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Showing 1881–1900 of 2058 publications.
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Hazell, Linda J.; Arnold, Leslie; Flowers, Dimity; Waeg, Georg; Malle, Ernst; Stocker, RolandOxidation of LDL may contribute to atherogenesis, though the nature of the in vivo oxidant(s) remains obscure. Myeloperoxidase, the enzyme responsible for hypochlorous acid/hypochlorite (HOCl) production in vivo, is present in active form in human atherosclerotic lesions, and HOCl aggregates and transforms LDL into a high-uptake form for macrophages in vitro. Here we demonstrate HOCl-modified proteins in human lesions using an mAb raised against HOCl-modified LDL that recognizes HOCl-oxidized proteins but does not cross-react with Cu2+-, malondialdehyde-, or 4-hydroxynonenal-modified LDL. This antibody detected significantly more material in advanced atherosclerotic lesions than normal arteries, even though azide and methionine were included during sample work-up to inhibit myeloperoxidase and to scavenge HOCl. The epitope(s) recognized was predominantly cell associated and present in monocyte/macrophages, smooth muscle, and endothelial cells. The intima and cholesterol clefts stained more heavily than the center of the thickened vessels; adventitial staining was apparent in some cases. Immunostaining was also detected in a very early lesion from an accident victim, beside healthy areas that were unreactive. LDL oxidized by HOCl in vitro, but not native LDL, effectively competed with the epitopes in lesions for antibody binding. Density centrifugation of plaque homogenates and Western blot analysis showed that, in the apo B-containing lipoprotein fraction, the mAb recognized protein(s) of molecular mass greater than apo B, similar to those produced during oxidation of LDL with HOCl in vitro. Three major proteins were recognized by the anti-HOCl-modified protein antibody but not by an anti-apo B antibody in the apo B-free fraction. Together, these results demonstrate HOCl-oxidized proteins in human atherosclerotic lesions, implicating this oxidant in LDL modification in vivo.
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Brown, Andrew J.[No abstract available]
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Upston, Joanne M.; Neuil, Ji? Stocker, RolandVarious lipoxygenases (LO) oxidize low density lipoprotein (LDL) in vitro and 15-LO has been implicated in the development of atherosclerosis in vivo. Direct oxidation of phospholipids (PL) and cholesteryl esters (CE) by LO has been proposed as a mechanism whereby these enzymes cause or contribute to LDL lipid peroxidation. Herein we show that the extent to which recombinant human 15-LO (rhLO) caused peroxidation of LDL's esterified core and surface lipids depended on, and directly related to, the ?-tocopherol (?-TOH) content of the lipoprotein. Thus, CE and PL of in vivo ?-TOH-depleted LDL, isolated from a patient with familial isolated vitamin E deficiency, were resistant to oxidation by rhLO, whereas those in ?-TOH-containing LDL from the same patient receiving vitamin E supplements readily oxidized. The extent to which rhLO caused oxidation of CE and PL directly and linearly correlated with LDL's content of vitamin E, as demonstrated by studies with in vitro ?-TOH-depleted lipoproteins. The geometric isomers of oxidized cholesteryl linoleate formed in LDL during oxidation initiated by rhLO, matched those obtained during non-enzymic, peroxyl radical-initiated oxidation of LDL whilst ?-TOH was present. Ascorbate, an efficient co-antioxidant for ?-TOH, completely prevented rhLO-initiated oxidation of LDL's CE, but did not inhibit rhLO-mediated oxidation of unesterified linoleate. jlr These results are inconsistent with direct oxidation of LDL esterified lipids by rhLO. Isolated LDL contained free fatty acids (FFA), and its exposure to rhLO caused a rapid formation of linoleate hydroperoxide. To reconcile these data, we propose that during rhLO-initiated oxidation of LDL, enzymic oxidation of FFA preceeds the oxidation of CE and PL, which occurs largely via a tocopherol-dependent process.
