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High efficiency of hybrid implants based on calcium-magnesium silicate ceramic, diopside, as a carrier of recombinant BMP-2 and xenogenic demineralized bone matrix (DBM) as a scaffold for bone tissue regeneration was demonstrated previously using the model of critical size cranial defects in mice. In order to investigate the possibility of using these implants for growing autologous bone tissue using in vivo bioreactor principle in the patient's own body, effectiveness of ectopic osteogenesis induced by them in intramuscular implantation in mice was studied. At the dose of 7 μg of BMP-2 per implant, dense agglomeration of cells, probably skeletal muscle satellite precursor cells, was observed one week after implantation with areas of intense chondrogenesis, initial stage of indirect osteogenesis, around the implants. After 12 weeks, a dense bone capsule of trabecular structure was formed covered with periosteum and mature bone marrow located in the spaces between the trabeculae. The capsule volume was about 8-10 times the volume of the original implant. There were practically no signs of inflammation and foreign body reaction. Microcomputed tomography data showed significant increase of the relative bone volume, number of trabeculae, and bone tissue density in the group of mice with BMP-2-containing implant in comparison with the group without BMP-2. Considering that DBM can be obtained in practically unlimited quantities with required size and shape, and that BMP-2 is obtained by synthesis in E. coli cells and is relatively inexpensive, further development of the in vivo bioreactor model based on the hybrid implants constructed from BMP-2, diopside, and xenogenic DBM seems promising. [ABSTRACT FROM AUTHOR]

The aim of the study is to synthesize a recombinant protein in Escherichia coli cells that carries the receptor-binding domain (RBD) of Spike protein of SARS CoV-2, with antiviral activity comparable to the activity of RBD obtained in a eukaryotic cells (eRBD). 6His-RBDshort (24.4 kDa) and 6His-RBDlong (33.7 kDa) proteins were expressed in Escherichia coli strain BL21 (DE3). Chromatographic purification of the proteins was carried out on WorkBeads 40 Ni-NTA and WorkBeads 40S sorbents followed by multistage refolding. Enzyme immunoassay was performed using GamP2C5 and GamXRH19 humanized single-domain monoclonal antibodies specific for SARS-CoV-2 Spike protein RBD. Antiviral activity against the SARS-CoV-2 virus was studied using Vero E6 cells. 6His-RBDshort recombinant protein was synthesized in Escherichia coli cells, including the Spike protein RBM (receptor-binding motif) of SARS-CoV-2 virus (330–527 а.а.). Two-stage chromatographic purification of 6His-RBDshort recombinant protein was performed, followed by refolding. Enzyme immunoassay demonstrated effective interaction of 6His-RBDshort recombinant protein with virus neutralizing antibodies, comparable to eRBD. The study of antiviral activity showed inhibition of SARS-CoV-2 virus reproduction after treatment of Vero E6 cells with 6His-RBDshort (45.1%) and eRBD (42.8%) proteins. The 6His-RBDlong recombinant protein obtained in the same work, which included a longer fragment of RBD, did not interact with virus neutralizing antibodies and did not inhibit the replication of the SARS-CoV-2 virus. After conducting additional studies, the developed 6His-RBDshort recombinant protein can be considered a promising drug for therapeutic use as an ACE2 receptor blocker. [ABSTRACT FROM AUTHOR]

Some restriction–modification systems contain two DNA methyltransferases. In the present work, we have classified such systems according to the families of catalytic domains present in the restriction endonucleases and both DNA methyltransferases. Evolution of the restriction–modification systems containing an endonuclease with a NOV_C family domain and two DNA methyltransferases, both with DNA_methylase family domains, was investigated in detail. Phylogenetic tree of DNA methyltransferases from the systems of this class consists of two clades of the same size. Two DNA methyltransferases of each restriction–modification system of this class belong to the different clades. This indicates independent evolution of the two methyltransferases. We detected multiple cross-species horizontal transfers of the systems as a whole, as well as the cases of gene transfer between the systems. [ABSTRACT FROM AUTHOR]

Protein phylogeny is usually reconstructed basing on a multiple alignment of amino acid sequences. One of the problems of such alignments is the presence of regions with different degree of conservation, including those with a questionable quality of the alignment. This problem is often solved by filtering the alignment columns with a special software developed for this purpose. In this work, we investigated various approaches to the phylogeny reconstruction using proteins with two evolutionary domains as examples. The sequences of such proteins are inherently heterogeneous in the degree of conservation due to the presence of both evolutionary domains and linkers between them, as well as the N- and C-termini. It is shown that filtering the alignment columns on average improves the quality of reconstruction only when using the full-length sequences and only for eukaryotic proteins. Limiting the alignment to the evolutionary domains with rejection of less conserved linkers and terminal sequences on average worsened the quality of phylogenetic reconstruction. [ABSTRACT FROM AUTHOR]

