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- Einarsdottir, Ingibjörg, 1951, et al.
(författare)
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PITUITARY HORMONE PURIFICATION BY CONTINUOUS-ELUTION ELECTROPHORESIS
- 2006
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Ingår i: 7th International Congress on the Biology of Fishes, Book of Abstracts.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Widely used methods for peptide hormone purification, such as gel filtration chromatography combined with rpHPLC, are time-consuming and often give low yields and instable proteins. The Model 491 Prep Cell from Bio-Rad provides a continuous-elution electrophoresis technique to separate proteins. The protein mixture runs through a cylindrical gel, proteins are separated by size and charge, as they elute from the matrix and are collected using a fraction collector. Sub-samples are assessed by SDS-PAGE and protein identity is verified by Western blot analysis. We applied this technique to separate putative hormones from adult Atlantic halibut pituitaries. Soluble proteins from homogenized pituitaries were denatured and subjected to continuous-elution electrophoresis. The fractions sampled contained purified proteins with molecular sizes ranging from 10 to 33 kDa. Fractions containing proteins with molecular weights of approximately 21, 24, 28 and 32 kDa were identified as putative growth hormone, prolactin, somatolactin and gonadotropins. These were analyzed further by mass spectrometry (MS) and identified with peptide mass protein fingerprinting. Good yield of highly purified GH was obtained, identified, and found to be biologically stable. Subsequently, it was used for radioactive labeling and as standards in a homologous RIA. Using this assay, the GH was measured in halibut larvae from start-feeding through metamorphosis, showing a stable size-related GH content of the head region. Funded by the EU.
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- Einarsdottir, Ingibjörg, 1951, et al.
(författare)
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The growth hormone – IGF-I system is involved in cranial tissue remodeling in metamorphosing Atlantic halibut larvae
- 2008
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Ingår i: Sixth International Symposium on Fish Endocrinology (6ISFE) Program and Abstract Book.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The Atlantic halibut is a large, cold-water flatfish which inhabits the North Atlantic. The pelagic and symmetric larvae undergo metamorphosis as an adaptation to better suit them for a benthic life. Metamorphosis involves many dramatic biological changes including a major reconstruction of the cranium during which the left eye migrates to the right side of the head. The structural driving force of eye migration is assumed to be the growth and migration of the ethmoid plate and the migration of the frontal processes towards the future ocular side, and fibroblast proliferation ventrally to the migrating eye. Metamorphosis is under endocrine control and thyroid hormones are known to be the main regulatory hormones. Data on the role of other growth regulators, including the growth hormone (GH) - insulin-like growth factor I (IGF-I) system, in halibut metamorphosis are sparse. GH content in the head region increases steadily from first feeding throughout the larval phase until post-metamorphosis, while IGF-I content in the body increases until the onset of metamorphosis and decline thereafter. In situ hybridization and immunohistochemistry reveal that mRNA and proteins of the type I GH receptor (GHR), and mRNA of the type II GHR and the IGF-I receptor (IGF-IR) are expressed in fibroblasts in soft tissue between the eyes, frontal bone osteocytes and dorsal cranial chondrocytes during the metamorphic process. In fibroblasts and chondrocytes, the expression appears most intense at the start of metamorphosis (stage 8) and decreases during subsequent developmental stages. IGF-IR expression decreases in frontal bone osteocytes after the completion of metamorphosis (stage 10). The decrease in tissue IGF-I content during metamorphosis, and the localization and change in abundance of GHRs and IGF-IR in head structures undergoing reconstruction, indicate the involvement of the GH-IGF-I system in Atlantic halibut metamorphosis. Acknowledgement: Work financed by the Commission of the European Communities within the project “Arrested development: the molecular and endocrine basis of flatfish metamorphosis” (Q5RS-2002-01192).
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