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Biomedical subjects

V M Govorun

Publications and source records attributed to V M Govorun.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of genetic markers of N. gonorrhoeae resistance to beta-lactam antibiotics.

A complex method for detection of genetic markers of N. gonorrhoeae resistance to penicillin was developed. Mutations in penA and ponA genes were detected by minisequencing reaction with subsequent detection of reaction products by MALDI-TOF mass spectrometry. This approach was tested on 31 clinical strains of N. gonorrhoeae with minimum inhibitory concentration of penicillin from 0.03 to 8 microg/ml and higher. Mutations in penA and ponA genes in moderately resistant strains were shown (minimum inhibitory concentration up to 0.5 microg/ml) and mutations in penA, ponA, and penB genes in resistant strains (minimum inhibitory concentration more than 1.0 microg/ml). beta-Lactamase genes were detected in 4 strains with high resistance (minimum inhibitory concentration 4-8 and more microg/ml). Correlation between microbiological resistance and presence of respective mutations in the studied locuses was detected.

Bacterial Proteins↗
PubMed2006

Expression of Chlamydia trachomatis inclusion membrane protein genes IncB and IncC in Escherichia coli.

In this study, we have cloned the Chlamydia trachomatis genes incB and incC into the expression plasmid vectors from pET series for the subsequent isolation of recombinant proteins. As a result, we have obtained the first full-length recombinant C. trachomatis proteins IncB and IncC, which can be used for following antibody production and for study of their protein-protein interaction.

Amino Acid Sequence↗
PubMed2005

Effect of induced expression of an antimicrobial peptide melittin on Chlamydia trachomatis and Mycoplasma hominis infections in vivo.

A plasmid construct was designed in which the gene of antimicrobial peptide melittin is controlled by the tetracycline-responsive promoter of human cytomegalovirus, aided by a constitutively expressed trans-activator protein gene. Its vaginal administration and induction of melittin gene transcription with doxycycline markedly suppressed subsequent genital tract infection of mice by Mycoplasma hominis and Chlamydia trachomatis. At least half of the melittin-protected animals proved free of either pathogen within 3-4 weeks. Recombinant plasmids expressing genes of antimicrobial peptides hold much promise as agents for prevention and control of urogenital latent infections.

Animals↗
PubMed2005

Location of C. trachomatis Inc proteins during expression of their genes in HeLa cell culture.

Plasma constructions including genes encoding C. trachomatis inclusion membrane protein as composite proteins with reporter green fluorescent protein were obtained. After transfection of HeLa cell culture with the resultant plasmid constructions the location of inclusion membrane proteins in transfected cell was for the first time detected by confocal microscopy.

Bacterial Proteins↗
PubMed2005

Identification of serovar-specific single-nucleotide polymorphisms of C. Trachomatis omp1 gene.

Complete sequences of omp1 gene encoding chlamydial main outer membrane protein were analyzed in 76 clinical strains of C. trachomatis. Thirty-four serovar-specific single-nucleotide polymorphisms were identified, 20 of them meet two criteria: unique position of the nucleotide and unique nucleotide substitution. Evaluation of serovar-specific single-nucleotide polymorphisms of omp1 gene can appreciably simplify and accelerate genetic diagnosis of C. trachomatis serovars.

Chlamydia trachomatis↗
PubMed2005

Comparative analysis of transcription profiles of Helicobacter pylori clinical isolates.

The transcription profiles of four Helicobacter pylori clinical isolates (two cag-negative and two cag-positive) were compared in stationary growth phase using a cDNA-macroarray. The correlation coefficient value between total transcription profiles of clinical isolates H. pylori varied from 0.70 to 0.83. For 44 groups of genes (total number 66) belonging to various functional classes of H. pylori, the correlation coefficient value between these isolates exceeded 0.7, and for 14 groups the value exceeded 0.9. These groups included genes encoding components involved in cell division, adaptations to atypical conditions, electron transport, salvage of nucleosides and nucleotides, glycolysis/gluconeogenesis, folding and stabilization of proteins, translation factors, anaerobic metabolism, and amino acids and amine metabolism. Expression of 52 genes significantly differed between H. pylori clinical isolates. Some of these genes determine microorganism virulence. They include: cytotoxin-associated gene (cagA), genes encoding neutrophil-activating protein (napA), major flagellar protein (flaA), and vacuolizing cytotoxin (vacA), some genes encoding outer membrane proteins (omp), urease alpha and beta subunits (ureA and ureB), and some regulatory proteins, and genes encoding stress-related proteins, such as the chaperone and heat shock protein genes (groEL and dnaK).

