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PubMed1994

Spermine and polylysine enhanced phosphorylation of calmodulin and tubulin in an insect endocrine gland.

Spermine-stimulated and heparin-inhibited phosphorylation of both exogenous casein and endogenous protein substrates of the prothoracic gland were measured in prothoracic gland cytosolic fractions from fifth instar larvae and early pupae of the tobacco hornworm, Manduca sexta. The results reveal a striking increase in casein kinase II (CKII) activity, i.e. approximately 3-fold above basal level in the presence of 5 mM spermine, with the highest activity exhibited by gland fractions from day 0-2 larvae, newly pupated animals and day 1 pupae. These results were verified by the results from Western blot analysis using a CKII alpha-subunit specific antibody and a 10 a.a. synthetic peptide that is a specific substrate for CKII. Several endogenous proteins were found to be substrates for CKII when assayed in the presence of spermine or polylysine. A 19 kDa peptide was shown to be calmodulin (CaM) by using the purified Manduca brain CaM as an indicator, and was only phosphorylated in the presence of polylysine. A 52 kDa protein was identified as tubulin by immunoprecipitation with a tubulin-specific monoclonal antibody, and was shown to be phosphorylated in the presence of spermine and polylysine. The possible roles of phosphocalmodulin and phosphotubulin are discussed in the context of prothoracic gland function.

Amino Acid Sequence↗
PubMed1991

NMR imaging study of the pharmacodynamics of polylysine-gadolinium-DTPA in the rabbit and the rat.

The pharmacodynamics of polylysine-(Gd-DTPA) (Schering, Berlin, Germany), a new blood pooling contrast agent for MRI, were studied in the rabbit and the rat. Polylysine-(Gd-DTPA) is a compound with high LD50. Due to its high molecular weight (50.000) and physico-chemical properties, it remains in the vascular system; during the first hour, the plasma level is three times higher than for Gd-DTPA. MRI was performed at 1.5 T using a SE sequence with TR/TE = 300/15 or 20 msec. Signal intensities of muscle, liver and kidney were measured before and after intravenous injection of the contrast agent (0.1 mmol/kg) during 8 hours in the rat (n = 3) and up to 2 wk in the rabbit (n = 3). A dose response study in three additional rabbits confirmed that the 0.1 mmol/kg dose was optimal. The pharmacodynamics results show that the effects of polylysine-(Gd-DTPA) are similar in both the rabbit and the rat. The liver signal is enhanced by about 60% immediately after injection in both species. This enhanced signal decays to half its maximal value in about one hour, which makes the contrast agent useful for clinical applications at a dose of 0.1 mmol/kg. In the kidney medulla and cortex the signals are enhanced by much larger factors (about 3 to 4); it takes at least one day for the kidney to clear the contrast agent in both species.

Animals↗
PubMed2001

Polylysine-induced 2H NMR-observable domains in phosphatidylserine/phosphatidylcholine lipid bilayers.

The interaction of three polylysines, Lys(5) (N = 5), Lys(30) (N = 30), and Lys(100) (N = 100), where N is the number of lysine residues per chain, with phosphatidylserine-containing lipid bilayer membranes was investigated using 2H NMR spectroscopy. Lys(30) and Lys(100) added to multilamellar vesicles composed of (70:30) (mol:mol) mixtures of choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) produced two resolvable 2H NMR spectral components under conditions of low ionic strength and for cases where the global anionic lipid charge was in excess over the global cationic polypeptide charge. The intensities and quadrupolar splittings of the two spectral components were consistent with the existence of polylysine-bound domains enriched in POPS, in coexistence with polylysine-free domains depleted in POPS. Lys(5), however, yielded no 2H NMR resolvable domains. Increasing ionic strength caused domains to become diffuse and eventually dissipate entirely. At physiological salt concentrations, only Lys(100) yielded 2H NMR-resolvable domains. Therefore, under physiological conditions of ionic strength, pH, and anionic lipid bilayer content, and in the absence of other, e.g., hydrophobic, contributions to the binding free energy, the minimum number of lysine residues sufficient to produce spectroscopically resolvable POPS-enriched domains on the 2H NMR millisecond timescale may be fewer than 100, but is certainly greater than 30.

