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.
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).
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. Donzova1* 1Department of Chemistry, Moscow State University, 119992 Moscow 2Blokhin Oncological Science Centre, Russian Academy of Medical Science, 115478 Moscow
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.
Catalytic Bioscavengers Against Toxic Esters, an Alternative Approach for Prophylaxis and Treatments of Poisonings
Patrick Masson1,2*, Daniel Rochu1,3 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
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.
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
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.
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. Gabibov1,2 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
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.
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: email@example.com
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.
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
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.
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
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.
MALDI mass spectrometry, bacteria profiling, Neisseria gonorrhoeae, Helicobacter pylori.
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
O. Nekrasova, A. Tagway, A. Ignatova,
A. Feofanov, M. Kirpichnikov
Studying of Membrane Localization of Recombinant Potassium Channels in E.coli
O. Nekrasova1*, A. Tagway1, A. Ignatova1,2, A. Feofanov1,2, M. Kirpichnikov1,2 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
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.
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
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.
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. Pyrkov1,2*, D. V. Pyrkova1, E. D. Balitskaya1,3, 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
Computer Modeling of the Structure and Spectra of Fluorescent Proteins
A.V. Nemukhin1,2*, B.L. Grigorenko1, A.P. Savitsky1,3 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
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.
green fluorescent protein, molecular modeling, molecular dynamics, molecular mechanics
QM/MM - combined methods of quantum and molecular mechanics, MD - molecular dynamics, tD-DFt - the method of density functional theory depending on time.
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
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.
cancer therapy, antisense oligonucleotides, ribozymes, DNAzymes, small interfering RNA
V.V. Terskikh*, Ye. A. Vorotelyak, A.V. Vasiliev
N.K. Koltsov Institute of Developmental Biology, Russian Academy of Sciences
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.
asymmetric division, stem cells, aging of stem cells, self-renewal of stem cells, aggresomes, Drosophila neuroblasts.
Covalent Binding Antibodies Suppress Advanced Glycation: On the Innate Tier of Adaptive Immunity
T. Shcheglova2, S. P. Makker1, and A. Tramontano1* 1Department of Pediatrics, University of California, Davis – School of Medicine Davis 2Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences
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.
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. Mollenkopf2*, S.H.E. Kaufmann2, A.S. Kaprelyants1 1A.N. Bach Institute of Biochemistry, RAS 2Max Planck Institute for Infection Biology
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.
M. tuberculosis, latent tuberculosis, "non-culturable" cells, global gene expression profile
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. Gottikh3* 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
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.
HIV Human immunodeficiency virus, HFV Human foamy virus, integrase, catalytic activity
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
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.
yeast telomerase, biotin, biotinylation.
DEAE-fraction – telomerase isolated from yeast extract using chromatography on DEAE-cellulose
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,
Engelghardt Institute of Molecular Biology, Russian Academy of Sciences
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences
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.
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
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.
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
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.
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. Davydova1*, 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
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.
Pacific Institute of Bioorganic Chemistry, Far-eastern Branch of the Russian Academy of Science
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.
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.
Influenza Virus Neuraminidase: Structure and Function
Y.A. Shtyrya, L.V. Mochalova, N.V. Bovin*
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS
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.
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
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.
phage display, filamentous bacteriophages, phagemids, phage display libraries of antibody fragments, biopanning, single chain antibody fragments, Fab-fragments.
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
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 19th century through the “arthritismus” doctrine. In the first half of the 20th 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.
syntropy, dystropy, syntropic and dystropic genes, genome, phenome, HuGENet.
Achievements and Peculiarities in Studies of Ancient DNA and DNA from Complicated Forensic Specimens
A.P. Grigorenko1,2,3, S.A. Borinskaya1, N.K. Yankovsky1, E.I. Rogaev1,2,3* 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: firstname.lastname@example.org
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.
ancient DNA, methods, evolution, DNA identification, forensic examination.
mtDNA – mitochondrial DNA, cmtDNA – complete mtDNA sequence, STR – short tandem repeats
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
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.
Atomic Resolution Crystal Structure of NAD+-Dependent Formate Dehydrogenase from Bacterium Moraxella sp. C-1
I.G. Shabalin1, K.M. Polyakov2,1, V.I. Tishkov1, V.O. Popov1* 1A.N. Bach Institute of Biochemistry RAS 2V.A. Engelhardt Institute of Molecular Biology RAS
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.
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. Švedas1* 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
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.
Combining Two Technologies for Full Genome Sequencing of Human
K.G. Skryabin1, E.B. Prokhortchouk1*, 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
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 (SOLiDTM) (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.
human genome, sequencing technology, single-nucleotide polymorphism
Indel SNP – insertion/deletion type single-nucleotide polymorphism.
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
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.
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.
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.
Study of Molecular Mechanisms Involved in the Pathogenesis of Immune-Mediated Inflammatory Diseases, using Psoriasis As a Model
E.S. Piruzian1*, 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: email@example.com
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.
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
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.
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.
Genome Paths: a Way to Personalized and Predictive Medicine
Ott’s Institute of Obstetrics and Gynecology, Russian Academy of Medical Sciences
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.
Protein Tyrosine Kinase Panel As a Tool for Anticancer Drug Design
T.V. Rakitina1,2*, O.V. Yudkina3,4, E.V. Smirnova1, A.V. Lipkin3,4 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
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.
protein tyrosine kinases; small molecule inhibitors; screening; 2-hydroxyphenol group
Genotoxic Effects of Silver Nanoparticles on Mice in Vivo
C.G. Ordzhonikidze1, L.K. Ramaiyya1, E.M. Egorova2, A.V. Rubanovich1* 1Vavilov Institute of General Genetics, Russian Academy of Sciences 2Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences Science-Technology Company "Nanomet"
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.
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. Dontsova1,3* 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
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.
deamination of 5-methylcytosine, 5-methylcytosine demethylation, the reaction of primer extension, MALDI MS, thymine DNA glycosylase.
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.
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
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.
Regulation of Immunogen Processing: Signal Sequences and their Application for the New Generation of DNA-Vaccines
E.S. Starodubova1*, M.G. Isaguliants2,3*, 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: firstname.lastname@example.org , email@example.com
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.
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
A.V. Letarov, A.K. Golomidova, K.K. Tarasyan
Winogradsky Institute of Microbiology RAS
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.
bacteriophages, phage therapy, human body microbiota, animal body microbiota, bacteriophage ecology
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.
O.A. Patutina1, N.L. Mironova1, E.I. Ryabchikova1, N.A. Popova2, V.P. Nikolin2, V.I. Kaledin2, V.V. Vlassov1, and M.A. Zenkova1* 1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences 2Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences
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.
antimetastatic activity, DNase I, RNase A, Lewis lung carcinoma, hepatoma 1A.
Lewis lung carcinoma (LLC), hepatoma 1A (HA-1).
Assessment of Formate Dehydrogenase Stress Stability in vivo using Inactivation by Hydrogen Peroxide
S.S. Savin1,2, V.I. Tishkov1,2,3* 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
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.
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
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.
immunoproteasomes, 19S proteasome activator, chymotrypsin-like activity of proteasomes, Western blot analysis, nodular regenerative hyperplasia of the liver, adenoma, hepatocellular carcinoma, mouse liver.
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. Shmarov1*, 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
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.
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
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.
DNA complexes, mismatch, selectivity, DNA ligase, DNA polymerase, modified oligonucleotide probes.
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.
University of Texas Southwestern Medical Center, Dallas, Texas, USA
Institute of Cytology, Russian Academy of Sciences, St. Petersburg
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.
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
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.
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
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.
stereoselective enzymatic acylation in aqueous medium, direct condensation, enantiomerically pure compounds, penicillin acylase
New 5-Modified Pyrimidine Nucleoside Inhibitors of Mycobacterial Growth
L.A. Alexandrova1*, 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
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.
Induced Pluripotent Stem Cells: Problems and Advantages when Applying them in Regenerative Medicine
S. P. Medvedev1, A. I. Shevchenko1, S. M. Zakian1,2* 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
Received 10. 04. 2010
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.
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: firstname.lastname@example.org Received 18.03.2010
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.
I.A. Mikhailopulo1*, A.I. Miroshnikov2 1Institute of Bioorganic Chemistry, National Academy of Sciences, Belarus 2Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
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.
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
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.
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. Khaliullin1,2, D.A. Suplatov1, D.N. Shalaeva1, M. Otsuka3, Y. Asano3, V.К. Švedas1* 1Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University 2Department of Chemistry, Lomonosov Moscow State University 3Biotechnology Research Center, Toyama Prefectural University, Japan
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.
D-aminopeptidase, penicillin-binding protein family, bioinformatic analysis, catalytic mechanism.
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.Ravin1*, K.G. Skryabin1 1Centre “Bioengineering” Russian Academy of Sciences 2Research Institute of Influenza, Russian Academy of Medical Sciences
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.
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. Tishkov1,2,3*, S. V. Uglanova1,4, V. V. Fedorchuk1, S. S. Savin2,3 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
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.
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. Zakian1,4* 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: email@example.com
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.
Effects of Myosin “Essential” Light Chain A1 on the Aggregation Properties of the Myosin Head
D. I. Markov1, O. P. Nikolaeva2, D. I. Levitsky1,2* 1Bach Institute of Biochemistry, Russian Academy of Sciences 2Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University
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.
Anionic Lipids: Determinants of Binding Cytotoxins from Snake Venom on the Surface of Cell Membranes
A.G. Konshina1*, 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
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.
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
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.
Genomes, Populations and Diseases: Ethnic Genomics and Personalized Medicine
V. A. Stepanov
Research Institute for Medical Genetics, Siberian Branch, Russian Academy of Medical Sciences
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.
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.
The Role of p66shc in Oxidative Stress and Apoptosis
E. R. Galimov
Belozersky Institute of Physico-Chemical Biology, Moscow State University
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.
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
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.
gene polymorphism, malignant brain tumors in children, genes of xenobiotic detoxication, DNA repair genes.
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. Sokolenko1,2, A. G. Iyevleva1,2, N. V. Mitiushkina1, E. N. Suspitsin1,2, 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. Yanus1,2, A. V. Togo1, V. M. Moiseyenko1,3, S. Ya. Maximov1, V.F. Semiglazov1, E. N. Imyanitov1,2,3* 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
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.
breast cancer, ovarian cancer, hereditary cancer syndromes, BRCA1, CHEK2, NBS.
Dosage Compensation of Sex Chromosome Genes in Eukaryotes
E. V. Dementyeva1,2, S. M. Zakian1,2,3* 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: firstname.lastname@example.org Received 24.09.2010
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.
dosage compensation, sex chromosomes, gene expression, X-chromosome inactivation
Classification of G-Quadruplex DNA on the Basis of the Quadruplex Twist Angle and Planarity of G-Quartets
R. V. Reshetnikov1,4, A. M. Kopylov2,3,4, A. V. Golovin1,4* 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
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.
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: email@example.com
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
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. Apalko1*, M. L. Filipenko1,2, V. A. Matveeva1,2, A. Yu. Bakulina1, V. G. Lunin3, M. V. Kostyanko1,4 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
Received 07. 10. 2011
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.
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. Zagoskin1*, 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
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.
Modeling of the Full-Size 3D Structure of Human Chaperone Hsp70 and Study of Its Interdomain Interactions
К. А. Chernorizov1, V.K.Svedas1,2*,2* 1Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University 2Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University
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.
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. Shishkin1*, 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
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.
proteomics, prostate cancer, digital database
2DE—two-dimensional gel electrophoresis, BPH—benign prostate hyperplasia, PCa—prostate cancer, PCP—prostate cancer proteomics
A In Vitro and In Vivo Study of the Ability of NOD1 Ligands to Activate the Transcriptional Factor NF-kB
A.I. Tukhvatulin1*, 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
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.
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. Tsareva1,2, M.A. Sudomoina1,2, O.G. Kulakova1,2, B.V. Titov1,2, O.V. Bykova3, N.V. Gol’tsova3, L.M. Kuzenkova3, A.N. Boiko2, O.O. Favorova1,2* 1Russian Cardiology Scientific and Production Center 2Russian State Medical University 3Scientific Center of Children Health, Russian Academy of Medical Sciences
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.
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).
Comparative Bioinformatic Analysis of Active Site Structures in Evolutionarily Remote Homologues of α,β-Hydrolase Superfamily Enzymes
D. A. Suplatov1,2, V. K. Arzhanik1,V. K. Svedas1,2* 1Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University 2Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University
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.
bioinformatics, comparative analysis, active site, structural alignment, α,β-hydrolases
PDB - Protein Data Bank; CSA - Catalytic Site Atlas
Multi-walled Сarbon Nanotubes Penetrate into Plant Cells and Affect the Growth of Onobrychis arenaria Seedlings
E.A. Smirnova1*, A.A. Gusev2, O.N. Zaitseva2, E.M. Lazareva1, G.E. Onishchenko1, E.V. Kuznetsova3, A.G. Tkachev4, A.V. Feofanov1,5, M.P. Kirpichnikov1,5 1Biology Faculty, Lomonosov Moscow State University
2Derzhavin Tambov State University
3Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch, Russian Academy of Sciences
5Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
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.
multi-walled carbon nanotubes, light microscopy, electron microscopy, electron diffraction pattern, O. arenaria seedlings.
CNM – carbon nanomaterials; MWNT – multi-walled carbon nanotubes; SWNT – single-walled carbon nanotubes; TEM – transmission electron microscopy, SAED – selected area electron diffraction.
Modeling Myocardial Infarction in Mice: Methodology, Monitoring, Pathomorphology
A.A. Ovsepyan4, D.N. Panchenkov1,3, E.B. Prokhortchouk1*, G.B. Telegin4, N.A. Zhigalova1, E.P. Golubev2, T. E. Sviridova5, S.T. Matskeplishvili2, K.G. Skryabin1, U.I. Buziashvili1,2 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
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.
Molecular and Physiological Mechanisms of Membrane Receptor Systems Functioning
E. S. Severin1*, M. V. Savvateeva2 1All-RussiaResearch Center for Molecular Diagnostics and Therapy 2Biology Faculty, Lomonosov Moscow State University
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.
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. Safonova1*, A.N. Petrin1, S. D. Arutyunov1, V. N. Tsarev1, L. A. Akulenko1, A. O. Zorina2, D. V. Rebrikov3,4, A. V. Rubanovich3,4, S. A. Borinskaya1,4, 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
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.
T. A. Zdobnova*, E. N. Lebedenko, S.М. Deyev
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
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.
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
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.
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
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.
Promoters with Cancer Cell-Specific Activity for Melanoma Gene Therapy
V.V. Pleshkan1,2*, I.V. Alekseenko1,2, M.V. Zinovyeva1, T.V. Vinogradova1, E.D. Sverdlov1,2 1Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences 2Institute of Molecular Genetics, Russian Academy of Sciences
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.
melanoma; gene therapy; tissue-specific promoters; specific expression of a transgene
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
Gold Nanoparticles in Biology and Medicine: Recent Advances and Prospects
L. A. Dykman1, N. G. Khlebtsov1,2* 1Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences 2Saratov State University
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.
gold nanoparticles; plasmon resonance; biosensors; biomedical diagnostics; photothermal and photodynamic therapy; targeted drug delivery; nanotoxicology
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
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 Stepanov1,6*, O. P. Balanovsky2,5, A. V. Melnikov3, A. Yu. Lash-Zavada3, V. N. Khar’kov1,6, 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
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.
microsatellites; short tandem repeats; allelic frequencies; forensic medical examination; DNA identification; population of Russia; reference database; genetic diversity; gene geography
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
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. Samsonova1,2, K. S. Kudryashova1, A. V. Feofanov1,2* 1Biological Faculty, Lomonosov Moscow State University 2Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences
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.
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
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.
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
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.
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
Received 24.02. 2011
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.
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. Zorina1*, A. A. Kulakov1, O. A. Boriskina1, D. V. Rebrikov2,3 1Central Research Institute of Dentistry and Oral Surgery 2Vavilov Institute of General Genetics, Russian Academy of Sciences 3Scientific Production Enterprise «DNA Technology», JSC
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.
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
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.
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. Protopopova2,3, E. V. Dubrovin3, I. V. Yaminskii2,3, N. P. Rodionova1, O. V. Karpova1*, J. G. Atabekov1,4 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
Received 21.04. 2011
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.
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. Timakhov1,2,3*, 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
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.
DLX5; transcription factor; small molecules; cancer; molecular docking.
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. Naroditskii1*, A.L. Gintsburg1 1GamaleyaResearch Institute for Epidemiology and Microbiology 2nstitute of Gene Biology, Russian Academy of Sciences
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.
Rabin8 Protein Interacts with GTPase Rheb and Inhibits Phosphorylation of Ser235/Ser236 in Small Ribosomal Subunit Protein S6
А. А. Parkhitko1,2,3*, О. О. Favorova1, E. P. Henske2,3 1Pirogov Russian National Research Medical University 2Fox Chase Cancer Center, Philadelphia, USA 3Brigham and Women’s Hospital, Harvard Medical School, USA
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.
complex mTORC1; Rheb; Rabin8; small ribosomal unit protein S6.
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.
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. Goncharuk1,2*, M.V. Goncharuk1,2, M.L. Mayzel1, D.M. Lesovoy1, V.V. Chupin1, E.V. Bocharov1, A. S. Arseniev1, M. P. Kirpichnikov1,2 1Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science 2Biology Department, Lomonosov Moscow State University
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.
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. Koliasnikov1,2#, V.G. Grigorenko2*#, 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
#The authors provided equivalent contributions to the study.
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.
Atomic Force Microscopy Study of the Arrangement and Mechanical Properties of Astrocytic Cytoskeleton in Growth Medium
Yu.M. Efremov1*, E.V. Dzyubenko1,2, 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
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.
atomic force microscopy; dorsal root ganglia; force spectroscopy; confocal microscopy; cytoskeleton.
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. Rogozhin1,2*, 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
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.
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.
Clinical Use of Inhibitors of HIV-1 Integration: Problems and Prospects
S. P. Korolev1*, Yu. Yu. Agapkina1, M.B. Gottikh1,2 1Department of Chemistry, Lomonosov Moscow State University 2Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University
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.
HIV-1 integrase; inhibition; mechanism of action; raltegravir.
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%; Сlp – 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; Т1/2 – time by which the concentration of a drug in plasma decreases twice; WT – wild type.
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
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.
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. Dzhemileva1*, 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: firstname.lastname@example.org Received 23.03.2011
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.
hereditary nonsyndromic sensorineural hearing loss; GJB2 (Cx26) gene; c.35delG mutation; ancestral haplotype; populations of the Volga-Ural region.
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
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.
SELEX method; NA aptamers; escort aptamers; specific cell binding; addressed cell delivery; detection of cells.
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; Kd– apparent constant of dissociation of the aptamer–target complex; LNA – locked nucleic acids; SELEX – Systematic Evolution of Ligands by EXponential Enrichment.
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
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.
A.A. Alekseeva1,2,3, S.S. Savin2,3, 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
NADsup>+--dependent formate dehydrogenase (FDH, EC 22.214.171.124) 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.
