Poly Amino Acid Research Articles

Photoresponsive peptide and polypeptide systems 15*: Synthesis of photo-crosslinkable poly(amino acid)s by watery process and its application as a reinforcement for polyion complex fibers

The present article described the synthesis of the coumarin-containing polypeptides using the water-soluble active ester method and the reinforcement of the polyion complex (PIC) fibers by the photochemical cross-linking between the polypeptide-pendant coumaryloxyacetyl groups. Two kinds of cationic polypeptides, poly(α,L-lysine) (PLL) and poly(α,L-ornithine) (PLO) were treated with (4-hydroxyphenyl)dimethyl sulfonium methyl sulfate ester in water to incorporate the photoreactive coumaryloxyacetyl groups into the side chain amino groups of the PLL and PLO. The contents of the photoreactive groups in the produced coumarin-containing polypepides were controlled by the molar ratios of the amino groups to the active ester. The resulting coumarin-containing cationic polypeptides were spun into the PIC fibers with the anionic polysaccharide gellan in water. The tensile strengths of the coumarin-containing PIC fibers were superior to those of the intact PLL- and PLO-gellan PIC fibers. The mechanical strength of the coumarin-containing PIC fibers further increased upon light-irradiation. From these results, we concluded that the hydrophobic interactions and the covalent cross-linking due to the photochemical dimerization between the coumaryloxyacetyl groups significantly contributed to reinforcement of the PIC fibers. The present results afford a new methodology to the PIC reinforcement that was achieved by the polymer modification reaction in watery process.

Silicification and biosilicification - Part 7: Poly-L-arginine mediated bioinspired synthesis of silica

Precipitated silica is synthesized commercially by neutralizing sodium silicate solution under harsh conditions of pH and temperature. In contrast, the formation of ornate silica structures in biological systems (biosilicification) occurs at (or close to) pH 7 under ambient conditions and is thought to be mediated by proteins. Determination of the primary sequences of these proteins has led to the identification of various amino acids that have been proposed to be important in biosilicification. The corresponding synthetic polyamino acids are now being successfully used in bioinspired materials chemistry for developing new materials and processes. Here we report the formation of well-defined silica in vitro as facilitated by poly-L-arginine (PLAr) under ambient conditions and at neutral pH. Two different silica precursors were used in this investigation; tetramethoxysilane (TMOS) and water glass. Scanning Electron Microscopy (SEM) was used for studying the silica morphology and it was revealed that the silica spheres had typical diameters in the range 300–500nm. The PLAr is a cationically charged macromolecule at neutral pH and is believed to act as a catalyst/template/scaffold for the formation of silica in vitro in analogous fashion to certain biomacromolecules that are able to facilitate silicification/biosilicification. These results are discussed here in the context of the role(s) of (bio)macromolecules that facilitate (bio)mineralization.

Polyamino acids take amyloid form

![][1] Although lacking a specific side–chain pattern, polythreonine forms amyloid-like fibers. Dobson/EMBO The folding of a polypeptide is a tug-of-war with bonds within the main chain fighting against interactions between the side chains, according to new results from Marcus Fa

The behaviour of polyamino acids reveals an inverse side chain effect in amyloid structure formation.

Amyloid fibrils and prions are proteinaceous aggregates that are based on a unique form of polypeptide configuration, termed cross-beta structure. Using a group of chemically distinct polyamino acids, we show here that the existence of such a structure does not require the presence of specific side chain interactions or sequence patterns. These observations firmly establish that amyloid formation and protein folding represent two fundamentally different ways of organizing polypeptides into ordered conformations. Protein folding depends critically on the presence of distinctive side chain sequences and produces a unique globular fold. By contrast, the properties of different polyamino acids suggest that amyloid formation arises primarily from main chain interactions that are, in some environments, overruled by specific side chain contacts. This side chain effect can be thought of as the inverse of the one that characterizes protein folding. Conditions including Alzheimer's and Creutzfeldt-Jakob diseases represent, on this basis, pathological cases in which a natural polypeptide chain has aberrantly adopted the conformation that is primarily defined by main chain interactions and not the structure that is determined by specific side chain contacts that depend on the polypeptide sequence.

Induction of specific immune responses by polycation-based vaccines.

