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Showing 1901–1920 of 2058 publications.
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Grewal, Thomas; Bartlett, Anna L.; Burgess, James W.; Packer, Nicolle H.; Stanley, Keith K.We have compared the uptake of desialylated low density protein (LDL) with other modified forms of LDL in mouse peritoneal macrophages and PMA-activated human U937 monocytes. Neuraminidase-treated LDL (NT-LDL) caused significant cholesterol ester accumulation in both cell types, although the efficiency relative to loading with acetylated LDL (AcLDL) was markedly different, suggesting a very different complement of receptors in the cells. We therefore determined the effect of PMA-activation on lipoprotein receptor expression in U937 cells and found that while scavenger receptor concentration was elevated after PMA-activation, there was no significant change in the expression of the LDL receptor. Receptor specificity of NT-LDL uptake was examined by competition experiments using the degradation assay. This showed that 125I-labelled NT-LDL uptake in U937 cells could largely be accounted for by the persistent expression of the LDL receptor in these cells. In contrast, in mouse peritoneal macrophages where LDL receptor expression is very low, 125I-labelled NT-LDL degradation was effectively competed by AcLDL. Measurement of sialic acid content of AcLDL showed that approximately 14% of the LDL sialic acid, equivalent to 2 to 3 residues per particle, was lost during acetylation of LDL with acetic anhydride. Thus competition between 125I-labelled NT-LDL and AcLDL could be due to lectin receptor binding rather than competition for scavenger receptor binding.
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Garner, Brett; Jessup, Wendy K.Low-density lipoprotein (LDL) oxidation is believed to be causal in the process of atherosclerosis. Evidence suggests that this oxidation occurs in the arterial intima and is almost certainly mediated by the surrounding cells. Mechanisms by which cells could promote oxidation may be one or more of the following: 1. direct action of cell-derived oxidants (e.g. hydrogen peroxide, superoxide radical, hypochlorous acid, peroxynitrous acid); 2. transfer of cell-derived lipid hydroperoxides to LDL; 3. exposure of LDL to cell-derived oxidizing enzymes (e.g. lipoxygenase) which use LDL directly as a substrate; 4. maintenance of transition metals in a reduced and therefore highly reactive state (e.g. by thiols or other mechanisms); 5. generation of a local microenvironment (e.g. modulation of pH, pO<inf>2</inf>, antioxidants) which promotes metal-catalysed lipid peroxidation. In vitro cell-mediated LDL oxidation is absolutely metal-dependent and although the mechanism involved is not yet defined, evidence to date is most consistent with maintenance of metals in the reduced state as the predominant route. Pearson Professional Ltd 1996.
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Roginsky, Vitaly A.; Mohr, Detlef; Stocker, RolandTo address whether reduction by vitamin C may contribute to the in vivo maintenance of coenzyme Q in the reduced form, we studied the reduction of ubiquinone-1 by ascorbate at pH 7.4. Addition of ascorbate to ubiquinone-1 resulted in rapid O<inf>2</inf> consumption and an increase in the steady-state concentration of ascorbyl radical. The initial rate of O<inf>2</inf> consumption was proportional to the product of [ubiquinone-1] and [ascorbate] whereas [ascorbyl radical] was proportional to the square root of this parameter; both dependencies were in quantitative agreement with each other. The extent of O<inf>2</inf> consumption greatly exceeded the amounts of ubiquinone-1 initially present. Formation of ubiquinol-1 from ubiquinone-1 by ascorbate was reversible, moderate under aerobic conditions, but substantial in the absence or near absence of oxygen. At high O<inf>2</inf> concentration, ascorbate promoted the oxidation of ubiquinol-l to ubiquinone-1. Addition of sodium dodecyl sulphate dramatically decreased the rate of reaction between ubiquinone-1 and ascorbate, most likely as a result of phase separation of the reagents. A preliminary reaction scheme with putative rate constants for the relevant reactions is presented that quantitatively describes the kinetic behaviour of the process studied. The key reactions in the scheme are electron transfer from ascorbate to ubiquinone-1 with formation of the ascorbyl and ubisemiquinone radical. The reaction of the latter with O<inf>2</inf> is postulated to be responsible for O<inf>2</inf> consumption, with ubiquinone-1 acting as a catalyst. Together, the results demonstrate that the extent of reduction of ubiquinone-1 by ascorbate was controlled by the O<inf>2</inf> concentration and the physical availability of the reactants. As the O<inf>2</inf> concentration in human blood is relatively high and ubiquinone-10 is located exclusively within the lipid phase of lipoproteins where negatively charged ascorbate has little access, our results suggest that direct reduction by ascorbate is unlikely to be responsible for the high reduction percentage observed for plasma coenzyme Q. 1996 Pearson Professional Ltd.
