RESUMO
This paper investigates the effect of altering the protein expression dynamics of the bHLH transcription factor Her6 at the single-cell level in the embryonic zebrafish telencephalon. Using a homozygote endogenous Her6:Venus reporter and 4D single-cell tracking, we show that Her6 oscillates in neural telencephalic progenitors and that the fusion of protein destabilisation (PEST) domain alters its expression dynamics, causing most cells to downregulate Her6 prematurely. However, counterintuitively, oscillatory cells increase, with some expressing Her6 at high levels, resulting in increased heterogeneity of Her6 expression in the population. These tissue-level changes appear to be an emergent property of coupling between single-cells, as revealed by experimentally disrupting Notch signalling and by computationally modelling alterations in Her6 protein stability. Despite the profound differences in the single-cell Her6 dynamics, the size of the telencephalon is only transiently altered and differentiation markers do not exhibit significant differences early on; however, a small increase is observed at later developmental stages. Our study suggests that cell coupling provides a compensation strategy, whereby an almost normal phenotype is maintained even though single-cell gene expression dynamics are abnormal, granting phenotypic robustness.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Fenótipo , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Telencéfalo/metabolismo , Telencéfalo/embriologia , Análise de Célula Única , Transdução de Sinais , Receptores Notch/metabolismo , Receptores Notch/genética , Diferenciação CelularRESUMO
MicroRNAs (miRs) have an important role in tuning dynamic gene expression. However, the mechanism by which they are quantitatively controlled is unknown. We show that the amount of mature miR-9, a key regulator of neuronal development, increases during zebrafish neurogenesis in a sharp stepwise manner. We characterize the spatiotemporal profile of seven distinct microRNA primary transcripts (pri-mir)-9s that produce the same mature miR-9 and show that they are sequentially expressed during hindbrain neurogenesis. Expression of late-onset pri-mir-9-1 is added on to, rather than replacing, the expression of early onset pri-mir-9-4 and -9-5 in single cells. CRISPR/Cas9 mutation of the late-onset pri-mir-9-1 prevents the developmental increase of mature miR-9, reduces late neuronal differentiation and fails to downregulate Her6 at late stages. Mathematical modelling shows that an adaptive network containing Her6 is insensitive to linear increases in miR-9 but responds to stepwise increases of miR-9. We suggest that a sharp stepwise increase of mature miR-9 is created by sequential and additive temporal activation of distinct loci. This may be a strategy to overcome adaptation and facilitate a transition of Her6 to a new dynamic regime or steady state.
Assuntos
MicroRNAs , Peixe-Zebra , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Neurogênese/genética , Peixe-Zebra/metabolismoRESUMO
Here, we study the dynamical expression of endogenously labeled Hes1, a transcriptional repressor implicated in controlling cell proliferation, to understand how cell-cycle length heterogeneity is generated in estrogen receptor (ER)+ breast cancer cells. We find that Hes1 shows oscillatory expression with â¼25 h periodicity and during each cell cycle has a variable peak in G1, a trough around G1-S transition, and a less variable second peak in G2/M. Compared to other subpopulations, the cell cycle in CD44HighCD24Low cancer stem cells is longest and most variable. Most cells divide around the peak of the Hes1 expression wave, but preceding mitoses in slow dividing CD44HighCD24Low cells appear phase-shifted, resulting in a late-onset Hes1 peak in G1. The position, duration, and shape of this peak, rather than the Hes1 expression levels, are good predictors of cell-cycle length. Diminishing Hes1 oscillations by enforcing sustained expression slows down the cell cycle, impairs proliferation, abolishes the dynamic expression of p21, and increases the percentage of CD44HighCD24Low cells. Reciprocally, blocking the cell cycle causes an elongation of Hes1 periodicity, suggesting a bidirectional interaction of the Hes1 oscillator and the cell cycle. We propose that Hes1 oscillations are functionally important for the efficient progression of the cell cycle and that the position of mitosis in relation to the Hes1 wave underlies cell-cycle length heterogeneity in cancer cell subpopulations.
