[Proteomic analysis of mitochondria from the Bos taurus heart. II. Identification of mitochondrial soluble proteins].

A fraction of the so-called mitochondrial soluble proteins was obtained after the destruction of purified mitochondria by sonication according to the previously found approach to the identification of protein subsets of the Bos taurus heart proteome. A tryptic destruction of these proteins was achieved. Approximately half of the tryptic hydrolysate was separated into two fractions of cysteine-containing and cysteine-free peptides by covalent chromatography on Thiopropyl Sepharose 4B. The cysteine-containing peptides were modified by iodoacetamide. The peptides were mass-spectrometrically identified in all the three fractions of tryptic hydrolysate, and the proteins were searched for in the amino acid sequence databases. There were 213 unique proteins reliably identified.

[Evaluation of biochips for the rifampin resistance detection of M. tuberculosis in strains isolated at the Novosibirsk and Tomsk regions].

Recently in Russia biochips for rifampin resistance detection of M. tuberculosis were developed. To investigate the conformity between rifampin resistance results determined both by the routinely used absolute concentration method and USING the biochips, 272 DNA samples of M. tuberculosis isolated from TB patients at Novosibirsk and Tomsk regions in 2000-2005 were analyzed. The biochip can detect 30 mutations in rpoB gene. The mutations were also tested using the single stranded conformational polymorphism method (SSCP). In addition, 60 DNAs were randomly sampled and sequenced. The results of rifampin resistance detection using biochip and absolute concentration methods were congruent in 86% cases, and were different when analyzed samples consisted of the susceptible and resistant strains of M. tuberculosis mixture. The most frequent mutations in the rpoB gene were S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). In 94% of rifampin resistant strains, there was also resistance to isoniazid. Therefore, in Siberia the rifampin resistance is the reliable marker for MDR strains of M. tuberculosis, and biochips can be used also for their detection. To hybridize with biochip the fluorescent-labeled single-stranded DNAs were routinely synthesized by two PCR, and intermediary product after the first PCR should be transferred into another tube. The last stage included high risk of cross-contamination. To exclude the risk, primer concentrations and temperature-time profile of PCR reactions were improved, and both PCR were combined in one tube. The two methods were congruent in 100%. The one tube method would be especially attractive for the routine PCR laboratory.

[Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates].

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.

A prototype prophylactic anti-influenza preparation in aerosol form on the basis of Abies sibirica polyprenols.

This research investigates a promising antiviral compound based on polyprenols from Siberian silver fir (Abies sibirica). The physico-chemical characteristics of a preparation developed in aerosol form and an estimation of its protective efficacy against aerosol challenge of laboratory animals are presented. It is shown that (1) by using a simple ultrasonic disperser one can obtain aerosol of three formulations studied with about 70% of its mass accumulated in the size range below 1.8 microm; (2) 40-100% of aerosol particles contain preparation for different formulations; (3) after delivering under specified schedules, the preparations as developed can protect up to 100% of mice against 5 LD(50) of influenza A/Aichi/2/68 (H3N2) virus aerosol infection. Animals inhaled twice the preparation doses (which were 100 times lower than injection ones of the same efficacy) and did not exceed 10 microg/mouse. It was shown that the mode of action of this immunomodulating preparation was nonspecific stimulation of immune cells' various activities.

[Molecular and genetic characterization of rifampicin- and/or izoniazid-resistent strains of M. tuberculosis, isolated in Novosibirsk and Tomsk regions].

The purpose of the work was to study rifampicin- and izoniazid-resistent strains of M. tuberculosis, circulating in Western Siberia, by VNTR and IS6110 typing. The authors also studied genetic causes of resistance to these antibiotics and undertook a search of new VNTR loci, displaying polymorphism in genomes of closely related clonally-disseminated variants of Mycobacterium tuberculosis in W-Beijing family model analysis.

Mutagenesis directed by phosphotriester analogues of oligonucleotides: a way to site-specific mutagenesis in vivo.

A new approach to induce directed mutations in genes of study through simple cotransfection of E. coli cells by the mixture of primer and template was developed. This method is based on the use of synthetic phosphotriester analogues of oligonucleotides as site-specific mutagenic primers. The achieved yield of mutant clones was 2-3%.

Effect of intramuscularly injected polyprenols on influenza virus infection in mice.

