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Cold Spring Harbor Protocols Sep 2019Homemade microarrays are printed on polylysine-coated slides. The lysines form a positively charged surface that can bind nonspecifically to the acidic nucleic acids...
Homemade microarrays are printed on polylysine-coated slides. The lysines form a positively charged surface that can bind nonspecifically to the acidic nucleic acids during hybridization, resulting in significant background fluorescence. Thus, a key step in microarray processing is blocking all of the surface lysines not associated with the oligonucleotides in the microarray spots. In this protocol, the ε-amino group of lysine is succinylated by reacting with succinic anhydride. The procedure is straightforward. Microarrays are, if necessary, rehydrated; excess liquid is removed by drying at a moderate temperature; and the succinylation reaction is performed. After the reaction is complete, the slides are washed and dried with ethanol, at which point they are ready for hybridization or they can be stored in a desiccator.
Topics: Oligonucleotide Array Sequence Analysis; Polylysine; Succinic Acid; Water
PubMed: 31481489
DOI: 10.1101/pdb.prot096479 -
The Journal of Gene Medicine 2002Glycosylated polylysines and histidylated polylysines complexed with plasmid DNA (pDNA) were proposed to develop polymer-based gene delivery systems. The present work...
BACKGROUND
Glycosylated polylysines and histidylated polylysines complexed with plasmid DNA (pDNA) were proposed to develop polymer-based gene delivery systems. The present work has been undertaken in two steps to study the uptake and the intracellular processing of pDNA, which are still poorly understood in the polyfection pathway.
METHODS AND RESULTS
The kinetics of the uptake and the intracellular processing of pDNA complexed with lactosylated polylysine, histidylated polylysine or histidylated polylysine bearing lactosyl residues (polyplexes) into a CF human airway epithelial cell line were assessed by flow cytometry and confocal microscopy. Complexes formed from histidylated polylysine, even though they were less taken up by cells, show better transfection efficiency with compared with lactosylated complexes. Lactosylated polymers segregated more rapidly when compared with non-lactosylated polymers into compartments different from those containing pDNA on internalization. Intracellular location and pH measurements indicated that polymers ended up in compartments of pH approximately 6.2 while pDNA reached less acidic compartments of pH approximately 6.6. These compartments did not contain the LAMP-1 lysosomal marker.
CONCLUSIONS
The present study exhibits that, upon internalization, pDNA and polylysine conjugates underwent segregation with a rate depending on the polylysine substitution and polymer degradation. The better transfection efficiency of polyplexes with histidylated polylysine can be ascribed to their prolonged stability inside the endocytic vesicles that likely favored the pDNA escape in the cytosol.
Topics: Cell Line, Transformed; DNA; Flow Cytometry; Histidine; Humans; Lysosomes; Microscopy, Confocal; Plasmids; Polylysine
PubMed: 12112644
DOI: 10.1002/jgm.277 -
Advanced Healthcare Materials Sep 2022With the rapid growth of fungal infections and the emergence of multi-drug resistant (MDR) fungal strains, new antifungals with novel mechanisms are a pressing need to...
With the rapid growth of fungal infections and the emergence of multi-drug resistant (MDR) fungal strains, new antifungals with novel mechanisms are a pressing need to tackle this emerging health problem. Herein it is reported for the first time that hyperbranched polylysine (HPL) shows antifungal activities against Candida, especially for drug-sensitive and MDR C. albicans strains, and broad-spectrum antibacterial activities against both Gram-negative and Gram-positive bacteria. The high antimicrobial activities are ascribed to the high charge density and compact size of the globular structure of HPL. The in vitro antifungal activities of HPL3 are further enhanced by the modification of amine groups to form guanylated polylysines (HPL3-Gxs). Similar to antimicrobial peptides (AMPs), HPLs and HPL3-Gxs interact with and lyse the membranes of microbes, which mitigates the emergence of drug resistance. HPLs and HPL3-Gxs demonstrate excellent in vivo antimicrobial efficacies against both lethal C. albicans challenge in the invasive candidiasis model and lethal Methicillin resistant Staphylococcus aureus challenge in the peritonitis model, and have potentials as broad-spectrum antimicrobials.
