The dose-response ototoxic effects of cisplatin were studied in guinea pigs. Loss of Preyer reflex and suppression of the N1 amplitude occurred in cisplatin-treated animals and was described as dose-related. Drug-induced hair cell damage, as observed with scanning electron microscopy, occurred sporadically throughout the turns of the cochlea and the incidence increased with dose. Na, K-ATPase activity in the lateral wall tissues was not significantly different between treatment groups. The results reported here indicate that cisplatin ototoxicity was dose-dependent, but was not directly related to Na,K-ATPase activity in the lateral wall.

In the first part of this study, the kinetics of decay of TNP-nucleotide superfluorescence was investigated with a view to understanding the role of nucleotides and Lys492 in later steps in the catalytic cycle of the skeletal muscle Ca²⁺ATPase. It has been found previously, and verified here, that tethering TNP-8N₃-AMP to the Ca²⁺ATPase via Lys492 retarded the Ca²⁺ initiated decay of Pᵢ-induced superfluorescence 10-fold compared with untethered nucleotide. The rapidity of the decay upon addition of EDTA suggested that the E₂ ↔ E₁ → E₁Ca₂ steps were being monitored rather than dephosphorylation per se. Tethered diand triphospho species did not accelerate the decay. While monophasic kinetics was observed with untethered TNP-AMP and TNP-8N₃-AMP, complex kinetics were observed with the di- and triphospho TNP-nucleotides. This was shown to be due to the utilization of TNP-ADP and -ATP, and the azido derivatives, as coupled substrates of the Ca²⁺ATPase in the forward direction of catalysis in the presence of Ca²⁺. The hydrolysis rates of TNP-ADP, TNP-ATP, TNP-8N₃ -ADP, and TNP-8N₃ -ATP were 10, 5, 15 and 10 nomoles/min/mg of protein, respectively, at room temperature and pH 5.5. Ca²⁺ transport was supported by all four nucleotides. This is the first time that a diphosphonucleotide has been shown to support Ca²⁺ transport. A new nonhydrolysable triphospho TNPnucleotide, TNP-AMP-PCP was synthesized and shown to interact with the Ca²⁺ATPase in a similar way, in terms of superfluorescence, as the other TNP-nucleotides. It did not show the complex kinetics on inhibition of the Pcinduced superfluorescence by Ca²⁺, but neither did it accelerate the kinetics. It was concluded that TNP-nucleotides do not accelerate the E₂ ↔ E₁ transition under these conditions, possibly because of the presence of glycerol in the medium. In the second part of the study, it was shown that addition of small amounts of chelators EGTA, EDTA, BAPTA, DTPA, HEDTA and NTA to a Ca²⁺ transport assay in which the free Ca²⁺ concentration is monitored by Fluo-3 causes the Ca²⁺ATPase to pump to apparently lower levels as seen in the [Ca²⁺] lim fluorescence. Addition of chelator retards pump function in the sense that it takes longer for 50 nmols Ca²⁺ to be accumulated. Increased thermodynamic efficiency of the pump and contaminating heavy metal ions were considered as possible mechanisms. To some extend Zn²⁺ and Cd²⁺, but not Fe²⁺ and Cu²⁺, appeared to reverse the partial inhibition. While interpretation of the results is difficult, it is suggested that heavy metal ions interact with luminal loops of the Ca²⁺ATPase and enhance Ca²⁺ release under conditions of high luminal Ca²⁺ concentrations.

