叶绿体巯基亚硝基化修饰蛋白质组学研究进展

蛋白质S-亚硝基化是NO与蛋白质的半胱氨酸残基共价连接形成S-亚硝基硫醇的过程.叶绿体内快速变化的氧化还原环境容易导致蛋白质S-亚硝基化修饰发生.综述了植物逆境应答过程中叶绿体蛋白质S-亚硝基化参与调节的光合电子传递、卡尔文循环、抗氧化系统、蛋白质合成、蛋白质加工与周转、Ca~(2+)介导的信号转导、氮同化、硫同化,以及四吡咯化合物合成等途径,为全面理解植物逆境应答过程的NO调控网络机制提供了线索.     

强光下一氧化氮对三角褐指藻EPA产量的影响

二十碳五烯酸(EPA)是一种长链多不饱和脂肪酸,具有重要的医药价值.本文研究了光照强度和亚硝基铁氰化钠(SNP)分解产生的NO对三角褐指藻EPA产量的影响及机理.采用氧电极法探究光照强度与三角褐指藻光合速率的关系,结果显示三角褐指藻的光饱和点为126.3μmol/(m2·s).采用气相色谱法检测EPA的含量,结果显示外源NO可通过促进强光下三角褐指藻的快速生长来提高强光下三角褐指藻EPA的产量,而对单位生物量中EPA含量并没有影响;用血球计数板法检测三角褐指藻的生长速率,结果显示在光强200μmol/(m2·s)时三角褐指藻出现了光抑制,而SNP能解除这种抑制.探究NO促进三角褐指藻快速生长的机理,结果显示NO提高了乙醇酸氧化酶(GO)的活性.用实时荧光定量PCR(Real-time PCR)检测外源NO对GO基因表达量的影响,结果显示GO的两个同工酶"GOX和GLY"的基因表达量均增强了.采用AMCA开关法对纯化的GO进行S–亚硝基化鉴定,结果显示GOX和GLY均发生了S–亚硝基化修饰.分别采用ISNO-AAPair、GPS-SNO和ISNO-Pse AAC方法预测S–亚硝基化位点,结果显示GLY的238位、GOX的237位和332位很可能是NO的修饰位点.因此,NO提高GO活性的途径有两条:提高GO基因的表达量;对GO酶蛋白进行S–亚硝基化修饰.综上所述,NO通过提高GO活性促进强光下三角褐指藻的快速生长从而提高三角褐指藻EPA的产量.     

2-十三烷酮和槲皮素诱导棉铃虫谷胱甘肽 S-转移酶组织特异性表达

利用分光光度酶动力学和定量PCR的方法,从蛋白质水平和基因水平上研究了棉铃虫幼虫谷胱甘肽S-转移酶(GST)的组织特异性表达和植物次生物质的诱导表达作用.结果表明,以1-氯-2,4-二硝基苯(CDNB)为底物检测到的棉铃虫幼虫不同组织GST的活性与其GST mRNA相对表达量的趋势是一致的.酶动力学活性测定结果表明的棉铃虫各组织GST活性大小的顺序与定量PCR结果表明的各组织GST mRNA相对量大小的顺序都依次为:脂肪体、中肠、头和体壁.通过培养基混药法研究植物次生物质对棉铃虫幼虫GST的诱导表明,2-十三烷酮和槲皮素对棉铃虫幼虫GST活性的诱导与其对mRNA表达量的影响是一致的.2-十三烷酮对GST活性及其GST mRNA表达量的诱导作用比槲皮素强.说明mRNA的转录量增加是植物次生物质诱导GST活性增加的主要机制.该结果对于进一步研究植物次生物质诱导作用对棉铃虫对杀虫药剂敏感度的影响具有重要的意义.     

吲哚丁酸对怀牛膝幼苗生长及谷胱甘肽抗氧化酶系统的影响

对怀牛膝幼苗施加不同浓度的吲哚丁酸(IBA),研究IBA对其幼苗生长及谷胱甘肽抗氧化酶系统的影响.结果显示:1.0mg/L IBA能同时显著提高怀牛膝叶片中谷胱甘肽过氧化物酶(GPx)、谷胱甘肽还原酶(GR)、谷胱甘肽S-转移酶(GST)的活性及蛋白质含量(P<0.05),并能促进其幼苗株高、根长和根干重的增加.结论:1.0mg/L IBA促进了牛膝幼苗的生长,提高了叶片中谷胱甘肽抗氧化酶系统的活性及蛋白质含量,有效的增强了植株的抗逆性.     

