Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

No description available.

by Cheung Yeuk Siu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 141-153). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 摘要 --- p.iv / List of abbreviations --- p.v / Table of contents --- p.viii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Prolactin (PRL) --- p.1 / Chapter 1.1.1 --- General introduction --- p.1 / Chapter 1.1.2 --- Genomic organization of teleost PRL gene --- p.2 / Chapter 1.1.3 --- Conserved domains of fish PRL --- p.3 / Chapter 1.1.4 --- Structure of teleost PRL --- p.6 / Chapter 1.1.5 --- Tissue sources of PRL --- p.8 / Chapter 1.2 --- Prolactin receptor (PRLR) --- p.9 / Chapter 1.2.1 --- Tissue distribution in teleosts --- p.9 / Chapter 1.2.2 --- Receptor structure and multiple forms of PRLR --- p.11 / Chapter 1.2.3 --- Possible action mechanisms of PRL --- p.14 / Chapter 1.3 --- Control of PRL release --- p.17 / Chapter 1.4 --- Biological functions of PRL in vertebrates --- p.19 / Chapter 1.4.1 --- Biological effects on teleosts --- p.19 / Chapter 1.4.1.1 --- Osmoregulatory roles --- p.20 / Chapter 1.4.1.2 --- Non-osmoregulatory roles --- p.29 / Chapter 1.5 --- Aim of the present study --- p.32 / Chapter Chapter 2 --- "Recombinant goldfish (Carassius auratus) prolactin: subcloning, expression, purification and refolding of the recombinant protein" / Chapter 2.1 --- Introduction --- p.35 / Chapter 2.2 --- Materials --- p.38 / Chapter 2.3 --- Methods --- p.48 / Chapter 2.3.1 --- Subcloning of the gfPRL cDNA --- p.48 / Chapter 2.3.1.1 --- PCR cloning of gfPRL cDNA --- p.48 / Chapter 2.3.1.2 --- DNA sequencing of the subcloned fragment --- p.51 / Chapter 2.3.1.3 --- Subcloning of the gfPRL cDNA fragment into the expression vector --- p.52 / Chapter 2.3.2 --- Expression and purification of rgfPRL --- p.53 / Chapter 2.3.2.1 --- Transformation of pRSETA/gfPRL into BL21(DE3)pLysS cells --- p.53 / Chapter 2.3.2.2 --- Prokaryotic expression of rgfPRL --- p.53 / Chapter 2.3.2.3 --- Affinity purification of rgfPRL --- p.55 / Chapter 2.3.2.4 --- Western blot analysis of the purified rgfPRL --- p.57 / Chapter 2.3.2.5 --- Protein concentration determination of the rgfPRL --- p.59 / Chapter 2.3.3 --- Protein refolding --- p.60 / Chapter 2.4 --- Results --- p.61 / Chapter 2.4.1 --- Subcloning and DNA sequencing of the gfPRL --- p.61 / Chapter 2.4.2 --- Expression and purification of rgfPRL --- p.63 / Chapter 2.4.2.1 --- Prokaryotic expression of rgfPRL --- p.63 / Chapter 2.4.2.2 --- Affinity purification of rgfPRL --- p.66 / Chapter 2.4.2.3 --- Western blot analysis of the purified rgfPRL --- p.68 / Chapter 2.4.2.4 --- Protein concentration determination of the rgfPRL --- p.68 / Chapter 2.4.3 --- Protein refolding --- p.70 / Chapter 2.5 --- Discussion --- p.72 / Chapter Chapter 3 --- Production of polyclonal antibodies against rgfRPL / Chapter 3.1 --- Introduction --- p.81 / Chapter 3.2 --- Materials --- p.82 / Chapter 3.3 --- Methods --- p.84 / Chapter 3.3.1 --- Immunization of rabbits --- p.84 / Chapter 3.3.2 --- Collection of the polyclonal antisera --- p.85 / Chapter 3.3.3 --- Purification of IgG from the polyclonal antisera --- p.86 / Chapter 3.3.4 --- Enzyme linked immunosorbent assay (ELISA) --- p.87 / Chapter 3.3.5 --- Western blot analysis for cross-reactivity --- p.88 / Chapter 3.4 --- Results --- p.90 / Chapter 3.4.1 --- Isolation and purification of IgG from the polyclonal antisera --- p.90 / Chapter 3.4.2 --- ELISA --- p.93 / Chapter 3.4.3 --- Western blot analysis for cross-reactivity --- p.96 / Chapter 3.5 --- Discussion --- p.98 / Chapter Chapter 4 --- Isolation of native PRL from goldfish pituitaries / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Materials --- p.101 / Chapter 4.3 --- Methods --- p.103 / Chapter 4.3.1 --- Alkaline extraction --- p.103 / Chapter 4.3.2 --- Size exclusion chromatography --- p.104 / Chapter 4.3.3 --- Anion exchange chromatography --- p.104 / Chapter 4.4 --- Results --- p.106 / Chapter 4.4.1 --- Size exclusion chromatography --- p.106 / Chapter 4.4.2 --- Anion exchange chromatography --- p.109 / Chapter 4.4.3 --- SDS-PAGE analysis and immuno-detection of the purified protein --- p.112 / Chapter 4.5 --- Discussion --- p.114 / Chapter Chapter 5 --- Receptor binding assays / Chapter 5.1 --- Introduction --- p.115 / Chapter 5.2 --- Materials --- p.117 / Chapter 5.3 --- Methods --- p.119 / Chapter 5.3.1 --- Gill membrane preparation --- p.119 / Chapter 5.3.2 --- Radioactive labelling of the primary ligand --- p.120 / Chapter 5.3.3 --- Determination of the percentage of 125I incorporation and specific radioactivity of the radioligand --- p.121 / Chapter 5.3.4 --- Membrane protein dependence assay --- p.122 / Chapter 5.3.5 --- Receptor binding study using rgfPRL --- p.124 / Chapter 5.4 --- Results --- p.125 / Chapter 5.4.1 --- Radioactive labelling of the primary ligand --- p.125 / Chapter 5.4.2 --- Determination of the percentage of 125I incorporation and specific radioactivity of the radioligand --- p.127 / Chapter 5.4.3 --- Membrane protein dependence assay --- p.129 / Chapter 5.4.4 --- Receptor binding study using rgfPRL --- p.131 / Chapter 5.5 --- Discussion --- p.133 / Chapter Chapter 6 --- General discussion and conclusion --- p.136 / References --- p.141

