Stabilization of thylakoid membranes in isoprene-emitting plants reduces formation of reactive oxygen species

Isoprene is emitted by a significant fraction of the world''s vegetation. Isoprene makes leaves more thermotolerant, yet we do not fully understand how. We have recently shown that isoprene stabilizes thylakoid membranes under heat stress. Here we show that heat-stressed, isoprene-emitting transgenic Arabidopsis plants also produce a lower pool of reactive oxygen and reactive nitrogen species, and that this was especially due to a lower accumulation of H₂O₂ in isoprene emitting plants. It remains difficult to disentangle whether in heat stressed plants isoprene also directly reacts with and quenches reactive oxygen species (ROS), or reduces ROS formation by stabilizing thylakoids. We present considerations that make the latter a more likely mechanism, under our experimental circumstances.          

Bordetella bronchiseptica responses to physiological reactive nitrogen and oxygen stresses

Bordetella bronchiseptica can establish prolonged airway infection consistent with a highly developed ability to evade mammalian host immune responses. Upon initial interaction with the host upper respiratory tract mucosa, B. bronchiseptica are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS), effector molecules of the innate immune system. However, the responses of B. bronchiseptica to redox species at physiologically relevant concentrations (nM-microM) have not been investigated. Using predicted physiological concentrations of nitric oxide (NO), superoxide and hydrogen peroxide (H2O2) on low numbers of CFU of B. bronchiseptica, all redox active species displayed dose-dependent antimicrobial activity. Susceptibility to individual redox active species was significantly increased upon introduction of a second species at subantimicrobial concentrations. An increased bacteriostatic activity of NO was observed relative to H2O2. The understanding of Bordetella responses to physiologically relevant levels of exogenous RNS and ROS will aid in defining the role of endogenous production of these molecules in host innate immunity against Bordetella and other respiratory pathogens.     

Determination of mitochondrial reactive oxygen species: methodological aspects

The generation of Reactive Oxygen Species (ROS) as by-products in mitochondria Electron Transport Chain (ETC) has long been admitted as the cost of aerobic energy metabolism with oxidative damages as consequence. The purpose of this methodological review is to present some of the most widespread methods of ROS generation and to underline the limitations as well as some problems, identified with some experiments as examples, in the interpretation of such results. There is now no doubt that besides their pejorative role, ROS are involved in a variety of cellular processes for the continuous adaptation of the cell to its environment. Because ROS metabolism is a complex area (low production, instability of species, efficient antioxidant defense system, several places of production…) bias, variances and limitations in ROS measurements must be recognized in order to avoid artefactual conclusions, and especially to improve our understanding of physiological and pathophysiological mechanisms of such phenomenon.     

A reporter system for the individual detection of hydrogen peroxide and singlet oxygen: its use for the assay of reactive oxygen species produced in vivo

A reporter system for the assay of reactive oxygen species (ROS) was developed in Chlamydomonas reinhardtii, a plant model organism well suited for the application of inhibitors and generators of various types of ROS. This system employs various HSP70A promoter segments fused to a Renilla reniformis luciferase gene as a reporter. Transformants with the complete HSP70A promoter were inducible by both hydrogen peroxide and singlet oxygen. Constructs that lacked upstream heat-shock elements (HSEs) were inducible by hydrogen peroxide, indicating that this induction does not require such HSEs. Rather, downstream elements located between positions -81 to -149 with respect to the translation start site appear to be involved. In contrast, upstream sequences are essential for the response to singlet oxygen. Thus, activation by singlet oxygen appears to require promoter elements that are different from those used by hydrogen peroxide. ROS generated endogenously by treatment of the alga with metronidazole, protoporphyrin IX, dinoterb or high light intensities were detected by this reporter system, and distinguished as production of hydrogen peroxide (metronidazole) and singlet oxygen (protoporphyrin IX, dinoterb, high light). This system thus makes it possible to test whether, under varying environmental conditions including the application of abiotic stress, hydrogen peroxide or singlet oxygen or both are produced.     

