Vol 10, No 4 (2018)


The EIMB Hydrogel Microarray Technology: Thirty Years Later

Gryadunov D.A., Shaskolskiy B.L., Nasedkina T.V., Rubina A.Y., Zasedatelev A.S.


Biological microarrays (biochips) are analytical tools that can be used to implement complex integrative genomic and proteomic approaches to the solution of problems of personalized medicine (e.g., patient examination in order to reveal the disease long before the manifestation of clinical symptoms, assess the severity of pathological or infectious processes, and choose a rational treatment). The efficiency of biochips is predicated on their ability to perform multiple parallel specific reactions and to allow one to study the interactions of biopolymer molecules, such as DNA, proteins, glycans, etc. One of the pioneers of microarray technology was the Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences (EIMB), with its suggestion to immobilize molecular probes in the three-dimensional structure of a hydrophilic gel. Since the first experiments on sequencing by hybridization on oligonucleotide microarrays conducted some 30 years ago, the hydrogel microarrays designed at the EIMB have come a long and successful way from basic research to clinical laboratory diagnostics. This review discusses the key aspects of hydrogel microarray technology and a number of state-ofthe-art approaches for a multiplex analysis of DNA and the protein biomarkers of socially significant diseases, including the molecular genetic, immunological, and epidemiological aspects of pathogenesis.

Acta Naturae. 2018;10(4):4-18
pages 4-18 views

Multifaced Roles of the Urokinase System in the Regulation of Stem Cell Niches

Dergilev K.V., Stepanova V.V., Beloglazova I.B., Tsokolayeva Z.I., Parfenova E.V.


Proliferation, subsequent migration to the damaged area, differentiation into appropriate cell types, and/or secretion of biologically active molecules and extracellular vesicles are important processes that underlie the involvement of stem/progenitor cells in the repair and regeneration of tissues and organs. All these functions are regulated through the interaction between stem cells and the microenvironment in the tissue cell niches that control these processes through direct cell-cell interactions, production of the extracellular matrix, release of extracellular vesicles, and secretion of growth factors, cytokines, chemokines, and proteases. One of the most important proteolytic systems involved in the regulation of cell migration and proliferation is the urokinase system represented by the urokinase plasminogen activator (uPA, urokinase), its receptor (uPAR), and inhibitors. This review addresses the issues of urokinase system involvement in the regulation of stem cell niches in various tissues and analyzes the possible effects of this system on the signaling pathways responsible for the proliferation, programmed cell death, phenotype modulation, and migration properties of stem cells.

Acta Naturae. 2018;10(4):19-32
pages 19-32 views

Bacterial Enzymes and Antibiotic Resistance

Egorov A.M., Ulyashova M.M., Rubtsova M.Y.


The resistance of microorganisms to antibiotics has been developing for more than 2 billion years and is widely distributed among various representatives of the microbiological world. Bacterial enzymes play a key role in the emergence of resistance. Classification of these enzymes is based on their participation in various biochemical mechanisms: modification of the enzymes that act as antibiotic targets, enzymatic modification of intracellular targets, enzymatic transformation of antibiotics, and the implementation of cellular metabolism reactions. The main mechanisms of resistance development are associated with the evolution of superfamilies of bacterial enzymes due to the variability of the genes encoding them. The collection of all antibiotic resistance genes is known as the resistome. Tens of thousands of enzymes and their mutants that implement various mechanisms of resistance form a new community that is called “the enzystome.” Analysis of the structure and functional characteristics of enzymes, which are the targets for different classes of antibiotics, will allow us to develop new strategies for overcoming the resistance.

Acta Naturae. 2018;10(4):33-48
pages 33-48 views

“Noah’s Ark” Project: Interim Results and Outlook for Classic Collection Development

Kalyakin M.V., Seregin A.P., Solovchenko A.E., Kamenski P.A., Sadovnichiy V.A.


The “Noah’s Ark” project, afoot at M.V. Lomonosov Moscow State University since 2015 and aimed at studying biodiversity, is the largest ongoing Russian project in life sciences. During its implementation, several hundred new species have been described; a comprehensive genetic and biochemical characterization of these species, as well as that of the pre-existing specimens in Moscow University’s collections, has been performed. A consolidated IT system intended to house the knowledge generated by the project has been developed. Here, we summarize the investigations around the Moscow University classical biocollections which have taken place within the framework of the project and discuss future promise and the outlook for these collections.

