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.

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.

This paper deals with the development of polymeric nanocarriers based on amphiphilic copolymers of N-vinyl-2-pyrrolidone and acrylic acid of various molecular weights synthesized through the AIBN-initiated radical copolymerization of N-vinyl-2-pyrrolidone and acrylic acid in the presence of n-octadecyl mercaptan. The structure of the copolymers is characterized by H-1 NMR, C-13 NMR, IR and MALDI-TOF MS spectroscopy. It is shown that the length of the hydrophilic block defines the size of the nanoaggregates while impacting the steric stabilization efficiency and the probability of the interchain hydrogen bond formation. The hydrogen bonds formation between the residues of N-vinyl-2-pyrrolidone and acrylic acid is in agreement with the reduction of the zeta-potential of the nanoaggregates and the critical aggregation concentrations upon increasing the molecular weight. The presence of acrylic acid residues in the amphiphilic macromolecules leads to a higher affinity for doxorubicin and slow partial release of doxorubicin bonded with the aggregates' corona, which is helpful for reducing its cardiac toxicity. Nanoaggregates with a paclitaxel-loaded hydrophobic core are obtained, showing the possibility of dual loading. The amphiphilic copolymers of N-vinyl-2-pyrrolidone and acrylic acid containing an n-octadecyl thio end group are thus promising candidates for combination cancer therapy with immobilized anti-cancer drugs, paclitaxel, and doxorubicin.

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.

AIM:To evaluate the effect of hypernatremia on the organization of blood plasma high density lipoproteins (HDL) in goldfish; to compare the state of hypernatremia in fish and humans; to assess the possible risks and consequences of the effect of hypernatremia on human plasma lipoproteins.METHODS:The fish were acclimated for 20 days at a critical salinity of 11.5 g/L; after that the salt water was gradually "desalinated". The concentration of Na+ and the content of total water were determined in tissues, cells, and body fluids. The HDL organization was assessed by the number of apolipoprotein molecules per particle. The methods of flame spectrophotometry, electrophoresis and MALDI were used.RESULTS:In fresh water, the state of normonatremia was maintained in the fish body; at critical water salinity, the state of hypernatremia. Against the background of hypernatremia, the initial signs of muscle and erythrocyte dehydration appeared in fish, the total water content in the plasma did not change, and HDL disintegrated into small particles, which, upon restoration of normonatremia, were combined into the original large forms.CONCLUSION:In goldfish at the state of normonatremia, large forms of HDL are stable while at the state of hypernatremia, the small forms of HDL are stable. Under conditions of a hypertonic environment and plasma hypernatremia, the breakdown of HDL prevents the loss of water from the fish organism and reduces the threat of their dehydration. Human hypernatremia is characterized by plasma sodium levels comparable to that in goldfish, however accompanied by life-threatening metabolic changes. The results of this study may be useful for assessing the risks of HDL breakdown at hypernatremia and for the development of protocols for the treatment of pathological conditions in humans (Fig. 4, Ref. 45).

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.

Bacteriolytic enzymes are promising antimicrobial agents for developing new-generation drugs. Recently, we have isolated a β-lytic protease (BlpLc) from the culture liquid of Lysobacter capsici VKM B-2533T. This BlpLc possesses a valuable property, not described for β-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.

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.

In order to develop a new approach to MALDI-TOF-mass-spectrometry-based serum proteome profiling without any fractionation, several cancer and non-cancer human sera were treated by different concentrations of trypsin. Serum albumin digestion required much more trypsin in cancer sera than in non-cancer sera, the observation being explained by increased serum level of protease inhibitors in cancer. Moderate concentrations of externally introduced trypsin unexpectedly lead to digestion of proteins different from the serum albumin. Of them, in cancer sera an acute-phase serum amyloid A (SAA) was shown to be digested. Further, using a synthetic SAA peptide with a C-terminal amide as a standard for quantitative MALDI-TOF-MS analysis, it was found that this protein was digested substantially faster in the cancer serum than in the normal serum. Thus, the trypsin being introduced into cancer serum possessed a modified activity. The change in trypsin behavior should be explained by its binding with alpha-2-macroglobulin.

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-(CH2)6NH2 (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.

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.