Efficacy of the Technologically Processed Antibodies to the Brain-Specific S100 Protein in a Rat Model of Hemorrhagic Stroke.

We studied the ability of technologically processed antibodies to the brain-specific S100 protein (drug Prospekta) to reduce the brain lesion area, neurological disorders, and mortality in a rat model of hemorrhagic stroke. Technologically processed antibodies to S100 exerted a positive effect on all these parameters (brain lesion area, survival rate, neurological status according to the Menzies scale, and proportion of contralateral turns). This allows us to recommend further research into the spectrum of pharmacological activity and the mechanism of action of technologically processed antibodies to S100 in order to expand the indications for their use after the necessary clinical trials.

[DNA Probes for Analysis of the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells].

The important role of DNA damage in the occurrence of various diseases, including cancer, has led to study of the mechanisms of genetic information stability, that have been carried out since the discovery of DNA repair systems. The question of the relationship between the accumulation of DNA damage, disorders in DNA repair pathways, and increased risk of disease development is still relevant. Over the past few years, significant efforts have been made to develop methods for analyzing the activity of DNA repair enzymes in human cells. In this work, we developed fluorescent DNA probes that allow us to determine the activity of key enzymes of base excision DNA repair in cell extracts, namely the DNA glycosylases UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1, and OGG1 and the AP endonuclease APE1. The sensitivity of DNA probes was determined on pure enzyme preparations. Determination of the activity of repair enzymes in cell extracts of the human ovarian tumor lines TOV112, 79, OVCAR3, MESOV, SCOV3, and TOV21 revealed significant variability in the level of enzyme activity in these cell lines. These results may become a test system platform for analyzing the activity of the base excision DNA repair system in the human body.

Age and Sex Characteristics of the Blood Cytokine Profile in Rats Subjected to Prenatal Stress.

Parameters of blood cytokine profile in male and female rats subjected to prenatal stress on the model of swimming in cold water (10°C, 5 min, days 10-16 of gestation) were studied. Prenatal stress had no significant effects on the blood levels of IL-6 and IL-10 cytokines. The blood concentration of proinflammatory cytokine TNFα in 60-day-old rats was higher than in age-matched controls. Stress led to a lower level of anti-inflammatory IL-4 in the blood of 30-day-old males compared to controls. In female rats subjected to prenatal stress, the concentration of IL-4 decreased on day 21, but increased by day 60 of postnatal ontogeny. Specific effects of prenatal stress on the blood cytokine profile in male and female animals at different periods of ontogeny were revealed. Different and even opposite changes in blood cytokine levels could be largely mediated by sex- and age-specific features of immune functions after prenatal stress.

[Functional heterogeneity of the von Willebrand factor: pathogenetic significance and practical aspects of use in cardiology].

This review focuses on the participation of von Willebrand factor (VWF), that considerably contributes to thrombogenesis in damaged blood vessels, in the pathogenesis of atherosclerosis-induced cardiovascular pathology. Excessive formation and dysfunction of VWF leads to intravascular thrombosis and facilitates the development of endothelial dysfunction, vascular inflammation, and, thereby, the initiation and progression of atherosclerosis. The review presents information based on the analysis of full-text publications from PubMed that address the role of VWF in the development of atherosclerosis and its complications as well as the potential for influencing this index.

Lymphocyte Index of Peripheral Blood in Rats at Different Stages of the Post-Stress Period under Conditions of Antigenic Exposure and Injection of Lipopolysaccharide.

Changes in the Shaganin lymphocyte index (ratio of the number of lymphocytes to segmented neutrophils) in the peripheral blood of rats after intraperitoneal administration of LPS (100 µg/kg) at the end of a single stress exposure in a model of 24-h restraint stress were studied. The lymphocyte index was analyzed 3 h later, on the 1st and 8th days after the stress load. Immobilization was accompanied by a decrease in this parameter 3 h after exposure. One day after the stress load, an increase in the lymphocyte index was noted, which remained on the 8th day of observation. LPS injection did not affect the changes in this parameter caused by 24-h immobilization on the 1st and 8th days of the study, but prevented a pronounced increase in the lymphocyte index on the 1st day after the stress load. The data obtained expand the existing scientific understanding of the specificity of the involvement of immunomodulatory substances in the implementation of adaptive-compensatory processes in mammals under conditions of emotional stress.

Correlation of Physiological Parameters in Rats after Stress Exposure under Conditions of Antigenic Stimulation with Lipopolysaccharide Administration.

