The combined action of microwave radiation and hydrogen peroxide on the viability and ultrastructure of Pseudomonas aeruginosa cells

The comparative study of the action of microwave radiation and hydrogen peroxide, as well as their combined action, on the viability and ultrastructure of P. aeruginosa cells has been made. The combined use of microwave radiation and hydrogen peroxide has been shown to decrease the viability of P. aeruginosa 1.5-2 times in comparison with the isolated action of each factor. The electron microscopic study of the ultrastructure of cells have shown the deterioration of the surface structures, nuclear and ribosomal apparatus of the cells under the isolated action of each of the above-mentioned factors or under their combined action. The morphological picture of these changes has proved to be different. The maximum changes in the ultrastructure of P. aeruginosa cells have been registered after the combined action of these bactericidal factors.     

The development of 1,3-diphenylisobenzofuran as a highly selective probe for the detection and quantitative determination of hydrogen peroxide

1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species (ROS) such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene (DBB). In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which DPBF reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion, and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is DBB. Only under the action of H₂O₂ 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis, and cyclic voltammetry (CV). A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196–3.941?mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8?μM, respectively. The kinetics of the reaction between DBBF and H₂O₂ was also studied.     

Fluorimetric study of the pro-oxidant activity of EUK8 in the presence of hydrogen peroxide

The catalase mimetic complex Mn(III)-salen chloride (EUK8) was found to be pro-oxidant under low hydrogen peroxide concentrations. The increase in the fluorescence rate of the probe 1,2,3-dihydrorhodamine (DHR) in solution, as well as the carbonyl content of human serum albumin were found to be maximum at H2O2:EUK8 molar ratios ranging from 0 to 2, supporting previous findings regarding the mechanism of EUK8 catalase activity and the formation of highly oxidative Mn(V)-O2- species. This pro-oxidant effect is precluded by the presence of glutathione. Cytotoxicity to HeLa cells, as probed by increased rate of oxidation of intracellular DHR, was not observed. Our findings suggest that the combination of H2O2 and EUK8 at specific molar ratios, in the absence of reductants/antioxidants, induces the oxidation of organic molecules. It is shown that the fluorimetric determination of pro-oxidant activity of metal complexes is more sensitive than the colorimetric quantification of protein carbonyl content. The implications of our findings with respect to the somewhat confusing results arising from in vivo studies of EUK8 and other Mn(III) anti-oxidant metal complexes are discussed.     

Effect of H<Subscript>2</Subscript>O<Subscript>2</Subscript> on tyrosine phosphorylation of pea proteins

Changes in tyrosine phosphorylation of soluble polypeptides of pea (Pisum sativum L.) roots were revealed under the action of exogenous hydrogen peroxide in situ and in vitro. The polypeptides whose tyrosine phosphorylation in situ was vanadate-sensitive were identified. A thiol agent dithiothreitol and the antioxidant ascorbic acid reversed the effect of hydrogen peroxide in vitro. The results indicate that tyrosine phosphorylation of pea proteins is a subject to redox regulation.     

Stimulatory and Inhibitory Actions of Proteins and Amino Acids On Copper-Catalysed Free Radical Generation in the Bulk Phase

The effect of a variety of proteins and amino acids was investigated on oxygen free radical activity as assessed by copper/hydrogen peroxide induced benzoate hydroxylation as well as copper-catalysed ascor-bate autoxidation. Serum albumins from a variety of species (human, bovine and dog) had both inhibitory and stimulatory effects depending on the molar copper to protein ratio; low ratios were inhibitory and high stimulatory. Some other proteins tested (lysozyme, soybean trypsin inhibitor and conalbumin) also had dual (inhibitory and stimulatory) effects, as did both histidine and polyhistidine, but all effects occurred at different molar ratios presumably dependent on the relative affinities for the copper ions. In contrast, metallolhioncin and cacruloplasmin, proteins specialised to bind copper in vivo had no stimulatory effects. In this paper we show that in addition to their fairly well documented inhibitory effects, under certain conditions some proteins also stimulate radical reactions. The possible role of this phenomenon in vivo is discussed.     

