Reactive Oxygen Species Scavenging Enzymes and Down-Adjustment of Metabolism Level in Mitochondria Associated with Desiccation-Tolerance Acquisition of Maize Embryo

It is a well-known fact that a mature seed can survive losing most of its water, yet how seeds acquire desiccation-tolerance is not well understood. Through sampling maize embryos of different developmental stages and comparatively studying the integrity, oxygen consumption rate and activities of antioxidant enzymes in the mitochondria, the main origin site of reactive oxygen species (ROS) production in seed cells, we found that before an embryo achieves desiccation-tolerance, its mitochondria shows a more active metabolism, and might produce more ROS and therefore need a more effective ROS scavenging system. However, embryo dehydration in this developmental stage declined the activities of most main antioxidant enzymes and accumulated thiobarbituric acid-reactive products in mitochondria, and then destroyed the structure and functional integrity of mitochondria. In physiologically-matured embryos (dehydration-tolerant), mitochondria showed lower metabolism levels, and no decline in ROS scavenging enzyme activities and less accumulation of thiobarbituric acid-reactive products after embryo dehydration. These data indicate that seed desiccation-tolerance acquisition might be associated with down-adjustment of the metabolism level in the late development stage, resulting in less ROS production, and ROS scavenging enzymes becoming desiccation-tolerant and then ensuring the structure and functional integrity of mitochondria.     

Reactive Oxygen Species Scavenging Enzymes and Down-Adjustment of Metabolism Level in Mitochondria Associated with Desiccation-Tolerance Acquisition of Maize Embryo

It is a well-known fact that a mature seed can survive losing most of its water, yet how seeds acquire desiccation- tolerance is not well understood. Through sampling maize embryos of different developmental stages and comparatively studying the integrity, oxygen consumption rate and activities of antioxidant enzymes in the mitochondria, the main origin site of reactive oxygen species （ROS） production in seed cells, we found that before an embryo achieves desiccation-tolerance, its mitochondria shows a more active metabolism, and might produce more ROS and therefore need a more effective ROS scavenging system. However, embryo dehydration in this developmental stage declined the activities of most main antioxidant enzymes and accumulated thiobarbituric acid-reactive products in mitochondria, and then destroyed the structure and functional integrity of mitochondria. In physiologically-matured embryos （dehydration- tolerant）, mitochondria showed lower metabolism levels, and no decline in ROS scavenging enzyme activities and less accumulation of thiobarbituric acid-reactive products after embryo dehydration. These data indicate that seed desiccation- tolerance acquisition might be associated with down-adjustment of the metabolism level in the late development stage, resulting in less ROS production, and ROS scavenging enzymes becoming desiccation-tolerant and then ensuring the structure and functional integrity of mitochondria.     

杨桃提取物体外清除氧自由基作用

从杨桃果中提取得到三种提取物为匀浆提取物、蛋白提取物和多糖提取物。采用化学发光法测定这三种提取物清除氧自由基的活性,实验结果:匀浆提取物清除羟自由基(·OH)和H2O2的活性大小相近,而清除超氧阴离子自由基(O2–·)的活性较小,其IC50约为前两者的4倍。蛋白提取物清除O2–·和·OH的活性大小相近,而清除H2O2的活性明显小于前两者,IC50约为前两者的9倍。多糖提取物清除.OH的活性明显大于清除O2–·和H2O2的活性,其IC50约为O2–·的1/22,约为H2O2的1/65。结果表明,杨桃果具有清除O2–·、·OH和H2O2的作用,不同提取物对这些活性氧自由基的清除能力有所不同。     

