Vol 3, No 3 (2011)


Letter from the Editors

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Acta Naturae. 2011;3(3):1-1
pages 1-1 views

High-Throughput Methods for Postgenomic Research

Sergiev P.V.
Acta Naturae. 2011;3(3):6-11
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Clinical Use of Inhibitors of HIV-1 Integration: Problems and Prospects

Korolev S.P., Agapkina Y.Y., Gottikh M.B.


The HIV-1 integrase enzyme is responsible for one of the key stages of retroviral replication; it acts as a catalyst for the integration of viral cDNA into the cell’s genome. Inhibitors of HIV-1 integration have been under development for over 10 years; yet, only one integration inhibitor, raltegravir, has been approved for clinical use so far. Raltegravir binds two metal ions in the enzyme’s active centre and blocks one of the integration stages: the strand transfer. Unfortunately, the clinical use of raltegravir results in the development of viral resistance among some patients. Several more HIV-1 integration inhibitors are undergoing clinical trials at the moment. However, the structure and mechanism of action of those are similar to raltegravir, which results in the emergence of cross resistance with raltegravir. The present review is focused on the history of the development and clinical trials of raltegravir and its analogues, the problems connected with the emergence of viral resistance to integration inhibitors, and the prospect of their future clinical use.
Acta Naturae. 2011;3(3):12-28
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Silencing of Her2, CCNB1 and pKC Genes by siRNA Results in Prolonged Retardation of Neuroblastoma Cell Division

Akimov I.A., Chernolovskaya E.L., Spitsyna Y.E., Ryabchikova E.I., Zenkova M.A.


Deregulation of the expression of the genes that are involved in the control of the cell cycle impairs cellular differentiation and leads to cell death. This process can result in uncontrollable cell proliferation and, subsequently, cancer development. In this study, we examined the effect of the silencing of cancer-related genes by small interfering RNAs (siRNA) targeted at mRNA of Her2, cyclin B1 (CCNB1), and protein kinase C (PKC) on the proliferation of human cancer cells of different origins. Maximum silencing of CCNB1, Her2 (in KB-3-1, SK-N-MC, MCF-7 cells), and PKC (in MCF-7 cells) was achieved 72 h after transfection of the corresponding siR-NAs, and 12 days after the transfection, the initial levels of the target mRNAs were fully recovered. Silencing of Her2, CCNB1, and PKC differently effected the proliferation of the cell lines under study. The most pronounced antiproliferative action of the investigated siRNAs was observed in neuroblastoma SK-N-MC cells (3 - 10-fold reduction in the proliferation rate) even after the recovery of the initial levels of expression of the Her2, CCNB1, and PKC genes. The obtained data indicate that the CCNB1 and PKC genes can be used as targets in the development of drugs for neuroblastoma treatment.
Acta Naturae. 2011;3(3):29-39
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Characteristics of Artificial Virus-like Particles Assembled in vitro from Potato Virus X Coat Protein and Foreign Viral RNAs

Arkhipenko M.V., Petrova E.K., Nikitin N.A., Protopopova A.D., Dubrovin E.V., Yaminskii I.V., Rodionova N.P., Karpova O.V., Atabekov J.G.


Potato virus X (PVX) and some other potexviruses can be reconstituted in vitro from viral coat protein (CP) and RNA. PVX CP is capable of forming viral ribonucleoprotein complexes (vRNP) not only with homologous, but also with foreign RNAs. This paper presents the structure and properties of vRNP assembled in vitro upon incubation of PVX CP and RNAs of various plant and animal viruses belonging to different taxonomic groups. We have shown that the morphology and translational properties of vRNPs containing foreign (heterologous) RNA are identical to those of homological vRNP (PVX RNA - PVX CP). Our data suggest that the assembly of the “mixed” vRNP in vitro could be started at the 5’-proximal region of the RNA, producing a helical structure of vRNPs with foreign nucleic acids. The formation of heterologous vRNP in vitro with PVX CP appears not to require a specific 5’ end RNA nucleotide sequence, and the PVX CP seems to be able to pack foreign genetic material of various sizes and compositions into artificial virus-like particles.
Acta Naturae. 2011;3(3):40-46
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Transcription Factor DLX5 As a New Target for Promising Antitumor Agents

Timakhov R.A., Fedichev P.O., Vinnik A.A., Testa J.R., Favorova O.O.


The crystal structure of the human transcription factor DLX5 has been used for the screening of a library consisting of 10 6 compounds by the molecular docking technique. In vitro tests of the 14 top-rated ligands showed that compound Q12 displays the best ability to inhibit the proliferation of Dlx5 positive mouse lymphoma cells, which correlates with the down-regulation of c-myc expression. Compound Q12 has low toxicity on normal human ovarian epithelial cells and mouse lymphoma cells with absent expression of Dlx5, and can be used for further chemical optimization and for the development of novel, highly efficient cancer treatments.
Acta Naturae. 2011;3(3):47-51
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Haplotype Diversity and Reconstruction of Ancestral Haplotype Associated with the c.35delG Mutation in the GJB2 (Cx26) Gene among the Volgo-Ural Populations of Russia

Dzhemileva L.U., Posukh O.L., Barashkov N.A., Fedorova S.A., Teryutin F.M., Akhmetova V.L., Khidiyatova I.M., Khusainova R.I., Lobov S.L., Khusnutdinova E.K.


