Diphenylene Iodonium Research Articles

Identification and activity of superoxide-producing protein complexes of the plasma membrane of etiolated maize seedlings subjected to low positive temperature

Kinetics of superoxide anion generation by the isolated plasma membrane was determined by the rate of formazan formation from XTT in the presence of NADPH or NADH. The plasma membrane was prepared from (control) etiolated maize seedlings grown at 25°C and from (cooled) seedlings incubated at 6°C for the last day. Membrane vesicles from the control plants possessed superoxide-producing activity, and the rate of NADH oxidation was markedly higher than that of NADPH. The low-temperature incubation of the seedlings suppressed the NADPH-dependent activity, whereas the NADH-dependent one slightly increased. The solubilized by dodecyl maltoside (DDM) plasma membranes were separated into multiprotein complexes by high-resolution clear native electrophoresis (hrCN-PAGE). The aim was to find complexes exhibiting the superoxide-producing activity sensitive to inhibition by diphenylene iodonium. Several protein complexes from the plasma membrane capable of superoxide producion in the presence of NADPH or NADH were found. The maximum diphenylene iodonium-sensitive activity was found in the high-molecular weight complex, in which proteins reacting with antibodies against C-terminal peptide of phagocytic oxidase (gp91phox) were detectable. The activity of this complex was lower in the cooled than in the control seedlings and displayed higher affinity to NADPH than to NADH. To search for the cooling-induced changes in the polypeptide content of protein complexes, the two-dimensional difference gel electrophoresis (hrCN/SDS-PAGE) was used. Control and cooled samples, whose lysine had been labeled with fluorescent dyes Cy2 and Cy3, respectively, were separated by this method in one gel. Decrease in a temperature of plant growing affected the protein content of the complex so that some new proteins appeared and several polypeptides disappeared as compared with the control. There were no significant differences between the cooled and control counterparts in the content of proteins detectable with gp91phox antibodies. Therefore, the high-molecular complex containing NADPH oxidase looses proteins under low temperature that may decrease its superoxide-producing activity.

Flavin-containing enzymes as a source of reactive oxygen species in HEMA-induced apoptosis

ObjectiveOxidative stress induced by compounds of dental composites like 2-hydroxyethyl methacrylate (HEMA) due to excess formation of reactive oxygen species (ROS) disturbs vital cell functions leading to apoptosis. The sources of ROS in cells exposed to resin monomers are unknown. The present study investigates functions of flavin-containing ROS and RNS (reactive nitrogen species) producing enzymes in cells exposed to HEMA. MethodsThe formation of oxidative stress in RAW264.7 mouse macrophages exposed to HEMA (0–6–8mM) was determined by flow cytometry (FACS) after staining of cells with 2′7′-dichlorodihydrofluorescin diacetate (H2DCF-DA), dihydroethidium (DHE) or dihydrorhodamine 123 (DHR123). Cells in apoptosis or necrosis were identified by annexin-V-FITC/propidium iodide labeling followed by FACS analysis. Expression of ROS/RNS producing enzymes was analyzed by Western blotting. ResultsDCF fluorescence increased in cells exposed to HEMA for 1h suggesting the production of hydroxyl radicals, H2O2, or nitric oxide and superoxide anions which form peroxynitrite (ONOO-). Increased DHR123 fluorescence after 24h indicated the formation of mostly H2O2. The induction of apoptosis in the presence of HEMA was decreased by low concentrations of diphenylene iodonium (DPI), an inhibitor of flavin-containing enzymes. Expression of p47phox, a regulatory subunit of the superoxide producing Nox2, was downregulated, and the expression of NOS which produces nitric oxide (NO) was possibly inhibited by feedback loop mechanisms in HEMA-exposed cultures. Inhibition of HEMA-induced apoptosis by VAS2870 or apocynin further suggested a crucial function of Nox2. SignificanceThe present findings show the physiological relevance of flavin-containing enzymes in monomer-induced oxidative stress and apoptosis.