Adhesins, Bacterial↗
PubMed2005

[Resistance of Russian isolates of Neisseria gonorrhoeae to fluoroquinolones].

Fluoroquinolones still belong to the drugs of choice in the treatment of uncomplicated gonorrhea. At the same time, there have been more data on the spreading N. gonorrhoeae strains resistant to fluoroquinolones. A variety of mechanisms, like modification of the target of antibiotic's action (point mutations in genes gyrA and parC), a decreasing permeability of the bacterial cell membrane (amino-acid changes Por protein) and a growing efflux of antibiotic (mutations in the promoter or in the coding region of mtrR) mediate in the shaping resistance of the drugs. The MIC values for four fluoroquinolone-series antibiotics were determined and the gyrA, parC, por and mtrR genes were examined for resistance-responsible mutations in 32 studied clinical strains of N. gonorrhoeae. Strains with high resistance to fluoroquinolones were detected; 3 of them had no common changes in GyrA or ParC, however, amino acid changes and mutations were detected in Por protein and promoter or gene mtrR encoding region, respectively. The paper contains priority data on the detection (in Russia) of N. gonorrhoeae strains with high resistance to fluoroquinolones. Involvement of different mechanisms in the process of resistance shaping is discussed. The results are of practical importance for planning the antibacterial therapy of gonorrhoeae; they point out the need in regional testing of resistance in the N. gonorrhoeae population encountered in Russia.

Anti-Bacterial Agents↗
PubMed2005

[Using the mass spectrometry analysis for hepatitis C virus typing].

Determination of the hepatitis C virus (HCV) genotype has become the standard procedure in laboratory diagnostics of HCV infection. Genotype elucidation has prognostic value assignment helps in assessing disease prognosis and promotes establishing appropriate duration of treatment. Now 11 major genotypes and more than 70 subtypes of HCV have been identified using the sequence variability within 5' non-coding region (5' NCR). In Russia the most common subtypes are 1a, 1b, 2a, 3a and more rare - 4 and 5 types. While the "gold standard" for testing is nucleic acid sequencing, a variety of other assays, including the line probe assay or type-specific amplification, has been developed to provide more rapid and cheaper forms of testing. The aim of this study was to determine the type-specific single nucleotide polymorphism (SNP) in 5' NCR HCV by the classical three-step minisequencing method with followed MALDI-TOF mass spectrometry detection The fragments of 5'NCR of HCV genomes were amplified by the nested RT PCR. The removal of excess nucleotides and primers was performed. Three oligonucleotide primers were design to detect two sets of type-specific SNP in 5' NCR HCV. The primer extension reaction was performed using modified thermostable DNA polymerase and in the presence of ddNTP. The molecular weights of primers extension reaction products were analyzed using MALDI-TOF mass spectrometry. The HCV genotype was determined according the presence in analyses sample the molecules with expected molecular weights. The suggested method was used to type HCV from 69 HCV-positive sera. The 1a genotype was determine in 4.5% samples, 1b - 48%, 2a - 4.5% 3a - 29%, 4 - 1.5%. The mixes of two genotypes were found in 13% samples. All data confirmed by direct nucleic acid sequence. Thus, the new method for HCV typing has been developed using the minisequencing reaction and mass spectrometry for the determination of nucleic acid molecular weight.

DNA Fingerprinting↗
PubMed2005

[A2144G is the main mutation in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance].

