RESUMO
Cardiac function requires appropriate proteins in each chamber. Atria requires slow myosin to act as reservoirs, while ventricles demand fast myosin for swift pumping. Myosins are thus under chamber-biased cis-regulation, with myosin gene expression imbalances leading to congenital heart dysfunction. To identify regulatory inputs leading to cardiac chamber-biased expression, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives preferential expression to the atria. We show that SMyHC III gene states are orchestrated by a complex Nuclear Receptor Element (cNRE) of 32 base pairs. Using transgenesis in zebrafish and mice, we demonstrate that preferential atrial expression is achieved by a combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Using comparative genomics, we show that the cNRE might have emerged from an endogenous viral element through infection of an ancestral host germline, revealing an evolutionary pathway to cardiac chamber-specific expression.
Assuntos
Átrios do Coração , Peixe-Zebra , Camundongos , Animais , Peixe-Zebra/genética , Átrios do Coração/metabolismo , Ventrículos do Coração , Miosinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
The teratogenic mechanisms triggered by ZIKV are still obscure due to the lack of a suitable animal model. Here we present a mouse model of developmental disruption induced by ZIKV hematogenic infection. The model utilizes immunocompetent animals from wild-type FVB/NJ and C57BL/6J strains, providing a better analogy to the human condition than approaches involving immunodeficient, genetically modified animals, or direct ZIKV injection into the brain. When injected via the jugular vein into the blood of pregnant females harboring conceptuses from early gastrulation to organogenesis stages, akin to the human second and fifth week of pregnancy, ZIKV infects maternal tissues, placentas and embryos/fetuses. Early exposure to ZIKV at developmental day 5 (second week in humans) produced complex manifestations of anterior and posterior dysraphia and hydrocephalus, as well as severe malformations and delayed development in 10.5 days post-coitum (dpc) embryos. Exposure to the virus at 7.5-9.5 dpc induces intra-amniotic hemorrhage, widespread edema, and vascular rarefaction, often prominent in the cephalic region. At these stages, most affected embryos/fetuses displayed gross malformations and/or intrauterine growth restriction (IUGR), rather than isolated microcephaly. Disrupted conceptuses failed to achieve normal developmental landmarks and died in utero. Importantly, this is the only model so far to display dysraphia and hydrocephalus, the harbinger of microcephaly in humans, as well as arthrogryposis, a set of abnormal joint postures observed in the human setting. Late exposure to ZIKV at 12.5 dpc failed to produce noticeable malformations. We have thus characterized a developmental window of opportunity for ZIKV-induced teratogenesis encompassing early gastrulation, neurulation and early organogenesis stages. This should not, however, be interpreted as evidence for any safe developmental windows for ZIKV exposure. Late developmental abnormalities correlated with damage to the placenta, particularly to the labyrinthine layer, suggesting that circulatory changes are integral to the altered phenotypes.
Assuntos
Artrogripose/virologia , Modelos Animais de Doenças , Hidrocefalia/virologia , Complicações Infecciosas na Gravidez/virologia , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Artrogripose/embriologia , Artrogripose/imunologia , Artrogripose/patologia , Feminino , Humanos , Hidrocefalia/embriologia , Hidrocefalia/imunologia , Hidrocefalia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Placenta/anormalidades , Placenta/imunologia , Placenta/virologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/patologia , Teratogênicos/análise , Infecção por Zika virus/embriologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/patologiaRESUMO
Elucidating cardiac evolution has been frustrated by lack of fossils. One celebrated enigma in cardiac evolution involves the transition from a cardiac outflow tract dominated by a multi-valved conus arteriosus in basal actinopterygians, to an outflow tract commanded by the non-valved, elastic, bulbus arteriosus in higher actinopterygians. We demonstrate that cardiac preservation is possible in the extinct fish Rhacolepis buccalis from the Brazilian Cretaceous. Using X-ray synchrotron microtomography, we show that Rhacolepis fossils display hearts with a conus arteriosus containing at least five valve rows. This represents a transitional morphology between the primitive, multivalvar, conal condition and the derived, monovalvar, bulbar state of the outflow tract in modern actinopterygians. Our data rescue a long-lost cardiac phenotype (119-113 Ma) and suggest that outflow tract simplification in actinopterygians is compatible with a gradual, rather than a drastic saltation event. Overall, our results demonstrate the feasibility of studying cardiac evolution in fossils.