This study demonstrates the possibility of achieving a prophylactic effect by intramuscular injection of Abies sibirica polyprenols for the control of influenza virus infection in mice. One of the five polyprenol preparations tested, preparation N1, which had the lowest hydrophilic-lipophilic balance (8.6), produced a significant protective effect when injected in a dose of 2000 microg/mouse 2 days before aerosol infection of mice with influenza virus. A moderate protective effect was also observed using a second preparation, designated N2. One day after aerosol infection, animals pre-treated with 2000 microg doses of the polyprenol preparations or Hanks' solution showed no difference in the level of interferon accumulation in the lungs. Three days after injection of preparation N2 and N1, a significant decrease in spleen and thymus weights was, observed in the mice. One day after injection of these preparations, the number of lymphocytes in the bronchoalveolar tract of the mice exceeded almost twice that seen in mice treated with placebo. After 3 days, relative and absolute numbers of macrophages decreased, whereas those of lymphocytes increased significantly. Three days after the administration of preparations N1 and N2, macrophages became approximately twice as active in absorbing zymozan granules. Preparation N1 affected the system of superoxide radical anion production to a greater extent than preparation N2. The production of radical anions by the macrophages of the bronchoalveolar tract in the mice, 1 day after intramuscular injection of preparation N1, was significantly higher than that seen on day 3 and that induced by preparation N2 1 and 3 days after injection. These data indicate that emulsions of polyprenols that have relatively low hydrophilic-lipophilic balance, inhibit influenza virus infection in mice through a modulation of the host immune response.

[Mutagenic properties of phosphotriester analogs of oligodeoxyribonucleotides]. / Mutagennye svoistva fosfotriéfirnykh analogov oligodezoksiribonukleidov.

The high effectivity of using phosphotriester analogs of oligonucleotides for aimed mutagenesis in vitro and in vivo was shown. A general scheme, describing the mutagenic effects of phosphotriester analogs of oligonucleotides and their natural homologs, was derived by analysis of data on the structures of the obtained mutants. This scheme can serve as a foundation for selecting the structure of effective agents for aimed mutagenesis.

[Directed reconstruction of the influenza virus hemagglutinin gene]. / Napravlennaia rekonstruktsiia gena gemaggliutinina virusa grippa.

A new approach to create chimeric genes by directed exchange of oligonucleotide fragments was developed. By oligonucleotide-directed mutagenesis a few deletion mutants of the influenza virus hemagglutinin (HA) gene were obtained. These variants of HA gene contain unique restriction sites in DNA regions coding for the A and B epitopes of the HA molecule. The obtained special vectors may be used for cloning DNA fragments coding for new amino acid sequences in internal sites of the HA gene.

[Construction and expression in Escherichia coli of a gene of hybrid hemagglutinin H1-H3 of influenza virus]. / Konstruirovanie i ékspressiia v Escherichia coli gena gibridnogo gemaggliutinina podtipa H1-H3 virusa grippa.

The hybrid gene of influenza virus hemagglutinin (HA) of the H1-subtype, carrying the sequence coding for the fragment of H3-subtype antigenic site B, was constructed. The product of expression of this gene in E. coli was obtained as a fusion protein with beta-galactosidase. The chimeric protein was shown to retain the antigenic properties of HA of H1-subtype and to interact specifically with antibodies against the synthetic peptide corresponding to the B site fragment of HA of the H3-subtype.

[A new primer for sequencing DNA fragments cloned in M13mp vectors]. / Novyi praimer dlia sekvenirovaniia fragmentov DNK, klonirovannykh v M13mp-vektorakh.

Tridecadeoxyribonucleotide d(CCAGGGTTTTCCC) was prepared by solid phase crown-ether-catalyzed phosphotriester method and proposed a new sequencing primer. This primer expands capacities of the Sanger dideoxy-chain-terminating method to solve various sequencing problems as compared to well-known universal primers. Criteria of the choice of an oligonucleotide primer without computer analysis of nucleotide sequence are described.

[Mutagenesis directed by phosphotriester analogs of oligonucleotides]. / Mutagenez, napravlennyi fosfotriéfirnymi analogami oligonukleotidov.