The mutations in the GJB2 (Сх26) gene make the biggest contribution to hereditary hearing loss. The spectrum and prevalence of the GJB2 gene mutations are specific to populations of different ethnic origins. For several GJB2 mutations, their origin from appropriate ancestral founder chromosome was shown, approximate estimations of “age” obtained, and presumable regions of their origin outlined. This work presents the results of the carrier frequencies’ analysis of the major (for European countries) mutation c.35delG (GJB2 gene) among 2,308 healthy individuals from 18 Eurasian populations of different ethnic origins: Bashkirs, Tatars, Chuvashs, Udmurts, Komi-Permyaks, Mordvins, and Russians (the Volga-Ural region of Russia); Byelorussians, Ukrainians (Eastern Europe); Abkhazians, Avars, Cherkessians, and Ingushes (Caucasus); Kazakhs, Uzbeks, Uighurs (Central Asia); and Yakuts, and Altaians (Siberia). The prevalence of the c.35delG mutation in the studied ethnic groups may act as additional evidence for a prospective role of the founder effect in the origin and distribution of this mutation in various populations worldwide. The haplotype analysis of chromosomes with the c.35delG mutation in patients with nonsyndromic sensorineural hearing loss (N=112) and in population samples (N =358) permitted the reconstruction of an ancestral haplotype with this mutation, established the common origin of the majority of the studied mutant chromosomes, and provided the estimated time of the c.35delG mutation carriers expansion (11,800 years) on the territory of the Volga-Ural region.
Acta Naturae. 2011;3(3):52-63
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Effective Genetic Expression of Nanoantibodies by Recombinant Adenoviral Vector in vitro

Gribova I.Y., Tillib S.V., Tutykhina I.L., Shmarov М.М., Logunov D.Y., Verkhovskaya L.V., Naroditskii B.S., Gintsburg A.L.


The present study is devoted to the feasibility of expressing the single-domain mini-antibody (nanoantibody) selected from the library of sequences of the variable domains of special single-stranded antibodies derived from an immunized camel, a gene of which was introduced into eukaryotic cells within a recombinant adenoviral vector. A vector bearing the gene of a single-domain nanoantibody was obtained using the AdEasy Adenoviral Vector System (Stratagene). This method of delivering the nanoantibody gene facilitates efficient expression of this gene and functional activity of the nanoantibody. The results obtained can be used to produce passive immunizing tools against pathogens or new-generation immunobiological antitoxic medication.
Acta Naturae. 2011;3(3):64-70
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Rabin8 Protein Interacts with GTPase Rheb and Inhibits Phosphorylation of Ser235/Ser236 in Small Ribosomal Subunit Protein S6

Parkhitko А.А., Favorova О.О., Henske E.P.


The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that in association with Raptor, mLST8, PRAS40 and Deptor forms a complex (mTORCl) playing the key role in the regulation of protein biosynthesis, transcription, cellular metabolism, apoptosis and autophagy; mainly via direct phosphorylation of S6 kinases. mTORC1 is activated by growth factors and amino acids via the activation of Rheb GTPase. In the current study, we demonstrate for the first time that the over-expression of Rabin8, which functions as a guanine nucleotide exchange factor for Rab8 GTPase, suppresses phosphorylation of Ser235/Ser236 in ribosomal protein S6. Downregulation of Rabin8 using small interfering RNA (siRNA) increases the phosphorylation of Ser235/Ser236 in ribosomal protein S6. Furthermore, Rabin8 can be immunoprecipitated with Rheb GTPase. These results suggest the existence of a novel mechanism of mTORC1 regulation and its downstream processes. Since Rabin8 is a known regulator of ciliogenesis, a potential link can exist between regulation of Rheb/ mTORC1 and ciliogenesis.
Acta Naturae. 2011;3(3):71-76
pages 71-76 views

Bacterial Synthesis and Purification of Normal and Mutant Forms of Human FGFR3 Transmembrane Segment

Goncharuk S.A., Goncharuk M.V., Mayzel M.L., Lesovoy D.M., Chupin V.V., Bocharov E.V., Arseniev A.S., Kirpichnikov M.P.


