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1.  Mechanisms of Activation of Voltage-Gated Potassium Channels 
Acta Naturae  2014;6(4):10-26.
Voltage-gated potassium ion channels (Kv) play an important role in a variety of cellular processes, including the functioning of excitable cells, regulation of apoptosis, cell growth and differentiation, the release of neurotransmitters and hormones, maintenance of cardiac activity, etc. Failure in the functioning of Kv channels leads to severe genetic disorders and the development of tumors, including malignant ones. Understanding the mechanisms underlying Kv channels functioning is a key factor in determining the cause of the diseases associated with mutations in the channels, and in the search for new drugs. The mechanism of activation of the channels is a topic of ongoing debate, and a consensus on the issue has not yet been reached. This review discusses the key stages in studying the mechanisms of functioning of Kv channels and describes the basic models of their activation known to date.
PMCID: PMC4273088  PMID: 25558391
activation; potassium ion channels; modeling; structure
2.  Regulation of the Target Protein (Transgene) Expression in the Adenovirus Vector Using Agonists of Toll-Like Receptors 
Acta Naturae  2014;6(4):27-39.
Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88→ NF-kB, while the inhibition of transgene expression depends on TRIF→ IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88→ NF-kB signaling.
PMCID: PMC4273089  PMID: 25558392
gene therapy; gene vaccination; recombinant replication-defective adenovirus vectors; transgene expression; Toll-like receptor agonists
3.  STIM1 Protein Activates Store-Operated Calcium Channels in Cellular Model of Huntington’s Disease 
Acta Naturae  2014;6(4):40-47.
We have shown that the expression of full-length mutated huntingtin in human neuroblastoma cells (SK-N-SH) leads to an abnormal increase in calcium entry through store-operated channels. In this paper, the expression of the N-terminal fragment of mutated huntingtin (Htt138Q-1exon) is shown to be enough to provide an actual model for Huntington’s disease. We have shown that Htt138Q-1exon expression causes increased store-operated calcium entry, which is mediated by at least two types of channels in SK-N-SH cells with different reversal potentials. Calcium sensor, STIM1, is required for activation of store-operated calcium entry in these cells. The results provide grounds for considering the proteins responsible for the activation and maintenance of the store-operated calcium entry as promising targets for developing novel therapeutics for neurodegenerative diseases.
PMCID: PMC4273090  PMID: 25558393
Huntington’s disease; calcium; neurodegeneration; SOC; STIM1
4.  Specific Visualization of Tumor Cells Using Upconversion Nanophosphors 
Acta Naturae  2014;6(4):48-53.
The development of targeted constructs on the basis of photoluminescent nanoparticles with a high photo- and chemical stability and absorption/emission spectra in the “transparency window” of biological tissues is an important focus area of present-day medical diagnostics. In this work, a targeted two-component construct on the basis of upconversion nanophosphors (UCNPs) and anti-tumor 4D5 scFv was developed for selective labeling of tumor cells overexpressing the HER2 tumor marker characteristic of a number of human malignant tumors. A high affinity barnase : barstar (Bn : Bs) protein pair, which exhibits high stability in a wide range of pH and temperatures, was exploited as a molecular adapter providing self-assembly of the two-component construct. High selectivity for the binding of the two-component 4D5 scFv-Bn : UCNP-Bs construct to human breast adenocarcinoma SK-BR-3 cells overexpressing HER2 was demonstrated. This approach provides an opportunity to produce similar constructs for the visualization of different specific markers in pathogenic tissues, including malignant tumors.
PMCID: PMC4273091  PMID: 25558394
upconversion nanophosphors; biomarker imaging; anti-tumor antibodies; self-assembly; HER2
5.  Excessive Labeling Technique Provides a Highly Sensitive Fluorescent Probe for Real-time Monitoring of Biodegradation of Biopolymer Pharmaceuticals in vivo 
Acta Naturae  2014;6(4):54-59.
