RESUMEN
Homothorax (HTH) is a homeobox-containing protein, which plays multiple roles in the development of the embryo and the adult fly. HTH binds to the homeotic cofactor Extradenticle (EXD) and translocates it to the nucleus. Its function within the nucleus is less clear. It was shown, mainly by in vitro studies, that HTH can bind DNA as a part of ternary HTH/EXD/HOX complexes, but little is known about the transcription regulating function of HTH-containing complexes in the context of the developing fly. Here we present genetic evidence, from in vivo studies, for the transcriptional-activating function of HTH. The HTH protein was forced to act as a transcriptional repressor by fusing it to the Engrailed (EN) repression domain, or as a transcriptional activator, by fusing it to the VP16 activation domain, without perturbing its ability to translocate EXD to the nucleus. Expression of the repressing form of HTH in otherwise wild-type imaginal discs phenocopied hth loss of function. Thus, the repressing form was working as an antimorph, suggesting that normally HTH is required to activate the transcription of downstream target genes. This conclusion was further supported by the observation that the activating form of HTH caused typical hth gain-of-function phenotypes and could rescue hth loss-of-function phenotypes. Similar results were obtained with XMeis3, the Xenopus homologue of HTH, extending the known functional similarity between the two proteins. Competition experiments demonstrated that the repressing forms of HTH or XMeis3 worked as true antimorphs competing with the transcriptional activity of the native form of HTH. We also describe the phenotypic consequences of HTH antimorph activity in derivatives of the wing, labial and genital discs. Some of the described phenotypes, for example, a proboscis-to-leg transformation, were not previously associated with alterations in HTH activity. Observing the ability of HTH antimorphs to interfere with different developmental pathways may direct us to new targets of HTH. The HTH antimorph described in this work presents a new means by which the transcriptional activity of the endogenous HTH protein can be blocked in an inducible fashion in any desired cells or tissues without interfering with nuclear localization of EXD.
Asunto(s)
Drosophila melanogaster/genética , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Activación Transcripcional , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Núcleo Celular/metabolismo , Secuencia Conservada , Proteínas de Drosophila , Drosophila melanogaster/embriología , Evolución Molecular , Extremidades/embriología , Proteína Vmw65 de Virus del Herpes Simple/genética , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Proteínas de Homeodominio/metabolismo , Fenotipo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras , Factores de Transcripción/metabolismoRESUMEN
The evolutionarily conserved basic helix-loop-helix (bHLH) transcription factors play important roles during development. Here we report the identification of Nato3 (nephew of atonal fer3) orthologs in Drosophila, C. elegans, mouse, and man, all of which share a high degree of similarity within the bHLH domain. Expression analysis revealed Nato3 transcripts in the central nervous system of both fly and mouse embryos. In the fly, Dnato3 is highly expressed in 9-15h embryos in a few ventral nerve cord cells and a subset of neurons in the brain. In mouse, the MNato3 transcripts were detected from embryonic day 7 until 5 weeks postnatally, with highest levels in the midbrain, thalamus, hypothalamus, pons, and medulla oblongata. In contrast to the brain, expression in the spinal cord was limited to the embryonic stages.
