RESUMEN
Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by inflammatory cells, has been implicated in several inflammatory disease states. (E)-2(R)-[1(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2'-isobutyl-2'-(methanesulfonyl)-4-methylvalerohydrazide (Ro 32-7315), is a potent, orally active inhibitor of the TNF-alpha convertase (TACE), an enzyme responsible for proteolytic cleavage of the membrane bound precursor, pro-TNF-alpha. Ro 32-7315 inhibited a recombinant form of TACE (IC(50) = 5.2 nM) with selectivity over related matrix metalloproteinases. In a cellular assay system, THP-1 cell line, and in human and rat whole blood, Ro 32-7315 significantly reduced lipopolysaccharide (LPS)-induced TNF-alpha release with IC(50) values of 350 +/- 14 nM (n = 5), 2.4 +/- 0.5 microM (n = 5), and 110 +/- 18 nM (n = 5), respectively. Oral administration of Ro 32-7315 to Wistar rats caused a dose-dependent inhibition of LPS-induced release of systemic TNF-alpha with an ED(50) of 25 mg/kg. Treatment (days 0-14) of Allen and Hamburys hooded rats with Ro 32-7315 (2.5, 5, 10, and 20 mg/kg, i.p., twice daily) significantly reduced adjuvant-induced secondary paw swelling (42, 71, 83, and 93%, respectively) as compared with the vehicle group. In the Ro 32-7315-treated group, the reduced paw swelling was associated with improved lesion score and joint mobility. Furthermore, in a placebo-controlled, single-dose study, Ro 32-7315 given orally (450 mg) significantly suppressed ex vivo, LPS-induced TNF-alpha release in the whole-blood samples taken from healthy male and female volunteers (mean inhibition of 42% over a 4-h duration, n = 6). These data collectively support the potential use of such a compound for the oral treatment of inflammatory disorders.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácidos Hidroxámicos/farmacología , Metaloendopeptidasas/antagonistas & inhibidores , Sulfonamidas/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas ADAM , Proteína ADAM17 , Animales , Artritis Experimental/tratamiento farmacológico , Línea Celular , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacocinética , Femenino , Humanos , Ácidos Hidroxámicos/farmacocinética , Lipopolisacáridos/farmacología , Masculino , Inhibidores de la Metaloproteinasa de la Matriz , Ratas , Ratas Wistar , Proteínas Recombinantes/metabolismo , Sulfonamidas/farmacocinéticaRESUMEN
Interleukin-8 (IL-8) is a dimeric, C-X-C chemokine, produced by a variety of cells and which elicits proinflammatory responses from the neutrophil. As a prelude to drug design, we have investigated the interactions between IL-8 and its receptor by preparing a number of single-site mutants of IL-8 and determining their activity in receptor-binding and functional assays. In order to define the binding surface as precisely as possible, we have used chemical shifts obtained from nuclear magnetic resonance spectroscopy to screen mutant proteins for structural changes which affect regions of the IL-8 surface remote from the site of mutation. In addition to a previously recognized sequence, Glu4-Leu5-Arg6 in the N-terminal peptide, we have identified a second epitope comprising a contiguous group of non-sequential, solvent-exposed, hydrophobic residues, Phe17, Phe2l, Ile22, and Leu43. These two receptor-binding regions are separated by over 20 A in the IL-8 structure and are important both for receptor binding and function. In addition, we have shown through the production of a covalently linked IL-8 dimer, that subunit dissociation is not necessary for biological activity.
Asunto(s)
Antígenos CD/metabolismo , Interleucina-8/metabolismo , Neutrófilos/fisiología , Receptores de Interleucina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Calcio/sangre , Membrana Celular/inmunología , Clonación Molecular , Epítopos/análisis , Epítopos/metabolismo , Humanos , Interleucina-8/biosíntesis , Interleucina-8/química , Interleucina-8/genética , Isoleucina , Leucina , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neutrófilos/inmunología , Fenilalanina , Estructura Secundaria de Proteína , Receptores de Interleucina-8A , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Previous studies showed that the keratan sulfate-containing proteoglycans of bovine corneal stroma contain three unique core proteins designated 37A, 37B, and 25 (Funderburgh, J. L., Funderburgh, M. L., Mann, M. M., and Conrad, G. W. (1991) J. Biol. Chem. 266, 14226-14231). Degenerate oligonucleotides designed from amino acid sequences of the 37A protein were used to screen a cDNA expression library from cultured bovine keratocytes. A cDNA clone coding for keratocan, a 37A protein, was isolated and sequenced. The deduced keratocan amino acid sequence is unique but related to two other keratan sulfate-containing proteins, lumican (the 37B core protein) and fibromodulin. These three proteins share approximately 35% amino acid identity and a number of conserved structural features. Northern hybridization and immunoblotting of tissue extracts found keratocan distribution to be more limited than that of lumican or fibromodulin. Keratocan is abundant in cornea and sclera and detected in much lesser amounts in skin, ligament, cartilage, artery, and striated muscles. Only in cornea was keratocan found to contain large, sulfated keratan sulfate chains. Keratocan, like lumican, is a core protein of a major corneal proteoglycan but is present in non-corneal tissues primarily as a nonsulfated glycoprotein.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/sangre , Córnea/metabolismo , Proteínas de la Matriz Extracelular , Sulfato de Queratano/sangre , Proteoglicanos/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Bovinos , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/análisis , Proteoglicanos Tipo Condroitín Sulfato/química , Clonación Molecular , Córnea/citología , Fibromodulina , Expresión Génica , Biblioteca de Genes , Sulfato de Queratano/análisis , Sulfato de Queratano/química , Lumican , Datos de Secuencia Molecular , Especificidad de Órganos , Filogenia , Proteoglicanos/análisis , Proteoglicanos/química , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Homología de Secuencia de AminoácidoRESUMEN
Traditionally, the ill in Greece are supported by kin and neighbors, who often provide diagnosis, medication and the social support required by an invalid. In Australia, the Greek patient is likely to be faced with impersonal efficiency rather than the highly personalized sympathy he is used to. The absence of supporting kin and friends may combine with lack of familiarity in the Australian system to make illness in Australia an extremely anxious experience, perhaps much more anxious than it should ever be.