Search

Purpose: To generate and evaluate a modular recombinant transporter (MRT) for targeting 211 At to cancer cells overexpressing the epidermal growth factor receptor (EGFR).Methods and Materials: The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[211 At]astato-5-guanidinomethylbenzoate (SAGMB), its 125 I analogue SGMIB, or with 131 I using Iodogen. Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines.Results: The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [211 At]astatide for all three cell lines.Conclusion: The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted alpha-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this 211 At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor. [ABSTRACT FROM AUTHOR]

A new dual-functional implant based on gellan-xanthan hydrogel with calcium-magnesium silicate ceramic diopside and recombinant lysostaphin and bone morphogenetic protein 2 (BMP-2)-ray is developed. In this composite, BMP-2 is immobilized on microparticles of diopside while lysostaphin is mixed directly into the hydrogel, providing sustained release of BMP-2 to allow gradual bone formation and rapid release of lysostaphin to eliminate infection immediately after implantation. Introduction of diopside of up to 3% (w/v) has a negligible effect on the mechanical properties of the hydrogel but provides a high sorption capacity for BMP-2. The hydrogels show good biocompatibility and antibacterial activity. Lysostaphin released from the implants over a 3 h period efficiently kills planktonic cells and completely destroys 24 h pre-formed biofilms of Staphylococcus aureus. Furthermore, in vivo experiments in a mouse model of critically-sized cranial defects infected with S. aureus show a complete lack of osteogenesis when implants contain only BMP-2, whereas, in the presence of lysostaphin, complete closure of the defect with newly formed mineralized bone tissue is observed. Thus, the new implantable gellan-xanthan hydrogel with diopside and recombinant lysostaphin and BMP-2 shows both osteogenic and antibacterial properties and represents a promising material for the treatment and/or prevention of osteomyelitis after bone trauma., (© 2024 Wiley‐VCH GmbH.)

The development of new and effective antibacterials for pharmaceutical or cosmetic skin care that have a low potential for the emergence and expansion of bacterial resistance is of high demand in scientific and applied research. Great hopes are placed on alternative agents such as bactericidal peptidoglycan hydrolases, depolymerases, etc. Enzybiotic-based preparations are being studied for the treatment of various infections and, among others, can be used as topical formulations and dressings with protein-polysaccharide complexes. Here, we investigate the antibiofilm properties of a novel enzybiotic cocktail of phage endolysin LysSi3 and bacteriocin lysostaphin, formulated in the alginate gel matrix and its ability to control the opportunistic skin-colonizing bacteria Staphylococcus aureus , Pseudomonas aeruginosa , and Klebsiella pneumoniae , as well as mixed-species biofilms. Our results propose that the application of SiL-gel affects different components of biofilm extracellular polymeric substances, disrupts the matrix, and eliminates the bacteria embedded in it. This composition is highly effective against biofilms composed of Gram-negative and Gram-positive species and does not possess significant cytotoxic effects. Our data form the basis for the development of antibacterial skin care products with a gentle but effective mode of action.

Lysostaphin is a zinc-dependent endopeptidase that is effective against both antibiotic-sensitive and antibiotic-resistant strains of Staphylococcus aureus. Lysostaphin is typically purified on cation-exchange or metal-chelate affinity resins, and there are data indicating potential influence of the chromatographic resin on the lysostaphin activity. In this study, we systematically investigated the impact of the resin used to purify the recombinant lysostaphin on its activity. To this end, recombinant lysostaphin with an additional histidine tag at the C-terminus was purified using a cation-exchange resin, three types of nickel-chelate resins with different strength of metal ion binding, or a zinc-chelate resin. Lysostaphin samples purified on the cation-exchange resin (WorkBeads 40S), the nickel-chelate resin with a strong nickel ion binding (WorkBeads NiMAC), and the zinc-chelate resin (WorkBeads NTA with immobilized zinc ions) had equal activity. On the contrary, the activity of lysostaphin preparations purified on nickel-chelate resins with medium (WorkBeads Ni-NTA) and relatively weak (WorkBeads Ni-IDA) nickel ion binding was significantly reduced. The decrease in activity can be explained by the interaction of lysostaphin with the nickel ions leached from the resin and is caused by either the exchange of the zinc ion in the lysostaphin active center with a nickel ion from the resin, or binding of an additional ion that inhibits the enzymatic activity. Removal of the metal ions from the active site of lysostaphin and subsequent incorporation of the native zinc ions lead to complete restoration of the activity of the enzyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Endopeptidases IdeS and IdeZ (streptococcal virulence factors that specifically cleave IgG heavy chains) are of particular interest because of their potential use in biotechnology, medicine, and veterinary. Genes encoding these enzymes were cloned and expressed in Escherichia coli heterologous expression system (ideS was cloned from a Streptococcus pyogenes collection strain; ideZ from Streptococcus zooepidemicus was synthesized). The 6His-tag was introduced into the amino acid sequence of each endopeptidase, and IdeS and IdeZ were purified by metal affinity chromatography to an apparent homogeneity (according to polyacrylamide gel electrophoresis). Purified enzymes were active against human and animal IgGs; their specificity toward human IgGs was confirmed by polyacrylamide gel electrophoresis. Recombinant IdeZ was used for immunological analysis of equine strangles infection (diagnostics and determination of the titer of specific antibodies in blood). Hence, IdeZ can be used in veterinary and sanitary microbiology to diagnose infections caused by Streptococcus equi and S. zooepidemicus in addition to its application in medicine and biotechnology.

