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Showing 1941–1960 of 2058 publications.
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Raison, Robert L.; Hook, Jeffrey W.; Coverley, Jennifer A.; Newton, Rebecca A.; Geczy, Carolyn L.; Raftos, David Andrew[No abstract available]
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Malle, Ernst; Sattler, WolfgangUnder physiological conditions human blood platelets play a beneficial role in fibrinolysis and regulate the balance with prostacyclin and other factors derived from the endothelium. In response to endothelial injury, adherence of platelets to the denuded arterial surface, platelet aggregation, release of mitogens and subsequent cell proliferation characterize early fibrous plaque lesions. 'Native' atherogenic plasma lipoproteins which are abundant in hypercholesterolemia have been found to play a subtle role in the development of atherosclerosis. In addition, lipoproteins modulate platelet function and alter the susceptibility of platelets to different stimulating agents. The properties of 'modified' atherogenic lipoproteins also seem to be well documented with respect to atherogenesis. After uptake by macrophages, modified atherogenic plasma lipoproteins are thought to contribute to formation of fatty streak lesions. On the other hand, modified atherogenic lipoproteins may directly promote endothelial injury and thus favour enhanced endothelial-platelet interactions. However, the direct effects of modified atherogenic lipoproteins on platelet function have not been revealed in detail. Recent findings have documented that activated platelets themselves may promote modification of atherogenic plasma lipoproteins and thus contribute to enhanced foam cell formation. Therefore stimulation of thrombocytes, and their interaction with native and modified lipoproteins must be considered an important factor in the current concept of atherogenesis. 1994 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
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Zammit, Adrian; Dawes, JoanOrgaran is a LMW heparinoid composed of heparan sulphate (83% w/w) of which 4-5% has high affinity for antithrombin, dermatan sulphate (12% w/w) and chondroitin sulphate (5% w/w). To examine the contribution of the low-affinity fraction to Orgaran's antithrombotic activity we have quantitated the binding of plasma proteins to Orgaran and its component fractions in whole, hirudin-anticoagulated human plasma. Antithrombin, largely bound to the high-affinity fraction, and histidine-rich glycoprotein, interacting with low-affinity components, were the dominant proteins bound to Orgaran. Vitronectin, fibrinogen, fibronectin, heparin cofactor II, and apolipoprotein B were also detected in small amounts. The ratio of bound antithrombin, histidine-rich glycoprotein and vitronectin to GAG was negatively correlated with the Orgaran concentration in plasma, implying that the efficacy of Orgaran may not be linearly related to dose. Binding of antithrombin to the high-affinity fraction was not decreased by other plasma proteins or affected by addition of low-affinity material. Moreover, the antithrombin and anti-factor Xa activities of the high-affinity material were unaltered by low-affinity GAGs. On the basis of our results we conclude that the low-affinity material does not contribute to the antithrombotic activity of Orgaran by binding non-anticoagulant plasma proteins and releasing the high-affinity chains to interact with antithrombin and its target proteinases.
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Geczy, Carolyn L.This chapter describes the biology of the cellular activators of the extrinsic coagulation pathway. These procoagulants do not act in isolation. Upon stimulation of both Mos and endothelial cells, there is a concomitant upregulation of the antifibrinolytic system by decreased or unchanged plasminogen activator synthesis and increased production of inhibitors of plasminogen activator. Downregulation of procoagulant expression may occur via inhibitors, cytokines, and other agents which regulate gene transcription and translation, by availability of phospholipids and other cofactors, and by the physiochemical presentation of these factors within the cell membrane. Investigation in this complex area is likely to have important clinical relevance, particularly in the development of more specific and potent inhibitors, and should be extended to include the possible interactions of Tissue factor/factor VIIa/Mac-l/factor X on adhesive substrates and on the extracellular matrix (ECM). The sequestration of coagulation factors to the ECM may have additional functions in inflammation. Under these circumstances, their effects on cell migration and activation may be of major significance. 1994, Academic Press, Inc.
