Resistance to beta-lactam antibiotics by re-modelling the active site of an E. coli penicillin-binding protein

The beta-lactam antibiotics kill bacteria by inhibiting a set of penicillin-binding proteins (PBPs) that catalyse the final stages of peptidoglycan synthesis. In some bacteria the development of intrinsic resistance to beta-lactam antibiotics by the reduction in the affinity of PBPs causes serious clinical problems. The introduction of beta-lactam antibiotics that are resistant to hydrolysis by beta-lactamases may also result in the emergence of intrinsic resistance among the Enterobacteriaceae. The clinical problems that would arise from the emergence of resistant PBPs in enterobacteria have led us to examine the ease with which Escherichia coli can gain resistance to beta-lactams by the production of altered PBPs. The development of resistant PBPs also provides an interesting example of enzyme evolution, since it requires a subtle re-modeling of the enzyme active centre so that it retains affinity for its peptide substrate but excludes the structurally analogous beta-lactam antibiotics. We show here that only four amino-acid substitutions need to be introduced into PBP 3 of E. coli to produce a strain possessing substantial levels of resistance to a wide variety of cephalosporins. We also show that transfer of the gene encoding the resistant PBP 3 from the chromosome to a plasmid could result in the spread of intrinsic resistance not only to other strains of E. coli but also to other enterobacterial species.     

弹性蛋白酶抑制剂筛选方法的建立及天然产物对其抑制活性观察

发现弹性蛋白酶抑制剂是治疗肺气肿等疾病药物的重要研发方向之一.本研究用弹性蛋白酶建立微量、高效的体外抑制剂筛选模型,利用西维来司钠作为阳性对照,验证了筛选模型的稳定性和灵敏度,并筛选了33种中药单体对弹性蛋白酶的抑制活性,其中部分化合物表现一定的抑制活性.     

A linguistic model for the rational design of antimicrobial peptides

Antimicrobial peptides (AmPs) are small proteins that are used by the innate immune system to combat bacterial infection in multicellular eukaryotes. There is mounting evidence that these peptides are less susceptible to bacterial resistance than traditional antibiotics and could form the basis for a new class of therapeutic agents. Here we report the rational design of new AmPs that show limited homology to naturally occurring proteins but have strong bacteriostatic activity against several species of bacteria, including Staphylococcus aureus and Bacillus anthracis. These peptides were designed using a linguistic model of natural AmPs: we treated the amino-acid sequences of natural AmPs as a formal language and built a set of regular grammars to describe this language. We used this set of grammars to create new, unnatural AmP sequences. Our peptides conform to the formal syntax of natural antimicrobial peptides but populate a previously unexplored region of protein sequence space.     

Crystallographic study of a beta-lactam inhibitor complex with elastase at 1.84 A resolution

The continuing discovery and development of beta-lactams as antibiotics has had an unparalleled impact on the overall health and well-being of society. Recently, appropriately substituted cephalosporins were shown to be potent inhibitors of elastase, suggesting a novel therapeutic role for the beta-lactams in the control of emphysema and other degenerative diseases. We have now solved and partially refined at atomic resolution the structure of a complex of porcine pancreatic elastase with the time-dependent irreversible inhibitor 3-acetoxymethyl-7-alpha-chloro-3-cephem-4-carboxylate-1,1-dioxide tert-butyl ester (I), the most potent of the beta-lactam elastase inhibitors yet reported. (Porcine pancreatic elastase is a close relative of the desired drug target, human polymorphonuclear leukocyte elastase.) A mechanism of action is presented, based on the structure and on biochemical evidence (T.-Y.L. et al., in preparation), which clarifies the operational similarities and differences between beta-lactam elastase inhibitors and antibiotics. Features of the reaction include the expulsion of a leaving group at the cephalosporin 3' position and the formation of two covalent bonds with the active site of porcine pancreatic elastase at residues Ser 195 and His 57.     

Refined crystal structure of beta-lactamase from Citrobacter freundii indicates a mechanism for beta-lactam hydrolysis

Beta-Lactamases (EC 3.5.2.6, 'penicillinases') are a family of enzymes that protect bacteria against the lethal effects of cell-wall synthesis of penicillins, cephalosporins and related antibiotic agents, by hydrolysing the beta-lactam antibiotics to biologically inactive compounds. Their production can, therefore, greatly contribute to the clinical problem of antibiotic resistance. Three classes of beta-lactamases--A, B and C--have been identified on the basis of their amino-acid sequence; class B beta-lactamases are metalloenzymes, and are clearly distinct from members of class A and C beta-lactamases, which both contain an active-site serine residue involved in the formation of an acyl enzyme with beta-lactam substrates during catalysis. It has been predicted that class C beta-lactamases share common structural features with D,D-carboxypeptidases and class A beta-lactamases, and further, suggested that class A and class C beta-lactamases have the same evolutionary origin as other beta-lactam target enzymes. We report here the refined three-dimensional structure of the class C beta-lactamase from Citrobacter freundii at 2.0-A resolution and confirm the predicted structural similarity. The refined structure of the acyl-enzyme formed with the monobactam inhibitor aztreonam at 2.5-A resolution defines the enzyme's active site and, along with molecular modelling, indicates a mechanism for beta-lactam hydrolysis. This leads to the hypothesis that Tyr 150 functions as a general base during catalysis.     