Assuntos
Neoplasias da Mama/metabolismo , Ciclo Celular , Ritmo Circadiano , Receptores de Estrogênio/metabolismo , Fatores de Transcrição HES-1/metabolismo , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/fisiologiaRESUMO
Ultradian oscillations of HES Transcription Factors (TFs) at the single-cell level enable cell state transitions. However, the tissue-level organisation of HES5 dynamics in neurogenesis is unknown. Here, we analyse the expression of HES5 ex vivo in the developing mouse ventral spinal cord and identify microclusters of 4-6 cells with positively correlated HES5 level and ultradian dynamics. These microclusters are spatially periodic along the dorsoventral axis and temporally dynamic, alternating between high and low expression with a supra-ultradian persistence time. We show that Notch signalling is required for temporal dynamics but not the spatial periodicity of HES5. Few Neurogenin 2 cells are observed per cluster, irrespective of high or low state, suggesting that the microcluster organisation of HES5 enables the stable selection of differentiating cells. Computational modelling predicts that different cell coupling strengths underlie the HES5 spatial patterns and rate of differentiation, which is consistent with comparison between the motoneuron and interneuron progenitor domains. Our work shows a previously unrecognised spatiotemporal organisation of neurogenesis, emergent at the tissue level from the synthesis of single-cell dynamics.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Análise de Célula Única/métodos , Medula Espinal/crescimento & desenvolvimento , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Comunicação Celular , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Camundongos , Neurogênese , Receptores Notch/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Análise Espaço-Temporal , Medula Espinal/metabolismo , Ritmo UltradianoRESUMO
Noise is prevalent in biology and has been widely quantified using snapshot measurements. This static view obscures our understanding of dynamic noise properties and how these affect gene expression and cell state transitions. Using a CRISPR/Cas9 Zebrafish her6::Venus reporter combined with mathematical and in vivo experimentation, we explore how noise affects the protein dynamics of Her6, a basic helix-loop-helix transcriptional repressor. During neurogenesis, Her6 expression transitions from fluctuating to oscillatory at single-cell level. We identify that absence of miR-9 input generates high-frequency noise in Her6 traces, inhibits the transition to oscillatory protein expression and prevents the downregulation of Her6. Together, these impair the upregulation of downstream targets and cells accumulate in a normally transitory state where progenitor and early differentiation markers are co-expressed. Computational modelling and double smFISH of her6 and the early neurogenesis marker, elavl3, suggest that the change in Her6 dynamics precedes the downregulation in Her6 levels. This sheds light onto the order of events at the moment of cell state transition and how this is influenced by the dynamic properties of noise. Our results suggest that Her/Hes oscillations, facilitated by dynamic noise optimization by miR-9, endow progenitor cells with the ability to make a cell state transition.
Assuntos
Animais Geneticamente Modificados/embriologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Relógios Biológicos , MicroRNAs/metabolismo , Neurogênese , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína Semelhante a ELAV 3/genética , Proteína Semelhante a ELAV 3/metabolismo , MicroRNAs/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences.
Assuntos
Epiderme/embriologia , Epiderme/imunologia , Células Caliciformes/imunologia , Imunidade Inata/fisiologia , Xenopus/embriologia , Xenopus/microbiologia , Animais , Diferenciação Celular/fisiologia , Cílios/imunologia , Embrião não Mamífero , Epiderme/metabolismo , Glicoproteínas/análise , Glicoproteínas/metabolismo , Fator 3-alfa Nuclear de Hepatócito/fisiologia , Íons/metabolismo , Larva , Muco/química , Muco/metabolismo , Via Secretória/imunologia , Vesículas Secretórias/imunologia , Vesículas Secretórias/metabolismo , Xenopus/imunologiaRESUMO
Embryonic wound healing provides a perfect example of efficient recovery of tissue integrity and homeostasis, which is vital for survival. Tissue movement in embryonic wound healing requires two functionally distinct actin structures: a contractile actomyosin cable and actin protrusions at the leading edge. Here, we report that the discrete formation and function of these two structures is achieved by the temporal segregation of two intracellular upstream signals and distinct downstream targets. The sequential activation of ERK and phosphoinositide 3-kinase (PI3K) signalling divides Xenopus embryonic wound healing into two phases. In the first phase, activated ERK suppresses PI3K activity, and is responsible for the activation of Rho and myosin-2, which drives actomyosin cable formation and constriction. The second phase is dominated by restored PI3K signalling, which enhances Rac and Cdc42 activity, leading to the formation of actin protrusions that drive migration and zippering. These findings reveal a new mechanism for coordinating different modes of actin-based motility in a complex tissue setting, namely embryonic wound healing.
