Oxygen stress: a regulator of apoptosis in yeast.

Oxygen radicals are important components of metazoan apoptosis. We have found that apoptosis can be induced in the yeast Saccharomyces cerevisiae by depletion of glutathione or by low external doses of H2O2. Cycloheximide prevents apoptotic death revealing active participation of the cell. Yeast can also be triggered into apoptosis by a mutation in CDC48 or by expression of mammalian bax. In both cases, we show oxygen radicals to accumulate in the cell, whereas radical depletion or hypoxia prevents apoptosis. These results suggest that the generation of oxygen radicals is a key event in the ancestral apoptotic pathway and offer an explanation for the mechanism of bax-induced apoptosis in the absence of any established apoptotic gene in yeast.     

Environmental factors regulating the radial oxygen loss from roots of Myriophyllum spicatum and Potamogeton crispus

Myriophyllum spicatum and Potamogeton crispus are common species of shallow eutrophic lakes in north-eastern Germany, where a slow recovery of the submersed aquatic vegetation was observed. Thus, the characterisation of the root oxygen release (ROL) as well as its implication for geochemical processes in the sediment are of particular interest. A combination of microelectrode measurements, methylene blue agar and a titanium(III) redox buffer was used to investigate the influence of the oxygen content in the water column on ROL, diel ROL dynamics as well as the impact of sediment milieu. Oxygen gradients around the roots revealed a maximum oxygen diffusion zone of up to 250 μm. During a sequence with a light/dark cycle as well as alternating aeration of the water column, maximum ROL with up to 35% oxygen saturation at the root surface occurred under light/O₂-saturated conditions. A decrease to about 30% was observed under dark/O₂-saturated conditions, no ROL was detected at dark/O₂-depleted conditions and only a weak ROL with 5–10% oxygen saturation at the root surface was measured under light but O₂-depleted water column. These results indicate, that during darkness, ROL is supplied by oxygen from the water column and even during illumination and active photosynthesis production, ROL is modified by the oxygen content in the water column. Visualisation of ROL patterns revealed an enhanced ROL for plants which were grown in sulfidic littoral sediment in comparison to plants grown in pure quartz sand. For both plant species grown in sulfidic littoral sediment, a ROL rate of 3–4 μmol O₂ h⁻¹ plant⁻¹ was determined with the Ti(III) redox buffer. For plants grown in pure quartz sand, the ROL rate decreased to 1–2 μmol O₂ h⁻¹ plant⁻¹. Hence, aside from the oxygen content in the water column, the redox conditions and microbial oxygen demand in the sediment has to be considered as a further major determinant of ROL.     

Entamoeba histolytica: Oxygen resistance and virulence

Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins, amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this work we compared several in vitro and in vivo features of both virulent E. histolytica (vEh) and non-virulent E. histolytica (nvEh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemolytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O₂. The only difference observed was a higher in vitro susceptibility of nvEh to O₂. The molecular mechanism of that difference was analyzed in both groups of amoebae after high O₂ exposure. vEh O₂ resistance correlated with: (i) higher O₂ reduction ( and H₂O₂ production); (ii) increased H₂O₂ resistance and thiol peroxidase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the high level of carbonylated proteins in nvEh after O₂ exposure, membrane oxidation by reactive oxygen species was not observed. These results suggest that the virulent phenotype of E. histolytica is related to the greater ability to reduce O₂ and H₂O₂ as well as PFOR reactivation, whereas nvEh undergoes irreversible PFOR inhibition resulting in metabolic failure and amoebic death.     

