液相色谱-串联质谱法测定生物样本全基因组DNA甲基化

建立了基于液相色谱-电喷雾串联质谱的分析方法,对生物样本中全基因组DNA甲基化水平进行定量测定.首先将DNA从生物样本中提取出来,将DNA片段酶解为单核苷,利用液相色谱-串联质谱测定胞嘧啶核苷和5-甲基胞嘧啶核苷的含量,从而计算出其全基因组DNA甲基化率.利用该法研究了暴露于全氟辛烷磺酸的L-02细胞、10例原发性肝癌病例血浆样本和10例对照血浆样本的全基因组DNA甲基化水平,得出了它们的总甲基化率变化的初步结果.本方法操作简单,具有很高的灵敏度和稳定性,为研究生物样本,尤其是临床上易得但DNA含量极低的血浆样本的总甲基化水平提供了思路.     

脱氧核糖核酸甲基化分析方法在肿瘤诊断和治疗中的应用

脱氧核糖核酸( DNA)甲基化是表观遗传改变的主要作用方式,在基因表达调控、基因组印迹、胚胎发育、维持正常细胞功能等过程中起着极其重要的作用;异常甲基化可以导致肿瘤的发生、发展.因此,探讨甲基化形成与改变的可能机制,建立准确性好、灵敏度高、操作简单的DNA甲基化分析方法,可为某些肿瘤的早期诊断提供重要依据.本文综述了D...     

DNA甲基化检测研究进展

DNA甲基化可以在不改变DNA碱基序列的情况下改变基因活动和功能,影响基因印迹、细胞分化、细胞增殖、染色质重塑、胚胎发育等重要生命活动,是表观遗传的研究重点和热点。为了研究DNA甲基化的分布、状态以及调控机制,越来越多的DNA甲基化分析和检测技术已被开发。本文归纳了近年来的DNA甲基化检测方法,讨论了不同方法的优缺点,为未来深入研究DNA甲基化提供参考。     

DNA胞嘧啶的甲基化与去甲基化进展

DNA胞嘧啶甲基化调控基因的表达以及多种生物功能,如细胞分化。概念上,DNA去甲基化是将已甲基化DNA核苷转化为未修饰核苷,是DNA甲基化的逆向过程。但在生物体内,这是一个涉及多步反应的非常复杂的过程。本综述简要介绍了动植物中DNA胞嘧啶的甲基化与去甲基化的研究现状,并讨论了恶性肿瘤(癌症)、阿尔茨海默氏病、心血管疾病、肺纤维病变和进食障碍等多种疾病中的甲基化与去甲基化异常。     

面向DNA甲基化修饰分析的质谱技术

DNA甲基化作为表观遗传修饰中一种重要的调控方式,通过调控基因的表达,从而影响机体内一系列的生物学过程。色谱-质谱法是研究DNA甲基化修饰的重要研究手段。随着对哺乳动物DNA甲基化的生物学功能的深入研究,应用于研究表观遗传修饰的手段与仪器设备越来越先进。为了对DNA修饰进行定性与定量的分析检测,除了高效液相色谱整合不同种类质量分析器的质谱联用（HPLC-MS）技术外,目前还开发应用了基质辅助激光解析质谱技术（MALDI-ToF-MS）和气相色谱-质谱联用技术（GC-MS）,从而极大拓展了DNA甲基化修饰研究的手段。本文对分析表观遗传DNA甲基化修饰的质谱技术发展进行综述,希望为DNA甲基化修饰分析提供有价值的研究策略。     

DNA羟甲基化测序技术

5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)是继5-甲基胞嘧啶(5-methylcytosine, 5mC)之后发现的第六种碱基, 已在多种哺乳动物组织和细胞中检出. 与5mC相比, 5hmC的含量更低, 但5hmC也具有非常重要的生物学功能. 已有的研究表明, 5hmC参与了染色体重新编程、基因表达的转录调控, 并在DNA去甲基化过程中发挥重要作用. 另外, 5hmC可能与特定肿瘤的发生密切相关, 有可能成为肿瘤早期诊断的生物标志物. 因此, 发展可靠、灵敏和准确的5hmC检测技术至关重要. 本综述针对DNA羟甲基化的检测及测序技术进行了简要介绍.     

