Vol 9, No 4 (2017)


Non-coding RNAs As Transcriptional Regulators In Eukaryotes

Burenina O.Y., Oretskaya T.S., Kubareva E.A.


Non-coding RNAs up to 1,000 nucleotides in length are widespread in eukaryotes and fulfil various regulatory functions, in particular during chromatin remodeling and cell proliferation. These RNAs are not translated into proteins: thus, they are non-coding RNAs (ncRNAs). The present review describes the eukaryotic ncRNAs involved in transcription regulation, first and foremost, targeting RNA polymerase II (RNAP II) and/or its major proteinaceous transcription factors. The current state of knowledge concerning the regulatory functions of SRA and TAR RNA, 7SK and U1 snRNA, GAS5 and DHFR RNA is summarized herein. Special attention is given to murine B1 and B2 RNAs and human Alu RNA, due to their ability to bind the active site of RNAP II. Discovery of bacterial analogs of the eukaryotic small ncRNAs involved in transcription regulation, such as 6S RNAs, suggests that they possess a common evolutionary origin.

Acta Naturae. 2017;9(4):13-25
pages 13-25 views

The Contribution of Ribosomal Protein S1 to the Structure and Function of Qβ Replicase

Kutlubaeva Z.S., Chetverina Е.V., Chetverin A.B.


The high resolution crystal structure of bacterial ribosome was determined more than 10 years ago; however, it contains no information on the structure of the largest ribosomal protein, S1. This unusual protein comprises six flexibly linked domains; therefore, it lacks a fixed structure and this prevents the formation of crystals. Besides being a component of the ribosome, protein S1 also serves as one of the four subunits of Qβ replicase, the RNA-directed RNA polymerase of bacteriophage Qβ. In each case, the role of this RNA-binding protein has been thought to consist in holding the template close to the active site of the enzyme. In recent years, a breakthrough was made in studies of protein S1 within Qβ replicase. This includes the discovery of its paradoxical ability to displace RNA from the replicase complex and determining the crystal structure of its fragment capable of performing this function. The new findings call for a re-examination of the contribution of protein S1 to the structure and function of the ribosome.

Acta Naturae. 2017;9(4):26-30
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Genome Stability Maintenance in Naked Mole-Rat

Petruseva I.O., Evdokimov A.N., Lavrik O.I.


The naked mole-rat (Heterocephalus glaber) is one of the most promising models used to study genome maintenance systems, including the effective repair of damage to DNA. The naked mole-rat is the longest lived rodent species, which is extraordinarily resistant to cancer and has a number of other unique phenotypic traits. For at least 80% of its lifespan, this animal shows no signs of aging or any increased likelihood of death and retains the ability to reproduce. The naked mole-rat draws the heightened attention of researchers who study the molecular basis of lengthy lifespan and cancer resistance. Despite the fact that the naked mole-rat lives under genotoxic stress conditions (oxidative, etc.), the main characteristics of its genome and proteome are a high stability and effective functioning. Replicative senescence in the somatic cells of naked mole-rats is missing, while an additional p53/pRb-dependent mechanism of early contact inhibition has been revealed in its fibroblasts, which controls cell proliferation and its mechanism of arf-dependent aging. The unique traits of phenotypic and molecular adaptations found in the naked mole-rat speak to a high stability and effective functioning of the molecular machinery that counteract damage accumulation in its genome. This review analyzes existing results in the study of the molecular basis of longevity and high cancer resistance in naked mole-rats.

Acta Naturae. 2017;9(4):31-41
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Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Ryabichko S.S., Ibragimov A.N., Lebedeva L.A., Kozlov E.N., Shidlovskii Y.V.


In recent decades, novel microscopic methods commonly referred to as super- resolution microscopy have been developed. These methods enable the visualization of a cell with a resolution of up to 10 nm. The application of these methods is of great interest in studying the structure and function of the cell nucleus. The review describes the main achievements in this field.

Acta Naturae. 2017;9(4):42-51
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Research Articles

Translational Cross-Activation of the Encapsidated RNA of Potexviruses

Arkhipenko M.V., Nikitin N.A., Donchenko E.K., Karpova O.V., Atabekov J.G.


We had shown the genomic RNA of potexviruses potato virus X and the alternanthera mosaic virus to be inaccessible in vitro to ribosomes while in intact virion form, but the RNAs can be translationally activated following the binding of movement protein 1 (MP1) to virus particles. Here, we present the results of the follow-up study targeting two more potexvirus species - the Narcissus mosaic virus and the Potato aucuba mosaic virus. We found encapsidated potexviral RNA to share common translational features in vitro and the MP1 to be potent over homological virions of its “own” species and over heterological virions of other species, as well exhibiting selective specificity. Reciprocal cross-activation is observed among viral species phylogenetically either close or distant. There is direct evidence that MP1 binding to the end of the virion is necessary, but not sufficient, for translational activation of encapsidated RNA.