The s.c injection of tumor Ag-derived, MHC class I-binding peptides together with cationic poly-amino acids (e.g., poly-L-arginine; pR) has been shown to protect animals against a challenge with tumor cells expressing the respective peptide(s). Given our only restricted knowledge about immunogenic tumor-associated peptides, we sought to determine whether this pR-based vaccination protocol would also induce protective cancer immunity if large proteins were used instead of peptide epitopes. We found that the intracutaneous administration of the model Ag beta-galactosidase (beta-gal) together with pR (referred to as pR-based protein vaccine; pR-PV) was significantly more potent in protecting mice against the growth of beta-gal-expressing RENCA cells than the protein alone. Coadministration of pR enhanced both the beta-gal-induced specific humoral and CD8 response. The protective effect required CD8(+), but neither CD4(+) T lymphocytes nor beta-gal-specific Abs. beta-Gal priming of protective CD8(+) T lymphocytes was found to be CD4(+) T cell-independent, to take place within the draining lymph nodes, and to be accomplished by day 5 after vaccination. Ablation of the injection sites as early as 1.5 h after pR-PV administration still led to protection in a large proportion of the animals, indicating that certain protein Ags administered intradermally in the context of polycations are quickly transported to the draining nodes, where they induce molecular and cellular events resulting in the helper-independent priming and expansion of Tc1 cells. However, optimal protection required the prolonged presence of the injection site, suggesting that pR-PV injection facilitates the formation of a cutaneous depot of Ag-charged cells capable of migration and T cell activation.

The role of bacteriolysis in the pathophysiology of inflammation, infection and post-infectious sequelae.

The literature dealing with the biochemical basis of bacteriolysis and its role in inflammation, infection and in post-infectious sequelae is reviewed and discussed. Bacteriolysis is an event that may occur when normal microbial multiplication is altered due to an uncontrolled activation of a series of autolytic cell-wall breaking enzymes (muramidases). While a low-level bacteriolysis sometimes occurs physiologically, due to "mistakes" in cell separation, a pronounced cell wall breakdown may occur following bacteriolysis induced either by beta-lactam antibiotics or by a large variety of bacteriolysis-inducing cationic peptides. These include spermine, spermidine, bactericidal peptides defensins, bacterial permeability increasing peptides from neutrophils, cationic proteins from eosinophils, lysozyme, myeloperoxidase, lactoferrin, the highly cationic proteinases elastase and cathepsins, PLA2, and certain synthetic polyamino acids. The cationic agents probably function by deregulating lipoteichoic acid (LTA) in Gram-positive bacteria and phospholipids in Gram-negative bacteria, the presumed regulators of the autolytic enzyme systems (muramidases). When bacteriolysis occurs in vivo, cell-wall- and -membrane-associated lipopolysaccharide (LPS (endotoxin)), lipoteichoic acid (LTA) and peptidoglycan (PPG), are released. These highly phlogistic agents can act on macrophages, either individually or in synergy, to induce the generation and release of reactive oxygen and nitrogen species, cytotoxic cytokines, hydrolases, proteinases, and also to activate the coagulation and complement cascades. All these agents and processes are involved in the pathophysiology of septic shock and multiple organ failure resulting from severe microbial infections. Bacteriolysis induced in in vitro models, either by polycations or by beta-lactams, could be effectively inhibited by sulfated polysaccharides, by D-amino acids as well as by certain anti-bacteriolytic antibiotics. However, within phagocytic cells in inflammatory sites, bacteriolysis tends to be strongly inhibited presumably due to the inactivation by oxidants and proteinases of the bacterial muramidases. This might results in a long persistence of non-biodegradable cell-wall components causing granulomatous inflammation. However, persistence of microbial cell walls in vivo may also boost innate immunity against infections and against tumor-cell proliferation. Therapeutic strategies to cope with the deleterious effects of bacteriolysis in vivo include combinations of autolysin inhibitors with combinations of certain anti-inflammatory agents. These might inhibit the synergistic tissue- and- organ-damaging "cross talks" which lead to septic shock and to additional post-infectious sequelae.

Improved lectin-mediated immobilization of human red blood cells in superporous agarose beads

A new type of agarose bead, superporous agarose, was used as a gel support for immobilization of human red blood cells (RBCs) mediated by wheat germ lectin. The number of immobilized cells was similar to that obtained with commercial wheat germ lectin–agarose but the cell stability appeared to be superior. This allowed improved frontal affinity chromatographic analyses of cytochalasin B (CB)-binding to the glucose transporter GLUT1 which established a ratio of one CB-binding site per GLUT1 dimer for both plain RBCs or those treated with different poly amino acids. The measured dissociation constants, 70±14 n M for CB and 12±3 m M for glucose binding to GLUT1, are similar to those reported earlier.

