RESUMEN
BACKGROUND: Because of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals), supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans. RESULTS: In an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an egfp transgenic stable fish line that trapped tnks1bp1, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(tnks1bp1:EGFP) line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells. CONCLUSIONS: We present a Tg(tnks1bp1:EGFP) stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general.
Asunto(s)
Animales Modificados Genéticamente , Proteínas Fluorescentes Verdes/genética , Sistema de la Línea Lateral/citología , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Transcriptoma , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Expresión Génica , Genes Reporteros , Marcadores Genéticos , Proteínas Fluorescentes Verdes/biosíntesis , Hibridación in Situ , Larva/citología , Larva/metabolismo , Sistema de la Línea Lateral/crecimiento & desarrollo , Datos de Secuencia Molecular , Mutagénesis Insercional , Mucosa Olfatoria/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteína 1 de Unión a Repeticiones Teloméricas/química , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Transcripción Genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismoRESUMEN
In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.
Asunto(s)
Células Ciliadas Auditivas/fisiología , Sistema de la Línea Lateral/fisiología , Regeneración , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Proliferación Celular , Células Ciliadas Auditivas/química , Sistema de la Línea Lateral/química , Sistema de la Línea Lateral/citología , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Inhibition of heat shock protein 90 (HSP90) leads to inappropriate processing of proteins involved in cell survival pathways. We found that HSP90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG), is synergistic with radiation for non-small cell lung cancer cell lines, NCI-H460 and A549. To establish the optimal schedule for this combination, cells were radiated before, after, or simultaneously with DMAG, and survival was scored by clonogenic assay. The sequence of DMAG administration was critical for synergy with radiation, and pretreatment for 16 h led to maximal synergy. Similar radiosensitization was observed in isogenic cells in which expression of wild-type p53 was silenced by RNA interference, although p53 loss rendered cells overall less radiosensitive. The mechanistic basis for synergy was studied by Western blotting, cell cycle analysis, alkaline comet assay, and direct measurement of the activities of key base excision repair enzymes. Regardless of schedule of administration, DMAG led to degradation of proteins involved in activation of cell survival pathways after radiation, which did not explain the differences in the schedule of administration observed in clonogenic assays. In addition to previously reported decrease in activation of ATM, pretreatment with DMAG blocked activation of base excision repair machinery and activity of key enzymes, apurinic/apyrimidinic endonuclease, and DNA polymerase-beta. Similarly, pretreatment with specific apurinic/apyrimidinic endonuclease inhibitor, CRT0044876, reproduced the effects of DMAG. Thus, administration of HSP90 inhibitors before radiation is critical for optimizing their use as radiosensitizers.
Asunto(s)
Benzoquinonas/farmacología , Proteínas de Ciclo Celular/metabolismo , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Radiación , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Tolerancia a Radiación/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Bloom syndrome is a rare, autosomal recessive inherited disorder in humans. The product of the Bloom syndrome mutated gene, designated BLM, is a member of the RecQ helicase family. BLM has been proposed to function at the interface of replication and recombination, and to facilitate the repair of DNA damage. Here, we report in vivo physical interaction and colocalization of BLM and a DNA structure-specific endonuclease, Mus81, at sites of stalled replication forks outside the promyelocytic leukemia nuclear bodies during the S-phase arrest of the cell cycle. Amino acids 125 to 244 of Mus81 interact with the C-terminal region (amino acids 1,007-1,417) of BLM. Whereas Mus81 does not have any effect on the helicase activity of BLM, BLM can stimulate Mus81 endonuclease activity on the nicked Holliday junctions and 3' flap. This stimulation is due to enhanced binding of Mus81 to the DNA substrates. These data suggest a new function of BLM in cooperating with Mus81 during processing and restoration of stalled replication forks.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Adenosina Trifosfatasas/genética , Sitios de Unión , Línea Celular , ADN/biosíntesis , ADN/metabolismo , ADN Helicasas/genética , Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Fibroblastos/enzimología , Células HCT116 , Humanos , Mapeo Peptídico , RecQ Helicasas , TransfecciónRESUMEN
RATIONALE: Ethnopharmacology has documented hundreds of psychoactive plants awaiting exploitation for drug discovery. A robust and inexpensive in vivo system allowing systematic screening would be critical to exploiting this knowledge. OBJECTIVE: The objective of this study was to establish a cheap and accurate screening method which can be used for testing psychoactive efficacy of complex mixtures of unknown composition, like plant crude extracts. METHODS: We used automated recording of zebrafish larval swimming behavior during light vs. dark periods which we reproducibly altered with an anxiogenic compound, pentylenetetrazole (PTZ). First, we reversed this PTZ-altered swimming by co-treatment with a well-defined synthetic anxiolytic drug, valproic acid (VPA). Next, we aimed at reversing it by adding crude root extracts of Valeriana officinalis (Val) from which VPA was originally derived. Finally, we assessed how expression of neural activity-regulated genes (c-fos, npas4a, and bdnf) known to be upregulated by PTZ treatment was affected in the presence of Val. RESULTS: Both VPA and Val significantly reversed the PTZ-altered swimming behaviors. Noticeably, Val at higher doses was affecting swimming independently of the presence of PTZ. A strong regulation of all three neural-activity genes was observed in Val-treated larvae which fully supported the behavioral results. CONCLUSIONS: We demonstrated in a combined behavioral-molecular approach the strong psychoactivity of a natural extract of unknown composition made from V. officinalis. Our results highlight the efficacy and sensitivity of such an approach, therefore offering a novel in vivo screening system amenable to high-throughput testing of promising ethnobotanical candidates.
Asunto(s)
Antimaníacos/farmacología , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Pentilenotetrazol/farmacología , Extractos Vegetales/farmacología , Natación/fisiología , Valeriana , Ácido Valproico/farmacología , Animales , Ansiolíticos/farmacología , Biomarcadores/metabolismo , Encéfalo/metabolismo , Quimioterapia Combinada , Genes fos/efectos de los fármacos , Hipnóticos y Sedantes/farmacología , Larva , Proteínas Proto-Oncogénicas c-fos/metabolismo , Pez Cebra/fisiologíaRESUMEN
Mutation in the CSB gene results in the human Cockayne's syndrome (CS). Here, we provide evidence that CSB is found not only in the nucleoplasm but also in the nucleolus within a complex (CSB IP/150) that contains RNA pol I, TFIIH, and XPG and promotes efficient rRNA synthesis. CSB is active in in vitro RNA pol I transcription and restores rRNA synthesis when transfected in CSB-deficient cells. We also show that mutations in CSB, as well as in XPB and XPD genes, all of which confer CS, disturb the RNA pol I/TFIIH interaction within the CSB IP/150. In addition to revealing an unanticipated function for CSB in rRNA synthesis, we show that the fragility of this complex could be one factor contributing to the CS phenotype.