胞外ATP对AlCl3诱导的细胞死亡过程中胞内H2O2和Ca2+水平的影响及其生理机制分析

该实验以烟草悬浮细胞 BY 2 为材料,在烟草悬浮细胞中分别加入0.05、0.10、0.15、0.20 mmol·L^-1AlCl₃,以等体积去离子水处理的悬浮细胞液为对照,并依据前述实验结果选择0.15 mmol·L^-1 AlCl₃,分别添加5 mmol·L^-1 DMTU(H₂O₂ 抑制剂)、20 μmol·L^-1CaCl₂、15 μmol·L^-1 LaCl₃(Ca²⁺通道抑制剂)和50 μmol·L^-1 ATP设计多项处理,分析胞外ATP(eATP)对铝离子(Al³⁺)胁迫引起的植物细胞死亡及其胞内H₂O₂、Ca²⁺的影响,以揭示Al³⁺胁迫下植物调节细胞死亡的可能机制,进一步扩展对eATP功能的认知。结果显示：(1)随着 AlCl₃ 胁迫浓度的提高,细胞死亡水平和胞内H₂O₂水平上升,而胞内Ca²⁺和eATP水平则逐渐降低。(2)外援施加H₂O₂抑制剂 DMTU(二甲基硫脲)和Ca²⁺能够有效缓解AlCl₃诱导的细胞死亡水平的上升;而Ca²⁺通道抑制剂LaCl₃(三氯化镧)则加剧了AlCl₃胁迫下的细胞死亡。(3)在AlCl₃胁迫下对细胞添加外源ATP,能够缓解AlCl₃胁迫下胞内H₂O₂水平上升和Ca²⁺水平下降的同时,并显著降低AlCl₃胁迫导致的细胞死亡。研究表明, Al³⁺以剂量依赖的模式提升细胞死亡和细胞内H₂O₂的水平并降低胞内Ca²⁺和eATP水平,AlCl₃诱导的细胞死亡受到H₂O₂和Ca²⁺水平变化的调节,eATP可以通过调节H₂O₂与Ca²⁺水平缓解AlCl₃诱导的细胞死亡。推测Al³⁺胁迫可能通过抑制钙离子通道而破坏了细胞内H₂O₂和Ca²⁺之间的协同关系,外源ATP对Al³⁺诱导H₂O₂上升的缓解作用可能是由于其提升了细胞的抗氧化能力。     

PKCγ过表达诱导C3H10T1/2细胞生长失控的初探

通过DNA重组构建蛋白激酶C_γ(PKC_γ)亚类的重组质粒并经基因转染技术和DNA印迹、蛋白质印迹与PKC活性分析,获得了过表达PKC_γ的C₃H₁₀T_1/2细胞——NCP4.NCP4细胞生长速率提高,流式细胞光度术检测表明,NCP4细胞G1期百分率下降,S期和G2+M期百分率升高,与对照组细胞相比,血清依赖性明显下降,贴壁依赖性降低,在软琼脂中形成小集落,出现部分转化表型.进一步检测,首次观察到NCP4细胞中癌基因c-sis表达明显增强,这可能是NCP4细胞血清依赖性下降的分子机理之一.实验表明,在正常C₃H₁₀T_1/2细胞中PKC_γ的过表达可直接导致细胞增殖加速并可诱导出现部分转化特征.     

