How Plants Cope with Foreign Compounds. Translocation of xenobiotic glutathione conjugates in roots of barley (Hordeum vulgare) (9 pp)

Background, Aim and Scope Numerous herbicides and xenobiotic organic pollutants are detoxified in plants to glutathione conjugates. Following this enzyme catalyzed reaction, xenobiotic GS-conjugates are thought to be compartmentalized in the vacuole of plant cells. In the present study, evidence is presented for long range transport of these conjugates in plants, rather than storage in the vacuole. To our knowledge this is the first report about the unidirectional long range transport of xenobiotic conjugates in plants and the exudation of a glutathione conjugate from the root tips. This could mean that plants possess an excretion system for unwanted compounds which give them similar advantages as animals. Materials and Methods: Barley plants (Hordeum vulgare L. cv. Cherie) were grown in Petri dishes soaked with tap water in the greenhouse. - Fluorescence Microscopy. Monobromo- and Monochlorobimane, two model xenobiotics that are conjugated rapidly in plant cells with glutathione, hereby forming fluorescent metabolites, were used as markers for our experiments. Their transport in the root could be followed sensitively with very good temporal and spatial resolution. Roots of barley seedlings were cut under water and the end at which xenobiotics were applied was fixed in an aperture with a thin latex foil and transferred into a drop of water on a cover slide. The cover slide was fixed in a measuring chamber on the stage of an inverse fluorescence microscope (Zeiss Axiovert 100). - Spectrometric enzyme assay. Glutathione S-transferase (GST) activity was determined in the protein extracts following established methods. Aliquots of the enzyme extract were incubated with 1-chloro-2,4-dinitrobenzene (CDNB), or monochlorobimane. Controls lacking enzyme or GSH were measured. - Pitman chamber experiments. Ten days old barley plants or detached roots were inserted into special incubation chambers, either complete with tips or decapitated, as well as 10 days old barley plants without root tips. Compartment A was filled with a transport medium and GSH conjugate or L-cysteine conjugate. Compartments B and C contained sugar free media. Samples were taken from the root tip containing compartment C and the amount of conjugate transported was determined spectro-photometrically. Results: The transport in roots is unidirectional towards the root tips and leads to exsudation of the conjugates at rates between 20 and 200 nmol min-1. The microscopic studies have been complemented by transport studies in small root chambers and spectroscopic quantification of dinitrobenzene-conjugates. The latter experiments confirm the microscopic studies. Furthermore it was shown that glutathione conjugates are transported at higher rates than cysteine conjugates, despite of their higher molecular weights. This observation points to the existence of glutathione specific carriers and a specific role of glutathione in the root. Discussion: It can be assumed that long distance transport of glutathione conjugates within the plant proceeds like GSH or amino acid transport in both, phloem and xylem. The high velocity of this translocation of the GS-X is indicative of an active transport. For free glutathione, a rapid transport-system is essential because an accumulation of GSH in the root tip inhibits further uptake of sulfur. Taking into account that all described MRP transporters and also the GSH plasmalemma ATPases have side activities for glutathione derivatives and conjugates, co-transport of these xenobiotic metabolites seems credible. - On the other hand, when GS-B was applied to the root tips from the outside, no significant uptake was observed. Thus it can be concluded that only those conjugates can be transported in the xylem which are formed inside the root apex. Having left the root once, there seems to be no return into the root vessels, probably because of a lack of inward directed transporters. Conclusions: Plants seem to possess the capability to store glutathione conjugates in the vacuole, but under certain conditions, these metabolites might also undergo long range transport, predominantly into the plant root. The transport seems dependent on specific carriers and is unidirectional, this means that xenobiotic conjugates from the rhizosphere are not taken up again. The exudation of xenobiotic metabolites offers an opportunity to avoid the accumulation of such compounds in the plant. Recommendations and Perspectives: The role of glutathione and glutathione related metabolites in the rhizosphere has not been studied in any detail, and only scattered data are available on interactions between the plant root and rhizosphere bacteria that encounter such conjugates. The final fate of these compounds in the root zone has also not been addressed so far. It will be interesting to study effects of the exuded metabolites on the biology of rhizosphere bacteria and fungi.     

