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Jet engine exhaust is a significant source of ultrafine particles and aviation-related emissions can adversely impact air quality over large areas surrounding airports. We investigated outdoor and indoor ultrafine particle number concentrations (PNC) from 16 residences located in two study areas in the greater Boston metropolitan area (MA, USA) for evidence of aviation-related impacts. During winds from the direction of Logan International Airport, that is, impact-sector winds, an increase in outdoor and indoor PNC was clearly evident at all seven residences in the Chelsea study area (â¼4-5 km from the airport) and three out of nine residences in the Boston study area (â¼5-6 km from the airport); the median increase during impact-sector winds compared to other winds was 1.7-fold for both outdoor and indoor PNC. Across all residences during impact-sector and other winds, median outdoor PNC were 19â¯000 and 10â¯000 particles/cm3, respectively, and median indoor PNC were 7000 and 4000 particles/cm3, respectively. Overall, our results indicate that aviation-related outdoor PNC infiltrate indoors and result in significantly higher indoor PNC. Our study provides compelling evidence for the impact of aviation-related emissions on residential exposures. Further investigation is warranted because these impacts are not expected to be unique to Logan airport.
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Poluentes Atmosféricos , Poluição do Ar em Ambientes Fechados , Aviação , Aeroportos , Boston , Monitoramento Ambiental , Tamanho da Partícula , Material ParticuladoRESUMO
The goal of the ASACUSA-CUSP collaboration at the Antiproton Decelerator of CERN is to measure the ground-state hyperfine splitting of antihydrogen using an atomic spectroscopy beamline. A milestone was achieved in 2012 through the detection of 80 antihydrogen atoms 2.7 m away from their production region. This was the first observation of 'cold' antihydrogen in a magnetic field free region. In parallel to the progress on the antihydrogen production, the spectroscopy beamline was tested with a source of hydrogen. This led to a measurement at a relative precision of 2.7×10-9 which constitutes the most precise measurement of the hydrogen hyperfine splitting in a beam. Further measurements with an upgraded hydrogen apparatus are motivated by CPT and Lorentz violation tests in the framework of the Standard Model Extension. Unlike for hydrogen, the antihydrogen experiment is complicated by the difficulty of synthesizing enough cold antiatoms in the ground state. The first antihydrogen quantum states scan at the entrance of the spectroscopy apparatus was realized in 2016 and is presented here. The prospects for a ppm measurement are also discussed.This article is part of the Theo Murphy meeting issue 'Antiproton physics in the ELENA era'.
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Highly charged iron (Fe(16+), here referred to as Fe XVII) produces some of the brightest X-ray emission lines from hot astrophysical objects, including galaxy clusters and stellar coronae, and it dominates the emission of the Sun at wavelengths near 15 ångströms. The Fe XVII spectrum is, however, poorly fitted by even the best astrophysical models. A particular problem has been that the intensity of the strongest Fe XVII line is generally weaker than predicted. This has affected the interpretation of observations by the Chandra and XMM-Newton orbiting X-ray missions, fuelling a continuing controversy over whether this discrepancy is caused by incomplete modelling of the plasma environment in these objects or by shortcomings in the treatment of the underlying atomic physics. Here we report the results of an experiment in which a target of iron ions was induced to fluoresce by subjecting it to femtosecond X-ray pulses from a free-electron laser; our aim was to isolate a key aspect of the quantum mechanical description of the line emission. Surprisingly, we find a relative oscillator strength that is unexpectedly low, differing by 3.6σ from the best quantum mechanical calculations. Our measurements suggest that the poor agreement is rooted in the quality of the underlying atomic wavefunctions rather than in insufficient modelling of collisional processes.
