Search
Showing 1841–1860 of 2058 publications.
-
Bolton, Elaine J.; Jessup, Wendy K.; Stanley, Keith K.; Dean, Roger T.Lipid-loaded macrophages were produced in vitro by incubation with acetylated or copper-oxidized LDL. In order to establish whether cellular membrane traffic is generally perturbed by such loading, we assessed endocytosis of fluid; cell surface binding, internalisation and degradation of a soluble ligand and of a particulate preparation; and exocytosis of lysosmal enzymes. Fluid-phase pinocytosis of sucrose was unaffected by either form of loading. Binding, uptake and degradation of soluble (mannosylated-BSA) and particulate (zymosan) ligands by these lipid-loaded and by non-loaded cells were compared. Loading with oxidized LDL decreased the processing of both ligands, while loading with acetylated LDL had little effect. Loading with oxidized LDL (Ox-LDL) also decreased zymosan binding at 4C; and the internalisation and degradation of ligands in Ox-LDL loaded and non-loaded cells reflected the extent of surface binding. Changes in binding release of lysosomal ?-N-acetyl-D-glucosaminidase from the cells. Loading with Ox- but not Ac-LDL decreased ?-N-acetyl-D-glucosaminidase secretion. After incubation with zymosan, intracellular levels of the enzyme were increased in the Ox-LDL loaded cells. Zymosan uptake and ?-N-acetyl-D-glucosaminidase secretion were correlated, but enzyme-activity per culture rose more in the absence than in the presence of zymosan. We conclude that membrane traffic is perturbed in model foam cells, particularly those loaded with Ox-LDL.
-
Mark Herman, S.; Robinson, Jacqui T.C.; McCredie, Robyn J.; Adams, Mark R.; Boyer, Michael J.; Celermajer, David S.Male gender is an independent risk factor for coronary artery disease, and androgen administration has been associated with increased atherosclerosis in experimental animals. Since endothelial dysfunction is an important event in the atherogenic process, we hypothesized that androgen deprivation in adult men might be associated with enhanced arterial endothelial function. Using external vascular ultrasound, brachial artery diameter was measured at rest, after flow increase (causing endothelium- dependent dilatation) and after nitroglycerin (an endothelium-independent dilator). We studied 30 adult males aged 40 to 70 years: 10 had had bilateral orchidectomy and/or maximal androgen blockade for ?6 months for treatment of prostate cancer, and all were in complete remission (group 1). Ten healthy controls (group 2) and 10 controls who had remission from nonprostate cancers (group 3) were matched for age and smoking history. Testosterone levels were lower in men in group 1 versus groups 2 or 3 (0.80.1 versus 19.28.4 or 16.14.9 nmol/L, P<.001). By contrast, endothelium-dependent dilatation was markedly higher in group 1 than in groups 2 or 3 (6.23 versus 2.72 or 2.01.9%, P<.001). The nitroglycerin response was similar in all three groups (P=.92). On multivariate analysis, increased endothelium-dependent dilatation was significantly associated with low serum testosterone levels (P=.001) but not with cholesterol levels or with a past history of malignancy (P>.25). The withdrawal of male sex hormones may be associated with enhanced endothelial function in adult men. This is consistent with a deleterious effect of physiologic levels of male sex steroids on the arterial wall.
-
Garner, Brett; Baoutina, Anna; Dean, Roger T.; Jessup, Wendy K.The demonstration of lipid loaded macrophages in atherosclerotic tissue has led to the development of in vitro systems to elucidate the mechanisms involved in lipid accumulation. Here we have characterised the changes which occur in human monocyte-derived macrophage (MDM) lipids during culture in either human serum (HS) or foetal calf serum (FCS). MDM cultured in HS were rapidly converted to lipid filled foam cells, as assessed using HPLC analysis and oil red-O staining and compared with the same cells grown in FCS. However, the lipids which accumulated were predominantly triglycerides with smaller amounts of unesterified cholesterol (UC) and only traces of cholesteryl esters (CE). ?-Tocopherol (?-TocH) was present at higher levels in MDM cultured in HS compared to the same cells grown in FCS. MDM lipid accumulation was dependent on the triglyceride-rich lipoprotein (TGRL) fraction of human serum; accordingly, supplementation of FCS with human TGRL also induced MDM lipid accumulation. The relationships between cellular lipid accumulation and secretion of apolipoprotein E (apo E) and lipoprotein lipase (LPL) as well as expression of the low density lipoprotein receptor-related protein (LRP) were also examined. MDM lipid accumulation was associated with increased apo E secretion but did not alter extracellular LPL activity. The lipid accumulation which was induced by HS was potently inhibited (but not reversed) by the inflammatory cytokine interferon-? (IFN?), and this was associated with decreased apo E production, LPL secretion and expression of LRP. These studies reveal striking differences in the lipid composition of MDM cultured in either HS or FCS, and indicate that oil red-O staining is not necessarily associated with cholesteryl ester accumulation in human macrophages. Furthermore, the effect that serum-induced lipid accumulation has on the specific MDM functions studied should be appreciated when developing in vitro macrophage models.
