Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.10, #1)

Studies on experimental animals with knockout of the insulin receptor gene (Insr) in the whole body or in certain tissues and/or related genes encoding proteins involved in realization of insulin signal transduction in target cells, have made an important contribution to the elucidation of insulin regulation of metabolism, particularly fat metabolism. Since the whole insulin secreted by β-cells, together with the products of gastrointestinal tract digestion of proteins, fats, and carbohydrates reaches in the liver, the latter is the first organ on which this hormone acts. The liver employs released amino acids for synthesis of proteins, including apo-proteins for various lipoproteins. Glucose is used for synthesis of glycogen, fatty acids, and triglycerides, which enter all the organs in very low density lipoproteins (VLDL). The LIRKO mice with knockout of the insr gene in the liver demonstrated inhibition of synthesis of macromolecular compounds from amino acids, glucose, and fatty acids. Low molecular weight substances demonstrated increased entry to circulation, and together with other disorders induced hyperglycemia. In LIRKO mice blood glucose levels and glucose tolerance demonstrated time-dependent normalization and at later stages the increase in glucose levels was replaced by hypoglycemia. These changes can be well explained if we take into consideration that one of the main functions of insulin consists in stimulation of energy accumulation by means of activation of triglyceride deposition in adipose tissue. FIRKO mice with selective knockout of adipose tissue Insr were characterized by decreased uptake of glucose in adipocytes, and its transformation into lipids. However, the level of body fat in animals remained normal, possibly due to preserved insulin receptor in the liver and insulin-induced activation of triglyceride production which maintained normal levels of body fat stores, the effective functioning of adipose tissue and secretion of leptin by adipocytes during inhibition of glucose transformation into triglyceride in adipose tissue. Knockout of the Insr gene in muscles blocked glucose uptake by myocytes, but it did not induce hyperglycemia, probably due to the increase in glucose uptake by other organs, which retained the insulin receptor, and induced some increase in fat resources in adipose tissue. Similar results were obtained in mice with knockout the glucose transporter 4 GLUT4 in muscle and/or adipose tissue. Insulin microinjections in the brain, in the cerebral ventricle 4 (CVI) and mediobasal hypothalamus (MBH) did not affect the insulin levels in the general circulation, but effectively activate lipogenesis and inhibited lipolysis in adipose tissue. They induced obesity, similar to conventional obesity when the insulin levels increased. These results may serve as an additional confirmation of the importance of the adipogenic insulin function in mechanisms of regulation of general metabolism.
Keywords: glycemia; fatty acids; triglycerides; obesity; diabetes mellitus; insulin receptor; gene; knockout of gene

The central role that cytochromes P450 play in the metabolism of drugs and other xenobiotics makes these enzymes a major subject for studies of drug disposition, adverse drug effects and drug-drug interactions. Despite tremendous success in elucidating structures and mechanisms of cytochrome P450 function, the concept of the drug-metabolizing ensemble as a functionally integrated system remains undeveloped. However, eukaryotic cells typically possess a multitude of different cytochromes P450 that are co-localized in the membrane of endoplasmic reticulum (ER); they interact with each other through the formation of dynamic heteromeric complexes (mixed oligomers). There has been growing appreciation of the importance of developing an approach to study the ensemble of cytochromes P450 as an integral system inspired growing interest of researchers to the principles of molecular organization of the microsomal monooxygenase system. Academician Archakov and his colleagues made important contributions to this field during the initial period of studies. Subsequent exploration of the molecular organization of the microsomal monooxygenase system as an integral multienzyme and multifunctional system have had an essential impact on our understanding of the key factors that determine the changes in human drug metabolism and other cytochrome P450-related functions in development and aging, as well as under the influence of various pathologies and environmental factors.
Keywords: cytochrome P450; endoplasmic reticulum; multienzyme system; allostery; protein-protein interactions; oligomerization

