Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.8, #3)

Oxidative stress and its effect on cell functional activity in Alzheimer’s disease by E. E. Dubinina; L. V. Schedrina; N. G. Neznanov; N. M. Zalutskaya; D. V. Zakharchenko (181-191).
The review summarizes literature data on the importance of oxidative stress as one of the pathogenetic mechanisms in Alzheimer’s disease. Special attention is paid to the main specific and nonspecific ways of reactive oxygen species (ROS) generation in the course of the disease development. Generated ROS influence functional activity of cells, particularly, apoptosis and the mitotic cycle. Phosphorylation/dephosphorylation processes associated with intense phosphorylation of tau protein and mitosis-specific proteins play the nodal regulatory role in the cell. Alzheimer’s disease is accompanied by impairments of the regulatory functions of peptidyl-prolyl isomerases, particularly, Pin1 involved in maintaining a balanced state of phosphorylation/dephosphorylation processes. Taking into consideration the multifactorial impairment of the cell cycle control, this process should be considered from the viewpoint of the general state of metabolic processes, and oxidative stress has one of the key positions in aging.
Keywords: oxidative stress; reactive oxygen species; Alzheimer’s disease; beta amyloid; apoptosis; mitotic cycle

Pathogenesis of insulin resistance in metabolic obesity by L. S. Litvinova; E. V. Kirienkova; I. O. Mazunin; M. A. Vasilenko; N. S. Fattakhov (192-202).
This review considers molecular mechanisms of insulin resistance developed under conditions of metabolic inflammation; special attention is paid to analysis of the results of experimental and clinical studies work aimed at identifying molecular targets for the development of new methods for prevention and treatment of insulin resistance.
Keywords: obesity; type 2 diabetes mellitus; metabolic syndrome; insulin resistance

Extracellular nucleic acids (exNAs) were originally described in blood of healthy and sick people in 1948; however, little attention was paid to them until 1960th. exNAs are intensively studied now, especially during the last five years. The major attention is paid to exNAs as the source of a diagnostic material, whereas mechanisms of their generation, as well as mechanisms responsible for their long-term circulation in blood-stream still need better understanding. Some authors believe that the processes of apoptosis and necrosis represent the main source of blood circulating deoxyribonucleic acids (cirDNA), while others suggest nucleic acid secretion by healthy and tumor cells. Circulating DNA were found to be stable in blood for a long time, escaping from the action of DNA hydrolyzing enzymes, possibly, due to formation of various supramolecular complexes. This review summarizes literature data which support all the theories describing appearance of cirDNA; certain attention is paid to features of circulation and structure of the cirDNA and factors affecting time of DNA circulation in blood.
Keywords: circulating DNA; apoptosis; necrosis; active secretion; DNases; methylation

SOI-nanowire biosensor for detection of D-NFATc1 protein by K. A. Malsagova; Yu. D. Ivanov; T. O. Pleshakova; A. F. Kozlov; N. V. Krohin; A. L. Kaysheva; I. D. Shumov; V. P. Popov; O. V. Naumova; B. I. Fomin; D. A. Nasimov (220-225).
The nanowire (NW) detection is one of the fast-acting and high-sensitive methods, which can recognize potentially relevant protein molecules. A NW-biosensor based on the silicon-on-insulator (SOI)-structures has been used for biospecific label-free real time detection of the NFATc1 (D-NFATc1) oncomarker. For this purpose, SOI-nanowires (NWs) were modified with aptamers against NFATc1 used as molecular probes. It was shown that using this biosensor it is possible to reach sensitivity of 10−15 M. This sensitivity was comparable to that of the NW-biosensor with immobilized antibodies used as macromolecular probes. The results demonstrate that approaches used in this study are promising for development of sensor elements for high-sensitive diagnostics of diseases.
Keywords: aptamer; biosensor; D-NFATc1; silicon-on-insulator; nanowires.

The use of immobilized ubiquitin for biosensor analysis of the mitochondrial subinteractome by O. A. Buneeva; O. V. Gnedenko; M. V. Medvedeva; A. S. Ivanov; A. E. Medvedev (226-230).
Protein ubiquitination is an important mechanism responsible not only for specific labeling of proteins for their subsequent degradation; it also determines localization of proteins in the cell and regulation of protein-protein interactions. In the context of protein-protein interactions binding of (mono/poly)ubiquitinated molecules to proteins containing specific ubiquitin binding domains plays the decisive role. Formation of the ubiquitin interactome has been demonstrated for cytosol. Involvement of mitochondria and associated extramitochondrial proteins into such interactions still requires detailed investigation. In this study using an optical biosensor we have demonstrated binding of proteins of mouse brain mitochondrial lysates to immobilized monomeric ubiquitin. Model purified proteins, which are known to be associated with the outer mitochondrial compartment (glyceraldehyde-3-phosphate dehydorgenase, creatine phosphokinase), interacted with immobilized ubiquitin as well as with each other. This suggests that (poly)ubiquitinated chains may be involved in protein-protein interactions between ubiquitinated and non-ubiquitinated proteins and thus may contribute to formation of (mitochondrial) ubiquitin subinteractome.
Keywords: ubiquitination; mitochondrial protein-protein interactions; subinteractome; biosensor analysis

