RESUMEN
In mice, homozygous deletion of the cardiac sodium channel Scn5a results in defects in cardiac morphology and embryonic death before robust sodium current can be detected. In zebrafish, morpholino knockdown of cardiac sodium channel orthologs scn5Laa and scn5Lab perturbs specification of precardiac mesoderm and inhibits growth of the embryonic heart. It is not known which developmental processes are perturbed by sodium channel knockdown and whether reduced cell number is from impaired migration of cardiac progenitors into the heart, impaired myocyte proliferation, or both. We found that embryos deficient in scn5Lab displayed defects in primary cardiogenesis specific to loss of nkx2.5, but not nkx2.7. We generated kaede reporter fish and demonstrated that embryos treated with anti-scn5Lab morpholino showed normal secondary differentiation of cardiomyocytes at the arterial pole between 30 and 48 h post-fertilization. However, while proliferating myocytes were readily detected at 48 hpf in wild type embryos, there were no BrdU-positive cardiomyocytes in embryos subjected to anti-scn5Lab treatment. Proliferating myocytes were present in embryos injected with anti-tnnt2 morpholino to phenocopy the silent heart mutation, and absent in embryos injected with anti-tnnt2 and anti-scn5Lab morpholinos, indicating cardiac contraction is not required for the loss of proliferation. These data demonstrate that the role of scn5Lab in later heart growth does not involve contribution of the secondary heart field, but rather proliferation of cardiomyocytes, and appears unrelated to the role of the channel in cardiac electrogenesis.
Asunto(s)
Proliferación Celular , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Pez Cebra/metabolismo , Animales , Diferenciación Celular , Corazón/embriología , Proteína Homeótica Nkx-2.5 , Morfogénesis , Morfolinos , Miocitos Cardíacos/citología , Canal de Sodio Activado por Voltaje NAV1.5/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Integrins are a ubiquitous family of non-covalently associated alpha/beta transmembrane heterodimers linking extracellular ligands to intracellular signaling pathways [1] [Cell, 2002; 110: 673]. Platelets contain five integrins, three beta1 integrins that mediate platelet adhesion to the matrix proteins collagen, fibronectin and laminin, and the beta3 integrins alphavbeta3 and alphaIIbbeta3 [2] [J Clin Invest, 2005; 115: 3363]. While there are only several hundred alphavbeta3 molecules per platelet, alphavbeta3 mediates platelet adhesion to osteopontin and vitronectin in vitro [3] [J Biol Chem, 1997; 272: 8137]; whether this occurs in vivo remains unknown. By contrast, the 80,000 alphaIIbbeta3 molecules on agonist-stimulated platelets bind fibrinogen, von Willebrand factor, and fibronectin, mediating platelet aggregation when the bound proteins crosslink adjacent platelets [2] [J Clin Invest, 2005; 115: 3363]. Although platelet integrins are poised to shift from resting to active conformations, tight regulation of their activity is essential to prevent the formation of intravascular thrombi. This review focuses on the structure and function of the intensively studied beta3 integrins, in particular alphaIIbbeta3, but reference will be made to other integrins where relevant.
