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Showing 1221–1240 of 2058 publications.
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Kappelle, Paul Jan Willem Herman; Lambert, Gilles; Dullaart, Robin P.F.Purpose: Proprotein convertase subtilisin-kexin type 9 (PCSK9) promotes low density lipoprotein (LDL) receptor degradation, thereby providing a key pathway for LDL metabolism. PCSK9 mRNA expression may be upregulated by insulin in murine models. Here we examined effects of exogenous hyperinsulinemia on plasma PCSK9 levels in humans without and with type 2 diabetes mellitus. Methods: A 24. h moderately hyperinsulinemic glucose clamp (30. mU/kg/h) was performed in 8 healthy men and 8 male type 2 diabetic patients. Plasma PCSK9 was measured using a sandwich enzyme-linked immunosorbent assay. Results: Plasma LDL cholesterol and apolipoprotein B were lowered by insulin in healthy subjects and diabetic patients (p< 0.01 for all), whereas triglycerides were also decreased in healthy subjects (p< 0.01). Plasma PCSK9 levels remained unchanged in healthy subjects (median (interquartile range) change, -23 (-63 to 25) %, P = 0.50) and in diabetic patients (change, 4 (-17 to 44) %, P = 0.20). Individual absolute and relative changes in LDL cholesterol, apolipoprotein B and triglycerides after 24. h of insulin were unrelated to changes in PCSK9 (P> 0.15 for all). Conclusion: Plasma PCSK9 levels are not increased by exposure to moderate 24. h hyperinsulinemia in healthy and type 2 diabetic individuals. 2010 Elsevier Ireland Ltd.
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Wise, Steven G.; Byrom, Michael John; Waterhouse, Anna; Bannon, Paul Gerard; Ng, Martin K.C.; Weiss, Anthony StevenSmall-diameter synthetic vascular graft materials fail to match the patency of human tissue conduits used in vascular bypass surgery. The foreign surface retards endothelialization and is highly thrombogenic, while the mismatch in mechanical properties induces intimal hyperplasia. Using recombinant human tropoelastin, we have developed a synthetic vascular conduit for small-diameter applications. We show that tropoelastin enhances endothelial cell attachment (threefold vs. control) and proliferation by 54.7 1.1% (3 days vs. control). Tropoelastin, when presented as a monomer and when cross-linked into synthetic elastin for biomaterials applications, had low thrombogenicity. Activation of the intrinsic pathway of coagulation, measured by plasma clotting time, was reduced for tropoelastin (60.4 8.2% vs. control). Platelet attachment was also reduced compared to collagen. Reductions in platelet interactions were mirrored on cross-linked synthetic elastin scaffolds. Tropoelastin was subsequently incorporated into a synthetic elastin/ polycaprolactone conduit with mechanical properties optimized to mimic the human internal mammary artery, including permeability, compliance, elastic modulus and burst pressure. Further, this multilayered conduit presented a synthetic elastin internal lamina to circulating blood and demonstrated suturability and mechanical durability in a small scale rabbit carotid interposition model. 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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Davies, Michael J.Theretis considerable interest in the role that mammalian heme peroxidase enzymes, primarily myeloperoxidase, eosinophil peroxidase and lactoperoxidase, may play in a wide range of human pathologies. This has been sparked by rapid developments in our understanding of the basic biochemistry of these enzymes, a greater understanding of the basic chemistry and biochemistry of the oxidants formed by these species, the development of bio-markers that can be used damage induced by these oxidants in vivo, and the recent identification of a number of compounds that show promise as inhibitors of these enzymes. Such compounds offer the possibility of modulating damage in a number of human pathologies. This reviews recent developments in our understanding of the biochemistry of myeloperoxidase, the oxidants that this enzyme generates, and the use of inhibitors to inhibit such damage. 2011 JCBN.
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Zhang, Lei; Song, James; Cavigiolio, Giorgio; Ishida, Brian Y.; Zhang, Shengli; Kane, John P.; Weisgraber, Karl H.; Oda, Michael N.; Rye, Kerry Anne; Pownall, Henry J.; Ren, GangPlasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography. -Zhang, L., J. Song, G. Cavigiolio, B. Y. Ishida, S. Zhang, J. P. Kane, K. H. Weisgraber, M. N. Oda, K-A. Rye, H. J. Pownall, and G. Ren. The morphology and structure of lipoproteins revealed by an optimized negativestaining protocol of electron microscopy. 2011 by the American Society for Biochemistry and Molecular Biology, Inc.
