RESUMO
Enteral nutrition (EN) is considered to be a more appropriate method than parenteral feeding for providing nutrition to critically ill children. However, children who undergo cardiac surgery are at high risk of postoperative gastrointestinal complications during EN. The purpose of this study was to demonstrate the safety and efficacy of our EN feeding protocol after paediatric cardiac surgery through comparison between a single-centre prospective case series and historical cases. Forty-seven children who were admitted to the ICU after cardiac surgery were enrolled ('post group'). Data for these children were compared with a similar cohort of children who were admitted before the implementation of the feeding protocol (n=62; 'pre group'). The incidence of complications including vomiting, necrotising enterocolitis and hypoglycaemia; the time until the initiation of EN; and the changes in calories provided were compared between the groups. The frequency of vomiting was significantly lower in the post group than in the pre group (36.2% versus 58.0%, P=0.038), and necrotising enterocolitis did not occur in either group. The time until the initiation of EN and the total calories provided did not differ significantly; however, in the post group the proportion of energy provided by parenteral nutrition was significantly smaller (P <0.001), and provided by EN was significantly larger (P=0.003), than in the pre group. The frequency of hypoglycaemia was similar in both groups. This study showed that our EN protocol resulted in adjustments to calories provided via EN versus parenteral nutrition after paediatric cardiac surgery, and reduced the frequency of vomiting.
Assuntos
Procedimentos Cirúrgicos Cardíacos , Nutrição Enteral/métodos , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Protocolos Clínicos , Enterocolite Necrosante/prevenção & controle , Feminino , Humanos , Hipoglicemia/prevenção & controle , Lactente , Masculino , Complicações Pós-Operatórias/prevenção & controle , Estudos Prospectivos , Vômito/prevenção & controleAssuntos
Manuseio das Vias Aéreas/métodos , Síndrome de Goldenhar/diagnóstico por imagem , Síndrome de Goldenhar/terapia , Obstrução das Vias Respiratórias/diagnóstico por imagem , Anestesia por Inalação , Humanos , Hipospadia/cirurgia , Imageamento Tridimensional , Lactente , Masculino , Cuidados Pré-Operatórios , Tomografia Computadorizada por Raios X , Traqueia/diagnóstico por imagemRESUMO
BACKGROUND: End-tidal P(CO(2)) (Pe'(CO(2))) is routinely used in the clinical assessment of the adequacy of ventilation because it provides an estimate of Pa(CO(2)). How well Pe'(CO(2)) reflects Pa(CO(2)) depends on the gradient between them, expressed as ΔPa-e'(CO(2)). The major determinant of ΔPa-e'(CO(2)) is alveolar dead space (Vd(alv)). The fraction of inspired O(2) (Fi(O(2))) is not thought to substantially affect ΔPa-e'(CO(2)) in anaesthetized patients. We hypothesized that a high Fi(O(2)) may indeed increase ΔPa-e'(CO(2)) by preferentially vasodilating well-perfused alveoli, resulting in the redistribution of blood flow to these alveoli from poorly perfused alveoli and an increase in Vd(alv). We therefore investigated the effects of changes in Fi(O(2)) on ΔPa-e'(CO(2)) and Vd(alv). METHODS: With Institutional Review Board approval and informed consent, we studied 20 ASA I-II supine patients undergoing elective lower abdominal surgery under combined general and epidural anaesthesia. At constant levels of ventilation, Fi(O(2)) levels of 0.21, 0.33, 0.5, 0.75, and 0.97 were applied in a random order and ΔPa-e'(CO(2)) and Vd(alv) were calculated. RESULTS: The ΔPa-e'(CO(2)) values were, in order of ascending Fi(O(2)), {mean [standard error of the mean (SEM)]} 0.13 (0.04), 0.28 (0.08), 0.29 (0.09), 0.44 (0.11), and 0.53 (0.09) kPa. The corresponding values of Vd(alv) were 25.5, 33.8, 35.8, 48.9, and 47.4 ml. Each successive hyperoxic level showed a significant increase in ΔPa-e'(CO(2)) except between the 0.33-0.5 and 0.75-0.97 Fi(O(2)) levels. CONCLUSIONS: These data demonstrate that ΔPa-e'(CO(2)), in anaesthetized patients depends on Fi(O(2)).