Calcium-magnesium silicate ceramics, diopside, is a promising material for use in bone plastics, but until now the possibility of its use as a carrier of recombinant bone morphogenetic protein-2 (BMP-2) has not been studied, as well as the features of reparative osteogenesis mediated by the materials based on diopside with BMP-2. Powder of calcium-magnesium silicate ceramics was obtained by solid-state synthesis using biowaste – rice husks and egg shells – as source components. Main phase of the obtained ceramics was diopside. The obtained particles were irregularly shaped with an average size of about 2.3 μm and ~20% porosity; average pore size was about 24 nm, which allowed the material to be classified as mesoporous. Diopside powder adsorbs more than 150 μg of recombinant BMP-2 per milligram, which exceeds binding capacity of hydroxyapatite, a calcium-phosphate ceramic often used in hybrid implants, by more than 3 times. In vitro release kinetics of BMP-2 was characterized by a burst release in the first 2 days and a sustained release of approximately 0.4 to 0.5% of the loaded protein over the following 7 days. In vivo experiments were performed with a mouse model of cranial defects of critical size with implantation of a suspension of diopside powder with/without BMP-2 in hyaluronic acid incorporated into the disks of demineralized bone matrix with 73-90% volume porosity and macropore size from 50 to 650 μm. Dynamics of neoosteogenesis and bone tissue remodeling was investigated histologically at the time points of 12, 21, 48, and 63 days. Diopside particles were evenly spread in the matrix and caused minimal foreign body reaction. In the presence of BMP-2 by the day 63 significant foci of newly formed bone tissue were formed in the implant pores with bone marrow areas, moreover, large areas of demineralized bone matrix in the implant center and maternal bone at the edges were involved in the remodeling. Diopside could be considered as a promising material for introduction into hybrid implants as an effective carrier of BMP-2. [ABSTRACT FROM AUTHOR]

Peptidoglycan-degrading enzymes are a group of proteins intensively studied as novel antibacterials, with some of them having reached pre-clinical and clinical stages of research. Many peptidoglycan-degrading enzymes have modular organization and consist of a catalytic and a cell wall binding domain. This property has been exploited in enzyme engineering efforts, and many new peptidoglycan-degrading enzymes were generated through domain exchange. However, rational combination of domains from different enzymes is still challenging since relative contribution of every domain to the cumulative bacteriolytic activity is not yet clearly understood. In this work, we investigated the influence of ionic strength and pH on the catalytic efficiency and cell binding of peptidoglycan-degrading enzyme lysostaphin and how this influence is reflected in the lysostaphin bacteriolytic activity. Contrary to generally accepted view, lysostaphin domains are not completely independent and their combination within one protein leads to increased bacteriolytic activity with increasing NaCl concentration, despite both catalysis and cell binding being inhibited by NaCl. This effect is likely mediated by changes in conformation of bacterial cell wall peptidoglycan rather than the physical inter-domain interaction. Key points: • NaCl enhances bacteriolytic activity of lysostaphin but not of its catalytic domain. • Catalytic activity and cell binding of lysostaphin are inhibited by NaCl. • Peptidoglycan conformation likely affects lysostaphin bacteriolytic activity. [ABSTRACT FROM AUTHOR]

Nanopowders of complex oxides with the double perovskite structure La2Ni1 – xFexMnO6 (x = 0, 0.2, 0.4) were synthesized using the glycine-nitrate combustion method. Thin nanostructured layers based on the obtained materials were fabricated and studied by X-ray diffraction, scanning electron microscopy and UV-Vis optical spectroscopy. It was shown that introduction of Fe3+ ions into the La2NiMnO6 crystal structure at low concentration (x = 0.2) leads to an increase in the band gap value. To determine the photosensitivity of La2Ni1 – xFexMnO6 thin layers, Schottky diodes based on them were fabricated and their photovoltaic characteristics were studied in the dark and under standard AM1.5G illumination conditions. It has been found that cation doping with Fe3+ ions at low concentration increases the photosensitivity of La2NiMnO6 thin layer by ~7%. The presented results provide new possibilities for application of inorganic perovskite materials in solar photovoltaics. [ABSTRACT FROM AUTHOR]

Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein–protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections. [ABSTRACT FROM AUTHOR]

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS‑CoV‑2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotogamaritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS‑CoV‑2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS‑CoV‑2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS‑CoV‑2 virus and the Spike protein receptor-binding domain at high titers. [ABSTRACT FROM AUTHOR]

This review is devoted to current approaches to the combined use of BMP-2 with other protein factors to increase osteoinductivity of recently developed materials. The current trend is to create materials with a low BMP-2 concentration that are efficient in osteoinduction and provide continuous protein release. A number of medications have been developed in Russia and South Korea on the basis of BMP-2 synthesized in E. coli cells. The first experience of their use in orthopedics, traumatology, spinal surgery, and dental implantology showed promising results. Also, the possibility of the recombinant BMP-2 use in ophthalmology, and oncology, as well as osteoarthritis treatment, was studied. The databases of PubMed Central (U.S. National Institutes of Health's National Library of Medicine (NIH/NLM)), PubMed (NLM National Center for Biotechnology Information (NCBI)), and e-library (Science Electronic Library) were used to analyze published results. [ABSTRACT FROM AUTHOR]