蛋白质甲基化修饰的蛋白质组学分析方法新进展

蛋白质的甲基化修饰是一类重要的翻译后修饰。但与磷酸化、糖基化和泛素化等翻译后修饰相比,甲基化修饰的蛋白质组学分析方法开发还是一个较新的研究领域。近几年,由于甲基化修饰在表观遗传调控中的重要作用,这一修饰类型得到了越来越多的关注,相关的分析技术和分析方法也取得了较多进展。其中,基于质谱的蛋白质组学分析方法在甲基化修饰中发挥着关键的作用,实现了这一甲基化修饰的高通量分析。该综述将从甲基化修饰的分离富集、假阳性率控制以及定量蛋白质组学等方面对一些蛋白质甲基化修饰的分析技术和方法的最新进展进行介绍。     

甲基化作用增强了盐效应对M.L.DNA大分子构象的影响

本文用Hha I DNA甲基化酶为工具酶,制备了甲基化水平为2.39％的Micrococcus luteus DNA(天然的M.L.DNA是完全未甲基化的)。又以CD光谱、UV光谱及微量热法等手段,比较了甲基化和未甲基化的M.L.DNA在不同MgCl_2浓度下的[θ]值、A值及释热值的变化。发现随着MgCl_2浓度的增加,两种M.L.DNA的[θ]_(275)值均减少;而—[θ]_(220-250)值加大;A_(260)值则呈明显的减色效应;释热值却相应增加。这都说明MgCl_2的作用使M.L.DNA大分子在空间上趋于缩拢,且这个过程均持续约10min。同时发现:甲基化水平为2.39％的M.L.DNA对MgCl_2效应的敏感性要比末甲基化的M.L.DNA高50—70倍。     

p16基因甲基化的芯片定量检测

p16基因的失活与多种肿瘤相关,但p16基因缺失率较低,突变更为罕见,p16基因启动子区CpG岛甲基化与其蛋白表达密切相关．DNA甲基化已成为目前研究的热点,现有的技术包括：Southernblot法、限制性内切酶-PCR法、DNA测序法、甲基化特异性PCR(MSP)、     

核酸甲基化检测方法进展

核酸甲基化是表观遗传学领域最重要的现象之一,对于基因的活化、基因印迹、染色体稳定性等有重要影响.在全基因组范围内和特定基因片段中,甲基化水平均应维持在各自的正常范围内,任何的甲基化水平失常都将导致相应生理功能的失调.由于核酸甲基化在生命体系里的重要性,基于全基因和特定基因的甲基化检测在学术界引起了广泛关注.目前已有多种生物学和化学的方法用于核酸甲基化检测.这里,我们重点总结近年来新发展的基于化学修饰和分析化学的核酸甲基化检测方法.     

Structural study on a bioactive fructan from the root of Achyranthes bidentata Blume

The structure of a saccharide component (Abs),with pronounced activity of improving immunity system,isolated from the root of Achyranthes bidentata Blume,a traditional Chinese herbal medicine,was studied.Based on 13C NMR,HPLC,and methylation analyses,Abs was shown to be a mixture of short-chain fructans with an average dp of 8,containing more (2→6) than (2→1) linked β-D-fructofuranosyl residues,with branching at O-6 or O-1 of 18% of the D-fructofuranoeyl residues.     

Novel erythromycin A 9-tritylhydrazone: selective 6-O-methylation and conformational analysis

The straightforward synthesis of the novel 9-tritylhydrazone erythromycin A and its highly regioselective O-methylation at C(6)-OH are described. Preliminary conformational data based on X-ray diffraction, ¹H NMR and molecular mechanics is also presented with the aim of understanding the observed high regioselectivity. The facile synthesis of 6,12-O-dimethylerythromycin A, a standard to assess clarithromycin purity in quality control processes, is reported as well.     

