RESUMEN
BACKGROUND: We performed the first multiple dose study of remimazolam designed to assess both the feasibility of maintaining suitable sedation during colonoscopy and reversing the sedative effects of remimazolam with flumazenil. METHODS: Healthy volunteers received fentanyl followed by remimazolam for sedation during colonoscopy. Three dose groups of 15 volunteers each received remimazolam in increasing initial doses, plus top-up doses to maintain sedation for a 30-minute period. In a separate double-blind crossover part of the trial, 6 volunteers were sedated with a single high dose of remimazolam, followed by flumazenil or placebo to reverse the sedation. RESULTS: Successful sedation that was adequate for colonoscopy was achieved in >70% of subjects. After the procedure, subjects rapidly recovered to fully alert, with a median of <10 minutes overall. Failures were due to the inability to sedate or adverse events, with 1 subject failing due to hypotension (arterial blood pressure 80/40) and low SpO2 (<90%). There were no serious adverse events reported, and no events that were unexpected with the combination of a benzodiazepine and fentanyl. The study also showed that sedation was rapidly reversible (1.0 minutes flumazenil vs 10.5 minutes placebo) without resedation. CONCLUSIONS: Remimazolam has the attributes of a sedative drug, with success rates comparable with recent studies of other drugs. Remimazolam provided adequate sedation in 33 of 44 subjects undergoing colonoscopy, and its sedative effects were easily reversed with flumazenil.
Asunto(s)
Benzodiazepinas/farmacología , Colonoscopía/métodos , Antídotos/farmacología , Colonoscopía/instrumentación , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Estudios de Factibilidad , Femenino , Flumazenil/farmacología , Humanos , Hipnóticos y Sedantes/farmacología , MasculinoRESUMEN
Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevented TTP-initiated deadenylation in an in vitro macrophage assay of TNF poly(A) stability. Neomycin inhibited TTP-promoted deadenylation at concentrations shown to inhibit the deadenylases poly(A) ribonuclease and CCR4. Stably transfected RAW264.7 macrophages overexpressing PABP1 do not oversecrete TNF; instead they upregulate TTP protein without increasing TNF protein production. The PABP1 inhibition of deadenylation initiated by TTP does not require the poly(A) binding regions in RRM1 and RRM2, suggesting a more complicated interaction than simple masking of the poly(A) tail from a 3'-exonuclease. Like TTP, PABP1 is a substrate for p38 MAP kinase. Finally, PABP1 stabilizes cotransfected TTP in 293T cells and prevents the decrease in TTP levels seen with p38 MAP kinase inhibition. These findings suggest several levels of functional antagonism between TTP and PABP1 that have implications for regulation of unstable mRNAs like TNF.
Asunto(s)
Silenciador del Gen , Proteína I de Unión a Poli(A)/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Tristetraprolina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Inhibidores Enzimáticos/farmacología , Exorribonucleasas/antagonistas & inhibidores , Exorribonucleasas/metabolismo , Silenciador del Gen/efectos de los fármacos , Humanos , Inmunoprecipitación , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Neomicina/farmacología , Fosforilación , Proteína I de Unión a Poli(A)/genética , Ribonucleósido Difosfato Reductasa/metabolismo , Factores de Tiempo , Transfección , Factor de Necrosis Tumoral alfa/genética , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The MNK kinases are downstream of both the p38 and ERK MAP kinase pathways and act to increase gene expression. MNK inhibition using the compound CGP57380 has recently been reported to inhibit tumor necrosis factor (TNF) production in macrophage cell lines stimulated with Escherichia coli lipopolysaccharide (LPS). However, the range of receptors that signal through the MNK kinases and the extent of the resultant cytokine response are not known. We found that TNF production was inhibited in RAW264.7 macrophage cells by CGP57380 in a dose-responsive manner with agonists for Toll-like receptor (TLR) 2 (HKLM), TLR4 (Salmonella LPS), TLR6/2 (FSL), TLR7 (imiquimod), and TLR9 (CpG DNA). CGP57380 also inhibited the peak of TNF mRNA production and increased the rate of TNF mRNA decay, effects not due to the destabilizing RNA binding protein tristetraprolin (TTP). Similar to its effects on TNF, CGP57380 caused dose-responsive inhibition of TTP production from stimulation with either LPS or CpG DNA. MNK inhibition also blocked IL-6 but permitted IL-10 production in response to LPS. Studies using bone marrow-derived macrophages (BMDM) isolated from a spontaneous mouse model of Crohn's disease-like ileitis (SAMP1/YitFc strain) revealed significant inhibition by CGP57380 of the proinflammatory cytokines TNF, IL-6, and monocyte chemoattractant protein-1 at 4 and 24 h after LPS stimulation. IL-10 production was higher in CGP53870-treated BMDM at 4 h but was similar to the controls by 24 h. Taken together, these data demonstrate that MNK kinases signal through a variety of TLR agonists and mediate a potent innate, proinflammatory cytokine response.