Asunto(s)
Plaquetas/química , Integrina beta3/química , Integrinas/química , Integrinas/fisiología , Humanos , Complejo GPIIb-IIIa de Glicoproteína PlaquetariaRESUMEN
The National Institute for Occupational Safety and Health (NIOSH) conducted 21 field surveys in selected industries to characterize workers' exposures to hexavalent chromium-containing airborne particulate and to evaluate existing technologies for controlling these exposures. Hexavalent chromium Cr(VI) is a respiratory irritant and chronic inhalation may cause lung cancer. Primary evaluation methods included collection of full work shift, personal breathing-zone (PBZ) air samples for Cr(VI), measurement of ventilation system parameters, and documentation of processes and work practices. This study emphasized evaluation of engineering exposure control measures, so PBZ exposures were measured on the outside of personal protective equipment, for example, respirators. Field surveys were conducted in two chromium electroplating facilities, including one where full-shift PBZ exposures to Cr(VI) ranged from 3.0 to 16 times the 1 micro g/m(3)NIOSH recommended exposure limit (REL) despite several engineering controls on the plating tanks. At a painting and coating facility that used Cr(VI)-containing products, full-shift exposures of painters and helpers (2.4 to 55 micro g/m(3)) exceeded the REL, but LEV effectiveness was limited. Other operations evaluated included welding in construction; metal cutting operations on chromium-containing materials in ship breaking; chromate-paint removal with abrasive blasting; atomized alloy-spray coating; foundry operations; printing; and the manufacture of refractory brick, colored glass, prefabricated concrete products, and treated wood products. NIOSH researchers concluded that, in many of the evaluated processes, Cr(VI) exposures at or below the current NIOSH REL are achievable. However, for some processes, it is unclear whether controlling exposures to this range is consistently achievable without respirator use. Some operations involving the application of coatings and finishes may be among those most difficult to control to this range. Most operations judged to be moderately difficult to control to this range involve joining and cutting metals with relatively high chromium content. Nonetheless, exposures in a wide variety of other processes were judged more easily controllable to the current REL or below, or were found to be minimal, including some operations meeting the general descriptions named above but with different specific operating parameters producing lower Cr(VI) exposures.
Asunto(s)
Contaminantes Ocupacionales del Aire/análisis , Cromo/análisis , Exposición Profesional/análisis , Galvanoplastia , Monitoreo del Ambiente , Humanos , Industrias , Exposición por Inhalación/análisis , Exposición por Inhalación/prevención & control , National Institute for Occupational Safety and Health, U.S. , Exposición Profesional/prevención & control , Pintura , Dispositivos de Protección Respiratoria , Absorción Cutánea , Estados Unidos , VentilaciónRESUMEN
We have expressed two proteins that correspond to the transmembrane and cytoplasmic domains of integrin alphaIIb and beta3 subunits. Characterization of these proteins, dispersed in anionic and zwitterionic micelles, revealed that, rather than interacting with each other, the two proteins associated into homodimers and homotrimers respectively. Moreover, studies using the TOXCAT assay system confirmed that the alphaIIb and beta3 transmembrane domains can self-associate in biological cell membranes. Transmembrane domain-mediated homo-oligomerization provides a plausible structural basis for integrin clustering and could promote integrin activation as well. Indeed, replacing specific residues in the transmembrane helix of either alphaIIb or beta3 with an asparagine residue resulted in a facilitated homo-oligomerization of the mutated transmembrane helix, promoted the formation of integrin clusters on the cell surface and shifted alphaIIbbeta3 to its activated state. Thus these studies support the hypothesis that the transmembrane domains play a vital role in the function and regulation of alphaIIbbeta3.
Asunto(s)
Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Secuencias de Aminoácidos , Animales , Células CHO , Membrana Celular/metabolismo , Cricetinae , Citoplasma/metabolismo , Dimerización , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Ligandos , Micelas , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
The integrin alphavbeta3 mediates platelet adhesion to the matrix protein osteopontin and likely is the predominant integrin mediating platelet adhesion to the matrix protein vitronectin. To address the mechanism that regulates alphavbeta3 activity in platelets, we measured the effect of the P2Y1 antagonist adenosine 3'-phosphate-5'-phosphate (A3P5P) and the P2Y12 antagonist AR-C66096 on ADP-stimulated platelet adhesion to osteopontin and vitronectin. Each antagonist completely inhibited platelet adhesion, implying that concurrent stimulation of P2Y1 and P2Y12 was required to activate alphavbeta3. The reducing agent dithiothreitol and Mn2+ also induced platelet adhesion to osteopontin, but did so without stimulating platelet activation. Thus, these data suggest that ADP stimulation regulates alphavbeta3 activity by perturbing the conformation of its extracellular domain. The actin polymerization inhibitors cytochalasin D and latrunculin A also induced platelet adhesion to osteopontin and vitronectin. Thus, alphavbeta3 activity in resting platelets appears to be constrained by the platelet cytoskeleton. Moreover, the effect of these agents was inhibited by A3P5P and AR-C66096 at micromolar and subnanomolar concentrations, respectively, suggesting that subthreshold platelet stimulation by ADP was required. Our data suggest that signals from both Galphaq- and Galphai-coupled receptors converge to release cytoskeletal constraints on alphavbeta3. We propose that the release of cytoskeletal constraints and a concurrent increase in affinity for ligands is responsible for alphavbeta3-mediated platelet adhesion.