过氧化氢对培养心肌细胞损伤作用的研究

氧化应激时产生大量的自由基,造成心肌细胞的损伤.过氧化氢(H₂O₂)是有机体氧化代谢产物,同时是一种活性氧.应用不同浓度的H₂O₂,分别于不同作用时间,动态观察其对心肌细胞的损伤作用.从实验结果看到,低浓度的H₂O₂（＜0.1 mmol/L）作用2 h,使心肌细胞产生早期的生物化学的改变,如MDA产生堆积和细胞周期时相改变（G1期细胞增加,G2期细胞减少）,此时心肌酶基本无泄漏,心肌细胞的死亡率很低,HE形态学观察基本无改变;随着H₂O₂浓度的增加（1～5 mmol/L）和作用时间的延长,进一步诱导细胞损伤加剧,LDH释放和MDA积累明显升高,细胞死亡率也明显增加,已具有统计学意义.同时可观察到其病理形态学的坏死性改变;当10 mmol/L H₂O₂作用时,细胞大量死亡,形态学可见细胞极度收缩、脱落,形成大面积的细胞脱失区.因此,H₂O₂作为一种活性氧自由基,依其浓度和作用时间不同可造成不同程度的心肌细胞的损伤.辣根过氧化物酶作为一种自由基清除剂,可明显减少H₂O₂活性氧自由基对心肌细胞的损伤作用.     

Smac/DIABLO在过氧化氢所致C2C12肌原细胞凋亡中的作用

为探讨Smac/DIABLO在过氧化氢(H₂O₂）所致C₂C₁₂肌原细胞凋亡中的作用,采用Hoechst 33258染色,观察H₂O₂ (0.5 mmol/L)处理C₂C₁₂肌原细胞不同时间后,细胞核形态学改变并计算凋亡核百分率,DNA抽提及琼脂糖电泳观察凋亡特征性梯状带,利用细胞成分分离后蛋白质印迹分析H₂O₂是否导致Smac/DIABLO从线粒体释放,采用Caspase检测试剂盒及蛋白质印迹分析Caspase-3和Caspase-9的活化,转染Smac/DIABLO基因,观察Smac/DIABLO过表达对H₂O₂所致的C₂C₁₂肌原细胞凋亡的影响.结果表明:H₂O₂处理1 h后,Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放入胞浆,2 h更明显;H₂O₂处理4 h后,Caspase-3和Caspase-9活化,12 h达高峰;H₂O₂处理24 h后,C₂C₁₂肌原细胞显示特征性的凋亡形态改变,凋亡核百分率明显升高,DNA电泳出现明显“梯状”条带.与单纯过氧化氢损伤组相比,Smac/DIABLO高表达的C₂C₁₂肌原细胞经过氧化氢损伤组的Caspase-3和Caspase-9的活化、凋亡核百分率的升高、“梯状”条带的出现均更明显.结果表明,H₂O₂可导致Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放,促进Caspase-9和Caspase-3的活化而促进细胞凋亡的发生.     

NO和H2O2诱导大豆根尖和边缘细胞耐铝反应的作用

 NO和H₂O₂是参与植物抗非生物胁迫反应的重要信号分子, 为了确定NO和H₂O₂在大豆(Glycine max)根尖和根边缘细胞(root border cells, RBCs)耐铝反应中的作用及其相互关系, 以‘浙春3号’大豆为材料, 研究了铝毒胁迫下大豆根尖内源NO和H₂O₂的变化, 以及外源NO和H₂O₂诱导大豆根尖和RBCs的耐铝反应。结果表明, 50 μmol·L^–1 Al处理48 h显著抑制大豆根的伸长, 提高Al在根尖的积累, 同时显著增加根尖内源NO和H₂O₂含量。施加0.25 mmol·L^–1外源NO供体亚硝基铁氰化钠(Na₂[Fe(CN)₅NO]·2H₂O, sodium nitroprusside, SNP)和0.1 mmol·L^–1H₂O₂, 能有效地缓解Al对大豆根伸长的抑制、根尖Al积累和RBCs 的死亡, 该缓解作用可以被0.05 mmol·L^–1 NO清除剂2-(4- 羧基苯)-4,4,5,5- 四甲基咪唑-1- 氧-3- 氧化物, 钾盐(C₁₄H₁₆N₂O₄·K, carboxy-PTIO, cPTIO)和150 U·mL^–1 H₂O₂清除酶(catalase, CAT)逆转。并且外源NO能够显著促进根尖H₂O₂的积累, 而外源H₂O₂对根尖NO的含量无显著影响。这表明NO和H₂O₂是诱导大豆根尖及RBCs耐铝反应的两种信号分子, NO可能通过调控H₂O₂的形成, 进而诱导大豆根尖及RBCs的耐铝反应。     