Assuntos
Peixes/anatomia & histologia , Fósseis , Coração/anatomia & histologia , Animais , Evolução Biológica , Microtomografia por Raio-XRESUMO
Repulsive guidance molecules (RGMs) compose a family of glycosylphosphatidylinositol (GPI)-anchored axon guidance molecules and perform several functions during neural development. New evidence has suggested possible new roles for these axon guidance molecules during skeletal muscle development, which has not been investigated thus far. In the present study, we show that RGMa, RGMb and RGMc are all induced during skeletal muscle differentiation in vitro. Immunolocalization performed on adult skeletal muscle cells revealed that RGMa, RGMb and RGMc are sarcolemmal proteins. Additionally, RGMa was found to be a sarcoplasmic protein with a surprisingly striated pattern. RGMa colocalization with known sarcoplasmic proteins suggested that this axon guidance molecule is a skeletal muscle sarcoplasmic protein. Western blot analysis revealed two RGMa fragments of 60 and 33 kDa, respectively, in adult skeletal muscle samples. RGMa phenotypes in skeletal muscle cells (C2C12 and primary myoblasts) were also investigated. RGMa overexpression produced hypertrophic cells, whereas RGMa knockdown resulted in the opposite phenotype. RGMa knockdown also blocked myotube formation in both skeletal muscle cell types. Our results are the first to show an axon guidance molecule as a skeletal muscle sarcoplasmic protein and to include RGMa in a system that regulates skeletal muscle cell size and differentiation.
Assuntos
Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Animais , Diferenciação Celular/fisiologia , Crescimento Celular , Hipertrofia/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologiaRESUMO
Transcriptional regulation controlled by thyroid hormone receptor (TR) drives events such as development, differentiation, and metabolism. TRs may act either as homodimers or as heterodimers with retinoid X receptor (RXR). Thyroid hormone T3 preferentially binds TR-RXR heterodimers, which activate transcription through coactivator recruitment. However, it is unclear whether TR-RXR heterodimers may also be responsive to the canonical RXR agonist 9-cis retinoic acid (9C) in the context of physiological gene regulation. New structural studies suggest that 9C promotes the displacement of bound coactivators from the heterodimer, modifying TR-RXR activity. To shed light on the molecular mechanisms that control TR-RXR function, we used biophysical approaches to characterize coregulator recruitment to TR-TR or to TR-RXR in the presence of T3 and/or 9C as well as cell-based assays to establish the functional significance of biophysical findings. Using cell-based and fluorescence assays with mutant and wild-type TR, we show that 9C does indeed have a function in the TR-RXR heterodimer context, in which it induces the release of corepressors. Furthermore, we show that 9C does not promote detectable conformational changes in the structure of the TR-RXR heterodimer and does not affect coactivator recruitment. Finally, our data support the view that DNA binding domain and Hinge regions are important to set up NR-coactivator binding interfaces. In summary, we showed that the RXR agonist 9C can regulate TR function through its modulation of corepressor dissociation.
Assuntos
Proteínas Correpressoras/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Receptores X de Retinoides/agonistas , Tretinoína/farmacologia , Alitretinoína , Anisotropia , Cromatografia em Gel , Dicroísmo Circular , DNA/metabolismo , Difusão Dinâmica da Luz , Fluorescência , Células HEK293 , Humanos , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores dos Hormônios Tireóideos/química , Espalhamento a Baixo Ângulo , Ativação Transcricional/genética , Triptofano/metabolismo , Ultracentrifugação , Difração de Raios XRESUMO
Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr(253) in α-tubulin as a site that is phosphorylated by protein kinase C ßI (PKCßI). Thr(253) is not part of a linear PKC consensus motif. Instead, Thr(253) occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCßI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this "structurally formed" consensus motif and its importance for the protein kinase-substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.
