RESUMO
Objective To analyze the determinants of emergency contraception non-use among women in unplanned and ambivalent pregnancies. Method Cross-sectional study with a probabilistic sample of 366 pregnant women from 12 primary health care units in the city of São Paulo, Brazil. A multinomial logistic regression was performed, comparing three groups: women who used emergency contraception to prevent ongoing pregnancies (reference); women who made no use of emergency contraception, but used other contraceptive methods; and women who made no use of any contraceptive methods at all. Results Cohabitation with a partner was the common determinant of emergency contraception non-use. No pregnancy risk awareness, ambivalent pregnancies and no previous use of emergency contraception also contributed to emergency contraception non-use. Conclusion Apart from what is pointed out in the literature, knowledge of emergency contraception and the fertile period were not associated to its use. .
Objetivo Analizar los determinantes del no uso de la anticoncepción de emergencia entre las mujeres con embarazo no planeado o ambivalente. Método Estudio transversal en una muestra probabilística de 366 mujeres embarazadas de 12 Unidades Básicas de Salud de São Paulo. Mediante regresión logística multinomial, se comparó tres grupos de mujeres: aquellas que usaron la anticoncepción de emergencia para prevenir el embarazo en curso (referencia), aquellas que usaron algún método anticonceptivo, pero no la anticoncepción de emergência; y aquellas que no usaron ningún método. Resultados Los hallazgos mostraron que vivir com la pareja fue el determinante común del no uso de la anticoncepción de emergencia. No tener conciencia del riesgo de embarazo, estar en un embarazo ambivalente y nunca tener utilizado la anticoncepción de emergencia también fueron associados con su no uso para prevenir el embarazo en curso. Conclusión Contrariamente a lo que reporta la literatura, el conocimiento de la anticoncepción de emergencia y el período fértil no mostró asociación con el no uso. .
Objetivo Analisar os determinantes do não uso da anticoncepção de emergência entre mulheres com gravidez não planejada ou ambivalente. Método Estudo transversal com amostra probabilística de 366 gestantes de 12 Unidades Básicas de Saúde da cidade de São Paulo. Por meio de regressão logística multinomial, compararam-se três grupos de mulheres: as que usaram anticoncepção de emergência para prevenir a gravidez em curso (referência); as que usaram algum método contraceptivo, mas não anticoncepção de emergência; e as que não usaram nenhum método. Resultados Os achados mostraram que morar com o parceiro foi o determinante comum do não uso da anticoncepção de emergência. Não ter consciência do risco de engravidar, estar em uma gravidez ambivalente e nunca ter usado anticoncepção de emergência também foram associados ao seu não uso para prevenir a gravidez em curso. Conclusão Diferentemente do que relata a literatura, o conhecimento sobre anticoncepção de emergência e sobre o período fértil não mostrou qualquer associação ao não uso. .
Assuntos
Proteínas de Ligação a DNA , Escherichia coli/genética , Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-Híbrido , Bacteriófago lambda/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/biossíntese , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/fisiologia , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Genes Reporter/genética , Fosforilação , Plasmídeos/biossíntese , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Transcrição Gênica/genética , Transcrição Gênica/fisiologia , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/fisiologia , Proteínas Virais Reguladoras e Acessórias , beta-Galactosidase/biossíntese , beta-Lactamases/biossínteseRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATLL) as well as inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Transgenic (Tg) mice expressing HTLV-1 Tax also develop T-cell leukemia/lymphoma and an inflammatory arthropathy that resembles rheumatoid arthritis. We found that 8 of 297 Tax-Tg mice developed HAM/TSP-like disease with symmetrical paraparesis of the hind limbs, but these symptoms were absent in non-Tg littermates and in other mice strains at our animal facilities. We could perform detailed evaluations for five of these mice. These evaluations showed that the disease was not inflammatory, unlike that in HAM/TSP patients, but instead involved the invasion of histiocytic sarcoma cells into the lumbar spinal cord from the bone marrow where they had undergone extensive proliferation.