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Malle, Ernst; Ibovnik, Anton; Steinmetz, Armin; Kostner, Gerhard Max; Sattler, WolfgangLipoprotein(a) [Lp(a)] plays an important role in atherosclerosis. The amino acid sequence of apolipoprotein(a) [apo(a)] reveals an arginyl-glycyl-aspartate (RGD) tripeptide that is the consensus sequence for binding of adhesive plasma proteins of the fibrinolytic system, such as fibrinogen and von Willebrand factor, to the platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex. Therefore, we undertook the present study to further investigate the role of Lp(a) in hemostasis. Binding of 125I-Lp(a) to a single platelet membrane-associated protein (1376 kD) comigrating with platelet GPIIb (140 kD) was found to be specific, saturable, and Ca2+ independent. Binding of <inf>125</inf>I-Lp(a) to resting human blood platelets was saturable, insensitive to temperature, and independent of the apo(a) isoform (B, S1 through S3). Scatchard analysis revealed a K<inf>d</inf> of 7.21.80-9 mol/L, with 729313 Lp(a) molecules bound per platelet. Monoclonal anti-GPIIb IgG diminished Lp(a) binding by approximately 80%, monoclonal anti-GPIIb-IIIa IgG by 60%, and anti-GPIIIa IgG by just 15%. 125I-Lp(a) binding was competitively inhibited to the same extent by either unlabeled Lp(a) or fibrinogen. Low- and high-density lipoproteins were much weaker competitors. A polyclonal antibody raised against the RGDGQSYRGT sequence of apo(a) was used to verify the presence of an ROD sequence in the different Lp(a) preparations investigated. However, two lines of evidence indicated that the RGD sequence is not the binding domain mediating Lp(a) binding to platelets. First, incubation of platelets with isolated RGD tripeptide did not influence Lp(a) binding. Second, coincubation of 125I-Lp(a) with anti-CRGDGQSY-RGT IgG or anti-CRGDGQSYRGT IgG F(ab?)<inf>2</inf> fragments had no effect on the binding of 125I-Lp(a) to intact, resting platelets. These data strongly suggest that platelet GPIIb is the major Lp(a) binding protein, but binding of Lp(a) is apparently not mediated by the RGD peptide motif to GPIIb-IIIa.
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Kima, M. J.; Dawesb, J.; Jessup, Wendy K.Human umbilical vein endothelial cell monolayers were grown as monolayers on porous filters and their transcellular transport and degradation of 125I-labelled native and modified forms of LDL, supplied to either the intimal or the luminal face, were measured. Intact native, acetylated and oxidized LDL were all transported in both directions across the cell monolayers by receptor-independent mechanisms, and all forms of LDL were transported at similar rates. However, the mass of intact LDL transported from the intimal to the luminal face of the monolayer was always four-fold more than that transported in the opposite direction under similar conditions. In addition to LDL transport, endothelial cell monolayers also degraded native and modified forms of LDL by predominantly receptor-dependent routes, in that these could be inhibited ( > 70%) by the addition of a 20-fold excess of the same form of (but unlabelled) LDL. The measured amounts of lipoprotein degraded were the same whether supplied to the intimal or the luminal face. Incubation of endothelial cells with oxidized LDL led to intracellular accumulation of a pool of macromolecular apo B which was apparently resistant to lysosomal proteolysis. 1994.