石松属植物化学成分抗炎活性的筛选

为研究2种石松属植物化学成分的抗炎活性,筛选具有较强抗炎活性的化合物,用LPS诱导RAW264.7细胞建立了体外炎症模型,采用MTT法检测了化合物对小鼠巨噬细胞株RAW264.7细胞增殖的影响.在安全浓度范围内(细胞存活率大于80%)给予药物干预,用Griess法检测了培养上清液中一氧化氮(NO)水平,以各化合物抑制NO释放的能力为筛选指标,评价了化合物的抗炎活性.结果表明:乙酰基二氢石松生物碱(16)、对香豆酸甲酯(21)、豆甾烷-3-酮-21-酸(22)具有较好的NO抑制率,并呈一定剂量依赖关系,提示它们具有一定的抗炎活性.     

植物细胞NO,Ca~(2+)和蛋白激酶的信号交叉

一氧化氮(NO)是植物体内重要的信号分子,生物和非生物的刺激都能使NO与胞内第2信使Ca2+和蛋白激酶产生相互作用.以动物细胞NO - Ca2+信号途径为基础,列举了植物NO信号途径中Ca2+和多种蛋白激酶的可能作用,论述了植物细胞中NO,Ca2+和蛋白激酶的信号交叉.     

一氧化氮对紫外线-B辐射下小浮萍保护作用

以小浮萍为材料,研究紫外线-B(UV-B)辐射下NO对植物的保护作用.经紫外线处理过的小浮萍,叶绿素和可溶性蛋白质含量降低,而SOD、POD和CAT活性上升;加入NO后,可以阻止叶绿素含量下降,提高可溶性蛋白质含量,且增加SOD、POD和CAT的活性.NO可以提高小浮萍抗紫外线能力.     

植物细胞质雄性不育的育性恢复机理研究进展

植物细胞质雄性不育(Cytoplasmic Male Sterility,CMS)是一种母性遗传现象,表现为不能产生正常功能的花粉.大量研究表明线粒体基因发生分子内或分子间的重组形成嵌合基因,这些嵌合基因的表达产物对线粒体产生毒性,导致了线粒体功能异常,从而导致雄性不育.恢复基因(Restorer of fertility,Rf)对CMS育性的恢复表现为多重效应,即同一物种中存在多个同源的Rf基因,而且对CMS育性恢复的效应和作用方式不同.绝大多数Rf包含不同数目的PPR(Pentatricopeptide Repeat)基序,这些基序形成亲水性的超螺旋结构.Rf对CMS育性恢复的调控是多样的,有的改变CMS相关的线粒体DNA(mtDNA)的结构;有的对线粒体RNA转录本进行加工和编辑;有的影响线粒体蛋白质的丰度.     

温度对甜菜种子萌发速率和线粒体活性的影响

研究了不同温度对甜菜种子萌发速率和线粒体活性的影响。在 7,14和 2 0℃中甜菜种子萌发 5 0 %需要的时间分别是 7,3和 2 3d ;萌发 5 0 %的热时间大约是 46℃·d。在 7,14和 2 0℃萌发的种子和幼苗的线粒体中 ,总蛋白含量以及细胞色素氧化酶 (CCO)和苹果酸脱氢酶 (MDH)的活性随着萌发进程增加 ;相对分子质量大约为 1880 0 ,35 0 0 0和 36 0 0 0的蛋白质随着萌发进程降低 ;相对分子质量大约为 2 2 0 0 0的蛋白质在 7℃中逐渐增加 ,在 14和 2 0℃中则先少量增加然后下降。 2 0℃中萌发的种子和幼苗的线粒体的CCO和MDH的比活性和总活性比在 7和 14℃中高。这些结果表明 ,萌发温度显著地影响种子的萌发速率、线粒体的CCO和MDH活性以及较小相对分子质量热休克蛋白 2 2     

