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Actually, the epidemiology is a dynamically developing medical science located at the intersection of social and biological branches of knowledge and bio-informatics. The new sources of data, the new methods create unique opportunities for epidemiologist. The number of epidemiological studies carrying out at the junction of several adjacent disciplines is increasing that requires harmonious interaction of specialists of different branches of medical knowledge. The change of the structure of global mortality towards chronic non-communicable diseases significantly affected the vector of epidemiological studies. Many interventional epidemiological projects are targeted to evaluation of effectiveness of new methods of prevention of cardiovascular, metabolic and oncological diseases. However, in recent years, the fight against unremembered infections affecting about 1 billion of people and taking away lives of 0.5 million people annually gained new importance. The current COVID-19 pandemic also affected epidemiology of communicable and chronic non-communicable diseases. Great attention is also currently attended to studying influence of social, economic and environmental factors on human health. The increase of average life expectancy of population contributes to development of epidemiology of the elderly. The new projects are initiated in the field of pharmacoepidemiology targeted to studying effectiveness of medications. The review of national and foreign publications considering current trends and achievements in the field of epidemiology. The reference retrieval engines such as PubMed, Google Scholar, CyberLeninka were used. The current directions of epidemiological research are analyzed. The challenges and development prospects of development of modern epidemiology are highlighted.

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.

Adenine-DNA-glycosylase MutY is a monofunctional enzyme and catalyzes hydrolysis of N-glycosidic bonds with adenine residues located opposite 8-oxonuanine residues in DNA. Rational design was carried out to construct mutant enzyme forms with altered catalytic activity. Structures of the MutY mutants were calculated by molecular dynamics (MD). Their analysis showed that some of the MutY mutants may have AP lyase activity in addition to hydrolyzing the N-glycosidic bond, as is the case with bifunctional DNA glycosylases. MutY mutants with the A120K or S124K substitution were obtained by site-directed mutagenesis, and their catalytic activities were determined. The S120K substitution was shown to confer additional AP lyase activity, while the A124K substitution completely inactivated the enzyme.

Food allergy (FA) is a health problem that adversely affect the quality of life of children and their family members. The purpose of the study was to assess the quality of life in families with children affected FA. Material and methods . A cross-sectional study was conducted in a group of 75 children with a confirmed FA (at the age of Me 4.9 years [1.3; 7.1]). One of the caregivers of the child was asked to complete the Russian version of a specialized questionnaire «The Food hypersensitivity famiLy ImPact, FLIP» for assessing the life quality of families with children affected FA. Results . Diet organization is the main concern affecting quality of life, while the daily life of the family and the emotional sphere are less impacted. Age, type of food allergens and clinical manifestations do not significantly contribute the life quality indicators. Hypersensitivity to several food is statistically associated with changes in everyday life and emotions. Non-compliance with the diet is associated with a lower impact of FA on quality of life. 56% of respondents worried about the nutritional value of child's diet and 49.3% of caregivers reported that a child's FA significantly impacted grocery shopping behaviors (reading labels, etc.). At the same time, 73.3% noted that child's FA does not affect the diet of other family members. Also, 33.3% of the parents experienced anxiety due to child's FA and 38.7% are worried that FA might stay persistent. 30.7% of respondents are afraid of accidental consuming of allergenic products. Conclusion . The acquired results indicate the importance of quality of life assessment for understanding the social aspects of FA. Strategies to improve the quality of life include the development of informational and educational programs both for parents and patients. In order to estimate impact of FA to life quality from the patient's perspective further development of questionnaires adapted for children and adolescents is necessary., Competing Interests: The authors declare no conflicts of interest., (Copyright© GEOTAR-Media Publishing Group.)

