Structural appearance of linker histone H1/siRNA complexes.

Efficient non-viral vectors for the in vivo siRNA transfer are still being searched for. Comparing the differences of the structural appearance of siRNA and pDNA one would assume differences in the assembling behaviour between these polyanions when using polycationic vectors such as nuclear proteins. The spontaneous assembly of nuclear proteins such as histone H1 (H1) with pDNA as polyanion which has intensively been investigated over the last decade, showed a particulate structure of the resulting complexes. For an efficient in vivo use small almost monomolecular structures are searched for. Using siRNA as the polyanion might enforce this structural prerequisite lacking unwanted aggregation processes, exploiting the molecular size of siRNA. We therefore investigated the structure of H1/siRNA complexes. Five commonly used methods characterizing the resulting assemblies such as retardation gels, static and dynamic light scattering, reduction of ethidium bromide fluorescence, analytical ultracentrifugation, and electron microscopy were used. From analytical ultracentrifugation we learned that under physiological salt conditions the siRNA-H1 binding was not cooperative, even though the gel analysis showed disproportionation which would be an indication for a cooperative binding mode. H1 formed very small and stable complexes with siRNA at a molar ratio of 1:1 and 1:2. In order to find out if the observed structural appearance of the H1/siRNA complexes is due to unspecific charge effects only or to special features of H1, polylysine was included in the study. Low molecular weight polylysine (K(16)) showed also non-cooperative binding with siRNA.

Expression of decorin and collagens I and III in different layers of human skin in vivo: a laser capture microdissection study.

Extracellular matrix (ECM) organization is a complex process that requires the coordinated efforts of many molecules. For the regulation of collagen fiber diameter, the proteoglycan decorin appears to be of major relevance. To investigate the role of decorin in the process of (photo-)aging in more detail, full-thickness punch biopsies were isolated from human buttock skin. Single exposure with two minimal erythemal doses of solar simulated irradiation caused down-regulation of decorin mRNA in young (n = 5) and old subjects (n = 5) after 24 h. Interestingly, decorin mRNA was elevated with age. To test the hypothesis that a decreased collagen-to-decorin-ratio impairs collagen structure we also investigated collagens I and III gene expression. Both were down-regulated with increasing age and after single UV-irradiation. As determined by laser capture microdissection-quantitative real time-Polymerase chain reaction (n = 11), decorin is mostly present in the reticular dermis while being absent from the papillary dermis. Minor expression was also observed in the epidermis. However, in contrast to full-thickness skin biopsies age-dependent changes in collagens I, III, and decorin expression could not be observed with this methodology indicating technical limitations. Together with our finding that collagens I and III mRNA are similarly expressed in the reticular and papillary dermis and are down-regulated by UV, our studies support the idea of a major role of decorin in ECM organization. Altered expression of decorin mRNA in the different dermal strata and a decrease in the collagen-to-decorin ratio inflicted by both age and ultraviolet irradiation possibly affect collagen bundle diameter and subsequently the mechanical properties of human skin.

Occurrence of laccases in lichenized ascomycetes of the Peltigerineae.

Following our previous findings of high extracellular redox activity in lichens, the results of the work presented here identify the enzymes involved as laccases. Despite numerous data on laccases in fungi and flowering plants, this is the first report of the occurrence of laccases in lichenized ascomycetes. Extracellular laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from suborder Peltigerineae, 18 displayed laccase activity, while activity was absent in species tested from other lichen groups. Identification of the enzymes as laccases was confirmed by the ability of lichen leachates to readily metabolize substrates such as 2,2'-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and o-tolidine in the absence of hydrogen peroxide, sensitivity of the enzymes to cyanide and azide, the enzymes having typical laccase pH and temperature optima, and an absorption spectrum with a peak at 614nm. Desiccation and wounding stimulated laccase activity. Laccase activity was not increased after treatment with normal inducers of laccase synthesis, suggesting that they are constitutively expressed. Electrophoresis showed that the active form of laccase from Peltigera malacea was a tetramer with an unusually high molecular mass of 340kDa and an isoelectric point (pI) of 4.7. The finding of abundant extracellular redox enzymes known to actively produce reactive oxygen species suggest that their roles may include increasing nutrient supply to lichens by delignification, and deterring pathogens by contributing to the oxidative burst. Furthermore, once released into the environment, they may participate in the carbon cycle by facilitating the breakdown or formation of humic substances.

