Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants.

Plant defense responses to pathogen infection involve the production of active oxygen species, including hydrogen peroxide (H2O2). We obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. H2O2 levels were elevated in both leaf and tuber tissues of these plants. Transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp carotovora, and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H2O2, because the resistance could be counteracted by exogenously added H2O2-degrading catalase. The transgenic plants with increased levels of H2O2 also exhibited enhanced resistance to potato late blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of an active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.     

Polyoxometalates as peroxidase mimetics and their applications in H2O2 and glucose detection

Polyoxometalates (H(3)PW(12)O(40), H(4)SiW(12)O(40) and H(3)PMo(12)O(40)) have been proven to possess intrinsic peroxidase-like activity for the first time, which can catalyze oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) by H(2)O(2) to form a blue color in aqueous solution. Among them, H(3)PW(12)O(40) (PW(12)) exhibits higher catalytic activity to TMB than natural enzyme HRP and other two POMs. In addition, H(3)PW(12)O(40)/graphene exhibited higher activity than H(3)PW(12)O(40) in this catalytic oxidation reaction due to the effect of graphene in promoting the electron transfer between the substrate and catalyst. POMs/H(2)O(2)/TMB system provides a simple, accurate approach to colorimetric detection for H(2)O(2) or glucose. The colorimetric method based on POMs showed good response toward H(2)O(2) and glucose detection with a linear range from 1.34×10(-7) to 6.7×10(-5) mol/L and 1×10(-7) to 1×10(-4) mol/L, respectively. The results showed that it is a simple, cheap, more convenient, highly selective, sensitive, and easy handling colorimetric assay.     

一株产葡萄糖氧化酶菌株的筛选、鉴定及酶学性质表征

[背景]海洋中蕴藏着大量未被开发利用的微生物种质资源,而且海洋微生物产的酶类因其具有耐低温、耐高压和耐高盐等明显区别于陆地微生物所产酶类的特点而备受关注.[目的]从渤海海域海泥样品中分离筛选产葡萄糖氧化酶的菌株,并研究其酶学性质.[方法]通过平板初筛和酶活复筛,确定产葡萄糖氧化酶的菌株;通过形态学鉴定和构建系统发育树分...     

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O₂ into superoxide radical, later transformed into H₂O₂. This enzymatic activity requires previous activation with either 3 mM Mn²⁺ or guanosine 5′-0-(3-thiotriphosphate) (GTPγS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S_0.5 for NADPH was 44 μM; S_0.5 for FAD was 8 μM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPγS plus adrenaline was dose- and Ca²⁺-dependent and proceeded through α₁-adrenergic receptors (AR), whereas β-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H₂O₂ as additional transduction signal for adrenaline response in hepatic cells.     

Sources of glucose production in cirrhosis by 2H2O ingestion and 2H NMR analysis of plasma glucose

Plasma glucose 2H enrichment was quantified by 2H NMR in patients with cirrhosis (n=6) and healthy subjects (n=5) fasted for 16 h and given 2H(2)O to approximately 0.5% body water. The percent contribution of glycogenolysis and gluconeogenesis to glucose production (GP) was estimated from the relative enrichments of hydrogen 5 and hydrogen 2 of plasma glucose. Fasting plasma glucose levels were normal in both groups (87+/-7 and 87+/-24 mg/dl for healthy and cirrhotic subjects, respectively). The percent contribution of glycogen to GP was smaller in cirrhotics than controls (22+/-7% versus 46+/-4%, P<0.001), while the contribution from gluconeogenesis was larger (78+/-7% versus 54+/-4%, P<0.001). In all subjects, glucose 6R and 6S hydrogens had similar enrichments, consistent with extensive exchange of 2H between body water and the hydrogens of gluconeogenic oxaloacetate (OAA). The difference in 2H-enrichment between hydrogen 5 and hydrogen 6S was significantly larger in cirrhotics, suggesting that the fractional contribution of glycerol to the glyceraldehyde-3-phosphate (G3P)-moiety of plasma glucose was higher compared to controls (19+/-6% versus 7+/-6%, P<0.01). In all subjects, hydrogens 4 and 5 of glucose had identical enrichments while hydrogen 3 enrichments were systematically lower. This reflects incomplete exchange between the hydrogen of water and that of 1-R-dihydroxyacetone phosphate (DHAP) or incomplete exchange of DHAP and G3P pools via triose phosphate isomerase.     