To detect point mutations A2115C, A2143G/C, and A2143G in the 23S rRNA gene of Helicobacter pylori associated with resistance of the microorganism to clarithromycin, a new powerful way of analysis was used. This method involved the reaction of minisequencing followed by MALDI-TOF mass spectrometry of reaction products. In ten analyzed clarithromycin-resistant clinical isolates of H. pylori obtained in Russia, the resistance was found to be mediated only by mutation A2144G in the 23S rRNA gene.

Anti-Bacterial Agents↗
PubMed2004

Fluoroquinolone-resistant Neisseria gonorrhoeae isolates from Russia: molecular mechanisms implicated.

OBJECTIVES: During a longitudinal study of the prevalence of antimicrobial resistance in Neisseria gonorrhoeae, a number of high-level fluoroquinolone-resistant isolates were obtained from the sexually transmitted diseases clinic in the Moscow region in 2002. The aim of the present study was to determine the molecular mechanisms of resistance and to assess the clonal relationship of these strains METHODS: For the 32 clinical strains of N. gonorrhoeae studied, the MIC values were determined for four fluoroquinolones. The gyrA, parC, por and mtrR genes were studied for the presence of mutations associated with fluoroquinolone resistance. RESULTS: We detected strains of N. gonorrhoeae showing high-level resistance to fluoroquinolones (21 strains, with MICs 1-32 mg/L). Mutations in gyrA and parC known to cause fluoroquinolone resistance were detected in a majority of strains. There were four strains (among 21) without known changes in gyrA and parC. However, amino acid changes in the Por protein and mutations in the promoter or encoding region of the mtrR gene were detected in three of them. One strain had no alteration in gyrA, parC, por or mtrR. CONCLUSIONS: The present study documents the first case of fluoroquinolone-resistant N. gonorrhoeae in Russia.

Drug Resistance, Bacterial↗
PubMed2004

Experimental Mycoplasma hominis infection of the genital tract in BALB/c mice.

We studied the ability of laboratory and clinical strains of Mycoplasma hominis to colonize the genital tract mucosa in BALB/c mice. Colonization with mycoplasma occurred only in mice receiving estrogen. Mycoplasma hominis strains obtained after 3-fold passage through the vaginal mucosa in mice and administered intravaginally in a dose of 0.5 x 10(8) CFU/ml caused infection in 100% animals. Inflammation in the lower genital tract was reduced by the 8th week after infection.

Animals↗
PubMed2004

Structural organization of the genome of SARS-associated coronavirus (Strain SoD) isolated on the territory of the Russian Federation.

Complete nucleotide cDNA sequence (29715 nucleotides) of SARS-associated coronavirus (strain SoD) isolated for the first time in the territory of the Russian Federation was determined. Phylogenetic analysis revealed maximum similarity between strain SoD genome and Frankfurt 1 strain genome. Three nucleotide substitutions determining two amino acid substitutions were detected.

Amino Acid Substitution↗
PubMed2004

Comparative analysis of different typing methods for Helicobacter pylori clinical isolates.

The goal of the present work was to compare different techniques of molecular typing using as an example clinical isolates of Helicobacter pylori obtained from patients in different regions of Russia. DNA-macroarray genome scanning using individual genes was employed to set up our basic classification of isolates that did or did not contain pathogenicity islands. In parallel, DNA of the same isolates was used in the conventional random amplified polymorphic DNA (RAPD) PCR analysis, and the isolates were also genotyped (cagA, vacA, iceA, and babA status) and their proteomic maps were obtained by means of unidimensional SDS polyacrylamide gel electrophoresis (1D-SDS-PAGE) coupled with identification using peptide mass fingerprinting by MALDI-TOF mass spectrometry. A statistically significant correlation (coefficient of correlation r = 0.25, p = 0.005) was observed between the results of genome scanning and 1D-SDS-PAGE. No correlation was found between RAPD-PCR typing and genome scanning.

Bacterial Typing Techniques↗
PubMed2004

Mutations in a 23S rRNA gene of Chlamydia trachomatis associated with resistance to macrolides.