Nuclear proteins as gene-transfer vectors.

An interest in nuclear proteins as possible gene vectors arose as early as 1988. Nuclear proteins possess important features, such as DNA condensing capacity and the presence of NLSs (nuclear localization signals), which are thought to be perfect tools to combine cellular and nuclear uptake of DNA. The NLS of the proteins might support the intracellular trafficking of the complexes, whereas their condensing capacity enables DNase stability. Nuclear proteins, histones and also non-histones, such as the high-mobility-group proteins, have been successfully employed in in vitro gene transfer by a variety of workers over the last decade. In the pertinent published data, almost all nuclear proteins tested showed gene-transfer activity. The degree of activity of the individual nuclear proteins tested varied according to sequential and structural differences of the proteins, discriminating their DNA binding behaviour, but reported transfection efficiency differences are inconsistent. They do not reveal a logical pattern, such as a correlation with their DNA affinity. The differences in transfection efficiency of certain nuclear proteins reported by different authors could be based on a lot of factors, including the source or method of protein preparation, the cells used, the media or the transfection additives, such as lysosomotropic agents. The variances are quite random, which suggests an involvement of physicochemical parameters of the nuclear protein-DNA complexes in the efficiency of gene expression. Physicochemical features of the complexes are also determined by factors such as transfection additives (e.g. Ca(2+)). The present review therefore includes, in addition to an overview about the nuclear proteins used in gene-transfer experiments, some assumptions about the mechanism of the cellular uptake of nuclear protein-DNA complexes and their translocation to the nucleus.

Structural aspects of histone H1-DNA complexes and their relation to transfection efficiency.

During transfection, polycation-DNA complexes are normally diluted by the transfection medium, which often contains salt in the physiological concentration range and serum. It is not exactly known to what extent this dilution step influences the properties of the complexes, which in turn influence the transfection efficiency. In order to gain more insight into the size-structure-transfection activity relationship, we prepared histone H1-DNA complexes in NaCl solutions at various concentrations known to determine the size and structure of the resulting complexes. We characterized the complexes by physicochemical methods. Fluorescence correlation spectroscopy enabled relative measurements of complex sizes even under physiological conditions. The different appearances of the complexes were correlated with their transfection efficiency. When transfection was performed by dilution of the complexes in cell-cultivation media, the initial structure of H1-DNA complexes preformed under distinct salt conditions had no significant influence on the transfection efficiency. The dilution of the preformed complexes with cell-cultivation medium resulted in re-formation and aggregation of the complexes. The addition of the complexes to the cells without cell-cultivation medium, however, showed a direct correlation between the size of the complexes and the transfection efficiency (correlation coefficient 0.91). Small complexes did not contribute to the transfection.

Polyelectrolyte nanoparticles mediate vascular gene delivery.

PURPOSE: The purpose is to develop a non-viral gene delivery system that meets the requirements of colloidal stability of DNA complexes expressed in terms of no particle aggregation under physiologic conditions. The system should be used to transfect cardiovascular tissues. METHODS: We used a strategy based on the formation of polyelectrolyte nanoparticles by deposition of alternatively charged polyelectrolytes onto a DNA core. Polyelectrolytes were transfer RNA as well as the synthetic polyanion, polyvinyl sulfate (PVS), and the polycation polyethylenimine (PEI). The PEI/DNA complex formed the DNA core. RESULTS: We observed that the DNA is condensed by polycations and further packaged by association with a polyanion. These nanoparticles exhibited negative surface charge and low aggregation tendency. In vivo rat carotid artery experiments revealed high transfection efficiency, not only with the reporter gene but also with the gene encoding human urokinase plasminogen activator (Hu-uPA). Hu-uPA is one of the proteins involved in the recovery of the blood vessels after balloon catheter injury and therefore clinically relevant. CONCLUSIONS: A strategy for in vivo gene transfer is proposed that uses the incorporation of polyanions as RNA or PVS into PEI/DNA complexes in order to overcome colloidal instability and to generate a negative surface charge. The particles proved to be transfectionally active in vascular gene transfer.

Integrin specificity of the cyclic Arg-Gly-Asp motif and its role in integrin-targeted gene transfer.

