Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.10, #2)

The history of renalase from amine oxidase to α-NAD(P)H-oxidase/anomerase by I. S. Severina; V. I. Fedchenko; A. V. Veselovsky; A. E. Medvedev (97-109).
Renalase is a recently discovered secretory protein, which plays a certain (still poorly understood) role in regulation of blood pressure. The review summarizes own and literature data on structure and catalytic properties of renalase accumulated since the first publication on this protein (2005). Initial reports on FADdependent amine oxidase activity were not confirmed in independent experiments performed in different laboratories. In addition, proposed amine oxidase activity of circulating extracellular renalase requires the presence of FAD, which has not been detected either in blood or urinary renalase. Moreover, renalase excreted into urine lacks its N-terminal peptide, which is ultimately needed for accommodation of the FAD cofactor. Results of the Aliverti’s group on NAD(P)H binding by renalase and weak diaphorase activity of this enzyme stimulated further studies of renalase as NAD(P)H oxidase catalyzing reaction of catecholamine co-oxidation. However, physiological importance of such extracellular catecholamine-metabolizing activity remains unclear due to existence of much more active enzymatic systems (e.g., neutrophil NAD(P)H oxidase, xanthine oxidase/xanthine) in circulation, which can perform such co-oxidation reactions. Recently α-NAD(P)H oxidase/anomerase activity of renalase, which also promotes oxidative conversion of β-NADH isomers inhibiting activity of NAD-dependent dehydrogenases, has been described. However, its possible contribution to the antihypertensive effect of renalase remains unclear. Thus, the antihypertensive effect of renalase still remains a phenomenon with unclear biochemical mechanim(s) and functions of intracellular and extracellular (circulating) renalases obviously differ.
Keywords: renalase; N-terminal peptide; FAD-binding domain; catalytic functions; antihypertensive effect

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (A review of own results) by N. I. Solovyeva; O. S. Timoshenko; T. A. Gureeva; E. V. Kugaevskaya (110-121).
Own results of long-term studies of expression of matrix metalloproteinases (MMPs) and their endogenous regulators examined in fibroblasts transformed by oncogene E7 HPV16 (TF), immortalized fibroblasts (IF), cell lines associated with HPV16 and HPV18, and tumor tissue samples from patients with squamous cervical carcinoma (SCC) associated with HPV16 have been summarized. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. MMP expression correlated with the tumorigenic of transformed clones. Expression of MMP-9 was found only in TF. In TF expression mRNA TIMP-1 decreased, while expression of the genatinase inhibitor, TIMP-2, increased. Collagenase activity and expression of the MMP-14 (collagenase) mRNA increased, while gelatinase activity remained unchanged. The destructive potential of TF is associated with induction of collagenases, gelatinase MMP-9 and decreased levels of MMP inhibitors. MMP-9 may serve as a TF marker. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-2 activity significantly increased in Caski and HeLa cell lines, while MMP-9 expression in these cell lines was not detected. The comparative study of expression MMP of and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously associated with increased expression of collagenases MMP-1, MMP-14 and gelatinase MMP-9, as well as decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent with increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. The morphologically normal tissue adjacent to the tumor tissue is characterized by significant expression of MMP-1, MMP-2, and MMP-9. This also contributes to the increased destructive potential of the tumor.
Keywords: matrix metalloproteinases (MMP); tissue inhibitors of MMP (TIMP); urokinase-type plasminogen activator (uPA); human papilloma virus (HPV); squamous cervical carcinom

