拟南芥AtDAD1 超量表达植株对H2O2抗性的研究

构建拟南芥AtDAD1超量表达载体,以农杆菌介导的方法转化拟南芥哥伦比亚生态型,比较AtDAD1超量表达植株和野生型植株表现型的差异,以及两者对H2O2抗性的不同。实验显示,AtDAD1转基因拟南芥生长较野生型拟南芥更为强壮,对高浓度H2O2有较强的耐受力。测定两者糖含量,发现AtDAD1转基因拟南芥叶片糖的含量明显高于野生型拟南芥叶片。以上结果表明,AtDAD1基因可能参与植物生长发育,并可能在拟南芥抵抗凋亡的过程中发挥重要的作用。     

Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory

Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and tetrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and tetrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of Cbeta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gem-diamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.     

Cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity.

The enzyme 5-formyl tetrahydrofolate cyclodehydrase plays an important role in the conversion of 5-formyl tetrahydrofolate to 5,10-methenyl tetrahydrofolate. A second enzyme, cyclohydrolase, converts 5,10-methenyl tetrahydrofolate to 10-formyl tetrahydrofolate. These folate derivatives play a significant part in the biosynthesis of purines. A method has been devised for the cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity which uses 5-formyl tetrahydrofolate or 5,10-methenyl tetrahydrofolate as substrate respectively, blocking possible interferences by other enzymes, and allows the nonenzymatic reduction of nitro-blue tetrazolium by 5,10-methenyl tetrahydrofolate formed by the action of the cyclodehydrase on the substrate 5-formyl tetrahydrofolate, and by 10-formyl tetrahydrofolate formed by the action of cyclohydrolase on the substrate 5,10-methenyl tetrahydrofolate, thus revealing intracellular sites of enzyme activity. The methods appear to show only intracellular localization of the blue formazan deposits of reduced tetrazolium. The distribution of positivity in cells of human blood and bone marrow is described.     

植物中的H2O2信号及其功能

H2O2是植物细胞的信号分子,是细胞正常代谢的产物,生物和非生物胁迫促使植物细胞产生H2O2,通过H2O2信号应答胁迫.H2O2信号调控一系列重要的植物生理生化过程,如系统获得抗性(SAR)和高度敏感抗性(HR)、细胞衰老与程序化细胞死亡(PCD)、气孔关闭、根的向地性、根的生长和不定根形成、细胞壁的发育、柱头与花粉的发育及相互关系等.Ca2+流动和可逆蛋白磷酸化作用是H2O2下游信号,通过MAPK级联作用于转录因子,最终调控基因的表达.H2O2调控多种基因的表达,包括编码抗氧化酶基因、调控程序化细胞死亡相关蛋白基因、生物与非生物胁迫应答蛋白基因等.     

磷脂酶A2在诱导红豆杉细胞产生活性氧中的作用

对磷脂酶A2(PLA2)在真菌诱导中国红豆杉细胞产生活性氧中的作用进行研究,结果表明：PLA2非特异抑制剂可降低真菌诱导子诱导产生的H2O22通过钙离子螯合和PLA2特异抑制剂实验,表明参与H2O2产生的PLA2为胞质CaO^2 依赖型2对PLA2诱导H202产生的机理进行分析,发现亚油酸可缓解PLA2抑制剂对诱导的活性氧的抑制作用,而且亚油酸单独处理可导致H2O2的发生,其它的脂肪酸也具有类似诱导H2O2发生的作用.不同离子型的脂肪酸对H2O2产生的影响不同,阴离子型脂肪酸较非离子型脂肪酸更能促进活性氧的发生.这些结果表明,PLA2可能通过产生脂肪酸或其衍生物激活H2O2的产生酶系.     

Arabidopsis 10-formyl tetrahydrofolate deformylases are essential for photorespiration

In prokaryotes, PurU (10-formyl tetrahydrofolate [THF] deformylase) metabolizes 10-formyl THF to formate and THF for purine and Gly biosyntheses. The Arabidopsis thaliana genome contains two putative purU genes, At4g17360 and At5g47435. Knocking out these genes simultaneously results in plants that are smaller and paler than the wild type. These double knockout (dKO) mutant plants show a 70-fold increase in Gly levels and accumulate elevated levels of 5- and 10-formyl THF. Embryo development in dKO mutants arrests between heart and early bent cotyledon stages. Mature seeds are shriveled, accumulate low amounts of lipids, and fail to germinate. However, the dKO mutant is only conditionally lethal and is rescued by growth under nonphotorespiratory conditions. In addition, culturing dKO siliques in the presence of sucrose restores normal embryo development and seed viability, suggesting that the seed and embryo development phenotypes are a result of a maternal effect. Our findings are consistent with the involvement of At4g17360 and At5g47435 proteins in photorespiration, which is to prevent excessive accumulation of 5-formyl THF, a potent inhibitor of the Gly decarboxylase/Ser hydroxymethyltransferase complex. Supporting this role, deletion of the At2g38660 gene that encodes the bifunctional 5,10-methylene THF dehydrogenase/5,10-methenyl THF cyclohydrolase that acts upstream of 5-formyl THF formation restored the wild-type phenotype in dKO plants.     

