15 research outputs found
Analysis of the association of the polymorphism of the CLIC1, MSH5, C6orf26, C6orf25 genes with the expression level of the HSPA1B gene
Heat shock proteins (HSP, heat shock proteins) form one of the cellular molecular systems with chaperone activity, aimed at stabilizing the structure of intracellular proteins, ensuring the resistance of cells to stress, renaturation of incorrectly folded and elimination of denatured intracellular proteins.Our task was to look for an association between the presence of single nucleotide polymorphisms in selected regions of the genome and the basal level of transcriptional activity of the HSPA group genes, namely HSPA1A/B, HSPA1A, HSPA1B, HSPA6 and HSPA8 in mononuclear leukocytes (PBMC), analyzed in our experiments volunteers from the population of the middle part of Russia in order to analyze the universality of the biological effects of these SNPs.The study was performed on DNA and RNA isolated from peripheral blood lymphocytes of 16 donors. Genotyping was performed by the polymerase chain reaction (PCR) followed by sequencing of the PCR product. To assess the level of gene expression of the HSPA group, cDNA was synthesized on an RNA template isolated from PBMC cell fraction samples, followed by real-time polymerase chain reaction.The following types of polymorphisms were genotyped: rs400547 (A/G), rs1150793 (G/A), rs707936 (A/G), rs707915 (A/T), rs376510 (T/C) located in the CLIC1, MSH5, C6orf26, MSH5, C6orf25genes, respectively. We determined the basal level of transcription of genes of constitutively expressed and inducible proteins of the HSP70 family and searched for the association of their expression with polymorphisms.It was found that in PBMC cells, DNA that has the following genotype: AG/AA (SNP rs400547), AG/GG (rs1150793), AG (rs707936), TArs707915, TC (rs376510), in the regions we studied, is associated with a decrease in the transcription of the HSPA1B gene (p = 0.02) compared with homozygotes: GG (SNP rs400547), AA (rs1150793), GG (rs707936), TT (rs707915), CC (rs376510).Associations of these polymorphisms with gene expression of HSPA1A, HSPA6 and HSPA8 have not been identified.The CLIC1, MSH5, C6orf26, C6orf25 genes, in which the polymorphisms studied by us are present, are located in the same locus near the HSPA1B gene on the 6th chromosome. We found a decrease in HSPA1B gene expression in the presence of single nucleotide polymorphisms in nearby genes may indicate spatial interactions of this locus and the HSPA1B gene locus, and that a change in the genotype CLIC1, MSH5, C6orf26, C6orf25 may entail a change in the expression of closely arranged genes which are functionally significant for the cell
New insights into the mechanism of action of the thienopyrimidine antitubercular prodrug TP053
The thienopyrimidine TP053 is an antitubercular prodrug active against both replicating and non-replicating Mycobacterium tuberculosis cells, which requires activation by the mycothiol-dependent nitroreductase Mrx2. Investigation of the mechanism of action of TP053 revealed that Mrx2 releases nitric oxide from this drug both in the enzyme assays with purified Mrx2 and in mycobacterial cultures, which can explain its activity against non-replicating bacilli, similar to pretomanid activated by the nitroreductase Ddn. In addition, we identified a highly reactive metabolite, 2-(4-mercapto-6-(methylamino)-2-phenylpyrimidin-5-yl)ethan-1-ol, which can contribute to the antimycobacterial effects on replicating cells. In summary, we explained the mechanism of action of TP053 on both replicating and non-replicating M. tuberculosis and report a novel activity for Mrx2, which in addition to Ddn, represents another example of nitroreductase releasing nitric oxide from its substrate. These findings are particularly relevant in the context of drugs targeting non-replicating M. tuberculosis, which were shown to be killed by increased levels of nitric oxide
Genome sequencing in clinical microbiology
The remit of the clinical microbiology laboratory encompasses analysis of samples, detection and identification of microorganisms associated with infection, determination of antibiotic susceptibilities and monitoring of pathogen spread. Although several new molecular approaches, such as nucleic acid amplification, mass spectrometry and sequence typing, have been adopted to varying extents, many of the techniques in use today would have been familiar to a nineteenth-century microbiologist