虎纹镇痛肽(HWAP-I)在大鼠蜜蜂毒致炎模型中的镇痛作用

研究了在蜜蜂毒致炎模型中虎纹镇痛肽 (HWAP I)的镇痛作用 .HWAP I有 6个剂量组 (n =10 ) ,分别为 0 .1、1、10、2 5、5 0、75 μg/kg .生理盐水作空白对照 .从 10 μg/kg开始不同剂量组表现不同程度的镇痛效果 (P<0 .0 5 ) ,且有一定的量效关系 ;同时用 2 5 μg/kg芋螺毒素SNX III做阳性对照 ,结果表明 2 5 μg/kgSNX III的镇痛效果介于 2 5 μg/kg与 5 0 μg/kgHWAP I之间 .计算得到HWAP I在给药后 1h半有效剂量ED50 大约是 30 μg/kg .     

药用植物川东獐牙菜的组织培养

针对川东獐牙菜野生资源受到严重破坏的情况 ,系统地探讨了通过组织培养为手段进行人工繁殖的方法 ,旨在为川东獐牙菜的保护提供坚实的理论依据。研究结果表明 :在所有的实验方案中 ,幼茎和老叶是理想的外植体材料。对叶片来说 ,较适宜的诱导愈伤组织的激素组合是 Zt1.0 m g/L +NAA 0 .0 5m g/L +IBA0 .0 5mg/L、BA 0 .5m g/L+2 ,4 - D0 .5mg/L或 BA 0 .2 m g/L+2 ,4 - D0 .2 mg/L+IBA0 .1m g/L ,对茎段来说 ,较适宜的诱导愈伤组织的激素组合是 BA0 .0 5m g/L+kt0 .0 5mg/L+IBA0 .0 5m g/L;诱导不定芽的适宜激素组合是 BA2 .0 mg/L+N AA0 .0 5mg/L或 BA 2 .0 mg/L+IBA 0 .1m g/L ;而根的诱导则是 MS+BA 0 .0 5mg/L+kt1.0 mg/L+N AA0 .1m g/L或 MS+BA1.0 m g/L+kt0 .3mg/L +IA A0 .5mg/L培养基上进行。     

扇蕨孢子的组织培养

1 植物名称扇蕨[Neocheiropteris palmatopedata (Baker)Christ]. 2 材料类别成熟孢子. 3 培养条件孢子萌发培养基:(1)B5 20 g·L-1蔗糖.原叶体增殖培养基:(1)B5 20 g·L-1蔗糖;(2)1/2B5 20 g·L-1蔗糖;(3)1/485 20 g·L-1蔗糖;(4)1/885 20g·L-1蔗糖;(5)B5,(6)B5 10 g·L-1蔗糖;(7)B5 30 g·L-1蔗糖;(8)B5 40 g·L-1蔗糖;(9)B5 50 g·L-1蔗糖;(10)B5 60 g·L-1蔗糖.     

中西医结合治疗带状疱疹4例

自 2 0 0 0年 2月～ 2 0 0 3年 8月 ,我科应用中西医结合方法治疗带状疱疹 4例 ,疗效显著 ,治愈率达 1 0 0 % ,现介绍如下。1　临床资料1 .1　一般资料　本组中男 2例 ,女 2例 ,年龄 45～ 76岁 ,平均 60岁 ;病程 4～ 62天。病变部位于颈部 1例 ,胸部 1例 ,腹部 1例 ,手部 1例。1 .2　治疗方法　西药 :用 5 %葡萄糖盐水 (或 5 %葡萄糖液 ) 2 5 0 ml加蝮蛇抗栓酶 0 .5 u静滴 ,每天 1次。中药 (龙胆泻肝汤加减 )组成 :龙胆草 6g,当归 1 0 g,柴胡 6g,黄苓 1 0 g,生地 1 5 g,山栀 1 0 g,大青叶 5 0 g,板兰根 5 0 g,乳香 1 0 g,没药 1 0 g,泽泻 1 …     

