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Biomedical subjects

M E Murphy

Publications and source records attributed to M E Murphy.

At least 73 records · Page 4Linked to original sources
PubMed1991

Reversible conversion of nitroxyl anion to nitric oxide by superoxide dismutase.

Superoxide dismutase (SOD) rapidly scavenges superoxide (O2-) and also prolongs the vasorelaxant effects of nitric oxide (NO), thought to be the endothelium-derived relaxing factor. This prolongation has been ascribed to prevention of the reaction between O2- with NO. We report that SOD supports a reversible reduction of NO to NO-. When cyanamide and catalase were used to generate NO- in the presence of SOD, NO was measured by the conversion of HbO2 to MetHb. When SOD[Cu(I)] was exposed to NO anaerobically, NO- was trapped by MetHb forming nitrosylmyoglobin. When NO was generated by 3-morpholinosydnonimine hydrochloride in the presence of SOD, NO- or a similar reductant was formed, which reduced catalase compound II and promoted the formation of the catalase [Fe(III)]-NO complex. It is, therefore, conceivable that SOD may protect NO and endothelium-derived relaxing factor by a mechanism in addition to O2- scavenging and that NO- may be a physiologically important form of endothelium-derived relaxing factor.

Animals↗
PubMed1991

Nitric oxide production by cultured aortic endothelial cells in response to thiol depletion and replenishment.

The requirements and influence of thiols on the production of nitric oxide (NO) were examined in cultured porcine aortic endothelial cells. NO production was diminished when cells were pretreated with thiol-depleting agents (IC50: N-ethylmaleimide, 30 microM; 1-chloro-2,4-dinitrobenzene, 200 microM; diamide, 1.5 mM; diethyl maleate, 20 mM). The depletion of glutathione (45-99% loss at the various IC50 values) and protein thiols (3-25% loss at IC50) showed no consistent relationship to decreased NO production. The effects of the agents on NO production were not linked to altered sensitivity to the stimulant (calcium ionophore A23187; maximal effect at 10 microM), but roughly paralleled the appearance of cell damage (17-44% lactate dehydrogenase release at IC50). The decrease in NO production due to 1-chloro-2,4-dinitrobenzene was partially reversed by cysteine, dithioerythritol, and dihydrolipoate, whereas cystine partially reversed the decrease due to diamide or diethyl maleate. On the other hand, several thiols diminished NO production in control cells. Overall, alterations of NO production did not parallel the depletion or replenishment of either glutathione, protein thiol, or soluble thiol pools, and so the results argue against hypotheses that cellular thiols are either substrates or necessary cofactors in the pathway of NO synthesis in endothelial cells.

Animals↗
PubMed1991

Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols.

Reactive oxygen species occur in tissues and can damage DNA, proteins, carbohydrates, and lipids. These potentially deleterious reactions are controlled by a system of enzymatic and nonenzymatic antioxidants which eliminate prooxidants and scavenge free radicals. The ability of the lipid-soluble carotenoids to quench singlet molecular oxygen may explain some anticancer properties of the carotenoids, independent of their provitamin A activity. Tocopherols are the most abundant and efficient scavengers of hydroperoxyl radicals in biological membranes. Water-soluble antioxidants include ascorbate and cellular thiols. Glutathione is an important substrate for enzymatic antioxidant functions and is capable of nonenzymatic radical scavenging. Thiols associated with membrane proteins may also be important to the antioxidant systems. Interactions between the thiols, tocopherols, and other compounds enhance the effectiveness of cellular antioxidant defense.

Animals↗
PubMed1990

Physical and chemical scavenging of singlet molecular oxygen by tocopherols.

Singlet molecular oxygen (1O2) arising from the thermal decomposition of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate was used to assess the effectiveness of alpha-, beta-, gamma-, and delta-tocopherol in the physical quenching as well as the chemical reaction of 1O2. The relative physical quenching efficiencies of the tocopherol homologs were found to decrease in the order of alpha greater than or equal to beta greater than gamma greater than delta-tocopherol. The ability of physical quenching depends on a free hydroxyl group in position 6 of the chromane ring. Chemical reactivity of the tocopherol homologs with 1O2 was low, accounting for 0.1-1.5% of physical quenching with beta-tocopherol showing particularly low reactivity, resulting in the sequence alpha greater than gamma greater than delta greater than beta-tocopherol. Tocopheryl quinones were products of all tocopherol homologs, and in addition a quinone epoxide was a major product from gamma-tocopherol. This quinone epoxide was not cleaved by rat liver microsomal epoxide hydrolase; however, it reacted further with 1O2. It is concluded that methylation in position 5 of the chromane ring enhances physical quenching of 1O2, whereas chemical reactivity is favored by a methylated position 7. In view of the fact that beta-tocopherol is as effective as alpha-tocopherol in physical quenching of 1O2 but shows very low chemical reactivity, this tocopherol homolog might be particularly suitable for biological conditions in which an accumulation of oxidation products might weaken the antioxidant defense.