Assuntos
Fosfotransferases/química , Motivos de Aminoácidos , Animais , Catálise , Bovinos , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas de Fluorescência Verde/química , Células HEK293 , Células HeLa , Humanos , Lisina/química , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Dobramento de Proteína , Proteína Quinase C/química , Treonina/química , Tubulina (Proteína)/químicaRESUMO
BACKGROUND: Dact gene family encodes multifunctional proteins that are important modulators of Wnt and TGF-ß signaling pathways. Given that these pathways coordinate multiple steps of limb development, we investigated the expression pattern of the two chicken Dact genes (Dact1 and Dact2) from early limb bud up to stages when several tissues are differentiating. RESULTS: During early limb development (HH24-HH30) Dact1 and Dact2 were mainly expressed in the cartilaginous rudiments of the appendicular skeleton and perichondrium, presenting expression profiles related, but distinct. At later stages of development (HH31-HH35), the main sites of Dact1 and Dact2 expression were the developing synovial joints. In this context, Dact1 expression was shown to co-localize with regions enriched in the nuclear ß-catenin protein, such as developing joint capsule and interzone. In contrast, Dact2 expression was restricted to the interzone surrounding the domains of bmpR-1b expression, a TGF-ß receptor with crucial roles during digit morphogenesis. Additional sites of Dact expression were the developing tendons and digit blastemas. CONCLUSIONS: Our data indicate that Dact genes are good candidates to modulate and, possibly, integrate Wnt and TGF-ß signaling during limb development, bringing new and interesting perspectives about the roles of Dact molecules in limb birth defects and human diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Aviárias/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Membro Posterior/embriologia , Proteínas Nucleares/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Embrião de Galinha , Membro Posterior/citologia , Humanos , Membrana Sinovial/citologia , Membrana Sinovial/embriologiaRESUMO
Transgenesis refers to the molecular genetic techniques for directing specific insertions, deletions and point mutations in the genome of germ cells in order to create genetically modified organisms (GMO). Genetic modification is becoming more practicable, efficient and predictable with the development and use of a variety of cell and molecular biology tools and DNA sequencing technologies. A collection of plasmidial and viral vectors, cell-type specific promoters, positive and negative selectable markers, reporter genes, drug-inducible Cre-loxP and Flp/FRT recombinase systems are available which ensure efficient transgenesis in the mouse. The technologies for the insertion and removal of genes by homologous-directed recombination in embryonic stem cells (ES) and generation of targeted gain- and loss-of function alleles have allowed the creation of thousands of mouse models of a variety of diseases. The engineered zinc finger nucleases (ZFNs) and small hairpin RNA-expressing constructs are novel tools with useful properties for gene knockout free of ES manipulation. In this review we briefly outline the different approaches and technologies for transgenesis as well as their advantages and disadvantages. We also present an overview on how the novel integrative mouse and human genomic databases and bioinformatics approaches have been used to understand genotype-phenotype relationships of hundreds of mutated and candidate disease genes in mouse models. The updating and continued improvements of the genomic technologies will eventually help us to unraveling the biological and pathological processes in such a way that they can be translated more efficiently from mouse to human and vise-versa.
Assuntos
Técnicas de Transferência de Genes , Animais , DNA/administração & dosagem , DNA/metabolismo , Embrião de Mamíferos/metabolismo , Marcação de Genes , Vetores Genéticos/genética , Humanos , Camundongos , MicroinjeçõesRESUMO
The identification of subpharyngeal cardiac precursors has had a strong influence on the way we think about early cardiac development. From this discovery was born the concept of multiple heart fields. Early support for the concept came from gene expression, genetic retrospective fate mapping, and gene targeting studies, which collectively suggested the existence of a second heart field (SHF) on the basis of specific Islet-1 (Isl-1) expression, presence of two cardiac ancestral lineages, and compatible cardiac knockout phenotypes, respectively. A decade after the original studies, support for the SHF concept is dwindling. This is because in all bilaterian models studied, Isl expression in heart progenitors is not SHF-specific, because lineage data are best explained by alternative models including an older, truly ancestral, lineage of cardiac pioneers with unrestricted contribution to all cardiac segments and, finally, because the inflow-to-outflow segmental nature of the early vertebrate peristaltic heart has been reaffirmed with novel, less invasive, methodologies. Altogether, the paradigms derived from the discovery of subpharyngeal cardiac progenitors helped us shift from relatively simple models, which rely predominantly either on patterning, gene expression patterns or lineages, to a much more sophisticated body of knowledge in which all these parameters must be accounted. Thus, it is well possible that due consideration of the key elements contained in the inflow/outflow, pioneer/scaffold, ballooning, and SHF hypotheses may provide us with a unified framework of the early stages of cardiac development. Here, we advance into this direction by suggesting an intuitive model of early heart development based on the concept of an inflow/outflow scaffold erected by cardiac pioneers, one that is required to assemble all the subsequent cell contribution that emigrates from cardiac progenitor areas.
