Cloning and characterization of the mouse ret finger protein (rfp), a B box zinc finger protein

An important question in biology is to understand the role of specific gene products in regulating embryogenesis and cellular differentiation. Many of the regulatory proteins possess specific motifs, such as the homeodomain, basic helix-loop-helix structure, zinc finger, and leucine zipper. These sequence motifs participate in specific protein-DNA, protein-RNA, and protein-protein interactions, and are important for the function of these regulatory proteins.^ The human rfp (ret finger protein) belongs to a novel zinc finger protein family, the B box zinc finger family. Most of the B box proteins, including rfp, have a conserved tripartite motif, consisting of two novel zinc fingers (the RING finger and the B box) and a coiled-coil domain. Interestingly, a fusion protein between the tripartite motif of rfp and the tyrosine kinase domain of c-ret has transforming activity. In this study, we examined the expression of rfp during mouse development, and characterized the role of the tripartite motif in rfp function.^ We cloned the mouse rfp cDNA, which shares a 98.4% homology with the human sequence at amino acid level. Such strikingly high degree of homology indicates the high evolutionary pressure on the conservation of the sequence, suggesting that rfp may have an important function. Using the somatic cell hybrid system, we assigned the rfp gene to mouse chromosome 13 and human chromosome 6. Rfp transcripts and protein were ubiquitous in day 10.5-13.5 mouse embryos; however, they were restricted in adult mice, with the highest level of expression in the testis. Rfp expression in the testis is detected only in late pachytene spermatocytes and round spermatids. In both embryos and spermatogenic cells, rfp protein was distributed within cell nuclei in a punctate pattern, similar to the PODs (PML oncogenic domains) observed with another B box protein, PML. In cultured mammalian cells, we found that rfp was indeed co-localized to the PODs with PML. Using the yeast two-hybrid system, we showed that the rfp could specifically interact with PML, and that the interaction was dependent on the distal portion of the rfp coiled-coil domain.^ We also showed that rfp could form homodimers, and both the B box and coiled-coil domain were required for proper dimerization. It seems that the proximal portion of the coiled-coil domain provides the interacting interface, while the B box zinc finger orients the coil and maintains the correct structure of the whole molecule. Our data are consistent with the zinc-binding property and structural analysis of the B box. The RING finger seems to be involved in rfp nuclear localization through interaction with other proteins. We believe that homodimerization and interaction with PML are important for the normal interaction of rfp during development and differentiation. In addition, rfp homodimerization may also be essential for the oncogenic activation of the rfp-ret fusion protein. ^

Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^

The structure of the debate on EU integration issues. A comparative analysis of party communication in the EP 2014 elections

The results of Eurosceptic parties in the recent European parliament election provide further evidence that the “permissive consensus” on European integration blurred. This paper focuses on the structure of the debate on EU integration issues. Which EU integration issues and positions do parties put forward? Can the debate on EU integration issues be subsumed in one or several dimensions? Do they reflect national political conflicts such as the left-right and the ‘new politics’/cultural divide? Or do they form one unique or several EU-specific dimensions, e.g. national sovereignty versus integration? In order to address these questions, this paper departs from the assumption that debate on European integration is multidimensional in its nature and therefore entails a multitude of issue areas. In other words, it does not look at how socio-economic and cultural issues are related to European integration but focuses on its components, i.e. particular EU-specific policies such as EU-wide employment, environment, immigration and monetary policy. The paper departs from the cleavage theory on political di-visions and different approaches transferring them to EU politics. Two points should be noted; first, this paper does not compare the debate on European integration issues between the national level and the EU level, but whether domestic divisions are reflected at the EU level. Second, it is not concerned with the general ideo-logical profile of political parties on EU integration issues, but on EU issues that parties communicated through press releases. By doing this, the paper is concerned with the salient EU issues that parties touch upon.

The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein

The polypeptide composition of the U7 small nuclear ribonucleoprotein (snRNP) involved in histone messenger RNA (mRNA) 3' end formation has recently been elucidated. In contrast to spliceosomal snRNPs, which contain a ring-shaped assembly of seven so-called Sm proteins, in the U7 snRNP the Sm proteins D1 and D2 are replaced by U7-specific Sm-like proteins, Lsm10 and Lsm11. This polypeptide composition and the unusual structure of Lsm11, which plays a role in histone RNA processing, represent new themes in the biology of Sm/Lsm proteins. Moreover this structure has important consequences for snRNP assembly that is mediated by two complexes containing the PRMT5 methyltransferase and the SMN (survival of motor neurons) protein, respectively. Finally, the ability to alter this polypeptide composition by a small mutation in U7 snRNA forms the basis for using modified U7 snRNA derivatives to alter specific pre-mRNA splicing events, thereby opening up a new way for antisense gene therapy.