鱼体甲基汞的萃取富集和测定

研究了苯萃取和半胱氨酸滤纸富集再用苯洗脱的方法,使鱼肉中甲基汞得到富集和提取。建立了气相色谱测定甲基汞的方法并对所建立的方法进行评价。结果表明:加标回收率为87.10%～105.69%;变异系数为2.93%。符合要求,大量样品实验表明,该方法适用于痕量甲基汞的提取和分析。     

L-半胱氨酸改性甲壳素处理含铅、镉工业废水

研究了L 半胱氨酸改性甲壳素作为吸附剂对Pb2 + 、Cd2 + 的动态吸附性能 ,同时考察了流速、溶液中离子的含量、吸附剂的相对用量等因素对吸附效果的影响 ,以及洗脱液的种类和酸度、洗脱速度、洗脱液体积等因素对解吸率的影响。结果表明 ,用该吸附剂填装的吸附柱对含Pb2 + 、Cd2 + 工业废水直接进行处理 ,处理后的水达到了排放标准。     

Halogenated hydrocarbons and aryl hydrocarbons identified in human tissues

New methods for the preparation and purification of nephrotoxic cysteine conjugates of chloro‐ and fluoro‐alkenes are described. The methods are applicable to a wide range of cysteine, N‐acetyl cysteine and glutathione conjugates and examples have been given of the synthesis and characterisation of two novel glutathione conjugates.     

球形红细菌去除和转化铅的机理研究

通过X-射线衍射光谱分析,球形红细菌(Rhodobacter sphaeroides)H菌株对铅离子的转化产物为硫化铅.H菌株在不同浓度Pb2 中培养后,研究了该菌株生长细胞对Pb2 去除的动力学和该菌体产生半胱氮酸脱巯基酶活性的变化,用聚丙烯酰胺凝胶电泳法分析了其同工酶谱.结果表明,Pb2 浓度为25～150 mg·L-1时,去除速率常数k较大,半衰期T1/2较短,去除率较高;Pb2 浓度为75、100和150 mg·L-1时,对H菌株产生半胱氨酸脱巯基酶活力有明显的促进作用,Pb2 浓度为200 mg·L-1时,对该酶活力有抑制作用.该菌体细胞及亚细胞组分中铅含量测定结果显示,94%的Pb2 分布于细胞壁和细胞质内,仅少量存在于细胞膜上,并结合透射电镜观察和红外光谱分析,证明了该H菌株对铅离子去除和转化是在细胞质内进行的.     

绵农小麦种子巯基蛋白酶抑制剂的纯化及性质研究

绵农11号小麦种子磨粉后的胚和麸皮，经溶液浸取和硫酸铵分级沉淀获得巯基蛋白酶抑制剂（CPI）粗品，再经木瓜蛋白酶－Sepharose4B亲和层析，DEAE－Sepharose离子交换层析和SephadexG－100分子筛层析，可获得两种CPI．通过SDS－PAGE或凝胶过滤法测得其Mr分别为18800［简称CPI（H）］和10500［简称CPI（L）］．它们在PAGE，SDS－PAGE和HPLC上均为单一蛋白带．CPI（L）和CPI（H）对木瓜白酶的抑制活性（ID50）分别为7．8×10－6mol／L和3．40×10－6mol／L．对无花果蛋白酶的抑制活性分别为9．5×10－5mol／L和5．8×10－6mol／L；CPI（H）对菠萝蛋白酶有弱抑制作用，但CPI（L）则无抑制作用；无论是CPI（L）或CPI（H）对胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶均无抑制作用．CPI（H）的N－末端为Ile．CPI（H）和CPI（L）在pH2．0～11．0范围内和90℃处理5min，两种CPI的抑制活性均无变化；CPI（H）和CPI（L）对木爪蛋白酶的抑制曲线经Dixon作图法，得出CPI（H）为反竞争性抑制类型，而CPI（L）为竞争性抑制类型，其Ki值为8．32×10－6mol／L．     

