Vol 8, No 3 (2016)


Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells from Umbilical Cord Blood

Sotnezova E.V., Andreeva E.R., Grigoriev A.I., Buravkova L.B.


Transplantation of umbilical cord blood cells is currently widely used in modern cell therapy. However, the limited number of hematopoietic stem and progenitor cells (HSPCs) and prolonged time of recovery after the transplantation are significant limitations in the use of cord blood. Ex vivo expansion with various cytokine combinations is one of the most common approaches for increasing the number of HSPCs from one cord blood unit. In addition, there are protocols that enable ex vivo amplification of cord blood cells based on native hematopoietic microenvironmental cues, including stromal components and the tissue-relevant oxygen level. The newest techniques for ex vivo expansion of HSPCs are based on data from the elucidation of the molecular mechanisms governing the hematopoietic niche function. Application of these methods has provided an improvement of several important clinical outcomes. Alternative methods of cord blood transplantation enhancement based on optimization of HPSC homing and engraftment in patient tissues have also been successful. The goal of the present review is to analyze recent methodological approaches to cord blood HSPC ex vivo amplification.

Acta Naturae. 2016;8(3):6-16
pages 6-16 views

Molecular and Cellular Mechanisms of Antitumor Immune Response Activation by Dendritic Cells

Markov O.V., Mironova N.L., Vlasov V.V., Zenkova M.A.


Dendritic cells (DCs) play a crucial role in the initiation and regulation of the antitumor immune response. Already , DC-based antitumor vaccines have been thoroughly explored both in animal tumor models and in clinical trials. DC-based vaccines are commonly produced from DC progenitors isolated from peripheral blood or bone marrow by culturing in the presence of cytokines, followed by loading the DCs with tumor-specific antigens, such as DNA, RNA, viral vectors, or a tumor cell lysate. However, the efficacy of DC-based vaccines remains low. Undoubtedly, a deeper understanding of the molecular mechanisms by which DCs function would allow us to enhance the antitumor efficacy of DC-based vaccines in clinical applications. This review describes the origin and major subsets of mouse and human DCs, as well as the differences between them. The cellular mechanisms of presentation and cross-presentation of exogenous antigens by DCs to T cells are described. We discuss intracellular antigen processing in DCs, cross-dressing, and the acquisition of the antigen cross-presentation function. A particular section in the review describes the mechanisms of tumor escape from immune surveillance through the suppression of DCs functions.

Acta Naturae. 2016;8(3):17-30
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Stem Cells in the Treatment of InsulinDependent Diabetes Mellitus

Borisov M.A., Petrakova O.S., Gvazava I.G., Kalistratova E.N., Vasiliev A.V.


Diabetes affects over 350 million people worldwide, with the figure projected to rise to nearly 500 million over the next 20 years, according to the World Health Organization. Insulin-dependent diabetes mellitus (type 1 diabetes) is an endocrine disorder caused by an autoimmune reaction that destroys insulin-producing β-cells in the pancreas, which leads to insulin deficiency. Administration of exogenous insulin remains at the moment the treatment mainstay. This approach helps to regulate blood glucose levels and significantly increases the life expectancy of patients. However, type 1 diabetes is accompanied by long-term complications associated with the systemic nature of the disease and metabolic abnormalities having a profound impact on health. Of greater impact would be a therapeutic approach which would overcome these limitations by better control of blood glucose levels and prevention of acute and chronic complications. The current efforts in the field of regenerative medicine are aimed at finding such an approach. In this review, we discuss the time-honored technique of donor islets of Langerhans transplantation. We also focus on the use of pluripotent stem and committed cells and cellular reprogramming. The molecular mechanisms of pancreatic differentiation are highlighted. Much attention is devoted to the methods of grafts delivery and to the materials used during its creation.

Acta Naturae. 2016;8(3):31-43
pages 31-43 views

Bioreactor-Based Tumor Tissue Engineering

Guller A.E., Grebenyuk P.N., Shekhter A.B., Zvyagin A.V., Deyev S.M.