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O(2) into superoxide radical, later transformed into H(2)O(2). This enzymatic activity requires previous activation with either 3 mM Mn(2+) or guanosine 5'-0-(3-thiotriphosphate) (GTPgammaS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S(0.5) for NADPH was 44 microM; S(0.5) for FAD was 8 microM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPgammaS plus adrenaline was dose- and Ca(2+)-dependent and proceeded through alpha(1)-adrenergic receptors (AR), whereas beta-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H(2)O(2) as additional transduction signal for adrenaline response in hepatic cells.     

The role of H2O2 as a mediator of UVB-induced apoptosis in keratinocytes

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Previously, it has been reported that UVB-irradiation of keratinocytes leads to intracellular generation of hydrogen peroxide (H₂O₂) and that antioxidants can inhibit ROS-induced apoptosis. Although both UVB-irradiation and H₂O₂-incubation led to increased intracellular H₂O₂ levels, the antioxidants catalase and glutathione monoester (GME), inhibited apoptosis only when induced by H₂O₂, not by UVB. Furthermore, extracellular signal-regulated kinase (ERK), a prominent member of the mitogen-activated protein kinase (MAPK) family, was found to be activated by treatment with both UVB and H₂O₂. Inhibition of ERK phosphorylation by pre-treatment with PD98059 resulted in enhanced apoptosis after H₂O₂-exposure. However, no significant difference of apoptosis was observed between cells with and without inhibitor pre-treatment upon UVB-irradiation. DNA damage in the form of cyclobutane pyrimidine dimers was observed after exposure to UVB, but no photoproducts were found in H₂O₂-treated cells. These results suggest a ROS-independent pathway of UVB-induced apoptosis. Although UVB-irradiation causes moderate increase in H₂O₂, the generation of H₂O₂ does not contribute to the induction of apoptosis. Moreover, activation of ERK only blocks H₂O₂-dependent apoptosis but has no impact on UVB-induced apoptosis.     

H2O2诱导Neuro—2a细胞死亡机理的研究

Reactive oxygen species (ROS), such as H2O2, can be produced by enzymes involved in electron leakage of respiration chain in mitochondria, and by neurochemical enzymes such as monoamine oxidase in neural cells. ROS are toxic to cells, and can result in cell death. ROS also play an important role in some diseases, especially in neurodegenerative diseases by yet unknown mechanisms. In the current research, the N-2a neuroblastoma cell was treated with H2O2, and the morphological changes of cell death were characterized. Our results show that N-2a cell death is different from classical apoptosis, but belongs type II nerve cell programmed death, which shows condensed chromatin within intact nuclear envelope and no apoptotic body. The chromatin DNA of dead cells shows no internucleosomal cleavage, as well as no requirement for caspase-3, 1 activity. However, the H2O2-induced N-2a cell death can be inhibited by Bcl-XL. It can be concluded that type II nerve cell death is the result of cell toxicity mediated by ROS. The results pave the way for further research of type II nerve cell death.     

The role of H2O2 outer diffusion on the performance of implantable glucose sensors

The performance of an implantable glucose sensor is strongly dependent on the ability of their outer membrane to govern the diffusion of the various participating species. In this contribution, using a series of layer-by-layer (LBL) assembled outer membranes, the role of outwards of H(2)O(2) diffusion through the outer membrane of glucose sensors has been correlated to sensor sensitivity. Glucose sensors with highly permeable humic acids/ferric cations (HAs/Fe(3+)) outer membranes displayed a combination of lower sensitivities and better linearities when compared with sensors coated with lesser permeable outer membranes (namely HAs/poly(diallyldimethylammonium chloride) (PDDA) and poly(styrene sulfonate) (PSS)/PDDA). On the basis of a comprehensive evaluation of the oxygen dependence of these sensors in conjunction with the permeability of H(2)O(2) through these membranes, it was concluded that the outer diffusion of H(2)O(2) is crucial to attain optimized sensor performance. This finding has important implications to the design of various bio-sensing elements employing perm-selective membranes.     

Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non‐phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H₂O₂ on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti‐angiogenesis. Our results showed that H₂O₂ dose‐dependently increased the generation of O₂ ^? (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H₂O₂ at low concentrations (10 µM) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H₂O₂ at 4 nmol/cm² strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm²). Also, H₂O₂ (200～500 µM) could stimulate apoptosis in HUVECs. All the effects of H₂O₂ on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H₂O₂ to produce O₂ ^? and then to regulate angiogenesis. In summary, our results suggest that H₂O₂ as well as O₂ ^? mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.     

