Tat-MANF融合蛋白的制备及生物活性研究

目的：构建用于表达具有Tat序列的新型神经营养因子MANF（mesencephalic astrocyte-derived neurotrophic factor）融合蛋白（Tat-MANF）的重组质粒;利用原核细胞表达系统表达该重组蛋白,并检测其生物学活性。方法：以MANF cDNA为模板,利用PCR技术在上下游分别添加TAT序列和His标签及合适的限制性酶切位点,构建TAT-MANF融合基因。插入表达载体Pet22b⁺后,转化大肠杆菌BL21进行表达和纯化,用SDS-PAGE及Western印迹鉴定表达的重组蛋白。为了验证Tat-MANF的生物学活性,用30μmol/L浓度的6-羟多巴胺（6-OHDA）对神经母胶质瘤细胞（SH-SY5Y）进行毒性诱导,同时加入2μg/ml的TAT-MANF及对照MANF蛋白,24h后用流式细胞仪检测细胞的凋亡率。用脑微血管内皮细胞（B-endo3）体外模拟血脑屏障,与FITC标记的Tat-MANF共孵育4h,荧光显微镜下观察。结果：成功构建TAT-MANF融合基因,表达产物与目的蛋白大小相符,能与MANF抗体发生结合反应。重组蛋白可减少由6-OHDA导致的SH-SY5Y细胞凋亡,Tat-MANF-FITC与B-end3细胞共孵育4h后,可见细胞内明显荧光。结论：获得的重组蛋白Tat-MANF具有神经细胞保护作用及跨膜功能,为进一步开展帕金森症的体内治疗研究奠定了物质基础。     

遥感数据支持下不同地表覆盖的区域蒸散

将地表能量平衡系统(SEBS)扩展成遥感日蒸散估算模型,利用MODIS遥感数据估算了黄淮海地区的区域蒸散,并在地理信息系统的支持下分析了不同地表覆盖下的区域蒸散统计分布特征.在缺乏各地表覆盖类型相应蒸散量实测值进行对比的情况下,以2001年4月17日估算的日蒸散量为例,通过各地表覆盖类型日蒸散量间的相互对比分析表明,SEBS估算的区域日蒸散量具有一定的合理性.分析结果表明：在黄淮海地区,荒地具有最低的蒸散量;森林、灌木、草地等地表覆盖类型具有中等的蒸散量;而水体、湿地以及耕地具有较高的蒸散量.可能由于包含绿地和水面,城镇用地的蒸散量也较高.土壤含水量的空间差异性导致森林、灌木、草地和耕地等地表覆盖类型的蒸散量具有明显的空间差异性.耕地蒸散量的空间差异性可以为制定合理、高效的农田灌溉计划提供指示作用.SEBS遥感日蒸散模型的局限性在于有可能低估水体和湿地等地表覆盖类型的蒸散量.     

Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation

Accumulating evidences showed metformin and berberine, well‐known glucose‐lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP‐activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD⁺/NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD⁺/NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non‐AMPK pathway, which may relate to the suppression of the cellular NAD⁺/NADH ratio.     

地塞米松与硫酸镁对大鼠小肠缺血再灌注损伤的作用及机制

目的研究地塞米松和硫酸镁对大鼠小肠缺血再灌注（I/R）损伤的保护作用,并初步探讨其机制。方法制作小肠I/R模型,实验分为假手术阴性对照组、I/R组、硫酸镁治疗组、地塞米松治疗组、地塞米松和硫酸镁联合治疗组,比较五组血浆二胺氧化酶（DAO）、丙二醛（MDA）的含量,同时比较小肠的病理切片观察治疗效果。结果①I/R组小肠组织病理变化明显,血浆DAO、MDA比假手术阴性对照组显著升高;②硫酸镁治疗组和地塞米松治疗组小肠病理变化减轻,血浆DAO、MDA比I/R组显著降低,且两组无显著差别;③硫酸镁和地塞米松合用组的血浆MDA比I/R组显著升高,但是小肠病理变化和I/R组相比无明显区别,血浆DAO也和I/R组无明显差别。结论硫酸镁,地塞米松分别对大鼠小肠缺血再灌注有保护作用而二者合用却无明显的保护作用。     

