Biochemistry (Moscow) (v.80, #5)

Ancient systems of sodium/potassium homeostasis as predecessors of membrane bioenergetics by D. V. Dibrova; M. Y. Galperin; E. V. Koonin; A. Y. Mulkidjanian (495-516).
Cell cytoplasm of archaea, bacteria, and eukaryotes contains substantially more potassium than sodium, and potassium cations are specifically required for many key cellular processes, including protein synthesis. This distinct ionic composition and requirements have been attributed to the emergence of the first cells in potassium-rich habitats. Different, albeit complementary, scenarios have been proposed for the primordial potassium-rich environments based on experimental data and theoretical considerations. Specifically, building on the observation that potassium prevails over sodium in the vapor of inland geothermal systems, we have argued that the first cells could emerge in the pools and puddles at the periphery of primordial anoxic geothermal fields, where the elementary composition of the condensed vapor would resemble the internal milieu of modern cells. Marine and freshwater environments generally contain more sodium than potassium. Therefore, to invade such environments, while maintaining excess of potassium over sodium in the cytoplasm, primordial cells needed means to extrude sodium ions. The foray into new, sodium-rich habitats was the likely driving force behind the evolution of diverse redox-, light-, chemically-, or osmotically-dependent sodium export pumps and the increase of membrane tightness. Here we present a scenario that details how the interplay between several, initially independent sodium pumps might have triggered the evolution of sodium-dependent membrane bioenergetics, followed by the separate emergence of the proton-dependent bioenergetics in archaea and bacteria. We also discuss the development of systems that utilize the sodium/potassium gradient across the cell membranes.
Keywords: ATP synthase; sodium-motive force; proton-motive force; sodium symporter; membrane efflux pumps; abiogenesis; anoxic geothermal fields

Mitochondrial ROS metabolism: 10 Years later by A. Y. Andreyev; Y. E. Kushnareva; A. N. Murphy; A. A. Starkov (517-531).
The role of mitochondria in oxidative stress is well recognized, but many questions are still to be answered. This article is intended to update our comprehensive review in 2005 by highlighting the progress in understanding of mitochondrial reactive oxygen species (ROS) metabolism over the past 10 years. We review the recently identified or re-appraised sources of ROS generation in mitochondria, such as p66shc protein, succinate dehydrogenase, and recently discovered properties of the mitochondrial antioxidant system. We also reflect upon some controversies, disputes, and misconceptions that confound the field.
Keywords: mitochondria; reactive oxygen species; p66shc; succinate dehydrogenase; dihydroorotate dehydrogenase; ROS scavenging; oxidative stress

Mitodiversity by V. A. Popkov; E. Y. Plotnikov; K. G. Lyamzaev; D. N. Silachev; L. D. Zorova; I. B. Pevzner; S. S. Jankauskas; S. D. Zorov; V. A. Babenko; D. B. Zorov (532-541).
Here, in addition to the previously coined term “mitobiota”, we introduce the term “mitodiversity” for various phenotypic and genetic heterogeneities of mitochondria within the same cell or organ. Based on data on the mitochondrial transmembrane potential determined both in situ and in vitro under normal conditions and after organ ischemia/reperfusion, such heterogeneity is most evident under pathologic conditions. Herein, a part of the mitochondrial population with transmembrane potential typical of the normal state is sustained even under a pathological condition that, perhaps, underlies the development of ways of reversing pathology back to the normal state. The membrane potentials of isolated mitochondria were shown to directly correlate with the magnitude of side-scattered light depicting internal structure of mitochondria. We analyzed possible interpretations of data on mitochondrial membrane potential obtained using fluorescent probes. We suggest a possible mechanism underlying retention of fluorescent probes inside the cells and mitochondria.
Keywords: mitochondria; heterogeneity; diversity; chondriome; population analysis; membrane potential; ischemia; pathology; lesion

