Biomarkers of Exposure and Effects for Assessing Toxicological Risk of Endocrine Disrupters in Top Predators of the Mediterranean Sea

Abstract. Endocrine Disrupting Chemicals (EDCs) have the potential to alter hormone pathways that regulate reproductive processes in wildlife and fisheries. In this research the unexplored hypothesis that Mediterranean top predator species (such as large pelagic fish and marine mammals) are potentially at risk due to EDCs is investigated. In the Mediterranean environment, top predators accumulate high concentrations of polyhalogenated aromatic hydrocarbons (PHAHs) and toxic metals, incurring high toxicological risk. Here we illustrate the need to develop sensitive biomarkers for evaluation of toxicological risk in top marine predators (Xiphias gladius, Thunnus thynnus thynnus) and non-lethal techniques, such as non-destructive biomarkers, for the hazard assessment of threatened species exposed to EDCs, such as marine mammals ( Stenella coeruleoalba, Tursiops truncatus, Delphinus delphis and Balaenoptera physalus ).     

条斑紫菜酵母双杂交文库的构建及PyMAPK5互作蛋白的筛选

为进一步研究条斑紫菜促分裂原活化激酶家族PyMAPK5的下游互作蛋白,理解其生物学功能,本研究通过酵母双杂交的方法进行其相互作用蛋白的筛选。提取不同温度和失水逆境胁迫下的RNA,利用Invitrogen体系构建条斑紫菜酵母双杂交cDNA文库,其库容为1.44×107CFU,重组率为91.8%。以pGBKT7-PyMAPK5为诱饵蛋白载体,利用共转化方法,从文库中筛选得到26个与PyMAPK5互作的候选蛋白。候选蛋白集中在光系统II相关蛋白、捕光蛋白、微管蛋白、ATP酶、GTP结合蛋白及假设蛋白等。微管蛋白、捕光蛋白、光系统II蛋白一对一验证结果为阳性,表明在酵母体内存在互作。本研究为阐明条斑紫菜PyMAPK5与其互作蛋白的关系及解析PyMAPK5下游作用机制奠定了基础。     

重组藻胆蛋白与高等植物类囊体膜之间能量传递的研究

为了探究藻胆蛋白与高等植物类囊体膜之间的光能传递规律,验证重组藻胆蛋白是否可以与高等植物类囊体膜之间的光能传递,本研究分别提取了重组藻红蛋白、重组藻蓝蛋白以及菠菜和韭菜的类囊体进行混合孵育,然后进行了全波长吸收光谱以及荧光光谱检测。结果显示:混合孵育时间为10~20 min有利于重组藻红蛋白和藻蓝蛋白与类囊体膜结合并进行光能传递,最适混合孵育时间随重组藻胆蛋白和类囊体的种类不同而稍有不同;重组藻红蛋白不能与菠菜或韭菜的类囊体膜直接发生光能传递,但是当体系中添加重组藻蓝蛋白的时候就可以;重组藻蓝蛋白可以与类囊体中的叶绿素a直接进行光能传递,使得叶绿素a的荧光发射峰值显著提升,当藻红蛋白和藻蓝蛋白的比例是1∶2时,光能传递效率较高。本研究为藻胆蛋白在构建新型光合系统中的应用提供实验依据。     

米氏凯伦藻细胞表面膜蛋白质组及其对温度变化的响应研究

米氏凯伦藻(Karenia mikimotoi)近年在我国福建、浙江和广东沿海经常形成赤潮,其赤潮不仅影响到海洋生态系统的稳定,也严重威胁到水产养殖以及人类生命健康安全。本论文以米氏凯伦藻为研究对象,建立了米氏凯伦藻细胞表面膜蛋白质荧光标记技术和细胞膜蛋白质提取方法,运用荧光差异凝胶电泳技术(2-DDIGE)对膜蛋白质进行了分析,并研究了米氏凯伦藻的膜蛋白质组及其对环境温度变动的响应。实验共鉴定到44个细胞表面膜蛋白,其中有效注释27个,主要为转运蛋白、HSP70蛋白家族和捕光蛋白等。米氏凯伦藻在20°C条件下的细胞生长和光合作用要明显好于16°C和12°C,但16°C和12°C条件下的差别不大,表明低温限制了米氏凯伦藻的生长。当米氏凯伦藻从12°C快速转移至16°C和20°C时,藻细胞密度和光合作用效率短时间迅速降低,但细胞很快即适应温度变化。细胞膜上的转运蛋白和光合作用蛋白在其适应温度变化中起着重要作用。     

Expression of heat shock protein 70 in a permanent cell line (EPC) exposed to sediment extracts from the North Sea and the Baltic Sea

Heat shock proteins 70 (hsp70) are known to be induced by a great variety of chemical stressors. The effects of different environmental contaminants, which were identified in sediments of the North Sea and the Baltic Sea as main contaminants or as contaminants of special toxicological interest, on the extent of expression of hsp70 in a permanent cell line Epithelioma papulosum cyprini (EPC) were investigated. Concentrations of the single contaminants leading to elevated hsp70 levels were compared to the respective concentrations of these contaminants encountered in the sediment extracts thus allowing an assessment of their contribution to the toxicity of these sediments. Analysis of hsp70 was performed using western blotting techniques with subsequent comparative quantification by densitometry. Eleven of thirteen contaminants have shown a dose-dependent increase in hsp70 levels. An expression of hsp70 was observed in most cases between 0.1 and 10 microg/mL test solution. Some of the investigated stressors led to extremely high hsp70 contents even at low concentrations. However, in most cases contaminant concentrations in sediments were too low to cause an effect. For this reason, effects of the tested sediments are attributed to other contaminants or rather to mixtures of stressors.     

