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.     

Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.     

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.     

Lecithotrophic development and metamorphosis in the Indo-West Pacific brittle star Ophiomastix venosa (Echinodermata: Ophiuroidea)

Summary

The larval development of the ophiocomid ophiuroid Ophiomastix venosais described using SEM. The gastrula transforms into a uniformly ciliated early larva which progressively changes into a lecithotrophic late premetamorphic larva with a continuous bilateral ciliated band. This stage is short-lived and equivalent to a highly reduced ophiopluteus. Comparisons between O. venosa and other ophiuroid species whose development has been investigated suggest that, whatever the developmental mode (lecithotrophic or planktotrophic), a pluteus stage always occurs in ophiuroids with planktonic development. Two metamorphic stages were identified, the late metamorphic larva differing from the early one by the closure of the larval mouth. The appearance of the permanent mouth marks the end of the metamorphosis. The postlarva still possesses remnants of larval features. The transformation of the reduced ophiopluteus into a barrel-shaped metamorphic larva with transverse ciliated bands, a vitellaria larva, is followed. The possible occurrence of a unique type of metamorphic larva in non-brooding ophiuroids is discussed. Verification of this, however, needs further SEM investigations on metamorphic larva from species having “regular” planktotrophic development.     

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