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Ultrafine particles are emitted at high rates by jet aircraft. To determine the possible impacts of aviation activities on ambient ultrafine particle number concentrations (PNCs), we analyzed PNCs measured from 3 months to 3.67 years at three sites within 7.3 km of Logan International Airport (Boston, MA). At sites 4.0 and 7.3 km from the airport, average PNCs were 2- and 1.33-fold higher, respectively, when winds were from the direction of the airport compared to other directions, indicating that aviation impacts on PNC extend many kilometers downwind of Logan airport. Furthermore, PNCs were positively correlated with flight activity after taking meteorology, time of day and week, and traffic volume into account. Also, when winds were from the direction of the airport, PNCs increased with increasing wind speed, suggesting that buoyant aircraft exhaust plumes were the likely source. Concentrations of other pollutants [CO, black carbon (BC), NO, NO2, NOx, SO2, and fine particulate matter (PM2.5)] decreased with increasing wind speed when winds were from the direction of the airport, indicating a different dominant source (likely roadway traffic emissions). Except for oxides of nitrogen, other pollutants were not correlated with flight activity. Our findings point to the need for PNC exposure assessment studies to take aircraft emissions into consideration, particularly in populated areas near airports.
Assuntos
Poluentes Atmosféricos/análise , Aeronaves , Aeroportos , Monitoramento Ambiental , Boston , Meteorologia , Tamanho da Partícula , Material Particulado/análise , Silicones , Fuligem , Emissões de Veículos/análise , VentoRESUMO
BACKGROUND: Human sarcomas with a poor response to vascular endothelial growth factor-A (VEGF-A) inhibition and radiation therapy (RT) have upregulation of hypoxia-inducible factor 1α (HIF-1α) and HIF-1α target genes. This study examines the addition of the hypoxia-activated chemotherapy TH-302 to VEGF-A inhibition and RT (a.k.a. trimodality therapy). METHODS: Trimodality therapy was examined in two xenograft models and in vitro in tumour endothelial cells and sarcoma cell lines. RESULTS: In both mouse models, VEGF-A inhibition and radiation showed greater efficacy than either therapy alone in slowing sarcoma growth. When TH-302 was added, this trimodality therapy completely blocked tumour growth with tumours remaining dormant for over 3 months after cessation of therapy. Trimodality therapy caused 2.6- to 6.2-fold more endothelial cell-specific apoptosis than bimodality therapies, and microvessel density and HIF-1α activity were reduced to 11-13% and 13-20% of control, respectively. When trimodality therapy was examined in vitro, increases in DNA damage and apoptosis were much more pronounced in tumour endothelial cells compared with that in sarcoma cells, especially under hypoxia. CONCLUSIONS: The combination of TH-302, VEGF-A inhibition, and RT is highly effective in preclinical models of sarcoma and is associated with increased DNA damage and apoptosis in endothelial cells and decreased HIF-1α activity.
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Antineoplásicos/uso terapêutico , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Nitroimidazóis/uso terapêutico , Mostardas de Fosforamida/uso terapêutico , Sarcoma/tratamento farmacológico , Sarcoma/radioterapia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ativação Metabólica , Animais , Antineoplásicos/farmacocinética , Terapia Combinada , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nitroimidazóis/farmacocinética , Mostardas de Fosforamida/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this Letter, we introduce the concept of in-trap nuclear decay spectroscopy of highly charged radioactive ions and describe its successful application as a novel spectroscopic tool. This is demonstrated by a measurement of the decay properties of radioactive mass A=124 ions (here, ^{124}In and ^{124}Cs) in the electron-beam ion trap of the TITAN facility at TRIUMF. By subjecting the trapped ions to an intense electron beam, the ions are charge bred to high charge states (i.e., equivalent to the removal of N-shell electrons), and an increase of storage times to the level of minutes without significant ion losses is achieved. The present technique opens the venue for precision spectroscopy of low branching ratios and is being developed in the context of measuring electron-capture branching ratios needed for determining the nuclear ground-state properties of the intermediate odd-odd nuclei in double-beta (ßß) decay.
