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Biomedical subjects

J B Clark

Publications and source records attributed to J B Clark.

At least 91 records · Page 5Linked to original sources
PubMed1996

Hypothyroidism alters the effect of GTP on adenylyl cyclase in forebrain and hindbrain synaptosomal membranes from 15-day-old rats.

The effect of GTP concentration of forskolin-stimulated adenylyl cyclase activity was examined in synaptosomal membranes from 15-day-old rats that were hypothyroid owing to administration of propylthiouracil and a low-iodine diet to the mothers during pregnancy and suckling. In membranes from the forebrain hypothyroidism abolished the overall stimulatory effect of GTP, which was seen in the euthyroid case. In membranes from the hindbrain hypothyroidism had the opposite effect in that there was an enhancement of an overall stimulatory effect of GTP. It is suggested that these findings reflect changes during early development of the brain in the expression of various G-proteins and/or the expression of different isoforms of adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1996

Nitric oxide-mediated mitochondrial damage: a potential neuroprotective role for glutathione.

In this study we have investigated the mechanisms leading to mitochondrial damage in cultured neurons following sustained exposure to nitric oxide. Thus, the effects upon neuronal mitochondrial respiratory chain complex activity and reduced glutathione concentration following exposure to either the nitric oxide donor, S-nitroso-N-acetylpenicillamine, or to nitric oxide releasing astrocytes were assessed. Incubation with S-nitroso-N-acetylpenicillamine (1 mM) for 24 h decreased neuronal glutathione concentration by 57%, and this effect was accompanied by a marked decrease of complex I (43%), complex II-III (63%), and complex IV (41%) activities. Incubation of neurons with the glutathione synthesis inhibitor, L-buthionine-[S,R]-sulfoximine caused a major depletion of neuronal glutathione (93%), an effect that was accompanied by a marked loss of complex II-III (60%) and complex IV (41%) activities, although complex I activity was only mildly decreased (34%). In an attempt to approach a more physiological situation, we studied the effects upon glutathione status and mitochondrial respiratory chain activity of neurons incubated in coculture with nitric oxide releasing astrocytes. Astrocytes were activated by incubation with lipopolysaccharide/interferon-gamma for 18 h, thereby inducing nitric oxide synthase and, hence, a continuous release of nitric oxide. Coincubation for 24 h of activated astrocytes with neurons caused a limited loss of complex IV activity and had no effect on the activities of complexes I or II-III. However, neurons exposed to astrocytes had a 1.7-fold fold increase in glutathione concentration compared to neurons cultured alone. Under these coculture conditions, the neuronal ATP concentration was modestly reduced (14%). This loss of ATP was prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. These results suggest that the neuronal mitochondrial respiratory chain is damaged by sustained exposure to nitric oxide and that reduced glutathione may be an important defence against such damage.

Animals↗
PubMed1996

Detecting pulmonary abnormalities on magnetic resonance images in patients with usual interstitial pneumonitis: effect of varying window settings and gadopentetate dimeglumine.

RATIONALE AND OBJECTIVES: We examined the effect of varying window settings and contrast enhancement on detecting pulmonary abnormalities on magnetic resonance (MR) images in patients with usual interstitial pneumonitis (UIP). METHODS: HRCT scans and MR images from 10 patients with UIP were evaluated. T1-weighted MR images were obtained before and after administration of gadopentetate dimeglumine and were photographed at conventional windows and at windows chosen to increase the conspicuity of the lung parenchyma ("lung windows"). The four MR image configurations were mixed with the high-resolution computed tomography (HRCT) scans of these patients and randomized. Corresponding scan levels in each patient were evaluated conjointly by two thoracic radiologists for the presence of "honeycomb lung," ground-glass opacity, parenchymal bands, and reticular abnormalities. Lung signal intensity in areas containing ground-glass signal intensity was measured on MR images using regions of interest. RESULTS: The administration of gadopentetate dimeglumine significantly improved the detection of honeycomb lung on scans photographed at lung windows, but it did not significantly influence the detection of ground-glass abnormalities. The use of lung windows improved the detection of ground-glass abnormalities for both enhanced and unenhanced scans, but lung windows improved the detection of honeycomb lung only for enhanced scans. All MR image configurations were insensitive compared with HRCT scans for detecting parenchymal bands and reticular abnormalities. There was a good correlation between measured lung signal intensity and visual ground-glass profusion score. CONCLUSION: The visibility of pulmonary abnormalities on MR images of patients with UIP is limited compared with that of HRCT scans. The improved visibility of some parenchymal abnormalities after intravenous administration of gadopentetate dimeglumine and with the use of lung windows is insufficient to warrant their routine use in thoracic MR imaging.

Aged↗
PubMed1996

Threshold effects and control of oxidative phosphorylation in nonsynaptic rat brain mitochondria.

The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV ), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by approximately 72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by approximately 70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed.

Adenosine Triphosphate↗
PubMed1996

Intrasynaptosomal free calcium concentration during rat brain development: effects of hypoxia, aglycaemia, and ischaemia.