We studied correlation dependences between physiological parameters in rats in 3 h, 1 day, and 8 days after administration of LPS (100 µg/kg) at the end of 24-h immobilization stress. In 3 h after LPS administration against the background of stress exposure, significant correlations of metabolic parameters with the relative weight of the adrenal glands and the perceptual component of nociception in rats were revealed. A direct relationship between the concentration of the proinflammatory cytokine TNFα and anti-inflammatory IL-4 was also found in these animals. On the first day after LPS injection, correlations were revealed, predominantly positive, only between the indicators of the cytokine blood profile. In the late post-stress period after antigenic exposure, no correlations between the studied physiological parameters were found. It can be hypothesized that immune modulation through systemic administration of LPS prevents persistent excessive stress of physiological functions at the later stages after stress exposure.

[Comparative Analysis of the Activity of the Polymorphic Variants of Human Uracil-DNA-Glycosylases SMUG1 and MBD4].

The human N-glycosylases SMUG1 and MBD4 catalyze the removal of uracil residues from DNA resulting from cytosine deamination or replication errors. For polymorphic variants of SMUG1 (G90C, P240H, N244S, N248Y) and the MBD4^(cat) catalytic domain (S470L, G507S, R512W, H557D), the structures of enzyme-substrate complexes were obtained by molecular dynamic simulation. It was experimentally found that the SNP variants of SMUG1, N244S and N248Y, had increased catalytic activity compared to the wild-type enzyme, probably due to the acceleration of the dissociation of the enzyme-product complex and an increase in the enzyme turnover rate. All other SNP variants of SMUG1 (G90C, P240H) and MBD4^(cat), in which amino acid substitutions disrupted the substrate binding region and/or active site, had significantly lower catalytic activity than the wild-type enzymes.

The role of active-site amino acid residues in the cleavage of DNA and RNA substrates by human apurinic/apyrimidinic endonuclease APE1.

BACKGROUND: Human apurinic/apyrimidinic endonuclease APE1 is one of participants of the DNA base excision repair pathway. APE1 processes AP-sites and many other types of DNA damage via hydrolysis of the phosphodiester bond on the 5' side of the lesion. APE1 also acts as an endoribonuclease, i.e., can cleave undamaged RNA. METHODS: Using pre-steady-state kinetic analysis we examined the role of certain catalytically important amino acids in APE1 enzymatic pathway and described their involvement in the mechanism of the target nucleotide recognition. RESULTS: Comparative analysis of the cleavage efficiency of damaged DNAs containing an abasic site, 5,6-dihydrouridine, or α-anomer of adenosine as well as 3'-5'-exonuclease degradation of undamaged DNA and endonuclease hydrolysis of RNA substrates by mutant APE1 enzymes containing a substitution of an active-site amino acid residue (D210N, N212A, T268D, M270A, or D308A) was performed. Detailed pre-steady-state kinetics of conformational changes of the enzyme and of DNA substrate molecules during recognition and cleavage of the abasic site were studied. CONCLUSIONS: It was revealed that substitution T268D significantly disturbed initial DNA binding, whereas Asn212 is critical for the DNA-bending stage and catalysis. Substitution D210N increased the binding efficacy and blocked the catalytic reaction, but D308A decreased the binding efficacy owing to disruption of Mg2+ coordination. Finally, the substitution of Met270 also destabilized the enzyme-substrate complex but did not affect the catalytic reaction. SIGNIFICANCE: It was found that the tested substitutions of the active-site amino acid residues affected different stages of the complex formation process as well as the catalytic reaction.

Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1.

Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme's tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.

Blood Cytokine Concentration in Rats during Antigenic Treatment after a Single Long-Term Stress Exposure.

We studied changes in the blood cytokine profile of rats 3 h, 1 day, and 8 days after acute stress on the model of 24-h immobilization followed by LPS administration (100 µg/kg intraperitoneally). The concentration of proinflammatory cytokines (particularly of IL-1ß and TNFα) significantly decreased at the early stage after stress exposure and physiological saline injection, but increased in the follow-up period and practically did not differ or even surpassed the control level by the end of observations. Under these conditions, the blood content of anti-inflammatory cytokine IL-10 increased most significantly on day 1 of the post-stress period. Restraint stress followed by LPS administration was accompanied by a decrease in the level of proinflammatory cytokines at the early (IFNγ and TNFα) and late stages (IL-1ß) of the experiment. Directed modulation of the immune status in animals after acute stress was followed by a significant increase in the content of IL-10 on days 1 and 8, as well as by a tendency toward elevation of IL-4 concentration by the end of the study. The directionality and degree of changes in the cytokine profile of mammalian tissues depend on the type of extreme exposure, duration of the post-stress period, and specific effects of exogenous pathogenic factors in the whole body.

