Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)_n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.     

Complete Plastid Genome of the Brown Alga Costaria costata (Laminariales,Phaeophyceae)

Costaria costata is a commercially and industrially important brown alga. In this study, we used next-generation sequencing to determine the complete plastid genome of C. costata. The genome consists of a 129,947 bp circular DNA molecule with an A+T content of 69.13% encoding a standard set of six ribosomal RNA genes, 27 transfer RNA genes, and 137 protein-coding genes with two conserved open reading frames (ORFs). The overall genome structure of C. costata is nearly the same as those of Saccharina japonica and Undaria pinnatifida. The plastid genomes of these three algal species retain a strong conservation of the GTG start codon while infrequently using TGA as a stop codon. In this regard, they differ substantially from the plastid genomes of Ectocarpus siliculosus and Fucus vesiculosus. Analysis of the nucleic acid substitution rates of the Laminariales plastid genes revealed that the petF gene has the highest substitution rate and the petN gene contains no substitution over its complete length. The variation in plastid genes between C. costata and S. japonica is lower than that between C. costata and U. pinnatifida as well as that between U. pinnatifida and S. japonica. Phylogenetic analyses demonstrated that C. costata and U. pinnatifida have a closer genetic relationship. We also identified two gene length mutations caused by the insertion or deletion of repeated sequences, which suggest a mechanism of gene length mutation that may be one of the key explanations for the genetic variation in plastid genomes.     

Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library

A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.     

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10⁶ to 10¹⁰ members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.     

草原沙漠化过程中植物的耐胁迫类型研究

以内蒙古锡林郭勒盟多伦县草原沙漠化过程中不同梯度共有种群为研究对象 ,探讨植物的受损机理 ,综合 2 0 0 1～ 2 0 0 3年的研究结果表明 :随着沙漠化的进展 ,共有种群的 1叶片含水量及叶绿素 a、b和总叶绿素含量均呈降低趋势。叶片含水量高低顺序为扁蓿豆 (Melilotoides ruthenica) >冷蒿 (Artemisia frigida) >羊草 (L eymus chinensis) >糙隐子草 (Cleistogenessquarrosa) ,反映了共有种群水分状况的不同。2质膜相对透性、游离脯氨酸含量均呈上升趋势 ,其中羊草、糙隐子草和冷蒿变化表现出先升后降再升的规律。3MDA含量变化均表现出先升后降再升的规律 ,不同植物不同阶段 MDA积累程度 (积累率大小顺序为沙漠化初期 :冷蒿 2 9.5 9% >羊草 18.14 % >扁蓿豆 13.6 8% >糙隐子草 3.77% ;沙漠化后期 :糙隐子草 80 .11% >冷蒿 77.2 9% >羊草 39.31% >扁蓿豆 8.5 6 % )的不同反映了不同阶段细胞受伤害程度的差异。4 SOD、CAT活性变化总体上均呈上升趋势 ,其中羊草 CAT活性呈降低趋势 ;酶活性均梯度间差异显著 (P<0 .0 0 1)。 5内源激素 ABA含量的变化因不同种群抗逆性强弱而异。糙隐子草、冷蒿和扁蓿豆 ABA增幅小 ,而羊草 ABA增幅大。综合各种生理特征及其对沙漠化环境的响应 ,对共有种群分类 :羊草为敏感型     

Kinetic resolution of ibuprofen catalyzed by Candida rugosa lipase in ionic liquids

Candida rugosa lipase-catalyzed esterification of ibuprofen with 1-propanol was conducted in seven ionic liquids and the results were compared with those in isooctane. Although the enzyme showed comparable or higher activity in some ionic liquids compared to that in isooctane, only in the case of [BMIM]PF6 was the enantioselectivity (E = 24.1) almost twice that (E = 13.0) of isooctane. In another six ionic liquids the enzyme enantioselectivity was much poorer (E = 1.1-6.4). At the same conversion of 30%, E of [BMIM]PF6 is more than triple that of isooctane. The lipase stability in [BMIM]PF6 was improved by 25% of that in isooctane. It was concluded that [BMIM]PF6 could be applied to substitute the conventional organic solvent (isooctane) in the esterification of ibuprofen.     

Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example

Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1–2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.     

百里香无性系的克隆生长特征

植物克隆生长及其与生态适应性的关系是当今植物种群生态学研究的热点和前沿课题,但目前小半灌木克隆生长的研究开展不多。百里香(Thymus serpyllum var. asiaticus)是一种具有地面匍匐茎的草本状小半灌木,可在土壤侵蚀剧烈、基岩大面积裸露的砒砂岩区形成百里香单优群落,在维持生态系统稳定方面具有重要的生态学作用。皇甫川流域是砒砂岩大面积分布的典型区域,在这一地区对百里香无性系的克隆生长进行研究,不仅具有重要的学术价值,而且在生态环境建设方面也具有一定的现实意义。在皇甫川流域选择含三级分株的百里香无性系,对其各级分株的总生物量、各构件生物量及数量、各构件生物量占总生物量的百分比及其月变化进行了研究。结果表明: 1)母株与子代相比,在总生物量、构件生物量及数量上占有绝对优势,而且具有体型大、结构复杂的特点; 2)对生物量分配格局的研究显示,母株根的生物量在总生物量中所占的比例最大,其叶所占的比例较低。子代叶的生物量在总生物量中所占的比例最大,其根所占的比例较低;3)不同级别分株在生物量分配上的差异,揭示了相互连接的分株在功能上的差别,母株可能更侧重于养分和水分的吸收,子株则更侧重于光合生产;4)构件枝、茎、花生物量分配比月变化显示,子1代各构件的生长规律与母株的基本一致,子2代与母株和子1代的相比差异较大,分析认为这可能是分株间不同程度的生理整合作用造成的结果。