A comparison of chemical systems for luminometric determination of antioxidant capacity towards individual reactive oxygen species.

The aim of the study was to investigate the reactive oxygen species (ROS) production in the hypoxanthine-xanthinoxidase (HX-XO), hydrogen peroxide-ferrous sulphate (H2O2-FeSO4) and hydrogen peroxide (H2O2) systems by using various concentrations of ROS scavengers, such as superoxide dismutase (SOD), dimethylthiourea (DMTU) or catalase (CAT). Luminol (0.8 mmol/L) was dissolved in a borate buffer, pH 9.0, and was used as a luminophor in the chemiluminescence (CL) measurements. In the HX-XO system SOD, CAT and DMTU deepened the CL signal, whereas in the H2O2-FeSO4 system, only CAT and DMTU deepened the CL signal, and in the H2O2 system SOD and CAT increased and DMTU deepened the CL signal. Electron spin resonance (ESR) measurements were performed only in the H2O2-FeSO4 system. 5,5-dimethyl-pyrroline-N-oxide (DMPO) was used as a spin trap. According to typical ESR spectra, .OH was produced in this chemical system. It can be concluded that the chemical systems do not produce single reactive oxygen species but a mixture of them.     

Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants.     

Diacylglycerol kinase alpha regulates globular adiponectin-induced reactive oxygen species

Abstract:

It has previously been reported that the globular form of adiponectin (gAd), mature adipocyte-derived cytokine, induced generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated whether diacylglycerol kinases (DGKs), enzymes functioning in sub-cellular signalling pathways, had a role on gAd-induced ROS generation in RAW 264 cells. Administration of R59022, a specific inhibitor for DGK, reduced gAd-induced ROS generation and NO release. RAW 264 cell expressed DGKα mRNA. Depression of DGKα mRNA by RNA interference significantly reduced the ROS generation in response to gAd treatment. Interestingly, transfection with the DGKα-specific small interfering RNA attenuated the expression level of Nox1 mRNA in gAd-treated RAW 264 cells. In addition, the DGKα knockdown with siRNA suppressed gAd-induced NO release.     

线粒体呼吸链与活性氧

已知有氧真核生物细胞吸收的氧分子绝大部分都是在线粒体呼吸链末端细胞色素氧化酶上通过四步单电子还原生成水。但同时也有1％-2％的氧可在呼吸链中途接受单电子或双电子被部分还原生成超氧（O2·^-和过氧化氢（H2O2）作为呼吸作用的正常代谢产物。此种来源于线粒体呼吸链的O2·^-和H2O2不但在多种病理的氧化损伤中起关键作用,同样它们也是正常生理条件下对多种细胞过程具有基本调控意义的氧还信号。基于Chance实验室约自20世纪70到90年代的早期研究贡献以及20世纪90年代后其他各实验室的研究新进展,我们聚焦于下述四个相关问题的评述和讨论：（1）由于线粒体内膜面积及其含有的呼吸链复合体酶活力远远高出细胞中所有膜系数量和相关酶活力之总和,因而线粒体呼吸链产生的O2·^-和H2O2构成生物体内最大数量ROS的恒定来源;（2）线粒体呼吸链复合体III的Q循环中Qo位点中半醌自由基（UQH·）已明确是O2·^-的单电子来源;还原细胞色素C-P66^SHC是生成H2O2的双电子供体。虽然复合体I也是产生线粒体基质内O2·^-的主要来源,但由于其确切生成位点尚未明确,在invivo条件下能否产生大量O2·^-也尚有争议;（3）线粒体呼吸链产生O2·^-后的分配和跨膜转移涉及其生理病理作用机制和作用靶点等复杂而重要的问题,直到目前尚未意见一致。“质子和O2·^-循环双回路解偶联模型”整合了目前提出的几种假说的联系点,指出H^＋和O2·^-相互作用生成HO2·及其跨膜很可能是这一复杂问题的中心环节,并与O2·^-对“脂肪酸shuttling model”或O2·^-对“UCPS激活”模型形成了内在的联系;（4）线粒体呼吸形成的△P（△ψ和△pH）能直接控制呼吸链的ROS生成,并以非线性（非欧姆）相关方式通过影响Q循环中的Qo半醌的氧还态和寿命来调节O2·^-生成的急速?     

