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A study of the theoretical and methodological aspects of balancing the interests of producers and consumers of recreational services in the regions of Ukraine is carried out. It is noted that the development of the recreational and tourist sphere for many countries is a very important task today, since the tourism industry is one of the main sources of income to the budget for them. Ukraine, located in the center of Europe at the crossroads of transport routes, with favorable natural and climatic conditions for the progressive advance of its recreational sphere, as well as significant historical and cultural potential, the necessary human and material resources, significantly lags behind most European countries in terms of development of recreational and tourist services. The factors influencing the price and quality of tourist services are defined. It is determined that the management of the balance of interests between the producer of tourist services (tour operator) and recreational needs of the population should be materialized on the basis of a set of properties and characteristics of products or services that provide them with the opportunity to meet the agreed or predictable needs of consumers. As result of the study, it is concluded that the price and quality of the tour package affect the balance of interests between the tour operator and the recreational needs of the population. To consider the pricing and proper quality, the factors influencing them are analyzed. It is determined that the most influential factor is: the theory of consumer behavior. The formation of the price of tourist services is influenced by the costs of the tour operator, which largely depend on contractors. To increase the balance of interests, it is necessary to better study the tourist demand and offer a variety of services at diversified prices. Managing the balance of interests of producers and consumers of recreational services in Ukraine should be based on the understanding that a tourist product is a specific one, and the quality determination largely depends on the factors of influence: volatility of quality, seasonality, etc.

The publication considers the issues regarding creation of a favorable investment and business climate in order to attract foreign direct investment (FDI), which constitute an integral part of an open and efficient economic system. On the other hand, direct investors do not have the opportunity to leave the market quickly, as they risk the long-term large volumes of financial investments. The FDI is therefore recognized as the main catalyst for the economies of developing countries and countries with transition economy. This aspect reveals the need to create an enabling environment for the foreign capital’s interest in the domestic market, as the progressive share of the FDI in the national capital structure can improve competitiveness of the country, aimed at an efficient integration into the global economic system. It is identified that the presence of such economic advantages as a favorable natural-geographical position, highly skilled labor force, high potential for structural distribution of the economy, developed infrastructure may be insufficient for the investor’s positive decision. A proper status of the institutional environment in the recipient country is a compelling factor for the FDI. A favorable institutional environment helps to avoid the transaction costs that result from the bureaucratic and judicial barriers, low levels of property rights protection and performance of contracts, corruption, political and economic instability, etc.

The publication discusses the issues of organization of financial monitoring - a considerably new method of scientific-practical activity, the purpose of which is to obtain and process warning information about the status of enterprise and tendencies of its development. Essence of concepts of «strategy» and «strategic management» is clarified; components of the decision-making cycle in the implementation of the enterprise’s strategy are allocated. It is specified that financial indicators only estimate the economic impact of the measures taken and are the indicators of compliance of the strategy with the overall plan of the enterprise’s development. In these terms the need for carrying out financial monitoring is substantiated together with its objectives and tasks. The organization of financial monitoring (which provides for displaying changes in the financial performance of firm not only regarding their compliance with the previously defined strategic guidelines, but also in creating an operational response mechanism for an efficient management of the long-term development of enterprise) is a direction of further analysis. It is concluded that in order to implement the sustainable development strategy of enterprise, one of the key directions of strategic management should be a regular, effective and flexible financial monitoring mechanism for obtaining an adequate estimate of the financial condition and supporting for the strategic orientation alternatives.

Listerias of the phylogenetic lineage II (PLII) are common in the European environment and are hypovirulent. Despite this, they caused more than a third of the sporadic cases of listeriosis and multi-country foodborne outbreaks. L. monocytogenes ST37 is one of them. During the COVID-19 pandemic, ST37 appeared in clinical cases and ranked second in occurrence among food isolates in the Moscow region. The aim of this study was to describe the genomic features of ST37 isolates from different sources. All clinical cases of ST37 were in the cohort of male patients (age, 48-81 years) with meningitis-septicemia manifestation and COVID-19 or Influenza in the anamnesis. The core genomes of the fish isolates were closely related. The clinical and meat isolates revealed a large diversity. Prophages (2-4/genome) were the source of the unique genes. Two clinical isolates displayed pseudolysogeny, and excided prophages were A006-like. In the absence of plasmids, the assortment of virulence factors and resistance determinants in the chromosome corresponded to the hypovirulent characteristics. However, all clinical isolates caused severe disease, with deaths in four cases. Thus, these studies allow us to speculate that a previous viral infection increases human susceptibility to listeriosis.

