RESUMEN
A study was performed to analyze whether local denervation of the medial branch of the dorsal ramus of the lumbar spinal nerve occurs in a patient with postoperative failed-back surgery syndrome/postdiscotomy syndrome (FBSS/PDS). We investigated the effect of the loss of innervation of the multifidus muscle on neuronal nitrite oxide synthetase (n-NOS) and endothelial nitrite oxide synthetase (e-NOS) applying realtime RT-PCR and immunohistochemistry. Our study demonstrates a substantial reduction of n-NOS expression, supporting the view that local denervation of the multifidus is involved in the pathology of FBSS. No regulation of e-NOS was detectable. Interestingly, this change is region-specific and does not occur throughout the entire multifidus segment. This result supports the hypothesis that local denervation of the multifidus muscle is involved in the pathology of FBSS/ PDS.
Asunto(s)
Laminectomía/efectos adversos , Dolor de la Región Lumbar/etiología , Músculo Esquelético/inervación , Atrofia Muscular Espinal/etiología , Anciano , Biopsia , Regulación hacia Abajo , Humanos , Desplazamiento del Disco Intervertebral/cirugía , Vértebras Lumbares/cirugía , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Atrofia Muscular Espinal/patología , Neuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Traumatismos de los Nervios Periféricos , SíndromeRESUMEN
The expression and functional properties of connexin36 (Cx36) have been investigated in two neuroblastoma cell lines (Neuro2A, RT4-AC) and primary hippocampal neurons transfected with a Cx36-enhanced green fluorescent protein (EGFP) expression vector. Transfected cells express Cx36-EGFP mRNA, and Cx36-EGFP protein is localized in the perinuclear area and cell membrane. Upon differentiation of cell lines, Cx36-EGFP protein was detectable in processes with both axonal and dendritic characteristics. Small gap junction plaques were found between adjacent cells, and electrophysiological recordings demonstrated that the electrical properties of these gap junctions were virtually indistinguishable from those reported for native Cx36. Mutagenesis of Cx36 led to the identification of a structural element that interferes with normal protein localization. In contrast, site directed mutagenesis of putative protein phosphorylation motifs did not alter subcellular localization. This excludes phosphorylation/dephosphorylation as a major regulatory step in Cx36 protein transport.
Asunto(s)
Comunicación Celular/fisiología , Compartimento Celular/fisiología , Sistema Nervioso Central/metabolismo , Conexinas/metabolismo , Proteínas del Ojo/metabolismo , Uniones Comunicantes/metabolismo , Neuronas/metabolismo , Transporte de Proteínas/fisiología , Animales , Animales Recién Nacidos , Sistema Nervioso Central/ultraestructura , Conexinas/genética , Proteínas del Ojo/genética , Técnica del Anticuerpo Fluorescente , Proteína GAP-43/metabolismo , Uniones Comunicantes/ultraestructura , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/metabolismo , Mutagénesis Sitio-Dirigida/genética , Neuronas/ultraestructura , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Proteína delta-6 de Union ComunicanteRESUMEN
Human renal cell carcinoma (RCC) is a common malignant disease of the kidney characterized by dedifferentiation of renal epithelial cells. Our previous experiments showed that most RCCs have a loss of function of the tissue-specific transcription factor hepatocyte nuclear factor (HNF) 1alpha. Detailed analyses of the 10 exons encoding HNF1alpha in 32 human RCCs by single-strand conformation polymorphism analysis and direct DNA sequencing revealed no tumor-associated mutation, whereas with the same probes we frequently found mutations in the von Hippel-Lindau tumor suppressor gene. No mutation leading to loss of HNF1alpha function was detected by analyzing the integrity of the HNF1alpha transcripts in the RNA derived from RCCs by the protein truncation test. Investigating human RCC cell lines by western blotting and gel retardation assays showed a dramatic loss in the expression of the tissue-specific transcription factor HNF1alpha in eight of 10 cell lines. As the HNF1alpha-related transcription factor HNF1beta was expressed in all these tumor cell lines, the loss of HNF1alpha expression was a specific event and was maintained in RCC cell lines. The loss of HNF1alpha expression in RCC cell lines on the RNA level was confirmed by reverse transcription polymerase chain reaction. We propose that tumor-associated mutations in the HNF1alpha gene do not occur in human RCC and that the loss of function is partially due to a transcriptional inactivation of the HNF1alpha gene.