Acta Naturae. 2017;9(4):52-57
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Synthesis and Characterization of Hybrid Core-Shell Fe3O4/SiO2 Nanoparticles for Biomedical Applications

Zelepukin I.V., Shipunova V.O., Mirkasymov A.B., Nikitin P.I., Nikitin M.P., Deyev S.M.


The creation of markers that provide both visual and quantitative information is of considerable importance for the mapping of tissue macrophages and other cells. We synthesized magnetic and magneto-fluorescent nanomarkers for the labeling of cells which can be detected with high sensitivity by the magnetic particle quantification (MPQ) technique. For stabilization under physiological conditions, the markers were coated with a dense silica shell. In this case, the size and zeta-potential of nanoparticles were controlled by a modified Stober reaction. Also, we developed a novel facile two-step synthesis of carboxylic acid-functionalized magnetic SiO2 nanoparticles, with a carboxyl polymer shell forming on the nanoparticles before the initiation of the Stober reaction. We extensively characterized the nanomarkers by transmission electron microscopy, electron microdiffraction, and dynamic and electrophoretic light scattering. We also studied the nanoparticle cellular uptake by various eukaryotic cell lines.

Acta Naturae. 2017;9(4):58-65
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A Novel Hybrid Promoter ARE-hTERT for Cancer Gene Therapy

Kalinichenko S.V., Shepelev M.V., Vikhreva P.N., Korobko I.V.


describe a novel hybrid tumor-specific promoter, ARE-hTERT, composed of the human TERT gene promoter (hTERT) and the antioxidant response element (ARE) from the human GCLM gene promoter. The hybrid promoter retains the tumor specificity of the basal hTERT promoter but is characterized by an enhanced transcriptional activity in cancer cells with abnormal activation of the Nrf2 transcription factor and upon induction of oxidative stress. In the in vitro enzyme-prodrug cancer gene therapy scheme, ARE-hTERT promoter-driven expression of CD : UPRT (yeast cytosine deaminase : uracil phosphoribosyltransferase) chimeric protein induced a more pronounced death of cancer cells either upon treatment with 5-fluorouracil (5FC) alone or when 5FC was combined with chemotherapeutic drugs as compared to the hTERT promoter. The developed hybrid promoter can be considered a better alternative to the hTERT promoter in cancer gene therapy schemes.

Acta Naturae. 2017;9(4):66-73
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Multilocus Analysis of Genetic Susceptibility to Myocardial Infarction in Russians: Replication Study

Kukava N.G., Titov B.V., Osmak G.J., Matveeva N.A., Kulakova O.G., Favorov A.V., Shakhnovich R.M., Ruda M.Y., Favorova O.O.


In search of genetic markers of myocardial infarction (MI) risk, which have prognostic significance for Russians, we performed a replication study of MI association with genetic variants of PCSK9 (rs562556), APOE (epsilon polymorphism, rs7412 and rs429358), LPL (rs320), MTHFR (rs1801133), eNOS (rs2070744), and the 9p21 region (rs1333049) in 405 patients with MI and 198 controls. Significant MI association was observed with variants of the lipid metabolism genes (PCSK9, APOE and LPL), and of eNOS. The SNPs in the MTHFR gene and the 9p21 region were not significantly associated with MI one by one but were included in several different MI-associated allelic combinations identified by multilocus analysis. Since we have not revealed nonlinear epistatic interactions between the components of the identified combinations, we postulate that the cumulative effect of genes that form a combination arises from the summation of their small independent contributions. The prognostic significance of the additive composite model built from the PCSK9, APOE, LPL, and eNOS genes as genetic markers was assessed using ROC analysis. After we included these markers in the previously published composite model of individual genetic risk of MI, the prognostic efficacy in our sample reached AUC = 0.676. However, the results obtained in this study certainly need to be replicated in an independent sample of Russians.

Acta Naturae. 2017;9(4):74-83
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Recombinant Antibodies to the Ebola Virus Glycoprotein

Panina A.A., Dementieva I.G., Aliev T.K., Toporova V.A., Balabashin D.S., Bokov M.N., Pozdnyakova L.P., Shchemchukova O.B., Dolgikh D.A., Sveshnikov P.G., Kirpichnikov M.P.


Currently, there are no approved therapies for targeted prevention and treatment of Ebola hemorrhagic fever. In the present work, we describe the development of a eukaryotic expression system for the production of three full-length chimeric antibodies (IgG1-kappa isotypes) GPE118, GPE325, and GPE534 to the recombinant glycoprotein of the Ebola virus (EBOV GP), which is a key factor in the pathogenicity of the disease. The immunochemical properties of the obtained antibodies were studied by immunoblotting and indirect, direct, and competitive ELISA using the recombinant EBOV proteins rGPdTM, NP, and VP40. The authenticity of the antibodies and the absence of cross-specificity with respect to the structural proteins NP and VP40 of the Ebola virus were proved. The epitope specificity of the resulting recombinant antibodies was studied using commercial neutralizing antibodies against the viral glycoprotein. The recombinant antibodies GPE118, GPE325, and GPE534 were shown to recognize glycoprotein epitopes that coincide or overlap with the epitopes of three well-studied neutralizing anti-Ebola virus antibodies.