This review focuses on modeling of cancer tumors using tissue engineering technology. Tumor tissue engineering (TTE) is a new method of three-dimensional (3D) simulation of malignant neoplasms. Design and development of complex tissue engineering constructs (TECs) that include cancer cells, cell-bearing scaffolds acting as the extracellular matrix, and other components of the tumor microenvironment is at the core of this approach. Although TECs can be transplanted into laboratory animals, the specific aim of TTE is the most realistic reproduction and long-term maintenance of the simulated tumor properties in vitro for cancer biology research and for the development of new methods of diagnosis and treatment of malignant neoplasms. Successful implementation of this challenging idea depends on bioreactor technology, which will enable optimization of culture conditions and control of tumor TECs development. In this review, we analyze the most popular bioreactor types in TTE and the emerging applications.

Acta Naturae. 2016;8(3):44-58
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Hyaluronic Acid in Vascular and Immune Homeostasis during Normal Pregnancy and Preeclampsia

Ziganshina M.M., Pavlovich S.V., Bovin N.V., Sukhikh G.T.


Preeclampsia (PE) is a multisystem pathologic state that clinically manifests itself after the 20th week of pregnancy. It is characterized by high maternal and perinatal morbidity and mortality. According to modern concepts, the impairment of trophoblast invasion into maternal spiral arteries, leading to the development of ischemia in placenta, is considered to be the major pathogenetic factor of PE development. Ischemic lesions initiate the development of a systemic inflammatory response (SIR) and endothelial dysfunction, which is the main cause of the multiple organ failure in PE. Some data has appear indicating the importance of a glycans-forming endothelial glycocalyx and extracellular matrix (ECM) for placenta morphogenesis, as well as their role in the regulation of vascular permeability and vascular tone in hypertension disorders and, in particular, PE. Since intact glycocalyx and ECM are considered to be the major factors that maintain the physiological vascular tone and adequate intercellular interactions, their value in PE pathogenesis is underestimated. This review is focused on hyaluronic acid (HA) as the key glycan providing the organization and stabilization of the ECM and glycocalyx, its distribution in tissues in the case of presence or absence of placental pathology, as well as on the regulatory function of hyaluronic acids of various molecular weights in different physiological and pathophysiological processes. The summarized data will provide a better understanding of the PE pathogenesis, with the main focus on glycopathology.

Acta Naturae. 2016;8(3):59-71
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Epigenetics of Ancient DNA

Zhenilo S.V., Sokolov A.S., Prokhortchou E.B.


Initially, the study of DNA isolated from ancient specimens had been based on the analysis of the primary nucleotide sequence. This approach has allowed researchers to study the evolutionary changes that occur in different populations and determine the influence of the environment on genetic selection. However, the improvement of methodological approaches to genome-wide analysis has opened up new possibilities in the search for the epigenetic mechanisms involved in the regulation of gene expression. It was discovered recently that the methylation status of the regulatory elements of the HOXD cluster and MEIS1 gene changed during human evolution. Epigenetic changes in these genes played a key role in the evolution of the limbs of modern humans. Recent works have demonstrated that it is possible to determine the transcriptional activity of genes in ancient DNA samples by combining information on DNA methylation and the DNAaseI hypersensitive sequences located at the transcription start sites of genes. In the nearest future, if a preserved fossils brain is found, it will be possible to identify the evolutionary changes in the higher nervous system associated with epigenetic differences.

Acta Naturae. 2016;8(3):72-76
pages 72-76 views

Research Articles

Biosynthesis of poly(3-hydroxybutyrateco-3-hydroxy-4-methylvalerate) by Strain Azotobacter chroococcum 7B

Bonartsev A.P., Bonartseva G.A., Myshkina V.L., Voinova V.V., Mahina T.K., Zharkova I.I., Yakovlev S.G., Zernov A.L., Ivanova E.V., Akoulina E.A., Kuznetsova E.S., Zhuikov V.A., Alekseeva S.G., Podgorskii V.V., Bessonov I.V., Kopitsyna M.N., Morozov A.S., Milanovskiy E.Y., Tyugay Z.N., Bykova G.S., Kirpichnikov M.P., Shaitan K.V.


