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Biomedical subjects

D R Thorburn

Publications and source records attributed to D R Thorburn.

At least 55 records · Page 3Linked to original sources
PubMed1996

Leigh syndrome: clinical features and biochemical and DNA abnormalities.

We investigated the etiology of Leigh syndrome in 67 Australian cases from 56 pedigrees, 35 with a firm diagnosis and 32 with some atypical features. Biochemical or DNA defects were determined in both groups, ie, 80% in the tightly defined group and 41% in the "Leigh-like" group. Eleven patients had mitochondrial DNA point mutations (nucleotide [nt] 8993 T to G, nt 8993 T to C, or nt 8344 A to G) and 1 Leigh-like patient had a heteroplasmic deletion. Twenty-nine patients had enzyme defects, ie, 13 respiratory chain complex I, 9 complex IV, and 7 pyruvate dehydrogenase complex (PDHC). Complex I deficiency is more common than recognized previously. Six PDHC-deficient patients had mutations in the X-chromosomal gene encoding the E1alpha subunit of PDHC. Parental consanguinity suggested autosomal recessive inheritance in two complex IV-deficient sibships. We found no strong correlation between the clinical features and basic defects. An assumption of autosomal recessive inheritance (frequently made in the past) would have been wrong in nearly one-half (11 of 28 tightly defined and 18 of 41 total patients) of those in whom a cause was found. A specific defect must be identified if reliable genetic counseling is to be provided.

Brain↗
PubMed1995

mtDNA deletion in a patient with symptoms of mitochondrial cytopathy but without ragged red fibers.

We describe a heteroplasmic 4237-bp mitochondrial DNA (mtDNA) deletion in an 11-year-old girl who has suffered from progressive illness since birth. Her clinical features include global developmental delay with regression, brainstem dysfunction, lactic acidosis, and a history of pancytopenia and failure to thrive. The deletion spanned nt 9498 to nt 13734 and was flanked by a 12-bp direct repeat. Southern blot analysis also revealed an altered ApaI restriction site caused by a G --> A nucleotide substitution at nt 1462 in the 12S rRNA gene. This homoplasmic nucleotide change was presumed to be a mtDNA nucleotide variant. No abnormalities of mitochondrial ultrastructure or distribution were observed, although mild deficiencies were noted for complexes IV, II + III, and I of the mitochondrial respiratory chain. The absence of ragged red fibers and COX-negative fibers in this patient shows that mtDNA deletions do not always result in these classical hallmarks of mitochondrial cytopathies.

Base Sequence↗
PubMed1995

A topoisomerase II cleavage site is associated with a novel mitochondrial DNA deletion.

Mitochondrial myopathies and encephalopathies can be caused by nucleotide substitutions, deletions or duplications of the mitochondrial DNA (mtDNA). In one such disorder, Kearns-Sayre Syndrome (KSS), large-scale heteroplasmic mtDNA deletions are often found. We describe a 14-year-old boy with clinical features of KSS, plus some additional features. Analysis of the entire mitochondrial genome by the polymerase chain reaction and Southern blotting revealed a 7864-bp mtDNA deletion, heteroplasmic in its tissue distribution. DNA sequencing established that the deletion was between nucleotides 6238 and 14,103, and flanked by a 4-bp (TCCT) direct repeat sequence. Deletions between direct repeats have been hypothesised to occur by a slipped-mismatching or illegitimate recombination event, or following the DNA cleavage action of topoisomerase II. Analysis of the gene sequence in the region surrounding the mtDNA deletion breakpoint in this patient revealed the presence of putative vertebrate topoisomerase II sites. We suggest that direct repeat sequences, together with putative topoisomerase II sites, may predispose certain regions of the mitochondrial genome to deletions.

Adolescent↗
PubMed1995

An amino acid substitution in the pyruvate dehydrogenase E1 alpha gene, affecting mitochondrial import of the precursor protein.

A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1 alpha deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1 alpha immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1 alpha targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1 alpha sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease.

Amino Acids↗
PubMed1994

A novel mtDNA deletion in an infant with Pearson syndrome.

