Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

Salivary hydrogen peroxide produced by holding or chewing green tea in the oral cavity

Tea (Camellia sinensis) catechins have been studied for disease prevention. These compounds undergo oxidation and produce H₂O₂. We have previously shown that holding tea solution or chewing tea leaves generates high salivary catechin levels. Herein, we examined the generation of H₂O₂ in the oral cavity by green tea solution or leaves. Human volunteers holding green tea solution (0.1-0.6%) developed salivary H₂O₂ with C_max = 2.9-9.6 μM and AUC_0 → ∞ = 8.5-285.3 μM min. Chewing 2 g green tea leaves produced higher levels of H₂O₂ (C_max = 31.2 μM, AUC_0 → ∞ = 1290.9 μM min). Salivary H₂O₂ correlated with catechin levels and with predicted levels of H₂O₂ (C_{max(expected)} = 36 μM vs C_{max(determined)} = 31.2 μM). Salivary H₂O₂ and catechin concentrations were similar to those that are biologically active in vitro. Catechin-generated H₂O₂ may, therefore, have a role in disease prevention by green tea.     

Reactive oxidants regulate membrane repolarization and store-operated uptake of calcium by formyl peptide-activated human neutrophils

Although the rapid and considerable membrane depolarization response which accompanies activation of the phagocyte NADPH oxidase is due to transmembrane electron fluxes, little is known about the involvement of reactive oxidant species (ROS) in the subsequent repolarization response. In the current study, we have investigated the effects of superoxide dismutase (SOD), catalase, methionine, and the myeloperoxidase (MPO) inhibitors, sodium azide and 4-aminobenzoyl hydrazide (ABAH), as well as those of H₂O₂ and HOCl (both at 100 μM) on the alterations in membrane potential which accompany activation of human neutrophils with the chemoattractant, FMLP (1 μM), and on store-operated uptake of Ca²⁺. The generation of ROS by FMLP-activated neutrophils was monitored according to the magnitude of oxygen consumption and autoiodination, while spectrofluorimetric procedures were used to measure alterations in membrane potential and influx of Ca²⁺. Treatment of the cells with H₂O₂, and HOCl, significantly impeded membrane repolarization, while sodium azide, ABAH, methionine, and catalase exerted the opposite effects, potentiating both the rates and the magnitudes of membrane repolarization and store-operated uptake of Ca²⁺. These observations demonstrate that NADPH oxidase regulates neutrophil membrane potential and Ca²⁺ influx not only via its electrogenic activity, but also as a consequence of the generation of ROS.     

A comparative Mössbauer study of the mineral cores of human H-chain ferritin employing dioxygen and hydrogen peroxide as iron oxidants

Ferritins are ubiquitous iron storage and detoxification proteins distributed throughout the plant and animal kingdoms. Mammalian ferritins oxidize and accumulate iron as a ferrihydrite mineral within a shell-like protein cavity. Iron deposition utilizes both O₂ and H₂O₂ as oxidants for Fe²⁺ where oxidation can occur either at protein ferroxidase centers or directly on the surface of the growing mineral core. The present study was undertaken to determine whether the nature of the mineral core formed depends on the protein ferroxidase center versus mineral surface mechanism and on H₂O₂ versus O₂ as the oxidant. The data reveal that similar cores are produced in all instances, suggesting that the structure of the core is thermodynamically, not kinetically controlled. Cores averaging 500 Fe/protein shell and diameter  2.6 nm were prepared and exhibited superparamagnetic blocking temperatures of 19 and 22 K for the H₂O₂ and O₂ oxidized samples, respectively. The observed blocking temperatures are consistent with the unexpectedly large effective anisotropy constant K_eff = 312 kJ/m³ recently reported for ferrihydrite nanoparticles formed in reverse micelles [E.L. Duarte, R. Itri, E. Lima Jr., M.S. Batista, T.S. Berquó and G.F. Goya, Large Magnetic Anisotropy in ferrihydrite nanoparticles synthesized from reverse micelles, Nanotechnology 17 (2006) 5549–5555.]. All ferritin samples exhibited two magnetic phases present in nearly equal amounts and ascribed to iron spins at the surface and in the interior of the nanoparticle. At 4.2 K, the surface spins exhibit hyperfine fields, H_hf, of 436 and 445 kOe for the H₂O₂ and O₂ samples, respectively. As expected, the spins in the interior of the core exhibit larger H_hf values, i.e. 478 and 486 kOe for the H₂O₂ and O₂ samples, respectively. The slightly smaller hyperfine field distribution DH_hf for both surface (78 kOe vs. 92 kOe) and interior spins (45 kOe vs. 54 kOe) of the O₂ sample compared to the H₂O₂ samples implies that the former is somewhat more crystalline.     