A Novel Method for Analysis of Xanthate Group Distribution in Viscoses

Analytical monitoring of xanthation in the viscose process along with xanthate group analysis in the viscose material is a long-debated problem in cellulose chemistry. The task is rendered extremely intricate by the lability of the starting material and the harshness of the reaction medium, which adds to a lack of suitable analytical approaches. In a four-years' endeavor in our lab, a method is being developed which allows to analyze the distribution of xanthate groups in viscoses relative to the anhydroglucose units and along the cellulose chain. In a first step the xanthate groups are stabilized by alkylation, which was optimized towards quantitative conversion. In a second step, the remaining free hydroxyl groups are protected by carbanilation, followed by selective removal of the stabilized xanthate groups. Steps two and three thus generate an inverse image of the initial xanthate pattern. In the forth and fifth step, the liberated hydroxyl groups are methylated, and the carbanilates are removed, so that in the overall process the xanthates were replaced by methyl groups. All reaction steps have been comprehensively tested with regard to completeness of conversion and orthogonality of the protecting groups.     

Characterization of microwave-synthesized polydextrose and its radical-scavenging activity

Polydextrose (PD) was rapidly synthesized under microwave irradiation using glucose as substrate and phosphoric acid as catalyst. The reaction products were identified by methylation analysis, Fourier- Transform Infrared (FT-IR) and NMR spectroscopy. The FT-IR spectra confirmed the polymerization of glucose to form PD. Methylation analysis and ¹H NMR and ¹³C NMR assignments indicated that the PD was a highly branched polysaccharide. The results of radical scavenging activity assays showed that this PD exhibited a potent free hydroxyl radical scavenging activity. This synthetic method can be used to produce PD for the food industry.     

Tuning the Conformation and Color of Conjugated Polyheterocyclic Skeletons by Installing ortho‐Methyl Groups

ortho‐Methyl effects are exploited to tune steric hindrance between side‐chain N,N′‐diaryls and polycyclic dihydrodibenzo[a,c]phenazine, and in turn control the conformations of N,N′‐diphenyl‐dihydrodibenzo[a,c]phenazine (DPAC) and its ortho‐methyl derivatives M x ‐M y (x=0, 1 or 2, y=1 or 2, x and y correlate with the number of methyl groups in the ortho‐positiond of N,N′‐diphenyl). The magnitude of steric hindrance increases as x and y increase, and the V‐shaped dihydrodibenzo[a,c]phenazine skeleton is gradually tuned from a bent (DPAC) to planar ( M2‐M2 ) structure in the ground state. As a result, the relaxation of the excited‐state structure of DPAC and its numerous analogues could be mimicked by model structures M x ‐M y, demonstrating for the first time the the conformation change from bent‐to‐planar and hence a large range of energy‐gap tuning of polycyclic conjugated structures controlled by the steric hindrance.     

O6-鸟嘌呤和O4-胸腺嘧啶甲基化与DNA碱基异常氢键作用的理论研究

用密度泛函B3LYP方法在6-311＋G**基组水平上对顺(cis-)、反式(anti-)O⁶-甲基鸟嘌呤(O⁶-MeG)和O⁴-甲基胸腺嘧啶(O⁴-MeT)与DNA碱基(腺嘌呤A、鸟嘌呤G)的非Watson-Crick氢键二聚体进行了优化. 在MP2/cc-pVXZ (X＝D,T)//B3LYP/6-311＋G**水平上, 采用完全基组外推方法校正了氢键二聚体的相互作用能, 并用完全均衡校正法(CP)校正了基组重叠误差(BSSE). 此外, 在B3LYP/6-311＋G**水平上计算了各氢键碱基对的全电子波函数, 并用分子中的原子理论(AIM)和电子密度拓扑方法分析了碱基间的弱相互作用. 计算结果显示, 甲基化使碱基对间的氢键作用模式发生了明显的扭转和不同程度的位移, 碱基间的电子密度分布和氢键作用能明显减小, 甲基化对O⁶-MeG和O⁴-MeT与DNA碱基间的氢键作用是去稳定化的, 这种影响主要来自于大体积的甲基的空间效应和给电子效应, 且对顺式的影响明显大于反式. 计算结果与文献给出的实验结论基本一致.     

