山楂粉蝶NPV的形态发生及其宿主细胞器的超微病理变化

应用电子显微技术,对山楂粉蝶核型多角体病毒感染三龄幼虫后,该病毒在体内的形态发生过程及其宿主细胞病理超微结构的变化等进行了观察与分析。结果表明：病毒感染７２～１６８ｈ后,山楂粉蝶幼虫中肠上皮细胞内出现病毒发生基质、核衣壳、套膜、丝状纤维或称前多角体蛋白。病毒粒子、病毒束,以及病毒束嵌入多角体蛋白,装配成完全的病毒多角体。在相应的中肠上皮细胞内,细胞器如线粒体、粗面内质网、核糖体、溶酶体等均发生显著的病理变化。同时还应指出的是内质网呈指纹图谱型,板层体与示髓样小体等膜状结构的病理变化比较特殊。     

Nonreductive release of O-linked oligosaccharides from mucin glycoproteins for structure/function assignments as neoglycolipids: application in the detection of novel ligands for E-selectin

The neoglycolipid technology comprises several microproceduresinvolving the generation of lipid-linked oligosaccharide probesfor carbohydrate recognition studies in conjunction with oligosaccharidesequence determination by mass spectrometry. Although applicableto any desired oilgosaccharides, procedures are greatly facilitatedif the ohgosaccharides are nonreduced, as conjugation is byreductive amination of a reducing end aldehyde to a phosphatidylethanolamine.Using bovine submaxillary mucin as a model for release of O-glycansin the reducing state, and based on yields of neoglycolipidsand side-products from "peeling" reactions and degradation,aqueous ethylamine 70% w/v at 22°C for 48 h has been selectedin preference to other conditions, triethylainine, sodium hydroxide,and bydrazine. The integrity of the main acidic and neutraloligosaccharides released under these conditions, di- to octasaccharides,was established by analyses of free oligosaccharides by liquidsecondary ion mass spectrometry (LSIMS) and of the derived neoglycolipidsby TLCLSIMS; the repertoire compared favorably with that ofthe oligosaccharide alditols generated by conventional reductivealkaline borohydride treatment. More forcing conditions of ethylamine70% w/v at 65°C for 6 h were required to release oligosaccharidesfrom porcine gastric mucin; di- to nonasaccharides were obtainedof which about one-third had an intact core GalNAc. Relativeto yields after reductive alkaline hydrolysis, the overall yieldsfor these two glycoproteins were 20% and 40–50% for acidicand neutral oligosaccharides, respectively. Among O-glycansreleased from an ovarian cystadenoma glycoprotein using ethylamine,three variants of the sulfated Le^a/x sequences were identifiedas ligands for the endothelial adhesion molecule E-selectin,one of which is based on the unusual backbone Gal-3/4GlcNAc-3Gal-3Gal. mucins O-linked oligosaccharides TLC-LSIMS neoglycolipids E-selectin     

新型表达载体 pEC34 试用于人 γ 干扰素基因的高表达

构建了一个新的双顺反子表达载体ｐＥＣ３４,它的第一个顺反子是已高表达的ｅｒａ基因的部分序列,并带有原核翻译增强子。当检测ｐＥＣ３４的可用性时,获得了人γ－干扰素的极高表达。目的蛋白占菌体总蛋白的８５．５％,诱导后经ＳＤＳ－ＰＡＧＥ分析,出现分子量为１５．５ｋＤａ左右的蛋白表达带。Ｗｅｓｔｅｒｎ ｂｌｏｔ结果证实为人γ－干扰素。无压力传代３０次表达质粒仍然稳定,表明ｐＥＣ３４可能用作真核蛋白质高表达     

诺卡氏菌对油酸的羟基化作用

诺卡氏菌(Nocardia sp．N13)休止细胞对油酸(顾-9-十八碳烯酸)进行羟基化作用。用休止细胞转化时最适温度为30℃,最适pH为6．5,当菌体(干菌)与底物的最适比例为20 ：1对,对油酸的转化率为83．4％,不同的表面活性剂影响转化的效果。N13休止细胞在磷酸缓冲液中重复转化3次仍可保持50％左右的转化率。红外光谱、质谱、棱磁共振鉴定产物为10－羟基硬脂酸。     

高苯丙氨酸血症大鼠脑内单胺类递质的研究

以3d龄Sprague-Dawley大鼠腹腔注射苯丙氨酸(Phe)诱导高苯丙氨酸血症,荧光法测定大脑皮层及其突触体中去甲肾上腺素(NE)、多巴胺(DA)和5-羟色胺(5-HT)含量;Y型电迷宫法测其学习记忆能力.结果显示:高苯丙氨酸血症大鼠大脑皮层NE、DA及5-HT含量降低38.6%～67.4%,突触体中NE、DA和5-HT含量降低51.9%～70.2%,学习记忆能力明显低于对照组.结果提示,苯酮尿症智力障碍可能与大脑皮层及其突触体中某些单胺类递质含量降低相关.     

