Enhancement of transfection efficiency for HeLa cells via incorporating arginine moiety into chitosan

Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan (Arg-CS). Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and 13C NMR. Arg-CS/DNA polye- lectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering (DLS) and atomic force microscopy (AFM). The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2:1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candi- date as a safe and efficient vector for gene delivery and transfection.     

Enhancement of transfection efficiency for HeLa cells via incorporating arsinine moiety into chitosan

Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan （Arg-CS）. Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and ^13C NMR. Arg-CS/DNA polyelectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering （DLS） and atomic force microscopy （AFM）. The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2：1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candidate as a safe and efficient vector for gene delivery and transfection.     

Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells.

Replication of DNA occurs at discrete sites in eukaryotic cell nuclei, where replication proteins are clustered into large complexes, or 'replicases'. Similarly, viral DNA replication is a highly structured process, notably in herpes simplex virus type-1 (HSV-1; reviewed in ref. 4) in which large globular 'replication compartments' containing the viral replication machinery exist. Replicating cellular DNA redistributes to these compartments upon HSV-1 infection. We have now used antibodies raised against several cellular proteins to detect changes in their subnuclear localization on HSV-1 infection. We found that various proteins involved in cellular DNA replication move to sites of viral DNA synthesis, whereas a selection of non-replication proteins do not. The retinoblastoma protein and p53 (the products of two putative anti-oncogenes) relocate to the same sites as known DNA replication proteins, suggesting that they may be associated with DNA replication complexes in normal, uninfected cells.     

Non-viral gene delivery carrier of probe type host molecule ——Interactions between DNA and β-cyclodextrin derivative complexes (I)

GENE THERAPY IS A COMMON PROCESS TO DELIVER EXTRIN- SIC GENES INTO TARGET CELLS FOR FURTHER GENE EXPRESSION, FOR THE PURPOSE OF TREATING DISEASES. A WELL DESIGNED GENE DELIVERY CARRIER CAN EFFICIENTLY PACKAGE AND PRO- TECT NUCLEIC ACID FROM BEING DIGESTED BY A VARIETY OF ENZYMES IN VIVO AND SHOULD BE ABLE TO SPECIFICALLY LO- CATE EXTRINSIC GENES INTO THE TARGET ORGANISMS. S…     

电化学研究亚甲蓝与DNA的相互作用

The interaction of small molecules with DNA is of interest because it is important in the design of new and more efficient drugs targeted to DNA. The metal complexes, natural antibiotics, and a lot of other planar heterocyclic cations, have been investigated for their DNA     

Newly replicated DNA is associated with DNA topoisomerase II in cultured rat prostatic adenocarcinoma cells

DNA topoisomerases have been proposed to function in a variety of genetic processes in both prokaryotes and eukaryotes. Here, we have assessed the role of DNA topoisomerase II in mammalian DNA replication by determining the proximity of newly synthesized DNA to covalent enzyme-DNA complexes generated by treating cultured rat prostatic adenocarcinoma cells with teniposide. Teniposide (VM-26), an epipodophyllotoxin, is known to interact with mammalian DNA topoisomerase II so as to trap the enzyme in a covalent complex with DNA. We have found that the teniposide-induced trapping of such complexes requires MgCl2, is stimulated by ATP and is inhibited by novobiocin. The formation of covalent complexes seems to be reversible on removal of teniposide. Furthermore, analysis of the covalent complexes formed between 3H-thymidine pulse-labelled DNA and topoisomerase II following teniposide treatment reveals a direct association of the enzyme with nascent DNA fragments. Our results suggest that DNA topoisomerase II may interact with newly replicated daughter DNA molecules near DNA replication forks in mammalian cells.     

含萘环结构的Salen-稀土配合物与DNA的相互作用研究

参照文献以2-羟基-1-萘甲醛为基础合成了含萘环结构的4种Salen-稀土配合物,研究了这4种配合物与DNA的相互作用.实验结果表明,配合物是以插入方式与DNA结合,与DNA的作用强度呈一定规律变化,原子序数越大,与DNA结合常数Kb越小.同时发现,与Salen–过渡金属配合物不同,Salen-稀土配合物在固体及非水溶剂中具有荧光,在水溶液中荧光强度减弱,但随着DNA浓度的增加,配合物荧光呈增大的趋势.     