We have fabricated a series of potassium-doped (K-doped) perovskite solar cells (PSCs). Structural investigations of the developed PSCs have shown that alkai doping affects the perovskite layer structure and morphology with the increase of the crystallite size and surface uniformity. K-doped samples exhibite higher photovoltaic (PV) performance and improved operational stability compared to the control series of undoped PSCs, due to retarded recombination processes. [ABSTRACT FROM AUTHOR]

Here, we give an overview of purification of the recombinant BMP-2 produced in Escherichia coli and its efficacy in bone tissue regeneration as a constituent of different osteoplastic materials. Protein production in this heterologous system and its subsequent purification and refolding resulting in the active protein are described. The efficacy of BMP-2 originated from prokaryotic cells in osteogenesis induction, which is similar to the efficacy of that produced in eukaryotic cells, has been demonstrated in many studies with the variety of carriers and animal models. In this review, the databases PubMed Central (United States National Institutes of Health's National Library of Medicine, NIH/NLM), PubMed (NLM National Center for Biotechnology Information, NCBI), and e-library (Scientific Electronic Library) were used. [ABSTRACT FROM AUTHOR]

Using mouse model of regeneration of critical size cranial defects, we studied combined effect of 1 and 10 μg of BMP-2 of prokaryotic origin and recombinant erythropoietin (Epostim) injected subcutaneously in the area of bone defect in a total dose of 6000 U/kg. Erythropoietin considerably improved quantitative and qualitative characteristics of the bone tissue in the site of implantation when used in combination with BMP-2 in both concentrations. [ABSTRACT FROM AUTHOR]

Microorganisms residing within a biofilm become more tolerant to antibiotics and other types of adverse impact, and biofilm formation by pathogenic bacteria is an important problem of current medicine. Polysaccharides that prevent biofilm formation are among the promising candidates to help tackle this problem. Earlier we demonstrated the ability of a potato polysaccharide galactan to inhibit biofilm formation by a Pseudomonas aeruginosa clinical isolate. Here we investigate the effect of potato galactan on P. aeruginosa biofilms in more detail. Microscopic analysis indicated that the galactan did not interfere with the adhesion of bacterial cells to the substrate but prevented the build-up of bacterial biomass. Moreover, the galactan not only inhibited biofilm formation, but partially destroyed pre-formed biofilms. Presumably, this activity of the galactan was due to the excessive aggregation of bacterial cells, which prohibited the formation and maintenance of proper biofilm architecture, or due to some other mechanisms of biofilm structure remodeling. This led to an unexpected effect, i.e., P. aeruginosa biofilms treated with an antibiotic and the galactan retained more viable bacterial cells compared to biofilms treated with the antibiotic alone. Galactan is the first polysaccharide demonstrated to exert such effect on bacterial biofilms. [ABSTRACT FROM AUTHOR]

The aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (EPO) with additional protein domains: 6His-s-tag-EPO protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) at the N-terminus and HBD-EPO and EPO-HBD proteins containing heparin-binding protein domains (HBD) of the bone morphogenetic protein 2 from Danio rerio at the N- and C-termini, respectively. The commercial preparation Epostim (LLC Pharmapark, Russia) produced by synthesis in Chinese hamster ovary cells was used for comparison. The EPO variant with the C-terminal HBD domain connected by a rigid linker (EPO-HBD) possesses the best properties as compared to HBD-EPO with the reverse domain arrangement. It was ~13 times more active in vitro (i.e., promoted proliferation of human erythroleukemia TF-1 cells) and demonstrated a higher rate of association with the erythropoietin receptor. EPO-HBD also exhibited the greatest binding to the demineralized bone matrix (DBM) and more prolonged release from the DBM among the four proteins studied. Subcutaneous administration of EPO-HBD immobilized on DBM resulted in significantly more pronounced vascularization of surrounding tissues in comparison with the other proteins and DBM alone. Therefore, EPO-HBD displayed better performance with regard to all the investigated parameters than other examined EPO variants, and it seems promising to study the possibility of its medical use. [ABSTRACT FROM AUTHOR]

New recombinant carriers—modular nanotransporters (MNTs)—with N-terminal ligand module to the epidermal growth factor receptor (EGFR) were developed and characterized. Human epidermal growth factor (hEGF) and antibody-like protein Z1907 were used as a ligand module. We demonstrated that MNTs are able to internalize in a receptor-specific manner into the target cancer cells and to accumulate in the target cell nuclei. Conjugation of MNTs with the Auger electron emitter 111In significantly enhanced the cytotoxic effect of 111In on the target cells. It was found that the transfer of EGF from the C-terminus to the N-terminus of the MNT enhanced the proliferation of target cells, whereas the use of Z1907 did not have a similar effect. [ABSTRACT FROM AUTHOR]

