重组hPRL-1的表达纯化及其理化和酶学性质研究

目的:表达纯化hPRL-1重组蛋白,分析其理化性质及酶学特性。方法:热激法将重组pET15b质粒转化入E.coli BL21中,IPTG诱导表达出His-tagged hPRL-1蛋白。使用Ni-NTA亲和层析法结合Mono Q离子交换层析法纯化。用SDS-PAGE法和Western Blot法进行表达情况的定性定量分析,并使用HPLC法鉴定蛋白纯度,计算出蛋白分子量,圆盘等电聚焦电泳分析重组蛋白等电点。比较分析以pNPP、4-MUP和DiFMUP为底物时的酶促反应动力学。同时以pNPP为底物测定酶的最适pH值;以4-MUP为底物测定酶的最适温度,分析探讨缓冲液离子强度与蛋白酪氨酸酶通用抑制剂钒酸钠对酶活力的影响。结果:以亲和层析和离子交换层析结合,可以纯化得到纯度约为95%的蛋白。测得蛋白分子量为24.54kD,等电点为9.11。以pNPP、4-MUP和DiFMUP为底物时Km分别为3720μmol/L,130μmol/L和50μmol/L。酶的最适pH值为7.6,最适温度为34℃。结论:纯化所得蛋白为目的蛋白hPRL-1;两步纯化相结合可以得到纯度较高的蛋白;三种底物特异性依次为DiFMUP>4-MUP>pNPP。     

以MyD88 TIR二聚化为靶点的抗炎抑制剂筛选模型的建立

MyD88是IL-1R/TLR受体超家族向细胞内转导胞外信号时募集到受体胞浆尾部的重要接头蛋白．由TIR结构域介导的MyD88分子同源二聚化是它招募到受体胞浆尾部的前提,然后二聚化的MyD88再募集下游信号分子,传递信号,引发促炎基因的表达．本研究旨在建立一种模型,以实现活细胞原位的、基于荧光信号变化的MyD88二聚化抑制物的高通量筛选．我们分别构建了MyD88 TIR与GFP和RFP的融合蛋白表达质粒,瞬时转染HeLa细胞,在488 nm激发光下,转染GFP-MyD88 TIR和RFP-MyD88 TIR细胞,检测到绿色荧光与红色荧光间的共振能量转移(FRET)．而当细胞转染GFP-MyD88 TIR和RFP或RFP-MyD88 TIR和GFP,因TIR二聚化不能实现,FRET效率受到严重影响．实验结果提示,依赖双阳性表达GFP-MyD88 TIR和RFP-MyD88 TIR的细胞株,检测不同化合物对于荧光FRET效率的影响,可以建立MyD88 TIR二聚化抑制药物的筛选模型．此外,我们构建了原核表达质粒,利用纯化的His-MyD88 TIR分别与GST或GST-MyD88 TIR蛋白进行体外结合实验,发现GST-MyD88 TIR(而非GST)可以与His-MyD88 TIR相互结合．结果的差异性提示,利用His-MyD88 TIR和GST-MyD88 TIR体外结合实验分析,可以进一步确定抑制物是否直接阻断了TIR的相互作用．结合真核细胞的荧光FRET阻断结果和原核表达的重组蛋白相互作用分析,可确定MyD88 TIR二聚化的抑制物．利用这一模型可以对商品化的小分子库、自行制备的天然产物组分进行广泛的筛选,从中获得有效抑制MyD88二聚化的化合物,参与对MyD88信号通路依赖的慢性炎症、自身免疫性疾病的药物治疗．     

FREUDEIA KASZAB, A NEWLY RECORDED GENUS AND A NEW SPECIES FROM CHINA (COLEOPTERA, TENEBRIONIDAE,TENTYRIINI)

报道中国鳖甲族1新纪录属及1新种:异颚弗鳖甲Freudeia heteromaxillaria sp.nov.,描述了新种的形态特征并附鉴别特征图和整体照片.新种模式标本保存于中国科学院西北高原生物研究所和河北大学博物馆.     

中国鳖甲族一新纪录属及一新种记述(鞘翅目,拟步甲科)

报道中国鳖甲族1新纪录属及1新种:异颚弗鳖甲Freudeia heteromaxillaria sp.nov.,描述了新种的形态特征并附鉴别特征图和整体照片。新种模式标本保存于中国科学院西北高原生物研究所和河北大学博物馆。     

Neotropical Copestylum (Diptera,Syrphidae) breeding in Agavaceae and Cactaceae including seven new species

We studied 42 species of saprophagous, Neotropical Copestylum (Diptera, Syrphidae) reared from decaying Cactaceae and Agavaceae. Thirty‐three species were reared during fieldwork in Bolivia, Costa Rica, Ecuador, Mexico, Peru, and Trinidad from 1998–2007. Nine species came from museum and private collections. Seven were new species. We describe these new species and the third stage larva and/or puparium and breeding sites of 40 species. Not described are two apparent species related to Copestylum apicale (Loew, 1866) reared from Cactaceae. Resolution of their status was beyond the scope of this paper but reference is made to their distinctive larval morphology. Based on early stage characters all reared species can be placed in ten species groups, all but three of which have been recognized previously on the basis of adult characters. A high level of congruence was found between adult and larval characters in terms of these species groups. Eight of the groups appear to be related closely and may represent a monophyletic lineage within Copestylum that has diversified in xeric habitats. Early stage morphology varied within and amongst groups but two trends in functional morphology are recognizable. One trend is towards feeding in watery decay and the other towards feeding in firmer decay. The latter trend is characterized by species that scoop food and use grinding mills in their head skeletons to break it up. They also have armoured thoraces with varying arrangements of sclerotized spicules or stiffened setae for gripping and protection during tunnelling, a short anal segment, and a short posterior breathing tube for protecting the openings. The former trend is characterized by species with opposite and contrasting features. They filter food and have well‐developed pre‐oral setal filters but they lack grinding mills or only have poorly developed grinding mills. They have reduced thoracic armature, elongate anal segments, and posterior breathing tubes which facilitates simultaneous feeding and respiration. Comparison with 23 Copestylum species reared from bromeliads (Bromeliaceae) suggests a common pattern of diversification in that species groups with the largest body sizes are more specialized.     

