RESUMO
A 1.5-year-old, female-spayed mix-breed dog was presented with recurrent episodes of shaking and excessive panting attributed to drug-refractory ventricular arrhythmia (VA) characterized predominantly by incessant periods of ventricular bigeminy. The VA had a narrow QRS morphology, suggestive of an origin near the His bundle or fascicular system. Diagnostic evaluation found no structural heart disease or underlying etiology. Three-dimensional electroanatomic mapping and radiofrequency catheter ablation were pursued. Voltage mapping demonstrated normal bi-ventricular voltage (≥1.5 mV) without any fractionated or multicomponent electrograms, indicating the absence of ventricular myocardial scar. Pace mapping identified an endocardial origin of the VA at the basal anterior septum of the left ventricle, distal to the His bundle and near the left bundle branch. Two ablation lesions were delivered to this site, and a left bundle branch block was temporarily induced. The dog recovered uneventfully. One month later, the owners reported a remarkable improvement in clinical signs, and follow-up 48-h Holter monitor found complete resolution of VA.
Assuntos
Ablação por Cateter , Doenças do Cão , Taquicardia Ventricular , Complexos Ventriculares Prematuros , Animais , Bloqueio de Ramo/veterinária , Ablação por Cateter/veterinária , Doenças do Cão/diagnóstico por imagem , Doenças do Cão/cirurgia , Cães , Eletrocardiografia , Feminino , Sistema de Condução Cardíaco , Taquicardia Ventricular/cirurgia , Taquicardia Ventricular/veterinária , Complexos Ventriculares Prematuros/veterináriaRESUMO
BACKGROUND: Cardiac structure and function in dogs are commonly assessed using echocardiography. A variety of linear, area, and flow-based measurements can be used to calculate left ventricular (LV) total stroke volume (TSV) and forward stroke volume (FSV), but the reproducibility of many of these measurements has not been fully studied. We hypothesized that survey of echocardiographic variables would identify those with high reproducibility and inform future investigation of different methods to measure LV TSV and FSV. METHODS: The reproducibility of 25 geometric and flow-based echocardiographic measurements was prospectively evaluated in 23 healthy dogs by two experienced observers. Reproducibility (i.e., interobserver agreement) was described using intraclass correlation coefficients. The reproducibility of various methods to calculate LV TSV and FSV was explored. RESULTS: Reproducibility was generally good to excellent. Variables of LV width, length, and area and aortic and sinotubular junction diameter and velocity time integral were among measures with the highest reproducibility. Measurements of mitral annular diameter and mitral inflow velocity time integral possessed lower reproducibility. Calculation of LV TSV using measurements involved in the cube and bullet formulas demonstrated higher reproducibility than the Simpson's method of disks or mitral inflow methods. Calculation of LV FSV using LV outflow tract and aortic diameters from the right parasternal view generally demonstrated higher reproducibility compared with the left-sided view. CONCLUSIONS: The reproducibility of many simple geometric and flow-based echocardiographic measurements is high. Comparison of the reliability of different measurement informs future investigation of echocardiographic methods to determine LV TSV and FSV in dogs.
Assuntos
Cães/fisiologia , Ventrículos do Coração/diagnóstico por imagem , Volume Sistólico , Função Ventricular Esquerda/fisiologia , Animais , Ecocardiografia/veterinária , Feminino , Masculino , Valores de Referência , Reprodutibilidade dos TestesRESUMO
Glucocorticoid-induced tumour necrosis factor receptor (TNFR)-related protein (GITR) is one of the T cell co-stimulatory molecules and is associated with the pathogenesis of a number of autoimmune diseases. We investigated the expression patterns of GITR in human arthritic synovium and the role of GITR in the pathogenesis of rheumatoid arthritis (RA). Immunohistochemical analyses revealed the expression of GITR and its cognate ligand, GITRL, in macrophages in RA, but not in osteoarthritis (OA), synovium. To investigate the role of GITR in macrophage functions, primary macrophages from RA patients and a human macrophage cell line, THP-1, were analysed. Stimulation of the macrophages with anti-GITR monoclonal antibody induced up-regulation of intercellular adhesion molecule (ICAM)-1 and subsequent aggregation/adhesion, which was enhanced by the presence of extracellular matrix proteins and blocked by anti-ICAM-1 monoclonal antibody. The validity of these in vitro observations was confirmed by immunohistochemical analyses of RA synovium, which showed strong expression of ICAM-1 in GITR-positive macrophages. Additionally, GITR stimulation induced expression of proinflammatory cytokines/chemokines and matrix metalloproteinase-9 in synovial macrophages. These data indicate that GITR, expressed on macrophages in human RA synovium, may enhance inflammatory activation of macrophages by promoting cytokine gene expression and adhesion between cells and to extracellular matrix in RA synovium.