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Fatty acids, uric acid and glucose are thought to contribute to subclinical inflammation associated with diabetes mellitus. We tested whether co-incubation of free fatty acids and uric acid or glucose influences the secretion of immune mediators from stimulated human whole blood in vitro. Fresh whole blood samples from 20 healthy subjects, 20 patients with type 1 diabetes and 23 patients with type 2 diabetes were incubated for 24 h with palmitic acid (PAL), linolenic acid (LIN) or eicosapentaenoic acid (EPA) alone or together with elevated concentrations of uric acid or glucose. Concentrations of proinflammatory cytokines interleukin (IL)-1ß, IL-2, IL-12(p70), IL-18, IFN-γ, of regulatory cytokines IL-4, IL-10, IL-17 and chemokine CCL2 (MCP-1) were measured by multiplex-bead technology from supernatants. Co-incubation of fatty acids with uric acid resulted in a significant reduction of IL-10, IL-12(p70), IFN-γ and CCL2 (MCP-1) concentrations in supernatants compared to incubation with uric acid alone (P < 0·0001). In contrast, IL-18 was up-regulated upon co-stimulation with fatty acids and uric acid. Similarly, co-incubation of fatty acids with glucose diminished secretion of IL-10, IFN-γ and CCL2 (monocyte chemotactic protein-1), while IL-8 was up-regulated (P < 0·001). Samples from healthy and diabetic subjects did not differ after adjustment for age, sex, body mass index and diabetes type. All three fatty acids similarly influenced whole blood cytokine release in vitro and modulated uric acid or glucose-stimulated cytokine secretion. Although the ω-3-fatty acid EPA showed slightly stronger effects, further studies are required to elaborate the differential effects of PAL, LIN and EPA on disease risk observed previously in epidemiological studies.
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Quimiocinas/sangue , Citocinas/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ácidos Graxos não Esterificados/farmacologia , Adulto , Estudos de Casos e Controles , Interações Medicamentosas , Ácido Eicosapentaenoico/administração & dosagem , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos não Esterificados/administração & dosagem , Feminino , Glucose/administração & dosagem , Glucose/farmacologia , Humanos , Mediadores da Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Ácido Palmítico/administração & dosagem , Ácido Palmítico/farmacologia , Fito-Hemaglutininas/farmacologia , Ácido Úrico/administração & dosagem , Ácido Úrico/farmacologia , Ácido alfa-Linolênico/administração & dosagem , Ácido alfa-Linolênico/farmacologiaRESUMO
Development of definitive (fetal liver-derived) red cells is blocked by a targeted mutation in the gene encoding the transcription factor GATA-1. We used in vitro differentiation of GATA-1- mouse embryonic stem (ES) cells to reveal a requirement for GATA-1 during primitive (yolk sac-derived) erythropoiesis and to establish a rescue assay. We show that the block to development includes primitive, as well as definitive, erythroid cells and is complete at the level of globin RNA expression; that the introduction of a normal GATA-1 gene restores developmental potential both in vivo and in vitro; and that efficient rescue is dependent on a putative autoregulatory GATA-motif in the distal promoter. Use of in vitro differentiated ES cells bridges a gap between conventional approaches to gene function in cell lines and analysis of loss of function mutations in the whole animal.
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Proteínas de Ligação a DNA/genética , Eritropoese/genética , Células-Tronco/citologia , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos/citologia , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Humanos , Camundongos , Mutação , TransfecçãoRESUMO
A low energy proton source for non-neutral plasma experiments was developed. Electrons from a hot filament ionize H2 gas inside a geometrically compensated Penning trap to produce protons via dissociative ionization. A rotating wall electric field destabilizes the unwanted H2+ and H3+ generated in the process while concentrating protons at the center of the trap. The source produces bunches of protons with relatively low ion contamination (5.5% H2+ and 15.5% H3+), with energy tunable from 35 to 300 eV.
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We present precision Penning trap mass measurements of neutron-rich calcium and potassium isotopes in the vicinity of neutron number N=32. Using the TITAN system, the mass of 51K was measured for the first time, and the precision of the (51,52)Ca mass values were improved significantly. The new mass values show a dramatic increase of the binding energy compared to those reported in the atomic mass evaluation. In particular, 52Ca is more bound by 1.74 MeV, and the behavior with neutron number deviates substantially from the tabulated values. An increased binding was predicted recently based on calculations that include three-nucleon (3N) forces. We present a comparison to improved calculations, which agree remarkably with the evolution of masses with neutron number, making neutron-rich calcium isotopes an exciting region to probe 3N forces.