Recombinant proteins represent a large sector of the biopharma market. Determination of the main elimination pathways raises the opportunities to significantly increase their half-lives in vivo. However, evaluation of biodegradation of pharmaceutical biopolymers performed in the course of pre-clinical studies is frequently complicated. Noninvasive pharmacokinetic and biodistribution studies in living organism are possible using proteins conjugated with near-infrared dyes. In the present study we designed a highly efficient probe based on fluorescent dye self-quenching for monitoring of in vivo biodegradation of recombinant human butyrylcholinesterase. The maximum enhancement of integral fluorescence in response to degradation of an intravenously administered enzyme was observed 6 h after injection. Importantly, excessive butyrylcholinesterase labeling with fluorescent dye results in significant changes in the pharmacokinetic properties of the obtained conjugate. This fact must be taken into consideration during future pharmacokinetic studies using in vivo bioimaging.
PMCID: PMC4273092  PMID: 25558395
fluorescent probe; biodegradation; pharmacokinetics; in vivo bioimaging; self-quenching; butyrylcholinesterase; proteolysis
6.  Investigation of Channel-Forming Activity of Polyene Macrolide Antibiotics in Planar Lipid Bilayers in the Presence of Dipole Modifiers 
Acta Naturae  2014;6(4):67-79.
The role of membrane components, sterols, phospholipids and sphingolipids in the formation and functioning of ion-permeable nanopores formed by antifungal macrolide antibiotics, amphotericin B, nystatin and filipin in planar lipid bilayers was studied. Dipole modifiers, flavonoids and styryl dyes, were used as a tool to study the molecular mechanisms of polyene channel-forming activity. The introduction of dipole modifiers into the membrane bathing solutions was shown to change the conductance of single channels and the steadystate transmembrane current induced by polyene antibiotics in the sterol-containing phospholipid-bilayers. The conductance of single amphotericin B channels was found to depend on the dipole potential of the membrane. The experiments with various phospholipids, sterols, and polyenes led to the assumption that the shape of a phospholipid molecule, the presence of double bonds at the positions 7 and 22 of a sterol molecule, the number of conjugated double bonds, and the presence of an amino sugar in the polyene antibiotic molecule are important factors impacting the stability of polyene-lipid complexes forming ion-permeable pores. Experimental and literature data presented in the paper suggest that the channel-forming activity of polyene antibiotics is also affected by the physicochemical properties of polyene-enriched ordered membrane domains.
PMCID: PMC4273094  PMID: 25558397
planar lipid bilayers; polyene antibiotics; sterols; styryl dyes; sphingolipids; flavonoids; phospholipids
7.  A Cytofluorometric Study of Membrane Rafts in Human Monocyte Subsets in Atherosclerosis 
Acta Naturae  2014;6(4):80-88.
The peripheral blood monocytes of atherosclerotic patients are pre-activated and have some of the features of tissue macrophages. Their adhesion to the endothelium is 1.5 times higher than that of monocytes from healthy subjects, and they express a number of receptors and antigens typical of tissue macrophages. Additionally, earlier we showed that the biosynthesis of gangliosides, whose main function is the formation of membrane rafts, is significantly activated in blood monocytes from atherosclerotic patients, as well as during the in vitro differentiation of normal monocytes into macrophages. In this study, we investigated the expression of membrane rafts on various monocyte subsets from healthy subjects and atherosclerotic patients. Based on flow cytometry results, the monocytes in the examined atherosclerotic patients were found to differ from those in healthy subjects by a twofold increase in the proportion of the intermediate subset (CD14++/CD16+) and by enhancement in the expression of the fractalkine receptor CX3CR1 on the intermediate and non-classical subsets (CD14++/CD16+ and CD14+/CD16++) (2.3 and 1.8 times, respectively). This suggests a pre-activated state of monocytes in atherosclerotic patients. At the same time, the expression of the membrane raft marker on the monocyte subsets was similar in both studied groups. However, a study of the in vitro differentiation of monocytes into macrophages showed that the membrane raft expression increased 2 times as early as on the 1st day of culturing and 3 times on the 7th day compared to that in freshly isolated monocytes. Therefore, it is suggested that monocytes in atherosclerosis accumulate gangliosides that are used to form membrane rafts during the macrophage differentiation after the migration of monocytes into the arterial intima.
PMCID: PMC4273095  PMID: 25558398
monocyte subsets; macrophages; membrane rafts; flow cytometry; atherosclerosis
8.  Hct-A Is a New Actinoporin Family from the Heteractis Crispa Sea Anemone 
Acta Naturae  2014;6(4):89-98.
