Involvement of Rel/Nuclear Factor-κB Transcription Factors in Keratinocyte Senescence

After a finite doubling number, normal cells become senescent, i.e. nonproliferating and apoptosis resistant. Because Rel/nuclear factor (NF)-κB transcription factors regulate both proliferation and apoptosis, we have investigated their involvement in senescence. cRel overexpression in young normal keratinocytes results in premature senescence, as defined by proliferation blockage, apoptosis resistance, enlargement, and appearance of senescence-associated β-galactosidase (SA-β-Gal) activity. Normal senescent keratinocytes display a greater endogenous Rel/NF-κB DNA binding activity than young cells; inhibiting this activity in presenescent cells decreases the number of cells expressing the SA-β-Gal marker. Normal senescent keratinocytes and cRel-induced premature senescent keratinocytes overexpressed manganese superoxide dismutase (MnSOD), a redox enzyme encoded by a Rel/NF-κB target gene. MnSOD transforms the toxic O2.- into H2O2, whereas catalase and glutathione peroxidase convert H2O2 into H2O. Neither catalase nor glutathione peroxidase is up-regulated during cRel-induced premature senescence or during normal senescence, suggesting that H 2O2 accumulates. Quenching H2O2 by catalase delays the occurrence of both normal and premature cRel-induced senescence. Conversely, adding a nontoxic dose of H2O2 to the culture medium of young normal keratinocytes induces a premature senescence-like state. All these results indicate that Rel/NF-κB factors could take part in the occurrence of senescence by generating an oxidative stress via the induction of MnSOD.

Characterization of the human and mouse ETV1/ER81 transcription factor genes: Role of the two alternatively spliced isoforms in the human

The Ets transcription factors of the PEA3 group - E1AF/PEA3, ETV1/ER81 and ERM - are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1α) or the absence (ETV1β) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1β) and the full-length isoform (human ETV1α) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1α and human ETV1β expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.

The NRF-1/α-PAL transcription factor regulates human E2F6 promoter activity

E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoretic-mobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/α-PAL (nuclear respiratory factor-1/α-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/α-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/α-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/α-PAL is implicated in the regulation of basal E2F6 gene expression.

The 26S proteasome system degrades the ERM transcription factor and regulates its transcription-enhancing activity

ERM is a member of the ETS transcription factor family. High levels of the corresponding mRNA are detected in a variety of human breast cancer cell lines, as well as in aggressive human breast tumors. As ERM protein is almost undetectable in these cells, high degradation of this transcription factor has been postulated. Here we have investigated whether ERM degradation might depend on the proteasome pathway. We show that endogenous and ectopically expressed ERM protein is short-lived protein and undergoes proteasome-dependent degradation. Deletion mutagenesis studies indicate that the 61 C-terminal amino acids of ERM are critical for its proteolysis and serve as a degradation signal. Although ERM conjugates with ubiquitin, this post-translational modification does not depend on the C-terminal domain. We have used an Ets-responsive ICAM-1 reporter plasmid to show that the ubiquitin-proteasome pathway can affect transcriptional function of ERM. Thus, ERM is subject to degradation via the 26S proteasome pathway, and this pathway probably plays an important role in regulating ERM transcriptional activity. © 2007 Nature Publishing Group. All rights reserved.

The evolutionarily conserved transcription factor PRDM12 controls sensory neuron development and pain perception

PR homology domain-containing member 12 (PRDM12) is a highly evolutionary conserved member of the Prdm family of transcription factors that play essential roles in many cell fate decisions. In human, PRDM12 coding mutations have been recently identified in several patients with hereditary sensory and autonomic neuropathy (HSAN) (submitted elsewhere). Here we show that PRDM12 is involved in sensory neurogenesis in Xenopus and that several of the human Prdm12 mutants show altered structure, subcellular localization and function. In Drosophila, we demonstrate that the sensory neuron specific RNAi knockdown of the Prdm12 ortholog Hamlet induces impaired nociception and that a similar phenotype is observed in hypomorph hamlet mutants. In human fibroblasts of patients with PRDM12 mutations, we identified additional possible downstream target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE). Knock-down of fly TRHDE in sensory neurons resulted in altered nociceptive neurons and impaired nociception. Collectively, these findings provide the first evidence showing that Prdm12 plays an important role in sensory neuron development. They also suggest that it has a critical evolutionarily conserved role in pain perception via modulation of the TRH signaling pathway.

