Branchial carbonic anhydrase was purified from silver seabream (Sparus sarba) and antibody against the enzyme was obtained by immunization in rabbits. An assay for quantifying the activity of carbonic anhydrase was developed. Using enzymatic and immunological techniques, the activity, expression and distribution of branchial carbonic anhydrase of silver seabream acclimated to different salinities were studied. Fish gill is one of the most important organs involved in various homeostatic processes. The ability of euryhaline fish to maintain constant internal ionic balance is crucial for the survival of the fish upon change in salinity. The presence of carbonic anhydrase in the chloride cells was suggested to be an important enzyme involved in ion regulation of fish. / In the present study, branchial carbonic anhydrase and erythrocyte carbonic anhydrase were purified from the gill cells of silver seabream with p-aminomethylbenzenesulfonamide-agarose affinity column. They were predominantly cytosolic with a molecular size of 26.6 k Da for branchial carbonic anhydrase and 28.6 k Da for erythrocyte carbonic anhydrase. Investigation of kinetic properties towards the inhibitor acetazolamide has helped determine the inhibition constants (Ki of branchial carbonic anhydrase: 0.54 x 10-9; Ki of erythrocyte carbonic anhydrase: 0.22 x 10-9). The difference in molecular size and inhibition constant towards acetazolamide supported the view that branchial carbonic anhydrase and erythrocyte carbonic anhydrase were two different isozymes. Polyclonal antibody specific to seabream branchial carbonic anhydrase was obtained by immunization in rabbit. The distribution of branchial carbonic anhydrase in the gill of seabream acclimated to different salinities was studied with immunohistochemical method. The enzyme was mainly located at the interlamellar region. The effect of salinity (0, 6, 12, 33, 50 and 70 ‰) acclimation on the expression and activities of branchial carbonic anhydrase has shown a U-shape pattern from freshwater to double-strength seawater on the quantity of seabream branchial carbonic anhydrase. Higher amount of branchial carbonic anhydrase in freshwater was consistent with the current view that the enzyme was actively involved in the ion uptake process through the hydration of carbon dioxide to produce bicarbonate ion and proton for the exchange of chloride and sodium ions, respectively. An interesting finding was obtained with elevated amount of branchial carbonic anhydrase in seabream acclimated to double-strength seawater and the possible role of the enzyme in such extreme environment was discussed. / This study has provided useful information on the properties, localizations and activities of branchial carbonic anhydrase in silver seabream for the understanding of the involvement of the enzyme in salinity adaptation of silver seabream. / Ma, Wing Chi Joyce. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3250. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 127-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

In the second part of our study, cDNA of AQP-1 and pro-vasotocin were cloned from the silver sea bream. An AQP-1 full clone was isolated from kidney and intestine and it consists of 904 bp with an open reading frame of 774 bp. The deduced amino sequence of sea bream AQP-1 shares highest identity with AQP-1a of gilthead sea bream (97.7%) and AQP-1a of other fish species (83.6% to 95.8%), however, considerably low identity was found between the silver sea bream AQP-1 and AQP-1b of gilthead sea bream (56%). The silver sea bream AQP-1 possesses basic features of a functional aquaporin and AQP-1, which includes two channel-forming asparagine-proline-alanine (NPA) signature motifs, six transmembrane domains, residues of the pore-forming region and a potential mercurial inhibiting site (Cys-178). The water channel was ubiquitously expressed in gills, liver, intestine, rectum, kidney, heart, urinary bladder and blood cells. A partial fragment of pro-vasotocin was