操纵茶树类黄酮3′-羟基化酶生物合成B环-3′,4′-二羟基黄酮类化合物

【目的】操纵茶树类黄酮3′-羟基化酶,生物合成B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素。【方法】构建了4个茶树类黄酮3′-羟基化酶基因(CsF3′H)和拟南芥的P450还原酶基因(ATR)融合表达质粒:SUMO-CsF3'H[7-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[7-517]::ATR2[75-711]3 AA和SUMO-CsF3'H[28-517]::ATR2[75-711]3 AA,分别转化大肠杆菌菌株TOP10、DH5α和BL21,获得12个转化菌株S1–S12;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒p YES-Dest52-CsF3′H,转化酵母菌株WAT11,得到转化菌株S13;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒pES-URA-CsF3′H,及茶树黄烷酮3-羟基化酶基因CsF3H与拟南芥黄酮醇合成酶基因At FLS的融合表达质粒pES-HIS-CsF3H::At FLS 9AA,二者共转化酵母菌株WAT11,获得转化菌株S14。【结果】转化SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA质粒的TOP10菌株S6在25°C条件下发酵,转化效率最高,能将1000μmol/L柚皮素、二氢山奈酚和山奈酚,分别转化生成287.93μmol/L圣草酚、131.76μmol/L二氢槲皮素和188.62μmol/L槲皮素。发酵菌株S13能分别将1000μmol/L柚皮素、二氢山奈酚和山奈酚,最多能转化生成734.32μmol/L圣草酚、446.07μmol/L二氢槲皮素和594.64μmol/L槲皮素。喂食S14发酵菌株5 mmol/L的底物柚皮素,在发酵36–48 h中,最多能生成1412.16μmol/L圣草酚、490.25μmol/L山奈酚、445.75μmol/L槲皮素、66.75μmol/L二氢槲皮素和73.50μmol/L二氢山奈酚。【结论】本研究首次将茶树类黄酮3′-羟基化酶基因应用于B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素的生物合成。     

Loss of Yme1L perturbates mitochondrial dynamics

Yme1L is an AAA protease that is embedded in the mitochondrial inner membrane with its catalytic domain facing the mitochondrial inner-membrane space. However, how Yme1L regulates mammalian mitochondrial function is still obscure. We find that endogenous Yme1L locates at punctate structures of mitochondria, and that loss of Yme1L in mouse embryonic fibroblast (MEF) cells results in mitochondrial fragmentation and leads to significant increased ‘kiss-and-run'' type of mitochondrial fusion; however, Yme1L knockdown (shYme1L (short hairpin-mediated RNA interference of Yme1L)) cells still remain normal mitochondrial fusion although shYme1L mitochondria have a little bit less fusion and fission rates, and the shYme1L-induced fragmentation is due to a little bit more mitochondrial fission than fusion in cells. Furthermore, shYme1L-induced mitochondrial fragmentation is independent on optic atrophy 1 (OPA1) S1 or S2 processing, and shYme1L results in the stabilization of OPA1 long form (L-OPA1); in addition, the exogenous expression of OPA1 or L-OPA1 facilitates the shYme1L-induced mitochondrial fragmentation, thus this fragmentation induced by shYme1L appears to be associated with L-OPA1''s stability. ShYme1L also causes a slight increase of mitochondrial dynamics proteins of 49 kDa and mitochondrial fission factor (Mff), which recruit mitochondrial key fission factor dynamin-related protein 1 (Drp1) into mitochondria in MEF cells, and loss of Drp1 or Mff inhibits the shYme1L-induced mitochondrial fragmentation. In addition, there is interaction between SLP-2 with Yme1L and shYme1L cells retain stress-induced mitochondrial hyperfusion. Taken together, our results clarify how Yme1L regulates mitochondrial morphology.     

Crystallographic studies of drug-nucleic acid interactions: Proflavine intercalation between the non-complementary base-pairs of cytidilyl-3′,5′-adenosine

