The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins

The hydrogen peroxide (H₂O₂) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2·4 mmol l⁻¹ H₂O₂ pretreatment conferred protection against a lethal concentration (45 mmol l⁻¹) of this agent. The relatively high concentrations of H₂O₂ used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H₂O₂ cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H₂O₂, in addition to the impressive enhancement of synthesis of five H₂O₂ stress proteins. Combined results suggest that these proteins might play an important role in the H₂O₂ tolerance response.     

Changes in catalase activity and hydrogen peroxide concentration in plants in response to low temperature

Taxicity of oxygen species such as free radicals and H₂O₂ has been invoked to explain a number of degradative processes in plants, most involving photo-oxidation. Since catalase is a major protectant against accumulation and toxicity of H₂O₂, we examined alterations in catalase activity in several plant species ( Pisum sativum L. cv. Greenfeast, Vigna radiata (L.) R. Wilcz, Cucumis sativus L. cv. Heinz Pickling, and Passiflora spp.) during chilling, and compared this change to change in H₂O₂ content. Catalase activity was reduced in a range of chilling sensitive and tolerant species by exposure to low temperature. This reduction in catalase activity correlated better with the onset of visible symptoms than with the treatment itself. Visible injury in turn was dependent on light and temperature differences. Hydrogen peroxide concentrations invariably decreased with low temperatures.
Reduction in catalase activity therefore does not necessarily imply accumulation of H₂O₂ to damaging levels. The absence of a clear inverse relationship between catalase activity and H₂O₂ concentration suggests the continued activity of other reactions that remove H₂O₂ and these may be important in the tolerance of plants to oxidative attack. Loss of catalase activity may result from the inability of damaged peroxisomal membranes to transport catalase precursors into the peroxisome.     

Research note : Unsuitability of the MRS medium for the screening of hydrogen peroxide-producing lactic acid bacteria

The effect of MRS broth on the stability of hydrogen peroxide (H₂O₂) has been studied. Known concentrations (1–100 μg ml⁻¹) of H₂O₂ were prepared in distilled water, phosphate buffer (pH 7·0) and MRS broth (pH 6·2 and 3·9). H₂O₂ was very stable in aqueous and buffer solutions but it was rapidly degraded in MRS broth (pH 3·9). The presence of H₂O₂ in MRS broth (pH 6·2) could not be detected.     

Effect of oxygen and peroxide on Bacteroides fragilis cell and phage survival after treatment with DNA damaging agents

Abstract Bacteroides fragilis Bf-2 cells were more sensitive to far-UV radiation, N -methyl- N '-nitrosoguanidine, ethylmethane sulphonate, acriflavine and mitomycin C under aerobic conditions than under anaerobic conditions. The opposite effect was observed with H₂O₂-treated cells and exposure to O₂ enhanced the survival of H₂O₂-treated cells. Pretreatment of cells with sublethal concentrations of H₂O₂ also increased the survival of H₂O₂-treated cells. Reactivation of UV- and X-irradiated and methylmethane sulphonate and H₂O₂-treated phage b-1 was induced by O₂ and H₂O₂ in B. fragilis .     

Response of antioxidant systems to NaCl stress in the halophyte Cakile maritima

The effects of salt stress on antioxidative activities were investigated in a coastal halophyte, Cakile maritima . Two Tunisian accessions, Jerba and Tabarka, were compared. Plants were subjected to 100, 200, or 400 m M NaCl for 20 days. Parameters of oxidative stress [malondialdehyde (MDA), electrolyte leakage (EL), and hydrogen peroxide (H₂O₂) concentration], activities of several enzymes [superoxide dismutase (SOD), catalase (CAT), peroxydase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR)], and antioxidant molecules (ascorbate, ASC, and glutathione, GSH) were determined. Growth of Jerba plants was improved at 100 m M NaCl as compared to that of control. Tabarka growth was inhibited by salt at all NaCl concentrations. The relative salt tolerance of Jerba was associated with high antioxidant enzyme activities and glutathione content, together with low MDA content, EL, and H₂O₂ concentration. Lower antioxidant activities and higher MDA content, EL, and H₂O₂ concentration were found in Tabarka. As a whole, these data suggest that the capacity to limit oxidative damage is important for salt tolerance of C. maritima .     

