Human gamma-glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro.

A cDNA encoding human gamma-glutamyl hydrolase has been identified by searching an expressed sequence tag data base and using rat gamma-glutamyl hydrolase cDNA as the query sequence. The cDNA encodes a 318-amino acid protein of Mr 35,960. The deduced amino acid sequence of human gamma-glutamyl hydrolase shows 67% identity to that of rat gamma-glutamyl hydrolase. In both rat and human the 24 amino acids preceding the N terminus constitute a structural motif that is analogous to a leader or signal sequence. There are four consensus asparagine glycosylation sites in the human sequence, with three of them conserved in the rat enzyme. Expression of both the human and rat cDNA in Escherichia coli produced antigenically related proteins with enzyme activities characteristic of the native human and rat enzymes, respectively, when methotrexate di- or pentaglutamate were used as substrates. With the latter substrate the rat enzyme cleaved the innermost gamma-glutamyl linkage resulting in the sole production of methotrexate as the pteroyl containing product. The human enzyme differed in that it produced methotrexate tetraglutamate initially, followed by the triglutamate, and then the diglutamate and methotrexate. Hence the rat enzyme is an endopeptidase with methotrexate pentaglutamate as substrate, whereas the human enzyme exhibits exopeptidase activity. Another difference is that the expressed rat enzyme is equally active on methotrexate di- and pentaglutamate whereas the human enzyme has severalfold greater activity on methotrexate pentaglutamate compared with the diglutamate. These properties are consistent with the enzymes derived from human and rat sources.

Processing of the leader mRNA plays a major role in the induction of thrS expression following threonine starvation in Bacillus subtilis.

The threonyl-tRNA synthetase gene, thrS, is a member of a family of Gram-positive genes that are induced following starvation for the corresponding amino acid by a transcriptional antitermination mechanism involving the cognate uncharged tRNA. Here we show that an additional level of complexity exists in the control of the thrS gene with the mapping of an mRNA processing site just upstream of the transcription terminator in the thrS leader region. The processed RNA is significantly more stable than the full-length transcript. Under nonstarvation conditions, or following starvation for an amino acid other than threonine, the full-length thrS mRNA is more abundant than the processed transcript. However, following starvation for threonine, the thrS mRNA exists primarily in its cleaved form. This can partly be attributed to an increased processing efficiency following threonine starvation, and partly to a further, nonspecific increase in the stability of the processed transcript under starvation conditions. The increased stability of the processed RNA contributes significantly to the levels of functional RNA observed under threonine starvation conditions, previously attributed solely to antitermination. Finally, we show that processing is likely to occur upstream of the terminator in the leader regions of at least four other genes of this family, suggesting a widespread conservation of this phenomenon in their control.

Naturally occurring variation in copia expression is due to both element (cis) and host (trans) regulatory variation.

Significant differences in levels of copia [Drosophila long terminal repeat (LTR) retrotransposon] expression exist among six species representing the Drosophila melanogaster species complex (D. melanogaster, Drosophila mauritiana, Drosophila simulans, Drosophila sechellia, Drosophila yakuba, and Drosophila erecta) and a more distantly related species (Drosophila willistoni). These differences in expression are correlated with major size variation mapping to putative regulatory regions of the copia 5' LTR and adjacent untranslated leader region (ULR). Sequence analysis indicates that these size variants were derived from a series of regional duplication events. The ability of the copia LTR-ULR size variants to drive expression of a bacterial chloramphenicol acetyltransferase reporter gene was tested in each of the seven species. The results indicate that both element-encoded (cis) and host-genome-encoded (trans) genetic differences are responsible for the variability in copia expression within and between Drosophila species. This finding indicates that models purporting to explain the dynamics and distribution of retrotransposons in natural populations must consider the potential impact of both element-encoded and host-genome-encoded regulatory variation to be valid. We propose that interelement selection among retrotransposons may provide a molecular drive mechanism for the evolution of eukaryotic enhancers which can be subsequently distributed throughout the genome by retrotransposition.

An RNA structure involved in feedback regulation of splicing and of translation is critical for biological fitness.