Assuntos
Artrite Reumatoide/imunologia , Citocinas/metabolismo , Ativação de Macrófagos/imunologia , Receptores de Fator de Crescimento Neural/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Anticorpos Monoclonais/imunologia , Adesão Celular/imunologia , Agregação Celular/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Macrófagos/imunologia , Metaloproteinase 9 da Matriz/metabolismo , Osteoartrite/imunologia , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Membrana Sinovial/imunologia , Fatores de Necrose Tumoral/imunologia , Fatores de Necrose Tumoral/metabolismo , Regulação para Cima/imunologiaRESUMO
PURPOSE: To investigate the regulation of NADPH-producing isocitrate dehydrogenase (ICDH) in cytosol (IDPc) and mitochondria (IDPm) upon gamma-ray irradiation, and the roles of IDPc and IDPm in the protection against cellular damage induced by gamma-ray irradiation. MATERIALS AND METHODS: Changes of IDPc and IDPm proteins upon gamma-ray irradiation to NIH3T3 cells were analysed by immunoblotting. To increase or decrease the expression of IDPc or IDPm, NIH3T3 cells were stably transfected with mouse IDPc or IDPm cDNA in either the sense or the antisense direction. The transfected cells with either increased or decreased IDPc or IDPm were exposed to gamma-rays, and the levels of reactive oxygen species generation, protein oxidation and lipid peroxidation were measured. RESULTS: Both IDPc and IDPm activities were induced by gamma-ray in NIH3T3 cells. Cells with decreased expression of IDPc or IDPm had elevated reactive oxygen species generation, lipid peroxidation and protein oxidation. Conversely, overproduction of IDPc or IDPm protein partially protected the cells from oxidative damage induced by gamma-ray irradiation. CONCLUSIONS: The protective role of IDPc and IDPm against gamma-ray-induced cellular damage can be attributed to elevated NADPH, reducing equivalents needed for recycling reduced glutathione in the cytosol and mitochondria. Thus, a primary biological function of the ICDHs may be production of NADPH, which is a prerequisite for some cellular defence systems against oxidative damage.
Assuntos
Sobrevivência Celular/efeitos da radiação , Raios gama , Isocitrato Desidrogenase/metabolismo , Estresse Oxidativo/fisiologia , Estresse Oxidativo/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Animais , Relação Dose-Resposta à Radiação , Peroxidação de Lipídeos/efeitos da radiação , Camundongos , Células NIH 3T3 , Oxirredução/efeitos da radiação , Doses de RadiaçãoRESUMO
As a means toward understanding the neural bases of schizophrenic thought disturbance, we examined brain activation patterns in response to semantically and superficially encoded words in patients with schizophrenia. Nine male schizophrenic and 9 male control subjects were tested in a visual levels of processing (LOP) task first outside the magnet and then during the fMRI scanning procedures (using a different set of words). During the experiments visual words were presented under two conditions. Under the deep, semantic encoding condition, subjects made semantic judgments as to whether the words were abstract or concrete. Under the shallow, nonsemantic encoding condition, subjects made perceptual judgments of the font size (uppercase/lowercase) of the presented words. After performance of the behavioral task, a recognition test was used to assess the depth of processing effect, defined as better performance for semantically encoded words than for perceptually encoded words. For the scanned version only, the words for both conditions were repeated in order to assess repetition-priming effects. Reaction times were assessed in both testing scenarios. Both groups showed the expected depth of processing effect for recognition, and control subjects showed the expected increased activation of the left inferior prefrontal cortex (LIPC) under semantic encoding relative to perceptual encoding conditions as well as repetition priming for semantic conditions only. In contrast, schizophrenics showed similar patterns of fMRI activation regardless of condition. Most striking in relation to controls, patients showed decreased LIFC activation concurrent with increased left superior temporal gyrus activation for semantic encoding versus shallow encoding. Furthermore, schizophrenia subjects did not show the repetition priming effect, either behaviorally or as a decrease in LIPC activity. In patients with schizophrenia, LIFC underactivation and left superior temporal gyrus overactivation for semantically encoded words may reflect a disease-related disruption of a distributed frontal temporal network that is engaged in the representation and processing of meaning of words, text, and discourse and which may underlie schizophrenic thought disturbance.
