三氧化二砷对肝癌细胞系SMMC-7721凋亡及 Smac caspase-9、caspase-3 蛋白表达的影响

目的：研究三氧化二砷（As203）对人肝癌细胞SMMC-7721的促凋亡作用及对Smac、caspase-9、caspase-3表达的影响。方法：人肝癌细胞SMMC-7721经As20,处理,共分为四组,分别为空白对照组、低剂量组、中等剂量组、高剂量组。分别采用MTT、Hoechst33258染色法、Annexin V-FITC／PI双染法观察其对SMMC．7721细胞增殖的抑制,凋亡细胞核的形态学变化,以及诱导凋亡作用;采用Westemblot法检测凋亡相关蛋白Smac、caspase-9、caspase-3表达的变化。结果：MTT显示：As203在体外能明显抑制SMMC-7721的生长,具有时间剂量依赖关系,与空白对照组相比,其余三组细胞生存率明显下降,差异均有统计学意义（P〈0．05）;Hoechst33258显示细胞呈明显的凋亡细胞形态学特征,具有剂量依赖性;AnnexinV-FITC／PI双染法显示：As203作用24小时可诱导SMMC-7721细胞凋亡,且呈剂量依赖性,与空白对照组相比（2．69±0．58）,其余三组（4．01±0．58）、（5．99±1．69）、（9．26±2．34）差异均有统计学意义（P〈0．05）;Westernblot显示：As2O3作用SMMC-7721细胞24小时,Smac、caspase-9、caspase-3表达上升,呈剂量依赖性,与空白对照组相比,其余三组蛋白表达量明显增加,差异均有统计学意义（P〈0．05）。结论：-定量的As203能抑制SMMC-7721细胞增殖,促进其凋亡,其机制可能与调控Smac、caspase-9、caspase-3表达有关。     

犬细小病毒NS1 非结构蛋白可诱导细胞凋亡

【目的】研究犬细小病毒(Canine parvovirus,CPV)非结构蛋白NS1在CPV引起宿主细胞凋亡中的作用,初步探讨CPV引起细胞凋亡的机制。【方法】首先采用PCR方法从犬细小病毒基因组中扩增NS1编码基因,然后利用pcDNA3.1A质粒构建NS1真核表达载体pcDNA-NS1,并通过HEK293FT细胞瞬时表达NS1重组蛋白,用Western-blot检测以确定重组NS1蛋白能否在真核细胞中表达。然后用CPV感染和用pcDNA-NS1表达载体转染F81宿主细胞,通过AnnexinV/PI双染法检测磷脂酰丝氨酸外翻和通过化学发光法检测caspase-3/7活性,分析感染CPV或转染NS1基因对F81宿主细胞凋亡的影响。【结果】结果表明,本实验扩增的NS1基因序列与GenBank的序列一致,构建的表达载体结构正确,并能够介导NS1基因在真核细胞中表达。感染CPV和转染NS1基因均能诱导F81细胞膜磷脂酰丝氨酸外翻和明显提高细胞内caspase-3/7的活性,表明CPV和NS1蛋白均能引起细胞的凋亡。【结论】CPV诱导宿主细胞凋亡与其编码的NS1非结构蛋白有关。     

