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Showing 1501–1520 of 2058 publications.
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Glucocorticoids inhibit placental cytokines from cultured normal and preeclamptic placental explantsXu, Bei; Makris, Angela; Thornton, Charlene Eliza; Hennessy, AnnemarieGlucocorticoids are used in pregnancy to enhance fetal lung maturity as well as to ameliorate antepartum and postpartum HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome, but it is not clear if glucocorticoids can modulate placental cytokine production. The aim of this study is to examine the effect of glucocorticoids at equivalent doses used for fetal lung maturity on placental tissue production of cytokines (IL-10, IL-6 and TNF-?). Placental biopsies were taken from the decidual surface of term placentas of normal pregnancy (n = 5) and preeclampsia (n = 5). Villous explants were cultured with increasing concentrations of glucocorticoids (betamethasone and methyl-prednisolone, 0.0025 ?M, 0.25 ?M and 25 ?M). The dose effect of glucocorticoids on cytokines (TNF-?, IL-6 and IL-10) production was examined using ELISA. There was a stepwise reduction of TNF-? (23.6-97.5% reduction) and IL-6 (13.7-71% reduction) with increasing doses of betamethasone and methyl-prednisolone from placentas of women with preeclampsia and normal pregnancy. However, IL-10 was not altered in conditioned medium by increasing doses of glucocorticoids. Our data suggest that the ratio of pro-inflammatory to anti-inflammatory cytokine (Th1/Th2) is potentially altered by exogenous glucocorticoids. These changes have a favourable effect on the ratio in preeclampsia with a reduction in the potentially vascular active pro-inflammatory cytokines but without altering or decreasing the necessary anti-inflammatory cytokine IL-10 production in placental tissue. 2004 Elsevier Ltd. All rights reserved.
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Barter, Philip J.[No abstract available]
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Ozawa, Kiyoshi; Headlam, Madeleine J.; Mouradov, Dmitri; Watt, Stephen J.; Beck, Jennifer L.; Rodgers, Kenneth John; Dean, Roger T.; Huber, Thomas L.; Otting, Gottfried; Dixon, Nicholas E.An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean 15N-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains. 2005 FEBS.
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Barter, Philip J.; Ballantyne, Christie Mitchell[No abstract available]
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Furukawa, Ayako; Hiraku, Yusuke; Oikawa, Shinji; Luxford, Catherine; Davies, Michael J.; Kawanishi, ShousukeIn ?-irradiation, .OH is directly generated from water and causes DNA damage leading to carcinogenesis. Exposure of proteins to ?-irradiation, in the presence of oxygen, gives high yields of hydroperoxides. To clarify whether these hydroperoxides, particularly those formed on DNA-binding histone proteins, participate in ?-irradiation- induced carcinogenesis, experiments using 32P-labelled DNA fragments obtained from human cancer-related genes were undertaken. Histone protein-hydroperoxides induced significant DNA damage in the presence of Cu(I). Histone H1- and H3-hydroperoxides showed stronger DNA damage compared with histone H2A- and H4-hydroperoxides at 0.7 ?M. Histone H1-hydroperoxides caused Cu(I)-dependent DNA damage predominantly at guanine residues, especially at 5?-GGC-3?, 5?-GGA-3?, 5?-GGT-3? and single G bases. In contrast, histone H3-hydroperoxides/Cu(I) induced DNA damage at 5?-G in GG sequences; this sequence specificity is identical with that generated by 2,2?-azobis (2-amidinopropane) dihydrochloride, which is known to produce peroxyl radicals (RO<inf>2</inf>.). The difference in site specificity of DNA damage induced by histone H1- and H3-hydroperoxides may arise from their amino acid composition or their mode of binding to DNA. The histone H1-hydroperoxides/Cu(I) system also induced 8-oxo-7,8-dihydro-2?- deoxyguanosine formation in calf thymus DNA. It is concluded that histone protein-hydroperoxides can induce guanine-specific DNA damage, which may contribute to ?-irradiation-induced carcinogenesis. 2005 Biochemical Society.
