这项研究是由以色列科学基金会和拉帕波特医学科学院研究家庭研究所和德意志研究联合会（DFG）的资助的支持。

然后才开始实验的前提条件： <ul><li>无RNase的水是用于在实验室中的RNA处理，以减少风险的RNA被RNase的降解。为此，水通常是治疗至少1小时，0.1％V / V酸二乙酯（DEPC）在37℃，然后高压灭菌（至少15分钟），灭活的DEPC的痕迹。 </li><li> Pipetman移液器核糖核酸酶净化解决方案（核糖核酸灭虫，生物产业拜特哈Haemek）在使用前清洗。 </li><li> Eppendorf管和提示应认证DNase和RNase的自由，以保持样品的完整性。 </li></ul> 1。 tRNA的天冬氨酸的制备此过程介绍了可用于任何的tRNA合成的体外决胜转录。此过程是，此前公布的核酶的方法适应。9这个模板设计的T7启动子和自我切割的核酶和所需的tRNA的互补序列组成的目的。在产品模板转录裂解5的方式结束，全长tRNA的是获得了5`- OH，而不是一个磷酸5`年底。全长度的tRNA可切割的核酶和uncleaved产品纯化尿素页。 <ol><li> PCR引物和模板设计： 为了获得一个有效的模板，T7 RNA 聚合酶体外转录介导的三个方面的要求必须满足。 <ol><li>的T7启动子序列（5`- TAATACGACTCACTATA - 3&#39;）在模板的牵连。 </li><li>为了提高转录效率，前三转录RNA的核苷酸应鸟苷。 </li><li>特定的tRNA的正确的互补序列是必要的。 </li></ol>由于不是每个tRNA的满足第二个条件，我们在此描述模板此外在进行自我切割的核酶 ，9智人tRNA的ASP（黑色）和核酶的互补序列组成的模范DNA模板序列，如图1所示（紫色）tRNA的九个5`-基地（浅紫色）的补充。在5月底包括一个六核苷酸序列，使高效转录（浅橙色）是位于T7启动子序列（橙色）。转录后，核酶切割离开核酶的谈话内容和完整长度的tRNA（图1B）。后者可以从核酶和uncleaved产品尿素- PAGE纯化。正向引物设计的，它必须考虑到引物序列，这应延长至少5个核苷酸5&#39;启动子序列，使酶的结合。请注意，这些额外的核苷酸，可在3模板DNA年底失踪。在这里，我们用一个正向引物含有T7启动子序列，在转录开始后，其中的TATA序列结束。这底漆是普遍适用不同的tRNA含有互补的T7启动子序列（两个T7序列彩色图1A橙色）的模板。反向引物，有一个完整的正向引物，以确保引物和高效扩增的具有约束力的类似的熔融温度。 </li><li> PCR扩增 对于模板DNA扩增，我们用一个标准的PCR反应在50μL的最终体积。表1详细的最终浓度（反应准备上冰）和PCR技术方案。退火温度低，在最初的10个循环，唯一的正向引物部分DNA为模板的结合的帐户使用。为了减少非特异性结合退火温度的进一步增加了25个扩增循环。 模板扩增的成功是由1.3％琼脂糖凝胶预染1X GelRed解决方案（图2）测试。 </li><li> 转录 <ol><li> T7预混料可准备的5倍浓缩（200毫米的Tris - HCl，pH值8.1; MgCl 2的30毫米，1毫米的亚精胺，5毫米的数码地面电视，2μg/ mL的BSA和0.01％的Triton X - 100），并至少有一个使用星期或5个冻融。 </li><li>转录混合物在室温下配制。 </li><li>转录反应，准备用80μL的T7号预混料，80μLPCR产物，80μL的NTP混合（25毫米），并调整为385μLMilliQ水。 15μL（1.34毫克/毫升）T7 RNA聚合酶（终浓度为0.05微克/μL）被添加到启动反应。 </li><li>在37 ° C 4 H的反应进行了焦磷酸的白色沉淀，表示成功的转录。 </li></ol></li><li> 净化 <ol><li>焦磷酸纺1分钟，以10,000 rpm。 </li><li>上清液提供一个卷formamid样染料和PURified上使用2小时20瓦的12％尿素PAGE。卸除凝胶放在保鲜膜。 </li><li>染色的凝胶补充0.1 M氯化钠20分钟3XGelRed解决方案。