Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.8, #2)

Preparation of dendritic cells for cancer immunotherapy by Zh. K. Nazarkina; P. P. Laktionov (85-93).
Development of new effective method for cancer therapy is one of the most important trends in the modern medicine. Along with surgery, chemotherapy and radiotherapy, induction of an immune response against the tumor cells is a promising approach for therapy of cancer, particularly metastatic, slowly dividing tumors and cancer stem cells. Induction of the antitumor T-cell immune response involves activation of antigen-presenting cells, which can efficiently present the cancer antigens and activate T-lymphocytes. The immune response may be activated by dendritic cells (DC) loaded with tumor antigens, such as tumor-specific proteins, tumor cell lysates, apoptotic or necrotic tumor cells, as well as nucleic acids encoding tumor antigens. Regardless of the selected source of the tumor antigen, preparation of mature DC is a principal step in the development of anticancer vaccines aimed at the induction of the cytotoxic T-cell immune response. Recently, various research groups have proposed several strategies for producing mature DC, differed by the set of agents used. It has been shown that the maturation strategy influences both their phenotype and the ability to induce the immune response. In this review we have analyzed the results of studies on the various strategies of preparation of mature DCs.
Keywords: dendritic cells; cancer; anticancer therapy

The role of angiostatins in diabetic complications by A. A. Tykhomyrov; S. I. Shram; T. V. Grinenko (94-107).
Angiogenesis, a process of formation of new blood vessels form pre-existing vessels, is regulated by a large number of peptide factors. Imbalance of pro- and anti-angiogenic factors appears to be the major cause of vascular abnormalities leading to various complications in diabetes mellitus. Angiostatins, kringle-containing fragments of plasminogen/plasmin, are among of the most potent physiological inhibitors of neovascularization. In this review we focus on the colligation and analysis of the available data on peculiarities of production of angiostatins and their functioning in diabetes mellitus conditions. Special attention is paid to the role of angiostatins in the pathogenesis of typical diabetic complications, including retinopathies, nephropathies, and cardiovascular diseases.
Keywords: angiostatins; kringle domains; plasminogen; diabetes mellitus; vascular abnormalities

Analysis of proteomic profile changes of Danio rerio embryos during exposure to doxorubicin, incorporated in the phospholipid transport nanosystem by N. F. Samenkova; Yu. S. Kisrieva; N. A. Petushkova; G. P. Kuznetsova; O. V. Larina; O. P. Trifonova; I. I. Karuzina; O. M. Ipatova; A. V. Lisitsa (108-114).
The proteome profile of Danio rerio embryos grown in the medium containing doxorubicin, included in the phospholipid transport nanosystem (doxolip) has been investigated using combination of 1D-electrophoresis with subsequent MALDI-TOF-PMF mass spectrometry. Cultivation of growing of D. rerio embryos in the medium with doxolip caused a substantial increase in expression of the cytoskeletal proteins, a decrease in the number of nuclear proteins involved in DNA and RNA synthesis and disappearance of vitellogenin 2 in comparison with control (the cultivation medium containing the phospholipid transport nanosystem). Analysis of the proteomic profiles of doxolip-treated embryos suggests lower toxicity of doxorubicin incorporated in the phospholipid nanosystem.
Keywords: doxolip; Danio rerio ; one-dimensional gel electrophoresis; mass spectrometry; phospholipid transport nanosystem

Atomic force microscopy fishing of GP120 on immobilized aptamers and its mass spectrometry identification by N. S. Bukharina; Yu. D. Ivanov; T. O. Pleshakova; P. A. Frantsuzov; E. Yu. Andreeva; A. L. Kaysheva; A. A. Izotov; T. I. Pavlova; V. S. Ziborov; S. P. Radko; A. I. Archakov (115-124).
A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.
Keywords: gp120 HIV-1; aptamer; atomic force microscopy; fishing; mass spectrometry

