酵母双杂交技术构建重组人雌激素受体基因酵母

应用酵母双杂交技术构建重组人雌激素受体基因酵母,用以检测类/抗雌激素化合物和环境样品的类/抗雌激素活性.采用多聚酶链反应(PCR)法扩增人雌激素受体(hER)基因,构建诱饵质粒pGBKT7-ER LBD;分别提取并纯化两种含有ER共激活因子基因的靶质粒pGAD424-GRIP1和pGAD424-SRC1;诱饵质粒和靶质粒同时转染酵母细胞Y187,于营养缺陷型培养基(SD/-Trp/-Leu)上筛选阳性菌落,分别构建两种ER双杂交酵母ER+GRIP1和ER+SRC1.考察双杂交酵母与不同激素:17β-雌二醇(E2)、二氢睾酮(DHT)、孕酮(PG)以及三碘甲状腺原氨酸(T3)的结合情况,并考察了雌激素受体拮抗剂4-羟基他莫昔芬(4-OHT)与酵母的相互作用.结果表明:ER+GRIP1和ER+SRC1酵母均能够专一性的和E2结合,并存在显著的剂量-效应关系,E2对ER+GRIP1和ER+SRC1酵母β-半乳糖苷酶活性诱导的EC50值分别为7.3×10-11mol·L-1和1.5×10-10mol·L-1,其中ER+GRIP1酵母细胞诱导产生的酶活性值明显高于ER+SRC1酵母细胞.4-OHT能够抑制E2诱导ER+GRIP1酵母细胞产生的酶活性,并存在显著的剂量-效应关系,IC50值为1.0×10-7mol·L-1.表明重组人雌激素受体基因酵母可以用于检测化合物和环境样品的类/抗雌激素活性.     

镉、汞、锌3种重金属的雌激素样作用（Estrogenic Activity of Cadmium, Mercury, and Zinc）

为探讨镉、汞、锌3种重金属的雌激素样作用,采用双杂交酵母法测定了醋酸镉、氯化汞、醋酸锌单独作用时对重组基因酵母β-半乳糖苷酶诱导活性的EC50值.实验结果表明,醋酸镉浓度为1.0×10-5mol·L-(1实验浓度范围10-7~10-2mol·L-1),氯化汞浓度为5.0×10-7mol·L-(1实验浓度范围10-9~10-5mol·L-1),醋酸锌浓度为1.0×10-4mol·L-(1实验浓度范围10-9~10-2mol·L-1)时对酵母β-半乳糖苷酶诱导活性最大,分别达到1.3、0.95和1.1U.不能求出镉、汞、锌单独作用时对β-半乳糖苷酶诱导活性的EC50值.一味计算EC50值不仅难以评价重金属的雌激素活性,而且限制重组受体基因酵母法的普遍运用.一些重金属通过MCF-7细胞法(E-screen)呈阳性,通过重组hERα酵母法YES检测呈阴性,有可能是重金属通过与ERβ作用而产生效应.若构建出含ERβ基因的双杂交酵母菌株,与现有的方法互补将能完善环境雌激素的体外测评系统.     

代谢活化作用对多溴代联苯类污染物类/抗甲状腺激素活性的影响

选用大鼠肝匀浆(S9)和鼠肝癌细胞(H4ⅡE)两种体系代谢活化多溴代联苯醚混标(BDEs)和十溴联苯醚(BDE209),采用重组甲状腺激素受体基因酵母检测BDEs和BDE209母体及其代谢产物的类/抗甲状腺激素效应.结果表明,BDEs和BDE209母体均不表现甲状腺激素效应(p>0.05);但是经S9和H4ⅡE细胞代谢活化后,其代谢产物表现出明显的类甲状腺激素活性和抗甲状腺激素活性(p<0.05),BDEs和BDE209的干扰甲状腺激素效应需要经过代谢活化步骤.比较不同代谢活化体系,重组酵母细胞本身的代谢活化作用并不显著,而H4ⅡE细胞和S9代谢活化体系均能够导致活性中间体.     

