Bioaugmentation with Pseudomonas sp. strain MHP41 promotes simazine attenuation and bacterial community changes in agricultural soils

Bioremediation is an important technology for the removal of persistent organic pollutants from the environment. Bioaugmentation with the encapsulated Pseudomonas sp. strain MHP41 of agricultural soils contaminated with the herbicide simazine was studied. The experiments were performed in microcosm trials using two soils: soil that had never been previously exposed to s -triazines (NS) and soil that had >20 years of s -triazine application (AS). The efficiency of the bioremediation process was assessed by monitoring simazine removal by HPLC. The simazine-degrading microbiota was estimated using an indicator for respiration combined with most-probable-number enumeration. The soil bacterial community structures and the effect of bioaugmentation on these communities were determined using 16S RNA gene clone libraries and FISH analysis. Bioaugmentation with MHP41 cells enhanced simazine degradation and increased the number of simazine-degrading microorganisms in the two soils. In highly contaminated NS soil, bioaugmentation with strain MHP41 was essential for simazine removal. Comparative analysis of 16S rRNA gene clone libraries from NS and AS soils revealed high bacterial diversity. Bioaugmentation with strain MHP41 promoted soil bacterial community shifts. FISH analysis revealed that bioaugmentation increased the relative abundances of two phylogenetic groups ( Acidobacteria and Planctomycetes ) in both soils. Although members of the Archaea were metabolically active in these soils, their relative abundance was not altered by bioaugmentation.     

吡咯伯克霍尔德氏菌JK-SH007抗菌蛋白的分离纯化

对拮抗细菌吡咯伯克霍尔德氏菌(Burkholderia pyrrocinia)JK-SH007菌株抗菌蛋白进行了初步研究。研究发现,JK-SH007菌株的抗菌粗蛋白对热和蛋白酶K不敏感,碱性环境不利于抑菌蛋白活性的发挥。JK-SH007菌株的无菌发酵滤液经硫酸铵沉淀、透析(3.5kD)、冷丙酮沉淀(-20℃)、Sephadex G-50葡聚糖凝胶分子筛层析及DEAE-Sephadex A-25离子交换层析分离纯化,得到了蛋白组分A-Ⅱ-2,经SDS聚丙烯酰胺凝胶电泳检测不是单一组分,该复合组分具有明显抑制3种杨树溃疡病病原真菌金黄壳囊孢(Cytospora chrysosperma)、拟茎点霉(Phomopsis macrospore)、七叶树壳梭孢(Fusicoccum aesculi)生长的作用。     

γ-Irradiation induces P2X7 receptor-dependent ATP release from B16 melanoma cells

Background

Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma.

Methods

Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X₇ receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X₇ receptor.

Results

Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X₇ receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X₇ receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X₇ receptor channel plays a significant role in mediating the ATP release.

General Significance

We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.     

日本血吸虫Sj Cyclophilin B基因的克隆、表达及免疫保护效果分析

本研究克隆和表达了日本血吸虫Cyclophilin B(Sj CyPB)编码基因的cDNA,分析其在日本血吸虫不同发育阶段虫体的表达情况,评估该重组抗原在小鼠体内诱导的抗血吸虫免疫保护效果。本研究以日本血吸虫童虫cDNA为模板,RT-PCR扩增其基因全长cDNA,提交序列到NCBI,登录号为GQ403665。荧光实时定量PCR分析该基因在日本血吸虫不同发育阶段虫体的表达情况,构建重组表达质粒,表达纯化重组蛋白。利用Western blotting检测重组蛋白的抗原性。以重组抗原免疫小鼠,评估其对小鼠诱导的免疫保护效果。结果表明,RT-PCR获得了Sj CyPB编码基因的全长cDNA,其开放阅读框为672bp。经分析确定其为CyPs家族中的CyPB基因,命名为Sj CyPB。荧光实时定量PCR分析表明,该基因在18d童虫期表达量最高,32d次之。构建了重组表达质粒pGEX-6P-1-SjCyPB,并在大肠杆菌中成功表达,表达产物分子量为49.5kDa。Western blotting试验显示该重组蛋白具有良好的抗原性,在小鼠免疫试验中,与空白对照组比较,免疫组小鼠获得31.5%的减虫率和41.01%的肝脏减卵率。本研究获得了日本血吸虫童虫期高表达的Sj CyPB基因的全长cDNA,成功构建了Sj CyPB原核重组表达质粒,并在大肠杆菌中成功表达,证实该重组抗原在小鼠体内诱导产生了部分免疫保护效果。     

