Bioinformatics analysis of the primary and secondary structure of the Vibrio cholerae Cef (CHO cell elongating factor) protein was conducted. Similarity with triacylglycerol lipases and cytotonic toxins of other bacteria...Bioinformatics analysis of the primary and secondary structure of the Vibrio cholerae Cef (CHO cell elongating factor) protein was conducted. Similarity with triacylglycerol lipases and cytotonic toxins of other bacterial species was observed. Cef was predicted to be a heat-tolerant serine lipase with the Kunitz domain and leucine zipper. These data were confirmed experimentally. The Cefs of the two biotypes of V. cholerae O1, as well as O139 and nonO1/nonO139 serogroups, were purified from the recombinant Escherichia coli strains carrying corresponding cloned genes, and their physicochemical properties, biochemical and biological activities in vitro and in vivo were characterized. Biological activity against the cultured cells was not associated with estherase activity. Evidently, Cef is a bifunctional protein contributing both to pathogenicity of the cholera agent and to its competitive ability in different ecological niches.
Nucleic acid-based aptamers are widely accepted as promising tools for development of a plethora of diagnostic and therapeutic preparations, as well as means ofenvironmental monitoring. Aptamers can be regarded as fully...Nucleic acid-based aptamers are widely accepted as promising tools for development of a plethora of diagnostic and therapeutic preparations, as well as means ofenvironmental monitoring. Aptamers can be regarded as fully synthetic analogs of antibodies. At the same time, certain properties ofaptamers render them superior to antibodies in terms of development of new diagnostic and monitoring systems that combine high sensitivity and specificity with high reproducibility and inexpensive manufacturing. In particular, the aptamers tailored to bind biomolecules and live cells can be employed in solving the problem of combining short analysis time with high sensitivity and specificity in detection of pathogenic bacteria. The present review summarizes the current state of the techniques developed for aptamer-based detection of bacteria and their components and discusses the potential of their practical application.
Bacillus stearothermophilus C8 was grown up on the Luria agar at 37 degrees C. A new DNA-methylase was determined in cellular lysate. The methylation of the DNAs of bacteriophages lambda and T7 in the region of 5'-G(m5C)...Bacillus stearothermophilus C8 was grown up on the Luria agar at 37 degrees C. A new DNA-methylase was determined in cellular lysate. The methylation of the DNAs of bacteriophages lambda and T7 in the region of 5'-G(m5C)NNGC-3' blocked the activity of BstC8I. Specificity of M.BstC8I was analyzed on methylated lambda DNA. For this purpose, we used computer modeling and the data on the sensitivity of restrictases BstC8I, BsuRI, AjnI, and PvuII to methylation. The sensitivity of some restrictases to new methylation was studied. The results may be used for DNA methylation studying.
Genetic diversity and phylogeny of rhizobia that nodulate 18 species of wild-growing bean plants of South Urals from 8 genera belonging to 4 tribes (Loteae, Genisteae, Galegeaev and Hedysareae) was studied. It was demons...Genetic diversity and phylogeny of rhizobia that nodulate 18 species of wild-growing bean plants of South Urals from 8 genera belonging to 4 tribes (Loteae, Genisteae, Galegeaev and Hedysareae) was studied. It was demonstrated that for the wild-growing plants of Galegeae and Hedysareae tribes symbiotic interaction with various strains of nodule bacteria that closely related to bacteria of Mesorhizobium sp. was typical of the plants of Genisteae tribe--to bacteria of Bradyrhizobium sp. In the nodules of Lortus ucrainicus from Loteae tribe we have found a rhizobium that is closely related to the bacteria of Mesorhizobium sp., and at Coronilla varia rhizobia strains obtained by us were close by sequence of a 16S pRNA gene to Rhizobium sp. In the nodules of some kinds of the investigated plants we found also minor species of a rhizobia, which structure is under the great influence of conditions of the host plant growth.
An increase in the nitric oxide (NO) biosynthesis in Lactobacillus plantarum 8P-A3 cells takes place under strong stress influence, which leads to a considerable decrease in the microbial cell viability: heating at 70 de...An increase in the nitric oxide (NO) biosynthesis in Lactobacillus plantarum 8P-A3 cells takes place under strong stress influence, which leads to a considerable decrease in the microbial cell viability: heating at 70 degrees C and 80 degrees C, prolonged cultivation, toxic effect of hexylresorcinol. The factors, which do not lead to cell death, such as heating at 60 degrees C, 50 microg/ml homoserine lactone, Bacillus intermedius 7P ribonuclease (binase) in concentrations up to 300 microg/ml, do not induce NO synthesis. The activation of the NO biosynthesis in response to stress treatment evidences to universality of key-mechanisms of stress response in cells differing in the level of their organization as well as to important role of nitric oxide in them.
