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Showing 1821–1840 of 2058 publications.

  • Garner, Brett; Waldeck, A. Reginald; Witting, Paul Kenneth; Rye, Kerry Anne; Stocker, Roland
    Journal of Biological Chemistry (Vol. 273/11) – 1998
    Human high density lipoproteins (HDL) can reduce cholesteryl ester hydroperoxides to the corresponding hydroxides (Sattler W., Christison J. K., and Stocker, R. (1995) Free Radical Biol. and Med. 18, 421-429). Here we demonstrate that this reducing activity extended to hydroperoxides of phosphatidylcholine, was similar in HDL<inf>2</inf> and HDL<inf>3</inf>, was independent of arylesterase and lecithin:cholesteryl acyltransferase activity, was unaffected by sulfhydryl reagents, and was expressed by reconstituted particles containing apoAI or apoAII only, as well as isolated human apoAI. Concomitant with the reduction of lipid hydroperoxides specific oxidized forms of apoAI and apoAII formed in blood-derived and reconstituted HDL. Similarly, specific oxidized forms of apoAI accumulated upon treatment of isolated apoAI with authentic cholesteryl linoleate hydroperoxide. These specific oxidized forms of apoAI and apoAII have been shown previously to contain Met sulfoxide (Met(O)) at Met residues and are also formed when HDL is exposed to Cu2+ or soybean lipoxygenase. Lipid hydroperoxide reduction and the associated formation of specific oxidized forms of apoAI and apoAII were inhibited by solubilizing HDL with SDS or by pretreatment of HDL with chloramine T. The inhibitory effect of chloramine T was dose-dependent and accompanied by the conversion of specific Met residues of apoAI and apoAII into Met(O). Canine HDL, which contains apoAI as the predominant apolipoprotein and which lacks the oxidation-sensitive Met residues Met112 and Met148, showed much weaker lipid hydroperoxide reducing activity and lower extents of formation of oxidized forms of apoAI than human HDL. We conclude that the oxidation of specific Met residues of apoAI and apoAII to Met(O) plays a significant role in the 2-electron reduction of hydroperoxides of cholesteryl esters and phosphatidylcholine associated with human HDL.
  • Hawkins, Clare L.; Davies, Michael J.
    Journal of the Chemical Society, Perkin Transactions 2 (Vol. /12) – 1998
    Direct rapid-flow EPR experiments together with computer simulations have been used to examine the selectivity of hydroxyl radical (generated using a Ti3+/H<inf>2</inf>O<inf>2</inf> redox couple) attack on a number of aliphatic amino acids, amino acid derivatives and small peptides. For glycine, glycine derivatives and glycine peptides attack at the ?-carbon position predominates under all conditions; in peptides attack at the C-terminal site is preferred over mid-chain sites, which in turn are favoured over the N-terminal position. This behaviour is rationalised in terms of the destabilising effect of the protonated ?-amino group, which can exert both short- and long-range effects. With alanine peptides hydrogen atom abstraction at the side-chain methyl group predominates with free amino acid; significant levels of attack at the ?-carbon position are however observed with peptides. In contrast, with valine and leucine peptides side-chain attack always predominates irrespective of whether the backbone amino group is derivatized or not; the ratio of side-chain species is also only marginally affected. The preference for attack at tertiary side-chain sites over primary side-chain methyl groups in such peptides is small. These results support the hypothesis that the selective fragmentation of large proteins as a result of exposure to hydroxyl radicals in the presence of oxygen may occur primarily as a result of attack at the ?-carbon position of surface-exposed glycine and alanine residues.
  • Waldeck, A. Reginald; Xu, Arron S.L.; Roufogalis, Basil D.; Kuchel, Philip W. William
    European Biophysics Journal (Vol. 27/3) – 1998
    NMR-based assays for measuring the fluxes of Ca2+, H+, and ATP in liposomal systems are presented. The 19F NMR Ca2+-chelating molecule 5,5- difluoro-1,2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) was trapped inside large unilamellar vesicles and used to monitor passive and A23187-mediated Ca2+ transport into them. The data were analyzed using progress curves of the transport reaction. They demonstrated the general applicability of 5FBAPTA as a 19F NMR probe of active Ca2+ transport. 31P NMR time-courses were used to monitor simultaneously the ATP hydrolysing activity of the reconstituted human erythrocyte Ca2+-ATPase and the concomitant acidification of the reaction medium in a suspension of small unilamellar vesicles. Using an estimate of the extraliposomal buffering capacity, the H+/ATP coupling stoichiometry, in the presence of A23187, was estimated from the NMR-derived data at steady state; it amounted to 1.4 0.3. This result is discussed with respect to the issue of molecular 'slip' in the context of a non-equilibrium thermodynamics model of the pump (accompanying paper in this issue). Importantly, NMR, in contrast to optical detection methods, can potentially register all fluxes and (electro)chemical gradients involved in the Ca2+-ATPase-mediated H+/Ca2+counterport, in a single experiment.
