Search PubMed⌕ Search

Biomedical subjects

M P Murphy

Publications and source records attributed to M P Murphy.

At least 73 records · Page 4Linked to original sources
PubMed1997

The effect on mitochondrial function of the tRNA Ser(UCN)/COI A7445G mtDNA point mutation associated with maternally-inherited sensorineural deafness.

An A to G point mutation at nucleotide position 7445 in the mitochondrial DNA genome is associated with maternally-inherited sensorineural deafness in two separate human pedigrees. To determine whether this point mutation [tRNA(SER)UCN)/COI A7445G] led to sensorineural deafness by affecting cellular energy metabolism we investigated the bioenergetic function of mitochondria in lymphoblastoid cultures established from these patients. Even though essentially all of the mitochondrial DNA in these cells contained the mutation at nucleotide position 7445, there was no effect on a number of mitochondrial bioenergetic functions (mitochondrial content, membrane potential in both intact and digitonin-permeabilised cells, cellular ATP/ADP ratios and respiratory enzyme activity) when compared with control cells. The implications of these findings for both the aetiology of the sensorineural deafness associated with the A7445G mtDNA mutation, and the role of bioenergetic defects in mitochondrial DNA diseases in general are discussed.

Adenosine Triphosphate↗
PubMed1997

Characterization of the erythrocyte superoxide dismutase allozymes in the deer Cervus elaphus.

The cDNA sequences for Cu,Zn superoxide dismutase from two Cervus elaphus subspecies, North American wapiti and European red deer, were determined. The derived amino acid sequences showed two differences: residue 8 was Leu in wapiti and Met in red deer and residue 25 was His in wapiti and Asn in red deer. The extra positive charge at position 25 in the wapiti isoform accounted for its greater mobility towards the cathode during non-denaturing electrophoresis, a procedure widely used in the genetic analysis of deer. There was no difference in specific activity between the two Cu,Zn superoxide dismutase isoforms, but the wapiti isoform was slightly more susceptible to heat denaturation.

Amino Acid Sequence↗
PubMed1996

Alterations to glutathione and nicotinamide nucleotides during the mitochondrial permeability transition induced by peroxynitrite.

Peroxynitrite is a biologically important oxidant that damages mitochondria in a number of ways. We investigated the interaction of peroxynitrite with the mitochondrial glutathione pool by measuring the formation of oxidised glutathione and glutathione-protein mixed disulfides in mitochondria exposed to either peroxynitrite or tert-butylhydroperoxide. In contrast to tert-butylhydroperoxide, peroxynitrite converts 40-50% of mitochondrial glutathione to products other than disulfides, primarily higher oxidation states of sulfur. These data show that peroxynitrite interacts with mitochondria quite differently from oxidants commonly used in studying mitochondrial oxidative stress. Peroxynitrite also induces a permeability transition in the mitochondrial inner membrane, and here we show that this permeability transition is prevented by the NAD(P)H-linked substrates glutamate and malate and by the thiol reagent dithiothreitol. Glutamate and malate prevented complete oxidation of the NAD(P)H pool by peroxynitrite or tert-butylhydroperoxide but did not prevent oxidation of the mitochondrial glutathione pool or the formation of glutathione-protein mixed disulfides. This study is consistent with regulation of the permeability transition by critical protein thiol groups, whose redox state responds to that of the mitochondrial NAD(P)H pool, but which do not equilibrate directly with the mitochondrial glutathione pool.

Animals↗
PubMed1996

Altered mitochondrial function in fibroblasts containing MELAS or MERRF mitochondrial DNA mutations.

