Differential Expression of Glutathione Reductase and Cytosolic Glutathione Peroxidase,GPX1, in Developing Rat Lungs and Kidneys

A mutant rat GPX1 (a cytosolic predominant form), in which the selenocysteine residue in the catalytic center was replaced by cysteine, was prepared and an antibody against the mutant enzyme was raised. The resultant antibody specifically reacted with rat GPX1 and was, together with the Glutathione reductase (GR) antibody, used in a Western blot analysis and immunohistochemistry experiments. To elucidate the physiological coupling of these enzymes under oxidative stress which accompanies the birth, developmental changes of the protein levels and enzymatic activities of GR and GPX1 were examined for lungs and kidneys from prenatal fetus to adult rats. The expression of GR was already evident at the prenatal stage and remained high in lungs at all stages. However, GR activity in kidneys gradually increased after birth reaching maximal levels at adulthood. An immunohistochemical study showed that GR was strongly bound to the bronchial epithelia in lungs and the epithelial cells of renal tubes. GPX1 was expressed in the renal tube epithelial cells and its level gradually increased after birth in a manner similar to that of GR. The expression of GPX1 in the lungs was, on the other hand, variable and occurred in some alveolar cells and bronchial epithelia only at restricted periods. It preferentially localized in nuclei at a late stage of development. Thus, the expression of the two functionally coupled enzymes via GSH did not appear to coordinate with development, tissue localization or under oxidative stress. Since many gene products show GSH-dependent preoxidase activity, other peroxidase(s) may be induced to compensate for the low GPX1 levels at stages with high GR expression.     

谷胱甘肽硫转移酶基因表达的调控

催化内源性或外源性亲电子化合物与谷胱甘肽(GSH)结合的谷胱甘肽硫转移酶(GST)超基因家族是一族解毒功能蛋白.其基因的表达通过不同的机制受多种物质的调控.根据最近文献资料,对调控谷胱甘肽硫转移酶基因表达的基因结构、调控机制及氧化应激对谷胱甘肽硫转移酶基因表达的调控作用等作一简要综述.     

Genome-Wide Characterization,Evolution, and Expression Analysis of the Ascorbate Peroxidase and Glutathione Peroxidase Gene Families in Response to Cold and Osmotic Stress in Ammopiptanthus nanus

Ascorbate peroxidase (APX) and glutathione peroxidase (GPX) are two families of essential peroxidases that maintain redox balance in cells by catalyzing the reduction of hydrogen peroxide. Ammopiptanthus nanus is a rare broad-leaved evergreen shrub that lives in the temperate desert areas of Central Asia and exhibits strong resistance to low temperature and water stress. GPX and APX family members might contribute to the stress response of A. nanus by participating in reactive oxygen species scavenging. In the present study, APX and GPX family members in A. nanus were identified and their structure, evolution, and expression patterns under stress conditions were investigated. A total of 8 GPX genes, 6 APX genes, and 1 APX-like gene were identified in A. nanus, and these genes were unevenly distributed on 7 chromosomes. These APXs and GPXs showed conservation in amino acid sequence, three-dimensional structure, and intron–exon structure. The GPX gene family in A. nanus expanded in gene number, and the expansions were mainly driven by segmental duplication caused by large-scale duplication events in the evolution of Tribe Sophoreae and might play important roles in the freezing and drought tolerance in A. nanus. Expression profiling based on RNA-seq datasets and qRT-PCR analysis showed that most of the APX and GPX members were differentially expressed under osmotic and cold stress, which is in line with the high copies of stress and hormone response-related cis-acting elements predicted from the promoters of the APX and GPX family genes. The study provided new insight into the evolution of APX and GPX family and promoted the understanding of the molecular mechanism underlying the stress tolerance of A. nanus.

     

植物谷胱甘肽过氧化物酶研究进展

氧化胁迫可诱导植物多种防御酶的产生,其中包括超氧化物歧化酶(SOD,EC1.15.L1)、抗坏血酸过氧化物酶(APX,EC1.11.1.11)、过氧化氢酶(CAT,E.C.1.11.1.6)和谷胱甘肽过氧化物酶(GPXs,EC1.11.1.9).它们在清除活性氧过程中起着不同的作用.GPXs是动物体内清除氧自由基的主要酶类,但它在植物中的功能报道甚少.最近几年研究表明,植物体内也存在类似于哺乳动物的GPXs家族,并对其功能研究已初见端倪.本文综述了有关GPXs的结构以及植物GPXs功能的研究进展.     