Assuntos
Coração/crescimento & desenvolvimento , Miocárdio/metabolismo , Evolução Biológica , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Humanos , Modelos Biológicos , Miocárdio/citologiaRESUMO
Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Celulases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cálcio/metabolismo , Celulases/química , Celulases/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/fisiologia , Temperatura Alta , Cinética , Manganês/química , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (~998 Å(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/química , Miócitos Cardíacos/química , Miosinas/química , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Animais , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Hipertrofia/metabolismo , Camundongos , Modelos Moleculares , Miócitos Cardíacos/metabolismo , Miosinas/metabolismo , Estrutura Quaternária de Proteína , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification.
Assuntos
Aldeído Desidrogenase/genética , Padronização Corporal/fisiologia , Evolução Molecular , Modelos Moleculares , Filogenia , Conformação Proteica , Transdução de Sinais/genética , Tretinoína/metabolismo , Animais , Sequência de Bases , Teorema de Bayes , Análise por Conglomerados , Biologia Computacional , Perfilação da Expressão Gênica , Genes Duplicados/genética , Hibridização In Situ , Funções Verossimilhança , Modelos Genéticos , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal, and differentiation. However, the function of specific PKC isoenzymes have yet to be determined. Of the PKCs expressed in undifferentiated ESCs, ßIPKC was the only isoenzyme abundantly expressed in the nuclei. To investigate the role of ßΙPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one ßIPKC-specific inhibitor peptide. We identified 13 nuclear proteins that are direct or indirect ßΙPKC substrates in undifferentiated ESCs. These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation. Inhibiting ßΙPKC had no effect on DNA synthesis in undifferentiated ESCs. However, upon differentiation, many cells seized to express ßΙPKC and ßΙPKC was frequently found in the cytoplasm. Taken together, our results suggest that ßIPKC takes part in the processes that maintain ESCs in their undifferentiated state.
Assuntos
Células-Tronco Embrionárias/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular , Núcleo Celular/metabolismo , Eletroforese em Gel Bidimensional , Células-Tronco Embrionárias/citologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Peptídeos/farmacologia , Fosfoproteínas/genética , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C beta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Transcrição GênicaRESUMO
RATIONALE: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. OBJECTIVE: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. METHODS AND RESULTS: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. CONCLUSION: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels.
Assuntos
Diferenciação Celular , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericárdio/metabolismo , Transdução de Sinais , Tretinoína/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Animais , Apoptose , Comunicação Autócrina , Diferenciação Celular/genética , Células Cultivadas , Embrião de Galinha , Técnicas de Cocultura , Vasos Coronários/embriologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Morfogênese , Miócitos Cardíacos/metabolismo , Comunicação Parácrina , Pericárdio/embriologia , Codorniz , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Técnicas de Cultura de Tecidos , Transdução Genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Introdução: A voz é o principal instrumento na vida profissional do professor, requerendo uma adaptação precisa dos órgãos da fonação. O desconhecimento da disfonia em professores de nossa região motivou esta pesquisa.Objetivos: Avaliar a frequência de disfonia em professores do Ensino Fundamentalda rede municipal em Maceió-AL e identificar sintomas associados às queixas vocais e possíveis fatores de risco ao aparecimento de alterações vocais. Metodologia: Estudo transversal abrangendo 126 docentes selecionadosaleatoriamente, avaliados a partir de entrevista, com aplicação de questionário dirigido, em 2008. Resultados: Dos 126 professores avaliados, 87,3% referiram ocorrência de disfonia na docência. Observou-se relação entre carga horária semanal e presença de disfonia (p=0,0038). Em relação ao ambiente de trabalho, poeira e ambiente seco foram as queixas mais relatadas, ambas apresentando associação significativa (p<0,04). Os sintomas de obstrução nasal, prurido, tosse e dispepsia apresentaram relação com a presença de rouquidão. Não houve associação entre disfonia e tabagismo ou tabagismopassivo (p<0,6). Conclusão: O estudo permitiu concluir que existe elevada prevalência de disfonia no grupo estudado e que o comprometimento vocal na atividade docente está relacionado aos fatores ambientais, bem como asintomas clínicos associados à rinopatia e ao refluxo gastroesofágico.
