Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.9, #2)

Mass spectrometry analysis of blood plasma lipidome as the method of disease diagnostics, evalution of effectiveness and optimization of drug therapy by P. G. Lokhov; D. L. Maslov; E. E. Balashova; O. P. Trifonova; N. V. Medvedeva; T. I. Torkhovskaya; O. M. Ipatova; A. I. Archakov; P. P. Malyshev; V. V. Kukharchuk; E. A. Shestakova; M. V. Shestakova; I. I. Dedov (95-105).
A new method for the analysis of blood lipids based on direct mass spectrometry of lipophilic low molecular weight fraction of blood plasma has been considered. Such technique allows quantification of hundreds of various types of lipids and this changes existing concepts on diagnostics of lipid disorders and related diseases. Here we demonstrate the versatility and quickness of the method, which significantly simplify its wide use. This method is applicable for diagnostics of atherosclerosis, diabetes mellitus, cancer and other diseases. Detalization of plasma lipid composition at the molecular level by means of mass spectrometry allows to assess the effectiveness of therapy and to optimize the drug treatment of cardiovascular diseases by phospholipid preparations.
Keywords: mass spectrometry; blood plasma; diagnostics; personalization of therapy; lipidome; metabolome

Insulin resistance is the characteristic feature of type 2 diabetes mellitus. This condition is manifested in decreased sensitivity of peripheral tissues to the biological action of insulin and is expressed in the inhibition of cell glucose uptake and metabolism in response to the hormonal stimulation. At the cellular level, impairments realized both at the receptor and the postreceptor levels and associated with changes in the content or dysfunction of the main molecules of the signal cascade can serve a molecular prerequisite to the formation of insulin resistance. Thus, the insulin receptor, as well as the other related signaling molecules can be considered as ideal therapeutic targets for the correction of insulin resistance and low molecular weight effectors, which act on the individual links of the insulin signaling cascade, may be positioned as a new generation of anti-diabetic agents. This review summarizes current knowledge on regulators of the insulin receptor cascade, main advantages and disadvantages of their effects on biological targets and prospects for their therapeutic use as anti-diabetic agents.
Keywords: non-peptide insulin receptor activators; diabetes mellitus; insulin resistance; insulin receptor

Interaction of novel oxazoline derivatives of 17(20)E-pregna-5,17(20)-diene with cytochrome P450 17A1 by S. V. Stulov; N. O. Dugin; M. S. Zharkova; D. S. Shcherbinin; A. V. Kuzikov; V. V. Shumantseva; A. Yu. Misharin; A. V. Veselovsky (114-120).
In order to find novel inhibitors of 17α-hydroxylase-17,20-lyase (cytochrome P450 17A1, CYP17A1), a key enzyme of biosynthesis of androgens, molecular docking of six new oxazoline-containing derivatives 17(20)E-pregna-5,17(20)-diene has been carried out to the active site of the crystal structure of CYP17A1 (pdb 3ruk). Results of this study indicate that: (1) complex formation of docked compounds with CYP17A1 causes their isomerization in energetically less favorable 17(20)Z-isomer; (2) the localization of the steroid moiety of all compounds in the active site is basically the same; (3) the structure of the oxazoline moiety significantly influences its position relative to heme as well as the energy of complex formation; (4) coordination of the nitrogen atom of the oxazoline moiety and the heme iron is only possible in the 17(20)Z-conformation with anti oriented double bonds 17(20), and C=N; (5) the presence of two substituents at C4′ of the oxazoline moiety significantly impairs ligand binding; (6) oxazoline- and benzoxazole-containing derivatives 17(20)E-pregna-5,17(20)-diene can effectively inhibit the catalytic activity CYP17A1 and may be of interest as a basis for the development of new drugs for the treatment of androgen-dependent cancer.
Keywords: nitrogen-containing derivatives of 17(20)E-pregna-5,17(20)-diene; cytochrome P450 17A1; inhibitors; molecular modeling; electrochemistry; structure-activity relationships

AFM-based protein fishing in the pulsed electric field by Yu. D. Ivanov; T. O. Pleshakova; K. A. Malsagova; A. L. Kaysheva; A. T. Kopylov; A. A. Izotov; V. Yu. Tatur; S. G. Vesnin; N. D. Ivanova; V. S. Ziborov; A. I. Archakov (121-129).
A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b 5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell.
Keywords: AFM; MS; detection of low-copy proteins; protein fishing

