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Domesticated honeybees facilitate interspecific hybridization between two Taraxacum congeners
No full textInterspecific hybridization is common in plants under natural conditions, but the ecological mechanisms underlying when and how it happens have not fully been understood.Taraxacum calanthodium and Taraxacum lugubre are two herbaceous annals co-occurring in alpine meadows of the eastern Tibetan Plateau that share the same pollinators including domestic honeybees during their overlapping flowering times. Because honeybees tend to visit flowers less discriminatively when bee densities are high, we hypothesized that intense apiculture would facilitate hybridization between these two congeneric species. We tested this hypothesis by examining the frequencies of the two parent species occurrence and the hybrid (based on morphological and genetic differences) along three transects radiating from well-established apiaries. Experiments show that both T.calanthodium and T.lugubre produce seeds sexually and asexually, and that they can hybridize via pollen transfer. Bee visitation rates and the frequency of the hybrid were significantly higher in the sites nearest to apiaries compared to distant site along each of the three transects. The hybrids were consistently genetically intermediate between the two species, as indicated by simple sequence repeat-based analyses.Synthesis. These data indicate that domestic honeybees foster interspecific hybridization between the two Taraxacum species and that anthropogenic effects on pollen vectors can significantly influence species hybridization in nature. We suggest that more effort should be made to quantify the effects of environmental change on pollinators and their effects on species evolution
CH4 concentrations and fluxes in a subtropical metropolitan river network: Watershed urbanization impacts and environmental controls
No full textUrbanization and greenhouse gas emissions are of great global concern, especially in developing countries such as China. However, little is known about the relationship between the two. In this study, we examined the influences of the urbanization of Chongqing Municipality, which covers an area of 5494 km(2), in China, on the CH4 emissions of in its metropolitan river network. The results from 84 sampling locations showed an overall mean CH4 concentration of 0.69 +/- 1.37 mu mol.L-1 and a CH4 flux fromthe river network of 1.40 +/- 2.53mmol CH(4)m(-2)d(-1). The CH4 concentrations and fluxes presented