Search PubMed⌕ Search

Biomedical subjects

L A Bindoff

Publications and source records attributed to L A Bindoff.

At least 37 records · Page 2Linked to original sources
PubMed1995

Presentation and clinical investigation of mitochondrial respiratory chain disease. A study of 51 patients.

Defects of the mitochondrial respiratory chain are associated with a great variety of clinical disorders. Whilst recognition of these conditions is increasing, the need for sophisticated biochemical and molecular studies has tended to limit both their investigation and diagnosis to a few specialist centres. Using a group of 51 patients with histochemically, biochemically and/or genetically defined respiratory chain defects, we have examined both the clinical heterogeneity of these disorders and how they may be investigated most effectively in non-specialist centres. We evaluated the use of the following routinely available clinical investigations--fasting intermediary metabolites (lactate, pyruvate, ketone bodies, etc.) in blood and cerebrospinal fluid, serum creatine kinase estimation, EMG, EEG, CT, MRI and histological/histochemical muscle biopsy analysis. Our studies show that, in addition to well-recognized syndromes (e.g. chronic progressive external ophthalmoplegia, mitochondrial encephalopathy lactic acidosis and stroke like episodes, and myoclonus epilepsy with ragged red-fibres, a significant number of patients present with non-specific encephalopathic disorders. Furthermore, even within those categories of respiratory chain disease which have been genetically defined, a wide variation of presenting symptoms and signs were found. Where there was initial doubt concerning the diagnosis, the following clinical features were helpful in suggesting respiratory chain disease: ophthalmoplegia; a maternal pattern of inheritance; the presence of myopathy or deafness in association with encephalopathy. Of the clinical investigations we assessed, elevated lactate in blood or cerebrospinal fluid and low density lesions in the basal ganglia were helpful in identifying patients with respiratory chain dysfunction. Histochemical analysis of muscle was, however, the single most useful investigation being diagnostic in patients with chronic progressive external ophthalmoplegia, Kearns-Sayre syndrome and myopathy, and of significant importance in patients presenting primarily with central nervous system disease. The results of our study are used to discuss the most appropriate approach to diagnosis of this group of disorders.

Adolescent↗
PubMed1994

Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein-ubiquinone oxidoreductase.

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) in the inner mitochondrial membrane accepts electrons from electron-transfer flavoprotein which is located in the mitochondrial matrix and reduces ubiquinone in the mitochondrial membrane. The two redox centers in the protein, FAD and a [4Fe4S]+2,+1 cluster, are present in a 64-kDa monomer. We cloned several cDNA sequences encoding the majority of porcine ETF-QO and used these as probes to clone a full-length human ETF-QO cDNA. The deduced human ETF-QO sequence predicts a protein containing 617 amino acids (67 kDa), two domains associated with the binding of the AMP moiety of the FAD prosthetic group, two membrane helices and a motif containing four cysteine residues that is frequently associated with the liganding of ferredoxin-like iron-sulfur clusters. A cleavable 33-amino-acid sequence is also predicted at the amino terminus of the 67-kDa protein which targets the protein to mitochondria. In vitro transcription and translation yielded a 67-kDa immunoprecipitable product as predicted from the open reading frame of the cDNA. The human cDNA was expressed in Saccharomyces cerevisiae, which does not normally synthesize the protein. The ETF-QO is synthesized as a 67-kDa precursor which is targeted to mitochondria and processed in a single step to a 64-kDa mature form located in the mitochondrial membrane. The detergent-solubilized protein transfers electrons from ETF to the ubiquinone homolog, Q1, indicating that both the FAD and iron-sulfur cluster are properly inserted into the heterologously expressed protein.

Amino Acid Sequence↗
PubMed1994

An evaluation of the measurement of the activities of complexes I-IV in the respiratory chain of human skeletal muscle mitochondria.

The measurement of individual respiratory chain complexes is an important component of the investigation of diseases due to mitochondrial dysfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these assays to provide sensitive and reliable diagnostic techniques, particularly in situations where a partial interruption at a single complex needs to identified. Using several established methods of membrane disruption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium phosphate buffer, whereas complex III and IV activities are markedly increased by the addition of the detergent n-dodecyl-beta-D-maltoside. Complex I activity is measured in the presence of 2.5 mg.ml-1 bovine serum albumin, which increases rotenone sensitivity, and we have shown that NADH-cytochrome b5 reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Complex II activity is measured after preincubation of the mitochondrial fraction with succinate to fully activate the complex. Complex I and III activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been used to establish a control range.

