Protective effect of exogenous 8-oxo-2'-deoxyguanosine on Drosophila melanogaster larval stages under heat shock.

The influence of exogenous 8-oxo-2'-deoxyguanosine on the development of Drosophila melanogaster under normal conditions, and under the influence of short-term heat shock was studied. It was shown that 8-oxo-2'-deoxyguanosine was not toxic at concentrations of up to 1 µM. A tendency to accelerate larval development and fly emergence was observed under the influence of this compound in our experiments. Short-term heat shock causes a 50-80% decrease in the number of larvae that complete development. The addition of exogenous 8-oxo-2'-deoxyguanosine to the food before thermal influence negates this effect and brings the levels of the imago emergence indicators back to the basal level. The obtained results are further evidence of the possible bioregulatory and adaptogen functions of 8-oxo-2'-deoxyguanosine.

SkBQ - prooxidant addressed to mitochondria.

Oxidative stress and mitochondrial dysfunction are the key links in the chain of development of pathologies associated with the violation of cellular energy metabolism. Development of mitochondria-addressed compounds highly specific for chemical processes is one of the most promising ways to develop approaches to the treatment of inherited and age-related diseases with mitochondrial etiology. Correlation of structure and chemical activity of the test compounds from a class of lipophilic cations revealed the key role of substituents in the aromatic ring of 1,4-benzoquinones in the manifestation of high antioxidant properties. In this work, it is shown that a synthesized benzoquinone derivative conjugated in position 6 with membrane-penetrating cation of decyltriphenylphosphonium and with substituents at position 2, 3, and 5 (SkBQ) has much lower antioxidant and significantly higher prooxidant activity in comparison with similar derivatives of plasto- and toluquinone SkQ1 and SkQT1 in experiments on isolated mitochondria. At the same time, SkBQ, like SkQ1 and SkQT1, can be reduced by the respiratory chain in the center i of complex III and decrease the mitochondrial membrane potential. In cell cultures of human fibroblasts, it was revealed that SkBQ does not protect cells from apoptosis induced by hydrogen peroxide. Under the same conditions, SkQ1 and SkQT1 exhibit a powerful protective effect. Thus, SkBQ can be seen as a mitochondria-addressed prooxidant. The possibility of using SkBQ as an anticancer drug for the treatment of cancers such as prostate cancer whose cells are sensitive to mitochondrial reactive oxygen species is discussed.

[8-oxo-2'-deoxyguanosine accumulation in DNA from "stationary phase aging" cultured cells].

The 8-oxo-dG/dG ratio in DNA of cultured transformed Chinese hamster cells was analyzed during their "stationary phase aging". Amount of 8-oxo-dG and dG in DNA hydrolyzate was evaluated by HPLC-EC. The cells grew and "aged" for 15 days. As expected, the 8-oxo-dG/dG ratio increased with cell "age". It did not change significantly from 4th to 8th day (6.26 x 10(-5) and 4.42 x 10(-5), correspondingly) and then abruptly increased to 15th day of "age" (22.40 x 10(-5)). The results are in accordance with the conception of cell proliferation restriction as the starting mechanism of ageing and the method can be used for evaluation of cell culture biological age when testing new compounds for their geroprotector or geropromoter activity.

[Potential geroprotector properties of the extract of reindeer antler powder in experiments on cultured cells].

Preparations from deer antlers are well known by their multiple medicinal properties. In particular, their health-giving effect on senescing organism has been repeatedly shown. In the study we investigated effect of water extract of reindeer mature antler powder (ERAP) on the kinetics of growth and "stationary phase aging" of HeLa (clone 11) cell line. Cell suspension was placed in the wells of 24-well plastic tissue culture plates with seeding density of 15 10(3)/cm2. The growth medium contained ERAP at 0, 10 or 100 microl/ml. In every 1-3 days microscopic evaluation of live cell number in the wells has been made. It turned out, that ERAP at 10 microl/ml increased proliferation rate of HeLa cells as well as their saturation density, i.e. acted as a geroprotector. The result was also confirmed by the observed "stationary phase aging" slowing down leading to increase of the "average life span" of cell culture. However, effect of ERAP at 100 microl/ml was different. In that case the evident decrease of cell culture saturation density was observed indicating increase of the culture "biological age". Besides, the cell death began earlier leading to decrease of the "average life span" of the cell culture. We think that ERAP contains some compounds with geroprotector activity as well as some geropromoters, or cell proliferation inhibitors. At the lower ERAP concentration in growth medium content of geropromoter(s) is too low for its effect manifestation and the evident "rejuvenation" of the cell culture is observed. At the higher concentration of ERAP (100 microl/ml) the content of geropromoter(s) reaches the "working" value and this not only masks the effect of geroprotector(s) but also leads to the cell culture "senescence".