基于肝癌细胞线粒体功能受损和caspase-3信号通路探讨罗哌卡因促进肝癌细胞凋亡的作用机制研究

摘要 目的：基于肝癌细胞线粒体功能受损和天冬氨酸蛋白水解酶3（caspase-3）信号通路探讨罗哌卡因促进肝癌细胞凋亡的作用机制。方法：选用细胞株人肝癌细胞BEL-7402进行实验研究。用不同浓度罗哌卡因处理BEL-7402细胞后,采用溴化噻唑蓝四氮唑（MTT）法检测肝癌细胞的增殖情况,光镜及4,6-二苯胺-2-苯吲哚二盐酸盐（DAPI）溶液染色观察细胞形态,台盼蓝染色法测定细胞活力,流式细胞术分析BEL-7402细胞的凋亡情况,电子显微镜下观察细胞线粒体,激光共聚焦显微镜观察caspase-3在BEL-7402细胞中的细胞核迁移情况,蛋白免疫印迹试验评价罗哌卡因对细胞质凋亡相关蛋白、线粒体凋亡相关蛋白、BEL-7402细胞和线粒体凋亡相关蛋白表达的影响。结果：罗哌卡因能够抑制肝癌细胞的生长,并呈剂量依赖性和时间依赖性。罗哌卡因可诱导BEL-7402细胞发生凋亡,显著增加BEL-7402细胞的凋亡率。罗哌卡因能够损伤肝癌细胞线粒体功能。激光共聚焦显微镜观察显示caspase-3分子迁移到细胞核。罗哌卡因与caspase-3相互作用,促进caspase-3向细胞核内迁移,刺激caspase-3和聚腺苷二磷酸核糖聚合酶（PARP-1）、天冬氨酸蛋白水解酶9（caspase-9）蛋白的表达,抑制B细胞淋巴瘤-2基因（Bcl-2）的表达,促进凋亡酶激活因子（Apaf-1）的表达,促进线粒体释放细胞色素C（Cytochrome C）,激活caspase-3活性。结论：罗哌卡因具有促进肝癌细胞凋亡的作用,其作用机制可能与破坏肝癌细胞线粒体功能和激活caspase-3信号通路有关。     

NF-kB拮抗剂PDTC对HepG2细胞的抑制及对caspase-3表达的影响

目的：研究NF-kB拮抗剂PDTC诱导人肝癌HepG2细胞凋亡及其对凋亡相关基因caspase-3表达的影响,初步探讨其诱导凋亡的可能机制。方法：以不同浓度的PDTC处理人肝癌HepG2细胞,利用MTT法检测对人肝癌HepG2细胞凋亡的影响;利用RT-PCR和Western-blot检测caspase-3mRNA和蛋白的表达。结果：不同浓度PDTC作用人肝癌HepG2细胞不同时间后,能够显著抑制HepG2细胞的生长增殖,存在剂量和时间依赖性（P〈0.05）;PDTC能够上调HepG2细胞中caspase-3mRNA和蛋白的表达。结论：NF-kB拮抗剂PDTC对人肝癌HepG2细胞产生显著抑制,并能上调HepG2细胞中caspase-3mRNA和蛋白的表达。     

NF-kB 拮抗剂PDTC 对HepG2 细胞的抑制及对caspase-3 表达的影响

目的:研究NF-kB拮抗剂PDTC诱导人肝癌HepG2细胞凋亡及其对凋亡相关基因caspase-3表达的影响,初步探讨其诱导凋亡的可能机制。方法:以不同浓度的PDTC处理人肝癌HepG2细胞,利用MTT法检测对人肝癌HepG2细胞凋亡的影响;利用RT-PCR和Western-blot检测caspase-3mRNA和蛋白的表达。结果:不同浓度PDTC作用人肝癌HepG2细胞不同时间后,能够显著抑制HepG2细胞的生长增殖,存在剂量和时间依赖性(P<0.05);PDTC能够上调HepG2细胞中caspase-3mRNA和蛋白的表达。结论:NF-kB拮抗剂PDTC对人肝癌HepG2细胞产生显著抑制,并能上调HepG2细胞中caspase-3mRNA和蛋白的表达。     

羊栖菜多糖通过激活Caspase途径诱导Lovo细胞凋亡

研究了羊栖菜多糖（Sargassum Fusiforme Polysaccharides,SFPS）诱导人大肠癌lovo细胞凋亡及凋亡过程中caspase-3、caspase-8、caspase-9的活性变化。MTT法检测SFPS对lovo细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western印迹法测定caspase-3酶原和caspase-9的变化;RToPCR检测caspase-3 mRNA表达;caspase-3,caspase-8、caspase-9活性检测试剂盒观察caspase-3、caspase-8、caspase-9的活性改变。结果显示,SFPS对lovo细胞增殖有显著抑制作用,经形态变化、DNA条带和流式细胞分析,可见明显的细胞凋亡特征。SFPS处理lovo细胞后,发现caspase-3酶原蛋白表达降低,caspase-3 mRNA高表达,并具有剂量和时间的依赖性。而在检测蛋白中,也发现caspase-9被激活进而形成具有活性的片段。另外,caspase的活性检测也进一步发现caspase-3、caspase-9的活性逐步增高。实验结果提示SFPS在体外诱导lovo胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,并且是通过激活启动caspase-9,进而激活下游效应caspase-3的级联反应来实现的。     

