Reactive oxygen species generated in the presence of fine pyrite particles and its implication in thermophilic mineral bioleaching

In the tank bioleaching process, maximising solid loading and mineral availability, the latter through decreasing particle size, are key to maximising metal extraction. In this study, the effect of particle size distribution on bioleaching performance and microbial growth was studied through applying knowledge based on medical geology research to understand the adverse effects of suspended fine pyrite particles. Small-scale leaching studies, using pyrite concentrate fractions (106–75, 75–25, ?25 μm fines), were used to confirm decreasing performance with decreasing particle size (D ₅₀ <40 μm). Under equivalent experimental conditions, the generation of the reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals from pyrite was illustrated. ROS generation measured from the different pyrite fractions was found to increase with increasing pyrite surface area loading (1.79–74.01 m² L^?1) and Fe²⁺ concentration (0.1–2.8 g?L^?1) in solution. The highest concentration of ROS was measured from the finest fraction of pyrite (0.85 mM) and from the largest concentration of Fe²⁺ (0.78 mM). No ROS was detected from solutions containing only Fe³⁺ under the same conditions tested. The potential of ROS to inhibit microbial performance under bioleaching conditions was demonstrated. Pyrite-free Sulfolobus metallicus cultures challenged with hydrogen peroxide (0.5–2.5 mM) showed significant decrease in both cell growth and Fe²⁺ oxidation rates within the concentration range 1.5–2.5 mM. In combination, the results from this study suggest that conditions of large pyrite surface area loading, coupled with high concentrations of dissolved Fe²⁺, can lead to the generation of ROS, resulting in oxidative stress of the microorganisms.     

Light emission from the Fe2+–EDTA–ascorbic acid–H2O2 system strongly enhanced by plant phenolic acids

Oxidative reactions can result in the formation of electronically excited species that undergo radiative decay depending on electronic transition from the excited state to the ground state with subsequent ultra‐weak photon emission (UPE). We investigated the UPE from the Fe²⁺–EDTA (ethylenediaminetetraacetic acid)–AA (ascorbic acid)–H₂O₂ (hydrogen peroxide) system with a multitube luminometer (Peltier‐cooled photon counter, spectral range 380–630 nm). The UPE, of 92.6 μmol/L Fe²⁺, 185.2 μmol/L EDTA, 472 μmol/L AA, 2.6 mmol/L H₂O₂, reached 1217 ± 118 relative light units during 2 min measurement and was about two times higher (P < 0.001) than the UPE of incomplete systems (Fe²⁺–AA–H₂O₂, Fe²⁺–EDTA–H₂O₂, AA–H₂O₂) and medium alone. Substitution of Fe²⁺ with Cr²⁺, Co²⁺, Mn²⁺ or Cu²⁺ as well as of EDTA with EGTA (ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid) or citrate powerfully inhibited UPE. Experiments with scavengers of reactive oxygen species (dimethyl sulfoxide, mannitol, sodium azide, superoxide dismutase) revealed the dependence of UPE only on hydroxyl radicals. Dimethyl sulfoxide at the concentration of 0.74 mmol/L inhibited UPE by 79 ± 4%. Plant phenolics (ferulic, chlorogenic and caffec acids) at the concentration of 870 μmol/L strongly enhanced UPE by 5‐, 13.9‐ and 46.8‐times (P < 0.001), respectively. It is suggested that augmentation of UPE from Fe²⁺–EDTA–AA–H₂O₂ system can be applied for detection of these phytochemicals.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp.

The induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp. was investigated. H₂O₂ (0.1 mM) enhanced the total astaxanthin formation from 5.8 to 6.5 mg g^–1 cell dry wt. Fe²⁺ (0.5 mM) added to the medium with H₂O₂ (0.1 mM) further promoted astaxanthin formation to 7.1 mg g^–1 cell dry wt. Similarly, Fe²⁺ (0.5 mM) together with methyl viologen (0.01 mM) promoted astaxanthin formation to 6.3 mg g^–1 cell dry wt. In contrast, an addition of KI (1 mM), a specific scavenger for hydroxyl radicals (OH), together with H₂O₂ (0.1 mM) and Fe²⁺ (0.5 mM), to the medium decreased astaxanthin formation to 1.8 mg g^–1 cell dry wt. KI (1 mM) also inhibited the enhancement of carotenogenesis by superoxide anion radicals (O₂ ^–), with a decrease of astaxanthin formation to 1.7 mg g^–1 cell dry wt. This suggested that O₂ ^– might be transformed to OH before promoting carotenogenesis in Chlorococcum sp.     

