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| 2. |
- Caruso, Vanni, et al.
(författare)
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mRNA GPR162 changes are associated with decreased food intake in rat, and its human genetic variants with impairments in glucose homeostasis in two Swedish cohorts
- 2016
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Ingår i: Gene. - : Elsevier BV. - 0378-1119 .- 1879-0038. ; 581:2, s. 139-145
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Tidskriftsartikel (refereegranskat)abstract
- G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating intercellular interactions of fundamental physiological importance for survival including regulation of food intake, blood pressure, and hormonal sensing signaling, among other roles. Homeostatic alterations in the physiological status of GPCRs are often associated with underlying causes of disease, and to date, several orphan GPCRs are still uncharacterized. Findings from our previous study demonstrate that the Rhodopsin family protein GPR162 is widely expressed in GABAergic as well as other neurons within the mouse hippocampus, whereas extensive expression is observed in hypothalamus, amygdala, and ventral tegmental area, regions strictly interconnected and involved in the regulation of energy homeostasis and hedonic feeding. In this study, we provide a further anatomical characterization of GPR162 in mouse brain via in situ hybridization as well as detailed mRNA expression in a panel of rat tissues complementing a specie-specific mapping of the receptor. We also provide an attempt to demonstrate a functional implication of GPR162 in food intake-related behavior via antisense knockdown studies. Furthermore, we performed human genetic studies in which for the first time, variants of the GPR162 gene were associated with impairments in glucose homeostasis.
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| 3. |
- Caruso, Vanni, et al.
(författare)
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The Orphan G Protein-Coupled Receptor Gene GPR178 Is Evolutionary Conserved and Altered in Response to Acute Changes in Food Intake
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:6
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Tidskriftsartikel (refereegranskat)abstract
- G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating physiological functions fundamental for survival, including energy homeostasis. A few years ago, an amino acid sequence of a novel GPCR gene was identified and named GPR178. In this study, we provide new insights regarding the biological significance of Gpr178 protein, investigating its evolutionary history and tissue distribution as well as examining the relationship between its expression level and feeding status. Our phylogenetic analysis indicated that GPR178 is highly conserved among all animal species investigated, and that GPR178 is not a member of a protein family. Real-time PCR and in situ hybridization revealed wide expression of Gpr178 mRNA in both the brain and periphery, with high expression density in the hypothalamus and brainstem, areas involved in the regulation of food intake. Hence, changes in receptor expression were assessed following several feeding paradigms including starvation and overfeeding. Short-term starvation (12-48h) or food restriction resulted in upregulation of Gpr178 mRNA expression in the brainstem, hypothalamus and prefrontal cortex. Conversely, short-term (48h) exposure to sucrose or Intralipid solutions downregulated Gpr178 mRNA in the brainstem; long-term exposure (10 days) to a palatable high-fat and high-sugar diet resulted in a downregulation of Gpr178 in the amygdala but not in the hypothalamus. Our results indicate that hypothalamic Gpr178 gene expression is altered during acute exposure to starvation or acute exposure to palatable food. Changes in gene expression following palatable diet consumption suggest a possible involvement of Gpr178 in the complex mechanisms of feeding reward.
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| 4. |
- Debiais-Thibaud, Melanie, et al.
(författare)
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Skeletal Mineralization in Association with Type X Collagen Expression Is an Ancestral Feature for Jawed Vertebrates
- 2019
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Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 36:10, s. 2265-2276
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Tidskriftsartikel (refereegranskat)abstract
- In order to characterize the molecular bases of mineralizing cell evolution, we targeted type X collagen, a nonfibrillar network forming collagen encoded by the Col10a1 gene. It is involved in the process of endochondral ossification in ray-finned fishes and tetrapods (Osteichthyes), but until now unknown in cartilaginous fishes (Chondrichthyes). We show that holocephalans and elasmobranchs have respectively five and six tandemly duplicated Col10a1 gene copies that display conserved genomic synteny with osteichthyan Col10a1 genes. All Col10a1 genes in the catshark Scyliorhinus canicula are expressed in ameloblasts and/or odontoblasts of teeth and scales, during the stages of extracellular matrix protein secretion and mineralization. Only one duplicate is expressed in the endoskeletal (vertebral) mineralizing tissues. We also show that the expression of type X collagen is present in teeth of two osteichthyans, the zebrafish Danio rerio and the western clawed frog Xenopus tropicalis, indicating an ancestral jawed vertebrate involvement of type X collagen in odontode formation. Our findings push the origin of Col10a1 gene prior to the divergence of osteichthyans and chondrichthyans, and demonstrate its ancestral association with mineralization of both the odontode skeleton and the endoskeleton.