Hydrogen-Ion Concentration↗
PubMed2002

Linkage of boronated polylysine to glycoside moieties of polyclonal antibody; boronated antibodies as potential delivery agents for neutron capture therapy.

Among the ways to deliver comparatively large amounts of boron to cells in vitro for boron neutron capture studies is the linkage of a boronated macromolecule such as polylysine to an antibody. In order to reduce interference with immunoreactivity, boronated polylysine (BPL) was linked to oligosaccharide moieties on the IgG molecule distant from the antibody combining sites. The resultant bioconjugate was chromatographically separated from free BPL and unconjugated antibody using a Sephacryl S300 column. The total measured boron per BPL-IgG conjugate, determined by direct current plasma atomic emission spectroscopy, was estimated to be approximately 6 x 10(3) atoms. This, together with molecular weight estimations, indicated conjugation of about 3 polylysines to each IgG molecule. Immunoreactivity of the conjugate was found to be the same as that of the unconjugated polyclonal antibody. This was based on its concentration dependent interference with immunometric reactions for an antigen (TSH), whereas heat inactivated or non-specific antibody had no such inhibitory effects. The results support the hypothesis that the binding affinity of the conjugate for antigen was preserved after its linkage to BPL under the conditions described. The methodology described in this report may have applicability for the preparation of boronated antibodies as delivery agents for BNCT.

Antibodies↗
PubMed2000

Polylysine-porphycene conjugates as efficient photosensitizers for the inactivation of microbial pathogens.

Porphycenes are electronic isomers of porphyrins which, when neutral, display no appreciable photosensitizing action towards Gram-negative bacteria. The covalent binding of oligomeric polylysine moieties, which are cationic at physiological pH values, endows porphycenes with a significant phototoxic activity against Gram-negative bacteria while retaining their photoefficiency against a variety of microbial pathogens, including Gram-positive bacteria, fungi and mycoplasmas. The effect of the polylysine moiety is dependent on both the polylysine concentration and the degree of oligomerization. A suitable interplay among the various parameters opens the possibility to obtain either a broad spectrum of antimicrobial activity or a selective action toward a specific pathogen while minimizing the damage to human fibroblasts.

Anti-Bacterial Agents↗
PubMedDate not supplied

Histidylated polylysine as DNA vector: elevation of the imidazole protonation and reduced cellular uptake without change in the polyfection efficiency of serum stabilized negative polyplexes.

We have reported that polylysine substituted with histidyl residues (His) was suited to make complexes with plasmid DNA (pDNA) and to transfect cells in vitro in the presence of serum. The present study was performed to determine whether the acetylation of the alpha-amino group of histidyl residues (AcHis) had an influence on the size and the charge of polyplexes and on their transfection efficiency. We found that the presence of free alpha-amino groups allowed the formation of smaller polyplexes but did not modify the zeta potential of +17 mV. At a physiological salt concentration, the adsorption of many serum proteins on His- and AcHis-polyplexes reduced their size below 100 nm, inhibited their aggregation, and reversed their zeta potential to -25 mV. The acetylation of the alpha-amino groups reduced slightly the adsorption of serum proteins. The presence of the alpha-amino groups increased the pK of the imidazole protonation of histidine bound to polylysine from pH 5.8 to 6.9; in addition, the protonation was further elevated in the presence of pDNA. Serum stabilized negative histidylated polyplexes were less taken up by cells but their transfection efficiency did not decrease; depending on the cell line, His-polyplexes were more efficient than AcHis-polyplexes. The results indicate that (i) the alpha-amino groups of histidyl residues bound to polylysine favorably influence the size and the transfection efficiency of polyplexes, (ii) the alpha-amino groups also elevate the imidazole protonation of His-polyplexes, which is suited to destabilize the membrane of early endocytic vesicles in order to favor pDNA delivery in the cytosol, and (iii) the absorption of selective serum proteins on His-polyplexes could be a way for in vivo gene targeting.

Gene Transfer Techniques↗
PubMedDate not supplied

DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety.

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eukaryotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNA-binding domains. Successful transferrin-receptor-mediated delivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (greater than 100 mg) of conjugates.

Carbohydrate Sequence↗
PubMed2006

Conformation and assembly of polypeptide scaffolds in templating the synthesis of silica: an example of a polylysine macromolecular "switch".

