by Ka-bik Lai. / Thesis (M.Phil.)--Chinese University of Hong Kong, / Includes bibliographical references (leaves 59-67). / Chapter Chapter I --- Introduction / Chapter I.1 --- Anatomy and functions of the epididym --- p.is / Chapter I.1.1 --- Gross anatomy and functions --- p.1 / Chapter I.1.2 --- Histology of the epithelium and functions of different cell types --- p.3 / Chapter I.2 --- Regional difference / Chapter I.2.1 --- Epithelium --- p.4 / Chapter I.2.2 --- Electrolyte transport --- p.6 / Chapter I.3 --- Short circuit current studies on C1- secretion in the cultured rat epididymal epithelium --- p.7 / Chapter I.4 --- Physiological effects of vasopressin in the epididymis and the male reproductive tract --- p.10 / Chapter I.5 --- Objectives of the study --- p.14 / Chapter Chapter II --- Materials and methods / Chapter II. 1 --- Materials / Chapter II.1.1 --- Chemicals for cell culture --- p.16 / Chapter II. 1.2 --- Chemicals and drugs for short circuit current study --- p.16 / Chapter II. 1.3 --- Materials for preparing pervious supports --- p.16 / Chapter II.2 --- Preparation of solution for short circuit current study --- p.17 / Chapter II.3 --- Preparation of pervious supports for the cultured epithelium --- p.17 / Chapter II.4 --- Culture of rat epididymal epithelial cells / Chapter II.4.1 --- "Dissection of the efferent duct, the initial segment and the cauda epididymidis" --- p.17 / Chapter II.4.2 --- Culture procedures for the epithelium --- p.19 / Chapter II.4.3 --- Confluent epithelial monolayer --- p.22 / Chapter II.5 --- Short circuit current measurement / Chapter II.5.1 --- Mounting of cultured epithelium on Ussing chambers --- p.22 / Chapter II.5.2 --- Experimental setup for zero voltage clamp --- p.25 / Chapter Chapter III --- Results / Chapter III.1 --- Regional differences / Chapter III.1.1 --- Basal bioelectrical properties --- p.29 / Chapter III. 1.2 --- Effects of C1- replacement and C1- channel blocker --- p.29 / Chapter III.1.3 --- Effects of ion transportor inhibitors --- p.33 / Chapter III. 1.4 --- Effect of adenylate cyclase stimulator --- p.33 / Chapter III.1.5 --- Effect of exogenous PGE2 --- p.33 / Chapter III.1.6 --- Effect of Ca2+ ionophore --- p.36 / Chapter III.2 --- Effect of [arg8]-vasopressin / Chapter III.2.1 --- Effect of [arg8] -vasopressin on short circuit current --- p.36 / Chapter III.2.2 --- Effects of C1- replacement and C1- channel blocker --- p.40 / Chapter III.2.3 --- Effect of repeated stimulation with [arg8]- vasopressin --- p.40 / Chapter III.2.4 --- Effects of receptor antagonists --- p.40 / Chapter III.2.5 --- Effects of thapsigargin and trifluoperazine --- p.44 / Chapter III.2.6 --- Effects of indomethacin --- p.44 / Chapter III.2.7 --- Effects of forskolin --- p.47 / Chapter Chapter IV --- Discussion --- p.50 / Chapter Chapter V --- References --- p.59

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Thesis (M.D.)--University of Pennsylvania, 1805. / Microform version available in the Readex Early American Imprints series.