Intercellular transfer of mitochondria by E. Y. Plotnikov; V. A. Babenko; D. N. Silachev; L. D. Zorova; T. G. Khryapenkova; E. S. Savchenko; I. B. Pevzner; D. B. Zorov (542-548).
Recently described phenomenon of intercellular transfer of mitochondria attracts the attention of researchers in both fundamental science and translational medicine. In particular, the transfer of mitochondria results in the initiation of stem cell differentiation, in reprogramming of differentiated cells, and in the recovery of the lost mitochondrial function in recipient cells. However, the mechanisms of mitochondria transfer between cells and conditions inducing this phenomenon are studied insufficiently. It is still questionable whether this phenomenon exists in vivo. Moreover, it is unclear, how the transfer of mitochondria into somatic cells is affected by the ubiquitination system that, for example, is responsible for the elimination of “alien” mitochondria of the spermatozoon in the oocyte during fertilization. Studies on these processes can provide a powerful incentive for development of strategies for treatment of mitochondria-associated pathologies and give rise a new avenue for therapeutic approaches based on “mitochondrial transplantation”.
Keywords: mitochondria; stem cells; axonal transport; tunneling nanotubes; differentiation

Does mitochondrial fusion require transmembrane potential? by I. E. Karavaeva; K. V. Shekhireva; F. F. Severin; D. A. Knorre (549-558).
Dissipation of transmembrane potential inhibits mitochondrial fusion and thus prevents reintegration of damaged mitochondria into the mitochondrial network. Consequently, damaged mitochondria are removed by autophagy. Does transmembrane potential directly regulate the mitochondrial fusion machinery? It was shown that inhibition of ATP-synthase induces fragmentation of mitochondria while preserving transmembrane potential. Moreover, mitochondria of the yeast Saccharomyces cerevisiae retain the ability to fuse even in the absence of transmembrane potential. Metazoan mitochondria in some cases retain ability to fuse for a short period even in a depolarized state. It also seems unlikely that transmembrane potential-based regulation of mitochondrial fusion would prevent reintegration of mitochondria with damaged ATP-synthase into the mitochondrial network. Such reintegration could lead to clonal expansion of mtDNAs harboring deleterious mutations in ATP synthase. We speculate that transmembrane potential is not directly involved in regulation of mitochondrial fusion but affects mitochondrial NTP/NDP ratio, which in turn regulates their fusion.
Keywords: mitochondria; fusion; fission; membrane potential; mtDNA; regulation

Negative feedback of glycolysis and oxidative phosphorylation: Mechanisms of and reasons for it by S. S. Sokolov; A. V. Balakireva; O. V. Markova; F. F. Severin (559-564).
There are two main pathways of ATP biosynthesis: glycolysis and oxidative phosphorylation. As a rule, the two pathways are not fully active in a single cell. In this review, we discuss mechanisms of glycolytic inhibition of respiration (Warburg and Crabtree effects). What are the reasons for the existence of this negative feedback? It is known that maximal activation of both processes can cause generation of reactive oxygen species. Oxidative phosphorylation is more efficient from the energy point of view, while glycolysis is safer and favors biomass synthesis. This might be the reason why quiescent cells are mainly using oxidative phosphorylation, while the quickly proliferating ones — glycolysis.
Keywords: glycolysis; oxidative phosphorylation; mitochondria; Crabtree effect

Cytochrome bd protects bacteria against oxidative and nitrosative stress: A potential target for next-generation antimicrobial agents by V. B. Borisov; E. Forte; S. A. Siletsky; M. Arese; A. I. Davletshin; P. Sarti; A. Giuffrè (565-575).
Cytochrome bd is a terminal quinol oxidase of the bacterial respiratory chain. This tri-heme integral membrane protein generates a proton motive force at lower efficiency than heme-copper oxidases. This notwithstanding, under unfavorable growth conditions bacteria often use cytochrome bd in place of heme-copper enzymes as the main terminal oxidase. This is the case for several pathogenic and opportunistic bacteria during host colonization. This review summarizes recent data on the contribution of cytochrome bd to bacterial resistance to hydrogen peroxide, nitric oxide, and peroxynitrite, harmful species produced by the host as part of the immune response to microbial infections. Growing evidence supports the hypothesis that bd-type oxidases contribute to bacterial virulence by promoting microbial survival under oxidative and nitrosative stress conditions. For these reasons, cytochrome bd represents a protein target for the development of next-generation antimicrobials.
Keywords: antimicrobial agents; bacteria; oxidative stress; nitrosative stress; reactive oxygen and nitrogen species; respiratory chain; terminal oxidase; virulence