Asunto(s)
Citocinas/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Receptores Toll-Like/fisiología , Compuestos de Anilina/farmacología , Animales , Western Blotting , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Enfermedad de Crohn/metabolismo , Ensayo de Inmunoadsorción Enzimática , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos , Purinas/farmacología , ARN/biosíntesis , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
A(2A) adenosine receptors (A(2A)AR) inhibit inflammation, although the mechanisms through which adenosine exerts its effects remain unclear. Although the transfer of regulatory Th cells blocks colitis induced by pathogenic CD45RB(high) Th cells, we show that CD45RB(low) or CD25+ Th cells from A(2A)AR-deficient mice do not prevent disease. Moreover, CD45RB(high) Th cells from A(2A)AR-deficient mice were not suppressed by control CD45RB(low) Th cells. A(2A)AR agonists suppressed the production of proinflammatory cytokines by CD45RB(high) and CD45RB(low) T cells in association with a loss of mRNA stability. In contrast, anti-inflammatory cytokines, including IL-10 and TGF-beta, were minimally affected. Oral administration of the A(2A)AR agonist ATL313 attenuated disease in mice receiving CD45RB(high) Th cells. These data suggest that A(2A)AR play a novel role in the control of T cell-mediated colitis by suppressing the expression of proinflammatory cytokines while sparing anti-inflammatory activity mediated by IL-10 and TGF-beta.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Colitis/inmunología , Colitis/metabolismo , Receptor de Adenosina A2A/metabolismo , Agonistas del Receptor de Adenosina A2 , Animales , Células Cultivadas , Colitis/genética , Colitis/patología , Citocinas/biosíntesis , Citocinas/genética , Antígenos Comunes de Leucocito/metabolismo , Ratones , ARN Mensajero/genética , Receptor de Adenosina A2A/deficiencia , Receptor de Adenosina A2A/genética , Receptores de Interleucina-2/metabolismoRESUMEN
FLVCR, a member of the major facilitator superfamily of transporter proteins, is the cell surface receptor for feline leukemia virus, subgroup C. Retroviral interference with FLVCR display results in a loss of erythroid progenitors (colony-forming units-erythroid, CFU-E) and severe anemia in cats. In this report, we demonstrate that human FLVCR exports cytoplasmic heme and hypothesize that human FLVCR is required on developing erythroid cells to protect them from heme toxicity. Inhibition of FLVCR in K562 cells decreases heme export, impairs their erythroid maturation and leads to apoptosis. FLVCR is upregulated on CFU-E, indicating that heme export is important in primary cells at this stage. Studies of FLVCR expression in cell lines suggest this exporter also impacts heme trafficking in intestine and liver. To our knowledge, this is the first description of a mammalian heme transporter.