Recombinant human erythropoietin (EPO) with additional N-terminal heparin-binding protein domain (HBD) from bone morphogenetic protein 2 was synthesized in Escherichia coli cells. A procedure for HBD-EPO purification and refolding was developed for obtaining highly-purified HBD-EPO. The structure of recombinant HBD-EPO was close to that of the native EPO protein. HBD-EPO contained two disulfide bonds, as shown by MALDI-TOF mass spectrometry. The protein demonstrated in vitro biological activity in the proliferation of human erythroleukemia TF-1 cell test and in vivo activity in animal models. HBD-EPO increased the number of reticulocytes in the blood after subcutaneous injection and displayed local angiogenic activity after subcutaneous implantation of demineralized bone matrix (DBM) discs with immobilized HBD-EPO. We developed a quantitative sandwich ELISA method for measuring HBD-EPO concentration in solution using rabbit polyclonal serum and commercial monoclonal anti-EPO antibodies. Pharmacokinetic properties of HBD-EPO were typical for bacterially produced EPO. Under physiological conditions, HBD-EPO can reversibly bind to DBM, which is often used as an osteoplastic material for treatment of bone pathologies. The data on HBD-EPO binding to DBM and local angiogenic activity of this protein give hope for successful application of HBD-EPO immobilized on DBM in experiments on bone regeneration. [ABSTRACT FROM AUTHOR]

Many proteins need recognition of specific DNA sequences for functioning. The number of recognition sites and their distribution along the DNA might be of biological importance. For example, the number of restriction sites is often reduced in prokaryotic and phage genomes to decrease the probability of DNA cleavage by restriction endonucleases. We call a sequence an exceptional one if its frequency in a genome significantly differs from one predicted by some mathematical model. An exceptional sequence could be either under or over-represented, depending on its frequency in comparison with the predicted one. Exceptional sequences could be considered biologically meaningful, for example, as targets of DNA-binding proteins or as parts of abundant repetitive elements. Several methods to predict frequency of a short sequence in a genome, based on actual frequencies of certain its subsequences, are used. The most popular are methods based on Markov chain models. But any rigorous comparison of the methods has not previously been performed. We compared three methods for the prediction of short sequence frequencies: the maximum-order Markov chain model-based method, the method that uses geometric mean of extended Markovian estimates, and the method that utilizes frequencies of all subse-quences including discontiguous ones. We applied them to restriction sites in complete genomes of 2500 prokaryotic species and demonstrated that the results depend greatly on the method used: lists of 5% of the most under-represented sites differed by up to 50%. The method designed by Burge and coauthors in 1992, which utilizes all subsequences of the sequence, showed a higher precision than the other two methods both on prokaryotic genomes and randomly generated sequences after computational imitation of selective pressure. We propose this method as the first choice for detection of exceptional sequences in prokaryotic genomes. [ABSTRACT FROM AUTHOR]

Recombinant human bone morphogenetic protein-2 with an additional s-tag domain (s-tag-BMP-2) synthesized in E. coli is characterized by higher solubility and activity than the protein without additional s-tag domain, which increases the yield during purification and simplifies protein introduction into the osteoplastic materials. The high osteoinductivity of the demineralized bone matrix with s-tag-BMP-2 was shown on the model of regeneration of cranial defects of a critical size in mice and on the model of implantation of porous titanium matrix into defects of femoral and tibial bones in rabbits. [ABSTRACT FROM AUTHOR]

Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture. [ABSTRACT FROM AUTHOR]

Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains were s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4). The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity of s-tag-BMP-2 in vivo was shown in various experimental animal models. [ABSTRACT FROM AUTHOR]

Osteoinductive characteristics of new osteoplastic materials based on demineralized bone matrix of xenogenic origin with high and controlled degree of purification were studied on the model of regeneration of critical-size cranial defects in rats using modern approaches, including histological analysis, evaluation of morphological parameters of the bone tissue obtained by micro-computed tomography, and estimation of bone tissue growth rate using in vivo fluorochrome label. Demineralized bone matrix and, to a much greater extent, its activated form containing modified recombinant growth factor rhBMP-2 with high content of the dimeric form exhibited osteoinductive activity. [ABSTRACT FROM AUTHOR]

ZrO2/Y2O3 nanoparticles containing 0, 3, and 10 mol % Y2O3 were synthesized under hydrothermal conditions. The structural and optoelectronic properties of the nanoparticles were studied using appropriate measurement techniques. From the synthesized ZrO2–Y2O3 nanostructured systems, electron-conductive thin-film photoelectrodes were fabricated and used to develop perovskite solar cells (PSCs) with the device configuration glass/FTO/ZrO2–Y2O3/CH3NH3PbI3/Spiro-MeO-TAD/Au. Comparative study of the PSC photovoltaic characteristics under 1000 W/m2 illumination (AM1.5G) has shown that the devices based on ZrO2–Y2O3 photoelectrodes exhibit a higher power conversion efficiency than the PSC based on undoped ZrO2 photoelectrodes. [ABSTRACT FROM AUTHOR]

