寡聚核苷酸与中药化学成分的相互作用研究

药物分子与生物分子的相互作用是分子水平上研究药物构效关系及药物筛选的基础。测定形成的非共价复合物及其稳定性是研究药物与靶分子相互作用的关键。单链和双链DAN与小分子的相互作用提供了包括抗癌、抗病毒及抗菌在内的大量治疗药物的作用机理。本论文选择合成了与SARS病毒和肿瘤相关的寡聚核昔酸靶分子，利用质谱高灵敏、专一性、快速、化学计量的特点，研究靶分子与临床上确有疗效的抗病毒、抗肿瘤中药中几大类化学成分的相互作用，探讨它们相互作用的规律，通过相互作用强度的比较，考察这些中药化学成分在抗病毒、抗肿瘤方面的可能活性。在单链寡聚核营酸靶分子（RNA，DNA）与皂昔类化合物的相互作用的研究中，首先建立了区分皂普类异构体的高分辨电喷雾质谱及多级串联质谱的分析方法，通过皂普准分子离子在CID谱中产生的碎片离子，可以提供皂昔昔元、糖基类型、糖链的连接位点，为类似化合物的异构体的区分提供了一个简捷、灵敏、准确的分析方法。考察了实验条件对单链寡聚核营酸靶分子（RNA，DNA）与中药化学成分相互作用的影响；在皂营、黄酮类化合物与单链寡聚核昔酸靶分子的作用研究中，发现非共价作用强度的大小与普元、糖链的结构有关，包括昔元的类型、轻基的数目、糖链的长短，以及糖链上甲基取代的位置。单链寡聚核普酸靶分子与生物碱的相互作用研究表明，相互作用亲和力的大小与生物碱的碱性强弱有关，季胺型生物碱的作用最强，如巴马汀、药根碱、小巢碱与靶分子均生成了较强的非共价复合物。与昔类、内酩、酚类等成分的相互作用同样与其结构密切相关。双链DNA与生物碱类化合物的相互作用研究发现，它们与生物碱的作用强度与双链DNA的AF碱基对富有或GC-碱基对富有有关，且复合物离子的CID断裂途径不同。生物碱化合物的结构不同，其作用强度存在较大差异。述研究结果建立了核昔酸与中药化学成分相互作用研究的软电离质谱方法，利用该方法的研究结果，可以为中药活性成分的初步筛选提供一条可以借鉴的途径。