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Wise, Steven G.; Byrom, Michael John; Waterhouse, Anna; Bannon, Paul Gerard; Weiss, Anthony Steven; Ng, Martin K.C.[No abstract available]
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Preiss, David J.; Seshasai, Sreenivasa Rao Kondapally; Welsh, Paul I.; Murphy, Sabina A.; Ho, Jennifer E.; Waters, David D.; DeMicco, David A.; Barter, Philip J.; Cannon, Christopher Paul; Sabatine, Marc S.; Braunwald, Eugene B.; Kastelein, Johannes Jacob Pieter; de Lemos, James Andrew; Blazing, Michael A.; Pedersen, Terje R.; Tikkanen, Matti Juhani; Sattar, Naveed A.; Ray, Kausik KumarContext: A recent meta-analysis demonstrated that statin therapy is associated with excess risk of developing diabetes mellitus. Objective: To investigate whether intensive-dose statin therapy is associated with increased risk of new-onset diabetes compared with moderate-dose statin therapy. Data Sources: We identified relevant trials in a literature search of MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials (January 1, 1996, through March 31, 2011). Unpublished data were obtained from investigators. Study Selection: We included randomized controlled end-point trials that compared intensive-dose statin therapy with moderate-dose statin therapy and included more than 1000 participants who were followed up for more than 1 year. Data Extraction: Tabular data provided for each trial described baseline characteristics and numbers of participants developing diabetes and experiencing major cardiovascular events (cardiovascular death, nonfatal myocardial infarction or stroke, coronary revascularization). We calculated trial-specific odds ratios (ORs) for new-onset diabetes and major cardiovascular events and combined these using random-effects model meta-analysis. Between-study heterogeneity was assessed using the I2 statistic. Results: In 5 statin trials with 32 752 participants without diabetes at baseline, 2749 developed diabetes (1449 assigned intensive-dose therapy, 1300 assigned moderate-dose therapy, representing 2.0 additional cases in the intensive-dose group per 1000 patient-years) and 6684 experienced cardiovascular events (3134 and 3550, respectively, representing 6.5 fewer cases in the intensive-dose group per 1000 patient-years) over a weighted mean (SD) follow-up of 4.9 (1.9) years. Odds ratios were 1.12 (95% confidence interval [CI], 1.04-1.22; I2 = 0%) for new-onset diabetes and 0.84 (95% CI, 0.75-0.94; I2 = 74%) for cardiovascular events for participants receiving intensive therapy compared with moderate-dose therapy. As compared with moderate-dose statin therapy, the number needed to harm per year for intensive-dose statin therapy was 498 for new-onset diabetes while the number neededto treat per year for intensive-dose statin therapy was 155 for cardiovascular events. Conclusion: In a pooled analysis of data from 5 statin trials, intensive-dose statin therapy was associated with an increased risk of new-onset diabetes compared with moderate-dose statin therapy. 2011 American Medical Association. All rights reserved.
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Ong, Kwok Leung; Wu, Ben Jing; Cheung, Bernard Man Yung; Barter, Philip J.; Rye, Kerry AnneObjective: A low circulating level of bilirubin is associated with increased cardiovascular risk. As statins can stimulate heme oxygenase-1 (HO-1), which increases bilirubin production, we investigated whether statins in routine use increase total bilirubin levels in subjects at high cardiovascular risk. Methods: Data from 3290 subjects with self-reported history of hypercholesterolemia, diabetes, or cardiovascular diseases in the United States National Health and Nutrition Examination Survey (NHANES) 1999-2008 were analyzed. Results: Subjects taking statins (n= 1156) had lower total bilirubin levels than those not taking any lipid-lowering medication (n= 2134) after adjusting for age, sex, race/ethnicity, and survey period (adjusted mean = 0.699 vs 0.729. mg/dl respectively, P= 0.001). The association remained significant after adjusting for more covariates (P= 0.002), but was attenuated after further adjusting for glycosylated hemoglobin, insulin resistance index, and low-density lipoprotein (LDL) cholesterol (P= 0.043). The use of lovastatin, rosuvastatin, and cerivastatin was associated with lower total bilirubin levels in the full adjustment model (P< 0.05). Conclusion: The use of statins was associated unexpectedly with lower total bilirubin levels. This could be explained at least partly by the effect of statins on glycemia and LDL cholesterol. Our results do not suggest that the anti-oxidant and anti-inflammatory effects of statins are due to HO-1 induction and increased serum bilirubin levels. 2011 Elsevier Ireland Ltd.
