Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

Accurate mass-based analysis in human sport doping control : Application of liquid chromatography - time-of-flight mass spectrometry

Human sport doping control analysis is a complex and challenging task for anti-doping laboratories. The List of Prohibited Substances and Methods, updated annually by World Anti-Doping Agency (WADA), consists of hundreds of chemically and pharmacologically different low and high molecular weight compounds. This poses a considerable challenge for laboratories to analyze for them all in a limited amount of time from a limited sample aliquot. The continuous expansion of the Prohibited List obliges laboratories to keep their analytical methods updated and to research new available methodologies. In this thesis, an accurate mass-based analysis employing liquid chromatography - time-of-flight mass spectrometry (LC-TOFMS) was developed and validated to improve the power of doping control analysis. New analytical methods were developed utilizing the high mass accuracy and high information content obtained by TOFMS to generate comprehensive and generic screening procedures. The suitability of LC-TOFMS for comprehensive screening was demonstrated for the first time in the field with mass accuracies better than 1 mDa. Further attention was given to generic sample preparation, an essential part of screening analysis, to rationalize the whole work flow and minimize the need for several separate sample preparation methods. Utilizing both positive and negative ionization allowed the detection of almost 200 prohibited substances. Automatic data processing produced a Microsoft Excel based report highlighting the entries fulfilling the criteria of the reverse data base search (retention time (RT), mass accuracy, isotope match). The quantitative performance of LC-TOFMS was demonstrated with morphine, codeine and their intact glucuronide conjugates. After a straightforward sample preparation the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The hydrophilic interaction technique (HILIC) provided good chromatographic separation, which was critical for the morphine glucuronide isomers. A wide linear range (50-5000 ng/ml) with good precision (RSD<10%) and accuracy (±10%) was obtained, showing comparable or better performance to other methods used. In-source collision-induced dissociation (ISCID) allowed confirmation analysis with three diagnostic ions with a median mass accuracy of 1.08 mDa and repeatable ion ratios fulfilling WADA s identification criteria. The suitability of LC-TOFMS for screening of high molecular weight doping agents was demonstrated with plasma volume expanders (PVE), namely dextran and hydroxyethylstarch (HES). Specificity of the assay was improved, since interfering matrix compounds were removed by size exclusion chromatography (SEC). ISCID produced three characteristic ions with an excellent mean mass accuracy of 0.82 mDa at physiological concentration levels. In summary, by combining TOFMS with a proper sample preparation and chromatographic separation, the technique can be utilized extensively in doping control laboratories for comprehensive screening of chemically different low and high molecular weight compounds, for quantification of threshold substances and even for confirmation. LC-TOFMS rationalized the work flow in doping control laboratories by simplifying the screening scheme, expediting reporting and minimizing the analysis costs. Therefore LC-TOFMS can be exploited widely in doping control, and the need for several separate analysis techniques is reduced.

Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N-6-adenine DNA methyltransferases of Neisseria meningitidis

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N-6-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N-6-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5'-CGY(m6)AG-3'), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5'-AC(m6)ACC-3') and ModD1 (exemplified by M.Nme579I) (5'-CC(m6)AGC-3'). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5'-CGY(m6)AG-3'. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5'-GCGC(m6)AGG-3' sites, to 100% at 5'-ACGT(m6)AGG-3' sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.

Binding site size limitations of imidazole-pyrrole polyamides for recognition in the minor groove of DNA

The discovery that the three ring polyamide Im-Py-Py-Dp containing imidazole (Im) and pyrrole (Py) carboxamides binds the DNA sequence 5'-(A,T)G(A,T)C(A,T)-3' as an antiparallel dimer offers a new model for the design of ligands for specific recognition of sequences in the minor groove containing both G,C and A,T base pairs. In Chapter 2, experiments are described in which the sequential addition of five N- methylpyrrolecarboxamides to the imidazole-pyrrole polyamide Im-Py-Py-Dp affords a series of six homologous polyamides, Im-(Py)_2-7-Dp, that differ in the size of their binding site, apparent first order binding affinity, and sequence specificity. These results demonstrate that DNA sequences up to nine base pairs in length can be specifically recognized by imidazole-pyrrole polyamides containing three to seven rings by 2:1 polyamide-DNA complex formation in the minor groove. Recognition of a nine base pair site defines the new lower limit of the binding site size that can be recognized by polyamides containing exclusively imidazole and pyrrolecarboxamides. The results of this study should provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity.

