甘蓝型油菜脂肪酸链延长酶基因FAE1的克隆与原核表达

脂肪酸延长酶1(FAE1)是广泛存在于植物中并定位于内质网上的一种能催化脂肪酸碳链延长的酮脂酰CoA合成酶.根据Genbank上已知的植物FAE1基因设计引物,以从甘蓝型油菜叶片中提取的总DNA为模板,进行PCR扩增,获得了1380bp的片段.回收该片段,并连接到pMD18-T载体测序.序列比对结果说明了该片段与植物中已知的FAE1序列有极高的相似性,并且不存在内含子序列.将该片段通过高保真酶扩增,EcoRⅠ酶切消化后定向克隆到pGEX-2T表达载体中,在IPTG诱导下于28℃表达出Mr76×103的蛋白质条带.用兔抗GST多克隆抗体做第一抗体进行Western-blot检测,并获得阳性检测结果.这为甘蓝型油菜脂肪酸链延长酶基因FAE1功能的进一步研究奠定了基础.图4表1参14     

不同来源的苏云金芽孢杆菌aiiA基因表达及其抗病性分析

苏云金芽孢杆菌(Bacillus thuringiensis,Bt)aiiA基因所编码的AiiA蛋白是一种内酯酶,可以降解N-乙酰高丝氨酸内酯(N-acyl-homoserine lactones,AHLs),从而减弱致病菌产生的危害.本研究克隆了6株不同来源的苏云金芽孢杆菌aiiA基因,并对来自于苔藓(LLB15)和土壤(LLS9)的BtaiiA基因进行了生物信息学分析.结果表明,AiiA-B15分子量为27.97×103,等电点为4.59;AiiA-S9分子量为28.14×103,等电点为4.32,两者都是亲水性蛋白.AiiA蛋白具有很高的保守性,AiiA-B15和AiiA-S9同源性为90%.进化树结果表明,AiiA-B15与Bacillus thuringiensis subsp.kyushuensis进化距离最近,AiiA-S9与Bacillus cereus进化距离最近.将aiiA-B15基因克隆到pGEX-4T-3表达载体中,构建重组质粒pGEXaiiA-B15,IPTG诱导后可大量表达融合蛋白.对表达的条件进行优化.结果表明,0.6mmol/LIPTG,30℃下诱导3h为最佳诱导条件.致病性检测表明,该融合蛋白对胡萝卜欧文氏软腐病菌具有较强的抗病作用.图13表4参23     

宁南霉素诱导烟草酸性病程相关蛋白的研究

研究了宁南霉素对烟草酸性病程相关蛋白的诱导表达．结果表明，宁南霉素可系统性地诱导烟草叶片中酸性病程相关蛋白的高效表达．与水杨酸相比，宁南霉素诱导酸性病程相关蛋白的表达水平较高，持续时问较长．此外，在同一品种不同形态叶片烟草植株中，宁南霉素对植物酸性病程相关蛋白的诱导存在差异．图6表3参20     

German taxi drivers' experiences and expressions of driving anger: Are the driving anger scale and the driving anger expression inventory valid measures?

Objective: The objective of this article was 2-fold: firstly, we wanted to examine whether the original Driving Anger Scale (DAS) and the original Driving Anger Expression Inventory (DAX) apply to German professional taxi drivers because these scales have previously been given to professional and particularly to nonprofessional drivers in different countries. Secondly, we wanted to examine possible differences in driving anger experience and expression between professional German taxi drivers and nonprofessional German drivers.

Methods: We applied German versions of the DAS, the DAX, and the State–Trait Anger Expression Inventory (STAXI) to a sample of 138 professional German taxi drivers. We then compared their ratings to the ratings of a sample of 1,136 nonprofessional German drivers (Oehl and Brandenburg n.d. Oehl M, Brandenburg S. Driving anger in Germany: validation of the Driving Anger Scale (DAS). Saf Sci. n.d. submitted.  [Google Scholar]).

Results: Regarding our first objective, confirmatory factor analysis shows that the model fit of the DAS is better for nonprofessional drivers than for professional drivers. The DAX applies neither to professional nor to nonprofessional German drivers properly. Consequently, we suggest modified shorter versions of both scales for professional drivers. The STAXI applies to both professional and nonprofessional drivers. With respect to our second objective, we show that professional drivers experience significantly less driving anger than nonprofessional drivers, but they express more driving anger.

Conclusions: We conclude that the STAXI can be applied to professional German taxi drivers. In contrast, for the DAS and the DAX we found particular shorter versions for professional taxi drivers. Especially for the DAX, most statements were too strong for German drivers to agree to. They do not show behaviors related to driving anger expression as they are described in the DAX. These problems with the original American DAX items are in line with several other studies in different countries. Future investigations should examine whether (professional) drivers from further countries express their anger as proposed by the DAX. In addition, professional drivers experience less driving anger (DAS) and less general trait anger (STAXI) than nonprofessional drivers, but they report more driving anger expression (DAX) and more current general state anger (STAXI). Subsequent studies should therefore focus on different types of anger within the group of professional drivers.     