Several new actinoporin isoforms with molecular weights of 18995.5 to 19398.7 Da exhibiting a high hemolytic activity were isolated from the tropical sea anemone Heteractis crispa using a combination of liquid chromatography techniques. The actinoporins were demonstrated to occur as mono-, di-, and trimers in aqueous solutions. The sequences of the genes encoding actinoporins were identified, and the amino acid sequences of the new polypeptides belonging to the Hct-A actinoporin family were obtained. The new acinoporins differ in their isoelectric points, the number and localization of charged amino acid residues at the functionally important N-terminal fragment of the molecule, as well as in the charge of a tetrapeptide (amino acid residues 74–77) involved in an electrostatic interaction with the cytoplasmic membrane. A recombinant actinoporin, rHct-A2, with a molecular weight of 19141 Da, pI of 9.64, and hemolytic activity of 4.0 × 104 HU/mg, was obtained. The conductivity of the ion channels formed by rHct-A2 in the BLM was demonstrated to be similar to that of the native actinoporin from H. crispa. The obtained data expand knowledge on the structural and functional relationships of actinoporins and contribute to our understanding of the functioning mechanism of these molecules, which is the basis for the development of compounds with a high biomedical potential. Currently, they are considered as models for obtaining antitumor, antibacterial, and cardiac-stimulating agents.
PMCID: PMC4273096  PMID: 25558399
sea anemone; actinoporins; hemolytic activity; lipid membrane conductivity; structural and functional analysis
9.  Acipensins – Novel Antimicrobial Peptides from Leukocytes of the Russian Sturgeon Acipenser gueldenstaedtii 
Acta Naturae  2014;6(4):99-109.
Antimicrobial peptides (AMPs) play an important role in the innate defense mechanisms in humans and animals. We have isolated and studied a set of antimicrobial peptides from leukocytes of the Russian sturgeon Acipenser gueldenstaedtii belonging to a subclass of chondrosteans, an ancient group of bony fish. Structural analysis of the isolated peptides, designated as acipensins (Ac), revealed in leukocytes of the Russian sturgeon six novel peptides with molecular masses of 5336.2 Da, 3803.0 Da, 5173.0 Da, 4777.5 Da, 5449.4 Da, and 2740.2 Da, designated as Ac1–Ac6, respectively. Complete primary structures of all the isolated peptides were determined, and the biological activities of three major components – Ac1, Ac2, and Ac6 – were examined. The peptides Ac1, Ac2, Ac3, Ac4, and Ac5 were found to be the N-terminal acetylated fragments 1–0, 1–5, 1–9, 1–4, and 1–1 of the histone H2A, respectively, while Ac6 was shown to be the 62–5 fragment of the histone H2A. The peptides Ac1 and Ac2 displayed potent antimicrobial activity towards Gram-negative and Gram-positive bacteria (Escherichia coli ML35p, Listeria monocytogenes EGD, MRSA ATCC 33591) and the fungus Candida albicans 820, while Ac6 proved effective only against Gram-negative bacteria. The efficacy of Ac 1 and Ac2 towards the fungus and MRSA was reduced upon an increase in the ionic strength of the solution. Ac1, Ac2, and Ac6, at concentrations close to their minimum inhibitory concentrations, enhanced the permeability of the E.coli ML35p outer membrane to the chromogenic marker, but they did not affect appreciably the permeability of the bacterial inner membrane in comparison with a potent pore-forming peptide, protegrin 1. Ac1, Ac2, and Ac6 revealed no hemolytic activity against human erythrocytes at concentrations of 1 to 40 μM and had no cytotoxic effect (1 to 20 μM) on K-562 and U-937 cells in vitro. Our findings suggest that histone-derived peptides serve as important anti-infective host defense molecules.
PMCID: PMC4273097  PMID: 25558400
innate immunity; antimicrobial peptides; sturgeon leukocytes; histone H2A derivatives; acipensin
10.  The Mechanism of Choline-Mediated Inhibition of Acetylcholine Release in Mouse Motor Synapses 
Acta Naturae  2014;6(4):110-115.
