RESUMEN

PURPOSE: Multiparametric MRI (mpMRI) has a potential role for the identification of aggressive cancer that can be targeted for biopsy. We report the incidence and severity of discordant information between the pathology found on the transrectal ultrasound (TRUS)-guided biopsy and the mpMRI findings in patients with favorable or intermediate-risk prostate cancer referred for brachytherapy. METHODS AND MATERIALS: From March 2014 to September 2015, 10/44 consecutive patients with low- or intermediate-risk prostate cancer referred for brachytherapy presented an aggressive lesion on mpMRI and underwent an MRI-TRUS fusion-guided transperineal biopsy of the index lesion. RESULTS: A median of two intraprostatic lesions were detected by mpMRI for each patient. Three patients had bilateral disease, and seven had unilateral disease on mpMRI. The median number of cores obtained by MRI-TRUS-guided fusion of the index lesion was 3 (range 2-4). As a result of the re-evaluation consequent to additional information becoming available after the transperineal biopsy, upgrading of Gleason score occurred in 8 of the 10 patients, which changed the risk group in 9 patients. These changes resulted in modification of the proposed treatment in 8 patients. CONCLUSIONS: MpMRI-US fusion-targeted biopsy sampling allows detection and characterization of otherwise undetected aggressive disease, often placing men in higher risk groups and altering the treatment approach.

Asunto(s)

Adenocarcinoma/patología , Neoplasias de la Próstata/patología , Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/radioterapia , Anciano , Biopsia con Aguja Gruesa , Braquiterapia , Endosonografía , Humanos , Biopsia Guiada por Imagen , Imagen por Resonancia Magnética , Masculino , Clasificación del Tumor , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/radioterapia

RESUMEN

OBJECTIVES: Adult mesenchymal stem cells were recently found to suppress effector T cell and inflammatory responses and have emerged as attractive therapeutic candidates for immune disorders. In rheumatoid arthritis (RA), a loss in the immunological self-tolerance causes the activation of autoreactive T cells against joint components and subsequent chronic inflammation. The aim of this study is to characterise the immunosuppressive activity of human adipose-derived mesenchymal stem cells (hASCs) on collagen-reactive T cells from patients with RA. METHODS: The effects of hASCs on collagen-reactive RA human T cell proliferation and cytokine production were investigated, as well as effects on the production of inflammatory mediators by monocytes and fibroblast-like synoviocytes from patients with RA. RESULTS: hASCs suppressed the antigen-specific response of T cells from patients with RA. hASCs inhibited the proliferative response and the production of inflammatory cytokines by collagen-activated CD4 and CD8 T cells. In contrast, the numbers of IL10-producing T cells and monocytes were significantly augmented upon hASC treatment. The suppressive activity of hASCs was cell-to-cell contact dependent and independent. hASCs also stimulated the generation of FoxP3 protein-expressing CD4(+)CD25(+) regulatory T cells, with the capacity to suppress collagen-specific T cell responses. Finally, hASCs downregulated the inflammatory response and the production of matrix-degrading enzymes by synovial cells isolated from patients with RA. CONCLUSIONS: The present work identifies hASCs as key regulators of immune tolerance, with the capacity to suppress T cell and inflammatory responses and to induce the generation/activation of antigen-specific regulatory T cells.

Asunto(s)

Artritis Reumatoide/inmunología , Mediadores de Inflamación/metabolismo , Células Madre Mesenquimatosas/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Tejido Adiposo/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Femenino , Humanos , Tolerancia Inmunológica/inmunología , Activación de Linfocitos/inmunología , Masculino

RESUMEN

BACKGROUND AND AIMS: Inflammatory bowel diseases (IBDs) are associated with uncontrolled innate and adaptive immunity against normal constituents, including commensal bacteria and microbial products. Mesenchymal stem cells (MSCs) suppress effector T cell responses and have beneficial effects in various immune disorders. This work investigates the therapeutic effects of human adipose-derived MSCs (hASCs) in various models of IBD and sepsis. METHODS: Acute and chronic colitis was induced in mice with dextran sulfate sodium. Sepsis was induced by caecal ligation and puncture or by endotoxin injection. Colitic and septic mice were treated intraperitoneally with hASCs or murine ASCs, and diverse disease clinical signs and mortality were determined. The levels of various inflammatory cytokines and chemokines, T helper 1(Th1)-type response and generation of regulatory T cells (Treg) were determined in affected organs. RESULTS: Systemic infusion of ASCs significantly ameliorated the clinical and histopathological severity of colitis, abrogating weight loss, diarrhoea and inflammation, and increasing survival. The therapeutic effect was associated with downregulation of the Th1-driven inflammatory responses. ASCs decreased a wide panel of inflammatory cytokines and chemokines and increased interleukin 10 (IL10), acting on macrophages. hASCs also impaired Th1 cell activation in both colonic mucosa and draining lymph nodes. The induction of IL10-secreting Treg was partially involved in the therapeutic effect of hASCs. Moreover, ASCs protected from severe sepsis by reducing the infiltration of inflammatory cells in various target organs and by downregulating the production of various inflammatory mediators. CONCLUSIONS: hASCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for IBD and sepsis.

