Kinin B1 receptor homo-oligomerization is required for receptor trafficking to the cell surface.

The kinin B1 receptor (B1R) is a G protein-coupled receptor with pro-inflammatory activity that is latent in healthy tissues but induced by tissue insult. Here, we investigated if B1R homo-oligomerization is a possible mechanism regulating the presentation of this receptor at the level of maturation and trafficking to the cell surface. To this end, we used HEK293 cells stably expressing N-terminal FLAG and HA epitope-tagged wild-type human B1R and an N-terminal receptor fragment, B1stop135, which terminates at the C-terminal end of the third transmembrane domain and has previously been shown to oligomerize with B1R. Receptors were monitored by immunoblotting and immunoprecipitation, receptor function by agonist binding and agonist-promoted phosphoinositide hydrolysis, and receptor trafficking by confocal immunofluorescence microscopy. When expressed alone, B1R is core N-glycosylated and forms oligomers localized intracellularly and on the cell surface. B1stop135 also exists as core N-glycosylated oligomers but is localized exclusively intracellularly. When co-expressed, B1stop135 prevents specifically B1R homo-oligomerization by forming nonfunctional B1R-B1stop135 hetero-oligomers, retains B1R intracellularly at least in part in the endoplasmatic reticulum (ER), increases calnexin binding to the receptor, and increases receptor degradation. We conclude that B1R homo-oligomerization is necessary for B1R maturation and trafficking to the cell surface. Modulating this mechanism may be a novel therapeutic avenue in inflammatory disease

Challenge clusters facing LCA in environmental decision-making—what we can learn from biofuels

Purpose Bioenergy is increasingly used to help meet greenhouse gas (GHG) and renewable energy targets. However, bioenergy’s sustainability has been questioned, resulting in increasing use of life cycle assessment (LCA). Bioenergy systems are global and complex, and market forces can result in significant changes, relevant to LCA and policy. The goal of this paper is to illustrate the complexities associated with LCA, with particular focus on bioenergy and associated policy development, so that its use can more effectively inform policymakers. Methods The review is based on the results from a series of workshops focused on bioenergy life cycle assessment. Expert submissions were compiled and categorized within the first two workshops. Over 100 issues emerged. Accounting for redundancies and close similarities in the list, this reduced to around 60 challenges, many of which are deeply interrelated. Some of these issues were then explored further at a policyfacing workshop in London, UK. The authors applied a rigorous approach to categorize the challenges identified to be at the intersection of biofuels/bioenergy LCA and policy. Results and discussion The credibility of LCA is core to its use in policy. Even LCAs that comply with ISO standards and policy and regulatory instruments leave a great deal of scope for interpretation and flexibility. Within the bioenergy sector, this has led to frustration and at times a lack of obvious direction. This paper identifies the main challenge clusters: overarching issues, application and practice and value and ethical judgments. Many of these are reflective of the transition from application of LCA to assess individual products or systems to the wider approach that is becoming more common. Uncertainty in impact assessment strongly influences planning and compliance due to challenges in assigning accountability, and communicating the inherent complexity and uncertainty within bioenergy is becoming of greater importance. Conclusions The emergence of LCA in bioenergy governance is particularly significant because other sectors are likely to transition to similar governance models. LCA is being stretched to accommodate complex and broad policy-relevant questions, seeking to incorporate externalities that have major implications for long-term sustainability. As policy increasingly relies on LCA, the strains placed on the methodology are becoming both clearer and impedimentary. The implications for energy policy, and in particular bioenergy, are large

G protein-coupled receptor regulation: The role of protein interactions and receptor trafficking

