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At least 145 records · Page 8Linked to original sources
PubMed1993

Host reaction against empty alginate-polylysine microcapsules. Influence of preparation procedure.

Microcapsules, prepared with alginate and polylysine, were injected intraperitoneally into mice and the number of peritoneal leucocytes as well as the cells sticking to the capsule wall were counted after 4-28 days. A significant increase in host reaction was observed when the microcapsules contained an outer layer of polylysine as compared with calcium alginate beads without polylysine or microcapsules coated with an outer layer of alginate. The alginate sources influenced the host reaction significantly. After an intraperitoneal residence of 4 days, the microcapsules were mainly surrounded by macrophages. After 28 days, several cell layers surrounded the microcapsules; macrophages, multinucleate giant cells, fibroblasts and mesothelial cells.

Alginates↗
PubMedDate not supplied

Alginate polylysine microcapsules as immune barrier: permeability of cytokines and immunoglobulins over the capsule membrane.

Transplantation of pancreatic islets in alginate polylysine microcapsules is a potential useful method for treating type I diabetes. In this study, the permeability for alginate-polylysine microcapsules to cytokines an immunoglobulines has been investigated by a newly developed method. Magnetic monodisperse polymer particles (Dynabeads) coated with antibodies against selected proteins were encapsulated in 0.7 mm alginate polylysine microcapsules. The capsule membrane permeability to IgG (150 kDa), Transferrin (81 kDa), Tumor necrosis factor (TNF, 51 kDa), Interleukin-1 beta (IL-1 beta, 17.5 kDa), and insulin (5.8 kDa) was estimated by measuring the binding of 125I-labeled proteins to the encapsulated antibody coated Dynabeads. Capsules with an inhomogeneous solid gel core were made of alginates with high guluronic or high mannuronic acid content and poly-L (PLL)- or poly-D-lysine (PDL) of concentrations varied from 0.05-0.2%. The various capsules examined were all impermeable to IgG. The capsules made with a PLL-, but not PDL-membranes were permeable for transferrin. IL-1 beta was found to penetrate all of the different capsule types. The high-G capsules, however, could be made impermeable to TNF and still allowed transferrin to pass. The permeability of these capsules to IL-1 beta, but not to TNF was confirmed in an assay where mouse islets of Langerhans were incubated with TNF and IL-1 beta, and comparing the IL-6 for encapsulated and non-encapsulated islets.

Animals↗
PubMed1991

Effect of alginate-polylysine-alginate microencapsulation on in vitro insulin release from rat pancreatic islets.

We investigated the effect of alginate-polylysine-alginate microencapsulation on glucose-induced insulin secretion by rat islets. Applying the encapsulation method originally described by Lim, we found severely reduced in vitro insulin release (expressed as picomoles of insulin.10 islets-1.45 min-1 when incubated in 16.5 mM glucose), because the insulin release with encapsulated islets was 1.42 +/- 0.49 compared to 13.58 +/- 0.80 with free control islets. This could not be explained by inadequate permeability of the capsule, because insulin release was also severely reduced (2.12 +/- 0.61) when islets were subjected to the procedure but without the membrane-forming polylysine step. Therefore, islets were tested after having been subjected separately to each of the steps of the procedure. Insulin release was not affected by either alginate or CaCl2 but was severely reduced after prolonged suspension in saline or treatment with citrate. When saline and citrate were replaced by Ca2(+)-free Krebs-Ringer bicarbonate buffer (KRBB) and 1 mM EGTA, respectively, insulin release improved significantly both with complete and with incomplete (no polylysine step) encapsulation. This outcome was verified in a set of experiments run in parallel with islets derived from the same isolation procedure. Insulin release was 1.20 +/- 0.23 from islets encapsulated with the method of Lim and 10.73 +/- 1.04 from free control islets. With the modified procedure, insulin release was 9.17 +/- 0.52 vs. 9.61 +/- 1.27 for complete versus incomplete encapsulation, respectively. We conclude that Ca2(+)-free KRBB instead of saline and EGTA instead of citrate should be used to obtain an adequate insulin response from encapsulated islets and that the capsule membrane as such has no influence on glucose and insulin diffusion.

Alginates↗
PubMed1995

Further studies on targeted DNA transfer to cells using a highly efficient delivery system of biotinylated transferrin and biotinylated polylysine complexed to streptavidin.