紫外线激发后，标签上保鲜膜一个永久性标记以供日后切除tRNA的乐队。另外，如果预期收益率是50微克带以上可以看出，紫外线遮蔽。紫外线遮蔽，放在一个荧光薄层层析板凝胶和紫外手灯，照亮从上面。标签频段，作为非荧光的阴影，在凝胶（图3），后来切除，出现。 </li><li>用手术刀切除tRNA的频段为1.5毫升的Eppendorf杯。 </li><li>后加入450μL0.5M NH 4醋酸样品冷冻在-80 °孵育2小时，并随后在20 ° C，550上的Eppendorf Thermoshaker转速超过晚晃动。 6。 Nanosep管（0.45微米）过滤上清液中的NH 4 OAC解决方案，从第5步，在8500 rpm到删除页面碎片旋转90秒。 </li><li>洗脱tRNA的降水量增加2.5卷-80 °彗星纯乙醇（PA），储存在-20 ° C隔夜和2.5小时离心4℃，15500 RPM。 </li><li>取出后上清颗粒干Speed​​Vac MilliQ水悬浮。 </li><li>使用Nanodrop - ND1000的浓​​度进行了测定。一个400μL的转录产量约80-100微克。 </li></ol></li></ol>故障排除： <ul><li>无PCR产物。 →是兼容的引物和模板的顺序和反应混合物内的所有组件吗？ </li><li>无转录产物。 →PCR有可能不成功。正确的T7启动子序列。太冷的解决方案，同时准备反应。 T7预混料太旧。 </li><li>凝胶的产品，但没有洗脱产品。 →乙醇沉淀，不冷，不够纯，不正确的卷。 </li></ul> 2。重组大肠杆菌的表达及纯化阿米巴 Dnmt2（Ehmeth）在大肠杆菌中的蛋白质大肠杆菌 BL21 此协议也适用于编制Dnmt2 10人类和果蝇的蛋白质 11 。 <ol><li>的大肠杆菌阿米巴 Ehmeth基因经PCR扩增，克隆PGEX 4NT1（GE生命科学），测序验证</li><li>重组质粒转化E.表达的重组蛋白的大肠杆菌（BL21） 。 </li><li>卢里亚- Bertani（LB）琼脂氨苄青霉素（100微克/毫升）培养基上选择转化菌。四，五殖民地被拾起，接种到5 mL LB培养基中，用合适的选择标记（100μg/ mL氨苄青霉素）和轨道摇床孵育过夜在37 ° C。 </li><li>过夜文化接种到500毫升2X酵母提取蛋白胨（2XYT）中型，100μg/ mL氨苄青霉素，并在轨道摇床在37 ° C，直至文化的OD 600 0.8 。 </li><li> Dnmt2重组蛋白的诱导表达异丙基-β- D -硫代半乳糖苷（IPTG），加入终浓度为0.5毫米的文化。文化是进一步培养16小时，在24 ° C，在一个轨道摇床孵化温度和时间都得到了优化，以避免形成包涵体。 </li><li>文化是在4 ° C，在200毫升NALGENE管，15分钟6000转离心。弃上清液，沉淀在-70℃冻至少1小时。这冻结一步提高的细菌裂解。 </li><li>收获在30 mL裂解缓冲液（100毫米氯化钾，1mm的数码地面电视服务，为1mM PMSF，100微克/毫升溶菌酶和Leupeptine 100微克/毫升磷酸盐缓冲液（PBS））中的颗粒。从这一点来说，至关重要的是保持在冰上的裂解液，以防止重组蛋白的灭活。设置没有在冰水浴超声裂​​解液。 4一个微型和小型企业（英国伦敦）sonifier（休息5分钟，30秒，30秒脉冲功率高，振幅5）。细菌的DNA可能会附加到Ehmeth Benzonase核酸（Novagen公司）的治疗（5 U / mL的超声裂解）在冰上30分钟，被删除。此外BugBuster蛋白抽提试剂（Novagen公司）300μL30分钟完成的裂解是由在4 ° C与轨道摇床（100转）轻轻摇动。 </li><li>重组GST - Ehmeth蛋白质纯化谷胱甘肽琼脂糖树脂的天然条件下，根据制造商的说明（Sigma公司）。 25 mL裂解液中，分别加入500μL浆树脂和孵育24℃，1小时。 </li><li>珠裂解液单位体积珠裂解缓冲液（10卷）和6倍，用冷PBS洗两次。这些广泛的清洗步骤insurE的没有Benzonase保持洗脱步骤之前。 </li><li>重组Ehmeth蛋白谷胱甘肽琼脂糖珠洗脱孵化谷胱甘肽洗脱缓冲液5毫升（三盐酸50 mM的pH值8.0，谷胱苷肽（Sigma公司）10毫米），12小时在4℃与轻柔旋转的珠子。珠被拆除离心（2,000 RPM，5分钟），收集上清。 </li><li>洗脱缓冲液上清中被替换的存储缓冲区（氯化钾200毫米，EDTA的10毫米，0.1毫米的DTT，甘油60％的PBS）。为此，5毫升上清液装上顶部的一个微孔单片机YM - 30离心式过滤装置（切断列30 kDa的）和离心20分钟在8000转4 ° C。在存储缓冲区（5毫升）稀释，浓缩蛋白和过滤装置上的重载。同样的程序应至少重复3次，以确保完成更换洗脱缓冲液的存储缓冲区。最后浓缩蛋白应约300存储缓冲液稀释。 </li><li>由Bradford蛋白测定重组蛋白的含量测定。重组酶制剂存放在-20 ° C。酶的最终浓度不应超过3微克/μL作为适宜浓度的甲基化检测。 </li><li>经SDS - PAGE蛋白制备检查。 </li></ol>注意：投保的一种活性酶的制备，纯化步骤应在冰上和数码地面电视应新鲜配制和添加到存储缓冲区。这保证6个月的活性酶。 （3） 在体外 tRNA的甲基化检测解决方案和应准备的试剂： <ol><li> 1个M解决方案三（羟甲基）氨基甲烷。用浓盐酸（HCl）溶液的pH值调整到8.0。 </li><li> 5X甲基缓冲区（MB）：100毫米的Tris pH值8.0，100MM NH 4醋酸，EDTA的0.1毫米，10毫米MgCl 2的 。 MB可提前做好准备，并储存在-20 ° C，无二硫苏糖醇（DTT）。 </li><li>准备101.67 mL蒸馏水混合5μL，AdoMet（New England Biolabs公司，32毫米的浓度）的未标记的S -腺苷甲硫氨酸（AdoMet）股票（终浓度为1.5毫米）。 </li><li> 5％三氯乙酸溶液（TCA） </li><li> 100％的乙醇</li><li>闪烁液3毫升，每样品瓶。 </li></ol>该试剂盒： <ol><li>一个pipeting计划的准备（见表2）为40μL，甲基化检测的最终体积。 </li><li>在1.5 mL Eppendorf管中，tRNA的DEPC处理水稀释到5微米的终浓度为（1μgtRNA的是相当于40 pmol） </li><li> 4μL数码地面电视（原液浓度）8μL5XMB解决方案。 </li><li>孵育85 ° C为2分钟，在步骤3中准备的解决方案是立即添加到tRNA的解决方案。混合物被允许降温15分钟，在室温下让tRNA的正确折叠。 </li><li> AdoMet组合（终浓度为200μM）混合标记AdoMet未标注AdoMet 150微米50微米（250μCi，10.0次/毫摩尔，珀金埃尔默）。我们AdoMet适合10个标记反应的组合，混合5.2μL无标签AdoMet（稀释液），29μL标记AdoMet和5.8μL蒸馏水。 </li><li> 4μL准备以上的AdoMet混合添加到tRNA的解决方案，并在37 ° C孵育2分钟</li><li> tRNA的甲基化，Ehmeth（1-10微米的最终浓度）被添加到解决方案中包含的tRNA基材和标记的未标记的AdoMet辅酶。混合物在37℃孵育3小时定期在孵化时，轻轻翻转试管混合。采取从反应混合物超过三个小时，期间每隔20分钟一个样品（8μL）建一个酶促反应的动力学曲线。 </li><li>每个样本被立即发现用Whatman过滤器（直径0.5厘米的）的中心和过滤器在5％TCA溶液浸泡，搅拌10分钟冰（除AdoMet样品，表2的E列，不应该水洗）。 5％TCA溶液洗涤步骤重复两次</li><li>该过滤器是100％冰浴10分钟乙醇洗涤。 </li><li>该过滤器是空气干燥至少20分钟。 </li><li>每个干燥过滤器被放置在一个以前充满闪烁液3毫升（CytoScint）管（规格） </li><li>在tRNA的注册成立的放射性，是用闪烁计数器（计数器的Beta三- CARB 2100TR）。 </li></ol> 4。