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Among cereal crops, salinity tolerance is rare and complex. Multiple genes control numerous pathways, which constitute plant’s response to salinity. Cell cultures act as model system and are useful to investigate the salinity response which can possibly mimic a plant’s response to stress. In the present study two indica rice varieties, KS-282 and Super Basmati which exhibited contrasting sodium chloride (NaCl) stress response were used to establish cell cultures. The cell cultures showed a contrasting response to salt stress at 100 mM NaCl. High level of intracellular hydrogen peroxide (H2O2) and nitric oxide (NO) were observed in sensitive cell culture for prolonged period as compared to the tolerant cells in which an extracellular H2O2 burst along with controlled intracellular H2O2 and NO signal was seen. To evaluate the role of NO in inducing cell death under salt stress, cell death percentage (CDP) was measured after 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) pre-treatment. CDP was reduced significantly in both tolerant and sensitive cell cultures emphasizing NO’s possible role in programmed cell death. Expression analysis of apoplastic NADPH oxidase, i.e. OsRbohA and recently characterised OSCA family members i.e. OsOSCA 1.2 and OsOSCA 3.1 was done. Intracellular H2O2/NO levels displayed an interplay between Ca2+ influx and ROS/RNS signal. Detoxifying enzyme (i.e. ascorbate peroxidase and catalase) activity was considerably higher in tolerant KS-282 while the activity of superoxide dismutase was significantly prominent in the sensitive cells triggering greater oxidative damage owing to the prolonged presence of intracellular H2O2. Salt stress and ROS responsive TFs i.e. OsSERF1 and OsDREB2A were expressed exclusively in the tolerant cells. Similarly, the expression of genes involved in maintaining high [K+]/[Na+] ratio was considerably higher and earlier in the tolerant variety. Overall, we suggest that a control over ROS production, and an increase in the expression of genes important for potassium homeostasis play a dynamic role in salinity tolerance in rice cell cultures. [ABSTRACT FROM AUTHOR]

Aims: Interferon-γ (IFN-γ) exhibits hepatotoxicity through signal transducer and activator of transcription 1 (STAT1) activation. On the contrary, interleukin-11 (IL-11) shows tissue-protective effects on various organs including the liver through STAT3 activation. Here, we found that IL-11 pretreatment protects hepatocytes from IFN-γ-induced death and investigated the molecular mechanisms, particularly focusing on signal crosstalk. Methods and results: Primary culture mouse hepatocytes were treated with IL-11 prior to IFN-γ, and cell death was evaluated by lactate dehydrogenase release into media. As a result, IL-11 pretreatment effectively suppressed IFN-γ-induced hepatocyte death. Since IFN-γ-induced hepatocyte death requires STAT1 signaling, the activity of STAT1 was analyzed. IFN-γ robustly activated STAT1 with its peak at 1 hr after stimulation, which was significantly attenuated by IL-11 pretreatment. Consistently, IL-11 pretreatment impeded mRNA increase of STAT1-downstream molecules promoting cell death, i.e., IRF-1, caspase 1, bak, and bax. IL-11-mediated suppression of STAT1 signaling was presumably due to upregulation of the suppressor of cytokine signaling (SOCS) genes, which are well-known negative feedback regulators of the JAK/STAT pathway. Interestingly, however, IFN-γ pretreatment failed to affect the following IL-11-induced STAT3 activation, although IFN-γ also upregulated SOCSs. Finally, we demonstrated that IL-11 pretreatment mitigated oxidative stress through increasing expression of ROS scavengers. Conclusion: IL-11 protects hepatocytes from IFN-γ-induced death via STAT1 signal suppression and ROS scavenging. Further investigation into the mechanisms underlying selective negative feedback regulation of IFN-γ/STAT1 signaling compared to IL-11/STAT3 signaling may shed new light on the molecular biology of hepatocytes. [ABSTRACT FROM AUTHOR]

We investigated the effect of peroxisome proliferator-activated receptor δ (PPARδ) on angiotensin II (Ang II)-triggered hypertrophy of vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly inhibited Ang II-stimulated protein synthesis in a concentration-dependent manner, as determined by [3H]-leucine incorporation. GW501516-activated PPARδ also suppressed Ang II-induced generation of reactive oxygen species (ROS) in VSMCs. Transfection of small interfering RNA (siRNA) against PPARδ significantly reversed the effects of GW501516 on [3H]-leucine incorporation and ROS generation, indicating that PPARδ is involved in these effects. By contrast, these GW501516-mediated actions were potentiated in VSMCs transfected with siRNA against NADPH oxidase (NOX) 1 or 4, suggesting that ligand-activated PPARδ elicits these effects by modulating NOX-mediated ROS generation. The phosphatidylinositol 3-kinase inhibitor LY294002 also inhibited Ang II-stimulated [3H]-leucine incorporation and ROS generation by preventing membrane translocation of Rac1. These observations suggest that PPARδ is an endogenous modulator of Ang II-triggered hypertrophy of VSMCs, and is thus a potential target to treat vascular diseases associated with hypertrophic changes of VSMCs. [ABSTRACT FROM AUTHOR]

The regulatory subunit of human DNA primase has a C-terminal domain (p58C) that contains a [4Fe4S] cluster and binds DNA. Previous electrochemical analysis of a p58C construct revealed that its affinity for DNA is sensitive to the redox state of the [4Fe4S] cluster. Concerns about the validity of this conclusion have been raised, based in part on differences in X-ray crystal structures of the p58C272-464 construct used for that study and that of a N-terminally shifted p58C266-456 construct and consequently, an assumption that p58C272-464 has abnormal physical and functional properties. To address this controversy, a new p58C266-464 construct containing all residues was crystallized under the conditions previously used for crystallizing p58C272-464, and the solution structures of both constructs were assessed using circular dichroism and NMR spectroscopy. In the new crystal structure, p58C266-464 exhibits the same elements of secondary structure near the DNA binding site as observed in the crystal structure of p58C272-464. Moreover, in solution, circular dichroism and 15N,1H-heteronuclear single quantum coherence (HSQC) NMR spectra show there are no significant differences in the distribution of secondary structures or in the tertiary structure or the two constructs. To validate that the two constructs have the same functional properties, binding of a primed DNA template was measured using a fluorescence-based DNA binding assay, and the affinities for this substrate were the same (3.4 ± 0.5 μM and 2.7 ± 0.3 μM, respectively). The electrochemical properties of p58C266-464 were also measured and this p58C construct was able to engage in redox switching on DNA with the same efficiency as p58C272-464. Together, these results show that although p58C can be stabilized in different conformations in the crystalline state, in solution there is effectively no difference in the structure and functional properties of p58C constructs of different lengths. [ABSTRACT FROM AUTHOR]

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins that exhibit ectodomains containing the domain of unknown function 26 (DUF26). The CRKs form a large subfamily of receptor-like kinases in plants, and their possible functions remain to be elucidated. Several lines of evidence suggest that CRKs play important roles in plant defense responses to environmental stress, including plant immunity. We performed a genome-wide analysis of CRK encoding genes in soybean (Glycine max). We found 91 GmCRKs distributed in 16 chromosomes, and identified several tandem and segmental duplications, which influenced the expansion of this gene family. According to our phylogenetic analysis, GmCRKs are grouped in four clades. Furthermore, 12% of the members exhibited GmCRKs with a duplicated bi-modular organization of the ectodomains, containing four DUF26 domains. Expression analysis of GmCRKs was performed by exploring publicly available databases, and by RT-qPCR analysis of selected genes in soybean leaves responding to biotic stress signals. GmCRKs exhibited diverse expression patterns in leaves, stems, roots, and other tissues. Some of them were highly expressed in only one type of tissue, suggesting predominant roles in specific tissues. Furthermore, several GmCRKs were induced with PAMPs, DAMPs and the pathogens Phakopsora pachyrhizi and Phytophthora sojae. Expression profiles of several GmCRKs encoding highly similar proteins exhibited antagonist modes of regulation. The results suggest a fine-tuning control of GmCRKs transcriptional regulation in response to external stimuli, including PAMPs and DAMPs. This study offers a comprehensive view of the GmCRKs family in soybean, and provides a foundation for evolutionary and functional analysis of this family of plant proteins involved in the perception of pathogens and activation of plant immunity. [ABSTRACT FROM AUTHOR]

