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Showing 2041–2058 of 2058 publications.
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Jessup, Wendy K.; Darley-Usmar, Victor M.; O'Leary, Vanessa J.; Bedwell, S.The concentration-dependent effects of a series of lipoxygenase inhibitors and antioxidants on the macrophage-mediated oxidative modification of low-density lipoprotein (LDL) were measured. Their influence on macrophage 5-lipoxygenase pathway activity was also studied over the same concentration range. No correlation between inhibition of 5-lipoxygenase and of macrophage-mediated oxidation of LDL was observed. The capacity of the compounds to prevent cell-mediated modification of LDL could be explained in terms of their activity as either aqueous- or lipid-peroxyl radical scavengers. Two potent 5-lipoxygenase inhibitors (MK 886 and Revlon 5901), which had no radical-scavenging properties, were unable to block LDL modification. It is concluded that 5-lipoxygenase is not essential for LDL oxidation by macrophages.
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Iismaa, S. E.; Vquez, Ana Elena; Jensen, Gerard M.; Stephens, Philip J.; Butt, Julea N.; Armstrong, Fraser A.; Burgess, Barbara K.We have used site-directed mutagenesis to obtain two variants of Azotobacter vinelandii ferredoxin I (AvFdI), whose x-ray structures are now available. In the C20A protein, a ligand to the [4Fe-4S] cluster was removed whereas in the C24A mutant a free cysteine next to that cluster was removed. Like native FdI, both mutants contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. The structure of C24A is very similar to that of native FdI, while the structure of C20A is rearranged in the region of the [4Fe-4S] cluster to allow it to use the free Cys-24 as a replacement ligand. Here we compare the properties of the native, C20A, and C24A proteins. Although all three proteins are O<inf>2</inf> stable in vitro, the C20A protein is much less stable toward proteolysis than the other two in vivo. Spectroscopic results show that all three proteins exhibit the same general redox behavior during O<inf>2</inf>-oxidation and dithionite reduction. Electrochemical data show that the [3Fe-4S] clusters in all three proteins have the same pH-dependent reduction potentials (-425 mV versus SHE, pH 7.8), whereas the [4Fe-4S] cluster potentials vary over a ?150 mV range from -600 mV (C24A) to -647 mV (native) to -746 mV (C20A). Despite this variation in potential both the C20A and C24A proteins appear to be functional in vivo. Native FdI reacts with three equivalents of Fe(CN)<inf>6</inf>3- to form a paramagnetic species previously proposed to be a cysteinyl-disulfide radical. Neither the C20A nor the C24A variant undergoes this reaction, strongly suggesting that it involves the free Cys-24.
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Christen, Stephan; Peterhans, Ernst; Stocker, RolandThe antioxidant properties of tryptophan and some of its oxidative metabolites were examined by measuring how efficiently they inhibited peroxyl radical-mediated oxidation of phosphatidylcholine liposomes and B-phycoerythrin. Low micromolar concentrations of 5-hydroxytryptophan, 3-hydroxykynurenine, xanthurenic acid, or 3-hydroxyanthranilic acid, but not their corresponding nonhydroxylated metabolic precursors, scavenged peroxyl radicals with high efficiency. In particular, 3-hydroxykynurenine and 3-hydroxyanthranilic acid protected B-phycoerythrin from peroxyl radical-mediated oxidative damage more effectively than equimolar amounts of either ascorbate or Trolox (a water-soluble analog of vitamin E). Enzyme activities involved or related to oxidative tryptophan metabolism, as well as endogenous concentrations of tryptophan and its metabolites, were determined within tissues of mice suffering from acute viral pneumonia. Infection resulted in a 100-fold induction of pulmonary indoleamine 2,3-dioxygenase (EC 1.13.11.17) as reported [Yoshida, R., Urade, Y., Tokuda, M. & Hayaishi, O. (1979) Proc. Natl. Acad. Sci. USA 76, 4084-4086]. This was accompanied by a 16-and 3-fold increase in the levels of lung kynurenine and 3-hydroxykynurenine, respectively. In contrast, endogenous concentrations of tryptophan and xanthurenic acid did not increase and 3-hydroxyanthranilic acid could not be detected. The activity of the Superoxide anion (O<inf>2</inf>-)-producing enzyme xanthine oxidase increased 3.5-fold during infection while that of the O<inf>2</inf>--removing Superoxide dismutase decreased to 50% of control levels. These results plus the known requirement of indoleamine 2,3-dioxygenase for Superoxide anion for catalytic activity suggest that viral pneumonia is accompanied by oxidative stress and that induction of indoleamine 2,3-dioxygenase may represent a local antioxidant defence against this and possibly other types of inflammatory diseases.
