Effect of oxygen on steady-state product distribution in Bacillus polymyxa fermentations

Bacillus polymyxa ferments glucose to 1-2,3 butanediol, acetoin, ethanol, acetic acid, lactic acid, and formic acid. This research investigates product formation as a function of oxygen availability. A predictive model that simulates product distribution at known oxygen transfer rates is developed on the hypothesis that, in an energy-limited environment, B. polymyxa utilizes glucose and oxygen in the most efficient manner. The efficiency of utilization of glucose and oxygen is measured in terms of the ATP yields of each oxidative pathway. The identity of the products constituting the profile at the given oxygen transfer rate is determined by comparing the ATP production and consumption rates. While the ATP generated is calculated from a knowledge of the oxygen transfer rate and ATP yields of the oxidative pathways, the ATP consumption is estimated by the Pirt expression in terms of growth- and nongrowth-associated components. The product formation rates are obtained by solving ATP and NAD balance equations. They equate the production and consumption rates of these intermediates and are derived from the pseudo-steady-state hypothesis. The model is applied to continuous culture systems that are both open and closed with respect to biomass. At a given oxygen transfer rate, dilution rate, and inlet glucose concentration, the model predicts steady-state concentrations of two dominant fermentation endproducts with the help of four parameters that can be determined from independent experiments. In contrast with earlier approaches, the experimental studies are carried out in continuous culture. Product profiles are obtained at various oxygen transfer rates, fer rates, inlet glucose concentrations, and dilution rates. The effect of pH on the relative distribution of products is also demonstrated. Results indicate that the model is fairly successful in predicting product profiles as a function of oxygen availability. (c) 1992 John Wiley & Sons, Inc.     

Interaction of aromatic ionen oligomers with DNA

A novel polycationic ionen was synthesized and fractionated on carboxymethyl-Sephadex using a salt gradient in 7M urea. A series of oligomers of discrete length were characterised by ultraviolet spectra. The ultraviolet spectra of oligomers revealed a new band centred at 232.5 nm which was probably due to exciton splitting. Thermal denaturation studies indicated both stabilization of the helix conformation and a higher degree of cooperativity in the melting of DNA (oligomers)n complex as compared to native calf thymus DNA. Ionen oligomers exhibited large extrinsic Cotton effect at 232.5 nm which could be attributed to exciton interaction.     

Mass spectrometric characterization of the isoforms in Escherichia coli recombinant DNA-derived interferon alpha-2b

The isoforms Iso-2, Iso-3, and Iso-4 of Escherichia coli-derived recombinant human interferon alpha-2b (rhIFN α-2b), generated by posttranslational modifications of the protein during fermentation, present a major problem in terms of purification and the yield of the drug substance. We report here the structural characterization of these isoforms by mass spectrometry (MS) methods. An extensive MS study was conducted on Iso-4, which is composed of up to 75% of the in-process IFN, and on the native rhIFN α-2b. The trypsin-digested peptide mixtures generated from the two samples were analyzed by liquid chromatography (LC)–MS, and targeted peptides were further studied by LC–tandem MS (triple quadrupole mass spectrometer), high-resolution MSⁿ (LTQ Orbitrap), and matrix-assisted laser desorption/ionization MS (MALDI–MS). The structure of Iso-4 was elucidated as a novel pyruvic acid ketimine derivative of the N-terminal cysteine (Cys1) of IFN α-2b, where the disulfide bond between Cys1 and Cys98 was fully reduced and the other disulfide bond pair, Cys29-ss-Cys138, was partially reduced. Similarly, Iso-2 was identified as a correctly disulfide-folded rhIFN α-2b with acetylation on Cys1, and Iso-3 was identified as an S-glutathionylated form (Cys98) of partially reduced rhIFN α-2b that was pyruvated on Cys1. Based on the characterization work, a reproducible conversion procedure was successfully implemented to convert Iso-4 to rhIFN α-2b.     

Alignments grow, secondary structure prediction improves.

Using information from sequence alignments significantly improves protein secondary structure prediction. Typically, more divergent profiles yield better predictions. Recently, various groups have shown that accuracy can be improved significantly by using PSI-BLAST profiles to develop new prediction methods. Here, we focused on the influences of various alignment strategies on two 8-year-old PHD methods. The following results stood out. (i) PHD using pairwise alignments predicts about 72% of all residues correctly in one of the three states: helix, strand, and other. Using larger databases and PSI-BLAST raised accuracy to 75%. (ii) More than 60% of the improvement originated from the growth of current sequence databases; about 20% resulted from detailed changes in the alignment procedure (substitution matrix, thresholds, and gap penalties). Another 20% of the improvement resulted from carefully using iterated PSI-BLAST searches. (iii) It is of interest that we failed to improve prediction accuracy further when attempting to refine the alignment by dynamic programming (MaxHom and ClustalW). (iv) Improvement through family growth appears to saturate at some point. However, most families have not reached this saturation. Hence, we anticipate that prediction accuracy will continue to rise with database growth.     