副猪嗜血杆菌外膜囊泡激活caspase-11和NLRP3炎性体诱导炎性细胞因子IL-1β和IL-18分泌

【背景】副猪嗜血杆菌可引起多种炎性反应和较高死亡率,其致炎机制目前尚不清楚。【目的】探究副猪嗜血杆菌外膜囊泡(outer membrane vesicles,OMVs)诱导RAW264.7细胞caspase-11及NLRP3炎性体活化的功能,以及caspase-11在OMVs活化炎性体诱导炎性因子表达过程中的关键作用。【方法】副猪嗜血杆菌OMVs感染RAW264.7细胞,收集6、12和24h细胞,RT-PCR检测caspase-11、NLRP3、ASC和caspase-1 mRNA的表达;收集感染后48 h细胞,Western blotting检测caspase-11、NLRP3、ASC和caspase-1蛋白表达。收集感染后6、12、24、48和72h细胞上清,ELISA检测interleukin(IL)-1β和IL-18表达水平;不同浓度OMVs(0、2.5、10、25、50和100μg/mL)感染RAW264.7细胞24h,ELISA检测上清中IL-1β和IL-18水平。构建caspase-11沉默RAW264.7细胞株,收集OMVs感染后12、24和48h细胞培养上清检测IL-1β和IL-18水平。【结果】Caspase-11 mRNA转录水平在OMVs感染6、12和24h均极显著高于对照组(P<0.01);NLRP3 mRNA转录水平在感染后6、24h极显著高于对照组(P<0.01);ASC mRNA转录水平在感染后12、24h显著低于对照组(P<0.05);caspase-1 mRNA转录水平在感染后6、12和24h显著高于对照组(P<0.05)。Western blotting结果显示,与空白对照组相比,OMVs组caspase-11、NLRP3、ASC和caspase-1蛋白表达均明显升高。OMVs感染RAW264.7细胞后12、24、48和72h,IL-1β表达量极显著高于对照组(P<0.01),而且呈现时间依赖性升高,IL-18表达水平在感染后6、12、24、48和72h显著高于对照组(P<0.05);不同浓度OMVs感染细胞时,OMVs对细胞的致炎作用呈现剂量依赖性增强。Caspase-11沉默后,IL-1β水平在感染后12、24和48h显著低于未沉默组;Caspase-11沉默组IL-18表达量在感染后24、48h均显著低于未沉默组(P<0.05)。【结论】副猪嗜血杆菌OMVs在副猪嗜血杆菌诱导的炎症反应中发挥重要作用,OMVs可诱导RAW264.7细胞caspase-11介导的非经典NLRP3炎性体信号通路活化。     

猪胸膜肺炎放线杆菌感染小鼠所致炎症中TLR-4信号通路涉及细胞焦亡

摘要:【目的】探讨革兰氏阴性菌胸膜肺炎放线杆菌(Actinobacillus Pleuropneumoniae,APP)感染小鼠致肺病变的分子细胞机制,以改善猪传染性胸膜肺炎的防治。【方法】滴鼻法感染小鼠,观察肺部眼观病变,石蜡切片HE染色观察肺组织病变。RT-PCR和qPCR方法检测小鼠肺部的Caspase-1、Caspase-3、TNF-α、IL-1β、IL-6、IL-18、TLＲ-4表达量的变化。【结果】小鼠在感染后48h内死亡,解剖后观察小鼠肺部有严重出血和炎症。实验组小鼠肺组织IL-1β、IL-6、IL-18 和TNF-α的表达水平显著增加;TLR-4的表达量显著上调;肺部组织Caspase-1的表达水平明显升高,而Caspase-3的表达水平未见明显变化。【结论】TLR-4信号通路可能参与APP感染引起的肺部炎症的发生,APP感染后激活TLR-4信号通路使有关细胞释放炎症因子,引起的肺部炎症。炎症可能涉及Caspase-1参与的细胞焦亡。     

