RESUMO
European eel is critically endangered in Europe. Among other stressors, pathogens are well-known to harm eels' fitness. One hundred and eighty-two eels were captured in three Eel Management Units in Andalucía (SE Spain) and analysed for Anguillicoloides crassus, Anguillid herpesvirus 1 (AngHV1), the rhabdovirus Eel Virus European X (EVEX) and the aquabirnavirus Eel Virus European (EVE). A. crassus adults and preadults were isolated and morphometrically identified, and the eel swimbladders were artificially digested to count A. crassus larvae. Also, eel tissues were examined by PCRs for the presence of viruses. EVEX and EVE were not detected in any of the eels. The estimated prevalence (95% confidence limits) was 71 (64-78)% for A. crassus and 35 (28-42)% for AngHV-1, varying these prevalences significantly between and within EMUs. Moreover, A. crassus prevalence was highest in smaller eels, in sites closest to the sea and eels sampled in the autumn. By contrast, AngHV-1 prevalence was highest in biggest eels, in sites far from the sea and sampled in the summer or winter. However, in mixed effects logistic models including site as a random variable, the risk of infection was associated with distance to the sea in both A. crassus and AngHV-1 infections and also to winter sampling in the case of AngHV-1 and not to other variables. These results are evidence that both pathogens are highly endemic in eels from Andalusian habitats. Further studies are needed to better understand the risk factors associated with these pathogens on eel populations.
Assuntos
Anguilla , Aquabirnavirus , Dracunculoidea , Doenças dos Peixes , Rhabdoviridae , Animais , Rhabdoviridae/genética , Espanha/epidemiologia , Doenças dos Peixes/epidemiologia , Sacos AéreosRESUMO
BACKGROUND: Oral immunotherapy (OIT) is a promising approach to cow's milk and egg allergies, but reactions are frequent and some patients cannot be desensitized. OBJECTIVE: To evaluate long-term OIT outcomes with omalizumab (OMZ) in paediatric patients with severe egg and/or milk allergies. METHODS: This retrospective real-life study analysed findings in children with Immunoglobulin E-mediated allergy to cow's milk and/or hen eggs refractory to conventional OIT, who underwent OIT with OMZ in our department between 1 January 2010 and 31 December 2015. RESULTS: In all, 41 patients were included (median age: 7 years; interquartile range [IQR]: 5.5-9.5); 26/41 (63.4%) underwent OIT for milk, 8/41 (19.5%) for egg and 7/41 (17.1%) for both. The median time between initiation of OMZ and OIT was 27 weeks (IQR: 22-33). Forty (97.56%) patients reached the maintenance phase (200 mL of cow's milk and 30 mL of raw egg or 1 cooked egg) in a median time of 27 weeks (IQR: 18-37). The median total time with OMZ was 117 weeks (IQR: 88-144). During the OMZ period, 2.44% (1/41) of patients presented anaphylaxis. After discontinuation of OMZ, 29.3% (12/41) presented anaphylaxis, 50% of them had a poor adherence to daily ingestion. One patient (2.44%) was diagnosed with eosinophilic esophagitis after 2 years of discontinuation of OMZ. Currently, after a median time of 9 years (IQR: 7-10) since the initiation of OMZ, 75.6% (31/41) are desensitized (27/31 without OMZ). CONCLUSIONS: Omalizumab allows desensitisation of children with severe allergies to cow's milk and/or egg without developing severe reactions while receiving this treatment. However, discontinuation of OMZ leads to severe allergic reactions, and hence must be monitored carefully.