Kinetics↗
PubMed1990

Temporal relationships between the loss of vitamin E, protein sulfhydryls and lipid peroxidation in microsomes challenged with different prooxidants.

Microsomal membranes exposed to various prooxidants displayed distinct patterns in the temporal relationships between the loss of antioxidants and the onset of lipid peroxidation. Prooxidants included ADP-chelated iron with reductants (NADPH and ascorbate), or compounds that generate peroxyl radicals (tert-butyl hydroperoxide and 2,2'-azobis(2-amidinopropane)), or free metal ions (Fe2+ and Cu2+). Lipid peroxidation initiated by Fe2+ began simultaneously with the loss of alpha-tocopherol, while about 15% of alpha-tocopherol was lost during the lag phase preceding lipid peroxidation in systems containing chelated iron. A substantial depletion of alpha-tocopherol (49-69%) preceded peroxidation when initiated by peroxyl radicals, while 97% of the antioxidant disappeared preceding peroxidation initiated by copper. The loss of protein sulfhydryl groups also varied between the prooxidants (ranging from 0 to 82% loss prior to the onset of peroxidation) to a degree that roughly paralleled the loss of vitamin E. Therefore, important determinants of peroxidation are prooxidant-dependent, and not only include alpha-tocopherol and sulfhydryl groups, but also membrane integrity, propagation reactions and the availability of catalytic transition metals.

Chemical Phenomena↗
PubMed1989

Oxidation state of tissue thiol groups and content of protein carbonyl groups in chickens with inherited muscular dystrophy.

Indirect evidence suggests that oxidative stress may play a role in the pathogenesis of inherited muscular dystrophy, but the significance and precise extent of this contribution is poorly understood. Compared with normal muscle, significantly higher contents of glutathione, glutathione disulphide, protein-glutathione mixed disulphides and protein carbonyl groups, and significantly lower contents of free protein thiol groups, were found in pectoralis major muscle of genetically dystrophic chickens (the muscle affected by this disease) at 4 weeks of age. Other tissues did not show such marked disease-related differences. Interestingly, the protein pool in normal, but not dystrophic, pectoralis major muscle was relatively less oxidized in relation to the glutathione pool as compared with other tissues studied. The mechanisms by which this unique relationship between the thiol pools is maintained remain unknown. Although the physiological consequences of the increased content of protein carbonyl groups and the altered thiol pools in dystrophic muscle are not clear, the changes evident at such a young age are consistent with the occurrence of oxidative stress and may reflect significant damage to cellular proteins in this disease.

Animals↗
PubMed1989

Structure of recombinant human renin, a target for cardiovascular-active drugs, at 2.5 A resolution.

The x-ray crystal structure of recombinant human renin has been determined. Molecular dynamics techniques that included crystallographic data as a restraint were used to improve an initial model based on porcine pepsinogen. The present agreement factor for data from 8.0 to 2.5 angstroms (A) is 0.236. Some of the surface loops are poorly determined, and these disordered regions border a 30 A wide solvent channel. Comparison of renin with other aspartyl proteinases shows that, although the structural cores and active sites are highly conserved, surface residues, some of which are critical for specificity, vary greatly (up to 10A). Knowledge of the actual structure, as opposed to the use of models based on related enzymes, should facilitate the design of renin inhibitors.

Aspartic Acid Endopeptidases↗
PubMed1989

Antioxidant activity of dihydrolipoate against microsomal lipid peroxidation and its dependence on alpha-tocopherol.