Asunto(s)
Adenocarcinoma de Células Claras/genética , Carcinoma de Células Renales/genética , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Neoplasias Renales/genética , Ligasas , Proteínas de Neoplasias/genética , Proteínas Nucleares , Proteínas/genética , Factores de Transcripción/deficiencia , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Adenocarcinoma de Células Claras/patología , Carcinoma de Células Renales/patología , Diferenciación Celular , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Exones/genética , Predisposición Genética a la Enfermedad , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Neoplasias Renales/patología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/fisiología , Polimorfismo Conformacional Retorcido-Simple , Proteínas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción Genética , Células Tumorales Cultivadas , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
We have adopted RNA fingerprinting methods to screen for genes that are rapidly up- or down-regulated during normal mammalian development, comparing mRNA from early (embryo day 12) to late (embryo day 13) mouse Schwann cell precursors. The use of total RNA, a reduction of cDNA template for amplification, the detection of RT-PCR products with a sensitive DNA stain and polyacrylamide gel electrophoresis and rigid selection criteria involving three screening steps are significant improvements on previous methods. Of 19 differentially displayed bands, 15 represented novel genes. The four known cDNA fragments (interleukin enhancer binding factor 1, beta3 subunit of phospholipase C, brain beta-spectrin, and P21 polypeptide) consisted of coding sequences, indicating a high chance of obtaining coding regions. A semiquantitative RT-PCR analysis of three of the four known genes and a cDNA fragment randomly selected from the pool of 15 novel sequences, confirmed that they were regulated between embryo days 12 and 13, as predicted by the display gels. Our results suggest that the combination of methods described here will have wide applicability in studies of other developmental systems where precisely timed changes occur and where only small amounts of RNA can be obtained for analysis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Técnicas Genéticas , Proteínas del Tejido Nervioso/biosíntesis , Cresta Neural/citología , ARN Mensajero/análisis , Células de Schwann/metabolismo , Células Madre/metabolismo , Técnica de Sustracción , Transcripción Genética , Animales , Biomarcadores , Diferenciación Celular , ADN Complementario/genética , Edad Gestacional , Ratones , Ratones Endogámicos ICR , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Células de Schwann/citología , Nervio Ciático/citología , Nervio Ciático/embriología , Células Madre/citologíaRESUMEN
We show that in the rat, the major gene of PNS myelin, P0, is expressed long before myelination in the neural crest, Schwann cell precursors, and embryonic Schwann cells irrespective of whether they will myelinate or not. This myelin-independent P0 expression is constitutive and likely to serve as a specific marker for the Schwann cell lineage. The much higher P0 expression accompanying myelination is therefore not new gene expression but strong up-regulation of preexisting basal levels. We provide new evidence that the up-regulation to myelination-related levels depends on positive extrinsic signals and therefore does not represent a constitutive phenotype. P0 mRNA is not detectable in mature non-myelin-forming Schwann cells of the sympathetic trunk, but is detectable after transection, indicating that there is a P0-inhibitory signal associated with mature unmyelinated axons. Thus, the regulation of the P0 gene is complex, encompassing extrinsically signaled amplification superimposed on a highly lineage-specific and constitutive basal expression.
Asunto(s)
Axones/fisiología , Embrión de Mamíferos/metabolismo , Proteína P0 de la Mielina/fisiología , Vaina de Mielina/ultraestructura , Sistema Nervioso/embriología , Cresta Neural/metabolismo , Células de Schwann/ultraestructura , Animales , Animales Recién Nacidos/fisiología , División Celular , Línea Celular , Células Cultivadas , Embrión de Mamíferos/fisiología , Desarrollo Embrionario y Fetal , Proteína P0 de la Mielina/genética , Proteína P0 de la Mielina/metabolismo , ARN Mensajero/metabolismo , Ratas/embriología , Ratas Sprague-Dawley , Células de Schwann/citología , Transducción de SeñalRESUMEN
