The structure of EXTL3 helps to explain the different roles of bi-domain exostosins in heparan sulfate synthesis.

Heparan sulfate is a highly modified O-linked glycan that performs diverse physiological roles in animal tissues. Though quickly modified, it is initially synthesised as a polysaccharide of alternating ß-D-glucuronosyl and N-acetyl-α-D-glucosaminyl residues by exostosins. These enzymes generally possess two glycosyltransferase domains (GT47 and GT64)-each thought to add one type of monosaccharide unit to the backbone. Although previous structures of murine exostosin-like 2 (EXTL2) provide insight into the GT64 domain, the rest of the bi-domain architecture is yet to be characterised; hence, how the two domains co-operate is unknown. Here, we report the structure of human exostosin-like 3 (EXTL3) in apo and UDP-bound forms. We explain the ineffectiveness of EXTL3's GT47 domain to transfer ß-D-glucuronosyl units, and we observe that, in general, the bi-domain architecture would preclude a processive mechanism of backbone extension. We therefore propose that heparan sulfate backbone polymerisation occurs by a simple dissociative mechanism.

The bionomics of an invasive species Sitona lepidus during its establishment in New Zealand.

The egg, larval, pupal and adult abundance of the clover root weevil Sitona lepidus Gyllenhal (Coleoptera: Curculionidae) was monitored at three sites for the first ten years following the discovery of this exotic pest in the Waikato region of New Zealand. The species went through an initial boom and bust cycle at two sites, with populations reaching up to 1800 larvae m-2. Thereafter, winter larval populations were relatively stable, ranging between 450-750 m-2. Unlike in the Northern Hemisphere, S. lepidus was found to have two generations a year in the Waikato region of New Zealand. Pasture white clover content at the time of peak adult numbers was positively related to the subsequent peak larval populations for each generation. The factors contributing to the emergence of S. lepidus as one of the most important pasture pests in New Zealand are discussed.

Changes in vimentin in human macrophages during apoptosis induced by oxidised low density lipoprotein.

Macrophage apoptosis contributes to the development of human atherosclerotic lesions. Oxidised LDL may be involved in macrophage death in vivo. We examined morphological and biochemical changes to the vimentin filament network during apoptosis of human macrophages. Only oxidised LDL, but not native or acetylated LDL, induced apoptosis, wherein vimentin was cleaved into fragments of 48-50, 46, 29 and 26 kDa. The use of caspase inhibitors suggested that caspase-6 mediates the formation of the 26 and 46 kDa fragments of vimentin. We were unable to demonstrate any significant involvement of caspase-3 in vimentin cleavage. However, caspase-3 was clearly activated during apoptosis whilst caspase-6 expression in macrophages was minimal. Vimentin filament breakdown occurred early during apoptosis and vimentin immunoreactivity was present in apoptotic bodies. However, the application of caspase inhibitors had no effect on the morphology of the vimentin network in apoptotic cells, suggesting that filament breakdown is not mediated by caspase proteolysis. Similar changes in vimentin were also seen in gliotoxin-induced apoptosis.

Macrophage death and the role of apoptosis in human atherosclerosis.

The arterial disease atherosclerosis is responsible for severe morbidity and is the most common cause of death in the Western population. The complete pathogenesis of the disease is unknown, but multiple risk factors have been identified that correlate with the development of its complications such as heart attack and stroke. Evidence suggests that atherosclerosis is an inflammatory disease and the major cell types involved are smooth muscle cells, macrophages, and T lymphocytes. In this paper, we review the function of macrophages in the context of atherosclerosis and we also discuss the role and significance of macrophage death, including apoptosis. There is much evidence, certainly in vitro, suggesting that low-density lipoprotein becomes atherogenic when it undergoes cell-mediated oxidation within the artery wall. Besides inducing apoptosis in vitro, oxidized low-density lipoprotein may also cause extensive DNA damage in intimal cells, which might presage apoptosis. We review the results of experimental and clinical studies, which may indicate how the complications of atherosclerosis could be prevented by using different therapeutical strategies including bone marrow transplantation and gene therapy.

