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.     

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 .     

Hydrogen peroxide formation in cultured rose cells in response to UV-C radiation

Suspension-cultured rose ( Rosa damascena Mill. cv. Gloire de Guilan) cells irradiated with UV-C (254 nm. 558 J m⁻²) showed a transient production of H₂O₂ as measured by chemiluminescence of luminol in the presence of peroxidase (EC 1.1 1.1.7). The peak concentration of H₂O₂, which occurred at about 60–90 min after irradiation, was 8–9 μ M . The time course for the appearance of H₂O₂ matched that for UV–induced K⁺ efflux. Treatments that inhibited the UV-induced efflux of K⁺, including heat and overnight incubation with cycloheximide and diethylmaleate, also inhibited the appearance of H₂O₂. The converse was not always true, since catalase (EC 1.11.1.6. and salicylhydroxamic acid, which inhibited luminescence, did not stop K⁺ efflux. We conclude that H₂O₂ synthesis depends on K⁺ efflux. Because H₂.O₂ in the extracellular space is required for lignin synthesis in many plant tissues, we suggest that the UV–stimulated production of H₂O₂ is an integral part of a defensive lignin synthesis.     

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.     

Molecular masses of cAMP-binding proteins in yeasts

Abstract The cAMP-binding proteins of different yeasts were photoaffinity labeled using 8- N ₃-[³²P]cAMP, and the M _r values of the labeled proteins estimated by SDS-polyacrylamide gel electrophoresis. The M _r values of the cAMP-binding proteins may be grouped into two size classes: (A) M _r of about 50 000 represented by Saccharomyces cerevisiae and S. uvarum , and (B) M _r of about 60 000 represented by Kluyveromyces fragilis, K. lactis, K. marxianus, S. globosus and S. rouxii .     

Cytoprotective effect of polypeptide from Chlamys farreri on neuroblastoma (SH-SY5Y) cells following H2O2 exposure involves scavenging ROS and inhibition JNK phosphorylation

Oxidative stress has long been linked to cell death in many neurodegenerative conditions. Treatment with antioxidants is a promising approach for slowing disease progression. In this study, we used the neuroblastoma SH-SY5Y cells as an in vitro model to first assess the effect of polypeptide from Chlamys farreri (PCF), a natural marine antioxidant, on H₂O₂-induced neuronal cell death. Pre-treatment of SH-SY5Y cells with PCF inhibited H₂O₂-induced cell death in a concentration-dependent manner. In parallel, intracellular reactive oxygen species generation and lipid peroxidation were inhibited by PCF. Under severe H₂O₂ insult, PCF promoted endogenous antioxidant defense components including glutathione peroxidase, catalase, superoxide dismutase, and glutathione. PCF also protected DNA from oxidative damage and enhanced the removal of 8-oxo-7,8-dihydro-2'-deoxyguanosine from DNA. Further, we found that PCF potentially prevented H₂O₂–induced cell apoptosis. When investigated mitogen-activated protein kinase signaling pathway, we found that pre-treatment of cells with PCF significantly blocked H₂O₂–induced phosphorylation of c- Jun N-terminal kinase of the mitogen-activated protein kinase family. However, PCF had little inhibitory effect on the H₂O₂–induced activation of extracellular signal-regulated kinase. Taken together, these data demonstrate that PCF prevents oxidative stress-induced reactive oxygen species production and c- Jun N-terminal kinase activation and may be useful in the treatment of neurodegenerative diseases.     

Hydrogen peroxide release from bacterial spores during germination

The release of free H₂O₂ from spores of Clostridium perfringens and Bacillus megaterium during germination has been demonstrated using the scopoletin fluorescence assay. Scopoletin oxidation was markedly inhibited when exogenous catalase was added, and was also influenced by the concentration of spores. H₂O₂ release into the germination medium was observed to parallel the O₂ consumption during germination, suggesting that the H₂O₂ may arise from certain O₂-dependent metabolism associated with initiation of spore germination.     

Identification of three porins in the outer membrane of Bacteroides fragilis

Abstract Four outer membrane proteins were purified to homogeneity from isolated outer membranes of Bacteroides fragilis ; three ( M _r 51000, 92000 and 125 000) had pore-forming activity in reconstituted liposomes as determined by swelling assay. Membrane vesicles containing the M _rmr 55 000 outer membrane protein showed no detectable pore-forming activity. The three B. fragilis porins formed pores that allowed the penetration of uncharged saccharides of M _r lower than 340–400, even though the efficiency of solute diffusion showed slight differences. The diffusion rates of glucose through the porins appeared to be lower than those through Escherichia coli porins.     

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.     