中国汉族人群若干疾病相关基因的定位研究

1. 中国一个轴前多指 II/III 型家系候选基因的定位 轴前多指（PPD）是人类众多肢体异常症状中比较常见的一种。前人的研 究把它定位到染色体7q36 上450Kb 的区段内。在这个候选区段内，不同的遗传 背景的几个多指家系的致病突变已被发现，它们位于SHH 基因的顺式调控元件 （ZRS）上，这个调控元件调控SHH 正常的时空表达，对指/趾的正常发育起着 重要的作用。在本研究之中，我们分析了我国一个大的多指家系，这个家系的多 指疾病的遗传方式是常染色体显性遗传并有接近100%的疾病外显率。通过连锁 分析和单倍型构建，我们把这个家系的致病基因定位到染色体7q36 上微卫星标 记D7S2465 和 D7S2423 之间1.7cM 的区域内，这个区域包含了上述的450Kb。 为了确定这个家系的致病突变，我们采用了直接测序的方法，筛查了我们定位的 这个区段内的5 个基因（HLXB9, C7orf2, NOM1, RNF32 和C7orf4）的编码区， SHH 基因的ZRS，C7orf2 基因的第5 内含子，和18 个在多物种中保守的非编码 区段（CNS）。我们的结果表明，在这个中国多指家系中，不是上述的5 个基因 和18 个多物种中保守非编码区段（CNS）的突变导致了多指的表型，也不是其 他研究小组报道的SHH 基因的ZRS 突变导致了多指表型。是否在我们界定的这 1.7cM 的候选区段内还存在着SHH 基因的其他调控元件，它的突变也一样会引 起SHH 基因异常的时空表达，最终导致多指表型的产生还需要进一步的研究来 证实。 2. 中国一个单纯性小眼球家系致病基因的定位 先天性小眼球是一种在临床上具有异质性的眼球发育异常疾病，表型从单 侧眼球体积变小到两侧眼球组织的完全缺失。单纯性小眼球疾病指的是除了眼球 的异常不伴随身体其他组织的异常，其遗传学上的致病机理到目前为止还未完全 清楚。之前的研究表明，不同遗传背景的小眼球家系的致病基因被定位到完全不 同的染色体区段上。本研究中的这个中国单纯性小眼球家系，具有常染色体显性的遗传方式，而且是第一个在分子水平上被研究的中国小眼球家系。为了研究这 个家系的致病基因，我们使用了382 个微卫星标记对这个家系进行了全基因组的 扫描，结果表明这个中国小眼球家系的致病基因定位于2 号染色体长臂上，这个 结果不同于以往报道的任何其他小眼球家系。两点连锁分析在重组率为0 时在微 卫星标记D2S2265 上得到最大值3.290，单倍型分析表明所有的受累个体在染色 体2q11-14 上微卫星标记D2S1890 和 D2S347 之间共享相同的单倍型，所以将 这个家系的致病基因定位到染色体2q11-14 上15 cM 的区段内。我们的结果进一 步提示了小眼球疾病的遗传异质性，并且定位了一个新的关于眼球发育的相关基 因。 3. 19 号染色体C 型凝集素家族与中国南方汉族人群的SARS 易感性研究 2003 年SARS 在全球爆发时，不同的个体在感染后表现出不同的病程和 最终的治疗结果，这被认为是个体本身的遗传因素在这个过程中起了一定的作 用，即不同个体在一些基因上的多态影响了他们对一些疾病的易感性。CD209L 基因是19 号染色体上C 型凝集素家族中的一员，它被证明是SARS 病毒的受体， 并且它的第4 外显子的VNTR 多态被认为和宿主对病毒的易感性有关。但是这 个结论在其后其他两个研究小组的工作未被得到证实。可能的原因有：(1) CD209L 基因本身的多态和宿主对SARS 病毒的易感性无关，而是和它紧密连锁 的其它基因的多态发挥真正的作用；(2)可能是由于以前未检测到的群体分层现 象的存在，使最初的研究得到了一个假阳性的结果。为了探讨这个问题，我们对 19 号染色体上C 型凝集素家族的4 个成员(FCER2, CLEC4G, CD209 和 CD209L) 作了全面的研究，我们采用了检测tagSNPs 的方法，在C 型凝集素家族基因簇上 选取了23 个tagSNPs 位点来代表这个基因簇的多态。我们检测了来自中国香港 的181 个SARS 病人和172 个匹配的正常对照，结果表明C 型凝集素家族的4 个基因跟中国南方汉族群体对SARS 病毒的易感之间没有相关性。同时我们检测 了来自中国不同地区的1145 个汉族样本CD209L 基因VNTR 的多态分布情况， 结果表明这个位点在中国人群中存在群体分层现象，这也解释了之前的研究小组 得到的假阳性结果可能正是由于他们忽略了这个问题而导致的