Acta Naturae. 2018;10(4):49-58
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Gamma-Carbolines Derivatives As Promising Agents for the Development of Pathogenic Therapy for Proteinopathy

Skvortsova V.I., Bachurin S.O., Ustyugov A.A., Kukharsky M.S., Deikin A.V., Buchman V.L., Ninkina N.N.


Uncontrolled protein aggregation, accompanied by the formation of specific inclusions, is a major component of the pathogenesis of many common neurodegenerative diseases known as proteinopathies. The intermediate products of this aggregation are toxic to neurons and may be lethal. The development strategy of pathogenic therapy for proteinopathy is based on the design of drugs capable of both inhibiting proteinopathy progression and increasing the survival of affected neurons. The results of a decade-long research effort at leading Russian and international laboratories have demonstrated that Dimebon (Latrepirdine), as well as a number of its derivatives from a gamma-carboline group, show a strong neuroprotective effect and can modulate the course of a neurodegenerative process in both in vitro and in vivo model systems. The accumulated data indicate that gamma-carbolines are promising compounds for the development of pathogenic therapy for proteinopathies.

Acta Naturae. 2018;10(4):59-62
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Assessment of the Parameters of Adaptive Cell-Mediated Immunity in Naïve Common Marmosets (Callithrix jacchus)

Gordeychuk I.V., Tukhvatulin A.I., Petkov S.P., Abakumov M.A., Gulyaev S.A., Tukhvatulina N.M., Gulyaeva T.V., Mikhaylov M.I., Logunov D.Y., Isaguliants M.G.


Common marmosets are small New World primates that have been increasingly used in biomedical research. This report presents efficient protocols for assessment of the parameters of adaptive cell-mediated immunity in common marmosets, including the major subpopulations of lymphocytes and main markers of T- and B-cell maturation and activation using flow cytometry with a multicolor panel of fluorescently labelled antibodies. Blood samples from eight common marmosets were stained with fluorescently labeled monoclonal antibodies against their population markers (CD45, CD3, CD20, CD4, CD8) and lymphocyte maturation and activation markers (CD69, CD62L, CD45RO, CD107a and CD27) and analyzed by flow cytometry. Within the CD45+ population, 22.7±5.5% cells were CD3- CD20+ and 67.6±6.3% were CD3+CD20-. The CD3+ subpopulation included 55.7±5.5% CD3+CD4+CD8- and 34.3±3.7% CD3+CD4-CD8+ cells. Activation and maturation markers were expressed in the following lymphocyte proportions: CD62L on 54.0±10.7% of CD3+CD4+ cells and 74.4±12.1% of CD3+CD8+ cells; CD69 on 2.7±1.2% of CD3+CD4+ cells and 1.2±0.5% of CD3+CD8+ cells; CD45RO on 1.6±0.6% of CD3+CD4+ cells and 1.8±0.7% of CD3+CD8+ cells; CD107a on 0.7±0.5% of CD3+CD4+ cells and 0.5±0.3% of CD3+CD8+ cells; CD27 on 94.6±2.1% of CD3+ cells and 8.9±3.9% CD20+ cells. Female and male subjects differed in the percentage of CD3+CD4+CD45RO+ cells (1.9±0.5 in females vs 1.1±0.2 in males; p < 0.05). The percentage of CD20+CD27+ cells was found to highly correlate with animals’ age (r = 0.923, p < 0.005). The basal parameters of adaptive cell-mediated immunity in naïve healthy marmosets without markers of systemic immune activation were obtained. These parameters and the described procedures are crucial in documenting the changes induced in common marmosets by prophylactic and therapeutic immune interventions.

Acta Naturae. 2018;10(4):63-69
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Overexpression of Adenoviral E1A Sensitizes E1A+Ras-Transformed Cells to the Action of Histone Deacetylase Inhibitors

Igotti M.V., Svetlikova S.B., Pospelov V.A.


The adenoviral E1A protein induces cell proliferation, transformation, and tumor formation in rodents, on the one hand. On the other hand, E1A expression increases cell sensitivity to a number of cytotoxic agents. Therefore, E1A is a candidate for use as a component of combination therapy for malignant tumors. The highest augmentation in the cytotoxic effect was achieved by a combined use of E1A expression and histone deacetylases (HDAC) inhibitors. However, HDAC inhibitors do not induce apoptosis in cells transformed with E1A and cHa-ras oncogenes. In this study, it was shown that HDAC inhibitors reduce the expression of adenoviral E1A. However, under unregulated E1A overexpression, these cells undergo apoptosis in the presence of HDAC inhibitors. Treatment with a HDAC inhibitor, sodium butyrate (NaBut), was shown to activate the anti-apoptotic factor NF-kB in control cells. However, NaBut was unable to modulate the NF-kB activity in E1A overexpressed cells. Therefore, it is fair to postulate that cells transformed with E1A and cHa-ras oncogenes avoid the apoptosis induced by HDAC inhibitors thanks to a NaBut-dependent decrease in E1A expression.