A number of human diseases are caused by inherited mitochondrial DNA mutations. Two of these diseases, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres), are commonly caused by point mutations to tRNA genes encoded by mitochondrial DNA. Here we report on how these mutations affect mitochondrial function in primary fibroblast cultures established from a MELAS patient containing an A to G mutation at nucleotide 3243 in the tRNA(Leu(UUR) gene and a MERRF patient containing an A to G mutation at nucleotide 8344 in the tRNA(Lys) gene. Both mitochondrial membrane potential and respiration rate were significantly decreased in digitonin-permeabilized MELAS and MERRF fibroblasts respiring on glutamate/malate. A similar decrease in mitochondrial membrane potential was found in intact MELAS and MERRF fibroblasts. The mitochondrial content of these cells, estimated by stereological analysis of electron micrographs and from measurement of mitochondrial marker enzymes, was similar in control, MELAS and MERRF cells. Therefore, in cultured fibroblasts, mutation of mitochondrial tRNA genes leads to assembly of bioenergetically incompetent mitochondria, not to an alteration in their amount. However, the cell volume occupied by secondary lysosomes and residual bodies in the MELAS and MERRF cells was greater than in control cells, suggesting increased mitochondrial degradation in these cells. In addition, fibroblasts containing mitochondrial DNA mutations were 3-4-fold larger than control fibroblasts. The implications of these findings for the pathology of mitochondrial diseases are discussed.

Cells, Cultured↗
PubMed1996

Exposure to the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and nitric oxide simultaneously causes cyclosporin A-sensitive mitochondrial calcium efflux and depolarisation.

The effect of the parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP+) together with nitric oxide donors on mitochondrial calcium homeostasis and membrane potential was investigated. Simultaneous exposure of calcium-loaded mitochondria to MPP+ and nitric oxide donors led to Cyclosporin A-sensitive mitochondrial calcium efflux and depolarisation. When MPP+ was replaced with the respiratory inhibitor rotenone, mitochondrial calcium efflux and depolarisation also occurred. As both MPP+ and rotenone induce mitochondrial superoxide formation, the possibility that calcium efflux and depolarisation were due to peroxynitrite formation from reaction of superoxide with nitric oxide was investigated. It was shown that simultaneous exposure of mitochondrial membranes to nitric oxide donors and rotenone led to peroxynitrite formation. The possible roles of nitric oxide, peroxynitrite, mitochondrial depolarisation, and calcium efflux in MPP+ toxicity are discussed.

1-Methyl-4-phenylpyridinium↗
PubMed1996

Superoxide production by mitochondria in the presence of nitric oxide forms peroxynitrite.

The mitochondrial respiratory chain continually produces superoxide leading to high levels of mitochondrial oxidative stress. This oxidative damage has been attributed to the formation of hydroxyl radicals and hydrogen peroxide from superoxide. Alternatively, mitochondrial superoxide may react with nitric oxide forming the potent oxidant peroxynitrite, thus damaging mitochondrial protein, lipid and DNA. To test this hypothesis we induced mitochondrial superoxide formation in the presence of nitric oxide. Here we demonstrate that mitochondrial superoxide reacts with nitric oxide to form peroxynitrite, suggesting that mitochondria may be a significant intracellular source of peroxynitrite.

Animals↗
PubMed1996

Short intertrial intervals impair water maze performance in old Fischer 344 rats.

Male and female Fischer 344 rats (N = 55) aged approximately 18, 21, and 24 months were tested for spatial learning in the water maze with intertrial intervals of 1-4 min (Massed) or 23-33 min (Spaced). Animals tested in the Massed condition showed an age-related impairment on trials to criterion; rats aged 24 months performed more poorly than younger subjects. Spaced animals did not differ at any age nor did they differ from 18- or 21-month-old Massed subjects. The youngest rats in both groups were comparable to animals aged 7-8 months tested under Massed conditions. Tests on swim distance, swim speed, and escape latency produced similar results. Our data suggest that acquisition deficits in 24-month-old rats tested with long intertrial intervals are due at least in part to increased susceptibility to fatigue and/or thermal stress. Caution should be used, therefore, when interpreting age-related impairments in water maze performance.

Aging↗
PubMed1996

Enhanced mitochondrial radical production in patients which rheumatoid arthritis correlates with elevated levels of tumor necrosis factor alpha in plasma.

