Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization

It is often essential to focus the study on the small-size domains of large proteins in eukaryotic cells in the post-genomic era, but the low expression level, insolubility, and instability of the domains have been continuing to hinder the massive purification of domain peptides for structural and biological investigation. In this work, a highly efficient expression and purification system based on a small-size fusion partner GB1 and histidine tag was utilized to solve these problems. Two vectors, namely pGBTNH and pGBH, were constructed to improve expression and facilitate purification. The linker and thrombin cleavage site have been optimized for minimal degradation during purification process. This system has been tested for eight domain peptides varying in size, linker, hydrophobicity, and predicted secondary structure. The results indicate that this system is achievable to produce these domain peptides with high solubility and stability for further biochemical characterization. Moreover, the fusion protein without the linker and thrombin cleavage site is also suitable for spectroscopic studies especially for NMR structural elucidation, if the target peptide is prone to precipitation or easily degraded during purification. This system will be beneficial to the research field of structure and function of small domain and peptide fragment.     

高压快速柱分离纯化牛血球超氧化物歧化酶的研究

以牛血球为材料,经溶血等处理和丙酮沉淀,获得牛血球超氧化物歧化酶粗品。此粗酶可以通过ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ和ＣＭ－Ｓｅｐｈａｒｏｓｅ快速柱层析,获得超氧化物歧化酶纯品。纯化的酶比活可达１３５００ｕ／ｍｇ,经ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和快速蛋白液相色谱（ＦＰＬＣ）检测,结果表明,纯化酶是均一的Ｓｅｐｈａｄｅｘ Ｇ－１００凝胶过滤测得该酶分子量为３１,８００,ＳＤＳ－ＰＡＧＥ测得亚基分子量为１５     

Integrative taxonomy recognized a new cryptic species within Stipa grandis from Loess Plateau of China

Stipa shanxiensis, a cryptic species within Stipa grandis that originated from central and western China, is described based on morphological, genomic, and ecological data from field and common garden experiments. Stipa shanxiensis morphologically resembles S. grandis, although phylogenetically it is closely related to the less morphologically similar Stipa baicalensis and Stipa krylovii. Of the eight significant morphological differences between S. shanxiensis and S. grandis, the two, cauline ligules longer than 2 cm with a filiform apex, and hairs shorter than 0.2 mm on the adaxial surface of the cauline uppermost leaves can be used to distinguish the species. Results from a common garden experiment verified that the two diagnostic characteristics were relatively stable and less morphologically plastic in response to environmental variation. Furthermore, a significant ecological divergence was found between S. shanxiensis and S. grandis, such that the former preferred warmer and more humid climates, and their predicted distribution was generally separated. Taken together, our results highlight that the integrative taxonomic approach was valuable for recognizing a new cryptic species in Stipa. In particular, we find that common garden experiments involving the effects of growth stage and characteristic position helped to morphologically diagnose cryptic species. These findings may also facilitate our understandings of ecological adaption and phenotypic plasticity in response to environmental change.     

Possible role of the CYC2c gene in the cornflower-like ray floret phenotype of Gaillardia cultivars

Journal of Plant Research - Gaillardia plants have been widely cultivated in China and have become an important component of garden landscaping. Different from the common ligulate ray floret, the...     

High endemicity of alveolar echinococcosis in Yili Prefecture,Xinjiang Autonomous Region,the People’s Republic of China: Infection status in different ethnic communities and in small mammals

