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Biomedical subjects

J B Clark

Publications and source records attributed to J B Clark.

At least 19 recordsLinked to original sources
PubMed1995

Immunocytochemical evidence for a mitochondrially located nitric oxide synthase in brain and liver.

In this article we demonstrate the immunocytochemical localization of nitric oxide synthase in non-synaptosomal rat brain mitochondria and rat liver mitochondria, using a monoclonal antibody directed against the endothelial form of nitric oxide synthase. The possibility that nitric oxide synthase located in mitochondria is involved in the regulation of mitochondrial oxidative phosphorylation is discussed.

Amino Acid Oxidoreductases↗
PubMed1995

Nitric oxide produced by activated astrocytes rapidly and reversibly inhibits cellular respiration.

Cultured astrocytes, activated to express the inducible form of nitric oxide synthase, produced up to 1 microM nitric oxide (NO) measured by a NO-selective electrode, while non-activated cells produced no detectable NO. The production of NO was associated with an inhibition of cellular respiration, measured simultaneously by an oxygen electrode. The inhibition of respiration was rapidly reversed by inhibiting the NO synthase or by binding the NO with haemoglobin. The respiratory inhibition had an NO, oxygen and substrate dependence consistent with NO-inhibition at cytochrome oxidase. This is the first demonstration that cells can reversibly inhibit mitochondrial respiration via NO production. This inhibition is large and potentially important in a range of pathophysiological conditions.

Animals↗
PubMed1995

Late-infantile Batten disease: purification of the subunit c of the mitochondrial ATP synthase from storage material.

The accumulation of subunit c of the mitochondrial ATP synthase in late-infantile neuronal lipofuscinosis (LINCL) and juvenile neuronal lipofuscinosis (JNCL) is well documented. The purification of the subunit from diverse sources has been reported previously, although not from the brain of Batten disease patients. This proteolipid has now been purified from late-infantile Batten disease brain. The procedures used were an original combination of the conventional solubilisation, differential centrifugation, organic solvent extractions, preparative gel electrophoresis, and FPLC. Gel filtration of the purified protein indicated molecular mass equal to or greater than 2 x 10(6) Da; however, electrophoresis of this pure protein suggested a molecular mass of approximately 3,500 Da, which is a characteristic of subunit c. The pure protein may be solubilised in aqueous buffer containing < 1% lithium dodecyl sulphate (LDS). The protein binds dicyclohexylcarbodiimide (DCCD) and shows immunoreactivity to antibodies raised against ovine storage bodies.

Blotting, Western↗
PubMed1995

Mitochondrial DNA (mtDNA) diseases: correlation of genotype to phenotype.

This study examines the relationship of genotype to phenotype in 14 unselected patients who were found to harbour the A3243G transition in the mitochondrial transfer RNALeu(UUR) gene commonly associated with the syndrome of mitochondrial encephalopathy, lactic acidosis and strokes (MELAS). Only 6 of the 14 cases (43%) had seizures and recurrent strokes, the core clinical features of the MELAS phenotype. Of the remaining cases, four had an encephalomyopathy with deafness, ataxia and dementia, two had syndromes with progressive external ophthalmoplegia and two had limb weakness alone. Even within the MELAS subgroup, the majority of patients had one or more clinical manifestations considered to be atypical of the MELAS syndrome. They included developmental delay, ophthalmoparesis, pigmentary retinopathy and intestinal pseudo-obstruction. The proportion of mutant mitochondrial DNA (mtDNA) in muscle was generally higher in patients with recurrent strokes than in those without strokes, the highest levels being observed in MELAS cases with early onset disease. Studies of isolated muscle mitochondria identified a range of respiratory chain abnormalities mostly involving Complex I; immunoblots of Complex I in 3 of 10 cases showed selective loss of specific subunits encoded by nuclear genes. In the group as a whole, however, no clear correlations were observed between the severity or extent of the respiratory chain abnormality and clinical phenotype or the proportion of mutant mtDNA in biopsied skeletal muscle. These discrepancies suggest that, in patients harbouring the common MELAS3243 mutation, differences in heteroplasmy and the proportions of mutant mtDNA may not be the sole determinants of disease expression and that additional genetic mechanisms are involved in defining the range of clinical and biochemical phenotypes associated with this aberrant mitochondrial genome.