Asunto(s)
Eritrocitos/metabolismo , Eritropoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Hemo/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Transporte de Proteínas/fisiología , Receptores Virales/metabolismo , Apoptosis/fisiología , Diferenciación Celular/fisiología , Línea Celular , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/aislamiento & purificación , ARN Mensajero/metabolismo , Receptores Virales/genética , Receptores Virales/aislamiento & purificación , Regulación hacia Arriba/fisiologíaRESUMEN
MAPKAP kinase 2 (MK2) is required for tumor necrosis factor synthesis. Tristetraprolin (TTP) binds to the 3'-untranslated region of tumor necrosis factor mRNA and regulates its fate. We identified in vitro and in vivo phosphorylation sites in TTP using nanoflow high pressure liquid chromatography microelectrospray ionization tandem mass spectrometry and novel methods for direct digestion of TTP bound to affinity matrices (GSH-beads or anti-Myc linked to magnetic beads). MK2Delta3B, activated in Escherichia coli by p38alpha, phosphorylates TTP in vitro at major sites Ser(52) and Ser(178) (>10-fold in abundance) as well as at several minor sites that were detected after enriching for phosphopeptides with immobilized metal affinity chromatography. MK2 phosphorylation of TTP creates a functional 14-3-3 binding site. In cells, TTP was phosphorylated at Ser(52), Ser(178), Thr(250), and Ser(316) and at SP sites in a cluster (Ser(80)/Ser(82)/Ser(85)). Anisomycin treatment of NIH 3T3 cells increased phosphorylation of Ser(52) and Ser(178). Overexpression of MK2 sufficed to increase phosphorylation of Ser(52) and Ser(178) but not Ser(80)/Ser(82)/Ser(85) or Thr(250). Thus, Ser(52) and Ser(178) are putative MK2 sites in vivo. Identified phosphosite(s) may be biologic switches controlling mRNA stability and translation.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Serina/química , Tirosina 3-Monooxigenasa/química , Proteínas 14-3-3 , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Cromatografía , Cromatografía Líquida de Alta Presión , Cricetinae , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Riñón/citología , Magnetismo , Ratones , Proteína Quinasa 14 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Químicos , Datos de Secuencia Molecular , Células 3T3 NIH , Péptidos/química , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Factores de Tiempo , Transfección , Tristetraprolina , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Nup475 (also known as tristetraprolin and TIS11) includes two zinc-binding domains of the form Cys-X8-Cys-X5-Cys-X3-His. These domains are required for rapid degradation of tumor necrosis factor (TNF) and other mRNAs through the interaction with AU-rich elements in their 3'-untranslated regions. The three-dimensional solution structure of the first domain was determined by multidimensional nuclear magnetic resonance spectroscopy, revealing a novel fold around a central zinc ion. The core structure is disk-like with a diameter of approximately 25 A and a width of approximately 12 A. This structure provides a basis for evaluating the role of individual residues for structural stability and for nucleic acid binding.
Asunto(s)
Cisteína/química , Proteínas de Unión al ADN , Histidina/química , Proteínas Inmediatas-Precoces/química , Pliegue de Proteína , Dedos de Zinc , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Inmediatas-Precoces/genética , Ratones , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Soluciones , Tristetraprolina , Dedos de Zinc/genéticaRESUMEN
Regulation of messenger RNA stability by AU-rich elements is an important means of regulating genes induced by growth factors and cytokines. Nup475 (also known as tristetraprolin, or TIS11) is the prototype for a family of zinc-binding Cys(3)His motif proteins required for proper regulation of tumor necrosis factor mRNA stability in macrophages. We developed an Escherichia coli expression system to produce soluble Nup475 protein in quantity to study its RNA binding properties. Nup475 protein bound a tumor necrosis factor AU-rich element over a broad range of pH and salt concentrations by RNA gel shift. This binding was inhibited by excess zinc metal, providing a potential mechanism for previous reports of zinc stabilization of AU-rich element (ARE) containing messenger RNAs. Immobilized Nup475 protein was used to select its optimal binding site by RNA SELEX and revealed a strong preference for the extended sequence UUAUUUAUU, rather than a simple AUUUA motif. These findings were confirmed by site-directed mutagenesis of the tumor necrosis factor ARE and RNA gel shifts on c-fos, interferon-gamma, and interferon-beta ARE fragments. A weaker binding activity toward adenine-rich sites, such as a poly(A) tail RNA fragment, can partially disrupt the Nup475-tumor necrosis factor AU-rich element complex.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Secuencia de Bases , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/genética , Proteínas Inmediatas-Precoces/genética , Sondas ARN , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/metabolismo , TristetraprolinaRESUMEN
Inducing apoptosis of activated lymphocytes via Fas ligand (FasL, CD95) may be a useful strategy for the treatment of autoimmune diseases mediated by pathogenic T cells. We propose that B cells may be ideal tools for effective delivery of a FasL-mediated apoptotic signal to pathogenic T cells for a variety of reasons, including their unique ability to efficiently take up and present antigen to T cells that share the same specificity. Here, we demonstrate that B cell clones engineered to express CD95 can effectively suppress a systemic primed antigen-specific T cell response in vivo. Intravenous injection of antigen-pulsed FasL-expressing B cells eliminated antigen-specific (TCR transgenic) T cells from the draining lymph nodes within 12-60 h, and suppressed a delayed-type hypersensitivity response in an antigen-specific manner. These results indicate that B cells can be engineered to express FasL, and used to impair T cell function in vivo, suggesting that FasL-expressing B cells may be an effective tool for the treatment of established T cell-mediated autoimmune and inflammatory diseases.