by Wong Kwan Po, Gary. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 132-153). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Abbreviations --- p.vi / Abbrevation Table for Amino Acids --- p.ix / List of Figures --- p.x / List of Tables --- p.xiii / Table of Contents --- p.xiv / Chapter Chapte r One --- General Introduction --- p.1 / Chapter 1.1 --- Structures of PRL --- p.1 / Chapter 1.2 --- PRL receptor and its mechanism of action --- p.7 / Chapter 1.3 --- Biosynthesis of PRL --- p.11 / Chapter 1.4 --- Biological functions of PRL --- p.13 / Chapter 1.5 --- Organization and regulation of PRL gene --- p.16 / Chapter 1.6 --- Aims of this study --- p.25 / Chapter Chapter Two --- PCR Cloning of gfPRL Gene --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Buffers and Reagents --- p.27 / Chapter 2.2.2 --- Methods --- p.30 / Chapter 2.2.2.1 --- PCR of the 5'-flanking region of gfPRL gene --- p.30 / Chapter 2.2.2.2 --- Genomic PCR of gfPRL gene --- p.31 / Chapter 2.2.2.3 --- Spectrophotometric quantification and qualification of DNA and RNA --- p.31 / Chapter 2.2.2.4 --- Agarose gel electrophoresis of DNA --- p.31 / Chapter 2.2.2.5 --- DNA radioactive labeling by random priming --- p.32 / Chapter 2.2.2.6 --- Vacuum transfer of DNA fragments to a nylon membrane --- p.32 / Chapter 2.2.2.7 --- Southern blot analysis --- p.33 / Chapter 2.2.2.8 --- Molecular Imager Analysis --- p.33 / Chapter 2.2.2.8 --- Phosphorylation of PCR amplified DNA --- p.34 / Chapter 2.2.2.9 --- Ligation of DNA fragment to linearized vector --- p.34 / Chapter 2.2.2.10 --- Preparation of Escherichia coli competent cells --- p.34 / Chapter 2.2.2.11 --- Bacterial transformation by heat stock --- p.35 / Chapter 2.2.2.12 --- Automated PCR sequencing with Sequencing Ready Reaction Kit --- p.35 / Chapter 2.2.2.13 --- Primer extension using reverse transcription --- p.36 / Chapter 2.3 --- Results --- p.38 / Chapter 2.3.1 --- Cloning of the 5'-flanking region of gfPRL gene --- p.38 / Chapter 2.3.2 --- PCR cloning of gfPRL gene --- p.43 / Chapter 2.3.3 --- Identification of the transcription initiation site --- p.47 / Chapter 2.4 --- Discussion --- p.51 / Chapter 2.4.1 --- Sequence analysis of the gfPRL gene --- p.51 / Chapter 2.4.2 --- Analysis of the exon-intron boundaries --- p.53 / Chapter 2.4.3 --- Analysis of the 5'flanking region of gfPRL gene --- p.53 / Chapter 2.4.4 --- Identification of the transcription initiation site --- p.54 / Chapter 2.5 --- Conclusion --- p.54 / Chapter Chapter Three --- Promoter Analysis of the gfPRL Gene --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Preparation of Luciferase reporter constructs --- p.56 / Chapter 3.2.2 --- Preparation of frozen stock of culture cells --- p.56 / Chapter 3.2.3. --- Cell culture --- p.56 / Chapter 3.2.4 --- Transfection of mammalian cells for transient gene expression study --- p.57 / Chapter 3.2.5 --- Luciferase assay --- p.57 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- Tissue-specific transcription of gfPRL promoter --- p.58 / Chapter 3.3.2 --- Identification of regulatory regions of gfPRL gene promoter --- p.61 / Chapter 3.3.3 --- Inhibitory effect of DA on gfPRL promoter transcription activity --- p.63 / Chapter 3.3.4 --- GfPRL promoter sequences that specifically confer negative regulation by DA --- p.65 / Chapter 3.3.5 --- The action of TRH on gfPRL promoter --- p.67 / Chapter 3.3.6 --- Investigation of gfPRL promoter sequence responsiveness towards TRH --- p.69 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Tissue-specific transcription of gfPRL promoter --- p.71 / Chapter 3.4.2 --- Identification of regulatory regions of goldfish prolactin gene promoter --- p.72 / Chapter 3.4.3 --- Dopamine inhibits gfPRL promoter activity --- p.73 / Chapter 3.4.4 --- TRH action on gfPRL promoter --- p.76 / Chapter 3.5 --- Conclusion --- p.78 / Chapter Chapter Four --- Seasonal Study on gfPRL and gfGH expression --- p.80 / Chapter 4.1 --- Introduction --- p.80 / Chapter 4.2 --- Materials and Methods --- p.81 / Chapter 4.2.1 --- Blood samples and radioimmunoassay --- p.81 / Chapter 4.2.2 --- Preparation of ribonuclease free reagents and apparatus --- p.81 / Chapter 4.2.3 --- Isolation of total RNA --- p.81 / Chapter 4.2.4 --- Formaldehyde agarose gel electrophoresis of RNA --- p.82 / Chapter 4.2.5 --- First strand cDNA synthesis --- p.82 / Chapter 4.2.6 --- RT-PCR --- p.83 / Chapter 4.2.7 --- Analysis of RT-PCR --- p.86 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Tissue-specific expression of gfPRL transcript --- p.88 / Chapter 4.3.2 --- Sexual maturity of goldfish throughout the reproductive cycle --- p.90 / Chapter 4.3.3 --- Serum gfGH levels throughout the year of 2000 --- p.91 / Chapter 4.3.4 --- Serum gfPRL levels throughout the year of 2000 --- p.92 / Chapter 4.3.5 --- The variation of gfGHR and gfPRLR mRNA in the brain throughout the reproductive cycle --- p.93 / Chapter 4.3.6 --- The variation of gfGHR mRNA in the liver throughout the reproductive cycle --- p.94 / Chapter 4.3.7 --- The variation of gfGHR and gfPRLR mRNA in the kidney throughout the reproductive cycle --- p.95 / Chapter 4.3.8 --- The variation of gfGHR and gfPRLR mRNA in the gonads throughout the reproductive cycle --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.4.1 --- Tissue-specific expression of gfPRL transcript --- p.98 / Chapter 4.4.2 --- Sexual maturity of goldfish throughout the reproductive cycle --- p.98 / Chapter 4.4.3 --- Serum gfGH and gfPRL level throughout the reproductive cycle --- p.99 / Chapter 4.4.4 --- The variation of gfGHR and gfPRLR mRNA in the brain throughout the reproductive cycle --- p.100 / Chapter 4.4.5 --- The variation of gfGHR mRNA in the liver throughout ´Øthe reproductive cycle --- p.101 / Chapter 4.4.6 --- The variation of gfGHR and gfPRLR mRNA in the kidney throughout the reproductive cycle --- p.102 / Chapter 4.4.7 --- The variation of gfGHR and gfPRLR mRNA in the gonads throughout the reproductive cycle --- p.102 / Chapter 4.5 --- Conclusion --- p.105 / Chapter Chapter Five --- Recombinant gfPRL Production --- p.106 / Chapter 5.1 --- Introduction --- p.106 / Chapter 5.2 --- Materials and Methods --- p.108 / Chapter 5.2.1 --- Buffers and Reagents --- p.108 / Chapter 5.2.2 --- Methods --- p.112 / Chapter 5.2.2.1 --- Recombinant protein expression --- p.112 / Chapter 5.2.2.2. --- Purification of the recombinant protein by XpressTM System Protein Purification (Invitrogen) --- p.112 / Chapter 5.2.2.3 --- SDS-PAGE preparation --- p.112 / Chapter 5.2.2.4 --- SDS-PAGE analysis of proteins --- p.113 / Chapter 5.2.2.5 --- Western blot analysis --- p.114 / Chapter 5.2.2.6 --- Protein refolding --- p.114 / Chapter 5.2.2.7 --- Alkaline Extraction --- p.115 / Chapter 5.2.2.8 --- Size Exclusion Chromatography --- p.115 / Chapter 5.2.2.9 --- ELISA analysis of the fractions --- p.115 / Chapter 5.2.2.10 --- Anion Exchange Chromatography --- p.116 / Chapter 5.3 --- Results --- p.117 / Chapter 5.3.1 --- Prokaryotic expression of recombinant gfPRL --- p.117 / Chapter 5.3.2 --- "Purification of reombinant gfPRL: SDS-PAGE, western blot and BCA analysis of purified recombinant gfPRL" --- p.119 / Chapter 5.3.3 --- Purification of native gfPRL and gfGH: Native hormone purification by size exclusion chromatography --- p.119 / Chapter 5.3.4 --- Native gfPRL purification by anion exchange chromatography --- p.122 / Chapter 5.3.5 --- Study the biological activity of refolded recombinant gfPRL --- p.126 / Chapter 5.4 --- Discussion --- p.127 / Chapter 5.4.1 --- Prokaryotic expression of recombinant gfPRL --- p.127 / Chapter 5.4.2 --- Purification of recombinant gfPRL --- p.128 / Chapter 5.4.3 --- Refolding of recombinant gfPRL --- p.129 / Chapter 5.4.4 --- Purification of native gfPRL --- p.130 / Chapter 5.4.5 --- Study the biological activity of recombinant gfPRL --- p.130 / Chapter 5.5 --- Conclusion --- p.131 / References --- p.132