氧自由基与妇科肿瘤关系的研究

目的探讨超氧化物歧化酶(SOD)、丙二醛(MDA)、一氧化氮(NO)、谷胱甘肽(GSH-PX)与妇科常见肿瘤的关系.方法1)以20例正常健康人为对照组,子宫肌瘤30例、子宫颈癌20例、卵巢癌22例,分别用生物化学法测定血清中SOD,GSH-PX活性与MDA,NO的含量.2)以子宫肌瘤30例、子宫颈癌20例、卵巢癌22例,分别用生物化学法测定癌组织与癌旁组织、子宫肌瘤核组织与核旁组织中SOD,GSH-PX活性和MDA,NO含量.结果1)血清测定中,肿瘤患者的MDA含量、SOD活性比正常对照组高;GSH-PX活性比正常对照组低;良性肿瘤的NO含量高于正常对照组,恶性肿瘤中的NO含量低于正常组.2)组织测定中,肿瘤组织MDA含量,SOD,GSH-PX活性比肿瘤旁组织高,癌组织中NO含量比肿瘤旁组织低.结论子宫颈癌、卵巢癌、子宫肌瘤组织及血清中有明显的自由基代谢紊乱,而且良恶性肿瘤有明显差异,并且随着肿瘤恶化程度的变化,自由基也呈现规律性变化.     

S-nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration

Stress proteins located in the cytosol or endoplasmic reticulum (ER) maintain cell homeostasis and afford tolerance to severe insults. In neurodegenerative diseases, several chaperones ameliorate the accumulation of misfolded proteins triggered by oxidative or nitrosative stress, or of mutated gene products. Although severe ER stress can induce apoptosis, the ER withstands relatively mild insults through the expression of stress proteins or chaperones such as glucose-regulated protein (GRP) and protein-disulphide isomerase (PDI), which assist in the maturation and transport of unfolded secretory proteins. PDI catalyses thiol-disulphide exchange, thus facilitating disulphide bond formation and rearrangement reactions. PDI has two domains that function as independent active sites with homology to the small, redox-active protein thioredoxin. During neurodegenerative disorders and cerebral ischaemia, the accumulation of immature and denatured proteins results in ER dysfunction, but the upregulation of PDI represents an adaptive response to protect neuronal cells. Here we show, in brains manifesting sporadic Parkinson's or Alzheimer's disease, that PDI is S-nitrosylated, a reaction transferring a nitric oxide (NO) group to a critical cysteine thiol to affect protein function. NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leads to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. S-nitrosylation also abrogates PDI-mediated attenuation of neuronal cell death triggered by ER stress, misfolded proteins or proteasome inhibition. Thus, PDI prevents neurotoxicity associated with ER stress and protein misfolding, but NO blocks this protective effect in neurodegenerative disorders through the S-nitrosylation of PDI.     

Nitric oxide:A potential key point of the signaling network leading to plant secondary metabolite biosynthesis

The endogenous signaling network of plants plays important roles in mediating the exogenous factor-induced biosynthesis of secondary metabolites. Nitric oxide (NO) has emerged as a key signaling molecule in plants recently. Numerous studies demonstrated that the main signaling molecules such as salicylic acid (SA), jasmonic acid (JA), reactive oxygen species (ROS), and NO were not only involved in regulating plant secondary metabolite biosynthesis but also interacted to form a complex signaling network by mutual inhibition and/or synergy. The recent progress in the signal network of plant secondary metabolite biosynthesis has been discussed in this paper. Furthermore, we propose a hypothetical model to show that NO might act as a potential molecular switch in the signaling network leading to plant secondary metabolite biosynthesis.     

Nitric oxide: A potential key point of the signaling network leading to plant secondary metabolite biosynthesis

The endogenous signaling network of plants plays important roles in mediating the exogenous factor-induced biosynthesis of secondary metabolites. Nitric oxide (NO) has emerged as a key signaling molecule in plants recently. Numerous studies demonstrated that the main signaling molecules such as salicylic acid (SA), jasmonic acid (JA), reactive oxygen species (ROS), and NO were not only involved in regulating plant secondary metabolite biosynthesis but also interacted to form a complex signaling network by mutual inhibition and/or synergy. The recent progress in the signal network of plant secondary metabolite biosynthesis has been discussed in this paper. Furthermore, we propose a hypothetical model to show that NO might act as a potential molecular switch in the signaling network leading to plant secondary metabolite biosynthesis.     

