Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.3, #1)
Mass spectrometry methods in metabolomics by P. G. Lokhov; A. I. Archakov (1-9).
The review deals with metabolomics, a new and rapidly growing area directed to the comprehensive analysis of metabolites of biological objects. Metabolites are characterized by various physical and chemical properties, traditionally studied by methods of analytical chemistry focused on certain groups of chemical substances. However, current progress in mass spectrometry has led to formation of rather unified methods, such as metabolic fingerprinting and metabolomic profiling, which allow defining thousands of metabolites in one biological sample and therefore draw “a modern portrait of metabolomics.” This review describes basic characteristics of these methods, ways of metabolite separation, and analysis of metabolites by mass spectrometry. The examples shown in this review, allow to estimate these methods and to compare their advantages and disadvantages. Besides that, we consider the methods, which are of the most frequent use in metabolomics; these include the methods for data processing and the required resources, such as software for mass spectra processing and metabolite search database. In the conclusion, general suggestions for successful metabolomic experiments are given.
Keywords: metabolomics; metabolic fingerprinting; metabolic profiling; mass spectrometry
Identification of differentially expressed proteins using automated meta-analysis of proteomic articles by E. A. Ponomarenko; A. V. Lisitsa; J. Petrak; S. A. Moshkovskii; A. I. Archakov (10-16).
An automated method for searching for differentially expressed proteins (DEP) in proteomic articles has been developed and tested. Full-text proteomics-related articles were selected using an electronic version of the journal Proteomics and PubMedCentral. The list of proteins most frequently mentioned in the articles shares 86% identity with the list of human frequently identified DEPs published recently (Petrak et al., Proteomics (2008) 8, 1744). Regardless of the goal or design or methods identification of DEP delivers annexins and peroxiredoxins, as well as alpha-enolase, triosephosphate isomerase, and heat-shock protein HSP60. Among the most often mentioned proteins were also serum albumin, cathepsin D and vimentin. In regard to protein function the most often mentioned proteins were involved in inflammation and the immune response. According to GenRIF and UniProtKB annotations, most of these proteins are linked to the pathogenesis of tumor diseases, or diseases of the cardiovascular and nervous systems.
Keywords: proteomics; meta-analysis; text mining; 2-DE; LC-MS/MS
The astacin family of metalloproteinases by S. A. Semenova; G. N. Rudenskaya (17-32).
The review deals with the properties of astacin family of zinc-dependent metalloproteinases. These enzymes exhibit arylamidase activity, which is not typical for metalloproteinases. Special attention is paid to physiological functions of the astacin proteinases and to the influence of domain composition and posttranslational modifications on the activity and stability of these enzymes.
Keywords: astacin; metalloproteinase; extracellular matrix
Immunochemical and molecular-Genetic markers in diagnostics of gastric cancer by E. V. Elistratova; P. P. Laktionov; P. I. Shelestuk; S. A. Tuzikov; V. V. Vlassov; E. Y. Rykova (33-43).
Intensive studies of molecular mechanisms responsible for tumor transformation results in identification of new proteins and their genes involved into tumor development. These proteins may be used as markers of tumor transformation of cells and the level of their expression may be evaluated by means of modern highly sensitive and technological methods of analysis. This review summarized literature data on currently used immunohistochemical and molecular genetic markers of gastric cancer. It highlights genetic and epigenetic changes detected in nucleic acids of tumor tissue cells in malignant and benign gastric diseases as well as in the level of DNA circulating in blood of patients with gastric cancer.
Keywords: gastric cancer; diagnostics; tumor markers; circulating DNA
YC-1-like potentiation of nitric oxide-dependent activation of soluble guanylate cyclase by adrenochrom by I. S. Severina; N. V. Pyatakova; A. Y. Shchegolev; T. A. Sidorova (44-47).
The influence of adrenochrome and YC-1 activation of human platelet soluble guanylate cyclase was investigated. Adrenochrome (0.1–10.0 μM) had no effect on the basal activity, but it potentiated in a concentration- dependent manner the spermine NONO-induced activation of this enzyme. Adrenochrome also sensitized guanylate towards nitric oxide (NO) and produced the leftward shift of the spermine NONO concentration response curve. Addition of adrenochrome decreased the YC-1-induced leftward shift of the spermine NONO concentration response curve. Adrenochrome also inhibited enzyme activation byYC-1. Thus, synergistic activation of NO-stimulated guanylate cyclase activity by adrenochrome represents a new biochemical effect of this compound and indicates that adrenochrome may act as an endogenous regulator of the NO-dependent stimulation of soluble guanylate cyclase. This new property of adrenochrome, similar to YC-1 but more effective, should be taken into consideration especially under conditions of adrenochrome overproduction in the body.