Assuntos
Cognição/fisiologia , Imageamento por Ressonância Magnética , Leitura , Esquizofrenia/fisiopatologia , Psicologia do Esquizofrênico , Adulto , Comportamento/fisiologia , Humanos , Interpretação de Imagem Assistida por Computador , Masculino , Córtex Pré-Frontal/fisiopatologia , Lobo Temporal/fisiopatologiaRESUMO
We screened 139 herbal spices in search of the acetylcholinesterase (AChE) inhibitor from natural resources. AChE inhibitors, which enhance cholinergic transmission by reducing the enzymatic degradation of acetylcholine, are the only source of compound currently approved for the treatment of Alzheimer's Disease (AD). Among these herbs, edible plants and spices, the ethanol extract from Origanum majorana L. showed the highest inhibitory effect on AChE in vitro. By sequential fractionation of Origanum majorana L. the active component was finally identified as ursolic acid (3 beta-Hydroxyurs-12-en-28-oic acid). The ursolic acid of Origanum majorana L. inhibited AChE activity in a dose-dependent and competitive/non-competitive type. The Ki value (representing the affinity of the enzyme and inhibitor) of Origanum majorana L. ursolic acid was 6 pM, and that of tacrine was 0.4 nM. The concentration required for 50% enzyme inhibition of the active component (IC50 value) was 7.5 nM, and that of tacrine was 1 nM. This study demonstrated that the ursolic acid of Origanum majorana L. appeared to be a potent AChE inhibitor in Alzheimer's Disease.
Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Lamiaceae/química , Triterpenos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Animais , Inibidores da Colinesterase/isolamento & purificação , Inibidores da Colinesterase/uso terapêutico , Cromatografia , Humanos , Estrutura Molecular , Células PC12 , Fitoterapia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Plantas Medicinais , Ratos , Tacrina/farmacologia , Triterpenos/isolamento & purificação , Triterpenos/uso terapêutico , Ácido UrsólicoRESUMO
Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.
Assuntos
Isocitrato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Anticorpos , Apoptose/fisiologia , Bovinos , Sobrevivência Celular , Clonagem Molecular , Fragmentação do DNA , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Homeostase , Humanos , Isocitrato Desidrogenase/química , Cinética , Camundongos , Dados de Sequência Molecular , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Espécies Reativas de Oxigênio/fisiologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/metabolismo , SuínosRESUMO
Slit is expressed in the midline of the central nervous system both in vertebrates and invertebrates. In Drosophila, it is the midline repellent acting as a ligand for the Roundabout (Robo) protein, the repulsive receptor which is expressed on the growth cones of the commissural neurons. We have isolated cDNA fragments of the zebrafish slit2 and slit3 homologues and found that both genes start to be expressed by the midgastrula stage well before the axonogenesis begins in the nervous system, both in the axial mesoderm, and slit2 in the anterior margin of the neural plate and slit3 in the polster at the anterior end of the prechordal mesoderm. Later, expression of slit2 mRNA is detected mainly in midline structures such as the floor plate cells and the hypochord, and in the anterior margins of the neural plates in the zebrafish embryo, while slit3 expression is observed in the anterior margin of the prechordal plate, the floorplate cells in the hindbrain, and the motor neurons both in the hindbrain and the spinal cord. To study the role of Slit in early embryos, we overexpressed Slit2 in the whole embryos either by injection of its mRNA into one-cell stage embryos or by heat-shock treatment of the transgenic embryos which carries the slit2 gene under control of the heat-shock promoter. Overexpression of Slit2 in such ways impaired the convergent extension movement of the mesoderm and the rostral migration of the cells in the dorsal diencephalon and resulted in cyclopia. Our results shed light on a novel aspect of Slit function as a regulatory factor of mesodermal cell movement during gastrulation.
Assuntos
Glicoproteínas , Proteínas do Tecido Nervoso/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Padronização Corporal/genética , Movimento Celular/genética , Clonagem Molecular , Primers do DNA/genética , Anormalidades do Olho/embriologia , Anormalidades do Olho/genética , Gástrula/citologia , Gástrula/metabolismo , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular , Mesoderma/citologia , Mesoderma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/metabolismoRESUMO
Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through supply of NADPH for antioxidant systems. When exposed to various reactive oxygen species such as hydrogen peroxide, singlet oxygen generated by photoactivated dye, superoxide anion, and hydroxyl radical produced by metal-catalyzed Fenton reactions, ICDH was susceptible to oxidative modification and damage, which was indicated by the loss of activity, fragmentation of the peptide as well as by the formation of carbonyl groups. Oxidative damage to ICDH was inhibited by antioxidant enzymes, free radical scavengers, and spin-trapping agents. The structural alterations of modified enzymes were indicated by the increase in thermal instability and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonic acid (ANSA). The reactive oxygen species-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.