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.

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 Thermotoga maritima, 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.

Antibacterial lysins are enzymes that hydrolyze bacterial peptidoglycan, which results in the rapid death of bacterial cells due to osmotic lysis. Lysostaphin is one of the most potent and well-studied lysins active against important nosocomial pathogen Staphylococcus aureus . Similarly to most other lysins, lysostaphin is composed of enzymatic and peptidoglycan-binding domains, and both domains influence its antibacterial activity. It is thus desirable to be able to study the activity of both domains independently. Lysostaphin cleaves pentaglycine cross-bridges within the staphylococcal peptidoglycan. Here, we report the protocol to study the catalytic activity of lysostaphin on the isolated pentaglycine peptide that is based on the chromogenic reaction of peptide amino groups with ninhydrin. Unlike previously reported assays, this protocol does not require in-house chemical synthesis or specialized equipment and can be readily performed in most laboratories. We demonstrate the use of this protocol to study the effect of EDTA treatment on the lysostaphin enzymatic activity. We further used this protocol to determine the catalytic efficiency of lysostaphin on the isolated pentaglycine and compared it to the apparent catalytic efficiency on the whole staphylococcal cells. These results highlight the relative impact of enzymatic and peptidoglycan-binding domains of lysostaphin on its bacteriolytic activity.

The spread of bacterial strains resistant to commonly used antibiotics urges the development of novel antibacterial compounds. Ideally, these novel antimicrobials should be less prone to the development of resistance. Peptidoglycan-degrading enzymes are a promising class of compounds with a fundamentally different mode of action compared to traditionally used antibiotics. The difference in the mechanism of action implies differences both in the mechanisms of resistance and the chances of its emergence. To critically assess the potential of resistance development to peptidoglycan-degrading enzymes, we review the available evidence for the development of resistance to these enzymes in vitro , along with the known mechanisms of resistance to lysozyme, bacteriocins, autolysins, and phage endolysins. We conclude that genetic determinants of resistance to peptidoglycan-degrading enzymes are unlikely to readily emerge de novo. However, resistance to these enzymes would probably spread by the horizontal transfer between intrinsically resistant and susceptible species. Finally, we speculate that the higher cost of the therapeutics based on peptidoglycan degrading enzymes compared to classical antibiotics might result in less misuse, which in turn would lead to lower selective pressure, making these antibacterials less prone to resistance development.

Antibacterial lysins are promising proteins that are active against both antibiotic-susceptible and antibiotic-resistant bacterial strains. However, a major limitation of antibacterial lysins is their fast elimination from systemic circulation. PEGylation increases the plasma half-life of lysins but renders them inactive. Here we report the construction of a fusion protein of lysostaphin, a potent anti-staphylococcal lysin, and an albumin-binding domain from streptococcal protein G. The resulting fusion protein was less active than the parent enzyme lysostaphin, but it still retained significant antibacterial activity even when bound to serum albumin. The terminal half-life of the fusion protein in rats was five-fold greater than that of lysostaphin (7.4 vs. 1.5 h), and the area under the curve increased more than 115 times. Most importantly, this increase in systemic circulation time compensated for the decrease in activity. The plasma from rats that received an injection of the fusion protein retained bactericidal activity for up to 7 h, while plasma from rats that received plain lysostaphin lacked any detectable activity after 4 h. To the best of our knowledge, this is the first report of an antibacterial lysin with both improved pharmacokinetic parameters and prolonged bactericidal activity in the systemic circulation.

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.

