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J Biol Chem ; 290(40): 24626-35, 2015 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26269594


Postpartum mammary gland involution is the physiological process by which the lactating gland returns to its pre-pregnant state. In rodent models, the microenvironment of mammary gland involution is sufficient to induce enhanced tumor cell growth, local invasion, and metastasis. Therefore, a deeper understanding of the physiological regulation of involution may provide in-depth information on breast cancer therapy. We herein identified Nucling as an important regulator of involution of the mammary gland. A knock-out mouse model was generated and revealed that postpartum involution were impaired in mice lacking Nucling. Involution is normally associated with an increase in the activation of NF-κB and STAT3, which is required for the organized regulation of involution, and was observed in WT glands, but not in the absence of Nucling. Furthermore, the loss of Nucling led to the suppression of Calpain-1, IL-6, and C/EBPδ factors, which are known to be essential for normal involution. The number of M2 macrophages, which are crucial for epithelial cell death and adipocyte repopulation after weaning, was also reduced in Nucling-KO glands. Taken together, the results of the present study demonstrated that Nucling played an important role in mammary gland involution by regulating NF-κB and STAT3 signaling pathways.

Regulação da Expressão Gênica , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana/genética , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Adipócitos/citologia , Animais , Apoptose , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Calpaína/metabolismo , Receptor gp130 de Citocina/metabolismo , Feminino , Interleucina-6/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Transdução de Sinais
J Pharm Biomed Anal ; 116: 94-100, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-25749303


D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression.

D-Aminoácido Oxidase/análise , D-Aminoácido Oxidase/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel Bidimensional/métodos , Animais , D-Aminoácido Oxidase/genética , Humanos , Células LLC-PK1 , Espectrometria de Massas/métodos , Ligação Proteica/fisiologia , Suínos
J Biochem ; 157(5): 377-87, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25500505


D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes d-amino acids. Until now, the DAO expression mechanism is still unclear. Our assessment of human DAO (hDAO) promoter activity using luciferase reporter system indicated the proximal upstream region of exon1 (-237/+1) has promoter activity (P1). Interestingly, we identified an alternative promoter in the proximal upstream region of exon2 (+4,126/+4,929) (P2). This alternative promoter has stronger activity than that of P1. Our results also revealed a negative regulatory segment (+1,163/+1,940) in intron1; that would act in concert with P1 and P2. Bioinformatics analyses elucidated the conservation of transcription factor PAX5 family binding sites among species. These sites (-60/-31) and (+4,464/+4,493), locate in P1 and P2 of hDAO, respectively. Gel shift assays demonstrated P1 contains a site (-60/-31) for PAX5 binding while P2 has three sites for both paired box gene 2 (PAX2) and paired box gene 5 (PAX5) binding. The dual roles of PAX5 family in regulating hDAO transcription by modulating promoter activity of P1 and activating promoter activity of P2 were implicated based on the site-directed mutagenesis experiment. Altogether, our data suggested the differential regulation of hDAO expression by two promoters whose activities may be modulated by the binding of PAX2 and PAX5.

D-Aminoácido Oxidase/genética , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX5/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Humanos , Células LLC-PK1 , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Suínos
PLoS One ; 8(3): e58987, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23536844


