Cloning and Expression of Grass Carp (ctenopharyngodon Idella) Growth Hormone PDF Download
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Author: Jun Zou
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Languages : en
Pages :
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Author: Jun Zou
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Category :
Languages : en
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Author: Kit-Wa Anthea To
Publisher: Open Dissertation Press
ISBN: 9781361192443
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Languages : en
Pages :
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This dissertation, "Steriod Regulation of Growth Hormone Gene Expression and Molecular Cloning of Estrogen Receptors in Chinese Grass Carp" by Kit-wa, Anthea, To, 杜潔華, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled STEROID REGULATION OF GROWTH HORMONE GENE EXPRESSION AND MOLECULAR CLONING OF ESTROGEN RECEPTORS IN CHINESE GRASS CARP Submitted by TO KIT WA ANTHEA for the degree of Master of Philosophy at The University of Hong Kong in December 2002 Hormones from the steroid/ thyroid superfamily are key regulators for body growth and metabolism in animals. Part of this mechanism is mediated via steroid/ thyroid hormone modulation of growth hormone (GH) synthesis at the pituitary level. In this study, using a static incubation approach, a systematic survey was conducted in vitro to examine the effects of sex steroids, adrenocorticoids, and thyroid hormone on GH mRNA expression in grass carp (Ctenopharyngodon idella) pituitary cells. Sex steroids, including estradiol, testosterone, and the estrogen agonist diethylstilbestrol, increased GH mRNA levels in grass carp pituitary cells. These stimulatory actions reached their peaks at submicromolar concentrations. In a similar dose range, progesterone did not affect basal GH mRNA expression and a slight inhibition was observed when the doses were increased to micromolar levels. Glucocorticoids, including hydrocortisone, corticosterone, and the synthetic analog dexamethasone, suppressed GH mRNA levels when tested at high micromolar doses. Unlike glucocorticoids, a slight elevation in GH mRNA levels was noted when pituitary cells were exposed to submicromolar doses of aldosterone. In parallel studies, retinoic acid and thyroid hormone did not alter basal GH mRNA expression in grass carp pituitary cells. Data provides evidence that steroid hormones can differentially regulate GH gene expression in grass carp by acting directly at the pituitary cell level. Among the hormones tested, estradiol was a potent stimulant for GH mRNA expression. Molecular cloning of grass carp estrogen receptors (ER) was also performed. Using 5' and 3' RACE, three isoforms of grass carp ERs, namely ERα, ERβ-S and ERβ-L, were cloned. They were highly homologous to the ERα and ERβ reported in other fish species. The full-length cDNAs of grass carp ERα (1692 bp), ERβ-S (2091 bp) and ERβ-L (2707 bp) carried the open reading frames of ERs composing of 564, 605, and 697 amino acids, respectively. RT-PCR also revealed that transcripts for ERα and ERβs could be identified in a variety of tissues in the carp, including the brain, pituitary, muscle, gonad, gill, heart, spleen, liver, and intestine. To characterize the pharmacological properties of the cloned ERs, functional expression of grass carp ERα, ERβ-S and ERβ-L was conducted in CHO-K1 cells. In the presence of estradiol, the ERs expressed were effective in activating a luciferase reporter gene with estrogen-responsive promoter elements. These stimulatory actions were specific for estradiol and could not be mimicked by parallel treatments with testosterone, progesterone, hydrocortisone, or thyroid hormone. Furthermore, estradiol-stimulated luciferase expression in CHO-K1 cells with expressed ERα, ERβ-S and ERβ-L could be partially inhibited or abolished by a simultaneous treatment with the estrogen antagonists, tamoxifen and ICI182780. These results strongly indicate that the newly cloned ERα, ERβ-S and ERβ-L are functional receptors for estrogen in grass carp. The cloning studi
Author: Yonghua Jiang
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ISBN: 9781374674592
Category :
Languages : en
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This dissertation, "Molecular Cloning of AP-1 Transcription Factors in Chinese Grass Carp and Their Functional Roles in PACAP-stimulated Growth Hormone Gene Expression" by Yonghua, Jiang, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. DOI: 10.5353/th_b3124541 Subjects: Transcription factors Genetic transcription - Regulation Ctenopharyngodon idella - China - Genetics
Author: Yonghua Jiang (Ph.D.)
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Category : Ctenopharyngodon idella
Languages : en
Pages : 482
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Author: Guodong Fu (Ph. D.)
