Leishmania braziliensis:Characterisation of a Complex Specific Subtelomeric Repeat Sequence and Its Use in the Detection of Parasites

Fu, G., Perona-Wright, G., and Barker, D. C. 1998.Leishmania braziliensis: Characterisation of a complex specific subtelomeric repeat sequence and its use in the detection of parasites.Experimental Parasitology90, 236–243. A 1.6-kb tandem repeat sequence had previously been identified in the subtelomeric region of mini- and megabase chromosomes fromLeishmania braziliensis.Southern hybridisation was used to demonstrate that the repeat is complex specific. The sequence was characterised in strains representing four species of theL. braziliensiscomplex. This data allowed an assessment of the evolutionary relationship of the four species. PCR primers targeted to the repeat amplify only DNA from species of theL. braziliensiscomplex. Titration assays indicate that a minimum of 50 fg of parasite DNA can be detected by PCR alone. Southern hybridisation increases the limit of detection to 5 fg. Interspecies variation in the repeat sequence enabled restriction enzyme digestion of PCR products to distinguish individual species within theL. braziliensiscomplex.     

反刍兽月形单胞菌β-木糖苷酶基因在毕赤酵母中的高效表达及酶学性质

β-木糖苷酶(β-xylosidase,酶编号EC 3.2.1.37)是木聚糖降解酶系中的重要组成部分。本研究以毕赤酵母Pichia pastoris GS115为宿主菌尝试表达反刍兽月形单胞菌Selenomonas ruminantium中的β-木糖苷酶基因Sxa。根据毕赤酵母对密码子的偏爱性、mRNA二级结构、GC含量和稀有密码子,对Sxa基因进行优化;通过基因合成技术获得了全长基因mSxa并构建重组酵母表达载体pPIC9K-mSxa;以BglⅡ酶切重组载体pPIC9K-mSxa,电击转化将m Sxa基因导入毕赤酵母GS115中,获得的转化子经过表型和遗传霉素G418抗性筛选、PCR鉴定,得到表达β-木糖苷酶基因的工程菌GS115-pPIC9K-mSxa;通过活性测定获得高效表达β-木糖苷酶的重组酵母,并对重组β-木糖苷酶的酶学性质进行了初步研究。结果表明,重组β-木糖苷酶的分子量约为66 kDa。在发酵罐水平表达的酶活性达到了287.61 IU/mL。对酶学性质研究显示,该酶在温度为40-60℃,pH为5.0-7.0时较稳定,其最适反应温度和pH分别为55℃和6.0,专一性地作用于β-木糖苷键。Mn~(2+)和Ca~(2+)对该酶具有激活作用,而Fe~(3+)、Cu~(2+)、Co~(2+)、Mg~(2+)、EDTA及SDS抑制其酶活性。本研究首次将反刍兽月形单胞菌的β-木糖苷酶基因转化到毕赤酵母中获得表达,并具有较高活性,为进一步工业化应用奠定了基础。     

匍匐茎草本植物形态可塑性、整合作用与觅食行为研究进展

综述了匍匐茎型克隆植物在形态可塑性、整合作用及觅食行为方面的研究进展。资源斑块性分布是生境异质性的特征之一,适应于异质性生境,匍匐茎植物对环境资源表现了一系列可塑性反应。本文着重从匍匐茎植物对光、水、肥的可塑性反应及其整合作用以及觅食行为等方面的研究进行总结分析,以期对匍匐茎型克隆植物进行更广泛深入的研究。     

