Vol 6, No 3 (2014)


Cobra Cytotoxins: Structural Organization and Antibacterial Activity

Dubovskii P.V., Utkin Y.N.


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.

Acta Naturae. 2014;6(3):11-18
pages 11-18 views

TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

Nemudryi A.A., Valetdinova K.R., Medvedev S.P., Zakian S.M.


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.

Acta Naturae. 2014;6(3):19-40
pages 19-40 views

Mycoplasma Contamination of Cell Cultures: Vesicular Traffic in Bacteria and Control over Infectious Agents

Chernov V.M., Chernova O.A., Sanchez-Vega J.T., Kolpakov А.I., Ilinskaya О.N.


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.

Acta Naturae. 2014;6(3):41-51
pages 41-51 views

Structural Features of the Interaction between Human 8-Oxoguanine DNA Glycosylase hOGG1 and DNA

Koval V.V., Knorre D.G., Fedorova O.S.


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.

Acta Naturae. 2014;6(3):52-65
pages 52-65 views

The Proteome of a Healthy Human during Physical Activity under Extreme Conditions

Larina I.M., Ivanisenko V.A., Nikolaev E.N., Grigorev A.I.


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.

Acta Naturae. 2014;6(3):66-75
pages 66-75 views

Research Articles

Study of the Structure-Function-Stability Relationships in Yeast D-amino Acid Oxidase: Hydrophobization of Alpha-Helices

Golubev I.V., Komarova N.V., Ryzhenkova K.V., Chubar T.A., Savin S.S., Tishkov V.I.


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.

Acta Naturae. 2014;6(3):76-88
pages 76-88 views

Changes in Gene Expression Associated with Matrix Turnover, Chondrocyte Proliferation and Hypertrophy in the Bovine Growth Plate

Tchetina E.V., Mwale F., Poole A.R.


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.

Acta Naturae. 2014;6(3):89-97
pages 89-97 views

Effects of Neonatal Fluvoxamine Administration on the Physical Development and Activity of the Serotoninergic System in White Rats

Glazova N.Y., Merchieva S.A., Volodina M.A., Sebentsova E.A., Manchenko D.M., Kudrun V.S., Levitskaya N.G.


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.

Acta Naturae. 2014;6(3):98-105
pages 98-105 views

Short communications

Possible Function of the ribT Gene of Bacillus subtilis: Theoretical Prediction, Cloning, and Expression

Yakimov A.P., Seregina T.A., Kholodnyak A.A., Kreneva R.A., Mironov A.S., Perumov D.A., Timkovskii A.L.


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.

Acta Naturae. 2014;6(3):106-109
pages 106-109 views

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