The superfamily of G protein-coupled receptors (GPCR) is the largest gene family in the human genome. GPCR-mediated signaling operates in every human cell, and about 50% of existing clinically useful drugs act through GPCR. Kinins are proinflammatory peptides that are rapidly produced extracellularly following pathological insults and tissue damage. These peptides act through two GPCR subtypes, B1 (B1R) and B2 (B2R), to elicit numerous inflammatory responses including vasodilatiation, increased vascular permeability, and pain. GPER1 (G protein-coupled estrogen receptor 1) has recently been suggested as a novel estrogen receptor. Much controversy surrounds this receptor regarding its subcellular localization and activation. The aim of the present thesis was to explore the role of receptor trafficking and protein-interactions in kinin receptor regulation, as well as studying the receptor trafficking and signaling of GPER1. B1R forms homo-oligomers, which is required for proper B1R maturation and cell-surface expression. Furthermore, the endopeptidase EP24.15 is able to degrade BK intracellularly and attenuate maximal B2R responsiveness without influencing the potency of BK. Finally, GPER1 is localized both intracellularly and on the cell surface, where it is subject to rapid, constitutive endocytosis. Estrogen was found to elevate the level of cAMP in mouse myotube C2C12 cells, in a GPER1-dependent manner, but it is still not clear whether estrogen is the true agonist for GPER1. Taken together, the present thesis demonstrates the importance of protein-protein interactions and trafficking for B1R, B2R, and GPER1 regulation and activity. Understanding these mechanisms will be of great benefit in drug development by presenting novel drug targets

Teachers´ way of working with children´s motor development in preschool : Is music used as a tool?

BakgrundMotorisk utveckling delas in i grov- och finmotorik. De grundläggande grovmotoriska rörel-serna som barn utvecklar främst i förskolan är springa, klättra, balansera och hoppa. Finmo-toriken handlar främst om handen och fingrarnas rörelse. Barn tycker om att röra sig till musik. Vilket gör musiken till ett bra hjälpmedel i arbetet med motoriken.SyfteSyfte med studien är att undersöka hur pedagoger beskriver att de arbetar för att stimulera barns motoriska utveckling i förskolan. Särskilt intresserade är vi om och hur musik används som ett hjälpmedel i deras arbete.MetodI vår studie har vi använt oss av kvalitativa fokusgruppintervjuer med en öppen fråga plus några följdfrågor. Sammanlagt intervjuades sex pedagoger från tre olika förskolor.ResultatDet resultat som kommit fram är att pedagogers medvetenhet har stor betydelse för att kunna utmana barnen i deras motoriska utveckling. Pedagogerna ser musiken som ett bra hjälpmedel, genom exempelvis rörelsesånger och dans blir den motoriska träningen lustfylld.Program: Lärarutbildninge

"Om jag nu tänker rätt på vad en pedagogisk dokumentation är.." : En kvalitativ studie om barnskötarnas förutsättningar och föreställningar i arbetet med pedagogisk dokumentation

Förskolans verksamhet ska enligt skollagen systematiskt och kontinuerligt följas upp, utvärderas och analyseras för att utveckla den verksamhet som bedrivs. För att kartlägga verksamheten och identifiera eventuella utvecklingsområden kan pedagogisk dokumentation användas som en metod i det systematiska utvecklingsarbetet. Idag består mer än hälften av de pedagoger som bedriver utbildningen i de svenska förskolorna av barnskötare. Eftersom att det i tidigare forskning verkar finnas en kunskapslucka gällande barnskötarnas roll i den pedagogiska dokumentationen, är syftet med studien att undersöka vilka förutsättningar och föreställningar de har i arbetet med den pedagogiska dokumentationen.   I studien har vi använt en kvalitativ metod med semistrukturerade intervjuer där empirin är insamlad utifrån ett subjektivt urval med sex pedagogiskt verksamma från två olika kommuner. Studiens syfte var att synliggöra barnskötarnas villkor och uppfattningar i arbetet med pedagogisk dokumentation, samt hur dessa faktorer påverkar. För att öka kunskapen kring olika aspekter som kan påverka användes ramfaktorteorin. I resultaten framkommer det att barnskötarnas förutsättningar är begränsade gällande kompetens och möjlighet till reflektion, som i sin tur hindrar dem i framställandet av den pedagogiska dokumentationen. Resultaten visar även att de har föreställningar kring att en pedagogisk dokumentation ska framställas på ett visst sätt, som att den exempelvis ska innehålla ett korrekt akademiskt språk, vilket tenderar att leda till en osäkerhetskänsla hos studiedeltagarna. Studiens resultat visar att barnskötarnas drivkraft och intresse har lett till att de själva hittat strategier, såsom att studera andra kollegors arbete för att utveckla sitt pedagogiska dokumentationsarbete. Barnskötarna förväntas genomföra pedagogisk dokumentation, men verkar sakna kunskap om verktygets funktion till det systematiska kvalitetsarbetet, vilket är en anledning till att det används som metod. Sammantaget vittnar detta om vikten att investera i god kompetens, inte bara för barnskötarna, utan för alla anställda

