高温处理防治黄瓜霜霉病的生理生化机制

以黄瓜感病品种‘长春密刺’为试材,通过室内盆栽试验研究高温对已感染霜霉病菌的黄瓜幼苗生理生化特征的影响.结果显示:(1)在黄瓜幼苗接种霜霉菌后8h,用45℃高温处理90min防治效果最明显.(2)与只接种霜霉病菌的黄瓜幼苗相比,接种霜霉菌后进行高温处理可显著提高叶片叶绿素含量,降低丙二醛含量;与对照相比,接种霜霉菌后进行高温处理可显著提高叶片几丁质酶活性,Western blotting验证了几丁质酶的活性变化.(3)SDS-PAGE结果表明,霜霉菌侵染可诱导一种28kD蛋白表达,高温处理后28kD蛋白的表达量降低.研究结果表明高温处理可能在杀死病原菌的同时,还诱导黄瓜幼苗对霜霉病产生了部分抗性.     

硅和白粉菌诱导接种对黄瓜幼苗白粉病抗性影响的研究

研究了硅酸盐和诱导接种白粉菌对黄瓜活性氧代谢、SiO2含量和抗病性的影响．结果表明,诱导接种能使叶片的超氧自由基(O2^-)产生速率、H2O2和丙二醛(MDA)含量升高,加硅接种处理的O2^-产生速率、H202和MDA含量明显低于不加硅接种处理．诱导接种能使叶片的过氧化氢酶(CAT)、过氧化物酶(POD)活性升高,超氧化物歧化酶(SOD)活性降低．加硅接种处理植株叶片的CAT、POD和SOD活性明显高于不加硅接种处理．诱导接种提高叶片的抗坏血酸(AsA)和还原型谷胱甘肽(GSH)含量,加硅处理的AsA含量明显低于不加硅处理,GSH含量高于不加硅处理．无论接种与否,加硅处理的SiO2含量显著高于不加硅处理,病情指数明显低于不加硅处理．     

水杨酸对水分胁迫黄瓜幼苗叶片生理过程的影响

研究了外源水杨酸(salicylic acid,SA)对水分胁迫下黄瓜幼苗叶片主要生理过程的影响。1mmol／L的SA处理黄瓜幼苗24h后,叶片中POD活性剧增,SOD活性增加不明显,H2O2清除酶CAT和APX活性受抑制,H2O2含量上升引起膜脂过氧化产物MDA含量上升,造成轻度氧化胁迫;在随后水分胁迫过程中,经SA预处理积累的H2O2诱导APX和CAT活性上升并清除产生的H2O2;SA预处理后,叶片中Rubisco含量及其基因转录水平明显高于对照,光合作用受影响较小。这表明SA使黄瓜幼苗生理活性有较大改善,增强了植株对水分胁迫的抗性。     

水杨酸对水分胁迫黄瓜幼苗叶片生理过程的影响

研究了外源水杨酸 salicylic acid,SA 对水分胁迫下黄瓜幼苗叶片主要生理过程的影响 . 1m mol/L 的 SA处理黄瓜幼苗 2 4 h后 ,叶片中 POD活性剧增 ,SOD活性增加不明显 ,H2 O2 清除酶 CAT和 APX活性受抑制 ,H2 O2 含量上升引起膜脂过氧化产物 MDA含量上升 ,造成轻度氧化胁迫 ;在随后水分胁迫过程中 ,经 SA预处理积累的 H2 O2 诱导 APX和 CAT活性上升并清除产生的 H2 O2 ;SA预处理后 ,叶片中 Rubisco含量及其基因转录水平明显高于对照 ,光合作用受影响较小 .这表明 SA使黄瓜幼苗生理活性有较大改善 ,增强了植株对水分胁迫的抗性     

镍处理对水稻叶片H_2O_2 积累和抗病的诱导效应

2 .2 0mmol/L的硝酸镍处理水稻幼苗后第 3天用稻白叶枯菌 (Xanthomonasoryzaepv .oryzae)挑战接种 ,硝酸镍处理的稻苗病情比对照明显减轻 ,并且叶片中过氧化物酶 (POD)活性上升 ,过氧化氢酶 (CAT)和抗坏血酸过氧化物酶 (APX)活性明显下降 ,H2 O2 和丙二醛 (MDA)含量显著增加。这些结果表明 ,H2 O2 积累与镍诱导的抗病作用密切有关     