Production of novel polyhydroxyalkanoates (PHAs), biodegradable polymers for biomedical applications, and biomaterials based on them is a promising trend in modern bioengineering. We studied the ability of an effective strain-producer Azotobacter chroococcum 7B to synthesize not only poly(3-hydroxybutyrate) homopolymer (PHB) and its main copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), but also a novel copolymer, poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) (PHB4MV). For the biosynthesis of PHB copolymers, we used carboxylic acids as additional carbon sources and monomer precursors in the chain of synthesized copolymers. The main parameters of these polymers’ biosynthesis were determined: strain-producer biomass yield, polymer yield, molecular weight and monomer composition of the synthesized polymers, as well as the morphology of A.repair rolex replica chroococcum 7B bacterial cells. The physico-chemical properties of the polymers were studied using nuclear magnetic resonance spectroscopy (NMR), differential scanning calorimetry (DSC), contact angle test, and other methods. In vitro biocompatibility of the obtained polymers was investigated using stromal cells isolated from the bone marrow of rats with the XTT cell viability test. The synthesis of the novel copolymer PHB4MV and its chemical composition were demonstrated by NMR spectroscopy: the addition of 4-methylvaleric acid to the culture medium resulted in incorporation of 3-hydroxy-4-methylvalerate (3H4MV) monomers into the PHB polymer chain (0.6 mol%). Despite the low molar content of 3H4MV in the obtained copolymer, irolex replica deep seats physico-chemical properties were significantly different from those of the PHB homopolymer: it has lower crystallinity and a higher contact angle, i.e. the physico-chemical properties of the PHB4MV copolymer containing only 0.6 mol% of 3H4MV corresponded to a PHBV copolymer with a molar content ranging from 2.5% to 7.8%. In vitro biocompatibility of the obtained PHB4MV copolymer, measured in the XTT test,rolex replica deep sea was not statistically different from the cell growth of PHB and PHBV polymers, rolex datejust real or fake which make its use possible in biomedical research and development.

Acta Naturae. 2016;8(3):77-87
pages 77-87 views

Cleavage of Human Embryos: Options and Diversity

Doronin Y.K., Senechkin I.V., Hilkevich L.V., Kurcer M.A.


In order to estimate the diversity of embryo cleavage relatives to embryo progress (blastocyst formation), time-lapse imaging data of preimplantation human embryo development were used. This retrospective study is focused on the topographic features and time parameters of the cleavages, with particular emphasis on the lengths of cleavage cycles and the genealogy of blastomeres in 2- to 8-cell human embryos. We have found that all 4-cell human embryos have four developmental variants that are based on the sequence of appearance and orientation of cleavage planes during embryo cleavage from 2 to 4 blastomeres. Each variant of cleavage shows a strong correlation with further developmental dynamics of the embryos (different cleavage cycle characteristics as well as lengths of blastomere cycles). An analysis of the sequence of human blastomere divisions allowed us to postulate that the effects of zygotic determinants are eliminated as a result of cleavage, and that, thereafter, blastomeres acquire the ability of own syntheses, regulation, polarization, formation of functional contacts, and, finally, of specific differentiation. This data on the early development of human embryos obtained using noninvasive methods complements and extend our understanding of the embryogenesis of eutherian mammals and may be applied in the practice of reproductive technologies.

Acta Naturae. 2016;8(3):88-96
pages 88-96 views

ZAD-Domain Is Essential for Nuclear Localization of Insulator Proteins in Drosophila melanogaster

Zolotarev N.A., Maksimenko O.G., Georgiev P.G., Bonchuk A.N.


Many arthropod zinc-finger transcription factors contain a N-terminal domain called ZAD (Zinc-finger Associated Domain), which consists of four cysteines coordinating a single zinc ion. Dimerization ability has been shown for several ZAD-domains. The functional role of this domain is poorly understood. In this paper, we demonstrate that a point mutation within the ZAD-domain of the Zw5 insulator protein disrupts its nuclear localization without affecting its dimerization ability. The importance of the ZAD-domain for nuclear localization has also been shown for the Pita and Grauzone proteins. Therefore, one of the ZAD-domain functions is control of the nuclear localization of transcription factors.

Acta Naturae. 2016;8(3):97-102
pages 97-102 views

Whole-Genome DNA Methylation Analysis of Peripheral Blood Mononuclear Cells in Multiple Sclerosis Patients with Different Disease Courses

Kulakova O.G., Kabilov M.R., Danilova L.V., Popova E.V., Baturina O.A., Tsareva E.Y., Baulina N.M., Kiselev I.S., Boyko A.N., Favorov A.V., Favorova O.O., Vlassov V.V.