Conjugates consisting of biotinylated transferrin and biotinylated poly-L-lysine attached to streptavidin have been prepared and found to transfer luciferase plasmid DNA very efficiently to HeLa cells in the presence of chloroquine. Transfection was dependent on (i) use of biotinylated short chain polylysine containing 70 lysine residues, (ii) biotinylated transferrin containing 1-2 biotin moieties, (iii) reaction of biotinylated transferrin with streptavidin followed by isolation of the resulting conjugate on Sephadex G-200 and (iv) interaction of streptavidin-biotinylated transferrin with biotinylated polylysine giving a complex suitable for DNA transfection. It was found that if the above sequence of steps resulting in the formation of streptavidin-biotinylated transferrin/biotinylated polylysine was followed without isolation of intermediate conjugates by Sephadex G-200 chromatography, pRSVL DNA transfer was still very efficient. Transfer of luciferase DNA by the streptavidin conjugates and subsequent expression of luciferase activity was almost completely inhibited by excess free transferrin, showing that gene transfer was through the transferrin receptor pathway via receptor-mediated endocytosis. The streptavidin (bio2-transferrin) bio10-pLys70 conjugate used in the present experiments was approximately one hundred times more efficient in pRSVL DNA transfection with the HeLa cells than the previously described avidin-pLys460 (bio-transferrin) complex.

Bacterial Proteins↗
PubMed1988

Polylysine and polyamine stimulation of the phosphatidylinositol kinases of amphibian oocyte membranes.

Phosphatidylinositol kinase present in Xenopus laevis oocyte membranes catalyzes the formation of phosphatidylinositol 4-phosphate using phosphatidylinositol and ATP as substrates while the activity of a second enzyme, phosphatidylinositol-4-phosphate kinase, results in the synthesis of phosphatidylinositol 4,5-bisphosphate. Large (Mr greater than 20,000) homopolymers of L-lysine or L-ornithine can stimulate the activity of both of these enzymes by at least 2-fold at 10-20 microM concentrations. Under similar conditions poly-L-arginine fails to stimulate the reaction causing a partial inhibition. Smaller polylysine (25 lysines) or lysine-rich oligopeptides such as one corresponding to the last 14 amino acids of the carboxyl end of c-Ki-ras 2 protein produce appreciable stimulation of phosphatidylinositol but at concentrations of 300-500 microM. Spermine and spermidine at millimolar concentrations also stimulate exogenous phosphatidylinositol phosphorylation. The amino-glycoside antibiotic neomycin has a biphasic effect, stimulating the phosphatidylinositol kinase at concentrations below 0.5 mM and strongly inhibiting at higher concentrations. Polylysine also moderately stimulates the loss of radioactivity of phosphatidylinositol-4-[32P] phosphate observed in oocyte membranes. Polylysine and polyornithine do not change the apparent Km for ATP of the phosphatidylinositol kinase but increase the Vmax of the reaction.

1-Phosphatidylinositol 4-Kinase↗
PubMed1996

Polylysine enhances cationic liposome-mediated transfection of the hepatoblastoma cell line Hep G2.

Plasmid DNA condensed by polylysine enhanced cationic-liposome-mediated transfection of Hep G2 cells. The luciferase expression plasmid pCMVL was complexed with the polycation poly-L-lysine and mixed with liposomes that contained a 1:1 molar ratio of the cationic lipid 1,2-dioleoyloxy-3-trimethyl-ammoniumpropane, with the neutral phospholipid 1,2-di-(cis-9-octadecenoyl)-sn-glycero-3-phosphoethanolamine. Polylysine enhanced cationic-liposome-mediated transfection of the hepatoblastoma cell line Hep G2 9-fold compared with pCMVL complexed alone with liposomes. The ratio of cationic to anionic charge of the polylysine-pCMVL complexes, and the quantity of cationic liposomes, are important determinants for optimal transfection of Hep G2 cells.

Cations↗
PubMedDate not supplied

Folic acid-polylysine carrier improves efficacy of c-myc antisense oligodeoxynucleotides on human melanoma (M14) cells.

