Search

For testing goodness of fit, we consider a class of U-statistics of overlapping spacings of order two, and investigate their asymptotic properties. The standard U-statistic theory is not directly applicable here as the overlapping spacings form a dependent random sequence. The asymptotic distribution of the statistics under the null hypothesis and under a sequence of local alternatives are derived. In terms of the Pitman ARE, the U-statistic based on Gini's mean square difference of overlapping spacings is found to be the asymptotically locally most powerful. Interestingly, this test has the same efficacy as the Greenwood test based on overlapping spacings. • For testing goodness of fit, we consider U-statistics of overlapping spacings. • We derive asymptotic distribution of statistics for a sequence of local alternatives. • The asymptotically locally most powerful test is obtained in terms of the Pitman ARE. [ABSTRACT FROM AUTHOR]

Living organisms have developed finely regulated homeostatic networks to mitigate the effects of environmental fluctuations in transition metal micronutrients, including iron, zinc, and copper. In Saccharomyces cerevisiae , the tandem zinc-finger protein Cth2 post-transcriptionally regulates gene expression under conditions of iron deficiency by controlling the levels of mRNAs that code for non-essential ferroproteins. The molecular mechanism involves Cth2 binding to AU-rich elements present in the 3′ untranslated region of target mRNAs, negatively affecting their stability and translation. Arabidopsis thaliana has two TZF proteins homologous to yeast Cth2, C3H14 and C3H15, which participate in cell wall remodelling. The present work examines the expression of representative metal homeostasis genes with putative AREs in plants with altered levels of C3H14 and C3H15 grown under varying metal availabilities. The results suggest that C3H15 may act as a post-transcriptional plant modulator of metal adequacy, as evidenced by the expression of SPL7 , the main transcriptional regulator under copper deficiency, and PETE2 , which encodes plastocyanin. In contrast to S. cerevisiae , the plant C3H15 affects copper and zinc homeostasis rather than iron. When grown under copper-deficient conditions, adult C3H15 OE plants exhibit lower chlorophyll content and photosynthetic efficiency compared to control plants, suggesting accelerated senescence. Likewise, metal content in C3H15 OE plants under copper deficiency shows altered mobilization of copper and zinc to seeds. These data suggest that the C3H15 protein plays a role in modulating both cell wall remodelling and metal homeostasis. The interaction between these processes may be the cause of altered metal translocation. • C3H15 functions as a post-transcriptional regulator in Arabidopsis copper and zinc homeostasis. • A comparison of the TZF domains of S. cerevisiae Cth2 and higher plants reveals that the TZF1 is most conserved than TZF2. • Analysis of Arabidopsis genes with putative C3H15 target sequences reveals that metal homeostasis genes are overrepresented. • Under copper deficiency, C3H15 OE show an accelerated senescence as indicated by chlorophyll content and fluorescence. • Seeds of C3H15OE contain reduced zinc and copper levels pointing to their difficulty in mobilizing copper. [ABSTRACT FROM AUTHOR]

Nuclear factor erythroid 2-related factor-2 (Nrf2), and the less well characterised proteins Nrf1 and Nrf3, are member of the cap 'n' collar family of transcription factors. Nrf proteins regulate the expression of endogenous antioxidant enzymes and have recently become the targets for various therapeutic treatments. Recently, Nrf proteins have been of particular interest as a target in placental-derived oxidative stress induced pregnancy disorders. Here, we report the presence of Nrf1, Nrf2 and Nrf3 proteins in both human primary trophoblast and human trophoblast choriocarcinoma cell line (BeWo). We also detail the steps taken to successfully silence all Nrf proteins in both human primary trophoblast cells and BeWo via detection of mRNA and protein using quantitative PCR, and SDS-PAGE and Western Blotting respectively. [ABSTRACT FROM AUTHOR]

The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of growth arrest and DNA damage-inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We establish that the 3′ untranslated region of PPP1R15A mRNA contains an active AU-rich element (ARE) recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a component of the transient ISR memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions. [Display omitted] • Under normal conditions, TTP/Brf1 recognize PPP1R15A ARE promoting mRNA rapid decay • PPP1R15A mRNA turnover regulates the threshold at which the ISR is triggered • Cellular stress enhances PPP1R15A mRNA stability, favoring GADD34 translation • Decay of PPP1R15A mRNA enhances sensitivity to stress and antagonizes adaptation Magg et al. uncover a novel regulatory step of the integrated stress response at the level of PPP1R15A mRNA stability and establish AU-rich element-driven mRNA turnover as an important determinant controlling GADD34 expression, thereby shaping the dynamics of stress adaptation and sensitivity to repetitive stress exposure. [ABSTRACT FROM AUTHOR]

The oxidative polymerizations of 2-methyl- m -phenylenediamine were performed to synthesize PMPDIA-A and PMPDIA-B polymers by H 2 O 2 (35% aqueous solution) oxidant in acid-catalyzed ethanol and basic aqueous medium. Furthermore, PMPDIA-E polymer of 2-methyl- m -phenylenediamine was enzymatically synthesized by HRP (horseradish peroxidase) enzyme and H 2 O 2 (35% aqueous solution) in dioxane/0.1 M phosphate tampon solution (pH = 7) mixture. The structures of 2-methyl- m -phenylenediamine and polymers were confirmed by FT-IR, NMR and UV–Vis spectrometer measurements. Molecular weight distributions, surface morphologies, thermal and fluorescence properties of polymers were determined from GPC, SEM, TG-DTA, DSC and fluorescence spectra analyses, respectively. CV and UV–Vis analyses were performed to determine the HOMO–LUMO energy levels, electrochemical (E g ′) and optical (E g) band gaps values of MPDIA, PMPDIA-E, PMPDIA-A and PMPDIA-B. The electrochemical and optical band gaps values of polymers were lower than MPDIA, because of their polyconjugated structures. According to GPC measurements of polymers, the weight average molecular weights were between 5400 and 10400 Da. The fluorescence quantum yield of PMPDIA-E was calculated to be 9.15% in DMSO solution. The adsorption of methylene blue (MB) from aqueous solution on PMPDIA-B was studied. Adsorption isotherms and equilibrium adsorption capacities were determined by the fitting of the experimental data to two well-known isotherm models: Langmuir and Freundlich. [ABSTRACT FROM AUTHOR]

• iRIP-seq captures the RNA targets directly and indirectly bound by TTP. • Over-representation of AUUUA motif occurs not only in the 3'UTR but also in the intronic TTP binding peaks. • High frequencies of hnRNPA2/B1 and CELF1-binding motifs are presented in the 3'UTR and intronic TTP peaks. • The UGGAC motif overlapping with the m6A motif GGACU is over-represented in the CDS TTP peaks. • TTP upregulates RelB expression correlating with its multiple intronic and exonic sites in RelB. TTP up-regulates the expression of innate immunity genes in a RelB-dependent manner. Tristetraprolin (TTP) regulates inflammatory and immune responses by destabilizing target mRNAs via binding to their 3′-UTR AREs. We have recently reported that TTP preferentially up-regulates the expression level of innate immunity genes involved in the type I interferon-mediated signaling pathway and viral response in cancer cells. To elucidate the role of TTP-RNA interaction in TTP-mediated upregulation of gene expression, we performed iRIP-seq experiments to obtain the RNA interaction map consisting of direct and indirect binding sites of TTP in HeLa cells. We found substantial TTP binding signals in mRNA regions and the introns. ARE-motif AUUUA is over-represented in TTP binding peaks. Strikingly, AUUUA frequency is high both in 3′UTR and intronic regions, and the intronic peaks were more associated with TTP-regulated genes. Analysis of the over-represented motifs in TTP peaks revealed the high frequencies of UAGG and GUGUG motifs reported for hnRNPA2/B1 and CELF1 respectively in the 3′UTR and introns, and also the UGGAC motif overlapping with the m6A motif GGACU in the CDS regions. We further demonstrated that TTP binds to multiple intronic and exonic sites in the pre-mRNA/mRNA of the transcription factor RelB, correlating with the TTP-upregulated expression of RelB. TTP-up-regulated genes without a TTP binding site, but not those with, are highly enriched in innate immunity pathways and show higher tendency of harboring RelB binding sites in their promoter regions. These findings support a model in which TTP binding of RelB pre-mRNA/mRNA coordinates the RelB upregulation and activation of the innate immunity for antiviral response. [ABSTRACT FROM AUTHOR]