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Van Reyk, David M.; Dean, Roger T.It was shown that the iron-selective chelator desferal (desferrioxamine mesylate: DFO) can reduce Cu(II) as judged by measuring the formation of the complex between Cu(I) and a specific chelator for this species, neocuproine (NC), in phosphate buffer. It was found that under optimal conditions, 3 moles of Cu(II) could be reduced per mole of DFO. Studies of the kinetics of Cu(II) reduction by DFO revealed that the rate of Cu(II) reduction by DFO was considerably slower than that by ascorbate. In the case of both reductants, even in the absence of NC, Cu(I) complexes remained in aqueous solutions for at least 30 min. DFO could also reduce Cu complexed to histidine. The results presented highlight the interpretive dangers which can arise in studies involving multiple transition metals, especially in the presence of multiple chelators. Specifically, when desferal is used, it is important to be aware that any copper present may become reduced, and that any Cu(I) formed might participate in ongoing redox reactions.
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Hawkins, Clare L.; Davies, Michael J.HO attack on hyaluronic acid, related polymers and monomers has been studied by both direct, rapid-flow, EPR (ESR) and EPR spin trapping using a variety of traps. Evidence has been obtained, with the monomers, for essentially random hydrogen-atom abstraction at all the ring C-H bonds with glucuronic acid, and at all sites except the N-acetyl side chain and C(2) with N-acetylglucosamine. The initial radicals do not undergo rapid rearrangement reactions at pH 4; however at both lower and higher pH values, acid- and base-catalysed rearrangement processes, respectively, result in the loss of these species. The rate of loss of these species is dependent on the substrate, with those derived from N-acetylglucosamine undergoing slower acid-catalysed rearrangement than the glucuronic acid-derived species. This is rationalized in terms of a rearrangement reaction of 1,2-dihydroxyalkyl (1,2-diol) radicals involving an electron-deficient radical-cation intermediate; the formation of this species would be disfavoured by the electron-withdrawing N-acetyl substituent. The base-catalysed process, which is believed to involve a radical-anion intermediate, occurs rapidly at pH 7.4, and appears to be less substrate dependent. In the case of glucuronic acid- (but not N-acetylglucosamine-) derived species this latter process results in the detection of ring-opened semidione species. With equimolar mixtures of the two monomers essentially random attack occurs on the two rings. However with chondroitin sulphate A, attack appears to be much more selective, with a radical generated at C(5) on the glucuronic acid ring present at highest concentration. The initial radicals obtained with this polysaccharide also undergo base- and acid-catalysed rearrangements; this leads to strand-breakage and the formation of low-molecular-weight material. Spin-trapping experiments carried out with hyaluronic acid, and a number of other polysaccharides, resulted in the detection of a number of novel spin adducts, the formation of which are consistent with attack on both the sugar rings in the polymer. The pH dependence of the observed spectra, and the detection of additional species at some pH values, suggest that at least some of the initial radicals undergo base-catalysed rearrangement reactions which result in strand-breakage and the formation of low-molecular-weight fragments. The extent of fragmentation at a particular pH, is also affected by the radical flux, with high radical yields giving more low molecular weight material. These observations suggest that pH-independent processes also contribute to strand-cleavage; this may be due to ?-cleavage of the radicals formed at C(1) on either ring, C(3) on N-acetylglucosamine or C(4) on the glucuronic acid ring.
