Glycoprotein Interactions in the Assembly of Hantaviruses

Hantaviruses are one of the five genera of the vector-borne virus family Bunyaviridae. While other members of the family are transmitted via arthropods, hantaviruses are carried and transmitted by rodents and insectivores. Occasional transmission to humans occurs via inhalation of aerosolized rodent excreta. When transmitted to man hantaviruses cause hemorrhagic fever with renal syndrome (HFRS, in Eurasia, mortality ~10%) and hantavirus cardiopulmonary syndrome (HCPS, in the Americas, mortality ~40%). The single-stranded, negative-sense RNA genome of hantaviruses is in segments S, M and L that respectively encode for nucleocapsid (N), glycoproteins Gn and Gc, and RNA-dependent RNA-polymerase (RdRp or L protein). The genome segments, encapsidated by N protein to form ribonucleoprotein (RNP), are enclosed inside a lipid envelope decorated by spikes formed of Gn and Gc. The focus of this study was to understand the mechanisms and interactions through which the virion is formed and maintained. We observed that when extracted from virions both Gn and Gc favor homo- over hetero-oligomerization. The minimal glycoprotein complexes extracted from virion by detergent were observed, by using ultracentrifugation and gel filtration, to be tetrameric Gn and homodimeric Gc. These results led us to suggest a model where tetrameric Gn complexes are interconnected through homodimeric Gc units to form the grid-like surface architecture described for hantaviruses. This model was found to correlate with the three-dimensional (3D) reconstruction of virion surface created using cryo-electron tomography (cryo-ET). The 3D-density map showed the spike complex formed of Gn and Gc to be 10 nm high and to display a four-fold symmetry with dimensions of 15 nm times 15 nm. This unique square-shaped complex on a roughly round virion creates a hitch for the assembly, since a sphere cannot be broken into rectangles. Thus additional interactions are likely required for the virion assembly. In cryo-ET we observed that the RNP makes occasional contacts to the viral membrane, suggesting an interaction between the spike and RNP. We were able to demonstrate this interaction using various techniques, and showed that both Gn and Gc contribute to the interaction. This led us to suggest that in addition to the interactions between Gn and Gc, also the interaction between spike and RNP is required for assembly. We found galectin-3 binding protein (referred to as 90K) to co-purify with the virions and showed an interaction between 90K and the virion. Analysis of plasma samples taken from patients hospitalized for Puumala virus infection showed increased concentrations of 90K in the acute phase and the increased 90K level was found to correlate with several parameters that reflect the severity of acute HFRS. The results of these studies confirmed, but also challenged some of the dogmas on the structure and assembly of hantaviruses. We confirmed that Gn and RNP do interact, as long assumed. On the other hand we demonstrated that the glycoproteins Gn and Gc exist as homo-oligomers or appear in large hetero-oligomeric complexes, rather than form primarily heterodimers as was previously assumed. This work provided new insight into the structure and assembly of hantaviruses.