The peptide EDYGA was docked to the active pocket of Keap1 Kelch domain, three hydrogen bonds were found, the pocket of Keap1 Kelch domain are H bond donor, and EDYGA was hydrogen bond acceptor. • Peptide EDYGA was the most potent ARE-luciferase inducer. • EDYGA enhanced Nrf2 level by stabilizing Nrf2 by down regulating Keap1. • EDYGA was docked to the active pocket with three hydrogen bonds. The antioxidant response element (ARE) is a cis-acting enhancer sequence located in the region containing genes related to antioxidant and detoxification. Under oxidative stress, the induction of nuclear factor-E2-related factor 2 (Nrf2)/ARE is considered as a fundamental process involved in defending reactive oxygen species (ROS) and providing protection against toxic xenobiotics. In this study, we obtained seven antioxidant peptides from soft-shelled turtle and concluded that Glu-Asp-Tyr-Gly-Ala (EDYGA) is the most potent ARE-luciferase inducer. To gain fundamental insights into the role of EDYGA in oxidative stress, we evaluated the effects of EDYGA on the Nrf2/Keap1 system in HepG 2 cells. The results revealed that EDYGA modulated the Nrf2/ARE pathway by enhancing Nrf2 level through the stabilization of Nrf2, which was accomplished by a decrease in the level of Keap1. These actions eventually led to an increase in nuclear Nrf2 accumulation and ARE-binding activity. Moreover, silencing Nrf2 markedly reduced ARE-driven activity induced by EDYGA. Docking results proved that glutamate residues of peptide EDYGA directly bind to Arg 415 of Kelch domain receptor pocke. The results were helpful in understanding the antioxidant activity of peptides from soft-shelled turtle which have potential to be used in foods and drugs as functional ingredients. [ABSTRACT FROM AUTHOR]

While destination governance has grown into a mature research field, the dynamic analysis of destination governance transitions remains in its infancy. Therefore, this paper analyses and compares the destination governance transitions in the successful skiing destinations of Åre (Sweden), Whistler (Canada) and Dolomites (Italy). This multiple case study unveils development patterns in governance transitions and investigates the factors that trigger such transitions. In particular, the paper investigates whether leading skiing destinations get increasingly corporatised and 'resortisised'. It also scrutinises what could trigger processes of 'resortisation' and focuses on the impact of crises. The study finds that governance is an adaptive phenomenon that transforms over time, but no strictly uniform transition pattern is detected. While differences arise from varying starting conditions and crises, the commonality is to be found in an apparent convergence of destination governance towards an intermediate stage of 'socially licensed resorts' with a minimum of community acceptance. • Governance is changeable, evolves over time and passes through several transitions. • Crises and change moments have shaped the destination governance transitions. • A tendency for ski destinations of becoming concentrated and centralised is detected. • Success also depends on good community relations and collaborative governance. • A converging trend towards an intermediate state of "resortisation" is found. [ABSTRACT FROM AUTHOR]

Background: A large outbreak of three epidemic vancomycin-resistant Enterococcus faecium (VRE) clones affected the study hospital for almost two years.Aim: To describe the strategy to successfully control this outbreak and eradicate VRE from the study hospital.Methods: Infection control interventions started after detection of VRE in three patients. Hospital-wide surveillance was started after ongoing transmission despite isolation precautions, cleaning and contact tracing. Hygiene education and discipline were enhanced. Despite these interventions, additional measures were required to control the outbreak, such as ward disinfection with hydrogen peroxide vapour and the introduction of a VRE quarantine ward. Ultimately, ciprofloxacin prophylaxis for haematological patients on chemotherapy was abandoned.Findings: Over a 22-month period, 242 VRE carriers were identified. Of these, 128 (53%) patients were detected by hospital-wide surveillance alone. Three epidemic clones were detected: ST494-vanA (N = 160), ST78-vanA (N = 23) and ST117-vanB (N = 32). In total, 5614 possible contacts were identified. VRE transmission occurred on 13 out of 23 wards. VRE was cultured from clinical specimens in 22 patients (seven with bacteraemia). Since January 2014, no further transmission of these VRE clones has been observed.Conclusion: Infection control measures according to international guidelines were insufficient to expose the outbreak to its full extent and control it. Its full extent only became apparent after sustained hospital-wide screening. Successful control of this hospital-wide VRE outbreak was feasible, but required great effort. Final containment and eradication of the epidemic clones was achieved by environmental decontamination with hydrogen peroxide vapour, strict isolation precautions, a VRE quarantine ward and antimicrobial stewardship. [ABSTRACT FROM AUTHOR]

Arsenic poisoning is a worldwide endemic disease that affects thousands of people. Growing evidence from animal, cell, and human studies indicates that arsenic has deleterious effects on the liver. Oxidative stress is considered the primary mechanism for arsenic toxicity in liver damage. However, the mechanisms remain unclear. In light of this fact, the main objective of this study was to investigate the effects of the protein kinase C delta-nuclear factor E2-related factor 2-antioxidant response element (PKCδ-Nrf2-ARE) signalling pathway on oxidative stress in liver damage. In the present study, we used a model of liver damage induced by coal-burning arsenic in rats, which was set up by our research group. The oxidative stress index and the transcription and protein expression levels of PKCδ, Nrf2, Keap1, SOD1, and GPx1 were detected, and then their correlation analyses were carried out. The results demonstrated that coal-burning arsenic can cause oxidative stress liver damage in rats, which may be related to the PKCδ-Nrf2-ARE signalling pathway. This study may provide a new pathway for studies of the mechanisms of arsenism. [ABSTRACT FROM AUTHOR]

Age-related cataracts are closely associated with lens chronological aging, oxidation, calcium imbalance, hydration and crystallin modifications. Accumulating evidence indicates that misfolded proteins are generated in the endoplasmic reticulum (ER) by most cataractogenic stresses. To eliminate misfolded proteins from cells before they can induce senescence, the cells activate a clean-up machinery called the ER stress/unfolded protein response (UPR). The UPR also activates the nuclear factor-erythroid-2-related factor 2 (Nrf2), a central transcriptional factor for cytoprotection against stress. Nrf2 activates nearly 600 cytoprotective target genes. However, if ER stress reaches critically high levels, the UPR activates destructive outputs to trigger programmed cell death. The UPR activates mobilization of ER-Ca2+ to the cytoplasm and results in activation of Ca2+-dependent proteases to cleave various enzymes and proteins which cause the loss of normal lens function. The UPR also enhances the overproduction of reactive oxygen species (ROS), which damage lens constituents and induce failure of the Nrf2 dependent cytoprotection. Kelch-like ECH-associated protein 1 (Keap1) is an oxygen sensor protein and regulates the levels of Nrf2 by the proteasomal degradation. A significant loss of DNA methylation in diabetic cataracts was found in the Keap1 promoter, which overexpresses the Keap1 protein. Overexpressed Keap1 significantly decreases the levels of Nrf2. Lower levels of Nrf2 induces loss of the redox balance toward to oxidative stress thereby leading to failure of lens cytoprotection. Here, this review summarizes the overall view of ER stress, increases in Ca2+ levels, protein cleavage, and loss of the well-established stress protection in somatic lens cells. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play vital roles in regulating neuron survival during cerebral ischemia/reperfusion injury. miR-142-5p is reported to be an important regulator of cellular survival. However, little is known about the role of miR-142-5p in regulating neuron survival during cerebral ischemia/reperfusion injury. In this study, we aimed to investigate the precise function and mechanism of miR-142-5p in the regulation of neuron ischemia/reperfusion injury using a cellular model of oxygen-glucose deprivation and reoxygenation (OGD/R)-induced injury in hippocampal neurons in vitro . We found that miR-142-5p was induced in hippocampal neurons with OGD/R treatment. The inhibition of miR-142-5p attenuated OGD/R-induced cell injury and oxidative stress, whereas the overexpression of miR-142-5p aggravated them. Nuclear factor erythroid 2-related factor 2 (Nrf2) was identified as a target gene of miR-142-5p. Moreover, miR-142-5p regulated Nrf2 expression and downstream signaling. Knockdown of Nrf2 abolished the protective effects of miR-142-5p suppression. In addition, we showed an inverse correlation relationship between miR-142-5p and Nrf2 in an in vivo model of middle cerebral artery occlusion in rats. Taken together, these results suggest that miR-142-5p contributes to OGD/R-induced cell injury and the down-regulation of miR-142-5p attenuates OGD/R-induced neuron injury through promoting Nrf2 expression. Our study provides a novel insight into understanding the molecular pathogenesis of cerebral ischemia/reperfusion injury and indicates a potential therapeutic target for the treatment of cerebral ischemia/reperfusion injury. [ABSTRACT FROM AUTHOR]

