The conformation alteration of mouse hepatic histones after reacting with nicotine in 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 nicotine in 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。     

~1H NMR Investigation of Supramolecular Complex between β-Cyclodextrin and Cholesterol

A supramolecular complex between β-cyclodextrin and cholesterol was synthesized and characterized via proton 1H NMR spectroscopy. In the supramolecular complex,the stoichiometric proportion of β-cyclodextrin to cholesterol is 1:2. The possible conformation of the supramolecular complex was depicted according to the chemical shift variance of proton 1H NMR of the host and guest molecules inside the inclusion complex. Removal efficiency of cholesterol complexed by β-cyclodextrin in our work is increased to a remarkable extent. This result can be applied in the field of drug development to reduce cholesterol in blood and other human organs.     

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.     

Interaction between poly（amidoamine） dendrimers and DNA studied by spectroscopic methods

The interaction between amino-terminated, and ethylenedtamine core poly（amtdoamine） （PAMAM） dendrimers and herring sperm DNA was investigated by various spectroscopic methods including UV spectroscopy, fluorescence spectroscopy, microscopic FTIR- and circular dichroism （CD-） spectroscopy. Ethidium bromide （EB） is used as a nucleic acid probe for this study. Experimental results show that PAMAM dendrimers can form stable complexes with DNA and the dendrimers bind to DNA sufficiently strong which cannot be displaced by EB, and we also found that the formation of the complexes can cause the conformation change of the DNA secondary structure. According to the Scatchard analysis, the association constant of PAMAM to DNA is calculated to be 2.53×10^4 mol/L^-1.     

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。     

NMR and IR investigations of ternary inclusion complexes among β-cyclodextrin, rare earth metal ions, and naphthalenediamines

Ternary inclusion complexes β-cyclodextrin (β-CD), rare earth metal ions (YbCl₃, YCl₃), and 1,8-naphthalene- diamine/1,5-naphthalenediamine are synthesized in basic aqueous media, which are characterized via ¹H NMR and IR spectroscopy. The stoichiometric proportion of β-CD:YbCl₃:1,5-naphthalenediamine is 2:1:2, that of β-CD:YCl₃:1,8-naphthalenediamine is 2:1:1, and that of β-CD:YbCl₃:1,8-naphthalenediamine is 2:1:1. The IR spectroscopy of the ternary inclusion complexes in the range of 935–1 000 cm⁻¹ reveals the existence of the coordinate bond M—O or M—N. The possible conformations of the ternary inclusion complexes are depicted. Biography: JIANG Huiming(1972–), male, Associate professor, research direction: molecular complex chemistry, supramolecular chemistry.     

Induction of nicotine in tobacco by herbivory and its relation to glucose oxidase activity in the labial gland of three noctuid caterpillars

Tobacco Nicotiana tabacum L. is a host plant ofHelicoverpa armigera (Huibner), Helicoverpa assulta Guenee and Spodoptera litura (Fabricius) (Lepidoptera, Noctuidae). The difference in leaf nicotine response to the feeding by these three larvae and the mechanical simulation of their feeding was examined by HPLC. Results indicated that nicotine induction was suppressed by H. armigera and H. assulta larvae feeding or by simulated damage treated with their labial glands extracts. The production of nicotine was also suppressed by the glucose oxidase from Aspergillus niger when it was treated on mechanically wounded leaf area. On the contrary, the nicotine production was stimulated by S. litura larva feeding or by simulated damage treated with its labial gland extract. Heat denature can not counteract the stimulation effect of the S. litura labial gland extracts to tobacco nicotine production. The glucose oxidase activity was detected in labial gland extracts of both H. armigera and H. assulta, but the activity in H. armigera was significantly higher than that in H. assulta. No glucose oxidase activity was detected in labial gland extracts of S. litura. It is shown that the glucose oxidase activity in labial glands of caterpillars plays an important role in the nicotine response to herbivory. The glucose oxidase was mainly contained in the labial gland of H. armigera larva, and had the highest activity at pH 7.0. D-Glucose was the optimal substrate of the glucose oxidase. Labial gland glucose oxidase activities varied daily during larval development with high activities found when larvae were actively feeding.     

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.