基于SAFLP的我国常见索线虫科昆虫病原线虫亲缘关系分析

本研究建立并优化了索线虫科(Mermithidae)SAFLP体系,构建了我国常见索线虫科昆虫病原线虫6属11种/亚种的指纹图谱。采用EcoRⅠ和MseⅠ两种限制性内切酶单酶切,结果显示EcoRⅠ酶更适合作为索线虫科线虫SAFLP的内切酶。3个带有3个选择性碱基的EcoRⅠ引物进行扩增共得条带225个,片段大小为250～1 650 bp。通过NTsys-PC2.1软件计算了索线虫科6属11种/亚种的Nei-Li遗传距离(0.1980～034554)和相似系数,利用NTsys-PC2.1软件中的UPGMA方法构建其聚类图。索线虫科这6属11种/亚种从属级阶元可以分为两大类群：罗索属Romanomermis和八腱索属Octomyomermis聚为一支构成第一大类群;六索属Hexamermis与卵索属Ovomermis先聚在一起然后与多索属Agamermis聚在一起,再与两索属Amphimermis聚在一起构成第二大类群。在八腱索属和两索属中属内不同种/亚种间线虫采集地相距较近的其遗传距离较近。优化的索线虫科线虫SAFLP体系能够反映索线虫科线虫种属间的亲缘关系,并与形态学的研究结果基本相符,可以用于属种的分类和亲缘关系的研究。     

危型竹叶青蛇伤性DIC的救治

目的：通过比较单纯中医药治疗与抗蛇毒血清及中西医结合救治方法,观察竹叶青蛇伤性DIC患者的疗效。方法：用抗蛇毒血清、立止血及中西医结合的方法。从1996年8月至2002年9月,共收治危型竹叶青蛇伤性DIC患者25例。结果：25例均治愈。结论：抗蛇毒血清的早期应用是有效方法。抗五步蛇毒血清或抗蝮蛇毒血清和立止血合用,可以对竹叶青蛇毒的出血毒素和促凝毒性起较好的中和作用。     

大鼠肾虚自然流产模型的建立及相关因子的表达

目的建立大鼠肾虚自然流产模型,研究模型蜕膜细胞因子、协同刺激因子表达。方法雌性大鼠20只,雄性大鼠10只。将雌鼠与雄鼠按2∶1合笼,以阴道涂片出现大量精子为妊娠第1天,随机分为对照组、模型组每天灌服羟基脲450 mg/kg,第8天灌服米非司酮3.75 mg/kg,建立肾虚模型;统计饮食量、饮水量、肾、卵巢、胚胎直径指数;RT-PCR检查Th1型/Th2型细胞因子mRNA表达;流式细胞检测协同刺激因子CD80、CD86、CD28、CTLA-4表达。结果模型组动物饮食量、饮水量、体重增加量与对照组比较,第8天差异有显著性（P〈0.05）;胚胎直径指数显著减少（P〈0.01）;平均流产率显著升高（P〈0.01）;TNF-α、IFN-γ表达显著升高（P〈0.05）,IL-4I、L-10表达显著升高（P〈0.05）;CD80、CD86、CD28、CTLA-4显著性降低（P〈0.05）。结论灌服羟基脲与米非司酮可成功建立肾虚自然流产制模型。Th1型（TNF-αI、FN-γ）可能对妊娠有害,高表达有可能造成流产;Th2型（IL-4I、L-10）可能有利于妊娠,高表达维持妊娠。协同刺激因子CD80、CD86、CD28、CTLA-4表达与自然流产有关。     

尖吻蝮蛇蛇毒中一种新型酸性磷酯酶的纯化和表征

从尖吻蝮蛇蛇毒中, 通过嗜硫色谱、Sephadex G-75、Blue胶和POROS HQ20离子交换色谱,分离纯化得到纯组分AA-PLA₂-I,并证明其为新型酸性磷酸酯酶A₂ 经鉴定,该酶为分子量14.4 kD的单体蛋白,等电点5.66,不含中性糖基,N端序列为SLIQFETLIMKVVKK,其磷酸酯酶A2活性的最适温度和pH条件分别为50 ℃ 和 pH 10此外,该酶蛋白酶活性较弱,无出血毒性,但具有抗凝血活性,且其抗凝血活性耐热至70 ℃.     

内生黄曲霉对丹参悬浮细胞氧化酶活性的影响

目的:研究内生黄曲霉对丹参悬浮细胞次生代谢相关氧化酶活性影响,探讨丹参组织培养和次生代谢产物产生的有效诱导途径和诱导机理.方法:将丹参内生黄曲霉以活菌、发酵茵液及制作成诱导子方式分别添加到丹参悬浮细胞中,测定丹参悬浮细胞过氧化物酶、过氧化氢酶及多酚氧化酶活性.结果:不同处理方式,内生黄曲霉对丹参悬浮细胞氧化酶表达均有明显的诱导作用,其中将黄曲霉制作成诱导子对三种酶活诱导作用最明显.结论:内生黄曲霉诱导子能有效提高丹参悬浮细胞氧化酶活性.     

