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Quinone derivatives of triphenylphosphonium have proven themselves to be effective geroprotectors and antioxidants that prevent oxidation of cell components with participation of active free radicals - peroxide (RO 2 ·), alkoxy (RO·), and alkyl (R·) radicals, as well as reactive oxygen species (superoxide anion, singlet oxygen). Their most studied representatives are derivatives of plastoquinone (SkQ1) and ubiquinone (MitoQ), which in addition to antioxidant properties also have a strong antibacterial effect. In this study, we investigated antibacterial properties of other quinone derivatives based on decyltriphenylphosphonium (SkQ3, SkQT, and SkQThy). We have shown that they, just like SkQ1, inhibit growth of various Gram-positive bacteria at micromolar concentrations, while being less effective against Gram-negative bacteria, which is associated with recognition of the triphenylphosphonium derivatives by the main multidrug resistance (MDR) pump of Gram-negative bacteria, AcrAB-TolC. Antibacterial action of SkQ1 itself was found to be dependent on the number of bacterial cells. It is important to note that the cytotoxic effect of SkQ1 on mammalian cells was observed at higher concentrations than the antibacterial action, which can be explained by (i) the presence of a large number of membrane organelles, (ii) lower membrane potential, (iii) spatial separation of the processes of energy generation and transport, and (iv) differences in the composition of MDR pumps. Differences in the cytotoxic effects on different types of eukaryotic cells may be associated with the degree of membrane organelle development, energy status of the cell, and level of the MDR pump expression.

Introduction: The study aimed to investigate the effects of low concentrations of mitochondrial uncouplers in endothelial cells on the CpG dinucleotide methylation of the ICAM1 gene promoter. The excessive inflammatory response in the endothelium is responsible for the development of many cardiovascular diseases. Mitochondria are important regulators of endothelial cell functions. Mild uncoupling of oxidative phosphorylation and respiration in endothelial mitochondria exerts a long lasting anti-inflammatory effect. However, the detailed mechanism of the anti-inflammatory activity of mitochondrial uncouplers remains unclear.We hypothesized that mild mitochondrial uncoupling leads to epigenetic changes in genomic DNA contributing to the anti-inflammatory response., Methods: We studied the long-term effects of mitochondria-targeted compounds with the uncoupler's activities: the antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1), dodecyl-triphenylphosphonium (C12TPP), and 2,4-dinitrophenol (DNP). The mRNA expression of the intercellular adhesion molecule 1 (ICAM1), a marker of inflammatory activation of endothelial cells, was measured by RT-qPCR. Cytosine methylation in the CpG sites of the ICAM1 gene promoter was estimated by bisulfite sequencing of individual clones., Results: It was found that downregulation of ICAM1 expression caused by DNP and C12TPP was accompanied by an increase in the methylation of CpG sites in the ICAM1 gene promoter. None of the compounds affected intracellular or intramitochondrial ATP levels., Conclusion: Low concentrations of mitochondrial oxidative phosphorylation uncouplers are able to increase methylation of ICAM1 gene promoter, which corresponds to the observed decrease in the levels of mRNA of this gene. Thus, the change in methylation of the ICAM1 gene promoter may underlie the mechanism of decreased ICAM1 expression caused by mild mitochondrial depolarization. Mitochondrial uncouplers may be exploited as possible therapeutic candidates to treat excessive inflammation in endothelium, by changing the methylation status of genomic DNA., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Transcription factor NRF2 is involved in inflammatory reactions, maintenance of redox balance, metabolism of xenobiotics, and is of particular interest for studying aging. In the present work, the CRISPR/Cas9 genome editing technology was used to generate the NRF2 ΔNeh2 mice containing a substitution of eight amino acid residues at the N-terminus of the NRF2 protein, upstream of the functional Neh2 domain, which ensures binding of NRF2 to its inhibitor KEAP1. Heterozygote NRF2 wt/ΔNeh2 mice gave birth to homozygous mice with lower than expected frequency, accompanied by their increased embryonic lethality and visual signs of anemia. Mouse embryonic fibroblasts (MEFs) from the NRF2 ΔNeh2/ΔNeh2 homozygotes showed impaired resistance to oxidative stress compared to the wild-type MEFs. The tissues of homozygous NRF2 ΔNeh2/ΔNeh2 animals had a decreased expression of the NRF2 target genes: NAD(P)H:Quinone oxidoreductase-1 (Nqo1); aldehyde oxidase-1 (Aox1); glutathione-S-transferase A4 (Gsta4); while relative mRNA levels of the monocyte chemoattractant protein 1 (Ccl2), vascular cell adhesion molecule 1 (Vcam1), and chemokine Cxcl8 was increased. Thus, the resulting mutation in the Nfe2l2 gene coding for NRF2, partially impaired function of this transcription factor, expanding our insights into the functional role of the unstructured N-terminus of NRF2. The obtained NRF2 ΔNeh2 mouse line can be used as a model object for studying various pathologies associated with oxidative stress and inflammation.

