Vol 7, No 4 (2015)


Assessment of the Volume of State Funding for the Development of Biomedicine in Russia and in the USA

Petrov A.N., Kurakova N.G.


This article demonstrates that Russia’s funding for research and development is less than 2.5 % of global funding, whereas the amount of financing of just three countries, the USA, China, and Japan amounts to 50%. It is argued that the inadequacy of Russia’s domestic financing for the development of the science sector vis a vis that of developed countries allows the country to prioritize only a limited number of research fields in its scientific and technological development. We have compared and contrasted expenditures on research and development in biomedicine in the USA and Russia. It has been demonstrated that in 2014, basic funding for 27 research centers included in the US National Health Institutes’ network exceeded the amount of financing for 104 Russian medical scientific and research institutes subordinated to the Russian Ministry of Health and Federal Agency of Scientific Organizations by 173 times. We have concluded that a substantial increase in state funding for fundamental, exploratory, and applied research in the field of biomedicine is required if life sciences are to be preserved as one of the priorities in the scientific-technological and social development of Russia. It is also necessary to eliminate all administrative and tax barriers that prevent active participation of domestic industrial entities in the co-financing of the development of Russian drugs and medical equipment.

Acta Naturae. 2015;7(4):4-10
pages 4-10 views


Broadly Neutralizing Antibodies against HIV-1 As a Novel Aspect of the Immune Response

Shcherbakov D.N., Bakulina A.Y., Karpenko L.I., Ilyichev A.A.


The human immunodeficiency virus-1 (HIV-1) has the ability to evade the adaptive immune response due to high mutation rates. Soon after the discovery of HIV-1, it was originally proposed that neutralizing of antibodies to the virus occurs rarely or cannot be elicited at all. In the 1990s, there appeared reports that sera of select HIV-1-infected individuals contained antibodies capable of neutralizing different virus subtypes. Such antibodies were named broadly neutralizing antibodies (bNAbs). Since 2009, the development of new cell technologies has intensified research efforts directed at identifying new bNAbs with a neutralization potency of over 90% of primary HIV-1 isolates. These antibodies have unique characteristics which include high levels of somatic mutations and unusually long variable loops that penetrate through the glycan shield of HIV-1 Env to contact the protein surface. In this review, we will attempt to summarize the latest data on bNAbs against HIV-1 in terms of their interactions with the sites of vulnerability on HIV-1 glycoproteins.

Acta Naturae. 2015;7(4):11-21
pages 11-21 views

Survival guide: Escherichia coli in the stationary phase

Pletnev P.I., Osterman I.А., Sergiev P.V., Bogdanov A.А., Dontsova O.А.


This review centers on the stationary phase of bacterial culture. The basic processes specific to the stationary phase, as well as the regulatory mechanisms that allow the bacteria to survive in conditions of stress, are described.

Acta Naturae. 2015;7(4):22-33
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Study of Functional and Allosteric Sites in Protein Superfamilies

Suplatov D.А., Švedas V.К.


The interaction of proteins (enzymes) with a variety of low-molecular-weight compounds, as well as protein-protein interactions, is the most important factor in the regulation of their functional properties. To date, research effort has routinely focused on studying ligand binding to the functional sites of proteins (active sites of enzymes), whereas the molecular mechanisms of allosteric regulation, as well as binding to other pockets and cavities in protein structures, remained poorly understood. Recent studies have shown that allostery may be an intrinsic property of virtually all proteins. Novel approaches are needed to systematically analyze the architecture and role of various binding sites and establish the relationship between structure, function, and regulation. Computational biology, bioinformatics, and molecular modeling can be used to search for new regulatory centers, characterize their structural peculiarities, as well as compare different pockets in homologous proteins, study the molecular mechanisms of allostery, and understand the communication between topologically independent binding sites in protein structures. The establishment of an evolutionary relationship between different binding centers within protein superfamilies and the discovery of new functional and allosteric (regulatory) sites using computational approaches can improve our understanding of the structure-function relationship in proteins and provide new opportunities for drug design and enzyme engineering.