-
Puppo, Alain; Davies, Michael J.; Moreau, Sophie; Turnbull, Ruth; Frendo, Pierre; Mathieu, Christel; Houart, DidierLeghemoglobin, the oxygen-carrying hemoprotein present in great amounts in legume nodules, can react with hydrogen peroxide to form phenoxyl radicals. These radicals are quenched by at least two processes : the first one involves an intramolecular heme-protein cross-link and the second route results in the formation of intermolecular cross-links and hence dimeric forms of the protein. They can also interact with peribacteroid membrane fractions, leading to the generation of additional lipid-derived radicals. This transfer of damage may be of great biological significance during the nodule senescence process. Glutathione, which is present at high concentration in nodules, most probably exerts a very important protective role, as it can react with these radicals. Thus, glutathione synthesis is studied in Medicago truncatula : partial cDNAs corresponding to the two enzymes involved in this synthesis are cloned and show high homology with their Arabidopsis counterparts.
-
Witting, Paul Kenneth; Upston, Joanne M.; Stocker, RolandHeme-containing (per)oxidases including horse radish peroxidase (HRP)/H<inf>2</inf>O<inf>2</inf> have been shown to oxidatively modify isolated low-density lipoprotein (LDL) in vitro and oxidized LDL is implicated in the early events leading to atherosclerosis. The role of ?-tocopherol (?-TOH) in the oxidation of LDL by HRP/H<inf>2</inf>O<inf>2</inf> is unclear, although ?-tocopheroxyl radical (?-TO), which is formed during this process, can act as a chain transfer agent of lipid peroxidation in LDL. By combining HPLC and EPR spectroscopy, we hereby show that during HRP/H<inf>2</inf>O<inf>2</inf>-induced oxidation of human LDL: (i) the accumulation of cholesteryl linoleate hydroperoxides and hydroxides (CE- O(O)H) occurs concomitantly with the formation of ?-TO and consumption of ?-TOH in the absence of other detectable organic (g ? 2) radicals; (ii) the rates of ?-TO formation and subsequent decay reflect the rates of both ?- TOH consumption and CE-O(O)H accumulation; (iii) CE-O(O)H accumulation is directly dependent on the level of endogenous ?-TOH, and vitamin E supplementation results in increased lipid oxidizability; (iv) the inhibition of HRP activity by catalase plus urate results in a persistent ?-TO signal, the decay (t( 1/4 ) ? 20 min) of which is accompanied by continued accumulation of CE-O(O)H, with complete cessation of lipid peroxidation upon loss of the chromanoxyl signal. These results demonstrate a direct correlation between ?-TOH/?-TO and the extent of HRP/H<inf>2</inf>O<inf>2</inf>-induced LDL lipid peroxidation, and that this type of oxidative modification can occur in the absence of g ? 2 radicals other than ?-TO. Together, the results support a role for tocopherol-mediated peroxidation but not the involvement of a protein radical in the initiation of LDL lipid peroxidation induced by HRP/H<inf>2</inf>O<inf>2</inf>.