Raw single-walled carbon nanotube-induced cytotoxic effects in human bronchial epithelial cells: comparison to asbestos

Single-walled carbon nanotubes (SWCNT) are being developed to be used in many industrial and biomedical applications. However, SWCNT's durability and likely fibrous morphology have raised health concerns. The present investigations were focused on understanding the cellular and molecular mechanisms induced by raw SWCNT (SWCNT) in human bronchial-epithelial cells (BEAS-2B). Asbestos (crocidolite) was used as a positive control. Exposure of BEAS-2B cells to SWCNT induced apoptosis, DNA damage, and oxidative stress. The generation of hydroxyl radical (^?OH) and increase of superoxide dismutase (SOD) activity were concentration-dependent. The increase in apoptosis was associated with activation of caspase-3, caspase-7, and poly (ADP-ribose) polymerase-1 (PARP-1). A short recovery period of 6?h of cells from SWCNT exposure resulted in reversal of caspase-3 and caspase-7, and a partial reversal of PARP-1 activation. The activation of PARP-1, caspase-3, and caspase-7 was only partially diminished after a recovery of 6?h from the exposure to crocidolite. Exposure of BEAS-2B cells to SWCNT resulted in the phosphorylation of protein p42/44 (p42/44) and protein p38 (p38). SWCNT did not induce protein serine-threonine kinase (AKT) phosphorylation. For all the above end points, crocidolite induced a greater response compared to SWCNT. SWCNT induced a significant activation of activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB), and the effect was inhibited by mitogen-activated protein kinase (MAPK) inhibitors. SWCNT also induced significant increase in the expression levels of c-Jun, βIGH3, and CD44 genes. The results of this study show that the molecular mechanism for raw SWCNT-mediated toxicity in BEAS-2B cells is through the activation of caspase-3, caspase-7, and PARP-1. Furthermore, the mechanism of AP-1 and NF-κB activation is through MAPK. This bioactivity of raw SWCNT is associated with the generation of oxidative stress and DNA damage. Considering the role of airway epithelium as a critical barrier for normal pulmonary function and focal point for tumor development, this study demonstrates that raw SWCNT activate molecular events which may be linked to adverse biological responses implicated in pulmonary diseases.     

Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization

Vinclozolin, a dicarboximide fungicide, is an endocrine disrupting chemical that competes with an androgenic endocrine disruptor compound. Most research has focused on the epigenetic effect of vinclozolin in humans. In terms of ecotoxicology, understanding the effect of vinclozolin on non-target organisms is important. The expression profile of a comprehensive set of genes in the amphipod Hyalella azteca exposed to vinclozolin was examined. The expressed sequence tags in low-dose vinclozolin-treated and -untreated amphipods were isolated and identified by suppression subtractive hybridization. DNA dot blotting was used to confirm the results and establish a subtracted cDNA library for comparing all differentially expressed sequences with and without vinclozolin treatment. In total, 494 differentially expressed genes, including hemocyanin, heatshock protein, cytochrome, cytochrome oxidase and NADH dehydrogenase were detected. Hemocyanin was the most abundant gene. DNA dot blotting revealed 55 genes with significant differential expression. These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc-finger–containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein. Vinclozolin appears to upregulate stress-related genes and hemocyanin, related to immunity. Moreover, vinclozolin downregulated NADH dehydrogenase, related to respiration. Thus, even a non-lethal concentration of vinclozolin still has an effect at the genetic level in H. azteca and presents a potential risk, especially as it would affect non-target organism hormone metabolism.     

双酚A(BPA)暴露对WHHL家兔糖类和脂类代谢的影响

为利用和人类更为接近的动物模型来研究在高脂血症条件下,BPA暴露是否能促进脂类和糖类代谢异常,并探索潜在的毒性效应机制,选取渡边可遗传高脂血症家兔(WHHL)模型,通过灌胃的方式将其暴露于400μg·kg-1体重的BPA溶液长达12周。在持续暴露第8周,检测了在空腹状态下,家兔血浆中葡萄糖、胰岛素和脂肪含量的变化,并根据所得结果进行了静脉注射胰岛素耐受试验(IVTT)。在暴露第12周,同样对空腹状态下,家兔血浆中的葡萄糖,胰岛素和脂肪含量进行了检测,并测量了血压和心率。然后,将所有的家兔进行解剖,通过苏木精-伊红(HE)及糖原(PAS)染色分别对心脏和肝脏部位的脂肪及糖原蓄积情况进行了病理学切片分析。同时,检测了肝脏中与脂类和糖类代谢相关基因在m RNA水平上的表达变化。结果显示,BPA暴露8周后促使WHHL家兔发生了胰岛素抵抗现象,导致第12周血糖及胰岛素含量升高,同时也促进了高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)及游离脂肪酸(NEFA)含量的上升。BPA暴露第12周,暴露组WHHL家兔的冠状动脉粥样硬化损伤程度未发生明显增加,但心肌细胞发生了肿胀,胞质中出现了脂肪的蓄积,同时伴随着心律失常。肝脏重量发生增加,肝细胞中同时出现了脂肪和糖原的蓄积现象,相关基因的表达发生了显著上调。研究结果表明,BPA持续暴露可促进WHHL家兔发生脂类和糖类代谢异常,其毒性效应机制可能和胰岛素抵抗及相关基因的异常表达有关。     

多源土地利用地学知识的获取与应用

土地利用地学知识可应用于LIS智能化分析及辅助遥感影像解译,是国土信息化工程建设中值得深入研究的重要技术环节之一.明确了土地利用地学知识概念,构建了多数据源的土地利用地学知识体系,针对不同数据源分析了土地利用地学知识的获取及表达,并着重对土地利用地学知识辅助遥感影像解译进行了阐述,指出采用知识辅助高分辨率遥感影像解译是亟待深入开展研究的课题.     

分光光度法分析中增色、褪色反应标准曲线绘制的一般形式

介绍了在分光光度法分析中，增色反应和褪色反应的标准曲线绘制用朗伯-比耳定律可推导出一般数学表达形式。其中增色反应为Ai=kbCi，使用频率较高；褪色反应为A0-Ai=k′bCi，实际工作中应用较少。