Branding in Public Transportation: Campfires and Beyond

The purpose of this research is to study how organizations in public transportation are viewing branding. We aim for this thesis to acknowledge branding as a tool to create a strong brand. This thesis is of importance due to the influence public organizations within transportation hold for Sweden to reach their sustainability goals. By creating a strong brand public organizations could attract more consumers to use public transportation and ultimately decrease harmful gas emissions. We wish to bring forward potential challenges and advantages these organization are facing related to branding. To fulfil the purpose of this research four interviews were conducted with representatives from organizations in public transportation. Empirical material was gathered through the interviews concerning questions in the categories: marketing, branding, competition and consumers. When analyzing the empirical material, previous theories were taken into consideration to be able to explain the outcomes and increase validity of the findings. The findings have shown that organizations in public transportation are in a process of realizing the potential benefits branding can bring to their organization. However, several challenges are also posed. Not forgetting advantages and opportunities if these organizations are able to incorporate efficient branding practices. The need for internal structure and creation of a brand personality and identity is urged for these organizations. Lastly, we would like to make a theoretical contribution to the existing research gap of branding in public organizations

Kinin-Stimulated B1 Receptor Signaling Depends on Receptor Endocytosis Whereas B2 Receptor Signaling Does Not.

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca(2+) mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg(9)-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling

Kinin B2 Receptor-Mediated Bradykinin Internalization and Metalloendopeptidase EP24.15-Dependent Intracellular Bradykinin Degradation.

Kinins are potent proinflammatory peptides that are produced extracellularly and rapidly degraded by extracellular peptidases and by intracellular peptidases accessed by kinins via receptor-mediated endocytosis. Here, we developed model cell systems expressing the kinin B2 receptor (B2R) and the metalloendopeptidase thimet oligopeptidase (EC 3.4.24.15; EP24.15) either individually or together to address 1) the cellular and functional relationship between these proteins and 2) the participation of EP24.15 in the metabolism of bradykinin (BK) following BK internalization via B2R. B2R was localized almost exclusively in the plasma membrane, whereas EP24.15 was localized both intracellularly and on the cell surface, and secreted in the media. Intracellular EP24.15 was present throughout the cell, both cytosolic and particulate, with less nuclear localization and no co-localization with either the endoplasmatic reticulum marker calnexin or Golgi marker GM130. No direct co-localization of B2R and EP24.15 was observed using immunofluorescence microscopy. However, the two proteins co-immunoprecipitated specifically, and EP24.15 attenuated maximal B2R responsiveness without influencing the potency of BK to stimulate phosphoinositide hydrolysis and intracellular Ca(2+) mobilization. Cell surface-bound BK remained intact in cells overexpressing EP24.15 but was degraded intracellularly in an EP24.15-dependent manner upon B2R-mediated endocytosis. These results show that EP24.15 acts to negatively regulate B2R responsiveness and by serving as an intracellular peptidase in the degradation of BK specifically internalized via this receptor

IL-17A is Elevated in End-stage COPD and Contributes to Cigarette Smoke-induced Lymphoid Neogenesis.

End-stage chronic obstructive pulmonary disease (COPD) is associated with an accumulation of pulmonary lymphoid follicles. Interleukin (IL)-17A is implicated in COPD and pulmonary lymphoid neogenesis in response to microbial stimuli. We hypothesized that IL-17A is increased in peripheral lung tissue during end-stage COPD and also directly contributes to cigarette smoke-induced lymphoid neogenesis