RESUMEN
Evidence suggests that pregnant animals are more sensitive than nonpregnant animals to the systemic administration of endotoxin. Studies were undertaken to assess whether an enhanced sensitivity of the pulmonary system to aerosolized endotoxin might exist during pregnancy. Pregnant Sprague-Dawley female rats (17 d of gestation) or age-matched virgin female rats were exposed to air or endotoxin (lipopolysaccharide) by inhalation for 3 h. At 18 h following exposure to endotoxin, lactate dehydrogenase activity levels in bronchoalveolar lavage (BAL) fluid samples from pregnant rats were 1.5-fold greater than those from endotoxin-exposed virgin rats. BAL polymorphonuclear leukocyte (PMN) numbers were also approximately twofold greater in pregnant rats than in virgins following the inhalation of endotoxin. The increases in BAL PMNs in pregnant rats following endotoxin exposure were observed just following exposure to endotoxin as well as at 18 h following exposure. These results indicate that an increased pulmonary inflammatory response to inhaled endotoxin occurs during pregnancy in rats. Additional findings suggest that these pregnancy-linked pulmonary responses to endotoxin cannot be explained by the following potential mechanisms: changes in the inhaled dose of endotoxin, or alterations in the responsiveness of alveolar macrophages to endotoxin. To our knowledge this is the first study that has evaluated pulmonary responses to inhaled endotoxin during pregnancy. Our finding that pregnancy is associated with an increased lung inflammatory response to aerosolized endotoxin raises the possibility that there may be a generalized enhancement of pulmonary responses to inhaled toxic agents during pregnancy.
Asunto(s)
Endotoxinas/toxicidad , Inflamación , Exposición por Inhalación , Enfermedades Pulmonares/etiología , Aerosoles , Animales , Endotoxinas/administración & dosificación , Femenino , Embarazo , Complicaciones del Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
Muscle damage due to stretch-shortening cycles (i.e., cyclic eccentric/concentric muscle actions) is one of the major concerns in sports and occupational related activities. Mechanical responses of whole muscle have been associated with damage in neural motor units, in connective tissues, and the force generation mechanism. The objective of this study was to introduce a new method to quantify the real-time changes in skeletal muscle forces of rats during injurious stretch-shortening cycles. Male Sprague Dawley rats ( n=24) were selected for use in this study. The dorsi flexor muscle group was exposed to either 150 stretch-shortening cycles ( n=12) or 15 isometric contractions ( n=12) in vivo using a dynamometer and electrical stimulation. Muscle damage after exposure to stretch-shortening cycles was verified by the non-recoverable force deficit at 48 h and the presence of myofiber necrosis. Variations of the dynamic forces during stretch-shortening cycles were analyzed by decomposing the dynamic force signature into peak force ( F(peak)), minimum force ( F(min)), average force ( F(mean)), and cyclic force ( F(a)). After the 15th set of stretch-shortening cycles, the decrease in the stretch-shortening parameters, F(peak), F(min), F(mean), and F(a), was 50% ( P<0.0001), 26% ( P=0.0055), 68% ( P<0.0001), and 50% ( P<0.0001), respectively. Our results showed that both isometric contractions and stretch-shortening cycles induce a reduction in the isometric force. However, the force reduction induced by isometric contractions fully recovered after a break of 48 h while that induced by stretch-shortening cycles did not. Histopathologic assessment of the tibialis anterior exposed to stretch-shortening cycles showed significant myofiber degeneration and necrosis with associated inflammation, while muscles exposed to isometric contractions showed no myofiber degeneration and necrosis, and limited inflammation. Our results suggest that muscle damage can be identified by the non-recoverable isometric force decrement and also by the variations in the dynamic force signature during stretch-shortening cycles.