Recombinant bone morphogenetic protein-2 (rhBMP-2) has pronounced osteoinductive properties, as evidenced by the results of experimental and clinical practices. This applies to both the protein produced in eukaryotic cells and the protein synthesized in bacterial cells. In eukaryotic expression systems, production of the protein is extremely low and, consequently, the cost of materials on its basis is very high. Therefore, optimization of heterologous expression systems for rhBMP-2 production represents an important task. In the present work, optimization of codon composition of the rhBMP-2 gene nucleotide sequence and secondary structure of the transcript, as well as strain selection for efficient gene expression, were carried out. The producing strain based on Escherichia coli BL-21(DE3) provides a high level of rhBMP-2 synthesis (about 57% of total cell proteins). Biological activity of rhBMP-2 dimeric forms purified from the obtained producing strain was measured by induction of alkaline phosphatase activity in C2C12 cells. It is comparable with that of commercial rhBMP-2 expressed in E. coli (R&D Systems, United States). Purified rhBMP-2 does not contain impurities of E. coli endotoxin and can be used in experimental studies of osteoinduction in laboratory animals. [ABSTRACT FROM AUTHOR]

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity. [ABSTRACT FROM AUTHOR]

Comparison of structures of homological proteins often helps to understand functionally significant features of these structures. This concerns not only structures of separate protein chains, but also structures of macromolecular complexes. In particular, a comparison of complexes of homologous proteins with DNA is significant for analysis of the recognition of DNA by proteins. We present program LCore for detecting geometrical cores of a family of structures; a geometrical core is a set of amino acid residues and nucleotides that disposed similarly in all structures of the family. We describe the algorithm of the program, its web interface, and an example of its application to analysis of complexes of homeodomains with DNA. [ABSTRACT FROM AUTHOR]

Comparative analysis of structures of complexes of homologous proteins with DNA is important in the analysis of DNA-protein recognition. Alignment is a necessary stage of the analysis. An alignment is a matching of amino acid residues and nucleotides of one complex to residues and nucleotides of the other. Currently, there are no programs available for aligning structures of DNA-protein complexes. We present the program StructAlign, which should fill this gap. The program inputs a pair of complexes of DNA double helix with proteins and outputs an alignment of DNA chains corresponding to the best spatial fit of the protein chains. [ABSTRACT FROM AUTHOR]

Restriction-modification (R-M) systems are able to methylate or cleave DNA depending on methylation status of their recognition site. It allows them to protect bacterial cells from invasion by foreign DNA. Comparative analysis of a large number of available bacterial genomes and methylomes clearly demonstrates that the role of R-M systems in bacteria is wider than only defense. R-M systems maintain heterogeneity of a bacterial population and are involved in adaptation of bacteria to change in their environmental conditions. R-M systems can be essential for host colonization by pathogenic bacteria. Phase variation and intragenomic recombinations are sources of the fast evolution of the specificity of R-M systems. This review focuses on the influence of R-M systems on evolution and ecology of prokaryotes. [ABSTRACT FROM AUTHOR]

A synthetic gene encoding dispersin B of Aggregatibacter actinomycetemcomitans was cloned and expressed in Escherichia coli cells. Procedure for purification of recombinant dispersin B was developed, and its in vitro activity was determined. The enzyme was used in experiments on disruption of the biofilms formed by various microorganisms. It exhibited high activity against Staphylococcus epidermidis biofilms. The biofilms formed by Burkholderia cenocepacia and Achromobacter xylosoxidans were more resistant to the recombinant enzyme. [ABSTRACT FROM AUTHOR]

The content of multipotent stromal cells (MSC) in the bone marrow and efficiency of their cloning (ECF-MSC) increased by 3 times 1 day after administration of complex S. typhimurium antigens to CBA mice, while the relative content of alkaline phosphatase-positive MSC colonies (marker of osteogenesis; P colonies) decreased from 14% (control) to 3%. After administration of the complex S. typhimurium antigens to CBA mice 3 h after (or 3 h before) curettage or treatment with morphogenetic protein (BMP-2), the content of MSC and ECF-MSC decreased on the next day by ~3 times in comparison with animals receiving antigens alone and approached the control level. The relative content of P colonies increased to 20 and 35%, respectively, in comparison with animals receiving antigens (3%), but was signifi cantly lower than after curettage (34%) or BMP-2 (42%) administration. Expression of IL-1β, IL-6, IL-12, TNF-α, and IFN-γ genes in the primary cultures of stromal bone marrow cells induced by antigen administration was suppressed, while the concentrations of IL-12 and TNF-α in the culture medium sharply decreased after antigen treatment in combination with curettage or BMP-2 administration. Administration of complex S. typhimurium antigens after pretreatment with BMP-2 (3 h before) was associated with a decrease in serum levels of IL-2, IFN-γ, IL-12, and TNF-α in mice receiving BMP-2+ S. typhimurium group 4 h after treatment in comparison with the animals receiving only S. typhimurium antigens alone by 1.9, 4.4, 1.5, and 6 times, respectively, i.e. to normal level or below it, while the concentration of IL-10 increased by almost 2 times, which probably reflected anti-inflammatory properties of BMP-2. These data probably attest to competitive relations between osteogenesis and immune response at the level of MSC. [ABSTRACT FROM AUTHOR]