萝卜磷脂氢谷胱甘肽过氧化物酶基因结构及其调控序列分析

磷脂氢谷胱甘肽过氧化物酶 (PHGPx) 是谷胱甘肽过氧化物酶 (GPx) 家族的重要一员,是目前已知能直接保护生物膜免受过氧化损伤的唯一酶类 . 此前的研究表明,萝卜磷脂氢谷胱甘肽过氧化物酶基因 (RsPHGPx) 编码一个有生理功能的过氧化物酶 , 并且 RsPHGPx 基因的表达可能受发育和环境胁迫信号的复杂调控 . 要深入了解该基因的表达调控机制首先必须阐明 RsPHGPx 基因的结构及其上游调控序列 . DNA 印迹表明萝卜 RsPHGPx 基因以单拷贝的形式存在于基因组中 . 以基因组 DNA 为模板,通过常规 PCR 与染色体步行相结合的方法克隆到了一段 3.3 kb 长的 RsPHGPx 基因组序列 . 分析发现,该基因由 7 个外显子和 6 个内含子组成,所有内含子的剪切位点均符合真核生物 GT-AG 规则 . 另外还发现该基因的上游基因是生物素合成酶基因;位于 RsPHGPx 基因上游的调控序列只有不足 300 bp. 这些结构特征与拟南芥 AtGPX3 基因极其相似 . 顺式作用元件的数据库搜索发现 RsPHGPx 基因的上游调控序列含有多个响应激素 ( 如 E-Box 和 W-Box) 、胁迫 ( 如转录因子 MYB 和 MYC 的结合位点 ) 和光 ( 如 Box Ⅱ和Ⅰ -Box) 信号的元件 . RNA 印迹分析表明 RsPHGPx 基因的表达受到脱落酸 (ABA) 和连续光照 ( 在黄化苗中 ) 处理的负调控,受到冷胁迫 (4℃ ) 的正调控,这暗示了预测的顺式作用元件的调控作用 . 然而,除草剂 paraquat 对该基因表达的正调控作用,暗示了某些与氧化胁迫相关的未知元件的存在 . 这些结果进一步印证了 RsPHGPx 基因的表达受发育和环境胁迫信号复杂调控的推测 . 这是迄今为止首个关于植物 PHGPx 基因结构和上游调控序列的系统报道,为今后全面认识植物 PHGPx 基因的表达调控机制奠定了必要基础 .     

植物谷胱甘肽过氧化物酶研究进展

氧化胁迫可诱导植物多种防御酶的产生, 其中包括超氧化物歧化酶(SOD, EC1.15.1.1)、抗坏血酸过氧化物酶(APX, EC1.11.1.11)、过氧化氢酶(CAT, E.C.1.11.1.6 )和谷胱甘肽过氧化物酶(GPXs,EC1.11.1.9)。它们在清除活性氧过程中起着不同的作用。GPXs是动物体内清除氧自由基的主要酶类,但它在植物中的功能报道甚少。最近几年研究表明, 植物体内也存在类似于哺乳动物的GPXs家族, 并对其功能研究已初见端倪。本文综述了有关GPXs的结构以及植物GPXs功能的研究进展。     

重组萝卜磷脂氢谷胱甘肽过氧化物酶在毕赤酵母优化表达初步纯化与鉴定

将萝卜磷脂氢谷胱甘肽过氧化物酶（RsPHGPx）基因插入到分泌表达载体pPIC9K中,转化巴斯德毕赤酵母GS115细胞,筛选具有G418抗性的单拷贝转化子。经过优化表达条件,RsPHGPx在1%甲醇、pH6.0、28℃条件下诱导60h后得到最大表达量,产率约为102 mg/L。通过硫酸铵分级沉淀、脱盐柱脱盐、凝胶过滤等纯化步骤,得到了90%以上纯度的RsPHGPx．活性分析显示纯化获得的RsPHGPx具有依赖于GSH的还原活性, 比活性为4.2μmol/min·mg,为获得大量RsPHGPx而用于应用开发研究奠定了基础。     

Glutathione Peroxidase Activity in Porcine Blood

Determination of the seleno-enzyme glutathione peroxidase (GSH-Px) in blood from Danish Landrace pigs was done using a quantitative, spectrophotometric method and a simple “spot test”. A close correlation between the net reaction rate measured spectrophotometrically (Δ A/min.) and time for defluores-cence (minutes) was obtained (r2 = 0.72—0.77, P < 0.0005). From these results the factors used for a conversion of defluorescence time to u/g hemoglobin were evaluated. The results further showed that the “spot test” can be used as a screening method for detection of subnormal GSH-Px levels in pigs. While red cell GSH-Px seems independent of the sex, an elevation of both plasma and red cell GSH-Px was found with increasing age of pigs. The normal range of red cell GSH-Px activity was wide, contrasting the small variations observed in the individual pig. Some evidence that porcine red cell GSH-Px is under genetical control was found and discussed in relation to the possible use of GSH-Px as an indicator of the pig's selenium status.     