重金属诱导细胞凋亡的分子机制

重金属诱导的细胞凋亡是一个十分复杂的过程,不同种类的重金属以及同类重金属离子的不同价态所诱导的凋亡效应及其分子机制不尽相同。目前的研究表明,重金属与DNA形成加合物而导致DNA损伤可能是引发细胞凋亡的重要步骤：多种重金属能通过激活内质网、线粒体钙通道,使Ca^2 释放进入细胞质而引发凋亡;重金属还能使细胞中ROS升高,在直接导致DNA损伤的同时,启动与线粒体相关的细胞凋亡信号通路。此外,ROS还能通过MAPKs增强JNK介导的：FasL和Fas表达,最终使caspase-3和caspase-7激活,从而促进凋亡的发生。重金属诱导细胞凋亡还涉及一系列重要基因和蛋白质的参与,包括促进凋亡的Src家族酪氨酸激酶、bax、fas和p53等基因及相关蛋白,抑制凋亡的Sp1锌指转录因子、bcl-2和myc等基因及相关蛋白。部分重金属如镉、锌等对细胞凋亡具有诱导和拮抗双重效应,其中拮抗效应主要是通过与自由钙离子协同进行的,而诱导效应则可能是通过调节caspase-3活性而实现的。     

预热诱导心肌细胞表达Hsp70对Fas通路活化后细胞凋亡变化的研究

目的:研究Hsp70对Fas通路活化后细胞凋亡的作用及其可能机制.方法:水浴预热诱导培养新生大鼠心肌细胞Hsp70表达,Fas抗体活化Fas通路,Western blot测定Hsp70的表达,流式细胞仪检测细胞凋亡率,特异性底物测定caspase-8及caspase-3的活力.结果:预热可以使心肌细胞Hsp70表达升高,Fas抗体降低细胞凋亡率,高表达Hsp70可以降低Fas通路活化后心肌细胞凋亡率,同时相应使caspase-8及caspase-3活力升高的幅度降低.结论:Hsp70可能通过抑制caspase-8及caspase-3的活力而降低Fas通路活化后培养新生大鼠心肌细胞凋亡的发生.     

Use of flow cytometry in an apoptosis assay to determine pH and temperature stability of shiga-like toxin 1

Shiga toxins and Shiga-like toxins (Stx) are a relatively large group of cytotoxins produced by certain serotypes of Shigella and E. coli (STEC). These toxins are responsible for diarrhea, hemorrhagic colitis and may induce hemolytic uremic syndrome (HUS) with serious consequences in young children. The toxins are proteins made up of 5 small B subunits responsible for binding to an outer membrane ligand on host cells and surround the larger, biologically active A subunit. For Shiga-like toxin 1 (Stx1), the cellular receptor is the carbohydrate globotriose. Stx1was purified from STEC. We utilized induction of apoptosis in the human monocyte cell line THP-1, as a biological endpoint to test the stability of Stx1 activity added to fruit punch at different pH (2-9) and temperatures (4 and 20 degrees C). A flow cytometric method was used to test for early and late apoptotic events based on binding of R-phycoerytherin-labeled annexin V to exposed membrane phosphatidyl serine. Membrane permeability to 7-Amino-actinomycin corresponds with late apoptosis or necrosis. The combination of acid pH and higher storage temperature resulted in greatest degree of toxin inactivation. This approach provides a rapid and high throughput method to determine the functional activity of Stx1, and related toxins in a food matrix.     