The mechanism of action of tonically applied choline, the agonist of α7 nicotinic acetylcholine receptors (nAChRs), to the spontaneous and evoked release of a neurotransmitter in mouse motor synapses in diaphragm neuromuscular preparations using intracellular microelectrode recordings of miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) was studied. Exogenous choline was shown to exhibit a presynaptic inhibitory effect on the amplitude and quantal content of EPPs for the activity of neuromuscular junction evoked by single and rhythmic stimuli. This effect was inhibited either by antagonists of α7-nAChRs, such as methyllycaconitine and α-cobratoxin, or by blocking SK-type calcium-activated potassium (KCa) channels with apamin or blocking intraterminal ryanodine receptors with ryanodine. A hypothesis was put forward that choline in mouse motoneuron nerve terminals can activate presynaptic α7-nAChRs, followed by the release of the stored calcium through ryanodine receptors and activation of SK-type KCa channels, resulting in sustained decay of the quantal content of the evoked neurotransmitter release.
PMCID: PMC4273098  PMID: 25558401
quantal content; ryanodine receptors; choline; α7-nicotinic acetylcholine receptors; SK channels
11.  Phosphoryl Guanidines: A New Type of Nucleic Acid Analogues 
Acta Naturae  2014;6(4):116-118.
A new type of nucleic acid analogues with a phosphoryl guanidine group is described. Oxidation of polymer-supported dinucleoside 2-cyanoethyl phosphite by iodine in the presence of 1,1,3,3-tetramethyl guanidine yields a dinucleotide with an internucleoside tetramethyl phosphoryl guanidine (Tmg) group as the main product. The Tmg group is stable under conditions of solid-phase DNA synthesis and subsequent cleavage and deprotection with ammonia. Oligonucleotides with one or more Tmg groups bind their complementary DNA or RNA with affinity similar to that of natural oligodeoxyribonucleotides.
PMCID: PMC4273099  PMID: 25558402
nucleic acid analogue; modified oligonucleotide; solid-phase synthesis; tetramethylguanidine; phosphoryl guanidine; phosphite
12.  Human SLURP-1 and SLURP-2 Proteins Acting on Nicotinic Acetylcholine Receptors Reduce Proliferation of Human Colorectal Adenocarcinoma HT-29 Cells 
Acta Naturae  2014;6(4):60-66.
Human secreted Ly-6/uPAR related proteins (SLURP-1 and SLURP-2) are produced by various cells, including the epithelium and immune system. These proteins act as autocrine/paracrine hormones regulating the growth and differentiation of keratinocytes and are also involved in the control of inflammation and malignant cell transformation. These effects are assumed to be mediated by the interactions of SLURP-1 and SLURP-2 with the α7 and α3β2 subtypes of nicotinic acetylcholine receptors (nAChRs), respectively. Available knowledge about the molecular mechanism underling the SLURP-1 and SLURP-2 effects is very limited. SLURP-2 remains one of the most poorly studied proteins of the Ly-6/uPAR family. In this study, we designed for the first time a bacterial system for SLURP-2 expression and a protocol for refolding of the protein from cytoplasmic inclusion bodies. Milligram quantities of recombinant SLURP-2 and its 13C-15N-labeled analog were obtained. The recombinant protein was characterized by NMR spectroscopy, and a structural model was developed. A comparative study of the SLURP-1 and SLURP-2 effects on the epithelial cell growth was conducted using human colorectal adenocarcinoma HT-29 cells, which express only α7-nAChRs. A pronounced antiproliferative effect of both proteins was observed. Incubation of cells with 1 μM SLURP-1 and 1 μM SLURP-2 during 48 h led to a reduction in the cell number down to ~ 54 and 63% relative to the control, respectively. Fluorescent microscopy did not reveal either apoptotic or necrotic cell death. An analysis of the dose-response curve revealed the concentration-dependent mode of the SLURP-1 and SLURP-2 action with EC50 ~ 0.1 and 0.2 nM, respectively. These findings suggest that the α7-nAChR is the main receptor responsible for the antiproliferative effect of SLURP proteins in epithelial cells.
