Dual roles of NRF2 in tumor prevention and progression: Possible implications in cancer treatment

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.     

Involvement of Nrf2 and Keap1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent peptides from soft-shelled turtle

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 HepG₂ 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.     

基于模式生物斑马鱼Nrf2的相关研究进展

Nrf2/ARE信号通路是大多数生物体内抗氧化应激反应、抵抗内外界刺激的关键通路,在抗炎症、免疫、抗肿瘤、抗凋亡、神经保护等方面起着重要的作用。斑马鱼作为一种常见的模式动物,广泛地应用于发育生物学、遗传学和毒理学等研究领域。研究表明转录因子NF_E2相关因子2(NF-E2-related factor 2,Nrf2)不仅在哺乳动物体内存在,也在斑马鱼体内存在并且高度保守,并在抗氧化应激反应中发挥着重要作用。本文通过对斑马鱼Nrf2的结构、生物学功能及其信号通路等方面的最新研究进行阐述,以期为Nrf2及其信号通路引发的相关疾病的预防和治疗提供新的思路。     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因马铃薯的耐氧化和耐盐性研究

高盐等逆境可以加剧植物体内活性氧的产生,进而引起植物细胞死亡。为开发抗逆境作物,以置于氧化诱导型启动子下定位于叶绿体的转铜/锌超氧化物歧化酶（Cu/ZnSOD）和抗坏血酸过氧化物酶基因(APX)马铃薯为材料,研究了其对MV和 NaCl所引起的氧化胁迫的耐受性。结果表明, MV胁迫下,转基因马铃薯叶片膜的相对电导率明显低于对照; NaCl胁迫下,其叶绿素含量高于对照。 在含NaCl 的培养基上,转基因幼苗生根率明显大于对照。另外,NaCl胁迫下转基因马铃薯叶片的SOD和APX酶活性显著高于对照,与其耐盐性的提高相一致。这些研究表明,转入Cu/ZnSOD和APX基因的马铃薯清除活性氧的能力增强,抗逆性得到提高。本实验采用氧化诱导型启动子调控下的SOD和APX两个基因协同作用,使外源基因只有在逆境胁迫时才特异性表达,增强转基因植株的抗逆效果,为培育抗逆经济作物开阔了思路。     

The effect of menadione-induced oxidative stress on the in vivo reactive oxygen species and antioxidant response system of Phanerochaete chrysosporium

The antioxidant response system of Phanerochaete chrysosporium against menadione-induced oxidative stress was investigated in this study. The superoxide anion radical levels in tested menadione-supplemented conditions generally decreased over the incubation period. The level of hydrogen peroxide and the activities of NAD(P)H oxidase, superoxide dismutase (SOD) and catalase (CAT) were higher than those in the controls at all incubation times. The highest NADH and NADPH oxidase activities were determined to be 4.9- and 5.0-fold higher than those in the control, respectively in cells exposed to 0.75 mM menadione. The SOD and CAT activities increased with increasing menadione, and their highest activities were 5.4- and 5.1-fold higher than those in the control, respectively. In 0.1–0.5 mM menadione exposed cells, the lipid peroxidation levels did not change significantly when compared to each other, except 8th hour of incubation (p > 0.01). Our result shows that although menadione induces the formation of reactive oxygen species, the antioxidant response system of P. Chrysosporium is able to negate menadione-induced oxidative stress up to relatively high menadione concentrations, as 0.75 mM. These results are important to determine the effects of menadione, as a medicine, on the antioxidant response system of eukaryotic models and the resulting level of damage.     

Antioxidant responses of Propylaea japonica (Coleoptera: Coccinellidae) exposed to high temperature stress

Temperature is one of the most important environmental factors, and is responsible for a variety of physiological stress responses in organisms. Induced thermal stress is associated with elevated reactive oxygen species (ROS) generation leading to oxidative damage. The ladybeetle, Propylaea japonica (Thunberg) (Coleoptera: Coccinellidae), is considered a successful natural enemy because of its tolerance to high temperatures in arid and semi-arid areas in China. In this study, we investigated the effect of high temperatures (35, 37, 39, 41 and 43 °C) on the survival and activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidases (POD), glutathione-S-transferases (GST), and total antioxidant capacity (TAC) as well as malondialdehyde (MDA) concentrations in P. japonica adults. The results indicated that P. japonica adults could not survive at 43 °C. CAT, GST and TAC were significantly increased when compared to the control (25 °C), and this played an important role in the process of antioxidant response to thermal stress. SOD and POD activity, as well as MDA, did not differ significantly at 35 and 37 °C compared to the control; however, there were increased levels of SOD, POD and MDA when the temperature was above 37 °C. These results suggest that thermal stress leads to oxidative stress and antioxidant enzymes play important roles in reducing oxidative damage in P. japonica adults. This study represents the first comprehensive report on the antioxidant defense system in predaceous coccinellids (the third trophic level). The findings provide useful information for predicting population dynamics and understanding the potential for P. japonica as a natural enemy to control pest insects under varied environmental conditions.     

