猪Ⅱ型圆环病毒浙江株的分离及全基因组结构分析

用PCR方法对从浙江省猪场收集的、以淋巴结肿大和生长迟缓为特征的患猪腹股沟淋巴结进行PCV2检测.将检测为阳性的病料研磨、离心、过滤除菌后,接种PCV-free PK-15细胞进行病毒分离,分离获得3株病毒,命名为HZ0201、HZ0202、NB0301.PK-15细胞增殖所得病毒离心纯化后,在电镜下可观察到直径约为17～20nm,呈二十面体对称的病毒粒子.以组织和病毒的细胞DNA为模板进行PCV2的全基因扩增,测序结果显示,3株病毒分离物全基因序列均由1767bp组成,直接来自病料与细胞分离毒株的核苷酸同源性为100%.基因组内含有11个ORF.通过比较发现,分离毒株与PCV2参考株的同源性介于94.2%～99.7%之间;与PCV1参考株的同源性为77.2%～77.9%.     

9-polylysine protein transduction domain: enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin

Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), have been considered to have a beneficial effect against various diseases that are mediated by the reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against oxidative stresses, the lack of their transduction ability into cells resulted in a limited ability to detoxify intracellular ROS. To render the SOD enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant SOD proteins were generated. A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment that encodes the 9 amino acids Tat protein transduction domain (RKKRRQRRR) of HIV-1 and lysine rich peptide (KKKKKKKKK) in a bacterial expression vector in order to produce a genetic in-frame Tat-SOD and 9Lys-SOD fusion protein, respectively. The expressed and purified Tat-SOD and 9Lys-SOD fusion proteins can transduce into human fibroblast cells, and they were enzymatically active and stable for 24 h. The cell viability of the fibroblast cells that were treated with paraquat, an intracellular superoxide anion generator, was increased by the transduced Tat-SOD or 9Lys-SOD. The transduction efficacy of 9Lys-SOD was more efficient than that of Tat-SOD. We evaluated the ability of the SOD fusion pmteins to transduce into animal skin. This analysis showed that Tat-SOD and 9Lys-SOD fusion proteins efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin (judged by the immunohistochemistry and specific enzyme activities). The enzymatic activity of the transduced 9Lys-SOD was higher than that of Tat-SOD, indicating that the penetration of 9Lys-SOD was more efficient when put into the skin. These results suggest Tat-SOD and 9Lys-SOD fusion proteins can be used as anti-aging cosmetics, or in protein therapy, for various disorders that are related to this antioxidant enzyme and ROS.     

基于质谱技术筛选差异表达蛋白的统计学策略研究进展

随着质谱技术的快速发展,蛋白质组学已成为继基因组学、转录组学之后的又一研究热点,寻找可靠的差异表达蛋白对于生物标记物的发现至关重要.因此,如何准确、灵敏地筛选出差异蛋白已成为基于质谱的定量蛋白质组学的主要研究内容之一.目前,针对该问题的研究方法众多,但这些方法策略的适用范围不尽相同.总体来说,基于质谱技术筛选差异蛋白的统计学策略可以分为3类:基于经典统计学派的策略、基于贝叶斯学派的统计检验策略和其他策略,这3类方法有各自的应用范围、特点及不足.此外,筛选过程还将产生部分假阳性结果,可以采用其他方法对差异表达蛋白的质量进行控制,以提高统计检验结果的可靠性.     

生活在沼泽地 如今打井成了出路

<正>进入若尔盖县后,路边常常可见层层堆叠着的水泥井圈,标示着该地区打井之普遍。随后在对多家牧民的询问中也证实了这一点:除了居民自挖的土井,也有不少政府打的机井。我们在路边的草场上看到这样一幅情景:     

GTPase Activity of MxB Contributes to Its Nuclear Location,Interaction with Nucleoporins and Anti-HIV-1 Activity

The human myxovirus resistance 2(Mx2/Mx B) protein, a member of interferon(IFN)-inducible dynamin-like large GTPases, restricts a number of virus infections. Inhibition of these viruses occurs at poorly-defined steps after viral entry and has a common requirement for Mx B oligomerization. However, the GTPase activity is essential for the anti-viral effects of Mx B against herpesviruses and HBV but not HIV-1. To understand the role of Mx B GTPase activity, including GTP binding and GTP hydrolysis, in restriction of HIV-1 infection, we genetically separated these two functions and evaluated their contributions to restriction. We found that both the GTP binding and hydrolysis function of Mx B involved in the restriction of HIV-1 replication. The GTPase activity of Mx B contributed to its nuclear location, interaction with nucleoporins(NUPs) and HIV-1 capsids. Furthermore, Mx B disrupted the association between NUPs and HIV-1 cores dependently upon its GTPase activity. The function of GTPase activity was therefore multi-faceted, led to fundamentally distinct mechanisms employed by wild-type Mx B and GTPase activity defective Mx B mutations to restrict HIV-1 replication.     

利用假型反转录病毒对大部分胰腺切除后再生细胞的世系追踪

胰腺是一个重要的内外分泌混合腺, 胰腺发生损伤后能够再生。为了探讨胰腺活体细胞世系追踪的方法和胰腺损伤后再生细胞的来源,分别通过胰腺伤口涂抹并胰内注射、尾静脉注射及腹腔注射三种方法, 利用假型反转录病毒对成体小鼠大部分切除后胰腺的细胞进行世系追踪。结果发现在活体条件下, 与尾静脉注射及腹腔注射法相比, 胰腺伤口涂抹并胰腺内注射反转录病毒的方法能够更有效的标记胰腺细胞; 而且, 通过对标记细胞的世系追踪研究证明, 在胰腺损伤后, 胰腺腺泡细胞能够接受损伤信号刺激发生再生。为今后进一步利用反转录假病毒对活体胰腺进行细胞命运追踪研究奠定基础, 为利用反转录病毒载体进行胰腺疾病的基因治疗提供线索。     

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-β-d-glucose via blockade of NF-κB and STAT1 activation in the HaCaT cells

Keratinocytes, one of major cell types in the skin, can be induced by TNF-α and IFN-γ to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-α/IFN-γ induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-α/IFN-γ-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-α/IFN-γ-induced NF-κB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-γ. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-α and/or IFN-γ-induced activation of NF-κB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.     

Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry 'foreign' DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6?. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8-16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8-16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine-bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.