RESUMEN
BACKGROUND: Debriefing is a critical component to promote effective learning during simulation-based training. Traditionally, debriefing is provided only after the end of a scenario. A possible alternative is to debrief specific portions during an ongoing simulation session (stop-and-go debriefing). While this alternative has theoretical advantages, it is not commonly used due to concerns that interruptions disturb the fidelity and adversely affect learning. However, both approaches have not been rigorously compared, and effects on skill acquisition and learning experience are unknown. METHODS: We randomly assigned 50 medical students participating in a simulation-based cardiopulmonary resuscitation training to either a post-scenario debriefing or stop-and-go debriefing. After four weeks, participants performed a repeat scenario, and their performance was assessed using a generic performance score (primary outcome). A difference of 3 or more points was considered meaningful. A 5-item questionnaire was used to assess the subjective learning experience and the perceived stress level (secondary outcomes). RESULTS: There was no significant difference between the groups for the performance score (mean difference: -0.35, 95%CI: -2.46 to 1.77, P = 0.748, n = 48). The confidence limits excluding the specified relevant 3-point difference suggest equivalence of both techniques with respect to the primary outcome. No significant differences were observed for secondary outcomes. CONCLUSIONS: Stop-and-go debriefing does not adversely affect skill acquisition compared to the classic post-scenario debriefing strategy. This finding is reassuring when interruptions are deemed necessary and gives simulation instructors the latitude to tailor the timing of the debriefing individually, rather than adhering to the unsupported dogma that scenarios should not be interrupted. TRIAL REGISTRATION: As this study is not a clinical trial, it was not registered in a clinical trials register.
Asunto(s)
Reanimación Cardiopulmonar/educación , Aprendizaje Basado en Problemas , Entrenamiento Simulado , Competencia Clínica , Humanos , Aprendizaje , Análisis y Desempeño de Tareas , EnseñanzaRESUMEN
Due to the high rates of epidural failure (3-32%), novel techniques are required to objectively assess the successfulness of an epidural block. In this study we therefore investigated whether thermographic temperature measurements have a higher predictive value for a successful epidural block when compared to the cold sensation test as gold standard. Epidural anesthesia was induced in 61 patients undergoing elective abdominal, thoracic or orthopedic surgery. A thermographic picture was recorded at 5, 10 and 15 min following epidural anesthesia induction. After 15 min a cold sensation test was performed. Epidural anesthesia is associated with a decrease in skin temperature. Thermography predicts a successful epidural block with a sensitivity of 54% and a PPV of 92% and a specificity of 67% and a NPV of 17%. The cold sensation test shows a higher sensitivity and PPV than thermography (97 and 93%), but a lower specificity and NPV than thermography (25 and 50%). Thermographic temperature measurements can be used as an additional and objective method for the assessment of the effectiveness of an epidural block next to the cold sensation test, but have a low sensitivity and negative predictive value. The local decrease in temperature as observed in our study during epidural anesthesia is mainly attributed to a core-to-peripheral redistribution of body heat and vasodilation.
Asunto(s)
Anestesia Epidural/métodos , Frío , Temperatura Cutánea , Piel/patología , Termografía/métodos , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Umbral Sensorial , VasodilataciónRESUMEN
We utilized a cDNA expression library derived from the B6SutA(1) mouse myeloid progenitor cell line to search for novel oncogenes that promote growth transformation of NIH3T3 cells. A 2.2 kb transforming cDNA was recovered that encodes the wild type thrombin-stimulated G protein-coupled receptor PAR-1. In addition to its potent focus forming activity, constitutive overexpression of PAR-1 in NIH3T3 cells promoted the loss of anchorage- and serum-dependent growth. Although inhibitors of thrombin failed to block PAR-1 transforming activity, a PAR-1 mutant that cannot be cleaved by thrombin was nontransforming. Since the foci of transformed cells induced by PAR-1 bear a striking resemblance to those induced by activated RhoA, we determined if PAR-1 transformation was due to the aberrant activation of a specific Rho family member. Like RhoA, PAR-1 cooperated with activated Raf-1 and caused synergistic enhancement of transforming activity, induced stress fibers when microinjected into porcine aortic endothelial cells, stimulated the activity of the serum response factor and NF-kappaB transcription factors, and PAR-1 transformation was blocked by co-expression of dominant negative RhoA. Finally, PAR-1 transforming activity was blocked by pertussis toxin and by co-expression of the RGS domain of Lsc, implicating Galpha(i) and Galpha(12)/Galpha(13) subunits, respectively, as mediators of PAR-1 transformation. Taken together, these observations suggest that PAR-1 growth transformation is mediated, in part, by activation of RhoA.