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Moreau, Sophie; Davies, Michael J.; Mathieu, Christel; Houart, Didier; Puppo, AlainReaction of H<inf>2</inf>O<inf>2</inf> with ferric leghemoglobin (metLb, the monomeric, oxygen-carrying, heme protein from root nodules of nitrogen-fixing plants) has been previously shown to generate an iron(IV)-oxo (ferryl) species and at least one protein radical. The latter has been suggested to be a tyrosine-derived phenoxyl radical present at Tyr-133 in the soybean protein and Tyr-138 in the lupin protein. To obtain further information on these protein radicals and their potential interaction with the physiologically important peribacteroid membrane (which surrounds the microsynibiont in vivo), EPR spin trapping studies have been carried out with soybean metLb. Evidence has been obtained for at least two additional protein-derived radicals in addition to the phenoxyl radical; these radicals are transient and reactive in nature. These species are carbon-centered, and at least one is a tertiary species (.CR1R2R3); these radicals may be side chain- or ?-carbon-derived, their exact sites have not been determined. Some of these radicals are on the protein surface and may be key intermediates in the formation of protein dimers. These radicals have been shown to be capable of reacting with peribacteroid membrane fractions, with the consequent generation of lipid-derived radicals. The formation of such radicals may result in the depletion of membrane antioxidants and the initiation of lipid peroxidation. This transfer of damage from the heme center via the protein surface to neighboring membranes may be of considerable biological significance; the destruction of this membrane is one of the earliest observable events in root nodule senescence and is associated with the loss of nitrogen-fixing activity.
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Timmins, Graham S.; Wei, Xudong; Hawkins, Clare L.; Taylor, Richard J.K.; Davies, Michael J.We report the synthesis and use of d<inf>2</inf>-15N isotopically-labelled 3,5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS-d<inf>2</inf>-1W, as its sodium salt), a spin-trap possessing several advantages over non-labelled DBNBS. The simplification in the electron paramagnetic resonance spectra of radical adducts of DBNBS-d<inf>2</inf>-15Ncompared with those of DBNBS not only results in increased sensitivity, but also facilitates the assignment and analysis of complex spectra. An example of this simplification is given. Pearson Professional Ltd 1996.
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de Angelis, Elena M.; Moss, Stephen H.; Pouton, Colin W.Significant progress has been made in the isolation and culture of endothelial cells. Large vessel endothelial cell culture is well established, and the diversity of microvascular endothelial cell function is now being explored at the molecular level using primary cultures from individual organs. A variety of tests can be used to identify the purity of cultures, and these methods are discussed. The availability of cell culture models of the endothelium has advanced knowledge of endothelial cell pharmacology and immunological interactions at the molecular level. There is much work still to be done which can be accomplished adequately using traditional monolayer culture techniques. Brain microvascular endothelia can be isolated routinely, to produce a good filter-culture model for transport studies across polarized cell monolayers, though to obtain tight junctions with electrical resistance as low as that found in vivo, it is necessary to grow cells in conditioned medium and/or in the presence of cAMP. These treatments promote formation of tight junctions and the establishment of cytoskeletal components to form a tight monolayer. Brain microvascular endothelial in culture have been used to identify receptor-mediated transport systems for various plasma components at a qualitative level. The routine primary culture of microvascular endothelia from peripheral sites is often more difficult and labour intensive. The way forward for transport studies is likely to be establishment of immortalized lines, and development of strict protocols for growth, characterization and preparation of monolayers for transport studies. The long-term aims of correlating transport in vitro with transport in vivo, and subsequently using cell culture models to predict the nature of microvascular endothelial transport in vivo, require models of well-established validity. The technology of isolation, culture and characterization of endothelia needs to go through a period of considerable development and maturation before these long-term aims will become a realistic prospect.; Significant progress has been made in the isolation and culture of endothelial cells. Large vessels endothelial cell culture is well established, and the diversity of microvascular endothelial cell function is now being explored at the molecular level using primary cultures from individual organs. A variety of test can be used to identify the purity of cultures, and these methods are discussed. The availability of cell culture models of the endothelium has advanced knowledge of endothelial cell pharmacology and immunological interactions at the molecular level. Brain microvascular endothelia for use in filter-culture model is discussed in detail. The routine primary culture of microvascular endothelia from peripheral sites is often more difficult and labor-intensive. The way forward in this field is discussed.
