Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles.

Transgenic mice carrying heterologous genes directed by a 670-bp segment of the regulatory sequence from the human transferrin (TF) gene demonstrated high expression in brain. Mice carrying the chimeric 0.67kbTF-CAT gene expressed TF-CAT in neurons and glial cells of the nucleus basalis, the cerebrum, corpus callosum, cerebellum, and hippocampus. In brains from two independent TF-CAT transgenic founder lines, copy number of TF-CAT mRNA exceeded the number of mRNA transcripts encoding either mouse endogenous transferrin or mouse endogenous amyloid precursor protein. In two transgenic founder lines, the chloramphenicol acetyltransferase (CAT) protein synthesized from the TF-CAT mRNA was estimated to be 0.10-0.15% of the total soluble proteins of the brain. High expression observed in brain indicates that the 0.67kbTF promoter is a promising director of brain expression of heterologous genes. Therefore, the promoter has been used to express the three common human apolipoprotein E (apoE) alleles in transgenic mouse brains. The apoE alleles have been implicated in the expression of Alzheimer disease, and the human apoE isoforms are reported to interact with different affinities to the brain beta-amyloid and tau protein in vitro. Results of this study demonstrate high expression and production of human apoE proteins in transgenic mouse brains. The model may be used to characterize the interaction of human apoE isoforms with other brain proteins and provide information helpful in designing therapeutic strategies for Alzheimer disease.

Inflammatory skin disease in transgenic mice that express high levels of interleukin 1 alpha in basal epidermis.

Resting epidermal keratinocytes contain large amounts of interleukin 1 (IL-1), but the function of this cytokine in the skin remains unclear. To further define the role of IL-1 in cutaneous biology, we have generated two lines of transgenic mice (TgIL-1.1 and TgIL-1.2) which overexpress IL-1 alpha in basal keratinocytes. There was high-level tissue-specific expression of transgene mRNA and protein and large quantities of IL-1 alpha were liberated into the circulation from epidermis in both lines. TgIL-1.1 mice, which had the highest level of transgene expression, developed a spontaneous skin disease characterized by hair loss, scaling, and focal inflammatory skin lesions. Histologically, nonlesional skin of these animals was characterized by hyperkeratosis and a dermal mononuclear cell infiltrate of macrophage/monocyte lineage. Inflammatory lesions were marked by a mixed cellular infiltrate, acanthosis, and, in some cases, parakeratosis. These findings confirm the concept of IL-1 as a primary cytokine, release of which is able to initiate and localize an inflammatory reaction. Furthermore, these mice provide the first definitive evidence that inflammatory mediators can be released from the epidermis to enter the systemic circulation and thereby influence, in a paracrine or endocrine fashion, a wide variety of other cell types.

Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is expressed in the adrenal cortex and gonads and regulates the expression of several P450 steroid hydroxylases in vitro. We examined the role of SF-1 in the adrenal glands and gonads in vivo by a targeted disruption of the mouse SF-1 gene. All SF-1-deficient mice died shortly after delivery. Their adrenal glands and gonads were absent, and persistent Mullerian structures were found in all genotypic males. While serum levels of corticosterone in SF-1-deficient mice were diminished, levels of adrenocorticotropic hormone (ACTH) were elevated, consistent with intact pituitary corticotrophs. Intrauterine survival of SF-1-deficient mice appeared normal, and they had normal serum level of corticosterone and ACTH, probably reflecting transplacental passage of maternal steroids. We tested whether SF-1 is required for P450 side-chain-cleavage enzyme (P450scc) expression in the placenta, which expresses both SF-1 and P450scc, and found that in contrast to its strong activation of the P450scc gene promoter in vitro, the absence of SF-1 had no effect on P450scc mRNA levels in vivo. Although the region targeted by our disruption is shared by SF-1 and by embryonal long terminal repeat-binding protein (ELP), a hypothesized alternatively spliced product, we believe that the observed phenotype reflects absent SF-1 alone, as PCR analysis failed to detect ELP transcripts in any mouse tissue, and sequences corresponding to ELP are not conserved across species. These results confirm that SF-1 is an important regulator of adrenal and gonadal development, but its regulation of steroid hydroxylase expression in vivo remains to be established.

Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake.

Human intestinal epithelial cells express functional cytokine receptors sharing the common gamma c chain of the interleukin 2 receptor.