Nitric oxide is involved in the oxytetracycline-induced suppression of root growth through inhibiting hydrogen peroxide accumulation in the root meristem

Use of antibiotic-contaminated manure in crop production poses a severe threat to soil and plant health. However, few studies have studied the mechanism by which plant development is affected by antibiotics. Here, we used microscopy, flow cytometry, gene expression analysis and fluorescent dyes to study the effects of oxytetracycline (OTC), a widely used antibiotic in agriculture, on root meristem activity and the accumulation of hydrogen peroxide (H2O2) and nitric oxide (NO) in the root tips of tomato seedlings. We found that OTC caused cell cycle arrest, decreased the size of root meristem and inhibited root growth. Interestingly, the inhibition of root growth by OTC was associated with a decline in H2O2 levels but an increase in NO levels in the root tips. Diphenyliodonium (DPI), an inhibitor of H2O2 production, showed similar effects on root growth as those of OTC. However, exogenous H2O2 partially reversed the effects on the cell cycle, meristem size and root growth. Importantly, cPTIO (the NO scavenger) and tungstate (an inhibitor of nitrate reductase) significantly increased H2O2 levels in the root tips and reversed the inhibition of root growth by OTC. Out results suggest that OTC-induced NO production inhibits H2O2 accumulation in the root tips, thus leading to cell cycle arrest and suppression of root growth.

Zinc-alleviating effects on iron-induced phytotoxicity in roots of Triticum aestivum

The mechanisms of growth inhibition and antioxidative response were investigated in wheat roots exposed to 300 μM iron together with different zinc concentrations (0, 50, and 250 μM). All Zn concentrations decreased Fe content but increased Zn content in the roots and leaves of Fe-treated seedlings. Compared with Fe stress alone, 50 or 250 μM Zn + Fe treatment stimulated root growth, and increased cell viability but decreased malondialdehyde content, which were correlated with the decreases of total and apoplastic hydrogen peroxide and superoxide anion radical (O2 ·−) content along with apoplastic hydroxyl radical content. Generation of O2 ·− in response to 10 μM diphenylene iodonium suggested that NADPH oxidase activity was lower in Zn + Fe-treated roots than in other roots. In addition, cell wallbound peroxidase, diamine oxidase, and polyamine oxidase in Fe-treated roots were insensitive to Zn addition. Further study showed the stimulation of total superoxide dismutase and glutathione reductase (GR) activities as well as apoplastic catalase, ascorbate peroxidase, and GR in Zn + Fe-stressed roots in comparison with Fe-alone-treated ones. Taken together, Zn could alleviate iron-inhibitory effect on root growth, which might be associated with the decrease of lipid peroxidation, the increase of cell viability and the reductions of reactive oxygen species generation.

Methyl Jasmonate Regulates Podophyllotoxin Accumulation in Podophyllum hexandrum by Altering the ROS-Responsive Podophyllotoxin Pathway Gene Expression Additionally through the Down Regulation of Few Interfering miRNAs.

Podophylloxin (ptox), primarily obtained from Podophyllum hexandrum, is the precursor for semi-synthetic anticancer drugs viz. etoposide, etopophos, and teniposide. Previous studies established that methyl jasmonate (MeJA) treated cell culture of P. hexandrum accumulate ptox significantly. However, the molecular mechanism of MeJA induced ptox accumulation is yet to be explored. Here, we demonstrate that MeJA induces reactive oxygen species (ROS) production, which stimulates ptox accumulation significantly and up regulates three ROS-responsive ptox biosynthetic genes, namely, PhCAD3, PhCAD4 (cinnamyl alcohol dehydrogenase), and NAC3 by increasing their mRNA stability. Classic uncoupler of oxidative phosphorylation, carbonylcyanide m-chlorophenylhydrazone, as well as H2O2 treatment induced the ROS generation and consequently, enhanced the ptox production. However, when the ROS was inhibited with NADPH oxidase inhibitor diphenylene iodonium and Superoxide dismutase inhibitor diethyldithio-carbamic acid, the ROS inhibiting agent, the ptox production was decreased significantly. We also noted that, MeJA up regulated other ptox biosynthetic pathway genes which are not affected by the MeJA induced ROS. Further, these ROS non-responsive genes were controlled by MeJA through the down regulation of five secondary metabolites biosynthesis specific miRNAs viz. miR172i, miR035, miR1438, miR2275, and miR8291. Finally, this study suggested two possible mechanisms through which MeJA modulates the ptox biosynthesis: primarily by increasing the mRNA stability of ROS-responsive genes and secondly, by the up regulation of ROS non-responsive genes through the down regulation of some ROS non-responsive miRNAs.