-
Hawkins, Clare L.; Davies, Michael J.Degradation of collagen by oxidant species may play an important role in the progression of rheumatoid arthritis. Whilst the overall effects of this process are reasonably well defined, little is known about the sites of attack, the nature of the intermediates, or the mechanism(s) of degradation. In this study electron paramagnetic resonance spectroscopy with spin trapping has been used to identify radicals formed on collagen and related materials by metal ion - H<inf>2</inf>O<inf>2</inf> mixtures. Attack of the hydroxyl radical, from a Fe(II)-H<inf>2</inf>O<inf>2</inf> redox couple, on collagen peptides gave signals from both side chain (.CHR'R'), and ?-carbon [.C(R)(NH -)CO -,R = side-chain] radicals. Reaction with collagen gave both broad anisotropic signals, from high-molecular-weight protein-derived radicals, and isotropic signals from mobile species. The latter may be low-molecular-weight fragments, or mobile side-chain species; these signals are similar to those from the ?-carbon site of peptides and the side-chain of lysine. Enzymatic digestion of the large, protein-derived, species releases similar low-molecular-weight adducts. The metal ion employed has a dramatic effect on the species observed. With Cu(I)-H<inf>2</inf>O<inf>2</inf> cu(II)-H<inf>2</inf>O<inf>2</inf> instead of Fe(II)-H<inf>2</inf>O<inf>2</inf>, evidence has been obtained for: i) altered sites of attack and fragmentation, C-terminal decarboxylation, and iii) hydrogen abstraction at N-terminal ?-carbon sites. This altered behaviour is believed to be due to the binding of copper ions to some substrates and hence site-specific damage. This has been confirmed in some cases by electron paramagnetic resonance studies of the Cu(II) ions.
-
Adams, Mark R.; Jessup, Wendy K.; Celermajer, David S.Objectives. This study sought to assess the effect of cigarette smoking on adhesion of human monocytes to human endothelial cells and to measure the effect of L-arginine and vitamin C supplementation on this interaction. Background. Cigarette smoking has been associated with abnormal endothelial function and increased leukocyte adhesion to endothelium, both key early events in atherogenesis. Supplementation with both oral L-arginine (the physiologic substrate for nitric oxide) and vitamin C (an aqueous phase antioxidant) may improve endothelial function; however, their benefit in cigarette smokers is not known. Methods. Serum was collected from eight smokers (mean [ SD] age 33 5 years) with no other coronary risk factors and eight age- and gender-matched lifelong nonsmokers. The serum was added to confluent monolayers of human umbilical vein endothelial cells and incubated for 24 h. Human monocytes obtained by counterflow centrifugation elutriation were then added to these monolayers for 1 h, and adhesion then was measured by light microscopy. To assess reversibility, monocyte/endothelial cell adhesion was then measured for each subject 2 h after 2 g of oral vitamin C and 2 h after 7 g of oral L-arginine. Results. In smokers compared with control subjects, monocyte/endothelial cell adhesion was increased (46.4 4.5% vs. 27.0 5.2%, p < 0.001), endothelial expression of intercellular adhesion molecule (ICAM)-1 was increased (0.31 0.02 vs. 0.22 0.03, p = 0.004), and vitamin C levels were reduced (33.7 24.1 vs. 53.4 11.5 ?mol/liter, p = 0.028). After oral L-arginine, monocyte/endothelial cell adhesion was reduced in smokers (from 46.4 4.5% to 35.1 4.0%, p = 0.002), as was endothelial cell expression of ICAM-1 (from 0.31 0.02 to 0.27 0.01, p = 0.001). After vitamin C, there was no significant change in monocyte/endothelial cell adhesion or ICAM-1 expression from baseline in the smokers despite an increase in vitamin C levels (to 115 7 ?mol/liter). Conclusions. Cigarette smoking is associated with increased monocyte-endothelial cell adhesion when endothelial cells are exposed to serum from healthy young adults. This abnormality is acutely reversible by oral L-arginine but not by vitamin C.
-
Garner, Brett; Van Reyk, David M.; Dean, Roger T.; Jessup, Wendy K.Oxidation of low density lipoprotein (LDL) results in changes to the lipoprotein that are potentially atherogenic. Numerous studies have shown that macrophages cultured in vitro can promote LDL oxidation via a transition metal-dependent process, yet the exact mechanisms that are responsible for macrophage-mediated LDL oxidation are not understood. One contributing mechanism may be the ability of macrophages to reduce transition metals. Reduced metals (such as Fe(II) or Cn(I)) rapidly react with lipid hydroperoxides, leading to the formation of reactive lipid radicals and conversion of the reduced metal to its oxidized form. We demonstrate here the ability of macrophages to reduce extracellular iron and copper and identify a contributing mechanism. Evidence is provided that a proportion of cell- mediated metal reduction is due to direct transplasma membrane electron transport. Glucagon suppressed both macrophage-mediated metal reduction and LDL oxidation. Although metal reduction was augmented when cells were provided with a substrate for thiol production, thiol export was not a strict requirement for cell-mediated metal reduction. Similarly, while the metal- dependent acceleration of LDL oxidation by macrophages was augmented by thiol production, macrophages could still promote LDL oxidation when thiol export was minimized (by substrate limitation). This study identifies a novel mechanism that may contribute to macrophage-mediated LDL oxidation and may also reveal potential new strategies for the inhibition of this process.
