Biochemistry (Moscow) (v.79, #11)

Evolution of α- and β-Globin genes and their regulatory systems in light of the hypothesis of domain organization of the genome by O. V. Iarovaia; E. S. Ioudinkova; N. V. Petrova; K. V. Dolgushin; A. V. Kovina; A. V. Nefedochkina; Y. S. Vassetzky; S. V. Razin (1141-1150).
The α- and β-globin gene domains are a traditional model for study of the domain organization of the eucaryotic genome because these genes encode hemoglobin, a physiologically important protein. The α-globin and β-globin gene domains are organized in completely different ways, while the expression of globin genes is tightly coordinated, which makes it extremely interesting to study the origin of these genes and the evolution of their regulatory systems. In this review, the organization of the α- and β-globin gene domains and their genomic environment in different taxonomic groups are comparatively analyzed. A new hypothesis of possible evolutionary pathways for segregated α- and β-globin gene domains of warm-blooded animals is proposed.
Keywords: α- and β-globin gene domains; gene expression; evolution

Mutations in mitochondrial DNA and approaches for their correction by M. V. Patrushev; P. A. Kamenski; I. O. Mazunin (1151-1160).
Apart from the nucleus, the mitochondrion is the only organelle of an animal cell that contains its own genome. Mitochondrial DNA is much less in size than the nuclear one and codes for only several dozens of biological macromolecules. Nevertheless, mutations in mitochondrial genes often result in the occurrence of serious hereditary neuromuscular diseases. New mitochondrial DNA mutations and their relations to clinical symptoms are continuously reported in the scientific literature. In this review, we summarize existing data about such mutations, and also about contemporary gene therapy approaches that have been developed for their suppression.
Keywords: mitochondrial genome; mitochondrial DNA; mutations; mitochondrial diseases; gene therapy

Mammalian hibernation and regulation of lipid metabolism: A focus on non-coding RNAs by D. Lang-Ouellette; T. G. Richard; P. Morin Jr. (1161-1171).
Numerous species will confront severe environmental conditions by undergoing significant metabolic rate reduction. Mammalian hibernation is one such natural model of hypometabolism. Hibernators experience considerable physiological, metabolic, and molecular changes to survive the harsh challenges associated with winter. Whether as fuel source or as key signaling molecules, lipids are of primary importance for a successful bout of hibernation and their careful regulation throughout this process is essential. In recent years, a plethora of non-coding RNAs has emerged as potential regulators of targets implicated in lipid metabolism in diverse models. In this review, we introduce the general characteristics associated with mammalian hibernation, present the importance of lipid metabolism prior to and during hibernation, as well as discuss the potential relevance of non-coding RNAs such as miRNAs and lncRNAs during this process.
Keywords: hibernation; hypometabolism; lipid synthesis; fatty acid degradation; microRNAs; long non-coding RNAs

SOX2 overexpression affects neural differentiation of human pluripotent NT2/D1 cells by A. Klajn; D. Drakulic; M. Tosic; Z. Pavkovic; M. Schwirtlich; M. Stevanovic (1172-1182).
SOX2 is one of the key transcription factors involved in maintenance of neural progenitor identity. However, its function during the process of neural differentiation, including phases of lineage-specification and terminal differentiation, is still poorly understood. Considering growing evidence indicating that SOX2 expression level must be tightly controlled for proper neural development, the aim of this research was to analyze the effects of constitutive SOX2 overexpression on outcome of retinoic acid-induced neural differentiation of pluripotent NT2/D1 cells. We demonstrated that in spite of constitutive SOX2 overexpression, NT2/D1 cells were able to reach final phases of neural differentiation yielding both neuronal and glial cells. However, SOX2 overexpression reduced the number of mature MAP2-positive neurons while no difference in the number of GFAP-positive astrocytes was detected. In-depth analysis at single-cell level showed that SOX2 downregulation was in correlation with both neuronal and glial phenotype acquisitions. Interestingly, while in mature neurons SOX2 was completely downregulated, astrocytes with low level of SOX2 expression were detected. Nevertheless, cells with high level of SOX2 expression were incapable of entering in either of two differentiation pathways, neurogenesis or gliogenesis. Accordingly, our results indicate that fine balance between undifferentiated state and neural differentiation depends on SOX2 expression level. Unlike neurons, astrocytes could maintain low level of SOX2 expression after they acquired glial fate. Further studies are needed to determine whether differences in the level of SOX2 expression in GFAP-positive astrocytes are in correlation with their self-renewal capacity, differentiation status, and/or their phenotypic characteristics.
Keywords: SOX2 overexpression; NT2/D1; neurogenesis; gliogenesis; neural differentiation

