Biochemical speciation in chromium genotoxicity

The biochemical speciation of chromium compounds in mammalian cells is discussed with respect to uptake, metabolism, DNA binding and damaging. Whereas soluble hexavalent chromium is taken up rapidly and accumulated intracellularly after its reduction, compounds of trivalent chromium penetrate biomembranes about three orders of magnitude slower. Cr(VI) after its uptake is metabolised by electron donating compounds via Cr(V) to Cr(III) compounds. Chromium from various Cr(III) compounds, but not chromate, binds to chromatin in isolated cell nuclei. The DNA‐protein crosslinks and DNA strand breaks observed in rat liver and kidney after chromate administration are also found in vitro, when Cr(III) compounds (but not chromate) interacts with isolated nuclei. In the Chinese Hamster cell HGPRT mutation assay, three out of four tested Cr(III) complexes were found to be mutagenic. In a direct DNA strand break assay with supercoiled bacteriophage PM 2 DNA, neither chromate nor the four Cr(III) compounds tested caused nicks. However, the combined action of chromate plus glutathione as well as the isolated complex of pentavalent chromium, Na₄Cr(glutathione)₄, did cause DNA breaks. Reactive oxygen species are inferred to be the ultimate DNA nicking agents in this assay. In conclusion there appear to be two mechanisms of chromate genotoxicity; one with direct DNA damage caused by Cr(V) species and one via DNA‐protein crosslinks formed with Cr(III), the final reduction state of chromate.     

Impact of rhizobacteria on growth and chromium accumulation in Scirpus lacustris L. grown under chromium supplementation

Four chromate tolerant rhizobacterial strains viz., RZB-01, RZB-02, RZB-03 and RZB-04 were isolated from rhizosphere of Scirpus lacustris collected from Cr-contaminated area. These strains characterized at morphological and biochemical levels. The most efficient chromate tolerant strain RZB-03 was inoculated to fresh plant of S. lacustris and grown in 2 microg ml(-1) and 5 microg ml(-1) of Cr+6 supplemented nutrient solution under controlled laboratory condition. The effects of rhizobacterial inoculation on growth and chromium accumulation in S. lacustris were evaluated. The inoculation of rhizobacteria increased biomass by 59 and 104%, while total chlorophyll content by 1.76 and 15.3% and protein content increased by 23 and 138% under 2 microg ml(-1) and 5 microg ml(-1) concentrations of Cr+6, respectively after 14 d as compared to non-inoculated plant. Similarly, the Cr accumulation also increased by 97 and 75% in shoot and 114 and 68% in root of inoculated plants as compared to non inoculated plants at 2 microg ml(-1) and 5 microg ml(-1) Cr+6 concentrations, respectively after 14 d. The chromate tolerant rhizobacteria which play an important role in chromium uptake and growth promotion in plant may be useful in development of microbes assisted phytoremediation system for decontamination of chromium polluted sites.     

Model reactions of chromium compounds with mammalian and bacterial cells†

Chromate uptake, reduction, cytotoxicity and mutagenicity were studied with human red blood cells, Chinese hamster ovary (CHO) cells and/or Salmonella typhimurium mutant cells. All cell types rapidly took up chromates whereas chromium(III) salts were excluded under the experimental conditions. Red blood cells reduced and accumulated chromium from chromate. At concentrations above 0.1 mM, chromate inactivated the red cell chromate carrier. Chromate above 0.01 mM inhibited CHO cell proliferation irrespective of the cations present. Chromate and two chromium(III) complexes were mutagenic with Salmonella mutants in the Ames’ assay. A model for chromate metabolism and genotoxicity is proposed.     

Coordination chemistry and the carcinogenicity and mutagenicity of chromium(VI)

Chromate is a known carcinogen, it is only in recent years that the molecular mechanisms by which this toxicity may be expressed have been investigated. The toxicity of chromate may be mediated by the reaction of chromium(VI) with glutathione (GSH) to generate relatively stable chromium(V) complexes and other more reactive intermediates. The conditions favouring the formation of such complexes have been studied. Reactive intermediates generated during the reduction of chromate by GSH include thionyl radicals and at least two relatively stable chromium(V) species (g -1.996 and g -.986). Mixtures of chromium(VI) and glutathione and a chromium(V) complex of glutathione, which we have isolated from the reaction (g = 1.996), are capable of causing strand breaks in bacteriophage PM2 DNA. In contrast a chromium(III) complex of GSSG, one of the final products of the reaction between GSH and chromium(VI), does not damage DNA in closed circle assays. These observations support the suggestion that reactive intermediates generated during the reduction of chromium(VI) provide one route by which the genotoxicity of chromate may be expressed.     

