Physicochemical and Biological Properties of Vancomycin-Containing Antibacterial Polysaccharide Gels for Biocomposite Bone Implant Impregnation.

Polymeric drugs containing up to 60% by weight of the antibiotic vancomycin were synthesized based on dextran carriers activated with epichlorohydrin. Vancomycin was covalently bound, involving the primary amino group of the molecule through the hydroxypropyl radical to the C6 position of the anhydroglucose units of the dextran main chain. Covalent binding is necessary to prevent spontaneous release of the antibiotic from the gel, thereby reducing the risk of bacterial multiresistance. Antibacterial depot gels were obtained from those polymers, containing up to 17.5% by weight of polysaccharide with a cross-linking density of q = 3-5 nodes per macromolecule for the deposition of another type of drugs not covalently bound to the polymer gel. They were used to coat the surface of the internal pores of biocomposite bone implants based on bovine cancellous bone used in orthopedics. The chemical structure of the polymer was studied using 13C NMR spectroscopy and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The stiffness of the gels was evaluated by the values of the accumulation modulus G' = 170-270 kPa and the loss modulus G″ = 3.7-4.2 kPa determined on a rheometer. Their values are close to those typical for materials used to replace soft tissue in plastic surgery. The minimum inhibitory concentration of the gels against Staphylococcus aureus P209 depends on the antibiotic content in the polymer. It equals 2.5 mg/L for vancomycin we used and 100 mg/L for a polymer containing 50% by weight of covalently bound antibiotic. The cytotoxic concentration measured with cell culture HEK 293T exceeds 1200 mg/L in 24 h exposure. The release dynamics of drugs not covalently bound to dextran from the depot gel were studied using fluorescein as a model. The release time is independent of the gel density and lasts up to 6 days for a 2 mm thick layer. Both the gel and the bone implants impregnated with it maintained consistently high antibacterial activity throughout the experiment, up to its completion after 168 h, with the local concentration of the released antibiotic at the site of bacterial attack exceeding the therapeutic level by 200 times.

Glyphosate-Induced Phosphonatase Operons in Soil Bacteria of the Genus Achromobacter.

Achromobacter insolitus and Achromobacter aegrifaciens, bacterial degraders of the herbicide glyphosate, were found to induce phosphonatase (phosphonoacetaldehyde hydrolase, EC 3.11.1.1) when grown on minimal media with glyphosate as the sole source of phosphorus. The phosphonatases of the strains were purified to an electrophoretically homogeneous state and characterized. The enzymes differed in their kinetic characteristics and some other parameters from the previously described phosphonatases. The phosphonatase of A. insolitus was first revealed to separate into two stable forms, which had similar kinetic characteristics but interacted differently with affinity and ion-exchange resins. The genomes of the investigated bacteria were sequenced. The phosphonatase genes were identified, and their context was determined: the bacteria were shown to have gene clusters, which, besides the phosphonatase operon, included genes for LysR-type transcription activator (substrate sensor) and putative iron-containing oxygenase PhnHD homologous to monooxygenases PhnY and TmpB of marine organophosphonate degraders. Genes of 2-aminoethylphosphonate aminotransferase (PhnW, EC 2.6.1.37) were absent in the achromobacterial phosphonatase operons; instead, we revealed the presence of genes encoding the putative flavin oxidase HpnW. In silico simulation showed 1-hydroxy-2-aminoethylphosphonate to be the most likely substrate of the new monooxygenase, and a number of glycine derivatives structurally similar to glyphosate to be substrates of flavin oxidase.

Gel Based on Hydroxyethyl Starch with Immobilized Amikacin for Coating of Bone Matrices in Experimental Osteomyelitis Treatment.

A polysaccharide gel containing covalently bound amikacin, a broad-spectrum antibiotic, was produced by using epichlorohydrin-activated hydroxyethyl starch (HES). The structure of the polymers was analyzed by 13C and 1H nuclear magnetic resonance (13C NMR and 1H NMR) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The sites of covalent attachment of amikacin to the epoxypropyl substituent and the HES backbone were determined. The antibacterial activity of the polymer was evaluated in vitro using the agar well diffusion method with the Staphylococcus aureus P209 strain. It was demonstrated that the polymer retained activity in the presence of bacterial amylase, which is released upon bacterial attack. The gel was applied for coating pores and surfaces of a biocomposite material based on a xenogenic bovine bone matrix. In vivo experiments showed the effectiveness of utilizing amikacin-containing biocomposite bone-substitute materials in the treatment of experimental osteomyelitis in rats using objective histological control and X-ray tomography.

