Expression cloning of Forssman glycolipid synthetase: a novel member of the histo-blood group ABO gene family.

A phenotypic cloning approach was used to isolate a canine cDNA encoding Forssman glycolipid synthetase (FS; UDP-GalNAc:globoside alpha-1,3-N-acetylgalactosaminyltransferase; EC 2.4.1.88). The deduced amino acid sequence of FS demonstrates extensive identity to three previously cloned glycosyltransferases, including the enzymes responsible for synthesis of histo-blood group A and B antigens. These three enzymes, like FS, catalyze the addition of either N-acetylgalactosamine (GalNAc) or galactose (Gal) in alpha-1,3-linkage to their respective substrates. Despite the high degree of sequence similarity among the transferases, we demonstrate that the FS cDNA encodes an enzyme capable of synthesizing Forssman glycolipid, and demonstrates no GalNAc or Gal transferase activity when closely related substrates are examined. Thus, the FS cDNA is a novel member of the histo-blood group ABO gene family that encodes glycosyltransferases with related but distinct substrate specificity. Cloning of the FS cDNA will allow a detailed dissection of the roles Forssman glycolipid plays in cellular differentiation, development, and malignant transformation.

Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis.

The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha1-->2-linked N-acetylglucosamine. The biosynthesis of these chains probably occurs in the lumen of the Golgi apparatus, by analogy to S. cerevisiae. The sugar donors, GDP-mannose and UDP-GlcNAc, must first be transported from the cytosol, their site of synthesis, via specific Golgi membrane transporters into the lumen where they are substrates in the biosynthesis of these mannoproteins. A mutant of K. lactis, mnn2-2, that lacks terminal N-acetylglucosamine in its mannan chains in vivo, has recently been characterized and shown to have a specific defect in transport of UDP-GlcNAc into the lumen of Golgi vesicles in vitro. We have now cloned the gene encoding the K. lactis Golgi membrane UDP-GlcNAc transporter by complementation of the mnn2-2 mutation. The mnn2-2 mutant was transformed with a genomic library from wild-type K. lactis in a pKD1-derived vector; transformants were isolated and phenotypic correction was monitored following cell surface labeling with fluorescein isothiocyanate conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescent activated cell sorter. A 2.4-kb DNA fragment was found to restore the wild-type lectin binding phenotype. Upon loss of the plasmid containing this fragment, reversion to the mutant phenotype occurred. The above fragment contained an open reading frame for a multitransmembrane spanning protein of 328 amino acids. The protein contains a leucine zipper motif and has high homology to predicted proteins from S. cerevisiae and C. elegans. In an assay in vitro, Golgi vesicles isolated from the transformant had regained their ability to transport UDP-GlcNAc. Taken together, the above results strongly suggest that the cloned gene encodes the Golgi UDP-GlcNAc transporter of K. lactis.

Cells expressing the DG42 gene from early Xenopus embryos synthesize hyaluronan.

DG42 is one of the main mRNAs expressed during gastrulation in embryos of Xenopus laevis. Here we demonstrate that cells expressing this mRNA synthesize hyaluronan. The cloned DG42 cDNA was expressed in rabbit kidney (RK13) and human osteosarcoma (tk-) cells using a vaccinia virus system. Lysates prepared from infected cells were incubated in the presence of UDP-N-acetylglucosamine and UDP-[14C]glucuronic acid. This yielded a glycosaminoglycan with a molecular mass of about 200,000 Da. Formation of this product was only observed in the presence of both substrates. The glycosaminoglycan could be digested with testicular hyaluronidase and with Streptomyces hyaluronate lyase but not with Serratia chitinase. Hyaluronan synthase activity could also be detected in homogenates of early Xenopus embryos, and the activity was found to correlate with the expression of DG42 mRNA at different stages of development. Synthesis of hyaluronan is thus an early event after midblastula transition, indicating its importance for the ensuing cell movements in the developing embryo. Our results are at variance with a recent report (Semino, C. E. & Robbins, P. W. (1995) Proc. Natl. Acad. Sci. USA 92, 3498-3501) that DG42 codes for an enzyme that catalyzes the synthesis of chitin-like oligosaccharides.

Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.