故障排除<ul><li>在列A或B（表3）的高值，控制样本值，预计从M乙基化反应，在列C或D（见表3）→过滤器，冲不正常。 </li><li>在列C或D（见表3）低值，甲基化的实验也有类似的值来控制反应列A或B（见表3）→酶是不正确准备，也就是说，有没有Adomet纳入。另一种选择是tRNA的退化，并没有一个合适的酶底物，这可能是尿素的丙烯酰胺凝胶上运行tRNA的样本（见1.2），并用溴化乙锭染色凝胶研究的tRNA的完整性检查。 </li><li>低列D值（见表3）→自Ehmeth活动较弱然后hDnmt2的酶的性能，可通过增加存储缓冲区的数码地面电视的浓度（范围可以1至10毫米之间）或增加酶浓度提高缓和的酶的质量差。 </li><li>列E值低（见表3）→此列表示的总放射性标记AdoMet值（简称为100％），如果在此列的值是低的，这意味着，AdoMet不再是活动的，已经失去了放射性物质。 </li></ul> 5。代表性的成果<table border="1"><tbody><tr><td> 试剂 </td><td> 终浓度 </td><td colspan="3"> PCR -方案 </td><td colspan="3"></td></tr><tr><td> Gentherm PCR缓冲液</td><td> 1X </td><td> 1。 </td><td> 90℃ </td><td> 2分钟</td><td colspan="3"></td></tr><tr><td> MgCl 2的 </td><td> 3.0毫米</td><td colspan="3"> ------------------------------ </td><td colspan="3"> ------------------------------ </td></tr><tr><td> dNTP混合液</td><td> 1.2毫米</td><td> 2。 </td><td> 90℃ </td><td> 30秒</td><td> 5。 </td><td> 90℃ </td><td> 30秒</td></tr><tr><td>模板DNA </td><td> 15.0海里</td><td> 3。 </td><td> 54℃。 </td><td> 30秒</td><td> 6。 </td><td> 60℃ </td><td> 30秒</td></tr><tr><td>正向引物</td><td> 3.0μM </td><td> 4。 </td><td> 72℃ </td><td> 45秒</td><td> 7。 </td><td> 72℃ </td><td> 45秒</td></tr><tr><td>反向引物</td><td> 3.0μM </td><td colspan="2"></td><td> 2. - 4。 10个周期</td><td colspan="2"></td><td>五，7。 25个循环</td></tr><tr><td> Taq聚合酶</td><td> 0.1 U /μL </td><td colspan="3"> ------------------------------ </td><td colspan="3"> ------------------------------ </td></tr><tr><td> H 2 O </td><td>公元50μL </td><td colspan="3">盖95 ° C </td><td> 8。 </td><td> 72℃ </td><td> 3分钟</td></tr></tbody></table> 表1：移液器PCR和PCR方案的计划 。 <table border="1"><tbody><tr><td></td><td>一 阴性对照 （只Ehmeth） </td><td>乙 阴性对照 （只tRNA的） </td><td>彗星 阳性对照与人类Dnmt2 </td><td> ð 标准反应 （Enz + SUBST + COF） </td><td> Ë 在过滤器上的总价值的AdoMet </td></tr><tr><td> tRNA的</td><td> - </td><td> （3微克） </td><td> （3微克） </td><td> （3微克） </td><td> - </td></tr><tr><td> hDnmt2 </td><td></td><td> - </td><td> （2.48微克） </td><td></td><td> - </td></tr><tr><td> Ehmeth </td><td> （2.48微克） </td><td></td><td></td><td> （2.48微克） </td><td></td></tr><tr><td> MB </td><td> 8 </td><td> 8 </td><td> 8 </td><td> 8 </td><td> - </td></tr><tr><td>数码地面电视</td><td> 4 </td><td> 4 </td><td> 4 </td><td> 4 </td><td> - </td></tr><tr><td> AdoMet </td><td> 4 </td><td> 4 </td><td> 4 </td><td> 4 </td><td> 1 </td></tr><tr><td> DDW </td><td></td><td></td><td></td><td></td><td> - </td></tr><tr><td>共有</td><td> 40微升</td><td> 40微升</td><td> 40微升</td><td> 40微升</td><td> 1微升</td></tr></tbody></table> 表2：pipeting计划的例子在该计划中的每一列代表一个样本。 * Ehmeth和hDnmt2融合估计62 kDa的蛋白大小与GST标签。 一个阴性对照，包括Ehmeth只。 B超阴性对照只包括tRNA的。 C -阳性与人类Dnmt2的控制。这种酶有十个时间多了ctivity比Ehmeth 标准的D -的反应，其中包括的酶​​，底物和辅酶 电子的总价值在过滤器上的AdoMet。此值用于甲基集团股份有限公司的百分比计算。 <table border="1"><tbody><tr><td></td><td>一 阴性对照 （只Ehmeth） </td><td>乙 阴性对照 （只tRNA的） </td><td>彗星 阳性对照与人类Dnmt2 </td><td> ð 标准反应 （Enz + SUBST + COF） </td><td> Ë 在过滤器上的总价值的AdoMet </td></tr><tr><td> CPM </td><td> 200 </td><td> 300 </td><td> 6700 </td><td> 1284 </td><td> 51653 </td></tr></tbody></table> 表3：闪烁计数器读值的例子在该计划中的每一列从表1中的一个样本。 <img src="/files/ftp_upload/2390/2390fig1.jpg" alt="图1" /> 图1：（一）T7转录模板和引物在体外转录。所有序列都写在5&#39;到3&#39;方向。 Colorcode：看到文字。 （二） 计划的产品形成过程中的转录 。 T7 RNA聚合酶合成RNA转录。核酶和tRNA折叠成各自的二维结构的核酶切割留在他们的5&#39; -端（从9适应）羟基的tRNA。 <img src="/files/ftp_upload/2390/2390fig2.jpg" alt="图2" /> 图2：PCR扩增反应控制在1.3％琼脂糖凝胶电泳 PCR扩增1X GelRed反应，在两个不同的模板预染旁边一个100个碱基对，加上阶梯。控制包括一个“模板只有”巷和反向引物被省略的反应。 <img src="/files/ftp_upload/2390/2390fig3.jpg" alt="图3" /> 图3：紫外线遮蔽一个PAGE分离的两个转录混合物显示。带易制毒化学成绩单（前乳沟），全长tRNA和核酶作为阴影成为可见的，因为它们可以防止紫外线灯，从深远的荧光薄层板上。总的tRNA 从 E 大肠杆菌是使用大小标记。