Assuntos
Anestesia Geral , Dióxido de Carbono/sangue , Oxigenoterapia , Abdome/cirurgia , Adulto , Idoso , Anestesia Epidural , Feminino , Humanos , Hiperóxia/sangue , Masculino , Pessoa de Meia-Idade , Monitorização Intraoperatória , Oxigênio/sangue , Alvéolos Pulmonares/irrigação sanguínea , Circulação Pulmonar , Respiração Artificial , Decúbito Dorsal/fisiologia , Volume de Ventilação Pulmonar/fisiologiaRESUMO
Glucocorticoids, the most downstream effectors of the hypothalamus-pituitary-adrenal axis, are one of main mediators of the stress reaction. Indeed, exposure to high levels of stress-triggered glucocorticoids is detrimental to brain development associated with abnormal behaviors in experimental animals and the risk of psychiatric disorders in humans. Despite the wealth of this knowledge, the cellular and molecular mechanisms underlying the detrimental effects of glucocorticoids on brain development remain unclear. Here, we show that excess glucocorticoids retard the radial migration of post-mitotic neurons during the development of the cerebral cortex, and identify an actin regulatory protein, caldesmon, as the glucocorticoids' main target. The upregulation of caldesmon expression is mediated by glucocorticoid receptor-dependent transcription of the CALD1 gene encoding caldesmon. This upregulated caldesmon negatively controls the function of myosin II, leading to changes in cell shape and migration. The depletion of caldesmon in vivo impairs radial migration. The overexpression of caldesmon also causes delayed radial migration during cortical development, mimicking the excessive glucocorticoid-induced retardation of radial migration. We conclude that an appropriate range of caldesmon expression is critical for radial migration, and that its overexpression induced by excess glucocorticoid retards radial migration during cortical development. Thus, this study provides a novel insight into the underlying mechanism of glucocorticoid-related neurodevelopmental disorders.
Assuntos
Movimento Celular/efeitos dos fármacos , Dexametasona/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucocorticoides/farmacologia , Neurônios/efeitos dos fármacos , Fatores Etários , Animais , Animais Recém-Nascidos , Bromodesoxiuridina/metabolismo , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/patologia , Proteínas do Domínio Duplacortina , Embrião de Mamíferos , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Neuropeptídeos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/patologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Ratos , Fatores de Tempo , Transfecção/métodos , Tubulina (Proteína)/metabolismoRESUMO
The podosome and invadopodium are dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin-binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmon's involvement in podosome/invadopodium-mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Extensões da Superfície Celular/metabolismo , Invasividade Neoplásica , Neoplasias Experimentais/metabolismo , Animais , Neoplasias da Mama/metabolismo , Proteínas de Ligação a Calmodulina/genética , Carcinoma/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Transformação Celular Viral , Células Clonais , Neoplasias do Colo/metabolismo , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Técnica Direta de Fluorescência para Anticorpo , Humanos , Ligação Proteica , Ratos , Vírus do Sarcoma de Rous/metabolismo , TransfecçãoAssuntos
Dispositivos de Compressão Pneumática Intermitente/efeitos adversos , Neuroma Acústico/cirurgia , Traumatismos dos Nervos Periféricos , Meias de Compressão/efeitos adversos , Craniotomia/métodos , Edema/etiologia , Falha de Equipamento , Feminino , Humanos , Hipestesia/etiologia , Pessoa de Meia-Idade , Debilidade Muscular/etiologia , Dor/etiologia , Púrpura/etiologia , Fatores de TempoRESUMO
A monitoring system for tracking the electromyogram (EMG) of the vocal cords with wire electrodes embedded in an endotracheal tube was designed to identify the recurrent laryngeal nerve during thyroidectomy. Our recent experience in two cases suggests that vagal nerve activity can be correctly detected by recording of the EMG of the vocal cords using a special endotracheal tube embedded with wire electrodes.