一株溶藻细菌对铜绿微囊藻的溶藻机理初探

为确定溶藻细菌S7（Chryseobaterium）对铜绿微囊藻的溶藻方式,分别采用高温灭菌（121~123℃）、离心（10 000 r.min-1）、0.22μm滤膜过滤等方式对S7菌液进行处理,检测其对铜绿微囊藻的去除效果。并通过对溶藻过程中叶绿素a和丙二醛（MDA）含量的测定,藻细胞显微结构的观察和细胞成分的红外光谱分析,初步探讨菌株S7对铜绿微囊藻的作用机理。结果表明,S7是通过释放胞外活性物质间接溶藻,该物质具有很强的热稳定性,不属于蛋白质类物质。该活性物质对铜绿微囊藻的叶绿素a有明显的去除效果,并可导致藻细胞膜脂过氧化产物MDA积累量的显著提高和藻细胞解体。藻细胞红外光谱分析表明,经过溶藻物质作用的藻细胞,其蛋白质结构遭到破坏。通过试验结果,推测出菌株S7的溶藻机理：溶藻物质先损伤铜绿微囊藻的细胞壁和粘质胶被,然后通过改变膜的选择透过性进入藻细胞内部,分解叶绿素a,破坏蛋白质,造成藻体正常生理功能的丧失,最终导致藻细胞破裂。     

Transmembrane transport of polycyclic aromatic hydrocarbons by bacteria and functional regulation of membrane proteins

• Explaintheadsorption, uptake and transmembrane transport of PAHs by bacteria. • Analyze functional regulation of membrane proteins inthe transmembrane transport. • Proteomics technology such as iTRAQ labeling was used to access expressed proteins. • Single cell analysis technology wereused to study the morphological structure. In recent years, increasing research has been conducted on transmembrane transport processes and the mechanisms behind the microbial breakdown of polycyclic aromatic hydrocarbons (PAHs), including the role of membrane proteins in transmembrane transport and the mode of transmission. This article explains the adsorption, uptake and transmembrane transport of PAHs by bacteria, the regulation of membrane protein function during the transmembrane transport. There are three different regulation mechanisms for uptake, depending on the state and size of the oil droplets relative to the size of the microbial cells, which are (i) direct adhesion, (ii) emulsification and pseudosolubilization, and (iii) interfacial uptake. Furthermore, two main transmembrane transport modes are introduced, which are (i) active transport and (ii) passive uptake and active efflux mechanism. Meanwhile, introduce the proteomics and single cell analysis technology used to address these areas of research, such as Isobaric tags for relative and absolute quantitation (iTRAQ) technology and Nano Secondary ion mass spectrometry (Nano-SIMS). Additionally, analyze the changes in morphology and structure and the characteristics of microbial cell membranes in the process of transmembrane transport. Finally, recognize the microscopic mechanism of PAHs biodegradation in terms of cell and membrane proteins are of great theoretical and practical significance for understanding the factors that influence the efficient degradation of PAHs contaminants in soil and for remediating the PAHs contamination in this area with biotechnology.     

肺炎嗜衣原体主要外膜蛋白基因片段在大肠杆菌中的表达与鉴定

表达肺炎嗜衣原体（Chlamydia pneumoniae,Cpn）主要外膜蛋白（MOMP）的可变区VD2-VD3区．纯化产物并进行免疫活性分析,为探索重组蛋白在肺炎嗜衣原体血清学诊断中的应用提供资料．应用聚合酶联反应（PCR）技术,从肺炎嗜衣原体标准株AR-39的MOMP上扩增出抗原优势表住VD2-VD3区,将目的片段定向插入pET-30a载体,转化大肠杆菌B121,IPTG诱导表达并以Ni-NTA亲和层析柱纯化表达产物并行western-blot鉴定．成功构建了pET-30a-MOMPVD2-VD3的原核表达系统,表达并纯化出相对分子质量（Mr）为24×10^3Da的重组蛋白．Western-blot证实重组蛋白能与Cpn MOMP多克隆抗体发生特异性反应．肺炎嗜衣原体的MOMPVD2-VD3基因可以在大肠杆菌中得到表达,其表达产物能与相应的抗体结合,为肺炎嗜衣原体诊断候选抗原的研究奠定了基础．图4,参9．     