Asunto(s)
Plaquetas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Actinas/metabolismo , Citoesqueleto/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Humanos , Osteopontina , Sialoglicoproteínas/metabolismo , Vitronectina/metabolismoRESUMEN
Integrins are a family of alpha/beta heterodimeric membrane proteins, which mediate cell-cell and cell-matrix interactions. The molecular mechanisms by which integrins are activated and cluster are currently poorly understood. One hypothesis posits that the cytoplasmic tails of the alpha and beta subunits interact strongly with one another in a 1:1 interaction, and that this interaction is modulated in the course of the activation of alphaIIbbeta3 [Hughes, P. E., et al. (1996) J. Biol. Chem. 271, 6571-6574]. To examine the structural basis for this interaction, protein fragments encompassing the transmembrane helix plus cytoplasmic tails of the alpha and beta subunits of alphaIIbbeta3 were expressed and studied in phospholipid micelles at physiological salt concentrations. Analyses of these fragments by analytical ultracentrifugation, NMR, circular dichroism, and electrophoresis indicated that they had very little or no tendency to interact with one another. Instead, they formed homomeric interactions, with the alpha- and beta-fragments forming dimers and trimers, respectively. Thus, these regions of the protein structure may contribute to the clustering of integrins that accompanies cellular adhesion.
Asunto(s)
Citoplasma/química , Proteínas de la Membrana/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Secuencia de Aminoácidos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Espectroscopía de Resonancia Magnética , Micelas , Datos de Secuencia Molecular , UltrafiltraciónRESUMEN
Binding of fibrinogen to GPIIb-IIIa on agonist-stimulated platelets results in platelet aggregation, presumably by crosslinking adjacent activated platelets. Although unactivated platelets express numerous copies of GPIIb-IIIa on their surface, spontaneous, and potentially deleterious, platelet aggregation is prevented by tightly regulating the fibrinogen binding activity of GPIIb-IIIa. Preliminary evidence suggests that it is the submembranous actin or actin-associated proteins that constrains GPIIb-IIIa in a low affinity state and that relief of this constraint by initiating actin filament turnover enables GPIIb-IIIa to bind fibrinogen. Two regions of the fibrinogen alpha chain that contain an RGD motif, as well as the carboxyl-terminus of the fibrinogen gamma chain, represent potential binding sites for GPIIb-IIIa in the fibrinogen molecule. However, ultrastructural studies using purified fibrinogen and GPIIb-IIIa, and studies using recombinant fibrinogen in which the RGD and relevant gamma chain motifs were mutated indicate that sequences located at the carboxyl-terminal end of the gamma chain mediates fibrinogen binding to GPIIb-IIIa. There is evidence that fibrinogen itself binds to regions in the amino terminal portions of both GPIIb and GPIIIa and that the sites interacting with the fibrinogen gamma chain and with RGD-containing peptides are spatially distinct. Nonetheless, there appears to be allosteric linkage between these sites, accounting for the ability of RGD-containing peptides to inhibit platelet aggregation and arterial thrombosis.