Although basic helix-loop-helix (bHLH) proteins play an important role in transcriptional control in many cell types, the role of HLH proteins in Schwann cells has yet to be assessed. In this study, we have analyzed the expression of the dominant negative HLH genes, Id1 to Id4 and the class A gene REB, during Schwann cell development. We found that mRNA derived from these genes was present in the Schwann cell lineage throughout development including embryonic precursors and mature cells. The mRNA levels were not significantly regulated during development. Nevertheless, by using antibodies against the four different Id proteins, we found clear regulation of some of these genes at the protein level, in particular Id 2, 4, and REB, both in amount and nuclear/cytoplasmic localization. All these proteins are found in the nuclei of Schwann cell precursors but are not seen in nuclei of Schwann cells of newborn nerves. We observed extensive overlap in Id expression, especially in Schwann cell precursors that co-expressed all four Id proteins and REB. We also showed that Id 1 and 2 were up-regulated as Schwann cells progressed through the cell cycle. These data indicate that HLH transcription factors act as regulators of Schwann cell development and point to the existence of as yet unidentified cell type-specific bHLH proteins in these cells.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Proteínas de Plantas , Proteínas Represoras , Células de Schwann/fisiología , Factores de Transcripción/biosíntesis , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Células Cultivadas , Desarrollo Embrionario y Fetal , Edad Gestacional , Proteína 1 Inhibidora de la Diferenciación , Ratones , Ratones Endogámicos ICR , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Células Madre/metabolismo , Fracciones Subcelulares/metabolismo , Transcripción GenéticaRESUMEN
We review the data available on the structure, developmental appearance and embryonic regulation of the tissue-specific transcription factor HNF1 alpha (LFB1) in Xenopus. The expression of the HNF1 alpha gene starts early in embryogenesis shortly after mid-blastula transition and the protein accumulates in the region of the embryo where liver, pronephros and gut--tissues that contain HNF1 alpha in the adult--are developing. The cofactor DCoH, known to stabilize dimer formation of HNF1 alpha, is present as a maternal factor in the egg and has a partially distinct tissue distribution compared to HNF1 alpha. This implies that DCoH does not only modulate HNF1 alpha dimerization but may also cooperate with other transcription factors. By injecting HNF1 alpha promoter CAT constructs into fertilized Xenopus eggs we obtained activation of the injected gene restricted to the region of the developing larvae expressing endogenous HNF1 alpha. Deletion analysis allowed to define the OZ-element that is essential for embryonic activation. This element also occurs in other promoters activated at mid-blastula transition in the embryo and interacts with the maternal factor OZ-1. As the HNF1 alpha promoter also contains functional binding sites for HNF4 and HNF1, we postulate that all of these transcription factors contribute to the cascade leading to proper embryonic activation of the HNF1 alpha gene.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Nucleares , Factores de Transcripción/genética , Xenopus/embriología , Xenopus/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , ADN/genética , ADN/metabolismo , Dimerización , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Hidroliasas/genética , Hidroliasas/metabolismo , Estructura Molecular , Regiones Promotoras Genéticas , Distribución Tisular , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Xenopus/metabolismo , Proteínas de XenopusAsunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas de Unión al ADN , Histidina/inmunología , Fosfoproteínas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Factores de Transcripción/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Factor Nuclear 4 del Hepatocito , Histidina/aislamiento & purificación , Inmunohistoquímica , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Fosfoproteínas/química , Fosfoproteínas/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Factores de Transcripción/química , Factores de Transcripción/inmunología , XenopusRESUMEN
Human renal cell carcinoma is characterized by the loss of differentiation markers such as glutathione S-transferase alpha (GST-alpha). In this paper we show that the promoter of a GST-alpha gene contains a functional binding site for the cell-specific transcription factor LFB1 (HNF1). To investigate the potential role of LFB1 in the down-regulation of GST-alpha expression, we have compared the amount and the binding activity of the LFB1 protein between normal kidney and tumor tissue. By Western analysis and gel retardation assay using a monoclonal antibody specific for LFB1 we show that in 11 of 14 carcinomas the amount of LFB1 is clearly reduced compared to the corresponding normal tissue and that in all 14 renal carcinomas LFB1 binding activity is diminished. As in the same samples the abundance of GST-alpha mRNA is lower than in the normal tissue, we postulate that the loss of LFB1 binding activity might be responsible for the decreased expression of the GST-alpha gene in renal cell carcinoma.
