A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: its use in the simultaneous fluorimetric assay of cellular activation processes

A fluorimetric assay for the indirect determination of superoxide production during the respiratory burst of stimulated polymorphonuclear leukocytes was described. The method allowed the determination of submicromolar concentrations of superoxide, and was sufficiently sensitive that first-derivative kinetic analysis of the respiratory burst could be quickly analyzed. Conditions for the simultaneous fluorimetric analysis of superoxide production and intracellular calcium fluxes were described.     

Oxidative determination of 14C-labeled 2-oxo acids

A simple and rapid assay for the determination of 1-14C- or U-14C-labeled 2-oxo acids is described. It is based on the selective and complete oxidation of the carboxyl group to 14CO2. Preceding purification procedures are not necessary. In rat hindlimb perfusion studies, the procedure was used to develop an indirect method for the estimation of the intracellular dilution of [1-14C]pyruvate and to determine the relationship between the transamination and decarboxylation rates of leucine in the perfused tissue by the use of tracer doses of L-[1-14C]leucine.     

A modified technique for the measurement of sulfhydryl groups oxidized by reactive oxygen intermediates

This paper suggests a simple modification of the Ellman procedure when used to measure accurate changes in sulfhydryl (-SH) content induced by reactive oxygen intermediates (ROI). This modification became necessary when we found that the standard technique did not produce time invariant results in the presence of ROI-generating systems. Cysteine (cys; 20–100 μM) in 20 mM imidazole buffer (pH 7.0) containing 1.0 mM EDTA was reacted with excess (0.2 mM) 5,5′-dithiobis(2-nitrobenzoic acid), DTNB. The absorbance of the product (p-nitrothiophenol anion) was recorded at 412 nm (A₄₁₂). This A₄₁₂ was stable for 60 min and gave a linear relationship with cys concentrations used. ROI were generated either by 0.01 U xanthine oxidase (XO) + 0.01–1.0 mM hypoxanthine (HX), 0.01–1.0 mM H₂O₂, or H₂O₂ + 100 μM FeSO₄. In the presence of ROI, A₄₁₂ decreased with time and its rate of decrease was dependent upon the concentration of components of the ROI-generating system. This time-dependent decrease in A₄₁₂ was prevented completely by the addition of 100 U of catalase (CAT). Therefore, we modified the DTNB method as follows: -SH groups were reacted with ROI for 30 min; this was followed by the addition of 100 U of CAT to scavenge the excess unreacted ROI before the addition of DTNB to generate the product. Using this modification the ROI-induced decrease in A₄₁₂ was stable with time and was linearly related to the cys concentration. We further tested the modified procedure using metallothionein (MT) as a substrate for the ROI-induced changes in -SH content. MT, at concentrations of 2.5, 5.0, and 7.5 μM, was treated with XO + 100 μM HX. Using the modified procedure, an average decrease (as compared to the untreated control) of 15, 22, and 33 μM in -SH content was observed consistently at the respective MT concentrations. However, without the modification in the procedure, these average decrease were 20, 38, and 51 μM, respectively and continued to further increase with time. These discrepancies could give rise to errors ranging from 28 to 35% or higher in determination of the ROI-induced decrease in the -SH groups of MT. This data suggests that scavenging the unreacted H₂O₂ with C prior to the addition of DTNB to the assay mixture gives a stable and accurate estimate of the ROI-induced oxidative damage to -SH groups.     

Enzymic Pathways Involved in Cell Response to H2O2

An influence of possible interaction of glutathione peroxidase and cyclooxygenase on the clonogenic survival of epithelial cells exposed in vitro to H₂O₂ was investigated. Indomethacin served as the inhibitor of cyclooxygenase, and the use of alkaline (7.5) or acidic (6.5) pH combined with controlled supply of glucose modified glutathione peroxidase activity. Indomethacin affected survival of cells exposed to H₂O₂ in a biphasic manner, enhancing cytotoxicity at lower hydrogen peroxide concentrations, and diminishing it at higher concentrations. The turning point moved gradually to higher concentrations of H₂O₂ corresponding to the augmented decomposition of hydrogen peroxide caused by increased activity of glutathione peroxidase. The data revealed that both enzymic pathways interact in the presence of H₂O₂, resulting in the overall cell survival different from that obtained after inhibition of either.     

