循环包装回收技术在筛选肝癌细胞相关耐药基因中的应用

肝癌先天性多表达多药耐药基因,严重影响肝癌的化疗效果,筛选肝癌细胞中的耐药基因,研究其耐药机制有助于提高肝癌化疗效果,提高肝癌的治愈率。首先构建肝癌细胞逆转录病毒的cDNA文库,感染成纤维细胞,使得逆转录病毒基因整合进细胞,加药筛选,存活细胞中的基因再次包装成病毒,用于下一轮筛选。采用循环包装回收(Cyclical packaging rescue,CPR)技术进行肝癌细胞耐药基因的筛选即是通过病毒包装将基因从细胞中钓取出来,相比于常规筛选方法,仅通过一轮筛选可能会出现很多假阳性基因,采用CPR技术则是通过多轮筛选,很大程度减少了假阳性细胞的出现。通过该方法经过四轮筛选获得核糖体蛋白S11(RPS11)、核糖体蛋白L6(RPL6)、核糖体蛋白L11(RPL11)、核糖体蛋白L24(RPL24)等几种基因,经初步检测,增加了细胞的耐药性。     

嘌呤拟似物对豚鼠胃窦环行肌自发性收缩及电活动的影响

为探讨非肾上腺素能非胆碱能神经递质对胃窦环行肌功能的调节作用,在离体胃平滑肌上观察了嘌呤拟似物对胃窦环行肌自发性收缩活动和电活动的影响。电活动用传统的细胞内微电极记录,并和收缩活动同步描记于多道生理记录仪。结果表明,嘌呤能P2Y受体激动剂,三磷酸腺苷(ATP)和2-methylthio ATP(2-MeSATP)均增强胃窦平滑肌的收缩活动,但不影响电活动,而且ATP和2-MeSATP对胃平滑肌收缩活动的增强作用可被嘌呤能P2Y受体阻断剂,reactive blue-2和苏拉明(suramin)所阻断。用100μmol／L α,β-MeATP引起的脱敏感使P2X受体被阻断,ATP增强胃窦平滑肌收缩活动的效应不受影响。嘌呤能P2X受体激动剂,α,β-MeATP明显抑制胃窦环行肌自发性收缩活动,同时使膜电位明显超极化。ATP对胃窦平滑肌的收缩作用不被L型钙通道阻断剂尼卡地平(nicardipine)阻断,但细胞外用无钙液灌流时这种效应则完全被阻断。用前列腺素合成抑制剂消炎痛预先处理20min后,ATP和2-MeSATP仍能增强胃窦平滑肌的自发性收缩活动。以上结果提示：(1)ATP和2-MeSATP通过嘌呤能P2Y受体增强胃窦平滑肌的自发性收缩活动,而α,β-MeATP或β,γ-MeATP通过嘌呤能P2X受体使膜电位超极化,引起胃窦平滑肌的舒张;(2)ATP和2-MeSATP增强胃窦平滑肌自发性收缩活动的效应依赖于细胞外钙,但钙离子进入细胞的途径并不是电压依赖性钙通道;(3)ATP和2-MeSATP增强胃窦平滑肌自发性收缩活动的效应不通过前列腺素介导。     

Cryptic phenology in plants: Case studies,implications, and recommendations

Plant phenology—the timing of cyclic or recurrent biological events in plants—offers insight into the ecology, evolution, and seasonality of plant‐mediated ecosystem processes. Traditionally studied phenologies are readily apparent, such as flowering events, germination timing, and season‐initiating budbreak. However, a broad range of phenologies that are fundamental to the ecology and evolution of plants, and to global biogeochemical cycles and climate change predictions, have been neglected because they are “cryptic”—that is, hidden from view (e.g., root production) or difficult to distinguish and interpret based on common measurements at typical scales of examination (e.g., leaf turnover in evergreen forests). We illustrate how capturing cryptic phenology can advance scientific understanding with two case studies: wood phenology in a deciduous forest of the northeastern USA and leaf phenology in tropical evergreen forests of Amazonia. Drawing on these case studies and other literature, we argue that conceptualizing and characterizing cryptic plant phenology is needed for understanding and accurate prediction at many scales from organisms to ecosystems. We recommend avenues of empirical and modeling research to accelerate discovery of cryptic phenological patterns, to understand their causes and consequences, and to represent these processes in terrestrial biosphere models.     