Droplet digital PCR, a prospective technological approach to quantitative profiling of microRNA by Ya. Yu. Kiseleva; K. G. Ptitsyn; S. P. Radko; V. G. Zgoda; A. I. Archakov (22-30).
MicroRNA is a special type of regulatory molecules modulating gene expression. Circulating microRNAs found in blood and other biological body fluids are now considered as potential biomarkers of human pathology. Quantitative changes of particular microRNAs have been recognized in many oncological diseases and other disorders. A recently developed method of droplet digital PCR (ddPCR) possesses a number of advantages making this method the most suitable for verification and validation of perspective microRNA markers of various human pathologies. These advantages include high accuracy and reproducibility of microRNA quantification as well as possibility of direct high-throughput determination of the absolute number of microRNA copies within a wide dynamic range. The present review considers microRNA biogenesis, the origin of circulating microRNAs, and methods used for their quantification. The special technical features of ddPCR, which make this method especially attractive for studying microRNAs as biomarkers of human pathologies and for basic research devoted to aspects of gene regulation by microRNA molecules, are also discussed.
Keywords: microRNA; biomarkers; profiling; digital droplet PCR

Antiviral activity and pathogenetic targets for seaweed sulfated polysaccharides in herpesvirus infections by N. N. Besednova; I. D. Makarenkova; T. N. Zvyagintseva; T. I. Imbs; L. M. Somova; T. S. Zaporozhets (31-42).
The review summarizes results of studies of effects of sulfated polysaccharides from seaweed on herpesviruses and the course of herpesvirus infections. Importance of this problem is determined by the prevalence of herpesviruses that can persist in the human body and demonstrate a high degree of immune mimicry and resistance to antiviral drugs. A wide range of physiological action of sulfated polysaccharides, receptor agonists of innate and adaptive immune cells, which possess potent antiviral, antioxidant and anti-inflammatory activities, open the possibility of their use for creation of new generation pharmacological substances and drugs with associated activity for the treatment of herpesvirus infections.
Keywords: sulfated polysaccharides; fucoidans; herpes viruses; antiviral activity

Capacities of computer evaluation of hidden potential of phytochemicals of medicinal plants of the traditional Indian Ayurvedic medicine by A. A. Lagunin; D. S. Druzhilovsky; A. V. Rudik; D. A. Filimonov; D. Gawande; K. Suresh; R. Goel; V. V. Poroikov (43-54).
Applicability of our computer programs PASS and PharmaExpert for prediction of biological activity spectra of rather complex and structurally diverse phytocomponents of medicinal plants, both separately and in combinations has been evaluated. For this purpose we have created the web-resource containing known information about structural formulas and biological activity of 1906 phytocomponents of 50 Ayurvedic medicinal plants used in Traditional Indian Medicine (TIM) (http://ayurveda.pharmaexpert.ru). The PASS training set was updated by addition of information about structure and biological activity of 946 natural compounds; then the training procedure and validation were performed, to estimate the quality of PASS prediction. It was shown that the differences between the average accuracy of prediction obtained in leave-5%-out cross-validation (94.467%) and in leave-one-out cross-validation (94.605%) are very small thus demonstrating high predictive ability of the program. Results of biological activity spectra prediction for all phytocomponents included in our database coincided in 83.5% of cases with known experimental data. Additional types of biological activity predicted with high probability indicate further promising directions for further studies of certain phytocomponents of some medicinal plants. The analysis of prediction results of sets of phytocomponents in each of 50 medicinal plants was made by the PharmaExpert software. Based on this analysis, we found that the combination of phytocomponents from Passiflora incarnata may exhibit nootropic, anticonvulsant, and antidepressant effects. Experiments carried out in mice models confirmed the predicted effects of P. incarnata extracts.
Keywords: SAR; PASS; PharmaExpert; computational prediction; Ayurveda; medicinal plants; phytocomponents; biological activity

An optical biosensor employing surface plasmon resonance (SPR; SPR-biosensor) is a highly efficient instrument applicable for direct real time registration of molecular interactions without additional use of any labels or coupled processes. As an independent approach it is especially effective in analysis of various ligand receptor interactions. SPR-biosensors are used for validation of studies on intermolecular interactions in complex biological systems (affinity profiling of various groups of proteins, etc.). Recently, potential application of the SPR-biosensor for molecular fishing (direct affinity binding of target molecules from complex biological mixtures on the optical biosensor surface followed by their elution for identification by LCMS/MS) has been demonstrated. Using SPR-biosensors in such studies it is possible to solve the following tasks: (a) SPR-based selection of immobilization conditions required for the most effective affinity separation of a particular biological sample; (b) SPR-based molecular fishing for subsequent protein identification by mass spectrometry; (c) SPR-based validation of the interaction of identified proteins with immobilized ligand. This review considers practical application of the SPR technology in the context of recent studies performed in the Institute of Biomedical Chemistry on molecular fishing of real biological objects.
Keywords: optical surface plasmon resonance based biosensor; molecular fishing; amyloid-binding proteins; cytochrome b 5 ; isatin binding proteins