翼手目四个类群的比较分子细胞遗传学研究

翼手类(Order Chiroptera)是现生哺乳动物中唯一真正具有空中飞翔能力的动物；同时，它也是哺乳动物中为数不多的具有回声定位功能的几个分类类群之一。现存的翼手目动物约有1100种，占整个哺乳动物种类的20％以上，是哺乳动物中仅次于啮齿目的第二大目。尽管对于翼手目系统发育已经进行了大量的形态学和分子系统学等方面研究，但是翼手目中主要类群的系统发育关系尚存在着许多争议。进化过程中发生的染色体重排在揭示物种的系统发育关系中起到非常重要的作用。种间染色体涂色可以准确、快速、直观地鉴定物种进化过程中发生的大规模染色体重排，已成为在全基因组水平上比较研究哺乳动物核型多样性及其起源和演化的首选方法。利用染色体涂色可以构建不同物种间的比较染色体图谱，通过分析进化过程中保守的染色体片段在不同类群物种核型中的分布和排列方式，可以推导各类群可能的祖先核型，并重建伴随物种形成所发生的基因组变化历史，包括染色体重排的类型、速率和核型演化的趋势。但是，目前关于翼手目染色体涂色研究还很少，至今仍不能为翼手目各级分类水平上的系统发育关系研究提供系统完整的细胞遗传学证据。本研究利用大鼠耳蝠染色体特异涂色探针，通过种间染色体涂色，首次构建了狐蝠科、菊头蝠科、蹄蝠科和蝙蝠科代表物种间的比较染色体同源图谱，并且将它们与之前发表的用人的染色体探针构建的比较染色体图谱进行整合。通过分析这些物种之间保守染色体片段的分布，推测翼手目动物在核型进化过程中所发生的染色体重排的类型和数量，以期为研究翼手目的系统发育关系提供细胞遗传学证据。研究结果表明： 1. 翼手目动物的大多数染色体臂具有高度保守性，罗伯逊易位是其核型进化的主要机制。此外，倒位也是翼手目染色体进化过程中常见的染色体重排方式。 2. 小蝙蝠亚目的菊头蝠科和蹄蝠科与大蝙蝠亚目的狐蝠科亲缘关系更为接近，而与小蝙蝠亚目其它科的亲缘关系较远。 3. 菊头蝠科和蹄蝠科应该各自作为独立的科，但这两个科拥有一个共同的细胞遗传学衍生特征，这一特征在翼手目其它科中并未发现，表明它们之间有非常紧密的系统发育关系。 4. 菊头蝠科的祖先核型可能并不是前人所推测的全由端着丝粒染色体构成的2n＝62的核型；很有可能是包含有双臂染色体的，2n低于62的核型。 5. 三叶小蹄蝠比中蹄蝠保留更多的祖先染色体特征，其核型蹄蝠科中较为原始的核型。 6. 着丝粒融合是蝙蝠科物种核型演化的主要机制。蝙蝠科的祖先核型并不是类似于2n=44鼠耳蝠的核型，而可能更类似于棕蝠的2n＝50核型，由全端着丝粒染色体构成。 7. 除着丝粒融合外，异染色质的扩增也是蝙蝠科扁颅蝠属和山蝠属的染色体进化方式之一。 8. 绒山蝠与三种亚洲的伏翼共有两个同源染色体片段联合（MMY 8＋11和9＋13）,提示蝙蝠科的山蝠属和伏翼属有非常紧密的亲缘关系。 9. MMY 9 + 23和MMY 18＋19 这两个保守的同源染色体片段联合是蝙蝠科扁颅蝠属的细胞遗传学鉴定特征。 通过翼手目这些动物的染色体涂色研究，我们不但在全基因组的水平上揭示了翼手目几个主要类群代表动物的核型系统发育关系，而且所构建的比较染色体图谱将有助于基因组的序列信息从已测序物种（人）向序列信息较少的翼手目物种转移，这些研究结果也可以为翼手目的分类、物种鉴定、系统发育关系和生物医学研究（如SARS病毒）等提供基础性资料。

Studies on the Interaction between Single-strand Oligonucleotide and Ginsenosides by Electrospray Ionization Mass Spectrometry

Oligonucleotide from SARS virus was selected as a target molecule in the paper. The noncovalent complexes of ginsenosides with the target molecule were investigated by electrospray ionization mass spectrometry. The effects of experimental conditions were examined firstly on the formation of noncovalent complexes. Based on the optimized experimental conditions, the interaction of different ginsenosides with the target molecule was researched, finding that the interaction orders are relative with the structure of aglycons, the length and terminal sugar types of saccharide chains in the ginsenosides. There are certain rules for the interaction between the ginsenosides and DNA target molecule. For different type ginsenosides, the interaction intensity takes the orders 20-S-protopanaxatriol > 20-S-protopanaxadiol, and panaxatriol ginsenosides > panaxadiol ginsenosides. For the ginsenosides with the same type aglycone, tri-saccharide chain > di-saccharide chain > tetra-saccharide chain and single-saccharide chain > panaxatriol. For the ginsenosides with the same tetra-saccharide chain, the ginsenosides with smaller molecule masses > the ginsenosides with larger molecule masses.