Gene-Gun-Mediated Transfer of Reporter Genes to Somatic Zebrafish (Danio rerio) Tissues

We describe the use of gene-gun-mediated transfer of luciferase and green fluorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimization of DNA transfer parameters indicated highest overall luciferase expression in epidermis and dermis using 1-μm microcarriers and 1 μg of pCMVL plasmid DNA at a delivery pressure of 200 psi. Time course studies revealed luciferase activity peaking at 18 hours and decreasing to 30% of the maximum at day 8 after DNA transfer. Onset of reporter gene (GFP) expression was detected at 13 minutes after DNA delivery, and by 65 minutes approximately 100% of the cells in the target area exhibited GFP expression. No germline association or integration events were detected in a screen of approximately 250,000 zebrafish sperm cells by fluorescence in situ hybridization at 15 or 30 days after delivery of 1 μg of pCMVL DNA, suggesting incidental male germline integration should not be considered as a risk factor when using the biolistic DNA delivery parameters and target tissues described. Received August 20, 1999; accepted January 6, 2000.     

Antioxidant responses of the pest natural enemy Hylyphantes graminicola (Araneae: Linyphiidae) exposed to short-term heat stress

Temperature is a critical abiotic factor that causes physiological changes in arthropods. However, little is known about the effect of heat stress on the antioxidant responses of Araneae species. Hylyphantes graminicola is a dominant predator in many cropping systems in China. In the present study, the effect of short-term heat stress (36, 38, 40 or 42 °C) on the reactive oxygen species (ROS) levels, the activities of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], peroxidases [POD] and glutathione-S-transferases GST]), total antioxidant capacity (TAC), malondialdehyde (MDA) concentrations and survival of H. graminicola spiderlings and adults were investigated. The results showed that H. graminicola adults had a significantly higher survival rate compared to spiderlings at 40 °C. The heat stress increased ROS contents in H. graminicola. The SOD, CAT, POD and GST activities increased in spiderlings and adults under heat stress. These data suggest a defensive function for these enzymes in alleviating oxidative damage. Specifically, SOD plays a key role in reducing the high level of superoxide radicals in spiderlings and adults. Moreover, the POD and CAT capabilities for scavenging H₂O₂ in spiderlings were similar, and CAT may play a more important role than POD in scavenging H₂O₂ in adults at 42 °C. The spiderling TAC increased significantly at 40 and 42 °C, and the adult TAC was stable at 36–40 °C but decreased at 42 °C. These data suggest that TAC was insufficient in H. graminicola adults under more severe stress conditions. These results further our understanding of the physiological response of Araneae species exposed to heat stress.     

miR-22心肌特异转基因斑马鱼的构建及心肌肥厚的评估

目的:构建miR-22心肌特异转基因斑马鱼系,在体评估miR-22对于心肌肥厚的作用。方法:构建pTol2-CMLC2-miR-22-IRES-EGFP表达载体。通过显微注射的方法将tol2重组质粒于一细胞期注射入斑马鱼受精卵胚胎中,荧光筛选获得心肌特异表达绿色荧光的斑马鱼胚胎,并稳定表达传代。然后对稳定传代的成年斑马鱼心脏进行心肌肥厚及心功能的检测。结果:成功建立了miR-22心肌特异转基因斑马鱼系,通过定量PCR确定心肌中miR-22表达升高,荧光显微镜观察发现斑马鱼心肌出现绿色荧光。miR-22心脏特异过表达的转基因鱼系的成年鱼与野生对照组相比,出现了心肌肥厚的现象,心肌肥厚分子标志物nppa、myh7明显升高。斑马鱼心脏病理切片结果同样显示出miR-22心肌特异转基因斑马鱼出现了心肌肥厚的现象。结论:成功构建了miR-22心肌特异转基因斑马鱼,为研究心肌中miR-22的生物学功能提供了重要的工具,并证明miR-22心脏特异过表达会引起斑马鱼心肌肥厚。     