Expression of the Ets transcription factor Erm is regulated through a conventional PKC signaling pathway in the Molt4 lymphoblastic cell line.

Erm, a member of the PEA3 group within the Ets family of transcription factors, is expressed in murine and human lymphocytes. Here, we show that in the human Molt4 lymphoblastic cell line, the erm gene expression is regulated by the conventional PKC (cPKC) pathway. To better characterize the molecular mechanism by which cPKC regulates Erm transcription in Molt4 cells, we tested proximal promoter deletions of the human gene, and identified a specific cPKC-regulated region between positions -420 and -115 upstream of the first exon.

Heparin-binding hemagglutinin, from an extrapulmonary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis.

Interactions of Mycobacterium tuberculosis with macrophages have long been recognized to be crucial to the pathogenesis of tuberculosis. The role of non-phagocytic cells is less well known. We have discovered a M. tuberculosis surface protein that interacts specifically with non-phagocytic cells, expresses hemagglutination activity and binds to sulfated glycoconjugates. It is therefore called heparin-binding hemagglutinin (HBHA). HBHA-deficient M. tuberculosis mutant strains are significantly impaired in their ability to disseminate from the lungs to other tissues, suggesting that the interaction with non-phagocytic cells, such as pulmonary epithelial cells, may play an important role in the extrapulmonary dissemination of the tubercle bacillus, one of the key steps that may lead to latency. Latently infected human individuals mount a strong T cell response to HBHA, whereas patients with active disease do not, suggesting that HBHA is a good marker for the immunodiagnosis of latent tuberculosis, and that HBHA-specific Th1 responses may contribute to protective immunity against active tuberculosis. Strong HBHA-mediated immuno-protection was shown in mouse challenge models. HBHA is a methylated protein and its antigenicity in latently infected subjects, as well as its protective immunogenicity strongly depends on the methylation pattern of HBHA. In both mice and man, the HBHA-specific IFN-gamma was produced by both the CD4(+) and the CD8(+) T cells. Furthermore, the HBHA-specific CD8(+) T cells expressed bactericidal and cytotoxic activities to mycobacteria-infected macrophages. This latter activity is most likely perforin mediated. Together, these observations strongly support the potential of methylated HBHA as an important component in future, acellular vaccines against tuberculosis.

The mycobacterial heparin-binding hemagglutinin, virulence factor and antigen useful for diagnostics and vaccine development.

info:eu-repo/semantics/published

Ectopic expression of the Ets transcription factor ER81 in transgenic mouse mammary gland enhances both urokinase plasminogen activator and stromelysin-1 transcription

The PEA3 group members PEA3, ER81 and ERM, which are highly conserved transcription factors from the Ets family, are over-expressed in metastatic mammary tumors. In the current study, we present the characterization of a transgenic mouse strain which over-expresses ER81 in the mammary gland via the long terminal repeat of the mouse mammary tumor virus (LTR-MMTV). Although six genotypically positive transgenic lines were identified, only one expressed the ectopic transcript with an exclusive expression in the lactating and late-pregnancy (18th day) mammary glands. No mammary tumor or mammary deregulation appeared after 2 years of ectopic ER81 expression following lactation. We then sought to identify ER81 target genes, and the urokinase plasminogen activator (uPA) and the stromelysin-1, two enzymes involved in extracellular matrix degradation, were found to be transcriptionally upregulated in lactating mammary glands over-expressing ER81. Since these enzymes are involved in metastasis, this murine model could be further used to enhance mammary cancer metastatic process by crossing these animals with mice carrying non-metastatic mammary tumors. We thus created a transgenic mouse model permitting the over-expression of a functionally active Ets transcription factor in the mammary gland without perturbing its development.