玉米叶表皮短细胞发育过程的形态特征及栓质细胞作用研究

采用大田试验,直接撕表皮或对叶片进行固定处理,结合单染、复染、荧光染色等多种细胞学显色方法,利用光学显微镜、荧光显微镜和扫描电子显微镜系统观察玉米叶表皮短细胞的发生时期、发育过程、分布规律以及形态结构特征,研究K⁺和H₂O₂在栓质细胞中的分布变化与表皮其它细胞中K⁺和H₂O₂的分布及气孔器开关的关系,为进一步挖掘短细胞的新功能提供细胞学依据。结果表明：（1）短细胞是同步发生在玉米多叶位新表皮组织形成过程中,所有植株从第7新生叶,大部分第6叶,极少数第5叶的基部同时开始发生短细胞,之后新生的高位叶也均发生短细胞,并随着叶位的升高叶片各部位短细胞密度均增大,所有植株的1~4叶（因不再生长）均无短细胞出现。（2）初期发育的叶表皮细胞进行不对称分裂,生成相互交替的长、短细胞,有的短表皮细胞横（垂直叶脉）分裂,形成栓质细胞和硅质细胞对;栓质细胞基部与叶肉细胞相邻,硅质细胞嵌在栓质细胞和表皮细胞间偏上。（3）有短细胞发生的叶片,宏观背面发亮且覆有蜡质层,微观表皮细胞的着色特性发生了变化;栓质细胞为面包形柱状细胞,硅质细胞为哑铃形扁细胞。（4）气孔器张开时,栓质细胞中没有K⁺和H₂O₂的积累;气孔器关闭时,栓质细胞中积累了大量的K⁺和H₂O₂,且栓质细胞中K⁺和H₂O₂的积累始终与副卫细胞中K⁺和H₂O₂的积累变化一致,而硅质细胞和长细胞没有K⁺和H₂O₂的积累。该研究确定了玉米叶表皮短细胞发生的时期;展示了其发育过程的形态学变化特征;发现栓质细胞中K⁺和H₂O₂的积累随气孔器开关呈周期性变化,且与副卫细胞中K⁺和H₂O₂的积累变化保持一致。     

外源亚精胺对甜瓜幼苗白粉病抗性的影响

以高感白粉病甜瓜‘066’、感病品种‘0544’、抗病品种‘Yuntian-930’为试材,对白粉病菌胁迫下外源亚精胺（Spd）处理的甜瓜幼苗叶片超氧化物岐化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）和多酚氧化酶（PPO）活性,以及过氧化氢（H₂O₂）、丙二醛（ MDA）和光合色素含量进行了研究,探讨外源Spd对甜瓜白粉病抗性的诱导效应及其生理机制。结果表明：（1）白粉病菌接种后甜瓜叶片的防御酶活性、光合色素含量先升后降,MDA含量升高;（2）外源Spd处理缓解了白粉病菌胁迫下甜瓜叶片防御酶活性、光合色素含量的下降,降低MDA含量及甜瓜的病情指数,并以1.0 mmol/L Spd处理效果最佳;（3）外源Spd处理使甜瓜叶片产生了更多的H₂O₂;（4）外源Spd提高甜瓜对白粉病抗性具有一定广谱性,且可向上传导。研究认为,H₂O₂可能参与白粉病胁迫下信号的传递,外源Spd可通过缓解白粉病菌胁迫下甜瓜防御酶活性和光合色素含量的下降来诱导甜瓜对白粉病的抗性。     

