不同浓度盐和H_2O_2对海马齿PHGPx活性的影响

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发挥重要作用.本研究探讨了海马齿PHGPx活性的测定,检测了不同浓度盐和H_2O_3胁迫对PHGPx活性的影响.结果显示,以蒸馏水为缓冲液提取的叶片总蛋白效果较好;NaCl梯度浓度处理下,海马齿叶片PHGPx活性呈先降低后升高然后再降低的趋势,其中500 mmol/L NaCl处理可以诱导最大活性;H_2O_2梯度浓度处理下,海马齿叶片PHGPx活性呈先升高后降低再升高趋势,0.5 mmol/LH_2O_2处理获得最大活性;海马齿植株经H_2O_2清除剂DMTU处理后再用H_2O_2处理,PHGPx的活性降低,同时NaCl的诱导效果并不受到影响.这些研究结果表明,海马齿中PHGPx的活性受到盐和H_2O_2的调节,并且它们对PHGPx酶活的调节可能是两个独立的过程.     

干旱条件下大豆叶片H_2O_2代谢变化及其同抗旱性的关系

干旱条件下大豆叶片H_2O_2含量增加,AsA POD与 GR活性均表现“先上升后下降”的趋势。叶片AsA与GSH含量均随干旱时间的延长而逐渐下降,而PPOD活性则持续增加。抗旱性较强的小粒大豆品种7605在干旱条件下能维持较强的 H_2O_2清除能力,H_2O_2累积较少。     

Cd~（2＋）胁迫诱导烟草悬浮细胞脯氨酸积累的生化途径及外源脯氨酸对Cd~（2＋）胁迫下H_2O_2产生的抑制作用

Cd~（2＋）可提高烟草悬浮细胞脯氨酸的含量,顺序上调脯氨酸合成关键酶鸟氨酸转氨酶（OAT）、精氨酸酶、△~1-吡咯啉-5-羧酸合成酶（P5CS）和谷氨酸脱氢酶（GDH）的活性,降低脯氨酸降解关键酶脯氨酸脱氢酶（ProDH）的活性,表明Cd~（2＋）胁迫诱导烟草细胞脯氨酸的积累是脯氨酸合成的鸟氨酸途径和谷氨酸途径顺序激活、而脯氨酸降解途径显著抑制的综合结果。此外,Cd~（2＋）能导致烟草细胞H_2O_2的快速产生及H_2O_2产生相关酶（质膜NADPH氧化酶、细胞壁多胺氧化酶及共价结合与离子结合细胞壁过氧化物酶）活性升高和脂质过氧化产物丙二醛（MDA）增加,导致烟草细胞的氧化胁迫。外源脯氨酸预处理显著抑制了Cd~（2＋）诱导的烟草细胞H_2O_2的产生与MDA的增加,减轻了Cd~（2＋）诱导的氧化胁迫。而脯氨酸抑制Cd~（2＋）诱导的H_2O_2产生可能是由于脯氨酸抑制了H_2O_2产生相关酶的活性所致。     

茶多酚对盐胁迫下小麦幼苗叶片生理特性的影响

以春小麦"陇春30号"为实验材料,主要研究了150 mmol/L NaCl和不同浓度(25 mg/L和100 mg/L)茶多酚(tea polyphenols, TP)单独或复合处理对小麦幼苗叶片叶绿素含量、叶绿素荧光参数及过氧化氢(H_2O_2)产生等生理特性的影响。结果表明:(1)150 mmol/L NaCl单独处理导致小麦幼苗叶片叶绿素含量及光适应下实际光量子产量[actual light quantum yield,Y(II)]、光化学淬灭(photochemical quenching, qP)、光合电子传递效率(photosynthetic electron transfer efficiency, ETR)均降低,非光化学淬灭(non-photochemical quenching, NPQ)增大;TP单独处理不影响这些指标。(2)盐胁迫诱导细胞壁过氧化物酶(cell wall-peroxidase, cw-POD)、二胺氧化酶(diamine oxidase, DAO)和多胺氧化酶(polyamine oxidase, PAO)活性显著增高;低浓度TP使cw-POD活性显著增大,而DAO和PAO活性无显著变化;不同的是,高浓度TP不影响cw-POD活性,却使DAO和PAO活性显著减小。(3)与NaCl单独处理相比,TP的添加导致NaCl处理下小麦幼苗叶片叶绿素含量增加,最大光化学效率(maximal photochemical efficiency,F_v/F_m)和ETR值增大,而NPQ值、H_2O_2含量及cw-POD、DAO和PAO三种酶活性均降低。总之,TP有效地缓解了盐胁迫诱导的小麦幼苗叶绿素含量的减少及对PS II光合电子传递效率和光化学反应速率的抑制,增强了植物的光合能力,与此同时降低了cw-POD、DAO和PAO活性,减少了H_2O_2的产生,从而缓解盐胁迫对小麦幼苗造成的伤害,提高小麦幼苗对盐环境的耐受性。     