Caspases are reversibly inactivated by hydrogen peroxide

Hydrogen peroxide (H₂O₂) is known to both induce and inhibit apoptosis, however the mechanisms are unclear. We found that H₂O₂ inhibited the activity of recombinant caspase-3 and caspase-8, half-inhibition occurring at about 17 μM H₂O₂. This inhibition was both prevented and reversed by dithiothreitol while glutathione had little protective effect. 100–200 μM H₂O₂ added to macrophages after induction of caspase activation by nitric oxide or serum withdrawal substantially inhibited caspase activity. Activation of H₂O₂-producing NADPH oxidase in macrophages also caused catalase-sensitive inactivation of cellular caspases. The data suggest that the activity of caspases in cells can be directly but reversibly inhibited by H₂O₂.     

Activity of magnetite-immobilized catalase in hydrogen peroxide decomposition

The present work analyzes the activity in decomposition of H₂O₂ using magnetite-immobilized catalase. The support of catalase is a glutaraldehyde-treated magnetite (Fe₃O₄). The data obtained in the H₂O₂ decomposition are analyzed. The fitting of the initial rate of the H₂O₂ decomposition versus hydrogen peroxide concentration data is discussed using a specific program for enzyme kinetics modeling (Leonora). The free catalase from Aspergillus niger (3.5 or 10 U/mL) does not show substrate inactivation up to 0.4 M H₂O₂. The immobilized catalase at low catalyst concentration shows substrate inhibition. Using 1 mg/mL of supported catalase the predicted maximum activity is higher than in the case of the free catalase at similar catalase concentration, although the optimum temperature is lower (40 °C versus 60 °C).     

Iron prevents ascorbic acid (vitamin C) induced hydrogen peroxide accumulation in copper contaminated drinking water

Ascorbic acid (vitamin C) induced hydrogen peroxide (H₂O₂) formation was measured in household drinking water and metal supplemented Milli-Q water by using the FOX assay. Here we show that ascorbic acid readily induces H₂O₂ formation in Cu(II) supplemented Milli-Q water and poorly buffered household drinking water. In contrast to Cu(II), iron was not capable to support ascorbic acid induced H₂O₂ formation during acidic conditions (pH: 3.5-5). In 12 out of the 48 drinking water samples incubated with 2 mM ascorbic acid, the H₂O₂ concentration exceeded 400 μM. However, when trace amounts of Fe(III) (0.2 mg/l) was present during incubation, the ascorbic acid/Cu(II)-induced H₂O₂ accumulation was totally blocked. Of the other common divalent or trivalent metal ions tested, that are normally present in drinking water (calcium, magnesium, zinc, cobalt, manganese or aluminum), only calcium and magnesium displayed a modest inhibitory activity on the ascorbic acid/Cu(II)-induced H₂O₂ formation. Oxalic acid, one of the degradation products from ascorbic acid, was confirmed to actively participate in the iron induced degradation of H₂O₂. Ascorbic acid/Cu(II)-induced H₂O₂ formation during acidic conditions, as demonstrated here in poorly buffered drinking water, could be of importance in host defense against bacterial infections. In addition, our findings might explain the mechanism for the protective effect of iron against vitamin C induced cell toxicity.     