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McCrohon, Jane A.; Adams, Mark R.; McCredie, Robyn J.; Robinson, Jacqui T.C.; Pike, Anne; Abbey, Mavis; Keech, Anthony C.; Celermajer, David S.Objective. Oestrogen replacement therapy is associated with a marked reduction in coronary event rates in post-menopausal women. As older age is associated with progressive arterial endothelial damage, a key event in atherosclerosis, we assessed whether hormone replacement therapy (HRT) with oestrogen alone, or oestrogen and progesterone combined, is associated with improved endothelial function in healthy women after the menopause. Design. Using high resolution external vascular ultrasound, brachial artery diameter was measured at rest and in response to reactive hyperaemia, with increased flow causing endothelium-dependent dilatation (flow mediated dilatation). Patients. We investigated 135 healthy women; 40 were pre-menopausal (mean SD age/26 6 years, group 1), 40 were post-menopausal and had never taken HRT (aged 58 3 years; group 2) and 55 were age-matched postmenopausal women who had taken HRT for ? 2 years, from within 2 years of the menopause (aged 57 4 years; group 3). In group 3, 40 women were on combined oestrogen and progesterone and 15 on oestrogen-only HRT. Results. In group 2, flow-mediated dilatation was significantly reduced compared with group 1 (4.4 3.4 vs 9.6 3.6%, P < 0.001), consistent with a decline in arterial endothelial function after the menopause. In group 3, however, flow-mediated dilatation was significantly better than group 2 (6.2 3.3 vs 4.4 3.4%, P = 0.01), suggesting a protective effect of HRT. Flow mediated dilatation was similar in women taking oestrogen alone and in those on combined HRT (5.5 2.8 vs 6.5 3.4%, P = 0.40). Conclusions. Long-term HRT is associated with improved arterial endothelial function in healthy postmenopausal women. This benefit was observed in both the combined hormone replacement and unopposed oestrogen therapy groups. This may explain some of the apparent cardioprotective effect of HRT after the menopause.
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Jessup, Wendy K.Nitric oxide has an important biological role as endothelium-derived relaxing factor, a key agent in the maintenance of normal vascular tone. It can also suppress lipoprotein oxidation, a potential anti-atherogenic property. However, in arteries subject to hypercholesterolemia or atherosclerosis, whereas nitric oxide synthesis is normal its biological activity is attenuated. This may be caused by its inactivation in the intima by components of oxidized lipoproteins acting both directly (by reaction with nitric oxide) and indirectly (by simulation of release of nitric oxide scavengers). Thus in hypercholesterolemia the normal balance between nitric oxide availability and lipoprotein oxidation is shifted to favour a self-reinforcing cycle of nitric oxide depletion and accelerated lipoprotein oxidation that may ultimately lead to atherosclerosis.
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Brown, Andrew J.; Dean, Roger T.; Jessup, Wendy K.We have defined the lipid composition of copper-oxidized LDL (Cu-oxLDL) and a macrophage-foam cell model generated by the uptake of this modified lipoprotein. An HPLC method previously developed by our group for the measurement of lipid oxidation products of LDL was extended to permit the analysis of an array of 7-ketocholesteryl esters. Gas chromatography was used for the quantitation of oxysterols (free and esterified) in Cu-oxLDL and their subsequent uptake by macrophages. LDL (1.0 mg protein/ml) was oxidized using Cu(II) (20 ?M) for up to 48 h at 37C. Resident mouse peritoneal macrophages were incubated with 24 h Cu-oxLDL (50 ?g/ml) for 24 h. In 24 h Cu-oxLDL, cholesterol comprised approximately 50% of total sterols, 7- ketocholesterol comprised approximately 30%, with five other oxysterols comprising the remainder (7?- and 7?-hydroxycholesterol, cholesterol ?- and ?-epoxides, and 6?-hydroxycholesterol). Macrophages that were incubated with 24 h Cu-oxLDL displayed a profile of oxysterols remarkably similar to that of 24 h Cu-oxLDL itself. The majority of cholesteryl esters and 7- ketocholesteryl esters in Cu-oxLDL and in Cu-oxLDL-loaded macrophages contained fatty acyl chains which are presumed oxidized. This work represents a comprehensive survey of free and esterified oxysterols in Cu-oxLDL and Cu- oxLDL-loaded macrophages and provides a basis for exploring how oxysterols are metabolized by macrophages and authentic human foam cells, and how, in turn, these oxysterols influence cellular metabolism.