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Ang, C.; Dawes, JoanThe effects of hyperthermia on potentially prothrombotic endothelial function were investigated by measuring levels of von Willebrand factor, thrombospondin, tissue plasminogen activator and plasminogen activator inhibitor-1 secreted by unstimulated human umbilical vein endothelial cells cultured at 37C, 39C, 41C and 43C for 24 h. Endothelial barrier function at 43C was compared with that at 37C by measuring permeability to radiolabelled human serum albumin and low density lipoprotein. Thrombospondin levels were unaffected by a temperature of 39C; they increased after 3 h at 41C and subsequently declined to values significantly below the 37C control. At 43C, secretion exhibited a time-dependent decrease. Secretion of von Willebrand factor was not discernibly affected by exposure to 39C or 41C. Its response to 43C resembled that of thrombospondin to 41C. In contrast, elevated temperatures markedly increased plasminogen activator inhibitor-1 while decreasing t-PA secretion, though after prolonged exposure to 43C the levels of both returned to control values. After 12-24 h at 43C, endothelial permeability to both albumin and low density lipoprotein increased markedly. Vascular endothelium may contribute to the thrombotic tendency associated with heat stroke by increasing access to the prothrombotic subendothelium and reducing fibrinolysis.
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Bolton, Elaine J.; Jessup, Wendy K.; Stanley, Keith K.; Dean, Roger T.Foam cells were produced in vitro by incubation of mouse peritoneal macrophages with acetylated or copper oxidized LDL. Nitric oxide synthesis was stimulated by exposure of the cells to IFNy and LPS. Nitric oxide production, detected by measurement of nitrite in the culture medium, was unchanged in Ac-LDL loaded cells as compared with non-loaded cells. However, Ox-LDL foam cells produced 68-99% less nitrite than non-loaded cells. Failure to detect nitric oxide synthase (NOS) products from macrophages previously loaded with Ox-LDL appeared to result from lack of NOS activity, as little active enzyme could be recovered from Ox-LDL loaded cells. However, addition of Ox-LDL to an active cell-free NOS preparation had no direct effect on enzymic activity. When native LDL was subsequently incubated with these various IFNy/LPS stimulated cells, cells pre-loaded with Ox-LDL promoted, on average, a 2-fold greater increase in oxidative modification of the LDL added than either non-loaded or Ac-LDL loaded cells. That is, there was an inverse correlation between NOS activity and the ability of the cells to promote LDL oxidation. Unstimulated Ox-LDL loaded foam cells also oxidized LDL better than unstimulated non-loaded or Ac-LDL loaded foam cells, and the extent of oxidative modification was generally greater than seen with the equivalent IFN?/LPS stimulated cells. This suggests that Ox-LDL loading also affects some additional factor(s) responsible for cell-mediated LDL oxidation. 1994.
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Vquez, Ana Elena; Shen, Binghui; Negaard, Kari; Iismaa, S. E.; Burgess, Barbara K.Azotobacter vinelandii has recently been used for a variety of genetic experiments which take advantage of its facile transformation system and its high-frequency homologous recombination. One gene that has been cloned and sequenced is the fdxA gene that encodes a small Fe-S protein called A. vinelandii ferredoxin I (AvFdI). Because this protein has been extensively characterized by X-ray crystallography and spectroscopic methods, we are using it as a model to address some general questions in Fe-S biochemistry. AvFdI is not a very abundant protein in wild-type cells, so to facilitate our biochemical studies we have developed the overexpression system described herein. The results show that AvFdI can be easily overproduced ca. 50-fold in its native background, by introducing multiple copies of the fdxA gene into A. vinelandii, on the broad-host-range multicopy plasmid, pKT23O. The protein can be expressed from its own constitutive promotor or from the controlled nifH promoter. The overproduced protein has no deleterious effects on the organism and is identical to the protein produced by wild-type cells. This A. vinelandii-hased system should be generally useful for the overproduction of other A. vinelandii proteins or for the expression of genes from thermophilic or other organisms with similarly high G-C contents, or for the expression of O<inf>2</inf>-sensitive metalloproteins that are unstable in other systems. 1994 Academic Press. All rights reserved.
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Stocker, RolandThe oxidation of LDL is now commonly implicated as an initiator of atherosclerosis and a standard in-vitro LDL 'oxidizability' test is required. This review will discuss current problems and advances that have been made in our understanding of the molecular mechanisms of radical-mediated LDL oxidation and antioxidation, how they relate to the in-vitro assessment of the 'oxidizability' of LDL and how they may be relevant to in-vivo LDL oxidation. Tocopherol-mediated peroxidation is used as a novel model of LDL lipid oxidation to discuss why terms such as 'lag time' are features of the in-vitro oxidation conditions, rather than being inherent to LDL oxidation per se. In addition, we will also cover why it is premature, at present, to use one particular LDL oxidizability test as a standard.
