The major aim of the experiments described in this thesis was to investigate the putative mechanisms that are involved in the control of B- endorphin (B-END) secretion into the peripheral blood of the Soay ram. In addition, the effect of 8-END and adrenocorticotrophin (ACTE1) on cortisol secretion was investigated. A series of experiments was carried out to assess the influence of season, photoperiod, melatonin implantation, pinealectomy, castration and testosterone replacement on B-END secretion. There were significant changes in the plasma concentrations of B-END related to season and photoperiod in rams kept outside and inside, respectively. In outdoor rams B-END levels were highest in autumn and lowest in winter; in indoor rams the levels were highest during short days and lowest during long days. Melatonin implantation in outdoor rams from May to August caused a significant increase in B-END secretion, indicating a melatonin-induced short-day effect inspite of the prevailing long days. Pinealectomy disrupted the seasonal cycle in B-END secretion. Castration and testostrone replacement in indoor rams did not influence B-END secretion. These results indicate that B-END secretion is strongly influenced by season and photoperiod (via melatonin from the pineal gland) and that testosterone plays no role in B-END secretion. In another series of experiments, the roles of arginine vasopressin (AVP), corticotrophin releasing factor (CRF), the synthetic glucocorticoid, dexamethasone (DEX) and the synthetic glucocorticoid antagonist, RU 486, in the control of the seasonal cycle in B-END secretion were investigated in spring, summer, autumn and winter. AVP and CRF given alone or in combination significantly stimulated 8-END secretion at all seasons and acted synergistically when given together. The responses were greater in summer and autumn than in winter and spring. DEX suppressed 8-END secretion at all seasons and the responses were also greater in summer and autumn. DEX also blocked the AVP-induced increase in 8-END secretion, indicating an action of DEX at the pituitary gland. RU 486 given in summer and winter significantly stimulated 8-END secretion only in winter, indicating a seasonal variation in the negative feedback action of endogenous glucocorticoids. In addition, ACTH, but not 8-END, significantly stimulated cortisol secretion at all seasons, with the greatest response in spring. These studies indicate that AVP, CRF and glucocorticoids are involved in the control of the seasonal cycle in 8-END secretion; and that ACTH rather than 8-END constitutes the " drive " to cortisol secretion. The roles of dopamine (DA) and endogenous opioid peptides (EOP) in the control of 8-END secretion were also investigated. The mixed DA antagonist, pimozide, significantly increased 8-END secretion under long and short days; with a greater effect under long days. The D2 agonist, bromocriptine, and the D2 antagonist, sulpiride, significantly decreased and increased, respectively, 8-END secretion both under long and short days. The opioid antagonist, naloxone, had no effect on 8-END secretion. These studies indicate that DA exerts an inhibitory control over 8-END secretion while the EOP play no role in 8-END secretion. Based on the current results and a survey of the relevant literature, a model is proposed in which AVP, CRF, glucocorticoids on one hand, and DA on the other hand, are involved in the control of the secretory activity of corticotrophs and melanotrophs in the pituitary gland. These central mechanisms are influenced by changes in photoperiod and other environmental factors to dictate the seasonal cycle in 6-END secretion in the Soay ram, which is low in winter and high in autumn. To fully assess the importance of AVP, CRF and DA in the seasonal control of 6-END secretion, it will be necessary to directly measure the concentration of these hormones in the hypophysial portal circulation of Soay rams at different seasons.

