RESUMEN
We develop a coherent beam splitter for single electrons driven through two tunnel-coupled quantum wires by surface acoustic waves (SAWs). The output current through each wire oscillates with gate voltages to tune the tunnel coupling and potential difference between the wires. This oscillation is assigned to coherent electron tunneling motion that can be used to encode a flying qubit and is well reproduced by numerical calculations of time evolution of the SAW-driven single electrons. The oscillation visibility is currently limited to about 3%, but robust against decoherence, indicating that the SAW electron can serve as a novel platform for a solid-state flying qubit.
RESUMEN
WHAT IS KNOWN AND OBJECTIVE: There have been several reports describing rectovaginal fistula development after bevacizumab treatment, and these fistulas were diagnosed by CT scan or colonoscopy. We report a case of sigmoid-vaginal fistula diagnosed by fistulography. CASE DESCRIPTION: The case is a 53-year-old woman who was treated for chronic myelogenous leukaemia and gynaecological cancers 8 years previously. At 52 years of age, she was diagnosed with colon cancer and had a partial colectomy performed. One year after surgery, colon cancer recurred, and she was treated with anticancer agents, including bevacizumab. During chemotherapy, she complained of a foul smelling discharge from the vagina. Fistulography revealed a sigmoid-vaginal fistula. WHAT IS NEW AND CONCLUSION: This is the first report of vaginal fistulography performed on a patient who was treated with bevacizumab. Fistulography may be useful for detecting sigmoid-vaginal fistula.
Asunto(s)
Inhibidores de la Angiogénesis/efectos adversos , Bevacizumab/efectos adversos , Colon Sigmoide/efectos de los fármacos , Fístula Vaginal/inducido químicamente , Inhibidores de la Angiogénesis/uso terapéutico , Bevacizumab/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Femenino , Humanos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/tratamiento farmacológicoRESUMEN
We report on the direct observation of the transmission phase shift through a Kondo correlated quantum dot by employing a new type of two-path interferometer. We observed a clear π/2-phase shift, which persists up to the Kondo temperature TK. Above this temperature, the phase shifts by more than π/2 at each Coulomb peak, approaching the behavior observed for the standard Coulomb blockade regime. These observations are in remarkable agreement with two-level numerical renormalization group calculations. The unique combination of experimental and theoretical results presented here fully elucidates the phase evolution in the Kondo regime.
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Apraxias/congénito , Síndrome de Cogan/genética , Enzimas Reparadoras del ADN/genética , Microcefalia/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Convulsiones/genética , Adulto , Edad de Inicio , Apraxias/diagnóstico por imagen , Apraxias/genética , Apraxias/fisiopatología , Encéfalo/diagnóstico por imagen , Encéfalo/fisiopatología , Niño , Síndrome de Cogan/diagnóstico por imagen , Síndrome de Cogan/fisiopatología , Femenino , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Microcefalia/diagnóstico por imagen , Microcefalia/fisiopatología , Mutación , Linaje , Convulsiones/diagnóstico por imagen , Convulsiones/fisiopatologíaRESUMEN
The Syrian cardiomyopathic hamster (BIO14.6) has an inherited form of progressive myocardial necrosis and congestive heart failure. Although widely studied as an animal model for human hypertrophic cardiomyopathy, further genetic analysis has been limited by a scarcity of DNA markers. Until now, only six autosomal linkage groups have been described and the number of polymorphic loci was extremely limited. In this study, we applied the restriction landmark genome scanning (RLGS) spot-mapping method to construct a genetic map of the Syrian hamster (Mesocricetus auratus) using 72 back-cross progeny. Although the polymorphic rate is very low (3-7%) between the strains, 531 polymorphic spots/loci were mapped, showing the power of this approach and reasonable applicability to other organisms lacking a well-defined genetic map. Further, the spot markers which flank the cardiomyopathy (cm) locus were cloned to determine the chromosomal location of cm by fluorescent in situ hybridization (FISH) analysis, resulting in the assignment of the locus to the centromeric region of hamster chromosome 9qa2.1-b1. Several candidate genes responsible for hypertrophic cardiomyopathy in humans have been excluded.
