Two domains of the V protein of virulent canine distemper virus selectively inhibit STAT1 and STAT2 nuclear import

Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-alpha/beta)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-alpha/beta-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-alpha/beta-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-alpha/beta-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-alpha/beta-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-alpha/beta-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-alpha/beta evasion among the morbilliviruses.

Interethnic Interaction Dynamics in the Russian Federation

The collapse of the Soviet Union at the beginning of the 1990s also meant the end of the idea of a common soviet identity incarnated in the "soviet man" and the new "historic community of the soviet people". While this idea still lives on in the generations of the 1920s to 1940s, the younger generations tend to prefer identification with family, profession, ethnic group or religion. Ms. Alexakhina set out to investigate different interethnic interaction strategies in the multi-ethnic context of the Russian Federation, with an emphasis on analysing the role of cultural and ethno-demographic characteristics of minority ethnic groups. It aimed to identify those specific patterns of interaction dynamics that have emerged in response to the political and economic transformation at present under way. The basic supposition was that the size and growth of an ethnic population are defined not only by demographic features such as fertility, mortality and net migration, but are also dependent on processes interethnic interaction and ethnic transition. The central hypothesis of the project was that the multi-ethnic and multi-cultural composition of Russia is apparently manifesting itself in the ethnic minority groups in various forms, but particularly in the form of ethnic revival and/or assimilation. The results of these complex phenomena are manifested as changes in ethnic attachments (national re-identification and language behaviour (multi-lingualism, language transition and loss of the mother tongue). The stress of the political and economic crisis has stimulated significant changes in ethnographic, social and cultural characteristics of inter-ethnic dynamics such as the rate of national re-identification, language behaviour, migration activity and the spread of mixed marriages, among both those minorities with a long history of settlement in Russia and those that were annexed during the soviet period. Patterns of language behaviour and the spread of mixed marriages were taken as the main indicators of the directions of interethnic interaction described as assimilation, ethnic revival and cultural pluralism. The first stage of the research involved a statistical analysis of census data from 1959 to 1994 in order to analyse the changing demographic composition of the largest ethnic groups of the Russian Federation. Until 1989 interethnic interaction in soviet society was distinguished by the process of russification but the political and economic transformation has stimulated the process of ethnic revival, leading to an apparent fall in the size of the Russian population due to ethnic re-identification by members of other ethnic groups who had previously identified themselves as Russian. Cross-classification of nationalities by demographic, social and cultural indicators has shown that the most important determinants of the nature of interethnic interaction are cultural factors such as religion and language affiliation. The analysis of the dynamics of language shift through the study of bilingualism and the domains of language usage for different demographic groups revealed a strong correlation between recognition of Russian as a mother tongue among some non-Russian ethnic groups and the declining size of these groups. The main conclusion from this macro-analysis of census data was the hypothesis of the growing importance of social and political factors upon ethnic succession, that ethnic identity is no longer a stable characteristic but has become dynamic in nature. In order to verify this hypothesis Ms. Alexakhina conducted a survey in four regions showing different patterns of interethnic interaction: the Karelian Republic, Buryatiya, the Nenezkii Autonomous Region and Tatarstan. These represented the west, east, north and south of the Russian Federation. Samples for the survey were prepared on the basis of census lists so as to exclude mono-Russian families in favour of mixed and ethnic-minority families. The survey confirmed the significant growth in the importance of ethnic affiliation in the everyday lives of people in the Federation following the de-centralisation of the political and economic spheres. Language was shown to be a key symbol of the consciousness of national distinction, confirmed by the fact that the process of russification has been reversed by the active mastering of the languages of titular nationalities. The results also confirmed that individual ethnic identity has ceased to be a fixed personal characteristic of one's cultural and genetic belonging, and people's social adaptation to the current political, social and economic conditions is also demonstrated in changes in individual ethnic self-identification. In general terms, the dynamic nature of national identity means that ethnic identity is at present acquiring the special features of overall social identity, for which the frequent change of priorities is an inherent feature of a person's life cycle. These are mainly linked with a multi-ethnic environment and high individual social mobility. From her results Ms. Alexakhina concludes that the development of national languages and multi-lingualism, together with the preservation of Russian as a state language, seems to be the most promising path to peaceful coexistence and the development of the national cultures of different ethnic groups within the Russian Federation.