湖南会同杉木人工林生态系统碳素密度

利用定位观测数据 ,对杉木人工林生态系统的碳素密度进行了探讨。结果表明 :不同年龄的杉木枝、叶中碳素密度的季节变化规律均表现为冬季 >秋季 >夏季 >春季。叶的碳素密度平均为 0 .5 0 4 4 g C· g- 1 ,变异系数 2 .0 8% ,枝的碳素密度平均为0 .4 4 79g C· g- 1 ,变异系数为 2 .2 5 %。不同层次的叶碳素密度的变化范围在 0 .4 6 12 g C· g- 1 ～ 0 .5 5 2 4 g C· g- 1 之间 ,平均值的大小排列顺序为 :上层叶 >中层叶 >下层叶。不同层次枝条的碳素密度在 0 .3917g C·g- 1～ 0 .4 96 5 g C· g- 1之间 ,平均值的大小次序为 :中层枝 >上层枝 >下层枝。 10年生杉木各器官的碳素密度变化范围为 0 .4 5 2 9g C·g- 1～ 0 .4 972 g C·g- 1 ,11年生的为 0 .4 5 80 g C· g- 1 ～ 0 .5 0 2 2 g C· g- 1 ,14年生的为 0 .4 5 80 g C· g- 1 ～ 0 .5 0 93g C· g- 1 之间 ,变异系数范围为 1.6 8%～ 8.4 4 %。不同器官的碳素密度按高低排列基本上为树叶 >树皮 >树根 >树干 >树枝 >球果。随着杉木林年龄的增长 ,林下植被各组分、死地被物的碳素密度变化规律不明显。同一林分中各层次植物的碳素密度高低排列顺序为 :乔木层 >灌木层 >草本层。 10年生和 14年生杉木林土壤各层的碳素密度随土壤深度的增加而逐渐下     

红酵母类胡萝卜素形成的生理学研究

从数株红酵母中选出 1株产类胡萝卜素能力较强的红酵母RY 98(生物量、类胡萝卜素含量和产量分别为19.9g/L ,334 .8μg/ g和 6 .7mg/L) ;研究了该菌株产类胡萝卜素的最适营养与环境条件 ,获得了最佳的发酵生理学条件 :葡萄糖 40 g/L ,(NH4 ) 2 SO4 10 g/L ,酵母膏 3g/L ,蕃茄汁 2mL/L ,花生油 0 .5mL/L ,接种量 30mL/L ,初始pH 6 .0和通气量 (培养基装量 ) 4 0mL/ 2 5 0mL。在此初步优化的培养条件下 ,红酵母RY 98经 72h摇瓶发酵其生物量、类胡萝卜素含量和产量分别可达 2 6 .8g/L ,386 .9μg/ g和 10 .4mg/L ,依次比初筛中提高了 34 .7% ,15 .6 %和 5 5 .2 %。     

利用竞争型ELISA法鉴定硒诱导的金属硫蛋白

利用竞争型ELISA法鉴定硒诱导的金属硫蛋白 (MT) ,发现对照组小鼠肝脏MT含量为 (2 .47± 0 .90 )μg/g湿重组织 ,硫酸锌组小鼠肝脏MT含量为 (8.15± 2 .2 0 ) μg/ g湿重组织 ,硒麦芽组小鼠肝脏MT含量为(12 .80± 1.44 ) μg/ g湿重组织。锌组和硒组的MT含量与对照组相比有显著性差异 (P <0 .0 5 ,P <0 .0 5 )。硒组MT含量要显著高于锌组的MT含量 (P <0 .0 5 )。     

云南两种食用地衣营养成分研究

测定云南两种食用地衣的主要营养成分 ,并与常见蔬菜及食用菌进行比较。测定结果 ,东方肺衣粗蛋白质含量为 11 0 2 g/ 10 0 g .DW ,粗脂肪 5 5 6 g/ 10 0g .DW ,总糖、可溶性糖、灰分含量分别为 0 6 2g/ 10 0 g、0 37g/ 10 0g、2 6 4g/ 10 0 g .DW ;裂髓树花 (新拟 )的粗蛋白质含量为 3 43g/ 10 0g .DW ,粗脂肪3 12 g/ 10 0g .DW ,总糖、可溶性糖、灰分含量分别为 0 79g/ 10 0 g、0 35g/ 10 0 g、2 99g/ 10 0 g .DW ;两种地衣均含有 17种氨基酸 (色氨酸未测 ) ,多种矿物元素 ,具有一定营养价值 ;且两种地衣营养成分的含量存在差异     

牛磺酸对蟾蜍离体心脏活动及蝌蚪抗缺氧能力的影响

研究牛磺酸对蟾蜍离体心脏活动及蝌蚪抗缺氧能力的影响。结果表明 :在气温为 (2 0± 1)℃时 ,浓度为 5g·L- 1 ,10g·L- 1 的牛磺酸任氏液使离体心脏跳动时间明显延长 (p <0 .0 5 ) ,浓度为 1g·L- 1 、5g·L- 1 的牛磺酸水溶液对蝌蚪抗缺氧能力有极显著 (p <0 .0 1)、显著 (p <0 .0 5 )改善     