The increasing prevalence of antibiotic-resistant strains of pathogenic bacteria is a major healthcare problem. Antibacterial lysins are enzymes that cleave the peptidoglycan of the bacterial cell wall. These proteins hold potential as a supplement or an alternative to traditional antibiotics since they are active against antibiotic resistant strains. However, antibacterial lysins are rapidly eliminated from the systemic circulation, which limits their application. Dimerization of an anti-pneumococcal lysin Cpl-1 has been demonstrated to decrease the clearance rate of this protein in mice. In the present work, we constructed a dimer of an anti-staphylococcal lysin lysostaphin by fusing it with an anti-parallel α-helical dimerization domain. Lysostaphin dimer had a more favorable pharmacokinetic profile with increased terminal half-life and area under the curve (AUC) values compared to monomeric lysostaphin. However, the staphylolytic activity of dimerized lysostaphin was decreased. This decrease in activity was likely caused by the dimerization; since the catalytic efficacy of lysostaphin dimer towards pentaglycine peptide was unaltered. Our results demonstrate that, although dimerization is indeed beneficial for the pharmacokinetics of antibacterial lysins, this approach might not be suitable for all lysins, as it can negatively affect the lysin activity.

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.

We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C-terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N-terminal domain. rsMutL demonstrated the highest activity in the presence of Mn2+. The extent of plasmid DNA hydrolysis declined in the row Mn2+ > Co2+ > Mg2+ > Cd2+; Ni 2+ and Ca2+ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn2+ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa.

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.

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.

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.

New innovative vaccines are highly needed to combat the global threat posed by tuberculosis. Efficient components-antigens and adjuvants-are crucial for development of modern recombinant TB vaccines. This study describes a new vaccine (GamTBvac) consisting of two mycobacterial antigen fusions (Ag85A and ESAT6-CFP10)-with dextran-binding domain immobilized on dextran and mixed with an adjuvant consisting of DEAE-dextran core, and with CpG oligodeoxynucleotides (TLR9 agonists). GamTBvac and its components were assessed for immunogenicity and protective efficacy in GamTBvac-prime/boost and BCG-prime/ GamTBvac-boost in murine and guinea pig TB models. Results show that in both infectious models, GamTBvac has a strong immunogenicity and significant protective effect against Mycobacterium tuberculosis strain H37Rv under aerosol and intravenous challenges. GamTBvac showed a particularly strong protective effect as a BCG booster vaccine.

Background: The control of genome stability is relevant for the worldwide BCG vaccine preventing the acute forms of childhood tuberculosis. BCG sub-strains whole genome comparative analysis and revealing the triggers of sub-strains transition were the purpose of our investigation., Results: Whole genome sequencing of three BCG Russia seed lots (1963, 1982, 2006 years) confirmed the stability of vaccine sub-strain genome. Comparative analysis of three Mycobacteruim bovis and nine M. bovis BCG genomes shown that differences between "early" and "late" sub-strains BCG genomes were associated with specific prophage profiles. Several prophages common to all BCG genomes included ORFs which were homologues to Caudovirales. Surprisingly very different prophage profiles characterized BCG Tice and BCG Montreal genomes. These prophages contained ORFs which were homologues to Herpesviruses. Phylogeny of strains cohort based on genome maps restriction analysis and whole genomes sequence data were in agreement with prophage profiles. Pair-wise alignment of unique BCG Tice and BCG Montreal prophage sequences and BCG Russia 368 genome demonstrated only similarity of fragmetary sequences that suggested the contribution of prophages in genome mosaic structure formation., Conclusions: Control of the extended sequences is important for genome with mosaic structure. Prophage search tools are effective instruments in this analysis.

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.

Background: E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV., Methods: Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model., Results: All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection., Conclusions: Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference "Tick-E-Vac" vaccine.

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 Sigma-Aldrich (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.

The investigation of the bacterial populations' heterogeneity contributes to the control of natural foci, causative agents of nosocomial infections, to the analysis of the microbial evolution. Multilocus sequence typing (MLST) was employed for the analysis of the diversity and features of the distribution of polyhostal ubiquitous microorganisms of the genera Burkholderia, Leptospira, and Listeria, which belong to three bacterial phyla: Proteobacteria, Spirochaetes, and Firmicutes. According to the bacterial samples analysis microbial genotypes prevalent and unique to Russia were identified; their occurrence in different Federal Regions was investigated; their similarity with global spread genotypes was appreciated. Obtained results allowed identifying common regularities of the selection of the microorganisms capable to cause the diseases of human and animals. The formation of genotypes that are most pathogenic for the host was demonstrated for all groups of bacteria. Leptospira spp. and Listeria monocytogenes strains with these genotypes have been circulating for a long time, being supported by natural foci. The formation of a wide variety of genotypes with different pathogenicity was demonstrated in the local geographic areas. In Russia, the zonal difference in all three groups of bacteria is most clearly traced to the Far Eastern Federal Region. The results are thought to contribute to analyzing the factors of selection and the phylogeny of the taxa under study.