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Penning trap mass measurements of short-lived nuclides have been performed for the first time with highly charged ions, using the TITAN facility at TRIUMF. Compared to singly charged ions, this provides an improvement in experimental precision that scales with the charge state q. Neutron-deficient Rb isotopes have been charge bred in an electron beam ion trap to q=8-12+ prior to injection into the Penning trap. In combination with the Ramsey excitation scheme, this unique setup creating low energy, highly charged ions at a radioactive beam facility opens the door to unrivaled precision with gains of 1-2 orders of magnitude. The method is particularly suited for short-lived nuclides such as the superallowed ß emitter 74Rb (T(1/2)=65 ms). The determination of its atomic mass and an improved Q(EC) value are presented.
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Spi-B and PU.1 are hematopoietic-specific transcription factors that constitute a subfamily of the Ets family of DNA-binding proteins. Here we show that contrary to previous reports, PU.1 and Spi-B have very different expression patterns. PU.1 is expressed at high levels in B cells, mast cells, megakaryocytes, macrophages, neutrophils, and immature erythroid cells and at lower levels in mature erythrocytes. PU.1 is completely absent from peripheral T cells and most T cell lines based on sensitive RT-PCR assays. In contrast, Spi-B is expressed exclusively in lymphoid cells and can be detected in early fetal thymus and spleen. In situ hybridizations of adult murine tissues demonstrate Spi-B mRNA in the medulla of the thymus, the white pulp of the spleen, and the germinal centers of lymph nodes. Spi-B expression is very abundant in B cells and both Spi-B mRNA and protein are detected in some T cells. In situ hybridization and Northern blot analysis suggest that Spi-B gene expression increases during B cell maturation and decreases during T cell maturation. Gel-retardation experiments show that Spi-B can bind to all putative PU.1 binding sites, but do not reveal any preferred Spi-B binding site. Finally, both PU.1 and Spi-B function as transcriptional activators of the immunoglobulin light-chain enhancer E lambda 2.4 when coexpressed with Pip (PU.1-interaction partner) in NIH-3T3 cells. Taken together, these data suggest that differences in patterns of expression between Spi-B and PU.1 distinguish the function of each protein during development of the immune system.
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Linfócitos B/fisiologia , Proteínas de Ligação a DNA/metabolismo , Linfócitos T/fisiologia , Transativadores , Fatores de Transcrição/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular , Primers do DNA/química , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Humanos , Cadeias kappa de Imunoglobulina/genética , Hibridização In Situ , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genéticaRESUMO
Photoionization (PI) of Fe14+ in the range from 450 to 1100 eV was measured at the BESSY II storage ring using an electron beam ion trap achieving high target-ion area densities of 10(10) cm(-2). Photoabsorption by this ion is observed in astrophysical spectra and plasmas, but until now cross sections and resonance energies could only be provided by calculations. We reach a resolving power E/ΔE of at least 6500, outstanding in the present energy range, which enables benchmarking and improving the most advanced theories for PI of ions in high charge states.
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BACKGROUND: Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) can cause profound and prolonged illness and disability, and poses significant problems of uncertainty for healthcare professionals in its diagnosis and management. The aim of this qualitative study was to explore the nature of professional 'best practice' in working with people with CFS/ME. METHODS: The views and experiences of health care practitioners (HCPs) were sought, who had been judged by people with CFS/ME themselves to have been particularly helpful and effective. Qualitative semi-structured interviews following a topic guide were carried out with six health care practitioners. Interviews were audio-recorded, transcribed and subject to thematic analysis. RESULTS: Five main themes were developed: 1) Diagnosis; 2) Professional perspectives on living with CFS/ME; 3) Interventions for treatment and management; 4) Professional values and support for people with CFS/ME and their families; 5) Health professional roles and working practices. Key findings related to: the diagnostic process, especially the degree of uncertainty which may be shared by primary care physicians and patients alike; the continued denial in some quarters of the existence of CFS/ME as a condition; the variability, complexity, and serious impact of the condition on life and living; the onus on the person with CFS/ME to manage their condition, supported by HCPs; the wealth of often conflicting and confusing information on the condition and options for treatment; and the vital role of extended listening and trustful relationships with patients. CONCLUSIONS: While professional frustrations were clearly expressed about the variability of services both in primary and specialist care and continuing equivocal attitudes to CFS/ME as a condition, there were also strong positive messages for people with CFS/ME where the right services are in place. Many of the findings from these practitioners seen by their patients as helping them more effectively, accord with the existing literature identifying the particular importance of listening skills, respect and trust for establishing a therapeutic relationship which recognises key features of the patient trajectory and promotes effective person-centred management of this complex condition. These findings indicate the need to build such skills and knowledge more systematically into professional training informed by the experience of specialist services and those living with the condition.
