RESUMEN
SB-219383 and its analogues are a class of potent and specific inhibitors of bacterial tyrosyl-tRNA synthetases. Crystal structures of these inhibitors have been solved in complex with the tyrosyl-tRNA synthetase from Staphylococcus aureus, the bacterium that is largely responsible for hospital-acquired infections. The full-length enzyme yielded crystals that diffracted to 2.8 A resolution, but a truncated version of the enzyme allowed the resolution to be extended to 2.2 A. These inhibitors not only occupy the known substrate binding sites in unique ways, but also reveal a butyl binding pocket. It was reported that the Bacillus stearothermophilus TyrRS T51P mutant has much increased catalytic activity. The S. aureus enzyme happens to have a proline at position 51. Therefore, our structures may contribute to the understanding of the catalytic mechanism and provide the structural basis for designing novel antimicrobial agents.
Asunto(s)
Inhibidores Enzimáticos/química , Staphylococcus aureus/enzimología , Tirosina-ARNt Ligasa/química , Secuencia de Aminoácidos , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Cristalización , Cristalografía por Rayos X , Dipéptidos/química , Dipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Furanos/química , Furanos/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Piperidinas/química , Piperidinas/farmacología , Conformación Proteica , Homología de Secuencia de Aminoácido , Tirosina-ARNt Ligasa/antagonistas & inhibidoresRESUMEN
Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.
Asunto(s)
Apoptosis , Inhibidores de Caspasas , Inhibidores Enzimáticos/química , Clorometilcetonas de Aminoácidos/farmacología , Animales , Sitios de Unión , Camptotecina/farmacología , Caspasa 3 , Caspasa 7 , Condrocitos/efectos de los fármacos , Colágeno/genética , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Isatina/análogos & derivados , Ratones , Modelos Moleculares , Estructura Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Osteoartritis/tratamiento farmacológico , Regiones Promotoras Genéticas , Proteínas Recombinantes/química , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
SK&F 107647, a previously described synthetic immunomodulatory peptide, indirectly stimulates bone marrow progenitor cells and phagocytic cells, and enhances host defense effector mechanisms in bacterial and fungal infection models in vivo. In vitro, SK&F 107647 induces the production of a soluble mediator that augments colony forming cell (CFU-GM) formation in the presence of CSFs. In this paper we purified and sequenced the stromal cell-derived hematopoietic synergistic factors (HSF) secreted from both murine and human cell lines stimulated with SK&F 107647. Murine HSF is an N-terminal 4-aa truncated form of the CXC chemokine, KC, while human HSF was identified as an N-terminal 4-aa truncated form of the CXC chemokine, GRO beta. In comparison to their full-length forms, truncated KC and truncated GRO beta were 10 million times more potent as synergistic growth stimulants for CFU-GM. Enhanced potency of these novel truncated chemokines relative to their full-length forms was also demonstrated in respiratory burst assays, CD11b Ag expression, and intracellular killing of the opportunistic pathogen, Candida albicans. Administration of truncated KC significantly enhanced survival of mice lethally infected with C. albicans. The results reported herein delineate the biological mechanism of action of SK&F 107647, which functions via the induction of unique specific truncated forms of the chemokines KC and GRO beta. To our knowledge, this represents the first example where any form of KC or GRO beta were purified from marrow stromal cells. Additionally, this is the first demonstration of in vivo efficacy of a CXC chemokine in an animal infectious fungal disease model.
Asunto(s)
Antifúngicos/aislamiento & purificación , Quimiocinas CXC/aislamiento & purificación , Factores Quimiotácticos/aislamiento & purificación , Sustancias de Crecimiento/aislamiento & purificación , Péptidos y Proteínas de Señalización Intercelular , Fragmentos de Péptidos/aislamiento & purificación , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Secuencia de Aminoácidos , Animales , Antifúngicos/sangre , Antifúngicos/inmunología , Células de la Médula Ósea/química , Células de la Médula Ósea/inmunología , Candidiasis/inmunología , Candidiasis/mortalidad , Candidiasis/prevención & control , Línea Celular , Quimiocina CXCL1 , Quimiocinas CXC/sangre , Quimiocinas CXC/genética , Quimiocinas CXC/inmunología , Factores Quimiotácticos/sangre , Factores Quimiotácticos/genética , Factores Quimiotácticos/inmunología , Sinergismo Farmacológico , Femenino , Sustancias de Crecimiento/sangre , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/inmunología , Humanos , Sueros Inmunes/farmacología , Inyecciones Intraperitoneales , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Activación Neutrófila/inmunología , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes/química , Células del Estroma/química , Células del Estroma/inmunologíaRESUMEN
The three-dimensional structure of a novel four amino acid truncated form of the CXC chemokine GRObeta [5-73] isolated from bone marrow stromal cells with potent hematopoietic and anti-infective activities has been determined by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy in solution. On the basis of 1878 upper distance constraints derived from nuclear Overhauser effects (NOE) and 314 dihedral angle constraints, a group of 20 conformers representing the solution structure of the human GRObeta [5-73] was computed with the program DYANA. At the concentrations used for NMR study, GRObeta [5-73] forms a dimer in solution that is architectured by a six-stranded antiparallel beta-sheet (residues 25 to 29, 39 to 44, 49 to 52) and a pair of helices (residues 58 to 68) with 2-fold symmetry, while the C terminus of the protein is disordered. The average of the pairwise root-mean-square deviations of individual NMR conformers relative to the mean coordinates for the backbone atoms N, C(alpha) and C' of residues 5 to 68 is 0.47 A. Overall, the global fold of GRObeta [5-73] is similar to that of the previously reported NMR structure of GROalpha and the NMR and X-ray structures of interleukin-8. Among these three CXC chemokines, GRObeta [5-73] is most similar in structure to GROalpha. Significant differences between GRObeta [5-73], GROalpha and interleukin-8 are in the N-terminal loop comprising residues 12 to 19. The N-terminal arm containing the conserved ELR motif and the loop of residues 30 to 38 containing the GPH motif are different among these three CXC chemokines. The structural differences in these two regions may be responsible for the specificity of the receptor binding and biological activity of different chemokines.