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.

Factor structure of the Bern Psychopathology Scale in a sample of patients with schizophrenia spectrum disorders.

BACKGROUND The Bern Psychopathology Scale (BPS) is based on a system-specific approach to classifying the psychopathological symptom pattern of schizophrenia. It consists of subscales for three domains (language, affect and motor behaviour) that are hypothesized to be related to specific brain circuits. The aim of the study was to examine the factor structure of the BPS in patients with schizophrenia spectrum disorders. METHODS One hundred and forty-nine inpatients with schizophrenia spectrum disorders were recruited at the Department of Psychiatry II, Ulm University, Germany (n=100) and at the University Hospital of Psychiatry, Bern, Switzerland (n=49). Psychopathology was assessed with the BPS. The VARCLUS procedure of SAS(®) (a type of oblique component analysis) was used for statistical analysis. RESULTS Six clusters were identified (inhibited language, inhibited motor behaviour, inhibited affect, disinhibited affect, disinhibited language/motor behaviour, inhibited language/motor behaviour) which explained 40.13% of the total variance of the data. A binary division of attributes into an inhibited and disinhibited cluster was appropriate, although an overlap was found between the language and motor behaviour domains. There was a clear distinction between qualitative and quantitative symptoms. CONCLUSIONS The results argue for the validity of the BPS in identifying subsyndromes of schizophrenia spectrum disorders according to a dimensional approach. Future research should address the longitudinal assessment of dimensional psychopathological symptoms and elucidate the underlying neurobiological processes.

Rotational constants and structure of para-difluorobenzene determined by femtosecond Raman coherence spectroscopy: A new transient type

Femtosecond Raman rotational coherence spectroscopy (RCS) detected by degenerate four-wave mixing is a background-free method that allows to determine accurate gas-phase rotational constants of non-polar molecules. Raman RCS has so far mostly been applied to the regular coherence patterns of symmetric-top molecules, while its application to nonpolar asymmetric tops has been hampered by the large number of RCS transient types, the resulting variability of the RCS patterns, and the 10³–10⁴ times larger computational effort to simulate and fit rotational Raman RCS transients. We present the rotational Raman RCS spectra of the nonpolar asymmetric top 1,4-difluorobenzene (para-difluorobenzene, p-DFB) measured in a pulsed Ar supersonic jet and in a gas cell over delay times up to ~2.5 ns. p-DFB exhibits rotational Raman transitions with ΔJ = 0, 1, 2 and ΔK = 0, 2, leading to the observation of J −, K −, A −, and C–type transients, as well as a novel transient (S–type) that has not been characterized so far. The jet and gas cell RCS measurements were fully analyzed and yield the ground-state (v = 0) rotational constants Aₒ = 5637.68(20) MHz, Bₒ = 1428.23(37) MHz, and Cₒ = 1138.90(48) MHz (1σ uncertainties). Combining the Aₒ, Bₒ, and Cₒ constants with coupled-cluster with single-, double- and perturbatively corrected triple-excitation calculations using large basis sets allows to determine the semi-experimental equilibrium bond lengths rₑ(C₁–C₂) = 1.3849(4) Å, rₑ(C₂–C³) = 1.3917(4) Å, rₑ(C–F) = 1.3422(3) Å, and rₑ(C₂–H₂) = 1.0791(5) Å.