油菜菌核病菌(Sclerotinia sclerotiorum)与细菌间的基因水平转移

油菜菌核病（Sclerotinia sclerotiorum）是油菜生产上最重要的病害之一,其致病性可能来源于基因水平转移（Horizontal gene transfer,HGT）.为认识其致病原理和寻找新的真菌抑制剂的靶点,首先通过BLASTp发现其基因XM₀01585458.1编码蛋白XP₀01585508.1与细菌比对结果中出现低E值3.23e-109和高SCORE值436,暗示存在HGT现象;进一步通过系统进化树的建立,发现该蛋白在进化分枝上更接近于细菌中由Streptomyces sp.C的NZ_CM000832.1基因编码的蛋白ZP₀7291173;同时核苷酸组成分析也发现该基因与油菜菌核病菌基因组的碱基组成有较大差别,GC含量提高了14.95%.这些结果证明了XM₀01585458.1的确存在基因水平转移事件.结构分析和COG蛋白功能分类显示该HGT序列编码蛋白XP₀01585508.1具有FA58C₃（Coagulation factors 5/8 type C domain）、Kelch repeat type 1、Galactose-binding domain-like、Galactose oxidase/kelch,beta-propeller等保守结构域,应为一个膜蛋白并参与多糖代谢,推测该水平转移基因与S.sclerotiorum在侵染植物时进行细胞壁水解和致病性有关.     

Chromate reduction by Serratia marcescens immobilized on activated carbon

Serratia marcescens, isolated from tannery effluents, was resistant to chromate (0.5 mM) under both aerobic and anaerobic conditions. A stable biofilm was developed, using activated carbon as a support. Extracellular precipitate suggests that insoluble chromium hydroxide was formed. The strain showed an enhanced cell growth in an aerobic culture medium, although under anaerobic conditions chromate reduction was carried out more efficiently. About 95% of chromate was reduced in 4 days.     

趋磁细菌磁小体形成机制研究进展

磁小体(Magnetosome)是由趋磁细菌(Magnetotactic bacteria,MTB)体内合成并有生物膜包裹的纳米级磁性晶体,其形成过程是一个包括多种基因和蛋白参与并且受基因表达调控的酶催化过程.本文主要从铁离子吸收、磁小体膜形成、铁离子转运、生物矿化和磁小体链组装等5个方面来详细阐述磁小体的形成过程,重点介绍近年来分离和鉴定的相关基因和蛋白,并分析其在磁小体形成过程中的作用机制,同时还介绍了氧气、铁源和培养基成分等环境因素对磁小体形成的影响.尽管已经鉴别了大部分磁小体形成的相关基因,但是运用遗传学和生化分析手段阐明其作用机理仍然是一个挑战.磁小体形成机制的进一步研究和阐明将为其在生物技术领域的应用奠定基础.     

纳米二氧化铈对蛋白核小球藻的生物学效应研究

纳米二氧化铈(CeO_2)在被广泛使用的同时,其潜在的环境效应也受到人们越来越多的关注。以蛋白核小球藻(Chlorella pyrenoidosa)为实验材料,研究纳米CeO_2的生物学效应,为探索纳米材料对微藻的生物学效应提供理论基础和数据支持。研究结果显示:1)纳米CeO_2在低浓度(≤80 mg·L~(-1))时可促进蛋白核小球藻的生长及色素、可溶性蛋白等的合成,但在高浓度(80 mg·L~(-1))下具有毒性效应;2)低浓度纳米CeO_2可诱导藻细胞合成超氧化物歧化酶(superoxide dismutase,SOD)等可溶性蛋白,以抵御纳米CeO_2的胁迫;但在高浓度时又会降低SOD活力;3)随着纳米CeO_2浓度的升高,藻细胞中丙二醛(malondialdehyde,MDA)含量显著增加,说明藻细胞中活性氧自由基(reactive oxygen species,ROS)过量积累,这将破坏藻细胞的膜结构与功能,使细胞遭受严重损伤。     

Detection of p34cdc2- and cyclin B-like proteins in Dunaliella tertiolecta (Chlorophyceae)

p34cdc2 and cyclin B are two key proteins in the eukaryotic cell cycle control machinery. They thus could be important cell cycle markers for studies of environmental effects on cell cycle progression and on growth rate of marine phytoplankton. From July 1993 to March 1995, we used commercially available antibodies to examine the presence of their homologs in a marine phytoplankton species, Dunaliella tertiolecta Butcher. A p34cdc2-like protein was detected on the Western blots, with an apparent molecular mass as expected (34 kDa). Anti-cyclin B detected a protein of 63 kDa, a size similar to that of cyclin B in other organisms. The two proteins decreased from the exponential to the stationary growth phase. As determined on the Western blots, their abundance only changed slightly during the cell cycle, being slightly more abundant prior to cell division. Immunofluorescence performed for a partially synchronized culture showed that the fraction of the cell population that was positively stained by anti-p34cdc2 was highest at the time when the culture was mainly in the late G1 or early S phase, and in the late G2 or early M phase, respectively. The fraction was low when the culture was mainly in the S phase. Although further characterization is required to verify their identities, these two growth phase-related proteins appear to be p34cdc2 and cyclin B homologs, which may be useful in studying the cell cycle and growth rates of phytoplankton.     