Introduction: Voice is teachers main tool and demands an accurate adaptation of the phonation organs. Teachers unawareness of dysphonia in our region lead to this survey. Objectives: Assess frequency of dysphonia in elementary public school teachers from Maceió, state of Alagoas, Brazil, and identify symptoms associated to vocal complaints and possible risk factors for changes in voice. Methodology: Transversal study with 126 teachers selected randomly, who have been assessed through interviews, and structured questionnaires in 2008. Results: From 126 teachers assessed, 87,3% reported dysphonia. A relation between weekly working hours and dysphonia (p=0,0038) was observed. In relation to work environment, dust and air dryness were the most reported complaints, both showing significant association (p<0,04). Nasal obstruction, itching, cough, and dyspepsia were the symptoms related to hoarseness. There was no association between dysphonia and smoking, either active or passive (p<0,6). Conclusion: There is a high prevalence of vocal problems in the studied group, and dysphonia in teaching activity is related to environmental factors, as well as to clinical symptoms associated to allergic rhinitis and gastroesophageal reflux.
Assuntos
Docentes/estatística & dados numéricos , Saúde Ocupacional , Distúrbios da VozRESUMO
Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.
Assuntos
Aldeído Oxirredutases/fisiologia , Medula Espinal/fisiologia , Animais , Sítios de Ligação , Galinhas , Sequência Conservada , Evolução Molecular , Fator 1-alfa Nuclear de Hepatócito , Proteínas de Homeodomínio , Interneurônios , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Transgênicos , Proteínas Repressoras , Fator 1 de Transcrição de Linfócitos T , Fatores de Transcrição , Tretinoína/fisiologiaRESUMO
BACKGROUND: Cardiac development is a complex and multifactorial biological process. Heterozygous mutations in the transcription factor NKX2.5 are between the first evidence of a genetic cause for congenital heart defects in human beings. In this study, we evaluated the presence and frequency of mutations in the NKX2.5 gene on 159 unrelated patients with a diverse range of non-syndromic congenital heart defects (conotruncal anomalies, septal defects, left-sided lesions, right-sided lesions, patent ductus arteriosus and Ebstein's anomaly). METHODS: The coding region of the NKX2.5 locus was amplified by polymerase chain reaction and mutational analysis was performed using denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. RESULTS: We identified two distinct mutations in the NKX2.5 coding region among the 159 (1.26%) individuals evaluated. An Arg25Cys mutation was identified in a patient with Tetralogy of Fallot. The second mutation found was an Ala42Pro in a patient with Ebstein's anomaly. CONCLUSIONS: The association of NKX2.5 mutations is present in a small percentage of patients with non-syndromic congenital heart defects and may explain only a few cases of the disease. Screening strategies considering the identification of germ-line molecular defects in congenital heart disease are still unwarranted and should consider other genes besides NKX2.5.
Assuntos
Deformidades Congênitas da Mão/genética , Proteínas de Homeodomínio/genética , Mutação Puntual , Fatores de Transcrição/genética , Sequência de Aminoácidos , Análise Mutacional de DNA , Permeabilidade do Canal Arterial/genética , Anomalia de Ebstein/genética , Defeitos dos Septos Cardíacos/genética , Proteína Homeobox Nkx-2.5 , Humanos , Reação em Cadeia da Polimerase , Tetralogia de Fallot/genéticaRESUMO
BACKGROUND: Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus. METHODS: One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay. RESULTS: We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls. CONCLUSION: In summary, our screen indicates that ALDH1A2 genetic variation is present in TOF patients, suggesting a possible causal role for this gene in rare cases of human CHD, but does not support the hypothesis that variation at the ALDH1A2 locus is a significant modifier of the risk for CHD in humans.
Assuntos
Variação Genética , Cardiopatias Congênitas/genética , Família Aldeído Desidrogenase 1 , Linhagem Celular , Cromossomos Humanos Par 15 , Éxons , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Desequilíbrio de Ligação , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Dobramento de Proteína , Retinal Desidrogenase/genética , Tetralogia de Fallot/genéticaRESUMO
Dapper (Dpr) proteins are context-dependent regulators of Wnt and Tgfbeta signaling. However, although inroads into their molecular properties have been made, their expression and biological function are not understood. Searching for avian Dpr genes, we found that the chicken harbors a Dpr1 and a Dpr2 paralogue only. The genes are expressed in distinct patterns at gastrulation, neurulation, and organogenesis stages of development with key expression domains being the posterior primitive streak, anterior node and notochord, presomitic mesoderm (segmental plate), lateral and cardiac mesoderm, limb mesenchyme, and neurogenic placodes for Dpr1, and anterior primitive streak, node, epithelial somites, embryonic muscle stem cells, oral ectoderm and endoderm, neural crest cells, limb ectoderm, and lung buds for Dpr2. Expression overlaps in a few tissues; however, in several tissues, expression is complementary.