Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

ANTIOXIDATIVE PROTECTION OF PERIDINIUM GATUNENSE IN LAKE KINNERET: SEASONAL AND DAILY VARIATION1

A comprehensive antioxidative mechanism was found in the freshwater dinoflagellate Peridinium gatunense Lemm. during the spring bloom in Lake Kinneret. Ascorbate was present throughout the bloom period and was responsible, together with catalase, for the elimination of photosynthetically produced H₂O₂. As glutathione concentrations and ascorbate regenerative enzymes were negligible during mid-spring, ascorbate was presumably biosynthesized during the photosynthetically active period. Antioxidative activity increased overall at the end of the spring in conjunction with elevated ambient stress conditions, for example high light. Under such circumstances, ascorbate was regenerated. Ascorbate levels doubled when cells were exposed to an increase in irradiance from 60 to 600 μmol photons·m^?2·s^?1, and on addition of H₂O₂, concentrations increased a further 20-fold. Significant antioxidative activity was also noted in the dark, although this was dependent on the presence of H₂O₂. Diurnal changes in antioxidants and their regenerative enzymes were observed. The activities of mono-dehydroascorbate reductase, glutathione reductase, and ascorbate concentrations showed ultraradian periodicity and were completely in phase throughout the day/night period. Dehydroascorbate reductase activity and glutathione concentrations were also in phase but showed aperiodic variation, as did ascorbate peroxidase activity. Superoxide dismutase and catalase activities were generally out of phase during the 24-h period but did show ultraradian periodicity. Lake samples entrained under constant light revealed an inate 12-h rhythm for catalase activity, during at least 36 h.     

In vitro Damage to Rat Lens by Lumazine and Xanthine Oxidase: Prevention by Superoxide Dismutase

Intact rat lenses incubated with lumazine and xanthine oxidase are physiologically damaged as evidenced by a decrease in the net accumulation of rubidium ions against a concentration gradient. Superoxide dismutase protected the tissue against this damage. These experiments, therefore, demonstrate the susceptibility of the lens tissue to O₂^?_? injury under ambient and nonphotochemical conditions, suggesting a possible implication of this radical in the tissue in vivo and eventual cataract formation. The lumazine/xanthine oxidase system which is known to cause oxygen reduction predominantly by the monovalent route, producing superoxide, appears quite suitable to evaluate the toxicity of O₂^?_? to the tissues in vitro.     

The scavenging of superoxide radical by manganous complexes: in vitro

Dialyzable manganese has been shown to be present in millimolar concentrations within cells of Lactobacillus plantarum and related lactic acid bacteria. This unusual accumulation of Mn appears to serve the same function as Superoxide dismutase (SOD), conferring hyperbaric oxygen and Superoxide tolerance on these SOD-free organisms. The form of the Mn in the lactic acid bacteria and the mechanisms whereby it protects the cell from oxygen damage are unknown. This report examines the mechanisms by which Mn catalytically scavenges O₂^?, both in the xanthine oxidase/cytochrome c SOD assay and in a number of in vitro systems relevant to the in vivo situation. In all the reaction mixtures examined, Mn(II) is first oxidized by O₂^? to Mn(III), and H₂O₂ is formed. In pyrophosphate buffer the Mn(III) thus formed is re-reduced to Mn(II) by a second O₂^?, making the reaction a true metal-catalyzed dismutation like that catalyzed by SOD. Alternatively, if the reaction takes place in orthophosphate or a number of other buffers, the Mn(III) is preferentially reduced largely by reductants other than O₂^?, such as thiols, urate, hydroquinone, or H₂O₂. H₂O₂, a common product of the lactic acid bacteria, reacted rapidly with Mn(III) to form O₂, apparently without intermediate O₂ release. Free hexaquo Mn(II) ions were shown by electron spin resonance spectroscopy and activity assays in noncomplexing buffers to be poorly reactive with O₂^?. In contrast, Mn(II) formed complexes having a high catalytic activity in scavenging O₂^? with a number of organic acids, including malate, pyruvate, propionate, succinate, and lactate, with the Mn-lactate complex showing the greatest activity.     