Asunto(s)
Sistema Nervioso Central/embriología , Drosophila/embriología , Expresión Génica , Secuencias Hélice-Asa-Hélice , Proteínas del Tejido Nervioso/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Encéfalo/embriología , Encéfalo/metabolismo , Sistema Nervioso Central/metabolismo , Drosophila/genética , Proteínas de Drosophila , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/metabolismo , Evolución Molecular , Perfilación de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Neuronas/metabolismo , Proteínas Represoras , Médula Espinal/embriología , Médula Espinal/metabolismo , Factores de Transcripción/químicaRESUMEN
The homothorax (hth) gene is involved in multiple aspects of embryonic and adult fly development. It encodes a homeodomain-containing protein of the MEIS family and was shown to regulate the subcellular localization of the homeotic protein cofactor Extradenticle (EXD). The HTH protein contains a TALE class homeodomain and a conserved MH domain, which is required for its interaction with EXD. In this work, we describe the structure of the hth locus, characterize at the molecular level a collection of mutant alleles of hth, and discuss the correlation between the identified structural defects and their consequent phenotypes. The hth locus spans more than 100 kb and contains 14 exons. Several of the exon-intron boundaries within the homeodomain and the MH domain-coding regions are conserved between Drosophila and Caenorhabditis elegans. The analysis of hth mutations demonstrates that the homeodomain of HTH is not required for nuclear localization of EXD and that the MH domain-containing first 240 residues are sufficient for nuclear localization of both EXD and HTH. Mutations that alter or delete the homeodomain cause only partial homeotic transformations in the PNS, whereas mutations affecting the MH domain cause distinct and more severe PNS phenotypes. These observations may suggest that driving nuclear localization of EXD is the main role of HTH in patterning the embryonic PNS. They may also suggest that homeodomain-defective HTH protein retains some of its transcription-regulating functions by binding DNA via its interaction with EXD.
Asunto(s)
Análisis Mutacional de ADN , Proteínas de Homeodominio/genética , Alelos , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Metanosulfonato de Etilo , Etilnitrosourea , Exones , Inmunohistoquímica , Intrones , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis , Mutagénesis Insercional , Mutágenos , Mutación , Fenotipo , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Calicheamicin is a hydrophobic enediyne antibiotic that binds noncovalently to DNA and causes sequence-selective oxidation of deoxyribose. While the drug makes several base contacts along the minor groove, the diversity of binding-site sequences and the effects of DNA conformation on calicheamicin-induced DNA cleavage suggest that sequence recognition per se is not the primary determinant of target selection. We now present evidence that calicheamicin bends its DNA targets. Using a gel mobility assay, we observed that polymers of oligonucleotide constructs containing AGGA and ACAA binding sites for calicheamicin did not possess intrinsic curvature. Binding of calicheamicin epsilon, the aromatized form of the parent calicheamicin gamma(1)(I), to oligonucleotide constructs containing binding sites in phase with the helical repeat caused a shift to smaller circle sizes in T4 ligase-mediated circle formation assays, with a much smaller shift observed with constructs containing out-of-phase binding sites. It was also observed that binding of calicheamicin epsilon to a 273 bp construct with phased binding sites caused an increase in the molar cyclization factor, J, from 8 x 10(-8) to 9 x 10(-6) M. These results are consistent with DNA bending as part of an induced-fit mechanism of DNA target recognition and with the hypothesis that the preferred targets of calicheamicin, the 3' ends of oligopurine tracts, are characterized by unique conformational properties.
Asunto(s)
Antibacterianos/metabolismo , ADN/química , Conformación de Ácido Nucleico , Aminoglicósidos , Antibacterianos/química , Secuencia de Bases , Sitios de Unión , Catálisis , ADN Ligasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Estructura MolecularRESUMEN
BACKGROUND: Placement of central venous catheters, although often considered to be a relatively safe and "junior"-level procedure, may be associated with life-threatening complications. METHODS: A recent surgical death associated with placement of a central venous catheter at this Institution led to submission of a questionnaire to pediatric surgeons referenced through the American Pediatric Surgical Association directory regarding knowledge of similar incidents and information regarding catheter placement-related complications. RESULTS: Results to this response, although anecdotal, provided data regarding complications of an acute nature, which fell into the categories of pneumothorax, hydrothorax, cardiac tamponade, and hemothorax. Of 10 children with cardiac tamponade, 7 were infants, and most complications were associated with needle stick for access, with symptoms developing within minutes up to 12 hours after the procedure. Drainage of the tamponade was performed by aspiration alone in 3 cases; surgical drainage in 6 children resulted in survival in 9 of the 10 patients. Hemothorax was described in 19 patients and appeared to be more common in children in the 1- to 6-year age group, usually associated with percutaneous access techniques. Thoracotomy for hemothorax was performed in 16 children with 11 survivors. Vascular injury to subclavian artery, vein, or superior vena caval were noted in most at operation. CONCLUSIONS: Although data included in this review are entirely anecdotal and not subject to scientific scrutiny or analysis, certain conclusions appear evident. Inherent risks of central venous catheters are intrinsic and should be discussed with the family in obtaining preoperative consent, including life-threatening risks that may necessitate urgent surgical intervention (by thoracotomy or other means). Certain technical aspects of the procedure should be rigidly followed with an experienced surgeon in attendance throughout the procedure. Rapid evaluation should be performed for any unexplained problems that occur in the operating theatre or during the early postoperative period.