Several polycations such as polylysine polymers are efficient transfection agents due to their capacity to bind DNA at physiological pH. By linking a ligand for a cell surface receptor to the polycation domain, a selective delivery of polyanionic compounds such as oligodeoxynucleotides (ODNs) without cell membrane-disruption can be achieved. Therefore, we aimed to develop this strategy to improve the uptake of oligomers in cancer cells. In particular, cationic polymers polylysine were covalently linked to a molecule of Folic Acid (FA) to deliver complexed ODNs in human melanoma (M-14) cells which express FA receptors. Since in these cells c-myc oncogene seems to play a crucial role in tumor growth, we used a c-myc antisense ODNs (15mer base antisense c-myc ODNs phosphorothioate) to inhibit its expression. The cellular uptake of the complexed ODNs was improved compared to the cellular uptake of free ODNs with a significant decrease in the intracellular c-myc protein level, resulting in a reduction of the growth rate and colony-forming capacity of the cells. No such effect was observed when ODNs in scrambled sequences were administered under the same experimental conditions. The efficacy of the uptake of the complex is receptor-related since a Transferrin-polylysine carrier produced no significant biological effects (in melanoma cells the Fe uptake is mediated by an oxidoreductase present in the cell membrane and not by Transferrin receptor pathway). Our results demonstrate that: a) By choosing the appropriate ligand for the membrane receptor present on the target cells, selective targeting of ODNs can be achieved. b) The uptake of the ODNs can be improved by receptor-mediated endocytosis. c) In a model system the complexed ODNs are capable of impairing gene product synthesis and function.

Drug Carriers↗
PubMed1975

Different effects of polylysine and polyarginine on the transition to a condensed state of DNA in polyethyleneglycol/salt solution.

Circular dichroism spectroscopy has been used to investigate the influence of polylysine and polyarginine on the transition to a condensed state of DNA brought about by high concentrations of polyethyleneglycol and salt. From the dependence on DNA concentration of the CD signals, the anomalous CD of free DNA in polyethyleneglycol/salt solution was attributed to the intermolecular association of DNA molecules. The CD spectral changes in polyethyleneglycol/salt solution of the DNA - polylysine complex were indistinguishable from those of free DNA while the DNA-polyarginine complex suffered much smaller spectral changes as compared with free DNA, at low DNA concentrations where time-independent CD spectra were observed in polyethyleneglycol/salt solution for both the complexed and free DNA. The repression of the spectral change by the latter complex was more remarkable at higher ratios of polyarginine to DNA. The facts indicate that, whereas polylysine binding has little influence on the intermolecular structural transition of double-stranded DNA into a compact molecular configuration in polyethyleneglycol/salt solution, polyarginine binding has an effect of inhibiting the transition.

Animals↗
PubMed1998

Polylysine-based transfection systems utilizing receptor-mediated delivery.

Receptor-mediated gene transfer is a promising gene delivery technique. It employs a DNA-binding polycation, such as polylysine, to compact plasmid DNA to a size that can be taken up by cells (<100-200 nm). To allow internalization by receptor-mediated endocytosis, cell binding ligands, such as asialoglycoproteins or galactose for hepatocytes, anti-CD3 and anti-CD5 for T-cells, and transferrin, have been covalently attached to polylysine. Intracellular barriers for successful gene transfer include release of DNA complexes from endosomes or lysosomes, nuclear import of DNA complexes, and disassembly of the DNA-polylysine particles. Release of particles from internal vesicles has been achieved by the addition of lysosomotropic agents or glycerol to the transfection medium, or by the incorporation of endosomolytic compounds, such as viruses or membrane active peptides. This technique has already been used to transfect certain organs in vivo, including liver and lung.

Journal Article↗
PubMed1975

DNA ligase activity in chromatin and its analogs. Rejoining of DNA strands in polylysine-DNA complexes and in reconstituted chromatins.