20-mer oligodeoxyribonucleotides d-ACGACGG (R') CCAG (R'') TGATCCGTA, where R' = R'' = H (20), F' = Et, R'' = H (20-Et), or R' = R'' = Et (20-Et2) were synthesized by modified triester method. Ethylated dinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. Structures of oligonucleotides were confirmed by Maxam - Gilbert method. Mutagenesis induced by oligonucleotides was studied on DNA of M13mpB phage. Oligonucleotides were not totally complementary to this DNA in the region of 4-11 codons of Z'-gene. They all were shown to direct the formation of the designed deletion mutants, phosphotriester analogues (20-Et) and (20-Et2) being more effective mutagens. The specificity of oligonucleotides: DNA binding and mutant DNA structure were shown by Sanger method.

[Site-localized mutagenesis directed by phosphotriester analogs of oligonucleotides]. / Sait-lokalizovannyi mutagenez, napravliaemyi fosfotriéfirnymi analogami oligonukleotidov.

17- and 20-mer oligodeoxyribonucleotides and their analogues, containing one to four phosphate groups esterified with ethyl alcohol in different positions of oligonucleotide chain, were synthesized by modified triester method. Ethylated di- and trinucleotide blocks were prepared by transesterification method from chlorophenyl derivatives. The structures of the oligonucleotides were confirmed by Maxam-Gilbert sequencing method. Oligonucleotides were not totally complementary to the N-terminal region of lac Z'gene (coding for N-terminal fragment of beta-galactosidase) of phage M13mpB DNA and induced the formation of the proposed deletion mutant DNA M13mp1 delta T. Phosphotriester analogues were more effective mutagens as compared to phosphodiester oligonucleotides due to their stability to nucleases. The use of E. coli DNA-polymerase I provided the increase in the mutant yields in case of the phosphotriester analogues. The stability of the analogues to 5'----3'----5'-endonuclease action, the specificity of oligonucleotide: DNA binding and the structure of mutant DNA were studied by the Sanger sequencing method.

[Primary structure of the mutant genes of human leukocyte interferon alpha2]. / Opredelenie pervichnoi struktury mutantnykh genov leikotsitarnogo interferona alpha2 cheloveka.

Nucleotide sequences of 10 mutant genes of human leukocyte interferon alpha 2 (IFN) with the use of 4 oligonucleotide primers containing ethyl substituents at phosphate groups were determined. To design primer sequences, an approach based on the local similarity profile of the IFN gene and M13mp7 vector DNA is described.

[Effectiveness of substituting E. coli DNA-polymerase for DNA-polymerase A in oligonucleotide-directed mutagenesis]. / Effektivnost' zameny DNK-polimerazy A DNK-polimerazoi I E. coli v oligonukleotidnapravlennom mutageneze.

The possibility to use the E. coli intact DNA polymerase I in the oligonucleotide-directed site-specific mutagenesis of DNA has been studied. Optimal conditions of the extension activity of this enzyme were found. We have shown that the substitution of the Klenow fragment of the E. coli DNA polymerase by the intact DNA polymerase I did not decrease the efficiency and fidelity of the oligonucleotide-directed mutagenesis.

[Variants of phage M13 DNA containing a fragment of the beta-galactosidase gene--a convenient mutation system for the study of oligonucleotide-directed mutagenesis]. / Proizvodnye DNK faga M13, soderzhashchie fragment gena beta-galaktozidazy--udobnaia mutatsionnaia sistema dlia issledovaniia napravlennogo oligonukleotidami mutageneza.

A model system is developed to test oligonucleotide-directed mutations: T----C transition, T and C deletions (delta T and delta C), C insertion, double mutations (A----G, delta T), (T----C, A----G), and large oligonucleotide deletions (36 or 44 nucleotides). The system includes 9 variants of the phage M13 DNA carrying fragment of beta-galactosidase gene, and oligodeoxyribonucleotides partially noncomplementary to DNA sequence of this gene. Six variants are obtained by the site-localized mutagenesis, the other were described earlier. Induced mutations are easily tested by phenotype change of transformed bacteria (Lac+----Lac-); by formation or loss of the sites for BamHI and EcoRI restrictases; by DNA hybridization with 32P-labeled oligonucleotides; and by DNA sequencing by the Sanger method. The system is used to study the role of some factors, such as completeness of RF DNA synthesis, thermal stability of the oligonucleotide: DNA complex, quality of enzymes and substrates used in polymerase reaction, mutation type or the efficiency of mutagenesis. A number of unexpected mutations were observed in the course of oligonucleotide-directed mutagenesis. Lower yields of some mutants induced by oligonucleotides are shown to be due to the action of repair systems of bacteria.

