[Dimeric bisberzimidazoles: cytotoxicity and effects on DNA methylation in normal and cancer human cells].

Cancer cells are characterized by the hypermethylation of promoter regions of tumor suppressor genes. DNA methyltransferase inhibitors cause re-activation of these genes that allows considering DNA methyltransferases as targets for anticancer therapy. As it was previously shown by us, dimeric bisbenzimidazoles, DB(n), differing in length of the oligomethylene linker between the two bisbenzimidazole fragments (n--number of methylene groups in linker) effectively inhibit the methylation of DNA duplexes by murine methyltransferase Dnmt3a. Here, the cytotoxicity of some of these compounds, their penetration into cells and influence on the methylation of genomic DNA in fetal lung fibroblasts line F-977 and cervical cancer cells HeLa have been studied. In the 0-60 microM concentration range, only the DB(11) displayed a significant toxic effect on the normal cells, whereas the effect of DB(n) investigated on the cancer cells was not significant. Interestingly, the DB(1) and DB(3) to a small extent stimulate the proliferation of HeLa and F-977 cells, respectively. DB(1) and DB(3) display ability to penetrate into the nucleus of HeLa and F-977 cells and accumulate in various parts of the nuclei. DB(11) is not able to penetrate into the nuclei of these cells. The incubation of F-977 cells with 26 microM of DB(1) or DB(3) led to a decrease of the methylation of 18S rRNA gene, which is located in the region of DB(1) and DB(3) accumulation. A similar effect produces the same concentration of DB (3) in the F-977 cells. However, the overall level of genomic DNA methylation was not changed. These data suggest that DB(n) can be directed to act on specific genes demethylation and in the future may selectively inhibit the proliferation of cancer cells.

[Multidrug resistance P-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1].

Mitochondria-targeted antioxidants of the SkQR1 family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQRI is ejected from chemotherapy-resistant cells by P-glycoprotein - one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQRI from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.

Chimeric lactase capable of spontaneous and strong immobilization on cellulose and development of a continuous-flow system for lactose hydrolysis at high temperatures.

Recombinant plasmids containing fusion proteins composed of two different modules were constructed and expressed in Escherichia coli. The modules encoded the lactase LacA (LacZ) from the thermophilic bacterium Thermoanaerobacter ethanolicus and the cellulase CelD, a cellulose-binding module (CBM) from Anaerocellum thermophilum. The CelD CBM provides a spontaneous and strong sorption of the fusion proteins onto a cellulose carrier. The enzymatic activities of both the free LacA protein and LacA-CelD CBM fusion proteins immobilized onto the cellulose carrier were assessed. The LacA activity of the fusion protein was dependent upon its position with respect to the CBM. The highest level of lactase activity and stability was observed when the lactase domain was localized at its N terminus. A continuous-flow column reactor of lactase immobilized on a cellulose carrier was constructed, and its activity was assessed. The lactose hydrolysis rate for a 150 mM (5%) solution at a flow rate of 1 reactor volume per min was 75%, which is a value optimal for further whey transformation into glucose/galactose syrup.

[HIV-1 integrase inhibition by dimeric bisbenzimidazoles having different spacers].

HIV-1 integrase is responsible for one of the key steps of the viral replication, integration of the viral cDNA into the host cell genome. Integration inhibition leads to complete block of the virus replication. In this study inhibition of integration by dimeric bisbenzimidazoles DBBI(7) with heptamethylene and DBBI(8) with tri(ethylene glycol) spacers was examined, and it was learned out that IC50 for DBBI(7) was about 0.03 microM, and IC50 for DBBI(8) was about 10 microM. By using cross-linking assays, it was shown that both compounds impeded a proper disposition of DNA-substrate at the active centre of integrase. Dissociation constants for complexes between either DBBI and DNA-substrate of integrase were determined. Calculated Kd values were 270 nM and 140 nM for complexes formed by DBBI(7) and DBBI(8), respectively. Therefore, inhibition of integration does not directly result from the binding of DBBIs with DNA. The dependence of initial rates of enzymatic reaction on the DNA-substrate concentration in presence of different concentrations of inhibitors was found, and inhibition constants were determined. All the data obtained allow us to suppose that the different inhibition activity of DBBI(7) and DBBI(8) results from the different mechanism of their binding: DBBI(7) is a competitive inhibitor of integrase whereas DBBI(8) is assumed to show a more complex mechanism of inhibition.