水稻土中半胱氨酸分解产生含硫气体的研究

在室内培养条件下,测定了水稻土中含硫气体的释放．结果表明,该土壤中有硫化氢(Ｈ２Ｓ),羰基硫(ＣＯＳ),二甲基硫(ＤＭＳ)三种气体释放．当土壤中加入半胱氨酸后,ＣＯＳ和Ｈ２Ｓ气体的浓度有了明显增加,并有ＣＳ２和ＣＨ３ＳＨ测出,而ＤＭＳ的浓度变化不大．这些结果表明半胱氨酸的分解可能是 ＣＯＳ,Ｈ２Ｓ,ＣＳ２和 ＣＨ３ＳＨ的产生源之一．在好氧(正常大气)条件下,Ｈ２Ｓ,ＣＯＳ,ＣＳ２和ＣＨ３ＳＨ的释放量低于厌氧(氮气氛围)条件下的释放量,ＤＭＳ则高于厌氧条件下．这表明水稻土中半胱氨酸分解产生 Ｈ２Ｓ,ＣＯＳ,ＣＳ２和 ＣＨ３ＳＨ需较强的还原条件,产生这四种气体的微生物需要严格的厌氧条件．产生ＤＭＳ的微生物则比前者需要高一些的含氧量．在光照条件下,各含硫气体的释放量普遍高于无光照条件下的释放量．释放含硫气体的大多数微生物适宜的含水率为５０%且对土壤的ｐＨ值有一定要求．     

外源含硫化合物对土壤挥发性有机硫化合物交换通量的影响

通过静态箱采样和Entech7100预浓缩仪-GC-MS分析了半胱氨酸、硫化钠和硫酸钠对土壤吸收或释放羰基硫（COS）、二甲基硫醚（DMS）、二硫化碳（CS2）和二甲二硫醚（DMDS）等4种挥发性有机硫化合物（VOSCs）的影响.结果表明,添加半胱氨酸后,土壤由COS和CS2汇转变为源,DMS和DMDS通量显著增加,且...     

增塑剂DIDP对肝脏氧化应激损伤及VitE拮抗作用研究

研究增塑剂邻苯二甲酸二异癸酯(didecyl phthalate, DIDP)致雄性小鼠肝损伤作用及其机理。以雄性BALB/c小鼠为受试动物,随机分为7组,包括溶剂对照组(生理盐水)、4个DIDP染毒组(0.15、1.5、15和150 mg·kg~(-1))、维生素E(vitamin E, VitE)(100 mg·kg~(-1))处理组和DIDP+维生素E处理组(150 mg·kg~(-1)DIDP+100 mg·kg~(-1)VitE),连续灌胃14 d。以肝组织匀浆测定活性氧(reactive oxygen species, ROS)、还原型谷胱甘肽(glutathione, GSH)、丙二醛(malondialdehyde, MDA)和细胞凋亡因子半胱氨酸天冬氨酸蛋白酶3(cysteine aspartic proteinase 3, Caspase-3)水平。采用动物自动生化分析仪检测肝功能指标血清中丙氨酸氨基转移酶(alanine aminotransferase, ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase, AST)、白蛋白(albumin, ALB)水平,并同时观察肝组织的病理变化与荧光染色结果。随着DIDP染毒剂量的增加,小鼠肝组织ROS、MDA和Caspase-3含量逐渐上升,血清ALT和AST水平也逐渐上升,GSH含量逐渐降低,血清ALB水平也逐渐降低,差异具有统计学意义(P 0.05,P 0.01); VitE处理组ROS、MDA和Caspase-3含量相应降低,血清ALT和AST水平也相应降低,GSH含量逐渐上升,血清ALB水平也相应上升。小鼠肝组织形态观察结果表明,随着DIDP染毒剂量的增加,小鼠肝组织的病理损伤程度呈上升趋势。研究表明,较高剂量(≥15 mg·kg~(-1))的DIDP能造成小鼠的肝脏损伤与细胞凋亡,抗氧化剂VitE可使肝脏损伤与细胞凋亡减轻,对小鼠肝组织起保护作用,说明氧化应激介导了DIDP对机体的损伤。