由已知核苷酸序列的DNA克隆产生的感染性乙型肝炎病毒

质粒pTKH10和质粒pHBV14-1经EcoRI或PstI消化和低溶点琼脂糖凝胶电泳分离等程序,分别产生了环状双股HBV-2DNA和环状HBV-14DNA.对6只黑猩猩肝内或静脉接种HBV-2DNA,10周后,3只黑猩猩出现了典型的乙型肝炎血清学和生化特征,血清中检测到HBsAg和HBeAg,肝组织活检显示轻度     

女性人乳头瘤病毒基因亚型分型情况分析

人乳头瘤病毒(human papilloma virus,HPV)是一种特异感人表皮与黏膜的双链环状DNA,已发现100多种亚型,期中约30种与人类肿瘤有关[1]。目前已有研究证实HPV感染是宫颈癌的主要危险因素[2]。     

血中游离DNA与肿瘤

血中游离DNA指血中游离于细胞之外的一类DNA,简称循环核酸,于1947年由Mandel和Metais发现。肿瘤患者外周血DNA水平高于正常人且血中游离DNA具有肿瘤细胞DNA的特征,如肿瘤相关基因的突变、微卫星改变、甲基化异常和线粒体DNA突变等。随着肿瘤分子生物学的深入研究,血中游离DNA定性定量分析已开始应用于肿瘤的早期诊断、个体化治疗和预后判断等。血中游离DNA结合各类分子生物学检测技术,有望成为具有一定敏感性及高特异性的临床诊断方法。此外,血中游离DNA还有可能成为非细胞形式的肿瘤转移的新途径。     

在质体pBR322中无性繁殖的乙型肝炎病毒的DNA顺序在大肠杆菌中的表现

乙型肝炎患者血清中含有一种42nm的 Dane颗粒,在其核中有一个双链环状DNA分子,分子量约为2×10~6,其中的一股链有一个大小不定的缺口和一个内源性DNA依赖性DNA聚合酶,此酶在体外反应中可填补这个缺口。鉴于乙型肝炎病毒(HBV)只能感染人和类人猿     

慢性乙型肝炎肝组织与血清中病毒标志物关系的研究

乙型病毒性肝炎在我国发生率较高,人群中乙型肝炎表面抗原(HBsAg)携带率高达10%。患者血清乙型肝炎病毒(HBV)DNA和肝组织HBsAg、乙型肝炎核心抗原(HBcAg)的免疫组织化学检测是HBV感染及复制的特异性标志。HBV闭合环状DNA(HBV     

浅谈慢性乙型肝炎的预防

1病原学 乙型肝炎病毒（HBV）属嗜肝DNA病毒科（hepadnaviridae）,基因组长约3．2kb,为部分双链环状DNA。HBV侵入人体后,与肝细胞膜上的受体结合,脱去包膜,穿入肝细胞质内,然后脱去衣壳,部分双链环状HBVDNA进入肝细胞核内,在宿主酶的作用下,以负链DNA为模板延长正链,修补正链中的裂隙区,     

DNA甲基化与肿瘤诊断

DNA甲基化异常是人类肿瘤中常见的表观遗传变化之一，伴随着肿瘤的发生和发展，且该异常现象在肿瘤患者的外周循环血清、血浆和尿液等体液中都可检测到。因此，利用DNA甲基化分析技术检测体液中特定分子DNA甲基化水平是肿瘤早期诊断、病程监控和疗效评估的潜在手段，对临床肿瘤的诊治意义重大。     

循环游离线粒体DNA作为新型肿瘤液体活检标志物的研究进展

恶性肿瘤是导致人类死亡的主要原因之一，早期诊断和精准治疗能够显著改善肿瘤患者预后。随着液体活检技术的迅速发展，循环肿瘤细胞、外泌体、循环肿瘤DNA等新型肿瘤标志物被发现并展现出巨大的临床应用潜力，为肿瘤的诊治提供了更为精准的检测手段，逐渐成为研究热点。和循环游离核DNA相比，循环游离线粒体DNA(cf-mt DNA)具有基因组小、拷贝数多、突变频率高的特点，在肿瘤诊断和治疗检测中具有独特的优势，显示出良好的应用前景。综述系统介绍了循环cf-mt DNA的特点、检测技术、分析指标，以及cf-mt DNA在肿瘤早期诊断、治疗监测、预后评估中的应用价值。     