The fibroblast growth factor receptor 3 (FGFR3) is a protein belonging to the family of receptor tyrosine kinases. FGFR3 plays an important role in human skeletal development. Mutations in this protein, including Gly380Arg or Ala391Glu substitutions in the transmembrane (TM) region, can cause different disorders in bone development. The determination of the spatial structure of the FGFR3 TM domain in a normal protein and in a protein with single Gly380Arg and Ala391Glu mutations is essential in order to understand the mechanisms that control dimerization and signal transduction by receptor tyrosine kinases. The effective system of expression of eukaryotic genes in bacteria and the purification protocol for the production of milligram amounts of both normal TM fragments of FGFR3 and those with single pathogenic mutations Gly380Arg and Ala391Glu, as well as their 15N- and [ 15N, 13C]-isotope-labelled derivatives, were described. Each peptide was produced in Escherichia coli BL21(DE3)pLysS cells as a C-terminal extension of thioredoxin A. The purification protocol involved immobilized metal affinity chromatography and cation- and anion-exchange chromatography, as well as the fusion protein cleavage with the light subunit of human enterokinase. The efficiency of the incorporation of target peptides into DPC/SDS and DPC/DPG micelles was confirmed using NMR spectroscopy. The described methodology of production of the native FGFR3 TM domain in norma and with single Gly380Arg and Ala391Glu mutations enables one to study their spatial structure using high-resolution heteronuclear NMR spectroscopy.
Acta Naturae. 2011;3(3):77-84
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Recombinant Production of Horseradish Peroxidase Conjugates with Fab Antibodies in Pichia pastoris for Analytical Applications

Koliasnikov O.V., Grigorenko V.G., Egorov A.M., Lange S., Schmid R.D.


Recombinant immunoconjugates of marker enzymes with antigens or antibodies present considerably more advantages than those obtained by conventional methods of chemical synthesis; i.e. they are homogeneous, have a strictly determined stoichiometry, and retain the functional activity of both a marker protein and an antigen/antibody. Based on the pPICZαB shuttle vector, we first managed to obtain a recombinant conjugate of key marker enzyme horseradish peroxidase (HRP) with Fab fragments of antibodies against atrazine. The resulting genetic construction allows us to switch to any other antibody sequence, via the simple re-cloning of variable parts and an additional reporter enzyme. Conjugates were successfully produced in the Pichia pastoris methylotrophic yeast expression system. The target activity of the conjugates (both enzymatic and antigen-binding) has been demonstrated by ELISA method.
Acta Naturae. 2011;3(3):85-92
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Atomic Force Microscopy Study of the Arrangement and Mechanical Properties of Astrocytic Cytoskeleton in Growth Medium

Efremov Y.M., Dzyubenko E.V., Bagrov D.V., Maksimov G.V., Shram S.I., Shaitan K.V.


Astrocytes are quite interesting to study because of their role in the development of various neurodegenerative disorders. The present work describes an examination of the arrangement and mechanical properties of cytoskeleton of living astrocytes using atomic force microscopy (AFM). The experiments were performed with an organotypic culture of dorsal root ganglia (DRG) obtained from a chicken embryo. The cells were cultivated on a gelatinous substrate and showed strong adhesion. AFM allows one to observe cytoskeleton fibers, which are interpreted as actin filaments and microtubules. This assumption is supported by confocal microscopy fluorescence imaging of α-tubulin and fibrillar actin. Mapping of the local Young’s modulus of a living astrocyte showed that the stiff areas correspond to the sites where the cytoskeleton fibers are located. Thus, the data obtained indicate that AFM is a promising method to study neural cells cytoskeleton integrity and arrangement in in vitro models of neurodegeneration.
Acta Naturae. 2011;3(3):93-99
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An Efficient Method for the Delivery of the Interleukin-2 Gene to Human Hematopoietic Cells using the Fiber-Modified Recombinant Adenovirus

Rogozhin V.N., Logunov D.Y., Shchebliakov D.V., Shmarov M.M., Khodunova E.E., Galtseva I.V., Belousova R.V., Naroditsky B.S., Gintsburg A.L.


Recombinant human adenovirus serotype 5 (Ad5/35F-IL2) with modified fibres containing the C-terminal domain fiber-knob of human adenovirus serotype 35, carrying the gene of recombinant human IL-2, has been designed. As a result of the fiber modification, the adenovirus can efficiently deliver the genetic information to bone marrow leukocytes and the tumor blood cells KG-1A (human myeloblastic leukemia cells) and U937 (human histiocytic lymphoma cells), which are normally resistant to Ad5 infection. The flow cytometry data reveal that the modified Ad5/35F penetrates into a population of monocytes, granulocytes, and blast cells of human bone marrow. The expression of interleukin-2 in CAR-negative bone marrow leukocytes (3682.52 ± 134.21 pg/ml) and the cell lines KG-1A (748.3 ± 32.8 pg/ml) and U937 (421.5 ± 59.4 pg/ ml) transduced with adenovirus Ad5/35F-IL2 is demonstrated. The fiber-modified adenovirus can be used as a vector for the efficient gene delivery of interleukin-2 to human normal and tumor hematopoietic cells. KEYWORDS adenovial vector; pseudotyping; interleukin-2; CD46; capsid modification.
Acta Naturae. 2011;3(3):100-106
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Guidelines for Authors

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Acta Naturae. 2011;3(3):107-108
pages 107-108 views

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