锰对锰超积累植物美洲商陆抗氧化系统的影响

采用水培法,研究了美洲商陆在不同锰浓度下的生长、锰积累、H₂O₂含量、脂质过氧化以及抗氧化系统的响应.结果表明：植株锰含量随锰浓度增大而显著增加,依次为叶＞茎＞根.低浓度锰（5 mmol·L^-1）显著促进植株生长,叶片中H₂O₂含量明显降低,丙二醛（MDA）含量与对照相当;高浓度锰（≥10 mmol·L^-1）抑制植株生长,叶片中H₂O₂和MDA水平显著增加,表明叶片中发生了明显的氧化损伤.抗坏血酸过氧化物酶、脱氢抗坏血酸还原酶、谷胱甘肽还原酶的活性和还原型抗坏血酸水平随锰处理浓度上升;超氧化物歧化酶活性在低浓度锰时显著降低,高浓度锰时则显著上升;过氧化氢酶和过氧化物酶活性、还原型谷胱甘肽含量在锰浓度为5～10 mmol·L^-1时显著上升,而在20 mmol·L^-1时明显回落.说明氧化损伤及锰积累可能是高浓度锰下美洲商陆生长受抑的生理原因.锰处理下,抗氧化系统效率提高可以部分解释美洲商陆耐锰和超积累锰的特性,不同抗氧化剂活性或含量随介质锰浓度变化的模式不同,反映了其在美洲商陆耐受不同浓度范围锰时的作用不同.     

Drought-Stimulated Activity of Plasma Membrane Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Its Catalytic Properties in Rice

The activity of plasma membrane (PM) nicoUnamide adenine dinucleotide phosphate (NADPH) oxidase and Its catalytic properties in rice was investigated under drought stress conditions. Drought stress led to decreased leaf relative water content (RWC) and, as a result of drought-induced oxidative stress, the activities of antioxidant enzymes increased significantly. More interestingly, the intensity of applied water stress was correlated with increased production of H_2O_2and O_2~- and elevated activity of PM NADPH oxidase, a key enzyme of reactive oxygen species generation in plants.Histochemlcal analyses also revealed increased H_2O_2 and O_2~- production in drought-stressed leaves. Application of dlphenylene iodonium (DPI), an Inhibitor of PM NADPH oxidasa, did not alleviate drought-induced production of H_2O_2 and O_2~-. Catalysis experiments indicated that the dce PM NADPH oxidass was partially fiavin-dependent. The pH and temperature optima for this enzyme were 9.8 and 40 ℃, respectively. In addition, drought stress enhanced the activity under alkaline pH and high temperature conditions. These results suggest that a complex regulatory mechanism, associated with the NADPH oxidase-H_2O_2 system, is involved in the response of rice to drought stress.     

NO和H2O2在光／暗调控蚕豆气孔运动中的作用及其相互关系

借助表皮条分析和激光扫描共聚焦显微镜技术,对NO和H_2O_2在光/暗调控蚕豆(Vicia faba L.)气孔运动中的作用及其相互关系进行了探索。结果显示,光下外源NO供体硝普钠(SNP)和H_2O_2促进气孔关闭的效应明显大于暗中,暗中NO专一性清除剂2,4-羧基苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)、一氧化氮合酶(NOS)抑制剂N~G-氮-L-精氨酸-甲酯(L-NAME)和H_2O_2清除剂抗坏血酸(Vc)、过氧化氢酶(CAT)对气孔开度的效应明显大于光下,而且光下蚕豆保卫细胞NO和H_2O_2水平比暗中明显降低。上述结果表明,光/暗通过影响保卫细胞NO和H_2O_2的水平调控气孔运动。研究还发现,光下H_2O_2既诱导NO水平增加,也诱导气孔关闭,cPTIO和L-NAME有效地逆转H_2O_2的这些效应;光下SNP既诱导H_2O_2水平增加,也诱导气孔关闭,SNP的上述效应又被Vc和CAT有效逆转。这些结果表明,NO和H_2O_2在生成及效应上均存在明显的相互作用。另外,L-NAME显著逆转暗和光下H_2O_2处理对气孔关闭和NO生成的效应表明,蚕豆保卫细胞中可能存在NOS,暗和光下H_2O_2处理可能通过提高NOS的活性促进NO水平增加,进而诱导气孔关闭。     