Asunto(s)
Cardiomiopatías/genética , Mapeo Cromosómico , Insuficiencia Cardíaca/genética , Mesocricetus/genética , Animales , Secuencia de Bases , Cardiomiopatía Hipertrófica/genética , Cricetinae , Cruzamientos Genéticos , Cartilla de ADN , Femenino , Ligamiento Genético , Humanos , Hibridación Fluorescente in Situ , Masculino , Datos de Secuencia Molecular , Polimorfismo Genético , Mapeo RestrictivoRESUMEN
Bloom Syndrome (BS) is a genetic DNA repair disorder, caused by mutations in the BLM gene. The clinical phenotype includes growth retardation, immunodeficiency and a strong predisposition to different types of malignancies. Treatment of malignancies in BS patients with radiotherapy or chemotherapy is believed to be associated with increased toxicity, but clinical and laboratory data are lacking. We collected clinical data of two Dutch BS patients with solid tumors. Both were treated with radiotherapy before the diagnosis BS was made and tolerated this treatment well. In addition, we collected fibroblasts from BS patients to perform in vitro clonogenic survival assays to determine radiosensitivity. BS fibroblasts showed less radiosensitivity than the severely radiosensitive Artemis fibroblasts. Moreover, studies of double strand break kinetics by counting 53BP1 foci after irradiation showed similar patterns compared to healthy controls. In combination, the clinical cases and laboratory experiments are valuable information in the discussion whether radiotherapy is absolutely contraindicated in BS, which is the Case in other DNA repair syndromes like Ataxia Telangiectasia and Artemis.
Asunto(s)
Síndrome de Bloom/complicaciones , Carcinoma/radioterapia , Radioterapia/efectos adversos , Adulto , Síndrome de Bloom/genética , Carcinoma/complicaciones , Células Cultivadas , Roturas del ADN de Doble Cadena , Reparación del ADN , Femenino , Fibroblastos/efectos de la radiación , Humanos , Masculino , Persona de Mediana Edad , Tolerancia a Radiación , RecQ Helicasas/genéticaRESUMEN
We have shown previously that CD8+ T cells proliferate upon exposure to autologous, antigen primed CD4+ T cells, and suppress the response of fresh T cells to the priming antigen but not irrelevant antigens. The stimulus and target of suppression in this system appears to be the antigen receptor on the surface of CD4+ cells, rather than the nominal antigen. In the current study, alloantigen primed CD4+ inducer cells and IL-2-containing medium were used to generate clones of suppressor cells from several individuals. The clones inhibited the response of fresh autologous T cells only to the original allogeneic stimulator cell and to stimulator cells that shared HLA-DR antigens with the priming cell. The clones were also genetically restricted, since they inhibited the response of HLA-A,B-compatible but not HLA-A,B-incompatible individuals. The availability of a method for reproducibly generating antigen receptor-specific suppressor T cell clones in vitro should make it possible to clarify the mechanism, whereby such cells are activated and exert their suppressive effect.
