RESUMEN
Low-dose ionizing radiation is known to induce radioadaptive responses in cells in vitro as well as in mice in vivo. Low-dose radiation decreases the incidence and increases latency for spontaneous and radiation-induced tumors in mice, potentially as a result of enhanced cellular DNA repair efficiency or a reduction in genomic instability. In this study, the cytokinesis-block micronucleus (CBMN) assay was used to examine dose response and potential radioadaptive response for cytogenetic damage and cell survival in C57BL/6 and BALB/c spleen cells exposed in vitro or in vivo to low-dose 60Co gamma radiation. The effects of genetic background, radiation dose and dose rate, sampling time and cell cycle were investigated with respect to dose response and radioadaptive response. In C57BL/6 mice, a linear-quadratic dose-response relationship for the induction of micronuclei (MN) was observed for doses between 100 mGy and 2 Gy. BALB/c mice exhibited increased radiosensitivity for MN induction compared to C57BL/6 mice. A 20 mGy dose had no effect on MN frequencies in splenocytes of either mouse strain, however, increased spleen weight and a reduced number of dead cells were noted in the C57BL/6 strain only. Multiple experimental parameters were investigated in radioadaptive response studies, including dose and dose rate of the priming dose (20 mGy at 0.5 mGy/min and 100 mGy at 10 mGy/min), time interval (4 and 24 h) between priming and challenge doses, cell cycle stage (resting or proliferating) at exposure and kinetics after the challenge dose. Radioadaptive responses were not observed for MN induction for either mouse strain under any of the experimental conditions investigated. In contrast, a synergistic response for radiation-induced micronuclei in C57BL/6 spleen was detected after in vivo 20 mGy irradiation. This increase in the percentage of cells with cytogenetic damage was associated with a reduction in the number of nonviable spleen cells, suggesting that low-dose irradiation led to a reduction in the turnover of damaged cells within the spleen of C57BL/6 mice. Overall, these results indicate that long-term protective effects against tumor latency and other beneficial health outcomes observed after low-dose irradiation are not mediated by a reduction of the proportion of cells harboring radiation-induced cytogenetic damage.
Asunto(s)
Adaptación Fisiológica/efectos de la radiación , Rayos gamma , Bazo/citología , Bazo/efectos de la radiación , Animales , Supervivencia Celular/efectos de la radiación , Citocinesis/efectos de la radiación , Reparación del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Femenino , Ratones , Pruebas de Micronúcleos , Tolerancia a Radiación/efectos de la radiaciónRESUMEN
Nitrobenzene is a major environmental pollutant, and its degradation is difficultto achieve. Hence, a chemical reduction pretreatment is sought in this research, before the resulting aniline can be treated by enzyme-mediated oxidative polymerization. Zerovalent iron (Fe0) has been successfully employed to reduce nitrobenzene to aniline in synthetic wastewater in both batch and continuous flow reactors. The concentration of nitrobenzene studied was thatwhich would be present in industrial wastewater streams (millimolar, 123 ppm), a concentration range considerably higher than those studied previously with groundwater by other researchers. Anaerobic conditions were maintained in the reactors by including Na2SO3 as an oxygen scavenger in the presence of CoCl2.6H2O, which acted as a catalyst. Batch reactors exhibited adsorption of aniline on the Fe0, which could be described by a langmuir isotherm. A 200 g Fe0 (particle size: 1-2 mm) bed completely converted 1 mM of nitrobenzene flowing upward for about 600 pore-volumes before experiencing flow reduction due to clogging due to corrosion products. Green-black precipitates (Fe0 corrosion products) were formed at the influent end of the column which were identified as maghemite.
