Expression cloning of cDNA encoding a human β-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis

The structure and biosynthesis of poly-N-acetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Lex. Herein we describe the isolation and functional expression of a cDNA encoding β-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-N-acetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by fluorescence-activated cell sorting because introduced plasmids containing Epstein–Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAcβ1 → 3Galβ1 → 4GlcNAc → R structure and poly-N-acetyllactosamine extension.

Somatic and germ cell parasitism in a colonial ascidian: Possible role for a highly polymorphic allorecognition system

A colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction in the wild that results alternatively in colony fusion (chimera formation) or inflammatory rejection. A single, highly polymorphic histocompatibility locus (called Fu/HC) is responsible for rejection versus fusion. Gonads are seeded and gametogenesis can occur in colonies well after fusion, and involves circulating germ-line progenitors. Buss proposed that colonial organisms might develop self/non-self histocompatibility systems to limit the possibility of interindividual germ cell “parasitism” (GCP) to histocompatible kin [Buss, L. W. (1982) Proc. Natl. Acad. Sci. USA 79, 5337–5341 and Buss, L. W. (1987) The Evolution of Individuality (Princeton Univ. Press, Princeton]. Here we demonstrate in laboratory and field experiments that both somatic cell and (more importantly) germ-line parasitism are a common occurrence in fused chimeras. These experiments support the tenet in Buss’s hypothesis that germ cell and somatic cell parasitism can occur in fused chimeras and that a somatic appearance may mask the winner of a gametic war. They also provide an interesting challenge to develop formulas that describe the inheritance of competing germ lines rather than competing individuals. The fact that fused B. schlosseri have higher rates of GCP than unfused colonies additionally provides a rational explanation for the generation and maintenance of a high degree of Fu/HC polymorphism, largely limiting GCP to sibling offspring.

The Malthusian parameter of ascents: What prevents the exponential increase of one’s ancestors?

The reason that the indefinite exponential increase in the number of one’s ancestors does not take place is found in the law of sibling interference, which can be expressed by the following simple equation:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\begin{matrix}{\mathit{N}}_{{\mathit{n}}} \enskip & \\ {\mathit{{\blacksquare}}} \enskip & \\ {\mathit{ASZ}} \enskip & \end{matrix} {\mathrm{\hspace{.167em}{\times}\hspace{.167em}2\hspace{.167em}=\hspace{.167em}}}{\mathit{N_{n+1},}}\end{equation*}\end{document} where Nn is the number of ancestors in the nth generation, ASZ is the average sibling size of these ancestors, and Nn+1 is the number of ancestors in the next older generation (n + 1). Accordingly, the exponential increase in the number of one’s ancestors is an initial anomaly that occurs while ASZ remains at 1. Once ASZ begins to exceed 1, the rate of increase in the number of ancestors is progressively curtailed, falling further and further behind the exponential increase rate. Eventually, ASZ reaches 2, and at that point, the number of ancestors stops increasing for two generations. These two generations, named AN SA and AN SA + 1, are the most critical in the ancestry, for one’s ancestors at that point come to represent all the progeny-produced adults of the entire ancestral population. Thereafter, the fate of one’s ancestors becomes the fate of the entire population. If the population to which one belongs is a successful, slowly expanding one, the number of ancestors would slowly decline as you move toward the remote past. This is because ABZ would exceed 2. Only when ABZ is less than 2 would the number of ancestors increase beyond the AN SA and AN SA + 1 generations. Since the above is an indication of a failing population on the way to extinction, there had to be the previous AN SA involving a far greater number of individuals for such a population. Simulations indicated that for a member of a continuously successful population, the AN SA ancestors might have numbered as many as 5.2 million, the AN SA generation being the 28th generation in the past. However, because of the law of increasingly irrelevant remote ancestors, only a very small fraction of the AN SA ancestors would have left genetic traces in the genome of each descendant of today.

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.