-
Kopp, Christoph W.; Siegel, Jonathan B.; Hancock, Wayne W.; Anrather, Josef; Winkler, Hans; Geczy, Carolyn L.; Kaczmarek, Elzbieta; Bach, Fritz Heintz; Robson, Simon ChristopherBackground. Delayed xenograft rejection (DXR) is characterized by inflammation and vascular thrombosis. Activation of coagulation may occur as a result of tissue factor (TF) expression on both activated donor endothelial cells (EC) and recipient infiltrating monocytes (Mo). In addition, natural anticoagulants associated with porcine endothelial cells may not function adequately across species. Methods. In the present study, we examined the interaction of the TF pathway of coagulation with the natural anticoagulant TF pathway inhibitor, in xenogeneic leukocyte-EC cultures in vitro, and during rejection of discordant xenografts in vivo. Results. Coculture of human Mo with pig aortic EC (PAEC) resulted in 1.7-fold and 2-fold higher induction of Mo TF and Mo intercellular adhesion molecule-1, respectively, when compared with coculture with human aortic endothelial cells (HAEC). In addition, TF-dependent and -independent activation of coagulation factor X was higher on PAEC than on HAEC. Low levels of mRNA for tissue factor pathway inhibitor (TFPI) and its variant, TFPI-2, in resting PAEC were up-regulated by stimulation with tumor necrosis factor ?. Procoagulant activity of recombinant human TF complexed to activated factor VII was inhibited by PAEC and HAEC-associated TFPI by 22% and 56%, respectively. In contrast, human activated factor X (factor Xa) activity was inhibited by human, but not porcine, EC-associated TFPI, suggesting functional incompatibility of PAEC for human factor Xa. Endothelial TFPI was detected in pig control organs and after hyperacute rejection, but was lost from the vasculature during DXR. Conclusions. Lack of appropriate human factor Xa inhibition by porcine EC during hyperacute rejection and loss of porcine EC TFPI during DXR could promote the development of a procoagulant environment leading to xenograft rejection.
-
Adams, Mark R.; McCredie, Robyn J.; Jessup, Wendy K.; Robinson, Jacqui T.C.; Sullivan, David R.; Celermajer, David S.L-Arginine is the physiological substrate for nitric oxide synthesis by the vascular endothelium. In hypercholesterolaemic rabbits, oral L-arginine reduces atheroma, improves endothelium-dependent dilatation and reduces monocyte/endothelial cell adhesion. The effect of oral L-arginine on endothelial physiology is unknown, however, in humans with established atherosclerosis. In a prospective, double-blind, randomised crossover trial, ten men aged 41 2 years with angiographically proven coronary atherosclerosis took L-arginine (7 g three times per day) or placebo for 3 days each, with a washout period of 10 days. After L-arginine, compared to placebo, plasma levels of arginine were increased (318 18 vs. 124 9 ?mol/l, P < 0.01) and endothelium-dependent dilatation of the brachial artery (measured as the change in diameter in response to reactive hyperaemia, using external vascular ultrasound) was improved (4.7 1.1 vs. 1.8 0.7%, P < 0.04). No changes were seen in endothelium-independent dilatation of the brachial artery (measured as the change in diameter in response to sublingual nitroglycerine), blood pressure, heart rate or fasting lipid levels. Serum from six of the ten subjects after L-arginine and placebo was en added to confluent monolayers of human umbilical vein endothelial cells for 24 h, before human monocytes obtained by counter current centrifiguation elutriation were added and cell adhesion assessed by light microscopy. Adhesion was reduced following L-arginine compared to placebo (42 2 vs. 50 1%, P < 0.01). In young men with coronary artery disease, oral L-arginine improves endothelium-dependent dilatation and reduces monocyte/endothelial cell adhesion.