  • Kritharides, Leonard; Christian, Aimee E.; Stoudt, Genevieve W.; Morel, Diane W.; Rothblat, George H.
    Arteriosclerosis, Thrombosis, and Vascular Biology (Vol. 18/10) – 1998
    This study has investigated in detail factors regulating accumulation, esterification, and mobilization of cholesterol in human THP-1 macrophages. Human THP-1 monocytes were differentiated into macrophages and then cholesterol enriched by exposure to acetylated LDL (AcLDL), together with [3H]free cholesterol (FC). Although THP-1 macrophages accumulated FC and esterified cholesterol (EC), assessed by both mass and radioactivity, cellular EC always demonstrated a much lower specific activity (cpm/?g) than did cellular FC, and several potential causes of this finding were investigated. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) during loading decreased cell [3H]EC by 951.4% but decreased cell EC mass by only 66.04.0%, indicating that some intracellular undegraded AcLDL- derived EC was present in these cells. Esterification of [3H]oleate to EC in THP-1 cells loaded with AcLDL was 2.0 nmol mg-1 h-1 consistent with previous literature. However, EC, triglyceride, and phospholipid fractions respectively contained 1.00.07%, 80.00.5%, and 18.90.3% of cell [3H]oleate, indicating triglycerides were much more metabolically active than EC. In addition, the mass of triglyceride in THP-1 macrophages exceeded that of EC both before and after exposure to AcLDL. Esterification of nonlipoprotein-derived cholesterol was compared in THP-1 cells and nonhuman Fu5AH, CHO, and RAW macrophage cells. Whereas the nonhuman cell lines all esterified over 30% of 2-hydroxypropyl-?-cyclodextrin (hp-?-CD)-delivered cholesterol within 6 hours, THP-1 cells esterified <8.0% of incorporated cholesterol. Kinetics of cholesterol efflux from AcLDL-loaded THP, 1 cells were first investigated after loading with only FC, and interactions between efflux and EC hydrolysis were further assessed after loading cells with both EC and FC. Over 24 hours, human apolipoprotein (apo) A-I, apoHDL reconstituted with phosphatidylcholine, and HDL<inf>3</inf> respectively removed 46.63.7%, 61.33.4%, and 76.410.1% of [3H]FC from FC-enriched THP-1 cells. Cholesterol efflux to apoA-I was saturated by 24 hours and was enhanced by using apoA-I-phospholipid instead of pure apoA-I. Kinetic modeling identified that 97% of effluxed FC derived from a slow pool, with a T 1/4 ranging from 27.7 hours for HDL to 69.3 hours for apoA-I. Although efflux enhanced net clearance of EC, hydrolysis of EC during concurrent inhibition of ACAT was unaffected by cholesterol efflux. Supplementation of THP-1 cultures with cAMP to stimulate hormone-sensitive lipase did not significantly enhance net hydrolysis of EC or cholesterol efflux. In conclusion, human THP-1 macrophages contain a large and metabolically active pool of triglyceride and a relatively inactive pool of EC. The low specific activity of EC relative to FC is contributed to by reduced esterification of FC, slow hydrolysis of EC, and accumulated lipoprotein EC. The relative inactivity of the EC pool may further contribute to already impaired cholesterol efflux from these cells. Net cholesterol efflux from human macrophages is achieved by pure apoA-I and is substantially further enhanced by the presence of phospholipid in acceptor particles.
  • James, Michael John; Van Reyk, David M.; Rye, Kerry Anne; Dean, Roger T.; Cleland, Leslie Glen; Barter, Philip J.; Jessup, Wendy K.