Asunto(s)
Quimiocinas CXC/química , Factores Quimiotácticos/química , Sustancias de Crecimiento/química , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/química , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Secuencias de Aminoácidos , Médula Ósea , Línea Celular , Quimiocina CXCL1 , Quimiocina CXCL2 , Secuencia Conservada , Cristalografía por Rayos X , Dimerización , Disulfuros/metabolismo , Humanos , Modelos Moleculares , Estructura Secundaria de Proteína , Eliminación de Secuencia , Soluciones , Electricidad Estática , Células del Estroma , Relación Estructura-ActividadRESUMEN
The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily that mediates cell attachment on arginine-glycine-aspartic acid (RGD)-containing adhesive proteins. A solid-phase microtiter assay was developed to investigate the binding properties of purified alpha v beta 3, using tritiated [3H]SK&F-107260 as the radiolabeled ligand. alpha v beta 3, purified from human platelets, human placenta, and chicken osteoclasts, bound [3H]SK&F-107260 saturably and specifically. Saturation binding studies using platelet alpha v beta 3 revealed a single class of high affinity binding sites, exhibiting a Kd of 1.44 nM and Bmax of 0.20 mol of [3H]SK&F-107260/mol of alpha v beta 3. [3H]SK&F-107260 binding was inhibited by a variety of RGD-containing peptides and by the snake venom protein echistatin, whereas an RGE-containing peptide and four nonpeptide fibrinogen receptor (alpha IIb beta 3) antagonists failed to do so. This study shows that alpha v beta 3 exhibits distinct ligand specificity from the structurally homologous fibrinogen receptor, alpha IIb beta 3. The relative potencies of the RGD-containing peptides in inhibiting [3H]SK&F-107260 binding to alpha v beta 3 were the same as their relative potencies in inhibiting biotinylated-fibrinogen binding to the receptor. alpha v beta 3 purified from chicken osteoclasts and human placenta bound [3H]SK&F-107260 with similar affinities and displayed the same pharmacological profile as the platelet vitronectin receptor. The alpha v beta 3 antagonists inhibited the attachment of MG63 human osteosarcoma cells or rat osteoclasts to recombinant rat osteopontin. The rank order of potency of the antagonists in the cell adhesion assays was similar to that of the receptor binding assay, suggesting that the purified alpha v beta 3-[3H]SK&F-107260 binding assay is a valid reflection of the ligand binding to alpha v beta 3 on cell systems.
Asunto(s)
Adhesión Celular , Oligopéptidos/farmacología , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Inhibidores de Agregación Plaquetaria/metabolismo , Receptores de Vitronectina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Plaquetas/metabolismo , Pollos , Cromatografía de Afinidad , Femenino , Humanos , Ligandos , Datos de Secuencia Molecular , Oligopéptidos/química , Osteoclastos/metabolismo , Osteopontina , Osteosarcoma , Placenta/metabolismo , Embarazo , Ensayo de Unión Radioligante , Ratas , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Sialoglicoproteínas/metabolismo , TritioRESUMEN
Osteopontin (OPN), a 41-kDa phosphorylated glycoprotein, has been detected in rat aorta and carotid arteries, and expression of its mRNA in blood vessels is strongly increased in response to vascular injury. To investigate the potential role of OPN in vascular pathophysiology, we studied the effect of rat OPN on aortic smooth muscle cell migration and proliferation in vitro. OPN enhanced the migration of rat smooth muscle cells in a time- and concentration-dependent manner with an EC50 value of 46 +/- 11 nmol/liter (n = 5). The maximal increase in cell migration by OPN was 29-fold over basal levels. OPN-induced smooth muscle cell migration was inhibited in a concentration-dependent manner by the monoclonal antibody F11, which recognizes the rat integrin subunit beta 3. In contrast, polyclonal antiserum recognizing the rat integrin beta 1 subunit did not inhibit smooth muscle cell migration in response to OPN, but did block fibronectin-promoted migration. Moreover, OPN-induced smooth muscle cell migration was dependent on the presence of extracellular divalent cations and was significantly inhibited by anti-OPN antibodies. OPN did not stimulate [3H]thymidine incorporation into cultured smooth muscle cells, indicating that it selectively enhanced migration. In view of the pathological significance of arterial smooth muscle cell migration in the formation of intimal thickening, our results suggest that smooth muscle cell recognition of OPN, probably through the vitronectin receptor, alpha v beta 3, could play a role in the cells' response to vascular injury and especially neointima formation.