Behavior of Transplanted Multipotent Cells after in Vitro Transplantation into the Damaged Retina
S.A. Sergeev1*, Y.V. Khramova1, M.L Semenova1, I.N. Saburina2, N.V. Kosheleva1,2 1Biology Faculty, Lomonosov Moscow State University 2Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences
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.
Effect of Sodium Chloride on Aggregation of Merocyanine 540 and Photosensitized Inactivation of Staphylococcus aureus and Pseudomonas aeruginosa
T.A. Shmigol1, V.A. Bekhalo2*, 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
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.
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. Rubtsov1*, 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
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.
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. Prassolov1* 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
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.
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. Atabekov1* 1Faculty of Biology, Lomonosov Moscow State University 2Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University
*E-mail: email@example.com Received 20.06.2011
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.
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
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.
Inhibition of DNA Gyrase by Levofloxacin and Related Fluorine-Containing Heterocyclic Compounds
V.L. Tunitskaya1*, A.R. Khomutov1, S.N. Kochetkov1, S.K. Kotovskaya2,3, V.N. Charushin2,3 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
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.
fluoroquinolones; levofloxacin; derivatives; bacterial DNA gyrase; enzymatic activity; inhibition.
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: firstname.lastname@example.org Received 10.06.2011
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.
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. Petrenko1* 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
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.
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. Stepanov1*, 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
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.
small nucleolar box C/D RNAs; post-transcriptional RNA modification; alternative splicing of premRNA.
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. Stepanov1* 1Research Institute of Medical Genetics, Siberian Branch, Russian Academy of Medical Sciences 2Buryat State University 3University of California, Berkley, USA
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.
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
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.
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: email@example.com Received 08.01.2012
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.
Targeted Therapy: A New Approach for the Treatment of Locally Advanced Oropharyngeal Cancer
L.Z. Velsher1,2, A.A. Kosmynin1*, M.Yu. Byakhov1,2, T.K. Duditskaya1,2, D.N. Reshetov1,2 1Moscow State Medical and Dental University 2Cancer Center JSC “RZD”, Moscow
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.
oropharyngeal cancers; targeted therapy; quality of life; gefitinib.
SCCHN – squamous-cell carcinoma of the head and neck; EGFR – epidermal growth factor receptor.
Cytotoxic and Immunochemical Properties of Viscumin Encapsulated in Polylactide Microparticles
E. S. Kolotova1*, S. G. Egorova1, A. A. Ramonova1, S. E. Bogorodski2, V. K. Popov2, I. I. Agapov1,3, 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
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.
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. Zalesov1,2, E.V.Olseeva3, A.R.Maksimov4, S.I. Kutsev5, M.M. Garaev6, A.V. Rubanovich1, N.K. Yankovsky1,2,7 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: firstname.lastname@example.org
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.
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
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.
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.
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. Levitskaya1*, A. A. Kamensky2, N. F. Myasoedov1 1Institute of Molecular Genetics, Russian Academy of Sciences 2Biological Faculty, Lomonosov Moscow State University
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.
chronic stress; neonatal isolation; Semax; body weight; corticosterone; rat.
Stable Expression of Recombinant Factor VIII in CHO Cells Using MethotrexateDriven Transgene Amplification
N. A. Orlova1,2#, S. V. Kovnir1,2#, I. I. Vorobiev1,2*, 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 : email@example.com
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.
Physicochemical Biology: Conquered Boundaries and New Horizons
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, prosp. Akademika Lavrent’eva, 8, 630090, Novosibirsk, Russia
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.
DNA structure; enzyme active sites; unstructured proteins; dynamics of biochemical processes; single molecule studies of enzymatic processes and biopolymer tertiary structure formation.
Telomere Lengthening and Other Functions of Telomerase
M.P. Rubtsova1,2*, D.P. Vasilkova1, А.N. Malyavko1, Yu.V.Naraikina3, M.I. Zvereva1,2, О.А. Dontsova1,2 1Lomonosov Moscow State University, Chemistry Department, 2Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University 3Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics
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.
telomerase; reverse transcriptase; telomeres; mitochondria; DNA damage; gene expression.
TER – telomerase RNA; hTR – human telomerase RNA; TERT – telomerase reverse transcriptase.
N. A. Orlova, S. V. Kovnir, I. I. Vorobiev, A.G.Gabibov
Coagulation Factor IX for Hemophilia B Therapy
N. A. Orlova1,2, S. V. Kovnir1,2, I. I. Vorobiev1,2*, 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
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.
сoagulation factor IX; hemophilia B; heterologous protein expression systems
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
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.
Construction of a Full-Atomic Mechanistic Model of Human Apurinic/Apyrimidinic Endonuclease APE1 for Virtual Screening of Novel Inhibitors
I.G. Khaliullin1, D.K. Nilov1,2, I.V. Shapovalova2, V.K. Svedas1,2* 1Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University 2Lomonosov Moscow State University, Faculty of Bioengineering and Bioinformatics
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).
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. Morozov1*, 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
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.
recombinant human cyclophilin A; spatial dynamics of clot growth; anticoagulant activity.
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.
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
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.
thiohydrazones; thiohydrazides; thiadiazines; type III secretion system; citotoxicity; Chlamydia; inhibitors; microscopy; electron microscopy; morphology.
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.
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
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.
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
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.
cytokinins; receptors; sensor histidine kinases; two-component systems; signal transduction.
HK – histidine kinase; HP – phosphotransmitter; RR – response regulator; ER – endoplasmic reticulum.
Lipopolysaccharide of Yersinia pestis, the Cause of Plague: Structure, Genetics, Biological Properties
Lipopolysaccharide of Yersinia pestis, the Cause of Plague: Structure, Genetics, Biological Properties
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.
A Polygenic Approach to the Study of Polygenic Diseases
Scientific Center of Russian Federation Research Institute for Genetics and Selection of Industrial
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.
medical genomics; pharmacogenomics; polygenic analysis; epistasis
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.
Cardiological Biopharmaceuticals in the Conception of Drug Targeting Delivery: Practical Results and Research Perspectives
Institute of Experimental Cardiology, Russian Cardiology Research and Production Complex
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.
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.
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
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.
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
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.
α-tocopheryl succinate; apoptosis; mitochondrial pathway; ROS; cytochrome c
α-TS – α-tocopheryl succinate; ROS – reactive oxygen species; AI – apoptotic index
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
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.
gene expression; regulation of cell proliferation and differentiation; cells containing Ph chromosome; chronic myeloid leukemia; RT-PCR, cell cycle; apoptosis.
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.
Recombinant vaccine; influenza; immunization.
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
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.
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.
Carbocyclic nucleosides; competitive inhibition; inosine-5’-monophosphate; human IMPDH II, Mycobacterium tuberculosis.
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
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.
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.
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
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
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
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.
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.
ribosome; initiation; self-assembly; ribosomal protein S7; UV– induced cross-linking.
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.
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
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
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.
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.
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
ABSTRACT Butyrylcholinesterase (BChE) is a serine hydrolase (EC 126.96.36.199) 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
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
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.
ABSTRACT RNA interference is a convenient tool for modulating gene expression. The widespread application of RNA interference is made difficult because of the imperfections of the methods used for efficient target cell delivery of whatever genes are under study. One of the most convenient and efficient gene transfer and expression systems is based on the use of lentiviral vectors, which direct the synthesis of small hairpin RNAs (shRNAs), the precursors of siRNAs. The application of these systems enables one to achieve sustainable and long-term shRNA expression in cells. This review considers the adaptation of the processing of artificial shRNA to the mechanisms used by cellular microRNAs and simultaneous expression of several shRNAs as potential approaches for producing lentiviral vectors that direct shRNA synthesis. Approaches to using RNA interference for the treatment of cancer, as well as hereditary and viral diseases, are under active development today. The improvement made to the methods for constructing lentiviral vectors and the investigation into the mechanisms of processing of small interfering RNA allow one to now consider lentiviral vectors that direct shRNA synthesis as one of the most promising tools for delivering small interfering RNAs.
ABSTRACT Dosage compensation of the X chromosomes in mammals is performed via the formation of facultative heterochromatin on extra X chromosomes in female somatic cells. Facultative heterochromatin of the inactivated X (Xi), as well as constitutive heterochromatin, replicates late during the S-phase. It is generally accepted that Xi is always more compact in the interphase nucleus. The dense chromosomal folding has been proposed to define the late replication of Xi. In contrast to mouse pluripotent stem cells (PSCs), the status of X chromosome inactivation in human PSCs may vary significantly. Fluorescence in situ hybridization with a whole X-chromosome-specific DNA probe revealed that late-replicating Xi may occupy either compact or dispersed territory in human PSCs. Thus, the late replication of the Xi does not depend on the compactness of chromosome territory in human PSCs. However, the Xi reactivation and the synchronization in the replication timing of X chromosomes upon reprogramming are necessarily accompanied by the expansion of X chromosome territory.
KEYWORDS reprogramming; ESCs; iPS cells; chromosome territories; the X chromosome; late replication
ABBREVIATIONS BrdU – 5-bromo-2’-deoxyuridine; DAPI – 4’, 6-diamidino-2-phenylindole; ESCs – embryonic stem cells; FISH – fluorescent in situ hybridization; H3K27me3 – trimethylation of histone H3 at lysine 27; H3K4me2 – dimethylation of histone H3 at lysine 4; H3K9me3 – trimethylation of histone H3 at lysine 9; HUVEC – human umbilical vein endothelial cells; iPS cells – induced pluripotent stem cells; Xa – active X chromosome; Xi – inactive X chromosome, PSC - pluripotent stem cells
ABSTRACT The aim of this study was to identify small molecule compounds that inhibit the kinase activity of the IGF1 receptor and represent novel chemical scaffolds, which can be potentially exploited to develop drug candidates that are superior to the existing experimental anti-IGF1R therapeuticals. To this end, targeted compound libraries were produced by virtual screening using molecular modeling and docking strategies, as well as the ligand-based pharmacophore model. High-throughput screening of the resulting compound sets in a biochemical kinase inhibition assay allowed us to identify several novel chemotypes that represent attractive starting points for the development of advanced IGF1R inhibitory compounds.
ABSTRACT Whole transcriptome profiling is now almost routinely used in various fields of biology, including microbiology. In vivo transcriptome studies usually provide relevant information about the biological processes in the organism and thus are indispensable for the formulation of hypotheses, testing, and correcting. In this study, we describe the results of genome-wide transcriptional profiling of the major human bacterial pathogen M. tuberculosis during its persistence in lungs. Two mouse strains differing in their susceptibility to tuberculosis were used for experimental infection with M. tuberculosis. Mycobacterial transcriptomes obtained from the infected tissues of the mice at two different time points were analyzed by deep sequencing and compared. It was hypothesized that the changes in the M. tuberculosis transcriptome may attest to the activation of the metabolism of lipids and amino acids, transition to anaerobic respiration, and increased expression of the factors modulating the immune response. A total of 209 genes were determined whose expression increased with disease progression in both host strains (commonly upregulated genes, CUG). Among them, the genes related to the functional categories of lipid metabolism, cell wall, and cell processes are of great interest. It was assumed that the products of these genes are involved in M. tuberculosis adaptation to the host immune system defense, thus being potential targets for drug development.
KEYWORDS Mycobacterium tuberculosis; transcriptome in vivo; next generation sequencing; RNA-seq; tuberculosis
ABSTRACT Recombinant blood clotting factor VIII is one of the most complex proteins for industrial manufacturing due to the low efficiency of its gene transcription, massive intracellular loss of its proprotein during post-translational processing, and the instability of the secreted protein. Improvement in hemophilia A therapy requires a steady increase in the production of factor VIII drugs despite tightening standards of product quality and viral safety. More efficient systems for heterologous expression of factor VIII can be created on the basis of the discovered properties of its gene transcription, post-translational processing, and behavior in the bloodstream. The present review describes the deletion variants of factor VIII protein with increased secretion efficiency and the prospects for the pharmaceutical development of longer acting variants and derivatives of factor VIII.
KEYWORDS blood clotting factor VIII; hemophilia A; heterologous protein expression systems
ABBREVIATIONS FVIII – blood clotting factor VIII; BDD – B domain deleted; IU – international unit; FVIII:Ag – concentration of FVIII antigen; FVIII:C – procoagulant activity of FVIII; vWF – von Willebrand factor. Addition of “a” to the number of the corresponding clotting factor denotes the activated factor
ABSTRACT X chromosome inactivation is a complex process that occurs in marsupial and eutherian mammals. The process is thought to have arisen during the differentiation of mammalian sex chromosomes to achieve an equal dosage of X chromosome genes in males and females. The differences in the X chromosome inactivation processes in marsupial and eutherian mammals are considered, and the hypotheses on its origin and evolution are discussed in this review.
KEYWORDS mammals; X chromosome inactivation; Xist
ABBREVIATIONS XIC – X inactivation center; PAR – pseudoautosomal region of the mammalian X chromosome
of Cytology, Russian Academy of Sciences, Tikhoretsky ave., 4, St. Petersburg, 194064
ABSTRACT Most neurodegenerative pathologies stem from the formation of aggregates of mutant proteins, causing dysfunction and ultimately neuronal death. This study was aimed at elucidating the role of the protein factors that promote aggregate formation or prevent the process, respectively, glyceraldehyde-3-dehydrogenase (GAPDH) and tissue transglutaminase (tTG) and Hsp70 molecular chaperone. The siRNA technology was used to show that the inhibition of GAPDH expression leads to a 4550% reduction in the aggregation of mutant huntingtin, with a repeat of 103 glutamine residues in a model of Huntingtons disease (HD). Similarly, the blockage of GAPDH synthesis was found for the first time to reduce the degree of aggregation of mutant superoxide dismutase 1 (G93A) in a model of amyotrophic lateral sclerosis (ALS). The treatment of cells that imitate HD and ALS with a pharmacological GAPDH inhibitor, hydroxynonenal, was also shown to reduce the amount of the aggregating material in both disease models. Tissue transglutaminase is another factor that promotes the aggregation of mutant proteins; the inhibition of its activity with cystamine was found to prevent aggregate formation of mutant huntingtin and SOD1. In order to explore the protective function of Hsp70 in the control of the aggregation of mutant huntingtin, a cell model with inducible expression of the chaperone was used. The amount and size of polyglutamine aggregates were reduced by increasing the intracellular content of Hsp70. Thus, pharmacological regulation of the function of three proteins, GAPDH, tTG, and Hsp70, can affect the pathogenesis of two significant neurodegenerative diseases.
ABSTRACT Transcription regulation in bacterial restrictionmodification (RM) systems is an important process, which provides coordinated expression levels of tandem enzymes, DNA methyltransferase (MTase) and restriction endonuclease (RE) protecting cells against penetration of alien DNA. The present study focuses on (cytosine-5)-DNA methyltransferase Ecl18kI (M.Ecl18kI), which is almost identical to DNA methyltransferase SsoII (M.SsoII) in terms of its structure and properties. Each of these enzymes inhibits expression of the intrinsic gene and activates expression of the corresponding RE gene via binding to the regulatory site in the promoter region of these genes. In the present work, complex formation of M.Ecl18kI and RNA polymerase from Escherichia сoli with the promoter regions of the MTase and RE genes is studied. The mechanism of regulation of gene expression in the Ecl18kI RM system is thoroughly investigated. M.Ecl18kI and RNA polymerase are shown to compete for binding to the promoter region. However, no direct contacts between M.Ecl18kI and RNA polymerase are detected. The properties of M.Ecl18kI and M.SsoII mutants are studied. Amino acid substitutions in the N-terminal region of M.Ecl18kI, which performs the regulatory function, are shown to influence not only M.Ecl18kI capability to interact with the regulatory site and to act as a transcription factor, but also its ability to bind and methylate the substrate DNA. The loss of methylation activity does not prevent MTase from performing its regulatory function and even increases its affinity to the regulatory site. However, the presence of the domain responsible for methylation in the M.Ecl18kI molecule is necessary for M.Ecl18kI to perform its regulatory function.
Metagenomic Analysis of the Dynamic Changes in the Gut Microbiome of the Participants of the MARS-500 Experiment, Simulating Long Term Space Flight
A metagenomic analysis of the dynamic changes of the composition of the intestinal microbiome of five participants of the MARS-500 experiment was performed. DNA samples were isolated from the feces of the participants taken just before the experiment, upon 14, 30, 210, 363 and 510 days of isolation in the experimental module, and two weeks upon completion of the experiment. The taxonomic composition of the microbiome was analyzed by pyrosequencing of 16S rRNA gene fragments. Both the taxonomic and functional gene content of the microbiome of one participant were analyzed by whole metagenome sequencing using the SOLiD technique. Each participant had a specific microbiome that could be assigned to one of three recognized enterotypes. Two participants had enterotype I microbiomes characterized by the prevalence of Bacteroides, while the microbiomes of two others, assigned to type II, were dominated by Prevotella. One participant had a microbiome of mixed type. It was found that (1) changes in the taxonimic composition of the microbiomes occurred in the course of the experiment, but the enterotypes remained the same; (2) significant changes in the compositions of the microbiomes occurred just 14-30 days after the beginning of the experiment, presumably indicating the influence of stress factors in the first stage of the experiment; (3) a tendency toward a reversion of the microbiomes to their initial composition was observed two weeks after the end of the experiment, but complete recovery was not achieved. The metagenomic analysis of the microbiome of one of the participants showed that in spite of variations in the taxonomic compositions of microbiomes, the “functional” genetic composition was much more stable for most of the functional gene categories. Probably in the course of the experiment the taxonomic composition of the gut microbiome was adaptively changed to reflect the individual response to the experimental conditions. A new, balanced taxonomic composition of the microbiome was formed to ensure a stable gene content of the community as a whole without negative consequences for the health of the participants.
Transfer of Silver Nanoparticles through the Placenta and Breast Milk during in vivo Experiments on Rats
Silver nanoparticles (NPs), widely used in the manufacture of various types of consumer products and for medical applications, belong to novel types of materials that pose potential risks to human health. The potential negative effects of the influence of these NPs on reproduction are insufficiently researched. A quantitative assessment of the transfer of metallic silver nanoparticles through the placenta and breast milk was carried out during an in vivo experiment. We used 34.9 ± 14.8 nm in size silver NPs that were stabilized by low-molecularweight polyvinylpyrrolidone and labeled with the 110mAg radioactive isotope using thermal neutron irradiation in a nuclear reactor. [110mAg]-labeled NPs preparations were administered intragastrically via a gavage needle to pregnant (20th day of gestation) or lactating (14–16th day of lactation) female rats at a dose of 1.69–2.21 mg/kg of body weight upon conversion into silver. The accumulation of NPs in rat fetuses and infant rats consuming their mother’s breast milk was evaluated using a low-background semiconductor gamma-ray spectrometer 24 and 48 hours following labeling, respectively. In all cases, we observed a penetration of the [110mAg]-labeled NPs through the placenta and ther entry into the mother’s milk in amounts exceeding by 100-1,000 times the sensitivity of the utilized analytical method. The average level of accumulation of NPs in fetuses was 0.085–0.147% of the administered dose, which was comparable to the accumulation of the label in the liver, blood, and muscle carcass of adult animals and exceeded the penetration of NPs across the hematoencephalic barrier into the brain of females by a factor of 10-100. In lactating females, the total accumulation of [110mAg]-labeled NPs into the milk exceeded 1.94 ± 0.29% of the administered dose over a 48 h period of lactation; not less than 25% of this amount was absorbed into the gastrointestinal tract of infant rats. Thus, this was the first time experimental evidence of the transfer of NPs from mother to offspring through the placenta and breast milk was obtained.
