ActaNaturae ActaNaturae

Acta Naturae, 2009. V. 1.
№ 1.

S. M. Deyev and E. N. Lebedenko

Modern Technologies for Creating Synthetic Antibodies for Clinical Application

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow

E-mail: deyev@ibch.ru

ABSTRACT The modular structure and versatility of antibodies enables one to modify natural immunoglobulins in different ways for various clinical applications. Rational design and molecular engineering make it possible to directionally modify the molecular size, affinity, specificity, and immunogenicity and effector functions of an antibody, as well as to combine them with other functional agents. This review focuses on up-to-date methods of antibody engineering for diagnosing and treating various diseases, particularly on new technologies meant to refine the effector functions of therapeutic antibodies.

KEYWORDS monoclonal antibodies, humanized antibodies, single-chain antibodies, multivalency, bispecificity, target-specific delivery, barnase:barstar module, and immunodibarnase

ABBREVIATIONS ADcc (antibody-dependent cellular cytotoxicity); cDc (complement-dependent cytotoxicity); MAb (monoclonal antibodies); cH and cL (constant domains of antibody heavy and light chains); СНО cells (chinese hamster ovary cells); eGFr (Her1) (epidermal growth factor receptor, cancer marker); Fab (antigen-binding fragment of antibody); Fc (constant (crystallizable) antibody fragment); Fc?r (cell receptor of antibody Fc-fragments); Fcrn (neonatal receptor of antibody Fc-fragments); Her1 and Her2/neu (cancer markers of tyrosine kinase receptor group); IgA, IgG, IgD, Ige, IgM (A, G, D, e, M immunoglobulins (antibodies of the A, G, D, e, M classes)); scFv (single chain fragment variable); PSMA (prostate-specific membrane antigen); VeGF (vascular endothelial growth factor); VH and VL (variable domains of heavy and light antibody chains).
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D. A. Skvortzov, M. P. Rubzova, M. E. Zvereva, F. L. Kiselev, O. A. Donzova

The Regulation of Telomerase in Oncogenesis

D. A. Skvortzov¹, M. P. Rubzova¹, M. E. Zvereva¹, F. L. Kiselev², O. A. Donzova^1*
¹Department of Chemistry, Moscow State University, 119992 Moscow
²Blokhin Oncological Science Centre, Russian Academy of Medical Science, 115478 Moscow
*E-mail: dontsova@genebee.msu.su

ABSTRACT Telomerase is a complex ribonucleoprotein that completes the telomeres' ends in eukaryotic cells which shorten due to DnA underreplication. The core enzyme consists of a protein catalytic subunit—telomerase reverse transcriptase (tert)—and telomeric rnA (telomerase rnA (tr)); a small region of this rnA serves as a template for the telomeric repeats synthesis. Apart from rare exceptions, telomerase is not active in the somatic cells and tissues of the human body. However, the activation of telomerase activity in cancer cells was shown for certain in 80–90 % of cases. Understanding the mechanism of telomerase functioning and the mechanisms of its regulation could be used in oncodiagnostics. Telomerase itself and its regulators could be important targets for anticancer therapy. The activity of telomerase in a cell is affected by proteins with multiple functions, and this influence is not necessarily specific. There are also cases when telomerase regulators act together or when several regulators are organised in the cascade. The aim of this review is to generalize and systemize data about the regulation of telomerase in ontogenesis.

KEYWORDS telomerase, telomerase reverse transcriptase, telomerase rnA, regulation, cancer.

ABBREVIATIONS 5azadc (5-aza-2'-deoxicitidine), AV (adenoviruses), HBV (Hepatitis B Virus) HIV (Human Immunodeficiency Virus), HPV (Human papillomavirus), eBV (epstein–Barr Virus), np (nucleotide pair), OB-motive (oligonecleotide/oligosaccharide binding motive), rt (reverse transcription), Pcr (polymerase chain reacion), rnAse (ribonuclease), tert (telomerase reverse transcriptase), htert (human tert), mtert (mouse tert), Dn-htert (dominantnegative mutated htert), erβ (estrogen β receptor), HtLV-I (Human t-lymphotropic virus), tr (telomerase rnA), trAP telomeric repeat Amplification Protocol), PHA (phytohemagglutinin), Hre (Hypoxia response element), neS (nuclear export signal).
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Patrick Masson, Daniel Rochu

Catalytic Bioscavengers Against Toxic Esters, an Alternative Approach for Prophylaxis and Treatments of Poisonings

Patrick Masson^1,2*, Daniel Rochu^1,3
¹Centre de Recherches du Service de Sante des Armees, Toxicology department, La Tronche, France
²Institute of Structure Biology, Molecular Biophysics Laboratory, Grenoble, France
³Bundeswehr Institute of Toxicology and Pharmacology, Munich, Germany
*E-mail: pmasson@unmc.edu

ABSTRACT Bioscavengers are biopharmaceuticals that specifically react with toxicants. Thus, enzymes reacting with poisonous esters can be used as bioscavengers for neutralization of toxic molecules before they reach physiological targets. Parenteral administration of bioscavengers is, therefore, intended for prophylaxis or pre-treatments, emergency and post-exposure treatments of intoxications. These enzymes can also be used for application on skin, mucosa and wounds as active components of topical skin protectants and decontamination solutions. Human butyrylcholinesterase is the first stoichiometric bioscavenger for safe and efficient prophylaxis of organophosphate poisoning. However, huge amounts of a costly enzyme are needed for protection. Thus, the bioscavenger approach will be greatly improved by the use of catalytic bioscavengers. Catalytic bioscavengers are enzymes capable of degrading toxic esters with a turnover. Suitable catalytic bioscavengers are engineered mutants of human enzymes. Efficient mutants of human butyrylcholinesterase have been made that hydrolyze cocaine at a high rate. Mutants of human cholinesterases capable of hydrolyzing OPs have been made, but so far their activity is too low to be of medical interest. Human paraoxonase a promiscuous plasma enzyme is certainly the most promising phosphotriesterase. However, its biotechnology is still in its infancy. Other enzymes and proteins from blood and organs, and secondary biological targets of OPs and carbamates are potential bioscavengers, in particular serum albumin that reacts with OPs and self-reactivates. Lastly, non-human enzymes, phosphotriesterases and oxidases from various bacterial and eukaryotic sources could be used for external use against OP poisoning and for internal use after modifications for immunological compatibility.

KEYWORDS bioscavengers, carbamates, cocaine, enzyme engineering, enzymotherapy, organophosphorus compounds

ABBREVIATIONS Ache, acetylcholinesterase; Bche, butyrylcholinesterase; cae, carboxylesterase; che, cholinesterase; DFPase, diisopropylfluorophosphate hydrolase; GMP, good manufacturing practice; GMP, good manufacturing practice, GSt, glutathione S-transferase; OP, organophosphorus compound; OPAA, organophosphoric acid anhydrolase; OPAH, organophosphorus acid anhydride hydrolase; PeG, polyethylene glycol; POn1, paraoxonase 1; Pte, phosphotriesterase
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Inna N. Lavrik, Peter H. Krammer

Decisions in the CD95 System: Main Pro- and Anti- Apoptotic Modulators

Inna N. Lavrik*, Peter H. Krammer
Division of Immunogenetics, Tumorimmunology Program German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
*E-mail: i.lavrik@dkfz-heidelberg.de

ABSTRACT Apoptosis is common to all multicellular organisms. Apoptosis can be triggered by the extrinsic (death receptor (DR)) or the intrinsic (mitochondrial) death pathways. CD95 (APO-1/Fas) is a prototypic member of the DR family. This review is focused on the mechanisms of CD95 (APO-1/Fas)-mediated apoptosis and the role in the apoptosis of the death effector domain (DED)-containing proteins: pro-apoptotic protein procaspase-8 and anti-apoptotic protein c-FLIP. Gaining insights into these processes will improve our understanding of the pathogenesis of diseases such as cancer, autoimmunity and AIDS, and will open new approaches to rational treatment strategies.

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A. V. Bacheva, A. A. Belogurov, N. A. Ponomarenko, V. D. Knorre, V. M. Govorun, M. V. Serebryakova, A. G. Gabibov

Analysis of Myelin Basic Protein Fragmentation by Proteasome

A. V. Bacheva¹, A. A. Belogurov², N. A. Ponomarenko², V. D. Knorre², V. M. Govorun², M. V. Serebryakova³, and A. G. Gabibov^1,2
¹Chemistry Department, Moscow State University
²Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
³Proteomic Center, Russian Academy of Medical Sciences, Institute of Physical–Chemical Medicine

ABSTRACT The proteasome is a high molecular protein complex whose purpose is specific protein degradation in eukaryotic cells. One of the proteasome functions is to produce peptides, which will then be presented on the outer cell membrane using main histocompatibility complex (MHc) molecules of the first or second class. There are definite reasons to believe that proteasome directly takes part in the specific degradation of myelin basic protein (MBP), which make up to 30% of all proteins in the myelin sheath of neuronal axons. The details of the proteasomal degradation of MBP are still unclear. In this work, the features of specific MBP degradation by proteasome were studied. It was demonstrated that MBP (non-ubiquitinated) is a good substrate for 20S and for the 26S proteasome. This is the first work on detecting the sites of MBP proteolysis by proteasome from brains of SJL/J/J and Balb/c mice’s lines. Substantial differences in the degradation pattern of this neuroantigen were found, which could indicate the better presentation MBP parts on MHc molecules in the case of mice predisposed to the development of experimental autoimmune encephalomyelitis.

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E.N. Lyukmanova, G.S. Kopeina, M.A. Shulepko, Z.O. Shenkarev, A.S. Arseniev, D.A. Dolgikh, M.P. Kirpichnikov

Cell-free Production of the Extracellular Domain of the Nicotinic Acetylcholine Receptor

E.N. Lyukmanova¹, G.S. Kopeina², M.A. Shulepko², Z.O. Shenkarev¹, A.S. Arseniev¹, D.A. Dolgikh^1,2, M.P. Kirpichnikov^1,2
¹Shemyakin-Ovchinnikov Insitute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10, Miklucho-Maklaya str., Moscow, Russia
²Moscow State University, Leninskie Gory, GSP-1, Moscow, 119991
*E-mail: genchem@ibch.ru

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V.A. Mordvinov, А.V. Mardanov, N.V. Ravin, S.V. Shekhovtsov, S.А. Demakov, А.V. Katokhin, N.А. Kolchanov, and K.G. Skryabin

Complete Sequencing of the Mitochondrial Genome of Opisthorchis felineus, Causative Agent of Opisthorchiasis

V.A. Mordvinov1, А.V. Mardanov2, N.V. Ravin2, S.V. Shekhovtsov1, S.А. Demakov1, А.V. Katokhin1, N.А. Kolchanov1, and K.G. Skryabin2*
1Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 10 Lavrentieva Ave, Novosibirsk, 630090, Russia
2Bioengineering Center, Russian Academy of Sciences, 7/1 60?letiya Oktyabrya Ave, Moscow, 117312, Russia
* E-mail: office@biengi.ac.ru

ABSTRACT Opisthorchis felineus, a hepatic trematode, is the causative agent of opisthorchiasis, a dangerous disease in both human beings and animals. Opisthorchiasis is widespread in Russia, especially Western Siberia. The purpose of the present study was to determine the complete mitochondrial DnA sequence of this flatworm. two parallel methods were employed: (1) capillary electrophoresis to sequence the mitochondrial genome fragments obtained through specific PCR amplification, and (2) high throughput sequencing of the DnA sample. Both methods made possible the determination of the complete nucleotide sequence of the O. felineus mitochondrial genome. the genome consists of a ring molecule 14,277 nt in length that contains 35 genes coding 2 rrnA, 22 trnA, and 12 proteins: 3 subunits of cytochrome-c-oxidase, 7 subunits of nADH-dehydrogenase, B apocytochrome, and subunit 6 of AtP-synthetase. Like many other flatworms, O. felineus is characterized by the absence of the AtP-synthetase subunit 8 gene. Nineteen out of the 22 trnAs have a typical “clover leaf” structure. The trnA(AGc) and trnA-cys genes lack DHu-loops, while the trnA-Ser(ucA) has 2 alternative structures: one with a DHu-loop, and one without it. Analyzing the results obtained from the high throughput sequencing revealed 45 single-nucleotide polymorphisms within the mitochondrial genome. The results obtained in this study may be used in the development of molecular diagnostic methods for opisthorchiasis. This study shows that high throughput sequencing is a fast and effective method for decoding the mitochondrial genome of animals.

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E.S. Philonenko, A.A. Gavrilov, S.V. Ravin, O.V. Iarovaia

The Organization in Micro-Loops of an Extended Fragment of Chicken Chromosome 14, Including the Alpha Globin Gene Cluster in the Erythroid Cells

E.S. Philonenko*, A.A. Gavrilov, S.V. Ravin, O.V. Iarovaia
Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, 119344 Moscow, Russia
*E-mail: len_soap@mail.ru

ABSTRACT It has been shown that the activation of tissue-specific gene transcription during the course of cell differentiation is associated with a spatial reorganization of the genomic domains harboring those specific genes. This reorganization consists of the relocation to the nuclear matrix of the whole genomic domain containing one or more of the genes being transcribed. However, it remains unclear whether, during this process, extended areas of the genome also become attached to the nuclear matrix. We studied the genome's pattern of interaction with the nuclear matrix in both erythroid and non-erythroid cells of chickens, using a 220Kb region of chromosome #14, which contains the alpha-globin gene cluster and some surrounding house-keeping genes. the results show that in erythroid cells, the fragment of the genome containing the alpha-globin gene domain became spatially arranged into micro-loops which could not be detected by mapping experiments.

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E. N. Ilina

Direct Matrix-Assisted Laser Desorption–Ionisation (MALDI) Mass-Spectrometry Bacteria Profiling for Identifying and Characterizing Pathogens

E. N. Ilina
Scientific Research Institute of Physical-Chemical Medicine
E-mail: ilinaEN@gmail.com

ABSTRACT This study examines the features and limitations of direct Matrix-Assisted Laser Desorption–Ionisation (MALDI) mass-spectrometry profiling of bacterial cells for investigating a microbial population. The optimal laboratory protocol, including crude bacteria lyses by a solution of 50% acetonitrile, 2.5% trifluoroacetic acid, and using α-cyano-4-hydroxy cinnamic acid as the MALDI matrix, has been developed. Two different bacteria species were under investigation, and representative mass spectra from 278 strains of Neisseria gonorrhoeae and 22 strains of Helicobacter pylori have been analyzed. It’s known that both bacteria demonstrate a variable degree of polymorphism. For N. gonorrhoeae, the MALDI mass spectra that was collected possessed about 70 peaks, 20 of which were good reproducible ones. In spite of the fact that three peaks were found with differing spectra in some strains, little diversity in the N. gonorrhoeae population was revealed. This fact indicates the prospects in using direct MALDI mass-spectrometry profiling for gonococcus identification. In the case of H. pylori strains, the variety in the collected mass-spectra was shown to be essential. Only five peaks were present in more than 70% of strains, and a single mass value was common for all spectra. While these data call into question the possibility of the reliable species identification of H. pylori using this approach, the intraspecies differentiation of strains was offered. Good association between MALDI profile distributions and the region of strain isolation have been found. Thus, the suggested direct MALDI mass-spectrometry profiling strategy, coupled with special analysis software, seems promising for the species identification of N. gonorrhoeae but is assumed insufficient for H. pylori species determination. At the same time, this would create a very good chance for an epidemiological study of such variable bacteria as H. pylori.

KEYWORDS MALDI mass spectrometry, bacteria profiling, Neisseria gonorrhoeae, Helicobacter pylori.

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V. N. Lazarev

The Genes of Antimicrobial Peptides for the Therapy of Intracellular Infections

V. N. Lazarev
Scientific Research Institute of Physical–Chemical Medicine, ul. Malaya Pirogovskaya 1a, Moscow, 119992, Russia
E-mail: lazar0@mail.ru

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K. А. Konduktorov, G. S. Lyudva, А. V. Ivanov, V. L. Tunitskaya, and S. N. Kochetkov

The Interaction between the RNA - Dependent RNA - Polymerase of the Hepatitis Virus and RNA Matrices

K. А. Konduktorov, G. S. Lyudva, А. V. Ivanov, V. L. Tunitskaya, and S. N. Kochetkov*
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilova 32, Moscow 119991, Russia
*E-mail: kochet@eimb.ru

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O. Nekrasova, A. Tagway, A. Ignatova, A. Feofanov, M. Kirpichnikov

Studying of Membrane Localization of Recombinant Potassium Channels in E.coli

O. Nekrasova^1*, A. Tagway¹, A. Ignatova^1,2, A. Feofanov^1,2, M. Kirpichnikov^1,2
¹Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997, Moscow, Russia
²Biological Faculty, Lomonosov Moscow State University, Vorobyevi Gori 1, Moscow, 119992, Russia
*E-mail: genchem@ibch.ru

ABSTRACT
The effective expression of recombinant membrane proteins in E.coli depends upon the targeting and insertion of proteins into the cellular membrane, as well as on those proteins adopting the correct spatial structure. A significant technological problem involves the design of approaches for detecting the location of target proteins within a host cell. Using a hybrid potassium channel KcsA-Kv1.3 as a model, we developed a technological scheme which is suitable for the study of membrane localization in E.coli cells of recombinant proteins containing voltage-gated eukaryotic potassium channels as the functional active site. The scheme involves both biochemical and fluorescent methods for detecting target proteins in the cytoplasmic membrane of E.coli, as well as the study of the ligand-binding activity of membrane-embedded proteins.

KEYWORDS membrane proteins; KcsA; cell fractionation; fluorescent methods

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E. L. Arsenieva, I. V. Kuzmin, E. S. Manuilova, E. V. Novosadova, E. V. Murkin, G. V. Pavlova, V. Z. Tarantul, and I. A. Grivennikov

The Production and Characteristics of a Mouse’s Embryonic Stem Cell Lineage, Transfected by the Glia Neurotrophic Factor and Gene Fused with the Green Fluorescent Protein Gene

E. L. Arsenieva, I. V. Kuzmin, E. S. Manuilova, E. V. Novosadova, E. V. Murkin, G. V. Pavlova, V. Z. Tarantul, and I. A. Grivennikov
Molecular Genetics Institute, Russian Academy of Sciences, Moscow
Gene Biology Institute, Russian Academy of Sciences, Moscow
*E-mail: igorag@img.ras.ru

ABSTRACT The influence that the expression of the human (glial-derived neurotrophic factor (GDnF)) neurotrophic factor has on the morphology and proliferative activity of embryonic stem cells (Sc) of a mouse with r1 lineage, as well as their ability to form embroid bodies (eB), has been studied. Before that, using a Pcr (polymerase chain reaction) coupled with reverse transcription, it was shown that, in this very lineage of the embryonic Sc, the expression of the receptors’ genes is being fulfilled for the neurotropfic ret and GFrα1 glia factor. The mouse's embryonic Sc lineage has been obtained, transfected by the human GDnF gene, and has been fused with the “green” fluorescent protein (GFP) gene. the presence of the expression of the human GDnF gene in the cells was shown by northern hybridization and the synthesis of its albuminous product by immunocitochemical coloration with the use of specific antibodies. The reliable slowing-down of the embriod-body formation by the embryonic Sc transfected by the GDnF gene has been shown. no significant influence of the expression of the GDnF gene on the morphology and the proliferative activity of the transfected embryonic Scs has been found when compared with the control ones.

KEYWORDS embryonic stem cells, glial-derived neurotrophic factor, transfection, proliferation, immunocitochemistry, emroid bodies

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T. V. Pyrkov, D. V. Pyrkova, E. D. Balitskaya, and R. G. Efremov

The Role of Stacking Interactions in Complexes of Proteins with Adenine and Guanine Fragments of Ligands

T. V. Pyrkov^1,2*, D. V. Pyrkova¹, E. D. Balitskaya^1,3, and R. G. Efremov¹
¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russia.
²Moscow Institute of Physics and Technology (State University), Institutskii per., 9, Dolgoprudny, Moscow oblast, 141700, Russia.
³Moscow State University, Moscow, 119991, Russia
*E-mail: pyrkov@nmr.ru

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Acta Naturae, 2009. V. 1.
№ 2.

A.V. Nemukhin, B.L. Grigorenko, A.P. Savitsky

Computer Modeling of the Structure and Spectra of Fluorescent Proteins

A.V. Nemukhin^1,2*, B.L. Grigorenko¹, A.P. Savitsky^1,3
¹Department of Chemistry, M.V. Lomonosov Moscow State University
²N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences
³A.N. Bach Institute of Biochemisty, Russian Academy of Sciences
*E-mail: anemukhin@yahoo.com

ABSTRACT Fluorescent proteins from the family of green fluorescent proteins are intensively used as biomarkers in living systems. The chromophore group based on the hydroxybenzylidene-imidazoline molecule, which is formed in nature from three amino-acid residues inside the protein globule and well shielded from external media, is responsible for light absorption and fluorescence. Along with the intense experimental studies of the properties of fluorescent proteins and their chromophores by biochemical, X-ray, and spectroscopic tools, in recent years, computer modeling has been used to characterize their properties and spectra. We present in this review the most interesting results of the molecular modeling of the structural parameters and optical and vibrational spectra of the chromophorecontaining domains of fluorescent proteins by methods of quantum chemistry, molecular dynamics, and combined quantum-mechanical–molecular-mechanical approaches. The main emphasis is on the correlation of theoretical and experimental data and on the predictive power of modeling, which may be useful for creating new, efficient biomarkers.

KEYWORDS green fluorescent protein, molecular modeling, molecular dynamics, molecular mechanics

ABBREVIATIONS QM/MM - combined methods of quantum and molecular mechanics, MD - molecular dynamics, tD-DFt - the method of density functional theory depending on time.
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O.A. Patutina, N.L. Mironova, V.V. Vlassov, M.A. Zenkova

New Approaches for Cancer Treatment: Antitumor Drugs Based on Gene-Targeted Nucleic Acids

O.A. Patutina, N.L. Mironova, V.V. Vlassov, M.A. Zenkova*
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
*E-mail: marzen@niboch.nsc.ru

ABSTRACT Currently, the main way to fight cancer is still chemotherapy. This method of treatment is at the height of its capacity, so, setting aside the need for further improvements in traditional treatments for neoplasia, it is vital to develop now approaches toward treating malignant tumors. This paper reviews innovational experimental approaches to treating malignant malformations based on the use of gene-targeted drugs, such as antisense oligonucleotides (asON), small interfering rnA (siRNA), ribozymes, and DNAzymes, which can all inhibit oncogene expression. The target genes for these drugs are thoroughly characterized, and the main results from pre-clinical and first-step clinical trials of these drugs are presented. It is shown that the gene-targeted oligonucleotides show considerable variations in their effect on tumor tissue, depending on the target gene in question. The effects range from slowing and stopping the proliferation of tumor cells to suppressing their invasive capabilities. Despite their similarity, not all the antisense drugs targeting the same region of the mRNA of the target-gene were equally effective. the result is determined by the combination of the drug type used and the region of the target-gene mRNA that it complements.

KEYWORDS cancer therapy, antisense oligonucleotides, ribozymes, DNAzymes, small interfering RNA

ABBREVIATIONS antisense oligonucleotides (asON), small interfering rnA (siRNA), RNA interference (RNAi)
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V.V. Terskikh, Ye. A. Vorotelyak, A.V. Vasiliev

Self-Renewal of Stem Cells

V.V. Terskikh*, Ye. A. Vorotelyak, A.V. Vasiliev N.K. Koltsov Institute of Developmental Biology, Russian Academy of Sciences *E-mail: terskikh@bk.ru

ABSTRACT Asymmetric division is one of the most fundamental characteristics of adult stem cells , which ensures one daughter cell maintains stem cell status and the other daughter cell becomes committed to differentiation. New data emerged recently that allow us to conclude that asymmetric division has another important aspect: it enables self-maintenance of stem cells.

KEYWORDS asymmetric division, stem cells, aging of stem cells, self-renewal of stem cells, aggresomes, Drosophila neuroblasts.

ABBREVIATIONS Hemopoietic stem cells (HSC), mutant protein huntingtin Htt, tumor suppressor Lgl (lethal giant larvae), stem cells (SC), tumor suppressor Discslarge (Dlg), Partitioning defective protein (Par)
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T. Shcheglova, S.P. Makker, and A. Tramontano

Covalent Binding Antibodies Suppress Advanced Glycation: On the Innate Tier of Adaptive Immunity

T. Shcheglova², S. P. Makker¹, and A. Tramontano^1*
¹Department of Pediatrics, University of California, Davis – School of Medicine Davis
²Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences
*E-mail: tramontano@ucdavis.edu

ABSTRACT Non-enzymatic protein glycation is a source of metabolic stress that contributes to cytotoxicity and tissue damage. Hyperglycemia has been linked to elevation of advanced glycation Endproducts, which mediate much of the vascular pathology leading to diabetic complications. enhanced glycation of immunoglobulins and their accelerated vascular clearance is proposed as a natural mechanism to intercept alternative advanced glycation endproducts, thereby mitigating microvascular disease. We reported that antibodies against the glycoprotein KLH have elevated reactivity for glycopeptides from diabetic serum. These reactions are mediated by covalent binding between antibody light chains and carbonyl groups of glycated peptides. Diabetic animals that were immunized to induce reactive antibodies had attenuated diabetic nephropathy, which correlated with reduced levels of circulating and kidney-bound glycation products. Molecular analysis of antibody glycation revealed the preferential modification of light chains bearing germline-encoded lambda V regions. We previously noted that antibody fragments carrying V regions in the germline configuration are selected from a human Fv library by covalent binding to a reactive organophosphorus ester. These Fv fragments were specifically modified at light chain V region residues, which map to the combining site at the interface between light and heavy chains. These findings suggest that covalent binding is an innate property of antibodies, which may be encoded in the genome for specific physiological purposes. This hypothesis is discussed in context with current knowledge of the natural antibodies that recognize altered self molecules and the catalytic autoantibodies found in autoimmune disease.

KEYWORDS natural autoantibodies, covalent binding, reactive antibody, advanced glycation, carbonyl group, diabetic complications, hydrolytic abzymes

ABBREVIATIONS Nabs, Natural antibodies; KLH, Keyhole limpet hemocyanin; VL, light chain variable region; VH, heavy chain variable region; CDR, complementarity determining region; LDL, low-density lipoprotein; AGEs, advanced glycation endproducts; RAGE, receptor of AGE; OP, organophosphorus ester; scFv, single-chain variable fragment; FR, framework region; PC, phosphorylcholine; PAMPs, pathogen-associated molecular patterns; TLR, toll-like receptor.
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E.G. Salina, H.J. Mollenkopf, S.H.E. Kaufmann, A.S. Kaprelyants

M. tuberculosis Gene Expression during Transition to the “Non-Culturable” State

E.G. Salina¹, H.J. Mollenkopf^2*, S.H.E. Kaufmann², A.S. Kaprelyants¹
¹A.N. Bach Institute of Biochemistry, RAS
²Max Planck Institute for Infection Biology
*E-mail: mollenkopf@mpiib-berlin.mpg.de

ABSTRACT
We analyzed the gene expression profile under specific conditions during reversible transition of M. tuberculosis cells to the “non-culturable” (nc) state in a prolonged stationary phase. More than 500 genes were differentially regulated, while 238 genes were upregulated over all time points during nc cell formation. Approximately a quarter of these upregulated genes belong to insertion and phage sequences indicating a possible high intensity of genome modification processes taking place under transition to the nc state. Besides the high proportion of hypothetical/conserved hypothetical genes in the cohort of upregulated genes, there was a significant number of genes belonging to intermediary metabolism, respiration, information pathways, cell wall and cell processes, and genes encoding regulatory proteins. We conclude that nc cell formation is an active process involved in the regulation of many genes of different pathways. A more detailed analysis of the experimental data will help to understand the precise molecular mechanisms of dormancy/latency/persistence of M. tuberculosis in the future. The list of upregulated genes obtained in this study includes many genes found to be upregulated in other models of M. tuberculosis persistence. Thirteen upregulated genes, which are common for different models, can be considered as potential targets for the development of new anti-tuberculosis drugs directed mainly against latent tuberculosis.

KEYWORDS M. tuberculosis, latent tuberculosis, "non-culturable" cells, global gene expression profile

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E.S. Knyazhanskaya, M.A. Smolov, O.V. Kondrashina, M.B. Gottikh

Relative Comparison of Catalytic Characteristics of Human Foamy Virus and HIV-1 Integrases

E. S. Knyazhanskaya¹, M. A. Smolov², O. V. Kondrashina², M. B. Gottikh^3*
¹Chemistry Department of MSU
²Department of Bioengineering and Bioinformatics of MSU
³A. N. Belozersky Institute of Physico-Chemical Biology, M. V. Lomonosov Moscow State University
*E-mail: gottikh@libro.belozersky.msu.ru

ABSTRACT Due to their ability to integrate into the host cell’s genome, retroviruses represent an optimal basis for the creation of gene therapy vectors. The integration reaction is carried out by a viral enzyme integrase: thus, a detailed research of this enzyme is required. In this work, the catalytic properties of human foamy virus integrase were studied. This virus belongs to the retroviridae family. The dissociation constant was determined, together with the kinetics of integrase catalytic activity. The data obtained were compared to those for the human immunodeficiency virus integrase and a considerable similarity in the activity of the two enzymes was observed.

KEYWORDS HIV Human immunodeficiency virus, HFV Human foamy virus, integrase, catalytic activity

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D.M. Shcherbakova, M.I. Zvereva, and O.A. Dontsova

Telomerase Complex from Yeast Saccharomyces cerevisiae Contains a Biotinylated Component

D.M. Shcherbakova, M.I. Zvereva,* and O.A. Dontsova
Department of Chemistry, Moscow State University
*E-mail: zvereva@genebee.msu.su

ABSTRACT
Telomerase adds telomeric repeats to single-stranded DNA at the ends of the chromosomes. This enzyme is a ribonucleoprotein complex. Telomerase from yeast Saccharomyces cerevisiae consists of TLC1 RNA, which serves as a template for the synthesis of telomeric repeats, telomerase reverse transcriptase Est2p, and a number of accessory proteins (Est1p, Est3p, Ku70/Ku80, and Sm-complex). We found that the yeast telomerase complex contains a biotinylated component. The telomerase fraction containing biotinylated protein is active in vitro and constitutes a small part of the total amount of active telomerase isolated from cells. We speculate about the nature of the biotinylated component.

KEYWORDS yeast telomerase, biotin, biotinylation.

ABBREVIATIONS DEAE-fraction – telomerase isolated from yeast extract using chromatography on DEAE-cellulose
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P.V. Spirin, D. Baskaran, P.M. Rubtsov, M.A. Zenkova, V.V. Vlassov, E.L. Chernolovskaya, V.S. Prassolov

A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA that Express Recombinant Lentiviral Vectors

P.V. Spirin, D. Baskaran, P.M. Rubtsov, M.A. Zenkova, V.V. Vlassov, E.L. Chernolovskaya,
V.S. Prassolov*
Engelghardt Institute of Molecular Biology, Russian Academy of Sciences
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
*E-mail: prasolov@eimb.ru

ABSTRACT RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it’s widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ETO by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented.

KEYWORDS retroviral vectors, rnA interference, embryonic mouse fibroblasts.

ABBREVIATIONS AML – Acute Myeloid Leukemia, CBF – Core Binding Factor, EGFP – Enhanced Green Fluorescent Protein, GTU – GFP transducing units, HDAC– Histone deacetylase, IRES –Internal ribosome entry site, NHR – Nervy Homology Region, RHD –Runt Homology Domain, VSV-G –Vesicular stomatitis virus G glycoprotein
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M.V. Shutova, A.N. Bogomazova, M.A. Lagarkova, S.L. Kiselev

Generation and Characterization of Human Induced Pluripotent Stem Cells

M.V. Shutova, A.N. Bogomazova, M.A. Lagarkova, S.L. Kiselev*
Vavilov Institute of General Genetics, Russian Academy of Sciences
*E-mail: kiselev@vigg.ru

ABSTRACT Cell biology is one of the most rapidly developing branches in modern biology. The most interesting stages in early embryonic development for cell biology are those when a large number of cells are pluripotent. Inner-cell mass of blastocyst can be cultivated in vitro, and these cells are called embryonic stem cells. They are able to differentiate into different types of cells and tissues. But the greatest interest for practical application is the return (reprogramming) of adult cells into the pluripotent state. In our study for the first time induced pluripotent cells were derived from human umbilical vein endothelial cells by genetic reprogramming. We showed that these cells are similar to embryonic stem cells in their morphology, function, and molecular level. We are the first to show that reprogramming sufficiently changes X-chromosome chromatin state, which is normally inactive in female endothelial cells, towards its activation, providing evidence that endothelial cells are reprogrammed at an epigenetic level.

KEYWORDS pluripotency, cell reprogramming, X-chromosome reactivation.

ABBREVIATIONS embryonic stem cells (eSc), cells with induced pluripotency (iPS)
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E.V. Novosadova, E.S. Manuilova, E.L. Arsenieva, A.N. Lebedev, N.V. Khaidarova, V.Z. Tarantul, and I.A. Grivennikov

Influence of pub Gene Expression on Differentiation of Mouse Embryonic Stem Cells into Derivatives of Ecto-, Meso-, and Endoderm in vitro

E.V. Novosadova, E.S. Manuilova, E.L. Arsenieva, A.N. Lebedev, N.V. Khaidarova, V.Z. Tarantul, and I.A. Grivennikov*
Institute of Molecular Genetics, Russian Academy of Sciences
*E-mail: igorag@img.ras.ru</p

ABSTRACT
The influence of low and high pub gene expression on the initial stages of the differentiation of mouse embryonic stem cells into derivatives of ecto-, meso-, and endoderm in vitro was investigated. As follows from the results of a RT-PCR analysis, the expression of the vimentin, somatostatin, GATA 4, and GATA 6 genes, being the markers of endodermal differentiation, does not vary in both the cells with high pub gene expression and the cells with low pub gene expression, as well as in the corresponding control lines. The cells with high pub gene expression are characterized by an increase in the expression of mesodermal differentiation gene-markers (trI card, trI skel, c-kit, and IL-7), whereas the cells with low pub gene expression are specified by a decrease in their expression. According to the analyses carried out, the reverse is characteristic of the expression of ectodermal differentiation gene-markers (nestin, β-III tubulin, gfap, and th). Expression of these genes decreases in cell lines with high pub gene expression, whereas their expression increases with the decrease in pub gene expression. Hence, it is suggested that the variations in the pub gene expression in the embryonic stem cells influence significantly the mesodermal and ectodermal differentiation of these cells.