Asunto(s)
Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Células Clonales , Antígenos HLA/análisis , Antígenos HLA-DR , Antígenos de Histocompatibilidad Clase II/análisis , Humanos , Prueba de Cultivo Mixto de LinfocitosRESUMEN
The outer dynein arm of Chlamydomonas flagella, when isolated under Mg(2+)-free conditions, tends to dissociate into an 11 to 12S particle (12S dynein) containing the gamma heavy chain and a 21S particle (called 18S dynein) containing the alpha and beta heavy chains. We show here that functional outer arms can be reconstituted by the addition of 12S and 18S dyneins to the axonemes of the outer armless mutants oda1-oda6. A third factor that sediments at integral 7S is required for efficient reconstitution of the outer arms on the axonemes of oda1 and oda3. However, this factor is not necessary for reconstitution on the axonemes of oda2, oda4, oda5, and oda6. SDS-PAGE analysis indicates that the axonemes of the former two mutants lack a integral of 70-kD polypeptide that is present in those of the other mutants as well as in the 7S fraction from the wild-type extract. Furthermore, electron micrographs of axonemal cross sections revealed that the latter four mutants, but not oda1 or oda3, have small pointed structures on the outer doublets, at a position in cross section where outer arms normally occur. We suggest that the 7S factor constitutes the pointed structure on the outer doublets and facilitates attachment of the outer arm. The discovery of this structure raises a new question as to how the attachment site for the outer arm dynein is determined within the axoneme.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Dineínas/metabolismo , Animales , Movimiento Celular , Chlamydomonas reinhardtii/ultraestructura , Flagelos/metabolismo , Flagelos/ultraestructura , Microscopía Electrónica , MutaciónRESUMEN
A new allele of the Chlamydomonas oda4 flagellar mutant (oda4-s7) possessing abnormal outer dynein arms was isolated. Unlike the previously described oda4 axoneme lacking all three (alpha, beta, and gamma) outer-arm dynein heavy chains, the oda4-s7 axoneme contains the alpha and gamma heavy chains and a novel peptide with a molecular mass of approximately 160 kD. The peptide reacts with a mAb (18 beta B) that recognizes an epitope on the NH2-terminal part of the beta heavy chain. These observations indicate that this mutant has a truncated beta heavy chain, and that the NH2-terminal part of the beta heavy chain is important for the stable assembly of the outer arms. In averaged electron microscopic images of outer arms from cross sections of axonemes, the mutant outer arm lacks its mid-portion, producing a forked appearance. Together with our previous finding that the mutant oda11 lacks the alpha heavy chain and the outermost portion of the arm (Sakakibara, H., D. R. Mitchell, and R. Kamiya. 1991. J. Cell Biol. 113:615-622), this result defines the approximate locations of the three outer arm heavy chains in the axonemal cross section. The swimming velocity of oda4-s7 is 65 +/- 8 microns/s, close to that of oda4 which lacks the entire outer arm (62 +/- 8 microns/s) but significantly lower than the velocities of wild type (194 +/- 23 microns/s) and oda11 (119 +/- 17 microns/s). Thus, the lack of the beta heavy chain impairs outer-arm function more seriously than does the lack of the alpha heavy chain, suggesting that the alpha and beta chains play different roles in outer arm function.
Asunto(s)
Chlamydomonas reinhardtii/fisiología , Dineínas/fisiología , Alelos , Animales , Western Blotting , Centrifugación por Gradiente de Densidad , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Dineínas/química , Dineínas/genética , Dineínas/ultraestructura , Flagelos/fisiología , Genes Protozoarios , Microscopía Electrónica , Mutación , Supresión GenéticaRESUMEN
We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the uter ynein rm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737- 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83. 4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme.
Asunto(s)
Chlamydomonas reinhardtii/genética , Dineínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chlamydomonas reinhardtii/química , Clonación Molecular , ADN Complementario/aislamiento & purificación , ADN de Plantas/aislamiento & purificación , Dineínas/química , Flagelos/química , Flagelos/ultraestructura , Genes de Plantas/fisiología , Datos de Secuencia Molecular , Mutagénesis Insercional/fisiología , Análisis de Secuencia de ADNRESUMEN
S100beta is a soluble protein released by glial cells mainly under the activation of the 5-HT1A receptor. It has been reported as a neuro-trophic and -tropic factor that promotes neurite maturation and outgrowth during development. This protein also plays a role in axonal stability and the plasticity underlying long-term potentiation in adult brains. The ability of S100beta to rapidly regulate neuronal morphology raises the interesting point of whether there are daily rhythm or gender differences in S100beta level in the brain. To answer this question, the S100beta expression in adult female and male rats, as well as in adult female CD-21 and S100beta -/- female mice, were investigated. Scintillation counting and morphometric analysis of the immunoreactivity of S100beta, showed rhythmic daily expression. The female and male rats showed opposite cycles. Females presented the highest value at the beginning of the rest phase (5:00 h), while in males the maximum value appeared in the beginning of the motor activity period (21:00 h). These results confirm previous S100beta evaluations in human serum and cerebrospinal fluid reporting the protein's function as a biomarker for brain damage (Gazzolo et al. in Clin Chem 49:967-970, 2003; Clin Chim Acta 330:131-133, 2003; Pediatr Res 58:1170-1174, 2005), similar behavior was also observed for GFAP in relation to Alzheimer Disease (Fukuyama et al. in Eur Neurol 46:35-38, 2001). The data should be taken into account when considering S100beta as a biomarker of health condition. In addition, the results raise questions on which structure or condition imposes these rhythms as well as on the physiological meaning of the observed gender differences.