The transcription factor NRF2 is a major regulator of cell antioxidant defense. NRF2 is activated by various stimuli, such as oxidants and electrophiles, to induce transcription of a number of genes whose products are involved in xenobiotic metabolism and contribute to the reduction of oxidative stress. NRF2 is one of the key transcription factors that ensure the endothelial cell function. The endothelium is a cell layer that lines the lumens of blood vessels and performs various homeostatic functions, controlling migration of leukocytes, regulating thrombosis and vascular tone, and playing a role in angiogenesis. Endothelial dysfunction is often accompanied by inflammation and oxidative stress, which may lead to cell aging and cell death by apoptosis, necrosis, or ferroptosis. Endothelial dysfunction contributes to the development of diabetes and common cardiovascular disorders, such as hypertension and atherosclerosis. Many pathophysiological processes in the endothelium, including senile changes, are associated with decreased NRF2 activity, leading to inflammatory activation and decreasing activity of the cell antioxidant defense systems. Activation of the NRF2 signaling pathway generally contributes to the resolution of inflammation and oxidative stress. The review focuses on the role that NRF2 plays in basic functions of the endothelium in normal and pathological conditions. Advantages and disadvantages of NRF2 activation as a way to prevent and treat cardiovascular diseases are discussed additionally.

For a long time Nrf2 transcription factor has been attracting attention of researchers investigating phenomenon of aging. Numerous studies have investigated effects of Nrf2 on aging and cell senescence. Nrf2 is often considered as a key player in aging processes, however this needs to be proven. It should be noted that most studies were carried out on invertebrate model organisms, such as nematodes and fruit flies, but not on mammals. This paper briefly presents main mechanisms of mammalian aging and role of inflammation and oxidative stress in this process. The mechanisms of Nrf2 activity regulation, its involvement in aging and development of the senescence-associated secretory phenotype (SASP) are also discussed. Main part of this review is devoted to critical analysis of available experimental data on the role of Nrf2 in mammalian aging.

Pathogenesis of the novel coronavirus infection COVID-19 is the subject of active research around the world. COVID-19 caused by the SARS-CoV-2 is a complex disease in which interaction of the virus with target cells, action of the immune system and the body's systemic response to these events are closely intertwined. Many respiratory viral infections, including COVID-19, cause death of the infected cells, activation of innate immune response, and secretion of inflammatory cytokines. All these processes are associated with the development of oxidative stress, which makes an important contribution to pathogenesis of the viral infections. This review analyzes information on the oxidative stress associated with the infections caused by SARS-CoV-2 and other respiratory viruses. The review also focuses on involvement of the vascular endothelium in the COVID-19 pathogenesis.

Background: Targeting negatively charged mitochondria is often achieved using triphenylphosphonium (TPP) cations. These cationic vehicles may possess biological activity, and a docking study indicates that TPP-moieties may act as modulators of signaling through the estrogen receptor α (ERα). Moreover, in vivo and in vitro experiments revealed the estrogen-like effects of TPP-based compounds. Here, we tested the hypothesis that TPP-based compounds regulate the activity of ERα., Methods: We used ERa-positive and ERα-negative human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231, respectively). Cell proliferation was measured using a resazurin cell growth assay and a real-time cell analyzer assay. Cell cycle progression was analyzed using flow cytometry. Real-time PCR was used to assess mRNA expression of endogenous estrogen-responsive genes. Luciferase activity was measured to evaluate transcription driven by estrogen-responsive promoters in cells transfected with an estrogen response element (ERE) 3 -luciferase expression vector., Results: The TPP-based molecules SkQ1 and C 12 TPP, as well as the rhodamine-based SkQR1, did not increase the proliferation or alter the cell cycle progression of MCF-7 cells. In contrast, 17β estradiol increased the proliferation of MCF-7 cells and the proportion of cells in the S/G2/M-phases of the cell cycle. TPP-based compounds did not affect the induction of transcription of an ERE-luciferase expression vector in vitro, and SkQ1 did not alter the levels of expression of estrogen-dependent genes encoding GREB1, TFF1, COX6, and IGFBP4., Conclusion: TPP-based compounds do not possess properties typical of ERα agonists., Competing Interests: The authors declare there are no competing interests., (©2020 Zinovkina et al.)