Geobacillus thermoglucosidasius (GT) is a thermophilic, ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. One strategy to improve fermentation yields would be to engineer GT strains to secrete hydrolases to increase the amount of available sugars from various feedstocks. Therefore, optimised protein secretion would be vital to improve feedstock utilisation. Secretion in the related mesophile Bacillus subtilis (BS) has been well studied, and several strategies have been developed to improve secretion of heterologous proteins in BS, one such strategy being the manipulation or changing of the signal peptide. One aim is to identify any differences in the secretion machinery and signal sequences between GT and BS. Another aim is to analyse any effects of overproduction of hydrolases and to identify any bottlenecks in protein secretion in GT. Using bio-informatics tools we find that although GT is a thermophile, the signal peptides in this organism do not differ significantly from those in BS. From a shotgun mass spectrometry approach it was also observed that unlike BS, GT undergoes significant cell lysis during growth releasing cytoplasmic proteins into the extracellular milieu, which could have implications on the levels of secreted hydrolases. A model enzyme was selected and over-produced at high levels in order to stress the secretion system in GT so as to identify any bottlenecks in secretion. The results thus far indicate that the rate limiting step in secretion could be post-translocation where the enzyme is degraded by proteases in the cell wall and extracellular milieu. The addition of protease inhibitor to growth media, increases the activity and abundance of the enzyme, suggesting that proteolysis may be a major factor when over-producing secreted enzymes at high levels.

A plasmid-encoded Type III Secretion System (T3SS) is employed by human pathogenic yersiniae to inject effector proteins, termed Yops, directly into host cells. The secretion of Yops is tightly regulated, and occurs only upon contact with a eukaryotic cell in vivo or in media devoid of calcium in vitro. A complex containing the secreted protein YopN, its heterodimeric chaperone SycN/YscB, and TyeA is required to prevent secretion of effector Yops until the appropriate secretion-triggering signals are encountered. The mechanism by which these proteins regulate the T3S process is unknown. A mutational analysis of YopN and TyeA was performed to identify regions and residues of these proteins that are required to regulate Yop secretion. Amino-acid residues of TyeA were identified that were specifically required for the interaction of TyeA with YopN, confirming that the YopN/TyeA interaction is essential for the regulation of Yop secretion. Furthermore, analysis of TyeA mutants identified a surface-exposed region that was critical for the regulation of Yop secretion, but not required for interaction with YopN. YopN residues critical for the regulation of secretion clustered within the N- and C-terminal regions of YopN that were required to interact with the SycN/YscB chaperone and TyeA, respectively. No residues critical for the regulation of secretion were identified in the central region of YopN, suggesting that this region acts primarily to maintain proper positioning of the functional N- and C-terminal regions of this complex. A novel role for the chaperone binding domain (CBD) of YopN in the regulation of Yop secretion was identified. This role was separate from its role in binding the SycN/YscB chaperone and targeting YopN for secretion. Finally, it was demonstrated that the SycN/YscB chaperone is dispensable for the regulation of secretion if the expression of both YopN and TyeA is increased, indicating that these chaperones have no direct role in the regulation of Yop secretion. These results indicate that the YopN secretion signal and SycN/YscB chaperone function to efficiently target the YopN/TyeA complex to the T3S apparatus, whereas the YopN CBD and C-terminal region of YopN complexed with TyeA mediate the block in Yop secretion.