KEYWORDS embryonic stem cells, differentiation, polymerase chain reaction, pub gene, mesoderm, endoderm, and ectoderm.

ABBREVIATIONS ES - embryonic stem cells, FBS - foetal bovine serum
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D.A. Davydova, E.A.Vorotelyak, Yu.A. Smirnova, R.D. Zinovieva, Yu. A. Romanov, N. V. Kabaeva, V.V. Terskikh, and A.V. Vasiliev

Cell Phenotypes in Human Amniotic Fluid

D.A. Davydova^1*, E.A.Vorotelyak¹, Yu.A. Smirnova¹, R.D. Zinovieva¹, Yu.A. Romanov², N.V. Kabaeva², V.V. Terskikh¹, and A.V. Vasiliev¹
¹Koltzov Institute of Developmental Biology, Russian Academy of Sciences
²Russian Cardiology Research-and-Production Complex
*E-mail: davydovad@gmail.com

ABSTRACT
Stem cells capable of long-term proliferation and differentiation into different cell types may be a promising source of cells for regenerative medicine. Recently, much attention has been paid to fetal stem cells, among which are cells from amniotic fluid (AF). We have isolated amniotic stem cells from 3 AF samples. Flow cytometry, RT-PCR and immunohistochemistry have shown that these cells express mesenchymal (CD90, CD73, CD105, CD13, CD29, CD44, and CD146), neural (β3-tubulin, nestin, and Pax6), epithelial (keratin 19 and p63) markers and also markers of pluripotency (Oct4, Nanog, and Rex-1). Transplantation of the cells to nude mice does not lead to tumor formation. Thus, putative stem/progenitor cells from AF are capable of long-term proliferation in vitro and the profile of gene expression led us to speculate that they have greater differentiation potential than mesenchymal stem cells and may be useful for cell therapy.

KEYWORDS amniotic fluid, cell culture, epithelial markers, mesenchymal stem cells, neural markers, stem cells.

ABBREVIATIONS amniotic fluid (AF); mesenchymal stem cells (MScs); embryonic stem cells (eScs).
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V.A. Stonik

Marine Natural Products: A Way to New Drugs

V.A. Stonik
Pacific Institute of Bioorganic Chemistry, Far-eastern Branch of the Russian Academy of Science
E-mail: Stonik@piboc.dvo.ru

ABSTRACT The investigation of marine natural products (low molecular weight bioregulators) is a rapidly developing scientific field at the intersection of biology and chemistry. Investigations aimed at detecting, identifying, and understanding the structure of marine natural products have led to the discovery of 20,000 new substances, including those characterized by an extremely high physiological activity. Some results and prospects of works aimed at creating new drugs on the basis of marine natural products are discussed herein.

KEYWORDS marine organisms, natural products, physiological activity, trabectidin, prialt, histochrome, collagenase KK.

ABBREVIATIONS HIV- human immunodeficiency virus, AIDS – acquired immune deficiency syndrome, PIBOc – Pacific Institute of Bioorganic chemistry, EC₅₀ – effective concentration that provokes a response halfway between the baseline and maximum response, IC₅₀ – concentration that provokes 50% inhibition, VEGF – vascular endothelial growth factor.
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Y.A. Shtyrya, L.V. Mochalova, N.V. Bovin

Influenza Virus Neuraminidase: Structure and Function

Y.A. Shtyrya, L.V. Mochalova, N.V. Bovin*
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS
*E-mail: professorbovin@yandex.ru

ABSTRACT The structure of the influenza virus neuraminidases, the spatial organization of their active site, the mechanism of carbohydrate chains desialylation by neuraminidase, and its role in the influenza virus function at different stages of the viral infectious cycle are considered in this review. Data on the neuraminidase substrate specificity and different approaches in studying the activity of this enzyme are summarized. In addition, data on neuraminidase inhibitors (as antivirals) are provided, along with considerations on the mechanisms of resistance of modern influenza viruses to those antivirals.

KEYWORDS influenza virus, neuraminidase, substrate specificity

ABBREVIATIONS a.a. – amino acid residue; HA – haemagglutinin; nA – neuraminidase; neu5Ac - n-acetylneuraminic acid; 3`SiaLac – neuAcα2-3Galβ1-4Glc; 6`SiaLac – neuAcα2-6Galβ1-4Glc; Mu-nAnA – methylumbelliferyl-α-neuraminic acid; BODIPY - 6-((4,4-difluoro-5,7- dimethyl -4- bora -3a,4a-diaza-sindacene-3-propionyl)amino)-hexaonic acid, succinimidyl ester; MDcK – Madin-Darby canine Kidney cells; VerO – kidney epithelial cells from African green monkey ; neu5Gc – n-glycolylneuraminic acid; 3`SiaLacnAc – neu5Acα2-3Galβ1-4GlcnAc; 6`SiaLacnAc – neu5Acα2-6Galβ1-4GlcnAc.
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Acta Naturae, 2009. V. 1.
№ 3.

N.V. Tikunova, V.V. Morozova

Phage Display on the Base of Filamentous Bacteriophages: Application for Recombinant Antibodies Selection

N.V. Tikunova*, V.V. Morozova
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Science
*E-mail: tikunova@niboch.nsc.ru

ABSTRACT The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

KEYWORDS phage display, filamentous bacteriophages, phagemids, phage display libraries of antibody fragments, biopanning, single chain antibody fragments, Fab-fragments.

ABBREVIATIONS
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V.P. Puzyrev, M.B. Freidin

Genetic View on the Phenomenon of Combined Diseases in Man

V.P. Puzyrev*, M.B. Freidin
Research Institute for Medical Genetics, Siberian Branch, Russian Academy of Medical Sciences
*E-mail: valery.puzyrev@medgenetics.ru

ABSTRACT
In clinical medicine, the phenomenon of polypathy, as a particular object of investigation, was first put forth by French clinicians at the end of the 19^th century through the “arthritismus” doctrine. In the first half of the 20^th century, German paediatricians singled out “syntropias,” which are combinations of diseases with common pathophysiological mechanisms, and “dystropias,” which are diseases that rarely co-occur in one individual. In the present paper, syntropy/dystropy is defined as a natural generic nonrandom phenomenon with an evolutionary-genetic basis. The genes involved in the development of syntropy are called “syntropic genes,” whereas the genes that co-participate in pathophysiological mechanisms and prevent the co-occurrence of particular phenotypes are called “dystropic genes.” Prospects for studying the genetic basis of this phenomenon are highlighted. The publicly available database HuGENet can be used in order to identify syntropic genes, as will be shown as examples in an analysis of cardiovascular diseases.

KEYWORDS syntropy, dystropy, syntropic and dystropic genes, genome, phenome, HuGENet.

ABBREVIATIONS
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A.P. Grigorenko, S.A. Borinskaya, N.K. Yankovsky, E.I. Rogaev

Achievements and Peculiarities in Studies of Ancient DNA and DNA from Complicated Forensic Specimens

A.P. Grigorenko^1,2,3, S.A. Borinskaya¹, N.K. Yankovsky¹, E.I. Rogaev^1,2,3*
¹Vavliov Institute of General Genetics, Russian Academy of Sciences
²Research Center of Mental Health, Russian Academy of Medical Sciences
³University of Massachusetts Medical School, Worcester, U.S.A.
* E-mail: rogaev@vigg.ru

ABSTRACT Studies of ancient DNA specimens started 25 years ago. At that time short mitochondrial DNA (mtDNA) fragments were the main targets in ancient DNA studies. The last three years were especially productive in the development of new methods of DNA purification and analysis. Complete mtDNA molecules and relatively large fragments of nuclear DNA are the targets of ancient DNA studies today. Ancient DNA studies allowed us to study organisms that went extinct more than ten thousand years ago, to reconstruct their phenotypic traits and evolution. Ancient DNA analyses can help understand the development of ancient human populations and how they migrated. A new evolutionary hypothesis and reconstruction of the biota history have been re-created from recent ancient DNA data. Some peculiarities and problems specific to the study of ancient DNA were revealed, such as very limited amounts of DNA available for study, the short length of the DNA fragments, breaks and chemical modifications in DNA molecules that result in “postmortem” mutations or complete blockage of DNA replication in vitro. The same specific features of DNA analysis were revealed for specimens from complicated forensic cases that result in the lack of experimental data or interpretation problems. Here, we list the specific features of ancient DNA methodology and describe some achievements in fundamental and applied research of ancient DNA, including our own work in the field.

KEYWORDS ancient DNA, methods, evolution, DNA identification, forensic examination.

ABBREVIATIONS mtDNA – mitochondrial DNA, cmtDNA – complete mtDNA sequence, STR – short tandem repeats
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N.M. Novozhilova, N.V. Bovin

D-Arabinose Methabolism: Characterization of Bifunctional Arabinokinase/ Pyrophosphorylase of Leishmania major

N.M. Novozhilova*, N.V. Bovin
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS
*E-mail: nnovogilov@gmail.com

ABSTRACT
In this work we describe an unusual enzyme from Leishmania major (Arabinokinase / Pyrophosphorylase) that catalyzes the synthesis of GDP-Darabinopyranose (GDP-D-Arap) via a D-arabinose-1-phosphate intermediate in the presence of ATP and GTP. Our data indicate GDP-D-Arap transport in vivo by the LPG2 multispecific nucleotide sugar transporter into the Leishmania Golgi apparatus, in which it can be used by glycosyltransferases as a donor substrate for glycosylation.

KEYWORDS D-arabinopyranose, Leishmania, bifunctional enzyme, kinase, pyriphosphorylase, lipophosphoglycan.

ABBREVIATIONS D-Arap – D-arabinopyranose; L-Fuc – L-fucose; K_m – the Michaelis-Menten constant; V_max – the maximum reaction velocity
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I.G. Shabalin, K.M. Polyakov, V.I. Tishkov, V.O. Popov

Atomic Resolution Crystal Structure of NAD⁺-Dependent Formate Dehydrogenase from Bacterium Moraxella sp. C-1

I.G. Shabalin¹, K.M. Polyakov^2,1, V.I. Tishkov¹, V.O. Popov^1*
¹A.N. Bach Institute of Biochemistry RAS
²V.A. Engelhardt Institute of Molecular Biology RAS
*E-mail: vpopov@inbi.ras.ru

ABSTRACT
The crystal structure of the ternary complex of NAD⁺-dependent formate dehydrogenase from the methylotrophic bacterium Moraxella sp. С-1 with the cofactor (NAD⁺) and the inhibitor (azide ion) was established at 1.1 Å resolution. The complex mimics the structure of the transition state of the enzymatic reaction. The structure was refined with anisotropic displacements parameters for non-hydrogen atoms to a R factor of 13.4%. Most of the nitrogen, oxygen, and carbon atoms were distinguished based on the analysis of the temperature factors and electron density peaks, with the result that side-chain rotamers of histidine residues and most of asparagine and glutamine residues were unambiguously determined. A comparative analysis of the structure of the ternary complex determined at the atomic resolution and the structure of this complex at 1.95 Å resolution was performed. In the atomic resolution structure, the covalent bonds in the nicotinamide group are somewhat changed in agreement with the results of quantum mechanical calculations, providing evidence that the cofactor acquires a bipolar form in the transition state of the enzymatic reaction.

KEYWORDS formate dehydrogenase, X-ray diffraction study, atomic resolution, enzymatic catalysis.

ABBREVIATIONS
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I.V. Shapovalova, W.B.L. Alkema, O.V. Jamskova, E. de Vries, D.T. Guranda, D.B. Janssen, V.K. Švedas

Mutation of Residue ßF71 of Escherichia coli Penicillin Acylase Results in Enhanced Enantioselectivity and Improved Catalytic Properties

I.V. Shapovalova¹, W.B.L. Alkema², O.V. Jamskova¹, E. de Vries², D.T. Guranda¹, D.B. Janssen², V.K. Švedas^1*
¹Belozersky Institute of Physicochemical Biology and Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Russia
²Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Netherlands
*E-mail: vytas@belozersky.msu.ru

ABSTRACT
Residue phenylalanine 71 of the β-chain of penicillin acylase from E. coli is involved in substrate binding and chiral discrimination of its enantiomers. Different amino acid residues have been introduced at position βF71, and the mutants were studied with respect to their enantioselectivity and substrate specificity. Some mutants demonstrated remarkably improved catalytic activity. Moreover, mutation of βF71 residue allowed to enhance penicillin acylase enantioselectivity. The catalytic activity to the specific substrates was improved up to 36 times, most notably for K, R, and L mutants. Increased activity to a D-phenylglycine derivative – a valuable specificity improvement for biocatalytic synthesis of new penicillins and cephalosporins – was shown for βF71r and βF71L mutants. The synthetic capacity of penicillin acylase with 6-aminopenicillanic acid as an external nucleophile was especially sensitive to mutation of the β71 residue in contrast to the synthesis with 7-aminodeacetoxycephalosporanic acid.

KEYWORDS penicillin acylase; βF71 mutants; enantioselectivity; improved catalysis;

ABBREVIATIONS PA – penicillin acylase; Ntn – n-terminal nucleophile; WT – wild type.
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K.G. Skryabin, E.B. Prokhortchouk, A.M. Mazur, E.S. Boulygina, S.V. Tsygankova, A.V. Nedoluzhko, S.M. Rastorguev, V.B. Matveev, N.N. Chekanov, D.A. Goranskaya, A.B. Teslyuk, N.M. Gruzdeva, V.E. Velikhov, D.G. Zaridze, M.V. Kovalchuk

Combining Two Technologies for Full Genome Sequencing of Human

K.G. Skryabin¹, E.B. Prokhortchouk^1*, A.M. Mazur¹, E.S. Boulygina¹, S.V. Tsygankova¹, A.V. Nedoluzhko¹, S.M. Rastorguev¹, V.B. Matveev², N.N. Chekanov³, D.A. Goranskaya³, A.B. Teslyuk¹, N.M. Gruzdeva¹, V.E. Velikhov¹, D.G. Zaridze², M.V. Kovalchuk¹
¹Russian Research Centre Kurchatov Institute
²Institute of Carcinogenesis, Blokhin Cancer Research Center, Russian Academy of Medical Sciences
³Bioengineering Center, Russian Academy of Sciences
*E-mail: prokhortchouk@biengi.ac.ru

ABSTRACT
At present, the new technologies of DNA sequencing are rapidly developing allowing quick and efficient characterisation of organisms at the level of the genome structure. In this study, the whole genome sequencing of a human (Russian man) was performed using two technologies currently present on the market - Sequencing by Oligonucleotide Ligation and Detection (SOLiD^TM) (Applied Biosystems) and sequencing technologies of molecular clusters using fluorescently labeled precursors (Illumina). The total number of generated data resulted in 108.3 billion base pairs (60.2 billion from Illumina technology and 48.1 billion from SOLiD technology). Statistics performed on reads generated by GAII and SOLiD showed that they covered 75% and 96% of the genome respectively. Short polymorphic regions were detected with comparable accuracy however, the absolute amount of them revealed by SOLiD was several times less than by GAII. Optimal algorithm for using the latest methods of sequencing was established for the analysis of individual human genomes. The study is the first Russian effort towards whole human genome sequencing.

KEYWORDS human genome, sequencing technology, single-nucleotide polymorphism

ABBREVIATIONS Indel SNP – insertion/deletion type single-nucleotide polymorphism.
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N.I. Grineva, T.V. Akhlynina, L.P. Gerasimova, T.E. Manakova, N.G. Sarycheva, D.A.Schmarov, A.M. Tumofeev, N.M. Nydenova, L.Yu. Kolosova, T.I. Kolosheynova, L.G. Kovaleva, S.V. Kuznetsov, A.V. Vorontsova, A.G. Turkina

Cell Regulation of Proliferation and Differentiation ex vivo for Cells Containing Ph Chromosome in Chronic Myeloid Leukemia

N.I. Grineva*, T.V. Akhlynina, L.P. Gerasimova, T.E. Manakova, N.G. Sarycheva, D.A.Schmarov, A.M. Tumofeev, N.M. Nydenova, L.Yu. Kolosova, T.I. Kolosheynova, L.G. Kovaleva, S.V. Kuznetsov, A.V. Vorontsova, A.G. Turkina
GU National Research Center for Hematology, Russian Academy of Medical Sciences
*E-mail: nigrin27@mail.ru

ABSTRACT
Cell regulation of Ph⁺ cell proliferation and differentiation has been studied ex vivo in various chronic myeloid leukemia (CML) patients. The regulation is provided by alternation of effective stages of proliferation and maturation with inhibition of Ph⁺ cell proliferation by accumulating neutrophils under apoptosis blockage. The alternation of stages consists of switching stage 1 (effective proliferation) to stage 2 (effective maturation) and proceeds according to the 1/2 -1/2/1 or 2/1-2/1/2/1 schemes. The kinetic plots of alternations pass through control points of crossing plots, where the parameters of proliferation and maturation are equal. The indices of P/D efficiency (ratio of proliferation and maturation rates) are 1.06±0.23 and don’t depend on time, alternation order, or sources of Ph⁺ cells – CML patients. During stages alternation, conversely, the parameters of Ph⁺ cell proliferation and maturation vary. The proliferation stages are characterized by increased proliferating cells content, a decreased number of neutrophils, and apoptosis induction. At the maturation stages, conversely, apoptosis is inhibited, the number of mature neutrophils increases, while immature Ph⁺ cells decrease. High content neutrophils inhibit the proliferation of Ph⁺ cells and impair their own maturation by inversion of maturation order, probably through a feedback mechanism. The regulation differences ex vivo reveal three types of Ph⁺ cells from various individual CML patients, distinguished by the number and duration of alternating stages of proliferation and maturation. Ph⁺ cells types 1 and 2 have one prolonged stage of effective proliferation or effective maturation with efficiency indices P/D1 = 1-20 or P/D2 ≤ 1. At the same time period, the proliferation and differentiation of the Ph⁺ cells type 3 proceeds with repeated alternations of stages with P/D1 = 1-4 or P/D2 ≤ 1. Type 1 Ph⁺ cells (~20%) were isolated from patients in advanced stages of CML, while Ph⁺ cells types 2 and 3 (30 and 50% correspondingly) were isolated from CML chronic phase patients sensitive to chemotherapy.

KEYWORDS regulation of Ph⁺ cell proliferation and Ph⁺ cell by mature cells, cultivation of hematopoietic Ph⁺ mononuclear cells, kinetics of Ph⁺ cell proliferation and differentiation in vitro, Ph⁺ cells apoptosis, Ph⁺ cell distribution in cell cycle phases, Ph⁺ cell proliferation and differentiation efficacy, inversion of accumulation order for maturating neutrophils, chronic myeloid leukemia.

ABBREVIATIONS P&D – proliferation and differentiation; CML – chronic myeloid leukemia; Ph – Philadelphia chromosome; MM, B, S, – metamyelocytes, band and segmented neutrophils; Ph⁺ cells – hematopoietic cells with Philadelphia chromosome; PB – peripheral blood ; BM – bone marrow; CP, AP, BP – chronic phase, accelerated phase and blastic phase of CML; FCS – fetal calf serum.
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E.S. Piruzian, V.V. Sobolev, R.M. Abdeev, A.D. Zolotarenko, A.A. Nikolaev, M.K. Sarkisova, M.E. Sautin, A.A. Ishkin, An.L. Piruzyan, S.A. Ilyina, I.M. Korsunskaya, O.Y. Rahimova, S.A. Bruskin

Study of Molecular Mechanisms Involved in the Pathogenesis of Immune-Mediated Inflammatory Diseases, using Psoriasis As a Model

E.S. Piruzian^1*, V.V. Sobolev¹, R.M. Abdeev², A.D. Zolotarenko¹, A.A. Nikolaev¹, M.K. Sarkisova¹, M.E. Sautin¹, A.A. Ishkin¹, An.L. Piruzyan², S.A. Ilyina², I.M. Korsunskaya², O.Y. Rahimova³, S.A. Bruskin¹
¹Vavilov Institute of General Genetics, Russian Academy of Sciences
²Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences
³Moscow Municipal Hospital № 24, Department of Health
* E-mail: eleopiru@vigg.ru

ABSTRACT Psoriasis was used as a model to analyze the pathogenetic pathways of immune-mediated inflammatory diseases, and the results of bioinformatic, molecular-genetic and proteomic studies are provided. Cell mechanisms, common for the pathogenesis of psoriasis, as well as Crohn’s disease, are identified. New approaches for immune-mediated diseases are discussed.

KEYWORDS Psoriasis, immune-mediated diseases, gene expression, proteomic analysis, gene interactions, bioinformatic analysis.

ABBREVIATIONS
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D.G. Knorre, N.V. Kudryashova, T.S. Godovikova

Chemical and Functional Aspects of Posttranslational Modification of Proteins

D.G. Knorre, N.V. Kudryashova, T.S. Godovikova*
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
*E-mail: godov@niboch.nsc.ru

ABSTRACT This paper reviews the chemical and functional aspects of the posttranslational modifications of proteins, which are achieved by the addition of various groups to the side chain of the amino acid residue backbone of proteins. It describes the main prosthetic groups and the interaction of these groups and the apoenzyme in the process of catalysis, using pyridoxal catalysis as an example. Much attention is paid to the role of posttranslational modification of proteins in the regulation of biochemical processes in live organisms, and especially to the role of protein kinases and their respective phosphotases. Methylation and acetylation reactions and their role in the “histone code,” which regulates genome expression on the transcription level, are also reviewed. this paper also describes the modification of proteins by large hydrophobic residues and their role in the function of membrane-associated proteins. Much attention is paid to the glycosylation of proteins, which leads to the formation of glycoproteins. We also describe the main non-enzymatic protein modifications such as glycation, homocysteination, and desamidation of amide residues in dibasic acids.

KEYWORDS proteins, enzymes, posttranslational modification, prosthetic groups, posphorylation, regulation, signal transduction, acylation, alkylation, ubiquitinilation, histone code, non-fermentative modification.

ABBREVIATIONS
CoA – coenzyme A, EGFR – epidermal growth factor receptor, JNK – Jun N-terminal kinase, SAPK – stress activated protein kinase, МАРК – mutagen-activated protein kinase, IF – inositoltriphosphate, DAG – diacylglycerol, JAK – Janus kinase, STAT - signal transducer and activator of transcription, Fyn, Lck – non-receptor tyrosinekinases of the Src family, Ub – ubiquitin residue, ULP – ubiquitin-like protein, ras, rab, rho – protein products of the protooncogenes ras, rab, rho, which play a role in cell growth and differentiation, SАМ – S-adenosylmethinone, PARP – poly(ADP-ribose)polymerase, VRAP – telomerase, found to be a part of vault particles, GSH – glutathione, HIF – hypoxia inducible factor, Gla – γ-carboxyglutamic acid, AGE – advanced glycation end products, CML – Nε-carboxymethyl-lysine, CEL – Nε-carboxyethyl-lysine, HSA – human serum albumin, GFP – green fluorescent protein, РIMT – protein isoaspartyl-О-methyltransferase, DNT – dermonecrotic toxin.

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V.S. Baranov

Genome Paths: a Way to Personalized and Predictive Medicine

V.S. Baranov
Ott’s Institute of Obstetrics and Gynecology, Russian Academy of Medical Sciences
E-mail: baranov@vb2475.spb.edu

ABSTRACT The review is devoted to the impact of human genome research on progress in modern medicine. Basic achievements in genome research have resulted in the deciphering of the human genome and creation of a molecular landmarks map of the human haploid genome (HapMap Project), which has made a tremendous contribution to our understanding of common genetic and multifactorial (complex) disorders. current genome studies mainly focus on genetic testing and gene association studies of multifactorial (complex) diseases, with the purpose of their efficient diagnostics and prevention . Identification of candidate (“predisposition”) genes participating in the functional genetic modules underlying each common disorder and the use of this genetic background to elaborate sophisticated measures to efficiently prevent them constitutes a major goal in personalized molecular medicine. The concept of a genetic pass as an individual DNA databank reflecting inherited human predisposition to different complex and monogenic disorders, with special emphasis on its present state, and the numerous difficulties related to the practical implementation of personalized medicine are outlined. The problems related to the uncertainness of the results of genetic testing could be overcome at least partly by means of new technological achievements in genome research methods, such as genome-wide association studies (GWAS), massive parallel DNA sequencing, and genetic and epigenetic profiling. The basic tasks of genomic today could be determined as the need to properly estimate the clinical value of genetic testing and its applicability in clinical practice. Feasible ways towards the gradual implementation of personal genetic data, in line with routine laboratory tests, for the benefit of clinical practice are discussed.

KEYWORDS genomics, genetic testing, multifactorial diseases, genetic passport, genome wide association studies.

ABBREVIATIONS GP – genetic polymorphism, FGU –a functional genetic unit; GT – genetic testing, genetic testing, MD – multifactorial diseases; GWAS – genome wide association studies; SNP – single nucleotide polymorphism, PS – polymorphic sites; STR – short tandem repeats; HapMap – haploid genome; VNTR – variable number tandem repeats; CNV – copy number variation.
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T.V. Rakitina, O.V. Yudkina, E.V. Smirnova, A.V. Lipkin

Protein Tyrosine Kinase Panel As a Tool for Anticancer Drug Design

T.V. Rakitina^1,2*, O.V. Yudkina^3,4, E.V. Smirnova¹, A.V. Lipkin^3,4
¹Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
²A.N. Bakh Institute of Biochemistry, Russian Academy of Sciences
³Institute of Crystallography, Russian Academy of Sciences
⁴Russian Research Center Kurchatov Institute
*E-mail: taniarakitina@yahoo.com

ABSTRACT
The discovery of the pharmaceutical potential of small molecule inhibitors of oncogenic protein tyrosine kinases is one of the directions in target therapy in oncology. Presently, investigations aiming at developing new therapeutically important inhibitors have to be based on a combination of computational and experimental approaches including biochemical, cell-based or in silico screening and the study of the three-dimensional structure of the kinase active center, in complex with an inhibitor, using crystallography and X-ray analysis or molecular modeling. this work is an example of a combination of inhibitor experimental search with the computational analysis of the potential mechanism of the inhibitors’ action, which allowed to propose the 2-hydroxyphenol group as a scaffold for the design of new tyrosine kinase inhibitors.

KEYWORDS protein tyrosine kinases; small molecule inhibitors; screening; 2-hydroxyphenol group

ABBREVIATIONS
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C.G. Ordzhonikidze, L.K. Ramaiyya, E.M. Egorova, A.V. Rubanovich

Genotoxic Effects of Silver Nanoparticles on Mice in Vivo

C.G. Ordzhonikidze¹, L.K. Ramaiyya¹, E.M. Egorova², A.V. Rubanovich^1*
¹Vavilov Institute of General Genetics, Russian Academy of Sciences
²Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences Science-Technology Company "Nanomet"
*E-mail: rubanovich@vigg.ru

ABSTRACT
The toxic and genotoxic effects of silver nanoparticles were studied on injected mice (BALB/c line) in vivo. A water solution of silver nanoparticles (SNP) with particle sizes of 9±6 nm was obtained by means of the original method of biochemical synthesis. The effect of the SNP solution was compared to those of AOT (anionic surfactant used as SNP stabilizer) and silver nitrate (i.e. Ag+ ions) introduced as water solutions. In studies of the toxic effects, the death of mice was registered 12-24 hours after injection only at two maximum dozes of SNP (equivalent to 0.54 and 0.36 gAg/l). It is shown that the toxic effect decreases in the sequence SNP>AOT>>AgNO3. The LE50/30 values for SNP and AOT are equal to 0.30±0.07 gAg/l and 13.3±2.1 gAg/l, respectively. Genotoxic effects were assessed by the abnormal sperm heads test and neutral comet assay. the frequencies of abnormal sperm heads (ASHs) did not differ after treatment by SNP and AOT, but both were significantly higher than those found with AgNO₃ and in control mice. Comet assay showed an increase of the DNA percentage in the comet tail in spleen cells after the injection of SNP and AOT in concentrations of ? Le50/30. Tail DNA % was 32.8±1.3 and 26.3±1.7%, respectively, vs 16.2±0.7% for the untreated control. To sum up, these tests showed that the genotoxic effects of the SNP solution are associated with the presence of AOT rather than SNP.

KEYWORDS silver nanoparticles; mice; lethal effect; germ cells; primary DNA breaks.

ABBREVIATIONS silver nanoparticles – SNP; Aerosol -ОТ – АОТ; lethal effect- LE; abnormal sperm heads - ASH
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E.V. Gromenko, P.V. Spirin, E.A. Kubareva, E.A. Romanova, V.S. Prassolov, O.V. Shpanchenko, O.A. Dontsova

Deamination of 5-Methylcytosine Residues in Mammalian Cells

E.V. Gromenko¹, P.V. Spirin², E.A. Kubareva³, E.A. Romanova³, V.S. Prassolov², O.V. Shpanchenko¹, O.A. Dontsova^1,3*
¹Department of Chemistry, Lomonosov Moscow State University
²Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
³Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University
*E-mail: olgash@genebee.msu.ru

ABSTRACT DNA demethylation in mammalia occurs after fertilization and during embryogenesis and accompanies cell aging and cancer transformation. With the help of the primer extension reaction, MALDI MS and DNA cleavage by thymine DNA glycosylase deamination of 5-methylcytosine residues has been shown to take place when the model methylated DNA duplexes are treated with nuclear extracts from the cell lines СНO, HeLa, and Skov3. The hypothesis that deamination of 5-methylcytosine is the first stage of demethylation in mammalia has been postulated.

KEYWORDS deamination of 5-methylcytosine, 5-methylcytosine demethylation, the reaction of primer extension, MALDI MS, thymine DNA glycosylase.

ABBREVIATIONS mC – 5-methyl-2’-deoxycytidine, PCR – polymerase chain reaction, PAAG – polyacrylamide gel, TDG – thymine DNA glycosylase, MALDI MS – matrix-assisted laser desorption/ionisation mass spectrometry.
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Acta Naturae, 2010. V. 2.
№ 1 (4).

L.B. Buravkova, P.M. Gershovich, J.G. Gershovich, A.I. Grigor'ev

Mechanisms of Gravitational Sensitivity of Osteogenic Precursor Cells

L.B. Buravkova, P.M. Gershovich, J.G. Gershovich*, A.I. Grigor'ev
State Scientific Centre of Russian Federation – Institute for biomedical problems RAS
*E-mail: juliet_g@mail.ru

ABSTRACT This report is a detailed review of the current data on mechanic and gravitational sensitivity of osteoblasts and osteogenic precursor cells in vitro. It summarizes the numerous responses of cells with an osteoblastic phenotype and osteogenic precursor cells and especially their responses to the alteration of their mechanic or gravitational surroundings. The review also discusses the osteogenic cell’s pathways of signal transduction and the mechanisms of gravitational sensitivity. It was shown, that the earliest multipotent stromal precursor cells of an adult organism’s bone marrow can sense changes of intensity in a gravitational or mechanic field in model conditions, which may play a certain role in the development of osteopenia in microgravity.

KEYWORDS osteopenia, gravitational sensitivity of cells, microgravity, osteoblasts, osteogenic precursor cells, multipotent mesenchymal stromal cells, differentiation, cytoskeleton.

ABBREVIATIONS
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E.S. Starodubova, M.G. Isaguliants, V.L. Karpov

Regulation of Immunogen Processing: Signal Sequences and their Application for the New Generation of DNA-Vaccines

E.S. Starodubova^1*, M.G. Isaguliants^2,3*, V.L. Karpov¹
¹Engelhardt Institute of Molecular Biology, Russian Academy of Science
²Karolinska Institutet, Sweden.
³Ivanovsky Institute of Virology, Russian Academy of Medical Science
* E-mail: estarodubova@yandex.ru , maria.isaguliants@smi.se

ABSTRACT Immunization with naked genes (DNA-immunization) is a perspective modern approach to prophylactic as well as therapeutic vaccination against pathogens, as well as cancer and allergy. A panel of DNA immunogens has been developed, some are already in the clinical trials. However, the immunogenicity of DNA vaccines, specifically of those applied to humans, needs a considerable improvement. There are several approaches to increase DNA vaccine immunogenicity. One approach implies the modifications of the encoded immunogen that change its processing and presentation, and thus the overall pattern of anti-immunogen response. For this, eukaryotic expression vectors are constructed that encode the chimeric proteins composed of the immunogen and specialized targeting or signal sequences. The review describes a number of signals that if fused to immunogen, target it into the predefined subcellular compartments. The review gives examples of their application for DNA-immunization.

KEYWORDS DNA-vaccines, МНС-I, MHC-II, antigen presentation.

ABBREVIATIONS ER - endoplasmic reticulum, Ub - ubiquitin, HCV - human hepatitis C virus, ODC - Ornithine decarboxylase, RT - HIV-1 reverse transcriptase, CRT - Ca²⁺ - binding protein calreticulin, HVP-16 - human papilloma virus 16, LAMP-1 - lysosome-associated protein 1, sarsN - nucleocapsid SARS coronavirus protein, LCMV – lymphocytic choriomeningitis virus, MHC I - major histocompatibility complex class I, MHC II - major histocompatibility complex class II
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A.V. Letarov, A.K. Golomidova, K.K. Tarasyan

Ecological Basis for Rational Phage Therapy

A.V. Letarov, A.K. Golomidova, K.K. Tarasyan
Winogradsky Institute of Microbiology RAS

ABSTRACT Understanding the mutual interactions of bacterial and phage populations in the environment of a human or animal body is essential in any attempt to influence these complex processes, particularly for rational phage therapy. Current knowledge on the impact of naturally occurring bacteriophages on the populations of their host bacteria, and their role in the homeostasis maintenance of a macro host, is still sketchy. The existing data suggest that different mechanisms stabilize phage-bacteria coexistence in different animal species or different body sites. The defining set of parameters governing phage infection includes specific physical, chemical, and biological conditions, such as pH, nutrient densities, host prevalence, relation to mucosa and other surfaces, the presence of phage inhibiting substances, etc. Phage therapy is also an ecological process that always implies three components that form a complex pattern of interactions: populations of the pathogen, the bacteriophages used as antibacterial agents, and the macroorganism. We present a review of contemporary data on natural bacteriophages occuring in human- and animal- body associated microbial communities, and analyze ecological and physiological considerations that determine the success of phage therapy in mammals.