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Celermajer, David S.; Adams, Mark R.; Clarkson, Peter; Robinson, Jacqui T.C.; McCredie, Robyn J.; Donald, Ann E.; Deanfield, John EricBackground. Passive smoking has been linked to an increased risk of dying from atherosclerotic heart disease. Since endothelial dysfunction is an early feature of atherogenesis and occurs in young adults who actively smoke cigarettes, we hypothesized that passive smoking might also be associated with endothelial damage in healthy young-adult nonsmokers. Methods. We studied 78 healthy subjects (39 male and 39 female) 15 to 30 years of age (mean SD, 224): 26 control subjects who had never smoked or had regular exposure to environmental tobacco smoke, 26 who had never smoked but had been exposed to environmental tobacco smoke for at least one hour daily for three or more years, and 26 active smokers. Using ultrasonography, we measured the brachial-artery diameter under baseline conditions, during reactive hyperemia (with flow increase causing endothelium-dependent dilatation), and after sublingual administration of nitroglycerin (an endothelium-independent dilator). Results. Flow-mediated dilatation was observed in all control subjects (8.23.1 percent; range, 2.1 to 16.7) but was significantly impaired in the passive smokers (3.12.7 percent; range, 0 to 9; P<0.001 for the comparison with the controls) and in the active smokers (4.43.1 percent; range, 0 to 10; P<0.001 for the comparison with the controls; P=0.48 for the comparison with the passive smokers). In the passive smokers, there was an inverse relation between the intensity of exposure to tobacco smoke and flow-mediated dilatation (r=-0.67, P<0.001). In contrast, dilatation induced by nitroglycerin was similar in all groups. Conclusions. Passive smoking is associated with dose-related impairment of endothelium-dependent dilatation in healthy young adults, suggesting early arterial damage.
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Cornish, Coralie J.; Devery, Jannine M.; Poronnik, Philip; Lackmann, Martin; Cook, David I.; Geczy, Carolyn L.Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, 'classical chemoattractants' such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and 'pure chemoattractants' such as TGF-?1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine 5100 chemoattractant protein, CP-10, belongs to the 'non-classical' group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca2+](i) in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca2+-independent G-protein-coupled pathway for postreceptor signal transduction triggered by 'pure chemoattractants.' The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds.
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Drake, Ian M.; Davies, Michael J.; Mapstone, Nicholas P.; Dixon, Michael F.; Schorah, Christopher J.; White, Kay L.M.; Chalmers, Douglas M.; Axon, Anthony Thomas Roger A.High dietary ascorbic acid intake appears to protect against gastric cancer. This may be due to its action as a scavenger of reactive radical species formed in the gastric mucosa, resulting in a reduced level of radical-mediated DNA damage. We have studied 82 patients, of whom 37 had Helicobacter pylori-associated gastritis, a condition which predisposes to gastric cancer. Using electron paramagnetic resonance (EPR) spectroscopy we have demonstrated, for the first time, that ascorbyl radicals are generated in human gastric mucosa, presumably as a result of scavenging of free radicals by ascorbic acid. Quantification of ascorbyl radicals demonstrates that there is a higher concentration in those patients with H.pylori gastritis compared with subjects with normal histology (P < 0.01). We also found gastric mucosal luminol-enhanced chemiluminescence and malondialdehyde concentrations (which are believed to be markers of radical generation and tissue damage) to be higher in patients with H.pylori gastritis compared with those with normal histology (P < 0.001 and P < 0.01 respectively). The observed concentrations of the ascorbyl radical correlate with the level of luminol-enhanced chemiluminescence (r = 0.41, P < 0.001), but not with malondialdehyde concentrations (r = 0.08, P = 0.47). Mucosal ascorbic acid and total vitamin C concentrations did not vary between histological groups, nor did they correlate with mucosal levels of the ascorbyl radical, chemiluminescence or malondialdehyde. These data suggest that ascorbic acid is acting as a scavenger of free radicals generated in human gastric mucosa. The experiments therefore provide direct supportive evidence for the hypothesis that ascorbic acid protects against gastric cancer by scavenging reactive radical species which would otherwise react with DNA, with resultant genetic damage.