养殖美洲鲥的生长特性

通过对各周年生长阶段的养殖美洲鲥(Alosa sapidissima)体长、体重的测量与分析,研究了工厂化养殖美洲鲥的周年生长特性。养殖美洲鲥雌雄鱼的体长与体重关系均呈幂函数增长相关,其方程分别为,雌:W=0.8062×10~(-5) L~(3.1113)(n=122,R~2=0.997 5,P0.01);雄:W=1.0047×10~(-5) L~(3.0574)(n=125,R~2=0.997 5,P0.01),b均接近于3,呈等速生长;雌雄鱼生长均可分快速生长期(0~+龄)、稳定生长期(1~+龄)、生长衰老期(2~+龄)三个时期,雌鱼生长快于雄鱼。拟合出von Bertallanffy生长方程,雌:Lt=467.92(1﹣e~(-0.5748(t+0.1710))),Wt=1637.72(1﹣e~(-0.5748(t+0.1710)))~(3.1113);雄:Lt=389.21(1﹣e~(-0.7374(t+0.1975))),Wt=834.08(1﹣e~(-0.7374(t+0.7189)))~(3.0574)。雌雄鱼体重生长拐点分别位于1.517 8 a和1.224 7 a,属性成熟拐点,拐点前生长较快;拐点后,特别是性成熟后(2龄),2~+龄鱼的生长速度明显降低;同时,2龄鱼繁殖季节过后会出现大量死亡。建议商业化工厂化养殖美洲鲥以2龄前上市为宜。     

九种蹄盖蕨科植物配子体的酯酶同工酶分析

本文培养了蹄盖蕨科９个种即中华蹄盖蕨（Ａｔｈｙｒｉｕｍｓｉｎｅｎｓｅ）带岭蹄盖蕨（Ａ．ｄａｌｉｎｇｅｎｓｅ）、多齿蹄盖蕨（Ａ．ｍｕｌｔｉｄｅｎｔａｔｕｍ）（包括青柄和紫柄２种带岭蹄蕨类型）、东北蛾眉蕨（Ｌｕｎａｔｈｙｒｉｕｍｐｙｃｎｏｓｏｒｕｍ）、朝鲜介蕨（Ｄｒｙｏａｔｈｙｒｉｕｍｃｏｒｅａｎｕｍ）、山冷蕨（Ｃｙｓｔｏｐｔｅｒｉｓｓｕｄｅｔｉｃａ）、假冷蕨（Ｐｓｅｕｄｏｃｙｓｔｏｐｔｅｒｉｓｓｐｉｎｕｌｏｓａ）、欧洲羽节蕨（Ｇｙｍｎｏｃａｒｐｉｕｍｄｒｙｏｐｔｅｒｉｓ）和黑鳞短肠蕨（Ａｌａｎｔｏｄｉａｃｒｅｎａｔａ）的配子体为材料,进行酯酶同工酶的分析,以表明它们的种间差异,其中多齿蹄盖蕨的青柄和紫柄两种类型的酶谱存在明显差异,应考虑紫柄为多齿蹄盖蕨的变型。     

Diazoxide Pretreatment Prevents Aβ1–42 Induced Oxidative Stress in Cholinergic Neurons Via Alleviating NOX2 Expression

The aggregation and accumulation of amyloid-β (Aβ) plays a significant role in the pathogenesis of Alzheimer’s disease. Aβ is known to increase free radical production in neuronal cells, leading to oxidative stress and cell death. Diazoxide (DZ), a highly selective drug capable of opening mitochondrial ATP-sensitive potassium channels, has neuroprotective effects against neuronal cell death. However, the mechanism through which DZ protects cholinergic neurons against Aβ-induced oxidative injury is still unclear. The present study was designed to investigate the effects of DZ pretreatment against Aβ_1–42 induced oxidative damage and cytotoxicity. Through measures of DZ effects on Aβ_1–42 induced cellular damage, reactive oxygen species (ROS) and MDA generation and expressions of gp91phox and p47phox in cholinergic neurons, new insights into the neuroprotective mechanisms can be derived. Aβ_1–42 significantly decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide levels and increased ROS and MDA production; all effects were attenuated by pretreatment with DZ or diphenyleneiodonium chloride (a NOX2 inhibitor). Pretreatment with DZ also attenuated the upregulation of NOX2 subunits (gp91phox and p47phox) induced by Aβ_1–42. Since NOX2 is one of the main sources of free radicals, these results suggest that DZ can counteract Aβ_1–42 induced oxidative stress and associated cell death by reducing the level of ROS and MDA, in part, by alleviating NOX2 expression.     