The Regioselective Conjugation of the 15-nt Thrombin Aptamer with an Optimized Tripeptide Sequence Greatly Increases the Anticoagulant Activity of the Aptamer.

Currently, oligonucleotide therapy has emerged as a new paradigm in the treatment of human diseases. In many cases, however, therapeutic oligonucleotides cannot be used directly without modification. Chemical modification or the conjugation of therapeutic oligonucleotides is required to increase their stability or specificity, improve their affinity or inhibitory characteristics, and address delivery issues. Recently, we proposed a conjugation strategy for a 15-nt G-quadruplex thrombin aptamer aimed at extending the recognition interface of the aptamer. In particular, we have prepared a series of designer peptide conjugates of the thrombin aptamer, showing improved anticoagulant activity. Herein, we report a new series of aptamer-peptide conjugates with optimized peptide sequences. The anti-thrombotic activity of aptamer conjugates was notably improved. The lead conjugate, TBA-GLE, was able to inhibit thrombin-induced coagulation approximately six-fold more efficiently than the unmodified aptamer. In terms of its anticoagulant activity, the TBA-GLE conjugate approaches NU172, one of the most potent G-quadruplex thrombin aptamers. Molecular dynamics studies have confirmed that the principles applied to the design of the peptide side chain are efficient instruments for improving aptamer characteristics for the proposed TBA conjugate model.

Recognition of 3' nucleotide context and stop codon readthrough are determined during mRNA translation elongation.

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3' contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3' stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3' nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3' nucleotides. Moreover, the efficiency of translation termination in weak 3' contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3' nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.

Synthesis of Amphiphilic Copolymers of N-Vinyl-2-pyrrolidone and Allyl Glycidyl Ether for Co-Delivery of Doxorubicin and Paclitaxel.

Co-delivery of chemotherapeutics in cancer treatment has been proven essential for overcoming multidrug resistance and improving the outcome of therapy. We report the synthesis of amphiphilic copolymers of N-vinyl-2-pyrrolidone and allyl glycidyl ether of various compositions and demonstrate that they can form nanoaggregates capable of simultaneous covalent immobilization of doxorubicin by the epoxy groups in the shell and hydrophobic-driven incorporation of paclitaxel into the core of nanoparticles. The structure of the obtained copolymers was characterized by 13C NMR, IR, and MALDI spectroscopy, as well as adsorption at the water/toluene interface. A linear increase in the number-average molecular weight of amphiphilic copolymers and a decrease in the number-average diameter of macromolecular aggregates with an increase in the ratio N-vinyl-2-pyrrolidone/allyl glycidyl ether were observed. The assembled nanocarriers were characterized by DLS. The reported novel nanocarriers can be of interest for delivery and co-delivery of a wide range of pharmacological preparations and combined therapy for cancer and other deceases.

Formation of Hydrogels Based on a Copolymer of N-Vinyl-2-pyrrolidone and Glycidyl Methacrylate in the Presence of the Reaction Product of 1,3-Dimethylmidazolium Dimethylphosphate and Elemental Sulfur.

The aim of the study is to search for a reaction that provides the possibility of tandem "one-pot" formation of polymer networks during radical copolymerization of N-vinyl-2-pyrrolidone and glycidyl methacrylate. It was shown that the addition of recently synthesized 1,3-dimethylimidazolium (phosphonooxy-)oligosulfanide makes it possible to obtain a cross-linked copolymer in one stage as a result of radical copolymerization of N-vinyl-2-pyrrolidone and glycidyl methacrylate with a molar ratio of monomers less than 1.4. The structure of the copolymerization products of N-vinyl-2-pyrroldione and glycidyl methacrylate formed in the presence of 1,3-dimethylimidazolium (phosphonooxy-)oligosulfanide was characterized by 1H NMR, FTIR and MALDI spectroscopy. 1H NMR spectroscopy revealed an interaction under moderate heating between glycidyl methacrylate and 1,3-dimethylimidazolium (phosphonooxy-)oligosulfanide, accompanied by the formation of a mixture of unsaturated products of complex structure, presumably acting as crosslinking agents. It is shown that when the molar ratio of N-vinyl-2-pyrroldione/glycidyl methacrylate comonomers is 0.89, a densely crosslinked copolymer is formed, capable of limited swelling in water with a velocity constant of 5.06 × 10-2 min-1 and an equilibrium degree of swelling of about 227%.