-
Martinic, Gary; Wells, Xanthe E.Complete hepatectomy enables definitive assessment of the role of the liver in the in vivo metabolism of pharmacologic agents, such as the widely used anticoagulant heparin. The methodology described here was developed during a study on the role of the liver in the catabolism of heparin in rats (1). Initially a reticuloendothelial system (RES) blocker was used; however, it was not suitable for studying heparin breakdown, because it has a similar chemical structure (1). Partial hepatectomy often is used as a means of studying hepatic biochemical and pathophysiologic factors (2); however, because some functional liver tissue remains, the method was not suitable for our needs, as we wished to ascertain the definitive role of the liver in heparin breakdown. Complete removal of the liver was, therefore, the most suitable procedure to fulfil the aims of the study. In rats, this surgical procedure can only be performed successfully as a 2-stage operation, otherwise the animal dies immediately (3). The first stage involves the placement of loosely tied ligatures around the hepatic portal vein and caudal vena cava of young animals so that, as the animals grow, these major vessels are slowly occluded and collateral vessels develop. In the second stage of the procedure, the blood supply to the liver is totally occluded, and the organ can be removed completely. In our report, we describe a modified procedure developed from the technique described by Bollman and Van Hook (4), which uses 3 ligatures during the final stage. In the study reported here, we used 2 ligatures, thus reducing the time required and the risk of damage to vessels.
-
Dean, Roger T.; Fu, Shanlin; Stocker, Roland; Davies, Michael J.Radical-mediated damage to proteins may be initiated by electron leakage, metal-ion-dependent reactions and autoxidation of lipids and sugars. The consequent protein oxidation is O<inf>2</inf>-dependent, and involves several propagating radicals, notably alkoxyl radicals. Its products include several categories of reactive species, and a range of stable products whose chemistry is currently being elucidated. Among the reactive products, protein hydroperoxides can generate further radical fluxes on reaction with transition-metal ions; protein-bound reductants (notably dopa) can reduce transition-metal ions and thereby facilitate their reaction with hydroperoxides; and aldehydes may participate in Schiff-base formation and other reactions. Cells can detoxify some of the reactive species, e.g. by reducing protein hydroperoxides to unreactive hydroxides. Oxidized proteins are often functionally inactive and their unfolding is associated with enhanced susceptibility to proteinases. Thus cells can generally remove oxidized proteins by proteolysis. However, certain oxidized proteins are poorly handled by cells, and together with possible alterations in the rate of production of oxidized proteins, this may contribute to the observed accumulation and damaging actions of oxidized proteins during aging and in pathologies such as diabetes, atherosclerosis and neurodegenerative diseases. Protein oxidation may also sometimes play controlling roles in cellular remodelling and cell growth. Proteins are also key targets in defensive cytolysis and in inflammatory self-damage. The possibility of selective protection against protein oxidation (antioxidation) is raised.
-
Fu, Shanlin; Dean, Roger T.We have previously reported the formation of valine hydroperoxides and aldehydes from hydroxyl-radical attack on free valine and protein molecules. We have also demonstrated that the major degradation products of valine hydroperoxides by several biochemical and cellular systems are the corresponding hydroxides, and therefore proposed that hydroxyvalines may serve as useful in vivo markers for studying protein oxidation. Here we have undertaken the structural elucidation of the oxidation products of leucine, another amino acid which is very susceptible to peroxidation. Hydroxyl-radical (H?) attack on L-leucine in the presence of oxygen, followed by NaBH<inf>4</inf> reduction, gave rise to five major oxidation products which have been isolated and identified. On the basis of chemical and spectroscopic evidence, the five products have been identified as (2S)-?-hydroxyleucine, (2S,4S)-?-hydroxyleucine, (2S,4R)-?-hydroxyleucine, (2S,4R)-4-methylproline (trans-4-methyl-L-proline) and (2S,4S)-4-methylproline (cis-4-methyl-L-proline). The three hydroxyleucines have been confirmed to be the reduction products of the corresponding hydroperoxyleucines, while the two proline analogues are from reduction of their corresponding cyclic Schiff bases. By HPLC analysis using post-column o-phthaldialdehyde derivatization, we have detected hydroxyleucines in the hydrolysates of tripeptides and proteins which had been ?-radiolysed and treated with NaBH<inf>4</inf>. Furthermore, we demonstrate the occurrence of the hydroxyleucines on proteins in physiological and pathological samples. Hydroxyleucines, like hydroxyvalines, may provide useful in vivo markers for studying protein oxidation. In the present study we also investigated the competition between leucine, valine and phenylalanine for H?, and proposed a possible radical-transfer process in such free-radical reactions.