Targeting of MPEG-protected polyamino acid carrier to human E-selectin in vitro.

Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab')(2). We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1 beta inducible proinflammatory marker E-selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab')(2), or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20-30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1 beta.

Helix–coil transitions re-visited

The thermally-induced helix–coil transition in polyamino acids is a good model for determining the helix-forming propensities of amino acids but not for the two-state folding/unfolding transition in globular proteins. The equilibrium and kinetic treatments of the helix–coil transition are summarized here together with a description of applications to various types of homopolymers and copolymers. Attention is then focused on the helix–coil transition in poly- l-alanine as an example of a non-polar polyamino acid. To render such a non-polar polymer water soluble, it is necessary to introduce polar amino acids such as lysines, but care must be taken as to the location of such polar residues. If they are attached as end groups, as in a triblock copolymer, they do not perturb the helix-forming tendency of the central poly- l-alanine block significantly, but if they are introduced within the sequence of alanine residues, then the hydration properties of the lysines dominate the behavior of the resulting copolymer, thereby leading to erroneous values of the parameters characterizing the helix-forming tendency of the alanines. Neutral but polar residues, such as glutamines, also exhibit hydration-dominating properties but less so than charged lysines. Some details of the calculations for an alanine/glutamine copolymer are presented here. It is concluded that random copolymers based on a neutral water-soluble host provide reliable information about the helix-forming tendencies of amino acid residues that are introduced as guests among such neutral host residues.

Improvements to the enzyme-developed radial immunodiffusion technique

An enzyme-developed radial immunodiffusion technique, previously known as the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA), has been improved in two ways: (a) antibody-containing spots have been made larger and more distinct by revealing them with a mixture of hydrogen peroxide, 3,3′-diaminobenzidine and nickel, and further intensification of the ensuing spots with silver; (b) the reliability of the method has been enhanced by chemically coupling the antigen to a layer of a polyamino acid (poly(lysine, phenylalanine)) adsorbed to the bottom of the polystyrene petri dish. The usefulness of the improved technique is illustrated by reference to the measurement of serum concentrations of IgM and IgG, and in the assessment of antibody levels against a particulate antigen (erythrocytes).

A strong inhibitor of chymotrypsin/elastase is highly antimetabolic to <i>Helicoverpa armigera</i> larvae

Chymostatin is a small nonprotein polyamino acid that specifically inhibits chymotrypsinlike enzymes by covalently binding to their active site It is therefore a model for tightbinding proteinaceous protease inhibitors of chymotrypsin/elastase specificity The Lepidopteran Helicoverpa armigera was raised on an artificial diet containing 003 chymostatin After 14 days the experimental animals were approximately twentyfold smaller than the controls although there was no increase in mortality The RNA expression levels of the main trypsin gene group were found to decrease while that of the chymotrypsin/elastase gene group increased Changes in activity of the midgut protease enzymes did not correlate to these changes in RNA expression

Targeting of Polyelectrolyte RNA Complexes to Cell Surface Integrins as an Efficient Cytoplasmic Transfection Mechanism

This is the first demonstration of receptor-mediated delivery of mRNA and establishes a new approach to gene therapy. Messenger RNA (mRNA) provides a promising alternative to plasmid DNA as a genetic material for delivery in non-viral gene therapy strategies. Since it does not require access to the nucleus and is less dependent on the cell cycle for expression, mRNA delivered using cationic lipids or short cationic polymers can be effectively translated within target cells. In this study, mRNA formed discrete nanoparticles following self assembly with a range of cationic polymers. Based on transfection activities, the low molecular weight polycations were more efficient than high molecular weight, while protamine and polyethylenimine were far more efficient than poly(L-lysine). Receptor-mediated delivery of mRNA was demonstrated using the synthetic polyamino acid (K)16GACDCRGDCFCA designed to promote cell entry following interaction with cell surface αυ integrins. RGD-bearing mRNA complexes showed very high levels of expression, reaching over 60% transduction of B16F10 cells.