Non-Viral Delivery and Therapeutic Application of Small Interfering RNAs
RNA interference (RNAi) is a powerful method used for gene expression regulation. The increasing knowledge about the molecular mechanism of this phenomenon creates new avenues for the application of the RNAi technology in the treatment of various human diseases. However, delivery of RNA interference mediators, small interfering RNAs (siRNAs), to target cells is a major hurdle. Effective and safe pharmacological use of siRNAs requires carriers that can deliver siRNA to its target site and the development of methods for protection of these fragile molecules from in vivo degradation. This review summarizes various strategies for siRNA delivery, including chemical modification and non-viral approaches, such as the polymer-based, peptide-based, lipid-based techniques, and inorganic nanosystems. The advantages, disadvantages, and prospects for the therapeutic application of these methods are also examined in this paper.
RNA interference; small interfering RNA; non-viral delivery.
S.A. Perevoztchikova, E.A. Romanova, T.S. Oretskaya, P. Friedhoff3, E.A. Kubareva*
Modern Aspects of the Structural and Functional Organization of the DNA Mismatch Repair System
This review is focused on the general aspects of the DNA mismatch repair (MMR) process. The key proteins of the DNA mismatch repair system are MutS and MutL. To date, their main structural and functional characteristics have been thoroughly studied. However, different opinions exist about the initial stages of the mismatch repair process with the participation of these proteins. This review aims to summarize the data on the relationship between the two MutS functions, ATPase and DNA-binding, and to systematize various models of coordination between the mismatch site and the strand discrimination site in DNA. To test these models, novel techniques for the trapping of short-living complexes that appear at different MMR stages are to be developed.
DNA mismatch repair system, structure of proteins, protein-protein and protein-DNA interactions, MutS, MutL, MutH.
S.A. Borinskaya*, A.A. Kim, A.V. Rubanovich, N.K. Yankovsky
The Impact of ADH1B Alleles and Educational Status on Levels and Modes of Alcohol Consumption in Russian Male Individuals
Alcohol abuse is one of the main reasons behind the low life span in Russia. Both social and genetic factors affect the alcohol consumption level. The genetic factors are alleles of the alcohol dehydrogenase ADH1B and aldehyde dehydrogenaseALDH2 genes. We have typed and found frequencies for the alleles in a cohort of 642 men, ethnic Russians. The individuals of the cohort were asked to complete a questionnaire in the framework of the Izhevsk Family Study (Leon et al., 2007, 2009) regarding the amount of alcohol consumed and on the type of hazardous alcohol consumption (nonbeverage alcohol consumption and the so-called “zapoï” which is a Russian term for a heavy drinking bout lasting for at least 2 days, when an individual is withdrawn from the normal social life). The ADH1B*48His allele was found among heterozygous individuals only (N=68, 10.6% of the cohort). The ALDH2*504Lys allele was also found among heterozygous individuals only (N=2, 0.3%) The effect of ADH1B alleles and the influence of the education level on the amount and type of alcohol consumed had not previously been studied in Russians. We have found that the amount of consumed alcohol is 21.6% lower (1733 g of ethanol per year) for ADH1B*48His allele carriers in the cohort of Russian men. The amount of consumed alcohol was found to be 9.8% lower (793 g of ethanol per year) in the case when individuals had a higher education as compared to those who had a secondary- or elementary school education level in the same cohort. Hence, the protective effect of the genetic factor (ADH1B*48His allele carriage) has proven to be more pronounced than the influence of the social factor (education level) at the individual level in the cohort of Russian men. Both factors have also proven to have a protective effect against hazardous types of alcohol consumption. Zapoï was not scored among individuals of the cohort with ADH1B*48His allele carriage (OR=12.6, P=0.006), as compared to 8.4% of “zapoï” individuals who did not carry the ADH1B*48His allele (genotype Arg/Arg).The percentage of individuals who consume non-beverage alcohol is lower (0.6%) in the subcohort of people with a higher education degree. This percentage is higher (6.0%, OR=10.0, P=0.004) in the subcohort of people without a higher education degree.
alcohol consumption level; genetic polymorphisms; ADH1B gene; social factors.
Original Nerve Growth Factor Mimetic Dipeptide GK-2 Restores Impaired Cognitive Functions in Rat Models of Alzheimer’s Disease
Dipeptide mimetic of the nerve growth factor (NGF) loop 4, hexamethylenediamide bis-(N-monosuccinyl- glutamyl-lysine) (GK-2), was synthesized at the V.V. Zakusov Scientific Research Institute of Pharmacology of the Russian Academy of Medical Sciences. GK-2 exhibited in vitro neuroprotective activity at nanomolar concentrations, was efficient in animal models of the Parkinson’s disease, ischemic and hemorrhagic stroke, and global cerebral ischemia at doses of 0.01–5 mg/kg (intraperitoneally) and 10 mg/kg (per os). The mnemotropic effects of subchronic intraperitoneal administration of GK-2 on rat models of the Alzheimer’s disease are described in this paper. Dipeptide GK-2 at a dose of 1 mg/kg is found to decrease the habituation deficit induced by the septo-hippocampal pathway transsection and, at a dose of 0.5 mg/kg, to significantly prevent spatial memory impairment in Morris water maze induced by intracerebral injection of streptozotocin. Thus, GK-2, an original dipeptide mimetic of NGF, acts on models of the Alzheimer’s disease upon systemic administration.
low molecular mimetic of NGF; GK-2; septo-hippocampal transsection; streptozotocin model of Alzheimer’s disease; habituation; Morris water maze.
K. N. Kashkin*, I. P. Chernov, E. A. Stukacheva, E. P. Kopantzev, G. S. Monastyrskaya, N. Ya. Uspenskaya, E. D. Sverdlov
Cancer Specificity of Promoters of the Genes Involved in Cell Proliferation Control
Core promoters with adjacent regions of the human genes CDC6, POLD1, CKS1B, MCM2, and PLK1 were cloned into a pGL3 vector in front of the Photinus pyrails gene Luc in order to study the tumor specificity of the promoters. The cloned promoters were compared in their ability to direct luciferase expression in different human cancer cells and in normal fibroblasts. The cancer-specific promoter BIRC5 and non-specific CMV immediately early gene promoter were used for comparison. All cloned promoters were shown to be substantially more active in cancer cells than in fibroblasts, while the PLK1 promoter was the most cancer-specific and promising one. The specificity of the promoters to cancer cells descended in the series PLK1, CKS1B, POLD1, MCM2, and CDC6. The bidirectional activity of the cloned CKS1B promoter was demonstrated. It apparently directs the expression of the SHC1 gene, which is located in a “head-to-head” position to the CKS1B gene in the human genome. This feature should be taken into account in future use of the CKS1B promoter. The cloned promoters may be used in artificial genetic constructions for cancer gene therapy.
promoter; cloning; cancer-specific; cancer gene therapy.
V. A. Glazunova, K. V. Lobanov, R. S. Shakulov, A. S. Mironov, A. A. Shtil
Acadesine Triggers Non-apoptotic Death in Tumor Cells
We studied the cytotoxicity of acadesine (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) for tumor and normal cells of various species and tissue origin. In tumor cells, acadesine triggered non-apoptotic death; the potency of the compound to normal cells was substantially lower. Acadesine was toxic for tumor cells with multidrug resistant phenotypes caused by the transmembrane transporter Р-glycoprotein or lack of proapoptotic p53. Activity of adenosine receptors was required for acadesine-induced cell death, whereas functioning of АМР-dependent protein kinase was not required. A more pronounced cytotoxicity for tumor cells, as well as the non-canonical death mechanism(s), makes acadesine a promising candidate for antitumor therapy.
AZT 5’-Phosphonates: Achievements and Trends in the Treatment and Prevention of HIV Infection
Despite the numerous drawbacks, 3’-azido-3’-deoxythymidine (AZT, Zidovudine, Retrovir) remains one of the key drugs used in the treatment and prevention of HIV infection in both monotherapy and HAART. A strategy in searching for new effective and safe AZT agents among latent (depot) forms of AZT has yielded its first positive results. In particular, the sodium salt of AZT 5’-H-phosphonate (Nikavir, phosphazide) has demonstrated clinical advantages over parent AZT: first and foremost, lower toxicity and better tolerability. It can be effectively used for the prevention of vertical transmission from mothers to babies and as an alternative drug for HIV-infected patients with low tolerance to Zidovudine. Preclinical studies of another phosphonate, AZT 5’-aminocarbonylphosphonate, have demonstrated that it releases AZT when taken orally. Pharmacokinetic studies have shown a prolonged action potential. Based on the analysis of both toxicological and pharmacological data, AZT 5’-aminocarbonylphosphonate has been recommended for clinical trials.
anti-HIV therapy; AZT 5'-phosphonates; depot forms; Nikavir; preclinical trials.
HIV RT – human immunodeficiency virus reverse transcriptase; AZT – 3’-azido-3’-deoxythymidine; 3ТС – (-)-β-L-2’,3’-dideoxy-3’-tiacytidine; L-FTC – (-)-β-L-2’,3’-dideoxy-3’-thia-5-fluoro-cytidine; НААRТ – high active antiretroviral therapy; LD50 – mean lethal dose; AUC – area under the concentration-time curve; MRT – mean residence time; CL – total clearance; Vss – steady state volume of distribution; F – bioavailability.
Bibliometric Indicators of Russian Journals by JCR-Science Edition, 1995-2010
A representative empirical bibliometric analysis of Russian journals included in the Journal Citation Reports-Science Edition (JCR-SE) for the time period 1995–2010 was conducted at the macro level (excluding the subject categories). It was found that the growth in the number of articles covered by JCR (a 1.8-fold increase compared to 1995) is ahead of the growth rates of Russian publications (1.2-fold increase). Hence, the share of Russian articles covered by JCR-SE was down from 2.5% in 1995 to 1.7% in 2010. It was determined that the number of articles published in an average Russian journal reduced by 20% as compared to the number of articles in an average journal of the full data set. These facts could partly shed light on the question why Russian research performance is staggering (approximately 30,000 articles per year), although the coverage of Russian journals has expanded to 150 titles. Over the past 15 years, a twofold increase in the impact factor of the Russian journals has been observed, which is higher than that for the full data set of journals (a 1.4-fold increase). Measures to improve the quality of Russian journals are proposed.
article; impact factor; Russian journal; full data set; bibliometric indicators; expected citation rate; JCR.
Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine ?-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii
The steady-state kinetic parameters of pyridoxal 5’-phosphate-dependent recombinant methionine γ-lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in β- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4–1.3 U/ml), PC-3 (IC50=0.1–0.4 U/ml), and MCF7 (IC50=0.04–3.2 U/ml) turned out to be the most sensitive cell lines.
Clathrin-Mediated Endocytosis and Adaptor Proteins
Macromolecules gain access to the cytoplasm of eukaryotic cells using one of several ways of which clathrin-dependent endocytosis is the most researched. Although the mechanism of clathrin-mediated endocytosis is well understood in general, novel adaptor proteins that play various roles in ensuring specific regulation of the mentioned process are being discovered all the time. This review provides a detailed account of the mechanism of clathrin-mediated internalization of activated G protein-coupled receptors, as well as a description of the major proteins involved in this process.
D.V. Shchepkin, A. M. Matyushenko, G.V. Kopylova, N.V. Artemova, S.Y. Bershitsky, A.K. Tsaturyan, D.I. Levitsky*
Stabilization of the Central Part of Tropomyosin Molecule Alters the Ca2+-sensitivity of Actin-Myosin Interaction
We show that the mutations D137L and G126R, which stabilize the central part of the tropomyosin (Tm) molecule, increase both the maximal sliding velocity of the regulated actin filaments in the in vitro motility assay at high Са2+ concentrations and the Са2+-sensitivity of the actin-myosin interaction underlying this sliding. Based on an analysis of the recently published data on the structure of the actin–Tm–myosin complex, we suppose that the physiological effects of these mutations in Tm can be accounted for by their influence on the interactions between the central part of Tm and certain sites of the myosin head.
actin-myosin interaction; in vitro motility assay; regulation of muscle contraction; tropomyosin.
For the Russian scientific community working in the field of life sciences, the year 2013 was marked by a high-profile event, namely the 38th Congress of the Federation of European Biochemical Societies (FEBS), held in St. Petersburg on July 6–11. The event was covered by the media (it should be mentioned that the RIA Novosti news agency was the official partner of the Congress); however, many things remained behind the scenes. Acta Naturae and its staff took an active part in the organization of the forum. In this year’s final issue, we would like to share our impressions “from inside the event” and to analyze it.
“Epigenetic Memory” Phenomenon in Induced Pluripotent Stem Cells
To date biomedicine and pharmacology have required generating new and more consummate models. One of the most perspective trends in this field is using induced pluripotent stem cells (iPSCs). iPSC application requires careful high-throughput analysis at the molecular, epigenetic, and functional levels. The methods used have revealed that the expression pattern of genes and microRNA, DNA methylation, as well as the set and pattern of covalent histone modifications in iPSCs, are very similar to those in embryonic stem cells. Nevertheless, iPSCs have been shown to possess some specific features that can be acquired during the reprogramming process or are remnants of epigenomes and transcriptomes of the donor tissue. These residual signatures of epigenomes and transcriptomes of the somatic tissue of origin were termed “epigenetic memory.” In this review, we discuss the “epigenetic memory” phenomenon in the context of the reprogramming process, its influence on iPSC properties, and the possibilities of its application in cell technologies.
The Role of Integrins in the Development and Homeostasis of the Epidermis and Skin Appendages
Integrins play a critical role in the regulation of adhesion, migration, proliferation, and differentiation of cells. Because of the variety of the functions they play in the cell, they are necessary for the formation and maintenance of tissue structure integrity. The trove of data accumulated by researchers suggests that integrins participate in the morphogenesis of the epidermis and its appendages. The development of mice with tissue-specific integrin genes knockout and determination of the genetic basis for a number of skin diseases in humans showed the significance of integrins in the biology, physiology, and morphogenesis of the epidermis and hair follicles. This review discusses the data on the role of different classes of integrin receptors in the biology of epidermal cells, as well as the development of the epidermis and hair follicles.
Antiviral Activity of Binase against the Pandemic Influenza A (H1N1) Virus
The lack of effective antiviral drugs restricts the control of the dangerous RNA-containing influenza A (H1N1) virus. Extracellular ribonuclease of Bacilli (binase) was shown to manifest antiviral activity during single- and multi-cycle viral replication in the range of concentrations non-toxic to epithelial cells and 0.01–0.1 multiplicity of infection. During antiviral treatment for 15–30 min, the concentration of 1 μg/ml binase reduced the amount of focus-forming units of viruses by a factor of 3–10 and suppressed the virus-induced cytopathic effect in A549 human lung cells. The possible mechanisms of interaction between the virus and enzyme are discussed. Positive charges in both binase and viral hemagglutinin cause electrostatic interaction with negatively charged sialic acid on the host cell’s surface followed by its penetration into the cell. Capsid elimination and release of viral RNA from endosome to the cytoplasm allows catalytic RNA cleavage by internalized binase. The data obtained confirm that binase is an effective antiviral agent against the pandemic influenza A (H1N1) virus. Certain progress in this field is associated with clarifying the detailed mechanism underlying the antiviral action of binase and development of the most effective way for its practical use.
3D Structure Modeling of Alpha-Amino Acid Ester Hydrolase from Xanthomonas rubrilineans
Alpha-amino acid ester hydrolase (EC 188.8.131.52, AEH) is a promising biocatalyst for the production of semi-synthetic β-lactam antibiotics, penicillins and cephalosporins. The AEH gene from Xanthomonas rubrilineans (XrAEH) was recently cloned in this laboratory. The three-dimensional structure of XrAEH was simulated using the homology modeling method for rational design experiments. The analysis of the active site was performed, and its structure was specified. The key amino acid residues in the active site – the catalytic triad (Ser175, His341 and Asp308), oxyanion hole (Tyr83 and Tyr176), and carboxylate cluster (carboxylate groups of Asp209, Glu310 and Asp311) – were identified. It was shown that the optimal configuration of residues in the active site occurs with a negative net charge –1 in the carboxylate cluster. Docking of different substrates in the AEH active site was carried out, which allowed us to obtain structures of XrAEH complexes with the ampicillin, amoxicillin, cephalexin, D-phenylglycine, and 4-hydroxy-D-phenylglycine methyl ester. Modeling of XrAEH enzyme complexes with various substrates was used to show the structures for whose synthesis this enzyme will show the highest efficiency.
alpha-amino acid ester hydrolase; Xanthomonas rubrilineans; computer simulation; docking; enzymatic synthesis of antibiotics, protein engineering.
A.D. Chernousov, M.F. Nikonova, N.I. Sharova, А.N. Mitin, М.М. Litvina, P.E. Sadchikov, I.L. Goldman, А.А. Yarilin, Е.R. Sadchikova
Neolactoferrin As a Stimulator of Innate and Adaptive Immunity
The effect of the innovative product Neolactoferrin, a natural combination of recombinant human lactoferrin (90%) and goat lactoferrin (10%) isolated from the milk of transgenic goats carrying the full-length human lactoferrin gene, on human immune system cells was studied. Neolactoferrin enhanced the production of IL-1β. Neolactoferrin saturated with iron ions increased the synthesis of pro-inflammatory cytokine TNFα. It determined the direction of the differentiation of precursor dendrite cells. Under the action of T cells, Neolactoferrin amplified the expression of the transcription factors responsible for the differentiation of Th- and Treg-cells and stimulated the production of both IFNγ and IL-4. The results suggest that Neolactoferrin exhibits an immunotropic activity and hinders the development of immune inflammatory processes. Iron saturation of Neolactoferrin increases its pro-inflammatory activity.
Recombinant human lactoferrin; Neolactoferrin; immunity; inflammation; cytokines; transcription factors.
Intensity of Free Radical Processes in Rat Liver under Type 2 Diabetes and Introduction of Epifamin
The effect of epifamin on free radical processes, the activity of caspase-1 and -3, aconitate hydratase and citrate content in rat’s liver at experimentally induced type 2 diabetes mellitus (T2DM) was studied. The action of epifamin at T2DM leads to a decrease in biochemiluminescence parameters, characterizing the intensity of free radical processes, and changes in aconitase activity and citrate level towards the control. Activities of caspase-1 and caspase-3 in the tissue decreased by a factor of 2.4 and 1.6 in comparison with the levels at the disease. Apparently, epifamin-mediated correction of the level of melatonin, providing a significant antioxidant effect, promotes positive action on the free radical homeostasis.
type 2 diabetes mellitus, biochemiluminescence, aconitate hydratase, citrate, caspase, epifamin.