Acta Naturae. 2015;7(4):34-45
pages 34-45 views

Cancer Stem Cells: Plasticity Works against Therapy

Vinogradova T.V., Chernov I.P., Monastyrskaya G.S., Kondratyeva L.G., Sverdlov E.D.


Great successes in identification and deciphering of mechanisms of the adult stem cells regulation have given rise to the idea that stem cells can also function in tumors as central elements of their development, starting from the initial stage and continuing until metastasis. Such cells were called cancer stem cells (CSCs). Over the course of intense discussion, the CSCs hypothesis gradually began to be perceived as an obvious fact. Recently, the existence of CSCs has been indeed confirmed in a number of works. However, when are CSCs universal prerequisites of tumors and to what extent their role is essential for tumor evolution remains an issue far from resolved. Likewise, the problem of potential use of CSCs as therapeutic targets remains unsolved. The present review attempts to analyze the issue of cancer stem cells and the potential of targeting them in tumor therapy.

Acta Naturae. 2015;7(4):46-55
pages 46-55 views

Research Articles

Induced Pluripotent Stem Cells of Microtus levis x Microtus arvalis Vole Hybrids: Conditions Necessary for Their Generation and Self-Renewal

Grigor’eva E.V., Shevchenko A.I., Medvedev S.P., Mazurok N.A., Zhelezova A.И., Zakian S.M.


Every year, the list of mammalian species for which cultures of pluripotent stem cells (PSCs) are generated increases. PSCs are a unique tool for extending the limits of experimental studies and modeling different biological processes. In this work, induced pluripotent stem cells (iPSCs) from the hybrids of common voles Microtus levis and Microtus arvalis, which are used as model objects to study genome organization on the molecular-genetic level and the mechanisms of X-chromosome inactivation, have been generated. Vole iPSCs were isolated and cultured in a medium containing cytokine LIF, basic fibroblast growth factor (bFGF), ascorbic acid, and fetal bovine serum. Undifferentiated state of vole iPSCs is maintained by activation of their endogenous pluripotency genes - Nanog, Oct4, Sox2, Sall4, and Esrrb. The cells were able to maintain undifferentiated state for at least 28 passages without change in their morphology and give rise to three germ layers (ectoderm, mesoderm and endoderm) upon differentiation.

Acta Naturae. 2015;7(4):56-69
pages 56-69 views

Modifiers of the Dipole Potential of Lipid Bilayers

Efimova S.S., Ostroumova O.S.


This paper assesses the magnitude of change in the dipole potential (φd) of membranes caused by the adsorption of modifiers on lipid bilayers of various compositions. We tested flavonoids, muscle relaxants, thyroid hormones, and xanthene and styrylpyridinium dyes in order to assess their dipole-modifying properties. A quantitative description of the modifying action of flavonoids, muscle relaxants, thyroid hormones, and xanthene dyes is shown as the ratio of the maximum change in the bilayer dipole potential upon saturation and the absolute φd value of the unmodified membrane. The slopes of the linear relationship between the increase in the dipole potential of phospholipid bilayers and the concentration of styrylpyridinium dyes in membrane-bathing solutions were found. We described the relationships between the change in φd and the chemical structure of modifiers, as well as the charge and spontaneous curvature of lipid monolayers.

Acta Naturae. 2015;7(4):70-79
pages 70-79 views

Valproic Acid Increases the Hepatic Differentiation Potential of Salivary Gland Cells

Petrakova O.S., Ashapkin V.V., Shtratnikova V.Y., Kutueva L.I., Vorotelyak E.A., Borisov M.A., Terskikh V.V., Gvazava I.G., Vasiliev A.V.