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Gaa, Bruno A.; Borthwick, Iain A.; Stanley, Keith K.We have sequenced a genomic DNA fragment containing the promoter and 5?-flanking region of the ?2MR/LRP. A cluster of five Sp1 sites situated over 600 base pairs away from the putative transcription start site doubles the activity of the promoter. A similar increase in activity was observed when this region was replaced by the SV40 enhancer, but the presence of both the cluster of Sp1 sites and SV40 enhancer gave no more transcription than either region alone. Within the previously described promoter region we have shown that only the most proximal Sp1 binding site influences transcription in CHO cells. The Sp 1 site situated 346 bp upstream of the putative transcription start site and previously described DNAse protection footprints had no effect on promoter activity in CHO cells. We also describe an NRF-1 binding site situated 143 bp upstream of the putative transcription start site. Deletion of the central 4 bp of this site caused a 60% decrease in transcription. No sterol regulatory (SRE-1) sites, used in the LDL receptor promoter for control of expression by cholesterol, were found in the ?2MR/LRP 5?-flanking region. However, one SRE-1 site was identified in the 5?-untranslated region of ?2MR/LRP. 1994.
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O'Connell, Anita M.; Gieseg, Steven Paul; Stanley, Keith K.When purified low density lipoprotein (LDL) or lipoprotein(a) (Lp[a]) was oxidized in vitro using concentrations of hypochlorite (50-500 ?M) which might be achieved by activated neutrophils in vivo, high molecular weight species were observed on SDS polyacrylamide gels. The reaction was concentration-, temperature- and time-dependent. The high molecular weight apoprotein complexes were resistant to heating in SDS and DTT, suggesting covalent, but non-disulfide bond, cross-linking. Negligible amounts of lower molecular weight degradation produts were formed. Bityrosine formation, measured by fluorescence and HPLC analysis, was found to increase with the amount of hypochlorite added. However, the molar concentration of bityrosine could not account for cross-linking, even if it was assumed that every bityrosine was intermolecular. Hypochlorite-oxidized Lp(a) and LDL were both effective as ligands for loading mouse peritoneal macrophages in vitro. We conclude that hypochlorite produced in inflammatory reactions might be important in the generation of atherogenic forms of lipoproteins. 1994.
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Burgess, James W.; Stanley, Keith K.We have measured the hepatobiliary transcytosis of asialofetuin (ASF) and low density lipoprotein (LDL) in rats following treatment with 5 mg of 17?- ethinylestradiol/kg of body weight for 3 days. The rate of appearance of both ligands in bile was increased 5-fold in estradiol-treated rats compared with controls. In contrast, no increase was observed in the transcytosis of dimeric IgA. The majority of the ASF recovered from the bile of estradiol- treated rats was undegraded, indicating transcytic delivery rather than lysosomal discharge. No increase in the rate of endocytosis of ASF was observed. When transcytosis of 125I-LDL was measured in the presence of excess ASF the rate decreased by approximately 70%. Moreover, sialidase treatment of LDL in vitro increased the biliary appearance of LDL 2-fold in estradiol-treated rats, suggesting that the effect of estradiol on both ligands was mediated via the asialoglycoprotein receptor. We conclude that 17?-ethinylestradiol increases the transcytosis of ASF and LDL through mechanisms that alter the intracellular trafficking of the asialoglycoprotein receptor.