A brief description of the principal directions for searching and investigating the model of local coupling between respiration and phosphorylation proposed by R. Williams is given in this paper. We found conditions where it was possible to reveal typical functional special features of the mitochondrial phosphorylating system. According to the theory, such special features should be observed experimentally if the mitochondrial phosphorylating system operated in the state of a super-complex. It was proved that the phosphorylating system is able to operate in two states: P. Mitchell state and R. Williams state. It was demonstrated that in the ATP synthesis reaction, ATP-synthase (F1F0) was able to use thermodynamic potential of Bronsted acids as a source of energy. It was shown using a double-inhibitor titration technique that when the phosphorylating system operated in the supercomplex state, the electron transfer system and ATP-synthesis system were docked rigidly. A model system of chemical synthesis of membrane-bound proton fraction (Bronsted acids), carrying a free energy excess, was developed on the model of bilayer lipid membrane. Catalysts selectively accelerating proton detachment of this fraction were also found. The formation of a Bronsted acids fraction carrying free energy excess was recorded during the operation of proton pumps on mitochondrial and mitoplastic membranes. In the experimental part of the work, a brief description is given of studies on new uncouplers that transfer the phosphorylation system from the local coupling state to the state of transmembrane proton transfer. Thus, they accelerated the respiration of mitochondria and decreased the ADP/O parameter.
Keywords: local coupling; phosphorylating system of mitochondria; membrane-bound protons carrying free energy excess; uncouplers deactivating local coupling

Chronic progressive nephropathy is a common noninfectious disease in aging (mice, rats) and non-aging (naked mole rat) rodents, sometimes resulting in death. The etiology and pathogenesis of the disease remain mysterious. For instance, it remains unclear what is the immediate cause of the disease and where exactly in the kidneys, glomerular or tubulointerstitial compartment, do primary and secondary changes occur. Here, I propose a potential scenario for development of progressive nephropathy that is based on an assumption that the disease is caused by occurrence and spread of mutant cellular clones from tubular epithelium secreting proinflammatory and prosclerotic cytokines. The hypothesis considers some features of the disease that have never been discussed earlier. According to the proposed concept, a clone of mutant cells secretes cytokines inducing chronic inflammation, proliferation of fibroblasts, and active collagen production that eventually results in sclerosis and thickening of tubular basement membranes. Sclerosis of interstitium and thickening of tubular basement membranes cause narrowing of some parts of the nephron, especially collecting ducts, which hinders passage of the urine, elevates tubular hydrostatic pressure, and impairs filtration and reabsorption in the kidneys. High hydrostatic pressure and reabsorption-induced elevated concentration of macromolecular substances in the primary urine result in development of large cysts and glomerular hyalinosis followed by renal failure. Based on this, it might be concluded that chronic progressive nephropathy in rodents represents a special type of tubulointerstitial dysplasia (or “non-tumorous neoplasia”) in kidneys with secondary glomerular disorder at late stage of the disease. The concept for development of the disease proposed here may be of special importance from the viewpoint of toxicological pathology and gerontology, particularly for analysis of pathological features resulting in death of non-aging animals (naked mole rats).
Keywords: chronic progressive nephropathy; age-related diseases; naked mole rat; somatic mutations; laboratory animals

