RESUMO
The goal of the present study was to compare six transposing signal-processing algorithms based on different principles (Fourier-based and modulation based), and to choose the algorithm that best enables identification of environmental sounds, i.e. improves the ability to monitor events in the surroundings. Ten children (12-15 years) and 10 adults (21-33 years) with normal hearing listened to 45 representative environmental (events) sounds processed using the six algorithms, and identified them in three different listening experiments involving an increasing degree of experience. The sounds were selected based on their importance for normal hearing and deaf-blind subjects. Results showed that the algorithm based on transposition of 1/3 octaves (fixed frequencies) with large bandwidth was better (p<0.015) than algorithms based on modulation. There was also a significant effect of experience (p<0.001). Adults were significantly (p<0.05) better than children for two algorithms. No clear gender difference was observed. It is concluded that the algorithm based on transposition with large bandwidth and fixed frequencies is the most promising for development of hearing aids to monitor environmental sounds.
Assuntos
Algoritmos , Percepção Auditiva , Audição/fisiologia , Som , Adolescente , Adulto , Fatores Etários , Criança , Meio Ambiente , Feminino , Auxiliares de Audição , Humanos , Modelos Logísticos , Masculino , Fatores Sexuais , Adulto JovemAssuntos
Segurança de Equipamentos , Vigilância de Produtos Comercializados , Sistemas de Notificação de Reações Adversas a Medicamentos , Ensaios Clínicos como Assunto , Qualidade de Produtos para o Consumidor , União Europeia , Humanos , Ciência de Laboratório Médico/legislação & jurisprudência , Ciência de Laboratório Médico/normas , Papel do Médico , Gestão de RiscosRESUMO
The coxsackie- and adenovirus receptor (CAR) is a cell adhesion molecule predominantly associated with epithelial tight junctions in adult tissues. CAR is also expressed in cardiomyocytes and essential for heart development up to embryonic day 11.5, but not thereafter. CAR is not expressed in vascular endothelial cells but was recently detected in neonatal lymphatic vessels, suggesting that CAR could play a role in the development of the lymphatic system. To address this, we generated mice carrying a conditional deletion of the CAR gene (Cxadr) and knocked out CAR in the mouse embryo at different time points during post-cardiac development. Deletion of Cxadr from E12.5, but not from E13.5, resulted in subcutaneous edema, hemorrhage and embryonic death. Subcutaneous lymphatic vessels were dilated and structurally abnormal with gaps and holes present at lymphatic endothelial cell-cell junctions. Furthermore, lymphatic vessels were filled with erythrocytes showing a defect in the separation between the blood and lymphatic systems. Regionally, erythrocytes leaked out into the interstitium from leaky lymphatic vessels explaining the hemorrhage detected in CAR-deficient mouse embryos. The results show that CAR plays an essential role in development of the lymphatic vasculature in the mouse embryo by promoting appropriate formation of lymphatic endothelial cell-cell junctions.
Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Junções Intercelulares/metabolismo , Vasos Linfáticos/embriologia , Receptores Virais/metabolismo , Animais , Western Blotting , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Genótipo , Técnicas Histológicas , Vasos Linfáticos/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Receptores Virais/genética , TamoxifenoRESUMO
To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.
Assuntos
Inativação Gênica , Fenótipo , Receptores Virais/deficiência , Receptores Virais/genética , Animais , Bloqueio Atrioventricular/genética , Atrofia/genética , Comportamento Animal/efeitos dos fármacos , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Feminino , Fertilidade/efeitos dos fármacos , Fertilidade/genética , Técnicas de Inativação de Genes , Inativação Gênica/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/patologia , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/metabolismo , Pâncreas Exócrino/patologia , Receptores Virais/metabolismo , Tamoxifeno/farmacologia , Timo/citologia , Timo/efeitos dos fármacos , Timo/metabolismoAssuntos
Pesquisa Biomédica/tendências , Apoio à Pesquisa como Assunto , Distinções e Prêmios , Disciplinas das Ciências Biológicas/tendências , Pesquisa Biomédica/economia , Pesquisa Biomédica/organização & administração , Ensaios Clínicos como Assunto , Europa (Continente) , Previsões , Editoração , Transferência de Tecnologia , Estados UnidosRESUMO
Epithelial-mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state. Transforming growth factor-beta (TGF-beta) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight-junction protein, and E-cadherin during TGF-beta-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a mechanism of gene repression during EMT.
Assuntos
Proteína Smad3/metabolismo , Proteína Smad4/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Western Blotting , Caderinas/genética , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Junções Intercelulares/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Regiões Promotoras Genéticas/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad3/genética , Proteína Smad4/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
The coxsackie- and adenovirus receptor (CAR) is a transmembrane protein belonging to the immunoglobulin superfamily. The function of CAR as a virus receptor has been extensively analyzed, while its physiological role and expression pattern in adult tissues have remained less clear. CAR associates with epithelial tight junctions in vitro and mediates cell-cell adhesion. Using a set of affinity-purified antibodies, we show that CAR is predominantly expressed in epithelial cells lining the body cavities in adult mice, where it specifically co-localizes with the tight junction components ZO-1 and occludin. Notably, CAR could not be detected in endothelial cells of the vasculature, including brain capillaries. CAR expression correlated positively with the maturity of tight junctions and inversely with permeability. With a few exceptions, the two known CAR isoforms were co-expressed in most epithelial cells analyzed. A CAR mutant lacking the intracellular tail over-expressed in transgenic mice was diffusely localized over the plasma membrane, showing the importance of this domain for correct subcellular localization in vivo. We conclude that CAR is localized to epithelial tight junctions in vivo where it may play a role in the regulation of epithelial permeability and tissue homeostasis.