[Multidrug resistance p-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1].

Mitochondria-targeted antioxidants of the SkQRI family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQR1 is ejected from chemotherapy-resistant cells by P-glycoprotein--one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQR1 from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.

[Determination of the qualitative and quantitative composition of antocyan pigments as components of dietary supplements and drugs for vision].

Bilberry has been long used in folk medicine and credited for an ability to improve vision, primarily night vision. The major active ingredients of bilberries are antocyans. Experimental and clinical studies confirmed the ability of bilberry antocyans to accelerate the regeneration of the photosensitive pigment rhodopsin, to improve nutrition of the retina, and to restore the tissue mechanisms of its protection. The authors studied the level of bilberry antocyans in 5 samples of dietary supplements and medicines for eyes, which had been bought in Moscow drugstores. The total content of antocyans was determined by pH-differential spectrophotometry. All the test samples were shown to contain antocyan pigments; however, their concentration in different samples varied in a wide range of 0.01 to 4.2%. The maximum content was found in the drug "Focus". The qualitative composition of antocyan pigments was estimated by reverse-phase high performance liquid chromatography. All the test samples other than Vitrum vision forte turned out to contain just bilberry antocyans. The chromatographic profile of a Vitrum vision forte sample was inconsistent with bilberry antocyan pigments and the agent was likely to have another source.

[Interactions of HIV-1 DNA heterocyclic bases with viral DNA]. / Izuchenie vzaimodeistvii geterotsiklicheskikh osnovanii DNK VICH-1 s virusnoi integrazoi.

Human immunodeficiency virus type 1 integrase is one of three viral enzymes, and it realizes a key process of the viral replication cycle, i.e. viral DNA integration into infected cell genome. Integrase recognizes nucleotide sequences located at the ends of the viral DNA U3 and U5 LTRs and catalyzes 3'-processing and strand transfer reactions. To study the interactions between integrase and viral DNA at present work, we used modified integrase substrates mimicking the terminal U5 LTR sequence and containing non-nucleoside insertions in one or/and both strands. It is shown that the substrate modifications have no influence on the integrase binding rate, while the heterocyclic bases removal in the 5th and 6th substrate positions and in the 3rd position of the substrate processed strand distinctly inhibits the integrase catalytic activity. This fact demonstrates these bases significance for the active enzyme/substrate complex formation. On the contrary, modification of the 3rd position within substrate non-processed strand stimulates 3'-processing. Since heterocyclic base elimination results in disruption of the DNA complementary and staking interactions, this result shows that DNA double helix destabilization close to the cleaved bond promotes the 3'-processing.

[Preparation of DNA-duplexes, containing internucleotide thiophosphate groups in various positions of the recognition site for the EcoRII restriction endonuclease]. / Poluchenie DNK-dupleksov, soderzhashchikh v razlichnykh polozheniiakh uchastka uznavaniia éndonukleazy restriktsii EcoRII khiral'nye mezhnukleotidnye tioforfatnye gruppy.

Twenty-four 12-mer DNA duplexes, each containing a chiral phosphorothioate group successively replacing one of the internucleotide phosphate groups either in the EcoRII recognition site (5'CCA/TGG) or near to it, were obtained for studying the interaction of the restriction endonuclease EcoRII with internucleotide DNA phosphates. Twelve of the 12-mer oligonucleotides were synthesized as Rp and Sp diastereomeric mixtures. Six of them were separated by reversed-phase HPLC using various buffers. Homogeneous diastereomers of the other oligonucleotides were obtained by enzymatic ligation of the Rp and Sp diastereomers of 5- to 7-mer oligonucleotides preliminarily separated by HPLC with the corresponding short oligonucleotides on a complementary DNA template. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

[Synthesis of modified oligonucleotides and use of a duplex from them with covalently bound chains]. / Sintez modifitsirovannykh oligonukleotidov i poluchenie iz nikh dupleksa s kovalentno sviazannymi tsepiami.

