RESUMO
UNLABELLED: Thyroid volume measurement by ultrasonography (US) is essential in numerous clinical diagnostic and therapeutic fields. While known to be limited, the accuracy and precision of two-dimensional (2D) US thyroid volume measurement have not been thoroughly characterized. OBJECTIVE: We sought to assess the intra- and interobserver variability, accuracy and precision of thyroid volume determination by conventional 2D US in healthy adults using reference volumes determined by three-dimensional (3D) US. Design, METHODS: In a prospective blinded trial, thyroid volumes of ten volunteers were determined repeatedly by nine experienced sonographers using conventional 2D US (ellipsoid model). The values obtained were statistically compared to the so-called true volumes determined by 3D US (multiplanar approximation), the so-called gold standard, to estimate systematic errors and relative deviations of individual observers. RESULTS: The standard error of measurement (SEM) for one observer and successive measurements (intraobserver variability), was 14%, and for different observers and repeated measurements (interobserver variability), 17%. The minimum relative thyroid volume change significantly different at the 95% level was 39% for the same observer and 46% for different observers. Regarding accuracy, the mean value of the differences showed a significant thyroid volume overestimation (17%, p < 0.01) by 2D relative to 3D US. CONCLUSION: 2D US is appropriate for routine thyroid volumetry. Nevertheless, the so-called human factor (random error) should be kept in mind and correction is needed for methodical bias (systematic error). Further efforts are required to improve the accuracy and precision of 2D US thyroid volumetry by optimizing the underlying geometrical modeling or by the application of 3D US.
Assuntos
Glândula Tireoide/anatomia & histologia , Glândula Tireoide/diagnóstico por imagem , Ultrassonografia/métodos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Valores de ReferênciaRESUMO
A novel thyroid ultrasound phantom with tissue-equivalent characteristics was designed consisting of two lobes with three lesions each. One set of lesions is manufactured with a -5 dB echo difference to the surrounding tissue, the other with -10 dB. This phantom was used as a standardized measuring object for reproducibility of two-dimensional and three-dimensional ultrasound volumetry and for an interobserver and intraobserver variability study. For the variability study, nine experienced physicians scanned all specimen three times. Each time the volumes were calculated using the ellipsoid method. A three-dimensional ultrasound scan of each specimen was performed to evaluate all volumes by multiplanar volume approximation. The results of these volume data were compared to the known true volumes. The interobserver variability ranged from -13.4% to 11.9% (median, 0.7%); the intraobserver variability from -9.1% to 16.4% (median, 3.6%). The systematic error as calculated from the total mean of all specimens is 0.5% for the interobserver variability and 4.1% for the intraobserver variability. The phantom can be used for training purposes, to improve the skills of the examining physicians by simulating real thyroid morphology, to provide a standardized reference object for long-term quality control of conventional ultrasound scanners, and the determination of the accuracy and reproducibility of volumetry using three-dimensional ultrasound systems.
Assuntos
Modelos Anatômicos , Glândula Tireoide/diagnóstico por imagem , Algoritmos , Interpretação Estatística de Dados , Humanos , Variações Dependentes do Observador , Controle de Qualidade , Reprodutibilidade dos Testes , UltrassonografiaRESUMO
Here we present novel information on non-classical functions of cholinesterases and on a cross-talk linking the two enzymes AChE and BChE. The first part of the article is focussed on the regulation of ChEs and the effects acquired when one of the proteins is knocked down (siRNA for BChE, AChE knock-out mouse). In the second part evidence is presented showing that AChE may exert adhesive properties through its binding to laminin, thus being involved in cell-matrix or cell-cell communication.