Asunto(s)
Plaquetas/metabolismo , Fibrinógeno/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Humanos , Oligopéptidos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión ProteicaRESUMEN
The polymorphism responsible for the Pl(A2) alloantigen on the beta(3)-component of beta(3)-containing integrins is reported to be a risk factor for coronary thrombosis. This study examined the effect of Pl(A2) on the function of beta(3)-integrins using platelets from subjects homozygous and heterozygous for Pl(A1) and Pl(A2). There was overlap in the distribution of the dissociation constant (K(d)) and maximum fibrinogen binding (B(max)) values for fibrinogen binding to alpha(IIb)beta(3) on platelets from Pl(A1) and Pl(A2) homozygotes and Pl(A1)/Pl(A2) heterozygotes. However, whereas there was no statistical difference in these values for the Pl(A1) homozygotes and Pl(A2) heterozygotes, the K(d) for the Pl(A2) homozygotes was significantly lower than that for the Pl(A1)/Pl(A2) heterozygotes, but was not statistically different from that for the Pl(A1) homozygotes. No differences were detected in ADP sensitivity between platelets from Pl(A1) homozygotes and Pl(A1)/Pl(A2) heterozygotes, in the IC(50) for RGDS inhibition of fibrinogen binding to alpha(IIb)beta(3), in the alpha(v)beta(3)-mediated adhesion of platelets to osteopontin and vitronectin, and in the phorbol ester-stimulated adhesion to fibrinogen of B lymphocytes expressing alpha(IIb)beta(3) containing either the Pl(A1) or the Pl(A2) polymorphism. Finally, no differential effects of Pl(A2) on turbidometric platelet aggregation, platelet secretion, or platelet thrombus formation were found as measured in the PFA-100. Because no differences were detected in the ability of beta(3)-integrins to interact with ligands based on the presence or absence of the Pl(A2) polymorphism, the results suggest that factors unrelated to beta(3)-integrin function may account for the reported association of the Pl(A2) allele with coronary thrombosis.
Asunto(s)
Antígenos CD/genética , Antígenos CD/fisiología , Antígenos de Plaqueta Humana/genética , Antígenos de Plaqueta Humana/fisiología , Plaquetas/fisiología , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/fisiología , Polimorfismo Genético , Adenosina Difosfato/farmacología , Adulto , Anciano , Linfocitos B/fisiología , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Trombosis Coronaria/genética , Femenino , Fibrinógeno/metabolismo , Heterocigoto , Homocigoto , Humanos , Integrina beta3 , Masculino , Persona de Mediana Edad , Osteopontina , Adhesividad Plaquetaria , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/fisiología , Sialoglicoproteínas/metabolismo , Transfección , Vitronectina/metabolismoAsunto(s)
Inhibidores de Agregación Plaquetaria/uso terapéutico , Humanos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/clasificación , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismoRESUMEN
To determine the molecular basis for the insensitivity of rat alpha(IIb)beta(3) to inhibition by RGD-containing peptides, hybrids of human and rat alpha(IIb)beta(3) and chimeras of alpha(IIb)beta(3) in which alpha(IIb) was composed of portions of human and rat alpha(IIb) were expressed in Chinese hamster ovary cells and B lymphocytes, and the ability of the tetrapeptide RGDS to inhibit fibrinogen binding to the various forms of alpha(IIb)beta(3) was measured. These measurements indicated that sequences regulating the sensitivity of alpha(IIb)beta(3) to RGDS are located in the seven amino-terminal repeats of alpha(IIb). Moreover, replacing the first three or four (but not the first two) repeats of rat alpha(IIb) with the corresponding human sequences enhanced sensitivity to RGDS, whereas replacing the first two or three repeats of human alpha(IIb) with the corresponding rat sequences had little or no effect. Nevertheless, RGDS bound to Chinese hamster ovary cells expressing alpha(IIb)beta(3) regardless whether the alpha(IIb) in the heterodimers was human, rat, or a rat-human chimera. These results indicate that the sequences determining the sensitivity of alpha(IIb)beta(3) to RGD-containing peptides are located in the third and fourth amino-terminal repeats of alpha(IIb). Because RGDS binds to both human and rat alpha(IIb)beta(3), the results suggest that differences in RGDS sensitivity result from differences in the allosteric changes induced in these repeats following RGDS binding.