Induced differentiation of leukemia cells is in the focus of basic and applied biomedical studies for more than 30 years. During this period specific regulatory molecules involved in the maturation process have been identified by biochemical and molecular biological methods. Recent developments of high-throughput transcriptomic and proteomic techniques made it possible to analyze large sets of mRNA and proteins; this resulted in identification of functionally important signal transduction pathways and networks of molecular interactions, and thus extent existing knowledge on the molecular mechanisms of induced differentiation. Despite significant achievements in studies of induced differentiation, many problems associated with the molecular mechanism of cell maturation, a phenomenon of therapeutic resistance of leukemic cells still need better understanding and thus require further detailed study. Transcriptomics and proteomics methods provide a suitable methodological platform for the implementation of such studies. This review highlights the use of transcriptomic and proteomic methods in studies aimed at various aspects of the induced differentiation. Special attention is paid to the employment of the systems approach for investigation of various aspects of cell maturation. The use of the systems approach in studies of induced differentiation is an important step for transition from the formal data accumulation on expression of mRNA and proteins towards creating models of biological processes in silico.
Keywords: HL60 cell line; induced differentiation; transcriptomics; proteomics; systems biology

The effect of resveratrol and dihydroquercetin inclusion into phospholipid nanopatricles on their bioavalability and specific activity by D. A. Guseva; Yu. Yu. Khudoklinova; N. V. Medvedeva; V. S. Baranova; T. S. Zakharova; E. B. Artyushkova; T. I. Torkhovskaya; O. M. Ipatova (138-144).
Bioavailability and activity of natural polyphenols, resveratrol (RES) and dihydroquercetin (DHQ), included in phospholipid nanoparticles have been investigated. Specific features of RES and DHQ in these compositions have been analyzed in vivo and in vitro experiments in comparison with free substances. Preincubation of low density lipoproteins (LDL), isolated from plasma of healthy donors, with RES or DHQ included in phospholipid nanoparticles caused a more pronounced delay in Cu2+ induced lipid oxidation compared with the free substances, and reduced the formation of lipid peroxidation (LPO) products. In phospholipid formulations bioavailability of RES and DHQ orally administered to rats were 1.5–2-fold higher than that of free substances. Prophylactic administration of phospholipid formulations containing RES or DHQ for two weeks resulted in the 25% increase of animal survival in the acute hypoxia model and 1.5-fold increase of catalase activity assayed in brain homogenates as compared with free substances. Using the model of endothelial dysfunction in rats induced by L-NAME, nitric oxide synthase inhibitor, it was shown, that RES markedly attenuated the inhibitory effect of L-NAME on NO synthesis. RES administered in phospholipid nanoparticles demonstrated the same efficiency at a dose one order of magnitude lower compared than that of free RES. The load test with resistance (clamping of the ascending aorta for 30 s) showed that the phospholipid formulation of RES exhibited a more pronounced protective effect due to stimulation of endothelial NO-synthase.
Keywords: resveratrol; dihydroquercetin; phospholipid nanoparticles; bioavailability; hypoxia; endothelial dysfunction

Molecularly imprinted polymers (MIP) in electroanalysis of proteins by V. V. Shumyantseva; T. V. Bulko; I. H. Baychorov; A. I. Archakov (145-151).
The review summarizes current knowledge on the main approaches used for creation of high affinity polymer analogs of antibodies (known as molecularly imprinted polymers, MIP) applicable for electroanalysis of functionally important proteins such as myoglobin, troponin T, albumin, ferritin, lysozyme, calmodulin. The main types of monomers for MIP preparation as well as methods convenient for analysis of MIP/protein interactions, such as surface plasmon resonance (SPR), nanogravimetry with the use of a quartz crystal resonator (QCM), spectral and electrochemical methods have been considered. Special attention is paid to experimental data on electrochemical registration of myoglobin by means of o-phenylenediaminebased MIP electrodes. It was shown that the imprinting factor calculated as a ratio of the myoglobin signal obtained after myoglobin insertion in MIP to the myoglobin signal obtained after myoglobin insertion in the polymer lacking the molecular template (NIP) is 2–4.
Keywords: molecular imprinting; molecularly imprinted polymers (MIP); nanomaterials; electroanalysis; electrochemical sensors; MIP sensor; proteins