SelS高表达保护人脐静脉内皮细胞免于H2O2诱导的细胞损伤

采用以下方法探讨SelS在内皮细胞中的表达和作用：将SelS基因克隆到真核表达载体pLNCX2，RT-PCR、XhoⅠ/ClaⅠ双酶切以及DNA序列分析验证目的基因；利用脂质体转染技术将pLNCX2-SelS或pLNCX2转染至人脐静脉内皮细胞(ECV₃₀₄细胞)，RT-PCR检测重组基因SelS的表达；MTT方法检测转染后过氧化氢(H₂O₂)对内皮细胞增殖能力的影响；硫代巴比妥酸法测定暴露于H₂O₂中不同转染组细胞脂质过氧化产物丙二醛含量. 结果表明：成功构建真核表达载体pLNCX2-SelS；转染后重组SelS mRNA表达水平是内源性水平的1.76倍；H₂O₂对ECV₃₀₄细胞损伤后，高表达SelS组细胞活性增强、H₂O₂诱导产生的丙二醛减少. 上述结果表明，高表达SelS可保护内皮细胞免于H₂O₂诱导的细胞损伤，其作用机制与抗氧化有关.     

H2O2诱发人成纤维细胞衰老样变化的基因表达谱

以50 μmol/L H₂O₂作用体外培养的人胚肺二倍体成纤维细胞4次,使之出现不可逆的衰老表型.提取年轻细胞及H₂O₂处理早老细胞的mRNA,以荧光物Cy3标记年轻细胞cDNA,Cy5标记H₂O₂处理的细胞cDNA,并与点有4 096条人类基因的芯片杂交,利用计算机数据处理判断基因是否存在表达差异.结果显示：有123种基因的表达变化较显著,这些基因参与细胞周期进程、细胞代谢及蛋白质修饰、细胞外基质及细胞骨架蛋白的形成和调节、炎症反应、调节受体酪氨酸蛋白激酶和G蛋白耦联受体信号转导.     

植物叶片中过氧化氢含量测定方法的改进

Ti（Ⅳ）-H₂O₂比色法因背景物质干扰而测得的植物叶片内H₂O₂含量偏高，5％三氯乙酸抽提，活性炭脱色，Ti（Ⅳ）-4-(2-吡啶偶氮)间苯二酚(PAR)比色法测得的H₂O₂含量偏低.萃取法有效地脱去丙酮提液中的色素，且H₂O₂的回收率在95％以上.用过氧化氢酶(CAT）处理作空白对照，利用H₂O₂与Ti（Ⅳ）-PAR的显色反应，建立了一种简便、快速、准确的植物叶片内的H₂O₂含量测定方法，H₂O₂的最低检测浓度为0.25 μmol·L^－1.用该方法测得多种植物叶片中H₂O₂的含量在0.1～0.8 μmol·g^－1.     

On the proper use of sigma analysis in the study of flash-induced oxygen evolution

Analysis of oxygen yield by photosynthetic higher plants systems submitted to flash sequence using the so-called sigma method as proposed in Lavorel (1976), J Theor Biol 57: 171–185 has been recently criticized in this Journal by Meunier and Popovic (1989), Photosynth Res 22: 131–136. By properly applying the above method—in particular using Meunier and Popovic's own data—it is argued that the criticism is not justified.     

Purification of Rabbit Liver Guanine Aminohydroiase

Abstract

Rabbit liver guanine aminohydrolase has been purified 1250-fold by utilization of an affinity chromatographic separation on 9-(p-aminoethoxyphenyl)guanine-Sepharose with 50% recovery of activity. Polyacrylamide gel electrophoresis of the purified preparations revealed several protein bands which corresponded to regions of enzyme activity measured on gels which had been run under the same conditions. Gel concentration studies of the protein migration rate showed that the protein bands differed in molecular size. The minimum molecular weight was 100, 000 from gel permeation chromatography studies. The pH optimum was near pH 8 and the Km, with guanine as substrate was 5.6 × 10^?6M. The latter values are in close agreement with partially purified preparations described in the literature.     