NO和钙信使系统在草酸诱导黄瓜叶片抗霜霉病中的作用

通过草酸及其与不同抑制剂亚甲基蓝、EGTA、氯丙嗪和Li+组合处理黄瓜叶片,研究了草酸与抑制剂不同处理组合方式对黄瓜叶片POD活性和叶片病情指数的影响,探讨NO、钙信使系统在草酸诱导叶片抗霜霉病中的作用.结果显示,10～70mmol/L草酸均能不同程度诱导黄瓜叶片POD活性的升高,提高叶片对黄瓜霜霉病的抗病性,降低叶片病情指数,并以30mmol/L效果最好.4种抑制剂分别与30mmol/L草酸同时或先于草酸处理,或草酸处理后一定时间再用抑制剂处理,均明显抑制黄瓜叶片POD活性的升高及病情指数的降低.研究表明,NO、Ca2+、钙调素(CaM)和磷酸肌醇均可能参与了草酸诱导黄瓜霜霉病抗性的信号转导过程.     

黄瓜叶片胞间隙几丁质酶与抗霜霉菌侵染关系的研究

采用高抗霜霉病的‘津春4号’和易感霜霉病的‘长春密刺’黄瓜的温室穴盘幼苗为材料,研究了接种霜霉菌对黄瓜叶片胞间隙几丁质酶累积变化及幼苗生长的影响.结果显示:(1)与易感品种相比,抗病品种叶片胞间隙液蛋白浓度升高快、含量高;同时几丁质酶活性上升的速度快、幅度大,于接种后第2天达到峰值,且高活性维持至第7天;(2) SDS-PAGE电泳分析发现,抗病品种有一种27 kD的酸性几丁质酶在接种后第2天迅速积累,并在接种后第4、7天呈现出较高的表达量;Western blotting的结果也证实了上述胞间隙几丁质酶积累的变化.(3)与未接种对照相比,接种处理后第4、7天,易感黄瓜幼苗鲜重、干物质积累量均出现显著降低,但在接种后第7天抗病品种的上述生长指标要显著高于易感病品种.研究表明,接种霜霉菌后,抗病黄瓜叶片胞间隙几丁质酶迅速累积且活性快速升高,以减轻霜霉病对幼苗生长的侵害,这可能是黄瓜抗霜霉菌侵染的一种防御机制.     

渗透胁迫对山黧豆幼苗H_2O_2及毒素积累的影响

用PEG经根部处理 15d龄的山黧豆幼苗 ,取幼苗叶片为实验材料 ,测定过氧化氢酶 (CAT)、过氧化物酶 (POD)活性、过氧化氢 (H2 O2 )和毒素 ( β N oxalyl α ,β diaminopropionicacid ,ODAP)的含量。结果表明 ,随着PEG处理时间的延长 ,POD和CAT活性降低 ,而H2 O2 和ODAP含量显著升高 ;在PEG处理液中加入二乙基二硫代氨基甲酸钠 (diethyldithiocarbamate ,DDC)和氨基三唑 (aminotriazole ,AT)后分别抑制和促进H2 O2 的产生 ,DDC可降低叶片中ODAP的含量 ,AT则使ODAP积累。由此我们推测 ,水分胁迫条件下活性氧代谢与ODAP的积累有关     

镍处理对水稻叶片H2O2积累和抗病的诱导效应

2.20mmol/L的硝酸镍处理水稻幼苗后第3天用稻白叶枯菌（Xanthomonas oryzae pv.oryzae）挑战接种,硝酸镍处理的稻苗病情比对照明显减轻,并且叶片中过氧化物酶（POD）活性上升,过氧化氢酶（CAT）和抗坏血酸过氧化物酶（APX）活性明显下降,H2O2和丙二醛（MDA）含量显著增加。这些结果表明,H2O2积累与镍诱导的抗病作用密切有关。     