A new modular nanotransporter (MNT) for the delivery of anticancer agents into the nuclei of cells with folate receptor overexpression was created. An effective method for acceding labeling of this MNT with Auger electron emitter In has been developed. A significant therapeutic effect was observed after a single intratumoral injection of the new In-labeled MNT to mice grafted with human cervical carcinoma characterized by folate receptor overexpression. [ABSTRACT FROM AUTHOR]

Test system ELISA-BMP-2 is developed for measuring recombinant human bone morphogenetic protein-2 in human and laboratory animal serum and plasma by sandwich ELISA. The test system has been used for studies of the kinetics of bone morphogenetic protein-2 release from collagen carrier in the presence of plasma proteins. [ABSTRACT FROM AUTHOR]

We have compared (LnZr)ZrO (Ln = Nd, Sm) pyrochlore-like solid solutions with interstitial oxide ion conduction and Ln(ZrLn)O (Ln = Nd, Sm) pyrochlore-like solid solutions with vacancy-mediated oxide ion conduction in the symmetric systems NdO-ZrO (NdZrO) and SmO-ZrO (SmZrO). We have studied their structure, microstructure, and transport properties and determined the excess oxygen content of the (SmZr)ZrO ( x = 0.2) material using thermal analysis and mass spectrometry in a reducing atmosphere (H/Ar-He). The LnZrO (Ln = Nd, Sm) solid solutions have almost identical maximum oxygen vacancy and interstitial conductivities: (3-4) × 10 S/cm at 750°C. The lower oxygen vacancy conductivity of the Ln(ZrLn)O (Ln = Nd, Sm; 0 < x ≤ 0.3) solid solutions is due to the sharp decrease in it as a result of defect association processes, whereas the interstitial oxide ion conductivity of the (LnZr)ZrO (Ln = Nd, Sm; 0.2 ≤ x < 0.48) pyrochlore-like solid solutions is essentially constant in a broad range of LnO concentrations. [ABSTRACT FROM AUTHOR]

The release kinetics of recombinant human bone morphogenic factor 2 (rhBMP-2) from collagen hydrogel in the presence of human blood plasma have been studied. The expulsion of rhBMP-2 from the collagen-BMP-2 complex by the competitive adhesion of collagen-binding proteins penetrating from plasma was firstly recognized. It was experimentally proven that that blood plasma fibronectin is the main collagen-binding protein, which is responsible for the controlled release of rhBMP-2. As a result, a new collagen hydrogel with the incorporation of fibronectin was created which retained rhBMP-2 for a twice longer period as compared to the ordinary collagen hydrogel. A distinctive feature of this new collagen-fibronectin matrix is the slow release of rhBMP-2 in the first three days which allows for the avoiding of adverse effects in clinics caused by the rapid release of large amounts of rhBMP-2 from collagen hydrogel. [ABSTRACT FROM AUTHOR]

We studied the effect of BMP-2 added to the culture medium on osteogenic and proliferative properties of multipotent stromal cells (MSC) and on the expression of cytokine genes induced by immunization of experimental animals with bacterial antigens. It is shown that the presence of BMP-2 in the culture medium stimulates proliferation of bone marrow MSC and especially spleen MSC (which was seen from enlargement of MSC colonies); improves the efficiency of MSC cloning; increases osteogenic activity of mouse bone marrow MSC; induces osteogenic differentiation of splenic MSC (osteogenesis is normally not observed in the spleen); reduces the number of macrophages in cultures; inhibits synthesis of mRNA for proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) that typically occurs in cultures of the bone marrow and spleen from animals immunized with S. typhimurium or group A streptococcus antigens. Bearing in mind that proinflammatory cytokines negatively affect osteogenic activity of the bone marrow, we can hypothesize that BMP-2 not only stimulates osteogenesis, but also provides optimal conditions for its realization by suppressing the expression of genes encoding these cytokines. [ABSTRACT FROM AUTHOR]

We have studied terbium substitution for ytterbium in (YbTbCa)TiO ( x = 0.1, 0.2, 0.3, 0.4) pyrochlore solid solutions synthesized through coprecipitation followed by firing at 1550°C. The results indicate that only a small amount of terbium (less than 10%) can be incorporated into the pyrochlore structure of (YbCa)TiO because of the large difference in ionic radius between the terbium and ytterbium cations: Δ r = r(Tb) − r(Yb) = 0.055 Å. The oxygen ion conductivity of the (YbTbCa)TiO solid solutions has been determined by impedance spectroscopy in air in the temperature range 300 to 900°C. At high temperatures ( t > 640°C), their bulk conductivity was essentially independent of the Yb/Tb ratio. The observed decrease in density and microstructural changes were insignificant. At relatively low temperatures ( t < 640°C), the bulk conductivity decreased slightly, and the decrease depended little on terbium concentration. [ABSTRACT FROM AUTHOR]