The safety of food production as concerns Listeria is the key to the sanitary wellbeing of manufactured products. Molecular-genetic methods for the analysis of Listeria , including whole-genome sequencing, are effective in monitoring persistent contaminants and in the epidemic investigation of cases of foodborne infections. They have been adopted in the European Union, United States, and Canada. In Russia, multilocus and whole-genome sequencing has proven itself in the analysis of clinical food isolates and Listeria from the environment. The objective of the study was molecular-genetic characterization of Listeria detected in the industrial environment of meat processing. To characterize the Listeria isolates, microbiological methods were used according to GOST (State Standard) 32031-2012, as well as multilocus sequencing, including the analysis of seven housekeeping genes and four virulence genes, as well as whole-genome sequencing. In swabs that were positive for the presence of Listeri a spp. taken at two meat-processing plants in Moscow, Listeria monocytogenes constituted 81% and L. welshimeri 19%. The predominant genotype (Sequence Type, ST) of L. monocytogenes was ST8. The variety was supplemented with ST321, ST121, and ST2330 (CC9 (Clonal Complex 9)). L. welshimeri , which prevailed in the second production, was represented by ST1050 and ST2331. The genomic characteristics of L. welshimeri isolates confirmed that they have high adaptive capabilities both as concerns production conditions (including resistance to disinfectants) and the metabolic peculiarities of the gastrointestinal tract of animals. L. monocytogenes CC9 and CC121 are also correlated with food production in other countries. However, L. monocytogenes CC8 and CC321 can cause invasive listeriosis. The concordance in the internalin profile of the ST8 isolates from the industrial environment with the clinical isolates ST8 and ST2096 (CC8) is a cause for concern. The study showed the effectiveness of molecular-genetic methods in determining the diversity of Listeria detected in the production environment of meat processing, and laid the foundation for monitoring of persistent contaminants., Competing Interests: Conflict of Interest The authors declare that they have no conflicts of interest., (© Allerton Press, Inc. 2023, ISSN 0891-4168, Molecular Genetics, Microbiology and Virology, 2023, Vol. 38, No. 1, pp. 21–28. © Allerton Press, Inc., 2023.Russian Text © The Author(s), 2023, published in Molekulyarnaya Genetika, Mikrobiologiya i Virusologiya, 2023, No. 1, pp. 24–31.)

Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma, Delta and Omicron variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Favorskaya, Shcheblyakov, Esmagambetov, Dolzhikova, Alekseeva, Korobkova, Voronina, Ryabova, Derkaev, Kovyrshina, Iliukhina, Botikov, Voronina, Egorova, Zubkova, Ryzhova, Aksenova, Kunda, Logunov, Naroditsky and Gintsburg.)

Totally, 45 L. monocytogenes strains isolated from meat, poultry, dairy, and fish products in the Central European part of Russia in 2001-2005 and 2019-2020 were typed using a combined MLST and internalin profile (IP) scheme. Strains belonged to 14 clonal complexes (CCs) of the phylogenetic lineages I and II. Almost half of the strains (20 of 45) belonged to six CCs previously recognized as epidemic clones (ECs). ECI and ECV strains were isolated during both studied periods, and ECII, ECIV, ECVI, and ECVII strains were isolated in 2001-2005, but not in 2019-2020. ECI, ECIV, ECV, and ECVII strains were isolated from products of animal origin. ECII and ECVI were isolated from fish. Testing of invasion efficiencies of 10 strains isolated in different years and from different sources and belonging to distinct CCs revealed a statistically significant difference between phylogenetic lineage I and II strains but not between ECs and non-EC CCs or strains differing by year and source of isolation. Strains isolated in 2001-2005 were characterized by higher phylogenetic diversity and greater presentation of ECs and CCs non-typical for natural and anthropogenic environments of the European part of Russia comparatively to isolates obtained in 2019-2020.Closing of the Russian market in 2019-2020 for imported food might be responsible for these differences.

Susceptibility of 117 L. monocytogenes strains isolated during three time periods (1950-1980; 2000-2005, and 2018-2021) to 23 antibiotics was tested by the disk diffusion method. All strains were sensitive to aminoglycosides (gentamicin, kanamycin, neomycin, streptomycin), glycopeptides (vancomycin and teicoplanin), clarithromycin, levofloxacin, amoxicillin/clavulanic acid, and trimethoprim/sulfamethoxazole. Resistance to clindamycin was observed in 35.5% of strains. Resistance to carbapenems, imipenem and meropenem was found in 4% and 5% of strains, respectively. Resistance to erythromycin, penicillin G, trimethoprim, and ciprofloxacin was found in 4%, 3%, 3%, and 2.5% of strains, respectively. Resistance to tylosin, ampicillin, enrofloxacin, linezolid, chloramphenicol, and tetracycline was found in less than 2%. Three strains with multiple antibiotic resistance and 12 strains with resistance to two antibiotics were revealed. Comparison of strains isolated in different time periods showed that the percentage of resistant strains was the lowest among strains isolated before 1980, and no strains with multiple antibiotic resistance were found among them. Statistical analysis demonstrated that the temporal evolution of resistance in L. monocytogenes has an antibiotic-specific character. While resistance to some antibiotics such as ampicillin and penicillin G has gradually decreased in the population, resistance to other antibiotics acquired by particular strains in recent years has not been accompanied by changes in resistance of other strains.