[Determination of oligonucleotide molecular masses by MS-MALDI].

MALDI mass spectrometry (MS) of 14- to 42-mer homogeneous oligonucleotides and their mixtures was carried out using a Vision 2000 instrument (Thermo BioAnalysis, Finnigan, United States). Conditions for the determination of oligonucleotide molecular masses were optimized by applying various matrices and operation modes. The most reproducible results with minimal uncontrolled decomposition of the oligonucleotides including their apurinization during the MALDI MS registration were obtained using 2,4,6-trihydroxyacetophenone as a matrix instead of 3-hydroxypicolinic acid, usually employed in the mass spectrometry of oligonucleotides. Our approach allows the determination of molecular masses of oligonucleotides obtained by chemical synthesis and the evaluation of their component composition and purity. It was applied to the mass spectrometric analysis of oligonucleotides containing a 3'-(methyl-C-phosphonate) group or a modified 1,N6-ethenodeoxyadenosine unit.

[Modified oligonucleotides containing 1-beta-D-galactopyranosylthymine: synthesis and substrate properties]. / Modifitsirovannye oligonukleotidy, soderzhashchie 1-beta-D-galaktopiranoziltimin: sintez i substraktnye svoistva.

A convenient method of regioselective introduction of 1-beta-D-galactopyranosylthymine into oligonucleotides was developed and the substrate properties of the modified oligonucleotides were investigated in the enzymic reaction of formation and hydrolysis of internucleotide bonds. The English version of the paper.

[Synthesis of phosphoceramide azidothymidine and phosphoceramide didehydrodeoxythymidine]. / Sintez tseramidfosfoazidotimidina i tseramidfosfodidegidrodezoksitimidina.

The synthesis of two novel lipophosphonucleoside potential antiviral agents, 2-stearoyl-rac-sphinganine-1-phosphoryl-5'-(3'-deoxy-3'-azido)thymidine and 2-stearoyl-rac-sphinganine-1-phosphoryl-5'-(2',3'-didehydro-2', 3'-dideoxy)thymidine, is reported. The phosphoester linkages between the primary hydroxyl group of rac-ceramide and the 5'-hydroxyl group of the corresponding 3'-deoxythymidine derivative were formed using either the H-phosphonate or the phosphite triester method. The H-phosphonate approach was shown to be the method of choice for the synthesis of ceramide phospho-3'-azidothymidine.

[Human tumor necrosis factor mutants: preparation and some properties]. / Mutanty faktora nekroza opukholei cheloveka: poluchenie i nekotorye svoistva.

Using polymerase chain reaction, a number of mutant genes encoding human tumor necrosis factor (TNF-alpha) with amino acid substitutions and a deletion were obtained. The mutant proteins (muteins) contained point mutations R32H, A33S, F144L, I118M, and I118A; double mutation R32H-F144L; and deletion of four amino acid residues 67-70. The mutant genes were expressed in E. coli under the control of constitutive promoters. A simple purification method for the muteins was developed and their physicochemical properties were studied. All the muteins obtained, except F144L and I118A, were shown by CD and cross-linking to from a spatial structure similar to that of the native TNF-alpha. The collection of muteins was characterized by their biological activity. Mutants R32H and A33S exerted a decreased cytotoxicity against murine fibroblast cell line L929, whereas point mutant F144L and double mutant R32H-F144L were essentially inactive.