Gas-phase fragmentation and conformation of the sugar-modified antisense oligonucleotide tcDNA

Tricyclo-DNA (tcDNA) is a sugar- and backbone-modified analogue of DNA that is currently tested as antisense oligonucleotide for the treatment of Duchenne muscular dystrophy. The name tricyclo-DNA is derived from the modified sugar-moiety: the deoxyribose is extended to a three-membered ring system. This modification is designed to limit the flexibility of the structure, thus giving rise to entropically stabilized hybrid duplexes formed between tcDNA and complementary DNA or RNA oligonucleotides. While the structural modifications increase the biostability of the therapeutic agent, they also render the oligonucleotide inaccessible to enzyme-based sequencing methods. Tandem mass spectrometry constitutes an alternative sequencing technique for partially and fully modified oligonucleotides. For reliable sequencing, the fragmentation mechanism of the structure in question must be understood. Therefore, the presented work evaluates the effect of the modified sugar-moiety on the gas-phase dissociation of single stranded tcDNA. Moreover, our experiments reflect the exceptional gas-phase stability of hybrid duplexes that is most noticeable in the formation of truncated duplex ions upon collision-induced dissociation. The stability of the duplex arises from the modified sugar-moiety, as the rigid structure of the tcDNA single strand minimizes the change of the entropy for the annealing. Moreover, the tc-modification gives rise to extended conformations of the nucleic acids in the gas-phase, which was studied by ion mobility spectrometry-mass spectrometry.

Hypofibrinogenemia and liver disease: a new case of Aguadilla fibrinogen and review of the literature.

INTRODUCTION Fibrinogen storage disease (FSD) is characterized by hypofibrinogenemia and hepatic inclusions due to impaired release of mutant fibrinogen which accumulates and aggregates in the hepatocellular endoplasmic reticulum. Liver disease is variable. AIM We studied a new Swiss family with fibrinogen Aguadilla. In order to understand the molecular peculiarity of FSD mutations, fibrinogen Aguadilla and the three other causative mutations, all located in the γD domain, were modelled. METHOD The proband is a Swiss girl aged 4 investigated because of fatigue and elevated liver enzymes. Protein structure models were prepared using the Swiss-PdbViewer and POV-Ray software. RESULTS The proband was found to be heterozygous for fibrinogen Aguadilla: FGG Arg375Trp. Familial screening revealed that her mother and maternal grandmother were also affected and, in addition, respectively heterozygous and homozygous for the hereditary haemochromatosis mutation HFE C282Y. Models of backbone and side-chain interactions for fibrinogen Aguadilla in a 10-angstrom region revealed the loss of five H-bonds and the gain of one H-bond between structurally important amino acids. The structure predicted for fibrinogen Angers showed a novel helical structure in place of hole 'a' on the outer edge of γD likely to have a negative impact on fibrinogen assembly and secretion. CONCLUSION The mechanism by which FSD mutations generate hepatic intracellular inclusions is still not clearly established although the promotion of aberrant intermolecular strand insertions is emerging as a likely cause. Reporting new cases is essential in the light of novel opportunities of treatment offered by increasing knowledge of the degradation pathway and autophagy.

Causes, forms and consequences of the professionalization of international sport federa-tions in Switzerland: Conceptual framework and first results