Assuntos
Acetilcolinesterase/metabolismo , Butirilcolinesterase/metabolismo , Acetilcolinesterase/genética , Amidoidrolases/metabolismo , Animais , Butirilcolinesterase/genética , Adesão Celular , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Laminina/metabolismo , Camundongos , Ligação Proteica , RatosRESUMO
We have isolated a cDNA encoding a member of the Tlx/Hox11 family of homeodomain factors from the zebrafish, most closely related to the vertebrate Tlx-1/Hox11 and Tlx-3/Hox11L2 proteins. The gene is expressed in a set of early differentiating neurons that project to a common tract, the lateral longitudinal fascicle. We show that the gene is specifically expressed in spinal cord Rohon Beard neurons, in nucleus of the posterior commissure neurons of the midbrain, in a set of hindbrain neurons that include RoL3 reticulospinal interneurons, and in the trigeminal, statoacoustic, anterior lateral line, glossopharyngeal, and vagal cranial sensory ganglia. Timing of expression of the gene in these neurons correlates with the phase of axonal outgrowth and target innervation. Expression of the gene is also observed in several non-neural tissues, including the pharyngeal arches, budding gill filaments, outgrowing semicircular protrusions in the otic vesicle, and in the pectoral fin buds.
Assuntos
Gânglios Sensitivos/embriologia , Expressão Gênica , Proteínas de Homeodomínio/genética , Neurônios/fisiologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Sequência de Aminoácidos/genética , Animais , Diferenciação Celular , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Embrião não Mamífero/fisiologia , Proteínas de Homeodomínio/isolamento & purificação , Dados de Sequência Molecular , Neurônios/citologia , Proteínas Oncogênicas/genética , Fatores de Tempo , Proteínas de Peixe-Zebra/isolamento & purificaçãoRESUMO
The eyes absent-like genes encode a group of putative transcriptional coactivators with a sole representative in Drosophila and several members in mammals. Haploinsufficiency of the human EYA1 gene results in branchio-oto-renal syndrome characterized by developmental anomalies of the branchial arches, the three compartments of the ear and the kidney. As a first step towards a functional analysis of this gene in lower vertebrates, we isolated its zebrafish homologue, eya1, and studied its expression pattern during embryogenesis. The eya1 cDNA predicts a protein with 84.7% identity with the human homologue. Transcripts are first detected at the tailbud stage in presumptive cranial placodal precursor cells. Thereafter, eya1 expression continues in anterior pituitary, olfactory, otic, and lateral line placodes. Aside from these placodal sites of expression, eya1 transcripts were observed in the somites, developing pectoral fins, and branchial arches. No expression was found in pronephros or Wolffian duct of the zebrafish renal system. Within the developing ear, eya1 expression becomes confined to the ventral part of the otic vesicle from where the acoustic ganglion precursor cells arise and the sensory patches differentiate. In the lateral line, eya1 is expressed in the placodes, ganglia, migrating primordia, and receptive organs at all developmental stages, including both the differentiating hair and supporting cells. Taken together, these results indicate a remarkable similarity in both the structure and expression pattern of eya1 between higher and lower vertebrates, suggesting that the function of this gene has been conserved throughout vertebrate evolution.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Transativadores/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Drosophila/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Proteínas Nucleares , Especificidade de Órgãos , Proteínas Tirosina Fosfatases , Alinhamento de Sequência , Peixe-Zebra/embriologia , Proteínas de Peixe-ZebraRESUMO
In the zebrafish embryo, cells fated to give rise to the rostral brain move in a concerted fashion and retain tissue coherence during morphogenesis. We demonstrate here that Otx proteins have a dramatic effect on cell-cell interactions when expressed ectopically in the zebrafish embryo. Injection of zebrafish Otx1 or Drosophila otd RNAs into a single cell at the 16-cell stage results in aggregation of descendants of the injected cell. The Otx/Otd homeodomain is necessary for aggregation and appears to be sufficient for the effect when substituted for the homeodomain of an unrelated homeodomain protein. When cells containing injected zOtx1 RNA are limited to the area that is normally fated to become the anterior brain and neural retina, the induced aggregates contribute to anterior brain and retina tissues. In many other embryonic regions, which do not express endogenous zOtx1, the aggregates appear to be incompatible with normal development and do not integrate into developing tissues. By using an activatable Otx1-glutocorticoid receptor fusion protein that results in the stimulation of cell association, we demonstrate that cell aggregates can form as a result of Otx1 activity even after gastrulation is completed. Time-lapse analysis of cell movements show that cell aggregation occurs with only a slight inhibition of the rate of convergence. These results suggest that promotion of cell adhesion or mediation of cell repulsion may be one of the normal functions of the Otx proteins in the establishment of the anterior brain.