Nitric oxide acts downstream of hydrogen peroxide in the regulation of ascorbate and glutathione metabolism by jasmonic acid in Agropyron cristatum leaves

The relationship between hydrogen peroxide (H2O2) and nitric oxide (NO) in the regulation of ascorbate and glutathione metabolism by jasmonic acid (JA) in Agropyron cristatum leaves were studied. Results showed that JA increased the production of H2O2 and NO, the activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), L-galactono-1,4-lactone dehydrogenase (GalLDH), and γ-glutamylcysteine synthetase (γ-ECS), as well as transcription of the respective genes and also the content of reduced ascorbate (AsA) and reduced glutathione (GSH). Above increases were suppressed by pre-treatments with H2O2 synthesis inhibitor diphenylene iodonium (DPI), H2O2 scavenger dimethylthiourea (DMTU), NO synthesis inhibitor N G-nitro-L-Arg methyl ester (L-NAME), and NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Pre-treatments with DPI and DMTU reduced H2O2 and NO production. Pre-treatments with L-NAME and cPTIO reduced NO production, but did not reduce the H2O2 production induced by JA. Our results suggested that NO acted downstream of H2O2 in JA signalling in the up-regulation of ascorbate and glutathione metabolism in A. cristatum leaves.

Expression of blood vessel growth inhibitory factor and platelet endothelial adhesion molecules 34 in hepatocellular carcinoma tissue and its clinical significance

Objective To investigate the effect and mechanism of advanced oxidation protein products (AOPP) on the proliferation of CD4+ CD25+ regulatery T cells (Tregs). Methods Tregs and CD4+ CD25- T cells (worked as effective T cells, Teff) were isolated, and dendrtic cells (DCs) were induced from the human peripheral blood mononuclear cells. Tregs (5×104/well) were co-cultured with Mitomycin-inactivated DCs loaded with allogeneic antigen and IL-2 (100 U/ml), and according to the presence of AOPP into 3 different groups: Control group (200 μg/ml serum albumin), AOPP group (200 μg/ml AOPP), Antioxidant group [pretreated with 100 μmol/L diphenyleneiodonium (DPI) 1 h before AOPP was added]. The incorporation of [3H]thymidine was used to evaluate the proliferation of Tregs and the suppressive function of Tregs on the Teff. Western blotting was used to observe the expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated protein kinase B (p-Akt), phosphorylated signal transducer and activators of transcription 5 (p-STAT5) and p27kip1. Results In AOPP group, the incorporation of [3H]thymidine was (36213.3±3 385.9) cpm, which was significantly lower than that [(65 557.0±3 981.6) cpm] of Control group (P=0.001). In Antioxidant group, the value was (44 827.0±3 694.6) cpm, which was significantly higher than that of AOPP group (P=0.041), but lower than that of Control group (P=0.003). When Tregs and Teff were incubated at the ratio of 1∶1 and 1/2∶1, the suppressive ratios of Tregs on the Teff in AOPP group were (27.3±4.0)% and (23.9±3.7)%, which were significantly lower than those [(68.5±7.9)% and (62.9±5.0)%] of Control group (P=0.001, 0.001). Compared with Control group, addition of AOPP significantly downregulated the expression of p-ERK, p-Akt, p-STAT5 and upregulated the expression of p27kip1 in Tregs (P=0.009, 0.021, 0.036, 0.036). After antioxidants was added, the expression of p-ERK, p-Akt, p-STAT5 was significantly upregulated compared with AOPP group (P=0.034, 0.030, 0.042) and lower than that of Control group (P=0.043, 0.039, 0.007); the expression of p27kip1 was significantly doenregulated compared with AOPP group (P=0.048) and higher than that of Control group (P=0.047). Conclusion AOPP significantly inhibits the proliferation of Tregs through downregulating p-ERK, p-Akt and p-STAT5, and upregulating p27kip1 via oxidative stress in vitro. Key words: Advanced oxidation protein products; Regulatory T cells; Proliferation

Nanosecond pulsed electric fields trigger cell differentiation in Chlamydomonas reinhardtii

Nanosecond pulsed electric fields (nsPEFs) have great potential for biotechnological and medical applications. However, the biological mechanisms causing the cellular responses are still far from understood. We used the unicellular green algae Chlamydomonas reinhardtii as experimental model to dissect the immediate consequences of electroporation from the developmental cellular responses evoked by nsPEFs. We observe that nsPEFs induce a short-term permeabilization of the membrane, accompanied by swelling and oxidative burst. These response are transient, but are followed, several days later, by a second wave of oxidative burst, arrested cell division, stimulated cell expansion, and the formation of an immobile palmella stage. This persistent oxidative burst can be suppressed by specific inhibitor diphenyl iodonium (DPI), but not by the unspecific antioxidant ascorbic acid (Asc). Treated with natural and artificial auxins allow to modulating the cell cycle and cell expansion, and natural auxin can suppress the spontaneous formation of palmella stages. However, when administered prior to the nsPEFs treatment, auxin cannot mitigate the elevated formation of palmella stages induced by nsPEFs. We interpret our findings in terms of a model, where nsPEFs generate a developmental signal that persists, although the other immediate responses remain transient. This signal will initiate, several days later, a developmental programme comprising halted cell cycle, stimulation of cell expansion, a persistent activation of NADPH oxidase activity causing a second wave of oxidative burst, and the irreversible initiation of palmella stages. Thus, a short transient nsPEFs treatment can initiate a stable response of cellular differentiation in Chlamydomonas reinhardtii.