Structure of coenzyme F420H2 oxidase (FprA), a di-iron flavoprotein from methanogenic Archaea catalyzing the reduction of O2 to H2O

The di-iron flavoprotein F(420)H(2) oxidase found in methanogenic Archaea catalyzes the four-electron reduction of O(2) to 2H(2)O with 2 mol of reduced coenzyme F(420)(7,8-dimethyl-8-hydroxy-5-deazariboflavin). We report here on crystal structures of the homotetrameric F(420)H(2) oxidase from Methanothermobacter marburgensis at resolutions of 2.25 A, 2.25 A and 1.7 A, respectively, from which an active reduced state, an inactive oxidized state and an active oxidized state could be extracted. As found in structurally related A-type flavoproteins, the active site is formed at the dimer interface, where the di-iron center of one monomer is juxtaposed to FMN of the other. In the active reduced state [Fe(II)Fe(II)FMNH(2)], the two irons are surrounded by four histidines, one aspartate, one glutamate and one bridging aspartate. The so-called switch loop is in a closed conformation, thus preventing F(420) binding. In the inactive oxidized state [Fe(III)FMN], the iron nearest to FMN has moved to two remote binding sites, and the switch loop is changed to an open conformation. In the active oxidized state [Fe(III)Fe(III)FMN], both irons are positioned as in the reduced state but the switch loop is found in the open conformation as in the inactive oxidized state. It is proposed that the redox-dependent conformational change of the switch loop ensures alternate complete four-electron O(2) reduction and redox center re-reduction. On the basis of the known Si-Si stereospecific hydride transfer, F(420)H(2) was modeled into the solvent-accessible pocket in front of FMN. The inactive oxidized state might provide the molecular basis for enzyme inactivation by long-term O(2) exposure observed in some members of the FprA family.     

The acid-induced state of glucose oxidase exists as a compact folded intermediate

A systematic investigation of the acid-induced unfolding of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase) (GOD) from Aspergillus niger was made using steady-state tryptophan fluorescence, circular dichroism (CD), and ANS (1-anilino 8-naphthalene sulfonic acid) binding. Intrinsic tryptophan fluorescence studies showed a maximally unfolded state at pH 2.6 and the presence of a non-native intermediate in the vicinity of pH 1.4. Flavin adenine dinucleotide (FAD) fluorescence measurements indicate that the bound cofactors are released at low pH. In the pH range studied, near- and far-UV CD spectra show maximal loss of tertiary as well as secondary structure (40%) at pH 2.6 although glucose oxidase at this pH is relatively less denatured as compared to the conformation in 6M GdnHCl. Interestingly, in the vicinity of pH 1.4, glucose oxidase shows a refolded conformation (A-state) with approximately 90% of native secondary structure and native-like near-UV CD spectral features. ANS fluorescence studies, however, show maximal binding of the dye to the protein at pH 1.4, indicating a "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a more compact conformation at low pH. Thermal stability of this state was assessed by ellipticity changes at 222 nm relative to native protein. While native glucose oxidase showed a completely reversible thermal denaturation profile, the state at pH 1.4 showed approximately 50% structural loss and the denatured state appeared to be in a different conformation exhibiting prominent beta-sheet structure (around 85 degrees C) that was not reversible. To summarize; the A-state of GOD exists as a compact folded intermediate with "molten-globule"-like characteristics, viz., native-like secondary structure but with non-native cofactor environment, enhanced hydrophobic surface area and non-cooperative thermal unfolding. That the A-state also possesses significant tertiary structure is an interesting observation made in this study.     

Prevention of H2O2 generation by monoamine oxidase protects against CNS O2 toxicity.

Toxicity to the central nervous system (CNS) by hyperbaric oxygen (HBO) presumably relates to increased production of reactive oxygen species. The sites of generation of reactive oxygen species during HBO, however, have not been fully characterized in the brain. We investigated the relationship between regional generation of hydrogen peroxide (H2O2) in the brain in the presence of an irreversible inhibitor of catalase, aminotriazole (ATZ), and protection from CNS O2 toxicity by a monoamine oxidase (MAO) inhibitor, pargyline. At 6 ATA of oxygen, pargyline significantly protected rats from CNS O2 toxicity whereas ATZ enhanced O2 toxicity. In animals pretreated with ATZ, HBO inactivated 21-40% more catalase than air exposure in the six brain regions studied. Because ATZ-mediated inactivation of catalase was H2O2 dependent, the decrease in catalase activity during hyperoxia was proportional to the intracellular production of H2O2. Pargyline, administered 30 min before HBO, inhibited MAO by greater than 90%, prevented ATZ inhibition of catalase activity during HBO, and reversed the augmentation of CNS O2 toxicity by ATZ. These findings indicate that H2O2 generated by MAO during hyperoxia is important to the pathogenesis of CNS O2 toxicity in rats.     