甜菜碱对干旱胁迫下湖北海棠超微弱发光及抗氧化能力的影响

以盆栽4年生的苹果砧木湖北海棠（Malus hupehensis）为试材,叶面喷施100 mmol·L^-1的甜菜碱,研究外源甜菜碱对干旱胁迫下湖北海棠叶片超微弱发光（UWL）、丙二醛(MDA)、超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢（H₂O₂）、脯氨酸（Pro）及甜菜碱（GB）含量的影响.结果表明,轻度干旱胁迫下,叶片UWL强度提高,但随着干旱胁迫程度的加重而下降,至严重干旱胁迫时UWL明显低于正常供水处理;H₂O₂、MDA含量随干旱胁迫加重而升高;SOD和POD活性随胁迫加重而升高,但至重度干旱时下降.与单独干旱处理相比,叶片喷施甜菜碱显著提高了轻度和中度干旱胁迫叶片的UWL（分别提高35.27%和43.95%）、SOD和POD活性,降低了H₂O₂和MDA含量;促进了Pro和GB的积累.表明甜菜碱及通过甜菜碱诱导的脯氨酸积累,能够提高干旱胁迫下叶片的抗氧化能力,减轻过氧化损伤,对叶片细胞起到保护作用.     

A three-dimensional structure of Plasmodium falciparum serine hydroxymethyltransferase in complex with glycine and 5-formyl-tetrahydrofolate. Homology modeling and molecular dynamics

Cytosolic Plasmodium falciparum serine hydroxymethyltransferase (pfSHMT) is a potential target for antimalarial chemotherapy. Contrasting with the other enzymes involved in the parasite folate cycle, little information is available about this enzyme, and its crystallographic structure is unknown yet. In this paper, we propose a theoretical low-resolution 3D model for pfSHMT in complex with glycine and 5-formyl tetrahydrofolate (5-FTHF) based on homology modeling by multiple alignment followed by intensive optimization, validation and dynamics simulations in water. Comparison between the active sites of our model and that of crystallographic Human SHMT (hSHMT) revealed key differences that could be useful for the design of new selective inhibitors of pfSHMT.     

外源抗坏血酸对臭氧胁迫下水稻叶片膜保护系统的影响

在田间原位条件下,运用OTCs（open top chamber）装置研究了外源抗坏血酸（exogenous ascorbate acid,ExAsA）对臭氧（O3）胁迫下水稻（Oryza Sativa L.）叶片膜保护系统的影响.研究发现,O3胁迫下的水稻叶片经过ExAsA处理后叶绿素a含量显著升高,而叶绿素b含量变化不明显;相对于对照,经ExAsA处理后的水稻叶片过氧化氢（H2O2）和丙二醛（MDA）含量及相对电导率（REC）均降低,超氧化物岐化酶（SOD）和抗坏血酸过氧化物酶（APX）活性明显提高,抗氧化剂类胡萝卜素（Carotene）含量升高.这表明,ExAsA改善了O3胁迫下水稻叶片的抗氧化系统功能,减少了叶片中活性氧（activity oxygen species,AOS）的积累,抑制了脂质过氧化（lipid peroxidation,LP）,延迟了O3对水稻叶片的老化作用,提高了水稻叶片对O3危害的抗性.     

大丽轮枝菌毒素与拟南芥互作反应中SA、NO与H2O2信号的相关性

本文研究了大丽轮枝菌毒素(VD-toxin)与拟南芥互作反应中外源SA、NO供体、NO合酶抑制剂等对拟南芥幼苗H2O2含量的影响,并对H2O2的积累部位进行了DAB组化染色检测.大丽轮枝菌毒素、外源SA、NO供体处理拟南芥幼苗均能诱导H2O2的积累,NO供体的诱导作用最强;NO合酶抑制剂处理则未表现出H2O2含量的增强;H2O2的积累部位主要在叶片的表皮毛和维管束组织.结果表明,在大丽轮枝菌毒素与拟南芥互作反应中,H2O2可能作为信号分子参与了SA和NO调控的拟南芥防卫反应,NO信号与H2O2信号间的关系可能更密切.     

Effects of thyroxine on the synthesis of folate coenzymes in rat liver

1. The effects of thyroidectomy and of ;acute' and ;chronic' administration of thyroxine on the synthesis of folate coenzymes were studied by determining the liver contents of folate active derivatives and the enzymic activities involved in their biosynthesis. The effect of thyroxine on the same enzymes in vitro was also studied. 2. In thyroidectomized rats the liver contents of folate coenzymes did not change except for a slight decrease in the contents of 5-formyltetrahydrofolate and tetrahydrofolate compared with those in control rats. 3. In the same animals serine hydroxymethyltransferase and formyltetrahydrofolate synthetase activities decreased markedly. 4. The ;chronic' administration of thyroxine to thyroidectomized rats caused more evident variations in the liver contents of folate coenzymes and in particular a decrease in the contents of 5-formyltetrahydrofolate, tetrahydrofolate, 5(or 10)-formyl derivatives of tetrahydropteroylpolyglutamate and of 5(or 10)-formyl derivatives of pteroylpolyglutamate. 5. The enzymic activities did not show significant variations. 6. The ;acute' administration of thyroxine caused changes in the liver contents of some folate derivatives such as 10-formyldihydrofolate, 10-formylfolate, tetrahydrofolate and the 10-formyl derivative of dihydropteroylpolyglutamate. In these animals also the enzymic activities were unchanged. 7. No effect of thyroxine on enzymic activities in vitro was observed.     