外源ABA对大蒜试管苗玻璃化发生和抗氧化系统的影响

以大蒜品种‘二水早’试管苗为材料,从活性氧代谢的角度研究了外源ABA、H_2O_2和H_2O_2+ABA处理下的试管苗玻璃化率、活性氧积累与组织定位和抗氧化系统的响应特征,探讨ABA缓解试管苗玻璃化过程的机理。结果表明:(1)外源H_2O_2处理可诱导大蒜试管苗玻璃化发生,外源ABA处理下玻璃化率最低,可以缓解H_2O_2诱导的玻璃化的发生。(2)试管苗O_2~产生速率和H_2O_2含量在H_2O_2处理下最高,在ABA处理下最低;在添加H_2O_2的培养基中同时添加ABA能显著减少因外源H_2O_2处理引起的O_2~产生和H_2O_2积累。(3)试管苗CAT、POD和APX活性在外源H_2O_2处理前期(0~8d)均上升并显著高于对照,但其CAT、APX活性在处理后期(8~16d)下降,其同期POD活性也增加缓慢;各抗氧化酶的活性在外源ABA与H_2O_2+ABA处理前期(0~8d)均呈直线上升趋势,而它们在H_2O_2+ABA处理后期(8~16d)均显著高于H_2O_2处理。(4)各处理试管苗抗坏血酸和谷胱甘肽含量随处理时间先升高后降低,并以外源ABA处理下最高,外源H_2O_2处理下最低。(5)试管苗O_2~和H_2O_2产生部位主要在基部和叶尖,且外源ABA处理下组织中ROS的积累最少。(6)在ABA+H_2O_2处理下,大蒜试管苗内丙二醛含量和膜相对透性显著低于对照和H_2O_2处理。研究发现,外源ABA处理可有效降低大蒜试管苗的内源O_2~产生速率和H_2O_2含量,提高抗氧化酶活性和抗氧化物质含量,抑制活性氧在试管苗内的产生和运输,显著降低试管苗玻璃化率;外源ABA可通过增强大蒜试管苗抗氧化能力来抑制玻璃化发生。     

过氧化氢对铜锌超氧化物歧化酶结构损伤作用的进一步研究

我们曾观察到H_2O_2可使牦牛红细胞超氧化物歧化酶的活性下降及某些理化性质发生变化。在此基础上,进一步确证H_2O_2可使该酶结构受到损伤,其依据为H_2O_2作用后,酶的圆二色谱变化显示其β-折叠含量减少,无规则卷曲含量增加;酶分子中有二酪氨酸生成;组氨酸、精氨酸、赖氨酸、丝氨酸和苏氨酸含量相对减少,而天冬氨酸、谷氨酸和甘氨酸含量相对增加;用2.4-二硝基苯肼试剂可测得羰基生成;SDS-PAGE测出酶的部分亚基被降解为小肽段;用荧光胺法测得自由氨基量的增加。本文对上述变化的机理进行了探讨。     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H_2O_2的产生(简报)

逆境下,植物细胞内ABA含量急剧增加,同时植物也可通过一些酶代谢反应积累活性氧,如H_2O_2,O_2~-。ABA作为逆境信号对气孔运动的显著调节作用已被诸多实验所证实,但关于其对气孔运动调节的细节还知之甚少。H_2O_2作为氧化信号分子在植物抗病信号转导中已得到广泛研究,但H_2O_2是否介导保卫细胞的气孔运动还缺乏直接的证据。我们已初步发现H_2O_2可参与外源ABA诱     

