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【目的】开发出与铜绿假单胞菌粘肽合成酶PBP3有高亲和力,具有全新结构的先导化合物。【方法】以铜绿假单胞菌粘肽合成酶PBP3为靶点,通过分子对接软件DOCK6.5,对含有104万个小分子化合物的数据库进行了大规模虚拟筛选,选取结构相对简单的中选化合物进行合成,得到先导化合物,并验证其抑菌活性。【结果】通过grid score进行第一轮初筛,筛选出grid score分值小于–30 kcal/mol的6万个化合物,接着以amber score进行第二轮筛选,筛出amber score分值小于–20 kcal/mol的化合物约200个。最终,经过观察分析,从中挑选出4种打分高并且结构新颖的小分子化合物作为先导化合物。合成出的先导化合物及其衍生物对铜绿假单胞菌等常见微生物的最小抑菌浓度(MIC)在175–275μg/m L之间,对革兰氏阴性菌和阳性菌均有效,MIC值比作为阳性对照的磺胺嘧啶更低,说明先导化合物具有较好的抗菌活性。【结论】这些先导化合物可以进一步开发为新型抗菌药物,用于解决铜绿假单胞菌的耐药性问题。 相似文献
913.
【目的】阿尔茨海默症治疗药物石杉碱甲(Huperzine A,Hup A)的生物合成途径起始于赖氨酸脱羧酶(Lysine decarboxylase,LDC)。本研究克隆及表达了来源于产Hup A的植物内生真菌的LDC基因,并研究了其功能。【方法】采用RT-PCR扩增法,从一株产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14获得LDC基因,构建表达质粒p ET-22b-LDC与p ET-32a-LDC,转化感受态细胞E.coli BL21,加入IPTG至终浓度为1×10~(–3) mol/L,于24°C、200 r/min培养8 h,诱导表达LDC蛋白质;通过Ni~(2+)金属亲和层析纯化重组LDC并建立酶促反应体系,利用TLC检测了LDC催化活性。利用生物信息学软件分析了LDC的理化性质及蛋白质的空间结构。【结果】成功克隆并异源表达出重组蛋白LDC与Trx-LDC,经SDS-PAGE电泳鉴定分子量分别为24.4 k Da和42.7 k Da,与预计大小相符。TLC结果表明LDC与Trx-LDC均具有赖氨酸脱羧酶活性。【结论】本研究从产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14中成功克隆到LDC基因并进行了异源表达,检测到了其催化活性,为丰富LDC分子信息及阐明内生真菌中Hup A生物合成机制提供参考数据。 相似文献
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A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B–natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility. 相似文献
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Day vs. night predation on artificial caterpillars in primary rainforest habitats – an experimental approach
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Carlo L. Seifert Christian H. Schulze Tobias C. T. Dreschke Heinrich Frötscher Konrad Fiedler 《Entomologia Experimentalis et Applicata》2016,158(1):54-59
The influence of natural enemies has led to the evolution of various predator avoidance strategies in herbivorous insects. Many caterpillars are exclusively active at night and rest during the day. It is widely assumed that nocturnal activity in caterpillars reduces their risk of falling prey to their natural enemies. To test this hypothesis, we compared predation pressure between day and night in tree‐fall gaps and closed‐canopy forest sites in an Amazonian primary lowland rainforest. Artificial clay caterpillars, showing camouflaged colouration (green), were exposed as potential prey to a natural predator community. Attacks were significantly more frequent during daytime and were reduced by about a quarter at night in tree‐fall gaps, and by a third in closed‐canopy forest sites. This supports the idea of time‐dependent activity in caterpillars as an antipredatory adaptation. Further, independent of the time of day, predation pressure on caterpillars was significantly higher in tree‐fall gaps compared to closed‐canopy forest habitats. Nearly all predation events were caused by arthropods, whereas birds played a negligible role. Across both habitat types and time scales, ants acted as major predator group, emphasising their important role in population control of herbivorous insects in lowland rainforest ecosystems. This is the first experimental study using artificial caterpillars to examine whether time‐scheduling of exposition might influence predation risk amongst undefended, solitary, free‐living lepidopteran larvae. 相似文献
920.
This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac0-BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac3-BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9 μM−1 min−1 for BK and Toac3-BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE-hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations. 相似文献