Assuntos
Actinas/metabolismo , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Cicatrização , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/fisiologia , Animais , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Wound healing is essential for survival. We took advantage of the Xenopus embryo, which exhibits remarkable capacities to repair wounds quickly and efficiently, to investigate the mechanisms responsible for wound healing. Previous work has shown that injury triggers a rapid calcium response, followed by the activation of Ras homolog (Rho) family guanosine triphosphatases (GTPases), which regulate the formation and contraction of an F-actin purse string around the wound margin. How these processes are coordinated following wounding remained unclear. Here we show that inositol-trisphosphate 3-kinase B (Itpkb) via its enzymatic product inositol 1,3,4,5-tetrakisphosphate (InsP4) plays an essential role during wound healing by modulating the activity of Rho family GTPases and F-actin ring assembly. Furthermore, we show that Itpkb and InsP4 modulate the speed of the calcium wave, which propagates from the site of injury into neighboring uninjured cells. Strikingly, both overexpression of itpkb and exogenous application of InsP4 accelerate the speed of wound closure, a finding that has potential implications in our quest to find treatments that improve wound healing in patients with acute or chronic wounds.
Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Cicatrização/fisiologia , Proteínas de Xenopus/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio/fisiologia , Ectoderma/embriologia , Ectoderma/fisiologia , Epiderme/embriologia , Epiderme/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Fosfatos de Inositol/fisiologia , Oócitos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/fisiologiaRESUMO
Primary vascular tumors of lymph nodes are extremely rare with the exception of AlDS-related Kaposi's sarcoma. The diagnosis of epithelioid hemangio-endothelioma (EH) is difficult to make without ancillary studies, since it is devoid of morphological features indicating its vascular nature and it may be overlooked when it appears as a primary tumor of lymph nodes. Spindle and epithelioid hemangio-endothelioma (SEH) is considered to be a variant of EH, which has been reported to occur exclusively in lymph nodes and the spleen. We report a 70-year-old male with chronic lymphocytic leukemia (CLL) and left cervical lymphadenopathy. An excisional biopsy was performed, and microscopically the lymph node showed effacement of nodal architecture by a tumor composed of spindle cells disposed in intersecting fascicles, and characterized by abundant eosinophilic cytoplasm, elongated nuclei and conspicuous nucleoli. A second population of cells had an epithelioid appearance with intracyto-plasmic vacuoles containing red blood cells. lmmunohistochemically, the tumor cells were positive for CD31 and CD34. The final diagnosis was SEH of the lymph node.
Assuntos
Hemangioendotelioma/patologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , Idoso , Hemangioendotelioma/complicações , Humanos , Achados Incidentais , Leucemia Linfocítica Crônica de Células B/complicações , MasculinoRESUMO
Background: Primary vascular tumors of lymph nodes are extremely rare with the exception of AlDS-related Kaposi's sarcoma. The diagnosis of epithelioid hemangio-endothelioma (EH) is difficult to make without ancillary studies, since it is devoid of morphological features indicating its vascular nature and it may be overlooked when it appears as a primary tumor of lymph nodes. Spindle and epithelioid hemangio-endothelioma (SEH) is considered to be a variant of EH, which has been reported to occur exclusively in lymph nodes and the spleen. We report a 70-year-old male with chronic lymphocytic leukemia (CLL) and left cervical lymphadenopathy. An excisional biopsy was performed, and microscopically the lymph node showed effacement of nodal architecture by a tumor composed of spindle cells disposed in intersecting fascicles, and characterized by abundant eosinophilic cytoplasm, elongated nuclei and conspicuous nucleoli. A second population of cells had an epithelioid appearance with intracyto-plasmic vacuoles containing red blood cells. lmmunohistochemically, the tumor cells were positive for CD31 and CD34. The final diagnosis was SEH of the lymph node.
