番茄八氢番茄红素合成酶基因的克隆及超量表达载体构建

八氢番茄红素合成酶是植物类胡萝卜素生物合成途径中促进番茄红素合成的关键酶,根据番茄该酶的编码基因序列设计一对引物,通过RT-PCR在番茄中扩增出一个约1500 bp的全长cDNA片段.测序结果表明,此片段包含一个长为1239 bp的完整的编码框,其氨基酸序列与辣椒、向日葵、万寿菊、枸杞、番木瓜、温州蜜柑、拟南芥八氢番茄红素合成酶基因编码的氨基酸序列的一致率分别为88%、75%、75%、74%、73%、73%、7l%.对这个全长片段构建了超量表达载体,酶切检测表明该片段已插入植物表达载体.     

西瓜八氢番茄红素合成酶基因全长的克隆与表达分析

通过RT-PCR和RACE技术从西瓜果实中克隆八氢番茄红素合成酶（PSY）基因的cDNA全长,用生物信息学方法对其cDNA序列及推测氨基酸序列进行分析,并用实时荧光定量PCR技术研究PSY在不同瓤色西瓜果实发育过程中的表达情况.结果表明,PSY基因cDNA全长1 561 bp（GenBank登录号为KC166870）,其开放阅读框为1 266 bp,编码421个氨基酸.该基因及其推导的氨基酸序列与同为葫芦科的其他植物的PSY及氨基酸序列的同源性分别为87％和91％以上;序列分析显示,PSY氨基酸序列N末端存在转运肽信号序列.不同瓤色西瓜果实发育过程中PSY的表达存在明显差异,红瓤果实中的表达量最高,这表明PSY可能与红瓤果实中积累较多的类胡萝卜素有关;PSY的表达量均高于PSY-A,推测PSY基因主要负责果实中类胡萝卜素的合成.     

大鼠端脑神经元型一氧化氮合酶免疫阳性神经元的分布

目的 :观察大鼠端脑神经元型一氧化氮合酶 (nNOS)免疫阳性神经元的分布。方法 :ABC免疫细胞化学法显示大鼠端脑nNOS阳性神经元。结果 :nNOS阳性神经元呈棕褐色 ,胞体的形态多样化 ,呈梭形、三角形、圆形等多种形状 ,突起有一个或多个 ;阳性纤维呈棕色串珠状 ,个别脑区阳性纤维相互交织成网。端脑nNOS免疫阳性神经元主要分布于嗅结节 (包括海马回 )、梨状区 (包括梨状皮质和梨状核 )、斜角带、隔区、杏仁复合体、海马结构、尾壳核和苍白球 ,以及大脑皮质等各个部位 ,其中以大脑皮质、梨状区和斜角带、尾壳核等部位最为丰富。结论 :大鼠端脑内分布有丰富的nNOS阳性神经元 ,它们可能通过调节神经递质的分泌 ,参与脑的高级整合功能。     

亚栖热菌产海藻糖合酶培养基的优化

对亚栖热菌CBS-01菌株发酵生产海藻糖合酶的培养基进行了优化.首先通过碳源、氮源实验,选出了适宜的碳、氮源,随后采用Plackett-Burman设计法对培养基中的组分进行了筛选,找出影响产酶的主要因素为蛋白胨和酵母膏,最后利用中心组合设计及响应面分析法求出了主要影响因素的最佳浓度,得到优化的发酵培养基配方(g/L):可溶性淀粉5,蛋白胨8.6,酵母膏1.725,NaNO3 0.7,氨三乙酸0.5,Na2HPO4 0.1,MgSO4·7H2O 0.2,CaCl2 0.1.10 L罐发酵实验表明,CBS-01菌株海藻糖合酶的合成属生长关联型,利用优化的发酵培养基,CBS-01菌株的产酶水平由0.2 U/mL提高到0.41 U/mL.     

