Zinc improves gene transfer mediated by DNA/cationic polymer complexes

Background

The weak efficiency of plasmid transfer into the cytosol remains one of the major limiting factors to achieve an efficient transfection with DNA/cationic polymer complexes. We found that divalent metal Zn²⁺ can improve the polyfection efficiency, especially with DNA/histidylated polylysine (His‐pLK) complexes.

Methods and results

The supplementation of the transfection medium with 250 µM ZnCl₂ increased the polyfection of human hepatocarcinoma (HepG2) cells with a plasmid encoding EGFP complexed with pLK, polyethyleneimine and His‐pLK. Zn²⁺ is more efficient on DNA/His‐pLK complexes: the number of EGFP‐positive cells increased from 1% to more than 40%. This phenomenon is selective to Zn²⁺ because no effect was obtained with other divalent cations. The effect of zinc varies from cell to cell. The binding of Zn²⁺ to histidyl residues might increase zinc endosomal concentration favoring membrane fusion. Flow cytometry and confocal microscopy studies clearly indicate that with His‐pLK, the plasmid is better delivered in the cytosol as well as in the cell nucleus in zinc‐treated cells. An investigation conducted with the histidine‐rich peptide H5WYG showed that zinc inhibits membrane permeabilization but promotes membrane fusion as evidenced by resonance energy transfer.

Conclusions

Data reported here imply that the addition of zinc ions in the transfection medium can trigger an increase of the fusion of endosomes containing polyplexes which is more effective in the presence of histidine‐rich molecules. Consequently, the amount of plasmid in the cytosol available to reach the nucleus is increased leading to an improvement of polyfection. Copyright © 2002 John Wiley & Sons, Ltd.

     

Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-l-lysine (degree of polymerization=51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 11 microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered thein vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.     

激光扫描共聚焦显微镜术检测单个血小板钙波动方法的探讨

目的 探讨血小板贴壁的简易方法,同时,观察单个血小板激活前后细胞内钙波动及形态变化的规律。方法 采用多种粘附剂固定血小板,利用钙荧光探针（Fluo-3/AM)（终浓度10－20μmol)进行染色,在激光扫描共聚焦显同镜检测下加入二磷酸腺苷,观察和分析血小板内Ca^2 浓度及形态变化。结果 多聚赖氨酸促进血小板贴壁固定的效果最佳;单个血小板活后细胞内Ca^2 浓度为激活前的128％,形态圆形或椭形转为不规则形,有空泡,突起形成。结论 多聚赖氨酸是血小板贴壁的理想粘附剂,本方法能简易,快捷地监测血小板激活过程中胞浆内钙离子动态变化及形态改变,有助于血小板功能的深入研究。     

PrP的胞内运输与朊病毒病

朊病毒病,即传染性海绵状脑病（transmissible spongiform encephalopathies,TSEs）,是一类致死性的神经退行性疾病,存在散发性、感染性和遗传性3种形式。在朊病毒病的病理过程中,细胞正常朊蛋白PrPc（cellular PrP）转化为异常构象的PrP^Sc（scrapie PrP）是至关重要的,但是朊病毒的增殖如何导致神经元凋亡仍不清楚。PrPc的胞内运输在朊病毒病中发挥重要作用,朊病毒感染后PrP^C转化为PrP^Sc,及遗传性朊病毒病中PrP突变可能影响PrP的生物合成、亚细胞定位及转运过程,通过干扰PrP^C的正常功能或产生毒性中间体而导致神经系统病变。现对近年来关于PrP胞内运输在朊病毒病中的作用进行综述。     

Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action

BACKGROUND: Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes. METHODS AND RESULTS: Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell. CONCLUSIONS: A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.     

Gene therapy of cystic fibrosis: the glycofection approach

Many cells express surface membrane lectins that selectively bind and carry glycoconjugates into intracellular endosomes; in addition, various intracellular membrane and soluble lectins act as shuttles between different compartments. On this basis, we developed glycosylated polycations, now called glycofectins (glycosylated polylysine and polyethyleneimine). Recently, we set up a simple way to transform oligosaccharides into glycosynthons suitable to substitute proteins or polymers. Glycofectins bind plasmid DNA leading to compact glycoplexes. Glycoplexes prepared with glycofectins were found to be much more active than naked plasmid to transfer genes to various types of cells including human airway epithelial and serous cells. The gene transfer efficiency was found to depend on the nature of the sugars borne by glycofectins. It appeared that the sugar-dependent efficiency was not only related to the uptake but also to the intracellular traffic of glycoplexes.     