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Barter, Philip J.[No abstract available]
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Morgan, Philip E.; Sturgess, Allan D.; Davies, Michael J.Objective. To examine protein oxidation in systemic lupus erythematosus (SLE) and to correlate levels of protein oxidation products with disease activity. Methods. Serum was collected from SLE patients and healthy control subjects. Protein-bound carbonyls and the pro-oxidant enzyme myeloperoxidase (MPO) were quantified by enzyme-linked immunosorbent assay. Protein thiols were quantified using 5,5?-dithionitrobenzoic acid. Protein-bound amino acids and methionine, tyrosine, and phenylalanine oxidation products were quantified by acid hydrolysis and high-performance liquid chromatography. Disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Levels of anti-double-stranded DNA (anti-dsDNA) antibodies were measured by radioimmunoassay. Results. Compared with control subjects, SLE patients exhibited elevated levels of protein carbonyls (0.108 0.078 versus 0.064 0.028 nmoles/mg of protein; P = 0.046), decreased levels of protein thiols (3.9 1.1 versus 4.9 0.7 nmoles/mg of protein; P = 0.003), decreased levels of protein-bound methionine (P = 0.0007), and increased levels of protein-bound methionine sulfoxide (P = 0.0043) and 3-nitrotyrosine (P = 0.0477). SLE patients with high SLEDAI scores or elevated anti-dsDNA antibody levels exhibited increased oxidation compared with patients with low SLEDAI scores or low antibody levels. Serum MPO levels were decreased in SLE patients (P = 0.03), suggesting that this enzyme is not responsible for the enhanced protein oxidation. Conclusion. We found elevated levels of multiple markers of protein oxidation in sera from SLE patients compared with controls, and these levels correlated with disease activity. The findings suggest that protein oxidation may play a role in the pathogenesis of chronic organ damage in SLE. 2005, American College of Rheumatology.
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Barter, Philip J.The cholesterol required by peripheral tissues, including vascular cells, is provided both by new synthesis in the cells and by a delivery from low-density lipoproteins (LDLs). When the level of LDLs is high, they accumulate in the artery wall where they are oxidized and taken up by foam cells in a process that leads to the development and progression of atherosclerosis. High-density lipoproteins (HDLs) oppose atherosclerosis directly, by removing cholesterol from foam cells, by inhibiting the oxidation of LDLs, and by limiting the inflammatory processes that underlie atherosclerosis. HDLs also have antithrombotic properties. Thus, HDL-cholesterol interrupts the process of atherogenesis at several key stages. The European Society of Cardiology 2005. All rights reserved.
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Choy, Katherine J.; Beck, Konstanze; Png, Francoise Y.; Wu, Ben Jing; Leichtweis, Steve B.; Thomas, Shane R.; Hou, Jingyun; Croft, Kevin D.; Mori, Trevor A.; Stocker, RolandObjective - To elucidate processes by which the antioxidant probucol increases lesion size at the aortic sinus and decreases atherosclerosis at more distal sites in apolipoprotein E-deficient (apoE-/-) mice. Methods and Results - Male apoE-/- mice were fed high-fat chow with 1% (w/w) probucol or without (controls) for 6 months, before aortic sinus, arch, and descending aorta were analyzed separately for lesion size and composition. Compared with control, probucol significantly increased lesion size by 33% at the sinus, but it inhibited atherosclerosis at the descending aorta by 94%. Sites where atherosclerosis was inhibited contained substantially fewer macrophages, less lipids (cholesterol and cholesteryl esters), and endogenous antioxidant (?-tocopherol), but not oxidized lipids, and the extent to which probucol metabolism occurred was increased. Compared with control, aortic sinus lesions of probucol mice contained a substantially increased content of extracellular matrix, but decreased total cell and macrophage density, comparable levels of lipids and ?-tocopherol, and decreased concentrations of oxidized lipids (cholesteryl ester hydroperoxides, F<inf>2</inf>- isoprostanes, and 7-ketocholesterol). Conclusions - Probucol affects atherosclerosis in apoE-/- mice independent of the accumulation of arterial lipid oxidation products, thereby dissociating the 2 processes. Rather, probucol exerts antiinflammatory activity by decreasing accumulation of macrophages in lesions, and it promotes a more stable lesion composition at the aortic sinus. 2005 American Heart Association, Inc.