Genetic diagnostics is widely used for detection of risk factors of hereditary thrombophilias caused by molecular defects in the coagulation system. The hereditary thrombophilias are frequently associated with higher incidences of point mutations in hemostasis (F2 20210G>A, F5 1691G>A) and folate metabolism (MTHFR 677C>T, MTHFR 1298A>C) genes. Combinations of gene abnormalities in F2 and/or MTHFR with Leiden mutation (F5 1691G>A) significantly increase risk of thrombosis. Thus, simultaneous analysis of allele polymorphism of these genes is of clinical importance. This study has demonstrated high efficiency of microchip-based multiplex real time PCR for analysis of allele specific polymorphism in hemostasis and folate metabolism genes. Using this test it is possible to analyze polymorphism of the three genes (four point mutations) in a short time; it requires a minimal quantity of DNA template and PCR reagents including DNA polymerase, and thus can be recommended for clinical laboratory diagnostics.
Keywords: thrombophilia; polymorphism; F2; F5; MTHFR; PCR; microchip

Selection of DNA aptamers for breast cancer by G. S. Zamay; I. V. Belyanina; A. S. Zamay; M. A. Komarova; A. V. Krat; E. N. Eremina; R. A. Zukov; A. E. Sokolov; T. N. Zamay (158-164).
A method of selection of DNA aptamers to breast tumor tissue based on the use of postoperative material has been developed. Breast cancer tissues were used as the positive target; the negative targets included benign tumor tissue, adjacent healthy tissues, breast tissues from mastopathy patients, and also tissues of other types of malignant tumors. During selection a pool of DNA aptamers demonstrating selective binding to breast cancer cells and tissues and insignificant binding to breast benign tissues has been obtained. These DNA aptamers can be used for identification of protein markers, breast cancer diagnostics, and targeted delivery of anticancer drugs.
Keywords: SELEX; DNA aptamers; oligonucleotides; breast cancer

Exosomes in tears of healthy individuals: Isolation, identification, and characterization by A. E. Grigor’eva; S. N. Tamkovich; A. V. Eremina; A. E. Tupikin; M. R. Kabilov; V. V. Chernykh; V. V. Vlassov; P. P. Laktionov; E. I. Ryabchikova (165-172).
Exosomes represent a sort of extracellular vesicles, which transfer molecular signals in the body and contain markers of the exosome-producing cell. This study was aimed at search of exosomes in the tears of healthy humans, validation of their nature and examination of their morphological and molecular-biological characteristics. Samples of the tears individually collected from 24 healthy donors (aged 45–60 years) were centrifuged at 20000 g for 15 min to pellet cell debris. The supernatants were examined in an electron microscope using negative staining and were also used for isolation and purification of exosomes by filtration (100 nm pore-size) and double ultracentrifugation (90 min at 100000 g, 4°C). Resultant pellets were investigated by electron microscopy and immunolabeling, RNA and DNA were isolated and their sizes were determined by capillary electrophoresis, concentration and localization of nucleic acids in the isolated exosomes were studied. DNA sequencing was performed using MiSeq (Illumina, USA), data were analyzed using CLC GW 7.5 (Qiagen, USA). Sequences were mapped on human genome (hg19). Supernatants of the tears contained cell debris, spherical microparticles (20–40 nm), and vesicles; some of the vesicles had morphology and sizes corresponding to exosomes. The pellets obtained after ultracentrifugation of tears contained microparticles (17%), spherical and cup-shaped vesicles (40–100 nm, 83%), which were positive for CD63, CD9, and CD24 receptors (specific markers of exosomes). The study revealed high concentrations of exosomes in human tears; these exosomes contained both RNA (of less than 200 nucleotides in size) and DNA (of 3–9 kb in size). DNA sequencing demonstrated that about 92% of the reads was mapped to human genome.
Keywords: tears; electron microscopy; extracellular vesicles; exosomes; DNA