甲氰菊酯与阿维菌素的混剂防治美洲斑潜蝇田间试验

田间试验表明 ,1 9%甲氰·阿维EC 9 5g (a .i.) 667m2 对美洲斑潜蝇LiriomyzasativaeBlanchard的校正防效药后 3～ 1 1d为 87 2 2 %～ 91 3 2 % ;7 1 2 5g (a.i.) 667m2 药后 3～ 1 1d为 81 2 9%～ 85 1 0 % ;9 5g (a .i.) 667m2 药后 3～ 1 1d防效显著优于单剂甲氰菊酯 ,药后 3～ 7d显著优于单剂阿维菌素 ,药后11d防效与阿维菌素无显著区别。 1 5 %甲氰·阿维EC 7 5g (a .i.) 667m2 对美洲斑潜蝇的校正防效药后3～ 1 1d为 85 5 9%～ 89 0 9% ;5 62 5g (a .i.) 667m2 药后 3～ 1 1d为 77 1 5 %～ 85 95 %。 1 0 %甲氰·阿维EC 7 5g (a .i.) 667m2 对美洲斑潜蝇的校正防效药后 3～ 1 1d为 85 5 9%～ 89 0 9% ;5 62 5g (a .i.) 667m2 药后 3～ 1 1d为 77 1 5 %～ 85 95 %。混剂的药效基本随有效成分含量的提高而提高。     

Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips

Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ～35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ～2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.     

91 Kinetics of DNA overstretching: melting vs. B-to-S transition

Single B-form DNA molecules undergo an overstretching transition at force F_ov to a ～1.7-fold longer form when stretched. The nature of overstretched DNA has been debated for over 10?years. Either peeled (PL DNA), internally melted (M DNA), or unwound double-helical (S DNA) forms of overstretched DNA have been suggested. Here, we characterize the kinetics of the overstretching transition in polymeric torsionally unconstrained double-stranded (ds) DNA molecules. We pull ～50?Kbp λ–DNA molecules using optical tweezers with rates ν ～10?nm/s to 5?×?10⁴?nm/s, (overstretching time between 0.2 and 10³?s). The F_ov(ν, [Na⁺]) dependence measured over a broad range of rates and solution ionic strength suggests the existence of all three forms of the overstretched DNA. Thus, at [Na⁺]?>?50?mM and the stretching time >>1?s, internal melting dominates overstretching. This B-to-M transition is highly cooperative (involves ～100?bp), and slow (on/off time ～1000?s). Faster overstretching during ?1?s leads to B-to-S DNA transition, which is less cooperative (involves ～10?bp) and faster (on/off time ～1?s). In contrast, in lower salt ([Na⁺]?<?50?mM), the overstretching during >1?s leads to DNA peeling. However, on the faster time scale of 0.2–1?s, even in low salt, the DNA overstretches into S DNA, as peeling becomes kinetically prohibited. Our conclusions are supported by several independent lines of evidence, including the salt and rate dependence of both the slope of the overstretched DNA force-extension curve and the value of the second transition force (from M or PL DNA into S DNA).     

Reusable Solid-Phase Supports for Oligonucleotide Synthesis Using Hydroquinone-O,O'-diacetic Acid (“Q-Linker”)

Abstract

Reusable solid-phase supports for large scale oligonucleotide synthesis have been prepared by converting amino derivatized supports into hydroxyl supports. Rapid nucleo side attachment, via a Q-linker arm, was automatically performed on the DNA synthesizer using HBTU and DMAP as the coupling reagents. All steps were suitable for automation and ～ 1.5 h was required to prepare the supports for reuse. Up to twelve consecutive syntheses of a 20-mer phosphorothioate were performed on a synthesis column.     