published_or_final_version / Zoology / Master / Master of Philosophy

A Prolactina (PRL) é um dos hormônios mais versáteis em termos de ação biológica. Sua ação mais conhecida está relacionada com o estímulo da lactação e regulação do crescimento e da diferenciação da glândula mamária; também apresenta importante aplicação diagnóstica. Somando os crescentes estudos sobre suas possíveis aplicações terapêuticas, fica cada vez mais notória a necessidade da obtenção desse hormônio puro, biologicamente ativo e na sua forma autêntica.O objetivo fundamental desse projeto foi a produção de hPRL em escala laboratorial a partir de bactérias (E.coli) modificadas geneticamente, utilizando um sistema de expressão baseado no promotor Lambda () PL, o mesmo utilizado com sucesso em nosso laboratório na expressão do hGH. Descrevemos nesse trabalho um processo de cultivo em biorreator, onde não foi utilizado o repressor cIts, uma proteína termo-sensível que usualmente é utilizada para inibir o funcionamento do promotor PL durante crescimento a 30ºC. O processo de cultivo apresenta basicamente três etapas: na primeira etapa o crescimento é realizado sem adição contínua de nutrientes (cultivo em batch), na segunda etapa ocorre adição contínua de nutrientes e carboidrato (cultivo em fed-batch) e na última etapa é realizada a ativação, caracterizada pelo aumento da temperatura mantendo-se a adição de nutrientes e carboidrato. Esse processo de fermentação rápido e flexível, com duração média de 20 horas, permitiu obter uma biomassa final correspondente à densidade óptica de aproximadamente 30 A600nm (unidades ópticas de absorbância em 600nm) e com uma expressão da ordem de 1g de hPRL mL-1 A600 -1, as mais altas já relatadas para secreção de prolactina no espaço periplásmico. A hPRL monomérica foi purificada e caracterizada por métodos físico-químicos e biológicos, os quais confirmaram a sua atividade biológica e imunológica, o seu correto processamento e uma massa molecular relativa (Mr) de 22.906. / Prolactin (PRL) is one of the most versatile hormones in terms of biological action. His best known action is related to the stimulation of lactation and regulation of growth and differentiation of the mammary gland; it also has wide important diagnostic applications. Considering all the increasing studies on its potential therapeutic applications, the need for obtaining this hormone in its pure, biologically active and authentic form becomes clearer and clearer. The fundamental objective of this project was the production of hPRL on the laboratory scale, from genetically modified bacteria (E.coli), using an expression system based on Lambda () PL promoter, the same successfully used in our laboratory for the expression of hGH. We set up a cultivation process in bioreactor, where the repressor (cIts), a thermo-sensitive protein that is usually used to inhibit the PL promoter during the growth phase (30°C). The cultivation process presents basically three stages: the first step in was not used the growth is carried out without the continuous addition of nutrients (batch cultivation), the second step in which a continuous addition of nutrients and carbohydrate occurs (fed-batch cultivation) and a final step when activation is carried out. The latter is characterized by an increased temperature, still maintaining the addition of nutrients and carbohydrate. This fast and flexible process of fermentation, with the average duration of 20 hours, led to a final biomass of approximately 30 A600nm (units of optical absorbance at 600nm), with the expression of about 1g of hPRL mL-1A600 -1, the highest ever reported for the secretion of prolactin in the periplasmic space. Monomeric hPRL was purified and characterized by physical-chemical methods and biological assays, which confirmed its biological and immunological activity, correct processing and a relative molecular mass (Mr) of 22,906.