Tert-butyl hydroperoxide (t-BHP) induced apoptosis and necroptosis in endothelial cells: Roles of NOX4 and mitochondrion

Oxidative stress causes endothelial death while underlying mechanisms remain elusive. Herein, the pro-death effect of tert-butyl hydroperoxide (t-BHP) was investigated with low concentration (50μM) of t-BHP (t-BHPL) and high concentration (500μM) of t-BHP (t-BHPH). Both t-BHPL and t-BHPH induced endothelial cell death was determined. T-BHPL induced caspase-dependent apoptosis and reactive oxygen species (ROS) generation, which was inhibited by N-acetyl-L-cysteine (NAC). Furthermore, NADPH oxidase inhibitor diphenyleneiodonium (DPI), NOX4 siRNA, and NOX4 inhibitor GKT137831 reduced t-BHPL-induced ROS generation while mitochondrial respiratory chain inhibitors rotenone (Rot), 2-thenoyltrifluoroacetone (TTFA), and antimycin A (AA) failed to do so. NOX4 overexpression resulted in increased ROS generation and Akt expression but decreased sensitivity to t-BHPL. In contrast, T-BHPH induced LDH release, PI uptake, and cell translucent cytoplasm. RIP1 inhibitor necrostatin-1 (Nec-1), MLKL inhibitor necrosulfonamide (NSA) and silencing RIP1, RIP3, and MLKL inhibited t-BHPH-induced cell death while pan-caspase inhibitor Z-VAD-FMK showed no effect. T-BHPH-induced ROS production was inhibited by TTFA, AA and Rot while DPI showed no effect. T-BHPH induced RIP1/RIP3 interaction, which was decreased by Rot, TTFA, and AA. Silence RIP1 and RIP3 but not MLKL inhibited t-BHPH-induced mitochondrial membrane potential (MMP) decrease and ROS production. Moreover, P38MAPK inhibitor SB203580 reversed both t-BHPL and t-BHPH-induced cell death while inhibitors for ERKs and JNKs showed no obvious effect. These data suggested that t-BHP induced both apoptosis and necroptosis in endothelial cells which was mediated by ROS and p38MAPK. ROS derived from NADPH oxidase and mitochondria contributed to t-BHPL and t-BHPH-induced apoptosis and necroptosis, respectively.

Reactive oxygen species inhibit catalytic activity of peptidylarginine deiminase

Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H2O2 and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H2O2 at concentrations above 40 µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H2O2 at concentrations up to 1000 µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.

Synthesis and interconversions of reduced, alkali-metal supported iron-sulfur-carbonyl complexes.

We report the synthesis, interconversions and X-ray structures of a set of [mFe-nS]-type carbonyl clusters (where S = S2-, S22- or RS-; m = 2-3; n = 1-2). All of the clusters have been identified and characterized by single crystal X-ray diffraction, IR and 13C NMR. Reduction of the parent neutral dimer [μ2-(SPh)2Fe2(CO)6] (1) with KC8 affords an easily separable ∼1 : 1 mixture of the anionic, dimeric thiolate dimer K[Fe2(SPh)(CO)6(μ-CO)] (2) and the dianionic, sulfido trimer [K(benzo-15-crown-5)2]2[Fe3(μ3-S)(CO)9] (3). Oxidation of 2 with diphenyl-disulfide (Ph2S2) cleanly returns the starting material 1. The Ph-S bond in 1 can be cleaved to form sulfide trimer 3. Oxidation of sulfido trimer 3 with [Fc](PF6) in the presence of S8 cleanly affords the all-inorganic persulfide dimer [μ2-(S)2Fe2(CO)6] (4), a thermodynamically stable product. The inverse reactions to form 3 (dianion) from 4 (neutral) were not successful, and other products were obtained. For example, reduction of 4 with KC8 afforded the mixed valence Fe(i)/Fe(ii) species [((FeS2)(CO)6)2FeII]2- (5), in which the two {Fe2S2(CO)6}2- units serve as bidendate ligands to a Fe(ii) center. Another isolated product (THF insoluble portion) was recrystallized in MeCN to afford [K(benzo-15-crown-5)2]2[((Fe2S)(CO)6)2(μ-S)2] (6), in which a persulfide dianion bridges two {2Fe-S} moieties (dimer of dimers). Finally, to close the interconversion loop, we converted the persulfide dimer 4 into the thiolate dimer 1 by reduction with KC8 followed by reaction with the diphenyl iodonium salt [Ph2I](PF6), in modest yield. These reactions underscore the thermodynamic stability of the dimers 1 and 4, as well as the synthetic and crystallization versatility of using the crown/K+ counterion system for obtaining structural information on highly reduced iron-sulfur-carbonyl clusters.