Comparative analysis of serum cytokine profiles of CBA mice was carried out 1, 5, 24, and 48 h after intraperitoneal injection of killed culture of different streptococcus A types. The production of cytokines in response to different streptococcus types varied. The highest level was recorded in response to types 1M and 3T+M, more often detected in invasive streptococcal infection. The highest levels of IL-2 were recorded in response to 1M (47-fold increase in comparison with the control) and 3T+M streptococcus types (more than 10-fold increase). Injections of these types also led to an increase of IFN-γ level (15.6 and 11.3 times, respectively). The level of TNF-α increased less (3.6 times in response to 3T+M and 2.6 times in response to 1M type). The levels of IL-5, IL-10, and IL-12 increased 2-3-fold. Injections of 1T and 5M types led to just a 2-fold increase of cytokine levels. These data indicated induction of the immune response trend by mainly Th1 or mixed Th1/Th2 pattern in response to group A streptococcus antigens.

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 significantly 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.

Background and Aim: The order Burkholderiales became more abundant in the healthcare units since the late 1970s; it is especially dangerous for intensive care unit patients and patients with chronic lung diseases. The goal of this investigation was to reveal the real variability of the order Burkholderiales representatives and to estimate their phylogenetic relationships., Methods: 16S rDNA and genes of the Burkholderia cenocepacia complex (Bcc) Multi Locus Sequence Typing (MLST) scheme were used for the bacteria detection., Results: . A huge diversity of genome size and organization was revealed in the order Burkholderiales that may prove the adaptability of this taxon's representatives. The following variability of the Burkholderiales in Russian healthcare units has been revealed: Burkholderiaceae (Burkholderia, Pandoraea, and Lautropia), Alcaligenaceae (Achromobacter), and Comamonadaceae (Variovorax). The Burkholderia genus was the most diverse and was represented by 5 species and 16 sequence types (ST). ST709 and 728 were transmissible and often encountered in cystic fibrosis patients and in hospitals. A. xylosoxidans was estimated by 15 genotypes. The strains of first and second ones were the most numerous., Conclusions: Phylogenetic position of the genus Lautropia with smaller genome is ambiguous. The Bcc MLST scheme is applicable for all Burkholderiales representatives for resolving the epidemiological problems.

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.

Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 μm) and activated factor XI (Ki = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The release kinetics of recombinant human bone morphogenic factor 2 (rhBMP-2) from collageneous 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 collageneous hydrogel with the incorporation of fibronectin was created which retained rhBMP-2 for a twice longer period as compared to the ordinary collageneous 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 collageneous hydrogel.

Background and Aim: Leptospira, the causal agent of leptospirosis, has been isolated from the environment, patients, and wide spectrum of animals in Russia. However, the genetic diversity of Leptospira in natural and anthropurgic foci was not clearly defined., Methods: The recent MLST scheme was used for the analysis of seven pathogenic species. 454 pyrosequencing technology was the base of the whole genome sequencing (WGS)., Results: The most wide spread and prevalent Leptospira species in Russia were L. interrogans, L. kirschneri, and L. borgpetersenii. Five STs, common for Russian strains: 37, 17, 199, 110, and 146, were identified as having a longtime and ubiquitous distribution in various geographic areas. Unexpected properties were revealed for the environmental Leptospira strain Bairam-Ali. WGS of this strain genome suggested that it combined the features of the pathogenic and nonpathogenic strains and may be a reservoir of the natural resistance genes. Results of the comparative analysis of rrs and rpoB genes and MLST loci for different Leptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separate Leptospira species., Conclusions: Thus, the natural and anthropurgic foci supported ubiquitous Leptospira species and the pool of genes important for bacterial adaptivity to various conditions.

The research carried out for 30 years from the moment of hepatitis E virus (HEV) discovery has proved the presence of the autochthonous HEV in non-endemic areas: Europe and Russia. Monitoring of the HEV antibodies (anti-HEV) among the Russian population has revealed regions with increased seroprevalence that testifies to high probability of local HEV infection in these areas. Contact with HEV can represent special danger for patients of the risk groups. In this work, the blood sera testing was carried out in order to assess the anti-HEV presence among these contingents (groups). Seropositive sera from the patients from the regions with high anti-HEV seroprevalence, risk groups patients, samples with high probability of HEV occurrence including the animals as possible reservoir, have been used for RNA extraction. The developed system of HEV RNA detection both in real-time RT-PCR and in a nested PCR variant has confirmed its sensitivity to the synthetic reference templates and positive control samples in commercial test system (Genesig, Great Britain). HEV RNA was absent in all tested samples. This indicates a low frequency of the autochthonous HEV carriage occurrence. Sampling enlargement to tens of thousands persons is necessary for significant HEV RNA detection.