Reexamination of the electrophile response element sequences and context reveals a lack of consensus in gene function

The electrophile response element (EpRE) is essential for regulation of many genes involved in protection against toxic agents. Putative EpRE core sequences (TGAnnnnGC) are localized in 5′-flanking regions (5′-UTR) of these genes but specificity of the internal bases and whether location affects function has not been refined. The catalytic subunit of human glutamate cysteine ligase (GCLC) gene is well documented to be under EpRE regulation and four sequences having an EpRE “consensus” sequence were reported with only one (EpRE 4) responsive to electrophiles. Using GCLC as a model, we asked whether the internal variable or flanking nucleotides and the location of the sequence were required for functional activity in response to 4-hydroxenonenal (HNE). We found that thirteen putative EpRE core sequences (TGAnnnnGC) were localized in 5′-UTR of GCLC and confirmed that EpRE 4 showed both constitutive and HNE-inducible activity. Four other sequences exhibited only constitutive activity while other putative EpREs demonstrated no activity. Nucleotide mutagenesis demonstrated specific requirements for internal and flanking nucleotides that were specific for the electrophilic response and that a TRE-like sequence within EpRE was essential for basal (non-electrophile-dependent) activity. Furthermore, EpRE 4 relocated to positions of other putative EpREs maintained activity but moving other EpREs to the EpRE 4 location did not. Thus in GCLC, specific flanking and internal nucleotides within EpRE were far more important for function than previously described while location did not influence activity. These two findings bring into question the meaning of the phrase, “consensus sequence” for this important cis element.     

NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector

NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) is a widely-distributed FAD-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes, at rates that are comparable with NADH or NADPH. These reductions depress quinone levels and thereby minimize opportunities for generation of reactive oxygen intermediates by redox cycling, and for depletion of intracellular thiol pools. NQO1 is a highly-inducible enzyme that is regulated by the Keap1/Nrf2/ARE pathway. Evidence for the importance of the antioxidant functions of NQO1 in combating oxidative stress is provided by demonstrations that induction of NQO1 levels or their depletion (knockout, or knockdown) are associated with decreased and increased susceptibilities to oxidative stress, respectively. Furthermore, benzene genotoxicity is markedly enhanced when NQO1 activity is compromised. Not surprisingly, human polymorphisms that suppress NQO1 activities are associated with increased predisposition to disease. Recent studies have uncovered protective roles for NQO1 that apparently are unrelated to its enzymatic activities. NQO1 binds to and thereby stabilizes the important tumor suppressor p53 against proteasomal degradation. Indeed, NQO1 appears to regulate the degradative fate of other proteins. These findings suggest that NQO1 may exercise a selective “gatekeeping” role in regulating the proteasomal degradation of specific proteins, thereby broadening the cytoprotective role of NQO1 far beyond its highly effective antioxidant functions.     

Methods for Detection of Conjugative Plasmid Transfer in Aquatic Environments

Donor and recipient counter selection was evaluated by selecting bacteria that received plasmid RP4 by conjugation on filters and in lake water microcosms. Three counter selection systems were compared; (i) Use of antibiotic-resistant recipients, (ii) use of an auxotrophic donor, and (iii) use of a donor with chromosomal suicide genes. Transfer efficiencies of transconjugants per recipient obtained with the three different counter selection systems in filter-matings were not significantly different. Some nalidixic acid-resistant recipients became partly sensitive to nalidixic acid after receiving the plasmid. Use of an auxotrophic donor was a feasible and easy way to recover indigenous transconjugants. A strain with two copies of the suicide gene gef was successfully eliminated in filter-matings, but elimination of the donor in microcosms by induction of the suicide genes did not succeed. Thus, this counter selection system was not usable in microcosm experiments. Received: 3 March 1998 / Accepted: 15 May 1998     

