RESUMEN
Splice variant and authentic mRNAs for procathepsin E were measured at a ratio of 5:1 and 1:2 in Kato 3 and AGS cells, two human gastric adenocarcinoma cell lines. As a result of the precise splicing of the 3'-end of exon 6 to the 5'-end of exon 8, the variant lacked the 142 bp of exon 7 which encodes the second of the Asp residues that operate the catalytic mechanism of aspartic proteinases.
Asunto(s)
Adenocarcinoma/metabolismo , Catepsina E/genética , Catepsinas/genética , Precursores Enzimáticos/genética , Neoplasias Gástricas/metabolismo , Empalme Alternativo , Catepsina E/metabolismo , Catepsinas/metabolismo , Precursores Enzimáticos/metabolismo , Exones , Variación Genética , Humanos , Intrones , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
The two human pronapsin genes which are located immediately adjacent to one another on chromosome 19 were shown, using quantitative RT-PCR, to have distinctly different expression patterns. Transcription of the pronapsin A gene in normal human lung tissue was not appreciably altered in lung carcinomas and comparable pronapsin A mRNA levels were also quantified in lung epithelial cell lines and in tumour cell lines originating from human lung epithelium. Pronapsin A may thus have considerable diagnostic value as a marker for primary lung cancer. In contrast, the pronapsin B gene, which lacks an in-frame stop codon and so may be a transcribed pseudogene, is expressed at comparable levels in normal human spleen, thymus, cytotoxic and helper T-lymphocytes, natural killer (NK) cells and B-lymphocytes. The mRNA levels quantified in B-cells from adults with chronic lymphoblastic leukaemia were not significantly different from those measured in B-cells from healthy individuals. Similarly, comparable levels of expression were quantified in monocytes from healthy individuals and from a patient with acute myeloid leukaemia, whereas in alveolar macrophages, which are terminally differentiated myeloid cells, transcription of the pronapsin B gene was down-regulated by approximately one order of magnitude. Reciprocally, an approximately 20-fold up-regulation in expression of the procathepsin D gene in the macrophages relative to the circulating monocytes was revealed by quantitative measurements made in parallel throughout all of this study for the respective mRNAs encoding the intracellular aspartic proteinases, procathepsin D and procathepsin E. Thus, while there appears to be little difference in expression of the pronapsin A and B genes between healthy and malignant human cells and tissues, the reciprocal alterations in the levels of transcription of the pronapsin B and procathepsin D genes may have significance in the developmental processes associated with myeloid cell differentiation into macrophages.
Asunto(s)
Ácido Aspártico Endopeptidasas/biosíntesis , Carcinoma/enzimología , Precursores Enzimáticos/biosíntesis , Leucocitos Mononucleares/enzimología , Neoplasias Pulmonares/enzimología , Pulmón/enzimología , Enfermedad Aguda , Ácido Aspártico Endopeptidasas/genética , Carcinoma/genética , Catepsina D/biosíntesis , Catepsina D/genética , Catepsina E/biosíntesis , Catepsina E/genética , Células Cultivadas , Precursores Enzimáticos/genética , Humanos , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Mieloide/enzimología , Leucemia Mieloide/genética , Neoplasias Pulmonares/genética , Linfocitos/enzimología , Células Mieloides/enzimología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , ARN Mensajero/biosíntesis , Bazo/enzimología , Timo/enzimología , Transcripción GenéticaRESUMEN
The 1.2 mm Electric Coring Tool (e-Core™) was developed to increase the throughput of FTA(™) sample collection cards used during forensic workflows and is similar to a 1.2 mm Harris manual micro-punch for sampling dried blood spots. Direct short tandem repeat (STR) DNA profiling was used to compare samples taken by the e-Core tool with those taken by the manual micro-punch. The performance of the e-Core device was evaluated using a commercially available PowerPlex™ 18D STR System. In addition, an analysis was performed that investigated the potential carryover of DNA via the e-Core punch from one FTA disc to another. This contamination study was carried out using Applied Biosystems AmpflSTR™ Identifiler™ Direct PCR Amplification kits. The e-Core instrument does not contaminate FTA discs when a cleaning punch is used following excision of discs containing samples and generates STR profiles that are comparable to those generated by the manual micro-punch.