Asunto(s)
Envejecimiento/metabolismo , Factores de Crecimiento Nervioso/biosíntesis , Proteínas S100/biosíntesis , Animales , Ritmo Circadiano/fisiología , Femenino , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Humanos , Inmunoensayo , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Actividad Motora/fisiología , Factores de Crecimiento Nervioso/genética , Prosencéfalo/metabolismo , Ratas , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/genética , Caracteres SexualesRESUMEN
To identify the precise molecular processes to induce DNA lesions, we attempt a novel spectroscopy of X-ray induced luminescence (XIL) using soft X-ray synchrotron radiation, which is a non-destructive analysis of the reaction intermediates in the elementary reaction pathway of damage induction and self-organized restoration. Using a liquid micro-jet technique to introduce aqueous samples in a vacuum chamber, we measure UV-visible luminescence from nucleotide solution as a function of the soft X-ray energy from the nitrogen to oxygen K-edge region. The XIL intensities for the nucleotide solutions are significantly enhanced in the soft X-ray region (410-530 eV) which is ascribed to the K-shell excitation/ionization of nitrogen atoms in the nucleobases. Furthermore, the XIL spectra do not show any signature of X-ray absorption near-edge structure (XANES) of the nucleobases. This is because the luminescence intensities collected from the integral area of the micro-jet only reflect the quantum yield of luminescence of the absorbed X-ray into UV-visible light irrespective of the absorption cross sections, i.e. of XANES. Thus the present result is the first evidence of luminescence as a result of X-ray absorption of aqueous nucleotides.
Asunto(s)
ADN/química , ADN/efectos de la radiación , Desoxirribonucleótidos/química , Desoxirribonucleótidos/efectos de la radiación , Diseño de Equipo , Concentración de Iones de Hidrógeno , Luminiscencia , Nitrógeno/química , Oxígeno/química , Sincrotrones , Agua/química , Espectroscopía de Absorción de Rayos XRESUMEN
Adult T-cell leukemia (ATL) is an intractable malignancy of CD4+ T cells that is etiologically associated with infection by human T-cell leukemia virus-type I. Most individuals in the chronic stage of ATL eventually undergo progression to a highly aggressive acute stage. To clarify the mechanism responsible for this stage progression, we isolated CD4+ cells from individuals in the chronic (n=19) or acute (n=22) stages of ATL and subjected them to profiling of gene expression with DNA microarrays containing >44,000 probe sets. Changes in chromosome copy number were also examined for 24 cell specimens with the use of microarrays harboring approximately 50,000 probe sets. Stage-dependent changes in gene expression profile and chromosome copy number were apparent. Furthermore, expression of the gene for MET, a receptor tyrosine kinase for hepatocyte growth factor (HGF), was shown to be specific to the acute stage of ATL, and the plasma concentration of HGF was increased in individuals in either the acute or chronic stage. HGF induced proliferation of a MET-positive ATL cell line, and this effect was blocked by antibodies to HGF. The HGF-MET signaling pathway is thus a potential therapeutic target for ATL.