V-ATPasen kommen bei Saccharomyces cerevisiae in den Membranen der Vakuole und des Golgiapparats vor. Durch ihre Aktivität wird das Lumen dieser Organellen, wenn auch in unterschiedlichem Maße, angesäuert. Da die V-ATPase ATP verbraucht, erscheint eine strikte Regulierung der Enzymaktivität unter Hungerbedingungen zwingend notwendig. Der Hauptregulationsmechanismus zur Deaktivierung der V-ATPase ist die reversible Dissoziation. In der vorliegenden Doktorarbeit wurden einige Faktoren hinsichtlich ihrer Beteiligung an der reversiblen Dissoziation der V-ATPase durch in vivo Beobachtungen an einzelnen Hefezellen untersucht. Fluoreszenzmikroskopische Beobachtungen unter in vivo-Bedingungen zeigten, dass nur die V1-Untereinheit C dem Mechanismus der reversiblen Dissoziation unterliegt und nicht, wie bisher angenommen, der gesamte V1-Komplex. Die restlichen V1-Untereinheiten verbleiben weiterhin an oder in der Nähe der Vakuolenmembran. Durch Depolymerisierung des Zytoskeletts zeigte sich eine Beteiligung der Mikrotubuli an der Dissoziation, während Aktin keinen Einfluss auf die reversible Dissoziation der Untereinheit C hatte. Mit Hilfe von Overlayblots und Co-Pelletierungsversuchen konnte eine direkte Interaktion der Untereinheit C mit den Mikrotubuli nachgewiesen werden. Fluoreszenzmikroskopische Lokalisationsstudien der Untereinheit C erbrachten bei Behandlung der Zellen mit unterschiedlichen Agenzien den Beweis, dass für die Dissoziation der Untereinheit C die V-ATPase nicht nur aktiv sein, sondern dass auch das intrazelluläre Lumen der Vakuole einen sauren pH-Wert haben muss. Die Reassoziation erfolgt jedoch unabhängig von diesen Faktoren. Ein anscheinend weiterer wichtiger Faktor bei der reversiblen Dissoziation der Untereinheit C ist der cAMP-PKA-Signalweg. Es konnte gezeigt werden, dass der Glucosesensor Gpr1 dabei keine Rolle spielt, während die Deletion der PKA Isoformen die Dissoziation von C inhibiert. Eine überaktive PKA durch Deletion der regulatorischen Untereinheit Bcy1 hatte hingegen eine Inaktivierung der V-ATPase zur Folge, was zum Verlust der Dissoziationsfähigkeit führte. Die Behandlung mit dem PKA Inhibitor H89 erbrachte den Nachweis, dass die PKA für die Reassoziation der Untereinheit C nicht gebraucht wird. Eine Phosphorylierung der Untereinheit C im Holoenzym durch die PKA zur Initiierung der Dissoziation scheint jedoch unwahrscheinlich. Stattdessen ist eine indirekte Beteiligung der PKA durch Regulierung von Stoffwechselwegen wahrscheinlicher, nachdem durch die TAP-Reinigung Enzyme der Glykolyse als Interaktionspartner von C gefunden wurden. Alle in der vorliegenden Arbeit gefundenen Faktoren spielen anscheinend eine Rolle bei der Dissoziation, nicht aber bei der Reassoziation. Im Gegensatz dazu ergab die Deletion des RAVE-Komplexes einen ersten Hinweis für eine Beteiligung an der Reassoziation bzw. Assemblierung, da Untereinheit C in diesem Stamm eine zytoplasmatische Lokalisation zeigte und die anderen V1-Untereinheiten eine Lokalisation an der Vakuole und im Zytoplasma.

Several bacterial pathogens including Shigella (shigellosis), Escherichia coli (urinary tract infections), Pseudomonas (lung infections), Salmonella (food poisoning), and Yersinia (plague) critically rely on a complex type three secretion system (T3SS) for infection. With the rise in multi-antibiotic resistant strains of several of these pathogens, we turn to the T3SS as a promising target for the development of novel therapeutics. The Dickenson lab at Utah State University has been the first to identify and characterize the ATPase Spa47, the energy source of the Shigella infection system. We show that Spa47 is necessary for proper T3SS formation and function, being ultimately responsible for overall Shigella virulence. We find that proper ATPase function and in turn T3SS apparatus formation can be affected by something as simple as a single mutation to the removal of a non-catalytic domain. The insights gained from this work expands our understanding of the powerhouse that fuels these infection systems and brings us a step closer to developing novel therapeutics to combat infection.

The mechanisms mediating the hypertension of preeclampsia (PE) are unclear. Endogenous digitalis-like factors (EDLFs) are specific sodium pump (SP) inhibitors implicated in essential and experimental hypertension, but they have not been fully explored in the setting of PE. This study uses a digoxin antibody Fab fragment to address the question of whether such factors are present and increased in PE, to investigate a possible treatment of PE, and to isolate and characterize all EDLFs present in PE. Sera and placenta from women with PE did show a significant increase in SP inhibition in comparison to women with normal pregnancy and Digibind® was found to bind EDLFs and essentially block or reverse SP inhibition. Sera were collected in a Phase II, double-blind, placebo controlled clinical study in which women with severe preeclampsia were dosed with Digibind®, as a therapeutic, and the SP activity measured. Sera and placenta from women with PE was also investigated for their inhibitory effects on the SP. Known candidates for EDLFs were investigated for their SP inhibitory effects, as well as how digitalis antibody immune Fab fragments, Digibind® and DigiFab™, bound them and affected the SP activity. Digibind® is also a sufficient affinity material used to isolate and purify PE EDLFs. Additionally, the placentas of preeclamptic women have high levels of similar EDLFs. These studies provide evidence for the existence of EDLFs that circulate in women with PE, and Digibind® is an effective and novel tool to bind, isolate and purify EDLFs in PE.

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