In order to get insights into the binding of dyes and mutagens with denatured and single-stranded nucleic acids and the possible implications in frameshift mutagenesis, a 1:1 complex between the non-self-complementary dinucleoside monophosphate cytidilyl-3′,5′-adenosine (CpA) and proflavine was crystallized. The crystals belong to the tetragonal space group P4₂2₁2 with cell constants $\text{a = b = 19.38(1)}\text{A}\text{?}\text{and}\text{c = 27.10(1)}\text{A}\text{?}$. The asymmetric unit contains one CpA, one proflavine and nine water molecules by weight. The structure was determined using Patterson and direct methods and refined to an R-value of 11% using 2454 diffractometer intensities.The non-self-complementary dinucleoside monophosphate CpA forms a selfpaired parallel chain dimer with a proflavine molecule intercalated between the protonated cytosine-cytosine (C · C) pair and the neutral adenine-adenine (A · A) pair. The dimer complex exhibits a right-handed helical twist and an irregular girth. The neutral A · A pair is doubly hydrogen-bonded through the N(6) and N(7) sites (C(1′)C(1′) distance: 10.97(2) Å) and the protonated C · C pair is triply hydrogen-bonded with a proton shared between the N(3) sites (C(1′)C(1′) distance: 9.59(2) Å). To accommodate the intercalating dye, the sugars of successive nucleotide residues adopt the two fundamental conformations (5′ end: 3′-endo, 3′ end: 2′-endo), the backbone adopts torsion angle values that fluctuate within their preferred conformational domains: the PO bonds (ω, ω′) adopt the characteristic helical (gauche^?-gauche^?) conformation, the CO bonds (φ, φ′) are both in the trans domain and the C(4′)C(5′) bonds (ψ) are in the gauche⁺ region. The bases of both residues are disposed in the preferred anti domain with the glycosyl torsion angles (χ) correlated to the puckering mode of the sugar so that the cytidine residue is C(3′)-endo, low χ (12 dg), and the adenosine residue is C(2′)-endo, high χ (84 °). The intercalated proflavine stacks more extensively with the C · C pair than the A · A pair. Between 4₂-related CpA proflavine units there is a second proflavine which stacks well with both the A · A and the C · C pairs sandwiching it. Both proflavine molecules are positionally disordered. In each of its two disordered sites, the intercalated proflavine forms hydrogen-bonded interactions with only one sugar-phosphate backbone. A total of 26 water sites has been characterized of which only two are fully occupied. These hydration sites are involved in an intricate network of hydrogen bonds with both the dye and CpA and provide insights on the various modes of interactions between water molecules and between water molecules and nucleic acids.The structure of the proflavine-CpA complex shows that intercalation of planar drugs can occur between non-complementary base-pairs. This result can be relevant for understanding the strong binding of acridine dyes to denatured DNA, single-stranded RNA, and single-stranded polynucleotides. Also, the ability of proflayine to promote self-pairs of adenine and cytosine bases could provide a chemical basis for an alternative mechanism of frameshift mutagenesis.     

Comparison of full-length genomics sequences between dengue virus serotype 3, parental strain,and its derivatives,and B-cell epitopes prediction from envelope region

Biological markers are normally used to evaluate the candidate of live-attenuated dengue vaccines. D3V 16562 Vero 23 and D3V 16562 Vero 33 which were derivatives of D3V 16562, parental strain, showed the similar biological data. We used molecular techniques and computational tools to evaluate these derivatives. The nucleotide and amino acid sequences of the derivatives were compared to their parent. The secondary structures of untranslated regions and B-cell epitopes were predicted. The results showed that nucleotide substitutions mostly occurred in NS5 and NS5 of V2 was unusual because of amino acid change at 3349 (tryptophan →stop codon). The nucleotide substitutions in 5''UTR, prM, E, NS1, NS2A, NS3, and 3''UTR were 4, 1, 2, 2, 1, 3, and 2, respectively. The secondary structure of 5''UTR of V2 was different from P and V1. The secondary structure of 3''UTR of V2 was similar to P and certainly distinct from V1. Furthermore, B-cell epitopes prediction revealed that there were 21 epitopes of envelope and the interesting epitope was at position 297-309 because it was in domain III in which the neutralizing antibody is induced. For this study, the attenuation of derivatives was caused by the nucleotide substitutions in 5''UTR, 3''UTR, and NS5 regions. The genotypic data and B-cell epitope make the derivatives attractive for the chimeric and peptide DENV vaccine development.     