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Christison, Julie K.; Karjalainen, Ari; Brauman, Julie N.; Bygrave, Fyfe L.; Stocker, RolandTo test whether high-density lipoproteins (HDL) could aid in the removal in vivo of potentially atherogenic oxidized lipids, we perfused rat liver in situ with buffer supplemented with isolated human HDL containing small amounts of cholesteryl linoleate hydro(pero)xides [Ch18:2-O(O)H]. Perfusion resulted in the rapid removal of Ch18:2-O(O)H from HDL with a half-life (t( 1/2 ) of 11.4 min, faster than that of unoxidized cholesteryl linoleate, and dependent on the presence of the liver. In addition, the liver enhanced the reduction of Ch18:2-OOH associated with HDL remaining in the perfusate buffer. Perfusion resulted in the release of a hepatic activity that enhanced the reduction of HDL-associated Ch18:2-OOH and was resistant to heat treatment. In contrast with the situation with HDL, low-density lipoprotein (LDL)-associated Ch18:2-O(O)H were neither removed nor reduced by perfused rat liver within the time course studied, in support of a possible role for HDL in the detoxification of circulating lipid hydroperoxides in vivo.
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Geczy, Carolyn L.[No abstract available]
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Gatto, Lissa M.; Hallen, Gregory K.; Brown, Andrew J.; Samman, SamirThe aim of this study was to determine the effect of ascorbic acid (AA) supplements on plasma lipids and lipoproteins in healthy, young women. Ten women were recruited to participate in a randomized double-blind cross-over trial and supplemented with 1000 mg AA daily for 4 weeks, followed by placebo, and vice versa. Plasma AA concentrations were significantly higher at 2 weeks (p < 0.0001) and at 4 weeks (p < 0.001), compared with baseline. Plasma AA levels appeared to peak after 2 weeks of supplementation. Plasma concentrations of LDL-C were found to be 16% lower at 4 weeks compared with baseline (p < 0.05) and although HDL-C levels did not change significantly with AA supplementation, the change in HDL-C was positively associated with the change in plasma AA (p < 0.05). Significant decreases were observed in the total cholesterol (TC) to HDL-C at 2 weeks and LDL-C to HDL-C ratios at 2 and 4 weeks supplementation (p < 0.05). Our findings agree with those from epidemiological studies and suggest that increases in AA intake may favorably alter the lipoprotein profile in young women. 1996 American College of Nutrition.
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Clarkson, Peter; Adams, Mark R.; Powe, Amanda J.; Donald, Ann E.; McCredie, Robyn J.; Robinson, Jacqui T.C.; McCarthy, Susan N.; Keech, Anthony C.; Celermajer, David S.; Deanfield, John EricIn hypercholesterolemic rabbits, oral L-arginine (the substrate for endothelium derived nitric oxide) attenuates endothelial dysfunction and atheroma formation, but the effect in hypercholesterolemic humans is unknown. Using high resolution external ultrasound, we studied arterial physiology in 27 hypercholesterolemic subjects aged 295 (19-40) years, with known endothelial dysfunction and LDL-cholesterol levels of 23843 mg/dl. Each subject was studied before and after 4 wk of L-arginine (7 grams x 3/day) or placebo powder, with 4 wk washout, in a randomized double-blind crossover study. Brachial artery diameter was measured at rest, during increased flow (causing endothelium-dependent dilation, EDD) and after sublingual glyceryl trinitrate (causing endothelium-independent dilation). After oral L-arginine, plasma L-arginine levels rose from 115103 to 231125 ?mol/liter (P < 0.001), and EDD improved from 1.71.3 to 5.63.0% (P < 0.001). In contrast there was no significant change in response to glyceryl trinitrate. After placebo there were no changes in endothelium-dependent or independent vascular responses. Lipid levels were unchanged after L-arginine and placebo. Dietary supplementation with L-arginine significantly improves EDD in hypercholesterolemic young adults, and this may impact favorably on the atherogenic process.
