转染E1B55K基因提高Hep2细胞包装肠腺病毒Ad41的能力

人F组腺病毒Ad40、Ad41难以在体外培养的细胞中传代,被称为难养腺病毒(Fastidious adenovirus).本研究观察了在Hep2细胞表达Ad41 E1B55K基因对Ad41复制的促进作用.从Ad41阳性粪便标本中用PCR的方法获得E1B55K基因,构建真核表达载体,转染Hep2细胞,筛选单克隆,用RT-PCR检测了E1B55K基因的表达.用引起293细胞完全CPE比较产毒量的方法对所得细胞克隆进行初步筛选,获得一株产毒相对较强的细胞Hep2-E1B#4.与对照细胞Hep2、Hep2-DNA3相比,等量Ad41接种Hep2-E1B#4产生的细胞病变效应(CPE)程度明显加深.用免疫细胞化学的方法测定产毒的感染滴度,等量Ad41接种后,Hep2-E1B#4产生的子代腺病毒滴度大于对照的9倍;半定量PCR测得Hep2-E1B#4子代病毒基因组拷贝数约为对照细胞的4倍.结果说明转染E1B55K基因促进了Ad41在Hep2细胞的复制,获得的Hep2-E1B#4细胞株可用于Ad41的分离、培养和体外扩增.     

Structure based virtual screening of anticanerous polyphenolic phytocompounds against G-protein coupled receptor and identification of potent antagonist ligand(s) through molecular docking

Design of potential drug-like candidates for cancer is of interest in recent years. We used 60 compounds which are known to have the potential to down regulate Nuclear Factor kappaB (NFκB) for this study. The compounds were assessed for Lipinski's RO5 and ADMET properties. Allixin, anethole, capsaicin, linearol and syringic acid satisfied both Lipinski's RO5 and ADMET properties. These compounds showed strong molecular interaction with receptor GPCR55 indicating they have ability to block GPCR55. Thus, their role in anticellular proliferation and induction of apoptosis is implied.     

GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB(2) receptor (CB(2)R), but additional modulatory sites distinct from CB(2)R have recently been suggested to impact CB(2)R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB(2)R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB(2)R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB(2)R interfere with each other's signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB(2)R, while it synergizes with CB(2)R in recruiting neutrophils to sites of inflammation.     

Splice variants of mda-7/IL-24 differentially affect survival and induce apoptosis in U2OS cells

Interleukin-24 (mda-7/IL-24) is a cytokine in the IL-10 family that has received a great deal of attention for its properties as a tumor suppressor and as a potential treatment for cancer. In this study, we have identified and characterized five alternatively spliced isoforms of this gene. Several, but not all of these isoforms induce apoptosis in the osteosarcoma cell line U2OS, while none affect the survival of the non-cancerous NOK cell line. One of these isoforms, lacking three exons and encoding the N-terminal end of the mda-7/IL-24 protein sequence, caused levels of apoptosis that were higher than those caused by the full-length mda-7/IL-24 variant. Additionally, we found that the ratio of isoform expression can be modified by the splice factor SRp55. This regulation suggests that alternative splicing of mda-7/IL-24 is under tight control in the cell, and can be modified under various cellular conditions, such as DNA damage. In addition to providing new insights into the function of an important tumor suppressor gene, these findings may also point toward new avenues for cancer treatment.     

CD59、CD55在T细胞活化信号转导中的协同作用

目的：研究糖基磷脂酰肌醇（GeD）锚固蛋白CD59、CD55在脂筏介导T细胞信号转导通路中的协同效应。方法：应用siRNA技术,构建特异性针对CD55与CD59基因的重组载体pSUPER—siCD55,pSUPER—siCD59。实验分为未转染的Jurkat细胞组（I组）、转染pSUPER空质粒的Jurkat细胞组（Ⅱ组）、转染pSUPER—siCD59重组质粒的Jurkat细胞组（Ⅲ组）及转染pSUPER—siCD55重组质粒的Jurkat细胞组（Ⅳ组）。RT—PCR检测转染细胞中CD55和CD59基因的表达。噻唑蓝（MTT）比色法和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对4组Jurkat细胞的增殖效应以及细胞内钙离子的变化。结果：稳定转染后,Ⅲ组细胞CD59分子的表达和Ⅳ组细胞CD55分子的表达被成功抑制。Ⅰ组和Ⅱ组细胞CD55与CD59联合作用后增殖能力和钙离子浓度均明显高于Ⅲ组、Ⅳ组（P〈0．05）,Ⅰ组和Ⅱ组之间无差异。结论：CD59和CD55在T细胞活化信号转导通路中存在协同效应。     

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes^1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.     

Introducing shear stress in the study of bacterial adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm(2). Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm(2)). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes (1). On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm(2)(2). The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium (3). To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber (4). Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells (1), to proliferate on the cell surface (5)and to detach to colonize new sites (6) (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience(1) and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.     

Changes of enzyme activity in lipid signaling pathways related to substrate reordering

The static fluid mosaic model of biological membranes has been progressively complemented by a dynamic membrane model that includes phospholipid reordering in domains that are proposed to extend from nanometers to microns. Kinetic models for lipolytic enzymes have only been developed for homogeneous lipid phases. In this work, we develop a generalization of the well-known surface dilution kinetic theory to cases where, in a same lipid phase, both domain and nondomain phases coexist. Our model also allows understanding the changes in enzymatic activity due to a decrease of free substrate concentration when domains are induced by peptides. This lipid reordering and domain dynamics can affect the activity of lipolytic enzymes, and can provide a simple explanation for how basic peptides, with a strong direct interaction with acidic phospholipids (such as beta-amyloid peptide), may cause a complex modulation of the activities of many important enzymes in lipid signaling pathways.