Addition Of Polyanions Research Articles

Major Differences between the Self-Assembly and Seeding Behavior of Heparin-Induced and in Vitro Phosphorylated Tau and Their Modulation by Potential Inhibitors.

Self-assembly of the microtubule-associated protein tau into neurotoxic oligomers, fibrils, and paired helical filaments, and cell-to-cell spreading of these pathological tau species are critical processes underlying the pathogenesis of Alzheimer’s disease and other tauopathies. Modulating the self-assembly process and inhibiting formation and spreading of such toxic species are promising strategies for therapy development. A challenge in investigating tau self-assembly in vitro is that, unlike most amyloidogenic proteins, tau does not aggregate in the absence of posttranslational modifications (PTM), aggregation inducers, or preformed seeds. The most common induction method is addition of polyanions, such as heparin; yet, this artificial system may not represent adequately tau self-assembly in vivo, which is driven by aberrant phosphorylation and other PTMs, potentially leading to in vitro data that do not reflect the behavior of tau and its interaction with modulators in vivo. To tackle these challenges, methods for in vitro phosphorylation of tau to produce aggregation-competent forms recently have been introduced (Despres et al. (2017) Proc. Natl. Acad. Sci. U.S.A., 114, 9080−908528784767). However, the oligomerization, seeding, and interaction with assembly modulators of the different forms of tau have not been studied to date. To address these knowledge gaps, we compared here side-by-side the self-assembly and seeding activity of heparin-induced tau with two forms of in vitro phosphorylated tau and tested how the molecular tweezer CLR01, a negatively charged compound, affected these processes. Tau was phosphorylated by incubation either with activated extracellular signal-regulated kinase 2 or with a whole rat brain extract. Seeding activity was measured using a fluorescence-resonance energy transfer-based biosensor-cell method. We also used solution-state NMR to investigate the binding sites of CLR01 on tau and how they were impacted by phosphorylation. Our systematic structure–activity relationship study demonstrates that heparin-induced tau behaves differently from in vitro phosphorylated tau. The aggregation rates of the different forms are distinct as is the intracellular localization of the induced aggregates, which resemble brain-derived tau strains suggesting that heparin-induced tau and in vitro phosphorylated tau have different conformations, properties, and activities. CLR01 inhibits aggregation and seeding of both heparin-induced and in vitro phosphorylated tau dose-dependently, although heparin induction interferes with the interaction between CLR01 and tau.

Polyanion based controlled release system for the GnRH-receptor antagonist degarelix

The aim of this study was to investigate the interaction between the positively charged gonadotropin releasing hormone receptor antagonist degarelix and the two polyanions alginate and carboxymethyl cellulose (CMC). Light as well as transmission electron microscopy revealed that complexes formed by simple mixing of the peptide with one of the polymers had a nano-structure consisting of twisted fibers. The remarkable unique process of complex formation could be followed by isothermal titration calorimetry: We found that peptide self-aggregates dissolved upon the addition of polyanion and peptide-polymer-complexes formed thereafter with the anionic polymer as a template. Peptide release from the complexes was tested in vitro and in vivo and compared to the dissolution of drug from self-aggregates. In vitro the release was monitored over a period of three months. We could find only slight differences in the release kinetics for the alginate and the CMC complexes compared to the pure drug. An in vivo study in Sprague Dawley rats showed similar degarelix plasma concentration levels for the complex formulations and an aqueous degarelix solution following subcutaneous injection. Overall, our findings suggest a competition between complex formation and peptide aggregation, which did not increase the availability of free drug.

Polymeric Switch on Lysozyme Activity: Role of Hydrophobic and Electrostatic Interactions

Enzymes have attracted potential applications in both medicine and biotechnology. In our present study, we show a strategy for switching the enzymatic activity of lysozyme by the complex formation with a cationic smart copolymer. PAMA- g -PEG graft copolymers suppressed the enzymatic activity of lysozyme without any conformational change, indicating the formation of complex and covering the active site of lysozyme by copolymers. The addition of polyanion, poly(acrylic acid) (PAAc), recovered the suppressed enzymatic activity of the lysozyme/polymer complex efficiently. These finding suggest that that hydrophobic interaction coupled with electrostatic interactions has a great role for the complexation and decomplexation of the lysozyme/polymer complex. Circular dichroism (CD) spectral analysis indicated that the conformation of the enzymes maintained largely during the course of the complexation.

