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Biomedical subjects

M P Murphy

Publications and source records attributed to M P Murphy.

At least 55 records · Page 3Linked to original sources
PubMed1999

gamma-Secretase, evidence for multiple proteolytic activities and influence of membrane positioning of substrate on generation of amyloid beta peptides of varying length.

gamma-Secretase activity is the final cleavage event that releases the amyloid beta peptide (Abeta) from the beta-secretase cleaved carboxyl-terminal fragment of the amyloid beta protein precursor (APP). No protease responsible for this highly unusual, purportedly intramembranous, cleavage has been definitively identified. We examined the substrate specificity of gamma-secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and then analyzing Abeta production from cells transfected with these mutant APPs by enzyme-linked immunosorbent assay and mass spectrometry. Abeta production was also analyzed from a subset of transmembrane domain APP mutants that showed dramatic shifts in gamma-secretase cleavage in the presence or absence of pepstatin, an inhibitor of gamma-secretase activity. These studies demonstrate that gamma-secretase's cleavage specificity is primarily determined by location of the gamma-secretase cleavage site of APP with respect to the membrane, and that gamma-secretase activity is due to the action of multiple proteases exhibiting both a pepstatin- sensitive activity and a pepstatin-insensitive activity. Given that gamma-secretase is a major therapeutic target in Alzheimer's disease these studies provide important information with respect to the mechanism of Abeta production that will direct efforts to isolate the gamma-secretases and potentially to develop effective therapeutic inhibitors of pathologically relevant gamma-secretase activities.

Amino Acid Sequence↗
PubMed1999

Protein carbonyl formation and tyrosine nitration as markers of oxidative damage during ischaemia-reperfusion injury to rat sciatic nerve.

We have investigated the role of oxidative damage in peripheral nerve ischaemia-reperfusion injury using a rat sciatic nerve model. After 5 h ischaemia blood flow to the sciatic nerve was restarted and markers of oxidative damage measured after various times of reperfusion. As a marker of protein oxidative damage, protein carbonyl formation was measured using a sensitive enzyme-linked immunosorbent assay. Protein carbonyl content was unaffected by ischaemia alone, but increased by 55% after 12-18 h reperfusion, correlating with the onset of nerve pathology. Pretreatment with the xanthine oxidase inhibitor allopurinol prevented these abnormalities, suggesting that xanthine oxidase activity is proximal to oxidative damage during reperfusion injury. To determine whether formation of the potent oxidant peroxynitrite from nitric oxide and superoxide contributed to ischaemia-reperfusion injury, we measured the accumulation of 3-nitrotyrosine residues in proteins. Only one protein of 49,000 mol. wt contained significant amounts of 3-nitrotyrosine residues which was shown to be glial fibrillary acidic protein, an abundant cytoskeletal protein in Schwann cells. However glial fibrillary acidic protein contained 3-nitrotyrosine residues prior to ischaemia-reperfusion, and the amount of nitrated tyrosine residues in total glial fibrillary acidic protein did not increase significantly during reperfusion, therefore it was not possible to draw conclusions about the role of peroxynitrite in nerve reperfusion injury.

Animals↗
PubMed1999

Decreased ATP synthesis is phenotypically expressed during increased energy demand in fibroblasts containing mitochondrial tRNA mutations.

Mutations in the tRNA genes of mitochondrial DNA (mtDNA) cause the debilitating MELAS (mitochondrial, myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres) syndromes. These mtDNA mutations affect respiratory chain function, apparently without decreasing cellular ATP concentration [Moudy et al. (1995) PNAS, 92, 729-733]. To address this issue, we investigated the role of mitochondrial ATP synthesis in fibroblasts from MELAS and MERRF patients. The maximum rate of mitochondrial ATP synthesis was decreased by 60-88%, as a consequence of the decrease in the proton electrochemical potential gradient of MELAS and MERRF mitochondria. However, in quiescent fibroblasts neither ATP concentration or the ATP/ADP ratio was affected by the lowered rate of ATP synthesis. We hypothesized that the low ATP demand of quiescent fibroblasts masked the mitochondrial ATP synthesis defect and that this defect might become apparent during higher ATP use. To test this we simulated high energy demand by titrating cells with gramicidin, an ionophore that stimulates ATP hydrolysis by the plasma membrane Na+/K+-ATPase. We found a threshold gramicidin concentration in control cells at which both the ATP/ADP ratio and the plasma membrane potential decreased dramatically, due to ATP demand by the Na+/K+-ATPase outstripping mitochondrial ATP synthesis. In MELAS and MERRF fibroblasts the corresponding threshold concentrations of gramicidin were 2-20-fold lower than those for control cells. This is the first demonstration that cells containing mtDNA mutations are particularly sensitive to increased ATP demand and this has several implications for how mitochondrial dysfunction contributes to disease pathophysiology. In particular, the increased susceptibility to plasma membrane depolarization will render neurons with dysfunctional mitochondria susceptible to excitotoxic cell death.