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Arsenault, Beno J.; Barter, Philip J.; DeMicco, David A.; Bao, Weihang; Preston, Gregory M.; LaRosa, John C.; Grundy, Scott M.; Deedwania, Prakash ?.; Greten, Heiner; Wenger, Nanette Kass; Shepherd, James; Waters, David D.; Kastelein, Johannes Jacob PieterObjectives The aim of this study was to investigate the relationship between lipid and nonlipid biomarker levels achieved during statin therapy and the incidence of major cardiovascular events (MCVEs) in patients with stable coronary heart disease (CHD). Background Several plasma nonlipid biomarkers have been shown to predict MCVEs in population studies. Methods This is a nested case-control study in the TNT (Treating to New Targets) study population, a randomized trial that compared the efficacy of high- (80 mg) versus low-dose (10 mg) atorvastatin for the secondary prevention of CHD. Fasting plasma levels of standard lipids and of 18 nonlipid biomarkers were obtained after an 8-week run-in period on atorvastatin 10 mg and again 1 year after being randomized to 10 or 80 mg atorvastatin in 507 patients who experienced MCVEs during the 4.9 years of study follow-up and in 1,020 control subjects. An MCVE was defined as CHD death; nonfatal, nonprocedure-related myocardial infarction; resuscitated cardiac arrest; or fatal or nonfatal stroke. Results Low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides were all predictive of recurrent MCVEs (p ? 0.009). Concentrations of many of the 18 nonlipid biomarkers were lowered by atorvastatin therapy (independent of dose). However, almost none of the nonlipid biomarker levels, whether measured after the 8-week run-in period or after 1 year of treatment with 10 or 80 mg atorvastatin, were predictive of recurrent MCVEs. Conclusions In patients with stable CHD, atorvastatin improved plasma levels of an expanded panel of nonlipid biomarkers. However, independently of atorvastatin dose, the achieved levels of the vast majority of nonlipid biomarkers did not predict MCVEs. (A Study to Determine the Degree of Additional Reduction in CV Risk in Lowering LDL Below Minimum Target Levels [TNT]; NCT00327691) 2011 American College of Cardiology Foundation.
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Hennessy, Annemarie; Makris, Angela[No abstract available]
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Gracanin, Michelle; Lam, Magdalena A.; Morgan, Philip E.; Rodgers, Kenneth John; Hawkins, Clare L.; Davies, Michael J.Proteins are major biological targets for oxidative damage within cells because of their high abundance and rapid rates of reaction with radicals and singlet oxygen. These reactions generate high yields of hydroperoxides. The turnover of both native and modified/damaged proteins is critical for maintaining cell homeostasis, with this occurring via the proteasomal and endosomal-lysosomal systems; the former is of particular importance for intracellular proteins. In this study we have examined whether oxidation products generated on amino acids, peptides, and proteins modulate 26S proteasome activity. We show that oxidation products, and particularly protein hydroperoxides, are efficient inhibitors of the 26S proteasome tryptic and chymotryptic activities, with this depending, at least in part, on the presence of hydroperoxide groups. Removal of these species by reduction significantly reduces proteasome inhibition. This loss of activity is accompanied by a loss of thiol residues, but an absence of radical formation, consistent with molecular, rather than radical, reactions being responsible for proteasome inhibition. Aldehydes also seem to play a role in the inhibition of chymotryptic activity, with this prevented by treatment with NaBH<inf>4</inf>, which reduces these groups. Inhibition occurred at hydroperoxide concentrations of ? 1 ?M for oxidized amino acids and peptides and ? 10 ?M for oxidized proteins, compared with ca. 100 ?M for H<inf>2</inf>O<inf>2</inf>, indicating that H<inf>2</inf>O<inf>2</inf> is a much less effective inhibitor. These data indicate that the formation of oxidized proteins within cells may modulate cell function by interfering with the turnover of native proteins and the clearance of modified materials. 2010 Elsevier Inc. All rights reserved.
