Nitric oxide (NO), a free radical released by macrophages (a subset of white blood cells) as a response to infection, is noxious to organisms due to its ability to disable crucial biomolecules such as lipids, proteins and DNA. Although normally effective at eradicating invading bacteria, several pathogens have developed mechanisms to detoxify NO and its toxic by-products, reactive nitrogen species (RNS). While some of these detoxification processes have been characterized, very little is known about the metabolic changes that enable microbes to survive this deleterious environment. Investigations into the effects of RNS on microbial physiology have shown that these harmful radicals inactivate the citric acid cycle and oxidative phosphorylation, the series of reactions responsible for making energy aerobically. The central aim of this thesis was to determine how the organism counteracts the detrimental effects of RNS, while bypassing the ineffective central metabolic pathways. The findings presented herein show that P. fluorescens engineers an elaborate metabolic network to generate ATP whilst withstanding the injurious effects of nitrosative stress. Crucial to this adaptation is the ability to produce energy via substrate level phosphorylation, a necessity that arises out of the cells’ inability to produce a substantial amount of ATP using the electron transport chain (ETC). The up-regulation of the enzymes citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate, phosphate dikinase (PPDK) helps the organism accomplish this feat. Blue native polyacrylamide gel electrophoresis (BN-PAGE), high performance liquid chromatography (HPLC) as well as co-immunoprecipitation (CO-IP) studies were applied to demonstrate that these proteins form a metabolon, a transient complex of enzymes that ensures citrate is converted into its desired end products, pyruvate and ATP. In order to gauge the individual contributions iv of phosphoenolpyruvate-dependent kinases, a novel in-gel activity assay was developed to probe these enzymes under disparate conditions. These results suggest that the organism switches from an ATP-dependent metabolism to one based on the utilization of pyrophosphate (PPi). The rationale for this appears to be energy efficiency, as pyrophosphate-dependent glycolysis can theoretically produce five ATP rather than the two yielded by Embden-Meyerhof glycolysis. Additionally, the up-regulation in activity of the enzymes adenylate kinase, nucleoside diphosphate kinase and acetate kinase seem to ensure that ATP generated by PPDK is properly shuttled and stored when aerobic metabolism is defective. The lower activity of inorganic pyrophosphatase likely ensures an adequate supply of pyrophosphate for the activity of PPDK. Taken together, this research reveals the critical role metabolism plays in the survival of microbes under the onslaught of NO and RNS. As several of these enzymes are absent in mammalian systems, they present themselves as novel targets for the development of new antibacterial agents. A comprehensive awareness of bacterial defense systems in response to NO may lay the groundwork to developing more effective treatments to impede microbial infections.

A key step in malignant progression is the acquired ability of tumour cells to escape immune-mediated lysis. A potential mechanism by which tumour cells avoid immune destruction involves the shedding of MHC Class I Chain-Related Protein A (MICA), a Natural Killer (NK) cell-activating ligand, from the tumour cell membrane. Hypoxia has been shown to cause increased MICA shedding; however, this hypoxia-induced effect can be attenuated by pharmacological activation of the cyclic guanosine monophosphate (cGMP)-dependent nitric oxide (NO)-signalling pathway in cancer cells. The primary objective of the present study was to determine whether treatment of tumour-bearing nude mice with the NO-mimetic glyceryl trinitrate (GTN) attenuates in vivo tumour growth and if so, whether this effect is dependent on the presence of an intact NK cell compartment. Results indicated that continuous transdermal administration of GTN (1.8 µg/h) can significantly attenuate the growth of transplanted human DU-145 prostate tumours but that this effect of GTN is lost in mice whose NK-cells have been depleted. Tumours and serum from the mice in this study were analysed to determine whether GTN treatment had any effect on the expression levels of proteins integral to the proposed MICA shedding mechanism; however, the results of these studies were inconclusive. As phosphodiesterase (PDE) inhibition represents a potential method to enhance NO-signalling, experiments were performed to determine whether treatment with the PDE5/6 inhibitor zaprinast could also attenuate hypoxia-induced MICA shedding and decrease in vivo growth of DU-145 tumours. Results demonstrated that treatment with zaprinast (10 mg/kg) significantly attenuates MICA shedding in DU-145 cancer cells and significantly decreases in vivo tumour growth. Taken together, the results of these experiments indicate that GTN attenuates tumour growth by sensitising tumour cells to innate immunity, likely by increasing membrane-associated tumour cell MICA levels through the reactivation of NO-signalling, and that zaprinast decreases tumour growth likely through a similar mechanism. These findings are important because they indicate that agents capable of reactivating NO-signalling, such as NO-mimetics and PDE inhibitors, can potentially be used as immunosensitisers in the treatment and/or prevention of cancer. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2011-04-18 10:43:28.077