Ebola fever is an acute, highly contagious viral disease with a mortality rate that can reach 90%. There are currently no licensed therapeutic agents specific to Ebola in the world. Monoclonal antibodies (MAbs) with viral-neutralizing activity and high specificity to the Ebola virus glycoprotein (EBOV GP) are considered as highly effective potential antiviral drugs. Over the past decade, nanobodies (single-domain antibodies, non-canonical camelid antibodies) have found wide use in the diagnosis and treatment of various infectious and non-infectious diseases. In this study, a panel of nanobodies specifically binding to EBOV GP was obtained using recombinant human adenovirus 5, expressing GP (Ad5-GP) for alpaca (Vicugna pacos) immunization, for the first time. Based on specific activity assay results, affinity constants, and the virus-neutralizing activity against the recombinant vesicular stomatitis virus pseudotyped with EBOV GP (rVSV-GP), the most promising clone (aEv6) was selected. The aEv6 clone was then modified with the human IgG1 Fc fragment to improve its pharmacokinetic and immunologic properties. To assess the protective activity of the chimeric molecule aEv6-Fc, a lethal model of murine rVSV-GP infection was developed by using immunosuppression. The results obtained in lethal model mice have demonstrated the protective effect of aEv6-Fc. Thus, the nanobody and its modified derivative obtained in this study have shown potential protective value against Ebola virus., (Copyright ® 2021 National Research University Higher School of Economics.)

Since the beginning of the 2021 year, all the main six vaccines against COVID-19 have been used in mass vaccination companies around the world. Virus neutralization and epidemiological efficacy drop obtained for several vaccines against the B.1.1.7, B.1.351 P.1, and B.1.617 genotypes are of concern. There is a growing number of reports on mutations in receptor-binding domain (RBD) increasing the transmissibility of the virus and escaping the neutralizing effect of antibodies. The Sputnik V vaccine is currently approved for use in more than 66 countries but its activity against variants of concern (VOC) is not extensively studied yet. Virus-neutralizing activity (VNA) of sera obtained from people vaccinated with Sputnik V in relation to internationally relevant genetic lineages B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.3 and Moscow endemic variants B.1.1.141 (T385I) and B.1.1.317 (S477N, A522S) with mutations in the RBD domain has been assessed. The data obtained indicate no significant differences in VNA against B.1.1.7, B.1.617.3 and local genetic lineages B.1.1.141 (T385I), B.1.1.317 (S477N, A522S) with RBD mutations. For the B.1.351, P.1, and B.1.617.2 statistically significant 3.1-, 2.8-, and 2.5-fold, respectively, VNA reduction was observed. Notably, this decrease is lower than that reported in publications for other vaccines. However, a direct comparative study is necessary for a conclusion. Thus, sera from "Sputnik V"-vaccinated retain neutralizing activity against VOC B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.617.3 as well as local genetic lineages B.1.1.141 and B.1.1.317 circulating in Moscow.

Introduction: The surveillance of influenza viruses in ARVI structure and study of their properties in epidemic season 2019-2020 in Russian Federation are actual for investigations due to tasks of Global Influenza Strategy initiated by WHO in 2019., Material and Methods: The data of epidemiological surveillance on influenza- and ARVI-associated morbidity and hospitalization in different age groups of population were analyzed; virological, genetic and statistical methods were used., Results: Preschool children were involved in epidemic the most. Meanwhile, the highest rate of hospitalization was observed in patients of 18-40 years old. Influenza A(H1N1)pdm09 virus dominated in etiology of ARVI in hospitalized patients and pneumonia. The role of respiratory viruses in severe cases of pneumonia and bronchoalveolar syndrome in children was shown. The differences in spectrum of circulating viruses caused ARVI in different regions of Russia were found. Influenza A(H1N1)pdm09 and B/Victoria-like viruses were the main etiological agents that caused of epidemic; its activity among all ARVI was 7.3 and 8.0%, respectively. The differences in antigenic properties of influenza A(H3N2) and B epidemic strains compared to vaccine viruses were found. The populations of epidemic strains were presented by following dominant genetic groups: 6B1.A5/183P for A(H1N1)pdm09, 3С.2а1b+137F for A(H3N2) and V1A.3 line B/Victoria-like for B viruses. The good profile of epidemic strains susceptibility to anti-neuraminidase inhibitors has been saved. The most of the studied influenza strains had the receptor specificity characteristic of human influenza viruses., Conclusions: Obtained results identified the peculiarities of viruses caused the influenza and ARVI in epidemic season 2019-2020 in different regions of Russia. These results suggested the important role of influenza A(H1N1) pdm09 in severe cases and pneumonia in adults 18-40 years old. The continuing drift in influenza viruses was found, which, apparently, could not but affect the efficacy of vaccine prophylaxis and was also considered in the recommendations of WHO experts on the composition of influenza vaccines for the countries of the Northern Hemisphere in the 2020-2021 season.

