Lipoxygenase may be involved in cationic liposome-induced macrophage apoptosis

The purpose of this study was to determine the source of reactive oxygen species (ROS) generation and the contribution of ROS to the apoptosis of RAW264.7 cells induced by cationic liposomes. Cationic liposome-induced apoptosis was inhibited by lipoxygenase inhibitors, but not inhibitors of NADPH-oxidase, xanthine oxidase or cyclooxygenase. ROS generation induced by cationic liposomes was also inhibited by the lipoxygenase inhibitor NDGA. Furthermore, lipid peroxidation was observed following liposome treatment, but the apoptosis was not inhibited by the antioxidant alpha-tocopherol. These findings suggested that lipoxygenase is responsible for ROS generation, and ROS but not lipid peroxidation acts as a key mediator in the progress of apoptosis induced by cationic liposomes.     

Oxidative stress and eicosanoids in the kidneys of hyperglycemic rats treated with dehydroepiandrosterone

Oxidative stress plays a crucial role in the pathogenesis of chronic diabetic complications. Normoglycemic and streptozotocin-diabetic rats were treated with dehydroepiandrosterone (DHEA) (4 mg/d per rat) for 3 weeks. At the end of treatment, hydroxynonenal, hydroperoxyeicosatetraenoic acids and antioxidant levels, as well as Na/K-ATPase activity and membrane fatty acids composition were evaluated in kidney homogenates. Chronic hyperglycemia caused a marked increase of both hydroxynonenal and lipoxygenase pathway products and a drop in both GSH levels and membrane Na/K-ATPase activity. DHEA treatment restored the antioxidant levels to close to the control value and considerably reduced hydroxynonenal and hydroperoxyeicosatetraenoic acid levels. Moreover, DHEA counteracted the detrimental effect of hyperglycemia on membrane function: the drop of Na/K-ATPase activity in diabetic animals was significantly inhibited by DHEA treatment. These results show that DHEA reduces oxidative stress and the consequent increase of lipoxygenase pathway products induced by experimental diabetes in rat kidney; they also suggest that, by reducing the inflammatory response to oxidative stress, DHEA treatment might delay the progression of diabetic kidney disease.     

12(R)-hydroxyeicosatetraenoic acid synthesis by 3-methylcholanthrene- and clofibrate-inducible cytochrome P450 in porcine ciliary epithelium.

Porcine ciliary epithelial microsomes synthesized 12[S]-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (12[S]-HETE) from arachidonic acid by a membrane-bound lipoxygenase and 12[R]-isomer by the cytochrome P450-dependent monooxygenase system. The activity to form 12(R)-isomer was markedly enhanced by 3-methylcholanthrene and clofibrate. Both basal and induced levels of 12(R)-HETE synthesizing activity were considerably higher in nonpigmented epithelial cells than in pigmented cells of the ciliary processes. The induced activity was suppressed by polyclonal antibodies raised against purified cytochrome P450 IA1 and NADPH-P450 reductase but not by substrates for clofibrate-inducible omega/omega-1 hydroxylases (P450 IVA-mediated). These results suggest that 12(R)-HETE synthesis by porcine ciliary microsomes may be mediated by a cytochrome P450 of the IA family.     

Casein kinase 2 binds and phosphorylates the nucleosome assembly protein-1 (NAP1) in Drosophila melanogaster.

