Now showing 1 - 6 of 6
  • Publication
    Migration of Fasciola hepatica newly excysted juveniles is inhibited by high-mannose and oligomannose-Type N-glycan-binding lectins
    (Cambridge University Press, 2017-11-01) ; ;
    Fasciola hepatica has both zoonotic importance and high economic impact in livestock worldwide. After ingestion by the definitive host, the Newly Excysted Juveniles (NEJ) penetrate the intestine before reaching the peritoneal cavity. The role of some NEJ-derived proteins in invasion has been documented, but the role of NEJ glycans or lectin-binding receptors during initial infection in the gut is still unknown. To address these questions, the migration of NEJ through rat intestine was recorded at 30 min intervals up to 150 min by two ex vivo methods. Firstly, jejunal sheets were challenged with NEJ incubated with biotinylated lectins. Secondly, untreated NEJ were incubated with distal jejunum pre-Treated with lectins. Both Concanavalin A (ConA) and Galanthus nivalis (GNL), which recognize mannose-Type N-glycans, significantly inhibited NEJ migration across the jejunum. Most of the lectins bound to the tegument and oral sucker of the NEJ, but only ConA and GNL maintained this interaction over 150 min. None of the lectins examined significantly reduced NEJ migration when pre-incubated with jejunal sheets, suggesting that host glycans might not be essential for initial binding/recognition of the gut by NEJ. Agents capable of blocking mannose-Type N-glycans on the NEJ tegument may have potential for disrupting infection.
    Scopus© Citations 10  320
  • Publication
    Mechanisms of Action of Zinc on Intestinal Epithelial Electrogenic Ion Secretion: Insights into its Anti-Diarrheal Actions
    Objectives  Zinc is a useful addition to oral rehydration therapy for acute diarrhoea. We have assessed the mechanism of its epithelial antisecretory action when intestinal epithelial tight junctions were pharmacologically opened. Methods  Rat isolated ileal and colonic mucosae were mounted in Ussing chambers and exposed to ZnSO4 (Zn2+) in the presence of secretagogues and inhibition of short circuit current (Isc) was measured. Key findings  Pre-incubation with basolateral but not apical Zn2+ reduced Isc stimulated by forskolin, carbachol and A23187. In the presence of the tight junction-opener, cytochalasin D, antisecretory effects of apically-applied Zn2+ were enabled in colon and ileum. The apparent permeability coefficient (Papp) of Zn2+ was increased 1.4- and 2.4-fold across rat ileum and colon, respectively, by cytochalasin D. Basolateral addition of Zn2+ also reduced the Isc stimulated by nystatin in rat colon, confirming K channel inhibition. In comparison with other inhibitors, Zn2+ was a relatively weak blocker of basolateral KATP and K Ca2+ channels. Exposure of ileum and colon to Zn2+ for 60 min had minimal effects on epithelial histology. Conclusions  Antisecretory effects of Zn2+ on intestinal epithelia arose in part through nonselective blockade of basolateral K channels, which was enabled when tight junctions were open.
    Scopus© Citations 13  382
  • Publication
    Investigation of facilitative urea transporters in the human gastrointestinal tract
    The symbiotic relationship between humans and their intestinal microbiomeis supported by urea nitrogen salvaging. Previous studies have shown thatcolonic UT-B urea transporters play a significant role in this important physi-ological process. This current study investigated UT-A and UT-B urea trans-porter expression along the human gastrointestinal tract. Initial end-pointPCR experiments determined that UT-A RNA was predominantly expressedin the small intestine, while UT-B RNA was expressed in stomach, small intes-tine, and colon. Using western blotting experiments, a strong 40–60 kDa UT-B signal was found to be abundant in both ileum and colon. Importantly, thissignal was deglycosylated by PNGaseF enzyme treatment to a core protein of30 kDa in both tissues. Further immunolocalization studies revealed UT-Btransporter proteins were present at the apical membrane of the villi in theileum, but predominantly at the basolateral membrane of the colonic surfaceepithelial cells. Finally, a blind scoring immunolocalization study suggestedthat there was no significant difference in UT-B abundance throughout thecolon (NS, ANOVA,N=5–21). In conclusion, this current study suggestedUT-B to be the main human intestinal urea transporter. Intriguingly, thesedata suggested that the same UT-B isoform was present in all intestinalepithelial cells, but that the precise cellular location varied.
      417Scopus© Citations 7
  • Publication
    High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs
    Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development.Wedescribe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic-helical peptide, melittin, and selected structural analogs. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.
      1653Scopus© Citations 24
  • Publication
    Stomaching drug delivery
    (Massachusetts Medical Society, 2019-04-25) ;
    Oral ingestion remains the preferred method of drug administration. Its disadvantages with regard to biologic agents reflect the fact that systemic bioavailability is influenced by the chemical properties of the drug and by the gastrointestinal physiological and pathologic features of the patient. Unfortunately, many candidate biologic agents are labile or cannot readily cross the gastrointestinal epithelium.
      689Scopus© Citations 6