Differential Gene Expression and Infection Profiles of Cutaneous and Mucosal Leishmania braziliensis Isolates from the Same Patient

BackgroundLeishmaniasis is a complex disease in which clinical outcome depends on factors such as parasite species, host genetics and immunity and vector species. In Brazil, Leishmania (Viannia) braziliensis is a major etiological agent of cutaneous (CL) and mucosal leishmaniasis (MCL), a disfiguring form of the disease, which occurs in ~10% of L. braziliensis-infected patients. Thus, clinical isolates from patients with CL and MCL may be a relevant source of information to uncover parasite factors contributing to pathogenesis. In this study, we investigated two pairs of L. (V.) braziliensis isolates from mucosal (LbrM) and cutaneous (LbrC) sites of the same patient to identify factors distinguishing parasites that migrate from those that remain at the primary site of infection.Conclusions/SignificanceDespite sharing high similarity at the genome structure and ploidy levels, the parasites exhibited divergent expressed genomes. The proteome and metabolome results indicated differential profiles between the cutaneous and mucosal isolates, primarily related to inflammation and chemotaxis. BALB/c infection revealed that the cutaneous isolates were more virulent than the mucosal parasites. Furthermore, our data suggest that the LbrPGF2S protein is a candidate to contribute to parasite virulence profiles in the mammalian host.     

盐芥谷胱甘肽过氧化物酶基因（ThGPX6）的克隆及表达分析

谷胱甘肽过氧化物酶在植物响应盐胁迫中具有重要作用。依据盐芥EST序列进行RACE实验,获得1个新的谷胱甘肽过氧化物酶基因,命名为ThGPX6(GenBank注册号为FJ357244)。该基因的cDNA全长892 bp,包含1个长为702bp的开放读码框,编码234个氨基酸。生物信息学分析表明,该蛋白具有植物GPX的典型结构,即GPX催化活性区(NVASKCGLT)和标志性基序(ILAFPCNQF),以及PHGPX特有序列(KWNF(S/T)KFL)。实时荧光定量PCR分析结果表明,ThGPX6在盐芥叶片和根中表达,其表达受NaCl诱导,显示ThGPX6在植物高盐响应中发挥作用。亚细胞定位结果表明,ThGPX6存在于线粒体和内体中的可能性最大,预示着ThGPX6在清除ROS过程中起着重要作用。     

亚硒酸钠对小麦幼苗中谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及谷胱甘肽含量的影响

用1.0 mg·L-1的亚硒酸钠根施小麦幼苗,测定亚硒酸钠对谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及还原性谷胱甘肽含量的结果表明,外源亚硒酸钠对麦苗地上部的谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性均有诱导作用,使麦苗体内的谷胱甘肽含量水平增加.     

水稻磷脂氢谷胱甘肽过氧化物酶在蛋白质水平上的组织和诱导表达特征

蛋白质是生命活动的主要承担分子,了解蛋白质在有机体中的时空分布对于正确解析蛋白质的功能十分重要．磷脂氢谷胱甘肽过氧化物酶 (PHGPx) 是目前发现的唯一能够直接还原膜上脂类过氧化物的抗氧化酶,在保护生物膜免受过氧化损伤方面有着重要作用．采用Western blot技术,分析了水稻PHGPx (OsPHGPx) 在水稻不同组织以及多种胁迫条件下的蛋白质表达特征．结果表明,OsPHGPx在成熟水稻植株内主要分布于叶组织中,以旗叶中含量最高,而在水稻幼苗中则在茎及叶组织中均检测到较强的杂交信号．OsPHGPx在幼苗中的表达受到H₂O₂和NaCl的强烈诱导,但植物激素对其表达的影响较弱．H₂O₂和NaCl的诱导效果呈现出时间及剂量的相关性,当用0.5 mmol/L H₂O₂处理12 h或用500 mmol/L NaCl处理24 h,此时OsPHGPx表达量达到最大值．对H₂O₂清除剂二甲基硫脲处理的水稻幼苗,外源H₂O₂的再处理并不能诱导OsPHGPx的表达,而NaCl的诱导效果并不受影响,说明H₂O₂可能并不介导NaCl诱导OsPHGPx的表达．这些结果为进一步研究OsPHGPx在水稻中生物学功能奠定了基础．     