渗透胁迫对山黧豆幼苗H2O2及毒素积累的影响

用PEG经根部处理15d龄的山黧豆幼苗,取幼苗叶片为实验材料,测定过氧化氢酶（CAT）、过氧化物酶（POD）活性、过氧化氢（H2O2）和毒素（β-N-oxalyl-α,β-diaminopropionic acid,ODAP）的含量。结果表明,随着PEG处理时间的延长,POD和CAT活性降低,而H2O2和ODAP含量显著升高;在PEG处理液中加入二乙基二硫代氨基甲酸钠(diethyldithiocarbamate,DDC)和氨基三唑(aminotriazole,AT)后分别抑制和促进H2O2的产生,DDC可降低叶片中ODAP的含量,AT则使ODAP积累。由此我们推测,水分胁迫条件下活性氧代谢与ODAP的积累有关。     

Inhibition of thymidylate synthase by 2′,2′-difluoro-2′-deoxycytidine (Gemcitabine) and its metabolite 2′,2′-difluoro-2′-deoxyuridine

2′,2′-Difluoro-2′-deoxycytidine (dFdC, gemcitabine) is a cytidine analogue active against several solid tumor types, such as ovarian, pancreatic and non-small cell lung cancer. The compound has a complex mechanism of action. Because of the structural similarity of one metabolite of dFdC, dFdUMP, with the natural substrate for thymidylate synthase (TS) dUMP, we investigated whether dFdC and its deamination product 2′,2′-difluoro-2′-deoxyuridine (dFdU) would inhibit TS. This study was performed using two solid tumor cell lines: the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000. The specific TS inhibitor Raltitrexed (RTX) was included as a positive control. Using the in situ TS activity assay measuring the intracellular conversion of [5-³H]-2′-deoxyuridine or [5-³H]-2′-deoxycytidine to dTMP and tritiated water, it was observed that dFdC and dFdU inhibited TS. In A2780 cells after a 4 h exposure to 1 μM dFdC tritium release was inhibited by 50% but did not increase after 24 h, Inhibition was also observed following dFdU at 100 μM. No effect was observed in the dFdC-resistant cell line AG6000; in this cell line only RTX had an inhibitory effect on TS activity. In the A2780 cell line RTX inhibited TS in a time dependent manner. In addition, DNA specific compounds such as 2′-C-cyano-2′-deoxy-1-beta-D-arabino-pentafuranosylcytosine and aphidicoline were utilized to exclude DNA inhibition mediated down regulation of the thymidine kinase.Inhibition of the enzyme resulted in a relative increase of mis-incorporation of [5-³H]-2′-deoxyuridine into DNA. In an attempt to elucidate the mechanism of in situ TS inhibition the ternary complex formation and possible inhibition in cellular extracts of A2780 cells, before and after exposure to dFdC, were determined. With the applied methods no proof for formation of a stable complex was found. In simultaneously performed experiments with 5FU such a complex formation could be demonstrated. However, using purified TS it was demonstrated that dFdUMP and not dFdCMP competitively inhibited TS with a Ki of 130 μM, without ternary complex formation. In conclusion, in this paper we reveal a new target of dFdC: thymidylate synthase.     

A concise synthesis of 4-nitrophenyl 2-azido-2-deoxy- and 2-acetamido-2-deoxy-D-mannopyranosides

4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha- and beta-D-mannopyranosides were prepared from methyl 4,6-O-benzylidene-alpha-D-glucopyranoside and 1,3,4,6-tetra-O-acetyl-alpha-D-glucopyranose, respectively. Chemoselective reduction of both azides with hydrogen sulfide readily afforded 4-nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D- and -beta-D-mannopyranosides in higher yields than reduction with triphenylphosphine or a polymer-supported triarylphosphine. Subsequent de-O-acetylation yielded 4-nitrophenyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside and 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-mannopyranoside in 20% and 44% overall yields, respectively.     