Food allergy (FA) is an actual problem in pediatric practice. The gut microbiota plays a crucial role in food sensitization development, since the maturation of immune system occurs under the influence of intestinal microorganisms. Immunoregulatory activity of gut microbiota is associated with the increase of IgA production and promotion of the barrier function of intestinal epithelium. Gut microbiota influence the activity of T-regulatory cells, as well. Violation of gut biocenosis, which occurs under the influence of various factors (artificial feeding, past diseases, the use of antibiotics, etc.), can lead to a shift in the balance of the immune system towards the increase of Th2-profile cytokines and the subsequent formation of hypersensitivity to food allergens. In this regard, the correction of the gut microbiome is a promising method of FA control, due to the ability of intestinal bacteria influence the production of T-regulatory cells and thus suppress allergy immune response. The aim of the review is to analyze experimental and clinical studies exploring effectiveness of methods modifying intestinal microbiota in order to treat and prevent FA. Material and methods . The analysis of the literature in eLIBRARY, MedLine and PubMed databases was carried out. Results . The analysis revealed the lack of rigorous evidence that pre-, pro- and synbiotics significantly increase the effectiveness of standard therapy of FA. However, the use of bifidobacteria, lactobacilli, lactic acid bacteria, in combination with the basic therapy of FA has general positive effect on the clinical outcome, especially in case of gastrointestinal symptoms. Also, the results of some studies indicate the effectiveness of synbiotics (Bifidobacterium breve M-16V, Lactobacillus rhamnosus GG in combination with oligosaccharides) for the prevention of FA in patients at risk of developing allergic diseases in the long-term period. Conclusion . At present, fecal microbiota transplantation is promising method for FA treatment. Polysaccharides fermented by the microflora, are also actively studied. Experimental studies and clinical trials are required to obtain substantiated conclusions about feasibility of these methods for treatment and prevention of FA., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

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.

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg^(2+)-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg^(2+) ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

In mammalian cells, base excision repair (BER) is the main pathway responsible for the correction of a variety of chemically modified DNA bases. DNA packaging in chromatin affects the accessibility of damaged sites to the enzymes involved in repair processes. This review presents data concerning the enzymes involved in BER. Within the nucleosome core particle (NCP), the accessibility of damaged DNA to enzymes is hindered by the presence of a histone octamer. This means that the removal of DNA lesions largely depends on their rotational and translational positioning in the NCP, as well as on the specific features of each enzyme.

Binase is an extracellular guanyl-preferring ribonuclease from Bacillus pumilus. The main biological function of binase is RNA degradation with the formation of guanosine-2',3'-cyclic phosphate and its subsequent hydrolysis to 3'-phosphate. Extracellular RNases are believed to be key agents that affect the functional activity of the body, as they directly interact with epithelial and immune cells. The biological effects of the enzyme may consist of both direct RNA degradation, and the accumulation of 2',3'-cGMP in the human body. In this work, we have performed a comparative analysis of the cleavage efficiency of model RNA substrates, i.e., short hairpin structures that contain guanosine at various positions. It has been shown that the hydrolysis efficiency of the model RNA substrates depends on the position of guanosine. We have also demonstrated the influence of various divalent metal ions and low molecular weight nucleotide compounds on the binase-catalyzed endoribonucleolytic reaction.