Protein-protein interactions in the systems of cytochromes P450 3A4 and 3A5 by O. V. Gnedenko; A. S. Ivanov; E. O. Yablokov; S. A. Usanov; D. V. Mukha; G. V. Sergeev; A. V. Kuzikov; N. E. Moskaleva; T. V. Bulko; V. V. Shumyantseva; A. I. Archakov (231-236).
Molecular interactions between protein partners of the monooxygenase system involved in drug biotransformation (cytochromes P450 3A4, 3A5 and cytochrome b 5) have been investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various forms of cytochrome b 5, including microsomal (b 5 mc) and mitochondrial (b 5 om) forms of this protein, as well as two chimeric constructs (b 5(om-mc),(b 5(mc-om). Interestingly, significant differences were observed only during interactions with b 5 om. Electroanalytical characteristics of electrodes with immobilized hemoproteins have been determined for CYP3A4, CYP3A5, b 5 mc, b 5 om, b 5 mc(om-mc), and b 5 mc(mc-om). The electrochemical analysis revealed that these proteins are characterized by close reduction potentials ranged from −0.435 V to −0.350 V (vs. Ag/AgCl). Cytochrome b 5 mc stimulated the electrocatalytic activity of CYP3A4.
Keywords: SPR biosensor; protein-protein interactions; cytochromes P450; cytochrome b 5 ; electrochemistry; electrocatalysis; electron transfer

New types of organic-inorganic hybrid nanocomposites based on nanosized titanium oxide(IV) (TiO2, particle size <100 nm) and carbon nanotubes (CNT, outer diameter of 10–15 nm, inner diameter of 2–6 nm, and length of 0.1–10 μm) and phosphatidylcholine were elaborated for improvement of analytical characteristics of screen printed electrodes. These nanomaterials were employed as an interface for immobilization of skeletal myoglobin. Electroanalytical and electrokinetic behavior of myoglobin on such interfaces was characterized with cyclic voltammetry (CV) and square wave voltammetry (SWV). Direct unmediated electron transfer between heme of immobilized myoglobin and electrodes modified with titanium oxide or carbon nanotubes was registered. The midpoint (redox) potential of the myoglobin Fe3+/Fe2+ E 1/2 = −0.263 V for electrodes modified with CNT and E 1/2 = −0.468 V for electrodes modified with TiO2 was observed (vs. Ag/AgCl reference electrode).
Keywords: enzyme electrodes; nanomaterials; titanium oxide; carbon nanotubes; myoglobin; electron transfer

Development of barcode and proteome profiling of glioblastoma by S. N. Naryzhny; N. L. Ronzhina; M. A. Mainskova; N. V. Belyakova; R. A. Pantina; M. V. Filatov (243-251).
High grade glioma (glioblastoma) is the most common brain tumor. Its malignancy makes it the fourth biggest cause of cancer death. In our experiments, we used several glioblastoma cell lines to obtain proteomics information specific for this disease. 2DE separation with following imaging, immunochemistry, spot picking, and mass-spectrometry allowed us to detecting more than 600 protein spots and identifying more than 130 of them. Proteome profiles in normal and glioblastoma cell lines are very similar but levels of several proteins have prominent differences between norm and cancer. Among these proteins are alpha-enolase (ENOA_HUMAN), pyruvate kinase M1/M2 (KPYM_HUMAN), cofilin 1 (COF1_HUMAN), translationally-controlled tumor protein TCTP_HUMAN, annexin 1 (ANXA1_HUMAN), PCNA (PCNA_HUMAN), p53 (TP53_HUMAN) and others. Most interesting results were obtained about protein p53. Its level was dramatically up-regulated and enriched by multiple additional isoforms in all glioblastoma cell lines. An immunological analysis (Western blot) of three hub-proteins (p53, 14-3-3, PCNA) allowed us to creating the minimal barcode of glioblastoma cell lines. These preliminary data point to this barcode as a promising diagnostic tool for testing of the biological fluids from patients.
Keywords: proteomics; glioblastoma; p53; PCNA