Asunto(s)
Compuestos Férricos/química , Nitrobencenos/química , Eliminación de Residuos Líquidos/métodos , Compuestos de Anilina/química , Industrias , Oxidación-Reducción , Movimientos del Agua , Contaminación del Agua/prevención & controlRESUMEN
The binding of polypeptide growth factors to their appropriate cell surface transmembrane receptors triggers numerous biochemical responses, including the transcriptional activation of specific genes. We have used a differential display approach to identify fibroblast growth factor-1-inducible genes in murine NIH 3T3 cells. Here, we report that the fibroblast growth factor-inducible-14 (Fn14) gene is a growth factor-regulated, immediate-early response gene expressed in a developmental stage- and adult tissue-specific manner in vivo. This gene, located on mouse chromosome 17, is predicted to encode an 129-amino acid type Ia membrane protein with no significant sequence similarity to any known protein. We have used two experimental approaches, direct fluorescence microscopy and immunoprecipitation analysis of biotinylated cell surface proteins, to demonstrate that Fn14 is located on the plasma membrane. To examine the biological consequences of constitutive Fn14 expression, we isolated NIH 3T3 cell lines expressing variable levels of epitope-tagged Fn14 and analyzed their phenotypic properties in vitro. These experiments revealed that Fn14 expression decreased cellular adhesion to the extracellular matrix proteins fibronectin and vitronectin and also reduced serum-stimulated cell growth and migration. These results indicate that Fn14 is a novel plasma membrane-spanning molecule that may play a role in cell-matrix interactions.
Asunto(s)
Proteínas de la Membrana/genética , Receptores del Factor de Necrosis Tumoral , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Adhesión Celular/genética , División Celular/genética , Movimiento Celular/genética , Mapeo Cromosómico , Clonación Molecular , Factor de Crecimiento Epidérmico/genética , Matriz Extracelular/metabolismo , Factor 1 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hemaglutininas/genética , Hibridación in Situ , Proteínas de la Membrana/química , Ratones , Microscopía Fluorescente , Mitógenos/farmacología , Datos de Secuencia Molecular , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Receptor de TWEAK , TransfecciónRESUMEN
In mammalian cells, the octamer motif (ATGCAAAT) binding proteins, Oct-1 and Oct-2, play an important role in the transcriptional transactivation of several ubiquitously expressed genes as well as cell-specifically expressed genes. To date, a role of the octamer binding proteins in damage-stimulated response is not known. In this report, we demonstrate that DNA-binding activity of Oct-1, as demonstrated by the electrophoretic mobility shift assay, is significantly induced in a dose-dependent manner upon treatment of human head and neck squamous carcinoma cells (PCI-04A) with ionizing radiation (5 Gy: 5-fold; 15 Gy: 11-fold). By comparison, activities of other transcription factors were modestly increased (15 Gy: AP-1, 2.5-fold; NF-kappaB, 2.6-fold; SP-1, 5-fold). Radiation stimulation of Oct-1 activity was also noted in two other human cancer cell lines, albeit to a lesser extent (MDA-MB231 breast carcinoma cells and PC-3 prostate carcinoma cells (5 Gy: approximately 2-fold). These data represent the first report of the activation of an octamer factor DNA binding activity in response to environmental cues and suggest a novel role of Oct-1 in the radiation signaling cascade in these cancer cells.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Proteínas de Unión al ADN/efectos de la radiación , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/radioterapia , Factores de Transcripción/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Histonas/genética , Factor C1 de la Célula Huésped , Humanos , FN-kappa B/metabolismo , FN-kappa B/efectos de la radiación , Factor 1 de Transcripción de Unión a Octámeros , Radiación Ionizante , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp1/efectos de la radiación , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/efectos de la radiación , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
In this study we have analyzed short- and long-term changes in extracellular signal-regulated kinase (ERK) 1 and 2 activity during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of human promyelocytic leukemia cells. Immunoprecipitation of HL-60 cellular extracts with an ERK antibody followed by in vitro myelin basic protein phosphorylation demonstrated a rapid reduction in total ERK activity by 70%. Mitogen-activated protein kinase substrate peptide phosphorylation also demonstrated that this reduction was sustained during differentiation. Immunoblot analysis revealed that ERK1 and ERK2 are the predominant ERK isoforms present in HL-60 cells and that over a 96-h period ERK1 protein was gradually reduced by 60% while ERK2 protein showed only a small, insignificant reduction. Therefore, the