The post-denervation hypertrophy of the rat hemidiaphragm has been studied for up to 15 days after nerve section. The denervated tissue increases in wet weight to a maximum at 5 days of 40 % over its initial weight; by the 15th day the tissue has atrophied to below the control value. Measurements have been made throughout the hypertrophy period of the activities of some enzymes characteristic of particular aspects of muscle metabolism. In additions concentrations of glycogen from fed, and overnight fasted rats; of myoglobin; and of free amino acid concentrations in the denervated tissue have also been studied. Methods are described for the use in rats of the anaesthetic Halothane so as to preserve muscle glycogen concentrations and to prevent stress-mediated activation of phosphorylase a levels. Unilateral phrenicectomy causes a decrease in glycogen concentration and contents a decrease of total glycogen phosphorylase activity is also found, but total content of phosphoglucorautase increases slightly, Hexokinase and phosphorylase contents increase so that their concentrations are maintained. The validity of the hexokinase/ phosphorylase ratio as an indicator of fibre composition in pathological tissues is questioned. Phosphohexoisomerase activity remains constant for 1 week after denervation, then decreases, but glyceraldehyde phosphate dehydrogenase & lactate dehydrogenase demonstrate increased contents at 7 days before they decrease; the responses of the dehydrogenases may be related to the reported proliferation of the sarcoplasmic reticulum. No significant change is observed in the lactate dehydrogenase isoenzyme proportions until 7 days when a decrease of H-type subunits is indicated; 3 days later though control proportions are regained. Content of malate dehydrogenase, VAD-, & NADP-specific dehydro-genases demonstrate rapid decreases after nerve section to roughly half the initial levels at 3 days after nerve section. Glutamate dehydrogenase concentration also decreases in the early stages of the hypertrophy, but later increases when protein catabolism becomes a significant process. These decreases art in 'accord with the reported fragmentation of the mitochondria. Glucose G-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase concentrations increase dramatically to a peak within the first few days after nerve section. The increased enzyme capacity could be responsible for provision of nentose phosphates for nucleotide and nucleic acid synthesis. Total creatine kinase activity remains constant for up to 5 days before a decline in observed; adenylate kinase & adenylate deaminase show increased contents. The responses are interpreted in terms of possible involvement in increased adenine nucleotide production. The validity of the adenylate kinase/creatine kinase activity- ratio for the identification of physiologically distinct muscles in questioned. Content of glutathione reductase shows two small peaks of increased activity at 3 and 10 days after denervation. Thus unlike other denervated or dystrophic muscles, the responses o NADP-linked dehydrogenoses are not similar. Myoglobin content increases slowly during the hypertrophy only reaching a peak of 20 % over control levels at 10 days. Concentration in the early stages is thus decreased but rises after 5 days. The response, as well as the changes in lactate dehydrogenase isoenzyme proportions, is interpreted in terms of an increased blood flow through the denervated tissue. Total free amino acid concentration in the denervated tissue is increased; responses of the individual species are interpreted in terms of possible modes of metabolism in the tissue. The responses in the denervated hemidiaphragm are interpreted in terms of a hypertrophy of the "red" &/or "intermediate" fibre types; suggestions are made as to possible causes for the responses.

No description available.

No description available.

A sensitive and reproducible double antibody radioimmunoassay technique, requiring 50ul of unknown serum or plasma per assay tube, is described for use with 125I and rabbit anti-rat LH serum. The assay system was applied to the study of LH secretion in rats under both normal and experimentally manipulated conditions. Particular attention was focussed upon comparison of circulating LH levels in conscious, unstressed animals with those in anaesthetized animals, with or without surgical stress. Thereafter, the effects of acoustic stimulation and of exogenous LRH administration were studied in conscious and anaesthetized animals. Urethane anaesthesia exerted a profound effect upon the LR-secretory response to exogenous LRH in male rats. Available evidence suggests that the blood sampling method, surgical stress and anaesthesia are each capable of significantly influencing LH secretion, thereby emphasizing the value of studies using conscious, unstressed animals. While a direct effect of urethane on the pituitary gland cannot be excluded, attention is drawn to the possible mediation of a urethane-sensitive inhibitory influence in the mechanism controlling LH secretion in the rat.

