RESUMEN
BACKGROUND: Opioid agonist activation at the mu opioid receptor (MOR) can lead to a wide variety of physiological responses. Many opioid agonists share the ability to selectively and preferentially activate specific signaling pathways, a term called biased agonism. Biased opioid ligands can theoretically induce specific physiological responses and might enable the generation of drugs with improved side effect profiles. METHODS: Dynorphins, enkephalins, and endomorphins are endogenous opioid agonist peptides that may possess distinct bias profiles; biased agonism of endogenous peptides could explain the selective roles of these ligands in vivo. Our purpose in the present study was to investigate biased signaling and potential underlying molecular mechanisms of bias using 35S-GTPγS and cAMP assays, specifically focusing on the role of adenylyl cyclases (ACs) and regulators of G-protein signaling proteins (RGSs) in CHO, N2a, and SH-SY5Y cell lines, all expressing the human MOR. RESULTS: We found that endomorphin-1/2 preferentially activated cAMP signaling, while dynorphin-B preferentially activated 35S-GTPγS signaling in most cell lines. Experiments carried out in the presence of an isoform selective RGS-4 inhibitor, and siRNA knockdown of AC6 in N2a cells did not significantly affect the bias properties of endomorphins, suggesting that these proteins may not play a role in endomorphin bias. CONCLUSION: We found that endomorphin-1/2 and dynorphin-B displayed contrasting bias profiles at the MOR, and ruled out potential AC6 and RGS4 mechanisms in this bias. This identified signaling bias could be involved in specifying endogenous peptide roles in vivo, where these peptides have low selectivity between opioid receptor family members.
Asunto(s)
Dinorfinas/farmacología , Endorfinas/farmacología , Oligopéptidos/farmacología , Receptores Opioides mu/agonistas , Adenilil Ciclasas/genética , Animales , Células CHO , Técnicas de Cultivo de Célula , Línea Celular , Cricetulus , AMP Cíclico/metabolismo , Técnicas de Silenciamiento del Gen , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Proteínas RGS/antagonistas & inhibidores , Receptores Opioides mu/genética , Transducción de SeñalRESUMEN
As percutaneous cardiac interventions continue to evolve, high-risk procedures are being performed on patients deemed too ill for surgery. What were once considered lower-risk procedures compared with open cardiac interventions may no longer be so because of the complex nature of the interventions and the complex comorbidities of the patients on whom these are being performed. We present a case involving high-risk percutaneous cardiac interventions (left main coronary angioplasty and aortic balloon valvuloplasty), facilitated by the use of extracorporeal membrane oxygenation in a critically ill patient with severe aortic stenosis, left main coronary disease, and ischemic cardiomyopathy.
Asunto(s)
Fibrosis Quística/complicaciones , Arteria Hepática , Trasplante de Hígado/métodos , Trasplante de Pulmón/métodos , Trombosis/terapia , Disfunción Ventricular Derecha/complicaciones , Anestesia , Antifibrinolíticos/uso terapéutico , Femenino , Humanos , Complicaciones Intraoperatorias/tratamiento farmacológico , Complicaciones Intraoperatorias/terapia , Complicaciones Posoperatorias/tratamiento farmacológico , Complicaciones Posoperatorias/terapia , Trombosis/complicaciones , Trombosis/tratamiento farmacológico , Función Ventricular Derecha , Adulto JovenRESUMEN
The pathogen group A Streptococcus (GAS) produces a wide spectrum of infections including necrotizing fasciitis (NF). Streptolysin S (SLS) produces the hallmark beta-haemolytic phenotype produced by GAS. The nine-gene GAS locus (sagA-sagI) resembling a bacteriocin biosynthetic operon is necessary and sufficient for SLS production. Using precise, in-frame allelic exchange mutagenesis and single-gene complementation, we show sagA, sagB, sagC, sagD, sagE, sagF and sagG are each individually required for SLS production, and that sagE may further serve an immunity function. Limited site-directed mutagenesis of specific amino acids in the SagA prepropeptide supports the designation of SLS as a bacteriocin-like toxin. No significant pleotrophic effects of sagA deletion were observed on M protein, capsule or cysteine protease production. In a murine model of NF, the SLS-negative M1T1 GAS mutant was markedly diminished in its ability to produce necrotic skin ulcers and spread to the systemic circulation. The SLS toxin impaired phagocytic clearance and promoted epithelial cell cytotoxicity, the latter phenotype being enhanced by the effects of M protein and streptolysin O. We conclude that all genetic components of the sag operon are required for expression of functional SLS, an important virulence factor in the pathogenesis of invasive M1T1 GAS infection.
Asunto(s)
Proteínas Bacterianas/fisiología , Operón , Infecciones Estreptocócicas/microbiología , Streptococcus/genética , Streptococcus/patogenicidad , Estreptolisinas/fisiología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Prueba de Complementación Genética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fagocitosis/genética , Enfermedades Cutáneas Bacterianas/microbiología , Estreptolisinas/genética , Estreptolisinas/metabolismo , Virulencia/genéticaRESUMEN
In Drosophila, the Toll pathway establishes the embryonic dorsoventral axis and triggers innate immune responses to infection. The transmembrane receptor Toll acts through three death domain-containing proteins, the kinase Pelle and the adapters Tube and MyD88, in signaling to downstream NF-kappaB-like transcription factors. Here, we delineate the critical events in the earliest stages of Toll signaling. Mutational studies based on structural modeling reveal that the direct interaction of the bivalent Tube death domain with MyD88 is critical for signaling in vivo. The complex of MyD88 and Tube forms prior to signaling and is localized to the embryonic plasma membrane by MyD88. Upon Toll homodimerization, this complex is rapidly recruited to Toll. Binding of Pelle to the MyD88-Tube complex promotes Pelle activation, leading to degradation of the IkappaB-like inhibitor, Cactus. Together, these experiments convert a linear picture of gene function into a dynamic mechanistic and structural understanding of signaling complex assembly and function.