The studies of cell plasticity and differentiation abilities are important problems in modern cellular biology. The use of histone deacetylase inhibitor - valproic acid is a promising approach to increasing the differentiation efficiency of various cell types. In this paper we investigate the ability of mouse submandibular salivary gland cells to differentiate into the hepatic direction and the effect of valproic acid on the efficiency of this differentiation. It was shown that the gene expression levels of hepatocyte markers (Aat, Afp, G6p, Pepck, Tat, Cyp3a13) and liver-enriched transcription factors (Hnf-3α, Hnf-3β, Hnf-4α, Hnf-6) were increased after differentiation in salivary gland cells. Valproic acid increases the specificity of hepatic differentiation, reducing the expression levels of the ductal (Krt19, Hhex1, Cyp7a1) and acinar (Ptf1a) markers. After valproic acid exposure, the efficiency of hepatic differentiation also increases, as evidenced by the increase in the gene expression level of Alb and Tdo, and increase in urea production by differentiated cells. No change was found in DNA methylation of the promoter regions of the genes; however, valproic acid treatment and subsequent hepatic differentiation largely affected the histone H3 methylation of liver-enriched genes. Thus, mouse submandibular salivary gland cells are capable of effective differentiation in the hepatic direction. Valproic acid increases the specificity and efficiency of the hepatic differentiation of these cells.

Acta Naturae. 2015;7(4):80-92
pages 80-92 views

Recombinant Immunotoxin 4D5scFv-PE40 for Targeted Therapy of HER2-Positive Tumors

Sokolova E.A., Stremovskiy O.A., Zdobnova T.A., Balalaeva I.V., Deyev S.M.


Recombinant immunotoxins are extremely promising agents for the targeted therapy of tumors with a certain molecular profile. In this work, we studied the properties of a new recombinant HER2-specific immunotoxin composed of the scFv antibody and a fragment of Pseudomonas exotoxin A (4D5scFv-PE40). High affinity of the immunotoxin for the HER2 tumor marker, its selective cytotoxicity against HER2-overexpressing cells, and its storage stability were demonstrated. The 50% inhibitory concentration (IC50) of the 4D5scFv-PE40 immunotoxin for HER2-overexpressing cancer cells was 2.5-3 orders of magnitude lower compared to that for CHO cells not expressing this tumor marker and was 2.5-3 orders of magnitude lower than IC50 of free PE40 for HER2-overexpressing cancer cells. These findings provide a basis for expecting in the long run high therapeutic index values of the 4D5scFv-PE40 immunotoxin for its use in vivo.

Acta Naturae. 2015;7(4):93-96
pages 93-96 views

A Model System in S2 Cells to Test the Functional Activities of Drosophila Insulators

Tikhonov M.V., Gasanov N.B., Georgiev P.G., Maksimenko O.G.


Insulators are a special class of regulatory elements that can regulate interactions between enhancers and promoters in the genome of high eukaryotes. To date, the mechanisms of insulator action remain unknown, which is primarily related to the lack of convenient model systems. We suggested studying a model system which is based on transient expression of a plasmid with an enhancer of the copia transposable element, in Drosophila embryonic cell lines. We demonstrated that during transient transfection of circle plasmids with a well-known Drosophila insulator from the gypsy retrotransposon, the insulator exhibits in an enhancer-blocking assay the same properties as in Drosophila stable transgenic lines. Therefore, the Drosophila cell line is suitable for studying the main activities of insulators, which provides additional opportunities for investigating the functional role of certain insulator proteins.

Acta Naturae. 2015;7(4):97-106
pages 97-106 views

Profiling of Mycoplasma gallisepticum Ribosomes

Fisunov G.Y., Evsyutina D.V., Arzamasov A.A., Butenko I.O., Govorun V.M.


The development of high-throughput technologies is increasingly resulting in identification of numerous cases of low correlation between mRNA and the protein level in cells. These controversial observations were made on various bacteria, such as E. coli, Desulfovibrio vulgaris, and Lactococcus lactis. Thus, it is important to develop technologies, including high-throughput techniques, aimed at studying gene expression regulation at the level of translation. In the current study, we performed proteomic profiling of M. gallisepticum ribosomes and identified high abundant noncanonical proteins. We found that binding of mRNAs to ribosomes is mainly determined by two parameters: (1) abundance of mRNA itself and (2) complimentary interactions between the 3’ end of 16S rRNA and the ribosome binding site in the 5’-untranslated region of mRNA.

Acta Naturae. 2015;7(4):107-112
pages 107-112 views

Attenuation of Vaccinia Virus

Yakubitskiy S.N., Kolosova I.V., Maksyutov R.A., Shchelkunov S.N.