For six clinical isolates of Chlamydia trachomatis, in vitro susceptibility to erythromycin, azithromycin, and josamycin has been determined. Four isolates were resistant to all the antibiotics and had the mutations A2058C and T2611C (Escherichia coli numbering) in the 23S rRNA gene. All the isolates had mixed populations of bacteria that did and did not carry 23S rRNA gene mutations.

Anti-Bacterial Agents↗
PubMed2004

[Allele Frequency Analysis of Four Single Nucleotide Polymorphisms Locating in Promoter and 5'-Untranslated Regions of ABCAI Gene in Young Men - Survivors From Myocardial Infarction].

Analysis of allele distribution of four single nucleotide polymorphisms (C-17G, C69T, G-191C and 319insG) of promoter and 5'-untranslated regions of the ABCA1 gene was carried out in a sample of 171 men, who had survived myocardial infarction before 45 years, and in controls. Two-fold increase of T69 and C-191 allele frequencies were observed in Russian population in comparison to Dutch one. While comparing allele and genotype distributions of the polymorphisms in the samples under study no statistically significant differences were found, so as no influence of different alleles on lipid spectrum data was observed. Role of polymorphisms under study appears to be insignificant in formation of genetic susceptibility to myocardial infarction in young men.

5' Untranslated Regions↗
PubMed2004

[Analysis of point mutations in the ygeD, gyrA and parC genes in fluoroquinolones resistant clinical isolates of Chlamydia trachomatis].

Resistance of 14 clinical isolates of C. trachomatis to fluoroquinolones, i.e. of ciprofloxacin, pefloxaxin and ofloxacin, was assayed. Three isolates with a high resistance degree to all 3 drugs (MIC equal or above 64 microg/ml) were detected. MIC was found to be equal to or below 4 microg/ml for 3 isolates. The remaining isolates had an intermediate resistance level. The nucleotide sequence was established for the Quinolone-Resistance Determining Region (QRDR) genes coding the DNA-gyrase subunit A (gyrA) and DNA-topoisomerase IV subunit C (parC) as well as for the 3'-region of ygeD coding, presumably, the efflux protein. In none of the isolates, the gyrA and gyrC QRDR differed from the corresponding regions in the published C. trachomatis genome sequence. Several silent mutations and mutations resulting in amino acid substitutions were observed in the ygeD 3' region of 2 isolates resistant to high FQ concentrations and in 1 isolate with the intermediate resistance level.

Anti-Bacterial Agents↗
PubMed2004

[Structural-functional motifs of sterol 14-alpha demethylases (CYP51)].

CYP51 family of cytochromes P450 (sterol 14-alpha-demethylases) comprises the representatives from different kingdoms of living world, thus positioning itself as the most ancient member of the superfamily. In the course of the present research the collection of 36 full-length CYP51 amino acid sequences was submitted to cluster analysis. Each node of the clustering dendrogram corresponds to the groups of proteins, located on the branches descending from the node. By making the multiple alignment of each group of protein sequences we obtained the node-specific consensus sequences. The informational content of the consensus was defined as the presence of the compact conserved sites, the motifs. The assessment of informational content was computed using Sherman's non-parametric statistical criterion. The high informational content was observed for the 100% conserved consensus sequences of the following CYP51,s groups: fungi, animal+plant, plant+protista and bacteria. These selected consensus sequences were next aligned all together to get the final consensus for the whole family. To enrich the informational content of the CYP51 consensus the level of its conservation was dropped to 75%. Regions of statistically significant conservation were unraveled in the CYP51 consensus sequence. These regions (motifs) were then correlated with the information on secondary structure elements and substrate recognition sites reported for CYP51 from Mycobacterium tuberculosis. Seven motifs appeared to be obligatory for every CYP51 protein. The motifs thus obtained were searched for among all the known cytochrome P450 proteins. Some motifs were found to be absolutely specific for 14-alpha-demethylases, whereas others were common to different species of cytochromes P450.

Amino Acid Motifs↗