Mitochondrial dysfunction contributes to cell damage in a number of human diseases. One significant mechanism by which mitochondria damage cells is by producing reactive oxygen species from the respiratory chain. In this study we measured the production of reactive oxygen species by leukocyte mitochondria in blood from rheumatoid arthritis patients. To do this we used the chemiluminescence of lucigenin, which is accumulated by mitochondria within cells and reacts with superoxide to form a chemiluminescent product. By using specific inhibitors we could distinguish between the production of reactive oxygen species by mitochondria and by NADPH oxidase. There was a five-fold increase in mitochondrial reactive oxygen species production in whole blood and monocytes from patients with rheumatoid arthritis, when compared to healthy subjects or patients with non-rheumatic diseases. There was no increases in mitochondrial reactive oxygen species production by neutrophils from rheumatoid arthritis patients. The enhanced mitochondrial radical production in rheumatoid arthritis patients correlated significantly with increased levels of tumor necrosis factor alpha in plasma (p < 0.0001). As tumor necrosis factor alpha is known to increase mitochondrial reactive oxygen species production the elevated mitochondrial radical formation seen in rheumatoid arthritis patients may be due to activation of the mitochondrial radical production. These data suggest that elevated mitochondrial oxidative stress contributes to the pathology of rheumatoid arthritis.

Acridines↗
PubMed1995

Peroxynitrite formed by simultaneous nitric oxide and superoxide generation causes cyclosporin-A-sensitive mitochondrial calcium efflux and depolarisation.

Nitric oxide reacts rapidly with superoxide to form the potent oxidant peroxynitrite. Mitochondria in vivo produce superoxide and in pathological situations the amount of superoxide produced increases; therefore, in the presence of nitric oxide, mitochondria will be a major site of peroxynitrite formation. Oxidative stress induces cyclosporin-A-sensitive mitochondrial calcium efflux and depolarisation which may contribute to tissue damage in pathological situations. To determine whether peroxynitrite could induce calcium efflux and depolarisation we exposed mitochondria to both nitric oxide and superoxide simultaneously, thus subjecting the mitochondria to a continual flux of peroxynitrite similar to that found in pathological situations. Our results show that: (a) exposure of mitochondria to nitric oxide plus superoxide induces cyclosporin-A-sensitive mitochondrial calcium efflux and depolarisation; (b) neither nitric oxide nor superoxide on their own induce calcium efflux or depolarisation under these conditions; (c) calcium efflux and depolarisation occur when peroxynitrite production (from nitric oxide and superoxide) exceeds about 0.99 +/- 0.03 nmol peroxynitrite.min-1 mg mitochondrial protein-1. This rate of production of peroxynitrite is similar in magnitude to superoxide formation by mitochondria in pathological situations, suggesting that mitochondria can produce sufficient peroxynitrite in vivo to cause mitochondrial calcium efflux and depolarisation. Our experiments suggest a plausible model for tissue damage when mitochondrial superoxide production increases, for example in ischaemia-reperfusion injury or following exposure to neurotoxins. In the presence of nitric oxide the mitochondrial superoxide production will lead to peroxynitrite formation which induces cyclosporin-A-sensitive mitochondrial calcium efflux and depolarisation. This disruption to mitochondrial function may in turn contribute to cell damage and death.

Animals↗
PubMed1995

Synthesis and characterization of thiobutyltriphenylphosphonium bromide, a novel thiol reagent targeted to the mitochondrial matrix.

Mitochondria are continually exposed to oxidative stress due to superoxide formation by the respiratory chain which increases in pathological situations such as ischemia reperfusion and neurodegeneration. During oxidative stress there are a number of changes in mitochondrial low-molecular-weight and protein thiols. In particular, the mitochondrial glutathione pool becomes oxidized and forms mixed disulfides with protein thiols. To investigate changes in the redox state and conjugation of mitochondrial glutathione, and other mitochondrial thiols, we designed and characterized a thiol probe specifically targeted to the mitochondrial matrix. This molecule, thiobutyltriphenylphosphonium bromide, contains a thiol group linked to a lipophilic triphenylphosphonium cation which causes it to accumulate in the negatively charged mitochondrial matrix. Using [14C]thiobutyltriphenylphosphonium bromide we confirmed that it was selectively accumulated by isolated mitochondria. In the mitochondrial matrix the thiol group equilibrated with endogenous thiols and during oxidative stress became disulfide-bonded to protein and nonprotein thiols. Therefore, this novel thiol probe can be used to label protein thiol groups and to investigate changes in conjugation and redox state of mitochondrial thiols during oxidative stress.

Animals↗
PubMed1995

Inhibition of complex I by hydrophobic analogues of N-methyl-4-phenylpyridinium (MPP+) and the use of an ion-selective electrode to measure their accumulation by mitochondria and electron-transport particles.

