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Trans-differentiation is the term applied where one fully differentiated cell type changes into another fully differentiated cell type. The AR432J-B13 cell line has been shown to demonstrate this phenomenon in vitro. These cells of exocrine pancreatic lineage, can trans-differentiate into hepatocytes upon treatment with the synthetic glucocorticoid dexamethasone. This thesis demonstrates that the generation of these cells (B13-H) from AR42J-B13 cells could prove to be a novel source of hepatocytes in culture. B13-H cells express functionally active and inducible CYP enzymes for at least 30 days in culture, an attribute that primary hepatocytes do not hold. In addition, B13-H cells have shown to be a useful alternative to primary hepatocytes in the investigation of protective mechanisms against paracetamol toxicity. The pancreas and liver have a close association developmentally which helps to explain their relationship in adulthood. As primary hepatocytes rapidly dedifferentiate in culture, the use of these or other pancreas-derived hepatocytes would be beneficial, both in a clinical (e.g. bioartificial liver device as a 'bridge' until transplant) and pharmacological (e.g. drug metabolism studies) settings. Another liver cell affected by disease is the hepatic stellate cell. These cells trans-differentiate into a myofibroblast-like cell and are pivotal in the formation of liver fibrosis. By inhibiting this trans-differentiation event, liver fibrosis can resolve, thereby preventing the terminal state of cirrhosis. PCN is such a compound capable of achieving this outcome both in vitro using isolated hepatic stellate cells and in vivo carbon tetrachloride-induced rodent liver-injury models. Transgenic mice enabled the determination that this effect is both dependent and independent upon the nuclear receptor PXR of which PCN is a ligand.

Previous studies have led to the proposal that elevated levels of secondary bile acids contribute to the pathogenesis of conditions such as secretory diarrhoea, inflammatory bowel disease and colon cancer. Increasing evidence suggests that these conditions arise in part from bile acid modulation of cellular signalling pathways. Studies on intestinal cell lines suggest that secondary bile acids, taurolithocholate (LCT) and taurodeoxycholate (OCT) in particular, may act directly on the intestinal epithelium by modulating calcium signalling, possibly via activation of acetylcholine receptors. AIMS: To investigate secondary bile acid modulation of calcium signalling, the canonical Wnt pathway and crypt cell proliferation in the native human colonic epithelium. METHODS: Colonic crypts were isolated from tissue biopsies obtained at sigmoidoscopy from healthy subjects (Ethical approval). Isolated crypts were attached to collagen-coated coverslips and cultured for 24 hours - 3 days in serum-free OMEM (5%C02/3rC). For calcium experiments colonic crypts were loaded with the calcium-sensitive dye Fura2-AM. For proliferation experiments crypts were incubated at 5%C02/37°C for 24 hours in the presence of BrdU. RESULTS: Application of LCT or LCA (1 00-300 ~M) failed to elicit a calcium response in any part of the crypt or modulate ACh- induced calcium signals (n=16). In contrast, OCT (0.5-1 mM) elicited oscillations of intracellular calcium levels along the entire crypt-axis (n=27). The highest oscillation frequencies were observed in the basal regions of the crypt. OCT-induced calcium oscillations were still evident in the absence of calcium in the bathing medium (n=3). Depletion of calcium from intracellular stores by thapsigargin (2 ~M) (n=3) abolished the onset of OCT-induced calcium oscillations as did pre-incubation with 2-APB (1 00 ~M) (n=3), TMB-8 (1 00 ~M) (n=3) and caffeine (20 mM) (n=3). The pacemaker region for OCT- induced oscillations did not reside at the crypt base. In contrast, acetylcholine (ACh) (1 0 ~M) induced non-oscillatory biphasic calcium signals at the crypt base that propagated in a unidirectional manner along the crypt axis (n=57). Atropine (1 ~M) inhibited the ACh response (n=3), but did not affect OCT- induced calcium oscillations. Finally, crypts treated with OCT exhibited increased levels of nuclear l3-catenin and Ki67 labelling suggesting activation of the proliferative Wnt signalling pathway. CONCLUSION: (Patho)physiologicallevels of OCT are sufficient to set in to motion a train of intracellular calcium oscillations along the crypt axis and stimulate crypt cell proliferation. The consequences to human colonic crypt fluid secretion and tissue renewal are expected to be significant and will impact on conditions such as secretory diarrhoea and colon cancer. The molecular target for OCT remains to be determined, but is unlikely to be a member of the muscarinic receptor family.

Glycolate oxidase (GO) is a peroxisomal flavoenzyme which catalyses the oxidation of short chain a-hydroxy acids, notably glycolate. The reaction product, glyoxylate, is an oxalate precursor and GO is thus of potential interest for its role in the pathogenesis of the primary hyperoxalurias. The project aims were to identify human GO, characterise the kinetics and substrate specificity of the enzyme and establish methods for the analysis of relevant metabolic pathways in vitro. The gene for human GO was cloned from liver and expressed in bacterial cells. The cDNA is 1128 bp in length and has a 1113 bp open reading frame encoding a 372 amino acid protein. The genomic sequence comprises eight exons and spans —57 kb of chromosome 20p12. Recombinant human GO protein shares 53% and 89% sequence similarity to GO from spinach and rat respectively, shows a-hydroxy acid oxidase activity in vitro and has been purified to homogeneity. Polyclonal anti-GO antibody detects a band of 43 kDa in human liver and, consistent with northern blot analysis, expression is not detected in other tissues including kidney and leucocytes. Kinetic analysis with a range of a-hydroxy acids indicates GO has highest affinity for glycolate as substrate (Km = 0.54 mM) and 10 fold less affinity for glyoxylate (Km = 5.1 mM). Site directed mutagenesis of active site residues demonstrates the importance of chain length for substrate affinity. Thus mutation of a Trp residue, conserved between spinach and human GO to a less bulky amino acid, permits the catalysis of longer chain length a-hydroxy acids. HPLC methods were developed for the separation and quantitation of glyoxylate, hydroxypyruvate and pyruvate, enabling analysis of metabolites produced by GO and neighboring enzymes in the metabolic pathway. These assays will be invaluable for future studies in which the pathways of glyoxylate metabolism are constructed in vitro.

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