黄土丘陵区9种群落共存种干旱胁迫下抗氧化酶的响应

通过盆栽试验,以9种黄土丘陵区撂荒演替中期群落共存种(猪毛蒿、中华隐子草、小花棘豆、冰草、无芒隐子草、铁杆蒿、达乌里胡枝子、茭蒿和白羊草)为研究对象,测定其生长季中(8~10月)重度和中度干旱胁迫及适宜水分条件下(分别为45%~50%、60%~65%和75%~80%的田间持水量)活性氧清除酶(SOD、CAT、POD、APX)活性以及MDA含量的变化,分析土壤干旱程度、植物和生长季三因素对上述各生理指标的影响,研究群落共存种对土壤水分变化的响应,揭示水分变化后群落共存种的群体效应和植被演替的植物生理生态机制。结果表明:(1)9种群落共存种的SOD、CAT、POD、APX活性以及MDA含量存在显著的种间差异,生长季中(8~10月)以及不同水分条件下也存在显著差异。(2)达乌里胡枝子SOD活性、白羊草CAT活性、中华隐子草POD活性以及小花棘豆的CAT、POD和APX活性均相对较高,而且达乌里胡枝子的MDA含量较低;生长季中(8~10月)9种群落共存种的SOD、CAT和POD活性逐月下降且MDA含量进入10月后也有所下降,说明生长旺季耐旱性指标的生理活性较高;与POD和CAT活性相比,9种群落共存种的SOD和APX活性在重度干旱胁迫下显著提高,显示SOD和APX活性对于干旱胁迫具有较好的响应能力。(3)达乌里胡枝子和小花棘豆具有较高的SOD、APX活性和较低的MDA含量,说明达乌里胡枝子和小花棘豆的耐旱性相对较强,在土壤水分较低时,其在群落中的相对多度可能会有所上升。(4)9种群落共存种在演替序列上以前期种的SOD和APX活性相对较低,而演替后期种相对较高,表明演替后期的植物较为耐旱。     

Response of Chinese Wampee Axes and Maize Embryos to Dehydration at Different Rates

Survival of wampee （Clausena lansium Skeels） axes and maize （Zea mays L.） embryos decreased with rapid and slow dehydration. Damage of wampee axes by rapid dehydration was much less than by slow dehydration, and that was contrary to maize embryos. The malondialdehyde contents of wampee axes and maize embryos rapidly increased with dehydration, those of wampee axes were lower during rapid dehydration than during slow dehydration, and those of maize embryos were higher during rapid dehydration than during slow dehydration. Activities of superoxide dismutase （SOD）, ascorbate peroxidase （APX） and catalase （CAT） of wampee axes markedly increased during the early phase of dehydration, and then rapidly decreased, and those of rapidly dehydrated axes were higher than those of slow dehydrated axes when they were dehydrated to low water contents. Activities of SOD and APX of maize embryos notable decreased with dehydration. There were higher SOD activities and lower APX activities of slowly dehydrated maize embryos compared with rapidly dehydrated maize embryos. CAT activities of maize embryos markedly increased during the early phase of dehydration, and then decreased, and those of slowly dehydrated embryos were higher than those of rapidly dehydrated embryos during the late phase of dehydration.     

Response of Chinese Wampee Axes and Maize Embryos to Dehydration at Different Rates

Survival of wampee (Clausena lansium Sksels) axes and maize (Zea mays L.) embryos decreased with rapid and slow dehydration. Damage of wampee axes by rapid dehydration was much less than by slow dehydration, and that was contrary to maize embryos. The malondialdehyde contents of wampee axes and maize embryos rapidly increased with dehydration, those of wampee axes were lower during rapid dehydration than during slow dehydration, and those of maize embryos were higher during rapid dehydration than during slow dehydration. Activities of superoxide dismutsse (SOD), ascorbate peroxidase (APX) and catalase (CAT) of wampee axes markedly increased during the sady phase of dehydration, and then rapidly decreased, and those of rapidly dehydrated axes were higher than those of slow dehydrated axes when they were dehydrated to low water contents. Activities of SOD and APX of maize embryos notable decreased with dehydration. There were higher SOD activities and lower APX activities of slowly dehydrated maize embryos compared with rapidly dehydrated maize embryos. CAT activities of maize embryos markedly increased during the eady phase of dehydration, and then decreased, and those of slowly dehydrated embryos were higher than those of rapidly dehydrated embryos during the late phase of dehydration.     