Identification and characterization of superoxide dismutase in Phytophthora cinnamomi

Superoxide dismutase (SOD) activities of the oomycete Phytophthora cinnamomi were examined. Five polypeptides with manganese superoxide dismutase (MnSOD) activity were found in mycelium growing in liquid culture with relative molecular weights ranging from approximately 25 to 100 kDa. Comparison with characterized avocado SODs showed no evidence for the presence of either iron or copper/zinc SODs in P. cinnamomi. The level of activity of the MnSOD polypeptides decreased in the presence of avocado root or cell wall components. Growth of P. cinnamomi, measured as dry weight, increased when the mycelium was grown in the presence of superoxide anion (O₂ ^?), which was added exogenously. Our results suggest that the metabolism of O₂ ^? has an important role in the development of P. cinnamomi.     

Some kinetic properties of Mucor rouxii phosphofructokinase. Effect of cyclic adenosine 3', 5'-monophosphate.

Intact and pure parenchymal and nonparenchymal cells were isolated from rat liver. The activities of Superoxide dismutase in these cell types were determined by two different methods. With both methods the specific activity of this enzyme is 1.5 times higher in parenchymal than in nonparenchymal liver cells. It can be calculated that about 7% of the total rat liver Superoxide dismutase activity is localized in the nonparenchymal liver cells. Electrophoresis on polyacrylamide gels indicates that the isolated parenchymal cells contain both cytosolic and mitochondrial isoenzymes, whereas with nonparenchymal cells only the cytosolic enzyme could be detected. The mitochondrial band observed in isolated parenchymal cells is absent in the original total liver homogenate. This isoenzyme seems to be activated during the parenchymal cell isolation procedure. Isoelectrofocusing indicates that the cytosolic Superoxide dismutase consists in four different isoelectric forms in both parenchymal and nonparenchymal cells. With the mitochondrial isoenzyme two bands are obtained. The possibility that O₂^? is an important intermediate in H₂O₂ formation in nonparenchymal liver cells is discussed. In this respect, Superoxide dismutase might not only protect the cell against a toxic reagent as O₂^t-, but might also help to regulate the level of the important antimicrobial agent, H₂O₂.     

Effects of non-steady-state iron limitation on nitrogen assimilatory enzymes in the marine diatom thalassiosira weissflogii (BACILLARIOPHYCEAE)

Since the recognition of iron‐limited high nitrate (or nutrient) low chlorophyll (HNLC) regions of the ocean, low iron availability has been hypothesized to limit the assimilation of nitrate by diatoms. To determine the influence of non‐steady‐state iron availability on nitrogen assimilatory enzymes, cultures of Thalassiosira weissflogii (Grunow) Fryxell et Hasle were grown under iron‐limited and iron‐replete conditions using artificial seawater medium. Iron‐limited cultures suffered from decreased efficiency of PSII as indicated by the DCMU‐induced variable fluorescence signal (F_v/F_m). Under iron‐replete conditions, in vitro nitrate reductase (NR) activity was rate limiting to nitrogen assimilation and in vitro nitrite reductase (NiR) activity was 50‐fold higher. Under iron limitation, cultures excreted up to 100 fmol NO₂^?·cell^?1·d^?1 (about 10% of incorporated N) and NiR activities declined by 50‐fold while internal NO₂^? pools remained relatively constant. Activities of both NR and NiR remained in excess of nitrogen incorporation rates throughout iron‐limited growth. One possible explanation is that the supply of photosynthetically derived reductant to NiR may be responsible for the limitation of nitrogen assimilation at the NO₂^? reduction step. Urease activity showed no response to iron limitation. Carbon:nitrogen ratios were equivalent in both iron conditions, indicating that, relative to carbon, nitrogen was assimilated at similar rates whether iron was limiting growth or not. We hypothesize that, diatoms in HNLC regions are not deficient in their ability to assimilate nitrate when they are iron limited. Rather, it appears that diatoms are limited in their ability to process photons within the photosynthetic electron transport chain which results in nitrite reduction becoming the rate‐limiting step in nitrogenassimilation.     