江苏某地健康绵羊群产志贺毒素大肠杆菌体内分离株的分子流行病学及致病力

【目的】探讨江苏某羊场健康绵羊体内产志贺毒素大肠杆菌的带菌和流行情况,同时就分离株的致病力和对Vero细胞的毒性作用作了研究。【方法】基于本实验室已经建立的EHEC O157:H7 EDL933W株的stx1、stx2、eaeA、hlyA四个基因的多重PCR检测并配合选择性增菌、平板筛选等方法对STEC进行分离鉴定。【结果】在为期6个月的连续跟踪调查中,共分离到STEC菌株107株,分离率为19.4%(107/550)。分离株属于41种O血清型、62种O:H血清型,未定型(ONT)有22株,粗糙型(OR)1株。其中属于绵羊STEC的优势血清型有O5(2株)、O91(1株)、O103(1株)。本文检测到的优势血清型为O93,stx2阳性菌株的分离率较stx1阳性菌株的分离率高,LD50测定结果表明分离株对小鼠致病力不高,受试的3个分离株均不能致小鼠死亡。对107株stx阳性分离株噬菌斑试验表明,71株阳性菌株携带噬菌体(66.3%,71/109)。受试分离株进行Vero细胞毒性试验,其中有一个菌株stx基因阳性但不能使Vero细胞产生病变。【结论】绵羊是STEC的天然宿主,可健康带菌。虽然STEC分离株对小鼠的致病力较弱,但不能排除其对人类安全的威胁。STEC携带志贺毒素基因并不意味着一定表达志贺毒素,需对志贺毒素的表达及调控机理做进一步的研究。     

Promiscuous Shiga toxin 2e and its intimate relationship to Forssman

Shiga toxin (Stx) 2e of Stx-producing Escherichia coli (STEC) represents the major virulence factor responsible for the pig edema disease which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Stx2e-producing strains from the intestine of slaughtered pigs (n = 3), feces of piglets with postweaning diarrhea or edema disease (n = 12) and feces of humans with asymptomatic infections or mild diarrhea (n = 13) were comparatively analyzed for the binding specificities of Stx2e to glycosphingolipids (GSLs) of the globo-series. Besides equivalent binding towards globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), we could demonstrate specific interaction of Stx2e preparations from human and porcine STEC isolates with Forssman GSL. Notably, Forssman GSL was recognized neither by structurally closely related Stx2 nor by Stx1 derived from human STEC isolates conferring Stx2e a unique recognition feature. Noteworthy, 7 (54%) of the 13 human and 8 (53%) of the 15 pig Stx2e samples exhibited cytotoxic action towards human brain microvascular endothelial cells. Our findings provide a basis for further exploring the functional role of the promiscuous receptor repertoire of Stx2e and the exact nature of the mechanisms that underlie different pathological outcomes of Stx2e-producing STEC in humans and pigs.     

SARS—CoV感染Vero E6细胞诱导细胞凋亡

为了确定SARS冠状病毒(SARS—CoV)感染Vero E6是否引起细胞凋亡,我们利用细胞DNA琼脂糖电泳,感染细胞的间接荧光染色和Hoechst 33258细胞核染色,以及流式细胞仪分析等方法证明了SARS-CoV感染的Veto E6具有典型的凋亡细胞学和生物化学特征。实验证明具有细胞凋亡特征的所有细胞均为处于感染晚期的细胞。表现明显细胞病变(CPE)的细胞大多已经出现核质凝缩或形成凋亡小体进入细胞凋亡的过程。可以断定SARS-CoV感染Vero E6细胞诱发了细胞凋亡。     

Effects of Escherichia Coli Subtilase Cytotoxin and Shiga Toxin 2 on Primary Cultures of Human Renal Tubular Epithelial Cells

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubA_A272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.     

Detection and Long-Term Existence of Shiga Toxin (Stx)-Producing Escherichia coli in Sheep

The isolation and characterization of Shiga-like toxin (Stx)-producing Escherichia coli (STEC) from sheep are described. The distribution of stx genes in E. coli isolates was detected by PCR. When brain heart infusion (BHI) broth and novobiocin supplemented m-EC broth (N-mEC) were used as enrichment culture for the isolation of STEC, N-mEC, compared to BHI, showed clearly lower efficiency. Finally, 5 STEC isolates from 4 sheep were isolated and characterized by biochemical and genetical analysis. All of them were confirmed by ELISA and Vero cell cytotoxicity assay for the production of Stx. Moreover, some strains carried hemolysin and eaeA genes and harbored large plasmids. Based on their plasmid profiles, antibiotic patterns and PCR-based DNA fingerprinting analysis using random amplified polymorphic DNA (RAPD), all isolates were different from each other. Three of the isolates were identified to belong to serogroups O2, O153 and O165, respectively, and the STEC strains belonging to these serogroups had been isolated from STEC outbreaks in humans. Four months after the first isolation in July 1997, STEC from sheep #1 was isolated again. A new isolate, HI-11, was identified as STEC O2: Hnt. Simultaneously, 2 STEC, which were genetically and phenotypically different from each other, were isolated from the same sheep at intervals of 4 months. These results demonstrate that sheep may be an important animal for studying human STEC infections, and that further epidemiological surveys on STEC are necessary.     

