Search
Showing 1721–1740 of 2058 publications.
-
Thomas, Shane R.; Stocker, RolandThe oxidation theory of atherosclerosis proposes that the oxidative modification of low-density lipoproteins (LDL) plays a central role in the disease. Although a direct causative role of LDL oxidation for atherogenesis has not been established, oxidized lipoproteins are detected in atherosclerotic lesions, and in vitro oxidized LDL exhibits putative pro-atherogenic activities. ?-Tocopherol (?-TOH; vitamin E), the major lipid-soluble antioxidant present in lipoproteins, is thought to be antiatherogenic. However, results of vitamin E interventions on atherosclerosis in experimental animals and cardiovascular disease in humans have been inconclusive. Also, recent mechanistic studies demonstrate that the role of ?-TOH during the early stages of lipoprotein lipid peroxidation is complex and that the vitamin does not act as a chain-breaking antioxidant. In the absence of co-antioxidants, compounds capable of reducing the ?-TOH radical and exporting the radical from the lipoprotein particle, ?-TOH exhibits anti- or pro-oxidant activity for lipoprotein lipids depending on the degree of radical flux and reactivity of the oxidant. The model of tocopherol-mediated peroxidation (TMP) explains the complex molecular action of ?-TOH during lipoprotein lipid peroxidation and antioxidation. This article outlines the salient features of TMP, comments on whether TMP is relevant for in vivo lipoprotein lipid oxidation, and discusses how co-antioxidants may be required to attenuate lipoprotein lipid oxidation in vivo and perhaps atherosclerosis. (C) 2000 Elsevier Science Inc.
-
Davies, Michael J.; Hawkins, Clare L.Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-,thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.
-
Thomas, Shane R.; Stocker, Roland[No abstract available]
-
Baoutina, Anna; Dean, Roger T.; Jessup, Wendy K.Murine and human macrophages rapidly decreased the level of cholesteryl ester hydroperoxides in low density lipoprotein (LDL) when cultured in media non-permissive for LDL oxidation. This process was proportional to cell number but could not be attributed to the net lipoprotein uptake. Macrophage- mediated loss of lipid hydroperoxides in LDL appears to be metal ion- independent. Degradation of cholesteryl linoleate hydroperoxides was accompanied by accumulation of the corresponding hydroxide as the major product and cholesteryl keto-octadecadienoate as a minor product, although taken together these products could not completely account for the hydroperoxide consumption. Cell-conditioned medium possessed a similar capacity to remove lipid hydroperoxides as seen with cellular monolayers, suggesting that the activity is not an integral component of the cell but is secreted from it. The activity of cell-conditioned medium to lower the level of LDL lipid hydroperoxides is associated with its high molecular weight fraction and is modulated by the availability of free thiol groups. Cell- mediated loss of LDL cholesteryl ester hydroperoxides is facilitated by the presence of ?-tocopherol in the lipoprotein. Together with our earlier reports on the ability of macrophages to remove peroxides rapidly from oxidized amino acids, peptides, and proteins as well as to clear selectively cholesterol 7-?-hydroperoxide, results presented in this paper provide evidence of a potential protective activity of the cell against further LDL oxidation by removing reactive peroxide groups in the lipoprotein.