Cytochromes b5↗
PubMed1994

Biochemical and molecular studies of mitochondrial function in diabetes insipidus, diabetes mellitus, optic atrophy, and deafness.

OBJECTIVE: To determine if diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD) combined with a cerebellar syndrome is associated with a systemic disorder of respiratory chain function as found in similar genetic syndromes. CASE: A muscle biopsy was taken from a patient with DIDMOAD, and a mitochondrial fraction was prepared. Respiratory chain function was assessed by analysis of intermediary metabolites, histochemical analysis of muscle biopsy, measurement of the activity of individual respiratory chain complexes, oxidative flux through the respiratory chain, and cytochrome concentration and compared with a population with normal respiratory chain function. Mitochondrial DNA from skeletal muscle, brain, and pancreas was examined for major rearrangements and specific point mutations. Brain tissue was examined neuropathologically for abnormalities, particularly those previously described in association with DIDMOAD. RESULTS: No abnormality was found in mitochondrial oxidation, individual complex activity, or cytochrome concentration. Histochemical analysis and electron microscopy showed no abnormality known to be associated with mitochondrial dysfunction. A single-base substitution at position 12308 of the mitochondrial genome was found, but no major rearrangement of mitochondrial DNA was demonstrated. Neuropathological examination revealed severe demyelination and gliosis in the optic nerves and loss of Purkinje cells associated with gliosis in the white matter in the cerebellum. CONCLUSIONS: We have found no evidence that DIDMOAD is associated with a systemic abnormality of respiratory chain function. The mitochondrial DNA single-base substitution noted is likely to be a polymorphism rather than a pathogenic point mutation. We have confirmed that DIDMOAD may be associated with a neurodegenerative disorder, but the cause of this remains undetermined.

Adult↗
PubMed1993

An antisense oligodeoxynucleotide approach to investigate the function of the nuclear-encoded subunits of human cytochrome c oxidase.

On treatment of human liver Hep G2 cells with thiamphenicol, intramitochondrial levels of cytochrome c oxidase (COX) nuclear gene products were shown to decrease in tandem with the mitochondrially-encoded subunits except, however, for subunit IV which proved to be remarkably stable. This nonspecific decrease in subunit level was utilized to assess the function of subunit VIIa-L. Thiamphenicol-pretreated cells were bathed in anti-VIIa-L oligonucleotides and the recovery rate of COX activity compared to control oligomer-untreated cells or cells with a similar concentration of a randomized oligomer. No difference in recovery rate was noted for the first two days post oligomer addition, after which a sharp decrease in recovery was noted for the anti-VIIa-L treated cells only. We conclude: (i) subunit IV is stable in the absence of other COX components, (ii) subunit VIIa-L is required for maintaining normal levels of COX activity, (iii) an antisense oligodeoxynucleotide approach for assessing the function of COX nuclear gene products is made feasible by first reducing levels of the complex and then assaying its recovery rate in the presence of transcript-specific antisense oligonucleotides.

Base Sequence↗
PubMed1993

Abnormal RNA processing associated with a novel tRNA mutation in mitochondrial DNA. A potential disease mechanism.

A patient with a mitochondrial myopathy and biochemically proven profound complex I deficiency has a new mutation in mtDNA. This A-to-G transition at position 3302, involving the aminoacyl stem of tRNA(Leu(UUR)), is associated with abnormal mitochondrial RNA processing. Northern analysis demonstrates marked accumulation of a polycistronic RNA precursor containing sequence for 16 S rRNA, tRNA(Leu(UUR)), and ND1. Comparison of skeletal muscle and skin fibroblasts suggests that the processing error may be quantitatively less severe in this tissue, and biochemical analysis shows that fibroblasts do not express a biochemical defect despite containing the mutation. Important qualitative differences in the processing of this RNA precursor were found when comparing muscle and skin fibroblasts. In muscle, processing appears to occur first at the 5'-end of the tRNA, generating 16 S rRNA plus a tRNA + ND1 intermediate. In fibroblasts, processing occurs at the 3'-end of the tRNA, generating a 16 S rRNA + tRNA intermediate. We suggest that the mutation at position 3302 induces abnormal mitochondrial RNA processing that is linked to the biochemical defect (profound loss of complex I activity), either by qualitative or quantitative abnormalities in the ND1 message. The restriction to skeletal muscle of both the processing error and the biochemical defect suggests that the observed tissue differences in RNA processing play a protective role in skin fibroblasts.