Acta Naturae. 2018;10(4):70-78
pages 70-78 views

Variants of Mitochondrial Genome and Risk of Multiple Sclerosis Development in Russians

Kozin M.S., Kulakova O.G., Kiselev I.S., Balanovsky O.P., Boyko A.N., Favorova O.O.


For the first time in the history of ethnic Russians, an association analysis the development of multiple sclerosis (MS) was performed for the mitochondrial haplogroups H, J, K, and U, as well as for the individual mitochondrial DNA (mtDNA) polymorphisms discriminating these haplogroups (m.1719G > A, m. 7028C > T, m.9055G > A, m.10398A > G, m.12308A > G). A total of 283 unrelated patients with the relapsing-remitting form of MS and 290 healthy controls were enrolled in the study. Association of haplogroup J with MS was observed (P = 0.0055, OR = 2.00 [95% CI 1.21-3.41]). After gender stratification, the association remained significant in women (P = 0.0083, OR = 2.20 [95% CI 1.19-4.03]). A multilocus analysis of the association between combinations of mtDNA haplogroups with variants of 38 nuclear immune-related genes and MS risk was carried out. MS-associated biallelic combinations of haplogroup J with the alleles CCL5 rs2107538*A, PVT1 rs2114358*G, TNFSF14 rs1077667*C, and IL4 rs2243250*C, which were not associated with MS individually, were identified. For the combination of haplogroup J and the CCL5*A allele (P = 0.00043, OR = 5.47 [95% CI 1.85-16.15]), a epistatic (synergistic) interaction between the components was established using two statistical criteria: the PFLINT value in the Fisher-like interaction numeric test and the synergy factor, SF (PFLINT = 0.025, SF = 4.32 [95% CI 1.20-15.60]). The combination of haplogroup J and the PVT1*G allele is characterized by PFLINT = 0.084; SF = 3.05 [95% CI 1.00-9.31] and can also be epistatic. Thus, interaction between nuclear and mitochondrial genome components in the risk of developing MS was demonstrated for the first time.

Acta Naturae. 2018;10(4):79-86
pages 79-86 views

The Mechanism of Fluorescence Quenching of Protein Photosensitizers Based on miniSOG During Internalization of the HER2 Receptor

Kuzichkina E.O., Shilova O.N., Deyev S.M.


The protein photosensitizer miniSOG is a promising agent for photodynamic therapy. The genetically encoded phototoxins 4D5scFv-miniSOG and DARPin-miniSOG specifically bind to the HER2 receptor overexpressed on the surface of cancer cells and promote receptor-mediated internalization of HER2. We show that ingestion of proteins in a complex with the receptor reduces the fluorescent signal of the phototoxic module in endosomes. In order to clarify the mechanism of decrease in the fluorescence intensity of miniSOG-based proteins as they enter a cancer cell during internalization, we analyzed the influence of different factors, including low pH, proteolysis, cofactor reduction, and shielding, on changes in the fluorescence of photosensitizers. Shielding and absorption of miniSOG fluorescence by cell fluorophores, including cytochrome c, were found to contribute significantly to the changes in the fluorescent properties of miniSOG.

Acta Naturae. 2018;10(4):87-94
pages 87-94 views

The TLR4 Agonist Immunomax Affects the Phenotype of Mouse Lung Macrophages during Respiratory Syncytial Virus Infection

Nikonova A.A., Pichugin A.V., Chulkina M.M., Lebedeva E.S., Gaisina A.R., Shilovskiy I.P., Ataullakhanov R.I., Khaitov M.R., Khaitov R.M.