Plasma myeloperoxidase activity as a criterion of therapeutic effectiveness for patients with cardiovascular diseases by D. V. Grigorieva; I. V. Gorudko; V. A. Kostevich; A. V. Sokolov; I. V. Buko; V. B. Vasilyev; L. Z. Polonetsky; O. M. Panasenko; S. N. Cherenkevich (173-179).
A significant increase in the myeloperoxidase (MPO) activity has been found in plasma of patients with stable angina and with acute coronary syndrome (ACS) in comparison with the control group. MPO concentration was significantly increased in plasma of ACS patients. Reduced MPO activity in the treated ACS patients correlated with a favorable outcome of the disease. Generally, changes in plasma MPO concentration coincided with changes in lactoferrin concentration thus confirming the role of neutrophil degranulation in the increase of plasma concentrations of these proteins. The increase in MPO activity was obviously determined by modification of the MPO protein caused by reactive oxygen species and halogen in the molar ratio of 1: 25 and 1: 50. The decrease in plasma MPO activity may be associated with increased plasma concentrations of the physiological inhibitor of its activity, ceruloplasmin, and also with modification of the MPO protein with reactive oxygen species and halogen at their molar ratio of 1: 100 and higher. Thus, MPO activity may be used for evaluation of effectiveness of the treatment of cardiovascular diseases.
Keywords: myeloperoxidase; acute coronary syndrome; peroxidase activity; neutrophils; ceruloplasmin; lactoferrin

Primary screening of candidate RNA biomarkers for diagnostics of prostate cancer by A. S. Nikitina; V. V. Babenko; K. A. Babalyan; A. O. Vasiliev; A. V. Govorov; E. A. Prilepskaya; S. A. Danilenko; O. V. Selezneva; E. I. Sharova (180-183).
Applicability of prostate cancer candidate RNA biomarkers screening in plasma and urine obtained by minimally invasive or noninvasive methods has been demonstrated. In patients with benign prostatic hyperplasia (BPH) significant amounts of RNAs known in the literature as biomarkers associated with prostate cancer have been detected in plasma and urine samples. Detectable amounts of the markers depended on the methods used for transcriptome profiling. Detection of known RNA biomarkers associated with prostate cancer in urine and plasma samples by the RNA-seq method shows its applicability for the search of new RNA biomarkers for minimally invasive diagnostics. However, the fact of the presence of known prostate cancer RNA biomarkers in samples from BPH patients suggests their insufficient specificity and indicates a clear need for further research in this field.
Keywords: prostate cancer; benign prostatic hyperplasia; RNA biomarkers; RNA-seq

Metagenomic analysis of taxonomic and functional changes in gut microbiota of patients with the alcohol dependence syndrome by V. B. Dubinkina; A. V. Tyakht; E. N. Ilina; D. S. Ischenko; B. A. Kovarsky; K. S. Yarygin; A. V. Pavlenko; A. S. Popenko; D. G. Alexeev; A. E. Taraskina; R. F. Nasyrova; E. M. Krupitski; L. O. Skorodumova; A. K. Larin; E. S. Kostryukova; V. M. Govorun (184-190).
The first metagenomic study of gut microbiota in patients with the alcohol dependence syndrome (ADS) has been performed in the whole-genome sequencing (“shotgun”) format. Taxonomic analysis revealed changes in the relative abundance of the predominant bacteria associated with inflammatioln (including increased levels of Ruminococcus gnavus and R. torques, and decreased levels of Faecalibacterium and Akkermansia genera). The microbiota of ADS patients was characterized by the presence of opportunistic pathogens rarely detected in metagenomes of healthy individuals from different countries. Comparative analysis of total metabolic potential revealed increased relative abundance of KEGG pathways associated with the response to oxidative stress. ADS patients also had increased levels of two specific groups of genes encoding enzymes involved in the metabolism of alcohol, as well as virulence factors. It is possible that gut microbiota of ADS patients demonstrating changes in both taxonomic and functional composition plays a role in modulating the effects of alcohol on the host body
Keywords: metagenome; gut microbiota; alcohol dependence syndrome; metabolic potential; microbial alcohol metabolism; virulence factors