Assuntos
Peróxido de Hidrogênio/farmacologia , Radical Hidroxila/farmacologia , Isocitrato Desidrogenase/antagonistas & inibidores , Oxigênio Singlete/farmacologia , Naftalenossulfonato de Anilina/química , Animais , Antioxidantes/farmacologia , Azidas/farmacologia , Catalase/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Corantes Fluorescentes/química , Sequestradores de Radicais Livres/farmacologia , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/metabolismo , Manitol/farmacologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína , Espécies Reativas de Oxigênio/farmacologia , Superóxido Dismutase/farmacologia , Suínos , TemperaturaRESUMO
HuC encodes an RNA binding protein homologous to Drosophila elav that serves as an excellent early marker for differentiating neurons. We have characterized the promoter of the zebrafish HuC gene by examining the ability of 5'-upstream fragments to drive expression of green fluorescent protein (GFP) in live embryos. We determined that 2.8 kb of the 5'-flanking sequence is sufficient to restrict GFP gene expression to neurons. The core promoter spans 251 base pairs and contains a CCAAT box and one SP1 sequence but no TATA box is present near the transcription start site. A putative MyT1 binding site and at least 17 E-box sequences are necessary to maintain the neuronal specificity of HuC expression. Interestingly, sequential removal of the putative MyT1 binding site and 14 distal E boxes does not appear to abolish neuronal expression; rather, it leads to a progressive expansion of GFP expression into muscle cells. Further removal of the three proximal E boxes eliminates neuronal and muscle specificity of GFP expression and leads to ubiquitous expression of GFP in the whole body. Identification of key components of the HuC promoter has led to the establishment of a stable zebrafish transgenic line (HuC-GFP) in which GFP is expressed specifically in neurons. We crossed mind bomb (mib) fish with this line to visualize their neurogenic phenotype in live mib(-/-) mutant embryos. This cross illustrates how HuC-GFP fish could be used in the future to identify and analyze zebrafish mutants with an aberrant pattern of early neurons.
Assuntos
Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/genética , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação/genética , Diferenciação Celular , DNA/genética , Primers do DNA/genética , Proteínas ELAV , Proteína Semelhante a ELAV 3 , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Neurônios/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ribonucleoproteínas/genética , Peixe-Zebra/metabolismoRESUMO
Coatomer is a major component of COPI vesicles and consists of seven subunits. The gamma-COP subunit of the coatomer is believed to mediate the binding to the cytoplasmic dilysine motifs of membrane proteins. We characterized cDNAs for Copg genes encoding gamma-COP from mouse, zebrafish, Drosophila melanogaster and Bombyx mori. Two copies of Copg genes are present in vertebrates and in B. mori. Phylogenetic analysis revealed that two paralogous genes had been derived from a single ancestral gene by duplication independently in vertebrates and in B. mori. Mouse Copg1 showed ubiquitous expression with the highest level in testis. Zebrafish copg2 was biallelically expressed in hybrid larvae in contrast to its mammalian ortholog expressed in a parent-of-origin-specific manner. A phylogenetic analysis with partial plant cDNA sequences suggested that copg gene was also duplicated in the grass family (Poaceae).