Anchored hybrid enrichment for massively high-throughput phylogenomics

The field of phylogenetics is on the cusp of a major revolution, enabled by new methods of data collection that leverage both genomic resources and recent advances in DNA sequencing. Previous phylogenetic work has required labor-intensive marker development coupled with single-locus polymerase chain reaction and DNA sequencing on clade-by-clade and locus-by-locus basis. Here, we present a new, cost-efficient, and rapid approach to obtaining data from hundreds of loci for potentially hundreds of individuals for deep and shallow phylogenetic studies. Specifically, we designed probes for target enrichment of >500 loci in highly conserved anchor regions of vertebrate genomes (flanked by less conserved regions) from five model species and tested enrichment efficiency in nonmodel species up to 508 million years divergent from the nearest model. We found that hybrid enrichment using conserved probes (anchored enrichment) can recover a large number of unlinked loci that are useful at a diversity of phylogenetic timescales. This new approach has the potential not only to expedite resolution of deep-scale portions of the Tree of Life but also to greatly accelerate resolution of the large number of shallow clades that remain unresolved. The combination of low cost (～1% of the cost of traditional Sanger sequencing and ～3.5% of the cost of high-throughput amplicon sequencing for projects on the scale of 500 loci × 100 individuals) and rapid data collection (～2 weeks of laboratory time) are expected to make this approach tractable even for researchers working on systems with limited or nonexistent genomic resources.     

Cooperative cluster formation, DNA bending and base-flipping by O6-alkylguanine-DNA alkyltransferase

O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O(6)-alkylguanine and O(4)-alkylthymine adducts in DNA, protecting the genome and also contributing to the resistance of tumors to chemotherapeutic alkylating agents. AGT binds DNA cooperatively, and cooperative interactions are likely to be important in lesion search and repair. We examined morphologies of complexes on long, unmodified DNAs, using analytical ultracentrifugation and atomic force microscopy. AGT formed clusters of ≤11 proteins. Longer clusters, predicted by the McGhee-von Hippel model, were not seen even at high [protein]. Interestingly, torsional stress due to DNA unwinding has the potential to limit cluster size to the observed range. DNA at cluster sites showed bend angles (～0, ～30 and ～60°) that are consistent with models in which each protein induces a bend of ～30°. Distributions of complexes along the DNA are incompatible with sequence specificity but suggest modest preference for DNA ends. These properties tell us about environments in which AGT may function. Small cooperative clusters and the ability to accommodate a range of DNA bends allow function where DNA topology is constrained, such as near DNA-replication complexes. The low sequence specificity allows efficient and unbiased lesion search across the entire genome.     

Kinetic characterization of single strand break ligation in duplex DNA by T4 DNA ligase

T4 DNA ligase catalyzes phosphodiester bond formation between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA in three steps: 1) enzyme-adenylylate formation by reaction with ATP; 2) adenylyl transfer to a 5'-phosphorylated polynucleotide to generate adenylylated DNA; and 3) phosphodiester bond formation with release of AMP. This investigation used synthetic, nicked DNA substrates possessing either a 5'-phosphate or a 5'-adenylyl phosphate. Steady state experiments with a nicked substrate containing juxtaposed dC and 5'-phosphorylated dT deoxynucleotides (substrate 1) yielded kcat and kcat/Km values of 0.4±0.1 s(-1) and 150±50 μm(-1) s(-1), respectively. Under identical reaction conditions, turnover of an adenylylated version of this substrate (substrate 1A) yielded kcat and kcat/Km values of 0.64±0.08 s(-1) and 240±40 μm(-1) s(-1). Single turnover experiments utilizing substrate 1 gave fits for the forward rates of Step 2 (k2) and Step 3 (k3) of 5.3 and 38 s(-1), respectively, with the slowest step ～10-fold faster than the rate of turnover seen under steady state conditions. Single turnover experiments with substrate 1A produced a Step 3 forward rate constant of 4.3 s(-1), also faster than the turnover rate of 1A. Enzyme self-adenylylation was confirmed to also occur on a fast time scale (～6 s(-1)), indicating that the rate-limiting step for T4 DNA ligase nick sealing is not a chemical step but rather is most likely product release. Pre-steady state reactions displayed a clear burst phase, consistent with this conclusion.     

A critical evaluation of models for complex molecular dynamics: application of NMR studies of double- and single-stranded DNA