The nucleosome assembly protein-1 (NAP1) was originally identified in HeLa cells as a factor facilitating the in vitro assembly of nucleosomes. However, in yeast cells NAP1 is required in the control of mitotic events induced by the Clb2/p34(CDC28). Here, we show that Drosophila NAP1 is a phosphoprotein that is associated with a kinase able to phosphorylate NAP1. By using an in-gel kinase assay we found that this kinase displays a molecular mass of 38 kDa. Following purification and peptide microsequencing, we identified the kinase phosphorylating NAP1 as the alpha subunit of casein kinase 2 (CK2). With the help of a series of NAP1 segments and synthetic peptides, we assigned the CK2 phosphorylation sites to residues Ser118, Thr120, and Ser284. Interestingly, Ser118 and Thr120 are located within a PEST domain, while Ser284 is adjacent to the nuclear localization signal. Substitution of the identified phosphoresidues by alanine was found to reduce considerably the ability of CK2 to phosphorylate NAP1. The enhanced ability of CK2 to phosphorylate phosphatase-treated NAP1 extracted from Drosophila embryos and the similar tryptic phospho-peptide pattern of in vivo labelled NAP1 and in vitro labelled NAP1 with CK2 indicate that NAP1 is a natural substrate of CK2. Further analysis revealed that both CK2alpha and beta subunits are associated with NAP1 but we found that only the catalytic alpha subunit establishes direct contact with NAP1 on two distinct domains of this protein. The location of CK2 phosphorylation sites in NAP1 suggests that their phosphorylation can contribute to a PEST-mediated protein degradation of NAP1 and the translocation of NAP1 between cytoplasm and nucleus.     

Purification and characterization of two cyclic AMP-independent protein kinases from AH-66 hepatoma ascites cells

Casein kinase 1 (CK 1) and casein kinase 2 (CK 2) were purified from the cytosol fraction of AH-66 cells to electrophoretic homogeneity by a simple procedure based on our finding that CK 1 and CK 2 are chromatographically distinct on phosvitin-Sepharose. The amino acid composition of CK 2 resembles those of cyclic AMP-dependent and cyclic GMP-dependent protein kinases but is considerably different from that of CK 1. Both CK 1 and CK 2 were markedly stimulated by low concentrations of spermine and spermidine but were practically unaffected by putrescine. When CK 1 and CK 2 were added back to AH-66 cytosol, they promoted the phosphorylation of the same cytosolic proteins that were phosphorylated endogenously. Although most of the cytosolic proteins phosphorylated by CK 1 and CK 2 were common, some proteins were preferentially phosphorylated by either CK 1 or CK 2. Interestingly, CK 1 was able to phosphorylate the plasma membrane proteins of AH-66 cells. In contrast, enhancement of the phosphorylation of the membrane proteins by CK 2 was practically undetectable.     

Programmed cell death induced by glutathione depletion in PC 12 cells is blocked by inhibitors of 12 lipoxygenase, but does not appear to be mediated through the formation of 12 HETE derivatives

Lipoxygenase metabolites have been postulated to be involved in the degenerative events provoked by oxidative stress in neuronal and nonneuronal targets, but their roles remain controversial. In the present work, we investigated the putative role of 12 lipoxygenase metabolites in the programmed cell death induced by glutathione depletion in PC 12 cells. Determinations of 12 lipoxygenase expression and activity reveal the presence of the enzyme in PC 12 cells, but the formation of arachidonate metabolites appears rather low and is not influenced by glutathione depletion. In addition, although the death induced by buthionine sulfoximine (BSO) treatment is abolished by known inhibitors of lipoxygenase enzymes, dexamethasone, a potent steroidal inhibitor of both cyclooxygenase and lipoxygenase pathways, fails to protect the cells from BSO-induced degeneration. Finally, incubation of the cells for 24 h in the presence of exogenous 12 HETE did not induce any significant decrease in cell viability. Our results indicate that 12 lipoxygenase is unlikely to play a major role in the process of cell degeneration provoked by glutathione depletion.     

