部分酶解酵母高效电击转化研究

以酵母质粒YCp50为外源DNA,电击转化部分酶解酵母宿主菌AB1380,转化效率稳定在10~6转化子/μg质粒DNA左右,比不酶解酵母或酵母原生质球作受体的电击转化效率高一个数量级以上,也比PEG介导的酵母原生质球转化高3～5倍,而且适合于大片段DNA如水稻YAC分子的转化。达最佳转化时的有关技术参数为:新接菌种通气培养至细胞密度1×10~8～1.5×10~8个/ml;转化时细胞密度控制在1×10~9～1.5×10~9个/ml;每毫升酶解缓冲液加15u溶菌酶(lyticase),30℃下处理酵母5min进行部分酶解;电击时,电场设置在6.25kV/cm、电容25μF,电击后直接铺板。     

Generation and ex vivo expansion of HTLV-1 specific CD8+ cytotoxic T-lymphocytes for adoptive immunotherapy

CD8(+) cytotoxic T-lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide-specific CD8(+) T-lymphocytes from the peripheral blood of healthy, human trophic leukemia virus-1 (HTLV-1) seronegative human leucocyte antigen (HLA)-A*0201 individuals. A combination of density-based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen-presenting cells (APCs), and T-lymphocytes. The DCs were pulsed with HLA-A*0201 binding peptides and cultured with autologous T-lymphocytes to generate peptide-specific CTLs. The CTLs were generated against a nine-amino-acid peptide from the Tax protein of HTLV-1. The CTLs were expanded according to a restimulation schedule employing peptide-pulsed autologous monocytes and low-dose interleukin-2 (IL-2) to numbers in excess of 100 x 10(6) cells following 5 weeks of culture. Expanded cells contained primarily CD3(+) T-cells, of which CD8(+) T-lymphocytes constituted greater than two-thirds of the cell population. Obtained CTLs exhibited potent antigen-specific lysis of peptide-pulsed target cells in a dose-dependent fashion in in vitro (51)Cr release cytotoxicity assay. This antigen-specific killing was shown to be HLA class I restricted and mediated by CD8(+) T-lymphocytes. Since the T-lymphocytes were obtained from HTLV-1 seronegative donors, the generation of peptide-specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. (c) 1996 John Wiley & Sons, Inc.     

Predictive models for phosphorus retention in wetlands

The potential of wetlands to efficiently remove (i.e., act as a nutrient sink) or to transform nutrients like phosphorus under high nutrient loading has resulted in their consideration as a cost-effective means of treating wastewater on the landscape. Few predictive models exist which can accurately assess P retention capacity. An analysis of the north American data base (NADB) allowed us to develop a mass loading model that can be used to predict P storage and effluent concentrations from wetlands. Phosphorus storage in wetlands is proportional to P loadings but the output total phosphorus (TP) concentrations increase exponentially after a P loading threshold is reached. The threshold P assimilative capacity based on the NADB and a test site in the Everglades is approximately 1 g m^–2 yr^–1. We hypothesize that once loadings exceed 1 g m^–2 yr^–1 and short-term mechanisms are saturated, that the mechanisms controlling the uptake and storage of P in wetlands are exceeded and effluent concentrations of TP rise exponentially. We propose a One Gram Rule for freshwater wetlands and contend that this loading is near the assimilative capacity of wetlands. Our analysis further suggests that P loadings must be reduced to 1 g m^–2 yr^–1 or lower within the wetland if maintaining long-term low P output concentrations from the wetlands is the central goal. A carbon based phosphorus retention model developed for peatlands and tested in the Everglades of Florida provided further evidence of the proposed One Gram Rule for wetlands. This model is based on data from the Everglades areas impacted by agricultural runoff during the past 30 years. Preliminary estimates indicate that these wetlands store P primarily as humic organic-P, insoluble P, and Ca bound P at 0.44 g m^–2 yr^–1 on average. Areas loaded with 4.0 g m^–2 yr^–1 (at water concentrations>150 g·L^–1 TP) stored 0.8 to 0.6 g m^–2 yr^–1 P, areas loaded with 3.3 g m^–2 yr^–1 P retained 0.6 to 0.4 g m^–2 yr^–1 P, and areas receiving 0.6 g m^–2 yr^–1 P retained 0.3 to 0.2 g m^–2 yr^–1. The TP water concentrations in the wetland did not drop below 50 g·L^–1 until loadings were below 1 g m² yr^–1 P.     