Phytophthora cinnamomi Rands (Pc) is a hemibiotrophic oomycete and the causal agent of Phytophthora root rot (PRR) of the commercially important fruit crop avocado (Persea americana Mill.). Plant defense against pathogens is modulated by phytohormone signaling pathways such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), auxin and abscisic acid. The role of specific signaling pathways induced and regulated during hemibiotroph-plant interactions has been widely debated. Some studies report SA mediated defense while others hypothesize that JA responses restrict the spread of pathogens. This study aimed to identify the role of SA- and JA- associated genes in the defense strategy of a resistant avocado rootstock, Dusa in response to Pc infection. Transcripts associated with SA-mediated defense pathways and lignin biosynthesis were upregulated at 6 hours post-inoculation (hpi). Results suggest that auxin, reactive oxygen species (ROS) and Ca2+ signaling was also important during this early time point, while JA signaling was absent. Both SA and JA defense responses were shown to play a role during defense at 18 hpi. Induction of genes associated with ROS detoxification and cell wall digestion (β-1-3-glucanase) was also observed. Most genes induced at 24 hpi were linked to JA responses. Other processes at play in avocado at 24 hpi include cell wall strengthening, the formation of phenolics and induction of arabinogalactan, a gene linked to Pc zoospore immobility. This study represents the first transcriptome wide analysis of a resistant avocado rootstock treated with SA and JA compared to Pc infection. The results provide evidence of a biphasic defense response against the hemibiotroph, which initially involves SA-mediated gene expression followed by the enrichment of JA-mediated defense from 18 to 24 hpi. Genes and molecular pathways linked to Pc resistance are highlighted and may serve as future targets for manipulation in the development of PRR resistant avocado rootstocks. [ABSTRACT FROM AUTHOR]

Glutathione peroxidase 3 (GPx3), a major scavenger of reactive oxygen species (ROS) in plasma, acts as a redox signal modulator. However, the mechanism underlying GPx3-mediated suppression of cancer cell growth is unclear. The aim of this study was to identify these mechanisms with respect to lung cancer. To enhance the redox modulating properties of GPx3, lung cancer cells were subjected to serum starvation for 12 h, resulting in ROS generation in the absence of oxidant treatment. We then investigated whether suppression of tumorigenesis under conditions of oxidative stress was dependent on GPx3. The results showed that GPx3 effectively suppressed proliferation, migration, and invasion of lung cancer cells under oxidative stress. In addition, GPx3 expression led to a significant reduction in ROS production by cancer cells and induced G2/M phase arrest. We also found that inactivation of cyclin B1 significantly suppressed by nuclear factor-κB(NF-κB) inactivation in lung cancer cells was dependent on GPx3 expression. To further elucidate the mechanism(s) underlying GPx3-medited suppression of tumor proliferation, we next examined the effect of GPx3-mediated redox signaling on the ROS-MKP3-extracellular signal-regulated kinase (Erk)-NF-κB-cyclin B1 pathway and found that GPx3 strongly suppressed activation of the Erk-NF-κB-cyclin B1 signaling cascade by protecting MKP3 (an Erk-specific phosphatase) from the effects of ROS. Thus, this study demonstrates for the first time that the GPx3 suppresses proliferation of lung cancer cells by modulating redox-mediated signals. [ABSTRACT FROM AUTHOR]

Copper (Cu) is a necessary trace element participated in many physiological processes in plants. But excessive Cu2+ is toxic, which can activate intracellular signals that lead to cellular damage. The mitogen-activated protein kinase (MAPK) cascade is at the center of cell signal transduction and has been reported to be involved in stress-related signaling pathways. ZmMPK3, a kind of MAPKs in maize cells, can be activated by diverse abiotic stresses. In the present study, we investigated the effects of Cu2+ on hydrogen peroxide (H2O2) level, ZmMPK3 activity as well as the activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbic acid peroxidase (APX) using maize leaf as an experimental model. The results demonstrated that acute Cu2+ exposure for 24 hours led to rapid increases of H2O2 level and the increase in ZmMPK3 activity as well as the total activities of antioxidant enzymes SOD, CAT and APX. H2O2 scavenger, dimethylthiourea (DMTU), effectively inhibited the Cu2+-increased H2O2 level and the activity of ZmMPK3 as well as the activities of the antioxidant enzymes SOD, CAT and APX. Pre-treatment with the MAPK inhibitor, PD98059, significantly blocked the Cu2+-increased activities of ZmMPK3, CAT, APX and SOD, but didn’t affect the accumulation of H2O2. Our results suggest that Cu2+ causes oxidative stress to the maize leaves which then activates defense antioxidant enzymes via MAPK pathway. Thus, the signaling pathway is Cu2+—H2O2—ZmMPK3—antioxidant enzymes. [ABSTRACT FROM AUTHOR]

The development of new dosimeters with good dosimetric properties is important for quality control in radiation applications. A new practical electron spin resonance (ESR) dosimeter based on carbonated hydroxyapatite that simulated the composition and structure of tooth enamel was specially synthesized. The synthesized material was investigated by transmission electron microscope, X-ray diffraction, fourier transform infrared spectroscopy and X-ray photo electron spectroscopy to confirm to the main composition of carbonated hydroxyapatite with CO32- successfully doped into the crystal lattice through optimizing the synthesis process of C/P molar ratio, pH value dynamical adjustment, annealing temperature and time. The dosimetric properties were systematically investigated by ESR spectroscopy. The results indicated that the radiation induced signal had a good dose response within a relatively wide dose range. The dose response was linear in the dose range of 0–400 Gy with a correlation coefficient of 0.9999 and had dose accumulative effect in the experimental dose range of 0–100 Gy. In a wider dose range up to 30 kGy, the dose response also presented linear feature in double-logarithmic coordinate system with a correlation coefficient of 0.9970. The dose detection limit was about 0.34Gy with a given probability of 95% confidence level depending upon a rigid calculation algorithm. The signal was extremely stable in the observation time of 360 days with a variation coefficient of 3.8%. The radiation sensitivity of the material showed no remarkable variation against photon energy from 662 KeV to 1.25 MeV and dose rate from 0.86 Gy/min to 12.17 Gy/min. The material showed more sensitive in lower photon energy range below 662 keV, which hint additional calibration may need when using in special photon energy condition. The preliminary results suggested that this newly developed dosimeter was potential to become a practical dosimeter that would expand the application fields of ESR dosimetry. [ABSTRACT FROM AUTHOR]

It is now well accepted that radiation induced bystander effects can occur in cells exposed to media from irradiated cells. The aim of this study was to follow the bystander cells in real time following addition of media from irradiated cells and to determine the effect of inhibiting these signals. A human keratinocyte cell line, HaCaT cells, was irradiated (0.005, 0.05 and 0.5 Gy) with γ irradiation, conditioned medium was harvested after one hour and added to recipient bystander cells. Reactive oxygen species, nitric oxide, Glutathione levels, caspase activation, cytotoxicity and cell viability was measured after the addition of irradiated cell conditioned media to bystander cells. Reactive oxygen species and nitric oxide levels in bystander cells treated with 0.5Gy ICCM were analysed in real time using time lapse fluorescence microscopy. The levels of reactive oxygen species were also measured in real time after the addition of extracellular signal-regulated kinase and c-Jun amino-terminal kinase pathway inhibitors. ROS and glutathione levels were observed to increase after the addition of irradiated cell conditioned media (0.005, 0.05 and 0.5 Gy ICCM). Caspase activation was found to increase 4 hours after irradiated cell conditioned media treatment (0.005, 0.05 and 0.5 Gy ICCM) and this increase was observed up to 8 hours and there after a reduction in caspase activation was observed. A decrease in cell viability was observed but no major change in cytotoxicity was found in HaCaT cells after treatment with irradiated cell conditioned media (0.005, 0.05 and 0.5 Gy ICCM). This study involved the identification of key signaling molecules such as reactive oxygen species, nitric oxide, glutathione and caspases generated in bystander cells. These results suggest a clear connection between reactive oxygen species and cell survival pathways with persistent production of reactive oxygen species and nitric oxide in bystander cells following exposure to irradiated cell conditioned media. [ABSTRACT FROM AUTHOR]