The antioxidant effect of dihydrolipoate and lipoate was examined in microsomal fractions obtained from normal and alpha-tocopherol-deficient animals after initiation of lipid peroxidation with an NADPH/iron/ADP system. Dihydrolipoate prolonged the lag phase before the onset of low-level chemiluminescence and before the rapid accumulation of thiobarbituric acid-reactive substances in normal but not in vitamin E-deficient microsomes. Lipoate did not show such an antioxidant effect. It is concluded that the dihydrolipoate-mediated protection against lipid peroxidation by prolonging the lag phase is dependent on alpha-tocopherol. Likewise, dihydrolipoate prolonged the lag phase before the onset of the rapid loss of vitamin E during lipid peroxidation. Dihydrolipoate, like other biological thiols such as GSH, also affects the peroxidative process after the lag period. The effects included a smaller slope of the chemiluminescence increase, a lower maximal level of chemiluminescence, a slower loss of alpha-tocopherol and a slower accumulation, but unchanged maximal levels, of thiobarbituric acid-reactive substances. The biological significance may be most prominent in the mitochondrial matrix space, where lipoamide-containing ketoacid dehydrogenases are located. A potential pharmacological use of this biological dithiol in conditions associated with oxidative stress could be based on the antioxidant activity of dihydrolipoate.

Animals↗
PubMed1989

Lipid peroxidation inhibitory factors in liver and muscle of rat, mouse, and chicken.

Glutathione- or sulfhydryl-dependent antioxidant factors that act to prevent lipid peroxidation have been reported in both microsomes and cytoplasm from rat liver. The cytoplasmic factor has been identified in several other tissues and species, but the distribution of the microsomal factor has not been reported. Chicken and mouse livers had much lower activities of the glutathione-dependent membrane-associated and cytoplasmic antioxidant factors than rat liver. Peroxidative damage to membranes has been hypothesized as a mechanism of tissue damage in muscular dystrophy. However, neither the chicken, mouse, nor rat had significant activities of the antioxidant factors in muscle. There was also no significant difference between normal and dystrophic chicken livers in the activity of the antioxidant factors associated with the microsomes or the cytoplasm, nor of the liver microsomal factor in normal and dystrophic mice. The results do not support an important role for the antioxidant factors in the pathogenesis of muscular dystrophy, and raise questions as to whether such factors are physiologically important in species other than rat or in tissues other than liver.

Animals↗
PubMed1989

Oxidative stress and muscular dystrophy.

Oxidative stress may be the fundamental basis of many of the structural, functional and biochemical changes characteristic of the inherited muscular dystrophies in animals and humans. The presence of by-products of oxidative damage, and the compensatory increases in cellular antioxidants, both indicate oxidative stress may be occurring in dystrophic muscle. Changes in the proportions and metabolism of cellular lipids, abnormal functions of cellular membranes, altered activity of membrane-bound enzymes such as the SR Ca2+-ATPase, disturbances in cellular protein turnover and energy production and a variety of other changes all indicate that these inherited muscular dystrophies appear more like the results of oxidative stress to muscle than any other type of underlying muscle disturbance. Particular details of these altered characteristics of dystrophic muscle, in combination with current knowledge on the processes of oxidative damage to cells, may provide some insight into the underlying biochemical defect responsible for the disease, as well as direct research towards the ultimate goal of an effective treatment.

Animals↗
PubMed1989

Sparrows discriminate between diets differing in valine or lysine concentrations.

White-crowned Sparrows (WCS) were given free access to pairs of semisynthetic diets that were either adequate or subadequate (25% of requirement) in valine or lysine. Within 2 to 4 days WCS chose a ratio of the paired diets that allowed them to maintain body mass or restore any losses quickly. On the initial choice days the birds transiently reduced total daily food intake (TDFI) roughly in proportion to their intake of the subadequate diet. The initial decrease of TDFI was greater and the latency in choosing an effective ratio of the paired diets was 2-3 days longer with valine than with lysine diets in well-nourished test birds. In malnourished birds fed only the subadequate test diet for 3 days, valine-deficient birds increased TDFI and body mass more promptly than did lysine-deficient birds when offered a choice of adequate and subadequate diets. The form of the test amino acid (CAA = crystalline, PB = protein-bound) had little effect on choice behavior, but sudden transfer of WCS from a PB acclimation diet to test diets with a large total CAA concentration increased the latency of effective choice by 2-3 days. A brief acclimation (2-3 days) to a CAA diet precludes any bias between nutritionally equivalent CAA and PB diets. The small differences in choice dynamics between valine and lysine and between dietary forms may help to identify mechanisms involved in food choice but are probably ecologically insignificant to free-living WCS. These birds are very adept at selecting diets that satisfy their amino acid requirements.

Animals↗
PubMed1989

Altered contents of tocopherols in chickens with inherited muscular dystrophy.