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Wraight, Christopher J.; Endert, Peter Van; Moller, Peter; Lipp, Joachim; Ling, Noel R.; MacLennan, Ian C.M.; Koch, Norbert; Moldenhauer, GerhardClass II major histocompatibility complex antigens are intracellularly associated with a nonpolymorphic polypeptide referred to as the invariant chain. Before the class II heterodimer appears on the cell surface, the invariant chain dissociates but it has so far been unclear as to whether or not a proportion of the invariant chain also appears on the plasma membrane. We describe a study with three monoclonal antibodies which recognize an extracytoplasmic determinant present on all forms of the invariant chain and use them to demonstrate its presence on the surface of the intact cells. The determinants recognized by two of the antibodies were found to be located within the 60 amino acids at the extreme C-terminal (extracytoplasmic) end of the invariant chain. The invariant chain-specific monoclonal antibody, VIC-Y1, was found to bind a determinant located between amino acids 1 and 73, which correspond to mainly cytoplasmic residues. Using the C-terminal specific antibodies, the number of antibody binding sites on the surface of two B lymphoma lines was estimated to be 105 per cell. The results of this study appear to resolve the highly disputed question of whether or not the invariant chain can appear as a plasma membrane protein. The results are discussed in the context of a possible role for the invariant chain in antigen processing and presentation.
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Dawes, Joan; Freeman, L.; Dawson, Nicholas J.; Pepper, Duncan S.; Barrowcliffe, Trevor W.Abstract. The molecular?weight distribution of proteins in factor VIII concentrates has been analysed by fast?protein liquid chromatography. The proportion of high?molecular?weight (HMW) aggregates in one product increased on freeze?drying and heating, with fibrinogen and fibronectin being the main protein components of the HMW peak. In all other concentrates, the HMW peak was less than or equal to 5% of the total protein content and there were no differences in HMW content according to purity or method of viral inactivation. 1990 S. Karger AG, Basel
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Devery, Jannine M.; Geczy, Carolyn L.; Declarle, D.; Skerritt, John H.; Krillis, Steven A.Gluten and casein digests were tested for their ability to induce a cellular immune response on peripheral blood mononuclear cells (PBM) obtained from coeliac patients as well as healthy volunteers and disease control patients using the macrphage procoagulant assay. PBM from coeliac patients who had been on a gluten-free diet for less than 2 years responded strongly to gluten peptides, while coeliac patients at diagnosis or who had maintained a strict gluten-free diet for longer than 5 years showed weaker responses. PBM from healthy volunteers did not respond to gluten or casein peptides, whereas those from patients with Crohn's disease displayed weak reactivity to gluten and casein peptides. Our study using the macrophage procoagulant assay confirms previous findings that lymphoid cells from patients with coeliac disease exhibit a specific cellular immune response to gluten. This assay represents an alternative measure for cell-mediated immunity and is technically much simpler than the previously described leucocyte migration inhibition assay. Macrophage procoagulant activity is measured using a simple plasma recalcification time assay or spectrophotometrically using commercially available chromogenic substrates.