Acidic residues in the membrane-proximal stalk region of vaccinia virus protein B5 are required for glycosaminoglycan-mediated disruption of the extracellular enveloped virus outer membrane

The extracellular enveloped virus (EEV) form of vaccinia virus (VACV) is surrounded by two lipid envelopes. This presents a topological problem for virus entry into cells, because a classical fusion event would only release a virion surrounded by a single envelope into the cell. Recently, we described a mechanism in which the EEV outer membrane is disrupted following interaction with glycosaminoglycans (GAGs) on the cell surface and thus allowing fusion of the inner membrane with the plasma membrane and penetration of a naked core into the cytosol. Here we show that both the B5 and A34 viral glycoproteins are required for this process. A34 is required to recruit B5 into the EEV membrane and B5 acts as a molecular switch to control EEV membrane rupture upon exposure to GAGs. Analysis of VACV strains expressing mutated B5 proteins demonstrated that the acidic stalk region between the transmembrane anchor sequence and the fourth short consensus repeat of B5 are critical for GAG-induced membrane rupture. Furthermore, the interaction between B5 and A34 can be disrupted by the addition of polyanions (GAGs) and polycations, but only the former induce membrane rupture. Based on these data we propose a revised model for EEV entry.

A mycelium with polyelectrolyte complex-bunched hyphae: Preparation and fermentation performance

We studied the immobilization of a mycelium ( Aspergillus niger) using the working hypothesis as follows: (a) when polycation was added to the cell suspension, a few parts of it would bind on the surface of a hypha, allowing to gather the hyphae in part but not all; (b) upon further addition of polyanion, such a gathering of the hyphae is tightly bunched by the polyelectrolyte complex (PEC) which is resulted from the remaining polycation; (c) as a result, a mycelium with partially bunched hyphae can be obtained. Potassium poly(vinyl alcohol) sulfate and trimethylammonium glycol chitosan iodide [6-O-(2-hydroxyethyl-2-(trimethylamonio)-chitosan iodide) were used as the polyanion and the polycation, respectively. The optical and electron microscopic analyses showed that our immobilized cell contains many of PEC-bunched hyphae. The sedimentation rate increased with the weight ratio of PEC to dry cells and leveled off at the weight ratio larger than 0.5. The gluconic acid production from glucose was studied by a semi-large scale (1 l) cultivation of the imobilized and free cells using a jar fermentor. It was found that an apparent specific activity of the immobilized cells for glucose oxidation becomes 1.44 times that of the free cells even at a high cell density of 40 g/l.

Production of Fab Fragments of Monoclonal Antibodies Using Polyelectrolyte Complexes

A new method for the production of monovalent Fab fragments of antibodies has been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyceraldehyde-3-phosphate dehydrogenase, digestion with papain resulted in significant damage of the binding sites of the Fab fragments. Antigen was covalently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen–polycation conjugate followed by (i) precipitation induced by addition of polyanion, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen–polycation conjugate than that of free antibodies in solution. The digestion of antibodies bound to the antigen–polyelectrolyte complex was less damaging, suggesting that binding to the antigen–polycation conjugate not only protected binding sites of monoclonal antibodies from proteolytic damage but also facilitated the proteolysis probably by exposing antibody molecules in a way convenient for proteolytic attack by papain.

Water-soluble polyion complex associates of DNA and poly(ethylene glycol)-poly(L-lysine) block copolymer.

Complex formation of poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) AB type block copolymer with salmon testes DNA or Col E1 plasmid DNA in aqueous milieu was studied. The PLL segment of PEG-PLL interacts with nucleic acid through an electrostatic force to form a water-soluble complex associate with a diameter of ca. 50 nm. PEG segments surrounding the core of the polyion complex prevented the complex from precipitation even under stoichiometric conditions, at which the unit ratio of L-lysine in PEG-PLL and phosphate in the DNA is equal. The profile of the thermal melting curve revealed a higher stabilization of DNA structure in PEG-PLL/DNA complexes compared to that in the complex made from DNA and PLL homopolymer with the same molecular weight as the PLL segment in PEG-PLL. This stabilizing effect on the DNA structure may be due to the compartmentalization of DNA into the microenvironment of PEG with low permittivity. The reversible nature of the PEG-PLL/DNA complex was further verified through the addition of polyanion [poly-(L-aspartic acid)]: Poly(L-aspartic acid) replaced DNA in the complex with PEG-PLL, resulting in the release of free DNA in the medium. Furthermore, the PEG-PLL/DNA complex showed high resistance against DNase I attack, suggesting DNA protection through the segregation into the core of the associate having PEG palisade.