Localization of pheromonal sexual dimorphism in Drosophila melanogaster and its effect on sexual isolation.

Drosophila melanogaster is sexually dimorphic for cuticular hydrocarbons, with males and females having strikingly different profiles of the long-chain compounds that act as contact pheromones. Gas-chromatographic analysis of sexual mosaics reveals that the sex specificity of hydrocarbons is located in the abdomen. This explains previous observations that D. melanogaster males display the strongest courtship toward mosaics with female abdomens. We also show that males of the sibling species Drosophila simulans preferentially court D. melanogaster mosaics with male abdomens. Because the primary male hydrocarbon in D. melanogaster is also the primary female hydrocarbon in D. simulans, this supports the idea that interspecific differences in cuticular hydrocarbons contribute to sexual isolation.

Expression cloning of a human polysialyltransferase that forms the polysialylated neural cell adhesion molecule present in embryonic brain.

Polysialic acid is a developmentally regulated posttranslational modification of the neural cell adhesion molecule (N-CAM). It has been suggested that this large anionic carbohydrate modulates the adhesive property of N-CAM, but the precise function of polysialic acid is not known. Here we describe the isolation and functional expression of a cDNA encoding a human polysialyltransferase. For this expression cloning, COS-1 cells were cotransfected with a human fetal brain cDNA library and a cDNA encoding human N-CAM. Transfected COS-1 cells were stained with a monoclonal antibody specific for polysialic acid and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of polysialic acid on the cell surface. The deduced amino acid sequence indicates that the polysialyltransferase shares a common sequence motif with other sialyltransferases cloned so far. The polysialyltransferase is, however, distinct by having two clusters of basic amino acids. The amount of the polysialyltransferase transcripts correlates well with the formation of polysialic acid in various human tissues, and is abundant in the fetal brain but not in the adult brain. Moreover, HeLa cells stably expressing polysialic acid and N-CAM promoted neurite outgrowth and sprouting. These results indicate that the cloned polysialyltransferase forms polysialylated, embryonic N-CAM, which is critical for plasticity of neural cells.

Construction and characterization of a highly complex retroviral library for lineage analysis.

Replication-incompetent retroviral vectors encoding histochemical reporter genes have been used for studying lineal relationships in a variety of species. A crucial element in the interpretation of data generated by this method is the identification of sibling relationships, or clonal boundaries. The use of a library of viruses in which each member is unique can greatly facilitate this aspect of the analysis. A previously reported murine retroviral library containing about 80 members demonstrated the utility of the library approach. However, the relatively low number of tags in the murine library necessitated using low infection rates in order to give confidence in clonal assignments. To obviate the need for low infection rates, a far more complex library was created and characterized. The CHAPOL library was constructed such that each member encodes a histochemical reporter gene and has a DNA tag derived from a degenerate oligonucleotide pool synthesized to have a complexity of > 1 x 10(7). The library was tested after infection of cells in vitro or in vivo. The DNA tag from each histochemically labeled cell or clone of cells was recovered by PCR and sequenced for unambiguous identification. Three hundred and twenty tags have been identified after infection, and so far no tag has been seen to result from more than one independent infection. Thus, an equal distribution of inserts is suggested, and Monte Carlo analysis predicts a complexity of > 10(4) members.

Rivalidad en el mercado español de depósitos bancarios: la teoría de los grupos estratégicos

El objetivo de este trabajo consiste en examinar la rivalidad intra y entre grupos estratégicos de una industria. La literatura sobre la materia sugiere que las evidencias empíricas detectadas pueden verse afectadas por problemas operativos relacionados con la medición de la rivalidad. Como novedad, la metodología aplicada propone la utilización de dos enfoques de medición de la rivalidad, uno indirecto mediante la estimación de la variación conjetural, y una evaluación directa a través de las noticias sobre acciones y reacciones estratégicas detectadas en las publicaciones. La aplicación empírica realizada en el mercado español de depósitos bancarios no permite asegurar nada acerca de la mayor o menor rivalidad intra que entre grupos; pero los grupos estratégicos permiten predecir la forma en que cada entidad compite con las demás.