Asunto(s)
Trastornos de Traumas Acumulados/patología , Trastornos de Traumas Acumulados/fisiopatología , Contracción Isométrica , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Enfermedades Musculares/patología , Enfermedades Musculares/fisiopatología , Animales , Estimulación Eléctrica , Masculino , Músculo Esquelético/lesiones , Músculo Esquelético/inervación , Periodicidad , Ratas , Ratas Sprague-Dawley , Estrés MecánicoRESUMEN
Results from previous studies indicate that hyperthyroidism increases the risk of ozone-induced lung toxicity. To better understand the processes that might contribute to the increased pulmonary inflammatory response to ozone in hyperthyroidism, we evaluated bronchoalveolar lavage fluid levels of selected cytokines in control and hyperthyroid rats after exposure to air or ozone. In addition, we assessed whether there is a relative increase in nuclear factor-kappa B (NF-kappaB) binding activity in cells harvested by bronchoalveolar lavage from hyperthyroid rats following the inhalation of ozone. A hyperthyroid condition was induced by the administration of thyroxine (0.5 mg/kg body weight) for 7 days. Control rats received vehicle injections. The animals were then exposed by inhalation to air or ozone (2 ppm for 3 h) and studied 18 h following the exposure. Bronchoalveolar lavage levels of MIP-2 and MCP-1 were increased in both control and hyperthyroid rats by ozone exposure. However, the increases in hyperthyroid rats were much greater, MIP-2 1.5-fold and MCP-1 11-fold, when compared to levels in controls following ozone. These changes appeared to be relatively specific; bronchoalveolar lavage fluid levels of interleukin (IL)-6, IL-4, and IL-10 were generally low or nondetectable across all of the studied groups at the 18-h postexposure time point. We also found that NF-kappaB binding activity was increased at both 4 and 18 h following ozone exposure in bronchoalveolar lavage cell extracts from hyperthyroid rats relative to the activity in control samples. Collectively, these results suggest that mechanisms contributing to the enhanced pulmonary inflammatory response to ozone in a hyperthyroid state include an increase in NF-kappaB activation and an upregulation of chemokine production.
Asunto(s)
Quimiocinas CXC , Quimiocinas/biosíntesis , Hipertiroidismo/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Enfermedades Pulmonares Intersticiales/metabolismo , Pulmón/metabolismo , FN-kappa B/biosíntesis , Ozono/toxicidad , Administración por Inhalación , Animales , Líquido del Lavado Bronquioalveolar/citología , Recuento de Células , Quimiocina CCL2/biosíntesis , Quimiocina CXCL2 , Modelos Animales de Enfermedad , Hipertiroidismo/inducido químicamente , Hipertiroidismo/complicaciones , Pulmón/efectos de los fármacos , Enfermedades Pulmonares Intersticiales/inducido químicamente , Enfermedades Pulmonares Intersticiales/complicaciones , Masculino , Monocinas/biosíntesis , FN-kappa B/efectos de los fármacos , Ozono/administración & dosificación , Ratas , Ratas Sprague-Dawley , Organismos Libres de Patógenos Específicos , Tiroxina/sangre , Tiroxina/farmacologíaRESUMEN
Transdermal therapy receives increasing attention as an attractive alternative to traditional drug delivery. Unfortunately the exact algorithm of transdermal permeation that could guide medicinal chemists towards delivery optimization at an early stage of the drug design process still remains to be decoded. This paper discusses some major hurdles on the way to full understanding of Quantitative Structure-Activity Relationships (QSAR) of skin permeation. From the statistical perspective, a recently published combined data set is found to be inappropriate with respect to the distribution of major molecular descriptors, and therefore should be approached cautiously as a source for QSAR model training and in modelling of occupational and environmental skin exposures.