Formation of nitric oxide metabolites during growth of transplanted tumors with different metastatic potential by V. P. Deryagina; N. I. Ryzhova; L. V. Krivosheeva; I. S. Golubeva (130-136).
The dynamics of the endogenous production of NO metabolites, nitrites, nitrates, and volatile nitrosamines, has been investigated in the body, a tumor tissue, and peritoneal macrophages of mice F1 (C57Bl × CBA), Balb/c and BDF with subcutaneously transplanted tumors: Ehrlich carcinoma (EC) and Lewis metastatic lung carcinoma (LC). It has been shown that during the first three weeks, growth of EC is accompanied by a statistically significant increase in the total concentration of NO metabolites (NOx; nitrites + nitrates) up to levels (7.3 ± 3.49) × 10−6−(7.8 ± 2.57) × 10−5 mol/kg in the tumor tissue and a sharp increase in NOx excretion with urine. At the same time, in the LC tumor tissue the maximal level of NOx (3.6 ± 0.46) × 10−5 mol/kg was registered on day 7, and later it decreased demonstrating negative correlation with the tumor mass. At the stage of intensive LC growth (14, 21 days) there was significant inhibition of nitrite secretion by peritoneal macrophages. In all the controlled time-intervals the EC tumor tissue contained carcinogenic N-nitrosodimethylamine and N-nitrosodiethylamine; however, their concentrations significantly varied. Thus, the ability of the tumor tissue to produce NO metabolites was more pronounced in EC than in LC and depended on the time parameters of tumor growth.
Keywords: biosynthesis of nitrites; nitrates; N-nitrosamines; tumor tissue; macrophages; Ehrlich carcinoma; Lewis lung carcinoma

Identification and quantitative determination of baclofen in human blood by HPLC with mass spectrometry detection by O. A. Dukova; M. Yu. Kotlovsky; A. A. Pokrovsky; E. V. Suvorova; T. G. Shivrina; E. A. Krasnov; A. A. Efremov (137-142).
A method of identification and quantitative determination of baclofen in blood by HPLC with mass spectrometry detection has been developed. It is characterized by high sensitivity, specificity, linearity, accuracy, reproducibility, and a low detection for quantitative determination. The method has been used for diagnostics of acute baclofen poisoning in patients.
Keywords: baclofen; poisoning; diagnostics; HPLC-MS/MS

The level of circulating PGC1α in cardiovascular diseases by A. A. Zhloba; T. F. Subbotina; E. S. Alekseevskaya; O. M. Moiseeva; N. D. Gavrilyuk; O. B. Irtyuga (143-150).
The levels of PGC1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) have been investigated in plasma samples from patients with cardiovascular diseases (n = 110) including aortic aneurysm (n = 69), aortic stenosis (n = 25), patients without aortal pathology and also from healthy individuals (n = 34). In patients with cardiovascular diseases the PGC1α concentration was higher than in healthy individuals, and tended to decrease with age. Increased concentrations of lactate, total homocysteine and asymmetric dimethylarginine in the blood of examined patients suggested a parallel development of endothelial and secondary mitochondrial dysfunctions. The increase in concentrations of lactate and pyruvate above the reference limits was accompanied by the decrease of PGC1α concentrations.
Keywords: PGC1α; diagnostics of mitochondrial dysfunction; endothelial dysfunction; lactic acid; pyruvic acid; blood plasma

Biochemical and immunological markers of autoimmune thyroiditis by E. M. Biktagirova; L. I. Sattarova; G. R. Vagapova; Y. V. Skibo; E. N. Chuhlovina; O. A. Kravtsova; Z. I. Abramova (151-158).
Correlations between biochemical and immunological markers of programmed cell death (apoptosis), and the functional state of the thyroid gland (hyperthyroidism, euthyroidism, hypothyroidism) have been investigated in autoimmune thyroiditis (AT) (also known as chronic autoimmune thyroiditis). Annexin V, TRAIL and TNFα, as well as DNA-hydrolyzing antibodies were used as the main markers. Increased levels of TRAIL were found in the serum of AT patients (hyperthyroidism > hypothyroidism > euthyroidism) compared with healthy individuals. The highest frequency of antibodies to denatured DNA (Abs-dDNA) had the highest frequency in AT patients (97%) compared with healthy controls. Among these patients, 75% had hyperthyroidism, 85% had hypothyroidism, and 84.7% had euthyroidism. Abs hydrolyzing activity demonstrated correlation dependence with symptoms of the thyroid dysfunction.
Keywords: apoptosis; autoantibodies; antibodies to DNA; abzymes; antibodies to thyroid tissue components; autoimmune thyroiditis