An inducible release of reactive oxygen radicals in four species of gorgonian corals

The capability for physical injury or heat stress to elicit the production of reactive oxygen species was examined in four species of gorgonian corals. The sea plumes Pseudopterogorgia elisabethae, Pseudopterogorgia americana, the sea rod Eunicea fusca and the azooxanthellate red branching gorgonian Lophogorgia chilensis were physically injured using sonic sound cavitations and heat shocked by incubation in 33°C sea water. The pharmacological probe, diphenylene iodonium chloride (DPI), an inhibitor of NAD(P)H oxidase and peroxidases was used to identify an enzymatic surrogate of the oxidative burst. Both injury and heat stress were capable of inducing the release of reactive oxygen species (ROS) in all gorgonians tested, yet the kinetics and amplitude of ROS release varied among genera. In both the treatments, P. americana demonstrated the largest oxidative burst among the other corals tested.     

Reactive oxygen species, nitric oxide, and their interactions play different roles in Cupressus lusitanica cell death and phytoalexin biosynthesis

Beta-thujaplicin Is a natural troponoid with strong antifungal, antiviral, and anticancer activities. Beta-thujaplicin production in yeast elicitor-treated Cupressus lusitanica cell culture and its relationships with reactive oxygen species (ROS) and nitric oxide (NO) production and hypersensitive cell death were investigated. Superoxide anion radical (O2*-) induced cell death and inhibited beta-thujaplicin accumulation, whereas hydrogen peroxide (H2O2) induced beta-thujaplicin accumulation but did not significantly affect cell death. Both elicitor and O2*- induced programmed cell death, which can be blocked by protease inhibitors, protein kinase inhibitors, and Ca2+ chelators. Elicitor-induced NO generation was nitric oxide synthase (NOS)-dependent. Inhibition of NO generation by NOS inhibitors and NO scavenger partly blocked the elicitor-induced beta-thujaplicin accumulation and cell death, and NO donors strongly induced cell death. Interaction among NO, H2O2, and O2*- shows that NO production and H2O2 production are interdependent, but NO and O2*- accumulation were negatively related because of coconsumption of NO and O2*-. NO- and O2*- -induced cell death required each other, and both were required for elicitor-induced cell death. A direct interaction between NO and O2*- was implicated in the production of a potent oxidant peroxynitrite, which might mediate the elicitor-induced cell death.     

Cancer-derived immunoglobulin G promotes tumor cell growth and proliferation through inducing production of reactive oxygen species

Cancer cells have been found to express immunoglobulin G (IgG), but the exact functions and underlying mechanisms of cancer-derived IgG remain elusive. In this study, we first confirmed that downregulation of IgG restrained the growth and proliferation of cancer cells in vitro and in vivo. To elucidate its mechanism, we carried out a co-immunoprecipitation assay in HeLa cells and identified 27 potential IgG-interacting proteins. Among them, receptor of activated protein kinase C 1 (RACK1), ras-related nuclear protein (RAN) and peroxiredoxin 1 (PRDX1) are closely related to cell growth and oxidative stress, which prompted us to investigate the mechanism of action of IgG in the above phenomena. Upon confirmation of the interactions between IgG and the three proteins, further experiments revealed that downregulation of cancer-derived IgG lowered levels of intracellular reactive oxygen species (ROS) by enhancing cellular total antioxidant capacity. In addition, a few ROS scavengers, including catalase (CAT), dimethylsulfoxide (DMSO), n-acetylcysteine (NAC) and superoxide dismutase (SOD), further inhibited the growth of IgG-deficient cancer cells through suppressing mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK) signaling pathway induced by a low level of intracellular ROS, whereas exogenous hydrogen peroxide (H₂O₂) at low concentration promoted their survival via increasing intracellular ROS levels. Similar results were obtained in an animal model and human tissues. Taken together, our results demonstrate that cancer-derived IgG can enhance the growth and proliferation of cancer cells via inducing the production of ROS at low level. These findings provide new clues for understanding tumor proliferation and designing cancer therapy.     