Assuntos
Proteínas de Transporte/genética , Proteína Coatomer , Evolução Molecular , Duplicação Gênica , Filogenia , Sequência de Aminoácidos , Animais , Bombyx/genética , Proteínas de Transporte/química , Drosophila melanogaster/genética , Humanos , Insetos , Masculino , Camundongos , Dados de Sequência Molecular , Plantas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Testículo/metabolismo , Vertebrados , Peixe-Zebra/genéticaRESUMO
During limb development, epithelial cells in the apical ectodermal ridge keep the underlying mesenchymal cells in a proliferative state preventing differentiation by secreting signaling molecules such as epidermal growth factor (EGF). We investigated the molecular mechanism of the EGF effect on the regulation of micromass culture-induced chondrogenesis of chick limb bud mesenchymal cells as a model system. We found that expression and tyrosine phosphorylation of the EGF receptor was increased transiently during chondrogenesis. Exogenous EGF inhibited chondrogenic differentiation of mesenchymal cells, and this effect was reversed by the EGF receptor inhibitor AG1478. EGF treatment also inhibited the expression and activation of protein kinase C-alpha, whereas it activated Erk-1 and inhibited p38 mitogen-activated protein kinase, all of which appeared to be involved in the EGF-induced inhibition of chondrogenesis. Stimulation of the EGF receptor blocked precartilage condensation and altered the expression of cell adhesion molecules such as N-cadherin and integrins alpha(5) and beta(1). All these EGF effects were reversible by AG1478. The data indicate that EGF negatively regulate chondrogenesis of chick limb bud mesenchymal cells by inhibiting precartilage condensation and by modulating signaling pathways including those of protein kinase C-alpha, Erk-1, and p38 mitogen-activated protein kinase.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Condrócitos/fisiologia , Fator de Crescimento Epidérmico/fisiologia , Isoenzimas/metabolismo , Mesoderma/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Animais , Adesão Celular , Divisão Celular , Células Cultivadas , Embrião de Galinha , Ativação Enzimática , Proteína Quinase 3 Ativada por Mitógeno , Proteína Quinase C-alfa , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The present communication reports the isolation and characterization of three new zebrafish elav/Hu (Kim, C.-H., Ueshima, E., Muraoka, O., Tanaka, H., Yeo, S.-Y., Huh, T.-L. and Miki, N., Zebrafish elav/HuC homologue as a very early neuronal marker. Neurosci. Lett., 216 (1996) 109-112) homologues, HuA, HuD and HuG. While HuA and HuG showed weak and ubiquitous expressions, HuD, as well as HuC, were specifically expressed in the neuronal cells. The first expression of HuD was detectable of the 10-somite stage, that is, several hours later than HuC. After 24 h of embryonic development, although HuD and HuC expressions overlapped overall, the cells expressing HuD were restricted to subsets of the HuC-positive neuronal cells in the brain and spinal cord. These differentially regulated spatial and temporal expression patterns implied distinct roles for HuC and HuD in neuronal determination and neuronal differentiation, respectively.
Assuntos
Antígenos de Superfície/genética , Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Proteínas de Ligação a RNA/genética , Medula Espinal/embriologia , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Antígenos de Superfície/química , Proteínas ELAV , Proteína Semelhante a ELAV 3 , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas de Ligação a RNA/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
To understand the interactions and functional role of each of the three mitochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH) subunits (alpha, beta, and gamma), we have characterized human cDNAs encoding two beta isoforms (beta(1) and beta(2)) and the gamma subunit. Analysis of deduced amino acid sequences revealed that beta(1) and beta(2) encode 349 and 354 amino acids, respectively, and the two isoforms only differ in the most carboxyl 28 amino acids. The gamma cDNA encodes 354 amino acids and is almost identical to monkey IDHgamma. Northern analyses revealed that the smaller beta(2) transcript (1.3 kilobases) is primarily expressed in heart and skeletal muscle, whereas the larger beta(1) mRNA (1.6 kilobases) is prevalent in nonmuscle tissues. Sequence analysis of the IDHbeta gene indicates that the difference in the C-terminal 28 amino acids between beta(1) and beta(2) proteins results from alternative splicing of a single transcript. Among the various combinations of human IDH subunits co-expressed in bacteria, alphabetagamma, alphabeta, and alphagamma combinations exhibited significant amounts of IDH activity, whereas subunits produced alone and betagamma showed no detectable activity. These data suggest that the alpha is the catalytic subunit and that at least one of the other two subunits plays an essential supporting role for activity. Substitution of beta(1) with beta(2) in the co-expression system lowered the pH optimum for IDH activity from 8.0 to 7.6. This difference in optimal pH was analogous to what was observed in mouse kidney and brain (beta(1) prevalent; optimal pH 8.0) versus heart (beta(2) prevalent; pH 7.6) mitochondria. Experiments with a specially designed splicing reporter construct stably transfected into HT1080 cells indicate that acidic conditions favor a splicing pattern responsible for the muscle- and heart-specific beta(2) isoform. Taken together, these data indicate a regulatory role of IDHbeta isoforms in determining the pH optimum for IDH activity through the tissue-specific alternative splicing.