Changes in elemental concentrations are associated with early stages of apoptosis in human monocyte-macrophages exposed to oxidized low-density lipoprotein: an X-ray microanalytical study.

This study examines ion homeostasis in monocyte-macrophages committed to death by apoptosis. X-ray microanalysis has been used to demonstrate that intracellular concentrations of potassium decreased whilst those of sodium increased following 3 h of exposure to 100 microg/ml of oxidized low-density lipoprotein (LDL) in vitro. In contrast, the maximal incidence of cell death, as determined by the inability to exclude trypan blue, was not seen until 24 h of exposure. At 12 h, less than 1 per cent of cells were stained using terminal transferase-mediated DNA nick-end labelling, which is generally accepted as a marker of late stages in the apoptotic pathway. This is the first demonstration of early perturbations of ion homeostasis in monocyte-macrophages exposed to concentrations of oxidized LDL known to cause apoptosis.

Carotenoids inhibit DNA synthesis in human aortic smooth muscle cells.

Quiescent, serum-starved human aortic smooth muscle cells were restimulated with 20% foetal calf serum in Dulbecco's modified Eagle medium, in the presence and absence of beta-carotene, canthaxanthin, zeaxanthin, lycopene, lutein or beta-cryptoxanthin, at final concentrations up to 23 microM. Concentration-dependent inhibition of DNA synthesis, measured by [methyl-3H]thymidine incorporation, was observed for the carotenoids, except for canthaxanthin and lutein which had no effect. Lycopene was the most potent of the carotenoids tested. The results suggest that antiproliferative effects of dietary carotenoids might be of significance in vivo.

Glutathione (GSH) and the toxicity of oxidised low-density lipoprotein to human monocyte-macrophages.

Macrophage death, believed to be an important event in the pathogenesis of human atherosclerosis, can be induced by oxidised low-density lipoprotein (LDL) in vitro. Supplementation of the culture medium with 5 mM GSH significantly protected human monocyte-macrophages in vitro against the toxicity of copper-oxidised LDL. Oxidation products of LDL include the aldehyde 4-hydroxynonenal (HNE). We present evidence that conjugation of HNE by GSH contributes to this protection. In the absence of cells, HPLC analysis showed there were marked reductions in the levels of both pure HNE and HNE in copper-oxidised LDL in the presence of GSH. However, GSH did not reverse protein modification, as judged by agarose gel electrophoresis, nor did it influence the depletion of polyunsaturated fatty acids, which were assessed using gas chromatography. The possible implications for human atherosclerosis are discussed.

Toxicity of polyunsaturated fatty acid esters for human monocyte-macrophages: the anomalous behaviour of cholesteryl linolenate.

We have investigated the toxicity to human monocytemacrophages, and susceptibility to oxidation, of different individual dietary fatty acids in cholesterol esters and triglycerides, added to the cell cultures as coacervates with bovine serum albumin. Toxicity was assessed using release of radioactivity from cells preloaded with tritiated adenine. Lipid oxidation was measured by gas chromatography (GC). The triglycerides showed a direct relationship between toxicity and increasing unsaturation, which in turn correlated with increasing susceptibility to oxidation. Triolein (18:1; omega-9) and trilinolein (18:2; omega-6) were non-toxic. Trilinolenin (18:3; omega-3) was toxic only after prolonged incubation. Triarachidonin (20:4; omega-6), trieicosapentaenoin (20:5; omega-3) and tridocosahexaenoin (22:6; omega-3) were profoundly and rapidly toxic. There was a similar relationship between toxicity and increasing unsaturation for most of the cholesterol esters, but cholesteryl linolenate was apparently anomalous, being non-toxic in spite of possessing three double bonds and being extensively oxidised. Probucol and DL-alpha-tocopherol conferred protection against the toxicity of cholesteryl arachidonate and triarachidonin. The oxidation in these experiments was largely independent of the presence of cells. GC indicated that formation of 7-oxysterols might contribute to the toxicity of cholesteryl linoleate. The toxicity of triglycerides suggests that polyunsaturated fatty acid peroxidation products are also toxic. Possible mechanisms of cytotoxicity and relevance to atherosclerosis are discussed.