Salsolinol,a catechol neurotoxin,induces oxidative modification of cytochrome c

Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), an endogenous neurotoxin, is known to perform a role in the pathogenesis of Parkinson’s disease (PD). In this study, we evaluated oxidative modification of cytochrome c occurring after incubation with salsolinol. When cytochrome c was incubated with salsolinol, protein aggregation increased in a dosedependent manner. The formation of carbonyl compounds and the release of iron were obtained in salsolinol- treated cytochrome c. Salsolinol also led to the release of iron from cytochrome c. Reactive oxygen species (ROS) scavengers and iron specific chelator inhibited the salsolinol-mediated cytochrome c modification and carbonyl compound formation. It is suggested that oxidative damage of cytochrome c by salsolinol might induce the increase of iron content in cells, subsequently leading to the deleterious condition which was observed. This mechanism may, in part, provide an explanation for the deterioration of organs under neurodegenerative disorders such as PD. [BMB Reports 2013; 46(2): 119-123]     

Aluminum induces changes in oxidative burst scavenging enzymes in Coffea arabica L. suspension cells with differential Al tolerance

The accumulation of reactive oxygen species (ROS) and concomitant oxidative stress have been considered deleterious consequences of aluminum toxicity. However, several lines of evidence suggest that ROS can function as important signaling molecules in the plant defense system for protection from abiotic stress and the acquisition of tolerance. The role of ROS-scavenging enzymes was assayed in two different coffee cell suspension lines. We treated L2 (Al-sensitive) and LAMt (Al-tolerant) Coffea arabica suspension cells with 100 μM AlCl₃ and observed significant differences in catalase activity between the two cell lines. However, we did not observe any differences in superoxide dismutase or glutathione reductase activity in either cell line following Al treatment. ROS production was diminished in the LAMt cell line. Taken together, these results indicate that aluminum treatment may impair the oxidative stress response in L2 cells but not in LAMt cells. We suggest a possible role for Al-induced oxidative bursts in the signaling pathways that lead to Al resistance and protection from Al toxicity.     

Tolerance to drug-induced cell death favours the acquisition of multidrug resistance in Leishmania

The control of the protozoan parasite Leishmania relies on few drugs with unknown cellular targets and unclear mode of action. Several antileishmanials, however, were shown to induce apoptosis in Leishmania and this death mechanism was further studied in drug-sensitive and drug-resistant Leishmania infantum. In sensitive parasites, antimonials (SbIII), miltefosine (MF) and amphotericin B (AMB), but not paromomycin (PARO), triggered apoptotic cell death associated with reactive oxygen species (ROS). In contrast, Leishmania mutants resistant to SbIII, MF or AMB not only failed to undergo apoptosis following exposure to their respective drugs, but also were more tolerant towards apoptosis induced by other antileishmanials, provided that these killed Leishmania via ROS production. Such tolerance favored the rapid acquisition of multidrug resistance. PARO killed Leishmania in a non-apoptotic manner and failed to produce ROS. PARO resistance neither protected against drug-induced apoptosis nor provided an increased rate of acquisition of resistance to other antileishmanials. However, the PARO-resistant mutant, but not SbIII-, MF- or AMB-resistant mutants, became rapidly cross-resistant to methotrexate, a model drug also not producing ROS. Our results therefore link the mode of killing of drugs to tolerance to cell death and to a facilitated emergence of multidrug resistance. These findings may have fundamental implications in the field of chemotherapeutic interventions.     

Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps

The activity and composition of leafhopper saliva are important in interactions with the host rice plant, and it may play a physiological role in detoxifying toxic plant substances or ingesting sap. We have characterized diphenoloxidase in the salivary glands of Nephotettix cincticeps, its activity as a laccase, and its presence in the watery saliva with the objective of understanding its function in feeding on rice plants. Nonreducing SDS-PAGE of salivary gland homogenates with staining by the typical laccase substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroquinone or syringaldazine revealed a band at a molecular mass of approximately 85 kDa at pH 5. A band also appeared at a molecular mass of approximately 200 kDa when the gels were treated with dopamine, L-3,4-dihydroxyphenylalanine (DOPA) or catechol at pH 7. The ABTS-oxidizing activity of the homogenates was drastically inhibited by N-hydroxyglycine, a specific inhibitor of laccase. However, the dopamine-oxidizing activity was not inhibited by N-hydroxyglycine, while it was inhibited by phenylthiourea (PTU). Thus, the salivary glands of N. cincticeps contain two types of phenoloxidases: a laccase (85 kDa) and a phenoloxidase (200 kDa). Laccase activity was detected in a holidic sucrose diet that was fed on for 16 h by two females, but only a trace of catechol oxidase activity was observed, suggesting that the laccase-type phenoloxidase was the predominant phenoloxidase secreted in watery saliva. The laccase exhibited an optimum pH of 4.75-5 in McIlvaine buffer and had a PI of 4.8. Enzyme activity was histochemically localized in V cells of the posterior lobe of the salivary glands. It remained at the same level throughout the adult stage from 2 days after eclosion. A possible function of N. cincticeps salivary laccase may be rapid oxidization of potentially toxic monolignols to nontoxic polymers during feeding on the rice plant. This is the first report proving that laccase occurs in the salivary glands of Hemiptera species and is secreted in the watery saliva.     