    Lipids (Vol. 33/11) – 1998
    Oxidatively modified low density lipoprotein (LDL) has many biological activities which could contribute to the pathology of the atherosclerotic lesion. Because atherosclerosis has an inflammatory component, there has been much interest in the extent to which LDL could be oxidatively modified in vivo by inflammation. The present study examined LDL present in an accessible inflammatory site, the inflamed synovial joint, for evidence of compositional change and oxidative modification. LDL was isolated from knee joint synovial fluid (SF) from subjects with inflammatory arthropathies and also from matched plasma samples. SF and plasma LDL had similar free cholesterol and ?-tocopherol content, but SF LDL had a lower content of esterified cholesterol. On electrophoresis, SF LDL was slightly more electronegative than LDL from matched plasma samples, but the changes were much less than those resulting from Cu2+-treatment of LDL. Oxidized cholesterol was not detected in any samples, but cholesterol ester hydroperoxide levels were greater in SF than in plasma LDL. When samples from three subjects were incubated with macrophages, the SF LDL did not cause significant loading of the cells with cholesterol or cholesterol esters, in contrast to the situation with acetylated LDL. Overall, the SF LDL displayed evidence of slightly increased oxidation by comparison with matched plasma samples. Despite their isolation from an environment with active inflammation, changes were modest compared with those resulting from Cu2+ treatment. Thus, extensive LDL oxidation is not a necessary correlate of location in a chronic inflammatory site, even though it is characteristic of atherosclerotic lesions.
  • Su, Tao; Stanley, Keith K.
    Journal of Cell Science (Vol. 111/9) – 1998
    We have transfected a polarised endothelial cell line, ECV 304, and an epithelial cell line, MDCK, with a well characterised epithelial protein, the rat polymeric immunoglobulin receptor (pIgR), in order to study the protein sorting and transcytosis in endothelial cells. The expressed protein was normally processed and the steady state distribution between apical and basolateral surfaces was similar in both cell types. MDCK cells, however, showed a marked polarity in the delivery of newly synthesised pIgR to the cell surface, and in the release of secretory component. 88% of newly synthesised pIgR in MDCK cells was first delivered to the basolateral surface and 99% of secretory component was released from the apical surface. In contrast the basolateral targeting signal of pIgR was only partially recognised in endothelial cells, with 63% of the newly synthesised pIgR being first delivered to the basolateral surface. At steady state only 43% of the pIgR was found on the basolateral membrane. The direction of dimeric IgA transcytosis in endothelial cells was from apical to basolateral surfaces, opposite to that in MDCK cells. These data suggest that endothelial cells poorly recognise the targeting signals of proteins from epithelial cells, and that the direction of transcytosis is linked to the biological role of the cells.
  • Neuil, Ji? Christison, Julie K.; Iheanacho, Eugene N.; Fragonas, Jean Charles; Zammit, Vivienne C.; Hunt, Nicholas H.; Stocker, Roland
    Journal of Lipid Research (Vol. 39/2) – 1998
    Exposure of plasma from apolipoprotein E gene knockout (apoE(-/-)) and control (CBA or C57BL/6J) mice plasma to a constant rate of aqueous peroxyl radicals (ROO.) resulted in the depletion of ascorbate, urate and ?- tocopherol (?-TOH), with substantial and little lipid peroxidation, respectively. ?-TOH levels were 3-times higher in plasma from apoE(-/-) than control mice and its addition enhanced the oxidizability of control mouse plasma. In apoE(-/-) mouse plasma, ?-TOH was associated primarily with very low density lipoprotein (VLDL), whereas in plasma from control mice, the vitamin was located largely in high density lipoproteins. Oxidation of isolated lipoproteins by ROO* resulted in the accumulation of lipid hydroperoxides to an extent that reflected the plasma concentration and ?- TOH content of the different lipoprotein fractions. Oxidation of 'plasma' reconstituted from components of apoE(-/-) mice and/or human plasma showed that human and apoE(-/-) mouse lipoproteins peroxidized with similar kinetics, although the initiation of lipid peroxidation was greater in the presence of mouse than human lipoprotein-deficient plasma. Also, the chain length of lipid peroxidation in apoE(-/-) mouse plasma after ascorbate depletion appeared to be independent of the rate of ROO* generation. Together, these results show that the ROO* induced peroxidation of plasma lipoproteins in atherogenesis-susceptible apoE(-/-) mice exhibits some, though not all, features of tocopherol-mediated peroxidation (TMP). Therefore, apoE(-/-) mice may represent a suitable animal model to test a role for TMP in atherogenesis and the prevention of this disease by anti-TMP agents.