Assuntos
Células Epiteliais/química , Homeostase/fisiologia , Receptores Virais/análise , Junções Íntimas/química , Animais , Linhagem Celular , Permeabilidade da Membrana Celular/fisiologia , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Imunofluorescência , Trato Gastrointestinal/química , Trato Gastrointestinal/citologia , Humanos , Rim/química , Rim/citologia , Fígado/química , Fígado/citologia , Masculino , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Imunoeletrônica , Ocludina , Fosfoproteínas/análise , Próstata/química , Próstata/citologia , Receptores Virais/genética , Receptores Virais/fisiologia , Sistema Respiratório/química , Sistema Respiratório/citologia , Junções Íntimas/fisiologia , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1RESUMO
The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein important for viral binding to target cells. Using RT-PCR, Western analysis, GST pull-down assay and indirect immunofluorescence, it was shown that CAR is expressed in male germ cells from mice, rats, and humans. CAR was detected in round spermatids in the testis as well as in purified, mature spermatozoa. The two membrane-bound isoforms of CAR occupied different subcellular sites in the acrosomal region of the spermatozoa. CAR was exposed on the surface of acrosome-reacted, but not acrosome-intact cells. Two CAR-binding proteins belonging to the ligand-of-numb protein-X (LNX) family also occupied distinct regions in spermatozoa. Finally, co-immunoprecipitation experiments demonstrated an interaction between CAR and JAM-C, a protein required for spermatid differentiation. Together, these findings imply a function for CAR in male fertility. The results also suggest that CAR in spermatozoa is inaccessible to adenovirus-based gene therapy vectors, and that the risk of germ line infection therefore is low.
Assuntos
Antígenos de Diferenciação/metabolismo , Moléculas de Adesão Celular/fisiologia , Regulação da Expressão Gênica , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Testículo/citologia , Acrossomo/metabolismo , Animais , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Epididimo/química , Epididimo/fisiologia , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Receptores Virais/genética , Receptores Virais/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Túbulos Seminíferos/química , Túbulos Seminíferos/citologia , Túbulos Seminíferos/fisiologia , Espermatogênese/genética , Espermatogênese/fisiologia , Espermatozoides/química , Espermatozoides/citologia , Espermatozoides/fisiologia , Testículo/química , Testículo/fisiologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The coxsackievirus and adenovirus receptor (CAR) is a cell surface protein that is proposed to be involved in cell-cell adhesion. Based on a yeast two-hybrid screen, co-immunoprecipitation and binding experiments, the intracellular tail of CAR was found to interact both in vivo and in vitro with the Ligand-of-Numb Protein-X2 (LNX2). The interacting domains between the two proteins were identified by truncation analyses and affinity chromatography. CAR and LNX2 protein expression in embryonic mouse tissues was analyzed by immunohistochemistry. The results suggest that CAR is a partner in a protein complex organized at specific subcellular sites by LNX2.
Assuntos
Proteínas de Transporte/metabolismo , Receptores Virais/metabolismo , Adenoviridae/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Embrião de Mamíferos/metabolismo , Enterovirus/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Junções Íntimas/metabolismoRESUMO
The sensitivity of human tissues and tumors to infection with type C adenoviruses correlates with the expression of the human coxsackie B- and adenovirus receptor, hCAR. HCAR is heterogeneously expressed in various tissues and types of human cancer cells, which has implications for the use of adenoviruses as vectors in cancer gene therapy. Using immunoblotting, real-time PCR, FACS-analysis and sensitivity to infection with adenovirus-lacZ, we analyzed the expression level of hCAR in glioma Grade IV cell lines. With real-time PCR, we also analyzed hCAR expression in primary human astrocytomas of different malignancy grades, as well as in their xenograft derivatives. Analysis of a set of 10 cell lines showed great variation in hCAR expression. Susceptibility to Ad5lacZ correlated well with hCAR expression, whereas no correlation was observed with the expression of alphavbeta3/alphavbeta5 integrins, proposed to function as co-receptors for adenoviruses. A great variation of CAR expression was also observed in primary astrocytomas of different malignancy grades. The mean value of CAR expression was significantly lower in 22 Grade IV tumors as compared to the values for 6 Grade II (p = 0.01) and 6 Grade III (p = 0.01) tumors. When the hCAR expression in 11 xenografts derived from Grade IV gliomas were compared to the levels detected in the original parental tumors, a mean 12-fold higher expression was seen in the xenografts (P = 0.01). Two xenografts with low hCAR expression grew considerably faster than the hCAR-expressing cells. Our results have relevance for the use of adenoviruses in gene therapy against astrocytomas.
Assuntos
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Receptores Virais/metabolismo , Adenoviridae/fisiologia , Animais , Astrocitoma/virologia , Neoplasias Encefálicas/virologia , Calnexina/metabolismo , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Enterovirus/fisiologia , Feminino , Fibroblastos/metabolismo , Imunofluorescência , Terapia Genética , Humanos , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia , RNA Mensageiro/metabolismo , Receptores Virais/genética , Receptores de Vitronectina/metabolismo , Pele/metabolismo , Transplante Heterólogo , Células Tumorais Cultivadas , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown. A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR. LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind to and regulate the expression level of the cell-fate determinant Numb. CAR was able to bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream sequences. The CAR binding region in LNX was mapped to the second PDZ domain. CAR and LNX were also shown to colocalize in vivo in mammalian cells. We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.