Chemical Oxidative Polymerization of Methylene Blue: Reaction Mechanism and Aspects of Chain Structure.

The kinetic regularities of the initial stage of chemical oxidative polymerization of methylene blue under the action of ammonium peroxodisulfate in an aqueous medium have been established by the method of potentiometry. It was shown that the methylene blue polymerization mechanism includes the stages of chain initiation and growth. It was found that the rate of the initial stage of the reaction obeys the kinetic equation of the first order with the activation energy 49 kJ × mol-1. Based on the proposed mechanism of oxidative polymerization of methylene blue and the data of MALDI, EPR, and IR spectroscopy methods, the structure of the polymethylene blue chain is proposed. It has been shown that polymethylene blue has a metallic luster, and its electrical conductivity is probably the result of conjugation over extended chain sections and the formation of charge transfer complexes. It was found that polymethylene blue is resistant to heating up to a temperature of 440 K and then enters into exothermic transformations without significant weight loss. When the temperature rises above 480 K, polymethylene blue is subject to endothermic degradation and retains 75% of its mass up to 1000 K.

The New Approach to the Preparation of Polyacrylamide-Based Hydrogels: Initiation of Polymerization of Acrylamide with 1,3-Dimethylimidazolium (Phosphonooxy-)Oligosulphanide under Drying Aqueous Solutions.

The new initiator of the polymerization of acrylamide, leading to the formation of crosslinked polyacrylamide, was discovered. The structure of the synthesized polyacrylamide was characterized by XRD, 1Н NMR, and 13С NMR spectroscopy. It was shown that 1,3-dimethylimidazolium (phosphonooxy-)oligosulphanide is able to initiate radical polymerization under drying aqueous solutions of acrylamide, even at room temperature. According to XRF data, the synthesized polyacrylamide gel contains 0.28 wt% of sulphur. The formed polymer network has a low crosslinking density and a high equilibrium degree of swelling. The swelling rate of polyacrylamide gel in water corresponds to the first order kinetic equation with the rate constant 6.2 × 10-2 min-1. The initiator is promising for combining acrylamide polymerization with the processes of gel molding and drying.

ß-Lytic Protease of Lysobacter capsici VKM B-2533T.

Bacteriolytic enzymes are promising antimicrobial agents for developing new-generation drugs. Recently, we have isolated a ß-lytic protease (BlpLc) from the culture liquid of Lysobacter capsici VKM B-2533T. This BlpLc possesses a valuable property, not described for ß-lytic proteases (Blps) earlier, of hydrolyzing living cells of Staphylococcus aureus 55 MRSA clinical isolate. This work phylogenetically characterized the BlpLc and investigated its properties. Analysis revealed a variability of pre-/pro-parts of Blp precursors. The mature BlpLc is the closest to the earlier annotated but not isolated Blp from Lysobacter sp. Root690. The biochemical characterization found conditions for the BlpLc general bacteriolytic activity relative to autoclaved S. aureus 209P cells to differ from that of earlier isolated Blp. Unexpected was the effect of serine (phenylmethylsulfonyl fluoride (PMSF)) and cysteine (p-chloromercuribenzoate (p-CMB)) protease inhibitors on BlpLc bacteriolytic and proteolytic activities. The specificity of BlpLc proteolytic action relative to hemoglobin, elastin, gelatin, collagen, azofibrin, myoglobin, ovalbumin, and ovamucoid was found. New types of peptide bonds-Gly-X, Ser-X, Lys-X, Ala-X, Val-X, Glu-X, and Phe-X-hydrolyzed by the enzyme in protein substrates were first revealed using MALDI-TOF. Turbidimetrically, the BlpLc was found to lyze living cells of S. aureus 209P, Micrococcus luteus B1819, and M. roseus B1236, which is important for expanding the enzyme's applied properties.