Cytokines and growth factors regulate cell proliferation, differentiation, migration and apoptosis, and play important roles in coordinating growth signal responses during development. The expression of cytokine genes and the signals transmitted through cytokine receptors are tightly regulated at several levels, including transcriptional and post-transcriptional levels. A majority of cytokine mRNAs, including growth factor transcripts, contain AU-rich elements (AREs) in their 3′ untranslated regions that control gene expression by regulating mRNA degradation and changing translational rates. In addition, numerous proteins involved in transmitting signals downstream of cytokine receptors are regulated at the level of mRNA degradation by GU-rich elements (GREs) found in their 3′ untranslated regions. Abnormal stabilization and overexpression of ARE or GRE-containing transcripts had been observed in many malignancies, which is a consequence of the malfunction of RNA-binding proteins. In this review, we briefly summarize the role of AREs and GREs in regulating mRNA turnover to coordinate cytokine and growth factor expression, and we describe how dysregulation of mRNA degradation mechanisms contributes to the development and progression of cancer. [ABSTRACT FROM AUTHOR]

Inflammation is the most common feature of many chronic diseases and complications, while playing critical roles in carcinogenesis. Several studies have demonstrated that Nrf2 contributes to the anti-inflammatory process by orchestrating the recruitment of inflammatory cells and regulating gene expression through the antioxidant response element (ARE). The Keap1 (Kelch-like ECH-associated protein)/Nrf2 (NF-E2 p45-related factor 2)/ARE signaling pathway mainly regulates anti-inflammatory gene expression and inhibits the progression of inflammation. Therefore, the identification of new Nrf2-dependent anti-inflammatory phytochemicals has become a key point in drug discovery. In this review, we discuss the members of the Keap1/Nrf2/ARE signal pathway and its downstream genes, the effects of this pathway on animal models of inflammatory diseases, and crosstalk with the NF-κB pathway. In addition we also discuss about the regulation of NLRP3 inflammasome by Nrf2. Besides this, we summarize the current scenario of the development of anti-inflammatory phytochemicals and others that mediate the Nrf2/ARE signaling pathway. [ABSTRACT FROM AUTHOR]

The Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor regulating a wide array of genes for antioxidant and detoxification enzymes in response to oxidative and xenobiotic stress. A large number of Nrf2-antioxidant response element (ARE) activators have been screened for use as chemopreventive agents in oxidative stress-related diseases and even cancer. However, constitutive activation of Nrf2 occurs in a variety of cancers. Aberrant activation of Nrf2 is correlated with cancer progression, chemoresistance, and radioresistance. In this review, we examine recent studies of Nrf2-ARE inhibitors in the context of cancer therapy. We enumerate the possible Nrf2-inhibiting mechanisms of these compounds, their effects sensitizing cancer cells to chemotherapeutic agents, and the prospect of applying them in clinical cancer therapy. [ABSTRACT FROM AUTHOR]

The Jun dimerization protein 2 (JDP2) is part of the family of stress-responsible transcription factors such as the activation protein-1, and binds the 12- O -tetradecanoylphorbol-13-acetateresponse element and the cAMP response element. It also plays a role as a histone chaperone and participates in diverse processes, such as cell-cycle arrest, cell differentiation, apoptosis, senescence, and metastatic spread, and functions as an oncogene and anti-oncogene, and as a cellular reprogramming factor. However, the molecular mechanisms underlying these multiple functions of JDP2 have not been clarified. This review summarizes the structure and function of JDP2, highlighting the specific role of JDP2 in cellular-stress regulation and prevention. [ABSTRACT FROM AUTHOR]

The discovery of Keap1–Nrf2 protein–protein interaction (PPI) inhibitors has become a promising strategy to develop novel lead molecules against variety of stress. Hence, Keap1–Nrf2 system plays an important role in oxidative/electrophilic stress associated disorders. Our earlier studies identified pterostilbene (PTS), a natural analogue of resveratrol, as a potent Nrf2 activator and Keap1–Nrf2 PPI inhibitor as assessed by luciferase complementation assay. In this study, we further identified the potential of PTS in Nrf2 activation and ARE-driven downstream target genes expression by nuclear translocation experiments and ARE-luciferase reporter assay, respectively. Further, the luciferase complementation assay identified that PTS inhibits Keap1–Nrf2 PPI in both dose and time-dependent manner. Computational studies using molecular docking and dynamic simulation revealed that PTS directly interacts with the basic amino acids of kelch domain of Keap1 and perturb Keap1–Nrf2 interaction pattern. This manuscript not only shows the binding determinants of Keap1–Nrf2 proteins but also provides mechanistic insights on Nrf2 activation potential of PTS. [ABSTRACT FROM AUTHOR]

Nrf2, a basic leucine zipper transcription factor encoded by the gene NFE2L2, is a master regulator of the transcriptional response to oxidative stress. Nrf2 is structurally and functionally conserved from insects to humans, and it heterodimerizes with the small MAF transcription factors to bind a consensus DNA sequence (the antioxidant response element, or ARE) and regulate gene expression. We have used genome-wide chromatin immunoprecipitation and gene expression data to identify direct Nrf2 target genes in Drosophila and humans. These data have allowed us to construct the deeply conserved ancient Nrf2 regulatory network—target genes that are conserved from Drosophila to human. The ancient network consists of canonical antioxidant genes, as well as genes related to proteasomal pathways and metabolism and a number of less expected genes. We have also used enhancer reporter assays and electrophoretic mobility-shift assays to confirm Nrf2-mediated regulation of ARE activity at a number of these novel target genes. Interestingly, the ancient network also highlights a prominent negative feedback loop; this, combined with the finding that Nrf2-mediated regulatory output is tightly linked to the quality of the ARE it is targeting, suggests that precise regulation of nuclear Nrf2 concentration is necessary to achieve proper quantitative regulation of distinct gene sets. Together, these findings highlight the importance of balance in the Nrf2–ARE pathway and indicate that Nrf2-mediated regulation of xenobiotic metabolism, glucose metabolism, and proteostasis has been central to this pathway since its inception. [ABSTRACT FROM AUTHOR]

Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder with increased oxidative stress, the underlying vital process contributing to cell death. Tanshinone IIA (Tan IIA), a major bioactive diterpene quinone of Salva miltiorrhiza, had been proved effective in the MPTP model through its anti-inflammatory activity. Here in this research, we found that Tan IIA prevented the loss of nigrostriatal dopaminergic neurons by activating the NF-E2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway. The cytotoxicity of 6-hydroxydopamine (6-OHDA) was attenuated by the treatment of Tan IIA in SH-SY5Y cells, which significantly reduced 6-OHDA-induced lactic dehydrogenase release and reactive oxygen species production. Further study indicated that Tan IIA contributed to the nuclear accumulation of Nrf2, which bound to the ARE sequence, and activated ARE-regulated genes, including heme oxygenase-1, glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM). Tan IIA also protected against damage to mitochondrial membrane potential, reduced the translocation of cytochrome c from the mitochondria to the cytoplasm and the activation of Caspase-9 and Caspase-3. Moreover, we demonstrated the above effects were performed in Nrf2-dependent manner. Further studies revealed that Tan IIA reduced the enhancement of miR-153 by 6-OHDA, which targeted the 3′-UTR of Nrf2, and suppressed its expression and activation. Additionally, neurodegeneration caused by in vivo stereotaxic injection of 6-OHDA could also be ameliorated by the administration of Tan IIA. Taken together, our results strongly suggest that Tan IIA may be beneficial for the treatment of PD, and also confirm that targeting the Nrf2/ARE pathway is a promising strategy for therapeutic intervention in PD. [ABSTRACT FROM AUTHOR]