Chromobacterium species are common in tropical and subtropical zones in environmental samples according to numerous studies. Here, we describe an environmental case of resident Chromobacterium vaccinii in biofilms associated with Carex spp. roots in Moscow region, Russia (warm-summer humid continental climate zone). We performed broad characterization of individual properties as well as surrounding context for better understanding of the premise of C. vaccinii survival during the winter season. Genome properties of isolated strains propose some insights into adaptation to habit and biofilm mode of life, including social cheaters carrying ΔluxR mutation. Isolated C. vaccinii differs from previously described strains in some biochemical properties and some basic characteristics like fatty acid composition as well as unique genome features. Despite potential to modulate membrane fluidity and presence of several genes responsible for cold shock response, isolated C. vaccinii did not survive during exposure to 4 °C, while in the complex biofilm sample, it was safely preserved for at least half a year in vitro at 4 °C. The surrounding bacterial community within the same biofilm with C. vaccinii represented a series of psychrophilic bacterial species, which may share resistance to low temperatures with other species within biofilm and provide C. vaccinii an opportunity to survive during the cold winter season.

Marinomonas primoryensis KMM 3633 T , extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633 T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin., Competing Interests: The authors declare that they have no conflict of interest.

Influenza A virus is a serious human pathogen that assembles enveloped virions on the plasma membrane of the host cell. The pleiomorphic morphology of influenza A virus, represented by spherical, elongated, or filamentous particles, is important for the spread of the virus in nature. Using fixative protocols for sample preparation and negative staining electron microscopy, we found that the recombinant A/WSN/33 (H1N1) (rWSN) virus, a strain considered to be strictly spherical, may produce filamentous particles when amplified in the allantoic cavity of chicken embryos. In contrast, the laboratory WSN strain and the rWSN virus amplified in Madin-Darby canine kidney cells exhibited a spherical morphology. Next-generation sequencing (NGS) suggested a rare Ser126Cys substitution in the M1 protein of rWSN, which was confirmed by the mass spectrometric analysis. No structurally relevant substitutions were found by NGS in other proteins of rWSN. Bioinformatics algorithms predicted a neutral structural effect of the Ser126Cys mutation. The mrWSN_M1_126S virus generated after the introduction of the reverse Cys126Ser substitution exhibited a similar host-dependent partially filamentous phenotype. We hypothesize that a shortage of some as-yet-undefined cellular components involved in virion budding and membrane scission may result in the appearance of filamentous particles in the case of usually "nonfilamentous" virus strains.

Microbiota as an integral component of human body is actively investigated, including by massively parallel sequencing. However, microbiomes of lungs and sinuses have become the object of scientific attention only in the last decade. For patients with cystic fibrosis, monitoring the state of respiratory tract microorganisms is essential for maintaining lung function. Here, we studied the role of sinuses and polyps in the formation of respiratory tract microbiome. We identified Proteobacteria in the sinuses and samples from the lower respiratory tract (even in childhood). In some cases, they were accompanied by potentially dangerous basidiomycetes. The presence of polyps did not affect formation of the sinus microbiome. Proteobacteria are decisive in reducing the biodiversity of lung and sinus microbiomes, which correlated with the worsening of the lung function indicators. Soft mutations in the CFTR gene contribute to the formation of safer microbiome even in heterozygotes with class I mutations.

Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2: 186-200, 406-411; PB1: 135-143, 538-546; PA: 515-523; NP: 61-68; M1: 76-84; M2: 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords: avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.