Assuntos
Idoso , Humanos , Masculino , Hemangioendotelioma/patologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , Hemangioendotelioma/complicações , Achados Incidentais , Leucemia Linfocítica Crônica de Células B/complicaçõesRESUMO
The molecular mechanisms that underlie the development of primitive myeloid cells in vertebrate embryos are not well understood. Here we characterize the role of cebpa during primitive myeloid cell development in Xenopus. We show that cebpa is one of the first known hematopoietic genes expressed in the embryo. Loss- and gain-of-function studies show that it is both necessary and sufficient for the development of functional myeloid cells. In addition, we show that cebpa misexpression leads to the precocious induction of myeloid cell markers in pluripotent prospective ectodermal cells, without the cells transitioning through a general mesodermal state. Finally, we use live imaging to show that cebpa-expressing cells exhibit many attributes of terminally differentiated myeloid cells, such as highly active migratory behavior, the ability to quickly and efficiently migrate toward wounds and phagocytose bacteria, and the ability to enter the circulation. Thus, C/EPBalpha is the first known single factor capable of initiating an entire myelopoiesis pathway in pluripotent cells in the embryo.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Células-Tronco Embrionárias/fisiologia , Mielopoese/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Primers do DNA/genética , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Mielopoese/genética , Fenótipo , Células-Tronco Pluripotentes/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Xenopus/embriologia , Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
Vertebrate blood formation occurs in 2 spatially and temporally distinct waves, so-called primitive and definitive hematopoiesis. Although definitive hematopoiesis has been extensively studied, the development of primitive myeloid blood has received far less attention. In Xenopus, primitive myeloid cells originate in the anterior ventral blood islands, the equivalent of the mammalian yolk sac, and migrate out to colonize the embryo. Using fluorescence time-lapse video microscopy, we recorded the migratory behavior of primitive myeloid cells from their birth. We show that these cells are the first blood cells to differentiate in the embryo and that they are efficiently recruited to embryonic wounds, well before the establishment of a functional vasculature. Furthermore, we isolated spib, an ETS transcription factor, specifically expressed in primitive myeloid precursors. Using spib antisense morpholino knockdown experiments, we show that spib is required for myeloid specification, and, in its absence, primitive myeloid cells retain hemangioblast-like characteristics and fail to migrate. Thus, we conclude that spib sits at the top of the known genetic hierarchy that leads to the specification of primitive myeloid cells in amphibians.
Assuntos
Células Mieloides/citologia , Proteínas Proto-Oncogênicas c-ets/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Animais , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Embrião não Mamífero/citologia , Microscopia de Vídeo , Proteínas Proto-Oncogênicas c-ets/isolamento & purificação , CicatrizaçãoRESUMO
Resumen: La mortalidad hospitalaria ha sido propuesta como uno de los indicadores de calidad asistencial más frecuentemente utilizado. Objetivos: Caracterizar la mortalidad hospitalaria observada en un servicio de medicina interna. Pacientes y métodos: Estudio basado en el análisis descriptivo retrospectivo de la mortalidad hospitalaria de 267 pacientes del servicio de medicina interna del Hospital Dr. Eduardo Pereira de Valparaíso de Junio de 2005 hasta Junio de 2006. Se confeccionó un protocolo de mortalidad hospitalaria, con el propósito de incluir cada uno de los aspectos a evaluar. Resultados: La tasa de mortalidad global obtenida en el período fue de un 8,2 por ciento. La tasa de mortalidad femenina corresponde al 3.62 por ciento y la masculina al 4,57 por ciento. La mortalidad precoz fue de 1.44 por ciento y la tardía de 6.41 por ciento. La edad media obtenida fue de 74.6 años en un rango de 26 a 95 años. Las causas de muerte observadas fueron enfermedades respiratorias (30 por ciento), cardiocirculatorias (27 por ciento) y digestivas (18 por ciento). Un 90 por ciento de los pacientes fallecidos ingresa desde el servicio de urgencia. Discusiones y Conclusiones: El porcentaje de fallecimientos es superior al estándar internacionalmente aceptado, siendo la mayoría hombres, similar a lo reportado en otros estudios. Las principales causas de muerte descritas coinciden con las estadísticas nacionales, aunque defieren en el orden que algunas de ellas ocupan. La mortalidad a edades avanzadas está en directa relación con el padecimiento de múltiples enfermedades o factores de riesgo para éstas.