α-萘乙酸对低温胁迫下油菜幼苗抗寒性的影响

为探究α-萘乙酸(NAA)对植物抗寒性的影响,以白菜型冬油菜‘陇油6号’为试验材料,经4℃、NAA+4℃、NAA+4℃+DPI(NADPH氧化酶抑制剂)、NAA+4℃+DMTU(H_2O_2清除剂)、NAA+4℃+U0126(MAPK抑制剂)和NAA+4℃+Tungstate(NO生成抑制剂)处理后,研究其对‘陇油6号’油菜的活性氧(H_2O_2和O_2~(-·))含量,抗氧化酶活性,丙二醛(MDA)、可溶性糖、脯氨酸和叶绿素含量,抗氧化酶基因(APX、CAT、GR、SOD)、Rboh A-F、MAPK3/4/6、CBF和ICE1基因表达量的影响。结果表明:与4℃低温处理相比,NAA+4℃处理下油菜根系中的细胞活性、H_2O_2和O_2~(-·)含量以及叶片中的MDA含量均降低;根系中的抗氧化酶(CAT、SOD、APX和POD)活性、叶片中的可溶性糖及脯氨酸含量、叶绿素含量、上述相关基因的表达量均升高,说明α-萘乙酸处理油菜可显著提高低温胁迫下油菜幼苗的抗氧化能力、光合能力和相关基因的表达,增强油菜幼苗的抗寒性。与NAA+4℃处理相比,NAA+4℃+抑制剂(DPI、DMTU、U0126和Tungstate)处理下油菜幼苗中叶绿素含量、抗氧化酶基因表达量、Rboh A-F、MAPK3/4/6、CBF和ICE1基因表达量均呈不同程度降低,说明H_2O_2和NO信号分子、NADPH氧化酶和MAP激酶级联途径均参与了α-萘乙酸增强油菜幼苗耐寒性过程的调控。     

外源H_2O_2对混合盐碱胁迫下燕麦幼苗叶片脯氨酸积累和代谢途径的影响

为探讨H_2O_2对盐碱胁迫下植物脯氨酸代谢的调控机理,以燕麦新品种‘定莜6号’幼苗为材料,采用水培法研究了外源H_2O_2对混合盐碱胁迫下燕麦脯氨酸积累和代谢途径的影响。结果表明,75 mmol·L-1混合盐碱(Na Cl∶Na_2SO_4∶Na HCO_3∶Na_2CO_3=12∶8∶9∶1)胁迫可促进燕麦幼苗叶片脯氨酸的积累,提高脯氨酸合成的鸟氨酸途径关键酶鸟氨酸δ-氨基转移酶(δ-OAT)活性,抑制脯氨酸合成的谷氨酸途径关键酶Δ1-吡咯啉-5-羧酸合成酶(P5CS)及脯氨酸降解限速酶脯氨酸脱氢酶(Pro DH)活性。在75 mmol·L-1混合盐碱胁迫下添加0.01~1 000μmol·L-1H_2O_2可显著提高燕麦幼苗叶片的脯氨酸含量,其中10μmol·L-1H_2O_2的作用最明显;10μmol·L-1H_2O_2上调了75 mmol·L-1混合盐碱胁迫下燕麦幼苗叶片的P5CS和δ-OAT活性,降低了Pro DH活性。此外,10μmol·L-1H_2O_2使75 mmol·L-1混合盐碱胁迫下燕麦幼苗叶片内源性H_2O_2含量急剧升高后迅速降低。表明外源H_2O_2能够提高混合盐碱胁迫下燕麦幼苗内源H_2O_2的含量,并通过活化脯氨酸合成的谷氨酸途径和鸟氨酸途径,抑制脯氨酸的降解,促进混合盐碱胁迫下燕麦幼苗脯氨酸的积累。     

外源H_2O_2对低温胁迫下柑橘叶片抗寒性的影响

以柑橘品种‘日南1号’离体秋稍为试材,采用Hoagland营养液培养方法,研究添加不同浓度H_2O_2处理对4℃低温胁迫下柑橘生长状态和叶片细胞相对电导率(REC)、丙二醛(MDA)含量、脯氨酸含量以及过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和过氧化物酶(POD)活性等生理指标的影响,筛选缓解柑橘低温伤害的最佳H_2O_2处理浓度,探讨外源H_2O_2处理对柑橘耐寒能力影响机制。结果显示:随低温胁迫时间的延长,各处理组柑橘叶片卷曲和叶片细胞膜伤害程度均逐渐加重;外源施加0.2和1.0mmol·L-1 H_2O_2处理均能缓解低温胁迫引起的叶片卷曲和萎蔫,降低叶片中REC和MDA的升高,减少叶片细胞中内源H_2O_2的积累,提高渗透调节物质脯氨酸的含量和抗氧化酶SOD、CAT和POD的活性,并以1.0mmol·L-1 H_2O_2缓解效果更为显著。研究表明,4℃低温能够引起柑橘离体秋梢叶片卷曲、枯萎、脱落和细胞膜伤害症状,外源1.0mmol·L-1 H_2O_2可以通过提高叶片的脯氨酸含量和SOD、CAT和POD抗氧化酶活性,有效缓解低温对柑橘叶片细胞膜的伤害,从而增强其抗寒性。     