Nickel (II) Complexes of Histidyl-Peptides as Fenton-Reaction Catalysts

Addition of histidyl-peptides containing the glycyl-glycyl-L-histidyl sequence stimulated the catalysis of Ni(II) hydrogen peroxide reduction. Maximum bleaching of murexide or nitrosodimethylaniline was obtained with glycyl-glycyl-L-histidine. A decrease in the bleaching rates was observed upon addition of SOD or hydroxyl radical scavengers, showing that the hydrogen peroxide/Ni(II)/glycyl-glycyl-L-histidine system generated superoxide anions as well as hydroxyl radicals. In contrast, addition of glycyl-glycyl-L-histidine inhibited the Cu(II) hydrogen peroxide reduction.

When peptides or proteins were exposed to oxygen radicals produced by Ni(II)/glycyl-glycyl-L-histidine catalysis of hydrogen peroxide reduction, the observed effects were similar to those produced by oxygen radicals generated by water radiolysis or by Fe(II) or Cu(II) mediated Fenton-reactions: hydroxylation of phenylalanine, interchange of disulfides, destruction of tryptophans and dityrosine formation.     

State of functionally essential Trp-29 in snake venom neurotoxins: A proton nuclear magnetic resonance study

Proton nuclear magnetic resonance (NMR) spectra have been recorded of various neurotoxins from snake venoms.pH dependence of the chemical shifts and resonance intensity has been followed for the functionally essential Trp-29. The indole N-1 proton of Trp-29 in -bungarotoxin, toxin B, and cobrotoxin exhibits appreciably large upfield shifts as thepH is lowered and the suppressed exchange with the solvent hydrogen atpH 3–4, but not inNaja haje annulifera 10 where Asp-31 is replaced with Gly-31. This observation strongly suggests the presence of a hydrogen bond between Trp-29 and Asp-31 that is probably important in stabilizing the arrangement of the functionally essential residues to form a distinct binding region for the receptor.     

Pyruvate oxidase activity dependent on thiamine pyrophosphate, flavin adenine dinucleotide and orthophosphate in Streptococcus sanguis

Abstract Oxygen uptake by Streptococcus sanguis ATCC10556 in the presence of pyruvate was studied. In permeabilized cells pyruvate oxidase activity dependent on thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD) and orthophosphate was demonstrated. The activity was ten times higher in cells grown aerobically than in cells grown anaerobically. Acetyl phosphate was a product, and 1.1 mol of acetyl phosphate was formed per mol of oxygen taken up. No pyruvate dehydrogenase activity dependent on NAD, coenzyme A (CoA) and TPP was detected.     

结合代谢组学和转录组学分析恶臭假单胞菌Y-9主动稳定胞内外pH的机制

【目的】揭示恶臭假单胞菌(Pseudomonas putida) Y-9在氨氧化过程中主动调节胞外和胞内pH稳态机制。【方法】在初始pH为7.19和9.40的硝化培养基中培养Y-9生长48 h，利用代谢组学对比分析Y-9氨氧化过程中的显著差异代谢产物并预测解离常数(pKa)；结合转录组学对比分析Y-9氨氧化过程中的显著差异调控基因。【结果】Y-9在初始pH为7.19的相对酸性条件下，产生麦芽糖醇提高胞外pH；通过上调脱氨酶、脱亚胺酶和阳离子转运相关基因在相对酸性环境中的表达来维持细胞内pH稳定性。在初始pH为9.40的碱性条件下，产5-氨基戊酸3和草氨酸等有机酸及酸性物质降低胞外pH；通过调控NADH脱氢酶、细胞色素、ATP合酶和氨基酸转运相关基因的表达来维持细胞内酸度，应对碱性环境。【结论】本研究结果首先发现了Y-9具有稳定胞外pH的能力，探讨了其胞内pH稳态机制，拓展了对微生物与环境相互作用的认知，为进一步认识微生物脱氮过程中系统pH稳定机理提供了理论依据。     

Induction of Abiotic Stress Tolerance by Salicylic Acid Signaling

The role of salicylic acid (SA) as a key molecule in the signal transduction pathway of biotic stress responses has already been well described. Recent studies indicate that it also participates in the signaling of abiotic stresses. The application of exogenous SA could provide protection against several types of stresses such as high or low temperature, heavy metals, and so on. Although SA may also cause oxidative stress to plants, partially through the accumulation of hydrogen peroxide, the results published so far show that the preliminary treatment of plants with low concentrations of SA might have an acclimation-like effect, causing enhanced tolerance toward most kinds of abiotic stresses due primarily to enhanced antioxidative capacity. The effect of exogenous SA depends on numerous factors such as the species and developmental stage of the plant, the mode of application, and the concentration of SA and its endogenous level in the given plant. Recent results show that not only does exogenous SA application moderate stress effects, but abiotic stress factors may also alter the endogenous SA levels in the plant cells. This review compares the roles of SA during different abiotic stresses.