Grupos estratégicos en el sector hotelero español: diferencias de desempeño intra e intergrupo

El mundo es cada vez más pequeño debido a los avances tecnológicos y a la mayor experiencia de los directivos hoteleros y de los turistas, lo que provoca que la rivalidad entre destinos en el sector turístico sea cada vez mayor. Ante esta situación, este estudio se centra en el sector hotelero español y aplica los grupos estratégicos para determinar de qué forma compiten los hoteles y cómo influyen sus comportamientos estratégicos en su desempeño. Asimismo, se ofrecen acciones para mejorar el desempeño de un hotel en función del grupo estratégico al que pertenezca. La relevancia de este estudio radica en que clarifica la complejidad estratégica a la que están sometidos los directivos de los hoteles y les sirve de guía para la toma de decisiones estratégicas.

Estrategias de fijación de precios de las aerolíneas de bajo coste: una aproximación al modelo de la rivalidad ampliada

En la presente tesis doctoral se realiza una aproximación a las estrategias de fijación de precios de las aerolíneas de bajo coste europeas a partir del estudio de su entorno (demanda, proveedores, rivales, productos sustitutos, etcétera). Para ello se emplea el marco del modelo de las cinco fuerzas descrito por Michael Porter. Para su análisis, se utiliza un método combinado de Análisis Factorial (ACP) y datos de panel.

Who performs a stronger response to whom? Detecting individual competitive actions and reactions

This study analyzes the degree of competition through individual actions and reactions. Empirical support for this analysis has derived mainly from structural econometric models describing the nature of competition. This analysis extends the existing literature by empirically considering a direct measurement of competition through the analysis of the competitive actions and responses, and describing how firms compete within and between strategic groups. We estimate the firms’ conduct in the Spanish deposits market with 146 firms and 18,888 observations. This is a specially compelling context for the banking industry, in which a deregulation process gives rise to the adoption of aggressive strategies seeking to increase the market shares of deposit accounts; thus, producing a turbulent situation of increasing rivalry. Our results offer a deeper understanding of the firms’ competitive behavior, since we identify different patterns of actions and reactions depending upon the strategic group the firm belongs to.

Understanding Dropout and Prolonged Engagement in Adolescent Competitive Sport

Objectives: The purpose of this study is to gain understanding of training patterns and roles of significant others (i.e. coaches, parents, peers, and siblings) in adolescent swimmers’ sport participation patterns. Design: The developmental model of sport participation [Côté, J., Baker, J., & Abernethy, B. (2003). From play to practice: A developmental framework for the acquisition of expertise in team sport. In J. Starkes, & K. A. Ericsson (Eds.), Recent advances in research on sport expertise (pp. 89–114). Champaign, IL: Human Kinetics; Côté, J., & Fraser-Thomas, J. (2007). Youth involvement in sport. In P. R. E. Crocker (Ed.), Introduction to sport psychology: A Canadian perspective (pp. 266–294). Toronto: Pearson Prentice Hall] was used as a framework. Method: Ten dropout and 10 engaged swimmers, matched on key demographic variables participated in a semi-structured qualitative interview. Results: Groups had many similar experiences (e.g. early training, supportive and unsupportive coaches, involved parents). However, only dropouts spoke of early peak performances, limited one-on-one coaching, pressuring parents during adolescence, lack of swimming peers during adolescence, and sibling rivalries. In contrast, only engaged athletes spoke of clubs’ developmental philosophies, coaches’ and parents’ open communication, school friends’ support, and siblings’ general positive influences. Conclusions: Findings highlight the importance of appropriately structured programs and the fragility of athletes’ relationships with significant others during the adolescent years. Implications for sport programmers, coaches, and parents are discussed.