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Elliott, David A.; Tsoi, Kayan; Holinkova, Sandra; Chan, Sharon L.; Kim, Woojin Scott; Halliday, Glenda Margaret; Rye, Kerry Anne; Garner, BrettApolipoprotein-E (apoE) plays important roles in neurobiology and the apoE4 isoform increases risk for Alzheimer's disease (AD). ApoE peptides are biologically active and may be produced in the brain. It is unclear if apoE proteolysis is dependent on isoform or AD status and this was addressed here. Hippocampus, frontal cortex, occipital lobe and cerebellum samples were homogenized into fractions that were soluble in Tris-buffered saline (TBS), Triton X-100 or guanidine hydrochloride and analysed for apoE fragmentation by Western blotting. Approximately 20% of apoE3 was detected as fragments and this was predominantly as a 25. kDa peptide in TBS-soluble fractions. The concentration of TBS-soluble apoE fragments was two- to three-fold higher in apoE3 compared to apoE4 subjects. This difference was observed in all areas of the brain examined and was not related to AD status. Cathepsin-D treatment generated apoE fragments that were very similar to those detected in brain, however, no apoE isoform-specific differences in susceptibility to cathepsin-D proteolysis were detected. This indicates that proteolytic processing of apoE to form soluble fragments in the human brain is dependent on apoE isoform but not AD status. 2009 Elsevier Inc.
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Kappelle, Paul Jan Willem Herman; Lambert, Gilles; Dahlbk, Bjn D.; Nielsen, Lars Bo; Dullaart, Robin P.F.Purpose: Apolipoprotein M (apoM) retards atherosclerosis development in murine models, and may be regulated by pathways involved in LDL metabolism. Proprotein convertase subtilisin-kexin type 9 (PCSK9) plays a key role in LDL receptor processing. We determined the extent to which plasma apoM is related to PCSK9 levels in subjects with varying degrees of obesity. Methods: We sought correlations between plasma apoM and PCSK9, measured using recently developed ELISAs, in 79 non-diabetic subjects. Results: ApoM and PCSK9 levels were both correlated positively with total cholesterol, non-HDL cholesterol, LDL cholesterol and apoB (p< 0.05 to p< 0.001). ApoM correlated positively with PCSK9 in lean individuals (n = 37, r = 0.337, P = 0.041), but not in overweight subjects (n = 32, r = 0.125, P = 0.50) and in obese subjects (n = 10, r = -0.055, P = 0.88). Conclusions: The PCSK9 pathway may contribute to plasma apoM regulation in humans. The influence of PCSK9 on circulating apoM appears to be modified by adiposity. 2010.