Adult↗
PubMed1993

Cytochrome c oxidase activity in single muscle fibers: assay techniques and diagnostic applications.

Microphotometric enzyme assay was used to study cytochrome c oxidase activity in single human skeletal muscle fibers. The assay techniques combine the precise localization of enzyme activity provided by histochemical methodology with the precise quantitation of a sensitive assay system. Abnormalities of cytochrome c oxidase were investigated using microphotometric enzyme assay in 12 patients with Kearns-Sayre syndrome, chronic progressive external ophthalmoplegia, or Leigh's syndrome. Control values were obtained using muscle biopsy specimens from 20 juvenile and 18 adult subjects with no evidence of neuromuscular disease. In the patients with Leigh's syndrome due to cytochrome c oxidase deficiency, the abnormality was found to be expressed uniformly throughout the muscle fiber population. In contrast, patients with Kearns-Sayre syndrome or chronic progressive external ophthalmoplegia showed abnormal heterogeneity of cytochrome c oxidase activity. In many cases, extreme degrees of variability were seen, with fibers containing high activity adjacent to fibers with no detectable activity. Mitochondrial DNA analysis showed that most of the patients with Kearns-Sayre syndrome and chronic progressive external ophthalmoplegia had major rearrangements of mitochondrial DNA. It was concluded that the extreme variability of cytochrome c oxidase activity detected using microphotometric enzyme assay was an indicator of a probable abnormality of mitochondrial DNA. Conversely, cytochrome c oxidase defects in muscle which show a homogeneous distribution are more likely to be associated with defects of the nuclear genome.

Adolescent↗
PubMed1992

The sequence of the flavoprotein subunit of bovine heart succinate dehydrogenase.

The cDNA sequence of the flavoprotein subunit of bovine heart succinate dehydrogenase is reported. This is the first complete eukaryotic sequence of the flavoprotein subunit to be characterized, and it encodes a 665-amino acid protein that consists of a presequence and a 621-residue mature protein. The deduced bovine sequence shows homology to the corresponding peptides of prokaryotic succinate dehydrogenase and the related fumarate reductases; in particular, there is good overall homology (48%) to the flavoprotein subunit of Escherichia coli succinate dehydrogenase. The conserved sequences comprising the active site and those involved in FAD binding are also found in the bovine protein. The active site of the bovine polypeptide contains a cysteine that confers sensitivity of the enzyme to sulfhydryl reagents; this cysteine is only present in some sequences and thus provides a discriminatory biochemical marker. A putative flavoprotein subunit of human placental succinate dehydrogenase (partial sequence) that lacks this critical cysteine (Malcovati, M., Marchetti, T., Zanelli, T., and Tenchini, M. L. (1991) in Flavins and Flavoproteins 1990 (Curti, B., Ronchi, S., and Zanetti, G., eds) pp. 727-730, Walter de Gruyter & Co., Berlin) has only 16% homology to the bovine heart flavoprotein subunit. However, we show that the enzyme from human placenta is as sensitive to N-ethylmaleimide as that from bovine tissues. In addition, a transcript in human placenta and muscle hybridizes to the bovine heart flavoprotein cDNA and is the same size as that in bovine tissues.

Amino Acid Sequence↗
PubMed1992

Leigh syndrome associated with a deficiency of the pyruvate dehydrogenase complex: results of treatment with a ketogenic diet.

A one-year-old boy suffering from intermittent lactic acidosis, muscular hypotonia, horizontal gaze paralysis and spasticity in both legs had low activity of the pyruvate dehydrogenase complex associated with low amounts of immunoreactive E 1 alpha and E 1 beta. Leigh syndrome was diagnosed on the basis of the clinical and biochemical abnormalities and the typical lesions observed on MRI of the brain. Treatment with a ketogenic diet was associated with clinical and biochemical amelioration. A striking improvement of the cerebral lesions was observed by neuro-imaging.

Brain↗
PubMed1992

Combined enzyme defect of mitochondrial fatty acid oxidation.