DNA甲基转移酶抑制剂guadecitabine

DNA甲基化过程广泛存在于哺乳动物细胞内,在表观遗传修饰和染色质稳定性中都起着关键的作用。DNA高甲基化修饰通常在肿瘤发展的进程中有着重要影响。guadecitabine是由Astex公司研发的新型的DNA甲基转移酶抑制剂。作为地西他滨的前药,guadecitabine在体内经磷酸酯酶水解得到地西他滨,掺入DNA链中,与DNA甲基转移酶共价结合,不可逆地抑制DNA甲基化过程。临床研究表明,guadecitabine在针对骨髓增生异常综合征和急性骨髓性白血病等肿瘤的治疗中具有明显的有效性和安全性。     

放线菌的无性繁殖载体

前言目前在基因工程中使用的大部分技术来源于对大肠杆菌及其噬菌体的分子生物学研究.处于分子遗传学中心地位的大肠杆菌作为其研究材料,具有其它生物不可比拟的优越性.像大肠杆菌那样的原核微生物由环状双链DNA承担遗传信息.原核微生物DNA也称为染色体,以基因操作法将含有特定基因的DNA片段插入独立复制的质粒或噬菌体DNA中,然后移入活细胞内进行复制,这种     

Interaction between A 3-nitrobenzothiazolo (3,2-a) quinolinium antitumour drug and deoxyribonucleic acid

The interaction of 3-nitrobenzothiazolo (3,2-a) quinolinium (NBQ) perchlorate with DNA was studied by u.v.-visible and fluorescence spectrophotometry as well as by hydrodynamic methods. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts, and the fluorescence was quenched. Binding parameters, determined from spectrophotometric measurements by Scatchard analysis according to an excluded-site model, indicated a binding constant of 2.4 × 10⁵ M^?1 for calf thymus DNA at ionic strength 0.01. The interaction was markedly suppressed by increasing the salt concentration. Binding to the GC-rich DNA of Micrococcus lysodeikticus was weaker than the binding to calf thymus DNA at ionic strength 0.01. NBQ increased the viscosity of sonicated rod-like DNA fragments, producing a calculated increment in length of 2.4 Å/bound drug molecule. It removed and reversed the supercoiling of closed circular duplex plasmid pBR322 DNA by virtue of a helix-unwinding angle estimated as approximately 13°/bound ligand molecule. We conclude that the binding of NBQ to DNA occurs by a mechanism of intercalation, which probably accounts for its reported antitumor activity.     

A new ionizable chromophore of 1,4-bis(alkylamino)benzo[g]phthalazine which interacts with DNA by intercalation

The tricyclic heteroaromatic nucleus of 1,4-bis(alkylamino)benzo[g]phthalazine can be protonated at physiological pH, depending on the nature of the side chains. The interaction of the 3-methoxypropyl derivative with calf thymus and closed, circular DNA has been studied with UV-vis spectroscopy and NMR. The effect of drug binding on the topology of closed, circular DNA was determined by topoisomerase-I catalyzed relaxation of the complex followed by gel electrophoresis. The results strongly support intercalative binding and suggest that this series of compounds are promising targets for anticancer activity evaluation.     

Steric constraints for DNA binding and biological activity in the amsacrine series

To determine the relation between DNA binding mode and biological activity in compounds related to the clinical anti-leukaemia drug amsacrine, a series of acridine-substituted derivatives has been synthesized and compared for lipophilicity, DNA-binding affinity, DNA-binding geometry and anti-leukaemia activity in vitro and in vivo. DNA-binding affinity, as estimated either by ethidium displacement or equilibrium dialysis, decreased progressively as the bulk of the substituent increased. Substitution at the 2 position provided the largest effects. The DNA unwinding angles, estimated by changes in viscosity of closed circular duplex DNA in the presence of drug, decreased significantly with ethyl and isopropyl substituents. However, with tertbutyl groups in the 2, 3 or 4 position of the acridine ring, unwinding was not observed even though DNA binding was measurable. Anti-leukaemia activity in vivo was also abolished when the acridine ring was substituted with a tertbutyl group. The results suggest that methyl substitution of the acridine ring inhibits DNA binding at the 2 position but not at the 3 and 4 positions, that ethyl and isopropyl substitution inhibits intercalative binding at all positions and that tertbutyl substitution abolishes intercalative binding. Biological activity in vitro is dependent on both lipophilicity and DNA binding and activity in vivo requires intercalative DNA binding.     