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Thomas, Shane R.; Neuil, Ji? Stocker, RolandThere is considerable interest in the ability of antioxidant supplementation, in particular with vitamin E, to attenuate LDL oxidation, a process implicated in atherogenesis. Since vitamin E can also promote LDL lipid peroxidation, we investigated the effects of supplementation with vitamin E alone or in combination with coenzyme Q on the early stages of the oxidation of isolated LDL. Isolated LDL was obtained from healthy subjects before and after in vitro enrichment with vitamin E (D-?-tocopherol, ?-TOH) or dietary supplementation with D-?-TOH (1 g/d) and/or coenzyme Q (100 mg/d). LDL oxidation initiation was assessed by measurement of the consumption of ?-TOH and cholesteryl esters containing polyunsaturated fatty acids and the accumulation of cholesteryl ester hydroperoxides during incubation of LDL in the transition metal-containing Ham's F-10 medium in the absence and presence of human monocyte-derived macrophages (MDMs). Native LDL contained 8.52 molecules of ?-TOH and 0.5 to 0.8 molecules of ubiquinol-10 (CoQ<inf>10</inf>H<inf>2</inf>, the reduced form of coenzyme Q) per lipoprotein particle. Incubation of this LDL in Ham's F-10 medium resulted in a time-dependent loss of ?-TOH with concomitant stoichiometric conversion of the major cholesteryl esters to their respective hydroperoxides. MDMs enhanced this process. LDL lipid peroxidation occurred via a radical chain reaction in the presence of ?-TOH, and the rate of this oxidation decreased on ?-TOH depletion. In vitro enrichment of LDL with ?-TOH resulted in an LDL particle containing sixfold to sevenfold more ?-TOH, and such enriched LDL was more readily oxidized in the absence and presence of MDMs compared with native LDL. In vivo ?-TOH-deficient LDL, isolated from a patient with familial isolated vitamin E deficiency, was highly resistant to Ham's F-10-initiated oxidation, whereas dietary supplementation with vitamin E restored the oxidizability of the patient's LDL. Oral supplementation of healthy individuals for 5 days with either ?-TOH or coenzyme O increased the LDL levels of ?-TOH and CoQ<inf>10</inf>,H<inf>2</inf>, by two to three or three to four times, respectively. ?-TOH-supplemented LDL was significantly more prone to oxidation, whereas CoQ<inf>10</inf>H<inf>2</inf>-enriched LDL was more resistant to oxidation initiation by Ham's F-10 medium than native LDL. Cosupplementation with both ?-TOH and coenzyme O resulted in LDL with increased levels of ?-TOH and CoQ<inf>10</inf>H<inf>2</inf>, and such LDL was markedly more resistant to initiation of oxidation than native or ?-TOH-enriched LDL. These results demonstrate that oral supplementation with ?-TOH alone results in LDL that is more prone to oxidation initiation, whereas Cosupplementation with coenzyme Q not only prevents this prooxidant activity of vitamin E but also provides the lipoprotein with increased resistance to oxidation.
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Cope, Ranald B.; Bosnic, Meira; Boehm-Wilcox, Christa; Mohr, Detlef; Reeve, Vivienne ElizabethDietary fats modulate a wide variety of T cell functions in mice and humans. This study examined the effects of four different dietary fats, predominantly polyunsaturated sunflower oil, margarine, and predominantly saturated butter, clarified butter, on the T cell-mediated, systemic suppression of contact hypersensitivity by ultraviolet radiation in the Skh:HR-1 hairless mouse. Diets containing either 200 g/kg or 50 g/kg butter or clarified butter as the sole fat source protected against systemic photoimmunosuppression, whether the radiation source was unfiltered ultraviolet B (280-320 rim) or filtered solar simulated ultraviolet radiation (290400 nm), in comparison with diets containing either 200 or 50 g/kg margarine or sunflower oil. There was a linear relationship (r > 0.9) between protection against photoimmunosuppression and the proportion of clarified butter in mice fed a series of 200 g/kg mixed fat diets that provided varying proportions of clarified butter and sunflower oil. The dietary fats did not modulate the contact hypersensitivity reaction in unirradiated animals. The observed phenomena were not primarily due to the carotene, tocopherol, cholecalciferol, retinol, lipid hydroperoxide or the nonfat solid content of the dietary fats used and appeared to be a result of the different fatty acid composition of the fats.