Controlled Permeability and Ion Exclusion in Microporous Membranes Functionalized with Poly(l-glutamic acid)

The functionalization of materials with polyamino acids provides opportunities where electrostatic and conformational properties can be utilized for selective separations and controlled transport applications. The influence of covalently attached poly(l-glutamic acid) (PLGA) on the performance characteristics of a microporous cellulosic support has been investigated to determine its effect on the transport of water and charged solutes. The presence of these charged multifunctional groups promotes electrostatic interactions with ionic species far removed from the pore surface. This allows for nonsteric ion exclusion involving membrane materials that offer less resistance to solvent (water) transport. In addition, the helix−coil transitions of this polypeptide and “core leakage” effects have been shown to affect both the permeability and solute retention in a reversible fashion upon variations in the solution pH. Following PLGA attachment, the permeability could be adjusted from 50 to 90% of the value of th...

Surprises and revelations in biochemistry: 1950–2000

Surprises and revelations in biochemistry: 1950–2000

Amino and polyamino acids as corrosion inhibitors for aluminium and zinc pigments

A severe problem with water‐borne paints containing aluminium or zinc pigments is hydrogen corrosion of these metals in the aqueous alkaline paint media. The subject of the present study is the examination of corrosion inhibition of aluminium and zinc pigments in aqueous alkaline media by different amino and polyamino acids. Aspartic acid inhibits this corrosion reaction of aluminium pigment only at pH 8 (protection factor 96 per cent) whereas polyaspartic acids do not. In contrast, aspartic acid stimulates the corrosion reaction of zinc pigment whereas polyaspartic acids show a moderate corrosion inhibiting effect. So, corrosion inhibition by aspartic and polyaspartic acids is completely different on aluminium and zinc pigment.

Dipeptide utilization by the periodontal pathogens Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum

Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum, which can frequently be isolated from periodontal pockets, preferentially utilize proteins and peptides as growth substrates. In this study, we determined the size of peptide that is preferentially utilized as a source of energy and material for cell growth by P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum using various sizes of poly amino acids consisting of two to approximately 100 molecules of aspartate or glutamate. Resting cells of P. gingivalis, P. intermedia and P. nigrescens utilized aspartylaspartate, while cells of P. gingivalis and F. nucleatum utilized glutamylglutamate. The addition of aspartylaspartate to the culture medium increased the growth of P. gingivalis, P. intermedia and P. nigrescens, while the addition of glutamylglutamate promoted the growth of P. gingivalis and F. nucleatum. These results clearly indicate that dipeptides such as aspartylaspartate and glutamylglutamate can be utilized as growth substrates for P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum.

Deterioration of polyamino acid-coated alginate microcapsules in vivo

The implantation of immuno-isolated recombinant cell lines secreting a therapeutic protein in alginate microcapsules presents an alternative approach to gene therapy. Its clinical efficacy has recently been demonstrated in treating several genetic diseases in murine models. However, its application to humans will depend on the long-term structural stability of the microcapsules. Based on previous implantations in canines, it appears that survival of alginate–poly-L-lysine–alginate microcapsules in such large animals is short-lived. This article reports on the biological factors that may have contributed to the degradation of these microcapsules after implantation in dogs. Alginate microcapsules coated with poly-L-lysine or poly-L-arginine were implanted in subcutaneous or intraperitoneal sites. The retrieved microcapsules showed a loss of mechanical stability, as measured by resistance to osmotic stress. The polyamino acid coats were rendered fragile and easily lost, particularly when poly-L-lysine was used for coating and the intraperitoneal site was used for implantation. Various plasma proteins were associated with the retrieved microcapsules and identified with western blotting to include Factor XI, Factor XII, prekallikrein, HMWK, fibrinogen, plasminogen, ATIII, transferrin, alpha-1-antitrypsin, fibronectin, IgG, alpha-2-macroglobulin, vitronectin, prothrombin, apolipoprotein A1, and particularly albumin, a major Ca-transporting plasma protein. Complement proteins (C3, Factor B, Factor H, Factor I) and C3 activation fragments were detected. Release of the amino acids from the microcapsule polyamino acid coats was observed after incubation with plasma, indicating the occurrence of proteolytic degradation. Hence, the loss of long-term stability of the polyamino acid-coated alginate microcapsules is associated with activation of the complement system, degradation of the polyamino acid coating, and destabilization of the alginate core matrix, probably through loss of calcium-mediated ionic cross-linking of the guluronic acid polymers in the alginate. These destructive forces may be slightly mitigated by using poly-L-arginine instead of polyL-lysine for coating and by implanting in a subcutaneous instead of an intraperitoneal site. However, the long-term stability of such devices may require significant improvements in the microcapsule polymer chemistry to withstand such biological impediments.

