Реферат: Seven novel human leukocyte antigen (HLA)-DRB1 alleles were identified during routine sequence-based typing (SBT) of healthy Kazakh individuals.
Exploring the use of dimethylsulfate for in vivo proteome footprinting
Реферат: Protein footprinting is a new methodology that is based on probing, typically with the use of MS, of reactivity of different amino acid residues to a modifying reagent. Data thus obtained allow one to make inferences about protein conformations and their intermolecular interactions. Most of the protein footprinting studies so far have been performed on individual proteins in vitro. We explore whether a similar approach is possible with the proteins inside of living cells, employing dimethylsulfate (DMS), a reagent widely used for the in vivo footprinting of nucleic acids. DMS can induce methylation of the lysine, histidine and glutamate residues on proteins. Using models of the histone H2B/H2AZ heterodimer assembled in vitro and from chromatin treated in vivo, we show that the methylation by deuterated DMS allows one to distinguish the accessibility of a particular residue in and out of the protein\'s environmental/structural context. The detection of changes in protein conformations or their interactions in vivo can provide a new approach to the identification of proteins involved in various intracellular pathways and help in the search for perspective drug targets and biomarkers of diseases.
Molecular characterization of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Kazakhstan
Реферат: Kazakhstan is one of the 14 countries with a high rate of morbidity due to multidrug-resistant tuberculosis (MDR TB) in WHO European region. The aim of our study was to characterize mutations associated with drug resistance to rifampicin and isoniazid in Mycobacterium tuberculosis isolates from Kazakhstan. M. tuberculosis strains were isolated from TB patients in different regions of Kazakhstan. A drug susceptibility test was performed on Lowenstein-Jensen medium using the absolute concentration method. Sequencing analysis was performed of the rpoB rifampicin resistance-determining region and the katG gene, the oxyR-ahpC intergenic region, and the inhA promoter region in 259 MDR M. tuberculosis isolates, in 51 isoniazid-resistant isolates, and in 13 rifampicin-resistant isolates. The mutational analysis revealed that the most frequent mutations associated with rifampicin and isoniazid resistance in M. tuberculosis are the substitutions at codons 531 (82.7%) and 315 (98.4%) in the rpoB and katG genes, respectively. In addition, we have found mutations with lower frequency at codon 526 (8.4%), 533 (1.5%), and 516 (1.1%) in the rpoB gene. In 6.2% of the isolates, no mutations were found in the rpoB gene. The findings of this study provide useful data for a better understanding of the mutation spectrum of isoniazid and rifampicin resistance among strains isolated from patients in Kazakhstan. Our results are also useful for the development of diagnostic tests of MDR M. tuberculosis.
Bottom-up signaling from HGF-containing surfaces promotes hepatic differentiation of mesenchymal stem cells
Автор(ы): Ghaedi M.*Tuleuova N.*Zern M. A.*Wu J.*Revzin A.*
Реферат: The capacity of stem cells to differentiate into specific cell types makes them very promising in tissue regeneration and repair. However, realizing this promise requires novel methods for guiding lineage-specific differentiation of stem cells. In this study, hepatocyte growth factor (HGF), an important morphogen in liver development, was co-printed with collagen I (Col) to create arrays of protein spots on glass. Human adipose stem cells (ASCs) were cultured on top of the HGF/Col spots for 2 weeks. The effects of surface-immobilized HGF on hepatic differentiation of ASCs were analyzed using RT-PCR, ELISA and immunocytochemistry. Stimulation of stem cells with HGF from the bottom-up caused an upregulation in synthesis of alfa-fetoprotein and albumin, as determined by immunocytochemistry and ELISA. RT-PCR results showed that the mRNA levels for albumin, alfa-fetoprotein and alfa1-antitrypsin were 10- to 20-fold higher in stem cells cultured on the HGF/Col arrays compared to stem cells on Col only spots. Our results show that surfaces containing HGF co-printed with ECM proteins may be used to differentiate mesenchymal stem cells such as ASCs into hepatocyte-like cells. These results underscore the utility of growth factor-containing culture surfaces for stem cell differentiation.
PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo
Реферат: The common techniques to study protein-protein proximity in vivo are not well adapted to the capabilities and the expertise of a standard proteomics laboratory, typically based on the use of mass spectrometry. With the aim of closing this gap, we have developed PUB-MS (for proximity utilizing biotinylation and mass spectrometry), an approach to monitor protein-protein proximity, based on biotinylation of a protein fused to a biotin-acceptor peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The biotinylation status of the BAP can be further detected by either Western analysis or mass spectrometry. The BAP sequence was redesigned for easy monitoring of the biotinylation status by LC-MS/MS. In several experimental models, we demonstrate that the biotinylation in vivo is specifically enhanced when the BAP- and BirA-fused proteins are in proximity to each other. The advantage of mass spectrometry is demonstrated by using BAPs with different sequences in a single experiment (allowing multiplex analysis) and by the use of stable isotopes. Finally, we show that our methodology can be also used to study a specific subfraction of a protein of interest that was in proximity with another protein at a predefined time before the analysis.