We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylation step of glycosphingolipid synthesis and the product, glucosylceramide, serves as the core of more than 300 glycosphingolipids. The cDNA has a G+C-rich 5' untranslated region of 290 nucleotides and the open reading frame encodes 394 amino acids (44.9 kDa). A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequence. In addition, considerable hydrophobicity was detected in the regions close to the C terminus, which may interact with the membrane. A catalytically active enzyme was produced from Escherichia coli transfected with the cDNA. Northern blot analysis revealed a single transcript of 3.5 kb, and the mRNA was widely expressed in organs. The amino acid sequence of ceramide glucosyltransferase shows no significant homology to ceramide galactosyltransferase, which indicates different evolutionary origins of these enzymes.

T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating O-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc-Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of Oglycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte O-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by O-glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate O-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific O-glycan formation governed by distinct polypeptide GalNAc-Ts.

A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants.

Sucrose synthase (SuSy; EC 2.4.1.13; sucrose + UDP reversible UDPglucose + fructose) has always been studied as a cytoplasmic enzyme in plant cells where it serves to degrade sucrose and provide carbon for respiration and synthesis of cell wall polysaccharides and starch. We report here that at least half of the total SuSy of developing cotton fibers (Gossypium hirsutum) is tightly associated with the plasma membrane. Therefore, this form of SuSy might serve to channel carbon directly from sucrose to cellulose and/or callose synthases in the plasma membrane. By using detached and permeabilized cotton fibers, we show that carbon from sucrose can be converted at high rates to both cellulose and callose. Synthesis of cellulose or callose is favored by addition of EGTA or calcium and cellobiose, respectively. These findings contrast with the traditional observation that when UDPglucose is used as substrate in vitro, callose is the major product synthesized. Immunolocalization studies show that SuSy can be localized at the fiber surface in patterns consistent with the deposition of cellulose or callose. Thus, these results support a model in which SuSy exists in a complex with the beta-glucan synthases and serves to channel carbon from sucrose to glucan.

Regulation of glycolipid synthesis in HL-60 cells by antisense oligodeoxynucleotides to glycosyltransferase sequences: effect on cellular differentiation.

Treatment of the human promyelocytic leukemia cell line HL-60 with antisense oligodeoxynucleotides to UDP-N-acetylgalactosamine:beta-1,4-N-acetylgalactosaminyl-transferase (GM2-synthase; EC 2.4.1.92) and CMP-sialic acid:alpha-2,8-sialyltransferase (GD3-synthase; EC 2.4.99.8) sequences effectively down-regulated the synthesis of more complex gangliosides in the ganglioside synthetic pathways after GM3, resulting in a remarkable increase in endogenous GM3 with concomitant decreases in more complex gangliosides. The treated cells underwent monocytic differentiation as judged by morphological changes, adherent ability, and nitroblue tetrazolium staining. These data provide evidence that the increased endogenous ganglioside GM3 may play an important role in regulating cellular differentiation and that the antisense DNA technique proves to be a powerful tool in manipulating glycolipid synthesis in the cell.

Electrophile and antioxidant regulation of enzymes that detoxify carcinogens.

Detoxication (phase 2) enzymes, such as glutathione S-transferases (GSTs), NAD(P)H:(quinone-acceptor) oxidoreductase (QR), and UDP-glucuronsyltransferase, are induced in animal cells exposed to a variety of electrophilic compounds and phenolic antioxidants. Induction protects against the toxic and neoplastic effects of carcinogens and is mediated by activation of upstream electrophile-responsive/antioxidant-responsive elements (EpRE/ARE). The mechanism of activation of these enhancers was analyzed by transient gene expression of growth hormone reporter constructs containing a 41-bp region derived from the mouse GST Ya gene 5'-upstream region that contains the EpRE/ARE element and of constructs in which this element was replaced with either one or two consensus phorbol 12-tetradecanoate 13-acetate (TPA)-responsive elements (TREs). When these three constructs were compared in Hep G2 (human) and Hepa 1c1c7 (murine) hepatoma cells, the wild-type sequence was highly activated by diverse inducers, including tert-butylhydroquinone, Michael reaction acceptors, 1,2-dithiole-3-thione, sulforaphane,2,3-dimercapto-1-propanol, HgCl2, sodium arsenite, and phenylarsine oxide. In contrast, constructs with consensus TRE sites were not induced significantly. TPA in combination with these compounds led to additive or synergistic inductions of the EpRE/ARE construct, but induction of the TRE construct was similar to that induced by TPA alone. Transfection of the EpRE/ARE reporter construct into F9 cells, which lack endogenous TRE-binding proteins, produced large inductions by the same compounds, which also induced QR activity in these cells. We conclude that activation of the EpRE/ARE by electrophile and antioxidant inducers is mediated by EpRE/ARE-specific proteins.