Interleukin (IL) 2 signaling requires the dimerization of the IL-2 receptor beta (IL-2R beta) and common gamma (gamma c) chains. The gamma is also a component of the receptors for IL-4, IL-7, and IL-9. To assess the extent and role of the receptor signal transducing system utilizing the gamma c chain on human intestinal epithelial cells, the expression of gamma c, IL-2R beta, and receptor chains specific for IL-4, IL-7, and IL-9 was assessed by reverse transcription-coupled PCR on human intestinal epithelial cell lines and on isolated primary human intestinal epithelial cells. Caco-2, HT-29, and T-84 cells were found to express transcripts for the gamma c and IL-4R chains constitutively. IL-2R beta chain expression was demonstrated in Caco-2 and HT-29 but not in T-84 cells. None of the cell lines expressed mRNA for the IL-2R alpha chain. After stimulation with epidermal growth factor for 24 h Caco-2, HT-29, and T-84 cells expressed transcripts for IL-7R. In addition, Caco-2 and HT-29 cells expressed mRNA for the IL-9R. Receptors for IL-2, IL-4, IL-7, and IL-9 on intestinal epithelial cells lines appeared to be functional; stimulation with these cytokines caused rapid tyrosine phosphorylation of proteins. The relevance of the observations in intestinal epithelial cell lines for intestinal epithelial function in vivo was supported by the demonstration of transcripts for gamma c, IL-2R beta, IL-4R, IL-7R, and IL-9R in primary human intestinal epithelial cells.

Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.

Levels and subcellular distribution of connexin 43 (Cx43), a gap junction protein, were studied in hamster leukocytes before and after activation with endotoxin (lipopolysaccharide, LPS) both in vitro and in vivo. Untreated leukocytes did not express Cx43. However, Cx43 was clearly detectable by indirect immunofluorescence in cells treated in vitro with LPS (1 micrograms/ml, 3 hr). Cx43 was also detected in leukocytes obtained from the peritoneal cavity 5-7 days after LPS-induced inflammation. In some leukocytes that formed clusters Cx43 immunoreactivity was present at appositional membranes, suggesting formation of homotypic gap junctions. In cell homogenates of activated peritoneal macrophages, Cx43, detected by Western blot analysis, was mostly unphosphorylated. A second in vivo inflammatory condition studied was that induced by ischemia-reperfusion of the hamster cheek pouch. In this system, leukocytes that adhered to venular endothelial cells after 1 hr of ischemia, followed by 1 hr of reperfusion, expressed Cx43. Electron microscope observations revealed small close appositions, putative gap junctions, at leukocyte-endothelial cell and leukocyte-leukocyte contacts. These results indicate that the expression of Cx43 can be induced in leukocytes during an inflammatory response which might allow for heterotypic or homotypic intercellular gap junctional communication. Gap junctions may play a role in leukocyte extravasation.

Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide.

Mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play important roles in tissue inflammation and in neuroimmune interactions. Very little is known about endogenous molecules and mechanisms capable of modulating mast cell activation. Palmitoylethanolamide, found in peripheral tissues, has been proposed to behave as a local autacoid capable of downregulating mast cell activation and inflammation. A cognate N-acylamide, anandamide, the ethanolamide of arachidonic acid, occurs in brain and is a candidate endogenous agonist for the central cannabinoid receptor (CB1). As a second cannabinoid receptor (CB2) has been found in peripheral tissues, the possible presence of CB2 receptors on mast cells and their interaction with N-acylamides was investigated. Here we report that mast cells express both the gene and a functional CB2 receptor protein with negative regulatory effects on mast cell activation. Although both palmitoylethanolamide and anandamide bind to the CB2 receptor, only the former downmodulates mast cell activation in vitro. Further, the functional effect of palmitoylethanolamide, as well as that of the active cannabinoids, was efficiently antagonized by anandamide. The results suggest that (i) peripheral cannabinoid CB2 receptors control, upon agonist binding, mast cell activation and therefore inflammation; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for the CB2 receptor on mast cells; (iii) modulatory activities on mast cells exerted by the naturally occurring molecule strengthen a proposed autacoid local inflammation antagonism (ALIA) mechanism; and (iv) palmitoylethanolamide and its derivatives may provide antiinflammatory therapeutic strategies specifically targeted to mast cells ("ALIAmides").

Versiones en español de películas de género fantástico en el Hollywood de los años treinta del siglo XX

A finales de la década de los años veinte el cine mudo estaba llegando a su fin y comenzaron a producirse masivamente películas habladas. Los grandes estudios de Hollywood temen perder público fuera de Estados Unidos y antes de decidirse por el doblaje, ya que este sistema presentaba en aquella época problemas de sincronización, comienzan filmar versiones múltiples de un mismo guion en diferentes idiomas para exportar a los países de habla no inglesa. Dentro de todas las películas en español producidas por Hollywood, esta tesis doctoral está centrada únicamente en las de género fantástico. Es cierto que en las versiones en español encontramos pocas de dicho género, pero dentro de las existentes hay películas muy variadas, desde películas totalmente de género como las dos versiones de Drácula hasta otras que contienen elementos fantásticos lo suficientemente interesantes como para ser incluidas en esta investigación. La mayoría de las versiones en español que se produjeron en la década de los años treinta eran comedias o melodramas, los dos géneros más populares de la época. Pero de las más de cien películas en español que se filmaron en Hollywood durante la década de los años treinta algunas pueden catalogarse dentro del género fantástico. Estas películas son: ¡Pobre infeliz! (Charles Rogers, 1930), versión en español de The Shrimp (Ídem, Charles Rogers ,1930); Noche de duendes (James Parrott, 1930), versión en español de The Laurel-Hardy Murder Case (Ídem, James Parrot, 1930); La voluntad del muerto (George Melford, 1930), versión en español de The Cat Creeps (Rupert Julian, John Willard, 1930); Wu Li Chang (Nick Grinde, 1930), versión en español de Mr. Wu (William Night, 1927); Drácula (George Melford, 1931), versión en español de Drácula (Dracula, Tod Browning, 1931); Cheri-Bibi (Carlos F. Borcosque, 1931), versión en español de The Phantom of Paris (Ídem, John S. Robertson, 1931) y El último varón sobre la tierra (James Tinling, 1933), versión en español de It's Great to Be Alive (Alfred L. Werker, 1933)...