In an attempt to elucidate the mechanism by which ethanol affects vertebrate thermoregulation, the effect of ethanol on temperature selection was studied in the goldfish, Carassius auratus. Ethanol was administered to 10 to 15 g fish by mixing it in the water of a temperature gradient. The dose response curve was very steep between 0.5% (v/v) ethanol (no response) and 0.7% (significant lowering of selected temperature in treated fish). Fish were exposed to concentrations of ethanol as high as 1.7%, at which concentration most experimental fish lost their ability to swim upright in the water. At concentrations higher than 0.7%, the magnitude of the effect did not increase with increasing concentration of ethanol; treated animals continued to select temperatures about 2 C below temperatures selected by controls. Experiments alternating exposure to 1.0% ethanol and water showed that the rate of onset and disappearance of the ethanol effect was rapid (within 10 min). Other experiments exposing fish to 1.0% ethanol for up to 3 hr showed that the effect remained stable for this period of time. The thermoregulatory responses of fish are behavioral, and therefore relatively easy to observe and quantify. Ethanol produces a prompt, stable and reproducible depression of selected temperature in the goldfish. Because the temperature at which fish regulate is controlled by a central nervous system set point and not altered by effects on peripheral effector systems, it appears that ethanol may cause hypothermia in goldfish by directly acting to lower the set point.

Thyrotropin (TSH) is a glycoprotein hormone released from the pituitary gland to promote the synthesis and secretion of thyroid hormone. The existence of well-established peripheral mechanisms for regulation of thyroid hormone delivery to targets has called into question the significance of TSH as a primary regulator of circulating thyroid hormone concentrations in fish. However, relatively little is known about the regulation or action of endogenously secreted teleost TSH, largely due to lack of purified TSH suitable for biological testing and immunoassay development. I developed a red drum in vivo bioassay to aid in the production and purification of recombinant TSH from the red drum, a perciform fish demonstrating dynamic daily thyroxine (T4) cycles hypothesized to be driven by TSH. Exogenous bovine TSH injection resulted in a time and dose-dependent increase in circulating TSH and T4 in red drum. However, the sensitivity of the red drum thyroid gland to stimulation by bovine TSH was lost during growth under controlled laboratory conditions, even when circulating levels of exogenously-administered mammalian TSH remained elevated. The insensitivity of the thyroid was not due to prior TSH injection or feed source. Because insensitivity of the Thyrotropin (TSH) is a glycoprotein hormone released from the pituitary gland to promote the synthesis and secretion of thyroid hormone. The existence of well-established peripheral mechanisms for regulation of thyroid hormone delivery to targets has called into question the significance of TSH as a primary regulator of circulating thyroid hormone concentrations in fish. However, relatively little is known about the regulation or action of endogenously secreted teleost TSH, largely due to lack of purified TSH suitable for biological testing and immunoassay development. I developed a red drum in vivo bioassay to aid in the production and purification of recombinant TSH from the red drum, a perciform fish demonstrating dynamic daily thyroxine (T4) cycles hypothesized to be driven by TSH. Exogenous bovine TSH injection resulted in a time and dose-dependent increase in circulating TSH and T4 in red drum. However, the sensitivity of the red drum thyroid gland to stimulation by bovine TSH was lost during growth under controlled laboratory conditions, even