Multiple sclerosis (MS) is a severe neurodegenerative disease of polygenic etiology affecting the central nervous system. In addition to genetic factors, epigenetic mechanisms, primarily DNA methylation, which regulate gene expression, play an important role in MS development and progression. In this study, we have performed the first whole-genome DNA methylation profiling of peripheral blood mononuclear cells in relapsing-remitting MS (RRMS) and primary-progressive MS (PPMS) patients and compared them to those of healthy individuals in order to identify the differentially methylated CpG-sites (DMSs) associated with these common clinical disease courses. In addition, we have performed a pairwise comparison of DNA methylation profiles in RRMS and PPMS patients. All three pairwise comparisons showed significant differences in methylation profiles. Hierarchical clustering of the identified DMS methylation levels and principal component analysis for data visualization demonstrated a clearly defined aggregation of DNA samples of the compared groups into separate clusters. Compared with the control, more DMSs were identified in PPMS patients than in RRMS patients (67 and 30, respectively). More than half of DMSs are located in genes, exceeding the expected number for random distribution of DMSs between probes. RRMS patients mostly have hypomethylated DMSs, while in PPMS patients DMSs are mostly hypermethylated. CpG-islands and CpG-shores contain 60% of DMSs, identified by pairwise comparison of RRMS and control groups, and 79% of those identified by pairwise comparison of PPMS and control groups. Pairwise comparison of patients with two clinical MS courses revealed 51 DMSs, 82% of which are hypermethylated in PPMS. Overall, it was demonstrated that there are more changes in the DNA methylation profiles in PPMS than in RRMS. The data confirm the role of DNA methylation in MS development. We have shown, for the first time, that DNA methylation as an epigenetic mechanism is involved in the formation of two distinct clinical courses of MS: namely, RRMS and PPMS.

Acta Naturae. 2016;8(3):103-110
pages 103-110 views

The Effect of Dopamine Secreted by the Brain into the Systemic Circulation on Prolactin Synthesis by the Pituitary gland in Ontogenesis

Nikishina Y.O., Sapronova A.Y., Ugrumov M.V.


This research was aimed at studying the brain’s endocrine function in ontogenesis. It has been previously shown in our laboratory that the brain serves as the source of dopamine in the systemic circulation of rats prior to the formation of the blood-brain barrier. This paper provides direct evidence that dopamine secreted by the brain directly into the systemic circulation in this period of ontogenesis has an inhibitory effect on prolactin secretion by pituitary cells. These results provide the basis for a fundamentally new understanding of the brain’s role in the neuroendocrine regulation of the development and function of peripheral target organs and, particularly in this study, the pituitary gland.

Acta Naturae. 2016;8(3):111-117
pages 111-117 views

Identification of New Structural Fragments for the Design of Lactate Dehydrogenase A Inhibitors

Nilov D.K., Kulikov A.V., Prokhorova E.A., Švedas V.K.


Human lactate dehydrogenase A plays an important role in the glucose metabolism of tumor cells and constitutes an attractive target for chemotherapy. Molecular fragments able to bind in the active site of this enzyme and form hydrogen bonds with the Arg168 guanidinium group, as well as additional interactions with the loop 96-111 in the closed conformation, have been identified by virtual screening of sulfonates and experimental testing of their inhibitory effect. The sulfo group can occupy a similar position as the carboxyl group of the substrate and its structural analogs, whereas the benzothiazole group attached via a linker can be located in the coenzyme (NADH) binding site. Thus, the value of merging individual structural elements of the inhibitor by a linker was demonstrated and ways of further structural modification for the design of more effective inhibitors of lactate dehydrogenase A were established.

Acta Naturae. 2016;8(3):118-122
pages 118-122 views

Analysis of the Domains of Hepatitis C Virus Core and NS5A Proteins that Activate the Nrf2/ARE Cascade

Smirnova O.A., Ivanova O.N., Mukhtarov F.S., Tunitskaya V.L., Jansons J., Isaguliants M.G., Kochetkov S.N., Ivanov A.V.


