Chemical modifications of carboxylated chitosan

C-6-carboxylated chitosan obtained by oxidation of chitosan was selectively modified in order to obtain derivatives similar to bacterial antigens. Selective O-acetylation of 6-carboxyl chitosan afforded a modified polysaccharide with the 2-amino group available for further modifications to create carbonyl groups. A deaminative degradation reaction allowed the formation of oligosaccharides with terminal aldehyde groups. Reductive alkylation with lactose introduced lactityl branches which were oxidized with galactose oxidase to give aldehyde groups in its -galactose residues.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

迟缓爱德华氏菌两种保护性抗原及AcrAB耐药系统的分析

迟缓爱德华氏菌是危害水产养殖业发展的重要病原菌之一，因而其免疫防治研究具有重要意义。论文分析了9种具有保护潜能的迟缓爱德华氏菌蛋白，经过牙鲆免疫保护实验，筛选出EseD和Et18两种有显著性保护效应的抗原。为了提高其保护效应，论文使用基因工程技术将这两种抗原融合到一起，构建重组融合蛋白EEH。结果表明，融合蛋白EEH保护效应较EseD和Et18分别免疫时有所提高。ELISA和Western blotting 结果显示，三种蛋白都能诱导牙鲆产生特异抗体。这些研究为开发迟缓爱德华氏菌疫苗提供了理论基础。 论文克隆分析了迟缓爱德华氏菌AcrAB耐药系统，采用定点突变确定了acrAB、acrR的启动子序列和AcrR在acrAB启动子的结合位点。启动子分析显示，AcrR对acrAB启动子有300倍抑制效应, 对acrR启动子有3倍抑制效应。定点突变显示，K39和R45对AcrR功能具重要性；缺失突变表明，N端205个氨基酸残基是其功能必需。实验筛选出Acriflavine、Ethidium Bromide、Methyl Viologen、Sodium Dodecyl Sulfate等四种AcrR诱导物。分析AcrR过量表达菌株结果显示，其耐药性、生长状况和毒力水平较阴性对照组降低。这些研究加深了我们对迟缓爱德华氏菌耐药机制及其与毒力关系的了解。

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis

Background: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. Objectives: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. Methods: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. Results: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD 106). Coronary MaVEC released significantly less EMP than MiVEC. Conclusion: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium. (C) 2003 Elsevier Science Ltd. All rights reserved.

