Infection with SIVmac251 in some rhesus monkeys (Macaca mulatta) leads to B-cell non-Hodgkin's lymphomas (B-NHL) clinically similar to that of HIV-infected AIDS patients. To further characterize the SIV-associated B-NHL we have generated genetic profiles of malignant cells by subtractive hybridization and Northern blot analysis. We have analyzed 21 clones of a subtracted cDNA library corresponding to overexpressed genes in diffuse large B-cell (DLBCL) SIV-associated monkey lymphoma. Eight of these clones represent a sequence homologous to an abundant transcript from KG-1 cells originally established from a human myelogenous leukemia. The protein encoded has a 60% similarity to a hypothetical glycine-rich transmembrane signal protein of Caenorhabditis elegans and 25% similarity to the ret finger protein. The other cDNA clones contained sequences of the serum amyloid A gene (SAA), the alpha1-acid glycoprotein gene (AGP), the ribosomal protein S3a (RPS3a) and L8 (RPL8) genes, the interferon-inducible gene (INF-ind), the metastasin gene (mts1), and the NADH dehydrogenase I gene (ND-I). The remaining cDNA clones consisted of yet unknown sequences. In addition, we detected an up-regulation of the cytochrome c oxidase II gene (COX-II), the ND-IV gene, and the SET oncogene by Northern blot hybridization in three SIV-associated NHLs of different histomorphological classification. All these genes have not previously been found to be overexpressed in B-NHL.
Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.
The aim of the study was to obtain induced pluripotent stem cells (iPSCs) from patients with various forms of Parkinson's disease (PD), and to create on this basis a platform for studying the pathogenesis of the disease at the molecular and cellular level with the development of a protocol of the stem cell differentiation.Materials and Methods. iPSCs were derived from cultured skin fibroblasts, taken from five patients with various forms of PD (PARK8, PARK2, GBA-associated and sporadic forms), and reprogrammed with the help of lentiviral vectors and on the basis of Sendai virus. The obtained iPSCs clones were cultured to the stage of embryonic bodies and, after spontaneous differentiation, stained immunocytochemically. Gene expression and neural markers in these iPSCs lines were analysed using reverse transcription polymerase chain reaction.Results. The obtained iPSCs clones had a normal 46 XY karyotype, stained specifically with Oct4, Nanog, TRA-1-81 and SSEA-4 antibodies, and expressed marker genes responsible for maintaining the pluripotent condition. In the cultures of differentiated iPSCs, cells positively stained for the markers of the three primary germ layers (ectoderm, mesoderm, and endoderm) have been revealed. An effective protocol of iPSCs differentiation into dopaminergic neurons has been worked out, and confirmed by the expression of the specific markertyrosine hydroxylase enzyme.Conclusion. On the basis of explicitly characterized iPSCs from patients with various forms of PD and the developed cellular protocol, a platform for studying the pathogenesis of PD at the molecular and cellular level has been created. Obtaining cell population enriched with dopaminergic neurons opens a perspective for their application for personalized cell replacement PD therapy.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.