Properties of hybrid hybrid complexes composed of photosynthetic reaction centers from the purple bacterium Rhodobacter sphaeroides and quantum dots in lecithin liposomes by V. E. Zagidullin; E. P. Lukashev; P. P. Knox; N. Kh. Seifullina; O. S. Sokolova; E. V. Pechnikova; H. Lokstein; V. Z. Paschenko (1183-1191).
Quantum dots (QDs) can absorb ultraviolet and long-wavelength light energy much more efficiently than natural light-harvesting proteins and transfer the excitation energy to photosynthetic reaction centers (RCs). Inclusion into liposomes of RC membrane pigment-protein complexes combined with QDs as antennae opens new opportunities for using such hybrid systems as a basis for artificial energy-transforming devices that potentially can operate with greater efficiency and stability than devices based only on biological components. RCs from Rhodobacter sphaeroides and QDs with fluorescence maximum at 530 nm (CdSe/ZnS with hydrophilic covering) were embedded in lecithin liposomes by extrusion of a solution of multilayer lipid vesicles through a polycarbonate membrane or by dialysis of lipids and proteins dispersed with excess detergent. The dimensions of the resulting hybrid systems were evaluated using dynamic light scattering and by transmission cryoelectron microscopy. The efficiency of RC and QD interaction within the liposomes was estimated using fluorescence excitation spectra of the photoactive bacteriochlorophyll of the RCs and by measuring the fluorescence decay kinetics of the QDs. The functional activity of the RCs in hybrid complexes was fully maintained, and their stability was even increased.
Keywords: purple bacteria; photosynthetic reaction center; quantum dots; liposomes; energy transfer

Unexpectedly strong effect of caffeine on the vitality of western honeybees (Apis mellifera) by A. Strachecka; M. Krauze; K. Olszewski; G. Borsuk; J. Paleolog; M. Merska; J. Chobotow; M. Bajda; K. Grzywnowicz (1192-1201).
We examined the influence of caffeine on honeybee lifespan, Nosema resistance, key enzyme activities, metabolic compound concentrations, and total DNA methylation levels. Caffeine slowed age-related metabolic tendencies. Bees that consumed caffeine lived longer and were not infested with Nosema spp. Caffeine-treated workers had higher protein concentrations. The levels increased with aging but they then decreased in older bees. Caffeine increased the activities of antioxidant enzymes (SOD, GPx, CAT, GST), AST, ALT, ALP, neutral proteases, and protease inhibitors, and the concentrations of uric acid, triglycerides, cholesterol, glucose, and Ca2+. Acidic and alkaline protease activities were lower in the bees treated with caffeine. Creatinine and Mg2+ concentrations were higher in the caffeine-treated workers but only up to 14 days of age. Caffeine significantly decreased DNA methylation levels in older bees. The compound could be considered as a natural diet supplement increasing apian resistance to stress factors. Our studies will enhance possibilities of using Apis mellifera as a model organism in gerontological studies.
Keywords: honeybee; caffeine; vitality; antioxidant; proteolysis; DNA methylation

