Vol 14, No 1 (2022)


Fifty Years of Research on Protonophores: Mitochondrial Uncoupling As a Basis for Therapeutic Action

Kotova E.A., Antonenko Y.N.


Protonophores are compounds capable of electrogenic transport of protons across membranes. Protonophores have been intensively studied over the past 50 years owing to their ability to uncouple oxidation and phosphorylation in mitochondria and chloroplasts. The action mechanism of classical uncouplers, such as DNP and CCCP, in mitochondria is believed to be related to their protonophoric activity; i.e., their ability to transfer protons across the lipid part of the mitochondrial membrane. Given the recently revealed deviations in the correlation between the protonophoric activity of some uncouplers and their ability to stimulate mitochondrial respiration, this review addresses the involvement of some proteins of the inner mitochondrial membrane, such as the ATP/ADP antiporter, dicarboxylate carrier, and ATPase, in the uncoupling process. However, these deviations do not contradict the Mitchell theory but point to a more complex nature of the interaction of DNP, CCCP, and other uncouplers with mitochondrial membranes. Therefore, a detailed investigation of the action mechanism of uncouplers is required for a more successful pharmacological use, including their antibacterial, antiviral, anticancer, as well as cardio-, neuro-, and nephroprotective effects.

Acta Naturae. 2022;14(1):4-13
pages 4-13 views

Eukaryotic Ribosome Biogenesis: The 40S Subunit

Moraleva A.A., Deryabin A.S., Rubtsov Y.P., Rubtsova M.P., Dontsova O.A.


The formation of eukaryotic ribosomes is a sequential process of ribosomal precursors maturation in the nucleolus, nucleoplasm, and cytoplasm. Hundreds of ribosomal biogenesis factors ensure the accurate processing and formation of the ribosomal RNAs’ tertiary structure, and they interact with ribosomal proteins. Most of what we know about the ribosome assembly has been derived from yeast cell studies, and the mechanisms of ribosome biogenesis in eukaryotes are considered quite conservative. Although the main stages of ribosome biogenesis are similar across different groups of eukaryotes, this process in humans is much more complicated owing to the larger size of the ribosomes and pre-ribosomes and the emergence of regulatory pathways that affect their assembly and function. Many of the factors involved in the biogenesis of human ribosomes have been identified using genome-wide screening based on RNA interference. This review addresses the key aspects of yeast and human ribosome biogenesis, using the 40S subunit as an example. The mechanisms underlying these differences are still not well understood, because, unlike yeast, there are no effective methods for characterizing pre-ribosomal complexes in humans. Understanding the mechanisms of human ribosome assembly would have an incidence on a growing number of genetic diseases (ribosomopathies) caused by mutations in the genes encoding ribosomal proteins and ribosome biogenesis factors. In addition, there is evidence that ribosome assembly is regulated by oncogenic signaling pathways, and that defects in the ribosome biogenesis are linked to the activation of tumor suppressors.

Acta Naturae. 2022;14(1):14-30
pages 14-30 views

Chemiluminescence Detection in the Study of Free-Radical Reactions. Part 2. Luminescent Additives That Increase the Chemiluminescence Quantum Yield

Romodin L.A.


The present review examines the use of chemiluminescence detection to evaluate the course of free radical reactions in biological model systems. The application of the method is analyzed by using luminescent additives that enhance the luminescence thanks to a triplet–singlet transfer of the electron excitation energy from radical reaction products and its emission in the form of light with a high quantum yield; these additives are called chemiluminescence enhancers or activators. Examples of these substances are provided; differences between the so-called chemical and physical enhancers are described; coumarin derivatives, as the most promising chemiluminescence enhancers for studying lipid peroxidation, are considered in detail. The main problems related to the use of coumarin derivatives are defined, and possible ways of solving these problems are presented. Intrinsic chemiluminescence and the mechanism of luminescence accompanying biomolecule peroxidation are discussed in the first part of the review.

Acta Naturae. 2022;14(1):31-39
pages 31-39 views

Immunogenic Cell Death in Cancer Therapy

Troitskaya O.S., Novak D.D., Richter V.A., Koval O.A.


Apoptosis plays a crucial role in chemotherapy-induced cell death. The conventional theory holding that apoptosis needs to be immunologically silent has recently been revised, and the concept of immunogenic cell death (ICD) has been proposed. This review describes the main features of ICD induction. These ICD markers are important for the effectiveness of anticancer therapy, as well as for basic research into cell death regulation. The mechanism of the “vaccination effect” of dying cancer cells undergoing ICD has been fully described, including the activation of specific antitumor response after re-challenge by the same living tumor cells. This review also discusses the whole set of molecular events attributing cell death to immunogenic type: the exposure of calreticulin and the heat shock protein HSP70 to the outer surface of the cell membrane and the release of the nuclear protein HMGB1 and ATP into the extracellular space. ICD inducers of various nature (chemotherapy drugs, cytotoxic proteins, and oncolytic viruses), as well as physical methods, are classified in the current review.