Keywords: guanylate cyclase; nitric oxide (NO); adrenochrome
Liposomal formulations of protein proteinase inhibitors: Preparation and specific activity by A. S. Balkina; A. A. Selischeva; N. I. Larionova (48-53).
Possibility of encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) was investigated. The influence of the PC/PE ratio (w/w) on efficiency of incorporation of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in the central part of the spherical particle and also between bilayers. The study of biological (antitrypsin and antichymotrypsin) activity demonstrated partial spatial shielding of active sites of proteins entrapped in liposomes. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI is consistent with this hypothesis and suggests that this shielding is reversible. Stability of liposomes was examined using three various media modeling gastrointestinal fluids (gastric and intestinal juices and fluids). Data obtained indicate that the prepared liposomes seem to be promising formulations for BBI and BPTI delivery.
Keywords: phosphatidylcholine; phosphatidylethanolamine; liposomes; aprotinin; Bowman-Birk soybean proteinase inhibitor
The influence of LIF (Leukemia Inhibitory Factor) on the functional status of R1 mouse embryonic stem cells by E. S. Lobanok; L. M. Mezhevikina; L. M. Belyanovich; R. R. Petrova; I. B. Vasilevich; I. D. Volotovsky; E. E. Fesenko (54-57).
The influence of cytokine LIF (Leukemia Inhibitory Factor) on the viability, and proliferation of mouse embryonic stem cells (ESC) (R1 cell line) and their distribution by cell cycle stages has been investigated. LIF (5–20 ng/ml) increased growth of colonies and maintained high proliferative and pluripotent properties of R1 cells. LIF was also involved into the inhibition of spontaneous cell differentiation and apoptotic cell death; it also decreased the rations of S/G2 + M cell cycle and doubling-time of cell population.
Keywords: embryonic stem cells; cytokine LIF; proliferation; cell cycle
Biospecific properties of lysine-containing polyelectrolyte coatings for blood contacting devices by N. A. Samoilova; M. A. Krayukhina; S. P. Novikova; L. I. Moukhametova; R. B. Aisina; I. A. Yamskov (58-63).
Biospecific properties of thromboresistant bilayer and multilayer coatings based on polyelectrolyte complexes of modified copolymer of N-vinylpyrrolidone and maleic acid (VPMA) with chitosan, amphiphilic chitosan or albumin were investigated. VPMA contained an affinity ligand towards plasminogen, α-amino coupled lysine residues. Polyethylene and polystyrene surfaces were investigated before and after their covering by protective polyelectrolyte coatings. The specific adsorption of plasminogen (precursor of the fibrinolytic enzyme plasmin) from its solutions and from human plasma was investigated using these model systems. It was found that all coatings with the outer contact layer of the lysine-containing affinity polymer exhibited affinity towards plasminogen. However, multilayer polyelectrolyte coatings were more efficient than the bilayer coatings with a single layer application and the affinity polymer coatings without interlayer. The decrease in the degree of thrombogenicity of the materials modified by polyelectrolyte coatings has been demonstrated in vitro and ex vivo. Employment of the proposed modification of surfaces will improve hemocompatibility of medical devices.
Keywords: affinity polymer; lysine; polyelectrolyte coating; plasminoigen adsorption; thromboresistance
Production of reactive oxygen species by monocyte-derived macrophages from blood of healthy donors and patients with ischemic heart disease by M. V. Bilenko; Yu. A. Vladimirov; S. A. Pavlova; Nguyen Thi Thu Thuy; Tran Thi Hai Yen (64-70).