A highly purified DNA ligase from rat liver nuclei has been tested on DNA containing single-strand breaks ("nicks"); the DNA was present in several types of complexes which were chosen to serve as models for chromatin. These model systems included complexes of polylysine or histones with DNA as well as reconstituted chromatin preparations. In all these cases, the limit of ligase sealing was measured as a function of the ratio of polypeptide or protein to DNA. With an excess of either polylysine or histones, the ligase is totally prevented from sealing nicks in the DNA. However, at ratios of histones to DNA similar to those occurring in chromatin, about half of the nicks are accessible to the ligase. In the reconstitution of chromatin, the proteins are dissociated from the DNA by exposure to high ionic strength either with or without urea. If such procedures are carried out in the presence of labeled nicked DNA, the proteins will redistribute over this ligase substrate as well. When the chromatin is reconstituted at protein/DNA ratios similar to those occurring in chromatin, once more only about half of the nicks are accessible to the ligase. Similar results were obtained with preparations reconstituted with either rat liver or duck reticulocyte chromatin. The rate of ligase action has been measured on a variety of the complexes. While the rate falls as the DNA is increasingly covered with polylysine or histones, this is largely or entirely due to the decrease in concentration of sealable sites. At saturating concentrations of these DNA complexes, the original rate on uncovered DNA is approached.

Animals↗
PubMed1998

Positively charged polymer polylysine-induced cell adhesion molecule redistribution in K562 cells.

We have recently demonstrated that if human K562 erythroleukemic cells, which normally grow in suspension, are grown in polylysine-coated culture flasks for 48 h, they adhere to these flasks and grow in an anchorage-dependent like manner. Important changes in both membrane conductivity (ionic transport across the cell membrane) and membrane permittivity (static distribution of charges across the cell membrane) were also observed, indicating perturbations in membrane lipids, proteins and polysaccharides. In order to better understand the changes occurring in K562 cells exposed to polylysine and because of the important role played by cell adhesion molecules (CAMs) in cell/cell and cell/substratum interactions, and in cellular adaptation to the surrounding environment, the possible redistribution of these molecules after exposure to polylysine were investigated. In particular, the CD54 (ICAM-1), CD58 (LFA-3) and alphaVbeta3 (vitronectin receptor) molecules were investigated at different times of growth both quantitatively and qualitatively utilizing flow cytometry and immunofluorescence microscopy, respectively. The data indicate that there were no significant quantitative variations in the CAMs examined at all the times tested. In addition, no qualitative changes were observed at 48 h (as well as 24 h) of exposure. However, shorter treatment times (30 min, 1 and 2 h) did induce important CAM reorganization. The results seem to demonstrate that this cycle of CAM redistribution may, in part, be responsible for cellular adaptation to the new growth environment of K562 cells and for the variations in membrane electrical properties observed.

Journal Article↗
PubMed1969

The preferential loss of the polylysine- or polyornithine-stimulated activity of Clostridium perfringens polynucleotide phosphorylase during proteolysis.

1. An improved method for the purification of Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate-polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is described. The product was stable and was highly stimulated by polylysine or polyornithine. 2. It migrated as a single enzyme during sucrose-density-gradient centrifugation, and no separation of polymerization and phosphorolytic activities was observed. 3. Trypsin digestion caused a rapid, preferential loss of the polylysine- or polyornithine-stimulated activity, which was prevented by low concentrations of polyornithine. 4. The protection by polyornithine was not specific. 5. It is concluded that charge effects on the clostridial polynucleotide phosphorylase itself are primarily responsible for the stimulation of this enzyme by polylysine or polyornithine.

Centrifugation, Density Gradient↗
PubMed1990

Greatly improved activity of staphylococcal ribosomes in polyadenylate directed polylysine synthesis: as an assay system for investigating their sensitivity to macrolide antibiotics.

In polyadenylate directed polylysine synthesis, homologously cell-free extracts containing ribosomes and S-100 (105,000 x g supernatant) from staphylococcal cells have less than one-half (one-tenth, when the extracts were stored at -80 degrees C within a few weeks) of the activity of the extracts from Escherichia coli Q13. The present study is concerned with further improving the activity of staphylococcal ribosomes. The polylysine-synthesizing ability by staphylococcal ribosomes increased up to about two times as much as that by E. coli Q13 ribosomes, when S-100 from E. coli Q13 was mixed with staphylococcal ribosomes which had been washed with a high salt HEPES buffer containing 10 mM HEPES, 1 mM EGTA, 16 mM magnesium acetate, 1.0 M ammonium chloride and 0.1 mM dithiothreitol (pH7.6). Polylysine synthesis by the heterologous extracts has an advantage over polyuridylate-directed polyphenylalanine synthesis in the analysis of ribosome sensitivity for macrolide antibiotics, especially erythromycin.

