IGF2基因组印迹系统人重组腺病毒载体的构建与鉴定

目的:构建携带IGF2印迹系统的腺病毒载体,并验证其在肿瘤细胞及正常细胞中的功效,为IGF2印迹在肿瘤靶向治疗中的应用提供理论基础.方法:将人源的IGF2印迹系统启动子H19、增强子enhancer及甲基化区域CTCF克隆至穿梭质粒pDC-312中构建IGF2基因印迹系统,从pDC-315-EGFP质粒中扩增出EGFP片段插入到构建好的IGF2印迹系统中,然后与腺病毒骨架Ad5通过脂质体Lipofectamine2000介导共转染HEK293细胞,包装成有感染能力的腺病毒Ad-H19-CTCF-enlmncer-EGFP,命名为Ad-EGFP;构建好的腺病毒分别感染IGF2基因印记保持的细胞MCF-7和GES-1及IGF2基因印迹丢失的细胞HRT-18,荧光显微镜下观察EGFP在三种细胞中表达的差异.结果:转染腺病毒载体的HEK293细胞表达EGFP随着时间逐渐增强,并且出现明显的细胞病变效应,EGFP在HRT-18细胞中有大量表达,在MCF-7和GES-1细胞中不表达或仅有少量表达.结论:成功构建了携带IGF2基因印迹系统的腺病毒载体,证明其在IGF2基因印迹丢失的肿瘤细胞中特异性的表达,在正常细胞及IGF2基因印迹保持细胞中不表达,为IGF2基因印迹系统应用于肿瘤细胞的靶向治疗提供了理论基础.     

Genome-Wide Analysis of Small RNA and Novel MicroRNA Discovery in Human Acute Lymphoblastic Leukemia Based on Extensive Sequencing Approach

Background

MicroRNAs (miRNAs) have been proved to play an important role in various cellular processes and function as tumor suppressors or oncogenes in cancers including leukemia. The identification of a large number of novel miRNAs and other small regulatory RNAs will provide valuable insights into the roles they play in tumorgenesis.

Methodology/Principal Findings

To gain further understanding of the role of miRNAs relevant to acute lymphoblastic leukemia (ALL), we employed the sequencing-by-synthesis (SBS) strategy to sequence small RNA libraries prepared from ALL patients and normal donors. In total we identified 159 novel miRNAs and 116 novel miRNA*s from both libraries. Among the 159 novel miRNAs, 42 were identified with high stringency in our data set. Furthermore, we demonstrated the different expression patterns of 20 newly identified and several known miRNAs between ALL patients and normal donors, suggesting these miRNAs may be associated with ALL and could constitute an ALL-specific miRNA signature. Interestingly, GO “biological process” classifications revealed that a set of significantly abnormally expressed miRNAs are associated with disease relapse, which implies that these dysregulated miRNAs might promote the progression of ALL by regulating genes involved in the pathway of the disease development.

Conclusion/Significance

The study presents a comprehensive picture of the expression of small RNAs in human acute lymphoblastic leukemia and highlights novel and known miRNAs differentially expressed between ALL patients and normal donors. To our knowledge, this is the first study to look at genome-wide known and novel miRNA expression patterns in in human acute lymphoblastic leukemia. Our data revealed that these deregulated miRNAs may be associated with ALL or the onset of relapse.     

细菌群体感应系统的研究

群体感应是细菌根据细胞密度变化进行基因表达调控的一种生理行为.细菌通过群体感应与周围环境进行信息交流,参与多种生理过程.就细菌群体感应系统的组成、作用机制、类型、特点及细菌中群体感应的最新进展作以综述.     

2007年中国植物科学若干领域重要研究进展

2007年在中国整体科研实力迅猛增长的大背景下,我国植物科学研究继续呈现飞跃发展的态势,更加高产,与世界的联系也更加广泛。这不仅仅反映在大量发表在国际主流刊物的原创性研究论文上,而且中国科学家在国际相关研究领域的影响力也日益增强。如华中农业大学张启发院士当选为美国国家科学院外籍院士,     

A cell strain cloned from Spodoptera exigua cell line (IOZCAS-Spex-II) highly susceptible to S. exigua nucleopolyhedrovirus infection

A cell strain (IOZCAS-Spex-II-A) cloned from IOZCAS-Spex-II, a cell line established from the fat body of Spodoptera exigua (Lepidoptera: Noctuidae) larva, was characterized, and its capability to produce S. exigua nucleopolyhedrovirus was high with infection rate exceeding 90% compared with its parental cell line IOZCAS-Spex-II that scored only 50%. Growth curve of budded virus (BV) in the strain was analyzed and the titer of BV reached the highest of 3.7?×?10⁴ pfu/mL by 96 h after inoculation. Concentration of occlusion bodies (OBs) produced by the cloned cell strain (IOZCAS-Spex-II-A) was 7.1?×?10⁷ OBs/mL, while the parental cell line produced 2.4?×?10⁷ OBs/mL. The average yield of the virus was 176 OBs/cell of IOZCAS-Spex-II-A compared with 211 OBs/cell that of the parental cell line. Significant differences were observed in virus production, growth characters, cell shape, between the parental cell line, and its clone. The cell lines (IOZCAS-Spex-II and IOZCAS-Spex-II-A) were also susceptible to Autographa californica multiple nucleopolyhedrovirus infection. In addition, they were characterized with regard to their growth rates and DNA amplification fingerprinting technique employing polymerase chain reaction.     

Helicobacter pylori proteins response to nitric oxide stress

Helicobacter pylori is a highly pathogenic microorganism with various strategies to evade human immune responses. Nitric oxide (NO) and reactive nitrogen species (RNS) generated via nitric oxide synthase pathway are important effectors during the innate immune response. However, the mechanisms of H. pylori to survive the nitrosative stress are not clear. Here the proteomic approach has been used to define the adaptive response of H. pylori to nitrosative stress. Proteomic analysis showed that 38 protein spots were regulated by NO donor, sodium nitroprusside (SNP). These proteins were involved in protein processing, anti-oxidation, general stress response, and virulence, as well as some unknown functions. Particularly, some of them were participated in iron metabolism, potentially under the control of ferric uptake regulator (Fur). Real time PCR revealed that fur was induced under nitrosative stress, consistent with our deduction. One stress-related protein up-regulated under nitrosative conditions was thioredoxin reductase (TrxR). Inactiva-tion of fur or trxR can lead to increased susceptivity to nitrosative stress respectively. These studies described the adaptive response of H. pylori to nitric oxide stress, and analyzed the relevant role of Fur regulon and TrxR in nitrosative stress management.     

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways

Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived microvesicles or microparticles; (4) PM blebbing; (5) cell–cell fusion resulting in formation of multinucleate cells; (6) phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes from multivesicular bodies. This work was supported by NIH grants R01-GM36387 and P01-HLHL18708 (G.R.D.).