Protective Effect of Dextran Sulfate and Heparin on Adult Rat Cardiomyocytes Damaged by Free Radicals

Studies in our laboratory have demonstrated that addition of polyanions, heparin or dextran sulfate, to endothelial cell cultures decrease free radical injury. To determine if these polyanions had a similar effect on cardiomyocytes, models of free radical injury were produced in adult rat cardiomyocyte cultures by: (1) addition of hydrogen peroxide; (2) exposure to hypoxia followed by reoxygenation; or (3) exposure to medium conditioned by endothelial cells that were damaged by hypoxia–reoxygenation. All three procedures significantly decreased cell viability as measured by trypan blue exclusion and increased release of enzymes, lactate dehydrogenase and creatine kinase, into cell medium when compared to control cultures. Addition of dextran sulfate or heparin to medium (5μg/ml) prior to cell injury significantly increased cell viability and decreased release of enzymes when cells were damaged by the above protocols. Heparin was significantly less protective than dextran sulfate when cells were injured by hydrogen peroxide or hypoxia–reoxygenation but not when conditioned medium from damaged endothelial cells was added to cardiomyocytes. These results indicate that polyanions are protective of cardiomyocytes as well as endothelium. This protective effect may contribute to the beneficial effects of heparin on the cardiovascular system.

Rapid Removal of Heparin from Plasma by Affinity Filtration

Coagulation test abnormalities caused by the presence of heparin are not uncommon and frequently result in additional laboratory investigation and unwarranted therapy. Methods to neutralize the effect of heparin include the addition of polyanions, enzymes, or resins to the clinical samples. These techniques are time consuming and cumbersome and produce inconsistent results. As an alternative, use of a positively charged porous filtration medium to remove heparin from the sample is described. The filtration procedure requires less than one minute and provides a consistent and total removal of heparin at plasma concentrations in excess of therapeutic levels. Chromogenic substrate analysis for residual heparin activity confirms total removal of the drug at levels greater than one unit/mL of plasma. In clinical trials, filtration normalized the activated partial thromboplastin time (APTT) of all patient specimens containing heparin (n = 41). Filtration did not shorten the coagulation assay times of patients receiving warfarin (n = 36) or of those with a variety of acquired coagulopathies (n = 14). The level of coagulation factors adsorbed to the filter medium compares favorably to that adsorbed by other commercially available heparin-binding resins. Routine use of the filter in the clinical laboratory allows for the rapid and definitive identification of heparin in specimens with prolonged coagulation times and provides clinically meaningful data.

Complex Formation in Cationic Dye–Organic Anion Systems in Aqueous Solution

Abstract The interaction between a cationic dye, Methylene Blue (MB), and organic anions (1-naphthalenesulfonate, 2-naphthalenesulfonate, 1-naphthaleneacetate, 2-anthracenesulfonate, 2,7-naphthalenedisulfonate, and 1,3,6-naphthalenetrisulfonate) in aqueous solution has been investigated by means of absorption and fluorescence spectra. The red shifts of the absorption and fluorescence spectra of MB caused by the addition of the organic monoanion are attributed to the 1:1 complex formation between MB and the organic anion. The thermodynamic quantities as well as the equilibrium constants for the complex formation have been determined. From the finding that the absorption spectrum of MB is not affected by the addition of ethanesulfonate, it is concluded that the complex formation is due to the interaction between the MB cation and the aromatic ring in the organic anion. The addition of polyanion (e.g., 2,7-naphthalenedisulfonate) in the low-concentration range accelerates the dimerization of MB; whereas, in the high-concentration range, the formation of the 1:1 complex rather than the MB dimer is dominant. Studies on the complex formation with monoanions have also been made for Rhodamine 6G, Pyronine G, Safranin T, and Crystal Violet, and similar results to that for MB have been obtained.

Interaction between Dyes and Polyelectrolytes. XV. Effect of Mixed Dimer Formation on the Excitation Energy Transfer between Bound Dyes

Abstract The effect of polyanions on the formation of mixed dimers of methylene blue (MB) and trypaflavine (TF), MB and phenosafranine (PhS), and MB and pyronine G (PG) was investigated spectrophotometrically. The following polyanions were used: potassium poly(vinyl sulfate) (PVSK), potassium poly(styrenesulfonate) (PSSK), sodium poly(methacrylate) (PMANa), and sodium poly-(acrylate) (PAANa). The formation of mixed dimers was dependent on the kind of polyanion and polyanion-dye ratio. MB-photo-sensitized isomerization of cis-p-(phenylazo)-phenyltrimethyl-ammonium iodide (cis-PTA) to the trans-isomer was used advantageously to investigate the triplet excitation energy transfer between cationic dyes bound to polyanions. Although the efficiency of the excitation energy transfer between MB and cis-PTA was enhanced by the addition of polyanions, the formation of mixed dimer of MB with TF, PhS, or PG reduced the efficiency of the excitation energy transfer. Correlation with the formation of mixed dimer of MB wi...