PMCID: PMC4273093  PMID: 25558396
nicotinic acetylcholine receptor; bacterial expression; refolding; Lynx; colon cancer
13.  Cobra Cytotoxins: Structural Organization and Antibacterial Activity 
Acta Naturae  2014;6(3):11-18.
Cardiotoxins (cytotoxins, CT) are β-structured proteins isolated from the venom of cobra. They consist of 59–61 amino acid residues, whose antiparallel chains form three ‘fingers’. In contrast to neurotoxins with an overall similar fold, CTs are amphiphilic. The amphiphilicity is caused by positively charged lysine and arginine residues flanking the tips of the loops that consist primarily of hydrophobic amino acids. A similar distribution of amino acid residues is typical for linear (without disulfide bonds) cationic cytolytic peptides from the venoms of other snakes and insects. Many of them are now considered to be lead compounds in combatting bacterial infections and cancer. In the present review, we summarize the data on the antibacterial activity of CTs and compare it to the activity of linear peptides.
PMCID: PMC4207557  PMID: 25349711
antibacterial activity; lipopolysaccharide; peptidoglycan; plasma membrane; three-finger cardiotoxins (cytotoxins); cytolytic cationic peptides
14.  TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery 
Acta Naturae  2014;6(3):19-40.
Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isn’t enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared – this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.
PMCID: PMC4207558  PMID: 25349712
TALEN; CRISPR/Cas9; genome editing
15.  Mycoplasma Contamination of Cell Cultures: Vesicular Traffic in Bacteria and Control over Infectious Agents 
Acta Naturae  2014;6(3):41-51.
Cell cultures are subject to contamination either with cells of other cultures or with microorganisms, including fungi, viruses, and bacteria. Mycoplasma contamination of cell cultures is of particular importance. Since cell cultures are used for the production of vaccines and physiologically active compounds, designing a system for controlling contaminants becomes topical for fundamental science and biotechnological production. The discovery of extracellular membrane vesicles in mycoplasmas makes it necessary to take into consideration the bacterial vesicular traffic in systems designed for controlling infectious agents. The extracellular vesicles of bacteria mediate the traffic of proteins and genes, participate in cell-to-cell interactions, as well as in the pathogenesis and development of resistance to antibiotics. The present review discusses the features of mycoplasmas, their extracellular vesicles, and the interaction between contaminants and eukaryotic cells. Furthermore, it provides an analysis of the problems associated with modern methods of diagnosis and eradication of mycoplasma contamination from cell cultures and prospects for their solution.
PMCID: PMC4207559  PMID: 25349713
diagnosis and eradication; cell cultures; mycoplasma contamination
16.  Structural Features of the Interaction between Human 8-Oxoguanine DNA Glycosylase hOGG1 and DNA 
Acta Naturae  2014;6(3):52-65.
The purpose of the present review is to summarize the data related with the structural features of interaction between the human repair enzyme 8-oxoguanine DNA glycosylase (hOGG1) and DNA. The review covers the questions concerning the role of individual amino acids of hOGG1 in the specific recognition of the oxidized DNA bases, formation of the enzyme–substrate complex, and excision of the lesion bases from DNA. Attention is also focused upon conformational changes in the enzyme active site and disruption of enzyme activity as a result of amino acid mutations. The mechanism of damaged bases release from DNA induced by hOGG1 is discussed in the context of structural dynamics.
PMCID: PMC4207560  PMID: 25349714
protein-nucleic acid recognition; human 8-oxoguanine DNA glycosylase; repair enzymes; loss-offunction mutants; structural analysis of hOGG1
17.  The Proteome of a Healthy Human during Physical Activity under Extreme Conditions 
Acta Naturae  2014;6(3):66-75.
The review examines the new approaches in modern systems biology, in terms of their use for a deeper understanding of the physiological adaptation of a healthy human in extreme environments. Human physiology under extreme conditions of life, or environmental physiology, and systems biology are natural partners. The similarities and differences between the object and methods in systems biology, the OMICs (proteomics, transcriptomics, metabolomics) disciplines, and other related sciences have been studied. The latest data on environmental human physiology obtained using systems biology methods are discussed. The independent achievements of systems biology in studying the adaptation of a healthy human to physical activity, including human presence at high altitude, to the effects of hypoxia and oxidative stress have been noted. A reasonable conclusion is drawn that the application of the methods and approaches used in systems biology to study the molecular pattern of the adaptive mechanisms that develop in the human body during space flight can provide valuable fundamental knowledge and fill the picture of human metabolic pathways.