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Wheeler, Helen Ruth; Geczy, Carolyn L.Cisplatin, doxorubicin and daunorubicin (drugs which intercalate with DNA) influenced the membrane?bound procoagulant potential of murine thioglycollate?induced peritoneal exudate (TG?PEC) macrophages and the monocytoid cell line WEHI 265, whereas the antimetabolites 5?fluorouracil and methotrexate had no effect. Enhanced procoagulant was not caused by non?specific toxicity of these agents. Cisplatin directly increased the procoagulant expressed on WEHI 265 cells, whereas MPCA on TG?PEC was enhanced only when cisplatin was combined with a second stimulant, either bacterial lipopolysaccharide (LPS) or interferon (IFN?). WEHI 265 cells failed to respond to the anthracycline drugs, either alone or in combination with LPS, whereas they enhanced the IFN? response. Doxorubicin and daunorubicin increased the LPS response of TG?PEC by approximately 4?fold and the IFN? response by approximately 10?fold. Pulsing experiments suggested that the anthracyclines enhanced procoagulant expression by a mechanism different from cisplatin. Daunorubicin primed TG?PEC within 4 hr to respond to low levels of LPS, whereas either LPS or cisplatin primed these cells to respond to cisplatin or LPS respectively. Furthermore, the procoagulant expressed by TG?PEC stimulated by LPS/cisplatin had properties of tissue factor (TF: 50% total activity) and Factor VIIa (50% total procoagulant)?like activities, whereas the predominant procoagulant on LPS/anthracycline activated TG?PEC was TF?like (70% total activity) with weak Factor VIIa and prothrombinase?like properties. Copyright 1990 Wiley?Liss, Inc., A Wiley Company
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Stocker, RolandCells respond to metabolic perturbations by producing specific stress proteins. Exposure of mammalian cells to various forms of oxidative stress induces haem oxygenase, the rate-limiting enzyme in haem degradation. This response is proposed to represent an antioxidant defence operating at two different stages simultaneously. It (i) decreases the levels of the potential pro-oxidants haem and haem proteins such as cytochrome P-450 and protoporphyrinogen oxidase, and (ii) increases the tissue concentrations of antio-xidatively active bile pigments. 1990 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
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Kakakios, Alyson M.; Ryan, Jane; Geczy, Carolyn L.Inflammatory reactions involved in delayed-type hypersensitivity (DTH) are associated with extravascular coagulation and fibrin deposition. Heparin and other anticoagulants administered systemically inhibit DTH reactions but the direct effect of intradermally injected heparin on the development of DTH skin responses has not been reported. The effects of heparin on the DTH reaction elicited by ovalbumin (OVA) in guinea pigs 1-3 weeks after sensitization were examined. Unfractionated heparin, low affinity heparin (LAH; non-anticoagulant) and high affinity heparin (HAH; anticoagulant) were injected together with suboptimal amounts of OVA. Heparin and LAH enhanced skin induration, LAH (0.5 ?g) by an average of 50% above that due to OVA alone at 24 h (p < 0.01). In contrast, HAH (0.5 ?g) significantly reduced skin induration at 24 h. Heparin and LAH also significantly increased cellular infiltration with LAH having the greater effect. At 4 h the infiltrate consisted mainly of neutrophils whereas at 24 h mononuclear cells predominated. Fibrin deposition, assessed both by immunohistology and quantitation of radioactive fibrin extracted from skin test sites, was increased by 30% when OVA was tested in the presence of LAH. Mast cell heparin released locally at sites of DTH has the potential to modulate these reactions in either a pro- or anti-inflammatory manner. This study is the first to demonstrate differences in the capacities of LAH and HAH to modulate cell-mediated inflammation.
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Simpson, Jeremy A.; Dean, Roger T.The effect of a variety of proteins and amino acids was investigated on oxygen free radical activity as assessed by copper/hydrogen peroxide induced benzoate hydroxylation as well as copper-catalysed ascor-bate autoxidation. Serum albumins from a variety of species (human, bovine and dog) had both inhibitory and stimulatory effects depending on the molar copper to protein ratio; low ratios were inhibitory and high stimulatory. Some other proteins tested (lysozyme, soybean trypsin inhibitor and conalbumin) also had dual (inhibitory and stimulatory) effects, as did both histidine and polyhistidine, but all effects occurred at different molar ratios presumably dependent on the relative affinities for the copper ions. In contrast, metallolhioncin and cacruloplasmin, proteins specialised to bind copper in vivo had no stimulatory effects. In this paper we show that in addition to their fairly well documented inhibitory effects, under certain conditions some proteins also stimulate radical reactions. The possible role of this phenomenon in vivo is discussed. 1990 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