保护性耕作对农田碳、氮效应的影响研究进展

作物产量的高低主要取决于土壤肥力,如何保持并提高土壤肥力是确保我国粮食安全和农业可持续发展的重要任务,也是众多学者关注的焦点。土壤有机碳和氮素是评价土壤质量的重要指标,其动态平衡直接影响土壤肥力和作物产量。随着全球气候变化及环境污染问题的愈加突出,农田土壤固碳及提高氮效率成为各界科学家研究的热点。目前,保护性耕作已成为发展可持续农业的重要技术之一,对土壤固碳及氮素的利用具有很大的影响。深入了解保护性耕作对土壤有机碳固持与氮素利用效率提高的影响机制,对于正确评价土壤肥力有着重要意义。但由于气候、土壤及种植制度等条件不一致,关于保护性耕作对农田碳、氮效应结论不一。阐述了国际上保护性耕作对农田系统土壤有机碳含量变化及其分解排放(如CO₂和CH₄)、氮素变化及其矿化损失(如NH₃挥发、N₂O排放与氮淋失)和碳氮素相互关系(如C/N层化率)影响的研究进展,并分析了其影响因素和相关机理。尽管国内保护性耕作的研究已进行30 多年,但在土壤有机碳与氮素方面与国外相比依然有较大的差距。保护性耕作对土壤固碳与氮素利用的影响机制,碳素和氮素在土壤-植株-大气系统中的转移变化,及结合农事管理等综合评价其生态效应的研究很少。在此基础上,提出未来我国保护性耕作在土壤有机碳固定和氮素利用方面的重点研究方向:(1)在定位试验基础上进一步探讨保护性耕作对土壤有机碳及氮素利用的影响机制;(2)深入研究土壤有机碳和氮素的相互关系及其对土壤肥力的影响;(3)结合环境保护与土壤可持续管理对保护性耕作农田土壤固碳及氮素高效利用的系统评价研究;(4)加强保护性耕作对农田碳、氮效应的宏观研究,合理评价保护性耕措施下对农田碳、氮综合效应。     

人Leptin基因在大肠杆菌中的高效表达、纯化及其生物学活性研究

将人Leptin表达质粒pBV220-OB转化E.coliJM109,经热诱导获得了目的蛋白的表达。经SDS-PAGE鉴定分析,表达产物以包涵体形式存在,目的蛋白表达量占菌体总蛋白的40%以上。通过包涵体分离,Sephacryl S200HR凝胶和DEAE52离子交换层析及Hypersil C18柱反相色谱纯化,获得纯度在95%以上,内毒素含量小于10EU/mg的高纯度的重组人Leptin。Western-blot鉴定表明,纯化表达产物能和抗Leptin抗体特异性结合;蛋白质N端15个氨基酸序列分析结果和预期的序列一致。纯化产物经复性处理,其分子中Cys96和Cys146形成二硫键。体内活性检测显示,纯化和复性的rh-Leptin明显抑制BALB/c小鼠的进食和体重增长,提示其具有明显的生物学活性。     

Quality,bioactivity study,and preclinical acute toxicity,safety pharmacology evaluation of PEGylated recombinant human endostatin (M2ES)

Endostar, a potent endogenous antiangiogenic factor, is wildly used in clinics. However, it was easily degraded by enzymes and rapidly cleared by the kidneys. To overcome these shortcomings, PEGylated recombinant human endostatin was developed. In this study, the purity of M₂ES was evaluated by silver stain and reversed‐phase high‐performance liquid chromatography. Ultraviolet spectrum was used to examine the structural of M₂ES and endostar. The bioactivity and antitumor efficacy of M₂ES were evaluated using an in vitro endothelial cell migration model and athymic nude mouse xenograft model of a heterogeneous lung adenocarcinoma, respectively. A preclinical study was performed to evaluate the acute toxicity and safety pharmacology in rhesus monkeys. The purity of M₂ES was more than 98%; PEG modification has no effect on endostatin structure. Compared with the control group, M₂ES dramatically retards endothelial cell migration and tumor growth. After intravenous (IV) infusions of M₂ES at a dose level of three and 75 mg/kg in rhesus monkeys, there was no observable serious adverse event in both acute toxicity and safety pharmacology study. On the basis of the quality and bioactivity study data of M₂ES and the absence of serious side effect in rhesus monkeys, M₂ES was authorized to initiate a phase I clinical trial.