牙鲆(Paralichthys olivaceus)β诺达病毒衣壳蛋白重组表达及复性条件优化 组表达及复性条件优化

β诺达病毒利用其缺乏校正功能的聚合酶进行基因组复制, 易导致突变, 因而具有广泛的宿主感染性, 而且可引发致死率极高的病毒性神经坏死症, 需要有针对性地研究检测及防御方法。本研究以感染牙鲆(Paralichthys olivaceus)的β诺达病毒为对象, 利用引物设计, 将His标签编码序列连接到完整病毒衣壳蛋白C端, 构建原核表达载体; 采用SDS-PAGE及质谱对表达产物进行分离和鉴定; 重组衣壳蛋白经镍离子亲和柱纯化后进行复性条件的正交优化。结果表明4个肽段经质谱鉴 定与预期一致; 纯化产物产量可达36mg/L; 4°C条件下, 复性缓冲液中尿素和PBS的最适浓度为0.8mol/L和0.05mol/L。本研究建立的制备方案可为研制相关疫苗以及开发针对该病毒的快速检测产品等提供有价值的参考。     

牙鲆NPY的体外表达及体内检测

神经肽Y(Neuropeptide Y,NPY)被普遍认为是一种重要的促食因子,在调节鱼类的摄食行为方面起着至关重要的作用。为进一步研究牙鲆NPY蛋白的生物学功能,作者克隆了牙鲆NPY成熟肽序列(m-NPY)及含有信号肽的全长序列(f-NPY),利用原核表达系统分别进行体外重组表达,筛选出诱导剂IPTG最佳诱导浓度为0.8 mmol/L及最佳诱导时间3 h;此外,根据牙鲆NPY第49-64位氨基酸序列制备了多克隆抗体并通过Western blot验证该多抗能够有效检验重组NPY及牙鲆体内NPY的表达。研究结果为研究重组牙鲆NPY蛋白在牙鲆水产养殖产业中的应用及检测提供了依据。     

Metallothioneins and trace metals in the dogwhelk Nucella lapillus (L.) collected from Icelandic coasts

Different sizes of the dogwhelk Nucella lapillus were collected from eight locations along the southwest and north coasts of Iceland. Concentrations of total metallothioneins (MTs) and heavy metals (Cd, Cu, Cr, Mn, Ni, and Zn) were analysed using the silver saturation method and inductively coupled plasma-atomic emission spectrometry. The level of tributyltin (TBT) contamination was also assessed using imposex indices, the vas deferens stage index (VDSI) and relative penis size index (RPSI). Gufunes N. lapillus presented the highest values of VDSI (4.0) and RPSI (11.1), followed by Grenivík individuals (VDSI = 3.0; RPSI = 0.9), while the Strandakirkja population showed the lowest VDSI (0.3) and zero RPSI. At a standardised size (0.25 g dry soft-body weight), Grenivík N. lapillus exhibited significantly higher concentrations of all metals whereas overall metal concentrations were significantly lower in individuals from Strandakirkja and Garðskagi compared to other study sites. Partial correlation analyses with size correction indicated that MT concentrations were better correlated with Cd and Cu concentrations than with other metals. At the standard size, the pattern of MT concentrations in N. lapillus from different sites was, however, very different from those of metal profiles. Such discrepancies between the patterns of MT and metals in N. lapillus might be explained by the fact that MT induction could be influenced by various factors such as temperature, dietary metal intake, growth rate and co-existence of other MT-inducing chemicals.     

东风螺、弯竹蛏和斑节对虾的壳基质蛋白组学分析

从软体动物中的腹足纲、双壳纲以及节肢动物中的甲壳纲分别选取了3种中国沿海常见的品种——东风螺、弯竹蛏、斑节对虾,提取并分析它们的壳基质蛋白。利用液相色谱串联质谱（LC-MS/MS）技术对蛋白质组学进行分析发现特定的蛋白质具有明显的种属差异：①N66蛋白（一种碳酸酐酶）通常存在于弯竹蛏中,但不存在于东风螺和斑节对虾中,揭示N66蛋白质具有特异性,在未来的物种鉴定中可能具有重要的参考意义;②角质层蛋白AM1199和CP14.6存在于斑节对虾的表皮结构中,在东风螺和弯竹蛏中未检测到;③血蓝蛋白同时在3种动物外壳的基质蛋白中被鉴定出来,揭示了其在不同物种的外壳形成与发育过程中的重要作用。该研究为壳基质蛋白在软体动物或节肢动物外壳中的鉴定提供了基础,也为探索不同物种的生物矿化过程提供了重要参考。