A method for the synthesis of DNA duplex with covalently linked strands was elaborated, and the thermal and hydrolytic stability of the duplex was studied. The strands were connected via an amide bond between carboxyl and aliphatic amino groups in the presence of water-soluble carbodiimide. For this purpose, a series of modified 5- to 26-mer oligonucleotides with primary amino or carboxyl group were prepared, and their properties were investigated.

[Synthesis of modified oligodeoxyribonucleotides containing 9-(2'-amino-2'-deoxy-beta-D-arabinofuranosyl)adenine]. / Sintez modifitsirovannykh oligodezoksiribonukleotidov, soderzhashchikh 9-(2'-amino-2'-dezoksi-beta-D-arabinofuranozil)adenin.

A method for preparing a modified derivative of 2'-amino-2'-deoxy-arabino-adenosine ready for directed insertion into an oligonucleotide chain during solid-phase synthesis was elaborated. A series of the title oligonucleotides (6-25-mers) containing a 2'-amino-2'-deoxy-arabino-adenosine fragment were prepared. A high reactivity of the 2'-amino group during the acylation with carboxylic acid anhydrides was demonstrated. It was shown that the insertion of the modified fragments into oligonucleotides did not inhibit the formation of the DNA duplex.

[Oligodeoxyribonucleotides containing 1-(beta-D-3'-deoxy-threo-pentofuranosyl)pyrimidine chains]. / Oligodezoksiribonukleotidy, soderzhashchie 1-(beta-D-3'-dezoksi- treo-pentofuranozil)pirimidinovye zven'ia.

Effective methods of multiple incorporations of nucleotides with the inverted configuration of C2' hydroxyl group have been developed. The presence of tU and tC as 3'-terminal oligonucleotide modifications is shown to increase their resistance to snake venom phosphodiesterase. The obtained compounds are promising for the use in antisense biotechnology.

[Antisense oligonucleotides containing 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine at the 3'- and 5'-end]. / Antisensovye oligonukleotidy, soderzhashchie po 3'- i 5'-kontsam 1-(beta-D'-dezoksi-treo-pentofuranozil)timin.

Oligonucleotides with 3'- and 5'-terminal 1-(beta-D-2'-deoxy-threo-pentafuranosyl)thymin residues (inversion at C3'-atom) were obtained by the automatic phosphoramidite synthesis. The modifications protect oligonucleotides from the nuclease degradation and do not affect the stability of nucleic acid duplexes.

[Interaction of RNAase H from E. coli with modified hybrid duplexes. I. Duplexes modified at the carbohydrate residue]. / Vzaimodeistvie RNKazy H iz E. coli s modifitsirovannymi gibridnymi dupleksami. I. Dupleksy, soderzhashchie modifikatsii po uglevodnomu ostatku.

The influence of oligodeoxyribonucleotide probes containing 1-(D-beta-2'-deoxythreo-pentofuranosyl)thymine or 1-(D-beta-2'-deoxy-2'-fluoro-pentofuranosyl)uracil on the ability of the hybrid duplexes to interact with RNase H from E. coli was studied. A kinetic approach was used to measure of the modification effect. The hybrid duplex, prA18/d(TTflU)6TT, was shown not to interact with RNase H, whereas prA18/d(xTTT)6 inhibited the RNase H activity (Ki = 0.67 mkM). The thermostability of the modified duplexes was estimated. The present technique may lead to the use of some modified oligonucleotides as antisences.

[Synthesis and properties of DNA duplexes with covalent bonds between cross-links]. / Sintez i svoistva DNK-dupleksov s kovalentnymi sviaziami mezhdu tiazhami.

Synthesis of cross-linked modified DNA duplexes is described. The structure of the duplexes was confirmed by digestion with the AluI restriction endonuclease. Thermostability and resistance to enzymatic hydrolysis of the cross-linked duplexes were studied.

