Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.7, #1)

Polymorphism of the plasminogen activator inhibitor type 1 gene, plasminogen level and thromboses in patients with the antiphospholipid syndrome by R. B. Aisina; L. I. Mukhametova; E. V. Ostryakova; N. V. Seredavkina; L. I. Patrushev; N. L. Patrusheva; T. M. Reshetnyak; D. A. Gulin; K. B. Gershkovich; E. L. Nasonov; S. D. Varfolomeyev (1-15).
The frequency of venous and arterial thromboses and plasminogen level have been investigated in 78 patients with the antiphospholipid syndrome (APS), including 35 patients with systemic lupus erythematosus (SLE + APS) and 43 patients with primary APS (PAPS). The levels and genotypes of plasminogen activator inhibitor type 1 (PAI-1) were determined in 45 patients with APS (21 patients with SLE + APS and 24 patients with PAPS). A control group included 10 individuals without autoimmune disease signs and thromboses during the observation period and in anamnesis. It has been shown for the first time that for one third of 67 patients with APS and thromboses, high-positive levels of antiphospholipid antibodies (aPL) are associated with low plasminogen levels. The levels of PAI-1 antigen measured by the ELISA method, which detects active, latent and bound to plasminogen activator PAI-1, were compared with frequency of thromboses in APS patients. In one third of 43 patients with APS and thromboses the high and increased levels of PAI-1 were associated with high-positive aPL levels. One of possible mechanisms of this interrelationship was considered. It was shown that arterial and, to a greater extent, venous thromboses are associated with the 4G/5G polymorphism of the PAI-1 gene and high plasma level of the inhibitor in 79% of APS patients. In the presence of the 4G allele SLE + APS patients had higher PAI-1 levels than PAPS patients. The data obtained show that measuring the levels of plasminogen and PAI-1 as well as the 4G/5G polymorphism of the PAI-1 gene associated with thromboses may have the practical importance for identification of high risk of thrombosis in APS patients.
Keywords: plasminogen; plasminogen activator inhibitor type 1; polymorphism; antiphospholipid syndrome; thromboses

Using a novel NO-specific reagent, the complex of Cu2+ with a fluorescein derivative (Cu-FL), stimulation of NO production by the medicinal leech salivary cell secretion (SCS) has been demonstrated for the first time in cultures of human endothelial cells (HUVEC) and rat cardiomyocytes (RCM). NO was detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in the culture fluid by the fluorescence method. SCSstimulated NO synthesis in HUVEC but not in RCM cells was accompanied by NO release into the intercellular space thus determining its subsequent distribution. Localization of intracellular NO synthesis centers is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In the endothelial cells SCS-activated nitrosylation processes, estimated by the increase of nitrite-ion content in the culture medium. It is therefore important to use Cu-FL, rather than Griss-reagent, during the first hour of analysis of NO synthesis. It is possible that the NO-depended mechanism of the SCS action on endothelial cells may be a factor responsible for the positive effect of SCS during hirudotheraphy.
Keywords: nitric oxide; medicinal leech salivary gland cell secretion; NO-probe Cu-FL; fluorescent microscopy; transmission electron microscopy; Griss-reagent; NO-synthases

The activity of the glutathione system and conjugated diene content (CD) have been investigated in the liver and blood serum of rats with experimental hyperthyroidism treated with melaxen and valdoxan. The study of glutathione reductase (GR), glutathione peroxidase (GP) and glutathione transferase (GST) activities increased under this pathology has shown that administration of these compounds decreased these activities towards control levels. Melaxen and valdoxan administration increased reduced glutathione (GSH) content as compared with untreated hyperthyroid rats. This increase may be associated with its decreased utilization for detoxification of toxic products of free radical oxidation (FRO). Administration of the melatonin correcting drugs also tended to normalize the CD level increased in the liver and blood serum of hyperthyroid rats. Results of this study indicate that melaxen and valdoxan exhibit positive effect on free radical homeostasis. This appears to be accompanied by a decrease in the load of the glutathione antioxidant system in comparison with the examined pathology.
Keywords: hyperthyroidism; free-radical oxidation; glutathione antioxidant system; melaxen; valdoxan