Oxidative damage has been hypothesized as the basis for some of the changes in enzymatic functions and physical properties of membranes in inherited muscular dystrophy. The contents of alpha- and gamma-tocopherol (vitamin E) and their oxidation products, the tocopheryl quinones, were measured at 1 to 4 weeks after hatching in the muscle and other tissues of chickens with inherited muscular dystrophy. Analyses at these early ages minimized the potential influence of pathological changes on the measured parameters. The affected muscle (pectoralis major) of dystrophic birds contained significantly higher levels of alpha-tocopheryl quinone and a decreased ratio of alpha- to gamma-tocopherol. Consistent changes in these parameters were not observed in other tissues. Although their basis remains unclear, these changes in the tocopherols are suggestive of oxidative stress in dystrophic muscle membranes. Lipid extracts of tissues of normal and dystrophic birds exhibited no significant differences in the content of conjugated dienes or lipofuscins, two other indices of oxidative stress. These data do not consistently support the hypothesis that oxidative stress plays a causal role in damage to dystrophic muscle, although it remains possible that free-radical damage is involved in the secondary alterations associated with muscular dystrophy.

Aging↗
PubMed1988

Comparative molecular model building of two serine proteinases from cytotoxic T lymphocytes.

Two genes that are expressed when precursor cytotoxic T lymphocytes are transformed to T killer cells have been cloned and sequenced. The derived amino acid sequences, coding for cytotoxic cell protease 1 (CCP1) and Hannuka factor (HF) are highly homologous to members of the serine proteinase family. Comparative molecular model building using the known three-dimensional structures and the derived amino acid sequences of the lymphocyte enzymes has provided useful structural information, especially in predicting the conformations of the substrate binding sites. In applying this modelling procedure, we used the X-ray structures of four serine proteinases to provide a structurally based sequence alignment: alpha-chymotrypsin (CHT), bovine trypsin (BT), Streptomyces griseus trypsin (SGT), and rat mast cell protease 2 (RMCP2). The root mean square differences in alpha-carbon atom positions among these four structures when compared in a pairwise fashion range from 0.79 to 0.97 A for structurally equivalent residues. The sequences of the two lymphocyte enzymes were then aligned to these proteinases using chemical criteria and the superimposed X-ray structures as guides. The alignment showed that the sequence of CCP1 was most similar to RMCP2, whereas HF has regions of homology with both RMCP2 and BT. With RMCP2 as a template for CCP1 and the two enzymes RMCP2 and BT as templates for HF, the molecular models were constructed. Intramolecular steric clashes that resulted from the replacement of amino acid side chains of the templates by the aligned residues of CCP1 and HF were relieved by adjustment of the side chain conformational angles in an interactive computer graphics device. This process was followed by energy minimization of the enzyme model to optimize the stereochemical geometry and to relieve any remaining unacceptably close nonbonded contacts. The resulting model of CCP1 has an arginine residue at position 226 in the specificity pocket, thereby predicting a substrate preference for P1 aspartate or glutamate residues. The model also predicts favorable binding for a small hydrophobic residue at the P2 position of the substrate. The primary specificity pocket of HF resembles that of BT and therefore predicts a lysine or arginine preference for the P1 residue. The arginine at position 99 in the model of HF suggests a preference for aspartate or glutamate side chains in the P2 position of the substrate. Both CCP1 and HF have a free cysteine in the segment of polypeptide 88 to 93.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1987

Simultaneous measurement of tocopherols and tocopheryl quinones in tissue fractions using high-performance liquid chromatography with redox-cycling electrochemical detection.

Tocopherols and tocopheryl quinones in lipid extracts of biological samples have primarily been measured using relatively insensitive ultraviolet detection methods. Oxidative electrochemical detection increases both the sensitivity and selectivity when measuring the tocopherols. We have developed an electrochemical detection system which sequentially reduces and oxidizes tocopheryl metabolites eluted from a reversed-phase high-performance liquid chromatography column, achieving sensitivities of about 0.05 pmol for both the tocopherols and their quinones. Using a rapid and mild extraction procedure, endogenous levels of alpha- and gamma-tocopherol as well as their respective quinones were measured in homogenates of chicken liver and muscle, and in dilute preparations of rat liver microsomes. The principle of the detection system could be applied to the determination of tocopheryl dihydroquinones, ubiquinols and ubiquinones with slight modifications to the mobile phase buffer and the electrode potentials of the detector.

Animals↗