Assuntos
Neoplasias da Medula Óssea/patologia , Modelos Animais de Doenças , Produtos do Gene tax/genética , Sarcoma Histiocítico/patologia , Paraparesia Espástica Tropical/etiologia , Medula Espinal/patologia , Animais , Quimiocinas/sangue , Cruzamentos Genéticos , Citocinas/sangue , Feminino , Produtos do Gene tax/fisiologia , Membro Posterior/fisiopatologia , Sarcoma Histiocítico/complicações , Vírus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T do Adulto/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Invasividade Neoplásica , Paraparesia Espástica Tropical/patologia , Proteínas Recombinantes de Fusão/fisiologiaRESUMO
Survivin is overexpressed in most of human cancer cells and tissues. Its overexpression is associated with apoptosis inhibition, drug resistance, and poor prognosis. In this study, we investigated the effect of adeno-associated virus (AAV)-mediated survivin mutant Cys84Ala [rAAV-Sur-Mut(C84A)] on gastric cancer growth. Sur-Mut(C84A) was subcloned into the AAV expression vector pAM/CAG to generate recombinant (r)AAV-Sur-Mut(C84A) virus. Cell survival was determined by the MTT method. Apoptosis was measured by FACS analysis and TUNEL. Tumor growth was assessed using a xenograft mouse model. Results showed that treatment of rAAV-Sur-Mut(C84A) virus significantly reduced cell survival, induced apoptosis, and sensitized gastric cancer cells to 5-fluorouracil in vitro. Furthermore, treatment of rAAV-Sur-Mut(C84A) virus markedly induced apoptosis and inhibited gastric cancer growth in vivo. Moreover, rAAV-Sur-Mut(C84A) treatment strongly enhanced the antitumor activity of 5-fluorouracil. Our results suggest that the combination of rAAV-Sur-Mut(C84A) with chemotherapy may be a promising strategy for gastric cancer therapy.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos/uso terapêutico , Proteínas Inibidoras de Apoptose/genética , Mutação de Sentido Incorreto , Mutação Puntual , Neoplasias Gástricas/terapia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/patologia , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Feminino , Fluoruracila/farmacologia , Vetores Genéticos/genética , Humanos , Proteínas Inibidoras de Apoptose/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Survivina , Transdução Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vitro and in vivo. The tumor-suppressing effects of maspin depend in part on its ability to enhance cell adhesion to extracellular matrix. Although the molecular mechanism of maspin's action is still unclear, its functional domain is believed to be located at the reactive center loop (RCL). We have elucidated the role of maspin RCL on adhesion, migration, and invasion by transfecting the highly invasive human breast carcinoma MDA-MB-231 cell line with pcDNA3.1-His/FLAG containing wild-type maspin, ovalbumin, or maspin/ovalbumin RCL chimeric mutants in which maspin RCL is replaced by ovalbumin (MOM) and vice versa (OMO). MDA-MB-231 cells transfected with maspin- or OMO-containing recombinant expression plasmid manifested significant increase in adhesion to fibronectin and reduction in in vitro migration and invasion through Matrigel compared with mock transfection or cells transfected with ovalbumin or MOM. Proteomics analysis of maspin- or OMO-transfected MDA-MB-231 cells revealed reduction in contents of proteins known to promote cancer metastasis and those of ubiquitin-proteasome pathway, while those with tumor-suppressing properties were increased. Furthermore, MDA-MB-231 cells containing maspin or OMO transgene have significantly higher levels of ubiquitin and ubiquitinated conjugates, but reduced 20S proteasome chymotrypsin-like activity. These results clearly demonstrate that the tumor-suppressive properties of maspin reside in its RCL domain.
Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Invasividade Neoplásica/fisiopatologia , Proteínas de Neoplasias/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/genética , Serpinas/fisiologia , Adenocarcinoma/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Neoplasias da Mama/genética , Domínio Catalítico , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/patologia , Feminino , Humanos , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Invasividade Neoplásica/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Ovalbumina/química , Ovalbumina/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serpinas/química , Serpinas/genética , Transgenes , UbiquitinaçãoRESUMO
Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.