-
McCrohon, Jane A.; Heather, Alison Kay; Nakhla, Shirley; Jessup, Wendy K.; Handelsman, David J.; Stanley, Keith K.; Celermajer, David S.Background - Male sex is an independent risk factor for the extent and severity of atherosclerosis. The influence of androgens on foam cell formation, a key event in atherogenesis, has not yet been investigated. Methods and Results - Primary human monocytes were allowed to differentiate into macrophages. RNA was then extracted from healthy male-donor (n=8) and premenopausal female-donor (n=8) macrophages, and message for the androgen receptor (AR) was examined by RT-PCR. There was a significantly higher level of AR mRNA in macrophages isolated from men than in those from women (0.640.06 versus 0.150.02 amol/?g total RNA; P<0.001). AR mRNA levels were similar in macrophages from postmenopausal and premenopausal women (P=0.16). The functional consequence of this sex difference was then explored. Lipid-loading studies were performed on male (n=9) macrophages treated with the androgen dihydrotestosterone (DHT) and/or the AR antagonist hydroxyflutamide. These showed that DHT caused a dose-dependent and receptor- mediated increase in macrophage cholesteryl ester content (10910%, 1173%, and 1204% for 4, 40, and 400 nmol/L DHT, respectively, as a percentage of control, P=0.002; 958% for DHT with hydroxyflutamide, P=0.58 versus controls). By contrast, there was no significant effect of androgen on lipid loading in female-donor macrophages (P>0.2 versus controls). Conclusions-Sex differences in androgen-mediated macrophage lipid loading may contribute to the greater prevalence and severity of atherosclerosis in men.
-
Edwards, Stacey L.; Watts, Ralph Neal; Richardson, Des RaymondThe iron-regulatory protein 1 (IRP1) regulates the expression of several molecules involved in iron (Fe) metabolism by reversibly binding to iron- responsive elements (IREs) in the untranslated regions (UTR) of particular mRNA transcripts. Several studies have indicated that nitrogen monoxide (NO) may influence IRP1 RNA-binding activity by a direct effect on the [4Fe-4S] cluster of the protein. It has also been suggested that NO may act indirectly on IRP1 by affecting the intracellular Fe pools that regulate the function of this protein [Pantopoulous et al. (1996) Mol. Cell. Biol. 16, 3781-3788]. There is also the possibility that NO may S-nitrosate sulfhydryl groups that are crucial for mRNA binding and decrease IRP1 activity by this mechanism. We have examined the effect of a variety of NO donors [e.g., S-nitroso-N- acetylpenicillamine (SNAP), spermine-NONOate (SperNO), and S- nitrosoglutathione (GSNO)] on IRP1 RNA-binding activity in both LMTK- fibroblast lysates and whole cells. In cell lysates, the effects of NO at increasing RNA-binding activity were only observed when cells were made Fe- replete. Under these circumstances, IRP1 contains an [4Fe-4S] cluster that was susceptible to NO. In contrast, when lysates were prepared from cells treated with the Fe chelator desferrioxamine (DFO), NO had no effect on the RNA-binding activity of IRP1. The lack of effect of NO under these conditions was probably because this protein does not have an [4Fe-4S] cluster. In contrast to the NO generators above, sodium nitroprusside (SNP) decreased IRP1 RNA binding when cells were incubated with this compound. However, SNP had no effect on IRP1 RNA-binding activity in lysates, suggesting that the decrease after incubation of cells with SNP was not due to S-nitrosation of critical sulfhydryl groups. Apart from the direct effect of NO on IRP1 in Fe- replete cells, we have shown that NO generated by SNAP, SperNO, and GSNO could also mobilize Fe from cells. When NO generation was induced in RAW 264.7 macrophages, an increase in IRP1 RNA-binding activity occurred but there was only a small increase in Fe release. Our results suggest that NO could activate IRP1 RNA-binding by two possible mechanisms: (1) its direct effect on the [4Fe-4S] cluster and (2) mobilization of 59Fe from cells resulting in Fe depletion, which then increases IRP1 RNA-binding activity.