Acta Naturae. 2022;14(1):40-53
pages 40-53 views

Artificial Scaffold PolypeptidesAs an Efficient Tool for the Targeted Delivery of Nanostructures In Vitro and In Vivo

Shipunova V.O., Deyev S.M.


The use of traditional tools for the targeted delivery of nanostructures, such as antibodies, transferrin, lectins, or aptamers, often leads to an entire range of undesirable effects. The large size of antibodies often does not allow one to reach the required number of molecules on the surface of nanostructures during modification, and the constant domains of heavy chains, due to their effector functions, can induce phagocytosis. In the recent two decades, targeted polypeptide scaffold molecules of a non-immunoglobulin nature, antibody mimetics, have emerged as much more effective targeting tools. They are small in size (3–20 kDa), possess high affinity (from subnano- to femtomolar binding constants), low immunogenicity, and exceptional thermodynamic stability. These molecules can be effectively produced in bacterial cells, and, using genetic engineering manipulations, it is possible to create multispecific fusion proteins for the targeting of nanoparticles to cells with a given molecular portrait, which makes scaffold polypeptides an optimal tool for theranostics.

Acta Naturae. 2022;14(1):54-72
pages 54-72 views

Research Articles

Tandem Exon Duplications Expanding the Alternative Splicing Repertoire

Ivanov T.M., Pervouchine D.D.


Tandem exon duplications play an important role in the evolution of eukaryotic genes, providing a generic mechanism for adaptive regulation of protein function. In recent studies, tandem exon duplications have been linked to mutually exclusive exon choice, a pattern of alternative splicing in which one and only one exon from a group of tandemly arranged exons is included in the mature transcript. Here, we revisit the problem of identifying tandem exon duplications in eukaryotic genomes using bioinformatic methods and show that tandemly duplicated exons are abundant not only in the coding parts, but also in the untranslated regions. We present a number of remarkable examples of tandem exon duplications, identify unannotated duplicated exons, and provide statistical support for their expression using large panels of RNA-seq experiments.

Acta Naturae. 2022;14(1):73-81
pages 73-81 views

Effect of Additional Amino Acid Replacements on the Properties of Multi-point Mutant Bacterial Formate Dehyderogenase PseFDH SM4S

Pometun A.A., Parshin P.D., Galanicheva N.P., Shaposhnikov L.A., Atroshenko D.L., Pometun E.V., Burmakin V.V., Kleymenov S.Y., Savin S.S., Tishkov V.I.


Formate dehydrogenase from Pseudomonas sp. 101 bacterium (PseFDH, EC is a research model for the elucidation of the catalytic mechanism of 2-oxyacid D-specific dehydrogenases enzyme superfamily. The enzyme is actively used for regeneration of the reduced form of NAD(P)H in chiral synthesis with oxidoreductases. A multi-point mutant PseFDH SM4S with an improved thermal and chemical stability has been prepared earlier in this laboratory. To further improve the properties of the mutant, additional single-point replacements have been introduced to generate five new PseFDH mutants. All new enzymes have been highly purified, and their kinetic properties and thermal stability studied using analysis of thermal inactivation kinetics and differential scanning calorimetry. The E170D amino acid change in PseFDH SM4S shows an increase in thermal stability 1.76- and 10-fold compared to the starting mutant and the wild-type enzyme, respectively.

Acta Naturae. 2022;14(1):82-91
pages 82-91 views

3D Models of Cellular Spheroids As a Universal Tool for Studying the Cytotoxic Properties of Anticancer Compounds In Vitro

Sogomonyan A.S., Shipunova V.O., Soloviev V.D., Larionov V.I., Kotelnikova P.A., Deyev S.M.