<ol>
	<li>Banerjee, S., Fisher, O., Lohia, A., Ankri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entamoeba+histolytica+DNA+methyltransferase+(Ehmeth)+is+a+nuclear+matrix+protein+that+binds+EhMRS2,+a+DNA+that+includes+a+scaffold/matrix+attachment+region+(S/MAR).">Entamoeba histolytica DNA methyltransferase (Ehmeth) is a nuclear matrix protein that binds EhMRS2, a DNA that includes a scaffold/matrix attachment region (S/MAR).</a> Mol Biochem Parasitol. 139, 91-97 (2005).</li><li>Fisher, O., Siman-Tov, R., Ankri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cytosine+methylated+regions+and+5-cytosine+DNA+methyltransferase+(Ehmeth)+in+the+protozoan+parasite+Entamoeba+histolytica.">Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica.</a> Nucleic Acids Res. 32, 287-297 (2004).</li><li>Harony, H., Bernes, S., Siman-Tov, R., Ankri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+and+targeting+of+LINE+retrotransposons+in+Entamoeba+histolytica+and+Entamoeba+invadens.">DNA methylation and targeting of LINE retrotransposons in Entamoeba histolytica and Entamoeba invadens.</a> Mol Biochem Parasitol. 147, 55-63 (2006).</li><li>Kuhlmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+retrotransposons+in+Dictyostelium+by+DNA+methylation+and+RNAi.">Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi.</a> Nucleic Acids Res. 33, 6405-6417 (2005).</li><li>Katoh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmentally+regulated+DNA+methylation+in+Dictyostelium+discoideum.">Developmentally regulated DNA methylation in Dictyostelium discoideum.</a> Eukaryot Cell. 5, 18-25 (2006).</li><li>Phalke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrotransposon+silencing+and+telomere+integrity+in+somatic+cells+of+Drosophila+depends+on+the+cytosine-5+methyltransferase+DNMT2.">Retrotransposon silencing and telomere integrity in somatic cells of Drosophila depends on the cytosine-5 methyltransferase DNMT2.</a> Nat Genet. 41, 696-702 (2009).</li><li>Tovy, A., Siman Tov, R., Gaentzsch, R., Helm, M., Ankri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+nuclear+function+of+the+Entamoeba+histolytica+glycolytic+enzyme+enolase:+the+metabolic+regulation+of+cytosine-5+methyltransferase+2+(Dnmt2)+activity.">A new nuclear function of the Entamoeba histolytica glycolytic enzyme enolase: the metabolic regulation of cytosine-5 methyltransferase 2 (Dnmt2) activity.</a> PLoS Pathog. 6, e1000775-e1000775 (2010).</li><li>Jeltsch, A., Nellen, W., Lyko, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+substrates+are+better+than+one:+dual+specificities+for+Dnmt2+methyltransferases.">Two substrates are better than one: dual specificities for Dnmt2 methyltransferases.</a> Trends Biochem Sci. 31, 306-308 (2006).</li><li>Fechter, P., Rudinger, J., Giege, R., Theobald-Dietrich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribozyme+processed+tRNA+transcripts+with+unfriendly+internal+promoter+for+T7+RNA+polymerase:+production+and+activity.">Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity.</a> FEBS Lett. 436, 99-103 (1998).</li><li>Hermann, A., Schmitt, S., Jeltsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+Dnmt2+has+residual+DNA-(Cytosine-C5)-methyltransferase+activity.">The human Dnmt2 has residual DNA-(Cytosine-C5)-methyltransferase activity.</a> J Biol Chem. 278, 31717-31721 (2003).</li><li>Narsa Reddy, M., Tang, L. Y., Lee, T. L., &amp;amp, T. L., James Shen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+candidate+gene+for+Drosophila+genome+methylation.">A candidate gene for Drosophila genome methylation.</a> Oncogene. 22, 6301-6303 (2003).</li></ol>

