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Biomedical subjects

Christine C Winterbourn

Publications and source records attributed to Christine C Winterbourn.

14 recordsLinked to original sources
PubMed2003

Characterization of non-covalent oligomers of proteins treated with hypochlorous acid.

Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1-20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.

Amino Acids↗
PubMed2003

Radical-radical reactions of superoxide: a potential route to toxicity.

Superoxide reacts with many radicals, such as phenoxyl radicals, at near diffusion-controlled rates. These reactions are usually considered to be repair processes and have received little biological attention. However, addition of superoxide to give hydroperoxides and secondary oxidation products can also occur. The relative contributions of addition and repair vary depending on the properties of the phenol. With tyrosine, addition to give tyrosine hydroperoxide predominates, but in peptides the efficiency of hydroperoxide formation depends on the proximity of free amine groups. Radicals from other phenolic compounds, such as alpha-tocopherol and serotonin, also undergo addition reactions with superoxide. Physiologically, these reactions are likely to be more significant than dimerization when both radicals are generated together. They warrant attention as potential contributors to superoxide toxicity.

Animals↗
PubMed2003

3-Chlorotyrosine as a marker of protein damage by myeloperoxidase in tracheal aspirates from preterm infants: association with adverse respiratory outcome.

Oxidative injury is implicated in the development of chronic lung disease in preterm infants with respiratory distress. However, direct evidence of a causal role is limited and the source of reactive oxidants has not been identified. We have previously shown that protein carbonyl levels in tracheal aspirates correlate positively with myeloperoxidase, suggesting that neutrophil oxidants could be the source of this protein injury. We have extended these observations by measuring 3-chlorotyrosine, a specific biomarker of the neutrophil oxidant, hypochlorous acid, in tracheal aspirate proteins (144 samples) from 69 infants with birth weight <1500 g. 3-Chlorotyrosine levels were higher in these infants than in larger infants without respiratory distress (median 83 compared with 13 micromol/mol tyrosine). They correlated strongly with myeloperoxidase activity (correlation coefficient 0.75, p < 0.0001) and to a lesser extent with protein carbonyls. 3-Chlorotyrosine levels (at 1 wk after birth) correlated negatively with birth weight or gestational age. They were significantly higher in infants who developed chronic lung disease (oxygen requirement at 36 wk postmenstrual age) than in those who did not (median 88 and 49 micromol/mol tyrosine, respectively) and correlated with days of supplemental oxygen. 3-Chlorotyrosine was also significantly higher in infants who had lung infection or were Ureaplasma urealyticum positive. Our results are the first evidence that chlorinated proteins are produced in the lungs of premature infants and that they are higher in infection. The higher 3-chlorotyrosine levels in infants who develop chronic lung disease suggest that neutrophil oxidants contribute to the pathology of this disease.

Biomarkers↗
PubMed2002

Biological reactivity and biomarkers of the neutrophil oxidant, hypochlorous acid.

Free radicals or reactive oxygen species are thought to contribute to the pathology of many diseases. These include inflammatory conditions, where neutrophils accumulate in large numbers and are stimulated to produce superoxide and other reactive oxidants. Hypochlorous acid (HOCl), produced by myeloperoxidase-catalysed oxidation of chloride by hydrogen peroxide, is the major strong oxidant generated by these cells. Neutrophil-mediated injury may also be important in toxicology when an initial insult is followed by an inflammatory response. It is important to characterize the inflammatory component of such injury and the extent to which it involves reactive oxidants. On the one hand, this requires an understanding of how neutrophil oxidants react with cells and tissue constituents. On the other, specific biomarkers are needed so that oxidative damage can be quantified in clinical material and related to disease severity. This presentation considers biologically relevant reactions of HOCl and the biomarker assays that can be applied to probing the pathological role of myeloperoxidase and its products.

Animals↗
PubMed2002

Interaction with substrate sensitises caspase-3 to inactivation by hydrogen peroxide.

