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Author: Kit-Wa Anthea To
Publisher: Open Dissertation Press
ISBN: 9781361192443
Category :
Languages : en
Pages :
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Book Description
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: Kit-Wa Anthea To
Publisher: Open Dissertation Press
ISBN: 9781361192443
Category :
Languages : en
Pages :
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Book Description
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: Sai-Kit Fung
Publisher: Open Dissertation Press
ISBN: 9781374727168
Category : Science
Languages : en
Pages : 102
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This dissertation, "Grass Carp Activin: Molecular Cloning and Functional Role in Regulating Growth Hormone Gene Expression in Grass Carp Pituitary Cells" by Sai-kit, Fung, 馮世傑, 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 GRASS CARP ACTIVIN: MOLECULAR CLONING AND FUNCTIONAL ROLE IN REGULATING GROWTH HORMONE GENE EXPRESSION IN GRASS CARP PITUITARY CELLS Submitted by Fung Sai Kit for the degree of Master of Philosophy at The University of Hong Kong in August 2004 Previous studies have shown that activin can regulate growth hormone (GH) release in mammals. However, the role of activin in modulating GH synthesis at the pituitary level is largely unknown. In this study, molecular cloning of two types of activin β subunits, namely activin βA and βB, was conducted in Chinese grass carp and their functional roles in regulating GH gene expression were examined in vitro in primary cultures of grass carp pituitary cells. The full-length cDNAs of grass carp activin βA and βB were isolated by nested PCR coupled to 5'/3' RACE. Both cDNAs contain the coding sequences with the signal peptide, pro-peptide, and mature peptide of activin β subunit. The ORF of these cDNAs, especially in the region of mature peptide, are highly homologous to activin cDNAs reported in other species. Protein modeling also revealed that the 3-D structures of activin βA and βB are highly conserved between the fish and human. To confirm the functionality of these newly cloned cDNAs, functional expression of activin βA and βB was conducted in CHO cells and the conditioned media containing grass carp activin A and B were effective in stimulating the promoter activity of a pAR3-Lux reporter construct through the Mix2 activin-responsive elements. In the grass carp, the transcripts for activin βA and βB are widely expressed in various tissues and the brain areas. At the pituitary level, activin βB was found to be the dominant form and its transcript expression could be detected in gonadotrophs and lactotrophs but not in somatotrophs. Furthermore, treatment of grass carp pituitary cells with human activin A and B resulted in a dose-dependent decrease in steady-state GH mRNA levels. In contrast, removal of endogenous activin using the activin-binding protein follistatin increased GH mRNA expression in a dose-related manner. To test the effects of activin on GH transcript stability, GH mRNA clearance studies was performed in grass carp pituitary cells pretreated with the transcription inhibitor actinomycin D. In this case, treatment with human activin B rather than activin A enhanced the degradation rate of GH mRNA with a drop in the half-life (T ) of GH transcripts. 1/2 By real-time PCR, activin A and B also reduced the levels of GH primary transcripts in carp pituitary cells, suggesting that GH gene transcription was suppressed during activin treatment. In parallel studies with GH3 cells transfected with a luciferase reporter construct carrying the 5' promoter of grass carp GH gene, activin A and B were both effective in reducing the promoter activity of GH gene and these effects could be mimicked by over-expression of SMAD and SMAD, the downstream 2 3 effectors of activin's actions. Besides, activin inhibition of GH promoter activity could be reverted by over-expression of a dominant negative SMAD . These results, as a whole, indicate that activin is expressed locally in the pituitary of grass carp and functions as a paracrine inhibitor of GH gene expression, presumably via stimulation
Author: Guodong Fu
Publisher: Open Dissertation Press
ISBN: 9781374674530
Category :
Languages : en
Pages :
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This dissertation, "Grass Carp CREB: Molecular Cloning, Regulation of Gene Expression and Functional Implications at the Pituitary Level" by Guodong, Fu, 傅國棟, 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 GRASS CARP CREB: MOLECULAR CLONING, REGULATION OF GENE EXPRESSION AND FUNCTIONAL IMPLICATIONS AT THE PITUITARY LEVEL Submitted by Fu Guodong for the degree of Doctor of Philosophy at The University of Hong Kong in January 2007 2+ The transcription factor CREB can be phosphorylated through cAMP- and Ca - dependent signaling cascades to induce target gene transcription. CREB binding to CRE sites present in the growth hormone (GH) promoter represents a key mechanism for cAMP induction of GH gene expression. The previous studies have largely restricted to mammals and very little is known about the implications of CREB gene expression in lower vertebrates. In this study, using grass carp as an animal model, the functional roles and the mechanisms of CREB gene expression in GH regulation were examined at the pituitary level. The structural identity of carp CREB was firstly established by molecular cloning. Carp CREB encoded by a single-copy gene, was widely expressed in various tissues. The newly cloned CREB was functional to bind to the canonical CRE and transactivate the gene promoter containing CRE site(s). In the carp pituitary cells, the possible involvement of CREB in GH expression was tested with treatment of somatostatin (SRIF) and insulin-like growth factor (IGF). After SRIF stimulation, CREB mRNA expression and protein synthesis were increased with a lagging drop in GH transcript levels. The differential actions of SRIF on CREB and GH gene expression were mediated by inhibiting the AC/cAMP/PKA pathway. Besides, SRIF stimulation or CREB over-expression