4/2015

Author

page: 4-9 pp
DOI: 10.11134/btp.4.2015.1
Kulmambetova G.N.2, Bekenova E.E.1, Tujakova A.K.1, Kozhakhmetov S.S.1, Logvinenko A.A.3, Sukashev A.T.3, Almagambetov K.K.1
1Republic Collection of Microorganisms
13/1, Valikhanov str., Astana, 010000, Kazahstan
2National Center for Biotechnology
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
3National Research Medical Center
42, Abylai Khan ave., Astana, 010000, Kazakhstan
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Abstract

Helicobacter pylori strains can be resistant to important triple combination therapies for H. pylori eradication. The aim of this study was to investigate the rate of resistance to clarithromycin, metronidazole, amoxicillin, tetracycline, and rifampicin, as well as to detect antibiotic resistance-associated mutations in H. pylori isolates from patients in Kazakhstan. Susceptibility of 20 H. pylori strains was tested using the E test method. Genes associated with resistance and susceptible clinical isolates were sequenced in order to assess resistance and non-resistance associated genetic alterations. Of the 20 clinical isolates examined, 8 (40%) showed phenotypic resistance to metronidazole (MIC > 256 mg/L), 13 (65%) to clarithromycin (MIC > 256 mg/L), and 1 (5%) to amoxicillin (MIC > 6 mg/L). The majority of resistant strains had point mutations in the 23S rRNA gene and rdxA gene, and one strain had mutations in the pbp1A gene. The remaining isolates with moderate resistant isolates (MIC <0.016 mg/L) demonstrated a drug-susceptible phenotype, and did not harbour any mutation in the gene sequences evaluated. In the Kazakh population of existing clarithromycin and metronidazole resistance, tetracycline, amoxicillin, and rifampicin could prove useful for rescue regimens in patients with previously unsuccessful H. pylori eradication regimens.

Keywords: Helicobacter pylori, clarithromycin, metronidazole, amoxicillin, tetracycline, rifampicin

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Author

page: 10-20 pp
DOI: 10.11134/btp.4.2015.2
Starovoitova S.A., Karpov A.V.
National University of Food Technologies
68, Vladimirskaya str., Kiev, 0160, Ukraine
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Abstract

This article summarizes previous studies investigating the influence of the microbiota on the immune system in health and disease. The main stages of formation of the human intestinal microflora and related factors, as well as its normal physiological functions are discussed. The diseases associated with disruption of the microbiota, in which the use of probiotics results in immunological disorders of varying nature and severity is also discussed. Moreover, we outline the mechanisms that regulate the diversification of immune responses to pathogenic and commensal microorganisms. The effects of normal flora microorganisms on innate and adaptive immunity are characterized. In addition, human inflammatory diseases associated with microbiota disorders are described. The biological properties of probiotics are reviewed in the context of their modulatory effect on the inflammatory response. Accordingly, the immunomodulatory potential of probiotic microorganisms is analysed. In conclusion, this article offers recommendations on strategies for effective application of the immunomodulatory activity of probiotic microorganisms and probiotics.

Keywords: gut microbiota, immunomodulation, immunobiotics, inflammation, probiotic functions.

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Author

page: 21-27 pp
DOI: 10.11134/btp.4.2015.3
Sergazy S.S.1,Shulgau Z.T. 2,Gulyayev A.E.1,Kabdulina A.A.1,Nurgozhin T.S.1
1 Nazarbayev University, Private institution «National Laboratory Astana»
53, Kabanbay batyr ave. Astana, Kazakhstan, 010000
2National Center for Biotechnology
13/5, Korgalzhyn Rd, Astana, Kazakhstan, 010000
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Abstract

Acute radiation syndrome (ARS) is one of the major problems in treating cancer patients using radiation therapy. ARS is caused by the death of rapidly dividing cells with short-term radiation. There is a strong correlation between ARS and the dose of ionizing radiation received by patients. Here, we describe the effect of a polyphenol concentrate of Cabernet Sauvignon grapes obtained from the Kazakhstan region on radiation sickness in rats. Acute radiation sickness in rats was induced by a single exposure to radiation at a dose of 8 Gray (Gy) by using a Rocus-AM equipment. The occurrence of radiation sickness was monitored by haematological analysis of blood collected from the tail vein of the rats one week after irradiation. The animals received a concentrate of polyphenols from grapes in therapeutic (after exposure) and prophylactic (one week before irradiation) forms. The oxidative status of rat plasma, measured as the amount of free radicals and the total antioxidant activity, was examined. The results demonstrate that a concentrate of grape polyphenols has radioprotective properties due to the observed increase in total plasma antioxidant activity. In addition, the polyphenols concentrate limited the development of pancytopenia.

Keywords: radiation sickness, polyphenols, radiation rays, grapes, radioprotective properties, antioxidant properties.