A new pathway to aspartic acid from urea and maleic acid affected by ultraviolet light.

The photochemistry of a mixture of urea and maleic acid, which are thought to have been widely present on the primitive Earth, was studied in order to examine a possibility of the formation of amino acids. When an aqueous solution of urea and maleic acid was irradiated with an ultraviolet light of wavelength 172 nm, urea was revealed to be rather resistant to photochemical decomposition. In contrast, maleic acid was completely decomposed within 4 h, reflecting the reactivity of a C-C double bond in the molecule. In the reaction mixture, 2-isoureidosuccinic acid was detected. The acid was considered to be formed by addition of an isoureido radical which had been produced from urea by the action of a hydroxyl radical, to a C-C double bond of maleic acid. The isoureido group of the product was revealed to undergo thermal rearrangement to afford 2-ureidosuccinic acid (N-carbamoylaspartic acid). The result suggested a novel pathway leading to the formation of aspartic acid from non-amino acid precursors, possibly effected by UV-light on the primitive Earth. The formation of ureidocarboxylic acids is of another significance, since they are capable of undergoing thermal polymerization, resulting in formation of polyamino acids.

Synthesis and Complexation of a Chiral Sexidentate Pendant-Arm Macropolycyclic Polyamino Acid

The reaction of the acyclic complex ion (methyl (SS,SS)-3-[(2�-aminocyclohexyl)amino]-2-[(2�-aminocyclohexyl)-aminomethyl]propionate)copper(II) with formaldehyde and nitroethane in basic solution yields the pendant-arm macrocyclic complex (SS,SS)-(methyl-15-methyl-15-nitro-2,6,13,17-tetraazatricyclo[16.4.0.07,12]docosane-4-carboxylate)copper(ii) ion. Reduction with zinc in hydrochloric acid yields the hydrochloride salt of the pendant-arm macrocycle (SS,SS)-15-amino-15-methyl-2,6,13,17-tetraazatricyclo[16.4.0.07,12]docosane-4-carboxylic acid (1), separated into and isolated as the major trans (1a) and minor cis (1b) isomers. Co-ordination of (1) to several metal ions [CoIII, CrIII, NiII, ZnII, CuII] and comparison with a previously reported analogue without the cyclohexane rings, trans-13-amino-13-methyl-1,4,8,11-tetraazacyclotetradecane-6-carboxylic acid (2a) salt, is described. The [Ni{(1a) – H+}](ClO4) crystallizes in the monoclinic space group P21, a 9.710(2), b 14.442(1), c 10.317(2) Å, β 96.02(2)°. The nickel displays a distorted octahedral geometry, with all four secondary amines coordinated (Ni–N range 2.044(7)–2.063(6) Å), in addition to pendant primary amine (Ni–N, 2.109(6) Å) and carboxylate (Ni–O, 2.100(6) Å) groups. Protonation constants for the trans (1a) and cis (1b) isomers and stability constants of these isomers with the labile metal ions ZnII, CdII, HgII, PbII, MnII, and CoII were determined by potentiometric titrations. The log K values for 1 : 1 complexes show small variations between geometric isomers.

Polylysine and Polyornithine Gene Transfer Complexes: A Study of Complex Stability and Cellular Uptake as a Basis for their Differential in-vitro Transfection Efficiency

Gene transfer vectors formed between the cationic polyamino acid, poly- (l) -ornithine (PLO) and plasmid DNA (pDNA) have demonstrated superior transfection efficiency (up to × 10-fold) compared to equivalent polylysine-based systems in-vitro. The mechanism(s) underlying this observation remains to be elucidated. We previously reported no significant difference in colloidal particle size or zeta potential of polycation/pDNA complexes formed with poly- (l) -lysine (PLL), poly- (d) -lysine (PDL) or PLO. Here we report spectrofluorometric analysis indicating that PLO condenses pDNA at lower charge (+/ −) ratios than PLL or PDL (cf. 0.8:1, 1.2:1 and 1.5:1). Moreover, PLO/pDNA complexes proved more stable to disruption by the polyanions, poly- (l) -aspartic acid (PAA) and heparin. There were no qualitative differences in the ability of the polycations to protect complexed pDNA from enzymatic degradation both in the presence and in the absence of polyanions. The superior transfection efficiency of PLO/pDNA complexes did not appear to be mediated by an increased cellular delivery of pDNA. The data suggests a greater affinity of PLO for pDNA as an important parameter for the observed superior in-vitro transfection efficiency.