Asunto(s)
Transformación Celular Neoplásica , Receptores de Trombina/fisiología , Transducción de Señal/fisiología , Proteína de Unión al GTP rhoA/fisiología , Células 3T3/citología , Células 3T3/metabolismo , Células 3T3/fisiología , Actinas/metabolismo , Animales , Adhesión Celular/fisiología , División Celular/fisiología , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , ADN Complementario/genética , Proteínas de Unión al ADN/fisiología , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Proteínas de Unión al GTP Heterotriméricas/fisiología , Ratones , Células Mieloides/fisiología , Receptor PAR-1 , Receptores de Trombina/biosíntesis , Receptores de Trombina/genética , Transfección , Proteínas de Unión al GTP rho/metabolismoRESUMEN
G2A is a heptahelical cell surface protein that has recently been described as a potential tumor suppressor, based on its ability to counteract transformation of pre-B cells and fibroblasts by Bcr-Abl, an oncogenic tyrosine kinase. We have isolated cDNAs encoding G2A in the course of screening libraries for clones that cause oncogenic transformation of NIH3T3 fibroblasts. When expressed at high levels in NIH3T3 cells by retroviral transduction, G2A induced a full range of phenotypes characteristic of oncogenic transformation, including loss of contact inhibition, anchorage-independent survival and proliferation, reduced dependence on serum, and tumorigenicity in mice. When expressed by transfection, G2A greatly enhanced the ability of a weakly oncogenic form of Raf-1 to transform NIH3T3 cells. These results demonstrate that G2A is potently oncogenic both on its own and in cooperation with another oncogene. Expression of G2A in fibroblasts and endothelial cells resulted in changes in cell morphology and cytoskeleton structure that were equivalent to those induced by the G protein subunit Galpha13. Transformation of NIH3T3 cells via G2A expression was completely suppressed by co-expression of LscRGS, a GTPase activating protein that suppresses signaling by Galpha12 and Galpha13. Hyperactivity of Galpha12 or Galpha13 has previously been shown to result in activation of Rho GTPases. G2A expression resulted in activation of Rho, and transformation via G2A was suppressed by a dominant negative form of RhoA. These results indicate that G2A may be directly coupled to Galpha13, and that it is the activation of this Rho-activating Galpha protein which is responsible for the ability of G2A to transform fibroblasts.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Proteínas de Unión al GTP/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Células 3T3/patología , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Citoesqueleto/genética , Citoesqueleto/ultraestructura , ADN Complementario , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endotelio Vascular/patología , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas de Unión al GTP/genética , Genes ras , Células Madre Hematopoyéticas/fisiología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Hibridomas , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Receptores de Superficie Celular/genética , Factor de Respuesta Sérica , Transducción de Señal , Transcripción Genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The congenital fibrosarcoma t(12;15)(p13;q25) rearrangement splices the ETV6 (TEL) gene on chromosome 12p13 in frame with the NTRK3 (TRKC) neurotrophin-3 receptor gene on chromosome 15q25. Resultant ETV6-NTRK3 fusion transcripts encode the helix - loop - helix (HLH) dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. We show here that ETV6-NTRK3 homodimerizes and is capable of forming heterodimers with wild-type ETV6. Moreover, ETV6-NTRK3 has PTK activity and is autophosphorylated on tyrosine residues. To determine if the fusion protein has transforming activity, NIH3T3 cells were infected with recombinant retroviral vectors carrying the full-length ETV6-NTRK3 cDNA. These cells exhibited a transformed phenotype, grew macroscopic colonies in soft agar, and formed tumors in severe combined immunodeficient (SCID) mice. We hypothesize that chimeric proteins mediate transformation by dysregulating NTRK3 signal transduction