The nature of internal and overall motions in native (double-stranded) and denatured (single-stranded) DNA fragments 120–160 base pairs (bp) long is examined by molecular-dynamics modeling using ¹³C-nmr spin-relaxation data obtained over the frequency range of 37–125 MHz. The broad range of ¹³C frequencies is required to differentiate among various models. Relatively narrow linewidths, large nuclear Overhauser enhancements (NOEs), and short T₁ values all vary significantly with frequency and indicate the presence of rapid, restricted internal motions on the nanosecond time scale. For double-stranded DNA monomer fragments (147 bp, 24 Å diam at 32°C), the overall motion is that of an axially symmetric cylinder (τ_x = ～10^?6 s;τ_Z = ～1.8 × 10^?8s), which is in good agreement with values calculated from hydrodynamic theory (τ_x = ～1.8 × 10^?6 s; τ_Z = ～2.7 × 10^?8 s). The DNA internal motion can be modeled as restricted amplitude internal diffusion of individual C? H vectors of deoxyribose methine carbons C1′, C3′, and C4′, either with conic boundary conditions (τ_w = ～4 × 10^?9 s, θ_cone = ～21°) or as a bistable jump (τ_A = τ_B = ～2 × 10^?9 s, θ = ～15°). We discuss the critical role in molecular-dynamics modeling played by the angle (β) that individual C? H vectors make with the long axis of the DNA helix. Heat denaturation brings about increases in both the rate and amplitude of the internal motion (described by the wobble model with τ_W = ～0.2 × 10^?9 s, θ_cone = ～50°), and overall motion is affected by becoming essentially isotropic (τ_x = τ_Z = ～5 × 10^?8 s) for the single-stranded molecules. Since ¹³C-nmr data obtained at various DNA concentrations for C2′ of the deoxyribose ring is not described well by the above models, a new model incorporating an additional internal motion is proposed to take into account the rapid, extensive, and weakly coupled motion of C2′.     

Impediment of E. coli UvrD by DNA‐destabilizing force reveals a strained‐inchworm mechanism of DNA unwinding

Escherichia coli UvrD is a non‐ring‐shaped model helicase, displaying a 3′—5′ polarity in DNA unwinding. Using a transverse magnetic tweezer and DNA hairpins, we measured the unwinding kinetics of UvrD at various DNA‐destabilizing forces. The multiform patterns of unwinding bursts and the distributions of the off‐times favour the mechanism that UvrD unwinds DNA as a dimer. The two subunits of the dimer coordinate to unwind DNA processively. They can jointly switch strands and translocate backwards on the other strand to allow slow (～40 bp/s) rewinding, or unbind simultaneously to allow quick rehybridization. Partial dissociation of the dimer results in pauses in the middle of the unwinding or increases the translocation rate from ～40 to ～150 nt/s in the middle of the rewinding. Moreover, the unwinding rate was surprisingly found to decrease from ～45 to ～10 bp/s when the force is increased from 2 to 12 pN. The results lead to a strained‐inchworm mechanism in which a conformational change that bends and tenses the ssDNA is required to activate the dimer.     

人皮肤成纤维细胞中α1(Ⅰ)前胶原基因转录调控研究

为寻找纤维化形成中调控人Ⅰ型前胶原基因高水平转录的启动序列及其DNA结合蛋白 ,以人皮肤成纤维细胞α1(Ⅰ )前胶原基因转录起始点上游 - 2 5kb至 + 4 2bp的片段为靶序列 ,采用PCR、基因重组、报告基因测活、细胞基因转染技术比较不同长短启动子活性 .凝胶滞留实验 (EM SA)研究高启动活性片段相应的DNA结合蛋白 .基因转染高活性转录因子识别序列至靶细胞 ,探讨前胶原基因激活阻断的新手段 .结果表明 ,- 2 4 83～ + 4 2bp、 - 2 6 8～ + 4 2bp序列具有强启动调控活性 ,而 - 10 5～ + 4 2bp片段启动活性最低 .EMSA对高启动活性小片段DNA结合蛋白的分析提示 ,- 2 6 8～ + 4 2bp序列中存在转录因子Ap 1、Sp 1、NF 1的特异结合位点 .转染高活性转录因子识别序列Ap 1、Sp 1至靶细胞可竞争性阻断胶原基因启动转录激活 .研究提示 ,人α1(Ⅰ )前胶原基因 - 2 4 83～ + 4 2bp、 - 2 6 8～ + 4 2bp片段有高启动活性 .转录因子Ap 1、Sp 1、NF 1与 - 2 6 8～ + 4 2bp序列中相应识别序列的结合与其基础高转录活性有关 .转染高活性转录因子识别序列Ap 1、Sp 1可从转录水平阻断胶原基因的激活     

一种提取麦红吸浆虫基因组DNA的方法

介绍一种可从单头麦红吸浆虫Sitodiplosis mosellana(Gehin)成虫、幼虫和蛹成功地提取基因组DNA的方法。经检测提取DNA样品浓度为100～200ng/μL,纯度在1.4～1.6范围之间,以此为模板,可顺利地进行RAPD引物扩增。该提取方法简单、安全、经济,可为小型昆虫基因组DNA的提取提供参考。