Alveolar echinococcosis (AE) is a life-threatening disease in humans caused by the larval stage of Echinococcus multilocularis. The tapeworm is transmitted between small mammals and dogs/foxes in the Northern Hemisphere. In this study 286 AE cases were reported from eight counties and one city in Yili Prefecture, Xinjiang Autonomous Region, the People’s Republic of China from 1989 to 2015 with an annual incidence (AI) of 0.41/100,000. Among the patients, 73.08% were diagnosed in the last 11 years. Four counties in the high mountainous areas showed higher AI (0.51–1.22 cases/100,000 residents) than the four counties in low level areas (0.19–0.29/100,000 residents). The AI of AE in Mongolian (2.06/100,000 residents) and Kazak (0.93/100,000 residents) ethnic groups was higher than the incidence in other ethnic groups indicating sheep-farming is a risk for infection given this activity is mainly practiced by these two groups in the prefecture. A total of 1411 small mammals were captured with 9.14% infected with E. multilocularis metacestodes. Microtus obscurus was the dominant species in the mountain pasture areas with 15.01% of the voles infected, whereas Mus musculus and Apodemus sylvaticus were the dominant small mammals in the low altitude areas. Only 0.40% of A. sylvaticus were infected with E. multilocularis. PCR amplification and sequencing analysis of the mitochondrial cox1 gene showed that E. multilocularis DNA sequences from the small mammals were identical to isolates of local human AE cases. The overall results show that Yili Prefecture is a highly endemic area for AE and that the high-altitude pasture areas favorable for M. obscurus may play an important role in its transmission in this region.     

兔肺静脉肌袖心肌细胞动作电位的特性和一些离子流机制

研究兔肺静脉肌袖心肌细胞（cardiomyocytes from rabbit pulmonary vein sleeves, PVC）动作电位的特性和一些离子流机制——内向整流钾电流（IKl）、瞬时外向钾电流（ITo）和非选择性阳离子流（I NSCC）,并与左心房心肌细胞（left atrial cardiomyocytes,LAC）进行比较。采用全细胞膜片钳技术,记录动作电位和上述各离子流。发现PVC动作电位时程（action potential duration,APD）较LAC的明显延长,并可以诱发出第二平台反应。PVC上存在I NSCC. PVC的IKl、I To和I NSCC电流密度均较LAC的明显减小。PVC和LAC存在复极离子流的差异,这种差异构成了两者动作电位差异的基础,进而可能成为肺静脉肌袖致心律失常特性的重要离子流机制。     

高通量测序分析实验性脑脊髓炎肠道微生物变化及与IL-17、IFN-γ相关性

【目的】通过建立实验性脑脊髓膜炎(EAE)小鼠模型,观察小鼠肠道菌群在不同发病时间点的变化和炎症因子IL-17、IFN-γ的表达情况,探讨肠道菌群的变化在EAE发病中的免疫调节作用。【方法】将48只C57BL/6小鼠按照随机数字表法分为正常对照组、EAE模型组各24只。EAE组采用MOG35-55与完全弗氏佐剂的混合物制备模型,进行神经功能评分,记录体重变化。分别取免疫后7、14、21、30 d的小鼠粪便,对样本DNA的16S rDNA V3/V4区基因测序。ELASE法检测IL-17、IFN-γ的表达。【结果】EAE组小鼠血液中IL-17、IFN-γ的表达从第7天开始逐渐升高,21 d时达到高峰。14 d时,EAE组与正常对照组相比,物种丰度有显著性差异(P0.05)的菌种有:Alistipes、布牢特氏菌属、毛螺菌科_NK4A136_group等。30dEAE组与正常对照组相比,物种丰度有显著性差异(P0.05)的菌种有:Allobaculum、真细菌属、螺杆菌等。通过LefSe分析,在7、14、21 d中分别主要作用的微生物菌种逐渐减少,在21d时最少。Odoribacter在21d时起了主要作用。【结论】与正常对照组相比,14、21、30dEAE小鼠肠道菌种的丰度均发生了变化,产生了肠道菌群的紊乱;其中普雷沃氏菌属_NK3B31_group的丰度均较正常对照组降低,与IFN-γ呈负相关(r=–0.537,P0.01)。普雷沃氏菌属_NK3B31_group可能是导致MS脱髓鞘发生的关键菌属。EAE组各个时间点相比起主要作用的肠道菌群种类减少,多样性降低。其中,Odoribacter是在21 d高峰期起主要作用的菌种,但其作用机制需要深入研究。EAE组中炎症因子IL-17、IFN-γ表达的升高,促进了MOG35-55诱发的炎症反应。     

Beta-xylosidase activity of a GH3 glucosidase/xylosidase from yak rumen metagenome promotes the enzymatic degradation of hemicellulosic xylans