Asunto(s)
Portadores de Fármacos , Fenómenos Fisiológicos de la Piel , Permeabilidad de la Membrana Celular , Portadores de Fármacos/química , Humanos , Técnicas In Vitro , Modelos Estadísticos , Permeabilidad , Absorción Cutánea , Relación Estructura-ActividadRESUMEN
Genotoxic stress triggers the activation of checkpoints that delay cell-cycle progression to allow for DNA repair. Studies in fission yeast implicate members of the Rad family of checkpoint proteins, which includes Rad17, Rad1, Rad9 and Hus1, as key early-response elements during the activation of both the DNA damage and replication checkpoints. Here we demonstrate a direct regulatory linkage between the human Rad17 homologue (hRad17) and the checkpoint kinases, ATM and ATR. Treatment of human cells with genotoxic agents induced ATM/ATR-dependent phosphorylation of hRad17 at Ser 635 and Ser 645. Overexpression of a hRad17 mutant (hRad17AA) bearing Ala substitutions at both phosphorylation sites abrogated the DNA-damage-induced G2 checkpoint, and sensitized human fibroblasts to genotoxic stress. In contrast to wild-type hRad17, the hRad17AA mutant showed no ionizing-radiation-inducible association with hRad1, a component of the hRad1-hRad9-hHus1 checkpoint complex. These findings demonstrate that ATR/ATM-dependent phosphorylation of hRad17 is a critical early event during checkpoint signalling in DNA-damaged cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Mutágenos/toxicidad , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular , Línea Celular , Daño del ADN , Proteínas de Unión al ADN , Doxiciclina/farmacología , Humanos , Ratones , Fosforilación , Serina/metabolismo , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
The risk of lung injury from ozone exposure has been well documented. It is also known that various factors may significantly influence the susceptibility of animals to the toxic effects of ozone. In the present study, we investigated the possibility that hyperthyroidism might be associated with increases in ozone-induced pulmonary toxicity. To create a hyperthyroid condition, mature male Sprague--Dawley rats were given injections of thyroxine (dose range: 0.1 to 1 mg/kg body wt daily for 7 days). Control rats received vehicle injections. The animals were then exposed to air or ozone (dose range: 0.5 to 3 ppm for 3 h). At 18 h postexposure, bronchoalveolar lavage fluid and cells were harvested. In hyperthyroid animals, ozone exposure was associated with three- to sixfold increases in bronchoalveolar lavage fluid lactate dehydrogenase activities and albumin levels as well as the number of polymorphonuclear leukocytes harvested by bronchoalveolar lavage above levels observed in ozone-exposed control rats. Additional results from the present study suggest that these thyroid hormone-linked effects cannot be fully explained by differences in whole-body metabolic rate or changes in the inhaled dose of ozone. These findings indicate that the risk of ozone-induced lung toxicity is substantially increased in a hyperthyroid state and suggest that the susceptibility of the lung to damage from ozone exposure may be significantly influenced by individual thyroid hormone status.
Asunto(s)
Hipertiroidismo/complicaciones , Enfermedades Pulmonares/inducido químicamente , Ozono/toxicidad , Albúminas/análisis , Animales , Metabolismo Basal , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Edema/inducido químicamente , Hipertiroidismo/inducido químicamente , L-Lactato Deshidrogenasa/análisis , Recuento de Leucocitos , Masculino , Neutrófilos , Ozono/administración & dosificación , Ratas , Ratas Sprague-Dawley , Respiración/efectos de los fármacos , Tiroxina/administración & dosificación , Tiroxina/sangre , Volumen de Ventilación PulmonarRESUMEN
The BRCA1 gene encodes a tumor suppressor that is mutated in 50% of familial breast cancers. The BRCA1 protein has been implicated in the DNA damage response, as DNA damage induces the phosphorylation of BRCA1 and causes its recruitment into nuclear foci that contain DNA repair proteins. The ataxia-telangiectasia-mutated (ATM) gene product controls overall BRCA1 phosphorylation in response to gamma-irradiation (IR). In this study, we show that BRCA1 phosphorylation is only partially ATM dependent in response to IR and ATM independent in response to treatment with UV light, or the DNA replication inhibitors hydroxyurea (HU) and aphidicolin (APH). We provide evidence that the kinase responsible for this phosphorylation is the ATM-related kinase, ATR. ATR phosphorylates BRCA1 on six Ser/Thr residues, including Ser 1423, in vitro. Increased expression of ATR enhanced the phosphorylation of BRCA1 on Ser 1423 following cellular exposure to HU or UV light, whereas doxycycline-induced expression of a kinase-inactive ATR mutant protein inhibited HU- or UV light-induced Ser 1423 phosphorylation in GM847 fibroblasts, and partially suppressed the phosphorylation of this site in response to IR. Thus, ATR, like ATM, controls BRCA1 phosphorylation in vivo. Although ATR isolated from DNA-damaged cells does not show enhanced kinase activity in vitro, we found that ATR responds to DNA damage and replication blocks by forming distinct nuclear foci at the sites of stalled replication forks. Furthermore, ATR nuclear foci overlap with the nuclear foci formed by BRCA1. The dramatic relocalization of ATR in response to DNA damage points to a possible mechanism for its ability to enhance the phosphorylation of substrates in response to DNA damage. Together, these results demonstrate that ATR and BRCA1 are components of the same genotoxic stress-responsive pathway, and that ATR directly phosphorylates BRCA1 in response to damaged DNA or stalled DNA replication.