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Síndrome de Fadiga Crônica/terapia , Médicos de Família , Qualidade de Vida , Adulto , Atitude do Pessoal de Saúde , Síndrome de Fadiga Crônica/diagnóstico , Feminino , Nível de Saúde , Humanos , Entrevistas como Assunto , Masculino , Relações Médico-Paciente , Médicos de Família/psicologia , Médicos de Família/estatística & dados numéricos , Padrões de Prática Médica , Pesquisa QualitativaRESUMO
The transcriptional response to hypoxia is primarily mediated by two hypoxia-inducible factors--HIF-1alpha and HIF-2alpha. While these proteins are highly homologous, increasing evidence suggests they have unique transcriptional targets and differential impact on tumor growth. Furthermore, non-transcriptional effects of the HIF-alpha subunits, including effects on the Notch and c-Myc pathways, contribute to their distinct functions. HIF-2alpha transcriptional targets include genes involved in erythropoiesis, angiogenesis, metastasis, and proliferation. Therefore, HIF-2alpha contributes significantly to both normal physiology as well as tumorigenesis. Here, we summarize the function of HIF-2alpha during development as well as its contribution to pathologic conditions, such as tumors and vascular disease.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Hipóxia/genética , Camundongos , Camundongos Knockout , Neoplasias/genética , Fenótipo , Distribuição Tecidual , Transcrição GênicaRESUMO
The transcription factor PU.1 is a hematopoietic-specific member of the ets family. Mice carrying a mutation in the PU.1 locus were generated by gene targeting. Homozygous mutant embryos died at a late gestational stage. Mutant embryos produced normal numbers of megakaryocytes and erythroid progenitors, but some showed an impairment of erythroblast maturation. An invariant consequence of the mutation was a multilineage defect in the generation of progenitors for B and T lymphocytes, monocytes, and granulocytes. Thus, the developmental programs of lymphoid and myeloid lineages require a common genetic function likely acting at the level of a multipotential progenitor.
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Proteínas de Ligação a DNA/fisiologia , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Eritropoese , Feminino , Rearranjo Gênico , Células-Tronco Hematopoéticas/citologia , Linfócitos/citologia , Linfócitos/fisiologia , Macrófagos/citologia , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/fisiologia , Mutação , Neutrófilos/citologia , Neutrófilos/fisiologia , Proteínas Oncogênicas de Retroviridae , Fatores de Transcrição/genéticaRESUMO
The emergence of antimicrobial-resistant microorganisms, in both humans and animals, calls for the development of new strategies for administering antibiotics in order to bring these microorganisms under control and, most importantly, to prevent the risk of resistant bacteria being transmitted to animals and humans. One such microorganism, Staphylococcus aureus, especially its methicillin-resistant strains, is particularly hazardous to public health. This study, conducted in intensive rabbit farms in Spain, has demonstrated a high prevalence of S. aureus strains, 17.2% of which were found to be methicillin-resistant. The study also unveiled very high resistance to quinolones and to some forms of penicillin. This source of multidrug resistance merits special attention in view of the problems it poses for choosing suitable treatment and the risk of this resistance being transmitted to humans, either from direct contact or via the food chain.
