A universal procedure for primer labelling of amplicons.

Detection and visualisation of nucleic acids is integral to genome analyses. Exponential amplification procedures have provided the means for the manipulation of nucleic acid sequences, which were otherwise inaccessible. We describe the development and application of a universal method for the labelling of any PCR product using a single end-labelled primer. Amplification was performed in a single reaction with the resulting amplicon labelled to a high specific activity. The method was adapted to a wide range of PCRs and significantly reduced the expense of such analyses.     

用酵母双杂交系统筛选影响apo-AI和SR-BI相互作用的苦豆子活性成分

本文通过将苦豆子总提取物及其分离得到的10个组分加入含有AH109菌株(携带大鼠apo-AI及其受体SR-BI全基因)的培养基中,利用报告基因半乳糖苷酶活力有无变化来寻找能够促进大鼠载脂蛋白apo-AI及其受体SR-BI相互作用的激动剂.经测试,我们观察到苦豆子总提取物(K-1),在上述系统中能显著增强这两种蛋白的相互作用.继续用硅胶柱层析方法从k-1中分出10个组分后,利用酵母双杂交系统再次筛选,发现有五个组分能增强两种蛋白的相互作用,(与空白对照组相比其中有两个组分有极显著差异)另外三个组分显著地抑制上述两种蛋白的相互作用,剩下的两个组分则没有差异.该实验还提示该酵母双杂交系统可能成为一种跟踪具有调脂功能的先导化合物的有效方法.     

不同温度、气体微环境对红托竹荪干品储藏品质的影响

本研究针对红托竹荪干品在储藏过程中易发生褐变、降低商品性问题,探究了不同储藏条件(温度、气体微环境)对红托竹荪干品储藏品质的影响。以红托竹荪干品为原材料,考察了在气体微环境(空气、N₂、CO₂和脱氧)和不同储藏温度(5、25和45 ℃)下红托竹荪干品储藏品质的动态变化。在60 d的储藏期内,所有样品的褐变指数、剪切力、多酚氧化酶、过氧化氢酶、总酚、还原糖和5-羟甲基糠醛含量均增加,游离氨基酸、白度值、复水比均降低。与25、45 ℃相比,以上指标在5 ℃条件下均表现最优,5 ℃储藏条件下呈味氨基酸和挥发性成分指标更接近于0 d;在不同气体微环境比较下,CO₂储藏环境下干品品质保持最好,通过综合评分得出5 ℃低温结合CO₂充气条件下干品品质最优,其次为N₂结合5 ℃低温。结合经济成本,5 ℃低温结合CO₂或N₂充气可以作为红托竹荪干品延长货架期的推荐储藏技术。     

Peroxide reduction by a metal-dependent catalase in Nostoc punctiforme (cyanobacteria)

This study investigated the role of a novel metal-dependent catalase (Npun_R4582) that reduces hydrogen peroxide in the cyanobacterium Nostoc punctiforme. Quantitative real-time PCR showed that npun_R4582 relative mRNA levels were upregulated by over 16-fold in cells treated with either 2 μM added Co, 0.5 μM added Cu, 500 μM Mn, 1 μM Ni, or 18 μM Zn. For cells treated with 60 μM H₂O₂, no significant alteration in Npun_R4582 relative mRNA levels was detected, while in cells treated with Co, Cu, Mn, Ni, or Zn and 60 μM peroxide, relative mRNA levels were generally above control or peroxide only treated cells. Disruption or overexpression of npun_R4582 altered sensitivity to cells exposed to 60 μM H₂O₂ and metals for treatments beyond the highest viable concentrations, or in a mixed metal solution for Npun_R4582⁻ cells. Moreover, overexpression of npun_R4582 increased cellular peroxidase activity in comparison with wild-type and Npun_R4582⁻ cells, and reduced peroxide levels by over 50%. The addition of cobalt, manganese, nickel, and zinc increased the capacity of Npun_R4582 to reduce the rate or total levels of peroxide produced by cells growing under photooxidative conditions. The work presented confirms the function of NpunR4582 as a catalase and provides insights as to how cells reduce potentially lethal peroxide levels produced by photosynthesis. The findings also show how trace elements play crucial roles as enzymatic cofactors and how the role of Npun_R4582 in hydrogen peroxide breakdown is dependent on the type of metal and the level available to cells.