The Aim: Estimate of efficiency of newborns with severe birth asphyxia treatment using systemic therapeutic hypothermia., Materials and Methods: a retrospective open-controlled clinical research with a resolution of the Ethics;Committee performed in 33 neonates born in asphyxia and treated at the NICU All newborns due to the presence of indications, overall controlled hypothermia was conducted according to the approved protocol, using the apparatus Allon 2001"., Results: The neurological status at admission: Sarnat II was detected in 60.6% (20) children; Sarnat III was detected in 39.4% (13) children. The correlation ofApgar scores with the most severe hypoxic ischemic encephalopathy. Pupillary reflexes admission absent in 48.5% (16) newborns. Convulsions within first hours of lfe were observed in 57.6% (19) newborns. Convulsions within I day of life were observed in 81.8 % (27) newborns. The correlation of the amniotic infection (AI) and meconium aspiration syndrome (MAS) treatment using high-frequency mechanical ventilation (p<0,05) and prolongation of mechanical ventilation for more than 5 days (p<0,01). At the beginning of systemic hypothermia fraction of inspired oxygen (FiO2), mean airway pressure (MAP), respiratory rate (RR) were down to the 12th hour of life and reaches a minimum at the end of the third day. These trends have been observed in children with MAS. The correlation of the use of high-frequency mechanical ventilation using high doses ofcardiotonics and the transition to the infusion of epinephrine or norepinephrine (p <0.01).There were no deaths in the studying group., Conclusions: 1. Reliable predictor of the clinical outcomes severity of hypoxic-ischemic encephalopathy (HIE) by the end of the first month lf is the presence of convulsions within the first hour of lf (p<0. 03). 2. When comparing the evaluation on Apgar scale with the data of acid-base balance and severity of HIE a significant correlation between the estimation at the first minute is I point and at the fifth minute 3 point and more severe pH shift, base deficiency, hyperlactatacidosis and severe HIE. 3. MAS and AI are the most frequent accompanying diseases that complicates the severity of newborn status causing prolonged artificial ventilation and the infusion ofcardiotonics. 4. There is a decrease in all parameters of artficial lung ventilation to the twelfth hour life in early systemic hypothermia and reached minimum by the end of the third day.

Food allergy to peanuts and nuts is an actual problem of practical health care, associated with significant prevalence of this disease, severe clinical symptoms and difficulty of diet organization. Purpose of the study--to study the prevalence of food allergy to peanut and hazelnut in Russian children, the investigation of clinical characteristics of this disease, and the mechanisms of sensitization to allergen components. The cross-sectional study was performed in the framework of the EuroPrevall (No FP6-2006-TTC-TU-5 Proposal 045879). The first stage was performed in random samples of primary schoolchildren aged 7-10 years (n = 13 010) from the Tomsk Region, Russia using a standardized questionnaire. The case-control sample was recruited for the second stage (n = 1288). Thus who reported adverse reactions to food in the screening stage were considered as cases (n = 652), children without reported reactions were controls (n = 636). The case-control stage included the completion of a clinical questionnaire, skin-prick test (ALK-Abelly, Spain), serum specific IgE measurement and component-resolved diagnostic: IgE measurement of allergen components of peanut (Ara h1, Ara h26, Ara h34, Ara h8), hazelnut (Cor a1, Cor a8, Cor a11) and birch allergen Bet v1 (ImmunoCAP, Phadia, Sweden). The prevalence of food allergy to peanut and hazelnut in children aged 7-10 years in the Tomsk region is 0.08 and 0.09%, respectively. The manifestation of the food allergy to nuts occurs in the preschool years, main reactions associated with allergy to nuts were oral allergy syndrome (75-80%), gastrointestinal disorders (60-80%) and itching skin rash (20-50%). Sensitization to birch is significantly correlated with the level of specific IgE to hazelnut (r = 0.53, p < 0.05) and peanut (r = 0.56, p < 0.05). Sensitization to heat-labile proteins peanut Ara h8 (12.3%) and hazelnut Cor a1 (8.8%) (homologues of Bet v1) dominates in the sample of children with food sensitization, that determines the cross-reactivity mechanism in the formation of food sensitization in the studied sample. The prevalence of allergies to peanut and hazelnut in Russia is much lower than in Europe and North America. Sensitization to these foods develops by the mechanism of cross-reactivity with birch pollen allergen. This type of sensitization determines mild clinical symptoms of allergy to hazelnut and peanut.

