Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references (leaves 102-113).

Turgor regulation is the process by which walled organisms alter their internal osmotic potential to adapt to osmotic changes in the environment. Much of what we know regarding turgor regulation and osmotic adjustment in algae is limited to the green characean and chlorphytan algae. This thesis is an investigation of turgor regulation in two species of the yellow-green xanthophycean alga, Vaucheria. The first part of this study involved the collection and identification of species of Vaucheria from contrasting habitats in New Zealand. Seven species of Vaucheria were identified based on the morphology of their reproductive structures. Two were described as new species (V. aestuarii and V. edaphica) and two others were reported for the first time from New Zealand (V. erythrospora and V. litorea). The genetic variation and phylogenetic position of these species were studied using phylogenetic analyses of rbcL sequences. Two of the species from contrasting habitats were selected for a comparative study on turgor regulation. These were Vaucheria erythrospora, isolated from an estuarine habitat, and Vaucheria repens, isolated from a freshwater habitat. Using a single cell pressure probe to directly measure turgor after hyperosmotic shock, V. erythrospora was found to recover turgor after a larger shock than V. repens. Threshold shock values for this ability were > 0.5 MPa for V. erythrospora and < 0.5 MPa for V. repens. Recovery was more rapid in V. erythrospora than V. repens after comparable shocks. Growth studies showed that V. erythrospora was able to grow and maintain turgor over a wider range of NaCl concentrations. These responses are thought to underlie the ability of V. erythrospora to survive in an estuarine habitat and restrict V. repens to freshwater. xi The final part of this study investigated the mechanisms underlying turgor regulation in V. erythrospora. Different responses were observed depending on whether NaCl or sorbitol was used to elicit the shock. Membrane potential (Em) measurements showed a rapid depolarization of the plasma membrane in response to a NaCl-induced hyperosmotic shock, followed by a slower repolarization, and recovery almost back to the resting Em. MIFE recordings indicate a net K+ efflux, a response that has been reported in other systems. While recordings of Na+ fluxes were not possible due to the high external Na+, these may account for the depolarisation and recovery of turgor as turgor recovery was inhibited by the non-selective cation channels (NSCCs) inhibitor Gd3+ and was dependant on the external Na+ concentration. An equivalent sorbitol-induced hyperosmotic shock hyperpolarized the Em, followed by depolarization and recovery back to the resting Em. Net flux recordings showed that both K+ and Na+ were taken up in response to a sorbitol shock when there was a low external Na+ concentration (1mM). K+ was possibly taken up through inward rectifying K+ channels activated by membrane hyperpolarization. The ability of V. erythrospora to rapidly regulate turgor by taking up ions during hyperosmotic stress is the possible reason for its survival in an estuarine habitat.

5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) was normally recognised as a Cl- channel inhibitor, but its specificity is in question, since an inhibitory effect against K⁺ channels has been reported. To identify the significance of the molecules structural components, NPPB and related compounds, such as 2-(3-phenylpropylamino) benzoic acid (PPAB), 5- nitro-2-heptylamino benzoic acid (HANB) and 2-nitro-5-heptylamino benzoic acid (HANB-2) were synthesised by reductive amination using various aldehydes and amines. Using internodal cells of the giant green Characean algae, Nitella hookeri, the effects of NPPB and related compounds on cytoplasmic streaming and turgor regulation were determined. Previous experiments stated that cytoplasmic streaming was sensitive to NPPB, PPAB and HANB with IC₅₀ values of 24µmol/L, 455µmol/L, and 6.4mmol/L, respectively. In this report, the IC₅₀ values of purchased NPPB and niflumic acid were found to be 88.65µmol/L and 121.82µmol/L, respectively. Although the IC₅₀ value of purchased NPPB showed a slight difference from that of synthesised NPPB, the results of the cytoplasmic streaming experiment indicated the possibility of this analysis to be a simple assay system for analysing the effects of structural modification to ion channel inhibitors on their biological activity. Moreover, NPPB and PPAB seem to stimulate regulation of turgor pressure under hyperosmotic shock, which can be explained by a blockage of K⁺ efflux during osmotic stress leading to faster recovery of turgor regulation. Additionally, the results of cytosolic free Ca²⁺ analysis using aequorin technology also suggested that the possibility of this analysis to be used as a more direct measure of the inhibitory effect, while the cytoplasmic streaming analysis is a more indirect method. The preliminary results from this research suggest the significance of the simple assay systems for analysing the effects of structural modification ion channel inhibitors, which can be used for future study regarding ion channel structures.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (leaves 273-295).