In Chapter 3 the design and synthesis of the hairpin polyamide Im-Py-Im-Py-γ-Im- Py-Im-Py-Dp is described. Quantitative DNase I footprint titration experiments reveal that Im-Py-Im-Py-γ-Im-Py-Im-Py-Dp binds six base pair 5'-(A,T)GCGC(A,T)-3' sequences with 30-fold higher affinity than the unlinked polyamide Im-Py-Im-Py-Dp. The hairpin polyamide does not discriminate between A•T and T•A at the first and sixth positions of the binding site as three sites 5'-TGCGCT-3', 5'-TGCGCA-3', and 5 'AGCGCT- 3' are bound with similar affinity. However, Im-Py-Im-Py-γ-Im-Py-Im-PyDp is specific for and discriminates between G•C and C•G base pairs in the 5'-GCGC-3' core as evidenced by lower affinities for the mismatched sites 5'-AACGCA-3', 5'- TGCGTT-3', 5'-TGCGGT-3', and 5'-ACCGCT-3'.

In Chapter 4, experiments are described in which a kinetically stable hexa-aza Schiff base La³⁺ complex is covalently attached to a Tat(49-72) peptide which has been shown to bind the HIV-1 TAR RNA sequence. Although these metallo-peptides cleave TAR site-specifically in the hexanucleotide loop to afford products consistent with hydrolysis, a series of control experiments suggests that the observed cleavage is not caused by a sequence-specifically bound Tat(49-72)-La(L)³⁺ peptide.

核酸四碱基体的量子化学计算方法比较

对9种四碱基体进行了6种半经验方法和HF／sT0—3G的计算，结果表明PM3方法对四 碱基体体系的计算比较适宜，其结果与从头算结果接近，而且大大缩短了计算时问。计算 还说明，四碱基体极有可能是通过碱基对形成．由稳定能的顺序看，除已报道的3种四碱 基体G4、G·c·G·c和u4外，一种新的G·c·G·c四碱基体也有稳定存在的可能。

长白山白眉蝮蛇蛇毒中酶类的纯化和表征

本论文系统研究了长白山白眉蝮蛇蛇毒中四种具有抗栓活性及可以作为基础理论研究的工具酶的分离纯化，并进行了扩大化试验，得到了可直接用于工业生产的分离工艺流程。并采用SDS－聚丙烯酰胺凝胶电泳（SDS－PAGE）、基质辅助激光解吸电离飞行时间质谱（MALDI－TOFMS）等分析手段确认了这四种酶的纯度及分子量，共获得以下几种质谱纯和电泳纯的酶A_2，其分子量为14.0kDa；三种精氨酸酯酶（AEasel、 AEase2和AEase3），它们的分子量分别为29.9kDa、27.5kDa、和28.8kDa；L－氨基酸氧化酶，其分子量为92.8 kDa；以及纤溶酶，其分子量为23.3 kDa。并对几种酶的质谱行为进行了系统研究，考察了酶的浓度等因素对多聚体多电荷离子峰形成的影响，并给出了它们的形成机理。上述酶中，我们按其分子量的大小、亚基组成以及氨基酸残基数目与文献报道的已知的酶相比较，认为L－氨基酸氧化酶、磷脂酶A_2和精氨酸酯酶2(Aease2)和精氨酸酯酶3(AEase3)是酶的新的类型。系统研究了所获得的四种酶的金属离子的含量，指出了磷脂酶A_2和纤溶酶均为Ca~(2+)离子结合蛋白，Ca~(2+)离子是酶分子活性所必需的。精氨酸酯酶和L－氨基酸氧化酶则为含Zn~(2+)酶，每个酶分子中分别含有两个和四个Zn~(2+)离子，它们仅起到稳定蛇毒酶的高级结构的作用，去除后对活性影响不大，并考察了多种金属离子对酶的活性和荧光光谱的作用。本文系统研究了四种酶的荧光光谱，用同步荧光光谱法了解了酶分子中略氨酸和色氨酸残基的微环境；用荧光淬灭方法，研究了其淬灭作用及机理，并讨论了荧光基团所处的环境。另外，还系统表征了几种酶的物理化学性质、酶学性质，得到了许多有意义的结果。

