Migration and habitat use of sea trout Salmo trutta in relation to a marine protected area A study conducted in Beiarfjorden national salmon fjord

Kystområder er viktige leveområder for anadrom brun ørret (sjøørret, Salmo trutta), men også sårbare for menneskeskapte påvirkninger, slik som oppdrettsanlegg, på grunn av den frie bevegelsen av patogener mellom oppdrettsmær og vannmassene rundt. Regjeringen har utpekt 29 fjorder for spesiell beskyttelse av viktige laksebestander. Fisk som oppholder seg i slike beskyttede områder, kjent som "nasjonale laksefjorder", forventes å ha lavere nivåer av lakselus og dermed være mindre påvirket av de negative effektene forbundet med påslag av selus. Det å undersøke sjøørretens vandringer i relasjon en nasjonal laksefjord er viktig for å evaluere i hvilken grad sjøørret bruker habitatene som er beskyttet. Målet med denne oppgaven var å undersøke sjøørretens atferd i forhold til plasseringen og dekningen av Beiarfjorden nasjonale laksefjord. Studien ble utført over en toårsperiode, hvor akustisk telemetri ble brukt til å undersøke hvordan sjøørret fra Beiarelva benyttet fjordsystemet. Merket sjøørret ble registrert i alle deler i fjordsystemet. Totalt brukte sjøørreten mer tid i det beskyttet området (63%) sammenlignet med det ikke-beskyttede fjordområdet utenfor. To forskjellige vandringsatferder ble observert: sjøørret som oppholdt seg kun i Beiarfjorden (mellomdistansevandrere) og sjøørret som forlot Beiarfjorden (langdistansevandrere). Av fisk som ble registrert i fjorden, gjennomførte 84% lang-distansevandringer (> 17 km). Ingen individer holdt seg kun i nærhet til Beiarelva (< 4 km) gjennom hele sommeren. Individer med en lengre kroppslengde forlot Beiarfjorden til mer åpen sjø og oppholdt seg en kortere tid i det beskyttede området sammenlignet med mindre individer. Kondisjonsfaktor, kjønn og alder hadde en begrenset påvirkning i beslutningen på å bli i Beiarfjorden, og i forhold til tiden brukt i det beskyttede området versus det ubeskyttede området. Sjøørret hadde en tendens til å bruke en høyere andel tid i det beskyttede området i 2020, enn i 2019. Resultatene viser at den nasjonale laksefjorden Beiarfjorden inkluderer viktige habitat for sjøørreten, selv om det er variasjon blant individer i andelen tid de oppholder seg i det beskyttede området. Graden beskyttelse gitt av den nasjonale laksefjorden virker ikke tilfeldig, men å avhenge av lengden på fisken, hvor lengre individer ser ut til å være dårligere beskyttet enn kortere og mindre individer. Beiarfjorden nasjonale laksefjord er en av de minste nasjonale laksefjordene i Norge, men har mange langdistansevandrere. Derfor, for å beskytte hele bestanden, bør det beskyttede området være større. Kunnskap om hvor godt nasjonale laksefjorder beskytter sjøørret mot potensielle negative påvirkning fra opprett, vil være viktig når man skal vurdere funksjonen til disse marine verneområdene.Coastal areas are important habitats for anadromous brown trout (sea trout, Salmo trutta), but vulnerable to anthropogenic activities, such as aquaculture, due to the free movement of pathogens between the net pen and the surrounding water masses. The Norwegian government has designated 29 fjords for protection of important Atlantic salmon populations. Fish residing in these protected areas, known as “national salmon fjords” are expected to have lower levels of salmon lice and thus be less affected by the negative effects by lice infestations. Investigating the sea trout ́s migration in relation to a national salmon fjord is important to evaluate to what extent the sea trout utilizes the protected habitats. The aim of this thesis was to investigate individual sea trout migratory behavior in relation to the location and coverage of Beiarfjorden national salmon fjord. The study was conducted over a two-year period, and acoustic telemetry was used to examine how the sea trout originating from river Beiarelva utilized the fjord system. Tagged sea trout were recorded in all parts of the fjord system. More time was spent in the protected area (63%) compared to the non-protected areas. Two different migratory behavioral traits were observed: sea trout either stayed in Beiarfjorden (medium-distance migrants, 17 km). Of the fish entering the fjord, 84% were classified as long-distance migrants. No individuals remained close to the river where they were tagged (< 4 km) throughout the summer. Individuals of a greater body length were more likely to leave Beiarfjorden to more open sea habitats and stayed a lower proportion of time in the protected area compared to smaller individuals. Body condition, sex, and age had limited influence on the decision on whether to stay in Beiarfjorden, and also limited effect on the proportion of time spent within versus the non-protected areas. Sea trout tended to use greater proportions of their time at sea within the protected area in 2020 than in 2019. The results indicates that Beiarfjorden national salmon fjord includes habitats that are important for sea trout, although there was variation among individuals in the proportion of time resided within the protected area. The degree of protection given did not seem random, but depended on the length of the fish, where larger individuals appeared to be less protected than smaller individuals. Beiarfjorden national salmon fjord is one of the smallest national salmon fjords in Norway, but is a fjord with many long-distance migrating sea trout. Hence, to protect the entire sea trout population, the protected area should be of a larger size. Knowledge of how well national salmon fjords protects sea trout from potential negative impact from aquaculture will be important when evaluating the function of these marine protected areas

