Specificity of Pyrrolysyl-tRNA Synthetase for Pyrrolysine and Pyrrolysine Analogs

Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG = UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA^Pyl for amber decoding as pyrrolysine. PylS and tRNA^Pyl have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K_m. The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA^Pyl was similar to in vitro results, with l-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.     

Development of software for human muscle force estimation

Muscle force estimation (MFE) has become more and more important in exploring principles of pathological movement, studying functions of artificial muscles, making surgery plan for artificial joint replacement, improving the biomechanical effects of treatments and so on. At present, existing software are complex for professionals, so we have developed a new software named as concise MFE (CMFE). CMFE which provides us a platform to analyse muscle force in various actions includes two MFE methods (static optimisation method and electromyographic-based method). Common features between these two methods have been found and used to improve CMFE. A case studying the major muscles of lower limb of a healthy subject walking at normal speed has been presented. The results are well explained from the effect of the motion produced by muscles during movement. The development of this software can improve the accuracy of the motion simulations and can provide a more extensive and deeper insight in to muscle study.     

西部高寒湿地系统的草地资源保护与优化利用模式研究

西部高寒湿地系统的草地资源保护与优化利用问题,是我国生态环境建设中倍受重视的科学问题。本研究以甘南的高寒湿地为研究对象,分析了高寒草地资源变化的时空分布特征及其在环境条件影响下的增长动态,采用生物控制论方法,组建了草地资源保护模型及草地放牧系统的最优控制模型。依据在甘南玛曲县高寒草地的调查结果,分别对其山地草原化草甸、山地典型草甸和沼泽草甸等草地资源的利用,建立了相应的保护指标。确定了该草地放牧系统的最优牧草资源水平和最优控制量,提出了草地资源优化利用的管理对策与措施,为草地放牧管理提供了优化模式和定量依据。     

mRNA差别显示技术及其在生命科学中的应用

ｍＲＮＡ表达水平的变化决定细胞的功能状态,个体发育、细胞增殖分化与凋谢、生理刺激和药物治疗等过程,都会出现ｍＲＮＡ 表达水平的变化。阐明这种变化有助于揭示细胞生理过程的分子机制。该技术无需更多的背景资料,可快速有效地分离表达水平出现差别的基础,这些基因编剧编码的功能多肽在细胞生理过程中扮演着重要角色。     

Investigation of a His-rich arabinogalactan-protein for micronutrient biofortification of cereal grain

The micronutrient content of most cereal grains is low and responsible for malnutrition deficiencies in millions of people who rely on grains as their primary food source. Any strategy that can increase the micronutrient content of grain will have significant benefits to world health. We identified a gene from barley encoding a cell wall protein with multiple histidine (His)-rich motifs interspersed with short arabinogalactan-protein (AGP) domains and have called it Hordeum vulgare His-rich AGP (HvHRA1). Sequence analysis shows that His-rich AGPs are rare in plants and that the number of His-rich and AGP domains differ between cereals and dicots. The barley and wheat encoded proteins have more than 13 His-rich domains, whereas the putative rice orthologue has only 5 His-rich regions. His-rich motifs are well-established metal-binding motifs; therefore, we developed transgenic (Tx) rice plants that constitutively overexpress barley HvHRA1. There was no significant effect on plant growth or grain yield in Tx plants. Purification of AGPs from wild-type and Tx plants showed that only Tx plants contained detectable levels of a His-rich AGP. Calcein assay shows that the AGP fraction from Tx plants had increased binding affinity for Cu(2+) . Micronutrient analysis of brown and white rice showed that the grain nutrient yield for Fe, Zn and Cu was higher in two Tx lines compared to their respective nulls, although the differences were not statistically significant. This approach highlights the potential of the plant apoplast (cell wall) for storage of key nutrients through overexpression of genes for metal-binding proteins.     

中国沿海浒苔属rbcL和ITS序列比较及系统进化分析

为阐明中国沿海浒苔的亲缘关系及地理分布特点,采集青岛栈桥、盐城弶港、宁波象山、温州平阳四地浒苔样本,克隆测序得到ITS1、5.8SrDNA和ITS23种不同长度序列片段。四个地区的rbcL目的片段,长度均为1201bp。分析核苷酸差异和遗传距离,采用邻接法建立系统发生树。结果显示,ITS序列进化速率较快,rbcL序列相当保守。ITS区较短,GC含量均在65%以上,5.8SrDNA的CG含量在50%左右,ITS1区的序列差异大于ITS2区。四个地区的浒苔存在一定的地理差异,盐城和青岛的样本间的亲缘关系较近;宁波和温州的样本间的亲缘关系较近。石莼属(Ulva)和浒苔属(Enteromorpha)的物种没有聚成各自独立的分枝,而是相互混合在一起,应是两个亲缘关系相近的属。引起青岛绿潮的海藻很可能是来自盐城海域的Enteromorpha linza或Enteromorpha prolifera。     