-
Richardson, Des RaymondMelanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue with several characteristics in common with serum Tf. MTf is found at high levels in melanoma cells and previous studies have shown that MTf can bind Fe. In addition, Chinese hamster ovary cells transfected with MTf transport Fe from 59Fe-citrate at greater rates than control cells. However, the role of MTf in the Fe uptake process of human melanoma cells remains unknown. In the present study we have characterized the role of MTf in Fe uptake by SK- Mel-28 melanoma cells in order to understand its function. Initial studies examined whether modulation of intracellular Fe levels using the Fe chelator desferrioxamine (DFO) or the Fe donor ferric ammonium citrate (FAC) could change MTf mRNA levels. In contrast to transferrin receptor (TfR) mRNA that increased after exposure to DFO and decreased after incubation with FAC, there was no change in MTf mRNA levels. In addition, compared to control cells, there was no alteration of 125I-labelled anti-MTf mAb-binding in cells exposed to DFO or FAC, suggesting no change in the number of MTf sites. Further studies examined the ability of DFO and FAC to modulate Fe uptake from 59Fe-citrate which is bound by MTf. In contrast to the effect of DFO or FAC at increasing and decreasing Fe uptake from 59Fe-Tf, respectively, DFO had no influence on 59Fe-citrate uptake, whereas FAC markedly increased it. Collectively, these studies suggest that MTf is not regulated in a manner similar to the TfR in response to cellular Fe levels. MTf can be removed from the membrane by phosphatidylinositol-specific phospholipase C (PtdIns-PLC). Preincubation of melanoma cells with PtdIns-PLC reduced anti-MTf mAb binding to 3% of the control, while PtdIns-PLC only slightly reduced 59Fe uptake from 59Fe-citrate. These results suggest that MTf played only a minor role in Fe uptake from 59Fe-citrate by these cells. The expression of MTf mRNA (poly A+) was also examined in 50 human tissues and found to be markedly different to Tf mRNA or TfR mRNA. Surprisingly, MTf mRNA expression was widespread in normal tissues, and was observed at its highest levels in the salivary gland. In contrast to expectations, MTf mRNA expression was generally greater in adult than fetal tissues.
-
Raitakari, Olli T.; McCredie, Robyn J.; Witting, Paul Kenneth; Griffiths, Kaye A.; Letters, Jacinta M.; Sullivan, David R.; Stocker, Roland; Celermajer, David S.Oxidative modification of low-density lipoprotein (LDL) may cause arterial endothelial dysfunction in hyperlipidemic subjects. Antioxidants can protect LDL from oxidation and therefore improve endothelial function. Dietary supplementation with coenzyme Q (CoQ<inf>10</inf>) raises its level within LDL, which may subsequently become more resistant to oxidation. Therefore, the aim of this study was to assess whether oral supplementation of CoQ<inf>10</inf> (50 mg three times daily) is effective in reducing ex vivo LDL oxidizability and in improving vascular endothelial function. Twelve nonsmoking healthy adults with hypercholesterolemia (age 34 10 years, nine women and three men, total cholesterol 7.4 1.1 mmol/l) and endothelial dysfunction (below population mean) at baseline were randomized to receive CoQ<inf>10</inf> or matching placebo in a double-blind crossover study (active/placebo phase 4 weeks, washout 4 weeks). Flow-mediated (FMD, endothelium-dependent) and nitrate- mediated (NMD, smooth muscle-dependent) arterial dilatation were measured by high-resolution ultrasound. CoQ<inf>10</inf> treatment increased plasma CoQ<inf>10</inf> levels from 1.1 0.5 to 5.0 2.8 ?mol/l (p = .009) but had no significant effect on FMD (4.3 2.4 to 5.1 3.6 %, p = .99), NMD (21.6 6.1 to 20.7 7.8 %, p = .38) or serum LDL-cholesterol levels (p = .51). Four subjects were selected randomly for detailed analysis of LDL oxidizability using aqueous peroxyl radicals as the oxidant. In this subgroup, CoQ<inf>10</inf> supplementation significantly increased the time for CoQ<inf>10</inf>H<inf>2</inf> depletion upon oxidant exposure of LDL by 41 19 min (p = .04) and decreased the extent of lipid hydroperoxide accumulation after 2 hours by 50 37 ?mol/l (p = .04). We conclude that dietary supplementation with CoQ<inf>10</inf> decreases ex-vivo LDL oxidizability but has no significant effect on arterial endothelial function in patients with moderate hypercholesterolemia. (C) 2000 Elsevier Science Inc.