Role of apoplastic ascorbate and hydrogen peroxide in the control of cell growth in pine hypocotyls

The growth cessation of plant axis has been related with the formation of diphenyl bridges among the pectic components of the cell wall caused by the action of apoplastic peroxidases using hydrogen peroxide as electron acceptor. The formation of diphenyl bridges is prevented by the presence of ascorbate in the apoplastic fluid which acts as a hydrogen peroxide scavenger. The current work focuses on the role of the apoplastic ascorbate and hydrogen peroxide in the cell growth. The addition of hydrogen peroxide caused an inhibition of the auxin-induced growth as well as a significant decrease in the cell wall creep induced by acid-pH solutions. The hydrogen peroxide content in apoplastic fluid increased with the hypocotyl age and along the hypocotyl axis of 10-day-old pine seedlings, as the growth capacity decreased. On the other hand, the ascorbate content in the apoplastic fluid decreased with the hypocotyl age and along the hypocotyl axis of 10-day-old seedlings. A very significant correlation between the hydrogen peroxide apoplastic level and the growth rate as well as between the ascorbate/hydrogen peroxide molar ratio and the growth rate of hypocotyls have been found suggesting that the redox state is the main factor controlling the cell wall stiffening mechanism and thus growth in pine hypocotyls.     

Stimulatory and Inhibitory Actions of Proteins and Amino Acids On Copper-Catalysed Free Radical Generation in the Bulk Phase

The effect of a variety of proteins and amino acids was investigated on oxygen free radical activity as assessed by copper/hydrogen peroxide induced benzoate hydroxylation as well as copper-catalysed ascor-bate autoxidation. Serum albumins from a variety of species (human, bovine and dog) had both inhibitory and stimulatory effects depending on the molar copper to protein ratio; low ratios were inhibitory and high stimulatory. Some other proteins tested (lysozyme, soybean trypsin inhibitor and conalbumin) also had dual (inhibitory and stimulatory) effects, as did both histidine and polyhistidine, but all effects occurred at different molar ratios presumably dependent on the relative affinities for the copper ions. In contrast, metallolhioncin and cacruloplasmin, proteins specialised to bind copper in vivo had no stimulatory effects. In this paper we show that in addition to their fairly well documented inhibitory effects, under certain conditions some proteins also stimulate radical reactions. The possible role of this phenomenon in vivo is discussed.     

Interaction between immunoglobulin G and peroxidase-like iron oxide nanoparticles: Physicochemical and structural features of the protein

The study is devoted to the oxidative modification of immunoglobulin G (IgG) on the surface of peroxidase-like iron oxide magnetic nanoparticles (MNPs) under conditions of induced reactive oxygen species (ROS) generation and without them. A pronounced change of thermodynamic parameters of denaturation has been detected for IgG in solutions containing MNPs under hydrogen peroxide action during 24 h of incubation. Dynamic light scattering measurements and UV–Visible spectrophotometry have been used to show aggregation in these solutions. Ferromagnetic resonance (FMR) was used to compare IgG coating thickness on individual MNPs under conditions of induced ROS generation and without them. The similarity between IgG adsorption on MNPs under these conditions after 24 h of incubation has been confirmed by the fluorescence measurements. The sites of IgG oxidative modifications that take place on MNPs surface and some evidences of the influence of oxidative modification and adsorption on the chemical structure of IgG were revealed by HPLC MS/MS analysis.     

Measurement of cytochrome f in thylakoids of higher plants using quantitative gel electrophoresis

A quantitative method was developed to estimate the concentration of cytochrome (cyt) f in isolated thylakoids, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with a heme-specific reagent containing 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide. This densitometric technique was at least as sensitive as difference spectroscopy. Analysis of thylakoid preparations by densitometry of stained bands using cyt c as standard gave molar ratios of cyt/chlorophyll which were identical to ratios obtained by difference spectroscopy. Densitometric assays demonstrated that the molar ratio of cyt f/chlorophyll decreased during leaf aging in seven higher plants; however, there was a marked difference in the rate at which cyt f was lost from the leaves of different species.     