Assuntos
Proteínas de Bactérias/fisiologia , Doenças Cardiovasculares/genética , Desoxirribonucleases de Sítio Específico do Tipo II/fisiologia , Desoxirribonucleases/fisiologia , Engenharia Genética/métodos , Genômica , Células-Tronco Pluripotentes Induzidas/citologia , Mutagênese Sítio-Dirigida/métodos , Medicina de Precisão/tendências , Xanthomonas/enzimologia , Animais , Proteínas de Bactérias/genética , Sítios de Ligação , Doenças Cardiovasculares/terapia , Diferenciação Celular , Células Cultivadas/citologia , Células Cultivadas/transplante , DNA/genética , DNA/metabolismo , Desoxirribonucleases/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Previsões , Genes Reporter , Engenharia Genética/tendências , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Modelos Cardiovasculares , Modelos Genéticos , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Reparo de DNA por Recombinação , Especificidade por Substrato , Xanthomonas axonopodis/enzimologia , Peixe-Zebra/genéticaRESUMO
The hypothesis that mitochondrial dysfunction and increased superoxide levels in thymocytes over expressing Bax (Lck-Bax1 and Lck-Bax38&1) contributes to lymphomagenesis after low-dose radiation was tested. Lck-Bax1 single-transgenic and Lck-Bax38&1 double-transgenic mice were exposed to single whole-body doses of 10 or 100 cGy of (137)Cs or iron ions (1,000 MeV/n, 150 keV/µm) or silicon ions (300 MeV/n, 67 keV/µm). A 10 cGy dose of (137)Cs significantly increased the incidence and onset of thymic lymphomas in female Lck-Bax1 mice. In Lck-Bax38&1 mice, a 100 cGy dose of high-LET iron ions caused a significant dose dependent acceleration of lymphomagenesis in both males and females that was not seen with silicon ions. To determine the contribution of mitochondrial oxidative metabolism, Lck-Bax38&1 over expressing mice were crossed with knockouts of the mitochondrial protein deacetylase, Sirtuin 3 (Sirt3), which regulates superoxide metabolism. Sirt3(-/-)/Lck-Bax38&1 mice demonstrated significant increases in thymocyte superoxide levels and acceleration of lymphomagenesis (P < 0.001). These results show that lymphomagenesis in Bax over expressing animals is enhanced by radiation exposure in both an LET and gender dependent fashion. These findings support the hypothesis that mitochondrial dysfunction leads to increased superoxide levels and accelerates lymphomagenesis in Lck-Bax transgenic mice.
Assuntos
Íons Pesados/efeitos adversos , Transferência Linear de Energia , Linfoma/etiologia , Mitocôndrias/efeitos da radiação , Neoplasias Induzidas por Radiação/etiologia , Estresse Oxidativo , Caracteres Sexuais , Superóxidos/metabolismo , Neoplasias do Timo/etiologia , Irradiação Corporal Total/efeitos adversos , Proteína X Associada a bcl-2/fisiologia , Animais , Radioisótopos de Césio , Relação Dose-Resposta à Radiação , Feminino , Dosagem de Genes , Ferro , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Linfoma/genética , Linfoma/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mitocôndrias/metabolismo , Neoplasias Induzidas por Radiação/genética , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/fisiopatologia , Fosforilação Oxidativa/efeitos da radiação , Doses de Radiação , Tolerância a Radiação/genética , Proteínas Recombinantes de Fusão/fisiologia , Silício , Sirtuína 3/deficiência , Sirtuína 3/genética , Sirtuína 3/fisiologia , Timócitos/metabolismo , Timócitos/patologia , Timócitos/efeitos da radiação , Neoplasias do Timo/genética , Neoplasias do Timo/fisiopatologia , Proteína X Associada a bcl-2/genéticaRESUMO
Cortical inhibitory neurons contact each other to form a network of inhibitory synaptic connections. Our knowledge of the connectivity pattern underlying this inhibitory network is, however, still incomplete. Here we describe a simple and complementary interaction scheme between three large, molecularly distinct interneuron populations in mouse visual cortex: parvalbumin-expressing interneurons strongly inhibit one another but provide little inhibition to other populations. In contrast, somatostatin-expressing interneurons avoid inhibiting one another yet strongly inhibit all other populations. Finally, vasoactive intestinal peptide-expressing interneurons preferentially inhibit somatostatin-expressing interneurons. This scheme occurs in supragranular and infragranular layers, suggesting that inhibitory networks operate similarly at the input and output of the visual cortex. Thus, as the specificity of connections between excitatory neurons forms the basis for the cortical canonical circuit, the scheme described here outlines a standard connectivity pattern among cortical inhibitory neurons.
