Monitoring Changes in Membrane Polarity,Membrane Integrity,and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC₄(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca²⁺, K⁺, and H⁺, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.     

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.     

围垦滩涂湿地旱生耐盐植物的入侵和影响

以上海市崇明东滩1998年围垦湿地为例,基于彩红外航片的景观格局分析以及样地群落调查,分析了湿地人工排水后,旱生耐盐植物在湿地原生芦苇群落中的入侵和影响.结果表明：以碱菀为建群种的旱生耐盐植物群落在围垦湿地区从东北向西南扩散,速度达143 m·a^-1,原生芦苇斑块迅速收缩、破碎,缀块密度指数和形状指数分别增长22.7倍和1.3倍;2006和2007年两个生长季后群落中碱菀生物量高达2783.8 g·m^-2,占群落总生物量的98％,重要值达88%;芦苇生物量和重要值则分别下降98％和83%.表明侵入过程与地下0～20 cm土壤水分和土壤盐度密切相关.应深入研究旱生耐盐植物的侵入机制,探讨合理、动态的湿地排水管理策略,以便控制旱生耐盐植物群落的迅速入侵.     

重金属污染可能改变稻田土壤团聚体组成及其重金属分配

采集了太湖地区污染与非污染稻田表土,采用原状土低能量分离-分散技术提取土壤团聚体粒组,分析土壤中不同粒径团聚体颗粒组质量组成和Pb、Cd、Hg、As等重金属元素的含量, 讨论重金属污染下土壤团聚体组成和重金属团聚体分配的变化.结果表明：在重金属污染下,供试水稻土砂粒级团聚体减少,而较细粒径团聚体相对增多;4种重金属元素在不同粒径团聚体颗粒组中的含量存在差异,但随粒径的变化趋势基本一致,即在<0.002 mm粒径的颗粒组中最高, 其次是0.2～2 mm粒径的颗粒组,而在0.02～0.2 mm和0.002～0.02 mm粒径的团聚体中呈现亏缺现象（富集系数为0.56～0.96）.表明重金属污染可能减弱了较大土壤团聚体的形成,导致细粒径团聚体相对增多,从而明显提高了重金属元素在粉砂和粘粒组团聚体中的分配,这可能进一步提高了污染农田重金属的水迁移和大气颗粒物迁移的风险.对于重金属污染对稻田土壤生物物理和生物化学过程的影响及其机制还需要进一步研究.     

五节芒定居对尾矿砂重金属形态、微生物群落功能及多样性的影响

选择尾矿砂裸地和4个五节芒(Miscanthus floridulus)定居地(RI、RII、RIII和RIV)为样地, 分别研究了五节芒定居对尾矿砂重金属形态转化和微生物功能参数的影响。结果表明: 五节芒自然定居显著地提高了尾矿砂碳酸盐结合态和硫化物-有机物结合态重金属比例(p＜0.05), 降低了尾矿砂残渣态重金属的比例(p＜0.05)。土壤微生物群落的纤维素分解作用、酚转化作用、固氮作用、氨化作用、硝化作用、有机磷转化作用、功能多样性、4类不同碳源(碳水化合(CH)、聚合物(PL)、胺类化合物(AM)和杂合物(ML))均随着五节芒自然定居显著提高(p＜0.05)。而氨基酸(AA)的利用强度却显著下降(p＜0.05)。典范相关分析(CCA)表明: 土壤微生物功能参数的总体变化与土壤碳酸盐结合态和硫化物-有机物结合态重金属的含量呈显著正相关, 与残渣态重金属含量呈显著负相关。该研究结果表明, 五节芒定居不仅促进了尾矿砂重金属朝着沉淀态和螯合态方面转化, 而且还显著地改善了尾矿砂微生物群落的功能发挥。因而, 五节芒在重金属矿业废弃地恢复实践中具有较大的应用潜力。     