Aptamers are short single-stranded oligonucleotides that are capable of binding various molecules with high affinity and specificity. When the technology of aptamer selection was developed almost a quarter of a century ago, a suggestion was immediately put forward that it might be a revolutionary start into solving many problems associated with diagnostics and the therapy of diseases. However, multiple attempts to use aptamers in practice, although sometimes successful, have been generally much less efficient than had been expected initially. This review is mostly devoted not to the successful use of aptamers but to the problems impeding the widespread application of aptamers in diagnostics and therapy, as well as to approaches that could considerably expand the range of aptamer application.
3D Structure Modeling of Alpha-Amino Acid Ester Hydrolase from Xanthomonas rubrilineans
Alpha-amino acid ester hydrolase (EC 184.108.40.206, AEH) is a promising biocatalyst for the production of semi-synthetic β-lactam antibiotics, penicillins and cephalosporins. The AEH gene from Xanthomonas rubrilineans (XrAEH) was recently cloned in this laboratory. The three-dimensional structure of XrAEH was simulated using the homology modeling method for rational design experiments. The analysis of the active site was performed, and its structure was specified. The key amino acid residues in the active site – the catalytic triad (Ser175, His341 and Asp308), oxyanion hole (Tyr83 and Tyr176), and carboxylate cluster (carboxylate groups of Asp209, Glu310 and Asp311) – were identified. It was shown that the optimal configuration of residues in the active site occurs with a negative net charge –1 in the carboxylate cluster. Docking of different substrates in the AEH active site was carried out, which allowed us to obtain structures of XrAEH complexes with the ampicillin, amoxicillin, cephalexin, D-phenylglycine, and 4-hydroxy-D-phenylglycine methyl ester. Modeling of XrAEH enzyme complexes with various substrates was used to show the structures for whose synthesis this enzyme will show the highest efficiency.
alpha-amino acid ester hydrolase; Xanthomonas rubrilineans; computer simulation; docking; enzymatic synthesis of antibiotics, protein engineering.
Depolarization-Induced Calcium- Independent Synaptic Vesicle Exo- and Endocytosis at Frog Motor Nerve Terminals
The transmitter release and synaptic vesicle exo- and endocytosis induced by constant current depolarization of nerve terminals were studied by microelectode extracellular recording of miniature endplate currents and fluorescent microscopy (FM 1-43 styryl dye). Depolarization of the plasma membrane of nerve terminals in the control specimen was shown to significantly increase the MEPC frequency (quantal transmitter release) and exocytotic rate (FM 1-43 unloading from the synaptic vesicles preliminarily stained with the dye), which was caused by a rise in the intracellular Са2+ concentration due to opening of voltage-gated Ca channels. A slight increase in the MEPC frequency and in the rate of synaptic vesicle exocytosis was observed under depolarization in case of blockade of Ca channels and chelating of intracellular Са2+ ions (cooperative action of Cd2+ and EGTA-AM). The processes of synaptic vesicle endocytosis (FM 1-43 loading) were proportional to the number of synaptic vesicles that had undergone exocytosis both in the control and in case of cooperative action of Cd2+ and EGTA-AM. A hypothesis has been put forward that Ca-independent synaptic vesicle exo- and endocytosis that can be induced directly by depolarization of the membrane exists in the frog motor terminal in addition to the conventional Ca-dependent process.
motor nerve terminals; exocytosis; endocytosis; calcium; constant depolarization current; cadmium.
EGTA-АМ – ethylene glycol-O, O’-bis(2-aminoethyl)-N, N, N’, N’-tetraacetic acid acetoxymethyl ester; MEPC – miniature end plate currents.
Antidepressant Effect of Dimeric Dipeptide GSB-106, an Original Low- Molecular-Weight Mimetic of BDNF
A large amount of clinical and experimental data suggest the involvement of neurotrophins, in particular the brain-derived neurotrophic factor (BDNF), in depression pathogenesis. However, the therapeutic use of BDNF is limited because of its instability in biological fluids, poor blood-brain barrier (BBB) permeability, and the presence of side effects. A low-molecular-weight mimetic GSB-106, which is a substituted dimeric dipeptide bis(N-monosuccinyl-L-seryl-L-lysine)hexamethylenediamide, was designed and synthesized based on the BDNF fourth loop structure at the V.V. Zakusov Institute of Pharmacology (RAMS). GSB-106 was found to exhibit an antidepressant activity in various models of depressive-like state when administered intraperitoneally to outbred mice and rats. An effect for the substance, when administered daily for 4–5 days, was detected in the Porsolt forced swimming test (0.1 and 1.0 mg/kg) and in the tail suspension test in mice (1.0 and 1.5 mg/ kg). An effect for GSB-106 at doses of 0.1 and 0.5 mg/kg was observed after a single application in experiments on rats in the Nomura water wheel test. The obtained evidence supports the hypothesis on the involvement of BDNF in the pathogenesis of various depression conditions, thus opening prospects for searching for new original antidepressants.
Competition within Introns: Splicing Wins over Polyadenylation via a General Mechanism
Most eukaryotic messenger RNAs are capped, spliced, and polyadenylated via co-transcriptional processes that are coupled to each other and to the transcription machinery. Coordination of these processes ensures correct RNA maturation and provides for the diversity of the transcribed isoforms. Thus, RNA processing is a chain of events in which the completion of one event is coupled to the initiation of the next one. In this context, the relationship between splicing and polyadenylation is an important aspect of gene regulation. We have found that cryptic polyadenylation signals are widely distributed over the intron sequences of Drosophila melanogaster. As shown by analyzing the distribution of genes arranged in a nested pattern, where one gene is fully located within an intron of another gene, overlapping of putative polyadenylation signals is a fairly common event affecting about 17% of all genes. Here we show that polyadenylation signals are silenced within introns: the poly(A) signal is utilized in the exonic but not in the intronic regions of the transcript. The transcription does not end within the introns, either in a transient reporter system or in the genomic context, while deletion of the 5'-splice site restores their functionality. According to a full Drosophila transcriptome analysis, utilization of intronic polyadenylation signals occurs very rarely and such events are likely to be inducible. These results confirm that the transcription apparatus ignores premature polyadenylation signals for as long as they are intronic.
Alu- and 7SL RNA Analogues Suppress MCF-7 Cell Viability through Modulating the Transcription of Endoplasmic Reticulum Stress Response Genes
11% of the human genome is composed of Alu-retrotransposons, whose transcription by RNA polymerase III (Pol III) leads to the accumulation of several hundreds to thousands of Alu-RNA copies in the cytoplasm. Expression of Alu-RNA Pol III is significantly increased at various levels of stress, and the increase in the Alu-RNA level is accompanied by a suppression of proliferation, a decrease in viability, and induction of apoptotic processes in human cells. However, the question about the biological functions of Pol III Alu-transcripts, as well as their mechanism of action, remains open. In this work, analogues of Alu-RNA and its evolutionary ancestor, 7SL RNA, were synthesized. Transfection of human breast adenocarcinoma MCF-7 cells with the Alu-RNA and 7SL RNA analogues is accompanied by a decrease in viability and by induction of proapoptotic changes in these cells. The analysis of the combined action of these analogues and actinomycin D or tamoxifen revealed that the decreased viability of MCF-7 cells transfected with Alu-RNA and 7SL RNA was due to the modulation of transcription. A whole transcriptome analysis of gene expression revealed that increased gene expression of the transcription regulator NUPR1 (p8), as well as the transcription factor DDIT3 (CHOP), occurs under the action of both the Alu- and 7SL RNA analogues on MCF-7 cells. It has been concluded that induction of proapoptotic changes in human cells under the influence of the Alu-RNA and 7SL RNA analogues is related to the transcriptional activation of the genes of cellular stress factors, including the endoplasmic reticulum stress response factors.
Alu-repeats; Alu-RNA; 7SL RNA; MCF-7 human breast adenocarcinoma cells.
Y. A. Lomakin, M. Yu. Zakharova, A. A. Belogurov, N. A. Bykova, M. A. Dronina, A. E. Tupikin, V. D. Knorre, A. N. Boyko, A. V. Favorov, M. R. Kabilov, N. A. Ponomarenko, A. G. Gabibov
Polyreactive Monoclonal Autoantibodies in Multiple Sclerosis: Functional Selection from Phage Display Library and Characterization by Deep Sequencing Analysis
Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system that primarily affects young and middle-aged people. It is widely accepted that B lymphocyte activation is required for MS progression. Despite the fact that the exact triggering mechanisms of MS remain enigmatic, one may suggest that MS can be induced by viral or bacterial infection in combination with specific genetic and environmental factors. Using deep sequencing and functional selection methodologies we characterized clones of poly- and cross-reactive antibodies that are capable of simultaneous recognition of viral proteins and autoantigens. The latter, in turn, possibly may trigger MS progression through molecular mimicry. It was identified that two cross-reactive antigens are probably recognized by light or heavy chains individually. According to the high structural homology between selected autoantibodies and a number of various antiviral IgGs, we suggest that a wide range of pathogens, instead of a single virus, be regarded as possible triggers of MS.
Multiple sclerosis, deep sequencing, cross-reactivity, autoreactive B cells, myelin basic protein, viral triggers.
Three-Dimensional Model of Mouse Epidermis for Experimental Studies of Psoriasis
Three-dimensional models of skin and epidermis imitate the structure of real tissues and provide accurate information about certain skin conditions, such as psoriasis. A three-dimensional model of mouse epidermis was generated from the epidermal keratinocytes of newborn mice and treated with cytokines. The aim of this study was to evaluate this model as an experimental model of psoriasis and to assess the changes occurring in its structure and gene expression after the exposure to proinflammatory cytokines. Treatment of the three-dimensional model with either interleukin 17 or a combination of tumor necrosis factor and interferon γ was shown to produce morphological changes, which were similar to acanthosis in psoriatic skin. The observed changes in gene expression of metalloproteinases and certain psoriasis biomarkers, such as mki67, krt16 and fosl1, were similar to the changes in patients’ skin. Notably, changes caused by interleukin 17 were less evident than those caused by the combination of interferon γ and tumor necrosis factor. On the contrary, HaCaT cells exhibited no significant changes in the expression of fosl1 and had decreased levels of mki67 after being treated with a combination of TNF and IFNG. Moreover, treatment with IL17 had no significant effect on krt16 and mki67 expression and even reduced the fosl1 levels. The findings suggest that artificially generated three-dimensional models of murine skin can be used to study psoriasis.
The composition of the intestinal microbiota is regulated by the immune system. This paper discusses the role of cytokines and innate immunity lymphoid cells in the intestinal immune regulation by means of IgA.
Е. А. Nikitina, A. V. Medvedeva, G. А. Zakharov, Е. V. Savvateeva-Popova
Williams Syndrome As a Model for Elucidation of the Pathway Genes – the Brain – Cognitive Functions: Genetics and Epigenetics
Genomic diseases or syndromes with multiple manifestations arise spontaneously and unpredictably as a result of contiguous deletions and duplications generated by unequal recombination in chromosomal regions with a specific architecture. The Williams syndrome is believed to be one of the most attractive models for linking genes, the brain, behavior and cognitive functions. It is a neurogenetic disorder resulting from a 1.5 Mb deletion at 7q11.23 which covers more than 20 genes; the hemizigosity of these genes leads to multiple manifestations, with the behavioral ones comprising three distinct domains: 1) visuo-spatial orientation; 2) verbal and linguistic defect; and 3) hypersocialisation. The shortest observed deletion leads to hemizigosity in only two genes: eln and limk1. Therefore, the first gene is supposed to be responsible for cardiovascular pathology; and the second one, for cognitive pathology. Since cognitive pathology diminishes with a patient’s age, the original idea of the crucial role of genes straightforwardly determining the brain’s morphology and behavior was substituted by ideas of the brain’s plasticity and the necessity of finding epigenetic factors that affect brain development and the functions manifested as behavioral changes. Recently, non-coding microRNAs (miRs) began to be considered as the main players in these epigenetic events. This review tackles the following problems: is it possible to develop relatively simple model systems to analyze the contribution of both a single gene and the consequences of its epigenetic regulation in the formation of the Williams syndrome’s cognitive phenotype? Is it possible to use Drosophila as a simple model system?
Williams syndrome; LIMK1; non-coding RNAs; Drosophila
A. V. Orlov, A. G. Burenin, V. O. Shipunova, A. A. Lizunova, B. G. Gorshkov, P. I. Nikitin
Development of Immunoassays Using Interferometric Real-Time Registration of Their Kinetics
A method for effective development of solid-phase immunoassays on a glass surface and for optimization of related protocols by highly sensitive quantitative monitoring of each assay step has been proposed and experimentally implemented. The method is based on the spectral correlation interferometry (SCI) that allows real-time measuring of the thickness of a biomolecular layer bound to the recognition molecular receptors on the sensor chip surface. The method is realized with compact 3-channel SCI?biosensors that employ as the sensor chips standard cover glass slips without deposition of any additional films. Different schemes for antibody immobilization on a glass surface have been experimentally compared and optimized toward a higher sorption capacity of the sensor chips. Comparative characterization of the kinetics of each immunoassay stage has been implemented with the optimized protocols: i) covalent immobilization of antibody on an epoxylated surface and ii) biotinylated antibody sorption on a biotinylated surface via a high-affinity biotin-streptavidin bond. We have shown that magnetic nanoparticles employed as labels with model detection of cardiac troponin I further amplify the SCI signal, resulting in 100-fold improvement of the detection limit. The developed protocols can also be used with the alternative immunoassay platforms, including the label methods based on registration of only the final assay result, which is the quantity of bound labels.
Label-free biosensors, interferometry, sensor chips, surface functionalization, surface epoxylation, surface biotinylation, efficiency of biomolecular immobilization, immunoassay, magnetic nanoparticles, cardiac troponin I
D. V. Pankratov, Y. S. Zeifman, А. V. Dudareva, G. K. Pankratova, M. E. Khlupova, Y. M. Parunova, D. N. Zajtsev, N. F. Bashirova, V. O. Popov, and S. V. Shleev
Impact of Surface Modification with Gold Nanoparticles on the Bioelectrocatalytic Parameters of Immobilized Bilirubin Oxidase
We unveil experimental evidence that put into question the widely held notion concerning the impact of nanoparticles on the bioelectrocatalytic parameters of enzymatic electrodes. Comparative studies of the bioelectrocatalytic properties of fungal bilirubin oxidase from Myrothecium verrucaria adsorbed on gold electrodes, modified with gold nanoparticles of different diameters, clearly indicate that neither the direct electron transfer rate (standard heterogeneous electron transfer rate constants were calculated to be 31±9 s-1) nor the biocatalytic activity of the adsorbed enzyme (bioelectrocatalytic constants were calculated to be 34±11 s-1) depends on the size of the nanoparticles, which had diameters close to or larger than those of the enzyme molecules.
gold nanoparticle; bilirubin oxidase; direct electron transfer; bioelectrocatalysis
3D – three-dimensional; Areal – real/electrochemical surface area; CV – cyclic voltammogram; ET – electron transfer; DET – direct electron transfer; k0 – standard heterogeneous electron transfer rate constant; kcat app – apparent bioelectrocatalytic constant; MCO – blue multicopper oxidase; MvBOx – Myrothecium verrucaria bilirubin oxidase; AuNPn – gold nanoparticles with diameter of n nm; AuNPn/Au – gold electrode modified with AuNPn before and after cycling in sulfuric acid (m-AuNP/Au u-AuNP/Au, respectively); ThLc – Trametes hirsuta laccase; PBS – phosphate buffered saline; NHE – normal hydrogen electrode; SEM – scanning electron microscopy; Aspr – absorbance maximum; А450 – absorbance at the wavelength of 450 nm; Γ – enzyme surface concentration; jmax – maximum current density
I. A. Muchkaeva, E. B. Dashinimaev, A. S. Artyuhov, E. P. Myagkova, E. A. Vorotelyak, Y. Y. Yegorov, K. S. Vishnyakova, J. E. Kravchenko, S. P. Chumakov, V. V. Terskikh, and A. V. Vasiliev
Generation of iPS Cells from Human Hair Follice Dermal Papilla Cells
Dermal papilla (DP) cells are unique regional stem cells of the skin that induce formation of a hair follicle and its regeneration cycle. DP are multipotent stem cells; therefore we supposed that the efficiency of DPC reprogramming could exceed that of dermal fibroblasts reprogramming. We generated induced pluripotent stem cells from human DP cells using lentiviral transfection with Oct4, Sox2, Klf4, and c-Myc, and cultivation of cells both in a medium supplemented with valproic acid and at a physiological level of oxygen (5%). The efficiency of DP cells reprogramming was ~0.03%, while the efficiency of dermal fibroblast reprogramming under the same conditions was ~0.01%. Therefore, we demonstrated the suitability of DP cells as an alternative source of iPS cells.