Crystal structure of human GGA1 GAT domain complexed with the GAT-binding domain of Rabaptin5

GGA proteins coordinate the intracellular trafficking of clathrin-coated vesicles through their interaction with several other proteins. The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5. The GGA-Rabaptin5 interaction is believed to function in the fusion of trans-Golgi-derived vesicles to endosomes. We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT-binding domain. In this structure, the Rabaptin5 domain is a 90-residue-long helix. At the N-terminal end, it forms a parallel coiled-coil homodimer, which binds one GAT domain of GGA1. In the C-terminal region, it further assembles into a four-helix bundle tetramer. The Rabaptin5-binding motif of the GGA1 GAT domain consists of a three-helix bundle. Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle-helix bundle interaction. The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis. The four-helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles.     

Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non-polarized and polarized cells

BACKGROUND: Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. METHODS: After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles. YOYO-labelled plasmid fluorescence intensity and luciferase activity were used as readouts for uptake and gene expression, respectively. RESULTS: PEI-polyplexes were more efficiently taken up than His-polyplexes by both non-polarized (2-fold) and polarized HLMEC (10-fold). They were mainly internalized by a clathrin-dependent pathway whatever the cell state. In non-polarized cells, His-polyplexes entered also mainly via a clathrin-dependent pathway but with an involvement of cholesterol. The cell polarization decreased this way and a clathrin-independent pathway became predominant. PEI-polyplexes transfected more efficiently HLMEC than His-polyplexes (10(7) vs. 10(5) relative light units (RLU)/mg of proteins) with a more pronounced difference in polarized cells. In contrast, no negative effect of the cell polarization was observed with tracheal epithelial cells in which both polyplexes had comparable efficiency. CONCLUSIONS: We show that the efficiency of polyplex uptake by HLMEC and their internalization mechanism are polymer-dependent. By contrast with His-polyplexes, the HLMEC polarization has little influence on the uptake process and on the transfection efficiency of PEI-polyplexes.     

Intracellular fate and nuclear targeting of plasmid DNA

One of the major steps limiting nonviral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of the transfected cells. The nuclear localization signal (NLS) of the SV40 large T antigen is known to efficiently induce nuclear targeting of proteins. We have developed two chemical strategies for covalent coupling of NLS peptides to plasmid DNA. One method involves a site-specific labeling of plasmid DNA by formation of a triple helix with an oligonucleotide–NLS peptide conjugate. After such modification with one NLS peptide per plasmid molecule, plasmid DNA remained fully active in cationic lipid-mediated transfection. In the other method, we randomly coupled 5–115 p-azidotetrafluorobenzyllissamine–NLS peptide molecules per plasmid DNA by photoactivation. Oligonucleotide–NLS and plasmid–lissamine–NLS conjugates interacted specifically with the NLS-receptor importin . Plasmid–lissamine–NLS conjugates were not detected in the nucleus, after cytoplasmic microinjection. Plasmids did not diffuse from the site of injection and plasmid–lissamine–NLS conjugates appeared to be progressively degraded in the cytoplasm. The process of plasmid DNA sequestration/degradation stressed in this study might be as important in limiting the efficiency of nonviral gene transfer as the generally recognized entry step of plasmid DNA from the cytoplasm into the nucleus     

Potocytosis and cellular exit of complexes as cellular pathways for gene delivery by polycations

BACKGROUND: Although polycations are among the most efficient nonviral vectors for gene transfer, the gene expression they allow is still too low for in vivo applications. To engineer more potent polycationic vectors, the factors governing the intracellular trafficking of a plasmid complexed with current polycations need to be identified. METHODS AND RESULTS: The trafficking of plasmid DNA complexed to glycosylated polylysines or polyethylenimine (PEI) derivatives was studied by electron microscopy of human airway epithelial cells. The cellular processing of complexes varied with their size and the polycation derivative used: large complexes (> 200 nm) made with all polycationic vectors studied were internalized by macropinocytosis. In contrast, intermediate (100-200 nm) ligand-coupled polylysine and PEI complexes primarily entered through clathrin-coated pits. Complexes were then found in endosomal vesicles, accumulated in lysosomes or vesicles near the nucleus and their nuclear entry was limited. For the population of small complexes (< or = 100 nm) obtained with PEI derivatives, they were internalized through caveolae and pursued a traffic pattern of potocytosis to the endoplasmic reticulum where their fate remains unclear. Finally, some complexes exited the cells either by regurgitation when PEI derivatives were used or through an exosome-like pathway for glycosylated-polylysine complexes. CONCLUSIONS: The different pathways of complex trafficking observed in relation with complex size imply the development and study of vectors forming complexes with definite size. Moreover, the complex exit we describe may contribute to the well-established short-term efficiency of gene transfer based on synthetic vectors. It favors the engineering of vectors allowing repeated treatment.     