利用斑马鱼胚胎快速鉴定真核质粒中目的基因表达的研究

目的建立利用斑马鱼胚胎快速鉴定真核质粒中目的基因表达的实验体系。方法选20枚斑马鱼受精卵,在显微镜下每隔1h记录胚胎的发育情况。另选250枚单细胞期斑马鱼胚胎,平均分成5组,一组胚胎作为对照,剩余4组分别向胚胎的单细胞内注射pEGFP-N1(真核表达质粒)、pCMV-DsRed-Express2(真核表达质粒)、pET28-GFP(原核表达质粒)、pET28-RFP(原核表达质粒)质粒,在不同时间点连续观察绿色荧光及红色荧光的表达情况。另选600枚单细胞期斑马鱼胚胎,平均分成3组,一组胚胎作为对照,一组向胚胎单细胞内注射pEGFP-N1质粒,另外一组向胚胎单细胞内注射pEGFP-N1-MUC1外源基因融合重组质粒,注射4h后在荧光显微镜下观察绿色荧光的表达情况,并用RT-PCR的方法检测目的基因MUC1mRNA的转录情况。结果注射pEGFP-N1、pCMV-DsRed-Express2真核表达质粒的胚胎,注射4h后分别观察到很强的绿色荧光及红色荧光;注射pET28-GFP、pET28-RFP原核表达质粒的胚胎,10h内都未观察到绿色荧光及红色荧光;注射pEGFP-N1-MUC1外源基因融合质粒,注射4h后同样...     

Inhibition of monoterpene biosynthesis accelerates oxidative stress and leads to enhancement of antioxidant defenses in leaves of rubber tree (Hevea brasiliensis)

This paper mainly studies the possible antioxidant of monoterpene and effects of its absence on other antioxidant defense. The leaves of rubber tree (Hevea brasiliensis) were fed with fosmidomycin through transpiration stream, in the dark, at room temperature for 2 h, and were then exposed to bright illumination (1,500 μmol m⁻² s⁻¹) and moderately high temperature (30°C) for 1 h. The results showed that monoterpene biosynthesis in leaves was considerably inhibited by fosmidomycin, and the elevated levels of both hydrogen peroxide and malondialdehyde were observed in the leaves fed with fosmidomycin (LFF). Compared to the control leaves (CK), ∆F/F _m′ in the LFF was markedly lower during the first 20 min; however, there were no significant differences in non-photochemical quenching and photosynthetic pigments (chlorophylls and carotenoids). In contrast, the activities of antioxidant enzymes (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase) were enhanced in the LFF. Meanwhile, the contents of antioxidant metabolites (ascorbate and glutathione) were also elevated in the LFF, when compared with the CK. The results obtained here suggest that monoterpene may be very effective molecule in protecting plants against oxidative stress, the absence of monoterpene leads to the increased responses of the enzymatic and non-enzymatic antioxidant defenses to oxidative stress, and the enhancement of the enzymatic and non-enzymatic antioxidant defenses may, in part, compensate for the loss of antioxidant conferred by monoterpene.     

斑马鱼行为学实验在神经科学中的应用

斑马鱼作为新型模式动物的优势正在逐渐被人们所认识,其应用的领域也越来越宽广．斑马鱼在神经生物学中的应用,除了在发育方面比其他模式动物更具优势外,在行为学方面的应用也更加丰富．由于斑马鱼幼体在受精后前两天通体透明,眼睛大小占到大脑体积的二分之一以上,成鱼昼夜节律明显,对光反应强烈,因此斑马鱼在视觉领域应用的优势十分明显．斑马鱼的嗅觉、听觉器官都在体表可见,可以很容易地用行为学实验手段对嗅觉和听觉功能进行检测．斑马鱼习性好动,利用斑马鱼进行运动方面的行为学观察也非常便利．斑马鱼具有群聚习性,在社会生物学研究方面正得到越来越多的关注．斑马鱼行为学是一种比较简单而又有效地分析神经整合功能的方法,并形成了许多相关的实验模型．     

Amyloid-β Deposition in Alzheimer Transgenic Mice Is Associated with Oxidative Stress

Abstract: Increased awareness for a role of oxidative stress in the pathogenesis of Alzheimer's disease has highlighted the issue of whether oxidative damage is a fundamental step in the pathogenesis or instead results from disease-associated pathology. In vitro experiments support both possibilities: Oxidative stress increases amyloid-β production, and, conversely, amyloid-β increases oxidative damage. To address the relationship between amyloid-β and oxidative stress in vivo, we examined, using an array of oxidative markers, transgenic mice that overexpress amyloid-β precursor protein and, as in Alzheimer's disease, develop characteristic amyloid-β deposits within the brain parenchyma. Transgenic animals show the same type of oxidative damage that is found in Alzheimer's disease, and it is important that this damage directly correlates with the presence of amyloid-β deposits. The significance of these studies is twofold. First, they provide evidence that amyloid-β and oxidative damage are inextricably linked in vivo. Second, they support the use of transgenic animals for the development of antioxidant therapeutic strategies.