DNA profiling of complex bacterial populations: toxic cyanobacterial blooms

Cyanobacteria are prokaryotic photosynthetic living organisms that inhabit our planet for over three billion years. With a worldwide distribution, they can be found in all types of environments: fresh, brackish and saltwater as well as terrestrial. Though beneficial in the development of life on earth, they also constitute a serious risk to our ecosystems since they can biologically produce harmful secondary metabolites named cyanotoxins. When studying cyanobacteria and their cyanotoxins, several methodologies have been applied with an increasing relevance to molecular methods. Therefore, the aim of this review is to describe alternative molecular methods that can be used as alternative methods for the identification of cyanobacteria. More traditional chemotaxonomic methods are discussed briefly as are the standard and somewhat dated techniques for assessing genome content for taxonomic classification schemes. The use of DNA amplification technology has been applied to the systematics and phylogeny of many bacterial groups, and the optimisation of methods for rapid identification and classification of cyanobacteria are presented. Together with novel methods developed for these photosynthetic microorganisms, the generated DNA profiles have been utilised to study cyanobacterial bloom population diversity and prediction of strain toxigenicity. Finally, the genotypes found were applied to a variety of phylogenetic analyses; trees were reconstructed and compared to the current morphological system of classification. The ecology and diversity of the cyanobacteria is discussed with respect to the derived molecular phylogenies and systematics.     

BR-TRG-Ⅰ型体腔热灌注治疗系统安全性评估的动物实验

目的探讨应用BR-TRG-I型体腔热灌注治疗系统进行持续循环腹腔热灌注治疗（continuous circulatory hyperthermic intraperitoneal perfusion treatment,CCHIP）对实验动物的生命体征、肝肾功能及内脏器官病理形态的影响。方法选用家猪作为实验动物,应用BR-TRG-I型体腔热灌注治疗系统建立CCHIP动物模型,分别用44℃、45℃的设定温度进行CCHIP。在CCHIP期间记录实验动物的生命体征变化;CCHIP前及治疗后1d、3d、7d和14d抽取外周血液保存备检;CCHIP结束后即刻及2周后处死动物,观察内脏器官的大体形态学改变,切取肝、肾、小肠等器官进行病理检查。结果44℃CCHIP持续1.5h对猪的生命体征、肝肾功能无明显影响,肝、肾、小肠等脏器损伤较轻,2周后恢复正常;45℃CCHIP持续1.5h对猪的生命体征影响严重,肝肾功能损害持续2周尚不能恢复正常,肝、肾、小肠等脏器病理损伤明显。结论44℃温度CCHIP1.5h安全可行,可以作为CCHIP的安全温度;45℃温度CCHIP1.5h可对实验动物肝肾功能造成严重损害,不适合作为CCHIP的治疗温度。     

Detection of saxitoxin-producing cyanobacteria and Anabaena circinalis in environmental water blooms by quantitative PCR

Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by marine "red tide" dinoflagellates and several species of freshwater filamentous cyanobacteria, including Anabaena circinalis, Aphanizomenon spp., Lyngbya wollei, and Cylindrospermopsis raciborskii. A specific quantitative PCR (qPCR) method based on SYBR green chemistry was developed to quantify saxitoxin-producing Anabaena circinalis cyanobacteria, which are major bloom-forming freshwater cyanobacteria. The aim of this study was to infer the potential toxigenicity of samples by determining the copy number of a unique and unusual polyketide synthase (PKS) sequence (sxtA) in the STX biosynthesis gene cluster identified in cyanobacteria. Our qPCR approach was applied to water samples collected from different Australian lakes, dams, and rivers. The STX concentration and cyanobacterial cell density of these blooms were also determined by high-pressure liquid chromatography (HPLC) and microscopic cell counting, respectively. STX concentrations correlated positively with STX gene copy numbers, indicating that the latter can be used as a measure of potential toxigenicity in Anabaena circinalis and possibly other cyanobacterial blooms. The qPCR method targeting STX genes can also be employed for both monitoring and ecophysiological studies of toxic Anabaena circinalis blooms and potentially several other STX-producing cyanobacteria.     

生物软件在序列分析过程中的运用

介绍了组合使用BLAST、FASTA/BLASTScan3.2,或用多序列比对软件,从数据库中快速提取大数量目标序列,最后用MEGA4快捷编辑整理大数量序列的方法。还介绍了一种生成核酸序列与其氨基酸序列相似性百分率整合表格的方法。简述了对引物设计的基本认识并介绍了多重引物兼容性筛选软件;对构建系统发育树的认识并引出分子进化树构建软件MEGA4的使用和PAUP 4.0常用建树命令模块。期望这些方法和软件的使用能解决生物序列分析过程的常见问题。