Asunto(s)
Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Reparación del ADN , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1/genética , Catálisis , Línea Celular Transformada , Núcleo Celular , Daño del ADN , Expresión Génica , Humanos , Células K562 , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Conejos , Serina/metabolismoAsunto(s)
Mapeo Cromosómico/veterinaria , Cromosomas , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Porcinos/genética , Animales , Bovinos , Ciclooxigenasa 2 , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Phosphorylation at Ser-15 may be a critical event in the up-regulation and functional activation of p53 during cellular stress. In this report we provide evidence that the ATM-Rad3-related protein ATR regulates phosphorylation of Ser-15 in DNA-damaged cells. Overexpression of catalytically inactive ATR (ATRki) in human fibroblasts inhibited Ser-15 phosphorylation in response to gamma-irradiation and UV light. In gamma-irradiated cells, ATRki expression selectively interfered with late-phase Ser-15 phosphorylation, whereas ATRki blocked UV-induced Ser-15 phosphorylation in a time-independent manner. ATR phosphorylated p53 at Ser-15 and Ser-37 in vitro, suggesting that p53 is a target for phosphorylation by ATR in DNA-damaged cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN/fisiología , Proteínas Serina-Treonina Quinasas , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Daño del ADN/efectos de la radiación , Proteínas de Unión al ADN , Doxiciclina/farmacología , Fibroblastos , Rayos gamma , Humanos , Células K562 , Mutación , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Pruebas de Precipitina , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Serina/metabolismo , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Rayos UltravioletaRESUMEN
The Rac1 guanine nucleotide exchange factor, Vav, is activated in hematopoietic cells in response to a large variety of stimuli. The downstream signaling events derived from Vav have been primarily characterized as leading to transcription or transformation. However, we report here that Vav and Rac1 in natural killer (NK) cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing. In addition, overexpression of Vav, but not of a mutant lacking exchange factor activity, enhances both forms of killing by NK cells. Furthermore, dominant-negative Rac1 inhibits the development of NK cell-mediated cytotoxicity by two mechanisms: (a) conjugate formation between NK cells and target cells is decreased; and (b) those NK cells that do form conjugates have decreased ability to polarize their granules toward the target cell. Therefore, our results suggest that in addition to participating in the regulation of transcription, Vav and Rac1 are pivotal regulators of adhesion, granule exocytosis, and cellular cytotoxicity.
Asunto(s)
Proteínas de Ciclo Celular , Citotoxicidad Inmunológica , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Células Asesinas Naturales/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Células Cultivadas , Proteínas de Unión al GTP/genética , Humanos , Células Asesinas Naturales/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-vav , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tirosina/metabolismo , Proteínas de Unión al GTP racRESUMEN
Significant progress has been made in our understanding of the basic signaling mechanisms regulating NK cell activation. Advances have been fueled in part by the molecular characterization of specific activating receptors (e.g., the Fc gamma RIII multi-subunit complex) and inhibitory receptors (e.g., novel MHC-recognizing inhibitory receptors). However, certain aspects of these analyses are complicated by the heterogeneous nature of the receptor-ligand interactions utilized during the development of a cytotoxic response. Future advances will depend in part on the further molecular characterization of the involved receptors and second messengers and on the development of experimental models for genetically manipulating the signaling elements. It will remain important to understand both activating and inhibitory signaling pathways as the emerging theme is that the balance of these two opposing forces determines the functional outcome of an NK cells interaction with its target.
