RESUMO
A human melanoma cell line (Bowes), which secretes extrinsic (tissue-type) plasminogen activator, was used as a source for the preparation of mRNA for extrinsic plasminogen activator. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)-rich RNA fraction was isolated by affinity chromatography on oligo(dT)-cellulose. This preparation was translated by oocytes into (a) protein(s) that had biological activity in the assay for extrinsic plasminogen activator. On sucrose gradient centrifugation the translatable fraction of the RNA migrated with a sedimentation coefficient of approximately 19 S, a value compatible with the molecular weight of extrinsic plasminogen activator (70 000). The translation product was characterized as being similar to or identical with authentic extrinsic plasminogen activator by the following criteria: (a) serological cross-reactivity with purified extrinsic plasminogen activator in neutralization and immunoprecipitation reactions; (b) plasminogen dependency of fibrinolytic activity and (c) apparent molecular weight of 70 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis.
Assuntos
Ativadores de Plasminogênio/genética , RNA Mensageiro/isolamento & purificação , Animais , Linhagem Celular , Feminino , Humanos , Melanoma/metabolismo , Neoplasias Experimentais/metabolismo , Oócitos/metabolismo , Biossíntese de Proteínas , RNA Neoplásico/isolamento & purificação , Xenopus laevisAssuntos
Interferons/genética , Linfócitos/imunologia , RNA Mensageiro/isolamento & purificação , Animais , Cromatografia de Afinidade , Feminino , Humanos , Linfócitos/efeitos dos fármacos , Oócitos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Acetato de Tetradecanoilforbol/farmacologia , XenopusRESUMO
Interferon was induced in leukocyte suspensions from human buffy coats by exposure to phytohemagglutinin P, concanavalin A (Con A) and staphylococcal enterotoxin A, under a variety of cell culture conditions. Con A was found to rapidly (within 12 h) induce high yields of antiviral activity (1.5 units/1000 cells). Lesser yields were obtained with the other two mitogens studied. The interferon was partially purified to a spec. act. around 10(5.3) units/mg protein, by batch adsorption on controlled-pore glass (CPG) beads and desorption by ethylene glycol. This material was characterized as containing mainly gamma-type interferon. Specifically on gel filtration, a fraction of 45000 daltons was obtained which could account for virtually all antiviral activity present in the starting material. Furthermore, the ethylene glycol-eluted antiviral activity was acid-labile, serologically distinct from alpha and beta-type interferon and strictly species-specific (no activity detectable on any of the available cell species sensitive to alpha and beta-type interferon). The crude culture supernatant also contained some antiviral activity which resembled beta-type interferon in that it adsorbed to CPG, could be desorbed by pH 2 buffer, was acid-resistant and could be neutralized by a specific anti-fibroblast interferon antiserum. The CPG/ethylene glycol-purified gamma-type interefron preparation was found to inhibit the growth of certain lymphoblastoid cells (Daudi and Molt-4). It also potentiated natural killer activity of fresh donor lymphocytes. In both respects, the gamma-type interferon preparation was not significantly more active than preparations of alpha and beta-interferon of similar antiviral potency.
