Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

大肠杆菌亮氨酰－tRNA合成酶LeuRS67R的纯化及其动力学研究

本实验室已得到的亮氨酰－ｔＲＮＡ合成酶（ＬｅｕＲＳ）基因,与文献相比,６７位氨基酸残基由Ｈｉｓ变为Ａｒｇ,此酶被定名为ＬｅｕＲＳ６７Ｒ。我们从该基因与ｐＵＣ１９重组质粒的大肠杆菌ＴＧ１转化子ＴＧ１－９１中得到ＬｅｕＲＳ的高表达,粗抽液中ＬｅｕＲＳ的表达量在转化子中比在宿主菌ＴＧ１中高２０倍以上。用三步拉层析得到电泳一条带的酶,其比活为１７８９单位／毫克。测定其动力学常数,氨酰化活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０２７ｍｍｏｌ／Ｌ、０．４７ｍｍｏｌ／Ｌ,Ｋｃａｔ值分别为３．５～５．１ｓ－１。ＡＴＰ－ＰＰｉ交换活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０３ｍｍｏｌ／Ｌ、１．０ｍｍｏｌ／Ｌ,Ｌｃａｔ值分别为１４０～１５５ｓ－１。此结果与从野生型大肠杆菌Ｋ－１２中提纯的ＬｅｕＲＳ的动力学常数差别很小,６７位氨基酸残基在与活性中心无直接关系的域可能是大肠杆菌的种间差异。     

大肠杆菌精氨酰－tRNA合成酶的变种ArgRS306KR的纯化和基本性质

本文从含ＡｒｇＲＳ３０６ＫＲ基因ａｒｇｓ３０６ＫＲ的ｐＵＣ１８重组质粒的大肠杆菌ＴＧ１转化子中经ＤＥＡＥ－Ｓｅｐｈａｃｅｌ和Ｂｌｕｅ－Ｓｅｐｈａｒｏｓｅ两步柱层析,得到电泳一条带的ＡｒｇＲＳ３０６ＫＲ。纯酶的比活为２７９０单位／毫克。该酶氨酰化和ＡＴＰ～ＰＰｉ交换活力的最适ｐＨ分别为ｐＨ８．３和ｐＨ７．５。氨酰化活力对ＡＴＰ、Ａｒｇ和ｔＲＮＡ的Ｋｍ分别为２．６ｍｍｏｌ／Ｌ、１４．０μｍｏｌ／Ｌ和５．０μｍｏｌ／Ｌ：Ｖｍａｘ为７６３０单位／毫克;ｋｏａｔ为９Ｓ－１。ＡＴＰ～ＰＰｉ交换活力对ＡＴＰ和Ａｒｇ的Ｋｍ分别为８．３ｍｍｏｌ／Ｌ和９９μｍｏｌ／Ｌ;Ｖｍａｘ为１６３２０单位／毫克;ｋｃａｔ为１８Ｓ－１。     

戊肝病毒活性肽的选择,合成与应用

对戊型肝炎病毒（ＨＥＶ）编码蛋白序列进行了亲水性分析及二级结构预测,选择亲水性强、具有β－转角与β－折叠的区段,采用多肽固相合成法合成了ＨＥＶ基因组３个开读框架（ＯＲＦ１,ＯＲＦ２和ＯＲＦ３）中可能的抗原表位,以免疫学方法进行鉴定并选出了分别来自ＨＥＶ３个ＯＲＦ的、具有重要生物活性与应用前景的３段肽（ＥＨ１７４、ＥＨ２６５、ＥＨ３６２）。在此基础上,进行了抗ＨＥＶＥＬＩＳＡ新型检测试剂盒的实验室研究及临床试用。结果表明,所研究的戊肝抗体检测试剂盒特异性高、临床符合性好、具有可重复性,在戊肝辅助诊断及流行病学调查中具有重要意义。     

使用MUG培养基定量检测植物药中大肠杆菌时荧光猝灭现象的发生与克服方法

实验中观察到,用ＭＵＧ培养基对植物药中的大肠杆菌定量时多发生荧光猝灭现象,影响检测结果。本文对此现象产生的原因与克服方法进行了系统的考察,发现以一种简便的转接方法可排除植物药介质对菌检的干扰。该方法由两组检验系列构成,当怀疑正常稀释系列（第一系列）４０ｈ培养液的荧光结果可能因猝灭现象呈假阴性时,立即分别将该系列的１—３号管培养液以０．５ｍｌ的接种量转接入新鲜的ＭＵＧ培养基（第二系列）,重新培养２４ｈ,荧光猝灭现象即可克服。综合两系列的荧光、产气和吲哚三项生化特征得出检品中大肠杆菌含量。实际应用表明,此法能显著提高使用该培养基时菌检结果的可靠性。     