[Stability of human immunodeficiency virus to azidothymidine. II. Kinetic characteristics of "AZT-resistant" mutant forms of reverse transcriptase]. / Issledovaniia ustoichivosti virusa immunodefitsita cheloveka k azidotimidinu. II. Kineticheskie kharakteristiki "AZT-ustoichivykh" mutantnykh form obratnoi transkriptazy.

Prolonged treatment of AIDS patients with azidothymidine results in the development of resistance to the drug which correlates with the appearance of point mutations in the reverse transcriptase (RT) coding region within the HIV-1 pol gene. Kinetic studies of interactions of wild type RT and its mutants harbouring the above mutations with substrates and azidothymidine 5'-triphosphate (AZTTP) have been carried out. The complete mutant containing all the above described mutations possess the highest resistance on all the templates tested. Significant increases in resistance for mutants 67,70,215 and 67,215 on all the templates have also been observed. Inhibition of mutant enzymes by AZTTP depends on the template used.

[Selectively cleaved synthetic oligodeoxyribonucleotides for reverse immobilization of DNA]. / Spetsifichno otshchepliaemye sinteticheskie oligodezoksiribonukleotidy dlia obratimoi immobilizatsii DNK.

A streptavidin-coated TSK-gel support, with the loading capacity of 50-70 nmol of biotinylated substance per gram of dry support, and biotinylated oligonucleotides, containing the 4,9-dithiadodecane-6,7-dihydroxy-1,12-diphosphate insert, were prepared for the reversible immobilization of DNA. A non-nucleotide link can be located either at 5'- or 3'-end of the DNA fragment between the biotin moiety and the nucleotide sequence and is subjected to the selective periodate cleavage at the glycol group, which takes 45 min in solution and 3 h in heterophase. For the incorporation of the cleavable and biotin moieties into synthetic oligonucleotides, the corresponding phosphoramidite reagents and biotinylated CPG support were synthesized.

[Studies of the resistance of the human immunodeficiency virus to azidothymidine. I. Kinetic studies of the interaction between reverse transcriptase and a series of its mutant forms with substrates and azidothymidine-5'-trisphosphate]. / Issledovaniia ustoichivosti virusa immunodefitsita cheloveka k azidotimidinu. I. Kineticheskie issledovaniia vzaimodeistviia obratnoi transkriptazy i riada ee mutantnykh form s substratami i azidotimidin-5'-trifosfatom.

Prolonged therapy of AIDS patients with azidothymidine results in the development of resistance to the drug. This phenomenon is accompanied by the appearance of point mutations in the pol gene coding for reverse transcriptase (RT). Kinetic studies of interactions of wild type RT and its forms containing the above-mentioned mutations with substrates and azidothymidine 5'-triphosphate have been carried out. Considerable differences in the affinities of RT and the mutants for RNA and DNA heteropolymer templates were established. The mutations did not affect the RT affinity for dNTP; however, its location influenced considerably the inhibition of the reaction with azidothymidine 5'-triphosphate.

[Hybrid human lymphokine genes. I. Design of recombinant plasmids coding hybrids of immune interferon and human tumor necrosis factor]. / Geny gibridnykh limfokinov cheloveka. I. Konstruirovanie rekombinantnykh plazmid, kodiruiushchikh gibridy immunnogo interferona i faktorov nekroza opukholei cheloveka.

Recombinant plasmids coding for hybrid proteins between human interferon gamma and human tumour necrosis factor alpha or beta have been constructed using site-directed mutagenesis. The genes were fused via a synthetic oligonucleotide linker coding for tetrapeptide Pro-Val-Gly-Pro. The fused genes were expressed in Escherichia coli under control of early promoters of bacteriophage T7. E. coli cells harbouring the plasmids with the hybrid genes gave rather high level of the fused proteins biosynthesis. The hybrid recombinant proteins proved to be unstable in E. coli cells.

Construction and properties of fusion proteins between human interferon-gamma and human tumour necrosis factors alpha and beta.