在此演示中，我们说明如何准备tRNA 的 E.甲基转移酶活性的措施中的活性成分阿米巴 Ehmeth酶。这个程序可以作为一个起点和衡量其他的tRNA甲基很容易适应。重要的是要遵循的程序，维护tRNA的衬底和tRNA的甲基蛋白质的质量和完整性，以确保酶活性的最佳措施。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Taq Polymerase (5 U/&mu;l)</td>
<td>&nbsp;</td>
<td>Rapidozym, Berlin</td>
<td>GEN-003-1000</td>
<td>&nbsp;</td>
<tr>
<td>10X Gentherm PCR Buffer</td>
<td>&nbsp;</td>
<td>Rapidozym, Berlin</td>
<td>With GEN-003-1000</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2 (50 mM)</td>
<td>&nbsp;</td>
<td>Rapidozym, Berlin</td>
<td>With GEN-003-1000</td>
<td>&nbsp;</td>
<tr>
<td>dNTP mix (10 mM)</td>
<td>&nbsp;</td>
<td>Fermentas, St. Leon-Rot</td>
<td>R0191</td>
<td>&nbsp;</td>
<tr>
<td>Oligo nucleotides</td>
<td>&nbsp;</td>
<td>IBA, G&ouml;ttingen</td>
<td>Customized</td>
<td>&nbsp;</td>
<tr>
<td>NTP mix (25mM)</td>
<td>&nbsp;</td>
<td>Fermentas, St. Leon-Rot</td>
<td>R0481</td>
<td>&nbsp;</td>
<tr>
<td>GelRed Nucleic Acid Stain 3X in water</td>
<td>&nbsp;</td>
<td>Biotium</td>
<td>41001</td>
<td>&nbsp;</td>
<tr>
<td>Dithiotreitol</td>
<td>&nbsp;</td>
<td>Fermentas, St. Leon-Rot</td>
<td>R0861</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma, Germany</td>
<td>S0266</td>
<td>&nbsp;</td>
<tr>
<td>Pall Nanosep 0.45 &mu;m</td>
<td>&nbsp;</td>
<td>Sigma, Germany</td>
<td>Z722014</td>
<td>&nbsp;</td>
<tr>
<td>Dichlorodimethylsilane</td>
<td>&nbsp;</td>
<td>Sigma, Germany</td>
<td>40140</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol (Ph. Eur.)</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>5054.3</td>
<td>&nbsp;</td>
<tr>
<td>Tris HCl</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>9090.3</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>AE15.1</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Fermentas, St. Leon-Rot</td>
<td>B14</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma, Germany</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>TEMED</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>2367.1</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Peroxodisulfate</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>9592.2</td>
<td>&nbsp;</td>
<tr>
<td>Rotiphorese Sequencing gel concentrate</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>3043.1</td>
<td>&nbsp;</td>
<tr>
<td>Rotiphorese Sequencing gel diluent</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>3047.1</td>
<td>&nbsp;</td>
<tr>
<td>Rotiphorese Sequencing gel buffer concentrate</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>3050.1</td>
<td>&nbsp;</td>
<tr>
<td>Rotiphorese 10X TBE-buffer</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>3061.2</td>
<td>&nbsp;</td>
<tr>
<td>DEPC</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5758</td>
<td>&nbsp;</td>
<tr>
<td>RNAse ExTERMINATOR</td>
<td>&nbsp;</td>
<td>Biological Industry Beit Haemek</td>
<td>01-895-1B</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0166</td>
<td>&nbsp;</td>
<tr>
<td>IPTG</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I6758</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L7651</td>
<td>&nbsp;</td>
<tr>
<td>Leupeptine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L2884</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase Nuclease</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70746-3</td>
<td>&nbsp;</td>
<tr>
<td>BugBuster protein extraction reagent</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70921-3</td>
<td>&nbsp;</td>
<tr>
<td>Glutathione-agarose resin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G4510</td>
<td>&nbsp;</td>
<tr>
<td>L-glutathione reduced</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G4251</td>
<td>&nbsp;</td>
<tr>
<td>AdoMet</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>B9003</td>
<td>&nbsp;</td>
<tr>
<td>AdoMet 3H</td>
<td>&nbsp;</td>
<td>PerkinElmer</td>
<td>NET155250UC</td>
<td>&nbsp;</td>
<tr>
<td>Scintillation Cocktail</td>
<td>&nbsp;</td>
<td>CytoScint</td>
<td>882453</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in vitro trna methylation assay with the entamoeba histolytica dna and trna methyltransferase dnmt2 (ehmeth) enzyme