Resveratrol monomer ( Res ) and its oligomers are considered as nutritional components distributed in edible plants. Three naturally occurring resveratrol dimers, namely parthenocissin A ( Par ), quadrangularin A ( Qua ) and pallidol ( Pal ), were synthesized and evaluated for their ability to scavenge reactive oxygen species (ROS) and to activate the transcription factor Nrf2, which regulates cellular antioxidant systems. In vitro studies with different ROS and radical assay models showed that all the three dimers are strong DPPH quenchers and selective singlet oxygen ( 1 O 2 ) scavengers (IC 50 = 4.90, 1.05 and 5.50 μM, respectively). However, they were ineffective against hydroxyl radical ( OH) or superoxide anion (O 2 − ). Exposing the dimers to an antioxidant response element (ARE) reporter cell line revealed that only pallidol was able to activate Nrf2 at 30 μM, while parthenocissin A and quadrangularin A had no significant effect on Nrf2. Our data demonstrates the distinct difference between reservatrol monomer and its dimers in activating the Nrf2/ARE signalling pathway. [ABSTRACT FROM AUTHOR]

The cap’n’collar (CNC) family serves as cellular sensors of oxidative and electrophilic stresses and shares structural similarities including basic leucine zipper (bZIP) and CNC domains. They form heterodimers with small MAF proteins to regulate antioxidant and phase II enzymes through antioxidant response element (ARE)-mediated transactivation. Among the CNC family members, NRF2 is required for systemic protection against redox-mediated injury and carcinogenesis. On the other hand, NRF2 is activated by oncogenic pathways, metabolism, and hypoxia. Constitutive NRF2 activation is observed in a variety of human cancers and it is highly correlated with tumor progression and aggressiveness. In this review, we will discuss how NRF2 plays dual roles in cancer prevention and progression depending on the cellular context and environment. Therefore, a better understanding of NRF2 will be necessary to exploit this complex network of balancing antioxidant pathways to inhibit tumor progression. [ABSTRACT FROM AUTHOR]

Abstract: Background: PEPT1 is a rheogenic transport protein in the apical membrane of intestinal epithelial cells capable of transporting essentially all possible di- and tripeptides that are generated from the luminal protein breakdown. In addition, several anticancer, antimicrobial and antiviral drugs are taken up from the intestinal lumen via PEPT1 and therefore PEPT1 is a target for efficient drug delivery via prodrug approaches. Thus, understanding PEPT1 gene regulation is not only of importance for dietary adaptation but also for drug treatment. Methods: In silico analysis of the Pept1 promoter was performed using MatInspector. Pept1 promoter constructs were generated and cotransfected with an Nrf2 expression plasmid. Caco-2 cells were stimulated with Nrf2 inducers followed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Biological relevance was investigated using western blot analysis and transport activity assays. Results: Reporter gene assays showed transcriptional activation of the Pept1 promoter in response to Nrf2 overexpression. EMSA as well as ChIP analysis validated Nrf2 binding to the ARE located closest to the start codon (Pept1-ARE1). Induction of the Nrf2 pathway resulted in increased endogenous PEPT1 protein abundance as well as transport activity. Moreover, we demonstrate that also the induction of autophagy by MG132 resulted in elevated Nrf2 binding to Pept1-ARE1 and increased PEPT1 protein expression. Conclusion: In summary, we identified a biologically active Nrf2 binding site within the Pept1 promoter which links Pept1 to the cellular defense program activated by Nrf2. General significance: This study identifies Pept1 as an inducible target gene of the Nrf2 pathway. [Copyright &y& Elsevier]

Abstract: Activation of the aryl hydrocarbon receptor (AhR) ultimately leads to the induction of the carcinogen-activating enzyme cytochrome P450 1A1 (CYP1A1), and activation of the nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) in addition to the AhR pathway induces the expression of the NADP(H):quinone oxidoreductase (NQO1). Therefore, the aim of this study was to examine the effect of As(III) pentavalent metabolites, MMA(V), DMA(V), and TMA(V), on AhR and Nrf2 activation and on the expression of their prototypical downstream targets CYP1A1 and NQO1, respectively. Our results showed that treatment of HepG2 cells with MMA(V), DMA(V), or TMA(V) in the absence and presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin or sulforaphane significantly induced both CYP1A1 and NQO1 at the mRNA, protein, and catalytic activity levels. Furthermore, these metabolites increased the AhR-dependent XRE-driven and the Nrf2-dependent ARE-driven luciferase reporter activities, which coincided with increased nuclear accumulation of both transcription factors. However, none of these metabolites were shown to be AhR ligands. The induction of CYP1A1 by these metabolites seems to be ligand-independent, possibly through a decrease in HSP90 protein expression levels. The metabolites also increased ROS production, which was significantly higher than that produced by As(III). Upon knockdown of AhR and Nrf2 the MMA(V)-, DMA(V)-, and TMA(V)-mediated induction of both CYP1A1 and NQO1 proteins was significantly decreased. In conclusion, this study demonstrates for the first time that methylated pentavalent arsenic metabolites are bifunctional inducers, as they increase CYP1A1 by activating the AhR/XRE signaling pathway and they increase NQO1 by activating the Nrf2/ARE signaling pathway in addition to the AhR/XRE pathway. [Copyright &y& Elsevier]

Abstract: The basic leucine zipper transcription factor Nrf2 has emerged as a master regulator of intracellular redox homeostasis by controlling the expression of a battery of redox-balancing antioxidants and phase II detoxification enzymes. Under oxidative stress conditions, Nrf2 is induced at the protein level through redox-sensitive modifications on critical cysteine residues in Keap1, a component of an E3 ubiquitin ligase complex that targets Nrf2 for proteasomal degradation. Poly(ADP-ribose) polymerase-1 (PARP-1) is historically known to function in DNA damage detection and repair; however, recently PARP-1 has been shown to play an important role in other biochemical activities, such as DNA methylation and imprinting, insulator activity, chromosome organization, and transcriptional regulation. The exact role of PARP-1 in transcription modulation and the underlying mechanisms remain poorly defined. In this study, we report that PARP-1 forms complexes with the antioxidant response element (ARE) within the promoter region of Nrf2 target genes and upregulates the transcriptional activity of Nrf2. Interestingly, PARP-1 neither physically interacts with Nrf2 nor promotes the expression of Nrf2. In addition, PARP-1 does not target Nrf2 for poly(ADP-ribosyl)ation. Instead, PARP-1 interacts directly with small Maf proteins and the ARE of Nrf2 target genes, which augments ARE-specific DNA-binding of Nrf2 and enhances the transcription of Nrf2 target genes. Collectively, these results suggest that PARP-1 serves as a transcriptional coactivator, upregulating the transcriptional activity of Nrf2 by enhancing the interaction among Nrf2, MafG, and the ARE. [Copyright &y& Elsevier]

Abstract: Background: Environmental factors such as solar ultraviolet (UV) radiation and other external aggressors provide an oxidative challenge that is detrimental to skin health. The levels of endogenous antioxidants decrease with age, thus resulting in less protection and a greater potential for skin damage. The NF-E2-related factor-2 (Nrf2) – antioxidant response element (ARE) pathway is a primary defense mechanism against oxidative stress, and induces the expression of antioxidant, detoxification and repair genes. Activation of ARE-Nrf2 can help restore oxidative homeostasis of the skin and play a role in inflammatory response and DNA repair mechanisms. Objective: To evaluate the role of a purified parthenolide-depleted Feverfew (PD-Feverfew) extract on the ARE-Nrf2 pathway and DNA repair in skin cells. Methods: These studies were undertaken in primary human keratinocytes or KB cells using Luciferase Promoter assay, siRNA transfection studies, Western blot analyses, Immunofluorescence microscopy, comet assay and quantitative real-time PCR. Results: PD-Feverfew was found to induce Nrf2 nuclear translocation and to increase ARE activity in a dose dependent manner. Furthermore, knockdown of Nrf2 resulted in suppression of PD-Feverfew-induced ARE activity. PD-Feverfew was also found to induce phosphorylation of Akt, a kinase downstream of PI3K. Inhibition of PI3K via pre-treatment with the selective pharmacological inhibitor, LY294002, abolished PD-Feverfew-induced Nrf2/ARE activation. PD-Feverfew also reduced UV-induced DNA damage in a PI3K and Nrf2-dependent manner. Conclusions: Therefore, by increasing endogenous defense mechanisms and aid in DNA repair of damaged skin cells via activation of a PI3K-dependent Nrf2/ARE pathway, PD-Feverfew may help protect the skin from numerous environmental aggressors. [Copyright &y& Elsevier]