Membrane lipid peroxidation, free radical scavangers and ethylene evolution in Amaranthus as affected by lead and cadmium

Membrane lipid peroxidation, activity of free radical scavangers and ethylene evolution of Amaranthus lividus seedlings were used to determine the lead and cadmium (1, 10, 100 and 1000 μM) induced phytotoxicity. Malondialdehyde (MDA) accumulation and higher lipoxygenase activity (LOX) was found in the 7-d-old treated seedlings. The activities of free radical scavangers like peroxidase, catalase and superoxide dismutase declined considerably with the concomitant rise in hydrogen peroxide level. Heavy metal treatment also caused decline in ethylene evolution in germinating seedlings. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of zinc on creatine kinase: activity changes, conformational changes, and aggregation

The effects of zinc on creatine kinase (CK) are very distinctive compared with other bivalent metal ions. Zinc up to 0.1 mM induced increases in CK activity, accompanied by significant hydrophobic surface exposure and increase in a-helix content of CK. Zinc over 0.1 mM denatured and inactived CK. In the presence of 0.1 mM zinc, the CK activity was very close to that of the native CK, but its conformation changed greatly. The kinetic courses of CK inactivation and conformational change in the presence of 1 mM zinc were measured to determine apparent rate constants of inactivation and conformational change. Zinc over 0.05 mM induced CK aggregation at 37°C, and the aggregation was dependent on zinc concentration, CK concentration, and temperature. The inactivation and aggregation can be reversed by EDTA. An explanation for CK aggregation induced by zinc is proposed, as well as a mechanism for CK abnormality in Alzheimer's disease.To whom correspondence should be addressed.     

Salicylate fails to prevent the inhibitory effect of 5,8,11,14-eicosatetraynoic acid on human platelet cyclo-oxygenase and lipoxygenase activities

Sodium salicylate is inactive both on cyclo-oxygenase and lipoxygenase prepared from human platelets. It prevents the inhibition of cyclo-oxygenase induced by aspirin, but does not counteract the inhibitory effect of 5,8,11,14-eicosatetraynoic acid on both enzymes. It also fails to interfere with the inhibitory activity of nordihydroguaiaretic acid on lipoxygenase. These data indicate that, unlike eicosatetraynoic acid, non-steroidal anti-inflammatory drugs interact with a site on cyclo-oxygenase distinct from the catalytic site, although related to it. Such a supplementary binding site is lacking on lipoxygenase.     

Analysis of 15-Lipoxygenase Activity in Irradiated Thymocytes

We studied the effect of -irradiation on 15-lipoxygenase activity in rat thymocytes. The enzyme activity was determined as the rate of linoleic acid oxidation by the protein fraction isolated from the control and irradiated thymocytes under standard conditions. We demonstrate lipoxygenase activation immediately after irradiation of thymocytes. High lipoxygenase activity is observed in the cells for no more than an hour after irradiation. No high lipoxygenase activity later indicates that its synthesis is not directly induced by irradiation. Irradiation-induced generation of lipid peroxides can be the factor of lipoxygenase activation.     

Inhibition of CK2 binding to BMPRIa induces C2C12 differentiation into osteoblasts and adipocytes

BMP2 is a growth factor that regulates the cell fate of mesenchymal stem cells into osteoblast and adipocytes. However, the detailed signaling pathways and mechanism are unknown. We previously reported a new interaction of Casein kinase II (CK2) with the BMP receptor type-Ia (BMPRIa) and demonstrated using mimetic peptides CK2.1, CK2.2 and CK2.3 that the release of CK2 from BMPRIa activates Smad signaling and osteogenesis. Previously, we showed that mutation of these CK2 sites on BMPRIa (MCK2.1 (476S-A), MCK2.2 (324S-A) and MCK2.3 (214S-A)) induced osteogenesis. However, one mutant MCK2.1 induced osteogenesis similar to overexpression of wild type BMPRIa, suggesting that the effect of this mutant on mineralization was due to overexpression. In this paper we investigated the signaling pathways involved in the CK2-BMPRIa mediated osteogenesis and identified a new signaling pathway activating adipogenesis dependent on the BMPRIa and CK2 association. Further the mechanism for adipogenesis and osteogenesis is specific to the CK2 interaction site on BMPRIa. In detail our data show that overexpression of MCK2.2 induced osteogenesis was dependent on Caveolin-1 (Cav1) and the activation of the Smad and mTor pathways, while overexpression of MCK2.3 induced osteogenesis was independent of Caveolin-1 without activation of Smad pathway. However, MCK2.3 induced osteogenesis via the MEK pathway. The adipogenesis induced by the overexpression of MCK2.2 in C2C12 cells was dependent on the p38 and ERK pathways as well as Caveolin-1. These data suggest that signaling through BMPRIa used two different signaling pathways to induce osteogenesis dependent on CK2. Additionally the data supports a signaling pathway initiated in caveolae and one outside of caveolae to induce mineralization. Moreover, they reveal the signaling pathway of BMPRIa mediated adipogenesis.     