Nitric oxide involved in signal transduction of Jasmonic acid-induced stomatal closure of Vicia faba L.

Nitric oxide (NO) and Jasmonic acid (JA) are two key signaling molecules involved in many and diverse biological pathways in plants. Growing evidence suggested that NO signaling interacts with JA signaling. In this work, Our experiment showed that NO exists in guard cell of Vicia faba L., and NO is involved in signal transduction of JAinduced stomata closuring: ( i ) JA enhances NO synthesis in guard cell; ( ii ) both JA and NO induced stomatal closure, and had dose response to their effects; ( iU ) there are synergetic correlation between JA and lower NO concentration in regulation of stomatal movement; (iV) JA-induced stomatal closure was largely prevented by 2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (PTIO), a specific NO scavenger. An inhibitor of NO synthase (NOS) in mammalian cells, N^G-nitro-L-Arg-methyl eater (L-NAME) also inhibits plant NOS, repressing JA-induced NO generation and JA-induced stomatal closure. We presumed that NO mainly comes from NOS after JA treatment.     

Involvement of carbon monoxide produced by heme oxygenase in ABA-induced stomatal closure in Vicia faba and its proposed signal transduction pathway

Carbon monoxide (CO) has recently proven to be an important bioactive or signaling molecule in mammalian cells. Its effects are mainly mediated by nitric oxide (NO) and cyclic GMP (cGMP). In Vicia faba leaves, CO production and heme oxygenase (HO) activity, an important CO synthetic enzyme, are first reported to increase in response to ABA treatment, which could result in stomatal closure. Inter- estingly, ABA-induced stomatal closure in V. faba guard cells is partially blocked when the synthetic CO inhibitor ZnPP, or the CO/NO scavenger Hb is added. Furthermore, we show that, exogenously applied CO donor, hematin, and CO aqueous solution not only result in the enhancement of CO release, but also time-dependently induce stomatal closure, and the latter is mimicked by the application of an NO donor SNP. The above-mentioned stomatal closure effects are differentially reversed by the addition of tungstate, a potent inhibitor of NO synthetic enzyme nitrate reductase (NR), the specific NO scavenger cPTIO, ZnPP, or Hb. During treatment for 4 h, SNP, 0.01% CO aqueous solution or hematin significantly triggers NO synthesis, whereas cPTIO, or tungstate approximately fully inhibits NO fluorescence. Ad- ditionally, application of the GC inhibitor ODQ blocks CO-induced stomatal closure. This inhibition could be reversed when 8-Br-cGMP is added. Thus, the above results suggest that CO produced by HO is involved in ABA-induced stomatal closure, and NO and cGMP may function as downstream interme- diates in the CO signaling responsible for stomatal closure.     

NO对新生大鼠心肌细胞PKC活性的影响

利用培养新生大鼠心肌细胞，检测NO前体L-精氨酸(L-arginine,L-Arg)和NO供体硝普钠(sodium nitroprusside,SNP)对PKC活性的影响，并探讨内、外源性NO在PKC激动剂佛波指(phorbol 12-myristate 13-acetate,PMA)激活PKC中的作用.实验结果表明：培养基中加入L-Arg，PKC活性呈剂量依赖性降低；用L-Arg进行预处理，30 min后加入PMA，PKC活性明显降低，与单纯PMA组相比有显著差异；NOS抑制剂L-NAME本身对基础状态PKC活性无明显影响，但可阻断L-Arg对上述2个效应的影响；培养液中加入NO供体SNP，PKC活性呈剂量依赖性降低；用SNP预处理心肌细胞，5 min后加入PMA，PKC活性与单纯PMA组相比有显著性差异.以上结果表明，内、外源性NO均具有剂量依赖性抑制PKC活性的作用，PKC可能是NO对心肌细胞作用的胞内信号传导通路的关键部位或重要信号分子之一；L-Arg通过NOS先生成NO，NO再对PKC起抑制作用.