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 105-113). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Membranproteine spielen eine wichtige Rolle in vielen biochemischen Prozessen der Zelle, wie zum Beispiel der Signaltransduktion, der Zelladhesion oder auch der Erkennung von Krankheitserregern. Viele dieser Proteine sind von Bedeutung für die Entwicklung neuer innovativer Medikamente. Somit hat auch die Entwicklung von Sensoren, die die Untersuchung von Membranproteinen in ihrer natürlichen Umgebung erlauben an Bedeutung gewonnen [1]. Thema dieser Doktorarbeit war die Entwicklung von Analysekonzepten die es ermöglichen unterschiedliche Aspekte von Membraninteraktionen zu untersuchen und zu quantifizieren. Als Analysemethode wurde dafür reflektometrische Interferenz Spektroskopie (RIfS) eine markierungsfreie, optische Methode verwendet. RIfS erlaubt es die Höhe dünner transparenter Filme zu bestimmen, indem das Weißlicht-Reflexionspektrum eines solchen Films aufgezeichnet wird. Durch die Überlagerung der in dem Film mehrfach reflektierten Teilstrahlen entsteht ein Interferenzmuster im Reflexionsspektrum, welches Aufschluß gibt über die Schichtdicke und den Brechungsindex des transparenten Films. Es wurde bereits gezeigt, dass RIfS eine geeignete Methode zur Untersuchung von Protein-ProteinWechselwirkungen ist [2]. Aus diesem Grund wurde RIfS als Detektionsverfahren für die Entwicklung eines Membransensors gewählt. Im Laufe dieser Arbeit entstanden zwei Aufbauten für reflektometrische Messungen. Ein Standard RIfS Aufbau und ein Instrument das die Methode mit Fluoreszenz-Mikroskopie kombiniert. Um dieWechselwirkung von Proteinen selbst und Proteinen mit Membranbestandteilen wie Lipiden zu untersuchen, wurde ein Konzept basierend auf festkörperunterstützten Membranen entwickelt. Dieses Experiment erlaubt es die Wechselwirkungen auf artifiziellen Membranen, sowie auf rekonstituierten Zellmembranen zu untersuchen. Zudem wurde ein Analysekonzept mit Nano-BLMs entwickelt, dass es erlaubt den simultanen Transport von Molekülen in ein membranverschlossenes Kompartiment hinein als auch heraus zu beobachten. Neben diesen membranbasierten Experimenten wurde auch ein Konzept entwickelt, welches es erlaubt die molekulare Erkennungsreaktion von sehr kleiner Analyten direkt zu messen. Dieses Messkonzept erlaubt es die Bindung von Molekülen mit sehr kleinem Molekulargewicht an einen auf dem Sensor immobilisierten Partner direkt zu quantifizieren.

The glucose transport protein, GLUT1, is highly expressed in rapidly proliferating cells, including cancer cells, while decreased GLUT1 levels are found in diseases such as GLUT1 deficiency syndrome and Alzheimer’s. There is increased interest in developing GLUT1 inhibitors as novel anticancer therapeutics, and the discovery of compounds that directly stimulate GLUT1 function. This work investigates how small molecules stimulate and/or inhibit GLUT1-mediated glucose transport, either directly or through the AMPK pathway. Using sugar transport assays and docking analyses to explore Ligand–GLUT1 interactions and specificity of binding, we show that: 1) Ligands inhibit GLUT1 by competing with glucose for binding to the exofacial or endofacial sugar binding sites; 2) Subsaturating inhibitor concentrations stimulate sugar uptake; 3) Ligands inhibit GLUT1–, GLUT3– and GLUT4–mediated sugar uptake in HEK293 cells; and 4) Inclusion of a benzonitrile head group on endofacial GLUT1 inhibitors confers greater inhibitory potency. Furthermore, we investigated AMPK-regulated GLUT1 trafficking in cultured blood-brain barrier endothelial cells, and show that inhibition of GLUT1 internalization is not responsible for increased cell surface levels of GLUT1 observed with AMPK activation in these cells. This study provides a framework for screening candidate GLUT1 inhibitors for specificity, and for optimizing drug design and delivery. Our data on transport stimulation at low inhibitor concentrations support the idea that GLUT1 functions as a cooperative oligomer of allosteric alternating access subunits.