-
Fu, Shanlin; Wang, Hongjie; Davies, Michael J.; Dean, Roger T.The toxicity of hypochlorous acid (HOCl) generated from activated neutrophils has been associated with several pathological processes such as atherosclerosis. Formation of 3-chlorotyrosine (Cl-Tyr) has been used as a marker for assessing the involvement of HOCl in such processes. In this study, we aimed to investigate the formation of Cl-Tyr from reaction of HOCl with tyrosine (both free and peptide-bound) and the fate of Cl-Tyr under such conditions. Tyrosine, N-acetyltyrosine, bovine serum albumin, and human low density lipoproteins were incubated with a range of reagent hypochlorite concentrations for varying periods in 10 mM phosphate buffer (pH 7.4) at 22 C. The reaction products, and several biological samples, were hydrolyzed (in the case of proteins), isolated, and purified by high pressure liquid chromatography and characterized or quantitated by mass spectrometry and NMR. A significant amount of 3,5-dichlorotyrosine (diCl-Tyr) was obtained from the bovine serum albumin, low density lipoprotein, and some biological samples, in addition to Cl-Tyr, indicating that Cl-Tyr competes effectively for HOCl even when tyrosine is present in great excess. Cl-Tyr and diCl-Tyr were also formed from free tyrosine but then reacted further with HOCl. This finding differs from a claim in the literature that Cl-Tyr was not formed in such a system. The further reaction products of Cl-Tyr and diCl-Tyr with HOCl were elucidated as their corresponding mono- and dichlorinated 4- hydroxyphenylacetaldehydes. These results indicate the importance of assessing other products of HOCl action in addition to Cl-Tyr.
-
Raitakari, Olli T.; Adams, Mark R.; McCredie, Robyn J.; Griffiths, Kaye A.; Stocker, Roland; Celermajer, David S.Objectives. To test the hypothesis that antioxidant therapy would improve endothelial function in smokers. Background. Several studies have documented a beneficial effect of short-term oral or parenteral vitamin C on endothelial physiology in subjects with early arterial dysfunction. Possible long-term effects of vitamin C on endothelial function, however, are not known. Methods. We studied the effects of short- and long-term oral vitamin C therapy on endothelial function in 20 healthy young adult smokers (age 36 6 years, 8 male subjects, 21 10 pack-years). Each subject was studied at baseline, 2 h after a single dose of 2 g vitamin C and 8 weeks after taking 1 g vitamin C daily, and after placebo, in a randomized double-blind crossover study. Blood samples were analyzed for plasma ascorbate levels and endothelial function was measured as flow-mediated dilation of the brachial artery, using high resolution ultrasound. Nitroglycerin-mediated dilation (endothelium-independent) was also measured at each visit. Results. At baseline, plasma ascorbate level was low in the smokers (42 21 ?mol/liter; normal range, 50 to 150 ?mol/liter), increased with vitamin C therapy after 2 h to 120 54 ?mol/liter (p < 0.001) and remained elevated after eight weeks of supplementation at 92 32 ?mol/liter (p < 0.001, compared with placebo). Flow-mediated dilation, however, increased at 2 h (from 2.8 2.0% to 6.3 2.8%, p < 0.001), but there was no sustained beneficial effect after eight weeks (3.9 3.2%, p = 0.26). Nitroglycerin-mediated dilation was unchanged throughout. Conclusion. Oral vitamin C therapy improves endothelial dysfunction in the short term in healthy young smokers, but it has no beneficial long-term effect, despite sustained elevation of plasma ascorbate levels. (C) 2000 by the American College of Cardiology.