a clear seasonal pattern, with the highest value in the spring and the lowest in the summer. Such seasonal variations were probably co-regulated by the dilution effect, temperature and supply of fresh organicmatter by algal blooms. Another important result was that the CH4 concentrations and fluxes increased with the degree of urbanization or the proportion of urban land use, being approximately 3-13 times higher in urban and suburban areas than in rural ones. The total nitrogen, dissolved oxygen (O%) and possible sewage discharge, which could affect the in situ CH4 production and exogenous CH4 input respectively, were important factors that influenced the spatial patterns of CH4 in human-dominated river networks, while the nitrogen (N) and phosphorus (P) could be good predictors of the CH4 emissions in urban watersheds. Hydrologic drivers, including bottom sediment type, flow velocity and river width, were strongly correlated with the CH4 concentrations and could also affect the spatial variance and predict the CH4 hotspots in such metropolitan river networks. With increasing urbanization, we should pay more attention to the increasing greenhouse gas emissions associated with urbanization. (c) 2017 Elsevier B. V. All rights reserved
The effects of experimental warming and CO2 concentration doubling on soil organic carbon fractions of a montane coniferous forest on the eastern Qinghai-Tibetan Plateau
No full textTo explore the effects of elevated temperature (ET), elevated atmospheric CO2 concentration (EC) and ET plus EC (ETC) on different fractions of soil organic carbon (SOC) is significant for understanding the interactions between SOC and environmental variables. Up to date, little information is available in montane forests of the eastern Qinghai-Tibetan Plateau, which is a key region for studying global climate change especially for high altitude areas. This work applied an automatic gas exchange system to investigate the responses of different fractions of SOC to these factors (ET: + 2.5 A degrees C; EC: + 350 ppm; ETC: + 2.5 A degrees C + 350 ppm) after two and a half years' treatments. Results showed that both ET and EC significantly increased the average amount of SOC and not-readily oxidizable carbon (NROC), while significantly decreased the readily oxidizable carbon (ROC) in soil. The dramatic contrast between the content of NROC and ROC provided evidence that trees exposed to either ET or EC would employ more labile nutrient to meet their growth demands. Soil microbial biomass carbon (SMBC) was significantly