Topics: Amines; Anti-Bacterial Agents; Anti-Infective Agents; Antifungal Agents; Candida albicans; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Polylysine
PubMed: 35775877
DOI: 10.1002/adhm.202201091 -
The Journal of Experimental Medicine Nov 2011This issue of the Journal of Experimental Medicine celebrates and honors the life of Ralph Steinman (1943-2011), winner of the 2011 Nobel Prize in Physiology or...
This issue of the Journal of Experimental Medicine celebrates and honors the life of Ralph Steinman (1943-2011), winner of the 2011 Nobel Prize in Physiology or Medicine. Ralph's science was rooted in fundamental discovery with the goal of translating these findings into clinical medicine. He recognized the power of immunology in treating human disease and passionately championed studies on vaccine design, immune therapy, and human immunology. One particular collaborative effort between the Steinman and Sekaly laboratories resulted in a paper published in this issue of the journal.
Topics: Adjuvants, Immunologic; Carboxymethylcellulose Sodium; Gene Expression Regulation; Humans; Immunity, Innate; Poly I-C; Polylysine; Signal Transduction
PubMed: 22110181
DOI: 10.1084/jem.20112321 -
Nuclear Medicine and Biology Feb 2002Among 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...
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.
Topics: Antibodies; Boron Compounds; Boron Neutron Capture Therapy; Carrier Proteins; Fluorescence; Immunoglobulin G; Molecular Weight; Polylysine; Radiotherapy Dosage
PubMed: 11823120
DOI: 10.1016/s0969-8051(01)00297-9 -
ChemMedChem Sep 2014Electrostatic interactions play a major role in protein-DNA interactions. As a model system of a cationic protein, herein we focused on a comb-type copolymer of a...
Electrostatic interactions play a major role in protein-DNA interactions. As a model system of a cationic protein, herein we focused on a comb-type copolymer of a polycation backbone and dextran side chains, poly(L-lysine)-graft-dextran (PLL-g-Dex), which has been reported to form soluble interpolyelectrolyte complexes with DNA strands. We investigated the effects of PLL-g-Dex on the conformation and thermodynamics of DNA oligonucleotides forming various secondary structures. Thermodynamic analysis of the DNA structures showed that the parallel conformations involved in both DNA duplexes and triplexes were significantly and specifically stabilized by PLL-g-Dex. On the basis of thermodynamic parameters, it was further possible to design DNA switches that undergo structural transition responding to PLL-g-Dex from an antiparallel duplex to a parallel triplex even with mismatches in the third strand hybridization. These results suggest that polycationic molecules are able to induce structural polymorphism of DNA oligonucleotides, because of the conformation-selective stabilization effects.
Topics: DNA; Dextrans; Genes, Switch; Nucleic Acid Conformation; Oligonucleotides; Polylysine; Thermodynamics
PubMed: 25045164
DOI: 10.1002/cmdc.201402157 -
Journal of Microbiology and... 2013We recently showed that polylysine, the polymer of lysines, retains anti-prion activity. Although the effectiveness of prion inhibition by polylysine was demonstrated...
We recently showed that polylysine, the polymer of lysines, retains anti-prion activity. Although the effectiveness of prion inhibition by polylysine was demonstrated with the regimen tolerated in mice, a determination of quantitative polylysine toxicity is necessary to precisely address the in vivo toxicity level of polylysine. In this communication, we report the results of body weight monitoring and hematologic tests performed in CD-1 mice that received two different tolerable dosages of polylysine for an either 5-day or 4-week period. We found that there was no significant alteration in overall serum chemistry, blood cell count, and body weight gain compared with controls. The only notable quantitative change with statistical significance was the decrease of platelet numbers in mice subchronically administered with polylysine. Our results suggest that polylysine is harmless in mice if administered for a short period, but administrations of polylysine in mice may require considerate attention for safety in future investigations as mice chronically receive tolerable doses of polylysine.
Topics: Animals; Blood Chemical Analysis; Body Weight; Mice; Platelet Count; Polylysine
PubMed: 23711515
DOI: 10.4014/jmb.1302.02055 -
Biochimica Et Biophysica Acta. General... Oct 2022The antimicrobial activity of ε-poly-l-lysine (EPL) has been documented, but its antifungal activity on yeast is not well defined and its mechanism of action has been...