Cell death in atherosclerotic plaques.

The investigation of the mechanisms of cell death in atherosclerosis has recently received added impetus with the realization that apoptosis is probably the predominant mechanism. This review examines the preliminary data on mechanisms of cell death in the atherosclerotic plaque.

Apoptosis in human monocyte-macrophages exposed to oxidized low density lipoprotein.

This study has demonstrated the toxicity to human monocyte-macrophages of low-density lipoprotein (LDL) which had been artificially oxidized using copper sulphate. The assays of cell damage used were tritiated adenine release, neutral red staining, lactate dehydrogenase leakage, and MTT dye reduction. Toxicity was concentration- and time-dependent. Exposure to native LDL under the same conditions did not result in toxicity. Transmission electron microscopy of cells exposed to oxidized LDL showed characteristic changes of apoptosis, including chromatin condensation and a decrease in cell volume. There was extensive loss of cell surface protrusions and evidence of the phagocytosis of apoptotic cells by neighbouring monocyte-macrophages. Apoptotic features preceded the increased membrane permeability revealed by the release of radioactivity from cells preloaded with tritiated adenine and by lactate dehydrogenase leakage. DNA fragmentation was indicated by nick end-labelling using the terminal transferase enzyme (TUNEL). The number of TUNEL-positive cells was markedly greater in cells exposed to oxidized LDL, compared with those incubated as no-additions controls. Inhibition of de novo protein synthesis with cycloheximide and of Ca2+/Mg(2+)-activated endonuclease activity with aurintricarboxylic acid or zinc ion did not inhibit the toxicity produced by oxidized LDL.

Cytotoxic and chemotactic potencies of several aldehydic components of oxidised low density lipoprotein for human monocyte-macrophages.

We have investigated the cytotoxic and chemotactic potencies of malondialdehyde (MDA), hexanal, 4-hydroxyhexenal (HHE), 4-hydroxynonenal (HNE) and 4-hydroxyoctenal (HOE), which are aldehydes found in oxidised low density lipoprotein (LDL), for human monocyte-macrophages. They were toxic in the following order: hexanal

Oxidation of low-density lipoprotein by human monocyte-macrophages results in toxicity to the oxidising culture.

Human monocyte-macrophage cultures were exposed to native low density lipoprotein (LDL) for up to 24 h in Ham's F10 medium and the extent of cell-mediated LDL oxidation was determined by measurement of electrophoretic mobility on agarose gels and measurement of lipids and oxidised lipids (including 7 beta-hydroxycholesterol) by GC. After an initial lag phase, which varied from 2-8 h, there was a steady increase in oxidation over 24 h. No-cell control incubations showed minimal increases in oxidation over 24 h. Significant toxicity, measured as release of radioactivity from macrophages pre-loaded with tritiated adenine, was observed in the cells when they oxidised LDL and the extent of radioactivity release correlated closely with the extent of LDL oxidation. Inhibition of oxidation using alpha-tocopherol or probucol reduced toxicity within the oxidising culture. This self-inflicted toxicity may help to explain the origin and enlargement of the lipid core of advanced atherosclerotic lesions.

Toxicity of oxysterols to human monocyte-macrophages.