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Hu, Shengping; Harrison, Craig A.; Xu, Ken; Cornish, Coralie J.; Geczy, Carolyn L.The murine S100 protein CP-10 is a potent chemotactic factor for murine and human myeloid cells in vivo and in vitro. This is the first report describing regulation of the CP-10 gene by a proinflammatory stimulus, lipopolysaccharide (LPS), in cells of the monocyte/macrophage lineage. Murine monocyte/macrophage-like WEHI 265 and RAW 264.7 cells preexposed to 5 to 50 ng/mL LPS expressed significant levels of CP-10 mRNA 4 hours, and maximal at 20 hours, after a secondary LPS challenge. This was accompanied by increasing levels of cell-associated and released CP-10 protein. In contrast, a single dose of LPS upregulated CP-10 mRNA in elicited peritoneal macrophages, whereas mRNA and protein levels decreased following LPS challenge. The state of macrophage differentiation may control responsiveness as LPS had no effect on CP-10 basal levels in bone marrow derived macrophages. LPS-induced CP-10 expression was controlled at the transcriptional level and nuclear run-on and protein synthesis inhibition assays indicated that LPS priming and challenge of RAW cells occurred via distinct pathways. MRP14, another S100 protein generally coordinately expressed with human MRP8, was not induced by LPS under the same conditions. We propose that CP-10 may play a key role in recruitment of leukocytes into tissues in response to gram-negative bacterial infection.
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Raftery, Mark J.; Harrison, Craig A.; ALEWOOD, PAUL F.; Jones, Alun; Geczy, Carolyn L.MRP14 (macrophage migration-inhibitory factor-related protein of molecular mass 14 kDa) is an S100 calcium binding protein constitutively expressed in human neutrophils which may be associated with cellular activation/inflammation. Murine MRP14 expression was up-regulated following concanavalin A activation of spleen cells, and the protein was isolated from conditioned medium in high yield (approx. 500 ng/ml). MRP14 had a mass of 12,972 2 Da by electrospray ionization MS, whereas the theoretical mass derived from the cDNA sequence, after removal of the initiator Met, was 12,918 Da, suggesting that the protein was post-translationally modified. We identified four post-translational modifications of MRP14: removal of the N-terminal Met, N-terminal acetylation, disulphide bond formation between Cys79 and Cys90, and 1-methylation of His106; the calculated mass was then 12,971.8 Da. Methylation of His106 was further characterized after incubation of spleen cells with L-[methyl-3H]Met during concanavalin A stimulation. Sequential analysis of a peptide (obtained by digestion with Lys C) containing methylated His indicated that > 80% of the label in the cycle corresponded to His106, suggesting that the methyl residue was transferred from S-adenosyl-L-methionine. Comparison of the C18 reverse-phase HPLC retention times of phenylthiocarbamoyl derivatives of a hydrolysed digest peptide of MRP14 with those of standards confirmed methyl substitution on the 1-position of the imidazole ring. MRP14 bound more 65Zn2+ than the same amounts of the 10 kDa chemotactic protein (CP10) or S100?. Ca2+ decreased Zn2+ binding in S100? but it did not influence binding to MRP14, suggesting that the Zn2+ binding site was distinct from and independent of the two Ca2+ binding domains.