平板影印与AHBA合成酶基因筛选用于安莎类抗生素产生菌的分离

基因筛选是筛选具有抗生素产生潜力微生物的新方法.针对筛选中单菌落分离纯化不仅工作量大,而且耗费时间,采用一种将平板影印技术与PCR扩增技术相结合从土壤中快速获得具有安莎类抗生素产生潜力的放线菌的方法.根据安莎类抗生素合成途径中的3-氨基-5-羟基苯甲酸合成酶(AH BAs)基因的保守性,通过放线菌的培养、影印、AH-BAs基因的PCR扩增,已从33份土样中获得8株AHBAs阳性菌株.结果表明:该方法是一种非常有效的能够快速获得AHBAs基因阳性菌株的方法,并且该方法可扩展用于从土壤中分离其他有价值的抗生素产生菌.     

A new type of synthetic peptide library for identifying ligand-binding activity

Our aim was to improve techniques for drug development by facilitating the identification of small molecules that bind with high affinity to acceptor molecules (for example, cell-surface receptors, enzymes, antibodies) and so to mimic or block their interaction with the natural ligand. Previously such small molecules have been characterized individually on a serial basis. The systematic synthesis and screening of peptide libraries of defined structure represents a new approach. For relatively small libraries, predetermined sequence variations on solid-phase supports have been used, and large libraries have been produced using a bacteriophage vector into which random oligodeoxynucleotide sequences have been introduced, but these techniques have severe limitations. Here we investigate an alternative approach to synthesis and screening of peptide libraries. Our simple methodology greatly enhances the production and rapid evaluation of random libraries of millions of peptides so that acceptor-binding ligands of high affinity can be rapidly identified and sequenced, on the basis of a 'one-bead, one-peptide' approach.     

电化学研究亚甲蓝与DNA的相互作用

The interaction of small molecules with DNA is of interest because it is important in the design of new and more efficient drugs targeted to DNA. The metal complexes, natural antibiotics, and a lot of other planar heterocyclic cations, have been investigated for their DNA     

来源于天然产物的α-葡萄糖苷酶抑制剂筛选研究进展概述

α-葡萄糖苷酶抑制剂是一种具有糖结构的新型抗糖尿病药物,它通过与α-葡萄糖苷酶发生竞争性抑制作用而达到降低餐后血糖的目的.目前以天然植物和药物为来源的α-葡萄糖苷酶抑制剂研究较多,现对以天然植物为来源的α-葡萄糖苷酶抑制剂的筛选国内外研究进展进行综述.     

GPR41稳定细胞株的建立及受体激动剂筛选

构建GPR41稳定细胞株,从RNA、蛋白水平验证了GPR41的表达并利用cAMP和钙流检测验证了GPR41的生物活性.实验结果表明,该细胞株可以用于筛选受体激动剂.从海藻来源的黄曲霉中提取到的次级代谢产物用于筛选GPR41的激动剂.实验结果还表明,2-吡喃酮类化合物(37号)在1μmol/L浓度条件下即可具有GPR41受体激动活性.这是首次报道2-吡喃酮类化合物具有GPR41受体激动活性.     

Tertiary structural similarity between a class A beta-lactamase and a penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase

beta-Lactam antibiotics--the penicillins, cephalosporins and related compounds--act by inhibiting enzymes that catalyse the final stages of the synthesis of bacterial cell walls. Recent crystallographic studies of representative enzymes are beginning to reveal the structural bases of antibiotic specificity and mechanism of action, while intensive efforts are being made to understand the beta-lactamase enzymes that are largely responsible for bacterial resistance to these antibiotics. It has been suggested that the beta-lactamases and beta-lactam target enzymes may be evolutionarily related and some similarity of amino-acid sequence around a common active-site serine residue supports this idea. We present here the first evidence from a comparison of three-dimensional structures in support of this hypothesis: the structure of beta-lactamase I from Bacillus cereus is similar to that of the penicillin-sensitive D-alanyl-D-alanine carboxypeptidase-transpeptidase from Streptomyces R61.     

日本和美国的基础设施管理经验及教训研究(英文)

经过20世纪后期大量的建设后,面临着诸如桥梁破坏等新问题。同时,由于全球气候变暖现象,21世纪的基础设施管理制度必须建立在符合总成本效益比最小和防止全球变暖的条件下。文章介绍了日本和美国对社会基础设施的一些管理制度和经验。     

A transglutaminase homologue as a condensation catalyst in antibiotic assembly lines

