ABSTRACT
Herein, we demonstrate that purified Stat1 binds to its tyrosine-phosphorylated docking site on the IFN gamma receptor alpha chain in a direct, specific, and reversible manner. Using surface plasmon resonance, we determine the affinity (KD = 137 nM) and specificity of the interaction and define the minimum affinity needed for receptor-mediated Stat1 activation. In addition, we quantitate the relative ability of purified Stat1 to interact with tyrosine-phosphorylated binding sites on other Stat proteins. Finally, we describe experiments that imply that the unidirectional release of activated Stat1 from the IFN gamma receptor reflects the preference of free tyrosine-phosphorylated Stat1 monomers to form high avidity reciprocal homodimers rather than reassociating with the receptor binding site. Our results demonstrate that IFN gamma-induced Stat1 activation is an ordered and affinity-driven process and we propose that this process may serve as a paradigm for Stat activation by other cytokine receptors.
Subject(s)
DNA-Binding Proteins/metabolism , Phosphopeptides/metabolism , Receptors, Cytokine/metabolism , Trans-Activators/metabolism , Amino Acid Sequence , Binding, Competitive , Biosensing Techniques , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/isolation & purification , Humans , Interferon-gamma/physiology , Molecular Sequence Data , Phosphorylation , Protein Binding/physiology , Protein-Tyrosine Kinases/metabolism , Receptors, Interferon/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , STAT1 Transcription Factor , Signal Transduction , Trans-Activators/chemistry , Trans-Activators/isolation & purification , Interferon gamma ReceptorABSTRACT
O teste ELISA ant-HTLVI/II foi introduzido na triagem sorlógica de doadores de sangue na Fundaçäo Pró-sangue Hemocentro de Säo Paulo (FPS/HSP) em julho de 1991. NO período compreendido entre julho de 1991 e julho de 1994 foram submetidos à triagem serológica 597.727 doadores. Destes, 7682 foram recusados por terem apresentado reatividade no teste ELISA anti-HTLVI/II. A positividade observada, para o referido teste, foi diminuindo com o correr do tempo: 2,12 por cento em 1991; 1,6 por cento em 1992; 0,8 por cento em 1993 e 1,0 por cento em 1994, sendo esse fato atribuido a melhora da especialidade e reprodutividade dos kits comerciais. Foi utilizado o teste suplementar de Western Blot para confirmar os resultados dos testes ELISA. Em 249 amostras de soros de doadores, com resultado repetidamente positivo (RRP) no teste ELISA (hemobio), o poder confirmatório do Western Blot (Cambridge Biotech) foi de 24.9 por cento (IC/90 por cento: 20,4 por cento-29,39 por cento). Baseados nesses dados, considera-se uma expectativa de prevalência de indivíduos infectados pelo HTLVI/II, na populaçäo de doadores de sangue da FPS/HSP de 0,142 por cento (IC/90 por cento; 0,116 por cento-0,167 por cento). Em 437 amostras de soro de doadores que retornaram ao Banco de Sangue, para confirmar o resultado inicial e apresentaram RRP no teste ELISA, o poder confirmatório do Western Blot foi de 34,55 por cento (IC/90 por cento: 30,82 por cento-38,28 por cento)...
Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Blood Donors , HTLV-I Antibodies/isolation & purification , HTLV-II Antibodies/isolation & purification , HTLV-I Infections/epidemiology , HTLV-II Infections/epidemiology , Blotting, Western , Enzyme-Linked Immunosorbent Assay , PrevalenceABSTRACT
We present measurements of the intensity autocorrelation function of the output of a free-running AlGaAs diode laser. To our knowledge these are the first such measurements performed on the output of a semiconductor laser. Our data display large structure at the relaxation oscillation frequency and reveal features that depend on the number of secondary modes lasing. This provides evidence that, even in so-called single-mode diode lasers, these side modes have a significant effect on the fluctuations of the total output of the laser. The autocorrelation function in fact can be used to measure how many modes contribute to the fluctuating intensity. The major features in the measured intensity autocorrelation function are in good agreement with the predictions of a simple multimode phase-diffusion model.