Aim: To determine the mechanism of food sensitization in children with chronic Opisthorchis felineus invasion., Patients and Methods: During the epidemiological study the groups of patients (7-10 years) with chronic opisthorchiasis (n = 237) and children without chronic opisthorchiasis (n = 496) were formed. The investigation included interviewing of parents/guardians, measurement of total IgE, specific IgE to food allergens and component-resolved diagnostic, real-time PCR in stool samples., Results: The chronic opisthorchiasis invasion in children is associated with reduced risk of food sensitization, compared with non-infected group (9.7 vs 16.94%, OR = 0.53; 95% CI 0.31-0.88; p = 0.01). Opisthorchiasis of high intensity negatively correlated with increased level of specific IgE to food allergens in serum (OR = 0.46; 95% CI 0.24-0.91; p = 0.023). The association between level of total IgE and intensity of opisthorchiasis combinated with food sensitization was found. The sensitization to allergens class I (cyp c1, cor a11, gal d2, pru p3) is more prevalent in patients with opisthorchiasis, in uninfected children the sensitization to bet v1-homologues (mal d1, pru p1, cor a1, ara h8) is most common., Conclusions: The epidemiological data on the effect of the Opisthorchis felineus invasion on mechanisms of food sensitization in children were obtained.

Accordingly to numerous surveys food allergy affects up to 2-6% of population in different countries and varies due to numerous factors including the prevalence of helminths infestation. The food allergy prevalence data in Russia are based on official medical statistics. The study was performed as part of "The Prevalence, Cost and Basis of Food Allergy Across Europe" (EU funded project No FP6-2006-TTC-TU-5 Proposal 045879 EuroPrevall). The aim is to create the concept offood allergy in children in opisthorchiasis endemic area based on epidemiological study of the prevalence, risk factors and clinical features. The main objective of the study will be the working out of the food safety regulations. This article describes the study design and its methodology.

Genome-wide association studies are currently considered as one of the most powerful tools to establishing the genetic basis of complex diseases. A number of such studies were carried out for allergic diseases; however, in Russian population this analysis has not been performed so far. For the first time, we performed genome-wide association study of allergic diseases in Russian inhabitants of Western Siberia. Two new loci associated with childhood bronchial asthma were identified (20q13.12, rs2425656, P = 1.99 x 10(-7); 1q32.1, rs3817222, rs12734001, P = 2.18 x 10(-7) and 2.79 x 10(-7), respectively) as well as one locus, associated with allergic rhinitis (2q36.1, rs1597167, P = 3.69 x 10(-7)). Genes located in the loci, YWHAB and PPP1R12B for asthma and KCNE4 for allergic rhinitis, are new genes for these diseases. It was found that BAT1 (6p21.33), MAGI2 (7q21.11) and ACPL2 (3q23) genes are, likely, common (syntropic) genes of allergic disease and a topic sensitisation. It was shown that RIT2 (18q12.3) and (5q31.1) genes can be involved in the control of lung function. The results of the study enlarge the body of data on genetic factors of allergy and expand the list of genes underlying these diseases.

Opisthorchiasis is one of the significant naturofocal diseases in Russia. The diagnosis of opisthorchiasis may mask some diseases, such as opisthorchiasis, metorchiasis, and clonorchiasis - biohelminthoses that are induced by various representatives of the family Opisthorchiidae-Opisthorchis felineus/O.viverrini, Metorchis bilis, and Clonorchis sinensis, respectively. Coproovoscopy and serologic methods fail to accurately define the species-specific affiliation; in this connection the identification of opisthorchids, by using DNA diagnostic techniques, becomes urgent. The present paper gives the results of development of DNA diagnosticum, which differentiates parasitic diseases induced by O. felineus and M. bills. The ribosomal RNA gene cluster fragment incorporating internal transcribed spacer 2 (ITS2) was used as a diagnostic marker. A system for diagnosing opisthorchiasis was developed as a multiplex polymerase chain analysis and tested on 37 patients infected with various species of opisthorchids.