Asunto(s)
Plaquetas/metabolismo , Fibrinógeno/química , Fibrinógeno/metabolismo , Oligopéptidos/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Adenosina Difosfato/metabolismo , Sitio Alostérico , Animales , Linfocitos B/metabolismo , Células CHO , Carcinógenos , Adhesión Celular , Cricetinae , ADN Complementario/metabolismo , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Ligandos , Agregación Plaquetaria , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Acetato de Tetradecanoilforbol , TransfecciónRESUMEN
Platelet-inhibitory drugs are of proven benefit to individuals who suffer from atherosclerotic cardiovascular disease. Despite substantial effort to identify more potent platelet-inhibitory agents, aspirin, an irreversible inhibitor of platelet cyclooxygenase activity, remains the standard against which other drugs are judged. Drugs that appear to be at least as efficacious as aspirin in specific clinical settings include the thienopyridines ticlopidine and clopidogrel, specific inhibitors of ADP-stimulated platelet function, and the phosphodiesterase 3 inhibitor cilostazol. Ligand binding to the platelet integrin alphaIIbbeta3 (GPIIb-IIIa), a prerequisite for platelet thrombus formation, has been a prominent target for drug development. Currently, three types of alphaIIbbeta3 antagonists are available: the monoclonal antibody Fab fragment abciximab, cyclic peptides based on the Arg-Gly-Asp (RGD) or related amino acid motifs, and RGD-based peptidomimetics. The efficacy of each type of alphaIIbbeta3 antagonist in the setting of acute coronary artery disease has been confirmed in multicenter clinical trials.
Asunto(s)
Arteriosclerosis/tratamiento farmacológico , Enfermedades Cardiovasculares/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Ticlopidina/análogos & derivados , Aspirina/farmacología , Aspirina/provisión & distribución , Aspirina/uso terapéutico , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Clopidogrel , Humanos , Adhesividad Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/fisiología , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/provisión & distribución , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Ticlopidina/farmacología , Ticlopidina/provisión & distribución , Ticlopidina/uso terapéutico , Resultado del TratamientoRESUMEN
Pleckstrin is a 40-kD phosphoprotein containing NH(2)- and COOH-terminal pleckstrin homology (PH) domains separated by a disheveled-egl 10-pleckstrin (DEP) domain. After platelet activation, pleckstrin is rapidly phosphorylated by protein kinase C. We reported previously that expressed phosphorylated pleckstrin induces cytoskeletal reorganization and localizes in microvilli along with glycoproteins, such as integrins. Given the role of integrins in cytoskeletal organization and cell spreading, we investigated whether signaling from pleckstrin cooperated with signaling pathways involving the platelet integrin, alphaIIbbeta3. Pleckstrin induced cell spreading in both transformed (COS-1 & CHO) and nontransformed (REF52) cell lines, and this spreading was regulated by pleckstrin phosphorylation. In REF52 cells, pleckstrin-induced spreading was matrix dependent, as evidenced by spreading of these cells on fibrinogen but not on fibronectin. Coexpression with alphaIIbbeta3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant alphaIIbbeta3 Ser753Pro, or beta3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that alphaIIbbeta3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading. Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of beta3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of beta3 Ser753Pro or alphaIIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin beta-chain is also required for pleckstrin-induced spreading. Thus, expressed phosphorylated pleckstrin promotes cell spreading that is both matrix and integrin dependent. To our knowledge, this is the first example of a mutated integrin functioning as a dominant negative inhibitor.
Asunto(s)
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal/fisiología , Animales , Células CHO , Células COS , Línea Celular Transformada , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Cricetinae , Matriz Extracelular/metabolismo , Fibrinógeno/farmacología , Expresión Génica/fisiología , Mutagénesis/fisiología , Fosforilación , Plásmidos , TransfecciónRESUMEN
Cleavage of osteopontin by thrombin has been reported to enhance cell adhesion. We asked whether thrombin could regulate the alpha(v)beta(3)-mediated adhesion of platelets and B lymphocytes to this substrate. Although there was no difference in the extent or the avidity of thrombin- and ADP-stimulated platelet adhesion to intact or thrombin-cleaved human osteopontin, both the extent and avidity of phorbol ester-stimulated B cell adhesion to thrombin-cleaved osteopontin was significantly increased. Thus, these data suggest that the ability of alpha(v)beta(3) to recognize osteopontin can be differentially regulated in a cell-specific manner. To localize the alpha(v)beta(3) binding site on osteopontin, we measured cell adhesion to the two thrombin cleavage products of osteopontin and to a series of nested RGD-containing osteopontin peptides cross-linked to albumin. Whereas ADP-stimulated platelets adhered to the amino-terminal but not the carboxyl-terminal osteopontin fragment and to the osteopontin peptide RGDSVVYGLR, phorbol ester-stimulated B cells did not adhere to this peptide, although they did so in the presence of 1 mm Mn(2+). Thus, our data confirm that thrombin cleavage enhances the accessibility of the binding motif for alpha(v)beta(3) on osteopontin, but this enhancement is also a function of the activation state of alpha(v)beta(3). Moreover, they indicate that the sequence RGDSVVYGLR contains sufficient information to specify activation-dependent alpha(v)beta(3)-mediated platelet and lymphocyte adhesion.