Intracellular trafficking of Shiga-toxin-B-subunit-functionalized spherulites

Background information. Spherulites are multi‐lamellar lipidic vesicles that can encapsulate biomolecules and may be used as carriers for drug delivery. STxB (Shiga toxin B‐subunit) is known to bind the glycosphingolipid Gb3 (globotriaosyl ceramide), which is overexpressed by various human tumours. After Gb3 binding, the toxin enters the cytoplasm via the retrograde route, bypassing the degrading environment of the late endosomes/lysosomes. STxB is non‐toxic and has been identified as a promising tool for drug delivery. So far, applications have relied on direct coupling with therapeutic agents. In the present study, we have investigated the functionalization of spherulites by STxB and the intracellular trafficking of these structures. Results. We demonstrate that STxB‐spherulites (ST×B‐functionalized spherulites) are internalized into HeLa cells in a receptor‐dependent manner. The intracellular distribution was studied by confocal microscopy for lipids, ligand and content. We observed an early separation between spherulites and STxB, leading to a late endosomal/lysosomal localization of lipids and content, whereas STxB remained partially at the plasma membrane. Conclusions. Although recognition of Gb3 is the cause of their specific adhesion to cell membranes, STxB‐spherulites do not follow the retrograde transport route. Our results strongly suggest that STxB‐spherulites are, at least in part, disrupted at the plasma membrane, leading to lipid and content targeting to the classical endocytic pathway. We discuss how these findings influence the development of innovative delivery strategies.     

新月旋孢腔菌胞内漆酶的活性测定及漆酶基因ClLac1克隆

漆酶是一种含铜的多酚氧化酶,与植物病原菌致病性、黑色素合成及降解木质素等方面相关。为明确漆酶在新月旋孢腔菌的催化作用及其催化活性,以2,2′-连氮-双(3-乙基苯并噻唑-6-磺酸)（简称ABTS）为底物,利用分光光度计在420nm下测定胞内漆酶活力,结果表明酶活测定最佳反应条件为缓冲液pH2.8、Cu2+浓度500μmol/L和0.6mmol/L ABTS。根据漆酶Cu2+结合保守结构域设计了1条引物,对新月旋孢腔菌漆酶基因进行克隆,并通过RACE技术克隆了其全长cDNA序列。开放阅读框长1,803bp,     

胞内抗体技术及医学应用

胞内抗体技术是近年来随抗体工程技术发展起来的一项新型基因治疗方法．它将单链抗体表达于非淋巴细胞内,并使之定向分布于细胞核、细胞浆或内质网等部位,特异性阻断、干扰分布于该部位的大分子物质生物活性或加工分泌过程．实验证明该技术能抑制生长因子受体的表达,灭活原癌基因蛋白,抑制病毒复制．在生物科学及医学研究中具有很大应用潜力．     

Stimulation of enzymatic activity in filament preparations of casein kinase II by polylysine,melittin, and spermine

Summary Casein kinase II (CKII) has been purified from bovine heart tissue. Under conditions of low salt (0.05 M NaCl, 10 MM MgCl₂), CKII forms structured aggregates that appear as filaments similar to results obtained withDrosophila CKII [C.V.C. Glover (1986) J. Biol. Chem. 261:14349]. The aggregates have been analyzed by sucrose density gradients and electron microscopy. Filament preparations of the enzyme have reduced but measurable kinase activity. The addition of salt restores activity. Various modulators of CKII activity have been examined with the enzyme in the low salt, polymerized form. The polyamines spermine or spermidine stimulated CKII activity as much as six fold; putrescine had no effect. Polylysine of varying lengths activated CKII 4–6 fold. Melittin, the basic polypeptide from bee venom, was also an effective activator. Activation of filament preparations was also observed if the CKII specific peptide (RRREEETEEE) was used as the substrate in place of casein. These results with filament preparations provide an alternative in vitro system for the study of possible regulatory aspects of CKII.     

Intracellular trafficking and secretion of cerebral dopamine neurotrophic factor in neurosecretory cells

Cerebral dopamine neurotrophic factor (CDNF) is a novel evolutionary conserved protein which can protect and restore the function of dopaminergic neurons in the rat model of Parkinson's disease, suggesting that CDNF might be beneficial for the treatment of Parkinson's disease. CDNF is widely expressed in neurons in several brain regions including cerebral cortex, hippocampus, substantia nigra, striatum and cerebellum. Human CDNF is glycosylated and secreted from transiently transfected cells; however, the mechanism underlying CDNF secretion is currently unclear. In this study, we found that CDNF could be secreted primarily via the regulated secretion pathway in PC12 cells. The glycosylation of CDNF is not required for its secretion. Moreover, we identified two key subdomains in CDNF which are important for its intracellular localization and secretion. Disrupting helix-1 of CDNF significantly reduces its constitutive and regulated secretion and the helix-1 mutant is retained in the endoplasmic reticulum. Although helix-7 mutation only decreases CDNF regulated secretion and has no effect on its constitutive secretion, which is further supported by the reduction in co-localization of helix-7 mutant with secretory granules. In all, these findings will advance our understanding of the molecular mechanism of CDNF trafficking and secretion.     