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Hazlewood, Clare; Davies, Michael J.Benzoyl peroxide is a known tumor promoter and progression agent in mouse skin, though it is not an initiator or complete carcinogen. Previous studies have suggested that this activity may be due to the generation of strand breaks in cells exposed to this compound. This may be as a result of free radical generation, though there is controversy as to which radicals are responsible for this damage; previous workers have variously implicated benzoyloxyl (PhCO<inf>2</inf>, phenyl (Ph), and hydroxyl radicals (HO) as the initiating agent. In the present study a detailed examination of the radicals generated on reaction of benzoyl peroxide with Cu(I) has been carried out by electron paramagnetic resonance (EPR) spectroscopy and spin trapping; the results obtained are consistent with the formation of PhCO<inf>2</inf> and Ph but not HO. The subsequent reactions of these benzoyl peroxide-derived radicals with nucleobases, sugars, nucleosides, nucleotides, RNA, and DNA have been examined and the intermediate species have been identified in many cases. Comparison of these data with those obtained with Ph alone has allowed the reactions of PhCO<inf>2</inf> and Ph to be distinguished. Evidence has been obtained which is consistent with both the addition of these radicals to the C<inf>5</inf>-C<inf>6</inf> double bond of the pyrimidines to give adduct species, and hydrogen abstraction from the sugar rings. The former process is the major reaction for nucleosides and nucleotides. Studies with RNA and DNA also provide strong evidence for the formation of base adducts, though the exact identity of the species detected in these cases could not be determined due to the complexity of the spectra. Hydrogen abstraction at the sugar-phosphate backbone is also believed to occur with these substrates as strand breakage is observed; the extent of the latter is dependent on the radical flux and the attacking species, with PhCO<inf>2</inf> appearing to be a much more effective inducer of fragmentation than Ph. The nature of the species detected with all the substrates examined, with the exception of the isolated sugars where essentially random attack by both radicals is observed, suggests that of the two possible radicals generated by benzoyl peroxide, PhCO<inf>2</inf> and Ph, it is the former which is responsible for the majority of the observed degradation. The resuits obtained in this study are consistent with the genetic damage produced by this compound being due to the formation of both strand breaks and high yields of altered bases via the formation of base adducts.
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Gelissen, Ingrid C.; Brown, Andrew J.; Mander, Erin L.; Kritharides, Leonard; Dean, Roger T.; Jessup, Wendy K.The aim of the present study was to investigate whether impairment of cholesterol efflux previously found from mouse peritoneal macrophages loaded with oxidized low density lipoprotein (OxLDL) could be ascribed to the presence of oxysterols in these cells. 7-Ketocholesterol (7KC), the major oxysterol present in OxLDL-loaded cells, was selectively incorporated into unoxidized LDL, which was subsequently acetylated to produce a high uptake form. Mouse macrophages incubated with 7KC-enriched acetylated LDL (7kAcLDL) did not reveal cytotoxicity judged by cell protein and trypan blue exclusion. A large proportion of cellular 7KC was esterified, indicating that it is a substrate for acyl CoA: cholesterol acyltransferase. Cholesterol efflux from mouse macrophages loaded with 7kAcLDL, using apoA-I as a sterol acceptor, was impaired in cells containing >50 nmol of 7KC/mg of cell protein compared with cells loaded with oxysterol-free acetylated LDL. Thus impairment of cholesterol efflux could be reproduced in cells loaded with 7kAcLDL containing similar proportions of 7KC as OxLDL. 7KC itself was exported very poorly, even when the levels of 7KC in the cells were low. These results suggest that oxysterols present in foam cells in vitro can affect reverse sterol transport and may be potentially important in foam cell formation in vivo.
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Langton, Paul E.The foramen ovale, between the right and left atria. exists in the foetal heart as a vital physiological communication. Haemodynamic closure occurs in the neonatal period with most people having permanent fusion of the foramen. In up to a third of adults the closure is functional only and a potential right to left atrial communication persists as a patent foramen ovale. Studies in patients with decompression illness after diving suggest a consistent increase in the prevalence of patent foramen ovale, as detected by transthoracic contrast echocardiography. The association is strongest for those patients with early onset of neurological decompression illness, particularly those cases occurring in the absence of other risk factors traditionally associated with decompression illness. However, patent foramen ovale is a common finding in the general population and the absolute risk of decompression illness, even in the presence of a patent foramen ovale, remains very low.