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Zeng, Jingmin; Davies, Michael J.Non enzymatic covalent adduction of glucose, or aldehydes derived from glucose or oxidation reactions, to proteins (glycation) has been proposed as a key factor in the vascular complications of diabetes. In conditions of chronic glucose elevation, ?-dicarbonyl compounds, including glyoxal and methylglyoxal, are also present at elevated levels. These carbonyls react rapidly with nucleophilic groups on Lys and Arg side chains and the N-terminal amino group, to give poorly defined products, often called advanced glycation endproducts. These are present at elevated levels in tissue samples from people with diabetes and have been linked with disease development. As the thiol group of Cys is a powerful nucleophile, we hypothesized that adduction should occur rapidly and efficiently at Cys residues. It is shown here that Cys residues react with dicarbonyl compounds to give thiol-aldehyde adducts, which have been detected by electrospray ionization mass spectrometry. This process is accompanied by loss of the thiol group and formation of stable products. In the case of glyoxal, these reactions give S-(carboxymethyl)cysteine. The percentage conversion of thiol lost to product is substrate-dependent and ?32%. S-(Carboxymethyl)cysteine has been quantified by HPLC on thiol-containing, protected amino acids, peptides, and proteins, after exposure to glyoxal. The yield of this product has been shown to increase in a time- and dose-dependent manner with higher glyoxal concentrations and to also be formed on extended incubation of serum albumin with glucose. This novel, stable, advanced glycation endproduct is a potential marker of glycation. 2005 American Chemical Society.
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Weichselbaum, Markus; Sparrow, Malcolm P.; Hamilton, Elisha J.; Thompson, Philip J.; Knight, Darryl A.Background: Pulmonary neuroendocrine cells (PNEC) are specialized epithelial cells that are thought to play important roles in lung development and airway function. PNEC occur either singly or in clusters called neuroepithelial bodies. Our aim was to characterize the three dimensional morphology of PNEC, their distribution, and their relationship to the epithelial nerves in whole mounts of adult human bronchi using confocal microscopy. Methods: Bronchi were resected from non-diseased portions of a lobe of human lung obtained from 8 thoracotomy patients (Table 1) undergoing surgery for the removal of lung tumors. Whole mounts were stained with antibodies to reveal all nerves (PGP 9.5), sensory nerves (calcitonin gene related peptide, CGRP), and PNEC (PGP 9.5, CGRP and gastrin releasing peptide, GRP). The analysis and rendition of the resulting three-dimensional data sets, including side-projections, was performed using NIH-Image software. Images were colorized and super-imposed using Adobe Photoshop. Results: PNEC were abundant but not homogenously distributed within the epithelium, with densities ranging from 65/mm2 to denser patches of 250/mm2, depending on the individual wholemount. Rotation of 3-D images revealed a complex morphology; flask-like with the cell body near the basement membrane and a thick stem extending to the lumen. Long processes issued laterally from its base, some lumenal and others with feet-like processes. Calcitonin gene-related peptide (CGRP) was present in about 20% of PNEC, mainly in the processes. CGRP-positive nerves were sparse, with some associated with the apical part of the PNEC. Conclusions: Our 3D-data demonstrates that PNEC are numerous and exhibit a heterogeneous peptide content suggesting an active and diverse PNEC population. 2005 Weichselbaum et al; licensee BioMed Central Ltd.