-
McCrohon, Jane A.; Walters, William Allen Willcox; Robinson, Jacqui T.C.; McCredie, Robyn J.; Turner, Leo A.; Adams, Mark R.; Handelsman, David J.; Celermajer, David S.Objectives. We sought to assess whether high dose estrogen treatment is associated with enhanced arterial reactivity in genetic males. Background. Although estrogens have been shown to enhance arterial reactivity in women, and are thereby thought to confer cardiovascular benefit, the vascular effects of long-term estrogen therapy in genetic males is unknown. Methods. We studied the arterial physiology of 30 genetic males-15 male to female transsexuals receiving long-term high dose estrogen therapy and 15 healthy male control subjects matched for age, smoking history and vessel size. Using external vascular ultrasound, brachial artery diameter was measured at rest, after flow increase (causing endothelium-dependent dilation [EDD]) and after nitroglycerin (GTN), an endothelium-independent dilator. Blood pressure, cholesterol and testosterone levels were also measured in each subject. Results. Total testosterone and free testosterone index levels were lower in the transsexuals compared with the control subjects (p < 0.001). In contrast, EDD was significantly higher in the transsexuals than in the control males (mean [SD] 7.1 3.1% vs. 3.2 2.8%, p = 0.001), as was the GTN response (21.2 6.7% vs. 14.6 3.3%, p = 0.002). Total and high density lipoprotein cholesterol, blond pressure levels and baseline vessel size were similar in the two groups. On multivariate analysis, enhanced EDD was associated independently with estrogen therapy (p = 0.02) and with low total cholesterol (p = 0.04). An enhanced GTN response was also significantly associated with estrogen therapy (p = 0.03). Conclusions. Long-term treatment with high dose estrogens is associated with enhanced arterial reactivity in genetic males, which may be due to the effects of estrogen excess or androgen deprivation, or both.
-
Neuil, Ji? Witting, Paul Kenneth; Stocker, RolandAs the oxidation of low density lipoprotein (LDL) lipids may be a key event in atherogenesis, there is interest in antioxidants as potential anti- atherogenic compounds. Here we report that ?-tocopheryl hydroquinone (?- TQH<inf>2</inf>) strongly inhibited or completely prevented the (per)oxidation of ubiquinol-10 (CoQ<inf>10</inf>H<inf>2</inf>), ?-tocopherol (?-TOH), and both surface and core lipids in LDL exposed to either aqueous or lipophilic peroxyl radicals, Cu2+, soybean lipoxygenase, or the transition metal-containing Ham's F-10 medium in the absence or presence of human monocyte-derived macrophages. The antioxidant activity of ?-TQH<inf>2</inf> was superior to that of several other lipophilic hydroquinones, including endogenous CoQ<inf>10</inf>H<inf>2</inf>, which is regarded as LDL's first line of antioxidant defence. At least three independent activities contributed to the antioxidant action of ?-TQH<inf>2</inf>. First, ?- TQH<inf>2</inf> readily associated with LDL and instantaneously reduced the lipoprotein's ubiquinone-10 to CoQ<inf>10</inf>H<inf>2</inf>, thereby maintaining this antioxidant in its active form. Second, ?-TQH<inf>2</inf> directly intercepted aqueous peroxyl radicals, as indicated by the increased rate of its consumption with increasing rates of radical production, independent of LDL's content of CoQ<inf>10</inf>H<inf>2</inf> and ?-TOH. Third, ?-TQH<inf>2</inf> rapidly quenched ?-tocopheroxyl radical in oxidizing LDL, as demonstrated directly by electron paramagnetic resonance spectroscopy. Similar antioxidant activities were also seen when ?-TQH<inf>2</inf> was added to high-density lipoprotein or the protein-free intralipid, indicating that the potent antioxidant activity of ?-TQH<inf>2</inf> was neither lipoprotein specific nor dependent on proteins. These results suggest that ?-TQH<inf>2</inf> is a candidate for a therapeutic lipid-soluble antioxidant. As ?-tocopherylquinone is formed in vivo at sites of oxidative stress, including human atherosclerotic plaque, and biological systems exist that reduce the quinone to the hydroquinone, our results also suggest that ?- TQH<inf>2</inf> could be a previously unrecognized natural antioxidant.