DNA与生物碱类化合物非共价作用的负离子电喷雾质谱研究(Ⅰ)

目前 ,临床上使用的许多抗病毒药物均是通过与 DNA,RNA发生相互作用破坏其结构 ,进而影响基因调控与表达的功能 ,表现出抗病毒活性 [1,2 ] .因此 ,核酸与药物分子相互作用的研究对阐述抗病毒药物的作用机理 ,以及对药物的体外筛选都具有重要的意义 .电喷雾电离质谱作为一种软电离手段 ,可将溶液中生物分子与药物分子的非共价复合物转为气相进行分析 ,再现其生理状态 ,使其成为分子水平上进行药物筛选的最佳方法 [3～ 6 ] 和在分子水平上筛选中药抗病毒活性成分的理想工具 .本文选择合成了与 SARS病毒相关的 DNA片段作为抗病毒药物筛选的靶分子 ,用电喷雾质谱技术 ,通过对靶分子与 5种生物碱的非共价复合作用 ,探讨了生物质谱方法用于药物筛选的可行性 .1　实验部分1 .1　材料及样品制备　 DNA分子系人工合成 ,由 1 5个碱基组成 ,分子量为 470 4 ,结构为 GGTAA-GATGGAGAGC( 1 5 - mer) ;腺苷 ( Adenosine,Mw=2 67)、鸟苷 ( Guanosine,Mw=2 83)和胞苷 ( Cyti-dine,Mw=2 4 3)购自 Sigma公司 ;小檗碱、...

核酸靶分子与苦参中生物碱成分的非共价作用研究

本文选择合成了与SARS病毒相关的DNA片段作为抗病毒药物筛选的靶分子.利用生物质谱技术,通过对该核酸分子与常见中药苦参中的两种主要生物碱成分的非共价复合作用的研究,探讨该方法作为药物筛选方法的可行性.

Population-Dynamics And Production Of Euphausiids. IV Euphausia-Krohni Nematoscelis-Megalops And Thysanoessa-Gregaria And 8 Rare Species In The North-Atlantic Ocean

Seasonal changes in the abundance, size and occurrence of furciliae of Euphausia krohni (Brandt), Nematoscelis megalops (G. O. Sars) and Thysanoessa gregaria G. O. Sars are described from samples taken at 10 m depth with the Continuous Plankton Recorder (CPR) over a period of 2 yr (January 1966 to December 1967) in the North Atlantic Ocean. E. krohni and T. gregaria were found to breed through most of the year but N. megalops bred only in spring and summer. Annual mean biomass was calculated directly from the data and production was estimated from published P:B ratios. The seasonal occurrences of E. brevis Hansen, E. hemigibba Hansen, E. mutica Hansen, E. tenera Hansen, Stylocheiron longicorne G. O. Sars, S. maximum Hansen, Thysanopoda acutifrons Holt and Tattershall and T. aequalis Hansen in the samples are described.

Continuous Plankton Records - Geographical Variations In Numerical Abundance Biomass And Production Of Euphausiids In The North-Atlantic Ocean And The North-Sea

Geographical variations in the numbers, biomass and production of euphausiids and the contribution of common species to the total are described from samples taken during 1966 and 1967 in the North Atlantic Ocean and the North Sea by the Continuous Plankton Recorder at 10 m depth. Euphausiids were most abundant in the central and western North Atlantic Ocean and the Norwegian Sea. Thysanoessa longicaudata (Krøyer) was numerically dominant. Biomass was greatest in the Norwegian Sea and the north-eastern North Sea where Meganyctiphanes norvegica (M. Sars) accounted for 81 and 59%, respectively, of the total biomass. Production was highest off Nova Scotia and in Iberian coastal waters; the dominant species were T. raschi (M. Sars) in the former area and Nyctiphanes couchi (Bell) in the latter. The mean P:B ratios were correlated with temperature.