Lipid A biosynthesis in Rhizobium leguminosarum: role of a 2-keto-3-deoxyoctulosonate-activated 4' phosphatase.

Lipid A from several strains of the N2-fixing bacterium Rhizobium leguminosarum displays significant structural differences from Escherichia coli lipid A, one of which is the complete absence of phosphate groups. However, the first seven enzymes of E. coli lipid A biosynthesis, leading from UDP-GlcNAc to the phosphorylated intermediate, 2-keto-3-deoxyoctulosonate (Kdo2)-lipid IVA, are present in R. leguminosarum. We now describe a membrane-bound phosphatase in R. leguminosarum extracts that removes the 4' phosphate of Kdo2-lipid IVA. The 4' phosphatase is selective for substrates containing the Kdo domain. It is present in extracts of R. leguminosarum biovars phaseoli, viciae, and trifolii but is not detectable in E. coli and Rhizobium meliloti. A nodulation-defective strain (24AR) of R. leguminosarum biovar trifolii, known to contain a 4' phosphatase residue on its lipid A, also lacks measurable 4' phosphatase activity. The Kdo-dependent 4' phosphatase appears to be a key reaction in a pathway for generating phosphate-deficient lipid A.

"Empezar es haber andado la mitad" : Organización social y condiciones laborales en la Sociedad Vitícola Uruguaya

La Sociedad Vitícola Uruguaya (SVU) se fundó en marzo de 1887 fue parte del proyecto modernizador y diversificador del agro uruguayo impulsado por la Asociación Rural del Uruguay (ARU), entidad corporativa similar a la Sociedad Rural Argentina. Este trabajo analiza los orígenes de la SVU, única entidad de este tipo en Sudamérica, en el marco del desarrollo agrario uruguayo de finales del siglo XIX. También se estudian las condiciones de la mano de obra asalariada, la división de las tareas y la estructura organizacional en los orígenes del establecimiento. El objetivo es analizar cómo un sector de la élite uruguaya, reunida en la ARU, impulsó la creación de la SVU como parte de un modelo de producción capitalista diversificada y basada en una sociedad por acciones en el agro a finales del siglo XIX. La hipótesis que guía este trabajo es que dicho proyecto no sólo tenía como objetivo el desarrollo de la vitivinicultura, sino también, el de servir de modelo sociocultural con el fin de motorizar relaciones sociales capitalistas en el agro y de afincar al "gaucho" en el entorno rural

Avaliação in vivo da efetividade e do pH de géis clareadores no clareamento em consultório em 12 meses de acompanhamento