Effects of Respiratory Burst Inhibitors on Nitric Oxide Production by Human Neutrophils

Human neutrophils (PMN) activated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) simultaneously release nitric oxide (.NO), superoxide anion (O₂-) and its dismutation product, hydrogen peroxide (H₂O₂). To assess whether NO production shares common steps with the activation of the NADPH oxidase, PMN were treated with inhibitors and antagonists of intracellular signaling pathways and subsequently stimulated either with fMLP or with a phorbol ester (PMA). The G-protein inhibitor, pertussis toxin (1-10 μg/ml) decreased H₂O₂ yield without significantly changing. NO production in fMLP-stimulated neutrophils; no effects were observed in PMA-activated cells. The inhibition of tyrosine kinases by genistein (1-25 μg/ml) completely abolished H₂O₂ release by fMLP-activated neutrophils; conversely, NO production increased about 1.5- and 3-fold with fMLP and PMA, respectively. Accordingly, orthovanadate, an inhibitor of phosphotyrosine phosphatase, markedly decreased -NO production and increased O_2;^- release. On the other hand, inhibition of protein kinase C with staurosporine and the use of burst antagonists like adenosine, cholera toxin or dibutyryl-cAMP diminished both H₂O₂ and NO production. The results suggest that the activation of the tyrosine kinase pathway in stimulated human neutrophils controls positively O₂^- and H₂O₂ generation and simultaneously maintains -NO production in low levels. In contrast, activation of protein kinase C is a positive modulator for O_2;^-and *NO production.     

The mechanism of the oxidation of ascorbate and Mn by chloroplasts: The role of the radical superoxide

1. 1. The mechanism of the photooxidation of ascorbate and of Mn²⁺ by isolated chloroplasts was reinvestigated.

2. 2. Our results suggest that ascorbate or Mn²⁺ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn²⁺ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin.

3. 3. In the absence of ascorbate or Mn²⁺ the superoxide formed dismutases either spontaneously or enzymatically producing O₂ and H₂O₂. In the presence of ascorbate or Mn²⁺, however, the superoxide is reduced to H₂O₂ with no formation of O₂. Consequently, in the absence of reducing compounds, in the reaction H₂O to low potential acceptor one O₂ (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn²⁺ or other suitable reductants up to three molecules O₂ can be taken up per four electrons transported.

4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O₂; the addition of ascorbate or Mn²⁺ to this system results in a two fold stimulation of O₂ uptake, with no stimulation of NADPH oxidation. The stimulation of O₂ uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O₂ uptake in the system H₂O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase.

5. 5. In Class II chloroplasts in the system H₂O → NADP upon the addition of ascorbate or Mn²⁺ an apparent inhibition of O₂ evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts.

6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP/2e in this system (when the rate of electron flow is based on the rate of O₂ uptake) should be revised in the upward direction.

Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.     

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g^-1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8-9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a K_m of 6.6 mM for H₂O₂ and V_max of 707 mM H₂O₂ min^-1 g^-1 wet wt. cells, and showed saturation kinetics at 50 mM H₂O₂. The cells were entrapped in calcium alginate and used for H₂O₂ degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.     

Enhancing effect of melatonin on chemiluminescence accompanying decomposition of hydrogen peroxide in the presence of copper