-
Edwards, Stacey L.; Richardson, Des RaymondThe natural resistance associated macrophage protein 2 (Nramp2) is a transporter that is involved in iron (Fe) uptake from transferrin (Tf) and low molecular mass Fe complexes. Here we describe the effect of the inflammatory mediators interferon-? (IFN-?) and lipopolysaccharide (LPS) on the expression of Nramp2 mRNA and Fe uptake by cells of the macrophage lineage. After incubation of the RAW264.7 macrophage cell line with LPS there was a sevenfold increase in the expression of the 2.3 kb Nramp2 mRNA transcript when compared with the control, but little effect on the Nramp2 3.1 kb transcript. These results indicate differential regulation of the two transcripts. Treatment with LPS resulted in an increase in 59Fe uptake from 59Fe-nitrilotriacetic acid, while transferrin receptor (TfR) mRNA levels and 59Fe uptake from 59Fe-Tf were decreased. Paradoxically, at the same time, an increase in iron regulatory protein (IRP)1 RNA-binding activity was observed. Incubation with IFN-? (50 U mL-1) resulted in a marked decrease in TfR mRNA levels but had no effect on Nramp2 mRNA expression. Exposure of RAW264.7 cells to both IFN-? and LPS resulted in a fourfold increase in the Nramp2 2.3-kb transcript and a four to fivefold decrease in the 3.1-kb transcript when compared with the control. Furthermore, there was a decrease in TfR mRNA levels despite an increase in IRP1 RNA-binding activity and a marked increase in inducible nitric oxide synthase mRNA expression. Hence, TfR and Nramp2 mRNA expression did not appear to be regulated in a concerted manner. Similar responses to those found above for RAW264.7 cells were also observed in the J774 macrophage cell line and also for primary cultures of mouse peritoneal macrophages. These results are of interest as the TfR and Nramp2 are thought to act together during Fe uptake from Tf. This is the first report to demonstrate regulation of the Nramp2 mRNA transcripts by inflammatory mediators.
-
Pattison, David I.; Lay, Peter Andrew; Davies, Michael J.The reductions of K<inf>2</inf>Cr<inf>2</inf>O<inf>7</inf> by catecholamines, DOPA, DOPA-?,?d<inf>2</inf>, N-acetyl-DOPA, ?-methyl-DOPA, dopamine, adrenaline, noradrenaline, catechol, 1,2-dihydroxybenzoic acid (DHBA), and 4-tert-butylcatechol (TBC), produce a number of Cr(V) electron paramagnetic resonance (EPR) signals. These species are of interest in relation to the potential role of oxidized proteins and amino acids in Cr-induced cancers. With excess organic ligand, all of the substrates yield Cr species with signals at g(iso) ~ 1.972 (A(iso)(53Cr) > 23.9 x 10-4 cm-1). These are similar to signals reported previously but have been reassigned as octahedral Cr(V) species with mixed catechol-derived ligands, [Cr(V)(semiquinone)<inf>2</inf>(catecholate)]+. Experiments with excess K<inf>2</inf>Cr<inf>2</inf>O<inf>7</inf> show complex behavior with the catecholamines and TBC. Several weak Cr(V) signals are detected after mixing, and the spectra evolve over time to yield relatively stable substrate-dependent signals at g(iso) ~ 1.980. These signals have been attributed to [Cr(O)L<inf>2</inf>]- (L = diolato) species, in which the Cr is coordinated to two cyclized catecholamine ligands and an oxo ligand. Isotopic labeling studies with DOPA (ring or side chain deuteration or enrichment with 15N), and simulation of the signals, show that the superhyperfine couplings originate from the side chain protons, confirming that the catecholamine ligands are cyclized. At pH 3.5, a major short-lived EPR signal is observed for many of the substrates at g(iso) ~ 1.969, but the species responsible for this signal was not identified. Several other minor Cr signals are detected, which are attributed (by comparison with isoelectronic V(IV) species) to Cr(V) complexes coordinated by a single catecholamine ligand (and auxiliary ligands e.g. H<inf>2</inf>O), or to [Cr(O)L<inf>2</inf>]- (L = diolato) species with a sixth ligand (e.g. H<inf>2</inf>O). Addition of catalase or deoxygenation of the solutions did not affect the main EPR signals. When the substrates were in excess (pH > 4.5), primary and secondary (cyclized) semiquinones were also detected. Semiquinone stabilization by Zn(II) complexation yielded stronger EPR signals (g(iso) ~ 2.004).