Matrix-assisted laser desorption/ionization mass spectrometry using porous silicon and silica gel as matrix

Porous silicon powder and silica gel particles have been applied as inorganic matrices for the analysis of small molecules in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS). In contrast to conventional MALDI-TOFMS, the signal interference of low-molecular analytes by the matrix has been eliminated. Almost no fragmentations of the analytes were observed. Effects of various factors, such as the particle and pore size, the suspending solution, and sample preparation procedures, on the intensity of mass spectra have been investigated. The pore structure of the inorganic matrix and penetration of the analytes into the pores must be optimized for effective desorption and ionization of the analytes. Matrices (DHB and HCCA) were covalently bound to silica gel for improvement of spectrum intensity. Copyright (C) 2001 John Wiley & Sons, Ltd.

利用MALDI-TOF质谱技术研究MPEG-b-PCL两嵌段共聚物的嵌段长度及嵌段分布

利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOFMS)结合源后分解(PSD)技术对甲氧基封端的聚乙二醇-b-聚己内酯(MPEG-b-PCL)两嵌段共聚物进行了结构分析.根据得到的MALDI-TOFMS谱图和PSD碎片信息清晰地确定了嵌段共聚物的嵌段长度和嵌段分布.结果表明,采用MALDI-TOFMS结合PSD技术研究这类嵌段共聚物链结构非常有效.这为更好地认识和应用这类嵌段共聚物提供了重要的依据,同时也建立了分析这类嵌段共聚物的方法.

三聚氰胺的基质辅助激光解吸/电离飞行时间质谱研究

Oxidized carbon nanotubes are tested as the matrix for analysis of the melamine by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Traditional MALDI matrix are not suit for analysis of the low molecular compounds due to the interference associated to the matrix clusters. Oxidized carbon nanotubes can transfer energy to the analyte under the laser irradiation, which makes analyte well ionized or desorbed. Moreover, the interference of the intrinsic matrix ions can be eliminated. Melamine as the a toxic additive which had been added in the milk powder, then it is necessary to establish a new method for detection of the melamine rapid and sensitive.

基质辅助激光解吸电离飞行时间质谱表征细胞色素C

A matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF—MS) technique was used for analysis of moleculear weight of cytochrome C.The effects of three kinds of matrix,such as 2,5-dihydroxybenzoic acid(DHB),a-cyano-4-hydroxycinnamic acid(a-CHC) and sinapinic acid(SA),were used to compared and a suitable a-CHC was found.Experimental data showed that this method was properable to analysis of the congeneric biochemical samples.

马鹿茸多肽的提取分离及基质辅助激光解吸飞行时间质谱分析

In this paper,active components of velvet antler polypeptide were extracted and separated.The molecular weight and purity of velvet antler polypeptide were determined by MALDI-TOFMS.The different influence factors such as matrix,sample concentration and laser energy were studied.This method is convenient and suitable for the identification of congener biochemical samples.