Highlights: [•] A novel cryptoquinone derivative inhibited neuronal cell death with a very broad of safety zone. [•] They activated ARE, induced HO-1 and NQO1 proteins and increased GSH. [•] The activation of phase 2 enzymes may, at least in part, have mediated the inhibition of neuronal cell death. [ABSTRACT FROM AUTHOR]

β-Lapachone is a phytochemotherapeutic originally isolated from Lapacho tree whose extract has been used medicinally for centuries. It is well known that NAD(P)H:quinone oxidoreductase (NQO1) activity is the principal determinant of β-Lapachone cytotoxicity. As NQO1 is overexpressed in most common carcinomas, recent investigations suggest its potential application against cancer. Photodynamic therapy (PDT) is a clinically approved and rapidly developing cancer treatment. PDT involves the administration of photosensitizer (PS) followed by local illumination with visible light of specific wavelength. In the presence of oxygen molecules, the light illumination of PS can lead to a series of photochemical reactions and consequently the generation of cytotoxic reactive oxygen species (ROS). It has been reported that β-Lapachone synergistically interacts with ionizing radiation, hyperthermia and cisplatin and that the sensitivity of cells to β-Lapachone is closely related to the activity of NQO1. So, the present study aimed to investigate the feasibility of PDT to increase the anticancer effect of β-Lapachone by up-regulating NQO1 expression on breast cancer MCF-7c3 cells. NQO1 expression was evaluated by Western blot analysis at different times after PDT using ME-ALA as PS. The cytotoxicity of the photodynamic treatment and β-Lapachone alone or in combination was determined by MTT assay and the combination index (CI)-isobologram method and the dose reduction index (DRI) analysis were used to assess the effect of drug combinations. Our studies for the first time demonstrated that the expression of NQO1 is induced 24h after photodynamic treatment. The sensitivity of cancer cells to β-Lapachone treatment increased 24h after PDT and a synergistic inhibitory effect on MCF-7c3 cells was showed. Taken together, these results lead us to conclude that the synergistic interaction between β-Lapachone and PDT in killing cells was consistent with the up-regulation of NQO1. The combination of β-Lapachone and PDT is a potentially promising modality for the treatment of cancer. [ABSTRACT FROM AUTHOR]

Abstract: When exposed to electrophiles, human colorectal cancer cells (HCT116) counteract oxidative stress through activating NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. To identify new activators, luciferase reporter gene assay was used to screen in-house database of our laboratory, leading to a novel α-pyrone compound 1 as a hit. 2 with 2-fluoro phenyl group exhibited the strongest ARE inductive activity in the first round structure–activity relationship (SAR) study. Biological studies showed the compound induced nuclear translocation of Nrf2 preceded by phosphorylation of ERK1/2. The data encouraged us to use 2 as lead and 20 derivatives were synthesized to discuss a more detailed SAR, leading to a more potent compound 9, which can be the starting compound for further modification. [Copyright &y& Elsevier]

Abstract: Objective: 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) has pleiotropic and beneficial effects on metabolic disorders. However, the effects of AICAR on low density lipoprotein (LDL) metabolism are poorly understood. Methods and results: AICAR induces increased LDLR mRNA levels and increased LDLR protein production in hepatocytes. The AICAR-dependent LDLR mRNA increase was partially mediated by mRNA stabilization in an extracellular signal-regulated kinase1/2 (ERK1/2)-dependent manner, but not by the AMP-activated protein kinase (AMPK) activation. Reporter assays using a variety of constructs harboring the 3′-untranslated region (UTR) of human LDLR mRNA revealed that the most upstream AU-rich element (ARE) was critical for these AICAR effects. Using UV cross-linking assays, we found increased binding of three cytoplasmic proteins to this ARE region in response to AICAR and identified a 35-kDa protein as Human antigen R (HuR). Blocking ERK signaling pathway activation resulted in attenuated HuR binding. Silencing HuR expression by RNA interference hindered AICAR-induced LDLR mRNA stability, whereas its overexpression stabilized this mRNA. Conclusions: AICAR-dependent LDLR mRNA stabilization is mediated, at least in part, by HuR binding to the ARE1 region. Given that AICAR enhanced LDL uptake in hepatocytes, our findings warrant further studies using animal models to develop a novel LDL-cholesterol lowering agent as a possible strategy to treat atherosclerosis-related cardiovascular diseases. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Baicalein is a flavone originally isolated from the roots of traditional Chinese medicinal herb, Scutellaria baicalensis, which has been proved as a promising chemopreventive compound for many chronic human diseases. Aim of the study: The present study aimed to clarify the molecular mechanism targeted by baicalein. Materials and methods: Gene expression profiling of HepG2 cells treated with baicalein was carried out, using the Affymetrix 42K oligonucleotide microarray in the present study. Microarray data analyzed by Ingenuity Pathway Analysis (IPA), further study performed by real time PCR, reporter gene assay, and Western blot. Results: Among total 42K gene probes, baicalein treatment up-regulated the signals of 440 gene probes (1.04% of total gene probes) and down-regulated signals of 254 gene probes (0.6% of total gene probes) by ≥2-fold. These genes were categorized into 35 groups and hit for biological processes, molecular functions, and signaling pathways. The network and pathway analyses of these data further revealed that an Nrf2 (nuclear factor-erythroid 2 p45-related factor 2)-mediated ARE (antioxidant response element) pathway is involved in baicalein-induced gene expression of hepatic metabolic enzymes. The representative enzymes involved in Nrf2/ARE pathway were further confirmed at mRNA level by real time PCR and at protein level by Western blot analysis. Moreover, the ARE-reporter gene assay demonstrated that baicalein stimulated Nrf2-mediated ARE transactivation. Conclusions: Our results provide a comprehensive data for understanding the hepatic metabolism, bioactive role and the molecular mechanisms of baicalein. [Copyright &y& Elsevier]

Abstract: Endosulfan is an organochlorine insecticide and has been implicated in neurotoxicity, hepatotoxicity, immunosuppression and teratogenicity. However, the molecular mechanism of endosulfan toxicity is not yet clear. Recent studies demonstrated that oxidative stress induced by endosulfan is involved in its toxicity and accumulating evidence suggests that endosulfan can modulate the activities of stress-responsive signal transduction pathways including extracellular signal regulated kinases (ERK) 1/2. However, none of the previous studies investigated the ability of endosulfan to modulate activating protein-1 (AP-1) binding and antioxidant response element (ARE)-mediated transcription as an underlying mechanism of endosulfan toxicity. In this report, we show that treatment of HepG2 cells with endosulfan significantly increased oxidative stress-responsive transcription via AP-1 activation. In addition, endosulfan-induced transcription was enhanced in cells depleted of glutathione by buthionine sulfoximine (BSO) treatment. Exposure to endosulfan resulted in a significant increase in the activities of MAPKs, ERK1/2 and p38. Endosulfan-induced increases in enzymatic activities of these MAPKs were consistent with MAPK phosphorylation. Endosulfan exposure also caused an increase in c-Jun phosphorylation. These results suggest a model for endosulfan toxicity in which endosulfan increases ERK1/2 and p38 activities and these activated MAPKs then increase c-Jun phosphorylation. Phosphorylated c-Jun, in turn, increases AP-1 activity, which results in activation of ARE-mediated transcription. [Copyright &y& Elsevier]