-
Panzenboeck, Ute; Kritharides, Leonard; Raftery, Mark J.; Rye, Kerry Anne; Stocker, RolandThe initial stage of oxidation of high density lipoproteins (HDL) is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three Met residues of apolipoprotein A-I (apoA-I) to Met sulfoxides (Met(O)). Formation of such selectively oxidized apoA-I (i.e. apoA-I<inf>+32</inf>) may affect the antiatherogenic properties of HDL, because it has been suggested that Met86 and Met112 are important for cholesterol efflux and Met148 is involved in the activation of lecithin:cholesterol acyl transferase (LCAT). We therefore determined which Met residues were oxidized in apoA-I<inf>+32</inf> and how such oxidation of apoA-I affects its secondary structure, the affinity for lipids, and its ability to remove lipids from human macrophages. We also assessed the capacity of discoidal reconstituted HDL containing apoA-I<inf>+32</inf> to act as substrate for LCAT, and the dissociation of apoA-I and apoA-I<inf>+32</inf> from reconstituted HDL. Met<inf>86</inf> and Met112 were present as Met(O), as determined by amino acid sequencing and mass spectrometry of isolated peptides derived from apoA-I<inf>+32</inf>. Selective oxidation did not alter the ?-helicity of lipid-free and lipid-associated apoA-I as assessed by circular dichroism, and the affinity for LCAT was comparable for reconstituted HDL containing apoA-I or apoA-I<inf>+32</inf>. Cholesteryl ester transfer protein mediated the dissociation of apoA-I more readily from reconstituted HDL containing apoA-I<inf>+32</inf> than unoxidized apoA- I. Also, compared with native apoA-I, apoA-I<inf>+32</inf> had a 2- to 3-fold greater affinity for lipid (as determined by the rate of clearance of multilamellar phospholipid vesicles) and its ability to cause efflux of [3H]cholesterol, [3H]phospholipid, and [14C]?-tocopherol from lipid-laden human monocyte- derived macrophages was significantly enhanced. By contrast, no difference was observed for cholesterol and ?-tocopherol efflux to lipid-associated apolipoproteins. Together, these results suggest that selective oxidation of Met residues enhances rather than diminishes known antiatherogenic activities of apoA-I, consistent with the overall hypothesis that detoxification of lipid hydroperoxides by HDL is potentially antiatherogenic.
-
Luxford, Catherine; Dean, Roger T.; Davies, Michael J.Exposure of individual histone proteins (H1, H2A, H2B, H3, or H4) and histone octamers (consisting of two molecules each of H2A, H2B, H3, and H4) to hydroxyl radicals, generated by ?-irradiation, in the presence of O<inf>2</inf> generates protein-bound hydroperoxides in a dose-dependent fashion; this is in accord with previous studies with other proteins. These histone hydroperoxides are stable in the absence of exogenous catalysts (e.g., heat, light, and transition metal ions), but in the presence of these agents decompose rapidly to give a variety of radicals which have been identified by EPR spin trapping. Histone hydroperoxide-derived radicals generated on decomposition of the hydroperoxides with Cu+ react with both pyrimidine and purine nucleobases. Thus, with uridine the histone hydroperoxide-derived radicals undergo addition across the C<inf>5</inf>-C<inf>5</inf> double bond of the pyrimidine ring to give cross-linked adduct species which have been identified by EPR spectroscopy. HPLC analysis of the products generated on reaction of histone hydroperoxide-derived radicals with 2'-deoxyguanosine, or intact calf thymus DNA, has shown that significant levels of the mutagenic oxidized DNA base 8- oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) are formed, with the yield dependent on the individual histone protein, the presence of hydroperoxide functions, and the concentration of metal ion. These studies demonstrate that initial oxidative damage to individual histone proteins or histone octamers can result in the transfer of oxidative damage to associated DNA via the formation and subsequent decomposition of protein hydroperoxides to reactive radicals, and provide a novel route for the formation of mutagenic lesions in DNA.