Highly pathogenic avian influenza viruses (HPAIV) A(H5N8) of group B (Gochang1-like) have emerged in the Tyva Republic of eastern Russia in May 2016. Since November 2016, HPAIV A(H5N8) has spread throughout the European part of Russia. Thirty-one outbreaks were reported in domestic, wild and zoo birds in 2017. The present study aimed to perform a comparative analysis of new HPAIV A(H5N8) strains. Phylogenetic analysis revealed four genetically distinct subgroups in HPAIV A(H5N8) from the 2016-2017 season. Russian strains consisted of three subgroups with differences between isolates from Tyva, Siberia (Chany Lake), and the European part of Russia. Strains from the European part of Russia showed the beginnings of divergent evolution. Slight differences of the Voronezh strains were suggested by sensitivity to antiviral compounds. Testing for host-specific mutations in sequenced strains revealed the absence of mutations associated with possible increased tropism/virulence in mammalian species, including humans. Only one residue of polymerase basic-1, 13P, is discussed, because the L13P mutation increased complementary RNA synthesis in mammalian cells. We concluded that the evolution of HPAIV A(H5N8) is continuous. Surveillance in Russia revealed new cases of HPAIV A(H5N8) and led to the elaboration of prevention strategies, which should be implemented., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Microbes infecting cystic fibrosis patients' respiratory tract are important in determining patients' functional status. Representatives of Burkholderiales order are the most dangerous. The goal of our investigation was to reveal the diversity of Burkholderiales, define of their proportion in the microbiome of various parts of respiratory tract and determine the pathogenicity of the main representatives., Results: In more than 500 cystic fibrosis patients, representing all Federal Regions of Russia, 34.0% were infected by Burkholderia cepacia complex (Bcc), 21.0% by Achromobacter spp. and 12.0% by Lautropia mirabilis. B. cenocepacia was the most numerous species among the Bcc (93.0%), and A. ruhlandii was the most numerous among Achromobacter spp. (58.0%). The most abundant genotype in Bcc was sequence type (ST) 709, and in Achromobacter spp. it was ST36. These STs constitute Russian epidemic strains. Whole genome sequencing of strains A. ruhlandii SCCH3:Ach33-1365 ST36 and B. cenocepacia GIMC4560:Bcn122 ST709 revealed huge resistomes and many virulence factors, which may explain the difficulties in eradicating these strains. An experience of less dangerous B. cenocepcia ST710 elimination was described. Massively parallel sequencing of 16S rDNA amplicons, including V1-V4 hypervariable regions, was used to definite "healthy" microbiome characteristics. Analysis of maxillary sinus lavage of 7 patients revealed infection with Proteobacteria of the same ST as pathogens from sputum, suggesting that the maxillary sinus is a source of infection in cystic fibrosis patients., Conclusions: Characterization of the Russian epidemic bacterial strains in the sputum and sinuses of cystic fibrosis patients have better defined the importance of Burkholderiales bacteria. This information may aid in the development of effective approaches for treatment of this disease.

The Ebola virus disease (EVD) is one of the most dangerous infections affecting humans and animals. The first EVD outbreaks occurred in 1976 in Sudan and Zaire. Since then, more than 20 outbreaks have occurred; the largest of which (2014-2016) evolved into an epidemic in West Africa and claimed the lives of more than 11,000 people. Although vaccination is the most effective way to prevent epidemics, there was no licensed vaccine for EVD at the beginning of the latest outbreak. The development of the first vaccines for EVD started in 1980 and has come a long technological way, from inactivated to genetically engineered vaccines based on recombinant viral vectors. This review focuses on virus-vectored Ebola vaccines that have demonstrated the greatest efficacy in preclinical trials and are currently under different phases of clinical trial. Particular attention is paid to the mechanisms of immune response development, which are important for protection from EVD, and the key vaccine parameters necessary for inducing long-term protective immunity against EVD.

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.

Biofilm formation by Burkholderia spp. is a principal cause of lung chronic infections in cystic fibrosis patients. A "lacking biofilm production" (LBP) strain B. contaminans GIMC4587:Bct370-19 has been obtained by insertion modification of clinical strain with plasposon mutagenesis. It has an interrupted transcriptional response regulator (RR) gene. The focus of our investigation was a two-component signal transduction system determination, including this RR. B. contaminans clinical and LBP strains were analyzed by whole genome sequencing and bioinformatics resources. A four-component operon (BiofilmReg) has a key role in biofilm formation. The relative location (i.e., by being separated by another gene) of RR and histidine kinase genes is unique in BiofilmReg. Orthologs were found in other members of the Burkholderiales order. Phylogenetic analysis of strains containing BiofilmReg operons demonstrated evidence for earlier inheritance of a three-component operon. During further evolution one lineage acquired a fourth gene, whereas others lost the third component of the operon. Mutations in sensor domains have created biodiversity which is advantageous for adaptation to various ecological niches. Different species Burkholderia and Achromobacter strains all demonstrated similar BiofilmReg operon structure. Therefore, there may be an opportunity to develop a common drug which is effective for treating all these causative agents., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

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.

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.

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.