O objetivo deste estudo in vivo, internacional, randomizado e duplo cedo foi avaliar comparativamente a efetividade e o pH de diferentes géis clareadores na técnica de clareamento em consultório, com e sem o emprego de fonte de luz híbrida em função do grau de alteração de cor, sensibilidade e manutenção do tratamento ao longo de 12 meses de acompanhamento. Foram selecionados 48 voluntários de acordo com os critérios de inclusão e exclusão. Os pacientes foram divididos, de forma randomizada, em 4 grupos de 12 participantes cada, onde: Grupo EXP10 5 aplicações do gel de peróxido de hidrogênio a 10% (Gel Experimental DMC Equipamentos) e ativação de luz híbrida de LED (violeta)/Laser (Experimental DMC Equipamentos) com 7′ e 30″ por aplicação, com tempo total de 37′30; Grupo LP15 5 aplicações do gel de peróxido de hidrogênio 15% (Lase Peroxide Lite DMC Equipamentos) seguindo mesmo protocolo do grupo EXP10; Grupo TB35LH 3 aplicações do gel de peróxido de hidrogênio a 35% (Total Blanc Office - DFL) e ativação de luz híbrida de LED (azul)/Laser (Whitening Lase II DMC Equipamentos) de 7′ e 30″ por aplicação, com tempo total de 22′30″; Grupo TB35 3 aplicações do gel de peróxido de hidrogênio a 35% (Total Blanc Office - DFL) sem ativação com fonte de luz, totalizando 45″. A determinação dos valores de pH foi realizada com o peagômetro digital (Sentron Model 1001, Sentron) nos tempos inicial e após o término do protocolo clareador. A aferição da cor foi feita com espectofotômetro VITA Easyshade antes do clareamento, após 24 horas, 1 semana, 1, 6 e 12 meses. A sensibilidade dentária e grau de satisfação dos pacientes foram avaliados por meio do questionário VAS e IPS antes, imediatamente após o clareamento, 24 horas e uma semana após. Os resultados da alteração do pH receberam tratamento estatístico pela ANOVA e teste de Bonferroni a 0,05%. Os resultados indicaram que o pH aumentou do momento inicial para o final para todos os protocolos. Não houve diferenças significativas entre os protocolos TB35 e TB35LH em nenhum dos momentos, e o pH médio do grupo EXP10 foi maior em comparação aos outros três grupos nos dois momentos avaliados. Os resultados do ΔE receberam tratamento estatístico pela ANOVA e teste de Bonferroni a 0,05%. Os resultados indicaram que não houve diferença significativa entre os grupos LP15, TB35 e TB35LH. O ΔE médio observado após 24 horas foi estatisticamente maior que para os outros tempos (inicial, 1 semana, 1 mês, 6 e 12 meses). Para análise da sensibilidade foi construído um modelo linear misto e atribuídos postos (ranks) aos valores de Δ e teste de Bonferroni a 0,05% para comparações pareadas. Não houve diferença nos valores da sensibilidade imediatamente e 24 horas após o tratamento, com relação ao momento inicial. Houve diferença significativa entre Δ1 e Δ3 indicando que a sensação de dor após uma semana do tratamento foi menor do que as observadas nos instantes imediato e após 24 horas. Para os resultados de satisfação foi construído um modelo linear misto e atribuídos postos (ranks) e o Método de Bonferroni (0,05%) foi utilizado para as comparações pareadas do efeito de tempo. Os resultados indicam queda nos níveis de satisfação entre os períodos imediato e um ano e entre os períodos 24 horas e um ano. Todos os géis clareadores apresentaram mínima variação do pH nos tempos avaliados, entretanto houve um aumento do pH da primeira para a última aplicação em todos os grupos estudados e o grupo EXP10 apresentou os maiores valores de pH seguido do LP15, TB35LH e TB35 apresentaram os valores mais baixos de pH. Os grupos LP15, TB35 e TB35LH apresentaram menor variação da cor ao longo de 12 meses de acompanhamento. O efeito do protocolo clareador não influenciou a sensibilidade dos pacientes e após uma semana a sensibilidade retornaram aos níveis normais. O nível de satisfação dos pacientes foi significativo em relação ao tempo e não aos protocolos clareadores, os pacientes do grupo TB35 mostraram-se mais insatisfeitos ao longo da pesquisa.

Responsabilidad social corporativa: el rol del Estado como prescriptor

La Responsabilidad Social Corporativa (RSC), como resultado de un proceso que lleva ya varias décadas, hoy y cada vez más, es asumida e incorporada por las organizaciones como un valioso instrumento estratégico, en el cual la RSC engloba todas las decisiones organizacionales que son adoptadas y que tienen en cuenta tanto la dimensión económica como la dimensión social y la ambiental, de manera que sea posible generar valor económico y contribuir simultáneamente al bienestar de las generaciones presentes y futuras. De alguna manera, los principios y prácticas de RSC no serán desarrollados ni adoptados espontáneamente por las organizaciones sino en la medida de que las organizaciones dominantes (una élite, en el sentido del modelo del nuevo institucionalismo) encuentren interesante imponer su concepción particular sobre la calidad de la RSC (en el sentido de análisis de calidad de economía industrial). Existen estudios que demuestran que el respeto a mayores exigencias en materia de desarrollo sostenible por parte de una organización tiene un efecto positivo sobre su productividad, ya que existe una correlación positiva entre la Performance Social (PS) de las organizaciones y su Performance Financiera (PF) (Hitchens et al., 2001; Orlitzky et al., 2003; Allouche and Laroche, 2005; Margolis and Elfenbein, 2008). Y aunque los estudios empíricos no han encontrado un vínculo simple entre PS y PF, la hipótesis del “círculo virtuoso” prevalece: una mejor PS (prácticas de RSC) conduce a una mejor PF (Waddock and Graves, 1997). Por lo tanto la RSC se consolida como fuente de ventaja competitiva y herramienta de protección en momentos de crisis (Gomez Carrasco, 2012). Es así como, gracias a la mayor rentabilidad resultante de la implementación a nivel institucional de la RSC en el seno de su organización, las organizaciones dominantes del mercado impondrán sus principios y prácticas al resto de los actores del mercado...