Activation of ERK1/2 implies the phosphorylation of tyrosine (pTyr) and threonine (pThr) by MEK1/2; both reactions were thought to be cytoplasmic, promoting ERK to reach the nucleus where it activates several transcription factors. In addition, H2O2 concentrations are known to modulate ERK intracellular translocation, which impacts on cellular proliferation. In this context, the objective of this work was to study the sequence of ERK phosphorylation under two redox conditions and to analyze a putative mitochondrial contribution to this process, in LP07 murine lung cells. A time-course of H2O2 administration was used and ERK phosphorylation was analyzed in cytosol, mitochondria and nuclei. At 1μM H2O2, a proliferative redox stimulus, immunoblot revealed a fast and transient increase in cytosol pTyr and a sustained increase in mitochondrial pTyr content. The detection for pThr/pTyrERK (2pERK) showed in cytosol a marked increase at 5 minutes with a fast dephosphorylation after that time, for both H2O2 concentrations. However, at 50 μM H2O2, an anti-proliferative condition, 2pERK was gradually retained in mitochondria. Interestingly, these results were confirmed by in vivo experiments using mice treated with a highly oxidizing agent [H2O2]. By the use of two ERK2 mutant constructions, where Tyr and Thr were replaced by alanine, we confirmed that 2pERK relied almost completely on pThr183. Confocal microscopy confirmed ERK subcellular distribution dependence on the incidence of cytosolic pTyr and mitochondrial pThr at 1μM H2O2. This work shows for the first time, both in vitro and in vivo, an ERK cycle involving a cross-talk between cytosol and mitochondria phosphorylation events, which may play a significant role in cell cycle progression, proliferation or differentiation under two different redox conditions. [ABSTRACT FROM AUTHOR]

In this study, the differences in reactive oxygen species (ROS) generation and abscisic acid (ABA) accumulation in senescing leaves were investigated by early-senescence-leaf (esl) mutant and its wild type, to clarify the relationship among ABA levels, ROS generation, and NADPH oxidase (Nox) in senescing leaves of rice (Oryza sativa). The temporal expression levels of OsNox isoforms in senescing leaves and their expression patterns in response to ABA treatment were determined through quantitative real-time reverse transcription PCR (qRT-PCR). Results showed that the flag leaf of the esl mutant generated more O2 - concentrations and accumulated higher ABA levels than the wild-type cultivar did in the grain-filling stage. Exogenous ABA treatment induced O2 - generation; however, it was depressed by diphenyleneiodonium chloride (DPI) pretreatment in the detached leaf segments. This finding suggested the involvement of NADPH oxidase in ABA-induced O2 - generation. The esl mutant exhibited significantly higher expression of OsNox2, OsNox5, OsNox6, and OsNox7 in the initial of grain-filling stage, followed by sharply decrease. The transcriptional levels of OsNox1, OsNox3, and OsFR07 in the flag leaf of the esl mutant were significantly lower than those in the wild-type cultivar. The expression levels of OsNox2, OsNox5, OsNox6, and OsNox7 were significantly enhanced by exogenous ABA treatments. The enhanced expression levels of OsNox2 and OsNox6 were dependent on the duration of ABA treatment. The inducible expression levels of OsNox5 and OsNox7 were dependent on ABA concentrations. By contrast, exogenous ABA treatment severely repressed the transcripts of OsNox1, OsNox3, and OsFR07 in the detached leaf segments. Therefore, OsNox2, OsNox5, OsNox6, and OsNox7 were probably involved in the ABA-induced O2 - generation in the initial stage of leaf senescence. Subsequently, other oxidases activated in deteriorating cells were associated with ROS generation and accumulation in the senescing leaves of the esl mutant. Conversely, OsNox1, OsNox3, and OsFR07 were not associated with ABA-induced O2 - generation during leaf senescence. [ABSTRACT FROM AUTHOR]

PITX2 is a homeobox transcription factor involved in embryonic left/right signaling and more recently has been associated to cardiac arrhythmias. Genome wide association studies have pinpointed PITX2 as a major player underlying atrial fibrillation (AF). We have previously described that PITX2 expression is impaired in AF patients. Furthermore, distinct studies demonstrate that Pitx2 insufficiency leads to complex gene regulatory network remodeling, i.e. Wnt>microRNAs, leading to ion channel impairment and thus to arrhythmogenic events in mice. Whereas large body of evidences has been provided in recent years on PITX2 downstream signaling pathways, scarce information is available on upstream pathways influencing PITX2 in the context of AF. Multiple risk factors are associated to the onset of AF, such as e.g. hypertension (HTN), hyperthyroidism (HTD) and redox homeostasis impairment. In this study we have analyzed whether HTN, HTD and/or redox homeostasis impact on PITX2 and its downstream signaling pathways. Using rat models for spontaneous HTN (SHR) and experimentally-induced HTD we have observed that both cardiovascular risk factors lead to severe Pitx2 downregulation. Interesting HTD, but not SHR, leads to up-regulation of Wnt signaling as well as deregulation of multiple microRNAs and ion channels as previously described in Pitx2 insufficiency models. In addition, redox signaling is impaired in HTD but not SHR, in line with similar findings in atrial-specific Pitx2 deficient mice. In vitro cell culture analyses using gain- and loss-of-function strategies demonstrate that Pitx2, Zfhx3 and Wnt signaling influence redox homeostasis in cardiomyocytes. Thus, redox homeostasis seems to play a pivotal role in this setting, providing a regulatory feedback loop. Overall these data demonstrate that HTD, but not HTN, can impair Pitx2>>Wnt pathway providing thus a molecular link to AF. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) from the cell envelope of Gram-negative bacteria is a principal cause of the symptoms of sepsis. LPS has been reported to modulate the function of platelets although the underlying mechanisms of LPS action in these cells remain unclear. Platelets express the Toll-like receptor 4 (TLR4) which serves as a receptor for LPS, although the potential role of TLR4 and associated cell signalling in controlling platelet responses to LPS has not been extensively explored. In this study, we therefore investigated the actions of LPS prepared from different strains of Escherichia coli on platelet function, the underlying signalling mechanisms, and the potential role of TLR4 in orchestrating these. We report that LPS increased the aggregation of washed platelets stimulated by thromboxane (U46619) or GPVI collagen receptor agonists, effects that were prevented by a TLR4 antagonist. Associated with this, LPS enhanced fibrinogen binding, P-selectin exposure and reactive oxygen species (ROS) release. Increase of ROS was found to be important for the actions of LPS on platelets, since these were inhibited in the presence of superoxide dismutase or catalase. The effects of LPS were associated with phosphorylation of Akt, ERK1/2 and PLA2 in stimulated platelets, and inhibitors of PI3-kinase, Akt and ERK1/2 reduced significantly LPS enhanced platelet function and associated ROS production. Furthermore, inhibition of platelet cyclooxygenase or the thromboxane receptor, revealed an important role for thromboxane A2. We therefore conclude that LPS increases human platelet activation through a TLR4-PI3K-Akt-ERK1/2-PLA2 -dependent pathway that is dependent on ROS and TXA2 formation. [ABSTRACT FROM AUTHOR]

Mechanical damage can induce fruiting body production in fungi. In this study, the antioxidant kojic acid (KA) was found to enhance injured mycelial regeneration and increase fruiting body production in Hypsizygus marmoreus. KA reduced the level of reactive oxygen species (ROS), which are harmful to mycelia when excessively generated by mechanical damage. Moreover, KA increased catalase and superoxide dismutase activities and glutathione and ascorbic acid contents by up-regulating antioxidant gene expression. These results suggest that KA promotes mycelial regeneration in response to damage by activating a “stress signal” and enhances the ability of H. marmoreus to resist oxidative damage by invoking the antioxidant system. In addition, KA increased the content of extracellular ATP, which serves as a “stress signal” in response to injury, and modulated ROS signaling, decreasing NADPH oxidase gene expression and ROS levels in the mycelial-regeneration stage. KA treatment also up-regulated the MAPK, Ca2+ and oxylipin pathways, suggesting their involvement in the damage response. Furthermore, laccase and cellulase activities were stimulated by KA at different developmental stages. These results demonstrate that KA regulates gene expression and activates pathways for mycelial wound healing, regeneration of damaged mycelia and reproductive structure formation in the basidiomycete H. marmoreus. [ABSTRACT FROM AUTHOR]