Asunto(s)

Tejido Adiposo/citología , Colitis/cirugía , Enfermedades Inflamatorias del Intestino/cirugía , Trasplante de Células Madre Mesenquimatosas , Sepsis/cirugía , Linfocitos T Reguladores/inmunología , Células Madre Adultas/inmunología , Animales , Células Cultivadas , Colitis/inmunología , Regulación hacia Abajo , Endotoxemia/prevención & control , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Células Madre Mesenquimatosas/inmunología , Ratones , Ratones Endogámicos C57BL , Sepsis/inmunología

RESUMEN

Wave-front sensing from artificial beacons is normally performed by formation of a focused spot in the atmosphere and sensing of the wave-front distortions produced during the beam's return passage. We propose an alternative method that senses the distortions produced during the outgoing path by forming an intensity pattern in the atmosphere that is then viewed from the ground. A key advantage of this method is that a parallel beam is used, and therefore the wave-front measurements will not suffer from the effects of focal anisoplanatism. We also envisage other geometries, all based on the concept of projecting a pupil pattern onto the atmosphere.

RESUMEN

A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Here, we show that three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm and the lateral plate mesoderm in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, we also show that the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo.

Asunto(s)

Embrión de Pollo/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Esbozos de los Miembros/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transactivadores , Proteínas de Pez Cebra , Animales , Proteínas Aviares , Proteínas del Citoesqueleto/fisiología , Ectodermo/fisiología , Factor 10 de Crecimiento de Fibroblastos , Factor 8 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/fisiología , Mitógenos/fisiología , Morfogénesis , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Alas de Animales/embriología , Proteínas Wnt , Proteína wnt2 , beta Catenina

RESUMEN

Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.

Asunto(s)

Filamentos Intermedios/ultraestructura , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Vimentina/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Activación Enzimática , Datos de Secuencia Molecular , Mapeo Peptídico , Fosfopéptidos/aislamiento & purificación , Fosforilación , Unión Proteica

RESUMEN

We present results of the isoplanatic performance of an astronomical adaptive optics system in the laboratory, by using a dual layer turbulence simulator. We describe how the performance of adaptive correction degrades with off--axis angle. These experiments demonstrate that it is now possible to produce quantifiable multi-layer turbulence in the laboratory as a precursor to constructing multi-conjugate adaptive optics.

Asunto(s)

Amalgama Dental/farmacocinética , Intoxicación por Mercurio/orina , Mercurio/orina , Carga Corporal (Radioterapia) , Exposición a Riesgos Ambientales , Femenino , Humanos , Mercurio/farmacocinética , Intoxicación por Mercurio/diagnóstico , Modelos Biológicos , Porfirinas/orina , Distribución Tisular

RESUMEN

IkappaB kinases (IKKs) IKK1 and IKK2 are two putative IkappaBalpha kinases involved in NF-kappaB activation. To examine the in vivo functions of IKK1, we generated IKK1-deficient mice. The mutant mice are perinatally lethal and exhibit a wide range of developmental defects. Newborn mutant mice have shiny, taut, and sticky skin without whiskers. Histological analysis shows thicker epidermis, which is unable to differentiate. Limbs and tail are wrapped inside the skin and do not extend properly out of the body trunk. Skeleton staining reveals a cleft secondary palate, split sternebra 6, and deformed incisors. NF-kappaB activation mediated by TNFalpha and IL-1 is diminished in IKK1-deficient mouse embryonic fibroblast (MEF) cells. The IKK complex in the absence of IKK1 is capable of phosphorylating IkappaBalpha and IkappaBbeta in vitro. Our results support a role for IKK1 in NF-kappaB activation and uncover its involvement in skin and skeleton development. We conclude further that the two related kinases IKK1 and IKK2 have distinct functions and can not be substituted for each other's functions.