Asunto(s)
Taponamiento Cardíaco/mortalidad , Cateterismo Venoso Central/efectos adversos , Cateterismo Venoso Central/mortalidad , Hemotórax/mortalidad , Distribución por Edad , Taponamiento Cardíaco/etiología , Niño , Preescolar , Recolección de Datos , Femenino , Hemotórax/etiología , Humanos , Incidencia , Lactante , Recién Nacido , Masculino , Pediatría , Factores de Riesgo , Distribución por Sexo , Encuestas y Cuestionarios , Tasa de Supervivencia , Estados Unidos/epidemiologíaRESUMEN
A homologue of the Drosophila homothorax (hth) gene, Xenopus Meis3 (XMeis3), was cloned from Xenopus laevis. XMeis3 is expressed in a single stripe of cells in the early neural plate stage. By late neurula, the gene is expressed predominantly in rhombomeres two, three and four, and in the anterior spinal cord. Ectopic expression of RNA encoding XMeis3 protein causes anterior neural truncations with a concomitant expansion of hindbrain and spinal cord. Ectopic XMeis3 expression inhibits anterior neural induction in neuralized animal cap ectoderm explants without perturbing induction of pan-neural markers. In naive animal cap ectoderm, ectopic XMeis3 expression activates transcription of the posteriorly expressed neural markers, but not pan-neural markers. These results suggest that caudalizing proteins, such as XMeis3, can alter A-P patterning in the nervous system in the absence of neural induction. Regionally expressed proteins like XMeis3 could be required to overcome anterior signals and to specify posterior cell fates along the A-P axis.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Homeodominio/fisiología , Sistema Nervioso/embriología , Receptores de Factores de Crecimiento , Proteínas de Xenopus , Xenopus/embriología , Secuencia de Aminoácidos , Animales , Northern Blotting , Receptores de Proteínas Morfogenéticas Óseas , Proteínas Portadoras , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Proteína 2 de la Respuesta de Crecimiento Precoz , Inducción Embrionaria , Expresión Génica , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Mutagénesis , Proteínas del Tejido Nervioso/metabolismo , Proteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
The putative origin of replication in prokaryotic genomes can be located by a new method that finds short oligomers whose orientation is preferentially skewed around the origin. The skewed oligomer method is shown to work for all bacterial genomes and one of three archaeal genomes sequences to date, confirming known or predicted origins in most cases and in three cases (H. pylori, M. thermoautotrophicum, and Synechocystis sp.), suggesting origins that were previously unknown. In many cases, the presence of conserved genes and nucleotide motifs confirms the predictions. An algorithm for finding these skewed seven-base and eight-base sequences is described, along with a method for combining evidence from multiple skewed oligomers to accurately locate the replication origin. Possible explanations for the phenomenon of skewed oligomers are discussed. Explanations are presented for why some bacterial genomes contain hundreds of highly skewed oligomers, whereas others contain only a handful.