pathways via ligand-independent dimerization and PTK activation. To test this hypothesis, we expressed a series of ETV6-NTRK3 mutants in NIH3T3 cells and assessed their transformation activities. Deletion of the ETV6 HLH domain abolished dimer formation with either ETV6 or ETV6-NTRK3, and cells expressing this mutant protein were morphologically non-transformed and failed to grow in soft agar. An ATP-binding mutant failed to autophosphorylate and completely lacked transformation activity. Mutants of the three NTRK3 PTK activation-loop tyrosines had variable PTK activity but had limited to absent transformation activity. Of a series of signaling molecules well known to bind to wild-type NTRK3, only phospholipase-Cgamma (PLCgamma) associated with ETV6-NTRK3. However, a PTK active mutant unable to bind PLCgamma did not show defects in transformation activity. Our studies confirm that ETV6-NTRK3 is a transforming protein that requires both an intact dimerization domain and a functional PTK domain for transformation activity. Oncogene (2000) 19, 906 - 915.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Línea Celular Transformada/enzimología , Proteínas de Unión al ADN/genética , Receptor trkC/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Represoras , Factores de Transcripción/genética , Translocación Genética , Células 3T3 , Animales , Línea Celular Transformada/metabolismo , Proteína Adaptadora GRB2 , Secuencias Hélice-Asa-Hélice/genética , Humanos , Isoenzimas/metabolismo , Ratones , Ratones SCID , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa C gamma , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Receptor trkC/biosíntesis , Receptor trkC/química , Receptor trkC/metabolismo , Fosfolipasas de Tipo C/metabolismo , Dominios Homologos src/genética , Proteína ETS de Variante de Translocación 6RESUMEN
We used genetic strategies which have been proven valuable to decipher signaling pathways in comparatively simple organisms such as Drosophila and Caenorhabditis elegans, to dissect signaling network activated by tyrosine kinases in mammals. The strategy was developed further towards a generally applicable expression cloning system to identify signal transducers in tyrosine kinase pathways. This system is based on the ability of downstream acting genes to rescue the transformation phenotype of partial loss-of-function mutants of BCR-ABL which still retain tyrosine kinase activity. Using this strategy we have previously shown that overexpression of c-Myc and Cyclin D1 can rescue a signaling defective SH2 mutant of BCR-ABL for transformation. In an unbiased approach to identify new compensating genes, a cDNA library was introduced by retroviral infection into fibroblasts which express the BCR-ABL SH2 mutant. CDNA clones, capable of rescuing the SH2 mutant for transformation should result in colony formation in soft agar. A PCR approach was used to recover these compensating genes from the genomic DNA of the transformed fibroblasts. Sequencing analysis of the initial cDNAs identified three known genes, the adapter molecule Shc, the kinases SPRK and p38 MAPK. These genes have been found to interact functionally with BCR-ABL for fibroblast and hematopoietic cell transformation. Currently, we are constructing and screening new libraries to identify novel genes which complement the BCR-ABL SH2 mutant. Our results demonstrate that this cloning approach is an effective means of identifying and characterizing signaling molecules that function in specific signaling pathways. This in turn may identify specific targets for mechanism-based therapeutic intervention to block altered signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Proteínas de Fusión bcr-abl/genética , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/genética , Animales , Bioensayo , Caenorhabditis elegans , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Transformación Celular Viral/genética , Drosophila melanogaster , Proteínas de Fusión bcr-abl/fisiología , Ratones , Oncogenes/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas/genética , Proteínas/fisiología , Retroviridae/genética , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.