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Brieger, David B.; Dawes, JoanWe have previously reported (Brieger D, Dawes J. Thromb Haemost 1994; 72: 275-80) that the prolonged anti-Xa amidolytic activity following intravenous administration of the low molecular weight heparin Enoxaparin sodium is mediated by small molecules derived from the injected drug, and an antithrombin binding penta/hexasaccharide can be detected in the circulation as late as 1 week after administration. To investigate the mechanism underlying this persistence we administered 125I-labelled fractions of Enoxaparin sodium and unfractionated 125I-heparin to rabbits. Both 125I-heparin and the radiolabeled high molecular weight (>6000 Da) Enoxaparin sodium were more effectively cleared from the circulation than the smaller components of LMW heparin. However, our data suggest that the circulating biologically active penta/hexasaccharide was not an unmodified component of the injected drug but was derived from a subpopulation of molecules of intermediate molecular weight (1800-6000 Da) which was retained in the tissues. Significant quantities of both Enoxaparin sodium and unfractionated heparin were retained in the internal organs. We propose that the sequestered subpopulations of Enoxaparin sodium and unfractionated heparin follow different catabolic routes. After administration of both unfractionated and LMW heparin additional anlithrombin binding material was released into the circulation by a bolus dose of heparin. This material was not contained on circulating blood cells and was probably sequestered on the endothelium.
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Stanley, Keith K.Membrane proteins can contain short sequence motifs that determine their intracellular location, either by a retention or a retrieval mechanism. In both cases the targeting signal is essentially a specific binding site for other proteins that effect the localization. The folding of targeting motifs is often robust leading to a dominant effect in molecular cut and paste experiments designed to identify them. However regulation can also occur, allowing a single membrane protein to express different targeting signals at different locations in the cell. Regulation can be achieved by phosphorylation of the cytoplasmic domain leading to changes in binding affinity for effector proteins, or by masking of the targeting signal by complex formation.
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Witting, Paul Kenneth; Westerlund, Christer; Stocker, RolandWe report a rapid and convenient method for screening potential inhibitors of the initiation of low density lipoprotein (LDL) lipid peroxidation. The method uses positively and negatively charged micelles of either cetyltrimethyl ammonium chloride or sodium dodecyl sulfate with added ?-tocopherol. It is based on the capacity of an antioxidant to attenuate ?-tocopheroxyl radicals, generated by irradiation of the ?-tocopherol-containing micelles with UV light, and measured directly by electron spin resonance spectroscopy. To establish the reliability of the method, we compared the ?-tocopheroxyl radical attenuating ability (TRAA) of 53 natural and synthetic potential antioxidants with their respective ability to inhibit the early stages of LDL lipid peroxidation initiated by a low flux of water-soluble peroxyl radicals. The relationship between the measured TRAA and corresponding LDL antioxidation activity was highly significant (P < 0.00005, Rank test). Thus, the potency of a co-antioxidant for LDLs ?-tocopherol could be predicted with > 98% probability by the TRAA test alone. The results suggest that this relatively simple method represents an effective and simple screening test that can be used for large numbers of potential inhibitors of the early stages of LDL lipid oxidation.
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Brieger, David B.; Dawes, JoanDermatan sulphate does not catalyse the inactivation of factor Xa. However, the low molecular weight (LMW) dermatan sulphate Desmin 370 has been shown to generate circulating anti-Xa activity following administration to humans. Using a single batch of Desmin 370, we measured 3 U/mg of anti-Xa activity by amidolytic assay in vitro. The material responsible for this activity had a lower molecular weight range (6000 and 1800 Da) than Desmin 370 and was more highly sulphated than the bulk of the drug. Heparinase digestion of Desmin 370 eliminated 90% of the in vitro anti-Xa activity without significantly interfering with its ability to potentiate inactivation of thrombin by HCII, suggesting that the anti-Xa activity is not due to dermatan sulphate and is probably heparin. When 125I-labelled Desmin 370 together with 40 mg/kg carrier drug was administered intravenously to a rabbit, anti-Xa activity was readily detectable in the plasma for up to 10 h and had a longer half-life than the sulphated radiolabel. Most of this anticoagulant activity was recovered from the plasma by Polybrene affinity chromatography and was probably a sulphated glycosaminoglycan. Administration of the heparinase-digested drug to a rabbit resulted in 70% less anti-Xa activity than the undigested drug. We conclude that Desmin 370 contains detectable quantities of biologically active low molecular weight heparin, which is responsible for persistent anti-Xa activity following intravenous administration.