State of Stress-Marker Organs in Rats after a Single Exposure to Long-Term Stress and Treatment with Lipopolysaccharide.

We studied the effect of LPS on the state of stress-marker organs in rats at various periods after a single exposure to long-term stress on the model of 24-h immobilization. The animals were intraperitoneally injected with LPS in a dose of 100 µg/kg immediately after the negative emotiogenic exposure. Changes in physiological parameters were evaluated 3 h, 1 day, and 8 days after immune stimulation. Acute stress was accompanied by a decrease in the weight of the thymus during all stages of the post-stress period. An increase in the relative weight of theadrenal glands in animals under these conditions was observed only on day 8 after restraint stress. The induction of immune reactions due to systemic treatment with LPS was shown to prevent involution of the spleen in the late stage after a single exposure to long-term stress (day 8). Hypertrophy of the adrenal glands, which serves as one of the typical reactions of mammals to negative emotiogenic factors, was not revealed during the post-stress period after antigenic stimulation. These data hold much promise for the development of new approaches to the use of immunoactive substances to prevent or reduce the severity of physiological changes after emotiogenic loads.

[DYNAMICS OF CYTOKINE CONCENTRATION IN THE BLOOD OF RATS WITH VARIOUS BEHAVIORAL CHARACTERISTICS AFTER ACUTE EMOTIONAL STRESS].

This work was designed to study a change in cytokine content in the peripheral blood of behaviorally passive and active Wistar rats at various time intervals after acute stress on the model of night-time immobilization. A decrease in the concentration of most pro- and anti-inflammatory cytokines in passive animals was most pronounced immediately and, particularly, 3 days after stress exposure. Variations in the blood cytokine profile after experimental stress were lower in behaviorally active specimens. A statistically significant decrease was observed only in the amount of a proinflammatory cytokine IL-1α and anti-inflammatory cytokines IL-4 and IL-13. As differentiated from passive rats, these changes in active specimens were most pronounced 1 day after negative emotiogenic exposure. Our results illustrate a specific involvement of immunoactive substances in the systemic regulation of physiological functions and development of individual resistance to the negative consequences of stress.

Dynamics of locomotor activity and heat production in rats after acute stress.

The dynamics of locomotor activity and heat production were studied in rats demonstrating passive and active behavior in the open field test at different time after exposure to acute emotional stress caused by 12-h immobilization during dark hours. The most pronounced changes in behavior and heat production followed by disturbances in circadian rhythms of these parameters were detected within the first 2 days after stress. In contrast to behaviorally active rats, the most significant decrease in locomotor activity and heat production of passive animals subjected to emotional stress was observed during dark hours. Circadian rhythms of behavior and heat production in rats tended to recover on day 3 after immobilization stress. These data illustrate the specificity of metabolic and behavioral changes reflecting the shift of endogenous biological rhythms in individuals with different prognostic resistance to stress at different terms after exposure to negative emotiogenic stimuli.

[Effect of melatonin on lipid peroxidation in the blood of rats with various behavioral characteristics during acute emotional stress].

This work was designed to study the effect of melatonin on lipid peroxidation in the peripheral blood of behaviorally passive and active Wistar rats. Immobilization of rats with simultaneous electrocutaneous stimulation (1 h) served as a model of acute stress. After intraperitoneal injection of melatonin (2 mg/kg) the intensity of lipid peroxidation in the blood plasma remained practically unchanged in passive specimens, but increased in active animals. Stress exposure was followed by specific variations in free radical processes in the blood (passive rats, inhibition; active specimens, no changes). Administration of melatonin contributed to a decrease in the intensity of lipid peroxidation in the blood of stressed rats (as compared to control specimens receiving this neurohormone). Therefore, the effect of melatonin on free radical processes depends on the initial behavioral characteristics and physiological state of animals. These data illustrate the importance of an individual approach to studying the systemic mechanisms for organization of functions in mammals. Key words: melatonin, emotional stress, lipid peroxidation, blood plasma, rats with various behavioral characteristics.