Interaction between dyes and polyelectrolytes. XIII. Effect of dye aggregation on the excitation energy transfer between bound dyes

AbstractEffect of polyanions on the aggregation of methylene blue (MB) was investigated spectrophotometrically. The following polyanions were used: potassium poly(vinyl sulfate), potassium poly(styrenesulfonate), sodium poly(methacrylate), and sodium poly(acrylate). The state of aggregation was largely dependent on the kind of polyanion and polyanion‐MB ratio. MB‐photo‐sensitized isomerization of cis‐p‐(phenylazo)phenyltrimethylammonium iodide(cis‐PTA) to the transisomer was used advantageously to investigate the effect of dye aggregation on the triplet excitation energy transfer between cationic dyes bound to polyanions. Although the efficiency of the excitation energy transfer between MB and cis‐PTA was enhanced by the addition of polyanions, the formation of highly aggregated MB reduced the efficiency of the excitation energy transfer. Correlation with the dye aggregation induced by polyanions and the efficiency of excitation energy transfer between dyes was discussed.

Polyelectrolyte complexes, 1. The effect of pH and ionic strength on the stoichiometry of model polycation—polyanion complexes

AbstractThe interactions between model polycations and polyanions have been studied using the fluorescent dye acridine orange as a probe. The dye emission at 540 nm is quenched by the addition of polyanions, a process which is reversed by the addition of certain polycations. Techniques have been developed which enable a determination of the stoichiometry of polyelectrolyte complexes so formed between three polyanions {carboxymethylcellulose, κ‐carrageenan and poly(styrenesulfonate)} and two polycations {poly(L‐lysine hydrobromide) and poly(p‐xylylviologen dibromide)}. A one to one pairing of acidic sites with basic groups was observed over a wide range of temperature, pH and ionic strength. Deviations from unit stoichiometry in the presence of multi‐charged metal ions have been interpreted as due to conformational changes in the polyanion. Results from viscosity measurements are included which suggest the neutralisation of charged groups in the polyelectrolyte complex.

Interaction between dyes and polyelectrolytes, 9. Effect of polyanions on alkaline fading reaction of methylene blue

AbstractThe effect of polyanions poly(potassium vinyl sulfate) (PVS), poly(sodium acrylate) (PANa), and poly(potassium p‐styrenesulfonate) (PSS), on the alkaline fading reaction of methylene blue (1) was investigated. The fading rate of 1 was extremely retarded on addition of polyanions. The binding type of 1 to polyanions was investigated spectrophotometrically by following the changes in the molar absorption coefficient (ε) at 664nm band. The relationship between the binding type and the fading rate of 1 in aqueous alkaline solution of polyanions is discussed.

Interaction between dyes and polyelectrolytes, 8. Effect of polyanions on the mixed dimer formation of cationic dyes

AbstractThe effect of polyanions on the formation of mixed dimers of methylene blue (1) and trypaflavine (2), methylene blue (1) and phenosafranine (3), and methylene blue (1) and pyronine G (4) was investigated spectrophotometrically. The following polyanions were used: poly(potassium styrenesulfonate) (PSS), poly(potassium vinyl sulfate) (PVS), and poly(sodium acrylate) (PAA). On addition of polyanions, the formation of mixed dimers was enhanced largely. Thermodynamic parameters inferred that the enhancement of the formation of mixed dimers in the presence of polyanions resulted from an entropic factor.

Killed Listeria monocytogenes vaccine becomes protective on addition of polyanions

IN the interaction between macrophages and intracellular parasites, microbial components which prevent phagosome–lysosome fusion1,2 have a decisive role. The resistance to faculative intracellular parasite Listeria monocytogenes is a form of cell-mediated immunity3, as it can be passively transferred with viable lymphoid cells4. Resistance to listeria infection can be induced by sublethal numbers of viable listeria5,6. Induction of resistance by non-viable listeria, however, is effective only after repeated injections of listeria preparations in combination with lipopolysaccharides7. Lipopolysaccharide is cytotoxic against macro-phages8,9, suggesting that the difference in processing of viable and dead microorganisms by macrophages might explain the induction of resistance by vaccine of live and not of dead bacteria. Impairing macrophage activity might result in a processing of dead listeria advantageous for the induction of resistance. We show here that killed L. monocytogenes vaccine becomes protective when the polyanions dextran sulphate (DS 500, molecular weight 500,000, Serva) and suramin (Bayer) are added. These polyanions are known to inhibit phagosome–lysosome fusion in macrophages2,10. Moreover, dextran sulphate is a potent adjuvant for both humoral11 and cell-mediated responses12. The latter might be particularly important in view of the requirement of cell-mediated immunity for resistance to listeria3.