A new approach to the regulation of catalytic properties of medically relevant enzymes has been proposed using the novel recombinant preparation of L-asparaginase from Erwinia carotovora (EwA), a promising antitumor agent. New branched co-polymers of different composition based on chitosan modified with polyethylene glycol (PEG) molecules, designated as PEG-chitosan, have been synthesized. PEG-chitosan copolymers were further conjugated with EwA. In order to optimize the catalytic properties of asparaginase two types of conjugates differing in their architecture have been synthesized: (1) crown-type conjugates were synthesized by reductive amination reaction between the reducing end of the PEG-chitosan copolymer and enzyme amino groups; (2) multipoint-conjugates were synthesized using the reaction of multipoint amide bond formation between PEG-chitosan amino groups and carboxyl groups of the enzyme in the presence of the Woodward’s reagent. The structure and composition of these conjugates were determined by IR spectroscopy. The content of the copolymers in the conjugates was controlled by the characteristic absorption band of C-O-C bonds in the PEG structure at the frequency of 1089 cm−1. The study of catalytic characteristics of EwA preparations by conductometry showed that at physiological pH values the enzyme conjugates with PEG-chitosan with optimized structure and the optimal composition demonstrated 5–8-fold higher catalytic efficiency (k cat/K m) than the native enzyme. To certain extent, this can be attributed to favorable shift of pH-optima in result of positively charged amino-groups introduction in the vicinity of the active site. The proposed approach, chito-pegylation, is effective for regulating the catalytic and pharmacokinetic properties of asparaginase, and is promising for the development of prolonged action dosage forms for other enzyme therapeutics.
Keywords: L-asparaginase; PEG-chitosan; branched copolymers; catalytic activity; conductometry; IR spectroscopy

The effect of pyruvate on antibody interaction with group-specific erythrocyte antigens by F. N. Gylmiyarova; V. M. Radomskaya; O. A. Gusyakova; E. A. Ryskina; N. A. Kolotyeva; E. A. Shahnovich; N. S. Nefedova; I. F. Sidorova; G. M. Baisheva; Yu. Y. Pervova; A. P. Pleten (260-266).
Using the AB0 antibody-antigen model the influence of natural metabolite pyruvate on the antibody interaction with of erythrocyte antigens, defining their group specificity has been investigated. Before agglutination reaction erythrocytes of A (II)–AB (IV) blood groups, monoclonal anti-A and anti-B antibodies were incubated with sodium pyruvate. Visualization of agglutinates was performed by means of flow cytometry and laser scanning confocal microscopy. Computer-aided prediction of the spectrum of biological activity of pyruvate by a PASS program proposed major regulatory pathways, in which pyruvate may be involved. It has been demonstrated that pyruvate can regulate the intensity of antigen-antibody interaction. These results suggest the possibility of using small molecules, for example pyruvate, as molecular probes and prospects of the use of erythrocytes with antigenic determinants of the ABO system expressed on their membranes for studies of protein-protein interactions due to convenient visualization and possibility of quantitative evaluation of this process.
Keywords: protein-protein interaction; AB0 antigens; monoclonal antibodies; pyruvate; confocal microscopy

Redox status and pharmacokinetics of coenzyme Q10 in rat plasma after its single intravenous administration by E. I. Kalenikova; E. V. Kharitonova; E. A. Gorodetskaya; O. G. Tokareva; O. S. Medvedev (267-272).
The pharmacokinetics of the total pool of coenzyme Q10 (CoQ10), its oxidized (ubiquinone) and reduced (ubiquinol, CoQ10H2) forms have been investigated in rats plasma during 48 h after a single intravenous injection of a solution of solubilized CoQ10 (10 mg/kg) to rats. Plasma levels of CoQ10 were determined by HPLC with spectrophotometric and coulometric detection. In plasma samples taken during the first minutes after the CoQ10 intravenous injection, the total pool of coenzyme Q10 and proportion of CoQ10H2 remained unchanged during two weeks of storage at −20°C. The kinetic curve of the total pool of coenzyme Q10 corresponds to a one-compartment model (R 2 = 0.9932), while the corresponding curve of its oxidized form fits to the two-compartment model. During the first minutes after the injection a significant portion of plasma ubiquinone undergoes reduction, and after 7 h the concentration of ubiquinol predominates. The decrease in total plasma coenzyme Q10 content was accompanied by the gradual increase in plasma ubiquinol, which represented about 90% of total plasma CoQ10 by the end of the first day. The results of this study demonstrate the ability of the organism to transform high concentrations of the oxidized form of CoQ10 into the effective antioxidant (reduced) form and justify prospects of the development of parenteral dosage forms of CoQ10 for the use in the treatment of acute pathological conditions.
Keywords: coenzyme Q10 ; CoQ10 ; ubiquinone; ubiquinol; redox status; intravenous administration; pharmacokinetics; HPLC