SILAC-MS Based Characterization of LPS and Resveratrol Induced Changes in Adipocyte Proteomics:Resveratrol as Ameliorating Factor on LPS Induced Changes

Adipose tissue inflammation is believed to play a pivotal role in the development obesity-related morbidities such as insulin resistance. However, it is not known how this (low-grade) inflammatory state develops. It has been proposed that the leakage of lipopolysaccharides (LPS), originating from the gut microbiota, through the gut epithelium could drive initiation of inflammation. To get a better understanding of which proteins and intracellular pathways are affected by LPS in adipocytes, we performed SILAC proteomic analysis and identified proteins that were altered in expression. Furthermore, we tested the anti-inflammatory compound resveratrol. A total of 927 proteins were quantified by the SILAC method and of these 57- and 64 were significantly up- and downregulated by LPS, respectively. Bioinformatic analysis (GO analysis) revealed that the upregulated proteins were especially involved in the pathways of respiratory electron transport chain and inflammation. The downregulated proteins were especially involved in protein glycosylation. One of the latter proteins, GALNT2, has previously been described to regulate the expression of liver lipases such as ANGPTL3 and apoC-III affecting lipid metabolism. Furthermore, LPS treatment reduced the protein levels of the insulin sensitizing adipokine, adiponectin, and proteins participating in the final steps of triglyceride- and cholesterol synthesis. Generally, resveratrol opposed the effect induced by LPS and, as such, functioning as an ameliorating factor in disease state. Using an unbiased proteomic approach, we present novel insight of how the proteome is altered in adipocytes in response to LPS as seen in obesity. We suggest that LPS partly exerts its detrimental effects by altering glycosylation processes of the cell, which is starting to emerge as important posttranscriptional regulators of protein expression. Furthermore, resveratrol could be a prime candidate in ameliorating dysfunctioning adipose tissue induced by inflammatory stimulation

Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix

Incorporation of Pentraxin 3 into Hyaluronan Matrices Is Tightly Regulated and Promotes Matrix Cross-linking

Mammalian oocytes are surrounded by a highly hydrated hyaluronan (HA)-rich extracellular matrix with embedded cumulus cells, forming the cumulus cell·oocyte complex (COC) matrix. The correct assembly, stability, and mechanical properties of this matrix, which are crucial for successful ovulation, transport of the COC to the oviduct, and its fertilization, depend on the interaction between HA and specific HA-organizing proteins. Although the proteins inter-α-inhibitor (IαI), pentraxin 3 (PTX3), and TNF-stimulated gene-6 (TSG-6) have been identified as being critical for COC matrix formation, its supramolecular organization and the molecular mechanism of COC matrix stabilization remain unknown. Here we used films of end-grafted HA as a model system to investigate the molecular interactions involved in the formation and stabilization of HA matrices containing TSG-6, IαI, and PTX3. We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6. This long pentraxin also failed to bind to products of the interaction between IαI, TSG-6, and HA, among which are the covalent heavy chain (HC)·HA and HC·TSG-6 complexes, despite the fact that both IαI and TSG-6 are ligands of PTX3. Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films. Moreover, we demonstrated that this ternary protein mixture made of IαI, PTX3, and TSG-6 is sufficient to promote formation of a stable (i.e. cross-linked) yet highly hydrated HA matrix. We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking

Clearance kinetics and matrix binding partners of the receptor for advanced glycation end products

Elucidating the sites and mechanisms of sRAGE action in the healthy state is vital to better understand the biological importance of the receptor for advanced glycation end products (RAGE). Previous studies in animal models of disease have demonstrated that exogenous sRAGE has an anti-inflammatory effect, which has been reasoned to arise from sequestration of pro-inflammatory ligands away from membrane-bound RAGE isoforms. We show here that sRAGE exhibits in vitro binding with high affinity and reversibly to extracellular matrix components collagen I, collagen IV, and laminin. Soluble RAGE administered intratracheally, intravenously, or intraperitoneally, does not distribute in a specific fashion to any healthy mouse tissue, suggesting against the existence of accessible sRAGE sinks and receptors in the healthy mouse. Intratracheal administration is the only effective means of delivering exogenous sRAGE to the lung, the organ in which RAGE is most highly expressed; clearance of sRAGE from lung does not differ appreciably from that of albumin. Copyright: © 2014 Milutinovic et al

Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)

<p>Abstract</p> <p>Background</p> <p>TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI.</p> <p>Results</p> <p>The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein.</p> <p>Conclusion</p> <p>The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.</p

The subunit composition of human extracellular superoxide dismutase (EC-SOD) regulate enzymatic activity

<p>Abstract</p> <p>Background</p> <p>Human extracellular superoxide dismutase (EC-SOD) is a tetrameric metalloenzyme responsible for the removal of superoxide anions from the extracellular space. We have previously shown that the EC-SOD subunit exists in two distinct folding variants based on differences in the disulfide bridge pattern (Petersen SV, Oury TD, Valnickova Z, Thøgersen IB, Højrup P, Crapo JD, Enghild JJ. Proc Natl Acad Sci USA. 2003;100(24):13875–80). One variant is enzymatically active (aEC-SOD) while the other is inactive (iEC-SOD). The EC-SOD subunits are associated into covalently linked dimers through an inter-subunit disulfide bridge creating the theoretical possibility of 3 dimers (<it>aa</it>, <it>ai </it>or <it>ii</it>) with different antioxidant potentials. We have analyzed the quaternary structure of the endogenous EC-SOD disulfide-linked dimer to investigate if these dimers in fact exist.</p> <p>Results</p> <p>The analyses of EC-SOD purified from human tissue show that all three dimer combinations exist including two homo-dimers (<it>aa </it>and <it>ii</it>) and a hetero-dimer (<it>ai</it>). Because EC-SOD is a tetramer the dimers may combine to generate 5 different mature EC-SOD molecules where the specific activity of each molecule is determined by the ratio of aEC-SOD and iEC-SOD subunits.</p> <p>Conclusion</p> <p>This finding shows that the aEC-SOD and iEC-SOD subunits combine in all 3 possible ways supporting the presence of tetrameric enzymes with variable enzymatic activity. This variation in enzymatic potency may regulate the antioxidant level in the extracellular space and represent a novel way of modulating enzymatic activity.</p

Developmental expression of the receptor for advanced glycation end-products (RAGE) and its response to hyperoxia in the neonatal rat lung

BACKGROUND: The receptor for advanced glycation end products (mRAGE) is associated with pathology in most tissues, while its soluble form (sRAGE) acts as a decoy receptor. The adult lung is unique in that it expresses high amounts of RAGE under normal conditions while other tissues express low amounts normally and up-regulate RAGE during pathologic processes. We sought to determine the regulation of the soluble and membrane isoforms of RAGE in the developing lung, and its expression under hyperoxic conditions in the neonatal lung. RESULTS: Fetal (E19), term, 4 day, 8 day and adult rat lung protein and mRNA were analyzed, as well as lungs from neonatal (0–24 hrs) 2 day and 8 day hyperoxic (95% O(2)) exposed animals. mRAGE transcripts in the adult rat lung were 23% greater than in neonatal (0–24 hrs) lungs. On the protein level, rat adult mRAGE expression was 2.2-fold higher relative to neonatal mRAGE expression, and adult sRAGE protein expression was 2-fold higher compared to neonatal sRAGE. Fetal, term, 4 day and 8 day old rats had a steady increase in both membrane and sRAGE protein expression evaluated by Western Blot and immunohistochemistry. Newborn rats exposed to chronic hyperoxia showed significantly decreased total RAGE expression compared to room air controls. CONCLUSION: Taken together, these data show that rat pulmonary RAGE expression increases with age beginning from birth, and interestingly, this increase is counteracted under hyperoxic conditions. These results support the emerging concept that RAGE plays a novel and homeostatic role in lung physiology

A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies

The SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to-express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of the catalytic domain, residues 403–621, is used for obtaining soluble and biologically active Ulp1. However, we have observed that the soluble and catalytically active Ulp1403-621 inhibits the growth of E. coli host cells. In the current study, we demonstrate an alternative route for producing active Ulp1 catalytic domain from a His-tagged N-terminally truncated variant, residues 416–621, which is expressed in E. coli inclusion bodies and subsequently refolded. Expressing the insoluble Ulp1416-621 variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1416-621 was recovered from inclusion bodies isolated from 1 L of high cell-density E. coli batch fermentation culture. After Ni2+-affinity purification of inactive and denatured Ulp1416-621 in 7.5 M urea, different refolding conditions with varying L-arginine concentration, pH, and temperature were tested. We have successfully refolded the enzyme in 0.25 M L-arginine and 0.5 M Tris-HCl (pH 7) at room temperature. Approximately 80 mg of active Ulp1416-621 catalytic domain can be produced from 1 L of high cell-density E. coli culture. We discuss the applicability of inclusion body-directed expression and considerations for obtaining high expression yields and efficient refolding conditions to reconstitute the active protein fold.</p