Formation of hydrogen peroxide and hydroxyl radicals in aqueous solutions of L-amino acids by the action of X-rays and heat

The action of 1 mM solutions of L-amino acids in 5 mM phosphate buffer, pH 7.4, on the production of hydrogen peroxide and hydroxyl radicals under the action of X-rays and heating has been studied. Hydrogen peroxide was estimated by the method of enhanced luminescence in a system luminol-paraiodophenol-peroxidase and hydroxyl radicals were determined by using the fluorescence probe coumarin-3-carboxylic acid. It was shown that amino acids can be divided by their influence on H202 formation into three groups: those that reduce the yield of H202, that do not influence it, and that increase it. A similar action of amino acids was observed upon heating, but the composition of the groups was different. All amino acids lowered the formation of hydroxyl radicals under the action of X-rays, and the most effective among them were Cys > His > Phe = Met = Trp > Tyr. Met, His and Phe lowered the amount of hydroxyl radicals by heating, Ser raised it, whereas Tyr and Pro did not change it. Thus, amino acids differently influence the formation of reactive oxygen species by the action of X-rays and heat, and some of amino acids reveal themselves as effective natural antioxidants.     

The Action of Nine Chelators on Iron-Dependent Radical Damage

Nine iron chelators were tested in five systems for their effects on radical-generation and conversion at chelator: iron molar ratios from 0.1 to 10. Stimulatory actions might distinguish toxic from safer chelators. Radical-generating reactions which represent different aspects of iron (ferrous and ferric) availability were studied: a) the reaction with hydrogen peroxide to hydroxylate benzoate; b) the oxidation of ascorbate; c) the reaction with hydrogen peroxide to fragment proteins; d) the reaction with hydrogen peroxide to permit amplified chemiluminescence; and e) the induction of peroxidation of mitochondrial membrane lipids. The compounds used were HBED, CP130, Desferal, EDTA, pyridine-hydrazone (CGP 43'902B), Ferrozine, CP 94 (CGP 46'700), LI (CGP 37 391) and rhodotorulic acid (CGP 45 274). Only the hexadentate compounds HBED, CP130 and Desferal were uniformly inhibitory (“protective”). The protective compounds were also apparently more stable during radical fluxes than the other chelators.     

Changes in chlorophyll <Emphasis Type="Italic">a</Emphasis> and β-carotene fluorescence caused by hydrogen peroxide

Hydrogen peroxide is shown to change the fluorescence of β-carotene and chlorophyll a as well as various mixtures of the pigments in Langmuir films and in complex with bovine serum albumin in water solution. Similar effects were shown for zinc etioporphyrin II in the films. No shifts of the fluorescence peaks were registered in any case.     

Inhibition kinetics of hydrogen peroxide on beta-n-acetyl-D-glucosaminidase from prawn (Penaeus vannamei)

The effects of hydrogen peroxide (H2O2) on prawn NAGase activity for the hydrolysis of pNP-beta-D-GlcNAc have been studied. The results show that H2O2 can reversible inhibit the enzyme (IC50 = 0.85 M) and the inhibition is of a mixed type. The kinetics show that k+o is much larger than k+0, indicating the free enzyme is more susceptible than the enzyme-substrate complex in the H2O2 solution. It is suggested that the presence of the substrate offers marked protection against inhibition by H202. Changes of activity and conformation of the enzyme in different concentrations of H202 have been compared by measuring the fluorescence spectra and residual activity and show that the change of conformation is more rapidly than that of the residual activity, which implies that the whole conformation of the enzyme changes more rapidly than the conformation of the active centre of the enzyme in the H2O2 solution.     