A Prolactina (PRL) é um dos hormônios mais versáteis em termos de ação biológica. Sua ação mais conhecida está relacionada com o estímulo da lactação e regulação do crescimento e da diferenciação da glândula mamária; também apresenta importante aplicação diagnóstica. Somando os crescentes estudos sobre suas possíveis aplicações terapêuticas, fica cada vez mais notória a necessidade da obtenção desse hormônio puro, biologicamente ativo e na sua forma autêntica.O objetivo fundamental desse projeto foi a produção de hPRL em escala laboratorial a partir de bactérias (E.coli) modificadas geneticamente, utilizando um sistema de expressão baseado no promotor Lambda () PL, o mesmo utilizado com sucesso em nosso laboratório na expressão do hGH. Descrevemos nesse trabalho um processo de cultivo em biorreator, onde não foi utilizado o repressor cIts, uma proteína termo-sensível que usualmente é utilizada para inibir o funcionamento do promotor PL durante crescimento a 30ºC. O processo de cultivo apresenta basicamente três etapas: na primeira etapa o crescimento é realizado sem adição contínua de nutrientes (cultivo em batch), na segunda etapa ocorre adição contínua de nutrientes e carboidrato (cultivo em fed-batch) e na última etapa é realizada a ativação, caracterizada pelo aumento da temperatura mantendo-se a adição de nutrientes e carboidrato. Esse processo de fermentação rápido e flexível, com duração média de 20 horas, permitiu obter uma biomassa final correspondente à densidade óptica de aproximadamente 30 A600nm (unidades ópticas de absorbância em 600nm) e com uma expressão da ordem de 1g de hPRL mL-1 A600 -1, as mais altas já relatadas para secreção de prolactina no espaço periplásmico. A hPRL monomérica foi purificada e caracterizada por métodos físico-químicos e biológicos, os quais confirmaram a sua atividade biológica e imunológica, o seu correto processamento e uma massa molecular relativa (Mr) de 22.906. / Prolactin (PRL) is one of the most versatile hormones in terms of biological action. His best known action is related to the stimulation of lactation and regulation of growth and differentiation of the mammary gland; it also has wide important diagnostic applications. Considering all the increasing studies on its potential therapeutic applications, the need for obtaining this hormone in its pure, biologically active and authentic form becomes clearer and clearer. The fundamental objective of this project was the production of hPRL on the laboratory scale, from genetically modified bacteria (E.coli), using an expression system based on Lambda () PL promoter, the same successfully used in our laboratory for the expression of hGH. We set up a cultivation process in bioreactor, where the repressor (cIts), a thermo-sensitive protein that is usually used to inhibit the PL promoter during the growth phase (30°C). The cultivation process presents basically three stages: the first step in was not used the growth is carried out without the continuous addition of nutrients (batch cultivation), the second step in which a continuous addition of nutrients and carbohydrate occurs (fed-batch cultivation) and a final step when activation is carried out. The latter is characterized by an increased temperature, still maintaining the addition of nutrients and carbohydrate. This fast and flexible process of fermentation, with the average duration of 20 hours, led to a final biomass of approximately 30 A600nm (units of optical absorbance at 600nm), with the expression of about 1g of hPRL mL-1A600 -1, the highest ever reported for the secretion of prolactin in the periplasmic space. Monomeric hPRL was purified and characterized by physical-chemical methods and biological assays, which confirmed its biological and immunological activity, correct processing and a relative molecular mass (Mr) of 22,906.

No description available.