The duration of the persistence and dynamics of accumulation of insertion bvg- Bordetella pertussis mutants were studied in lungs of laboratory mice after intranasal and intravenous challenge by virulent bacteria of the causative agent of whooping cough. The capability of the virulent B. pertussis bacteria to long-term persistence in the body of mice was tested. Using the real-time PCR approximately hundred genome equivalents of the B. pertussis DNA were detected in lungs of mice in two months after infection regardless of the way of challenge. Using the bacterial test bacteria were identified during only four weeks after challenge. Bvg- B. pertussis avirulent mutants were accumulated for the infection time. The percentage of the avirulent bacteria in the B. pertussis population reached 50% in 7-9 weeks after challenge. The obtained results show that the laboratory mice can be used for study of the B. pertussis insertion mutant formation dynamics in vivo and confirm the hypothesis about insertional bvg- B. pertussis virulent mutants accumulation during development of pertussis infection in human.

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.

Until recently food proteins were considered to be an energy source and a source of essential and nonessential amino acids required for protein synthesis and precursors of many vital biomolecules. However, we assumed earlier that food protein fragments might perform some regulatory functions. The theoretical justification for this assumption is advanced in this work. In the present work, the primary structures of protein fragments were compared with amino acid sequences of known natural regulatory oligopeptides in silico. It is shown that fragments formed as a result of animal food protein cleavage by proteolytic enzymes can exist in the gastrointestinal tract for a long time. Many of them are enzyme inhibitors, regulators of nervous, endocrine, and immune system, and possess antimicrobial and other activities. It has also been shown that the lifetime of fragments before their cleavage in the gastrointestinal tract could be enough for performing corrective functions. Thus, as a result of food protein fragmentation a dynamic pool of exogenous regulatory oligopeptides with functions changing as shorter fragments are generated may form. The detection of an endogenous-exogenous pool of regulatory molecules expands the significance and content of the Ashmarin-Obukhova hypothesis on a functional continuum of natural oligopeptides. The possible practical importance of these results is noted.

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.

Structures and functions of about 700 oligopeptides of various plants are presently known. However, only one polypeptide has been isolated from grapes and characterized. At the same time, tens of thousands of uncharacterized amino acid sequences have been revealed in this plant, among which there can also be precursors of oligopeptide regulators. Due to the scientific and practical importance of innate immunity of agricultural plants, we have undertaken structural and functional investigation of these sequences to identify new regulatory oligopeptides including antimicrobial agents. For this purpose, we elaborated a special method of computer analysis enabling comparison of primary structures of putative precursors of grape oligopeptides with amino acid sequences of known oligopeptides of other plants. Structural similarity served as the basis for prediction of potential functional properties. As a result, over 20 new structures of antimicrobial and other grape oligopeptides have been found.

Aim: To analyze prevalence and variability of Legionella strains isolated in town Verkhnaya Pyshma located in Sverdlovsk region during prophylactic surveillance of potentially dangerous water objects in 2007 - 2008., Materials and Methods: Sequencing of mip gene was conducted for identification of species of Legionella. Multi-locus sequence typing was used for describing of allelic profiles of Legionella pneumophila strains., Results: Five firstly identified on Russian territory strains of Legionella species were deposited in institute's collection. Sixty-three strains of L. pneumophila belonging to 28 sequence types were characterized. Relation between strains isolated in industrial building and from water supply system was demonstrated., Conclusion: Observations made on the basis of study of L. pneumophila strains isolated from cooling stacks of industrial plants confirmed potential danger of these objects as a source of dissemination of Legionella infection.

Strains of Legionella pneumophila Pyshma-1 and Pyshma-2 were typed according to the international standard - STB protocol developed by EGWLI. Allelic profile of the strains was determined. Identity of strains on the locus pilE, which codes protein important for virulence of bacterium, was shown. Close similarity of nucleotide sequences in Pyshma-1 and Pyshma-2 strains (mainly, 98-100%) was noted. Both strains differed more from reference strain Philadelphia-1 ATCC 33152. Maximal differences (5-6%) were observed in fragment of mompS gene. The study revealed considerable need for conducting of systemic analysis of collected and newly isolated strains in order to get information picture on endemic strains of L. pneumophila in Russia.

The study is concerned with proapoptotic properties of chimera peptides which incorporate sequences of inhibitors of cyclin kinases p161NK4a and p21CIP/WAF1 as well as internalized sequences (Antp and tat). Sequences of the p16 type appeared to be more cytotoxic than the p21 one. Cytotoxic effect proved dependent on orientation with respect to the C or N terminal point of a polypeptide chain rather than on chimera sequence extent. Although p16 endogenous synthesis did not influence chimera peptide levels, apoptosis did not take place in certain cellular lines. Due to the rather unsophisticated nature of such synthesis, it might be used in designing individually-tailored chemotherapeutic drugs.

