ABSTRACT
Analysis of variations in DNA structure using a low-density microarray technology for routine diagnostic in evidence-based medicine is still relevant. In this work the applicability of 3-D macroporous monolithic methacrylate-based platforms for detection of different pathogenic genomic substitutions was studied. The detection of nucleotide replacements in F5 (Leiden G/A, rs6025), MTHFR (C/T, rs1801133) and ITGB3 (T/C, rs5918), involved in coagulation, and COMT (C/G, rs4818), TPH2 (T/A, rs11178997), PON1 (T/A rs854560), AGTR2 (C/A, rs11091046) and SERPINE1 (5G/4G, rs1799889), associated with pregnancy complications, was performed. The effect of such parameters as amount and type of oligonucleotide probe, amount of PCR product on signal-to-noise ratio, as well as mismatch discrimination was analyzed. Sensitivity and specificity of mutation detections were coincided and equal to 98.6%. The analysis of SERPINE1 and MTHFR genotypes by both NGS and developed microarray was performed and compared.
Subject(s)
Genome, Human , Methacrylates/chemistry , Oligonucleotide Array Sequence Analysis , Pregnancy Complications/genetics , Aryldialkylphosphatase/genetics , Base Sequence , Catechol O-Methyltransferase/genetics , Ethylene Glycols , Female , Genotype , Humans , Integrin beta3/genetics , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , Mutation , Plasminogen Activator Inhibitor 1/genetics , Porosity , Pregnancy , Tryptophan Hydroxylase/geneticsABSTRACT
The participation of proteasome in the programmed cells death is now extensively investigated. Studies using selective inhibitors of proteasomes have provided a direct evidence of both pro- and anti-apoptotic functions of proteasomes. Such opposite roles of 26S proteasomes in regulation of apoptosis may be defined by the proliferative state of cell. The induction of apoptosis in K562 cells by diethylmaleate was used as a model to investigate changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes undergoing the programmed cell death. Here we have shown that proteasomes isolated from the cytoplasm of control and diethylmaleate treated K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. It has been shown for the first time that proteolytic activity of 26S proteasomes is decreased, and endoribonuclease activity of 26S proteasomes is affected under diethylmaleate action on K562 cells. Treatment of K562 cells with an inductor of apoptosis--diethylmaleate--leads to modification of a proteasomal subunit (zeta/alpha5) associated with RNase activity of proteasomes. These data suggest the subunit composition and enzymatic activities of 26S proteasomes to be changed in K562 cells undergoing apoptosis, and that specific subtypes of 26S proteasomes participate in execution of programmed death of these cells.
Subject(s)
Apoptosis , Proteasome Endopeptidase Complex/metabolism , Humans , K562 Cells/drug effects , K562 Cells/metabolism , K562 Cells/physiology , Maleates/pharmacology , Molecular Weight , Phosphorylation , Proteasome Endopeptidase Complex/chemistry , Ribonucleases/metabolism , Threonine , TyrosineABSTRACT
In eukaryotic cells the population of proteasomes is heterogeneous. Here we have shown that proteasomes from nuclei and cytoplasm of rat liver cells differ in their subunit patterns. The subunit pattern of alpha-RNP differs from that of proteasomes, however, alpha-RNP particles contain the number of 26S proteasome subunits. Moreover, the proteasomes contain subunits of alpha-RNP. We have shown for the first time that nuclear proteasomes and alpha-RNP are hyperphosphorylated on threonine residues. Differences in phosphorylation state of subunits of nuclear and cytoplasmic proteasomes and alpha-RNP on threonine and tyrosine residues have been revealed. A suggestion is put forward that hyperphosphorylation of subunits may determine nuclear localization of these complexes in rat liver cells. The results obtained suggest that a highly specialized system of protein kinases and phosphatases may be involved in the regulation of phosphorylation state of different populations of proteasomes and alpha-RNP in rat liver cells.