[Solid phase synthesis and chromatographic characteristics of nucleophilic agents conjugated with oligonucleotides containing 5'-terminal carboxyl group]. / Kon''iugaty oligonukleotidov, soderzhashchikh 5'-kontsevuiu karboksil'nuiu gruppu, s razlichnymi nukleofil'nymi agentami: tverdofaznyi sintez i khromatograficheskoe povedenie.

Conjugates of amines or short peptides with oligonucleotides containing 5'-terminal carboxyl group were prepared by solid phase chemical synthesis. A correlation between the physicochemical parameters and retention times of the synthesized conjugates was established by ion-pair reversed-phase HPLC.

[Oligodeoxyribonucleotides containing 2'-amino-2'-deoxypyrimidine nucleosides]. / Oligodezoksiribonukleotidy, soderzhashchie 2'-amino-2'-dezoksipirimidinovye nukleozidy.

The synthesis, by means of the standard phosphoramidite chemistry, of modified oligodeoxynucleotides (5-20 residues) containing 2'-amino-2'-deoxypyrimidine nucleosides has been carried out, and their ability to form duplexes with complementary DNA has been investigated. A high reactivity of such compounds in N-acylation was shown.

[Theoretical approaches for evaluation of DNA-protein interactions]. / Teoreticheskie podkhody k otsenke spetsificheskogo DNK-belkovogo vzaimodeistviia.

The notion of the DNA-recognizing segment of a protein for three-dimensional structures of DNA-protein complexes was formalized. Algorithms for calculating two parameters, hydrogen bonds and hydrophobic contacts, that characterize the interaction of the DNA-recognizing segment of the protein with the groove DNA major were proposed. DNA-recognizing protein segments in three-dimensional structures (of complexes) were classified according to these two parameters. These data were compared with the classification of the corresponding DNA-recognizing domains according to structure. The contribution of each pair amino acid residue-base to the interaction of protein with the DNA major groove was calculated.

[Kinetic modeling of the mechanism of allosteric interactions of restriction endonuclease EcoRII with two DNA segments]. / Kineticheskoe modelirovanie mekhanizma allostericheskogo vzaimodeistviia éndonukleazy restriktsii EcoRII s dvumia uchastkami DNK.

The effect of correlations between kinetic parameters of two inducible substrates on allosteric activation of EcoRII endonuclease hydrolysis of one substrate was studied. The pairs of DNA duplexes were constructed that were the substrates of EcoRII restriction endonuclease or their analogs and had different kinetic constants of interaction with the enzyme; the effects of their concentrations on mutual hydrolysis induction were studied. A kinetic mechanism is suggested considering the allosteric effects of two DNA recognition sites on dimeric molecule of EcoRII. Mathematic modeling was used to analyze the kinetic mechanism and evaluate optimal characteristics of the inductor. Thus, activation increases when (i) substrate concentration decreases, (ii) enzyme binding of two inductor or substrate molecules decreases, (iii) binding of one substrate molecule increases versus binding of one inductor molecule, and (iv) kcat of the enzyme-substrate complex including on substrate and one inductor increases.

[Hybridase cleavage of RNA. III. Use of UV-spectroscopy for studying the kinetic properties of E. coli RNAase properties]. / Gibridaznoe rasshcheplenie RNK. III. Ispol'zovanie UF-spektroskopii dlia izucheniia kineticheskikh svoistv RNKazy N iz E. coli.

A one-step procedure for estimating the activity of ribonuclease H from E. coli has been developed. This method is based on continuous registration of the increment in the UV adsorption of the substrate in the course of the enzymatic reaction. The heteroduplex Am.dT20 (m = 18-24) was found to be the optimal substrate for the enzyme. A comparative analysis of the rates of the enzymatic reaction as determined by UV spectroscopy and ion-pair HPLC was carried out. The kinetic parameters of the Am hydrolysis in Am.dT20 catalyzed by E. coli RNase have been determined for the first time (Km = 44 +/- 11 nM, Vmax = 0.0363 +/- 0.0053 E). The method sensitivity is 0.01-0.05 E which makes it possible to determine the RNAse H within the concentration range of 0.5-2.5 u./ml.