Involvement of apolipoprotein A in maintaining tissue fluid balance in goldfish Carassius auratus.

Mammalian plasma proteins play a key role in maintaining tissue fluid balance because they are retained within capillaries and thus create colloid osmotic pressure. Likewise, fish plasma contain a considerable concentration oligomeric proteins which likely serve a similar role. To elucidate the functions of these oligomeric proteins, we analyzed blood serum (BS) and interstitial fluid (IF) complexes in goldfish from the wild and under experimental conditions using 2D electrophoresis and matrix-assisted laser desorption/ionization (MALDI). We detected protein compounds with MWs ranging from 50 to 155 kDa, organized as oligomeric complexes. The protein compounds consisted of apolipoproteins АроА-I and Аро-14 which are homological to mammalian АроА-I and АроА-II, respectively. The 155-kDa and 50-125-kDa oligomer complexes were located very low-density lipoproteins (LDL) and high-density lipoproteins (HDL) areas on the BS/IF proteomic maps, respectively. The latter resembled mammalian HDL plasma particles by size and contained lipids, so we considered them as HDL particle populations. Investigation of the uniform dissociation/association mechanism for HDL and LDL oligomers in goldfish, from the wild and under critical salinity conditions, showed the "125/110 → 85/60 kDa" reorganization. This was associated with overcoming physiological stress during spawning and under critical salinity conditions. Opposite reorganization "85/60 → 125/110 kDa" was associated with restoration of metabolic processes after stress. The association/dissociation reorganizations promoted equilibration of BS and IF osmolarities in all fish groups. We discuss the connection of these reorganizations with total protein distribution across the capillary wall and salinity, as well as the role of oligomeric apolipoproteins as universal metabolic regulators.

Paip2 is localized to active promoters and loaded onto nascent mRNA in Drosophila.

Paip2 (Poly(A)-binding protein - interacting protein 2) is a conserved metazoan-specific protein that has been implicated in regulating the translation and stability of mRNAs. However, we have found that Paip2 is not restricted to the cytoplasm but is also found in the nucleus in Drosophila embryos, salivary glands, testes, and tissue culture cells. Nuclear Paip2 is associated with chromatin, and in chromatin immunoprecipitation experiments it maps to the promoter regions of active genes. However, this chromatin association is indirect, as it is RNA-dependent. Thus, Paip2 is one more item in the growing list of translation factors that are recruited to mRNAs co-transcriptionally.

Oligomeric forms of bacterial malate dehydrogenase: a study of the enzyme from the phototrophic non-sulfur bacterium Rhodovulum steppense A-20s.

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the phototrophic purple non-sulfur bacterium Rhodovulum steppense A-20s. According to gel-chromatography and electrophoretic studies, malate dehydrogenase is present as a dimer, tetramer and octamer depending on cultivation conditions. In phototrophic aerobic conditions only the tetrameric form was present, in chemotrophic aerobic conditions all three forms were detected, while in the absence of oxygen the octameric form disappeared. The malate dehydrogenase oligomers are encoded by a single gene and composed of the same 35 kDa polypeptide but differ in pH and temperature optimum, in affinities to malate, oxaloacetate, NADH and NAD+ and in regulation by cations and citrate. By modulating the cultivation conditions, it has been established that the dimer participates in the glyoxylate cycle; the tetramer operates in the tricarboxylic acid cycle, and the octamer may be involved in the adaptation to oxidative stress.

Human aqueous humor proteome in cataract, glaucoma, and pseudoexfoliation syndrome.

Twenty-nine human aqueous humor samples from patients with eye diseases such as cataract and glaucoma with and without pseudoexfoliation syndrome were characterized by LC-high resolution MS analysis. In total, 269 protein groups were identified with 1% false discovery rate including 32 groups that were not reported previously for this biological fluid. Since the samples were analyzed individually, but not pooled, 36 proteins were identified in all samples, comprising the constitutive proteome of the fluid. The most dominant molecular function of aqueous humor proteins as determined by GO analysis is endopeptidase inhibitor activity. Label-free protein quantification showed no significant difference between glaucoma and cataract aqueous humor proteomes. At the same time, we found decrease in the level of apolipoprotein D as a marker of the pseudoexfoliation syndrome. The data are available from ProteomeXchange repository (PXD002623).

Cell surface display of Yarrowia lipolytica lipase Lip2p using the cell wall protein YlPir1p, its characterization, and application as a whole-cell biocatalyst.

The Yarrowia lipolytica lipase Lip2p was displayed on the yeast cell surface via N-terminal fusion variant using cell wall protein YlPir1p. The hydrolytic activity of the lipase displayed on Y. lipolytica cells reached 11,900 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. The calculated number of recombinant enzyme displayed on the cell surface corresponds to approximately 6 × 10(5) molecules per cell, which is close to the theoretical maximum (2 × 10(6) molecules/cell). Furthermore, the leaking enzyme was presented as three N-glycosylated proteins, one of which corresponds to the whole hybrid protein. Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, the surface-displayed lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. Cell-bound lipase retained 74 % of its original activity at 60 °C for 5 min of incubation, and 83 % of original activity after incubation at 50 °C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1 and 71.0 % methyl esters after 33- and 45-h reactions, respectively.

Immunolocalization of Picornavirus RNA in infected cells with antibodies to Tyr-pUp, the covalent linkage unit between VPg and RNA.

The genomic RNA of picornaviruses is attached to a small protein (VPg) via a covalent bond between a tyrosine and a 5'-terminal uridine phosphate. The same structure is present in potyvirus and calicivirus families. VPgs play a key role in initiation of viral replication by acting as primers for RNA synthesis. The model compound [N(Ac),CO(NHMe)]Tyr-(5'P→O)Up-O-(CH(2))(6)NH(2) (mCLU), mimicking this 'covalent linkage unit' (CLU) and containing Tyr-pUp was synthesized in solution following the phosphoramidite scheme and used to raise antibodies for studying picornavirus infection. The antibodies recognized CLU-containing mengovirus RNA and showed minimal cross-reactivity with RNAs lacking CLU. Immunofluorescence staining of cells infected with a human rhinovirus demonstrated co-localization of the signals from anti-mCLU and from anti-VPg antibodies. Efficient synthesis of mCLU and anti-mCLU antibodies might be of great utility for investigating viral replication and identifying yet unknown viral and cellular CLU-containing RNA-protein complexes.

The N-domain of angiotensin-converting enzyme specifically hydrolyzes the Arg-5-His-6 bond of Alzheimer's Abeta-(1-16) peptide and its isoAsp-7 analogue with different efficiency as evidenced by quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Chronic imbalance between production and degradation of the human amyloid-beta peptide (Abeta) is assumed to play an important role in pathogenesis of Alzheimer's disease (AD). Post-translational modifications of Abeta could influence its interactions with specifically cleaving proteases and, therefore, perturb the Abeta homeostasis. The angiotensin-converting enzyme (ACE) was previously shown to degrade non-modified Abeta in vitro and in cells. In the presented work, we investigated the effect of isomerization of Asp-7, a common non-enzymatic age-related modification found in AD-associated Abeta species, on hydrolysis of Abeta by ACE. Two synthetic peptides corresponding to the Abeta region 1-16 with either Asp or isoAsp residues in position 7 were examined as monomeric soluble substrates for the N- as well as for the C-domain of ACE. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with the (18)O-labeled internal standard approach has allowed us to show that (i) the N-domain of ACE (N-ACE), but not the C-domain, selectively cleaves the Arg-5-His-6 bond in both peptides, and that (ii) N-ACE hydrolyzes the isoAsp-7 analogue more efficiently than the non-modified one. Our results demonstrate a new endopeptidase activity of N-ACE as well as high preference of the domain to recognize and hydrolyze the isomerized Abeta species that were earlier suggested to promote AD pathogenesis. The results suggest the need for further analysis of biological effects of isomerized Abeta and its interaction with ACE in AD pathogenesis.