     

Hybridization between diploid Centaurea pseudophrygia and tetraploid C. jacea (Asteraceae): the role of mixed pollination,unreduced gametes,and mentor effects

We studied hybridization between the diploid Centaurea pseudophrygia and the tetraploid C. jacea by performing crossing experiments and screening natural populations using flow cytometry. The experiments confirm that the studied species exhibit strong reproductive isolation. Interspecific hybrids were formed at a low frequency, including triploids (originating from reduced gametes) and tetraploids (involving unreduced gametes of the diploids). In contrast, hybrids were almost absent among seeds and adult plants of natural mixed populations and among the offspring from experimental pollinations with a mixture of pollen of both ploidy levels. We found that mixed pollination is an important mechanism for preventing hybridization between plants of different ploidy levels and sustaining the reproduction of the tetraploids. A mentor effect (induced selfing in the presence of pollen of different ploidy levels) was observed in both diploids and tetraploids, reinforcing the reproductive isolation between cytotypes. Higher ploidy levels (pentaploid, hexaploid) involving unreduced gametes of the tetraploid species were identified. Notably, pentaploids were discovered for the first time in Centaurea sect. Jacea. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 93–106.     

重组hPRL-1的表达纯化及其理化和酶学性质研究

目的:表达纯化hPRL-1重组蛋白,分析其理化性质及酶学特性。方法:热激法将重组pET15b质粒转化入E.coli BL21中,IPTG诱导表达出His-tagged hPRL-1蛋白。使用Ni-NTA亲和层析法结合Mono Q离子交换层析法纯化。用SDS-PAGE法和Western Blot法进行表达情况的定性定量分析,并使用HPLC法鉴定蛋白纯度,计算出蛋白分子量,圆盘等电聚焦电泳分析重组蛋白等电点。比较分析以pNPP、4-MUP和DiFMUP为底物时的酶促反应动力学。同时以pNPP为底物测定酶的最适pH值;以4-MUP为底物测定酶的最适温度,分析探讨缓冲液离子强度与蛋白酪氨酸酶通用抑制剂钒酸钠对酶活力的影响。结果:以亲和层析和离子交换层析结合,可以纯化得到纯度约为95%的蛋白。测得蛋白分子量为24.54kD,等电点为9.11。以pNPP、4-MUP和DiFMUP为底物时Km分别为3720μmol/L,130μmol/L和50μmol/L。酶的最适pH值为7.6,最适温度为34℃。结论:纯化所得蛋白为目的蛋白hPRL-1;两步纯化相结合可以得到纯度较高的蛋白;三种底物特异性依次为DiFMUP>4-MUP>pNPP。     

以MyD88 TIR二聚化为靶点的抗炎抑制剂筛选模型的建立

MyD88是IL-1R/TLR受体超家族向细胞内转导胞外信号时募集到受体胞浆尾部的重要接头蛋白．由TIR结构域介导的MyD88分子同源二聚化是它招募到受体胞浆尾部的前提,然后二聚化的MyD88再募集下游信号分子,传递信号,引发促炎基因的表达．本研究旨在建立一种模型,以实现活细胞原位的、基于荧光信号变化的MyD88二聚化抑制物的高通量筛选．我们分别构建了MyD88 TIR与GFP和RFP的融合蛋白表达质粒,瞬时转染HeLa细胞,在488 nm激发光下,转染GFP-MyD88 TIR和RFP-MyD88 TIR细胞,检测到绿色荧光与红色荧光间的共振能量转移(FRET)．而当细胞转染GFP-MyD88 TIR和RFP或RFP-MyD88 TIR和GFP,因TIR二聚化不能实现,FRET效率受到严重影响．实验结果提示,依赖双阳性表达GFP-MyD88 TIR和RFP-MyD88 TIR的细胞株,检测不同化合物对于荧光FRET效率的影响,可以建立MyD88 TIR二聚化抑制药物的筛选模型．此外,我们构建了原核表达质粒,利用纯化的His-MyD88 TIR分别与GST或GST-MyD88 TIR蛋白进行体外结合实验,发现GST-MyD88 TIR(而非GST)可以与His-MyD88 TIR相互结合．结果的差异性提示,利用His-MyD88 TIR和GST-MyD88 TIR体外结合实验分析,可以进一步确定抑制物是否直接阻断了TIR的相互作用．结合真核细胞的荧光FRET阻断结果和原核表达的重组蛋白相互作用分析,可确定MyD88 TIR二聚化的抑制物．利用这一模型可以对商品化的小分子库、自行制备的天然产物组分进行广泛的筛选,从中获得有效抑制MyD88二聚化的化合物,参与对MyD88信号通路依赖的慢性炎症、自身免疫性疾病的药物治疗．