Assuntos
Anafilaxia , Hipersensibilidade a Leite , Administração Oral , Anafilaxia/etiologia , Animais , Bovinos , Galinhas , Criança , Dessensibilização Imunológica/efeitos adversos , Feminino , Humanos , Fatores Imunológicos , Leite/efeitos adversos , Hipersensibilidade a Leite/terapia , Omalizumab/uso terapêutico , Estudos RetrospectivosRESUMO
OBJECTIVES: We sought to compare the procedural implications of using bioresorbable everolimus-eluting scaffolds (BVS) and Pt-Cr everolimus-eluting stent with abluminal bioabsorbable polymer (Synergy). BACKGROUND: There are important differences in the respective platforms, which could impact on procedural performance, complications and outcomes. METHODS: A prospective, randomized single center study including consecutive patients in stable clinical condition and with lesions amenable to be treated with BVS according to predefined criteria. Patients were randomized to either treatment with BVS or Synergy. All procedural data were collected and 12 months clinical follow up conducted. Primary objectives were fluoroscopy time, median dose-area product, contras agent volumen, and peri-procedural troponin release. RESULTS: A total of 200 patients were included, 100 in BVS group and 100 in Synergy group. No significant differences were observed in baseline clinical and angiographic characteristics. Predilatation (97.6 vs. 25.4%; P < 0.001), postdilatation (64.8 vs. 38.4%: P < 0.01), and use of 2 wires (20.8 vs. 10%; P = 0.02) were more frequent with BVS. The BVS group showed a significant increase in fluoroscopy time (18%), dose-area product (20%), and contrast volume (10%). Post-procedural increase of creatinine was similar and amount of TnI release was significantly higher with BVS but incidence of peri-procedural infarction was comparable. Clinical outcomes at 12 months were similar with definite thrombosis being 1% with BVS and 0% with Synergy. CONCLUSIONS: The use of BVS in comparison with the Synergy stent in a similar lesional setting is associated with a higher use of resources in the procedure, more radiation, and higher TnI release. © 2016 Wiley Periodicals, Inc.
Assuntos
Implantes Absorvíveis , Fármacos Cardiovasculares/administração & dosagem , Materiais Revestidos Biocompatíveis , Doença da Artéria Coronariana/terapia , Stents Farmacológicos , Everolimo/administração & dosagem , Intervenção Coronária Percutânea/instrumentação , Polímeros/química , Idoso , Fármacos Cardiovasculares/efeitos adversos , Meios de Contraste/administração & dosagem , Angiografia Coronária , Doença da Artéria Coronariana/diagnóstico por imagem , Trombose Coronária/etiologia , Everolimo/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/etiologia , Intervenção Coronária Percutânea/efeitos adversos , Estudos Prospectivos , Desenho de Prótese , Doses de Radiação , Exposição à Radiação , Radiografia Intervencionista , Fatores de Risco , Espanha , Fatores de Tempo , Resultado do TratamentoRESUMO
The phytohormone abscisic acid (ABA) regulates the expression of many genes in plants; it has critical functions in stress resistance and in growth and development. Several proteins have been reported to function as ABA receptors, and many more are known to be involved in ABA signalling. However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear. Here we show that by combining the recently identified ABA receptor PYR1 with the type 2C protein phosphatase (PP2C) ABI1, the serine/threonine protein kinase SnRK2.6/OST1 and the transcription factor ABF2/AREB1, we can reconstitute ABA-triggered phosphorylation of the transcription factor in vitro. Introduction of these four components into plant protoplasts results in ABA-responsive gene expression. Protoplast and test-tube reconstitution assays were used to test the function of various members of the receptor, protein phosphatase and kinase families. Our results suggest that the default state of the SnRK2 kinases is an autophosphorylated, active state and that the SnRK2 kinases are kept inactive by the PP2Cs through physical interaction and dephosphorylation. We found that in the presence of ABA, the PYR/PYL (pyrabactin resistance 1/PYR1-like) receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases. Our results reveal new insights into ABA signalling mechanisms and define a minimal set of core components of a complete major ABA signalling pathway.