Acta Naturae. 2017;9(4):84-91
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Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase

Svirid A.V., Ershov P.V., Yablokov E.O., Kaluzhskiy L.A., Mezentsev Y.V., Florinskaya A.V., Sushko T.A., Strushkevich N.V., Gilep A.A., Usanov S.A., Medvedev A.E., Ivanov A.S.


Thromboxane synthase (TBXAS1) catalyzes the isomerization reaction of prostaglandin H2 producing thromboxane A2, the autocrine and paracrine factor in many cell types. A high activity and metastability by these arachidonic acid derivatives suggests the existence of supramolecular structures that are involved in the regulation of the biosynthesis and directed translocation of thromboxane to the receptor. The objective of this study was to identify TBXAS1 protein partners from human liver tissue lysate using a complex approach based on the direct molecular fishing technique, LC-MS/MS protein identification, and protein-protein interaction validation by surface plasmon resonance (SPR). As a result, 12 potential TBXAS1 protein partners were identified, including the components regulating cytoskeleton organization (BBIP1 and ANKMY1), components of the coagulation cascade of human blood (SERPINA1, SERPINA3, APOH, FGA, and FN1), and the enzyme involved in the metabolism of xenobiotics and endogenous bioregulators (CYP2E1). SPR validation on the Biacore 3000 biosensor confirmed the effectiveness of the interaction between CYP2E1 (the enzyme that converts prostaglandin H2 to 12-HHT/thromboxane A2 proantagonist) and TBXAS1 (Kd = (4.3 ± 0.4) × 10-7 M). Importantly, the TBXAS1•CYP2E1 complex formation increases fivefold in the presence of isatin (indole-2,3-dione, a low-molecular nonpeptide endogenous bioregulator, a product of CYP2E1). These results suggest that the interaction between these hemoproteins is important in the regulation of the biosynthesis of eicosanoids.

Acta Naturae. 2017;9(4):92-100
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YABBY3-Orthologous Genes in Wild Tomato Species: Structure, Variability, and Expression

Filyushin M.A., Slugina M.A., Shchennikova A.V., Kochieva E.Z.


Evolution of the genes encoding YABBY transcription factors is believed to be one of the key reasons for flat leaf emergence from the radially symmetrical stem and gynoecium diversity. YABBY genes determine the identity of the abaxial surface of all aboveground lateral organs in seed plants. In the present study, complete sequences of YABBY3-orthologous genes were identified and characterized in 13 accessions of cultivated and wild tomato species with diverse morphophysiology of leaves, flowers, and fruits. The obtained gene sequences showed high homology (95-99%) and an identical exon-intron structure with the known S. lycopersicum YABBY3 gene, and they contained sequences that encode the conserved HMG-like YABBY and Cys2Cys2-zinc-finger domains. In total, in the analyzed YABBY3 genes, 317 variable sites were found, wherein 8 of 24 exon-specific SNPs were nonsynonymous. In the vegetative and reproductive organs of red-fruited and green-fruited tomato species, YABBY3 gene expression was similar to that in S. pimpinellifolium described earlier, but it demonstrated interspecies differences at the leaf-, bud- and flower-specific expression levels.

Acta Naturae. 2017;9(4):101-109
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Short communications

CaMKII Is Involved in the Choline-Induced Downregulation of Acetylcholine Release in Mouse Motor Synapses

Gaydukov A.E., Balezina O.P.


We investigated the involvement of calcium-dependent enzymes, protein kinase C (PKC) and calcium-calmodulin-dependent protein kinase II (CaMKII), in the signaling pathway triggered by the activation of presynaptic alpha7-type nicotinic acetylcholine receptors by exogenous choline, leading to downregulation of the evoked acetylcholine (ACh) release in mouse motor synapses. Blockade of PKC with chelerythrine neither changed the evoked release of ACh by itself nor prevented the inhibitory effect of choline. The CaMKII blocker KN-62 did not affect synaptic activity but fully prevented the choline-induced downregulation of ACh release.

Acta Naturae. 2017;9(4):110-113
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40 Years without Smallpox

Shchelkunova G.A., Shchelkunov S.N.


The last case of natural smallpox was recorded in October, 1977. It took humanity almost 20 years to achieve that feat after the World Health Organization had approved the global smallpox eradication program. Vaccination against smallpox was abolished, and, during the past 40 years, the human population has managed to lose immunity not only to smallpox, but to other zoonotic orthopoxvirus infections as well. As a result, multiple outbreaks of orthopoxvirus infections in humans in several continents have been reported over the past decades. The threat of smallpox reemergence as a result of evolutionary transformations of these zoonotic orthopoxviruses exists. Modern techniques for the diagnostics, prevention, and therapy of smallpox and other orthopoxvirus infections are being developed today.

Acta Naturae. 2017;9(4):4-12
pages 4-12 views

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