Influence of SkQ1 on expression of Nrf2 transcription factor gene, ARE-controlled genes of antioxidant enzymes and their activity in rat blood leukocytes by V. V. Vnukov; O. I. Gutsenko; N. P. Milutina; A. A. Ananyan; A. O. Danilenko; S. B. Panina; I. V. Kornienko (586-591).
This study demonstrated that pretreatment of rats with mitochondria-targeted antioxidant SkQ1 (50 nmol/kg during 5 days) significantly increased the mRNA levels of Nrf2 transcription factor and Nrf2-induced genes encoding antioxidant enzymes SOD1, SOD2, CAT, and GPx4 in rat peripheral blood leukocytes. The increase in expression of these genes with SkQ1 addition was accompanied by increased activities of catalase, glutathione peroxidase, and glutathione-S-transferase in leukocytes. These results indicate that antioxidant properties of SkQ1 might be realized via induction of expression of the genes regulating activity of antioxidant system elements.
Keywords: gene expression; antioxidant enzymes; leukocytes; mitochondria-targeted antioxidant

Mitochondria-targeted plastoquinone antioxidant SkQR1 has positive effect on memory of rats by E. V. Stelmashook; A. V. Stavrovskaya; N. G. Yamshchikova; A. S. Ol’shanskii; N. A. Kapay; O. V. Popova; L. G. Khaspekov; V. G. Skrebitsky; N. K. Isaev (592-595).
A single intraperitoneal injection to rats of the mitochondria-targeted plastoquinone antioxidant SkQR1 at dose 1 μmol/kg significantly improved reproduction by the rats of the passive avoidance conditional reflex. In vitro experiments on hippocampal slices showed that a single intraperitoneal injection of SkQR1 24 h before the preparation of the slice significantly increases the synaptic transmission efficiency of the pyramidal neurons of the CA1 field. The findings indicate that SkQR1 has a positive effect on memory processes.
Keywords: memory; long-term potentiation; hippocampus; mitochondria-targeted antioxidants; SkQR1

Influence of antioxidant SkQ1 on accumulation of mitochondrial DNA deletions in the hippocampus of senescence-accelerated OXYS rats by P. S. Loshchenova; O. I. Sinitsyna; L. A. Fedoseeva; N. A. Stefanova; N. G. Kolosova (596-603).
Reduction of efficiency of oxidative phosphorylation associated with aging and the development of neurodegenerative diseases including Alzheimer’s disease is thought to be linked to the accumulation of deletions in mitochondrial DNA (ΔmtDNA), which are seen as a marker of oxidative damage. Recently, we have shown that mitochondria-targeted antioxidant SkQ1 (10-(6′-plastoquinonyl)decyltriphenylphosphonium) can slow the development of signs of Alzheimer’s disease in senescence-accelerated OXYS rats. The purpose of this study was to explore the relationship between the development of neurodegenerative changes in the brain of OXYS rats and changes in the amount of mtDNA and the 4834-bp mitochondrial DNA deletion (ΔmtDNA4834) as well as the effect of SkQ1. We studied the relative amount of mtDNA and ΔmtDNA4834 in the hippocampus of OXYS and Wistar (control) rats at ages of 1, 2, 6, 10, and 20 days and 3, 6, and 24 months. During the period crucial for manifestation of the signs of accelerated aging of OXYS rats (from 1.5 to 3 months of age), we evaluated the effects of administration of SkQ1 (250 nmol/kg) and vitamin E (670 mmol/kg, reference treatment) on the amount of mtDNA and ΔmtDNA4834 and on the formation of the behavioral feature of accelerated senescence in OXYS rats — passive type of behavior in the open field test. In OXYS rats, the level of ΔmtDNA4834 in the hippocampus is increased compared to the Wistar rats, especially at the stage of completion of brain development in the postnatal period. This level remains elevated not only at the stages preceding the manifestation of the signs of accelerated brain aging and the development of pathological changes linked to Alzheimer’s disease, but also during their progression. However, at age of 24 months, there were no detectable differences between the two strains. SkQ1 treatment reduced the level of ΔmtDNA4834 in the hippocampus of Wistar and OXYS rats and slowed the formation of passive behavior in OXYS rats. These results support the possible use of SkQ1 for prophylaxis of brain aging.
Keywords: aging; hippocampus; mitochondria; 4834-bp mitochondrial DNA deletion; mitochondria-targeted antioxidant SkQ1

