[Sweet protein brazzein as a promising sweetener].

The excessive consumption of sugar-containing foods contributes to the development of a number of diseases, including obesity, diabetes mellitus, etc. As a substitute for sugar, people with diabetes mellitus and obesity most often use sweeteners. Sweet proteins, in particular brazzein, are an alternative to synthetic sweeteners that have natural origin, are broken down in the intestines along with food proteins, and do not affect blood sugar and insulin levels. The purpose of the review was to analyze the available data on the sweet protein brazzein, its physical and chemical properties, existing biotechnological methods of production, and prospects for application in the food industry in order to further develop an optimized heterologous expression system. Material and methods. Google Scholar, Scopus, Web of Science, PubMed, RSCI and eLibrary.ru databases were used for collecting and analyzing literature. Search depth - 30 years. Results. Numerous studies of the physical and chemical properties of brazzein have demonstrated its high potential for use in the food industry. In particular, a short amino acid sequence, thermal stability, the ability to maintain its structure and sweet properties in a wide pH range, hypoallergenicity, lack of genotoxicity, and an extremely high level of sweetness compared to sucrose allow us to conclude that its use is promising. Mutant variants of brazzein have been generated, the sweetest of which (with three amino acid substitutions H31R/E36D/E41A) exceeds sucrose sweetness by 22 500 times. To date, various systems for the expression of recombinant brazzein have already been developed, in which bacteria (Escherichia coli, Lactococcus lactis, Bacillus licheniformis), yeast (Komagataella phaffii, Kluyveromyces lactis, Saccharomyces cerevisiae), plants (Zea mays, Oryza sativa, Lactuca sativa, Nicotiana tabacum, Daucus carota) and animals (Mus musculus) have been used. Conclusion. Due to its high sweetness, organoleptic properties and long history of human consumption, brazzein can be considered as a promising natural sweetener. Despite the short peptide sequence, the production of the recombinant protein faced a number of problems, including low protein yield (for example, it could only be detected in mouse milk by Western blot hybridization) and loss of sweetness. Thus, further optimization of the process is necessary for widespread brazzein use in the food industry, which includes the selection of an adequate producer and the use of extracellular expression systems to reduce the final cost of the product.

[Role of syndecan-2 in amyloid plaque formation].

The famous phrase of F. Engels "Life is the mode of existence of protein bodies", has deeply insinuated itself in our mind. However at a more profound insight, the form of protein bodies is associaited not only with the fact of their existence, but also with the time changes. What unites all of us in our oldage? The answer is clear: it is the change in the way of existence of protein molecules, and more precisely, their uncontrolled aggregation that can take place in any organ and be associated with any protein. In spite of different clinical presentations, all diseases associated with pathological accumulation of aggregated proteins are combined in a general group called amyloisosis. Depen- dent on the place of formation, it is possible to distinguish an infinite number of pathologies from neurodegen- erative and oncologic ones to arthritis and tuberculosis. There is no doubt that provided all clandestine mechanisms are clarified at which an absolutely normal functioning.protein can transform into a pathological aggregated form, it will give us a chance to prevent protein aggregation and create a new form of drugs for prolongation of life. In this review we considered the function of syndecan-2, the structure of syndecan-2 and its role in the formation of amyloid plaques.

[EFFECTIVENESS OF FULLERENE-(TRIS-AMINOCAPRONIC ACID) HYDRATE IN THE MODEL OF EXPERIMENTAL VIRAL-BACTERIAL PNEUMONIA OF MICE].

AIM: Study the effectiveness of the substance and various drug formulations of fullerene-(tris-aminocapronic acid) hydrate (FTAAH onwards) in the model of experimental viral-bacterial pneumonia of mice. MATERIALS AND METHODS: BALB/c mice were infected with influenza virus A/California/04/2009 and subsequently infected with Staphylococcus aureus. The animals were treated after viral infection with the substance and various drug forms of FTAAH, as well as comparative preparations--oseltamivir and arbidol. Therapy effectiveness was evaluated by clinical indicators (survival, lifespan, animal mass decrease reduction), virological (virus titer), microbiological (density of bacteria in lungs) parameters, confirmed by pathomorphological characteristics of lungs. RESULTS: FTAAH therapy in injectable form was effective in the model of a combined viral-bacterial pneumonia of mice by all the studied criteria: treatment increased mice survival, reduced the decrease of their body weight, resulted in a reduction of virus titers and density of bacteria in lungs, that correlated with the data from morphological study and signs of bronchopneumonia resolution in mice. FTAAH therapy in rectal form depended on animal infection schemes, as well as preparation dose, increasing with its increase. CONCLUSION: FTAAH substance is effective in the model of experimental viral-bacterial pneumonia of mice.