Although the role of polycationic macromolecules in catalyzing the synthesis of silica structures is well established, detailed understanding of the mechanisms behind the production of silica structures of controlled morphologies remains unclear. In this study, we have used both poly-L-lysine (PLL) and/or poly-D-lysine (PDL) for silica synthesis to investigate mechanisms controlling inorganic morphologies. The formation of both spherical silica particles and hexagonal plates was observed. The formation of hexagonal plates was suggested, via circular dichroic spectroscopy (CD), to result from the assembly of helical polylysine molecules. We confirm that the formation of PLL helices is a prerequisite to the hexagonal silica synthesis. In addition, we present for the first time that the handedness of the helicity of the macromolecule does not affect the formation of hexagonal silica. We also show, by using two different silica precursors, that the precursor does not have a direct effect on the formation of hexagonal silica plates. Furthermore, when polylysine helices were converted to beta-sheet structure, only silica particles were obtained, thus suggesting that the adoption of a helical conformation by PLL is required for the formation of hexagonally organized silica. These results demonstrate that the change in polylysine conformation can act as a "switch" in silica structure formation and suggest the potential for controlling morphologies and structures of inorganic materials via control of the conformation of soft macromolecular templates.

Circular Dichroism↗
PubMed1998

Enhancement by polylysine of transient, but not stable, expression of genes carried into cells by polyoma VP1 pseudocapsids.

Gene transfer to provide long-term expression of a therapeutic product, without introducing unwelcome genetic information, is a goal being sought for therapy of both hereditary and acquired diseases. Polyoma virus pseudocapsids, generated from a VP1-expressing recombinant baculovirus, lack viral DNA and have been successfully used to introduce small exogenous genes stably into cells in vitro by a process designated 'pseudofection'; although pseudocapsids protect only about 3 kbp of exogenous DNA, low efficiency transfer of a larger fragment (6.2 kbp) has been observed. Here, expression of a 7.2 kbp plasmid (pCMV beta) encoding the beta-galactosidase gene was assessed to monitor not only efficiency, but the ability of pseudocapsids to transfer larger-sized DNA on their own, or in the presence of the polycation, poly-L-lysine, added to protect nonencapsidated DNA. When complexed to pseudocapsids only, the efficiency of expression of the transferred beta-galactosidase gene (in human or rodent cells), although low, appeared to stabilise with time. In the presence of polylysine, unencapsidated DNA was shown to be protected against DNase activity, but electron microscopy (EM) revealed the formation of large mixed aggregates. The addition of pseudocapsids to these aggregates, and measurement of mobilities of the complexes in CsCl equilibrum centrifugation, indicated that they contained negligible amounts of VP1. For subsequent pseudofection experiments, DNA was complexed first with pseudocapsids, then polylysine was added. The latter did not appear to displace pseudocapsids from DNA, and was found to increase the efficiency of short-term expression both in in vitro and in vivo experiments. Gene expression, analysed histochemically or by the polymerase chain reaction, revealed transcriptional activity of the input gene, with expression first diminishing, then stabilising over time. The presence of pseudocapsids, in complexes with DNA with or without polylysine, allowed for stable and persistent gene expression.

Animals↗
PubMed1997

The carboxy terminus of p53 mimics the polylysine effect of protein kinase CK2-catalyzed MDM2 phosphorylation.

The oncogene product MDM2 can be phosphorylated by protein kinase CK2 in vitro 0.5-1 mol of phosphate were incorporated per mol MDM2 protein. The catalytic subunit of protein kinase CK2 (alpha-subunit) catalyzed the incorporation of twice as much phosphate into the MDM2 protein as it was obtained with the holoenzyme. Polylysine stimulated MDM2 phosphorylation by CK2 holoenzyme threefold in contrast to the alpha-subunit-catalyzed MDM2 phosphorylation which was reduced by about 66% when polylysine was added. Full length p53, but also a peptide representing a C-terminal fragment of the tumor suppressor gene product p53 (amino acids 264-393 which also harbors the CK2beta interaction site at amino acids 287-340) mimicked the polylysine effect in all respects, ie. stimulation of phosphate incorporation by CK2 holoenzyme and inhibition in the presence of the catalytic CK2 alpha-subunit. Stimulation by p53(264-393) was on the average close to twofold and inhibition in the case of the alpha-subunit-catalyzed MDM2 phosphorylation was about 40%. Phosphorylation of MDM2 by CK2 holoenzyme in the presence of the p21(WAF1/CIP1), known to be a potent inhibitor of cyclin-dependent protein kinases, also led to a significant reduction of phosphate incorporation into MDM2 indicating that p21(WAF1/CIP1) does not exclusively inhibit cell cycle kinases. Furthermore, these data add new insight into the autoregulatory loop which include p21(WAF1/CIP1), MDM2 protein, CK2 and p53.