The formation of a new horseradish peroxidase binding rare earth

The charge distribution, the isolation, purification, and characterization of horseradish peroxidase (HRP) were investigated. A new HRP protein binding La (La-HRP) was found for the first time in vivo. The molecular weight of the La-HRP protein is about 43,833 Da. The activity index (Rz) of the La-HRP protein (Rz = 2.4) is lower than that of HRP (Rz = 3.1). The La-HRP protein is absorbed in the plasma membrane of the plant and animal, leading to the change in the function of the cell membrane. Therefore, the La-HRP protein is harmful to living organisms.     

Application of proteomics in environmental science

Proteomics involves the separation of proteins, identification of the amino acid sequence of the interested or target proteins, study of the function of the proteins, modification, structure and ultimate assignments to functional pathways in the cell. The proteomic investigations have contributed greatly to human diseases studies, new drugs discovery researches, and environmental science in recent years. This article provides a review on the development of the main proteomic technologies, including both the gel based and non-gel based technologies, and their applications in environmental science. Proteomic technologies have been utilized in the environmental stresses studies to analyze the induction or reduction of proteins at expression level and identify the target proteins to investigate their function in response to environmental stresses, such as high or low pH, oxidation stress, and toxic chemicals. Such protein responses are also helpful to understand the mechanisms of some cellular activities and the functions of some proteins.     

Amelioration of acute mercury toxicity by a novel,non-toxic lipid soluble chelator N,N′bis-(2-mercaptoethyl)isophthalamide: effect on animal survival,health, mercury excretion,and organ accumulation

The toxic effects of mercury are known to be complex with specific enzyme inhibitions and subsequent oxidative stress adding to the damaging effects. There are likely other factors involved, such as the development of impaired metal ion homeostasis and depletion of thiol- and selenium-based metabolites such as cysteine and selenium. Much of the toxicity of mercury occurs at the intracellular level via binding of Hg²⁺ to thiol groups in specific proteins. Therefore, amelioration of mercury toxicity by the use of chelation would likely be enhanced by the use of a chelator that could cross the cell membrane and the blood brain barrier. It would be most favorable if this compound was of low toxicity, had appropriate pharmacokinetics, bound and rendered mercury cation non-toxic and had antioxidant properties. Herein we report on such a chelator, N,N′-bis(2-mercaptoethyl)isophthalamide (NBMI), and, using an animal model, show that it prevented the toxic effects associated with acute exposure induced by injected mercury chloride.     

Cloning and expression analysis of ScBAK1 gene and its alternative spliceosome in sugarcane北大核心CSCD

Alternative splicing (AS) is an important part of regulation of eukaryotic gene expression. BAK1 (Brassinosteroid insensitive1-associated receptor kinase 1) is a specific type of plant serine/threonine protein kinases, and can regulate growth and development and natural immunization. To reveal the responses of sugarcane BAK1 gene to the adverse environment, a ScBAK1 gene and its alternative spliceosome, termed ScBAK1 (GenBank accession number: KP032226) and ScBAK1 S1 (GenBank accession number: KP032227), were cloned from leaf cDNA of Yacheng 05-179 utilizing the methods of electronic cloning and RT-PCR. The open reading frame (ORF) length of ScBAK1/ScBAK1 S1 gene was 1 860bp/1 770bp, encoding 619/589 amino acids residues. The predicted molecular weight of the protein was 69.28 kDa/ 65.76 kDa. Both proteins were located in plasma membrane, estimated as acid, hydrophikic and secretive proteins. Random coil and alpha helix gave priority to extended strand in their secondary structure without beta turn. The most important protein function was cell envelope, secondly biosynthesis of amino acids and cofactors. Real-time quantitative PCR analysis revealed that the expression of sugarcane ScBAK1 S1 gene exhibited the reduced expression trend under smut fungus stress and various abiotic exogenous stresses, including SA, CuCl2, PEG, ABA, NaCl and JA, while the expression of ScBAK1 gene was induced by SA, CuCl2, PEG, NaCl and smut fungus stresses. The phenomenon showed that the absent sequences or amounts of ScBAK1 S1 gene plays a key role in the response of ScBAK1 to the stress of sugarcane smut fungus, osmotic stress and cell growth. The differential expression of ScBAK1 and ScBAK1 S1 lays a foundation for further research on the function of ScBAK1 gene under biotic and abiotic stress.     