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Kocher, Markus; Kenny, Peter A.; Farram, Eunice; Abdul-Majid, Khairul Bariah; Finlay-Jones, John J.; Geczy, Carolyn L.Murine abscesses induced by intraperitoneal injection of a mixture of Escherichia coli, Bacteroides fragilis, and bran are established models for the study of localized infectious and inflammatory lesions. Chemotactic factors are thought to mediate the directed migration of large numbers of leukocytes into the abscess. Microorganisms located within the encapsulated lesion are not readily eliminated by the leukocytes, but their numbers are controlled over many weeks. We report the presence of large amounts of two murine S100 proteins. CP-10 and migration inhibition factor-related protein 14 (MRP-14), in abscesses as demonstrated by immunohistochemistry and measured by enzyme-linked immunosorbent assay and Western blotting (immunoblotting). High levels of CP-10 (7.7 1 mg/ml) and MRP-14 (5.5 1 mg/ml) were found throughout the time course of abscess development from early acute-phase lesions, which are predominantly neutrophilic, to late chronic-phase lesions, which contained more mononuclear cells. Approximately one-third of these amounts occurred as monomers (2.0 mg/ml for MRP 14 and 2.2 mg/ml for CP-10). Abscess fluid was strongly chemotactic, and a portion of the activity was due to CP-10, indicating its important role in leukocyte recruitment. CP-10-MRP-14 complexes were present in abscess fluid, and the proteins were immunoabsorbed together. In analogy with the related human MRP- 8-MRP-14 complex, these proteins could be involved in the inhibition of microbial growth. No growth inhibition occurred with 20 ?g of CP-10 or MRP- 14 per ml or with mixtures of both, but these concentrations may have been insufficient and were not representative of the high concentrations found within abscesses. CP-10 may contribute indirectly to the antimicrobial response in abscesses by virtue of its strong chemotactic properties and its capacity to modulate the activation state of recruited leukocytes.
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Brown, Andrew J.Smokers have a lower antioxidant status than nonsmokers which may contribute to their higher risk of vascular disease. To determine if antioxidant status improves 84 hr after smoking cessation, nine middle-aged fasting male smokers were studied. Six stopped smoking after the first day while three maintained their usual smoking pattern as ascertained by blood carboxyhemoglobin measurements. After smoking cessation, significant increases were seen in total plasma vitamin C levels (mean SEM, 19 7%; P < 0.05) and in low density lipoprotein (LDL) ?-and ?-carotene. No change after smoking cessation was detected in other parameters including plasma lipids, ?-tocopherol, and copper-induced LDL oxidizability. However, there was a 34 13% (P < 0.05) increase in plasma ?-tocopherol after smoking cessation explicable by increases in LDL and high density lipoprotein (HDL) ?-tocopherol. That this increase was related to smoking status was supported by the correlation between the rise in plasma ?-tocopherol and the selfreported daily cigarette consumption at entry into the study (r = 0.874, P = 0.016). Moreover, plasma ?-tocopherol levels were significantly lower at baseline in the smokers studied (n = 9) when compared with a matched group of nonsmokers (1.43 0.19 vs. 2.87 0.51 ?M). These results point to a special relation between ?-tocopherol and smoking status and indicate that increases in plasma antioxidant vitamins can be detected very soon after the cessation of smoking. (J. Nutr. Biochem. 7:29-39, 1996.).