large, rapid decrease in total ERK activity could not be attributed to the gradual reductions in ERK1 or ERK2 amounts. Immunoblot analysis with two different phosphotyrosine antibodies revealed a rapid decrease in ERK1 phosphotyrosine and a concurrent transient increase in ERK2 phosphotyrosine. These contrasting changes in phosphorylated ERKs were paralleled by respective shifts in mobility during SDS-PAGE analysis. Together these results indicate that the rapid reduction in total ERK activity is due to rapid tyrosine and possible threonine dephosphorylation of ERK1 but not of ERK2. These results also indicate that ERK1 and ERK2 are regulated by distinct mechanisms during TPA-induced HL-60 differentiation, suggesting that their biological roles are nonredundant.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células HL-60/enzimología , Proteínas Quinasas Activadas por Mitógenos , Acetato de Tetradecanoilforbol/farmacología , Diferenciación Celular/efectos de los fármacos , Células HL-60/citología , Humanos , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Proteína Básica de Mielina/metabolismo , Péptidos/metabolismo , Fosforilación , Treonina/metabolismo , Tirosina/metabolismoRESUMEN
PO-B was originally characterized as a transcriptional regulatory factor of the pro-opiomelanocortin (POMC) gene; however, it has become increasingly clear that this protein may be active in tissues outside the pituitary, since it is present in diverse cell types, including differentiated HL-60 promyelocytic leukemia cells. We previously showed that PO-B DNA-binding is progressively induced during differentiation of promyelomonocytic leukemic HL-60 cells to the macrophage-like lineage (with phorbol esters). We now report that PO-B DNA-binding in HL-60 cells is similarly induced during differentiation to the granulocytic lineage (with either retinoic acid or dimethylsulfoxide). Either a genetic or pharmacologic blockade of HL-60 differentiation prohibited these inductive effects. These studies have prompted our interest in the dynamics of other transcription factor changes during HL-60 differentiation. Of these, we observed that another transcription factor (AP-1) is also robustly induced at the DNA-binding level during macrophage-like HL-60 differentiation, but not during granulocytic differentiation. Conversely, the DNA-binding of the transcription factor AP-2 was slightly reduced by TPA-induced HL-60 differentiation but unchanged during granulocyte differentiation. From these data, we conclude that the induction of PO-B DNA binding is a general marker of HL-60 myelomonocytic differentiation, but that qualitative aspects of the induction of additional distinct transcription factors, such as AP-1, may contribute to lineage-specific determinants of cell fate.
Asunto(s)
Diferenciación Celular , ADN/metabolismo , Lactonas/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/metabolismo , Tretinoina/farmacología , Antineoplásicos/farmacología , Brioestatinas , Diferenciación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Dimetilsulfóxido/farmacología , Granulocitos , Células HL-60 , Humanos , Macrólidos , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-2RESUMEN
Expression of several muscle-specific genes was monitored during chicken muscle development and myoblast differentiation in primary cultures. The individual patterns of expression for many muscle-specific genes are well documented in ovo and in other model systems of muscle development. However, comparison of aldolase A to other muscle-specific genes in one system has not been reported. Both sarcomeric and cytosolic genes important for the adult muscle fiber were examined in order to elucidate their timing of expression and its relationship to cell fusion. Steady-state mRNA expression was measured using RNase protection assays with cRNA probes generated from cDNA clones for muscle creatine kinase, fast skeletal troponin-T, embryonic myosin heavy chain, and aldolase A. Nonmuscle genes expressed largely in the embryo, aldolase C and beta-actin, were used as controls. The expression of all six genes revealed differences in temporal expression patterns between limb and axial muscle. The temporal expression patterns of all six genes were also monitored in primary myoblast cultures relative to myoblast fusion. In both axial and limb myoblast cultures most of the muscle-specific genes were expressed prior to fusion. During the differentiation of myoblasts to myotubes there was a biphasic pattern in the expression of the muscle-specific genes. The appearance of measurable mRNA was detected by 16 hr in culture, prior to appreciable fusion of the cells. During further differentiation the expression increased gradually and then more rapidly at 96 hr, once fusion was complete. Meanwhile, the nonmuscle embryonic gene expression declined only slightly. For one gene, aldolase A, expression was delayed relative to the other muscle-specific genes, both in the appearance of measurable mRNA and in the later rapid increase in mRNA.