Synopsis: Sport organisations are facing multiple challenges originating from an increasingly complex and dynamic environment in general, and from internal changes in particular. Our study seeks to reveal and analyse the causes for professionalization processes in international sport federations, the forms resulting from it, as well as related consequences. Abstract: AIM OF ABSTRACT/PAPER - RESEARCH QUESTION Sport organisations are facing multiple challenges originating from an increasingly complex and dynamic environment in general, and from internal changes in particular. In this context, professionalization seems to have been adopted by sport organisations as an appropriate strategy to respond to pressures such as becoming more “business-like”. The ongoing study seeks to reveal and analyse the internal and external causes for professionalization processes in international sport federations, the forms resulting from it (e.g. organisational, managerial, economic) as well as related consequences on objectives, values, governance methods, performance management or again rationalisation. THEORETICAL BACKGROUND/LITERATURE REVIEW Studies on sport as specific non-profit sector mainly focus on the prospect of the “professionalization of individuals” (Thibault, Slack & Hinings, 1991), often within sport clubs (Thiel, Meier & Cachay, 2006) and national sport federations (Seippel, 2002) or on organisational change (Griginov & Sandanski, 2008; Slack & Hinings, 1987, 1992; Slack, 1985, 2001), thus leaving broader analysis on governance, management and professionalization in sport organisations an unaccomplished task. In order to further current research on above-mentioned topics, our intention is to analyse causes, forms and consequences of professionalisation processes in international sport federations. The social theory of action (Coleman, 1986; Esser, 1993) has been defined as appropriate theoretical framework, deriving in the following a multi-level framework for the analysis of sport organisations (Nagel, 2007). In light of the multi-level framework, sport federations are conceptualised as corporative actors whose objectives are defined and implemented with regard to the interests of member organisations (Heinemann, 2004) and/or other pressure groups. In order to understand social acting and social structures (Giddens 1984) of sport federations, two levels are in the focus of our analysis: the macro level examining the environment at large (political, social, economic systems etc.) and the meso level (Esser, 1999) examining organisational structures, actions and decisions of the federation’s headquarter as well as member organisations. METHODOLOGY, RESEARCH DESIGN AND DATA ANALYSIS The multi-level framework mentioned seeks to gather and analyse information on causes, forms and consequences of professionalization processes in sport federations. It is applied in a twofold approach: first an exploratory study based on nine semi-structured interviews with experts from umbrella sport organisations (IOC, WADA, ASOIF, AIOWF, etc.) as well as the analysis of related documents, relevant reports (IOC report 2000 on governance reform, Agenda 2020, etc.) and important moments of change in the Olympic Movement (Olympic revenue share, IOC evaluation criteria, etc.); and secondly several case studies. Whereas the exploratory study seeks more the causes for professionalization on an external, internal and headquarter level as depicted in the literature, the case studies rather focus on forms and consequences. Applying our conceptual framework, the analysis of forms is built around three dimensions: 1) Individuals (persons and positions), 2) Processes, structures (formalisation, specialisation), 3) Activities (strategic planning). With regard to consequences, we centre our attention on expectations of and relationships with stakeholders (e.g. cooperation with business partners), structure, culture and processes (e.g. governance models, performance), and expectations of and relationships with member organisations (e.g. centralisation vs. regionalisation). For the case studies, a mixed-method approach is applied to collect relevant data: questionnaires for rather quantitative data, interviews for rather qualitative data, as well as document and observatory analysis. RESULTS, DISCUSSION AND IMPLICATIONS/CONCLUSIONS With regard to causes of professionalization processes, we analyse the content of three different levels: 1. the external level, where the main pressure derives from financial resources (stakeholders, benefactors) and important turning points (scandals, media pressure, IOC requirements for Olympic sports); 2. the internal level, where pressure from member organisations turned out to be less decisive than assumed (little involvement of member organisations in decision-making); 3. the headquarter level, where specific economic models (World Cups, other international circuits, World Championships), and organisational structures (decision-making procedures, values, leadership) trigger or hinder a federation’s professionalization process. Based on our first analysis, an outline for an economic model is suggested, distinguishing four categories of IFs: “money-generating IFs” being rather based on commercialisation and strategic alliances; “classical Olympic IFs” being rather reactive and dependent on Olympic revenue; “classical non-Olympic IFs” being rather independent of the Olympic Movement; and “money-receiving IFs” being dependent on benefactors and having strong traditions and values. The results regarding forms and consequences will be outlined in the presentation. The first results from the two pilot studies will allow us to refine our conceptual framework for subsequent case studies, thus extending our data collection and developing fundamental conclusions. References: Bayle, E., & Robinson, L. (2007). A framework for understanding the performance of national governing bodies of sport. European Sport Management Quarterly, 7, 249–268 Chantelat, P. (2001). La professionnalisation des organisations sportives: Nouveaux débats, nouveaux enjeux [Professionalisation of sport organisations]. Paris: L’Harmattan. Dowling, M., Edwards, J., & Washington, M. (2014). Understanding the concept of professionalization in sport management research. Sport Management Review. Advance online publication. doi: 10.1016/j.smr.2014.02.003 Ferkins, L. & Shilbury, D. (2012). Good Boards Are Strategic: What Does That Mean for Sport Governance? Journal of Sport Management, 26, 67-80. Thibault, L., Slack, T., & Hinings, B. (1991). Professionalism, structures and systems: The impact of professional staff on voluntary sport organizations. International Review for the Sociology of Sport, 26, 83–97.