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Cannon, Christopher Paul; Shah, Sukrut; Dansky, Hayes M.; Davidson, Michael H.; Brinton, Eliot A.; Gotto, Antonio M.; Stepanavage, Michael E.; Liu, Sherry Xueyu; Gibbons, Patrice H.; Ashraf, Tanya B.; Zafarino, Jennifer; Mitchel, Yale B.; Barter, Philip J.BACKGROUND: Anacetrapib is a cholesteryl ester transfer protein inhibitor that raises high-density lipoprotein (HDL) cholesterol and reduces low-density lipoprotein (LDL) cholesterol. METHODS: We conducted a randomized, double-blind, placebo-controlled trial to assess the efficacy and safety profile of anacetrapib in patients with coronary heart disease or at high risk for coronary heart disease. Eligible patients who were taking a statin and who had an LDL cholesterol level that was consistent with that recommended in guidelines were assigned to receive 100 mg of anacetrapib or placebo daily for 18 months. The primary end points were the percent change from baseline in LDL cholesterol at 24 weeks (HDL cholesterol level was a secondary end point) and the safety and side-effect profile of anacetrapib through 76 weeks. Cardiovascular events and deaths were prospectively adjudicated. RESULTS: A total of 1623 patients underwent randomization. By 24 weeks, the LDL cholesterol level had been reduced from 81 mg per deciliter (2.1 mmol per liter) to 45 mg per deciliter (1.2 mmol per liter) in the anacetrapib group, as compared with a reduction from 82 mg per deciliter (2.1 mmol per liter) to 77 mg per deciliter (2.0 mmol per liter) in the placebo group (P<0.001) - a 39.8% reduction with anacetrapib beyond that seen with placebo. In addition, the HDL cholesterol level increased from 41 mg per deciliter (1.0 mmol per liter) to 101 mg per deciliter (2.6 mmol per liter) in the anacetrapib group, as compared with an increase from 40 mg per deciliter (1.0 mmol per liter) to 46 mg per deciliter (1.2 mmol per liter) in the placebo group (P<0.001) - a 138.1% increase with anacetrapib beyond that seen with placebo. Through 76 weeks, no changes were noted in blood pressure or electrolyte or aldosterone levels with anacetrapib as compared with placebo. Prespecified adjudicated cardiovascular events occurred in 16 patients treated with anacetrapib (2.0%) and 21 patients receiving placebo (2.6%) (P = 0.40). The prespecified Bayesian analysis indicated that this event distribution provided a predictive probability (confidence) of 94% that anacetrapib would not be associated with a 25% increase in cardiovascular events, as seen with torcetrapib. CONCLUSIONS: Treatment with anacetrapib had robust effects on LDL and HDL cholesterol, had an acceptable side-effect profile, and, within the limits of the power of this study, did not result in the adverse cardiovascular effects observed with torcetrapib. (Funded by Merck Research Laboratories; ClinicalTrials.gov number, NCT00685776.) Copyright 2010 Massachusetts Medical Society.
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Beck, Konstanze; Wu, Ben Jing; Ni, Jun; Santiago, Fernando S.; Malabanan, Kristine P.; Li, Cheng; Wang, Yutang; Khachigian, Levon M.; Stocker, RolandRationale: Induction of heme oxygenase (HO)-1 protects against experimental atherosclerotic diseases, and certain pharmacological HO-1 inducers, like probucol, inhibit the proliferation of vascular smooth muscle cells and, at the same time, promote the growth of endothelial cells in vivo and in vitro. Objective: Because such cell-specific effects are reminiscent of the action of the transcription factor Yin Yang (YY)1, we tested the hypothesis that there is a functional relationship between HO-1 and YY1. Methods and results: We report that probucol increases the number of YY1 cells in rat carotid artery following balloon injury at a time coinciding with increased HO-1 expression. The drug also induces the expression of YY1 mRNA and protein in rat aortic smooth muscle cells (RASMCs) in vitro, as do other known HO-1 inducers (tert-butylhydroquinone and hemin) and overexpression of HO-1 using a human HMOX1 cDNA plasmid. Conversely, overexpression of YY1 induces expression of HO-1 in RASMCs. Induction of YY1 expression is dependent on HO-1 enzyme activity and its reaction product CO, because pharmacological inhibition of heme oxygenase activity or CO scavenging block, whereas exposure of RASMCs to a CO-releasing molecule increases, YY1 expression. Furthermore, RNA interference knockdown of YY1 prevents probucol or adeno-HO-1 from inhibiting RASMC proliferation in vitro and neointimal formation in vivo. Conclusions: Our findings show, for the first time, that HO-1 functionally interplays with the multifunctional transcription factor YY1 and that this interplay explains some of the protective activities of HO-1. 2010 American Heart Association, Inc.