Superoxide dismutase: a comparison of rate constants

O₂^?was introduced, at a constant rate, into buffered aqueous solutions, either by mechanical infusion of KO₂, dissolved in tetrahydrofuran, or by the in situ action of xanthine oxidase on xanthine plus oxygen. This O₂^? was allowed to react with ferricytochrome c or with tetranitromethane and the formation of the reaction products, ferrocytochrome c or nitroform, respectively, was monitored spectrophotometrically. That concentration of Superoxide dismutase, which competed equally with given levels of cytochrome c or tetranitromethane and which thus caused 50% inhibition of the rates of accumulation of ferrocytochrome c or of nitroform, was determined. The rate constant for the enzymatic dismutation of O₂^? by the copper and zinc containing enzyme from bovine erythrocytes was then calculated from the known rate constants for the reaction of O₂^? with ferricytochrome c and with tetranitromethane and was found to be 2 × 10⁹m^?1 sec^?1 at pH 7.8 and 8.5. This rate constant was obtained at steady-state concentrations of O₂^? in the 10^?8m → 10^?13m range and is in full agreement with the results of pulse radiolytic investigations which were performed at O₂^? concentrations in the 10^?5m range. The second order rate constant for the enzymatic dismutation of O₂^? is thus independent of the concentration of O₂^? in the range 10^?5 → 10^?13m.Several distinct types of Superoxide dismutase have been described. These include the mangano-enzymes from Escherichia coli and from chicken liver mitochondria and the iron-enzyme from E. coli. The rate constants for the dismutations catalyzed by these enzymes have also been investigated as a function of pH.     

A reaction of the superoxide radical with tetrapyrroles

Bilirubin and biliverdin were bleached during exposure to the aerobic xanthine oxidase reaction. Enzymic scavenging of O₂^?, by Superoxide dismutase, inhibited, whereas enzymic scavenging of H₂O₂, by catalase, did not. Increasing the rate of production of O₂^? without increasing the turnover rate of xanthine oxidase, by increasing pO₂, accelerated the bleaching of the biliverdin. Moreover, a scavenger of OH·, such as benzoate, or an inactivating chelating agent for iron, such as diethylenetriamine pentaacetate or desferrioxamine mesylate, did not inhibit. It follows that O₂^? can directly attack these tetrapyrroles. Kinetic competition between Superoxide dismutase and bilirubin yielded a value for k_{bilirubin, O2^?} = 2.3 × 10⁴ M^?1s^?1 at pH 8.3 and at 23 °C. A similar experiment for biliverdin yielded a value for k_{bilirubin, O2^?} = 7 × 10⁴ M^?1s^?1.     

Oxidative Stress Responses in the Unicellular Cyanobacterium Synechococcus Pcc 7942

Oxidative stress responses were tested in the unicellular cyanobacterium synechococcus PCC 7942 (R-2). Cells were exposed to hydrogen peroxide, cumene hydroperoxide and high light intensities. The extent and time course of oxidative stress were related to the activities of ascorbate peroxidase and catalase. Ascorbate peroxidase was found to be the major enzyme involved in the removal of hydrogen peroxide under the tested oxidative stresse. Catalase activity was inhibited in cells, treated with high H₂O₂ concentrations, and was not induced under photooxidative stress. Catalase was specifically induced in cells treated with cumene hydroperoxide.

Superoxide dismutase activity increased under conditions generating superoxide, such as high light intensities. The induction of the antioxidative enzymes was light dependent and was inhibited by chloramphenicol.     

Immediate response of Chara braunii exposed to zinc and hydrogen peroxide

The immediate effect of zinc (Zn) and hydrogen peroxide (H₂O₂) in Chara braunii was analyzed in short-time exposure experiments. The exposure concentrations were 12.3, 18.4, and 24.5 μmol L^?1 H₂O₂, 12, 60, and 120 mg L^?1 Zn, and 12.3 μmol L^?1 H₂O₂ + 12 mg L^?1 Zn, 12.3 μmol L^?1 H₂O₂ + 60 mg L^?1 Zn, and 18.4 μmol L^?1 H₂O₂ + 12 mg L^?1 Zn. The stress response of C. braunii was analyzed by measuring photosynthetic photosystem II activity, chlorophyll a and b and carotenoid contents, the H₂O₂ concentration, and antioxidant enzyme activities of ascorbic peroxidase, catalase, and guaiacol peroxidase. The short-term addition of Zn reduced pigment contents in C. braunii. Chlorophyll a and b and carotenoid contents in H₂O₂-exposed C. braunii were as high as in control plants. Photosynthesis was reduced in H₂O₂-treated C. braunii and the short-term addition of Zn did not affect the electron transport rate. H₂O₂ concentration and antioxidant enzyme activities in C. braunii were not significantly different between control and exposed plants. Trends of enzymatic adaptation were described: the H₂O₂-induced stress response was characterized by increased antioxidant enzyme activities, whereas Zn inactivated catalase in C. braunii.     