The origin of red cell fluorescence caused by hydrogen peroxide treatment

Fluorescence in red cells following hydrogen peroxide treatment has been attributed to lipid peroxidation of the membrane. The putative relationship between lipid peroxidation and fluorescence was questioned by the finding that BHT and alpha-tocopherol, which are thought to inhibit lipid peroxidation, do not inhibit the fluorescence detected by flow cytometry. Furthermore, lipid peroxidation induced in red cells by the Fe(III)-ADP-ascorbate system did not produce fluorescence. These results require an alternative explanation for the hydrogen peroxide-induced fluorescence. A role for reduced hemoglobin is indicated by the inhibition of fluorescence by pretreatment of cells with CO that binds strongly to ferrohemoglobin and nitrite that oxidizes ferrohemoglobin. Our earlier studies have shown the formation of fluorescent heme degradation products during the reaction of purified hemoglobin with hydrogen peroxide, which was also inhibited by CO and nitrite pretreatment. The fluorescence produced in red cells after the addition of hydrogen peroxide can, therefore, be attributed to fluorescent heme degradation products.     

An amperometric hydrogen peroxide sensor based on immobilization of hemoglobin in poly(o-aminophenol) film at iron-cobalt hexacyanoferrate-modified gold electrode

A series of hybrid iron-cobalt hexacyanoferrate (FeCoHCF) films were electrodeposited on gold electrodes from solutions containing 6mM Fe(CN)(6)(3-) with different concentrations of Co(2+) and Fe(3+). FeCoHCF films deposited from solutions with different molar ratios of iron were studied by cyclic voltammetry, and their solid states were characterized by Fourier transform infrared spectroscopy. The kind of FeCoHCF film that deposited from a solution with a molar ratio of iron of 0.4 showed the largest response current to H(2)O(2) and was characterized by energy-dispersive X-ray spectroscopy. Therefore, the optimized FeCoHCF film was combined with nonconducting poly(o-aminophenol) (POAP) film that entrapped the hemoglobin (Hb) to construct hydrogen peroxide biosensor. The response current of the Hb/POAP/FeCoHCF/Au electrode (29.8 nA) was nearly 40 and was 1.5 times that of the Hb/POAP/Au (0.7 nA) and POAP/FeCoHCF/Au (20 nA) electrodes, respectively. The Michaelis-Menten constant of Hb in the Hb/POAP/FeCoHCF/Au film was 9.31 mM. These results show that the immobilized Hb in the Hb/POAP/FeCoHCF/Au film exhibits higher catalytic activity and larger response current to H(2)O(2) by the mediation of FeCoHCF. In addition, effects of applied potential, solution pH, and electroactive interferent on the response current of the Hb/POAP/FeCoHCF/Au electrode were investigated in detail.     

鲜切加工加速荸荠组织衰老与H2O2累积的关系

以荸荠为材料,研究了鲜切加工加速组织衰老与活性氧代谢的关系.结果表明:鲜切加工提高了荸荠切片抗氧化酶(超氧化物歧化酶、抗坏血酸-过氧化物酶和过氧化氢酶)的活性;但同时明显刺激了O2-产生,促进了H2O2累积,加速了抗坏血酸在贮藏后期的损失,加强了膜脂过氧化作用和增加了电解质渗出率.统计分析表明H2O2含量、丙二醛含量、电解质渗出率三者之间存在正相关性.H2O2组织定位结果也证实鲜切加速组织衰老与H2O2累积密切相关.完整荸荠组织O2-产生比较平稳,抗氧化酶活性维持稳定,H2O2未有明显累积.     

Tetrabromohydroquinone and riboflavin are possibly responsible for green luminescence in the luminous acorn worm, Ptychodera flava.

2,3,5,6-Tetrabromohydroquinone was isolated as a luminous substance from Ptychodera flava. This compound emitted light after addition of hydrogen peroxide under basic conditions. Since hydroquinone had no fluorescence, further investigation by spectral analysis revealed that riboflavin was the only possible light emitter having green fluorescence. In the presence of both tetrabromohydroquinone and riboflavin under a basic condition containing 70% 1,4-dioxane, green light emission was observed following the addition of hydrogen peroxide. We succeeded in recording the same emission spectrum as that in the bioluminescence caused by the addition of aqueous diluted hydrogen peroxide solution in P. flava.     