The genetic reprogramming technology allows one to generate pluripotent stem cells for individual patients. These cells, called induced pluripotent stem cells (iPSCs), can be an unlimited source of specialized cell types for the body. Thus, autologous somatic cell replacement therapy becomes possible, as well as the generation of in vitro cell models for studying the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss of upper and lower motor neurons. About 10% of cases are genetically inherited, and the most common familial form of ALS is associated with mutations in the SOD1 gene. We used the reprogramming technology to generate induced pluripotent stem cells with patients with familial ALS. Patient-specific iPS cells were obtained by both integration and transgene-free delivery methods of reprogramming transcription factors. These iPS cells have the properties of pluripotent cells and are capable of direct differentiation into motor neurons.
induced pluripotent stem cells; amyotrophic lateral sclerosis; differentiation; motor neurons
A. A. Khutornenko, A. A. Dalina, B. V. Chernyak, P. M. Chumakov, A. G. Evstafieva
The Role of Dihydroorotate Dehydrogenase in Apoptosis Induction in Response to Inhibition of the Mitochondrial Respiratory Chain Complex III
A mechanism for the induction of programmed cell death (apoptosis) upon dysfunction of the mitochondrial respiratory chain has been studied. Previously, we had found that inhibition of mitochondrial cytochrome bc1, a component of the electron transport chain complex III, leads to activation of tumor suppressor p53, followed by apoptosis induction. The mitochondrial respiratory chain is coupled to the de novo pyrimidine biosynthesis pathway via the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). The p53 activation induced in response to the inhibition of the electron transport chain complex III has been shown to be triggered by the impairment of the de novo pyrimidine biosynthesis due to the suppression of DHODH. However, it remained unclear whether the suppression of the DHODH function is the main cause of the observed apoptotic cell death. Here, we show that apoptosis in human colon carcinoma cells induced by the mitochondrial respiratory chain complex III inhibition can be prevented by supplementation with uridine or orotate (products of the reaction catalyzed by DHODH) rather than with dihydroorotate (a DHODH substrate). We conclude that apoptosis is induced in response to the impairment of the de novo pyrimidine biosynthesis caused by the inhibition of DHODH. The conclusion is supported by the experiment showing that downregulation of DHODH by RNA interference leads to accumulation of the p53 tumor suppressor and to apoptotic cell death.
apoptosis; p53 tumor suppressor; mitochondrial respiratory chain; dihydroorotate dehydrogenase; de novo pyrimidine biosynthesis
V. V. Makarov, A. J. Love, O. V. Sinitsyna, S. S. Makarova, I. V. Yaminsky, M. E. Taliansky, N. O. Kalinina
“Green” Nanotechnologies: Synthesis of Metal Nanoparticles Using Plants
While metal nanoparticles are being increasingly used in many sectors of the economy, there is growing interest in the biological and environmental safety of their production. The main methods for nanoparticle production are chemical and physical approaches that are often costly and potentially harmful to the environment. The present review is devoted to the possibility of metal nanoparticle synthesis using plant extracts. This approach has been actively pursued in recent years as an alternative, efficient, inexpensive, and environmentally safe method for producing nanoparticles with specified properties. This review provides a detailed analysis of the various factors affecting the morphology, size, and yield of metal nanoparticles. The main focus is on the role of the natural plant biomolecules involved in the bioreduction of metal salts during the nanoparticle synthesis. Examples of effective use of exogenous biomatrices (peptides, proteins, and viral particles) to obtain nanoparticles in plant extracts are discussed.
biomatrices; bioreduction; metal nanoparticles; plant metabolites; plant extracts
E. S. Starodubova, М. G. Isaguliants, Y. V. Kuzmenko, A. A. Latanova, О. А. Krotova, V. L. Karpov
Fusion to the Lysosome Targeting Signal of the Invariant Chain Alters the Processing and Enhances the Immunogenicity of HIV-1 Reverse Transcriptase
Intracellular processing of the antigen encoded by a DNA vaccine is one of the key steps in generating an immune response. Immunization with DNA constructs targeted to the endosomal-lysosomal compartments and to the MHC class II pathway can elicit a strong immune response. Herein, the weakly immunogenic reverse transcriptase of HIV-1 was fused to the minimal lysosomal targeting motif of the human MHC class II invariant chain. The motif fused to the N-terminus shifted the enzyme intracellular localization and accelerated its degradation. Degradation of the chimeric protein occurred predominantly in the lysosomal compartment. BALB/c mice immunized with the plasmid encoding the chimeric protein demonstrated an enhanced immune response, in the form of an increased antigen-specific production of Th1 cytokines, INF-γ and IL-2, by mouse splenocytes. Moreover, the majority of the splenocytes secreted both cytokines; i.e., were polyfunctional. These findings suggest that retargeting of the antigen to the lysosomes enhances the immune response to DNA vaccine candidates with low intrinsic immunogenicity.
D. N. Shcherbinin, I. B. Esmagambetov, A. N. Noskov, Yu. O. Selyaninov, I. L. Tutykhina, M. M. Shmarov, D. Yu. Logunov, B. S. Naroditskiy, A. L. Gintsburg
Protective Immune Response against Bacillus anthracis Induced by Intranasal Introduction of a Recombinant Adenovirus Expressing the Protective Antigen Fused to the Fc-fragment of IgG2a
Anthrax is a particularly dangerous infectious disease that affects humans and livestock. It is characterized by intoxication, serosanguineous skin lesions, development of lymph nodes and internal organs, and may manifest itsself in either a cutaneous or septic form. The pathogenic agent is Bacillus anthracis, a grampositive, endospore-forming, rod-shaped aerobic bacterium. Efficacious vaccines that can rapidly induce a long-term immune response are required to prevent anthrax infection in humans. In this study, we designed three recombinant human adenovirus serotype-5-based vectors containing various modifications of the fourth domain of the B. anthracis protective antigen (PA). Three PA modifications were constructed: a secretable form (Ad-sPA), a non-secretable form (Ad-cPA), and a form with the protective antigen fused to the Fc fragment of immunoglobulin G2a (Ad-PA-Fc). All these forms exhibited protective properties against Bacillus anthracis. The highest level of protection was induced by the Ad-PA-Fc recombinant adenovirus. Our findings indicate that the introduction of the Fc antibody fragment into the protective antigen significantly improves the protective properties of the Ad-PA-Fc adenovirus against B. anthracis.
M. M. Moisenovich, A. Yu. Arkhipova, A. A. Orlova, M. S Drutskaya, S. V. Volkova, S. E. Zacharov, I. I. Agapov, Academician M. P. Kirpichnikov
Composite Scaffolds Containing Silk Fibroin, Gelatin, and Hydroxyapatite for Bone Tissue Regeneration and 3D Cell Culturing
Three-dimensional (3D) silk fibroin scaffolds were modified with one of the major bone tissue derivatives (nano-hydroxyapatite) and/or a collagen derivative (gelatin). Adhesion and proliferation of mouse embryonic fibroblasts (MEF) within the scaffold were increased after modification with either nano-hydroxyapatite or gelatin. However, a significant increase in MEF adhesion and proliferation was observed when both additives were introduced into the scaffold. Such modified composite scaffolds provide a new and better platform to study wound healing, bone and other tissue regeneration, as well as artificial organ bioengineering. This system can further be applied to establish experimental models to study cell-substrate interactions, cell migration and other complex processes, which may be difficult to address using the conventional two-dimensional culture systems.
Molecular Mechanism of Global Genome Nucleotide Excision Repair
Nucleotide excision repair (NER) is a multistep process that recognizes and eliminates a wide spectrum of damage causing significant distortions in the DNA structure, such as UV-induced damage and bulky chemical adducts. The consequences of defective NER are apparent in the clinical symptoms of individuals affected by three disorders associated with reduced NER capacities: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). These disorders have in common increased sensitivity to UV irradiation, greatly elevated cancer incidence (XP), and multi-system immunological and neurological disorders. The eucaryotic NER system eliminates DNA damage by the excision of 24–32 nt single-strand oligonucleotides from a damaged strand, followed by restoration of an intact double helix by DNA repair synthesis and DNA ligation. About 30 core polypeptides are involved in the entire repair process. NER consists of two pathways distinct in initial damage sensor proteins: transcription-coupled repair (TC-NER) and global genome repair (GG-NER). The article reviews current knowledge on the molecular mechanisms underlying damage recognition and its elimination from mammalian DNA.
nucleotide excision repair; repair factors; molecular mechanisms of damage recognition and elimination
Disruption of the Colonization Resistance Syndrome in Humans in Altered Habitats and Its Prevention
Exposure of human subjects to environments with modified parameters is associated with reduced colonization resistance of the intestine and epithelial tissue, which leads to dysbiotic changes. Probiotics – preparations based on protective microflora – are used to correct dysbacteriosis of different etiologies and localizations. However, the effectiveness of probiotics largely depends on the adhesive ability of a probiotic strain and lack of competitive relations with the indigenous microflora, which can be achieved by individual selection of a preparation. We propose to use autochtonous microflora as a probiotic drug to optimize the prevention and treatment results. A personalized approach to probiotic selection will improve therapy efficiency and reduce the risk of adverse effects in each individual patient.
autostrains; autoprobiotics; dysbacteriosis; altered habitat; disruption of colonization resistance
O. V. Arkova, N. A. Kuznetsov, O. S. Fedorova, N. A. Kolchanov, L. K. Savinkova
Real-Time Interaction between ТВР and the TATA Box of the Human Triosephosphate Isomerase Gene Promoter in the Norm and Pathology
The TATA-binding protein (ТВР) is a key part of the transcription complex of RNA polymerase II. Alone or as a part of the basal transcription factor TFIID, TBP binds the TATA box located in the core region of the TATA-containing promoters of class II genes. Previously, we studied the effects of single nucleotide polymorphisms (SNPs) on ТВР/ТАТА-box interactions using gel retardation assay. It was demonstrated that most SNPs in the ТАТА boxes of some human gene promoters cause a 2- to 4-fold decrease in ТВР/ТАТА affinity, which is associated with an increased risk of hereditary diseases, such as β thalassemias of diverse severity, hemophilia B Leyden, myocardial infarction, thrombophlebitis, lung cancer, etc. In this work, the process of ТВР/ТАТА complex formation has been studied in real time by a stopped-flow technique using recombinant human TBP and duplexes, which were identical to the TATA box of the wild-type and a SNP-containing triosephosphate isomerase gene promoter and were fluorescently labeled by the Cy3/Cy5 FRET pair. It has been demonstrated for the first time that real-time binding of ТВР to the TATA box of the TPI gene promoter is complete within 10 s and is described by a single-stage kinetic model. The complex formation of ТВР with the wild-type TATA box occurs 5.5 times faster and the complex dissociation occurs 31 times slower compared with the SNPcontaining TATA box. Within the first seconds of the interaction, ТВР binds to and simultaneously bends the TATA box. Importantly, the TATA box of the wild-type TPI gene promoter requires lower TBP concentrations compared to the TATA box containing the -24Т → G SNP, which is associated with neurological and muscular disorders, cardiomyopathy, and other diseases.
B. B. Vartapetian, Y. I. Dolgikh, L. I. Polyakova, N. V. Chichkova , А. B. Vartapetian
Biotechnological approaches to creation of hypoxia and anoxia tolerant pl
The present work provides results of a number of biotechnological studies aimed at creating cell lines and entire plants resistant to anaerobic stress. Developed biotechnological approaches were based on earlier fundamental researches into anaerobic stress in plants, so “Introduction” briefly covers the importance of the problem and focuses on works considering two main strategies of plants adaptation to anaerobic stress. Those are adaptation at molecular level where key factor is anaerobic metabolism of energy (true tolerance) and adaptation of the entire plant via formation of aerenchyma and facilitated transportation of oxygen (apparent tolerance). Thus, sugarcane and wheat cells resistant to anaerobic stress were obtained through consecutive in vitro selection under conditions of anoxia and absence of exogenous carbohydrates. Tolerant wheat cells were used to regenerate entire plants of higher resistance to root anaerobiosis. It has been demonstrated that cells tolerance to anoxia is significantly supported by their ability to utilize exogenous nitrate. Cells tolerance established itself at the genetic level and was inherited by further generations. Apart from that, other successful attempts to increase tolerance of plants to anaerobic stress by means of stimulation of glycolysis and overexpression of genes responsible for cytokinin synthesis and programmed cell death are also discussed. The presented data proved the notion of two main strategies of plants adaptation to anaerobic stress proposed earlier on the base of fundamental studies.
A. V. Nedoluzhko, E. S. Boulygina, A. S. Sokolov, S. V. Tsygankova, N. M. Gruzdeva, A. D. Rezepkin, E. B. Prokhortchouk
Analysis of the Mitochondrial Genome of a Novosvobodnaya Culture Representative using Next-Generation Sequencing and Its Relation to the Funnel Beaker Culture
The Novosvobodnaya culture is known as a Bronze Age archaeological culture in the North Caucasus region of Southern Russia. It dates back to the middle of the 4th millennium B.C. and seems to have occurred during the time of the Maikop culture. There are now two hypotheses about the emergence of the Novosvobodnaya culture. One hypothesis suggests that the Novosvobodnaya culture was a phase of the Maikop culture, whereas the other one classifies it as an independent event based on the material culture items found in graves. Comparison between Novosvobodnaya pottery and Funnelbeaker (TRB) pottery from Germany has allowed researchers to suggest that the Novosvobodnaya culture developed under the influence of Indo-European culture. Nevertheless, the origin of the Novosvobodnaya culture remains a matter of debate. We applied next-generation sequencing to study ~5000-year-old human remains from the Klady kurgan grave in Novosvobodnaya stanitsa (now the Republic of Adygea, Russia). A total of 58,771,105 reads were generated using Illumina GAIIx with a coverage depth of 13.4х over the mitochondrial (mt) DNA genome. The mtDNA haplogroup affiliation was determined as V7, suggesting a role of the TRB culture in the development of the Novosvobodnaya culture and supporting the model of sharing between Novosvobodnaya and early Indo-European cultures.
S. A. Evfratov, E. M. Smekalova, A. V. Golovin, N. A. Logvina, M. I. Zvereva, O. A. Dontsova
Structural Features of the Telomerase RNA Gene in the Naked Mole Rat Heterocephalus glaber
Telomere length, an important feature of life span control, is dependent on the activity of telomerase (a key enzyme of the telomere-length-maintaining system). Telomerase RNA is a component of telomerase and, thus, is crucial for its activity. The structures of telomerase RNA genes and their promoter regions were compared for the long-living naked mole rat and different organisms. Two rare polymorphisms in Heterocephalus glaber telomerase RNA (hgTER) were identified: A→G in the first loop of pseudoknot P2b-p3 (an equivalent of 111nt in hTR) and G→A in the scaRNA domain CR7-p8b (an equivalent of 421nt in hTR). Analysis of TER promoter regions allowed us to identify two new transcription factor binding sites. The first one is the ETS family site, which was found to be a conserved element for all the analyzed TER promoters. The second site is unique for the promoter region of TER of the naked mole rat and is a binding site for the SOX17 transcription factor. The absence of one Sp1 site in the TER promoter region of the naked small rat is an additional specific feature of the promoter area of hgTER. Such variation in the hgTER transcription regulation region and hgTER itself could provide increased telomerase activity in stem cells and an extended lifespan to H. glaber.
H. glaber; telomerase RNA; bioinformatics; promoter analysis; comparative genomics.
TER – telomerase RNA; hTR – human telomerase RNA; TERT – telomerase catalytic subunit; hTERT – human telomerase catalytic subunit; hgTER – Heterocephalus glaber telomerase RNA.
D. V. Kapustin, A. I. Prostyakova, Ya. I. Alexeev, D. A. Varlamov, V. P. Zubov, S. K. Zavriev
High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis
The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation.
Е. А. Nikitina, A. V. Medvedeva, G. А. Zakharov, Е. V. Savvateeva-Popova
The Drosophila agnostic Locus: Involvement in the Formation of Cognitive Defects in Williams Syndrome
The molecular basis of the pathological processes that lead to genome disorders is similar both in invertebrates and mammals. Since cognitive impairments in Williams syndrome are caused by LIMK1 hemizygosity, could the spontaneous and mutant variants of the Drosophila limk1 gene serve as a model for studying two diagnostic features from three distinct cognitive defects of the syndrome? These two symptoms are the disturbance of visuospatial orientation and an unusualy strong fixation on the faces of other people during pairwise interaction with a stranger. An experimental approach to the first cognitive manifestation might be an analysis of the locomotor behavior of Drosophila larvae involving visuospatial orientation during the exploration of the surrounding environment. An approach to tackle the second manifestation might be an analysis of the most natural ways of contact between a male and a female during courtship (the first stage of this ritual is the orientation of a male towards a female and following the female with constant fixation on the female’s image). The present study of locomotor activity and cognitive repertoire in spontaneous and mutant variants of the Drosophila agnostic locus allows one to bridge alterations in the structure of the limk1 gene and behavior.
Williams syndrome; LIMK1; Drosophila; locomotor activity; learning; memory.
CRSC – conditioned reflex suppression of courtship; LI – learning index; CI – courtship index.
M. S. Syrkina, M. A. Rubtsov, D. M. Potashnikova, Y. D. Kondratenko, A. A. Dokrunova, V. P. Veiko
Cell Models for the Investigation of the Role of the Mucin MUC1 Extracellular Domain in Metastasizing
The speculations on the role of MUC1, a substance which is overexpressed in glandular cancer cells, on the metastatic potential of such cells are rooted in data that seem to indicate that cell malignization correlates with a change from the apical localization of mucin MUC1 to a peripheral one. Nonetheless, the role of MUC1 in cancer metastasizing remains far from clear. The major hurdle remains the absence of adequate cell models. The aim of the present study was to create cell models that present different fragments of the human mucin MUC1 extracellular domain on their surface. Genetic constructions were generated on the basis of the plasmid vector pEGFP-N3. These constructions contain fusion genes coding for chimeric proteins composed of different combinations of mucin MUC1 functional domains and identification markers (FLAG-epitope, located at the N-terminus, and EGFP, located at the C-terminus of the chimeric proteins). These constructions were used for a stable transformation of HT-29 human cancer cells. The transformants obtained were characterized by flow cytometry. The low expression level of endogenous mucin MUC1 and the high expression level of recombinant proteins were confirmed by real-time PCR. The microscopic examination of the transformed cells confirmed the membrane localization of the fusion proteins. The resulting cell models could be used to investigate the role of the mucin MUC1 domains in cancer cell metastasizing. The obtained cells are used as an applicable model of MUC1-expressing cancers and might be used to study the role of different functional fragments of mucin MUC1 in metastasizing.
Z. O. Shenkarev, E. N. Lyukmanova, A. S. Paramonov, P. V. Panteleev, S. V. Balandin, M. A. Shulepko, K. S. Mineev, T. V. Ovchinnikova, M. P. Kirpichnikov, A. S. Arseniev
Lipid–Protein Nanodiscs Offer New Perspectives for Structural and Functional Studies of Water-Soluble Membrane- Active Peptides
Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution by the apolipoprotein or a special membrane scaffold protein (MSP). In this work, the applicability of LPN-based membrane mimetics in the investigation of water-soluble membrane-active peptides was studied. It was shown that a pore-forming antimicrobial peptide arenicin-2 from marine lugworm (charge of +6) disintegrates LPNs containing both zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 : 1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs containing zwitterionic lipids (POPC), and it weakly interacts with the protein component of nanodiscs, MSP (charge of –6). The neurotoxin II (NTII, charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine receptor, shows a comparatively low affinity to LPNs containing anionic lipids (POPC/DOPG, 3 : 1 or POPC/DOPS, 4 : 1), and it does not bind to LPNs/POPC. The obtained data show that NTII interacts with the LPN/POPC/DOPS surface in several orientations, and that the exchange process among complexes with different topologies proceeds fast on the NMR timescale. Only one of the possible NTII orientations allows for the previously proposed specific interaction between the toxin and the polar head group of phosphatidylserine from the receptor environment (Lesovoy et al., Biophys. J. 2009. V. 97. № 7. P. 2089–2097). These results indicate that LPNs can be used in structural and functional studies of water-soluble membrane-active peptides (probably except pore-forming ones) and in studies of the molecular mechanisms of peptide-membrane interaction.
V. V. Dolgikh, I. V. Senderskiy, G. V. Tetz, V. V. Tetz
Optimization of the Protocol for the Isolation and Refolding of the Extracellular Domain of HER2 Expressed in Escherichia coli
Receptor 2 of the human epidermal growth factor (HER2/neu, c-erbB2) is a 185 kDa proto-oncogene protein characterized by an overexpression in some oncological diseases, including 30% of mammary glands cancers, as well as tumors in the ovary, stomach and other organs of the human body. Since HER2- tumor status testing is the essential part of a successful cancer treatment, the expression and purification of substantial amounts of the extracellular domain (ECD) of HER2 is an important task. The production of ECD HER2 in Escherichia coli has several advantages over the use of eukaryotic expression systems, but the bulk of the recombinant product in bacteria accumulates as insoluble protein inclusion bodies. In this study, we obtained ECD HER2 in Escherichia coli as insoluble inclusion bodies and elaborated a simple, efficient, and fast protocol for the solubilization, refolding, and isolation of the protein in soluble form.