Caspases have an active site cysteine whose oxidation blocks catalytic activity. Caspase activity, measured in lysates of apoptotic cells, was inhibited by H2O2 with an IC50 of 7 microM. Recombinant caspase-3 was directly inhibited by H2O2, with an estimated second-order rate constant of 750 M-1 s-1. These values were determined when H2O2 was added while the caspases were cleaving a peptide substrate. There was a 40-fold decrease in sensitivity to inactivation if the substrate was absent at the time of H2O2 addition. These results rationalise conflicting reports of the sensitivity of caspase-3 to H2O2, and identify a novel mechanism for sensitising a thiol enzyme to oxidative inactivation.

Binding Sites↗
PubMed2002

Detection of oxidant sensitive thiol proteins by fluorescence labeling and two-dimensional electrophoresis.

Oxidants can activate signaling pathways and modulate a variety of cellular activities. Their action at a molecular level involves the post-translational modification of protein thiols. We have developed a proteomic method to monitor the reduction and oxidation of protein thiols, and identify those thiol proteins most sensitive to oxidation. Cells were disrupted in the presence of N-ethylmaleimide to block the reduced thiol proteins and dithiothreitol was added to reduce the oxidized thiol proteins before labeling with 5-iodoacetamidofluorescein. Two-dimensional (2-D) electrophoresis was used to resolve the labeled samples. We applied the method to Jurkat T lymphocytes and examined the effect of diamide on the oxidized and reduced thiol protein profiles. A small percentage of protein thiols were already oxidized in untreated cells. Exposure of cells to 2 mM diamide for ten minutes led to a dramatic increase in thiol protein oxidation as seen in the oxidized thiol protein map. However, it was difficult to detect any change in the pattern of reduced thiol proteins. Separation of proteins by 2-D electrophoresis revealed approximately 200 thiol proteins that were oxidized by diamide treatment. This method will be valuable in elucidating redox signaling pathways.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2002

Markers of oxidative injury in the cerebrospinal fluid of a premature infant with meningitis and periventricular leukomalacia.

Free radicals have been hypothesized to play a key role in the evolution of periventricular leukomalacia, although direct evidence of oxidative injury in the human infant is lacking. This case report is the first to demonstrate a marked elevation in the levels of lipid and protein oxidative products in the cerebrospinal fluid during the evolution of periventricular leukomalacia in a premature infant with meningitis.

Biomarkers↗
PubMed2002

The effect of hypochlorous acid on the expression of adhesion molecules and activation of NF-kappaB in cultured human endothelial cells.

Exposure to oxidants can up-regulate the expression of adhesion molecules in endothelial cells with a consequent increase in neutrophil attachment. Similarly, the transcription factor nuclear factor-kappaB (NF-kappaB), which controls the expression of the intercellular adhesion molecules (ICAMs), can also be activated by oxidants in some cells. We have investigated whether hypochlorous acid (HOCl), the major strong oxidant produced by neutrophils, can affect the expression of adhesion molecules on human umbilical vein endothelial cells (HUVEC) and promote neutrophil adhesion. We found that HOCl could induce an increase in neutrophil adhesion to the endothelial cells after 60 min of treatment. Activation of NF-kappaB could be detected under similar conditions. However, the dose of HOCl required for this effect resulted in considerable longer-term toxicity to the cells. Treatment of HUVEC with sublethal doses of HOCl had no effect on NF-kappaB activation, neutrophil adhesion, or the surface expression of E-selectin, ICAM-1, or P-selectin. However, pretreatment with low concentrations of HOCl prevented phorbol myristate acetate-induced von Willebrand factor expression (a marker for P-selectin). These results show that, unlike H(2)O(2), HOCl does not significantly enhance neutrophil attachment to the endothelium. Rather it may be able to inhibit the expression of adhesion molecules with important consequences for endothelial function and inflammatory vascular disease.

Cell Adhesion↗
PubMed2002

Oxidant-mediated phosphatidylserine exposure and macrophage uptake of activated neutrophils: possible impairment in chronic granulomatous disease.