DNA replication in human mitochondria has been studied for several decades; however, its mechanism still remains unclear. During the last 15 years, many new experimental data on the mitochondrial replication have appeared, although extremely contradictory. Two asynchronous (strand displacement and RITOLS) and one synchronous (strand-coupled) replication models have been proposed. In the asynchronous models, replication from the origin in the H-chain starts earlier, so that the replication of the two chains ends at different times. The synchronous model is more traditional and implies two replication forks with leading and lagging strands initiated at the same origin. For each of the three models, both confirming and contradicting experimental data exist. Most likely, there is no single model of mitochondrial replication. It is possible that the unique mitochondrial replication machinery that has originated as a results of endosymbiosis has an unexpected variety of replication strategies to maintain the mitochondrial genome. An unusual combination of enzymes of different origin (phage, bacterial, eukaryotic) and unique features of the mitochondrial genome (existance of heavy and light chains, insertions of ribonucleotides, a variety of origins) can allow replication through different mechanisms. In human mitochondria, asynchronous replication seems to dominate; however, synchronous replication is also possible under certain conditions. In the human heart mitochondria, circular mitochondrial DNA (mtDNA) molecules can rearrange in a network of rapidly replicating linear genomes, thereby suggesting possible existence of a wide range of replication mechanisms in the mitochondria. The review describes the main stages of mtDNA replication and enzymes involved in this process, as well as discusses the prospects of mitochondrial replication studies.

Accumulation of mutations in mitochondrial DNA leads to the development of severe, currently untreatable diseases. The contribution of these mutations to aging and progress of neurodegenerative diseases is actively studied. Elucidation of DNA repair mechanisms in mitochondria is necessary for both developing approaches to the therapy of diseases caused by mitochondrial mutations and understanding specific features of mitochondrial genome functioning. Mitochondrial DNA repair systems have become a subject of extensive studies only in the last decade due to development of molecular biology methods. DNA repair systems of mammalian mitochondria appear to be more diverse and effective than it had been thought earlier. Even now, one may speak about the existence of mitochondrial mechanisms for the repair of single- and double-stranded DNA lesions. Homologous recombination also takes place in mammalian mitochondria, although its functional significance and molecular mechanisms remain obscure. In this review, I describe DNA repair systems in mammalian mitochondria, such as base excision repair (BER) and microhomology-mediated end joining (MMEJ) and discuss a possibility of existence of mitochondrial DNA repair mechanisms otherwise typical for the nuclear DNA, e.g., nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination, and classical non-homologous end joining (NHEJ). I also present data on the mechanisms for coordination of the nuclear and mitochondrial DNA repair systems that have been actively studied recently.

Objective: To measure the release of plasma nuclear deoxyribonucleic acid (DNA) and to assess the relationship between nuclear DNA level and acute kidney injury occurrence in patients undergoing cardiac surgery., Setting: Cardiovascular anesthesiology and intensive care unit of a large tertiary-care university hospital., Design: Prospective observational study., Participants: Fifty adult patients undergoing cardiac surgery., Interventions: Nuclear DNA concentration was measured in the plasma. The relationship between the level of nuclear DNA and the incidence of acute kidney injury after coronary artery bypass grafting was investigated., Measurements and Main Results: Cardiac surgery leads to significant increase in plasma nuclear DNA with peak levels 12 hours after surgery (median [interquartile range] 7.0 [9.6-22.5] µg/mL). No difference was observed between off-pump and on-pump surgical techniques. Nuclear DNA was the only predictor of acute kidney injury between baseline and early postoperative risk factors., Conclusions: The authors found an increase of nuclear DNA in the plasma of patients who had undergone coronary artery bypass grafting, with a peak after 12 hours and an association of nuclear DNA with postoperative acute kidney injury., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Extracellular plasma DNA is thought to act as a damage-associated molecular pattern causing activation of immune cells. However, purified preparations of mitochondrial and nuclear DNA were unable to induce neutrophil activation in vitro. Thus, we examined whether granulocyte-macrophage colony-stimulating factor (GM-CSF) acting as a neutrophil priming agent can promote the activation of neutrophils by different types of extracellular DNA. GM-CSF pretreatment greatly increased p38 MAPK phosphorylation and promoted CD11b/CD66b expression in human neutrophils treated with mitochondrial and, to a lesser extent, with nuclear DNA. Our experiments clearly indicate that GM-CSF-induced priming of human neutrophils is necessary for their subsequent activation by extracellular DNA.

DNA methylation is a key epigenetic process involved in the regulation of nuclear gene expression. Progress in the study of genomic DNA methylation led to the precise identification of methylation sites reflecting biological age of cells and tissues. However, the functional significance of mitochondrial DNA (mtDNA) methylation remains unknown. Growing evidence suggests that mtDNA methylation is linked to aging and oxidative stress. This mini-review summarizes information about the methylation of nuclear and mtDNA in mammals, indicating the connection of these processes to programmed aging.

