从cDNA文库中筛选分析阳性克隆的简便方法

cDNA文库中阳性克隆的传统筛选分析法既费时又费力.利用微波炉加热的方法,简化了原位杂交中噬菌斑裂解、DNA变性与固定的程序;进一步运用PCR扩增技术,特异扩增克隆载体中插入的cDNA片段,加快了阳性克隆分析的进程.     

表达人高致病性禽流感病毒H5N1(安徽株)结构基因的DNA疫苗在小鼠中诱导的免疫应答分析

本研究旨在研发经济、高效的人高致病性禽流感病毒H5N1实验疫苗并优化免疫方案。首先优化并合成H5N1(安徽株)的血凝素基因(HAop)和神经氨酸酶基因(NAop)并证实其正确表达,再构建含有H5N1(安徽株)结构基因的多个单顺反子(HAwt、HAop和NAop)和双顺反子(HAop/M2和NAop/M1)DNA疫苗质粒。随后,采用不同途径的体内电转法在第0周和第3周2次免疫Balb/C小鼠,初步分析了抗原特异性体液免疫(HA血凝抑制抗体,NA特异性抗体,中和抗体)和细胞免疫应答(IFN-γELISpot)的特点。结果表明:含有优化血凝素基因(HAop)和优化神经氨酸酶基因(NAop)的DNA疫苗可以快速激发较强的免疫应答,尤其是细胞免疫应答;皮内电转所激发的体液免疫应答强于肌肉电转。本研究为新型H5N1疫苗的研发以及免疫方案的优化奠定了基础。     

Distinct responses of soil respiration to experimental litter manipulation in temperate woodland and tropical forest

Global change is affecting primary productivity in forests worldwide, and this, in turn, will alter long‐term carbon (C) sequestration in wooded ecosystems. On one hand, increased primary productivity, for example, in response to elevated atmospheric carbon dioxide (CO₂), can result in greater inputs of organic matter to the soil, which could increase C sequestration belowground. On other hand, many of the interactions between plants and microorganisms that determine soil C dynamics are poorly characterized, and additional inputs of plant material, such as leaf litter, can result in the mineralization of soil organic matter, and the release of soil C as CO₂ during so‐called “priming effects”. Until now, very few studies made direct comparison of changes in soil C dynamics in response to altered plant inputs in different wooded ecosystems. We addressed this with a cross‐continental study with litter removal and addition treatments in a temperate woodland (Wytham Woods) and lowland tropical forest (Gigante forest) to compare the consequences of increased litterfall on soil respiration in two distinct wooded ecosystems. Mean soil respiration was almost twice as high at Gigante (5.0 μmol CO₂ m^?2 s^?1) than at Wytham (2.7 μmol CO₂ m^?2 s^?1) but surprisingly, litter manipulation treatments had a greater and more immediate effect on soil respiration at Wytham. We measured a 30% increase in soil respiration in response to litter addition treatments at Wytham, compared to a 10% increase at Gigante. Importantly, despite higher soil respiration rates at Gigante, priming effects were stronger and more consistent at Wytham. Our results suggest that in situ priming effects in wooded ecosystems track seasonality in litterfall and soil respiration but the amount of soil C released by priming is not proportional to rates of soil respiration. Instead, priming effects may be promoted by larger inputs of organic matter combined with slower turnover rates.     

Regulation of enzymes involved in C4 photosynthesis and the antioxidant metabolism by UV-B radiation in Egeria densa,a submersed aquatic species

Egeria densa, a submersed aquatic species, was exposed to different treatments under UV-B radiation, and the response of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME) was determined. Exposure to UV-B radiation for 4 h per day over 7–16 days caused an increase in both enzymes, together with an increase in the activity of some isoforms of several enzymes involved in the antioxidant metabolism, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD). The content of chlorophylls and carotenoids was considerably decreased, suggesting that degradation or repression of the synthesis of these molecules may be occurring after UV-B exposure. Reactive oxygen species (ROS) were also required for UV-B induction of PEPC and NADP-ME, as the addition of ascorbic acid before UV-B treatment prevented the induction of these enzymes, while salicylic acid was not effective in inducing NADP-ME but increased the expression of the lower molecular mass isoform of PEPC. On the other hand, damage to the photosynthetic machinery may be occurring after exposure to UV-B radiation for 8 per day over 1–2 days, as indicated by a decrease in the levels of Rubisco, PEPC and NADP-ME. Some of the enzymes involved in the antioxidant metabolism, such as CAT and APX, were also sensitive to continuous exposure, evidenced by a decrease in their activity. In this way, in E. densa, several enzymes involved in different metabolic pathways showed a distinct response, depending on the UV-B treatment. This revised version was published online in June 2006 with corrections to the Cover Date.     