-
Proudfoot, Julie M.; Puddey, Ian B.; Beilin, Lawerence Joseph; Stocker, Roland; Croft, Kevin D.Oxidative modification of low-density lipoprotein (LDL) may be an important factor in atherogenesis. The susceptibility of LDL to oxidation is usually determined in isolation by exposing LDL to oxidative stress induced by Cu ions or a free radical initiator. In these cases oxidation is carried out in the absence of water-soluble vitamins or serum proteins that may be present at the site of oxidation in vivo. We have examined the Cu2+- induced oxidation of lipoproteins in diluted serum. When oxidizing isolated LDL, there is a decrease in lag time with increasing concentration of Cu2+ until a minimum 'lag time' is reached at a Cu:LDL ratio of about 50:1. In serum, we have shown an initial decrease in 'lag time' with increasing Cu concentration up to 12.5 ?M. However, with higher Cu concentrations 'lag time' to oxidation increases, contrary to expectation, until a maximum is reached at about 50 ?M Cu. This dose response observed for Cu oxidation of diluted serum was highly reproducible in a number of individual subjects. When serum was gel-filtered to remove low molecular weight compounds, the resulting filtrate behaved the same as isolated LDL. Uric acid was found to be an important component of the low molecular weight fraction responsible for the paradoxical effect of Cu concentration on serum oxidation. The same paradoxical effect was found when isolated LDL was incubated with uric acid in the presence of human serum albumin (HSA) and Cu. The incubation of HSA with reducing agents such as uric acid or bilirubin in the presence of high Cu concentrations, produces a 'peroxidase-like' activity, capable of breaking down hydrogen peroxide as well as lipid hydroperoxides. The decomposition of lipid peroxides is a likely explanation for the longer serum oxidation lag times seen at higher Cu concentrations. Our study highlights the possible importance of interactions between uric acid and serum proteins in the presence of high metal ion concentrations.
-
Hazell, Linda J.; Stocker, RolandThe amount of ?-tocopherol (?-TOH) can dramatically alter the extent of radical-induced oxidation of low density lipoprotein (LDL) lipids, a process generally thought to be important in atherogenesis. However, LDL with atherogenic features can also be formed in vitro by exposure to the strong non-radical oxidant hypochlorite (HOCl), which preferentially oxidises LDL apolipoprotein B-100. Here we show that varying LDL content of ?-TOH by vitamin supplementation or depletion has no effect on the extent of HOCl-induced oxidation of apolipoprotein B-100 as measured by the loss of lysine and tryptophan residues, and the alteration in relative electrophoretic mobility of the lipoprotein particle.
-
Wardell, Mark R.; Wayne Chang, Wun Shaing; Bruce, David B.; Skinner, Richard; Lesk, Arthur M.; Carrell, Robin W.The inhibitory mechanism of the serpin family of serine protease inhibitors is characterized by a remarkable degree of conformational flexibility. Various conformational states have been elucidated by X-ray crystallography and indicate that the inhibitory loop, the central A-?- sheet, and the outside edge of the C-?-sheet are particularly mobile. However, no crystal structure of a serpin-enzyme complex is yet available, and the likely nature of the protease-complexed serpin remains for biochemical and biophysical researchers to examine. Here, we show that the biochemical induction of the latent state of antithrombin is slow relative to polymer formation, and infer that this may reflect structural features that are important for the regulation of the initial docking and subsequent locking of serpins with cognate proteases. L-Antithrombin was induced by incubation of native antithrombin at 60 C for 10 h in the presence of citrate to prevent polymerization. L-Antithrombin was more stable to denaturation by both heat and urea than native antithrombin. Whereas native antithrombin formed binary complexes with synthetic peptide homologues of the inhibitory loop, biochemically induced L-antithrombin did not, indicating that the inhibitory loop of L-antithrombin is probably fully inserted into the A-?-sheet as in the crystal structure. This was confirmed by limited proteolysis studies which demonstrated that the inhibitory loop of L- antithrombin could not he cleaved by five proteases which do cleave the loop of native antithrombin. The limited proteolysis studies also indicated that the 'gate' region (residues 236-248) of the biochemically induced L- antithrombin was in a conformation substantially different from that of the native antithrombin. This again is similar to L-antithrombin in the crystal structure in which the gate has 'opened' away from the body of the molecule by a rotation of 24to facilitate the relocation of strand 1C from its ordered position in the C-?-sheet to a disordered surface loop. At 60 C in the absence of citrate, antithrombin (and other serpins) rapidly polymerizes. In the presence of citrate, the formation of L-antithrombin is slow and increases with time, indicating that the inhibition of polymer formation by citrate allows the time necessary for the much slower formation of the L form. We therefore suggest that L-antithrombin formation is a two-step process: an initial rapid conformational change, probably including partial incorporation of the reactive loop into the A-sheet (as in the active molecule in the crystal structure) and displacement of sic from the C-?- sheet which supports polymer formation, and a much slower transition to complete loop insertion within the A-?-sheet. It is likely that both the first rapid transitional step and the structural features that impose resistance to the second more extensive conformational change reflect the optimization of the unique inhibitory function in the serpins.