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Posner, Barry I.; Faure, Robert L.; Burgess, James W.; Bevan, A. Paul; Lachance, Danielle; Zhang-Sun, Guiyi; Fantus, Ivan George; Ng, Jesse B.; Hall, David A.; Soo Lum, Bernadette; Shaver, Alan G.Twelve peroxovanadium (pV) compounds, each containing an oxo ligand, one or two peroxo anions, and an ancillary ligand in the inner coordination sphere of V, were synthesized, crystallized, and characterized by 51V NMR as >95% pure. These compounds activated the insulin receptor kinase (IRK) of cultured hepatoma cells, stimulated lipogenesis in adipocytes, and inhibited the in situ dephosphorylation of autophosphorylated IRs and epidermal growth factor receptors of rat liver endosomes. The phosphotyrosine phosphatase inhibitory and IRK activating potencies of these compounds were linearly correlated (r = 0.74; p < 0.003), decayed in parallel in solution, and varied considerably with the ancillary ligands within these compounds. In vivo administration activated rat liver IRK in parallel with its tyrosine phosphorylation. Co-administration of insulin plus pV was markedly synergistic in both respects. pV administration significantly decreased circulating insulin and plasma glucose concentrations; the latter to levels seen after a dose of insulin yielding ?50% occupancy of IRs in vivo. Two compounds (mpV(pic) and mpV(2,6-pdc)) displayed relative specificity as phosphotyrosine phosphatase inhibitors by inhibiting IR dephosphorylation to a significantly greater degree than epidermal growth factor receptor dephosphorylation. Thus, pV compounds are the most potent phosphotyrosine phosphatase inhibitors described to date. Their capacity to activate IRK appears to derive from their phosphotyrosine phosphatase inhibitory activity. Their hypoglycemic action is due to a direct tissue effect.
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Devery, Jannine M.; King, Nicholas J.C.; Geczy, Carolyn L.The murine S100 chemotactic protein of m.w. 10,000 termed (CP-10), has potent chemotactic activity for murine and human myeloid cells. We examined the ability of a synthetic CP-10 hinge region peptide CP-10((42-35)) and rCP- 10 to act as chemotactic agents and induce expression of the adhesion molecule Mac-1 (CD 11b/CD 18) in vivo. Maximal neutrophil (PMN) accumulation occurred between 2 to 8 h after mouse footpad injection of rCP-10 (10-7 M) or CP-10 peptide (10-6 M). The infiltrating PMN expressed high levels of Mac-1, and low levels of the murine L-selectin Mel-14. Injection of CP-10 peptide i.p. also induced infiltration of PMNs that expressed high levels of Mac-1. Cell suspensions obtained after i.p. injection of CP-10 peptide could be significantly inhibited from adhering to fibrinogen-coated plates when incubated with anti-Mac-1 antibody. The chemotactic activity of CP-10 peptide toward murine inflammatory PMN in vitro was also inhibited by anti-Mac-1 antibody. Neither CP-10 analogue stimulated or primed murine inflammatory or human blood neutrophils for superoxide production or granular enzyme release. The localization of CP-10 in vivo was examined using murine footpads injected with LPS and was found to be concentrated around the endothelial cells of the small blood vessels. This distribution suggests that the accumulated CP-10 may contribute to the generation of a chemotactic gradient.