Mechanisms of amyloid fibril formation.

Amyloid and amyloid-like aggregates are elongated unbranched fibrils consisting of ß-structures of separate monomers positioned perpendicular to the fibril axis and stacked strictly above each other. In their physicochemical properties, amyloid fibrils are reminiscent of synthetic polymers rather than usual proteins because they are stable to the action of denaturing agents and proteases. Their mechanical stability can be compared to a spider's web, that in spite of its ability to stretch, is stronger than steel. It is not surprising that a large number of diseases are accompanied with amyloid fibril depositing in different organs. Pathologies provoked by depositing of incorrectly folded proteins include Alzheimer's, Parkinson's, and Huntington's diseases. In addition, this group of diseases involves mucoviscidosis, some types of diabetes, and hereditary cataracts. Each type of amyloidosis is characterized by aggregation of a certain type of protein that is soluble in general, and thus leads to specific distortions of functions of the corresponding organs. Therefore, it is important to understand the process of transformation of "native" proteins to amyloid fibrils to clarify how these molecules acquire such strength and what key elements of this process determine the pathway of erroneous protein folding. This review presents our analysis of complied information on the mechanisms of formation and biochemical properties of amyloid fibrils.

[Umifenovir (Arbidol) efficacy in experimental mixed viral and bacterial pneumonia of mice].

Pneumonia often occurs as a secondary infection after influenza and accounts for a large proportion of the morbidity and mortality associated with seasonal and pandemic influenza outbreaks. The efficacy of umifenovir (Arbidol) was investigated on a murine model of S. aureus pneumonia following A/California/04/2009 (H1N1) influenza virusinfection. Oral treatment with umifenovir (40 and 60 mg/kg/day) in all the contamination schemes increased the survival rate in the mice from 0% to 90% and lowered the animal weight loss. The umifenovir treatment also decreased the virus titer by ≥ 2 logs and the viable bacteria counts in the lungs of the mice. The lungs of the mice treated with umifenovir had less severe histopathologic lesions compared to the control group.

Structure and functions of syndecans in vertebrates.

Syndecans constitute a family of transmembrane proteoglycans that perform multiple functions during development, damage repair, tumor growth, angiogenesis, and neurogenesis. Through mediating binding of a great number of extracellular ligands to their receptors, these proteoglycans trigger a cascade of reactions regulating, thereby, various processes in a cell: cytoskeleton formation, proliferation, differentiation, adhesion, and migration. In fibroblasts, syndecans are responsible for cell adhesion by modulating functions of integrins through interaction with fibronectin at the external side of a cell and with cytoskeleton and signaling molecules inside the cell. The extracellular domain of syndecans is subjected to periodic shedding from the cell membrane. This process may be stimulated in response to inflammation, tissue damage, and other pathological manifestations. Cleaved domain may act as either competitive inhibitor or activator of signaling cascades. This review summarizes and analyzes the available data regarding structure, main biochemical properties, and functions of syndecans in vertebrates.

[Comparative characteristics of the structure and function for syndecan-1 from animal organisms].

Syndecan-1 is a general representative of transmembrane proteoglycans known for their ability to perform diverse functions in multicellular organisms: to influence on cell interactions, to be act as a co-receptor and bind with to a large number ofligands and integrate them on the receptors, to fulfill the protective function, and to affect on wound repair and oncogenesis. Moreover, syndecan-1 as similar to the extracellular matrix plays an essential role in the development of organisms. Such a diversity of functions is associated with its structure. In this work we demonstrate that intracellular and intercellular domains of syndecan-1 are intrinsically disordered regions. Such structural feature allows them to bind with adapter proteins in the cytoplasm and be extended with glycosaminoglicans in the extracellular matrix, and to take part in the diverse and important cellular processes. The comparison of the occurrence of 20 amino acids for syndecan-1 from 32 animal organisms and 17 animal proteomes demonstrated that for the first such amino acids as glycine, treonine, glutamine, glutamic acid, and proline predominate on amount in the content of the former that results to the appearance of disordered regions in the proteins.

[Formation of spatial structure of RNA molecules].