-
Burgess, James W.; Stanley, Keith K.Previous studies have shown that treatment of rats with 17 ?-ethynylestradiol (EE) causes the appearance in bile of intravenously injected, desialylated ligands, including asialofetuin and low density lipoprotein (LDL). Here we show that activated ?<inf>2</inf>-macroglobulin (?<inf>2</inf>-M*), but not insulin, transferrin or acetylated LDL, shows the same phenomenon. ?<inf>2</inf>-M* appearance in bile in EE-treated rats was inhibited by receptor associated protein, but not unlabelled asialofetuin, strongly implicating the ?<inf>2</inf>-macroglobulin receptor (?<inf>2</inf>MR/LRP) receptor in this process. Asialofetuin, apolipoprotein B (ApoB) of LDL and ?<inf>2</inf>-M* appeared undegraded in the bile of EE treated but not control rats. When LDL was injected, not only was intact apolipoprotein B detected in bile, but the profile of cholesterol esters appearing in bile was characteristic of the injected human LDL rather than rat lipoproteins. After floatation of the bile on KBr gradients, intact Apo B and cholesterol esters characteristic of human LDL were found at the normal density of LDL suggesting that the majority of the lipoprotein particle remains intact. Stimulation of transcytosis was specific to estrogens, and was highest with 17?-ethynylestradiol. After subcutaneous injection of 0.05 mg/kg body weight of ethynylestradiol, sufficient to give a measurable increase in transcytosis, the plasma concentration of ethynylestradiol rose to 2.2 nM. Thus estrogen-stimulated transcytosis of desialylated ligands and ?<inf>2</inf>-M* would be expected at physiological estrogen concentrations.
-
Upston, Joanne M.; Neuil, Ji? Witting, Paul Kenneth; Alleva, Renata; Stocker, Roland15-Lipoxygenase has been implicated in the in vivo oxidation of low density lipoprotein (LDL) a process thought to be important in the origin and/or progression of human atherogenesis. We have suggested previously that oxidation of LDL's cholesteryl esters (CE) and phospholipids by soybean (SLO) or human recombinant 15-lipoxygenase (rhLO) can be ascribed largely to ?- tocopherol (?-TOH)-mediated peroxidation (TMP). In this study we demonstrate that addition to LDL of unesterified linoleate (18:2), other free fatty acid (FFA) substrates, or phospholipase A<inf>2</inf> (PLA<inf>2</inf>) significantly enhanced the accumulation of CE hydro(pero)xides (CE-O(O)H) induced by rhLO, whereas the corresponding CE and nonsubstrate FFA were without effect. The enhanced CE- O(O)H accumulation showed a dependence on the concentration of free 18:2 in LDL. In contrast, addition of 18:2 had little effect on LDL oxidation induced by aqueous peroxyl radicals or Cu2+ ions. Analyses of the regio- and stereoisomers of oxidized 18:2 in SLO-treated native LDL demonstrated that the small amounts of 18:2 associated with the lipoprotein were oxidized enzymically and within minutes, whereas cholesteryl linoleate (Ch18:2) was oxidized nonenzymically and continuously over hours. ?-Tocopheroxyl radical (?-TO) formed in LDL exposed to SLO was enhanced by addition of 18:2 or PLA<inf>2</inf>. With rhLO and 18:2-supplemented LDL, oxidation of 18:2 was entirely enzymic, whereas that of Ch18:2 was largely, though not completely, nonenzymic. The small extent of enzymic Ch18:2 oxidation increased with increasing enzyme to LDL ratios. Ascorbate and the reduced form of coenzyme Q, ubiquinol-10, which are both capable of reducing ?-TO and thereby preventing TMP, inhibited nonenzymic Ch18:2 oxidation induced by rhLO. Trolox and ascorbyl palmitate, which also inhibit TMP, ameliorated both enzymic and nonenzymic oxidation of LDL's lipids, whereas probucol, a radical scavenger not capable of preventing TMP, was ineffective. These results demonstrate that rhLO-induced oxidation of CE is largely nonenzymic and increases with LDL's content of FFA substrates. We propose that conditions which increase LDL's FFA content, such as the presence of lipases, increase 15-LO-induced LDL lipid peroxidation and that this process requires only an initial, transient enzymic activity.