Targeting of NADPH oxidase in vitro and in vivo suppresses fibroblast activation and experimental skin fibrosis.

Although there is increasing evidence that oxidative stress is involved in collagen synthesis and myofibroblast activation, the NADPH oxidase (Nox) system is incompletely investigated in the context of human dermal fibroblasts (HDFs) and skin fibrosis. Using the pan-Nox inhibitor diphenyleneiodonium (DPI) as an initial tool, we show that gene expression of collagen type I, α-smooth muscle actin (α-SMA) and fibronectin 1 is suppressed in HDFs. Detailed expression analysis of all Nox isoforms and adaptors revealed expression of RNA and protein expression of Nox4, p22phox and Poldip2 but neither Nox1 nor Nox2. Nox4 could be immunolocalized to the endoplasmic reticulum. Importantly, TGF-β1 had a dose- and time-dependent upregulating effect on NADH activity and Nox4 gene expression in HDFs. Genetic silencing of Nox4 as demonstrated by siRNA in HDFs as well as in murine fibroblasts established from Nox4 knockout mice confirmed that TGF-β1 -mediated collagen type I gene, α-SMA and fibronectin 1 gene expressions were Nox4-dependent. This TGF-β1 effect was mediated by Smad3 as shown by in silico promoter analysis, pharmacological inhibition and gene silencing of Smad3. The relevance of these findings is highlighted in the bleomycin-induced scleroderma mouse model. DPI treatment attenuated skin fibrosis and myofibroblast activation. Moreover, Nox4 knockdown by siRNA reduced skin collagen synthesis, α-SMA and fibronectin 1 expression in vivo. Finally, analyses of HDFs from patients with systemic sclerosis confirmed the expression of Nox4 and its adaptors, whereas Nox1 and Nox2 were not detectable. Our findings indicate that Nox4 targeting is a promising future treatment for fibrotic skin diseases.

Arsenic-induced genotoxic responses and their amelioration by diphenylene iodonium, 24-epibrassinolide and proline in Glycine max L.

Presence of the toxic metalloid, “arsenic (As)” is ubiquitous in the environment especially in the soil and water. Its excess availability in the soil retards growth and metabolism of plants via (a) slowing down the cell division/elongation, (b) overproduction of reactive oxygen species (ROS), (c) modulation of antioxidant enzymes, and (d) alteration of DNA profile/genomic template stability (GTS). In the current study, diphenylene iodonium (DPI), 24-epibrassinolide (EBL) and proline (Pro) were used to analyze their roles in eliminating the adverse effects of As. Glycine max L. (variety JS 335) seeds were subjected to As (75 μM, Sodium arsenite was used as source of As), and in combination with DPI (10 μM), EBL (0.5 μM) or Pro (10 mM), for five consecutive days, and effects of these treatment combinations were analyzed on germination percentage, biomass, membrane stability, GTS and expressions of defensive genes. In addition, the levels of As, ROS, malondialdehyde, DNA content, oxidation, fragmentation, polymorphism, DNase activity, endogenous Pro and pyrroline-5-carboxylate synthetase activity were evaluated. The results indicated that the treatments of DPI, EBL or Pro are capable to alleviate detrimental effects of As, gauged from above variables, but with different magnitudes. Apropos As-stress mitigation, Pro was found to be the most effective under the confines of the study protocol. This study certainly provides new ideas for intensifying studies to unravel elusive central mechanism of amelioration involving use of DPI, EBL or Pro in plants with confirmed As-toxicity.

The protective mechanism of QGC in feline esophageal epithelial cells by interleukin-1β treatment.