Abstract: Prostatic secretory protein 94 (PSP94) is a prostatic protein found in both humans and rodents. As with other prostatic proteins, expression of this protein is regulated by androgens. In order to understand the androgen-responsive transcriptional regulation mechanisms involved, the present study aimed to identify and characterize the promoter activity of the gene. The 5′flanking (5′f) region of mouse PSP94 (mPSP94) gene was cloned and introduced into a vector upstream of the luciferase reporter gene. A Chinese hamster ovarian cell line, CHO, and a human prostate adenocarcinoma cell line, LNCaP, were transiently transfected with our reporter constructs along with an androgen receptor expression vector, and treated with dihydrotestosterone. Reporter gene assay revealed that the 5′f region of mPSP94 gene was indeed responsible for the androgen-dependent transcription. Subsequent deletion and mutation analysis indicated that the androgen responsive element (ARE)-like sequence at position −93 from the transcription start site was primarily responsible for androgen dependency. Interestingly, when estrogen receptor (ER) α was co-transfected, the androgen-dependent transcription was substantially increased. However, ERα-dependent enhancement of androgen responses was not observed when estrogen responsive element (ERE)-like motifs of the promoter region were deleted. Administration of estrogen did not influence the enhancement associated with ERα, although an anti-estrogen suppressed such effects. Collectively, these data suggest that the androgen-dependent transcription of the mPSP94 gene was co-regulated/modulated by the presence of ERα via ERE-like motifs. [Copyright &y& Elsevier]

Abstract: Inducible Keap1/Nrf2/ARE pathway determines the ability of multicellular organisms to adapt to conditions of stress caused by oxidants and electrophiles through upregulating proteins with versatile cytoprotective functions. Para- and ortho-hydroquinones were among the first identified small-molecule inducers of this pathway. Their oxidative lability strongly suggested that the electrophilic quinone metabolites, and not the hydroquinones themselves, were the ultimate inducers. Molecular orbital calculations re-enforced this notion by showing linear correlations between inducer potency and: (i) the ability of diphenols to release electrons, and (ii) the electron affinity of their corresponding quinones. Consequently, a two-step mechanism was proposed which involves oxidation of the diphenols to their corresponding quinone derivatives, followed by modification of specific cysteine residues of the sensor protein Keap1. Our finding that Cu2+, as well as other transition metals, enhanced induction by oxidizable diphenols provided a rationale to test this hypothesis. We found that hypoxia inhibits the potentiation of diphenolic inducer activity afforded by copper as oxygen is required to oxidize Cu+ and regenerate Cu2+. In the stably transfected AREc32 reporter cell line, exposure to 2-tert-butyl-1,4-hydroquinone (tBHQ) for 30min induced ARE-luciferase (measured 24h later) only in the presence of copper (Cu2+ or Cu+), whereas induction by tert-butyl-1,4-quinone (tBQ) was copper-independent. tBQ, but not tBHQ, reacts with cysteine residues of Keap1. Other para- and ortho-hydroquinones, such as catechol estrogens, dopamine, and l-DOPA, also induce ARE-driven transcription in a Cu2+–dependent manner. Thus, based on theoretical and experimental evidence, the oxidation of para- and ortho-hydroquinones to their corresponding electrophilic quinones is a requisite step for the activation of the Keap1/Nrf2/ARE pathway. [Copyright &y& Elsevier]

Abstract: Reactive oxygen species (ROS) generated by vascular endothelial and smooth muscle cells contribute to the development and progression of vascular diseases. We have recently shown that hyperoxia enhances NADPH oxidase 4 (Nox4) expression, which regulates lung endothelial cell migration and angiogenesis. Regulation of Nox4 in the vasculature is poorly understood. The objective of this study was to identify the transcriptional factor(s) involved in regulation of endothelial Nox4. We found that hyperoxia-induced Nox4 expression was markedly reduced in Nrf2 −/− mice, compared to Nrf2 +/+ mice. Exposure of human lung microvascular endothelial cells (HLMVECs) to hyperoxia stimulated Nrf2 translocation from the cytoplasm to the nucleus and increased Nox4 expression. Knockdown of Nrf2 expression using an siRNA approach attenuated basal Nox4 expression; however, it enhanced superoxide/ROS generation under both normoxia and hyperoxia. In silico analysis revealed the presence of at least three consensus sequences for the antioxidant response element (ARE) in the promoter region of Nox4. In transient transfections, hyperoxia stimulated Nox4 promoter activity in HLMVECs, and deletion of the −438 to −458 and −619 to −636 sequences markedly reduced hyperoxia-stimulated Nox4 promoter activation. ChIP analysis revealed an enhanced recruitment of Nrf2 to the endogenous Nox4 promoter spanning these two AREs after hyperoxic insult. Collectively, these results demonstrate, for the first time, a novel role for Nrf2 in regulating hyperoxia-induced Nox4 transcription via AREs in lung endothelium. [Copyright &y& Elsevier]

Abstract: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor regulating the expression of a battery of cytoprotective genes. Constitutive Nrf2 activation in many tumors enhances cell survival and resistance to anticancer drugs. Using a cell-based ARE-reporter assay we discovered that the flavonoid luteolin is a potent Nrf2 inhibitor. Luteolin inhibited ARE-driven gene expression redox-independently. In non-small-cell lung cancer A549 cells, which possess constitutively active Nrf2, luteolin elicited a dramatic reduction in Nrf2 at both the mRNA and the protein levels, leading to decreased Nrf2 binding to AREs, down-regulation of ARE-driven genes, and depletion of reduced glutathione. After transcription was blocked with actinomycin D, 1μM luteolin decreased the Nrf2 mRNA level by 34% in 30min, indicating its role in accelerating Nrf2 mRNA turnover. At physiological concentrations, luteolin significantly sensitized A549 cells to the anticancer drugs oxaliplatin, bleomycin, and doxorubicin. However, knockdown of Nrf2 using siRNA essentially abolished the induced sensitivity by the flavonoid, implying the importance of inhibiting Nrf2 for its activity. Our study demonstrates that an Nrf2 inhibitor can enhance the responsiveness of cancer cells to chemotherapeutic drugs and indicates the potential application of luteolin as a natural sensitizer in chemotherapy. [Copyright &y& Elsevier]

Abstract: Carbonyl reductase/20β-hydroxysteroid dehydrogenase (CR/20β-HSD) serves both as a key enzyme in the gonadal synthesis of maturing-inducing hormone in salmonids, and as an enzyme protecting against certain reactive oxygen species. We have previously shown that mRNA of the hepatic CR/20β-HSD B isoform is increased in rainbow trout caged downstream from a Swedish sewage treatment plant. Here, we report an increase of both the A as well as B form in fish kept downstream from a second sewage treatment plant. The two mRNAs were also induced in fish hepatoma cells in vitro after exposure to effluent extract. This indicates that the effects observed in vivo could be a direct effect on the liver, i.e. the mRNA induction does not require a signal from any other organ. When fish were exposed in vivo to several effluents treated with more advanced methods (ozone, moving bed biofilm reactor or membrane bioreactor) the expression of hepatic mRNA CR/20β-HSD A and B was significantly reduced. Their abundance did not parallel the reduction of estrogen-responsive transcripts, in agreement with our previous observations that ethinylestradiol is not a potent inducer. Treatment with norethisterone, methyltestosterone or hydrocortisone in vivo did not induce the hepatic CR/20β-HSD A and B mRNA expression. In contrast, both isoforms were markedly induced by the aryl hydrocarbon receptor agonist β-naphthoflavone as well as by the pro-oxidant herbicide paraquat. We hypothesize that the induction of CR/20β-HSD A and B by sewage effluents could be due to anthropogenic contaminants stimulating the aryl hydrocarbon receptor and/or causing oxidative stress. [Copyright &y& Elsevier]

Abstract: This study was conducted to investigate the effects of mechanical stress, particularly cyclic strain, on proinflammatory cytokines as well as antioxidant properties and their interactions with cellular defense systems in human dental pulp (HDP) cells. Exposure of HDP cells to mechanical strain induced inflammatory cytokines such as interleukin-1β, tumor necrosis factor–α, and interleukin-6, as well as antioxidant genes such as heme oxygenase-1, superoxide dismutases, reduced nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1, and glutathione peroxidases. In addition, treatment with N-acetylcysteine, indomethacin, and heme oxygenase–1 inhibitors blocked reactive oxygen species production, antioxidant response element (ARE) gene expression, and Nrf2 accumulation that occurred in response to mechanical stress. These data demonstrate that mechanical strain activates inflammatory cytokines and oxidative stress, which then act in concert to induce the Nrf2-/ARE-mediated antioxidant enzymes. Therefore, we suggest that the activation of a compensatory adaptation or defense antioxidant system might represent a novel mechanism for protecting HDP cells against mechanical stress. [Copyright &y& Elsevier]