Assuntos
Neoplasias do Tronco Encefálico/cirurgia , Eletrodos , Monitorização Intraoperatória , Nervo Vago/fisiologia , Adulto , Criança , Craniotomia , Eletromiografia , Feminino , Humanos , Resultado do Tratamento , Nervo Vago/fisiopatologiaAssuntos
Obstrução das Vias Respiratórias/terapia , Intubação Intratraqueal/instrumentação , Complicações Pós-Operatórias/terapia , Traqueia/cirurgia , Adulto , Obstrução das Vias Respiratórias/etiologia , Anastomose Cirúrgica , Broncoscopia , Feminino , Humanos , Intubação Intratraqueal/métodos , Edema Laríngeo/etiologia , Edema Laríngeo/terapiaRESUMO
Simple manual ventilation facilitates removal of intra-alveolar accumulations. High-frequency jet ventilation can be performed through a narrow lumen like that of a fibreoptic bronchoscope. Accordingly, we expected that high frequency jet ventilation through a fibreoptic bronchoscope channel would facilitate lung lavage, and we developed a new bronchoalveolar lavage system, in which high-frequency jet ventilation through the channel of a fibreoptic bronchoscope was combined with conventional bronchoalveolar lavage. We describe a case in which this new lavage system, unlike conventional bronchoalveolar lavage, successfully removed the lipid material in the alveoli associated with pulmonary alveolar proteinosis.
Assuntos
Lavagem Broncoalveolar/instrumentação , Tecnologia de Fibra Óptica , Ventilação em Jatos de Alta Frequência/instrumentação , Proteinose Alveolar Pulmonar/terapia , Adulto , Lavagem Broncoalveolar/métodos , Desenho de Equipamento , Ventilação em Jatos de Alta Frequência/métodos , Humanos , Masculino , Tomografia Computadorizada por Raios XRESUMO
PSD-Zip45 (Homer 1c) and PSD-95 are postsynaptic density (PSD) proteins containing distinct protein-interacting motifs. Green fluorescent protein (GFP)-tagged PSD-Zip45 and PSD-95 molecules were targeted to the PSD in hippocampal neurons. We analyzed dynamic behavior of these GFP-tagged PSD proteins by using time-lapse confocal microscopy. In contrast to the less dynamic properties of PSD-95, PSD-Zip45 showed rapid redistribution and a higher steady-state turnover rate. Differential stimulation protocols were found to alter the direction of PSD-Zip45 assembly-disassembly. Transient increases in intracellular Ca(2+) by voltage-dependent Ca(2+) channel activation induced PSD-Zip45 clustering. In contrast, NMDA receptor-dependent Ca(2+) influx resulted in the disassembly of PSD-Zip45 clusters. Thus, neuronal activity differentially redistributes a specific subset of PSD proteins, which are important for localization of both surface receptors and intracellular signaling complexes.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Animais , Cádmio/farmacologia , Cálcio/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Citoesqueleto/metabolismo , Proteína 4 Homóloga a Disks-Large , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Fluorescência Verde , Guanilato Quinases , Hipocampo , Proteínas de Arcabouço Homer , Líquido Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/genética , Substâncias Macromoleculares , Proteínas de Membrana , Camundongos , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neuropeptídeos/genética , Transporte Proteico/fisiologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Estimulação QuímicaRESUMO
Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens, that increase plasma membrane water permeability in secretory and absorptive cells. In astrocytes of the central nervous system (CNS), using the reverse transcription-polymerase chain reaction (RT-PCR), we previously detected AQP3, 5 and 8 mRNAs in addition to the reported AQP4 and 9. However the mechanisms regulating the expression of these AQPs are not known. In this study, we investigated the effects of a protein kinase C (PKC) activator on the expression of AQP4, 5 and 9 in cultured rat astrocytes. Treatment of the cells with TPA caused decreases in AQP4 and 9 mRNAs and proteins in time- and concentration-dependent manners. The TPA-induced decreases in AQP4 and 9 mRNAs were inhibited by PKC inhibitors. Moreover, prolonged treatment of the cells with TPA eliminated the subsequent decreases in AQP4 and 9 mRNAs caused by TPA. Pretreatment of cells with an inhibitor of protein synthesis, cycloheximide, did not inhibit the decreases in AQP4 and 9 mRNAs induced by TPA. These results suggest that signal transduction via PKC may play important roles in regulating the expression of AQP4 and 9.