Assuntos
Interneurônios/fisiologia , Inibição Neural/fisiologia , Córtex Visual/fisiologia , Animais , Biomarcadores , Channelrhodopsins , Feminino , Genes Reporter , Potenciais Pós-Sinápticos Inibidores/efeitos da radiação , Interneurônios/química , Interneurônios/classificação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/análise , Optogenética , Compostos Organofosforados/farmacologia , Parvalbuminas/análise , Técnicas de Patch-Clamp , Estimulação Luminosa , Análise de Componente Principal , Células Piramidais/fisiologia , Quinoxalinas/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Somatostatina/análise , Transmissão Sináptica/fisiologia , Transmissão Sináptica/efeitos da radiação , Peptídeo Intestinal Vasoativo/análiseRESUMO
The primary somatosensory cortex (S1) contains a complete body map that mirrors the subcortical maps developed by peripheral sensory input projecting to the sensory hindbrain, the thalamus and then S1. Peripheral changes during development alter these maps through 'bottom-up' plasticity. Unknown is how S1 size influences map organization and whether an altered S1 map feeds back to affect subcortical maps. We show that the size of S1 in mice is significantly reduced by cortex-specific deletion of Pax6, resulting in a reduced body map and loss of body representations by an exclusion of later-differentiating sensory thalamocortical input. An initially normal sensory thalamus was repatterned to match the aberrant S1 map by apoptotic deletion of thalamic neurons representing body parts with axons excluded from S1. Deleted representations were rescued by altering competition between thalamocortical axons using sensory deprivation or increasing the size of S1. Thus, S1 size determined the resolution and completeness of body maps and engaged 'top-down' plasticity that repatterned the sensory thalamus to match S1.
Assuntos
Plasticidade Neuronal/fisiologia , Núcleos Posteriores do Tálamo/fisiologia , Córtex Somatossensorial/fisiologia , Animais , Apoptose , Axônios/fisiologia , Imagem Corporal , Proteínas do Olho/genética , Proteínas do Olho/fisiologia , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Vias Neurais/fisiologia , Neurônios/fisiologia , Especificidade de Órgãos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/deficiência , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/fisiologia , Núcleos Posteriores do Tálamo/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Rombencéfalo/fisiologia , Sensação/fisiologia , Córtex Somatossensorial/patologia , Vibrissas/inervaçãoRESUMO
Defects in DNA repair have been linked to cognitive decline with age and neurodegenerative disease, yet the mechanisms that protect neurons from genotoxic stress remain largely obscure. We sought to characterize the roles of the NAD(+)-dependent deacetylase SIRT1 in the neuronal response to DNA double-strand breaks (DSBs). We found that SIRT1 was rapidly recruited to DSBs in postmitotic neurons, where it showed a synergistic relationship with ataxia telangiectasia mutated (ATM). SIRT1 recruitment to breaks was ATM dependent; however, SIRT1 also stimulated ATM autophosphorylation and activity and stabilized ATM at DSB sites. After DSB induction, SIRT1 also bound the neuroprotective class I histone deacetylase HDAC1. We found that SIRT1 deacetylated HDAC1 and stimulated its enzymatic activity, which was necessary for DSB repair through the nonhomologous end-joining pathway. HDAC1 mutations that mimic a constitutively acetylated state rendered neurons more susceptible to DNA damage, whereas pharmacological SIRT1 activators that promoted HDAC1 deacetylation also reduced DNA damage in two mouse models of neurodegeneration. We propose that SIRT1 is an apical transducer of the DSB response and that SIRT1 activation offers an important therapeutic avenue in neurodegeneration.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Proteínas de Ligação a DNA/fisiologia , Instabilidade Genômica , Histona Desacetilase 1/fisiologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Sirtuína 1/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Acetilação , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Córtex Cerebral/citologia , Ensaio Cometa , Ativação Enzimática/efeitos dos fármacos , Etoposídeo/farmacologia , Vetores Genéticos , Células HEK293 , Hipocampo/citologia , Histona Desacetilase 1/genética , Histona Desacetilase 1/imunologia , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fosforilação , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genéticaRESUMO