The genes of ESAT-6, CFP-10 and Ag85A secreted antigens of Mycobacterium tuberculosis were cloned in Escherichia coli. Fusion proteins Esat6-CBD, Ag85A-CBD and CFP10-CBD with thermostable cellulose binding domain (CBD) of Anaerocellum thermophilum providing high affinity to cellulose were constructed. Content of the fused proteins in overproducing strains was 20% of total protein for CFP10-CBD and 15% for both Esat6-CBD and Ag85A-CBD. Immobilization of the fusion proteins on cellulose is a one-stage process that provides simultaneous antigen purification and absorption on cellulose. The proteins retain their antigenic properties and can be used as the components for subunit tuberculosis vaccine production or in diagnostic kits. [ABSTRACT FROM AUTHOR]

Low meadow-rue ( Thalictrum minus L.) antioxidant complex was studied in cell extracts and culture medium. Its activity was expressed as total polyphenol content in ferulic acid equivalents. In these model systems (cell extracts and culture medium) the inhibition of lipid oxidation and diphenylpicrylhydrazine reduction (EC = 12-15 μg/ml) were observed. At the phenolic compound concentration of 8-15 μg/ml, the reducing capacity of cell extracts was equivalent to 1.5 mM ascorbic acid. At the same time, berberine, a major alkaloid synthesized by the culture, manifested a low antioxidant activity. The analysis of phenolic acid composition in low meadow-rue showed that one of the main components of its antioxidant system were caffeic, gallic, chlorogenic, and ferulic acids. [ABSTRACT FROM AUTHOR]

Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein-protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein-2 (rhBMP-2) is an osteoinductive protein factor which plays a dominant role in growth and regeneration of bone tissue. In clinical practice, bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials have not been produced yet. The fragment of the bmp-2 gene encoding a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on the basis of E. coli BL21(DE3). The level of rhBMP-2 production was approximately 25% of total cell protein. Biologically active dimeric form of rhBMP-2 was obtained as a result of isolation and purification of protein from inclusion bodies with subsequent refolding. The obtained rhBMP-2 sample contained more than 80% of the dimeric form and was able to interact with specific antibodies to BMP-2. Biological activity of the rhBMP-2 samples was verified in in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On a model of ectopic osteogenesis, it was shown that the obtained rhBMP-2 exhibited biological activity in vivo, causing tissue calcification in the site of injection. The protein activity in vivo depends on the way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The elaborated method of rhBMP-2 isolation and purification results in an increased common protein yield and ensures the maintenance of biologically active dimeric form compared to the analogues described in the literature. [ABSTRACT FROM AUTHOR]

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) has a long N-terminal region (1-71 residues) preceding the sequence with conservative motifs, which are characteristic for all DNA methyltrans-ferases of such kind. The presence of this region provides M.SsoII capability to act as a transcription regulator in SsoII restriction-modification system. To perform its regulatory function, M.SsoII binds specifically to a 15-mer inverted repeat in the promoter region of SsoII restriction-modification system genes. In the present work, properties of the protein Δ(72-379)M.Ecl18kI are studied, which is a deletion mutant of the SsoII-like DNA-methyltransferase M.Ecl18kI and is homologous to M.SsoII N-terminal region. Δ(72-379)M.Ecl18kI capability to bind specifically a DNA duplex containing the regulatory site is demonstrated. However, such a binding takes place only in the presence of high protein excess relative to DNA, which could indicate an altered structure in the deletion mutant in comparison with the full-length M.SsoII. Circular dichroism spectroscopy demonstrated that Δ(72-379)M.Ecl18kI has a strongly pronounced secondary structure and contains 32% α-helices and 20% β-strands. Amino acid sequences alignment of M.SsoII N-terminal region and transcription factors of known spatial structure is made. An assumption is made how α-helices and β-strands are arranged in M.SsoII N-terminal region. [ABSTRACT FROM AUTHOR]

This review summarizes the recently published data on the molecular mechanisms of Chlamydiae-host cell interaction, first of all, on chlamydial effector proteins. Such proteins, along with type III transport system proteins, which transfer many effector proteins into the host cytoplasm, are attractive targets for drug therapy of chlamydial infections. The majority of the data concerns two species, Chlamydia trachomatis and Chlamydophila pneumoniae. The C. trachomatis protein TARP, which is presynthesized in elementary bodies, plays an essential role in the initial stages of infection. The pathogen proteins that are involved in the next stage, which is the intracellular inclusion traffic to the centrosome, are C. trachomatis CT229 and C. pneumoniae Cpn0585, which interact with cell Rab GTPases. In C. trachomatis, IncA plays a key role in the fusion of chlamydial inclusions, CT847 modulates the life cycle of the host cell, and LDA3 is essential for the acquisition of nutrients. The protease CPAF and the inclusion membrane proteins IncG and CADD are involved in suppressing apoptosis of infected cells. The proteases CPAF and CT441 and the deubiquitinating protein ChlaDub1 help the pathogen to evade the immune response. [ABSTRACT FROM AUTHOR]