Use of some HIV-1 nucleoside reverse transcriptase inhibitors (NRTI) is associated with severe adverse events. However, the exact mechanisms behind their toxicity has not been fully understood. Mitochondrial dysfunction after chronic exposure to specific NRTIs has predominantly been assigned to mitochondrial polymerase-γ inhibition by NRTIs. However, an increasing amount of data suggests that this is not the sole mechanism. Many NRTI induced adverse events have been linked to the incurrence of oxidative stress, although the causality of events leading to reactive oxygen species (ROS) production and their role in toxicity is unclear. In this study we show that short-term effects of first generation NRTIs, which are rarely discussed in the literature, include inhibition of oxygen consumption, decreased ATP levels and increased ROS production. Collectively these events affect fitness and longevity of C. elegans through mitohormetic signalling events. Furthermore, we demonstrate that these effects can be normalized by addition of the anti-oxidant N-acetylcysteine (NAC), which suggests that ROS likely influence the onset and severity of adverse events upon drug exposure. [ABSTRACT FROM AUTHOR]

Plants interpret their immediate environment through perception of small molecules. Microbe-associated molecular patterns (MAMPs) such as flagellin and chitin are likely to be more abundant in the rhizosphere than plant-derived damage-associated molecular patterns (DAMPs). We investigated how the Arabidopsis thaliana root interprets MAMPs and DAMPs as danger signals. We monitored root development during exposure to increasing concentrations of the MAMPs flg22 and the chitin heptamer as well as of the DAMP AtPep1. The tissue-specific expression of defence-related genes in roots was analysed using a toolkit of promoter::YFPN lines reporting jasmonic acid (JA)-, salicylic acid (SA)-, ethylene (ET)- and reactive oxygen species (ROS)- dependent signalling. Finally, marker responses were analysed during invasion by the root pathogen Fusarium oxysporum. The DAMP AtPep1 triggered a stronger activation of the defence markers compared to flg22 and the chitin heptamer. In contrast to the tested MAMPs, AtPep1 induced SA- and JA-signalling markers in the root and caused a severe inhibition of root growth. Fungal attack resulted in a strong activation of defence genes in tissues close to the invading fungal hyphae. The results collectively suggest that AtPep1 presents a stronger danger signal to the Arabidopsis root than the MAMPs flg22 and chitin heptamer. [ABSTRACT FROM AUTHOR]

The rhizosphere, the narrow zone of soil around plant roots, is a complex network of interactions between plants, bacteria, and a variety of other organisms. The absolute dependence on host-derived signals, or xenognosins, to regulate critical developmental checkpoints for host commitment in the obligate parasitic plants provides a window into the rhizosphere’s chemical dynamics. These sessile intruders use H2O2 in a process known as semagenesis to chemically modify the mature root surfaces of proximal host plants and generate p-benzoquinones (BQs). The resulting redox-active signaling network regulates the spatial and temporal commitments necessary for host attachment. Recent evidence from non-parasites, including Arabidopsis thaliana, establishes that reactive oxygen species (ROS) production regulates similar redox circuits related to root recognition, broadening xenognosins’ role beyond the parasites. Here we compare responses to the xenognosin dimethoxybenzoquinone (DMBQ) between the parasitic plant Striga asiatica and the non-parasitic A. thaliana. Exposure to DMBQ simulates the proximity of a mature root surface, stimulating an increase in cytoplasmic Ca2+ concentration in both plants, but leads to remarkably different phenotypic responses in the parasite and non-parasite. In S. asiatica, DMBQ induces development of the host attachment organ, the haustorium, and decreases ROS production at the root tip, while in A. thaliana, ROS production increases and further growth of the root tip is arrested. Obstruction of Ca2+ channels and the addition of antioxidants both lead to a decrease in the DMBQ response in both parasitic and non-parasitic plants. These results are consistent with Ca2+ regulating the activity of NADPH oxidases, which in turn sustain the autocatalytic production of ROS via an external quinone/hydroquinone redox cycle. Mechanistically, this chemistry is similar to black and white photography with the emerging dynamic reaction-diffusion network laying the foundation for the precise temporal and spatial control underlying rhizosphere architecture. [ABSTRACT FROM AUTHOR]

4'-Hydroxywogonin (4'-HW), a flavonoid, has been isolated from various plants and shown to inhibit NO production in macrophages. However, the molecular mechanisms and its in vivo activity have not been determined. Our study aimed to investigate the mechanisms underlying the anti-inflammatory effects of 4'-HW in vitro and in vivo. We showed that 4'-HW potently reduced the expression levels of COX-2 and iNOS as well as their products, prostaglandin E2 (PGE2) and nitric oxide (NO) respectively, in LPS-stimulated RAW 264.7 macrophages. 4'-HW also suppressed LPS-induced pro-inflammatory cytokines at mRNA and protein levels. Moreover, 4'-HW blocked the interaction of TAK1 and TAB1 in LPS-stimulated RAW 264.7 macrophages, resulting in an inhibition of the TAK1/IKK/NF-κB signaling pathway. Furthermore, 4'-HW also reduced the phosphorylation of MAPKs and PI3/Akt signaling pathways in LPS-stimulated RAW 264.7 macrophages. 4'-HW was also significantly decreased the intracellular reactive oxygen species (ROS) level. The effect of 4'-HW was confirmed in vivo. 4'-HW exhibited potent protective effect against LPS-induced ALI in mice. These findings indicate that 4'-HW suppresses the LPS-induced response in vitro and in vivo. It is likely that the inhibition of the TAK1/IKK/NF-κB, MAPKs and PI3/AKT signaling pathways contribute to the anti-inflammatory effects of 4'-HW. Our study suggests that 4'-HW may be an important functional constituent in the plants and has the potential value to be developed as a novel anti-inflammatory agent. [ABSTRACT FROM AUTHOR]

Cotton is a pioneer of saline land crop, while salt stress still causes its growth inhibition and fiber production decrease. Phenotype identification showed better salt tolerance of a wild diploid cotton species Gossypium klotzschianum. To elucidate the salt-tolerant mechanisms in G. klotzschianum, we firstly detected the changes in hormones, H2O2 and glutathione (GSSH and GSH), then investigated the gene expression pattern of roots and leaves treated with 300 mM NaCl for 0, 3, 12, 48 h, and each time control by RNA-seq on the Illumina-Solexa platform. Physiological determination proved that the significant increase in hormone ABA at 48 h, while that in H2O2 was at 12 h, likewise, the GSH content decrease at 48 h and the GSSH content increase at 48 h, under salt stress. In total, 37,278 unigenes were identified from the transcriptome data, 8,312 and 6,732 differentially expressed genes (DEGs) were discovered to be involved in salt stress tolerance in roots and leaves, respectively. Gene function annotation and expression analysis elucidated hormone biosynthesis and signal transduction, reactive oxygen species (ROS), and salt overly sensitive (SOS) signal transduction related genes revealed the important roles of them in signal transmission, oxidation balance and ion homeostasis in response to salinity stress. This is a report which focuses on primary response to highly salty stress (upto 300 mM NaCl) in cotton using a wild diploid Gossypium species, broadening our understanding of the salt tolerance mechanism in cotton and laying a solid foundation of salt resistant for the genetic improvement of upland cotton with the resistance to salt stress. [ABSTRACT FROM AUTHOR]

The immune system is necessary for protecting against various pathogens. However, under certain circumstances, self-reactive immune cells can drive autoimmunity, like that exhibited in type 1 diabetes (T1D). CD4+ T cells are major contributors to the immunopathology in T1D, and in order to drive optimal T cell activation, third signal reactive oxygen species (ROS) must be present. However, the role ROS play in mediating this process remains to be further understood. Recently, cellular metabolic programs have been shown to dictate the function and fate of immune cells, including CD4+ T cells. During activation, CD4+ T cells must transition metabolically from oxidative phosphorylation to aerobic glycolysis to support proliferation and effector function. As ROS are capable of modulating cellular metabolism in other models, we sought to understand if blocking ROS also regulates CD4+ T cell activation and effector function by modulating T cell metabolism. To do so, we utilized an ROS scavenging and potent antioxidant manganese metalloporphyrin (MnP). Our results demonstrate that redox modulation during activation regulates the mTOR/AMPK axis by maintaining AMPK activation, resulting in diminished mTOR activation and reduced transition to aerobic glycolysis in diabetogenic splenocytes. These results correlated with decreased Myc and Glut1 upregulation, reduced glucose uptake, and diminished lactate production. In an adoptive transfer model of T1D, animals treated with MnP demonstrated delayed diabetes progression, concurrent with reduced CD4+ T cell activation. Our results demonstrate that ROS are required for driving and sustaining T cell activation-induced metabolic reprogramming, and further support ROS as a target to minimize aberrant immune responses in autoimmunity. [ABSTRACT FROM AUTHOR]