isolated from hypothalamus of silver sea bream and consists of 184 bp, including encoding regions for the processing and amidation signal, vasotocin hormone and part of the neurophysin. / Lastly, single doses of cortisol (50 microg/g tissue) or vasotocin (1 microg/g tissue) were administered to seawater-acclimated sea bream with further three-day stabilizing period in seawater followed by an abrupt 6‰ exposure or administered to seawater transfer controls for three days. Cortisol markedly stimulated intestinal expression of AQP-1 in both the seawater transfer control and abrupt 6‰ transfer groups. Vasotocin treatment did not significantly modify AQP-1 expression in all tested organs. Hypothalamic pro-vasotocin expression levels were similar among different treatment groups. / Semi-quantitative RT-PCR analysis was used for studying the effect of salinity and hormones on expression of AQP-1 and pro-vasotocin. In the long-term salinity acclimation experiment, the sea bream were acclimated to six different salinity regimes (0‰, 6‰, 12‰, 33‰, 50‰, 70‰) for four weeks. The abundance of AQP-1 transcript was the highest in intestine of 70‰-acclimated fish among different salinity groups and there was also a statistically significant increase in 12‰-acclimated fish. Branchial AQP-1 expression was significantly upregulated in sea bream acclimated to freshwater. In contrast, the hypothalamic pro-vasotocin expression was significantly downregulated during freshwater acclimation. In addition, the sea bream were also subjected to an abrupt 6%o transfer at different time intervals (2, 6, 12, 72 and 168 hours). RT-PCR analysis revealed there was a transient decrease in branchial AQP-1 expression two hours after abrupt hypo-osmotic exposure and the expression levels subsequently returned to the seawater control levels. The expression levels of hypothalamic pro-vasotocin were not significantly altered by the abrupt exposure treatment. / The present experiments investigated the effects of salinity and hormones on the relative expression of hypothalamic pro-vasotocin, and aquaporin-1 (AQP-1) in intestine, gills and kidney of the silver sea bream Sparus sarba. With the use of immunohistochemical techniques, immunoreactivity of AQP-1 was detected at the basal side of enterocytes and gill chloride cells, and at the apical brush border of kidney tubules whereas AQP-3 was only localized in similar positions in the gills and intestines. AQP-1 was relatively more ubiquitous than AQP-3 and was localized with same cell types as the electrogenic Na+-K+-ATPase in gills and kidney. / The present study had demonstrated the responsiveness of intestinal and branchial AQP-1 expressions of the silver sea bream to environmental salinity perturbations. Further to this, cortisol was observed to upregulate the transcription of AQP-1 in the intestine. Pro-vasotocin expression was altered by long-term salinity adaptation, however, the linkage of this alteration to AQP-1 functioning in different osmoregulatory organs is yet to be elucidated. / Luk, Chun Yin. / Adviser: Norman Y. S. Woo. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 200-222). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wong, Sze Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 87-106). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract in Chinese --- p.iv / Table of contents --- p.V / List of figures --- p.ix / List of abbreviations --- p.X / Chapter Chapter 1. --- General introduction --- p.1 / Chapter Chapter 2. --- Literature review --- p.6 / Chapter 2.1. --- Pathogenic mechanisms of Vibrio species in fish --- p.7 / Chapter 2.1.1. --- Introduction --- p.7 / Chapter 2.1.2. --- Adhesion --- p.7 / Chapter 2.1.3. --- Invasion --- p.8 / Chapter 2.1.4. --- Proliferation --- p.9 / Chapter 2.2. --- Vibrio virulence factors --- p.12 / Chapter 2.2.1. --- Introduction --- p.12 / Chapter 2.2.2. --- Hemolysin --- p.12 / Chapter 2.2.3. --- Protease --- p.14 / Chapter 2.2.4. --- Siderophore --- p.15 / Chapter 2.2.5. --- Lipopolysaccharide --- p.15 / Chapter 2.3. --- General apoptotic pathways --- p.17 / Chapter 2.3.1. --- Introduction --- p.17 / Chapter 2.3.2. --- Extrinsic apoptotic pathway --- p.17 / Chapter 2.3.2.1. --- Death receptor signaling apoptosis --- p.17 / Chapter 2.3.2.1.1. --- Fas signaling pathway --- p.18 / Chapter 2.3.2.1.2. --- TNF-R1 signaling pathway --- p.19 / Chapter 2.3.2.1.3. --- TRAIL receptors signaling pathway --- p.20 / Chapter 2.3.2.2. --- Growth factor receptor signaling apoptosis --- p.21 / Chapter 2.3.3. --- Intrinsic apoptotic pathway --- p.21 / Chapter 2.3.3.1. --- Mitochondrial apoptotic pathway --- p.21 / Chapter 2.3.3.1.1. --- Cyto c --- p.22 / Chapter 2.3.3.1.2. --- Smac/DIABLO --- p.22 / Chapter 2.3.3.1.3. --- Omi/HtrA2 --- p.22 / Chapter 2.3.3.1.4. --- AIF and endo G --- p.23 / Chapter 2.3.3.1.5. --- Bcl-2 family --- p.23 / Chapter 2.3.3.1.6. --- Mitochondrial membrane permeabilization (MMP) --- p.23 / Chapter 2.3.3.2. --- p53-regulated apoptotic pathway --- p.24 / Chapter 2.3.3.3. --- Endoplasmic reticulum (ER) stress-induced apoptotic pathway --- p.25 / Chapter 2.4. --- Membrane vesiculation in erythrocytes --- p.26 / Chapter 2.4.1. --- Introduction --- p.26 / Chapter 2.4.2. --- Induction of vesiculation --- p.26 / Chapter 2.4.3. --- Contents of vesicles --- p.28 / Chapter 2.4.4. --- Mechanisms involved during vesiculation --- p.29 / Chapter 2.4.5. --- Correlation between apoptosis and membrane vesiculation in erythrocytes --- p.31 / Chapter 2.4.6. --- Reasons for vesiculation --- p.31 / Chapter Chapter 3. --- "Induction of apoptosis by Vibrio alginolyticus thermolabile hemolysin (TLH) in blood cells of silver sea bream, Sparus sarba" --- p.33 / Chapter 3.1. --- Abstract --- p.34 / Chapter 3.2. --- Introduction --- p.34 / Chapter 3.3. --- Materials and methods --- p.36 / Chapter 3.3.1. --- Experimental fish --- p.36 / Chapter 3.3.2. --- Whole blood preparation --- p.37 / Chapter 3.3.3. --- Preparation of V. alginolyticus TLH --- p.37 / Chapter 3.3.4. --- "Caspase-3, -8, -9/6 fluorescent assay" --- p.38 / Chapter 3.3.5. --- TUNEL assay --- p.39 / Chapter 3.3.6. --- Apoptotic DNA ladder assay --- p.40 / Chapter 3.3.7. --- Statistical analysis --- p.41 / Chapter 3.4. --- Results --- p.42 / Chapter 3.4.1. --- "Increase of caspase-3, -8, -9/6 activities" --- p.42 / Chapter 3.4.2. --- Detection of DNA fragmentation by TUNEL assay --- p.44 / Chapter 3.4.3. --- Detection of DNA fragmentation by apoptotic DNA ladder assay --- p.44 / Chapter 3.5. --- Discussion --- p.46 / Chapter Chapter 4. --- "Occurrence of membrane vesiculation, apoptosis and post-apoptotic necrosis after exposure to Vibrio alginolyticus thermolabile hemolysin (TLH) in erythrocytes of silver sea bream, Sparus sarba" --- p.51 / Chapter 4.1. --- Abstract --- p.52 / Chapter 4.2. --- Introduction --- p.52 / Chapter 4.3. --- Materials and methods --- p.54 / Chapter 4.3.1. --- Experimental fish --- p.54 / Chapter 4.3.2. --- Whole blood preparation --- p.54 / Chapter 4.3.3. --- Preparation of V. alginolyticus TLH --- p.55 / Chapter 4.3.4. --- Light microscopy --- p.55 / Chapter 4.3.5. --- Transmission electron microscopy (TEM) --- p.56 / Chapter 4.3.6. --- Measurement of membrane vesiculation - acetylcholinesterase (AChE) assay --- p.56 / Chapter 4.3.7. --- Measurement of necrosis - hemoglobin colorimetric assay --- p.57 / Chapter 4.3.8. --- Apoptotic DNA ladder assay --- p.58 / Chapter 4.3.9. --- Flow cytometry --- p.59 / Chapter 4.3.10. --- Statistical analysis --- p.59 / Chapter 4.4. --- Results --- p.60 / Chapter 4.4.1. --- Ultrastructural changes in red blood cells after exposure to TLH --- p.60 / Chapter 4.4.2. --- Changes of cell population in size and granularity after exposure of TLH --- p.67 / Chapter 4.4.3. --- Effect of TLH dosage on necrosis and DNA fragmentation --- p.72 / Chapter 4.4.4. --- "Occurrence of membrane vesiculation, necrosis and DNA fragmentation in cells exposed to TLH" --- p.72 / Chapter 4.5. --- Discussion --- p.76 / Chapter Chapter 5. --- General conclusions --- p.82 / References --- p.87