The impact of the position effect on the factorial structure of the Culture Fair Test (CFT)

The Culture Fair Test (CFT) is a psychometric test of fluid intelligence consisting of four subtests; Series, Classification, Matrices, and Topographies. The four subtests are only moderately intercorrelated, doubting the notion that they assess the same construct (i.e., fluid intelligence). As an explanation of these low correlations, we investigated the position effect. This effect is assumed to reflect implicit learning during testing. By applying fixed-links modeling to analyze the CFT data of 206 participants, we identified position effects as latent variables in the subtests; Classification, Matrices, and Topographies. These position effects were disentangled from a second set of latent variables representing fluid intelligence inherent in the four subtests. After this separation of position effect and basic fluid intelligence, the latent variables representing basic fluid intelligence in the subtests Series, Matrices, and Topographies could be combined to one common latent variable which was highly correlated with fluid intelligence derived from the subtest Classification (r=.72). Correlations between the three latent variables representing the position effects in the Classification, Matrices, and Topographies subtests ranged from r=.38 to r=.59. The results indicate that all four CFT subtests measure the same construct (i.e., fluid intelligence) but that the position effect confounds the factorial structure

Purification and cloning of the novel phosphoprotein copine III and characterization of its associated kinase activity

The copines, named and first described by Creutz et al. (1998), comprise a two C2 domain-containing protein family that can aggregate phosphatidylserine membranes in a calcium-dependent manner. Although no enzymatic function has been attributed to copines, their carboxyl terminus shows homology to the A domain found in integrins that allows binding of magnesium ions. The secondary structure of A domains resembles a Rossmann fold, which can bind dinucleotides and is present in a number of intracellular enzymes. Due to a crossreacting activity of Mik b 1, an antibody to the IL-2R b chain, we were able to serendipitously clone human copine III (CIII). CIII is 65% identical to copine I (CI) and the 5 kb CIII transcript is expressed ubiquitously as determined by a multitissue Northern blot. A polyclonal antibody generated against the carboxyl terminus of CIII recognized CIII in immunoblots and immunoprecipitations. Phosphorylation of CIII was observed on serine and threonine residues, as determined by phosphoamino acid analysis. ^ Experiments were designed to determine whether or not any enzymatic activity, specifically kinase activity, was intrinsic to or associated with CIII. In vitro and in gel kinase assays were performed using transfected HA-tagged CI and CIII, immunoprecipitated endogenous CIII and purified endogenous CIII. The exogenous substrate MBP was phosphorylated in all in vitro kinase assays containing CIII protein purification and column chromatography expertise with me. ^

Mapping and characterization of the Xlsirt P11 RNA cis-acting localization element

In many organisms, polarity of the oocyte is established post-transcriptionally via subcellular RNA localization. Many RNAs are localized during oogenesis in Xenopus laevis, including Xlsirts ( Xenopus laevis short interspersed repeat transcripts) [Kloc, 1993]. Xlsirts constitute a large family defined by highly homologous repeat units 79–81 nucleotides in length. Endogenous Xlsirt RNAs use the METRO (Message Transport Organizer) pathway of localization, where RNAs are transported from the nucleus to the mitochondrial cloud in stage I oocytes. Secondly, RNAs anchor at the vegetal pole in stage II oocytes. Exogenous Xlsirt RNAs can also utilize the Late pathway of localization, which involves localization to the vegetal cortex during stage III of oogenesis and results in RNAs anchored in the cortex of the entire vegetal hemisphere. ^ The Xlsirts localization signal is contained within the repeat region. This study was designed to test the hypothesis that there are cis -acting localization elements in Xlsirts, and that higher order structure plays a role. Results of experiments on Xlsirt P11, a 1700 basepair (bp) family member, led to the conclusion that a 137-bp fragment of the repetitive region is necessary and sufficient for METRO and Late pathway localization. This analysis definitively demonstrates that the Xlsirt localization signal for the METRO and Late pathways reside within the repetitive region and not within the flanking regions. Analysis of Xlsirt linker scanning mutations revealed two METRO-pathway specific subelements, and one Late-pathway specific subelement. Functional, computer, and biochemical evidence relates the higher order structure of this element to its ability to function as a localization element. ^ Xlsirt 137 is 99% identical to the Xlsirt consensus sequence identified in this study, suggesting that it is the localization element for all localized Xlsirt family members. The repeat unit was reframed based on function, rather than arbitrarily based on sequence. This work supports the hypothesis presented in 1981 by George Spohr, who originally isolated the Xlsirts, which stated that the highly conserved repetitive elements must be constrained from variability due to some unknown function of the repeats themselves. These studies shed light on the mechanism of RNA localization, linking structure and function. ^