Production of reactive oxygen species (ROS) by macrophages derived from blood monocytes of healthy donors (MPN) and patients with ischemic heart disease (IHD) (MPIHD) before, during, and after their incubation with low-density lipoprotein (LDL) isolated from blood plasma of healthy donors (LDLN) and patients with a high cholesterol level (LDLH) was investigated by the method of luminol-dependent (spontaneous) and stimulated chemiluminescence (CL) using opsonized zymosan (OZ) or phorbol-12-myristate-13-acetate (PMA) as the CL stimulators. It was shown that proper, luminol-dependent, and zymosan-or PMA-stimulated chemiluminescence of MPIHD was 1.4-, 1.8-, 2.7-, and 1.6-fold higher than the same types of chemiluminescence of MPN, respectively, (p<0.05–0.01). Although the effect of OZ on MPN and MPIHD was more potent than that of PMA (by 4.3- and 3.2-fold, respectively), but it appeared in 2.5–3.0 times slower than that of PMA. LDLN and LDLH incubated with MPN for the first 15 and 60 min caused the 1.4- and 2.5-increase of the luminol-dependent CL of MPN; the same treatment of MPIHD did not influence ROS production by these cells. Repeated increase in the OZ-stimulated CL of MPN was also observed after preincubation for 15–180 min with LDLN and LDLH followed by LDL removal, subsequent MPN washing and addition of Hanks solution and OZ; the repeated increase in OZ-stimulated CL of MPN was only observed after incubation with LDLH than with LDLN. No increase of CL was observed in experiments with MPIHD. Thus, more intensive chemiluminescence of macrophages obtained from blood of patients with IHD suggests their in vivo stimulation. LDLN and LDLH may cause both primary and secondary (after preincubation) stimulating effect on CL of MPN but not of MPIHD. Thus, the analysis of macrophage chemiluminescence is a sensitive test for evaluation the degree of macrophage stimulation; it may be effectively used for monitoring of effectiveness of medical treatment of patients.
Keywords: human blood monocyte-derived macrophages; ROS; LDL; chemiluminescence; ischemic heart disease; atherosclerosis
Substituted 1,5,6,7-tetrahydro-4H-benzimidazol-4-ones as urease inhibitors by E. I. Tarun; T. A. Zheldakova; D. I. Metelitza (71-76).
A comparative kinetic study of the inhibition of urea hydrolysis by 9 substituted 1,5,6,7-tetrahydro-4H-benzimidazol-4-ones (BI I–IX) has been carried out. The inhibition had reversible competitive mode; the inhibition constants K i, varied from 29 to 754 μM in dependence of structure of BI I–IX. Three BI I–IX, characterized by the K i values ranged from 29 to 82 μM, may be used as potential therapeutic agents in gastroenterology for treatment of gastroduodenal ulcers.
Keywords: soybean urease; urea; inhibition; substituted benzimidazols; inhibition constants
Anticoagulant activity of fucoidans from brown algae by N. A. Ushakova; G. E. Morozevich; N. E. Ustyuzhanina; M. I. Bilan; A. I. Usov; N. E. Nifantiev; M. E. Preobrazhenskaya (77-83).
The anticoagulant activity of polysaccharide fucoidans from 11 species of brown algae was studied. The anticoagulant activity was measured by the activated partial thromboplastin time (APTT), prothrombin time, and thrombin time. Inhibitory action of these fucoidans significantly varied from one species to another. Fucoidans from Laminaria saccharina and Fucus distichus exhibited high anticoagulant activity, while fucoidans from Cladosiphon okamuranus and Analipus japonicus were almost inactive. Other fucoidans exhibited intermediate inhibitory activity. The inhibitory effect of fucoidans on thrombin and factor Xa was investigated in the presence or in the absence of natural thrombin inhibitor, antithrombin III (AT III). In contrast to the best-studied anticoagulant, heparin, most of these fucoidans inhibited thrombin in the absence of AT III. In the presence of AT III the inhibitory effect of fucoidans considerably increased. In contrast to heparin, fucoidans weakly influenced factor Xa activity in the presence of AT III and their inhibitory effect was not observed in the absence of AT III. There was no correlation between the anticoagulant activities of this series of fucoidans and their anti-inflammatory action, studied earlier. It is suggested that these two types of fucoidan activities depend on different structural features of fucoidans. Results of this study demonstrate a possibility of preparation of fucoidans with high anti-inflammatory activity but low anticoagulant activity. Anticoagulant activity of the fucoidans did not exhibit direct dependence on the content of fucose, the other neutral sugars and sulfates; no dependence was also found between the anticoagulant activity and the structure of the backbone of their molecules.
Keywords: fucoidan; heparin; blood coagulation; thrombin; factor Xa; antithrombin
Prognostic significance of lysosomal cysteine proteases in the estimation of the effectiveness of the antitumor therapy by S. Ya. Zhanaeva; A. I. Dyakov; T. A. Alekseenko; T. A. Korolenko (84-88).
The influence of the antitumor drugs, cyclophosphamide (CPA) and nitrosomethylurea (NMU) on the activity of lysosomal cysteine proteases cathepsin B and L in tumor tissue has been investigated using CPA-sensitive (LS) and CPA-resistant mouse lymphosarcomas (RLS). (These drugs exhibit high and low antitumor efficiency towards LS and RLS mouse lymphosarcomas, respectively). Regression or reduction in the growth rate of LS and RLS lymphosarcomas caused by CPA or NMU administration was accompanied by the increase in the activity of cysteine proteases cathepsin B and L in the tumor tissue. The increase of cathepsin B and L activity in tumor tissue correlated with the therapeutic effect of these drugs. Data obtained suggest that activity of cathepsin B and L in tumor tissue has a prognostic significance for the estimation of the effectiveness of antitumor therapy.