The Sociolinguistics of Hungarian Outside Hungary

This study describes the sociolinguistic situation of the indigenous Hungarian national minorities in Slovakia (c. 600,000), Ukraine (c. 180,000), Romania (c. 2,000,000), Yugoslavia (c. 300,000), Slovenia (c. 8,000) and Austria (c. 6,000). Following the guidelines of Hans Goebl et al, the historical sociolinguistic portrait of each minority is presented from 1920 through to the mid-1990s. Each country's report includes sections on geography and demography, history, politics, economy, culture and religion, language policy and planning, and language use (domains of minority and/or majority language use, proficiency, attitudes, etc.). The team's findings were presented in the form of 374 pages of manuscripts, articles and tables, written in Hungarian and English. The core of the team's research results lies in the results of an empirical survey designed to study the social characteristics of Hungarian-minority bilingualism in the six project countries, and the linguistic similarities and differences between the six contact varieties of Hungarian and Hungarian in Hungary. The respondents were divided by age, education, and settlement group - city vs. village and local majority vs. local minority. The first thing to be observed is that Hungarian is tending to be spoken less to children than to parents and grandparents, a familiar pattern of language shift. In contact varieties of Hungarian, analytic constructions may be used where monolingual Hungarians would use a more synthetic form. Mr Kontra gives as an example the compound tagdij, which in Standard Hungarian means "membership fee" but which is replaced in contact Hungarian by the two-word phrase tagsagi dij. Another similar example concerns the synthetic verb hegedult "played the violin" and the analytic expression hegedun jatszott. The contrast is especially striking between the Hungarians in the northern Slavic countries, who use the synthetic form frequently, and those in the southern Slavic countries, who mainly use the analytic form. Mr. Kontra notes that from a structural point of view, there is no immediate explanation for this, since Slovak or Ukrainian are as likely to cause interference as is Serbian. He postulates instead that the difference may be attributable to some sociohistoric cause, and points out that the Turkish occupation of what is today Voivodina caused a discontinuity of the Hungarian presence in the region, with the result that Hungarians were resettled in the area only two and a half centuries ago. However, the Hungarians in today's Slovakia and Ukraine have lived together with Slavic peoples continuously for over a millennium. It may be, he suggests, that 250 years of interethnic coexistence is less than is needed for such a contact-induced change to run its course. Next Mr. Kontra moved on to what he terms "mental maps and morphology". In Hungarian, the names of cities and villages take the surface case (eg. Budapest-en "in Budapest") whereas some names denoting Hungarian settlements and all names of foreign cities take the interior case (eg. Tihany-ban "in Tihany" and Boston-ban "in Boston). The role of the semantic feature "foreign" in suffix-choice can be illustrated by such minimal pairs as Velence-n "in Velence, a village in Hungary" versus Velence-ben "in Velence [=Venice], a city in Italy", and Pecs-en "in Pecs, a city in Hungary" vs. Becs-ben "in Becs, ie. Vienna". This Hungarian vs. foreign distinction is often interpreted as "belonging to historical (pre-1920) Hungary" vs. "outside historical Hungary". The distinction is also expressed in the dichotomy "home" vs. "abroad'. The 1920 border changes have had an impact on both majority and minority Hungarians' mental maps, the maps which govern the choice of surface vs. interior cases with placenames. As there is a growing divergence between the mental maps of majority and minority Hungarians, so there will be a growing divergence in their use of the placename suffixes. Two placenames were chosen to scratch the surface of this complex problem: Craiova (a city in Oltenia, Romania) and Kosovo (Hungarian Koszovo) an autonomous region in southeast Yugoslavia. The assumption to be tested was that both placenames would be used with the inessive (interior) suffixes categorically by Hungarians in Hungary, but that the superessive suffix (showing "home") would be used near-categorically by Hungarians in Romania and Yugoslavia (Voivodina). Minority Hungarians in countries other than Romania and Yugoslavia would show no difference from majority Hungarians in Hungary. In fact, the data show that, contrary to expectation, there is considerable variation within Hungary. And although Koszovo is used, as expected, with the "home" suffix by 61% of the informants in Yugoslavia, the same suffix is used by an even higher percentage of the subjects in Slovenia. Mr. Kontra's team suggests that one factor playing a role in this might be the continuance of the former Yugoslav mentality in the Hungarians of Slovenia, at least from the geographical point of view. The contact varieties of Hungarian show important grammatical differences from Hungarian in Hungary. One of these concerns the variable use of Null subjects (the inclusion or exclusion of the subject of the verb). When informants were asked to insert either megkertem or megkertem ot - "I asked her" - into a test sentence, 54.9% of the respondents in the Ukraine inserted the second phrase as opposed to only 27.4% in Hungary. Although Mr. Kontra and his team concentrated more on the differences between Contact Hungarian and Standard Hungarian, they also discovered a number of similarities. One such similarity is demonstrable in the distribution of what Mr. Kontra calls an ongoing syntactic merger in Hungarian in Hungary. This change means effectively that two possibilities merge to form a third. For instance, the two sentences Valoszinuleg kulfoldre fognak koltozni and Valoszinu, hogy kulfoldre fognak koltozni merge to form the new construction Valszinuleg, hogy kulfoldre fognak koltozni ("Probably they will move abroad."). When asked to choose "the most natural" of the sentences, one in four chose the new construction, and a chi-square test shows homogeneity in the sample. In other words, this syntactic change is spreading across the entire Hungarian-speaking region in the Carpathian Basin Mr. Kontra believes that politicians, educators, and other interested parties now have reliable and up-to-date information about each Hungarian minority. An awareness of Hungarian as a pluricentric language is being developed which elevates the status of contact varieties of Hungarian used by the minorities, an essential process, he believes, if minority languages are to be maintained.