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Grewal, Thomas; Evans, Rachel; Rentero, Carles; Tebar, Francesc; Cubells, Laia Garrigos; de Diego, Iki; Kirchhoff, Matthias F.; Hughes, William E.; Heeren, Jrg; Rye, Kerry Anne; Rinninger, Franz; Daly, Roger J.; Pol, Albert; Enrich, CarlosAnnexin A6 is a calcium-dependent membrane-binding protein that interacts with signalling proteins, including the GTPase-activating protein p120GAP, one of the most important inactivators of Ras. Since we have demonstrated that annexin A6 inhibits EGF- and TPA-induced Ras signalling, we investigated whether modulation of Ras activity by annexin A6 was mediated via altered subcellular localization of p120GAP. First, we exploited our observation that high-density lipoproteins (HDL) can activate the Ras/MAP kinase pathway. Expression of annexin A6 caused a significant reduction in HDL-induced activation of Ras and Raf-1. Annexin A6 promoted membrane binding of p120GAP in vitro, and plasma membrane targeting of p120GAP in living cells, both in a Ca2+- dependent manner, which is consistent with annexin A6 promoting the Ca 2+-dependent assembly of p120GAP-Ras at the plasma membrane. We then extended these studies to other cell types and stimuli. Expression of annexin A6 in A431 cells reduced, while RNAi-mediated suppression of annexin A6 in HeLa cells enhanced EGF-induced Ras and Erk activation. Importantly, the enhancement of Ras activation following RNAi-mediated reduction in p120GAP levels was more marked in annexin A6-expressing A431 cells than controls, indicating that the effect of annexin A6 on Ras was mediated via p120GAP. Finally, we demonstrated that annexin A6 promotes plasma membrane targeting of p120GAP in A431 cells in response to a variety of stimuli, resulting in colocalization with H-Ras. These findings demonstrate an important role for annexin A6 in regulating plasma membrane localization of p120GAP and hence Ras activity. 2005 Nature Publishing Group. All rights reserved.
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Crossley, Ellen L.; Caiazza, Daniela; Rendina, Louis M.A highly water-soluble platinum (II) complex containing a thiopropyl-1,2-dicarba-closo-dodecaborane (12) ligand, functionalized with a pendant glycerol group, was demonstrated. The precursor bromopropyl-closo-1,2- carborane derivative bearing a pendant benzyl-protected glycerol group was prepared in five steps by an adaptation of the method described by Malmquist and Sjerg and Yamamoto. It was demonstrated that platinum (II)-carborane complexes are a novel means of delivering boron atoms close to DNA, an important criterion in the development of new classes of BNCT agents. It was concluded that the functionalization of a metal-carborane complex with a glycerol moiety is a feasible strategy for increasing its aqueous solubility.
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Birinyi-Strachan, Liesl C.; Davies, Michael J.; Lewis, Richard James; Nicholson, Graham M.The basis for the neuroprotectant effect of D-mannitol in reducing the sensory neurological disturbances seen in ciguatera poisoning, is unclear. Pacific ciguatoxin-1 (P-CTX-1), at a concentration 10 nM, caused a statistically significant swelling of rat sensory dorsal root ganglia (DRG) neurons that was reversed by hyperosmolar 50 mM D-mannitol. However, using electron paramagnetic resonance (EPR) spectroscopy, it was found that P-CTX-1 failed to generate hydroxyl free radicals at concentrations of toxin that caused profound effects on neuronal excitability. Whole-cell patch-clamp recordings from DRG neurons revealed that both hyper- and iso-osmolar 50 mM D-mannitol prevented the membrane depolarisation and repetitive firing of action potentials induced by P-CTX-1. In addition, both hyper- and iso-osmolar 50 mM D-mannitol prevented the hyperpolarising shift in steady-state inactivation and the rise in leakage current through tetrodotoxin (TTX)-sensitive Na<inf>v</inf> channels, as well as the increased rate of recovery from inactivation of TTX-resistant Na <inf>v</inf> channels induced by P-CTX-1. D-Mannitol also reduced, but did not prevent, the inhibition of peak TTX-sensitive and TTX-resistant I<inf>Na</inf> amplitude by P-CTX-1. Additional experiments using hyper- and iso-osmolar d-sorbitol, hyperosmolar sucrose and the free radical scavenging agents Trolox and L-ascorbic acid showed that these agents, unlike D-mannitol, failed to prevent the effects of P-CTX-1 on spike electrogenesis and Na <inf>v</inf> channel gating. These selective actions of D-mannitol indicate that it does not act purely as an osmotic agent to reduce swelling of nerves, but involves a more complex action dependent on the Na<inf>v</inf> channel subtype, possibly to alter or reduce toxin association. 2005 Elsevier Ltd. All rights reserved.