  • Hailstones, Deborah L.; Sleer, Leanne S.; Parton, Robert G.; Stanley, Keith K.
    Journal of Lipid Research (Vol. 39/2) – 1998
    We have examined the expression of caveolin in MDCK cells under conditions that vary cellular cholesterol concentration. Caveolin mRNA levels dropped to one-sixth of control levels after treatment with simvastatin, an inhibitor of cholesterol synthesis, or ?-trimethyl cyclodextrin (CD), a cholesterol sequestering drug. Both simvastatin and CD treatment decreased total cellular cholesterol levels to about 50% of control values. The potent activator of the sterol regulatory element, 25-hydroxycholesterol, showed no direct regulation of caveolin mRNA levels. Caveolin protein concentration was also decreased to 50% of control values in cholesteroldepleted cells, giving rise to a severe attenuation of caveolin expression detected by indirect immunofluorescence labeling. Quantitative electron microscopy showed a total loss of morphologically recognizable invaginated caveolae after these cholesterol depletion treatments. When the number of invaginated caveolae per cell was expressed as a function of the cellular cholesterol content, a threshold phenomenon was observed, suggesting that caveolae only form when the steady state cellular cholesterol is above 50% of control values. These findings indicate that caveolins, and caveolae, may play an important part in cellular cholesterol homeostasis.
  • Kopp, Christoph W.; Robson, Simon Christopher; Siegel, Jonathan B.; Anrather, Josef; Winkler, Hans; Grey, Shane T.; Kaczmarek, Elzbieta; Bach, Fritz Heintz; Geczy, Carolyn L.
    Thrombosis and Haemostasis (Vol. 79/3) – 1998
    The regulation of tissue factor (TF) activity by the cell associated tissue factor pathway inhibitor (TFPI) during monocyte (Mo) and endothelial cell (EC) interactions is not fully understood. This report describes co-ordinate induction of TF antigen (TF-Ag) and membrane-associated TFPI-Ag on human Mo following coculture with human aortic (HAEC) or porcine aortic EC (PAEC) or after stimulation with TNF?. We show that both allo- and xenogeneic EC induce human Mo-TF antigen in short-term culture. However, the TF activity of TNF?-primed Mo is suppressed when these cells are cocultured with HAEC [by 40.3 6.3% (p < 0.02)] or PAEC [by 50.5 10.6% (p < 0.001)]. Antibody (Ab) blocking studies confirm that TFPI is the principal anticoagulant associated with this suppression of TF-activity. Our data indicate that anti-TF activity originates, at least in part, from the activated human Mo in the coculture; additionally, specific generation of TFPI by Mo is observed under the xenogeneic culture conditions. As Mo associated TF, induced by allo- or xenogeneic EC interactions, is regulated by cell-associated TFPI, we propose that infiltrating Mo may modulate the thrombotic process at sites of vascular injury in association with both allo- and xenograft rejection.
  • Kumar, Rakesh K.; Harrison, Craig A.; Cornish, Coralie J.; Kocher, Markus; Geczy, Carolyn L.
    Pathology (Vol. 30/1) – 1998
    The murine S-100 protein designated CP-10 is a potent chemotactic factor for phagocytic cells, exhibiting optimal activity in the picomolar range. We assessed the role of this cytokine in the inflammatory response to pulmonary injury following intratracheal administration of bleomycin to mice. In the lungs of normal animals, strong cytoplasmic immunostaining for CP-10 was demonstrable in all recognisable neutrophil leucocytes sequestered within alveolar capillaries. Following induction of pulmonary inflammation in susceptible C57BL/6 mice, numerous CP-10-positive neutrophils were observed, but many of the recruited neutrophils did not exhibit staining for CP-10. No other cells were immunoreactive. The concentration of CP-10 in bronchoalveolar lavage (BAL) fluids from normal mice and mice administered intratracheal saline was below the level of detection by enzyme immunoassay. In contrast, nanomolar levels of CP-10 were detected in unconcentrated BAL fluids from C57BL/6 mice after bleomycin-induced injury, and the presence or monomeric CP-10 was demonstrable by Western blotting. Elevation of CP-10 levels correlated with the influx of inflammatory cells in C57BL/6 mice, but was not demonstrable in BAL fluids from BALB/c mice, which are resistant to pulmonary injury by bleomycin. We conclude that CP-10 may contribute to the recruitment of inflammatory cells in bleomycin-induced lung damage.