Asunto(s)
Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Proteínas Nucleares , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Secuencia de Bases , Sitios de Unión , Carcinoma de Células Renales/enzimología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Riñón/metabolismo , Riñón/fisiología , Neoplasias Renales/enzimología , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Factores de Transcripción/genéticaRESUMEN
Liver factor B1 [LFB1, also called hepatocyte nuclear factor 1 (HNF1)] is a tissue-specific vertebrate transcription factor that is present in the liver, intestine, stomach and kidney. The LFB1 protein contains an unusual homeobox that is characterized by an insertion of 21 amino acids (aa) not found in any other homeodomain protein. We have isolated and characterized the genomic sequences encoding the LFB1 of Xenopus laevis. By comparing the genomic sequences with the cDNA clones, we could identify nine exons. In general, the position of the introns is identical to the one previously found in the rat. However, the C-terminal activation domain of LFB1 contains, in each species, an exon that is split in two in the other species. The homeobox of the X. laevis LFB1 contains an intron at exactly the position where the 21 aa typical for LFB1 are inserted. This is in agreement with the structure found in the rat gene and supports the notion that the LFB1 homeobox evolved separately from the other genes encoding homeodomain proteins.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Nucleares , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , Exones , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Intrones , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Proteínas de Xenopus , Xenopus laevisRESUMEN
LFB1 (HNF1) is a tissue-specific transcription factor found in the livers, stomachs, intestines, and kidneys of vertebrates. By analyzing the promoter of the Xenopus LFB1 gene, we identified potential autoregulation by LFB1 and regulation by HNF4, a transcription factor with a tissue distribution similar to that of LFB1. Injection of LFB1 promoter-chloramphenicol acetyltransferase constructs into Xenopus eggs revealed embryonic activation that is restricted to the region of the developing larvae expressing endogeneous LFB1. Proper embryonic activation was also observed with a rat LFB1 promoter. Deletion analysis of the Xenopus and rat promoters revealed that in both promoters embryonic activation is absolutely dependnet on the presence of an element that contains CCNCTCTC as the core consensus sequence. Since this element is recognized by the maternal factor OZ-1 previously described by N. Ovsenek, A. M. Zorn, and P. A. Krieg (Development 115:649-655, 1992), we might have identified the main constituents of a hierarchy that leads via LFB1 to the activation of tissue-specific genes during embryogenesis.
Asunto(s)
Proteínas de Unión al ADN , Hígado/metabolismo , Proteínas Nucleares , Fosfoproteínas , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión , Clonación Molecular , ADN , Embrión no Mamífero/metabolismo , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Factor Nuclear 4 del Hepatocito , Hígado/embriología , Mamíferos , Ratones , Microinyecciones , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Regiones Promotoras Genéticas , Ratas , Homología de Secuencia , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteínas de Xenopus , Xenopus laevisRESUMEN
The transcription factor LFB1 (HNF1) was initially identified as a regulator of liver-specific gene expression in mammals. It interacts with the promoter element HP1, which is functionally conserved between mammals and amphibians, suggesting that a homologous factor, XLFB1, also exists in Xenopus laevis. To study the role of LFB1 in early development, we isolated two groups of cDNAs coding for this factor from a Xenopus liver cDNA library by using a rat LFB1 cDNA probe. A comparison of the primary structures of the Xenopus and mammalian proteins shows that the myosin-like dimerization helix, the POU-A-related domain, the homeo-domain-related region, and the serine/threonine-rich activation domain are conserved between X. laevis and mammals, suggesting that all these features typical for LFB1 are essential for function. Using monoclonal antibodies, we demonstrate that XLFB1 is present not only in the liver but also in the stomach, intestine, colon, and kidney. In an analysis of the expression of XLFB1 in the developing Xenopus embryo, XLFB1 transcripts appear at the gastrula stage. The XLFB1 protein can be identified in regions of the embryo in which the liver diverticulum, stomach, gut, and pronephros are localized. The early appearance of XLFB1 expression during embryogenesis suggests that the tissue-specific transcription factor XLFB1 is involved in the determination and/or differentiation of specific cell types during organogenesis.
Asunto(s)
Proteínas de Unión al ADN , Regulación de la Expresión Génica , Proteínas Nucleares , Factores de Transcripción/metabolismo , Xenopus laevis/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Evolución Biológica , ADN/genética , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Hígado/fisiología , Datos de Secuencia Molecular , Mapeo Restrictivo , Alineación de Secuencia , Distribución Tisular , Factores de Transcripción/inmunología , Transcripción Genética , Activación Transcripcional , Proteínas de XenopusRESUMEN
The A2 vitellogenin gene of Xenopus laevis, which is expressed liver specifically, contains an A-activator-binding site (AABS) that mediates high in vitro transcriptional activity in rat liver nuclear extracts. Footprint experiments with DNase I and gel retardation assays revealed the binding of several proteins to AABS. Using binding sites of known DNA-binding proteins as competitors in the gel retardation assay, we found that the transcription factor C/EBP and/or one of its "iso-binders" as well as LFB1/HNF1 bound AABS. These interactions were confirmed by in vitro transcription experiments using various oligonucleotides as competitors. However, saturating amounts of C/EBP- and LFB1/HNF1-binding sites as competitors only partially blocked AABS-mediated transcriptional activity. This finding implies that at least a third distinct transcription factor interacts with AABS. In vitro transcription experiments revealed that AABS was present not only in the closely related Xenopus A1 vitellogenin gene but also in acute-phase genes as a liver-specific regulatory element known to confer the interleukin-6 response. Both AABS and the interleukin-6 response element are promoter modules interacting with at least three distinct transcription factors, including C/EBP and LFB1/HNF1.