Ipso-substitution by cytochrome P450 with conversion of p-hydroxybenzene derivatives to hydroquinone: evidence for hydroperoxo-iron as the active oxygen species.

Evidence for multiple functional active oxidants in cytochrome P450-catalyzed reactions was previously obtained in this laboratory with mutants in which proton delivery was perturbed by replacement of the highly conserved threonine residue in the active site by alanine, thus apparently interfering with the conversion of the peroxo-iron to the hydroperoxo-iron and the latter to the oxenoid-iron species. These enzymes have now been employed to examine the reaction in which cytochrome P450 in liver microsomes is known to effect ipso-substitution, the elimination of p-substituents in phenols to yield hydroquinone. As shown with purified NH(2)-truncated cytochromes in a reconstituted enzyme system, the reaction exhibits an absolute requirement for cytochrome P450 and NADPH-cytochrome P450 reductase. Under optimal conditions truncated cytochrome P450 2E1 is active with 10 of the p-substituted phenols examined. Of particular interest, the corresponding cytochrome with threonine-303 replaced by alanine is from 1.5- to 50-fold higher in activity with the p-chloro, -bromo, -nitro, -cyano, -hydroxymethyl, -formyl, and -acetyl derivatives, and the reaction with the p-benzoyl, -methyl, and -t-butyl compounds is catalyzed by the mutant enzyme only. The results implicate the hydroperoxo-iron species as an electrophilic active oxidant in cytochrome P450-catalyzed aromatic ipso-substitution.     

Aging defect at the QO site of complex III augments oxyradical production in rat heart interfibrillar mitochondria

Complex III in the mitochondrial electron transport chain is a proposed site for the enhanced production of reactive oxygen species that contribute to aging in the heart. We describe a defect in the ubiquinol binding site (Q(O)) within cytochrome b in complex III only in the interfibrillar population of cardiac mitochondria during aging. The defect is manifested as a leak of electrons through myxothiazol blockade to reduce cytochrome b and is observed whether cytochrome b in complex III is reduced from the forward or the reverse direction. The aging defect increases the production of reactive oxygen species from the Q(O) site of complex III in interfibrillar mitochondria. A greater leak of electrons from complex III during the oxidation of ubiquinol is a likely mechanism for the enhanced oxidant production from mitochondria that contributes to aging in the rat heart.     

菹草种群内外水质日变化

对菹草(Potamogeton crispus)种群内外水体进行了昼夜连续监测,分析菹草种群内外水质的日变化趋势.结果显示,种群内DO自日出后增加,日落后持续下降,且与水温变化一致,于19:30左右出现1次明显低谷,后略有恢复,后一直降至日出前后;开阔水域DO变化与水温呈现一定相关性,但变化幅度较小;交界处DO含量兼有种群内与开阔水域的变化特点.种群内水体pH值自日出前后升高,日落时达最高值,后开始下降,直至次日出前后;交界处与种群变化趋势一致;开阔水域pH总体变化幅度较小,白天高于夜间,总体DO含量及pH分布:种群区＞交界处＞开阔水域.种群及交界处TN含量均在日出前后达最高,日出后递减,开阔水域夜间含量较高,日出前后达最高;各点NH+4-N变化规律不显著;种群内水体TP含量夜间较高,日出前后达最高值,日出后递减,日落后递增;开阔水域TP含量夜间较高,白天略低;交界处正午前后达最低值,傍晚达最高值,TN、TP总体分布:种群＜交界处＜开阔水域.菹草种群存在对水体DO、pH等环境因子均产生重要影响,继而影响水体内源性氮磷的迁移,其中对pH影响较小,其变化未能影响水体氮磷迁移,而DO昼夜变化较大,对水体内源性氮磷的迁移起重要影响.     