  • Baoutina, Anna; Dean, Roger T.; Jessup, Wendy K.
    Journal of Lipid Research (Vol. 39/1) – 1998
    We have investigated the effect of ?-tocopherol-loading of mouse peritoneal macrophages and human monocytes on their ability to oxidize human low density lipoprotein (LDL). Mouse peritoneal macrophages incorporated ?- tocopherol (?-TOH) from culture medium supplemented with the vitamin in a time- and concentration-dependent manner. Subcellular fractionation by density gradient ultracentrifugation showed that the distribution of incorporated ?-TOH within the cell was similar to that of free cholesterol. Most (?88%) of ?-TOH partitioned into the membrane fractions (plasma membrane ?41%, mitochondria and lysosomes ?26%, and endosomes plus endoplasmic reticulum ?21%). Cellular ?-TOH was stable for at least 24 h in serum- or LDL-free media whether permissive (Ham's F-10) or non-permissive (Dulbecco's minimum essential medium, DMEM) for LDL oxidation. When incubated with LDL in DMEM, ?-TOH-preloaded cells transferred small amounts of ?-TOH (approximately 1 nmol/mg LDL protein after 9 h) to the lipoprotein. However, enrichment of the cells with ?-TOH did not change the kinetics of oxidation of either normal or TOH-depleted LDL in Ham's F-10 medium compared with non- loaded cells, as assessed by ?-TOH consumption, cholesteryl ester degradation, and cholesteryl ester hydroperoxide and 7-ketocholesterol accumulation. Nor did it alter superoxide release by the cells or their ability to reduce extracellular copper(II). Similar to mouse macrophages, enrichment of human monocytes with ?-TOH did not change the kinetics of cell-mediated LDL oxidation. B We conclude that elevated cellular levels of ?-TOH in mouse peritoneal macrophages and in human monocytes do not affect their ability to oxidize LDL lipids in vitro. This suggests that either cell- mediated oxidation of LDL under the conditions of this study is not dependent on cell-derived radical species or that cellular ?-TOH is unable to affect their formation.
  • Mathieu, Christel; Moreau, Sophie; Frendo, Pierre; Puppo, Alain; Davies, Michael J.
    Free Radical Biology and Medicine (Vol. 24/7-Aug) – 1998
    Electron paramagnetic resonance spectroscopy has been employed to examine the nature of the metal ions and radicals present in intact root nodules of soybean plants grown in the absence of nitrate. The spectra obtained from nodules of different ages using this non-invasive technique show dramatic differences, suggesting that there are both qualitative and quantitative changes in the metal ion and radical species present. A major component of the spectra obtained from young nodules is assigned to a complex (Lb-NO) of nitric oxide (NO*) with the heme protein leghemoglobin (Lb). This Lb-NO species, which has not been previously detected in intact root nodules of plants grown in the absence of nitrate, is thought to be formed by reaction of nitric oxide with iron(II) leghemoglobin. The nitric oxide may he generated from arginine via a nitric oxide synthase-like activity present in the nodules of the soybean plants, in a manner analogous to that recently described for Lupinus albus. This Lb-NO complex is present at lower concentrations in older nodules, and is almost completely absent from senescent nodules. Exposure of young and mature nodules to oxidant stress, in the form of hydrogen peroxide, results in changes in the EPR spectra, with the loss of the signals from the Lb-NO complex and appearance of absorptions similar to those from untreated senescent nodules. These results suggest that there are characteristic changes in both the metal ion complexes and radicals present in intact root nodules of different ages, and support the theory that nitric oxide and other radicals play a significant role in determining the nitrogen fixing activity of root nodules; the modulatory activity of NO* may involve regulation of gene activity.
  • Witting, Paul Kenneth; Upston, Joanne M.; Stocker, Roland
    Sub-cellular Biochemistry (Vol. 30) – 1998
    [No abstract available]
  • Kritharides, Leonard; Upston, Joanne M.; Jessup, Wendy K.; Dean, Roger T.