Shcherbakova NS, Chikaev AN, Karpenko LI
… +1 more, Il'ichev AA
Mol Gen Mikrobiol Virusol
· 2012 · PMID 22702140
The impact of monoclonal antibodies (mAb) biotinylation on the output and the repertoire of selected peptides in the biopanning procedure were tested. A comparative analysis of the peptides selected from phage library us...The impact of monoclonal antibodies (mAb) biotinylation on the output and the repertoire of selected peptides in the biopanning procedure were tested. A comparative analysis of the peptides selected from phage library using the biotinylated and non-biotinylated mAb 2F5 was performed. It was shown that the output of peptides homologous to the native epitope was 1.7-fold higher for biotinylated antibodies, whereas the binding capacity of the selected phages with mAb 2F5 in ELISA was higher in the case of using non-biotinylated antibodies. It should be noted that the phages exposing peptides, which have 4-5 amino acid sequence similarity with the native epitope, demonstrate the highest binding affinity. The phages that expose peptides with 3 amino acid sequence similarity demonstrate different binding affinity: from the smallest to the largest. Based on the obtained data, it is safe to suggest that the rational biopanning may proceed in accordance with the task.
Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons...Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.
The latest data on selection and construction of poxviruses capable of specifically lysing tumor cells of different genesis, inducing antitumor immunity and apoptosis of malignant cells are discussed. The review concerns...The latest data on selection and construction of poxviruses capable of specifically lysing tumor cells of different genesis, inducing antitumor immunity and apoptosis of malignant cells are discussed. The review concerns several directions: virus attenuation, insertion of immunomodulatory protein genes, and anti-tumor protein genes. Thymidine kinase and viral growth factor genes make the greatest contribution to the virus attenuation as their inactivation results in the virus inability to replicate in non-dividing cells, thereby contributing to increased selectivity with respect to tumor cells. Among the immunomodulatory proteins, interleukins 2, 12, and granulocyte-macrophage colony-stimulating factor proved to be most promising for oncolytic virotherapy. An attempt to use p53 protein gene expressed by vaccinia virus for addressed apoptosis of tumor cells was reported. The use of the double and triple viral recombinants carrying genes of multidirectional action seems to be most promising. Encouraging results were obtained using vaccinia virus in the oncotherapy with prodrugs and angiogenesis inhibitors. At present, two poxviral strains are undergoing Phase III clinical trials as anti-tumor preparations in the USA.
Maianskiĭ AN, Chebotar' IV, Rudneva EI
… +1 more, Chistiakova VP
Mol Gen Mikrobiol Virusol
· 2012 · PMID 22702137
Definition of the biofilm process as one of the types of intercellular bacterial communications is presented. The modern data concerning the structure of the Pseudomonas aeruginosa biofilm matrix and genetic mechanisms n...Definition of the biofilm process as one of the types of intercellular bacterial communications is presented. The modern data concerning the structure of the Pseudomonas aeruginosa biofilm matrix and genetic mechanisms necessary for its production are described. Active and passive rejections of biofilm bacteria, which are the basis of bacterial spreading to new surfaces, are discussed. The complexity and chain type of the reactions associated with biofilm formation are emphasized.
The study of basic biological properties of H5N1 subtype strain isolated during an outbreak among wild birds in Russia in 2010 was presented. The study was carried out using conventional methods according to the WHO reco...The study of basic biological properties of H5N1 subtype strain isolated during an outbreak among wild birds in Russia in 2010 was presented. The study was carried out using conventional methods according to the WHO recommendations. H5N1 influenza virus isolated in Siberia belonged to clade 2.3.2 of the hemagglutinin gene; the phylogenetic analysis was performed. The antigenic characteristics and the basic genetic markers of biological properties were studied. It was shown that all strains were highly pathogenic for chickens and white mice. Thus, it was shown that in Russia in the 2010 H5N1 virus phylogenetically closely related to Asian variants caused epizootic among wild birds. The potential danger of this variant of the virus for humans was confirmed by different methods. We discussed the possibility of formation of H5N1 influenza natural focus.
Kiseleva IV, Voeten JT, Teley LC
… +7 more, Larionova NV, Dubrovina IA, Berdygulova ZhA, Bazhenova EA, van den Bosch H, Heldens JG, Rudenko LG
Mol Gen Mikrobiol Virusol
· 2011 · PMID 22312898
The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza v...The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6:2 reassortant viruses containing the surface antigens hemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, and conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6:2 reassortant viruses were selected from co-infections between master donor virus and wild type viruses that theoretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6:2 reassortant viruses are allowed as vaccine viruses, screening needs to be both rapid and unambiguous. The screening of the reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets was described to select both influenza A and influenza B 6:2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine.