哈氏弧菌谷胱甘肽还原酶基因克隆及原核表达

经克隆哈氏弧菌谷胱甘肽还原酶(GR)基因,并构建其原核表达载体,以获得相应的表达蛋白。将GR和p ET-32a(+)通过Bam H I和Xho I双酶切后,体外用T4连接酶连接,构建重组质粒p ET-GR;然后转化至大肠杆菌BL21(DE3)中,利用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,应用SDS-PAGE分析表达情况和表达条件。SDS-PAGE电泳获得分子量约为68.9 k D融合蛋白条带。在E.coli BL21(DE3)中重组质粒p ET-GR的表达条件为28℃,0.7 mmol/L的IPTG浓度诱导4 h表达量最高,且主要以包涵体形式表达。哈氏弧菌谷胱甘肽还原酶基因在大肠杆菌中获得了高效表达。     

Inactivation of Glutathione Peroxidase by Peroxynitrite

Glutathione peroxidase (GSH-Px) is inactivated on exposure to peroxynitrite under physiologically relevant conditions. Stopped-flow kinetic studies show that the reaction between peroxynitrite and GSH-Px is first-order in each of the reactants, with an apparent second-order rate constant of 4.5 ± 0.2 × 10⁴M⁻¹s⁻¹per monomer unit of enzyme. In good agreement with this value, GSH-Px inactivation experiments afford an apparent second-order rate constant of 1.8 ± 0.1 × 10⁴M⁻¹s⁻¹per monomer unit of enzyme. The hydroxyl radical scavengers mannitol, DMSO, and benzoate (at 100 mM) afford only 8–12% protection of the enzyme, while addition of 25 mM bicarbonate results in 55% protection. The minimal protection by hydroxyl radical scavengers indicates, as expected, that hydroxyl radicals are not involved in the inactivation. Protection by bicarbonate occurs because peroxynitrite is rapidly trapped by CO₂to form the adduct nitrosoperoxycarbonate (ONOOCO⁻₂), and/or other reactive species that preferentially decompose to nitrate rather than react with GSH-Px. The close agreement between the rate constants obtained from enzyme inactivation and from stopped-flow kinetics experiments suggests that the mechanism of the reaction between peroxynitrite and GSH-Px involves the oxidation of the ionized selenol of the selenocysteine residue in the enzyme's active site (E-Se⁻) by peroxynitrite. This reaction does not simply involve formation of the selenenic acid, E-SeOH, because E-SeOH is an intermediate in the catalytic cycle of the enzyme, and thus its formation cannot explain the inactivation we observe. Thus, the ionized selenol in the active site is transformed into a form of selenium that cannot easily be reduced back to the selenol.     

苦瓜谷胱甘肽磷脂氢过氧化物酶cDNA的克隆及其特征分析

根据谷胱甘肽磷脂氢过氧化物酶(PHGPX)氨基酸序列中高度保守的区段设计引物,采用RACE-PCR从苦瓜中克隆到一个全长927 bp的cDNA片段.DNA序列的数据库分析比较表明,该cDNA编码167个氨基酸,含有动植物PHGPX的特征结构,是一个新发现的苦瓜PHGPX基因（mocPHGPX）.RNA印迹结果显示,该基因在苦瓜幼苗的根中表达相对较弱,茎的信号较强,叶中最强.这些结果将有助于深入研究植物PHGPX的功能以及全面了解植物抗氧化体系.     

Progenitor Stage-Specific Activity of a cis-Acting Double GATA Motif for Gata1 Gene Expression

GATA1 is a master regulator of erythropoiesis, expression of which is regulated by multiple discrete cis-acting elements. In this study, we examine the activity of a promoter-proximal double GATA (dbGATA) motif, using a Gata1 bacterial artificial chromosome (BAC)-transgenic green fluorescent protein (GFP) reporter (G1BAC-GFP) mouse system. Deletion of the dbGATA motif led to significant reductions in GFP expression in hematopoietic progenitors, while GFP expression was maintained in erythroblasts. Consistently, in mice with a germ line deletion of the dbGATA motif (Gata1^ΔdbGATA mice), GATA1 expression in progenitors was significantly decreased. The suppressed GATA1 expression was associated with a compensatory increase in GATA2 levels in progenitors. When we crossed Gata1^ΔdbGATA mice with Gata2 hypomorphic mutant mice (Gata2^fGN/fGN mice), the Gata1^ΔdbGATA::Gata2^fGN/fGN compound mutant mice succumbed to a significant decrease in the progenitor population, whereas both groups of single mutant mice maintained progenitors and survived to adulthood, indicating the functional redundancy between GATA1 and GATA2 in progenitors. Meanwhile, the effects of the dbGATA site deletion on Gata1 expression were subtle in erythroblasts, which showed increased GATA1 binding and enhanced accumulation of active histone marks around the 1st-intron GATA motif of the ΔdbGATA locus. These results thus reveal a novel role of the dbGATA motif in the maintenance of Gata1 expression in hematopoietic progenitors and a functional compensation between the dbGATA site and the 1st-intron GATA motif in erythroblasts.