The shared bacteria Burkholderia capacia complex and Achromobacter sp. infect the respiratory tract of patients with mucoviscidosis brining on disorders of respiratory patency. Burkholderia capacia complex is characterized by transmissivity and higher lethality of patients infected by Burkholderia. Hence, the importance of differentiation of these phenotypically similar microorganisms is obvious. The developed express technique of diagnostic includes the separation of DNA from phlegm amplification and sequenation was fragments of genes recA, gltB, gyrB, 16S rDNA. The evaluation of products of amplification of genes recA, gltB makes it possible to differentiate Burkholderia capacia complex and Achromobacter sp. The analysis of successions of recA, gltB, gyrB makes it possible to identify genotype of Burkholderia capacia complex on the basis of data of allele profiles of strains of Burkholderia capacia complex circulating in Russia. The succession of gene 16S rDNA makes it possible to determine the taxonomic position of microorganism dominating in phlegm and not belonging to Burkholderia capacia complex or Achromobacter sp. The real time polymerase chain reaction in presence of intercalating dye Sybr Green I, DMSO and D(+)-trehalose makes it possible to differentiate Burkholderia capacia complex from other microorganisms infecting respiratory tract of patients with mucoviscidosis. This approach provides additional reduction of diagnostic duration and decrease possibility of contamination.

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.

The nucleotide sequence of a chromosome fragment of the thermophilic anaerobic bacterium Caldicellulosiruptor bescii (syn. Anaerocellum thermophilum) has been determined. The fragment contains four open reading frames with the second one of 749 aa encoding a multimodular endo-1,4-beta-glucanase CelD (85019 Da). N-terminal region of the protein includes the signal peptide and the catalytic module of glycoside hydrolase family 5 (GH5), followed by the substrate-binding module of family 28 (CBM28). The C-terminal region bears three SLH modules. The recombinant endoglucanase and its two separate modules, the catalytic one and CBM28, were produced in E. coli cells and purified to homogeneity. Analysis of the catalytic properties showed CelD to be endo-1,4-beta-glucanase whose maximum activity was exhibited on beta-glucan of barley at pH 6.2 and 70 degrees C. The enzyme was stable at 50 degrees C for 30 days. Upon removal of the C-terminal CBM28, the activity of GH5 decreased on cellulose substrates, and its thermostability was dropped. Binding of CBM28 to amorphous cellulose was almost irreversible as it could not be removed from this substrate in a range of pH 4-11, temperatures--of 0-75 degrees C, and NaCl concentration--of 0-5 M. Only 100% formamide or 1% SDS were able to remove the protein.

88 cultures of microorganisms referred to the Burkholderia cepacia complex (Bcc) during initial identification were analyzed by multilocus sequencing (Multilocus Sequence Typing, MLST). 13 genotypes (sequence type, ST) were detected, 9 of them (708, 709, 710, 711, 712, 714, 727, 728, 729) were identified for the first time. Two new alleles for the gene trpB (357, 358), one of the genes atpD (306) and gltB (352) were detected and registered. It was found that strains of 2 genotypes (711, 712) belong to the species B. multivorans, 1 (ST102) - B. contaminans, 1 (ST51) - B. stabilis, 1 (ST729) - B. vietnamiensis. Most strains of the sample, representing 8 genotypes (208, 241, 728, 727, 708, 709, 710, 714), belong to the species B. cenocepacia. Identified genotypes differ in the global spread of the world: 4 genotype (51, 102, 208, 241) have intercontinental distribution, 1 (712) - intra. It is shown that strains causing nosocomial infections, in most cases refer to genotypes 728 and 708. Epidemiologically significant in respect of patients with cystic fibrosis should recognize genotype 709, detected in strains isolated from patients in seven federal districts (FD) of Russia. The Bcc strains of genotypes 241 (B. cenocepacia) and 729 (B. vietnamiensis) were isolated from the patients of the Far Eastern FD. They are not typical for other FD Russia. The possibility of concomitant infection in cystic fibrosis patient with two genotypes 709 - epidemiologically significant and 708 - nosocomial, was indicated. The long-termpersistence of a single genotype strain in the organism of patients with cystic fibrosis was demonstrated as for Bcc species B. cenocepacia (ST 709), so for B. multivorans (ST712). The possibility of transferring the strain Bcc, typical for nosocomial environment to patient with cystic fibrosis at surgery was observed.