While studies of the regulation of gene expression have generally concerned qualitative changes in the selection or the level of expression of a gene, much of the regulation that occurs within a cell involves the continuous subtle optimization of the levels of proteins used in macromolecular complexes. An example is the biosynthesis of the ribosome, in which equimolar amounts of nearly 80 ribosomal proteins must be supplied by the cytoplasm to the nucleolus. We have found that the transcript of one of the ribosomal protein genes of Saccharomyces cerevisiae, RPL32, participates in such fine tuning. Sequences from exon I of the RPL32 transcript interact with nucleotides from the intron to form a structure that binds L32 to regulate splicing. In the spliced transcript, the same sequences interact with nucleotides from exon II to form a structure that binds L32 to regulate translation, thus providing two levels of autoregulation. We now show, by using a sensitive cocultivation assay, that these RNA structures and their interaction with L32 play a role in the fitness of the cell. The change of a single nucleotide within the 5' leader of the RPL32 transcript, which abolishes the site for L32 binding, leads to detectably slower growth and to eventual loss of the mutant strain from the culture. Experiments designed to assess independently the regulation of splicing and the regulation of translation are presented. These observations demonstrate that, in evolutionary terms, subtle regulatory compensations can be critical. The change in structure of an RNA, due to alteration of just one noncoding nucleotide, can spell the difference between biological success and failure.

Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses.

Equine rhinovirus 1 (ERhV1) is a respiratory pathogen of horses which has an uncertain taxonomic status. We have determined the nucleotide sequence of the ERhV1 genome except for a small region at the 5' end. The predicted polyprotein was encoded by 6741 nucleotides and possessed a typical picornavirus proteolytic cleavage pattern, including a leader polypeptide. The genomic structure and predicted amino acid sequence of ERhV1 were more similar to those of foot-and-mouth disease viruses (FMDVs), the only members of the aphthovirus genus, than to those of other picornaviruses. Features which were most similar to FMDV included a 16-amino acid 2A protein which was 87.5% identical in sequence of FMDV 2A, a leader (L) protein similar in size to FMDV Lab and the possibility of a truncated L protein similar in size to FMDV Lb, and a 3C protease which recognizes different cleavage sites. However, unlike FMDV, ERhV1 had only one copy of the 3B (VPg) polypeptide. The phylogenetic relationships of the ERhV1 sequence and nucleotide sequences of representative species of the five genera of the family Picornaviridae were examined. Nucleotide sequences coding for the complete polyprotein, the RNA polymerase, and VP1 were analyzed separately. The phylogenetic trees confirmed that ERhV1 was more closely related to FMDV than to other picornaviruses and suggested that ERhV1 may be a member, albeit very distant, of the aphthovirus genus.

Enhancer-dependent interaction between 5' and 3' splice sites in trans.

Splice-site selection and alternative splicing of nuclear pre-mRNAs can be controlled by splicing enhancers that act by promoting the activity of upstream splice sites. Here we show that RNA molecules containing a 3' splice site and enhancer sequence are efficiently spliced in trans to RNA molecules containing normally cis-spliced 5' splice sites or to normally trans-spliced spliced leader RNAs from lower eukaryotes. In addition, we show that this reaction is stimulated by (Ser + Arg)-rich splicing factors that are known to promote protein-protein interactions in the cis-splicing reaction. Thus, splicing enhancers facilitate the assembly of protein complexes on RNAs containing a 3' splice site, and this complex is sufficiently stable to functionally interact with 5' splice sites located on separate RNAs. This trans-splicing is mediated by interactions between (Ser + Arg)-rich splicing factors bound to the enhancer and general splicing factors bound to the 5' and 3' splice sites. These same interactions are likely to play a crucial role in alternative splicing and splice-site selection in cis.

Degradation of the cleaved leader peptide of thiolase by a peroxisomal proteinase.

A peroxisomal location for insulin-degrading enzyme (IDE) has been defined by confocal immunofluorescence microscopy of stably transfected CHO cells overexpressing IDE and digitonin-permeabilization studies in normal nontransfected fibroblasts. The functional significance of IDE in degrading cleaved leader peptides of peroxisomal proteins targeted by the type II motif was evaluated with a synthetic peptide corresponding to the type II leader peptide of prethiolase. The peptide effectively competed for degradation and cross-linking of the high-affinity substrate 125I-labeled insulin to IDE. Direct proteolysis of the leader peptide of prethiolase was confirmed by HPLC; degradation was inhibited by immunodepletion with an antibody to IDE. Phylogenetic analysis of proteinases related to IDE revealed sequence similarity to mitochondrial processing peptidases.