Effect of ozone produced from antibody-catalyzed water oxidation on pathogenesis of atherosclerosis

Recent studies have suggested that antibodies can catalyze the generation of unknown oxidantsincluding hydrogen peroxide (H_2O_2) and ozone (O_3) from singlet oxygen (~1O_2) and water.This study is aimedto detect the effect of antibody-catalyzed water oxidation on atherosclerosis.Our results showed that bothH_2O_2 and O_ were produced in human leukemia THP-1 monocytes incubated with human immunoglobulin Gand phorbol myristate acetate.In the THP-1 monocytes incubated with human immunoglobulin G,phorbolmyristate acetate and low density lipoprotein the intracellular total cholesterol,free cholesterol,cholesterylester and lipid peroxides clearly increased,and a larger number of foam cells were observed by oil red Ostaining.The accumulation of all intracellular lipids was significantly inhibited by vinylbenzoic acid,and onlyslightly affected by catalase.These findings suggested that the production of O_3,rather than H_2O_2,might beinvolved in the pathogenesis of atherosclerosis through the antibody-catalyzed water oxidation pathway.     

模拟酸雨和铜复合污染对白花泡桐生理特性的影响及其解毒机制

采用盆栽方法,研究了模拟酸雨(pH分别为4.0、5.0)和Cu(0～200mg·kg-1)复合污染对白花泡桐生理特性的影响及其解毒机制.结果表明:未加Cu处理时,不同酸雨处理间,白花泡桐的叶绿素、类胡萝卜素、超氧阴离子、过氧化氢和丙二醛含量均差异不显著;100和200mg·kg-1Cu处理时,pH4.0处理的叶绿素和类胡萝卜素含量显著低于,而超氧阴离子、过氧化氢和丙二醛含量显著高于pH5.0处理.Cu处理的叶绿素a/b值明显高于未加Cu处理.随着酸雨酸度增加,叶片铜含量明显减少,而根部铜含量明显增加.pH5.0时,随Cu浓度增加,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性均持续增强,植物络合素和谷胱甘肽(GSH)总量均明显增加;而在pH4.0时,SOD、POD、CAT和APX活性呈先升后降,而GSH总量在Cu浓度为200mg·kg-1时明显下降.模拟酸雨加剧了高浓度Cu对白花泡桐的氧化胁迫.     

一株蛹拟青霉(Paecilomyces militaris )深层发酵产物中抗肿瘤活性物质分析

摘要： 【目的】初步搞清一株蛹拟青霉（Paecilomyces militaris ）菌株RCEF0927在发酵罐发酵条件下发酵液的抗中国仓鼠卵巢瘤（CHO）细胞活性及活性成分的具体组成。【方法】用刃天青（Resazurin）法测定样品对CHO细胞的抑制率;用高效液相色谱-高分辨质谱和活性测定联用的方法进行活性成分分析和鉴定。【结果】活性测定结果表明菌株RCEF0927经发酵罐发酵的发酵液具有较强的抗CHO细胞活性;提取实验结果表明抗肿瘤活性物质能较好地被乙酸乙酯提取出来;液相色谱-质谱-活性测定分析表明     

Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter.

Leaf senescence is an active process involving remobilization of nutrients from senescing leaves to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, supplemental cytokinin delays senescence. Plants that overexpress isopentenyl transferase (ipt), a cytokinin-producing gene, or knotted1 (kn1), a homeobox gene, have many phenotypes in common. Many of these phenotypes are characteristic of altered cytokinin physiology. The effect of kn1 on leaf senescence was tested by driving its expression using the promoter of the senescence-associated gene SAG12. SAG:kn1 tobacco plants showed a marked delay in leaf senescence but otherwise developed normally. The delay in senescence was revealed by an increase in chlorophyll content in SAG:kn1 leaves relative to leaves of the control plants and by a decrease in the number of dead leaves. Senescence was also delayed in detached leaves of SAG:kn1 plants. Delayed senescence was accompanied by increased leaf cytokinin content in older leaves expressing kn1. These experiments extend the current understanding of kn1 function and suggest that in addition to mediating meristem maintenance, kn1 is capable of regulating the onset of senescence in leaves.