KEYWORDS bacteriophages, phage therapy, human body microbiota, animal body microbiota, bacteriophage ecology

ABBREVIATIONS GIT – gastro-intestinal tract, PFU – plaque-forming unit, which corresponds to a one viable bacteriphage particle, if the efficiency of infection in these conditions and in this strain is close to 1; CFU – colony-forming unit, which corresponds to a one viable bacterial cell.
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O.A. Patutina, N.L. Mironova, E.I. Ryabchikova, N.A. Popova, V.P. Nikolin, V.I. Kaledin, V.V. Vlassov, M.A. Zenkova

Tumoricidal Activity of RNase A and DNase I

O.A. Patutina¹, N.L. Mironova¹, E.I. Ryabchikova¹, N.A. Popova², V.P. Nikolin², V.I. Kaledin², V.V. Vlassov¹, and M.A. Zenkova^1*
¹Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
²Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences
*E-mail: marzen@niboch.nsc.ru

ABSTRACT
In our work the antitumor and antimetastatic activities of RNase A and DNase I were studied using two murine models of pulmonary (Lewis lung carcinoma) and liver (hepatoma A-1) metastases. We found that intramuscular administration of RNase A at the dose range of 0.1–50 μg/kg retarded the primary tumor growth by 20–40%, and this effect disappeared with the increase in RNase A dose over 0.5 mg/kg. DNase I showed no effect on the primary tumor growth. The intramuscular administration of RNase A (0.35–7 μg/kg) or DNase I (0.02–2.3 mg/kg) resulted in a considerable decrease in the metastasis number into the lungs of animals with Lewis lung carcinoma and a decrease of the hepatic index of animals with hepatoma 1A. A histological analysis of the organs occupied by metastases revealed that the administration of RNase A and DNase I induced metastasis pathomorphism as manifested by the destruction of oncocytes, an increase in necrosis and apoptosis foci in metastases, and mononuclear infiltration. Our data indicated that RNase A and DNase I are highly promising as supplementary therapeutics for the treatment of metastasizing tumors.

KEYWORDS antimetastatic activity, DNase I, RNase A, Lewis lung carcinoma, hepatoma 1A.

ABBREVIATIONS Lewis lung carcinoma (LLC), hepatoma 1A (HA-1).
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S.S. Savin, V.I. Tishkov

Assessment of Formate Dehydrogenase Stress Stability in vivo using Inactivation by Hydrogen Peroxide

S.S. Savin^1,2, V.I. Tishkov^1,2,3*
¹A.N. Bach Institute of Biochemistry, Russian Academy of Sciences
²Innovations and High Technologies MSU Ltd.
³Division of Chemical Enzymology, Department of Chemistry, M.V. Lomonosov Moscow State University
*E-mail: vitishkov@gmail.com

ABSTRACT
Kinetic studies on hydrogen peroxide-induced inactivation of mutant formate dehydrogenase from Pseudomonas sp. 101 (PseFDH Cys255Ala) suggest a simple bimolecular mechanism for enzyme reaction with the inactivation agent. In the excess of hydrogen peroxide, the decrease in enzyme activity follows first-order kinetics. Therefore, the first-order effective inactivation kinetic constants determined for various FDH forms at a constant H₂O₂ concentration can be used as a quantitative measure of the enzyme stability. It was shown that two cysteine residues located in the active site formate- and coenzyme-binding domains (Cys145 and Cys255, respectively) make similar contributions to the enzyme stability, while the contribution of Cys354 is insignificant. The inactivation kinetics of wild-type PseFDH, mutant PseFDH Cys145Ser/Cys255Ala, and FDH produced under stress conditions by bacterium Staphylococcus aureus, higher plants Arabidopsis thaliana, and soya Glycine max, was studied. It was found that the stress-induced FDHs are at least 20 times more stable than the nonstress-induced PseFDH from Pseudomonas sp. 101 grown on methanol.

KEYWORDS formate dehydrogenase, hydrogen peroxide, inactivation, stress, mutant enzyme.

ABBREVIATIONS
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T.M. Astakhova, G.V. Delone, Yu.V. Lyupina, E.B. Abramova, I.V. Uryvaeva, N.P. Sharova

Changes in the Proteasome Pool During Malignant Transformation of Mouse Liver Cells

T. M. Astakhova, G. V. Delone, Yu. V. Lyupina, E. B. Abramova, I. V. Uryvaeva, N. P. Sharova*
Koltsov Institute for Developmental Biology, Russian Academy of Sciences, 26 Vavilova St., 119334 Moscow, Russia
E-mail: npsharova@bk.ru

ABSTRACT
Multiple forms of proteasomes regulate cellular processes by destroying proteins or forming the peptides involved in those processes. Various pathologies, including carcinogenesis, are related to changes in functioning the proteasome forms. In this study, we looked at the changes in the pool of liver proteasomes during nodular regenerative hyperplasia and formation of adenoma and hepatocellular carcinoma in mice treated with Dipin, followed by partial liver resection. The relative content of various proteasome forms was determined using Western blot analysis. The chymotrypsin-like activity of proteasomes was assessed from the hydrolysis of the commercial Suc-LLVY-AMC substrate. It was found that changes in the proteasome pool appeared already during the formation of diffuse nodules, the changes being the increased expression of the X(β5) constitutive subunit and the LMP7(β5i) and LMP2(β1i) immune subunits, accompanied by the increase of the total proteasome pool and the decrease in the chymotrypsin-like activity.
These changes were more pronounced in hepatocellular carcinoma. The content of the total proteasome pool and the LMP2(β1i) immune subunit and the chymotrypsin-like activity in adenoma were intermediate compared to those in the samples of liver with diffuse nodules and carcinoma. In addition, the level of the Rpt6 subunit present in the 19S proteasome activator was increased in carcinoma. Our results indicate that nodular regenerative hyperplasia and adenomatosis may be stages preceding carcinogenesis. We also conclude that there is a need to find signalling pathways that change the expression of various proteasome subunits during carcinogenesis. The 19S proteasome activator, which is overexpressed in malignant tumours, can be a promising target for the development of new anticancer drugs.

KEYWORDS immunoproteasomes, 19S proteasome activator, chymotrypsin-like activity of proteasomes, Western blot analysis, nodular regenerative hyperplasia of the liver, adenoma, hepatocellular carcinoma, mouse liver.

ABBREVIATIONS nNOS - neuronal nitric oxide synthase, dipin - 1,4-Bis-[N,N’-di(Ethylene)-phosphamide]piperazine, Suc-LLVY-AMC - N-succinyl-leu-leu-val-tyr7-amido-4-methyl coumarin, MG132 - Z-leucyl-leucyl-leucinal, mAb - monoclonal antibody, pAb - polyclonal antibody
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М.М. Shmarov, E.S. Sedova, L.V. Verkhovskaya, I.A. Rudneva, E.А. Bogacheva, Yu.А. Barykova, D.N. Shcherbinin, А.А. Lysenko, I.L. Tutykhina, D.Y. Logunov, Yu.А. Smirnov, B.S. Naroditsky and A.L. Gintsburg

Induction of a Protective Heterosubtypic Immune Response Against the Influenza Virus by Using Recombinant Adenoviral Vectors Expressing Hemagglutinin of the Influenza H5 Virus

М.М. Shmarov^1*, E.S. Sedova¹, L.V. Verkhovskaya¹, I.A. Rudneva², E.А. Bogacheva¹, Yu.А. Barykova¹, D.N. Shcherbinin¹, А.А. Lysenko¹, I.L. Tutykhina¹, D.Y. Logunov¹, Yu.А. Smirnov², B.S. Naroditsky¹ and A.L. Gintsburg¹
¹Gamaleya Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences
²Ivanovsky Virology Research Institute, Russian Academy of Medical Sciences
*E-mail: mmshmarov@gmail.com

ABSTRACT Influenza viruses are characterized by a high degree of antigenic variability, which causes the annual emergence of flu epidemics and irregularly timed pandemics caused by viruses with new antigenic and biological traits. Novel approaches to vaccination can help circumvent this problem. One of these new methods incorporates genetic vaccines based on adenoviral vectors. Recombinant adenoviral vectors which contain hemagglutinin-encoding genes from avian H5N1 and H5N2 (Ad-HA5-1 and Ad-HA5-2) influenza viruses were obtained using the AdEasy Adenoviral Vector System (Stratagene). Laboratory mice received a double intranasal vaccination with Ad-HA5-1 and Ad-HA5-2. This study demonstrates that immunization with recombinant adenoviruses bearing the Н5 influenza virus hemagglutinin gene induces a immune response which protect immunized mice from a lethal dose of the H5 influenza virus. Moreover, it also protects the host from a lethal dose of H1 virus, which belongs to the same clade as H5, but does not confer protection from the subtype H3 influenza virus, which belongs to a different clade. Our data allow us to conclude that adenoviral vectors may become a universal platform for obtaining vaccines against seasonal and pandemic strains of the influenza virus.

KEYWORDS adenoviral vector, influenza virus, hemagglutinin, immunization, heterosubtypic protection

ABBREVIATIONS
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O.A. Vinogradova, D.V. Pyshnyi

Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DNA

O.A. Vinogradova, D.V. Pyshnyi*
Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian Academy of Sciences, prosp. Lavrentieva 8, Novosibirsk 630090

ABSTRACT The analysis of DNA nucleotide polymorphisms is one of the main goals of DNA diagnostics. DNA-dependent enzymes (DNA polymerases and DNA ligases) are widely used to enhance the sensitivity and reliability of systems intended for the detection of point mutations in genetic material. In this article, we have summarized the data on the selectiveness of DNA-dependent enzymes and on the structural factors in enzymes and DNA which influence the effectiveness of mismatch discrimination during enzymatic conversion of oligonucleotide probes on a DNA template. The data presented characterize the sensitivity of a series of DNA-dependent enzymes that are widely used in the detection of noncomplementary base pairs in nucleic acid substrate complexes. We have analyzed the spatial properties of the enzyme-substrate complexes. These properties are vital for the enzymatic reaction and the recognition of perfect DNA-substrates. We also discuss relevant approaches to increasing the selectivity of enzyme-dependent reactions. These approaches involve the use of modified oligonucleotide probes which “disturb” the native structure of the DNA-substrate complexes.

KEYWORDS DNA complexes, mismatch, selectivity, DNA ligase, DNA polymerase, modified oligonucleotide probes.

ABBREVIATIONS PCR – polymerase chain reaction, NA – nucleic acid, AdD – nucleotidyltransferase domain of DNA ligases, OB – oligonucleotide/oligosaccharide binding domain of DNA ligases, DBD – DNA binding domain of DNA ligases, HhH – motif of DNA ligases helix-hairpin-helix, Zn – zinc-fingers, BRCT – C-terminal domain of DNA ligases, PNA – Peptide Nucleic Acids, LNA – Locked Nucleic Acid, ENA – Ethylene Nucleic Acid, dNTP – deoxyribonucleosidetriphosphate, PPi – inorganic pyrophosphate, mc – main chain of protein backbone.
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I.B. Bezprozvanny

Calcium Signaling and Neurodegeneration

I.B. Bezprozvanny
University of Texas Southwestern Medical Center, Dallas, Texas, USA
Institute of Cytology, Russian Academy of Sciences, St. Petersburg
E-mail: Ilya.Bezprozvanny@UTSouthwestern.edu

ABSTRACT Neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and spinocerebellar ataxias (SCA) are very important both for fundamental science and for practical medicine. Despite extensive research into the causes of these diseases, clinical researchers have had very limited progress and, as of now, there is still no cure for any of these diseases. One of the main obstacles in the way of creating treatments for these disorders is the fact that their etiology and pathophysiology still remain unclear. This paper reviews results that support the so-called “calcium hypothesis of neurodegenerative diseases.” The calcium hypothesis states that the atrophic and degenerative processes in the neurons of AD, PD, ALS, HD, and SCA patients are accompanied by alterations in calcium homeostasis. Moreover, the calcium hypothesis states that this deregulation of calcium signaling is one of the early-stage and key processes in the pathogenesis of these diseases. Based on the results we reviewed, we conclude that the calcium channels and other proteins involved in the neuronal calcium signaling system are potential drug targets for AD, PD, ALS, HD, and SCA therapy.

KEYWORDS Alzheimer’s disease, Parkinson’s disease (PD), amyotrophic lateral sclerosis, Huntington’s disease, spinocerbellar ataxias, calcium channels, calcium signaling, mitochondria, transgenic mice, clinical trials, imaging, memantine, dimebon, riluzole.

ABBREVIATIONS endoplasmic reticulum (ER), mitochondrial Ca2⁺ uniporter (MCU), Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), spinocerbellar ataxias (SCA), huntingtin protein (Htt), N-methyl-D-aspartate receptors (NMDAR), medium spiny neurons (MSN), tetrabenazine (TBZ), heritable Alzheimer’s disease (HAD), phosphatidyl serine (PtdS). InsP3 R1 - type 1 inositol (1,4,5)-trisphosphate receptor, mPTP mitochondrial permeability transition pore
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A.S. Isaeva, E.E. Kulikov, K.K. Tarasyan, A.V. Letarov

A Novel High-Resolving Method for Genomic PCR-fingerprinting of Enterobacteria

A.S. Isaeva, E.E. Kulikov, K.K. Tarasyan, A.V. Letarov*
Winogradsky Institute of Microbiology, Russian Academy of Sciences
*E-mail: letarov@gmail.com

ABSTRACT We developed a novel PCR-fingerprinting system for differentiation of enterobacterial strains using a single oligonucleotide primer IS1tr that matches the inverted terminal repeats of the IS1 insertion element. Compared to widely used BOX-PCR and ribotyping methods, our system features higher resolution allowing differentiation of closely related isolates that appear identical in BOX-PCR and ribotyping but differ in their phage sensitivity. The IS1-profiling system is less sensitive to the quality of the material and equipment used. At the same time, BOX-PCR is more universal and suitable for bacterial strain grouping and reconstruction of the low-distance phylogeny. Thus, our system represents an important supplement to the existing set of tools for bacterial strain differentiation; it is particularly valuable for a detailed investigation of highly divergent and rapidly evolving natural bacterial populations and for studies on coliphage ecology. However, some isolates could not be reliably differentiated by IS1-PCR, because of the low number of bands in their patterns. For improvement of IS1-fingerprinting characteristics, we offer to modify the system by introducing the second primer TR8834 hybridizing to the sequence of a transposase gene that is widely spread in enterobacterial genomes.

KEYWORDS genomic fingerprinting, whole-cell PCR fingerprinting, insertion element, Enterobacterial diversity, strain differentiation.

ABBREVIATIONS IS – insertion sequence, ERIC – enterobacterial repetitive intergenic consensus, REP – repetitive extragenic palindromic sequence, dNTP – deoxyribonucleotide triphosphate, OTUs – operational taxonomic units.
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D.T. Guranda, G.A. Ushakov, V.K. Svedas

Penicillin Acylase-Catalyzed Effective and Stereoselective Acylation of 1-phenylethylamine in Aqueous Medium using Non-Activated Acyl Donor

D.T. Guranda, G.A. Ushakov, V.K. Svedas*
Belozersky Institute of Physicochemical Biology, Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Russia
*E-mail: vytas@belozersky.msu.ru

ABSTRACT Until recently the biocatalytic preparation of enantiomerically pure amines was based on stereoselective acyl transfer in an organic medium using activated acyl donors. The possibility of performing an effective and enantioselective enzymatic acylation of amines in an aqueous medium without using activated acyl donors was demonstrated for the first time as the example of direct condensation of phenylacetic acid and racemic 1-phenylethylamine. Direct condensation of the acid and the amine took place at mild reaction conditions with a high initial rate (3.3 µmole/(l•h)), degree of conversion (80% acylation of active amine enantiomer), and enantioselectivity (enantiomeric excess of the product was more than 95%). The suggested approach has remarkable advantages compared to enzymatic reactions in organic media and is of practical value for the biocatalytic preparation of enantiomerically pure compounds at mild conditions using readily available reagents.

KEYWORDS stereoselective enzymatic acylation in aqueous medium, direct condensation, enantiomerically pure compounds, penicillin acylase

ABBREVIATIONS
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L.A. Alexandrova, E.R. Shmalenyuk, S.N. Kochetkov, V.V. Erokhin, T.G. Smirnova, S.N. Andreevskaia, and L.N. Chernousova

New 5-Modified Pyrimidine Nucleoside Inhibitors of Mycobacterial Growth

L.A. Alexandrova^1*, E.R. Shmalenyuk¹, S.N. Kochetkov¹, V.V. Erokhin², T.G. Smirnova², S.N. Andreevskaia², and L.N. Chernousova²
¹Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991, Russia
²Central Tuberculosis Research Institute, Russian Academy of Medical Sciences, Yauzskaya alley 2, Moscow, 107564, Russia
*E-mail: ala2004_07@mail.ru

ABSTRACT
The WHO has declared tuberculosis (TB) a global health emergency. Therefore, there is an urgent need to discover and develop new anti-TB drugs. Here we report a new category of 5-substituted pyrimidine nucleosides as potent inhibitors of Mycobacterium tuberculosis growth in vitro. A series of 2'-deoxy-, 3'-azido-2',3'-dideoxy-, and 3'-amino-2',3'-dideoxypyrimidine nucleoside analogues bearing lengthy flexible alkyloxymethyl substituents exhibited marked inhibitory activity against M tuberculosis in vitro. 5-Dodecyloxymethyl-2'-deoxyuridine was found to be a potent inhibitor of M. tuberculosis propagation in vitro. In contrast, monophosphates of the tested nucleosides were devoid of antimycobacterial activity. This new class of inhibitors seems to be a promising chemotherapeutic agent against TB and merits further studies.

KEYWORDS tuberculosis; Mycobacterium tuberculosis; anti-TB drug; nucleoside; 5-substituted pyrimidine; inhibitor

ABBREVIATIONS TB - tuberculosis, HIV - human immunodeficiency virus, MDR - multi-drug resistance, MIC – minimal inhibitory concentration, CFU - colony-forming unit
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Acta Naturae, 2010. V. 2.
№ 2 (5).

S. P. Medvedev, A. I. Shevchenko, S. M. Zakian

Induced Pluripotent Stem Cells: Problems and Advantages when Applying them in Regenerative Medicine

S. P. Medvedev¹, A. I. Shevchenko¹, S. M. Zakian^1,2*
¹Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences
²Research Center of Clinical and Experimental Medicine, Siberian Branch, Russian Academy of Medical Sciences
*E-mail: zakian@bionet.nsc.ru
Received 10. 04. 2010

ABSTRACT Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells that can be obtained by reprogramming animal and human differentiated cells. In this review, issues related to the nature of iPSCs are discussed and different methods of iPSC production are described. We particularly focused on methods of iPSC production without the genetic modification of the cell genome and with means for increasing the iPSC production efficiency. The possibility and issues related to the safety of iPSC use in cell replacement therapy of human diseases and a study of new medicines are considered.

KEYWORDS cell reprogramming, induced pluripotent stem cells, directed stem cell differentiation, cell replacement therapy

ABBREVIATIONS ESCs – embryonic stem cells; iPSC_s – induced pluripotent stem cells; Chd1 – chromodomain helicase DNA-binding protein; BAF – rg/Brahma-associated factors; miRNA, miR – microRNA; TERRA – telomeric-repeat-containing RNA; Cdkn – cyclin-dependent kinase inhibitor; VPA – valproic acid; siRNA – small interfering RNA; KMOS – Klf4, c-Myc, Oct4, Sox2; KOS – Klf4, Oct4, Sox2; LNOS – Lin28, Nanog, Oct4, Sox2; GSK-3 – glycogen synthase kinase 3; ROS – reactive oxygen species; HIF1 – hypoxia-inducible factor 1; VEGF – vascular endothelial growth factor.
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Yu. S. Ovodov

Bioglycans and Natural Glycosides As a Promising Research Topic in Bioorganic Chemistriy

Yu. S. Ovodov
Institute of Physiology, Komi Science Center, The Urals Branch, Russian Academy of Sciences
E-mail: ovoys@physiol.komisc.ru
Received 18.03.2010

ABSTRACT This review defines bioorganic chemistry as one of the most important constituents of physico-chemical biology, which is a fundamental life science. The problems and goals of bioorganic chemistry are examined through a comparatively small number of examples. Bioorganic chemistry is supposed to be a logical continuation of the chemistry of the natural substances that arose many years ago. Bioorganic chemistry has contributed some achievements in solving the problems of the chemical structure, biological function, and physiological activity of biopolymers and lowmolecular-weight bioregulators, as well as in the elucidation of the molecular mechanisms of different life processes. The most striking achievements in bioorganic chemistry are discussed in this paper. However, this review discusses not only the general achievements in this field of science, but also research data obtained by scientists from the Pacific Institute of Bioorganic Chemistry, Far East Branch, Russian Academy of Sciences (Vladivostok, Russia), and the Institute of Physiology, Komi Science Centre, The Urals Branch, Russian Academy of Sciences (Syktyvkar, Russia). Particular attention is focused on comprehensive research into polysaccharides and biopolymers (bioglycans) and some natural glycosides that the author of this review has studied for a long time. The author has worked in these institutes for a long time and was honored by being chosen to head one of the scientific schools in the field of bioorganic chemistry and molecular immunology.

KEYWORDS bioglycans, natural glycosides, low-molecular-weight bioregulators.

ABBREVIATIONS LPS – lipopolysaccharide, PSA – prostate specific antigen, LPPC - lipopolysaccharide-protein complex, CEA – carcino-embryonic antigen, PC – prostate cancer.
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I.A. Mikhailopulo, A.I. Miroshnikov

New Trends in Nucleoside Biotechnology

I.A. Mikhailopulo^1*, A.I. Miroshnikov²
¹Institute of Bioorganic Chemistry, National Academy of Sciences, Belarus
²Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
*E-mail: igor_mikhailo@yahoo.de
Received 29.04.2010

ABSTRACT
This review focuses on new trends in nucleoside biotechnology, which have emerged during the last decade. Continuously growing interest in the study of this class of compounds is fueled by a number of factors: (i) a growing need for large-scale production of natural 2'deoxy-β-D-ribonucleosides as well as their analogs with modifications in the carbohydrate and base fragments, which can then be used for the synthesis and study of oligonucleotides, including short-interfering RNA (siRNA), microRNA (miRNA), etc.; (ii) a necessity for the development of efficient practical technologies for the production of biologically important analogs of natural nucleosides, including a number of anticancer and antiviral drugs; (iii) a need for further study of known and novel enzymatic transformations and their use as tools for the efficient synthesis of new nucloside analogs and derivates with biomedical potential. This article will review all of these aspects and also include a brief retrospect of this field of research.

KEYWORDS nucleosides, nucleic acid metabolism enzymes, chemoenzymaticsynthesis, bio-mimetic synthesis.

ABBREVIATIONS
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B. S. Shenkman, O. V.Turtikova, T. L. Nemirovskaya, A. I. Grigoriev

Skeletal Muscle Activity and the Fate of Myonuclei

B. S. Shenkman*, O. V.Turtikova, T. L. Nemirovskaya, A. I. Grigoriev
Institute for Biomedical Problems, Russian Academy of Sciences
*E-mail: shenkman@imbp.ru
Received 28.12.2009

ABSTRACT Adult skeletal muscle fiber is a symplast multinuclear structure developed in ontogenesis by the fusion of the myoblasts (muscle progenitor cells). The nuclei of a muscle fiber (myonuclei) are those located at the periphery of fiber in the space between myofibrils and sarcolemma. In theory, a mass change in skeletal muscle during exercise or unloading may be associated with the altered myonuclear number, ratio of the transcription, and translation and proteolysis rates. Here we review the literature data related to the phenomenology and hypothetical mechanisms of the myonuclear number alterations during enhanced or reduced muscle contractile activity. In many cases (during severe muscle and systemic diseases and gravitational unloading), muscle atrophy is accompanied by a reduction in the amount of myonuclei. Such reduction is usually explained by the development of myonuclear apoptosis. A myonuclear number increase may be provided only by the satellite cell nuclei incorporation via cell fusion with the adjacent myofiber. It is believed that it is these cells which supply fiber with additional nuclei, providing postnatal growth, work hypertrophy, and repair processes. Here we discuss the possible mechanisms controlling satellite cell proliferation during exercise, functional unloading, and passive stretch.

KEYWORDS skeletal muscle, myonuclei apoptosis, physical training, working hypertrophy, satellite cells, growth factors, gravitational unloading, muscle stretch.

ABBREVIATIONS IGF – insulin-like growth factor, AIF – apoptosis-inducing factor, GFP – green fluorescent protein, BrdU – 5-bromo-2-deoxyuridine, CD34, 45, 54 - clusters of differentiation, c-Met – HGF receptor, HGF – hepatocyte growth factor, FGF fibroblast growth factor, MMPs – matrix metalloproteinases, MGF – mechano-growth factor.
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I.G. Khaliullin, D.A. Suplatov, D.N. Shalaeva, M. Otsuka, Y. Asano, V.К. Švedas

Bioinformatic Analysis, Molecular Modeling of Role of Lys65 Residue in Catalytic Triad of D-aminopeptidase from Ochrobactrum anthropi

I.G. Khaliullin^1,2, D.A. Suplatov¹, D.N. Shalaeva¹, M. Otsuka³, Y. Asano³, V.К. Švedas^1*
¹Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University
²Department of Chemistry, Lomonosov Moscow State University
³Biotechnology Research Center, Toyama Prefectural University, Japan
*E-mail: vytas@belozersky.msu.ru
Received 23.04.2010

ABSTRACT
A bioinformatic and phylogenetic study has been performed on a family of penicillin-binding proteins including D-aminopeptidases, D-amino acid amidases, DD-carboxypeptidases, and β-lactamases. Significant homology between D-aminopeptidase from Ochrobactrum anthropi and other members of the family has been shown and a number of conserved residues identified as S62, K65, Y153, N155, H287, and G289. Three of those (Ser62, Lys65, and Tyr153) form a catalytic triangle – the proton relay system that activates the generalized nucleophile in the course of catalysis. Molecular modeling has indicated the conserved residue Lys65 to have an unusually low pKa value, which has been confirmed experimentally by a study of the pH-profile of D-aminopeptidase catalytic activity. The resulting data have been used to elucidate the role of Lys65 in the catalytic mechanism of D-aminopeptidase as a general base for proton transfer from catalytic Ser62 to Tyr153, and vice versa, during the formation and hydrolysis of the acylenzyme intermediate.

KEYWORDS D-aminopeptidase, penicillin-binding protein family, bioinformatic analysis, catalytic mechanism.

ABBREVIATIONS
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R.Y. Kotlyarov, V.V.Kuprianov, A.I. Migunov, L.A. Stepanova, L.M. Tsybalova, O.I. Kiselev, N.V.Ravin, K.G. Skryabin

Development of Recombinant Vaccine against A(H1N1) 2009 Influenza Based on Virus-like Nanoparticles Carrying the Extracellular Domain of M2 Protein

R.Y. Kotlyarov¹, V.V.Kuprianov¹, A.I. Migunov², L.A. Stepanova², L.M. Tsybalova², O.I. Kiselev², N.V.Ravin^1*, K.G. Skryabin¹
¹Centre “Bioengineering” Russian Academy of Sciences
²Research Institute of Influenza, Russian Academy of Medical Sciences
E-mail: nravin@biengi.ac.ru
Received 23.04.2010

ABSTRACT The conventional vaccines currently being used to deal with influenza are based on a virus obtained in chicken embryos or its components. The high variability of the major immunogenic surface proteins – hemagglutinin and neuraminidase–require the development of strain-specific vaccines that match the antigenic specificity of a newly emerging virus. Recombinant vaccines based on single viral proteins that could be easily produced in standard expression systems are attractive alternatives to traditional influenza vaccines. We constructed recombinant nanosized virus-like particles based on a nuclear antigen of the hepatitis B virus. These particles expose on the surface the extracellular domain of the M2 protein of the highly pathogenic A(H1N1) 2009 influenza virus. The methods of production of these virus-like particles in Escherichia coli and their purification were developed. Experiments on animals show that M2sHBc particles are highly immunogenic in mice and provide complete protection against the lethal influenza challenge.

KEYWORDS influenza, vaccine, M2 protein, nanoparticle, HBc antigen.

ABBREVIATIONS M2e – extracellular domain of Influenza virus M2 protein, HBc – nuclear antigen of hepatitis B virus, M2sHBc – hybrid protein including M2e of the swine flu virus and HBc, PCR – polymerase chain reaction, , PBS-phosphate buffered saline, TMB – tetramethylbenzidine, LD50 – dose corresponding to 50% lethality rate, ELISA – enzymelinked immunosorbent assay, FCS – fetal calf serum
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V. I. Tishkov, S. V. Uglanova, V. V. Fedorchuk, S. S. Savin

Influence of Ion Strength and pH on Thermal Stability of Yeast Formate Dehydrogenase

V. I. Tishkov^1,2,3*, S. V. Uglanova^1,4, V. V. Fedorchuk¹, S. S. Savin^2,3
¹Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov Moscow State University
²Innovations and High Technologies MSU Ltd.
³Bach Institute of Biochemistry, Russian Academy of Sciences
⁴Emanuel Institute of Biochemical Physics, Russian Academy of Sciences
*E-mail: vitishkov@gmail.com

ABSTRACT
The kinetics of the thermal inactivation of recombinant wild-type formate dehydrogenase from Candida boidinii yeast was studied in the temperature range of 53–61oC and pH 6.0, 7.0, and 8.0. It was shown that the loss of the enzyme’s activity proceeds via a monomolecular mechanism. Activation parameters ΔН ≠ and ΔS ≠ were calculated based on the temperature relations dependence of inactivation rate constants according to the transition state theory. Both parameters are in a range that corresponds to globular protein denaturation processes. Optimal conditions for the stability of the enzyme were high concentrations of the phosphate buffer or of the enzyme substrate sodium formate at pH = 7.0.

KEYWORDS formate dehydrogenase, Candida boidinii, thermal inactivation, ionic strength, stabilization

ABBREVIATIONS FDH – NAD+-dependent formate dehydrogenase, PseFDH – FDH from Pseudomonas sp.101 bacteria, PseFDH GAV – mutant FDH from Pseudomonas sp.101 GAV version, CboFDH – FDH from Candida boidinii yeast
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S. P. Medvedev, A. A. Malakhova, E. V. Grigor’eva, A. I. Shevchenko, E. V. Dementyeva, I. A. Sobolev, I. N. Lebedev, A .G. Shilov, I. F. Zhimulev, S. M. Zakian

Derivation of Induced Pluripotent Stem Cells from Fetal Human Skin Fibroblasts

S. P. Medvedev¹, A. A. Malakhova¹, E. V. Grigor’eva¹, A. I. Shevchenko¹, E. V. Dementyeva¹, I.A. Sobolev¹, I. N. Lebedev², A .G. Shilov¹, I. F. Zhimulev³, S. M. Zakian^1,4*
¹Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences
²Research Institute of Medical Genetics, Siberian Branch, Russian Academy of Medical Sciences
³Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
⁴Research Center of Clinical and Experimental Medicine, Siberian Branch, Russian Academy of Medical Sciences
* E-mail: zakian@bionet.nsc.ru
Received 10.04.2010

ABSTRACT The isolation and study of autologous human stem cells remain among the most urgent problems in cell biology and biomedicine to date. Induced pluripotent stem cells can be derived from human somatic cells by the overexpression of a number of genes. In this study we reprogrammed fetal human skin fibroblasts by transduction with retroviral vectors carrying murine Oct4, Sox2, Klf4, and c-Myc cDNAs. As a result, cells with the protein expression and gene transcription pattern characteristic of human embryonic stem cells were derived. These induced pluripotent cells are capable of differentiation in vitro into the ectoderm, mesoderm, and endoderm derivatives.

KEYWORDS induced pluripotent stem cells, reprogramming, retroviral vectors

ABBREVIATIONS ESCs – embryonic stem cells, HEFs – human embryonic fibroblasts, iPSCs – induced pluripotent stem cells, PCR – polymerase chain reaction, RT-PCR – reverse transcription-polymerase chain reaction
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D. I. Markov, O. P. Nikolaeva, D. I. Levitsky

Effects of Myosin “Essential” Light Chain A1 on the Aggregation Properties of the Myosin Head

D. I. Markov¹, O. P. Nikolaeva², D. I. Levitsky^1,2*
¹Bach Institute of Biochemistry, Russian Academy of Sciences
²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University
*E-mail: levitsky@inbi.ras.ru
Received 24.05.2010

ABSTRACT We compared the thermal aggregation properties of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different “essential” (or “alkali”) light chains, A1 or A2. Temperature dependencies for the aggregation of these two S1 isoforms, as measured by the increase in turbidity, were compared with the temperature dependencies of their thermal denaturation obtained from differential scanning calorimetry (DSC) experiments. At relatively high ionic strength (in the presence of 100 mM KCl) close to its physiological values in muscle fibers, we have found no appreciable difference between the two S1 isoforms in their thermally induced aggregation. Under these conditions, the aggregation of both S1 isoforms was independent of the protein concentration and resulted from their irreversible denaturation, which led to the cohesion of denatured S1 molecules. In contrast, a significant difference between these S1 isoforms was revealed in their aggregation measured at low ionic strength. Under these conditions, the aggregation of S1 containing a light chain A1 (but not A2) was strongly dependent on protein concentration, the increase of which (from 0.125 to 2.0 mg/ml) shifted the aggregation curve by ~10 degrees towards the lower temperatures. It has been concluded that the aggregation properties of this S1 isoform at low ionic strength is basically determined by intermolecular interactions of the N-terminal extension of the A1 light chain (which is absent in the A2 light chain) with other S1 molecules. These interactions seem to be independent of the S1 thermal denaturation, and they may take place even at low temperature.