Asunto(s)
Proteínas de Unión al ADN/genética , Factores de Intercambio de Guanina Nucleótido , Acetato de Tetradecanoilforbol/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Análisis Mutacional de ADN , ADN Complementario/genética , Activación Enzimática/fisiología , Expresión Génica/genética , Humanos , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación Puntual/genética , ARN Mensajero/genética , Retroviridae/genética , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Transformación Genética/genéticaRESUMEN
We have characterized a flat cellular variant of HTLV-1 Tax-transformed rat fibroblasts, 5R, which is unresponsive to all tested NF-kappaB activating stimuli, and we report here its genetic complementation. The recovered full-length cDNA encodes a 48 kDa protein, NEMO (NF-kappaB Essential MOdulator), which contains a putative leucine zipper motif. This protein is absent from 5R cells, is part of the high molecular weight IkappaB kinase complex, and is required for its formation. In vitro, NEMO can homodimerize and directly interacts with IKK-2. The NEMO cDNA was also able to complement another NF-kappaB-unresponsive cell line, 1.3E2, in which the protein is also absent, allowing us to demonstrate that this factor is required not only for Tax but also for LPS, PMA, and IL-1 stimulation of NF-kappaB activity.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Prueba de Complementación Genética , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/química , Factores de Transcripción/fisiología , Activación Transcripcional/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular Transformada , Clonación Molecular/métodos , ADN Complementario/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Fibroblastos , Productos del Gen tax , Virus Linfotrópico T Tipo 1 Humano , Quinasa I-kappa B , Leucina Zippers , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/biosíntesis , Ratas , Análisis de Secuencia de ADN , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Substitution of phenylalanine for tyrosine at codon 809 (Y809F) of the human colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) impairs ligand-stimulated tyrosine kinase activity, prevents induction of c-MYC and cyclin D1 genes, and blocks CSF-1-dependent progression through the G1 phase of the cell cycle. We devised an unbiased genetic screen to isolate genes that restore the ability of CSF-1 to stimulate growth in cells that express mutant CSF-1R (Y809F). This screen led us to identify a truncated form of the murine type Ibeta phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta). This truncated protein lacks residues 1 to 238 of mPIP5K-Ibeta and is catalytically inactive. When we transfected cells expressing CSF-1R (Y809F) with mPIP5K-Ibeta (delta1-238), CSF-1-dependent induction of c-MYC and cyclin D1 was restored and ligand-dependent cell proliferation was sustained. CSF-1 normally triggers the rapid disappearance of CSF-1R (Y809F) from the cell surface; however, transfection of cells with mPIP5K-Ibeta (delta1-238) stabilized CSF-1R (Y809F) expression on the cell surface, resulting in elevated levels of ligand-activated CSF-1R (Y809F). These results suggest a role for PIP5K-Ibeta in receptor endocytosis and that the truncated enzyme compensated for a mitogenically defective CSF-1R by interfering with this process.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptores del Factor Estimulante de Colonias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular , Línea Celular , Factores Estimulantes de Colonias/farmacología , Ciclina D1/genética , Cartilla de ADN/genética , ADN Complementario/genética , Endocitosis , Genes myc , Prueba de Complementación Genética , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Transducción de SeñalRESUMEN
The role of the C-terminal domain of CTP: phosphocholine cytidylyltransferase (CT) was explored by the creation of a series of deletion mutations in rat liver cDNA, which were expressed in COS cells as a major protein component. Deletion of up to 55 amino acids from the C-terminus had no effect on the activity of the enzyme, its stimulation by lipid vesicles or on its intracellular distribution between soluble and membrane-bound forms. However, deletion of the C-terminal 139 amino acids resulted in a 90% decrease in activity, loss of response to lipid vesicles and a significant decrease in the fraction of membrane-bound enzyme. Identification of the domain that is phosphorylated in vivo was determined by analysis of 32P-labelled CT mutants and by chymotrypsin proteolysis of purified CT that was 32P-labelled in vivo. Phosphorylation was restricted to the C-terminal 52 amino acids (domain P) and occurred on multiple sites. CT phosphorylation in vitro was catalysed by casein kinase II, cell division control 2 kinase (cdc2 kinase), protein kinases C alpha and beta II, and glycogen synthase kinase-3 (GSK-3), but not by mitogen-activated kinase (MAP kinase). Casein kinase II phosphorylation was directed exclusively to Ser-362. The sites phosphorylated by cdc2 kinase and GSK-3 were restricted to several serines within three proline-rich motifs of domain P. Sites phosphorylated in vitro by protein kinase C, on the other hand, were distributed over the N-terminal catalytic as well as the C-terminal regulatory domain. The stoichiometry of phosphorylation catalysed by any of these kinases was less than 0.2 mol P/mol CT, and no effects on enzyme activity were detected. This study supports a tripartite structure for CT with an N-terminal catalytic domain and a C-terminal regulatory domain comprised of a membrane-binding domain (domain M) and a phosphorylation domain (domain P). It also identifies three kinases as potential regulators in vivo of CT, casein kinase II, cyclin-dependent kinase and GSK-3.