Poly (lactide-co-glycolide) acid nanoencapsulation of a synthetic coumarin: cytotoxicity and bio-distribution in mice, in cancer cell line and interaction with calf thymus DNA as target

Several naturally occurring coumarin compounds, including scopoletin (7 hydroxy-6 methoxycoumarin), of plant origin have been reported to have anti-cancer potentials. A related but chemically synthesized coumarin, 4-methyl-7-hydroxy coumarin (SC), was also shown to have similar anti-cancer potentials. In the present study, to test if nano-encapsulated SC could be a more potent anti-cancer agent, we encapsulated SC with poly lactide-co-glycolide acid (PLGA) nanoparticles (Nano Coumarin; NC) and tested its potentials with a variety of protocols. NC demonstrated greater efficiency of drug uptake and showed anti-cancer potentials in melanoma cell line A375, as revealed from scanning electronic and atomic force microscopies. To test its possible interaction with target DNA, the combined data of circular dichroism spectra (CD) and melting temperature profile (T_m) of calf thymus DNA treated with NC were analyzed. Results indicated a concentration dependent interaction of NC with calf thymus DNA, bringing in effective change in structure and conformation, and forming a new complex that increased its stability. Particle size and morphology of NC determined through polydispersity index and zeta potential using dynamic light scattering qualified NC to be a more potent anti-cancer agent than SC. Further, SC and NC showed negligible cytotoxic effects on normal skin cells and peripheral blood mononuclear cells of mice. Distribution assay of PLGA nanoparticles in different tissues like brain, heart, kidneys, liver, lungs, and spleen in mice revealed the presence of nanoparticles in different tissues including brain, indicating that the particles could cross the blood brain barrier, significant information for drug design.     

Intestinal absorption of human insulin in pigs using delivery systems based on superporous hydrogel polymers

The interaction of 8-methoxypsoralen (8-MOP) with calf thymus DNA was studied in darkness at 25 degrees C and pH 7.4. The enthalpy curve for 8-MOP-DNA interaction was obtained by isothermal titration calorimetry and showed a two-step process for the interaction. According to the spectrophotometric data, it was suggested that some compaction may occur in the DNA structure at higher [8-MOP](t)/[DNA] ratio. Using the fluorescence quenching data, the Scatchard analysis was performed for 8-MOP-DNA interaction at the extended ranges of drug concentration. The results indicated that the first set of binding sites was occupied by 1 mol of drug bound per near eight base pairs of DNA. Also 8-MOP caused the quenching of the fluorescence emission of DNA-ethidium bromide complex. The Scatchard analysis of these data indicated the non-competitive manner for quenching. A non-displacement based quenching mechanism has been suggested for this behavior. The circular dichroism spectra also confirmed the non-intercalative binding of 8-MOP at higher concentrations accompanied by some conformational changes in DNA structure. It has been suggested that at low drug load, 8-MOP binds to DNA as an intercalator, which is an endothermic process, whereas at higher ratios of [8-MOP](t)/[DNA], it binds to the outside of DNA, probably in the minor groove and causes some compaction in DNA, which is the exothermic process.     

Development of DNA tetrahedron-based drug delivery system

Nanocarriers of drugs have attracted significant attention to tackle the problems of drug resistance or nucleic acid drug delivery, which can optimize pharmaceutical parameters and enhance the cellular uptake efficiency. Nowadays, DNA nanostructure presents an opportunity in the field of nanomaterial due to its precise control in shape and size, excellent biocompatibility, as well as multiple sites for targeting decoration. DNA tetrahedron, which is stable and easily synthesized, is used for various applications, including nuclear magnetic resonance imaging, molecular diagnosis, targeting drug delivery, and so on. In this review, we will discuss the applications of DNA tetrahedron about drug delivery, intracellular routes and its fates. Also challenges and possible solutions for developing DNA tetrahedron-based drug delivery system are detailed.     

Interactions of gold coordination complexes with DNA

The interactions of certain gold(I) and gold(III) complexes with isolated plasmid pBR322 DNA were defined and compared to those of cis-diamminedichloroplatinum(II), CDDP, using an agarose gel electrophoresis assay. Trichloro(pyridine)gold(III) appeared to bind to DNA as evidenced by its ability to produce dose-dependent changes in the electrophoretic mobilities of closed circular, supercoiled, closed circular, relaxed, and open circular plasmid DNAs. These effects suggest that the gold containing complex induces conformational changes in the plasmid as a result of the compound binding to the DNA and the subsequent unwinding of the double helix and shorting of the DNA. Auranofin [(2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S)-triethyl phosphine gold(I)] did not appear to interact with DNA under any conditions. However, its analog chloro(triethylphosphine) gold(I) interacted with DNA at pH 9.5 in borate buffer and produced electrophoretic mobility changes in pBR322 DNA which were different from those produced by the gold(III) complexes that were evaluated. Binding of chloro(triethylphosphine) gold(I) was inhibited by the co-addition of the thiosugar portion of auranofin suggesting preferential binding of the gold moiety to thiosugar, which results in the production of auranofin (or a sugar containing) gold complex and inhibition of gold binding to DNA. The interactions of a number of gold compounds with DNA were also evidenced by their abilities to inhibit the binding of ethidium bromide to DNA. The results from these studies indicate that: gold containing complexes can bind to, and produce conformational changes in, DNA; gold(I) and gold(III) complexes may interact with DNA via different chemical mechanisms to produce different conformational changes in DNA; and certain coordinating ligands in gold complexes (e.g. Cl, Br and SCN) can be exchanged for binding sites on DNA by gold.     