缓/控释肥料研究进展

缓/控释肥料对作物产量的影响因作物种类、肥料种类和试验条件而异,大多数植物增产比较明显,如大豆使用长效尿素,与普通尿素相比增产幅度最高可达33%。各种类型的缓/控释肥料,均可不同程度的提高肥料利用率。缓/控释肥料在农业上的应用能有效地保护生态环境,如抑制土壤NH4 向NO3-氧化,减少土壤NO3-的积累,从而减少氮肥以NO3-形式淋溶损失,减少了施肥对环境的污染;可以减少土壤N2O的释放等。提出了目前缓/控释肥料在农业应用中存在的问题及今后的发展方向。     

贝伐珠单抗联合化疗治疗晚期非鳞非小细胞肺癌的临床观察

目的:观察贝伐珠单抗联合化疗对晚期非小细胞肺癌患者的疗效、安全性及影像学改变。方法:对2007年至2014年于我院治疗的晚期NSNSCLC(非鳞非小细胞肺癌non-squamous non-small cell lung cancer)患者,给予贝伐珠单抗(15 mg/kg或7.5mg/kg)联合化疗(紫杉醇175 mg/m~2,d1,卡铂AUC=5或6,d1,q3 w)6周期及贝伐珠单抗维持治疗(15 mg/kg或7.5 mg/kg,d1,q3w)。观察疗效、不良反应、肺部病灶空洞改变的情况、恶性胸腔积液的治疗效果及部分患者EGFR、KRAS基因突变状况。结果:共观察26例患者,均接受贝伐珠单抗联合化疗,17例行贝伐珠单抗维持治疗。部分缓解(partial response,PR)、疾病稳定(stable disease,SD)、疾病进展(disease progression,PD)率分别为53.8%、42.3%、3.8%。中位无进展生存期(progression free survival,PFS)为11.0个月,中位总生存期(overall survival,OS)达25.8个月。26例患者中15.4%治疗后病变发生空洞改变,空洞组的2年、3年生存率略高于无空洞组,但无统计学差异(P值分别为0.586、0.509)。13例患者伴有恶性胸腔积液,胸腔积液的疾病控制率为100%。11例患者标本可进行EGFR基因检测,敏感突变占36.4%,未突变占63.6%。对10例患者标本行KRAS基因检测,均为突变阴性。不良反应包括骨髓抑制、消化道反应、鼻衄、咯血、高血压、蛋白尿等。大多数不良反应程度较轻,可控制。结论:贝伐珠单抗联合化疗治疗晚期NSNSCLC患者疗效确切,副反应可耐受,控制恶性胸腔积液效果较好。肺部病灶空洞改变的临床意义有待进一步研究。     

油橄榄果渣抗氧化部位化学成分的研究北大核心CSCD

为了开展油橄榄果渣中主要抗氧化活性成分的研究,该文通过1,1-二苯基-2-苦肼基自由基(DPPH·)法测定了油橄榄果渣不同提取部位清除DPPH·的能力,并采用硅胶、ODS、Sephadex LH-20等柱色谱方法对抗氧化活性部位的化合物进行分离,同时运用核磁共振波谱等方法鉴定了化合物的结构。结果表明:(1)油橄榄果渣的乙酸乙酯部位具有显著的抗氧化活性,其清除DPPH·的半数抑制浓度(IC_(50))为119.11μg·mL^(-1)。(2)从该活性部位分离得到17个化合物,分别鉴定为没食子酸(1)、羟基酪醇(2)、原儿茶酸(3)、酪醇(4)、儿茶素(5)、香草酸(6)、咖啡酸(7)、香草醛(8)、丁香酸(9)、木犀草苷(10)、橄榄苦苷(11)、丁香酚(12)、槲皮素(13)、木犀草素(14)、芦丁(15)、山楂酸(16)、齐墩果酸(17)。其中,化合物1、6、7、9、15为首次从油橄榄果渣中分离得到。该研究明确了油橄榄果渣的抗氧化物质基础,为油橄榄果渣进一步的高值化利用提供了科学依据。     