    Journal of Lipid Research (Vol. 39/12) – 1998
    Cholesteryl linoleate hydroperoxide (CLOOH) and hydroxide (CLOH) are present in human atheroma. The intracellular metabolism of low density lipoprotein (LDL)-derived CLOOH and CLOH remain undefined because extensive free radical-mediated LDL oxidation, which modifies LDL apolipoprotein B sufficiently to allow endocytosis by the scavenger receptor (ScR), also degrades CLOOH and CLOH. This problem was approached by first acetylating LDL lysine residues (AcLDL) to achieve protein modification, then exposing AcLDL to the aqueous radical donor 2,2'-azobis(2-amidinopropane) HCl (AAPH), to generate mildly oxidized AcLDL (OxAcLDL). Murine peritoneal macrophages incubated with OxAcLDL accumulated large quantities of CE and small, non- toxic quantities of CLOOH and CLOH in a time- and concentration-dependent manner, and accumulation was inhibited by fucoidin. Inhibition of acyl CoA: cholesterol acyltransferase during loading did not inhibit the accumulation of either CLOOH or CLOH, whereas NH<inf>4</inf>Cl decreased intracellular clearance of accumulated CLOOH from 68.3 1.7% to 35.3 1.0% over 12 h, suggesting lysosomal or pre-lysosomal accumulation. Intracellular clearance of unoxidized lipoprotein-derived CE decreased from 84.0 5.9% to 43.1 2.3% over 12 h when cells were loaded with AcLDL or OxAcLDL, respectively. Aggregation of mildly oxidized LDL, even without acetylation, also promoted cellular accumulation of CLOOH and CLOH. We conclude that intracellular accumulation of cholesteryl linoleate hydroperoxide and cholesteryl linoleate hydroxide can follow charge modification or aggregation of mildly oxidized LDL, and that LDL-derived oxidation products may inhibit hydrolysis of LDL- derived CE in foam cell macrophages.
  • Hawkins, Clare L.; Davies, Michael J.
    Journal of the Chemical Society, Perkin Transactions 2 (Vol. /9) – 1998
    EPR spin trapping together with UV-VIS spectroscopy has been employed to examine the reaction of HOCl with amino acids and some small peptides. Evidence has been obtained for the formation and subsequent decomposition of short-lived chloramine derivatives from free amine groups present on both amino acid side-chains and at the N-terminus. Radical formation, detected by EPR spin trapping, occurs concurrently with chloramine decomposition. This process is enhanced by, but does not require, the presence of added Fe2+. With some substrates nitrogen-centred (aminyl, RNH or RNH<inf>2</inf>+) radicals, formed from cleavage of the N-Cl bond of the chloramine, can be detected. These initial aminyl radicals undergo a variety of hydrogen atom abstraction, rearrangement and fragmentation reactions to give carbon-centred species. Evidence has been obtained for both inter- and intra-molecular (1,2- and 1,5-) hydrogen atom abstraction reactions, decarboxylation and ?-scission processes. The last of these only occurs to a significant extent where the resulting radical is highly stabilised, for example, by aromatic substituents, and results in loss of the amino acid side-chain. Studies with N-acetyl derivatives and peptides are consistent with reaction of HOCl at amide (peptide) bonds to give transient chloramides which rapidly decompose to give (undetected) amidyl [N(R)C(O)R'] radicals. These species undergo rapid 1,2-hydrogen atom shift reactions to give (stabilised) ?-carbon radicals with most peptides. Evidence has also been obtained for the occurrence of hydrogen atom abstraction and decarboxylation reactions with these substrates.
  • Sanni, Latifu A.; Thomas, Shane R.; Tattam, Bruce N.; Moore, Douglas Edwin; Chaudhri, Geeta; Stocker, Roland; Hunt, Nicholas H.