Effects of iron and oxygen species scavengers on Listeria spp. chemiluminescence

Listeria monocytogenes and Listeria innocua are able, under certain conditions, to produce chemiluminescence (CL), which is amplified by luminol. Kinetic studies of CL by L. monocytogenes and L. innocua show a close parallelism between CL and growth curves during the exponential phase, with a maximum of CL reached just before entrance of bacteria into the stationary phase. CL is tightly correlated with the release of oxygen compounds. The reactive oxygen species scavengers tryptophan, mannitol, and tiron, as well as cellobiose and high temperature, were assessed with regard to CL in the two Listeria species. Only tiron strongly reduced the CL emitted by L. monocytogenes and L. innocua. On the other hand, charcoal pretreatment of the growth medium inhibited the CL, whereas ferric citrate strongly increased the CL of L. monocytogenes and L. innocua. These data suggest that iron and superoxide radical are implicated in the CL produced by these bacteria, but this phenomenon is not correlated to virulence.     

Photosynthetic circadian rhythmicity patterns of Symbiodium,the coral endosymbiotic algae

Biological clocks are self-sustained endogenous timers that enable organisms (from cyanobacteria to humans) to anticipate daily environmental rhythms, and adjust their physiology and behaviour accordingly. Symbiotic corals play a central role in the creation of biologically rich ecosystems based on mutualistic symbioses between the invertebrate coral and dinoflagellate protists from the genus Symbiodinium. In this study, we experimentally establish that Symbiodinium photosynthesis, both as a free-living unicellular algae and as part of the symbiotic association with the coral Stylophora pistillata, is ‘wired’ to the circadian clock mechanism with a ‘free-run’ cycle close to 24 h. Associated photosynthetic pigments also showed rhythmicity under light/dark conditions and under constant light conditions, while the expression of the oxygen-evolving enhancer 1 gene (within photosystem II) coincided with photosynthetically evolved oxygen in Symbiodinium cultures. Thus, circadian regulation of the Symbiodinium photosynthesis is, however, complicated as being linked to the coral/host that have probably profound physiochemical influence on the intracellular environment. The temporal patterns of photosynthesis demonstrated here highlight the physiological complexity and interdependence of the algae circadian clock associated in this symbiosis and the plasticity of algae regulatory mechanisms downstream of the circadian clock.     

Proliferation of Toxoplasma gondii in inflammatory macrophages in vivo is associated with diminished oxygen radical production in the host cell

While reactive oxygen species (ROS) can kill Toxoplasma gondii in vitro the role these molecules play in vivo is not known. We used a flow cytometry-based assay to investigate the relationship between intracellular infection and ROS production during acute peritoneal toxoplasmosis in mice. A distinct population of ROS(+) inflammatory macrophages, detected by the oxidation of hydroethidine, was observed to increase progressively in frequency during the course of infection, and to be inversely correlated with the degree of cell parasitization. These data imply that either intracellular parasites inhibit ROS synthesis or, alternatively, ROS-producing cells contain anti-Toxoplasma activity. The latter interpretation was supported by the finding that uninfected ROS-producing inflammatory macrophages were resistant to infection in vivo. However, in the same animals, ROS-producing macrophages that had previously been parasitized could readily be infected with additional parasites, suggesting that the difference in ROS production between highly infected and less infected cells was not due to ROS-associated killing of parasites within these cells. In addition, macrophages infected with T. gondii in vitro and then briefly transferred to acutely infected mice upregulated ROS production in a manner that was again inversely correlated with the degree of intracellular parasitization. Taken together, these findings suggest that both ROS-associated anti-Toxoplasma activity and parasite-driven inhibition of ROS production underlie the observed pattern of ROS production. ROS function and parasite evasion of this function may contribute significantly to the balance between host defense and disease progression during acute infection.     

Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells

Physalis angulata and P. peruviana are herbs widely used in folk medicine. In this study, the aqueous and ethanol extracts prepared from the whole plant of these species were evaluated for their antihepatoma activity. Using XTT assay, three human hepatoma cells, namely Hep G2, Hep 3B and PLC/PRF/5 were tested. The results showed that ethanol extract of P. peruviana (EEPP) possessed the lowest IC50 value against the Hep G2 cells. Interestingly, all extracts showed no cytotoxic effect on normal mouse liver cells. Treatment with carbonyl cyanide m-chlorophenyl hydrazone, a protonophore, caused a reduction of membrane potential (Deltapsim) by mitochondrial membrane depolarization. At high concentrations, EEPP was shown to induce cell cycle arrest and apoptosis through mitochondrial dysfunction as demonstrated by the following observations: (i) EEPP induced the collapse of Deltapsim and the depletion of glutathione content in a dose dependent manner; (ii) pretreatment with the antioxidant (1.0 microg/ml vitamin E) protected cells from EEPP-induced release of ROS; and (iii) at concentrations 10 to 50 microg/ml, EEPP displayed a dose-dependent accumulation of the Sub-G1 peak (hypoploid) and caused G0/G1-phase arrest. Apoptosis was elicited when the cells were treated with 50 microg/ml EEPP as characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane. The results conclude that EEPP possesses potent antihepatoma activity and its effect on apoptosis is associated with mitochondrial dysfunction.     

Effect of copper exposure on GST activity and on the expression of four GSTs under oxidative stress condition in the monogonont rotifer, Brachionus koreanus

Glutathione S-transferases (GSTs; EC 2.5.1.18) are major enzymes that function in Phase II detoxification reactions by catalyzing the conjugation of reduced glutathione through cysteine thiol. In this study, we cloned and sequenced four GST genes from the monogonont rotifer Brachionus koreanus. The domain regions of four Bk-GSTs showed a high similarity to those of other species. In addition, to evaluate the potential of GST genes as an early warning signal for oxidative stress, we exposed sublethal concentrations of copper (Cu) to B. koreanus and measured glutathione (GSH) contents and several antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione reductase (GR; EC 1.8.1.7). The reactive oxygen species (ROS) at 12 h and 24 h after copper exposure increased significantly. GSH contents however did not increase significantly and even it decreased at 0.24 mg/L at 12 h. The activities of several antioxidant enzymes, particularly GPx and GR, showed a dramatic increase in 0.24 mg/L of CuCl₂. Messenger RNAs of each Bk-GST showed different patterns of modulations according to GST types, and particularly, Bk-GST-omega, Bk-GST-sigma, and Bk-GST zeta genes were highly sensitive to Cu. These results indicate that Bk-GSTs, functioning as one of the enzymatic defense mechanisms particularly in the early stage of oxidative stress response, were induced by Cu exposure. This also suggests that these genes and related enzymes have a potential as biomarkers for a more sensitive initial stress response.     

Basal superoxide as a sex-specific immune constraint

There is increasing evidence that reactive oxygen species (ROS), a group of unstable and highly reactive chemical molecules, play a key role in regulating and maintaining life-history trade-offs. Upregulation of ROS in association with immune activation is costly because it may result in an imbalance between pro- and antioxidants and, hence, oxidative damage. Previous research aimed at quantifying this cost has mostly focused on changes in the pro-/antioxidant balance subsequent to an immune response. Here, we test the hypothesis that systemic ROS may constrain immune activation. We show that systemic, pre-challenge superoxide (SO) levels are negatively related to the strength of the subsequent immune response towards the mitogen phytohaemagglutinin in male, but not female painted dragon lizards (Ctenophorus pictus). We therefore suggest that systemic SO constrains immune activation in painted dragon males. We speculate that this may be due to sex-specific selection pressures on immune investment.     