In this review we consider several experimental and theoretical approaches for investigation of RNA folding and determination of nucleotides that play an important role upon folding of such molecules as tRNA and several classes of ribozymes. It has been shown that nucleotides in the D- and T-loop regions are the last to be involved in tRNA structure or they are not included in the folding nucleus of tRNA. Using the specially elaborated method SHAPE it has been demonstrated that the model of hierarchical folding which was recognized for a long time is not correct for tRNA folding. In the second part of the given review the algorithms and programs used for the prediction of secondary structures of RNA as well as for modeling of RNA folding are considered.

[Production of hybrid protein OprF-OprL of Pseudomonas aeruginosa].

AIM: Production of preparation consisting of amino acid sequences of 2 proteins of outer membrane--OprF and OprI--of P. aeruginosa and study of its protective properties from experimental P. aeruginosa infection. MATERIALS AND METHODS: Nucleotide sequences coding OprF protein (1 kb) as well as its C-terminal region (0.6 kb) and OprI protein (0.25 kb) were integrated into pQE-30 plasmid (QIAGEN). And oprF gene (C-terminal region of oprF in variant 2) and oprI gene were combined and cloned sequentially into a single vector. E. coli M15 strain cells (QIAGEN) were used for the production of producent strains of recombinant proteins. Protein products were analyzed by electrophoresis in polyacrylamide gel by Lammle. Purification of recombinant proteins was performed by affinity chromatography in Ni-sepharose columns. Live virulent culture P. aeruginosa PA-170015 strain was used for the analysis of protective properties of recombinant proteins. RESULTS: 2 hybrid recombinant proteins were produced including amino acid sequences of F and I proteins of outer membrane (OprF and OprI) of P. aeruginosa. Recombinant protein 1 included whole size sequences of OprF and OprI and protein 2--C-terminal region (including amino acid residues 192-342) of OprF and whole size sequence of OprI. These recombinant proteins after 2 immunizations protected mice from the experimental intraperitoneal infection with P. aeruginosa. Hybrid protein consisting of whole size sequences had the best protective effect. CONCLUSION: The results obtained open a perspective for further immunobiological testing of hybrid recombinant protein OprF-OprI with the aim of creating immunopreparations for prophylaxis of P. aeruginosa infection.

[Analysis of biological properties of strains-producers of a number of Pseudomonas aeruginosa recombinant proteins].

AIM: Study of biological properties of strains-producers of a number of Pseudomonas aeruginosa recombinant proteins compared with the initial strains. MATERIALS AND METHODS: 5 Escherichia coli strains (initial strains E. coli M15 and E. coli BL21 (DE3) and strains-producers of OprL, OprF, aTox3 recombinant proteins (E. coli M15/oprF, E. coli M 15/oprL, E. coli BL21-aTox3) were studied. Toxicity, toxigenicity and virulence determination were carried out in experiments in non-linear mice. Enzymatic properties of the initial strains and strains-producers were compared in a number of biochemical tests. RESULTS: The studied strains-producers of the most immunogenic P. aeruginosa recombinant proteins were confirmed to be biosafe, belong to Enterobacteriaceae family and Escherichia genus. As a result of genetic engineering manipulations carried out with E. coli M15 and E. coli BL21(DE3) alterations ofbiochemical and growth properties, virulence, toxicity and toxigenicity in the constructed strains-producers of recombinant proteins - E. coli M 15/oprF, E. coli M15/oprL, E. coli BL21-aTox3 - were not detected. CONCLUSION: The results obtained allow to consider the possibility of use of E. coli M 15/oprF, E. coli M15/oprL and E. coli BL21-aTox3 strains-producers for production and isolation of candidate proteins for inclusion into vaccine against pseudomonas infection.

Prediction of folding nuclei in tRNA molecules.

Prediction of folding nuclei in RNA molecules allows one to look in a new way at the problem of possible RNA base sequence folding and at problems associated with incorrect RNA folding, as well as at RNA structure stability. We have chosen a model and energy parameters for description of RNA structure. The algorithm for studying processes including protein folding/unfolding was successfully applied to calculations on tRNA. Four tRNA molecules were considered whose structures were obtained in the free state (tRNA(Phe), tRNA(Asp), tRNA(fMet), and tRNA(Lys)). The calculated Φ-values for tRNA molecules correlate with experimental data showing that nucleotide residues in the D and T hairpin regions are involved in tRNA structure last, or more exactly, they are not included in the tRNA folding nucleus. High Φ-values in the anticodon hairpin region show that the nucleus of tRNA folding is localized just in that place.