Study on induction of apoptosis on HeLa and Vero cells by recombinant shiga toxin and its subunits

Verotoxin (VT) or shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae is AB5 holotoxin with potent protein synthesis inhibitor. VT can induce both apoptosis and necrosis depending on the cell type, it has been shown that VT-induced apoptosis and cytotoxicity are distinct processes, and the A subunit can be necessary for apoptosis. In other words, the precise role of each subunit in apoptosis signaling has yet to be established. In this study, induction of apoptosis has been examined by using both recombinant A and B subunits, and recombinant Stx (rStx) with different doses in HeLa and Vero cells. For this purpose, the polymyxin B extract of constructs expressing A, B and AB5 recombinant proteins was used. Therefore, amounts greater than normally reported were used to induce desire effects on cell lines. The apoptotic effect of A and B subunits appear at higher doses than that of rStx. The highest apoptotic effect was observed for rStx at low concentration, compared to A and B subunits. A or B subunits separately cannot induce the signaling pathway stimulated by holotoxin though A subunit, does induce laddering pattern similar to holotoxin. We concluded that both subunits are important in complete death signaling pathway. Since different concentration of A and B subunits and rStx was required in different assay, therefore, it could be emphasized that cell death or even apoptosis caused by either of the subunits or holotoxin depends on sensitivity or specificity of the assay and cell types used.     

Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources

AIMS: To evaluate the suitability of the commercially distributed Ridascreen Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity. METHODS AND RESULTS: The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains. CONCLUSIONS: The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.     

Characterisation of Shiga toxin-producing Escherichia coli (STEC) isolated from seafood and beef

Shiga toxin-producing Escherichia coli (STEC) strains isolated in Mangalore, India, were characterised by bead-enzyme-linked immunosorbent assay (bead-ELISA), Vero cell cytotoxicity assay, PCR and colony hybridisation for the detection of stx1 and stx2 genes. Four strains from seafood, six from beef and one from a clinical case of bloody diarrhoea were positive for Shiga toxins Stx1 and Stx2 and also for stx1and stx2 genes. The seafood isolates produced either Stx2 alone or both Stx1 and Stx2, while the beef isolates produced Stx1 alone. The stx1 gene of all the beef STEC was found to be of recently reported stx1c type. All STEC strains and one non-STEC strain isolated from clam harboured EHEC-hlyA. Interestingly, though all STEC strains were negative for eae gene, two STEC strains isolated from seafood and one from a patient with bloody diarrhoea possessed STEC autoagglutinating adhesion (saa) gene, recently identified as a gene encoding a novel autoagglutinating adhesion.     

Regulated expression of the Shiga toxin B gene induces apoptosis in mammalian fibroblastic cells

Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli may induce colonic ulceration, bloody diarrhoea and acute renal failure. The A subunit (StxA) is known to inhibit protein synthesis, whereas the B subunits (StxB) bind to Gb3 on the cell surface. However, the mechanisms by which Stxs kill target cells remain unclear. Stx1A or Stx1B genes were introduced into pcDNA3.1 vectors and transfected into NIH3T3 and HeLa cells. The Stx1B gene-transfected cells became apoptotic with accompanying DNA fragmentation, whereas the Stx1A gene-transfected cells were found to be necrotic and no DNA fragmentation occurred. The HeLa/C4 cells integrated with the Stx1B gene with a tetracycline-inducible promoter eventually produced cytoplasmic Stx1B, leading to DNA fragmentation on the addition of doxycycline. These apoptotic changes were abrogated by pretreatment with Z-VAD-fmk. These results suggest that the transfected Stx1B gene induces apoptosis by activating the caspase cascade after Stx1B expression in the cytoplasm.