The aim of this work is to develop a 3D cell culture model based on cell spheroids for predicting the functional activity of various compounds in vivo. Agarose gel molds were made using 3D printing. The solidified agarose gel is a matrix consisting of nine low-adhesive U-shaped microwells of 2.3 × 3.3 mm for 3D cell spheroid formation and growth. This matrix is placed into a single well of a 12-well plate. The effectiveness of the cell culture method was demonstrated using human ovarian carcinoma SKOVip-kat cells stably expressing the red fluorescent protein Katushka in the cytoplasm and overexpressing the membrane-associated tumor marker HER2. The SKOVip-kat cell spheroids were visualized by fluorescence microscopy. The cell concentration required for the formation of same-shape and same-size spheroids with tight intercellular contacts was optimized. To verify the developed model, the cytotoxicity of the targeted immunotoxin anti-HER2 consisting of the anti-HER2 scaffold DARP 9_29 and a fragment of the Pseudomonas aeroginosa exotoxin, DARP-LoPE, was studied in 2D and 3D SKOVip-kat cell cultures. The existence of a difference in the cytotoxic properties of DARP-LoPE between the 2D and 3D cultures has been demonstrated: the IC50 value in the 3D culture is an order of magnitude higher than that in the monolayer culture. The present work describes a universal tool for 3D cultivation of mammalian cells based on reusable agarose gel molds that allows for reproducible formation of multicellular spheroids with tight contacts for molecular and cell biology studies.

Acta Naturae. 2022;14(1):92-100
pages 92-100 views

Analysis of the TREC and KREC Levels in the Dried Blood Spots of Healthy Newborns with Different Gestational Ages and Weights

Cheremokhin D.A., Shinwari K., Deryabina S.S., Bolkov M.A., Tuzankina I.A., Kudlay D.A.


ABSTRACT Inborn errors of immunity can be detected by evaluating circular DNA (cDNA) fragments of T- and B-cell receptors (TREC and KREC) resulting from the receptor gene rearrangement in T and B cells. Maturation and activation of the fetal immune system is known to proceed gradually according to the gestational age, which highlights the importance of the immune status in premature infants at different gestational ages. In this article, we evaluated TREC and KREC levels in infants of various gestational ages by real-time PCR with taking into account the newborn’s weight and sex. The 95% confidence intervals for TREC and KREC levels (expressed in the number of cDNA copies per 105 cells) were established for different gestational groups. The importance of studying immune system development in newborns is informed by the discovered dependence of the level of naive markers on the gestational stage in the early neonatal period.

Acta Naturae. 2022;14(1):101-108
pages 101-108 views

Genomic Estimated Breeding Valueof Milk Performance and Fertility Traits in the Russian Black-and-White Cattle Population

Sharko F.S., Khatib A., Prokhortchouk E.B.


A breakthrough in cattle breeding was achieved with the incorporation of animal genomic data into breeding programs. The introduction of genomic selection has a major impact on traditional genetic assessment systems and animal genetic improvement programs. Since 2010, genomic selection has been officially introduced in the evaluation of the breeding and genetic potential of cattle in Europe, the U.S., Canada, and many other developed countries. The purpose of this study is to develop a system for a genomic evaluation of the breeding value of the domestic livestock of Black-and-White and Russian Holstein cattle based on 3 milk performance traits: daily milk yield (kg), daily milk fat (%), and daily milk protein content (%) and 6 fertility traits: age at first calving (AFC), calving interval (CI), calving to first insemination interval (CFI), interval between first and last insemination (IFL), days open (DO), and number of services (NS). We built a unified database of breeding animals from 523 breeding farms in the Russian Federation. The database included pedigree information on 2,551,529 cows and 69,131 bulls of the Russian Holstein and Black-and-White cattle breeds, as well as information on the milk performance of 1,597,426 cows with 4,771,366 completed lactations. The date of birth of the animals included in the database was between 1975 and 2017. Genotyping was performed in 672 animals using a BovineSNP50 v3 DNA Analysis BeadChip microarray (Illumina, USA). The genomic estimated breeding value (GEBV) was evaluated only for 644 animals (427 bulls and 217 cows) using the single-step genomic best linear unbiased prediction – animal model (ssGBLUP-AM). The mean genetic potential was +0.88 and +1.03 kg for the daily milk yield, -0.002% for the milk fat content, and –0.003 and 0.001% for the milk protein content in the cows and bulls, respectively. There was negative genetic progress in the fertility traits in the studied population between 1975 and 2017. The reliability of the estimated breeding value (EBV) for genotyped bulls ranged from 89 to 93% for the milk performance traits and 85 to 90% for the fertility traits, whereas the reliability of the genomic estimated breeding value (GEBV) varied 54 to 64% for the milk traits and 23 to 60% for the fertility traits. This result shows that it is possible to use the genomic estimated breeding value with rather high reliability to evaluate the domestic livestock of Russian Holstein and Black-and-White cattle breeds for fertility and milk performance traits. This system of genomic evaluation may help bring domestic breeding in line with modern competitive practices and estimate the breeding value of cattle at birth based on information on the animal’s genome.

Acta Naturae. 2022;14(1):109-122
pages 109-122 views

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