A number of recombinant plasmids coding for fusion proteins between human interferon-gamma (IFN-gamma) and human tumour necrosis factor alpha (TNF alpha) or beta (TNF beta) were constructed by using site-directed mutagenesis and ligation of the respective genes. In these proteins the whole IFN-gamma sequence of the molecule is linked at the N terminus via a short polypeptide linker to the TNF alpha sequence lacking two N-terminal amino acid residues or to the whole TNF beta sequence. A series of mutants with deletions in the interferon part of the fusion proteins were also produced. All the fusion genes obtained were efficiently expressed in Escherichia coli under the control of early promoters of bacteriophage T7. The recombinant fusion proteins were found to be unstable inside bacterial cells. Bacterial cell lysates expressing these fusion genes or their deletion mutants showed both biological activities in vitro: the antiviral activity of IFN-gamma and the cytotoxic activity of TNF.

[Enzymatic ligation of DNA fragments containing phosphoamide modification of the internucleotide bonds]. / Enzimaticheskoe ligirovanie fragmentov DNK, soderzhashchikh fosfamidnuiu modifikatsiiu mezhnukleotidnykh sviazei.

DNA fragments with the point amidophosphate (cyclohexylamido- or morpholido-) modification in the sugar-phosphate backbone were synthesized and separated into individual diastereoisomer. The isomers were separated by the reversed-phase HPLC (RPC), and chirality at phosphorus was assigned by a stereochemical correlation scheme using phosphorothioate standards. The RPC-retention time values for Rp-isomers were found to be lower than for Sp-analogues. Amidophosphate DNA fragments were used as P- and OH-components in the T4 DNA-ligation. The enzyme does not ligate amidated fragments with modified internucleotide linkage near 5'- or 3'-end, independently of the amidophosphate chirality. When an unmodified phosphodiester linkage separates the amidophosphate group from 3'-end in O-component, the ligation occurs only with Sp-isomer, whereas Rp-analogue does not give the ligation product. In the P-component of the ligation, configuration of the modified linkage separated from 5'-phosphate by an unmodified linkage does not affect the result of the enzymatic reaction: both Sp-and Rp-stereomers do take part in the ligation. As a result of the ligation of the modified fragments on unmodified templates a set of 31-mers was obtained. They contain FokI and EcoRI recognition sites with the cleavage points of both endonucleases coinciding and being amidated. Upon treatment of duplex DNA consisted of unmodified and amidated strands with these endonucleases Sp-configuration did not hinder the cleavage of the unmodified strand, whereas Rp-configuration inhibited the EcoRI and did not affect the FokI cleavage.

[Solid phase ligation of synthetic DNA fragments]. / Tverdofaznoe ligirovanie sinteticheskikh fragmentov DNK.

A synthetic oligodeoxyribonucleotide (oligo) covalently bound by an internucleotide linkage to the succinylated Sephacryl S-500 support through 1.9-diaminononane spacer was used as starting compound to assemble the E. coli rec A promoter DNA fragment from synthetic oligos by means of T4 DNA ligase. The solid-phase assembly of the designed DNA was performed by two ways: stepwise ligation of two pairs of oligos (2 dyads) or simultaneous ligation of four oligos (tetrad). Both ways gave equal results with some preference in the tetrad case. The reliability of E. coli promoter DNA fragment assembly was demonstrated by cloning it in a plasmid vector and sequencing the cloned DNA by the solid-phase Maxam--Gilbert technique.

[Synthesis of 5'-phosphorylated oligodeoxyribonucleotides by the H-phosphonate method]. / Sintez 5'-fosforilirovannykh oligodezoksiribonukleotidov H-fosfonatnym metodom.

N,O-protected 2'-deoxyribonucleotide-3'-H-phosphonates and 2-(p-nitrophenylethyl)-H-phosphonate were prepared and used in solid-phase synthesis of 5'-phosphorylated DNA. H-Phosphonate condensation is performed with 1:2 ratio of P-component to activator (pivaloyl chloride). Protection groups were removed either by sequential treatment with conc. NH3 and DBU or by combination of these reagents.