    American Journal of Pathology (Vol. 152/2) – 1998
    The pathogenesis of human cerebral malaria (CM) remains unresolved. In the most widely used murine model of CM, the presence of T lymphocytes and/or interferon (IFN)-? is a prerequisite. IFN-? is the key inducer of indoleamine 2,3-dioxygenase (IDO), which is the catalyst of the first, and rate-limiting, step in the metabolism of tryptophan (Trp) along the kynurenine (Kyn) pathway. Quinolinic acid (QA), a product of this pathway, is a neuro-excitotoxin, like glutamic acid (Glu) and aspartic acid (Asp). Kynurenic acid (KA), also produced from the Kyn pathway, antagonizes the neuro-excitotoxic effects of QA, Glu, and Asp. We therefore examined the possible roles of IDO, metabolites of the Kyn pathway, Glu, and Asp in the pathogenesis of fatal murine CM. Plasmodium bergbei ANKA infection was studied on days 6 and 7 post-inoculation (p.i.), at which time the mice exhibited cerebral symptoms such as convulsions, ataxia, coma, and a positive Wooly/White sign and died within 24 hours. A model for noncerebral malaria (NCM), P. bergbei K173 infection, was also studied on days 6 and 7 and 13 to 17 p.i. to examine whether any changes were a general response to malaria infection. Biochemical analyses were done by high-pressure liquid chromatography and gas chromatography/mass spectrometry/mass spectrometry (GC/MS/MS). IDO activity was low or absent in the brains of uninfected mice and NCM mice (days 6 and 7 p.i.) and was induced strongly in the brains of fatal murine CM mice (days 6 and 7 p.i.) and NCM animals (days 13 to 17 p.i.). This induction was inhibited greatly by administration of dexamethasone, a treatment that also prevented CM symptoms and death. Furthermore, IDO induction was absent in IFN-? gene knockout mice, which were also resistant to CM. Brain concentrations of Kyn, 3-hydroxykynurenine, and the neuro-excitotoxin QA were significantly increased in both CM mice on days 6 and 7 p.i. and NCM mice on days 13 to 17 p.i., whereas an increase in the ratio of brain QA to KA occurred only in the CM mice at the time they were exhibiting cerebral symptoms. Brain concentrations of Glu and Asp were significantly decreased in CM and NCM mice (days 13 to 17 p.i.). The results imply that neuro-excitation induced by QA may contribute to the convulsions and neuro-excitatory signs observed in CM.
  • Silvester, Julie A.; Timmins, Graham S.; Davies, Michael J.
    Archives of Biochemistry and Biophysics (Vol. 350/2) – 1998
    Porphyrin-sensitized photo-oxidation of bovine serum albumin results in oxidation at specific sites to produce protein radical species: at the Cys- 34 residue (to give a thiyl radical) and at one or both tryptophan residues (Trp-134 and Trp-214) to give tertiary carbon-centered radicals and cause disruption of the indole ring system. This study shows that these photooxidation processes also consume oxygen and give rise to hydrogen peroxide, protein hydroperoxides, and carbonyl functions. The yield of hydrogen peroxide, protein hydroperoxides, and carbonyl functions is shown to be dependent on illumination time, the nature of the sensitizer, and the concentration of oxygen; the yield of hydroperoxides can also be markedly diminished by the presence of a spin trap which reacts with the initial protein radicals. The mechanism of formation of the protein hydroperoxides is suggested to be primarily through type I processes (i.e., independent of singlet oxygen), while type II (singlet oxygen) mechanisms may play a significant role in protein carbonyl formation. Reaction of the protein hydroperoxide species with metal ion complexes is shown to produce further protein-derived radicals which are predominantly present on amino acid side chains.
  • Fu, Shanlin; Fu, Minxin; Baynes, John W.; Thorpe, Suzanne R.; Dean, Roger T.
    Biochemical Journal (Vol. 330/1) – 1998
    Glycation and subsequent Maillard or browning reactions of glycated proteins, leading to the formation of advanced glycation end products (AGEs), are involved in the chemical modification of proteins during normal aging and have been implicated in the pathogenesis of diabetic complications. Oxidative conditions accelerate the browning of proteins by glucose, and AGE proteins also induce oxidative stress responses in cells bearing AGE receptors. These observations have led to the hypothesis that glycation-induced pathology results from a cycle of oxidative stress, increased chemical modification of proteins via the Maillard reaction, and further AGE-dependent oxidative stress. Here we show that the preparation of AGE-collagen by incubation with glucose under oxidative conditions in vitro leads not only to glycation and formation of the glycoxidation product N?-(carboxymethyl)lysine (CML), but also to the formation of amino acid oxidation products on protein, including m-tyrosine, dityrosine, dopa, and valine and leucine hydroperoxides. The formation of both CML and amino acid oxidation products was prevented by anaerobic, anti-oxidative conditions. Amino acid oxidation products were also formed when glycated collagen, prepared under anti-oxidative conditions, was allowed to incubate under aerobic conditions that led to the formation of CML. These experiments demonstrate that amino acid oxidation products are formed in proteins during glycoxidation reactions and suggest that reactive oxygen species formed by redox cycling of dopa or by the metal-catalysed decomposition of amino acid hydroperoxides, rather than by redox activity or reactive oxygen production by AGEs on protein, might contribute to the induction of oxidative stress by AGE proteins.