Cryptochromes are evolutionarily conserved blue-light absorbing flavoproteins which participate in many important cellular processes including in entrainment of the circadian clock in plants, Drosophila and humans. Drosophila melanogaster cryptochrome (DmCry) absorbs light through a flavin (FAD) cofactor that undergoes photoreduction to the anionic radical (FAD•-) redox state both in vitro and in vivo. However, recent efforts to link this photoconversion to the initiation of a biological response have remained controversial. Here, we show by kinetic modeling of the DmCry photocycle that the fluence dependence, quantum yield, and half-life of flavin redox state interconversion are consistent with the anionic radical (FAD•-) as the signaling state in vivo. We show by fluorescence detection techniques that illumination of purified DmCry results in enzymatic conversion of molecular oxygen (O2) to reactive oxygen species (ROS). We extend these observations in living cells to demonstrate transient formation of superoxide (O2•-), and accumulation of hydrogen peroxide (H2O2) in the nucleus of insect cell cultures upon DmCry illumination. These results define the kinetic parameters of the Drosophila cryptochrome photocycle and support light-driven electron transfer to the flavin in DmCry signaling. They furthermore raise the intriguing possibility that light-dependent formation of ROS as a byproduct of the cryptochrome photocycle may contribute to its signaling role. [ABSTRACT FROM AUTHOR]

It is well established that the formation of radical species centered on various atoms is involved in the mechanism leading to the development of several diseases or to the appearance of deleterious effects of toxic molecules. The detection of free radical is possible using Electron Paramagnetic Resonance (EPR) spectroscopy and the spin trapping technique. The classical EPR spin-trapping technique can be considered as a “hypothesis-driven” approach because it requires an a priori assumption regarding the nature of the free radical in order to select the most appropriate spin-trap. We here describe a “data-driven” approach using EPR and a cocktail of spin-traps. The rationale for using this cocktail was that it would cover a wide range of biologically relevant free radicals and have a large range of hydrophilicity and lipophilicity in order to trap free radicals produced in different cellular compartments. As a proof-of-concept, we validated the ability of the system to measure a large variety of free radicals (O-, N-, C-, or S- centered) in well characterized conditions, and we illustrated the ability of the technique to unambiguously detect free radical production in cells exposed to chemicals known to be radical-mediated toxic agents. [ABSTRACT FROM AUTHOR]

Mouse F9 cells differentiate to primitive endoderm (PrE) when treated with retinoic acid (RA). Differentiation is accompanied by increased reactive oxygen species (ROS) levels, and while treating F9 cells with antioxidants attenuates differentiation, H2O2 treatment alone is sufficient to induce PrE. We identified the NADPH oxidase (NOX) complexes as candidates for the source of this endogenous ROS, and within this gene family, and over the course of differentiation, Nox1 and Nox 4 show the greatest upregulation induced by RA. Gata6, encoding a master regulator of extraembryonic endoderm is also up-regulated by RA and we provide evidence that NOX1 and NOX4 protein levels increase in F9 cells overexpressing Gata6. Pan-NOX and NOX1-specific inhibitors significantly reduced the ability of RA to induce PrE, and this was recapitulated using a genetic approach to knockdown Nox1 and/or Nox4 transcripts. Interestingly, overexpressing either gene in untreated F9 cells did not induce differentiation, even though each elevated ROS levels. Thus, the data suggests that ROS produced during PrE differentiation is dependent in part on increased NOX1 and NOX4 levels, which is under the control of GATA6. Furthermore, these results suggest that the combined activity of multiple NOX proteins is necessary for the differentiation of F9 cells to primitive endoderm. [ABSTRACT FROM AUTHOR]

The free-living soil nematode Caenorhabditis elegans adapts its development to the availability of food. When food is scarce and population density is high, worms enter a developmentally arrested non-feeding diapause stage specialized for long-term survival called the dauer larva. When food becomes available, they exit from the dauer stage, resume growth and reproduction. It has been postulated that compound(s) present in food, referred to as the “food signal”, promote exit from the dauer stage. In this study, we have identified NAD+ as a component of bacterial extract that promotes dauer exit. NAD+, when dissolved in alkaline medium, causes opening of the mouth and ingestion of food. We also show that to initiate exit from the dauer stage in response to NAD+ worms require production of serotonin. Thus, C. elegans can use redox cofactors produced by dietary organisms to sense food. [ABSTRACT FROM AUTHOR]

The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute β-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: β-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to β-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after β-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute β-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels. [ABSTRACT FROM AUTHOR]

EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt’s lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10–20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5–10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy. [ABSTRACT FROM AUTHOR]

Norepinephrine (NE) produces multifaceted regulatory patterns in T-lymphocytes. Recently, we have shown that NE utilizes redox signaling as evidenced by increased superoxide (O2●-) causally linked to the observed changes in these cells; however, the source of this reactive oxygen species (ROS) remains elusive. Herein, we hypothesized that the source of increased O2●- in NE-stimulated T-lymphocytes is due to disruption of mitochondrial bioenergetics. To address this hypothesis, we utilized purified mouse splenic CD4+ and CD8+ T-lymphocytes stimulated with NE and assessed O2●- levels, mitochondrial metabolism, cellular proliferation, and cytokine profiles. We demonstrate that the increase in O2●- levels in response to NE is time-dependent and occurs at later points of T-lymphocyte activation. Moreover, the source of O2●- was indeed the mitochondria as evidenced by enhanced MitoSOX Red oxidation as well as abrogation of this signal by the addition of the mitochondrial-targeted O2●--scavenging antioxidant MitoTempol. NE-stimulated T-lymphocytes also demonstrated decreased mitochondrial respiratory capacity, which suggests disruption of mitochondrial metabolism and the potential source of increased mitochondrial O2●-. The effects of NE in regards to redox signaling appear to be adrenergic receptor-dependent as specific receptor antagonists could reverse the increase in O2●-; however, differential receptors regulating these processes were observed in CD4+ versus CD8+ T-lymphocytes. Finally, mitochondrial O2●- was shown to be mechanistic to the NE-mediated T-lymphocyte phenotype as supplementation of MitoTempol could reverse specific changes in cytokine expression observed with NE treatment. Overall, these studies indicate that mitochondrial metabolism and O2●--mediated redox signaling play a regulatory role in the T-lymphocyte response to NE. [ABSTRACT FROM AUTHOR]

T cells reach a state of replicative senescence characterized by a decreased ability to proliferate and respond to foreign antigens. Calcium release associated with TCR engagement is widely used as a surrogate measure of T cell response. Using an ex vivo culture model that partially replicates features of organismal aging, we observe that while the amplitude of Ca2+ signaling does not change with time in culture, older T cells exhibit faster Ca2+ rise and a faster decay. Gene expression analysis of Ca2+ channels and pumps expressed in T cells by RT-qPCR identified overexpression of the plasma membrane CRAC channel subunit ORAI1 and PMCA in older T cells. To test whether overexpression of the plasma membrane Ca2+ channel is sufficient to explain the kinetic information, we adapted a previously published computational model by Maurya and Subramaniam to include additional details on the store-operated calcium entry (SOCE) process to recapitulate Ca2+ dynamics after T cell receptor stimulation. Simulations demonstrated that upregulation of ORAI1 and PMCA channels is not sufficient to explain the observed alterations in Ca2+ signaling. Instead, modeling analysis identified kinetic parameters associated with the IP3R and STIM1 channels as potential causes for alterations in Ca2+ dynamics associated with the long term ex vivo culturing protocol. Due to these proteins having known cysteine residues susceptible to oxidation, we subsequently investigated and observed transcriptional remodeling of metabolic enzymes, a shift to more oxidized redox couples, and post-translational thiol oxidation of STIM1. The model-directed findings from this study highlight changes in the cellular redox environment that may ultimately lead to altered T cell calcium dynamics during immunosenescence or organismal aging. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 3 kinase—protein kinase B (PI3K-AKT) pathway has been considered as major drug target site due to its frequent activation in cancer. AKT regulates the activity of various targets to promote tumorigenesis and metastasis. Accumulation of reactive oxygen species (ROS) has been linked to oxidative stress and regulation of signaling pathways for metabolic adaptation of tumor microenvironment. Hydrogen peroxide (H2O2) in this context is used as ROS source for oxidative stress preconditioning. Antioxidants are commonly considered to be beneficial to reduce detrimental effects of ROS and are recommended as dietary supplements. Quercetin, a ubiquitous bioactive flavonoid is a dietary component which has attracted much of interest due to its potential health-promoting effects. Present study is aimed to analyze PI3K-AKT signaling pathway in H2O2 exposed Dalton’s lymphoma ascite (DLA) cells. Further, regulation of PI3K-AKT pathway by quercetin as well as PI-103, an inhibitor of PI3K was analyzed. Exposure of H2O2 (1mM H2O2 for 30min) to DLA cells caused ROS accumulation and resulted in increased phosphorylation of PI3K and downstream proteins PDK1 and AKT (Ser-473 and Thr-308), cell survival factors BAD and ERK1/2, as well as TNFR1. However, level of tumor suppressor PTEN was declined. Both PI-103 & quercetin suppressed the enhanced level of ROS and significantly down-regulated phosphorylation of AKT, PDK1, BAD and level of TNFR1 as well as increased the level of PTEN in H2O2 induced lymphoma cells. The overall result suggests that quercetin and PI3K inhibitor PI-103 attenuate PI3K-AKT pathway in a similar mechanism. [ABSTRACT FROM AUTHOR]