Functional regulation of the Neurofibromatosis 2 tumor suppressor merlin

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder manifested by the formation of multiple benign tumors of the nervous system. Affected individuals typically develop bilateral vestibular schwannomas which lead to deafness and balance disorders. The syndrome is caused by inactivation of the NF2 tumor suppressor gene, and mutation or loss of the NF2 product, merlin, is sufficient for tumorigenesis in both hereditary and sporadic NF2-associated tumors. Merlin belongs to the band 4.1 superfamily of cytoskeletal proteins, which also contain the related ezrin, radixin, and moesin (ERM) proteins. The ERM members provide a link between the cell cytoskeleton and membrane by connecting membrane-associated proteins to actin filaments. By stabilizing complexes in the cell cortex, the ERMs modulate morphology, growth, and migration of cells. Despite their structural homology, overlapping subcellular distribution, direct molecular association, and partial overlap of molecular interactions, merlin and ezrin exert opposite effects on cell proliferation. Merlin suppresses cell proliferation, whereas ezrin expression is linked to oncogenic activity. We hypothesized that the regions which differ between the proteins might explain merlin s specificity as a tumor suppressor. We therefore analyzed the regions, which are most diverse between merlin and ezrin; the N-terminal tail and the C-terminus. To determine the properties of the C-terminal region, we studied the two most predominant merlin isoforms together with truncation variants similar to those found in patients. We also focused on the evolutionally conserved C-terminal residues, E545-E547, that harbor disease causing mutations in its corresponding DNA sequence. In addition to inhibiting cell proliferation, merlin regulates cytoskeletal organization. The morphogenic properties of merlin may play a role in tumor suppression, since patient-derived tumor cells demonstrate cytoskeletal abnormalities. We analyzed the mechanisms of merlin-induced extension formation and determined that the C-terminal region of amino acids 538-568 is particularly important for the morphogenic activity. We also characterized the role of C-terminal merlin residues in the regulation of proliferation, phosphorylation, and intramolecular associations. In contrast to previous reports, we demonstrated that both merlin isoforms are able to suppress cell proliferation, whereas C-terminally mutated merlin constructs showed reduced growth inhibition. Phosphorylation serves as a mechanism to regulate the tumor suppressive activity of merlin. The C-terminal serine 518 is phosphorylated in response to both p21-activated kinase (PAK) and protein kinase A (PKA), which inactivates the growth inhibitory function of merlin. However, at least three differentially phosphorylated forms of the protein exist. In this study we demonstrated that also the N-terminus of merlin is phosphorylated by AGC kinases, and that both PKA and Akt phosphorylate merlin at serine 10 (S10). We evaluated the impact of this N-terminal tail phosphorylation, and showed that the phosphorylation state of S10 is an important regulator of merlin s ability to modulate cytoskeletal organization but also regulates the stability of the protein. In summary, this study describes the functional effect of merlin specific regions. We demonstrate that both S10 in the N-terminal tail and residues E545-E547 in the C-terminus are essential for merlin activity and function.

Visuaalisen työmuistin vapaasti jaettavat resurssit : muistiedustusten lukumäärä ja tarkkuus ääriviivakuvioilla

According to the most prevalent view, there are 3-4 fixed "slots" in visual working memory for temporary storage. Recently this view has been challenged with a theory of dynamic resources which are restricted in their totality but can be freely allocated. The aim of this study is to clarify which one of the theories better describes the performance in visual working memory tasks with contour shapes. Thus in this study, the interest is in both the number of recalled stimuli and the precision of the memory representations. Stimuli in the experiments were radial frequency patterns, which were constructed by sinusoidally modulating the radius of a circle. Five observers participated in the experiment and it consisted of two different tasks. In the delayed discrimination task the number of recalled stimuli was measured with 2-interval forced choice task. Observer was shown serially two displays with 1, 5 s ISI (inter stimulus interval). Displays contained 1-6 patterns and they differed from each other with changed amplitude in one pattern. The participant s task was to report whether the changed pattern had higher amplitude in the first or in the second interval. The amount of amplitude change was defined with QUEST-procedure and the 75 % discrimination threshold was measured in the task. In the recall task the precision of the memory representations was measured with subjective adjustment method. First, observer was shown 1-6 patterns and after 1, 5 s ISI one location of the previously shown pattern was cued. Observer s task was to adjust amplitude of a probe pattern to match the amplitude of the pattern in working memory. In the delayed discrimination task the performance of all observes declined smoothly when the number of presented patterns was increased. The result supports the resource theory of working memory as there was no sudden fall in the performance. The amplitude threshold for one item was 0.01 0.05 and as the number of items increased from 1 to 6 there was a 4 15 -fold linear increase in the amplitude threshold (0.14 0.29). In the recall adjustment task the precision of four observers performance declined smoothly as the number of presented patterns was increased. The result also supports the resource theory. The standard deviation for one item was 0.03 0.05 and as the number of items increased from 1 to 6 there was a 2 3 -fold linear increase in the amplitude threshold (0.06 0.11). These findings show that the performance in a visual working memory task is described better according to the theory of freely allocated resources and not to the traditional slot-model. In addition, the allocation of the resources depends on the properties of the individual observer and the visual working memory task.