Characterization of ThOX proteins as components of the thyroid H(2)O(2)-generating system

We have recently cloned two thyroid-specific cDNAs encoding new members of the NADPH oxidase family. ThOX1 and ThOX2 proteins are colocalized with thyroperoxidase at the apical membrane of human thyroid cells. In the present study we have determined their subcellular localization and maturation in relation to their enzymatic activity. A majority of ThOX proteins accumulated inside the cell and only a small fraction was expressed at the surface. Western blots demonstrated that ThOX's are glycoproteins of 180,000 and 190,000. When totally deglycosylated the molecular weight of both ThOX1 and ThOX2 drops to 160,000. Ca(2+) stimulates the basal H(2)O(2) generation in PC Cl3 cells at a level corresponding to 20% of the leukocyte H(2)O(2) production stimulated by PMA. Nonthyroid cell lines transfected with ThOX1 and ThOX2 show only a single immunoreactive band in Western blot analysis, corresponding to the protein of 180,000. This "immature" protein remains exclusively intracellular and does not present any enzymatic activity. This is not modified by coexpression of thyroperoxidase and p22(Phox). Transfection of ThOX cDNAs into PLB-XCGD cells does not reconstitute their NADPH oxidase activity. We conclude that (1) the thyroid contains some elements of the leukocyte H(2)O(2)-generating system but not all of them; (2) ThOX's are predominantly or exclusively located inside the cell in thyrocytes or in transfected cells, respectively, and as such they are inactive; (3) ThOX's cannot replace gp91(Phox) in the leukocyte; and (4) the thyroid H(2)O(2)-generating system is analogous to the leukocyte system with regard to ThOX's and gp91(Phox) but very different in other aspects. Additional thyroid-specific components are probably required to get complete protein processing and full enzymatic activity in the thyroid.     

Plant sulfite oxidase as novel producer of H2O2: combination of enzyme catalysis with a subsequent non-enzymatic reaction step

Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome.     

Underestimation of D-glucose phosphorylation as measured by 3H2O production from D-[2-3H]glucose

In rat pancreatic islets, tumoral islet cells (RINm5F line), parotid gland, and in human erythrocytes, but not in rat hepatocytes, the production of 3H2O from D-[2-3H]glucose is 20-30% lower than from D-[5-3H]glucose. This coincides with the production of tritiated lactic acid from D-[2-3H]glucose and may be attributable to an intramolecular hydrogen transfer in the phosphoglucoisomerase reaction. It is concluded that the production of 3H2O from D-[2-3H]glucose is not a reliable tool to assess the total rate of hexose phosphorylation.     

Tuning the redox and enzymatic activity of glucose oxidase in layered organic films and its application in glucose biosensors

Glucose oxidase was embedded in organic films through a layer-by-layer approach, where the enzyme demonstrated significantly enhanced electron-transfer reactivity and finely tuned enzymatic activity. An unmediated, reagentless glucose biosensor was accordingly prepared with two polyethylenimine/glucose oxidase bilayers-modified pyrolytic graphite electrode. A calibration linear range of glucose was 0.5-8.9 mM with a detection limit of 50 microM and sensitivity of 0.76 microA mM(-1).     

Weak O2 binding and strong H2O2 binding at the non-heme diiron center of trypanosome alternative oxidase

Alternative oxidase (AOX) catalyzes the four-electron reduction of dioxygen to water as an additional terminal oxidase, and the catalytic reaction is critical for the parasite to survive in its bloodstream form. Recently, the X-ray crystal structure of trypanosome alternative oxidase (TAO) complexed with ferulenol was reported and the molecular structure of the non-heme diiron center was determined. The binding of O₂ was a unique side-on type compared to other iron proteins. In order to characterize the O₂ binding state of TAO, the O₂ binding states were searched at a quantum mechanics/molecular mechanics (QM/MM) theoretical level in the present study. We found that the most stable O₂ binding state is the end-on type, and the binding states of the side-on type are higher in energy. Based on the binding energies and electronic structure analyses, O₂ binds very weakly to the TAO iron center (ΔE =6.7 kcal mol^?1) in the electronic state of Fe(II)…OO, not in the suggested charge transferred state such as the superoxide state (Fe(III)OO· ^–) as seen in hemerythrin. Coordination of other ligands such as water, Cl^?, CN^?, CO, N₃^? and H₂O₂ was also examined, and H₂O₂ was found to bind most strongly to the Fe(II) site by ΔE = 14.0 kcal mol^?1. This was confirmed experimentally through the measurement of ubiquinol oxidase activity of TAO and Cryptosporidium parvum AOX which was found to be inhibited by H₂O₂ in a dose-dependent and reversible manner.