Asunto(s)
Integrinas/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Sialoglicoproteínas/farmacología , Animales , Aorta/citología , Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Integrina beta3 , Integrinas/inmunología , Masculino , Oligopéptidos , Osteopontina , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ratas , Ratas Sprague-Dawley , Sialoglicoproteínas/inmunologíaRESUMEN
The gene coding for a soluble form of human E-selectin (sE-selectin) has been expressed in Chinese hamster ovary (CHO) cells. Cells seeded into a hollow fiber reactor secreted protein at a level of 160 mg/liter. The protein was purified to > 95% pure and low endotoxin (< 2 ng/mg), using physiological pH and buffers. The amino acid composition and N-terminal sequence were as predicted from the cDNA sequence. HL-60 cells bound to sE-selectin-coated plates in a dose-dependent manner, and this binding could be blocked up to 100% by pretreatment of HL60 cells with sE-selectin. The concentration of sE-selectin required for 50% inhibition was 1 microM. This value puts an upper limit for the affinity of E-selectin for its natural receptor. sE-selectin also inhibited inflammatory migration of neutrophils in a selective fashion. Purified sE-selectin exhibited a broad band of M(r) approximately 75,000 on nonreducing SDS-PAGE. sE-selectin eluted with M(r) approximately 310,000 from size exclusion chromatography at physiological pH and buffers, suggesting an oligomeric state. Matrix-assisted laser-desorption MS gave a molecular weight of 80,000, while the minimum monomer molecular weight from the gene sequence should be 58,571, demonstrating that the monomeric molecule thus expressed had 27% carbohydrate. Equilibrium analytical ultracentrifugation gave an average solution molecular weight of 81,600 (+/- 4,500). Velocity ultracentrifugation gave a sedimentation coefficient of 4.3 S and, from this, an apparent axial ratio of 10.5:1, assuming a prolate ellipsoid of revolution. An analysis of the NMR NOESY spectra of sE-selectin, sialyl-Lewis X, and sE-selectin with sialyl-Lewis X demonstrates that the recombinant protein binds sialyl-Lewis X productively. Hence, in solution, sE-selectin is a functional elongated monomer.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Células CHO , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/aislamiento & purificación , Movimiento Celular , Células Cultivadas , Cromatografía en Gel , Cricetinae , Cricetulus , ADN , Selectina E , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Inflamación/metabolismo , Espectroscopía de Resonancia Magnética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , UltracentrifugaciónRESUMEN
A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.
Asunto(s)
Interleucina-1/biosíntesis , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Bioensayo , Cromatografía , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Humanos , Interleucina-1/genética , Interleucina-1/aislamiento & purificación , Ratones , Mitosis , Peso Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Compuestos de Sulfhidrilo/análisis , Linfocitos T/citologíaRESUMEN
Strategic marketing management in the hospital industry is maturing through a series of stages, from a passive, reactive posture toward a more aggressive, proactive stance. However, a survey of 80 hospitals shows that most have not yet made the critical transition from the traditional production orientation to the more progressive marketing orientation.
Asunto(s)
Administración Hospitalaria , Comercialización de los Servicios de Salud/organización & administración , Modelos Teóricos , Innovación Organizacional , Estados UnidosRESUMEN
This paper reports the results of a survey of acute care hospitals which was undertaken to: (1) identify and establish the organizational positioning of key hospital marketing personnel; (2) measure the role of these personnel in influencing the traditional marketing mix decisions; and, (3) identify tactical marketing activities most frequently undertaken.
Asunto(s)
Administradores de Instituciones de Salud , Administradores de Hospital , Comercialización de los Servicios de Salud/organización & administración , Recolección de Datos , Toma de Decisiones , Humanos , Proyectos de Investigación , Estados UnidosRESUMEN
Purified bovine factor XII was radiolabeled with iodine-125 and its binding to kaolin studied. Binding was rapid and was not readily reversible upon adding unlabeled factor XII. The optimum pH for binding was in the region of pH 5-7. The isoelectric point of factor XII was pH 5.7. High concentrations of urea or increasing the ionic strength of the medium did not inhibit binding. Polyvalent macromolecules, such as Polybrene and polylysine, were effective inhibitors of factor XII binding to kaolin. Polylysine caused the release of factor XII that had bound to the kaolin surface.