Inactivation of Escherichia coli by superoxide radicals and their dismutation products.

Escherichia coli cells are inactivated by the products of the reaction between dialuric acid and oxygen, of which the primary product is Superoxide. The rate of inactivation is decreased by Superoxide dismutase, by catalase, and by EDTA, whereas it is increased by addition of cupric ions or hydrogen peroxide. It is concluded that a toxic product is formed in a reaction involving Superoxide, hydrogen peroxide, and metal ions, which might be the Haber-Weiss reaction, O₂^? + H₂O₂ → OH + OH^? + O₂. In radiation chemical experiments it is shown that this reaction does not occur in the absence of metal ions.     

Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

We have reported previously that dihydropyridine-type calcium-channel antagonists (DTCCA) such as nifedipine decrease plasma markers of oxidative stress damage in systemic sclerosis (SSc). To clarify the cellular basis of these beneficial effects, we investigated the effects in vivo and in vitro of nifedipine on superoxide anion (O₂ ^•-) production by peripheral blood monocytes. We compared 10 healthy controls with 12 patients with SSc, first after interruption of treatment with DTCCA and second after 2 weeks of treatment with nifedipine (60 mg/day). O₂ ^•- production by monocytes stimulated with phorbol myristate acetate (PMA) was quantified by the cytochrome c reduction method. We also investigated the effects in vitro of DTCCA on O₂ ^•- production and protein phosphorylation in healthy monocytes and on protein kinase C (PKC) activity using recombinant PKC. After DTCCA had been washed out, monocytes from patients with SSc produced more O₂ ^•- than those from controls. Nifedipine treatment considerably decreased O₂ ^•- production by PMA-stimulated monocytes. Treatment of healthy monocytes with nifedipine in vitro inhibited PMA-induced O₂ ^•- production and protein phosphorylation in a dose-dependent manner. Finally, nifedipine strongly inhibited the activity of recombinant PKC in vitro. Thus, the oxidative stress damage observed in SSc is consistent with O₂ ^•- overproduction by primed monocytes. This was decreased by nifedipine treatment both in vivo and in vitro. This beneficial property of nifedipine seems to be mediated by its cellular action and by the inhibition of PKC activity. This supports the hypothesis that this drug could be useful for the treatment of diseases associated with oxidative stress.     

The effects of La(III) on the peroxidation of membrane lipids in wheat seedling leaves under osmotic stress

The physiological effects of the rare earth ion La³⁺ on the peroxidation of membrane lipids in wheat (Triticum aestivum L.) seedling leaves under osmotic stress were determined. With the passage of time under osmotic stress, the inhibition ability of lanthanum ions to the relative membrane permeability and concentration of malondialdehyde, Superoxide radicals, and hydrogen peroxide caused by osmotic stress increased substantially, but no changes were noted in ferrous and relative water content. It indicated that lanthanum ions could not retain the water content because of osmotic stress. However, La³⁺ appears to decrease the production of OH by reducing the content of O₂ and H₂O₂ of Haber-Weiss and Fenton reactions, which efficiently alleviated peroxidation of membrane lipids under osmotic stress and, to some degree, protected the membrane from injury of free radicals. Thus, La³⁺ increased the tolerance ability of plant to osmotic stress, which could assure the function of membrane normal temporally after stressed.     