related to the amount of ROC. Unlike ROC, the trends of particulate organic carbon (POC) and mineral-associated organic carbon (MOC) under different treatments were consistent with that of SOC. Under ET, EC and ETC, the alterations of %ROC were more obvious than that of %POC, and positive correlations were observed between SOC, POC, MOC and NROC, with the exception of ROC and SMBC. These results indicated that the biotical and chemical labile components were more sensitive than the physical active fraction of soil organic matter. Meanwhile, the physical protection and chemical recalcitrance effects were important protection mechanisms from the response to global climate change
Bioactive halogenated dihydroisocoumarins produced by the endophytic fungus Lachnum palmae isolated from Przewalskia tangutica
No full textGuided by the UPLC-ESIMS profile, seven previously undescribed halogenated dihydroisocoumarins, palmaerones A-G, along with eleven known dihydroisocoumarins, were isolated from Lachnum palmae, an endophytic fungus from Przewalskia tangutica by exposure to a histone deacetylase inhibitor SAHA. Structures of the isolates were elucidated by analysis of their NMR, MS and optical rotation values. The antimicrobial, anti-inflammatory and cytotoxic activities of palmaerones A-G were evaluated. Palmaerones A-G showed antimicrobial activities against the strains (C. neoformans, Penicillium sp., C. albicans, B. subtilis and S. aureus), and palmaerone E exhibited potential antimicrobial activities against all the test strains with the MIC value in the range of 10-55 mu g/mL. Palmaerones A and E exhibited moderate inhibitory effects on NO production in LPS-induced RAW 264.7 cells, with the IC50 values of 26.3 and 38.7 mu M, respectively and no obvious toxicities were observed at 50 mu M. Palmaerone E showed weak cytotoxicity against HepG2 with the IC50 value of 42.8 mu M. This work provides an effective strategy for expanding natural product resource. (C) 2018 Elsevier Ltd. All rights reserved
Effect and Mechanism of Purple Sweet Potato Anthocyanins on Liver Cancer
No full text为研究紫甘薯花青素对体外培养的人肝癌细胞SNU-387的影响及其可能的机制。在不同终浓度紫甘薯花青素处理下,利用MTT法和Hoechst 33258染色法,测定人肝癌细胞SNU-387增殖活力,通过测定细胞TNF-alpha含量来考察死亡受体TNFR1介导的外源性凋亡通路的作用,通过测定细胞内Caspase-8表达量变化来考察NF-kappaB和MAPK信号通路的作用。结果表明在细胞凋亡实验中,不同浓度紫甘薯花青素作用下,人肝癌细胞SNU-387增殖活力降低,出现核固缩等凋亡特征,细胞内TNF-alpha含量和Caspase-8表达量提高;在阻断剂PDTC作用下,Caspase-8表达量无差异;在阻断剂SB203580作用下,Caspase-8表达量下降。综上,紫甘薯花青素可以通过死亡受体TNFR1介导的外源性凋亡通路和MAPK信号通路参与介导肝癌细胞SNU-387的凋亡过程
微管解聚药物加速番茄果实成熟机制的研究