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| 5. |
- Dennhag, Nils, 1989-
(författare)
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Genetic studies of zebrafish muscles : clues to protection in muscle disease
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Muscular dystrophies (MDs) are caused by dysregulation of over 40 proteins but commonly share features of muscle weakness, myofiber death and regeneration, loss of ambulation and premature death. A MD involves a broken link anywhere in the connection from extracellular matrix through the sarcolemma to the sarcomere. Thus, any protein which is a part of this link causes MD if misfolded, dysregulated or absent. In MD, the most common causes of death are cardiac or respiratory failure, when the muscles involved in these processes fail. Although MDs affect 1:3500-5000 births worldwide there are currently no cures available. Extraocular muscles (EOMs) are strikingly not affected by MDs, however, the mechanisms behind this native resistance remain elusive. We have recently shown that the EOMs cytoskeleton differs significantly from that of other muscles and hypothesized that investigation of their cytoskeleton in MD models would provide important clues. Furthermore, we hypothesized that application of the EOMs strategies to trunk muscle tissue would decrease the detrimental impact of MD overall.The zebrafish model system has recently increased vastly in popularity, and has quickly become a MD model. Due to its compatibility with the CRISPR/Cas9 method, genetic knockout studies can be utilized to generate novel mutant lines tailored to fit various aspects in studies of the zebrafish skeletal muscle. In this thesis I present nine new zebrafish lines which I used to study muscle biology processes, including muscle regeneration and the EOM cytoskeleton. Our results clearly demonstrate the need for understanding compensatory mechanisms in biology. Interestingly, pax3 and pax7 were shown to functionally compensate for each other both in appendicular muscle formation and in muscle regeneration, respectively, two processes where these individual genes have great impact in other organisms. This finding would also prove to be important in aiding our understanding of the EOM biology in adaptive strategies towards MDs. Furthermore, our results show that zebrafish EOMs are a good model to study cytoskeletal composition, as they share important features with human EOMs. Utilizing the CRISPR/Cas9 genome editing technique, I developed several knockout models of cytoskeletal proteins (desmin, obscurin, plectin) and studied their importance for the function of the EOMs. In studies of zebrafish EOMs lacking obscurin, we found that EOMs functionally adapt their myosin composition over time via upregulation of myh7, a cardiac specific myosin. Furthermore, an RNA-sequencing screen on a CRISPR/Cas9 induced desminopathy model (desma; desmb double mutant) identified several protective genes of interest. We show that a four and a half LIM-domain protein (Fhl2) is upregulated in EOMs in several muscular dystrophy models and that fhl2b protects EOMs from excessive myonuclei turnover and hypertrophy. Furthermore, its ectopic expression in trunk muscle can also protect an additional muscle dystrophy model (dmd) from acute early death, improve myofiber function and stabilize neuromuscular junctions. Importantly, this protein was also detected in both human and mouse EOMs, indicating a potentially conserved role in the EOMs across species.In summary, we identified several novel strategies of adaptation to disease progression in the EOMs. Together, these findings have contributed significantly to a better understanding of the EOMs and suggest new treatment strategies for MD that may have important future clinical applications.
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| 6. |
- Dierker, Tabea, et al.
(författare)
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Nematodes join the family of chondroitin sulfate-synthesizing organisms : Identification of an active chondroitin sulfotransferase in Caenorhabditis elegans
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycans are proteins that carry sulfated glycosaminoglycans (GAGs). They help form and maintain morphogen gradients, guiding cell migration and differentiation during animal development. While no sulfated GAGs have been found in marine sponges, chondroitin sulfate (CS) and heparan sulfate (HS) have been identified in Cnidarians, Lophotrocozoans and Ecdysozoans. The general view that nematodes such as Caenorhabditis elegans, which belong to Ecdysozoa, produce HS but only chondroitin without sulfation has therefore been puzzling. We have analyzed GAGs in C. elegans using reversed-phase ion-pairing HPLC, mass spectrometry and immunohistochemistry. Our analyses included wild type C. elegans but also a mutant lacking two HS sulfotransferases (hst-6 hst-2), as we suspected that the altered HS structure could boost CS sulfation. We could indeed detect sulfated CS in both wild type and mutant nematodes. While 4-O-sulfation of galactosamine dominated, we also detected 6-O-sulfated galactosamine residues. Finally, we identified the product of the gene C41C4.1 as a C. elegans CS-sulfotransferase and renamed it chst-1 (CarboHydrate SulfoTransferase) based on loss of CS-4-O-sulfation in a C41C4.1 mutant and in vitro sulfotransferase activity of recombinant C41C4.1 protein. We conclude that C. elegans indeed manufactures CS, making this widely used nematode an interesting model for developmental studies involving CS.
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| 7. |
- Filipek-Gorniok, Beata, et al.