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Yu, Yi; Wyszynski, Diego F.; Waterworth, Dawn M.; Wilton, Steve D.; Barter, Philip J.; Kesaniemi, YrjAntero; Mahley, Robert W.; McPherson, Ruth M.; Waeber, Gard; Bersot, Thomas P.; Ma, Qianli; Sharma, Sanjay S.; Montgomery, Douglas S.; Middleton, Lefkos T.; Sundseth, Scott S.; Mooser, Vincent E.; Grundy, Scott M.; Farrer, Lindsay A.We conducted a genome-wide scan using variance components linkage analysis to localize quantitative-trait loci (QTLs) influencing triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol, and total cholesterol (TC) levels in 3,071 subjects from 459 families with atherogenic dyslipidemia. The most significant evidence for linkage to TG levels was found in a subset of Turkish families at 11q22 [logarithm of the odds ratio (LOD) = 3.34] and at 17q12 (LOD = 3.44). We performed sequential oligogenic linkage analysis to examine whether multiple QTLs jointly influence TG levels in the Turkish families. These analyses revealed loci at 20q13 that showed strong epistatic effects with 11q22 (conditional LOD = 3.15) and at 7q36 that showed strong epistatic effects with 17q12 (conditional LOD = 3.21). We also found linkage on the 8p21 region for TG in the entire group of families (LOD = 3.08). For HDL-C levels, evidence of linkage was identified on chromosome 15 in the Turkish families (LOD = 3.05) and on chromosome 5 in the entire group of families (LOD = 2.83). Linkage to QTLs for TC was found at 8p23 in the entire group of families (LOD = 4.05) and at 5q13 in a subset of Turkish and Mediterranean families (LOD = 3.72 ). These QTLs provide important clues for the further investigation of genes responsible for these complex lipid phenotypes. These data also indicate that a large proportion of the variance of TG levels in the Turkish population is explained by the interaction of multiple genetic loci. Copyright 2005 by the American Society for Biochemistry and Molecular Biology, Inc.