Population-Dynamics And Production Of Euphausiids. III Meganyctiphanes-Norvegica And Nyctiphanes-Couchi In The North-Atlantic Ocean And The North-Sea

Seasonal changes in abundance, size and aspects of the population structure of Meganyctiphanes norvegica (M. Sars) and Nyctiphanes couchi (Bell) are described from samples taken with the “Continuous Plankton Recorder” at 10 m depth over a 2 yr period (1966 and 1967) in the North Atlantic Ocean and the North Sea. M. norvegica lived for a maximum of just over 2 yr, and adults of both year-classes spawned during a limited breeding season in the spring or summer. N. couchi spawned over a prolonged breeding season, giving rise to a complex of cohorts with overlapping size ranges. It was concluded that 3 or 4 cohorts were spawned in each year and that the maximum life span was probably greater than 1 yr, although maturity may be attained in less than a year. Estimated annual production at 10 m depth for M. norvegica ranged from 0.80 to 18.74 mg m-3yr-1 and for N. couchi from 0.67 to 8.23 mg m-3yr-1. P:B ratios ranged from 1.3:1 to 6.3:1 for M. norvegica and 4.0:1 to 5.5:1 for N. couchi.

Population-Dynamics And Production Of Euphausiids .2. Thysanoessa-Inermis And Thysanoessa-Raschi In The North-Sea And American Coastal Waters

Results from the Continuous Plankton Recorder (CPR) survey for 1966 and 1967 are used to describe seasonal changes in abundance, size and aspects of the population structure of Thysanoessa inermis (Krøyer) and T. raschi (M. Sars) at a depth of 10 m in the North Sea and in American coastal waters from the Grand Banks to the Gulf of Maine. Production and dry weight were estimated from these data. Two year-groups were usually present in the breeding population, the proportion surviving into a second year being higher in American waters than in the North Sea. Annual production for each species was within the range 0.69 to 4.66 mg m-3 and the ratio between production and biomass (P:B) was between 1.3 and 4.2; values outside these ranges were obtained only for American coastal waters in 1967, when the frequency of sampling was low.

Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase

Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5'-to-3' direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5'-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5' cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5'-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.

Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis

Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (CoV) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled ssRNA substrates were processed to final products of 8–12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-labeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs.

Structural and functional basis for ADP-ribose and poly(ADP-ribose) binding by viral macro domains.

Macro domains constitute a protein module family found associated with specific histones and proteins involved in chromatin metabolism. In addition, a small number of animal RNA viruses, such as corona- and toroviruses, alphaviruses, and hepatitis E virus, encode macro domains for which, however, structural and functional information is extremely limited. Here, we characterized the macro domains from hepatitis E virus, Semliki Forest virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). The crystal structure of the SARS-CoV macro domain was determined at 1.8-Å resolution in complex with ADP-ribose. Information derived from structural, mutational, and sequence analyses suggests a close phylogenetic and, most probably, functional relationship between viral and cellular macro domain homologs. The data revealed that viral macro domains have relatively poor ADP-ribose 1"-phosphohydrolase activities (which were previously proposed to be their biologically relevant function) but bind efficiently free and poly(ADP-ribose) polymerase 1-bound poly(ADP-ribose) in vitro. Collectively, these results suggest to further evaluate the role of viral macro domains in host response to viral infection.

A new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods.

The coronavirus main protease, Mpro, is considered to be a major target for drugs suitable for combating coronavirus infections including severe acute respiratory syndrome (SARS). An HPLC-based screening of electrophilic compounds that was performed to identify potential Mpro inhibitors revealed etacrynic acid tert-butylamide (6a) as an effective nonpeptidic inhibitor. Docking studies suggested a binding mode in which the phenyl ring acts as a spacer bridging the inhibitor's activated double bond and its hydrophobic tert-butyl moiety. The latter is supposed to fit into the S4 pocket of the target protease. Furthermore, these studies revealed etacrynic acid amide (6b) as a promising lead for nonpeptidic active-site-directed Mpro inhibitors. In a fluorimetric enzyme assay using a novel fluorescence resonance energy transfer (FRET) pair labeled substrate, compound 6b showed a Ki value of 35.3 M. Since the novel lead compound does not target the S1', S1, and S2 subsites of the enzyme's substrate-binding pockets, there is room for improvement that underlines the lead character of compound 6b.