模拟空气潜水对大鼠脾组织氧自由基生成的影响

目的:观察模拟空气潜水对大鼠脾组织氧自由基(OFR)生成的影响。方法:腹腔注射自旋捕捉剂,高气压处理后检测大鼠脾组织生成的OFR。结果:模拟空气潜水后大鼠脾组织OFR生成增多,并检测到了.OH信号。结论:空气模拟潜水时呼吸气中高分压氧可促进脾组织OFR生成,主要类型可能为羟自由基。     

Effect of Light on Scavenging Capacity for Organic Free Radical in Leaves of Four Woody Plants

Change of antioxidant capacity of the organic-free-radical scavengers to 1.1-diphenyl-2-picrylhydrazyl (DPPH·) reagent in the leaf extracts of the seedlings and young trees of four woody species (Schima superba Gardn. et Champ., Castanopsis fissa (Champ. ex Benth.) Rehd. et Wils., Psychotria rubra (Lour.) Poir., Ardisia quinquegona Bl.) in exposureto different light intensities was investigated. The organic free radical scavenging capacity (ORSC) expressed as the percentage of decreasing DPPH· was 16%-59% (pot seedlings) or 48%-88% (young trees in the forest). The highest ORSC was observed in plants grown under natural light, and the ORSC reduced with the decreasing light intensity. Similar trend was observed through the assay of inhibition to linoleic acid oxidation. The ORSC of P. rufra, an understory shrub, was more sensitive to the change of light intensity. A linear relationship was found between ORSC and AsA (ascorbic acid) content (r=0.92) or the absorption around the wavelength of 204-227 nm. It is proposed that ORSC of leaf was regulated by incident light intensity, and the contents of AsA and, flavonoid phenolics might be the important components contributed to ORSC and total antioxidant activity of leaves. The increasing ORSC is likely to be a protective strategy of plant in response to strong light.     

cDNA RDA在类似普通2型糖尿病大鼠肾脏组织基因差异表达筛查中的应用

应用代表性差异分析 (cDNARDA)技术 ,对类似普通 2型糖尿病大鼠肾脏组织基因差异表达进行筛查 ,初步探讨类似普通 2型糖尿病大鼠肾脏损害发病的分子机制 .首先以类似普通 2型糖尿病大鼠肾脏组织作为实验组 (Tester) ,正常大鼠肾脏作为对照组或驱动组 (Driver)通过cDNARDA进行基因差异表达筛查 ;最终的差异产物亚克隆到Puc 18载体 ,测序及并进行生物信息学分析 ;半定量RT PCR对筛查到新的基因进行初步的鉴定 .结果发现 9个新ESTs ,2个新基因 .这 2个新基因分别与人及小鼠的丝氨酸蛋白酶抑制因子F ,及真核细胞转录启动因子 3亚单位 5 (EIF 3epsilon)基因有高度的相似性 (>90 % )并在类似普通 2型糖尿病大鼠肾脏组织表达上调 .推测 2个新基因分别是大鼠的丝氨酸蛋白酶抑制因子F及真核细胞转录启动因子 3亚单位 5 .两个新基因在类似普通 2型糖尿病大鼠肾脏组织表达上调 ,可能与类似普通 2型糖尿病大鼠肾脏损害相关 .同时 ,对新基因RS91进行了全长cDNA克隆     

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC. Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.     

库克诺你果汁提取物体外清除自由基及抗氧化活性研究

本文对诺你果汁多糖、乙醇溶出物和乙酸乙酯萃取物体外对超氧阴离子(O2·)、羟自由基(·OH)、DPPH和脂质过氧化(LPO)的抑制作用进行了研究。超氧阴离子(O2·)由邻苯三酚自氧化产生;羟自由基(·OH)由Fenton反应产生;利用Fe2 诱发卵黄脂蛋白产生丙二醛(MDA),TBA法测定。所有测定均为分光光度法。结果表明,与已知抗氧化剂L抗坏血酸相比,乙醇溶出物和乙酸乙酯萃取物均有明显的捕捉自由基和抗氧化能力,而多糖捕捉自由基和抗氧化能力很低,且对O2·没有抑制作用,反而会增加其生成速度。     