芳香环状低聚物组分分布与单体结构关系的研究

根据Jacoson-Stockmayer(J-S)环化理论,应用基质辅助激光解吸离子化飞行时间质谱(MAIDL-TOFMS),对一系列芳香环状低聚物组分分布进行分析,研究了芳香环状低聚物的组分分布与单体结构的关系,结果表明,在芳香聚酯、聚膦酸酯及芳香聚醚环状低聚物系列中,Inc_n与Inn呈良好的线性关系,符合J-S理论分布,在环状聚酯及聚膦酸酯系列中,低聚物的组分分布受双酚单体的中心键角影响,单体的中心键角在100°～120°之间,其中心键角愈小,γ值愈大,而在聚醚系列中,在固定一种单体的前提下,环化物的组分分布受另一单体键角的影响与环状聚酯和聚膦酸酯相一致。

Studies on enzymological properties of antibody elicited by meso-tetra(alpha, alpha, alpha, alpha-O-phenylacetyl benzene)porphyrin

meso-Tetra (alpha, alpha, alpha, alpha-O-phenylacetyl benzene) porphyrin was used as a complete antigen to elicit monoclonal antibody 1F2 through the immunization and cell fusion techniques. McAb 1F2 obtained was demonstrated very pure by HPLC and MALDI/TOFMS. The retention time of McAb 1F2 was 2. 63 min. The subtype of McAb 1F2 was IgG2a. The relative molecular weight was 156 678. 8. When the McAb 1F2-porphyrin was formed, the maximal absorption of the porphyrin soret region had a redshift from 408 to 416 nm and hyperchromical effect, showing that the antigen-antibody combination was rigid and intense, and the abzyme constancy was high. But compared with HRP, the activity of the abzyme was only 4. 687 5 U/mg and 1. 899 % of that of HRP. Its K-m was 20. 29 mmol/L, k(cat) 396. 82 min(-1), k(cat)/K-m. 1. 955 7 X 10(4) L . mol(-1) . min(-1).

Use of nitrocellulose films for affinity-directed mass spectrometry for the analysis of antibody/antigen interactions

Combination of affinity extraction procedures with mass spectrometric analyses is termed affinity-directed mass spectrometry, a technique that has gained broad interest in immunology and is extended here with several improvements from methods used in previous studies. A monoclonal antibody was immobilized on a nitrocellulose (NC) membrane, allowing the corresponding antigen to be selectively captured from a complex solution for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method was also used to rapidly determine the approximate binding region responsible for the antibody/antigen interaction. The tryptic fragments of antigen protein in buffer were applied to the antibody immobilized on NC film and allowed to interact. The NC film was then washed to remove salts and other unbound components, and subjected to analysis by MALDI-TOFMS. Using interferon-alpha (2a) and anti-interferon-alpha (2a) monoclonal antibody IgG as a model system, we successfully extracted the antigen protein and determined the approximate binding region for the antigen/antibody interaction (i.e., the tryptic fragment responsible). Copyright (C) 2001 John Wiley & Sons, Ltd.

Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus

Rabies virus was used as the antigen to immunize laying chickens. Anti-rabies virus immunoglobulin Y(IgY) was isolated from yolks of the eggs laid by these chickens using a two-step salt precipitation and one-step gel filtration protocol. The purified IgY was reduced with dithiothreitol, and heavy chains (HC) and light chains (LC) were obtained. In addition, the purified IgY was digested with pepsin and the fragment with specific antigen binding properties (Fab) was produced. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS), the average molecular weights of IgY, HC, LC, and Fab were determined as 167 250, 65 105, 18 660, and 45,359 Da, respectively. IgY has two structural differences compared with mammalian IgGs. First, the molecular weight of the heavy chain of IgY is larger than that of its mammalian counterpart, while the molecular weight of the light chain of IgY is smaller. Second, upon pepsin digestion, anti-rabies virus IgY is degraded into Feb, in contrast to mammalian IgG, which has been reported to be degraded into F(ab')(2) under the same conditions. Copyright (C) 2001 John Wiley & Sons, Ltd.