The conformation alteration of mouse hepatic histones after reacting with nicotinein vitro

UV differential spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy assays have been applied to studying the conformation alteration of mouse hepatic histones H1 and H3 after reacting with nicotinein vitro. The results indicate that their conformation changes from regular form to random form with the increasing reaction dose of nicotine. The adduction of nicotine or its metabolites with histones H1 and H3 accounts for the conformation alteration. Nicotine may affect the structure, function and expression of genes of chromosome by changing the conformation of histones.     

生物加速器质谱法研究尼古丁与生物大分子的加合

自1992年以来,使用北京大学重离子物理研究所的2×6 MV EN型串列静电加速器上的加速器质谱仪(AMS),较系统地研究了¹⁴C标记的尼古丁和鼠肝、肺细胞核DNA的加合,以及和鼠肝细胞核组蛋白的加合。在这些体内生物大分子中均观察到肯定的加合作用。加合物数目随尼古丁的动物剂量的增加而增加,剂量响应呈指数直线关系或非指数直线或曲线关系。实验结果表明尼古丁不仅是公认的吸烟上瘾的主要因子,而且可能是致癌物质。AMS测量灵敏度达很高水平：每10¹⁰个核苷酸可检测的最低限量为1个加合物和每mg(毫克)H1蛋白质可检测的最低限量为4.6×10^-17mol加合物,后者高于前人报道过的灵敏度。生物加速器质谱法可以为基因毒性研究提供极灵敏的生物标志物。     

盐透析体外组装核小体及检测方法

核小体是构成真核生物染色质的基本结构单位,体内研究核小体及染色质结构受到诸多因素限制,体外重构核小体结构是研究与核小体及染色质结构相关课题的一种重要的方法手段.实验将ES1,CS1以及601DNA序列克隆到载体中,通过PCR大量扩增回收得到目的DNA条带,表达纯化了4种组蛋白且装配成组蛋白八聚体,在盐透析的条件下组装形成核小体结构,利用EB染色以及Biotin标记的方法分析检测了形成核小体的效率.结果显示,在盐透析的条件下,可以有效的组装形成核小体结构,而且随着组蛋白八聚体与DNA的比例增加,核小体的形成效率显著提高.本实验为核小体定位、染色质重塑及组蛋白变体等表观遗传学以及结构生物学领域的研究奠定一定的基础.     

The RCAF complex mediates chromatin assembly during DNA replication and repair

Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.     

六元瓜环对尼古丁捕集作用的测试

作者通过利用^1H NMR技术,荧光光谱和紫外吸收光谱方法,考察尼古丁(gN)与六元瓜环(Q[6]的相互作用情况,三种方法得到的结果相同。结果表明,六元瓜与尼古丁环能相互作用而形成配合物,是一个快速反应,且在pH=3～10时,相互作用效果均很好。     

Structural determinants for generating centromeric chromatin

Mammalian centromeres are not defined by a consensus DNA sequence. In all eukaryotes a hallmark of functional centromeres--both normal ones and those formed aberrantly at atypical loci--is the accumulation of centromere protein A (CENP-A), a histone variant that replaces H3 in centromeric nucleosomes. Here we show using deuterium exchange/mass spectrometry coupled with hydrodynamic measures that CENP-A and histone H4 form sub-nucleosomal tetramers that are more compact and conformationally more rigid than the corresponding tetramers of histones H3 and H4. Substitution into histone H3 of the domain of CENP-A responsible for compaction is sufficient to direct it to centromeres. Thus, the centromere-targeting domain of CENP-A confers a unique structural rigidity to the nucleosomes into which it assembles, and is likely to have a role in maintaining centromere identity.     

Spectroscopic studies on interaction of hemoglobin and serum albumin with nicotine

The interactions of nicotine and Hb/SA were studied in vitro by UV/Vis, fluorescence, 1H NMR and FT-IR spectroscopies. The UV/Vis absorbance of Hb/SA (200 nm)shifted to red and decreased gradually with the addition of nicotine, indicating that the protein conformational change resulted from the chemical interaction. With increasing nicotine concentration, incubation of SA with nicotine caused the quenching of fluorescence typical of protein tryptophan residues, which meant that the vicinity of the tryptophan residues of SA was changed because of nicotine. FT-IR spectra showed that α-helix component of Hb/SA decreased, turn and β-structure components of Hb/SA increased in the presence of nicotine. In the 1H NMR spectra of nicotine, all proton peaks on pyrrolidinyl ring moved to downfield and the resonance emanating from nicotine was preferentially broadened while the concentration of Hb/SA increased. All these results indicate that nicotine and Hb/SA in vitro interact on each other, forming a new complex and inducing the protein conformational change.     