The effects of hypoxia, aglycaemia, and hypoxia-aglycaemia on intrasynaptosomal free Ca2+ concentration ([Ca2+]i) have been investigated in rat brain synaptosomes prepared from animals aged 5, 10, 15, 20, 25, and 60 days. After 60 min of hypoxia there was no significant difference, when compared with controls, in basal [Ca2+]i or [Ca2+]i following depolarisation in all of the ages studied. Following 60 min of aglycaemia there was no significant difference from controls in [Ca2+]i of synaptosomes prepared from pups of < or = 20 days, although a significant rise in [Ca2+]i was seen in preparations from animals > 20 days old. Sixty minutes of hypoxia-aglycaemia led to a significant rise in [Ca2+]i only in preparations from animals 15-60 days old. With both aglycaemia and hypoxia-aglycaemia a progressive increase in the magnitude of the rise in [Ca2+]i was seen with development. These data suggest increases in [Ca2+]i in adult nerve terminals following prolonged aglycaemia and hypoxia-aglycaemia but no change following prolonged hypoxia. In contrast, no significant changes in [Ca2+]i values were apparent in neonatal nerve terminals under any of these conditions. In control synaptosomes with glucose and oxygen freely available, a decrease in resting and depolarised [Ca2+]i during development was seen, suggesting a change in calcium homeostasis within the nerve terminal as the brain develops. It is suggested that the mechanism underlying the relative resistance to ischaemic damage of neonatal brain as compared with adult brain may be related to the regulation of calcium at the nerve ending.

Age Factors↗
PubMed1996

Investigations into the mechanism of action of a novel nitric oxide generator on cellular respiration.

Nitric oxide may regulate cellular respiration by competition with oxygen at mitochondrial cytochrome oxidase. Using an astrocyte-derived cell line, we have compared the mechanism of action of the nitric oxide-generating compound Roussin's black salt with that of sodium nitroprusside on cellular oxygen consumption. Intense light exposure induced the release of large quantities of nitric oxide from both of the donor compounds. However, in room light only Roussin's black salt generated low levels of the radical. Simultaneous measurement of oxygen consumption and of nitric oxide production demonstrated that sodium nitroprusside only had inhibitory actions when exposed to intense light (nitric oxide release), whereas Roussin's black salt had inhibitory actions in room light. Extracellular haemoglobin did not prevent the inhibition of respiration rate induced by Roussin's black salt even though stimulation of nitric oxide release on light exposure was markedly reduced. Preincubation of cells with Roussin's black salt and subsequent measurement of levels of light-liberated nitric oxide demonstrated that the compound was rapidly internalised. The uptake of sodium nitroprusside was minimal. These data suggest that, in contrast to sodium nitroprusside, the cellular internalisation of Roussin's black salt allows site-directed nitric oxide release and very effective inhibition of cellular respiration.

Animals↗
PubMed1996

A simple snare for transscleral fixation of dislocated intraocular lenses.

PURPOSE: The construction and use of a surgical snare in retrieving and securing posterior chamber intraocular lenses is described. METHODS: The snare consists of a 10-O polypropylene suture threaded into a 25-gauge retrobulbar needle. This is used after a pars plana vitrectomy to retrieve the dislocated intraocular lens. The needle attached to the polypropylene suture is used to secure the lens haptic 1.5 mm behind the limbus. Two snares are used to secure the two lens haptics. CONCLUSIONS: The snare is easy to make with readily available materials. The use of this snare involves less intraocular and extraocular manipulations than other described methods.

Foreign-Body Migration↗
PubMed1996

Signal transduction processes in the developing brain: perturbations of G protein alpha-subunit abundances by perinatal hypothyroidism.

The abundances of G protein alpha-subunits (Gi1 alpha, Gi2 alpha, G0 alpha and Gq/ll alpha) were measured in synaptosomal membranes isolated from forebrain and hindbrain regions of euthyroid and hypothyroid neonatal rats at 10, 15, 20 and 25 days post-partum. The findings show that hypothyroidism causes a distinct perturbation of the normal developmental profile of these signalling components. It is suggested that these changes may contribute to some of the neurological deficits arising from hypothyroidism in early development.

Animals↗
PubMed1996

Glutathione protects astrocytes from peroxynitrite-mediated mitochondrial damage: implications for neuronal/astrocytic trafficking and neurodegeneration.

In this study we have examined the susceptibility of the mitochondrial respiratory chain of astrocytes and astrocytes depleted of glutathione to peroxynitrite exposure. Astrocytes, as reported previously by us, appeared resistant to the actions of peroxynitrite. In contrast, depletion (-94%) of astrocytic glutathione rendered the cells susceptible with mitochondrial complexes I and II/III being decreased in activity by 80 and 64%, respectively, after peroxynitrite exposure. Furthermore, cell death, as judged by lactate dehydrogenase release, was significantly increased (+81%) in the glutathione-depleted astrocytes exposed to peroxynitrite. Glutathione depletion alone had no effect on any of the measured parameters. It is concluded that glutathione is an important intracellular defence against peroxynitrite and that when glutathione levels are compromised the mitochondrial respiratory chain is a vulnerable target and cell death ensues. In view of the relative paucity of neuronal glutathione, it is possible that astrocyte-derived peroxynitrite may, in certain pathological conditions, be released and diffuse into neighboring neurones where mitochondrial damage may occur.

Animals↗
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↗