The effects of structural variations of thiophene-containing Ru(II) complexes on the acid–base and DNA binding properties

A phenylthiophenyl-bearing Ru(II) complex of [Ru(bpy)₂(Hbptip)](PF₆)₂ {bpy?=?2,2′-bipyridine, Hbptip?=?2-(4-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline} was synthesized and characterized by elemental analysis, ¹H NMR spectroscopy, and electrospray ionization mass spectrometry. The ground- and excited-state acid–base properties of the complex were studied by UV–visible absorption and photoluminescence spectrophotometric pH titrations and the negative logarithm values of the ground-state acid ionization constants were derived to be pK _a1?=?1.31?±?0.09 and pK _a2?=?5.71?±?0.11 with the pK _a2 associated deprotonation/protonation process occurring over 3 pK _a units more acidic than thiophenyl-free parent complex of [Ru(bpy)₂(Hpip)]²⁺ {Hpip?=?2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline}. The calf thymus DNA-binding properties of [Ru(bpy)₂(Hbptip)]²⁺ in Tris–HCl buffer (pH 7.1 and 50?mM NaCl) were investigated by DNA viscosities and density functional theoretical calculations as well as UV–visible and emission spectroscopy techniques of UV–visible and luminescence titrations, steady-state emission quenching by [Fe(CN)₆]^4?, DNA competitive binding with ethidium bromide, DNA melting experiments, and reverse salt effects. The complex was evidenced to bind to the DNA intercalatively with binding affinity being greater than those for previously reported analogs of [Ru(bpy)₂(Hip)]²⁺, [Ru(bpy)₂(Htip)]²⁺, and [Ru(bpy)₂(Haptip)]²⁺ {Hip?=?1H-imidazo[4,5-f][1,10]phenanthroline, Htip?=?2-thiophenimidazo[4,5-f][1,10]phenanthroline, Haptip?=?2-(5-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline}.     

Mechanisms of Saccharomyces Cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H⁺-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H₂O₂, Fe²⁺, Fe- and Cu-Fenton reagents. Inactivation by Fe²⁺ required the presence of oxygen and hence involved auto-oxidation of Fe²⁺ to Fe³⁺. The highest Fe^2- (100 μM) and H₂O₂ (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe²⁺ content than on H₂O₂ concentration, occurred only when Fe²⁺ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN₃, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDs, bipyridine, 1, 10-phenanthroline). Inactivation by Fe- and Cu- Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH^·. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H⁺-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe²⁺ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe²⁺, blocked H⁺-ATPase inactivation by Fe²⁺ and the Fenton reagent but not that caused by H₂O₂, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H⁺-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.     

Reversible sporicidal action of cuprous ions

At 10 mM, Cu⁺ was highly protective against killing of spores of Bacillus megaterium ATCC 19213 by H₂O₂, while at higher concentrations, from 15–100 mM, killing was augmented. In contrast, Cu²⁺, Fe²⁺, Fe³⁺, Co²⁺ or Co³⁺ ions acted only protectively. Cu⁺ itself was sporicidal in the absence of H₂O₂ or ascorbate, and its sporicidal action did not depend on generation of highly reactive oxygen species. It appeared that killing involved either inhibition of germination or copper toxicity to germinated cells in that Cu⁺-inactivated spores did not germinate readily but chemical decoating of the cells prior to plating on a solid medium resulted in reversal of the sporicidal effect. Received 12 July 1996/ Accepted in revised form 03 November 1996     

Inactivation of the plasma membrane ATPase ofSchizosaccharomyces pombe by hydrogen peroxide and by the fenton reagent (Fe2+/H2O2): Nonradicalvs. Radical-induced oxidation

In the absence of added Fe²⁺, the ATPase activity of isolatedSchizosaccharomyces pombe plasma membranes (5–7 μmolP _i per mg protein per min) is moderately inhibited by H₂O₂ in a concentration-dependent manner. Sizable inactivation occurs only at 50–80 mmol/L H₂O₂. The process, probably a direct oxidative action of H₂O₂ on the enzyme, is not induced by the indigenous membrane-bound iron (19.3 nmol/mg membrane protein), is not affected by the radical scavengers mannitol and Tris, and involves a decrease of both theK _m of the enzyme for ATP and theV of ATP splitting. On exposing the membranes to the Fenton reagent (50 μmol/L Fe²⁺ +20 mmol/L H₂O₂), which causes a fast production of HO⁻ radicals, the ATPase is 50–60% inactivated and 90% of added Fe²⁺ is oxidized to Fe³⁺ within 1 min. The inactivation occurs only when Fe²⁺ is added before H₂O₂ and can thus bind to the membranes. The lack of effect of radical scavengers (mannitol, Tris) indicates that HO⁻ radicals produced in the bulk phase play no role in inactivation. Blockage of the inactivation by the iron chelator deferrioxamine implies that the process requires the presence of Fe²⁺ ions bound to binding sites on the enzyme molecules. Added catalase, which competes with Fe²⁺ for H₂O₂, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide radical that gives rise to delayed HO⁻ production.     