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Dawes, Joan; James, Keith K.; Lane, David AnthonyA murine monoclonal antibody (MAb) raised against a covalent antithrombin-heparin complex was used to probe the conformational change resulting when the serpin antithrombin binds to heparin. This MAb completely inhibited the progressive activity of antithrombin against thrombin. However, although the MAb remained bound to antithrombin in the presence of heparin, it did not significantly inhibit heparin cofactor activity against thrombin, and increasing concentrations of the antithrombin-binding pentasaccharide progressively unblocked the inhibitory action of the MAb. The MAb bound to antithrombin without affecting either heparin-binding affinity or heparin-induced fluorescence enhancement, and it did not convert antithrombin from inhibitor to substrate. The MAb failed to interact with reduced and S-carboxymethylated antithrombin, indicating the conformational nature of its epitope. Antithrombin variants with N-terminal substitutions (Arg47?Cys or His, Leu99?Phe, Arg 129?Gln) modifying heparin binding, and C-terminal substitutions affecting the reactive site (Arg393?Cys) or resulting in substrate-variant antithrombin (Ala384?Pro), were all recognized normally, as were normal reactive site cleaved antithrombin and the thrombin-antithrombin complex. However, interaction of the MAb with antithrombin was reduced by several substitution mutations (Phe402?Cys, Phe402?Ser, Phe402?Leu, Ala404?Thr, Pro407?Thr) in the 402407 sequence which codes for amino acid residues of strand 1C and the polypeptide leading to strand 4B. Pro429?Leu also blocks recognition [Olds et al. (1992) Blood 79, 12061212], and this residue is believed to be spatially approximated to strand 1C. These observations support the concept of a mobile reactive loop which is conformationally linked to the heparin-binding site, but is independently mobile of the adjacent strand 1C/4B region. When heparin binds to antithrombin, spatial separation of a thrombin-binding site and the 1C/4B region appears to increase. 1994, American Chemical Society. All rights reserved.
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Mander, Erin L.; Dean, Roger T.; Stanley, Keith K.; Jessup, Wendy K.We have studied the intracellular fate of the apolipoprotein B of copper-oxidized LDL in cultured J774 macrophages, using subcellular fractionation and immunofluorescence techniques. The oxidized apolipoprotein B, using cell fractionation, was located primarily in secondary lysosomes (identified using the lysosomal marker-enzyme aryl sulfatase). Light microscopy using antibodies to the mannose-6-phosphate receptor, the lysosomai membrane protein lgp 120, and oxidized LDL (biotinylated) confirmed that apo B of oxidized LDL did accumulate in secondary lysosomes rather than in endosomes. We conclude from these results that the oxidized apolipoprotein B of LDL reaches the secondary lysosomes, but is not efficiently degraded, leading to intracellular accumulation within this compartment. If this occurs in vivo it may influence the physiology of the macrophage and their subsequent roles in forming foam cells and the development of the fatty streaks of early atherosclerosis. 1994.
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Thomas, Shane R.; Mohr, Detlef; Stocker, RolandIndoleamine 2,3-dioxygenase (IDO) and nitric oxide synthase are part of the anti-tumor and antimicrobial activities of mononuclear phagocytes induced by interferon-? (IFN?). As IDO is a heme-containing enzyme and NO, the product of nitric oxide synthase-initiated arginine degradation, is a regulator of heme enzymes, we investigated whether NO is capable of modulating IDO activity in IFN?-primed mononuclear phagocytes. Authentic NO gas or the NO-generating compound, diethylamine dinitric oxide adduct, dose-dependently inhibited IDO activity in cell lysates prepared from IFN?-primed human peripheral blood mononuclear cells, as assessed by the ascorbate/methylene blue assay for IDO. In contrast, neither nitrite nor nitrate affected IDO activity. Exposure of intact IFN?-primed human peripheral blood mononuclear cells or monocyte-derived macrophages to any of the NO-generating compounds, sodium nitroprusside, glyceryl trinitrate, S-nitroso-N-acetylpenicillamine, or diethylamine dinitric oxide adduct, resulted in inhibition of both the consumption of tryptophan from and formation of its metabolite, kynurenine, in the culture medium. The observed inhibition of IDO activity was not due to toxicity of the NO generators and was abrogated by the co-addition of oxyhemoglobin, an antagonist of NO function. Comparable concentrations of nitrite or nitrate did not inhibit IDO activity in intact cells. In contrast to human cells, addition of IFN? to murine macrophages, cultured in complete RPMI 1640 medium, readily induced nitric oxide synthase. Others have reported that such treatment does not induce IDO activity in these cells. However, induction of IDO activity was observed in murine macrophages when the synthesis of reactive nitrogen species was inhibited, by using arginine-free medium and/or the nitric oxide synthesis inhibitor, NG-monomethyl-L-arginine. Together, these results demonstrate that both exogenous and endogenous NO inhibit IDO activity and that oxidative arginine and tryptophan metabolism in IFN?-primed mononuclear phagocytes are functionally related. Our study thereby provides an insight into how these cells may regulate some of their antimicrobial and anti-tumor activities.