Identifying protein construct variants with increased crystallization propensity--a case study

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.     

双向电泳结合质谱分析长双歧杆菌XY01分泌蛋白质图谱

菌体的分泌蛋白质在宿主和菌体的相互作用之间起着重要的作用. 本研究采用双向凝胶电泳的方法建立了长双歧杆菌XY01分泌蛋白质图谱,通过MALDI-TOF/TOF质 谱鉴定和数据库搜索,对鉴定到的分泌蛋白进行了分析. 共检测到21个蛋白质点, 成功鉴定18个蛋白质点,分别代表14个不同的蛋白质,等电点分布在4.5～7.0之间 ,分子质量分布在20 ～65 kD之间;通过COGs分类和功能分析,信号肽和细胞定位及KEGG代谢通路分析. 结果表明,这些蛋白质对菌体细胞壁/膜的形成、生物信号传导和物质代谢等起着重要作用. 研究结果为长双歧杆菌蛋白质组学和基因组学的研究提供了参考.     

复杂疾病靶基因识别与网络重建的计算系统生物研究

复杂疾病相关靶基因的识别、构建疾病驱使相关基因网络及进行疾病机制研究,是功能基因组学研究中非常重要的科学问题。文章以计算系统生物学的观点和三维的角度,综述了基于生物谱（SNP遗传谱、芯片表达谱和2D-PAGE蛋白质谱等）的复杂疾病靶基因识别、多水平（SNPs虚拟网络、基因调控网络、蛋白质互作网络等）遗传网络逆向重构方法,及不同水平的网络之间在生物学和拓扑学上的纵向映射关系,并给出复杂疾病靶基因识别与网络关系的计算系统生物方法研究的未来展望。     

ZmGns,a maize class I β-1,3-glucanase,is induced by biotic stresses and possesses strong antimicrobial activity

Plant b-1,3-glucanases are members of the pathogenesis-related protein 2(PR-2) family,which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses.One of the differentially expressed proteins(spot 842) identified in a recent proteomic comparison between five pairs of closely related maize(Zea mays L.) lines differing in aflatoxin resistance was further investigated in the present study.Here,the corresponding cDNA was cloned from maize and designated as ZmGns.ZmGns encodes a protein of338 amino acids containing a potential signal peptide.The expression of Zm Gns was detectible in all tissues studied with the highest level in silks.ZmGns was significantly induced by biotic stresses including three bacteria and the fungus Aspergillus flavus.ZmGns was also induced by most abiotic stresses tested and growth hormones including salicylic acid.In vivo,ZmGns showed a significant inhibitory activity against thebacterial pathogen Pseudomonas syringae pv.tomato DC3000 and fungal pathogen Botrytis cinerea when it overexpressed in Arabidopsis.Its high level of expression in the silk tissue and its induced expression by phytohormone treatment,as well as by bacterial and fungal infections,suggest it plays a complex role in maize growth,development,and defense.     

Gene expression profiles of endothelial progenitor cells by oligonucleotide microarray analysis

Among the many tissue stem or progenitor cells recently being unveiled, endothelial progenitor cells (EPCs) have attracted particular attention, not only because of their cardinal role in vascular biology and embryology but also because of their potential use in the therapeutic development of a variety of postnatal diseases, including cardiovascular and peripheral vascular disorders and cancer. The aim of this study is to provide some basic and comprehensive information on gene expression of EPCs to characterize the cells in molecular terms. Here, we focus on EPCs derived from CD34-positive mononuclear cells of human umbilical cord blood. The EPCs were purified and expanded in culture and analyzed by a high-density oligonucleotide microarray and real-time RT-PCR analysis. We identified 169 up-regulated and 107 down-regulated genes in the EPCs compared with three differentiated endothelial cells of human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (LMEC) and human aortic endothelial cells (AoEC). It is expected that the obtained list include key genes which are critical for EPC function and survival and thus potential targets of EPC recognition in vivo and therapeutic modulation of vasculogenesis in cancer as well as other diseases, in which de novo vasculogenesis plays a crucial role. For instance, the list includes Syk and galectin-3, which encode protein tyrosine kinase and β-galactoside-binding protein, respectively, and are expressed higher in EPCs than the three control endothelial cells. In situ hybridization showed that the genes were expressed in isolated cells in the fetal liver at E11.5 and E14.5 of mouse development.