Heat shock induces production of reactive oxygen species and increases inner mitochondrial membrane potential in winter wheat cells by A. V. Fedyaeva; A. V. Stepanov; I. V. Lyubushkina; T. P. Pobezhimova; E. G. Rikhvanov (1202-1210).
Heat shock leads to oxidative stress. Excessive ROS (reactive oxygen species) accumulation could be responsible for expression of genes of heat-shock proteins or for cell death. It is known that in isolated mammalian mitochondria high protonic potential on the inner membrane actuates the production of ROS. Changes in viability, ROS content, and mitochondrial membrane potential value have been studied in winter wheat (Triticum aestivum L.) cultured cells under heat treatment. Elevation of temperature to 37–50°C was found to induce elevated ROS generation and increased mitochondrial membrane potential, but it did not affect viability immediately after treatment. More severe heat exposure (55–60°C) was not accompanied by mitochondrial potential elevation and increased ROS production, but it led to instant cell death. A positive correlation between mitochondrial potential and ROS production was observed. Depolarization of the mitochondrial membrane by the protonophore CCCP inhibited ROS generation under the heating conditions. These data suggest that temperature elevation leads to mitochondrial membrane hyperpolarization in winter wheat cultured cells, which in turn causes the increased ROS production.
Keywords: Triticum aestivum L.; reactive oxygen species; mitochondrial membrane potential; heat stress

PPX1 gene overexpression has no influence on polyphosphates in Saccharomyces cerevisiae by L. P. Lichko; M. A. Eldarov; M. V. Dumina; T. V. Kulakovskaya (1211-1215).
The role of exopolyphosphatase PPX1 in polyphosphate metabolism in yeasts has been studied in strains of Saccharomyces cerevisiae with inactivated PPX1 and PPN1 genes transformed by the expression vector carrying the yeast PPX1 gene. Exopolyphosphatase activity in transformant strains increased 90- and 40-fold compared to the ΔPPX1 and ΔPPN1 strains, respectively. The purified recombinant exopolyphosphatase PPX1 was similar to the PPX1 of wild strains in its substrate specificity and requirement for divalent metal cations. It was more active with tripolyphosphate and low molecular mass polyphosphates than with high molecular mass polyphosphates and required Mg2+ for its activity. The high level of recombinant PPX1 expression caused no decrease in polyphosphate content in the cells of the transformant. This fact suggests the restricted role of PPX1 in polyphosphate metabolism in yeasts.
Keywords: polyphosphates; exopolyphosphatase; PPX1 gene; Saccharomyces cerevisiae ; overexpression

Effect of naphthalene on photosystem 2 photochemical activity of pea plants by A. V. Lankin; V. D. Kreslavski; A. Yu. Khudyakova; S. K. Zharmukhamedov; S. I. Allakhverdiev (1216-1225).
The effect of a typical polyaromatic hydrocarbon, naphthalene (Naph), on photosystem 2 (PS-2) photochemical activity in thylakoid membrane preparations and 20-day-old pea leaves was studied. Samples were incubated in water in the presence of Naph (0.078, 0.21, and 0.78 mM) for 0.5–24 h under white light illumination (15 μmol photons·m−2·s−1). The PS-2 activity was determined by studying fast and delayed chlorophyll (Chl) a fluorescence. Incubation of samples in water solutions at Naph concentrations of 0.21 and 0.78 mM led to a decrease in the maximum PS-2 quantum efficiency (Fv/Fm), noticeable changes in the polyphasic induction kinetics of fluorescence (OJIP), and a decrease in the amplitudes of the fast and slow components of delayed fluorescence of Chl a. The rate of release of electrolytes from leaves that were preliminarily incubated with Naph (0.21 mM) was also increased. Significant decrease in the fluorescence parameters in thylakoid membrane preparations was observed at Naph concentration of 0.03 mM and 12-min exposure of the samples. Chlorophyll (a and b) and carotenoid content (mg per gram wet mass) was insignificantly changed. The quantum yields of electron transfer from QA to QBET2o) and also to the PS-1 acceptors (φRE1o) were reduced. These results are explained by the increase in the number of QB-non-reducing centers of PS-2, which increased with increasing Naph concentration and exposure time of leaves in Naph solution. The suppression of PS-2 activity was partly abolished in the presence of the electron donor sodium ascorbate. Based on these results, it is suggested that Naph distorts cell membrane intactness and acts mainly on the PS-2 acceptor and to a lesser degree on the PS-2 donor side.
Keywords: naphthalene; photosystem 2; variable and delayed fluorescence of chlorophyll; detached pea leaves; thylakoid membranes; photosynthetic pigments