Multiple effects of salinity on photosynthesis of the protist Euglena gracilis

The effect of NaCl in the culture medium on growth, photosynthesis and cell content of chlorophyll, K⁺, Na⁺, Ca²⁺ and Mg²⁺ in Euglena gracilis was studied. O₂ production, quantum yield of photosystem II (PSII), the non-photochemical quenching of chlorophyll fluorescence (q_N) and the chlorophyll alb ratio all diminished by 0.2 M NaCl. Respiration and chlorophyll a and b increased, whereas the photochemical quenching (q_p) of chlorophyll fluorescence was not affected by 0.2 M NaCl. Salt stress also induced an increase in cell volume and in K⁺ and Na⁺ concentrations, but decreased the concentrations of Ca²⁺ and Mg²⁺. Except for a protective effect on O₂ production, additional Ca²⁺ in the culture medium did not attenuate the salt effect on the parameters measured. The addition of HCO₃^? restored the PSII quantum yield of O₂ production in cells grown in high salt. Salt stress promoted a decrease in the apparent rate of quinone A (Q_A) reduction and an apparent obstruction of Q_B reduction, which were not prevented by excess HCO₃^?; the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not increase chlorophyll fluorescence in salt-grown cells. These results indicate that photosynthesis in Euglena grown under salt stress exhibits: (1) diminution of the HCO₃^? dependent water-splitting activity of PSII; (2) inhibition of the electron transfer at the quinone pool level; (3) probable increase in thylakoid stacking (as indicated by the effect on the chlorophyll alb ratio); and (4) dissipation of the H⁺ gradient across the thylakoid membranes (as indicated by the decrease of q_N).     

Mechanisms of nitric oxide crosstalk with reactive oxygen species scavenging enzymes during abiotic stress tolerance in plants

Nitric oxide (NO) acts in a concentration and redox-dependent manner to counteract oxidative stress either by directly acting as an antioxidant through scavenging reactive oxygen species (ROS), such as superoxide anions (O₂^?*), to form peroxynitrite (ONOO^?) or by acting as a signaling molecule, thereby altering gene expression. NO can interact with different metal centres in proteins, such as heme-iron, zinc–sulfur clusters, iron–sulfur clusters, and copper, resulting in the formation of a stable metal–nitrosyl complex or production of varied biochemical signals, which ultimately leads to modification of protein structure/function. The thiols (ferrous iron–thiol complex and nitrosothiols) are also involved in the metabolism and mobilization of NO. Thiols bind to NO and transport it to the site of action whereas nitrosothiols release NO after intercellular diffusion and uptake into the target cells. S-nitrosoglutathione (GSNO) also has the ability to transnitrosylate proteins. It is an NO˙ reservoir and a long-distance signaling molecule. Tyrosine nitration of proteins has been suggested as a biomarker of nitrosative stress as it can lead to either activation or inhibition of target proteins. The exact molecular mechanism(s) by which exogenous and endogenously generated NO (or reactive nitrogen species) modulate the induction of various genes affecting redox homeostasis, are being extensively investigated currently by various research groups. Present review provides an in-depth analysis of the mechanisms by which NO interacts with and modulates the activity of various ROS scavenging enzymes, particularly accompanying ROS generation in plants in response to varied abiotic stress.     

Zinc‐induced oxidative stress in Verbascum thapsus is caused by an accumulation of reactive oxygen species and quinhydrone in the cell wall

Oxidative stress is one aspect of metal toxicity. Zinc, although unable to perform univalent oxido‐reduction reactions, can induce the oxidative damage of cellular components and alter antioxidative systems. Verbascum thapsus L. plants that were grown hydroponically were exposed to 1 and 5 mM Zn²⁺. Reactive oxygen species (ROS) accumulation was demonstrated by the fluorescent probe H₂DCFDA and EPR measurements. The extent of zinc‐induced oxidative damage was assessed by measuring the level of protein carbonylation. Activities and isoform profiles of some antioxidant enzymes and the changes in ascorbate and total phenolic contents of leaves and roots were determined. Stunted growth because of zinc accumulation, preferentially in the roots, was accompanied by H₂O₂ production in the leaf and root apoplasts. Increased EPR signals of the endogenous oxidant quinhydrone, ^?CH₃ and ^?OH, were found in the cell walls of zinc‐treated plants. The activities of the antioxidative enzymes ascorbate peroxidase (APX) (EC 1.11.1.11), soluble superoxide dismutase (SOD) (EC 1.15.1.1), peroxidase (POD), (EC 1.11.1.7) and monodehydroascorbate reductase (EC 1.6.5.4) were increased; those of glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1) and ascorbate oxidase (AAO) (EC 1.10.3.3) were decreased with zinc treatment. Zinc induced a cell‐wall‐bound SOD isoform in both organs. Leaves accumulated more ascorbate and phenolics in comparison to roots. We propose a mechanism for zinc‐promoted oxidative stress in V. thapsus L. through the generation of charge transfer complexes and quinhydrone because of phenoxyl radical stabilisation by Zn²⁺ in the cell wall. Our results suggest that the SOD and APX responses are mediated by ROS accumulation in the apoplast. The importance of the POD/Phe/AA (ascorbic acid) scavenging system in the apoplast is also discussed.     