带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法,然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性,影响外植体的再生和转化频率。将一个植物的内含子插入卡那霉素抗性基因编码区的N端,合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达栽体pYP1202并转化烟草,受侵外植体在含卡那霉素50～200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响,然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍,高达91.4％,然而具GUS活性的转化子占抗性芽的比例仅有26.7％,在200m/L的卡那霉素筛选下,比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。     

Identification of tumor metastasisrelated gene TMSG-1 by mRNA differential display

To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression of two cancer sublines derived from prostate carcinoma cell PC-3M that had different metastatic potentials. The differentially expressed genes were confirmed by Northern blot, and sequenced. The full-length cDNA of a tumor metastasis suppressor gene (TMSG-1) was obtained by using EST assembling and verified by RT-PCR and sequencing. The results showed that expression levels of TMSG-1 were lower in the highly metastatic cell line 1E8, compared with the non-metastatic cell line 2B4. The difference was significant. Full-length cDNA of TMSG-1 was about 2 kb, containing an open reading frame that encoded a protein of 230 amino acids. GenBank Blastn showed no marked homology with known genes. The functional prediction of amino acids sequence encoded by TMSG-1 gene indicated TMSG-1 protein was transmembrane protein, with 3 transmembrane domains, 3 putative protein kinase phosphorylatio     

两种野生花卉的扦插繁殖研究

开展野生花卉车轮梅(Raphiolepis indica)和赤楠(Syzygium buxifolium)扦插试验,结果表明:车轮梅硬枝扦插需一定浓度的外源激素方能生根;对激素浓度大小不敏感;总体上NAA组合生根质量优于IBA组合。综合不同处理生根率、根数和不定根根长3个指标,以800 mg/L NAA或800 mg/L IBA作为车轮梅生产上扦插的激素种类和浓度。赤楠生根率较低,最高扦插率达66.7%,生根时间长,约需45 d始生根。综合生根率、不定根根数和不定根根长3个生根指标,试验的4种激素均能较好促进赤楠生根,200×根太阳在生根率和根数上效果最好,生产上可用200×根太阳浸泡2 h,也可用50-100 mg/L NAA或100-400 mg/L IBA浸泡2 h后进行扦插。     

基于集对分析的城市生态系统健康评价—以兰州为例

城市生态系统是城市社会经济发展的基础和载体,但城市生态系统健康状况问题中包含多种不确定性因素,而集对分析可以有效处理这种不确定性.论文以特大型综合性城市兰州为研究案例,基于集对分析方法,构建包括生产力、生活态、生态势和生机度的城市生命力指数,诊断兰州城市生态系统2002至2008年间的健康状态.研究结果表明:2002～2008年,兰州市城市生态系统健康状况呈上升趋势,其健康联系度由2002年的0.451增至2008年的0.519,但总体水平仍不高.该研究为兰州市城市规划和生态建设推荐了重点行动领域,同时也可为兰州市实施可持续发展战略提供决策依据.     

The altered activity of complex III may contribute to the high penetrance of Leber's hereditary optic neuropathy in a Chinese family carrying the ND4 G11778A mutation

The ND4 G11778A mutation is the most common mitochondrial DNA mutation leading to Leber's hereditary optic neuropathy (LHON). Despite considerable clinical evidences, the modifier role of nuclear background and mitochondrial haplotypes in phenotypic manifestation of LHON remains poorly understood. We investigated the effect of these modifiers on bioenergetics in lymphoblastoid cell lines derived from five affected subjects of one Chinese family carrying the G11778A mutation and five Chinese controls. Significant reductions in the activities of complexes I and III were observed in mutant cell lines from the Chinese family, whereas the mutant cell lines from other families carrying the same mutation exhibited only reduced activity of complex I. The reduced activities of complexes I and III caused remarkably higher reductions of ATP synthesis in mutant cell lines from the Chinese family than those from other families. The deficient respiration increased generation of reactive oxygen species. The defect in complex III activity, likely resulting from the mitochondrial haplotype or nuclear gene alteration, worsens mitochondrial dysfunction caused by the G11778A mutation, thereby causing extremely high penetrance and expressivity of optic neuropathy in this Chinese family. Our data provide the first experimental evidence that altered activity of complex III modulates the phenotypic manifestation of LHON-associated G11778A mutation. Thus, our findings may provide new insights into the pathophysiology of LHON.