两湖泊轮虫群落结构的时空动态对水体氮磷含量及“下行效应”的响应

为探讨氮和磷、"上行效应"和"下行效应"在轮虫群落结构时空变动调控中的相对重要性,于2011年7月至2012年6月每月两次采集了芜湖市九莲塘和汀棠湖中的轮虫样品,利用相关分析法和典范对应分析法(CCA)分析了轮虫群落结构的时空变动与水体理化因子及其潜在捕食者密度间的关系。卡尔森营养状态指数表明,两湖均处在富营养化初期。除了总氮和硝态氮的年均浓度、晶囊轮虫(Asplanchna)和剑水蚤(Cyclopoidea)无节幼体的年均密度存在显著差异外(P0.05),两湖泊的其他水环境因子间并未表现出明显的差异(P0.05)。实验室鉴定发现九莲塘中共有轮虫48种,隶属于15科21属;汀棠湖中共有轮虫55种,隶属于15科24属。两湖泊中,疣毛轮虫(Synchaeta)、臂尾轮虫(Brachionus)和异尾轮虫(Trichocerca)均是主要轮虫类群,而密度优势种分别有7种和4种。相关分析结果显示,P含量与九莲塘轮虫群落的均匀度和物种多样性指数间均呈现显著的负相关(P0.05),而N含量却与汀棠湖轮虫群落的物种多样性指数呈现显著的正相关(P0.05);两湖泊中的轮虫物种多样性指数均与水体中的TN∶TP(质量比)间呈现显著的正相关(P0.05)。CCA分析结果显示,九莲塘中桡足类和其无节幼体密度显著高于其他采样批次时的轮虫群落聚成Ⅰ类,汀棠湖中晶囊轮虫密度显著高于其他批次时的轮虫群落聚成Ⅱ类,而其他的轮虫潜在捕食者密度很低或未出现时的轮虫群落聚成Ⅲ类。研究结果表明,湖泊中的TN∶TP(质量比)可能是反映N和P含量对轮虫群落物种多样性影响程度的较为合适的指标。当水体中的轮虫潜在捕食者密度较高时,两湖泊轮虫群落结构的变动均主要取决于"下行效应";而当轮虫潜在捕食者密度很低时,"上行效应"可能是调控两湖泊轮虫群落结构变动的主要因素。     

喜盐鸢尾花色形成关键基因的克隆及表达分析

喜盐鸢尾(Iris halophila Pall.)及其变种蓝花喜盐鸢尾(I.halophila Pall.var.sogdiana(Bung)Grubov)因耐盐碱及其多种花色而具有盐碱地园艺开发价值。本文根据喜盐鸢尾内轮花被转录组测序结果,利用基因特异性引物从这2种植物中分别克隆了编码查尔酮合成酶(CHS)、查尔酮异构酶(CHI)、类黄酮-3',5'-羟基化酶类(F3'5'H-like)等基因的部分片段,并对它们在内轮花被中的表达水平进行实时定量PCR分析。序列分析结果确认在喜盐鸢尾中所克隆的CHS(311 bp)、CHI(457 bp)、F3'5'H-like(496 bp)3个基因(部分)未见文献报道与NCBI等数据库记录。其中F3'5'H-like基因与经典的属于细胞色素P450CYP75A亚家族的F3'5'H不同,而与万带兰的F3'5'H-like同属于CYP76AB亚家族,为一类新的蓝花相关基因。实时定量PCR表达分析结果表明,与黄花的喜盐鸢尾相比,蓝花喜盐鸢尾中CHS与F3'5'H-like显著上调表达,可能是其花色不同于喜盐鸢尾的主要原因。     

Sulfolobus acidocaldarius DHH超家族核酸酶Saci0542的酶学特征研究

【目的】以嗜酸嗜热硫化叶菌Sulfolobus acidocaldarius的DHH超家族核酸酶（Saci0542）为例,研究其核酸外切酶活性特点,为阐明其在DNA代谢中的具体功能提供生化基础。【方法】将嗜酸嗜热硫化叶菌DHH超家族核酸酶Saci0542基因在大肠杆菌中重组表达,经亲和层析纯化得到电泳纯的重组蛋白;利用荧光标记的寡核苷酸作为底物,用尿素变性聚丙烯酰胺凝胶电泳技术,鉴定Saci0542的酶学特征。【结果】重组表达的DHH超家族核酸酶Saci0542具有典型的单链核酸特异性的3’-5’外切酶活性。进一步酶学特征表征结果如下:酶活性依赖于二价金属离子Mn²⁺,而Ca²⁺、Mg²⁺、Zn²⁺等二价金属离子对活性没有明显的促进作用;Saci0542在pH5.5–10的广泛范围内均表现出较高酶活性;高于200 mmol/L的NaCl强烈抑制酶活性;最适反应温度为50–55℃;末端磷酸基团抑制3’-5’外切酶活性。【结论】本研究证实,Saci0542是一种Mn²⁺依赖型3’-...     

Hamilton's rule meets the Hamiltonian: kin selection on dynamic characters

Many biological characters of interest are temporal sequences of decisions. The evolution of such characters is often modelled using dynamic optimization methods such as the maximum principle. A quantity central to these analyses is the ''Hamiltonian'' function, named after the mathematician William R. Hamilton. On the other hand, evolutionary models in which individuals interact with relatives are usually based on Hamilton''s rule, named after the evolutionary biologist William D. Hamilton. In this article we present a generalized maximum principle that includes the effects of interactions among relatives and we show that a time-dependent (dynamic) version of Hamilton''s rule holds involving the Hamiltonian. This result brings together the power and generality of both the maximum principle and Hamilton''s rule thereby providing a natural framework for understanding the evolution of ''dynamic'' characters under kin selection.     