-
Gray, David F.; Bundgaard, Henning; Hansen, Peter S.; Buhagiar, Kerrie A.; Mihailidou, Anastasia Susie; Jessup, Wendy K.; Kjeldsen, Keld Per; Rasmussen, Helge H.Objectives: HMG CoA reductase inhibitors reduce cellular availability of mevalonate, a precursor in cholesterol synthesis. Since the cholesterol content of cell membranes is an important determinant of Na+-K+ pump function we speculated that treatment with HMG CoA reductase inhibitors affects Na+-K+ pump activity. Methods: We treated rabbits and rats for 2 weeks with the HMG CoA reductase inhibitor lovastatin and measured Na+-K+ pump current (I(p)) in isolated rabbit cardiac myocytes using the whole cell patch-clamp technique, K-dependent p-nitrophenyl phosphatase (p-NPPase) activity in crude myocardial and skeletal muscle homogenates, and vanadate- facilitated 3H-ouabain binding in intact skeletal muscle samples from rats. Results: Treatment with lovastatin caused statistically significant reductions in I(p), myocardial and skeletal muscle K-dependent p-NPPase activity and 3H-ouabain binding in the myocardium and skeletal muscle. The lovastatin-induced decrease in I(p) was eliminated by parenteral co- administration of mevalonate. However, this was not related to cardiac cholesterol content. Conclusions: Treatment with lovastatin reduces Na+-K+ pump activity and abundance in rabbit and rat sarcolemma. (C) 2000 Elsevier Science B.V.
-
Van Reyk, David M.; Sarel, Shalom; Hunt, Nicholas H.The capacity of three novel iron chelators, namely 1-[N-ethoxycarbonylmethylpyridoxylidenium]-2-[2'-pyridyl]hydrazine bromide (EPH), 1-[5'-bromosalicylidene]-2-[2''-pyridyl]hydrazine (BsPH), and 1-pyridoxylidene-2-[1'-phthalazyl]hydrazine dihydrochloride (PPhH), to inhibit the proliferation of mitogen-stimulated murine lymph node cells was examined in vitro. All three are of the aryl hydrazone class, the prototype of which is pyridoxal isonicotinoyl hydrazone. The chelators inhibited lymphoproliferation at low micromolar concentrations. EPH and PPhH had an inhibitory capacity comparable to that of desferrioxamine (IC<inf>50</inf>: 3 and 2 ?M, respectively), whereas BsPH was more potent (IC<inf>50</inf> < 1 ?M). The inhibitory effects of the chelator were not due to cell cytotoxicity and could be abrogated by pretreating the chelator with iron. Time-course studies established a site of action for the chelators at the G<inf>1</inf>/S phase transition. These agents warrant further investigation for their potential as immunosuppressants. Copyright (C) 2000 Elsevier Science Inc.