KEYWORDS myosin subfragment 1, “essential” light chains, aggregation, thermal denaturation, differential scanning calorimetry

ABBREVIATIONS
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A.G. Konshina, I.A. Boldyrev, A.V. Omelkov, Yu.N. Utkin, R.G. Efremov

Anionic Lipids: Determinants of Binding Cytotoxins from Snake Venom on the Surface of Cell Membranes

A.G. Konshina^1*, I.A. Boldyrev¹, A.V. Omelkov², Yu.N. Utkin¹, R.G. Efremov¹
¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
²Faculty of Technology of Organic Substances and Chemical Pharmaceutical Compounds, Mendeleev University of Chemical Technology of Russia
*E-mail: nastya@nmr.ru
Received 19.05.2010.

ABSTRACT The cytotoxic properties of cytotoxins (CTs) from snake venom are mediated by their interaction with the cell membrane. The hydrophobic pattern containing the tips of loops I–III and flanked by polar residues is known to be a membrane-binding motif of CTs. However, this is not enough to explain the difference in activity among various CTs which are similar in sequence and in 3D structure. The mechanism of further CT–membrane interaction leading to pore formation and cell death still remains unknown. Published experimental data on the specific interaction between CT and low molecular weight anionic components (sulphatide) of the bilayer point to the existence of corresponding ligand binding sites on the surface of toxin molecules. In this work we study the membrane-lytic properties of CT I, CT II (Naja oxiana), and CT 4 (Naja kaouthia), which belong to different structural and functional types (P- and S-type) of CTs, by measuring the intensity of a fluorescent dye, calcein released from liposomes containing a phosphatidylserine (PS) lipid as an anionic component. Using molecular docking simulations, we find and characterize three sites in CT molecules that can potentially bind the PS polar head. Based on the data obtained, we suggest a hypothesis that CTs can specifically interact with one or more of the anionic lipids (in particular, with PS) contained in the membrane, thus facilitating the interaction between CTs and the lipid bilayer of a cell membrane.

KEYWORDS cytotoxins, phosphatidylserine, lipid membranes, molecular docking, fluorescent spectroscopy

ABBREVIATIONS CT — cytotoxin, PS — phosphatidylserine, PKC — protein kinase C, SGC—sulphatide, POPC — palmitoyl oleyl phosphatidylcholine, 6-CF — 6-carboxyfluorescein, L/P — lipid/protein ratio, DPC — dodecylphosphocholin, NMR — nuclear magnetic resonance, hPS — phosphatidylserine polar head, PDB — Protein Data Bank
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D. Riess, I. Lavrik

2D-gel Electrophoresis As a Tool to Investigate the Composition of CD95 DISC

D. Riess, I. Lavrik*
Division of Immunogenetics, German Cancer Center, Heidelberg, Germany
E-mail: i.lavrik@dkfz-heidelberg.de
Received 25.05.2010

ABSTRACT Stimulation of CD95 (APO-1/Fas) leads to apoptosis induction in multicellular organisms. CD95-mediated apoptosis starts with the formation of the protein complex at the receptor CD95 (APO-1/Fas), which was named DISC (death-inducing signaling complex). In this work, the composition of the CD95 DISC in two different cell types was analyzed using proteomics approaches. Using 2D gels, the composition of the CD95 DISC was analyzed in the so-called Type I and Type II cells, which are characterized by different kinetics of apoptosis. The detailed analysis of the CD95 DISC performed by 2D gels demonstrated that, besides the well-established components of the CD95 DISC, which are present in both cell types (CD95, FADD and procaspase-8), there are a number of differential spots detected at the CD95 DISC of Type I versus Type II cells. Taken together, this work demonstrates the differential composition of the CD95 DISC of Type I versus Type II cells.

KEYWORDS apoptosis, receptor CD95, 2D-gel electrophoresis

ABBREVIATIONS
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Acta Naturae, 2010. V. 2.
№ 3 (6).

Acta Naturae, 2010. V. 2.
№ 4 (7).

V.A. Stepanov

Genomes, Populations and Diseases: Ethnic Genomics and Personalized Medicine

V. A. Stepanov
Research Institute for Medical Genetics, Siberian Branch, Russian Academy of Medical Sciences
E-mail: vadim.stepanov@medgenetics.ru
Received 19.09.2010

ABSTRACT This review discusses the progress of ethnic genetics, the genetics of common diseases, and the concepts of personalized medicine. We show the relationship between the structure of genetic diversity in human populations and the varying frequencies of Mendelian and multifactor diseases. We also examine the population basis of pharmacogenetics and evaluate the effectiveness of pharmacotherapy, along with a review of new achievements and prospects in personalized genomics.

KEYWORDS

ABBREVIATIONS ADD – autosomal dominant diseases, ARD – autosomal recessive diseases, HVSI – hypervariable segment I, mtDNA – mitochondrial DNA, CD – common diseases, RFLP – restriction fragments length polymorphism, IHD – ischemic heart disease, COLD – chronic obstructive lung disease, SNP – single nucleotide polymorphism, CNV – copy number variation, STR – short tandem repeats, HapMap – Haplotype Map of the Human Genome, CEU – population of Central European origin, YRI – population of Yoruba from Ibadan, Nigeria, CHB – Chinese population from Beijing, JPT – Japanese population from Tokyo, CMT1 – Charcot-Marie-Tooth disease, type 1, GWAS – genome-wide associations search, OR – odds ratio, OMIM – on-line Mendelian Inheritance in Man catalogue.
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E. R. Galimov

The Role of p66shc in Oxidative Stress and Apoptosis

E. R. Galimov
Belozersky Institute of Physico-Chemical Biology, Moscow State University
E-mail: e.r.galimov@gmail.com
Received 03.11.2010

ABSTRACT p66shc is a gene that regulates the level of reactive oxygen species (ROS), apoptosis induction, and lifespan in mammals. Mice knocked out for p66shc have a lifespan ~30% longer and demonstrate an enhanced resistance to oxidative stress and age-related pathologies such as hypercholesterolemia, ischemia, and hyperglycemia. In this respect, p66shc is a promising pharmacological target for the treatment of age-related diseases. In this review, an attempt has been made to survey and put to a critical analysis data concerning the involvement of p66shс in the different signaling pathways that regulate oxidative stress and apoptosis.

KEYWORDS apoptosis, reactive oxygen species, p66shc, mitochondria.

ABBREVIATIONS
Akt – protein kinase B, Cdc42 (cell division control protein 42 homolog) – a small GTPase of the Rho-subfamily, Cyt C – cytochrome c, DMTU – dimethylthiourea, Grb2 – growth factor receptor-bound protein 2, ERK – extracellular signal-regulated kinases, HIV-1 – human immunodeficiency virus 1, IMS – intermembrane space of mitochondria, JNK – c-Jun N-terminal kinases, MAPK – mitogen-activated protein kinases, mHSP70 – mitochondrial 70 kilodalton heat shock proteins, MnSOD – mitochondrial superoxide dismutase, PKCβ – protein kinase Cβ, Pin-1 – peptidyl-prolyl cis/trans isomerase, PP2A – Protein phosphatase 2, Prx1 – peroxiredoxin 1, PTP – permeability transition pore, PTP-PEST – protein tyrosine phosphatase that contains a C-terminal PEST motif, Rac-1 – ras-related C3 botulinum toxin substrate 1, RAS – rat sarcoma viral oncogene, REF-1 – redox factor 1, ROS – reactive oxygen species, SOS1 (Son of Sevenless) – guanine nucleotide-binding protein, TIM – translocase of the inner membrane of mitochondria, TMPD – N,N,N’,N’- tetramethyl-p-phenyldiamine, TOM – translocase of the outer membrane of mitochondria.

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L.E. Salnikova, N.I. Zelinskaya, O. B. Belopolskaya, M.M. Aslanyan, A. V. Rubanovich

Association Study of Xenobiotic Detoxication and Repair Genes with Malignant Brain Tumors in Children

L. E. Salnikova¹, N. I. Zelinskaya², O. B. Belopolskaya¹, M. M. Aslanyan³, A. V. Rubanovich¹
¹Vavilov Institute of General Genetics, Russian Academy of Sciences
²Federal State Center “Russian Scientific Center of Roentgenoradiology”
³Lomonosov Moscow State University
*E-mail: rubanovich@vigg.ru
Received 31.08.2010

ABSTRACT
This study presents the results of research on DNA polymorphism in children with malignant brain tumors (172 patients, 183 in the control group). Genotyping was performed using an allele-specific tetraprimer reaction for the genes of the first (CYP1A1 (2 sites)) and second phases of xenobiotic detoxication (GSTM1, GSTT1, GSTP1, GSTM3), DNA repair genes XRCC1, XPD (2 sites), OGG1, as well as NOS1 and MTHFR. The increased risk of disease is associated with a minor variant of CYP1A1 (606G) (p = 0.009; OR = 1.50) and a deletion variant of GSTT1, (p = 0.013, OR = 1.96). Maximum disease risk was observed in carriers of double deletions in GSTT1-GSTM1 (p = 0.017, OR = 2.42). The obtained results are discussed in reference to literary data on the risk of malignant brain tumor formation in children and adults.

KEYWORDS gene polymorphism, malignant brain tumors in children, genes of xenobiotic detoxication, DNA repair genes.

ABBREVIATIONS
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A.P. Sokolenko, A.G. Iyevleva, N.V. Mitiushkina, E.N. Suspitsin, E.V. Preobrazhenskaya, E.Sh. Kuligina, D.A. Voskresenskiy, O.S. Lobeiko, N.Yu. Krylova, T.V. Gorodnova, K.G. Buslov, E.M. Bit-Sava, G.D. Dolmatov, N.V. Porhanova, I.S. Polyakov, S.N. Abysheva, A.S. Katanugina, D.V. Baholdin, G.A. Yanus, A.V. Togo, V.M. Moiseyenko, S.Ya. Maximov, V. F. Semiglazov, E. N. Imyanitov

Hereditary Breast-Ovarian Cancer Syndrome in Russia

A. P. Sokolenko^1,2, A. G. Iyevleva^1,2, N. V. Mitiushkina¹, E. N. Suspitsin^1,2, E.V. Preobrazhenskaya¹, E.Sh. Kuligina¹, D. A. Voskresenskiy², O. S. Lobeiko², N. Yu. Krylova¹, T. V. Gorodnova¹, K. G. Buslov², E. M. Bit-Sava¹, G. D. Dolmatov⁴, N. V. Porhanova⁵, I. S. Polyakov⁵, S. N. Abysheva¹, A. S. Katanugina¹, D. V. Baholdin¹, G. A. Yanus^1,2, A. V. Togo¹, V. M. Moiseyenko^1,3, S. Ya. Maximov¹, V.F. Semiglazov¹, E. N. Imyanitov^1,2,3*
¹Petrov Institute of Oncology, St. Petersburg
²State Pediatric Medical Academy, St. Petersburg
³Medical Academy for Postgraduate Studies, St. Petersburg
⁴City Oncological Hospital, St. Petersburg
⁵Kuban State Medical University, Krasnodar
*E-mail: evgeny@imyanitov.spb.ru
Received 20.09.2010

ABSTRACT
Hereditary breast-ovarian cancer syndrome contributes to as much as 5–7% of breast cancer (BC) and 10–15% of ovarian cancer (OC) incidence. Mutations in the “canonical” genes BRCA1 and BRCA2 occur in 20–30% of affected pedigrees. In addition to BRCA1 and BRCA2 mutations, germ-line lesions in the CHEK2, NBS1, and PALB2 genes also contribute to familial BC clustering. The epidemiology of hereditary breast-ovarian cancer in Russia has some specific features. The impact of the “founder” effect is surprisingly remarkable: a single mutation, BRCA1 5382insC, accounts for the vast majority of BRCA1 defects across the country. In addition, there are two other recurrent BRCA1 alleles: BRCA1 4153delA and BRCA1 185delAG. Besides BRCA1, in Russia breast cancer is often caused by germ-line alterations in the CHEK2 and NBS1 genes. In contrast to BRCA1 and BRCA2, the CHEK2 and NBS1 heterozygosity does not significantly increase the OC risk. Several Russian breast cancer clinics recently started to investigate the efficacy of cisplatin in the therapy of BRCA1-related cancers; initial results show a unique sensitivity of BRCA1-associated tumours to this compound.

KEYWORDS breast cancer, ovarian cancer, hereditary cancer syndromes, BRCA1, CHEK2, NBS.

ABBREVIATIONS
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E. V. Dementyeva, S. M. Zakian

Dosage Compensation of Sex Chromosome Genes in Eukaryotes

E. V. Dementyeva^1,2, S. M. Zakian^1,2,3*
¹Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences
²Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
³Research Center of Clinical and Experimental Medicine, Siberian Branch, Russian Academy of Medical Sciences
*E-mail: zakian@bionet.nsc.ru
Received 24.09.2010

ABSTRACT
Sex chromosome evolution is accompanied by significant divergence in morphology and gene content and results in most genes of one of the sex chromosomes being present in two dosages in one sex and in one dosage in the other. To eliminate the difference in the expression levels of these genes between sexes and to restore equal expression levels of the genes between sex chromosomes and autosomes, mechanisms of dosage compensation have appeared. Studies of three classical objects, Drosophila melanogaster, Caenorhabditis elegans, and mammals, have shown that dosage compensation of X-linked genes can be achieved through completely different chromosome-wide mechanisms. New data on sex chromosome gene expression demonstrating that many sex chromosome genes can be expressed at different levels in males and females were recently obtained from birds and butterflies. In this review, dosage compensation mechanisms in D. melanogaster, C. elegans, and mammals are considered and the data on sex chromosome gene expression in birds and butterflies, and their influence on our view of dosage compensation, are discussed.

KEYWORDS dosage compensation, sex chromosomes, gene expression, X-chromosome inactivation

ABBREVIATIONS
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R.V. Reshetnikov, A.M. Kopylov, A.V. Golovin

Classification of G-Quadruplex DNA on the Basis of the Quadruplex Twist Angle and Planarity of G-Quartets

R. V. Reshetnikov^1,4, A. M. Kopylov^2,3,4, A. V. Golovin^1,4*
¹Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University
²Faculty of Chemistry, Lomonosov Moscow State University
³Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University
⁴Apto-Pharm LLC
*E-mail: golovin@belozersky.msu.ru
Received 8.09.2010

ABSTRACT The present work is devoted to the analysis of the G-quadruplex DNA structure using the bioinformatics method. The interest towards quadruplex DNAs is determined by their involvement in the functioning of telomeres and onco-promoters as well as by the possibility to create on their basis aptamers and nanostructures. Here, we present an algorithm for a general analysis of the polymorphism of the G-quadruplex structure from the data bank PDB using original parameters. 74 structures were grouped according to the following parameters: the number of DNA strands, the number of G-quartets, and the location and orientation of the connecting loops. Two quantitative parameters were used to describe the quadruplex structure: the twist angle between two adjacent quartets (analogous to that for the complementary pair in the duplex DNA) and the quartet planarity (an original parameter). The distribution patterns of these values are specific for each group of quadruplex structures and are dependent upon the type of connecting loops used (diagonal, lateral or propeller). The tetramolecular loopless parallel quadruplex was used as a comparison template. The lateral loops introduce the strongest distortion into the structure of quadruplexes: the values of the twist angles are the lowest and are not typical for the other quadruplex groups. The loops of the diagonal type introduce much weaker deformation into quadruplexes; the structures with propeller loops are characterized by the optimum geometry of G-quartets. Hence, the correlation between the twist angle and the tension in the structure of quadruplex DNA is revealed.

KEYWORDS G-quadruplex, G-quartet, twist angle, loops, structure.

ABBREVIATIONS
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A.V. Maksimenko, A.V. Vavaev, L.I. Bouryachkovskaya, V.P. Mokh, I.A. Uchitel, V.L. Lakomkin, V.I. Kapelko, E.G. Tischenko

Biopharmacology of Enzyme Conjugates: Vasoprotective Activity of Supramolecular Superoxide Dismutase-Chondroitin Sulfate-Catalase Derivative

A.V. Maksimenko*, A.V. Vavaev, L.I. Bouryachkovskaya, V.P. Mokh, I.A. Uchitel, V.L. Lakomkin, V.I. Kapelko, E.G. Tischenko
Institute of Experimental Cardiology, Russian Cardiology Research-and-Production Complex
* E-mail: alexmak@cardio.ru
Received 07.10.2010

ABSTRACT Bienzyme conjugate was obtained by the covalent connection of superoxide dismutase with catalase through endothelial glycocalyx glycosaminoglycan – chondroitin sulfate (SOD-CHS-CAT). This SOD-CHS-CAT conjugate has vasoprotective activity in respect to platelet interactions, tonus of the ring arterial fragment of a rat blood vessel, as well as normalization of hemodynamic parameters in rats and rabbits in conditions of oxidative stress caused by the administration of hydrogen peroxide. The SOD-CHS-CAT conjugate had antiplatelet potential due to its antiaggregation action manifested through the combination of enzyme activities and an acquired supramolecular structure. The influence on arterial fragment tonus was equivalent for SOD and CAT in native and conjugated form. Blood pressure and heart rate were significant and effectively normalized with SOD-CHS-CAT conjugate in rats and rabbits (after hydrogen peroxide administration as a perturbance stimulus). We have discovered the possibility of using the antioxidant bienzyme conjugate in chronic prophylaxis. It is important for a real development of the oral form of the SOD-CHS-CAT conjugate. These results indicate that the development of enzyme conjugates can be medically significant, as a promising approach for the creation of new drugs.

KEYWORDS antioxidant therapy, superoxide dismutase, catalase, chondroitin sulfate, vascular wall, oxidative stress, hydrogen peroxide, bienzyme conjugate, platelets, ring arterial fragment, hemodynamics, vasoprotective activity.

ABBREVIATIONS SOD – superoxide dismutase, CAT – catalase, CHS – chondroitin sulfate, SOD-CHS-CAT – bienzyme conjugate superoxide dismutase-chondroitin sulfate-catalase, L-NNA – N? -nitro-L-arginine, SNP – sodium nitroprusside, ADP – adenosine diphosphate, TRAP – thrombin receptor-activation peptide, NA – noradrenaline, BP – mean blood pressure, HR – heart rate, ECG – electrocardiogram, PRP – platelet-rich plasma, ROS – reactive oxygen species.
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A. N. Glushkov, S. V. Apalko, M. L. Filipenko, V. A. Matveeva, A. Yu. Bakulina, V. G. Lunin, M. V. Kostyanko

A Novel Approach to the Development of Anticarcinogenic Vaccines

A. N. Glushkov¹, S. V. Apalko^1*, M. L. Filipenko^1,2, V. A. Matveeva^1,2, A. Yu. Bakulina¹, V. G. Lunin³, M. V. Kostyanko^1,4
¹Institute of Human Ecology, Siberian Branch, Russian Academy of Sciences
²Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
³Gamaleya Research Institute of Epidemiology and Microbiology
⁴Kemerovo State University
*E-mail: apalko@ngs.ru
Received 07. 10. 2011

ABSTRACT Human exposure to chemical carcinogens is an important etiological factor in cancer diseases. In this article, we will discuss a new approach to the development of anticarcinogenic vaccines. The main task in our research was to select a benzo[a]pyrene immunomimetic peptide considered as a hapten-specific component. For this purpose, we synthesized carcinogen-protein conjugates and prepared mono- and polyclonal antibodies to benzo[a]pyrene. Phage display technology was used to select the benzo[a]pyrene immunomimetic peptide, followed by an evaluation of the immunological properties of the obtained peptide. The obtained benzo[a]pyrene immunomimetic peptide could only simulate chemical carcinogens in the frame of the pIII protein. As a result, we prepared a recombinant protein composed of the benzo[a]pyrene immunomimetic peptide and pIII-encoding sequences. Using ELISA, we demonstrated that the recombinant protein specifically interacts with the antibenzo[a]pyrene monoclonal antibody (mAB B2). Using molecular modeling, we predicted the 3-D structure of the mAB B2 active center and analyzed the characteristics of its interaction with different polycyclic aromatic hydrocarbons, as well as with the benzo[a]pyrene immunomimetic peptide. Thus, a comprehensive analysis of the results of the obtainment of hapten-specific components of anticarcinogenic vaccines allowed us to outline a strategy for future development in this direction.

KEYWORDS benzo[a]pyrene, anticarcinogenic vaccine, immunomimetic peptide, phage display, molecular modeling

ABBREVIATIONS CBD – cellulose-binding domain; OD – optical density; BP – benzo[a]pyrene; BSA – bovine serum albumin; AB – antibody; mAB – monoclonal antibody; ELISA – enzyme-linked immunosorbent assay; PAH – polycyclic aromatic hydrocarbon
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M. V. Zagoskin, T. L. Marshak, D. V. Mukha, A. K. Grishanin

Chromatin Diminution Process Regulates rRNA Gene Copy Number in Freshwater Copepods

M. V. Zagoskin^1*, T. L. Marshak², D. V. Mukha¹, A. K. Grishanin¹
¹Vavilov Institute of General Genetics, Russian Academy of Sciences
²Koltsov Institute of Developmental Biology, Russian Academy of Sciences
*E-mail: zagoskin_mv@mail.ru
Received 20.09.2010

ABSTRACT
The results of quantitative PCR (qPCR) presented in the paper clearly demonstrate that the sixteenfold genome reduction in Cyclops kolensis during chromatin diminution (from 15.3 pg to 0.98 pg) results in a dramatic decrease in ribosomal RNA gene copy numbers in the genome of a somatic cell line by more than two orders of magnitude. The results presented allow for the consideration of the chromatin diminution as a mechanism of rDNA copy number regulation.

KEYWORDS Chromatin diminution, ribosomal RNA genes, Copepoda, real-time PCR, gene copy number

ABBREVIATIONS
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К.А. Chernorizov, V.K.Svedas

Modeling of the Full-Size 3D Structure of Human Chaperone Hsp70 and Study of Its Interdomain Interactions

К. А. Chernorizov¹, V.K.Svedas^1,2*,2*
¹Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University
²Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University
*E-mail: vytas@belozersky.msu.ru
Received 02.11.2010

ABSTRACT
Hsp70 is a chaperone protein that participates in the folding of de novo synthesized proteins, protection of the hydrophobic regions of denaturated proteins, the regulation of apoptosis, the immune response, and several other cellular processes. Despite the large number of publications devoted to the functioning and structure of Hsp70, a reliable full-size 3D structure of this protein remains currently unavailable. Several probable full-size models of human Hsp70 have been constructed based on the structures of individual domains and their components from different organisms and using molecular modeling methodology. The stability of the obtained structures was studied using molecular dynamics. As a result of such an analysis, the most adequate model was selected. The model was built on the basis of Hsp70 elements from Bos Taurus and Caenorhabditis elegans. Using the method of steered molecular dynamics, the key salt bridges responsible for the interdomain interactions were identified: Arg171: Glu516 and Arg416: Glu218. Based on the performed molecular modeling, the scheme of the mechanism triggering ATP hydrolysis and leading to the separation of ATPase and the substrate-binding domains was proposed.

KEYWORDS chaperone, Hsp70, model, tertiary structure, ATPase and substrate-binding domains, molecular dynamics.

ABBREVIATIONS
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S.S. Shishkin, L.I. Kovalyov, M.A. Kovalyova, K.V. Lisitskaya, L.S. Eremina, A.V. Ivanov, E.V. Gerasimov, E.G. Sadykhov, N.Y. Ulasova, O.S. Sokolova, I.Y. Toropygin, V.O. Popov

“Prostate Cancer Proteomics” Database

S. S. Shishkin^1*, L. I. Kovalyov¹, M. A. Kovalyova¹, K. V. Lisitskaya¹, L. S. Eremina¹, A. V. Ivanov¹, E. V. Gerasimov¹, E. G. Sadykhov¹, N. Y. Ulasova², O. S. Sokolova², I. Y. Toropygin³, V. O. Popov¹
¹Bach Institute of Biochemistry, Russian Academy of Sciences
²Lomonosov Moscow State University
³Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences
*E-mail: shishkin@inbi.ras.ru
Received 28.09.2010

ABSTRACT A database of Prostate Cancer Proteomics has been created by using the results of a proteomic study of human prostate carcinoma and benign hyperplasia tissues, and of some human-cultured cell lines (PCP, http://ef.inbi.ras.ru). PCP consists of 7 interrelated modules, each containing four levels of proteomic and biomedical data on the proteins in corresponding tissues or cells. The first data level, onto which each module is based, is a 2DE proteomic reference map where proteins separated by 2D electrophoresis, and subsequently identified by mass-spectrometry, are marked. The results of proteomic experiments form the second data level. The third level contains protein data from published articles and existing databases. The fourth level is formed with direct Internet links to the information on corresponding proteins in the NCBI and UniProt databases. PCP contains data on 359 proteins in total, including 17 potential biomarkers of prostate cancer, particularly AGR2, annexins, S100 proteins, PRO2675, and RO2044. The database will be useful in a wide range of applications, including studies of molecular mechanisms of the aetiology and pathogenesis of prostate diseases, finding new diagnostic markers, etc.

KEYWORDS proteomics, prostate cancer, digital database

ABBREVIATIONS 2DE—two-dimensional gel electrophoresis, BPH—benign prostate hyperplasia, PCa—prostate cancer, PCP—prostate cancer proteomics
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Acta Naturae, 2011. V. 3.
№ 1 (8).

A.I. Tukhvatulin, D.Y. Logunov, I.I. Gitlin, M.M. Shmarov, P.V. Kudan, А.А. Adzhieva, A.F.Moroz, N.N. Kostyukova,L.G.Burdelya,B.S.Naroditsky,A.L.Gintsburg, A.V.Gudkov

A In Vitro and In Vivo Study of the Ability of NOD1 Ligands to Activate the Transcriptional Factor NF-kB

A.I. Tukhvatulin^1*, D.Y. Logunov¹, I.I. Gitlin², M.M. Shmarov¹, P.V. Kudan¹, А.А. Adzhieva¹, A.F. Moroz¹, N.N. Kostyukova¹, L.G. Burdelya², B.S.Naroditsky¹, A.L.Gintsburg¹, A.V.Gudkov²
¹Gamaleya Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences
²Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, New York, USA
*E-mail: amir_tuhvatulin@yahoo.com
Received 31.01.2011

ABSTRACT
Pattern-recognition receptors (PRR) play a crucial role in the induction of the defense reactions of the immune system against pathogenic bacterial and viral infections. The activation of PRR by specific, highly conserved pathogen-associated molecular patterns (PAMPs) induces numerous immune reactions related both to innate and adaptive immunity. In addition to the well-studied Toll-like receptors, pathogens can be recognized by the receptors belonging to the other PRR families; including NOD-like receptors (NLR). Stimulation of members of NOD-like receptors (NOD1, 2) and Toll-like receptors results in the activation of the transcriptional factor NF-kB regulating gene expression in numerous molecules implicated in the development of proinflammatory reactions. As opposed to Toll-like receptors, the NF-kB-activating ability of NLRs has not been fully studied. In this work, we examine the ability of one member of the NLR family – NOD1 – to activate the main proinflammatory transcriptional factor NF-kB. We also compare the NF-kB-activating ability of NOD1 ligands of a different structure with TLR4,5 ligands in vitro and in vivo.

KEYWORDS pattern-recognition receptors, Toll-like receptors, NOD1-receptor, transcriptional factor NF-kB, peptidoglycan of gram-negative bacteria.

ABBREVIATIONS NF-kB – nuclear factor kappa-light-chain-enhancer of activated B cells); AP-1 – activator protein 1; TLR – Toll-like receptor; PAMPs pathogen-associated molecular patterns; DAMPs – damage-associated molecular patterns; PRR – pattern recognition receptor; TNF-α – tumor necrosis factor alpha; RT-PCR – reverse transcription polymerase chain reaction; IRF – interferon regulatory factor; NRL – NOD-like-receptor; RLR – RIG-like receptors; LPS – lipopolysaccharide.
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O.Yu. Makarycheva, E.Yu. Tsareva, M.A. Sudomoina, O.G. Kulakova, B.V. Titov, O.V. Bykova, N.V. Gol’tsova, L.M. Kuzenkova, A.N. Boiko, O.O. Favorova

Family Analysis of Linkage and Association of HLA-DrB1, CTLa4, TGFB1, IL4, CCr5, ranTES, MMP9 and TIMP1 Gene Polymorphisms with Multiple Sclerosis

O.Yu. Makarycheva¹, E.Yu. Tsareva^1,2, M.A. Sudomoina^1,2, O.G. Kulakova^1,2, B.V. Titov^1,2, O.V. Bykova³, N.V. Gol’tsova³, L.M. Kuzenkova³, A.N. Boiko², O.O. Favorova^1,2*
¹Russian Cardiology Scientific and Production Center
²Russian State Medical University
³Scientific Center of Children Health, Russian Academy of Medical Sciences
*E-mail: olga_favorova@mail.ru
Received 14.02.2011

ABSTRACT
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Proteins of the immune system, as well as proteins that are involved in the infiltration of activated immune cells in the CNS, play an important role in the pathogenesis of MS. We investigated the association and linkage with MS of the following immune-system genes polymorphisms: HLA-DRB1, CTLA4, TGFB1, IL4, CCR5 and RANTES, as well as of the matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) genes polymorphisms. For this purpose we used the transmission disequilibrium test (TDT). The group investigated was comprised of 100 nuclear families of Russian ethnicity, each consisting of an affected offspring and his nonaffected parents. It was found that HLA-DRB1*15 allele and MMP9*-1562C allele were transmitted from healthy heterozygous parents to affected children more frequently than alternative alleles (p = 0.02 and p = 0.04, respectively). Another family-based method, AFBAC (affected family-based control), showed MS association with HLA-DRB1*15, but not with the MMP9*-1562C allele.

KEYWORDS functional genomics, human, multiple sclerosis, genotyping, CCR5 gene, CTLA4 gene, HLA-DRB1 gene, IL4 gene, MMP9 gene, RANTES gene, TGFB1 gene, TIMP1 gene, allelic polymorphism, TDT, AFBAC.

ABBREVIATIONS
MS – multiple sclerosis; PCR – polymerase chain reaction; CNS – central nervous system; AFBAC (affected family-based control) – the method of family analysis, in which the control group consists of the alleles, that were not transmitted from parents to affected children; CCR5 (CCR5) – CC chemokine receptor 5 (its gene); CTLA4 (CTLA4) – cytotoxic T lymphocyte antigen-4 (its gene); HLA-DRB (HLA-DRB1) – ? chain of human HLA-DR antigen (its gene 1); IL-4 (IL4) – interleukin-4 (its gene); MMP (MMP) – matrix metalloproteinase (its gene); RANTES (RANTES) – chemokine regulated on activation, normal T cell expressed and secreted (its gene); SNP – single-nucleotide polymorphism; TDT – transmission disequilibrium test; TGFβ1 (TGFB1) – transforming growth factor ?1 (its gene); TIMP (TIMP) – tissue inhibitor of matrix metalloproteinases (its gene).

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D. A. Suplatov, V. K. Arzhanik,V. K. Svedas

Comparative Bioinformatic Analysis of Active Site Structures in Evolutionarily Remote Homologues of α,β-Hydrolase Superfamily Enzymes

D. A. Suplatov^1,2, V. K. Arzhanik¹,V. K. Svedas^1,2*
¹Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University
²Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University
E-mail: vytas@belozersky.msu.ru
Received 25.02.2011

ABSTRACT Comparative bioinformatic analysis is the cornerstone of the study of enzymes’ structure-function relationship. However, numerous enzymes that derive from a common ancestor and have undergone substantial functional alterations during natural selection appear not to have a sequence similarity acceptable for a statistically reliable comparative analysis. At the same time, their active site structures, in general, can be conserved, while other parts may largely differ. Therefore, it sounds both plausible and appealing to implement a comparative analysis of the most functionally important structural elements – the active site structures; that is, the amino acid residues involved in substrate binding and the catalytic mechanism. A computer algorithm has been developed to create a library of enzyme active site structures based on the use of the PDB database, together with programs of structural analysis and identification of functionally important amino acid residues and cavities in the enzyme structure. The proposed methodology has been used to compare some α,β-hydrolase superfamily enzymes. The insight has revealed a high structural similarity of catalytic site areas, including the conservative organization of a catalytic triad and oxyanion hole residues, despite the wide functional diversity among the remote homologues compared. The methodology can be used to compare the structural organization of the catalytic and substrate binding sites of various classes of enzymes, as well as study enzymes’ evolution and to create of a databank of enzyme active site structures.

KEYWORDS bioinformatics, comparative analysis, active site, structural alignment, α,β-hydrolases

ABBREVIATIONS PDB - Protein Data Bank; CSA - Catalytic Site Atlas
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E.A. Smirnova, A.A. Gusev, O.N. Zaitseva, E.M. Lazareva, G.E. Onishchenko, E.V. Kuznetsova, A.G. Tkachev, A.V. Feofanov, M.P. Kirpichnikov

Multi-walled Сarbon Nanotubes Penetrate into Plant Cells and Affect the Growth of Onobrychis arenaria Seedlings

E.A. Smirnova^1*, A.A. Gusev², O.N. Zaitseva², E.M. Lazareva¹, G.E. Onishchenko¹, E.V. Kuznetsova³, A.G. Tkachev⁴, A.V. Feofanov^1,5, M.P. Kirpichnikov^1,5
¹Biology Faculty, Lomonosov Moscow State University
²Derzhavin Tambov State University
³Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch, Russian Academy of Sciences
⁴NanoTechCenter Ltd.
⁵Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
*E-mail: kinggobi@yandex.ru Received 21.02.2011

ABSTRACT
Engineered nanoparticles (ENPs) are now being used in many sectors of industry; however, the impact of ENPs on the environment still requires further study, since their use, recycling, and accidental spill can result in the accumulation of nanoparticles in the atmosphere, soil, and water. Plants are an integral part of ecosystems; hence their interaction with ENPs is inevitable. It is important to understand the consequences of this interaction and assess its potential effects. The present research is focused on studying the effects of the industrial material Taunit, containing multi-walled carbon nanotubes (MWNTs), on plants, and testing of its ability to penetrate into plant cells and tissues. Taunit has been found to stimulate the growth of roots and stems and cause an increase in peroxidase activity in Onobrychis arenaria seedlings. Peroxidase activity increases with decreasing concentration of Taunit from 1,000 to 100 mg/l. MWNTs from Taunit were detected in the cells and tissues of seedling roots and leaves, implying the ability of MWNTs to penetrate into roots and accumulate there, as well as their ability to be transported into seedling leaves. Thus, the changes in the physiological parameters of plants are associated not only with MWNT adsorption on the root surface, as previously believed, but also with their penetration, uptake and accumulation in the plant cells and tissues.