BACKGROUND: Under the threat of global climatic change and food shortages, it is essential to take the initiative to obtain a comprehensive understanding of common and specific defence mechanisms existing in plant systems for protection against different types of biotic invaders. We have implemented an integrated approach to analyse the overall transcriptomic reprogramming and systems-level defence responses in the model plant species Arabidopsis thaliana (A. thaliana henceforth) during insect Brevicoryne brassicae (B. brassicae henceforth) and bacterial Pseudomonas syringae pv. tomato strain DC3000 (P. syringae henceforth) attacks. The main aim of this study was to identify the attacker-specific and general defence response signatures in A. thaliana when attacked by phloem-feeding aphids or pathogenic bacteria. RESULTS: The obtained annotated networks of differentially expressed transcripts indicated that members of transcription factor families, such as WRKY, MYB, ERF, BHLH and bZIP, could be crucial for stress-specific defence regulation in Arabidopsis during aphid and P. syringae attack. The defence response pathways, signalling pathways and metabolic processes associated with aphid attack and P. syringae infection partially overlapped. Components of several important biosynthesis and signalling pathways, such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and glucosinolates, were differentially affected during the two the treatments. Several stress-regulated transcription factors were known to be associated with stress-inducible microRNAs. The differentially regulated gene sets included many signature transcription factors, and our co-expression analysis showed that they were also strongly co-expressed during 69 other biotic stress experiments. CONCLUSIONS: Defence responses and functional networks that were unique and specific to aphid or P. syringae stresses were identified. Furthermore, our analysis revealed a probable link between biotic stress and microRNAs in Arabidopsis and, thus gives indicates a new direction for conducting large-scale targeted experiments to explore the detailed regulatory links between them. The presented results provide a comparative understanding of Arabidopsis - B. brassicae and Arabidopsis - P. syringae interactions at the transcriptomic level.

Arabidopsis/genética , Perfilação da Expressão Gênica , Animais , Afídeos , Arabidopsis/imunologia , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Infecções Bacterianas/genética , Biologia Computacional , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Redes Reguladoras de Genes , Redes e Vias Metabólicas , MicroRNAs/genética , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Oxilipinas/metabolismo , Pseudomonas syringae/metabolismo , Processamento Pós-Transcricional do RNA , Reprodutibilidade dos Testes , Ácido Salicílico/farmacologia , Metabolismo Secundário , Transdução de Sinais , Estresse Fisiológico/genética , Estresse Fisiológico/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
BMC Genomics ; 11: 190, 2010 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-20307264


BACKGROUND: Glutamate plays a central position in the synthesis of a variety of organic molecules in plants and is synthesised from nitrate through a series of enzymatic reactions. Glutamate synthases catalyse the last step in this pathway and two types are present in plants: NADH- or ferredoxin-dependent. Here we report a genome wide microarray analysis of the transcriptional reprogramming that occurs in leaves and roots of the A. thaliana mutant glu1-2 knocked-down in the expression of Fd-GOGAT1 (GLU1; At5g04140), one of the two genes of A. thaliana encoding ferredoxin-dependent glutamate synthase. RESULTS: Transcriptional profiling of glu1-2 revealed extensive changes with the expression of more than 5500 genes significantly affected in leaves and nearly 700 in roots. Both genes involved in glutamate biosynthesis and transformation are affected, leading to changes in amino acid compositions as revealed by NMR metabolome analysis. An elevated glutamine level in the glu1-2 mutant was the most prominent of these changes. An unbiased analysis of the gene expression datasets allowed us to identify the pathways that constitute the secondary response of an FdGOGAT1/GLU1 knock-down. Among the most significantly affected pathways, photosynthesis, photorespiratory cycle and chlorophyll biosynthesis show an overall downregulation in glu1-2 leaves. This is in accordance with their slight chlorotic phenotype. Another characteristic of the glu1-2 transcriptional profile is the activation of multiple stress responses, mimicking cold, heat, drought and oxidative stress. The change in expression of genes involved in flavonoid biosynthesis is also revealed. The expression of a substantial number of genes encoding stress-related transcription factors, cytochrome P450 monooxygenases, glutathione S-transferases and UDP-glycosyltransferases is affected in the glu1-2 mutant. This may indicate an induction of the detoxification of secondary metabolites in the mutant. CONCLUSIONS: Analysis of the glu1-2 transcriptome reveals extensive changes in gene expression profiles revealing the importance of Fd-GOGAT1, and indirectly the central role of glutamate, in plant development. Besides the effect on genes involved in glutamate synthesis and transformation, the glu1-2 mutant transcriptome was characterised by an extensive secondary response including the downregulation of photosynthesis-related pathways and the induction of genes and pathways involved in the plant response to a multitude of stresses.

Aminoácido Oxirredutases/genética , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Mutação , Estresse Fisiológico , Arabidopsis/genética , Perfilação da Expressão Gênica , Ácido Glutâmico/biossíntese , Folhas de Planta/enzimologia , Folhas de Planta/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Transcrição Genética