Assuntos
Isocitrato Desidrogenase/genética , Isoenzimas/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Células Cultivadas , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/fisiologia , Isoenzimas/química , Isoenzimas/fisiologia , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/análiseRESUMO
The zebrafish is widely used as a model system for studying mammalian developmental genetics and more recently, as a model system for carcinogenesis. Since there is mounting evidence that selenium can prevent cancer in mammals, including humans, we characterized the selenocysteine tRNA[Ser]sec gene and its product in zebrafish. Two genes for this tRNA were isolated and sequenced and were found to map at different loci within the zebrafish genome. The encoding sequences of both are identical and their flanking sequences are highly homologous for several hundred bases in both directions. The two genes likely arose from gene duplication which is a common phenomenon among many genes in this species. In addition, zebrafish tRNA[Ser]sec was isolated from the total tRNA population and shown to decode UGA in a ribosomal binding assay.
Assuntos
Aminoacil-RNA de Transferência/genética , Peixe-Zebra/genética , Animais , Sequência de Bases , Biblioteca Gênica , Genes Duplicados , Modelos Genéticos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Xenopus/genéticaRESUMO
Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in radiation damage. NADP+-dependent isocitrate dehydrogenase (ICDH) in Escherichia coli produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of ICDH against ionizing radiation in E. coli was investigated in wild-type and ICDH-deficient strains. Upon exposure to ionizing radiation, the viability was lower and the lipid peroxidation was higher in mutant cells compared to wild-type cells. Activities of key antioxidant enzymes such as superoxide dismutase, catalase, glutathione reductase, and glucose-6-phosphate dehydrogenase were decreased by irradiation in both cells. Results suggest that ICDH plays an important role as an antioxidant enzyme in cellular defense against ionizing radiation.
Assuntos
Escherichia coli/efeitos da radiação , Isocitrato Desidrogenase/genética , NADP/metabolismo , Tolerância a Radiação , Antioxidantes/metabolismo , Catalase/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Glucosefosfato Desidrogenase/metabolismo , Glutationa Peroxidase/metabolismo , Peroxidação de Lipídeos , Superóxido Dismutase/metabolismo , Raios XRESUMO
We have isolated and characterized two complete cDNA clones, Zfz8a and Zfz8b, which encode zebrafish Frizzled (Fz) homologues. The predicted protein sequences, spanning 579 and 576 amino acid residues for ZFz8a and ZFz8b, respectively, were highly homologous (78%) to each other and contained an extracellular cysteine-rich domain and seven transmembrane domains that are well conserved in Fz receptor protein members. In comparison with other Fz family members, ZFz8a and ZFz8b showed the highest homology with mouse Fz8 (MFz8), sharing 84 and 76% amino acid identity, respectively. The presence of Zfz8a and Zfz8b transcripts was detected by in situ hybridization in zebrafish embryos from the 512 cell stage, and their appearance in the future dorsal region could be observed before embryos reached the 30% epiboly stage. At shield stage, Zfz8a transcripts were expressed in both epiblast and shield whereas expression of Zfz8b was only detected in the embryonic shield. During gastrula stages, both Zfz8a and Zfz8b transcripts were found in anterior dorsal regions of the involuting mesendoderm (future prechordal plate). By the 2- to 3-somite stage, expression of both Zfz8a and Zfz8b was restricted to the prechordal plate and prospective anterior neurectoderm, although expression of the Zfz8a gene was no longer present in the most anterior portion of the prechordal plate, the polster. In one-eyed pinhead mutant embryos, which lack prechordal plate, both Zfz8a and Zfz8b transcripts were reduced, confirming the prechordal plate specificity of Zfz8a and Zfz8b gene expression. These results provide an additional evidence supporting the role of Wnt signaling in organizer-mediated axial patterning.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genes , Isoformas de Proteínas/genética , Receptores de Superfície Celular/genética , Proteínas de Xenopus , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , DNA Complementar/genética , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Morfogênese/genética , Isoformas de Proteínas/biossíntese , Receptores de Superfície Celular/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Peixe-Zebra/embriologiaRESUMO
Several basic helix-loop-helix (bHLH) transcription factors are known to be involved in vertebrate neurogenesis. To investigate their roles in zebrafish neurogenesis, we isolated cDNAs for homologues of neurogenin and Math(-1)/atonal. The transcription of neurogenin was first detectable in zebrafish nervous system at late gastrulation stage. The expression of zebrafish neurogenin precedes and overlaps that of HuC, one of the earliest neuronal precursor markers. Injection of neurogenin mRNA into early stage zebrafish embryos induced ectopic expression of HuC. These results suggest that neurogenin may participate in the generation of HuC-expressing cells, implying its role in neuronal determination in zebrafish.