N-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, kills dopaminergic neurons after its accumulation in mitochondria where it inhibits Complex I of the respiratory chain. MPP+ inhibits respiration by binding to both a hydrophobic and a hydrophilic site on Complex I and this inhibition is increased by the lipophilic tetraphenylboron anion (TPB-) which facilitates movement of MPP+ through membranes and its penetration to the hydrophobic binding site on Complex I. To investigate the inhibition of respiration by MPP(+)-like compounds, we have measured simultaneously NADH-linked mitochondrial respiration and the uptake and accumulation of the N-benzyl-4-styrylpyridinium and N-ethyl-4-styrylpyridinium cations in mitochondria using ion-selective electrodes. The data provide direct evidence that TPB- increases the inhibition not by increasing matrix concentration but by facilitating access to the inhibitory sites on Complex I. We have also compared the rates of uptake of MPP+ analogues of varied lipophilicity by the inner membrane and the development of inhibition of NADH oxidation, using an inverted mitochondrial inner membrane preparation and appropriate ion-selective electrodes. These experiments demonstrated that the amount of MPP+ analogue bound to the inner membrane greatly exceeded the quantity required for complete inhibition of NADH oxidation. Moreover, binding to the membrane occurred much more rapidly than the development of inhibition with all MPP+ analogues tested. This suggests that the attainment of a correct orientation of these compounds within the membrane and the binding site may be a rate-limiting step in the development of inhibition.

1-Methyl-4-phenylpyridinium↗
PubMed1995

Development of spontaneous seizures over extended electrical kindling. II. Persistence of dentate inhibitory suppression.

The effect of an extended program of perforant path or amygdala kindling on paired-pulse suppression in the dentate gyrus was studied in male hooded rats. Repeated kindling stimulations were delivered twice or three times daily until either 300 stimuli had been delivered or generalized convulsions had been observed to occur spontaneously. Paired-pulse suppression was monitored prior to and over the course of kindling using a standard variable interval paradigm. We also used a variable intensity paradigm in which the intensity of the conditioning pulse was varied while the test pulse intensity was fixed at 600 microA and the interpulse interval was fixed at 30 ms. Both procedures revealed progressive increases in paired-pulse suppression which persisted over the course of kindling. This increased inhibition also persisted in animals which developed spontaneous seizures. The variable intensity paired-pulse procedure also allowed us to monitor facilitation effects which were relatively uncontaminated by recurrent inhibition (when the conditioning pulse intensity was low). Kindling was found to increase paired-pulse facilitation. With the standard variable interval paradigm, these increases in facilitation masked the increases in suppression.

Amygdala↗
PubMed1995

A simple and rapid test of sensorimotor function in the aged rat.

A total of 107 Fischer 344 rats (male and female, 2 to 29 months old) were tested on three tasks designed to measure sensorimotor function. In the first task, a new test of sensorimotor function, animals were required to maintain their balance on a stationary inclined Plexiglas plane for 5 s. Performance was evaluated with a series of increasing angles ranging from 25 degrees to 50 degrees in 1 degree increments (four trials/angle). The animals were also evaluated on two common tests of sensorimotor function: suspension from a wire by the forepaws and traversing a narrow, horizontal bar (three trials each). Age effects were significant (p < .043 to p < .0001) for each of the 11 dependent measures (first fall angle, threshold angle, and total falls for the inclined plane, and first, best, mean, and median latencies to fall for the remaining two tests). No relationship was observed between performance, sex, and body weight. Coefficients of variation differed between tests, with the inclined plane being the least variable and the horizontal bar the most. Importantly, the inclined plane was least likely to violate the ANOVA assumption of homogeneity of variance, a problem often encountered when measuring age-related variables. Since the inclined plane test has a relatively low degree of variability, and since the between-group variation is homogeneous, it should serve as both a useful and robust test for sensorimotor function in the aged rat.

Aging↗
PubMed1994

Peroxynitrite causes calcium efflux from mitochondria which is prevented by Cyclosporin A.