Morphometric examination of mitochondrial ultrastructure in aging cardiomyocytes by Ch. M. El’darov; V. B. Vays; I. M. Vangeli; N. G. Kolosova; L. E. Bakeeva (604-609).
Mitochondrial ultrastructure in cardiomyocytes from 3- and 24-month-old Wistar and OXYS rats was investigated using a new approach designed for morphometric analysis. The data fully confirm the electron microscopy data: the area of the inner mitochondrial membrane per unit volume of mitochondria was significantly decreased with age, as found on heart muscle section. In 3-month-old Wistar rats from the control group, this parameter was 41.3 ± 1.52 μm2/μm3, where-as in OXYS rats it was decreased to 30.57 ± 1.74 μm2/μm3. With age, an area of the inner mitochondrial membrane per unit volume of mitochondria declined in both rat strains: Wistar — from 41.3 ± 1.52 to 21.47 ± 1.22 μm2/μm3, OXYS — from 30.57 ± 1.74 to 16.3 ± 0.89 μm2/μm3. A new method that we designed and used for morphometric analysis notably simplifies the process of morphometric measurements and opens up good opportunities for its further optimization using image recognition technology.
Keywords: morphometry; aging; ultrastructure; cardiomyocyte

Low concentrations of uncouplers of oxidative phosphorylation prevent inflammatory activation of endothelial cells by tumor necrosis factor by V. P. Romaschenko; R. A. Zinovkin; I. I. Galkin; V. V. Zakharova; A. A. Panteleeva; A. V. Tokarchuk; K. G. Lyamzaev; O. Yu. Pletjushkina; B. V. Chernyak; E. N. Popova (610-619).
In endothelial cells, mitochondria play an important regulatory role in physiology as well as in pathophysiology related to excessive inflammation. We have studied the effect of low doses of mitochondrial uncouplers on inflammatory activation of endothelial cells using the classic uncouplers 2,4-dinitrophenol and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole, as well as the mitochondria-targeted cationic uncoupler dodecyltriphenylphosphonium (C12TPP). All of these uncouplers suppressed the expression of E-selectin, adhesion molecules ICAM1 and VCAM1, as well as the adhesion of neutrophils to endothelium induced by tumor necrosis factor (TNF). The antiinflammatory action of the uncouplers was at least partially mediated by the inhibition of NFκB activation due to a decrease in phosphorylation of the inhibitory subunit IκBα. The dynamic concentration range for the inhibition of ICAM1 expression by C12TPP was three orders of magnitude higher compared to the classic uncouplers. Probably, the decrease in membrane potential inhibited the accumulation of penetrating cations into mitochondria, thus lowering the uncoupling activity and preventing further loss of mitochondrial potential. Membrane potential recovery after the removal of the uncouplers did not abolish its antiinflammatory action. Thus, mild uncoupling could induce TNF resistance in endothelial cells. We found no significant stimulation of mitochondrial biogenesis or autophagy by the uncouplers. However, we observed a decrease in the relative amount of fragmented mitochondria. The latter may significantly change the signaling properties of mitochondria. Earlier we showed that both classic and mitochondria-targeted antioxidants inhibited the TNF-induced NFκB-dependent activation of endothelium. The present data suggest that the antiinflammatory effect of mild uncoupling is related to its antioxidant action.
Keywords: inflammation; endothelium; adhesion molecules; mitochondria; uncoupling of oxidative phosphorylation; penetrating cations of SkQ family