Assuntos
Aquaporinas/metabolismo , Astrócitos/metabolismo , Proteínas de Membrana , Proteína Quinase C/fisiologia , Animais , Aquaporina 4 , Aquaporina 5 , Aquaporinas/genética , Western Blotting , Células Cultivadas , Regulação da Expressão Gênica , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , RatosRESUMO
The phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state to the dedifferentiated one is critically involved in the development and progression of atherosclerosis. Although many cytokines and growth factors have been reported as atherogenic factors, the critical pathogens for inducing atherosclerosis remain unknown, largely because proper examining systems of them have not been developed. We recently established primary culture systems for visceral SMCs and VSMCs in which both SMCs, when cultured on laminin with insulin-like growth factor-I, show a differentiated phenotype, as indicated by a spindle-like shape, ligand-induced contractility, and a high level of SMC differentiation marker gene expression. In this study, we searched for critical dedifferentiation factors for these SMCs using our culture system. We found that polar lipids extracted from human serum markedly induced VSMC dedifferentiation, and this activity was solely present in the lysophosphatidic acid (LPA) fraction. Among several LPA species detected in human serum lipids, unsaturated LPAs were identified as major contributors to the induction of VSMC dedifferentiation. Signaling and phenotype analyses revealed that unsaturated LPA-induced VSMC dedifferentiation is mediated through the coordinated activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. Thus, this report demonstrates the first finding that unsaturated LPAs, but not saturated LPAs, specifically induce VSMC phenotypic modulation, suggesting that these molecules could function as atherogenic factors.
Assuntos
Lisofosfolipídeos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Receptores Acoplados a Proteínas G , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia em Camada Fina , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Substâncias de Crescimento/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Laminina/farmacologia , Lisofosfolipídeos/sangue , Lisofosfolipídeos/isolamento & purificação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Fenótipo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/biossíntese , Ratos , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Ácidos Lisofosfatídicos , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [Cell 39 (1984) 49], that increase plasma membrane water permeability in secretory and absorptive cells. In the central nervous system (CNS), we detected the transcripts of AQP3, 5 and 8 in addition to the previously reported transcripts of AQP4 and 9 in astrocytes, of AQP3, 5 and 8 in neurons, of AQP8 in oligodendrocytes, and none of them in microglia using RNase protection assay and the reverse transcription-polymerase chain reaction (RT-PCR). Hypoxia evoked a marked decrease in the expression levels of AQP4, 5 and 9, but not of AQP3 and 8 mRNAs, and in astrocytes in vitro subsequent reoxygenation elicited the restoration of the expression of AQP4 and 9 to their basal levels. Interestingly, AQP5 showed a transient up-regulation (about 3-fold) and subsequent down-regulation of its expression within 20 h of reoxygenation after hypoxia. The changes in the profiles of AQP expression during hypoxia and reoxygenation were also observed by Western blot analysis. These results suggest that AQP5 may be one of the candidates for inducing the intracranial edema in the CNS after ischemia injury.