No full text研究背景和目的：番茄因具有营养品质高、用途广泛和环境适应力强等特点，在国内外市场上的需求量和栽培面积都在逐年增加。同时，番茄也成为研究果实发育的模式植物。研究番茄果实成熟的调节机制，不仅可以从分子水平上阐述植物果实发育成熟和衰老相关的生物学机理，而且可以为延长番茄采后贮藏时间的遗传改良提供新的思路和方法。目前国内外对番茄果实成熟分子机理的研究主要集中在乙烯的生物合成、信号感知和信号转导、乙烯下游应答基因的表达和功能分析。植物微管是一种高度动态性的细胞骨架，参与细胞的分裂和延伸，细胞壁合成，而且有研究者发现微管参与植物的衰老过程和对环境胁迫的应对，但是微管是否影响果实成熟的研究还未见报道。本文将粗略阐述微管参与调控果实成熟的可能机制。研究方法：以番茄植株(Solanum lycopersicum cv. MicroTom)为实验材料，对开花后22 天的果实注射微管解聚药物戊炔草胺（Propyzamide），对照组注射相应的缓冲液，观察两组间表型差异。同时分别收获注射前，注射后5 天，14 天，20 天的果实，液氮保存，分别对三组处理组和三组对照组进行RNA-seq 分析，比较同一时期两组间基因表达的差异。同时Western-blot 检测果实中微管蛋白Tubulin 的含量，抽提叶绿素和番茄红素，分别采用紫外分光光度计法和HPLC检测果实中色素含量，研究微管异常后对番茄果实成熟的影响。研究结果：微管解聚药物戊炔草胺处理相同时期番茄果实后，观察表型，发现较对照组而言，处理组果实更早进入破色期，提前3 天左右；同时分析果实处理后不同阶段叶绿素和番茄红素含量的变化情况，发现处理组比对照组叶绿素含量降低更快，番茄红素合成启动更早，积累更快，表明处理组果实加速成熟；提取果实总蛋白，用Western-blot 检测果实各个时期微管蛋白alpha-Tubulin (TUA)、微丝Actin 蛋白和谷氨酰胺合成酶GS 蛋白含量，发现在果实成熟过程中Actin和TUA 蛋白含量都逐渐降低，但是TUA 蛋白含量降低的更明显。比较同一时期的处理组与对照组可以发现，药物对Actin 丰度影响较小，但显著降低TUA丰度，说明药物处理确实破坏了微管，加速TUA 蛋白的降解。此外，比较转录组数据表明处理组乙烯合成和应答途径的基因如ACO1，ETR 等表达明显提高，且胡萝卜素合成信号途径中如GGPS1、PSY1、Z-ISO 和ZDS 基因表达也显著提高。结论：综合上述结果，我们发现使用微管干扰药物破坏微管动态稳定性能够促使番茄果实早熟，并且初步推断微管对果实成熟调控方式的机理是通过促进乙烯的合成，激活乙烯信号途径，并使类胡萝卜素合成途径的基因表达升高，从而加速番茄果实的成熟。本研究首次揭示了微管骨架参与调控番茄果实的成熟过程，不仅为进一步深入研究调节果实成熟的分子机制提供了新的方向，而且为操控番茄果实成熟过程或遗传改良番茄果实成熟时间提供了新的靶点和思路
万古霉素及阿尼芬净相关杂质分离鉴定与活性研究
No full text本论文的研究工作包含两个部分：第一部分是万古霉素相关杂质分离提纯、结构鉴定与活性研究；第二部分是阿尼芬净原料药相关杂质分离提纯与结构鉴定。第一部分是万古霉素相关杂质分离提纯、结构鉴定与活性研究。万古霉素是从东方链霉菌的发酵液中分离得到的一种糖肽类抗生素，是目前临床上用于治疗由MRSA引起的严重感染疾病的首选药物。万古霉素中包含多种不易分离纯化的微量杂质，尽管欧洲药典和中国药典已经标注出5种可能存在的相对杂质，但依然有一些小组分杂质没有被分离纯化和结构鉴定，这些发酵或降解产生的相关杂质可能存在严重的毒副作用，也有可能是高活性的新化合物。目前万古霉素的研究主要集中在寻找创造高活性化合物和药物质量控制上，对万古霉素相关杂质进行研究有助于发现毒副作用小、抗菌活性高的新抗生素，也为完善产品质量信息提供基础。因此本文首先对万古霉素产品中相关杂质进行HPLC定位，再从粗品中对相关杂质进行富集纯化，通过反向柱中压层析和高效液相色谱各种分离纯化技术，从万古霉素产品中分离纯化出12个相关杂质，且HPLC纯度大于93%，结合质谱、一维核磁（1H NMR、13C NMR、H-D NMR、DEPT135、DEPT90）、二维核磁（COSY、TOCSY、NOESY、HSQC、HMBC）对12个相关杂质进行结构鉴定，发现5个N-甲基亮氨酸部位发生改变的万古霉素类似物（杂质A、G、J、E、F），4个天冬酰胺部位发生改变的万古霉素类似物（杂质C、D、H、I），1个N-甲基亮氨酸部位、天冬酰胺部位均有所改变的万古霉素类似物（杂质B），2个糖基部位改变的万古霉素类似物（杂质L、O）。其中杂质B、C、H、E、F、J、L、O为已知相关杂质：去甲基及脱氨基万古霉素、脱氨基万古霉素（两种异构体杂质C和杂质H）、去甲基万古霉素（两种异构体杂质E和杂质F）、N，N-二甲基万古霉素、无糖万古霉素和脱万古糖胺单糖万古霉素。杂质A、D、G、I为首次发现的新化合物：二醛基万古霉素、亚甲基万古霉素、醛基万古霉素和开环中间体。对8个已知相关杂质和4个新结构的相关杂质进行抑菌测试，结果显示杂质F对金黄色葡萄球菌、枯草芽孢杆菌的抑菌活性与万古霉素相当。随后采用人肾胚细胞测试12个相关杂质对人体潜在的肾胚细胞毒性，结果显示杂质A、B对人肾胚细胞的抑制作用小于万古霉素，体外肾胚细胞毒性较低。最后，对相关杂质的来源进行了简单的分析。本研究首次对万古霉素相关杂质进行系统的分离纯化、结构鉴定和活性研究，共分离鉴定12个相关杂质，其中4个为新发现的化合物，活性测试表明2个相关杂质抑菌活性与万古霉素相当，2个相关杂质的体外肾胚细胞毒性低于万古霉素，这一研究进一步完善了国产万古霉素产品的质量信息，并为寻找抑菌活性高，毒副作用小的新万古霉素类抗生素提供了有利的参考。第二部分是阿尼芬净原料药相关杂质分离提纯与结构鉴定。阿尼芬净是第三代棘白菌素类抗真菌药物，在我国目前处于临床实验阶段，其是从发酵产物半合成而来，产品中可能存在影响药物质量的相关杂质。中国药典规定，在我国生产上市的新药需要进行杂质鉴定研究，而目前尚未有阿尼芬净中相关杂质研究的报道。