The Influence of the Family in the Development of Talent in Sport

The purpose of the present study was to describe patterns in the dynamics of families of talented athletes throughout their development in sport. Four families, including three families of elite rowers and one family of an elite tennis player were examined. The framework provided by Ericsson, Krampe, and Tesch- Römer (1993) to explain expert performance served as the theoretical basis for the study. Ericsson et al. suggested that the acquisition of expert performance involves operating within three types of constraints: motivational, effort, and resource. In-depth interviews were conducted with each athlete, parent, and sibling to explore how they have dealt with these three constraints. A total of 15 individual interviews were conducted. Results permitted the identification of three phases of participation from early childhood to late adolescence: the sampling years, the specializing years, and the investment years. The dynamics of the family in each of these phases of development is discussed

50 years of the ‘twin engine’. Franco-German reconciliation, European integration and reflections for Asia. EU Centre Singapore Background Brief No. 8, March 2013

On 22 January 2013, French President François Hollande and German Chancellor Angela Merkel gathered in Berlin to celebrate the 50th anniversary of the signing of the Élysée Treaty, the document that ended centuries of rivalry and warfare between their two countries. It is all too easy to forget the importance of Franco-German reconciliation. The 1950 Schuman Declaration, which led to the creation of the European Union’s (EU) predecessor, the European Coal and Steel Community (ECSC), sought to render the prospect of war between France and Germany ‘not only unthinkable but materially impossible’. Over 60 years later, when the EU was awarded the Nobel Peace Prize, the Norwegian Nobel Committee noted that indeed, ‘war between Germany and France is unthinkable’. Halfway around the world in Asia, the other theatre of World War II, tensions between China and Japan have arisen, with Taiwan and South Korea also in the fray. Nationalist movements in these countries have grown. This background brief lays out the issues for a timely reappraisal of the applicability, or otherwise, of the European integration and reconciliation processes to East Asia. The brief seeks to outline the contours of the historic act of Franco-German reconciliation, and its consequences ever since. Starting from a brief look at the history of rivalry and war between the two countries, the brief examines the events leading to the signing of the Élysée Treaty in 1963, and the development of Franco-German exchanges that have cemented the relationship. Difficulties between the countries are also raised. A timescale analysis of the opinion of the two publics is considered, as a measure of the success of Franco-German reconciliation.

Hostages to Moscow, clients of Beijing. Security in Central Asia as the role of the West diminishes. OSW Study 10/2014

The goal of this publication is to attempt to assess the thirteen years (2001- -2014) of the West’s military presence in the countries of post-Soviet Central Asia, closely associated with the ISAF and OEF-A (Operation Enduring Freedom – Afghanistan) missions in Afghanistan. There will also be an analysis of the actual challenges for the region’s stability after 2014. The current and future security architecture in Central Asia will also be looked at closely, as will the actual capabilities to counteract the most serious threats within its framework. The need to separately handle the security system in Central Asia and security as such is dictated by the particularities of political situation in the region, the key mechanism of which is geopolitics understood as global superpower rivalry for influence with a secondary or even instrumental role of the five regional states, while ignoring their internal problems. Such an approach is especially present in Russia’s perception of Central Asia, as it views security issues in geopolitical categories. Because of this, security analysis in the Central Asian region requires a broader geopolitical context, which was taken into account in this publication. The first part investigates the impact of the Western (primarily US) military and political presence on the region’s geopolitical architecture between 2001 and 2014. The second chapter is an attempt to take an objective look at the real challenges to regional security after the withdrawal of the coalition forces from Afghanistan, while the third chapter is dedicated to analysing the probable course of events in the security dimension following 2014. The accuracy of predictions time-wise included in the below publication does not exceed three to five years due to the dynamic developments in Central Asia and its immediate vicinity (the former Soviet Union, Afghanistan, Pakistan, Iran), and because of the large degree of unpredictability of policies of one of the key regional actors – Russia (both in the terms of its activity on the international arena, and its internal developments).