快速测定厚朴中厚朴酚及其含量的方法

目的:通过建立系统的HPLC快速检测方法,分析厚朴中主要药用成分厚朴酚与和厚朴酚的含量,确定厚朴的药用价值及开发利用价值.方法:用HPLC法对张家界、隆回及恩施地区的厚朴植株的不同部位的药材中厚朴酚与和厚朴酚的含量进行了含量的分析.结果:结果快速、准确、重现性好、HPLC分析图谱理想.结论:本文所运用的HPLC分析条件能快速准确的鉴定厚朴酚及和厚朴酚的含量,确定厚朴药材的品质,并对混乱的药材市场有一定的监控作用.     

Variability and divergence in Pongamia pinnata (L.) Pierre germplasm – a candidate tree for biodiesel

Three explorations were undertaken in South East Coastal zone of India covering parts of Andhra Pradesh (AP) and Orissa states to collect Pongamia pinnata (L.) Pierre germplasm during March–June 2007. A total of 123 accessions were collected and seed data recorded were analyzed for morphometric traits viz., seed length, seed width, seed thickness, 100‐seed weight and oil content. Variation in the collected germplasm was analyzed using anova , simple measures of variation and D² statistics. Significant genetic variability between seed traits and oil content and association among the seed traits was recorded. Phenotypic variance was higher than genotypic variance for all the characters indicating dominant role of environment. High heritability (broad sense) for 100‐seed weight (97.6%) and oil content (86.7%) indicated the reliability of these characters as selection criteria for plus trees. Genetic gain was maximum for 100‐seed weight (62.6%) followed by oil content (30.5%). D² analysis grouped the accessions into 12 clusters. Cluster XII and cluster IX were the most diverse based on the intercluster distance. Based on the observed diversity, Chittoor, Srikakulam and Adilabad districts of AP are most suitable for collecting diverse germplasm lines and also for in situ conservation.     

云前胡的萜类成分研究

从云前胡(Peucedanum rubricauleSheh et Shan)的乙醇提取物中首次分离并鉴定了4个萜类化合物,分别为sinodielides A(1)、B(2),积雪草酸(asiatic acid,3)和乌苏酸(ursolic acid,4)。     

Induction of diabetes with signs of autoimmunity in primates by the injection of multiple-low-dose streptozotocin

Aim

To develop a preclinical large animal model of autoimmune diabetes to facilitate the translational research of autoimmune diabetes in human.

Materials and methods

Nine young rhesus monkeys received multiple-low-dose (MLD) intravenous injections of streptozotocin for five consecutive days, followed by two additional boosting injections of STZ given 1 week apart. The induction of autoimmune diabetes was evaluated by regular metabolic testing, serological assessment of islet-reactive autoantibodies and histological examination of pancreatic tissues.

Results

Seven of nine treated animals became diabetic with moderate hyperglycemia initially and more severe hyperglycemia thereafter. All diabetic animals exhibited severely impaired glucose tolerance, limited islet function, and required insulin therapy to maintain relatively normal glucose metabolism and healthy status. Serological tests showed that all diabetic monkeys developed autoantibodies specifically against insulin and islet antigens. Furthermore, histological examination of the pancreata from diabetic animals revealed evidence of specific destruction of islet β cells and islets infiltrated with T lymphocytes. Overt and persistent diabetes can be induced in young rhesus monkeys by the injection of MLD-STZ, and autoimmune responses to pancreatic islet cells seem to be involved in the development of glucose intolerance and diabetes.

Conclusion

These data indicate for the first time that autoimmune diabetes can be induced in primates; this may serve as a valuable preclinical model for studying the pathogenesis of and potential therapies for autoimmune diabetes in humans.     

Production and characterization of LEA29Y,a variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin,in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Blocking the CD28/B7 costimulatory pathway is a promising strategy in the treatment of graft rejection, graft-versus-host disease and autoimmune diseases. LEA29Y, a high-affinity variant of cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4Ig), is a more potent inhibitor of the interaction between CD28 and B7 than is CTLA4Ig. In a previous study, LEA29Y was produced in a mammalian cell system, which is time-consuming and expensive. To obtain LEA29Y more efficiently and cost effectively, we attempted to produce LEA29Y using a Pichia pastoris expression system. The gene encoding LEA29Y, with an additional 6-His tag at the N-terminus, was cloned into the yeast vector pPIC9K and expressed in the P. pastoris strain GS115. Under the optimized induction conditions for protein expression (inoculum density, OD₆₀₀ = 80; methanol concentration added daily, 1.0–3.0%; induction time point, 72–96 h; culture medium pH = 6.0), the yield of purified LEA29Y was approximately 30 mg l⁻¹ by one-step Ni-agarose affinity chromatography. PNGase F treatment showed the purified LEA29Y to be post-translational modified by N-linked glycosylation. In biological function assays, LEA29Y expressed in P. pastoris demonstrated specific binding to B7-1/B7-2-positive Raji cells and also suppressed lymphocyte proliferation in a dose-dependent manner. These results suggest that LEA29Y produced in P. pastoris is biologically active and will be useful for experimental therapy on immunotherapy for transplant rejection and autoimmune diseases.