was effective in suppressing the GH promoter activity via a negative response sequence, -742 to -656 of GH promoter. These results suggest that SRIF may inhibit GH gene transcription via up-regulation of CREB expression by the cAMP/PKA-dependent mechanisms. Regarding IGF treatment, a parallel drop in CREB and GH mRNA levels was noted via activation of 2+ the Ca /CaM/calcineurin pathway. IGF stimulation also attenuated CREB protein and GH primary transcripts in carp somatotrophs. CREB over-expression, interestingly, could stimulate GH promoter activity via downstream of -55 of GH promoter. These findings raise the possibility that IGF may inhibit GH gene expression by reducing the stimulatory input of CREB acting on the proximal region 2+ of GH gene promoter via the Ca /CaM-dependent mechanisms. To further examine the functional interactions between CREB and CRE sites in fish model, we also examined the involvement of CREB in Jun-B gene transcription. Pituitary adenylate cyclase-activating polypeptide (PACAP) can induce carp Jun-B gene expression to nullify PACAP stimulation of GH gene expression. In this study, PACAP also stimulated the Jun-B promoter activity. Apparently, this stimulatory effect of PACAP was mediated through the cAMP-dependent mechanisms, CREB phosphorylation, and CREB binding to two non-canonical CRE sites with CGTCA motif in the proximal region of carp Jun-B promoter. These results, taken together, provide evidence that modulation of CREB gene expression and subsequent binding with CRE sites on a target promoter can act as the site of modulation to regulate GH gene expression in the carp species. DOI: 10.5353/th_b3843751 Subjects: Transcription factors Molecular cloning Somatotropin
Author: Yonghua Jiang
Publisher: Open Dissertation Press
ISBN: 9781374674585
Category :
Languages : en
Pages :
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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: Sai-kit Fung
Publisher:
ISBN:
Category : Activin
Languages : en
Pages : 262
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Author: Xinyan Wang
Publisher: Open Dissertation Press
ISBN: 9781374674707
Category :
Languages : en
Pages :
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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: Philippa Melamed
Publisher: World Scientific
ISBN: 9814482730
Category : Science
Languages : en
Pages : 308
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Book Description
Research on the molecular aspects of fish reproduction has progressed swiftly over the past few years. With the availability of wide-ranging molecular tools, fish researchers have elucidated many of the molecular mechanisms regulating reproduction which operate in the brain, pituitary and gonad. This research has revealed novel variants of reproductive hormones and their receptors, and has shed new light on the mechanisms through which many of these genes can be activated. Several of the findings, which are reported in this book, have formed the basis for subsequent mammalian research and will also constitute the platform on which new approaches to reproductive management in aquaculture can be developed.
Author:
Publisher:
ISBN:
Category : Medicine
Languages : en
Pages : 1872
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Author: Mike Ludwig
Publisher: John Wiley & Sons
ISBN: 111939094X
Category : Medical
Languages : en
Pages : 460
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Book Description
Model Animals in Neuroendocrinology: From Worm to Mouse to Man offers a masterclass on the opportunities that different model animals offer to the basic understanding of neuroendocrine functions and mechanisms of action and the implications of this understanding. The authors review recent advances in the field emanating from studies involving a variety of animal models, molecular genetics, imaging technologies, and behavior assays. These studies helped unravel mechanisms underlying the development and function of neuroendocrine systems. The book highlights how studies in a variety of model animals, including, invertebrates, fish, birds, rodents and mammals has contributed to our understanding of neuroendocrinology. Model Animals in Neuroendocrinology provides students, scientists and practitioners with a contemporary account of what can be learnt about the functions of neuroendocrine systems from studies across animal taxonomy. This is the seventh volume in the Masterclass in Neuroendocrinology Series, a co-publication between Wiley and the INF (International Neuroendocrine Federation) that aims to illustrate highest standards and encourage the use of the latest technologies in basic and clinical research and hopes to provide inspiration for further exploration into the exciting field of neuroendocrinology.
Author: Patrick J. Babin
Publisher: Springer Science & Business Media
ISBN: 1402062338
Category : Nature
Languages : en
Pages : 513
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Book Description
This book addresses the growing needs in deciphering the biological processes associated with fish reproduction, in view of the growth of aquaculture and the dwindling natural stocks of commercially important fish. It presents a comprehensive overview on egg production in fish, from the standpoint of the oocyte. With this view in mind, the book includes chapters on oocyte development (oogenesis), hormonal regulation and hormone receptors, formation of the egg envelopes, growth, accumulation of nutrients and maternal transcripts, maturation, hydration, ovulation and fertilization. A special emphasis is placed on using state-of-the-art tools including electron microscopy for discerning the ultra-structure of the follicle and genomic/proteomic tools to fully understand biological basis of fish reproduction. Studies on promoting oocyte maturation, ovulation and spawning in farmed fish and preservation of fish oocytes at low temperatures are also included. The book will appeal to University lecturers, students, research scientists and those associated with culture of fish in freshwater or marine aquaculture.