In a previous study, Quercetin-3-O-β-D-glucuronopyranoside (QGC) has anti-oxidative and anti-inflammatory effects in vivo. QGC is a flavonoid glucoside extracted from Rumex Aquaticus. We investigated the downstream target proteins involved in IL-1β-stimulated ROS production and the ability of QGC to inhibit ROS production. Cell viability was determined using the MTT reduction assay. Western blot analysis was performed with antibodies to investigate the activation of three MAPKs, NF-κB, and phosphorylated IκB-α (pIB), and the expression of COX-2. 2',7'-dichlorofluorescin diacetate was used to detect the generation of intracellular ROS species. When the cells were exposed to media containing IL-1β for 18h, cell viability was not affected. QGC did not reduce the COX-2 expression induced by IL-1β. However; QGC attenuated the production of intracellular ROS induced by IL-1β. IL-1β increased the expression of ERK, p38 MAPK, and pIB, and nuclear translocation of NF-κB were recovered by the ROS scavenger N-acetyl-L-cysteine (NAC) and QGC, but not by the NADPH oxidase inhibitor diphenylene iodonium. Pretreatment of cells with the ERK inhibitor PD98059, the p38 MAPK inhibitor SB202190, NAC, and QGC attenuated nuclear translocation of NF-κB and activation of pIB. QGC has a scavenging effect on cytokine-induced ROS production, thereby preventing its downstream effects, nuclear translocation of NF-κB, and activation of pIB is mediated by activation of ERK and p38 MAPK, although QGC does not inhibit IL-1β-stimulated COX-2 expression in feline esophageal epithelial cells. The data suggest that QGC exerts anti-oxidative effects and inhibitory effects against esophageal epithelial cells signals by the action of IL-1β treatment.

Coronatine Inhibits Stomatal Closure through Guard Cell-Specific Inhibition of NADPH Oxidase-Dependent ROS Production.

Microbes trigger stomatal closure through microbe-associated molecular patterns (MAMPs). The bacterial pathogen Pseudomonas syringae pv. tomato (Pst) synthesizes the polyketide toxin coronatine, which inhibits stomatal closure by MAMPs and by the hormone abscisic acid (ABA). The mechanism by which coronatine, a jasmonic acid-isoleucine analog, achieves this effect is not completely clear. Reactive oxygen species (ROS) are essential second messengers in stomatal immunity, therefore we investigated the possible effect of coronatine on their production. We found that coronatine inhibits NADPH oxidase-dependent ROS production induced by ABA, and by the flagellin-derived peptide flg22. This toxin also inhibited NADPH oxidase-dependent stomatal closure induced by darkness, however, it failed to prevent stomatal closure by exogenously applied H2O2 or by salicylic acid, which induces ROS production through peroxidases. Contrary to what was observed on stomata, coronatine did not affect the oxidative burst induced by flg22 in leaf disks. Additionally, we observed that in NADPH oxidase mutants atrbohd and atrbohd/f, as well as in guard cell ABA responsive but flg22 insensitive mutants mpk3, mpk6, npr1-3, and lecrk-VI.2-1, the inhibition of ABA stomatal responses by both coronatine and the NADPH oxidase inhibitor diphenylene iodonium was markedly reduced. Interestingly, coronatine still impaired ABA-induced ROS synthesis in mpk3, mpk6, npr1-3, and lecrk-VI.2-1, suggesting a possible feedback regulation of ROS on other guard cell ABA signaling elements in these mutants. Altogether our results show that inhibition of NADPH oxidase-dependent ROS synthesis in guard cells plays an important role during endophytic colonization by Pst through stomata.

The combined effect of resveratrol and diphenyleneiodonium on irradiation-induced injury to the hematopoietic system

Both resveratrol(Res) and diphenyleneiodonium(DPI) have been shown to have radioprotective effects on hematopoietic system injury. However, the cooperative effect of Res and DPI are unknown. In this study, we explored the radioprotective effect of the combination of Res and DPI both in vitro and in vivo. Our results showed that the combined treatment of Res and DPI was more effective in protecting irradiated BMMNCs in terms of cell viability, colony-forming ability, and reconstitution ability in vitro compared with Res or DPI treatment alone. However, in mice, the combination of Res and DPI had no enhanced protection on 4Gy total body irradiation (TBI)-induced hematopoietic system injury, including TBI-induced myelosuppression, induction of the splenic index, and increases in HSC/HPC numbers and the colony-forming ability of BMCs,compared to Res or DPI alone. An exception was the number of BMCs. These studies illustrated the inconsistency between experiments carried out in vitro and in vivo and suggest an interaction between Res or DPI in vivo.

