Localization of NOS-like protein in guard cells of Vicia faba L. and its possible function

Using the immuno-fluorescence and immuno-gold electron microscope technology, localization of ni- tric oxide synthase (NOS)-like proteins was determined in guard cells of Vicia faba L. NOS is mainly localized in nucleus, cytoplasm, chloroplast, mitochondria and the cell wall of guard cells. Scorch and exogenous JA can enhance the level of nitric oxide (NO) and increase NOS activity in both leaf and epidermis, and the changing pattern of NOS activity was consistent with the change of NO. NOS in- hibitor, L-NAME, inhibited JA-induced NO generation. From the results, we presumed that NO genera- tion from NOS pathway is the main pathway in the stress and JA responses. The pharmacological ex- periment showed that increasing the Ca2 at a suitable concentration promoted leaf NOS activity and the NO level, indicating that NOS activity together with the distribution of NO is Ca2 -dependent. NOS and NO are possibly involved in the regulation of stomatal movement thus playing an important role in plant stress responses.     

QSAR analysis of substituted benzylamino- and heterocyclylmethylamino-carbodithioate derivatives of 4-（3H）-quinazolinone using CoMFA and SCORE2.0

Thymidylate synthase (TS) is a critical enzyme for DNA biosynthesis and many nonclassical lipophilic antifolates targeting this enzyme are quite efficient and encouraging as antitumor drugs. In this paper, the binding model of 14 antifolates of substituted benzylamino- and heterocyclylmethylamino- carbodithioate derivatives of 4-(3H)-quinazolinone with TS is examined using molecular simulation methods―― FlexiDock and SCORE2.0. The resulting conformation and orientation of these antifolates are directly applied to CoMFA study. The robust QSAR model, its three-dimensional contour map, and binding score of these antifolates derived from SCORE2.0 provide guidelines for structural optimiza- tion of current antifolates. The experiment indicates that deletion of cancer chemopreventive structure of dithiocarbamate is unfavorable for interaction between TS and antifolates.     

LPS和IL-2对合浦珠母贝血细胞iNOS活性的影响

建立了一种合浦珠母贝血细胞原代培养方法,为贝类免疫学研究提供实验平台。通过台盼兰染色法检测细胞活力,结果显示:7d内细胞生长状态稳定,并且培养基中的FBS对细胞生长活力没有显著影响。以细胞中iNOS活性为检测指标,详细研究了LPS和IL-2对合浦珠母贝血细胞的激活作用。结果表明:LPS和IL-2对iNOS活性的诱导作用表现出浓度和时间依赖的特征。刺激后36h左右iNOS活性达到最大,LPS和IL-2的最佳诱导剂量范围分别为1～10g/mL和10～100ng/mL,并且IL-2对iNOS活性的激活作用较LPS快。     

F1F0-ATP酶的超分子结构和功能的研究

通过X衍射晶体学法、荧光标记显微镜法、负染色体电子显微镜与单克隆抗体技术等对F1F0 ATP酶结构和功能的研究发现,在大肠杆菌、叶绿体和牛心线粒体中,F1F0 ATP酶复合体分别由8、9和16种不同的亚基组成.所有F1F0 ATP酶都有类似的结构,球状的F1和F0是由一个中心转轴和一个外围柄连接在一起.其中,中心转轴由γ和ε亚基组成,外围柄由b2(Ⅰ,Ⅱ)和δ亚基组成.在酶的催化过程中,α3β3亚基通过γ亚基与膜上的c亚基环相互作用,驱动ATP合成酶的中心转轴旋转.F1F0 ATP酶利用电化学质子梯度的能量,催化ADP(腺苷二磷酸)和Pi(无机磷酸盐)形成ATP(腺苷三磷酸).     

甘蔗蔗糖磷酸合成酶基因中间缺失突变体的构建

根据NCBI中EU278617序列设计引物,利用甘蔗SPSG2菌液为模板,扩增SPS8-11并克隆到T载体后测序.通过排除野生型DNA的定点突变PCR方法获得SPS8-11中第8内含子缺失TGCATG的突变体pMD-SPS8-11-A,酶切后测序鉴定.成功构建了甘蔗SPS8-11中间缺失突变体pMD-SPS8-11-A.     