Assuntos
Aquaporinas/biossíntese , Astrócitos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas do Tecido Nervoso/biossíntese , Oxigênio/farmacologia , RNA Mensageiro/biossíntese , Animais , Aquaporinas/genética , Astrócitos/efeitos dos fármacos , Western Blotting , Hipóxia Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Córtex Cerebral/citologia , Perfilação da Expressão Gênica , Infarto da Artéria Cerebral Média/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteínas do Tecido Nervoso/genética , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Tropomyosin (TM) is a regulatory protein of actomyosin system. Muscle type-specific expression of TM isoforms is generated from different genes and by alternative splicing. beta-TM isoforms in chicken skeletal and smooth muscles are encoded by a single gene and transcribed from the same promoter. We previously reported a smooth muscle cell (SMC) phenotype-dependent change in beta-TM expression (Kashiwada, K., Nishida, W., Hayashi, K., Ozawa, K., Yamanaka, Y., Saga, H., Yamashita, T., Tohyama, M., Shimada, S., Sato, K., and Sobue, K. (1997) J. Biol. Chem. 272, 15396-15404), and identified beta-TM as an SMC-differentiation marker. Here, we characterized the transcriptional machinery of the beta-TM gene in SMCs. Promoter and gel mobility shift analyses revealed an obligatory role for serum response factor and its interaction with the CArG box sequence in the SMC-specific transcription of the beta-TM gene in differentiated SMCs. We further isolated a novel homologue of the Barx homeoprotein family, Barx1b, from chicken gizzard. Barx1b was exclusively localized to SMCs of the upper digestive organs and their attached arteries and to craniofacial structures. Serum response factor and Barx1b bound each other directly, coordinately transactivated the beta-TM gene in differentiated SMCs and heterologous cells, and formed a ternary complex with a CArG probe. Taken together, these results suggest that SRF and Barx1b are coordinately involved in the SMC-specific transcription of the beta-TM gene in the upper digestive organs and their attached arteries.
Assuntos
Proteínas Aviárias , Proteínas de Ligação a DNA/genética , Proteínas de Homeodomínio/genética , Músculo Liso/fisiologia , Proteínas Nucleares/genética , Tropomiosina/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Proteínas de Homeodomínio/metabolismo , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Fator de Resposta Sérica , Ativação TranscricionalRESUMO
Aquaporin-4 (AQP4), a mercury-insensitive water channel protein, is abundant in the central nervous system and is localized in astrocytes and ependymal cells. AQP4 is speculated to maintain the homeostasis of intracellular and extracellular water in the brain, but little is known about the mechanism of induction of its expression. To investigate the expressional regulation of AQP4, we analyzed changes in its expression during chemically induced differentiation of embryonal carcinoma cells (P19) to neuronal and astrocytic cells, and during the cell cycle of glioma cells. After exposure to retinoic acid for 4 days AQP4 mRNA expression started at the initiation of astrocytic differentiation of P19 cells at 6 days, and increased markedly by 21 days. AQP4 expression was parallel to that of GFAP, a marker intermediate filament of astrocytes. In glioma cell lines, AQP4 mRNA was not detected in the growing phase, but was induced when the cell cycle was arrested at G0/G1 by transient expression of p21. Although quiescent astrocytes in the G0/G1-phase cultured under the serum-free condition exhibited a high expression of AQP4, serum supplement moved them to the S-phase and markedly decreased the AQP expression. These results suggest that AQP4 expression may be induced not only at the initiation of astrocytic differentiation of neural stem cells, but also at the G0/G1-phase during the cell cycle of astrocytes.
Assuntos
Aquaporinas/genética , Astrócitos/fisiologia , Animais , Antineoplásicos/farmacologia , Aquaporina 4 , Astrócitos/citologia , Astrocitoma , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Córtex Cerebral/citologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Células-Tronco de Carcinoma Embrionário , Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/fisiologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Transfecção , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