The JcERF1 gene, which is related to the ERF family (ethylene responsive factor coding genes), was isolated and characterized from the oil tree Jatropha curcas. The JcERF1 protein contains conserved an AP2/EREBP DNA-binding domain of 58 amino acid residues. The JcERF1 gene could be induced by abscisic acid, high salinity, hormones, and osmotic stress, suggesting that JcERF1 is regulated by certain components of the stress-signaling pathway. The full-length and C-terminus of JcERF1 driven by the GAL4 promoter functioned effectively as a transactivator in yeast, while its N-terminus was completely inactive. Transient expression analysis using a JcERF1-mGFP fusion gene in onion epidermal cells revealed that the JcERF1 protein is targeted to the nucleus. Transgenic tobacco plants carrying CaMV35S::JcERF1 fragments were shown to be much more salt tolerant compared to wild-type plants. Our results indicate that JcERF1 is a new member of the ERF transcription factors family that may play an important role in tolerance to environmental stress.
Keywords: ethylene response factor (ERF); Jatropha curcas ; JcERF1 ; subcellular localization; overexpression; salt stress

Microbial/bacterial resistance against antibiotics is considered as a potentially serious threat to public health. Further, as these antibiotics elicit side effects, there is interest in developing new molecules with novel modes of action from diverse organisms. Along these lines, in this study the antibacterial potential of the basic protein VRV-PL-V (Vipera russellii venom phospholipase A2 fraction V) of Daboia russellii pulchella venom was evaluated. VRV-PL-V demonstrated a potent antibacterial activity against all the human pathogenic strains tested. It inhibited more effectively Gram-positive bacteria like Staphylococcus aureus and Bacillus subtilis when compared to Gram-negative bacteria like Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae, and Salmonella paratyphi. It inhibited bacterial growth with MIC values ranging from 13 to 24 μg/ml. The antibacterial potential of VRV-PL-V was comparable to the standards used like gentamycin, chloramphenicol, and streptomycin. There was a strong correlation between PLA2 activities and hemolytic and antibacterial activity. It was found that even in the presence of p-bromophenacyl bromide (an inhibitor of PLA2 enzymatic activity), there was marked antibacterial activity, suggesting dissociation or partial overlapping of the bactericidal/antimicrobial domains. Therefore, this study shows that although there is a strong correlation between enzymatic and antimicrobial activities of VRV-PL-V, it may also possess other properties that mimic bactericidal/membrane permeability-increasing protein.
Keywords: snake venom; phospholipase A2 ; antimicrobial; Daboia russellii pulchella ; human pathogenic bacteria

