High-capacity purification of His-tagged proteins by affinity membranes containing functionalized polymer brushes

Porous membrane absorbers are attractive for increasing the rate of protein purification, but their binding capacity is low relative to porous beads. Modification of membranes with functionalized polymer brushes, however, can greatly enhance capacity. This work demonstrates that membrane modification with poly(2-hydroxyethyl methacrylate) (PHEMA) brushes derivatized with nitrilotriacetate-Ni2+ (NTA-Ni2+) complexes allows purification of polyhistidine-tagged ubiquitin (HisU) in less than 30 min with a binding capacity of 120 mg of HisU/cm3 of porous alumina membrane. Adsorption isotherms show that saturation of the brushes occurs at HisU concentrations as low as 0.04 mg/mL and that these brushes can bind up to 23 monolayers of HisU. Gel electrophoresis reveals that the purity of eluted HisU is more than 99%, even when the initial feed solution contains 10% bovine serum or a 20-fold excess of BSA. Thus, reusable porous membranes modified by PHEMA-NTA-Ni2+ brushes are attractive candidates for rapid purification of polyhistidine-tagged proteins.     

Design and synthesis of quinolin-2(1H)-one derivatives as potent CDK5 inhibitors

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine protein kinase and its deregulation is implicated in a number of neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and ischemic stroke. Using active site homology modeling between CDK5 and CDK2, we explored several different chemical series of potent CDK5 inhibitors. In this report, we describe the design, synthesis, and CDK5 inhibitory activities of quinolin-2(1H)-one derivatives.     

A short-form C-type lectin from amphioxus acts as a direct microbial killing protein via interaction with peptidoglycan and glucan

To investigate the evolution and immune function of C-type lectin in amphioxus, the primitive representative of the chordate phylum, we identified three C-type lectins consisting solely of a carbohydrate recognition domain and N-terminal signal peptide and found that they had distinct express patterns in special tissues and immune response to stimulations analyzed by quantitative real-time PCR. We characterized the biochemical and biological properties of AmphiCTL1, which was dramatically up-regulated in amphioxus challenged with Staphylococcus aureus, Saccharomyces cerevisiae, and zymosan. Immunohistochemistry demonstrated that the localization of AmphiCTL1 protein was exclusively detected in the inner folding tissues of the hepatic diverticulum. Recombinant AmphiCTL1 was characterized as a typical Ca2+-dependent carbohydrate-binding protein possessing hemagglutinating activity, preferentially bound to all examined four Gram-positive bacteria and two yeast strains, but had little binding activity toward four Gram-negative bacteria we tested. It aggregated S. aureus and S. cerevisiae in a Ca2+-dependent manner and specifically bound to insoluble peptidoglycan and glucan, but not to LPS, lipoteichoic acid, and mannan. Calcium increased the intensity of the interaction between AmphiCTL1 and those components, but was not essential. This lectin directly killed S. aureus and S. cerevisiae in a Ca2+-independent fashion, and its binding to microorganism cell wall polysaccharides such as peptidoglycan and glucan preceded microbial killing activity. These findings suggested that AmphiCTL1 acted as a direct microbial killing C-type lectin through binding microbial targets via interaction with peptidoglycan and glucan. Thus, AmphiCTL1 may be an evolutionarily primitive form of antimicrobial protein involved in lectin-mediated innate immunity.     

植物硼营养研究的重要进展与展望

硼是高等植物必需的矿质营养元素, 但是人们对硼行使生理功能及其分子机理的认识远落后于其它必需营养元素。近几年国际上植物硼营养的研究取得了一些重要的突破。首先是进一步明确了B-RG-II复合物的形成及其影响细胞壁结构和功能的分子机制, 并且发现B-RG-II的形成及其含量与陆生植物的进化密切相关。其次是在拟南芥中克隆了第一个植物硼转运子基因 BOR1, 并揭示了它的作用机理; 通过转基因实验证明了植物硼的高效吸收与水通道基因NIP5;1密切相关。进而通过大量的种质筛选, 从油菜、小麦、大麦及棉花等农作物中获得一批硼高效吸收利用的优异种质材料, 并开展了硼高效QTL定位和克隆。本文详细综述了以上几个方面的研究进展, 并对进一步的研究做了展望。     

甘草粗提物对鲫的免疫调节作用

本研究对中药甘草(Glycyrrhiza uralensis Fiseh)进行了粗提,将粗提物按0.5%和2%的质量分数制成药饵投喂鲫鱼(Carassius auratus)后,于第14天、28天、35天、42天5、6天采样检测其免疫指标。结果显示,低剂量组试验鱼血清溶菌酶活性逐渐升高,高剂量组在35d达到最大值(205.5±28.8)U/mL,之后略有下降。低剂量组和高剂量组试验鱼血清杀菌活性均有所波动,波动范围分别为62.0%—73.1%和75.0%—83.3%,就各期而言,对照组、低剂量组和高剂量组试验鱼的血清杀菌活性依次升高。另外,在淋巴细胞计数方面,高剂量组仅在第42天采样时显著高于对照组(p<0.05),在其他采样期,试验组与对照组均无统计学差异。此外,低剂量组试验鱼血液中白细胞吞噬活性有所增强(p<0.05),第42天达到最大值(66.5±9.4)%,高剂量组先升后降,在第35天达到峰值(74.7±5.6)%。最后,低剂量组试验鱼脾脏重量指数在第56天显著高于对照组(p<0.05),结果分别为(1.08±0.09)×10-3和(0.71±0.09)×10-3,高剂量组在第28天与对照组差异极显著(p<0.01),结果分别为(1.12±0.13)×10-3和(0.63±0.08)×10-3。实验表明,该甘草粗提物对鲫免疫功能有良好的调节作用,具有广阔的应用前景。     