Superoxide reacts with nitric oxide to form peroxynitrite, a potent oxidising agent which may contribute to tissue damage in pathological situations such as inflammation and ischaemia/reperfusion. One mechanism by which oxidative stress damages tissues is the induction of a specific Cyclosporin A-sensitive mitochondrial calcium efflux pathway. Here we show that peroxynitrite induces calcium efflux from mammalian mitochondria and that this efflux is blocked by Cyclosporin A. These data suggest that disruption of mitochondrial calcium efflux may contribute to tissue damage when superoxide and nitric oxide are present together in vivo.

Animals↗
PubMed1994

Cyclosporin A blocks 6-hydroxydopamine-induced efflux of Ca2+ from mitochondria without inactivating the mitochondrial inner-membrane pore.

Oxidative stress causes Ca(2+)-loaded mitochondria to release Ca2+. The mechanism of this efflux is unclear, but it appears to be associated with the opening of a pore in the mitochondrial inner membrane. Pore opening depolarizes the mitochondria, letting solutes enter the mitochondrial matrix, causing swelling. Cyclosporin A (CsA) prevents opening of this pore. The neurotoxin 6-hydroxydopamine (6HD) autoxidizes, producing free radicals, which cause oxidative stress. In this paper it is shown that 6HD-induced efflux from Ca(2+)-loaded mitochondria was prevented by CsA. The 6HD-induced Ca2+ efflux was not accompanied by mitochondrial swelling, depolarization of the mitochondrial inner membrane or movement of radiolabelled sucrose into the mitochondrial matrix. In agreement with others [Schlegel, Schweizer and Richter (1992) Biochem. J. 285, 65-69], these findings suggest that the mitochondrial pore remained closed during pro-oxidant-induced Ca2+ efflux. However, the implication that CsA blocks pro-oxidant-induced Ca2+ efflux by some mechanism other than inactivating the mitochondrial pore, suggests that the interaction of CsA with mitochondria may be more complex than is currently supposed.

Animals↗
PubMed1994

Right latissimus dorsi cardiomyoplasty improves left ventricular function by increasing peak systolic elastance (Emax).

BACKGROUND: Dynamic cardiomyoplasty remains a promising but poorly understood surgical modality for selected patients with dilated cardiomyopathy. Despite encouraging clinical results, objective evidence of enhanced ventricular function using traditional indexes (cardiac output, ejection fraction, and dP/dt) has been difficult to document after cardiomyoplasty. Several investigators have suggested that cardiomyoplasty acts partly by unloading the left ventricle. These indexes all depend somewhat on loading conditions, however, and might not detect such an interaction. The time-varying elastance model provides an index of contractility, Emax, that is relatively insensitive to changes in loading conditions. We applied this model to study the effect of right latissimus dorsi cardiomyoplasty on left ventricular function in an acute canine model. METHODS AND RESULTS: Five dogs underwent acute cardiomyoplasty using untrained right latissimus dorsi muscle. Instrumentation included Millar pressure transducers in the left ventricle and aortic root, an electromagnetic flow probe around the ascending aorta, and a volume conductance catheter in the left ventricle. A cuffed nerve electrode around the thoracodorsal nerve and a right ventricular sensing lead were connected to a Medtronic Cardiomyostimulator (5 V, 30 Hz, 1:1 synchronization). Transient caval occlusions were performed with the stimulator both off and on to calculate Emax and the slope of the end-systolic pressure-volume relationship (Ees). Turning the stimulator on significantly increased peak systolic elastance (Emax) and end-systolic elastance (Ees) in all five dogs by an average of 56% and 78%, respectively (P < .05). End-diastolic volume and end-systolic volume decreased by 18% and 28%, respectively (P < .05). All other measured hemodynamic parameters, including peak left ventricular pressure, mean arterial pressure, cardiac output, stroke volume, stroke work, ejection fraction, preload-recruitable stroke work, and dP/dt, did not change significantly. CONCLUSIONS: These results show that, in this acute canine model, right latissimus dorsi cardiomyoplasty significantly improves left ventricular function while decreasing left ventricular volumes. The results are consistent with the theory that cardiomyoplasty increases contractility while unloading the ventricle by decreasing end-diastolic volume. This increase in Emax despite inconsistent changes in other indexes underlies the importance of using load-insensitive indexes of ventricular function when studying cardiomyoplasty.

Animals↗