The present research studied the regulation of duodenal bicarbonate secretion in the anaesthetized guinea-pig, using a model that permitted the study of active transport of bicarbonate. It was determined that dibutyryl 3' ,5'-cyclic adenosine monophosphate, vasoactive intestinal polypeptide, prostaglandin E2, carbachol and theophylline are the chief agonists of duodenal bicarbonate secretion. Vasoactive intestinal polypeptide and prostaglandin E2 act directly via distinct receptors on the duodenal enterocytes, activating adenylate cyclase and protein kinase A in sequence to initiate bicarbonate secretion. In addition, there is good evidence that the inositol phospholipid and protein kinase C cascade is also involved, possibly to a lesser extent, since tetradecanoyl-phorbolacetate and prostaglandin F2a were agonists of bicarbonate secretion. Carbachol, using a m-cholinoceptor pathway, stimulates duodenal bicarbonate secretion by releasing vasoactive intestinal polypeptide. Consistent with this finding is the observation that carbachol has no receptors on duodenal enterocytes. The role of the nicotinic pathway in bicarbonate secretion, however, remains uncertain. Duodenal bicarbonate secretion can be inhibited by somatostatin and acetazolamide. Somatostatin selectively suppresses carbachol-stimulated and VIP-stimulated duodenal bicarbonate secretion, but not PGE2-stimulated bicarbonate secretion. Receptors for somatostatin coupled to adenylate cyclase could not be detected on isolated duodenal enterocytes, which strengthens the hypothesis that carbachol does not act directly on these epithelial cells, but via a second transmitter, vasoactive intestinal polypeptide. Carbonic anhydrase activity is necessary for secretion of bicarbonate, since acetazolamide-inhibition of this enzyme decreased bicarbonate secretion, both basal and stimulated by many different agonists. Carbonic anhydrase serves as a common final step in the generation of bicarbonate in duodenal enterocytes. This enzyme was located in the cytoplasm of cells in the villus as well as the crypt cells, implying that bicarbonate secretion occurs along the length of the villus and crypt. In summary, the present research has shown direct stimulation of duodenal bicarbonate secretion by vasoactive intestinal polypeptide, which participates also in themcholinergic pathway, and by prostaglandin E2. Adenylate cyclase and protein kinase A appear to be the intracellular messengers with the primary function of initiating duodenal bicarbonate secretion. However, there is convincing evidence that the inositol phospholipid and protein kinase C cascade also activates this secretion. Somatostatin selectively stops duodenal bicarbonate secretion. Carbonic anhydrase activity in the crypt and villus is required as the final common step in bicarbonate production.

No description available.

Graduate

Pyruvate carboxylase (PC), converting pyruvate to oxaloacetate (OAA), is a critical contributor to anaplerosis in pancreatic β-cell, the process that can replenish the intermediates in Krebs cycle. The level of PC is markedly high in pancreatic β-cell, about 7-fold higher than that in α-cell. PC activity is reduced in the islets of animals with type 2 diabetes. Moreover, the rate of pyruvate carboxylation catalyzed by PC is well correlated with glucose-stimulated insulin secretion (GSIS), further supporting the important role of PC in insulin secretion. Tumor protein p53 or p53, best known for its role as a tumor suppressor, has been studied extensively for its broad influence and complex regulation. p53 suppresses tumor progression by responding to a wide variety of intrinsic and extrinsic cellular stress signals. Recent studies have revealed novel roles of p53 in response to metabolic stress, such as oxidative or nutrient stress. MDM2 is the major E3 ubiquitin ligases to control p53 activity negatively. p53 and MDM2 form a negative-feedback loop, where p53 stimulates the expression of MDM2, in turn MDM2 inhibits not only the stability but also the transcriptional activity of p53. In the previous study, mice lacking Mdm2 specifically in pancreatic β-cells have been generated, and display impaired insulin secretion and glucose metabolism. Further study has suggested that the impaired insulin secretion is caused by downregulation of pyruvate carboxylase (PC). To explore the interaction of PC with MDM2-p53 pathway in regulating insulin secretion, we inserted human PC DNA into a shuttle vector pShuttle-CMV to generate a recombinant adenovirus containing human PC gene, that is then used to restore the level of PC in Mdm2 KO islet. The recovery of insulin secretion confirmed that the downregulation of PC leads to β-cell dysfunction. Given that MDM2 is the main E3 ubiquitin protein ligase to restrain p53 activity, abnormal high level of p53 is suggested to suppress the activity of PC in Mdm2 β-cell KO mice. The putative p53 response element is found in a sequence of PC intron gene, indicating that PC can be a downstream target of p53. Using pGL3 Luciferase Reporter Vector, it is verified that p53 suppresses the activity of PC by directly targeting PC. / published_or_final_version / Medicine / Master / Master of Medical Sciences