The unrelenting emergence of antibiotic-resistant bacterial pathogens demands the investigation of antibiotics with new modes of action. The pseudopeptide antibiotic andrimid is a nanomolar inhibitor of the bacterial acetyl-CoA carboxylase that catalyses the first committed step in prokaryotic fatty acid biosynthesis. Recently, the andrimid (adm) biosynthetic gene cluster was isolated and heterologously expressed in Escherichia coli. This establishes a heterologous biological host in which to rapidly probe features of andrimid formation and to use biosynthetic engineering to make unnatural variants of this important and promising new class of antibiotics. Bioinformatic analysis of the adm cluster revealed a dissociated biosynthetic assembly system lacking canonical amide synthases between the first three carrier protein domains. Here we report that AdmF, a transglutaminase (TGase) homologue, catalyses the formation of the first amide bond, an N-acyl-beta-peptide link, in andrimid biosynthesis. Hence, AdmF is a newly discovered biosynthetic enzyme that acts as a stand-alone amide synthase between protein-bound, thiotemplated substrates in an antibiotic enzymatic assembly line. TGases (enzyme class (EC) 2.3.2.13) normally catalyse the cross-linking of (poly)peptides by creating isopeptidic bonds between the gamma-carboxamide group of a glutamine side chain of one protein and various amine donors, including lysine side chains. To the best of our knowledge, the present study constitutes the first report of a TGase-like enzyme recruited for the assembly of an antibiotic. Moreover, genome mining using the AdmF sequence yielded additional TGases in unassigned natural product biosynthetic pathways. With many more microbial genomes being sequenced, such a strategy could potentially unearth biosynthetic pathways producing new classes of antibiotics.     

海藻共附生真菌活性天然产物研究进展

海藻共附生真菌次级代谢产物结构类型多样、骨架新颖、活性丰富,为新型海洋药物先导化合物的发现提供了宝贵的来源。本文以生物活性对海藻共附生真菌来源的天然产物进行分类,从抗菌、抗肿瘤、抗氧化、抗炎、浮游生物抑制等方面,对2006年以来报道的海藻共附生真菌来源的112个活性天然产物进行概述,为海藻及其共附生微生物的进一步研究与开发提供参考。     

规范化自然梯度ICA算法

对基于李群不变性的自然梯度ICA算法进行了改进,提出了一种规范化自然梯度ICA算法.该算法通过引入规范化因子,保证参数矩阵的行列式的绝对值在学习过程中恒为1,避免了参数矩阵剧烈变化,使得学习过程更稳定更快速,这种改进还起到简化目标函数的作用,使得规范化自然梯度ICA算法更加简单便利.在BSS模拟实验中,把常规梯度的ICA算法、自然梯度ICA算法与规范化自然梯度ICA算法进行比较,结果表明新算法的信号恢复精度更高,收敛速度更快.     

食用菌栽培中常见杂菌有效杀菌剂的筛选

为了解决食用菌生产中杂菌污染问题。我们从19种杀菌剂和抗生素中筛选出粉锈宁有较强的抗霉菌作用,并对其抗菌范围及抑菌的有效剂量进行初步的探讨。本文为食用菌栽培防治杂菌污染提供了依据。     

Genetic basis of fitness differences in natural populations

Genomics profoundly influences current biology. One of many exciting consequences of this revolution is the potential for identifying and studying the genetic basis of those traits affecting fitness that are key to natural selection. Recent studies using a multitude of genomic approaches have established such genotype-phenotype relationships in natural populations, giving new insight into the genetic architecture of quantitative variation. In parallel, an emerging understanding of the quantitative genetics of fitness variation in the wild means that we are poised to see a synthesis of ecological and molecular approaches in evolutionary biology.     

关于自然资源与环境可持续发展的认识论飞跃

人类满足于物欲,片面追求消费,对物质和金钱的关爱远远胜过对自然和环境的关爱,人类无情地向自然索取,破坏自然,使自然生态环境千疮百孔。我国自然资源的大量耗损,生态环境的恶化,严重地影响了人类生存和生活的质量。人类在充分享受现代物质文明的同时,饱尝着资源匮乏和环境灾难酿成的苦果,人类社会长期以来形成的价值观在新文明的冲击下面临危机。环境道德:人类的普遍道德,在本世纪自然资源和环境可持续发展中将发挥着重要的作用。人与自然的和谐与发展,珍爱生命,珍视对资源和环境的保护,是人类对于人与自然、环境关系的新发展。     

从链霉菌中发现新抗生素的趋势分析

到目前为止，链霉菌是微生物中产生抗生素最多的菌种，据报道，从20世纪40年代后期到70年代，每年由链霉菌产生的抗生素几乎呈现指数增长，并在20世纪70年代达到最高峰，20世纪80年代后期到90年代增加幅度下降。收集到的数据显示一条S形曲线，比对数方程预测的曲线要平缓得多，在不断优化参数后为这条曲线建立1个较好的数学模型，根据这一模型可以估计链霉菌中还有多少没有被发现的抗生素，同时也便于预测不远的将来新抗生素产生的趋势。此模型估计在这类菌种中能产生的抗菌化合物的总数量约100000多种，而这只是迄今为止未发现抗生素中很微小的一部分。曲线中斜率的减少是由于筛选方法的减少，而不是由于新抗生素的枯竭，如果这种趋势任其发展下去，在近10～20年内斜率将会趋近于零，但如果不断地探索新的筛选方法，在未来的几十年里发现新抗生素的速度是不会下降的。