A stress-induced oxidative burst in Eucheuma platycladum (Rhodophyta)

A hurst of hydrogen peroxide has been found in the red macroalga Eucheuma platycladwn Schmitz as a response to mechanical stress. After exposure of pieces of thalli (2 cm) broken from the plant and stirred with a magnetic bar an oxidative burst was registered, as measured by luminol dependent chemiluminescence (LDC). The burst was totally inhibited by cataluse (EC 1.11.1.6). showing the generation of H_2:O_2; Ten g of seaweed in 300 ml sea water caused a maximal medium concentration of LDC corresponding to 7 u .M H₂O_2; The burst decayed after about 30 min. The decay is probably caused by increased catalase aciivity of the sea water. due to leakage of catalasc from the seaweed. Addition of NaN₃ caused a dramatic increase in LDC. probably due to inhibition of catalase. Similar bursts of active oxygen, involving active oxygen species such as O₂, H₂O₂ and OH. have been reported as pan of the hypcrsensitive reaction in some higher plants, e.g. tobacco. potato and soybean. Exposure of plants or cell suspension cultures to some pathogenic bacteria, fungi, inorganic elicitors or physical damage causes an oxidalive burst that is often followed by necrosis. The production ot active oxygen is thought to he a first defence against invading pathogens. We assume that the oxidative burst from E. platycladum is of a defensive nature, providing a protection against grazers and pathogenic organisms. To our knowledge this is the first repoil of an oxidalive burst from seaweeds.     

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.     

Selective protection by phosphatidic acid against staurosporine-induced neuronal apoptosis

Phosphatidic acid, the main product of lipid breakdown through phospholipase D activation, has been implicated in important signal transduction pathways able to influence cell fate in many ways. The purpose of this work was to determine possible effects of phosphatidic acid on neuronal cell death pathways. Here we used cerebellar granular cell cultures and cell death was triggered with either staurosporine or H₂O₂. Cell viability was quantified by spectrophotometry, using the 3- (4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl-tetrazolium bromide (MTT) test. Staurosporine (1-3 μM) or H₂O₂ (50-800 μM) induced cell death in a dose-dependent manner. Using fluorescent staining (propidium iodide or annexin V-Cy3/6-carboxyfluorescein) we showed that cell death was mostly apoptotic in staurosporine treated cells and mostly non-apoptotic (necrotic) in H2O2 treated cells. Phosphatidic acid was able to increase cell viability in staurosporine-, but not in H₂O₂ - treated cells. We therefore conclude that phosphatidic acid has neuroprotective potential in neurons exposed to stimuli that trigger apoptosis.     

Oxidant Injury in PC12 Cells—A Possible Model of Calcium "Dysregulation" in Aging: II. Interactions with Membrane Lipids

Abstract: In a model recently developed to study the parameters altering vulnerability to oxidative stress, it was shown via image analysis that H₂O₂-exposed PC12 cells exhibited increased levels of intracellular Ca²⁺ (baseline), decreases in K⁺-stimulated Ca²⁺ levels (peak), and decreased poststimulation Ca²⁺ clearance (recovery). The present experiments were performed to determine if the response patterns in these parameters to oxidative stress would be altered after modification of membrane lipid composition induced by incubating the PC12 cells with 660 µ M cholesterol (CHL) in the presence or absence of 500 µ M sphingomyelin (SPH) before low (5 µ M ) or high (300 µ M ) H₂O₂ exposure. Neither CHL nor SPH had synergistic effects with high concentrations of H₂O₂ on baseline. However, CHL in the presence or absence of SPH reversed the effect of low concentrations of H₂O₂ on baseline. SPH decreased significantly the cell's ability to clear excess Ca²⁺ in the presence or absence of H₂O₂ and increased significantly the level of conjugated dienes (CDs). It is surprising that in the cells pretreated with CHL, the CD levels were not significantly different from controls. However, in the presence of SPH, the effects of CHL on CDs were altered. These results suggest that the ratios of membrane lipids could be of critical importance in determining the vulnerability to oxidative stress and Ca²⁺ translocation in membranes. This may be of critical importance in aging where there is increased membrane SPH and significant loss of calcium homeostasis.     

Effect of broad-band ultraviolet and visible radiation on hydrogen peroxide formation by cultured rose cells

Broad-band radiation from a high-pressure Hg-vapor lamp, including ultraviolet wavelengths from 290 to 400 nm, blue, green and red wavelengths, did not induce the synthesis of H₂O₂ in cultured rose cells. This was in contrast to the effects of shortwave (254 nm) ultraviolet radiation, even though, like shortwave ultraviolet radiation, the UV-B component of the broadband radiation induced a striking K⁺ efflux from the cells, and this efflux has been associated with H₂O₂ synthesis in a previous report. The UV-A and visible wavelengths were shown to inhibit the synthesis of H₂O₂. This effect was associated with inhibition of peroxidase, an enzyme reported to be involved in the synthesis of H₂O₂ in cell walls. UV-B radiation inhibited the alternate pathway for mitochondrial electron transport, but there was no evidence that this effect contributed to the inhibition of H₂O₂ synthesis in cells treated with broad-band radiation.     