Assuntos
Ácido Abscísico/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Transdução de Sinais , Estresse Fisiológico/fisiologia , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mutação , Fenótipo , Fosforilação , Protoplastos/fisiologiaRESUMO
The plant hormone abscisic acid (ABA) plays a crucial role in the control of the stress response and the regulation of plant growth and development. ABA binding to PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS intracellular receptors leads to inhibition of key negative regulators of ABA signaling, i.e. clade A protein phosphatases type 2C (PP2Cs) such as ABA-INSENSITIVE1 and HYPERSENSITIVE TO ABA1 (HAB1), causing the activation of the ABA signaling pathway. To gain further understanding on the mechanism of hormone perception, PP2C inhibition, and its implications for ABA signaling, we have performed a structural and functional analysis of the PYR1-ABA-HAB1 complex. Based on structural data, we generated a gain-of-function mutation in a critical residue of the phosphatase, hab1(W385A), which abolished ABA-dependent receptor-mediated PP2C inhibition without impairing basal PP2C activity. As a result, hab1(W385A) caused constitutive inactivation of the protein kinase OST1 even in the presence of ABA and PYR/PYL proteins, in contrast to the receptor-sensitive HAB1, and therefore hab1(W385A) qualifies as a hypermorphic mutation. Expression of hab1(W385A) in Arabidopsis (Arabidopsis thaliana) plants leads to a strong, dominant ABA insensitivity, which demonstrates that this conserved tryptophan residue can be targeted for the generation of dominant clade A PP2C alleles. Moreover, our data highlight the critical role of molecular interactions mediated by tryptophan-385 equivalent residues for clade A PP2C function in vivo and the mechanism of ABA perception and signaling.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Cristalografia por Raios X , Germinação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Complexos Multiproteicos , Mutação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2C , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Sementes/efeitos dos fármacos , Sementes/enzimologia , Sementes/genética , Sementes/fisiologia , Triptofano , Técnicas do Sistema de Duplo-HíbridoRESUMO
The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Biologia Computacional , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Imunoprecipitação , Fosfoproteínas Fosfatases/genética , Fosforilação/efeitos dos fármacos , Proteínas Quinases/genética , Proteína Fosfatase 2C , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Abscisic acid (ABA) is a key phytohormone involved in adaption to environmental stress and regulation of plant development. Clade A protein phosphatases type 2C (PP2Cs), such as HAB1, are key negative regulators of ABA signaling in Arabidopsis. To obtain further insight into regulation of HAB1 function by ABA, we have screened for HAB1-interacting partners using a yeast two-hybrid approach. Three proteins were identified, PYL5, PYL6 and PYL8, which belong to a 14-member subfamily of the Bet v1-like superfamily. HAB1-PYL5 interaction was confirmed using BiFC and co-immunoprecipitation assays. PYL5 over-expression led to a globally enhanced response to ABA, in contrast to the opposite phenotype reported for HAB1-over-expressing plants. F(2) plants that over-expressed both HAB1 and PYL5 showed an enhanced response to ABA, indicating that PYL5 antagonizes HAB1 function. PYL5 and other members of its protein family inhibited HAB1, ABI1 and ABI2 phosphatase activity in an ABA-dependent manner. Isothermal titration calorimetry revealed saturable binding of (+)ABA to PYL5, with K(d) values of 1.1 mum or 38 nm in the absence or presence of the PP2C catalytic core of HAB1, respectively. Our work indicates that PYL5 is a cytosolic and nuclear ABA receptor that activates ABA signaling through direct inhibition of clade A PP2Cs. Moreover, we show that enhanced resistance to drought can be obtained through PYL5-mediated inhibition of clade A PP2Cs.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fosfoproteínas Fosfatases/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Secas , Regulação da Expressão Gênica de Plantas , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2C , RNA de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
INTRODUCTION: The aetiological agent of erythema infectiosum is Erythrovirus B19 (also known as parvovirus B19), frequently found in children and adolescents, but also associated with arthropathy, aplastic crisis, and abortion in adults. MATERIAL AND METHODS: A retrospective study of Erythrovirus B19 cases in the years 2010-2015. RESULTS: Of the 56 cases of Erythrovirus B19 diagnosed, 34 were adults (32 women and 2 men) and 22 younger than 18 years (12 girls and 10 boys). Six cases were in pregnant women. Infections mainly occurred between spring and summer. In childhood, fever (64%), rash (50%), and anaemia (55%) were the most frequent symptoms. However, arthralgia (59%) was the most frequent symptom in adults, and less frequent were anaemia (41%), fever (32%), and rash (29%). CONCLUSIONS: The characteristic clinical presentation in childhood was rash and fever, whereas in adults it was arthralgia. Anaemia is also frequent, but only severe in previous haematological disease. It should be pointed out that Erythrovirus B19 infection during pregnancy could severely affect the foetus.