母乳喂养与婴儿HCV感染关系的研究

目的：探讨母乳喂养与婴儿HCV感染的关系。方法：采用Meta分析方法对中国生物医学数据库、雏普数据库、方正数据库、PUBMED数据库和MEDLINE报道的文献进行分析。纳入标准依据Abdolmaleky HM方法。用RevMan4．2软件对纳入文献计算特异OR值。x^2test检验OR值异质性。联系的强度采用0R值进行评价。结果：共有120篇文献,37篇为综述,只有6篇文献符合纳入标准。Meta分析OR值为0．60（95％CI=0．22-1．60）,证实母乳喂养与婴儿HCV感染无关。结论：母乳喂养不是婴儿感染HCV的危险因素。     

Bacillus halodurans菌株xynM基因的克隆、表达和序列分析

目的:用2株新分离鉴定的细菌克隆与最近源种(Bacillus halodurans C-125)木聚糖酶基因的同源序列,并对其进行序列分析。方法:根据已发表的近源种(C-125)的保守区序列设计引物,扩增出与其同源的木聚糖酶基因,进而用pQE31载体对该基因进行表达。同时对表达蛋白的三维空间结构进行网络模拟分析,对蛋白同源性进行比对分析。结果:克隆的木聚糖酶基因片段分别为1284bp和1236bp,而且该基因也成功表达;该蛋白是(α/α)6折叠构象,与C-125三者同属于一个分支上。结论:通过分析、表达蛋白编码外切-1,4-β-木聚糖酶,属于M家族。这该新分离菌株木聚糖酶的研究和应用提供了重要线索。     

pH、温度、NH4+和盐度对圆果雀稗内生固氮菌固氮特性的影响

采用乙炔还原法对从圆果雀稗(Paspalum orbiculare)根、茎中分离到的5株内生固氮菌(POJy11、POG12、POGy22、POGy612、POG4)的固氮特性进行了研究。结果表明,POGy612、POG4固氮的pH适应范围是6.0-10.0,其余3株菌为6.0-8.0;POGy22固氮的最适温度为35℃,其余4株菌为30℃;随着NH4 浓度和NaCl浓度的升高,菌株固氮活性总体呈下降趋势,其中POGy22的固氮活性受NH4 抑制最明显,NH4 浓度为1.0 mmol/L时,已无固氮活性,但POGy612在NH4 浓度为2.5 mmol/L时的固氮活性最高,达到1 045.0 nmol ml-1h-1,POJy11、POGy22在NaCl浓度为0.5%时的固氮活性最高,分别达到735.5 nmol ml-1h-1、703.0 nmol ml-1h-1。     

ELISA 法用于马抗狂犬病毒抗体的检测

本文建立了ＥＬＩＳＡ间接法,用以检测精制（马）抗狂犬病血清（或血浆）制造过程中的中和抗体效价,并与传统的小鼠脑内中和法比较。结果表明（１）两种检测方法对不同水平抗体的检出是一致的。它们之间有很好的线性关系（ｙ＝２．１７ｘ＋２１,ｒ＝０．９９,ｐ＜０．０１）。（２）应用ＥＬＩＳＡ间接法测定马抗狂犬病血清效价简便、快速、特异性及重复性好,可代替小鼠脑内中和法。     

A novel cysteine‐free venom peptide with strong antimicrobial activity against antibiotics‐resistant pathogens from the scorpion Opistophthalmus glabrifrons

Antibiotic‐resistant bacteria, such as methicillin‐resistant Staphylococcus aureus and vancomycin‐resistant Enterococcus, pose serious threat to human health. The outbreak of antibiotic‐resistant pathogens in recent years emphasizes once again the urgent need for the development of new antimicrobial agents. Here, we discovered a novel antimicrobial peptide from the scorpion Opistophthalmus glabrifrons, which was referred to as Opisin. Opisin consists of 19 amino acid residues without disulfide bridges. It is a cationic, amphipathic, and α‐helical molecule. Protein sequence homology search revealed that Opisin shares 42.1–5.3% sequence identities to the 17/18‐mer antimicrobial peptides from scorpions. Antimicrobial assay showed that Opisin is able to potently inhibit the growth of the tested Gram‐positive bacteria with the minimal inhibitory concentration (MIC) values of 4.0–10.0 μM; in contrast, it possesses much lower activity against the tested Gram‐negative bacteria and a fungus. It is interesting to see that Opisin is able to strongly inhibit the growth of methicillin‐ and vancomycin‐resistant pathogens with the MICs ranging from 2.0 to 4.0 μM and from 4.0 to 6.0 μM, respectively. We found that at a concentration of 5 × MIC, Opisin completely killed all the cultured methicillin‐resistant Staphylococcus aureus. These results suggest that Opisin is a promising therapeutic candidate for the treatment of the antibiotic‐resistant bacterial infections. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.