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Sourris, Karly C.; Harcourt, Brooke E.; Penfold, Sally Anne; Yap, Felicia Y.T.; Morley, Amy L.; Morgan, Philip E.; Davies, Michael J.; Baker, Scott Thomas; Jerums, George J.; Forbes, Josephine MareeBackground. Advanced glycation end-products (AGEs) and their receptors are prominent contributors to diabetic kidney disease. Methods. Flow cytometry was used to measure the predictive capacity for kidney impairment of the AGE receptors RAGE, AGE-R1, and AGE-R3 on peripheral blood mononuclear cells (PBMCs) in experimental models of type 2 diabetes (T2DM) fed varied AGE containing diets and in obese type 2 diabetic and control human subjects. Results. Diets high in AGE content fed to diabetic mice decreased cell surface RAGE on PBMCs and in type 2 diabetic patients with renal impairment (RI). All diabetic mice had elevated Albumin excretion rates (AERs), and high AGE fed dbdb mice had declining Glomerular filtration rate (GFR). Cell surface AGE-R1 expression was also decreased by high AGE diets and with diabetes in dbdb mice and in humans with RI. PBMC expression of AGE R3 was decreased in diabetic dbdb mice or with a low AGE diet. Conclusions. The most predictive PBMC profile for renal disease associated with T2DM was an increase in the cell surface expression of AGE-R1, in the context of a decrease in membranous RAGE expression in humans, which warrants further investigation as a biomarker for progressive DN in larger patient cohorts. Copyright 2010 Karly C. Sourris et al.
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Kamili, Alvin; Wat, Elaine C.L.; Chung, Rosanna W.S.; Tandy, Sally; Weir, Jacquelyn M.; Meikle, Peter J.; Cohn, Jeffrey S.Background. Milk phospholipids (PLs) reduce liver lipid levels when given as a dietary supplement to mice fed a high-fat diet. We have speculated that this might be due to reduced intestinal cholesterol uptake. Methods. Mice were given a high-fat diet for 3 or 5 weeks that had no added PL or that were supplemented with 1.2% by wt PL from cow's milk. Two milk PL preparations were investigated: a) a PL-rich dairy milk extract (PLRDME), and b) a commercially-available milk PL concentrate (PC-700). Intestinal cholesterol uptake was assessed by measuring fecal and hepatic radioactivity after intragastric administration of [14C]cholesterol and [ 3H]sitostanol. Fecal and hepatic lipids were measured enzymatically and by ESI-MS/MS. Results. Both PL preparations led to significant decreases in total liver cholesterol and triglyceride (-20% to -60%, P < 0.05). Hepatic accumulation of intragastrically-administered [14C]cholesterol was significantly less (-30% to -60%, P < 0.05) and fecal excretion of [ 14C]cholesterol and unlabeled cholesterol was significantly higher in PL-supplemented mice (+15% to +30%, P < 0.05). Liver cholesterol and triglyceride levels were positively correlated with hepatic accumulation of intragastrically-administered [14C]cholesterol (P < 0.001) and negatively correlated with fecal excretion of [14C]cholesterol (P < 0.05). Increased PL and ceramide levels in the diet of mice supplemented with milk PL were associated with significantly higher levels of fecal PL and ceramide excretion, but reduced levels of hepatic PL and ceramide, specifically, phosphatidylcholine (-21%, P < 0.05) and monohexosylceramide (-33%, P < 0.01). Conclusion. These results indicate that milk PL extracts reduce hepatic accumulation of intestinal cholesterol and increase fecal cholesterol excretion when given to mice fed a high-fat diet. 2010 Kamili et al; licensee BioMed Central Ltd.
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Dunlop, Rachael Anne[No abstract available]
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Peskin, Alexander V.; Cox, Andrew G.; Nagy, Per Literi; Morgan, Philip E.; Hampton, Mark B.; Davies, Michael J.; Winterbourn, Christine C.Prxs (peroxiredoxins) are a ubiquitous family of cysteinedependent peroxidases that react rapidly with H<inf>2</inf>O<inf>2</inf> and alkyl hydroperoxides and provide defence against these reactive oxidants. Hydroperoxides are also formed on amino acids and proteins during oxidative stress, and they too are a potential cause of biological damage. We have investigated whether Prxs react with amino acid, peptide and protein hydroperoxides, and whether the reactions are sufficiently rapid for these enzymes to provide antioxidant protection against these oxidants. Isolated Prx2, which is a cytosolic protein, and Prx3, which resides within mitochondria, were reacted with a selection of hydroperoxides generated by ?-radiolysis or singlet oxygen, on free amino acids, peptides and proteins. Reactions were followed by measuring the accumulation of disulfide-linked Prx dimers, via non-reducing SDS/PAGE, or the loss of the corresponding hydroperoxide, using quench-flow and LC (liquid chromatography)/MS. All the hydroperoxides induced rapid oxidation, with little difference in reactivity between Prx2 and Prx3. N-acetyl leucine hydroperoxides reacted with Prx2 with a rate constant of 404 M-1 s-1. Hydroperoxides present on leucine, isoleucine or tyrosine reacted at a comparable rate, whereas histidine hydroperoxides were ? 10-fold less reactive. Hydroperoxides present on lysozyme and BSA reacted with rate constants of ? 100 M -1 s-1. Addition of an uncharged derivative of leucine hydroperoxide to intact erythrocytes caused Prx2 oxidation with no concomitant loss in GSH, as did BSA hydroperoxide when added to concentrated erythrocyte lysate. Prxs are therefore favoured intracellular targets for peptide/protein hydroperoxides and have the potential to detoxify these species in vivo. The Authors Journal compilation 2010 Biochemical Society.