Substitution at the F-ring N-imide of the indolocarbazole antitumor drug NB-506 increases the cytotoxicity, DNA binding, and topoisomerase I inhibition activities.

The antitumor drug NB-506, which is currently undergoing phase I/II clinical trials, contains a DNA-intercalating indolocarbazole chromophore substituted with a glucose residue. In addition to interacting with DNA, the drug stabilizes the topoisomerase I-DNA covalent complex. To reinforce the DNA binding and anti-topoisomerase I activities of NB-506, an analogue containing a new substituent on the naphthalimide ring F was synthesized. The N-formylamino group of NB-506 has been replaced with a more hydrophilic group, N-bis(hydroxymethyl)methylamino. In this study we show that the incorporation of a longer substituent on the N6 position effectively reinforces both the interaction with DNA and the capacity of the drug to maintain the integrity of the topoisomerase I-DNA covalent complexes. The strength and the mode of binding of the drugs to DNA were studied by complementary biophysical techniques including absorption, fluorescence, and circular and linear dichroism. Various biochemical procedures were applied to investigate the effects on human topoisomerase I using plasmid DNA as well as restriction fragments. The drug binding sites and the positions of the topoisomerase I-mediated cleavage sites were mapped with nucleotide resolution using footprinting and sequencing techniques. Cytotoxicity measurements performed with various human cancer cell lines (HCT-116, DLD-1, MKN-45) indicate that the newly designed drug is 3 to 4 times more toxic to colon and gastric cancer cells than NB-506. Therefore, the results suggest that the antitumor activity of indolocarbazole-based drugs can be enhanced by incorporating DNA and/or topoisomerase I reactive groups. They also support the hypothesis that the substituent on the imide nitrogen on the F ring of NB-506 has direct interaction with the molecular target. The study helps to define the structure-activity relationships in the indolocarbazole series of antitumor agents targeting topoisomerase I.     

Interaction of a curcumin analogue dimethoxycurcumin with DNA

Dimethoxycurcumin (Dimc), a synthetic analogue of curcumin, that has been reported to exhibit better in vivo stability and anti-tumour activity, was investigated for its interaction with DNA, employing spectroscopic methods based on absorption, fluorescence, circular dichroism (CD), ethidium bromide (EtBr) competitive binding assay, 4'-6-diamidino-2-phenylindole (DAPI) displacement assay and fluorescence resonance energy transfer (FRET) assay. The mean binding constant for its interaction with calf thymus DNA (ct-DNA) was estimated to be 4.4±0.8 × 10(4) m(-1) . The studies using CD revealed that Dimc did not cause unwinding of the ct-DNA helix or induce major conformational changes. The EtBr and DAPI assays indicated that Dimc is not an intercalator but a minor groove binder. FRET assay also confirmed that Dimc interacts with DNA strands. Furthermore, viscosity measurements of ct-DNA solutions in the presence of Dimc supported these spectroscopic observations. Addition of Dimc to MCF-7 cells showed nuclear localization as visualized by confocal microscopy. In conclusion, the present studies addressed the mode of interaction of Dimc with biomolecules, which may have implications in developing Dimc as a DNA-targeted drug.     

Stimulation of topoisomerase II-mediated DNA cleavage by an indazole analogue of lucanthone.

Lucanthone is an antitumour drug used as an adjuvant in radiation therapy. The drug intercalates into DNA and inhibits topoisomerase II. An indazole analogue of lucanthone (IA-5) was examined for its ability to modulate topoisomerase II-DNA cleavable complex formation in vitro. The drug contains a methylbenzothiopyranoindazole chromophore instead of the methyl-thioxanthenone nucleus of lucanthone. Using a radiolabelled linear plasmid DNA as a substrate, both lucanthone and the indazole analogue were shown to promote the cleavage of DNA by human topoisomerase II. Sequencing experiments with different restriction fragments indicated that the indazole drug promoted DNA cleavage primarily at sites having a C on the 3' side of the cleaved bond (-1 position). By contrast, in the same sequencing methodology lucanthone exerted a much weaker effect on topoisomerase II. The sequence selectivity of IA-5 is reminiscent of that of the anticancer drug mitoxantrone and its anthrapyrazole analogue losoxantrone, which is structurally close to IA-5. Binding to DNA and topoisomerase II inhibition are two distinct processes contributing separately to the cytotoxic activity of the indazole drug.