Assuntos
Encéfalo/embriologia , Adesão Celular , Proteínas de Homeodomínio/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Retina/embriologia , Fatores de Transcrição , Animais , Proteínas de Drosophila , Gástrula , Proteínas de Homeodomínio/genética , Microinjeções , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição Otx , RNA Mensageiro/administração & dosagem , Transativadores/biossíntese , Peixe-Zebra/embriologia , Proteínas de Peixe-ZebraRESUMO
We have studied the role of Bmp signaling in patterning neural tissue through the use of mutants in the zebrafish that disrupt three different components of a Bmp signaling pathway: swirl/bmp2b, snailhouse/bmp7 and somitabun/smad5. We demonstrate that Bmp signaling is essential for the establishment of the prospective neural crest and dorsal sensory Rohon-Beard neurons of the spinal cord. Moreover, Bmp signaling is necessary to limit the number of intermediate-positioned lim1+ interneurons of the spinal cord, as observed by the dramatic expansion of these prospective interneurons in many mutant embryos. Our analysis also suggests a positive role for Bmp signaling in the specification of these interneurons, which is independent of Bmp2b/Swirl activity. We found that a presumptive ventral signal, Hh signaling, acts to restrict the amount of dorsal sensory neurons and trunk neural crest. This restriction appears to occur very early in neural tissue development, likely prior to notochord or floor plate formation. A similar early role for Bmp signaling is suggested in the specification of dorsal neural cell types, since the bmp2b/swirl and bmp7/snailhouse genes are only coexpressed during gastrulation and within the tail bud, and are not found in the dorsal neural tube or overlying epidermal ectoderm. Thus, a gastrula Bmp2b/Swirl and Bmp7/Snailhouse-dependent activity gradient may not only act in the specification of the embryonic dorsoventral axis, but may also function in establishing dorsal and intermediate neuronal cell types of the spinal cord.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Neurônios/citologia , Medula Espinal/embriologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Hibridização In Situ , Interneurônios/citologia , Interneurônios/metabolismo , Mutação , Neurônios/metabolismo , Transdução de Sinais , Medula Espinal/citologia , Medula Espinal/metabolismo , Peixe-Zebra/metabolismoRESUMO
We describe the isolation of the zebrafish MyoD gene and its expression in wild-type embryos and in two mutants with altered somite development, no tail (ntl) and spadetail (spt). In the wild-type embryo, MyoD expression first occurs in an early phase, extending from mid-gastrula to just prior to somite formation, in which cells directly adjacent to the axial mesoderm express the gene. In subsequent phases, during the anterior-to-posterior wave of somite formation and maturation, expression occurs within particular regions of each somite. In spt embryos, which lack normal paraxial mesoderm due to incorrect cell migration, early MyoD expression is not observed and transcripts are instead first detected in small groups of trunk cells that will develop into aberrant myotomal-like structures. In ntl embryos, which lack notochords and tails, the early phase of MyoD expression is also absent. However, the later phase of expression within the developing somites appears to occur at the normal time in the ntl mutants, indicating that the presomitogenesis and somitogenesis phases of MyoD expression can be uncoupled. In addition, we demonstrate that the entire paraxial mesoderm of wild-type embryos has the potential to express MyoD when Sonic hedgehog is expressed ubiquitously in the embryo, and that this potential is lost in some of the cells of the paraxial mesoderm lineage in no tail and spadetail embryos. We also show that MyoD expression precedes myogenin expression and follows or is coincident with expression of snaill in some regions that express this gene.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteína MyoD/genética , Transativadores , Fatores de Transcrição , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas Hedgehog , Hibridização In Situ , Dados de Sequência Molecular , Estrutura Molecular , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Mutação , Proteína MyoD/química , Miogenina/genética , Proteínas/genética , Homologia de Sequência de Aminoácidos , Fatores de Transcrição da Família Snail , Fatores de TempoRESUMO