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Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Saúde Pública , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/efeitos dos fármacos , Animais , Cruzamento , Cadeia Alimentar , Humanos , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Coelhos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/transmissão , ZoonosesAssuntos
Proteínas de Ligação a DNA/fisiologia , Regiões Promotoras Genéticas , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Diferenciação Celular/genética , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA2 , Fator de Transcrição GATA3 , Fator de Transcrição GATA4 , Fator de Transcrição GATA5 , Fator de Transcrição GATA6 , Hematopoese/genética , Humanos , Camundongos , Dados de Sequência Molecular , Família Multigênica , Regiões Promotoras Genéticas/genética , Dedos de Zinco/genética , Dedos de Zinco/fisiologiaRESUMO
Totipotent murine embryonic stem (ES) cells can be differentiated in vitro to form embryoid bodies (EBs) containing hematopoietic cells of multiple lineages, including erythroid cells. In vitro erythroid development parallels that which is observed in vivo. ES cells in which the gene for the erythroid transcription factor GATA-1 has been disrupted fail to produce mature erythroid cells either in vivo or in vitro. With the EB in vitro differentiation assay, constructs expressing heterologous GATA-binding proteins were tested for their abilities to correct the developmental defect of GATA-1-deficient ES cells. The results presented here show that the highly divergent chicken GATA-1 can rescue GATA-1 deficiency to an extent similar to that of murine GATA-1 (mGATA-1), as determined by size and morphology of EBs, presence of red cells, and globin gene expression. Furthermore, GATA-3 and GATA-4, which are normally expressed in different tissues, and a protein consisting of the zinc fingers of GATA-1 fused to the herpes simplex virus VP16 transcription activation domain were able to compensate for the GATA-1 defect. Chimeric molecules in which both zinc fingers of mGATA-1 were replaced with the zinc fingers of human GATA-3 or with the single finger of the fungal GATA factor areA, as well as a construct bearing the zinc finger region alone, displayed rescue activity. These results suggest that neither the transcription activation domains of mGATA-1 nor its zinc fingers impart erythroid cell specificity for its action in vivo. Rather, it appears that specificity is mediated through the cis-acting control regions which determine spatial and temporal expression of the GATA-1 gene. Furthermore, our results demonstrate that the zinc finger region may have a biological function in addition to mediating DNA binding.
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Proteínas de Ligação a DNA/metabolismo , Células-Tronco/citologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Fator de Transcrição GATA3 , Fator de Transcrição GATA4 , Humanos , Camundongos , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Células-Tronco/metabolismo , Transativadores/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , TransfecçãoRESUMO
The protein product of the c-fps/fes (c-fes) proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and neutrophils). mRNA for c-fes has been detected exclusively in myeloid cells and vascular endothelial cells in adult mammals. Although a 13-kilobase-pair (kb) human c-fes transgene exhibits high levels of expression in mice, the sequences that confer myeloid-cell-specific expression of the human c-fes gene have not been defined. Transient-transfection experiments demonstrated that plasmids containing 446 bp of c-fes 5'-flanking sequences linked to a luciferase reporter gene were active exclusively in myeloid cells. No other DNA element within the 13-kb human c-fes locus contained positive cis-acting elements, with the exception of a weakly active region within the 3'-flanking sequences. DNase I footprinting assays revealed four distinct sites that bind myeloid nuclear proteins (-408 to -386, -293 to -254, -76 to -65, and -34 to +3). However, the first two footprints resided in sequences that were largely dispensable for transient activity. Plasmids containing 151 bp of 5'-flanking sequences confer myeloid-cell-specific gene expression. Electrophoretic mobility shift analyses demonstrated that the 151-bp region contains nuclear protein binding sites for Sp1, PU.1, and/or Elf-1, and a novel factor. This unidentified factor binds immediately 3' of the PU.1/Elf-1 sites and appears to be myeloid cell specific. Mutation of the PU.1/Elf-1 site or the 3' site (FP4-3') within the context of the c-fes promoter resulted in substantially reduced activity in transient transfections. Furthermore, transient-cotransfection assay demonstrated that PU.1 (and not Elf-1) can transactivate the c-fes promoter in nonmyeloid cell lines. We conclude that the human c-fes gene contains a strong myeloid-cell-specific promoter that is regulated by Sp1, PU.1, and a novel transcription factor.