Singlet oxygen-mediated and EXECUTER-dependent signalling and acclimation of Arabidopsis thaliana exposed to light stress

Plants respond to environmental changes by acclimation that activates defence mechanisms and enhances the plant''s resistance against a subsequent more severe stress. Chloroplasts play an important role as a sensor of environmental stress factors that interfere with the photosynthetic electron transport and enhance the production of reactive oxygen species (ROS). One of these ROS, singlet oxygen (¹O₂), activates a signalling pathway within chloroplasts that depends on the two plastid-localized proteins EXECUTER 1 and 2. Moderate light stress induces acclimation protecting photosynthetic membranes against a subsequent more severe high light stress and at the same time activates ¹O₂-mediated and EXECUTER-dependent signalling. Pre-treatment of Arabidopsis seedlings with moderate light stress confers cross-protection against a virulent Pseudomonas syringae strain. While non-pre-acclimated seedlings are highly susceptible to the pathogen regardless of whether ¹O₂- and EXECUTER-dependent signalling is active or not, pre-stressed acclimated seedlings without this signalling pathway lose part of their pathogen resistance. These results implicate ¹O₂- and EXECUTER-dependent signalling in inducing acclimation but suggest also a contribution by other yet unknown signalling pathways during this response of plants to light stress.     

Effect of amphotericin B on the infection success of Schistosoma mansoni in Biomphalaria glabrata

In the present study, we examined the effect of amphotericin B on larval stages (miracidia and primary sporocyst) of the helminth Schistosoma mansoni, the causative agent of human schistosomiasis. Amphotericin B (AmB) is a polyene macrolide that disturbs the function of the cell membrane; it is widely used as prophylactic antimycotic agent in in vitro culture. We show for the first time that S. mansoni miracidia infectivity is considerably reduced after AmB treatment. Moreover we demonstrate that AmB does not affect the development, growth, viability, and behavior of miracidia and primary sporocysts. Our data indicate that AmB effects on S. mansoni sporocyst prevalence are linked to the oxidative properties of AmB. These may alter the capacity of sporocysts to respond to the oxidative stress generated by the snail immune defence system.     

Leaf variegation in the rice zebra2 mutant is caused by photoperiodic accumulation of tetra-Cis-lycopene and singlet oxygen

In field conditions, the zebra2 (z2) mutant in rice (Oryza sativa) produces leaves with transverse pale-green/yellow stripes. It was recently reported that ZEBRA2 encodes carotenoid isomerase (CRTISO) and that low levels of lutein, an essential carotenoid for non-photochemical quenching, cause leaf variegation in z2 mutants. However, we found that the z2 mutant phenotype was completely suppressed by growth under continuous light (CL; permissive) conditions, with concentrations of chlorophyll, carotenoids and chloroplast proteins at normal levels in z2 mutants under CL. In addition, three types of reactive oxygen species (ROS; superoxide [O₂ ⁻], hydrogen peroxide [H₂O₂], and singlet oxygen [¹O₂]) accumulated to high levels in z2 mutants grown under short-day conditions (SD; alternate 10-h light/14-h dark; restrictive), but do not accumulate under CL conditions. However, the levels of lutein and zeaxanthin in z2 leaves were much lower than normal in both permissive CL and restrictive SD growth conditions, indicating that deficiency of these two carotenoids is not responsible for the leaf variegation phenotype. We found that the CRTISO substrate tetra-Cis-lycopene accumulated during the dark periods under SD, but not under CL conditions. Its accumulation was also positively correlated with ¹O₂ levels generated during the light period, which consequently altered the expression of ¹O₂-responsive and cell death-related genes in the variegated z2 leaves. Taking these results together, we propose that the z2 leaf variegation can be largely attributed to photoperiodic accumulation of tetra-cis-lycopene and generation of excessive ¹O₂ under natural day-night conditions.