贵阳市表层土壤重金属污染元素环境地球化学基线研究

贵阳市位于贵州省中部云贵高原东斜坡地带，地处东经106°07′至107°17′，北纬262°11′，至272°22′之间，属东部平原向西部高原的过渡地带，地形地貌多样，海拔高，纬度低，具有亚热带湿润温和型气候的特点，资源丰富，能源充足，自然环境得天独厚。本论文详细研究了贵阳市8046平方公里面积内表层土壤中砷、福、铬、铜、汞、镍、铅、锌8种重金属污染元素的环境地球化学特征，确定了土壤环境地球化学基线，并建立了判别自然的和人为的环境影响的地球化学指标，进而计算污染参数：重金属累计指数和污染程度，在此基础上进行了土壤重金属污染来源分析。取得认识如下：1、贵阳市表层土壤中重金属污染元素的均值、标准差、极值、主要含量区间分别是：砷18·ogmg／kg、11·57 mg／kg、79.30mg/kg、AS95.9％＜40mg/kg；福0.302mg／kg、0.363mg／kg、2.620mg／lcg、Cd95.7％＜1.000mg／kg；铬75.3mg／kg、37.3mg／kg、271.0mg／kg、Cr95.9％＜150.0mg／1cg；铜43．lmg／kg、30.3mg/kg、213.0mg瓜g，Cu94.6o／＜100mg／kg；铅43.2mg／kg、31.3mg／kg、318.9mg/kg，Pb95.5％＜100.0mg/kg；汞0.222mg／kg、0.531mg／kg、7.030mg／kg，Hg98.2％＜1.00Omg/kg；镍38.3mg/kg、14.9mg／kg、102.5mh/kg，Ni95.8%＜70mg/kg；锌84.7mkg、49.8mg／kg、385.0mg/kg，Zn93.4％＜150.0mg/kg。2、利用相对累计总量分析方法和相对累计频率分析方法确定贵阳市表层土壤中重金属元素的基线值和受人为影响的下限：砷为9.04mk和29.0mg/kg；福为0.068mg／kg和1 ．OIOms／kg；铬为44.0mg／kg和100.2mg/kg；铜为18.8mg/kg和68.4mg/kg；铅为14.8mg/kg和70.lmg／kg；汞为0.045mg／kg和0.530mkg；镍为17.0mg/kg和57.0mg/kg；锌46.3mg/kg和112.0mg/kg。3、对贵阳市表层土壤重金属含量进行地质累计指数分析，结果显示贵阳市的表层土壤中：41％未受砷污染，43％的在无污染到中度污染之间，14％的中度污染，只有2％的介于中度污染到强污染之间；40％未受福污染，19％无污染到中度污染之间，14％的中度污染，19％的介于中度污染到强污染之间，7％的强污染，1％的表层土壤强污染到极强污染之间；46％未受铬污染，47％在无污染到中度污染之间，6.8％的中度污染，0.2％介于中度污染到强污染之间；38％未受铜污染，38％在无污染到中度污染之间，22％的中度污染，2％的介于中度污染到强污染之间；18％未受铅污染，47％在无污染到中度污染之间，28％的中度污染，3％介于中度污染到强污染之间；12％未受元素汞污染，37％在无污染到中度污染之间，36％的中度污染，11％介于中度污染到强污染之间，2％的强污染，1％的介于强污染与极强污染之间，1％的极强污染；19.2％未受镍污染，63.7％在无污染到中度污染之间，16.8％的中度污染，0.3％介于中度污染到强污染之间；41％未受锌污染，50％在无污染到中度污染之间，7％的中度污染，2％介于中度污染到强污染之间。4、贵阳市表层土壤中重金属元素含量的污染程度计算显示：砷的污染程度最大为4，27，50.2％的表层土壤未受污染，97％的表层土壤污染程度小于2，总污染程度略大于O，即受到轻微污染；蝠的污染程度最大为12.1，57.9％的表层土壤未受污染，％％的表层土壤污染程度小于4，总污染程度大于。，即受到污染；铬的污染程度最大为2.01，69％的表层土壤未受到重金属元素铬的污染，30.6％的表层土壤受到轻微污染，总污染程度小于O，即未受到污染；铜的污染程度最大为5.09，53.2％的表层土壤未受到重金属元素铜的污染，总污染程度略大于0，即受到轻微污染；铅的污染程度最大为8.11，49.9％的表层土壤未受到重金属元素铅的污染，总污染程度略大于0，即受到轻微污染；汞的污染程度最大为45.87，56.1％的表层土壤未受到重金属元 素汞的污染。总污染程度大于0，即受到污染；镍的污染程度最大为1.56，64％的表层土壤未受到重金属元素镍的污染，总污染程度小于O，即未受到污染；锌的污染程度最大为2.85，77.8％的表层土壤未受到重金属元素锌的污染，总污染程度小于0，即未受到污染。综合考虑8种重金属污染元素，贵阳市40.2％的表层土壤无重金属元素污染，巧％的表层土壤在无污染到轻微污染之间，36.1的表层土壤轻微污染，7.2％的中度污染，1.4％的表层土壤严重污染。5、福和镍只存在较少的污染区域，其余六种元素都存在局部的重金属含量受人为因素影响。砷的污染区域主要集中在筑东部的马场和西部清镇的暗流、王家院等地；铬的污染区域主要集中筑南部的花溪马林、西部的暗流一酒坪等地；铜的污染区域则主要集中在筑南部的燕楼马林处；汞的污染区域分布在筑北开阳双流、筑西北六广以及筑中扎佐三片；铅的污染区域分布在筑东北的宅吉、城关；筑西麦格等处；而锌的污染区则集中在筑东北面的开阳花梨和城关。总体来说，镍、铜的含量是处于最安全的位置，而汞、铅的警戒区域面积很大，几乎函盖了全部研究区域，福次之，砷、锌、铬适中。6、贵阳市表层土壤中重金属污染元素的含量主要与矿山、长期的农业措施和燃煤带来的污染有关。所有重金属含量超标区几乎都对应于矿区，其污染可能来自于母岩，也可能来自矿区生产而导致的“三废”污染。汞、铅、镉的“警戒”区域面积较大，主要是由长期的农业污染造成，特别是磷肥和含污染元素的农药、除草剂等的施用，汞、隔“警戒”区域面积较大的原因还与燃煤后重金属的大气沉降有关。另外砷的污染也和燃煤后大气沉降有关。贵阳市表层土壤没有受到重金属污染元镍的污染。7、在本研究区域内，重金属含量呈现不同含量等级的土类是由于长时间受人为影响的结果，而重金属含量处于单一等级区间的土类则受人为因素影响较小，其含量区间反映了背景值含量范围。在贵阳市的表层土壤中，黄壤、石灰土、水稻土受人为影响较多，石质土、粗骨土、紫色土、黄棕壤受人为影响适中，沼泽土和山地草甸土受人为影响较少。8、本文建议：（1）在乌当区金华镇三铺村、修文县久长镇新隆村、白云区牛场乡小山村、修文县扎佐镇丁官村、修文县大石乡天桥村、开阳县双流镇凉水井村、修文县六广镇滨江村这几个综合污染较为严重的区域采取政府宏观调控手段，指导当地农户进行绿化苗木、花卉等不进入食物链的植物，或者在合适的地方，进行退耕还林，种植生态林。同时建议有关部门在这些区域开展土壤重金属治理工作；（2）在清镇市的暗流一卫城、王家院、水塘以及开阳县马场一哨上等砷含量较高区域种植旱生禾谷类食用籽粒的作物，如小麦、玉米、高粱等，不能种植水生作物和叶菜类作物；在北部开阳双流片、西北部修文六广一大石片、筑中扎佐一牛场片汞含量较高区域推广竺麻的种植；在东北部开阳花梨一城关锌含量较高区域建议农户种植玉米；在开阳的宅吉一龙水、城关和清镇的麦格片区铅含量较高区域，大力发展烟草的种植；在修文久长、大石、开阳城关一马场、清镇麦格、乌当水田一新堡等搞含量较高地区推荐种植豆科食用籽粒作物。9、综合考虑国家《土壤环境质量标准（GB15618一1995）》中规定监测的8种重金属污染元素，污染程度（Cd）的判别指标可以定量化为：Cd≤O：无污染；O＜Cd燕1无污染到轻微污染；1＜Cd≤5：轻微污染：5＜Cd≤10中度污染；Cd＞10：严重污染。