Study of gene expression in thyrotropin-stimulated thyroid cells by cDNA expression array: ID3 transcription modulating factor as an early response protein and tumor marker in thyroid carcinomas.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with (32)P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

A New Approach to Media Cluster Research: Bringing the Human Factor back into Scope

This paper is part of a collaborative project being undertaken by the three leading universities of Brussels, VUB, ULB and USL-B supported by Innnoviris. The project called Media Clusters Brussels - MCB - started in October 2014 with the goal to analyze the development of a Media Park around the two public broadcasters at the site of Reyers in Brussels being host of a media cluster in the capital city. Not only policymakers but also many authors recognized in the last decade that the media industry is characterized from a geographical point of view by a heavy concentration to a limited number of large cities, where media clusters have emerged (Karlsson & Picard, 2011). The common assumption about media clusters is that locating inside a regional agglomeration of related actors brings advantages for these firms. Especially, the interrelations and interactions between the actors on a social level matter for the shape and efficiency of the agglomerations (Picard, 2008). However, even though the importance of the actors and their interrelations has been a common assumption, many authors solely focus on the macro-economical aspects of the clusters. Within this paper, we propose to realize a socio-economical analysis of media clusters to make informed decisions in the development and so, bring the social (human) factor back into scope. Therefore, this article focuses on the development of a novel valuable framework, the so-called 7P framework with a multilevel and interdisciplinary approach, which includes three aspects, which have been identified as emerging success-factors of media clusters: partnerships, (media) professionals and positive spillovers.

El capital social como factor de producción en los sistemas de agronegocios en Argentina : un análisis comparado de los sistemas aviar, vitivinícola y vacuno

El sector agroalimentario se ha convertido en el de mayor importancia durante los 90 para la economía argentina junto a los sectores del petróleo y acero. Sin embargo, el gran empuje proveniente del 'campo' no se traslada al resto de redes de abatecimiento de alimentos. El desarrollo veloz de las interrelaciones comerciales que conducen al establecimiento de distintas formas de relaciones para el abastecimiento de alimentos es un fenómeno clave en las economías agroalimentarias modernas. Los principales actores de las cadenas agroindustriales deben diseñar las mejores opciones respecto del diseño de sus relaciones comerciales. El objetivo principal del siguiente trabajo es identificar al capital social como un factor de producción para el desarrolo de los sistemas de agronegocios argentinos. Los objetivos específicos serán identificar el nivel de capital social del sistema de ganados y carnes vacuna, del sistema avícola y del sistema vitivinícola de la Argentina. El trabajo determina que el desarrollo global de los agronegocios en Argentina depende del grado de enforcement de la ley y los contratos y del nivel de acción colectiva. Dentro de las variables estudiadas 'confianza', 'acción colectiva' y 'cooperación y ética' presenta altos niveles en las tres variables, el sistema vitivinícola medios y el sistema de ganados y carnes vacuno los presenta bajos. El sistema de negocios avícola es la que marca un mayor desarrollo de capital social a lo largo de la muestra. El nivel de confianza expresado por sus miembros demuestra su capacidad para resolver los dilemas que presenta la acción colectiva en el negocio real, contrariamente al sistema vacuno, mientras que el sistema vitivinícola se coloca en una posición media. El sistema avícola presenta mayor cantidad de contratos formales y como vimos un mayor respeto por su cumplimiento más allá de la incompltitud de los mismos. La falta de contratos formales y la falta de control por parte del Estado, en mayor medida en el subsector vacuno por sobre el vitivinícola, favorece el doble estándar impositivo, comercial y sanitario (no en el caso del vino). En tal sentido, el no respeto por el conjunto de reglas de conducta formales (leyes, tradiciones, costumbres, sistema de valores, religiones, tendencias sociológicas, etc.), es decir las instituciones, que facilitan la coordinación o rigen las relaciones entre individuos o grupos, le agrega mayor incertidumbre a la interacción humana

Los contratistas de cosecha de plantaciones forestales : análisis de las funciones de producción y de la influencia de la innovación tecnológica como factor en las mismas