[Antiviral activity of mutant interferons. A new approach to the study of structural-functional organization of interferons]. / Antivirusnoe deistvie mutantnykh interferonov. Novyi podkhod k issledovaniiu strukturno-funktsional'noi organizatsii interferonov.

The developed approach to investing the structure-functional organization of interferon has been developed consisting in: 1) fusing genes of interferon and alpha-peptide of beta-galactosidase, the resultant protein having the interferon properties and being determined by the beta-galactosidase alpha-complementation test; 2) constructing mutant genes of interferon by the localized mutagenesis; 3) determining the mutant interferon activity; 4) deducing the amino acid sequence of mutant interferon by sequencing mutant genes; 5) analyzing structure-functional organization of interferon. In accordance with this approach, ten mutant interferons with up to 15 changes of amino acid substitutions are obtained and their antiviral activity is determined. The role of some amino acid residues in antiviral activity of interferon alpha 2 is revealed.

[Mutagenesis directed by phosphotriester analogs of oligonucleotides: a way to site-specific mutagenesis in vivo]. / Mutagenez, napravlennyi fosfotriéfirnymi analogami oligonukleotidov, - put' k sait-spetsificheskomu mutagenezu in vivo.

A new approach is proposed to obtain the directed mutations in the gene under study. The technique is based on using alkylphosphotriester analogues of oligodeoxyribonucleotides as site-specific mutagens. The deletion C in lacZ' gene of bacteriophage M13mpB was obtained by cotransfection of Escherichia coli cells with a mix of DNA and phosphotriester analogues of oligonucleotides.

[Isolation of mutant genes for human leukocyte alpha2-interferon by a method of localized mutagenesis]. / Poluchenie mutantnykh genov leikotsitarnogo alpha2-interferona cheloveka metodom lokalizovannogo mutageneza.

An efficient method to obtain the mutant genes for human leucocyte alpha 2-interferon (IFN) has been elaborated. The technique includes the following main stages: cloning of interferon gene in M13mp8 DNA; isolation of double-stranded hybrid DNA complex, containing IFN gene as a single-stranded fragment; selective modification of a single-stranded hybrid DNA by sodium bisulphite; the repair of hybrid DNA by DNA polymerase I from Escherichia coli, transformation of Escherichia coli JN103 cells by double-stranded circular DNA, containing the selectively modified gene IFN. The technique is based on the protection of bacteriophage M13 genome from mutagen induced damage by means of converting phage DNA into the double-stranded structure leaving the single-stranded fragment to be mutagenized prone to mutagen action. This is achieved by reannealing of single-stranded M13mpB DNA hydrolyzed by restriction endonuclease BamHI. The technique preserves the infectiousness of vector DNA under the conditions permitting modification of up to 10% cytosine residues in IFN gene. Every clone resulting from transformation of Escherichia coli by modified DNA carried mutations in IFN gene, identified by sequencing after Sanger.

[Specific modification of DNA at E. coli RNA-polymerase binding sites]. / Spetsificheskaia modifikatsiia DNK v mestakhh sviazyvaniia RNK-polimerazy E. coli.

Specific modification of promoter regions of DNA has been studied. Plasmid pK56B1 DNA has been used as a model to test RNA-polymerase binding with DNA under various conditions. RNA-polymerase is shown to form specific complexes with DNA which are stable in solutions with a moderate ionic strength (0.1-0.2 M NaCl), under pH 5-8 in the presence of 0.5 M O-methylhydroxylamine of O-delta-aminooxybutylhydroxylamine. Escherichia coli JM103 cells have been transfected with DNAs treated with 0.5 M O-methylhydroxylamine at 37 degrees C, pH 5.2. The inactivation effects of the mutagen on single-stranded DNA of bacteriophage M13 m p1, double-stranded form of this bacteriophage (replicative form-RF) and on the complex of RNA-polymerase with RF DNA have been compared. The obtained data confirmed the specificity of reagent action with DNA sites binding with the enzyme. Selectivity of promoters modification has been confirmed also by the analysis of M13 m p1 DNA mutations induced in lacZ' gene by delta-aminooxybutylhydroxylamine effect on the DNA complex with DNA-polymerase.