The oxidation of melatonin (MEL) using the Cu(II) + H₂O₂ + HO⁻ (the Fenton-like reaction) system was investigated by chemiluminescence (CL), fluorescence, spectrophotometric, and EPR spin trapping techniques. The reaction exhibits CL in the 400–730 nm region. The light emission from the Fenton-like reaction was greatly enhanced in the presence of MEL and was strongly dependent on its concentration. The spectrum measured with cut-off filters revealed maxima at around 460, 500, 580–590, 640–650, and 690–700 nm. The band at 460 nm may be due to the excited cleavage product, N¹-acetyl-N²-formyl-5-methoxykynuramine, whereas the bands at 500, 580–590, 640–650, and 700 nm were similar to those observed for singlet molecular oxygen (¹O₂). The effect of reactive oxygen species (ROS) scavengers on the light emission was studied. The CL was strongly inhibited by the ¹O₂ scavengers in a dose-dependent manner; at concentration 1 mM the potency of ¹O₂ scavenging was 5,5-dimethylcyclohexandione-1,3 > methionine > histidine > hydroquinone. The potency of HO^• scavenging by thiourea, tryptophan, cysteine at concentration 5 mM was 79–94%, by 1 mM glutathione and trolox 75 and 94%, respectively, and by 10 mM cimetidine 18%. Specific acceptors of O₂^•− such as p-nitroblue tetrazolium chloride and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) at concentration 5 mM decreased the CL by 51 and 95%, respectively, whereas superoxide dismutase (SOD) does not reduce the emission at concentration 2.8 U/ml. At higher concentration SOD substantially enhanced the light emission. Addition of 1360 U/ml catalase and 100 μM desferrioxamine strongly inhibited CL (96 and 90%, respectively). The increased generation of ¹O₂ from the Cu/H₂O₂ system in the presence of MEL was confirmed using the spectrophotometric method based on the bleaching of p-nitrosodimethylaniline and by trapping experiments with 2,2,6,6-tetramethylpiperidine (TEMP) and subsequent electron paramagnetic (EPR) spectroscopy. These findings suggest the increased production of reactive oxygen species (O₂^•−, HO^•, ¹O₂) from the Fenton-like reaction in the presence of MEL. This means that the hormone is not able to act as classical chain-breaking antioxidant even at low concentration, and may show clear prooxidant activity at higher concentrations. In addition, long-lived carbonyl product of the MEL transformation in the triplet state can also be toxic by transferring its energy to organelles and causing a photochemical process.     

过氧化氢缓解裸燕麦幼苗低温胁迫的主成分和隶属函数分析

为探明信号分子过氧化氢（H₂O₂）提高裸燕麦幼苗耐冷性的作用,以‘定莜6号’沙培幼苗为材料,在3叶期喷施10 μmol·L^-1 H₂O₂ 12 h后于8℃/5℃（昼/夜）条件下低温胁迫,以喷蒸馏水（H₂O）为对照,分别在低温处理的0、1、2、3、4、5 d取幼苗叶片测定超氧阴离子（O₂^-·）、H₂O₂、丙二醛（MDA）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）、抗坏血酸过氧化物酶（APX）、抗坏血酸（AsA）、谷胱甘肽（GSH）、可溶性糖（SS）、脯氨酸（Pro）、可溶性蛋白质（SP）和热稳定性蛋白质（HSP）13项与耐冷性有关的生理指标及低温处理5 d后植株株高和生物量增量,采用主成分和隶属函数分析综合评价H₂O₂对裸燕麦幼苗耐冷性的影响。结果表明,与对照相比,喷施H₂O₂显著提高了低温胁迫下裸燕麦幼苗株高和生物量增量,降低了裸燕麦幼苗叶片O₂^-·和MDA及低温胁迫2~5 d的H₂O₂含量,促进低温胁迫期间裸燕麦幼苗叶片SOD、CAT、POD和APX活性提高及AsA、GSH、SS、Pro、SP和HSP积累。主成分分析13项生理指标离差标准化数据,提取的前4个主成分累积方差贡献率达85.6%;隶属函数综合评价4个主成分得分值显示,喷施H₂O₂显著提高了低温胁迫0~5 d的综合评价值。表明喷施H₂O₂能够通过调控生理生化代谢提高裸燕麦幼苗的耐冷性。     

Protection against nuclear DNA damage offered by flavonoids in cells exposed to hydrogen peroxide: the role of iron chelation

The ability of a number of flavonoids belonging to the flavone, flavonol, flavanone, and flavan-3-ol subclasses to protect cellular DNA from H₂O₂-induced single-strand breaks and the underlying molecular mechanisms were investigated in this work. Formation of single-strand breaks on nuclear DNA, after exposure of Jurkat cells to continuously generated H₂O₂ in the presence or absence of the flavonoid compounds, was evaluated by the comet assay (single-cell gel electrophoresis). The results indicate the following structural requirements of flavonoids for effective DNA protection: (a) the ortho-dihydroxy structure in either ring A or ring B, (b) the hydroxyl moiety on position 3 in combination with the oxo group at position 4, and (c) the presence of a C₂, C₃ double bond in ring C. In contrast to free flavonoids, the ability of complexes of [Fe²⁺]/[flavonoid] to protect nuclear DNA was decreased as the ratio increased, and the complex was completely inactive when the ratio reached a certain value. Moreover, it was observed that several of the flavonoids tested were able to remove iron from calcein loaded into cells and that this property was in excellent correlation with their ability to protect DNA (Spearman's correlation coefficient, ρ = 0.9, p = 0.005). The antioxidant (electron donating) capacities of the same flavonoids were also evaluated by a conventional method, but no relation with their DNA-protective ability could be established even when their membrane-penetrating abilities were taken into account (p = 0.64). In conclusion, the results presented in this work strongly support the notion that intracellular binding of iron is responsible for the protection offered by flavonoids against H₂O₂-induced DNA damage.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Two new multi-cobalt-containing heteropolyoxotungstates