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Category : Ctenopharyngodon idella
Languages : en
Pages : 422
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Author: Xinyan Wang
Publisher: Open Dissertation Press
ISBN: 9781374674707
Category :
Languages : en
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This dissertation, "Pituitary Dopamine D1 Receptor and Growth Hormone Gene Expression in Chinese Grass Carp" by Xinyan, Wang, 汪新艷, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled PITUITARY DOPAMINE D1 RECEPTOR AND GROWTH HORMONE GENE EXPRESSION IN CHINESE GRASS CARP Submitted by Wang Xinyan for the degree of Doctor of Philosophy at The University of Hong Kong in April 2007 In fish models, dopamine (DA) serves as a potent stimulator for growth hormone (GH) release by direct action at the pituitary level via activation of DA D1 receptors. Although multiple subtypes of D1 receptors have been reported, the form(s) of D1 receptor expressed in the fish pituitary responsible for DA-stimulated GH release has not been clarified. Since previous studies have focused mainly on GH secretion, the mechanisms for DA D1 regulation of GH gene expression are largely unknown. In this study, using grass carp (Ctenopharyngodon idellus) as an animal model, the structural identity of the D1 receptor expressed in the carp pituitary was established by molecular cloning. Sequence alignment and phylogenetic analysis have revealed that the receptor is a member of the D1A receptor subfamily. Functional expression studies also confirmed that the receptor binding properties and signaling coupling via cAMP production by this grass carp D1A receptor were highly comparable to that of mammalian D1 receptors. This newly cloned receptor was found to be a single copy gene in the grass carp genome and expressed predominantly in the pituitary. At the pituitary level, D1A receptor expression could be detected in the somatotrophs and lactotrophs, but not in gonadotrophs. In carp pituitary cells, D1A receptor transcript levels could be elevated by GH treatment, whereas the opposite effect was noted by removing endogenous GH using immunoneutralization. In contrast to GH treatment, luteinizing hormone (LH) or its functional analog hCG was found to suppress D1A receptor mRNA expression. Similarly, the differential actions of GH and hCG were also observed in grass carp somatotrophs. In this case, GH-induced D1A receptor gene expression could be blocked by simultaneous treatment with hCG, suggesting that D1A receptor expression in carp somatotrophs is regulated by local interactions between GH and LH at the pituitary level. To further examine the functional role of D1 receptors in GH regulation, the mechanisms for DA D1 stimulation of GH gene expression were also elucidated by direct measurement of "steady-state" GH mRNA level, GH transcript stability, and GH primary transcript production in grass carp pituitary cells and indirect measurement of GH promoter activity in GH cells with stable expression of grass carp D1A receptors. In these studies, DA D1 stimulation was effective in increasing GH mRNA and GH primary transcript expression without affecting the half-life of GH transcripts. These stimulatory effects also occurred with parallel increase in GH promoter activity, suggesting that GH gene transcription can be activated through pituitary D1A receptors by actions acting at the promoter level. Using pharmacological and molecular manipulations perturbing individual signaling steps, MAPK MAPK activation of MAPK (P and P ) and PI3K/Akt cascades coupled to the 42/44 38 AC/cAMP/ PKA pathway was shown to be the key mechanisms mediating DA D1 stimulation of GH gene expression. Activation of these signaling events also induced subsequent phosphorylation of the transcription factor CREB to trigger GH gene transcription at the promoter level.
Author: Longfei Huo
Publisher:
ISBN: 9781374726284
Category :
Languages : en
Pages :
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This dissertation, "Molecular Cloning and Functional Studies of Cyprinid Calmodulin" by Longfei, Huo, 霍龍飛, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled MOLECULAR CLONING AND FUNCTIONAL STUDIES OF CYPRINID CALMODULIN Submitted by Huo Longfei for the degree of Doctor of Philosophy at The University of Hong Kong in May 2004 2+ Calmodulin (CaM) is a Ca -binding protein essential for eukaryotic cells. In mammals, modulation of CaM gene expression in regulating the functionality of the brain-pituitary-axis has not been characterized. In lower vertebrates, functional studies on CaM gene expression in the pituitary still await investigation. By using the members of the Cyprinid family as animal models, the role of CaM gene expression in the pituitary in mediating the signal input from the hypothalamus and the expression of growth hormone (GH) gene was examined. Firstly, CaM cDNAs were isolated from the pituitaries of goldfish and grass carp. The ORF of these cDNAs encodes the same CaM protein as that of mammals. These cDNAs can be grouped with the β-subtype of fish CaM cDNAs and are structurally related to the mammalian CaM-I gene subfamily. In both goldfish and grass carp, CaM mRNA is ubiquitously expressed. In goldfish pituitary cells, activation of cAMP- or PKC-dependent pathways increased CaM mRNA levels, whereas the opposite was true 2+ for the induction of Ca entry. Basal levels of CaM mRNA were accentuated by GnRH and PACAP but were suppressed by dopaminergic stimulation through pituitary D2 receptors. These results demonstrate for the first time that pituitary CaM gene expression can be regulated by functional interactions among hypothalamic factors. To study the role of CaM in pituitary hormone gene expression, the possible involvement of CaM expression in IGF feedback on GH gene transcription was tested in carp pituitary cells. In this case, IGF-I/-II increased the amount of CaM mRNA with a concurrent drop in the GH mRNA levels. The stimulatory effect was not mimicked by insulin. CaM antagonism also increased GH mRNA expression in grass carp pituitary cells whereas CaM over-expression could reduce GH promoter activity. These results, as a whole, imply that the attenuation of GH gene expression by IGF is mediated by CaM gene expression in grass carp pituitary cells. To further examine the mechanisms of regulating CaM gene expression in fish, the full-length CaM gene was cloned in grass carp by intron trapping and genome- walking. This CaM gene is 12-Kb in size and has the same structural organization as that of human CaM genes. The 5'-promoter of this carp CaM gene is AT rich rather than GC rich and contains a TATA box, indicating that the gene is not a typical "house- keeping" gene. 5'-Deletion analysis has revealed that a strong silencer element is located within the region from -1509bp to -1369bp in the CaM promoter. In addition, the 5'-UTR has strong influence on the carp CaM promoter activity. By the use of a reporter gene approach, the grass carp CaM promoter could be activated in α-T3-1 cells by IGFs, insulin, and PKC activators, but was suppressed by PACAP, forskolin, cAMP analog, CaM antagonist, and CaMK-II inhibitor. Transcription factors Sp1 and Sp3, but not Pit-1, were also effective in suppressing the CaM promoter activity. These results indicate that CaM gene transcription in the fish model is regulated by hormones, probably through various signaling pathways and transcription factors. DOI:
Author: Sinothai Poen
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Category : Pangasianodon hypophthalmus
Languages : en
Pages : 204
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Author: Margit Urbanek
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Category :
Languages : en
Pages : 376
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Author: J. M. Warwick
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Category :
Languages : en
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