The antimicrobial activity of ε-poly-l-lysine (EPL) has been documented, but its antifungal activity on yeast is not well defined and its mechanism of action has been vaguely explained. Our studies revealed that on both, Candida albicans and Saccharomyces cerevisiae, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were 250 μg·mL; EPL produced a K and Ca efflux, and at higher concentrations also an efflux of material absorbing at 260 nm, small peptides, and phosphate is produced, along with the inhibition of fermentation and extracellular acidification and respiration. Moreover, growth was inhibited, reactive oxygen species (ROS) production increased, and cell viability decreased. The polycation also produced plasma membrane potential hyperpolarization. The effects were dependent both on the cell quantity and polycation concentration, as well as the media used. The plasma membrane disruption was confirmed by TEM and PI staining.
Topics: Antifungal Agents; Candida albicans; Microbial Sensitivity Tests; Polylysine; Saccharomyces cerevisiae
PubMed: 35732210
DOI: 10.1016/j.bbagen.2022.130197 -
ACS Applied Materials & Interfaces Jun 2016We investigate the effect of stretching on the secondary structure of cross-linked poly(l-lysine)/hyaluronic acid (PLL/HA) multilayers. We show that stretching these...
We investigate the effect of stretching on the secondary structure of cross-linked poly(l-lysine)/hyaluronic acid (PLL/HA) multilayers. We show that stretching these films induces changes in the secondary structure of PLL chains. Our results suggest that not only α- but also 310-helices might form in the film under stretching. Such 310-helices have never been observed for PLL so far. These changes of the secondary structure of PLL are reversible, i.e., when returning to the nonstretched state one recovers the initial film structure. Using molecular dynamics simulations of chains composed of 20 l-lysine residues (PLL20), we find that these chains never adopt a helical conformation in water. In contrast, when the end-to-end distance of the chains is restrained to values smaller than the mean end-to-end distance of free chains, a distance domain rarely explored by the free chains, helical conformations become accessible. Moreover, the formation of not only α- but also 310-helices is predicted by the simulations. These results suggest that the change of the end-to-end distance of PLL chains in the stretched film is at the origin of the helix formation.
Topics: Hyaluronic Acid; Polylysine; Protein Structure, Secondary; Water
PubMed: 26646202
DOI: 10.1021/acsami.5b08302 -
Microscopy Research and Technique Apr 2022Bacterial sample preparation is crucial for its observation by scanning electron microscopy (SEM). However, the current polylysine (PLL) method leads to bacterial...
Bacterial sample preparation is crucial for its observation by scanning electron microscopy (SEM). However, the current polylysine (PLL) method leads to bacterial morphological changes. To overcome this problem, we employed chitosan (CS) to coat coverslips to prepare bacteria for SEM and compared it with the PLL method. Coverslips coated with 0.025% (w/v) CS showed satisfactory bacterial binding ability. Within 30 min of binding time, the number of bacteria on CS-coated and PLL-coated coverslips exhibited no differences. Four bacteria strains were employed to compare the differences in SEM images between the two methods. Most of the bacteria showed irregular surface or sticky substances after settling on PLL-coated coverslips, while bacteria with clear surface texture were observed on CS-coated coverslips. Transmission electron microscopy (TEM) images showed deformed bacterial envelope on PLL-coated coverslips; meanwhile, similar intact envelope was observed from the bacteria on CS-coated coverslips and the bacteria without any treatment. The TEM results verified the morphological differences of SEM between the two methods. Except for morphology, the length of the rod-shaped bacteria was longer on CS-coated coverslips than that on PLL-coated coverslips, less shrinkage of the sample was observed, and CS could preserve the length of the rod-shaped bacteria better than PLL in its preparation for SEM. It is demonstrated that the low-cost CS could be utilized in bacterial preparation for SEM to acquire preferable images. Bacterial suspension with optical density at 600 nm of about 0.5, deposited on 0.025% CS-coated coverslips for 30 min, and followed by routine fixation, dehydration, and drying are optimal parameters.
Topics: Bacteria; Chitosan; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Polylysine
PubMed: 34851006
DOI: 10.1002/jemt.23992