PMCID: PMC4207561  PMID: 25349715
integrative physiology; space flight; proteomics; systems biology
18.  Study of the Structure-Function-Stability Relationships in Yeast D-amino Acid Oxidase: Hydrophobization of Alpha-Helices 
Acta Naturae  2014;6(3):76-88.
Hydrophobization of alpha-helices is one of the general approaches used for improving the thermal stability of enzymes. A total of 11 serine residues located in alpha-helices have been found based on multiple alignments of the amino acid sequences of D-amino acid oxidases from different organisms and the analysis of the 3D-structure of D-amino acid oxidase from yeast Trigonopsis variabilis (TvDAAO, EC As a result of further structural analysis, eight Ser residues in 67, 77, 78, 105, 270, 277, 335, and 336 positions have been selected to be substituted with Ala. S78A and S270A substitutions have resulted in dramatic destabilization of the enzyme. Mutant enzymes were inactivated during isolation from cells. Another six mutant TvDAAOs have been highly purified and their properties have been characterized. The amino acid substitutions S277A and S336A destabilized the protein globule. The thermal stabilities of TvDAAO S77A and TvDAAO S335A mutants were close to that of the wild-type enzyme, while S67A and S105A substitutions resulted in approximately 1.5- and 2.0-fold increases in the TvDAAO mutant thermal stability, respectively. Furthermore, the TvDAAO S105A mutant showed on average a 1.2- to 3.0-fold higher catalytic efficiency with D-Asn, D-Tyr, D-Phe, and D-Leu as compared to the wild-type enzyme.
PMCID: PMC4207562  PMID: 25349716
D-amino acid oxidase from yeast Trigonopsis variabilis; protein engineering; hydrophobization of alpha-helices; site-directed mutagenesis; substrate specificity; thermal stability
19.  Changes in Gene Expression Associated with Matrix Turnover, Chondrocyte Proliferation and Hypertrophy in the Bovine Growth Plate 
Acta Naturae  2014;6(3):89-97.
The aim of the study is to investigate the interrelationships between the expression of genes for structural extracellular matrix molecules, proteinases and their inhibitors in the bovine fetal growth plate. This was analyzed by RT-PCR in microsections of the proximal tibial growth plate of bovine fetuses in relationship to expression of genes associated with chondrocyte proliferation, apoptosis, and matrix vascularization. In the resting zone the genes for extracellular matrix molecule synthesis were expressed. Extracellular matrix degrading enzymes and their inhibitors were also expressed here. Onset of proliferation involved cyclic upregulation of cell division-associated activity and reduced expression of extracellular matrix molecules. Later in the proliferative zone we noted transient expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with vascularization and apoptosis. With the onset of hypertrophy expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with vascularization and apoptosis were significantly upregulated. Terminal differentiation was characterized by high expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with apoptosis. This study reveals the complex interrelationships of gene expression in the physis that accompany matrix assembly, resorption, chondrocyte proliferation, hypertrophy, vascularization and cell death while principal zones of the growth plate are characterized by a distinct signature profile of gene expression.
PMCID: PMC4207563  PMID: 25349717
growth plate; gene expression; proteinases; chondrocyte differentiation
20.  Effects of Neonatal Fluvoxamine Administration on the Physical Development and Activity of the Serotoninergic System in White Rats 
Acta Naturae  2014;6(3):98-105.