Adenosine Diphosphate↗
PubMed1999

Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA.

Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed rho degrees cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in rho degrees cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP4- for ADP3- by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete FoF1-ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin-permeabilized mammalian rho degrees cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in rho degrees cells and an incomplete FoF1-ATP synthase was also required. The maximum value of this potential was approximately 110 mV in permeabilized cells and approximately 67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete FoF1-ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear-encoded proteins but approximately 65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of rho degrees mitochondria showed that the azide-sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete FoF1-ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete FoF1-ATP synthase in maintaining the mitochondrial membrane potential in rho degrees cells.

Adenosine Triphosphatases↗
PubMed1999

Selective targeting of an antioxidant to mitochondria.

Mitochondrial oxidative damage contributes significantly to a range of human disorders, including neurodegenerative diseases, ischaemia-reperfusion injury and ageing-associated dysfunction. To prevent this damage we have delivered a molecule containing the active antioxidant moiety of vitamin E to mitochondria. This was carried out by covalently coupling the antioxidant moiety to a lipophilic triphenylphosphonium cation. This mitochondrially targeted antioxidant, 2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2, 5,7,8-tetramethyl-2H-1-benzopyran-6-ol bromide (TPPB), accumulated several-hundred fold within the mitochondrial matrix, driven by the organelle's large membrane potential. When cells were incubated with micromolar concentrations of TPPB, they accumulated millimolar concentrations within their mitochondria. The amount of TPPB taken up by mitochondria was approximately 80-fold greater than endogenous levels of vitamin E. Consequently the targeted derivative of vitamin E protected mitochondrial function from oxidative damage far more effectively than vitamin E itself. The mitochondrially targeted antioxidant TPPB has potential as an antioxidant therapy for disorders involving mitochondrial oxidative damage. It also suggests a new family of mitochondrially targeted antioxidants, redox-active and pharmacologically active molecules designed to prevent damage or manipulate mitochondrial function.

Animals↗
PubMed1999

Clinimetric evaluation of the Sinonasal Outcome Test-16. Student Research Award 1998.

This study describes the reliability, validity, and responsiveness of the Sinonasal Outcome Test-16 (SNOT-16), a rhinosinusitis-specific health-related quality-of-life instrument, in the University of Washington Department of Otolaryngology-Head and Neck Surgery patient population. The SNOT-16 was completed by 47, 24, and 22 patients at weeks 0, 6, and 12, respectively. In addition, all 47 patients completed the Short-form 36-item Health Survey (SF-36) at week 0. Furthermore, an additional cohort of patients from the otology clinic who denied symptoms of rhinosinusitis or previous physician diagnoses of rhinosinusitis were asked to complete the SNOT-16. These scores were subsequently used to determine discriminant validity of the instrument. Cronbach's alpha was 0.89, indicating a high degree of homogeneity of the test items. The SNOT-16 demonstrated excellent discriminant validity, and mean total SNOT-16 scores were significantly correlated with patient-reported overall health, overall bother, and 7 of the 8 SF-36 subscales. The standardized response mean calculated between weeks 0 and 6 was 0.69, indicating moderate sensitivity to change. We conclude that the SNOT-16 is a reliable, valid, and responsive instrument for measuring rhinosinusitis-specific health-related quality of life.

Adolescent↗
PubMed1999

Measuring the effects of Meniere's disease: results of the Patient-Oriented Severity Index (MD POSI) version 1.

We describe the development and initial psychometric testing of a new self-report instrument for quantifying the impact of Meniere's disease (MD) on patients' lives: the Meniere's Disease Patient-Oriented Severity Index (MD POSI). Eighty-five volunteers with chronic MD took a self-administered MD POSI. The internal consistency form of reliability was determined by calculating Cronbach's alpha. The relationship between total MD POSI problem severity scores and responses to a global question concerning the degree to which his or her MD had changed the patient's life assessed construct-related validity. The total problem severity score yielded excellent reliability (Cronbach's alpha = .928). No significant age or gender effects were seen. A highly statistically significant association was noted between total problem severity scores and the degree to which the patient's life had been changed by his or her MD (Spearman rank correlation = .526, p < .0001), indicating excellent construct-related validity. The material included in the initial questionnaire provides important information that can be used clinically to quantify the impact of MD on patients' health status. A beta version of the instrument is in the process of validation and determination of its sensitivity to change in health status.

Adult↗
PubMed1998

Bioenergetic consequences of accumulating the common 4977-bp mitochondrial DNA deletion.

