Ganglioside-dependent factor, inhibiting lipid peroxidation in rat brain synaptosomes.

Preincubation of rat brain synaptosomes with GM1, GD1a or GT1b (10(-10)-10(-6) M), as well as with phorbol 12-myristate, 13-acetate (10(-10)-10(-6) M) was found to have dose dependent inhibitory effect on Fe(2+)-ascorbate induced lipid peroxidation, while penetrating analogue of c-AMP markedly decreased the inhibitory effect of these compounds. In liposomes made of lipids isolated from synaptosomal membranes the degree of inhibition of induced LPO by gangliosides was practically absent. The inhibitory effect of GM1 on lipid peroxidation could not be revealed after thermal denaturation of synaptosomes or after treatment with polymyxin B (inhibitor of lipid-dependent protein kinases). These results and some other data provide evidence for the existence of ganglioside-dependent factor inhibiting lipid peroxidation in brain tissue. It may be suggested to be a protein kinase modulated by gangliosides.     

Retinal pigments and carotenoids of the light-shading "spectacles" of the greenling Hexagrammus octogrammus]

Three light-sensitive pigments having lambdamax of 480, 505 and 540 nm which contain retinal as a chromophore were found in the digitonine extracts from the retina of H. octogrammus. In summer time, only one pigment (lambdamax equals 480 nm) was found, whereas during autumn and winter periods the other two pigments (lambdamax equals 505 and 540 nm) could be also observed together with the first one. The lambdamax 480 pigment is easily degraded when being exposed to light, although it is resistant to the effect of hydroxylamine. The other two pitments are less sensitive to the light, but are readily bleached by hydroxylamine. The yellow-orange coloured cells of the light-shading "spectacles" contain a mixture of beta-carotenoids. When extracted by petroleum ether, these beta-carotenoids display lambdamax at 425, 445 and 476 nm. Column chromatography on aluminium oxide revealed 6 fractions in the extracted carotenoids: light-yellow, dark-yellow, brown, reddish-brown, pink and pinkish ones. In the range from yellow to pink fractions, the contribution of the lambdamax 475 nm band increases, while that of two other ones-decreases.     

Amplification of leader proregions as a mean to increase the secretion of antibody fragments in the Pichia pastoris yeast

The dependence of secretion efficiency in Pichia pastoris cells on the copy number of proregions in leader polypeptides has been studied. The humanized light kappa-chain of the murine H3-1 antibody was used as a reporter protein. The leader pre-pro-polypeptides were composed of the signal peptide (preregion) from the α-factor precursors of Saccharomyces cerevisiae and a variable number of proregions from the prepro-precursors of the α-factor or the Hsp150p protein of S. cerevisiae or Hsp150p of P. pastoris. An increase in the proregion copy number either resulted in an almost 1.5-fold increase or a fivefold decrease in secretion depending on the proregion used. It was concluded that the enhancement of the proregion copy number could be of potential value for the intensification of protein secretion in P. pastoris.     

Nitrosative stress inhibits the aminophospholipid translocase resulting in phosphatidylserine externalization and macrophage engulfment: implications for the resolution of inflammation

Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of "nitrosatively" modified cells by RAW 264.7 macrophages. Using S-nitroso-L-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO. scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation.     

Myeloperoxidase-catalyzed redox-cycling of phenol promotes lipid peroxidation and thiol oxidation in HL-60 cells

Various types of cancer occur in peroxidase-rich target tissues of animals exposed to aryl alcohols and amines. Unlike biotransformation by cytochrome P450 enzymes, peroxidases activate most substrates by one-electron oxidation via radical intermediates. This work analyzed the peroxidase-dependent formation of phenoxyl radicals in HL-60 cells and its contribution to cytotoxicity and genotoxicity. The results showed that myeloperoxidase-catalyzed redox cycling of phenol in HL-60 cells led to intracellular formation of glutathionyl radicals detected as GS-DMPO nitrone. Formation of thiyl radicals was accompanied by rapid oxidation of glutathione and protein-thiols. Analysis of protein sulfhydryls by SDS-PAGE revealed a significant oxidation of protein SH-groups in HL-60 cells incubated in the presence of phenol/H2O2 that was inhibited by cyanide and azide. Additionally, cyanide- and azide-sensitive generation of EPR-detectable ascorbate radicals was observed during incubation of HL-60 cell homogenates in the presence of ascorbate and H2O2. Oxidation of thiols required addition of H2O2 and was inhibited by pretreatment of cells with the inhibitor of heme synthesis, succinylacetone. Radical-driven oxidation of thiols was accompanied by a trend toward increased content of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the DNA of HL-60 cells. Membrane phospholipids were also sensitive to radical-driven oxidation as evidenced by a sensitive fluorescence HPLC-assay based on metabolic labeling of phospholipids with oxidation-sensitive cis-parinaric acid. Phenol enhanced H2O2-dependent oxidation of all classes of phospholipids including cardiolipin, but did not oxidize parinaric acid-labeled lipids without addition of H2O2. Induction of a significant hypodiploid cell population, an indication of apoptosis, was detected after exposure to H2O2 and was slightly but consistently and significantly higher after exposure to H2O2/phenol. The clonogenicity of HL-60 cells decreased to the same extent after exposure to H2O2 or H2O2/phenol. Treatment of HL-60 cells with either H2O2 or H2O2/phenol at concentrations adequate for lipid peroxidation did not cause a detectable increase in chromosomal breaks. Detection of thiyl radicals as well as rapid oxidation of thiols and phospholipids in viable HL-60 cells provide strong evidence for redox cycling of phenol in this bone marrow-derived cell line.     

Redox regulation of copper-metallothionein

Copper (Cu) is an essential element whose localization within cells must be carefully controlled to avoid Cu-dependent redox cycling. Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects during metal exposure and oxidative stress. The specific role of MTs, however, in modulating Cu-dependent redox cycling remains unresolved. Our studies utilized a chemically defined model system to study MT modulation of Cu-dependent redox cycling under reducing (Cu/ascorbate) and mild oxidizing (Cu/ascorbate + H2O2) conditions. In the presence of Cu and ascorbate, MT blocked Cu-dependent lipid oxidation and ascorbyl radical formation with a stoichiometry corresponding to Cu/MT ratios 相似文献   

Appetizing rancidity of apoptotic cells for macrophages: oxidation,externalization, and recognition of phosphatidylserine

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.     

Quantitative analysis of phospholipid peroxidation and antioxidant protection in live human epidermal keratinocytes

To characterize oxidative stress in phospholipids of normal human epidermal keratinocytes we metabolically labeled their membrane phospholipids with a natural oxidation-sensitive fluorescent fatty acid, cis-parinaric acid, and exposed the cells to two different sources of oxidants—a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethyl-valeronitrile), AMVN, and a superoxide generator, xanthine oxidase/xanthine. We demonstrated that both oxidants induced pronounced oxidation of four major classes of cis-parinaric acid-labeled phospholipids—phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol—in normal human epidermal keratinocytes that was not detectable as any significant change of their phospholipid composition. Vitamin E was effective in protecting the cells against phospholipid peroxidation. Since viability of normal human epidermal keratinocytes was not changed either by labeling or exposure to oxidants the labeling protocol and oxidative stress employed are compatible with the quantitative analysis of phospholipid peroxidation in viable cells.     

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1

Summary.  The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H₂O₂-Fe²⁺. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.