ECD HER2 – extracellular domain of receptor 2 of the human epidermal growth factor; SDSPAGE – protein electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate as an anionic detergent.
E. A. Trifonova, T. V. Gabidulina, N. I. Ershov, V. N. Serebrova, A. Yu. Vorozhishcheva, V. A. Stepanov
Analysis of the Placental Tissue Transcriptome of Normal and Preeclampsia Complicated Pregnancies
Preeclampsia is one of the most severe gestational complications which is one of the leading causes of maternal and perinatal morbidity and mortality. A growth in the incidence of severe and combined forms of the pathology has been observed in recent years. According to modern concepts, inadequate cytotrophoblast invasion into the spiral arteries of the uterus and development of the ischemia-reperfusion syndrome in the placental tissue play the leading role in the development of preeclampsia, which is characterized by multipleorgan failure. In this regard, our work was aimed at studying the patterns of placental tissue transcriptome that are specific to females with PE and with physiological pregnancy, as well as identifying the potential promising biomarkers and molecular mechanisms of this pathology. We have identified 63 genes whose expression proved to differ significantly in the placental tissue of females with PE and with physiological pregnancy. A cluster of differentially expressed genes (DEG) whose expression level is increased in patients with preeclampsia includes not only the known candidate genes that have been identified in many other genome-wide studies (e.g., LEP, BHLHB2, SIGLEC6, RDH13, BCL6), but also new genes (ANKRD37, SYDE1, CYBA, ITGB2, etc.), which can be considered as new biological markers of preeclampsia and are of further interest. The results of a functional annotation of DEG show that the development of preeclampsia may be related to a stress response, immune processes, the regulation of cell-cell interactions, intracellular signaling cascades, etc. In addition, the features of the differential gene expression depending on preeclampsia severity were revealed. We have found evidence of the important role of the molecular mechanisms responsible for the failure of immunological tolerance and initiation of the pro-inflammatory cascade in the development of severe preeclampsia. The results obtained elaborate the concept of the pathophysiology of preeclampsia and contain the information necessary to work out measures for targeted therapy of this disease.
M. N. Garas, S. V. Tillib, O. V. Zubkova, V. N. Rogozhin, T. I. Ivanova, L. A. Vasilev, D. Yu. Logunov, M. M. Shmarov, I. L. Tutykhina, I. B. Esmagambetov, I. Yu. Gribova, A. S. Bandelyuk, B. S. Naroditsky, A. L. Gintsburg
Construction of a pIX-modified Adenovirus Vector Able to Effectively Bind to Nanoantibodies for Targeting
Current targeting strategies for genetic vectors imply the creation of a specific vector for every targeted receptor, which is time-consuming and expensive. Therefore, the development of a universal vector system whose surface can specifically bind molecules to provide efficient targeting is of particular interest. In this study, we propose a new approach in creating targeted vectors based on the genome of human adenovirus serotype 5 carrying the modified gene of the capsid protein pIX (Ad5-EGFP-pIX-ER): recombinant pseudoadenoviral nanoparticles (RPANs). The surfaces of such RPANs are able to bind properly modified chimeric nanoantibodies that specifically recognize a particular target antigen (carcinoembryonic antigen (CEA)) with high affinity. The efficient binding of nanoantibodies (аСЕА-RE) to the RPAN capsid surfaces has been demonstrated by ELISA. The ability of the constructed vector to deliver target genes has been confirmed by experiments with the tumor cell lines A549 and Lim1215 expressing CEA. It has been shown that Ad5-EGFP-pIX-ER carrying аСЕА-RE on its surface penetrates into the tumor cell lines A549 and Lim1215 via the CAR-independent pathway three times more efficiently than unmodified RPAN and Ad5-EGFP-pIX-ER without nanoantibodies on the capsid surface. Thus, RPAN Ad5-EGFP-pIX-ER is a universal platform that may be useful for targeted gene delivery in specific cells due to “nanoantibody–modified RPAN” binding.
adenoviral vector; pIX; leucine zipper; nanobody; CEA
RPAN – recombinant pseudoadenoviral nanoparticles; CEA – carcinoembryonic antigen; Ad – human adenovirus; Ad5 – Ad serotype 5; CAR – coxsackievirus and adenovirus receptor; a.a. – amino acid residue; pfu – plaque-forming unit.
S. S. Popov, K. K. Shulgin, A. N. Pashkov, A. A. Agarkov
The Effect of Melaxen on the Activity of Caspases and the Glutathione Antioxidant System in Toxic Liver Injury
A comparative study of the activity of caspase-1 and caspase-3, the glutathione antioxidant system and NADPH-generating enzymes (glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase) and a study of DNA fragmentation in the blood serum of patients with chronic alcoholic hepatitis during basic treatment and combination therapy including melaxen have been carried out. It was found that the blood serum level of reduced glutathione, which decreases in pathology, increased more significantly in patients receiving melaxen as compared to the group of patients receiving the standard treatment. More significant changes in the activity of caspase-1 and caspase-3, glutathione reductase, glutathione peroxidase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase toward the control values were observed during the combination therapy. The correction in the melatonin level under the influence of melaxen apparently had a positive effect on the free-radical homeostasis in patients, which resulted in more pronounced changes in the investigated parameters towards the normal values as compared to the basic treatment.
There Are Many Ways to Fight Aging. Results of the Third International Conference “Genetics of Aging and Longevity”
The 3rd International Conference “Genetics of Aging and Longevity” was held in Sochi, Russia, April 6 to 10, 2014, which was attended by over 300 delegates from 18 countries. Apart from 50 oral presentations given by world-leading gerontologists and an extensive poster session, four round tables dedicated to the development and integration of aging theories, the development of personalized medicine, and attracting venture capital to research were held. The conference participants signed an open letter to the World Health Organization with the appeal to organize the worldwide collection and integration of data on age-related diseases. The conference clearly demonstrated the progress in investigation of aging and the development of technologies for intervention in this process. In particular, identification of somatic mutations and DNA damages in individual cells has become possible; reliable markers of biological age have been characterized; many genes, alleles, processes, metabolites, intestinal bacteria, and external factors that influence the aging rate have been identified; online databases of age-related changes as well as the genomes of long-lived individuals and long-lived animal species have been generated. Preclinical trials of pharmacological agents that are likely capable of slowing aging, such as nicotinamide riboside, selective inhibitors of TORC1, and IGF-1 receptorblocking antibodies, have been carried out. Methods of cultivation and transplantation of artificial organs, targeted delivery of drugs to individual cells and organelles, targeted genome editing, and introduction of artificial chromosomes have been suggested.
Cobra Cytotoxins: Structural Organization and Antibacterial Activity
Cardiotoxins (cytotoxins, CT) are β-structured proteins isolated from the venom of cobra. They consist of 59–61 amino acid residues, whose antiparallel chains form three ‘fingers’. In contrast to neurotoxins with an overall similar fold, CTs are amphiphilic. The amphiphilicity is caused by positively charged lysine and arginine residues flanking the tips of the loops that consist primarily of hydrophobic amino acids. A similar distribution of amino acid residues is typical for linear (without disulfide bonds) cationic cytolytic peptides from the venoms of other snakes and insects. Many of them are now considered to be lead compounds in combatting bacterial infections and cancer. In the present review, we summarize the data on the antibacterial activity of CTs and compare it to the activity of linear peptides.
A. A. Nemudryi, K. R. Valetdinova, S. P. Medvedev, S. M. Zakian
TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery
Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isn’t enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared – this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.
V. M. Chernov, O. A. Chernova. T. Sanchez-Vega, А. I. Kolpakov, О. N. Ilinskaya
Mycoplasma Contamination of Cell Cultures: Vesicular Traffic in Bacteria and Control over Infectious Agents
Cell cultures are subject to contamination either with cells of other cultures or with microorganisms, including fungi, viruses, and bacteria. Mycoplasma contamination of cell cultures is of particular importance. Since cell cultures are used for the production of vaccines and physiologically active compounds, designing a system for controlling contaminants becomes topical for fundamental science and biotechnological production. The discovery of extracellular membrane vesicles in mycoplasmas makes it necessary to take into consideration the bacterial vesicular traffic in systems designed for controlling infectious agents. The extracellular vesicles of bacteria mediate the traffic of proteins and genes, participate in cell-to-cell interactions, as well as in the pathogenesis and development of resistance to antibiotics. The present review discusses the features of mycoplasmas, their extracellular vesicles, and the interaction between contaminants and eukaryotic cells. Furthermore, it provides an analysis of the problems associated with modern methods of diagnosis and eradication of mycoplasma contamination from cell cultures and prospects for their solution.
diagnosis and eradication, cell cultures, mycoplasma contamination
Structural Features of the Interaction between Human 8-Oxoguanine DNA Glycosylase hOGG1 and DNA
The purpose of the present review is to summarize the data related with the structural features of interaction between the human repair enzyme 8-oxoguanine DNA glycosylase (hOGG1) and DNA. The review covers the questions concerning the role of individual amino acids of hOGG1 in the specific recognition of the oxidized DNA bases, formation of the enzyme–substrate complex, and excision of the lesion bases from DNA. Attention is also focused upon conformational changes in the enzyme active site and disruption of enzyme activity as a result of amino acid mutations. The mechanism of damaged bases release from DNA induced by hOGG1 is discussed in the context of structural dynamics.
protein-nucleic acid recognition, human 8-oxoguanine DNA glycosylase, repair enzymes, loss-offunction mutants, structural analysis of hOGG1
AP – apurinic/apyrimidinic site; 8-oxoG – 7,8-dihydro-8-oxoguanine.
I. V.Golubev, N. V. Komarova, K. V. Ryzhenkova, T. A. Chubar, S. S. Savin, V. I. Tishkov
Study of the Structure-Function-Stability Relationships in Yeast D-amino Acid Oxidase: Hydrophobization of Alpha-Helices
Hydrophobization of alpha-helices is one of the general approaches used for improving the thermal stability of enzymes. A total of 11 serine residues located in alpha-helices have been found based on multiple alignments of the amino acid sequences of D-amino acid oxidases from different organisms and the analysis of the 3D-structure of D-amino acid oxidase from yeast Trigonopsis variabilis (TvDAAO, EC 220.127.116.11). As a result of further structural analysis, eight Ser residues in 67, 77, 78, 105, 270, 277, 335, and 336 positions have been selected to be substituted with Ala. S78A and S270A substitutions have resulted in dramatic destabilization of the enzyme. Mutant enzymes were inactivated during isolation from cells. Another six mutant TvDAAOs have been highly purified and their properties have been characterized. The amino acid substitutions S277A and S336A destabilized the protein globule. The thermal stabilities of TvDAAO S77A and TvDAAO S335A mutants were close to that of the wild-type enzyme, while S67A and S105A substitutions resulted in approximately 1.5- and 2.0-fold increases in the TvDAAO mutant thermal stability, respectively. Furthermore, the TvDAAO S105A mutant showed on average a 1.2- to 3.0-fold higher catalytic efficiency with D-Asn, D-Tyr, D-Phe, and D-Leu as compared to the wild-type enzyme.
D-amino acid oxidase from yeast Trigonopsis variabilis, protein engineering, hydrophobization of alpha-helices, site-directed mutagenesis, substrate specificity, thermal stability
Changes in Gene Expression Associated with Matrix Turnover, Chondrocyte Proliferation and Hypertrophy in the Bovine Growth Plate
The aim of the study is to investigate the interrelationships between the expression of genes for structural extracellular matrix molecules, proteinases and their inhibitors in the bovine fetal growth plate. This was analyzed by RT-PCR in microsections of the proximal tibial growth plate of bovine fetuses in relationship to expression of genes associated with chondrocyte proliferation, apoptosis, and matrix vascularization. In the resting zone the genes for extracellular matrix molecule synthesis were expressed. Extracellular matrix degrading enzymes and their inhibitors were also expressed here. Onset of proliferation involved cyclic upregulation of cell division-associated activity and reduced expression of extracellular matrix molecules. Later in the proliferative zone we noted transient expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with vascularization and apoptosis. With the onset of hypertrophy expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with vascularization and apoptosis were significantly upregulated. Terminal differentiation was characterized by high expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with apoptosis. This study reveals the complex interrelationships of gene expression in the physis that accompany matrix assembly, resorption, chondrocyte proliferation, hypertrophy, vascularization and cell death while principal zones of the growth plate are characterized by a distinct signature profile of gene expression.
N. Yu. Glazova, S. A. Merchieva, M. A. Volodina, E. A. Sebentsova, D. M. Manchenko, V. S. Kudrun, N. G. Levitskaya
Effects of Neonatal Fluvoxamine Administration on the Physical Development and Activity of the Serotoninergic System in White Rats
Selective serotonin reuptake inhibitors (SSRIs), including fluvoxamine, are widely used to treat depressive disorders in pregnant women. These antidepressants effectively penetrate through the placental barrier, affecting the fetus during the critical phase of neurodevelopment. Some clinical studies have linked prenatal exposure to SSRIs with increased neonatal mortality, premature birth, decreased fetal growth and delay in psychomotor development. However, the effects of prenatal exposure to SSRIs remain unknown. The administration of SSRIs in rodents during the first postnatal weeks is considered as an model for studying the effects of prenatal SSRIs exposure in human. The aim of this work was to study the acute effects of chronic fluvoxamine (FA) administration in white rat pups. The study was carried out in male and female rat pups treated with FA (10 mg/kg/day, intraperitoneally) from postnatal days 1 to 14. The lethality level, body weight, age of eye opening, and motor reflex maturation were recorded. The contents of biogenic amines and their metabolites in different brain structures were also determined. It was shown that neonatal FA administration led to increased lethality level, reduced body weight, and delayed maturation of motor reflexes. Furthermore, increased noradrenalin level in hypothalamus, serotonin level in hippocampus and serotonin metabolite 5-HIAA level in frontal cortex, hypothalamus, hippocampus, and striatum were observed in drug-treated animals compared to the control group. We can conclude that the altered activity of the serotoninergic system induced by fluvoxamine administration at early developmental stages leads to a delay in physical and motor development.
A. P. Yakimov, T. A. Seregina, A. A. Kholodnyak, R. A. Kreneva, A. S. Mironov, D. A. Perumov, A. L. Timkovskii
Possible Function of the ribT Gene of Bacillus subtilis: Theoretical Prediction, Cloning, and Expression
The complete decipherment of the functions and interactions of the elements of the riboflavin biosynthesis operon (rib operon) of Bacillus subtilis are necessary for the development of superproducers of this important vitamin. The function of its terminal ribT gene has not been established to date. In this work, a search for homologs of the hypothetical amino acid sequence of the gene product through databases, as well as an analysis of the homolgs, was performed; the distribution of secondary structure elements was theoretically predicted; and the tertiary structure of the RibT protein was proposed. The ribT gene nucleotide sequence was amplified and cloned into the standard high-copy expression vector pET15b and then expressed after induction with IPTG in E. coli BL21 (DE3) strain cells containing the inducible phage T7 RNA polymerase gene. The ribT gene expression was confirmed by SDS-PAGE. The protein product of the expression was purified by affinity chromatography. Therefore, the real possibility of RibT protein production in quantities sufficient for further investigation of its structure and functional activity was demonstrated.
I. M. Larina, V. A. Ivanisenko, E. N. Nikolaev, A. I. Grigorev
The Proteome of a Healthy Human during Physical Activity under Extreme Conditions
The review examines the new approaches in modern systems biology, in terms of their use for a deeper understanding of the physiological adaptation of a healthy human in extreme environments. Human physiology under extreme conditions of life, or environmental physiology, and systems biology are natural partners. The similarities and differences between the object and methods in systems biology, the OMICs (proteomics, transcriptomics, metabolomics) disciplines, and other related sciences have been studied. The latest data on environmental human physiology obtained using systems biology methods are discussed. The independent achievements of systems biology in studying the adaptation of a healthy human to physical activity, including human presence at high altitude, to the effects of hypoxia and oxidative stress have been noted. A reasonable conclusion is drawn that the application of the methods and approaches used in systems biology to study the molecular pattern of the adaptive mechanisms that develop in the human body during space flight can provide valuable fundamental knowledge and fill the picture of human metabolic pathways.
integrative physiology, space flight, proteomics, systems biology
OMICs – biological disciplines integrated in the group of post-genomic technologies, with the names ending in -omics; MALDI – matrix-assisted laser desorption/ionization; ESI – electrospray ionization; PCR – polymerase chain reaction; HUPO – Human Proteome Organization; C-HPP – Chromosome-Centric Human Proteome Project; HLPP – Human Liver Proteome Project; KEGGDB – Kyoto Encyclopedia of Genes and Genomes database; PGC-1α – peroxisome proliferator-activated receptor-γ coactivator 1α; HIF – hypoxiainducible factor; HSP70 – heat shock protein 70 kDA; PDIA3 – protein disulfide isomerase family A, member 3; ROS – reactive oxygen species; CV – coefficient of variation.
Mechanisms of Activation of Voltage- Gated Potassium Channels
Voltage-gated potassium ion channels (Kv) play an important role in a variety of cellular processes, including the functioning of excitable cells, regulation of apoptosis, cell growth and differentiation, the release of neurotransmitters and hormones, maintenance of cardiac activity, etc. Failure in the functioning of Kv channels leads to severe genetic disorders and the development of tumors, including malignant ones. Understanding the mechanisms underlying Kv channels functioning is a key factor in determining the cause of the diseases associated with mutations in the channels, and in the search for new drugs. The mechanism of activation of the channels is a topic of ongoing debate, and a consensus on the issue has not yet been reached. This review discusses the key stages in studying the mechanisms of functioning of Kv channels and describes the basic models of their activation known to date.
activation; potassium ion channels; modeling; structure.
Kv – voltage-gated potassium channel; aa – amino acid residue; VSD, voltage-sensing domain; SHM – sliding-helix model; TM – transport model; PM, paddle model; CMH, model of coordinated movement of helices; CM – consensus model; MCT – model of charge transfer; MKD – model of Kv deactivation; MMd – mechanistic model of Kv activation/deactivation; FRET – Förster resonance energy transfer; MM – molecular modeling; СTС – charge transfer center, MD – molecular dynamics; GC – gate channel.
A. V. Bagaev, A. V. Pichugin, E. S. Lebedeva, A. A. Lysenko, M. M. Shmarov, D. Yu. Logunov, B. S. Naroditsky, R. I. Ataullakhanov, R. M. Khaitov, A. L. Gintsburg
Regulation of the Target Protein (Transgene) Expression in the Adenovirus Vector Using Agonists of Toll-Like Receptors
Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88→ NF-kB, while the inhibition of transgene expression depends on TRIF→ IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88→ NF-kB signaling.