Asunto(s)

Huesos/anomalías , Proteínas Serina-Treonina Quinasas/fisiología , Anomalías Cutáneas/genética , Animales , Embrión de Mamíferos/anatomía & histología , Epidermis/anatomía & histología , Quinasa I-kappa B , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutagénesis , FN-kappa B/metabolismo , Fenotipo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Limb development has become one of the model systems for studying vertebrate development. One crucial aspect in limb development is the origin, differentiation and patterning of muscle. Much progress has been made in recent years towards understanding this process. One of the general observations is that the genes involved in limb muscle development appear to be very similar to those involved in muscle development in other regions of the embryo. In this review, we summarize some of the genes and mechanisms that regulate limb muscle development and discuss various avenues along which a deeper understanding can be gained of how muscle cells originate and differentiate in different tissues during vertebrate development.

Asunto(s)

Tipificación del Cuerpo , Extremidades/embriología , Músculo Esquelético/embriología , Vertebrados/embriología , Animales , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Modelos Biológicos , Proteínas Musculares/genética , Músculo Esquelético/citología

RESUMEN

The body plan is generated by cells and tissues that become arranged precisely in the embryo. This process, termed pattern formation, involves cell interactions in which a particular group of cells produce signals that specify new cell types or patterns of differentiation in responding cells. These patterning signals emanate from very discrete centers called "organizer centers," such as the Hensen's node or Spemann organizer, the midbrain-hindbrain junction, the notochord, or in the case of the limb, the zone of polarizing activity (ZPA) or the apical ectodermal ridge (AER). The developing vertebrate limb is an ideal model system for the study of pattern formation because, in addition to surgical manipulations, molecular manipulations are now feasible. In this review we summarize early experiments that established, by means of surgical manipulations, the different organizer centers of the vertebrate limb: the ectoderm covering the limb bud, the apical ectodermal ridge, the zone of polarizing activity, and the distal mesoderm (progress zone) underlying the AER. We then describe the domains of expression of various genes present during the development of the limb and discuss some of the functional approaches (overexpression and lack of function studies) undertaken to ascertain their role in limb outgrowth. The knowledge acquired in the last few years has had an enormous impact not only on our view of how limbs develop (perhaps now one of the most approachable vertebrate model systems) but also in a more general sense of how the embryo is organized in space and time.

Asunto(s)

Tipificación del Cuerpo/genética , Extremidades/embriología , Vertebrados/embriología , Animales , Tipificación del Cuerpo/fisiología , Embrión de Pollo , Ectodermo/citología , Ectodermo/trasplante , Factores de Crecimiento de Fibroblastos/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Mesodermo/trasplante , Vertebrados/genética

RESUMEN

We have determined that Strong's Luxoid (lstJ) [corrected] mice have a 16 bp deletion in the homeobox region of the Alx-4 gene. This deletion, which leads to a frame shift and a truncation of the Alx-4 protein, could cause the polydactyly phenotype observed in lstJ [corrected] mice. We have cloned the chick homologue of Alx-4 and investigated its expression during limb outgrowth. Chick Alx-4 displays an expression pattern complementary to that of shh, a mediator of polarizing activity in the limb bud. Local application of Sonic hedgehog (Shh) and Fibroblast Growth Factor (FGF), in addition to ectodermal apical ridge removal experiments suggest the existence of a negative feedback loop between Alx-4 and Shh during limb outgrowth. Analysis of polydactylous mutants indicate that the interaction between Alx-4 and Shh is independent of Gli3, a negative regulator of Shh in the limb. Our data suggest the existence of a negative feedback loop between Alx-4 and Shh during vertebrate limb outgrowth.

Asunto(s)

Tipificación del Cuerpo , Extremidades/embriología , Proteínas de Homeodominio/genética , Deformidades Congénitas de las Extremidades , Proteínas del Tejido Nervioso , Proteínas Represoras , Transactivadores , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Clonación Molecular , Proteínas de Unión al ADN , Extremidades/cirugía , Retroalimentación , Factores de Crecimiento de Fibroblastos , Proteínas Hedgehog , Factores de Transcripción de Tipo Kruppel , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Polidactilia/genética , Proteínas , Análisis de Secuencia de ADN , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Trasplante de Tejidos , Factores de Transcripción , Proteína Gli3 con Dedos de Zinc

Asunto(s)

Anomalías Múltiples/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas del Tejido Nervioso , Proteínas Represoras , Factores de Transcripción , Proteínas de Xenopus , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión al ADN/química , Humanos , Factores de Transcripción de Tipo Kruppel , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Transcripción Genética , Proteína Gli3 con Dedos de Zinc , Dedos de Zinc