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Sheil, Meredith L.K.; Luxford, Catherine; Davies, Michael J.; Peat, Jennifer Kay; Nunn, Graham R.; Celermajer, David S.Objective: Proteins are the major effectors of biological structure and function. Oxidation-induced changes to protein structure can critically impair protein function, with important pathologic consequences. This study was undertaken to examine whether oxidation-induced changes to protein structure occur during pediatric cardiopulmonary bypass and to examine the association with postoperative outcome. Methods: Elevation of the 3,4-dihydroxyphenylalanine content of a protein relative to its native tyrosine content indicates structural damage due to oxidation. Protein 3,4-dihydroxyphenylalanine/native tyrosine ratios were measured before surgery and up to 6 hours after institution of cardiopulmonary bypass in 24 children undergoing repair of congenital heart disease, who were prospectively selected to form a cyanotic and comparable acyanotic control group. Results were correlated with perioperative variables and postoperative outcomes. Results: Elevation of protein 3,4- dihydroxyphenylalanine/tyrosine ratios above baseline (0.48 mmol/mol [SD, 0.11 mmol/mol] vs 0.36 mmol/mol [SD, 0.13 mmol/mol]; P = .001) occurred within 30 minutes of initiating cardiopulmonary bypass in cyanotic but not in acyanotic children and correlated inversely with preoperative arterial oxygen saturation (R = -0.52; P = .03). Protein 3,4-dihydroxyphenylalanine/tyrosine ratios were also increased above baseline at 120 minutes (0.44 mmol/mol [SD, 0.12 mmol/mol]; P = .007) and 180 minutes (0.40 mmol/mol [SD, 0.14 mmol/mol]; P = .01) after the institution of cardiopulmonary bypass in children who underwent prolonged procedures. Elevation of 3,4-dihydroxyphenylalanine/tyrosine during prolonged procedures was associated with postoperative arrhythmias and the need for increased inotropic support (P = .001). Conclusions: Oxidative injury to proteins occurs during pediatric cardiopulmonary bypass. Cyanotic children are most at risk, particularly those undergoing prolonged procedures, in whom elevation of the protein 3,4-dihydroxyphenylalanine/tyrosine ratio is associated with increased postoperative morbidity. Copyright 2005 by The American Association for Thoracic Surgery.
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Rees, Martin D.; Pattison, David I.; Davies, Michael J.Activated phagocytes release the haem enzyme MPO (myeloperoxidase) and produce superoxide radicals and H<inf>2</inf>O<inf>2</inf> via an oxidative burst. MPO uses H<inf>2</inf>O<inf>2</inf> and Cl to form HOCl, the physiological mixture of hypochlorous acid and its anion present at pH 7.4. As MPO binds to glycosaminoglycans, oxidation of extracellular matrix and cell surfaces by HOCl may be localized to these materials. However, the reactions of HOCl with glycosaminoglycans are poorly characterized. The GlcNAc (N-acetylglucosamine), GlcNSO<inf>3</inf> (glucosamine-N-sulphate) and GlcNH<inf>2</inf> [(N-unsubstituted) glucosamine] residues of heparan sulphate are potential targets for HOCl. It is shown here that HOCl reacts with each of these residues to generate N-chloro derivatives, and the absolute rate constants for these reactions have been determined. Reaction at GlcNH<inf>2</inf> residues yields chloramines and, subsequently, dichloramines with markedly slower rates, k<inf>2</inf> ? 3.1 105 and 9 M -1 s-1 (at 37C) respectively. Reaction at GlcNSO<inf>3</inf> and GlcNAc residues yields N-chlorosulphonamides and chloramides with k<inf>2</inf> ?0.05 and 0.01 M-1 s -1 (at 37C) respectively. The corresponding monosaccharides display a similar pattern of reactivity. Decay of the polymer-derived chloramines, N-chlorosulphonamides and chloramides is slow at 37C and does not result in major structural changes. In contrast, dichloramine decay is rapid at 37C and results in fragmentation of the polymer backbone. Computational modelling of the reaction of HOCl with heparan sulphate proteoglycans (glypican-1 and perlecan) predicts that the GlcNH<inf>2</inf> residues of heparan sulphate are major sites of attack. These results suggest that HOCl may be an important mediator of damage to glycosaminoglycans and proteoglycans at inflammatory foci. 2005 Biochemical Society.