长江中下游环境激素效应的污染特征及生态风险

生活污水和养殖废水排放导致受纳水环境中激素类物质的污染,对水生生物产生不利影响。分别利用嵌入雌激素受体和雄激素受体的基因重组酵母菌测定了长江中下游流域不同时期水体和沉积物中4种环境激素效应,即雌激素效应、抗雌激素效应、雄激素效应和抗雄激素效应。结果表明,雌激素效应污染最为普遍,在地表水和沉积物中检出率均超过50%,水体和沉积物的最高浓度分别为2.05 ng·L~(-1)雌二醇当量(EEQ)和0.43 ng EEQ·g~(-1)。其他3种激素效应在水体和沉积物介质中的检出率均低于雌激素效应,按总体检出率来看:抗雄激素效应雄激素效应抗雌激素效应,3种激素效应在水体中最大检出浓度分别为144μg·L~(-1)氟他胺当量(FEQ)、37.9 ng·L~(-1)二氢睾酮当量(DEQ)和103μg·L~(-1)他莫西芬当量(TEQ),在沉积物中分别为53.6μg FEQ·g~(-1)、12.0 ng DEQ·g-1和51.5μg TEQ·g~(-1)。环境激素效应的浓度分布在水体中均呈现季节性的差异,雌激素效应的区域性高值位于武汉段、鄱阳湖口和芜湖-南京段,其他3种激素效应没有明显的高污染区域。环境激素效应与当地人口数量、有机质、氨氮等呈现一定相关性,表明环境激素效应与人类活动排放密切相关。雌激素效应仅在鄱阳湖口点位具有高风险,其他区域为中等风险,雄激素效应无高风险区域。研究结果有助于认识长江中下游区域的环境激素效应污染态势,为相关污染控制提供基础数据。     

中国虎纹捕鸟蛛神经毒素肽基因的克隆及在酵母中的表达

从中国珍稀毒蜘蛛种虎纹捕鸟蛛的粗毒中分离纯化的虎纹捕鸟蛛毒素-Ⅰ(HWTX-Ⅰ)是含33个氨基酸的多肽.有关实验已证实,HWTX-Ⅰ是一种作用于突触前膜的神经毒素和高阈值钙通道抑制剂,在电刺激时HWTX-Ⅰ也影响交感神经释放肾上腺素和迷走神经释放乙酰胆碱,这些结果表明,HWTX-Ⅰ具有潜在的镇痛活性.本文报道通过RT-PCR方法克隆、测定了HWTX-Ⅰ的cDNA序列.在此基础上,以pPIC9K为表达载体和GS115为宿主,筛选His Muts克隆,以甲醇为诱导剂,成功地构建并在毕赤酵母系统分泌表达HWTX-Ⅰ基因.进一步分析HWTX-Ⅰ表达产物,生物活性实验观察到酵母表达体系所获得的HWTX-Ⅰ产物具有与天然HWTX-Ⅰ相似的生理活性.图4参10     

全氟辛基磺酸及其替代产品的内分泌干扰效应评价

为研究全氟辛基磺酸(PFOS)及其替代品的内分泌干扰效应,将重组人雌激素/雄激素/孕激素/甲状腺激素/维甲酸受体基因的双杂交酵母暴露于不同浓度的PFOS及替代品中,并结合体外代谢活化技术检测直接和间接的类/抗激素活性。结果表明,被替代品PFOS表现为雄激素受体、甲状腺受体和维甲酸受体拮抗剂,且在代谢活化后新增了雌激素受体拮抗效应,其半数效应浓度(EC₅₀)在3.0×10^-4～4.8×10^-3 g·L^-1之间;同时,4种替代化学品没有任何效应检出,说明不会通过与核受体结合干扰天然激素的正常功能。研究结果说明,与原化学品相比,替代产品的应用在内分泌干扰效应方面可能具有更低的环境风险。     

类二噁英物质及芳香烃受体(AhR)介导的有害结局路径(AOP)研究进展

二噁英及类二噁英物质(dioxin-like compounds,DLCs)是一类高毒性化合物的统称,对其毒理学的研究一直都是备受关注的焦点。已有证据表明,高毒二噁英及DLCs主要通过激活芳香烃受体(aryl hydrocarbon receptor,AhR),进而导致一系列生物毒性。近年来越来越多的新型有机污染物被发现具有类二噁英分子结构并存在潜在生物毒性或活性。与此同时,如何评估二噁英及DLCs对本土生态生物的危害及其风险也受到更多关注。本文综述了近几年发现的新型二噁英物质、二噁英及DLCs的AhR致毒机制、相应的有害结局路径(adverse outcome pathway,AOP)及AOP在指导探索新型物质及物种敏感性方面上的新观点和发现,同时也展望了二噁英及DLCs在生态毒理及风险评估领域的未来研究方向。     

酵母基因报道系统检测环境二恶英类似物的污染

利用一套酵母基因报道检测系统，建立和标化了对二恶英类标准样品测定的方法，证明此系统可检测ng级的二恶英及其类似物，具有简单、快速的的特点．对广州市区工业和生活污水排放较为集中的水体进行的测定显示，二恶英及其类似物在部分地区超标，而各区经处理的自来水均无超标污染．对小鼠急性毒性实验的血清样品的测定同样证明系统能够测定未浓缩处理的复杂样本，具有一定的应用价值．图4参10     