We have investigated the toxicity of the cholesterol oxidation products (oxysterols), 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, 7-ketocholesterol, 25-hydroxycholesterol and 26-hydroxycholesterol to human monocyte-macrophages in vitro. The 7-position derivatives are present in low density lipoprotein (LDL) oxidised with copper (II) sulphate and macrophages, and in extracts of human atherosclerotic lesions, which also contain 26-hydroxycholesterol. We have also assessed 25-hydroxycholesterol for toxicity because it has often been used in studies of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition and LDL receptor down-regulation. Measurement of radioactivity release from monocyte-macrophages preloaded with tritiated adenine, as a means of assessing cytotoxicity that all the oxysterols showed time- and concentration-dependent toxicity. The cytotoxic potency of 26-hydroxycholesterol was the greatest. The 7-position derivatives also produced marked cell damage, though at higher concentrations than for 26-hydroxycholesterol. Of the oxysterols assessed, the toxicity of 25-hydroxycholesterol was the least. The cytotoxicity of 7 beta-hydroxycholesterol and 26-hydroxycholesterol was also shown using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay which confirmed that 26-hydroxycholesterol was more toxic than 7 beta-hydroxycholesterol. Incubation of monocyte-macrophages with cholesterol added to the different oxysterols gave varying results. Cholesterol, which was not itself toxic, inhibited the toxicity of 25-hydroxycholesterol and 26-hydroxycholesterol, but the toxicity of the 7-position derivatives was not affected. The possible relevance of these molecules to the death of macrophages seen in atherosclerosis is discussed.

Oxidized low-density lipoprotein is cytotoxic to human monocyte-macrophages: protection with lipophilic antioxidants.

Human monocyte-macrophages were incubated for 24 h with low-density lipoprotein (LDL) which had been previously oxidized for varying periods up to 24 h with copper ions, in the presence or absence of DL-alpha-tocopherol or probucol. The release of radioactivity from cells preloaded with tritiated adenine was used as an assay of toxicity. Toxicity of oxidized LDL increased with duration of copper oxidation and with increasing evidence of lipid oxidation, measured by assay of thiobarbituric acid-reactive substances and by gas chromatography. Oxidation and toxicity were inhibited by DL-alpha-tocopherol (200 microM) and probucol (50 microM).

Macrophages, lipid oxidation, ceroid accumulation and alpha-tocopherol depletion in human atherosclerotic lesions.

Necropsy samples of atherosclerotic lesions of different histological stages have been analysed. Ceroid was present in all the lesions, within lipid-laden macrophage foam cells and extracellularly in the atheromatous core of advanced lesions. Mean levels of 7 beta-hydroxycholesterol, 26-hydroxycholesterol and hydroxyoctadecadienoic acids were all significantly greater in lesions than in normal intima. Levels of hydroxycholesterols were very low or undetectable in normal intima. Fatty streaks showed the highest ratio of 7 beta-hydroxycholesterol to cholesterol, and the lowest ratio of linoleate to oleate, suggesting that this type of lesion experiences the greatest free radical activity. Levels of 26-hydroxycholesterol, a product of the cytochrome P-450 enzyme sterol 26-hydroxylase, and the ratio of 26-hydroxycholesterol to cholesterol were significantly higher in advanced lesions than in intermediate lesions or fatty streaks. The ratio of alpha-tocopherol to cholesterol levels varied widely in normal intima but was consistently low in lesions, especially those rich in macrophage foam cells, suggesting that oxidative activity in the lesion may lead to significant oxidation of the lesion constituents only after alpha-tocopherol has been depleted. Macrophage death was a characteristic feature of advanced lesions, with apoptotic bodies present, and occasionally, intact apoptotic cells were seen in lesions. These striking correlations between macrophages, lipid oxidation, alpha-tocopherol depletion, ceroid accumulation, and macrophage death in advanced lesions, strongly support a role for oxidative damage in atherosclerosis, and lend credence to the idea that alpha-tocopherol dietary supplementation may slow the progression of atherosclerosis in humans.

Adenosine accumulation as an indicator of cell-specific toxicity in rat lung slices.