To gain an insight into the cellular function of the unconventional myosin VIIA, we sought proteins interacting with its tail region, using the yeast two-hybrid system. Here we report on one of the five candidate interactors we identified, namely the type I alpha regulatory subunit (RI alpha) of protein kinase A. The interaction of RI alpha with myosin VIIA tail was demonstrated by coimmunoprecipitation from transfected HEK293 cells. Analysis of deleted constructs in the yeast two-hybrid system showed that the interaction of myosin VIIA with RI alpha involves the dimerization domain of RI alpha. In vitro binding assays identified the C-terminal "4.1, ezrin, radixin, moesin" (FERM)-like domain of myosin VIIA as the interacting domain. In humans and mice, mutations in the myosin VIIA gene underlie hereditary hearing loss, which may or may not be associated with visual deficiency. Immunohistofluorescence revealed that myosin VIIA and RI alpha are coexpressed in the outer hair cells of the cochlea and rod photoreceptor cells of the retina. Our results strongly suggest that myosin VIIA is a novel protein kinase A-anchoring protein that targets protein kinase A to definite subcellular sites of these sensory cells.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miosinas/metabolismo , Animais , Sítios de Ligação , Dineínas , Escherichia coli , Humanos , Camundongos , Miosina VIIa , Miosinas/análise , Ligação Proteica , Especificidade por SubstratoRESUMO
Defects in myosin VIIA are responsible for deafness in the human and mouse. The role of this unconventional myosin in the sensory hair cells of the inner ear is not yet understood. Here we show that the C-terminal FERM domain of myosin VIIA binds to a novel transmembrane protein, vezatin, which we identified by a yeast two-hybrid screen. Vezatin is a ubiquitous protein of adherens cell-cell junctions, where it interacts with both myosin VIIA and the cadherin-catenins complex. Its recruitment to adherens junctions implicates the C-terminal region of alpha-catenin. Taken together, these data suggest that myosin VIIA, anchored by vezatin to the cadherin-catenins complex, creates a tension force between adherens junctions and the actin cytoskeleton that is expected to strengthen cell-cell adhesion. In the inner ear sensory hair cells vezatin is, in addition, concentrated at another membrane-membrane interaction site, namely at the fibrillar links interconnecting the bases of adjacent stereocilia. In myosin VIIA-defective mutants, inactivity of the vezatin-myosin VIIA complex at both sites could account for splaying out of the hair cell stereocilia.
Assuntos
Caderinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Miosinas/química , Miosinas/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Caderinas/química , Linhagem Celular , Proteínas do Citoesqueleto/química , Surdez/genética , Surdez/metabolismo , Dineínas , Células Ciliadas Auditivas/metabolismo , Humanos , Técnicas In Vitro , Junções Intercelulares/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Mutação , Miosina VIIa , Miosinas/genética , Ligação Proteica , Estrutura Terciária de Proteína , alfa CateninaRESUMO
Spinocerebellar ataxia 7 (SCA7) is a neurodegenerative disease caused by expansion of a CAG repeat in the coding region of the SCA7 gene. The disease primarily affects the cerebellum and the retina, but also many other central nervous system (CNS) structures as the disease progresses. Ataxin-7, encoded by the SCA7 gene, is a protein of unknown function expressed in many tissues including the CNS. In normal brain, ataxin-7 is found in the cytoplasm and/or nucleus of neurons, but in SCA7 brain ataxin-7 accumulates in intranuclear inclusions. Ataxin-7 is expressed ubiquitously, but mutation leads to neuronal death in only certain areas of the brain. This selective pattern of degeneration might be explained by interaction with a partner that is specifically expressed in vulnerable cells. We used a two-hybrid approach to screen a human retina cDNA library for ataxin-7-binding proteins, and isolated R85, a splice variant of Cbl-associated protein (CAP). R85 and CAP are generated by alternative splicing of the gene SH3P12 which we localized on chromosome 10q23-q24. The interaction between ataxin-7 and the SH3P12 gene products (SH3P12GPs) was confirmed by pull-down and co-immunoprecipitation. SH3P12GPs are expressed in Purkinje cells in the cerebellum. Ataxin-7 colocalizes with full-length R85 (R85FL) in co-transfected Cos-7 cells and with one of the SH3P12GPs in neuronal intranuclear inclusions in brain from a SCA7 patient. We propose that this interaction is part of a physiological pathway related to the function or turnover of ataxin-7. Its role in the pathophysiological process of SCA7 disease is discussed.