Cytokine levels in rat blood and brain structures after administration of lipopolysaccharide.

We compared cytokine profile of rat serum and brain structures after immune status modulation by LPS (30 µg/kg intraperitoneally). The content of inflammatory (IL-1α, IL-1ß, IL-2, IL-6, IFN-γ, and TNF-α) and anti-inflammatory (IL-4 and IL-10) cytokines in biological samples of animals was measured on days 1 and 7 after antigenic stimulation. LPS administration reduced the levels of both inflammatory and anti-inflammatory cytokines in the peripheral blood of the rats, especially on the 1st day. LPS administration was also accompanied by specific changes in cytokine content in the dorsal hippocampus and anterior cingulate cortex. Antigenic stimulation increased the level of anti-inflammatory cytokines IL-4 and IL-10 in the examined brain tissues, the changes were most pronounced on day 1 after LPS injection. No significant changes in the levels of proinflammatory cytokines in the brain tissue of animals were found at the above terms after LPS injection. Thus, peripheral LPS administration to rats shifts the balance between the inflammatory and anti-inflammatory cytokines in the CNS structures towards the latter.

[Novel hybrid inhibitors of the phage T7 RNA polymerase: synthesis, docking and screening in vitro].

A number of new hybrid heteroaromatic compounds, consisting of tricyclic fragments (acridone, thioxanthone and phenazine) and bicyclic fragments (benzimidazole, benzothiazole and benzoxazole) were synthesized using the method, developed by the authors. As a result of screening against the transcription model system of the phage T7 DNA-dependent RNA polymerase three effective inhibitors of the RNA syntheses with the IC50 value of 8.9, 5.7 and 19.8 microM were detected. To cast light on the mode of interaction between the synthesized compounds and the target, the molecular docking was applied to the model pocket of the phage T7 RNA polymerase transcription complex. It was established that these ligands form networks of H-bonds with residues of the pocket conservative amino acids and pi-interaction with the Mg2+ ion. A planar geometry of the hybrid molecules, realized due to the intramolecular H-bonds, proved to be an important structural feature, which correlates with an efficacious inhibitory activity.

[Optimization of mobilization regimes of blood hemopoietic stem cells in patients with multiple myeloma].

AIM: To determine an optimal cyclophosphamide dose in the mobilization scheme providing adequate collection of CD34+ cells in patients with multiple myeloma (MM), to optimize the time of initiation of granulocytic colony-stimulating factor (G-CSF) administration, to study effects of induction therapy schemes on results of mobilization and collection of CD34+ cells. MATERIAL AND METHODS: Department of hemoblastoses chemotherapy and bone marrow transplantation of the Russian Hematological Center performed mobilization of autologous blood hemopoietic stem cells (BHSC) in 93 MM patients treated in 2001-2010. This was done with cyclophosphamide and G-CSF. The former was used in 59 cases in a dose 6 g/m2, in 34 cases - 4 g/m2. RESULTS: Myelotoxic agranulocytosis after cyclophosphamide administration developed in all the patients and was observed for 3-10 days (median 5 days). Agranulocytosis ran without documented infections in 51 (54.8%) patients, with febril fever - in 42 (45.2%) patients. Cepticemia, pneumonia, necrotic enteropathy, stomatitis, herpetic lesion of the skin were registered in 9, 4, 11, 14 and 6 cases, respectively. Severe thrombocytopenia (< 30 x 10(9)/l) occurred more frequently in administration of 6 g/m2 cyclophosphamide. It was corrected with 2-5 transfusions of thromboconcentrates, only 1 transfusion was needed after the dose 4 g/m2. Collection of CD34+ cells started in leukocyte level over 3.5 x 10(9)/l on mobilization day 12-20 (median day 15). The day of the first leukocytapheresis did not depend on the day of the first introduction of G-CSF. Duration of G-CSF administration was significantly shorter in the start of its use after leukocyte count decrease under 1.0 x 10(9)/l. Conduction of 1 to S (median 2) leukocytapheresis was needed for collection of BHSC. Sufficient for 2 autotransplantations number of BHSC were stored in 90 of 93 patients. Cyclophosphamide administration in a dose 6 g/m2 allowed collection of cells sufficient for one autotransplantation for the first leukapheresis in 52 (88.1) patients. A total number of CD34+ cells over 4 x 10(6) cells/kg were collected in 56 (94.9%) patients. In administration of cyclophosphamide in a dose 4 g/m2 mobilization was effective in all 34 patients. The first leukapheresis provided sufficient for one autotransplantation number of cells in 29 (85.3%) patients. CONCLUSION: Administration of high cyclophosphamide doses in combination with G-CSF is an effective and safe method of BHSC mobilization providing collection of adequate number of CD34+ cells for double autotransplantation in 96.8% patients. Cost effective is the start of G-CSF administration in the fall of leukocytes under 1.0 x 10(9)/l. Cyclophosphamide dose 4 g/m2 provides collection of CD34+ cells number sufficient for two autotransplantations in moderate thrombocytopenia and in less number of substitute transfusions in the absence of serious toxic complications.