Salicylhydroxamic acid-stimulated NADH oxidation by purified plasmalemma vesicles from wheat roots

Purified, right side-out plasmalemma vesicles were isolated from 7-day-old roots of dark-grown wheat ( Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. The oxygen consumption by these vesicles at pH 6.5 in the presence of 1 m M NADH [12–29 nmol (mg protein)⁻¹min⁻¹] was 66% inhibited by 1 m M KCN and ca 40% by 1 m M EDTA. It was unaffected by rotenone, antimycin A, carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), mersalyl, chlorotetracycline + Ca²⁺, and EGTA. Salicylhydroxamic acid (SHAM) and its analogue, m -chlorobenzhydroxamic acid, stimulated the rate of oxygen consumption 10–20 fold in the presence of 1 m M NAD(P)H with an apparent K_m (SHAM) of ca 40 μ M (with NADH). The dependence of O₂ consumption on NADH concentration in the presence of SHAM (2 m M ) was sigmoidal, possibly due to endogenous catalase activity, and half-maximal rate was obtained at 1.5 m M . In the absence of SHAM the rate increased with increasing acidity and no pH optimum was detectable between pH 4.5 and 8.5. In the presence of SHAM an optimum was observed at pH 6.5 and 0.8 mol of H₂O₂ was produced for every 1 mol O₂ consumed. Endogenous catalase converted this H₂O₂ to O₂ and after complete conversion the stoichiometry was 2 mol NADH consumed for every mol O₃. SHAM was not consumed in the reaction. The possible involvement of a cytochrome P-450/420 system is discussed.     

Temperature profile of oxygen activation and defense against oxygen toxicity in a psychrophilic member of the Bacteroidaceae

Abstract The temperature profiles have been determined for O₂ reduction by activating substrates for whole cells and cell extracts of the psychrophilic, obligately anaerobic bacterium, strain B6, belonging to the Bacteroidaceae. The profiles were similar whether the cells were grown at 15 or 1°C, and also for cells harvested in the exponential or stationary phase. The H₂O producing pyruvate oxidase displayed in cell-free extracts a considerably higher activity than the H₂O₂ producing NADH and NADPH oxidases at all temperatures in the range 30–1°C, and characteristically makes up a larger proportion of the total O₂ reduction capacity the lower the temperature. It thus seems that the O₂ scavenging property of the pyruvate oxidase, postulated to be utilized in a defense mechanism against the detrimental effects of the H₂O₂ producing pyridine nucleotide oxidases, is particularly well adapted to function at the low temperatures of the Barents Sea, from which this obligately anaerobic organism originates.     

Metabolism of hydrogen peroxide by the scavenging system in Chlamydomonas reinhardtii

The metabolism of hydrogen peroxide by the scavenging system was studied in Chlamydomonas grown in a selenium-lacking and a selenium-containing medium. In cells of the former, 40% of external hydrogen peroxide (H₂O₂) was scavenged by ascorbate peroxidase (AsAP; EC 1.11.1.11) and the residual H₂O₂ by catalase (EC 1.11.1.6). The enzymes involved in the ascorbate-glutathione cycle including AsAP. were localized in the chloroplast. In cells of the latter, glutathione peroxidase (GSHP; EC 1.11.1.9) functioned primarily in the removal of external H₂O₂. GSHP was located solely in the cytosol. The Chlamydomonas AsAP was relatively stable in ascorbate-depleted medium as compared with chloroplast AsAP of higher plants. No inactivation of the enzyme was found upon its incubation with hydroxyurea, an inhibitor of the chloroplast enzyme of higher plants. The enzyme showed higher specificity with pyrogallol than with ascorbate. The amino acid sequences in the N-terminal region of Chlamvdomonas AsAP showed no significant similarity to any other AsAP from higher plants and Euglena . The enzyme had a molecular mass of 34 kDa. The K_m values of the enzyme for ascorbate and H₂O₂ were 5.2±0.3 and 25±3.4 μ M , respectively. Hydrogen peroxide was generated at a rate of 6.1±0.8 μmol mg^-1 chlorophyll h^-1 in intact chloroplasts isolated from Chlamydomonas cells grown in the presence of Na-selenite, and it diffused from the organelles into the medium.     

Fusarium response to oxidative stress by H2O2 is trichothecene chemotype-dependent

The present study aims at clarifying the impact of oxidative stress on type B trichothecene production. The responses to hydrogen peroxide (H₂O₂) of an array of Fusarium graminearum and Fusarium culmorum strains were compared, both species carrying either the chemotype deoxynivalenol (DON) or nivalenol (NIV). In both cases, levels of in vitro toxin production are greatly influenced by the oxidative parameters of the medium. A 0.5 mM H₂O₂ stress induces a two- to 50-fold enhancement of DON and acetyldeoxynivalenol production, whereas the same treatment results in a 2.4- to sevenfold decrease in NIV and fusarenone X accumulation. Different effects of oxidative stress on toxin production are the result of a variation in Fusarium 's antioxidant defence responses according to the chemotype of the isolate. Compared with DON strains, NIV isolates have a higher H₂O₂-destroying capacity, which partially results from a significant enhancement of catalase activity induced by peroxide stress. A 0.5 mM H₂O₂ treatment leads to a 1.3- to 1.7-fold increase in the catalase activity of NIV isolates. Our data, which show the higher adaptation to oxidative stress developed by NIV isolates, are consistent with the higher virulence of these Fusarium strains on maize compared with DON isolates.