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Vassiliou, Gerard G.; Stanley, Keith K.We have investigated the proposal that the receptor-associated protein (RAP) of the low density lipoprotein receptor-related protein/?<inf>2</inf>- macroglobulin receptor binds to heparan sulfate proteoglycans (HSP). 125I- RAP binds to two sites on the surface of fibroblasts as follows: a high affinity site with a K(d) of 1.4 nM and a low affinity site (K(d) = 188 nM) with a capacity of more than 1000-fold the maximum amount of lipoprotein receptor-related protein/?<inf>2</inf>-macroglobulin receptor on the cell surface. 125I-RAP binding to the low affinity site was abolished by heparin or Suramin. However, maximal digestion of the glycosaminoglycan chains of HSP with heparinase or culturing the cells in chlorate, an inhibitor of proteoglycan sulfation, did not affect the binding of 125I-RAP or of 125I-labeled, methylamine-activated ?<inf>2</inf>-macroglobulin. Comparison of 125I-RAP degradation at two different concentrations suggests that the low affinity, high capacity site on the surface of human fibroblasts participates in the endocytosis of 125I-RAP. The nature of the low affinity site remains to be elucidated, but we can exclude the glycosaminoglycan chains of HSP.
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Neuil, Ji? Stocker, RolandPeroxidation of the lipid moieties of low density lipoproteins (LDL) is regarded as an early event in atherogenesis. Because bilirubin is a physiological reductant with antioxidant activities, we investigated its inhibitory action on the radical-mediated oxidation of LDL and plasma lipids. Exposing fresh human blood plasma to lipophilic peroxyl radicals generated from 2,2?-azobis(2,4-dimethylvaleronitrile) (AMVN) resulted in rapid oxidation of ubiquinol-10, followed by that of ascorbate and bilirubin. Plasma lipids were well protected from peroxidation as long as these three antioxidants were present, as assessed by the amounts of cholesterylester hydroperoxides formed during this period. Following consumption of these antioxidants, and in the presence of ?-tocopherol, the rate of hydroperoxide formation increased sharply with roughly 2 molecules of cholesterylester hydroperoxides being formed for each peroxidation initiating event. Supplementation of AMVN-oxidizing plasma with exogenous bilirubin at the onset of rapid lipid peroxidation, i.e. after depletion of endogenous ubiquinol-10, ascorbate, and bilirubin, led to a halt in both hydroperoxide formation and consumption of a-tocopherol. When isolated LDL was incubated with AMVN, ?9 molecules of cholesterylester hydroperoxides were formed per peroxidation initiating event and while ?-tocopherol was consumed. Addition of free or albumin-bound bilirubin to isolated LDL at the onset of oxidation resulted in a strong inhibition of hydroperoxide formation and ?-tocopherol consumption, the effect being more pronounced with the free pigment. Addition of the corresponding amounts of albumin alone was without effect. In the presence of albumin-bound bilirubin, some 30% of the pigment was initially converted into biliverdin, whereas formation of this oxidation product was not observed with the free pigment. Also, the presence of bilirubin oxidase partially reversed the inhibitory activity of bilirubin on AMVN-induced LDL oxidation in the absence but not presence of albumin. An attenuation of hydroperoxide formation and a temporary increase in LDL's ?-tocopherol concentration were observed when free- or albumin-bound bilirubin were added to AMVN-oxidizing, ?-tocopherol-containing LDL. In contrast, hydroperoxide formation was not inhibited significantly when the albumin-bound pigment was added to oxidizing LDL after complete consumption of its ?-tocopherol. Our results show that bilirubin inhibits oxidation of LDL lipids initiated within the lipoprotein core and indicate that this activity is mediated by interaction of the pigment with LDL's ?-tocopherol.