Assuntos
Eritema Infeccioso/epidemiologia , Parvovirus B19 Humano/isolamento & purificação , Complicações Infecciosas na Gravidez/virologia , Adulto , Fatores Etários , Artralgia/epidemiologia , Artralgia/virologia , Criança , Pré-Escolar , Eritema Infeccioso/fisiopatologia , Exantema/epidemiologia , Exantema/virologia , Feminino , Febre/epidemiologia , Febre/virologia , Seguimentos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Gravidez , Estudos RetrospectivosRESUMO
Introducción: la persistencia de microorganismos en los conductos radiculares es uno de los principales factores del fracaso endodóncico. Por ello la importancia de conocer las propiedades antimicrobianas de los distintos tipos de selladores. Objetivo: realizar una comparación con base en la evidencia disponible sobre la actividad antimicrobiana de los diferentes cementos selladores en endodoncia. Material y métodos: la información fue recopilada de las bases de datos PubMed y Google Académico en el idioma inglés y español, publicados en el periodo 2014-2023. Resultados: un gran número de microorganismos se encuentran presentes en los diferentes tipos de infecciones de origen endodóncico, se han identificado más de 500 especies microbianas, entre ellas bacterias, hongos, arqueas y virus. Los cementos selladores se pueden clasificar según su composición química, en cementos a base de óxido de zinc-eugenol, hidróxido de calcio, a base de ionómero de vidrio, silicona, resina y biocerámicos. Conclusión: los cementos selladores que mostraron mayor actividad antimicrobiana contra los microorganismos persistentes fueron los cementos a base de óxido de zinc-eugenol, resina y biocerámicos. Sin embargo, se identificó que cada autor utilizó diferentes métodos y tiempos, por lo tanto, no es posible lograr definir con exactitud qué cemento sellador posee la mejor capacidad antimicrobiana (AU)
Introduction: the persistence of microorganisms in root canals is one of the main factors of endodontic failure. Therefore, the importance of knowing the antimicrobial properties of the different types of sealants. Objective: to make a comparison based on the available evidence on the antimicrobial activity of the different endodontics sealers. Material and methods: the information was collected from PubMed and Google Academic databases in English and Spanish, published in the period 2014-2023. Results: a large number of microorganisms are present in the different types of infections of endodontic origin, more than 500 microbiological species have been identified, including bacteria, fungi, archaea and viruses. Sealer cements can be classified according to their chemical composition, into cements based on zinc oxide-eugenol, calcium hydroxide, based on glass ionomer, silicone, resin and bioceramics. Conclusion: sealer cements that showed the highest antimicrobial activity against persistent microorganisms were zinc oxide-eugenol, resin, and bioceramic-based cements. However, it was identified that each author used different methods and times, therefore, it is not possible to accurately define which sealer cement has the best antimicrobial capacity (AU)
Assuntos
Materiais Restauradores do Canal Radicular/química , Cavidade Pulpar/microbiologia , Cimento de Óxido de Zinco e Eugenol/química , Hidróxido de Cálcio/química , Bases de Dados Bibliográficas , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Cimentos de Resina/química , Cerâmicas Modificadas Organicamente/química , Cimentos de Ionômeros de Vidro/química , Antibacterianos/farmacologiaRESUMO
BACKGROUND: Cystic fibrosis is a degenerative disease characterized by progressive epithelial secretory gland dysfunction associated with repeated respiratory infections. Bacterial infections are very frequent in children with cystic fibrosis, but because rapid METHODS: for screening for the wide variety of potentially involved viruses were unavailable until recently, the frequency of viral presence is unknown. Multiplex PCR enables screening for many viruses involved in respiratory infections. OBJECTIVES: This study aimed to evaluate the frequency of viruses and bacteria in respiratory specimens from children with cystic fibrosis and to clarify the incidence and characteristics (seasonality and age of patients) of different viruses detected in children with cystic fibrosis. STUDY DESIGN: In this 2-year prospective study, we obtained paired nasopharyngeal-swab and sputum specimens from children with cystic fibrosis during clinical respiratory examinations separated by at least 14days. We analyzed viruses in nasopharyngeal-swab specimens with multiplex PCR and bacteria in sputum with standard methods. RESULTS: We analyzed 368 paired specimens from 33 children. We detected viruses in 154 (41.8%) and bacteria in 132 (35.9%). Bacteria were commoner in spring and summer; viruses were commoner in autumn and winter. In every season, Staphylococcus aureus was the commonest bacteria and rhinovirus was the commonest virus. Nearly all infections with Haemophilus influenzae occurred in autumn and winter. Viruses were more prevalent in children <5 years old, and bacteria were more prevalent in children ≥12 years old. CONCLUSIONS: Multiplex PCR screening for respiratory viruses is feasible in children with cystic fibrosis; the clinical implications of screening warrant further study.
Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/epidemiologia , Fibrose Cística/complicações , Reação em Cadeia da Polimerase Multiplex/métodos , Viroses/epidemiologia , Vírus/isolamento & purificação , Adolescente , Fatores Etários , Bactérias/classificação , Bactérias/genética , Técnicas Bacteriológicas , Criança , Pré-Escolar , Feminino , Humanos , Incidência , Lactente , Masculino , Nasofaringe/microbiologia , Nasofaringe/virologia , Prevalência , Estudos Prospectivos , Estações do Ano , Escarro/microbiologia , Escarro/virologia , Vírus/classificação , Vírus/genéticaAssuntos
Abscesso , Braço , Abscesso/tratamento farmacológico , Antibacterianos/uso terapêutico , HumanosRESUMO
Metalloproteins play major roles in cell metabolism and signalling pathways. In many cases, they show moonlighting behaviour, acting in different processes, depending on the physiological state of the cell. To understand these multitasking proteins, we need to discover the partners with which they carry out such novel functions. Although many technological and methodological tools have recently been reported for the detection of protein interactions, specific approaches to studying the interactions involving metalloproteins are not yet well developed. The task is even more challenging for metalloproteins, because they often form short-lived complexes that are difficult to detect. In this review, we gather the different proteomic techniques and biointeractomic tools reported in the literature. All of them have shown their applicability to the study of transient and weak protein-protein interactions, and are therefore suitable for metalloprotein interactions.
Assuntos
Metaloproteínas/metabolismo , Proteômica , Células/metabolismo , Cromatografia de Afinidade , Espectrometria de Massas , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Transdução de SinaisRESUMO
To gain further insight into abscisic acid (ABA) signaling and its role in growth regulation, we have screened for Arabidopsis (Arabidopsis thaliana) mutants hypersensitive to ABA-mediated root growth inhibition. As a result, we have identified a loss-of-function allele of BREVIS RADIX (BRX) in the Columbia background, named brx-2, which shows enhanced response to ABA-mediated inhibition of root growth. BRX encodes a key regulator of cell proliferation and elongation in the root, which has been implicated in the brassinosteroid (BR) pathway as well as in the regulation of auxin-responsive gene expression. Mutants affected in BR signaling that are not impaired in root growth, such as bes1-D, bzr1-D, and bsu1-D, also showed enhanced sensitivity to ABA-mediated inhibition of root growth. Triple loss-of-function mutants affected in PP2Cs, which act as negative regulators of ABA signaling, showed impaired root growth in the absence of exogenous ABA, indicating that disturbed regulation of ABA sensitivity impairs root growth. In agreement with this result, diminishing ABA sensitivity of brx-2 by crossing it with a 35S:HAB1 ABA-insensitive line allowed significantly higher recovery of root growth after brassinolide treatment. Finally, transcriptomic analysis revealed that ABA treatment negatively affects auxin signaling in wild-type and brx-2 roots and that ABA response is globally altered in brx-2. Taken together, our results reveal an interaction between BRs, auxin, and ABA in the control of root growth and indicate that altered sensitivity to ABA is partly responsible for the brx short-root phenotype.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Mutação/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/enzimologia , Arabidopsis/genética , Brassinosteroides , Colestanóis/farmacologia , Proteínas de Ligação a DNA , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Técnicas de Inativação de Genes , Genes de Plantas , Proteínas Nucleares/metabolismo , Fenótipo , Fosfoproteínas Fosfatases/metabolismo , Raízes de Plantas/genética , Proteína Fosfatase 2C , Esteroides Heterocíclicos/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