Organic cations (OCs) make up ~40% of all prescribed drugs and renal secretion plays a major role in clearing these (and other OCs), from the plasma. The active and rate-limiting step of renal OC secretion is mediated by luminal OC/H+ exchange, the molecular basis of which is suspected to involve two homologous transport proteins, Multidrug And Toxin Extruders 1&2-K (MATE1 and MATE2-K). This study has two aims to resolve outstanding issues dealing with the mechanism of MATE-mediated OC transport: (Aim 1) develop predictive models of ligand interaction with hMATE1; (Aim 2) establish the kinetic mechanism(s) of ligand interaction with MATE transporters and the extent to which inhibitory ligands serve as transported substrates of MATE transporters. Transport was measured using human MATE1 and MATE2-K stably expressed in Chinese Hamster Ovary cells. Both MATEs had similar affinities for the prototypic OC substrate, 1-methyl-4-phenylpyridinium (MPP), and had overlapping selectivity for most of the test inhibitors. The IC50 values for 59 structurally diverse inhibitory ligands were used to generate a common features (HIPHOP) pharmacophore and three quantitative pharmacophores for hMATE1 (each displaying a significant correlation between predicted and measured IC50 values). The models identified (i) structural features that influence ligand interaction with hMATE1, including hydrophobic regions, H-bond donor and acceptor sites and an ionizable feature; and (ii) novel high affinity inhibitors of MATE-mediated transport from 13 new drug classes. Whereas metformin and creatinine were shown to be competitive inhibitors of MPP, the inhibition of MATE1-mediated MPP transport produced by pyrimethamine (PYR) and related analogs was not competitive but, instead, had a "linear, mixed-type" inhibitory profile suggestive of a MATE binding surface rather than a singular binding site. "Competitive exchange diffusion" showed that selected inhibitory ligands (including quinidine, caffeine, and the organic anion, PAH) also serve as transported substrates for MATE1. In conclusion, these data are consistent with the presence of a MATE binding surface with multiple, non-overlapping binding sites that can display different kinetic interactions with structurally distinct substrates. The creation of hMATE1 pharmacophores offers insight into development and interpretation of predictive models of drug-drug interaction in the kidney.

Mammalian ABC (ATP-binding cassette) transporters are integral membrane proteins that translocate allocrites across biological membranes using ATP as a substrate. ABCB1 is a polyspecific efflux pump which can confer multidrug resistance in cancer. ABCB1 is also expressed in a variety of normal tissues where it functions to prevent the accumulation of toxic allocrites. Direct inhibition of ABCB1 can therefore have detrimental effects on patients. Identification of ABCB1-interacting partners that influence trafficking or function would therefore provide alternative targets for therapy which may be cell- or tissue-type specific. The “split-ubiquitin” yeast two-hybrid system, that can detect protein:protein interactions at the plasma membrane, was used to screen for ABCB1-interactors in a human liver library. All candidates isolated from the screen interacted with ABCB1 in a non-specific manner when subjected to strict testing. ABCB4, a close relative of ABCB1, is expressed primarily at the hepatocyte canalicular membrane where it flops phosphatidylcholine (PC) into the outer leaflet for extraction by bile salts. Many ABCB4 non-synonymous mutations have been linked to cholestatic liver diseases in humans, but data confirming an impact on ABCB4 function is lacking. Transient expression of wild-type (WT) ABCB4 in tissue culture has proved difficult because the protein is toxic to HEK293T cells. However, co-expression of the phosphatidylserine flippase ATP8B1 (FIC1) and its accessory protein CDC50A allowed the cells to tolerate ABCB4. To investigate the impact of SNPs on ABCB4 function, equivalent changes were introduced into the ABCB4 cDNA for transient expression in the presence or absence of ATP8B1/CDC50A. ABCB4 expression and targeting to the plasma membrane were monitored by western analysis and confocal microscopy, respectively, and, by “feeding” the transfected cells [methyl-3H]choline, PC efflux to added bile salt acceptor was measured. By thus mimicking the situation at the canalicular membrane I report the preliminary characterisation of nine variants of ABCB4 that have been linked to cholestatic liver disease.

The Tat protein export system transports folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. In Escherichia coli, the Tat system is composed of the TatA, TatB and TatC proteins. TatB and TatC assemble into a multimeric receptor complex that recognises and binds the substrate, before the TatA protomers cluster at the TatBC complex to facilitate substrate transport. A genetic screen was devised to explore the oligomeric state of TatC, reasoning that the isolation of dominant negative TatC variants that inactivate the Tat system in the presence of a functional copy of wild type TatC would provide strong evidence TatC is an obligate oligomer. Single dominant negative TatC substitutions were isolated that were located in the first and second periplasmic loops of TatC. These substitutions did not prevent TatC from interacting with TatB, TatA, itself or with a Tat substrate. Blue Native PAGE analysis showed that the TatC variants were unable to form the 440 kDa TatBC complex. Surprisingly, the substitutions did not prevent TatC:TatC self-interactions in the periplasmic regions, detected by disulphide cross-linking, but they did abolish a substrate-induced interaction at the fifth transmembrane helix of TatC. Fluorescence microscopy experiments revealed that the dominant negative TatC variants prevented the polymerisation of TatA-YFP in vivo. These results show that TatC possesses at least two interaction interfaces and imply that the periplasmic loops are critical for the transition between substrate binding and TatA polymerisation. Accessibility of single cysteine substitutions in TatC was probed by PEG-Mal labelling in intact cells. TatB was shown to be important for the proper insertion of TatC into the membrane. The absence of TatA led to accessibility changes in the vicinity of the fifth transmembrane domain of TatC, where both TatA and TatB are known to dock. This suggests that TatA and TatB may share an overlapping binding site.