Molecular forms of acetylcholinesterase present in the white and grey matter of pig brain

Extraction of the white matter of pig brain with EDTA, lysolecithin or Triton X-100 gave poor yields of soluble acetylcholinesterase although these agents had proved effective at solubilizing the enzyme in the grey matter. This finding, together with the observation that the strong detergent sodium deoxycholate, was needed to solubilize the enzyme, shows that it is more difficult to remove acetylcholinesterase from the white matter of brain than from the grey. This could mean that the enzyme in the white matter is more firmly bound to the membrane than the enzyme in the grey matter.The difference in binding of the enzyme from the two regions of the brain is also reflected in the affinity chromatography experiments which showed a lower recovery for the acetylcholinesterase of white matter compared with the enzyme from grey matter.Starch-block electrophoresis of acetylcholinesterase showed a single negatively charged peak of activity for both the naturally soluble and the deoxycholate solubilized preparations. The presence of only one form on electrophoresis suggests that the molecular species of acetylcholinesterase do not arise from differences in charge.Sucrose density gradient centrifugation of the two preparations from white matter gave a single peak of activity with a sedimentation constant of about 10 S. This corresponds closely to the major species of molecular weight 260,000 detected by gradient gel electrophoresis. Other forms detected in both enzyme preparations by gradient gel electrophoresis were species with molecular weights of 660,000, 180,000, 130,000 and 115,000. The significance of these species in terms of the formation of oligomers is discussed.A comparison was made with the corresponding preparations of acetylcholinesterase from the grey matter and the results showed that acetylcholinesterase from the white and grey matter of pig brain were very similar. The exception to this was the species with a molecular weight of 68,000 which was present in the grey but not the white matter of pig brain.     

抗呼吸道合胞病毒(RSV)单克隆抗体的制备及其对RSV病毒株抗原性的分析

用呼吸道合胞病毒R6(武汉地方株)活毒滴鼻加用戍二醛固定的病毒感染的Hela细胞免疫BALB/C鼠,取脾细胞与小鼠骨髓瘤SP_2/0细胞融合,培育出分泌抗呼吸道合胞病毒单克隆抗体的杂交瘤细胞13株,这些细胞的染色体数为94至104条,其分泌的抗体分别属于鼠IgC_1、IgG_(2a)、IgG_(2b)亚类。腹水荧光抗体滴度为1:10000～1:100000。其中五株单抗有中和病毒作用,尤其是两株中和放价达1:128。应用免疫转印法证实了这些单抗分别能识别RSV6种主要结构蛋白,用7株识别不同病毒结构蛋白的单抗对12株合胞病毒进行抗原性分析,可将这些病毒区分为二个血清型,即A亚型和B亚型。     

血红细胞超氧化物歧化酶(SOD)制备工艺的研究初报

本文报告了本实验室设计的由血红细胞自溶液60℃热变性, 乙醇——氯仿法除血红蛋白,旋转蒸发法减压浓缩抽去氯仿、乙醇,硫酸铵分级盐析法沉降SOD,Sepbadex G-75层析提纯SOD等步骤构成的一条成本低、设计合理、简便实用的分离纯化SOD的工艺路线。     

Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids.

Binding specificity of the major surfactant protein SP-A from human and dog lung has been investigated. Radiobinding experiments have shown that both proteins bind in a Ca(2+)-dependent manner to galactose, mannose, fucose, and glucose linked to bovine serum albumin. These results are in accord with a previous study in which monosaccharides were linked to agarose (Haagsman, H. P., Hawgood, S., Sargeant, T., Buckley, D., White, R. T., Drickamer, K., and Benson, B. J. (1987) J. Biol. Chem. 262, 13877-13880). Chromatogram overlays in conjunction with in situ liquid secondary ion mass spectrometry (TLC-LSIMS) of several purified glycosphingolipids and neoglycolipids as well as binding assays with glycolipids immobilized on plastic wells, demonstrate recognition of galactose (human and dog SP-A), glucose, and lactose (human SP-A) in association with specific lipids. In addition, the occurrence of several neutral and acidic glycosphingolipids in human and rat extracellular surfactants and rat alveolar type II cells is described. Selected components among the neutral glycolipids are bound by radiolabeled human SP-A; these are identified by TLC-LSIMS as predominantly ceramide mono- and disaccharides (human surfactant) and ceramide tri- and tetrasaccharides (rat surfactant and type II cells). A recombinant carbohydrate recognition domain (CRD) of human SP-A inhibits the binding of human SP-A to galactosyl ceramide and to galactose- and mannose-bovine serum albumin, indicating that the CRD is directly involved in the binding of SP-A to these ligands. These results provide evidence for a novel type of binding specificity for proteins that have Ca(2+)-dependent CRDs and raise the possibility that glycosphingolipids are endogenous ligands for SP-A.