Prokaryotic restriction-modification (R-M) systems defend the host cell from the invasion of a foreign DNA. They comprise two enzymatic activities: specific DNA cleavage activity and DNA methylation activity preventing cleavage. Typically, these activities are provided by two separate enzymes: a DNA methyltransferase (MTase) and a restriction endonuclease (RE). In the absence of a corresponding MTase, an RE of Type II R-M system is highly toxic for the cell. Genes of the R-M system are linked in the genome in the vast majority of annotated cases. There are only a few reported cases in which the genes of MTase and RE from one R-M system are not linked. Nevertheless, a few hundreds solitary RE genes are present in the Restriction Enzyme Database (http://rebase.neb.com) annotations. Using the comparative genomic approach, we analysed 272 solitary RE genes. For 57 solitary RE genes we predicted corresponding MTase genes located distantly in a genome. Of the 272 solitary RE genes, 99 are likely to be fragments of RE genes. Various explanations for the existence of the remaining 116 solitary RE genes are also discussed.

Aim: Study genetic diversity of P. aeruginosa strains persisting in patients of Federal Scientific Center of Transplantology and Artificial Organs, and main factors facilitating persistence of strains in the hospital., Materials and Methods: 136 P. aeruginosa strains isolated from patients of the center for 3 years 6 months were genotyped by RAPD-PCR and MLST methods and studied for antibiotics resistance and presence of integrons., Results: Genetic diversity of strains persisting in hospital was established. Strains of main genotypes ST235, ST446, ST598 were isolated from patients of various surgical departments. Patients were shown to be colonized by these strains during stay in reanimation and intensive therapy department (RITD) of the hospital. Strains of dominant genotype 235 were isolated from 47% of examined patients during more than 3 years. Only genotype 235 strains contained integron with cassettes of antibiotics resistance genes blaGES5 and aadA6 in the genome., Conclusion: The data obtained show that over the period of observation in the center 1 clone of P. aeruginosa that belonged to genotype 235 dominated. This clone was endemic for this hospital and in the process of prolonged persistence became more resistant to antibiotics. Colonization of patients with these strains occurs in RITD. This confirms the necessity of constant monitoring of hospital microflora for advance detection of potentially dangerous epidemic hospital strains able to cause hospital infections.

Aim: Revision of the species identification of collection lactobacilli strains based on 16S rDNA and rpoA gene sequencing., Materials and Methods: 52 lactobacilli cultures that present mostly Gamaleya Research Institute of Epidemiology and Microbiology (GIMC) collection were studied. 16S rDNA gene fragments were amplified by using Lb16a, Lb16b, 16S-midford, 16S-midrev primers. 2 different reverse primers were used for the analysis of rpoA gene depending on lactobacilli species. DNA fragments sequencing was performed with 3130 Genetic Analyzer (Applied Biosystems/Hitachi) with primers used for amplification., Results: The effectiveness of sequencing of 2 targets for differentiation of species within lactobacilli phylogenetic groups was shown. Species diversity was demonstrated for GIMC lactobacilli strain collection that includes members of 9 species. All the strains marked previously as L. acidophilus were determined to belong to L. helveticus. Strains belonging to recently discovered L. farraginis species that has promising application in agriculture were detected., Conclusion: Genetic passports of original strains of 9 species of lactobacilli that are promising for further research.

Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.

Immunization of CBA mice with killed group A streptococcus (type 5) vaccine changed the counts of stromal precursor cells (CFC-F) in bone marrow transplants at different donor-recipient combinations (normal, N, or immune, I). CFC-F counts in bone marrow transplants from normal mice transplanted to immunized animals decreased 4-6-fold depending on the transplant age in comparison with similar transplants in normal recipients. The percentage of CFC-F colonies with alkaline phosphatase (osteogenesis marker) activity decreased more than 2-fold. Similarly, the count of CFC-F in the transplants was 2-fold lower during delayed (7 months) period after bone marrow transplantation from immunized donors (8-12 days after the end of immunization) to intact recipients, while 2 months after transplantation it was 3-fold lower. The mean optical density of the bone capsule in preparations stained for glycogen and alkaline phosphatase was 1.5-3 times lower in the N-->I and I-->N experiments in comparison with the control (N-->N). On the other hand, CFC-F count in the femoral bone marrow of immunized animals was significantly (3.5-2.5 times) higher during the period from 8 days to 8 months after the end of immunization compared to CFC-F count in the femoral bone marrow of intact mice. These results attest to a significant prolonged effect of streptococcal antigens on the bone marrow stromal tissue. These data also indicate that not all CFC-F, the counts of which increased in response to antigens, are responsible for transplantability of the stromal tissue in heterotopic transplantation. Immunization by streptococcal antigens seemed to suppress transplantability and osteogenic activity of stromal stem cells. The efficiency of CFC-F cloning in mouse bone marrow cultures increased significantly (2-3-fold) in the presence of sera from immune mice. The levels of TNF-α and IFN-γ were low in this serum (2.7 and 6 times lower, respectively) in comparison with normal serum. Presumably, the effects of streptococcal antigens on stromal tissue were mediated through serum cytokines.