Studies on the lactoperoxidase system: reaction kinetics and antibacterial activity using two methods for hydrogen peroxide generation

D.A. DIONYSIUS, P.A. GRIEVE AND A.C. VOS. 1992. Components of the lactoperoxidase system were measured during incubation in Isosensitest broth, with enzymatic (glucose oxidase, GO) or chemical (sodium carbonate peroxyhydrate, SCP) means to generate H₂O₂. When low levels of thiocyanate (SCN^-) were used in the GO system, H₂O₂ was detected and lactoperoxidase (LP) was inactivated when SCN^- was depleted. With 10-fold higher SCN^-, LP remained active and H₂O₂ was not detectable. The oxidation product of the LP reaction, most likely hypothiocyanite, was present in low concentrations. When SCP was used for the immediate generation of H₂O₂ in a system employing low SCN^-, half the LP activity was lost within minutes but thereafter it remained stable. Low concentrations of oxidation product were measured and H₂O₂ was not detected during the course of the experiment. At high SCN^- levels, relatively high concentrations of oxidation product were produced immediately, with H₂O₂ undetectable. The results suggest that the final product of the LP reaction depends on the method of H₂O₂ generation and the relative proportions of the substrates. Antibacterial activity of the two LPS was tested against an enterotoxigenic strain of Escherichia coli. Both systems showed bactericidal activity within 4 h incubation at 37°C.     

Elicitation of H2O2 production in cucumber hypocotyl segments by oligo-1,4-α-D-galacturonides and an oligo-β-glucan preparation from cell walls of Phythophthora megasperma f. sp. glycinea

The production of H₂O₂ by cucumber hypocotyl segments ( Cucumis sativus L. cv. Wisconsin SMR 58) in response to α-1,4-linked oligomers of galacturonic acid and oligo-β-glucans from the cell walls of Phytophthora megasperma f. sp. glycinea was studied. Oligogalacturonides with degrees of polymerization of 9 to 13 elicited H₂O₂ production, the most effective being the deca-, undeca- and dodecamers. A similar relationship between size and effect was previously obtained when oligogalacturonides were tested for their ability to elicit lignification in cucumber hypocotyls. The oligogalacturonide-induced increase in H₂O₂ concentration was detected after 4 h, reaching a maximum after 10 h of incubation. The glucan elicitor induced lignification at a 100-fold lower concentration than the oligogalacturonides, but yielded only 10% of the maximum H₂O₂ accumulation seen with oligogalacturonides. The glucan elicitor-induced H₂O₂ production was detectable after 2 h, and reached a maximum after 4 to 6 h. Catalase abolished the elicitation of both phenol red oxidation and lignification in cucumber hypocotyls. At least part of the oligogalacturonide-induced H₂O₂ production appeared to be dependent upon de novo protein synthesis.     

Size, acetylation and concentration of chitooligosaccharide elicitors determine the switch from defence involving PAL activation to cell death and water peroxide production in Arabidopsis cell suspensions

Chitosan oligomers are known elicitors of plant defence mechanisms. In this work, chitooligosaccharides of different degrees of polymerization and degrees of acetylation were prepared and characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The effect of the degree of polymerization (DP), degree of acetylation and concentration of these chitooligosaccharides on defence activation in Arabidopsis thaliana suspension-cultured cells was studied. Our study results show that fully deacetylated chitooligosaccharides (chitosan oligomers) induce, depending on their DP and concentration, phenylalanine ammonia-lyase (PAL) activation, H₂O₂ synthesis and cell death in A. thaliana cell suspensions. The progressive reacetylation of the chitosan oligomer elicitors progressively impaired their ability to enhance H₂O₂ accumulation and cell death, but did not affect the activation of PAL.     

Inhibition of Methanosarcina barkeri strain 227 by cadmium

Abstract The effect of cadmium (Cd) on methane formation from methanol and/or H₂–CO₂ by Methanosarcina barkeri was examined in a defined growth medium and in a simplified buffer system containing 50 mM Tes with or without 2 mM dithiothreitol (DTT). No inhibition of methanogenesis by high concentrations of cadmium was observed in growth medium. Similarly, little inhibition of methanogenesis by whole cells in the Tes buffer system was observed in the presence of 430 μM Cd or 370 μM mercury (Hg) with 2 mM DTT. When the concentration of DTT was reduced to 0.4 mM, almost complete inhibition of methanogenesis from H₂–CO₂ and methanol by 600 μM Cd was observed. In the absence of DTT, 150 μM Cd inhibited methanogenesis from H₂–CO₂ completely and from methanol by 97%. Methanogenesis from H₂–CO₂ was more sensitive to Cd than that from methanol.     

Oxidative burst and expression of germin/oxo genes during wounding of ryegrass leaf blades: comparison with senescence of leaf sheaths

Two bursts of H₂O₂ production have been detected by in situ 3,3'-diaminobenzidine (DAB) staining after cutting of Lolium perenne L. leaf blades. The first burst, which occurred immediately after wounding was inhibited by Na-diethydithiocarbamate (DIECA), a Cu/Zn–superoxide dismutase (SOD) inhibitor. The second burst, which was initiated several hours later, coincided with the induction of oxalate oxidase (G-OXO) activity detected in vitro or visualized in situ by the α-chloronaphtol assay. Four genes encoding G-OXO have been identified from cDNA obtained from wounded L. perenne L . leaf blades. Comparison of protein sequences revealed more than 91% homology in the coding region between G-OXOs of the true cereals and G-OXOs of ryegrass, which is a Gramineae belonging to the tribe of Festucaceae. The wound-dependent increase of G-OXO activity in floated cut leaf blades was the result of differential induction of the four g-oxo genes. The involvement of G-OXOs in wound-induced H₂O₂ production coincided with the presence in leaf tissues of oxalate throughout the period of increase of G-OXO synthesis. Moreover, expression of g-oxo genes was enhanced by an exogenous supply of H₂O₂ or methyljasmonate (MeJa). Expression of the four g-oxo genes was also induced after in planta stinging of leaf blades. The pattern of their expression in planta was identical to that occuring in senescing leaf sheaths. These results emphasize the importance of G-OXOs in H₂O₂ production in oxalate-producing plant species such as ryegrass. G-OXOs might be crucial during critical events in the life of plants such as cutting and senescence by initiating H₂O₂-mediated defences against pathogens and foraging animals.     

Detection of hydrogen peroxide produced by meat lactic starter cultures

Twelve strains of meat lactic starter cultures (Pediococcus spp. and Lactobacillus plantarum) were found to produce hydrogen peroxide in vitro. The (cumulative) amounts of H₂O₂ produced were measured through the peroxidative action of catalase on H₂O₂ and oxidation of added formate to CO₂ by the H₂O₂-catalase complex formed. There was a problem in building a calibration curve for converting values of formate oxidation into amounts of H₂O₂, either by adding H₂O₂ directly to the assay mixture or having it produced via a glucose-glucose oxidase system.     

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H₂O₂ on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H₂O₂ (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca²⁺-dependent exocytosis of glutamate, induced by KCl or by the K⁺-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H₂O₂, although a transient decrease was observed after the treatment. H₂O₂ did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H₂O₂ did not modify significantly the KCl-induced increase of [Ca²⁺]_i. H₂O₂ inhibited exocytosis also when the latter was induced by increasing [Ca²⁺]_i with the Ca²⁺ ionophore ionomycin. The effects of H₂O₂ were unchanged in the presence of superoxide dismutase and the presence of the Fe³⁺ chelator deferoxamine. These results appear to indicate that H₂O₂, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.     

Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?

Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid-binding protein (SABP) isolated from tobacco was shown to have catalase activity. Based on this finding elevated levels of hydrogen peroxide (H₂O₂) were postulated to act as a second messenger of SA in the SAR signal transduction pathway. A series of experiments have been carried out to clarify the role of H₂O₂ in SAR-signaling. No increase of H₂O₂ was found during the onset of SAR. Induction of the SAR gene, PR-1, by H₂O₂ and H₂O₂-inducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H₂O₂ induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H₂O₂, a dose-dependent accumulation of total SA species was found, suggesting that H₂O₂ may induce PR-1 gene expression through SA accumulation. While the results do not support a role for H₂O₂ in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.     

Correlation of resistance and H2O2 production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with Ophiostoma novo-ulmi

The Dutch elm disease (DED) pathogen Ophiostoma novo-ulmi Buissm. elicited the production of H₂O₂ in cell suspension cultures of the resistant species Ulmus pumila L. This response was not observed in suspensions of the susceptible elm U. campestris Mill. H₂O₂ production started after a lag time of 30–40 min following inoculation, peaked between 4 and 6 h and lasted up to 24 h. Treatment of the suspensions with exogenously added H₂O₂ did not cause accumulation of the sesquiterpene phytoalexins mansonones nor of the coumarin scopoletin. Spore germination and growth of O. novo-ulmi were significantly delayed with different amounts of H₂O₂ (0.1–1 m M ). These results suggest that H₂O₂ production is an inducible defence response which may contribute to DED resistance by delaying the growth of the pathogen at the earliest stages of infection. Whether H₂O₂ is involved in other elm defence responses to the pathogen is presently unknown, but its production seems to be an independent event from phytoalexin formation.     

Detoxification of hydrogen peroxide maintains the water transport activity in figleaf gourd (Cucurbita ficifolia) root system exposed to low temperature

The figleaf gourd ( Cucurbita ficifolia Bouché) root system has the ability to take up water and nutrients at low soil temperatures, and in the present paper, we attempt to reveal some of the molecular mechanisms behind this low-temperature tolerance. Exposure of figleaf gourd root system to low temperature induced accumulation of H₂O₂ along the plasma membrane but not in the cytoplasm. H⁺-ATPase (EC 3.6.1.35) activity of isolated root plasma membranes and root hydraulic conductivity ( Lp_r ) were largely insensitive to externally applied H₂O₂. However, using bromocresol purple, it was shown that the acidification of the medium surrounding the root was strongly inhibited with low temperature- and H₂O₂-treated roots. Addition of catalase (EC 1.11.1.6) to the root medium during low-temperature exposure led to a recovery of H⁺-efflux along the root surface and increased Lp_r , demonstrating the importance of an H₂O₂ detoxification system in the root cells. Additional evidence for an increased Lp_r was obtained by the Fenton reaction wherein a warming of the solution increased the activity of the detoxification system. All available evidence suggests that the ability of figleaf gourd root system to maintain a low level of H₂O₂ in the cytoplasm and to detoxify reactive oxygen species is related to the maintenance of water transport activity at low temperatures.     

Peroxide inducible phage reactivation in Bacteroides fragilis

Abstract Reactivation of UV-irradiated phage b-1 was induced by H₂O₂ and UV in Bacteroides fragilis . The characteristics of H₂O₂ and UV induced phage reactivation differ from a previously reported oxygen induced reactivation system. The survival of B. fragilis cells after UV irradiation was also increased by pretreatment with H₂O₂. DNA synthesis was not inhibited in the host cells exposed to H₂O₂ concentrations which induced phage reactivation. The pattern of DNA degradation and synthesis after UV irradiation with and without H₂O₂ differed from the effect of O₂ on DNA synthesis in irradiated B. fragilis cells.     

Interaction of α-Phenyl-N-tert-Butyl Nitrone and Alternative Electron Acceptors with Complex I Indicates a Substrate Reduction Site Upstream from the Rotenone Binding Site

Abstract: Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O₂ to hydrogen peroxide (H₂O₂) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H₂O₂ formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI-mediated enzyme inactivation. The O₂ consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H₂O₂ generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH-coupled H₂O₂ flux was accelerated by rotenone. α-Phenyl- N-tert -butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex I-stimulated H₂O₂ flux and NBT reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H₂O₂ synthesis, possessing an "electron leak" site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H₂O₂ production by PBN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.