Cytokine production using membrane adsorbers: Human basic fibroblast growth factor produced by Escherichia coli

Basic fibroblast growth factor (FGF‐2) is a multifunctional cytokine that regulates various cellular processes both in vitro and in vivo. FGF‐2 is extensively used in embryonic stem cell cultures since it can maintain the cells in an undifferentiated state. However, the high price of FGF‐2 has limited its application in stem cell research. Here we present a fast and efficient process for the purification of FGF‐2 from recombinant Escherichia coli cultures using reusable membrane adsorbers. A high expression level of FGF‐2 (42 mg/g dry cell) was achieved by fed‐batch cultivation of E. coli BL21(DE3). A new combination of cation exchange membrane chromatography and heparin‐sepharose affinity chromatography was used for the purification of the protein. A novel anion exchange membrane chromatography was used in the polishing step to remove endotoxins and DNA. In this new process, about 200 mg soluble FGF‐2 was yielded from 1.9 L culture broth with a purity of 98%. The purified protein was identified to be endotoxin‐free and bioactive. It was successfully tested to keep primate embryonic stem cell and human‐induced pluripotent stem cell pluripotent. Our approach, in which a controlled cultivation process is combined with an optimized fast and versatile downstreaming process, is suitable for low‐cost preparation of bioactive FGF‐2 at bench‐scale and may be beneficial to the effective production of other cytokines.     

Construction of a bacterial artificial chromosome library from the spikemoss Selaginella moellendorffii: a new resource for plant comparative genomics

Background

The lycophytes are an ancient lineage of vascular plants that diverged from the seed plant lineage about 400 Myr ago. Although the lycophytes occupy an important phylogenetic position for understanding the evolution of plants and their genomes, no genomic resources exist for this group of plants.

Results

Here we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from the lycophyte Selaginella moellendorffii. Based on cell flow cytometry, this species has the smallest genome size among the different lycophytes tested, including Huperzia lucidula, Diphaiastrum digita, Isoetes engelmanii and S. kraussiana. The arrayed BAC library consists of 9126 clones; the average insert size is estimated to be 122 kb. Inserts of chloroplast origin account for 2.3% of the clones. The BAC library contains an estimated ten genome-equivalents based on DNA hybridizations using five single-copy and two duplicated S. moellendorffii genes as probes.

Conclusion

The S. moellenforffii BAC library, the first to be constructed from a lycophyte, will be useful to the scientific community as a resource for comparative plant genomics and evolution.     

Changes in morphology and biochemical indices in browning callus derived from <Emphasis Type="Italic">Jatropha curcas</Emphasis> hypocotyls

Callus browning is a typical feature of callus cultures derived from the hypocotyl of Jatropha curcas. Brown callus results in decreased regenerative ability, poor growth and even death. In this study, we investigated the effect of browning on callus morphology and biochemical indices. Light microscopy and scanning electron microscopy showed striking differences in callus morphology. During browning, chlorophylls and carotenoids concentrations decreased steadily. Polyphenol oxidase (PPO) and peroxidase (POD) enzymatic activities patterns were similar during callus culture with a higher activity level at week 3 compared to week 2 or later weeks. Grey relation degree analysis indicated that PPO played a more important role than POD in enzymatic callus browning. Polyacrylamide gel electrophoresis results showed differences between browning and non-browning callus. Gas chromatography–mass spectrometry results showed that saturated and unsaturated fatty acid quantities differed significantly but there was little difference in fatty acid composition between non-browning and browning callus. Differences in 17, 18.4 and 25 kDa protein concentrations were also observed in browning and non-browning callus using sodium dodecyl sulfate–polyacrylamide gel electrophoresis.     

盾盘亚纲二种盾盘吸虫Aspidogaster conchicola和Lophotaspis orientalis神经系统结构差异的研究

采用乙酰胆碱组织化学定位方法,分别对盾盘亚纲两种吸虫Aspidogaster conchicola和Lophotaspis orientalis的神经系统进行染色观察.经比较,两种吸虫的腩神经节及神经联合的位置郁位于咽前部;由脑神经节向前发出的神经干最终会合于围口神经环;脑神经节向后发出3对神经干,其中后腹神经干最为粗大,它通过多对神经与腹吸盘相连.二者存在的差别在于:第一,体前神经干的分支数目及分布位置不同;第二,腹吸盘部神经分布形式不同;第三,Aspidogaster conchicola的两侧排泄管均有明显着色,并显示螺纹状结构.     