Ein neuer Biotest mit der HefeSaccharomyces cerevisiae auf aquatische Toxizität

The fermentation activity of the yeastSaccharomyces cerevisiae is used as a bioindicator to detect aquatoxic effects. This is done by measuring the CO₂ production of yeast cells after a multiplication period affected by toxic substances. The concentration of toxicants diminishing the fermentation by 20% is taken as a characteristic value (EC₂₀). Organic compounds (unpolar and polar narcotics), inorganic salts (especially of heavy metals), surfactants and plant pesticides were tested. The results are compared so far as available with data of a ciliata assay usingTetrahymena pyriformis. A congruence of 90% and an equal sensibility of the assays resulted. The results of the yeast assay are therefore representative for ecotoxicological testing. The yeast assay is reproducible, easy to handle, and offers an alternative for sewage testing because sterile handling is not necessary.     

2,3,7,8-四氯苯并二噁英（TCDD）短期染毒可造成着床前胚胎丢失并伴随雌性生殖器官中毒物相关蛋白的诱导表达（Short Time Exposure of TCDD Causing Loss of Implantation Failure of Embryos and Its Associated with Induced Expression of Relevant Proteins）

为探明妊娠早期胚胎的丢失是否与卵巢、输卵管、子宫组织受到2,3,7,8-四氯苯并二噁英(TCDD)直接毒害有关,检测了NIH小鼠胚胎着床前期和后期TCDD暴露对胚胎毒性影响的敏感性,并利用免疫组化方法分析了模型动物肝脏、子宫、输卵管和卵巢组织中TCDD所引起的AhR、ARNT以及Cyp1a2分子标记物的变化.检测发现:妊娠第9d,100ng·kg-1·d-1剂量TCDD经口染毒,造成胚胎着床数量减少,且着床前期暴露的影响大于着床后期;子宫蜕膜反应受到明显抑制;胚胎迁移率没有明显变化,但胚胎数量减少.免疫组织化学分析发现正常组小鼠的肝脏、子宫、输卵管和卵巢组织中有AhR和Cyp1a2弱阳性信号表达,ARNT有细胞核的强阳性信号表达;妊娠第1～8d、第1～3d和第4～8d处理组小鼠肝脏、子宫、输卵管和卵巢组织中的AhR、Cyp1a2的阳性面积和光密度值均高于正常组;随处理时间和组织蓄积量的增加,ARNT在组织中的变化由胞核(妊娠第1～3d组)表达到胞浆(妊娠第4～8d组)表达,然后完全无表达(妊娠第1～8d组).以上研究结果表明:TCDD对早期妊娠小鼠子宫、输卵管和卵巢组织中的AhR、ARNT和Cyp1a2的激活和代谢方式与肝脏相同,说明雌性生殖系统中的组织有TCDD蓄积和代谢活性,这可能是导致早期胚胎迁移、着床等过程改变,造成胚胎丢失的重要原因.     

杂合抗菌肽LPCB在毕赤酵母中的表达条件优化

为获取能表达具有天然活性的杂合抗菌肽的重组酵母菌,确定其最佳表达条件,将构建好的重组酵母表达载体pPICZα-A-CecropinB(1～10)-Magainin2(1～12)(简称pPICZα-A-CBMA)和pPICZα-A-Lfcin-Pro-CecropinB(简称pPICZα-A-LPCB)经SacⅠ线性化处理,分别电转入巴斯德毕赤酵母Pichia pastoris X-33,利用抗生素Zeocin筛选阳性克隆,PCR扩增验证,甲醇诱导表达.采用抑菌圈法初步测定产物活性,优化杂合肽LPCB诱导时间、诱导剂浓度、诱导温度、培养基pH等表达条件,传代检测其质粒稳定性.结果显示,基因工程菌P.pastoris X-33/pPICZα-A-CBMA和P.pastoris X-33/pPICZα-A-LPCB成功构建.表达产物前者抑菌活性微弱,后者抑菌活性良好.杂合肽LPCB的最佳表达条件为:25℃,pH中性培养,每24 h添加0.5%(V/V,φ)甲醇,诱导表达72 h.重组酵母菌pPICZα-A-LPCB在培养超过100代后,未发生质粒丢失.     