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Rahmanto, Aldwin Suryo; Morgan, Philip E.; Hawkins, Clare L.; Davies, Michael J.Reaction of radicals and singlet oxygen (1O<inf>2</inf>) with proteins results in both direct damage and the formation of long-lived reactive hydroperoxides. Elevated levels of protein hydroperoxide-derived products have been detected in multiple human pathologies, suggesting that these secondary oxidants contribute to tissue damage. Previous studies have provided evidence for protein hydroperoxide-mediated inhibition of thiol-dependent enzymes and modulation of signaling processes in isolated systems. In this study 1O<inf>2</inf> and hydroperoxides have been generated in J774A.1 macrophage-like cells using visible light and the photosensitizer rose bengal, with the consequences of oxidant formation examined both immediately and after subsequent (dark-phase) incubation. Significant losses of GSH (?50%), total thiols (?20%), and activity of thiol-dependent proteins (GAPDH, thioredoxin, protein tyrosine phosphatases, creatine kinase, and cathepsins B and L; 10-50% inhibition) were detected after 1 or 2 min photo-oxidation. Non-thiol-dependent enzymes were not affected. In contrast, NADPH levels increased, together with the activity of glutathione reductase, glutathione peroxidase, and thioredoxin reductase; these increases may be components of a rapid global cytoprotective cellular response to stress. Neither oxidized thioredoxin nor radical-mediated protein oxidation products were detected at significant levels. Further decreases in thiol levels and enzyme activity occurred during dark-phase incubation, with this accompanied by decreased cell viability. These secondary events are ascribed to the reactions of long-lived hydroperoxides, generated by 1O<inf>2</inf>-mediated reactions. Overall, this study provides novel insights into early cellular responses to photo-oxidative damage and indicates that long-lived hydroperoxides can play a significant role in cellular damage. 2010 Elsevier Inc.
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Jansson, Patric J.; Hawkins, Clare L.; Lovejoy, David B.; Richardson, Des RaymondIron chelation therapy was initially designed to alleviate the toxic effects of excess iron evident in iron-overload diseases. However, some iron chelator-metal complexes have also gained interest due to their high redox activity and toxicological properties that have potential for cancer chemotherapy. This communication addresses the conflicting results published recently on the ability of the iron chelator, Dp44mT, to induce hydroxyl radical formation upon complexation with iron (B.B. Hasinoff and D. Patel, J Inorg. Biochem. 103 (2009), 1093-1101). This previous study used EPR spin-trapping to show that Dp44mT-iron complexes were not able to generate hydroxyl radicals. Here, we demonstrate the opposite by using the same technique under very similar conditions to show the Dp44mT-iron complex is indeed redox-active and induces hydroxyl radical formation. This was studied directly in an iron(II)/H<inf>2</inf>O<inf>2</inf> reaction system or using a reducing iron(III)/ascorbate system implementing several different buffers at pH 7.4. The demonstration by EPR that the Dp44mT-iron complex is redox-active confirms our previous studies using cyclic voltammetry, ascorbate oxidation, benzoate hydroxylation and a plasmid DNA strand-break assay. We discuss the relevance of the redox activity to the biological effects of Dp44mT. 2010.