Changes in proteome profiles of rat liver microsomes induced by silicon dioxide nanoparticles by O. N. Tananova; E. A. Arianova; I. V. Gmoshinskii; I. Yu. Toropygin; E. V. Khryapova; N. V. Trusov; S. A. Khotimchenko; V. A. Tutel’yan (125-129).
The effect of daily intragastric administration of an aqueous dispersion of silicon nanoparticles (NPs) (the dose range from 1.0 mg/kg to 100 mg/kg body weight for 28 days) to rats on the proteomic profile of liver microsomes has been investigated by 2D-electrophoresis followed by subsequent mass spectrometry identification. The liver microsomal fraction was isolated by differential centrifugation and its protein composition was analyzed by 2D-polyacrylamide gel electrophoresis. Identification of protein spots was carried out using MALDI-TOF mass spectrometric analysis. The mass spectrometry analysis revealed the protein GRP78 (78 kD glucose-regulated protein precursor), belonging to the family of heat shock proteins. This protein present in animals of the control group was not detected in NP-treated rats of group 2 (1 mg/kg body weight/day) and group 3 (10 mg/kg body weight/day). This protein predominantly localized in the liver cell endoplasmic reticulum and plasma membrane has the chaperone biological activity. Possible mechanisms of the effects of engineered nanoparticles on biosynthetic processes in the body are discussed.
Keywords: silicon dioxide; nanoparticles; rats; two-dimensional electrophoresis; heat shock proteins; GRP78

The effect of L-lysine alpha-oxidase from Trichoderma cf. aureoviride Rifai VKM F-4268D on the rat pheochromocytoma PC12 cell line by E. V. Lukasheva; Yu. S. Ribakova; T. N. Fedorova; M. G. Makletsova; A. Yu. Arinbasarova; A. G. Medentzev; T. T. Berezov (130-133).
L-Amino acid oxidases (L-AAO; EC comprise a group of flavoproteins that catalyze oxidative deamination of L-alpha amino acids to corresponding alpha-keto acids, NH3 and H2O2. Most of these enzymes are homodimers with molecular mass of 100–150 kDa that exhibit antiviral, antifungal, antibacterial, and anticancer activity. Among this group of enzymes L-lysine alpha-oxidase (LO) is especially important as its biological effects may differ from the effects of other L-AAO, because this enzyme preferentially oxidizes L-lysine, the essential amino acid for the human body, without any practical effect on other amino acids. Since molecular mechanisms of the cytotoxic action of LO still require better understanding, in this study we have investigated a possible mechanism of action of LO from Trichoderma cf. aureoviride Rifai VKMF-4268D. A rat pheochromocytoma PC12 cell culture was used as a model. Using flow cytometry a dose-dependent cell death induced by LO was shown. The increase in intracellular reactive oxygen species detected by the 2,7-dichlorodihydrofluorescein assay suggests that the oxidative pathway is one of mechanisms underlying the cytotoxic LO action; however, this does not rule out the involvement of other (previously demonstrated) mechanisms of LO effects on cell death.
Keywords: L-amino acid oxidase; L-lysine alpha-oxidase; mechanism of cytotoxicity; pheochromocytoma PC12; reactive oxygen species