Interfacial instability and the agglutination of erythrocytes by polylysine

Human erythrocytes have been exposed to poylysine of molecular weight range 4 to 220 kDa and concentration range 0.5 to 2,000 /ml at 37°C. Threshold concentrations for cell agglutination by the polycation have been determined for the samples of different molecular weight. Light and electron micrographs show that, in the erythrocyte agglutinates, cell-cell contact is generally made only at discrete, spatially periodic, regions which are distributed over a significant part of the cell surface. The average spacing between contact regions is 0.83 m. The cell membrane has a wavy profile between contact regions. Agglutination occurs only in cell samples whose electrophoretic mobility is significantly altered by polylysine and, in agreement with a previous report, occurs even when the electrophoretic mobility reaches high positive values. The electrophoretic mobility data implies that agglutination requires some protrusion of polylysine from the cell glycocalyx. We discuss how a resulting net attractive intercellular force could act to destabilize the aqueous layer between two cells, allowing surface wave growth which results in spatially periodic contact regions. Examples of situations where cell and membrane contact might be explained by the general concept of interfacial instability are discussed.     

Factors affecting the assay of histone H1 and polylysine by binding of Coomassie blue G

Rabbit hepatic microsomal suspensions were bound directly to nitrocellulose sheets using a "Hybridot" apparatus to ensure uniformity. Cytochrome P-450, form 2, was then detected by a modified immunochemical method wherein the nitrocellulose paper was incubated sequentially with antibody to form 2 for 1 h at 25 degrees C, rabbit anti-goat immunoglobulin G (IgG) at a 1:100 dilution for 15 min at 25 degrees C, goat peroxidase-antiperoxidase at a 1:2000 dilution for 15 min at 25 degrees C, and 3,3'-diaminobenzidine at 0.3 mg/ml plus 0.002% hydrogen peroxide for 30 min at 25 degrees C. These conditions, as opposed to those previously published, yielded less background staining. The density of the stain, scanned with a soft laser (Zeineh), increased linearly from 2 to 100 fmol for purified form 2. Cytochrome P-450, form 2, was detected and quantitated in microsomal samples containing 0.1 to 0.5 and 0.02 to 0.05 micrograms protein for preparations from untreated and phenobarbital-treated rabbits, respectively. The results agreed with those obtained by Western blotting and single radial immunodiffusion. This assay is more sensitive than either Western blotting or radial immunodiffusion and has significant advantages such as ease of operation, increased sample numbers, and reduced interference from extraneous proteins.     

The molecular weight cut-off of microcapsules is determined by the reaction between alginate and polylysine

Mammalian cells encapsulated in alginate-polylysine microcapsules are used as artificial organs in cancer research and in biotechnology. These applications require microcapsules with a reproducible mol. wt. cut-off. The high cost of the polycation, polylysine, requires an efficient preparation procedure. This article shows that the overall reported contact time of 5 minutes at ambient conditions should be increased several times in order to reach a maximal binding between the calcium alginate beads and 0.1% (w/v) polylysine solutions. An increase of the polylysine concentration from 0.0125% to 0.8% (w/v) resulted in a faster maximal binding, but the amount of polylysine bound increased also. Immersion of calcium alginate beads with a diameter of 750 mum, prepared from 1 mL alginate, in 30 mL of a 0.8% (w/v) polylysine solution, resulted in a polylysine spill of more than 89%. The time required to reach a maximal binding was related to the reaction temperature. The interaction zone between calcium alginate beads and fluorescein isothiocyanate-labeled polylysine solutions was visualized with a confocal laser scanning microscope as a function of time. Microcapsules, prepared at 40 degrees C with 0.1% (w/v) polylysine solutions with mol. wts. between 12 and 249.2 kD, were permeable for fluorescein isothiocyanate-labeled dextran, mol. wt. 4.7, but not for 40.5 kD. Higher polylysine concentrations resulted in a membrane with a mol. wt. cut-off lower than 4.7 kD. (c) 1993 John Wiley & Sons, Inc.     

Nuclear delivery of plasmid DNA determines the efficiency of gene expression

As a cationic non‐viral gene delivery vector, poly(agmatine/ N, N′‐cystamine‐bis‐acrylamide) (AGM‐CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM‐CBA/pDNA polyplexes was found to have a non‐linear relationship with AGM‐CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM‐CBA), we used pGL3‐control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM‐CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate‐limiting step for pLUC transfection expression. Further optimization of the non‐viral gene delivery system can be focused on the improvement of gene intracellular availability.