RATIONALE: Vascular calcification is a regulated process that involves osteoprogenitor cells and frequently complicates common vascular disease, such as atherosclerosis and diabetic vasculopathy. However, it is not clear whether the vascular endothelium has a role in contributing osteoprogenitor cells to the calcific lesions. OBJECTIVE: To determine whether the vascular endothelium contributes osteoprogenitor cells to vascular calcification. METHODS AND RESULTS: In this study, we use 2 mouse models of vascular calcification, mice with gene deletion of matrix Gla protein, a bone morphogenetic protein (BMP)-inhibitor, and Ins2Akita/+ mice, a diabetes model. We show that enhanced BMP signaling in both types of mice stimulates the vascular endothelium to contribute osteoprogenitor cells to the vascular calcification. The enhanced BMP signaling results in endothelial-mesenchymal transitions and the emergence of multipotent cells, followed by osteoinduction. Endothelial markers colocalize with multipotent and osteogenic markers in calcified arteries by immunostaining and fluorescence-activated cell sorting. Lineage tracing using Tie2-Gfp transgenic mice supports an endothelial origin of the osteogenic cells. Enhancement of matrix Gla protein expression in Ins2Akita/+ mice, as mediated by an Mgp transgene, limits the generation of multipotent cells. Moreover, matrix Gla protein-depleted human aortic endothelial cells in vitro acquire multipotency rendering the cells susceptible to osteoinduction by BMP and high glucose. CONCLUSIONS: Our data suggest that the endothelium is a source of osteoprogenitor cells in vascular calcification that occurs in disorders with high BMP activation, such as deficiency of BMP-inhibitors and diabetes mellitus.
Assuntos
Calcinose/fisiopatologia , Proteínas de Ligação ao Cálcio/fisiologia , Transdiferenciação Celular/fisiologia , Angiopatias Diabéticas/fisiopatologia , Células Endoteliais/patologia , Endotélio Vascular/fisiopatologia , Proteínas da Matriz Extracelular/fisiologia , Insulina/fisiologia , Células-Tronco Multipotentes/patologia , Doenças Vasculares/fisiopatologia , Animais , Aorta/citologia , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Linhagem da Célula , Células Cultivadas/efeitos dos fármacos , Diabetes Mellitus Tipo 2/genética , Angiopatias Diabéticas/genética , Modelos Animais de Doenças , Endotélio Vascular/patologia , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Glucose/farmacologia , Heterozigoto , Humanos , Insulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas dos Microfilamentos/fisiologia , Proteínas Musculares/fisiologia , RNA Interferente Pequeno/farmacologia , Receptor TIE-2/genética , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Proteína de Matriz GlaRESUMO
RATIONALE: A stable 40-kDa fragment is produced from cardiac myosin-binding protein C when the heart is stressed using a stimulus, such as ischemia-reperfusion injury. Elevated levels of the fragment can be detected in the diseased mouse and human heart, but its ability to interfere with normal cardiac function in the intact animal is unexplored. OBJECTIVE: To understand the potential pathogenicity of the 40-kDa fragment in vivo and to investigate the molecular pathways that could be targeted for potential therapeutic intervention. METHODS AND RESULTS: We generated cardiac myocyte-specific transgenic mice using a Tet-Off inducible system to permit controlled expression of the 40-kDa fragment in cardiomyocytes. When expression of the 40-kDa protein is induced by crossing the responder animals with tetracycline transactivator mice under conditions in which substantial quantities approximating those observed in diseased hearts are reached, the double-transgenic mice subsequently experience development of sarcomere dysgenesis and altered cardiac geometry, and the heart fails between 12 and 17 weeks of age. The induced double-transgenic mice had development of cardiac hypertrophy with myofibrillar disarray and fibrosis, in addition to activation of pathogenic MEK-ERK pathways. Inhibition of MEK-ERK signaling was achieved by injection of the mitogen-activated protein kinase (MAPK)/ERK inhibitor U0126. The drug effectively improved cardiac function, normalized heart size, and increased probability of survival. CONCLUSIONS: These results suggest that the 40-kDa cardiac myosin-binding protein C fragment, which is produced at elevated levels during human cardiac disease, is a pathogenic fragment that is sufficient to cause hypertrophic cardiomyopathy and heart failure.