La cosecha de plantaciones forestales ha crecido sostenidamente en Sudamérica en los últimos años. Esta se desarrolla mayoritariamente con empresas contratistas, que abastecen plantas industriales. Esta Tesis tiene por objetivos analizar por medio de funciones de producción y de la innovación, las empresas contratistas de cosecha en Misiones y Corrientes (Argentina), Paraná, Santa Catarina y Rio Grande do Sul (Brasil), y Uruguay. Los datos fueron obtenidos por encuestas personalizadas: 22 en Argentina, 35 en Brasil, y 10 en Uruguay, entre 2008 y 2012. Se evaluaron modelos de costos y producción. El crecimiento de las empresas fue analizado con modelos lineales mixtos. La producción relevada fue de 17,7 millones de metros cúbicos, (58,3 por ciento del total regional). Los modelos de producción del tipo Cobb-Douglas, mostraron muy alta necesidad de capital al inicio, con un cambio a partir de las 50.000 t/mes, y fueron asintóticos desde las 100.000 t/mes. Los contratistas en Argentina produjeron en promedio 13.636 t/mes, con 30,7 empleados por empresa. En Brasil, produjeron 25.649t/mes, con 65,12 empleados. Los modelos de costo por tonelada resultaron en 5,82 US$/t para 18.000 t/mes para Argentina. En Brasil el costo mínimo fue de 11,23 US$/t para 25.000 t/mes. En Uruguay fue de 18,43 US$/t para 15.000 t/mes. En los tres países existió preponderancia de contratos con celulósicas. No se encontró ninguna contratista que desarrolle los indicadores de producción o de costo por tonelada que obtuvieron estas empresas mutualistas. En los análisis de producción de los últimos 10 años, fueron significativos el número de empleados, y la mecanización. Cuando se consideraron la capacidad de hacer negocios y la innovación, fue solo significativa esta última. La inclusión de las variables aleatorias, país y edad de la empresa, produjo mejores AIC (Akaike Information Criterion). En el análisis del crecimiento de las empresas en los últimos 10 años, resultaron significativos el capital y el número de empleados. En los últimos 5 años, ocurrió lo contrario, fueron significativas la innovación y la capacidad de hacer negocios. Se puede concluir que el capital es el principal factor para predecir la capacidad productiva de una contratista. Existen diferencias de escala entre las empresas de los tres países, así como en los sectores de cada país. A los largo de los últimos 10 años, las empresas han ido creciendo basándose en diferentes estrategias, al principio en capital y empleados, y últimamente en la capacidad de hacer negocios y en la innovación. Sudamérica, Cosecha forestal.

Influencia de la temperatura en llenado de grano como factor determinante de la presencia de granos verdes en soja

Una de las características que define la calidad de los granos de soja es la presencia de granos verdes (GV). Estos exhiben una coloración verdosa, total o parcial, debida a la presencia de pigmentos clorofílicos no degradados durante el proceso de maduración de los granos. A pesar de que algunas evidencias sugieren que la presencia de GV se asocia a que parte del ciclo del cultivo coincida con temperaturas altas, no se encontraron en la bibliografía trabajos que confronten fehacientemente las condiciones ambientales con la presencia de GV en el cultivo de soja en condiciones de campo. Más aún, aunque se presume que la temperatura durante la fase de llenado está involucrada en modular la incidencia de GV, se desconoce si los efectos están asociados a los períodos tempranos del llenado, cuando predomina la formación de los granos, a la fase de crecimiento intermedia o a las fases finales, en las que se degrada la clorofila y se alcanza la madurez. El objetivo de este trabajo fue producir información que contribuya a desarrollar estrategias de manejo adecuadas para reducir la incidencia de GV en los sistemas de producción de soja, sobre la base de la identificación de las condiciones ambientales que determinan su aparición. Durante las campañas agrícolas 2004/05 a 2008/09, se realizaron ensayos a campo combinando diferentes fechas de siembra y genotipos, de modo de generar un escalonamiento de la etapa de maduración de los granos. Se realizaron además, dos ensayos a campo en condiciones semi-controladas. En todos los casos se evaluó el porcentaje de GV y se analizó su asociación con la temperatura y el déficit hídrico en diferentes momentos del llenado. Las altas temperaturas por sí solas no fueron determinantes de la aparición de granos verdes en el cultivo de soja ni en los experimentos de siembras escalonadas ni en los llevados a cabo en forma semi-controlada. Por el contrario, condiciones de altas temperaturas durante las etapas finales del llenado en combinación con estrés hídrico favorecieron la presencia de GV. Los genotipos más afectados fueron los de ciclo precoz (GM III y IV), sembrados en fechas tempranas, ya que ubicaron el llenado en condiciones conducentes de la aparición de GV. Los genotipos de GM largos no exploraron, en Rafaela, condiciones conducentes a la aparición de GV. La almacenabilidad de las semillas fue afectada en mayor medida por el GV que por el valor de PG inicial, luego de la cosecha.

Una propuesta para predecir el factor de erosividad R para el centro y norte de la Argentina

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