Asunto(s)
Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Transducción de Señal , Animales , HumanosRESUMEN
Cells may be protected from natural killer (NK)-cell-mediated killing by the expression of specific MHC class I complexes. This protective effect is due to the expression on NK cells of MHC class I-recognizing receptors which, upon ligation, transduce potent inhibitory signals into the NK cells. The molecular signalling mechanisms employed by the human NK-cell MHC-recognizing killer cell inhibitory receptors (KIR) and CD94 are the focus of this review. A sequential model of KIR signalling involving lck-dependent tyrosine phosphorylation of KIR and subsequent association of KIR with the SH2-containing tyrosine phosphatase, SHP-1, is presented. We explore how engagement of either KIR or CD94 modulates the protein tyrosine kinase-dependent biochemical signals responsible for activation of NK-cell cytotoxic function. Additionally, we discuss models of inhibitory signalling proposed for each of the lymphocyte lineages, emphasizing that disparate molecular mechanisms may be utilized by cells to produce similar biological responses.
Asunto(s)
Antígenos HLA/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal/inmunología , Antígenos HLA/inmunología , HumanosRESUMEN
Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70-deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Precursores Enzimáticos/metabolismo , Células Asesinas Naturales/enzimología , Proteínas Tirosina Quinasas/metabolismo , Animales , Calcio/metabolismo , Genes MHC Clase I , Fosfatos de Inositol/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Células Asesinas Naturales/inmunología , Cinética , Ratones , Transducción de Señal , Quinasa Syk , Transfección , Células Tumorales CultivadasRESUMEN
Recognition of major histocompatibility (MHC) class I complexes on target cells by killer cell inhibitory receptors (KIR) blocks natural killer (NK) and T cell cytotoxic function. The inhibitory effect of KIR ligation requires the phosphotyrosine-dependent association of KIR with the cytoplasmic SH2-containing protein tyrosine phosphatase SHP-1. Using a somatic genetic model, we first define a requirement for the Src family protein tyrosine kinase (PTK) Lck in mediating KIR tyrosine phosphorylation. We then investigate how KIR ligation interrupts PTK-dependent NK cell activation signals. Specifically, we show that KIR ligation inhibits the Fc receptor (FcR)-induced tyrosine phosphorylation of the FcR-associated zeta signaling chain, the PTK ZAP-70, and phospholipase C gamma. Overexpression of catalytically inactive SHP-1 (acting as a dominant negative) restores the tyrosine phosphorylation of these signaling events and reverses KIR-mediated inhibition of NK cell cytotoxic function. These results suggest sequential roles for Lck and SHP-1 in the inhibition of PTK following MHC recognition by NK cells.
Asunto(s)
Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Proteínas de Unión al GTP Monoméricas , Proteínas Tirosina Fosfatasas/metabolismo , Receptores Inmunológicos/metabolismo , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas de Unión al GTP/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Complejo Mayor de Histocompatibilidad , Ratones , Datos de Secuencia Molecular , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Quinasas/metabolismo , Receptores Fc/metabolismo , Transducción de Señal , Tirosina/metabolismoRESUMEN
Ligation of MHC class I-recognizing receptors on NK cells dramatically modulates their secretory and cytotoxic function. This study focuses on characterizing key signaling events regulating these activities after ligation of the C-type lectin superfamily member, CD94. We isolated separate clonal populations of human NK cells in which ligation of CD94 (kp43) either triggered cell-mediated cytotoxicity (group A clones) or potently inhibited NK cell activation (group B clones). We then evaluated the proximal signaling events that regulate these alternative responses. CD94 stimulation of group A clones induced the rapid activation of intracellular protein tyrosine kinases (i.e., lck and ZAP-70), phospholipase C, and phosphatidylinositol 3-kinase. In contrast, CD94 ligation on group B clones had none of the above noted effects and instead inhibited the FcR-induced tyrosine phosphorylations of ZAP-70 and phospholipase C-gamma 2, the formation of phospho-zeta/ZAP-70 complexes, and the release of inositol phosphates. These results define distinct proximal signaling events initiated after CD94 ligation and suggest that clonotypic differences in signaling generate fundamentally different NK cell-mediated responses.