Tyrosine derivatives labeled with a short-living fluorine 18 isotope (T(1/2) 110 min), namely 2-[(18)F]fluoro-L-tyrosine (FTYR) and O-(2'-[(18)F]fluoroethyl)-L-tyrosine (FET), promising radiopharmaceutical products (RPP) for positron emission tomography (PET), were obtained by asymmetric synthesis. Accumulation of FTYR and FET in the rat tumor "35 rat glioma" and in abscesses induced in Vistar mouse muscles was studied and compared with that of a well-known glycolysis radiotracer 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG). It was shown that the relative accumulation indices of amino acid RPP were considerably lower than those of FDG. At the same time, tumor/muscle ratios were high enough (2.9 for FET and 3.9 for FTYR 120 min after injection) for reliable tumor visualization. The data obtained indicated a possibility in principle to use FTYR and FET for differentiated PET diagnostics of brain tumors and inflammation lesions. Of the tyrosine derivatives studied, FET seems to be the most promising agent due to a simple and easily automated method of preparation based on direct nucleophilic substitution of the leaving tosyloxy group of an enantiomerically pure Ni-(S)-BPB-(S)-Tyr(CH2CH2OTs) precursor by an activated [(18)F]fluoride.

Catalytic oxidative modification of single-stranded DNA with hydrogen peroxide and molecular oxygen in the presence of a conjugate containing an oligonucleotide complementary to the DNA fragment and tetra-4-carboxyphthalocyanine Fe(II) was studied. The conjugate examined was found to be active in the reaction of oxidative DNA cleavage in the presence of hydrogen peroxide, like earlier studied oligonucleotide conjugates containing tetra-4-carboxyphthalocyanine Co(II) and 2,4-di-[2-(2-hydroxyethyl)]deuteroporphyrin IX Fe(III) metallocomplexes generating active oxygen forms. The new conjugate was more active in the case of oxidation with molecular oxygen. Kinetic regularities and optimal regimes of DNA oxidation with hydrogen peroxide were found.

The cobalt(II) tetracarboxyphthalocyanine-deoxyribonucleotide pd(TCTTCCCA) conjugate was synthesized. The phthalocyanine N-succinimide ester prepared from phthalocyanine using DCC was mixed in DMF with an aqueous solution of the oligonucleotide bearing a 1,3-diaminopropane linker at the 5'-phosphate. The resulting conjugate was tested in the intraduplex reaction with target 14-mer and 22-mer oligonucleotides containing conjugate-complementary sequences. In the presence of O2 and a thiol (2-mercaptoethanol or DTT) as a coupled reducer or H2O2, sequence-specific DNA modification was observed that caused the cleavage of the target upon treatment with piperidine.

The kinetics of photomodification of oligodeoxyribonucleotide pd(GTGTGA) with a derivative of the complementary oligodeoxyribonucleotide pd(CACACA) bearing a 3-(n-azidotetrafluorobenzoylamino)propylamine residue (ArN3) at the terminal phosphate group was studied at 20 degrees C. It was found that the target's G3 residue is preferentially modified. Along with the transformation of the arylazide moiety, degradation of the oligonucleotide fragment of the reagent occurred with a partial loss of affinity. With the use of the reagent labeled at the 5'-end, sites of photomodification were found. From the dependence of the modification level on the reagent concentration at the initial time of irradiation, the association constant (Kx = (1.40 +/- 0.24) x 10(5) M-1) was determined. From the dependence of the modification level on the concentration of the pre-irradiated reagent, the constant of the transformed reagent-target association in solution (Kr = (2.49 +/- 0.30) x 10(4) M-1) was determined. From the time-dependence of the modification level [PZ]/P0, the rate constant for the limiting step of photomodification (k0 = (5.31 +/- 0.28) x 10(-4) S-1) and the modification efficiency of the target in the complex with the reagent (gamma = 1) were found.