Ca2+位于H2O2上游参与H2S诱导的拟南芥气孔关闭过程

以拟南芥为材料,利用药理学实验,结合分光光度法和激光共聚焦显微技术,研究了Ca2+在硫化氢(H2S)诱导拟南芥气孔关闭过程中的作用及其与过氧化氢(H2O2)的关系。结果表明： H2S诱导气孔关闭, Ca2+螯合剂EGTA和质膜Ca2+通道阻断剂硝苯地平(Nif)能不同程度抑制H2S诱导的气孔关闭,而内质网钙泵阻断剂毒胡萝卜素(Thaps)对H2S的作用无显著影响。由此推测, Ca2+参与调节H2S诱导的拟南芥气孔关闭过程,且胞质中Ca2+来源于胞外Ca2+的内流。另外, H2S诱导拟南芥叶片NADPH氧化酶基因AtRBOHD和AtRBOHF以及细胞壁过氧化物酶基因AtPRX34表达增强,促进叶片和保卫细胞中H2O2积累, EGTA对此起抑制作用,而外源CaCl2处理上调AtRBOHD、AtRBOHF和AtPRX34的表达。表明Ca2+可能位于H2O2上游参与H2S诱导的拟南芥气孔关闭过程。     

Smurf2 is a TRAF2 binding protein that triggers TNF-R2 ubiquitination and TNF-R2-induced JNK activation

TRAF2 plays a central role in TNF-induced signalling to NF-κB and JNK/p38 MAPK. To better understand the molecular mechanisms that mediate this dual function of TRAF2, we performed a yeast two-hybrid screening for TRAF2 interacting proteins using the Sos recruitment system. This resulted in the identification of the E3 ubiquitin ligase Smurf2 as a TRAF2 binding protein. TRAF2 overexpression was shown to trigger Smurf2 ubiquitination and the formation of a TNF-R2/Smurf2 complex. Smurf2 on its turn promoted TNF-R2 ubiquitination and the relocalization of TNF-R2 as well as TRAF2 to a detergent-insoluble cell fraction. This was associated with enhanced TNF-R2-induced JNK activation, whereas TNF-R2-induced NF-κB activation remained unaffected. These results suggest an important role for Smurf2 binding to TRAF2 in determining specific signalling outputs of TNF-R2.     

Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

PPP2R2A在乳腺癌细胞中结合GFPT2并导致其去磷酸化

PPP2R2A是PP2A磷酸酶的调控亚基之一,以往的研究报道显示,PPP2R2A可促进肿瘤细胞生存和生长。本研究通过串联亲和纯化联合HPLC-Chip-ESI/MS/MS筛选PPP2R2A的相互作用蛋白质,分析结果显示,L-谷氨酰胺-D-果糖-6-磷酸转氨酶1(Glutamine-fructose-6-phosphate transaminase 1,GFPT1)和L-谷氨酰胺-D-果糖-6-磷酸转氨酶2(Glutamine-fructose-6-phosphate transaminase 2,GFPT2)是PPP2R2A可能的结合蛋白。通过免疫荧光共定位、GST Pull-down和免疫共沉淀等方法,进一步确认了PPP2R2A和GFPT1及GFPT2的相互结合。通过shRNA下调PPP2R2A后,GFPT2的磷酸化水平显著增加,但GFPT1的磷酸化水平改变不明显。GFPT2是O-GlcNAC糖基化修饰通路中的一个限速酶,在乳腺癌细胞MDA-MB-231中下调PPP2R2A后,蛋白质O-GlcNAC糖基化修饰水平增加。这些结果表明,PPP2R2A可直接结合GFPT2,并导致其去磷酸化,进而影响细胞内O-GlcNAC糖基化修饰。     

拟南芥血红蛋白1(AtGLB1)与过氧化氢的相互作用

拟南芥的血红蛋白1(AtGLB1)属于非共生的血红蛋白。在低氧胁迫中对植物细胞中过氧化氢(H2O2)内稳态的维持起了很重要的作用。为了检测AtGLB1与H2O2能否直接相互作用,我们扩增了拟南芥的AtGLB1基因,并将其克隆到原核表达质粒pET32a中,测序鉴定正确后转化大肠杆菌BL21。IPTG诱导目的蛋白表达后,镍离子亲和层析柱(Ni2+-NTA)纯化了靶蛋白。体外表达的氧合的AtGLB1能与H2O2直接相互作用。因此,与H2O2反应可能是AtGLB1清除低氧胁迫下产生的H2O2的一种方式。