Pearson syndrome is a multisystem mitochondrial disorder of infancy that is associated with deletions in the mitochondrial DNA (mtDNA) genome. We report a study on a male infant with Pearson syndrome. Assessment of oxidative phosphorylation activity indicated combined respiratory-chain defects in muscle, liver and fibroblasts; in particular, activity of complex I was reduced. Analysis of the patient's mtDNA identified a novel heteroplasmic 2.461 kb deletion, present at levels greater than 50% of the total mtDNA in the tissues examined. The deletion spanned nucleotides 10368 to 12828 and was flanked by a 3 bp GCC direct repeat sequence. Gene sequences affected are subunits 3, 4, 4L and 5 of complex I, and tRNAs for arginine, histidine, serine and leucine. Our findings correlate with the multiorgan involvement observed in Pearson syndrome.

Base Sequence↗
PubMed1993

A fluorimetric assay for succinic semialdehyde dehydrogenase activity suitable for prenatal diagnosis of the enzyme deficiency.

Succinic semialdehyde dehydrogenase (SSAD) is an enzyme involved in the turnover of the neurotransmitter 4-aminobutyrate (GABA). Deficiency of SSAD results in developmental delay, ataxia, seizures and 4-hydroxybutyric aciduria. We have developed a simple fluorimetric assay for the enzyme and applied it to measurement of SSAD activity in a range of cell types often used for prenatal and postnatal diagnosis of enzyme defects. Lymphocytes from children with SSAD deficiency were found to have < 5% of the activity found in lymphocytes from normal children. Heterozygotes are asymptomatic and have intermediate enzyme activities. Although SSAD activity has been detected previously in uncultured chorionic villi, we found that SSAD was not expressed in cultured chorionic villus cells nor in some fibroblast-like amniocytes from control fetuses. Lymphocytes from fetal blood and non-fibroblastic amniocytes have high SSAD activities, and should be suitable for prenatal diagnosis of SSAD deficiency.

Adolescent↗
PubMed1993

Malonyl coenzyme A decarboxylase deficiency.

Two new cases of malonyl coenzyme A (CoA) decarboxylase deficiency are described. Hitherto, the worldwide experience of the disorder has been confined to reports on two affected Australian children. The new cases are Scots born and are the offspring of consanguinous parents of Scots/Irish origin. They were investigated during episodes of vomiting and febrile convulsions associated with concomitant developmental delay. Malonic aciduria and grossly reduced malonyl CoA decarboxylase activity were demonstrated and the total ion current chromatograms of urinary organic acid profiles obtained by gas chromatography-mass spectrometry are presented. The clinical and biochemical features of the Scots and Australian patients are compared.

Adipates↗
PubMed1991

The loss of enzyme activity from erythroid cells during maturation.

Erythrocyte enzyme activities in patients with reticulocytosis or transient erythroblastopenia show that loss of age-dependent enzyme activity is not a simple exponential process occurring throughout the life-span of the cell. In vivo studies of reticulocyte maturation in rabbits indicate that there are multiple mechanisms of enzyme decay, and that proteolysis continues after the maturation of (morphologically recognisable) reticulocytes into young erythrocytes. Most reticulocyte hexokinase is degraded by lysosomal proteolysis, apparently triggered by an initial attack by lipoxygenase.

Adenosine Triphosphate↗
PubMed1990

13C and 31P NMR studies of the pentose phosphate pathway in human erythrocytes.

31P NMR was used to monitor the concentrations of some of the intermediates of the non-oxidative pentose phosphate pathway (PPP) during the dissimilation of inosine and phosphate in dilute haemolysates. The temperature dependence of the ketone/hydrate ratio in dihydroxyacetone phosphate and the relative proportions of the isomers of sedoheptulose 1,7-bisphosphate were measured. The 31P NMR time courses were compared with the simulations obtained with a computer model of the modified F-type PPP.

Aldose-Ketose Isomerases↗
PubMed1989

Decay of hexokinase during reticulocyte maturation: is oxidative damage a signal for destruction?

Proteolysis of hexokinase in cell-free systems prepared from rabbit reticulocytes has been shown previously to be ATP-dependent and apparently mediated by the ubiquitin system (Magnani et al. J. Biol.Chem.261, 8327-8333). We have investigated this phenomenon, but found no substantial loss of hexokinase in cell-free systems prepared from fresh lysates. Storage of lysates at -20 degrees C or addition of a free radical generating system was required to demonstrate rapid ATP-dependent decay. It appears that initial oxidative damage to hexokinase does not abolish its activity but allows it to be recognized by an ATP-dependent proteolytic system. The relevance of this mechanism to in vivo degradation of hexokinase is discussed.