Divergencias y convergencias de la disputa educativa por la construcción de la nación entre el liberalismo y el conservadurismo católico en México entre 1857-1982

La presente investigación trata de responder la siguiente pregunta: ¿hay un proceso de confluencia ideológica a lo largo de los siglos XIX y XX de los liberales y conservadores mexicanos en un proyecto educativo nacionalista? Para responderla, el trabajo analiza la disputa ideológica de tipo histórico en torno a la educación entre la élite liberal y la conservadora católica (incluida la jerarquía católica) mexicanas, con referencia a las relaciones Iglesia-Estado y al proceso de construcción de la nación. Dicho en otros términos, este trabajo analiza la construcción de la nación en México a través de la educación durante el periodo 1857-1982. En este estudio se considera que el origen de la nación mexicana es resultado de un proceso de construcción orquestado por las élites. El proceso se inició formalmente con la independencia de España en 1821. Desde entonces, las élites consideraron a la educación como una herramienta fundamental para trasmitir sus valores y construir los emblemas representativos de la nación. Las élites políticas percibieron que el monopolio de la educación les dotaba de ventajas sobre sus competidores, ya que proporcionaba diversos recursos institucionales para el ejercicio del poder y su conservación. En la disputa por el control de la educación, la Iglesia Católica (y el conservadurismo católico) proponía que la orientación moral del Estado, así como los valores de la democracia y la participación política, deberían sustentarse sobre una educación integral que incluyera la religión católica como parte de la regeneración moral de los individuos. Por su parte, la élite liberal dedicó el quehacer público a la secularización de la educación y a crear una sociedad racional que se liberara del oscurantismo y del fanatismo religioso. El conflicto se agudizó cuando la Iglesia Católica perdió el monopolio educativo con las Leyes de Reforma (1855-1863); entonces comenzó una fuerte competencia con el Estado (el Gobierno) por el control ideológico y político de la sociedad. Por su parte, la élite política liberal impidió el avance del conservadurismo católico y se hizo con los recursos institucionales (formales e informales) para conservar y legitimar su control del poder...

Ciudad de Vacaciones 1933: el concurso internacional para Playa de San Juan (Alicante) [PPT]

Presentación realizada para el Seminari Internacional Territoris del Turisme, Girona, 23-25 gener 2014.

Ciudad de Vacaciones 1933. El concurso internacional para Playa de San Juan (Alicante)

En 1933, por iniciativa municipal y con el apoyo del Gobierno, con la intención de captar un turismo extranjero y nacional de élite generando una nueva ‘industria’, se convoca el concurso de anteproyectos para la construcción de una ciudad satélite (a modo de ciudad jardín) para destinarla a ciudad de vacaciones en la Playa de San Juan (cvPSJ), Alicante, al que se presentan tres propuestas. Aquí se estudia el anteproyecto ganador (de P. Muguruza), que resulta pionero por las técnicas urbanísticas empleadas (información y zonificación), por la aplicación de la fotografía para la inserción de arquitecturas y equipamientos y por la sensibilidad desplegada en la protección del patrimonio cultural (medioambiental e histórico). Los referentes para este macro complejo turístico (de casi 10 km2), coetáneo a la Ciutat del Repós i Vacances (CRV) de Castelldefels, no proceden tanto de Europa como de EUA. Se realiza un análisis pormenorizado de la ordenación urbanística en atención a cómo el territorio existente la condiciona y se entrelaza con estrategias de promoción turística, donde se combinan la tríada: hotel, deporte y naturaleza (alojamiento, ocio y salud). Pero toda la ciudad está enfocada a un turismo burgués, para el que se prevé una arquitectura comercial que pronto envejecería en su repertorio. Veinticinco años después, en 1958, cuando las condiciones económicas y sociales fueron favorables al desarrollo de la zona, el mundo sería ya otro y el proyecto quedó obsoleto.