Interaction between dyes and polyelectrolytes. VI. Metachromatic behavior of methylene blue induced by potassium poly(vinyl sulfate) and its homologs

AbstractEffect of structure of potassium poly(vinyl sulfate) and its homologs on the metachromatic behavior of methylene blue was investigated spectrophotometrically. The metachromatic behavior with addition of polyanions was followed by the changes in the molar extinction coefficient of the 664 nm band of methylene blue and in the wavelength of metachromatic band. Important factors for metachromasy were the number of anionic sites per polymer molecule, the degree of polymerization, and the flexibility of polymer chain. It was found that the amount of KCl or urea required to destroy the metachromasy was a useful measure for estimating the binding strength of methylene blue to polyanions.

Chromatin as a template for RNA synthesis in vitro.

RNA transcribed in vitro from myeloblast chromatin by exogenously added RNA polymerase B predominantly consists of short chains that remain in hybrid structure with the template; the remainder of the product is free RNA of heterogeneous size. Addition of polyanions during synthesis caused an increase in the size and amount of free RNA with a concomitant decrease in the proportion of small RNA. The large molecular weight RNA is derived from the short RNA chains, which are synthesized de novo during the reaction in vitro. The effect of polyanions on the size and nature of the product may be related to structural changes induced in the template rather than to an inhibition of nuclease activity.

Lipid-polypeptide interactions in bilayer lipid membranes

The modifications of the electrical properties of bilayer lipid membranes (BLM) composed of cholesterol and an ionic surfactant upon interaction with charged polypeptides were studied. The addition of 10(-8) M polylysine (Ps(+)) to one side of anionic cholesterol dodecylphosphate BLM increases the specific membrane conductance over 1000-fold (from 10(-8) to 10(-5) mho/cm(2)) and develops a cationic transmembrane potential larger than 50 mV. This potential is reverted by addition of polyanions such as RNA, polyglutamic or polyadenilic acid to the same side on which Ps(+) is present, by addition of Ps(+) to the opposite side, or by addition of trypsin to either side. Both conductance and potential changes are hindered by increasing the ionic strength or by raising the pH of the bathing medium, disappearing above pH 11.5 where it is known that Ps(+) folds into an α-helix. The interaction of polyglutamic acid (PGA) with a cationic cholesterol-hexadecyltrimethylammonium bromide BLM results in increased membrane conductance and development of an anionic transmembrane potential which is reverted by addition of polycations to the same aqueous phase where PGA is present. Addition of either Ps(+) or PGA to one or both sides of a neutral BLM composed of 7-dehydrocholesterol induces no significant change. The observations suggest the formation of a lipid polymer membrane resultant from the interaction, predominantly electrostatic, of the isolated components. The implications of these results are discussed in terms of the current models of membrane structure.

Evidence for IgG-immunoglobulin synthesis by isolated rat spleen nuclei

Rat spleen nuclei, isolated with isotonic sucrose solution containing 3.0 mM MgCl 2, were found to incorporate radioactive amino acid into protein which was precipitable with rabbit antiserum against rat IgG-immunoglobulin. This finding was closely examined from various points of view. The antiserum-precipitable radioactivity was strongly inhibited by deoxyribonuclease, respiratory poisons, and puromycin. Ribonuclease did not significantly block this radioactivity Antiserum-precipitable radioactivity which was inhibited by deoxyribonuclease was completely restored by the addition of polyanion. These facts were all characteristic of nuclear protein synthesis in agreement with the observation on bulk acid-insoluble radioactivity and the results observed in other systems of nuclear protein synthesis. The method of identification by immunological technique was extensively examined excluding possible nonspecific inclusion of intrusive radioactive materials and excluding also possible spurious coprecipitation of irrelevant radioactive polypeptides. The ratio of IgG-immunoglobulin to whole labeled nuclear proteins was approx. 3%, judging from radioactivity counted in the supernatant after ultracentrifugation of incubated and sonicated nuclei.