Abstract: Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. In this study, we found that the expressions of anti-oxidant proteins (γ-glutamylcysteine ligase heavy chain (γ-GCLh), heme oxygenase-1, thioredoxin and peroxiredoxin1) in TAM-resistant MCF-7 (TAMR-MCF-7) cells were higher than control MCF-7 cells. Molecular analyses using antioxidant response element (ARE)-containing reporters and gel-shift supported the critical role of NF-E2-related factor2 (Nrf2)/ARE in the overexpression of antioxidant proteins in TAMR-MCF-7 cells. Intracellular peroxide production was significantly decreased in TAMR-MCF-7 cells and TAM resistance was partially reversed by Nrf2 siRNA. The basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase were increased in the TAMR-MCF-7 cells and the inhibition of ERK significantly decreased the activity of minimal ARE reporter and γ-GCLh protein expression in TAMR-MCF-7 cells. However, exposure of TAMR-MCF-7 cells to 17-β-estradiol or ICI-182,780 did not significantly change γ-GCLh expression. These results suggest that the persistent activation of Nrf2/ARE is critical for the enhanced expression of anti-oxidant proteins in TAM-resistant breast cancer cells and the pathway of ERK, but not of estrogen receptor signaling are involved in the up-regulation of Nrf2/ARE. [Copyright &y& Elsevier]

Abstract: Constitutively activated NF-κB occurs in many inflammatory and tumor tissues. Does it interfere with anti-inflammatory or anti-tumor signaling pathway? Here, we report that NF-κB p65 subunit repressed the Nrf2-antioxidant response element (ARE) pathway at transcriptional level. In the cells where NF-κB and Nrf2 were simultaneously activated, p65 unidirectionally antagonized the transcriptional activity of Nrf2. In the p65-overexpressing cells, the ARE-dependent expression of heme oxygenase-1 was strongly suppressed. However, p65 inhibited the ARE-driven gene transcription in a way that was independent of its own transcriptional activity. Two mechanisms were found to coordinate the p65-mediated repression of ARE: (1) p65 selectively deprives CREB binding protein (CBP) from Nrf2 by competitive interaction with the CH1-KIX domain of CBP, which results in inactivation of Nrf2. The inactivation depends on PKA catalytic subunit-mediated phosphorylation of p65 at S276. (2) p65 promotes recruitment of histone deacetylase 3 (HDAC3), the corepressor, to ARE by facilitating the interaction of HDAC3 with either CBP or MafK, leading to local histone hypoacetylation. This investigation revealed the participation of NF-κB p65 in the negative regulation of Nrf2-ARE signaling, and might provide a new insight into a possible role of NF-κB in suppressing the expression of anti-inflammatory or anti-tumor genes. [Copyright &y& Elsevier]

Abstract: Curcumin (diferuloymethane), a yellow colouring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), has been reported to possess anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic activities. Curcumin exerts its chemoprotective and chemopreventive effects via multiple mechanisms. It has been reported to induce expression of the antioxidant enzymes in various cell lines. Heme oxygenase-1 (HO-1) is an important antioxidant enzyme that plays a pivotal role in cytoprotection against noxious stimuli of both endogenous and exogenous origin. In the present study, we found that oral administration of curcumin at 200mg/kg dose for four consecutive days not only protected against dimethylnitrosamine (DMN)-induced hepatic injury, but also resulted in more than three-fold induction of HO-1 protein expression as well as activity in rat liver. Inhibition of HO-1 activity by zinc protoporphyrin-IX abrogated the hepatoprotective effect of curcumin against DMN toxicity. NF-E2-related factor 2 (Nrf2) plays a role in the cellular protection against oxidative stress through antioxidant response element (ARE)-directed induction of several phase-2 detoxifying and antioxidant enzymes including HO-1. Curcumin administration resulted in enhanced nuclear translocation and ARE-binding of Nrf2. Taken together, these findings suggest that curcumin protects against DMN-induced hepatotoxicity, at least in part, through ARE-driven induction of HO-1 expression. [Copyright &y& Elsevier]

Abstract: Interleukin-1β (IL-1β) is the prototypic pro-inflammatory cytokine. All the biological effects of IL-1β are mediated through interaction with type 1 IL-1 receptor (IL-1RI), whereas another receptor, called type 2 IL-1R (IL-1RII), lacks an intracellular signalling domain and acts as a decoy receptor that down-regulates responses to IL-1β. Although both receptors are present in bony fish, their expression and biological role in the regulation of IL-1β activity in non-mammalian vertebrates remain to be established. In this study, a homologue of mammalian IL-1RII was isolated and characterized in the gilthead seabream (Sparus aurata). The seabream IL-1RII harboured two Ig-like domains in its extracellular region and a short cytoplasmic tail lacking a signalling domain. The seabream IL-1RII cDNA showed an unexpectedly long 3′UTR compared with that from other species and contained three ATTTA instability motifs, which seem to be responsible for its relatively short half-life (less than 2h). The expression of seabream IL-1RII was dramatically up-regulated after infection with Vibrio anguillarum in all the immune tissues examined and was even more strongly induced than the IL-1β gene in the head kidney, spleen and liver. Strikingly, the mRNA levels of IL-1RII were 15-fold higher than those of IL-1β in the liver, suggesting a role for this organ in the neutralization of IL-1β leaking into the systemic circulation from the sites of inflammation. In vitro, bacterial DNA and flagellin increased the mRNA levels of IL-1RII in macrophages, while only flagellin was able to weakly induce its expression in acidophilic granulocytes. Finally, the seabream IL-1RII was localized in the plasma membrane when expressed in HEK293 cells and was able to bind IL-1β. [Copyright &y& Elsevier]

Abstract: The overproduction of cytokines, such us interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα), contributes to the pathological complications observed in many inflammatory diseases caused by bacterial endotoxins. The synthesis of these cytokines is tightly regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of gene expression depends on specific cis-acting sequences and trans-acting factors. Thus, the presence of adenylate- and uridylate-rich (AU-rich) elements (AREs) has been described in the 3′-untranslated regions (UTRs) of many unstable mammalian mRNAs. Although, it represents the most widespread, phylogenetically conserved and efficient determinant of mRNA stability among those so far characterized in mammalian cells, no studies are available on the functional relevance of this sequence in non-mammalian vertebrates. In this contribution, we study the enzymatic activity of various luciferase reporter constructs, containing or lacking the 3′UTR of IL-1β and TNFα from different fish species, and report the finding that bony fish AREs are able to decrease luciferase activity but are less potent than their mammalian counterparts. Surprisingly, the 3′UTR of the IL-1β from the cartilaginous fish small spotted catshark had the greatest ability to decrease luciferase activity. Lastly, the functional significance of the above was confirmed by measuring the half-life of IL-1β and TNFα mRNAs in gilthead seabream leukocytes by blocking transcription with actinomycin D. Both cytokine mRNAs were unstable with an estimated half-life of about 45min in control and activated cells. [Copyright &y& Elsevier]

Abstract: Oxidant/antioxidant imbalance, a major cause of cell damage, is the hallmark for lung inflammation. Glutathione (GSH), a ubiquitous tripeptide thiol, is a vital intra- and extra-cellular protective antioxidant against oxidative stress, which plays a key role in the control of signaling and pro-inflammatory processes in the lungs. The rate-limiting enzyme in GSH synthesis is glutamylcysteine ligase (GCL). GSH is essential for development as GCL knock-out mouse died from apoptotic cell death. The promoter (5′-flanking) region of human GCL is regulated by activator protein-1 (AP-1) and antioxidant response element (ARE), and are modulated by oxidants, phenolic antioxidants, growth factors, inflammatory and anti-inflammatory agents in various cells. Recent evidences have indicated that Nrf2 protein, which binds to the erythroid transcription factor (NF-E2) binding sites, and its interaction with other oncoproteins such as c-Jun, Jun D, Fra1 and Maf play a key role in the regulation of GCL. Alterations in alveolar and lung GSH metabolism are widely recognized as a central feature of many chronic inflammatory lung diseases. Knowledge of the mechanisms of GSH regulation could lead to the pharmacological manipulation of the production and/or gene transfer of this important antioxidant in lung inflammation and injury. This article describes the role of AP-1 and ARE in the regulation of cellular GSH biosynthesis and assesses the potential protective and therapeutic role of glutathione in oxidant-induced lung injury and inflammation. [Copyright &y& Elsevier]