No description available.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Tsoi Lo Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 99-109). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract in Chinese --- p.III / List of Abbreviations --- p.IV / List of Figures --- p.V / List of Tables --- p.VI / Table of Contents --- p.VII / Chapter Chapter 1 : --- Introduction / Chapter 1.1. --- Apolipoprotein and Lipoprotein Metabolism --- p.1 / Chapter 1.2. --- Molecular Information of ApoE --- p.2 / Chapter 1.3. --- Tissue Distribution of ApoE --- p.2 / Chapter 1.4. --- Functions of ApoE --- p.4 / Chapter 1.5. --- Genetic Polymorphism of ApoE --- p.7 / Chapter 1.6. --- Protein Structure and Characteristics of ApoE Isoforms --- p.9 / Chapter 1.7. --- Plasma and Cellular Expression Level of ApoE Isoforms --- p.12 / Chapter 1.8. --- Association between ApoE Isoforms and Plasma Lipid Profiles --- p.13 / Chapter 1.9. --- ApoE Polymorphisms and Pathophysiological Conditions / Chapter 1.9.1. --- Type III Hyperlipoproteinemia (Type III HLP) --- p.14 / Chapter 1.9.2. --- Alzheimer's Disease --- p.15 / Chapter 1.9.3. --- Atherosclerosis / Chapter 1.9.3.1. --- Atherosclerosis - An Inflammatory Process --- p.15 / Chapter 1.9.3.2. --- Role of ApoE in Atherosclerosis --- p.18 / Chapter (a) --- Functions Associated to Lipid Metabolism --- p.19 / Chapter (b) --- Functions Independent to Lipid Metabolism --- p.20 / Chapter 1.9.3.3. --- TNF-α and IL-6 in Atherosclerosis --- p.25 / Chapter 1.10. --- Macrophage Cytokine Expression and MAPKs / Chapter 1.10.1. --- Organization of MAPKs Signaling Pathway --- p.26 / Chapter 1.10.2. --- Lipopolysaccharide and MAPKs in Macrophage Cytokine Expression --- p.28 / Chapter 1.10.3. --- Regulation of Macrophage Cytokine Expression / Chapter 1.10.3.1. --- ERK1/2 and p38 MAPK Pathway --- p.30 / Chapter 1.10.3.2. --- Arachidonic Acid Metabolism --- p.30 / Chapter 1.11. --- Aim and Hypothesis --- p.31 / Chapter Chapter 2 : --- Materials and Methods / Materials / Chapter 2.1 --- Culture of ApoE-isoform-expressing J774A.1 Macrophage Cell Line --- p.32 / Chapter 2.2 --- RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.33 / Chapter 2.3 --- Protein Extraction and Quantification --- p.37 / Chapter 2.4 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.38 / Chapter 2.5 --- Western Blotting --- p.39 / Chapter 2.6 --- LPS Treatment --- p.42 / Chapter 2.7 --- MAPK Inhibitor Experiment --- p.43 / Methods / Chapter 2.8 --- Study on the Effect of Endogenously Expressed ApoE Isoforms on Macrophage Cytokine Secretion / Chapter 2.8.1. --- Establishment of ApoE-isoform-expressing Macrophages --- p.44 / Chapter 2.8.2. --- Semi-quantification of ApoE mRNA Level by RT-PCR / Chapter 1) --- Isolation of Total RNA --- p.45 / Chapter 2) --- RT-PCR --- p.46 / Chapter 2.8.3. --- Determination of ApoE Protein Expression Level by ELISA and Western Blot --- p.47 / Chapter 1) --- Quantification of Total Proteins --- p.48 / Chapter 2) --- ELISA --- p.48 / Chapter 3) --- Western Blot --- p.49 / Chapter 2.8.4. --- LPS Treatment --- p.51 / Chapter 2.8.5. --- MEK1/2 Inhibitor Experiment --- p.53 / Chapter 2.8.6. --- p38 Inhibitor Experiment --- p.54 / Chapter 2.9 --- Study on the Effect of Exogenous ApoE Isoform on Macrophage Cytokine Secretion --- p.55 / Chapter 2.10 --- Statistical Analysis --- p.55 / Chapter Chapter 3: --- Results / Changes of Inflammatory Properties Associated with Endogenous ApoE Isoform Expression in Macrophages / Chapter 3.1 --- Characterization of ApoE-isoform-expressing Macrophages --- p.56 / Chapter 3.1.1. --- Cell Lines with Stable Expression of ApoE Isoforms --- p.56 / Chapter 3.2 --- Cell Morphology Study --- p.58 / Chapter 3.3 --- Changes of IL-6 and TNF-α Secretion Associated with Endogenous ApoE Isoforms Expression / Chapter 3.3.1. --- In the Presence of Lipoproteins --- p.60 / Chapter 3.3.2. --- Serum/Lipoprotein-independent Effects of ApoE Isoforms --- p.63 / Chapter 3.4 --- The Effects of Endogenous ApoE Isoform Expression on the Activities of MAPK Signaling Pathways / Chapter 3.4.1. --- Study on the Activation Status and Expression of MAPKs --- p.66 / Chapter 1) --- ERK1/2 MAPK Pathway --- p.66 / Chapter 2) --- p38 MAPK