Effects of increasing the number of players and memory size in the iterated Prisoner's Dilemma: a numerical approach

The Prisoner''s Dilemma has become a paradigm for the evolution of altruistic behaviour. Here we present results of numerical simulations of the infinitely iterated stochastic simultaneous Prisoner''s Dilemma considering players with longer memory, encounters of more than two players as well as different pay-off values. This provides us with a better foundation to compare theoretical results to experimental data. We show that the success of the strategy Pavlov, regardless of its simplicity, is far more general by having an outstanding role in the iterated N-player N-memory Prisoner''s Dilemma. Besides, we study influences of increased memory sizes in the iterated two-player Prisoner''s Dilemma, and present comparisons to results of experiments with first-year students.     

Morphological variations in a widely distributed Eastern Asian passerine cannot be consistently explained by ecogeographic rules

Ecogeographic rules that describe quantitative relationships between morphologies and climate might help us predict how morphometrics of animals was shaped by local temperature or humidity. Although the ecogeographic rules had been widely tested in animals of Europe and North America, they had not been fully validated for species in regions that are less studied. Here, we investigate the morphometric variation of a widely distributed East Asian passerine, the vinous‐throated parrotbill (Sinosuthora webbiana), to test whether its morphological variation conforms to the prediction of Bergmann''s rule, Allen''s rules, and Gloger''s rule. We at first described the climatic niche of S. webbiana from occurrence records (n = 7838) and specimen records (n = 290). The results of analysis of covariance (ANCOVA) suggested that the plumage coloration of these parrotbills was darker in wetter/warmer environments following Gloger''s rule. However, their appendage size (culmen length, beak volume, tarsi length) was larger in colder environments, the opposite of the predictions of Allen''s rule. Similarly, their body size (wing length) was larger in warmer environments, the opposite of the predictions of Bergmann''s rule. Such disconformity to both Bergmann''s rule and Allen''s rule suggests that the evolution of morphological variations is likely governed by multiple selection forces rather than dominated by thermoregulation. Our results suggest that these ecogeographic rules should be validated prior to forecasting biological responses to climate change especially for species in less‐studied regions.     

Effects of Cycloate on Development of Heterodera schachtii and Growth of Three Beta Species

Greenhouse tests were set up to evaluate the effects of the herbicide, cycloate (S-ethyl cydohexylethylthiocarbamate), oil development of Heterodera schachtii and growth of three Beta species. Cycloate added to infested soil enhanced cyst development/gm root on B. vulgaris and larvae/gm of root in B. patellaris and B. procumbens at 4, 16, and 16 μg(a.i.)/gm of soil, respectively. Total numbers of nematodes/individual root system decreased because of poor root growth of seedlings in cycloate-amended soil. Penetration and larval development through stage three did occur in the wild Beta species in any treatment. Thus, resistance of B. patellaris and B. pocumbens to development of H. schachtii was not altered by cycloate. Cycloate also retarded growth (P = 0.05) of the sugarbeet cultivars and B. patellaris at 4 μg(a.i.)/gm and B. procumbens at 16 μg(a.i.)/gm of soil. Higher concentrations of nematodes/gm root in plants growing in cycloate-amended soil may be attributed to factors such as fewer roots available for penetration, possible effects of cycloate on egg hatch, greater attraction of nematodes to roots, and increased susceptibility of roots to larval penetration. Suppression of seedling growth in cycloate-amended soil may be attributed in part to higher nematode density and in part to direct root damage from cycloate.     

城市小尺度生态基础设施的设计方法——以雄安新区启动区为例

高速的城市化进程带来一系列生态破坏和环境污染等可持续发展挑战，需要在城市规划阶段尽量规避这些风险。生态基础设施建设倡导生态系统修复和环境协同治理的理念，是新兴城市化背景下指导城市规划的一种有效手段。目前，国内外生态基础设施建设在宏观尺度研究比较系统，而在小尺度的研究相对欠缺。我国社区等小尺度生态基础设施建设，由于规模较小，缺乏网络化构建，导致了建设后雨水内涝、面源污染问题仍层出不穷。构建了一种城市小尺度生态基础设施的设计方法和技术流程，以雄安新区启动区为研究区开展生态基础设施设计：（1）基于实际调研与理论研究分析场地现状，综合考虑自然要素、物理感知、心理感知、生态过程等相关因素构建"廊道为骨，斑块为节"的生态基础设施体系；（2）辨识区域主要动物活动、迁徙以及保护植物多样性等功能生态斑块，构建串联全城提供多种功能的系统性廊道、增加城市内部生态系统服务的结构性廊道、将生态系统服务渗入城市肌理的局部功能性廊道；（3）从景感满意度和年径流总量控制率2个方面进行生态基础设施建设预期效果评估。研究结果有助于缓解雄安新区建设所面临生态环境问题，保障人居生活环境品质，并为今后城市建设工作提供参考。     