Mean platelet volume: Interrelation with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels by S. G. Khaspekova; I. T. Zyuryaev; V. V. Yakushkin; Ya. A. Naimushin; O. V. Sirotkina; N. O. Zaytseva; M. Ya. Ruda; A. V. Mazurov (134-142).
Increased mean platelet volume (MPV) is an independent risk factor of thrombotic events in patients with cardiovascular diseases. Interactions of MPV with platelet aggregation activity and contents of glycoprotein (GP) IIb-IIIa (αIIb/β3 integrin, fibrinogen receptor) and GP Ib (von Willebrand factor receptor) have been investigated in this study. The study was performed in a group of healthy volunteers (n = 38) and a group of patients with acute coronary syndrome (ACS, n = 116). Patient’s blood was collected at days 1, 3–5 and 8–12 after ACS development. All patients received acetylsalicylic acid (ASA, inhibitor of thromboxane A2 synthesis) as the antiaggregant therapy and most of them also received clopidogrel (ADP receptor antagonist), except 44 patients who had not taken clopidogrel at day 1 before first blood collection. Aggregation of volunteers’ platelets was stimulated by 1.25, 2.5, 5 and 20 μM ADP, while aggregation of patients’ platelets was stimulated by 5 and 20 μM ADP. GP IIb-IIIa and GP Ib content on the platelet surface was measured using 125I-labelled monoclonal antibodies. GP IIb-IIIa and GP Ib genetic polymorphisms were determined in ACS patients. In healthy donors significant correlations between MPV and aggregation levels have been recognized at 1.25 μM and 2.5 μM ADP (correlation coefficient (r) values of 0.396 and 0.373, p < 0.05), while at 5 μM and 20 μM ADP these interactions did not reach the level of statistical significance (r values of 0.279 and 0.205, p > 0.05). Correlations between MPV and aggregation levels were observed at day 1 of ACS in a subgroup of patients receiving ASA but before the beginning of clopidogrel treatment (r values of 0.526, p < 0.001 and 0.368, p < 0.05 for 5 and 20 μM ADP, respectively). Correlations between these parameters were not found during combined treatment of patients with ASA and clopidogrel. Strong direct correlations between MPV and GP IIb-IIIa and GP Ib contents were detected in both healthy donors and ACS patients (at all time points): the r values ranged from 0.439 to 0.647 (p ≤ 0.001 for all correlations). Genetic polymorphisms of GP IIb-IIIa (GP IIIa Leu33Pro) and GP Ib ((−5)T/C (Kozak) and Thr145Met) identified in ACS patients did not affect expression levels of corresponding glycoproteins. The data obtained indicate that increased MPV values correlate with increased platelet aggregation activity and enhanced GP IIb-IIIa and GP Ib expression.
Keywords: mean platelet volume; platelet aggregation; glycoprotein IIb-IIIa; glycoprotein Ib; acute coronary syndrome

The NMR study of human biological fluids for detection of pathologies NMR study of biological fluids by P. M. Beskaravainy; M. V. Molchanov; A. V. Suslikov; S. I. Paskevich; V. P. Kutyshenko; S. I. Vorob’ev (143-149).
The paper deals with the NMR spectra obtained using preparations of five different human biological body fluids. Characteristic metabolite signals of blood, urine, tears, saliva, and sweat spectra have been determined and classified. The biological body fluid samples were used for search and identification of biomarkers of cardiovascular disease. Absolute functional biomarkers for diseases such as coronary heart disease (CHD) have not been recognized even in the case acute myocardial infarction. A hypothesis explaining reasons of lack of such markers has been formulated. The results of comparative analysis of blood and urine samples from humans and some laboratory animals are given. Identify and analyze signals of metabolites of pathogenic microflora and their dynamics in the urine from patients with urogenital diseases have been determined and analyzed and characteristic biomarkers have been recognized.
Keywords: biological fluids; NMR; biomarkers

The correcting effects of dihydroquercetin in cerebral ischemia-reperfusion injury by N. Ye. Maksimovich; I. K. Dremza; E. I. Troyan; Ya. N. Maksimovich; A. N. Borodinskii (150-154).
The dynamics of changes in the mitochondrial respiratory function, changes in the parameters of carbohydrate metabolism and some parameters of oxidative stress in the brain tissue have been investigated under conditions of ischemia-reperfusion and administration of dihydroquercetin. Dihydroquercetin (65 mg/kg) was administered per os 1 h before modeling of ischemia-reperfusion. Studies were carried 1 h after reperfusion. It was found that administration of dihydroquercetin caused a corrective effect to impairments of the respiratory function of mitochondria, indicators of carbohydrate metabolism and parameters of oxidative stress induced by ischemia-reperfusion.
Keywords: reperfusion; brain; mitochondrial respiration; dihydroquercetin; oxidative stress