Involvement of reactive oxygen species in PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm,Bombyx mori

In the present study, the possible involvement of reactive oxygen species (ROS) in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis of Bombyx mori prothoracic glands (PGs) was investigated. Results showed that PTTH treatment resulted in a rapidly transient increase in the intracellular ROS concentration, as measured using 2′,7′-dichlorofluorescin diacetate (DCFDA), an oxidation-sensitive fluorescent probe. The antioxidant, N-acetylcysteine (NAC), abolished PTTH-induced increase in fluorescence. Furthermore, PTTH-induced ROS production was partially inhibited by the NAD(P)H oxidase inhibitor, apocynin, indicating that NAD(P)H oxidase is one of the sources for PTTH-stimulated ROS production. Four mitochondrial oxidative phosphorylation inhibitors (rotenone, antimycin A, the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and diphenylene iodonium (DPI)) significantly attenuated ROS production induced by PTTH. These data suggest that the activity of complexes I and III in the electron transport chain and the mitochondrial inner membrane potential (ΔΨ) contribute to PTTH-stimulated ROS production. In addition, PTTH-stimulated ecdysteroidogenesis was greatly inhibited by treatment with either NAC or mitochondrial inhibitors (rotenone, antimycin A, FCCP, and DPI), but not with apocynin. These results indicate that mitochondria-derived, but not membrane NAD(P)H oxidase-mediated ROS signaling, is involved in PTTH-stimulated ecdysteroidogenesis of PGs in B. mori.     

Interaction between albumin-bound dinitrosyl iron complexes and reactive oxygen species

Dinitrosyl iron complexes (DNIC) bound to BSA are shown to be destroyed by superoxide radicals generated in the xanthine oxidase-xanthine system. Peroxynitrite is also efficient in this respect. By contrast, neither hydrogen peroxide nor tert-butyl hydroperoxide appreciably destroy BSA-DNIC even at a tenfold molar excess. Evidence is obtained for the vasodilatory properties of BSA-DNIC. It is suggested that in this way peroxynitrite and superoxide radical can affect the physiological functions of nitric oxide.     

Nucleotide receptor signalling and the generation of reactive oxygen species

Elevated levels of extracellular nucleotides are present at sites of inflammation, platelet degranulation and cellular damage or lysis. These extracellular nucleotides can lead to the activation of purinergic (nucleotide) receptors on various leukocytes, including monocytes, macrophages, eosinophils, and neutrophils. In turn, nucleotide receptor activation has been linked to increased cellular production and release of multiple inflammatory mediators, including superoxide anion, nitric oxide and other reactive oxygen species (ROS). In the present review, we will summarize the evidence that extracellular nucleotides can facilitate the generation of multiple ROS by leukocytes. In addition, we will discuss several potential mechanisms by which nucleotide-enhanced ROS production may occur. Delineation of these mechanisms is important for understanding the processes associated with nucleotide-induced antimicrobial activities, cell signalling, apoptosis, and pathology. This work was supported by National Institutes of Health Grants HL56396 and AI50500. The first author was supported by the Hematology Training Program NIH 5 T32 HL07899 at the University of Wisconsin.     