The phenotypic modulation of smooth muscle cells (SMCs) is closely associated with the development and progression of various SMC diseases. We investigated the molecular mechanism of phenotypic modulation triggered by EGF family ligands using a primary culture system of differentiated SMCs. Among four EGF-receptor (EGFR) family members, the EGFR was solely activated by EGF, heparin-binding EGF (HB-EGF), transforming growth factor alpha (TGF alpha), epiregulin (ER), and betacellulin (BTC), resulting in induction of phenotypic modulation of SMCs. This effect was mediated through the coordinated activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) pathways. These results suggest that EGF family ligand- and EGFR-triggered signaling pathways are critically involved in the phenotypic modulation of SMCs.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Músculo Liso/efeitos dos fármacos , Animais , Betacelulina , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação a Calmodulina/genética , Células Cultivadas , Embrião de Galinha , Ativação Enzimática/efeitos dos fármacos , Epirregulina , Receptores ErbB/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Proteínas dos Microfilamentos , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso/citologia , Músculo Liso/metabolismo , Fenótipo , Fator de Crescimento Transformador alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno , CalponinasRESUMO
Neurofibrillary tangles associated with Alzheimer's disease are composed mainly of paired helical filaments that are formed by the aggregation of abnormally phosphorylated microtubule-associated protein tau. 14-3-3, a highly conserved protein family that exists as seven isoforms and regulates diverse cellular processes is present in neurofibrillary tangles (Layfield, R., Fergusson, J., Aitken, A., Lowe, J., Landon, M., Mayer, R. J. (1996) Neurosci. Lett. 209, 57-60). The role of 14-3-3 in Alzheimer's disease pathogenesis is not known. In this study, we found that the 14-3-3zeta isoform is associated with tau in brain extract and profoundly stimulates cAMP-dependent protein kinase catalyzed in vitro phosphorylation on Ser(262)/Ser(356) located within the microtubule-binding region of tau. 14-3-3zeta binds to both phosphorylated and nonphosphorylated tau, and the binding site is located within the microtubule-binding region of tau. From brain extract, 14-3-3zeta co-purifies with microtubules, and tubulin blocks 14-3-3zeta-tau binding. Among four 14-3-3 isoforms tested, beta and zeta but not gamma and epsilon associate with tau. Our data suggest that 14-3-3zeta is a tau protein effector and may be involved in the abnormal tau phosphorylation occurring during Alzheimer's disease ontogeny.
Assuntos
Proteínas/fisiologia , Tirosina 3-Mono-Oxigenase , Proteínas tau/metabolismo , Proteínas 14-3-3 , Doença de Alzheimer , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Encéfalo/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Glutationa Transferase/metabolismo , Humanos , Immunoblotting , Microtúbulos/metabolismo , Mutagênese , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Isoformas de Proteínas , Proteínas/química , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Serina/química , Tubulina (Proteína)/farmacologia , Proteínas tau/químicaRESUMO
Neuronal Cdc2-like protein kinase (NCLK), a approximately 58-kDa heterodimer, was isolated from neuronal microtubules (Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T. and Imahori, K. (1992) J. Biol. Chem. 267, 10897-10901). The biochemical nature of NCLK-microtubule association is not known. In this study we found that NCLK is released from microtubules upon microtubule disassembly as a 450-kDa species. The 450-kDa species is an NCLK.tau complex, and NCLK-bound tau is in a nonphosphorylated state. Tau phosphorylation causes NCLK.tau complex dissociation, and phosphorylated tau does not bind to NCLK. In vitro, the Cdk5 subunit of NCLK binds to the microtubule-binding region of tau and NCLK associates with microtubules only in the presence of tau. Our data indicate that in brain extract NCLK is complexed with tau in a tau phosphorylation-dependent manner and that tau anchors NCLK to microtubules. Recently NCLK has been suggested to be aberrantly activated and to hyperphosphorylate tau in Alzheimer's disease brain (Patrick, G. N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P, and Tsai, L.-H. (1999) Nature 402, 615-622). Our findings may explain why in Alzheimer's disease NCLK specifically hyperphosphorylates tau, although this kinase has a number of protein substrates in the brain.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Neurônios/enzimologia , Animais , Sequência de Bases , Domínio Catalítico , Bovinos , Quinase 5 Dependente de Ciclina , Primers do DNA , Microtúbulos/metabolismo , Fosforilação , Ligação ProteicaRESUMO
We used mRNA subtraction of differentiated and dedifferentiated smooth muscle cells (SMCs) to reveal the molecular mechanisms underlying the phenotypic modulation of SMCs. With this approach, we found that a 10 kb mRNA encoding a homotypic cell adhesion molecule, cadherin 6B, was strongly expressed in differentiated vascular and visceral SMCs, but not in the dedifferentiated SMCs derived from them. In vivo, cadherin 6B was expressed in vascular and visceral SMCs, in addition to brain, spinal cord, retina and kidney, at a late stage of chicken embryonic development. These results suggest that cadherin 6B is a novel molecular marker for vascular and visceral SMC phenotypes and is involved in the late differentiation of SMCs.