Mutations and deletions in mitochondrial DNA (mtDNA) lead to a number of human diseases characterized by neuromuscular degeneration. Accumulation of truncated mtDNA molecules (delta-mtDNA) lacking a specific 4977-bp fragment, the common deletion, leads to three related mtDNA diseases: Pearson's syndrome; Kearns-Sayre syndrome; and chronic progressive external ophthalmoplegia (CPEO). In addition, the proportion of delta-mtDNA present increases with age in a range of tissues. Consequently, there is considerable interest in the effects of the accumulation of delta-mtDNA on cell function. The 4977-bp deletion affects genes encoding 7 polypeptide components of the mitochondrial respiratory chain, and 5 of the 22 tRNAs necessary for mitochondrial protein synthesis. To determine how the accumulation of delta-mtDNA affects oxidative phosphorylation we constructed a series of cybrids by fusing a human osteosarcoma cell line depleted of mtDNA (rho0) with enucleated skin fibroblasts from a CPEO patient. The ensuing cybrids contained 0-86% delta-mtDNA and all had volumes, protein contents, plasma-membrane potentials and mitochondrial contents similar to those of the parental cell line. The bioenergetic consequences of accumulating delta-mtDNA were assessed by measuring the mitochondrial membrane potential, rate of ATP synthesis and ATP/ADP ratio. In cybrids containing less than 50-55% delta-mtDNA, these bioenergetic functions were equivalent to those of cybrids with intact mtDNA. However, once the proportion of delta-mtDNA exceeded this threshold, the mitochondrial membrane potential, rate of ATP synthesis, and cellular ATP/ADP ratio decreased. These bioenergetic deficits will contribute to the cellular pathology associated with the accumulation of delta-mtDNA in the target tissues of patients with mtDNA diseases.

Adenosine Diphosphate↗
PubMed1998

Peroxynitrite: a biologically significant oxidant.

1. Peroxynitrite is a short-lived and damaging oxidant that forms rapidly from the reaction of superoxide with nitric oxide. 2. In 1990, Joseph Beckman proposed that peroxynitrite contributed significantly to pathological oxidative stress in living tissues, and subsequent evidence strongly supports this proposal. 3. In this review, we outline the properties of peroxynitrite and discuss how it can affect biological systems and contribute to human pathologies.

Animals↗
PubMed1998

Implementing a residency program for the acute care nurse practitioner.

A clinical residency program for acute care nurse practitioners (ACNPs) was developed to facilitate the strengthening of practice competencies and role performance. Strengthening collaboration between physicians and ACNPs and supporting the growth of ACNPs through supportive and individualized learning opportunities were key components to a highly successful initiative.

Acute Disease↗
PubMed1997

Release of apoptogenic proteins from the mitochondrial intermembrane space during the mitochondrial permeability transition.

The Bcl-2-sensitive release of proteins such as cytochrome c from the mitochondrial intermembrane space into the cytosol is a critical early event in apoptosis. The mitochondrial permeability transition is also an important event in many forms of apoptotic cell death. To determine whether the permeability transition led to the release of apoptogenic proteins from mitochondria we induced the permeability transition in isolated rat liver mitochondria and characterised the proteins which were released. The permeability transition led to a generalised, non-specific release of proteins, including cytochrome c, from the mitochondrial intermembrane space which was prevented by an inhibitor of the permeability transition. To determine the mechanism of this protein release we measured both mitochondrial matrix swelling and protein release during the permeability transition in media of different osmolarities. Protein release correlated with mitochondrial matrix swelling, therefore the permeability transition causes release of proteins from the intermembrane space by rupturing the mitochondrial outer membrane. Supporting an apoptotic role for the proteins released by this mechanism, supernatants from mitochondria undergoing the permeability transition caused apoptotic changes in isolated nuclei. These data support the proposal that the mitochondrial permeability transition can induce apoptosis by releasing apoptogenic proteins into the cytoplasm [Skulachev, V.P., FEBS Lett. 397 (1996) 7-10].

Animals↗
PubMed1997

Chloroquine administration in mice increases beta-amyloid immunoreactivity and attenuates kainate-induced blood-brain barrier dysfunction.

The anti-malarial drug chloroquine (CHL) has been reported to cause the accumulation of beta-amyloid peptide containing fragments (fA beta) of the amyloid precursor protein within lysosomes in vitro. However, the significance of this finding with regards to the development of Alzheimer's disease (AD) pathology in vivo is not known. Hence, we investigated the effects of chronic CHL administration in the mouse. Systemically administered CHL caused an astrocytic response and an increase in intracellular A beta immunoreactivity throughout the brain, but no plaque-like pathology. Pharmacological challenge with the excitotoxin kainic acid (KA) revealed a mild proconvulsant effect of CHL pretreatment (P < 0.06). Interestingly, CHL protected the blood-brain barrier from characteristic KA-induced dysfunction. Given the hypothesized involvement of both excitotoxic processes and the vascular system in AD, the observed interactions may assist in elucidating the pathogenesis of AD.