Xylarinase: a novel clot busting enzyme from an endophytic fungus Xylaria curta

Xylarinase is a bi-functional fibrinolytic metalloprotease isolated from the culture filtrate of endophytic fungus Xylaria curta which is monomeric with a molecular mass of ～33.76?kDa. The enzyme displayed both plasmin and tissue plasminogen activator like activity under in vitro conditions. It hydrolyses Aα and Bβ chains of the fibrinogen. Optimal fibrinolytic activity of xylarinase is observed at 35?°C, pH 8. Ca²⁺ stimulated the fibrinolytic activity of xylarinase while Fe²⁺ and Zn²⁺ inhibited suggesting it to be a metalloprotease. The K_m and V_max values of xylarinase were 240.9?μM and 1.10?U/ml for fibrinogen and 246?μM and 1.22?U/ml for fibrin, respectively. Xylarinase was found to prolong the activated partial thromboplastin time and prothrombin time. The N-terminal sequence of xylarinase (SNGPLPGGVVWAG) did not show any homology with previously known fibrinolytic enzymes. Thus xylarinase is a novel fibrinolytic metalloprotease which could be possibly used as a new clot busting enzyme.     

Fungal co-culture increases ligninolytic enzyme activities: statistical optimization using response surface methodology

Abstract

The optimization of ligninolytic enzyme (LE) activities in a novel fungal co-culture between Pycnoporus sanguineus and Beauveria brongniartii were studied using a Plackett–Burman experimental design (PBED) and a central composite design (CCD). In addition, H₂O₂ role was analyzed. Laccase (EC. 1.10.3.2) and MnP (EC 1.11.1.14) activities of P. sanguineus increased 6.0- and 2.3-fold, respectively, in the co-culture with B. brongniartii. The H₂O₂ content was higher in the co-culture (0.33–7.12-fold) than in the P. sanguineus monoculture. The PBED revealed that yeast extract (YE), FeSO_4, and inoculum amount were significant factors for laccase and MnP activities and H₂O₂ production in the co-culture, which increased by 8.2-, 5.2- and 1.03-fold, respectively. The YE and FeSO₄ were studied using a CCD to optimize the studied response variables. Laccase activity was enhanced 1.5-fold by CCD, the optimal amount of YE was 0.366?g L^?1. Quadratic term of FeSO₄ modulated MnP activity and was associated with a 4.28-fold increase compared to the PBED. Both YE and its quadratic term significantly affected H₂O₂ production; however, the CCD did not enable an increase in H₂O₂ production. Pearson correlation indicated an increase in laccase (r²=0.4411, p?=?0.0436) and MnP (r²=0.5186, p?=?0.0198) activities following increases in H₂O₂ in the co-culture system.     

Cobalt-chelated magnetic particles for one-step purification and immobilization of His6-tagged Escherichia coli γ-glutamyltranspeptidase

Cobalt-chelated magnetic (Fe₃O₄-AA-ANTA-Co^{2 +}) particles were prepared and one-step purification and immobilization of His₆-tagged Escherichia coli γ-glutamyltranspeptidase (His₆-EcGGT) using these particles were evaluated. The optimal conditions for the adsorption of His₆-EcGGT to Fe₃O₄-AA-ANTA-Co^{2 +} particles were found to be 24.7 U g^–1 adsorbent, pH 6.5, 300 mM NaCl and 30 min incubation at 4°C, while the elution solution was optimized to be 50 mM phosphate buffer (pH 6.5) containing 150 mM imidazole and 300 mM NaCl. The immobilized His₆-EcGGT was recycled five times without significant loss of GGT activity. The average yield rate for the synthesis l-theanine from glutamine and ethylamine reached 56.7%. These results indicate that one-step affinity purification and immobilization of His₆-EcGGT by Fe₃O₄-AA-ANTA-Co^{2 +} particles might serve as an effective process for industrial application.     

Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone

Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide (H₂O₂) in tobacco and tomato, and superoxide (O₂^–?) together with H₂O₂ in Arabidopsis accessions. Accumulation of these reactive oxygen species (ROS) preceded the induction of cell death, and both responses co‐occurred spatially in the periveinal regions of the leaves. Re‐current ozone exposure of the sensitive tobacco cv. Bel W3 in closed chambers or in the field led to an enlargement of existing lesions by priming the border cells for H₂O₂ accumulation. Open top chamber experiments with native herbaceous plants in the field showed that Malva sylvestris L. accumulates O₂^–? at those sites that later exhibit plant cell death. Blocking of ROS accumulation markedly reduced ozone‐induced cell death in tomato, Arabidopsis and M. sylvestris. It is concluded that ozone triggers an in planta generation and accumulation of H₂O₂ and/or O₂^–? depending on the species, accession and cultivar, and that both these reactive oxygen species are involved in the induction of cell death in sensitive crop and native plants.     

Effect of fungal co-cultures on ligninolytic enzyme activities,H2O2 production,and orange G discoloration

Abstract

In this study, the effects of Aspergillus niger in coculture with the basidiomycetes, Trametes versicolor, T. maxima, and Ganoderma spp., were studied to assess H₂O₂ production and laccase (Lac), Lignin Peroxidase (LiP), and manganese peroxidase (MnP) activities. The results indicated that maximum discoloration was of 97%, in the T. maxima and A. niger coculture, where the concentration of H₂O₂ was 5?mg/L and 6.3?mg/L in cultures without and with dye, respectively. These concentrations of H₂O₂ were 1.6- and 1.8-fold higher than monocultures of T. maxima (3.37?mg/L) and A. niger (3.87?mg/L), respectively. In the same coculture, the LiP and MnP enzyme activities also increased 12-fold, (from 0.08?U/mg to 0.99?U/mg), and 67-fold, (from 0.11?U/mg to 7.4?U/mg), respectively. The Lac activity increased 1.7-fold (from 13.46?U/mg to 24?U/mg). Further, a Box–Behnken experimental design indicated a 1.8-fold increase of MnP activity (from 7.4?U/mg to 13.3?U/mg). In addition, dye discoloration regression model obtained from the Box–Behnken experimental design showed a positively correlation with H₂O₂, (R²?=?0.58) and a negatively correlation with Lac activity (R² = –0.7).     

A novel hydrogen peroxide sensor based on immobilization of haemoglobin on nano-TiO2/DTAB composite film

Abstract

The direct electron transfer of immobilized haemoglobin (Hb) on nano-TiO₂ and dodecyltrimethylammonium bromide (DTAB) film modified carbon paste electrode (CPE) and its application as a hydrogen peroxide (H₂O₂) biosensor were investigated. On nano-TiO₂/DTAB/Hb/CPE, Hb displayed a rapid electron transfer process with participation of one proton and with an electron transfer rate constant which estimated as 0.29 s^??1. Thus, the proposed biosensor exhibited a high sensitivity and excellent electrocatalytic activity for the reduction of H₂O₂. The catalytic reduction current of H₂O₂ was proportional to H₂O₂ concentration in the range of 0.2–4.0 mM with a detection limit of 0.07 mM. The apparent Michaelis–Menten constant (K_m^app) of the biosensor was calculated to be 0.127 mM, exhibiting a high enzymatic activity and affinity. This sensor for H₂O₂ can potentially be applied in determination of other reactive oxygen species as well.     

An Integrated Mitochondrial ROS Production and Scavenging Model: Implications for Heart Failure

It has been observed experimentally that cells from failing hearts exhibit elevated levels of reactive oxygen species (ROS) upon increases in energetic workload. One proposed mechanism for this behavior is mitochondrial Ca²⁺ mismanagement that leads to depletion of ROS scavengers. Here, we present a computational model to test this hypothesis. Previously published models of ROS production and scavenging were combined and reparameterized to describe ROS regulation in the cellular environment. Extramitochondrial Ca²⁺ pulses were applied to simulate frequency-dependent changes in cytosolic Ca²⁺. Model results show that decreased mitochondrial Ca²⁺uptake due to mitochondrial Ca²⁺ uniporter inhibition (simulating Ru360) or elevated cytosolic Na⁺, as in heart failure, leads to a decreased supply of NADH and NADPH upon increasing cellular workload. Oxidation of NADPH leads to oxidation of glutathione (GSH) and increased mitochondrial ROS levels, validating the Ca²⁺ mismanagement hypothesis. The model goes on to predict that the ratio of steady-state [H₂O₂]_m during 3Hz pacing to [H₂O₂]_m at rest is highly sensitive to the size of the GSH pool. The largest relative increase in [H₂O₂]_m in response to pacing is shown to occur when the total GSH and GSSG is close to 1 mM, whereas pool sizes below 0.9 mM result in high resting H₂O₂ levels, a quantitative prediction only possible with a computational model.     