生活污水分散处理技术的进展

为了确保我国水体清洁,生活污水除了集中处理之外,分散式的净化槽系统也是不可缺少的。对生活污水分散处理的特点、难点及发展状况进行了论述,探讨了生活污水分散处理技术在应用中存在的问题及解决方法,指出厌氧和好氧生物膜联合处理的小型一体化生活污水净化槽的发展方向。     

网隔栽培法对生姜姜瘟病防治及产量的影响

姜瘟病是由青枯雷尔氏菌(Ralstoniasolanacearum)引起的一种细菌性病害,被称为生姜种植产业的“癌症”.本试验设计了一种“网隔栽培法”,并探索其对土传姜瘟病发病率和生姜产量的影响.结果表明,经过连续3年的田间验证,相比传统栽培法,“网隔栽培法”种植可显著降低姜瘟病的发病率,平均减少了6.08个百分点,平均防治效果达到了48.33%;同时,生姜产量平均增加了13.21%.这种“短行播种、纵横开沟、深沟隔病”的“网隔栽培法”为姜瘟病的绿色防控提供了新方案,也为其他蔬菜、中药材等植物的土传病害防治提供了新的借鉴思路.     

海洋微生物活性物质研究进展

由于海洋环境的特殊性和多样性,海洋生物产生了与陆地生物不同的代谢途径和防御体系,分泌出多种结构新颖、活性特异的物质,如抗生素、抗肿瘤活性物质、酶等,这些活性物质在化工、医药、食品以及生命科学等领域有着广阔的应用前景。本文主要介绍了海洋微生物活性物质的主要类型及研究现状。     

基于Red重组系统构建含Flag标签标记UL23基因的重组人巨细胞病毒

利用来源于λ噬菌体的Red系统,将Flag标签及两侧带有FRT位点的卡那霉素抗性基因片段插入原HCMV TowneBAC中UL23基因3 '末端区域,通过卡那抗性筛选带有抗性标记的重组菌株,并通过表达重组酶FLP的质粒pCP20去除卡那霉素抗性基因,得到带有Flag标签标记UL23基因和单一FRT位点的突变BAC.重组后的BAC分子同质粒pcDNA3.1(+)-pUL82共转染HFF细胞后重建重组HCMV.Western blotting检测证实所构建重组病毒能够表达含Flag标签标记的pUL23蛋白.此含有Flag标签标记UL23基因的重组HCMV的成功构建为了进一步研究人巨细胞病毒UL23基因及其产物的功能提供依据.     

γ-Secretase Processing and Effects of γ-Secretase Inhibitors and Modulators on Long Aβ Peptides in Cells

Understanding how different species of Aβ are generated by γ-secretase cleavage has broad therapeutic implications, because shifts in γ-secretase processing that increase the relative production of Aβx-42/43 can initiate a pathological cascade, resulting in Alzheimer disease. We have explored the sequential stepwise γ-secretase cleavage model in cells. Eighteen BRI2-Aβ fusion protein expression constructs designed to generate peptides from Aβ1–38 to Aβ1–55 and C99 (CTFβ) were transfected into cells, and Aβ production was assessed. Secreted and cell-associated Aβ were detected using ELISA and immunoprecipitation MALDI-TOF mass spectrometry. Aβ peptides from 1–38 to 1–55 were readily detected in the cells and as soluble full-length Aβ proteins in the media. Aβ peptides longer than Aβ1–48 were efficiently cleaved by γ-secretase and produced varying ratios of Aβ1–40:Aβ1–42. γ-Secretase cleavage of Aβ1–51 resulted in much higher levels of Aβ1–42 than any other long Aβ peptides, but the processing of Aβ1–51 was heterogeneous with significant amounts of shorter Aβs, including Aβ1–40, produced. Two PSEN1 variants altered Aβ1–42 production from Aβ1–51 but not Aβ1–49. Unexpectedly, long Aβ peptide substrates such as Aβ1–49 showed reduced sensitivity to inhibition by γ-secretase inhibitors. In contrast, long Aβ substrates showed little differential sensitivity to multiple γ-secretase modulators. Although these studies further support the sequential γ-secretase cleavage model, they confirm that in cells the initial γ-secretase cleavage does not precisely define subsequent product lines. These studies also raise interesting issues about the solubility and detection of long Aβ, as well as the use of truncated substrates for assessing relative potency of γ-secretase inhibitors.