Keywords: cysteine proteases; malignant tumors; prognostic significance
Cloning, expression, and purification of Helicobacter pylori L-asparaginase by Yu. A. Gladilina; N. N. Sokolov; J. V. Krasotkina (89-91).
Asparaginase from Helicobacter pylori (HpA) has been cloned and expressed in E. coli cells. The recombinant strain stably expressed catalytically active HpA. Optimization of culturing and expression conditions resulted in the expression level of the recombinant enzyme amounting up to 6% of total protein of the producer strain. A method developed for HpA purification included a single chromatographic stage and provided more than 60%-yield of the active enzyme. Specific asparaginase activity was 92 U/mg of protein, whereas the rate of glutamine hydrolysis was just 8.3 × 10−3 U/mg, respectively. Data obtained indicate that due to low glutaminase specificity HpA may be employed as a non-toxic enzyme preparation for treatment of leukemia.
Keywords: recombinant L-asparaginase; acute lymphoblastic leucosis; Helicobacter pylori
Variations of glycoprotein IIb–IIIa (αIIb/β3-integrin) content in healthy donors. The influence on platelet aggregation activity and efficacy of aspirin action by S. G. Khaspekova; O. V. Sirotkina; Y. V. Shimanova; A. V. Mazurov (92-98).
The effects of content of a fibrinogen receptor, glycoprotein (GP) IIb–IIIa (αIIb/β3-integrin), GP IIIa genetic polymorphism (substitution Leu33Pro), and fibrinogen concentration in blood plasma on platelet aggregation activity have been investigated in a group of healthy volunteers. In 35 examined donors the GP IIb–IIIa content on platelet surface varied from 40 to 71 × 103 per platelet. Repeated measurements revealed that the GP IIb–IIIa content coefficient of variation was 9.5%, and deviations from mean levels did not exceed 20%. The level and the rate of platelet aggregation induced by ADP (1.25–20 μM) correlated with GP IIb–IIIa number (r from 0.315 to 0.591) and were higher in the group of donors with high in comparison with low GP IIb–IIIa content (>60 and (40–50) × 10−3 per platelet, respectively). Aspirin, the inhibitor of thromboxane A2 synthesis, partially suppressed ADP-induced platelet aggregation. The level of residual aggregation in the presence of aspirin also correlated with GP IIb–IIIa content and increased in subjects with high receptor content. Parameters of ADP-induced aggregation did not differ in donors with genotypes GP IIIa Pro33(−) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotype. GP IIb–IIIa content was also not affected by GP IIIa polymorphism. No significant correlations were found between the level and rate of platelet aggregation and fibrinogen concentration in blood plasma. The data obtained indicate that the effects of variations of GP IIb–IIIa content on platelet aggregation are higher than GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration. High GP IIb–IIIa content is associated with increased platelet aggregation activity and decreased efficacy of aggregation inhibition by aspirin.
Keywords: platelets; glycoprotein IIb–IIIa; integrins; genetic polymorphism; fibrinogen
Glutathione system of erythrocyte and plasma in peptic ulcer by V. I. Kulinsky; A. V. Shcherbatykh; A. A. Bolsheshapov; V. I. Bakhtairova; O. A. Bulavintseva; I. E. Egorova; A. I. Suslova; M. V. Yasko; O. V. Kolbaseeva; L. K. Noskova (99-103).
A complex study of the blood glutathione system has been carried out for the first time in patients with peptic (gastric and duodenal) ulcer. In erythrocytes and blood plasma of patients with the complicated peptic ulcer and postgastroresection syndromes there was the increase of conjugated dienes (and in the second group the increase in antioxidant activity). Under these conditions the main change was the sharp and identical decrease in glutathione peroxidase activity. In patients with uncomplicated peptic ulcer there was sharp increase in erythrocite and plasma glutathione reductase activity and plasma GSH. In operated but basically healthy patients plasma glutathione peroxidase remained decreased but plasma GSH sharply increased. Evidently complicated peptic ulcer is characterized by decreased functioning of the glutathione system. Activation of this system and the decrease or disappearance of manifestations of oxidative stress are associated with a favorable course of this disease, especially at uncomplicated peptic ulcer. The revealed changes significantly differ from those observed in patients with viral hepatitis, blle excretory diseases and strokes.
Keywords: glutathione system; oxidative stress; peptic ulcer