“Let the American potter be a professional amateur" Laura Anne Fry and the American art pottery movement

"Artist Laura Anne Fry blended the concepts of professional and amateur, and helped raise the merit of ceramics in the United States. Fry influenced American art pottery with her contributions to Rookwood Pottery of Cincinnati—changing the course of the company. Her successful experiments with decorating techniques helped Rookwood become a national leader in art pottery, and eventually led to over a decade of controversy between Fry and Rookwood"

Knowledge spirals, situational leadership and informal learning applied on b-learning methodology

The methodology “b-learning” is a new teaching scenario and it requires the creation, adaptation and application of new learning tools searching the assimilation of new collaborative competences. In this context, it is well known the knowledge spirals, the situational leadership and the informal learning. The knowledge spirals is a basic concept of the knowledge procedure and they are based on that the knowledge increases when a cycle of 4 phases is repeated successively.1) The knowledge is created (for instance, to have an idea); 2) The knowledge is decoded into a format to be easily transmitted; 3) The knowledge is modified to be easily comprehensive and it is used; 4) New knowledge is created. This new knowledge improves the previous one (step 1). Each cycle shows a step of a spiral staircase: by going up the staircase, more knowledge is created. On the other hand, the situational leadership is based on that each person has a maturity degree to develop a specific task and this maturity increases with the experience. Therefore, the teacher (leader) has to adapt the teaching style to the student (subordinate) requirements and in this way, the professional and personal development of the student will increase quickly by improving the results and satisfaction. This educational strategy, finally combined with the informal learning, and in particular the zone of proximal development, and using a learning content management system own in our University, gets a successful and well-evaluated learning activity in Master subjects focused on the collaborative activity of preparation and oral exhibition of short and specific topics affine to these subjects. Therefore, the teacher has a relevant and consultant role of the selected topic and his function is to guide and supervise the work, incorporating many times the previous works done in other courses, as a research tutor or more experienced student. Then, in this work, we show the academic results, grade of interactivity developed in these collaborative tasks, statistics and the satisfaction grade shown by our post-graduate students.

Multimodal analysis of a sample of political posters in Ireland during and after the Celtic Tiger

The aim of this research paper is to analyse the key political posters made for the campaigns of Irish political party Fianna Fáil framed in the Celtic Tiger (1997-2008) and post-Celtic Tiger years (2009-2012). I will then focus on the four posters of the candidate in the elections that took place in 1997, 2002, 2007 and 2011 with the intention of observing first how the leader is represented, and later on pinpointing the similarities and possible differences between each. This is important in order to observe the main linguistic and visual strategies used to persuade the audience to vote that party and to highlight the power of the politician. Critical discourse analysis tools will be helpful to identify the main discursive strategies employed to persuade the Irish population to vote in a certain direction. Van Leeuwen’s (2008) social actor theory will facilitate the understanding of how participants are represented in the corpus under analysis. Finally, the main tools of Kress and van Leeuwen’s visual grammar (2006) will be applied for the analysis of the images. The study reveals that politicians are represented in a consistently positive way, with status and formal appearance so that people are persuaded to vote for the party they represent because they trust them as political leaders. The study, thus, points out that the poster is a powerful tool used in election campaigns to highlight the power of political parties.

Jamaican fossil Echini / by Benjamin Walworth Arnold and Hubert Lyman Clark. With descriptions of new species of Cainozoic Echinoidea / by Herbert L. Hawkins.

v.50:no.1 (1927)

De Gaulle and Europe: Historical Revision and Social Science Theory. Program for the Study of Germany and Europe Working Paper Series 8.5

The thousands of books and articles on Charles de Gaulle's policy toward European integration, whether written by historians, social scientists, or commentators, universally accord primary explanatory importance to the General's distinctive geopolitical ideology. In explaining his motivations, only secondary significance, if any at all, is attached to commercial considerations. This paper seeks to reverse this historiographical consensus by examining the four major decisions toward European integration during de Gaulle's presidency: the decisions to remain in the Common Market in 1958, to propose the Foucher Plan in the early 1960s, to veto British accession to the EC, and to provoke the "empty chair" crisis in 1965-1966, resulting in the "Luxembourg Compromise." In each case, the overwhelming bulk of the primary evidence-speeches, memoirs, or government documents-suggests that de Gaulle's primary motivation was economic, not geopolitical or ideological. Like his predecessors and successors, de Gaulle sought to promote French industry and agriculture by establishing protected markets for their export products. This empirical finding has three broader implications: (1) For those interesred in the European Union, it suggests that regional integration has been driven primarily by economic, not geopolitical considerations--even in the "least likely" case. (2) For those interested in the role of ideas in foreign policy, it suggests that strong interest groups in a democracy limit the impact of a leader's geopolitical ideology--even where the executive has very broad institutional autonomy. De Gaulle was a democratic statesman first and an ideological visionary second. (3) For those who employ qualitative case-study methods, it suggests that even a broad, representative sample of secondary sources does not create a firm basis for causal inference. For political scientists, as for historians, there is in many cases no reliable alternative to primary-source research.