Asunto(s)
Linfocitos B/fisiología , Adhesión Celular , Adhesividad Plaquetaria , Receptores de Vitronectina/fisiología , Sialoglicoproteínas/metabolismo , Trombina/metabolismo , Adenosina Difosfato/farmacología , Plaquetas/efectos de los fármacos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Herpesvirus Humano 4 , Humanos , Magnesio/farmacología , Espectroscopía de Resonancia Magnética , Osteopontina , Conformación ProteicaRESUMEN
For many years exposure to airborne contaminants has been estimated by air or biological monitoring. In occupational settings, mathematical models increasingly are employed as adjuncts to monitoring, for instance, during process design or in retrospective epidemiological studies. Models can make predictions in a wide variety of scenarios, can be used for rapid screening, and may reduce the need for monitoring in exposure assessment. However, models make simplifying assumptions regarding air flow and contaminant transport. The errors resulting from these assumptions have not been systematically evaluated. Here we compare exposure estimates from the single-zone completely mixed (CM-1), two-zone completely mixed (CM-2), and uniform diffusivity (UD) models with workroom concentration fields predicted by computational fluid dynamics (CFD). The room air flow, concentration fields, and the breathing zone concentration of a stationary worker were computed using Fluent V4.3 for factorial combinations of three source locations, three dilution air flow rates and two emission rate profiles, constant and time-varying. These numerical experiments were used to generate plausible concentration fields, not to simulate exactly the processes in a real workroom. Thus, "error" is defined here as difference between model and CFD predictions. For both constant and time-varying emission sources, exposure estimates depended on receptor and source location. For the constant source case, ventilation rate was shown to be inconsequential to CM-1 model error. CM-1, CM-2, and UD models differed in their agreement with CFD. UD was closest to CFD for estimating concentration in the simulated breathing zone (BZ) near the source, although large errors resulted when the model was applied to the plane of possible breathing zones. CM-1 performed better for this plane but underestimated the near-source BZ exposure. For the near-source BZ location, CM-2 replicated CFD predictions more closely than CM-1 did, but less closely than UD did. Error in CM-1 model estimation of short-term average exposure to a time-varying source was highly dependent on ventilation rate. Error decreased as ventilation rate increased.