(författare)
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Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development
- 2013
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Ingår i: Developmental Dynamics. - : Wiley. - 1058-8388 .- 1097-0177. ; 242:8, s. 964-975
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Tidskriftsartikel (refereegranskat)abstract
- Background: Chondroitin/dermatan sulfate (CS/DS) proteoglycans present in the extracellular matrix have important structural and regulatory functions. Results: Six human genes have previously been shown to catalyze CS/DS polymerization. Here we show that one of these genes, chpf, is represented by two copies in the zebrafish genome, chpfa and chpfb, while the other five human CS/DS glycosyltransferases csgalnact1, csgalnact2, chpf2, chsy1, and chsy3 all have single zebrafish orthologues. The putative zebrafish CS/DS glycosyltransferases are spatially and temporally expressed. Interestingly, overlapping expression of multiple glycosyltransferases coincides with high CS/DS deposition. Finally, whereas the relative levels of the related polysaccharide HS reach steady-state at around 2 days post fertilization, there is a continued relative increase of the CS amounts per larvae during the first 6 days of development, matching the increased cartilage formation. Conclusions: There are 7 CS/DS glycosyltransferases in zebrafish, which, based on homology, can be divided into the CSGALNACT, CHSY, and CHPF families. The overlap between intense CS/DS production and the expression of multiple CS/DS glycosyltransferases suggests that efficient CS/DS biosynthesis requires a combination of several glycosyltransferases.
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| 8. |
- Filipek-Gorniok, Beata, et al.
(författare)
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The Ndst Gene Family in Zebrafish : Role of Ndst1b in Pharyngeal Arch Formation
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate (HS) proteoglycans are ubiquitous components of the extracellular matrix and plasma membrane of metazoans. The sulfation pattern of the HS glycosaminoglycan chain is characteristic for each tissue and changes during development. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes catalyze N-deacetylation and N-sulfation during HS biosynthesis and have a key role in designing the sulfation pattern. We here report on the presence of five NDST genes in zebrafish. Zebrafish ndst1a, ndst1b, ndst2a and ndst2b represent duplicated mammalian orthologues of NDST1 and NDST2 that arose through teleost specific genome duplication. Interestingly, the single zebrafish orthologue ndst3, is equally similar to tetrapod Ndst3 and Ndst4. It is likely that a local duplication in the common ancestor of lobe-finned fish and tetrapods gave rise to these two genes. All zebrafish Ndst genes showed distinct but partially overlapping expression patterns during embryonic development. Morpholino knockdown of ndst1b resulted in delayed development, craniofacial cartilage abnormalities, shortened body and pectoral fin length, resembling some of the features of the Ndst1 mouse knockout.
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| 9. |
- Habicher, Judith, et al.
(författare)
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Chondroitin / Dermatan Sulfate Modification Enzymes in Zebrafish Development
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- Chondroitin/dermatan sulfate (CS/DS) proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS). Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.
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| 10. |
- Haitina, Tatjana, et al.
(författare)
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Cloning, tissue distribution, pharmacology and three-dimensional modelling of melanocortin receptors 4 and 5 in rainbow trout suggest close evolutionary relationship of these subtypes
- 2004
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 380:2, s. 475-486
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Tidskriftsartikel (refereegranskat)abstract
- The rainbow trout (Oncorhynchus mykiss) is one of the most widely used fish species in aquaculture and physiological research. In the present paper, we report the first cloning, 3D (three-dimensional) modelling, pharmacological characterization and tissue distribution of two melanocortin (MC) receptors in rainbow trout. Phylogenetic analysis indicates that these receptors are orthologues of the human MC4 and MC5 receptors. We created 3D molecular models of these rainbow trout receptors and their human counterparts. These models suggest greater divergence between the two human receptors than between their rainbow trout counterparts. The pharmacological analyses demonstrated that ACTH (adrenocorticotropic hormone) had surprisingly high affinity for the rainbow trout MC4 and MC5 receptors, whereas alpha-, beta- and gamma-MSH (melanocyte-stimulating hormone) had lower affinity. In second-messenger studies, the cyclic MSH analogues MTII and SHU9119 acted as potent agonist and antagonist respectively at the rainbow trout MC4 receptor, indicating that these ligands are suitable for physiological studies in rainbow trout. Interestingly, we found that the rainbow trout MC4 receptor has a natural high-affinity binding site for zinc ions (0.5 microM) indicating that zinc may play an evolutionary conserved role at this receptor. Reverse transcription PCR indicates that the rainbow trout receptors are expressed both in peripheral tissues and in the central nervous system, including the telencephalon, optic tectum and hypothalamus. Overall, this analysis indicates that the rainbow trout MC4 and MC5 receptors have more in common than their mammalian counterparts, which may suggest that these two receptors have a closer evolutionary relationship than the other MC receptor subtypes.
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