基于地高辛标记对小麦进行Southern杂交分析主要影响因素的优化和验证

以转基因小麦和野生型小麦DNA为材料,对利用地高辛标记对小麦基因组DNA进行Southern杂交分析的影响因素进行了优化研究,包括探针制备与纯化、样品DNA量、酶切体系、真空转印条件、杂交条件、免疫检测方法等。结果表明,对随机引物标记的模板和标记后的探针进行纯化可明显提高探针的标记效率,10μg高质量的DNA样品在80μl的体系中,酶切8～12h可获得良好的效果;真空转膜时使用碱性液比中性液获得的转膜效果更干净;试剂纯度、杂交温度及杂交炉转速等均对杂交效果产生重要影响;配合改进的CSPD涂布方法,使用化学发光检测系统比单纯使用X光片显像更易操作,背景更干净;本研究所优化的地高辛标记的小麦Southern杂交分析显示出较高的灵敏度和信噪比,结果稳定,可克服同位素标记对实验条件、设备及实验人员身体状况等限制,在普通实验室推广应用。     

GFP转基因裸鼠脾脏组织学观察

目的探讨GFP基因导入对BALB/c荧光裸鼠脾脏组织学及免疫功能的影响。方法取不同日龄（14日龄、28日龄、49日龄、70日龄）BALB/c荧光裸鼠及BALB/c普通裸鼠各32只,雌雄各半,处死取脾脏,对脾脏的绝对重量、脾脏指数进行测量分析,对脾脏的组织学改变进行观察,并对脾脏淋巴细胞数进行统计分析。结果与14日龄荧光裸鼠相比,28日龄荧光裸鼠脾脏指数明显较高（P〈0.05）。与14日龄荧光裸鼠相比,49日龄、70日龄荧光裸鼠淋巴细胞数明显变少（P〈0.05）。与普通裸鼠（14日龄、28日龄、49日龄、70日龄）相比较,相同日龄荧光裸鼠（14日龄、28日龄、49日龄、70日龄）淋巴细胞数明显减少（P〈0.05）。结论 GFP基因对不同日龄荧光裸鼠的脾脏发育及其功能有一定影响。     

SARS病毒s1基因片段真核表达载体的构建及免疫效果

本研究根据GenBank登录的BJ01株SARS-CoV序列合成801bpS1基因片段,该片段被亚克隆至真核表达载体pcDNA3.1( )得到重组质粒pcDNA3.1( )/S1;转染Hela细胞,SDS-PAGE、Western-Blotting鉴定蛋白表达;肌注免疫BALB/c小鼠,利用ELISA法检测免疫后小鼠的抗SARS-CoVIgG及IFN-γ水平,MTT法检测T细胞增殖活性。结果显示,重组质粒pcDNA3.1( )/S1可在Hela细胞内表达S1蛋白,免疫后小鼠的T细胞增殖活性增强,抗SARS-CoVIgG与IFN-γ水平升高。本实验说明pcDNA3.1( )/S1可诱导小鼠产生一定的体液免疫和细胞免疫应答。     

Conditions for pathogen elimination by immune systems

Summary A continuous harvest effort can lead a population to extinction. How an “unconscious” immune system would perpetrate such an effort in order to eliminate a self-replicating antigen (a pathogen) becomes an intriguing problem if the system responses are functions of the pathogen population: the responses cannot be a continuous effort as the pathogen vanishes. On theoretical grounds, we show some qualities an immune response must have to support pathogen elimination. Then, three specific mechanisms are addressed: a pathogen-independent positive feedback loop among the responding cells of the system (e.g., B-lymphocyte and T-helper); the persistence of antigen bound to presenting cells; and the programmed expansion/contraction of a pool of responding cells. The maintenance of responding cells due to these mechanisms is the essential feature to the effective clearance of self-replicating agents. Thus, evolutionarily, the primary function of a helper lymphocyte would be to amplify a response and the primary function of memory would be the very elimination of pathogens.     

C6／36细胞上登革2型病毒受体的免疫共沉淀

本文利用差速离心方法提纯的登革2型病毒、抗登革病毒单抗、病毒受体、耦合有G蛋白的琼脂糖珠之间特异性结合的作用,利用免疫共沉淀方法从C6／36细胞中分离鉴定出分子量约为35kDa受体蛋白;并用糖蛋白染色方法否定了此蛋白的糖基化特征。     

Importance of Lipids for Bacteriorhodopsin Structure, Photocycle, and Function

This review begins with a brief history of early studies on the involvement of lipids in certain bacteriorhodopsin (BR) properties. Such properties include the regulation of the pK for the purple to blue transition caused by deionization, and the reformation of trimers from monomers after exposure of the purple membrane to Triton X-100. Most of the review is devoted to newer studies which indicate an important role for the neutral lipid squalene in the functional stability of the fast-decaying M-intermediate, for its decay through a pathway involving the O-intermediate, and for the regulation of the relative amounts of slow-decaying and fast-decaying forms of M. Participation of a peripheral acidic amino acid in the overall expression of fast-decaying M is also discussed. Initial studies suggest that the acidic amino acid may be Asp36 and/or Asp38.     

Comparative Characterization of Methods for Removal of Cu(II) from the Active Sites of Fungal Laccases

Copper-containing sites of laccases isolated from the Basidiomycetes Coriolus hirsutus and Coriolus zonatus were characterized by optical methods and EPR spectroscopy. Methods for preparation of fungal laccase derivatives free from type 2 copper ions were compared. The data of EPR spectroscopy and spectrophotometric titration of copper sites showed that only a modified method based on the use of bathocuproine as a chelator for type 2 copper yielded laccase derivatives completely free from type 2 copper. The original enzymes can be reconstituted from the derivatives by dialysis under anaerobic conditions, resulting in complete recovery of native conformation of the protein molecule and the structure of the copper-containing site.     

Nuclear reconstitution of demembranated Orychophragmus violaceus sperm in Xenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon-densation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.     

CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc

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In vitro modulatory effects of functionalized pyrimidines and piperidine derivatives on Aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR) activities

The development of biologically active molecules based on molecular recognition is an attractive and challenging task in medicinal chemistry and the molecules that can activate/deactivate certain receptors are of great medical interest. In this contribution, selected pyrimidine/piperidine derivatives were synthesized and tested for the ability to activate/deactivate Aryl hydrocarbon receptor (AhR) and Glucocorticoid receptor (GR). Tested compounds are shown to activate the receptors but to much lesser extent than positive controls, dioxin and dexamethasone for Ahr and GR, respectively. However, some of them antagonized the positive controls action. Although further in vivo studies are needed to fully characterize the bioactivities of these compounds, the reported in vitro evidences demonstrate that they might be used as the modulators of AhR and GR activities.