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Pease, Richard J.; Milne, Ross W.; Jessup, Wendy K.; Law, Adam; Provost, Pierre R.; Fruchart, Jean Charles; Dean, Roger T.; Marcel, Yves L.; Scott, James M.Bacterial expression of apolipoprotein (apo) B cDNA constructs has been used to map a series of monoclonal antibodies (mAbs) to apoB by immunoblotting. In some cases assignments have been confirmed and refined by (i) semipurification of expressed protein, CNBr digestion, and assignment of the immunoreactive fragments; (ii) controlled digestion of the cDNA with the exonuclease Bal31 and bacterial expression of the truncated proteins that result; or (iii) expression of specific segments of cDNA amplified by the polymerase chain reaction. Forty mAbs were mapped to a minimum of 17 separate determinants on apoB. Tryptic fragments have been used to confirm the epitope assignments. In addition, this approach in conjunction with immunoassay, enables some deductions to be made about the trypsin-accessible regions in low density lipoprotein (LDL). The cleavage pattern obtained predicts retention of structure in the cysteine-rich domain of the amino terminus and also in the LDL receptor binding region. Trypsinized LDL was shown to bind to the LDL receptor by an authentic process, using monoclonal antibodies as competing ligands. In conjunction with the previous paper (Milne, R. W., Theolis, R., Maurice, R., Pease, R. J., Weech, P. K., Rassart, E., Fruchart, J.-C., Scott, J., and Marcel, Y. L. (1989) J. Biol. Chem. 265, 19754-19760) the mapped mAbs have been used to define the receptor-binding domain of apoB100 in LDL.
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Prigent, Sally A.; Stanley, Keith K.; Siddle, KennethThe sequential epitopes on the human insulin receptor recognized by polyclonal and monoclonal antibodies were investigated using a recombinant DNA technique. Short random fragments of receptor cDNA were cloned and expressed in Escherichia coli as ?-galactosidase fusion proteins by using the expression vector pUEX1. Immunoreactive peptides were detected by colony blotting and identified by sequencing the corresponding cDNA inserts. Eleven antigenic determinants were located with rabbit antisera, nine of these being on the ?-subunit and two on the ?-subunit of which one was intracellular. Two human autoantibodies reacted with the ?-subunit on blots, but no sequential epitopes could be located. In the rabbit sera, antibody reacting with these linear epitopes represented a substantial fraction (approximately 50%) of antibody reacting with reduced denatured receptor on blots, but a generally smaller fraction (5-40%) of antibody reacting with solubilized native receptor. Epitope-specific subfractions of antibodies were purified by binding to and elution from bacterial fusion proteins. All of these subfractions reacted with denatured receptor on nitrocellulose blots, but only three precipitated native receptor (epitopes between amino acids 190 and 231, 654 and 669, 954 and 982) and none inhibited insulin binding. (The numbering system used in this manuscript is that of Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A., and Rutter, W. J. (1985) Cell 40, 747-758). The binding sites of two monoclonal antibodies were also determined. One of these antibodies (83-14) is insulin-mimetic, but inhibits insulin binding and its epitope on the ?-subunit (between amino acids 469 and 592) may contribute to the insulin binding site in the folded protein. The other antibody (18-44) binds close to the N terminus of the ?-subunit (amino acids 765-770) and does not inhibit insulin binding, but does mimic insulin action. The identification of epitopes therefore provides information on receptor conformation and allows structural domains to be identified which are involved in the functional effects of different antibodies.
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Dawes, JoanThe pharmacokinetics of enoxaparin (Clexane) and unfractionated heparin were compared by crossover study in healthy volunteers, using three different assays. After intravenous administration, unfractionated heparin was cleared with a half-life of 35 min, irrespective of assay methods. However, the concentration of enoxaparin, measured by competitive binding assay, declined with the longer half-life of 60 min, and its anti-Factor IIa and anti-Factor Xa activities had half-lives of 40 and 275 min, respectively. These data may reflect more rapid clearance of longer chain molecules with anti-Factor IIa activity, or release by enoxaparin of an endogenous compound with anti-Factor Xa activity. Following subcutaneous injection, the bioavailability of enoxaparin was 3-fold greater than that of unfractionated heparin; unlike unfractionated heparin, enoxaparin was almost completely absorbed. Peak plasma concentrations occurred 3 h after injection. The pharmacokinetic parameters of enoxaparin were not affected by dose; by contrast, the half-life of unfractionated heparin was highly dose-dependent. The pharmacokinetics of both unfractionated heparin and enoxaparin did not display circadian variation, and neither preparation crossed the human placenta.