Iron metabolism after application of modified magnetite nanoparticles in rats by I. V. Milto; A. Yu. Grishanova; T. K. Klimenteva; I. V. Suhodolo; G. Yu. Vasukov; V. V. Ivanova (1245-1254).
The influence of modified nanosized magnetite (NSM) particles (magnetic microspheres coated with chitosan and magnetoliposomes) after a single intravenous infusion of their suspensions on iron metabolism in rats has been studied. Modern physical and chemical methods (X-ray fluorescence, dynamic light scattering, transmission electron microscopy) were used for standardization of the modified NSM particles (their size, structure, ζ-potential, and concentration were determined). Atomic emission spectroscopy was used to reveal the dynamics of iron content in rat liver, spleen, lungs, and kidneys during 120 days. Colorimetric and immunoturbidimetric methods were used to determine the concentrations of plasma iron and the proteins involved in its metabolism — ceruloplasmin, transferrin, and ferritin. Their dynamics through- out the experiments were studied.
Keywords: nanomagnetite; magnetoliposomes; chitosan-modified nanomagnetite; transferrin; ceruloplasmin; ferritin

pH-induced conformational isomerization of leghemoglobin from Arachis hypogea by P. Basak; R. Pattanayak; S. Nag; M. Bhattacharyya (1255-1261).
The pH dependence of proteins is related to the thermodynamic stability and electrostatic interactions in the native state of a protein. Here we report the pH-induced conformational transition of the heme protein leghemoglobin (Lb) isolated from root nodules of the leguminous plant Arachis hypogea. Unlike the other heme proteins myoglobin, hemoglobin, and cytochrome c, the structural characteristics and interactions of Lb is almost unknown, though its functional importance is already established since it binds oxygen to maintain the environment for N2 fixation. We investigated pH-induced unfolding of this protein and identified a number of conformational isomers using multiple fluorescence observables as a function of pH titration. We have characterized the acid- and base-induced conformational transitions among the structural states over the pH range 2–11. Depending on the solution conditions, Lb can exist in one of three phases: pH 2, 3, 4; pH 5, 6, 7; pH 8, 9, 10. The secondary structure as revealed by CD spectroscopy indicated the maximum percentage of α-helix to be present at pH 7, where the structure of Lb is also most rigid according to fluorescence anisotropy experiments. The fluorescence lifetime of tryptophan was observed to be maximum at pH 10 and minimum at pH 6, suggesting unfolding transitions of Lb. Thus, alteration of the microenvironment of the globin moiety during pH transition ultimately leads to the conformational change of this monomeric protein Lb.
Keywords: leghemoglobin; pH titration; protein conformation; unfolding; mean residual ellipticity; excited state lifetime; microenvironment

DNA methylation catalyzed by DNA methyltransferases (MTases) is widespread in prokaryotes. In an attempt to find EcoDam variants with enhanced preference for hemimethylated DNA, the L122, P134, and V133 residues were replaced with other amino acids using site directed mutagenesis, and the catalytic activity of all variants on unmethylated and hemimethylated substrates was studied. Our results showed that, in addition to L122A, the L122S and L122A/V133L EcoDam variants were able to sense the methylation status of the 5′-GATC-3′ double-stranded target recognition site and methylated only hemimethylated DNA.
Keywords: EcoDam; rational design; protein engineering

Expression of hp1 family genes and their plausible role in formation of flamenco phenotype in D. melanogaster by A. R. Lavrenov; L. N. Nefedova; N. I. Romanova; A. I. Kim (1267-1272).
Results of expression analysis of transcription of the flamenco locus that controls transposition of the mobile genetic element gypsy, RNA interference system genes ago3, zuc, aub, and HP1 heterochromatin protein family genes hp1a, hp1b, hp1c, hp1d (rhino), and hp1e in D. melanogaster SS strain mutant on the flamenco gene are presented. We show that the number of transcripts in the SS strain that are formed in the flamenco locus is unchanged in some freely chosen points, and this is different from the wild-type strain where a decreased number of transcripts is observed, which clearly is a result of processing of the flamenco locus primary transcript, a predecessor of piRNA. At the same time, expression of genes of the RNA interference system is not affected, but there is a reduced level of hp1d gene expression in ovary tissue. We suggest that the hp1d gene product is directly or indirectly involved in the flamenco locus primary transcript processing.
Keywords: gene expression; RNA interference; heterochromatin proteins; Drosophila ; flamenco ; mobile genetic elements