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Author

page: 28-37 pp
DOI: 10.11134/btp.4.2015.4
Tagimanova D.S., Novakovskaya A.P., Uvashov A.O., Khapilina O.N., Kalendar R.N.
National Center for Biotechnology
13/5 Korghalzhyn Road, Astana, 010000, Kazakhstan
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Abstract

The wild ancestor of all cultivated tetra- and hexaploid wheat, wild emmer wheat (Triticum dicoccoides), harbours considerable genetic diversity. This diversity might be expected to display eco-geographical patterns of variation, conflating gene flow, and local adaptation. Similarly, retrotransposons, as self-replicating entities comprising the bulk of genomic DNA in wheat, are expected to generate generally neutral variation due to their transpositional activity. Here, we examined the genetic diversity of 14 Israeli and one Turkish population of wild emmer wheat, based on the retrotransposon marker methods IRAP (Inter-Retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism). The level of genetic diversity we detected was in agreement with previous studies that have used a variety of marker systems to assay genes and other genomic components. The genetic distances failed to correlate with the geographical distances, suggesting local selection on geographically widespread haplotypes (“weak selection”). However, the proportion of polymorphic loci correlated with the latitude of the population and the genetic diversity correlated with the longitude. Principal component analysis of the marker data resulted in separation of some of the populations.

Keywords: Triticum dicoccoides, wild emmer wheat, IRAP, REMAP, genetic diversity.

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Author

page: 38-46 pp
DOI: 10.11134/btp.4.2015.5
Kulyyassov A.T.1, Zhubanova G.S.1, Ramanculov E.M.1, Ogryzko V.V.2
1National Center for Biotechnology
13/5, K orgalzhyn Rd, Astana, 010000,Kazakhstan
2Institut Gustave Roussy, CNRS UMR8126
94805, 39 Rue Camilles Desmoulin, Villejuif, France
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Abstract

The proximity utilizing biotinylation (PUB) method is based on co-expression within a single cell of recombinant proteins. It involves fusion of the protein of interest with biotin ligase BirA and that of its partner with the biotin acceptor peptide (BAP). The method allows an accurate quantitative assessment of the extent of their interaction in vivo.

The aim of this study was to construct a new plasmid vector containing human ubiquitin UbC fused with a BAP and to evaluate its expression in HEK293T cells.

To test the model system, we evaluated the protein expression of ubiquitin-BAP and BirA-HP1g (BirA-GFP, control) in HEK293T cells, and we observed a high specificity interaction (biotinylation) in the case of BirA-HP1g versus the control BirA-GFP. In another example of the model system, we observed higher levels of protein-protein interaction of BirA-Tip49 with different ubiquitinated proteins, compared to BirA-Tap54.

Keywords: protein-protein interactions, biotinylation, biotin ligase, biotin acceptor peptide, plasmids, transient transfection, western blot, ubiquitin.

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Author

page: 47-56 pp
DOI: 10.11134/btp.4.2015.6
Korotetskiy I.S., Zubenko N.V., Shilov S.V., Ivanova L.N., Shvidko S.V., Toxanbayev R.D.
Scientific Centre for Anti-infectious Drugs
84, Auezov srt. Almaty, Kazakhstan
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Abstract

Here, we describe the adaptation of influenza strains A/FPV/Weybridge/78 (H7N7) and A/Swine/Iowa/30 (H1N1) to the derivatives of adamantane (Rimantadine) and the neuraminidase inhibitor (Tamiflu). To determine the stability of the resistance phenotype, mutant strains were consecutively passaged in the absence of antiviral drugs for 5 passages. The IC50 value for FPV_RTam and Sw_RTam was >0.3 mg/ml for Tamiflu and >0.1 mg/ml for Rimantadine, respectively. Furthermore, the thermal stability of haemagglutinin and neuraminidase activity was determined in the study. Analysis of the amino acid sequence revealed the presence of a S31N substitution in the M2 protein of the FPV_RRim mutant strain, and an A30T substitution in Sw_RRim, which is responsible for the development of Rimantadine resistance. A specific substitution in position N207S of the amino acid sequences of the M1 protein in FPV_RTam and Sw_RTam mutants was also observed. Taken together, the results suggest that the nature of drug resistance is a multigene character.

Keywords: influenza virus, drug resistance, Tamiflu, Rimantadine, sequencing, mutation.

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Author

page: 57-65 pp
DOI: 10.11134/btp.4.2015.7
Kalimkulova M., Kiribayeva A., Mukhamedyarov D., Kulametov Zh., Akhmetollayev I., Silayev D., Khassenov B
National Center for Biotechnology
13/5, Korgalzhyn Road, Astana, 010000, Kazakhstan
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Abstract

Enzymes that mediate hydrolysis of starch-containing raw materials are widely used in the modern biotechnology industry. In the present study, a recombinant α-amylase, Amy1UA7, from Bacillus subtilis was generated in Escherichia coli, in order to study its biochemical parameters. The Amy1UA7 gene was synthesized by oligonucleotides and cloned in a pET-28c (+) vector under the control of the bacteriophage T7 promoter. Recombinant Amy1UA7 was obtained in BL21 (DE3) cells by plasmid gene expression; protein purification was carried out by metal affinity chromatography. During the study, the temperature and acid optimum of the recombinant enzyme was fixed, and the impact of metal ions and organic acids on enzymatic activity was determined. Analysis of the effect of temperature on the activity of Amy1UA7 demonstrated that the efficiency of starch hydrolysis by the recombinant α-amylase increased up to + 50°C to + 55°C, when amylase activity had a maximum value of 165 U. The results also demonstrated that amylase α-Amy1UA7 remains active in a wide pH range (4–9) while retaining more than 80% of its maximum activity. In addition, it is a calcium-independent enzyme that has tolerance to a range of metal ions and organic acids.

These data are essential for encouraging the use of recombinant enzymes in biotechnological processes involving the hydrolysis of polysaccharides.

Keywords: α-amylase, Bacillus, recombinant enzymes, amylase activity.

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