Two new multi-cobalt-containing polyoxotungstates K₄Na₆Co₂(H₂O)₁₂{Co(H₂O)₄[Co₂(H₂O)₁₀Co₄(H₂O)₂(B--SiW₉O₃₄)₂]₂} · 40H₂O (1) and K₁₀Na₂[Co₄(H₂O)₂(GeW₉O₃₄)₂] · 20H₂O (2) have been obtained by the routine synthetic reactions in aqueous solution. The polyoxoanion framework of 1 consists of two sandwich-type polyoxoanions [Co₄(H₂O)₂(B--SiW₉O₃₄)₂]¹²⁻ connected together by a [CoO₂(H₂O)₄] cluster to constitute the sandwich dimer, and then, four isolated Co(H₂O)₅ cations coordinate to the dimer through four μ₂-O atoms. The polyoxoanion 2 is isomorphic to the sandwich-type polyoxoanion [Co₄(H₂O)₂(B--SiW₉O₃₄)₂]¹²⁻ in 1. The magnetic property of compound 1 has been studied by measuring its magnetic susceptibility in the temperature range 2.0–300.0 K, indicating the existence of intramolecular ferromagnetic Co–Co interactions, and, the electrochemical properties of 1 and 2 are detected in the pH 4 buffer solution.     

香蕉不同成熟度果实活性氧、乙烯与淀粉酶活性相关性研究

采用各种活性氧诱发剂(0.5g/L百草枯、10mmol/L H₂O₂和10mmol/L H₂O₂+10mmol/L FeSO₄·7H₂O)及相应的清除剂(20mmol/L没食子酸丙酯、10个单位CAT和70mmol/L DMSO)、外源乙烯(750μl/L乙烯利)、乙烯生成先导物(合成前体,1mmol/L ACC)和乙烯合成抑制剂(10mmol/L AOA)处理香蕉果肉切片。结果表明,在不同成熟度的香蕉果实中,只有O₂^·存在及乙烯合成不受抑制时,淀粉酶活性才能呈现,果实才能软化、成熟;而O₂^·及OH^·可能在乙烯合成中所起的作用有所不同。     

胞外H2O2及NADPH氧化酶参与了铜胁迫对植物细胞死亡的诱导

以烟草悬浮细胞BY-2(Nicotiana tabacum L.cv.Bright Yellow-2)为材料,探讨了在铜离子胁迫下植物细胞死亡发生过程中胞外H₂O₂及NADPH氧化酶所扮演的角色。实验结果表明,随着外源CuCl₂浓度的上升(从0~700 μmol·L^-1),细胞死亡水平不断上升,且胞外H₂O₂的水平也不断增加。在300 μmol·L^-1的CuCl₂诱导细胞死亡的过程中,加入H₂O₂清除剂N-N-二甲基硫脲(DMTU)降低了胞外CuCl₂胁迫下H₂O₂含量增加的同时也降低了细胞死亡水平的上升,这一观察表明了铜离子胁迫所导致的细胞死亡的发生和胞外H₂O₂的增加有关。进一步的研究表明,300 μmol·L^-1 CuCl₂的胁迫导致了NADPH氧化酶活性的显著性上升,而加入NADPH氧化酶的抑制剂（二亚苯基碘,DPI,)则降低了CuCl₂胁迫所导致的细胞死亡和胞外H₂O₂含量的上升。上述结果表明,胞外H₂O₂和NADPH氧化酶参与了CuCl₂对植物细胞死亡的诱导作用。