In the study, the effect of the TLR4 agonist Immunomax was investigated in vitro and in vivo. In particular, Immunomax was shown to polarize mouse bone marrow macrophages from the M0 and M2 states into the M1 state (ARG1 and iNOS mRNA expression levels were used to identify the mouse M1 and M2 phenotypes). Next, we investigated the prophylactic antiviral effect of Immunomax in both a model of mouse respiratory syncytial virus (RSV) infection and a model of RSV-induced bronchial asthma (BA) exacerbation. In the experiment with RSV-induced BA exacerbation, Immunomax-treated mice were characterized by a significant decrease of the viral load in lung homogenates, an increased amount of M1 macrophages in the lung, a tendency toward Th2-dependent ovalbumin-specific IgG1 antibodies decrease in blood serum, a significant increase in RSV-activated CD4+ T cells secreting IFNγ (Th1 cells), and a simultaneous significant decrease in the amount of CD4+ cells secreting IL-4 (Th2 cells) in the mouse spleen, which were detected by ELISPOT 1.5 months after experiment. These findings suggest that treatment with the TLR4 agonist Immunomax polarizes the immune response towards antiviral Th1 and may be used for short-term antiviral prophylaxis to prevent acute respiratory viral infections in asthmatics.

Acta Naturae. 2018;10(4):95-99
pages 95-99 views

Identification of Novel Interaction Partners of AIF Protein on the Outer Mitochondrial Membrane

Fadeeva N.P., Antipova N.V., Shender V.O., Anufrieva K.S., Stepanov G.A., Bastola S., Shakhparonov M.I., Pavlyukov M.S.


In response to the wide variety of external and internal signals, mammalian cells undergo apoptosis, programmed cell death. Dysregulation of apoptosis is involved in multiple human diseases, including cancer, autoimmunity, and ischemic injuries. Two types of apoptosis have been described: the caspase-dependent one, leading to digestion of cellular proteins, and caspase-independent apoptosis, resulting in DNA fragmentation. The latter type of apoptosis is executed by AIF protein and is believed to have appeared first during evolution. The key step in the caspase-independent apoptosis program is the dissociation of AIF from the outer mitochondrial membrane (OMM). However, the molecular mechanism of interaction between AIF and OMM remains poorly understood. In this study, we demonstrated that AIF can bind to OMM via mortalin protein. We confirmed interaction between AIF and mortalin both in vitro and in vivo and mapped the amino acid sequences that are important for the binding of these proteins. Next, we showed that apoptosis induction by chemotherapy leads to downregulation of AIF-mortalin interaction and dissociation of AIF from the OMM. Finally, a bioinformatic analysis demonstrated that a high level of mortalin expression correlates with a worse survival prognosis for glioma patients. Altogether, our data revealed that mortalin plays an important role in the regulation of the caspase-independent apoptotic pathway and allowed us to speculate that inhibition of AIF-mortalin interaction may induce a dissociation of AIF from the OMM and subsequent apoptosis of cancer cells.

Acta Naturae. 2018;10(4):100-109
pages 100-109 views

Zinc Finger Protein CG9890 - New Component of ENY2-Containing Complexes of Drosophila

Fursova N.A., Nikolenko J.V., Soshnikova N.V., Mazina M.Y., Vorobyova N.E., Krasnov A.N.


In previous studies, we showed that the insulator protein Su(Hw) containing zinc finger domains interacts with the ENY2 protein and recruits the ENY2-containing complexes on Su(Hw)-dependent insulators, participating in the regulation of transcription and in the positioning of replication origins. Here, we found interaction between ENY2 and CG9890 protein, which also contains zinc finger domains. The interaction between ENY2 and CG9890 was confirmed. It was established that CG9890 protein is localized in the nucleus and interacts with the SAGA, ORC, dSWI/SNF, TFIID, and THO protein complexes.

Acta Naturae. 2018;10(4):110-114
pages 110-114 views

Tag7-Mts1 Complex Induces Lymphocytes Migration via CCR5 and CXCR3 Receptors

Sharapova T.N., Romanova E.A., Sashchenko L.P., Yashin D.V.


The discovery of new chemokines that induce the migration of lymphocytes to the infection site is important for the targeted search for therapeutic agents in immunotherapy. We recently showed that Tag7 (PGLYRP1), an innate immunity protein, forms a stable complex with the Ca2+ -binding protein Mts1 (S100A4), which is able to induce lymphocyte movement, although the individual Tag7 and Mts1 do not have this activity. The purpose of this study is to identify receptors that induce the migration of lymphocytes along the concentration gradient of the Tag7-Mts1 complex, and the components of this complex capable of interacting with these receptors. The study investigated the migration of human PBMC under the action of the Tag7-Mts1complex. PBMC of healthy donors were isolated using a standard Ficoll-Hypaque gradient centrifugation procedure. It has been established that the movement of PBMC along the concentration gradient of the Tag7-Mts1 complex is induced by the classical chemotactic receptors CCR5 and CXCR3. It has been shown that only Mts1 is able to bind to the extracellular domain of CCR5, however, this binding is not enough to induce cell movement. A comparative analysis of the primary and 3D structures of the three proteins revealed the homology of the amino acid sequence fragments of the Tag7-Mts1 protein complex with different sites of the CCR5 receptor ligand - MIP1α protein. In conclusion, it should be noted that the Tag7-Mts1 complex can be considered as a new ligand of the classical chemotactic receptors CCR5 and CXCR3.

Acta Naturae. 2018;10(4):115-120
pages 115-120 views

Short communications

The Preferable Binding Pose of Canonical Butyrylcholinesterase Substrates Is Unproductive for Echothiophate

Zlobin A.S., Zalevsky A.O., Mokrushina Y.A., Kartseva O.V., Golovin A.V., Smirnov I.V.


In this paper, we, for the first time, describe the interaction between the butyrylcholinesterase enzyme and echothiophate, a popular model compound and an analogue of the chemical warfare agents VX and VR, at the atomistic level. Competition between the two echothiophate conformations in the active site was found using molecular modeling techniques. The first one is close to the mode of binding of the substrates of choline series (butyrylcholine and butyrylthiocholine) and is inhibitory, since it is unable to react with the enzyme. The second one is characterized by a significantly worse estimated binding affinity and is reactive. Thus, echothiophate combines the features of two types of inhibitors: competitive and suicidal. This observation will help clarify the kinetic reaction scheme in order to accurately assess the kinetic constants, which is especially important when designing new butyrylcholinesterase variants capable of full-cycle hydrolysis of organophosphorus compounds.

Acta Naturae. 2018;10(4):121-124
pages 121-124 views

Mouse Model for Assessing the Subchronic Toxicity of Organophosphate Pesticides

Palikov V.A., Terekhov S.S., Palikova Y.A., Khokhlova O.N., Kazakov V.A., Dyachenko I.A., Panteleev S.V., Mokrushina Y.A., Knorre V.D., Shamborant O.G., Smirnov I.V., Gabibov A.G.


The development of antidotes to organophosphate poisons is an important aspect of modern pharmacology. Recombinant acetylcholinesterase and butyrylcholinesterase are effective DNA-encoded acceptors of organophosphate poisons and, in particular, pesticides. Here, we present the results of a study on the effectiveness of recombinant butyrylcholinesterase (BChE) in modeling organophosphate poisoning caused by oral administration of paraoxon at a dose of 2 mg / kg. The study showed a high activity of BChE as a protective agent for subchronic anticholinesterase poisoning in an in vivo model. The administration of BChE in a dose of 20 mg / kg allows one to avoid mortality, and also contributed to rapid recovery after model poisoning.

Acta Naturae. 2018;10(4):125-128
pages 125-128 views

Effect of Temperature, pH and Plasmids on In Vitro Biofilm Formation in Escherichia coli

Mathlouthi A., Pennacchietti E., Biase D.D.


Acid resistance (AR) in Escherichia coli is an important trait that protects this microorganism from the deleterious effect of low-pH environments. Reports on biofilm formation in E. coli K12 showed that the genes participating in AR were differentially expressed. Herein, we investigated the relationship between AR genes, in particular those coding for specific transcriptional regulators, and their biofilm-forming ability at the phenotypic level. The latter was measured in 96-well plates by staining the bacteria attached to the well, following 24-hour growth under static conditions, with crystal violet. The growth conditions were as follows: Luria Bertani (LB) medium at neutral and acidic pH, at 37°C or 25°C. We observed that the three major transcriptional regulators of the AR genes (gadX, gadE, gadW) only marginally affected biofilm formation in E. coli. However, a striking and novel finding was the different abilities of all the tested E. coli strains to form a biofilm depending on the temperature and pH of the medium: LB, pH 7.4, strongly supported biofilm formation at 25°C, with biofilm being hardly detectable at 37°C. On the contrary, LB, pH 5.5, best supported biofilm formation at 37°C. Moreover, we observed that when E. coli carried a plasmid, the presence of the plasmid itself affected the ability to develop a biofilm, typically by increasing its formation. This phenomenon varies from plasmid to plasmid, depends on growth conditions, and, to the best of our knowledge, remains largely uninvestigated.

Acta Naturae. 2018;10(4):129-132
pages 129-132 views

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