Selective serotonin reuptake inhibitors (SSRIs), including fluvoxamine, are widely used to treat depressive disorders in pregnant women. These antidepressants effectively penetrate through the placental barrier, affecting the fetus during the critical phase of neurodevelopment. Some clinical studies have linked prenatal exposure to SSRIs with increased neonatal mortality, premature birth, decreased fetal growth and delay in psychomotor development. However, the effects of prenatal exposure to SSRIs remain unknown. The administration of SSRIs in rodents during the first postnatal weeks is considered as an model for studying the effects of prenatal SSRIs exposure in human. The aim of this work was to study the acute effects of chronic fluvoxamine (FA) administration in white rat pups. The study was carried out in male and female rat pups treated with FA (10 mg/kg/day, intraperitoneally) from postnatal days 1 to 14. The lethality level, body weight, age of eye opening, and motor reflex maturation were recorded. The contents of biogenic amines and their metabolites in different brain structures were also determined. It was shown that neonatal FA administration led to increased lethality level, reduced body weight, and delayed maturation of motor reflexes. Furthermore, increased noradrenalin level in hypothalamus, serotonin level in hippocampus and serotonin metabolite 5-HIAA level in frontal cortex, hypothalamus, hippocampus, and striatum were observed in drug-treated animals compared to the control group. We can conclude that the altered activity of the serotoninergic system induced by fluvoxamine administration at early developmental stages leads to a delay in physical and motor development.
PMCID: PMC4207564  PMID: 25349718
biogenic amines; fluvoxamine; neonatal administration; psychomotor development; selective serotonin reuptake inhibitors
21.  Possible Function of the ribT Gene of Bacillus subtilis: Theoretical Prediction, Cloning, and Expression 
Acta Naturae  2014;6(3):106-109.
The complete decipherment of the functions and interactions of the elements of the riboflavin biosynthesis operon (rib operon) of Bacillus subtilis are necessary for the development of superproducers of this important vitamin. The function of its terminal ribT gene has not been established to date. In this work, a search for homologs of the hypothetical amino acid sequence of the gene product through databases, as well as an analysis of the homolgs, was performed; the distribution of secondary structure elements was theoretically predicted; and the tertiary structure of the RibT protein was proposed. The ribT gene nucleotide sequence was amplified and cloned into the standard high-copy expression vector pET15b and then expressed after induction with IPTG in E. coli BL21 (DE3) strain cells containing the inducible phage T7 RNA polymerase gene. The ribT gene expression was confirmed by SDS-PAGE. The protein product of the expression was purified by affinity chromatography. Therefore, the real possibility of RibT protein production in quantities sufficient for further investigation of its structure and functional activity was demonstrated.
PMCID: PMC4207565  PMID: 25349719
proteomics; bioinformatics; homology search; theoretical protein structure; gene cloning; inducible expression
22.  Disruption of the Colonization Resistance Syndrome in Humans in Altered Habitats and Its Prevention 
Acta Naturae  2014;6(2):10-18.
Exposure of human subjects to environments with modified parameters is associated with reduced colonization resistance of the intestine and epithelial tissue, which leads to dysbiotic changes. Probiotics – preparations based on protective microflora – are used to correct dysbacteriosis of different etiologies and localizations. However, the effectiveness of probiotics largely depends on the adhesive ability of a probiotic strain and lack of competitive relations with the indigenous microflora, which can be achieved by individual selection of a preparation. We propose to use autochtonous microflora as a probiotic drug to optimize the prevention and treatment results. A personalized approach to probiotic selection will improve therapy efficiency and reduce the risk of adverse effects in each individual patient.
PMCID: PMC4115221  PMID: 25093106
autostrains; autoprobiotics; dysbacteriosis; altered habitat; disruption of colonization resistance
23.  Biotechnological approaches to creation of hypoxia and anoxia tolerant plants 
Acta Naturae  2014;6(2):19-30.
The present work provides results of a number of biotechnological studies aimed at creating cell lines and entire plants resistant to anaerobic stress. Developed biotechnological approaches were based on earlier fundamental researches into anaerobic stress in plants, so “Introduction” briefly covers the importance of the problem and focuses on works considering two main strategies of plants adaptation to anaerobic stress. Those are adaptation at molecular level where key factor is anaerobic metabolism of energy (true tolerance) and adaptation of the entire plant via formation of aerenchyma and facilitated transportation of oxygen (apparent tolerance). Thus, sugarcane and wheat cells resistant to anaerobic stress were obtained through consecutive in vitro selection under conditions of anoxia and absence of exogenous carbohydrates. Tolerant wheat cells were used to regenerate entire plants of higher resistance to root anaerobiosis. It has been demonstrated that cells tolerance to anoxia is significantly supported by their ability to utilize exogenous nitrate. Cells tolerance established itself at the genetic level and was inherited by further generations. Apart from that, other successful attempts to increase tolerance of plants to anaerobic stress by means of stimulation of glycolysis and overexpression of genes responsible for cytokinin synthesis and programmed cell death are also discussed. The presented data proved the notion of two main strategies of plants adaptation to anaerobic stress proposed earlier on the base of fundamental studies.
