A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.     

妊娠期肝内胆汁淤积症患者血管内皮祖细胞生物学变化

目的:研究妊娠期肝内胆汁淤积症(Intrahepatic Cholestasis of Pregnancy,ICP)患者血管内皮祖细胞(Endothelial Progenitor Cells,EPC)的生物学变化并探讨其意义.方法:40例28-40周孕妇分为ICP组(n=20)、正常组(n=20).用酶联免疫吸附试验法测定两组血胆汁酸浓度;用密度梯度离心法从外周血分离两组单个核细胞培养后进行EPC数量、增殖率、黏附及迁移能力测定并对胆汁酸与EPC相关性进行分析.结果:ICP组孕妇的EPC黏附、增殖和迁移能力明显受损;数量较正常组减少(P=0.00).结论:ICP患者血管内皮祖细胞功能受损,可能与高胆汁酸造成的细胞膜损伤有关.     

Variation in habitat use along the freshwater–marine continuum by grey mullet Mugil cephalus at the southern limits of its distribution

In this study, habitat use by Mugil cephalus was investigated in the waters of the west coast of the North Island of New Zealand by analysing microchemical composition of otoliths (laser‐ablation inductively coupled plasma mass spectrometry) obtained from individuals from commercial fish stocks and research surveys. Results of this study show that M. cephalus at the southern limits of its distribution display highly flexible migratory behaviour with extensive use of freshwater and brackish habitats, potentially enabling them to maximize foraging opportunities. Mugil cephalus can tolerate a wide range of salinities and can therefore utilize higher productivity areas, such as estuaries and eutrophic riverine lakes. Finally, M. cephalus populations across a range of climates and latitudes appear to differ in the extent to which they utilize freshwater and brackish habitats, possibly with increasing penetration of fresh waters with increasing latitude.     

单纯疱疹病毒2型感染细胞多肽27真核表达质粒的构建及表达

为了构建单纯疱疹病毒2型(HSV-2)感染细胞多肽27(ICP27)真核表达质粒,应用PCR技术从HSV-2 333株的基因组中扩增ICP27基因,并连接至真核表达载体pEGFPC2,对阳性克隆进行菌落PCR、酶切和测序鉴定后,成功构建了重组质粒pEGFPC2-ICP27。用X fect转染试剂盒将重组质粒pEGFPC2-ICP27转染至Vero细胞中,并用RT-PCR及W estern b lot-ting检测其表达情况。结果显示,ICP27基因在Vero细胞中得到正确表达。真核表达质粒pEGFPC2-ICP27的构建成功,为进一步研究ICP27对宿主细胞的影响奠定了基础。     

Natural recombinant between equine herpesviruses 1 and 4 in the ICP4 gene

Equine herpesvirus 1 (EHV-1) is a pathogen causing rhinopneumonia in young horses, abortion in mares, and myeloencephalitis in adult horses. Two types, EHV-1 P and EHV-1 B, have recently been dominant among 16 electropherotypes. EHV-1 P and EHV-1 B viruses were compared by long and accurate polymerase chain reaction (LA-PCR) and restriction fragment length polymorphism (RFLP) analysis. Differences in restriction sites were found to be focused in ORF64, which encodes the infected cell protein 4 (ICP4), and downstream of the ICP4 gene. The 3 ' -end and downstream of ICP4 gene of EHV-1 B were found to be replaced by the corresponding region of EHV-4, indicating that EHV-1 B is a naturally occurring recombinant virus between progenitors of EHV-1 P and EHV-4. This is the first report showing a natural interspecies recombinant in alphaherpesviruses.     

Characterization of antiproliferative and cytotoxic properties of the HSV-1 immediate-early ICPo protein

BACKGROUND: The identification of novel proteins displaying cytostatic and/or cytotoxic actions that could eventually be used for gene therapy is a major issue in cancer research. Data from the literature suggested that the immediate-early ICP0 protein from herpes simplex virus type 1 (HSV-1) could fulfill these properties as it had been observed that this protein is involved in arrest of cell growth at the G1/S and G2/M phases of the cell cycle and that deletion of ICP0 from HSV-1 or HSV-1-recombinant vectors significantly reduced their cytotoxicity. The molecular basis of its action is likely related to the ability of ICP0, which possesses E3-ubiquitin ligase activity, to target destruction of key cellular proteins, including centromeric proteins, resulting in abnormal chromosome segregation, unusual cytokinesis, and emergence of nuclear morphological aberrations. However, neither the gene therapy potential of ICP0 on its own nor its action on primary quiescent cells has been assessed to date. The aim of this work was therefore to evaluate the antiproliferative and cytotoxic properties of ICP0 on a human glioblastoma cell line and on quiescent primary cells, and to explore whether this protein has a potential for gene therapy of cancer. METHODS: HSV-1-based amplicon particles were generated following a recently described method that produces relatively high titers of vector stocks that are essentially free of helper virus. These vectors express either wild-type ICP0 or FXE, a RING finger mutated inactive form of ICP0, together with reporter green fluorescent protein (GFP). The vectors were used to infect Gli36 cells, which derive from a human glioblastome, and cultured rat primary cardiomyocytes and brain cells, two well-established models of non-dividing cells. Expression and localization of ICP0 and FXE, as well as their action on centromeres and nuclear morphology, were evaluated by Western blotting, indirect immune fluorescence, and confocal microscopy using specific antibodies and DAPI labeling. The impact of ICP0 on cell growth, toxicity, and viability was evaluated in the different cells using a variety of methods, including FACS analysis after propidium iodide and AnnexinV staining, crystal violet staining, clonogenic capability, caspase 3 activation, MTT tests, and release of lactate dehydrogenase, after infection with the different vectors. RESULTS: The three cell types under study showed high levels of transduction by amplicons and strong expression of GFP, ICP0, and FXE transgenic proteins. Wild-type ICP0, but not FXE, induced centromeric disruption, appearance of micronuclei, arrest of proliferation, and significant cell death in glioblastoma Gli36 cells. In contrast, neither micronuclei formation nor any other sign of cell toxicity could be observed in cultured primary cardiomyocytes or brain cells, as evaluated by MTT tests and crystal violet staining. Furthermore, in the case of cardiomyocytes, expression of ICP0 did not interfere with beating as cells continued to beat at the same frequency as non-infected cells for several days post-infection. Neither AnnexinV early staining nor caspase 3 activation was observed in dying infected Gli36 cells, suggesting that these cells were not entering apoptosis. In contrast, release of lactate dehydrogenase by infected Gli36 cells suggested a necrotic way of death. CONCLUSIONS: ICP0 induced a strong cytostatic action followed by significant cell death on the glioblastoma Gli36 cell line. In contrast, neither cell death nor any other evidence of ICP0-induced toxicity affecting major physiological parameters was observed in primary cultured cardiomyoctes and brain cells, two models of primary non-cycling cells. These results suggest that ICP0 has gene therapy potential and could represent the first member of a novel family of directly acting proteins that could be used to treat cancers.