Animals↗
PubMed1987

Polylysine-containing peptides, including the carboxyl-terminal segment of the human c-Ki-ras 2 protein, affect the activity of some key membrane enzymes.

Polylysine-containing peptides are found to affect membrane protein kinases, phosphatidylinositol kinases, and adenylate cyclase. Poly(L-lysine), poly(D-lysine), random copolymers of lysine and serine or lysine and alanine, and poly(L-ornithine) produced large increases in the in vitro phosphorylation of some membrane proteins present in Xenopus laevis oocyte membranes. Poly(L-arginine) did not cause a similar stimulation. In these membranes the phosphorylation of polydisperse protein of approximately 25 kDa was also greatly increased by 1 mM spermine and spermidine, by 10 microM histone H1, or by 200 microM peptide containing the 14-residue sequence at the carboxyl terminus of the human c-Ki-ras 2 gene product, which has eight lysines. Similar specific stimulation of protein phosphorylation was observed with membranes of NG-108-15 nerve cells in culture. Polylysine peptides, including the c-Ki-ras 2 segment, also stimulate the in vitro phosphorylation of membrane inositolphospholipids, to produce mainly phosphatidylinositol 4-phosphate and less phosphatidylinositol 4,5-bisphosphate. Polylysine also alters the activity of oocyte adenylate cyclase, assayed in the presence of either F- or 5'-guanylyl imidodiphosphate.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

Adenovirus enhancement of transferrin-polylysine-mediated gene delivery.

Gene transfer may be accomplished by the receptor-mediated endocytosis pathway using transferrin-polylysine conjugates. For some target cells, however, gene transfer by this vector is extremely limited, despite the presence of the appropriate surface receptors, a phenomenon attributed to lysosomal degradation of endosome-internalized conjugate-DNA complexes. To enhance DNA escape from the cell vesicle system and thus augment gene transfer by this route, we have used the capacity of adenoviruses to disrupt endosomes as part of their entry mechanism. Adenoviral infection augmented levels of gene transfer by transferrin-polylysine conjugates in a dose-dependent manner: levels of gene transfer of greater than 2000-fold above baseline were achieved. Use of the adenovirus in this context allowed enhanced levels of gene transfer in a variety of target cells, including cell lines otherwise refractory to gene transfer by transferrin-polylysine conjugates. This augmentation was based on adenoviral-mediated vesicle disruption, a process independent of viral gene expression. Thus, the development of specific mechanisms to effect release from the endosome in combination with gene transfer by the receptor-mediated endocytosis pathway will increase the utility of this delivery system by allowing high levels of gene expression in target cells.

Adenoviridae↗
PubMed2000

The C-terminal polylysine region and methylation of K-Ras are critical for the interaction between K-Ras and microtubules.

After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease Rce1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking Rce1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.

Amino Acid Sequence↗
PubMed1979

Isolation of human platelet plasma membranes with polylysine beads.

Human platelet plasma membranes were isolated with polylysine beads according to the technique developed by Jacobson and Branton (1977, Science [Wash. D. C.] 195:302--304). Lactoperoxidase-catalyzed surface iodination revealed that ninefold greater 125I specific activity was associated with the membranes isolated on beads than with whole platelets. Enrichment in the bead membrane preparation of the activities of membrane marker enzymes, bis(p-nitrophenyl)phosphate phosphodiesterase and Na,K-ATPase, was 8.0 and 4.4, respectively. Contamination with enzymes of other organelles, cytochrome oxidase and beta-glucuronidase, was relatively low as compared with membranes isolated by sucrose gradient centrifugation. Analysis by SDS polyacrylamide gel electrophoresis showed that a full complement of surface glycoproteins was present on the membranes isolated with polylysine beads. The polylysine bead technique is a rapid, reproducible and efficient method for the preparation of relatively pure platelet plasma membranes.