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Hawkins, Clare L.; Davies, Michael J.Activated eosinophils, and hypobromous acid (HOBr) generated by these cells, have been implicated in the tissue injury in asthma, allergic reactions, and some infections. Proteins are major targets for this oxidant, but limited information is available on the mechanisms of damage and intermediates formed. Reaction of HOBr with proteins is shown to result in the formation of bromamines and bromamides, from side-chain and backbone amines and amides, and 3-bromo- and 3,5-dibromo-Tyr, from Tyr residues; these materials account for ca. 70% of the oxidant consumed. Protein carbonyls, dityrosine, and 3,4- dihydroxyphenylalanine are also formed, though these are minor products (<5% of HOBr added). With BSA, extensive (selective and nonspecific) protein fragmentation and limited aggregation are also observed. The bromamines/bromamides are unstable and induce further oxidation and free radical formation as detected by EPR spin trapping. Evidence was obtained for the generation of nitrogen-centered radicals on side-chain and backbone amide groups of amino acids, peptides, and proteins. These radicals readily undergo rearrangement reactions to give carbon-centered radicals. With proteins, ?-carbon (backbone) radicals are detected, which may play a role in protein fragmentation. A novel damage transfer pathway from Gln side-chain amide groups to backbone sites was also observed. 2005 Elsevier Inc. All rights reserved.
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Hawkins, Clare L.; Davies, Michael J.Excessive or misplaced activation of leukocytes causes host tissue damage which has been implicated in diseases such as atherosclerosis and chronic inflammation. This may arise via either the generation of oxidants such as hypochlorous acid (HOCl) by the heme enzyme myeloperoxidase, the action of released enzymes including lysozyme and proteases, or a combination of these two activities. Thus, oxidant-mediated inactivation of protease inhibitors that modulate tissue proteolysis by the released enzymes may exacerbate protease-induced degradation of host tissue. The role of myeloperoxidase-derived oxidants, such as HOCl, in the inactivation of Kunitz-type inhibitors and lysozyme is not well-characterized and is the subject of the current study. Exposure of both trypsin inhibitor and lysozyme to low molar excesses of HOCl compared to protein is shown to result in loss of function. With trypsin inhibitor, this loss of activity is associated with the selective oxidation of Trp, Tyr, and His residues, which results in protein unfolding and the disruption of complex formation with active trypsin. Oxidation of Met residues, a major target for HOCl, or the active site Arg, does not appear to play a key role in this loss of activity. In contrast, with lysozyme, oxidation of Met residues to Met sulfoxide appears to be the major process resulting in loss of enzyme activity. With both proteins, inactivation occurs in a time-dependent manner, consistent with both direct oxidation by HOCl and secondary reactions of protein chloramines formed from amine groups (e.g., from Lys and His) playing a role in loss of activity. 2005 American Chemical Society.
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Skilton, Michael R.; Nakhla, Shirley; Sieveking, Daniel P.; Caterson, Ian Douglas; Celermajer, David S.1. In the present study, we sought to determine whether physiological or pathophysiological concentrations of obesity related peptides influence the key early atherogenic events of monocyte adhesion to endothelial cells and adhesion molecule expression using primary human cells. 2. Human umbilical vein endothelial cells were grown to confluence and human monocytes were obtained by elutriation. Adhesion was assessed by automated cell counting and cell adhesion molecule expression (E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)) was assayed by ELISA. 3. Experimental conditions included untreated control, ghrelin (100, 150, 450 and 1350 pmol/L), resistin (15, 40 and 100 ng/mL) and combined leptin and insulin (combinations of 30 and 120 pmol/L insulin and 5, 50 and 500 ng/mL leptin). 4. Both resistin and ghrelin produced modest but significant increases in VCAM-1 expression (110 4 and 117 13% compared with controls, respectively; both P ? 0.01). Ghrelin also increased ICAM-1 expression (119 17% of control; P ? 0.01). 5. However, despite these increases in adhesion molecule expression, neither ghrelin nor resistin altered monocyte adhesion values. 6. Neither leptin nor insulin altered monocyte adhesion to endothelial cells or cell adhesion molecule expression. 7. Pathophysiologically relevant concentrations of ghrelin and resistin, within the range of concentrations exhibited by patients with anorexia nervosa or the Prader-Willi syndrome and type 2 diabetes, respectively, increase endothelial cell adhesion molecule expression, possibly contributing to increased atherosclerosis risk in such subjects.