Adolescent↗
PubMed1995

Tetrahydrobiopterin deficiency and brain nitric oxide synthase in the hph1 mouse.

Tetrahydrobiopterin (BH4) is the cofactor for the aromatic amino acid monoxygenase group of enzymes and for all known isoforms of nitric oxide synthase (NOS). Inborn errors of BH4 metabolism lead to hyperphenylalaninaemia and impaired catecholamine and serotonin turnover. The effects of BH4 deficiency on brain nitric oxide (NO) metabolism are not known. In this study we have used the hph-1 mouse, which displays GTP cyclohydrolase deficiency, to study the effects of BH4 deficiency on brain NOS. In the presence of exogenous BH4, NOS specific activity was virtually identical in the control and hph-1 preparations. However, omission of BH4 from the reaction buffer led to a significant 20% loss of activity in the hph-1 preparations only. The Km for arginine was virtually identical for the control and hph-1 NOS when BH4 was present in the reaction buffer. In the absence of cofactor, the Km for arginine was 3-fold greater for control and 5-fold greater for hph-1 preparations. It is concluded that (a) BH4 does not regulate the intracellular concentration of brain NOS; (b) less binding of BH4 to NOS occurs in BH4 deficiency states; (c) BH4 has a potent effect on the affinity of NOS for arginine; and (d) the availability of arginine for NOS activity may become severely limiting in BH4 deficiency states. Since, in the presence of suboptimal concentrations of BH4 or arginine, NOS may additionally form oxygen free-radicals, it is postulated that in severe BH4 deficiency states NO formation is impaired and the central nervous system is subjected to increased oxidative stress.

Amino Acid Oxidoreductases↗
PubMed1995

Depletion of brain glutathione is accompanied by impaired mitochondrial function and decreased N-acetyl aspartate concentration.

The effect of depletion of reduced glutathione (GSH) on brain mitochondrial function and N-acetyl aspartate concentration has been investigated. Using pre-weanling rats, GSH was depleted by L-buthionine sulfoximine administration for up to 10 days. In both whole brain homogenates and purified mitochondrial preparations complex IV (cytochrome c oxidase) activity was decreased, by up to 27%, as a result of this treatment. In addition, after 10 days of GSH depletion, citrate synthase activity was significantly reduced, by 18%, in the purified mitochondrial preparations, but not in whole brain homogenates, suggesting increased leakiness of the mitochondrial membrane. The whole brain N-acetyl aspartate concentration was also significantly depleted at this time point, by 11%. It is concluded that brain GSH is important for the maintenance of optimum mitochondrial function and that prolonged depletion leads also to loss of neuronal integrity. The relevance of these findings to Parkinson's disease and the inborn errors of glutathione metabolism are also discussed.

Animals↗
PubMed1995

High resolution proton NMR spectroscopy of multiple sclerosis lesions.

Tissue from postmortem multiple sclerosis and normal control brains was extracted with perchloric acid and analysed using proton NMR spectroscopy. The content of N-acetyl-derived groups (the sum of N-acetyl-aspartate, acetate, and N-acetylaspartylglutamate) was decreased in multiple sclerosis plaques compared with normal control white matter (mean, 4.36 vs. 6.64 mumol/g wet weight). In normal appearing white matter adjacent to plaques a corresponding decrease was seen, with no change in white matter distant from plaques. A decrease in the content of total creatine was observed in multiple sclerosis plaques in comparison with normal control white matter (mean, 4.64 vs. 6.56 mumol/g wet weight), which correlated strongly with the decrease in N-acetyl-derived groups. No changes in other metabolites such as total choline or myo-inositol were seen. The decreases in content of N-acetyl-derived groups are in agreement with observations from in vivo proton NMR spectroscopy in multiple sclerosis patients. The decrease in total creatine is in contrast to most of the observations made in vivo where total creatine is assumed to be unchanged and metabolite levels are often expressed as a total creatine ratio. The use of a total creatine ratio in vivo could lead to an underestimation of reductions in N-acetylaspartate and an apparent increase in other metabolites in the multiple sclerosis lesion.

Adult↗
PubMed1995

Effect of peroxynitrite on the mitochondrial respiratory chain: differential susceptibility of neurones and astrocytes in primary culture.