酿酒酵母乙醇脱氢酶2(ADH2)基因的克隆表达及活性验证

为了从酿酒酵母Saccharomyces cerevisiae中克隆出乙醇脱氢酶2(Alcoholdehy drogenase2,ADH2)基因并使之在大肠杆菌中高效表达。以酿酒酵母细胞中提取的总RNA为模板,通过反转录获得酿酒酵母乙醇脱氢酶2基因,连接到表达载体pTAT上,得到重组表达质粒pTAT-ADH2,将此重组质粒转化到大肠杆菌BL21中,重组工程菌株经IPTG诱导表达得到ADH2蛋白。将该蛋白纯化后,在体外进行活性检测和小鼠体内进行毒理试验,检测ADH2的酶活性。测序结果表明克隆的基因与GenBank中所报道的adh2基因序列有90%的同源性,经SDS-PAGE电泳分析,目的蛋白得到了有效表达,蛋白条带扫描分析表明,表达量占总蛋白的50%左右,纯化得到的蛋白在小鼠体内进行毒理试验,显示出一定的活性。酿酒酵母adh2基因的克隆正确,不仅在大肠杆菌中进行了高效表达而且表现出了较好的酶活性。     

Wood formation in Angiosperms

Wood formation is a complex biological process, involving five major developmental steps, including (1) cell division from a secondary meristem called the vascular cambium, (2) cell expansion (cell elongation and radial enlargement), (3) secondary cell wall deposition, (4) programmed cell death, and (5) heartwood formation. Thanks to the development of genomic studies in woody species, as well as genetic engineering, recent progress has been made in the understanding of the molecular mechanisms underlying wood formation. In this review, we will focus on two different aspects, the lignification process and the control of microfibril angle in the cell wall of wood fibres, as they are both key features of wood material properties.     

克隆植物蛇莓基株的分子鉴定

采用随机扩增序列区间(ISSR)分子标记技术对蛇莓基株进行鉴定,结果表明6个ISSR引物在22个分株中共扩增出62个条带,每个引物扩增的条带数为4～16条,平均每个引物扩增的条带数为10.3条。采用6种引物对22个克隆分株的DNA扩增共产生28种带谱类型,其中有9种带谱为特异性带谱。综合分析这些带谱,确知这22个克隆分株分属16个基株。由带型可知,通过6个引物中的4种引物就可以把所有的基株鉴定出来,表明ISSR技术在分子水平上鉴定蛇莓的克隆基株是一种行之有效的方法。同时每个引物扩增出来的条带的多态性比例也比较高,平均达到90.3%。     

解脂耶氏酵母脂肪酶基因lip1的克隆、密码子优化及功能表达

摘要：【目的】克隆解脂耶氏酵母（Yarrawia lipolytica）脂肪酶基因lip1,并通过密码子优化,首次实现其在毕赤酵母(Pichia pastoris)中的诱导型和组成型表达。【方法】通过PCR扩增Y. lipolytica脂肪酶基因lip1,根据P. pastoris密码子偏爱性,运用重叠延伸PCR合成改造后基因MLip1,将其分别克隆至诱导型分泌载体pPIC9K和新构建的组成型分泌载体pGAP9K上,电转至P. pastoris GS115中,G418抗性筛选得到高拷贝转化重组子,摇瓶发酵     

Highly efficient rescue of dengue virus using a co-culture system with mosquito/mammalian cells

The production rate of dengue viruses (DENVs), especially low-passage virus isolates, is low, and, therefore, the isolates are generally used only after several passages. However, in vitro passages could induce mutation(s). In this study, we established a system for the characterization of low-passage viral isolates using an infectious cDNA clone. We used R05-624, a plaque derived from type 2 (DENV-2) Thai strain, for the construction of the cDNA clone, named pmMW/R05-624. We found that transfection of both of mammalian Vero cells and mosquito C6/36 cells with viral RNA derived from the cDNA clone produced a significant amount of progeny virus: 3.2 × 10⁶ focus-forming units (FFU) production per ml of cultured fluid only 3 days after transfection with 2 μg RNA. Conversely, no detectable level of viruses was produced by conventional methods using a single cell line, Vero or C6/36. When this system was applied for the characterization of eight low-passage clinical viral isolates by placing their 5′-half or 3′-half in the above cDNA clone, we found that all the isolates, except for L04-225, produced similar levels of progeny virus. Among a total of eight cDNA clones reconstructed with the NS4A-3′NCR region derived from L04-225, one clone carried an insertion and produced a low level of progeny virus. Thus, our system to efficiently rescue clinical samples or low-passage viral isolates could be useful for assessing the virological and molecular characteristics of DENV that could be related to disease pathogenesis.