Metabolism of innate immune cells in bacterial infections by N. G. Plekhova; L. M. Somova; E. I. Drobot (155-163).
Metabolic activity of innate immune cells infected by various doses of Gram-negative (Yersinia pseudotuberculosis, Salmonella enteritidis) and Gram positive (Staphylococcus aureus, Listeria monocytogenes) bacteria has been investigated. Using various animal models we found that in during the initial period (up to 2 days) the changes in cellular responses depend on the type of the pathogen. In response to infection caused by Gram-negative bacteria predominant of neutrophil accumulation in the foci of inflammation was observed, while Gram-positive bacteria induced preferential accumulation of macrophages. The study of metabolism of these cells showed that the response of terminally differentiated primed phagocytes to pathogen appearance was higher than in cells circulating in blood. In addition to the priming state the phagocyte reactivity is influenced by the bacterial load. At a low phagocyte/microbe ratio the cells reaction is almost undetectable, while an excess of microorganisms causes (despite of the increase of the phagocytic parameters) the hyperactivation of cell metabolism and production of maximal amounts of bactericide agents, which exhibit a damaging effect on the cell itself.
Keywords: innate immune cells; enzymes; nitric oxide; bacterial infection

Streptavidin conjugates with gold nanoparticles for visualization of single DNA interactions on the silicon surface by G. V. Presnova; M. Yu. Rubtsova; D. E. Presnov; V. G. Grigorenko; I. V. Yaminsky; A. M. Egorov (164-167).
Applicability of scanning electron microscopy (SEM) for visualization of individual acts of DNA hybridization with oligonucleotide probes has been investigated using gold nanoparticles as a label. DNA or oligonucleotides were labeled with biotin molecules, which were then detected in DNA duplexes using a streptavidin conjugate with gold nanoparticles. Effective imaging of DNA duplexes was possible using the conjugate prepared by covalent binding. The detection limit of the model oligonucleotide of 19 bases was 20 pg.
Keywords: hybridization analysis of DNA; single biospecific interactions; gold nanoparticles; scanning electron microscopy

Molecular docking and 3D-QSAR of 16α,17α-cycloalkanoprogesterone derivatives as ligands of the progesterone receptor by I. V. Fedyushkina; V. S. Skvortsov; I. V. Romero Reyes; I. S. Levina (168-176).
A series of 42 (pregna-D′-pentarane) steroid ligands was used to generate models predicting ligand affinity to the progesterone receptor. The best result (Q 2 = 0.91) was obtained using a combination of molecular docking, molecular dynamics simulation and artificial neural networks. Good predictive power of the model was validated using a group of 8 pentaranes synthesized separately and tested in vitro (R test 2 = 0.77). This model can be used for determination of ligand-receptor binding affinity and accurate ranking of binding capacity of compounds tested.
Keywords: pentaranes; progesterone receptor; ligand-binding domain; affinity; QSAR; computational methods; COMFA; COMSIA

The rifampicin drug delivery system based on phospholipid nanoparticles by M. A. Sanzhakov; V. N. Prozorovskyi; O. M. Ipatova; E. G. Tikhonova; N. V. Medvedeva; T. I. Torkhovskaya (177-180).
Low bioavailability of rifampicin, one of the main antituberculous agents, stimulates searches of its new optimized formulations. The present study has shown a possibility of rifampicin incorporation into nanoparticles from plant phosphatidylcholine (diameter of 20–30 nm). Addition of sodium oleate to the phospholipid system caused a 2-fold increase in the percent of rifampicin incorporation. The maximal concentration of rifampicin assayed in plasma samples by LC/MS was observed 1 h after oral administration to rats (6 mg/kg) and represented 0.5 and 4.2 μg/mL for free rifampicin and rifampicin incorporated in the phospholipids-oleate nanoparticles, respectively. These levels were maintained for more than 2 h of the experiment. High rifampicin bioavailability in the oleate containing phospholipid nanosystem suggests its prospects for practical use.
Keywords: tuberculosis treatment; rifampicin; nanoparticles; phospholipids; oleate; bioavailability