Abstract: Polyphenols are wide variety of compounds that occur in fruits and vegetables, wine, tea, extra virgin olive oil, chocolate and other cocoa products. Several polyphenols have been demonstrated to have clear antioxidant properties in vitro, and many of their biological actions have been attributed to their intrinsic reducing capabilities. However, this concept appears now to be a simplistic way to conceive their activity. Evidence is indeed accumulating that polyphenols might exert several other specific biological effects that are as yet poorly understood. In this article we review the most recent data on the subject and describe the additional functions that polyphenols can have in biological systems, focusing on their effects on glutathione and its related enzymes. Experimental data indicate that polyhenols may offer an indirect protection by activating endogenous defense systems. Several lines of evidence suggest a tight connection between exogenous and endogenous antioxidants that appear to act in a coordinated fashion. It is reasonable to hypothesize that this is achieved, at least in part, through antioxidant responsive elements (AREs) present in the promoter regions of many of the genes inducible by oxidative and chemical stress. The latest studies strongly suggest that dietary polyphenols can stimulate antioxidant transcription and detoxification defense systems through ARE. [Copyright &y& Elsevier]

Abstract: Comparisons of the glandular kallikreins loci in human, mouse and rat revealed remarkable differences. For example, the mouse and the rat lack the genes encoding prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2). In contrast, the intergenic region between KLK1 and KLK15 is devoid of genes and spans only 1.5 kb in humans, but encompasses 23 KLK1-like genes spanning 290 kb in the mouse. To further elucidate the evolution of glandular kallikrein genes, we investigated the structure and organization of these genes in the cotton-top tamarin (Saguinus oedipus), a New World monkey. We conclude that this species has no PSA gene. Moreover, the ortholog of the hK2 gene is a pseudogene, as it contains several mutations that preclude formation of a functional serine protease. The expression of this gene was probably silenced by a 15-bp deletion observed in an androgen response element in the upstream promoter region. Replacing the deleted base pairs in vitro with nucleotides from the human counterpart dramatically restored the transcriptional activity to a level that even surpassed that of the human ortholog. We also determined the nucleotide sequence of KLK15 and the intergenic region between this gene and KLK1 in the cotton-top tamarin. The region between KLK1 and KLK15 is conserved between the cotton-top tamarin and humans, and there are no signs of the extension seen in the mouse. KLK15 appeared to be functional, thus, we predict that it generates a protease with specificity similar to that of the human ortholog. [Copyright &y& Elsevier]

Exposure of HepG2 cells to nonsteroidal anti-inflammatory drugs (i.e., indomethacin and ibuprofen; NSAIDs) as well as resveratrol, caused increased expression of the mRNAs coding for the catalytic (Gclc) and modifier (Gclm) subunits of the glutathione synthetic enzyme, γ-glutamylcysteine synthetase. In addition, indomethacin exposure increased intracellular glutathione content as well as inhibited glutathione depletion and cytotoxicity caused by diethyl maleate. Indomethacin-induced increases in the expression of γ-glutamylcysteine synthetase mRNA were preceded by increases in steady state levels of intracellular pro-oxidants and glutathione disulfide accumulation. Simultaneous incubation with the thiol antioxidant N-acetylcysteine (NAC) inhibited indomethacin-mediated increases in GCLC mRNA, suggesting that increases in GCLC message were triggered by changes in intracellular oxidation/reduction (redox) reactions. Indirect immunofluorescence using intact cells demonstrated that indomethacin induced the nuclear translocation of Nrf2, a transcription factor believed to regulate GCLC expression. Immunoprecipitation studies showed that indomethacin treatment also inhibited Nrf2 tethering to KIAA0132 (the human homolog of Keap1 accession #D50922), which is believed to be a negative regulator of Nrf2. Consistent with this idea, over-expression of Nrf2 increased GCLC reporter gene expression and over-expression of KIAA0132 inhibited GCLC reporter gene activity as well as inhibited indomethacin-induced increases in the expression of GCLC. Finally, simultaneous treatment with NAC inhibited both indomethacin-induced release of Nrf2 from KIAA0132 and indomethacin-induced nuclear translocation of Nrf2. These results demonstrate that NSAIDs and resveratrol cause increases in the expression of γ-glutamylcysteine synthetase mRNA and identify these agents as being capable of stimulating glutathione metabolism. These results also support the hypothesis that indomethacin-induced transcriptional activation of GCLC involves the redox-dependent release of KIAA0132 from Nrf2 followed by the nuclear translocation of Nrf2. [Copyright &y& Elsevier]

Fragile X-related protein-1 (FXR1) gene is highly amplified in patients with ovarian cancer, and this amplification is associated with increased expression of both FXR1 mRNA and protein. FXR1 expression directly associates with the survival and proliferation of cancer cells. Surface sensing of translation (SUnSET) assay demonstrates that FXR1 enhances the overall translation in cancer cells. Reverse-phase protein array (RPPA) reveals that cMYC is the key target of FXR1. Mechanistically, FXR1 binds to the AU-rich elements (ARE) present within the 3′ untranslated region (3′UTR) of cMYC and stabilizes its expression. In addition, the RGG domain in FXR1 interacts with eIF4A1 and eIF4E proteins. These two interactions of FXR1 result in the circularization of cMYC mRNA and facilitate the recruitment of eukaryotic translation initiation factors to the translation start site. In brief, we uncover a mechanism by which FXR1 promotes cMYC levels in cancer cells. [Display omitted] • CNVs of FXR1 associate with its expression in ovarian cancer • FXR1 promotes the survival and proliferation of ovarian cancer cells • FXR1 binds to AU-rich elements (ARE) within 3′UTR of cMYC • FXR1 promotes the recruitment of eIF4F complex to translation initiation site George et al. demonstrate that FXR1 binds to the AREs within the 3′UTR of MYC mRNA and improves its stability. The authors also show that the RGG domain of FXR1 interacts with eIF4A1 and eIF4E and facilitates recruitment of the eIF4F complex to translation initiation sites for cMYC translation. [ABSTRACT FROM AUTHOR]

This study was to evaluate the antifatigue effect of T. heterochaetus and explore the underlying mechanism of action. T. heterochaetus extract was treated to mice for 28 days. On the 28th day, after weight loaded swimming test. The levels of antioxidant enzymes and levels of pro- and anti-inflammatory cytokines in the liver and muscles of exercised mice were evaluated. mRNA and protein expression levels of Nrf2, SOD, HO-1, and Keap-1 were evaluated using RT-PCR and western blot analysis. The low (2.70 mg/0.5 ml/20 g) and medium (5.41 mg/0.5 ml/20 g) dose enhanced the activities of antioxidant enzymes like SOD, CAT and GPx in the liver and skeletal muscle thereby enhancing the antifatigue effect. The low and medium doses showed good anti-inflammatory effects by evaluating the levels of pro and anti-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-10 both in the liver and skeletal muscle. Furthermore, RT-PCR and western blot analysis showed increased expression of HO-1, SOD, Nrf2, and decreased expression of Keap-1 gene and proteins in liver and skeletal muscle of T. heterochaetus treated group mice. The current results indicate that T. heterochaetus exert the antifatigue effect through attenuating oxidative stress injury and inflammatory responses through the Nrf2/ARE-mediated signaling pathway. [Display omitted] • Hechong enhanced the activity of antioxidant enzymes SOD, CAT and GPx. • Hechong modulated the levels of TNF-α, IL-1β, IL-6, and IL-10. • Hechong increased expression of HO-1, SOD, Nrf2, and decreased expression of Keap-1. • Hechong exerted antifatigue effect through attenuating oxidative stress injury and inflammatory responses. • Hechong exerted antifatigue effect via Nrf2/ARE-mediated signaling pathway. [ABSTRACT FROM AUTHOR]