Asunto(s)
Perfilación de la Expresión Génica , Genoma Humano/genética , Factor de Crecimiento de Hepatocito/genética , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas Proto-Oncogénicas/genética , Receptores de Factores de Crecimiento/genética , Línea Celular Tumoral , Dosificación de Gen , Genómica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-met , Transcripción GenéticaRESUMEN
In giant, large internal carotid ophthalmic artery aneurysms, revised double lumen balloon catheters introduced directly into the cervical carotid arteries were used successfully to perform suction decompression methods. No general heparinization was used during these maneuvers except for continuous intraluminal irrigation of heparin contained saline. Intraoperative multi-directional portable DSA ascertained complete neck clippings and patency of parent vessels. These methods were tried for 8 cases of large, giant IC ophthalmic aneurysms. Among these 5 patients who complained of incomplete visual failures, visual acuity improved in 3 cases. Postoperative transient hemiparesis were seen in two cases which had partially thrombosed, calcified giant aneurysm and the other was due to prolonged proximal ICA occlusion at its cavernous portion. Neither dead nor severely disabled cases were seen, fortunately. Delayed postoperative conventional angiography disclosed complete neck clippings sparing parent arteries except for only one case. This revised direct carotid puncture method is simple and acceptable because of minimal morbidity.
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Aneurisma/patología , Aneurisma/terapia , Descompresión Quirúrgica/métodos , Arteria Oftálmica , Angiografía Cerebral/métodos , HumanosRESUMEN
Mutation or epigenetic silencing of mismatch repair genes, such as MLH1 and MSH2, results in microsatellite instability (MSI) in the genome of a subset of colorectal carcinomas (CRCs). However, little is yet known of genes that directly contribute to tumor formation in such cancers. To characterize MSI-dependent changes in gene expression, we have now compared transcriptomes between fresh CRC specimens positive or negative for MSI (n=10 for each) with the use of high-density oligonucleotide microarrays harboring >44,000 probe sets. Correspondence analysis of the expression patterns of isolated MSI-associated genes revealed that the transcriptome of MSI+ CRCs is clearly distinct from that of MSI- CRCs. Such MSI-associated genes included that for AXIN2, an important component of the WNT signaling pathway. AXIN2 was silenced, apparently as a result of extensive methylation of its promoter region, specifically in MSI+ CRC specimens. Forced expression of AXIN2, either by treatment with 5'-azacytidine or by transfection with AXIN2 cDNA, resulted in rapid cell death in an MSI+ CRC cell line. These data indicate that epigenetic silencing of AXIN2 is specifically associated with carcinogenesis in MSI+ CRCs.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Proteínas del Citoesqueleto/genética , Epigénesis Genética , Silenciador del Gen , Repeticiones de Microsatélite , Proteínas Adaptadoras Transductoras de Señales , Adulto , Anciano , Anciano de 80 o más Años , Proteína Axina , Azacitidina/farmacología , Benzotiazoles , Proteínas Portadoras/metabolismo , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Análisis por Conglomerados , Islas de CpG , Proteínas del Citoesqueleto/metabolismo , Metilación de ADN , Reparación del ADN , ADN Complementario/metabolismo , Diaminas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Compuestos Orgánicos/farmacología , Quinolinas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
The distal portion of mouse chromosome 12 is imprinted. To date, however, Gtl2 is the only imprinted gene identified on chromosome 12. Gtl2 encodes multiple alternatively spliced transcripts with no apparent open reading frame. Using conceptuses with maternal or paternal uniparental disomy for chromosome 12 (UPD12), we found that Gtl2 is expressed from the maternal allele and methylated at the 5' end of the silent paternal allele. A reciprocally imprinted gene, Delta-like (Dlk), with homology to genes involved in the Notch signalling pathway was identified 80kb upstream of Gtl2. Dlk was expressed exclusively from the paternal allele in both the embryo and placenta, but the CpG-island promoter of Dlk was completely unmethylated on both parental alleles. Rather, a paternally methylated region was identified in the last exon of the active Dlk allele. The proximity, reciprocal imprinting and methylation in this domain are reminiscent of the co-ordinately regulated Igf2-H19 imprinted domain on mouse chromosome 7. Like H19 and Igf2, Gtl2 and Dlk were found to be co-expressed in the same tissues throughout development, though not after birth. These results have implications for the regulation, function and evolution of imprinted domains.