The effect of the neurotoxic nitric oxide derivative, the peroxynitrite anion (ONOO-), on the activity of the mitochondrial respiratory chain complexes in cultured neurones and astrocytes was studied. A single exposure of the neurones to ONOO- (initial concentrations of 0.01-2.0 mM) caused, after a subsequent 24-h incubation, a dose-dependent decrease in succinate-cytochrome c reductase (60% at 0.5 mM) and in cytochrome c oxidase (52% at 0.5 mM) activities. NADH-ubiquinone-1 reductase was unaffected. In astrocytes, the activity of the mitochondrial complexes was not affected up to 2 mM ONOO-. Citrate synthase was unaffected in both cell types under all conditions studied. However, lactate dehydrogenase activity released to the culture medium was increased by ONOO- in a dose-dependent manner (40% at 0.5 mM ONOO-) from the neurones but not from the astrocytes. Neuronal glutathione concentration decreased by 39% at 0.1 mM ONOO-, but astrocytic glutathione was not affected up to 2 mM ONOO-. In isolated brain mitochondria, only succinate-cytochrome c reductase activity was affected (22% decrease at 1 mM ONOO-). We conclude that the acute exposure of ONOO- selectively damages neurones, whereas astrocytes remain unaffected. Intracellular glutathione appears to be an important factor for ameliorating ONOO(-)-mediated mitochondrial damage. This study supports the hypothesis that the neurotoxicity of nitric oxide is mediated through mitochondrial dysfunction.

Animals↗
PubMed1995

Changes of respiratory chain activity in mitochondrial and synaptosomal fractions isolated from the gerbil brain after graded ischaemia.

In this study we have examined (1) the integrated function of the mitochondrial respiratory chain by polarographic measurements and (2) the activities of the respiratory chain complexes I, II-III, and IV as well as the ATP synthase (complex V) in free mitochondria and synaptosomes isolated from gerbil brain, after a 30-min period of graded cerebral ischaemia. These data have been correlated with cerebral blood flow (CBF) values as measured by the hydrogen clearance technique. Integrated functioning of the mitochondrial respiratory chain, using both NAD-linked and FAD-linked substrates, was initially affected at CBF values of approximately 35 ml 100 g-1 min-1, and declined further as the CBF was reduced. The individual mitochondrial respiratory chain complexes, however, showed differences in sensitivity to graded cerebral ischaemia. Complex I activities decreased sharply at blood flows below approximately 30 ml 100 g-1 min-1 (mitochondria and synaptosomes) and complex II-III activities decreased at blood flows below 20 ml 100 g-1 min-1 (mitochondria) and 35-30 ml 100 g-1 min-1 (synaptosomes). Activities declined further as CBF was reduced below these levels. Complex V activity was significantly affected only when the blood flow was reduced below 15-10 ml 100 g-1 min-1 (mitochondria and synaptosomes). In contrast, complex IV activity was unaffected by graded cerebral ischaemia, even at very low CBF levels.

Animals↗
PubMed1995

Investigation into the role of N-acetylaspartate in cerebral osmoregulation.

Marked abnormalities of the magnetic resonance intensity of N-acetylaspartate (NAA) have been reported in patients with various neurological disorders, but the neurochemical consequences of these alterations are difficult to assess because the function of NAA remains speculative. The purpose of this study was to examine whether NAA plays a role in protecting neurons against osmotic stress. Intracerebral microdialysis was used to expose a small region of the rat dorsolateral striatum to an increasingly hyposmotic environment and to measure resulting changes in NAA extracellular concentrations. NAA changes in the extracellular fluid (ECF) were compared with those of the amino acids, in particular, taurine, known to be involved in brain osmoregulation. Stepped increases in cellular hydration produced by hyposmotic perfusion media induced a marked increase in ECF NAA, reflecting a redistribution of NAA from intra-to extracellular space. Parallel experiments showed that, of all the extracellular amino acids measured, only taurine markedly increased with hyposmolar perfusion medium, indicating that the ECF NAA increase associated with hyposmotic stress was a specific response and not passive leakage out of the cells. As NAA is predominantly neuronal, it may contribute to the protection of neurons against swelling (i.e., regulatory volume decrease). In conditions with impaired blood-brain barrier and cytotoxic oedema, efflux of intracellular NAA subsequent to sustained cellular swelling might lead to a reduction in total brain NAA detectable by magnetic resonance spectroscopy. Alternatively, redistribution of NAA from intra-to extracellular space implies changes in its chemical environment that may alter its magnetic resonance visibility.