Assuntos
Proteínas de Transporte/metabolismo , Animais , Butadienos/farmacologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Feminino , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/citologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Nitrilas/farmacologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Sarcômeros/químicaRESUMO
Alphaherpesviruses, including pseudorabies virus (PRV), spread directionally within the nervous systems of their mammalian hosts. Three viral membrane proteins are required for efficient anterograde-directed spread of infection in neurons, including Us9 and a heterodimer composed of the glycoproteins gE and gI. We previously demonstrated that the kinesin-3 motor KIF1A mediates anterograde-directed transport of viral particles in axons of cultured peripheral nervous system (PNS) neurons. The PRV Us9 protein copurifies with KIF1A, recruiting the motor to transport vesicles, but at least one unidentified additional viral protein is necessary for this interaction. Here we show that gE/gI are required for efficient anterograde transport of viral particles in axons by mediating the interaction between Us9 and KIF1A. In the absence of gE/gI, viral particles containing green fluorescent protein (GFP)-tagged Us9 are assembled in the cell body but are not sorted efficiently into axons. Importantly, we found that gE/gI are necessary for efficient copurification of KIF1A with Us9, especially at early times after infection. We also constructed a PRV recombinant that expresses a functional gE-GFP fusion protein and used affinity purification coupled with mass spectrometry to identify gE-interacting proteins. Several viral and host proteins were found to associate with gE-GFP. Importantly, both gI and Us9, but not KIF1A, copurified with gE-GFP. We propose that gE/gI are required for efficient KIF1A-mediated anterograde transport of viral particles because they indirectly facilitate or stabilize the interaction between Us9 and KIF1A.
Assuntos
Alphaherpesvirinae/fisiologia , Herpesvirus Suídeo 1/fisiologia , Cinesinas/fisiologia , Lipoproteínas/fisiologia , Neurônios/fisiologia , Neurônios/virologia , Fosfoproteínas/fisiologia , Proteínas do Envelope Viral/fisiologia , Proteínas Virais/fisiologia , Alphaherpesvirinae/genética , Alphaherpesvirinae/patogenicidade , Animais , Transporte Axonal/fisiologia , Linhagem Celular , Células Cultivadas , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/patogenicidade , Interações Hospedeiro-Patógeno , Peptídeos e Proteínas de Sinalização Intracelular , Lipoproteínas/genética , Células PC12 , Fosfoproteínas/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Suínos , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Vírion/fisiologiaRESUMO
Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO(3+)), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Animais , Biocatálise , Coenzimas/química , Coenzimas/fisiologia , Sistema Enzimático do Citocromo P-450/fisiologia , Humanos , Hidroxilação , Oxirredução , Prostaglandinas/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/fisiologiaRESUMO
KIR aid in the regulation of NK cell activity. In this study, the effect of the interaction between the KIR2DS and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when cotransfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12-specific siRNA. Treatment of the KIR2DS and DAP12-transfected cells with CHX or BFA repressed KIR2DS surface expression, revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS, indicating that the interaction likely initiates within the ER. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed that internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest that in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Células Matadoras Naturais/metabolismo , Proteínas de Membrana/fisiologia , Receptores KIR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Células Jurkat , Células Matadoras Naturais/imunologia , Lisossomos/fisiologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Receptores KIR/genética , Proteínas Recombinantes de Fusão/fisiologia , TransfecçãoRESUMO
Candida albicans, the most commonly isolated human fungal pathogen, is able to grow as budding yeasts or filamentous forms, such as hyphae. The ability to switch morphology has been attributed a crucial role for the pathogenesis of C. albicans. To mimic disseminated candidiasis in humans, the mouse is the most widely used model organism. Neutrophils are essential immune cells to prevent opportunistic mycoses. To explore potential differences between the rodent infection model and the human host, we compared the interactions of C. albicans with neutrophil granulocytes from mice and humans. We revealed that murine neutrophils exhibited a significantly lower ability to kill C. albicans than their human counterparts. Strikingly, C. albicans yeast cells formed germ tubes upon internalization by murine neutrophils, eventually rupturing the neutrophil membrane and thereby, killing the phagocyte. On the contrary, growth and subsequent escape of C. albicans are blocked inside human neutrophils. According to our findings, this blockage in human neutrophils might be a result of higher levels of MPO activity and the presence of α-defensins. We therefore outline differences in antifungal immune defense between humans and mouse strains, which facilitates a more accurate interpretation of in vivo results.