植物螯合肽及其在重金属耐性中的作用

综述植物螯合肽的生物合成及其在重金属耐性中的作用．有毒重金属在土壤中的积累不仅影响作物的生长和产量形成,而且严重威胁农产品的安全性．植物对重金属的耐性和积累在种间和基因型之间存在着很大的差异,在重金属胁迫条件下植物螯合肽(PC)的合成是植物对胁迫的一种适应性反应,耐性基因型合成较多的PC谷胱苷肽是合成PC的前体,PC可与重金属螯合,并进一步转运至液泡贮存,使细胞质的重金属浓度降低,从而达到解毒效果．重金属诱导植物合成PC的遗传机理和生化途径有赖于分子生物学的深入研究,cD-敏感型拟南芥突变体Cad1-1(缺失GSH)和Cad2-1(缺失PC合成诱导酶)的分离及相关研究,佐证了PC在Cd-解毒中起关键作用．对PC在重金属污染土壤或水体的植物修复和农作物安全生产中的意义进行了讨论．     

温度、盐度、光照对海洋卡盾藻生长和产毒的影响

在正交试验条件下,分析了盐度、温度、光照强度对海洋卡盾藻生长和产毒的影响.结果表明：在盐度22、33、45和温度20 ℃、25 ℃、30 ℃以及光照强度2000、3000、4500 lx条件下,三因素对海洋卡盾藻生长的影响均不显著,盐度是影响海洋卡盾藻产毒的主要因子;盐度45、温度30 ℃、光照2000 lx下海洋卡盾藻的比生长速率最大,盐度22、温度20 ℃、光照4500 lx下海洋卡盾藻的产毒能力最强;低盐条件不利于海洋卡盾藻的生长,但有利于溶血毒素的合成;当海洋卡盾藻生长受到限制时,其溶血毒素合成增多.     

斜纹夜幼虫对食物中重金属Ni2+的积累与排泄

为明确食物中重金属离子在昆虫体内的分布和转移情况,本文采用等离子体原子发射光谱仪检测了植食性昆虫斜纹夜蛾Spodoptera litura Fabricius连续3个世代6龄幼虫对食物中过量Ni²⁺的排泄和积累情况。结果表明：大部分Ni²⁺可通过粪便排出体外;沉积在体内的Ni²⁺主要积累在中肠,部分Ni²⁺可通过中肠上皮细胞基底膜进入血淋巴,经由血淋巴的转运作用积累在脂肪体和表皮等组织中。6龄幼虫不同组织中所积累Ni²⁺的含量为中肠>脂肪体>表皮,且不同组织和粪便中的Ni²⁺都随饲料中Ni²⁺浓度的增加而增加,并存在显著的剂量-反应关系。研究结果可为进一步研究过量Ni²⁺对斜纹夜蛾幼虫的生长发育和繁殖的影响,以及斜纹夜蛾幼虫不同组织对Ni²⁺的解毒能力等提供一定依据。     

重金属污染对土壤有机质积累的影响

采用田间采样分析与室内培养试验相结合的方法,研究了不同重金属污染土壤中有机质积累的差异及重金属污染强度对土壤有机质矿化动态变化的影响.结果表明：污染土壤中重金属的大量积累可减弱有机物质的矿化速率,增加土壤有机质的积累.土壤中颗粒态有机质及其占总有机碳的比例随重金属积累的增加而增加;而微生物生物量碳占总碳的比例却随土壤重金属污染水平的提高而下降.污染土壤中颗粒态有机质对重金属有显著的富集,这可能是影响土壤有机物质进一步矿化的原因之一.重金属污染可改变土壤有机质的矿化速率,影响土壤有机质的积累与分配.     

沈阳某冶炼厂废弃厂区的人类健康风险评价

以沈阳某冶炼厂废弃厂区重金属污染监测为依据,采用美国环保局最新的人类健康风险评价标准方法对沈阳某冶炼厂废弃地块污染土壤进行了评价,并且假设未来该土地利用类型为工业用地(Ⅰ)或休闲用地(Ⅱ).评价结果显示:工业用地(Ⅰ)和休闲用地(Ⅱ)的累积非致癌风险指数分别为2.65×10-2和3.67×10-2;工业用地(Ⅰ)和休闲用地(Ⅱ)由呼吸摄入Cd造成的潜在致癌风险指数分别为4.48×10-9和7.30×10-10,不会对在该地区工作和休闲的人们造成身体健康上的伤害;无论是工业用地假设还是休闲用地假设,由无机铜造成的人类健康风险在整个风险中所占的比例最大;由美国环保局的人类健康风险评价方法反推得出的冶炼厂地块未来为工业用地的土壤修复目标值均小于我国工业企业土壤环境质量风险评价基准值.