Summary: Hospital mortality study in internal medicine service from Dr. Eduardo Pereira Hospital's in Valparaíso from June 2005 to June 2006. Hospital mortality has been proposed as a frequently used quality indicator. Objectives: to characterize an internal medicine service's mortality. Patients and methods: a hospital mortality's descriptive retrospective study o 267 patients from the internal medicine service of Hospital Dr. Eduardo Pereira in Valparaíso from June 2005 to July 2006. A hospital mortality protocol that included related topics was prepared. Results: global mortality rate was 8.2 percent. Fermale mortality rate was 3.62 percent, and male mortality rate was 4.57 percent. Premature mortality rate was 1.44 percent and late mortality rate was 6.41 percent. Obtained middle age was 74.6 years. Observed mortality causes were respiratory (30 percent), cardiovascular (27 percent) and digestive deseases (16 percent). Ninety percent of the patients came from the emergency service. Discusions and conclusions: the death rate is higher than the accepted internationally standard, with a higher death rate in males, which is similar to other studies. The most frequent mortality causes are coincidental with national statistics, although they differ in the incidence by the organic system affected. Mortality at advanced ages is directly related with the presence of simultaneous multiple disease occurrence, and the presence of their risk factors.
Assuntos
Humanos , Masculino , Feminino , Causas de Morte , Doenças Respiratórias/mortalidade , Mortalidade Hospitalar/tendências , ChileRESUMO
Immature stage VI Xenopus oocytes are arrested at the G(2)/M border of meiosis I until exposed to progesterone, which induces meiotic resumption through a non-genomic mechanism. One of the earliest events produced by this hormone is inhibition of the plasma membrane enzyme adenylyl cyclase (AC), with the concomitant drop in intracellular cAMP levels and reinitiation of the cell cycle. Recently Gsalpha and Gbetagamma have been shown to play an important role as positive regulators of Xenopus oocyte AC, maintaining the oocyte in the arrested state. However, a question that still remains unanswered, is how the activated state of Gsalpha and Gbetagamma is achieved in the immature oocyte, since no receptor or ligand have been found to be required. Here we provide evidence that xRic-8 can act in vitro and in vivo as a GEF for Gsalpha. Overexpression of xRic-8, through mRNA injection, greatly inhibits progesterone induced oocyte maturation and endogenous xRic-8 mRNA depletion, through siRNA microinjection, induces spontaneous oocyte maturation. These results suggest that xRic-8 is participating in the immature oocyte by keeping Gsalpha-Gbetagamma-AC signaling complex in an activated state and therefore maintaining G2 arrest.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Meiose , Oócitos/citologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/genéticaRESUMO
The non-canonical Wnt/Ca2+ signaling pathway has been implicated in the regulation of axis formation and gastrulation movements during early Xenopus laevis embryo development, by antagonizing the canonical Wnt/beta-catenin dorsalizing pathway and specifying ventral cell fate. However, the molecular mechanisms involved in this antagonist crosstalk are not known. Since Galphaq is the main regulator of Ca2+ signaling in vertebrates and from this perspective probably involved in the events elicited by the non-canonical Wnt/Ca2+ pathway, we decided to study the effect of wild-type Xenopus Gq (xGalphaq) in dorso-ventral axis embryo patterning. Overexpression of xGalphaq or its endogenous activation at the dorsal animal region of Xenopus embryo both induced a strong ventralized phenotype and inhibited the expression of dorsal-specific mesoderm markers goosecoid and chordin. Dorsal expression of an xGalphaq dominant-negative mutant reverted the xGalphaq-induced ventralized phenotype. Finally, we observed that the Wnt8-induced secondary axis formation is reverted by endogenous xGalphaq activation, indicating that it is negatively regulating the Wnt/beta-catenin pathway.