Blood Platelets↗
PubMed1963

Studies on artifical antigens. I. Antigenicity of DNP-polylysine and DNP copolymer of lysine and glutamic acid in guinea pigs.

Dinitrophenyl conjugates of poly-L-lysine, varying in percentage conjugation and molecular weight have been found to induce skin reactivity and precipitating antibodies in guinea pigs. At best, 40 per cent of immunized animals developed delayed and immediate responses to DNP-polylysine, which is believed to reflect constitutional differences among the animals assayed. Only those animals capable of responding to DNP-polylysine, responded to an immunologically distinct poly-alpha-amino acid consisting of glutamyl and lysyl residues ("copolymer glu-lys"). The percentage of animals responding to the DNP-polylysine antigen decreased as the degree of DNP conjugation increased.

Animals↗
PubMed1999

High-efficiency transfer of cystic fibrosis transmembrane conductance regulator cDNA into cystic fibrosis airway cells in culture using lactosylated polylysine as a vector.

To find more efficient vectors for the transfer of CFTR cDNA, lactosylated polylysine was explored for transfer into airway epithelial cells in primary culture. The efficacy and high efficiency of transfection were shown by several criteria: expression of both mRNA and protein for CFTR and the functional correction of the Cl- channel activity. Using specific combinations of agents to enhance the transfection, an efficiency of 90% was obtained as detected by in situ hybridization with digoxigenin-labeled probes generated against exon 14 of CFTR. The highest efficiency was observed by adding E5CA peptide (10 microg) and 5% glycerol to the transfection mixture. The degree of transfection could be controlled by the enhancing agents, thus modulating the efficiency of transfection. The highest level of transfection efficiency is equivalent to that reported for viral vectors. None of the agents or their combinations in the concentrations used were cytotoxic to the primary cells. Antibody pAb3145 was used to detect the expression of the CFTR protein in the cells. When an N-terminal GFP-CFTR fusion gene was used to transfect the CF cells a functional correction of the CFTR Cl- channel was detected by patch-clamp electrophysiology. The high efficiency of CFTR gene transfer with lactosylated polylysine leads to the conclusion that lactosylated polylysine is a promising vector to transfer the CFTR gene into human airway cells in culture.

Base Sequence↗
PubMed1992

High-efficiency gene transfer mediated by adenovirus coupled to DNA-polylysine complexes.

Employment of recombinant viruses as gene transfer vectors is limited by constraints on the size and functional design of the genetic material to be transferred as well as potential safety hazards deriving from obligatory co-transfer of viral genetic elements. As an alternative strategy that capitalizes on the efficient cellular entry mechanisms of viruses, we have derived adenovirus-polylysine-DNA complexes whereby foreign DNA is transferred bound to the exterior of the virion. This linkage was accomplished utilizing an antibody bridge in which a monoclonal antibody was rendered competent to carry DNA by the attachment of a polylysine residue. Attachment of the antibody-polylysine to the virus was by virtue of the antibody's specificity for the virion. The resulting vector system mediates high-efficiency gene transfer to target cells in vitro. In addition, this vector design allows greatly enhanced flexibility in terms of the size and design of heterologous sequences that can be transferred. Since this strategy selectively exploits viral entry functions, which are independent of viral gene expression, the potential exists to derive vectors that avoid the hazards deriving from transfer of parent virus genome.

Adenoviridae↗
PubMed1994

Complement activation by alginate-polylysine microcapsules used for islet transplantation.

A foreign body reaction is frequently observed around implanted microcapsules of alginate-polylysine. Since complement activation can play a role in this reaction, we checked in vitro the ability of empty alginate-polylysine microcapsules to activate complement. Human serum was incubated with microcapsules, and complement activation was evaluated by two methods: the complement hemolytic activity (CH50) and the assay of the C3adesArg fragment. The occurrence of complement activation in the presence of microcapsules was suggested both by a CH50 decrease and by high C3adesArg levels despite C3adesArg adsorption to the capsule membrane. Capsule membrane protection against the cytotoxic effects of complement was also tested. No hemolysis occurred when microencapsulated sensitized sheep erythrocytes were incubated with activated complement. In conclusion, the microcapsule membrane can protect cells against activated complement fragments. Nevertheless, alginate-polylysine microcapsules do activate complement, and this effect must be considered for its use as an implant.

Alginates↗