Asunto(s)
Genoma Bacteriano , Genoma , Oligodesoxirribonucleótidos/química , Origen de Réplica , Algoritmos , Archaea/genética , Secuencia de Bases , Grupo Borrelia Burgdorferi/genética , Cianobacterias/genética , Haemophilus influenzae/genética , Helicobacter pylori/genética , Mycobacterium/genética , Alineación de SecuenciaRESUMEN
Mutations in the Drosophila gene pavarotti result in the formation of abnormally large cells in the embryonic nervous system. In mitotic cycle 16, cells of pav mutant embryos undergo normal anaphase but then develop an abnormal telophase spindle and fail to undertake cytokinesis. We show that the septin Peanut, actin, and the actin-associated protein Anillin, do not become correctly localized in pav mutants. pav encodes a kinesin-like protein, PAV-KLP, related to the mammalian MKLP-1. In cellularized embryos, the protein is localized to centrosomes early in mitosis, and to the midbody region of the spindle in late anaphase and telophase. We show that Polo kinase associates with PAV-KLP with which it shows an overlapping pattern of subcellular localization during the mitotic cycle and this distribution is disrupted in pav mutants. We suggest that PAV-KLP is required both to establish the structure of the telophase spindle to provide a framework for the assembly of the contractile ring, and to mobilize mitotic regulator proteins.
Asunto(s)
División Celular/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto , Proteínas de Insectos/fisiología , Proteínas de Microfilamentos , Proteínas Asociadas a Microtúbulos/fisiología , Sistema Nervioso/embriología , Huso Acromático/ultraestructura , Telofase/genética , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Tamaño de la Célula , Centrosoma/ultraestructura , Clonación Molecular , Proteínas Contráctiles/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Embrión no Mamífero/ultraestructura , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Mamíferos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Sistema Nervioso/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Fracciones Subcelulares/químicaRESUMEN
The homeotic genes of the bithorax complex are required, among other things, for establishing the patterns of sensory organs in the embryonic peripheral nervous system (PNS). However, the molecular mechanisms by which these genes affect pattern formation in the PNS are not understood and other genes that function in this pathway are not characterized. Here we report the phenotypic and molecular analysis of one such gene, homothorax (hth; also named dorsotonals). Mutations in the hth gene seem to alter the identity of the abdominal chordotonal neurons, which depend on Abd-A for their normal development. However, these mutations do not alter the expression of the abd-A gene, suggesting that hth may be involved in modulating abd-A activity. We have generated multiple mutations in the hth locus and cloned the hth gene. hth encodes a homeodomain-containing protein that is most similar to the murine proto-oncogene meis1. The hth gene is expressed throughout embryonic development in a spatially restricted pattern, which is modulated in abdominal segments by abd-A and Ubx. The spatial distribution of the HTH protein during embryonic development is very similar to the distribution of the Extradenticle (EXD) protein, a known modulator of homeotic gene activity. Here we show that the PNS phenotype of exd mutant embryos is virtually indistinguishable from that of hth mutant embryos and does not simply follow the homeotic transformations observed in the epidermis. We also show that the HTH protein is present in extremely low levels in embryos lacking exd activity as compared to wild-type embryos. In contrast, the EXD protein is present in fairly normal levels in hth mutant embryos, but fails to accumulate in nuclei and remains cytoplasmic. Ectopic expression of hth can drive ectopic nuclear localization of EXD. Based on our observations we propose that the genetic interactions between hth and exd serve as a novel mechanism for regulating homeotic protein activity in embryonic PNS development.