A young girl presented with recurrent episodes of muscle weakness culminating in a severe attack of generalized muscle weakness. In the muscle mitochondria from the patient there was an abnormal pattern of intermediates of beta-oxidation with an accumulation of 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters, and 3-oxoacylcarnitines. There was low activity of long-chain 3-hydroxyacyl-CoA dehydrogenase in mitochondria from all tissues. The activity of long-chain 2-enoyl-CoA hydratase was low in muscle mitochondria and 3-oxoacyl-CoA thiolase activity measured with 3-oxohexadecanoyl-CoA as substrate was low in fibroblast, muscle, and cardiac mitochondria but only partial deficiency was present when the activity was measured with 3-oxooctanoyl-CoA. The activity of the long-chain 3-hydroxyacyl-CoA dehydrogenase and long-chain 3-oxoacyl-CoA thiolase in fibroblasts from the patient's parents was intermediate between those of controls and the patient. The patient has a combined defect of the long-chain 3-hydroxyacyl-CoA dehydrogenase, long-chain 3-oxoacyl-CoA thiolase, and long-chain 2-enoyl-CoA hydratase which appears to be inherited in an autosomal recessive manner. This suggests there is a multifunctional enzyme catalyzing these activities in human mitochondria and that this enzyme is deficient in our patient.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1991

Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation.

We have used radio-high pressure liquid chromatography to study the acyl-CoA ester intermediates and the acylcarnitines formed during mitochondrial fatty acid oxidation. During oxidation of [U-14C]hexadecanoate by normal human fibroblast mitochondria, only the saturated acyl-CoA and acylcarnitine esters can be detected, supporting the concept that the acyl-CoA dehydrogenase step is rate-limiting in mitochondrial beta-oxidation. Incubations of fibroblast mitochondria from patients with defects of beta-oxidation show an entirely different profile of intermediates. Mitochondria from patients with defects in electron transfer flavoprotein and electron transfer flavoprotein:ubiquinone oxido-reductase are associated with slow flux through beta-oxidation and accumulation of long chain acyl-CoA and acylcarnitine esters. Increased amounts of saturated medium chain acyl-CoA and acylcarnitine esters are detected in the incubations of mitochondria with medium chain acyl-CoA dehydrogenase deficiency, whereas long chain 3-hydroxyacyl-CoA dehydrogenase deficiency is associated with accumulation of long chain 3-hydroxyacyl- and 2-enoyl-CoA and carnitine esters. These studies show that the control strength at the site of the defective enzyme has increased. Radio-high pressure liquid chromatography analysis of intermediates of mitochondrial fatty acid oxidation is an important new technique to study the control, organization and defects of the enzymes of beta-oxidation.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1991

Multiple defects of the mitochondrial respiratory chain in a mitochondrial encephalopathy (MERRF): a clinical, biochemical and molecular study.

We describe a young man with a progressive neurological disorder including myoclonus, mental retardation, muscle weakness and a mitochondrial myopathy (myoclonus epilepsy and ragged red fibres--MERRF). Multiple abnormalities of the mitochondrial respiratory chain in skeletal muscle are shown by direct measurement of the flux through the individual complexes, low-temperature redox spectroscopy and decreased immunodetectable subunits of complexes I and IV by immunoblotting. No abnormality of mitochondrial DNA was found. This is the first report of combined defects of complexes I, III and IV as a cause of this clinical syndrome. However, we propose that the occurrence of multiple respiratory chain defects may be more common than previously recognised and that this particular combination of defects, involving complexes I, III and IV, may be the predominant biochemical abnormality in MERRF.

Adult↗
PubMed1991

Respiratory chain abnormalities in skeletal muscle from patients with Parkinson's disease.

Parkinson's disease is one of the commonest neurodegenerative disorders in Western society. Although the neuropathological changes have been well documented, the underlying biochemical defect is unknown. Toxins may play a part in the aetiology of this disorder. It has been shown that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a Parkinson-like syndrome in both man and primates and 1-methyl-4-phenylpyridine (MPP+), a metabolite of MPTP, inhibits NADH-ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain. We studied mitochondrial respiratory chain function in skeletal muscle from patients with Parkinson's disease because, like brain, it has a high dependence on oxidative metabolism. Our results show low activity in all complexes studied (I, II and IV). The implications of these findings are discussed in relation to the aetiology of Parkinson's disease.

Adult↗