人白介素6受体功能区片段在E．coli中的表达

通过ＤＮＡ体外重组技术,以ｐＥＴ－３ｂ为表达载体,构建了重组表达质粒ｐＥＴ－６Ｒ（Ｂ）和ＰＥＴ－６Ｒ（Ｂ）４,分别编码２８ｋＤ的ｈＩＬ－６Ｒ配基结合区片段及其５３ｋＤ的二联体蛋白,并为酶切分析和ＤＮＡ序列分析所证实。ＳＤＳ－ＰＡＧＥ分析表明,含有重组表达质粒的菌株可分别表达出２８ｋＤ的蛋白ｒＩＬ６Ｒ－２８和５３ｋＤ的ｒＩＬ６Ｒ－５３。重组蛋白分别占菌体总蛋白的４５％和２９％左右。重组蛋白主要以包涵体形式存在,Ｗｅｓｔｅｒｎ印迹表明重组蛋白具有ＩＬ－６Ｒ的抗原性。     

昆虫杆状病毒晚期启动子在大肠杆菌中启动CAT基因表达

将油桐尺蠖（Ｂｕｚｕｒａｓｕｐｐｒｅｓｓａｒｉａ）核多角体病毒晚期基因──多角体蛋白基因启动子及５′端编码区,以两种不同方式置于缺乏启动子的氯霉素乙酰基转移酶（ＣＡＴ）基因上游,使其分别终止在不同翻译终止位点,其宿主菌具有明显不同的氯霉素抗性,最高达２００ｍｇ／Ｌ　ＬＢ培养基以上,表明昆虫病毒启动子能启动原核基因表达。对多角体蛋白基因启动子能在大肠杆菌中有效工作的原因进行了讨论。     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Eicosanoids and their role in immune modulation in fish—a brief overview

Eicosanoids have been demonstrated to play a central role in immune regulation in mammals brought about by their direct effects on cells such as macrophages and lymphocytes or by their indirect effects via cytokines. Studies have shown that fish mononuclear phagocytes, granulocytes and thrombocytes synthesize and release both cyclooxygenase- and lipoxygenase-derived products such as prostaglandin E₂, leukotriene B₄ and lipoxin A₄. Whether lymphocytes have the ability to generate leukotrienes and lipoxins is still unclear but they do appear to have 12-lipoxygenase activity that leads to the generation of 12-hydroxy fatty acid derivatives. As in mammals, leukotriene and lipoxin biosynthesis requires the presence of a 5-lipoxygenase activating protein-like molecule that is sensitive to the action of the specific inhibitor, MK-886. The prostaglandin-generating ability of trout macrophages can be altered by incubation with lipopolysaccharide suggesting the possible presence of an inducible cyclooxygenase activity. Prostaglandins have been found to suppress the mitogen-induced proliferation of trout leucocytes and the generation of humoral antibody and plasma cells both in vivo and in vitro. The lipoxygenase products, leukotriene B₄ and lipoxin A₄ have more variable effects ranging from inhibition to stimulation depending on the assay system employed. Overall, there is clear evidence that eicosanoids play a role in immune regulation in fish in a similar way to that reported in mammals.     

乌奴龙胆中五个新的环烯醚萜甙

从藏药乌奴龙胆（ＧｅｎｔｉａｎａｕｒｎｕｌａＳｍｉｔｈ）（龙胆科）的全草中分离到５个新的环烯醚萜甙,命名为乌奴龙胆甙（ｇｅｎｔｉｏｕｍｏｓｉｄｅ）Ａ－Ｅ;它们的结构主要通过光谱分析得以确定。其中,乌奴龙胆Ａ－Ｃ是二聚环烯醚萜甙,而乌奴龙胆甙Ｄ和Ｅ为马钱素型的环烯醚萜甙,所有这些化合物的分子中都具有一个２,３－二羟基苯甲酰基或其衍生物的取代基。