Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work, a tannase from Aspergillus oryzae has been cloned and expressed in Pichia pastoris. The catalytic activity of the recombinant enzyme was assayed. A secretory form of enzyme was made with the aid of Saccharomyces cerevisiae alpha-factor, and a simple procedure purification protocol yielded tannase in pure form. The productivity of secreted tannase achieved 7000 IU/L by fed-batch culture. Recombinant tannase had a molecular mass of 90 kDa, which consisted of two kinds of subunits linked by a disulfide bond(s). Our study is the first report on the heterologous expression of tannase suggesting that the P. pastoris system represents an attractive means of generating large quantities of tannase for both research and industrial purpose.     

Enhancement of butanol production and reducing power using a two-stage controlled-pH strategy in batch culture of Clostridium acetobutylicum XY16

The effects of pH control on the process of acetone/butanol/ethanol (ABE) production in batch cultures of Clostridium acetobutylicum XY16 have been investigated. Based on the specific acid- and solvent-forming rates in batch fermentation at different pH values (from 4.3 to 6.0), a two-stage controlled-pH strategy was developed in which the pH was shifted from 5.5 to 4.9 after a dry cell weight of 0.5 g L(-1) was achieved. By applying this strategy, the maximum ABE concentration and productivity reached 20.3 g L(-1) and 0.63 g L(-1) h (-1), and were significantly improved by 12.2 and 40.1 %, respectively, compared with the process with no pH control. In addition, reducing power capability was significantly enhanced by this strategy. The two-stage controlled-pH strategy was a convenient and rapid method for high intensity ABE production.     

Proteomic Analysis of Interactions Between the Generalist Herbivore <Emphasis Type="Italic">Spodoptera exigua</Emphasis> (Lepidoptera: Noctuidae) and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In this study, comparative proteomics was used to investigate the interaction of Spodoptera exigua and Arabidopsis thaliana. By using 2-D electrophoresis of differentially expressed proteins, combined with high-throughput matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF/TOF MS, the changes in the abundance of proteins induced by insect feeding were studied in A. thaliana. More than 1,100 protein spots were reproducibly detected on each gel. The intensities of 30 protein spots in particular changed significantly, showing differences in volume of at least twofold. Among these, 17 protein spots were upregulated, and 13 were downregulated following an 8-h insect feeding period. Nineteen insect-feeding-responsive proteins were identified, all of which were involved in metabolic regulation, binding functions or cofactor requirement of protein, cell rescue, and defense and virulence, as assessed by Munich Information Center for Protein Sequences function category. About 50% of these were involved in metabolism, including transketolase, S-adenosylmethionine synthase 3, 2,3-biphosphoglycerate-independent phosphoglycerate mutase, beta-ureidopropionase, GDP-d-mannose 3′,5′-epimerase, and fatty acid synthase. The identification of insect-feeding-responsive proteins on Arabidopsis provides not only new insights into insect stress but also a good start for further investigation of their functions. Understanding how the plant responses to insects in the proteomic level will provide tools for a better management of insect pest in the field.     

一种宫内节育器的染色体畸变试验研究

目的检测一种宫内节育器的体外细胞的染色体畸变作为遗传毒性评价的一部分。方法在加和不加S9活化系统条件下,试验组用三种不同浓度的节育器浸提液处理CHL细胞20h,对照组分别加入阴性、阳性进行交换,各组置37℃培养箱中培养。24h后采集细胞并分析中期细胞染色体畸变情况,计算染色体畸变率。结果在4g/20mL的浓度下受试物对细胞有明显的细胞毒性,其稀释浸提液的畸变率与阴性对照相比,在统计学上无显著性差异(P>0.05)。结论在该试验条件下,受试物稀释浸提液未诱发CHL细胞染色体畸变。     

载脂蛋白E在神经系统中的作用

载脂蛋白Ｅ（ａｐｏＥ）在中枢神经系统（ＣＮＳ）生长发育,成熟衰老和损伤修复过程中发挥重要作用。其分子机制是：（１）稳定神经细胞骨架系统;（２）通过ａｐｏＥ受体途径调节神经细胞中胆固醇脂的运输和突触末梢的再生;（３）调控神经元之间及神经细胞与介质之间的相互作用;（４）调节神经细胞的Ｃａ＾２＋离子的平衡。     

Homocysteine inhibits endothelial progenitor cells proliferation via DNMT1-mediated hypomethylation of Cyclin A

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.     

哺乳类细胞基因表达系统

真核基因的转录和转译调控是哺乳类细胞基因表达系统建立和发展的基础,外源基因的表达水平不仅决定于启动子/增强子的强弱,还与剪接信号、终止信号和poly(A)信号以及质粒的拷贝数等因素有关。另外,在基因治疗的研究中,也已寻找到多种具有组织或肿瘤特异性的启动子,来达到特异性肿瘤基因治疗的目的。     

Interactive effects of elevated CO2 and drought stress on leaf water potential and growth in Caragana intermedia

We studied the responses of leaf water potential (Ψ_w), morphology, biomass accumulation and allocation, and canopy productivity index (CPI) to the combined effects of elevated CO₂ and drought stress in Caragana intermedia seedlings. Seedlings were grown at two CO₂ concentrations (350 and 700 μmol mol⁻¹) interacted with three water regimes (60–70%, 45–55%, and 30–40% of field capacity of soil). Elevated CO₂ significantly increased Ψ_w, decreased specific leaf area (SLA) and leaf area ratio (LAR) of drought-stressed seedlings, and increased tree height, basal diameter, shoot biomass, root biomass as well as total biomass under the all the three water regimes. Growth responses to elevated CO₂ were greater in well-watered seedlings than in drought-stressed seedlings. CPI was significantly increased by elevated CO₂, and the increase in CPI became stronger as the level of drought stress increased. There were significant interactions between elevated CO₂ and drought stress on leaf water potential, basal diameter, leaf area, and biomass accumulation. Our results suggest that elevated CO₂ may enhance drought avoidance and improved water relations, thus weakening the effect of drought stress on growth of C. intermedia seedings.