Since 1980, in the post-smallpox vaccination era the human population has become increasingly susceptible compared to a generation ago to not only the variola (smallpox) virus, but also other zoonotic orthopoxviruses. The need for safer vaccines against orthopoxviruses is even greater now. The Lister vaccine strain (LIVP) of vaccinia virus was used as a parental virus for generating a recombinant 1421ABJCN clone defective in five virulence genes encoding hemagglutinin (A56R), the IFN-γ-binding protein (B8R), thymidine kinase (J2R), the complement-binding protein (C3L), and the Bcl-2-like inhibitor of apoptosis (N1L). We found that disruption of these loci does not affect replication in mammalian cell cultures. The isogenic recombinant strain 1421ABJCN exhibits a reduced inflammatory response and attenuated neurovirulence relative to LIVP. Virus titers of 1421ABJCN were 3 lg lower versus the parent VACV LIVP when administered by the intracerebral route in new-born mice. In a subcutaneous mouse model, 1421ABJCN displayed levels of VACV-neutralizing antibodies comparable to those of LIVP and conferred protective immunity against lethal challenge by the ectromelia virus. The VACV mutant holds promise as a safe live vaccine strain for preventing smallpox and other orthopoxvirus infections.

Acta Naturae. 2015;7(4):113-121
pages 113-121 views

Role of the Lipid Environment in the Dimerization of Transmembrane Domains of Glycophorin A

Kuznetsov A.S., Volynsky P.E., Efremov R.G.


An efficient computational approach is developed to quantify the free energy of a spontaneous association of the α-helices of proteins in the membrane environment. The approach is based on the numerical decomposition of the free energy profiles of the transmembrane (TM) helices into components corresponding to protein-protein, protein-lipid, and protein-water interactions. The method was tested for the TM segments of human glycophorin A (GpA) and two mutant forms, Gly83Ala and Thr87Val. It was shown that lipids make a significant negative contribution to the free energy of dimerization, while amino acid residues forming the interface of the helix-helix contact may be unfavorable in terms of free energy. The detailed balance between different energy contributions is highly dependent on the amino acid sequence of the TM protein segment. The results show the dominant role of the environment in the interaction of membrane proteins that is changing our notion of the driving force behind the spontaneous association of TM α-helices. Adequate estimation of the contribution of the water-lipid environment thus becomes an extremely urgent task for a rational design of new molecules targeting bitopic membrane proteins, including receptor tyrosine kinases.

Acta Naturae. 2015;7(4):122-127
pages 122-127 views

Sulfoxides, Analogues of L-Methionine and L-Cysteine As Pro-Drugs against Gram-Positive and Gram-Negative Bacteria

Anufrieva N.V., Morozova E.A., Kulikova V.V., Bazhulina N.P., Manukhov I.V., Degtev D.I., Gnuchikh E.Y., Rodionov A.N., Zavilgelsky G.B., Demidkina T.V.


The problem of resistance to antibiotics requires the development of new classes of broad-spectrum antimicrobial drugs. The concept of pro-drugs allows researchers to look for new approaches to obtain effective drugs with improved pharmacokinetic and pharmacodynamic properties. Thiosulfinates, formed enzymatically from amino acid sulfoxides upon crushing cells of genus Allium plants, are known as antimicrobial compounds. The instability and high reactivity of thiosulfinates complicate their use as individual antimicrobial compounds. We propose a pharmacologically complementary pair: an amino acid sulfoxide pro-drug and vitamin B6 - dependent methionine γ-lyase, which metabolizes it in the patient’s body. The enzyme catalyzes the γ- and β-elimination reactions of sulfoxides, analogues of L-methionine and L-cysteine, which leads to the formation of thiosulfinates. In the present work, we cloned the enzyme gene from Clostridium sporogenes. Ionic and tautomeric forms of the internal aldimine were determined by lognormal deconvolution of the holoenzyme spectrum and the catalytic parameters of the recombinant enzyme in the γ- and β-elimination reactions of amino acids, and some sulfoxides of amino acids were obtained. For the first time, the possibility of usage of the enzyme for effective conversion of sulfoxides was established and the antimicrobial activity of thiosulfinates against Gram-negative and Gram-positive bacteria in situ was shown.