Electrophoretic analysis of the products of chemical destruction at modified base residues was used to determine the site-directedness of self-alkylation of the 26-mer DNA fragment pTTGCCTTGAATGGGAAGAGGGTCATT (T26). This fragment possesses a 4-[N-methyl-N-(2-chloroethyl)amino]benzylamido group (CIR-), covalently attached to the 5'-terminal phosphate group both in the presence and in the absence of the oligonucleotide effector (Phn-L)pTGACCCTCp(L-Phn), where Phn is an N-(2-hydroxyethyl)phenazinium residue and L is an ethylenediamine linker. Molecular modeling with the method of molecular mechanics/dynamics (MM/D) was used to investigate the secondary structure of the CIR-T26 conjugate and to interpret the change of the alkylation site upon treatment with CIR-T26 in the presence of an effector.

Kinetics of photomodification of 26-meric deoxyribonucleotide pTTGCCTTGAATGGGAA-GAGGGTCATT with derivatives of the complementary oligonucleotides pTCTTCCCATTC, pTCTTCCCA, and pTTCCCA bearing a residue of (p-azidotetrafluorobenzoyl)aminopropylamine(-ArN3) attached to the terminal phosphate (reagents I, II, and III, respectively) was studied at 37 degrees C. It was established that during irradiation the reagents are inactivated, loosing their affinity to the target. A kinetic equation describing the modification was suggested. From the dependence of the time-limited modification level on the reagent concentration, the association constants of the reagents with the target were determined: [Kx = (9.9 +/- 0.4) x 10(4), (1.1 +/- 0.1) x 10(5), and (8.4 +/- 2.1) x 10(6) M-1 for reagents I, II, and III, respectively] and the efficiency of the modification in the complex gamma ef (ca. 0.3 for all the reagents) were determined. From the dependence of the modification level [PZ]/p0 on time for reagent II, the rate constant was determined for the rate-determining step of the photomodification k0 = (7.9 +/- 0.9) x 10(-3) s-1, which is close to the rate constant for the photolysis of p-azidotetrafluorobenzoic acid kp = (5.5 +/- 0.3) x 10(-3) s-1.

Quantitative characteristics of the modification of deoxyribooligonucleotide TTGCCTTGAATGG-GAAGAGGGTCATT (P) with 4-(N-2-chloroethyl-N-methylamino)benzyl phosphamide derivative of oligonucleotide pTTCCCA (X) were studied. The modification was performed in the presence of derivatives of the oligonucleotides (Phn-L)pTTCAAGGCp(L-Phn) (E1) and (Phn-L)pTGACCCTCp(L-Phn) (E2), where Phn is the residue of N-(2-hydroxyethyl)phenazinium, and L is ethylene diamine spacer. In PXE1, PXE2, and PXE1E2 complexes, E1, E2, and reagent X are bound with target P in tandem, with E1 near the 3'-end and E2 near the 5'-end of the reagent X. From the dependences of the maximum in time modification degree of target P and the shorter targets containing the complementary binding site for the reagent X on its concentration, the association constants of the complexes PX, PE1, and PE2 were determined as Kx = (4.2 +/- 0.6) x 10(4) M-1, Ke1 = (1.25 +/- 0.44) x 10(7) M-1, and Ke2 = (2.56 +/- 1.22) x 10(6) M-1, respectively. The cooperativity coefficients of joint binding the X, E1, and E2 with the target giving rise to the complexes PXE1, PXE2, and PXE1E2 were estimated as alpha 1 = 15.7 +/- 2.1, alpha 2 = 8.7 +/- 1.2, and alpha 12 = 136.5 +/- 2.6, respectively. The data obtained suggest that E2 is not only the effector of modification but it is also an inhibitor due to the formation of the complex PE2* with Ke2* = (1.97 +/- 1.27) x 10(7) M-1 not capable of adding the reagent X.