An Integrated Mitochondrial ROS Production and Scavenging Model: Implications for Heart Failure

It has been observed experimentally that cells from failing hearts exhibit elevated levels of reactive oxygen species (ROS) upon increases in energetic workload. One proposed mechanism for this behavior is mitochondrial Ca²⁺ mismanagement that leads to depletion of ROS scavengers. Here, we present a computational model to test this hypothesis. Previously published models of ROS production and scavenging were combined and reparameterized to describe ROS regulation in the cellular environment. Extramitochondrial Ca²⁺ pulses were applied to simulate frequency-dependent changes in cytosolic Ca²⁺. Model results show that decreased mitochondrial Ca²⁺uptake due to mitochondrial Ca²⁺ uniporter inhibition (simulating Ru360) or elevated cytosolic Na⁺, as in heart failure, leads to a decreased supply of NADH and NADPH upon increasing cellular workload. Oxidation of NADPH leads to oxidation of glutathione (GSH) and increased mitochondrial ROS levels, validating the Ca²⁺ mismanagement hypothesis. The model goes on to predict that the ratio of steady-state [H₂O₂]_m during 3Hz pacing to [H₂O₂]_m at rest is highly sensitive to the size of the GSH pool. The largest relative increase in [H₂O₂]_m in response to pacing is shown to occur when the total GSH and GSSG is close to 1 mM, whereas pool sizes below 0.9 mM result in high resting H₂O₂ levels, a quantitative prediction only possible with a computational model.     

3,4-Dihydroxybenzaldehyde purified from the barley seeds (Hordeum vulgare) inhibits oxidative DNA damage and apoptosis via its antioxidant activity

Barley is a major crop worldwide. It has been reported that barley seeds have an effect on scavenging ROS. However, little has been known about the functional role of the barley on the inhibition of DNA damage and apoptosis by ROS. In this study, we purified 3,4-dihydroxybenzaldehyde from the barley with silica gel column chromatography and HPLC and then identified it by GC/MS. And we firstly investigated the inhibitory effects of 3,4-dihydroxybenzaldehyde purified from the barley on oxidative DNA damage and apoptosis induced by H₂O₂, the major mediator of oxidative stress and a potent mutagen. In antioxidant activity assay such as DPPH radical and hydroxyl radical scavenging assay, Fe²⁺ chelating assay, and intracellular ROS scavenging assay by DCF-DA, 3,4-dihydroxybenzaldehyde was found to scavenge DPPH radical, hydroxyl radical and intracellular ROS. Also it chelated Fe²⁺. In in vitro oxidative DNA damage assay and the expression level of phospho-H2A.X, it inhibited oxidative DNA damage and its treatment decreased the expression level of phospho-H2A.X. And in oxidative cell death and apoptosis assay via MTT assay and Hoechst 33342 staining, respectively, the treatment of 3,4-dihydroxybenzaldehyde attenuated H₂O₂-induced cell death and apoptosis. These results suggest that the barley may exert the inhibitory effect on H₂O₂-induced tumor development by blocking H₂O₂-induced oxidative DNA damage, cell death and apoptosis.     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

Effects of ambient calcium concentration on morphological form of callus-like cells in Saccharina japonica (Phaeophyceae) sporophyte

Explants obtained from young sporophytes of Saccharina japonica were cultured in an artificial medium with different concentrations of Ca²⁺ (0–20?mM). The culture with 10?mM Ca²⁺ promoted the formation of unpigmented filamentous callus-like cells in the cortical layer. In contrast, explants cultured with 5?mM Ca²⁺ formed pigmented round callus-like cells in the epidermis at a high percentage. The thallus regeneration rate of explants in 5?mM Ca²⁺ was ten times higher than those of explants cultured in 10?mM Ca²⁺. Ambient Ca²⁺ concentrations also influenced the production of radical oxygen species (ROS) in explants. Explants cultured in 10?mM Ca²⁺ produced higher ROS than did those cultured in 5?mM. The ROS production was histologically observed mainly in the plasma membrane of callus-like cells using 2′, 7′-dichlorodihydrofluorescein diacetate. Moreover, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride, inhibited the ROS production with propagation of filamentous callus-like cells. These results suggest that Ca²⁺ concentration in medium influences the development of callus-like cells and thallus regeneration by affecting NADPH oxidase activity and ROS production in the plasma membrane of the callus-like cells. Therefore, the morphological form of callus-like cells and the development of thallus will be controlled by Ca²⁺ concentration in the medium.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.