Seasonal Variation in Schools’ Indoor Air Environments and Health Symptoms among Students in an Eastern Mediterranean Climate

The indoor air quality (IAQ) in classrooms highly affects the health and productivity of students. This article aims to clarify seasonal variation in indoor environment and sick building syndromes (SBS) symptoms in an Eastern Mediterranean climate. A series of field measurements were conducted during the fall and winter seasons from October 2011 to March 2012 in 12 naturally ventilated schools located in the Gaza Strip. Data on environmental perception and health symptoms were obtained from 724 students by using a validated questionnaire. The results showed that indoor PM₁₀ and PM_2.5 concentrations were 426.3 ± 187.6 μg/m³ and 126.6 ± 94.8 μg/m³, respectively. The CO₂ concentrations and ventilation rate widely exceeded their reference values during the winter season. The prevalence rates of general symptoms were relatively high at baseline assessment in the fall season and increased significantly during follow-up in the winter season. Significant increases in disease symptoms such as mucosal irritation and pre-existing asthma symptoms among students could be related to poor indoor air quality. Five distinct groups of SBS symptoms from factor analysis of students’ related symptoms were significantly correlated with PM₁₀ and PM_2.5, CO₂, ventilation rate, and indoor temperature. As vulnerable children, this situation negatively affects their school performance and health.     

A simple procedure to express results of soil analysis (1∶2 volume extract) on a volume basis

Summary A simple method of expressing the results of routine soil analysis by volume is described. A good agreement with the method estimating the volume-weight of the soil is achieved.     

Worldwide distribution of phosphoglucomutase 1 (PGM1) polymorphism detected by isoelectric focusing: A review

This paper reports an exhaustive and updated compilation of phenotype and allele frequency data for phosphoglucomutase locus 1 (PGM1), obtained with an analytical isoelectric focusing technique, in human populations. The analysis of the PGM1 allele frequency distributions within and among the major human groups together with the degree of diversification evaluated by Wright's F_st, computed per allele and averaged over alleles, are also presented.     

Study on air pollution in Beijing’s major industrial areas using multielements in biomonitors and NAA techniques

Three kinds of plant leaves, Chinese white aspen, arborvitae, and pine needles, have been sampled from the Yanshan Oil refinery complex, the Capital Iron and Steel Factory, and Badachu, a control area in Beijing, as biomonitors for air-pollution studies. Each sample was divided into two parts, washed, and unwashed. Thirty-one trace elements (As, Au, Br, Ca, Cd, K, La, Lu, Mo, Na, Sb, Sm, U, W, Yb, Ba, Ce, Co, Cr, Cs, Eu, Fe, Hf, Hg, Rb, Sc, Se, Sr, Ta, Tb, Th, and Zn) have been determined by using the relative and the K₀ methods of instrumental neutron activation analysis (NAA) techniques. The results indicated the following: (1) The concentration of trace elements in unwashed samples are much higher than these in washed samples; (2) the area around Capital Iron and Steel Factory is heavily polluted, and the Yanshan Oil refinery complex area is moderately polluted; (3) Chinese white aspen is a good biomonitor in particular seasons and pine needles are better than arborvitae for yearly monitoring; (4) elements As, Cd, Hg, Co, Rb, Sb, W, and Zn are highly absorbed by Chinese white aspen. Pine needles are sensitive for the absorption the elements Br, Cr, Cd, Fe, Sc, Cs and rare earth elements, but arborvitae is very sensitive for the absorption of Sr.     

Homeostatic regulation upon changes of enzyme activities in the Calvin cycle as an example for general mechanisms of flux control. What can we expect from transgenic plants?

In order to explain the mechanisms of Calvin-cycle regulation, the general properties of metabolic systems under homeostatic flux control are analyzed. It is shown that the main characteristic point for an enzyme in such a system can be the value of a sharp transition from some constant homeostatic flux to a limitation by this enzyme. A special method for the quantitative treatment of the experimental dependence of a metabolic flux such as photosynthesis on enzyme content is developed. It is pointed out that reactions close to a thermodynamic equilibrium under normal conditions can considerably limit the homeostatic fluxes with a decrease of the enzyme content. Calvin-cycle enzymes are classified as non-limiting, near-limiting and limiting. The deduced rules for the regulation of a homeostatic metabolic pathway are used to explain the data obtained for transgenic plants with reduced activities of Calvin-cycle enzymes. The role of compensating mechanisms that maintain the photosynthesis rate constant upon the changes of enzyme contents is analyzed for the Calvin cycle. The developed analysis explains the sharp transitions between limiting and non-limiting conditions that can be seen in transgenic plants with reduced content of some Calvin-cycle enzymes, and the limiting role of such reversible enzymes as aldolase, transketolase and others. The attempt is made to predict the properties of plants with increased enzyme contents in the Calvin cycle.     