Use of in vivo biotinylation for chromatin immunoprecipitation
Реферат: This unit describes a system for expression of biotinylated proteins in mammalian cells in vivo, and its application to chromatin immunoprecipitation (ChIP). The system is based on co-expression of the target protein fused to a short biotin acceptor domain, together with the biotinylating enzyme BirA from Escherichia coli. The superior strength of the biotin-avidin interaction in the modified ChIP protocol presented here allows one to employ more stringent washing conditions, resulting in a better signal/noise ratio. Methods for interpreting the data obtained from ChIP samples analyzed by qPCR, and methods for testing the efficiency of biotinylation using a streptavidin gel-shift are also presented. In addition, a complementary method, based on isothermal multiple strand displacement amplification (IMDA) of circular concatemers generated from the DNA fragments obtained after ChIP, is described. This method helps to decrease bias in DNA amplification and is useful for the analysis of complex mixtures of DNA fragments typically generated in miniscale ChIP experiments.
Francisella RNA polymerase contains a heterodimer of non-identical alfa subunits
Автор(ы): Mukhamedyarov D. A.*Makarova K. S.*Severinov K.*Kuznedelov K.*
Реферат: By in vitro assembly from fully denatured state, we determined that both Francisella alfa subunits are required for efficient dimerization; no homodimer formation was detected. Bacterial two-hybrid system analysis likewise indicated strong interactions between the alfa1 and alfa2 N-terminal domains (NTDs, responsible for dimerization). NTDs of alfa2 did not interact detectably, while weak interaction between alfa1 NTDs was observed. This weak homotypic interaction may explain low-level transcription activity observed in in vitro RNAP reconstitution reactions containing Francisella large subunits (beta\', beta) and alfa1. No activity was observed with RNAP reconstitution reactions containing alfa2, while robust transcription activity was detected in reactions containing alfa1 and alfa2. Phylogenetic analysis based on RpoA resulted in a tree compatible with standard bacterial taxonomy with both Francisella RpoA branches positioned within gamma-proteobacteria. The observed phylogeny and analysis of constrained trees are compatible with Francisella lineage-specific rpoA duplication followed by acceleration of evolutionary rate and subfunctionalization.
Identification of phenotypically and genotypically related Lactobacillus strains based on nucleotide sequence analysis of the groEL6 rpoB6 rplB6 and S rRNA genes
Автор(ы): Shevtsov A. B.*Kushugulova A. R.*Tynybaeva I. K.*Kozhakhmetov S. S.*Abzhalelov A. B.*Momynaliev K. T.*Stoyanova L. G.*
Реферат: Sixty_eight cultures of lactic acid bacteria were isolated and identified from national fermented milk drinks (airan, koumiss, kurunga, shubat) home_made in different regions of the Republic of Kazakhstan and the Buryat Republic of Russia. The cultures of lactic acid bacteria of the genus Lactobacillus were identified as L. paracasei and L. rhamnosus related to the L. casei group and as L. brevis, L. buchneri, L. diolivorans, and L. parabuchneri (the L. buchneri group) using the classical microbiological methods and on the basis of the S rRNA gene sequence analysis. The polymorphism of the nucleotide sequences of the genes groEL6 rpoB6 and rplB encoding specific proteins was studied for intraspecific differentiation of the lactobacilli. The analysis of these genes allowed a more accurate identification of the lactobacilli that are genetically and phenotypically related to the L. casei group as L. paracasei subsp. paracasei and L. paracasei subsp. tolerans. The gene nucleotide sequences of all the genotyped strains were deposited in the GenBank database.
Detection of Lactobacillus species using a gene fragment of the RNA polymerase beta subunit rpoB
Автор(ы): Shevtsov A. B.*Kushugulova A. R.*Tynybaeva I. K.*Kozhakhmetov S. S.*Oralbaeva S. S.*Stoyanova L. G.*Abzhalelov A. B.*Seidalina A. B.*Momynaliev K. T.*
Реферат: In this paper, we report on the comparative analysis of nucleotide sequences of the rpoB gene to assess its potential for use as a genetic marker for identification of Lactibacillus spp. recovered from acid milk products in different regions of Kazakhstan. The discriminatory power of the rpoB gene sequence was shown to be significantly higher for identification of closely related species of the Lactobacillus genus as compared to that of the S rRNA gene. The phylogeny based on the rpoB gene proved identical to that of the S rRNA gene and could be used as a supplement phylogenetic marker.
Synovial membrane derived mesenchymal stem cells in hyaluronic acid scaffold transplantation for cartilage defect repair
Автор(ы): Ogay V. B.*Bekbolsynov D. A.*Mukhambetova A. E.*Batpenov N. D.*Raimagambetov E. K.*Ramankulov Ye. M.*
Объем документа: С. 366
МРНТИ: 76.03.43
Ключевые слова: дефект хряща*трансплантация синовиальной оболочки*стволовые клетки мезенхимальные*
Реферат: Synovial membrane-derived mesenchymal stem cells (SM-MSCs) are known for their high proliferative and regenerative capacities6 thereby presenting an area of significant interest for applications in tissue engineering and regenerative medicine. The objective of this study was to evaluate the efficiency of the hyaluronic acid (HA) supplement OstenilR as a scaffold for noninvasive autologous SM-MSC transplantation to regenerate damaged cartilage in an animal model.