The phytohormone abscisic acid (ABA) is a key regulator of plant growth and development as well as plant responses to situations of decreased water availability. Protein phosphatases type 2C (PP2Cs) from group A, which includes the ABI1/HAB1 and PP2CA branches, are key negative regulators of ABA signaling. Specifically, HAB1, ABI1, ABI2, and PP2CA have been shown to affect both seed and vegetative responses to ABA. To further understand their contribution to ABA signaling and to unravel possible genetic interactions and functional redundancy among them, we have generated different combinations of double and triple mutants impaired in these PP2Cs. Interestingly, hab1-1pp2ca-1 and abi1-2pp2ca-1 double mutants showed reduced water loss and enhanced resistance to drought stress, which further supports the role of PP2CA in vegetative responses to ABA. Two triple hab1-1abi1-2abi2-2 and hab1-1abi1-2pp2ca-1 mutants were generated, which showed an extreme response to exogenous ABA, impaired growth, and partial constitutive response to endogenous ABA. Thus, transcriptomic analysis revealed a partial up-regulation/down-regulation of a subset of ABA-responsive genes in both triple mutants in the absence of exogenous ABA. Comparison of ABA responses in the different pp2c mutants showed that a progressive increase in ABA sensitivity could be obtained through combined inactivation of these PP2Cs. These results indicate that ABA response is finely tuned by the integrated action of these genes, which is required to prevent a constitutive response to endogenous ABA that might have a deleterious effect on growth and development in the absence of environmental stress.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Perfilação da Expressão Gênica , Mutagênese Sítio-Dirigida , Fosfoproteínas Fosfatases/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
Abscisic acid (ABA) has an important role for plant growth, development, and stress adaptation. HYPERSENSITIVE TO ABA1 (HAB1) is a protein phosphatase type 2C that plays a key role as a negative regulator of ABA signaling; however, the molecular details of HAB1 action in this process are not known. A two-hybrid screen revealed that SWI3B, an Arabidopsis thaliana homolog of the yeast SWI3 subunit of SWI/SNF chromatin-remodeling complexes, is a prevalent interacting partner of HAB1. The interaction mapped to the N-terminal half of SWI3B and required an intact protein phosphatase catalytic domain. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the interaction of HAB1 and SWI3B in the nucleus of plant cells. swi3b mutants showed a reduced sensitivity to ABA-mediated inhibition of seed germination and growth and reduced expression of the ABA-responsive genes RAB18 and RD29B. Chromatin immunoprecipitation experiments showed that the presence of HAB1 in the vicinity of RD29B and RAB18 promoters was abolished by ABA, which suggests a direct involvement of HAB1 in the regulation of ABA-induced transcription. Additionally, our results uncover SWI3B as a novel positive regulator of ABA signaling and suggest that HAB1 modulates ABA response through the regulation of a putative SWI/SNF chromatin-remodeling complex.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Montagem e Desmontagem da Cromatina , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Germinação , Mutação , Fosfoproteínas Fosfatases/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Mapeamento de Interação de Proteínas , RNA de Plantas/genética , Proteínas de Ligação a RNA/genética , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Coenzyme A (CoA) is an essential cofactor in the metabolism of both prokaryotic and eukaryotic organisms and a universal five-step pathway is utilized to synthesize CoA from pantothenate. Null mutations in two of the five steps of this pathway led to embryo lethality and therefore viable reduction-of-function mutations are required to further study its role in plant biology. In this article, we have characterized a viable Arabidopsis (Arabidopsis thaliana) T-DNA mutant affected in the penultimate step of the CoA biosynthesis pathway, which is catalyzed by the enzyme phosphopantetheine adenylyltransferase (PPAT). This ppat-1 knockdown mutation showed an approximately 90% reduction in PPAT transcript levels and was severely impaired in plant growth and seed production. The sum of CoA and acetyl-CoA levels was severely reduced (60%-80%) in ppat-1 seedlings compared to wild type, and catabolism of storage lipids during seedling establishment was delayed. Conversely, PPAT overexpressing lines showed, on average, approximately 1.6-fold higher levels of CoA + acetyl-CoA levels, as well as enhanced vegetative and reproductive growth and salt/osmotic stress resistance. Interestingly, dry seeds of overexpressing lines contained between 35% to 50% more fatty acids than wild type, which suggests that CoA biosynthesis plays a crucial role in storage oil accumulation. Finally, biochemical analysis of the recombinant PPAT enzyme revealed an inhibitory effect of CoA on PPAT activity. Taken together, these results suggest that the reaction catalyzed by PPAT is a regulatory step in the CoA biosynthetic pathway that plays a key role for plant growth, stress resistance, and seed lipid storage.