Selenium and oxidative stress in cancer patients by E. G. Gorozhanskaya; S. P. Sviridova; M. M. Dobrovolskaya; G. N. Zubrikhina; Sh. R. Kashiya (32-39).
In order to determine impairments of free-radical processes in blood serum of 94 untreated cancer patients with various localization of the tumors (gastric cancer, colon cancer, breast and ovarian cancers, hemoblastoses) selenium levels and parameters of oxidative stress (the sum of nitric oxide metabolites (NOx), superoxide dismutase (Cu/ZnSOD) and malondialdehyde (MDA), and catalase activity) have been investigated. A comparative evaluation of the efficacy of the selenium-containing drug Selenase (sodium selenite pentahydrate) has been carried out in 40 patients with malignant liver disease and clinical signs of liver failure during the early postoperative period. The selenium levels in cancer patients were 73.0 ± 2.6 μg/L; this is 25–30% lower than the normal value (110–120 μg/L). Decreased levels of NOx were detected in all patients regardless of tumor localizations (22.1 ± 1.1 μM, versus the normal range of 28.4 ± 0.9 μM). Only patients with extensive malignant process in the liver had the NOx levels, which significantly exceeded the normal values (p < 0.001). The high level of NOx had a toxic effect on hepatocytes and caused metabolic disorders and inflammatory-necrotic changes in the liver. All examined patients had increased levels of SOD and MDA in normal values of catalase activity. The use of Selenase in postoperative patients with tumors of the liver increased selenium levels by 10–12%. This was accompanied by a decrease in the content of SOD and NOx, and earlier recovery of detoxification and synthetic liver function. These findings point to intensification of oxidative stress and metabolic disorders in the malignant process, which is the basis for prescription of metabolic correction therapy.
Keywords: selenium; nitric oxide; oxidative stress; cancer patients

Computer search for molecular mechanisms of ulcerogenic action of non-steroidal anti-inflammatory drugs by S. M. Ivanov; A. A. Lagunin; A. V. Zakharov; D. A. Filimonov; V. V. Poroikov (40-45).
Peptic ulcers are the most frequent side effect of therapy with non-steroidal anti-inflammatory drugs (NSAIDs). Good experimental evidence exists that pathogenesis of peptic ulcers cannot be attributed only to inhibition of cyclooxygenases. The knowledge about other molecular mechanisms of drug action associated with development of peptic ulcers could be useful for design of new safer NSAIDs. However, considerable time and material resources are needed for corresponding experimental studies. For simplification of the experimental search, we have developed an approach for in silico identification of putative molecular mechanisms of drug actions associated with their side effects. We have generated a data set of 85 NSAIDs, which includes information about their structures and side effects. Unknown molecular mechanisms of action of these NSAIDs were evaluated by the computer program PASS (Prediction of Activity Spectra for Substances) predicting more than 3000 molecular mechanisms of action based on structural formula of sub-stances. Statistically significant associations have been found between predicted molecular mechanisms of action and development of peptic ulcers. Twenty six molecular mechanisms of action probably associated with development of peptic ulcers have been found: two of them were known previously and 24 were quite new. Analyzing Gene Ontology data, data on signal and metabolic pathways, and available MEDLINE publication data, we proposed hypotheses on the role of 10 molecular mechanisms of action in the pathogenesis of peptic ulcer.
Keywords: non-steroidal anti-inflammatory drugs; peptic ulcers; molecular mechanisms of action; computer-aided drug design

Irreversible chemical AFM-based fishing for detection of low-copied proteins by Yu. D. Ivanov; V. V. Danichev; T. O. Pleshakova; I. D. Shumov; V. S. Ziborov; N. V. Krokhin; M. N. Zagumenniy; V. S. Ustinov; L. P. Smirnov; A. V. Shironin; A. I. Archakov (46-61).
The atomic-force microscopy-based method of irreversible chemical AFM-fishing (AFM-IFchem) has been developed for the detection of proteins existing at ultra-low concentrations in solution. Using this method, a very low concentration of horseradish peroxidase (HRP) protein (10−17 M) was detected in solution. A theoretical model that allows the description of obtained experimental data has been proposed. This model takes into consideration both transport of the protein from the bulk solution onto the AFM-chip sur-face and its irreversible binding to the activated area.
Keywords: AFM-fishing; AFM-chip; low-copied proteins

Preparation of monospecific antibodies against isoform 2 of translation elongation factor 1A (eEF1A2) by E. F. Kolesanova; T. E. Farafonova; E. Yu. Aleshina; N. V. Pyndyk; M. V. Veremieva; A. V. Novosylnaya; M. I. Kovalenko; V. F. Shalak; B. S. Negrutskii (62-69).
Amino acid sequences of eukaryotic translation elongation factor isoform 1 (eEF1A1) and 2 (eEF1A2) were compared and two peptide fragments of eEF1A2 were chosen as linear antigenic determinants for generation of monospecific antipeptide antibodies. Synthesized peptides corresponded to the selected peptide fragments were conjugated to bovine serum albumin (BSA) and used for immunizations of mice. Antibodies, produced against the eEF1A2 fragment 330–343 conjugated to BSA, specifically recognized this isoform in the native and partially denatured states but did not interact with the eEF1A1 isoform. It was shown that these monospecific anti-eEF1A2 antibodies could be employed for eEF1A2 detection both by enzyme-linked immunosorbent assay and by immunoblotting.
Keywords: translation elongation factor 1A; antipeptide antibodies; peptide synthesis; enzyme-linked immunosorbent assay; immunoblotting