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Esser, Alfred F.; Tarnuzzer, Roy W.; Tomlinson, Stephen A.; Tatar, Laura D.; Stanley, Keith K.Lack of hemolytic activity of horse serum is an inherent property of horse C9. To understand the molecular reasons for this deficiency we have cloned C9 cDNA from a horse liver cDNA library and have sequenced the cDNA yielding the complete coding sequence for horse C9. Purification of C9 from horse plasma and microsequencing established the N-terminus of the mature protein and verified that the correct horse C9 cDNA clone had been isolated. The deduced amino acid sequence corresponds to a mature protein of 526 amino acids that is 77% identical to human C9. It has the same domain structure as human C9 and contains 22 cysteines and four invariant tryptophanes. The few differences include the N-terminus, which is an unblocked glycine in horse C9 but pyroglutamine in human C9, and three potential N-glycosylation sites compared to two in human C9. The N-terminal difference is unimportant since microsequencing of bovine C9, which is strongly hemolytic, established that it also has an unblocked glycine identical to horse C9. There are no obvious structural differences apparent that could resolve the differences in hemolytic potency between the two molecules. Aside from a few conservative replacements, both C9 sequences are identical between positions 250 and 360. This region includes the membrane interaction domain in C9 and the postulated transmembrane segment that is thought to constitute the wall of a putative transmembrane pore and, therefore, should be required for cytotoxicity. In agreement with this prediction we have observed that, in contrast to the marked decrease in hemolytic activity, horse C9 is very efficient in killing a variety of Gram-negative bacteria. These results demonstrate that horse C9 is a structurally competent molecule with efficient cytotoxic activity. Its inability to lyse erythrocytes may be related to the action of control proteins on target cell membranes.
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Witting, Paul Kenneth; Bowry, Vincent W.; Stocker, Roland?-Tocopherol (?-TOH) can act as a pro- or anti-oxidant for isolated ubiquinol-10-free human low density lipoprotein (LDL). We demonstrate that ?-TOH is a more potent pro-oxidant than other forms of vitamin E for LDL peroxidation initiated by mild fluxes of aqueous peroxyl radicals and low concentrations of Cu2+. A simple deuterium exchange test shows that ?-TOH switches from pro- to anti-oxidant at Cu2+: LDL ratios 2.5. The results suggest that this test may be useful to distinguish 'inhibited' perioxidation of emulsion lipids propagated via the lipid peroxyl radical from that mediated via the antioxidant radical. 1995.
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Adams, Mark R.; Nakagomi, Akihiro; Keech, Anthony C.; Robinson, Jacqui T.C.; McCredie, Robyn J.; Bailey, Brian P.; Freedman, Ben Ben; Celermajer, David S.Background: Intima-media thickness (IMT) of the common carotid artery (CCA), measured with external vascular ultrasound, has been widely used in clinical trials as a surrogate marker for coronary atherosclerosis. Despite this, the degree of correlation between carotid IMT and the extent and severity of coronary artery disease (CAD) is not known. Methods and Results: Common carotid IMT was measured by ultrasound in 350 consecutive subjects of age 6010 years (range, 30 to 85 years) on the day of coronary angiography. Carotid mean IMT was 0.830.20 mm (range, 0.43 to 1.80 mm), and maximum IMT was 1.040.27 mm (range, 0.49 to 2.19 mm). Coronary angiograms were analyzed by independent observers for disease severity (number of vessels with ?70% stenosis), extent score, and a modified Gensini score. Mean carotid IMT was weakly but significantly correlated with CAD severity (r = .26), extent (r = .23), and modified Gensini score(r = .29, P<.0001 for all correlations). Carotid IMT was not clinically useful, however, because it was not specific or sensitive enough to identify patients with or without significant CAD. Increasing age, male sex, and presence of diabetes were all associated with a significantly (P<.01) higher CAD score than the average for any level of carotid IMT, suggesting differential effects of these traditional risk factors on the coronary and common carotid arteries. Conclusions: Although carotid IMT is significantly correlated with extent and severity of CAD, the relationship is weak. This relatively poor correlation (r2<.10) should be considered in the interpretation of clinical trials that use carotid IMT as a surrogate end point for coronary atherosclerosis.
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Zehavi-Feferman, Revital; Burgess, James W.; Stanley, Keith K.TGN38/41 cycles between the trans-Golgi network (TGN) and plasma membrane, traversing three sorting compartments: the TGN, plasma membrane and early endosome. The targeting signals responsible for this complex itinerary reside in a short cytoplasmic domain of 33 amino acid residues. We show that phosphorylation of the cytoplasmic domain of TGN38 prevents binding of p62 - a cytoplasmic protein essential for exocytic vesicle formation. Thus the cycle of TGN38/41 traffic, and by implication the pathway of exocytosis, could be controlled by phosphorylation of the TGN38 cytoplasmic domain. 1995.