Asunto(s)
Hígado/enzimología , Nucleotidiltransferasas/metabolismo , Eliminación de Secuencia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Proteína Quinasa CDC2/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasa de la Caseína II , División Celular , Chlorocebus aethiops , Citidililtransferasa de Colina-Fosfato , Cartilla de ADN , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Isoenzimas/metabolismo , Riñón , Cinética , Datos de Secuencia Molecular , Mutagénesis , Mutagénesis Sitio-Dirigida , Nucleotidiltransferasas/análisis , Nucleotidiltransferasas/biosíntesis , Fosfopéptidos/análisis , Fosforilación , Mutación Puntual , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Human CTP:phosphocholine cytidylyltransferase (CT) cDNAs were isolated by PCR amplification of a human erythroleukemic K562 cell library. Initially two degenerate oligonucleotide primers derived from the sequence of the rat liver CT cDNA were used to amplify a centrally located 230 bp fragment. Subsequently overlapping 5' and 3' fragments were amplified, each using one human CT primer and one vector-specific primer. Two cDNAs encoding the entire translated domain were also amplified. The human CT (HCT) has close homology at the nucleotide and amino acid level with other mammalian CTs (from rat liver, mouse testis or mouse B6SutA hemopoietic cells and Chinese hamster ovary). The region which deviates most from the rat liver CT sequence is near the C-terminus, where 7 changes are clustered within 34 residues (345-359), of the putative phosphorylation domain. The region of the proposed catalytic domain (residues 75-235) is 100% identical with the rat liver sequence. Significant homology was observed between the proposed catalytic domain of CT and the Saccharomyces cerevisiae MUQ1 gene product, and between the proposed amphipathic alpha-helical membrane binding domains of CT and soybean oleosin, a phospholipid-binding protein. There are several shared characteristics of these amphipathic helices. An approx. 42,000 Da protein was over-expressed in COS cells using a pAX142 expression vector containing one of the full-length HCT cDNA clones. The specific activity of the HCT in COS cell homogenates was the same as that of analogously expressed rat liver CT. The activity of HCT was lipid dependent. The soluble form was activated 3 to 4-fold by anionic phospholipids and by oleic acid or diacylglycerol-containing PC vesicles.
Asunto(s)
Nucleotidiltransferasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citidililtransferasa de Colina-Fosfato , Cricetinae , Cricetulus , Cartilla de ADN/química , Activación Enzimática , Humanos , Metabolismo de los Lípidos , Ratones , Datos de Secuencia Molecular , Fosforilación , Estructura Secundaria de Proteína , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The murine interleukin 3 receptor (mIL-3R) is a heterodimer consisting of a 70-kDa alpha subunit and one of two alternative 120-kDa beta subunits termed beta IL-3 and beta c. beta IL-3 (originally called Aic2A) is capable of binding mIL-3 by itself, whereas beta c (Aic2B) does not bind any ligand on its own but increases the affinity of mIL-3, murine granulocyte/macrophage-colony-stimulating factor, and mIL-5 for their respective alpha subunits. Interestingly, although the mIL-3R does not possess tyrosine kinase activity, its beta IL-3 subunit does become tyrosine phosphorylated upon binding mIL-3. To further investigate the properties of this subunit, we have purified it from the cell line B6SUtA1, which expresses a high level of mIL-3R. Intriguingly, studies comparing the stability of the 140-kDa, tyrosine-phosphorylated form of this subunit with its 120-kDa, non-tyrosine-phosphorylated form reveal that the former is far less stable and is rapidly degraded to a 70-kDa fragment. Mixing experiments demonstrate that the differential stability of the two forms is due to an intrinsic difference in protease susceptibility. Phosphatase studies indicate that the higher protease susceptibility of the tyrosine-phosphorylated beta IL-3 is due to the presence of both phosphotyrosine and phosphoserine residues. Western analyses using an anti-N-terminal mIL-3R beta IL-3 chain antibody reveal that this proteolytic cleavage also occurs rapidly in intact cells following stimulation with mIL-3 and occurs at the cell surface, since it takes place within minutes at 37 degrees C, is observed with purified plasma membranes, and is not inhibited by chloroquine. This degradative step may play an important role in the mechanism of action of mIL-3.