Aim: Development of Staphylococcus haemolyticus strain typing method based on multilocus sequencing for resolving problems of molecular epidemiology., Materials and Methods: 102 strains of coagulase negative staphylococci (CNS) isolated in hospitals of various specialization in N. Novgorod and Moscow were studied. Species identification of strain was performed by using tuf gene fragment sequencing, S. haemolyticus strain differentiation--by MLST results. eBURST approach was used for cluster analysis of MLST data; structural changes in tagatose-6-phosphate kinase were studied by using InterProScan platform and SWISS-MODEL site programs; MLST scheme gene allele variability analysis was performed by using MEGA4.0 program package., Results: In the 102 strains sampled CNS was detected in 28 strains of the S. haemolyticus species. The MLST scheme developed for the first time for S. haemolyticus including mvaK, rphE, tphK, gtr, arcC, triA, aroE genes allowed the differentiation of the sampled strains by 11 genotypes. Strains with ST 3, 8, 6, 1, 4, 5 and 11 differed by highest epidemiologic significance. Cluster and phylogenetic analysis of the data obtained showed a high adaptive ability of the nosocomial S. haemolyticus strains. Multiresistance to antibacterial preparations was detected in the analyzed strains., Conclusion: The MLST method developed was effective in the differentiation of S. haemolyticus strains that circulate in hospitals and threaten both neonates and hospitalized adult patients.

Aim: Comparative analysis of species diversity of sample of coagulase-negative staphylococci (CNS) isolated in hospitals of different specializations., Materials and Methods: For identification of 102 CNS strains, biochemical systems manufactured by NPO "Diagnostic Systems", VITEK 2 Compact, and BBL Crystal as well as sequencing of fragments of tuf and gap genes were used., Results: Greater differentiating capability of genotyping compared with phenotyping methods for species identification of staphylococci was demonstrated. Six CNS species were identified in the sample: S. epidermidis, S. haemolyticus, S. hominis, S. warneri, S. capitis, and S. pasteuri. The largest species diversity was noted for strains from maternity hospitals in Nizhny Novgorod and Kulakov Scientific Center for Obstetrics, Gynecology and Perinatology. Strains isolated from blood of patients in Bakulev Center for Cardiovascular Surgery were represented mostly by S. epidermidis and S. haemolyticus. Differences in species diversity of CNS--causative agents of neonatal conjunctivitis and omphalitis--were observed., Conclusion: Two species of CNS: S. epidermidis and S. haemolyticus pose special threat as nosocomial pathogens both in hospitals for adults and obstetrical facilities. Additionally, in neonatal units it is necessary to control such species as S. warneri, S. capitis, S. pasteuri.

Two recombinant plasmids were constructed. The first plasmid contained the hybrid gene composed of immunomimetic peptide of benzo[a]pyrene, of the protein pIII of bacteriophage M13 and of cellulose binding domain encoding sequences. The second plasmid contained the hybrid gene composed of the signal peptide of the protein pIII of bacteriophage M13, of immunomimetic peptide of benzo[a]pyrene, of the protein pill of bacteriophage M13 and of cellulose binding domain sequences. The obtained recombinant plasmids were used in expression of chimeric protein containing immunomimetic peptide ofbenzo[a]pyrene based on strain E. coli M15. The lack of the recombinant protein expression using first plasmid was demonstrated. In the same time, it was shown that accumulation of recombinant protein contained immunomimetic peptide with signal peptide of the protein pIIIl of bacteriophage was present. This chimeric protein was produced in "mature" (without signal peptide) and "unprocessing" (with signal peptide) forms. Using the Western-blot analysis, it was shown that the "mature" form only specifically bound to the B2 monoclonal antibody against benzo[a]pyrene. Thus, we expressed, purified, and characterized the recombinant protein containing immunomimetic peptide of benzo[a]pyrene.