Ultrastructure and lipid alterations induced by cadmium in tomato (Lycopersicon esculentum) chloroplast membranes

The effects of cadmium (Cd) uptake on ultrastructure and lipid composition of chloroplasts were investigated in 28-day-old tomato plants (Lycopersicon esculentum var. Ibiza F1) grown for 10 days in the presence of various concentrations of CdCl2. Different growth parameters, lipid and fatty acid composition, lipid peroxidation, and lipoxygenase activity were measured in the leaves in order to assess the involvement of this metal in the generation of oxidative stress. We first observed that the accumulation of Cd increased with external metal concentration, and was considerably higher in roots than in leaves. Cadmium induced a significant inhibition of growth in both plant organs, as well as a reduction in the chlorophyll and carotenoid contents in the leaves. Ultrastructural investigations revealed that cadmium induced disorganization in leaf structure, essentially marked by a lowered mesophyll cell size, reduced intercellular spaces, as well as severe alterations in chloroplast fine structure, which exhibits disturbed shape and dilation of thylakoid membranes. High cadmium concentrations also affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the fatty acid content and a shift in the composition of fatty acids, resulting in a lower degree of fatty acid unsaturation in chloroplast membranes. The level of lipid peroxides and the activity of lipoxygenase were also significantly enhanced at high Cd concentrations. These biochemical and ultrastructural changes suggest that cadmium, through its effects on membrane structure and composition, induces premature senescence of leaves.     

The effect of BW775C on respiratory distress in the rat induced by arachidonic acid

BW775C, an inhibitor of the lipoxygenase and cyclo-oxygenase pathways, inhibits the respiratory distress induced by arachidonic acid in rats. The degree of respiratory distress was measured in terms of respiratory rate using electrodes implanted at each side of the thorax. Indomethacin, an inhibitor of the cyclooxygenase pathway, failed to influence the respiratory distress induced by arachidonic acid. The results implicate the lipoxygenase pathway, i.e. the leukotrienes synthesis inhibition, in the respiratory distress induced by arachidonic acid.     

Diabetes alters the reactivity of myocardium to a thromboxane analogue

Atrial contractile response to U-46619 was studied in auricles from normal and acutely diabetic rats. U-46619 induced an increment of dF/dt in diabetic atria, whereas nondiabetic auricles elicited a negative contractile effect. Blockers of arachidonic acid metabolism via cyclooxygenase inhibited the stimulatory action of U-46619. The stimulant action of the thromboxane A2 mimetic was attenuated when diabetic auricles were incubated with lipoxygenase(s) blocking agents. Results suggest that in diabetic atria, the abnormal inotropic effect induced by U-46619 may be associated with thromboxane formation and with lipoxygenase(s) metabolites.     