Preparation and characterization of magnetic microspheres for the purification of interferon alpha-2b

Magnetic agarose microspheres (MAMS), magnetic cellulose microspheres (MCMS), and magnetic poly(vinyl alcohol) microspheres (MPVAMS) were prepared by various different preparation methods. MCMS coupled with anti-IFN alpha-2b monoclonal antibodies (mAb) were selected for the purification of interferon alpha-2b (IFN alpha-2b) after performance characterization among microspheres. Parameters of immunomagnetic separation (IMS), including binding mAb, elution behavior, and sample pretreatment conditions, were optimized to improve the purification efficiency of the separation of IFN alpha-2b by MCMS. Size-exclusion HPLC (HPSEC) showed that the IFN alpha-2b was purified from crude cell lysate had an overall purity of 92.9%, while immunological and biological assays showed an activity recovery of 88.5% and specific antiviral activity of 2.7 x 10(8) IU/mg. Identity and molecular mass of purified IFN alpha-2b were confirmed by western blot and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. This study illustrated the favorable separation media which combined desired properties for the development of magnetic separation of biological materials.     

基因组数据库简介

本文以北京大学生物信息中心安装的3个国际著名基因组数据库GDB、GenoList和Ensembl为基础,介绍目前常用的基因组数据库,包括这些数据数据库的内容、数据格式、使用方法,以及用于构建上述数据库的数据库管理系统。 Abstract:A brief introduction to the genome databases GDB,GenoList and Ensembl is given.These databases,mirrored and maintained at the Centre of Bioinformatics,Peking University,provide useful information for genome research.     

Blockade of L-type voltage-gated Ca channel inhibits ischemia-induced neurogenesis by down-regulating iNOS expression in adult mouse

Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. The signals that regulate neurogenesis in the dentate gyrus following ischemic stroke insult are not well known. We have previously reported that inducible nitric oxide synthase (iNOS) expression is necessary for ischemia-stimulated neurogenesis in the adult dentate gyrus. Here, we show that mice subjected to 90 min of middle cerebral artery occlusion (MCAO) significantly increased the number of new neurons and up-regulated iNOS expression in the dentate gyrus. Blockade of the L-type voltage-gated Ca(2+) channel (L-VGCC) prevented neurogenesis in the dentate gyrus and subventricular zone (SVZ), and down-regulated iNOS expression in the dentate gyrus after cerebral ischemia. This study suggests that Ca(2+) influx through L-VGCC is involved in ischemia-induced neurogenesis by up-regulating iNOS expression.     

钙激活钾通道在黎芦碱致神经细胞损伤中的作用

目的:观察新生SD大鼠原代培养皮层神经元的钙激活钾通道(Kca)在黎芦碱致神经元损伤模型上的激活、抑制效应.方法:采用细胞贴附和内面向外两种膜片钳单通道记录方法记录新生SD大鼠原代培养皮层神经元的Kca电生理活动.结果:黎芦碱在胞外可激活Kca.在有钙浴液内,细胞贴附式,钳制膜电位 30 mV,加入不同浓度黎芦碱(μmol/L:15、25、50、75),通道开放概率由0.005分别增加为0.014±0.003、0.085±0.010、0.132±0.016、0.059±0.006(P＜0.01),在50μmol/L以内表现出浓度依赖性.无钙浴液内,细胞贴附式膜片上,钳制膜电位 50 mV,随药物浓度(μmol/L)增加为15、40、60、100时,通道开放概率由0.005分别增加为0.014±0.010、0.113±0.006、0.141±0.004、0 295±0.009(P＜0.05).6例内面向外式膜片上,钳制膜电位 40 mV,分别加入黎芦碱25 μmol/L、50μmol/L 3 min后,通道开放概率由0.011±0.008分别增加为0.010±0.010、0.012±0.007(P＞0.05).黎芦碱在胞内Kca开放概率,平均开放/关闭时间,电流幅值均无明显变化.结论:黎芦碱通过影响胞内游离钙水平间接调节Kca,在缺血缺氧早期,胞内游离钙增高激活Kca开放.