污灌与镉胁迫对菠菜几种抗氧化酶活性的影响（Effects of Sewage Irrigation and Cadmium Stresses on the Activities of Several Antioxidant Enzymes of Spinach）

为了探讨污水灌溉与镉胁迫对菠菜抗氧化酶活性的影响,采用盆栽试验的方法,分别用清水和生活污水配制不同浓度(0、1、5、10、50mg·L-1)的Cd2+溶液,对菠菜进行受试处理,每5d灌喷1次,共处理5次.分别采用邻苯二酚比色法、L-苯丙氨酸比色法、水杨酸比色法和高锰酸钾滴定法,对菠菜叶片多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)、β-1,3葡聚糖酶(β-1,3glucanase)和过氧化氢酶(CAT)活性进行了测定.结果表明:1)清水处理系列:随着镉处理浓度的增大,与清水对照相比,PPO、PAL活性呈先升高后降低的趋势,CAT活性略有升高,β-1,3葡聚糖酶活性略有降低,各暴露组与清水对照组相比,均呈现显著差异(p<0.05).2)污水处理系列:随着镉处理浓度的增大,与污水对照相比,PPO、PAL活性呈先升高后降低的趋势,CAT和β-1,3葡聚糖酶活性呈逐渐降低趋势;各暴露组与污水对照相比,均呈现显著差异(p<0.05).3)相同Cd2+浓度下,与清水处理相比,各浓度污水处理PAL和PPO活性均明显降低(p<0.05);而β-1,3葡聚糖酶明显升高(p<0.05).以上结果显示:单一镉污染和生活污水与镉复合污染对菠菜不同抗氧化系统酶具有不同影响,这可能与污水中各成分与镉的相互作用有关.     

Pathway of 17��-estradiol degradation by Nitrosomonas europaea and reduction in 17��-estradiol-derived estrogenic activity

In recent years, natural and synthetic estrogens have been recognized as endocrine disruptors in aquatic organisms. Although natural and synthetic estrogens are known to be degraded by microbes, only limited information about their degradation pathways is available. Here, we studied the degradation pathways of a natural estrogen, 17β-estradiol, by the nitrifying microorganism Nitrosomonas europaea, and we determined whether the degradation products of 17β-estradiol had estrogenic activity. To identify the degradation products, we subjected the culture solution to solid-phase extraction, and the extract was analyzed by gas chromatography–mass spectrometry. The potential estrogenic activity of the degradation products was investigated by means of a yeast two-hybrid assay. 1,3,5(10),16-Estratetraen-3-ol (estratetraenol) was newly identified as a degradation intermediate produced by dehydration of 17β-estradiol. Estratetraenol was also degraded by N. europaea, and its degradation rate was faster than that of 17β-estradiol. The two-hybrid assay confirmed that estratetraenol acted as a ligand for the estrogen receptor; estratetraenol thus has potential estrogenic activity. N. europaea eliminated the estrogenic activity derived from 17β-estradiol. This paper is the first to report dehydration as a mechanism of microbial estrogen degradation.     

In vitro evaluation of atmospheric particulate matter and sedimentation particles using yeast bioassay system.

Little information on the evaluation of airborne particulate matter (APM) and sedimentation particles from subway stations is available. The thermal metamorphism of train wheels generating toxic particles in subway stations is a possibility. In this study, the toxicity and physiological effects of particles from subway stations were evaluated using a yeast bioassay system. Estrogenic and antiestrogenic activities of APM in APM extracts from subway stations were determined. No estrogenic activity was found in the APM fractions and their S9-activated APM samples. Sedimentation dust samples also showed no estrogen activity. In contrast, extracts from sedimentation dust samples showed antiestrogen activity. Marked yeast toxicity was observed in the samples extracted from sedimentation dust. Potent yeast toxicity was also found in the S9-activated extracts from sedimentation dust. The results suggest that sedimentation dust from a semiclosed area of a subway system has antiestrogen activity, although both the origin and generation system of this activity are uncertain. These pollutants in sedimentation dust may change to a more toxic form in vivo by S9 activation.     

长江(南京段)环境雌激素的污染特征

利用固相萃取技术和重组基因酵母检测了长江(南京段)四个典型断面环境雌激素污染状况.结果表明,除秦淮新河断面外,江心洲断面、三汊河断面和大桥断面均有环境雌激素的检出,雌二醇当量分别为0.38、0.24和0.45 ng·L~(-1).与国内外地表水环境雌激素检测结果相比,长江(南京段)典型断面雌激素活性水平为较低至中等.对江心洲断面和大桥断面,甲醇洗脱组分的雌激素活性最大,其次为丙酮洗脱组分,二氯甲烷洗脱组分的雌激素活性最小;对三汊河断面,丙酮洗脱组分的雌激素活性最大,其次为二氯甲烷洗脱组分,甲醇洗脱组分的雌激素活性最小.这可能是污水来源不同所致,生活污水导致大量极性雌激素污染物的输入,而工业污水则主要引入了中等至弱极性雌激素污染物.三个雌激素活性阳性检出断面主要活性洗脱组分的雌激素活性均大于混合样的雌激素活性,这可能是由于样品混合后各种结构上不同的污染物之间的交互作用掩盖了实际的雌激素活性.     