Anti-Bacterial Agents↗
PubMed1999

Study of the interactions of D- and L-polylysine enantiomers withpectate in aqueous solutions.

The interaction between D- and L-enantiomers of polylysine and potassium pectate was studied by means of CD, microcalorimetry, and osmometry. Upon binding with pectate, only poly(L-lysine) undergoes a coil to alpha-helix transition, while poly(D-lysine) remains in the disordered state. This suggest that the energetics of the interaction is influenced by stereochemical constraints besides electrostatic forces. Experimental findings from microcalorimetry suggest that a contribution to the overall enthalpy of binding comes from the polysaccharidic moiety. Stoichiometry of the macromolecular complexes studied by osmometry gives a polylysine:pectate ratio of 3:1, in agreement with the respective degree of polymerization of the two polyelectrolytes.

Calorimetry↗
PubMed1986

Development and elimination of presynaptic elements on polylysine-coated beads implanted in neonatal rat cerebellum.

Polylysine-coated sepharose beads were implanted in the cerebellum of neonatal rats and examined at 3 hr, 3 days, 7 days, 14 days, and 21 days after surgery. Previous studies at 5 or 8 days after implantation showed that axons formed neuronal swellings that appeared to be presynaptic elements, with the bead surface in the position of a postsynaptic element. Results reported here show that no beads at 3 hr had presynaptic elements, whereas the number of beads with presynaptic elements increased to high levels at 3 and 7 days but dropped to low values at 14 and 21 days after implantation. Presynaptic elements were seen on beads regardless of their distance from cerebellar tissue except at 3 hr, when no axons were seen in the implant, indicating that axons first grew into the implant and then formed presynaptic elements. The morphological measurements of presynaptic elements on beads at 3 to 7 days after implantation showed increases in area and number of synaptic vesicles, which then decreased at 14 and 21 days after implantation. These results show that axons can grow into implants of polylysine-coated beads and form presynaptic elements that do not survive with increased time after implantation. The survival of presynaptic elements on beads can be used as a model for investigations into regeneration of axons and presynaptic elements in the injured brain.

Animals↗
PubMed1999

Detection of hybrid formation between peptide nucleic acids and DNA by fluorescence polarization in the presence of polylysine.

A new method for the detection of PNA/DNA hybrids is presented. In this method, short PNA probes (9-13 mer) are labeled with a fluorescent dye and allowed to hybridize to target DNA molecules. A cationic polyamino acid, such as polylysine, is then added to the reaction mixture, whereupon the DNA molecules bind electrostatically to this polycation. The PNA probes, which are uncharged or may carry only a small charge due to the fluorescent dye, do not bind to polylysine unless hybridized to the negatively charged DNA target. The binding of the labeled PNA/DNA hybrid to the high-molecular-weight polymer leads to a significant change in the rotational correlation time of the fluorophore attached to the PNA. This can be conveniently detected by measuring the fluorescence polarization of the latter. The method is completely homogeneous because no separation of free from bound PNA probe is required. The hybridization and dehybridization reactions can be followed in real time. The method has been applied to the typing of single-nucleotide polymorphisms in PCR products.

Base Sequence↗
PubMed1993

Polyamines as negative regulators of casein kinase-2: the phosphorylation of calmodulin triggered by polylysine and by the alpha[66-86] peptide is prevented by spermine.

Calmodulin and other protein substrates of casein kinase-2 (CK2) are not phosphorylated by CK2 holoenzyme under basal conditions. The non catalytic beta-subunit of CK2 is responsible for such a down-regulation which can be overcome by the addition of polylysine [Meggio, F. et al. (1992) Eur. J. Biochem. 205, 939-945]. Here we show that the peptide CVVKILKPVKKKKIKREIKILE, reproducing the basic insert 66-86 of CK2 catalytic subunit, can mimick polylysine in triggering the latent "calmodulin kinase" activity of CK2 holoenzyme, and that spermine and, to a lesser extent, spermidine, but not putrescine, can reversibly and dose-dependently counteract such an activation. Spermine also abolishes the stimulation by polybasic peptides of basal CK2 activity. These findings disclose the possibility that spermine may act in vivo as a negative regulator of CK2 activity toward a category of substrates, like calmodulin and ornithine decarboxylase, whose phosphorylation is dependent on polybasic peptides.

Amino Acid Sequence↗