原虫是最具毁灭性的传染性病原体对人类负责各种疾病，包括阿米巴病，寄生虫病的三种最常见的死亡原因之一。阿米巴剂是阿米巴寄生虫阿米巴存在下两个阶段：发现感染性囊肿的食物或水，并在肠道内侵入滋养体生活。阿米巴范围的临床表现从无症状大肠杆菌肠炎，痢疾或肝脓肿。 阿米巴是一个罕见的单细胞寄生虫，它的基因组与5 -甲基胞嘧啶（5mC）1，2，它包含一个单一的DNA甲基化，Ehmeth，属于Dnmt2家庭。2一个Dnmt2在重复元素的控制作用建立了在大肠杆菌中3， 阿米巴粘菌 4,5和果蝇 。6我们最近的工作表明，Ehmeth甲基化tRNA 的 ASP，这一发现表明，这种酶具有双重DNA / tRNA 的 ASP甲基转移酶活性。7这个观察的双重活动的协议已报告为D。 discoideum和 D。 果蝇 。8 Dnmt2酶的DNA / tRNA的特异性的功能意义，仍是未知数。为了解决这个问题，成立了一个方法来确定的tRNA Dnmt2蛋白甲基转移酶活性。在这段视频中，我们描述了一个简单的方法，准备Dnmt2足够的tRNA基板和方法来衡量它的tRNA甲基转移酶活性。

Watch this Scientific Journal Video about In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme at JoVE.com

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

这个协议描述为合成tRNA的衬底准备<em&gt;阿米巴</em&gt;的tRNA / DNA的甲基转移酶2（Dnmt2）的同源性Ehmeth和甲基转移酶活性的措施。这种实验方法可用于调查其他Dnmt2蛋白的活性。

在体外的tRNA与阿米巴 DNA和tRNA甲基Dnmt2（Ehmeth）酶甲基化含量

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

in-vitro-trna-methylation-assay-with-entamoeba-histolytica-dna-trna

Research

JoVE Journal

Immunology and Infection

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

14.2K 次观看.  Technion - Israel Institute of Technology. 这个协议描述为合成tRNA的衬底准备阿米巴的tRNA / DNA的甲基转移酶2（Dnmt2）的同源性Ehmeth和甲基转移酶活性的措施。这种实验方法可用于调查其他Dnmt2蛋白的活性。

视频: In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

一个负选择标记的发展阿米巴</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

科研

免疫与感染

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

限制性内切酶克隆方法，以评估<em&gt;在体外</em&gt; HIV-1 C亚型的Gag-MJ4嵌合病毒的复制能力

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.