Recombinant plasmids containing fusion proteins composed of two different modules were constructed and expressed in Escherichia coli. The modules encoded the lactase LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus and the cellulase CelD, a cellulose-binding module (CBM) from Anaerocellum thermophilum. The CelD CBM provides a spontaneous and strong sorption of the fusion proteins onto a cellulose carrier. The enzymatic activities of both the free LacA protein and LacA-CelD CBM fusion proteins immobilized onto the cellulose carrier were assessed. The LacA activity of the fusion protein was dependent upon its position with respect to the CBM. The highest level of lactase activity and stability was observed when the lactase domain was localized at its N terminus. A continuous-flow column reactor of lactase immobilized on a cellulose carrier was constructed, and its activity was assessed. The lactose hydrolysis rate for a 150 mM (5%) solution at a flow rate of 1 reactor volume per min was 75%, which is a value optimal for further whey transformation into glucose/galactose syrup.

Aim: To assess effect of continued immunization of mice from CBA line with inactivated group A streptococcal vaccine on levels of serum cytokines and number of stromal bone marrow progenitor cells in immunized mice and in heterotopic transplants after different variants of transplantation., Materials and Methods: CBA mice were immunized during 3 weeks with heat-killed vaccine prepared from group A streptococci type 5. Levels of pro- and antiinflammatory cytokines were measured with BioPlex device. Number of stromal progenitor cells was determined on quantity of colonies formed by cells explanted to monolayer cultures., Results: Significant 2.3-fold increase of number of stromal progenitor cells in femoral bone marrow of immune mice was demonstrated. Experiments with heterotopic transplants showed that bone marrow in transplants of mice immunized with streptococci--variant Normal-->Immune (N-->1)-- is defective both on efficacy of cloning and number of stromal progenitor cells. Even short-term presence of stromal tissue in immune organism (variant I -->N) significantly changed these parameters, especially at late time after transplantation. In serum of immune mice changes of cytokines levels, especially TNFalpha, were observed. The level of the latter was decreased (mean--2.5-fold) in all immune serum samples compared to normal serum., Conclusion: Immunization of mice with group A streptococci leads to changes in stromal tissue and, possibly, to damage of microenvironment functions including hemo- and lymphopoiesis.

Bone morphogenetic protein-2 (rhBMP-2) represents the osteoinductive protein factor which plays a dominant role in growth and regeneration of a bone tissue. In clinical practice the bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials are not produced. The fragment of the bmp2gene coding for a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on a basis of the E. coli BL21 (DE3). The rhBMP-2 production was about 25% of total cell protein. The biologically active dimeric form of rhBMP-2 was obtained by isolation and purification of protein from inclusion bodies with subsequent refolding. The rhBMP-2 sample with more than 80% of the dimeric form was obtained, which is able to interact with specific antibodies to BMP-2. Biological activity of the received rhBMP-2 samples was shown in the in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On model of the ectopic osteogenesis it was shown that received rhBMP-2 possesses biological activity in vivo, causing tissue calcification in the place of an injection. The protein activity in vivo depends on 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 offered method of rhBMP-2 isolation and purification results in increasing common protein yield as well as the maintenance of biologically active dimeric form in comparison with the analogues described in the literature.

Human exposure to chemical carcinogens is an important etiological factor in cancer diseases. In this article, we will discuss a new approach to the development of anticarcinogenic vaccines. The main task in our research was to select a benzo[a]pyrene immunomimetic peptide considered as a hapten-specific component. For this purpose, we synthesized carcinogen-protein conjugates and prepared mono- and polyclonal antibodies to benzo[a]pyrene. Phage display technology was used to select the benzo[a]pyrene immunomimetic peptide, followed by an evaluation of the immunological properties of the obtained peptide. The obtained benzo[a]pyrene immunomimetic peptide could only simulate chemical carcinogens in the frame of the pIII protein. As a result, we prepared a recombinant protein composed of the benzo[a]pyrene immunomimetic peptide and pIII-encoding sequences. Using ELISA, we demonstrated that the recombinant protein specifically interacts with the anti-benzo[a]pyrene monoclonal antibody (mAB B2). Using molecular modeling, we predicted the 3-D structure of the mAB B2 active center and analyzed the characteristics of its interaction with different polycyclic aromatic hydrocarbons, as well as with the benzo[a]pyrene immunomimetic peptide. Thus, a comprehensive analysis of the results of the obtainment of hapten-specific components of anticarcinogenic vaccines allowed us to outline a strategy for future development in this direction.