Asunto(s)
Interleucina-3/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Interleucina-3/metabolismo , Alcaloides/farmacología , Animales , Western Blotting , Línea Celular , Cloroquina/farmacología , Genisteína , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-3/farmacología , Interleucina-5/metabolismo , Isoflavonas/farmacología , Cinética , Ligandos , Sustancias Macromoleculares , Ratones , Peso Molecular , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores de Interleucina-3/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Estaurosporina , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
In this report we describe the purification of the murine interleukin 3 receptor (mIL-3R) to apparent homogeneity using a two-step procedure involving biotinylated mIL-3 (B-mIL-3) and affinity binding to immobilized antiphosphotyrosine and streptavidin agarose (SA). Purification was monitored using an assay for detergent solubilized-mIL-3Rs that utilized unglycosylated 125I-mIL-3 and concanavalin A (ConA)-Sepharose beads. The final material consisted of a 140-kDa tyrosine and serine phosphorylated protein that was greater than 98% pure as assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of either [35S]methionine-labeled, silver-stained, or radioiodinated preparations. Characterization of the purified receptor revealed that it migrated identically under reducing and nonreducing conditions in SDS gels, possessed 10 kDa of N-linked carbohydrate, and was cleaved upon storage at 4 degrees C to a 70-kDa form. These properties suggested that the purified mIL-3R was identical to that identified by cross-linking studies. The KD of the purified receptor was 1-5 nM, similar to estimates obtained using intact normal mouse bone marrow cells and mIL-3-dependent cell lines. The two-step purification procedure also isolated a 120-kDa serine phosphorylated but nontyrosine phosphorylated mIL-3R species. Apart from phosphorylation differences, the 140- and 120-kDa species were apparently identical, yielding, after alkaline phosphatase treatment, the same molecular mass on SDS gels and similar chymotryptic peptide maps. Amino acid sequences and composition data obtained from the more abundant and more stable serine phosphorylated 120-kDa mIL-3R, further purified by SDS-polyacrylamide gel electrophoresis, suggested that the purified mIL-3R may be identical to the predicted sequence of the recently isolated cDNA clone AIC2A. This was further suggested by comparing chymotryptic maps of the 120-kDa mIL-3R with the Aic2A protein and using antibodies corresponding to the amino and carboxyl termini of the AIC2A cDNA product. However, the Aic2A protein, when expressed on the surface of COS or 3T3 cells or following detergent solubilization and partial purification with biotinylated mIL-3 and SA, displayed a substantially lower affinity for mIL-3.
Asunto(s)
Interleucina-3/metabolismo , Receptores de Interleucina-3/aislamiento & purificación , Células 3T3 , Aminoácidos/análisis , Animales , Línea Celular , Membrana Celular/inmunología , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Interleucina-3/aislamiento & purificación , Cinética , Ratones , Peso Molecular , Mapeo Peptídico , Fosforilación , Receptores de Interleucina-3/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified, but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved, we performed Northern analyses on RNA extracted from human marrow LTC adherent layers, or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL-1 beta), IL-6, and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF, GM-CSF, and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast, transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC, and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures, respectively. Together, these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC, with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF, GM-CSF, and IL-6. Given the similarities between the LTC system and the marrow microenvironment, it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells.