Lysosomal Disruption Augments Obinutuzumab-Induced Direct Cell Death

Anti-CD20 mononclonal antibodies (mAbs) have a well-established role in the treatment of B-cell lymphoid malignancies. In addition to classic Fc-dependent mechanisms, including antibody-dependent and complement-mediated cytotoxicity, the so-called type II mAbs induce direct cell death. It has been shown that obinutuzumab, without Fc-crosslinking agents or effector cells, triggers non-apoptotic, lysosomal-dependent programmed cell death (PCD). The mechanism of PCD is characterized by actin reorganization, followed by permeabilization of the lysosomal membrane and subsequent generation of reactive oxygen species (ROS) through NADPH oxidase. Although, mechanisms of PCD are well-described, little is known about factors influencing sensitivity of malignant B-cells to obinutuzumab-mediated direct cell killing. Strategies to improve PCD could be potentially exploited to eliminate malignant cells, which are refractory to conventional immunotherapy. In this study, we aimed to investigate the influence of lysosomotropic agent, chloroquine, on the efficacy of obinutuzumab-mediated cytotoxicity. As PCD is dependent on lysosomal destabilization, we hypothesized that combination of obinutuzumab with lysosome-destabilizing agent would result in increased cell death.In our study, we used a Burkitt lymphoma Raji cell line that is widely employed as a model to assess the efficacy of obinutuzumab. Raji cells were incubated with obinutuzumab, alone or in combination with increasing concentrations of chloroquine, followed by annexin V/PI staining. Chloroquine, significantly increased direct cell death induced by obinutuzumab, without being toxic alone. Those observations were further corroborated by cell staining with other viability dies - TO-PRO-3 iodide and 7-AAD.The efficacy of the tested combination was completely abrogated by cytochalasin D - an inhibitor of actin polymerization and concanamycin A - inhibitor of vacuolar ATPases that activate acidic vacuoles including lysosomes, suggesting that chloroquine and obinutuzumab share a common mechanism of action. Since chloroquine has been reported to promote ROS generation, we analyzed the production of ROS with DCFDA staining. We observed that chloroquine potentiated the oxidative effect of obinutuzumab. Consistently, the effect of the combination was completely abrogated by a cell-permeable ROS scavenger - Tiron. As obinutuzumab has been shown to induce ROS production via activation of NADPH oxidase, we investigated the influence of NADPH oxidase inhibitor - diphenylene iodonium (DPI) on the combination's efficacy. DPI only partially reversed the effect of obinutuzumab, while strongly decreasing the effect of the combination.Altogether, we show for the first time that chloroquine sensitizes cell to lysosomal cell death induced by obinutuzumab. The results of our study provide a strong rationale for combining obinutuzumab with lysosomotropic agents. These findings may have particular importance given the fact that another lysomotropic agent - siramesine has recently been shown to induce selective cytotoxicity in chronic lymphocytic leukemia (CLL). DisclosuresNo relevant conflicts of interest to declare.

Neutrophil Extracellular Traps Accelerate Cholestatic Liver Injury through Bile Acids in Bile Duct Ligation Mice

Introduction: The mechanisms involved in cholestatic liver injury remain elusive although emerging evidence indicate neutrophils, rather than bile acids (BAs), are the predominant source of damage. Neutrophil extracellular traps (NETs) are double-edge swords that serve to ensnare and kill microbial pathogens but also contribute to excessive inflammation and tissue damage. However, the role of NETs during bile duct ligation (BDL)-induced cholestatic liver injury is largely unknown. Moreover, whether neutrophils and BAs cooperatively participate in this process also needs to be investigated. Our objectives were to localize and determine the magnitude of NETs in the hepatic microvasculature, define the role of NETs on hematological and histological changes, and characterize the role of BAs in the induction of NETs in BDL mice.Methods: BDL was induced in C57BL/6 mice by ligation of the common bile duct. After 3 days, liver tissue was collected and NETs were detected by immunostaining and western blot. Serum myeloperoxidase (MPO)-DNA complexes were measured by ELISA. Cholestatic liver injury was characterized by hematological and histological assessment. NETs were dismantled by DNase I or depleted with peptidylarginine deiminase 4 (PAD4) inhibitor Cl-amidine. The apoptosis of hepatocytes was measured by TUNEL and the induction of chemokines on Kupffer cells was analyzed by quantitative polymerase chain reaction (qPCR). BAs and inflammatory cytokines were utilized collaboratively to induce NETs formation both in vitro and in vivo. Diphenylene iodonium (DPI) was used to inhibit reactive oxygen species (ROS) formation. Plasma was obtained from healthy controls and patients with entrahepatic cholestasis.Results: We found that NETs formed in the sinusoids of cholestatic liver lobes in vivo, which was associated with significantly increased serum MPO-DNA complexes (P<0.01) and tissue levels of citrullinated-histone 3 (P<0.05) in BDL mice compared with sham operated mice. The formation of NETs exacerbated BDL-induced liver injury as evidenced by markedly increased levels of serum alamine transaminase (ALT) and percentage of necrotic tissue area (both P<0.05 vs. sham group, Figure 1). Depleting of NETs with DNase I or Cl-amidine decreased NETs formation, protected hepatocytes and dramatically alleviated cholestatic liver injury, indicating the pathophysiological role of NETs in BDL mice (Figure 1). In addition, NETs promoted the apoptosis of hepatocytes and induced the release of chemokines from Kupffer cells in BDL mice compared to sham group. Both effects could be inhibited by DNase I or Cl-amidine. Moreover, BAs induced mouse neutrophils, primed with inflammatory cytokines, to release NETs in vitro in a dose-dependent manner and mediated by PAD4 and ROS. Intravenously injection of BAs and inflammatory cytokines to healthy mice lead to the formation of NETs and exacerbated liver injury, suggesting that depleting NETs can prevent organ damage. Furthermore, we found NETs markers were significantly increased in the serum of cholestatic patients, correlating positively with increased levels of ALT and BAs.Conclusions: Our results suggest that BAs promote NETs formation via PAD4 and ROS. Development of NETs subsequently initiates inflammatory responses and exacerbates organ damage during BDL-induced cholestatic liver injury, and may therefore serve as a promising therapeutic target in cholestasis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fructose suppresses uric acid excretion to the intestinal lumen as a result of the induction of oxidative stress by NADPH oxidase activation