Site directed alkylation of three oligonucleotide targets: 41-mer (hairpin structure), 22-mer (loop part of this hairpin) and 10-mer (part of the loop) with 5'-p-(N-2-chloroethyl-N-methylamino)benzylamides of oligonucleotides complementary to the loop region was studied. Thermodynamic parameters of the interaction were estimated using the dependence of the limit modification extent on the reagent concentration at different temperatures. The stability of the complex increases much in the set: 302-mer carrying the above hairpin, 41-mer, 22-mer; data on 22-mer and 10-mer being almost identical. This indicates significant influence of the loop supporting structure on the interaction with antisense reagents.

We studied the effect of different factors (reagent concentration, temperature, presence of oligonucleotide-effector (3',5'-diphenazinium derivative of oligodeoxyribonucleotide) stabilizing duplex RNA.reagent) on the selectivity of the site-directed modification of 16S rRNA with 2,3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]-benzylidene derivative of oligonucleotide p(dTTTGCTCCCC)rA (reagent I) under conditions of secondary structure stability. The constant of cooperative binding of the reagent and oligonucleotide-effector with 16s rRNA was determined. The temperature rise from 20 to 40 degrees C brought about a 1.5-fold increase in the relative extent of modification at the target site 771-781. In the presence of oligonucleotide-effector, which is a full complementary copy of the 782-789 fragment of 16S rRNA (reagent concentration is 1 x 10(-6) M), the selectivity of the RNA modification at the target site is doubled and a high level of the modification is retained. When the reagent concentration in the reaction mixture was decreased down to 1 x 10(-7) M, the same level of selectivity was achieved without the oligonucleotide-effector. Under these conditions, however, a drastic (20-fold) drop of the level of the 16S rRNA alkylation was observed.

Effects of Cephalotaxus alkaloids (homoharringtonine and cephalotaxine) on the translation of endogenous mRNA in a cell-free system of rabbit reticulocyte lysate and on poly(U)-directed poly(Phe) synthesis on human placenta ribosomes was studied. The effect of the alkaloids on the activity of human placenta ribosomes in a template-dependent aminoacyl-tRNA binding, N-acetyl-phenylalanyl-puromycin and diphenylalanine formation was also studied. Homoharringtonine was shown to have little effect of codon-dependent Phe-tRNA(Phe) binding but the alkaloid strongly inhibited (Phe)2 formation as well as N-Ac-Phe-puromycin synthesis from the complex N-Ac-Phe-tRNA(Phe).poly(U).80S ribosomes. It was concluded that the site of homoharringtonine binding overlaps or coincides with the acceptor site of the ribosomal peptidyltransferase center. The association constant of homoharringtonine to the ribosomes was estimated to be (4.8 +/- 1.0) x 10(7) M-1. Cephalotaxine had no effect on the elongation steps.

A hairpin-shaped oligodeoxyribonucleotide d(pTTGGCACGAGCAGCCAA) (I) was alkylated with the reagent d(TTGGG) greater than UCHRCl (RCl = -C6H5-N(CH3)-CH2-CH2Cl) complementary to the hairpin's stem. Thermodynamic parameters for the hairpin structure estimated from melting curves were: delta Hh = -125 +/- 17 kJ/mol, delta Sh = -380 +/- 84 J/mol.K; and for the reagent - target complex delta Hpx = -155 +/- 8 kJ/mol, delta Spx = -427 +/- 21 J/mol.K. Effective constants of association Kx of the oligonucleotide with the reagent were determined at 30 and 50 degrees from the concentration dependence of the reaction yield and were 1988 +/- 83 and 1239 +/- 58 M-1, respectively. Experimental values of Kx agreed with the values of Kx = Kpx/(1 + Kh), calculated with the use of the thermodynamic parameters.

Hemin was attached covalently to the 5'-terminus of the 14-mer d(pTGACCCTCTT.CCC)rA and was shown, in the presence of oxygen and a reducing agent, to be active in the cleavage of the complementary sequence (position 261-274) in a 303 nucleotide-long DNA fragment. The yield of the cleavage products reached approximately 50%, the cleavage locus comprising two bases (G275 and G276).