细菌核糖体的结构和功能

二十多年来,国际上几家实验室独立地竞争性地应用高分辨率X-射线衍射技术在原子水平上绘制出了细菌完整核糖体及其亚基精细的三维结构图,为其生物功能的研究提供了清晰的结构基础。由于这项伟大的科学成果,美国科学家文卡特拉曼·拉马克里希南（Venkatraman Ramakrishnan）、托马斯．施泰茨（Thomas A．Steitz）和以色列女科学家阿达．约纳特（Ada E．Yonath）三人荣获2009年度诺贝尔化学奖。细菌核糖体是细胞中合成蛋白质的一种细胞器,包括大小不同的两个亚基,由3种RNA和50多种不同的蛋白质组成。     

基质金属蛋白酶-2/9敏感型可跨膜融合抗氧化酶的表达、纯化和表征北大核心CSCD

融合了跨膜肽的抗氧化酶可进入细胞,保护细胞免受放射损伤。然而跨膜肽的跨膜能力没有靶向性,其也可把抗氧化酶带入肿瘤细胞进而保护肿瘤细胞,降低放疗的效果。为此,根据多数肿瘤细胞微环境中存在活性基质金属蛋白酶(matrix metalloproteinase,MMP)-2或MMP-9的特点,在细胞跨膜肽R9与人铜、锌超氧化物歧化酶(superoxide dismutase 1,SOD1)和谷胱甘肽S-转移酶(glutathione S-transferase,GST)之间融合MMP-2/9的底物肽X,设计了融合蛋白GST-SOD1-X-R9。该蛋白在肿瘤微环境中可因MMP-2/9酶切底物肽X而失去跨膜肽,从而无法进入肿瘤细胞,进而只能进入正常细胞。全基因合成SOD1-X-R9序列,并将其插入原核表达载体pGEX-4T-1中,得到表达质粒,并实现了GST-SOD1-X-R9融合蛋白的可溶表达。GST-SOD1-X-R9经硫酸铵沉淀和GST亲和层析纯化,分子量约为47 kDa,与理论值一致。纯化的融合蛋白的SOD活性和GST活性分别为2954 U/mg和328 U/mg。GST-SOD1-X-R9的SOD活性或GST活性在生理条件下几乎没有变化。该融合蛋白在溶液中可被胶原酶Ⅳ部分水解。分别建立了2D和3D培养的HepG2细胞模型来检验肿瘤微环境中的MMP-2活力对该蛋白跨膜能力的影响。在2D培养模型中,HepG2的MMP-2活力极低,但在3D培养模型中,随着培养时间的增加,HepG2肿瘤球的体积变大,其胞外MMP-2活力也随之增强。GST-SOD1-X-R9在2D培养的HepG2细胞中具有和GST-SOD1-R9蛋白一样的跨膜效率,但在3D培养的HepG2细胞球中的跨膜能力大大降低。本研究为后续深入研究GST-SOD1-X-R9靶向防护正常细胞的氧化损伤效应奠定了基础。     

Methylparaben induces malformations and alterations on apoptosis,oxidant–antioxidant status,ccnd1 and myca expressions in zebrafish embryos

Methylparabens (MP) are widely used as preservatives in cosmetics, pharmacy, and food industry. Although acute toxicity studies in animals indicated that parabens are not significantly toxic, the effects of chronic exposure under sublethal doses are still unknown and the number of related studies is limited. Our aim was to evaluate the effects of MP on the development of zebrafish embryos focusing on development, locomotor activity, oxidant–antioxidant status, apoptosis, and ccnd1 and myca expressions. The expressions of ccnd1 and myca were determined by RT‐PCR. Lipid peroxidation (LPO), nitric oxide (NO), and glutathione‐S‐transferase (GST) activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Locomotor activity was measured using touch‐evoked movement test. MP exposure increased malformations, LPO, apoptosis, ccnd1 and myca expressions, and decreased GST activities and NO levels compared with the control group. Our findings will lead to further understanding of the mechanism of MP toxicity, and merit further research.