when circulating levels of exogenously-administered mammalian TSH remained elevated. The insensitivity of the thyroid was not due to prior TSH injection or feed source. Because insensitivity of the red drum thyroid precluded their use as a bioassay species, the plasma TSH and T4 response to exogenous TSH was next characterized in goldfish. The T4 response in goldfish was stable and repeatable, with T4 levels peaking at 5 hours and remaining elevated for more than 11 hours after bovine TSH injection. Plasma TSH peaked from 2-5 hours following TSH injection with more than 90 percent cleared by 11 hours. The goldfish bioassay was further utilized to evaluate the effects of structural modifications on TSH biological activity. Substitution of four positively charged amino acids at the n-recombinant human TSH, had the same effect in goldfish. The heterothyrotropic potency of mammalian follicle stimulating hormone in goldfish was also enhanced by the same amino acid substitutions. Finally, the importance of oligosaccharides to TSH bioactivity was also examined in goldfish. Deglycosylation abolished TSH bioactivity, even when immunoreactivity persisted in circulation. Furthermore, recombinant canine TSH was less potent when produced in cell lines generating insect-type glycosylation than when produced in a cell line capable of mammalian-type glycosylation. These studies utilizing recombinant mammalian demonstrated conservation of mammalian TSH hormone-receptor interactions in goldfish, suggesting TSH function might likewise be conserved. Thus, I have established goldfish as a sensitive and stable bioassay which can now be utilized to monitor the biological activity of teleost TSH expressed in vitro as well as to evaluate how structural modifications of the TSH molecule influence its vivo biological activity.

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

No description available.

Goldfish subjected to an intermittent short-term aerobic exercise training regime prior to acute hypoxic exposure demonstrated a shift in hypoxia response. Intermittent aerobic training enhanced the aerobic potential of goldfish in the red muscle by increasing maximal activity of citrate synthase by 72% and reduced pyruvate kinase activity by 21% in white muscle. Across red and white muscle tissue, aerobic training caused a decrease in glycogen storage by 19% and 32%, respectively. Liver glycogen stores remained unchanged by training during normoxia. Subsequent hypoxic exposure demonstrated a significant training effect with a77% glycogen depletion in the liver of trained fish compared to a 53% depletion in untrained fish. Hypoxia caused glycogen depletion, glucose mobilization, and ATP depletion in trained and untrained fish muscle tissue. Meanwhile, the liver of trained recovered ATP slower than untrained fish and both liver and plasma had greater lactate accumulation by 1 h hypoxic recovery in trained fish. Alcohol dehydrogenase maximal activity of trained fish responded to hypoxia with a 50% reduction and trained white muscle significantly reduced alcohol dehydrogenase activity during hypoxic recovery. Ethanol was produced with and without training preconditioning in response to hypoxia in red muscle; however, trained fish white muscle showed an ethanol accumulation trend following training and 12 h hypoxia that was significantly cleared during recovery. Ethanol accumulation in white muscle of trained fish may reveal greater perturbation caused by training and hypoxia and/or some developed mechanism for ethanol retention. In effect, this training regime created a very different metabolic profile in goldfish such that during environmental oxygen limitation, trained fish may experience an enhanced metabolic perturbation and greater glycogen depletion which may compromise hypoxic tolerance. / Thesis (Master, Biology) -- Queen's University, 2011-09-30 13:25:36.148

No description available.