Targeted gene transfer, addressing the alphavbeta3 integrin by coupling the appropriate ligand, cRGD (S(2)-bridged cyclic Arg-Gly-Asp containing peptide) motif, on to a DNA condensing sequence was described as early as 1995 by Hart, Harbottle, Cooper, Miller, Williamson and Coutelle [(1995) Gene Ther. 2, 552-554]. Their work was followed by a series of publications, introducing the cRGD motif in polycationic DNA carriers, such as peptides, proteins and liposomes. Polyethylenimine and even adenoviruses were additionally ligated using the cRGD motif. 'Integrin specificity' has been determined from the significantly improved transfection efficiency compared with the DNA carriers with control ligands, mainly the cRGE (S(2)-bridged cyclic-Arg-Gly-Glu-containing peptide) motif. However, by observing the physicochemical appearance of the resulting complexes and their controls such as the poly(L-lysine)-DNA complexes carrying the cRGD and the cRGE motifs, we doubted the integrin-mediated specificity of the increased transfection efficiency. To clarify this contradiction, we investigated the suitability of the cRGD motif for targeted gene transfer. We proved the specificity of the RGD motif and its controls using computational docking procedures and molecular modelling methods. Since we were confident of the motifs used, we improved our transfection method. Since aggregation of the RGD-ligated poly(L-lysine)-DNA complexes under physiological conditions caused an enormous amount of unspecific cell uptake and transfection, a method had to be designed to exclude aggregation processes of the motif-polycation-DNA complexes. Small complex sizes are necessary for receptor-specific uptake. The complexes were therefore recharged using poly(vinyl sulphate). Inhibited aggregation of the targeted DNA carriers under physiological conditions is a necessary prerequisite for successful in vivo gene transfer.

Histone H1-mediated transfection: role of calcium in the cellular uptake and intracellular fate of H1-DNA complexes.

Previously, we have shown that the transgene expression in the endothelial cell line ECV 304 strongly depends on the presence of low concentrations of Ca2+. However, it remained unclear, which transfection steps are controlled by Ca2+ ions. In the present study, we constructed transfection complexes of digoxigenin-labelled DNA and FITC-labelled histone H1. We monitored the pathway of these complexes with the use of anti-digoxigenin and anti-cathepsin B antibodies and immunofluorescence microscopy. Double labelling of DNA and cathepsin B permitted the localization of transfection complexes into endosomes/lysosomes which suggests an uptake of transfection complexes via endocytosis. It was also found that the uptake of transfection complexes by the cells was independent of the presence or absence of Ca2+ ions in the transfection medium. On the other hand, the presence of Ca2+ in the transfection medium dramatically changed the composition of the transfection complexes inside the endosome/lysosome compartment, which resulted in a strong reduction of H1 binding to DNA. Presence of Ca2+ in the postincubation medium for 24 h resulted in release of the transfection complexes with reduced H1 content from the endosomes/lysosomes into the cytosol. In the absence of Ca2+ the transfection complexes practically disappeared. These results allow us to come to the following conclusions: Ca2+ ions control the reorganization of the transfection complexes in endosomes/lysosomes and their release into the cytosol, which is an important prerequisite for transgene expression, whereas uptake of transfection complexes by the cells is not dependent on Ca2+.

Characterization of structure and mechanism of transfection-active peptide-DNA complexes.

We studied a number of physicochemical parameters of transfection-active peptide-DNA complexes including size, aggregation behaviour and circular dichroism (CD) spectra. These data were brought in relationship to the transfection activity of these peptides in order to better understand the mechanism of peptide-mediated gene transfer. A DNA binding oligolysine (K(16)) and a peptide comprising K(16) with an added peptide loop containing the arbitrary sequence RAD not known as a receptor ligand were used. Whereas the K(16)-DNA complex at 88% charge neutralization of the DNA phosphates collapsed into small toroidal particles with a diameter of 200 nm by dynamic light scattering, K(16)-cRAD did not. Instead, large aggregates were observed. CD spectra showed that the K(16)-DNA complexes were in a -psi state observed at liquid crystalline phases. Increasing positive charge by addition of further K(16) or disturbing the -psi state by introducing the RAD-peptide loop resulted in increasing instability indicated by aggregation and loss of the -psi CD spectrum of the complexes. Transfection experiments indicated that the aggregated material was the transfection-active component.