BackgroundA high intake of fructose increases the risk for hyperuricemia. It has been reported that long-term fructose consumption suppressed renal uric acid excretion and increased serum uric acid level. However, the effect of single administration of fructose on excretion of uric acid has not been clarified. MethodsWe used male Wistar rats, which were orally administered fructose (5g/kg). Those rats were used in each experiment at 12h after administration. ResultsSingle administration of fructose suppressed the function of ileal uric acid excretion and had no effect on the function of renal uric acid excretion. Breast cancer resistance protein (BCRP) predominantly contributes to intestinal excretion of uric acid as an active homodimer. Single administration of fructose decreased BCRP homodimer level in the ileum. Moreover, diphenyleneiodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), recovered the suppression of the function of ileal uric acid excretion and the Bcrp homodimer level in the ileum of rats that received single administration of fructose. ConclusionsSingle administration of fructose decreases in BCRP homodimer level, resulting in the suppression the function of ileal uric acid excretion. The suppression of the function of ileal uric acid excretion by single administration of fructose is caused by the activation of Nox. The results of our study provide a new insight into the mechanism of fructose-induced hyperuricemia.

The role of phytosphingosine-1-phosphate (Phyto-S1P) and its relationships with cytosolic pH and hydrogen peroxide (H2O2) during stomatal closure by darkness in broad bean

We examined the role of phytosphingosine-1-phosphate (Phyto-S1P) and its relationships with cytosolic pH and hydrogen peroxide (H2O2) during stomatal closure by darkness using broad bean as experimental material. Darkness caused cytoplasmic alkalization, induced H2O2 generation, and led to stomatal closure. DL-threo-dihydrosphingosine (DL-threo-DHS) and N,N-dimethylsphingosine (DMS) inhibited these effects of darkness. Exogenous Phyto-S1P promoted cytoplasmic alkalization, H2O2 synthesis, and caused stomatal closure. Butyric acid largely suppressed cytoplasmic alkalization and prevented stomatal closure by Phyto-S1P. The H2O2 modulators ascorbic acid (ASA), catalase (CAT), and diphenyleneiodonium (DPI) obviously restricted Phyto-S1P-promoted H2O2 synthesis and stomatal closure. These results suggest that Phyto-S1P was involved in darkness-induced stomatal closure by causing cytoplasmic alkalization and H2O2 synthesis. Furthermore, darkness-induced H2O2 synthesis was inhibited by butyric acid, suggesting that cytoplasmic alkalization caused by Phyto-S1P was probably necessary for H2O2 synthesis. The rise in cytosolic pH promoted by Phyto-S1P became noticeable at 6min and reached a maximum at 18min, while H2O2 levels sharply increased at 8min and peaked at 22min, and butyric acid prevented Phyto-S1P-promoted H2O2 synthesis, which confirmed the conclusion that Phyto-S1P-induced cytosolic alkalization acts upstream of the production of H2O2 during stomatal closure by darkness. We suggest that Phyto-S1P is involved in stomatal closure by darkness through cytoplasmic alkalization and H2O2 synthesis, and Phyto-S1P-promoted cytoplasmic alkalization acts upstream of H2O2 synthesis in the process.