多胺修饰?-环糊精:钕配合物对萘二胺的分子识别研究

将?-环糊精的6-OH用二乙烯三胺基取代，得到了6-脱氧-二乙烯三胺基?-环糊精(DTCD),在镧系金属NdCl3的存在下，分别与1,5-萘二胺和1,8-萘二胺反应，得到了2种三元超分子配合物，并用核磁共振氢谱对配合物进行了表征。其中主客体分子的化学计量比为2:1，客体分子等价氢原子的信号简并现象在包合前后没有发生变化，表明客体分子在主体空腔保持着对称的构象。2种三元超分子配合物均呈“三明治”式夹心结构。     

Reduced antinociception in mice lacking neuronal nicotinic receptor subunits

Nicotine exerts antinociceptive effects by interacting with one or more of the subtypes of nicotinic acetylcholine receptors (nAChRs) that are present throughout the neuronal pathways that respond to pain. To identify the particular subunits involved in this process, we generated mice lacking the alpha4 subunit of the neuronal nAChR by homologous recombination techniques and studied these together with previously generated mutant mice lacking the beta2 nAChR subunit. Here we show that the homozygous alpha4-/- mice no longer express high-affinity [3H]nicotine and [3H]epibatidine binding sites throughout the brain. In addition, both types of mutant mice display a reduced antinociceptive effect of nicotine on the hot-plate test and diminished sensitivity to nicotine in the tail-flick test. Patch-clamp recordings further reveal that raphe magnus and thalamic neurons no longer respond to nicotine. The alpha4 nAChR subunit, possibly associated with the beta2 nAChR subunit, is therefore crucial for nicotine-elicited antinociception.     

吡唑并[3,4-b]吡啶酮衍生物与牛血清白蛋白相互作用的研究

在模拟生理条件下,用荧光光谱法研究了吡唑并[3,4-b]吡啶酮衍生物4-(3-硝基苯基)-4,5-二氢-3-甲基-1-苯基-吡唑并[3,4-b]吡啶-6-(7H)-酮(NPPO)与牛血清白蛋白(BSA)的相互作用.NPPO对BSA的荧光猝灭属于动态猝灭,并获得了不同温度对应的猝灭常数.由热力学参数,推断了NPPO与BSA之间主要靠疏水作用力结合.按照Frster的偶极-偶极能量转移理论,推算得出NPPO与蛋白质的结合位置距离色氨酸残基2.72nm.研究了NPPO对BSA构象的影响,并探讨了一些金属离子对体系的影响.     

水杨醛甘氨酸Schiff碱的从头算构象研究

运用从头算HF方法对水杨醛甘氨酸Schiff碱的构象进行了理论研究.结果表明,C=N键与苯环的共轭以及分子内弱相互作用是分子能量降低的2个重要结构因素.在各存在形态的所有最稳定构象中c=N键均与苯环共轭,均至少存在1条H14的弱氢键.酸式结构最稳定构象羧基上羟基氢H22背离大共轭平面;酸根形态的稳定构象羧基与苯环共面形成平面分子,且存在2条氢键;钾盐酚式最稳定构象羧基螯合成键,而醌式最稳定构象为平面分子,羧基单齿成键,且存在H14的2条弱氢键.由于具有很大的能量优势,水杨醛甘氨酸Schiff碱在水中的主要存在形态为酸式,该结论与其水溶液的实验研究结果一致.     

超高效液相色谱法测定卷烟烟气中游离态和质子化尼古丁含量

采用超高效液相色谱法(UPLC)建立了一种快速测定卷烟烟气中游离态和质子化尼古丁含量的反相离子对液相色谱法.首先对游离态和质子化的尼古丁的样品提取分离条件进行了研究,该方法的流动相为含有4mmol/L的庚烷磺酸钠盐作为离子对试剂的三氟乙酸缓冲水溶液(pH=3)与乙腈混合(体积比86∶14).线性范围为0.06～129μg/mL,相关系数是0.9999,回收率为87%～99.5%.检测下限为60ng/mL.采用该方法测定了26种商品卷烟的游离尼古丁和质子化尼古丁的含量.最后,根据所测样品卷烟中游离态烟碱的数据,并结合卷烟的实际评吸结果数据进行了方差分析和相关性分析.结果表明,游离态烟碱的含量与评吸的劲头指标具有特别显著的正相关性,与喉部刺激、鼻腔刺激、口腔刺激和干躁感4个评吸指标具有一定的负相关性,而与余味评吸指标没有相关性.     

硫酸反萃取法从烟草中提取烟碱

采用改进的二次萃取蒸馏的方法,提取烟草中的烟碱,用正交实验和单因素实验考察了提取工艺和萃取分离过程对提取效率的影响.结果表明,优化工艺条件为:以0.5%NaOH溶液浸泡粉碎后的烟叶,置于65℃水浴中搅拌3 h,过滤后调节所得滤液pH至12,用正己烷按照体积比1∶2萃取3次,有机相用20%硫酸溶液反萃,调节pH到12后,用正己烷二次萃取,减压蒸馏后得到纯度为45%的烟碱,提取率可达72%.