Assuntos
Candida albicans/imunologia , Evasão da Resposta Imune , Camundongos/imunologia , Modelos Animais , Neutrófilos/imunologia , Animais , Candida albicans/crescimento & desenvolvimento , Feminino , Técnicas de Introdução de Genes , Humanos , Hifas/crescimento & desenvolvimento , Imunidade Inata , Masculino , Camundongos Endogâmicos C57BL , Neutrófilos/enzimologia , Neutrófilos/microbiologia , Peroxidase/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Explosão Respiratória , Especificidade da Espécie , alfa-Defensinas/genética , alfa-Defensinas/fisiologiaRESUMO
RATIONALE: Endothelial microRNA-126 (miR-126) modulates vascular development and angiogenesis. However, its role in the regulation of smooth muscle cell (SMC) function is unknown. OBJECTIVE: To elucidate the role of miR-126 secreted by endothelial cells (ECs) in regulating SMC turnover in vitro and in vivo, as well as the effects of shear stress on the regulation. METHODS AND RESULTS: Coculture of SMCs with ECs or treatment of SMCs with conditioned media from static EC monoculture (EC-CM) increased SMC miR-126 level and SMC turnover; these effects were abolished by inhibition of endothelial miR-126 and by the application of laminar shear stress to ECs. SMC miR-126 did not increase when treated with EC-CM from ECs subjected to inhibition of miR biogenesis, or with CM from sheared ECs. Depletion of extracellular/secreted vesicles in EC-CM did not affect the increase of SMC miR-126 by EC-CM. Biotinylated miR-126 or FLAG (DYKDDDDK epitope)-tagged Argonaute2 transfected into ECs was detected in the cocultured or EC-CM-treated SMCs, indicating a direct EC-to-SMC transmission of miR-126 and Argonaute2. Endothelial miR-126 represses forkhead box O3, B-cell lymphoma 2, and insulin receptor substrate 1 mRNAs in the cocultured SMCs, suggesting the functional roles of the transmitted miR-126. Systemic depletion of miR-126 in mice inhibited neointimal lesion formation of carotid arteries induced by cessation of blood flow. Administration of EC-CM or miR-126 mitigated the inhibitory effect. CONCLUSIONS: Endothelial miR-126 acts as a key intercellular mediator to increase SMC turnover, and its release is reduced by atheroprotective laminar shear stress.
Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica , MicroRNAs/fisiologia , Miócitos de Músculo Liso/citologia , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/fisiologia , Artéria Carótida Primitiva/patologia , Técnicas de Cultura de Células/instrumentação , Linhagem Celular , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Inativação Gênica , Genes bcl-2 , Hemorreologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Substratos do Receptor de Insulina/biossíntese , Proteínas Substratos do Receptor de Insulina/genética , Ligadura , Camundongos , MicroRNAs/genética , MicroRNAs/uso terapêutico , Músculo Liso Vascular/citologia , Neointima , Comunicação Parácrina , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Artérias Umbilicais/citologiaRESUMO
TMEM16A and TMEM16B proteins are CaCCs (Ca2+-activated Cl- channels) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a 10-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimaeric proteins in which different domains of the TMEM16A protein were replaced by the equivalent domains of TMEM16B. Replacement of the N-terminus, TMD (transmembrane domain) 1-2, the first intracellular loop and TMD3-4 did not change the channel's properties. Instead, replacement of intracellular loop 3 decreased the apparent Ca2+ affinity by nearly 8-fold with respect to wild-type TMEM16A. In contrast, the membrane currents derived from chimaeras containing TMD7-8 or the C-terminus of TMEM16B showed higher activation and deactivation rates without a change in Ca2+ sensitivity. Significantly accelerated kinetics were also found when the entire C-terminus of the TMEM16A protein (77 amino acid residues) was deleted. Our findings indicate that the third intracellular loop of TMEM16A and TMEM16B is the site involved in Ca2+-sensitivity, whereas the C-terminal part, including TMD7-8, affect the rate of transition between the open and the closed state.