Amino Acids↗
PubMed1995

Effect of reperfusion following cerebral ischaemia on the activity of the mitochondrial respiratory chain in the gerbil brain.

The effect of reperfusion following 30 min of cerebral ischaemia on brain mitochondrial respiratory chain activity has been studied in the gerbil. The state 3 respiration rates with both FAD- and NAD-linked substrates were reduced after ischaemia. After 5 min of reperfusion, state 3 respiration with FAD-linked substrates was restored, but levels of NAD-linked substrates did not return to control values until 30 min of reperfusion. By 120 min of reperfusion state 3 respiration decreased relative to control values with all substrates studied. Measurement of the individual respiratory chain complexes showed that complex I, complex II-III, and complex V activities were reduced after ischaemia. By 5 min of reperfusion complex II-III activity was restored, but the activities of complexes I and V did not return to control values until 30 min of reperfusion. In contrast, complex IV activity was unaffected by ischaemia or 5 and 30 min of reperfusion but was significantly reduced after 120 min of reperfusion, possibly owing to free radical production and lipid peroxidation.

Animals↗
PubMed1995

Molecular evolution of P transposable elements in the genus Drosophila. I. The saltans and willistoni species groups.

A phylogenetic survey using the polymerase chain reaction (PCR) has identified four major P element subfamilies in the saltans and willistoni species groups of Drosophila. One subfamily, containing about half of the sequences studied, consists of elements that are very similar to the canonical (and active) P element from D. melanogaster. Within this subfamily, nucleotide sequence differentiation among different copies from the same species and among elements from different species is relatively low. This observation suggests that the canonical elements are relatively recent additions to the genome or, less likely, are evolving slowly relative to the other subfamilies. Elements belonging to the three noncanonical lineages are distinct from the canonical elements and from one another. Furthermore, there is considerably more sequence variation, on the average, within the noncanonical subfamilies compared to the canonical elements. Horizontal transfer and the coexistence of multiple, independently evolving element subfamilies in the same genome may explain the distribution of P elements in the saltans and willistoni species groups. Such explanations are not mutually exclusive, and each may be involved to varying degrees in the maintenance of P elements in natural populations of Drosophila.

Animals↗
PubMed1995

Visually triggered migraine headaches affect spatial orientation and balance in a helicopter pilot.

The authors present a case of an attack helicopter pilot with recurrent spatial disorientation (SD) flying with night vision goggles (NVG's), diagnosed as having visually triggered migraine headaches. Serial Dynamic Platform Posturography testing during an acute migraine attack demonstrated balance dysfunction under visual and somatosensory deficient conditions, correlating with headache intensity. Vestibular symptoms are associated with migraine, and may be triggered by visual stimuli. NVG scintillations in susceptible individuals may act as a visual trigger for migraine. Migraine phenomenon may be a contributing factor to SD, especially during NVG operations. The association of visual and vestibular dysfunction with migraine and aeromedical disposition of migraine in aviators is discussed.

Adult↗
PubMed1994

Trolox protects mitochondrial complex IV from nitric oxide-mediated damage in astrocytes.

The efficacy of cystine, ascorbate and trolox, a vitamin E analogue, at protecting against nitric oxide-mediated mitochondrial complex IV damage has been investigated in cultured astrocytes. Of these compounds, only trolox afforded protection. It is suggested that lipid peroxidation is responsible for nitric oxide-mediated mitochondrial damage and that inhibitors of this process may be of therapeutic benefit in conditions where excessive nitric oxide production is implicated.

Amino Acid Oxidoreductases↗
PubMed1994

Identification of ethanolamine in rat and gerbil brain tissue extracts by NMR spectroscopy.

Some NMR resonances which have previously been observed but not identified in mammalian brain tissue extracts have been shown to arise from ethanolamine. This conclusion is drawn from a systematic study of the perchloric acid extracts of rodent brain tissue in which several NMR experiments were used to assign the peaks unambiguously. The extraction procedure used in this work gave samples with highly reproducible spectra, and ethanolamine was observed in all our extract samples. A localized increase in the concentration of ethanolamine was seen in the spectra of extracts produced from a cerebral infarct induced by occlusion of the middle cerebral artery in the rat.

Animals↗