Shikov AN, Sementsova AO, Demina OK
… +8 more, Sergeev AA, Berillo SA, Sergeeva EI, Vinokurova AV, Ishenina AV, Ternovoĭ VA, Agafonov AP, Drozdov IG
Mol Gen Mikrobiol Virusol
· 2011 · PMID 22312897
Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1...Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.
Seregin SV, Babkin IV, Petrova ID
… +3 more, Iashina LN, Malkova EM, Petrov VS
Mol Gen Mikrobiol Virusol
· 2011 · PMID 22312896
Molecular epidemiological study of novel strain of Rubella virus isolated during the outbreak in Western Siberia in 2004 was described. Detailed phylogenetic analysis performed based upon entire SP-region, which encodes...Molecular epidemiological study of novel strain of Rubella virus isolated during the outbreak in Western Siberia in 2004 was described. Detailed phylogenetic analysis performed based upon entire SP-region, which encodes all three Rubella structural proteins (C, E2, and E1), was implemented. This analysis provides characterization of this strain and classifies it as 1H genotype, thereby correcting previous classification of this strain based upon shorter nucleotide sequence, only encoding E1 protein. Therefore, this study identified the genotype of the Rubella virus not previously detected in Western Siberia (and even entire Russian Federation), which highlights the importance of more extensive characterization of genetic variability of the Rubella virus, especially with regard to potential influence of vaccination on the Rubella virus mutagenesis.
The comparative analysis of the symbiotic genes nifD, nifH, nodA of wild-growing Lathyrus L. species (Fabaceae) connected by genes sequences of 16S aRNA to Rhizobium leguminosarum bv. viceae, Rhizobium tropici, Agrobacte...The comparative analysis of the symbiotic genes nifD, nifH, nodA of wild-growing Lathyrus L. species (Fabaceae) connected by genes sequences of 16S aRNA to Rhizobium leguminosarum bv. viceae, Rhizobium tropici, Agrobacterium sp., and Phyllobacterium sp. was carried out. It was demonstrated that all tested genes of strains taken for analysis had high degree of homology with analogous genes of Rhizobium leguminosarum bv. viceae. It was suggested that symbiotic genes were introduced into Rhizobium tropici, Agrobacterium sp., and Phyllobacterium sp. strains by means of horizontal gene transfer over from Rhizobium leguminosarum bv. viceae strain. The recombinant strains were formed, capable to nodulate Lathyrus L. species that earlier was not considered characteristic for these plants.
Priamchuk SD, Fursova NK, Anisimova VA
… +4 more, Kovalev IuN, Abaev IV, Kuzhel'naia EN, Svetoch EA
Mol Gen Mikrobiol Virusol
· 2011 · PMID 22312894
The algorithm of the identification of the bla(CTX-M) genes coding CTX-M-type beta-lactamases providing resistance to cephalosporins III-IV was developed. This algorithm provides identification of 49 genes of 96 genes pr...The algorithm of the identification of the bla(CTX-M) genes coding CTX-M-type beta-lactamases providing resistance to cephalosporins III-IV was developed. This algorithm provides identification of 49 genes of 96 genes presented in the GenBank database so far. Remaining 47 genes can be identified as consisting of small sub-groups composed of 2-6 genes with the exception of sub-group of the bla(CTX-M-14)-like genes composed of 13 genes. The identification of the bla(CTX-M) genes is based on two-step restriction fragment length polymorphism analysis of 544 bp PCR-product (PCR-RFLP). In the first step, determination of subtype (cluster) of the bla(CTX-M) gene occurred using the restriction nuclease Alu I: cluster 1, -2, -8, -9 or -25. Moreover, four genes can be identified just at this step: bla(CTX-M)-59, (cluster 2); bla(CTX-M-63) (cluster 8), bla(CTX-M-45) (cluster 9), and bla(CTX-M-78) (hybrid gene between cluster 2 and cluster 25). At the second step gene identification goes on inside of each cluster separately using a set of 26 restriction nucleases. As a result of the PCR-RFLP-analysis, 23 bla(CTX-M) genes can be identified at the cluster 1, 11 genes--at the cluster 2, 4 genes--at the cluster 8, 9 genes--at the cluster 9, 1 gene--at the cluster 25, and 2 hybrid genes: bla(CTX-M-78) (between clusters 2 and 25), and bla(CTX-M-64) (between clusters 1 and 9). The described algorithm was used for identification of the blac(CTX-M) genes (n = 585) detected in Enterobacteriaceae nosocomial isolates (n = 877), collected from Russial hospitals in 2003-2007. It was shown that major genes belonged to cluster 1 (n = 543), namely--bla(CTX-M-15) gene (n = 515), bla(CTX-M-3) (n = 25), bla(CTX-M-22) (n = 1), bla(CTX-M-23) (n = 1), and bla(CTM-34) (n = 1). Moreover, the genes atributed to cluster 2 were identified: bla(CTX-M-2) (n = 1), and bla(CTX-M-5) (n = 4); and genes belonged to cluster 9: bla(CTX-M-9) (n = 2), and bla(CTX-M-14) (n = 35).