Microbial Degradation of α-Picolinic Acid, 2-Pyridinecarboxylic Acid

The biological effects of raw winged bean seeds were investigated with feeding experiments on rats, and the effects of lectin (phytohemagglutinin) present in the seeds are discussed. Administration of a 30% raw winged bean diet caused strong growth depression in young rats, and led to death within 10 ~ 20 days, inducing severe damage to the small intestine of the rats. Significant morphological changes of the intestinal mucosa were observed with a microscopic investigation. As the lethal effect was eliminated by autoclaving but not removed with supplementation of 0.5% l-methionine to the raw winged bean diet, the lectin was assumed to be closely related to the deleterious effects of raw winged bean. In vitro and in vivo digestion tests of the lectin revealed that the winged bean lectin had resistance to peptic, pancreatic and membrane digestions. The hemagglutinating activity was also detected in the intestinal mucosa and faeces from rats ingesting the raw winged bean or its purified lectin. The binding action of lection to mucosal epitheliums of the gastrointestinal tract is suggested to be the initial step of the deleterious effects induced by the winged bean lectin.     

基于工程化毕赤酵母一锅法合成硫酸软骨素A北大核心CSCD

硫酸软骨素(chondroitin sulfate,CS)是一种线性多糖,广泛应用于医疗和保健等领域。相比于传统动物组织提取法,微生物合成硫酸软骨素具有可控、易规模化放大等优势。为实现硫酸软骨素A(CSA)的高效合成,本研究首先通过整合软骨素合酶编码基因kfoC、kfoA以及UDP-葡萄糖脱氢酶编码基因tuaD至毕赤酵母GS115基因组中,构建了以甘油为唯一碳源发酵生产软骨素的毕赤酵母工程菌株。通过进一步优化软骨素合成途径,软骨素分批补料发酵水平达到2.6 g/L。在进一步整合表达软骨素-4-O-磺基转移酶的基础上,本研究通过向生产软骨素毕赤酵母工程菌株破碎液中添加3′-磷酸腺苷-5′-磷酰硫酸和软骨素-4-O-磺基转移酶,成功建立了CSA的一锅法生物合成体系。通过优化,最终实现0-40%不同磺酸化水平CSA的可控合成。本研究中CSA的一锅法生物合成体系操作简便、易放大,更适用于工业化大规模生产。本研究结果也为肝素等其他糖胺聚糖的合成提供了思路。     

Myotonia congenita: novel mutations in CLCN1 gene

Myotonia congenita belongs to the group of non-dystrophic myotonia caused by mutations of CLCN1gene, which encodes human skeletal muscle chloride channel 1. It can be inherited either in autosomal dominant (Thomsen disease) or recessive (Becker disease) forms. Here we have sequenced all 23 exons and exon-intron boundaries of the CLCN1 gene, in a panel of 5 unrelated Chinese patients with myotonia congenita (2 with dominant and 3 with recessive form). In addition, detailed clinical analysis was performed in these patients to summarize their clinical characteristics in relation to their genotypes. Mutational analyses revealed 7 different point mutations. Of these, we have found 3 novel mutations including 2 missense (R47W, V229M), one splicing (IVS19+2T>C), and 4 known mutations (Y261C,G523D, M560T, G859D). Our data expand the spectrum of CLCN1 mutations and provide insights for genotype–phenotype correlations of myotonia congenita in the Chinese population.     

NAD/NADP及其介导氧化应激在神经退行性疾病中的作用

神经退行性疾病如阿尔茨海默病、帕金森病、亨廷顿病等疾病的发生与氧化应激紧密相关。NAD和NADP是维持氧化系统和抗氧化系统平衡的两个关键物质。NAD和NADP的生物合成和降解有多种途径,参与其生物途径的物质如NAMPT、NADK、PARP1、SIRT1、CD38等,均报道在神经退行性疾病发挥一定的作用。因此,本文分别从NAD和NADP的合成和降解途径中的一些关键物质出发,结合氧化应激总结并探讨它们在神经退行性疾病的作用,以期为临床治疗神经退行性疾病提供新思路。