猪CAPN1基因部分外显子及3′UTR区的SNPs检测

以野猪、民猪和大白猪为研究对象, 根据网上公布的序列设计了7对引物, 采用测序、PCR-SSCP和PCR-RFLP方法对CAPN1基因的部分外显子和3′UTR区进行了单核苷酸多态性检测和基因型分析, 探讨CAPN1基因多态性与瘦肉率和嫩度的关系。研究发现11个SNPs, 其中5个位于外显子, 4个位于内含子, 2个位于3′UTR区, 外显子中的突变有一处是错义突变, 导致了蛋白质多肽链第260位氨基酸发生了M/V的替代。群体遗传学分析表明, 在所检测的各多态位点上, 野猪、民猪、大白猪3个品种间不同基因型的分布都存在着极显著的差异(P<0.01), 而野猪和民猪之间各基因型的分布差异不显著(P>0.05), 民猪和大白猪之间各基因型的分布存在着极显著的差异(P<0.01)。结合品种特性分析表明, P4、P6引物和3′ UTR区HinfⅠ位点所检测的不同基因型和瘦肉率具有一定的相关性。     

奶牛结核病诊断方法的比较研究

旨在比较目前实验室用于检测牛结核方法及PPD皮内变态反应检测牛结核的敏感性和特异性.对145头进行了PPD结核菌素实验的牛进行了IFN-γ体外释放试验,ELISA方法检测ESAT-6,CFP-10,MPB70,MPB83四个蛋白的抗体,胶体金检测两种相似蛋白MPB70和MPB83.结果表明,IFN-γ体外释放试验的敏感性和特异性最高,与PPD皮内变态反应有较高的符合性.在抗体检测方面,iELISA的敏感性高于胶体金方法.虽然抗体检测(iELISA和胶体金方法)比细胞介导的免疫方法(IFN-γ和PPD)敏感性要低,前者更适合于检测处于结核病程发展后期的样本,并且可以有效降低假阳性反应.     

甾醇C-24甲基转移酶基因在酿酒酵母中的内源表达及工程菌麦角甾醇的合成

通过高保真PCR克隆到含酿酒酵母甾醇C-24甲基转移酶基因编码序列及终止子序列的DNA片段ERG6, 以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体, 磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG6。通过同源重组, 以铜离子螯合蛋白基因CUP1替换染色体上ERG6基因内部序列获得ERG6破坏菌株YS58-erg6, 其中麦角甾醇的合成被阻断, 同时细胞的生长也受到明显抑制。表达质粒pPERG6转化破坏菌株YS58-erg6后, 不但使细胞恢复了合成麦角甾醇的能力, 细胞生物量也得到明显提高, 这说明表达质粒上的ERG6基因得到了功能性的表达。分别用载体质粒YEp352和表达质粒pPERG6转化酿酒酵母单倍体菌株YS58, 获得对照菌株YS58(YEp352)和重组菌株YS58(pPERG6)。重组菌株YS58(pPERG6) 生物量和麦角甾醇含量分别是对照菌YS58(YEp352)的1.23和1.32倍。可见甾醇C-24甲基转移酶基因的高表达可以增强酵母细胞麦角甾醇的合成能力。     

独叶草叶片性状表型多样性研究

独叶草是特产于中国的一种珍稀濒危植物.为研究独叶草自然居群的叶片形态变异特征及影响因素,选取分布于陕西、四川、甘肃的9个自然居群,对独叶草叶片的叶面积、叶柄长、总齿数、末级叶脉数、盲脉数、网结脉数等指标进行统计分析.结果表明:(1)独叶草叶片各属性间存在显著相关性,个别性状表现出与地理位置的相关性.(2)主成分分析显示独叶草的末级叶脉数、叶面积、总齿数对变异有较大贡献,而网结脉贡献很小.(3)独叶草叶片性状居群间变异程度大于居群内,其中盲脉数量在各个居群间变异程度最大,而末级叶脉数为最稳定的性状.(4)将本研究中形态变异结果与前期的遗传分析结果对比分析认为独叶草形态变异主要来自生态环境,而非遗传组成.(5)独叶草居群的形态及遗传变异聚类结果都显示独叶草形态变异并不具有地理格局规律,可能由于地质历史变化打断了独叶草原本连续的分布区,遗传保守性使其仍然保持分布区断裂前的遗传特性,但其表型随着生境的变化表现出可塑性.     

Ribosomal proteins S13 and L23 promote multidrug resistance in gastric cancer cells by suppressing drug-induced apoptosis

Ribosomal proteins (RP) S13 and RPL23 were previously identified as two upregulated genes in a multidrug-resistant gastric cancer cell line SGC7901/VCR compared to its parental cell SGC7901 by differential display PCR. The aim of this study was to explore the roles of RPS13 and RPL23 in multidrug resistance (MDR) in gastric cancer cells. RPS13 and RPL23 were genetically overexpressed in SGC7901 cells, respectively. Either RPS13 or RPL23 enhanced resistance of SGC7901 cells to vincristine, adriamycin, and 5-fludrouracil. RPL23 also enhanced resistance of SGC7901 cells to cisplatin. Overexpression of either RPS13 or RPL23 did not alter the population doubling time, [³H]leucine incorporation, and intracellular adriamycin accumulation of SGC7901 cells. However, either RPS13 or RPL23 could protect SGC7901 cells from undergoing vincristine-induced apoptosis. Western blot analysis revealed that both RPS13 and RPL23 significantly increased the expression level of Bcl-2 and Bcl-2/Bax ratio in SGC7901 cells. In addition, overexpression of RPL23 enhanced glutathione S-transferase (GST) activity and intracellular glutathione content in SGC7901 cells. Together, this work demonstrates that either RPS13 or RPL23 can promote MDR in gastric cancer cells by suppressing drug-induced apoptosis, and that RPL23 may also promote MDR in gastric cancer cells through regulation of glutathione S-transferase-mediated drug-detoxifying system.     

The lipid-droplet proteome reveals that droplets are a protein-storage depot

BACKGROUND: Lipid droplets are ubiquitous organelles that are among the basic building blocks of eukaryotic cells. Despite central roles for cholesterol homeostasis and lipid metabolism, their function and protein composition are poorly understood. RESULTS: We purified lipid droplets from Drosophila embryos and analyzed the associated proteins by capillary LC-MS-MS. Important functional groups include enzymes involved in lipid metabolism, signaling molecules, and proteins related to membrane trafficking. Unexpectedly, histones H2A, H2Av, and H2B were present. Using biochemistry, genetics, real-time imaging, and cell biology, we confirm that roughly 50% of certain embryonic histones are physically attached to lipid droplets, a localization conserved in other fly species. Histone association with droplets starts during oogenesis and is prominent in early embryos, but it is undetectable in later stages or in cultured cells. Histones on droplets are not irreversibly trapped; quantitation of droplet histone levels and transplantation experiments suggest that histones are transferred from droplets to nuclei as development proceeds. When this maternal store of histones is unavailable because lipid droplets are mislocalized, zygotic histone production starts prematurely. CONCLUSIONS: Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.     

Electrochemical biosensor for the detection of cauliflower mosaic virus 35 S gene sequences using lead sulfide nanoparticles as oligonucleotide labels

Lead sulfide (PbS) nanoparticles were synthesized in aqueous solution and used as oligonucleotide labels for electrochemical detection of the 35 S promoter from cauliflower mosaic virus (CaMV) sequence. The PbS nanoparticles were modified with mercaptoacetic acid and could easily be linked with CaMV 35 S oligonucleotide probe. Target DNA sequences were covalently linked on a mercaptoacetic acid self-assembled gold electrode, and DNA hybridization of target DNA with probe DNA was completed on the electrode surface. PbS nanoparticles anchored on the hybrids were dissolved in the solution by oxidation of HNO₃ and detected using a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used to monitor the hybridization reaction. The CaMV 35 S target sequence was satisfactorily detected with the detection limit as 4.38 × 10⁻¹² mol/L (3σ). The established method extends nanoparticle-labeled electrochemical DNA analysis to specific sequences from genetically modified organisms with higher sensitivity and selectivity.     

Activation of STAT6 by STING is critical for antiviral innate immunity

STAT6 plays a prominent role in adaptive immunity by transducing signals from extracellular cytokines. We now show that STAT6 is required for innate immune signaling in response to virus infection. Viruses or cytoplasmic nucleic acids trigger STING (also named MITA/ERIS) to recruit STAT6 to the endoplasmic reticulum, leading to STAT6 phosphorylation on Ser(407) by TBK1 and Tyr(641), independent of JAKs. Phosphorylated STAT6 then dimerizes and translocates to the nucleus to induce specific target genes responsible for immune cell homing. Virus-induced STAT6 activation is detected in all cell-types tested, in contrast to the cell-type specific role of STAT6 in cytokine signaling, and Stat6(-/-) mice are susceptible to virus infection. Thus, STAT6 mediates immune signaling in response to both cytokines at the plasma membrane, and virus infection at the endoplasmic reticulum.     

脑血管内皮细胞Prmt5在脑血管发育过程中的表达及其下游靶分子

目的:研究内皮细胞中蛋白精氨酸甲基转移酶5(PRMT5)在脑血管发育及血脑屏障建成的关键时期的表达变化及其潜在下游靶分子。方法:原位观察PRMT5在小鼠不同发育时期脑血管内皮上的分布;流式分选获得原代脑血管内皮,利用real-time PCR分析Prmt5的表达;体外培养小鼠内皮细胞敲降Prmt5后,利用Western印迹、real-time PCR、ChIP等方法检测其对经典下游分子的影响。结果:PRMT5在胚胎期各时间点的脑血管内皮细胞质均有表达,小鼠出生后主要表达在脑血管内皮细胞核,在胚胎期18.5 d时表达量显著升高;小鼠脑血管内皮细胞体外细胞系中基因敲降Prmt5后,其经典对称甲基化组蛋白产物H4R3me2s及H3R2me2s均明显下降,Bmp4表达显著上调;免疫共沉淀实验提示Bmp4启动子区域组蛋白具有H3R2me2s修饰,Prmt5基因敲降后,该组蛋白修饰显著减少。结论:脑血管发育过程中PRMT5在脑血管内皮细胞中表达的位置和水平均发生变化,脑血管内皮细胞中PRMT5可以调节H4R3和H3R2对称二甲基化水平,Bmp4启动子区域组蛋白具有H3R2me2s修饰,且PRMT5可以抑制Bmp4表达。