Asunto(s)
Proteínas de Drosophila , Drosophila/embriología , Drosophila/genética , Genes de Insecto , Nervios Periféricos/embriología , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , Hibridación in Situ , Proteínas de Insectos/genética , Masculino , Datos de Secuencia Molecular , Mutación , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/genética , Fenotipo , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: The objectives of this study were to compare vincristine/actinomycin D/cyclophosphamide/adriamycin (VACA) with VACA/plus imidazole carboxamide (DTIC) (VACAD) therapy in regards to complete/partial response and event free survival rates in children and adolescents with metastatic non-rhabdomyosarcoma soft tissue sarcomas (NRSTS) or previously chemotherapy-naive recurrent NRSTS or locally persistent gross residual tumor after surgery and radiation therapy. PROCEDURES: Between 1986 and March 1994, 75 patients entered this randomized study comparing VACA and VACAD, given at 3 week intervals. Sixty-one patients were considered eligible and received chemotherapy and radiation therapy to the primary tumor and areas of metastases. Thirty-six patients had regional unresected (Group III) disease, and 25 had metastatic disease (Group IV) at time of accession. Thirty-six patients received VACA (11 were not randomized), and 25 received VACAD. RESULTS: With a median follow-up of greater than 4 years, overall and event-free survival for all eligible patients are 30.6% and 18.4% respectively (S.E: 9.5% and 6.8%). There was insufficient evidence that DTIC offered any advantage to event free survival, but there was evidence for better outcome for patients in Group III disease in comparison to patients with Group IV disease, and for patients with a Grade 1 and 2 histology in comparison to Grade 3 lesions. CONCLUSIONS: Combination chemotherapy with VACA and VACAD were insufficient to prevent recurrent or progressive disease in children and adolescents with high stage NRSTS. The use of vincristine/ifosfamide/doxorubicin with cytokine support is under study.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Sarcoma/tratamiento farmacológico , Neoplasias de los Tejidos Blandos/tratamiento farmacológico , Adolescente , Adulto , Niño , Preescolar , Terapia Combinada , Ciclofosfamida/administración & dosificación , Dacarbazina/administración & dosificación , Dactinomicina/administración & dosificación , Supervivencia sin Enfermedad , Doxorrubicina/administración & dosificación , Femenino , Humanos , Infusiones Intravenosas , Inyecciones Intravenosas , Masculino , Recurrencia , Sarcoma/patología , Sarcoma/radioterapia , Sarcoma/cirugía , Neoplasias de los Tejidos Blandos/patología , Neoplasias de los Tejidos Blandos/radioterapia , Neoplasias de los Tejidos Blandos/cirugía , Vincristina/administración & dosificaciónRESUMEN
The Extradenticle (Exd) protein in Drosophila acts as a cofactor to homeotic proteins. Its nuclear localization is regulated. We report the cloning of the Drosophila homothorax (hth) gene, a homolog of the mouse Meis1 proto-oncogene that has a homeobox related to that of exd. Comparison with Meis1 finds two regions of high homology: a novel MH domain and the homeodomain. In imaginal discs, hth expression coincides with nuclear Exd. hth and exd also have virtually identical, mutant clonal phenotypes in adults. These results suggest that hth and exd function in the same pathway. We show that hth acts upstream of exd and is required and sufficient for Exd protein nuclear localization. We also show that hth and exd are both negative regulators of eye development; their mutant clones caused ectopic eye formation. Targeted expression of hth, but not of exd, in the eye disc abolished eye development completely. We suggest that hth acts with exd to delimit the eye field and prevent inappropriate eye development.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila , Drosophila/crecimiento & desarrollo , Ojo/crecimiento & desarrollo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Núcleo Celular/química , Clonación Molecular , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Drosophila/química , Drosophila/genética , Expresión Génica/genética , Expresión Génica/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto/genética , Proteínas de Homeodominio/análisis , Datos de Secuencia Molecular , Mutación/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/genética , Fenotipo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Distribución Tisular , Factores de Transcripción/análisis , Factores de Transcripción/genéticaRESUMEN
To identify novel genes and to isolate tagged mutations in known genes that are required for the development of the peripheral nervous system (PNS), we have screened a novel collection of 2460 strains carrying lethal or semilethal P element insertions on the third chromosome. Monoclonal antibody 22C10 was used as a marker to visualize the embryonic PNS. We identified 109 mutant strains that exhibited reproducible phenotypes in the PNS. Cytological and genetic analyses of these strains indicated that 87 mutations affect previously identified genes: tramtrack (n = 18 alleles), string (n = 15), cyclin A (n = 13), single-minded (n = 13), Delta (n = 9), neuralized (n = 4), pointed (n = 4), extra macrochaetae (n = 4), prospero (n = 3), tartan (n = 2), and pebble (n = 2). In addition, 13 mutations affect genes that we identified recently in a chemical mutagenesis screen designed to isolate similar mutants: hearty (n = 3), dorsotonals (n = 2), pavarotti (n = 2), sanpodo (n = 2), dalmatian (n = 1), missensed (n = 1), senseless (n = 1), and sticky ch1 (n = 1). The remaining nine mutations define seven novel complementation groups. The data presented here demonstrate that this collection of P elements will be useful for the identification and cloning of novel genes on the third chromosome, since >70% of mutations identified in the screen are caused by the insertion of a P element. A comparison between this screen and a chemical mutagenesis screen undertaken earlier highlights the complementarity of the two types of genetic screens.