Assuntos
Padronização Corporal , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Wnt/antagonistas & inibidores , Xenopus laevis/metabolismo , beta Catenina/antagonistas & inibidores , Animais , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Gastrulação , Xenopus laevis/embriologiaRESUMO
Cystic echinococcosis is the most prevalent zoonosis in Tierra del Fuego province, Argentina, with important economic, productive and public health consequences. The present work was performed to determine the ovine prevalence in Tierra del Fuego, Argentina, as well as to evaluate the quality of diagnostic systems in slaughterhouses. Moreover, genetic analyses to characterize the strain of Echinococcus granulosus involved in the region were done. The first actions to perform a diagnosis of the epidemiological situation of hydatidosis in Tierra del Fuego were done between 1976 and 1977. A canine prevalence of 80% and an ovine prevalence of 55% results were obtained. Since 1979 the control program of Hydatidosis of Tierra del Fuego was implemented. It was based on semiannual canine anthelmintic treatment with praziquantel at dose of 5mg/kg, and complemented with sanitary education and canine and ovine epidemiological surveillance. During May 1997-January 1999: 5,916 sheep coming from 20 farms of the programmatic area were evaluated. In the lamb category, hydatid cysts were not found. In the adults category, 62 infected animals were found (3.2%). The ovine prevalence was 1.1% and there was 100% of coincidence between diagnosis in the slaughterhouse, re-inspection in the laboratory and histopathological study. The marked decrease in the prevalence observed for sheep infection evidenced a destabilization of the biological cycle of the parasite. This could be explained by the application of a control program with uninterrupted systematic actions. Polymerase chain reaction-ribosomal ITS-1 DNA (rDNA) restriction fragment length polymorphism (PCR-RFLP) analysis and partial sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene were used to characterize E. granulosus isolates collected from different regions of Tierra del Fuego to determine which genotypes occurred in this region. The results revealed the presence of the G1 genotype (sheep-dog strain). This is the first time that a molecular analysis was performed for the E. granulosus isolates from Tierra del Fuego.
Assuntos
Equinococose/veterinária , Echinococcus granulosus/classificação , Doenças dos Ovinos/epidemiologia , Matadouros , Animais , Argentina/epidemiologia , DNA Mitocondrial/química , DNA Espaçador Ribossômico/genética , Cães , Equinococose/diagnóstico , Equinococose/epidemiologia , Echinococcus granulosus/genética , Echinococcus granulosus/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Fígado/parasitologia , Pulmão/parasitologia , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Prevalência , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/parasitologia , ZoonosesRESUMO
Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a nongenomic mechanism that implicates the inhibition of the effector system adenylyl cyclase (AC). Recently, it has been shown that the G protein betagamma heterodimer is involved in oocyte maturation arrest. Since AC is the proposed target for Gbetagamma action, we considered of importance to identify and characterize the Gbetagamma regulated AC isoform(s) that are expressed in the Xenopus oocyte. Through biochemical studies, we found that stage VI plasma membrane oocyte AC activity showed attributes of an AC2 isoform. Furthermore, exogenous Gbetagamma was capable to activate oocyte AC only in the presence of the activated form of Galphas (Galphas-GTPgammaS), which is in agreement with the Ggammabeta conditional activation reported for the mammalian AC2 and AC4 isotypes. In order to study the functional role of AC in oocyte maturation we cloned from a Xenopus oocyte cDNA library a gene encoding an AC with high identity to AC7 (xAC7). Based on this sequence, we constructed a minigene encoding the AC-Gbetagamma interacting region (xAC7pep) to block, within the oocyte, this interaction. We found that microinjection of the xAC7pep potentiated progesterone-induced maturation, as did the AC2 minigene. From these results we can conclude that a Gbetagamma-activated AC is playing an important role in Xenopus oocyte meiotic arrest in a Galphas-GTP dependent manner.