RESUMEN

Gli genes represent a small family, encoding zinc-finger proteins of the Krüppel-type. The family consists of Gli(1), Gli2, and Gli3, all of which are expressed in the developing mouse limb bud. To assess the role of the Gli family and Sonic hedgehog (Shh) in mouse limb development, we compared the expression domains of all three Gli genes and of Shh. Although each Gli gene has its own distinct expression pattern in limb buds, at 10.5-11.5 dpc all three genes were found not to be expressed in the posterior region, the presumptive Shh expression domain. This transient mutually exclusive expression suggested a potential interaction between Gli genes and Shh. To address this matter, we analysed the expression of Gli genes and Shh in two polydactyly mouse mutants, Extra toes (Xt) and Hemimelic-extra toes (Hx) which express Shh ectopically in the anterior region of the limb field. Since Xt mice lack Gli3 expression, the ectopic Shh expression is genetically linked to the absence of Gli3. In Hx mice we found a down-regulation of Gli3 in the anterior region of the limb bud. In both mutants Gli2 expression pattern was not altered, whereas Gli1 expression was anteriorly up-regulated adjacent to the ectopic Shh domain. These results strongly suggest a positive regulation of Gli1 by Shh and a negative interaction between Shh and Gli3.

Asunto(s)

Extremidades/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso , Proteínas Oncogénicas/genética , Proteínas/genética , Proteínas Represoras , Transactivadores , Factores de Transcripción/genética , Proteínas de Xenopus , Animales , Tipificación del Cuerpo/genética , Proteínas de Unión al ADN/genética , Proteínas Hedgehog , Factores de Transcripción de Tipo Kruppel , Esbozos de los Miembros , Ratones , Ratones Endogámicos C3H , Ratones Mutantes , Polidactilia/embriología , Polidactilia/genética , ARN Mensajero/análisis , Proteína con Dedos de Zinc GLI1 , Proteína Gli2 con Dedos de Zinc , Proteína Gli3 con Dedos de Zinc , Dedos de Zinc

RESUMEN

Sonic hedgehog (Shh) expression in the developing limb is associated with the zone of polarising activity (ZPA), and both are restricted to the posterior part of the limb bud. We show that the expression patterns of Shh and Gli3, a member of the Gli-family believed to function in transcriptional control, appear to be mutually exclusive in limb buds of mouse embryos. In the polydactyly mouse mutant extra toes (Xt), possessing a null mutation of Gli3, Shh is additionally expressed in the anterior region of the limb bud. The transcript of Ptc, the putative receptor for Shh protein, can be detected anteriorly as well. Other genes known to be involved in limb outgrowth and patterning, like Fibroblast growth factor (Fgf), Bone morphogenetic protein (Bmp), and Hoxd are misexpressed in relation to the ectopic Shh expression domain in Xt limb buds. This data suggest that Gli3 is a regulator of Shh expression in mouse limb development.

Asunto(s)

Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Extremidades/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso , Proteínas/metabolismo , Proteínas Represoras , Transactivadores , Factores de Transcripción , Factor de Crecimiento Transformador beta , Proteínas de Xenopus , Animales , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteína Morfogenética Ósea 7 , Extremidades/embriología , Factor 4 de Crecimiento de Fibroblastos , Factor 8 de Crecimiento de Fibroblastos , Sustancias de Crecimiento/metabolismo , Proteínas Hedgehog , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Proteínas de Insectos/metabolismo , Factores de Transcripción de Tipo Kruppel , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C3H , Mutación , Polidactilia/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Superficie Celular , Proteína Gli3 con Dedos de Zinc

RESUMEN

Ubiquitously expressed SH2-containing tyrosine phosphatases interact physically with tyrosine kinase receptors or their substrates and relay positive mitogenic signals via the activation of the Ras-mitogen-activated protein kinase (MAPK) pathway. Conversely, the structurally related phosphatase SHP-1 is predominantly expressed in hemopoietic cells and becomes tyrosine phosphorylated upon colony-stimulating factor 1 treatment of macrophages without associating with the colony-stimulating factor 1 receptor tyrosine kinase. Mice lacking functional SHP-1 (me/me and me(v)/me(v)) develop systemic autoimmune disease with accumulation of macrophages, suggesting that SHP-1 may be a negative regulator of hemopoietic cell growth. By using macrophages expressing dominant negative Ras and the me(v)/me(v) mouse mutant, we show that SHP-1 is activated in the course of mitogenic signal transduction in a Ras-dependent manner and that its activity is necessary for the Ras-dependent activation of the MAPK pathway but not of the Raf-1 kinase. Consistent with a role for SHP-1 as an intermediate between Ras and the MEK-MAPK pathway, Ras-independent activation of the latter kinases by bacterial lipopolysaccharide occurred normally in me(v)/me(v) cells. Our results sharply accentuate the diversity of signal transduction in mammalian cells, in which the same signaling intermediates can be rearranged to form different pathways.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Madre Hematopoyéticas/metabolismo , Macrófagos/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal , Animales , Activación Enzimática , Guanosina Trifosfato/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Mamíferos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Biológicos , Fosfotirosina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/deficiencia , Proteínas Tirosina Fosfatasas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf

RESUMEN

The zinc finger gene Gli3 plays a role in limb and brain development. To facilitate the molecular analysis of different mouse mutations of this gene, the murine cDNA was isolated and sequenced. This 5113 bp cDNA encodes a putative protein of 1596 amino acids. Comparison of the murine and human GLI3 cDNA revealed an overall homology of 85% between the deduced amino acid sequences. More importantly, several regions of the protein, including the zinc fingers, are more highly conserved ( > 95%), suggesting that these represent functional domains in the Gli3 protein.

Asunto(s)

ADN Complementario/química , Proteínas de Unión al ADN/genética , Proteínas del Tejido Nervioso , Proteínas Represoras , Factores de Transcripción/genética , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/química , Humanos , Factores de Transcripción de Tipo Kruppel , Ratones , Datos de Secuencia Molecular , Proteína Gli3 con Dedos de Zinc

RESUMEN

Many tyrosine kinase growth factor receptors activate the MAP Kinase (MAPK) pathway by stimulating the activity of the RAF kinase. In some, but not all cell types, the expression of activated RAF is sufficient to induce constitutive MAPK activation. In BAC-1.2F5 macrophages the expression of virally activated RAF does not correlate with constitutive MAPK activation; on the contrary, growth factor-mediated stimulation of MAPK activity is suppressed in these cells. Suppression correlates with v-RAF expression, as MAPK activation is normal in a revertant cell line that stopped expressing v-RAF. Inhibition of MAPK activation is associated with lack of ERK-2 tyrosine phosphorylation, and is not due to the suppression of CSF-1-mediated MEK activation. Pretreatment with vanadate restores growth factor-stimulated activation and tyrosine phosphorylation of MAPK in v-RAF-expressing macrophages, indicating the involvement of a tyrosine phosphatase. Interestingly, v-RAF-expressing macrophages contain low constitutive levels of MKP-1 mRNA, an immediate early gene that encodes a MAPK-specific phosphatase and is induced in the parental cell line by CSF-1 treatment. The restoration of MAPK activation by vanadate pretreatment and the presence of MKP-1 mRNA in v-RAF-expressing macrophages raise the intriguing possibility that in macrophages RAF may be feeding back on the MAPK pathway by participating in the control of MKP-1 expression.

Asunto(s)

Proteínas de Ciclo Celular , Proteínas Inmediatas-Precoces/metabolismo , Macrófagos/metabolismo , Fosfoproteínas Fosfatasas , Proteínas Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Oncogénicas de Retroviridae/fisiología , Secuencia de Aminoácidos , Línea Celular , Fosfatasa 1 de Especificidad Dual , Activación Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/enzimología , Datos de Secuencia Molecular , Proteínas Oncogénicas v-raf , Fosforilación , Proteína Fosfatasa 1 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Oncogénicas de Retroviridae/genética , Tirosina/metabolismo

RESUMEN

Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Lipopolisacáridos/farmacología , Quinasa 1 de Quinasa de Quinasa MAP , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/enzimología , Proteínas Quinasas Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Hidrocarburos Aromáticos con Puentes/farmacología , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Indoles/farmacología , Maleimidas/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Datos de Secuencia Molecular , Norbornanos , Péptidos/química , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Tiocarbamatos , Tionas/farmacología , Fosfolipasas de Tipo C/metabolismo , Proteína Elk-1 con Dominio ets

RESUMEN

We have measured power spectra of atmospheric phase fluctuations with the Mark III stellar interferometer on Mt. Wilson under a wide variety of seeing conditions. On almost all nights, the high-frequency portions of the temporal power spectra closely follow the form predicted by the standard Kolmogorov-Tatarski model. At lower frequencies, a variety of behavior is observed. On a few nights, the spectra clearly exhibit the low-frequency flattening characteristic of turbulence with an outer-scale length of the order of 30 m. On other nights, examination of individual spectra yields no strong evidence of an outer scale less than a few kilometers in size, but comparison of the spectra on different interferometer baselines shows a saturation of the spatial structure function on long baselines. This saturation is consistent with the assumption of an outer-scale length similar to that derived for the nights when low-frequency flattening of the spectra is clearly seen. We discuss possible explanations of this behavior and conclude that power spectra from a single interferometer baseline are a poor diagnostic for the effective outer scale compared with multiple-baseline spectra.