In an attempt to evaluate the role of gangliosides as receptors for fibronectin, a series of Balb/c 3T3 variant cell lines, with a reduced ability to bind the ganglioside-specific ligand cholera toxin (CT), were examined. Initial characterisation showed that the cell lines displayed a generalised reduction in the synthesis of gangliosides more complex than GM3, but not of cell surface glycoproteins. There was no reduction in the levels of fibronectin found at the surface of the variants as compared with the parental cell line and all were able to spread and form focal contacts on fibronectin-coated substrates. These results suggest that complex glycolipids of the 'ganglio' series are not essential for Balb/c 3T3 cells to interact with fibronectin. The role of gangliosides as receptors for heat-labile toxin of E.coli (H-LT) was investigated using CT as a ganglioside-specific control. Both toxins bound to ganglioside GM1 and to Balb/c 3T3 cell membranes. Binding of 125I-labelled toxins was inhibited by either unlabelled toxin. There was no evidence to suggest that CT or H-LT recognised receptor(s), in Balb/c 3T3 cells, in addition to GM1. In rabbit intestinal brush borders at 0°C, there were more binding sites for H-LT than CT and 125I-H-LT binding could not be inhibited by unlabelled CT. At higher temperatures there was some inhibition of 125I-H-LT binding by CT. In Western blots H-LT recognised proteins co-migrating with the major brush border galacto-proteins. Toxin binding to brush borders from the Wistar strain of rat was similar. One of the 125I-H-LT binding sites may be the sucrase-isomaltase complex, since the toxin bound to brush border fractions enriched for enzyme activity. The data suggest that the major binding sites for H-LT in brush borders are not ganglioside in nature but may be glycoproteins.

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Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 108-122).

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Kam Yiu-wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 200-215). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Publications --- p.iv / Abbreviations --- p.v / Contents --- p.vii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- G protein-coupled receptors --- p.1 / Chapter 1.1.1 --- Introduction --- p.1 / Chapter 1.1.2 --- G protein-coupled receptors --- p.2 / Chapter 1.1.3 --- Heterotrimeric G-proteins --- p.4 / Chapter 1.1.4 --- Second messenger systems --- p.5 / Chapter 1.1.5 --- Mechanism of GPCR activation --- p.6 / Chapter 1.1.6 --- GPCR cross talk --- p.8 / Chapter 1.2 --- Receptor theory --- p.10 / Chapter 1.2.1 --- Introduction --- p.10 / Chapter 1.2.2 --- Two-state model --- p.11 / Chapter 1.2.3 --- Three-state model --- p.12 / Chapter 1.2.4 --- Extended ternary complex model --- p.12 / Chapter 1.2.5 --- Multiple receptor state --- p.13 / Chapter 1.2.6 --- Constitutively active mutant receptors --- p.14 / Chapter 1.3 --- Agonist trafficking --- p.15 / Chapter 1.3.1 --- Introduction --- p.15 / Chapter 1.3.2 --- Effect of agonist efficacy on receptor coupling --- p.17 / Chapter 1.3.3 --- Effect of receptor expression level on receptor coupling --- p.17 / Chapter 1.3.4 --- Receptor promiscuity --- p.18 / Chapter 1.3.5 --- Agonist-induced conformational changes --- p.19 / Chapter 1.3.5.1 --- Conformational induction --- p.19 / Chapter 1.3.5.2 --- Conformational selection --- p.19 / Chapter 1.3.6 --- Receptor/G-protein system --- p.20 / Chapter 1.3.7 --- Implication of agonist trafficking --- p.21 / Chapter 1.4 --- Receptor switching --- p.22 / Chapter 1.4.1 --- Introduction --- p.22 / Chapter 1.4.2 --- Receptor switching --- p.23 / Chapter Chapter 2 --- Prostacyclin receptors --- p.34 / Chapter 2.1 --- General properties of prostacyclin --- p.34 / Chapter 2.1.1 --- Synthesis of prostacyclin --- p.34 / Chapter 2.1.2 --- Prostacyclin and its mimetics --- p.34 / Chapter 2.1.3 --- Characterization of IP-receptors --- p.36 / Chapter 2.1.3.1 --- Classification of IP-receptors --- p.36 / Chapter 2.1.3.2 --- Distribution of IP-receptors in the peripheral system --- p.36 / Chapter 2.1.3.3 --- Distribution of IP-receptors in the central nervous system --- p.38 / Chapter 2.1.3.4 --- Structure of IP-receptors --- p.39 / Chapter 2.1.4 --- Anti-thrombotic role of prostacyclin --- p.40 / Chapter 2.1.5 --- Cytoprotective role of