脂质体--基因载体

概述了基因载体的作用、类型和脂质体用于基因载体的发展历史,介绍了一类很有前途的基因载体．阳离子脂质体的组成结构和分类及其转基因的作用原理,讨论了影响阳离子脂质体／DNA复合物物理化学性质的参数及其目标复合物的制备方法．     

Stacking interaction in metal complexes with compositions of DNA and heteroaromatic N-bases

The current development in the intramolecular aromatic-ring stacking i nteractions in the complexes with compositions of DNA and heteroaromatic N-bases has been reviewed to a great extent, especially the significant contributions i n several important systems about ternary mixed-ligand complexes, including nucl eotide-metal ion-po- lyaromatic amine, amino acid-metal ion-polyaromatic amine, nucleotide-metal ion-pyridine-like aromatic amine, nucleotide-metal ion-amino ac id, nucleotide-metal ion-nucleic acid base, nucleic acid base-metal ion, and the important factors affecting the intramolecular aromatic-ring stacking interacti ons in the complexes. Based on the study of stacking interaction in the complexe s, the mechanism of interaction between DNA molecules and complexes of heteroaro matic N-bases has been established, which is crucial for the design and synthesi s of the complexes acting as molecular devices of DNA.     

Study of the interaction of DNA and histones by spin-stretching and droplet evaporation

Molecular combing is a powerful and simple method for aligning DNA molecules onto a surface. Using this technique combined with fluorescence microscopy, DNA-histone complexes are stretched on a hydrophobic polymethyl methacrylate (PMMA) surface and observed directly. We have developed a new method to stretch single DNA-histone complexes, termed spin-stretching. The results show that the histones markedly enhance DNA binding to the PMMA surface. DNA winds around the histones and therefore decreases in length. The number of histones that bind to each DNA molecule is found to correlate with the histone concentration. The combed DNA-histone complexes are found to depend on two factors: the binding force on the surface and the centrifugal force at its local position. Na+ ions should compete with histones for binding to DNA; however, the observed competitive binding effect of Na+ ions at low concentrations was negligible.     

三[（5-亚甲基-8-羟基喹啉）氨基乙基]胺及其稀土配合物的合成、表征和与DNA的作用

为研究三爪结构配体稀土配合物的组成、可能的配位状态及与DNA的作用方式,合成了新型三爪结构配体——三[（5-亚甲基-8-羟基喹啉）氨基乙基]胺及其5种稀土配合物.用元素分析、摩尔电导率、IR光谱和TG-DTA方法分析了该类化合物的组成和结构;通过UV光谱和荧光光谱研究了配合物与CT DNA的作用方式.结果表明,该系列配合物的组成为[REL（NO3）2]NO3.4 H2O（RE=Eu,Tb,Nd,La,Ce）,其中2个硝酸根以双齿形式与金属离子配位,总配位数为10;配合物与CT DNA之间存在着插入作用.     

含萘环结构的手性Salen-Mn（Ⅲ）配合物与DNA的相互作用

合成含萘环结构的手性Salen-Mn（III）配合物,[Mn（III）L]＋Cl（L=N,N’-bis-（2-hydroxynaphthalene-1-carbaldehyde）-（1R,2R）-（-）-diaminocyclohexane（R）和N,N’-bis-（2-hydroxynaphthalene-1-carbaldehyde）-（1S,2S）-（＋）-diamino-cyclohexane（S））,运用紫外光谱滴定和粘度实验,研究配合物与DNA的插入作用。并通过凝胶电泳观察配合物对pBR322DNA的断裂情况,结果表明配合物以插入模式与DNA结合,呈现出手性差异;在H2O2存在下,配合物能够使pBR322DNA断裂。     

甲壳三糖-g-壳聚糖/累托石纳米复合材料作为基因载体的研究

本文合成了甲壳三糖-g-壳聚糖（TCS），并与累托石插层复合制备纳米复合材料，采用XRD和TEM对其进行了表征。粒径分析结果表明，两种分子量的TCS与pDNA复合物的粒径大小都在75nm左右，而累托石加入后，其粒径都不同程度的增加，最大达到191nm。在PBS中的聚集动力学及琼脂糖凝胶电泳实验发现，高分子量TCS /pDNA复合物较低分子量复合物稳定，而累托石的加入降低了其稳定性。体外转染实验初步表明，甲壳三糖-g-壳聚糖/累托石-DNA复合物能介入肝癌细胞中并表达荧光。该纳米复合材料可作为潜在的非病毒基因载体。     