KEYWORDS
multi-walled carbon nanotubes, light microscopy, electron microscopy, electron diffraction pattern, O. arenaria seedlings.

ABBREVIATIONS CNM – carbon nanomaterials; MWNT – multi-walled carbon nanotubes; SWNT – single-walled carbon nanotubes; TEM – transmission electron microscopy, SAED – selected area electron diffraction.
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A.A. Ovsepyan, D.N. Panchenkov, E.B. Prokhortchouk, G.B. Telegin, N.A. Zhigalova, E.P. Golubev, T. E. Sviridova, S.T. Matskeplishvili, K.G. Skryabin, U.I. Buziashvili

Modeling Myocardial Infarction in Mice: Methodology, Monitoring, Pathomorphology

A.A. Ovsepyan⁴, D.N. Panchenkov^1,3, E.B. Prokhortchouk^1*, G.B. Telegin⁴, N.A. Zhigalova¹, E.P. Golubev², T. E. Sviridova⁵, S.T. Matskeplishvili², K.G. Skryabin¹, U.I. Buziashvili^1,2
¹Center “Bioengineering”, Russian Academy of Sciences
²Bakoulev Center for Cardiovascular Surgery, Russian Academy of Medical Sciences
³Moscow State University of Medicine and Dentistry
⁴The Branch of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Pushchino, Russian Academy of Sciences
⁵Semashko Railway Hospital
*E-mail: prokhortchouk@biengi.ac.ru
Received 12.11.2010

ABSTRACT Myocardial infarction is one of the most serious and widespread diseases in the world. In this work, a minimally invasive method for simulating myocardial infarction in mice is described in the Russian Federation for the very first time; the procedure is carried out by ligation of the coronary heart artery or by controlled electrocoagulation. As a part of the methodology, a series of anesthetic, microsurgical and revival protocols are designed, owing to which a decrease in the postoperational mortality from the initial 94.6 to 13.6% is achieved. ECG confirms the development of large-focal or surface myocardial infarction. Postmortal histological examination confirms the presence of necrosis foci in the heart muscles of 87.5% of animals. Altogether, the medical data allow us to conclude that an adequate mouse model for myocardial infarction was generated. A further study is focused on the standardization of the experimental procedure and the use of genetically modified mouse strains, with the purpose of finding the most efficient therapeutic approaches for this disease.

KEYWORDS myocardial infarction, coronary artery, ligation, controlled electrocoagulation, ECG (electrocardiogram).

ABBREVIATIONS
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E. S. Severin, M. V. Savvateeva

Molecular and Physiological Mechanisms of Membrane Receptor Systems Functioning

E. S. Severin^1*, M. V. Savvateeva²
¹All-RussiaResearch Center for Molecular Diagnostics and Therapy
²Biology Faculty, Lomonosov Moscow State University
E-mail: e.severin@mail.ru
Received 16.11.2010

ABSTRACT Molecular physiology is a new interdisciplinary field of knowledge that looks into how complicated biological systems function. The living cell is a relatively simple, but at the same time very sophisticated biological system. After the sequencing of the human genome, molecular physiology has endeavored to investigate the systems of cellular interactions at a completely new level based on knowledge of the spatial organization and functions of receptors, their ligands, and protein-protein interactions. In recent years, the achievements in molecular physiology have centered on the study of sensor reception mechanisms and intercellular data transfer, as well as the immune system physiology, amongst other processes.

KEYWORDS molecular physiology, receptor profile, secondary messengers, targeted delivery, epigenetic diagnostics, prostate cancer

ABBREVIATIONS BPA - Benign prostatic adenoma, PIN – Prostatic Intraepithelial Neoplasia, PSA – Prostatespecific antigen, PC – prostate cancer, cAMP – cyclic adenosine monophosphate, cGMP – Cyclic guanosine monophosphate, АМАCR - Alpha-methylacyl-coenzyme A Racemase, CGI – CpG-island, DAG – Diacylglycerol, DD3 - Differential Display Code 3, EPСA - Early Prostate Cancer Antigen, ERG4 – V-ets Erythroblastosis virus E26 oncogene homolog, ETS – transcription factor, ETV1 – ETS translocation variant 1, EGFR - Epidermal Growth Factor Receptor, GSTP1 - Glutathione-S-Transferase ω1, IP3 - Inositol Trisphosphate, RARß2 – Retinoic Acid Receptor Beta 2, RASSF1A - RAS Association Domain Family Protein 1A, TMPRSS2 - Transmembrane Serine Protease 2
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A. V. Safonova, A.N. Petrin, S. D. Arutyunov, V. N. Tsarev, L. A. Akulenko, A.O. Zorina, D. V. Rebrikov, A. V. Rubanovich, S. A. Borinskaya, N. K. Yankovsky

Association of Cytokine Gene Alleles with the Inflammation of Human Periodontal Tissue

A. V. Safonova^1*, A.N. Petrin¹, S. D. Arutyunov¹, V. N. Tsarev¹, L. A. Akulenko¹, A. O. Zorina², D. V. Rebrikov^3,4, A. V. Rubanovich^3,4, S. A. Borinskaya^1,4, N. K. Yankovsky⁴
¹Moscow State University of Medicine and Dentistry
²Central Research Institute of Dentistry and Oral Surgery, Federal Agency of Medical Technologies
³DNA-Technology JSC.
⁴Vavliov Institute of General Genetics, Russian Academy of Sciences
*E-mail: safnastia@yandex.ru
Received 29.10.2010

ABSTRACT Gingivitis and periodontitis are chronic inflammatory diseases of the periodontal tissue in humans caused by both environmental and genetic factors. The human cytokine genes that regulate the immune response may play an important role in the development of these chronic inflammatory diseases. The aim of this study is to analyze the allele status of eight human cytokine genes and to associate it with the inflammation of periodontal tissue in humans. A total of 296 unrelated males of Russian origin were studied. A significant association of the IL1B and IL6 minor alleles and gingivitis was found. In addition, we found a significant association of the OHI-S index with the IL18 gene alleles. The influence of genetic factors on gingivitis may contribute to the understanding of the mechanisms of interaction between genetic and environmental factors in periodontal conditions, and to the identification of risk groups for effective prevention and treatment.

KEYWORDS gingivitis, periodontitis, cytokine genes, genetic polymorphism.

ABBREVIATIONS
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T. A. Zdobnova, E. N. Lebedenko, S.М. Deyev

Quantum Dots for Molecular Diagnostics of Tumors

T. A. Zdobnova*, E. N. Lebedenko, S.М. Deyev
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
*E-mail: t.zdobnova@mail.ru
Received 15.10.2010

ABSTRACT Semiconductor quantum dots (QDs) are a new class of fluorophores with unique physical and chemical properties, which allow to appreciably expand the possibilities for the current methods of fluorescent imaging and optical diagnostics. Here we discuss the prospects of QD application for molecular diagnostics of tumors ranging from cancer-specific marker detection on microplates to non-invasive tumor imaging in vivo. We also point out the essential problems that require resolution in order to clinically promote QD, and we indicate innovative approaches to oncology which are implementable using QD.

KEYWORDS quantum dots, fluorescence imaging, multicolor labeling, nanoparticles.

ABBREVIATIONS AFP – alpha-fetoprotein; EGF – epidermal growth factor; EGFR - epidermal growth factor receptor; ELISA – enzyme-linked immunosorbent assay; HER2/neu - human epidermal growth factor receptor 2/neu; IGF1R – type 1 insulin-like growth factor receptor; IHC – immunohistochemistry; MAA – mercaptoacetic acid; QDs – quantum dots; PEG – polyethylene glycol; scFv – single-chain variable antibody fragment; PSCA – prostate stem cell antigen; PSMA – prostate-specific membrane antigen; RES –reticuloendothelial system; ROS – reactive oxygen species.
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D. A. Skvortsov, M. E. Zvereva, O.V. Shpanchenko, O. A. Dontsova

Assays for Detection of Telomerase Activity

D. A. Skvortsov, M. E. Zvereva, O.V. Shpanchenko, O. A. Dontsova
Faculty of Chemistry, Lomonosov Moscow State University
*E-mail: skvorratd@mail.ru
Received 02.11.2010

ABSTRACT Progressive loss of the telomeric ends of chromosomes caused by the semi-conservative mechanism of DNA replication is an important timing mechanism which controls the number of cells doubling. Telomerase is an enzyme which elongates one chain of the telomeric DNA and compensates for its shortening during replication. Therefore, telomerase activity serves as a proliferation marker. Telomerase activity is not detected in most somatic cells, with the exception of embryonic tissues, stem cells, and reproductive organs. In most tumor cells (80–90%), telomerase is activated and plays the role of the main instrument that supports the telomere length, which can be used for the diagnostics of neoplastic transformation. This is the primary reason why assays regarding the development of telomerase activity have attracted the attention of researchers. Telomerase activity testing may be useful in the search for telomerase inhibitors, which have the potential to be anti-cancer drugs. Moreover, telomerase activation may play a positive role in tissue regeneration; e.g., after partial removal of the liver or cardiac infarction. All telomerase activity detection assays can be divided into two large groups: those based on direct detection of telomerase products, and those based on different systems of amplification of the signals from DNA that yield from telomerase. The methods discussed in this review are suitable for testing telomerase activity in different samples: in protozoa and mammalian cells, mixed cellular populations, and tissues.

KEYWORDS telomerase, telomerase activity assay, tumor diagnostics, physicochemical methods, polymerase activity assay, DNA determination.

ABBREVIATIONS
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K. D. Nadezhdin, O. V. Bocharova, E. V. Bocharov, A. S. Arseniev

Structural and Dynamic Study of the Transmembrane Domain of the Amyloid Precursor Protein

K. D. Nadezhdin, O. V. Bocharova, E. V. Bocharov*, A. S. Arseniev
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
*E-mail: bon@nmr.ru
Received 28.10.10

ABSTRACT
Alzheimer’s disease affects people all over the world, regardless of nationality, gender or social status. An adequate study of the disease requires essential understanding of the molecular fundamentals of the pathogenesis. The amyloid β-peptide, which forms amyloid plaques in the brain of people with Alzheimer’s disease, is the product of sequential cleavage of a single-span membrane amyloid precursor protein (APP). More than half of the APP mutations found to be associated with familial forms of Alzheimer’s disease are located in its transmembrane domain. The pathogenic mutations presumably affect the structural-dynamic properties of the APP transmembrane domain by changing its conformational stability and/or lateral dimerization. In the present study, the structure and dynamics of the recombinant peptide corresponding to the APP fragment, Gln686-Lys726, which comprises the APP transmembrane domain with an adjacent N-terminal juxtamembrane sequence, were determined in the membrane mimetic environment composed of detergent micelles using NMR spectroscopy. The structure obtained in dodecylphosphocholine micelles consists of two α-helices: a short surface-associated juxtamembrane helix (Lys687-Asp694) and a long transmembrane helix (Gly700-Leu723), both connected via a mobile loop region. A minor bend of the transmembrane α-helix is observed near the paired residues Gly708-Gly709. A cholesterol-binding hydrophobic cavity is apparently formed under the loop region, where the juxtamembrane α-helix comes into contact with the membrane surface near the N-terminus of the transmembrane α-helix.

KEYWORDS Alzheimer’s disease, amyloid precursor protein, transmembrane domain, NMR spectroscopy, spatial structure, dynamics.

ABBREVIATIONS APP – amyloid precursor protein; Aβ –amyloid β-peptide; TM – transmembrane; JM – juxtamembrane, APPjmtm – APP686-726 fragment; DPC – dodecylphosphocholine; DSA – doxyl stearic acid; NOE – nuclear Overhauser effect; NOESY – NOE spectroscopy; HSQC – heteronuclear single-quantum correlation.
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Acta Naturae, 2011. V. 3.
№ 2 (9).

V.V. Pleshkan, I.V. Alekseenko, M.V. Zinovyeva, T.V. Vinogradova, E.D. Sverdlov

Promoters with Cancer Cell-Specific Activity for Melanoma Gene Therapy

V.V. Pleshkan^1,2*, I.V. Alekseenko^1,2, M.V. Zinovyeva¹, T.V. Vinogradova¹, E.D. Sverdlov^1,2
¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
²Institute of Molecular Genetics, Russian Academy of Sciences
*E-mail: vpleshkan@gmail.com
Received 01.04.2011

ABSTRACT Melanoma is one of the most aggressive tumors. It develops from pigment-forming cells (melanocytes) and results in a high number of lethal outcomes. The use of genetic constructs with the ability to specifically kill melanoma cells, but not normal cells, might increase the lifespan of patients, as well as improve their quality of life. One of the methods to achieve a selective impact for therapeutic genes on cancer cells is to utilize a transcriptional control mechanism using promoters that are specifically activated only in cancerous cells. In this review, promoters of the genes that are preferentially expressed in melanoma cells are described. These promoters, and other highly melanoma-specific regulatory elements, reduce the unspecific expression of therapeutic genes in normal tissues. Moreover, cancer-specific promoters and their elements are advantageous for the development of universal anticancer drugs. Examples of the use of double promoters that have a high potential as instruments in cancer gene therapy are also given in this review.

KEYWORDS melanoma; gene therapy; tissue-specific promoters; specific expression of a transgene

ABBREVIATIONS Ad – adenovirus; CRAds – conditionally replicative adenoviruses; DT-A – diphtheria toxin A chain; HSVtk – herpes simplex virus thymidine kinase; SCLC – small cell lung cancer; MC1R – melanocortin 1 receptor; MIA – melanoma inhibitory activity, MITF – microphthalmia-associated transcription factor; TERT – human telomerase reverse transcriptase; TSP – tumor specific promoter; TSS – transcription start site
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L. A. Dykman, N. G. Khlebtsov

Gold Nanoparticles in Biology and Medicine: Recent Advances and Prospects

L. A. Dykman¹, N. G. Khlebtsov^1,2*
¹Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences
²Saratov State University
*E-mail: khlebtsov@ibppm.sgu.ru
Received 21.02.2011

ABSTRACT Functionalized gold nanoparticles with controlled geometrical and optical properties are the subject of intensive studies and biomedical applications, including genomics, biosensorics, immunoassays, clinical chemistry, laser phototherapy of cancer cells and tumors, the targeted delivery of drugs, DNA and antigens, optical bioimaging and the monitoring of cells and tissues with the use of state-of-the-art detection systems. This work will provide an overview of the recent advances and current challenges facing the biomedical application of gold nanoparticles of various sizes, shapes, and structures. The review is focused on the application of gold nanoparticle conjugates in biomedical diagnostics and analytics, photothermal and photodynamic therapies, as a carrier for delivering target molecules, and on the immunological and toxicological properties. Keeping in mind the huge volume and high speed of the data update rate, 2/3 of our reference list (certainly restricted to 250 Refs.) includes publications encompassing the past 5 years.

KEYWORDS gold nanoparticles; plasmon resonance; biosensors; biomedical diagnostics; photothermal and photodynamic therapy; targeted drug delivery; nanotoxicology

ABBREVIATIONS GNP – gold nanoparticles; PR – plasmon resonance; PPTT – plasmonic photothermal therapy; PEG – polyethylenglycol; SEM – scanning electron microscopy; TEM – transmission electron microscopy; PDT – photodynamic therapy; TNF – tumor necrosis factor; CTAB – cetyltrimethylammonium bromide; SPIA – sol particle immunoassay
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V. A Stepanov, O. P. Balanovsky, A. V. Melnikov, A. Yu. Lash-Zavada, V. N. Khar’kov, T. V. Tyazhelova, V. L. Akhmetova, O. V. Zhukova, Yu. V. Shneider, I. N. Shil’nikova, S. A. Borinskaya, A. V. Marusin, M. G. Spiridonova, K. V. Simonova, I. Yu. Khitrinskaya, M. O. Radzhabov, A. G. Romanov, O. V. Shtygasheva, S. M. Koshel’, E. V. Balanovskaya, A. V. Rybakova, E. K. Khusnutdinova, V. P. Puzyrev, N. K. Yankovsky

Characteristics of Populations of the Russian Federation over the Panel of Fifteen Loci Used for DNA Identification and in Forensic Medical Examination

V. A Stepanov^1,6*, O. P. Balanovsky^2,5, A. V. Melnikov³, A. Yu. Lash-Zavada³, V. N. Khar’kov^1,6, T. V. Tyazhelova², V. L. Akhmetova⁴, O. V. Zhukova², Yu. V. Shneider², I. N. Shil’nikova², S. A. Borinskaya², A. V. Marusin¹, M. G. Spiridonova¹, K. V. Simonova¹, I. Yu. Khitrinskaya¹, M. O. Radzhabov⁷, A. G. Romanov⁵, O. V. Shtygasheva⁸, S. M. Koshel’⁹, E. V. Balanovskaya⁵, A. V. Rybakova³, E. K. Khusnutdinova⁴, V. P. Puzyrev¹, N. K. Yankovsky¹
¹Institute for Medical Genetics, Russian Academy of Medical Sciences
²Vavilov Institute of General Genetics, Russian Academy of Sciences
³Forensic Centre, Ministry of Interior of Russian Federation
⁴Institute of Biochemistry and Genetics, Ufa Research Centre, Russian Academy of Sciences
⁵Research Centre for Medical Genetics, Russian Academy of Sciences
⁶Genome Diagnostics, Ltd.
⁷Dagestan State University
⁸Katanov Khakas State University
⁹Geography Faculty, Lomonosov Moscow State University
*E-mail: vadim.stepanov@medgenetics.ru
Received 05.03.2011

ABSTRACT Seventeen population groups within the Russian Federation were characterized for the first time using a panel of 15 genetic markers that are used for DNA identification and in forensic medical examinations. The degree of polymorphism and population diversity of microsatellite loci within the Power Plex system (Promega) in Russian populations; the distribution of alleles and genotypes within the populations of six cities and 11 ethnic groups of the Russian Federation; the levels of intra- and interpopulation genetic differentiation of population; genetic relations between populations; and the identification and forensic medical characteristics of the system of markers under study were determined. Significant differences were revealed between the Russian populations and the U.S. reference base that was used recently in the forensic medical examination of the RF. A database of the allelic frequencies of 15 microsatellite loci that are used for DNA identification and forensic medical examination was created; the database has the potential of becoming the reference for performing forensic medical examinations in Russia. The spatial organization of genetic diversity over the panel of the STR markers that are used for DNA identification was revealed. It represents the general regularities of geographical clusterization of human populations over various types of genetic markers. The necessity to take into account a population’s genetic structure during forensic medical examinations and DNA identification of criminal suspects was substantiated.

KEYWORDS microsatellites; short tandem repeats; allelic frequencies; forensic medical examination; DNA identification; population of Russia; reference database; genetic diversity; gene geography

ABBREVIATIONS MI RF – Ministry of Interior of the Russian Federation; PCR – polymerase chain reaction; He – expected heterozygosity; AMOVA – Analysis of molecular variance; CODIS – combined DNA index system; EDNAP – the European DNA Profiling Group; ENFSI – European Network of Forensic Science Institutes; ESS – European Standard Set; MP – matching probability; PD – power of discrimination; PE – power of exclusion; PI – paternity index; SNP – single nucleotide polymorphism; STR – short tandem repeats; UPGMA – unweighted pair group method with arithmetic mean
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O. V. Samsonova, K. S. Kudryashova, A. V. Feofanov

N-Terminal Moiety of Antimicrobial Peptide Ltc1-K Increases its Toxicity for Eukaryotic Cells

O. V. Samsonova^1,2, K. S. Kudryashova¹, A. V. Feofanov^1,2*
¹Biological Faculty, Lomonosov Moscow State University
²Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
*E-mail: avfeofanov@yandex.ru
Received 11.02.2011

ABSTRACT The antimicrobial peptide Ltc1-K and its derivates without one, two, then three N-terminal amino acid residues were studied based on the hypothesis (backed by some experimental data) that the hydrophobic N-terminal moiety of linear cationic antimicrobial peptides defines their haemolytic activity. It was discovered that the excision of three N-terminal amino acid residues considerably decreases the peptide’s toxicity for eukaryotic cells and simultaneously increases the selectivity of antibacterial activity for some bacteria species. Studies performed with the model membrane systems and human erythrocytes revealed that the main reason for the observed effect is a multifold decrease in the peptide’s affinity to an eukaryotic cellular membrane enriched with zwitterionic phospholipids.

KEYWORDS antimicrobial peptides; latarcin; haemolytic activity; circular dichroism; fluorescent confocal microscopy

ABBREVIATIONS AMP – antimicrobial peptides; LSCM – laser scanning confocal microscopy; CD – circular dichroism; NMR – nuclear magnetic resonance; LML – large monolamellar liposomes; DOPC – 1,2-dioleoyl-snglycero-3-phosphocholine; LMPC – 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine; CF – carboxyfluorescein; FD70 and FD500 – fluorescein-labelled dextrans of 70 kDa and 500 kDa, respectively; EC₅₀ – peptide concentration required for 50% haemolysis or cell death; MIC – minimal inhibitory concentration; K_d– dissociation constant
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K.V. Lobanov, L. Errais Lopes, N.V. Korol’kova, B.V. Tyaglov, A.V. Glazunov, R.S. Shakulov, A.S. Mironov

Reconstruction of Purine Metabolism in Bacillus subtilis to Obtain the Strain Producer of AICAR: A New Drug with a Wide Range of Therapeutic Applications

K.V. Lobanov, L. Errais Lopes, N.V. Korol’kova, B.V. Tyaglov, A.V. Glazunov, R.S. Shakulov, A.S. Mironov*
State Research Institute for Genetics and Selection of Industrial Microorganisms
*E-mail: alexmir@genetika.ru
Received 24.02.2011

ABSTRACT
AICAR is a natural compound, an analogue and precursor of adenosine. As activator of AMP-activated protein kinase (AMPK), AICAR has a broad therapeutic potential, since it normalizes the carbohydrate and lipid metabolism and inhibits the proliferation of tumor cells. The synthesis of AICAR in Bacillus subtilis cells is controlled by the enzymes of purine biosynthesis; their genes constituting purine operon (pur-operon). Reconstruction of purine metabolism in B. subtilis was performed to achieve overproduction of AICAR. For this purpose, the gene purH, which encodes formyltransferase/IMP-cyclohydrolase, an enzyme that controls the conversion of AICAR to IMP, was removed from the B. subtilis genome, ensuring the accumulation of AICAR. An insertion inactivating the gene purR that encodes the negative transcriptional regulator of the purine biosynthesis operon was introduced into the B.subtilis chromosome in order to boost the production of AICAR; the transcription attenuator located in the leader sequence of pur-operon was deleted. Furthermore, the expression integrative vector carrying a strong promoter of the rpsF gene encoding the ribosomal protein S6 was designed. The heterologous Escherichia coli gene purF encoding the first enzyme of the biosynthesis of purines with impaired allosteric regulation, as well as the modified E.coli gene prs responsible for the synthesis of the precursor of purines — phosphoribosyl pyrophosphate (PRPP) — was cloned into this vector under the control of the rpsF gene promoter. The modified purF and prs genes were inserted into the chromosome of the B. subtilis strain. B. subtilis strain obtained by these genetic manipulations accumulates 11–13 g/L of AICAR in the culture fluid.

KEYWORDS anticancer agent AICAR; purine metabolism; genome reconstruction; Bacillus subtilis strain - producer of AICAR

ABBREVIATIONS AICAR – 5-aminoimidazole-4-carboxamide ribofuranoside; AICAR-P – nucleotide AICAR phosphate; IMP – inosine monophosphate; AMP – adenosine monophosphate; АМPK – 5’-adenosine monophosphate-activated protein kinase; PRPP – phosphoribosyl pyrophosphate; GAR – 5’- phosphoribosyl glycineamide ribonucleotide; GMP – guanosine monophosphate; PCR – polymerase chain reaction; CF – culture fluid
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M. V. Nesterchuk, P. V. Sergiev, O. A. Dontsova

Posttranslational Modifications of Ribosomal Proteins in Escherich ia coli

M. V. Nesterchuk*, P. V. Sergiev, O. A. Dontsova
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University
Faculty of Chemistry, Lomonosov Moscow State University
*E-mail: nesterchuk.mihail@gmail.com
Received 14.02.2011

ABSTRACT
А number of ribosomal proteins in Escherichia coli undergo posttranslational modifications. Six ribosomal proteins are methylated (S11, L3, L11, L7/L12, L16, and L33), three proteins are acetylated (S5, S18, and L7), and protein S12 is methylthiolated. Extra amino acid residues are added to protein S6. С-terminal amino acid residues are partially removed from protein L31. The functional significance of these modifications has remained unclear. These modifications are not vital to the cells, and it is likely that they have regulatory functions. This paper reviews all the known posttranslational modifications of ribosomal proteins in Escherichia coli. Certain enzymes responsible for the modifications and mechanisms of enzymatic reactions are also discussed.

KEYWORDS ribosomal proteins; posttranslational modification; Escherichia coli

ABBREVIATIONS RNA – ribonucleic acid; rRNA – ribosomal RNA; mRNA – messenger RNA; tRNA – transfer RNA; IF2 – initiation factor 2; EF-Tu – elongation factor Tu; EF-G – elongation factor G; RF3 – release factor 3; GTP – guanosine-5'-triphosphate
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K.S. Mineev, E.V. Bocharov, P.E. Volynsky, M.V. Goncharuk, E.N. Tkach, Ya.S. Ermolyuk, A.A. Schulga, V.V. Chupin, I.V. Maslennikov, R.G. Efremov, A.S. Arseniev

Dimeric Structure of the Transmembrane Domain of Glycophorin A in Lipidic and Detergent Environments

K.S. Mineev, E.V. Bocharov*, P.E. Volynsky, M.V. Goncharuk, E.N. Tkach, Ya.S. Ermolyuk, A.A. Schulga, V.V. Chupin, I.V. Maslennikov, R.G. Efremov, A.S. Arseniev
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
*E-mail: bon@nmr.ru
Received 24.02. 2011

ABSTRACT Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane ?-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties.

KEYWORDS bitopic membrane proteins; transmembrane domain; dimerization; spatial structure; molecular dynamics; NMR

ABBREVIATIONS ТМ – transmembrane; GpAtm – TM fragment 61-98 of human protein glycophorin A, GpA_61-98; DPC – dodecylphosphocholine; DMPC – dimyristoylphosphatidylcholine; DHPC – dihexanoylphosphatidylcholine; RMSD – root-mean-square deviation; NOE – nuclear Overhauser effect; NOESY – nuclear Overhauser enhancement spectroscopy experiment; HSQC – heteronuclear single quantum coherence experiment
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O. A. Zorina, A. A. Kulakov, O. A. Boriskina, D. V. Rebrikov

Relationship between the Pathogenic Representatives of Periodontal Pockets Microbiocenosis in Patients with Periodontitis with Varying Degrees of Severity

O. A. Zorina^1*, A. A. Kulakov¹, O. A. Boriskina¹, D. V. Rebrikov^2,3
¹Central Research Institute of Dentistry and Oral Surgery
²Vavilov Institute of General Genetics, Russian Academy of Sciences
³Scientific Production Enterprise «DNA Technology», JSC
*E-mail: zorina-cniis@yandex.ru
Received 09.02.2011

ABSTRACT
Periodontitis is a common disease that is considered to be a manifestation of the distortion of the ratio between the normal and conditionally pathogenic microflora of periodontal pockets. In this study, the ratio between the six most important periodontal pathogens and the total microflora of the periodontal pocket in healthy individuals and patients with varying severity of periodontitis was ascertained by quantitative real-time PCR. It was ascertained that the relative content of Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythensis (Bacteroides forsythus) persistently develops in the total microflora of the periodontal pocket upon progressing periodontitis; this value is higher than that in the control group by more than two orders of magnitude upon a severe degree of chronic generalized periodontitis.

KEYWORDS ecosystem; habitat; periodontitis; periodontopathogenic microflora of periodontal pockets; quantitative polymerase chain reaction

ABBREVIATIONS PCR – polymerase chain reaction; CGP – chronic generalized periodontitis
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Yu. G. Odnoshivkina, A. M. Petrov, A. L. Zefirov

The Effects of β2-Adrenoreceptor Activation on the Contractility, Ca-Signals and Nitric Oxide Production in the Mouse Atria

Yu. G. Odnoshivkina, A. M. Petrov*, A. L. Zefirov
Kazan State Medical University, Federal Agency for Health Care and Social Development
*E-mail: fysio@rambler.ru
Received 28.12.2010

ABSTRACT
The effects of the selective β2-adrenoreceptor agonist (fenoterol) on the functioning of mouse atrial were studied using both tensometry and fluorescent methods. It has been demonstrated that with the use of a high concentration of fenoterol (in the range of 1–50 μM), there is a more significant positive inotropic effect observed within a shorter period of time. In the case of relatively low doses of fenoterol (1 and 5 μM), its contractility effects are observed 20 min after the application of agonist, whereby in the case of high concentrations (25, 50 and 300 μM), the effects appear within the first minutes. During the first 10–15 min, 5 μM fenoterol causes an increase in the amplitude of Ca-signals in cardiomyocytes (this indicates an increase in the concentration of Ca ions during systole) and the activation of NO synthesis. However, after 20 min, the production of NO decreases; while the amplitude of Ca-signals remains high. The application of 50 μM fenoterol leads to a rapid increase in the amplitude of Ca-signals: at the same time, it causes a decrease in the production of NO, which we found to begin to increase after 10 min of agonist application. It is suggested that the dynamics of the positive inotropic effect occurring under pharmacological stimulation of β2-adrenoreceptors depend on the rate of increase in the amplitude of Ca-signals and on the degree of NO synthesis.

KEYWORDS β2-adrenoreceptor; fenoterol; calcium; nitrico xide; contractility; atrial cardiomyocytes

ABBREVIATIONS
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Acta Naturae, 2011. V. 3.
№ 3 (10).

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

Characteristics of Artificial Virus-likeParticles Assembled in vitro from Potato Virus X Coat Protein and Foreign Viral RNAs

M. V. Arkhipenko¹, E. K. Petrova¹, N. A. Nikitin¹, A. D. Protopopova^2,3, E. V. Dubrovin³, I. V. Yaminskii^2,3, N. P. Rodionova¹, O. V. Karpova^1*, J. G. Atabekov^1,4
¹Biology Department, Lomonosov Moscow State University
²Advanced Technologies Center
³Physical Department, Lomonosov Moscow State University
⁴Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University
*E-mail: okar@genebee.msu.su
Received 21.04. 2011

ABSTRACT
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 RNPs 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.

KEYWORDS plant viruses; RNA; viral ribonucleoproteins; translation activation.

ABBREVIATIONS PVX – Potato virus X; CP – coat protein; vRNP – viral ribonucleoprotein; MP – movement protein; BMV – Brome mosaic virus; PAMV – Potato aucuba mosaic virus; TMV – Tobacco mosaic virus; NMV – Narcissus mosaic virus; AltMV – Altenathera mosaic virus.
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R. A. Timakhov, P. O. Fedichev, A. A. Vinnik, J. R. Testa, O. O. Favorova

Transcription Factor DLX5 As a New Target for Promising Antitumor Agents

R. A. Timakhov^1,2,3*, P. O. Fedichev¹, A. A. Vinnik¹, J. R. Testa³, O. O. Favorova²
¹Quantum Pharmaceuticals, Russia
²Pirogov Russian National Research Medical University
³Fox Chase Cancer Centre, Philadelphia, USA
E-mail: timakhov@gmail.com
Received 11.05.2011

ABSTRACT
The crystal structure of the human transcription factor DLX5 has been used for the screening of a library consisting of 10⁶ 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.

KEYWORDS DLX5; transcription factor; small molecules; cancer; molecular docking.

ABBREVIATIONS DLX5 – human transcription factor, encoded by gene DLX5 (Distall-less homeobox gene 5); Dlx5 – mouse transcription factor encoded by gene Dlx5; K_d – binding (affinity) constant; NSCLC - non-small cell lung cancer; siRNA – small interfering RNA, RT-PCR – reverse transcription polymerase chain reaction.
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I.Yu. Gribova, S.V. Tillib, I.L. Tutykhina, М.М. Shmarov, D.Yu. Logunov, L.V. Verkhovskaya, B.S. Naroditskii, A.L. Gintsburg

Effective Genetic Expression of Nanoantibodies by Recombinant Adenoviral Vector in vitro

I.Yu. Gribova¹, S.V. Tillib², I.L. Tutykhina¹, М.М. Shmarov¹, D.Yu. Logunov¹, L.V. Verkhovskaya¹, B.S. Naroditskii^1*, A.L. Gintsburg¹
¹GamaleyaResearch Institute for Epidemiology and Microbiology
²nstitute of Gene Biology, Russian Academy of Sciences
*E-mail: bsnar1941@yahoo.com
Received 12.05.2011

ABSTRACT 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.

KEYWORDS recombinant adenoviral vector; nanoantibodies; genetic immunization.

ABBREVIATIONS HEK-293 – human embryonic kidney cell culture; His₆-tag - amino acid motif in proteins consisting of six histidines; НА-tag – epitope tag (YPYDVPDYA) derived from the haemagglutinin molecule; PFU – plaque-forming unit; PCR – polymerase chain reaction; PT-PCR – reverse transcription polymerase chain reaction; GAPDH - glyceraldehyde 3-phosphate dehydrogenase.
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А. А. Parkhitko, О. О. Favorova, E. P. Henske

Rabin8 Protein Interacts with GTPase Rheb and Inhibits Phosphorylation of Ser235/Ser236 in Small Ribosomal Subunit Protein S6

А. А. Parkhitko^1,2,3*, О. О. Favorova¹, E. P. Henske^2,3
¹Pirogov Russian National Research Medical University
²Fox Chase Cancer Center, Philadelphia, USA
³Brigham and Women’s Hospital, Harvard Medical School, USA
*E-mail: parhitko@mail.ru
Received 18.05.2011

ABSTRACT The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that in association with Raptor, mLST8, PRAS40 and Deptor forms a complex (mTORC1) 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 mTORС1 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.

KEYWORDS complex mTORC1; Rheb; Rabin8; small ribosomal unit protein S6.