The physiological effects of the pineal gland on the prolactin cells of the adenohypophysis were examined in short photoperiod-exposed male hamsters, as well as in blinded male and female hamsters. Pituitary storage of prolactin was assessed by monitoring radioimmunoassayable prolactin levels in the pituitaries in vivo and the total amount of immunoreactive prolactin in vitro. The effects of the pineal on prolactin secretion were estimated by measuring immunoassayable prolactin titers in the serum. Prolactin synthesis was measured by the ability of anterior pituitaries to incorporate ³H-leucine into prolactin in vitro. Finally, the effects of blinding and the activated pineal on hypothalamic hypophysiotrophic activity was assessed by incubating pituitaries in the presence of neutralized, acidic extracts of the stalk-medium eminence (SME) region of the mediobasal hypothalamus. In the male hamster, the pineal gland inhibits PRL cell function which encompasses reductions in the synthesis, storage and release of prolactin. The depressions in prolactin release and in pituitary storage are evident as early as three weeks after males are deprived of light. However, the inhibitory influence of the pineal on prolactin synthesis may be only partially apparent by eight to nine weeks after male hamsters are deprived of light, and is not fully evident until 12 weeks of light restriction. In the blinded female hamster, the synthesis, storage and release of prolactin are also markedly suppressed. However, all aspects of prolactin cell inhibition in the female may not be pineal-mediated. Furthermore, it appears that there may be some direct hypothalamic mechanism by which orbital enucleation inhibits prolactin cell function that is independent of the pineal gland. Based on the data presented in this dissertation, it is concluded that the SME region of the female hamster contains inhibitory activity which may be specifically responsible for the inhibition of prolactin synthesis. Furthermore, blinding and the pineal gland may independently exert rather specific influences upon hypophysiotrophic activity within the SME region of the female hamster.

In early pregnancy prolonged exposure to stress is known to have profound adverse effects on reproduction and is associated with suppressed progesterone secretion and consequent disturbance of the pregnancy-protective cytokine milieu, thus threatening early pregnancy maintenance. Maternal neuroendocrine responses to stress in early pregnancy are poorly understood. Therefore, we designed experiments to (1) study the hypothalamo-pituitary-adrenal (HPA) axis responses to stress in early pregnant mice, to discover whether and how responses change and (2) to determine the effect of stress in early gestation on pregnancy hormones, with a particular focus on the secretion and regulation of prolactin. To establish the effects of stress in early pregnancy (day 5.5) two different ethologically relevant stressors were used: lipopolysaccharide (LPS) or 24h fast stress, to mimic situations that may potentially arise during pregnancy in women: infection or hunger. HPA axis secretory responses to immune stress in early-mid pregnancy were robust and comparable to that in virgins. Vasopressin rather than the usual CRH neurone responses play a key role in maintaining this. However, the mode of action of glucocorticoids in mediating pregnancy complications is not yet established. Prolactin, and its hypothalamic control mechanisms, is a key candidate to mediate brain-to-body responses to stress. Prolactin has important roles in progesterone secretion, pregnancy establishment and immune regulation. We hypothesised that stress would negatively affect prolactin and its neuroendocrine control systems. Prolactin is mainly under the inhibitory control of dopamine, released predominantly from the tuberoinfundibular dopamine (TIDA) neurones. Prolactin also negatively feeds back on itself via prolactin receptors on the TIDA neurones and janus kinase (JAK)2/signal transducer and activator of transcription (STAT)5 signalling. Both immune and fasting stressors strongly inhibited basal prolactin secretion in early pregnancy, accompanied by a mild increase in activation of TIDA as shown by elevated Fos expression, compared to virgins. In addition, pregnancy attenuated LPS-induced recruitment of parvocellular paraventricular nucleus neurones and increased activation of brainstem noradrenergic nuclei which could potentially contribute to altered control of the dopamine-prolactin system. Following either immune or fast stress in early pregnancy ovine prolactin was able to drive enhanced expression of phosphorylated (p)STAT5. However, stress alone did not alter pSTAT5 implying it is not exclusively responsible for the stress-reduced prolactin observed in early pregnancy and another stress-induced stimulus must be activating TIDA neurones in these mice. LPS did not alter dopamine activity the median eminence (DOPAC: dopamine ratio) suggesting dopamine does not underlie stress-reduced prolactin secretion and other mechanisms must be considered. Direct effects of LPS, or its associated cytokines, on pituitary lactotrophs to inhibit prolactin secretion is a possible candidate. To investigate the effect of proinflammatory cytokines on the prolactin system in early pregnancy, d5.5 mice were administered TNF-alpha (a) or interleukin (IL)-6. Both cytokines increased TIDA activation, however, only TNF-a decreased plasma prolactin and progesterone, suggesting additional TNF-alpha action at the pituitary. As prolactin is anxiolytic we further proposed that stress would have a more profound effect on elevated plus maze performance in pregnant mice. However, early pregnant mice were generally more anxious vs. virgins regardless of LPS treatment. Taken together data show that stress in early pregnancy reduces prolactin and progesterone secretion, contributing to pregnancy complications/failure, but the neuroendocrine stress-related mechanism behind this suppression is yet to be determined.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy