Vol 12, No 1 (2020)


Spinal cord microglia in health and disease

Kolos E., Korzhevsky D.


The review summarizes data of recent experimental studies on spinal microglia, the least explored cells of the spinal cord. It focuses on the origin and function of microglia in mammalian spinal cord embryogenesis. The main approaches to the classification of microgliocytes based on their structure, function, and immunophenotypic characteristics are analyzed. We discuss the results of studies conducted on experimental models of spinal cord diseases such as multiple sclerosis, amyotrophic lateral sclerosis, systemic inflammation, and some others, with special emphasis on the key role of microglia in the pathogenesis of these diseases. The review highlights the need to detect the new microglia-specific marker proteins expressed at all stages of ontogeny. New sensitive and selective microglial markers are necessary in order to improve identification of spinal cord microgliocytes in normal and pathological conditions. Possible morphometric methods to assess the functional activity of microglial cells are presented.

Acta Naturae. 2020;12(1):4-17
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Stochastics of degradation: the autophagic-lysosomal system of the cell

Kudriaeva A., Sokolov A., Belogurov A.


Autophagy is a conservative and evolutionarily ancient process that provides transfer of various cellular substances, organelles and potentially dangerous cellular components to the lysosome for their degradation. This process is crucial for the recycling of energy and substrates, which are required for the cellular biosynthesis. Autophagy plays a major role not only in the survival of cells under stress conditions, but also is actively involved in maintaining of cellular homeostasis. It has multiple effects on immune system and cellular remodeling during organism development. The effectiveness of autophagy is ensured by the tightly managed interaction of two organelles – autophagosomes and lysosomes. Despite significant progress in description of molecular mechanisms underlying in autophagolysosomal system (ALS) functioning, many fundamental questions are still open. Namely, specialized functions of lysosomes and role of ALS in pathogenesis of human diseases are still enigmatic. Knowledge about mechanisms of subsequent stages of autophagolysosomal degradation, from the initiation of autophagy to the terminal stage of substrate destruction in the lysosome, may generate new approaches in order to orchestrate ALS and therefore selectively control cellular proteostasis.

Acta Naturae. 2020;12(1):18-32
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Genes that control vaccinia virus immunogenicity

Shchelkunov S., Shchelkunova G.A.


The live smallpox vaccine was a historical first and highly effective vaccine. However, along with high immunogenicity, the vaccinia virus (VACV) caused serious side effects in vaccinees, sometimes with lethal outcomes. Therefore, after global eradication of smallpox, VACV vaccination was stopped. For this reason, most of the human population worldwide lacks specific immunity against not only smallpox, but also other zoonotic orthopoxviruses. Outbreaks of diseases caused by these viruses have increasingly occurred in humans on different continents. However, use of the classical live VACV vaccine for prevention against these diseases is unacceptable because of potential serious side effects, especially in individuals with suppressed immunity or immunodeficiency (e.g., HIV-infected patients). Therefore, highly attenuated VACV variants that preserve their immunogenicity are needed. This review discusses current ideas about the development of a humoral and cellular immune response to orthopoxvirus infection/vaccination and describes genetic engineering approaches that could be utilized to generate safe and highly immunogenic live VACV vaccines.

Acta Naturae. 2020;12(1):33-41
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Research Articles

Simple recommendations for improving efficiency in generating genome-edited mice

Averina O.A., Vysokikh M.Y., Permyakov O.A., Sergiev P.V.


The generation of transgenic model organisms (primarily mice) is an integral part of modern fundamental and applied research. Simple techniques based on the biology of these laboratory rodents can often increase efficiency when generating genome-edited mouse strains. In this study, we share our three years of experience in the optimization of mouse genome editing based on microinjection of CRISPR/Cas9 components into ca. 10,000 zygotes. We tested a number of techniques meant to improve efficiency in generating knockout mice, such as optimization of the superovulation method and choosing the optimal mouse strains to be used as zygote donors and foster mothers. The presented results might be useful to laboratories aiming to quickly and efficiently create new mouse strains with tailored genome editing.

Acta Naturae. 2020;12(1):42-50
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Establishment of a FDC-P1 murine cell line with human KIT N822K gene overexpression

Vagapova E.R., Lebedev T.D., Popenko V.I., Leonova O.G., Spirin P.V., Prasolov V.S.


The mechanism of resistance of leukemia cells to chemotherapeutic drugs remains poorly understood. New model systems for studying the processes of malignant transformation of hematopoietic cells are needed. Based on cytokine-dependent murine acute myeloid leukemia (AML) FDC-P1 cells, we generated a new cell line with ectopic expression of the KIT gene encoding mutant human receptor tyrosine kinase (N822K). We investigated the role played by overexpression of the mutant KIT in the survival of leukemia cells and their sensitivity to therapeutic drugs. We also generated a co-culture system consisting of FDC-P1 murine leukemia cells and a HS-5 human stromal cell line. Our data can be used for a further comprehensive analysis of the role of KIT N822K mutation in the cellular response to anti-leukemic drugs, growth factors, and cytokines. These data are of interest in the development of new effective therapeutic approaches to the treatment of acute leukemia.

Acta Naturae. 2020;12(1):51-55
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The genome structure of ciprofloxacin-resistant Mycoplasma hominis clinical isolates

Kolesnikova E.A., Brusnigina N.F., Makhova M.A., Alekseeva A.E.


The genome structure of three ciprofloxacin-resistant Mycoplasma hominis clinical isolates was studied using next-generation sequencing on the Illumina platform. The protein sequences of the studied Mycoplasma strains were found to have a high degree of homology. Mycoplasma hominis (M45, M57, MH1866) was shown to have limited biosynthetic capabilities, associated with the predominance of the genes encoding the proteins involved in catabolic processes. Multiple single-nucleotide substitutions causing intraspecific polymorphism of Mycoplasma hominis were found. The genes encoding the efflux systems – ABC transporters (the ATP-binding cassette superfamily) and proteins of the MATE (multidrug and toxic compound extrusion) family – were identified. The molecular mechanism of ciprofloxacin resistance of the Mycoplasma hominis M45 and M57 isolates was found to be associated with the Ser83Leu substitution in DNA gyrase subunit A. In the Mycoplasma hominis MH1866 isolate it was related to the Lys144Arg substitution in topoisomerase IV subunit A.

Acta Naturae. 2020;12(1):56-62
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The behavioral and neurochemical aspects of the interaction between antidepressants and unpredictable chronic mild stress

Kudryashov N.V., Kalinina T.C., Shimshirt A.A., Volkova A.V., Narkevich V.B., Naplekova P.L., Kasabov K.A., Kudrin V.C., Voronina T.A., Fisenko V.P.


The behavioral and neurochemical effects of amitriptyline (10 mg/kg, i.p.) and fluoxetine (20 mg/kg, i.p.) after single and chronic administration in the setting of unpredictable mild stress in outbred mice ICR (CD-1) were studied. After 28-day exposure to stress we observed an increase in depressive reactions in forced swim test in mice and decrease in serotonin (5-HT) and 5-oxyindol acetic acid (5-HIAA) levels in hippocampus, an increase in noradrenaline (NA) concentration in hypothalamus. Single and chronic administration of amitriptyline and fluoxetine led to shortening of immobilization and increase in swimming in forced swim test. The antidepressant-like effect of fluoxetine, but not of amitriptyline, after single injection coincided with an increase of 5-HT turnover in hippocampus. The chronic administration of the antidepressants caused an increase in NA levels in hypothalamus. Thus, antidepressant-like effect of amitriptyline and fluoxetine may arise from enhancement of stress-dependent adaptive mechanisms depleted by chronic stress.

Acta Naturae. 2020;12(1):63-72
pages 63-72 views

Subtypes of neurohypophyseal nonapeptide receptors and their functions in rat kidneys

Kutina A.V., Makashov A.A., Balbotkina E.V., Karavashkina T.A., Natochin Y.V.


The nonapeptides of neurohypophysis, vasotocin and mesotocin, detected in most vertebrates, are replaced by vasopressin and oxytocin in mammals. Using bioinformatics methods, we determined the spectrum of receptor subtypes for these hormones in mammals and their physiological effects in the kidneys of rats. A search for sequences similar to the vertebrate vasotocin receptor by proteomes and transcriptomas of nine mammalian species and the rat genome revealed three subtypes of vasopressin receptors (V1a, V1b, and V2) and one type of oxytocin receptors. In the kidneys of non-anesthetized rats, which received a water load of 2 ml per 100 g of body weight, three effects of vasopressin were revealed: 1) increased reabsorption of water and sodium, 2) increased excretion of potassium ions, and 3) increased excretion of sodium ions. It has been suggested that each of the effects on the kidney is associated with selective stimulation of the vasopressin receptor subtypes V2, V1b, and V1a depending on the concentration of nonapeptide. In experiments on non-anaesthetized rats with a water load, the injection of oxytocin reduces the reabsorption of solute-free water in the kidneys and increases the excretion of sodium ions. The possible physiological mechanisms behind the realization of both effects with the participation of a single type of oxytocin receptors are being analyzed. Thus, the spectrum of activated receptor subtypes varies depending on the current concentration of neurohypophyseal hormones, as a result of which the predominant effect on renal function changes, which ensures precise regulation of water-salt homeostasis.

Acta Naturae. 2020;12(1):73-83
pages 73-83 views

The telomeric Cdc13 protein from yeast Hansenula polymorpha

Malyavko A.N., Dontsova O.A.


Telomeres are special structures at the ends of chromosomes that play an important role in the protection of the genetic material. Telomere composition is very diverse; noticeable differences can often be observed even among closely related species. Here, we identify the homolog of telomeric protein Cdc13 in the thermotolerant yeast Hansenula polymorpha. We show that it can specifically bind single-stranded telomeric DNA, as well as interact with the Stn1 protein. In addition, we have uncovered an interaction between Cdc13 and TERT (one of the core components of the telomerase complex), which suggests that Cdc13 is potentially involved in telomerase recruitment to telomeres in H. polymorpha.

Acta Naturae. 2020;12(1):84-88
pages 84-88 views

The mechanisms of cardiac protection using a synthetic agonist of galanin receptors during chronic administration of doxorubicin

Studneva I.M., Veselova O.М., Bahtin A.A., Konovalova G.G., Lankin V.Z., Pisarenko O.I.


The use of the anticancer drug doxorubicin (Dox) is limited by its cardiotoxic effect. The aim of this work was to study the effect of a new synthetic agonist of the galanin receptor GalR1-3 [βAla14, His15]-galanine (2–15) (G) on the metabolism, antioxidant enzyme activity, and cardiac function in rats with cardiomyopathy (CM) caused by chronic administration of Dox. Coadministration of peptide G and Dox significantly increased the fractional shortening (FS) and ejection fraction (EF) by an average of 30 ± 4% compared with the indices in the Dox group. The reduced severity of cardiac dysfunction under the action of G was accompanied by a 2.5-fold decrease in the activity of creatine kinase-MB (CK-MB) in blood plasma. The protective mechanism of the action of peptide G is caused by a reduced lipid peroxidation (LP) that is due to the increased activity of Cu,Zn superoxide dismutase (Cu,Zn-SOD) and glutathione peroxidase (GSH-Px) in the damaged heart. Administration of peptide G significantly increased the adenine nucleotide pool (ΣAH), ATP content, and the levels of phosphocreatine (PCr) and total creatine (ΣCr) in the damaged myocardium. It also reduced lactate accumulation relative to its content in the Dox group. The better energy supply of cardiomyocytes after treatment with peptide G prevented the accumulation of cytotoxic ammonia and disruption in the metabolism of the key myocardial amino acids (glutamic acid (Glu), aspartic acid (Asp), and alanine (Ala)). Peptide G significantly improved the morphological parameters of the heart in rats treated with Dox. The results show promise in using peptide G to efficiently correct functional, morphological, and metabolic damage to the heart caused by anthracycline chemotherapy.

Acta Naturae. 2020;12(1):89-98
pages 89-98 views

Lipophilic prodrug of methotrexate in the membrane of liposomes promotes their uptake by human blood phagocytes

Tretiakova D.C., Khaidukov S.V., Babayants A.A., Frolova I.S., Shcheglovitova O.N., Onishchenko N.R., Vodovozova E.L.


Previously, we showed that incorporation of methotrexate (MTX) in the form of a lipophilic prodrug (MTXDG) in 100-nm lipid bilayer liposomes of egg phosphatidylcholine can allow one to reduce toxicity and improve the antitumor efficiency of MTX in a mouse model of T-cell leukemic lymphoma. However, in our hemocompatibility tests in vitro, MTX liposomes caused complement (C) activation, obviously due to binding on the liposome surface and fragmentation of the C3 complement factor. In this work, we studied the interactions of MTX liposomes carrying stabilizing molecules phosphatidylinositol (PI), ganglioside GM1, or a lipid conjugate of N-carboxymethylated oligoglycine (CMG) in the bilayer with subpopulations of human blood leukocytes. Liposomes labeled with BODIPY-phosphatidylcholine were incubated with whole blood (30 min and 1 h, 37°C), blood cells were lysed with a hypotonic buffer, and the fluorescence of the liposomes bound but not internalized by the leukocytes was quenched by crystal violet. Cell suspensions were analyzed by flow cytometry. Incorporation of MTXDG dramatically enhanced the phagocytosis of liposomes of any composition by monocytes. Neutrophils consumed much less of the liposomes. Lymphocytes did not accumulate liposomes. The introduction of PI into MTX liposomes practically did not affect the specific consumption of liposomes by monocytes, while CMG was likely to increase the consumption rate regardless of the presence of MTXDG. The GM1 ganglioside presumably shielded MTX liposomes from phagocytosis by one of the monocyte populations and increased the efficiency of monocyte uptake by another population, probably one expressing C3b-binding receptors (C3b was detected on liposomes after incubation with blood plasma). MTX liposomes were shown to have different effects on TNF-α production by activated leukocytes, depending on the structure of the stabilizing molecule.

Acta Naturae. 2020;12(1):99-109
pages 99-109 views

Short communications

Relationship between the mRNA expression levels of calpains 1/2 and proteins involved in cytoskeleton remodeling

Kakurina G.V., Kolegova E.S., Shashova E.E., Cheremisina O.V., Choynzonov E.L., Kondakova I.V.


Remodeling of the cytoskeleton underlies various cellular processes, including those associated with metastasis. The role of the proteases and proteins involved in cytoskeletal reorganization is being actively studied. However, there are no published data on the relationship between the mRNA expression levels of calpains 1/2 (CAPN 1/2) and the proteins associated with cytoskeleton remodeling. Therefore, the purpose of our study was to establish the relationship between the mRNA expression levels of CAPN 1/2 and the proteins involved in cytoskeletal reorganization, such as cell motility markers (SNAI1, VIM, and RND3) and actin-binding proteins (CFN1, PFN1, EZR, FSCN1, and CAP1) using the model of laryngeal/laryngopharyngeal squamous cell carcinoma (LC). The gene expression level was determined by reverse transcriptase real-time PCR and calculated using the 2-ΔΔCt method in paired tissue samples of 44 patients with LC (T1-4N0-2M0). The patients were divided into two groups: those with low and those with high CAPN 1/2 expression levels. It was found that metastasis in LC patients was associated with decreased expression levels of VIM and CAP1, and increased levels of CAPN1. A high level of CAPN2 was accompanied by a high expression level of EZR, indicating the activation of invasion processes. The results obtained need to be confirmed in further studies using a larger sample of patients and target genes. Our study is important in elucidating the mechanisms that underlie cancer progression and metastasis, a development that could subsequently open the way to a search for new prognostic and predictive markers of laryngeal/laryngopharyngeal cancer progression.

Acta Naturae. 2020;12(1):110-113
pages 110-113 views

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