ABBREVIATIONS DMEM – Dulbecco’s Modified Eagle’s Medium; FBS – fetal bovine serum; GAP protein – GTPase-activating protein; mTOR – mammalian target of Rapamycin; Rheb – Ras homologue enriched in brain; siRNA – small interfering RNA; TBST – Tris-Buffered Saline and Tween 20.
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S. A. Goncharuk, M. V. Goncharuk, M. L. Mayzel, D. M. Lesovoy, V. V. Chupin, E.V. Bocharov, A.S. Arseniev, M.P. Kirpichnikov

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

S.A. Goncharuk^1,2*, M.V. Goncharuk^1,2, M.L. Mayzel¹, D.M. Lesovoy¹, V.V. Chupin¹, E.V. Bocharov¹, A. S. Arseniev¹, M. P. Kirpichnikov^1,2
¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science
²Biology Department, Lomonosov Moscow State University
*E-mail: ms.goncharuk@gmail.com
Received 17.05.2011

ABSTRACT
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.

KEYWORDS membrane protein; FGFR; bacterial expression; purification; detergent solubilization; NMR.

ABBREVIATIONS FGFR - Fibroblast growth factor receptor family; FGFR3 - Fibroblast growth factor receptor 3; RTK - receptor tyrosine kinase; TM - transmembrane (domain of membrane protein); DPC — dodecylphosphocholine; DPG — dodecylphosphoglycerol; SDS - sodium dodecyl sulfate; TFE — 2,2,2-trifluoroethanol.
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O.V. Koliasnikov, V.G. Grigorenko, A.M. Egorov, S. Lange, R.D. Schmid

Recombinant Production of Horseradish Peroxidase Conjugates with Fab Antibodies in Pichia pastoris for Analytical Applications

O.V. Koliasnikov^1,2#, V.G. Grigorenko^2*#, A.M. Egorov², S. Lange³, R.D. Schmid³
¹Kolmogorov Advanced Education and Science Center, Lomonosov Moscow State University
²Department of Chemistry, Lomonosov Moscow State University
³Institute of Technical Biochemistry, University of Stuttgart
*Е-mail: vitaly@immunotek.ru
#The authors provided equivalent contributions to the study.
Received 05.05.2011

ABSTRACT
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 pPICZ?B 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.

KEYWORDS horseradish peroxidase; antibodies; recombinant conjugates; Pichia pastoris expression.

ABBREVIATIONS HRP – horseradish peroxidase; ELISA – enzyme-linked immunosorbent assay; BSA – bovine serum albumin; PCR – polymerase chain reaction; TMB – 3,3’,5,5’-tetramethylbenzidine; ABTS – 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid).
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Yu.M. Efremov, E.V. Dzyubenko, D.V. Bagrov, G.V. Maksimov, S.I. Shram, K.V. Shaitan

Atomic Force Microscopy Study of the Arrangement and Mechanical Properties of Astrocytic Cytoskeleton in Growth Medium

Yu.M. Efremov^1*, E.V. Dzyubenko^1,2, D.V. Bagrov¹, G.V. Maksimov¹, S.I. Shram², K.V. Shaitan¹
1Biological Department, Lomonosov Moscow State University
2Institute of Molecular Genetics, Russian Academy of Sciences
*E-mail: yu.efremov@gmail.com
Received 23.05.2011

ABSTRACT 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 ?-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.

KEYWORDS atomic force microscopy; dorsal root ganglia; force spectroscopy; confocal microscopy; cytoskeleton.

ABBREVIATIONS AFM –atomic force microscopy; DRG – dorsal root ganglia; GFAP – glial fibrillary acidic protein.
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Logunov, D. V. Shchebliakov, M. M. Shmarov, E. E. Khodunova, I. V. Galtseva, R. V. Belousova, B.S. Naroditsky, A. L. Gintsburg

An Efficient Method for the Delivery of the Interleukin-2 Gene to Human Hematopoietic Cells using the Fiber-Modified Recombinant Adenovirus

V. N. Rogozhin^1,2*, D. Yu. Logunov¹, D. V. Shchebliakov¹, M. M. Shmarov¹, E. E. Khodunova³, I. V. Galtseva³, R. V. Belousova², B. S. Naroditsky¹, A. L. Gintsburg¹
¹Gamaleya Research Institute of Epidemiology and Microbiology, Ministry of Health and Social Development of the Russian Federation
²Moscow state academy of veterinary medicine and biotechnology named K.I. Skryabin
³National Research Center for Hematology, Ministry of Health and Social Development of the Russian Federation
*E-mail: Rogojin_V@mail.ru
Received 01.07.2011

ABSTRACT 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.

ABBREVIATIONS Ad – human adenovirus; Ad5 – Ad serotype 5; Ad35 – Ad serotype 35; Ad5/35F – fiber-modified recombinant Ad5; UV – ultraviolet radiation; RBM – red bone marrow; IL2 – human interleukin-2; CAR – coxsackievirus and adenovirus receptor; aa – amino acid residue; pfu – plaque-forming unit.
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S. P. Korolev, Yu. Yu. Agapkina, M.B. Gottikh

Clinical Use of Inhibitors of HIV-1 Integration: Problems and Prospects

S. P. Korolev^1*, Yu. Yu. Agapkina¹, M.B. Gottikh^1,2
¹Department of Chemistry, Lomonosov Moscow State University
²Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University
*E-mail: spkorolev@mail.ru
Received 04.04.2011

ABSTRACT 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.

KEYWORDS HIV-1 integrase; inhibition; mechanism of action; raltegravir.

ABBREVIATIONS HAART – highly active antiretroviral therapy; HIV-1 – human immunodeficiency virus type 1; IN – integrase; ST – strand transfer; AIDS – acquired immunodeficiency syndrome; AUC – area under the pharmacokinetic curve (concentration–time curve) – the change in concentration of the active component in blood plasma or serum over time; СIC₅₀ – the inhibitor concentration at which the cytopathogenic effect of the virus in the infected cells decreases by 50%; CIC₉₅ – the inhibitor concentration at which the cytopathogenic effect of the virus in the infected cells decreases by 95%; Сl_p – clearance, or the extraction ratio – the index showing the rate of extraction of a substance from blood plasma during the biotransformation of this substance, its redistribution in the organism, and excretion; Cmax – the maximum or peak of concentration of an active component in blood; EC₅₀ – inhibitor concentration, at which in vivo replication of the virus is suppressed by 50%; EC₉₀ – inhibitor concentration, at which in vivo replication of the virus is suppressed by 90%; F – bioavailability – the fraction of a dose of unchanged drug administered orally that reaches the systemic circulation; FBS – fetal bovine serum; FDA – Federal Drug Administration (United States); IC₅₀ – inhibitor concentration, at which the enzyme activity is suppressed by 50%; NHS – normal human serum; PPB – percentage plasma protein binding; Т_1/2 – time by which the concentration of a drug in plasma decreases twice; WT – wild type.
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I. A. Akimov, E. L. Chernolovskaya, Yu. E. Spitsyna, E. I. Ryabchikova, and M. A. Zenkova

Silencing of Her2, CCNB1 and PKC Genes by siRNA Results in Prolonged Retardation of Neuroblastoma Cell Division

I. A. Akimov, E. L. Chernolovskaya*, Yu. E. Spitsyna, E. I. Ryabchikova, M. A. Zenkova
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
*E-mail: elena_ch@niboch.nsc.ru
Received 15.04.2011

ABSTRACT 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, SKN-MC, MCF-7 cells), and PKC (in MCF-7 cells) was achieved 72 h after transfection of the corresponding siRNAs, 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 PKС genes. The obtained data indicate that the CCNB1 and PKC genes can be used as targets in the development of drugs for neuroblastoma treatment.

KEYWORDS neuroblastoma; siRNA; Her2; CCNB1; PKC; proliferation.

ABBREVIATIONS C – 2’-O-methylcytosine; U – 2’-O-methyluridine; siRNA – small interfering RNA; МТТ – 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PBS – phosphate buffered saline; FBS – fetal bovine serum.
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L. U. Dzhemileva, O. L. Posukh, N. A. Barashkov, S. A. Fedorova, F. M. Teryutin, V. L. Akhmetova, I. M. Khidiyatova, R. I. Khusainova, S. L. Lobov, E. K. Khusnutdinova

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

L. U. Dzhemileva^1*, O. L. Posukh², N. A. Barashkov³, S. A. Fedorova³, F. M. Teryutin³, V. L. Akhmetova¹, I. M. Khidiyatova¹, R. I. Khusainova¹, S. L. Lobov¹, E. K. Khusnutdinova¹
¹Institute of Biochemistry and Genetics, Ufa Research Center, Russian Academy of Sciences
²Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences
³Yakut Research Center of Complex Medical Problems, Siberian Branch, Russian Academy of Medical Sciences
*E-mail: dzhemilev@anrb.ru
Received 23.03.2011

ABSTRACT
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.

KEYWORDS hereditary nonsyndromic sensorineural hearing loss; GJB2 (Cx26) gene; c.35delG mutation; ancestral haplotype; populations of the Volga-Ural region.

ABBREVIATIONS
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Acta Naturae, 2011. V. 3.
№ 4 (11).

A. S. Davydova, M. A. Vorobjeva, A. G. Venyaminova

Escort Aptamers: New Tools for the Targeted Delivery of Therapeutics into Cells

A. S. Davydova, M. A. Vorobjeva*, A. G. Venyaminova
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
*E-mail: maria.vorobjeva@gmail.com
Received 08.07.2011

ABSTRACT Escort aptamers are DNA or RNA sequences with high affinity to certain cell-surface proteins, which can be used for targeted delivery of various agents into cells of a definite type. The peculiarities of the selection of escort aptamers are discussed in this review. The methods used in selection of escort aptamers via the SELEX technique are considered, including selection against isolated cell-surface proteins, cell fragments, living eukaryotic cells, and bacteria. Particular attention is given to the design and chemical modification of escort aptamers. The different fields of application of escort aptamers are described, including the targeted delivery of siRNAs, nanoparticles, toxins, and photoagents, as well as the identification of specific cell markers and the detection or isolation of cells of a definite type. The potential for the application of escort aptamers in the development of new therapeutic agents and diagnostic systems is also discussed.

KEYWORDS SELEX method; NA aptamers; escort aptamers; specific cell binding; addressed cell delivery; detection of cells.

ABBREVIATIONS dsDNA – double-stranded DNA; ssDNA – single-stranded DNA; siRNA – small interfering RNA; PSMA – prostate-specific membrane antigen; IC₅₀ – therapeutical concentration required to suppress the growth of 50% cells; K_d– apparent constant of dissociation of the aptamer–target complex; LNA – locked nucleic acids; SELEX – Systematic Evolution of Ligands by EXponential Enrichment.
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N. I. Kalinina, V. Yu. Sysoeva, K. A. Rubina, Ye. V. Parfenova, V. A. Tkachuk

Mesenchymal Stem Cells in Tissue Growth and Repair

N. I. Kalinina*, V. Yu. Sysoeva, K. A. Rubina, Ye. V. Parfenova, V. A. Tkachuk
Department of Fundamental Medicine, Lomonosov Moscow State University
*E-mail: n_i_kalinina@mail.ru
Received 14.07.2011

ABSTRACT
It has been established in the recent several decades that stem cells play a crucial role in tissue renewal and regeneration. Mesenchymal stem cells (MSCs) are part of the most important population of adult stem cells. These cells have hereby been identified for the very first time and subsequently isolated from bone marrow stroma. Bone marrow-derived MSCs have been believed to play the role of a source of cells for the renewal and repair of connective tissues, including bone, cartilage and adipose tissues. Cells similar to bone marrow-derived MSCs have now been identified in all postnatal tissues. Data on the distribution and function of MSCs in vivo collected using novel approaches pertaining to the identification of MSCs in situ, to their isolation from tissues, and finally to the determination of their biological properties have enabled successful revision of the role of MSCs in various organs and tissues. This review summarizes our own, as well as others’, data concerning the role of MSCs in the regulation processes of tissue repair and regeneration. In our opinion, MSCs provide the connection between the blood-vascular, immune, endocrine, and nervous systems and tissue-specific stem cells in the body.

KEYWORDS mesenchymal stem cells; tissue regeneration; differentiation; cell therapy.

ABBREVIATIONS HSC – hematopoietic stem cell; MSC – mesenchymal stem cell; MMSC –multipotent mesenchymal stromal cell; TSC – tissue-specific stem cell.
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A.A. Alekseeva, S.S. Savin, V.I. Tishkov

NAD⁺-dependent Formate Dehydrogenase from Plants

A.A. Alekseeva^1,2,3, S.S. Savin^2,3, V.I. Tishkov^,2,3*1
¹Chemistry Department, Lomonosov Moscow State University
²Innovations and High Technologies MSU Ltd
³Bach Institute of Biochemistry, Russian Academy of Sciences
*E-mail: vitishkov@gmail.com
Received 05.08.2011

ABSTRACT
NADsup>+--dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases.

KEYWORDS plant formate dehydrogenase; physiological role; properties; structure; expression; Escherichia coli; protein engineering.

ABBREVIATIONS FDH – formate dehydrogenase; PseFDH, CboFDH – formate dehydrogenases from bacteria Pseudomonas sp. 101 and yeast Candida boidinii, respectively; SoyFDH, AthFDH – plant formate dehydrogenases from soybean Glycine max and Arabidopsis thaliana, respectively.
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S.A. Sergeev, Y.V. Khramova, M.L Semenova, I.N. Saburina, N.V. Kosheleva

Behavior of Transplanted Multipotent Cells after in Vitro Transplantation into the Damaged Retina

S.A. Sergeev^1*, Y.V. Khramova¹, M.L Semenova¹, I.N. Saburina², N.V. Kosheleva^1,2
¹Biology Faculty, Lomonosov Moscow State University
²Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences
*E-mail: embryossa@gmail.com
Received 05.08.2011

ABSTRACT The use of stem cell technologies in retinal defect reparation therapy has produced beneficial results. Nowadays, numerous protocols exist which provide a neural differentiation of the stem cells transplanted into the retina. However, questions concerning the functional replacement of the missing retinal neurons by transplanted cells thus far remain unanswered. The organotypic culture protocol was used in this study in order to prove the possibility of transdifferentiation of bone marrow stromal cells (MMSCs) and neural stem/progenitor cells (NSPCs) from EGFP-positive mice and the functional integration of these cells. This technique enables a detailed characterization of cell behavior post-transplantation. Using atomic force microscopy, we reliably demonstrated the difference (p < 0.01) between the thickness of the outgrowths formed by glial and endothelial retina cells and the thickness of neurites and neuro-like transplanted MMSC outgrowths. MMSCs are also shown to form synapses up to 2.5 ± 0.06 µm in diameter on day 4 after the transplantation. Following electrical stimulation (20V, 0.5Hz, 200ms), clear depolarization of retinal neurons and their outgrowths is detected. It is shown that some of these GFP+ MMSCs, which changed their morphology after the transplantation in retinal explants to neuro-like MMSCs, are capable of depolarizing after exogenous stimulation.

KEYWORDS stem cells; in vitro transplantation; retinal organotypic cultures; stem cell plasticity; retinal reparation.

ABBREVIATIONS MMSC – bone marrow-derived mesenchymal stem cells; NSPC – neural stem/progenitor cells; EGFP – enhanced green fluorescent protein.
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T.A. Shmigol, V.A. Bekhalo, Е.V. Sysolyatina, E.V. Nagurskaya, S.A. Ermolaeva, A.Ya. Potapenko

Effect of Sodium Chloride on Aggregation of Merocyanine 540 and Photosensitized Inactivation of Staphylococcus aureus and Pseudomonas aeruginosa

T.A. Shmigol¹, V.A. Bekhalo^2*, Е.V. Sysolyatina², E.V. Nagurskaya², S.A. Ermolaeva², A.Ya. Potapenko¹
¹Pirogov Russian National Research Medical University
²Gamaleya Research Institute of Epidemiology and Microbiology, Ministry of Health and Social
Development of the Russian Federation
*E-mail: bekhalo@gamaleya.org
Received 07.07.2011

ABSTRACT
Merocyanine 540 (MC540) is used as a photosensitizer for the inactivation of microorganisms. The following is already known about MC540: firstly, MC540 exists in distilled water in both monomeric and dimeric forms, and the addition of salts into a MC540 solution leads to the formation of large aggregates that can be detected by the resonance light scattering technique. Secondly, singlet oxygen can only be photogenerated by MC540 monomers. In the present work, we studied the effect of MC540 in the aggregated state on the rate of photosensitized inactivation of Staphylococcus aureus and Pseudomonas aeruginosa. To this end, bacteria either in MC540-containing distilled water or in a 0.25 M sodium chloride aqueous solution also containing MC540 are irradiated (546 nm). The results show that, in the presence of salt, the aggregation of MC540 greatly increases the efficiency of the MC540-photosensitized inactivation of P. aeruginosa and S. aureus. In the presence of salt, the rates of P. aeruginosa and S. aureus inactivation increase by factors of 10 and 30, respectively, in comparison with the rate of inactivation observed in the case of distilled water. Our results suggest that a salt-induced photosensitization mechanism can switch from the singlet oxygen to the free-radical pathway.

KEYWORDS antimicrobial photodynamic therapy; merocyanine 540; Staphylococcus aureus; Pseudomonas aeruginosa.

ABBREVIATIONS CAC – critical aggregation concentration; MC540 – merocyanine 540; RLS – resonance light scattering; PS – photosensitizer; CFU – colony-forming unit.
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Y.P. Rubtsov, Y.G. Suzdaltseva, K.V. Goryunov, N.I. Kalinina, V. Y. Sysoeva, V. A. Tkachuk

Regulation of Immunity via Multipotent Mesenchymal Stromal Cells

Y. P. Rubtsov^1*, Y. G. Suzdaltseva², K. V. Goryunov¹, N. I. Kalinina¹, V. Y. Sysoeva¹, V. A. Tkachuk¹
¹Faculty of Fundamental Medicine, Lomonosov Moscow State University
²Institute of Experimental Cardiology
*E-mail: yrubtsov@gmail.com
Received 15.11.2011

ABSTRACT
Immune cells responsible for inflammation development are involved in tissue damage caused by wounding and various pathologies. Control of immune cell activation could be of significant benefit for regenerative medicine and the treatment of patients with autoimmune and degenerative diseases. It is a proven fact that MCSs (multipotent mesenchymal stromal cells) are capable of suppressing immune responses via the inhibition of dendritic cell maturation and via the restraining of the T, B, and NK cell function in the course of autoimmune diseases and various forms of inflammation. MSCs can be isolated easily from almost every type of tissue or organ and subsequently expanded in vitro. These cells are self-renewable and can be differentiated into various cell types of mesenchymal lineage. The current review contains a collection and critical analysis of data regarding the molecular mechanisms responsible for cross-talk between immune cells and MSCs. Some of these mechanisms can be used for the development of new practical approaches for the treatment of autoimmune diseases.

KEYWORDS immune system; multipotent mesenchymal stromal cells; inflammation; autoimmune disease; regeneration; immune suppression.

ABBREVIATIONS MSCs – multipotent mesenchymal stromal cells; CD – cluster of differentiation; SDF-1 – stem cell-derived factor-1; CXCR4 C-X-C chemokine receptor 4; VEGF – vascular endothelial growth factor; IGF-1 – insulin-like growth factor-1; BDNF – brain-derived neurotrophic factor; TGF-β – tumour growth factor; BMP– bone morphogenetic protein; IL-10 – interleukin-10; TNF-α– tumour necrosis factor; NK – natural killers; DC – dendritic cells; IFN-γ – interferon gamma; MHC – major histocompatibility complex; IDO – indoleamine-2,3-dioxygenase; PGE2 – prostaglandin E2; ICAM – intercellular adhesion molecule; VCAM – vascular cell adhesion protein; IL-1β – interleukin-1 beta; GVHD – graft versus host disease; EAE – experimental autoimmune encephalomyelitis; TLR – Toll-like receptor; HLA-G5 – non-classical molecule of histocompatibility complex class I antigen, G5 isoform.
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M.M. Prokofjeva, P.V. Spirin, D.V. Yanvarev, A.V. Ivanov, M.S. Novikov, O.A. Stepanov, M.B. Gottikh, S.N. Kochetkov, B. Fehse, C. Stocking, V.S. Prassolov

Screening of Potential HIV-1 Inhibitors/Replication Blockers Using Secure Lentiviral in Vitro System

M.M. Prokofjeva¹, P.V. Spirin¹, D.V. Yanvarev¹, A.V. Ivanov¹, M.S. Novikov², O.A. Stepanov¹, M.B. Gottikh³, S.N. Kochetkov¹, B. Fehse⁴, C. Stocking⁵, V.S. Prassolov^1*
¹Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
²Volgograd State Medical University
³Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University
⁴Research Department Cell and Gene Therapy, Department for Stem Cell Transplantation University Medical Center Hamburg-Eppendorf
⁵Heinrich-Pette-Institute for Experimental Virology and Immunology
*E-mail: prassolov45@mail.ru
Received 08.07.2011

ABSTRACT
The development and usage of safe cell systems for testing agents which possess anti-HIV activity is a very important factor in the design of new drugs. We have described in detail a system we designed that is based on lentiviral vectors (Prokofjeva et. al., Antiviral Therapy, in print) for swift and completely safe screening of potential HIV-1 replication inhibitors. The system enables one to test the efficiency of the inhibitory activity of compounds whose action is directed towards either wild-type HIV-1 reverse transcriptase or integrase, or mutant enzymes corresponding to the drug-resistant virus form. Testing results of a number of already known drugs, which correlate well with published data as well as data on newly synthesized compounds, were obtained. Application of this system substantially broadens the possibilities of preclinical anti-HIV drugs testing.

KEYWORDS HIV; lentiviral vectors; pseudo-HIV-1 particles; nucleoside reverse transcriptase inhibitors; nonnucleoside reverse transcriptase inhibitors; integrase inhibitors.

ABBREVIATIONS
HIV – human immunodeficiency virus; RT – reverse transcriptase; VSV – vesicular stomatitis virus; eGFP – enhanced green fluorescent protein; AZT – 3-azido-3- deoxythymidine; IC₅₀ – half maximal (50%) inhibitory concentration (IC) of a substance.

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L. G. Tyulkina, E. V. Skurat, O.Yu. Frolova, T.V. Komarova, E. M. Karger, I.G. Atabekov

New Viral Vector for Superproduction of Epitopes of Vaccine Proteins in Plants

L. G. Tyulkina¹, E. V. Skurat¹, O. Yu. Frolova², T. V. Komarova², E. M. Karger², I. G. Atabekov^1*
¹Faculty of Biology, Lomonosov Moscow State University
²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University
*E-mail: atabekov@genebee.msu.su
Received 20.06.2011

ABSTRACT
The novel viral vectors PVX-CP AltMV and PVXdt-CP AltMV are superexpressors of the capsid protein (CP). These viral vectors were constructed on the basis of the potato virus X (PVX) genome and Alternanthera mosaic virus (AltMV) CP gene. The expression, based on the hybrid viral vectors, is genetically safe, since the systemic transport and formation of infective viral particles are blocked. CP AltMV can self-assemble into virus-like particles (VLPs) in the absence of genomic RNA. The vectors can be used for the presentation of foreign peptides (including epitopes of human pathogens) on the surface of the VLP. The N-terminal extracellular domain (M2e) of the influenza virus A M2 protein and its truncated variant (ΔM2e) were used as model heterologous peptides for the construction of the chimeric CP AltMV. Chimeric CP AltMV retains its ability to self-assemble into VLP. The epitopes of the M2 influenza virus protein were not eliminated during the process of accumulation, polymerization and purification of chimeric VLP AltMV, providing evidence of the stability of chimeric VLP with C-terminal heterologous epitopes. It appears that VLP produced by the vectors PVXCP AltMV and PVXdt-CP AltMV can be used in the field of biotechnology for the presentation of the epitopes of vaccine proteins on their surfaces. The chimeric VLP AltMV with the presented foreign epitopes can be used as candidate vaccines.

KEYWORDS potexviruses; viral vector; foreign epitope; chimeric virus-like particles.

ABBREVIATIONS PVX – potato virus X; AltMV – Alternanthera mosaic virus; CP – capsid protein; dt – deletion of triple gene block; PVX-CP AltMV – hybrid viral vector based on PVX genome and CP AltMV; M2e – ectodomain of influenza A virus M2 protein; ΔM2e – truncated variant of M2e; CP-M2e AltMV, CP-ΔM2e AltMV – chimeric CP AltMV with epitope of M2 protein; VLP – virus-like particles; PCR – polymerase chain reaction; NTR – Nontranslated region.
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D.V. Shcheblyakov, D.Y. Logunov, I.V. Rakovskaya, M.M. Shmarov, B.S. Naroditsky, A.L. Ginzburg

Triggering of Toll-like Receptor-2 in Mouse Myelomonocytic Leukaemia Cells WEHI-3B Leads to the Suppression of Apoptosis and Promotes Tumor Progression in Vivo

D.V. Shcheblyakov*, D.Y. Logunov, I.V. Rakovskaya, M.M. Shmarov, B.S. Naroditsky, A.L. Ginzburg
Gamaleya Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences
*E-mail: sdmitryv@yahoo.com
Received 26.08.2011

ABSTRACT
Toll-like receptors are the essential components of innate immunity. It is shown that TLRs play an essential role in the immune resistance of an organism to bacterial and viral infections. The binding of TLR to its own ligands results in the activation of several adapter molecules and kinases, inducing the activation of the main pro-inflammatory transcriptional factors, which in turn induce the activation of the main pro-inflammatory transcriptional factors. This activation results in the development of both the innate immune response triggered by the enhanced expression of a number of pro-inflammatory cytokines and antimicrobial peptides and that of the adaptive immune response, via the activation of dendritic cells and enhancement of antigen presentation, etc. The ability of TLR agonists to bolster the immune reaction makes them promising for use in the therapy of infectious diseases and in the chemotherapy of malignant neoformations. However, different TLR ligands may have either antitumor activity (lipopolysaccharide, imiquimod, CpG) or, conversely, could beef up the resistance of tumor cells to apoptosis, stimulating their proliferation under certain conditions (lipopolysaccharide, lipopeptide). It has been shown that the TLR2-dependent signalling pathway in the myelomonocytic mouse leukaemia cell line WEHI-3B leads to the constitutive activation of the transcriptional factor NF-kB, suppression of apoptosis in tumor cells, and progression of myelomonocytic mouse leukaemia in vivo, upon the addition of TLR2 agonist (synthetic lipopeptide Pam2CSK4) or following the infection of tumor cells with Mycoplasma arginini.

KEYWORDS Toll-like receptor 2; synthetic diacylated lipopeptide Pam2CSK4; mouse myelomonocytic leukaemia cells WEHI-3B; transcription factor NF-kB; apoptosis; tumor progression.

ABBREVIATIONS TLR2 – Toll-like receptor 2; NF-kB – nuclear transcription factor-kB; PAMP – pathogen-associated molecular pattern; DAMP – damage-associated molecular pattern; ssRNA – single-stranded ribonucleic acid; RT-PCR – reverse transcription polymerase chain reaction; TNF-α – tumor necrosis factor α; IL – interleukin; MCP1– monocyte chemoattractant protein 1.
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V.L. Tunitskaya, A.R. Khomutov, S.N. Kochetkov, S.K. Kotovskaya, V.N. Charushin

Inhibition of DNA Gyrase by Levofloxacin and Related Fluorine-Containing Heterocyclic Compounds

V.L. Tunitskaya^1*, A.R. Khomutov¹, S.N. Kochetkov¹, S.K. Kotovskaya^2,3, V.N. Charushin^2,3
¹Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
²Postovsky Institute of Organic Synthesis, Ural Branch, Russian Academy of Sciences
³1Urals Federal University, Yekaterinburg, Russia
*E-mail: ve_tun@mail.ru
Received 28.09.2011

ABSTRACT Fluoroquinolones are an important class of modern and efficient antibacterial drugs with a broad spectrum of activity. Levofloxacin (the optically active form of ofloxacin) is one of the most promising fluoroquinolone drugs, and its antibacterial activity is substantially higher than the activity of other drugs of the fluoroquinolone family. Earlier, in the Postovsky Institute of Organic Synthesis, UB RAS, an original method of levofloxacin synthesis was developed, and now the pilot batch of the drug is being prepared. Bacterial DNA gyrase is a specific target of fluoroquinolones; hence, the study of the enzyme-drug interaction is of theoretical and practical importance. Moreover, the parameters of DNA gyrase inhibition may serve as a criterion for drug quality. Here, we present the results of studying the interaction of DNA gyrase with a number of fluoroquinolones and their analogs: intermediates and semi-products of the levofloxacin synthesis, and also samples from the pilot batches of this drug. The importance of two structural elements of the levofloxacin molecule for the efficiency of the inhibition is revealed. The data obtained may be useful for the design of new drugs derived from levofloxacin.

KEYWORDS fluoroquinolones; levofloxacin; derivatives; bacterial DNA gyrase; enzymatic activity; inhibition.

ABBREVIATIONS
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K.V. Shishova, О.О. Zharskaya, О.V. Zatsepina

The Fate of the Nucleolus during Mitosis: Comparative Analysis of Localization of Some Forms of Pre-rRNA by Fluorescent in Situ Hybridization in NIH/3T3 Mouse Fibroblasts

K.V. Shishova, О.О. Zharskaya, О.V. Zatsepina
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
*E-mail: kseniya.shishova@inbox.ru
Received 10.06.2011

ABSTRACT
Nucleolus is the major structural domain of the cell nucleus, which in addition to proteins contains ribosomal RNA (rRNA) at different stages of maturation (or pre-rRNA). In mammals, the onset of mitosis is accompanied by the inhibition of rRNA synthesis, nucleolus disassembly, and the migration of pre-rRNA to the cytoplasm. However, the precise role of cytoplasmic pre-rRNA in mitosis remains unclear, and no comparative analysis of its different forms at consequent mitotic stages has thus far been performed. The focus of this research was the study of the localization of pre-rRNA in mitotic NIH/3T3 mouse fibroblasts by fluorescent in situ hybridization (FISH) with probes to several regions of mouse primary 47S pre-rRNA transcripts and by confocal laser microscopy. The results reveal that all types of pre-rRNA appear in the cytoplasm at the beginning of mitosis, following the breakdown of the nucleolus and nuclear envelope. However, not all pre-rRNA are transported by chromosomes from maternal cells into daughter cells. At the end of mitosis, all types of pre-rRNA and 28S rRNA can be visualized in nucleolus-derived foci (NDF), structures containing many proteins of mature nucleoli the appearance of which indicates the commencement of nucleologenesis. However, early NDF are enriched in less processed pre-RNA, whereas late NDF contain predominantly 28S rRNA. Altogether, the results of this study strengthen the hypotheses that postulate that different forms of pre-rRNA may play various roles in mitosis, and that NDF can be involved in the maturation of pre-rRNA, remaining preserved in the cytoplasm of dividing cells.

KEYWORDS nucleolus; mitosis; nucleolus-derived foci (NDF); NIH/3Т3 mouse fibroblasts; fluorescence in situ hybridization (FISH).

ABBREVIATIONS rRNA – ribosomal RNA; 47S pre-rRNA - 47S precursor of rRNA; NDF – nucleolus-derived foci; PNB – prenucleolar bodies; 5’ETS, 3’ETS – 5’- and 3’-external transcribed spacers; ITS1, ITS2 - internal transcribed spacer 1 and 2; bp – base pairs; snoRNA – small nucleolar RNA; DAPI – 4’,6-diamidino-2-phenylindole; FISH –fluorescent in situ hybridization.
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I.E. Deyev, D.I. Rzhevsky, A.A. Berchatova, O.V. Serova, N.V. Popova, A.N. Murashev, A.G. Petrenko

Deficient Response to Experimentally Induced Alkalosis in Mice with the Inactivated insrr Gene

I.E. Deyev¹, D.I. Rzhevsky², A.A. Berchatova¹, O.V. Serova¹, N.V. Popova¹, A.N. Murashev², A.G. Petrenko^1*
¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
²Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Pushchino, Russian
Academy of Sciences
*E-mail: petrenkoag@gmail.com
Received 12.05.2011

ABSTRACT
Currently, the molecular mechanisms of the acid-base equilibrium maintenance in the body remain poorly understood. The development of alkalosis under various pathological conditions poses an immediate threat to human life. Understanding the physiological mechanisms of alkalosis compensation may stimulate the development of new therapeutic approaches and new drugs for treatment. It was previously shown that the orphan insulin receptor-related receptor (IRR) is activated by mildly alkaline media. In this study, we analyzed mutant mice with targeted inactivation of the insrr gene encoding IRR, and revealed their phenotype related to disorders of the acid-base equilibrium. Higher concentrations of bicarbonate and CO₂ were found in the blood of insrr knockout mice in response to metabolic alkalosis.

KEYWORDS alkalosis; IRR.

ABBREVIATIONS
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Acta Naturae, 2012. V. 4.
№ 1 (12).

G. A. Stepanov, D. V. Semenov, E. V. Kuligina, O. A. Koval, I. V. Rabinov, Y. Y. Kit, V. A. Richter

Analogues of Artificial Human Box C/D Small Nucleolar RNA As Regulators of Alternative Splicing of a pre-mRNA Target

G. A. Stepanov^1*, D. V. Semenov¹, E. V. Kuligina¹, O. A. Koval¹, I. V. Rabinov¹, Y.Y. Kit², V.A. Richter¹
¹Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
²Institute of Cell Biology, National Academy of Sciences of Ukraine
*E-mail: stepanovga@niboch.nsc.ru
Received 11.11.2011

ABSTRACT Small nucleolar RNAs (snoRNAs) play a key role in ribosomal RNA (rRNA) biogenesis. Box C/D snoRNAs guide the site-specific 2’-O-ribose methylation of nucleotides in rRNAs and small nuclear RNAs (snRNAs). A number of box C/D snoRNAs and their fragments have recently been reported to regulate posttranscriptional modifications and the alternative splicing of pre-mRNA. Artificial analogues of U24 snoRNAs directed to nucleotides in 28S and 18S rRNAs, as well as pre-mRNAs and mature mRNAs of human heat shock cognate protein (hsc70), were designed and synthesized in this study. It was found that after the transfection of MCF-7 human cells with artificial box C/D RNAs in complex with lipofectamine, snoRNA analogues penetrated into cells and accumulated in the cytoplasm and nucleus. It was demonstrated that the transfection of cultured human cells with artificial box C/D snoRNA targeted to pre-mRNAs induce partial splicing impairments. It was found that transfection with artificial snoRNAs directed to 18S and 28S rRNA nucleotides, significant for ribosome functioning, induce a decrease in MCF-7 cell viability.

