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Item Analysis of gate residues in the type 2 secretin PulD : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Biochemistry at Massey University, Manawatu, New Zealand(Massey University, 2012) Whitaker, RowanSecretins are gated outer-membrane channels with large internal pore sizes (6-10 nm). They are the outer membrane components of bacterial trans-envelope complexes that assemble/export filamentous bacteriophages as well as pili, complex protein toxins and virulence factors. 12-14 identical subunits form the radially symmetrical channels which share a common architecture - a 3-tiered barrel with middle septum. Secretins are essential components of Gram-negative Type 2/3 secretion systems, spanning the outer membrane and interacting with the inner membrane components of transport machinery. Since secretins have such large pore diameters a simple channel would allow noxious compounds through the normally impermeable outer membrane. The presence of a gate structure allows for the controlled opening and closing of secretin channels, in response to specific cues regulating protein export. Here I have determined gate-structural elements of the Klebsiella oxytoca Type 2 Secretin, PulD. Random mutagenesis coupled with selection for open or 'leaky'-gate phenotypes created a library of mutations which were mapped by DNA sequence analysis. Analysis of leaky mutants revealed 12 distinct missense point mutations in pulD. Additionally, two deletion mutants were isolated, spanning 5 and 9 amino acids, both conferring a leaky gate phenotype. Comparison of these pulD mutations with those previously identified in another secretin gene encoding the Escherichia coli filamentous phage f1 secretin pIV, reveals mutations in both are localised in two main clusters that correspond to regions within the secretin homology domain. Named GATE1 and GATE2, these clusters indicate functional gate regions in both secretins.Item A novel gastrin inhibitor in sheep abomasal contents : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physiology at Massey University, Palmerston North, New Zealand(Massey University, 2000) Simcock, David CrispinGastrin secretion was studied in vitro and in vivo in response to pharmacological agents and chemicals, as well as abomasal parasites and microbial products. The causes and effects of hypergastrinaemia, along with bacterial numbers and the presence of a gastrin secretion inhibitor in the abomasal contents of sheep infected with Ostertagia circumcincta were studied. The pharmacology of the gastrin secretion from the unparasitised antrum was shown to be similar to that in monogastric animals. In vitro gastrin secretion by ovine antral segments was stimulated by Gastrin Releasing Peptide, carbachol and nicotine, but not adrenaline. Basal gastrin release was unaffected by somatostatin or Vasoactive Intestinal Polypeptide, but these reduced the gastrin response to stimulants. Gastrin secretion was also stimulated by amino acids, ammonia and acetate. Hypergastrinaemia during O. circumcincta infection did not correlate well with decreased food intake or appear to affect parietal cell recovery. Serum gastrin concentrations correlated well with abomasal pH following adult O. circumcincta transplant, but poorly after larval infections. This suggests that other factors, such as inflammation and tissue damage, also affect gastrin secretion during abomasal parasitism. Anaerobic bacterial numbers in abomasal contents increased to near rumen levels when abomasal pH was 3.5 and above, but this did not affect serum gastrin concentrations. An inhibitor of in vitro gastrin secretion also started to appear in abomasal contents of pH3.5 and over, but did not have significant effects on in vitro gastrin secretion unless contents were pH4.5 and over. However, gastrin inhibitory activity in abomasal contents and serum gastrin levels were positively correlated, suggesting abomasal gastrin inhibitory activity has little effect on gastrin secretion in vivo. Three competing factors were present in rumen fluid and rumen incubates: an inhibitor and a stimulant of secretion and an elimination factor. The stimulant was resistant to acid degradation, had a molecular weight below 3000 Mr and was hydrophilic. Both the elimination factor and the inhibitor were sensitive to acidity and hydrophobic and are likely to be proteinaceous.