  • Garner, Brett; Witting, Paul Kenneth; Waldeck, A. Reginald; Christison, Julie K.; Raftery, Mark J.; Stocker, Roland
    Journal of Biological Chemistry (Vol. 273/11) – 1998
    The lipids of high density lipoproteins (HDL) are initially oxidized in preference to those in low density lipoprotein when human plasma is exposed to aqueous peroxyl radicals. In this work we report on the relative susceptibility of HDL protein and lipid to oxidation and on the role HDL's ?-tocopherol (?-TOH) plays in modulating protein oxidation. Exposure of isolated HDL to either low fluxes of aqueous peroxyl radicals, Cu2+ ions, or soybean lipoxygenase resulted in the oxidation of apoAI and apoAII during the earliest stages of the reaction, i.e. after consumption of ubiquinol-10 and in the presence of ?-TOH. Hydro(pero)xides of cholesteryl esters and phospholipids initially accumulated together with specific oxidized forms of apoAI and apoAII, separated by high pressure liquid chromatography. The specific oxidized forms of apoAI were 16 and 32 mass units heavier than those of the native apolipoproteins and contained 1 and 2 methionine sulfoxide residues per protein, respectively. The third methionine residue in apoAI, as well as Trp residues, remained unoxidized during the earliest stages of HDL oxidation examined. Exposure of isolated apoAI to peroxyl radicals, Cu2+, or soybean lipoxygenase resulted in nonspecific (for peroxyl radicals) or no discernible protein oxidation (Cu2+ and soybean lipoxygenase). This indicated that the formation of the specific oxidized forms of apoAI observed with native HDL was not the result of direct reaction of i these oxidants with the apolipoprotein. In vitro and in vivo enrichment of HDL with ?-TOH resulted in a dose dependent increase in the extent of peroxyl radical- induced formation of HDL cholesteryl ester hydroperoxides (r = 0.96) and cholesteryl ester hydroxides (r = 0.92), as well as the loss of apoAI (r = 0.96) and apoAII (r = 0.94). ?-TOH enrichment also enhanced HDL lipid and protein oxidation induced by Cu2+ or soybean lipoxygenase. These results indicate that the earliest stages of HDL oxidation are accompanied by the oxidation of specific methionine residues in apoAI and apoAII and that in the absence of co-antioxidants, ?-TOH can promote this process.
  • McQuillan, Brendan M.; Hung, Joseph C.; Nidorf, Stefan Mark; Beilby, John P.; Thompson, Peter Lindsay
    Australian Journal of Medical Science (Vol. 18/4) – 1997
    Oxidauve modification of LDL-cholesterol is thought to be an important process in the early development of atherosclerosis. The evidence for a beneficial effect of antioxidant vitamins cardiovascular disease stems largely from biochemical investigations, experimental animal studies as well as observational epidemiological trials. Large-scale prospective intervention trials to test natural antioxidants in atherosclerosis, however, are awaiting completion. We determined if the dietary intake or plasma levels of anu'-oxidant vitamins was associated with the extent of atherosclerosis as assessed by carotid intima-media wall thickness (IMT) in a randomly selected adult population. We studied 1,100 subjects (548 males, 552 females) aged 52 13 (mean SD; range 26 - 76) years recruited from a random electoral roll survey. Dietary and supplemental vitamin intake of carotenoids, vitamin (vit) E and vit C were assessed by a validated semi-quantitative food-frequency questionnaire. Conventional vascular risk factors were also assessed. Fasting plasma levels of vit E, vit C, lycopene, a and B carotene were measured by HPLC. Bilateral carotid B-mode ultrasound was performed according to a standardized protocol. Vitamin supplement intake was more prevalent among females (29% versus 8% of males, p <0.01 for difference). Dietary vitamin intake was correlated with the respective plasma levels (r =0.32 0.34, all p <0.01). Carotid IMT was negatively correlated with dietary vit E (r = -0.15, p <0.01), plasma lycopene (r = -0.21, p <0.01). On multivariate analysis age, systolic BP, smoking, HDL-cholesterol and dietary vit E, but not supplement use or plasma vitamin levels, were found to be independent predictors of carotid IMT (R2 for model =0.51, p <0.001). In a random adult population dietary intake of vitamin E, but not other anti-oxidant vitamins or their plasma levels, was associated with the extent of carotid atherosclerosis after adjustment for standard risk factors.

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