Producing interactivity : Does media convergence promote interactivity and audience participation?

This study investigates the process of producing interactivity in a converged media environment. The study asks whether more media convergence equals more interactivity. The research object is approached through semi-structured interviews of prominent decision makers within the Finnish media. The main focus of the study are the three big ones of the traditional media, radio, television and the printing press, and their ability to adapt to the changing environment. The study develops theoretical models for the analysis of interactive features and convergence. Case-studies are formed from the interview data and they are evaluated against the models. As a result the cases arc plotted and compared on a four-fold table. The cases are Radio Rock, NRJ, Biu Brother, Television Chat, Olivia and Sanoma News. It is found out that the theoretical models can accurately forecast the results of the case studies. The models are also able to distinguish different aspects of both interactivity and convergence so that a case, which at a first glance seems not to be very interactive is in the end found out to receive second highest scores on the analysis. The highest scores are received by Big Brother and Sanoma News. Through the theory and the analysis of the research data it is found out that the concepts of interactivity and convergence arc intimately intertwined and very hard in many cases to separate from each other. Hence the answer to the main question of this study is yes, convergence does promote interactivity and audience participation. The main theoretical background for the analysis of interactivity follows the work of Came Fleeter, Spiro Kiousis and Sally McMillan. Heeler's six-dimensional definition of interactivity is used as the basis for operationalizing interactivity. The actor-network theory is used as the main theoretical framework to analyze convergence. The definition and operationalization of the actor-network theory into a model of convergence follows the work of Michel Callon. Bruno Latour and especially John Law and Felix Stalder.

Importance of COMT in the regulation of prefrontal dopamine

The prefrontal cortex (PFC), located in the anterior region of the frontal lobe, is considered to have several key roles in higher cognitive and executive functions. In general, the PFC can be seen as a coordinator of thought and action allowing subjects to behave in a goal-directed manner. Due to its anatomical connections with a variety of cortical and subcortical structures, several neurotransmitters, including dopamine, are involved in the regulation of PFC activity. In general, the majority of released dopamine is cleared by the dopamine transporter (DAT). In the PFC however, the number of presynaptic DAT is diminished, emphasizing the relative importance of catechol-O-methyltransferase (COMT) in dopamine metabolism. As a result, the role of COMT in the etiology of psychotic disorders is under constant debate. The present study investigated the role of COMT in prefrontal cortical dopamine metabolism by different neurochemical methods in COMT knockout (COMT-KO) mice. Pharmacological tools to inhibit other dopamine clearing mechanisms were also used for a more comprehensive and collective picture. In addition, this study investigated how a lack of the soluble (S-) COMT isoform affects the total COMT activity as well as the pharmacokinetics of orally administered L-dopa using mutant mice expressing only the membrane-bound (MB-) COMT isoform. Also the role of COMT in striatal and accumbal dopamine turnover during Δ9-tetrahydrocannabinol (THC) challenge was studied. We found markedly increased basal dopamine concentrations in the PFC, but not the striatum or nucleus accumbens (NAcc), of mice lacking COMT. Pharmacological inhibition of the noradrenaline transporter (NET) and monoamine oxidase (MAO) elevated prefrontal cortical dopamine levels several-fold, whereas inhibition of DAT did not. The lack of COMT doubled the dopamine raising effects of NET and MAO inhibition. No compensatory expression of either DAT or NET was found in the COMT-KO mice. The lack of S-COMT decreased the total COMT activity by 50-70 % and modified dopamine transmission and the pharmacokinetics of exogenous Ldopa in a sex and tissue specific manner. Finally, we found that subsequent tolcapone and THC increased dopamine levels in the NAcc, but not in the striatum. Conclusively, this study presents neurochemical evidence for the important role of COMT in the PFC and shows that COMT is responsible for about half of prefrontal cortical dopamine metabolism. This study also highlights the previously underestimated proportional role of MB-COMT and supports the clinical evidence of a gene x environment interaction between COMT and cannabis.