3,5-二碘水杨醛缩-2-氨基苯并噻唑schiff碱过渡金属配合物的合成及与DNA的作用

合成3,5-二碘水杨醛缩-2-氨基苯并噻唑schiff碱及其过渡金属（Cu,Zn,Ni,Co）的配合物.用元素分析、UV和IR吸收光谱、TG-DTA分析和摩尔电导率方法表征了配合物的组成和基本性质;通过荧光发射光谱、黏度及其与溴化乙锭（EB）的竞争实验研究了配合物与CT DNA的作用情况.结果表明,该系列配合物的组成为M（NO3）L·H2O;配合物与CT DNA的作用方式为插入模式.另外,4种配合物对藤黄微球菌的抑菌作用明显高于配体.     

基于低分子量PEI的不同疏水链修饰的梳状低聚物基因载体

为了探究基于低分子量聚乙烯亚胺(polyethylenimine, PEI)基因载体的转染效率,通过Michael加成反应将PEI 600 Da接枝于含有疏水链的生物可降解聚酯上形成系列梳状聚合物,并将之应用于基因载体;利用核磁氢谱和凝胶渗透色谱对聚合物的化学结构与分子量进行了测定,此外,还利用凝胶电泳实验和绿色荧光蛋白实验研究了聚合物与DNA的结合能力及其复合物的转染性能。结果表明：本文方法成功合成了系列低聚物,并对DNA表现出较好的包裹能力;油酸修饰后的低聚物与DNA复合物在质量比为6.4时转染效果与PEI 25 kDa相当。可见,油酸修饰后的脂质体低聚物有作为非病毒基因载体的前景。     

镓(Ⅲ)—水杨醛氨基酸Schiff碱配合物与DNA的相互作用研究

应用紫外-可见光谱、循环伏安法和粘度法研究了3个镓(Ⅲ)—水杨醛氨基酸Schiff碱配合物(GaL2,GaLbipyCl,GaLphenCl,L为水杨醛谷氨酸Schiff碱,bipy为2,2'-联吡啶,phen为1,10-邻菲啰啉)与DNA分子的键合作用.紫外-可见光谱表明配合物与DNA发生插入作用;循环伏安法表明GaLbipyCl与DNA的作用涉及沟槽或静电结合方式,GaL2和GaLphenCl与DNA发生嵌入作用;粘度法表明配合物与DNA以沟槽模式或部分插入的方式结合.综上所述,推测3个配合物以沟槽模式或部分插入的方式与DNA结合.     

双吡啶单吡咯钌配合物与DNA的相互作用

三联吡啶钌配合物和DNA相互作用已有大量报道,但基于类三联吡啶配体,如双吡啶吡咯,稳定的金属配合物和DNA作用却鲜见报道.利用荧光光谱、圆二色谱(CD)和紫外-可见光谱等表征手段,研究了4个已知的双吡啶吡咯钌配合物与DNA结合性质： [(PDP)Ru(COD)X],其中HPDP＝2,5-bis(2’-pyridyl)pyrrolide,COD＝1,5-环辛二烯,X＝Cl－ (1), NO3(－) (2);[(PDP)Ru(COD)(H2O)](BF4) (3) ; [(PDPCl)Ru(COD)Cl] (HPDPCl＝2,5-bis(2’-pyridyl)-3,4-dichloropyrrolide(4).发现化合物1以经典嵌插方式和DNA结合,2和3可能为部分或非经典嵌插结合DNA,4主要以静电相互作用结合DNA.此外,通过MTT法考察了配合物1～4对人宫颈癌Hela细胞的体外细胞毒性.     

Amino-terminal fragments of Escherichia coli lac repressor bind to DNA.

The N-terminal fragments (residues 1-51 and 1-59) obtained by selective tryptic cleavage of native lac repressor retain the ability to bind DNA. These fragments (headpieces) are monomeric and form complexes which resemble those of tetrameric repressor with non-operator DNA. But, they do not show the high specificity of repressor for operator sequences. The DNA binding has been demonstrated by filter-binding assay as well as in solution using absorption, circular dichroism, and fluorescence measurements.