表没食子儿茶素没食子酸酯对IL-4刺激的小鼠腹腔巨噬细胞诱导型一氧化氮合酶和精氨酸酶-1表达的影响

为探讨表没食子儿茶素没食子酸酯(EGCG)对白介素-4(IL-4)刺激的巨噬细胞精氨酸酶1(Arg-1)和诱导型一氧化氮合成酶(iNOS)表达的影响.将6~8周龄Balb/c小鼠经无血清DMEM培养基刺激后,收集腹腔巨噬细胞用于体外培养.体外培养的巨噬细胞经20ng/mL IL-4和不同浓度EGCG处理后,用实时荧光定量PCR(RTqPCR)和酶联免疫吸附试验(ELISA)方法检测Arg-1和iNOS的mRNA表达水平和含量.结果显示IL-4和EGCG单独处理均可显著刺激Arg-1的表达和抑制iNOS的表达,而EGCG(12.5~50μM)增强IL-4刺激的Arg-1表达和IL-4对iNOS表达的抑制作用,且存在剂量依赖效应.上述结果提示EGCG以剂量依赖方式促进IL-4刺激的Arg-1表达、抑制iNOS的表达.     

一种与核黄素合酶α亚基具有结构相似性的板蓝根多肽的分离纯化与表征

利用离子交换色谱SP-5PW和疏水色谱POROS HP2从板蓝根鲜根水相抽提物中分离纯化多肽组分,并对其进行生化表征.获得一种达到电泳纯的多肽RIP2,其相对分子质量约为6.87 ku,等电点约为7.73,N端氨基酸序列依次为QIGEFATAPF.经数据库搜索比对,发现该多肽与核黄素合酶α亚基具有一定的同源性.细胞毒性实验表明该多肽具有一定抗病毒作用.     

Direct observation of the clockwise light-driven rotation of F<Subscript>0</Subscript>F<Subscript>1</Subscript>-ATP synthase complex

The purified thermophilic bacterium PS3 F₁ β _10×His-tag is inserted into the F_oF₁-ATP synthases of chro-matophores isolated from photosynthetic bacteria Rhodo-spirillum rubrum. The studies of biochemical properties of the hybrid chromatophores show that they have both protons-driving capability and photophosphorylation. The fluorescent actin filaments, as a marker of its orientation by video-microscopic experiment, are connected via Maleimido-C₃-NTA to the reconstituted β_10×His-tag of F_oF₁-ATP synthases. The clockwise rotation of F_oF₁-ATP synthases driven by light is observed directly when viewed from the F_o side to F₁. This system should be valuable for further studying the coupling property of F_oF₁-ATP synthase.     

Regulation between nitric oxide and MAPK signal transduction in mammals

Nitric oxide (NO) is an important biological messenger in the regulation of tissue homeostasis. It exhibits a wide range of effects during physiological and pathophysiological processes. Typical beneficial properties of NO include the regulation of vascular tone,the protection of cells against apoptosis, the modulation of immune responses, and the killing of microbial pathogens. On the other hand,NO may cause severe vasodilation and myocardial depression during bacterial sepsis or act as a cytotoxic and tissue-damaging molecule in autoimmune diseases. Mitogen-activated protein kinase (MAPK) is a family of serine/threonine protein kinases that are widely distributed in mammalian cells. MAPK cascade plays pivotal roles in gene expression, cell proliferation, differentiation, neuronal survival and programmed cell death under a variety of experimental conditions. MAPKs transduce the signal for the cellular response to extracellular stresses or stimuli. The relation between them, however, has never been reviewed. Based on our researches and other reports in the field, we review their reciprocal regulatory functions.     

Cloning and enzymology analysis of farnesyl pyrophosphate synthase gene from a superior strain of<Emphasis Type="Italic">Artemisia annua</Emphasis> L.

A cDNA(af1) encoding farnesyl pyrophosphate synthase AaFPS1(FPS,EC2.5.1.1/EC2.5.1.10) from a high yield Artemisia annua strain 025 has heen cloned from its cDNA library .Sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with molecular weight of 39 kD.Deduced aa sequence of the cDNA was similar to FPS from other plants,yeast and mammals,containing 5 conserved domains found in both prenyl trans-ferase and polyprenyl synthase,The expression of the cDNA in Escherichia coli showed measurable specific activity of FPS in vitro.The enzyme was purified by ion exchange chromatography and its kinetics was measured ,These results would further promote the molecular regulation of artemisnin biosynthesis.