RESUMO
Prion diseases are fatal neurodegenerative diseases which occur as sporadic, genetic, and transmissible disorders. A molecular hallmark of prion diseases is the conformational conversion of the host-encoded cellular form of the prion protein (PrPC) into its misfolded pathogenic isoform (PrPSc). PrPSc is the main component of the pathological and infectious prion agent. The study of the conversion mechanism from PrPC to PrPSc is a major field in prion research. PrPC is glycosylated and attached to the plasma membrane via its glycosyl phosphatidyl inositol (GPI)-anchor. In this study we established and characterised the expression of fully posttranslationally modified mammalian Syrian golden hamster PrPC in the yeast Pichia pastoris using native PrPC-specific N- and C-terminal signal sequences. In vivo as well as in vitro-studies demonstrated that the signal sequences controlled posttranslational processing and trafficking of native PrPC, resulting in PrPC localised in the plasma membrane of P. pastoris. In addition, the glycosylation pattern of native PrPC could be confirmed.
Assuntos
Pichia/química , Pichia/genética , Proteínas PrPSc/química , Proteínas PrPSc/genética , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular Transformada , Cricetinae , Vetores Genéticos , Glicosilação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mesocricetus , Pichia/metabolismo , Proteínas PrPSc/metabolismo , Processamento de Proteína Pós-Traducional/genética , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/genética , TransfecçãoRESUMO
BACKGROUND AIMS: Amongst different stem cell populations derived from human cord blood (CB), unrestricted somatic stem cells (USSC) are distinguished from CB mesenchymal stromal cells (CB MSC) by expression patterns of homeobox (HOX) genes, delta-like1 homolog (DLK1) expression and adipogenic differentiation potential. In this study we investigated the effects of oxygen tension on the generation, proliferation and expression of stem cell marker genes, which could be critical during large-scale cell culture for clinical applications. METHODS: We cultured CB-derived stem cells at 5% and 20% O(2). Telomere length shortening was analyzed and we investigated gene expression using reverse-transcription (RT)-polymerase chain reaction (PCR) and real-time PCR. Additionally we performed adipogenic and osteogenic in vitro differentiation. Results. Altering the cultivation conditions of USSC or CB MSC from 20% to 5% O(2) had no significant impact. In contrast, cell populations derived from primary cultures prepared at 5% O(2) qualified as neither USSC nor as CB MSC. When converted to 20%, their proliferation was diminished, telomere shortening was accelerated, and two of six cell lines ceased expression of HOX genes. The HOX code of the other cell populations was not been affected by culture conditions. CONCLUSIONS: Altering culture conditions during generation can impact cell characteristics such as the HOX code. These effects need to be considered when dealing with cell cultures for clinical applications.