Natural oligopeptides may regulate nearly all vital processes. To date, the chemical structures of nearly 6000 oligopeptides have been identified from >1000 organisms representing all the biological kingdoms. We have compiled the known physical, chemical and biological properties of these oligopeptides--whether synthesized on ribosomes or by non-ribosomal enzymes--and have constructed an internet-accessible database, EROP-Moscow (Endogenous Regulatory OligoPeptides), which resides at http://erop.inbi.ras.ru. This database enables users to perform rapid searches via many key features of the oligopeptides, and to carry out statistical analysis of all the available information. The database lists only those oligopeptides whose chemical structures have been completely determined (directly or by translation from nucleotide sequences). It provides extensive links with the Swiss-Prot-TrEMBL peptide-protein database, as well as with the PubMed biomedical bibliographic database. EROP-Moscow also contains data on many oligopeptides that are absent from other convenient databases, and is designed for extended use in classifying new natural oligopeptides and for production of novel peptide pharmaceuticals.

The structure of native and modified uracil DNA glycosylase from E. coli in solution was studied by synchrotron small-angle X-ray scattering. The modified enzyme (6His-uracyl DNA glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence amino acid residues including a block of six His residues. It was found that the conformations of these enzymes in solution at moderate ionic strength (60 mM NaCI) substantially differ in spite of minimal differences in the amino acid sequences and functional activity. The structure of native uracil DNA glycosylase in solution is close to that in crystal, showing a tendency for association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes a partial dissociation of associates and a compactization of protein structure. At the same time, 6His-uracyl DNA glycosylase has a compact structure essentially different from the crystal one. A decrease in the ionic strength of solution results in a partial disruption of compact structure of the modified protein, without changes in its functional activity.

Eleven wild measles virus isolated, in 1988 and in 1999-2001 in the European territory of the Russian Federation, were investigated. On the basis of an analysis of N-gene region sequences, encoding the COOH terminal end of nucleoprotein, the isolates were divided into 2 subgroups. According to the WHO classification, subgroup 1 was in line with genotype A and subgroup 2--with genotype D. Subgroup 2 was close to genotype D4 but differed from it according to its composition of nucleotides on the average by 2.8%, and according to its amino-acid composition--by 2.6%. with respect to the WHO criteria, the latter can be referred to preliminarily as an independent genotype. Finally, the measles viruses' strains of genetic groups A and D circulated in the Russian Federation in 1988, and in 1999-2001.

The search for new pharmaceuticals that are specific for diseased rather than normal cells in the case of cancer and viral disease has raised interest in locally acting drugs that act over short distances within the cell and for which different cell compartments have distinct sensitivities. Thus, photosensitizers (PSs) used in anti-cancer therapy should ideally be transported to the most sensitive subcellular compartments in order for their action to be most pronounced. Here we describe the design, production, and characterization of the effects of bacterially expressed modular recombinant transporters for PSs comprising 1) alpha-melanocyte-stimulating hormone as an internalizable, cell-specific ligand; 2) an optimized nuclear localization sequence of the SV40 large T-antigen; 3) an Escherichia coli hemoglobin-like protein as a carrier; and 4) an endosomolytic amphipathic polypeptide, the translocation domain of diphtheria toxin. These modular transporters delivered PSs into the nuclei, the most vulnerable sites for the action of PSs, of murine melanoma cells, but not non-MSH receptor-overexpressing cells, to result in cytotoxic effects several orders of magnitude greater than those of nonmodified PSs. The modular fusion proteins described here for the first time, capable of cell-specific targeting to particular subcellular compartments to increase drug efficacy, represent new pharmaceuticals with general application.

A gene (No. AF0497 GenBank, USA) was cloned from the archaeon Archaeoglobus fulgidus strain found in the water of hot springs. This gene contains an open reading frame of 2346 base pairs which encodes a thermostable DNA-polymerase (762 amino acid residues). A recombinant protein Afu-pol with molecular weight of 94 kD was isolated in an Escherichia coli strain used as a producer and characterized. By site-directed mutagenesis in the afu-pol gene the amino acid residue Glu170 was replaced with Ala; this resulted in a complete loss of the 3;-5;-exonuclease activity of the enzyme. Thus, the Glu170 residue was suggested to be directly involved in formation of the 3;-5;-exonuclease site. Physicochemical features of the exodeficient enzyme form were studied, and the possible use of Afu(exo(-))-pol in the polymerase chain reaction is shown.

A number of drugs are regarded as possessing local activity because their effects take place at an extremely short distance from their location site in the cell. The response of different cellular compartments to these effects is different. Such substances as photosensitizers (PSs), which are used in photodynamic cancer therapy, should be targeted to the cell compartments where their effect is the most pronounced. This study describes the construction and properties of the chimeric modular recombinant transporters (MRTs) expressed in Escherichia coli and used for PS targeting. These constructs include (1) the alpha-melanocyte-stimulating hormone as a ligand module, which is internalized by the target cells (mouse melanoma); (2) the optimized SV40 large T-antigen nuclear localization signal; (3) the hemoglobin-like protein from E. coli as a carrier module; (4) the endosomolytic module, the translocation domain of the diphtheria toxin. These MRTs were used for PS targeting to the mouse melanoma cell nuclei, the most PS-damaged intracellular compartment, which resulted in a PS photocytotoxic effect increase of several orders of magnitude. In our opinion, MRTs, which target locally active drugs into the desired cell compartment and thereby enhance the drug response, represent a new generation of the pharmacological agents.

