published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 10 uppsatser.

There is a need for a simple, safe, reproducible and non-invasive method for studying upper gastrointestinal motility in humans. Existing methods, measuring electrical contractions and intraluminal pressure changes have limitations in their correlation with the physiology of what is actually happening to ingested food. Transit time has been suggested as a more physiologic means of studying gut motility; therefore a method was developed to measure transit time and secretory changes in response to ingested liquids, using ileostomy patients. 2.5 gm of polyethylene glycol (PEG) was added to 500 ml of normal saline, and given orally to volunteers with ileostomies. The ileostomy effluent was collected for 2 hours in 10 minute aliquots. PEG assay was performed by the turbidimetric method of Hyden, using Malawer's modification with an emulsifier. The following were measured: most rapid, mode, median, mean and total transit t imes. A study was then performed to determine if different foodstuffs -carbohydrate, fat, and protein - produce measurable changes in transit time. 2.5 gm of PEG was added to 500 ml of (a) 90 ml Lipomul in 410 ml normal saline (b) 5% dextrose (c) 100 ml of Travasol 10% in 400 ml distilled water. The volumes were chosen to produce isoosmolar test feeds. Validating studies showed satisfactory reproducibility and individual variation (r = 0.68 for volume recovery, r = 0.69 for PEG recovery, p = < 0.5) The recovery pattern of a test feed of 500 ml normal saline was found to follow a skew distribution, with mode, median and mean transit times all different. The most reproducible and easily measured was mode, or peak, transit time (average 40 minutes for volume and PEG recovery). Significant delays in all transit times were found (p = < 0.01) using each of the test feeds: (a) for Lipomul a peak volume recovery of 60 minutes and PEG recovery of 70 minutes; (b) for 5% dextrose a peak volume recovery of 90 minutes and PEG recovery of 90 minutes; (c) with Travasol, negligible amounts of ileostomy output were obtained over 2 hours. The most rapid transit time was consistently less than 10 minutes, as measured by PEG appearance from the ileostomy. This is far less than previously described by standard methods, but is in accordance with transit times measured to the ileocaecal valve in intact gastrointestinal tracts using the recently-introduced breath hydrogen method following lactulose ingestion. Comparison of total volume recovery with total PEG recovery over 2 hours indicates whether net absorption or secretion has occurred: (a) with normal saline a volume recovery of 62% and PEG recovery of 48% indicates net secretion; (b) with Lipomul a volume recovery of 66% and PEG recovery of 58% also indicates net secretion, with no significant difference from normal saline (p = < 0.05); (c) with 5% dextrose a volume recovery of 4% and PEG recovery of 13% indicates net absorption, significantly different from normal saline (p = < 0.01); (d) for Travasol a volume recovery of 1% and PEG recovery of 1% indicates no net absorption or secretion, but confirms the above finding of a very large delay in transit time. These studies have shown that isotonic solutions of normal saline, glucose, fat and protein result in widely different peak transit times in ileostomy patients. They also result in widely different fluid outputs from the ileostomy due to net absorption or secretion. These differences have not been described before. / Surgery, Department of / Medicine, Faculty of / Graduate

flhE belongs to the flhBAE flagellar operon in Enterobacteria, whose first two members function in Type III secretion (T3S). In Salmonella enterica, absence of FlhE affects swarming but not swimming motility. Based on a chance observation of a ‘green’ colony phenotype of flhE mutants on pH indicator plates containing glucose, I have established that this phenotype is associated with lysis of flagellated cells in an acidic environment created by glucose metabolism. The flhE mutant phenotype of Escherichia coli is similar overall to that of S. enterica, but is seen in the absence of glucose and unlike in S. enterica, causes a substantial growth defect. flhE mutants have a lowered cytoplasmic pH in both bacteria, indicative of a proton leak. GFP reporter assays indicate that the leak is dependent on the flagellar system, is present before the T3S system switches to secretion of late substrates, but gets worse after the switch and upon filament assembly, leading to cell lysis. I show that FlhE is a periplasmic protein, which co-purifies with flagellar basal bodies. Also, co-localization of fluorescent fusion proteins suggests a plausible interaction between FlhE and FlhA, implicated in channeling protons for PMF-driven secretion. These results imply that FlhE may act as a plug or a chaperone to regulate proton flow through the flagellar T3S system. I have obtained crystals of the FlhE protein. X-ray data show that the FlhE crystal belongs to space group P212121 and is diffracted to 2.02 Å. Completion of this study will contribute to a better understanding of the proposed role of FlhE. / text

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