Aims: To characterize the duel activities of a glycosyl hydrolase family 3 β‐glucosidase/xylosidase from rumen bacterial metagenome and to investigate the capabilities of its β‐d ‐xylosidase activities for saccharification of hemicellulosic xylans. Methods and Results: A β‐glucosidase/xylosidase gene RuBGX1 was cloned from yak (Bos grunniens) rumen using the metagenomic technology. Recombinant RuBGX1, expressed in Escherichia coli, demonstrated high hydrolytic activities on both p‐nitrophenyl‐β‐d ‐glucopyranoside (pNP‐Glc) and p‐nitrophenyl‐β‐d ‐xylopyranoside (pNP‐Xyl) substrates. Analysis of the kinetic properties indicated that RuBGX1 had a lower affinity for pNP‐Glc substrate as the K_m was 0·164 mmol l^?1 for pNP‐Glc and 0·03 mmol l^?1 for pNP‐Xyl at pH 6·0 and 50°C, respectively. The capabilities of RuBGX1 β‐xylosidase for hydrolysis of xylooligosaccharide substrates were further investigated using an endoxylanase‐coupled assay. Hydrolysis time courses illustrated that a significant increase (about 50%) in the reducing sugars, including xylobiose, xylotriose and xylotetraose, was achieved by supplementing endoxylanase with RuBGX1. Enzymatic product analysis using high‐performance anion‐exchange chromatography‐pulsed amperometric detection showed that RuBGX1 could release xyloses from intermediate xylooligosaccharides produced by endoxylanase. Conclusions: The RuBGX1 shows β‐glucosidase activity in hydrolysis of cello‐oligosaccharides; meanwhile, it has β‐xylosidase activity and functions synergistically with endoxylanase to promote the degradation of hemicellulosic xylans. Significance and Impact of the study: This was the first to report the β‐xylosidase activity of family 3 β‐glucosidase/xylosidase functioned in the degradation of hemicellulosic xylans. The bifunctional β‐glucosidase/xylosidase property of RuBGX1 can be used in simultaneous saccharification of cellulose and xylan into fermentable glucose and xylose.     

Combination effects of chronic cadmium exposure and gamma-irradiation on the genotoxicity and cytotoxicity of peripheral blood lymphocytes and bone marrow cells in rats

This work investigated the effects of chronic cadmium (Cd) exposure combined with γ-ray irradiation on the cytotoxicity and genotoxicity of peripheral blood cells and bone marrow cells in rats. Results showed that when the rats were exposed to low dose (LD) Cd of 0.1mg CdCl?/(kgd) for 8 and 12 weeks, the Cd concentration in blood reached to 135-140 μg/L and no toxic effects on peripheral blood lymphocytes, white blood cells (WBC) and granulocyte-monocyte (GM) progenitor cells were observed except polychromatic erythrocytes (PCE) of bone marrow. Moreover, this chronic LD Cd exposure significantly decreased irradiation-induced micronucleus (MN) formation and hypoxanthine-guanine phosphoribosyl transferase (hprt) mutation in lymphocytes and PCE, while the combination of LD Cd exposure and irradiation induced the additive metallothionein (MT) protein expression in bone marrow cells. When the rats were exposed to a high dose (HD) Cd of 0.5mg CdCl/?(kgd) for 8 and 12 weeks, the blood Cd level approached to 458-613 μg/L and an inflammatory response was induced, meanwhile, MN formation and hprt mutation were markedly increased, and the ratio of PCE/NCE (normochromatic erythrocyte) was significantly decreased. Furthermore, when the rats were exposed to HD Cd plus 2 Gy irradiation, additive toxic effects on MN formation, hprt mutation, PCE damage and GM progenitor cell proliferation were observed, while this combination treatment resulted in an obvious reduction of MT protein compared to HD Cd group. In conclusion, chronic exposure to LD Cd induced the adaptive response to irradiation in the genotoxicity of peripheral blood lymphocytes and PCE of bone marrow by the up-regulation of Cd-induced MT protein, but the combination of HD Cd exposure and irradiation generated the additive effects on the cytotoxicity and genotoxicity in peripheral blood lymphocytes and bone marrow cells.