本文参照辉瑞公司报道的合成路线，合成4批次的阿尼芬净原料药，采用一种新的HPLC 方法和UPLC-MS 方法对自制原料药和辉瑞公司产品中相关杂质的分布情况进行分析，共发现8个潜在的相关杂质。随后利用HPLC定位杂质，并采用制备色谱方法从阿尼芬净粗品中分离纯化出4个相关杂质（杂质I、杂质II、杂质III和杂质VIII），结合质谱、一维核磁、二维核磁方法对分离纯化出的4 个相关杂质进行结构鉴定，发现杂质I为阿尼芬净开环产物，杂质II和杂质III分别为吡咯烷环部位或苏氨酸部位少一个甲基的阿尼芬净类似物，而杂质VIII为多一个甲醚结构的阿尼芬净类似物。结合文献及合成工艺路线分析，我们推断杂质I为阿尼芬净水解开环产物，杂质II、杂质III和杂质VIII为合成起始原料中棘白菌素类杂质随合成反应生成的过程杂质。本研究工作不仅首次采用HPLC方法系统分析比较了自制阿尼芬净原料药与辉瑞公司产品中相关杂质的分布情况，而且采用制备色谱方法从原料药中分离纯化出的4个相关杂质，并结合各种波谱学方法对其进行详细的结构鉴定，发现3个为未见报道的新化合物，通过本研究工作可以为阿尼芬净质量信息提供参考，同时对杂质形成来源进行分析也有利于改进生成工艺，提高阿尼芬净药物质量
铜催化苯乙烯不对称硼胺化反应研究
No full text在有机合成中，有机硼试剂是一类重要的化合物，在碳碳键和碳杂键的构建中具有很高的实用性，并且广泛存在于天然活性产物，药物和功能材料的合成中。过渡金属催化的碳碳不饱和键双官能化由于其直接通用的优点引起了广泛的关注。通过这种方法可以在一步合成操作中同时引进两个官能团，在引进不同的两个官能团时往往会遭遇区域选择性和立体选择性的问题。鉴于氨基在很多天然产物，药物和不对称催化剂中是一个常见片段，同时也是有机合成中的一个具有很高价值的构建模块，对于发展一种新的高效合成胺类化合物的方法仍然有很大的需求。发展烯烃的硼胺化在此显得非常重要，尽管对于烯烃的铜催化硼胺化也已经初步建立，但是简单苯乙烯的不对称硼胺化仍然具有挑战。 我们课题组一直致力于铜硼物种对于简单苯乙烯的不对称加成策略，并且由我们自己发展的手性亚砜膦配体显示出了优越的性能。根据课题组已有的工作基础和查阅相关文献，对苯乙烯的不对称硼胺化进行了尝试。通过系统地优化条件，最后确定以氯化亚铜和手性亚砜膦配体作为催化剂，O-苯甲酰-N,N-二苄基羟胺作为亲电性胺试剂，联双联频哪醇硼酸酯作为亲核硼源，使用叔丁醇锂，四氢呋喃和乙酸乙酯的混合溶剂为最优反应条件，随后在温和的氧化条件下以好的收率，优秀的区域以及好至优秀的立体选择性获得β-羟胺（收率高达92%,ee高达95%）。为了证明该反应的实用性，对这一反应进行了克级规模放大，取得了优秀的收率和选择性，并且顺利地完成了苄基的脱除
重组羰基还原酶ChKRED20的分子改造
No full text手性醇是合成多种手性药物的重要中间体，羰基还原酶可以高效地催化潜手性酮的还原，是制备手性醇的重要方法之一。来源于金黄杆菌CA49 (Chryseobac terium sp. CA49)的羰基还原酶ChKRED20对多种苯乙酮类衍生物及脂肪族酮酯类具有很高的催化活性及优异的立体选择性，该酶能够高效不对称生物合成上市药物替格瑞洛、立普妥及阿瑞匹坦等的手性醇中间体。本研究采用蛋白质工程技术，结合定向进化和基于晶体结构的半理性设计方法，对该酶的性能进行了多方位的提升：(1) 大幅增强了ChKRED20的热稳定性；(2) 提高了该酶对药物中间体2-氯-1-(3,4-二氟苯基)乙酮的活性；(3) 拓展了对大位阻底物的催化能力；(4)反转了对邻位苯乙酮衍生物的立体选择性。结合羰基还原酶ChKRED20全酶及ChKRED20/NAD+复合物的晶体结构，对酶活性、底物特异性和立体选择性与酶细微结构之间的关系进行了深入地分析。论文主要包括以下几部分工作：第一部分，通过定向进化提高羰基还原酶ChKRED20的热稳定性。(S)-4-氯-3-羟基丁酸乙酯((S)-CHBE)是具有重要工业意义的手性醇中间体，用于合成HMG-CoA的还原酶抑制剂，例如重磅炸弹药物立普妥。重组的野生型羰基还原酶ChKRED20可催化前体4-氯乙酰乙酸乙酯(COBE)的不对称还原生成高光学纯度的(S)-CHBE。为了进一步提高催化效率，我们试图利用耐热性较好的突变子在较高的反应温度下实施生物催化反应温度，使合成(S)-CHBE的时空转化率得以提高。通过对ChKRED20基因进行随机突变并对文库进行高通量筛选，并进一步整合3个有益的突变位点，获得了热稳定性大幅度提高的突变体MC135，其最适反应温度升高至65 °C，连续处理15天后残余活性大于95%，相比之下，野生型ChKRED20在65 °C的热失活半衰期(t1/2)仅有11.9 min。突变体MC135粗酶液(3g/l)在65 °C条件下可在1小时内完全转化300 g/L的底物，ee 值 >99.5 %，时空产率为1824 mM/h，而野生型ChKRED20在该温度下30min内便完全失去活力；野生型粗酶液(3 g/l)在50 °C条件下完全转化300 g/L底物则需要3小时，时空产率为608 mM/h。第二部分：通过定向进化增强该酶对药物中间体2-氯-1-(3,4-二氟苯基)乙酮(CFPO)的活性。在替格瑞洛手性醇中间体(S)-2-氯-1-(3,4-二氟苯基)乙醇 ((S)-CFPL)的生物合成中，我们筛选了同样的随机突变文库，获得了比活力较高的突变子H145L、L205M，并进一步构建和筛选了这两个位点的饱和突变文库，获得了酶活力大幅提高的突变子L205A。突变子L205A的比活力为178 U/mg，是野生型比活力的10倍，并且该突变体的催化效率(kcat/Km)是野生型的15倍，在50 °C的热失活半衰期(t1/2)增加了70%。在40 °C 条件下，突变体L205A粗酶液(3g/L)可在20小时内完全转化200 g/L的底物，生物合成光学纯(S)-CFPL，ee值>99.5%，而野生型ChKRED20不能耐受200 g/L的底物浓度，完全转化100和150g/L底物分别需要16小时和26小时。