豌豆外源凝集素基因的克隆及序列分析

从豌豆幼叶分离基因组ＤＮＡ,设计特异引物,用聚合酶链式反应方法扩增出豌豆外源凝集素基因并克隆到Ｅ．ｃｏｌｉ质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＥｃｏＲＶ位点。进一步亚克隆至ｐＵＣ１９。序列分析表明,克隆到的片段大小为８３２ｂｐ,包含了豌豆外源凝集素基因完整的编码序列。该基因无内含子,同报道的已知序列相比,其核苷酸序列及推测的氨基酸序列的同源率分别为９９．６％和９８．９％。     

华南胡椒化学成分的研究

从胡椒科植物华南胡椒（Ｐｉｐｅｒ ａｕｓｔｒｏｓｉｎｅｎｓｅ Ｃ．ＤＣ．）中分离出七个成分,经光谱分析和理化常数测定,分别鉴定为Ｎ－ｉｓｏｂｕｔｙｌ－３（３＇,４＇－ｍｅｔｈｙｌｅｎｅｄｉｏｘｙ－５＇－ｍｅｔｈｏｘｙｐｈｅｎｙｌ）－２Ｅ－ｔｒｉｍｏｎｏｉｅｎａｍｉｄｅ（Ｉ）、Ｎ－ｉｓｏｂｕｔｙｌ－７（３＇,４＇－ｍｅｔｙｌｅｎｅｄｉｏｘｙｐｈｅｎｙｌ）－２Ｅ,４Ｅ－ｈｅｐｔａｄｉｅｎａｍｉｄｅ（Ⅱ     

辽宁三角洲区域生态经济分区及其功能分析

应用主成分分析和系统聚类分析方法,将辽河三角洲划分为３个生态经济区和８个生态经济小区．通过对反映系统功能的３个表现属性的分析,评价了辽河三角洲农业生态经济系统的功能状况．     

编码人疱疹病毒－6型核糖核苷酸还原酶和P41蛋白基因的克隆和序列测定

本文报道人疱疹病豢－６型（ＨＨＶ－６）ｐＳＴＹ２８ＤＮＡ片段的序列测定。应用分子克隆、缺损突变体（Ｄｃｌｅｔｉｏｎｍｕｔａｎｔ）制备和序列测定等技术,完成了３．９ｋｂＨＨＶ－６ｐＳＴＹ２８ＤＮＡ片段的全序列测定。经ＤＮＡＳＩＳ核酸蛋白软件分析,该片段含有两个开读框架（ＯＲＦ）核糖核苷酸还原酶（ＲＩＲ）ＯＲＦ有２４１４个核苷酸,可编码８０５个氨基酸;Ｐ４１蛋白由１１００个核苷酸组成。与其他疱疹病毒作氨基酸同源性比较,ＨＨＶ－６ＲｉＲ与人巨细胞病毒（ＨＣＭＶ）有高度同源性,最适记分（Ｏｐｔｉｍｉｚｅｄｓｃｏｒｅ）达４５９。实验结果支持Ｅｓｆｔａｔｈｉｏｕ提出的论点,ＨＨＶ－６属于β－疱疹病毒。     

新的生物杀虫剂螨虫素对棉花、蔬菜害虫的毒力评价及应用技术研究

本文通过生物杀虫剂螨虫素对棉花、蔬菜害虫的毒力评价及田间应用研究表明,它对棉朱砂叶螨和菜青虫两种害虫杀伤力最强,其中LC90分别为0．078ppm和0．013ppm。兼有胃毒、触杀作用,残效期较长,分别达13天和9天。田间小区试验认为防治菜青虫和茄朱砂叶螨以2ppm浓度为宜。防治适期为卵孵化初期或低龄幼(若)虫（螨)期,其防效能达90%以上。该药特点用药量低、效果好、无公害、值得推广。