The bacterial pathogen Xanthomonas campestris pv. vesicatoria 85–10 (Xcv) translocates about 30 type-3 effector proteins (T3Es) into pepper plants (Capsicum annuum) to suppress plant immune responses. Among them is XopB which interferes with PTI, ETI and sugar-mediated defence responses, but the underlying molecular mechanisms and direct targets are unknown so far. Here, we examined the XopB-mediated suppression of plant defence responses in more detail. Infection of susceptible pepper plants with Xcv lacking xopB resulted in delayed symptom development compared to Xcv wild type infection concomitant with an increased formation of salicylic acid (SA) and expression of pathogenesis-related (PR) genes. Expression of xopB in Arabidopsis thaliana promoted the growth of the virulent Pseudomonas syringae pv. tomato (Pst) DC3000 strain. This was paralleled by a decreased SA-pool and a lower induction of SA-dependent PR gene expression. The expression pattern of early flg22-responsive marker genes indicated that MAPK signalling was not altered in the presence of XopB. However, XopB inhibited the flg22-triggered burst of reactive oxygen species (ROS). Consequently, the transcript accumulation of AtOXI1, a ROS-dependent marker gene, was reduced in xopB-expressing Arabidopsis plants as well as callose deposition. The lower ROS production correlated with a low level of basal and flg22-triggered expression of apoplastic peroxidases and the NADPH oxidase RBOHD. Conversely, deletion of xopB in Xcv caused a higher production of ROS in leaves of susceptible pepper plants. Together our results demonstrate that XopB modulates ROS responses and might thereby compromise plant defence. [ABSTRACT FROM AUTHOR]

Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H2O2 remain obscure. Here we demonstrate sustained live responses of H2O2 to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H2O2. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H2O2. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H2O2, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H2O2, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt. [ABSTRACT FROM AUTHOR]

A series of hemoglobin-based oxygen carrier candidates (HBOC), previously noted for their differences in prooxidative and physiological reactivity, were compared in terms of the negative effects displayed upon injection in Wistar rats. At the concentrations tested, antioxidant strategies based on albumin as well as based on rubrerythrin appear to offer observable physiological advantages. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphate dehydrogenase (G6PD) provides the reducing agent NADPH to meet the cellular needs for reductive biosynthesis and the maintenance of redox homeostasis. G6PD-deficient cells experience a high level of oxidative stress and an increased susceptibility to viral infections. Cyclooxygenase-2 (COX-2) is a key mediator in the regulation of viral replication and inflammatory response. In the current study, the role of G6PD on the inflammatory response was determined in both scramble control and G6PD-knockdown (G6PD-kd) A549 cells upon tumor necrosis factor-α (TNF-α) stimulation. A decreased expression pattern of induced COX-2 and reduced production of downstream PGE2 occurred upon TNF-α stimulation in G6PD-kd A549 cells compared with scramble control A549 cells. TNF-α-induced antiviral activity revealed that decreased COX-2 expression enhanced the susceptibility to coronavirus 229E infection in G6PD-kd A549 cells and was a result of the decreased phosphorylation levels of MAPK (p38 and ERK1/2) and NF-κB. The impaired inflammatory response in G6PD-kd A549 cells was found to be mediated through NADPH oxidase (NOX) signaling as elucidated by cell pretreatment with a NOX2-siRNA or NOX inhibitor, diphenyleneiodonium chloride (DPI). In addition, NOX activity with TNF-α treatment in G6PD-kd A549 cells was not up-regulated and was coupled with a decrease in NOX subunit expression at the transcriptional level, implying that TNF-α-mediated NOX signaling requires the participation of G6PD. Together, these data suggest that G6PD deficiency affects the cellular inflammatory response and the decreased TNF-α-mediated antiviral response in G6PD-kd A549 cells is a result of dysregulated NOX/MAPK/NF-κB/COX-2 signaling. [ABSTRACT FROM AUTHOR]

Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation. [ABSTRACT FROM AUTHOR]

Conidiophore development of fungi belonging to the genus Aspergillus involves dynamic changes in cellular polarity and morphogenesis. Synchronized differentiation of phialides from the subtending conidiophore vesicle is a good example of the transition from isotropic to multi-directional polarized growth. Here we report a small GTPase, RacA, which is essential for reactive oxygen species (ROS) production in the vesicle as well as differentiation of phialides in Aspergillus fumigatus. We found that wild type A. fumigatus accumulates ROS in these conidiophore vesicles and that null mutants of racA did not, resulting in the termination of conidiophore development in this early vesicle stage. Further, we found that stress conditions resulting in atypical ROS accumulation coincide with partial recovery of phialide emergence but not subsequent apical dominance of the phialides in the racA null mutant, suggesting alternative means of ROS generation for the former process that are lacking in the latter. Elongation of phialides was also suppressed by inhibition of NADPH-oxidase activity. Our findings provide not only insights into role of ROS in fungal cell polarity and morphogenesis but also an improved model for the developmental regulatory pathway of conidiogenesis in A. fumigatus. [ABSTRACT FROM AUTHOR]

Overproduction of free radicals during ischemia/reperfusion (I/R) injury leads to an interest in using antioxidant therapy. Activating an endogenous antioxidant signaling pathway is more important due to the fact that the free radical scavenging behavior in vitro does not always correlate with a cytoprotection effect in vivo. Caffeic acid (CA), an antioxidant, is a major phenolic constituent in nature. Pyrrolidinyl caffeamide (PLCA), a derivative of CA, was compared with CA for their antioxidant and cytoprotective effects. Our results indicate that CA and PLCA exert the same ability to scavenge DPPH in vitro. In response to myocardial I/R stress, PLCA was shown to attenuate lipid peroxydation and troponin release more than CA. These responses were accompanied with a prominent elevation in AKT and HO-1 expression and a preservation of mnSOD expression and catalase activity. PLCA also improved cell viability and alleviated the intracellular ROS level more than CA in cardiomyocytes exposed to H2O2. When inhibiting the AKT or HO-1 pathways, PLCA lost its ability to recover mnSOD expression and catalase activity to counteract with oxidative stress, suggesting AKT/HO-1 pathway activation by PLCA plays an important role. In addition, inhibition of AKT signaling further abolished HO-1 activity, while inhibition of HO-1 signaling attenuated AKT expression, indicating cross-talk between the AKT and HO-1 pathways. These protective effects may contribute to the cardiac function improvement by PLCA. These findings provide new insight into therapeutic approaches using a modified natural compound against oxidative stress from myocardial injuries. [ABSTRACT FROM AUTHOR]