Assuntos
Canais de Cloreto/química , Canais de Cloreto/genética , Proteínas de Membrana/química , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Recombinantes de Fusão/química , Anoctamina-1 , Anoctaminas , Canais de Cloreto/fisiologia , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Relação Estrutura-AtividadeRESUMO
Stromal-derived factor (SDF)-1α, insulin-like growth factor (IGF)-1 and hepatocyte growth factor (HGF) are potent mediators of cell migration. We studied the effect of combinations of these cytokines on the migration of myeloma cells. When SDF-1α was combined with either HGF or IGF-1, we found a striking synergy in the cytokines' ability to guide cells across a transwell membrane. Between HGF and IGF-1 there was no cooperativity. However, the effects of HGF and IGF-1 were not redundant. HGF and SDF-1 caused concentration gradient-directed migration, as opposed to IGF-1, which apparently caused randomly directed cell movement. The SDF-1α-driven migration of JJN-3 cells, a myeloma cell line secreting large amounts of HGF, was reduced when JJN-3 cells were given an inhibitor of the HGF receptor, demonstrating a cooperative activity between autocrine HGF and exogenous SDF-1α. There was a clear positive correlation between the degree of cytokine-induced migration and phosphorylation of p21-activated kinase (PAK) both in primary myeloma cells and in cell lines including INA-6 and IH-1. Downregulation of PAK with small interfering RNA in INA-6 cells resulted in decreased cytokine-driven migration. This study shows synergy between SDF-1α and HGF/IGF-1 in inducing migration of myeloma cells, yet each cytokine has distinct properties in the way it regulates cell migration. These findings are likely to be of clinical relevance because multiple myeloma cells are located in an environment containing HGF and IGF-1 and are exposed to an SDF-1α gradient between the bone marrow and peripheral blood.
Assuntos
Quimiocina CXCL12/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Mieloma Múltiplo/patologia , Quinases Ativadas por p21/fisiologia , Comunicação Autócrina , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/fisiologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Fator de Crescimento Insulin-Like I/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-met/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Receptores CXCR4/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Transfecção , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genéticaRESUMO
Recently discovered Sushi domain-containing protein 4 (SUSD4) contains several Sushi or complement control protein domains; therefore, we hypothesized that it may act as complement inhibitor. Two isoforms of human SUSD4, fused to the Fc part of human IgG, were recombinantly expressed in Chinese hamster ovary (CHO) cells. The secreted soluble isoform of SUSD4 (SUSD4b) inhibited the classical and lectin complement pathways by 50% at a concentration of 0.5 µM. This effect was due to the fact that 1 µM SUSD4b inhibited the formation of the classical C3 convertase by 90%. The membrane-bound isoform (SUSD4a) inhibited the classical and alternative complement pathways when expressed on the surface of CHO cells but not when expressed as a soluble, truncated protein. In all functional studies, we used known complement inhibitors as positive controls, while Coxsackie adenovirus receptor, which has no effect on complement, expressed with Fc tag, was a negative control. We also studied the mRNA expression of both isoforms of SUSD4 in a panel of human tissues using quantitative PCR and primarily found SUSD4a in esophagus and brain, while SUSD4b was highly expressed in esophagus, ovary, and heart. Overall, our results show that SUSD4 is a novel complement inhibitor with restricted expression.
Assuntos
C3 Convertase da Via Clássica do Complemento/antagonistas & inibidores , C3 Convertase da Via Clássica do Complemento/biossíntese , Proteínas Inativadoras do Complemento/fisiologia , Proteínas de Membrana/fisiologia , Animais , Células CHO , Complemento C3/metabolismo , Proteínas Inativadoras do Complemento/genética , Via Clássica do Complemento , Cricetinae , Cricetulus , Feminino , Humanos , Imunidade Inata/genética , Proteínas de Membrana/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Distribuição TecidualRESUMO
γ-protocadherins (γ-pcdhs) are transmembrane receptor proteins ubiquitously expressed in the postnatal and adult mouse brain. γ-pcdhs are required for normal neuronal development as shown for spinal cord interneurons, retinal ganglion cells and cortical neurons. To test the role of γ-pcdhs during development of subventricular zone progenitor cells and their subsequent differentiation into olfactory granule cells we generated a conditional γ-pcdh(lox/lox) allele (γ-pcdh(lox/lox)) allowing for functional γ-pcdh inactivation upon lentivirus-mediated Cre-recombinase expression selectively in subventricular zone progenitor cells. While γ-pcdh loss did not alter the proliferation of subventricular zone progenitors, γ-pcdh ko progenitors that reached the main olfactory bulb showed a significant reduction in dendritic arborization and failed to develop dendritic spines. Our results suggest that olfactory bulb granule cell maturation necessitates functional γ-pcdh expression.