Asunto(s)
Contaminación del Aire Interior/análisis , Exposición a Riesgos Ambientales/análisis , Modelos Teóricos , Movimientos del Aire , Humanos , Exposición Profesional , Reproducibilidad de los Resultados , VentilaciónRESUMEN
Decreased expression of functional alphaIIbbeta3 complexes on the platelet surface produces Glanzmann thrombasthenia. We have identified mutations of alphaIIb(P145) in 3 ethnically distinct families affected by Glanzmann thrombasthenia. Affected Mennonite and Dutch patients were homozygous and doubly heterozygous, respectively, for a P(145)A substitution, whereas a Chinese patient was doubly heterozygous for a P(145)L substitution. The mutations affect expression levels of surface alphaIIbbeta3 receptors on their platelets, which was confirmed by co-transfection of alphaIIb(P145A) and beta3 cDNA constructs in COS-1 cells. Each mutation also impaired the ability of alphaIIbbeta3 on affected platelets to interact with ligands. Moreover, when alphaIIb(P145A) and beta3 were stably coexpressed in Chinese hamster ovary cells, alphaIIbbeta3 was readily detected on the cell surface, but the cells were unable to adhere to immobilized fibrinogen or to bind soluble fluorescein isothiocyanate-fibrinogen after alphaIIbbeta3 activation by the activating monoclonal antibody PT25-2. Nonetheless, incubating affected platelets with the peptide LSARLAF, which binds to alphaIIb, induced PF4 secretion, indicating that the mutant alphaIIbbeta3 retained the ability to mediate outside-in signaling. These studies indicate that mutations involving alphaIIb(P145 )impair surface expression of alphaIIbbeta3 and that the alphaIIb(P145A) mutation abrogates ligand binding to the activated integrin. A comparative analysis of other alphaIIb mutations with a similar phenotype suggests that these mutations may cluster into a single region on the surface of the alphaIIb and may define a domain influencing ligand binding. (Blood. 2000;95:180188)
Asunto(s)
Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Mutación Puntual , Trombastenia/genética , Adolescente , Adulto , Sustitución de Aminoácidos , Animales , Sitios de Unión , Plaquetas/fisiología , Células CHO , Células COS , Preescolar , China/etnología , Cristianismo , Cricetinae , Etnicidad/genética , Femenino , Heterocigoto , Humanos , Ligandos , Sustancias Macromoleculares , Masculino , Modelos Moleculares , Países Bajos/etnología , Pennsylvania , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Transfección , Población Blanca/genéticaRESUMEN
Agonist-generated inside-out signals enable the platelet integrin alpha(IIb)beta(3) to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to alpha(IIb)beta(3). By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding. Cytochalasin D- and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating alpha(IIb)beta(3) function, cytochalasin D- and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains alpha(IIb)beta(3) in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on alpha(IIb)beta(3), allowing it to assume the high affinity conformation required for soluble ligand binding.
Asunto(s)
Actinas/metabolismo , Plaquetas/metabolismo , Plaquetas/ultraestructura , Citoesqueleto/metabolismo , Fibrinógeno/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Citoesqueleto/ultraestructura , HumanosRESUMEN
The simian virus 40 small t antigen (small-t) is required for optimal viral replication and transformation, especially during the infection of nondividing cells, suggesting that the function of small-t is to promote cell cycle progression. The mechanism through which small-t promotes cell growth reflects, in part, its binding and inhibition of protein phosphatase 2A (PP2A). The use of recombinant adenoviruses allows small-t expression in a majority of cells in a population, thus providing a convenient source of cells for biochemical analyses. In monkey kidney CV1 cells, small-t expressed from these adenovirus vectors activated the mitogen-activated protein kinase (MAPK) pathway, induced JNK activity, and increased AP-1 DNA-binding activity, all in a PP2A-dependent manner. Expression of small-t also caused an increase in the phosphorylation of the Na+/H+ antiporter, a mitogen-activated ion exchanger whose activity correlates with its phosphorylation. At least part of the antiporter phosphorylation induced by small-t reflected activation of the MAPK pathway, as suggested by results of assays using a chemical inhibitor of the MAPK-activating kinase, MEK. Finally, small-t expression from adenovirus vectors promoted efficient cell cycle progression by growth-arrested cells. These vectors should facilitate further analysis of effects of small-t on cell cycle mediators.
Asunto(s)
Adenoviridae/genética , Antígenos Virales de Tumores/genética , Vectores Genéticos , Quinasa 1 de Quinasa de Quinasa MAP , Proteínas Quinasas Activadas por Mitógenos , Virus 40 de los Simios/genética , Virus 40 de los Simios/inmunología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Ciclo Celular , División Celular , Línea Celular , Chlorocebus aethiops , Activación Enzimática , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Mutación , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Virus 40 de los Simios/fisiología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factor de Transcripción AP-1/metabolismo , Replicación ViralRESUMEN
We studied the function of the platelet integrin alphaIIb beta3 using a B lymphocyte model in which alphaIIb beta3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate alphaIIb beta3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and alphaIIb beta3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Galphai, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of alphaIIb beta3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that alphaIIb beta3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Galphai, protein kinase C, and the actin cytoskeleton.