V. A. Vigont, O. A. Zimina, L. N. Glushankova, J. A. Kolobkova, M. A. Ryazantseva, G. N. Mozhayeva, E. V. Kaznacheyeva
STIM1 Protein Activates Store-Operated Calcium Channels in Cellular Model of Huntington’s Disease
We have shown that the expression of full-length mutated huntingtin in human neuroblastoma cells (SK-N-SH) leads to an abnormal increase in calcium entry through store-operated channels. In this paper, the expression of the N-terminal fragment of mutated huntingtin (Htt138Q-1exon) is shown to be enough to provide an actual model for Huntington’s disease. We have shown that Htt138Q-1exon expression causes increased store-operated calcium entry, which is mediated by at least two types of channels in SK-N-SH cells with different reversal potentials. Calcium sensor, STIM1, is required for activation of store-operated calcium entry in these cells. The results provide grounds for considering the proteins responsible for the activation and maintenance of the store-operated calcium entry as promising targets for developing novel therapeutics for neurodegenerative diseases.
E. A. Grebenik, A. N. Generalova, A. V. Nechaev, E.V. Khaydukov, K. E. Mironova, O. A. Stremovskiy, E.N. Lebedenko, A. V. Zvyagin, S. M. Deyev
Specific Visualization of Tumor Cells Using Upconversion Nanophosphors
The development of targeted constructs on the basis of photoluminescent nanoparticles with a high photo- and chemical stability and absorption/emission spectra in the “transparency window” of biological tissues is an important focus area of present-day medical diagnostics. In this work, a targeted two-component construct on the basis of upconversion nanophosphors (UCNPs) and anti-tumor 4D5 scFv was developed for selective labeling of tumor cells overexpressing the HER2 tumor marker characteristic of a number of human malignant tumors. A high affinity barnase : barstar (Bn : Bs) protein pair, which exhibits high stability in a wide range of pH and temperatures, was exploited as a molecular adapter providing self-assembly of the two-component construct. High selectivity for the binding of the two-component 4D5 scFv-Bn : UCNP-Bs construct to human breast adenocarcinoma SK-BR-3 cells overexpressing HER2 was demonstrated. This approach provides an opportunity to produce similar constructs for the visualization of different specific markers in pathogenic tissues, including malignant tumors.
E. N. Lyukmanova, M. A. Shulepko, M. L. Bychkov, Z. O. Shenkarev, A. S. Paramonov, A. O. Chugunov, A. S. Arseniev, D. A. Dolgikh, M. P. Kirpichnikov
Human SLURP-1 and SLURP-2 Proteins Acting on Nicotinic Acetylcholine Receptors Reduce Proliferation of Human Colorectal Adenocarcinoma HT-29 Cells
Human secreted Ly-6/uPAR related proteins (SLURP-1 and SLURP-2) are produced by various cells, including the epithelium and immune system. These proteins act as autocrine/paracrine hormones regulating the growth and differentiation of keratinocytes and are also involved in the control of inflammation and malignant cell transformation. These effects are assumed to be mediated by the interactions of SLURP-1 and SLURP-2 with the α7 and α3β2 subtypes of nicotinic acetylcholine receptors (nAChRs), respectively. Available knowledge about the molecular mechanism underling the SLURP-1 and SLURP-2 effects is very limited. SLURP-2 remains one of the most poorly studied proteins of the Ly-6/uPAR family. In this study, we designed for the first time a bacterial system for SLURP-2 expression and a protocol for refolding of the protein from cytoplasmic inclusion bodies. Milligram quantities of recombinant SLURP-2 and its 13С-15N-labeled analog were obtained. The recombinant protein was characterized by NMR spectroscopy, and a structural model was developed. A comparative study of the SLURP-1 and SLURP-2 effects on the epithelial cell growth was conducted using human colorectal adenocarcinoma HT-29 cells, which express only α7-nAChRs. A pronounced antiproliferative effect of both proteins was observed. Incubation of cells with 1 μM SLURP-1 and 1 μM SLURP-2 during 48 h led to a reduction in the cell number down to ~ 54 and 63% relative to the control, respectively. Fluorescent microscopy did not reveal either apoptotic or necrotic cell death. An analysis of the dose-response curve revealed the concentration-dependent mode of the SLURP-1 and SLURP-2 action with EC50 ~ 0.1 and 0.2 nM, respectively. These findings suggest that the α7-nAChR is the main receptor responsible for the antiproliferative effect of SLURP proteins in epithelial cells.
Investigation of Channel-Forming Activity of Polyene Macrolide Antibiotics in Planar Lipid Bilayers in the Presence of Dipole Modifiers
The role of membrane components, sterols, phospholipids and sphingolipids in the formation and functioning of ion-permeable nanopores formed by antifungal macrolide antibiotics, amphotericin B, nystatin and filipin in planar lipid bilayers was studied. Dipole modifiers, flavonoids and styryl dyes, were used as a tool to study the molecular mechanisms of polyene channel-forming activity. The introduction of dipole modifiers into the membrane bathing solutions was shown to change the conductance of single channels and the steadystate transmembrane current induced by polyene antibiotics in the sterol-containing phospholipid-bilayers. The conductance of single amphotericin B channels was found to depend on the dipole potential of the membrane. The experiments with various phospholipids, sterols, and polyenes led to the assumption that the shape of a phospholipid molecule, the presence of double bonds at the positions 7 and 22 of a sterol molecule, the number of conjugated double bonds, and the presence of an amino sugar in the polyene antibiotic molecule are important factors impacting the stability of polyene-lipid complexes forming ion-permeable pores. Experimental and literature data presented in the paper suggest that the channel-forming activity of polyene antibiotics is also affected by the physicochemical properties of polyene-enriched ordered membrane domains.
M. A. Chelombitko, V. S. Shishkina, O. P. Ilyinskaya, A. I. Kaminnyi, T. O. Pavlunina, N. N. Samovilova, E. V. Gracheva, E. M. Tararak, N. V. Prokazova
A Cytofluorometric Study of Membrane Rafts in Human Monocyte Subsets in Atherosclerosis
The peripheral blood monocytes of atherosclerotic patients are pre-activated and have some of the features of tissue macrophages. Their adhesion to the endothelium is 1.5 times higher than that of monocytes from healthy subjects, and they express a number of receptors and antigens typical of tissue macrophages. Additionally, earlier we showed that the biosynthesis of gangliosides, whose main function is the formation of membrane rafts, is significantly activated in blood monocytes from atherosclerotic patients, as well as during the in vitro differentiation of normal monocytes into macrophages. In this study, we investigated the expression of membrane rafts on various monocyte subsets from healthy subjects and atherosclerotic patients. Based on flow cytometry results, the monocytes in the examined atherosclerotic patients were found to differ from those in healthy subjects by a twofold increase in the proportion of the intermediate subset (CD14++/CD16+) and by enhancement in the expression of the fractalkine receptor CX3CR1 on the intermediate and non-classical subsets (CD14++/CD16+ and CD14+/CD16++) (2.3 and 1.8 times, respectively). This suggests a pre-activated state of monocytes in atherosclerotic patients. At the same time, the expression of the membrane raft marker on the monocyte subsets was similar in both studied groups. However, a study of the in vitro differentiation of monocytes into macrophages showed that the membrane raft expression increased 2 times as early as on the 1st day of culturing and 3 times on the 7th day compared to that in freshly isolated monocytes. Therefore, it is suggested that monocytes in atherosclerosis accumulate gangliosides that are used to form membrane rafts during the macrophage differentiation after the migration of monocytes into the arterial intima.
E. V. Leichenko, M. M. Monastirnaya, E. A. Zelepuga, E. S. Tkacheva, M. P. Isaeva, G. N. Likhatskaya, S. D. Anastyuk, E. P. Kozlovskaya
Hct-A Is a New Actinoporin Family from the Heteractis Crispa Sea Anemone
Several new actinoporin isoforms with molecular weights of 18995.5 to 19398.7 Da exhibiting a high hemolytic activity were isolated from the tropical sea anemone Heteractis crispa using a combination of liquid chromatography techniques. The actinoporins were demonstrated to occur as mono-, di-, and trimers in aqueous solutions. The sequences of the genes encoding actinoporins were identified, and the amino acid sequences of the new polypeptides belonging to the Hct-A actinoporin family were obtained. The new acinoporins differ in their isoelectric points, the number and localization of charged amino acid residues at the functionally important N-terminal fragment of the molecule, as well as in the charge of a tetrapeptide (amino acid residues 74–77) involved in an electrostatic interaction with the cytoplasmic membrane. A recombinant actinoporin, rHct-A2, with a molecular weight of 19141 Da, pI of 9.64, and hemolytic activity of 4.0 × 104 HU/mg, was obtained. The conductivity of the ion channels formed by rHct-A2 in the BLM was demonstrated to be similar to that of the native actinoporin from H. crispa. The obtained data expand knowledge on the structural and functional relationships of actinoporins and contribute to our understanding of the functioning mechanism of these molecules, which is the basis for the development of compounds with a high biomedical potential. Currently, they are considered as models for obtaining antitumor, antibacterial, and cardiac-stimulating agents.
Phosphoryl Guanidines: A New Type of Nucleic Acid Analogues
A new type of nucleic acid analogues with a phosphoryl guanidine group is described. Oxidation of polymer-supported dinucleoside 2-cyanoethyl phosphite by iodine in the presence of 1,1,3,3-tetramethyl guanidine yields a dinucleotide with an internucleoside tetramethyl phosphoryl guanidine (Tmg) group as the main product. The Tmg group is stable under conditions of solid-phase DNA synthesis and subsequent cleavage and deprotection with ammonia. Oligonucleotides with one or more Tmg groups bind their complementary DNA or RNA with affinity similar to that of natural oligodeoxyribonucleotides.
S. S. Terekhov, I. V. Smirnov, O. G. Shamborant, M. A. Zenkova, E. L. Chernolovskaya, D. V. Gladkikh, A. N. Murashev, I. A. Dyachenko, V. D. Knorre, A. A. Belogurov, N. A. Ponomarenko, S. M. Deyev, V. V. Vlasov, A. G. Gabibov
Excessive Labeling Technique Provides a Highly Sensitive Fluorescent Probe for Real-time Monitoring of Biodegradation of Biopolymer Pharmaceuticals in vivo
Recombinant proteins represent a large sector of the biopharma market. Determination of the main elimination pathways raises the opportunities to significantly increase their half-lives in vivo. However, evaluation of biodegradation of pharmaceutical biopolymers performed in the course of pre-clinical studies is frequently complicated. Noninvasive pharmacokinetic and biodistribution studies in living organism are possible using proteins conjugated with near-infrared dyes. In the present study we designed a highly efficient probe based on fluorescent dye self-quenching for monitoring of in vivo biodegradation of recombinant human butyrylcholinesterase. The maximum enhancement of integral fluorescence in response to degradation of an intravenously administered enzyme was observed 6 h after injection. Importantly, excessive butyrylcholinesterase labeling with fluorescent dye results in significant changes in the pharmacokinetic properties of the obtained conjugate. This fact must be taken into consideration during future pharmacokinetic studies using in vivo bioimaging.
fluorescent probe, biodegradation, pharmacokinetics, in vivo bioimaging, self-quenching, butyrylcholinesterase, proteolysis.
O. V. Shamova, D. S. Orlov, S. V. Balandin, E. I. Shramova, E. V. Tsvetkova, P. V. Panteleev, Yu. F. Leonova, A. A. Tagaev, V. N. Kokryakov, T. V. Ovchinnikova
Acipensins – Novel Antimicrobial Peptides from Leukocytes of the Russian Sturgeon Acipenser gueldenstaedtii
Antimicrobial peptides (AMPs) play an important role in the innate defense mechanisms in humans and animals. We have isolated and studied a set of antimicrobial peptides from leukocytes of the Russian sturgeon Acipenser gueldenstaedtii belonging to a subclass of chondrosteans, an ancient group of bony fish. Structural analysis of the isolated peptides, designated as acipensins (Ac), revealed in leukocytes of the Russian sturgeon six novel peptides with molecular masses of 5336.2 Da, 3803.0 Da, 5173.0 Da, 4777.5 Da, 5449.4 Da, and 2740.2 Da, designated as Ac1–Ac6, respectively. Complete primary structures of all the isolated peptides were determined, and the biological activities of three major components – Ac1, Ac2, and Ac6 – were examined. The peptides Ас1, Ас2, Ас3, Ас4, and Ac5 were found to be the N-terminal acetylated fragments 1–50, 1–35, 1–49, 1–44, and 1–51 of the histone Н2А, respectively, while Ас6 was shown to be the 62–85 fragment of the histone Н2А. The peptides Ac1 and Ac2 displayed potent antimicrobial activity towards Gram-negative and Gram-positive bacteria (Escherichia coli ML35p, Listeria monocytogenes EGD, MRSA ATCC 33591) and the fungus Candida albicans 820, while Ac6 proved effective only against Gram-negative bacteria. The efficacy of Ac 1 and Ac2 towards the fungus and MRSA was reduced upon an increase in the ionic strength of the solution. Ac1, Ac2, and Ac6, at concentrations close to their minimum inhibitory concentrations, enhanced the permeability of the E.coli ML35p outer membrane to the chromogenic marker, but they did not affect appreciably the permeability of the bacterial inner membrane in comparison with a potent pore-forming peptide, protegrin 1. Ac1, Ac2, and Ac6 revealed no hemolytic activity against human erythrocytes at concentrations of 1 to 40 μM and had no cytotoxic effect (1 to 20 μM) on K-562 and U-937 cells in vitro. Our findings suggest that histone-derived peptides serve as important anti-infective host defense molecules.
A. E. Gaydukov, P. O. Bogacheva, E. O. Tarasova, O. P. Balezina
The Mechanism of Choline-Mediated Inhibition of Acetylcholine Release in Mouse Motor Synapses
The mechanism of action of tonically applied choline, the agonist of α7 nicotinic acetylcholine receptors (nAChRs), to the spontaneous and evoked release of a neurotransmitter in mouse motor synapses in diaphragm neuromuscular preparations using intracellular microelectrode recordings of miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) was studied. Exogenous choline was shown to exhibit a presynaptic inhibitory effect on the amplitude and quantal content of EPPs for the activity of neuromuscular junction evoked by single and rhythmic stimuli. This effect was inhibited either by antagonists of α7-nAChRs, such as methyllycaconitine and α-cobratoxin, or by blocking SK-type calcium-activated potassium (KCa) channels with apamin or blocking intraterminal ryanodine receptors with ryanodine. A hypothesis was put forward that choline in mouse motoneuron nerve terminals can activate presynaptic α7-nAChRs, followed by the release of the stored calcium through ryanodine receptors and activation of SK-type KCa channels, resulting in sustained decay of the quantal content of the evoked neurotransmitter release.
quantal content, ryanodine receptors, choline, α7-nicotinic acetylcholine receptors, SK channels.
One of the most important aspects of modern biochemistry is the detailing of the mechanisms of cell energy supply. Mitochondria have for a long time been considered the “power plant” of cellular metabolism. In 1961, Peter Mitchell, who was later awarded the Nobel Prize for his discovery, published in Nature a revolutionary paper  that laid the groundwork for the chemiosmotic theory. According to this theory, the electrochemical potential of protons (ΔμH +) plays a critical role in energy production by mitochondria. During respiration, protons are transferred from the mitochondrial matrix to the intermembrane space to form a potential on the inner mitochondrial membrane. This respiration-generated potential is used by H+-ATP synthase to convert ADP to ATP. Mitchell also assumed that uncoupling agents (such as dinitrophenol), which suppress the synthesis of ATP, do not inhibit ATP synthase directly, but their action is a result of the dissipation of the membrane potential - the driving force of ATP synthesis. This was a revolutionary interpretation of events at that time. It aroused great interest among leading scientists, many of whom expressed skepticism and offered their own hypotheses on mitochondrial ATP synthesis. A valuable contribution to the development of modern mitochondriology was made by the outstanding Russian biochemist V.P. Skulachev. In this issue, the Editorial Board has decided to devote the “Forum” section to a brief description of the major milestones in the development of this fascinating field of research.
Senescence has been the focus of research for many centuries. Despite significant progress in extending average human life expectancy, the process of aging remains largely elusive and, unfortunately, inevitable. In this review, we attempted to summarize the current theories of aging and the approaches to understanding it.
aging, life expectancy, reactive oxygen species, accumulation of damage, telomerase, advanced glycation end-product.
Model Systems of Motor Neuron Diseases As a Platform for Studying Pathogenic Mechanisms and Searching for Therapeutic Agents
Over the past 30 years, many molecular genetic mechanisms underlying motor neuron diseases (MNDs) have been discovered and studied. Among these diseases, amyotrophic lateral sclerosis (ALS), which causes the progressive degeneration and death of central and peripheral motor neurons, and spinal muscular atrophy (SMA), which is one of the inherited diseases that prevail among hereditary diseases in the pattern of child mortality, hold a special place. These diseases, like most nerve, neurodegenerative, and psychiatric diseases, cannot be treated appropriately at present. Artificial model systems, especially those that are based on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are of paramount importance in searching for adequate therapeutic agents, as well as for a deep understanding of the MND pathogenesis. This review is mainly focused on the recent advance in the development of and research into cell and animal models of ALS and SMA. The main issues concerning the use of cellular technologies in biomedical applications are also described.
P. V. Panteleev, I. A. Bolosov, S. V. Balandin, T. V. Ovchinnikova
Structure and Biological Functions of β-Hairpin Antimicrobial Peptides
Antimicrobial peptides (AMPs) are evolutionarily ancient factors of the innate immune system that serve as a crucial first line of defense for humans, animals, and plants against infection. This review focuses on the structural organization, biosynthesis, and biological functions of AMPs that possess a β-hairpin spatial structure. Representatives of this class of AMPs are among the most active antibiotic molecules of animal origin. Due to their wide spectrum of activity and resistance to internal environmental factors, natural β-hairpin AMPbased compounds might become the most promising drug candidates.
D. V. Popov, E. A. Lysenko, I. V. Kuzmin, O. L. Vinogradova, A. I. Grigoriev
Regulation of PGC-1α Isoform Expression in Skeletal Muscles
The coactivator PGC-1α is the key regulator of mitochondrial biogenesis in skeletal muscle. Skeletal muscle expresses several PGC-1α isoforms. This review covers the functional role of PGC-1α isoforms and the regulation of their exercise-associated expression in skeletal muscle. The patterns of PGC-1α mRNA expression may markedly differ at rest and after muscle activity. Different signaling pathways are activated by different physiological stimuli, which regulate the expression of the PGC-1α gene from the canonical and alternative promoters: expression from a canonical (proximal) promoter is regulated by activation of the AMPK; expression from an alternative promoter, via a β2-adrenergic receptor. All transcripts from both promoters are subject to alternative splicing. As a result, truncated isoforms that possess different properties are translated: truncated isoforms are more stable and predominantly activate angiogenesis, whereas full-length isoforms manly regulate mitochondrial biogenesis. The existence of several isoforms partially explains the broad-spectrum function of this protein and allows the organism to adapt to different physiological stimuli. Regulation of the PGC-1α gene expression by different signaling pathways provides ample opportunity for pharmacological influence on the expression of this gene. Those opportunities might be important for the treatment and prevention of various diseases, such as metabolic syndrome and diabetes mellitus. Elucidation of the regulatory mechanisms of the PGC-1α gene expression and their functional role may provide an opportunity to control the expression of different isoforms through exercise and/or pharmacological intervention.
alternative splicing, alternative promoter, skeletal muscle, PGC-1α, gene expression.