Effects of immersion in water containing high concentrations of CO2 (CO2-water) at thermoneutral on thermoregulation and heart rate variability in humans

Immersion in high concentrations of CO₂ dissolved in freshwater (CO₂-water) might induce peripheral vasodilatation in humans. In this study, we investigated whether such immersion could affect the autonomic nervous system in humans using spectral analysis of heart rate variability. Ten healthy men participated in this study. Tympanic temperature, cutaneous blood flow and electrocardiogram (ECG) were measured continuously during 20 min of immersion in CO₂-water. The ECG was analyzed by spectral analysis of R–R intervals using the maximal entropy method. The decrease in tympanic temperature was significantly greater in CO₂-water immersion than in freshwater immersion. Cutaneous blood flow at the immersed site was significantly increased with CO₂-water immersion compared to freshwater. The high frequency component (HF: 0.15–0.40 Hz) was significantly higher in CO₂-water immersion than in freshwater immersion, but the low frequency (LF: 0.04–0.15 Hz) /high frequency ratio (LF/HF ratio) was significantly lower in CO₂-water immersion than in freshwater immersion. The present study contributes evidence supporting the hypothesis that CO₂-water immersion activates parasympathetic nerve activity in humans.     

A sensitive, simple assay of mitochondrial ATP synthesis of cultured mammalian cells suitable for high-throughput analysis

A new assay has been developed to measure mitochondrial ATP synthesis of cultured mammalian cells. Cells in a microplate are exposed to streptolysin O to make plasma membranes permeable without damaging mitochondrial function and ATP synthesis is monitored by luciferase. Addition of inhibitors of F_oF₁-ATP synthase (F_oF₁), respiratory chain, TCA cycle and ATP/ADP translocator, as well as knockdown of β-subunit of F_oF₁, resulted in loss of ATP synthesis. Compared with the conventional procedures that need mitochondria fractionation and detergent, this assay is simple, sensitive and suitable for high-throughput analysis of genes and drugs that could affect mitochondrial functional integrity as represented by ATP synthesis activity.     

Modeling magnesium,phosphorus and potassium uptake by loblolly pine seedlings using a Barber-Cushman approach

The Barber-Cushman mechanistic nutrient uptake model, which has been utilized extensively to describe and predict nutrient uptake by crop plants, was evaluated for its ability to predict K, Mg, and P uptake by loblolly pine (Pinus taeda L.) seedlings. Sensitivity analyses were also used to investigate the impact of changes in soil nutrient supply, root morphological, and root uptake kinetics parameters on simulated nutrient uptake. Established experimental techniques were utilized to define the 11 parameters needed to model uptake by 1-0 seedlings of K, Mg, and P from a modified A horizon soil (Lilly series). Model predictions of K and P uptake over a 180-d growth period were underestimated by 6 and 11%, respectively. Estimates of Mg uptake were underestimated by 62%. While the level of agreement between predicted and observed K and P values was quite acceptable, analysis of parameter values and results of sensitivity analyses both indicated that the model underestimation of Mg uptake was the result of applying an I_max value developed under relatively low Mg concentration to a situation in which the functional I_max would be much higher due to the dominance of passive versus active uptake. Overall results of sensitivity analyses indicate that under the circumstances investigated, I_max, was the primary variable controlling plant uptake of K, Mg, and P. The dominance of this term over others was due to the relatively high C_li values for all three nutrients. Reducing (-50%) or increasing (+ 100%) other soil supply, root morphological, and remaining root uptake kinetics values did not substantially alter model estimates of nutrient uptake.