Assuntos
Arabidopsis/fisiologia , Metabolismo dos Lipídeos , Nucleotidiltransferases/metabolismo , Salinidade , Sementes/metabolismo , Acil Coenzima A/metabolismo , Arabidopsis/crescimento & desenvolvimento , Ácidos Graxos/metabolismo , Mutagênese Insercional , Nucleotidiltransferases/genéticaRESUMO
Once the plant coenzyme A (CoA) biosynthetic pathway has been elucidated by comparative genomics, it is feasible to analyze the physiological relevance of CoA biosynthesis in plant life. To this end, we have identified and characterized Arabidopsis (Arabidopsis thaliana) T-DNA knockout mutants of two CoA biosynthetic genes, HAL3A and HAL3B. The HAL3A gene encodes a 4'-phosphopantothenoyl-cysteine decarboxilase that generates 4'-phosphopantetheine. A second gene, HAL3B, whose gene product is 86% identical to that of HAL3A, is present in the Arabidopsis genome. HAL3A appears to have a predominant role over HAL3B according to their respective mRNA expression levels. The hal3a-1, hal3a-2, and hal3b mutants were viable and showed a similar growth rate as that in wild-type plants; in contrast, a hal3a-1 hal3b double mutant was embryo lethal. Unexpectedly, seedlings that were null for HAL3A and heterozygous for HAL3B (aaBb genotype) displayed a sucrose (Suc)-dependent phenotype for seedling establishment, which is in common with mutants defective in beta-oxidation. This phenotype was genetically complemented in aaBB siblings of the progeny and chemically complemented by pantethine. In contrast, seedling establishment of Aabb plants was not Suc dependent, proving a predominant role of HAL3A over HAL3B at this stage. Total fatty acid and acyl-CoA measurements of 5-d-old aaBb seedlings in medium lacking Suc revealed stalled storage lipid catabolism and impaired CoA biosynthesis; in particular, acetyl-CoA levels were reduced by approximately 80%. Taken together, these results provide in vivo evidence for the function of HAL3A and HAL3B, and they point out the critical role of CoA biosynthesis during early postgerminative growth.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Coenzima A/biossíntese , Plântula/crescimento & desenvolvimento , Acil Coenzima A/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/fisiologia , DNA Bacteriano/genética , Ácidos Graxos/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional , Panteteína/análogos & derivados , Panteteína/farmacologia , Fenótipo , RNA Mensageiro/metabolismo , Plântula/genética , Plântula/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sacarose/metabolismoRESUMO
La autoestima en los niños ha sido poco etudiada en México por no contar con los instrumentos apropiados para medirla, por lo que se probó un instrumento que ha sido muy utilizado e investigado en los países de habla inglesa, para comparar el comportamiento de la población mexicana. Este trabajo presenta el procedimiento para la elaboración de la primera versión en español del Inventario de Autoestima de Coopersmith, así como los datos normativos correspondientes posteriores. Se analizan las diferencias en relación con el sexo y el grado escolar, y se comparan los resultados obtenidos en la población norteamericana y mexicano-norteamericana de los Estados Unidos. La investigación se hizo en 292 niños, 128 hombres y 164 mujeres, de nivel socioeconómico medio y bajo, cuyas edades estaban entre los 8 y los 15 años de edad