"生存还是毁灭",发育还是阻滞--小鼠早胚体外发育2-细胞阻滞产生机制初析

在体外培养条件下,小鼠受精卵往往经一次分裂后就停滞在２－细胞,不能完成到达囊胚的后续发育过程,称作２－细胞阻滞。氧自由基伤害、培养液成分不平衡等外界因素都能引起阻滞。小鼠的２－细胞阻滞现象受细胞质内母型物质制约,具有品系依赖性。在阻滞品系小鼠胚胎的细胞质内可能缺乏某些重要的蛋白因子,在无外源信号的培养体系内不能继续分裂。发生阻滞的胚胎细胞内ＭＰＦ前体物虽然储备充足,但因无法去磷酸化激活而最终导致发     

重度干旱胁迫下燕麦叶片活性氧清除系统对喷施腐植酸水溶肥的响应

以燕麦品种‘燕科2号’为试验材料,采用盆栽方式,设置正常供水(CK)、正常供水下喷施腐植酸水溶肥(CKH)、重度干旱胁迫(SS)和重度干旱胁迫下喷施腐植酸水溶肥(SSH)4个处理,对燕麦叶片中活性氧水平、抗氧化酶活性、总抗氧化剂含量及产量等进行测定,以明确腐植酸水溶肥(HA)对重度干旱胁迫下燕麦叶片活性氧清除系统的调控效应,并探讨HA对燕麦耐旱性的影响及其作用机制。结果表明：(1)与CK相比,燕麦叶片超氧阴离子■、羟自由基(·OH)、过氧化氢(H₂O₂)和丙二醛(MDA)含量、以及超氧化物歧化酶(SOD)和过氧化物酶(POD)活性在重度干旱胁迫下显著提高,且均在喷施HA后比重度干旱胁迫处理显著降低,但此时活性氧的水平仍显著高于CK。(2)与CK相比,燕麦叶片过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)、谷胱氨肽还原酶(GR)和谷胱氨肽过氧化物酶(GPX)活性在重度干旱胁迫下显著降低,而其总抗氧化能力(T-AOC)显著提高,它们在喷施HA后均比重度干旱胁迫处理显著提高,但各酶活性仍不同程度低于CK。(3)与CK相比,燕麦籽粒产量和生物产量...     

筛选代表小鼠植入前胚胎紧密化相关基因的EST

分别收集181及241枚昆明白小鼠8细胞早期胚胎及8细胞紧密化胚胎,采用SMART PCR方法直接合成胚胎双链cDNA.进而运用抑制消减杂交技术(SSH)对8细胞早期胚胎及8细胞紧密化胚胎的基因表达进行研究,并将所获得的差异表达产物按片段大小分段分离纯化后克隆入pUCm-T载体中,经PCR鉴定后挑选阳性克隆进行测序,筛选出27个代表8细胞早期胚胎和紧密化8细胞胚胎差别表达基因的cDNA片段;经与GenBank中收录的序列进行同源性匹配分析,证实其中17个eDNA片段为新的EST,提交GenBank后被接受并给予了新序列编号.这1 7个片段均可能为与紧密化密切相关的新基因的表达片段,为今后进一步克隆新的紧密化相关基因的全长cDNA及后续新基因的结构和功能研究打下基础.通过采用不同长度大小片段分别克隆的方法,可获得较长片段的EST,避免差异表达大片段的丢失.     