Asunto(s)
Alelos , Elementos Transponibles de ADN , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Genes de Insecto , Animales , Anticuerpos Monoclonales/metabolismo , Axones , Mapeo Cromosómico , Cromosomas , Expresión Génica , Operón Lac , Mutagénesis , Neuronas/metabolismo , Sistema Nervioso Periférico/citología , Sistema Nervioso Periférico/embriología , FenotipoRESUMEN
Assessing the effects of non-covalently bound chemicals on DNA structure is particularly challenging. Traditional methods require the use of cumbersome electrophoretic techniques or that the compounds bind DNA with an extremely high affinity. Here we demonstrate that, by extending the use of the exonuclease Bal 31, we can rely on a standard cyclization assay technique and one dimensional gel electrophoresis to identify and quantitate chemical induced DNA bending. An important application of this method is to the study of small molecules that bind to DNA non-covalently and we illustrate the method with the antitumor antibiotic calicheamicin. Our results suggest that the distribution of circles resulting from the polymerization of a 21 bp DNA construct reflects the kinetics of the competing cyclization and dimerization reactions and provides a method for rapidly screening compounds for DNA bending.
Asunto(s)
Aminoglicósidos , Antibacterianos/metabolismo , ADN/química , Electroforesis en Gel de Poliacrilamida/métodos , Conformación de Ácido Nucleico , Sitios de Unión , ADN/metabolismo , ADN Ligasas/metabolismo , EndodesoxirribonucleasasRESUMEN
The gutfeeling (guf) gene was uncovered in a genetic screen for genes that are required for proper development of the embryonic peripheral nervous system. Mutations in guf cause defects in growth cone guidance and fasciculation and loss of expression of several neuronal markers in the embryonic peripheral and central nervous systems. guf is required for terminal differentiation of neuronal cells. Mutations in guf also affect the development of muscles in the embryo. In the absence or guf activity, myoblasts are formed properly, but myoblast fusion and further differentiation of muscle fibers is severely impaired. The guf gene was cloned and found to encode a 21-kD protein with a significant sequence similarity to the mammalian ornithine decarboxylase antizyme (OAZ). In mammals, OAZ plays a key regulatory role in the polyamine biosynthetic pathway through its binding to, and inhibition of, ornithine decarboxylase (ODC), the first enzyme in the pathway. The elaborate regulation of ODC activity in mammals still lacks a defined developmental role and little is known about the involvement of polyamines in cellular differentiation. GUF is the first antizyme-like protein identified in invertebrates. We discuss its possible developmental roles in light of this homology.
Asunto(s)
Diferenciación Celular , Drosophila/genética , Inhibidores Enzimáticos , Músculos/citología , Neuronas/citología , Inhibidores de la Ornitina Descarboxilasa , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sistema Nervioso Central/citología , ADN Complementario , Humanos , Datos de Secuencia Molecular , Mutagénesis , Sistema Nervioso Periférico/citología , Homología de Secuencia de AminoácidoRESUMEN
Using a multimethod multistage screening procedure, the authors interviewed 201 parents and their children with the Diagnostic Interview Schedule for Children (DISC 2.1). In addition, parents completed the Child Behavior Checklist (CBCL) and other survey measures, while their children completed self-report scales. Receiver operating characteristic (ROC) analyses were done to determine optimal cutpoints on the CBCL, referenced to DISC diagnostic "caseness." DISC diagnoses, DISC "stem" symptoms, CBCL scores, and CBCL ROC-outpoints were compared against "external validators," in order to determine the comparative advantages of each approach for assessing child psychopathology. Overall findings suggest that the controversies about "best" assessment strategies may be artificial: When both assessment approaches are compared using similar methods, they are reasonably comparable. However, highly specific diagnostic categories may show fewer relationships with external validators and may therefore need more systematic validational studies.