Pathway --- p.69 / Chapter 3.4.2. --- IL-6 and TNF-a Secretion Among ApoE Isoforms in the Presence of MEK1/2 mhibitor --- p.72 / Chapter 3.4.3. --- IL-6 and TNF-α Secretion Among ApoE Isoforms in the Presence of p38 Inhibitor --- p.75 / Chapter Chapter 4 : --- Discussions / Chapter 4.1. --- Mouse Peritoneal Macrophage Cell Line J774A.1 as Cell Model --- p.79 / Chapter 4.2. --- Inflammatory Properties Associated with Endogenous ApoE Isoform Expression in Macrophages / Chapter 4.2.1. --- Expression Level of ApoE Isoform Transgenes in Mouse Peritoneal Macrophages --- p.80 / Chapter 4.2.2. --- Macrophage Activation by LPS --- p.81 / Chapter 4.2.3. --- Effect of Endogenous ApoE Isoform Expression on Cytokine Secretion and Signal Transduction in Macrophages --- p.82 / Chapter 4.3. --- Conclusions and Future Prospects / Chapter 4.3.1. --- Conclusions --- p.90 / Chapter 4.3.2. --- Future Prospects --- p.91 / Chapter Chapter 5 : --- Appendices / Chapter 5.1 --- Changes of Inflammatory Properties of Macrophages Supplemented with Exogenous ApoE Isoforms / Chapter 5.1.1. --- Changes of IL-6 and TNF-a Secretion in Macrophages Supplemented with Exogenous ApoE Isoforms --- p.92 / Chapter 5.1.2. --- Changes of Signal Transduction in Macrophages Supplemented with Exogenous ApoE Isoforms / Chapter 5.1.2.1. --- Study on the Activation Status and Expression of MAPKs / Chapter 1) --- ERK1/2 MAPK Pathway --- p.95 / Chapter 2) --- p38 MAPK Pathway --- p.97 / Chapter Chapter 6: --- Bibliography --- p.99

Les bactéries à Gram négatif sont caractérisées par une organisation complexe de leur enveloppe,impliquant une membrane interne (ou cytoplasmique), un espace périplasmique et une membrane externe. Si le transport de petits composés chimiques se fait facilement, la sécrétion des protéines et des toxines nécessite par contre l’utilisation de machineries spécialisées : les systèmes de sécrétion.Chez Pseudomonas aeriginosa, une bactérie pathogène opportuniste, le système de sécrétion de Type II, ou sécrétion Xcp, constitue l’une des voies principales de la sécrétion. Ce sécréton Xcp est un complexe macromoléculaire de 12 protéines, nommées XcpAO et XcpPC-ZM, organisé en trois sous-complexes : une plateforme d’assemblage ancrée dans la membrane interne (XcpPC-SF etXcpYL-ZM), un pore localisé dans la membrane externe et formé par multimérisation de la sécrétine XcpQD, et un pseudopilus périplasmique impliquant les pseudopilines XcpTG-XK. Au travers de son introduction bibliographique, ce manuscrit présente les différents constituants de cette machinerie et leur implication dans la sécrétion. Un grand nombre de copies du constituant majoritaire de ce système, XcpTG, s’assemble sous forme d’un pseudopilus dont les cycles d’assemblage –désassemblage, semblables aux mouvements d’un piston, pourraient permettre la sécrétion des substrats à travers la membrane externe. Le travail effectué au cours de cette thèse a pour but d’approfondir la compréhension des conditions d’assemblage du pseudopilus, qui s’avère être une étape cruciale dans la sécrétion. Les résultats obtenus s’articulent autour de la détermination par RMN hétéronucléaire de la structure de XcpTG, le composant majoritaire du pseudopilus etre présente le premier constituant de la machinerie de type II de P. aeruginosa à voir sa structure résolue par RMN. / Gram negative bacteria are characterized by a complex organisation of their cell envelope, with aninner membrane (or cytoplasmic membrane), a periplasmic space and an outer membrane. Incontrast to the transport of chemical compounds, which is preformed usually by porins localized inthe impermeable cell envelop, secretion of proteins and toxins requires specialized machineries: thesecretion systems. In Pseudomonas aeruginosa, an opportunistic pathogen, among the wide rangeof section systems, the Type II secretion system, called Xcp secreton, is a major pathway for therelease of virulence factors. This Xcp secreton is a macromolecular complex involving 12 proteinscalled XcpA0 and XcpPC to XcpZM. This machinery is organized in 3 complexes, the assemblyplatform anchored in the inner membrane (implicating XcpPC,RE,SF,YL and ZM), the pore localizedin the outer membrane and formed