid turnover of unassembled subunits of the enzyme. In the present study, immunoblot analysis of blue native gels of yeast wild-type and Cox2p mutants revealed five cytochrome oxidase complexes or subcomplexes: a, b, c, d and f; a is likely to be the fully assembled enzyme; b lacks Cox6ap; d contains Cox7p and/or Cox7ap; f represents unassembled Cox1p; and c, observed only in the Cox2p mutants, contains Cox1p, Cox3p, Cox5p and Cox6p and lacks the other subunits. The identification of these novel cytochrome oxidase subcomplexes should encourage the reexamination of other yeast mutants.
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DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting... more
DGUOK [dG (deoxyguanosine) kinase] is one of the two mitochondrial deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA (mitochondrial DNA) replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of MDS (mtDNA depletion syndrome). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10 years old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14 and 10% residual activity as compared with controls with dG and deoxyadenosine as phosphate acceptors respectively. Similar apparent negative co-operativity in th...