[AntiEBV activity of 6-azacytidine and its derivatives].

Epstein-Barr virus (EBV) causes several lymphoproliferative diseases, lesions of the central and peripheral nervous systems. Spectrum of etiotropic medicines against EBV is limited. The paper presents the anti-virus research of modified nucleoside with a wide spectrum of action - 6-azacytidine and its derivatives (2'-3'-seco-5-methyl-6-AC, 2',3'-dideoxy-2',3'-didehydro-6-AC and 2'-deoxy-6-AC) on the model of Epstein-Barr virus in Raji lymphoblastoid cells. Parameters of cytotoxicity (CC,) for the investigated substances were determined which amounted to 120 microg/ml, 180 microg/ml, 500 microg/ml, 330 microg/ml, and the effective concentration (ECU)--0.5 microg/ml. 1 microg/ml, 4 microg/ml, 11 microg/ml, in accordance with the sequence of preparations listed above. The results indicate a high antiEBV activity since their selectivity indexes (SI) were 240, 180, 125, 30 that is 1.3-10 times higher than the reference substance acycloguanosine. Apoptosis-stimulating action of 6-azacytidine and its derivatives was revealed. An increase of percentage of apoptotic cells in EBV-infected Raji cells and those treated with investigated substances as compared to uninfected ones was observed after 24 hours. Thus, our results provide new biological properties of the azacytidine substances.

[The inhibitory action of 6-azacytidine on Mollicutes and its proposed mechanism]. / Ingibitornoe deistvie 6-azatsitidina na mollikuty i ego predpolagaemyi mekhanizm.

6-Azacytidine (6-AC) is shown to have an inhibitory effect on the Mollicutes of the different systematic position. The growth of type strains of Mollicutes (Acholeplasma laidlawii PG-8, Mycoplasma pneumoniae FH and M. fermentans PG-18) completely ceased in the nutrient medium at concentration of the above substance in it within the range of 125-250 micrograms/ml. 50% inhibiting concentration of 6-AC equaled: for M. fermentans PG-8: 23.43 micrograms/ml; M. pneumoniae FN: 46.8 micrograms/ml; Acholeplasma laidlawii PG-8: 62.5 micrograms/ml. 6-AC concentration 5 micrograms/ml decreased the process of DNA-dependent DNA synthesis in the in vitro system more than by 60%. 6-AC exerted less effect on the DNA-dependent RNA synthesis in the in vitro system: at different concentrations of 6-AC (up to 400 micrograms/ml) RNA synthesis decreased only by 20%. Translation on ribosomes of Mollicutes in the in vitro system completely ceased at 6-AC concentration 100 micrograms/ml. The results obtained indicate that for 6-AC in cells of Mollicutes and, possibly, for other microorganisms there are two targets: ribosomes and DNA-dependent DNA-polymerase. Total effect of blocking of the translation and replication processes by 6-azacytidine causes death of Mollicutes. Since 6-AC has no harmful effect on the human cells, it can be used as an efficient method for treatment of respiratory and urogenital diseases induced by Mollicutes.

[Use of xydiphone diphosphonate for correcting the course of posttraumatic bone regeneration in protracted hypodynamia]. / Primenenie difosfonata ksidifona dlia korrektsii techeniia posttravmaticheskoi regeneratsii kostnoi tkani pri dlitel'noi gipodinamii.

Experiments were conducted on rats to study the effect of daily injection of xydiphone diphosphonate in a dose of 5 mg/100 g on bone tissue in normal motor activity and in hypodynamia caused by amputation of a limb and posttraumatic regeneration. It was established that xydiphone inhibits thinning of the diaphyseal cortical layer induced by hypodynamia and normalizes the parameters which characterize posttraumatic bone tissue regeneration in an unsupported position of the limb.