Excessive activation of the innate immune system often leads to fatal consequences and can be considered as one of the phenoptotic events. After traumatic injury, various components of mitochondria are released into the circulation and stimulate myeloid cells of the innate immunity. Presumably, mitochondrial DNA (mtDNA) might activate immune cells (Zhang, Q., et al. (2010) Nature, 464, 104-107). In the present study, we investigated the role of mtDNA as a direct activator of human neutrophils, as well as a prognostic marker in patients with severe trauma. Quantitative determination of mtDNA in the plasma of these patients revealed its significant increase (p < 0.02) in the group of survivors compared to non-survivors. Highly purified mtDNA was not able to induce activation of human neutrophils, thus possibly indicating the existence of additional factor(s) ensuring the recognition of mtDNA as a damage-associated molecular pattern.

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-DeltaT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-DeltaT have normal dynactin "comets" at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.

LOSK (LOng Ste20-like Kinase) protein kinases of mammals belong to a recently identified family of GCK kinases which are involved in the induction of apoptosis. LOSK have an N-terminal acidic catalytic domain and a long C-terminal basic structural domain which is cleaved off in cells by caspases during apoptosis. To study the LOSK enzymatic activity and its dependence on the structural domain, two preparations of this protein kinase were prepared: a natural full-length protein immunoprecipitated from CHO-K1 cultured cells and a recombinant N-terminal catalytic fragment synthesized in E. coli. Both preparations displayed the ability for autophosphorylation and the ability for phosphorylation of MBP and of H1 histone, and their activities were comparable. H1 histone was a better substrate for LOSK than casein and ATP was a better substrate than other nucleotides. The pH dependence of the activity of the immunoprecipitated protein was more pronounced than the pH dependence of its recombinant fragment deprived of the C-terminal domain. The catalytic and the structural domains of LOSK can interact through electrostatic forces; therefore, effects were studied of various polyions at the concentration of 0.1 mg/ml on the activity. Heparin, protamine sulfate, and poly(L-Lys) decreased tenfold the ability of the full-length kinase to phosphorylate H1 histone. Heparin did not affect the activity of the recombinant fragment, whereas protamine sulfate and poly(L-Lys) had a slight effect. Moreover, protamine increased fourfold the autophosphorylation of the immunoprecipitated protein kinase. These data suggest that the structural C-terminal domain of LOSK should be involved in the regulation of its protein kinase activity: the LOSK protein kinase with C-terminal domain cleaved off could significantly less depend on conditions in the cell than the full-size enzyme.

We studied the possibility of using the spermatozoa of the loach Misgurnus fossilis L. for identification of centrosome proteins. It has been shown that the centrosome of the loach spermatozoa consists of a pair of centrioles of the standard structure and contains the marker protein gamma-tubulin, cytoplasmic microtubules branch out from it, and it does not contain any additional structures characteristic of the centrosomes of spermatozoa of many other fishes. A preparation enriched with intact centrosomes has been obtained from the loach spermatozoa. These centrosomes contained gamma-tubulin although they lost their ability to induce polymerization of microtubules. The preparation of loach centrosomes was successfully used to obtain a set of monoclonal antibodies against the mammalian centrosome. A new protein kinase LOSTEK was identified with the help of one of these monoclonal antibodies, SN2-3D2, which was localized in the centrosome and on then microtubules in both loach spermatozoa and cultured mammalian cells. Hence, the loach spermatozoa are a promising object for identification of new proteins of the mammalian centrosome.

Monoclonal antibody raised against a preparation of loach fish sperm centrosomes was used for screening of cDNA expressing library of Chinese hamster CHO-K1 cells. Two positive clones appeared to encode 628 amino acid protein fragment that was 72% identical to human KIAA0204 protein, i.e. putative protein kinase. Polyclonal antibodies raised against products of cDNA expression in E. coli recognized 210-kDa polypeptide in CHO-K1 cells and immunostained nuclear speckles, centrosomes and microtubules in these cells. The 210-kDa polypeptide (named MAK-L) co-sedimented with exogenous microtubules. Thus, one more protein kinase seems to be associated with the microtubule network in vertebrate cells.

The review summarizes recent data on structure and function of nearly two dozen centrosomal proteins of the animal cells; centrosome is an organelle that organizes cellular microtubules. Most of these data were obtained by molecular biology techniques including screening of phage expression cDNA libraries with centrosome-specific antibodies, bacterial expression of recombinant proteins recognized by these antibodies, analysis of multiple non-allelic gene of cytoskeletal proteins, etc. new classification of centrosomal proteins is suggested based on their possible centrosomal function comprising proteins of microtubule polymerization templates, structural proteins of pericentriolar material, microtubule severing proteins, microtubule-dependent transport-catalyzing motor proteins, regulatory proteins (specific protein kinases, phosphatases, etc), and centriolar proteins.