-
Brown, Andrew J.; Watts, Gerald F.; Burnett, John R.; Dean, Roger T.; Jessup, Wendy K.27-Hydroxycholesterol (27OH) is the major oxysterol in human atherosclerotic lesions, followed by 7-ketocholesterol (7K). Whereas 7K probably originates nonenzymically, 27OH arises by the action of sterol 27-hydroxylase, a cytochrome P450 enzyme expressed at particularly high levels in the macrophage and proposed to represent an important pathway by which macrophages eliminate excess cholesterol. We hypothesized and here show that 27-hydroxylated 7-ketocholesterol (270H-7K) is present in human lesions, probably generated by the action of sterol 27-hydroxylase on 7K. Moreover, [3H]27OH-7K was produced by human monocyte-derived macrophages (HMDMs) supplied with [3H]7K but not in HMDMs from a patient with cerebrotendinous xanthomatosis (CTX) shown to have a splice-junction mutation of sterol 27-hydroxylase. Whereas [3H]270H-7K was predominantly secreted into the medium, [3H]-27OH formed from [3H]-cholesterol was mostly cell-associated. The majority of supplied [3H]7K was metabolized beyond 27OH-7K to aqueous-soluble products (apparently bile acids derived from the sterol 27-hydroxylase pathway), Metabolism to aqueous-soluble products was ablated by a sterol 27-hydroxylase inhibitor and absent in CTX cells. Sterol 27-hydroxylase therefore appears to represent an important pathway by which macrophages eliminate not only cholesterol but also oxysterols such as 7K. The fact that 7K (and cholesterol) still accumulates in lesions and foam cells indicates that this pathway may be perturbed in atherosclerosis and affords a new opportunity for the development of therapeutic strategies to regress atherosclerotic lesions.
-
Sekyere, Eric Owusu; Richardson, Des RaymondMelanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue that is found at high levels in melanoma cells. MTf has many characteristics in common with serum Tf and previous studies have shown that it can bind Fe. This has led to speculation that MTf may be involved in Fe transport. Because Fe is required for a variety of metabolic reactions including ATP and DNA synthesis, MTf could play a role in proliferation. However, recently it has been shown that MTf plays very little role in Fe uptake by melanoma cells, and unlike other Fe transport molecules (e.g. the transferrin receptor), its expression is not controlled by Fe. In the present review the function of MTf is discussed in relation to data suggesting other roles apart from Fe uptake. Copyright (C) 2000 Federation of European Biochemical Societies.
-
Jessup, Wendy K.; Kritharides, LeonardOxidation products of lipids and proteins are found in atherosclerotic plaque and in macrophage foam cells. Macrophages avidly endocytose in-vitro oxidized LDL and accumulate sterols. What is the evidence that such a process is involved in in-vivo foam cell formation? The present review surveys current knowledge on the metabolism of oxidized LDL by macrophages, and the types, amounts and location of oxidation products that accumulate in these cells. Comparable studies of lesion lipoproteins and foam cells indicate that limited extracellular lipoprotein oxidation, perhaps followed by more extensive intracellular oxidation subsequent to uptake by macrophages, is a more likely scenario in vivo. (C) 2000 Lippincott Williams and Wilkins.
-
Bartlett, Anna L.; Grewal, Thomas; de Angelis, Elena M.; Myers, Simon J.; Stanley, Keith K.Desialylated low density lipoprotein (LDL) is rapidly taken up and accumulated by both peripheral blood monocytes and cells isolated from human arterial intima consisting predominantly of smooth muscle cells. It is shown that thioglycollate (TG)-elicited mouse macrophages and mouse peritoneal macrophages stimulated with lipopolysaccharide (LPS) show increased expression of a membrane-bound, galactose-specific lectin that could be responsible for this uptake. In LPS-stimulated macrophages accumulation of desialylated LDL is increased ca. 2.6-fold. Accumulation of acetylated LDL in the same cells is reduced, suggesting that the galactose-specific lectin might be responsible for the uptake of desialylated LDL. Transfection of cells with the mouse macrophage Gal/GalNAc-specific lectin (MMGL) increased their capacity to take up asialofetuin (ASF) and, to a smaller extent, desialylated LDL. The uptake of desialylated LDL was small, most likely due to the high k(d) of MMGL for biantennary oligosaccharides as found on LDL, and low concentration of LDL achieved in tissue culture experiments. The data suggest that the expression of galactose-specific lectins can be elevated under inflammatory conditions, and that these receptors could contribute to foam cell formation under conditions of high desialylated LDL concentration, as might be found in arterial intima. Copyright (C) 2000 Elsevier Science Ireland Ltd.