The secondary structure of DNA aptamer to Taq DNA polymerase was established as a hairpin. Both stem and loop structures of DNA ligand were shown to be involved in the interaction with Taq DNA polymerase. Moreover, the structure and sequence of DNA aptamer that was the most effective inhibitor of DNA polymerase activity were established. This crucial structure was evaluated as a GC-rich stem longer than 17 bp, and a loop consisting of 12 bases with strictly determined nucleotide sequence. It was demonstrated that nucleotide in position 23 counting from the 5;-end of DNA ligand was involved in direct contact with Taq DNA polymerase. The ability of optimized DNA aptamer TQ21-11 to form a complex with the enzyme was increased 5-fold in comparison to the initial aptamer.

A new method for the identification of point mutations is proposed. The method is based on ligase chain reaction (LCR) and it includes a procedure for correction of ligation by Cleavase. Reaction products are detected by a colorimetric method after adsorption of the resulting DNA duplexes to the solid phase. One strand of LCR products carries biotin to be bound on a streptavidin-coated microwell. Another strand contains a single-stranded region that is to be coupled with an oligonucleotide carrying a substrate for colorimetric detection. The suggested method has two advantages: (i) use of Cleavase increases the accuracy of ligation and (ii) a template independent ligation does not occur in LCR due to a special design of primers.

The common chemical features of oligopeptide releasing-hormones and release inhibiting hormones were investigated with the aid of computer methods. 339 regulatory molecules of such type have been extracted out of data from computer bank EROP-Moscow. They contain from 2 to 47 amino acid residues and their sequences include short sites, which play apparently a decisive role in realization of interactions with the receptors. The analysis of chemical radicals shows that all liberins and statins contain positively charged group and cyclic radical of some amino acids or hydrophobic group. Results of this study indicate that the most chemical radicals of hormones are open for the interaction with potential receptors of target-cells. The mechanism of hormone ligand and receptors binding and conceivable role of amino acid and neurotransmitter radicals in hormonal properties of liberins and statins is discussed.

Evidence for the localization of up to 25-30% of the total glutamate dehydrogenase activity in rabbit liver cell hyaloplasm has been obtained. Differences were revealed in the properties of glutamate dehydrogenase from the hyaloplasm and mitochondria. The ratios of activities in the amination/deamination reactions as measured at optimal values of pH for the mitochondrial and liver enzymes were 3.7 and 1.0, respectively. The enzyme preparations isolated from the both fractions appeared to be electrophoretically homogeneous and had a subunit molecular mass of about 56 +/- 2 kDa. Fresh preparations of mitochondrial and hyaloplasm liver glutamate dehydrogenase contained several oligomeric forms, predominantly with M of 350 and 280 kDa, respectively. Data from gel-filtration on Sephacryl S-300 are suggestive of a complex (M = 800 kDa) formation between liver hyaloplasm glutamate dehydrogenase (280 kDa) and NAD-kinase (440 kDa); this reaction is accompanied by simultaneous activation of these enzymes (3.5- and 2.4-fold, respectively).

It has been shown that rabbit liver NAD-kinase is a Ca(2+)-dependent, calmodulin-regulated enzyme. Differences were found between the sensitivity of the alpha- and beta-forms of the enzyme to calmodulin. The maximal activation of the NAD-kinase alpha-form was observed at a calmodulin/NAD-kinase ratio equal to 2, that of the beta-form--at 4. Further increases in the above parameter (up to 10) had no effect on the activity of the alpha-form, whereas that of the beta-form dropped down to the initial (basal) level.

An electrophoretically homogeneous preparation of a NAD-kinase activator from rabbit liver was obtained and its physico-chemical properties were investigated. The molecular mass of the monomer and oligomer, pI, number of SH-groups per enzyme subunit and some other factors were determined. The similarity of activator properties to those of glutamate dehydrogenase and the revealed glutamate dehydrogenase activity of the NAD-kinase activator permitted to identify the latter as glutamate dehydrogenase. It was demonstrated that the enzyme activates NAD-kinase 2-4 times already at the glutamate dehydrogenase: NAD-kinase ratio of 2:1. The effect of glutamate dehydrogenase on the enzyme consists in an increase of Vmax; the KmNAD value for the NAD-kinase reaction remains thereby unchanged. The physiological role of the interaction between the two enzymes is discussed.