High reactive oxygen species (ROS) levels and enhanced vascular smooth muscle cells (VSMC) proliferation are observed in numerous cardiovascular diseases. The mechanisms by which hormones such as angiotensin II (Ang II) acts to promote these cellular responses remain poorly understood. We have previously shown that the ADP-ribosylation factor 6 (ARF6), a molecular switch that coordinates intracellular signaling events can be activated by the Ang II receptor (AT1R). Whether this small GTP-binding protein controls the signaling events leading to ROS production and therefore Ang II-dependent VSMC proliferation, remains however unknown. Here, we demonstrate that in rat aortic VSMC, Ang II stimulation led to the subsequent activation of ARF6 and Rac1, a key regulator of NADPH oxidase activity. Using RNA interference, we showed that ARF6 is essential for ROS generation since in conditions where this GTPase was knocked down, Ang II could no longer promote superoxide anion production. In addition to regulating Rac1 activity, ARF6 also controlled expression of the NADPH oxidase 1 (Nox 1) as well as the ability of the EGFR to become transactivated. Finally, ARF6 also controlled MAPK (Erk1/2, p38 and Jnk) activation, a key pathway of VSMC proliferation. Altogether, our findings demonstrate that Ang II promotes activation of ARF6 to controls ROS production by regulating Rac1 activation and Nox1 expression. In turn, increased ROS acts to activate the MAPK pathway. These signaling events represent a new molecular mechanism by which Ang II can promote proliferation of VSMC. [ABSTRACT FROM AUTHOR]

Inflammation and reactive oxygen species (ROS) play important roles in the pathogenesis of atherosclerosis. Resveratrol has been shown to possess anti-inflammatory and antioxidative stress activities, but the underlying mechanisms are not fully understood. In the present study, we investigated the molecular basis associated with the protective effects of resveratrol on tumor necrosis factor-alpha (TNF-α)-induced injury in human umbilical endothelial cells (HUVECs) using a variety of approaches including a cell viability assay, reverse transcription and quantitative polymerase chain reaction, western blot, and immunofluorescence staining. We showed that TNF-α induced CD40 expression and ROS production in cultured HUVECs, which were attenuated by resveratrol treatment. Also, resveratrol increased the expression of sirtuin 1 (SIRT1); and repression of SIRT1 by small-interfering RNA (siRNA) and the SIRT1 inhibitor Ex527 reduced the inhibitory effects of resveratrol on CD40 expression and ROS generation. In addition, resveratrol downregulated the levels of p65 and phospho-p38 MAPK, but this inhibitory effect was attenuated by the suppression of SIRT1 activity. Moreover, the p38 MAPK inhibitor SD203580 and the nuclear factor (NF)-κB inhibitor pyrrolidine dithiocarbamate (PDTC) achieved similar repressive effects as resveratrol on TNF-α-induced ROS generation and CD40 expression. Thus, our study provides a mechanistic link between resveratrol and the activation of SIRT1, the latter of which is involved in resveratrol-mediated repression of the p38 MAPK/NF-κB pathway and ROS production in TNF-α-treated HUVECs. [ABSTRACT FROM AUTHOR]

The NRF2 signalling cascade provides a primary response against electrophilic chemicals and oxidative stress. The activation of NRF2-signaling is anticipated to have adverse clinical consequences; NRF2 is activated in a number of cancers and, additionally, its pharmacological activation by one compound can reduce the toxicity or efficiency of a second agent administered concomitantly. In this work, we have analysed systematically the ability of 152 research, pre-clinical or clinically used drugs to induce an NRF2 response using the MCF7-AREc32 NRF2 reporter. Ten percent of the tested drugs induced an NRF2 response. The NRF2 activators were not restricted to classical cytotoxic alkylating agents but also included a number of emerging anticancer drugs, including an IGF1-R inhibitor (NVP-AEW541), a PIM-1 kinase inhibitor (Pim1 inhibitor 2), a PLK1 inhibitor (BI 2536) and most strikingly seven of nine tested HDAC inhibitors. These findings were further confirmed by demonstrating NRF2-dependent induction of endogenous AKR genes, biomarkers of NRF2 activity. The ability of HDAC inhibitors to stimulate NRF2-signalling did not diminish their own potency as antitumour agents. However, when used to pre-treat cells, they did reduce the efficacy of acrolein. Taken together, our data suggest that the ability of drugs to stimulate NRF2 activity is common and should be investigated as part of the drug-development process. [ABSTRACT FROM AUTHOR]

Continuous free radical assault upsets cellular homeostasis and dysregulates associated signaling pathways to promote stress-induced cell death. In spite of the continuous development and implementation of effective therapeutic strategies, limitations in treatments for stress-induced toxicities remain. The purpose of the present study was to determine the potential therapeutic efficacy of bacterial fucose polysaccharides against hydrogen peroxide (H2O2)-induced stress in human lung fibroblast (WI38) cells and to understand the associated molecular mechanisms. In two different fermentation processes, Bacillus megaterium RB-05 biosynthesized two non-identical fucose polysaccharides; of these, the polysaccharide having a high-fucose content (∼42%) conferred the maximum free radical scavenging efficiency in vitro. Structural characterizations of the purified polysaccharides were performed using HPLC, GC-MS, and 1H/13C/2D-COSY NMR. H2O2 (300 µM) insult to WI38 cells showed anti-proliferative effects by inducing intracellular reactive oxygen species (ROS) and by disrupting mitochondrial membrane permeability, followed by apoptosis. The polysaccharide (250 µg/mL) attenuated the cell death process by directly scavenging intracellular ROS rather than activating endogenous antioxidant enzymes. This process encompasses inhibition of caspase-9/3/7, a decrease in the ratio of Bax/Bcl2, relocalization of translocated Bax and cytochrome c, upregulation of anti-apoptotic members of the Bcl2 family and a decrease in the phosphorylation of MAPKs (mitogen activated protein kinases). Furthermore, cellular homeostasis was re-established via stabilization of MAPK-mediated Nrf2/Keap1 signaling and transcription of downstream cytoprotective genes. This molecular study uniquely introduces a fucose-rich bacterial polysaccharide as a potential inhibitor of H2O2-induced stress and toxicities. [ABSTRACT FROM AUTHOR]

Intrauterine growth restriction (IUGR) is associated with impaired vascular function, which contributes to the increased incidence of chronic disease. The aim of this study was to investigate whether aerobic training improves AngII-induced vasoconstriction in IUGR rats. Moreover, we assess the role of superoxide dismutase (SOD) isoforms and NADPH oxidase-derived superoxide anions in this improvement. Female Wistar rats were randomly divided into two groups on day 1 of pregnancy. A control group was fed standard chow ad libitum, and a restricted group was fed 50% of the ad libitum intake throughout gestation. At 8 weeks of age, male offspring from both groups were randomly assigned to 4 experimental groups: sedentary control (SC), trained control (TC), sedentary restricted (SRT), and trained restricted (TRT). The training protocol was performed on a treadmill and consisted of a continuous 60-min session 5 days/week for 10 weeks. Following aerobic training, concentration–response curves to AngII were obtained in endothelium-intact aortic rings. Protein expression of SOD isoforms, AngII receptors and the NADPH oxidase component p47phox was assessed by Western blot analysis. The dihydroethidium was used to evaluate the in situ superoxide levels under basal conditions or in the presence of apocynin, losartan or PD 123,319. Our results indicate that aerobic training can prevent IUGR-associated increases in AngII-dependent vasoconstriction and can restore basal superoxide levels in the aortic rings of TRT rats. Moreover, we observed that aerobic training normalized the increased p47phox protein expression and increased MnSOD and AT2 receptor protein expression in thoracic aortas of SRT rats. In summary, aerobic training can result in an upregulation of antioxidant defense by improved of MnSOD expression and attenuation of NADPH oxidase component p47phox. These effects are accompanied by increased expression of AT2 receptor, which provide positive effects against Ang II–induced superoxide generation, resulting in attenuation of AngII-induced vasoconstriction. [ABSTRACT FROM AUTHOR]

Celastrol, a tripterine derived from the traditional Chinese medicine plant Tripterygium wilfordii Hook F. (“Thunder of God Vine”), has been reported to have multiple effects, such as anti-inflammation, suppression of tumor angiogenesis, inhibition of tumor growth, induction of apoptosis and protection of cells against human neurodegenerative diseases. However, the mechanisms that underlie these functions are not well defined. In this study, we reported for the first time that Celastrol could induce HIF-1α protein accumulation in multiple cancer cell lines in an oxygen-independent manner and that the enhanced HIF-1α protein entered the nucleus and promoted the transcription of the HIF-1 target genes VEGF and Glut-1. Celastrol did not influence HIF-1α transcription. Instead, Celastrol induced the accumulation of the HIF-1α protein by inducing ROS and activating Akt/p70S6K signaling to promote HIF-1α translation. In addition, we found that the activation of Akt by Celastrol was transient. With increased exposure time, inhibition of Hsp90 chaperone function by Celastrol led to the subsequent depletion of the Akt protein and thus to the suppression of Akt activity. Moreover, in HepG2 cells, the accumulation of HIF-1α increased the expression of BNIP3, which induced autophagy. However, HIF-1α and BNIP3 did not influence the cytotoxicity of Celastrol because the main mechanism by which Celastrol kills cancer cells is through stimulating ROS-mediated JNK activation and inducing apoptosis. Furthermore, our data showed that the dose required for Celastrol to induce HIF-1α protein accumulation and enhance HIF-1α transcriptional activation was below its cytotoxic threshold. A cytotoxic dose of Celastrol for cancer cells did not display cytotoxicity in LO2 normal human liver cells, which indicated that the novel functions of Celastrol in regulating HIF-1 signaling and inducing autophagy might be used in new applications, such as in anti-inflammation and protection of cells against human neurodegenerative diseases. Future studies regarding these applications are required. [ABSTRACT FROM AUTHOR]

Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth. [ABSTRACT FROM AUTHOR]

IL-6/STAT3 pathway is involved in a variety of biological responses, including cell proliferation, differentiation, apoptosis, and inflammation. In our present study, we found that CO releasing molecules (CORMs) suppress IL-6-induced STAT3 phosphorylation, nuclear translocation and transactivity in endothelial cells (ECs). CO is a byproduct of heme degradation mediated by heme oxygenase (HO-1). However, CORMs can induce HO-1 expression and then inhibit STAT3 phosphorylation. CO has been found to increase a low level ROS and which may induce protein glutathionylation. We hypothesized that CORMs increases protein glutathionylation and inhibits STAT3 activation. We found that CORMs increase the intracellular GSSG level and induce the glutathionylation of multiple proteins including STAT3. GSSG can inhibit STAT3 phosphorylation and increase STAT3 glutathionylation whereas the antioxidant enzyme catalase can suppress the glutathionylation. Furthermore, catalase blocks the inhibition of STAT3 phosphorylation by CORMs treatment. The inhibition of glutathione synthesis by BSO was also found to attenuate STAT3 glutathionylation and its inhibition of STAT3 phosphorylation. We further found that HO-1 increases STAT3 glutathionylation and that HO-1 siRNA attenuates CORM-induced STAT3 glutathionylation. Hence, the inhibition of STAT3 activation is likely to occur via a CO-mediated increase in the GSSG level, which augments protein glutathionylation, and CO-induced HO-1 expression, which may enhance and maintain its effects in IL-6-treated ECs. [ABSTRACT FROM AUTHOR]

Nox5 is an EF-hand containing, calcium-dependent isoform of the NADPH oxidase family of reactive oxygen species (ROS) generating enzymes. Altered expression and activity of Nox5 has been reported in cardiovascular diseases and cancers but the absence of Nox5 in rodents has precluded a greater understanding of its physiological and pathophysiological roles. Multiple polymorphisms have been identified within the coding sequence of human Nox5, but whether this translates into altered enzyme function is unknown. Herein, we have generated 15 novel mutants of Nox5β to evaluate the effect of exonic SNPs on basal and stimulated enzyme activity. Compared to the WT enzyme, ROS production was unchanged or slightly modified in the majority of mutants, but significantly decreased in 7. Focusing on M77K, Nox5 activity was dramatically reduced in unstimulated cells and following challenge with both calcium- and phosphorylation-dependent stimuli despite equivalent levels of expression. The M77K mutation did not influence the Nox5 phosphorylation or the ability to bind Hsp90, but in cell-free assays with excess co-factors and calcium, ROS production was dramatically reduced. A more conservative substitution M77V arising from another SNP yielded a different profile of enzyme activity and suggests a critical role of M77 in calcium-dependent ROS production. Two C-terminal mutants, R530H and G542R, were observed that had little to no activity and relatively high minor allele frequency (MAF). In conclusion, we have identified 7 missense SNPs in Nox5 that result in little or no enzyme activity. Whether humans with dysfunctional Nox5 variants have altered physiology or disease remains to be determined. [ABSTRACT FROM AUTHOR]

Studies have demonstrated that reactive oxygen species (ROS) generated by NADPH oxidase are essential for melanoma proliferation and survival. However, the mechanisms by which NADPH oxidase regulates these effects are still unclear. In this work, we investigate the role of NADPH oxidase-derived ROS in the signaling events that coordinate melanoma cell survival. Using the highly metastatic human melanoma cell line MV3, we observed that pharmacological NADPH oxidase inhibition reduced melanoma viability and induced dramatic cellular shape changes. These effects were accompanied by actin cytoskeleton rearrangement, diminished FAKY397 phosphorylation, and decrease of FAK-actin and FAK-cSrc association, indicating disassembly of focal adhesion processes, a phenomenon that often results in anoikis. Accordingly, NADPH oxidase inhibition also enhanced hypodiploid DNA content, and caspase-3 activation, suggesting activation of the apoptotic machinery. NOX4 is likely to be involved in these effects, since silencing of NOX4 significantly inhibited basal ROS production, reduced FAKY397 phosphorylation and decreased tumor cell viability. Altogether, the results suggest that intracellular ROS generated by the NADPH oxidase, most likely NOX4, transmits cell survival signals on melanoma cells through the FAK pathway, maintaining adhesion contacts and cell viability. [ABSTRACT FROM AUTHOR]

Patients with cystic fibrosis (CF) have elevated concentration of cytokines in sputum and a general inflammatory condition. In addition, CF cells in culture produce diverse cytokines in excess, including IL-1β. We have previously shown that IL-1β, at low doses (∼30 pM), can stimulate the expression of CFTR in T84 colon carcinoma cells, through NF-κB signaling. However, at higher doses (>2.5 ng/ml, ∼150 pM), IL-1β inhibit CFTR mRNA expression. On the other hand, by using differential display, we found two genes with reduced expression in CF cells, corresponding to the mitochondrial proteins CISD1 and MTND4. The last is a key subunit for the activity of mitochondrial Complex I (mCx-I); accordingly, we later found a reduced mCx-I activity in CF cells. Here we found that IB3-1 cells (CF cells), cultured in serum-free media, secrete 323±5 pg/ml of IL-1β in 24 h vs 127±3 pg/ml for S9 cells (CFTR-corrected IB3-1 cells). Externally added IL-1β (5 ng/ml) reduces the mCx-I activity and increases the mitochondrial (MitoSOX probe) and cellular (DCFH-DA probe) ROS levels of S9 (CFTR-corrected IB3-1 CF cells) or Caco-2/pRSctrl cells (shRNA control cells) to values comparable to those of IB3-1 or Caco-2/pRS26 cells (shRNA specific for CFTR). Treatments of IB3-1 or Caco-2/pRS26 cells with either IL-1β blocking antibody, IL-1 receptor antagonist, IKK inhibitor III (NF-κB pathway) or SB203580 (p38 MAPK pathway), restored the mCx-I activity. In addition, in IB3-1 or Caco-2/pRS26 cells, IL-1β blocking antibody, IKK inhibitor III or SB203580 reduced the mitochondrial ROS levels by ∼50% and the cellular ROS levels near to basal values. The AP-1 inhibitors U0126 (MEK1/2) or SP600125 (JNK1/2/3 inhibitor) had no effects. The results suggest that in these cells IL-1β, through an autocrine effect, acts as a bridge connecting the CFTR with the mCx-I activity and the ROS levels. [ABSTRACT FROM AUTHOR]

Resveratrol has been widely reported to reduce cancer progression in model systems and to selectively induce cell death in transformed cell lines. Many enzymes have been reported to respond to resveratrol in mammalian cells, including the Ataxia-Telangiectasia Mutated (ATM) protein kinase that acts in DNA damage recognition, signaling, and repair. Here we investigate the responses of ATM to resveratrol exposure in normal and transformed human cell lines and find that ATM autophosphorylation and substrate phosphorylation is stimulated by resveratrol in a manner that is promoted by reactive oxygen species (ROS). We observe direct stimulatory effects of resveratrol on purified ATM in vitro and find that the catalytic efficiency of the kinase on a model substrate is increased by resveratrol. In the purified system we also observe a requirement for oxidation, as the effect of resveratrol on ATM signaling is substantially reduced by agents that prevent disulfide bond formation in ATM. These results demonstrate that resveratrol effects on ATM are direct, and suggest a mechanism by which the oxidizing environment of transformed cells promotes ATM activity and blocks cell proliferation. [ABSTRACT FROM AUTHOR]