PIP2信号与PIP2的再合成

磷脂酰肌醇-4,5-二磷酸（phosphatidylinositol-4,5-bisphosphate,PIP2）是一种分布在细胞膜内侧面的微量磷脂。虽然含量很低,但PIP2在细胞信号转导以及膜蛋白功能调节等方面却起着十分重要的作用。细胞膜中PIP2的含量水平呈动态平衡,在其代谢调节改变时,PIP2局部浓度的变化可影响特定蛋白的功能。该文就近二十年来针对PIP2信号和PIP2代谢调节相关的研究作一综述。     

Absence of enantioselectivity in the pharmacodynamics of P450 2B induction by 5-ethyl-5-phenylhydantoin in the male rat liver or in cultured rat hepatocytes

To explore the enantioselectivity of ligand interaction with the putative phenobarbital receptor, the pharmacodynamics of cytochrome P450 2B (CYP2B) induction by racemic 5-ethyl-5-phen-ylhydantoin and its two enantiomers were investigated in the male F344/NCr rat and in cultured adult male rat hepatocytes. Steady-state serum drug concentrations, measured following 14 days of administration of the compounds in the diet (0-1320 ppm, n = 3 rats per group), were used as an approximation of intrahepatocellular drug concentration. The serum xenobiotic concentrations associated with half-maximal hepatic CYP2B induction were 5-10 μM, based on measurement of pentoxy- or benzyloxyresorufin O-dealkylation activities, or immunoreactive CYP2B1 protein. The corresponding potency values in the hepatocyte culture experiments were 8-12 μM, based on measurement of total cellular RNA coding for CYP2B1. In both the in vivo and the hepatocyte culture experiments, the potencies for CYP2B induction were essentially equivalent for the racemate and the individual enantiomers of 5-ethyl-5-phenylhydantion. In the case of this compound, there would appear to be no enantioselectivity for CYP2B induction. This finding may be interpreted as evidence against receptor mediation in the induction of CYP2B activity, although it is also possible that a receptor is involved that does not exhibit enantioselectivity.     

Effective cancer therapy with a single injection of interleukin-2 at the site of the tumour

Mice with a severe metastasized tumour burden can be cured with a single local injection of interleukin-2. Such a treatment can also be effective against ocular squamous cell carcinoma in cows and transmissible venereal tumours in dogs. We did not notice any toxic effects of this treatment. Received: 9 April 1999 / Accepted: 21 June 1999     

Modulation of death receptor-mediated apoptosis by CK2

Protein kinase CK2 has long been known to be involved in cell growth and proliferation. Recent work has also implicated its role in the suppression of apoptosis. We originally documented that removal of survival or growth stimuli resulted in rapid loss of CK2 from the nuclear matrix and chromatin which preceded induction of apoptosis. Further, we demonstrated that overexpression of CK2 in cells promotes suppression of drug-mediated apoptosis. In the present work, we have extended these observations to demonstrate that CK2 can influence apoptosis mediated via the death receptors. Overexpression of CK2 resulted in suppression of apoptosis mediated by TNF-α, TRAIL, and Fas-L in cells responsive to these ligands, whereas downregulation of CK2 resulted in augmentation of apoptosis mediated by these ligands. To our knowledge, this is the first report to show that receptor-mediated apoptosis can be modulated by changes in CK2 in prostate cancer cells. Based on our previous observations together with the evidence presented here, we propose that CK2 has an impact on the process of apoptosis mediated by diverse type of mechanisms thus playing a global role in regulation of apoptotic activity in cells.     