DNA Methylation: Site-Specific Methylations Cause Gene Inactivation

In the molecular biology of eukaryotic organisms, the elucidation of mechanisms involved in the regulation of gene expression has assumed an important role. All cells of an organism carry the same genes, but differ in the patterns of genes they express. There is an increasing amount of evidence that cancer cells exhibit a pattern of gene expression which can be very different from that of normal cells. One of the molecular signals that has been recognized in the regulation of gene expression in eukaryotes is the modified nucleotide 5-methylcytosine (5-mC). Through experiments in well-characterized eukaryotic systems, evidence has been adduced that the introduction of 5-mC into highly specific sequences, particularly into the 5′ and promoter regions of a gene, can cause gene inactivation. Viral and other eukaryotic systems have helped in the recognition of this cause-and-effect relationship. Inactive genes are frequently hypermethylated in the promoter region; active genes are hypomethylated. However, these correlations are not always as simple and straightforward. The biochemical mechanisms by which site-specific DNA methylations cause gene inactivation have not yet been determined. It is plausible to postulate that promoter methylations could somehow affect the binding of cellular enzymes involved in recognizing the promoter of a gene. Structural alterations of DNA promoter sequences arising from DNA methylations could also be important. DNA methylation is likely to represent a long-term inactivation signal, since it is presently thought that patterns of DNA methylation can be changed only by DNA replication and specific inhibition of post-replicative maintenance methylation.     

The Impact of Amino Acid Side Chain Mutations in Conformational Design of Peptides and Proteins

Local energetic effects of amino acid replacements are often considered to have only a moderate influence on the backbone conformation of proteins or peptides. As these effects are difficult to determine experimentally, no comparison has yet been performed. However, knowledge of the influence of side chain mutations is essential in protein homology modeling and in optimizing biologically active peptide ligands in medicinal chemistry. Furthermore, the tool of N‐methylation of peptides is of increasing importance for the design of peptidic drugs to gain oral availability or receptor selectivity. However, N‐methylation is often accompanied by considerable population of cis‐peptide bond structures, resulting in completely different conformations compared with the parent peptide. To retain a favored structure, it might be important to understand the effect of different side chains on the backbone conformation and to enable the introduction of an N‐methylation at the right position without disturbing a biologically active conformation. In order to detect even small energetic effects due to side chain mutations, we employed a trick to investigate the structural equilibrium of a selected cyclic pentapeptide in which two conformations are equally populated. Very small energetic differences between both conformations could easily be determined experimentally by identifying shifts in the population of both isomers.     

The pH effect on the naphthoquinone-photosensitized oxidation of 5-methylcytosine

The pH effect on the one-electron photooxidation of 5-methyl-2'-deoxycytidine (d(m)C) by sensitization with 2-methyl-1,4-naphthoquinone (NQ) was investigated. Photoirradiation of an aqueous solution containing d(m)C and NQ under slightly acidic conditions of pH 5.0 efficiently produced 5-formyl-2'-deoxycytidine, whereas similar NQ-photosensitized oxidation of d(m)C proceeded to a lesser extent under more acidic or basic conditions. Fluorescence-quenching experiments revealed that the less-efficient photooxidation at pH values below 4.5 is attributed to the decreased rate of one-electron oxidation of d(m)C owing to protonation at the N(3)-position. The NQ-photosensitized oxidation of an N(4)-dimethyl-substituted d(m)C derivative under various pH conditions also suggests that the pH change in the range of 5.0 to 8.0 may be responsible for a reversible deprotonation-protonation equilibrium at the N(4)-exocyclic amino group of the d(m)C radical cation. In accord with the photochemical reactivity of monomeric d(m)C, the 5-methylcytosine residue in oligodeoxynucleotides was oxidized efficiently by photoexcited NQ-tethered oligodeoxynucleotides under slightly acidic conditions to form an alkali-labile 5-formylcytosine residue.