EU actorness and effectiveness under political pressure at the Copenhagen climate change negotiations

This paper analyses the extent of European Union (EU) actorness and effectiveness at the fifteenth United Nations Framework Convention on Climate Change (UNFCCC) Conference of the Parties (COP) meeting in Copenhagen in December 2009. For over a decade the European Union has been characterised as a leader in international climate policy-making and as an important actor in international climate change negotiations. The COP15 meeting in Copenhagen has overall brought about disappointing outcomes, especially from the perspective of the European Union. This casts doubts on EU leadership and begs the question of what has happened to EU actorness and effectiveness in this field. In terms of actorness we take Jupille and Caporaso (1998) as a point of departure and then specify a more parsimonious actorness framework that consists of cohesion and autonomy. Effectiveness (i.e. goal attainment) is seen as conceptually separate from actorness. Effectiveness is conceptualised as the result of actorness conditioned by the ‘opportunity structure’, i.e. the external context (of other actors, events and ideas) that enables or constrains EU actions. We hold that the EU’s actorness has been only moderate, especially given somewhat limited preference cohesion. In terms of the opportunity structure in Copenhagen we argue that the high degree of politicisation constrained the EU’s ability to negotiate and thus to attain its goals. Another external factor that had a substantial adverse impact on the EU’s effectiveness at the Copenhagen negotiations was the strong involvement of other actors with rather different positions, namely the United States (US) and the BASIC countries (Brazil, South Africa, India, and China).

Georgia- between a dream and reality. OSW Commentary no. 133, 15.04.2014

Following several years of political turmoil triggered by constitutional reform (a shift from a presidential to a semi-presidential system) and electoral reshuffles (parliamentary elections in 2012; presidential elections in 2013), the political situation in Georgia has stabilised: key posts in the country are now in the hands of democratically elected members of the Geor-gian Dream coalition. Despite its mosaic-like structure and internaltensions, Georgian Dream remains strong and enjoys high levels of public support. This puts it in good stead to play a central role in Georgian politics in the foreseeable future, including securing victory in the local government elections scheduled for June. However, local billionaire Bidzina Ivanishvili does not currently hold a political office - despite the fact that he is the founder, sponsor and undisputed leader of the coalition, as well as former prime minister and the most popular public figure in Georgia (besides Patriarch Ilia II). This raises several questions, for example: Who is really at the helm of the Georgian state? What is the lon-g-term vision of the current government? The past achievements of the politically heterogeneo-us Georgian Dream - dominated by Mr Ivanishvili - offer little help in answering these questions. In addition to a series of challenges on the domestic front, the new Georgian leadership is also facing strategic geopolitical challenges, compounded by the current conflict in Ukraine. These include the future of Georgia’s relations with the West (including the process of EU and NATO integration) and with Russia (in response to repeated attempts to re-integrate the post-Soviet republics). The scale and dynamism of the changes in both the geopolitical order in the post-Soviet region and in the relations between Russia and the West are causing further questions to be raised about their impact on the position of the Georgian political elite and about their consequences for the entire country.

Od demokracji "wirtualnej" ku europejskiej: Geneze i konsekwencje przelomu politycznego w Moldawii. Prace OSW 32/2009. = From "virtual" to European democracy - the origins and consequences of the political breakthrough in Moldova. OSW Study 32/2009

2009 may become a turning point in the history of post-Soviet Moldova. The country’s political class and society are faced with a fundamental choice. On the one hand, if the Party of Communists of the Republic of Moldova, which has governed the country since 2001, remains in power, this would mean a consolidation of the authoritarian rule of the party leader Vladimir Voronin, perpetuation of the superficial nature of democratic institutions and a continuation of the manoeuvring between the European Union and Russia (with the increasing risk of falling into the latter’s exclusive zone of influence). On the other hand, the take over of political power by the opposition parties creates an opportunity for Moldova to resume building a democratic, pluralistic political system based on the rule of law and to develop closer links with the European Union within the framework of the European Neighbourhood Policy, including the Eastern Partnership.