Fate of prions in soil: Degradation of recombinant prion in aqueous extracts from soil and casts of two earthworm species

Prions represent the active agent in transmissible spongiform encephalopathy (TSE) diseases and can remain infective to mammals even after prolonged periods in soil. The influence of mesofauna on prion dispersal and degradation in soil, however, remains unknown. In this study the effect of earthworms on the retention/dissemination of TSEs in soil was evaluated using a model recombinant prion protein (recPrP) and aqueous extracts from soil and fresh casts of two earthworm species, Lumbricus terrestris and Aporrectodea caliginosa. Our results showed that earthworm gut-derived enzymes did not enhance the degradation of recPrP in comparison to soil, even though non-prion related proteolytic activity was higher in fresh worm excrements than in soil samples. Complete degradation of recPrP occurred in the aqueous extracts from all samples within up to 6 days at +15 °C. The proteolytic enzymes responsible for degrading recPrP were inhibited by aprotinin and leupeptin and studies in pure cultures suggested these were most probably of soil microbial origin.     

PrPc重组蛋白脑内接种金黄地鼠对mRNA表达的影响

为明确将牛PrPc重组蛋白接种金黄地鼠脑内是否引起异常临床表现、脑组织病理学变化及对其mRNA表达产生影响,为进一步研究PrPc蛋白与异构体PrPsc 的结构转换机制提供基础数据,将纯化的牛PrPc重组蛋白磷酸盐缓冲液制剂进行地鼠颅内接种,约3 μL/只,对照接种磷酸盐缓冲液（PBS）;102 d后取出脑组织：一部分福尔马林固定后进行常规H.E.染色观察, 另一部分提取脑组织总RNA,进行实时荧光定量RT-PCR检测.结果表明：处理未见临床异常表现;大脑、小脑、脑干组织病理学检测未见海绵状空泡变性和淀粉样斑;大脑PrPc mRNA表达水平无显著性差异.上述结果表明,用PrPc重组蛋白接种,在检测时间102 d内未引起金黄地鼠行为和脑组织的异常改变.     

羊痒病研究进展

牛的海绵状脑病（BSE）的爆发让人们意识到要控制传染性海绵状脑病（TSE）的流行来保护人和动物的健康。羊痒病是世界上传播最广的传染性海绵状脑病,为此,不同国家的兽医当局都有针对痒病的根除计划,除了增强对动物的监管外,还要培育对痒病有抗性的羊。笔者主要综述了当前羊痒病的研究进展。     

朊蛋白在物种内、物种间的致病机理

朊蛋白(prion)是传染性海绵状脑病(transmissible spongiform encephalopathy,TSE)的唯一致病因子。在细胞内存在两种形式的朊蛋白,即正常形式PrP~c和致病形式PrP~(sc)(PrP~(res))。PrP~(sc)的出现是TSE发生的关键因素。本文阐述了朊蛋白的发现与意义及其在物种内、物种间的致病机理。     

Scrapie infection alters the distribution of plasma metabolites in diseased Cheviot sheep indicating a change in energy metabolism

Nuclear magnetic resonance (NMR) spectroscopy has been used to profile the metabolic status of plasma from; sheep showing clinical signs of scrapie, those known to be infected with scrapie but yet to show clinical signs, and control animals. The NMR measurements have shown that energy metabolism in scrapie infected animals is altered before the onset of clinical symptoms. These metabolic changes may provide the foundation for a pre-clinical diagnostic test for scrapie in sheep.     

传染性海绵状脑病及其分子生物学诊断

传染性海绵状脑病是一种区别于普通流行病的人兽共患病 ,其病原体是一种具有传染性的、未知功能的糖蛋白 ,命名为蛋白质感染颗粒 ,即朊病毒。它是一个超出经典病毒学和生物学的全新概念。朊病毒是有机体的正常细胞蛋白质错误折叠的产物 ,其扩增机制和致病机制不同于一般病毒。传染性海绵状脑病潜伏期长 ,传染性强且无法治愈 ,机体免疫系统对朊病毒不识别。目前 ,传染性海绵状脑病的诊断已达到分子水平 ,国际上主要利用抗 Pr P抗体、脑脊液中蛋白质的改变以及实时 PCR和质谱分析法等手段来进行临床诊断和病原体的检测。     

PrPsc检测方法研究进展

海绵状脑病(prions)是人和动物中的一类致死性中枢神经系统疾病.现尚无理想的早期、快速的检测方法和防治措施.目前,组织病理学检测、动物试验和免疫学检测为较为常用的prions病检测方法,其它一些新兴的检测方法如毛细管电泳、光谱分析和高效液相色谱等也逐渐被开发研制,但因其本身缺陷,暂时无法得到广泛应用.     

牛、羊、人叠朊基因的克隆与分析

为了解我国哺乳动物叠朊基因Prnd的生物信息，本实验采用PCR方法克隆了秦川黄牛、甘肃土种绵羊和汉族人的Prnd，运用分子生物学软件对这些序列进行了分析比较。结果表明，获得的三种哺乳动物的Prnd均位于1个单一的外显子内，与GenBank登录的相应序列具有高度同源性。秦川黄牛与甘肃土种绵羊Prnd的同源性为96．8％，推导氨基酸序列同源性为93．3％。汉族人Prnd与秦川黄牛和甘肃土种绵羊Prod的同源性均为80％，推导的氨基酸序列同源性均为76．3％。氨基酸一级结构分析显示3种Prod编码的叠朊均由氨基端的信号肽、中间的成熟蛋白和羧基端的GPI锚结合区组成。二级结构预测表明，3种Prod编码的叠朊均由3个α-螺旋和2个β-片层组成。本研究获得的这3种主要哺乳动物的Prnd信息为一进步研究叠朊的结构与功能奠定了基础。     

Counting of single prion particles bound to a capture-antibody surface (surface-FIDA)

Hitherto accredited prion tests use the PK resistance of PrP(Sc), the pathogenic isoform of the prion protein, as a marker for the disease. Because of variations in the amount of disease-related aggregated PrP, which is not PK-resistant, these prion tests offer only limited sensitivity. Therefore, a prion detection method that does not rely on PK digestion would allow for the detection of both PK-resistant as well as PK-sensitive PrP(Sc). Furthermore, single particle counting is more sensitive than methods measuring an integrated signal. Our new test system is based on dual-colour fluorescence correlation spectroscopy (FCS). This method quantifies the number of protein aggregates that have been simultaneously labelled with two different antibodies using dual-colour fluorescence intensity distribution analysis (2D-FIDA). This only counts PrP aggregates, and not PrP monomers. To increase the sensitivity, PrP(Sc) was concentrated in a two-dimensional space by immobilizing it so that the antibodies could be captured on the surface of the slide (surface-FIDA). When the surface was systematically scanned, even single prion particles were detected. Using this new technique, the sensitivity to identify samples from scrapie-infected hamster as well as BSE-infected cattle can be dramatically increased in comparison with identification using FIDA in solution.