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Reginald Waldeck, A.; Stocker, RolandWe present kinetic models of various complexity for radical-initiated lipid peroxidation in low density lipoproteins (LDL). The models, comprised of simultaneous differential equations programmed in Mathematica, were used to evaluate the concentration profiles of the reactants of interest. Single- phase reaction schemes describing lipid peroxidation and antioxidation according to the 'conventional' and tocopherol-mediated peroxidation (TMP) model were simulated for conditions of low and high radical fluxes produced by thermolabile azo initiators. The results show that the particular dependencies of the rates of lipid peroxidation (R(p)) on the rates of initiation (R(i)) for the two reaction schemes were accurately predicted by the simulations. Both models qualitatively predicted inhibition of lipid peroxidation in the presence of ?-tocopherol (?-TOH) under high radical flux conditions, suggesting that both can describe inhibited lipid peroxidation in solution under these conditions. TMP, but not the conventional model, could also predict the experimentally observed complex behavior of LDL lipid peroxidation induced with different concentrations of azo initiators. Specifically, TMP faithfully reproduced the observed kinetic chain length of lipid peroxidation of >>1 at low and <<1 at high concentration of the initiator (i.e., 0.2 and 10 mM, respectively for LDL at 1 ?mol apoB-100/L) during the ?-TOH-containing period of oxidation. It also demonstrated the experimentally observed nondependence of R(p)/(TMP) on R(i). Kinetic analysis of radical generation and initiation of lipid peroxidation in an extended, two-compartment model of TMP showed that phase separation of bimolecular reactions in a suspension of LDL particles can lead to a ~400-fold increase in the rate of lipid hydroperoxide formation. The experimentally observed co-antioxidant action of water-soluble ascorbate and lipid-soluble ubiquinol-10 were verified using this model. A simple biophysical model constituting the reactions of TMP and incorporating the compartmental nature of an LDL suspension is proposed. Together, the results demonstrate that TMP is the only model that fits the experimental data describing the early stages of LDL lipid peroxidation under various oxidizing conditions. The implications of our findings are discussed in relation to atherogenesis and a recently proposed alternative model of LDL lipid peroxidation.
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Lass, Achim; Witting, Paul Kenneth; Stocker, Roland; Esterbauer, HerrmannThe effects of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one on human LDL lipid oxidation induced by different fluxes of aqueous peroxyl radicals and cupric ion (at a Cu2+:LDL ratio of 17:1) were investigated. Addition of ebselen to LDL oxidised with Cu2+ prolonged the duration of the lag-phase typical for this oxidising condition, with the increase being proportional to the square of the ebselen concentration. Ebselen also prevented the formation of lipid hydroperoxides and inhibited the consumption of endogenous antioxidants during the early period of Cu2+-induced oxidation, during which time the drug was converted stoichiometrically into ebselen oxide (2-phenyl-1,2-benzisoselenazol-3(2H)-one-Se-oxide). Ebselen oxide itself was antioxidant inactive. Ebselen also inhibited formation of lipid-hydroperoxides and spared ?-tocopherol during the initial stages of LDL oxidation mediated by low-flux of aqueous peroxyl radicals, where a lag-phase was not observed. When a higher flux of aqueous peroxyl radicals was used, ebselen increased the observed inhibited phase of peroxidation in a dose-dependent manner, though less pronounced than its prolongating effect on the lag-phase of Cu2+-induced LDL lipid oxidation. Ebselen was also able to directly interact with Cu1+, alkyl peroxyl radicals and ?-tocopheroxyl radicals, demonstrating that the drug has a number of potential antioxidant activities in addition to its well-known hydroperoxide-reducing activity. We conclude that the antioxidant activities of ebselen are complex and that their relative importance likely vary depending on the experimental system used.