Lactobacillus rhamnosus GG conditioned media modulates acute reactive oxygen species and nitric oxide in J774 murine macrophages

Phagocytes such as macrophages are capable of detecting and killing pathogenic bacteria by producing reactive oxygen and nitrogen species. Formation of free radicals in macrophages may be regulated by probiotics or by factors released by probiotics but yet to be identified. Thus, studies were carried out to determine whether cell-free conditioned medium obtained from cultures of Lactobacillus rhamnosus GG (LGG-CM) regulate production of reactive oxygen species (ROS) and/or nitric oxide (NO) in macrophages. J774 macrophages in culture were loaded with either H₂DCFDA for monitoring ROS or with DAFFM-DA for NO detection. Free radical production was measured on a fluorescence microplate reader and changes were analysed by Cumulative sum (CuSum) calculations. Low concentration of LGG-CM (10% LGG-CM) or LPS did not cause any significant change in basal levels of ROS or NO production. In contrast, high concentration of LGG-CM (75% and 100%) significantly enhanced ROS generation but also significantly reduced NO level. These findings are novel and suggest for the first time that probiotics may release factors in culture which enhance ROS production and may additionally reduce deleterious effects associated with excessive nitrogen species by suppressing NO level. These events may account, in part, for the beneficial bactericidal and anti-inflammatory actions ascribed to probiotics and may be of clinical relevance.     

Chilling-enhanced photooxidation: The production,action and study of reactive oxygen species produced during chilling in the light

Chilling-enhanced photooxidation is the light- and oxygen-dependent bleaching of photosynthetic pigments that occurs upon the exposure of chilling-sensitive plants to temperatures below approximately 10 °C. The oxidants responsible for the bleaching are the reactive oxygen species (ROS) singlet oxygen (¹O₂), superoxide anion radical (O ₂ ,hydrogen peroxide (H₂O₂), the hydroxyl radical (OH·), and the monodehydroascorbate radical (MDA) which are generated by a leakage of absorbed light energy from the photosynthetic electron transport chain. Cold temperatures slow the energy-consuming Calvin-Benson Cycle enzymes more than the energy-transducing light reactions, thus causing leakage of energy to oxygen. ROS and MDA are removed, in part, by the action of antioxidant enzymes of the Halliwell/Foyer/Asada Cycle. Chloroplasts also contain high levels of both lipid- and water-soluble antioxidants that act alone or in concert with the HFA Cycle enzymes to scavenge ROS. The ability of chilling-resistant plants to maintain active HFA Cycle enzymes and adequate levels of antioxidants in the cold and light contributes to their ability to resist chilling-enhanced photooxidation. The absence of this ability in chilling-sensitive species makes them susceptible to chilling-enhanced photooxidation. Chloroplasts may reduce the generation of ROS by dissipating the absorbed energy through a number of quenching mechanisms involving zeaxanthin formation, state changes and the increased usage of reducing equivalents by other anabolic pathways found in the stroma. During chilling in the light, ROS produced in chilling-sensitive plants lower the redox potential of the chloroplast stroma to such a degree that reductively-activated regulatory enzymes of the Calvin Cycle, sedohepulose 1,7 bisphosphatase (EC 3.1.3.37) and fructose 1,6 bisphosphatase (EC 3.1.3.11), are oxidatively inhibited. This inhibition is reversible in vitro with a DTT treatment indicating that the enzymes themselves are not permanently damaged. The inhibition of SBPase and FBPase may fully explain the inhibition in whole leaf gas exchange seen upon the rewarming of chilling-sensitive plants chilled in the light. Methods for the study of ROS in chilling-enhanced photooxidation and challenges for the future are discussed.Abbreviations ASP ascorbate-specific peroxidase - -TH reduced -tocopherol - DTT dithiothreitol - FBP fructose 1,6 bisphosphate - FBPase fructose 1,6 bisphosphatase (EC 3.1.3.11) - HFA Cycle the Halliwell/Foyer/Asada Cycle responsible for the enzymatic removal of ROS in the chloroplast stroma - MDA monodehydroascorbate radical - MDAR monodehydroascorbate reductase - ROS reactive oxygen species - SBP sedohepulose 1,7 bisphosphate - SBPase sedohepulose 1,7 bisphosphatase (EC 3.1.3.37) - SOD superoxide dismutase     