Strain differentiation of Staphylococcus aureus by means of direct MALDI TOF mass spectrometry profiling by M. A. Kornienko; E. N. Ilina; A. D. Borovskaya; M. V. Edelstein; M. V. Sukhorukova; M. Kostrzewa; V. M. Govorun (70-78).
Staphylococcus aureus is one of the most interesting microbial species in clinical studies. It is characterized by a wide extent of strain diversity, first of all, due to variability in virulence and pathogenicity. The aim of this study was to test the method of rapid Staphylococcus strain differentiation by a certain sign based on registration of characteristics features of MALDI mass spectra accumulated during direct protein profiling of the bacterial cell. The model signs registered as strain differences included production of β-lactamase and α-hemolysin encoded by blaZ and hla genes, respectively.The mathematical analysis of MALDI mass spectra accumulated for 53 S. aureus isolates using the clustering genetic algorithm resulted in generation of two independent classification models, which could differentiate the strains by the considered features. Using statistical contribution of each mass peak to the model, the most significant peaks (masses), which could be considered as the markers of Staphylococcus strain differences, were found. The generated diagnostic models were characterized by the following sensitivity and specificity coefficients: 97.5 and 82.5%, respectively, for strain differentiation by β-lactamase production and 90.0 and 88.7% by the presence of α-hemolysin.
Keywords: Staphylococcus ; strain differentiation; MALDI TOF mass spectrometry

Several parameters of the cytoplasmic enzymatic antioxidant system of the liver and brain of the rat have been investigated under conditions of immobilization stress and of an antioxidant preparation in the diet of animals. These included superoxide dismutase (SOD) and glutathione reductase (GR) activities and nonspecific NADPH oxidation. Only changes in the activity of SOD both in the liver and brain were revealed. In the liver of animals that receive no preparation, a decrease in the activity of SOD after 30-min immobilization and its restoration after a 360-min immobilization were observed. In the brain, the activity of SOD decreased only in preconditioned animals after 30 and 360 min of exposure to stress. In addition, the activity of SOD in the brain of preconditioned animals, both stressed and unstressed, was lower than in the corresponding groups of control animals. It is probable that, under the conditions of immobilization stress, the level of reactive oxygen species (ROS) and as a consequence the activity of SOD decrease. The intake of an antioxidant preparation under these conditions seems to be not correct.
Keywords: superoxide dismutase; glutathione reductase; immobilization stress; brain; liver

Estrogen and progestin (ethinylestradiol and levonorgestrel, respectively) in a dose dependent manner accelerated platelet lipid peroxidation (LPO), activated platelets, increased continuous intravascular coagulation and reduced tolerance to thrombin. Antioxidants limited these effects.
Keywords: ethinylestradiol; levonorgestrel; lipid peroxidation; hemostasis

Components of the adrenocortical system (adrenal and blood corticosteroid hormones and hepatic and renal 11β-hydroxysteroid dehydrogenase activity) and also activity of the most important enzyme of the renin-angiotensin system, tissue and blood angiotensin converting enzyme (ACE), have been investigated in dynamics of alloxan diabetes. The study has shown that the initial period of diabetes is characterized by activation of synthesis and secretion of adrenocortical hormones into blood. High blood glucose and glucocorticoid hormones increase activity of the renin-angiotensin system in lungs and decrease ACE secretion into blood. This is accompanied by a decrease of activity of the renin-angiotensin system in kidneys. Subsequent progression of diabetes resulted in impairments of physiologically determined correlations between the components of these systems. Development of experimental diabetes for 30 days was accompanied by sign of a decrease of the adrenal glucocorticoid function regardless of stable impairments of carbohydrate metabolism. Under these conditions increased adrenal and hepatic 11β-hydroxysteroid dehydrogenase activity may be responsible for maintenance of elevated levels of the main glucocorticoid in blood and tissues. Factor analysis revealed impairments in intersystem relationships between the adrenocortical and renin-angiotensin systems in experimental diabetes thus suggesting disintegration of regulatory systems.
Keywords: corticosteroid hormones; 11β-hydroxysteroid dehydrogenase; angiotensin-converting enzyme; alloxan diabetes; high performance liquid chromatography