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The present study was designed to investigate the hypothesis that interleukin-4 (IL-4) can induce apoptosis of human endothelial cells and to study regulatory pathways of this process. Indeed, DNA ladder assay and flow cytometry study showed that IL-4 can induce apoptosis of endothelial cells in a time- and dose-dependent manner. In addition, IL-4 markedly increased activity of caspase-3, and inhibition of this enzyme suppressed IL-4-induced apoptosis in a dose-dependent manner. These results provide the first evidence that IL-4 can induce apoptosis of human endothelial cells. In addition, the data indicate that the caspase-3-dependent pathway is critically involved in this process.

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases (PH-GPx) are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes appears to be important for the cellular peroxide tone regulating the expression of redox sensitive genes. In contrast to lipoxygenases the molecular biology of PH-GPx is less well investigated. In this study we cloned the PH-GPx cDNA from a mouse fibroblast cDNA library and the PH-GPx gene from a mouse genomic library. The gene spans approximately 4 kb which includes 1 kb of 5'-flanking region and consists of seven exons and six introns. The immediate promoter region does not contain a TATA box but there are binding sites for several transcription factors which also occur in the porcine gene. Our investigations provide useful tools for future targeted gene disruption studies.

It is generally accepted that the oxidation of pentadiene structures of polyunsaturated lipids by lipoxygenase (LOX) is regio- and enantio-specific, while the free radical-mediated lipid peroxidation gives stereo-random racemic products. It was confirmed that the oxidation of human low density lipoprotein (LDL) by 15-LOX from rabbit reticulocytes gave phosphatidylcholine (PC) and cholesteryl ester (CE) hydroperoxides regio-, stereo- and enantio-specifically. 15-LOX also oxidized human plasma to give specific PC and CE hydroperoxides in spite of the presence of high concentrations of antioxidants. More CE hydroperoxides were formed than PC hydroperoxides from LDL, but the reverse order was observed for plasma oxidation. The S/R ratio of the hydroperoxides decreased during long time incubation but remained significantly larger than one, while free radical-mediated oxidation of LDL and plasma gave racemic products.

Reticulocyte-type 15-lipoxygenase is known to dioxygenate phospholipids without preceding action of phospholipases A2 (PLA2). Therefore we studied the reaction of the secretory PLA2s (sPLA2) from pancreas and snake venom, and of the human cytosolic PLA2 (cPLA2) with 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) and their 15-lipoxygenated species (PAPC-OOH and PAPC-OH) either alone or as equimolar mixtures. These PLA2s cleaved PAPC-O(O)H with higher (sPLA2) or similar rates (cPLA2) as compared with native PAPC. In mixtures, however, PAPC proved to be the preferred, albeit not exclusive substrate for all three PLA2s. Thus, partial 15-lipoxygenation of phospholipids may also trigger liberation of arachidonic acid.

The germination process of oilseed plants is characterized by a mobilization of the storage lipids which constitute the major carbon source for the growing seedling. Despite the physiological importance of the lipid mobilization, the mechanism of this process is not well understood. Recently, it was found that a specific linoleate 13-lipoxygenase is induced during the stage of lipid mobilization in various oilseed plants and that this enzyme is translocated to the membranes of the lipid storage organelles, the so called lipid bodies. Lipoxygenase expression was paralleled by the occurrence of enantiospecific hydro(pero)xy polyenoic fatty acid derivatives in the storage lipids suggesting the in vivo action of the enzyme. Furthermore, it was reported that oxygenated polyenoic fatty acids, in particular as 13(S)-hydro(pero)xy-9(Z),11(E)-octadecanoic acid [13(S)-H(P)ODE], are cleaved preferentially from the storage lipids when compared with their non-oxygenated linoleate residues. These findings may suggest that 13(S)-H(P)ODE may constitute the endogenous substrate for beta-oxidation during lipid mobilization of oilseed plants.

Nitric oxide is known as an inhibitor of soybean lipoxygenase-1. Investigating the interaction of a mammalian 15-lipoxygenase with nitric oxide, we found that this enzyme is also inhibited reversibly when incubated with nitric oxide for a short time period (5 s) under anaerobic conditions. This inhibition may be due to the formation of a dissociable lipoxygenase-nitric oxide complex. With longer incubation periods the ferrous lipoxygenase is oxidised to a ferric form. This oxidation renders the enzyme more susceptible to peroxide activation, causing a time-dependent shortening of the kinetic lag phase.

The lipid body lipoxygenase of cucumber seedlings is at a high level expressed during the germinating process which is the stage of triglyceride mobilisation. This enzyme exhibits an unusual positional specificity which has not been described so far for any plant and animal lipoxygenase. The purified enzyme converts arachidonic acid to 15-S-hydroperoxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid (15S-HPETE), 12-S-hydroperoxy-5Z, 8Z, 10E, 14Z-eicosatetraenoic acid (12S-HPETE), and 8-S-hydroperoxy-5Z,9E,11Z,14Z-eicosatetraenoic acid (8S-HPETE) in a ratio of 76:4:20 with the corresponding R-isomers being only minor contaminants. Binding to the lipid bodies enhances the arachidonic acid dioxygenase activity more than 4-times and alters positional specificity of the enzyme in favour of 8-S-hydroperoxy-5Z,9E,11Z,14Z-eicosatetraenoic acid (8S-HPETE) formation.

The arachidonate 15-lipoxygenase from rabbit reticulocytes oxygenates cholesterol esters containing polyenoic fatty acids. Cholesterol esterified with saturated fatty acids is not oxygenated. The structures of the oxygenation products formed from various cholesterol esters have been identified by high pressure liquid chromatography, UV-spectroscopy and gas chromatography/mass spectroscopy. Oxygenated cholesterol esters have been detected in atherosclerotic plaques of human aortas.

Arrestin (also named '48 kDa protein' or 'S-antigen') is a soluble protein involved in controlling light-dependent cGMP phosphodiesterase activity in retinal rods, and is also known for its ability to induce autoimmune uveitis of the eye. We report a rapid and simple purification method based on the property of arrestin to bind specifically and reversibly to illuminated and phosphorylated rhodopsin [(1984) FEBS Lett. 176, 473-478]. This method does not require column chromatography and yields about 2-4 mg purified arrestin from 15 bovine retinas. Pure arrestin can be resolved by isoelectric focusing into at least 10 distinct bands, all of which stain with a monoclonal antibody specific for S-antigen.