Vascular endothelial cell-specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development

VE-PTP, a receptor-type phosphotyrosine phosphatase, associates with the tyrosine kinase receptor Tie-2 and VE-cadherin and enhances the adhesive function of the latter. Here, VE-PTP was found to be restricted to endothelial cells, with a preference for arterial endothelium. Mutant mice expressing a truncated, secreted form of VE-PTP lacking the cytoplasmic and transmembrane domains and the most membrane-proximal extracellular fibronectin type III repeat, showed severe vascular malformations causing lethality at 10 days of gestation. Although blood vessels were initially formed, the intraembryonic vascular system soon deteriorated. Blood vessels in the yolk sac developed into dramatically enlarged cavities. In explant cultures of mutant allantoides, endothelial cells were found next to vessel structures growing as cell layers. No signs for enhanced endothelial apoptosis or proliferation were observed. Thus, the activity of VE-PTP is not required for the initial formation of blood vessels, yet it is essential for their maintenance and remodeling.

Erosive tooth wear - a multifactorial condition of growing concern and increasing knowledge

Dental erosion is often described solely as a surface phenomenon, unlike caries where it has been established that the destructive effects involve both the surface and the subsurface region. However, besides removal and softening of the surface, erosion may show dissolution of mineral underneath the surface. There is some evidence that the presence of this condition is growing steadily. Hence, erosive tooth wear is becoming increasingly significant in the management of the long-term health of the dentition. What is considered as an acceptable amount of wear is dependent on the anticipated lifespan of the dentition and, therefore, is different for deciduous compared to permanent teeth. However, erosive damage to the permanent teeth occurring in childhood may compromise the growing child's dentition for their entire lifetime and may require repeated and increasingly complex and expensive restoration. Therefore, it is important that diagnosis of the tooth wear process in children and adults is made early and adequate preventive measures are undertaken. These measures can only be initiated when the risk factors are known and interactions between them are present. A scheme is proposed which allows the possible risk factors and their relation to each other to be examined.

Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have been identified as ligands with different effector functions of the vascular assembly and maturation-mediating receptor tyrosine kinase Tie-2. To understand the molecular interactions of the angiopoietins with their receptor, we have studied the binding of Ang-1 and Ang-2 to the Tie-2 receptor. Enzyme-linked immunosorbent assay-based competition assays and co-immunoprecipitation experiments analyzing the binding of Ang-1 and Ang-2 to truncation mutants of the extracellular domain of Tie-2 showed that the first Ig-like loop of Tie-2 in combination with the epidermal growth factor (EGF)-like repeats (amino acids 1-360) is required for angiopoietin binding. The first Ig-like domain or the EGF-like repeats alone are not capable of binding Ang-1 and Ang-2. Concomitantly, we made the surprising finding that Tie-2 exon-2 knockout mice do express a mutated Tie-2 protein that lacks 104 amino acids of the first Ig-like domain. This mutant Tie-2 receptor is functionally inactive as shown by the lack of ligand binding and receptor phosphorylation. Collectively, the data show that the first 104 amino acids of the Tie-2 receptor are essential but not sufficient for angiopoietin binding. Conversely, the first 360 amino acids (Ig-like domain plus EGF-like repeats) of the Tie-2 receptor are necessary and sufficient to bind both Ang-1 and Ang-2, which suggests that differential receptor binding is not likely to be responsible for the different functions of Ang-1 and Ang-2.

Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Here we report that two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. Our phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicate a requirement for ephrin-Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system.

Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

GENERATING PLANS IN CONCURRENT, PROBABILISTIC, OVER-SUBSCRIBED DOMAINS

Planning in realistic domains typically involves reasoning under uncertainty, operating under time and resource constraints, and finding the optimal subset of goals to work on. Creating optimal plans that consider all of these features is a computationally complex, challenging problem. This dissertation develops an AO* search based planner named CPOAO* (Concurrent, Probabilistic, Over-subscription AO*) which incorporates durative actions, time and resource constraints, concurrent execution, over-subscribed goals, and probabilistic actions. To handle concurrent actions, action combinations rather than individual actions are taken as plan steps. Plan optimization is explored by adding two novel aspects to plans. First, parallel steps that serve the same goal are used to increase the plan’s probability of success. Traditionally, only parallel steps that serve different goals are used to reduce plan execution time. Second, actions that are executing but are no longer useful can be terminated to save resources and time. Conventional planners assume that all actions that were started will be carried out to completion. To reduce the size of the search space, several domain independent heuristic functions and pruning techniques were developed. The key ideas are to exploit dominance relations for candidate action sets and to develop relaxed planning graphs to estimate the expected rewards of states. This thesis contributes (1) an AO* based planner to generate parallel plans, (2) domain independent heuristics to increase planner efficiency, and (3) the ability to execute redundant actions and to terminate useless actions to increase plan efficiency.

Flotillins interact with PSGL-1 in neutrophils and, upon stimulation, rapidly organize into membrane domains subsequently accumulating in the uropod

BACKGROUND: Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation. METHODOLOGY/PRINCIPAL FINDINGS: We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform. Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1. CONCLUSIONS/SIGNIFICANCE: These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.

Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1

Zyxin is a versatile component of focal adhesions in eukaryotic cells. Here we describe a novel binding partner of zyxin, which we have named LIM-nebulette. LIM-nebulette is an alternative splice variant of the sarcomeric protein nebulette, which, in contrast to nebulette, is expressed in non-muscle cells. It displays a modular structure with an N-terminal LIM domain, three nebulin-like repeats, and a C-terminal SH3 domain and shows high similarity to another cytoskeletal protein, Lasp-1 (LIM and SH3 protein-1). Co-precipitation studies and results obtained with the two-hybrid system demonstrate that LIM-nebulette and Lasp-1 interact specifically with zyxin. Moreover, the SH3 domain from LIM-nebulette is both necessary and sufficient for zyxin binding. The SH3 domains from Lasp-1 and nebulin can also interact with zyxin, but the SH3 domains from more distantly related proteins such as vinexin and sorting nexin 9 do not. On the other hand, the binding site in zyxin is situated at the extreme N terminus as shown by site-directed mutagenesis. LIM-nebulette and Lasp-1 use the same linear binding motif. This motif shows some similarity to a class II binding site but does not contain the classical PXXP sequence. LIM-nebulette reveals a subcellular distribution at focal adhesions similar to Lasp-1. Thus, LIM-nebulette, Lasp-1, and zyxin may play an important role in the organization of focal adhesions.