Asunto(s)
Trastornos de la Conducta Infantil/diagnóstico , Psiquiatría Infantil/métodos , Trastornos Mentales/diagnóstico , Escalas de Valoración Psiquiátrica/normas , Psicometría , Adolescente , Trastornos de Ansiedad/clasificación , Trastornos de Ansiedad/diagnóstico , Trastorno por Déficit de Atención con Hiperactividad/diagnóstico , Niño , Trastornos de la Conducta Infantil/clasificación , Preescolar , Trastorno Depresivo/diagnóstico , Análisis Discriminante , Análisis Factorial , Humanos , Trastornos Mentales/clasificación , Psiquiatría Militar/métodos , Valor Predictivo de las Pruebas , Escalas de Valoración Psiquiátrica/estadística & datos numéricos , Psicometría/métodos , Curva ROC , Reproducibilidad de los Resultados , Factores de Riesgo , MuestreoAsunto(s)
Genes , Invertebrados/embriología , Sistema Nervioso/embriología , Neuronas/fisiología , Vertebrados/embriología , Animales , Diferenciación Celular/genética , Drosophila/embriología , Drosophila/genética , Embrión no Mamífero/fisiología , Genes de Insecto , Humanos , Invertebrados/genética , Sistema Nervioso/citología , Neuronas/citología , Especificidad de la Especie , Vertebrados/genéticaRESUMEN
Using the Xenopus borealis 5S RNA gene, we have identified several new features of the interaction of calicheamicin (CAL), an enediyne antitumor agent, with nucleosomal and naked DNA targets. CAL-mediated DNA damage was generally reduced by incorporation of the DNA into a nucleosome. However, in one instance, the frequency of DNA damage was enhanced in the nucleosome compared to naked DNA. This increase in CAL damage may result from bending-induced changes in the target site, while the association of histone proteins with DNA in the nucleosome may generally reduce the affinity of CAL for its targets by imposing dynamic constraints on the DNA, by altering target structure, or by steric hindrance. One implication of these observations is that new structural features created by incorporation of DNA into chromatin may produce 'hot spots' for CAL-mediated DNA damage not apparent in naked DNA studies. In a second set of experiments, the orientation of CAL at damage sites in naked 5S rDNA was determined. The results suggest that minor groove width per se is not a major determinant of CAL target selection. Our studies support the generality of an oligopurine recognition element, with the additional requirement that the purine tract is interrupted at the 3'-end by a pyrimidine(s). To account for these observations, we propose a model in which CAL recognizes the unique structural and dynamic features associated with the 3'-end of an oligopurine tract. Finally, we conclude that the dyad axis of pseudosymmetry of the 5S rRNA gene nucleosome cannot be determined with any degree of certainty. This places significant limitations on the interpretation of results from the study of drug-DNA interactions with reconstituted nucleosomes.
Asunto(s)
Aminoglicósidos , Antibacterianos/farmacología , Antibióticos Antineoplásicos/farmacología , ADN/efectos de los fármacos , Nucleosomas/genética , Animales , Antibacterianos/química , Antibióticos Antineoplásicos/química , Secuencia de Bases , Sitios de Unión , Pollos , Daño del ADN , ADN Ribosómico/efectos de los fármacos , Enediinos , Modelos Moleculares , Datos de Secuencia Molecular , Nucleosomas/efectos de los fármacos , ARN Ribosómico 5S/genética , Relación Estructura-Actividad , XenopusRESUMEN
Decannulation of a tracheostomy generally results in spontaneous closure. Occasionally, epithelialization results in persistence of the fistula, which may be initially treated by local curettage or cautery. Failure of these methods constitutes an indication for surgical closure. Dissection of the entire tracheocutaneous tract permits fistula closure in juxtaposition to but outside the trachea and prevents any iatrogenic airway narrowing. Twelve patients have been so managed over the last 10 years, and there have been no immediate or long-term complications.