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Células de la Médula Ósea , Citocinas/fisiología , Sistema Hematopoyético/citología , Northern Blotting , Médula Ósea/metabolismo , Médula Ósea/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Sistema Hematopoyético/efectos de los fármacos , Sistema Hematopoyético/metabolismo , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) is a key regulatory enzyme in the synthesis of phosphatidylcholine in higher eukaryotes. This enzyme can interconvert between an inactive cytosolic form and an active membrane-bound form. To unravel the structure of the transferase and the mechanism of its interaction with membranes, we have cloned a cytidylyltransferase cDNA from rat liver by the oligonucleotide-directed polymerase chain reaction. Transfection of the rat clone into COS cells resulted in a 10-fold increase in cytidylyltransferase activity and content. The activity of the transfected transferase was lipid-dependent. The central portion of the derived protein sequence of the rat clone is highly homologous to the previously determined yeast cytidylyltransferase sequence [Tsukagoshi, Y., Nikawa, J. & Yamashita, S. (1987) Eur. J. Biochem. 169, 477-486]. The rat protein sequence lacks any signals for covalent lipid attachment and lacks a hydrophobic domain long enough to span a bilayer. However, it does contain a potential 58-residue amphipathic alpha-helix, encompassing three homologous 11-residue repeats. We propose that the interaction of cytidylyltransferase with membranes is mediated by this amphipathic helix lying on the surface with its axis parallel to the plane of the membrane such that its hydrophobic residues intercalate the phospholipids.
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Expresión Génica , Hígado/enzimología , Nucleotidiltransferasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Citidililtransferasa de Colina-Fosfato , Clonación Molecular , Biblioteca de Genes , Masculino , Modelos Estructurales , Datos de Secuencia Molecular , Nucleotidiltransferasas/aislamiento & purificación , Nucleotidiltransferasas/metabolismo , Sondas de Oligonucleótidos , Fosfatidilcolinas/biosíntesis , Reacción en Cadena de la Polimerasa , Conformación Proteica , Ratas , Ratas Endogámicas , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
Monoclonal antibody (mAb) 114/A10, raised against the murine bone marrow-derived multipotential hemopoietic progenitor cell line B6SUtA, identifies an antigen highly expressed by various interleukin-3 (IL-3)-dependent cell lines, the myelomonocytic cell line WEHI-3, and a large proportion of primary myeloid and erythroid colony-forming cells. Spleen- and bone marrow-derived 114/A10-positive cells were shown to selectively proliferate in vitro in response to pokeweed mitogen-stimulated spleen cell-conditioned medium or recombinant IL-3. Western blot analysis indicated that the antigen recognized by mAb 114/A10 has a mean relative molecular mass of approximately 150,000, although it is extremely heterogeneous in nature, and differs greatly in size range among different cell lines.
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Antígenos de Superficie/análisis , Células de la Médula Ósea , Factor de Crecimiento Epidérmico , Eritrocitos/citología , Interleucina-3/farmacología , Células Madre/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Médula Ósea/inmunología , División Celular , Línea Celular , Eritrocitos/inmunología , Ratones , Peso Molecular , Bazo/citologíaRESUMEN
Monoclonal antibody 114/A10, raised against the murine multipotential hemopoietic progenitor cell line B6SUtA, identifies an antigen highly expressed by primary myeloid progenitor cells, the myelomonocytic leukemia cell line WEHI-3, and various interleukin-3 (IL-3)-dependent cell lines. Western blotting studies indicate that the 114/A10 antigen has a mean relative molecular mass of 150,000 but varies greatly in size range between different cell types. cDNA clones encoding this protein were isolated from a plasmid-based expression vector library prepared from B6SUtA RNA. Three clones corresponding in size to the two major mRNA species detected in IL-3-dependent cell lines (3.0 and 2.2 kilobase pairs) and differing in their utilization of alternative polyadenylation signals were obtained. These clones contain a single long open reading frame of 573 amino acids possessing the typical characteristics of an integral membrane protein. A particularly striking feature of this sequence is the presence at the extracellular amino terminus of a series of eight highly conserved 27-amino acid, serine/threonine-rich (55%) tandem repeats that may serve as sites of extensive glycosylation. The extracellular domain also contains three epidermal growth factor-like cysteine-rich repeats. The distribution and structural characteristics of the 114/A10 antigen suggest a possible regulatory role in the cellular response to IL-3.