Amyloid beta-Peptides↗
PubMed1997

Labeling of mitochondrial proteins in living cells by the thiol probe thiobutyltriphenylphosphonium bromide.

Alterations to the redox state of mitochondrial thiols is a particularly important response to oxidative stress, because mitochondria are a major source of reactive oxygen species within cells. To investigate these changes we designed and synthesized a novel probe for mitochondrial thiols, thiobutyltriphenylphosphonium bromide [TBTP; R. J. Burns, R. A. J. Smith, and M. P. Murphy (1995) Arch. Biochem. Biophys. 322, 60-68]. This lipophilic cation was accumulated several hundred-fold into the negatively charged matrix of isolated mitochondria, where it equilibrated with endogenous thiols and during oxidative stress formed disulfide bonds to exposed protein thiols. In this paper we show that TBTP can also selectively react with mitochondrial protein thiols in living cells. Since TBTP localizes specifically to mitochondria and forms disulfide bonds selectively with mitochondrial proteins during oxidative stress, we conclude that TBTP has utility for investigating changes in mitochondrial thiols, independently of alterations occurring in the cytoplasm.

Animals↗
PubMed1997

Right latissimus dorsi cardiomyoplasty improves left ventricular energetics.

BACKGROUND: The mechanism by which cardiomyoplasty appears to enhance left ventricular (LV) function is not well understood. We applied the time-varying elastance model to study the effect of cardiomyoplasty on LV function, ventriculovascular coupling, and LV energetics in an acute canine model. METHODS: Right latissimus dorsi cardiomyoplasty was performed in 5 dogs. The end-systolic pressure-volume relation was generated by using brief caval occlusions. End-systolic elastance, effective arterial elastance, stroke work, internal work, total mechanical work, and stroke work efficiency (stroke work/total mechanical work) were calculated from these pressure-volume data. Myocardial oxygen consumption and overall mechanical efficiency (stroke work/myocardial oxygen consumption) were predicted using the myocardial oxygen consumption-total mechanical work relation. RESULTS: Skeletal muscle contraction significantly increased end-systolic elastance, an index of contractility. Although stroke work did not change significantly, the increase in end-systolic elastance led to a 29% decrease in total mechanical work, a 50% decrease in internal work, and an increase in stroke work efficiency from 53% to 66%. This was consistent with the observed 29% decrease in effective arterial elastance/end-systolic elastance, an indicator of ventriculovascular coupling that is related inversely to stroke work efficiency. Predicted myocardial oxygen consumption decreased by at least 22%, and predicted overall mechanical efficiency increased at a minimum from 16.1% to 18.4%. CONCLUSIONS: These results support the theory that cardiomyoplasty unloads the LV by decreasing LV volumes and increasing contractility. These effects appear to improve LV energetics by decreasing total mechanical work without significantly affecting stroke work, resulting in improved stroke work efficiency. The decrease in total mechanical work strongly suggests a decrease in myocardial oxygen consumption and an increase in overall mechanical efficiency.

Animals↗
PubMed1997

Effects of extended electrical kindling on exploratory behavior and spatial learning.

Short-term electrical kindling, a widely used experimental model of epilepsy, appears to have little effect on behavior. The effects of extended kindling are largely unknown. Rats implanted with kindling electrodes in amygdala (AM) or perforant path (PP) received 300 kindling trials over approximately 7 months, and were tested in the Morris watermaze after a 7-10 day recovery period. Kindled animals were impaired during the initial training on hidden-platform acquisition, but not in retention of platform location. No deficits were found in acquiring a new hidden-platform location, latency to reach a visible-platform, or in swim speed. Open-field activity showed a sustained increase when tested during kindling, but only a transient increase when tested following suspension of kindling. Similar results were obtained for both AM and PP kindled animals. Hence, long-term kindling of both of these sites produced behavioral changes that were transient in nature. Further, these results also indicate that propagation of seizure activity from remote sites can alter hippocampally-mediated or related behavior.

Animals↗
PubMed1997

Selective targeting of bioactive compounds to mitochondria.

Mitochondrial function can be manipulated selectively by targeting bioactive compounds to mitochondria in living cells. Current targeting mechanisms involve harnessing either the mitochondrial membrane potential or the mitochondrial protein-import machinery to take up molecules linked to lipophilic cations or mitochondrial signal peptides, respectively. Compounds localized like this have been used in studies of mitochondrial metabolism and show potential as anticancer drugs. In addition, because mitochondria play a role in many critical cell processes, mitochondrial targeting has the potential to protect, repair or kill cells selectively, and may become a key tool in the development of gene therapy for mitochondrial DNA diseases.

Antineoplastic Agents↗