PMCID: PMC4115222  PMID: 25093107
anaerobic stress; growth index; in vitro cell selection; programmed cell death; transgenic plants; mitochondrial ultrastructure
24.  Analysis of the Mitochondrial Genome of a Novosvobodnaya Culture Representative using Next-Generation Sequencing and Its Relation to the Funnel Beaker Culture 
Acta Naturae  2014;6(2):31-35.
The Novosvobodnaya culture is known as a Bronze Age archaeological culture in the North Caucasus region of Southern Russia. It dates back to the middle of the 4th millennium B.C. and seems to have occurred during the time of the Maikop culture. There are now two hypotheses about the emergence of the Novosvobodnaya culture. One hypothesis suggests that the Novosvobodnaya culture was a phase of the Maikop culture, whereas the other one classifies it as an independent event based on the material culture items found in graves. Comparison between Novosvobodnaya pottery and Funnelbeaker (TRB) pottery from Germany has allowed researchers to suggest that the Novosvobodnaya culture developed under the influence of Indo-European culture. Nevertheless, the origin of the Novosvobodnaya culture remains a matter of debate. We applied next-generation sequencing to study ~5000-year-old human remains from the Klady kurgan grave in Novosvobodnaya stanitsa (now the Republic of Adygea, Russia). A total of 58,771,105 reads were generated using Illumina GAIIx with a coverage depth of 13.4x over the mitochondrial (mt) DNA genome. The mtDNA haplogroup affiliation was determined as V7, suggesting a role of the TRB culture in the development of the Novosvobodnaya culture and supporting the model of sharing between Novosvobodnaya and early Indo-European cultures.
PMCID: PMC4115223  PMID: 25093108
Novosvobodnaya culture; Maikop culture; haplogroup; mitochondrial DNA; sequencing; genomics
25.  Real-Time Interaction between TBP and the TATA Box of the Human Triosephosphate Isomerase Gene Promoter in the Norm and Pathology 
Acta Naturae  2014;6(2):36-40.
The TATA-binding protein (TBP) is a key part of the transcription complex of RNA polymerase II. Alone or as a part of the basal transcription factor TFIID, TBP binds the TATA box located in the core region of the TATA-containing promoters of class II genes. Previously, we studied the effects of single nucleotide polymorphisms (SNPs) on TBP/TATA-box interactions using gel retardation assay. It was demonstrated that most SNPs in the TATA boxes of some human gene promoters cause a 2- to 4-fold decrease in TBP/TATA affinity, which is associated with an increased risk of hereditary diseases, such as β thalassemias of diverse severity, hemophilia B Leyden, myocardial infarction, thrombophlebitis, lung cancer, etc. In this work, the process of TBP/TATA complex formation has been studied in real time by a stopped-flow technique using recombinant human TBP and duplexes, which were identical to the TATA box of the wild-type and a SNP-containing triosephosphate isomerase gene promoter and were fluorescently labeled by the Cy3/Cy5 FRET pair. It has been demonstrated for the first time that real-time binding of TBP to the TATA box of the TPI gene promoter is complete within 10 s and is described by a single-stage kinetic model. The complex formation of TBP with the wild-type TATA box occurs 5.5 times faster and the complex dissociation occurs 31 times slower compared with the SNPcontaining TATA box. Within the first seconds of the interaction, TBP binds to and simultaneously bends the TATA box. Importantly, the TATA box of the wild-type TPI gene promoter requires lower TBP concentrations compared to the TATA box containing the -24T → G SNP, which is associated with neurological and muscular disorders, cardiomyopathy, and other diseases.
PMCID: PMC4115224  PMID: 25093109
TATA box; TBP; polymorphism; TBP/TATA-box interaction; stopped flow

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