by multimerization of the secretin XcpQD, and the periplasmicpseudopilus involving XcpTG-XK matured by the prepilin peptidase XcpAO. The introduction of thismanuscript presents all the components of the type II secretion system and their principal functionin the secretion process. XcpTG, the major components of this system, seems to polymerize to allowtransfer of secretion products across the outer membrane by a piston-like process. The workpresented in this manuscript is underlined by the idea of improving the understanding of themecanism of the type II secretion. The results articulate around the heteronuclear NMR solutionstructure determination of the XcpTG, which represents the first structure obtained for a componentof the type II secretion system of P. aeruginosa.

The cell envelope of Aggregatibacter actinomycetemcomitans, a Gram-negative pathogenic bacterium implicated in human oral and systemic disease, plays a critical role in maintenance of cellular homeostasis, resistance to external stress, and host'pathogen interactions. Our laboratory has identified a novel gene product, morphogenesis protein C (MorC), deletion of which leads to multiple pleotropic effects pertaining to membrane structure and function. The MorC sequence was determined to be conserved in Gammaproteobacteria. Based on this bioinformatic analysis, the functional conservation of this protein was investigated utilizing an A. actinomycetemcomitans morC mutant as a model system to express homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. MorC from all organisms restored at least one of the A. actinomycetemcomitans mutant phenotypes, implying that the protein is functionally conserved across Gammaproteobacteria. Further, deletion mutagenesis indicated that the last 10 amino acids of the carboxyl terminus were necessary to maintain the integrity of the membrane. The observed pleiotropic effects suggested alterations in the membrane protein composition of the morC mutant. Stable isotope dimethyl labeling in conjunction with mass spectrometry was employed to quantitatively determine the differences in the abundance of membrane proteins of the isogenic mutant and wild-type strains. A total of 665 envelope associated proteins were identified and functionally annotated using bioinformatic tools. All proteins, except MorC, were detected in the mutant strain. However, 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain. These proteins were ascribed functions associated with protein quality control, oxidative stress response, and protein secretion systems. One protein found to be reduced was a component of the fimbrial secretion system of A. actinomycetemcomitans. The significance of this finding was unclear due to the afimbriated nature of the laboratory strain used in the study. Therefore, the defect in fimbriation was identified and complemented in trans. The transformed strain displayed all of the hallmarks of a naturally fimbriated strain including: a distinct star-like colony morphology; robust biofilm formation; and presence of fimbriae as detected by electron microscopy. The isogenic morC mutant strain transformed with an identical plasmid did not display any fimbriated phenotypes. The role of MorC in fimbriae production of a naturally fimbriated strain was investigated by inactivation of morC in a clinical isolate. The mutant strain displayed phenotypes typically associated with inactivation of morC. However, fimbriae were still observed on the surface, although in lesser amounts on some individual bacteria, and this strain formed a biofilm with volume similar to the parent. Interestingly, significant changes in microcolony architecture of the biofilm were observed by confocal microscopy. MorC plays a critical role in maintaining secretion of major virulence determinants of A. actinomycetemcomitans. Specific changes in the protein composition of the cell envelope indicate a direct or compensatory role of these proteins in maintaining membrane physiology. The functional conservation of MorC also implies an important role for this protein in other Gram-negative bacteria. This work suggests a role of MorC as an accessory or a scaffold protein involved in secretion.