The removal of neutrophils from inflammatory sites is essential for the resolution of inflammation. Surface changes, including phosphatidylserine exposure, label neutrophils for phagocytosis by macrophages. Here, we demonstrate that externalization of phosphatidylserine and uptake by monocyte-derived macrophages occurred in human neutrophils ingesting Staphylococcus aureus. Both processes were dependent on oxidant production from the neutrophil NADPH oxidase. There was no requirement for myeloperoxidase, and H(2)O(2) was identified as the most likely trigger for PS exposure. We hypothesize that clearance of stimulated neutrophils would be delayed in chronic granulomatous disease (CGD) neutrophils, which lack a functional NADPH oxidase. To explore this possibility, heat-killed S. aureus were injected into the peritoneum of CGD and normal mice. Elevated neutrophil numbers were observed in the inflammatory exudate of the CGD animals, consistent with impaired recognition and clearance.

Adolescent↗
PubMed2001

Chlorination of bacterial and neutrophil proteins during phagocytosis and killing of Staphylococcus aureus.

Myeloperoxidase is proposed to play a central role in bacterial killing by generating hypochlorous acid within neutrophil phagosomes. However, it has yet to be demonstrated that these inflammatory cells target hypochlorous acid against bacteria inside phagosomes. In this investigation, we treated Staphylococcus aureus with varying concentrations of reagent hypochlorous acid and found that even at sublethal doses, it converted some tyrosine residues in their proteins to 3-chlorotyrosine and 3,5-dichlorotyrosine. To determine whether or not ingested bacteria were exposed to hypochlorous acid in neutrophil phagosomes, we labeled their proteins with [(13)C(6)]tyrosine and used gas chromatography with mass spectrometry to identify the corresponding chlorinated isotopes after the bacteria had been phagocytosed. Chlorinated tyrosines were detected in bacterial proteins 5 min after phagocytosis and reached levels of approximately 2.5/1000 mol of tyrosine at 60 min. Inhibitor studies revealed that chlorination was dependent on myeloperoxidase. Chlorinated neutrophil proteins were also detected and accounted for 94% of total chlorinated tyrosine residues formed during phagocytosis. We conclude that hypochlorous acid is a major intracellular product of the respiratory burst. Although some reacts with the bacteria, most reacts with neutrophil components.

Anti-Bacterial Agents↗
PubMed2001

Thiol oxidase activity of copper, zinc superoxide dismutase.

The ability of copper,zinc superoxide dismutase (Cu,Zn-SOD) to catalyze autoxidation of cysteine and other thiols was investigated by measuring thiol loss and oxygen consumption. The reaction occurred equally well with the bovine and human enzymes and produced hydrogen peroxide and the corresponding disulfide. It did not occur with manganese SOD and is not, therefore, due to the dismutase activity of the enzyme. Cysteine and cysteamine were highly reactive: the K(m) for cysteine was 1.4 mm and V(max) (with 40 microg/ml SOD) 35 microm/min; the equivalent values for cysteamine (with 20 microg/ml SOD) were 1.4 mm and 36 microm/min. With 1 mm thiol and 40 microg/ml SOD, rates of oxidation of other thiols (microm/min) were as follows: GSH, 1.0; dithiothreitol, 2.1; dihydrolipoic acid, 1.7; homocysteine, 1.6; cys-gly, 1.4; penicillamine, 0.6; and N-acetylcysteine, 0.1. SOD-mediated oxidation of cysteine, in the absence of chelating agents, proceeded only after a variable lag phase. The lag was decreased but not eliminated with Chelex-treated reagents and is attributed to interference by submicromolar concentrations of iron and possibly other transition metal ions. SOD-catalyzed oxidation of the other thiols was variably affected by adventitious metal ions and chelating agents. Reactions were all performed in the presence of desferrioxamine to obviate these effects. SOD-catalyzed oxidation of GSH and homocysteine was enhanced by cysteine through a thiol-disulfide exchange mechanism. This study characterizes a novel pro-oxidant thiol oxidase activity of Cu,Zn-SOD. It is a potential source of reactive oxidants and may contribute to the cytotoxicity of reactive thiols such as cysteine and cysteamine.

Animals↗