Obesity is associated with premature mortality, impaired quality of life, and large healthcare costs. However, treatment options remain quite limited. Here we studied potential anti-obesity effects of a novel cationic mitochondrial uncoupler, C4R1 (derivative of rhodamine 19) in C57Bl/6 mice. Obesity was induced by long-term (eight weeks) high fat diet feeding at thermoneutrality. The treated group of mice received consecutively two doses of C4R1 in drinking water (30 and 12–14 μmol/kg daily) during 30 days. Effects of C4R1 were dose-dependent. After six days of C4R1 treatment at dose 30 μmol/kg daily, food intake was reduced by 68%, body weight by 19%, and fat mass by 21%. Body weight decrease was explained partly by reduced food intake and partly by increased metabolism, likely resulting from uncoupling. Body fat reduction upon C4R1 treatment was associated with improved lipid utilization estimated from decrease in respiratory quotient to the minimal level (0.7). Interestingly, the classical uncoupler 2,4-dinitrophenol at similar dose (27 μmol/kg daily) did not have any effect. Our results are relevant to the search for substances causing mild uncoupling of mitochondria that could be a promising therapeutic strategy to treat obesity.
Keywords: cationic mitochondrial uncoupler; 2,4-dinitrophenol; obesity; resting metabolic rate; energy expenditure

Pure mitochondrial DNA does not activate human neutrophils in vitro by A. S. Prikhodko; A. K. Shabanov; L. A. Zinovkina; E. N. Popova; M. A. Aznauryan; N. O. Lanina; M. V. Vitushkina; R. A. Zinovkin (629-635).
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 nonsurvivors. 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.
Keywords: trauma; extracellular DNA; mitochondrial DNA; neutrophil activation; damage-associated molecular patterns (DAMPs)

By admitting the programmed organism death (phenoptosis) concept, it inevitably raises a question as to what advantages it gives to communities containing elderly and naturally weakened individuals. We believe that the broadest prevalence of the aging phenomenon is accounted for, particularly, by the fact that in certain situations occurrence of elderly individuals may guarantee not only evolution, but also the mere existence of populations. The goal of our study was to create a mathematical model illustrating the occurrence of situations when existence of elderly individuals accounts for population survival, whereas an ageless population would be completely extinguished. A logic basis for such model is as follows: 1) natural populations exist under conditions of uneven renewal of resources; 2) if resources are renewed at a high level and rapidly compensated by their restoration, then a population strives to achieve a maximum size, which is limited not by resource availability, but specific biological mechanisms; 3) rate of resource influx may decline down to zero very rapidly (e.g. during drought); 4) a capacity, at least, for some individuals to survive during resource shortage is crucial for survival of the entire population; 5) rapid extinction of the weaker elderly individuals saves resources for survival of the younger and stronger ones. A multi-agent simulation is used as a mathematical basis for the proposed model ( ). In silico experiments confirmed the lack of fatal contradictions in our logical construction. The presence of the aged individuals once the aging program has been turned at the age of 25–30 years results in a 24–26% increase in lifetime of the population.
Keywords: aging; phenoptosis; multi-agent simulation; extinction; resource shortage

Carbaryl is a low molecular weight insecticide that inhibits cholinesterase. Residues of carbaryl in food and the environment have damaged human health. A high-specificity scFv that can identify carbaryl is still lacking. In the present study, an anti-carbaryl scFv gene was prepared by cloning VL and VH genes from hybridoma cells secreting monoclonal antibody, then VH and VL were fused together using splicing by overlap extension (SOE) PCR with a flexible polypeptide linker connector (Gly4Ser)3, and then the scFv-pET-26b recombinant plasmid was constructed and transformed into E. coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was identified by SDS-PAGE and ELISA. The three-dimensional structure of the anti-carbaryl scFv was constructed by computer modeling, and carbaryl was docked to the scFv model to obtain the structure of the binding complex. The binding site was composed of Ala51, Ser52, Ile51, Gly54, Ser56, Arg98, and Gly100. This helps to understand the mechanism of interaction between anti-carbaryl antibody and antigen. Furthermore, it provides guidance for in vitro affinity maturation of anti-carbaryl antibody.
Keywords: anti-carbaryl scFv; ELISA; construction; binding complex