Kirillova NV, Fedosova EA, Naranbat N
… +5 more, Oyuntuya T, Buyankhishig B, Enkhjsaikhan D, Demkin VV, Nymadawa P
Mol Gen Mikrobiol Virusol
· 2011 · PMID 22312893
112 strains of M. tuberculosis isolated from lung tuberculosis patients in Mongolia were genotyped using RD9, RD7, TbD1, RD105, and RD750 loci. The genotypes of all the strains studied were characterized using the conser...112 strains of M. tuberculosis isolated from lung tuberculosis patients in Mongolia were genotyped using RD9, RD7, TbD1, RD105, and RD750 loci. The genotypes of all the strains studied were characterized using the conservation of RD7, RD9, and RD750 loci and the presence of the deletion in the locus TbD1. RD105 was detected in 65 isolates (58%). The isolate was classified into two groups--East-Asian and Euro-American.
Apal'ko SV, Lunin VG, Filipenko ML
… +7 more, Matveeva VA, Liashchuk AM, Lavrova NV, Sherina EA, Aver'ianov AV, Kostianko MV, Glushkov AN
Mol Gen Mikrobiol Virusol
· 2011 · PMID 21789801
Two recombinant plasmids were constructed. The first plasmid contained the hybrid gene composed of immunomimetic peptide of benzo[a]pyrene, of the protein pIII of bacteriophage M13 and of cellulose binding domain encodin...Two recombinant plasmids were constructed. The first plasmid contained the hybrid gene composed of immunomimetic peptide of benzo[a]pyrene, of the protein pIII of bacteriophage M13 and of cellulose binding domain encoding sequences. The second plasmid contained the hybrid gene composed of the signal peptide of the protein pIII of bacteriophage M13, of immunomimetic peptide of benzo[a]pyrene, of the protein pill of bacteriophage M13 and of cellulose binding domain sequences. The obtained recombinant plasmids were used in expression of chimeric protein containing immunomimetic peptide ofbenzo[a]pyrene based on strain E. coli M15. The lack of the recombinant protein expression using first plasmid was demonstrated. In the same time, it was shown that accumulation of recombinant protein contained immunomimetic peptide with signal peptide of the protein pIIIl of bacteriophage was present. This chimeric protein was produced in "mature" (without signal peptide) and "unprocessing" (with signal peptide) forms. Using the Western-blot analysis, it was shown that the "mature" form only specifically bound to the B2 monoclonal antibody against benzo[a]pyrene. Thus, we expressed, purified, and characterized the recombinant protein containing immunomimetic peptide of benzo[a]pyrene.
The Parkinson disease (PD) is the second most common progressive neurodegenerative disorder that arises due to degeneration of dopaminergic neurons. The causes of this disease are still unknown, but a number of genes inv...The Parkinson disease (PD) is the second most common progressive neurodegenerative disorder that arises due to degeneration of dopaminergic neurons. The causes of this disease are still unknown, but a number of genes involved in pathogenesis of familial and sporadic forms of PD has been identified. According to recent data of genome wide association studies (GWAS), single nucleotide polymorphisms (SNPs) in these genes (including MAPT locus) may play an important role in the development of PD. Therefore, we analyzed distribution of genotype frequencies of SNP rs415430 in the WNT3 gene in the Russian patients with sporadic PD and in the Russian population controls (OR = 0.84, Confidence Interval (95% CI) 0.58-1.23, p = 0.39). It was concluded that SNP rs415430 in the WNT3 gene was not associated with the risk of development of PD.
Efficacy of candidate DNA-vaccines based on the variola virus natural gene A30L and artificial gene A30Lopt with modified codon usage, optimized for expression in mammalian cells, was tested. The groups of mice were intr...Efficacy of candidate DNA-vaccines based on the variola virus natural gene A30L and artificial gene A30Lopt with modified codon usage, optimized for expression in mammalian cells, was tested. The groups of mice were intracutaneously immunized three times with three-week intervals with candidate DNA-vaccines: pcDNA_A30L or pcDNA_A30Lopt, and in three weeks after the last immunization all mice in the groups were intraperitoneally infected by the ectromelia virus K1 strain in 10 LD50 dose for the estimation of protection. It was shown that the DNA-vaccines based on natural gene A30L and codon-optimized gene A30Lopt elicited virus, thereby neutralizing the antibody response and protected mice from lethal intraperitoneal challenge with the ectromelia virus with lack of statistically significant difference.