RESUMO
S-Tubercidinylhomocysteine (STH) is a structural analog of S-adenosylhomocysteine and a potent inhibitor of S-adenosylmethionine-dependent methyltransferase reactions. We investigated the effects of STH on HeLa cell mRNA metabolism. Dual labeling studies reveal that STH dramatically inhibits the methylation of HeLa mRNA in a dose-dependent manner. Analysis of the modified nucleosides and 5'-terminal cap structures in radiolabeled mRNA by high-pressure liquid chromatography indicated that internal N6-methylation of adenosine was reduced by 65% at 50 microM STH and by 83% at 500 microM STH. The N6-methylation of adenosine contained in cap structures was similarly reduced at both concentrations of STH. Substantial amounts of cap structures lacking 2'-O-methylated nucleosides (m7GpppN, cap zero) were detected at the higher level of STH. To test the possibility that methylation affects mRNA stability, cytoplasmic mRNA half-life was measured in a pulse-chase experiment. The half-life of undermethylated mRNA, produced as a consequence of STH treatment, was unchanged compared with the control. To determine whether mRNA methylation is coupled to nuclear processing or transport, the time of cytoplasmic appearance of polyadenylated RNA in STH-treated HeLa cells was compared with untreated cells. STH caused a significant lag in the time of appearance of the polyadenylated RNA, suggesting that mRNA methylation may be required for efficient processing or transport.
Assuntos
Homocisteína/análogos & derivados , Poli A/metabolismo , RNA Mensageiro/metabolismo , RNA/metabolismo , S-Adenosil-Homocisteína/análogos & derivados , Cromatografia Líquida de Alta Pressão , Meia-Vida , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Cinética , Metilação , Poli A/isolamento & purificação , RNA/isolamento & purificação , RNA Neoplásico/metabolismo , S-Adenosil-Homocisteína/farmacologiaRESUMO
Block poly(Ala)16-poly(Lys)13.5 was synthesized by the Leuchs anhydride method. This polypeptide is water soluble in a largely monomeric form, but binds rapidly and spontaneously to unilamellar vesicles of dimyristoyl phosphatidylcholine at pH 7.4. The interaction is evidently of a hydrophobic nature since the complex is not disrupted by salt and no similar reaction is given by polylysine. Evidence for the interaction was obtained by ultrafiltration, chromatography on Sepharose 4B, and sedimentation velocity ultracentrifugation. While direct information on the molecular structure of the complex is still lacking, we propose that this amphipathic block copolymer binds to lipids in a similar manner as intrinsic membrane proteins and hence can be used to study the interactions of intrinsic proteins with lipids.
Assuntos
Proteínas de Membrana , Peptídeos , Polilisina , Peptídeos e Proteínas de Sinalização Intercelular , Lipídeos de Membrana , Modelos Biológicos , Fosfatidilcolinas , Ligação Proteica , Solubilidade , Ultracentrifugação , ÁguaRESUMO
Most attrition research has focused on dropping out once the patient has arrived at the clinic. However, many potential patients, who are in need of service, do not contact clinics. Such patients are said to be in the appraisal stage of treatment. Discontinuation of appraisal, for whatever reason, can be termed "attrition." Such attrition has not received adequate attention. A conceptual model of the appraisal process is given, and a review is made of current research bearing on the conditions under which appraisals continued or discontinued by an individual. Recommendations for future research are made.