A. A. Alekseeva, V. V. Fedorchuk, S. A. Zarubina, E. G. Sadykhov, A. D. Matorin, S. S. Savin, V. I. Tishkov
The Role of Ala198 in the Stability and Coenzyme Specificity of Bacterial Formate Dehydrogenases
It has been shown by an X-ray structural analysis that the amino acid residues Ala198, which are located in the coenzyme-binding domain of NAD+-dependent formate dehydrogenases (EC 18.104.22.168., FDH) from bacteria Pseudomonas sp.101 and Moraxella sp. C-1 (PseFDH and MorFDH, respectively), have non-optimal values of the angles ψ and φ. These residues were replaced with Gly by site-directed mutagenesis. The mutants PseFDH A198G and MorFDH A198G were expressed in E.coli cells and obtained in active and soluble forms with more than 95% purity. The study of thermal inactivation kinetics showed that the mutation A198G results in a 2.5- fold increase in stability compared to one for the wild-type enzymes. Kinetic experiments indicate that A198G replacement reduces the KM NAD+ value from 60 to 35 and from 80 to 45 μM for PseFDH and MorFDH, respectively, while the KM HCOO ? value remains practically unchanged. Amino acid replacement A198G was also added to the mutant PseFDH D221S with the coenzyme specificity changed from NAD+ to NADP+. In this case, an increase in thermal stability was also observed, but the influence of the mutation on the kinetic parameters was opposite: KM increased from 190 to 280 μM and from 43 to 89 mM for NADP+ and formate, respectively. According to the data obtained, inference could be drawn that earlier formate dehydrogenase from bacterium Pseudomonas sp. 101 was specific to NADP+, but not to NAD+.
O. S. Rogovaya, A. K. Fayzulin, A. V. Vasiliev, A. V. Kononov, V. V. Terskikh
Reconstruction of Rabbit Urethral Epithelium with Skin Keratinocytes
We have investigated the living skin equivalent (LSE) as an alternative source of plastic material for closing full-thickness epithelial-stromal urethral injuries. The possibility of transdifferentiation of epidermal keratinocytes, a component of 3D tissue constructs, was investigated in vivo in a model of the recovery of urethral injuries in laboratory rabbits. Autologous grafting of LSE in de-epithelialized urethra showed that skin keratinocytes placed in a specific in vivo microenvironment can be incorporated into the damaged area and function as urothelium. The use of EGFP transfected keratinocytes allowed us to identify transplanted cells. The reconstructed urethral tubes did not develop strictures or fistulas at the site of the grafted LSE. Immunohistochemical studies of neo-urothelium revealed EGFP-positive cells expressing the urothelial markers K7 and UP3.
O. A. Shadrina, T. S. Zatsepin, Yu. Yu. Agapkina, M. G. Isaguliants, M. B. Gottikh
Influence of Drug Resistance Mutations on the Activity of HIV-1 Subtypes A and B Integrases: a Comparative Study
Integration of human immunodeficiency virus (HIV-1) DNA into the genome of an infected cell is one of the key steps in the viral replication cycle. The viral enzyme integrase (IN), which catalyzes the integration, is an attractive target for the development of new antiviral drugs. However, the HIV-1 therapy often results in the IN gene mutations inducing viral resistance to integration inhibitors. To assess the impact of drug resistance mutations on the activity of IN of HIV-1 subtype A strain FSU-A, which is dominant in Russia, variants of the consensus IN of this subtype containing the primary resistance mutations G118R and Q148K and secondary compensatory substitutions E138K and G140S were prepared and characterized. Comparative study of these enzymes with the corresponding mutants of IN of HIV-1 subtype B strains HXB-2 was performed. The mutation Q148K almost equally reduced the activity of integrases of both subtypes. Its negative effect was partially compensated by the secondary mutations E138K and G140S. Primary substitution G118R had different influence on the activity of proteins of the subtypes A and B, and the compensatory effect of the secondary substitution E138K also depended on the viral subtype. Comparison of the mutants resistance to the known strand transfer inhibitors raltegravir and elvitegravir, and a new inhibitor XZ-259 (a dihydro-1H-isoindol derivative), showed that integrases of both subtypes with the Q148K mutation were insensitive to raltegravir and elvitegravir but were effectively inhibited by XZ-259. The substitution G118R slightly reduced the efficiency of IN inhibition by raltegravir and elvitegravir and caused no resistance to XZ_259.
integrase, HIV-1 subtype A, strain FSU-A, strand transfer inhibitor, drug resistance mutations.
HIV-1 – human immunodeficiency virus type 1; IN – integrase; INA – integrase of HIV-1 subtype A strain FSU-A; INB – integrase of HIV-1 subtype B strain HXB-2; RAL – raltegravir; EVG – elvitegravir; DTG – dolutegravir; IC50 – inhibitor concentration causing 50% decrease in enzymatic activity; FC – fold change in IC50 of a mutant protein compared to that of wild-type integrase, wt – wild-type integrase; PAAG – polyacrylamide gel; DTT – dithiothreitol, EDTA – ethylenediaminetetraacetic acid, ТBЕ – tris-borate-EDTA buffer.
O. V. Orlova, V. L. Drutsa, P. V. Spirin, V. S. Prasolov, P. M. Rubtsov, S. N. Kochetkov, S. N. Beljelarskaya
The Role of HCV E2 Protein Glycosylation in Functioning of Virus Envelope Proteins in Insect and Mammalian Cells
The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.
baculovirus expression vector system, hepatitis C virus envelope proteins E1 and E2, virus-like particles, N-linked protein glycosylation, Sf9 insect cells, mammalian HEK293T and Huh7.0 cells, oligonucleotide- directed mutagenesis.
CFU – colony forming units; HCV – hepatitis C virus; VLPs – virus-like particles; ER – endoplasmic reticulum.
D. V. Pankratov, E. González-Arribas, Yu. M. Parunova, M. A. Gorbacheva, Yu. S. Zeyfman, S. V. Kuznetsov, A. V. Lipkin, S. V. Shleev
New Nanobiocomposite Materials for Bioelectronic Devices
We have developed and synthesized nanobiocomposite materials based on graphene, poly(3,4-ethylenedioxythiophene), and glucose oxidase immobilized on the surface of various nanomaterials (gold nanoparticles and multi-walled carbon nanotubes) of different sizes (carbon nanotubes of different diameters). Comparative studies of the possible influence of the nanomaterial’s nature on the bioelectrocatalytic characteristics of glucose- oxidizing bioanodes in a neutral phosphate buffer solution demonstrated that the bioelectrocatalytic current densities of nanocomposite-based bioanodes are only weakly dependent on the size of the nanomaterial and are primarily defined by its nature. The developed nanobiocomposites are promising materials for new bioelectronic devices due to the ease in adjusting their capacitive and bioelectrocatalytic characteristics, which allows one to use them for the production of dual-function electrodes: i.e., electrodes which are capable of generating and storing electric power simultaneously.
S. V. Dentovskaya, S. A. Ivanov, P. Kh. Kopylov, R. Z. Shaikhutdinova, М. E. Platonov, T. I. Kombarova, T. V. Gapel’chenkova, S. V. Balakhonov, A. P. Anisimov
Selective Protective Potency of Yersinia pestis ΔnlpD Mutants
It has recently been shown that the NlpD lipoprotein is essential to Yersinia pestis virulence and that subcutaneous administration of the nlpD mutant could protect mice against bubonic and pneumonic plague better than the EV vaccine strain [PLoS One 2009. V. 4. № 9. e7023]. In this study, similar ΔnlpD mutants were generated on the basis of other Y. pestis parent strains, including strains from the subspecies microtus, which is avirulent to guinea pigs and humans. Comparative testing confirmed that immunization of mice with ΔnlpD mutants induces immunity 105 times more potent than the one induced by the administration of the EV vaccine strain. At the same time, NlpD- bacteria failed to protect guinea pigs in the case of a subcutaneous challenge with Y. pestis, inducing a 106 times less potent protection compared with that conferred by immunization with the EV vaccine strain. The possible causes of the observed phenomena are discussed.
Yersinia pestis, ΔnlpD mutant, selectivity of protective potency, live plague vaccine.
SCPM-Obolensk – State Collection of Pathogenic Microbes and Cell Cultures on the base of State Research Center for Applied Microbiology and Biotechnology; SRCAMB – State Research Center for Applied Microbiology and Biotechnology; II – index of immunity; ELISA – enzyme-linked immunosorbent assay; CFU – colony-forming unit; LPS – lipopolysaccharide; CS – current study; CCIARISFE– Culture Collection of Irkutsk Antiplague Research Institute of Siberia and Far East; PHAT – passive hemagglutination test; DCL (LD100) – absolutely lethal dose (dosis certa letalis); ImD50 – immunizing dose protecting 50% of infected animals from death; LB – Luria-Bertani broth; LD50 – dose lethal for 50 % of infected animals.
А. V. Аkimova, G. N. Rychkov, М. А. Grin, N. A. Filippova, G. V. Golovina, N. A. Durandin, A. M. Vinogradov, Т. А. Коkrashvili, А. F. Mironov, А. А. Shtil, V. А. Кuzmin
Interaction with Serum Albumin As a Factor of the Photodynamic Efficacy of Novel Bacteriopurpurinimide Derivatives
Optimization of the chemical structure of antitumor photosensitizers (PSs) is aimed at increasing their affinity to a transport protein, albumin and irreversible light-induced tumor cell damage. Bacteriopurpurinimide derivatives are promising PSs thanks to their ability to absorb light in the near infrared spectral region. Using spectrophotometry, we show that two new bacteriopurpurinimide derivatives with different substituents at the N atoms of the imide exocycle and the pyrrole ring A are capable of forming non-covalent complexes with human serum albumin (HSA). The association constant (calculated with the Benesi-Hildebrand equation) for N-ethoxybacteriopurpurinimide ethyloxime (compound 1) is higher than that for the methyl ether of methoxybacteriopurpurinimide (compound 2) (1.18×105 M-1 vs. 1.26×104 M-1, respectively). Molecular modeling provides details of the atomic interactions between 1 and 2 and amino acid residues in the FA1 binding site of HSA. The ethoxy group stabilizes the position of 1 within this site due to hydrophobic interaction with the protein. The higher affinity of 1 for HSA makes this compound more potent than 2 in photodynamic therapy for cultured human colon carcinoma cells. Photoactivation of 1 and 2 in cells induces rapid (within a few minutes of irradiation) necrosis. This mechanism of cell death may be efficient for eliminating tumors resistant to other therapies.
photosensitizers, albumin, association constant, photodynamic therapy, cancer, necrosis.
L. A. Balabanova, V. А. Golotin, I. Y. Bakunina, L. V. Slepchenko, V. V. Isakov, A. B. Podvolotskaya, V. А. Rasskazov
Recombinant α-N-Acetylgalactosaminidase from Marine Bacterium-Modifying A Erythrocyte Antigens
A plasmid based on pET-40b was constructed to synthesize recombinant α-N-acetylgalactosaminidase of the marine bacterium Arenibacter latericius KMM 426T (α-AlNaGal) in Escherichia coli cells. The yield of α-Al- NaGal attains 10 mg/ml with activity of 49.7 ± 1.3 U at 16°C, concentration of inductor 2 mM, and cultivation for 12 h. Techniques such as anion exchange, metal affinity and gel filtration chromatography to purify α-AlNaGal were applied. α-AlNaGal is a homodimer with a molecular weight of 164 kDa. This enzyme is stable at up to 50°C with a temperature range optimum activity of 20–37°C. Furthermore, its activity is independent of the presence of metal ions in the incubation medium. 1H NMR spectroscopy revealed that α-AlNaGal catalyzes the hydrolysis of the O-glycosidic bond with retention of anomeric stereochemistry and possesses a mechanism of action identical to that of other glycoside hydrolases of the 109 family. α-AlNaGal reduces the serological activity of A erythrocytes at pH 7.3. This property of α-AlNaGal can potentially be used for enzymatic conversion of A and AB erythrocytes to blood group O erythrocytes.
glycoside hydrolase GH109; Arenibacter latericius; 1H NMR spectroscopy; conversion of A erythrocytes.
N. V. Krakhmal, M. V. Zavyalova, E. V. Denisov, S. V. Vtorushin, V. M. Perelmuter
Cancer Invasion: Patterns and Mechanisms
Cancer invasion and the ability of malignant tumor cells for directed migration and metastasis have remained a focus of research for many years. Numerous studies have confirmed the existence of two main patterns of cancer cell invasion: collective cell migration and individual cell migration, by which tumor cells overcome barriers of the extracellular matrix and spread into surrounding tissues. Each pattern of cell migration displays specific morphological features and the biochemical/molecular genetic mechanisms underlying cell migration. Two types of migrating tumor cells, mesenchymal (fibroblast-like) and amoeboid, are observed in each pattern of cancer cell invasion. This review describes the key differences between the variants of cancer cell migration, the role of epithelial-mesenchymal, collective-amoeboid, mesenchymal-amoeboid, and amoeboid- mesenchymal transitions, as well as the significance of different tumor factors and stromal molecules in tumor invasion. The data and facts collected are essential to the understanding of how the patterns of cancer cell invasion are related to cancer progression and therapy efficacy. Convincing evidence is provided that morphological manifestations of the invasion patterns are characterized by a variety of tissue (tumor) structures. The results of our own studies are presented to show the association of breast cancer progression with intratumoral morphological heterogeneity, which most likely reflects the types of cancer cell migration and results from different activities of cell adhesion molecules in tumor cells of distinct morphological structures.
A. V. Grishin, M. S. Krivozubov, A. S. Karyagina, A. L. Gintsburg
Pseudomonas Aeruginosa Lectins As Targets for Novel Antibacterials
Pseudomonas aeruginosa is one of the most widespread and troublesome opportunistic pathogens that is capable of colonizing various human tissues and organs and is often resistant to many currently used antibiotics. This resistance is caused by different factors, including the acquisition of specific resistance genes, intrinsic capability to diminish antibiotic penetration into the bacterial cell, and the ability to form biofilms. This situation has prompted the development of novel compounds differing in their mechanism of action from traditional antibiotics that suppress the growth of microorganisms or directly kill bacteria. Instead, these new compounds should decrease the pathogens’ ability to colonize and damage human tissues by inhibiting the virulence factors and biofilm formation. The lectins LecA and LecB that bind galactose and fucose, as well as oligo- and polysaccharides containing these sugars, are among the most thoroughly-studied targets for such novel antibacterials. In this review, we summarize the results of experiments highlighting the importance of these proteins for P. aeruginosa pathogenicity and provide information on existing lectins inhibitors and their effectiveness in various experimental models. Particular attention is paid to the effects of lectins inhibition in animal models of infection and in clinical practice. We argue that lectins inhibition is a perspective approach to combating P. aeruginosa. However, despite the existence of highly effective in vitro inhibitors, further experiments are required in order to advance these inhibitors into pre-clinical studies.
In Vitro Mouse Ovarian Follicle Growth and Maturation in Alginate Hydrogel: Current State of the Art
This review describes the main factors affecting the in vitro development of mouse ovarian follicles under conditions of three-dimensional alginate hydrogel system. The factors discussed include concentration of alginate hydrogel, presence of additives (collagen, fibrin) influencing substrate rigidity; culture conditions; composition of culture media; substances that act like antioxidants (salts of ascorbic acid, glutathione) and contribute to the improvement of lipid metabolism (L-carnitine), hormones and growth factors. The methods for follicle group cultivation in alginate hydrogel and cocultivation of different cell populations with follicles encapsulated in alginate hydrogel are covered in the present article.
Search for Human Lactate Dehydrogenase A Inhibitors Using Structure-Based Modeling
The human lactate dehydrogenase isoform A plays an important role in the anaerobic metabolism of tumour cells and therefore constitutes an attractive target in the oncology field. Full-atom models of lactate dehydrogenase A (in complex with NADH and in the apo form) have been generated to enable structure-based design of novel inhibitors competing with pyruvate and NADH. The structural criteria for the selection of potential inhibitors were established, and virtual screening of a library of low-molecular-weight compounds was performed. A potential inhibitor, STK381370, was identified whose docking pose was stabilized through additional interactions with the loop 96-111 providing for the transition from the open to the closed conformation.
J. A. Filippova, G. A. Stepanov, D. V. Semenov, O. A. Koval, E. V. Kuligina, I. V. Rabinov, V. A. Richter
Modified Method of rRNA Structure Analysis Reveals Novel Characteristics of Box C/D RNA Analogues
Ribosomal RNA (rRNA) maturation is a complex process that involves chemical modifications of the bases or sugar residues of specific nucleotides. One of the most abundant types of rRNA modifications, ribose 2’-O-methylation, is guided by ribonucleoprotein complexes containing small nucleolar box C/D RNAs. Since the majority of 2’-O-methylated nucleotides are located in the most conserved regions of rRNA that comprise functionally important centers of the ribosome, an alteration in a 2’-O-methylation profile can affect ribosome assembly and function. One of the key approaches for localization of 2’-O-methylated nucleotides in long RNAs is a method based on the termination of reverse transcription. The current study presents an adaptation of this method for the use of fluorescently labeled primers and analysis of termination products by capillary gel electrophoresis on an automated genetic analyzer. The developed approach allowed us to analyze the influence of the synthetic analogues of box C/D RNAs on post-transcriptional modifications of human 28S rRNA in MCF-7 cells. It has been established that the transfection of MCF-7 cells with a box C/D RNA analogue leads to an enhanced modification level of certain native sites of 2’-O-methylation in the target rRNA. The observed effect of synthetic RNAs on the 2’-O-methylation of rRNA in human cells demonstrates a path towards targeted regulation of rRNA post-transcriptional maturation. The described approach can be applied in the development of novel diagnostic methods for detecting diseases in humans.
A. V. Stepanov, A. A. Belogurov Jr., P. Kothapalli, O. G. Shamborant, V. D. Knorre, G. B. Telegin, A. A. Ovsepyan, N. A. Ponomarenko, S. M. Deyev, S. V. Kaveri, A. G. Gabibov
Specific Depletion of Myelin-Reactive B Cells via BCR-Targeting
B cells play a crucial role in the development and pathogenesis of systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce antibodies, but also secrete pro-inflammatory cytokines and present specific autoantigens to T cells. The treatment of autoimmune diseases via the elimination of the majority of B cells using the monoclonal anti-CD19/20 antibody (Rituximab) causes systemic side effects and, thus, requires a major revision. Therapeutic intervention directed towards selective elimination of pathogenic autoreactive B cells has the potential to become a universal approach to the treatment of various autoimmune abnormalities. Here, we developed a recombinant immunotoxin based on the immunodominant peptide of the myelin basic protein (MBP), fused to the antibody Fc domain. We showed that the obtained immunotoxin provides selective in vivo elimination of autoreactive B cells in mice with experimental autoimmune encephalomyelitis. The proposed conception may be further used for the development of new therapeutics for a targeted treatment of multiple sclerosis and other autoimmune disorders.
multiple sclerosis, autoantigens, B cells, immunoglobulins, immunotoxins.