Asunto(s)
Metilación de ADN , Regulación del Desarrollo de la Expresión Génica , Impresión Genómica , Proteínas de Homeodominio/genética , Proteínas de la Membrana/genética , Alelos , Animales , Cromosomas , ADN/metabolismo , Embrión de Mamíferos/metabolismo , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Normal human peripheral blood T lymphocytes activated by concanavalin A (Con A) were fractionated into OKT4+ and OKT8+ populations by complement-dependent cell lysis using OKT8 and OKT4 antibodies, respectively. By using the preferential ability of some, but not all, Con A-activated T cells to form rosettes with autologous erythrocytes, each population was further divided into autorosetting cells and nonautorosetting cells, and thus Con A-activated OKT4+ autorosetting, OKT4+ nonautorosetting, OKT8+ autorosetting, and OKT8+ nonautorosetting cells were obtained. The immune regulatory function of these populations was then investigated using a pokeweed mitogen-driven B cell plaque-forming cell system. These studies demonstrated that (a) autorosetting cells can exert potent suppressor activity regardless of their phenotypes of OKT4+ and OKT8+ antigens, and fail to help B cell differentiation; suppressor function mediated by these cells is radiosensitive; moreover, receptors for autologous erythrocytes may constitute either the interleukin 2 (IL2) receptors themselves or a component of an IL2 receptor-effector complex involved in modulating the growth signal that IL2 transmits to T cells; (b) OKT4+ nonrosetting cells serve adequately as radioresistant helper cells, but are devoid of suppressor cells; and (c) OKT8+ nonrosetting cells are found to lack either suppressor or helper activity, suggesting that they may belong to a T lymphocyte subset distinct from the subsets related to immune regulation. The results lead us, therefore, to the conclusion that there may exist functional heterogeneities among both the OKT4+ and OKT8+ populations; these heterogeneities can be dissected by virtue of the autologous erythrocyte rosette technique.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Eritrocitos/inmunología , Linfocitos T/inmunología , Células Cultivadas , Concanavalina A/farmacología , Humanos , Interleucina-2/farmacología , Activación de Linfocitos , Monocitos/inmunología , Formación de Roseta , Linfocitos T/clasificación , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of beta-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax in Xenopus embryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not beta-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.
Asunto(s)
Proteínas Portadoras/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Represoras , Transducción de Señal , Transactivadores , Proteínas de Pez Cebra , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Proteína Axina , Tipificación del Cuerpo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN , Proteínas Dishevelled , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Proteínas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Factores de Transcripción , Técnicas del Sistema de Dos Híbridos , Proteínas Wnt , Xenopus/embriología , Proteínas de Xenopus , beta CateninaRESUMEN
The mammalian Ror family of receptor tyrosine kinases consists of two structurally related proteins, Ror1 and Ror2. We have shown that mRor2-deficient mice exhibit widespread skeletal abnormalities, ventricular septal defects in the heart, and respiratory dysfunction, leading to neonatal lethality (S. Takeuchi, K. Takeda, I. Oishi, M. Nomi, M. Ikeya, K. Itoh, S. Tamura, T. Ueda, T. Hatta, H. Otani, T. Terashima, S. Takada, H. Yamamura, S. Akira, and Y. Minami, Genes Cells 5:71-78, 2000). Here we show that mRor1-deficient mice have no apparent skeletal or cardiac abnormalities, yet they also die soon after birth due to respiratory dysfunction. Interestingly, mRor1/mRor2 double mutant mice show markedly enhanced skeletal abnormalities compared with mRor2 mutant mice. Furthermore, double mutant mice also exhibit defects not observed in mRor2 mutant mice, including a sternal defect, dysplasia of the symphysis of the pubic bone, and complete transposition of the great arteries. These results indicate that mRor1 and mRor2 interact genetically in skeletal and cardiac development.
Asunto(s)
Huesos/anomalías , Cardiopatías Congénitas/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Animales , Animales Recién Nacidos , Huesos/metabolismo , Hibridación in Situ , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Genéticos , Mutación , Fenotipo , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Factores de TiempoRESUMEN
Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared from Xenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopus sperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to gamma- and alpha/beta-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.