The fusion gene, which consists of fragments that code the dextran-binding domain of dextraninvertase of Leuconostoc mesenteroides, subsp. mesenteroides (DBD) and human recombinant interferon-β (INF-β) with the nucleotide sequence that codes the recognition site of hydrolysis of human enteropeptidase (DDDDK) between them, was cloned in Escherichia coli cells. The effective bacterial strain that producing the chimeric DBD-INF-β protein, which consists of the IFN-β sequence, a spacer that contains ten alternate glycine and serine residues, a human enteropeptidase recognition site, and a dextran-binding domain, was constructed. Free recombinant human interferon-β was obtained as a result of the treatment of chimeric DBD-IFN-β protein and immobilized on G-25 sephadex with human enteropeptidase. The ability of free and immobilized proteins to protect human cells from viral infection was demonstrated. The developed approach can be used to produce recombinant proteins with different biological activity. Thus, these proteins can be used as a basis for the construction of new immunomodulatory and antiviral drugs, growth factors, anticancer drugs, etc. [ABSTRACT FROM AUTHOR]

DNA fragments that encode two collagen-binding decapeptides from human von Willebrand factors vWF-H1 and vWF-H2 were cloned in Escherichia coli cells. The effective chimeric proteins vWF(H1)-CBD and vWF(H2)-CBD, which produce strains that contain corresponding decapeptide sequences, Gly-Ser spacer, and a cellulose-binding domain (CBD) from Anaerocellum thermophilum were constructed. Highly purified samples of vWF(H1)-CBD and vWF(H2)-CBD proteins were obtained using the one-stage purification method on cellulose and their ability to bind collagen was studied. The obtained constructions are planned to for use in the development of recombinant collagen-binding proteins with different biological activity, which will be used for the further development of a new generation of products and materials for medical purposes, e.g., various kids of implants and coatings. [ABSTRACT FROM AUTHOR]

ETS proteins are a family of widespread transcription factors that regulate the expression of many animal genes. Structurally, ETS proteins are characterized by a conserved DNA-binding ETS domain, which recognizes DNA sequences containing the trinucleotide GGA. The structural features of ETS domain-DNA complexes were analyzed, and conserved contacts important in terms of interaction stability and specificity were identified. The analysis revealed nine conserved hydrogen bonds with oxygens of DNA backbone phosphates, two bidentate hydrogen bonds with DNA major groove atoms, one conserved hydrophobic cluster located on the protein-DNA interface and important for binding site recognition, and 12 conserved water molecules presumably mediating the ETS domain-DNA interaction. The results are represented in specialized data bank of protein-DNA complexes (NPIDB). [ABSTRACT FROM AUTHOR]

The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Δ(72–379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyl-transferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating. [ABSTRACT FROM AUTHOR]

The goal of this work was to elucidate the mechanism of the direct interaction of bacteria cells with tumor necrosis factor (TNF-α, cytokine). It was shown previously that this interaction facilitated the activation of the bacterial growth and recultivation of noncultivated forms in vitro and in vivo. Mice deficient in genes that encode eukaryotic TNF-α receptors were infected with salmonella cells. When exogenous TNF-α was introduced into the suspension of the infected cells, the infection process (seeding of bacteria from spleens) started one day earlier in both the knockout and control mice, in comparison with that in the absence of cytokine. Bacteria are therefore able to interact with cytokine, even in the absence of eukaryotic receptors. The specificity of the bacteria-cytokine interaction was demonstrated and bacterial protein EF-Tu, which mediates the direct interaction of bacteria with cytokine, was identified using the method of immobilization of the recombinant protein TNF-α-spacer-CBD on cellulose. [ABSTRACT FROM AUTHOR]

Ho2Ti2O7 and LnYTi2O7 (Ln = Dy, Ho) pyrochlores have been synthesized using hydroxide coprecipitation, mechanical activation, and firing at 1600°C. The bulk and grain-boundary components of their conductivity have been determined for the first time by impedance spectroscopy. The 740°C bulk conductivity of Ho2Ti2O7 is 4 × 10−4 S/cm, and that of HoYTi2O7 is 1 × 10−3 S/cm, with activation energies E a = 1.01 and 1.17 eV, respectively, suggesting that these materials are new oxygen-ion conductors. The bulk conductivity of DyYTi2O7 (3 × 10−4 S/cm at 740°C, E a = 1.09 eV) is almost one order of magnitude lower than that of HoYTi2O7 (1 × 10−3 S/cm at 740°C, E a = 1.17 eV). [ABSTRACT FROM AUTHOR]