KEYWORDS small nucleolar box C/D RNAs; post-transcriptional RNA modification; alternative splicing of premRNA.

ABBREVIATIONS rRNAs – ribosomal RNAs; snoRNAs – small nucleolar RNAs; snRNAs – small nuclear RNAs; RT-PCR – reverse transcription polymerase chain reaction; RP-HPLC – reverse phase high-performance liquid chromatography; MTT – 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FAM – carboxyfluorescein.
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E.A. Trifonova, E.R. Eremina, F.D. Urnov, V.A. Stepanov

The Genetic Diversity and Structure of Linkage Disequilibrium of the MTHFR Gene in Populations of Northern Eurasia

E.A. Trifonova¹, E.R. Eremina², F.D. Urnov³, V.A. Stepanov^1*
¹Research Institute of Medical Genetics, Siberian Branch, Russian Academy of Medical Sciences
²Buryat State University
³University of California, Berkley, USA
*E-mail: vadim.stepanov@medgenetics.ru
Received 13.12.2011

ABSTRACT
The structure of the haplotypes and linkage disequilibrium (LD) of the methylenetetrahydrofolate reductase gene (MTHFR) in 9 population groups from Northern Eurasia and populations of the international HapMap project was investigated in the present study. The data suggest that the architecture of LD in the human genome is largely determined by the evolutionary history of populations; however, the results of phylogenetic and haplotype analyses seems to suggest that in fact there may be a common “old” mechanism for the formation of certain patterns of LD. Variability in the structure of LD and the level of diversity of MTHFR haplotypes cause a certain set of tagSNPs with an established prognostic significance for each population. In our opinion, the results obtained in the present study are of considerable interest for understanding multiple genetic phenomena: namely, the association of interpopulation differences in the patterns of LD with structures possessing a genetic susceptibility to complex diseases, and the functional significance of the pleiotropic MTHFR gene effect. Summarizing the results of this study, a conclusion can be made that the genetic variability analysis with emphasis on the structure of LD in human populations is a powerful tool that can make a significant contribution to such areas of biomedical science as human evolutionary biology, functional genomics, genetics of complex diseases, and pharmacogenomics.

KEYWORDS genome; linkage disequilibrium; populations of Northern Eurasia; methylenetetrahydrofolate reductase; haplotype.

ABBREVIATIONS CD – complex diseases; LD – linkage disequilibrium; MTHFR – methylenetetrahydrofolate reductase; SNP – single nucleotide polymorphism; HC – homocysteine.
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E. M. Smekalova, O. A. Petrova, M. I. Zvereva, O. A. Dontsova

Hansenula Polymorpha TERT: A Telomerase Catalytic Subunit Isolated in Recombinant Form with Limited Reverse Transcriptase Activity

E. M. Smekalova*, O. A. Petrova, M. I. Zvereva, O. A. Dontsova
Chemistry Department, Lomonosov Moscow State University
*E-mail: esmekalova@yahoo.com
Received 09.12.2011

ABSTRACT
Telomerase is a ribonucleoprotein, the main function of which is to synthesize telomeres, i.e. repetitive sequences which are localized at the ends of eukaryotic chromosomes. Telomerase maintains the stability of the genome in eukaryotic cells by replicating chromosomal ends. The structural and functional investigation of the telomerase complex is significantly restricted due to difficulties connected with the isolation of its main catalytic subunit in recombinant form. Herein, we describe a method developed for the isolation of the recombinant telomerase reverse transcriptase from thermotolerant yeast Hansenula polymorpha. A functional test performed for the isolated protein and the RNA/DNA duplex, simulating the interaction of telomerase RNA and telomere, reveals that the isolated catalytic subunit of telomerase possesses limited reverse transcriptase activity.

KEYWORDS telomerase reverse transcriptase; recombinant proteins; thermotolerant yeast Hansenula polymorpha.

ABBREVIATIONS
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O. V. Bondar, D. V. Saifullina, I. I. Shakhmaeva, I. I. Mavlyutova, T. I. Abdullin

Monitoring of the Zeta Potential of Human Cells upon Reduction in Their Viability and Interaction with Polymers

O. V. Bondar*, D. V. Saifullina, I. I. Shakhmaeva, I. I. Mavlyutova, T. I. Abdullin
Kazan (Volga Region) Federal University
*E-mail: oxanav.bondar@gmail.com
Received 08.01.2012

ABSTRACT The dynamic light scattering (DLS) technique was applied in order to assess the zeta potential of the plasma membrane of human cells. At pH 7.4, the cell zeta potential for different types of cells showed variations over a wide range and was equal to –19.4 ± 0.8 mV for HeLa cells and –31.8 ± 1.1 mV for erythrocytes. The difference could presumably be attributed to the differences in the biochemical composition of the cell plasma membrane. As a result of the heating of HeLa cells, the zeta potential shifted towards more negative voltages by 4.2 mV. An increase in the zeta potential correlated with an increase in the content of phosphatidylserine on the cell surface, which is considered to be an early marker of apoptosis. The DLS technique was also used to study the interactions between the cells and membranotropic polymers, such as polycations and nonionogenic Pluronic L121.

KEYWORDS dynamic light scattering; zeta potential; HeLa cells, MCF-7, mononuclear leukocytes, erythrocytes; apoptosis; phosphatidylserine; membranotropic polymers.

ABBREVIATIONS DLS – dynamic light scattering; PBS – phosphate-buffered saline.
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L.Z. Velsher, A.A. Kosmynin, M.Yu. Byakhov, T.K. Duditskaya, D.N. Reshetov

Targeted Therapy: A New Approach for the Treatment of Locally Advanced Oropharyngeal Cancer

L.Z. Velsher^1,2, A.A. Kosmynin^1*, M.Yu. Byakhov^1,2, T.K. Duditskaya^1,2, D.N. Reshetov^1,2
¹Moscow State Medical and Dental University
²Cancer Center JSC “RZD”, Moscow
*E-mail: kosmos-83@list.ru
Received 27.07.2011

ABSTRACT Presented herein is a clinical study comprising 48 patients (42 men and 6 women) of working age (40–70 years), all of whom are suffering from locally advanced oropharyngeal cancer. A modern approach is applied to treat these patients, i.e., neoadjuvant targeted therapy, taking into account the biological profile of the tumor. The use of gefitinib causes an antitumor effect in 90.5% of cases as opposed to 56.5% when no drug is applied.

KEYWORDS oropharyngeal cancers; targeted therapy; quality of life; gefitinib.

ABBREVIATIONS SCCHN – squamous-cell carcinoma of the head and neck; EGFR – epidermal growth factor receptor.
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E.S. Kolotova, S.G. Egorova, A.A. Ramonova, S.E. Bogorodski, V.K. Popov, I.I. Agapov, M.P. Kirpichnikov

Cytotoxic and Immunochemical Properties of Viscumin Encapsulated in Polylactide Microparticles

E. S. Kolotova^1*, S. G. Egorova¹, A. A. Ramonova¹, S. E. Bogorodski², V. K. Popov², I. I. Agapov^1,3, M. P. Kirpichnikov¹
¹Biological Faculty, Lomonosov Moscow State University
²Institute of Laser and Information Technologies, Russian Academy of Sciences
³Shumakov Federal Research Centre of Transplantology and Artificial Organs
*E-mail: ekaterinakolotova@mail.ru
Received 28.09.2011

ABSTRACT Biodegradable polylactide microparticles with encapsulated cytotoxic protein viscumin were obtained via the ultrasound-assisted supercritical fluid technique. The size of the microparticles was 10–50 µM, as shown by electron microscopy. The time course of viscumin release from microparticles was studied using an immunoenzyme test system with anti-viscumin monoclonal antibodies. It was found that 99.91% of the cytotoxic protein was incorporated into polymer microparticles. Only 0.08% of the initially encapsulated viscumin was released from the microparticles following incubation for 120 h in a phosphate-buffered saline at neutral pH. Importantly, the method of ultrasonic dry supercritical fluid encapsulation failed to alter both the cytotoxic potency and the immunochemical properties of the encapsulated viscumin. Thus, this procedure can be used to generate biodegradable polylactide microparticles with encapsulated bioactive substances.

KEYWORDS biodegradable microparticles; viscumin; polylactide.

ABBREVIATIONS MLI – viscumin; RIP – ribosome inactivating protein; SCF – supercritical fluid; sc-CO2 – supercritical carbon dioxide; PBS – phosphate buffered saline; TMB – tetramethylbenzidine; MSG – magnetostriction generator; SEM – scanning electron microscopy; BSA – bovine serum albumin; MTT – 3-(4,5-dimethylthiozolyl-2-yl)-2,5-diphenyltetrazolium bromide; LD50 – lethal dose for 50% of the cells.
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S.A. Borinskaya, Zh.M. Kozhekbaeva, A.V. Zalesov, E.V. Olseeva, A.R. Maksimov, S.I. Kutsev, M.M. Garaev, A.V. Rubanovich, N.K. Yankovsky

Risk of HIV Infection and Lethality Are Decreased in CCR5del32 Heterozygotes: Focus Nosocomial Infection Study and Meta-analysis

S.A.Borinskaya¹, Zh.M.Kozhekbaeva¹, A.V. Zalesov^1,2, E.V.Olseeva³, A.R.Maksimov⁴, S.I. Kutsev⁵, M.M. Garaev⁶, A.V. Rubanovich¹, N.K. Yankovsky^1,2,7
¹Vavilov Institute of General Genetics, Russian Academy of Sciences
²Moscow Institute of Physics and Technology
³Ministry of Health and Social Development of the Republic of Kalmykia
⁴Blood Centre of the Republic of Kalmykia
⁵Rostov State Medical University
⁶Ivanovsky Research Institute of Virology, Russian Academy of Medical Sciences
⁷Faculty of Biology, Lomonosov Moscow State University,
* E-mail: yankovsky@vigg.ru
Received 17.10.2011

ABSTRACT
CCR5del32 Homozygous deletion in the chemokine receptor R5 gene provides almost complete protection to individuals against HIV infection. However, data relating to the protective effect for CCR5del32 heterozygous individuals have been contradictory. The frequency of the CCR5del32 allele in population control cohorts was compared with that of a group of children (27 Kalmyks and 50 Russians) infected by G-subtype HIV-1 in a nosocomial outbreak. The frequency of the CCR5del32 allele was shown to be lower among the infected children in comparison with that of the control group; however, the difference was small and statistically insignificant. Similar results were obtained in a number of earlier studies. The insignificance of the small differences could be a result of one of two reasons. (i) The fact that there is no protective effect of the heterozygous state, and that the phenomenon depends only on the fluctuation of allele frequencies. In this case, there would be no differences even if the infected cohort is enlarged. (ii)The protective effect of the heterozygous state is real; however, the size of the studied cohort is insufficient to demonstrate it. In order to discern between these two reasons, a meta-analysis of data from 25 published articles (a total of 5,963 HIV-infected individuals and 5,048 individuals in the control group, including the authors’ own data) was undertaken. A conclusion was drawn from the meta-analysis that the CCR5del32 allele protects individuals against the HIV infection even in a heterozygous state (OR=1.22, 95%CI=1.10–1.36). The risk of HIV infection for CCR5 wt/del32 heterozygotes was lower by at least 13% as compared to that for wild type CCR5 wt/wt homozygotes. Prior to this study, no data of the type or any conclusions had been published for Caucasians. The mortality rate in the 15 years following the infection was found to be approximately 40% lower for CCR5del32 heterozygotes in comparison with that for the wild type homozygotes in the studied group. The size of the studied group was insufficient to claim difference validity (OR=2.0; p= 0.705), even though the effect quantitatively matched the published data. The features of the meta-analysis influencing the threshold level and the statistical validity of the effects are being discussed. The level of the CCR5del32 protective effect on the chances to be infected with HIV and on the outcome of the HIV infection was assessed for various ethnic groups.

KEYWORDS HIV; nosocomial infection; lethality risk; infection risk; chemokine receptor gene; allele CCR5del32; meta-analysis.

ABBREVIATIONS HIV – human immunodeficiency virus; AIDS – acquired immune deficiency syndrome; PCR – polymerase chain reaction.
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O. N. Solopova, L. P. Pozdnyakova, N. E. Varlamov, M. N. Bokov, E. V. Morozkina, Т. А. Yagudin, P. G. Sveshnikov

Conformational Differences between Active Angiotensins and Their Inactive Precursors

O. N. Solopova¹, L. P. Pozdnyakova¹, N. E. Varlamov¹, M. N. Bokov¹, E.V. Morozkina²,Т.А. Yagudin², P. G. Sveshnikov¹
¹Russian Research Center for Molecular Diagnostics and Therapy
²Bach Institute of Biochemistry, Russian Academy of Sciences
*E-mail: solopova@msn.com
Received 28.10.2011

ABSTRACT The peptide conformation in the context of a protein polypeptide chain is influenced by proximal amino acid residues. However, the mechanisms of this interference remain poorly understood. We studied the conformation of angiotensins 1, 2 and 3, which are produced naturally in a sequential fashion from a precursor protein angiotensinogen and contain an identical peptide core structure. Using the example of angiotensins 1, 2 and 3, it was shown that similar amino acid sequences may have significant conformational differences in various molecules. In order to assess the conformational changes, we developed a panel of high-affinity mouse monoclonal antibodies against angiotensins 1, 2 and 3 and studied their cross-reactivity in indirect and competitive ELISAs. It was found that the conformations of inactive angiotensin1 and the corresponding fragment of angiotensinogen are similar; the same is true for the conformations of active angiotensins 2 and 3, whereas the conformations of homologous fragments in the active and inactive angiotensins differ significantly.

KEYWORDS peptides; conformation; angiotensins; angiotensinogen; monoclonal antibodies.

ABBREVIATIONS Ang1 – human angiotensin 1; Ang2 – human angiotensin 2; Ang3 – human angiotensin 3; ELISA – enzyme-linked immunosorbent assay; PAG – polyacrylamide gel; Hsp70 – heat shock protein with a molecular mass of 70 kDa; Kd– dissociation constant.
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M.A. Volodina, E.A. Sebentsova, N.Yu. Glazova, D.M. Manchenko, L.S. Inozemtseva, O.V. Dolotov, L.A. Andreeva, N.G. Levitskaya, A.A. Kamensky, N.F. Myasoedov

Correction of Long-Lasting Negative Effects of Neonatal Isolation in White Rats Using Semax

M. A. Volodina², E. A. Sebentsova¹, N. Yu. Glazova¹, D. M. Manchenko², L. S. Inozemtseva¹, O.V. Dolotov¹, L. A. Andreeva¹, N. G. Levitskaya^1*, A. A. Kamensky², N. F. Myasoedov¹
¹Institute of Molecular Genetics, Russian Academy of Sciences
²Biological Faculty, Lomonosov Moscow State University
*E-mail: nglevitskaya@gmail.com
Received 26.01.2012

ABSTRACT
Adverse experience during the early postnatal period induces negative alterations in physiological and neurobiological functions, resulting in long-term disorder in animal behavior. The aim of the present work was to study the long-lasting effects of chronic neonatal stress in white rats and to estimate the possibility of their correction using Semax, an analogue of ACTH fragment (4–10). Early neonatal isolation was used as a model of early-life stress. Rat pups were separated from their mothers and littermates for 5 h daily during postnatal days 1–14. The pups of the control group were left undisturbed with the dams. Half of the rats subjected to neonatal isolation received an intranasal injection of Semax at a dose of 50 μg/kg daily, from postnatal day 15 until day 28. The other animals received intranasal vehicle injections daily at the same time points. It was shown that neonatal isolation leads to a delay in physical development, metabolic disturbances, and a decrease in the corticosterone stress response in white rats. These changes were observed during the first two months of life. Semax administration weakened the influence of neonatal isolation on the animals, body weight , reduced metabolic dysfunction, and led to an increase in stress-induced corticosterone release to the control values. So the chronic intranasal administration of Semax after termination of the neonatal isolation procedure diminishes the negative effects of neonatal stress.

KEYWORDS chronic stress; neonatal isolation; Semax; body weight; corticosterone; rat.

ABBREVIATIONS ACTH adrenocorticotropic hormone; MD – maternal deprivation; NI – neonatal isolation.
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N.A. Orlova, S.V. Kovnir, I.I. Vorobiev, A.S. Yuriev, A.G. Gabibov, A.I.Vorobiev

Stable Expression of Recombinant Factor VIII in CHO Cells Using MethotrexateDriven Transgene Amplification

N. A. Orlova^1,2#, S. V. Kovnir^1,2#, I. I. Vorobiev^1,2*, A.S. Yuriev², A.G. Gabibov¹, A.I.Vorobiev²
¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
²Hematology Research Centre Ministry of Healthcare and Social Development of the Russian Federation
^#Authors contributed equally to this work
*E-mail : ptichman@gmail.com
Received 30.12.2011

ABSTRACT Prophylaxis and treatment of inherited clotting disorder hemophilia A requires regular administration of factor VIII. Recombinant factor VIII, which is produced in CHO or BHK cells, is equivalent to the plasmaderived one and is prevalent in current clinical practice in developed countries. Development of a biosimilar recombinant FVIII requires the creation of a highly productive clonal cell line and generation of monoclonal antibodies suitable for affinity purification of the product. Methotrexate-driven transgene amplification of genetic cassettes that code full-length and truncated variants of FVIII under the control of the CMV promoter was studied. It was shown that the expression level of the truncated variant of FVIII is 6.5 times higher than that of the full-length molecule. The transgene amplification procedure was sufficient for a twofold increase of the expression level in the transfected cells pool and subsequent selection of the clonal line, stably producing truncated FVIII at the level of 0.52 IU/ml during cultivation in a chemically defined protein-free culture medium. Four generated mouse monoclonal antibodies toward the heavy chain of FVIII were found suitable for binding the truncated variant of FVIII directly from the conditioned medium and elution of the FVIII with a more than 85% yield and normal pro-coagulant activity. The producer cell line and monoclonal antibodies obtained are sufficient for the development of upstream and downstream processes of biosimilar FVIII production. Generation of more productive cell lines by the use of stronger, nonviral promoters and shorter cDNA of FVIII will be the subject of further studies.

KEYWORDS coagulation factor VIII; B-domain deleted factor VIII; hemophilia A; heterologous protein expression systems.

ABBREVIATIONS FVIII – blood clotting factor VIII; rhFVIII – recombinant human FVIII, BDD-FVIII – FVIII with deleted B-domain, MTX - methotrexate, EG - ethylene glycol, EMCV - Encephalomyocarditis virus, IRES - internal ribosome entry site, DHFR - dihydrofolate reductase (EC 1.5.1.3); IU – international unit (1 IU of FVIII corresponds to it’s content in 1 ml of pooled donor plasms), ORF – open reading frame, PBS – phosphate buffered solution.
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Acta Naturae. 2012. V. 4.
№ 2 (13)

D.G. Knorre

Physicochemical Biology: Conquered Boundaries and New Horizons

D.G. Knorre
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, prosp. Akademika Lavrent’eva, 8, 630090, Novosibirsk, Russia
E-mail: knorre@niboch.nsc.ru
Received 04.11.2011

ABSTRACT In this paper, we shall consider the main evolutionary stages that occurred within the field of physicochemical biology during the 20th century, following the determination of the tertiary structure of DNA by Watson and Crick and the subsequent successes in the X-ray structural analysis of biopolymers. The authors’ deas on the pre-emptive problems and the methods used in physicochemical biology in the 21st century are also presented, including an investigation of the dynamics of biochemical processes, studies of the functions of unstructured proteins, as well as single-molecule investigations of enzymatic processes and of biopolymer tertiary structure formation.

KEYWORDS DNA structure; enzyme active sites; unstructured proteins; dynamics of biochemical processes; single molecule studies of enzymatic processes and biopolymer tertiary structure formation.

ABBREVIATIONS
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M.P. Rubtsova, D.P. Vasilkova, А.N. Malyavko, Yu.V.Naraikina3, M.I. Zvereva, О.А. Dontsova

Telomere Lengthening and Other Functions of Telomerase

M.P. Rubtsova^1,2*, D.P. Vasilkova¹, А.N. Malyavko¹, Yu.V.Naraikina³, M.I. Zvereva^1,2, О.А. Dontsova^1,2
¹Lomonosov Moscow State University, Chemistry Department,
²Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University
³Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics
*E-mail: mprubtsova@gmail.com
Received 08.02.2012

ABSTRACT Telomerase is an enzyme that maintains the length of the telomere. The telomere length specifies the number of divisions a cell can undergo before it finally dies (i.e. the proliferative potential of cells). For example, telomerase is activated in embryonic cell lines and the telomere length is maintained at a constant level; therefore, these cells have an unlimited fission potential. Stem cells are characterized by a lower telomerase activity, which enables only partial compensation for the shortening of telomeres. Somatic cells are usually characterized by the absence of telomerase activity. Telomere shortening leads to the attainment of the Hayflick limit, the transition of cells to a state of senescence. The cells subsequently enter a state of crisis, accompanied by massive cell death. The surviving cells become cancer cells, which are capable both of dividing indefinitely and maintaining telomere length (usually with the aid of telomerase). Telomerase is a reverse transcriptase. It consists of two major components: telomerase RNA (TER) and reverse transcriptase (TERT). TER is a non-coding RNA, and it contains the region which serves as a template for telomere synthesis. An increasing number of articles focusing on the alternative functions of telomerase components have recently started appearing. The present review summarizes data on the structure, biogenesis, and functions of telomerase.

KEYWORDS telomerase; reverse transcriptase; telomeres; mitochondria; DNA damage; gene expression.

ABBREVIATIONS TER – telomerase RNA; hTR – human telomerase RNA; TERT – telomerase reverse transcriptase.
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N. A. Orlova, S. V. Kovnir, I. I. Vorobiev, A.G.Gabibov

Coagulation Factor IX for Hemophilia B Therapy

N. A. Orlova^1,2, S. V. Kovnir^1,2, I. I. Vorobiev^1,2*, A.G.Gabibov¹
¹Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
²Hematology Research Centre, Ministry of Healthcare and Social Development of the Russian Federation
*E-mail: ptichman@gmail.com
Received 28.12.2011

ABSTRACT Factor IX is a zymogen enzyme of the blood coagulation cascade. Inherited absence or deficit of the IX functional factor causes bleeding disorder hemophilia B, which requires constant protein replacement therapy. Reviewed herein are the current state in the manufacturing of FIX, improved variants of the recombinant protein for therapy, transgenic organisms for obtaining FIX, and the advances in the gene therapy of hemophilia B.

KEYWORDS сoagulation factor IX; hemophilia B; heterologous protein expression systems

ABBREVIATIONS FIX –Factor IX; rFIX – recombinant factor IX; rhFIX - recombinant human factor IX; FIXa - activated FIX; pdFIX - plasma-derived factor IX; PTM - posttranslational modification; VKD - vitamin K dependent; IU – international unit; EGF – epidermal growth factor
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R.M. Barsova, B.V. Titov, N.A. Matveeva, A.V. Favorov, T.S. Sukhinina, R.M. Shahnovich2, M.Ia. Ruda, O.O. Favorova

Contribution of the TGFB1 Gene to Myocardial Infarction Susceptibility

R.M. Barsova1,2, B.V. Titov1,2, N.A. Matveeva1,2, A.V. Favorov3,4, T.S. Sukhinina2, R.M. Shahnovich2, M.Ia. Ruda2, O.O. Favorova1,2
1Pirogov Russian National Research Medical University , Moscow
2Russian Cardiology Scientific and Production Center, Moscow
3Vavilov Institute of General Genetics, Russian Academy of Science, Moscow
4Oncology Biostatistics and Bioinformatics, Johns Hopkins School of Medicine, Baltimore, MD 21205, US
*E-mail: olga_favorova@mail.ru
Received 10.02.2012

ABSTRACT
Carriage frequencies of alleles and genotypes of the TGFB1 gene polymorphous loci –509C>T (rs1800469), 869T>C (rs1982073), 915G>C (rs1800471), which affect the level of cytokine TGF-β1 production, were analyzed in the patients of Russian ethnic descent with myocardial infarction (MI) (406 cases) and in the control group of the same ethnic descent (198 controls). Significant association with MI was observed in carriage frequencies of the allele TGFB1*–509T (p=0.046, OR =1.45, 95% CI: 1.02-2.06), genotypes TGFB1*869T/T (p=0.0024, OR =1.75, 95% CI: 1.22-2.51), and TGFB1*915G/G (p=0.048, OR=1.76, 95% CI: 1.05-2.97). Linkage disequilibrium analysis for these SNPs has shown that the associations revealed can be considered to be independent. A complex analysis of MI association with combinations of alleles/genotypes of said SNPs indicates their cumulative effect. An analysis of susceptibility to early-onset MI (? 50 years old) revealed a positive association of the allele TGFB1*–509T (p=0.002, OR=2.24, 95% CI: 1.35-3.71) and genotype TGFB1*869T/T (p=0.008, OR=1.93, 95% CI: 1.18-3.15), as well as their additivity. An analysis of susceptibility to recurrent MI revealed an association of the genotype TGFB1*–509T/T (p=0.0078, OR=2.60, 95% CI: 1.28-5.28). The results obtained indicate the important role of the TGFB1 gene in susceptibility to MI, including early-onset and recurrent MI, in Russians.

KEYWORDS myocardial infarction; Russians; genes; allelic polymorphism; transforming growth factor β1; TGFB1; APSampler.

ABBREVIATIONS dNTP – deoxynucleoside triphosphate; LD – linkage disequilibrium; SD – standard deviation; SNP – single nucleotide polymorphism; TGF-β1 – transforming growth factor beta 1; TGFB1 – gene encoding transforming growth factor beta 1; CI – confidence interval; IHD – ischemic heart disease; MI – myocardial infarction; OR – odds ratio; PCR – polymerase chain reaction; PCR-SSP – polymerase chain reaction with sequence-specific primers; CVD – cardiovascular diseases.
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I.G. Khaliullin, D.K. Nilov, I.V. Shapovalova, V.K. Svedas

Construction of a Full-Atomic Mechanistic Model of Human Apurinic/Apyrimidinic Endonuclease APE1 for Virtual Screening of Novel Inhibitors

I.G. Khaliullin¹, D.K. Nilov^1,2, I.V. Shapovalova², V.K. Svedas^1,2*
¹Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University
²Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics
*E-mail: vytas@belozersky.msu.ru
Received 18.04.2012

ABSTRACT A full-atomic molecular model of human apurinic/apyrimidinic endonuclease APE1, an important enzyme in the DNA repair system, has been constructed. The research consisted of hybrid quantum mechanics/molecular mechanics modeling of the enzyme-substrate interactions, as well as calculations of the ionization states of the amino acid residues of the active site of the enzyme. The choice of the APE1 mechanism with an Asp210 residue as a proton acceptor was validated by means of a generalization of modeling and experimental data. Interactions were revealed in the active site that are of greatest significance for binding the substrate and potential APE1 inhibitors (potential co-drugs of interest in the chemo- and radiotherapy of oncological diseases).

KEYWORDS apurinic/apyrimidinic endonuclease; QM/MM; enzymatic mechanism; molecular modeling; inhibition.

ABBREVIATIONS BER – base excision repair; AP – apurinic/apyrimidinic; APE1 – human apurinic/apyrimidinic endonuclease 1; QM/MM – quantum mechanics/molecular mechanics; MD – molecular dynamics.
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Yu. A. Morozov, L. M. Khromykh, I. I. Dementieva, M. A. Charnaia1, N. L. Kulikova, D.B. Kazansky

Recombinant Human Cyclophilin A in vitro Inhibits the Formation of Fibrin Clot

Yu. A. Morozov^1*, L. M. Khromykh², I. I. Dementieva¹, M. A. Charnaia¹, N. L. Kulikova³, D.B. Kazansky²
¹Petrovsky Russian Research Center of Surgery, Russian Academy of Medical Sciences
²Carcinogenesis Institute, Blokhin Cancer Research Center
³Institute of Immunological Engineering
*E-mail: moroz_111@rambler.ru
Receved 27.03.2012

ABSTRACT
The chemotactic properties of cyclophilin A are well-known. There exists however a poor level of understanding regarding the hemostatic effects of this protein. Herein it is shown that recombinant human cyclophilin A (rhСyA), in contrast to the granulocyte colony-stimulating factor, is capable of inhibiting in vitro the formation of a fibrin clot, thereby violating the spatial dynamics of clot growth; this effect is transient and dose-independent. Furthermore, the hypothesis that the conformational changes in the thrombin–rhCyA complex may mediate the anticoagulant effect of rhCyA on the autowave processes of blood clotting is postulated.

KEYWORDS recombinant human cyclophilin A; spatial dynamics of clot growth; anticoagulant activity.

ABBREVIATIONS rhCyA – recombinant human cyclophilin A; DCG – delay of clot growth, min; IGR – initial growth rate, min; SSGR – steady-state growth rate, min; CS-30 – clot size after 30-minute growth, μm; CD – the clot density; G-CSF – granulocyte colony-stimulating factor.
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N.A. Zigangirova, E.S. Zayakin, L.N. Kapotina, E.A. Kost, L.V. Didenko, D.Y. Davydova, J.P. Rumyanceva, A.L. Gintsburg

Development of Chlamydial Type III Secretion System Inhibitors for Suppression of Acute and Chronic Forms of Chlamydial Infection

N.A. Zigangirova, E.S. Zayakin*, L.N. Kapotina, E.A. Kost, L.V. Didenko, D.Y. Davydova, J.P. Rumyanceva, A.L. Gintsburg
Gamaleya Research Institute of Epidemiology and Microbiology
E-mail: e.s.zayakin@gmail.com
Received 17.01.2012

ABSTRACT The Type III secretion system (T3SS) is currently considered to be one of the main pathogenicity factors in Gram-negative bacteria, which exhibit different types of parasitizing activity. The presence of this structure is essential for the development of an acute infection; the chronicity of the infection is fundamentally dependent upon its functioning. In this regard, T3TS is one of the most promising targets for the development of broad-spectrum antimicrobial drugs that do not develop resistance and are efficacious for the acute and chronic forms of infection. The mechanism of action in drug development is based on the specific inhibition of T3SS, which should interrupt the infectious process, thereby enabling the immune system to eliminate the pathogen. As a result of pilot screening using specific cellular and bacterial tests, followed by chemical optimization and detailed characterization of the biological activity, a new class of chlamydial T3SS inhibitors was obtained. The selected compounds have obvious advantages over the currently available inhibitors of T3SS pathogens thanks to the high inhibitory activity of these compounds with minimal damaging effects on eukaryotic cells. Preclinical trials of the selected inhibitors are currently under way.

KEYWORDS thiohydrazones; thiohydrazides; thiadiazines; type III secretion system; citotoxicity; Chlamydia; inhibitors; microscopy; electron microscopy; morphology.

ABBREVIATIONS T3SS – type III secretion system; MOI – multiplicity of infection; МОМР – major outer membrane protein of Chlamydia; LPS – lipopolysaccharide; EB – elementary bodies; RB – reticular cells; IFU – inclusion forming units.
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Acta Naturae. 2012. V. 4. № 3 (14).

O. S. Petrakova¹, E. S. Chernioglo¹, V. V. Terskikh1, E. N. Kalistratova², A. V. Vasiliev¹^,2

The Use of Cellular Technologies in Treatment of Liver Pathologies

Koltzov Institute of Developmental Biology, Russian Academy of Sciences

ABSTRACT

Cell techniques find increasing application in modern clinical practice. The II and III phases of clinical trials are already under way for various cellular products used for the restoration of the functions of the cornea, larynx, skin, etc. However, the obtainment of functional cell types specific to different organs and tissues still remains a subject of laboratory research. Liver is one of the most important organs; the problems and prospects of cellular therapy for liver pathologies are currently being actively studied.

Cellular therapy of liver pathologies is a complex multistage process requiring a thorough understanding of the molecular mechanisms occurring

in liver cells during differentiation and regeneration. An analysis of the current cellular therapy for liver pathologies is presented, the use of various cell types is described, the main molecular mechanisms of hepatocyte differentiation are analyzed, and the challenges and prospects of cell therapy for liver disorders are discussed in this review.

KEYWORDS
cell transplantation; cellular therapy; differentiation; liver.

ABBREVIATIONS
ES cells – embryonic stem cells; iPS cells – induced pluripotent stem cells; HSC – haematopoietic stem cells, MSC – mesenchymal stem cells; SP cells – side population cells.

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S. N. Lomin, D.M. Krivosheev, M. Yu. Steklov, D. I. Osolodkin, G. A. Romanov

Receptor Properties and Features of Cytokinin Signaling

Receptor Properties and Features of Cytokinin Signaling

ABSTRACT

Cytokinins belong to one of the most important and well-known classes of plant hormones. Discovered over half a century ago, cytokinins have retained the attention of researchers due to the variety of the effects they have on the growth and development of vegetable organisms, their participation in a plant adaptation to external conditions, and the potential to be used in biotechnology, agriculture, medicine and even cosmetics.

The molecular mechanism by which cytokinins function remained unknown for a long time. Things started to change only in the 21st century, after the discovery of the receptors for these phytohormones. It appeared that plants found ways to adapt a two-component signal transduction system borrowed from prokaryotic organisms for cytokinin signalling. This review covers the recent advances in research of the molecular basis for the perception and transduction of the cytokinin signal. Emphasis is placed on cytokinin receptors, their domain and three-dimensional structures, subcellular localization, signalling activity, effect of mutations, ligand-binding properties, and phylogeny.

KEYWORDS

cytokinins; receptors; sensor histidine kinases; two-component systems; signal transduction.

ABBREVIATIONS
HK – histidine kinase; HP – phosphotransmitter; RR – response regulator; ER – endoplasmic reticulum.