Asunto(s)
Fructosa-Bifosfato Aldolasa/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Isoenzimas/biosíntesis , Músculos/embriología , ARN Mensajero/biosíntesis , Animales , Diferenciación Celular , Células Cultivadas , Embrión de Pollo , Fructosa-Bifosfato Aldolasa/genética , Isoenzimas/genética , Músculos/citología , Músculos/metabolismo , Factores de TiempoRESUMEN
In chickens, as in all vertebrates, tissue-specific expression of aldolase isozymes A, B, and C is developmentally coordinated. These developmental transitions in aldolase expression have been studied most extensively by charting enzyme activity during normal and abnormal development of specific vertebrate tissues. Indeed, aldolase expression has been a key marker for normal differentiation and for retrodifferentiation during carcinogenesis. Aldolase expression during chicken myoblast differentiation offers a model for investigating the regulatory mechanisms of these developmental transitions at the level of gene expression. For these studies, cDNAs encoding the most isozyme-specific regions of both chicken aldolase A and C were cloned. The chicken aldolase A cDNA represents the first report of this sequence. Aldolase steady-state mRNA expression was measured during chicken myoblast differentiation in primary cultures using RNase protection assays with cRNA probes generated from these aldolase cDNA clones. Steady-state mRNA for aldolase C, the predominant embryonic aldolase isozyme in chickens, did not significantly change throughout myoblast differentiation. In contrast, expression of steady-state mRNA for aldolase A, the only aldolase isozyme found in adult-skeletal muscle, was not detected until after myoblast fusion was approximately 50% completed. Aldolase A expression gradually increased throughout myoblast differentiation until approximately 48 h after fusion was completed when there was a dramatic increase. These results are contrasted with those of Turner et al. (1974) [Dev Biol 37:63-89] that showed a coordinated switch in isozyme activities between the embryonic aldolase C and the muscle-specific aldolase A. This discordant expression indicates that the aldolase A and C genes may employ different regulatory mechanisms during myoblast differentiation.
Asunto(s)
Fructosa-Bifosfato Aldolasa/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Músculo Esquelético/metabolismo , ARN Mensajero/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular/genética , Embrión de Pollo , Pollos , Clonación Molecular , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Músculo Esquelético/citología , Células Madre/metabolismoRESUMEN
The mechanisms responsible for emergence of resistance during antimicrobial therapy were investigated in isolates obtained from a patient suffering from Pseudomonas aeruginosa endocarditis. The strain was first isolated from blood cultures obtained upon admission of the patient. It was sensitive to piperacillin, ticarcillin and tobramycin. Piperacillin-tobramycin therapy was therefore instituted and led to a rapid improvement of the patient's condition. Unfortunately, the patient subsequently became febrile again and blood cultures continued to yield P. aeruginosa. However, the organism isolated was now resistant to piperacillin and other penicillins tested. Cell-free extracts of the pre- and post-therapy isolates were analyzed for their beta-lactamase activity. The susceptible pre-therapy strain did not produce significant levels of beta-lactamase. In contrast, the post-therapy strain produced high levels of the enzyme. Induction experiments indicated that the production of beta-lactamase was constitutive in the post-therapy isolate. Thus, piperacillin therapy has led to the selection of resistant mutants which produced high levels of beta-lactamase constitutively. This was associated with relapse of the infection and therapeutic failure.