Association of miR-21 and miR-155 with regulation of 15-HPGD mRNA in human breast cancer cells by Z. N. Nikiforova; M. A. Taipov; I. A. Kudryavtsev; V. E. Shevchenko (159-165).
Breast cancer (BC) is the most common form of cancer, leading to high mortality rates among women worldwide. In this study we have analyzed mRNA expression of 15-hydroxy-prostaglandin-dehydrogenases (15-HPGD), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) and miRNAs (miR-21, miR-155) in three cell lines of estrogen-positive human breast carcinomas (MCF-7, BT-474, ZR-75-1). Results of three independent experiments have demonstrated significantly higher levels of COX-2 and COX-1 mRNAs in the cell line ZR-75-1 cells than in MCF-7 and BT-474 cells. mRNA levels of total 15-HPGD and functional-15-HPGD were lower in BT-474 than in MCF-7 and in ZR-75-1 cells. Synthesis of the 15-HPGD enzyme in BT-474 cells was blocked at the level of nuclear processing of an immature pre-mRNA. High expression of miR-21 was detected in all the tumor cell lines (MCF-7, ZR-75-1 and BT-474). In the breast cancer cell lines, the expression level of miR-155 was significantly lower than that of miR-21. Correlations have been found between dysregulation of miR-21, miR-155 and mRNA levels of 15-HPGD, COX-1, COX-2. The results obtained in this study showed that miR-21 and miR-155 regulate activity of several genes in the tumor cell and on some genes they exhibited a cumulative effect. Based on these results, we concluded that the miR-21 and miR-155 inhibit activity of the tumor suppressor gene 15-HPGD and induce a potential oncogene COX-2, which contributes to malignancy and metastasis of breast cancer cells.
Keywords: breast cancer; cyclooxygenase 1; cyclooxygenase 2; miRNA; mRNA; 15-hydroxy-prostaglandin dehydrogenase

The dose-dependent effects of cytokines (IL-2, IL-7, and IL-15), which have a common γ-chain, on mRNA expression of U2afll4 and GFi1 genes involved in regulation of alternative splicing of the Ptprc gene, have been investigated in vivo using T-lymphocyte cultures with different degrees of differentiation. IL-2, IL-7, and IL-15 caused a similar unidirectional inhibitory effect of various severity on restimulated CD45RO+ T-cells exposed to an antigen-independent activation; they caused a dose-dependent decrease of the U2af1l4 gene expression, and an increase of Gfi1 gene expression. This may suggest formation of active forms of the CD45 receptor, and also limitation of the formation of low-molecular short splice variants of the CD45RO receptor. Under conditions of antigen-independent stimulation of naive CD45RA+ - cells rIL-7 and IL-15 exhibited opposite effects on U2af1l4 and Gfi1 gene expression. The increase of IL-7 concentrations in the incubation medium of naive cells was accompanied by a decrease in expression of both genes. IL-15 IL-7 exhibited opposite effects. Cytokines possessing a common Γ-chain (IL-2, IL-7, and IL-15) prevented antigen-independent differentiation of naive T-cells, by preventing the formation of polyclonal “surrogate” cells. In general, the study of the molecular mechanisms of genetic control determining homeostatic processes of T-cells in response to exposure to antigenic or non-antigenic treatments may be important in the construction of a general model of self-maintenance and differentiation of immune cells.
Keywords: cytokines; gene expression; T-cells; activation; alternative splicing