丹参提取液对球形红细菌菌体蛋白及几种酶的影响

为探索球形红细菌对丹参的生物转化机理,采用非变性聚丙烯酰胺凝胶电泳(N-PAGE)分别对丹参水提液、醇提液、醇水提液培养后球形红细菌菌体及纯球形红细菌(PRS)菌体蛋白质(Pro)、酯酶(EST)、细胞色素氧化酶(COD)、超氧化物歧化酶(SOD)和过氧化物酶(POD)进行分析,比较用丹参提取液培养前后球形红细菌菌体蛋白及4种酶的同工酶变化.结果表明:丹参提取液培养前后球形红细菌菌体蛋白及4种酶的同工酶谱带差别较大,同工酶的电泳迁移率、活性、所表达同工酶的数目及分布均有差异,培养d 2～6蛋白及酶表达量变化最大,d 14～20基本稳定.研究表明丹参能诱导球形红细菌生成新的蛋白质及酶,亦可抑制某些蛋白质和酶的合成;这些蛋白和酶可能参与了丹参化学成分的生物转化.图4参22     

六价铬诱导的肝细胞线粒体依赖性凋亡中VDAC1的作用

铬(chromium)是一种在工业生产过程中广泛使用的重金属,进入人体后可导致急慢性中毒,具有神经毒性、基因毒性、致癌性和免疫毒性。了解六价铬(hexavalent chromium,Cr(Ⅵ))的细胞毒性,进一步探究Cr(Ⅵ)的毒作用机制,可为防治Cr(Ⅵ)对人群健康的损害提供实验依据。电压依赖性阴离子通道蛋白1(voltage-dependent anion channel-1,VDAC1)参与调控线粒体外膜通透性,影响细胞凋亡;同时VDAC1也是BCL-2家族的重要结合位点,它可与BAX/BAK相互作用形成孔道,使线粒体内的凋亡相关蛋白,如细胞色素C等,进入胞浆,引起细胞凋亡。但VDAC1影响细胞凋亡的确切路径与分子机制,目前尚未完全研究清楚。使用L02人正常肝细胞作为实验对象,通过慢病毒包装法建立VDAC1低表达细胞系,检测在相同浓度Cr(Ⅵ)处理的条件下,与非染毒组相比,不同受试细胞的生存率、凋亡情况、活性氧簇(reactive oxygen species,ROS)生成量、线粒体功能和凋亡诱导因子(AIF)的变化情况是否存在差异。结果表明,与VDAC1正常表达的细胞相比,VDAC1低表达组在Cr(Ⅵ)染毒的情况下,细胞生存率升高,凋亡率下降,细胞内ROS生成量减少,MPTP活性增加不明显,胞浆内细胞色素C(Cytochrome C,Cyt C)和AIF含量降低。上述研究结果表明VDAC1参与了由六价铬诱导的线粒体依赖性肝细胞凋亡,并且抑制VDAC1的表达可减轻因Cr(Ⅵ)暴露而引起的L02肝细胞损伤。     

铬酸钠晶体中铝酸钠杂质的分离

重点研究了含有杂质氢氧化钠和铝酸钠的酸钠晶体中铬酸钠和铝酸钠溶解动力学,洗涤一定组成的混合晶体的结果表明,两者在溶解动学方面具有很大差别,铬酸钠能迅速达到溶解平衡,而铝酸钠极易形成过程饱和溶液,且有一定的稳定性。     

纳米二氧化钛暴露人胚肺细胞差异表达基因分析

为探讨纳米二氧化钛(Nano-TiO2)颗粒对人胚肺(HPF)细胞基因表达和基因功能的影响,使用粒径10nmTiO2暴露体外培养的人胚肺细胞24h,提取RNA,应用基因芯片方法,寻找差异表达基因,并对差异基因进行基因本体(GeneOntology,GO)分类.结果表明,纳米TiO2暴露人胚肺细胞,导致514条肺中表达的基因发生差异表达,基因分类显示400条基因涉及生物学过程;415条基因涉及分子学功能;391条基因涉及细胞构成.纳米TiO2作为外界刺激物质,与细胞膜上的受体结合,影响钙、钾离子通道,上调细胞免疫和炎症反应相关的细胞因子TNF、IL1B、IL1A等基因表达.