法呢基焦磷酸合酶基因的克隆及双元载体的构建

以留兰香(MenthaspicataL)为材料,利用RT PCR方法克隆法呢基焦磷酸合酶基因(fps)的cDNA,核酸序列分析表明,该基因的编码区长1050bp,编码349个氨基酸.进一步将fps基因插入植物表达载体BinAR,酶切鉴定及测序结果都证明fps的植物双元表达载体构建成功,为fps基因在烟草中的异源表达奠定了基础.     

Crystallization and preliminary X-ray crystallographic analysis of human geranylgeranyl pyrophosphate synthase

Human geranylgeranyl pyrophosphate synthase (GGPS) is an enzyme that catalyzes the synthesis of geranylgeranyl pyrophosphate (GGPP) from farnesyldiphosphate and isopentenyldiphosphate. Recombinant human GGPS was crystallized by the hanging-drop vapor diffusion method. Crystals were grown at 18℃ using PEG 4000 as precipitant. Diffraction data were obtained to a resolution of 2.8 ? from a single frozen crystal belonging to space group P1, with unit-cell parameters: a=68.9 ?, b=107.7 ?, c=137.4 ?, α=99.6°, β=97.6°, γ=97.8°.     

氯化钴对人eNOS基因启动子转录活性的影响

目的:建立氯化钴诱导的人脐静脉血管内皮细胞-12(HUVEC-12)缺氧模型,研究人血管内皮细胞一氧化氮合酶(eNOS)基因的启动子活性变化.方法:用含不同浓度氯化钴的培养基培养细胞,检测细胞培养上清中的乳酸脱氢酶(LDH)含量;将已构建的pGL2-eNOS-p质粒转染HUVEC-12细胞,利用双荧光素酶报告基因技术检测在不同浓度氯化钴和不同作用时间下的eNOS启动子转录活性.结果:氯化钴刺激下HUVEC-12细胞培养上清的LDH含量随氯化钴作用浓度增加而提高;氯化钴刺激后转染细胞的eNOS启动子活性呈现随氯化钴剂量增加而升高的趋势,随作用时间的延长而上升的趋势.结论:成功建立氯化钴诱导HUVEC-12细胞缺氧的体外模型.     

红树林土壤微生物模块化聚酮合酶基因(簇)及其多样性研究

聚酮类化合物(Polyketide)是一类重要的具有药用价值的生物活性分子.通过宏基因组学技术获得了红树林土壤微生物中模块化聚酮合酶(Polyketide Synthase,PKS)基因(簇),并对其多样性进行了研究.首先构建了该红树林土壤微生物的16SrDNA文库,初步分析了红树林土壤样本的细菌群落组成,发现许多菌群曾经被报道存在PKS基因簇,约占整个细菌群落的75%.利用相关菌群的PKS酮基合成酶结构域(Ketosynthase,KS)的保守区域序列信息设计简并引物,并构建了KS DNA序列文库.经测序获得131条新型KS序列,该结果表明红树林土壤微生物中蕴含了许多新型的聚酮类化合物合成酶.为了进一步获得聚酮合酶基因(簇),我们通过土壤微生物宏基因组Cosmid文库的构建与筛选,得到了13个属于trans-AT PKS,cis-AT PKS,NRPS/PKS Hybrid等类型的聚酮合酶的基因(簇),并通过构建KS序列的系统进化树分析了样本中聚酮合酶的多样性组成.     

太极拳锻炼对中老年人血清NO含量和NOS活性的影响

对长期坚持太极拳锻炼的中老年人血清NO含量及NOS活性进行测定.结果显示:(1)长期规律的太极拳锻炼可以纠正和延缓中老年人因增龄引起的血清NO含量下降,从而预防心血管疾病的发生;(2)长期规律的太极拳运动明显增强老年人血清NOS的活性,进而增加NO的合成.