抗禽流感病毒多表位DNA疫苗的构建及其免疫效力研究

多表位DNA疫苗是建立在常规DNA疫苗基础上的一种新型疫苗。它是用表位作免疫原,这样就比较容易在一个表达载体上克隆病原体的多个抗原基因中具有免疫活性的部分。本试验以H5N1亚型禽流感病毒的HA和NP基因及其表位为基础构建了4个重组质粒：1 pIRES/HA（表达全长的HA基因）;2 pIRES/tHA（只表达HA基因的主要抗原表位区）;3 pIRES/tHANpep（融合表达HA基因的抗原表位区和NP基因的3个CTL表位）;4 pIRES/tHANpep-IFN-γ（用鸡的IFN-γ基因取代质粒pIRES/tHANpep中的neo基因）。分别用这4个重组质粒和空载体质粒pIRES1neo肌注免疫30日龄SPF鸡。免疫3次,间隔为2周,每次每只鸡的剂量为200μg。第3次免疫后两周以高致病性禽流感病毒H5N1强毒攻击,免疫及攻毒前后均采血检测HI抗体效价和外周血CD4⁺、CD8⁺T细胞的变化。结果发现,攻毒前各质粒免疫组均检测不到HI抗体,攻毒后1周存活鸡HI抗体效价迅速升高到64～256。流式细胞仪检测显示外周血CD4⁺、CD8⁺T细胞在疫苗免疫后都有不同程度的升高。空载体质粒对照组鸡（10只）在攻毒后3～8 d内全部死亡,其他各重组质粒免疫组鸡都获得了部分保护,保护率分别是：pIRES/HA组为545%（6/11）,pIRES/tHA组为30%（3/10）,pIRES/tHANPep组为36.3%（4/11）, pIRES/tHANPepIFNγ组为50%（5/10）。这些结果表明我们构建的多表位DNA疫苗能够诱导机体产生特异性免疫应答,并在同型禽流感强毒攻击时对鸡只提供了一定的保护。     

The discovery of anthranilic acid-based MMP inhibitors. Part 3: incorporation of basic amines.

Anthranilic acid derivatives bearing basic amines were prepared and evaluated in vitro and in vivo as inhibitors of MMP-1, MMP-9, MMP-13, and TACE. Piperazine 4u has been identified as a potent, selective, orally active inhibitor of MMP-9 and MMP-13.     

Technical note phytoremediation of triazophos by Canna indica Linn. in a hydroponic system

The phytoremediation of triazophos (O, O-diethyl-O-(1-phenyl-1, 2, 4-triazole-3-base) sulfur phosphate, TAP) by Canna indica Linn. in a hydroponic system was studied. After 21 d of exposure, the removal kinetic constant (K) of TAP was 0.0229-0.0339 d(-1) and the removal percentage of TAP was 41-55% in the plant system and the K and removal percentage of TAP were about 0.002 d(-1) and 1%, respectively, in darkness and disinfected control. However, the K and removal percentage of TAP were 0.006 d(-1) and approximately 11%, respectively, in the treatment with eluate from the media of constructed wetland. The contribution of plant to the remediation of TAP was 74% and C. indica played the most important role in the hydroponic system. Under the stress of TAP and without inorganic phosphorus nutrient, the activity of phosphatase in the plant system increased and phytodegradation was observed. The production and release of phosphatase is seen as the key mechanism for C. indica to degrade TAP. C. indica, which showed the potential of phytoremediation of TAP, and is commonly used in constructed wetland, so the technique of phytoremediation of TAP from contaminated water can be developed with the combination of constructed wetland.     

高产申嗪霉素和吩嗪-1-酰胺的水稻根际铜绿假单胞菌PA1201分离、鉴定与应用潜力

【目的】从水稻根际筛选能高效抑制水稻病原菌的细菌,分析和鉴定其形态和生化特征,为开发新型绿色农药奠定基础。【方法】从水稻根际分离能以1-氨基环丙烷-1-羧酸(ACC)为唯一碳源的菌株,根据菌株形态和生化特性、16S r DNA序列比对和磷脂脂肪酸图谱,对该菌株进行鉴定。通过氯仿萃取抽提、高效液相色谱分析,确定菌株PA1201在PPM培养基和黄豆粉培养基中申嗪霉素和吩嗪-1-酰胺的产量。【结果】菌株PA1201能有效抑制水稻纹枯病菌和水稻白叶枯病菌的生长,属于铜绿假单胞菌(Pseudomonas aeruginosa sp.PA1201);PA1201产生两种抑菌代谢产物申嗪霉素和吩嗪-1-酰胺,在PPM和黄豆粉培养基中申嗪霉素的产量可达81.7 mg/L和926.9 mg/L,吩嗪-1-酰胺的产量亦可达18.1 mg/L和489.5 mg/L;PA1201产生大量胞外蛋白酶,对人肺腺癌细胞系A549和黑腹果蝇具有一定毒性。【结论】PA1201的申嗪霉素产量比现有生产菌株的出发菌株M18高3-4倍,还能产生另一种抑菌活性更高的衍生物吩嗪-1-酰胺,具有进一步开发的潜力。     

Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release

Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand‐full of laccases in plants have been functionally evaluated, and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here, we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall.