Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.     

2011年、2013年和2016年医院内获得性血流感染常见病原菌分布及其耐药性分析

动态监测2011年、2013年和2016年我国不同地区医院内获得性血流感染病原菌分布及耐药进展趋势。从全国10个城市回顾性收集血流感染病原菌非重复性株,采用琼脂稀释法或微量肉汤稀释法进行药物敏感性试验,采用Whonet 5.6软件对药敏试验结果进行分析。收集的2 248株血流感染病原菌中革兰阴性杆菌为1 657株 (占73.7%),革兰阳性球菌为591株 (占26.3%)。分离率排名前五的病原菌依次为大肠埃希菌 (32.6%,733株/2 248株)、肺炎克雷伯菌 (14.5%,327株/2 248株)、金黄色葡萄球菌 (10.0%,225株/2 248株)、鲍曼不动杆菌 (8.7%,196株/ 2 248株) 和铜绿假单胞菌 (6.2%,140株/2 248株)。血流感染分离的革兰阴性杆菌对抗菌药物体外敏感率较高的抗菌药物依次为粘菌素 (96.5%,1 525株/1 581株,不包括天然耐药菌株)、替加环素 (95.6%,1 375株/1 438株,不包括天然耐药菌株)、头孢他啶/克拉维酸 (89.2%,1 112株/1 246株)、阿米卡星 (86.4%,1 382株/1 599株) 和美罗培南 (85.7%,1 376株/1 605株);革兰阳性球菌对抗菌药物体外敏感率较高的抗菌药物依次为替加环素、替考拉宁和达托霉素 (敏感率均为100.0%)、万古霉素和利奈唑胺 (敏感率均为99.7%)。2011年、2013年和2016年产超广谱β-内酰胺酶肠杆菌科细菌分离率分别为50.6% (206株/407株)、49.8% (136株/273株) 和38.9% (167株/429株);碳青霉烯不敏感肠杆菌科细菌分离率分别为2.2% (9株/408株)、4.0% (16株/402株) 和3.9% (17株/ 439株);多重耐药鲍曼不动杆菌分离率分别为76.4% (55株/72株)、82.7% (43株/52株) 和87.5% (63株/72株),多重耐药铜绿假单胞菌分离率分别为9.8% (5株/51株)、20.0% (7株/35株) 和13.0% (7株/54株);甲氧西林耐药金黄色葡萄球菌的分离率分别为51.9% (41株/79株)、29.7% (19株/64株) 和31.7% (26株/82株)。屎肠球菌和粪肠球菌中高水平庆大霉素耐药株分离率分别为43.2% (48株/111株) 和40.9% (27株/66株)。碳青霉烯不敏感肠杆菌科细菌中肺炎克雷伯菌居首位,占57.1% (24株/42株) 。肠杆菌科细菌中分离出30株替加环素不敏感株,其中肺炎克雷伯菌占76.7% (23株/30株);分离出粘菌素耐药肠杆菌科细菌39株,其中大肠埃希菌、阴沟肠杆菌和肺炎克雷伯菌分别占43.6% (17株/39株)、35.9% (14株/39株) 和15.4% (6株/39株)。医院获得性血流感染病原菌主要为革兰阴性杆菌 (以大肠埃希菌和肺炎克雷伯菌为主),其对替加环素、粘菌素和碳青霉烯类药物的敏感率较高;革兰阳性球菌中分离率最高的为金黄色葡萄球菌,其次为屎肠球菌,这两种细菌对替加环素、达托霉素、利奈唑胺、万古霉素和替考拉宁的敏感率较高。粘菌素耐药肠杆菌科细菌、替加环素不敏感肠杆菌科细菌、利奈唑胺或万古霉素不敏感革兰阳性球菌的分离,警示临床高度关注,仍需动态监测耐药进展趋势。     

Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay

Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate.     

禽流感病毒NS1蛋白对细胞的影响

NS1蛋白为流感病毒非结构蛋白,只在病毒侵入宿主细胞后产生.目前NS1蛋白对细胞整体水平上的作用仍不清楚,为了解NS1蛋白在病毒感染细胞中的作用,构建了重组质粒pCMV-myc-NS1并将其转染A549细胞,利用双向电泳技术检测了受NS1蛋白调控的宿主蛋白,以期从蛋白质组水平上研究禽流感病毒与宿主细胞间的相互作用.同时,还检测了转染NS1对细胞增殖和细胞周期的影响.结果显示,NS1在细胞中的表达,能够明显引起宿主细胞代谢的变化,并通过阻滞细胞周期的正常进行而减缓细胞的增殖.     

Gene conjugation of molecular adjuvant C3d3 to hCGbeta increased the anti-hCGbeta Th2 and humoral immune response in DNA immunization

Human chorionic gonadotropin (hCG) has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. We have explored the use of three copies of C3d in DNA vaccine as molecular adjuvant to improve the immunogenicity of this hormone in previous work and found that the immune response induced by pcDNA3-hCGbeta-C3d3 has been enhanced 243-fold compared with pcDNA3-hCGbeta following DNA immunization in BALB/c mice. In the present study, a new functionally active DNA vaccine of hCGbeta-C3d3 chimera based on pCMV4 vector has been described. We compared the expression efficiency of pCMV4 and pcDNA3 eukaryotic vectors for hCGbeta and hCGbeta-C3d3 fusion protein and the immune response of mice immunized with pcDNA3-hCGbeta, pCMV4-hCGbeta, pcDNA3-hCGbeta-C3d3 and pCMV4-hCGbeta-C3d3, respectively, at 25, 50 and 100 pmol dose, and further analyzed the levels of Th1 and Th2 cytokines produced by spleen lymphocytes of the immunized mice upon hCG restimulation in vitro. It was found that pCMV4 vector achieved 1.3-1.5-fold higher protein expression and raised 1.1-1.2 (primary) and 1.2-1.3 (booster) logs higher titer of anti-hCGbeta IgG than pcDNA3. Mice vaccinated with 50 pmol of hCGbeta-C3d3-DNAs elicited the highest titer of hCGbeta-specific antibody among the serial doses and the immune response induced by pCMV4-hCGbeta-C3d3 were, respectively, 1.3, 1.3 and 1.2 logs higher than that of pcDNA3-hCGbeta-C3d3 and 2.2, 2.9 and 2.4 logs higher than that of pCMV4-hCGbeta at week 2 following the booster immunization. Moreover, we observed that the production of IL-4 and IL-10 increased in mice vaccinated with hCGbeta-C3d3-DNAs and the ratio of IL-4/IFN-(gamma) showed a Th2 bias of immune response in the mice immunized with hCGbeta-C3d3-DNAs. These findings indicated that gene fusion of C3d3 to hCGbeta, as a means of harnessing the adjuvant potential of the innate immune system, may improve the antigen-specific Th2 humoral immune response of the hCGbeta DNA vaccine and the pCMV4 vector is a more ideal eukaryotic vector for DNA vaccine than pcDNA3.     

Recombinant Galectins of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> Parasite Induces Apoptosis in the Peripheral Blood Lymphocytes of Goat

The effect of Haemonchus contortus galectin peptides rHco-gal-m/f to induce apoptosis in the peripheral blood lymphocytes (PBLCs) of goats was investigated. Analysis of apoptosis was carried out with agarose gel electrophoresis, flow cytometry and transmission electron microscopy. The results indicated that there were visible apoptosis bodies and typical DNA ladders by genomic DNA fragmentation. The quantitative analysis of apoptosis by flow cytometry indicated that rHco-gal-m/f peptides induced apoptosis was time and dose dependent. Ultrastructural studies of the PBLCs revealed that a large number of apoptotic cells were present in galectin-treated cells, which had the typical morphologic changes of apoptosis such as reduction of the cytoplasmic volume, loss of cell surface microvilli, chromatin condensation and fragmentation of the apoptotic cells into small apoptotic bodies.     

Secondary succession of plant communities in a subtropical mountainous region of SW China

Since 1985, originally forested mountainous areas of China have been allowed to return to their natural state after years of exploitation including agriculture, development, and logging. The reforms began earlier in less accessible locations, so that today the successional process is more advanced there. The vegetation in Luquan, Qiongzhusi, and Xishan near Kunming, central Yunnan, exhibits, in a limited area, a range of stages of plant succession that are widely encountered throughout the broader region, and thus affords a special opportunity for a comprehensive study. We analyzed the successional sequence of these various plant communities. They ranged from pioneer coniferous and/or pioneer deciduous broad-leaved stands to pre-mature semi-humid evergreen broad-leaved stands, through mixed coniferous and broad-leaved or mixed deciduous and evergreen broad-leaved stands. The succession proceeded from pioneer coniferous Pinus and Keteleeria, and deciduous Platycarya and Alnus, to late-successional evergreen broad-leaved Cyclobalanopsis and Castanopsis. Two regeneration types of woody species in either the early successional (15–50 years), the mid-successional (40–80 years), or the late-successional (80–180 years) stage were classified. Relatively high species diversity was found in the seral phase at the three study sites. The late-successional stage was commonest where human disturbance was least evident. Poor soil chemical properties under pioneer Pinus were seen as a limitation to plant growth, while the abundance of Alnus at the early stage led to an improved level of organic matter and nitrogen.     

Structure insights into mechanisms of ATP hydrolysis and the activation of human heat-shock protein 90

The activation of molecular chaperone heat-shock protein 90 (Hsp90) is dependent on ATP binding and hydrolysis, which occurs in the N-terminal domains of protein. Here, we have determined three crystal structures of the N-terminal domain of human Hsp90 in native and in complex with ATP and ATP analog, providing a clear view of the catalytic mechanism of ATP hydrolysis by Hsp90. Additionally, the binding of ATP leads the N-terminal domains to be an intermediate state that could be used to partially explain why the isolated N-terminal domain of Hsp90 has very weak ATP hydrolytic activity.