Assuntos
Adenilil Ciclases/metabolismo , Diferenciação Celular/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Xenopus laevis/metabolismo , Adenilil Ciclases/genética , Adenilil Ciclases/isolamento & purificação , Animais , Diferenciação Celular/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Guanosina 5'-O-(3-Tiotrifosfato)/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Dados de Sequência Molecular , Oócitos/citologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/farmacologia , Progesterona/metabolismo , Progesterona/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMO
Glycine receptors (GlyRs), together with GABA(A) and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein betagamma dimer (Gbetagamma). We found that the amplitude of the glycine-activated Cl- current was enhanced after application of purified Gbetagamma or after activation of a G protein-coupled receptor. Overexpression of three distinct G protein alpha subunits (Galpha), as well as the Gbetagamma scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gbetagamma increased the GlyR open probability (nP(o)). Our results indicate that this interaction of Gbetagamma with GlyRs regulates both motor and sensory functions in the central nervous system.
Assuntos
Glicina/farmacologia , Proteínas Heterotriméricas de Ligação ao GTP/farmacologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Animais , Células Cultivadas , Canais de Cloreto/fisiologia , Condutividade Elétrica , Eletrofisiologia , Proteínas de Ligação ao GTP/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Peptídeos/farmacologia , Receptores de Glicina/efeitos dos fármacos , Receptores de Glicina/metabolismo , Receptores de Glicina/fisiologia , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
Heterotrimeric G-proteins transduce signals from heptahelical transmembrane receptors to different effector systems, regulating diverse complex intracellular pathways and functions. In brain, facilitation of depolarization-induced neurotransmitter release for synaptic transmission is mediated by Gsalpha and Gqalpha. To identify effectors for Galpha-proteins, we performed a yeast two-hybrid screening of a human brain cDNA library, using the human Galphas protein as a bait. We identified a protein member of the synembryn family as one of the interacting proteins. Extending the study to other Galpha subunits, we found that Gqalpha also interacts with synembryn, and these interactions were confirmed by in vitro pull down studies and by in vivo confocal laser microscopy analysis. Furthermore, synembryn was shown to translocate to the plasma membrane in response to carbachol and isoproterenol. This study supports recent findings in C. elegans where, through genetic studies, synembryn was shown to act together with Gqalpha regulating neuronal transmitter release. Based on these observations, we propose that synembryn is playing a similar role in human neuronal cells.
Assuntos
Encéfalo/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Transporte Proteico/fisiologia , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Carbacol/farmacologia , Membrana Celular/efeitos dos fármacos , Agonistas Colinérgicos/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Isoproterenol/farmacologia , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Células PC12 , Transporte Proteico/efeitos dos fármacos , Ratos , Técnicas do Sistema de Duplo-HíbridoRESUMO
G-protein alpha subunits consist of two domains: a Ras-like domain also called GTPase domain (GTPaseD), structurally homologous to monomeric G-proteins, and a more divergent domain, unique to heterotrimeric G-proteins, called helical domain (HD). G-protein activation, requires the exchange of bound GDP for GTP, and since the guanine nucleotide is buried in a deep cleft between both domains, it has been postulated that activation may involve a conformational change that will allow the opening of this cleft. Therefore, it has been proposed, that interdomain interactions are playing an important role in regulating the nucleotide exchange rate of the alpha subunit. While constructing different Gs(alpha) quimeras, we identified a Gs(alpha) random mutant, which was very inefficient in stimulating adenylyl cyclase activity. The introduced mutation corresponded to the substitution of Ser(111) for Asn (S111N), located in the carboxi terminal end of helix A of the HD, a region neither involved in AC interaction nor in the interdomain interface. In order to characterize this mutant, we expressed it in bacteria, purified it by niquel-agarose chromatography, and studied its nucleotide exchange properties. We demonstrated that the recombinant S111N Gs(alpha) was functional since it was able to undergo the characteristic conformational change upon GTP binding, detected by the acquisition of a trypsin-resistant conformation. When the biochemical properties were determined, the mutant protein exhibited a reduced GDP dissociation kinetics and as a consequence a slower GTPgammaS binding rate that was responsible for a diminished adenylyl cyclase activation when GTPgammaS was used as activator. These data provide new evidence that involves the HD as a regulator of Gs(alpha) function, in this case the alphaA helix, which is not directly involved with the nucleotide binding site nor the interdomain interface.