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Antígenos de Superficie/genética , Células Madre Hematopoyéticas/fisiología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Northern Blotting , Línea Celular , Clonación Molecular , ADN/genética , Factor de Crecimiento Epidérmico/genética , Regulación de la Expresión Génica , Interleucina-3/farmacología , Datos de Secuencia Molecular , Peso MolecularRESUMEN
Exposure of the nematode Caenorhabditis elegans to a heat shock results in the induction of a number of genes not normally expressed in the animals under normal growth conditions. Among these are a family of genes encoding 16 kDa heat shock proteins (hsp16s). The major hsp16 genes have been cloned and characterized, and found to reside at two clusters in the C. elegans genome. One cluster contains two distinct genes, hsp16-1 and hsp16-48, arranged in divergent orientations separated by only 348 base pairs (bp). An identical pair, duplicated and inverted with respect to the first pair, is located 415 bp away. This cluster, located on chromosome V, therefore contains four genes as two identical pairs within less than 4 kilobases of DNA, and the pairs form the arms of a large inverted repeat. A second pair of genes, hsp16-2 and hsp16-41, constitutes a second hsp16 locus with an organization very similar to that of the hsp16-1/48 locus, except that it is not duplicated. Comparisons of the derived amino acid sequences show that hsp16-1 and hsp16-2 form a closely related pair, as do hsp16-41 and hsp16-48. These hsps show extensive sequence identity with the small hsps of Drosophila, as well as with mammalian alpha-crystallins. The coding region of each gene is interrupted by a single intron of approximately 50 bp, in a position homologous to that of the first intron in mouse alpha-crystallin gene.(ABSTRACT TRUNCATED AT 250 WORDS)
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Caenorhabditis/genética , Proteínas de Choque Térmico/genética , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis/embriología , Regulación de la Expresión Génica , Genes , Proteínas de Choque Térmico/biosíntesis , Calor , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
Splicing of a pair of intron-containing heat shock genes from Caenorhabditis elegans has been studied in transfected mouse cells. The hsp16-1 and hsp16-48 genes of C. elegans encode 16,000 Da heat shock polypeptides. Each gene contains a short intron of 52 (hsp16-1) or 55 (hsp16-48) base pairs. When these genes were introduced into mouse cells, they were efficiently induced following heat shock, but splicing of the introns was abnormal. In mouse cells, cleavage of the hsp16 transcripts occurred at the correct 5' splice sites, but the 3' splice sites were located at AG dinucleotides downstream of the correct sites. This aberrant splicing was not solely due to the small size of the C. elegans introns, since a hsp16-1 gene containing an intron enlarged by tandem duplication showed exactly the same splicing pattern. The mouse cells thus seem to be unable to recognize the natural 3' splice sites of the C. elegans transcripts. The efficiency of splicing was greatly reduced under heat shock conditions, and unspliced transcripts accumulated in the nucleus. During a subsequent recovery period at 37 degrees C, these transcripts were spliced and transported to the cytoplasm.
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Caenorhabditis/genética , Proteínas de Choque Térmico/biosíntesis , Empalme del ARN , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Animales , Proteínas de Choque Térmico/genética , Intrones , Ratones , Proteínas Recombinantes/genética , Especificidad de la Especie , TemperaturaRESUMEN
A divergently transcribed pair of Caenorhabditis elegans hsp16 genes was introduced into mouse fibroblasts by stable transfection with vectors containing bovine papillomavirus plasmid maintenance sequences and a selectable gene. The hsp16 genes were transcriptionally inactive in the mouse cells under normal growth conditions and were strongly induced by heat shock or arsenite. In a cell line with 12 copies of the gene pair, there were estimated to be more than 10,000 hsp16 transcripts in each cell after 2 h of heat shock treatment. The hsp16 transcript levels were more than 100 times higher than those of a gene with a herpes simplex virus thymidine kinase gene promoter carried on the same vector. A single heat shock promoter element (HSE) could activate bidirectional transcription of the two hsp16 genes when placed between the two TATA elements, but the transcriptional efficiency was reduced 10-fold relative to that of the wild-type gene pair. Four overlapping HSEs positioned between the two TATA elements resulted in inducible bidirectional transcription at greater than wild-type levels. The number of HSEs can therefore be a major determinant of the promoter strength of heat-inducible genes in mammalian cells. Partial disruption of an alternating purine-pyrimidine sequence between the two hsp16 genes had no significant effect on their transcriptional activity.