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Jones, Alun; Geczy, Carolyn L.Interleukin-1 (IL-1) plays a major role in inflammatory responses. Activation of coagulation and fibrin deposition typical of these reactions is mediated by macrophage procoagulants induced on stimulated macrophages. IL-1 activity in the supernatant of lipopolysaccharide (LPS)-stimulated guinea-pig macrophages was markedly enhanced by the presence of thrombin during macrophage activation. Although thrombin alone had no effect, inclusion of 1 mU/ml of thrombin with suboptimal levels of LPS produced a 200-fold increase in IL-1 activity, and further enhancement was observed with increasing doses of thrombin. The active site of thrombin was necessary for enhancement, as the serine esterase inhibitor di-isopropyl-fluorophosphate (DIP) and hirudin inhibited the synergy observed with LPS and thrombin. Prothrombin and Factor Xa also enhanced IL-1 production, although not to the same extent as thrombin. Factor Xa-like activity was demonstrated on the surface of LPS-stimulated macrophages. Both the Xa-like activity and IL-1 generated by LPS-stimulated cells were inhibited by heparin. Heparin with a high affinity for antithrombin III (anti-coagulant heparin; HAH) inhibited IL-1 generation, whereas low-affinity heparin (non-anticoagulant; LAH) had no effect. We show that protease of the extrinsic coagulation cascade enhance IL-1 generation and propose that a Factor Xa-like activity present in activated macrophages, together with thrombin, may be important in IL-1 processing.
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Dawes, Joan; Hodson, Benjamin A.; Pepper, Duncan S.An iodinated derivative of dermatan sulphate was administered by the intravenous, subcutaneous and oral routes to healthy human volunteers in conjunction with unlabelled dermatan sulphate. Following intravenous injection clearance of radiolabel and concentration as measured by competitive binding assay were highly correlated and displayed complex kinetics which were not dose-dependent. Intact 125I-dermatan sulphate was absorbed following both subcutaneous and oral administration, though there appeared to be selective uptake by the gut of a subfraction comprising the smaller or less sulphated molecules. The intact material was subsequently excreted unchanged in the urine. Degradation products of dermatan sulphate were not detected by either gel filtration or affinity chromatography on Polybrene-Sepharose at any time in either plasma or urine, indicating that administered dermatan sulphate is not catabolised by man.
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Beisiegel, Ulrike; Weber, Wilfried; Ihrke, Gudrun; Herz, Joachim; Stanley, Keith K.The low-density-lipoprotein (LDL) receptor is a cell-surface pro-tein that plays an important part in the metabolism of cholesterol by mediating the uptake of LDL from plasma into cells1. Although LDL particles bind to the LDL receptor through their apolipoprotein B (apo B) and apolipoprotein E (apo E) moieties, other apo E-containing particles, like chylomicron remnants, are not dependent on the LDL receptor for uptake into cells. Chylomicrons formed in the intestinal mucosa during the absorption of the products of digestion, are processed by the peripheral circula-tion by lipoprotein lipase, which catalyses the breakdown of triglycerides in chylomicrons to free fatty acids and glycerol. The resulting chylomicron remnants, which are cholesterol-rich liproproteins, are subsequently taken up in the liver2-6. A second distinct protein that binds to apo E-containing lipoproteins, but not to LDL, has been proposed to be the receptor mediating the clearance of chylomicron remnants from the plasma. This protein has a relative molecular mass (M<inf>r</inf>) of 56,000 (56K) 7. More recent studies have failed, however, to establish whether this protein is a cell-surface receptor8. Here we describe crosslinking experiments in which apo E liposomes were found to bind specifically to the cell surface of hepG2 cells and to human liver membranes. The size and immunological cross-reactivity of the protein to which the liposomes bound was indistinguishable from that of the recently cloned and sequenced LDL-receptor-related protein, LRP9. We therefore conclude that the LRP might function as an apo E receptor. 1989 Nature Publishing Group.
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Milford, David V.; Taylor, Clare Mark; Afaat, F. R.; Halloran, E. A.; Dawes, J.[No abstract available]
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Dean, Roger T.; Simpson, Jeremy A.[No abstract available]