该工作对生物合成法生产替格瑞洛手性醇中间体的实际应用具有重要推动作用。第三部分，羰基还原酶全酶及ChKRED20/NAD+复合物晶体结构的解析。我们分别通过镍柱/GST亲和层析柱、阴离子交换色谱、Superdex 75凝胶排阻色谱三步蛋白纯化获得了ChKRED20结晶级纯酶，然后采用悬滴法对ChKRED20的结晶条件进行筛选，最终利用X-ray 晶体衍射技术得到了分辨率为1.8?的ChKRED20晶体结构及1.6?的ChKRED20/NAD+复合物晶体结构。ChKRED20晶体结构的亚单元拓扑结构与其他同源结构类似，为Rossman-fold结构，主要由6个α/β二级结构单元构成，平行β片层位于内部，α螺旋位于外部。其中β片层较短，外侧的α螺旋较长，从而形成一个大的凹陷的口袋。ChKRED20/NAD+复合物晶体结构中，NAD+作为辅酶，平躺在底物口袋中，深入口袋底部，口袋入口处的柔性区域与ChKRED20晶体结构略有不同。第四部分，通过半理性设计拓展ChKRED20 酶的底物谱。基于对羰基还原酶ChKRED20晶体结构的分析，我们尝试对分子对接模拟结构中的底物邻位取代基附近9个氨基酸残基进行首轮丙氨酸扫描，然后对有益位点进行整合后发现突变体H145A/M201A在转化1-(2-氟苯基)乙酮具有更好的催化特性。我们进一步以H145A/M201A为模板，选择羰基还原酶ChKRED20的晶体结构中距离145 His和205Met在7?之内的9个氨基酸残基建立单点饱和突变文库，以1-(2-氯苯基)乙酮为底物，应用高通量筛选方法筛选的到底物特异性改变较大的H145A/ M201A/S153R和H145A/M201A /Y188L，其中，H145A/M201A/Y188L的立体选择性也发生了反转。然后，通过将底物口袋外部边缘的氨基酸残基进行饱和突变，并以1-(2，4-二甲基苯基)乙酮为底物进行高通量筛选，最终获得了底物特异性进一步改变的突变子H145A/M201A/S153R/Q97A/N39D，该突变子的底物谱比野生型ChKRED20更加广泛。综上，本研究将定向进化及半理性设计应用于羰基还原酶ChKRED20的分子改造，获得了若干功能改进的突变体，促进了该酶在多种手性药物中间体合成中的工业应用。并且，羰基还原酶ChKRED20/NAD+复合物晶体结构的解析不仅有助于我们了解其结构与功能的关系，而且为我们理解突变体酶功能改进的分子机制提供了一些线索
Influence of peripheral alkyl chain length on the mesomorphic behaviours of hexasubstituted triphenylene 2,3-dicarboxylic esters
No full textA comparative study of our established synthetic approaches to hexasubsituted triphenylenes 2,3-dicarboxylic esters containing four identical -alkoxy and two adjacent -alkoxycarbonyl side chains shows that the phase behaviours of small-sized discotic liquid crystals can be tailored over a wide range by simply varying the length of the peripheral alkyl chains. All the prepared esters in two series were observed to form a single hexagonal columnar phase, except for Tp4-1 having four -butyloxy and two adjacent -methoxycarbonyl chains which displays two columnar mesophase behaviours with a transition from the columnar plastic phase to hexagonal columnar phase. A significant difference between the two mesophase was observed in the variable temperature X-ray diffraction studies, and the mesophase assignment was also confirmed by polarising optical microscopy and differential scanning calorimetry. Moreover, the prepared esters in each series display the general trend of decreasing clearing temperature upon increasing alkoxy or alkoxycarbonyl chains length. The intermediate triphenylene 2,3-dicarboxylic acids were also found not only to exhibit columnar hexagonal mesophase over a narrower temperature range by maintaining high melting and clearing points but also to form organogel on mixing with toluene or dichloromethane with the assistance of hydrogen bonding. [GRAPHICS]