Previously, it was found that blood plasma extracellular DNA (ecDNA) of patients with rheumatoid arthritis (RA) is enriched with CpG-rich genomic DNA fragments, which contain TLR9 ligands (Veiko et al., 2006). In this study we have demonstrated that ecDNA of a RA patient and model fragments added to a cultivation medium of peripheral blood mononuclear cells (PBMC) of healthy donors stimulate expression of genes for the TLR9-MyD88-NFkB-signaling pathway; this leads to a significant increase in concentrations of the proinflammatory cytokines IL-6 and TNFα in the cultivation medium. Human genomic DNA non-enriched with the CpG sequences did not stimulate IL-6 and TNFα synthesis in PBMC. A scheme explaining the potential role ecDNA in the induction and maintenance of increased levels of the proinflammatory cytokines under conditions damaging the human cells has been proposed.
Keywords: rheumatoid arthritis (RA); extracellular DNA (ecDNA); TLR9; NF-κB

Oxidative modification of glyceraldehyde-3-phosphate dehydrogenase influences its interaction with endogenous neuroprotector isatin by O. A. Buneeva; O. V. Gnedenko; M. V. Medvedeva; A. S. Ivanov; A. E. Medvedev (185-188).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a classical glycolytic redox sensitive enzyme, exhibits various non-glycolytic functions, which are considered to be especially important for progression of various neurodegenerative diseases. GAPDH binds isatin (indole-dione-2,3), an endogenous indole often used as a parent component in numerous derivatives demonstrating diverse pharmacological (including neuroprotector) activities. In this study we have investigated binding of intact and mildly oxidized GAPDH to immobilized isatin, using an optical biosensor technique, employing surface plasmon resonance (SPR), and the effect of isatin as a probe for this binding. Mild GAPDH oxidation by 70 μM H2O2 increased enzyme dissociation from immobilized isatin. Since GAPDH is considered as a putative target for various neuroprotector agents, this suggests that its redox state determines sensitivity to neuroprotective agents, and oxidative stress typical for various neurodegenerative disorders may significantly reduce pharmacological effectiveness of such compounds.
Keywords: glyceraldehyde-3-phosphate dehydrogenase; actin; amyloid-beta; isatin; ligand-protein interaction; surface plasmon resonance optical biosensor

Tumor necrosis factor-alpha is a potential target for the neuroprotector Dimebon by A. V. Alessenko; S. O. Bachurin; S. V. Gurianova; Y. O. Karatasso; E. F. Shevtsova; L. N. Shingarova (189-198).
Since 1983, Dimebon (Dimebolin) is used clinically in Russia as an antihistamine drug. Recent interest in Dimebolin is associated with its therapeutic effect in patients with Alzheimer’s disease. Animal studies have shown that Dimebon activity is realized via multiple mechanisms. Our experiments performed on the fibroblast cell culture L929 and C57Bl mice have been shown that Dimebon may block cytotoxic signals induced by the proinflammatory cytokines, tumor necrosis factor α (TNFα). Dimebon (10 μg/mL) protected mouse fibroblast cells L929 against toxic action of TNFα. Pretreatment of mice with Dimebon prevented development of changes in molecular species of sphingomyelins and galactosylceramides induced by a single dose administration of TNAα. Dimebon itself did not induce changes in sphingolipids of the investigated brain structures.
Keywords: Dimebon; TNF-α; lipids; mouse brain; mouse fibroblasts L929

Activated protein C (APC) is a serine protease involved in hemostasis; APC also exhibits antiinflammatory and anti-apoptotic properties, which are independent of its anticoagulant activity; these properties determine a possibility of the protective effects of APC in various diseases, including sepsis and healing of chronic wounds. We have hypothesized that the cytoprotective effect of APC on the cells, involved in wound healing, may be mimicked by peptide analogues of PAR1 “tethered ligand” activating PAR1. In order to test this hypothesis experimentally, we have synthesized a peptide (AP9)—analogue of the PAR1 tethered ligand, released by APC, and demonstrated for the first time that the AP9 peptide (0.1–10 μM), like to APC (0.01–100 nM), stimulates the proliferative activity of human primary keratinocytes. Using a model of the epithelial layer wounds we found that peptide AP9, as well as protease APC, accelerates wound healing. Using specific antibodies we have investigated the receptor mechanism of the AP9 effect in wound healing compared with the APC action. The proliferative activity of agonists requires both PAR1 receptor and endothelial protein C receptor (EPCR). Imitation of APC effect on keratinocytes by peptide AP9, PAR1 ligand, found in this study suggests the possibility of the use of peptide AP9 for stimulation of tissue repair.
Keywords: peptide agonist of protease-activated receptor; protein C; wound healing; keratinocytes