Antioxidant role of astaxanthin in the green alga Haematococcus pluvialis

The green unicellular alga, Haematococcus pluvialis has two antioxidative mechanisms against environmental oxidative stress: antioxidative enzymes in vegetative cells and the antioxidative ketocarotenoid, astaxanthin, in cyst cells. We added a reagent that generates superoxide anion radicals (O₂ ⁻), methyl viologen, to mature and immature cysts of H. pluvialis. Tolerance to methyl viologen was higher in mature than in immature cysts. Mature (astaxanthin-rich) cysts showed high antioxidant activity against O₂ ⁻ in permeabilized cells, but not in astaxanthin-free cell extracts, while immature (astaxanthin-poor) cysts had very low antioxidant activities against O₂ ⁻ in both. The results suggested that astaxanthin accumulated in the cyst cells functions as an antioxidant against excessive oxidative stress. The same levels of antioxidant activities against O₂ ⁻ in both permeabilized cells and cell extracts from vegetative cells suggested the presence of antioxidative enzymes (superoxide dismutase). Received: 13 January 1997 / Received revision: 26 February 1997 / Accepted: 27 March 1997     

Comparison of oxidative stress induced by ciprofloxacin and pyoverdin in bacteria and in leukocytes to evaluate toxicity

Oxidative stress induced by ciprofloxacin and pyoverdin, a leukotoxic pigment, was studied by comparing their effect in bacteria and leukocytes. Chemiluminescence (CL) assays with lucigenin or luminol were adapted to measure the stimuli of superoxide anion (O₂^?) and other reactive species of oxygen (ROS) in bacteria. Ciprofloxacin principally induced the production of O₂^? in the three species studied: Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. Lucigenin CL assay showed high oxidative stress in S. aureus due to its low superoxide dismutase (SOD) activity, whereas E. coli exhibited important SOD activity, responsible for little production of O₂^? in absence or presence of ciprofloxacin. Reduction of nitroblue of tetrazolium (NBT) was applied. This assay indicated that there was higher oxidative stress in S. aureus and E. faecalis than in E. coli. The comparison of oxidative stress generated in bacteria and leukocytes was used to check the selective toxicity of ciprofloxacin in comparison with pyoverdin. Ciprofloxacin did not generate significant stimuli of O₂^? in neutrophils, while pyoverdin duplicated the production of O₂^?. CL and NBT were useful to study the leukotoxicity of ciprofloxacin. Oxidative stress caused by the antibiotic and the leukotoxic pigment was similar in bacteria. Copyright © 2003 John Wiley & Sons, Ltd.     

Molecular Characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica juncea</Emphasis> Cu/Zn-Superoxide Dismutases Reveals Their Regulation of Shoot Regeneration

Superoxide dismutases (SODs) are ubiquitous metalloenzymes that catalyze the dismutation of superoxide radicals (O₂^-) to molecular oxygen (O₂) and hydrogen peroxide (H₂O₂). In this study we characterized an Arabidopsis thaliana CuZnSOD (CSD1), a close ortholog of a previously identified Brassica juncea CuZnSOD (MSOD1). CSD1 and other two homologs CSD2 and CSD3 were spatially regulated in Arabidopsis, and CSD1 exhibited distinct expression patterns in response to different stress treatments. To investigate the in vivo function of SOD, transgenic Arabidopsis plants, expressing sense and antisense MSOD1 RNAs, were generated and those with altered SOD activity were selected for further characterization. Although SOD transgenic plants exhibited normal phenotypes, the shoot regeneration response in transgenic explants was significantly affected by the modulated SOD activity and the corresponding H₂O₂ levels. Transgenic explants with downregulated SOD activity were poorly regenerative, whereas those with upregulated SOD activity were highly regenerative. These results suggest that shoot regeneration in vitro is regulated by the SOD activity.