prostacyclin --- p.41 / Chapter 2.1.6 --- Role of prostacyclin in adipogenesis --- p.42 / Chapter 2.2 --- Coupling capacity of IP-receptors --- p.43 / Chapter 2.2.1 --- Introduction --- p.43 / Chapter 2.2.2 --- Interaction with Gs --- p.44 / Chapter 2.2.3 --- Interaction with Gi --- p.45 / Chapter 2.2.4 --- Interaction with Gq --- p.45 / Chapter 2.2.5 --- Interaction with PPARs --- p.43 / Chapter 2.2.6 --- IP-receptor isoprenylation --- p.49 / Chapter 2.3 --- Regulation of IP-receptors --- p.50 / Chapter 2.3.1 --- Mechanism of signal termination --- p.50 / Chapter 2.3.2 --- Desensitization of IP-receptors --- p.51 / Chapter 2.3.3 --- Internalization of IP-receptors --- p.53 / Chapter Chapter 3 --- Materials and solutions --- p.59 / Chapter 3.1 --- Materials --- p.59 / Chapter 3.2 --- "Culture media, buffers and solutions" --- p.53 / Chapter 3.2.1 --- Culture media --- p.63 / Chapter 3.2.2 --- Buffers --- p.64 / Chapter 3.2.3 --- Solutions --- p.65 / Chapter Chapter 4 --- Methods --- p.67 / Chapter 4.1 --- Cell culture --- p.67 / Chapter 4.2 --- Mammalian cell transfection --- p.68 / Chapter 4.2.1 --- Preparation of plasmid DNA --- p.68 / Chapter 4.2.2 --- Transient transfection of mammalian cells --- p.71 / Chapter 4.2.3 --- β-galactosidase assay --- p.73 / Chapter 4.2.3.1 --- Introduction --- p.73 / Chapter 4.2.3.2 --- Preparation of cell lysate --- p.73 / Chapter 4.2.3.3 --- Micro β-galactosidase assay --- p.73 / Chapter 4.3 --- Measurement of adenylate cyclase activity --- p.74 / Chapter 4.3.1 --- [3H]-adenine prelabelling method --- p.74 / Chapter 4.3.1.1 --- Preparation of columns --- p.74 / Chapter 4.3.1.2 --- Incubation of cells --- p.75 / Chapter 4.3.1.3 --- Measurement of [3H]-cyclic AMP production --- p.75 / Chapter 4.3.1.4 --- Data analysis --- p.76 / Chapter 4.3.2 --- cAMP [125I]-scintillation proximity assay (SPA) --- p.77 / Chapter 4.3.2.1 --- Introduction --- p.77 / Chapter 4.3.2.2 --- Cell lysis method --- p.77 / Chapter 4.3.2.3 --- cAMP [I25I]-scintillation proximity assay --- p.78 / Chapter 4.4 --- Measurement of phospholipase C activity --- p.78 / Chapter 4.4.1 --- Introduction --- p.78 / Chapter 4.4.1.1 --- Preparation of columns --- p.78 / Chapter 4.4.1.2 --- [3H]-inositol phosphate assay --- p.79 / Chapter 4.4.1.3 --- Measurement of [3H]-inositol phosphate production --- p.79 / Chapter 4.4.1.4 --- Data analysis --- p.80 / Chapter 4.4.2 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H] assay system" --- p.81 / Chapter 4.4.2.1 --- Introduction --- p.81 / Chapter 4.4.2.2 --- Sample preparation --- p.81 / Chapter 4.4.2.3 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H] assay system" --- p.82 / Chapter 4.5 --- Measurement of changes in intracellular Ca2+ concentration --- p.82 / Chapter 4.5.1 --- Introduction --- p.82 / Chapter 4.5.2 --- Cell preparation --- p.83 / Chapter 4.5.3 --- Measurement of Fura-2 fluorescence --- p.83 / Chapter 4.6 --- Radioligand binding --- p.84 / Chapter 4.6.1 --- Introduction --- p.84 / Chapter 4.6.2 --- [3H]-iloprost ligand binding --- p.85 / Chapter 4.6.3 --- Data analysis --- p.86 / Chapter 4.7 --- Cytotoxicity test using trypan blue exclusion test --- p.86 / Chapter Chapter 5 --- Multiple coupling capacity of prostacyclin receptors in CHO cells --- p.88 / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Method --- p.88 / Chapter 5.3 --- Results and Discussion --- p.89 / Chapter 5.3.1 --- IP agonist log concentration-response curves for [3H]-cAMP and [3H]-inositol phosphate production in mIP-CHO cells --- p.89 / Chapter 5.3.2 --- Effect of varying Gαs cDNA concentration on cicaprost-stimulated [3H]-cAMP and [3H]-inositol phosphate production in mlP-CHO cells --- p.90 / Chapter 5.3.3 --- Effect of varying Gαq cDNA concentration on cicaprost-stimulated [3H]-cAMP and [3H]-inositol phosphate production in mlP-CHO cells --- p.92 / Chapter 5.4 --- Conclusion --- p.95 / Chapter Chapter 6 --- Multiple coupling capacity of prostacyclin receptors in neuroblastoma cells --- p.113 / Chapter 6.1 --- Introduction --- p.113 / Chapter 6.2 --- Method --- p.114 / Chapter 6.3 --- Results and Discussion --- p.114 / Chapter 6.3.1 --- Characterization of prostanoid receptors in SK-N-SH cells --- p.114 / Chapter 6.3.2 --- Property of IP-receptor signaling in SK-N-SH cells --- p.116 / Chapter 6.3.3 --- Effect of Gαq subunits on PLC activation in SK-N-SH cells --- p.117 / Chapter 6.3.4 --- Coupling capacity of IP-receptorin rat/mouse neuroblastoma (NG108-15) cells --- p.119 / Chapter 6.4 --- Conclusion --- p.123 / Chapter Chapter 7 --- Agonist trafficking --- p.133 / Chapter 7.1 --- Introduction --- p.133 / Chapter 7.2 --- Method --- p.134 / Chapter 7.3 --- Results and Discussion --- p.134 / Chapter 7.3.1 --- Simultaneous measurement of AC and PLC activation in metabolically-labelled mIP-CHO cells --- p.134 / Chapter 7.3.1.1 --- Effect of IBMX on PLC activation --- p.135 / Chapter 7.3.1.2 --- Effect of Li+ ion on AC activation --- p.135 / Chapter 7.3.1.3 --- Separation of [3H]-adenine and [3H]-inositol using column chromatography method --- p.136 / Chapter 7.3.2 --- Measurement of AC and PLC activation using different assay systems --- p.137 / Chapter 7.3.2.1 --- cAMP 125I-Scintillation Proximity Assay System --- p.137 / Chapter 7.3.2.2 --- "D-myo-Inositol 1,4,5-trisphosphate (IP3) [3H]-assay system" --- p.138 / Chapter 7.4 --- Conclusion --- p.139 / Chapter Chapter 8 --- Regulation of prostacyclin receptor coupling --- p.147 / Chapter 8.1 --- Introduction --- p.147 / Chapter 8.2 --- Methods --- p.149 / Chapter 8.3 --- Results and Discussion --- p.150 / Chapter 8.3.1 --- Role of protein kinases on IP-receptor activity --- p.150 / Chapter 8.3.2 --- Effect of SQ22536 on IP-receptor activity --- p.155 / Chapter 8.3.3 --- Role of PKA phosphorylation site in mIP-receptor activity --- p.156 / Chapter 8.4 --- Conclusion --- p.157 / Chapter Chapter 9 --- Prostacyclin receptor isoprenylation --- p.171 / Chapter 9.1 --- Introduction --- p.171 / Chapter 9.2 --- Method --- p.172 / Chapter 9.3 --- Results and Discussion --- p.173 / Chapter 9.4 --- Conclusion --- p.175 / Chapter Chapter 10 --- IP/DP chimeric receptors --- p.178 / Chapter 10.1 --- Introduction --- p.178 / Chapter 10.2 --- Method --- p.179 / Chapter 10.3 --- Results and Discussion --- p.180 / Chapter 10.3.1 --- Property of IP/DP chimeric receptors --- p.180 / Chapter 10.3.2 --- "ID1 chimeric receptor mutant receptors (M4, M5, M6)" --- p.182 / Chapter 10.3.3 --- "Mutant mIP-receptors (Ml, M2, M3)" --- p.183 / Chapter 10.3.4 --- Comparison between M5 and ID1 receptors --- p.184 / Chapter 10.4 --- Conclusion --- p.184 / Chapter Chapter 11 --- General Discussion and Conclusions --- p.193 / Chapter 11.1 --- Introduction --- p.193 / Chapter 11.2 --- Multiple coupling capacity of prostacyclin receptors --- p.193 / Chapter 11.3 --- Factors influencing prostacyclin receptor coupling --- p.196 / Chapter 11.4 --- Prostacyclin receptor cross talk and regulation --- p.198 / References --- p.200

Gram-negative bacteria decorate their outermost surface structure, lipopolysaccharide, with elaborate chemical moieties, which effectively disguises them from immune surveillance and protects them from the onslaught of host defenses. Many of these changes occur on the lipid A component of lipopolysaccharide, which is crucial for host recognition of Gram-negative infection. Despite its highly inflammatory nature, LPS is a molecule with remarkable therapeutic potential. Lipid A is a glycolipid that serves as the hydrophobic anchor of LPS and constitutes a potent ligand of the TLR4/MD2 receptor of the innate immune system. A less toxic mixture of mono-phosphorylated lipid A species (MPL) recently became the first new FDA-approved adjuvant in over 70 years. Whereas wild-type E. coli LPS provokes strong inflammatory MyD88-mediated TLR4 signaling, MPL preferentially induces less inflammatory TRIF-mediated responses. Here, we developed a system for combinatorial structural diversification of E. coli lipid A yielding a spectrum of bioactive variants that display distinct TLR4 agonist activities and cytokine induction. Mice immunized with engineered lipid A/antigen emulsions exhibited robust IgG titers indicating the efficacy of these molecules as adjuvants. Other types of modification to the lipid A domain, such as altering the length of the fatty acyl chains that anchor LPS to the cell membrane, were found to affect bacterial fitness but not drastically influence detection by the TLR4/MD2 receptor. Overall, this combinatorial approach demonstrates how engineering lipid A can be exploited to generate a spectrum of immunostimulatory molecules for vaccine and therapeutics development as well as for a deeper understanding of bacterial membrane biogenesis. / text