Activity of Enzymes Related to H2O2 Generation and Metabolism in Leaf Apoplastic Fraction of Tomato Leaves Infected with Botrytis cinerea

Hydrogen peroxide generation rates of uninfected and infected leaves of two tomato (Lycopersicon esculentum) cultivars showing differential susceptibility to Botrytis cinerea were determined. The superoxide anion, hydroxyl radical, ascorbate contents and changes in NADH peroxidase, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities in the apoplast fraction were analysed. Infected leaves had an increased hydrogen peroxide level. It was greater and generally occurred earlier in plants of the less susceptible cv. Perkoz than in those of the more susceptible cv. Corindo. Induction of nitrotetrazolium blue reducing activity and SOD levels in apoplast were higher in cv. Perkoz 24 h after inoculation. In the controls, NADH peroxidase activity in apoplast was higher in the more susceptible cv. Corindo, but after infection it increased faster and to a higher level in the less susceptible cv. Perkoz. NADH oxidation was inhibited by only 15% by a specific inhibitor DPI (diphenylene‐iodonium) but was completely inhibited by KCN and NaN₃. Similar increases in APX activity after 48 h and a small increase in catalase activities were observed in both cultivars soon after infection. These results indicate that resistance of tomato plants to infection by the necrotrophic fungus B. cinerea may result from early stimulation of hydrogen peroxide and superoxide radical generations by NADH peroxidase and SOD in apoplastic space, and they confirm the important role of their enhanced production in apoplastic spaces of plants.     

Xenopus as a model to study alternative splicing in vivo

An increasing number of genes are being identified for which the corresponding mRNAs contain different combinations of the encoded exons. This highly regulated exon choice, or alternative splicing, is often tissue-specific and potentially could differentially affect cellular functions. Alternative splicing is therefore not only a means to increase the coding capacity of the genome, but also to regulate gene expression during differentiation or development. To both evaluate the importance for cellular functions and define the regulatory pathways of alternative splicing, it is necessary to progress from the in vitro or ex vivo experimental models actually used towards in vivo whole-animal studies. We present here the amphibian, Xenopus, as an experimental model highly amenable for such studies. The various experimental approaches that can be used with Xenopus oocytes and embryos to characterize regulatory sequence elements and factors are presented and the advantages and drawbacks of these approaches are discussed. Finally, the real possibilities for large-scale identification of mRNAs containing alternatively spliced exons, the tissue-specific patterns of exon usage and the way in which these patterns are modified by perturbing the relative amount of splicing factors are discussed.     

Physiological response of Clostridium carboxidivorans during conversion of synthesis gas to solvents in a gas-fed bioreactor

Clostridium carboxidivorans P7 is one of three microbial catalysts capable of fermenting synthesis gas (mainly CO, CO₂, and H₂) to produce the liquid biofuels ethanol and butanol. Gasification of feedstocks to produce synthesis gas (syngas), followed by microbial conversion to solvents, greatly expands the diversity of suitable feedstocks that can be used for biofuel production beyond commonly used food and energy crops to include agricultural, industrial, and municipal waste streams. C. carboxidivorans P7 uses a variation of the classic Wood–Ljungdahl pathway, identified through genome sequence‐enabled approaches but only limited direct metabolic analyses. As a result, little is known about gene expression and enzyme activities during solvent production. In this study, we measured cell growth, gene expression, enzyme activity, and product formation in autotrophic batch cultures continuously fed a synthetic syngas mixture. These cultures exhibited an initial phase of growth, followed by acidogenesis that resulted in a reduction in pH. After cessation of growth, solventogenesis occurred, pH increased and maximum concentrations of acetate (41 mM), butyrate (1.4 mM), ethanol (61 mM), and butanol (7.1 mM) were achieved. Enzyme activities were highest during the growth phase, but expression of carbon monoxide dehydrogenase (CODH), Fe‐only hydrogenases and two tandem bi‐functional acetaldehyde/alcohol dehydrogenases were highest during specific stages of solventogenesis. Several amino acid substitutions between the tandem acetaldehyde/alcohol dehydrogenases and the differential expression of their genes suggest that they may have different roles during solvent formation. The data presented here provide a link between the expression of key enzymes, their measured activities and solvent production by C. carboxidivorans P7. This research also identifies potential targets for metabolic engineering efforts designed to produce higher amounts of ethanol or butanol from syngas. Biotechnol. Bioeng. 2012; 109: 2720–2728. © 2012 Wiley Periodicals, Inc.