Activity of trypsin inhibitors in wheat seedlings exposed to pathogenic fungus Tilletia caries and phytohormones

We compared the effect of common bunt (caused by fungus Tilletia caries Tul.) and treatment with phytohormones IAA, ABA, and cytokinins (CK) on the activity of trypsin inhibitors (TI) in wheat seedlings. The experiments were conducted with pathogen-susceptible species of wheat Triticum aestivum L. (cv. Zhnitsa) and resistant species T. timopheevii Zhuk. (accession k-58666 from the collection of All-Russian Institute of Plant Industry). In the resistant wheat, the fungus elevated the activity of TI and the content of CK, whereas in the susceptible wheat, it induced accumulation of IAA. In the seedlings of wheat T. aestivum, TI activity increased under the effect of CK, same as upon the action of pathogen. ABA briefly increased the activity of TI, whereas IAA did not considerably affect it. It was concluded that among the investigated hormones, CK play a leading role in the regulation of defense responses of wheat plants involving TI.     

Reactive oxygen production associated with arachidonic acid metabolism by peritoneal macrophages

The nature of the calcium-dependent chemiluminescence observed in peritoneal macrophages after exposure to the calcium ionophore A23187 or during the phagocytosis of zymosan has been investigated. Eicosatetraynoic acid, an inhibitor of the lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism, inhibited the calcium-dependent chemiluminescence whereas indomethacin, a selective inhibitor of the cyclooxygenase pathway, did not. Arachidonic acid induced chemiluminescence only in phagocytosing cells, whilst 15-HPETE, an intermediate of the lipoxygenase pathway, generated a similar, transient chemiluminescent response in either unstimulated or phagocytosing cells. The results suggest that the lipoxygenase pathway may be a significant source of the reactive species of oxygen that give rise to chemiluminescence. Prostaglandin E₁ inhibited the chemiluminescence induced by zymosan and A23187, but did not affect that generated in response to 15-HPETE or arachidonic acid, suggesting that the inhibition is directed at a step either connected with or occurring prior to the release of free arachidonic acid by the cells.     

Expression, activity, and cellular accumulation of methyl jasmonate-responsive lipoxygenase in soybean seedlings

Exposure of soybean (Glycine max) seedlings to low levels of atmospheric methyl jasmonate induced the expression and accumulation of one or more lipoxygenase(s) in the primary leaves, hypocotyls, epicotyls, and cotyledons. In the primary leaf, the major site of lipoxygenase accumulation in response to methyl jasmonate was in the vacuoles of paraveinal mesophyll cells. In the other organs, however, most of the methyl jasmonate-responsive lipoxygenase(s) were associated with both the epidermal and cortical cells and were present in both vacuoles and plastids. In plastids, the methyl jasmonate-responsive lipoxygenase was sequestered into protein inclusion bodies; no lipoxygenase was evident in either the thylakoids or the stroma. Both spectrophotometric measurement of conjugated diene formation and thin layer chromatography of lipoxygenase product formation indicated that methyl jasmonate caused an increase in the amount of lipoxygenase activity. Electron microscopy of the methyl jasmonate-responsive lipoxygenase protein in the vacuoles showed that it was arranged into a stellate, paracrystalline structure in various cell types other than the paraveinal mesophyll cells. The paracrystals appeared to be composed of tubular elements of between 5 and 8 nm in diameter, were of variable length, and were observed in most cell types of the seedling organs.     

Arachidonic acid metabolim in rat pancreatic acinar cells: Calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [¹⁴C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromtography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8–10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonic derivative was stimulated by the calcium ionophore ionomycin. Stimulation of lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF_1α, PGF_2α, and PGE₂ were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

Eicosapentaenoic acid metabolism in human and rabbit anterior uvea

Eicosapentaenoic acid (EPA) metabolism into 3 series cyclooxygenase and 5 series lipoxygenase products was assessed in human and rabbit anterior uvea. Both tissues synthesized 3 series cyclooxygenase products such as delta17 6-keto-PGF1 (PGI3 metabolite), PGE3 alpha, PGE3, PGD3 and TxB3 (a stable product of TxA3) and lipoxygenase products 12-hydroxyeicosapentaenoic acid (HEPE), 5-HEPE and 5,12-diHEPE from 14C-EPA. EPA-derived cyclooxygenase product synthesis was considerably greater than the formation of lipoxygenase products from EPA in both tissues.