We have investigated the potential of adenosine uptake as a marker of chemically induced, cell-selective pulmonary injury using cell-selective toxicants. The administration of alpha-naphthylthiourea (ANTU), an agent which is known to damage the pulmonary endothelium, diminished spermidine and adenosine accumulation. In contrast, paraquat (a toxicant that selectively damages pulmonary epithelial cells) did not reduce adenosine uptake, although uptake of spermidine (a marker of pulmonary epithelial damage) was reduced. Taken together, these findings suggest that adenosine and spermidine are accumulated into different cell types. We characterized adenosine uptake and fate in rat lung slices. Accumulation was time and concentration dependent. In our experiments, the radiolabel retained within the slice comprised mainly nucleotides, primarily ATP. Adenosine-induced ATP elevation was initially a rapid event which reached a maximum. The use of a well characterized enzymatic assay for ATP confirmed the ATP elevation suggested by thin-layer chromatography (TLC). Adenosine uptake proved a more consistent marker of ANTU-induced pulmonary injury than measurement of 5-hydroxytryptamine (5-HT) uptake, which was slightly increased by ANTU administration to rats compared with control animals.

Fragmentation of DNA in P388D1 macrophages exposed to oxidised low-density lipoprotein.

Exposing a macrophage-like murine cell line to copper-oxidised low-density lipoprotein led to DNA fragmentation which was inhibited by the putative Ca2+/Mg2+ endonuclease inhibitor, zinc sulphate. DNA fragmentation preceded loss of membrane impermeability. These results suggest that apoptosis may be a mechanism of macrophage foam cell death in atherosclerotic lesions in the arterial wall.

Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro.

Elevation of cytosolic calcium ([Ca2+]i) has been reported to induce apoptosis in a number of cell types. However, in the neutrophil, which undergoes apoptosis constitutively during aging in vitro, activation by inflammatory mediators elevates [Ca2+]i and prolongs lifespan via inhibition of apoptosis. To examine this paradox, we investigated the effects of modulation of [Ca2+]i upon apoptosis of neutrophils in vitro. Calcium ionophores (A23187, ionomycin) retarded apoptosis in neutrophil populations after 20 h (P < 0.001). Conversely, intracellular Ca(2+)-chelation, using bis-(o-aminophenoxy)-N,N,N'N'-tetraacetic acid (BAPTA) acetoxymethyl ester (AM) promoted apoptosis (P < 0.02). W-7 (an inhibitor of calmodulin) also promoted apoptosis (P < 0.05). Measurements of [Ca2+]i, using fura-2, showed (a) increased apoptosis in neutrophil populations was not associated with elevated [Ca2+]i, (b) neutrophils cultured with ionophore at concentrations inhibiting apoptosis exhibited transient (< 1 h) elevations of [Ca2+]i, to levels previously reported with receptor-mediated stimuli, and (c) BAPTA was able to prevent the elevation of [Ca2+]i and the inhibition of apoptosis produced by ionophore. Modulation of apoptosis occurred without alterations in intracellular pH. Thus, in the neutrophil, unlike lymphoid cells, elevation of [Ca2+]i exerts an inhibitory effect upon apoptosis. Furthermore, these data suggest that transient elevation of [Ca2+]i elicits signaling events leading to prolonged inhibition of apoptosis.

Paracetamol toxicity and its prevention by cytoprotection with iloprost.

In a well-established two phase model of paracetamol toxicity in hamster hepatocytes cell death was accompanied, but not preceded, by a rise in cytosolic free calcium [Ca2+]i. Cell death appears to involve reversible oxidative damage, possibly to the cytoskeleton or mitochondria. In this model low concentrations (10(-8) to 10(-14) M) of iloprost, a stable analogue of prostacyclin, offered protection against the toxic effects of paracetamol. In preliminary studies with a rat liver epithelial cell line transduced with murine P4501A2 the toxicity of paracetamol was attenuated by iloprost. Inhibition of protein synthesis with cycloheximide had no effect on paracetamol toxicity but abolished the cytoprotective effect of iloprost.