The hepatitis C virus (HCV) triggers a chronic disease that is often accompanied by a spectrum of liver pathologies and metabolic alterations. The oxidative stress that occurs in the infected cells is considered as one of the mechanisms of HCV pathogenesis. It is induced by the viral core and NS5A proteins. It is already known that both of these proteins activate the antioxidant defense system controlled by the Nrf2 transcription factor. Here, we show that this activation is mediated by domain 1 of the NS5A protein and two fragments of the core protein. In both cases, this activation is achieved through two mechanisms. One of them is mediated by reactive oxygen species (ROS) and protein kinase C, whereas the other is triggered through ROS-independent activation of casein kinase 2 and phosphoinositide 3-kinase. In the case of the HCV core, the ROS-dependent mechanism was assigned to the 37-191 a.a. fragment, while the ROS-independent mechanism was assigned to the 1-36 а.a. fragment. Such assignment of the mechanisms to different domains is the first evidence of their independence. In addition, our data revealed that intracellular localization of HCV proteins has no impact on the regulation of the antioxidant defense system.

Acta Naturae. 2016;8(3):123-127
pages 123-127 views

Derivatization of Aminoglycoside Antibiotics with Tris(2,6-dimethoxyphenyl)carbenium Ion

Topolyan A.P., Belyaeva M.A., Bykov E.E., Coodan P.V., Rogozhin E.A., Strizhevskaya D.A., Ivanova O.M., Ustinov A.V., Mikhura I.V., Prokhorenko I.A., Korshun V.A., Formanovsky A.A.


Detection of aminoglycoside antibiotics by MS or HPLC is complicated, because a) carbohydrate molecules have low ionization ability in comparison with other organic molecules (particularly in MALDI-MS), and b) the lack of aromatics and/or amide bonds in the molecules makes common HPLC UV-detectors useless. Here, we report on the application of a previously developed method for amine derivatization with tris(2,6-dimethoxyphenyl)carbenium ion to selective modification of aminoglycoside antibiotics. Only amino groups bound to primary carbons get modified. The attached aromatic residue carries a permanent positive charge. This makes it easy to detect aminoglycoside antibiotics by MS-methods and HPLC, both as individual compounds and in mixtures.

Acta Naturae. 2016;8(3):128-135
pages 128-135 views

Minibactenecins ChBac7.Nα and ChBac7. Nβ - Antimicrobial Peptides from Leukocytes of the Goat Capra hircus.

Shamova O.V., Orlov D.S., Zharkova M.S., Balandin S.V., Yamschikova E.V., Knappe D., Hoffmann R., Kokryakov V.N., Ovchinnikova T.V.


Antimicrobial peptides (AMPs) of neutrophils play an important role in the animal and human host defenses. We have isolated two AMPs (average molecular masses of 2895.5 and 2739.3 Da), with potent antimicrobial activity from neutrophils of the domestic goat (Capra hircus). A structural analysis of the obtained peptides revealed that they encompass N-terminal fragments (1-21 and 1-22) of the proline-rich peptide bactenecin 7.5. The primary structure of caprine bactenecin 7.5 had been previously deduced from the nucleotide sequence, but the corresponding protein had not been isolated from leukocytes until now. The obtained caprine AMPs were designated as mini-batenecins (mini-ChBac7.5Nα and mini-ChBac7.5Nβ), analogously to the reported C-terminal fragment of the ovine bactenecin 7.5 named Bac7.5mini [Anderson, Yu, 2003]. Caprine mini-ChBac7.5Nα and mini-ChBac7.5Nβ exhibit significant antimicrobial activity against Gram-negative bacteria, including drug-resistant strains of Pseudomonas aeruginosa, Klebsiella spp., Acinetobacter baumannii at a range of concentrations of 0.5-4 μM, as well as against some species of Gram-positive bacteria (Listeria monocytogenes EGD, Micrococcus luteus). The eptides demonstrate lipopolysaccharide-binding activity. Similarly to most proline-rich AMPs, caprine peptides inactivate bacteria without appreciable damage of their membranes. Mini-ChBac7.5Nα and mini-ChBac7.5Nβ have no hemolytic effect on human red blood cells and are nontoxic to various cultured human cells. Therefore, they might be considered as promising templates for the development of novel antibiotic pharmaceuticals. Isolation of highly active fragments of the antimicrobial peptide from goat neutrophils supports the hypothesis that fragmentation of cathelicidin-related AMPs is an important process that results in the generation of potent effector molecules, which are in some cases more active than full-size AMPs. These truncated AMPs may play a crucial role in host defense reactions.

Acta Naturae. 2016;8(3):136-146
pages 136-146 views

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