Chemical composition and biological activity of triterpenes and steroids of chaga mushroom by S. A. Nikitina; V. R. Habibrakhmanova; M. A. Sysoeva (63-69).
Data on the chemical composition of triterpenic and steroid compounds, isolated from the chaga mushroom grown in natural environment or in a synthetic culture have been summarized. Special attention has been paid to the biological activity of chaga mushroom extracts and these particular compounds against various cancer cell lines in vitro and in vivo. This analysis has demonstrated some common features in inhibition of growth of various cell lines by chaga mushroom components. In this context, the most active are triterpene compounds containing OH group at C-22 and a side chain unsaturated bond.
Keywords: chaga mushroom; triterpenes; steroids; biological activity; in vivo; in vitro

The effect of fenofibrate on expression of genes involved in fatty acids beta-oxidation and associated free-radical processes by A. P. Gureev; M. L. Shmatkova; V. Yu. Bashmakov; A. A. Starkov; V. N. Popov (70-74).
Fenofibrate is a synthetic ligand for peroxisome proliferator-activated receptors subtype alpha (PPARα); it is used for the treatment of a wide range of metabolic diseases such as hypertriglyceridemia, dyslipidemia, diabetes and various neurodegenerative diseases. We have studied the effect of fenofibrate on β-oxidation of fatty acids and related free-radical processes. The most effective concentration of fenofibrate (0.3%) added to the chow caused a significant decrease of the body weight of mice due lipolysis. The data obtained by quantitative PCR demonstrated increased hepatic gene expression responsible for β-oxidation of fatty acids in peroxisomes and mitochondria. Enhancement of oxidative processes caused a 2-fold increase in the rate of reactive oxygen species (ROS) production, as evidenced by determination of the level of lipid peroxidation (LPO) products in the liver. Mitochondrial antioxidant systems are more sensitive to elevated ROS production, as they respond by increased expression of SOD2 and PRDX3 genes, than cytoplasmic and peroxisomal antioxidant systems, where expression of CAT1, SOD1, PRDX5 genes remained unaltered.
Keywords: fenofibrate; β-oxidation; peroxisome; mitochondria; antioxidant systems

Mammaglobin in peripheral blood and the tumor of breast cancer patients by V. K. Bozhenko; N. V. Kharchenko; E. F. Vaskevich; E. A. Kudinova; A. V. Oorzhak; N. I. Rozhkova; I. D. Trotsenko (75-80).
Currently, no molecular biological markers do exist for early diagnosis of breast cancer. One of the possible candidates for the marker of early breast cancer is mammaglobin (MGB1) or SCGB2A2 (secretoglobin, family 2A, member 2), characterized by the maximal expression level in early breast cancer. Using the RT-PCR method MGB1 mRNA expression was examined in 57 tumor tissue samples and 57 samples of morphologically non-malignant tissue (MNT) of breast cancer (BC) patients. Specificity and sensitivity of the MGB1 mRNA assay in peripheral blood of BC patients was evaluated by nested PCR. 169 blood samples (from 95 BC patients, 22 from patients with benign breast tumors, 28 from patients with tumors of other localizations, and 24 samples from healthy donors) have been analyzed. MGB1 expression was significantly higher in BC tissue samples compared to MNT (p = 0.0019). The maximal expression level was in the samples T1 (p = 0.013), stage I BC (p = 0.037), GI (p = 0.0019). MGB1 expression positively correlated with expression of estrogen (p = 0.034) and progesterone (p = 0.0004) receptors. Sensitivity and specificity of the MGB1 mRNA assay in peripheral blood were 60.6 and 92.3%, respectively. Expression of MGB1 was higher in BC than MNT and it decreased during BC progression. The sensitivity and specificity of the MGB1 mRNA assay may be used as an additional diagnostic method.
Keywords: breast cancer; mammaglobin mRNA; peripheral blood mammaglobin

The pathogenetic importance of C774T single nucleotide polymorphism of the endothelial nitric oxide synthase gene in the development of metabolic syndrome by N. S. Fattakhov; M. A. Vasilenko; D. A. Skuratovskaia; D. I. Kulikov; E. V. Kirienkova; P. A. Zatolokin; M. A. Beletskaya; L. S. Litvinova (81-86).
The relationship between nitric oxide production and metabolic disorders and the role of endothelial nitric oxide synthase (eNOS or NOS3) in metabolic syndrome (MS) remain poorly understood and need deeper investigation. In this context the role of the NOS3 gene in pathogenesis of MS is of special interest. The aim of the study was to investigate association of NOS3 single nucleotide polymorphism C774T with risk of MS in the Slavic population of the Kaliningrad region and the relationship of this polymorphic variant with some parameters of endothelial dysfunction. The study included 128 patients (48 men and 80 women aged from 36 to 52 years) with MS. The control group consisted of 126 healthy volunteers (60 men and 66 women aged from 30 to 40 years). Genotyping was performed by real-time PCR. Serum nitrite levels were determined spectrophotometrically by the Griess method. Serum levels of endothelin-1 and eNOS were evaluated by ELISA. The study has shown association of T allele (OR = 2.06; p = 0.0004; CI: 1.38–3.08) and CT genotype (OR = 1.97; p = 0.014; CI: 1.14–3.40 ) C774T polymorphism of the NOS3 gene with risk of MS in the Slavic population of the Kaliningrad region. Allele C (OR = 0.48; p = 0.0004; CI: 0.32–0.72) and homozygous CC genotype (OR = 0.41; p = 0.001; CI: 0.24–0.69) C774T polymorphism of the NOS3 gene were associated with reduced risk of the development of MS. Significant differences in serum levels of eNOS and endothelin-1 depended on the CT and TT genotypes of C774T polymorphism of the NOS3 gene in MS.
Keywords: metabolic syndrome; nitric oxide; endothelial NO synthase; single nucleotide polymorphism C774T

The combined action of binase and bleomycin on human lung adenocarcinoma cells by P. V. Zelenikhin; A. V. Makeeva; T. N. Nguen; Y. A. Siraj; O. N. Ilinskaya (87-90).
Some microbial ribonucleases (RNases) demonstrate selective cytotoxic effect against a wide range of tumor cells. In this context combined use of cytotoxic RNases in complex therapy with other chemotherapeutic agents appears to be especially promising. In this study we have investigated the apoptosisinduced effect of Bacillus pumilus RNase (binase) in combination with known antitumor antibiotic bleomycin on human lung adenocarcinoma A549 cells. The combined effect of high concentrations of these agents did not have any mutual increase in their apoptosisinduced action, while a combination of nonapoptotic concentrations resulted in the increase of proportion of apoptotic cells up to 22% as compared with individual effect of bleomycin (6%) and binase (12%) used separately. These results indicate that binase and bleomycin are effective in combination of their low concentrations and ineffective in combination of their high concentrations.
Keywords: cytotoxic ribonucleases; Bacillus pumilus ; binase; bleomycin; antitumor activity; lung adenocarcinoma

A simple method of prediction of visibility of peptides in mass spectrometry with electrospray ionization by A. V. Rybina; V. S. Skvortsov; A. T. Kopylov; V. G. Zgoda (91-95).
A new method for selection of essential peptides applicable for protein detection and quantification analysis in the targeted positive electrospray mass spectrometry has been proposed. It is based on the prediction of the normalized abundance of the mass spectrometric peaks by using a linear regression model. This method has the following a priori restrictions: first selection of peptides must be arranged so that at pH 2.5 the tested peptides must be presented mainly as the 2+ and 3+ ions. Only peptides containing C-terminal lysine or arginine residues should be considered. The amino acid composition of the peptide, the peptide concentration, the ratio of the polar surface of peptide to common surface and ratio of the polar volume to the common volume are used as independent variables. Among several considered combinations of variables the best linear regression model had a determination coefficient in leave-one-out cross-validation procedure of 0.54. This model confidently discriminated peptides with high response ability and peptides with low response ability, and therefore it is applicable for selection of the most favorable peptides among peptides selected by means of simple criteria. This simple and fast screening method can be successfully applied to reduce the list of observed peptides.
Keywords: peptide mass spectrometry; electrospray ionization; prediction of properties