Adenosine Triphosphate↗
PubMed1989

Comparison of computer simulations of the F-type and L-type non-oxidative hexose monophosphate shunts with 31P-NMR experimental data from human erythrocytes.

Mathematical modelling was used to predict the behaviour of the two most favoured schemes for the operation of the non-oxidative hexose monophosphate shunt (HMS), the F-type and the L-type pathways. The models simulate the time courses of sugar-phosphate concentrations when various substrates are metabolized via each pathway. A 31P-NMR technique, with which to observe time courses of concentrations of sugar phosphates in a human red cell lysate, was developed. The accuracy of each hypothesised scheme was then evaluated by comparing predicted with observed data. The results were more consistent with time courses of sugar-phosphate levels predicted by the F-type (classical) pathway than those predicted by the L-type model. However, the accumulation of sedoheptulose 1,7-bisphosphate when a haemolysate was incubated with ribose 5-phosphated showed that the F-type pathway is not a complete description of the system of reactions. Transaldolase was demonstrated to be essential for the normal metabolism of sugar phosphates by haemolysates. The effects of the heat-inactivation of transaldolase on the metabolism of sugar phosphates were accurately predicted by the F-type model. The relevance of attempting to describe the reaction of the non-oxidative HMS as a distinct 'pathway' or 'cycle' is discussed.

Computer Simulation↗
PubMed1987

Determination of erythrocyte glucose 1,6-bisphosphate--a comparison of two methods using a centrifugal analyzer.

Glucose 1,6-bisphosphate is a key effector of human erythrocyte glycolysis, yet to date its assay has been problematical. Two methods for measuring glucose 1,6-bisphosphate were modified and adapted to a centrifugal analyser and the inaccuracy and imprecision of each method were compared. One assay, based on stimulation of phosphoglucomutase, was shown to underestimate the erythrocyte levels by approximately 5% due to inhibition of the mutase by endogenous 2,3-bisphosphoglycerate. An alternative chemical/enzymic method, consisting of acid hydrolysis of glucose 1,6-bisphosphate to glucose 6-phosphate and subsequent determination of the monophosphate was modified by omitting an initial alkaline hydrolysis step and by increasing the duration of acid hydrolysis. The modified method also enabled the determination of erythrocyte glucose 6-phosphate. The normal concentration of glucose 1,6-bisphosphate in whole blood and in washed human erythrocytes, determined using the more accurate chemical/enzymic method was 83 +/- 5 mumol/l cells and 86 +/- 4 mumol/l cells, respectively; the corresponding concentrations of glucose 6-phosphate were 26 +/- 2 mumol/l cells and 15 +/- 3 mumol/l cells.

Centrifugation↗
PubMed1987

Glutathione regeneration in density-fractionated human erythrocytes.

When human erythrocytes were separated into fractions of increasing density, the measured rate of glutathione regeneration did not decline as much as predicted by computer simulation. This phenomenon can be explained by the heterogeneous distribution of hexokinase activity in the less-dense fractions.

Cell Separation↗
PubMed1987

Computer simulation of the metabolic consequences of the combined deficiency of 6-phosphogluconolactonase and glucose-6-phosphate dehydrogenase in human erythrocytes.

Computer simulation of the human erythrocyte hexose monophosphate shunt was used to investigate whether the severity of hemolysis, caused by deficiency of glucose-6-phosphate dehydrogenase, could be influenced by an accompanying deficiency of 6-phosphogluconolactonase. The known kinetics of the enzymes involved in the oxidative part of the hexose monophosphate shunt suggest that partial deficiency of 6-phosphogluconolactonase would not restrict the rate of reduced nicotinamide-adenine dinucleotide phosphate production significantly. It is therefore concluded that the metabolic consequences of a combined deficiency of the two enzymes are unlikely to be responsible for the interpatient variability of the hematologic response to deficiency of glucose-6-phosphate dehydrogenase.

Carboxylic Ester Hydrolases↗