Discovery and optimization of 1,7-disubstituted-2,2-dimethyl-2,3-dihydroquinazolin-4(1H)-ones as potent and selective PKCθ inhibitors

A high-throughput screening campaign helped us to identify an initial lead compound (1) as a protein kinase C-θ (PKCθ) inhibitor. Using the docking model of compound 1 bound to PKCθ as a model, structure-based drug design was employed and two regions were identified that could be explored for further optimization, i.e., (a) a hydrophilic region around Thr442, unique to PKC family, in the inner part of the hinge region, and (b) a lipophilic region at the forefront of the ethyl moiety. Optimization of the hinge binder led us to find 1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one as a potent and selective hinge binder, which resulted in the discovery of compound 5. Filling the lipophilic region with a suitable lipophilic substituent boosted PKCθ inhibitory activity and led to the identification of compound 10. The co-crystal structure of compound 10 bound to PKCθ confirmed that both the hydrophilic and lipophilic regions were fully utilized. Further optimization of compound 10 led us to compound 14, which demonstrated an improved pharmacokinetic profile and inhibition of IL-2 production in a mouse.     

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.     

两种新型白细胞介素-2的热稳定优越性

研制成两种新型白细胞介素-2(新型IL-2),其一为125Ser-IL-2,另一为125Ala-IL-2(即原型IL-2中125位Cys分别被Ser或Ala取代)。两种新型IL-2均已被高度纯化,并已完成了中试,获准临床应用。发现新型IL-2的热稳定优越性。在65℃加热,新型IL-2的稳定性高于原型,失活比原型慢,被保留的活性也较原型多。在0.1％SDS中能使失活的IL-2逐渐恢复一定的活性,也是新型IL-2恢复活性高。同时,比较了其它条件下这三种重组rIL-2的稳定性及SDS对其复性的影响,发现三者之间差异很大。上述结果均证明:125Ala-IL-2的热稳定性最好,125Ser-IL-2次之,原型较新型差。     

超声处理对PRC2相关蛋白染色质免疫共沉淀测序结果的影响

超声处理是染色质免疫共沉淀(Chromatin immunoprecipitation,Ch IP)实验过程中染色质片段化的重要手段之一。选用多梳抑制复合物2 (Polycomb repressive complex 2,PRC2)相关蛋白组蛋白甲基转移酶EZH2及其催化产物H3K27me3为代表,研究不同分子量大小的蛋白在不同超声处理时间下对染色质免疫共沉淀测序(Chromatin immunoprecipitation sequencing,Ch IP-seq)实验的影响,结果表明在启动子区或非启动子区,不同超声时间下小分子量组蛋白H3K27me3结合位点的注释基因均无明显差异,说明超声时间对组蛋白Ch IP-seq数据影响不大。与组蛋白不同,超声时间从10 min延长至20 min后启动子区EZH2新增结合位点的注释基因能够显著聚类在与肌动蛋白丝组装等相关通路上。超声20min相比10min,非启动子区EZH2新增基因的GO(Gene ontology)聚类通路要远多于丢失基因,且超声30 min相比20 min,非启动子区丢失基因的GO聚类通路要远多于新增基因,这些通路大多与RNA聚合酶Ⅱ(RNApolymeraseⅡ,RNAPII)、器官发育、细胞形态发生相关。这表明超声时间不足或过长均会导致EZH2的基因组定位信息的不全。另外,超声主要影响启动子区中的PRC2非结合区域及二价启动子区域的EZH2结合位点,还影响非启动子区中PRC2结合区域、PRC2不结合区域以及活化态增强子区域的EZH2结合位点。综上,建议对大分子量的染色质修饰相关蛋白优化超声处理时间,使形成的染色质片段聚集在100–500 bp可获得比较全面的基因组信息。对小分子量的组蛋白来说,超声时间对Ch IP-seq结果影响不大。     

Induction of proteins during phage reactivation induced by UV irradiation, oxygen and peroxide in Bacteroides fragilis

Abstract Phage reactivation systems in Bacteroides fragilis were induced by far-UV irradiation, O₂ and H₂O₂. These three treatments also induced the synthesis of 3, 6, and 4 protein bands, respectively, which were easily detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Two proteins with apparent M _r s of approx. 90 000 and 70 000 were induced by all three treatments. Caffeine completely inhibited UV- and O₂-induced phage reactivation and prevented the synthesis of the M _r 90 000 and M _r 70 000 proteins. The results suggest that these two proteins may be involved in phage reactivation processes induced by UV, O₂ and H₂O₂ in B. fragilis .