Geometry of Zn(II) complexes with dissolved organic matter: X-ray studies at variable pH

The structure of Zn(II) complexes with dissolved organic matter (DOM) is an important consideration in developing molecular-level models of Zn(II) speciation, but recent reports favoring the tetrahedral geometry differ from earlier findings that geometry was largely octahedral. In general, the presence of thiolate ligands favors the tetrahedral geometry, while O and N ligands favor the octahedral geometry. This work presents extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) spectroscopic results, indicating an octahedral geometry over the pH range of 5–9 for a freshwater DOM isolate. Changes in XANES derivatives as a function of pH can be explained in terms of ligand protonation and/or changing ligand groups. Tetrahedral Zn(II)–DOM geometry may be restricted to binding environments containing thiol groups.     

北京市污水厂污泥中的内分泌干扰效应物质

采用重组基因酵母法检测了北京市16个污水处理厂污泥的雌激素、雄激素和孕激素效应.结果表明,各污水厂污泥中均可检测到雌激素受体诱导效应,范围在0.587～6.76ngEEQ·g-(1dw)之间.各水厂污泥均未检测出雄激素诱导效应和孕激素诱导效应,大部分污水厂污泥样品表现出较明显的雄激素抑制效应和较强的孕激素抑制效应.与国外相比,北京市污水厂污泥中的雌激素活性值相对较低.堆肥处理不能有效去除雌激素类物质和孕激素抑制物质,但对雄激素抑制物质的去除明显.     

Assessment of estrogenic chemicals using an estrogen receptor α (ERα)- and ERβ-mediated reporter gene assay in fish

In vitro reporter gene assays using vertebrate cell lines or yeast have been used for assessment of the estrogenic chemicals. However, estrogen receptor α (ERα)- and ERβ-mediated reporter gene system in fish has yet to be developed. In the present study, we developed an ERα- and ERβ-mediated reporter gene assay in fish and estimated estrogenic activities of 17β-estradiol (E2), p-nonylphenol (NP), bisphenol A (BPA), p,p′-DDE, and genistein (Gen) using the in vitro reporter assay. The activity was intensely induced by transfection with either ERα or ERβ expression plasmid under E2 or Gen administration, whereas it was significantly induced by transfection with ERα expression plasmid, but not with ERβ expression plasmid under NP administration. On the other hand, the activity was induced more intensely by transfection with ERα expression plasmid than ERβ expression plasmid under BPA or p,p′-DDE administration. These results indicate that there are obvious differences in the activity through ERα and ERβ among the estrogenic chemicals examined in vitro. Thus, the in vitro reporter assay provides an excellent system for elucidating the action mechanism of estrogenic chemicals in fishes. Physical and Chemical Impacts on Marine Organisms, a Bilateral Seminar Italy–Japan held in November 2004     

Antimicrobial activities of marine sponges from the Mediterranean Sea

Extracts from marine sponges collected along the French coast of Mediterranean in 1983 have been tested for antibacterial and antifungal activities. The distribution of antimicrobial activities against five classes of microorganisms (5 Gram-positive and 5 Gram-negative terrestrial bacteria, 10 marine bacteria, 15 human pathogenic fungi and 5 phytopathogenic fungi) among 28 species of marine demosponges is reported. Antibacterial activity against Gram-positive bacteria (77%) is much more frequent than against Gram-negative bacteria (53%). Activity against marine bacteria and yeast is less frequent (32%). Those sponges which contain a large quantily of symbiotic bacteria display a weak antifungal activity.     

短小芽孢杆菌A—30耐碱性木聚糖酶基因的分子生物学研究

采用PCR方法对短小芽孢杆菌A－30菌株的耐碱性木聚糖酶基因进行克隆。在木聚糖选择平板上用刚果红染色法筛选出阳性克隆,提取阳性克隆的重组质粒进行酶切鉴定和测序。该基因在大肠杆菌中表达,过夜培养物胞外、胞内和周质空间的木聚糖酶酶活分别为0．159IUmL^-1、0.322IUmL^-1和0.007IUmL^-1。此木聚糖酶表现出较宽的pH作用范围,最适作用pH7左右,在pH9时仍有60％以上的酶活性。图4参14