Modulation of the GABA(A)-gated chloride channel by reactive oxygen species

The accumulation of reactive oxygen species during cellular injury leads to oxidative stress. This can have profound effects on ionic homeostasis and neuronal transmission. Gamma-aminobutyric acid (GABA) neurotransmission is sensitive to reactive oxygen species, but most studies have indicated that this is due to alterations in GABA release. Here, we determined whether reactive oxygen species can alter GABA(A) receptor-gated Cl- channels in the adult hippocampus. First, we measured the effects of hydrogen peroxide on intracellular Cl- using UV laser scanning confocal microscopy and the Cl(-)-sensitive probe, 6-methoxy-N-ethylquinolium iodide (MEQ). Superfusion of adult rat hippocampal slices with hydrogen peroxide for 10 min decreased MEQ fluorescence (elevation in [Cl-]i) significantly in area CA1 pyramidal cell soma. Alterations in [Cl-]i were prevented by the vitamin E analog Trolox, an antioxidant that scavenges free radicals. After exposure of slices to hydrogen peroxide, the ability of the GABA agonist muscimol to increase [Cl-]i was attenuated. To determine if GABA(A) receptors were sensitive to oxidative insults, the effect of hydrogen peroxide on the binding of [35S]t-butylbicyclophosphorothionate (TBPS) to GABA-gated Cl- channels was measured using receptor autoradiography and homogenate binding assays. Hydrogen peroxide inhibited [35S]TBPS binding in a regionally selective manner, with the greatest inhibition in cerebral cortex, hippocampus and striatum, areas vulnerable to oxidative stress. Similarly, xanthine and xanthine oxidase, which generate superoxide radicals, reduced [35S]TBPS binding in these regions. The effect of hydrogen peroxide on [35S]TBPS binding was non-competitive and was prevented by Trolox and the iron chelator, deferoxamine. We conclude that reactive oxygen species may compromise GABA(A)-mediated neuronal inhibition via interaction with pre and postsynaptic sites. A reduction in GABA(A)-gated Cl- channel function during periods of oxidative stress may contribute to the development of neuronal damage.     

Isolated respiring heart mitochondria release reactive oxygen species in states 4 and 3

Isolated mitochondria respiring on physiological substrates, both in state 4 and 3, are reported to be or not to be a source of reactive oxygen species (ROS). The cause of these discrepancies has been investigated. As protein concentration was raised in in vitro assays at 37°C, the rate of H₂O₂ release by rat heart mitochondria supplemented with pyruvate/malate or with succinate (plus rotenone) was shown to increase (0.03-0.15 mg protein/ml), to decrease (0.2-0.5 mg protein/ml) and to be negligible (over 0.5 mg protein/ml). The inhibition of mitochondrial respiration (with rotenone or antimycin A) or the increase in the oxygen concentration dissolved in the assay medium allowed an enhancement of ROS production rate throughout the studied range of protein concentrations. In mitochondria respiring in state 3 on pyruvate/malate or on succinate (plus rotenone), ROS release vanished for protein concentrations over 0.5 or 0.2 mg/ml, respectively. However, ROS production rates measured with low protein concentrations (below 0.1 mg/ml) or in oxygen-enriched media were similar or even slightly higher in the active respiratory state 3 than in the resting state 4 for both substrates. Consequently, these findings indicate that isolated mitochondria, respiring in vitro under conditions of forward electron transport, release ROS with Complex I- and II-linked substrates in the resting condition (state 4) and when energy demand is maximal (state 3), provided that there is sufficient oxygen dissolved in the medium.