Acta Naturae. 2015;7(4):128-135
pages 128-135 views

Chemical Polysialylation and In Vivo Tetramerization Improve Pharmacokinetic Characteristics of Recombinant Human Butyrylcholinesterase-Based Bioscavengers

Terekhov S.S., Smirnov I.V., Shamborant O.G., Bobik T.V., Ilyushin D.G., Murashev A.N., Dyachenko I.A., Palikov V.A., Knorre V.D., Belogurov A.A., Ponomarenko N.A., Kuzina E.S., Genkin D.D., Masson P., Gabibov A.G.


Organophosphate toxins (OPs) are the most toxic low-molecular compounds. The extremely potent toxicity of OPs is determined by their specificity toward the nerve system. Human butyrylcholinesterase (hBChE) is a natural bioscavenger against a broad spectrum of OPs, which makes it a promising candidate for the development of DNA-encoded bioscavengers. The high values of the protective index observed for recombinant hBChE (rhBChE) make it appropriate for therapy against OP poisoning, especially in the case of highly toxic warfare nerve agents. Nevertheless, large-scale application of biopharmaceuticals based on hBChE is restricted due to its high cost and extremely rapid elimination from the bloodstream. In the present study, we examine two approaches for long-acting rhBChE production: I) chemical polysialylation and II) in-vivo tetramerization. We demonstrate that both approaches significantly improve the pharmacokinetic characteristics of rhBChE (more than 5 and 10 times, respectively), which makes it possible to use rhBChE conjugated with polysialic acids (rhBChE-CAO) and tetrameric rhBChE (4rhBChE) in the treatment of OP poisonings.

Acta Naturae. 2015;7(4):136-141
pages 136-141 views

Short communications

2-(2,4-Dioxy-1,2,3,4-Tetrahydropyrimidin-1-yl)-N-(4-Phenoxyphenyl)-Acetamides as a Novel Class of Cytomegalovirus Replication Inhibitors

Babkov D.A., Paramonova M.P., Ozerov A.A., Khandazhinskaya A.L., Snoeck R., Andrei G., Novikov M.S.


A series of novel uracil derivatives, bearing N-(4-phenoxyphenyl)acetamide moiety at N3 of a pyrimidine ring, has been synthesized. Their antiviral activity has been evaluated. It has been found that the novel compounds possess high inhibitory activity against replication of human cytomegalovirus (AD-169 and Davis strains) in HEL cell cultures. In addition, some of the derivatives proved to be inhibitory against varicella zoster virus.

Acta Naturae. 2015;7(4):142-145
pages 142-145 views

Mutations in the Parkinson’s Disease-Associated PARK2 Gene Are Accompanied by Imbalance in Programmed Cell Death Systems

Konovalova E.V., Lopacheva O.M., Grivennikov I.A., Lebedeva O.S., Dashinimaev E.В., Khaspekov L.G., Fedotova E.Y., Illarioshkin S.N.


Parkinson’s disease is caused by the degeneration of midbrain dopaminergic neurons. A rare recessive form of the disease may be caused by a mutation in the PARK2 gene, whose product, Parkin, controls mitophagy and programmed cell death. The level of pro- and anti-apoptotic factors of the Bcl-2 family was determined in dopaminergic neurons derived from the induced pluripotent stem cells of a healthy donor and a Parkinson’s disease patient bearing PARK2 mutations. Western blotting was used to study the ratios of Bax, Bak, Bcl-2, Bcl-XL, and Bcl-W proteins. The pro-apoptotic Bak protein level in PARK2-neurons was shown to be two times lower than that in healthy cells. In contrast, the expression of the anti-apoptotic factors Bcl-XL, Bcl-W, and Bcl-2 was statistically significantly higher in the mutant cells compared to healthy dopaminergic neurons. These results indicate that PARK2 mutations are accompanied by an imbalance in programmed cell death systems in which non-apoptotic molecular mechanisms play the leading role.

Acta Naturae. 2015;7(4):146-149
pages 146-149 views

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