RESUMO
Escherichia coli vectors were constructed for the production of a protein complex that mimics the native ecdysone receptor (EcR) isolated from Drosophila. The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains. The recombinant receptor complex was able to mimic the pharmacology of the native receptor with respect to both synthetic and natural agonists. USP and EcR fusion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociation constant (K(D)) for Ponasterone A of 1.5 nM and a total yield of 1.9 pmol ligand binding sites/mg protein. Alternatively, the simultaneous coexpression of both receptors increased yields by several orders of magnitude to 6 nmol ligand binding sites/mg protein with a K(D) of 0.6 nM. Chromatographic analysis under native conditions showed that EcR, when expressed alone, migrated as a variety of complexes, mostly coming out in the void volume as denatured, insoluble, aggregate. In contrast, purified extracts of coexpressed EcR and USP eluted as a single peak with a mobility indicating a heterodimer. The majority of the coexpressed fusion receptors, following purification, formed functional steroid binding sites. A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Drosophila/metabolismo , Ecdisona/metabolismo , Receptores de Esteroides/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Fatores de Transcrição/biossíntese , Animais , Cromatografia Líquida , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Drosophila , Escherichia coli/metabolismo , Vetores Genéticos , Histidina/genética , Peptídeos/genética , Estrutura Terciária de Proteína , Receptores de Esteroides/química , Receptores de Esteroides/genética , Receptores de Esteroides/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Tetra-Hidrofolato Desidrogenase/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Faced with many patients with comorbid severe mental illness and substance use disorders, a university-affiliated, inner-city community mental health center and a psychosocial rehabilitation center initiated this clinical trial assessing a program to care for these patients. Fifty-four patients, age 18 to 40, with either schizophrenia or major affective disorder and a substance use disorder were randomly assigned to usual community mental health center and rehabilitation services with or without an innovative group and intensive case management program. One-year follow-ups detected no significant advantages on patient outcomes for adding the innovative program to usual services. Failure to engage patients in the experimental program posed a major and enduring barrier to treatment, despite intensive case management. Future efforts must give greater consideration to effective engagement techniques and patients' readiness for active treatment.