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Y. A. Knirel, A. P. Anisimov

Lipopolysaccharide of Yersinia pestis, the Cause of Plague: Structure, Genetics, Biological Properties

Lipopolysaccharide of Yersinia pestis, the Cause of Plague: Structure, Genetics, Biological Properties

ABSTRACT

The present review summarizes data pertaining to the composition and structure of the carbohydrate moiety (core oligosaccharide) and lipid component (lipid A) of the various forms of lipopolysaccharide (LPS), one of the major pathogenicity factors of Yersinia pestis, the cause of plague. The review addresses the functions and the biological significance of genes for the biosynthesis of LPS, as well as the biological properties of LPS in strains from various intraspecies groups of Y. pestis and their mutants, including the contribution of LPS to the resistance of bacteria to factors of the innate immunity of both insect-vectors and mammal-hosts. Special attention is paid to temperature-dependent variations in the LPS structure, their genetic control and roles in the pathogenesis of plague.

The evolutionary aspect is considered based on a comparison of the structure and genetics of the LPS of Y. pestis and other enteric bacteria, including other Yersinia species. The prospects of development of live plague vaccines created on the basis of Y. pestis strains with the genetically modified LPS are discussed.

KEYWORDS
lipopolysaccharide; lipid A; plague; Yersinia pestis; immune response; antibiotic resistance

ABBREVIATIONS
CAMP – cationic antimicrobial peptides; LPS – lipopolysaccharide; NHS – normal human serum; Ara4N – 4-amino-4-deoxy-L-arabinose; Gal – galactose; Glc – glucose; GlcN, GlcNAc – glucosamine, N-acetylglucosamine; DD-Hep, LD-Hep – D-glycero-, L-glycero-D-manno-heptose; Kdo – 3-deoxy-D-mannooct-2-ulosonic acid; Ko – D?glycero-D-talo-oct-2-ulosonic acid; PEtN – phosphoethanolamine; UndP, UndPP – undecaprenyl phosphate, diphosphate.

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D. Lvovs*, O.O. Favorova, A.V. Favorov

A Polygenic Approach to the Study of Polygenic Diseases

Scientific Center of Russian Federation Research Institute for Genetics and Selection of Industrial
Microorganisms “Genetika”

ABSTRACT

Polygenic diseases are caused by the joint contribution of a number of independently acting or interacting polymorphic genes; the individual contribution of each gene may be small or even unnoticeable. The carriage of certain combinations of genes can determine the occurrence of clinically heterogeneous forms of the disease and treatment efficacy. This review describes the approaches used in a polygenic analysis of data in medical genomics, in particular, pharmacogenomics, aimed at identifying the cumulative effect of genes.

This effect may result from the summation of gains of different genes or be caused by the epistatic interaction between the genes. Both cases are undoubtedly of great interest in investigating the nature of polygenic diseases. The means that allow one to discriminate between these two possibilities are discussed. The methods for searching for combinations of alleles of different genes associated with the polygenic phenotypic traits of the disease, as well as the

methods for presenting and validating the results, are described and compared. An attempt is made to evaluate the applicability of the existing methods to an epistasis analysis. The results obtained by the authors using the APSampler software are described and summarized.

KEYWORDS
medical genomics; pharmacogenomics; polygenic analysis; epistasis

ABBREVIATIONS

CDCV – common disease / common variant; CDRV – common disease / rare variant; CI – confidence interval; CMC – combined multivariate and collapsing; FDR – false discovery rate; GWAS – genome-wide association study; MCMC – Markov Chain Monte Carlo; MDR – multifactor dimensionality reduction; MS – multiple sclerosis; IS – ischemic stroke; OR – odds ratio; ORR – odds ratios ratio; RR – relative risk; SF – synergy factor; TDT – transmission disequilibrium test.

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A. V. Maksimenko

Cardiological Biopharmaceuticals in the Conception of Drug Targeting Delivery: Practical Results and Research Perspectives

Institute of Experimental Cardiology, Russian Cardiology Research and Production Complex

ABSTRACT

The results of the clinical use of thrombolytic and antithrombotic preparations developed on the basis of protein conjugates obtained within the framework of the conception of drug targeting delivery in the organism are considered. A decrease has been noted in the number of biomedical projects focused on these derivatives as a result of various factors: the significant depletion of financial and organizational funds, the saturation of the pharmaceutical market with preparations of this kind, and the appearance of original means for interventional procedures.

Factors that actively facilitate the conspicuous potentiation of the efficacy of bioconjugates were revealed: the biomedical testing of protein domains and their selected combinations, the optimization of molecular sizes for the bioconjugates obtained, the density of target localization, the application of cell adhesion molecules as targets, and the application of connected enzyme activities. Enzyme antioxidants and the opportunity for further elaboration of the drug delivery conception via the elucidation and formation of therapeutic targets for effective drug reactions by means of pharmacological pre- and postconditioning of myocardium arouse significant interest.

KEYWORDS
drug targeting delivery; protein bioconjugates; thrombolytics; antithrombotic agents; molecular size of bioconjugates; density of molecular targets; enzyme connected antioxidants; cell adhesion molecules; pharmacological pre- and post-conditioning of myocardium.

ABBREVIATIONS
EC-SOD – extracellular superoxide dismutase; CAT – catalase; EMA – emergency medical aid; SOD – superoxide dismutase; CHS – chondroitin sulphate; SOD-CHS-CAT – covalent bienzyme superoxide dismutase chondroitin sulphat–catalase conjugate; ECG – electrocardiogram.

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A. I. Tuhvatulin, E. V. Sysolyatina, D. V. Scheblyakov, D. Yu. Logunov, M. M. Vasiliev, M. A. Yurova, M. A. Danilova, O. F. Petrov, B. S. Naroditsky, G. E. Morfill, A. I. Grigoriev, V. E. Fortov, A. L. Gintsburg, S. A. Ermolaeva*

Non-thermal Plasma Causes p53-Dependent Apoptosis in Human Colon Carcinoma Cells

ABSTRACT

Non-thermal plasma (NTP) consists of a huge amount of biologically active particles, whereas its temperature is close to ambient. This combination allows one to use NTP as a perspective tool for solving different biomedical tasks, including antitumor therapy. The treatment of tumor cells with NTP caused dose-dependent effects, such as growth arrest and apoptosis. However, while the outcome of NTP treatment has been established, the molecular mechanisms of the interaction between NTP and eukaryotic cells have not been thoroughly studied thus far. In this work, the mechanisms and the type of death of human colon carcinoma HCT 116 cells upon application of non-thermal argon plasma were studied.

The effect of NTP on the major stress-activated protein p53 was investigated. The results demonstrate that the viability of HCT116 cells upon plasma treatment is dependent on the functional p53 protein. NTP treatment caused an increase in the intracellular concentration of p53 and the induction of the p53-controlled regulon. The p53-dependent accumulation of active proapoptotic caspase-3 was shown in NTP-treated cells. The study was the first to demonstrate that treatment of human colon carcinoma cells with NTP results in p53-dependent apoptosis. The results obtained contribute to our understanding of the applicability of NTP in antitumor therapy.

KEYWORDS
non-thermal plasma; protein p53; apoptosis.

ABBREVIATIONS
NTP – non-thermal plasma

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M. A. Savitskaya*, M. S. Vildanova, O. P. Kisurina-Evgenieva, E.A. Smirnova, G. E. Onischenko

Mitochondrial Pathway of ?-Tocopheryl Succinate-Induced Apoptosis in Human Epidermoid Carcinoma A431 Cells

ABSTRACT

Vitamin E derivatives are known to act as agents exhibiting cytotoxity against tumor cells. The effect of vitamin E succinate on human epidermoid carcinoma cell line A431 was investigated in this study using live imaging, immunocytochemistry, and transmission electron microscopy. α-Tocopheryl succinate-induced apoptotic cell death in A431 cells was shown to be both dose- and time-dependent.

The hyperproduction of reactive oxygen species, changes in size, shape and ultrastructural characteristics of mitochondria followed by the release of cytochrome c from mitochondria to cytosol were observed. These results suggest that α-tocopheryl succinate induces apoptosis that occurs via the mitochondrial pathway. Mitochondria are shown to be crucial targets in α-tocopheryl succinate-induced caspase-dependent cell death in human carcinoma A431 cells.

KEYWORDS

α-tocopheryl succinate; apoptosis; mitochondrial pathway; ROS; cytochrome c

ABBREVIATIONS
α-TS – α-tocopheryl succinate; ROS – reactive oxygen species; AI – apoptotic index

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N. I. Grineva*, E. A. Duchovenskay, A. M. Timofeev, T. V. Akhlynina, L. P. Gerasimova, T. E. Manakova., T. V. Borovkova, D. A. Schmarov, N. G. Sarycheva, N. M. Naydenova, A .R. Gavrichkova, L. Y. Kolosova, T. I. Kolosheynova, L. G. Kovaleva

Gene Expression upon Proliferation and Differentiation of Hematopoietic Cells with Ph Chromosome ex vivo

ABSTRACT

The genes p53, mdm2, p21, c-myc, bcr/abl, bcr, bcl2, bax, and gapdh participate in the regulation of cell proliferation and differentiation, apoptosis and cell distribution for the cell cycle ex vivo in the Ph+cells of chronic myeloid leukemia containing the Ph chromosome and bcr/abl oncogene. Expression of these genes correlates with regulation of cell proliferation and differentiation by alternating proliferation and maturation stages for three main Ph+cell types that occur under chronic myeloid leukemia. The p53, p21, mdm2, and gapdh genes overexpress in active proliferating myeloid cells in the cell cycle S+ G2/M phases and when the phases are coincident with the proliferation stage.

Expression of these genes decreases to a considerable level under alternation of the Ph+cell proliferation and maturation stages and whenever the expression is greatly diminished under significant neutrophil accumulation and especially under repeated alternation of the stages. In the course of neutrophil maturation, gene expression levels decrease in the range of gapdh > actin > c-myc, bcr/abl,p21 > p53 > bcl2 > bax. The expression levels of these genes in neutrophils are lower than those in myelocytes and lower by an order of magnitude than that in the cells with a prolonged proliferation stage. The Bcr/abl expression gene under prolonged maturation and neutrophil accumulation is inhibited; however it is enhanced by 2–3 times for the proliferation stage with myelocyte accumulation. Minimal bcr/abl expression is observed under overexpression of p53, mdm2, p21, c-myc, as well as under cell maximum at the S and G2/M phases. Bcr/abl overexpression is observed under low expression of the p53, p21, mdm2 genes.

In the Ph+ cells with a high P/D efficiency index (5–20), overexpression of the genes in the range of bcr> gapdh>bcr/abl, as well as a decreased expression of the p53, bcl2, mdm2, p21<< gapdh genes is observed for Ph+ cells from the CML blast crisis and CML acceleration phase. Low control of cell proliferation and cell cycle by gene-regulators presumably promotes bcr/abl overexpression and activаtes the production of bcr/abl+ cells. Apoptosis in the Ph+ cells is induced by expression of the bax > bcl2, р53, p21, c-myc and gapdh genes. The blocking of Ph+ cell apoptosis, neutrophil accumulation, and decrease in the expression of the p53, mdm2 and p21, c-myc, bcr/abl genes occur at the maturation stage.

KEYWORDS
gene expression; regulation of cell proliferation and differentiation; cells containing Ph chromosome; chronic myeloid leukemia; RT-PCR, cell cycle; apoptosis.

ABBREVIATIONS
GEL – gene expression level; CML – chronic myeloid leukemia; Ph+cells – hematopoietic cells containing Ph chromosome; PB – peripheral blood; BM – bone marrow; FBS – fetal bovine serum; RT-PCR – reverse transcription polymerase chain reaction; CPD – cell proliferation and differentiation.

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Acta Naturae. 2012. V. 4. № 4 (15).

E. S. Sedova, D. N. Shcherbinin, A. I. Migunov, Iu. A. Smirnov, D. Iu. Logunov, M. M. Shmarov, L. M. Tsybalova, B. S. Naroditskii, O. I. Kiselev, A. L. Gintsburg

Recombinant Influenza Vaccines

Gamaleya Research Institute of Epidemiology and Microbiology, Gamaleya Str., 18, Moscow, Russia, 123098

Research Institute of Influenza, prof. Popov Str., 15/17, Saint Petersburg, Russia, 197376

Ivanovsky Research Institute of Virology, Gamaleya Str., 16, Moscow, Russia, 123098

*E-mail: sedova-es@yandex.ru

ABSTRACT

This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains.

KEYWORDS
Recombinant vaccine; influenza; immunization.

ABBREVIATIONS
WHO – World Health Organization; DNA – deoxyribonucleic acid; HA – hemagglutinin; NA – neuraminidase; VLP – virus-like particles; RNA – ribonucleic acid; NP – nucleoprotein; DC – dendritic cell; APC – antigen-presenting cell; Ad – adenovirus; NK – natural killers.
S. P. Medvedev , E. A. Pokushalov, S. M. Zakian

Epigenetics of Pluripotent Cells

Department of Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Prospekt Lavrentyeva, 10, Novosibirsk, Russia, 630090

Meshalkin Novosibirsk State Research Institute of Circulation Pathology, Rechkunovskaja Str., 15, Novosibirsk, Russia, 630055

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Prospekt Lavrentyeva, 8, Novosibirsk, Russia, 630090

E-mail: zakian@bionet.nsc.ru

ABSTRACT
Pluripotency is maintained by a complex system that includes the genetic and epigenetic levels. Recent studies have shown that the genetic level (transcription factors, signal pathways, and microRNAs) closely interacts with the enzymes and other specific proteins that participate in the formation of the chromatin structure. The interaction between the two systems results in the unique chromatin state observed in pluripotent cells. In this review, the epigenetic features of embryonic stem cells and induced pluripotent stem cells are considered. Special attention is paid to the interplay of the transcription factors OCT4, SOX2, and NANOG with the Polycomb group proteins and other molecules involved in the regulation of the chromatin structure. The participation of the transcription factors of the pluripotency system in the inactivation of the X chromosome is discussed. In addition, the epigenetic events taking place during reprogramming of somatic cells to the pluripotent state and the problem of "epigenetic memory" are considered.

KEYWORDS
embryonic stem cells; induced pluripotent stem cells; pluripotency; covalent histone modifications; DNA methylation

ABBREVIATIONS

ESC – embryonic stem cells; iPSC – induced pluripotent stem cells; DMR – differentially methylated
regions; ICM – inner cell mass.
E.S. Matyugina, S.N. Andreevskaya, T.G. Smirnova, A.L. Khandazhinskaya

Carbocyclic Analogues of Inosine-5’-Monophosphate: Synthesis and Biological Activity

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilova Str., 32, Moscow, Russia, 119991

Central Tuberculosis Research Institute, Russian Academy of Medical Sciences, 2, Yauzskaya Alley, Moscow, Russia, 107564

*E-mail: khandazhinskaya@bk.ru

ABSTRACT

9-(4’-Phosphonomethoxy-2’-cyclopenten-1’-yl)hypoxanthine and 9-(4’-phosphonomethoxy-2’,3’-

dihydroxycyclopenten-1’-yl)hypoxanthine were synthesized as isosteric carbocyclic analogues of inosine-5’-monophosphate. The synthesized compounds were shown to be capable of inhibiting the activity of human type II inosine-5′-monophosphate dehydrogenase (IMPDH II) (IC50 = 500 μM) and to have no significant effects on

the growth of Mycobacterium tuberculosis.

KEYWORDS
Carbocyclic nucleosides; competitive inhibition; inosine-5’-monophosphate; human IMPDH II, Mycobacterium tuberculosis.

ABBREVIATIONS
Yu. M. Yevdokimov, V. I. Salyanov, E. I. Katz, S. G. Skuridin

Gold Nanoparticle Clusters in Quasinematic Layers of Liquid-Crystalline Dispersion Particles of Double-Stranded Nucleic Acids

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilova Str., 32, Moscow, Russia, 119991

Landau Institute for Theoretical Physics, Russian Academy of Sciences, Kosygina Str. 2, Moscow, Russia, 119334

E-mail: yevdokim@eimb.ru

ABSTRACT

The interaction between gold nanoparticles and particles of cholesteric liquid-crystalline dispersions
formed by double-stranded DNA and poly(I)Чpoly(C) molecules is considered. It is shown that small-sized (~ 2 nm) gold nanoparticles induce two different structural processes. First, they facilitate the reorganization of the spatial cholesteric structure of the particles into a nematic one. This process is accompanied by a fast decrease in the amplitude of an abnormal band in the CD spectrum. Second, they induce cluster formation in a “free space” between neighboring nucleic acid molecules fixed in the structure of the quasinematic layers of liquidcrystalline particles. This process is accompanied by slow development of the surface plasmon resonance band in the visible region of the absorption spectrum. Various factors influencing these processes are outlined. Some

assumptions concerning the possible mechanism(s) of fixation of gold nanoparticles between the neighboring double-stranded nucleic acid molecules in quasinematic layers are formulated.

KEYWORDS

DNA; poly(I)Чpoly(C); liquid-crystalline dispersions of nucleic acids; gold nanoparticles; circular dichroism; absorption spectroscopy; abnormal optical activity; surface plasmon resonance; structure of biopolymer lyotropic liquid crystals; cytotoxicity of nanoparticles.

ABBREVIATIONS

DAU – daunomycin; CD – circular dichroism; Au nanoparticles (nano-Au) – gold nanoparticles; SPR – surface plasmon resonance; PEG – poly(ethylene glycol); UV region – ultraviolet region; CLCD – cholesteric
liquid-crystalline dispersion.
E.N. Lyukmanova,*, Z.O. Shenkarev, N.F. Khabibullina, D.S. Kulbatskiy, M.A. Shulepko, L.E. Petrovskaya, A.S. Arseniev, D.A. Dolgikh, M.P. Kirpichnikov

N-Terminal Fusion Tags for Effective Production of G-Protein-Coupled Receptors in Bacterial Cell-Free Systems

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, Moscow, Russia, 117997

Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory 1/12, Moscow, Russia, 119991
*E-mail: ekaterina-lyukmanova@yandex.ru

ABSTRACT

G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins.

In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts from E. coli cells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cellfree production of the human β2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin from Exiguobacterium sibiricum (ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein from B. subtilis allows to increase the CF synthesis of the target GPCRs by 5–38 times, resulting in yields of 0.6–3.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies

KEYWORDS

Cell-free expression; GPCR; translation initiation.

ABBREVIATIONS
a.a. – amino acid residue; β2AR – human β2-adrenergic receptor; CF system – cell-free expression

system; ESR – bacteriorhodopsin from Exiguobacterium sibiricum; ESR-tag – 6 a.a. N-terminal fragment of the ESR; FM – feeding mixture; GPCR – G-protein-coupled receptor; M1-mAChR – human muscarinic acetylcholine receptor M1; MP – membrane protein; RM – reaction mixture; SSTR5 – human somatostatin receptor type 5;

S-tag – 16 a.a. N-terminal fragment of ribonuclease A; T7-tag – 11 a.a. N-terminal fragment of the leader peptide of the protein 10 of the bacteriophage T7; TM – transmembrane; TRX – thioredoxin from Escherichia coli.
O. S. Petrakova, V. V. Ashapkin, E. A. Voroteliak, E. Y. Bragin, V. Y. Shtratnikova, E. S. Chernioglo, Y. V. Sukhanov, V. V. Terskikh, A. V. Vasiliev

Effect of 3D Cultivation Conditions on the Differentiation of Endodermal Cells

Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Vavilova Str. 26, Moscow, Russia, 119334

Lomonosov Moscow State University, Faculty of Biology, Leninskie Gory 1/12, Moscow, Russia, 119991

Belozersky Institute, Moscow State University, Leninskie Gory, 1/40, Moscow, Russia, 119991

Center of Innovation and Technology of Biologically Active Compounds and Their Applications, Russian Academy of Sciences, Gubkina Str. 3/2, Moscow, Russia, 117312

*E-mail: PetrakovaOl@yandex.ru

ABSTRACT

Cellular therapy of endodermal organs is one of the most important issues in modern cellular biology and biotechnology. One of the most promising directions in this field is the study of the transdifferentiation abilities of cells within the same germ layer. A method for an in vitro investigation of the cell differentiation potential (the cell culture in a three-dimensional matrix) is described in this article. Cell cultures of postnatal salivary gland cells and postnatal liver progenitor cells were obtained; their comparative analysis under 2D and 3D cultivation conditions was carried out. Both cell types have high proliferative abilities and can be cultivated for more than 20 passages. Under 2D cultivation conditions, the cells remain in an undifferentiated state. Under 3D conditions, they undergo differentiation, which was confirmed by a lower cell proliferation and by an increase in the differentiation marker expression. Salivary gland cells can undergo hepatic and pancreatic differentiation under 3D cultivation conditions. Liver progenitor cells also acquire a pancreatic differentiation capability under conditions of 3D cultivation. Thus, postnatal salivary gland cells exhibit a considerable differentiation potential within the endodermal germ layer and can be used as a promising source of endodermal cells for the cellular therapy of liver pathologies. Cultivation of cells under 3D conditions is a useful model for the in vitro analysis of the cell differentiation potential.

KEYWORDS
3D conditions; collagen gel; differentiation; endoderm; submandibular salivary gland cells; liver progenitor cells

ABBREVIATIONS

2D conditions – two-dimensional conditions; 3D conditions – three-dimensional conditions; BrdU - 5-bromo-2’-deoxyuridine; cDNA – complementary deoxyribonucleic acid; DAPI - 4’,6-diamidino-2-phenylindole; DNA – deoxyribonucleic acid; EGF – epidermal growth factor; ITS – insulin–transferrin–selenium;
mRNA – messenger ribonucleic acid; PLPC – postnatal liver progenitor cells; PSGC – postnatal salivary gland cells; RNA – ribonucleic acid; Real-time PCR – real-time polymerase chain reaction; RT-PCR – reverse transcription polymerase chain reaction.
V. Golovin, G. A. Khayrullina, B. Kraa, А. М. Kopylov

Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3’-Terminal Fragment of 16S rRNA in E. Coli

Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Leninskie Gory, 1/73, Moscow, Russia, 119991

Chemistry Department, Lomonosov Moscow State University, Moscow, Leninskie Gory, 1/3, Moscow, Russia, 119992

Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, the Netherlands

*E-mail: kopylov.alex@gmail.com

ABSTRACT

For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNA–protein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNA–protein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified.

UV–induced RNA–protein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA– protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA – protein cross–link within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNA–protein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit.

KEYWORDS
ribosome; initiation; self-assembly; ribosomal protein S7; UV– induced cross-linking.

ABBREVIATIONS
XRD – X-ray diffraction analysis; RNP – ribonucleoprotein; EcoS7, TthS7, BstS7 –proteins S7 isolated from E. coli, T. thermophilus and B. stearothermophilus, respectively; Tth30S and Eco30S – small ribosomal subunits isolated from T. thermophilus and E. coli, respectively.

Acta Naturae. 2013. V. 5. № 1 (16).

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O. S. Fedorova, Yu. D. Tsvetkov

Pulsed Electron Double Resonance in Structural Studies of Spin-Labeled Nucleic Acids

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Lavrentyev Ave., 8, Novosibirsk, 630090

Institute of Chemical Kinetics and Combustion, Siberian Branch, Russian Academy of Sciences, Institutskaya Str. 3, Novosibirsk, 630090

E-mail: fedorova@niboch.nsc.ru

ABSTRACT
This review deals with the application of the pulsed electron double resonance (PELDOR) method to studies of spin-labeled DNA and RNA with complicated spatial structures, such as tetramers, aptamers, riboswitches, and three- and four-way junctions. The use of this method for studying DNA damage sites is also described.

KEYWORDS
pulsed electron double resonance (PELDOR); spin-labels; DNA; RNA; oligonucleotides

ABBREVIATIONS
Rus PDF Eng PDF
O. G. Maksimenko, A.V. Deykin, Yu. M. Khodarovich, P. G. Georgiev

Use of Transgenic Animals in Biotechnology: Prospects and Problems

Institute of Gene Biology of the Russian Academy of Sciences, Vavilov St., 34/5, Moscow, Russia, 119334

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklucho-Maklai St., 16/10, Moscow, Russia, 117997

E-mail: georgiev_p@mail.ru

ABSTRACT
During the past two decades, there have been numerous attempts at using animals in order to produce recombinant human proteins and monoclonal antibodies. However, it is only recently that the first two therapeutic agents isolated from the milk of transgenic animals, C1 inhibitor (Ruconest) and antithrombin (ATryn), appeared on the market. This inspires hope that a considerable number of new recombinant proteins created using such technology could become available for practical use in the near future. In this review, the methods applied to produce transgenic animals are described and the advantages and drawbacks related to their use for producing recombinant human proteins and monoclonal antibodies are discussed.

KEYWORDS
bioreactor; milk protein production; production of monoclonal antibodies; recombinant proteins; therapeutic drugs; transgenic animals

ABBREVIATIONS
mAb – monoclonal antibodies; MI – intranuclear microinjection of DNA; NT – nuclear transfer; RP – recombinant protein; RHA – recombinant human albumin; rhBChE – recombinant human butyrylcholinesterase; TA – transgenic animal; FDA – United States Food and Drug Administration; EMEA – European Medicines Evaluation Agency; CHO cells – Chinese hamster ovary cells; ES cells – embryonic stem cells; UTR – untranslated region of a gene
Rus PDF Eng PDF
V.I. Glazko, T.T. Glazko

Sources of Contradictions in the Evaluation of Population Genetic Consequences after the Chernobyl Disaster

The Ministry of Agriculture of the Russian Federation, Orlikov Pereulok, 1/11, Moscow, Russia, 107139

Russian State Agrarian University – Moscow Timiryazev Agricultural Academy, Timirjazevskaja Str., 49, Moscow, Russia, 127550

E-mail: vglazko@yahoo.com

ABSTRACT
The review covers the analysis of our own and published data pertaining to population and genetic consequences in various mammalian species under conditions of high levels of ionizing radiation as a result of the Chernobyl accident. The findings indicate that these conditions have promoted the reproduction of heterozygotes in polyloci spectra of molecular genetic markers and animals with a relatively increased stability of the chromosomal apparatus. The prospects of using the reproductive “success” of the carriers of these characteristics as an integral indicator of the selective influence of environmental stress factors are discussed.

KEYWORDS
ionizing radiation; molecular genetic markers; reproductive ”success”; cytogenetic anomalies; ecological stress

ABBREVIATIONS
Rus PDF Eng PDF
S. P. Korolev, O. V. Kondrashina, D. S. Druzhilovsky, A. M. Starosotnikov, M. D. Dutov, M. A. Bastrakov, I. L. Dalinger, D. A. Filimonov, S. A. Shevelev, V. V. Poroikov, Y. Y. Agapkina, M. B. Gottikh

Structural-Functional Analysis of 2,1,3-Benzoxadiazoles and Their N-oxides As HIV-1 Integrase Inhibitors

Department of Chemistry, Lomonosov Moscow State University, Leninskie gory, 1/3, Moscow, Russia, 119991

Belozersky Research Institute of Physicochemical Biology, Lomonosov Moscow State University, Leniskie gory, 1/40, Moscow, Russia, 119991

Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya Str., 10/8, Moscow, Russia, 119121

Zelinsky Institute of Organic Chemistry, Russian Academy of Science, Leninskiy prospekt, 47, Moscow, Russia, 119991

E-mail: spkorolev@mail.ru

ABSTRACT
Human immunodeficiency virus type 1 integrase is one of the most attractive targets for the development of anti-HIV-1 inhibitors. The capacity of a series of 2,1,3-benzoxadiazoles (benzofurazans) and their N-oxides (benzofuroxans) selected using the PASS software to inhibit the catalytic activity of HIV-1 integrase was studied in the present work. Only the nitro-derivatives of these compounds were found to display inhibitory activity. The study of the mechanism of inhibition by nitro-benzofurazans/benzofuroxans showed that they impede the substrate DNA binding at the integrase active site. These inhibitors were also active against integrase mutants resistant to raltegravir, which is the first HIV-1 integrase inhibitor approved for clinical use. The comparison of computer-aided estimations of the pharmacodynamic and pharmacokinetic properties of the compounds studied and raltegravir led us to conclude that these compounds show promise and need to be further studied as potential HIV-1 integrase inhibitors.

KEYWORDS
HIV-1 integrase; inhibition; nitrobenzofuroxan; nitrobenzofurazan; PASS; QikProp

ABBREVIATIONS
ADME – absorption, distribution, metabolism, and excretion; AIDS – acquired immunodeficiency syndrome; BFX – benzofuroxan; BFZ – benzofurozan; HIV-1 – human immunodeficiency virus type 1; IN – integrase; IC50 – inhibitor concentration at which the enzyme activity is suppressed by 50 %; IC95 – inhibitor concentration at which the enzyme activity is suppressed by 95 %; PASS – Prediction of Activity Spectra for Substances software program; QikProp – ADME prediction software program
Rus PDF Eng PDF
D.G. Ilyushin, O.M. Haertley, T.V. Bobik, O.G. Shamborant, E.A. Surina, V.D. Knorre, P. Masson, I.V. Smirnov, A.G. Gabibov, N.A. Ponomarenko

Recombinant Human Butyrylcholinesterase As a New-Age Bioscavenger Drug: Development of the Expression System

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, Moscow, Russia, 117997

Departement de Toxilogie, Centre de Recherches du Servise de Sante des Armes, BP 87, 38702 La Tronche Cedex, France

E-mail: IlyushinDenis@gmail.com

ABSTRACT
Butyrylcholinesterase (BChE) is a serine hydrolase (EC 3.1.1.8) which can be found in most animal tissues. This enzyme has a broad spectrum of efficacy against organophosphorus compounds, which makes it a prime candidate for the role of stoichiometric bioscavenger. Development of a new-age DNA-encoded bioscavenger is a vival task. Several transgenic expression systems of human BChE were developed over the past 20 years; however, none of them has been shown to make economic sense or has been approved for administration to humans. In this study, a CHO-based expression system was redesigned, resulting in a significant increase in the production level of functional recombinant human butyrylcholinesterase as compared to the hitherto existing systems. The recombinant enzyme was characterized with Elman and ELISA methods.

KEYWORDS
bioscavenger; butyrylcholinesterase; CHO cell line; recombinant protein; оrganophosphorus toxins

ABBREVIATIONS
a.a.r. – amino acid residue; BChE – butyrylcholinesterase; PAGE – polyacrylamide gel electrophoresis; OPT – organophosphate toxin; CHO – Chinese hamster ovary cells; DMEM – Dulbecco’s Modified Eagle Medium
Rus PDF Eng PDF
Z. Shevchuk, M. Y. Yurchenko, S. D. Darekar, I. Holodnuka-Kholodnyuk, V. I. Kashuba, E. V. Kashuba

Overexpression of MRPS18-2 in Cancer Cell Lines Results in Appearance of Multinucleated Cells

Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, 17177, Sweden

Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NASU, 45 Vasylkivska str., Kyiv-22, 03022, Ukraine

Kirchenstein Institute of Microbiology and Virology, Riga Stradins University, 5 Ratsupites, Riga, LV-1067, Latvia

of Molecular Biology and Genetics of NASU, 150 Zabolotnogo str., Kyiv-143, 03680, Ukraine

E-mail: Elena.Kashuba@ki.se

ABSTRACT
Human mitochondrial ribosomal protein MRPS18-2 (S18-2) is encoded by a cellular gene that is located on the human chromosome 6p21.3. We discovered that overexpression of the S18-2 protein led to immortalization and de-differentiation of primary rat embryonic fibroblasts. Cells showed anchorage-independent growth pattern. Moreover, pathways characteristic for rapidly proliferating cells were upregulated then. It is possible that the S18-2 overexpression induced disturbance in cell cycle regulation. We found that overexpression of S18-2 protein in human cancer cell lines led to an appearance of multinucleated cells in the selected clones.

KEYWORDS
Mitochondrial ribosomal protein S18-2 (MRPS18-2), multinucleated cells, cancer cell line, cell cycle, RB binding protein

ABBREVIATIONS
Rus PDF Eng PDF
N. V. Punina, V. S. Zotov, A. L. Parkhomenko, T. U. Parkhomenko, A. F. Topunov

Genetic Diversity of Bacillus thuringiensis from Different Geo-Ecological Regions of by Analyzing the 16S rRNA and gyrB Genes and by AP-PCR and saAFLP

Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospect, 33, bld. 2, Moscow, Russia, 119071

Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moskvorechje str.,1, Moscow, Russia, 115478

Institute of Agriculture of Crimea, National Academy of Agrarian Sciences of Ukraine, Kievskaya str., 150, Simferopol, Ukraine, 95453

E-mail: hin-enkelte@yandex.ru

ABSTRACT
The Bacillus cereus group consists of closely related species of bacteria and is of interest to researchers due to its importance in industry and medicine. However, it remains difficult to distinguish these bacteria at the intra- and inter-species level. Bacillus thuringiensis (Bt) is a member of the B. cereus group. In this work, we studied the inter-species structure of five entomopathogenic strains and 20 isolates of Bt, which were collected from different geo-ecological regions of Ukraine, using various methods: physiological and biochemical analyses, analysis of the nucleotide sequences of the 16S rRNA and gyrB genes, by AP-PCR (BOX and ERIC), and by saAFLP. The analysis of the 16S rRNA and gyrB genes revealed the existence of six subgroups within the B. cereus group: B anthracis, B. cereus I and II, Bt I and II, and Bt III, and confirmed that these isolates belong to the genus Bacillus. All strains were subdivided into 3 groups. Seventeen strains belong to the group Bt II of commercial, industrial strains. The AP-PCR (BOX and ERIC) and saAFLP results were in good agreement and with the results obtained for the 16S rRNA and gyrB genes. Based on the derived patterns, all strains were reliably combined into 5 groups. Interestingly, a specific pattern was revealed by the saAFLP analysis for the industrial strain Bt 0376 р.о., which is used to produce the entomopathogenic preparation “STAR-t”.

KEYWORDS
Bacillus cereus group; B. thuringiensis; 16S ribosomal RNA; gyrB; saAFLP; taxonomy; phylogeny

ABBREVIATIONS
Bt – Bacillus thuringiensis; ICPs – δ-endotoxins; b.p. – base pair; MLST – multilocus sequence typing; MEE – multilocus enzyme electrophoresis; saAFLP, AFLP – single adapter amplified fragment length polymorphism; RFLP – restriction fragment length polymorphism; AP-PCR – arbitrarily primed polymerase chain reaction; rep-PCR – repetitive sequence-based PCR; BOX, ERIC – DNA repeats: ME – minimum evolution; NJ – neighbor joining
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