Investigation of Ternary Complex Formations of Polyacrylic Acid with Bovine Serum Albumin in the Presence of Metal Ions by Fluorescence and Dynamic Light Scattering Measurements

In this paper, the complex formation of bovine serum albumin (BSA) and polyacrylic acid (PAA) in the presence metal ions at pH = 7 has been examined by using fluorescence and dynamic light scattering measurements. It has been observed that the most stable complexes of polyacrylic acid and bovine serum albumin have occurred in the presence of copper(II) ions. The other ions have the ability to form weak complexes between polyions and bovine serum albumin. To prior characterizing the interaction between bovine serum albumin and polyacrylic acid, the dynamic light scattering technique have been applied to determine the intensity-size distributions of the solutions of bovine serum albumin, polyacrylic acid, and ternary mixtures containing various molar ratios of bovine serum albumin to polyacrylic acid (the molar ratios of bovine serum albumin to polyacrylic acid has been taken equal to 0.5, 1.0, 1.5, 2.0 and 2.5) prepared at different molar ratios of copper(II) ions/acrylic acid unit. When the molar ratio of copper(II) ions to acrylic acid in the ternary mixtures has been lower than and equals to 0.3, two peaks have been observed in the curves of the intensity-size distributions due to contents of free bovine serum albumin and ternary complexes of polyacrylic acid-copper(II)-bovine serum albumin whereas when the molar ratio of copper(II) ions to acrylic acid equals to 0.4, the hydrodynamic diameter has pointed out only one peak. This result indicates that soluble and stable ternary complexes has occurred when the molar ratio of copper(II) ions to acrylic acid has been taken equal to 0.4.     

The bactericidal, fungicidal and sporicidal properties of hydrogen peroxide and peracetic acid

The antimicrobial properties of aqueous solutions of peracetic acid and hydrogen peroxide have been compared. Peracetic acid exhibited excellent antimicrobial properties, especially under acidic conditions. Reductions by a factor of 10⁶ in the numbers of vegetative bacteria are obtained within 1 min at 25°C using a solution containing 1.3 mmol/l of peracetic acid. Rapid activity against bacterial spores and yeasts also occurs. Hydrogen peroxide is more effective as a sporicide than as a bactericide, with sporicidal action being obtained using a solution containing 0.88 mol/l. Bactericidal action is poor but hydrogen peroxide was bacteriostatic at concentrations above 0.15 mmol/l.     

Bcl-2 and mitochondrial oxygen radicals. New approaches with reactive oxygen species-sensitive probes.

Investigations into the capacity of the Bcl-2 protein to prevent apoptosis have targeted mitochondria as key sites of the preventative action accorded by Bcl-2 to cells. Using novel approaches with fluorescence probes and autofluorescence detection of endogenous NAD(P)H, we have examined the effects of expressing Bcl-2 in the Bcl-2 negative Burkitt's lymphoma cell line Daudi. We evaluated for the first time the effect of Bcl-2 expression on the intracellular distribution and production of hydrogen peroxide, under basal conditions and after treatment with apoptosis inducing agents, ceramide analogs and tumor necrosis factor (TNF)-alpha. Increased availability of mitochondrial NAD(P)H was detected in Bcl-2-expressing cells and was correlated with an increased constitutive mitochondrial production of hydrogen peroxide. Although production of hydrogen peroxide was increased by either C(6)-ceramide or TNF-alpha in Bcl-2 negative Daudi cells commensurate with the early phases of apoptosis, this increase did not occur in Bcl-2-expressing cells. Thus, Bcl-2 appears to allow cells to adapt to an increased state of oxidative stress, fortifying the cellular anti-oxidant defenses and counteracting the radical overproduction imposed by different cell death stimuli. Furthermore, we report altered cytological features of mitochondria during the early phases of apoptosis induced by C(6)-ceramide and TNF-alpha. In particular, mitochondria changed in appearance, clustering in the perinuclear region and Bcl-2 expression prevented these changes from occurring.