La evolución de las actitudes del héroe en el cine de Hollywood: percepción de psicología del espectador

El cine ha ido pasando por diversas etapas que destacan por la existencia de características comunes, en el ámbito académico se suele dividir en clásico, moderno y postmoderno. Los diferentes elementos narrativos, tales como el espacio, el tiempo, la acción e, incluso, la construcción física de los personajes, se actualizan y evolucionan a lo largo de la historia, formando parte en el nacimiento de una nueva tipología de cine. Sin embargo, en el estudio de la caracterización psicológica de los protagonistas y, más concretamente, de los héroes no se ha explorado tanto como con el resto de elementos. El espectador se convierte en uno de los principales agentes quien no solo dota de significado a la obra sino que, también, actúa como juez y testigo privilegiado de las acciones del héroe, mismas que sirven como referente para crear un compendio de las actitudes que son definitorias del mismo. Además, el espectador forma parte de un colectivo, semejante al comportamiento del protagonista, que puede ser entendido en relación a otros personajes, por ende, la presente investigación se centra en determinar la evolución en las actitudes del héroe por medio de un estudio empírico aplicado al espectador con una metodología propia de la psicología social. Para este objetivo se escogieron tres películas que se corresponden con cada uno de los periodos cinematográficos y se elaboró una serie de proposiciones que son planteadas al espectador quien, por medio de su propia percepción, será el encargado final de determinar si los protagonistas pueden ser catalogados como héroes y cuál es el compendio de actitudes que los caracterizan.

El ciclo de acción de Hollywood (1980-2015): industria, autoría y cine de acción en la obra de Steven Seagal

Esta tesis doctoral analiza y evalúa el impacto, alcance e influencia de los filmes comprendidos en lo que ha dado en llamarse el ciclo de acción de Hollywood entre los años 1980 y 2015 —el ciclo posee etapas netamente diferenciadas: «musculosa», «acrobática» y «inhumana»—. Mediante el estudio de tres vertientes —industria, autoría y el cine de acción—, se ha puesto en contexto este tipo de cinematografía dentro de un escenario de entretenimiento multinacional —donde el cine es una de las múltiples for-mas artísticas de entretenimiento basadas en la narrativa, junto a los videojuegos, la música, la literatura, etc.— y globalizado —el cine de Hollywood, la industria más impor-tante del mundo en términos económicos, lo es no solo por sí misma, sino por su im-pacto en el mercado internacional—. En este sentido, el cine de acción, dadas sus carac-terísticas intrínsecas más elementales, se ha manifestado como un vehículo narrativo y artístico idóneo para la exportación del entretenimiento de Hollywood —así como su ideología— a lo largo de todo el mundo. A través del análisis de una de las obras cinematográficas más consistentes y coherentes del ciclo de acción Hollywood —la obra de Steven Seagal: actor, productor, guionista, director, compositor musical y coreógrafo—, la presente tesis ofrece una pro-funda reflexión sobre el fenómeno de la acción en el cine, su origen, su alcance y sus más habituales manifestaciones. Argumenta, además, la imposibilidad de concebir un cine narrativo sin acción —en mayor o menor medida— en su seno. El estudio de la obra de Steven Seagal —y su clasificación en etapas: clasicista, manierista y metadiscursivo— permite extraer algunas de las más relevantes caracterís-ticas y contradicciones de este tipo de filmes, al tiempo que ofrece un nuevo paradigma de análisis centrado en la influencia autoral del actor en esta cinematografía y/o en los datos de carácter cuantificable extraídos de los mismos —número de escenas de acción, duración de las mismas, porcentaje de acción por filme, etc.

SQL Server Management Studio Express Instalación

Instalación de SQL Server Management Studio Express en un MS Windows 7 Profesional.

SQL Server Management Studio Express 01

Primeros pasos en el uso de este programa cliente de acceso y administración de SQL Server. Este cliente, en su versión Express, es gratuito. El ejemplo del vídeo se conecta con un servidor remoto SQL Server 2008 R2.