Background: Anthracyclines, in particular Doxorubicin (DOX), are highly effective chemotherapeutic agents in the breast cancer setting, which are limited by their cardiotoxic side effects. Recently, the introduction of Trastuzumab (TRZ), a novel monoclonal antibody against the HER2 receptor, in the breast cancer setting compounds the issue of DOX mediated cardiac dysfunction. Amongst the potential mechanisms for the deleterious effects of this drug-induced cardiomyopathy, the relationship between nitric oxide synthase 3 (NOS3) and oxidative stress has gained recent attention. Objective: To determine the role of NOS3 in a clinically relevant female murine model of DOX+TRZ induced heart failure. Methods: A total of 120 C57Bl/6 female mice [60 wild type (WT) and 60 NOS3 knockout (NOS3-/-)] were treated with either 0.9% saline, DOX (20 mg/kg), TRZ (10 mg/kg), or DOX+TRZ. Serial echocardiography was performed daily for a total of 10 days, after which the mice were euthanized for histological and biochemical analyses. Results: As compared to WT, NOS3-/- mice demonstrated increased cardiotoxicity following treatment with DOX. This effect was potentiated with DOX+TRZ combination therapy. In WT female mice receiving DOX+TRZ, left ventricular ejection fraction (LVEF) decreased from 75±3% at baseline to 46±2% at day 10 (p<0.05). In the NOS3-/- group, LVEF decreased from 72±3% at baseline to 35±2% at day 10 (p<0.05). LVEF was significantly lower in NOS3-/- mice than WT at day 10 in those receiving DOX+TRZ (p<0.05). As compared to WT, NOS3-/- mice also demonstrated increased mortality following treatment with DOX+TRZ, corroborating the echocardiographic findings. Histological analysis using light and electron microscopy demonstrated increased loss of cell integrity including myofibrillar degradation, cytoplasmic vacuolization, and enlargement of the smooth endoplasmic reticulum in both the WT and NOS3-/- mice treated with DOX+TRZ. There was no significant difference, however, in the degree of cardiac remodeling between the WT and NOS3-/- groups. There was an increasing trend in the degree of cardiac apoptosis in both WT and NOS3-/- mice treated with DOX+TRZ therapy. Conclusion: Congenital absence of NOS3 potentiates the cardiotoxic effects of DOX+TRZ in an acute female murine model of chemotherapy-induced cardiomyopathy.

Ascorbic acid has been found to promote nitrosothiol decomposition via two pathways. In the first, ascorbic acid acts as a reducing agent for added or adventitious copper (II), producing copper (I). This reacts with the nitrosothiol, giving nitric oxide and disulfide as the ultimate products. The reaction requires only small quantities of ascorbic acid, and is catalytic in copper. The second pathway requires higher concentrations of ascorbic acid, the stoichiometry being one mole of ascorbic acid to two moles of nitrosothiol. The products are nitric oxide and thiol, and the reaction has been interpreted in terms of rate limiting nucleophihc attack by ascorbate at the nitroso nitrogen, followed by decomposition of the 0-nitroso ascorbate formed to nitric oxide and dehydroascorbic acid. The rate equation is first order in both the nitrosothiol and ascorbic acid, and the entropy of activation is significantly negative. pH - rate profiles reveal the ascorbate dianion is much more reactive than the monoanion, and that the neutral form has negligible reactivity. Nitrosation of thione-containing nitrogen heterocycles by nitrous acid leads to the equilibrium formation of =SNO(^+) species; large equilibrium constants are observed. The reactions exhibit many of the features generally observed in nitrosation, including catalysis by halides and thiocyanate, and some participation by dinitrogen trioxide as a nitrosating agent. The nitrosation rate constants are large, approaching values representing the encounter-controlled limit. The =SNO(^+) species are generally unstable, decomposing under acidic conditions to nitric oxide and a disulfide. Decomposition of S-nitrosated 4-thiopyridine showed hydrolysis occurs at pH 7.4, re-forming the thione. The nitroso species reacts rapidly with ascorbate, forming nitric oxide and thione.

Three bovine casein peptides, LLY, PGPIPN, and TTMPLW, have been reported to stimulate phagocytosis of sheep red blood cells by murine macrophages. TTMPLW also protected mice against Klebsiella pneumoniae infection. The purpose of this study was to investigate the effects of these three peptides on cytokine (TNF-$ alpha$ and IL-6) production and nitric oxide (NO) release by bone marrow macrophages (BMM). The peptides alone were incapable of stimulating cytokine production or NO release in naive or IFN-$ gamma$-primed BMM. However, when BMM were coincubated with the peptides (1.0 $ mu$M) and LPS (100 ng/ml), an augmentative effect on TNF-$ alpha,$ IL-6 and NO production was observed. The peptides increased the response of BMM to stimulation with LPS in a dose- and time-dependent manner. Of the three peptides, TTMPLW (0.01, or 1.0 $ mu$M) had the greatest augmentative effect on NO production by LPS-stimulated BMM. Tumor necrosis factor-$ alpha$ production peaked after 4 hr stimulation, and decreased rapidly thereafter. Among three peptides, TTMPLW induced the highest amount of TNF-$ alpha$ production at a concentration of 1.0 $ mu$M. When used at a concentration as low as 0.01 $ mu$M, TTMPLW and PGPIPN, but not LLY, potentiated TNF-$ alpha$ production. All the peptides (1.0 $ mu$M) stimulated IL-6 production by BMM, which plateaued after 12 hr. The auto/paracrine TNF-$ alpha$ produced by LPS-stimulated BMM was partially responsible for release of NO. After all the TNF-$ alpha$ was neutralized, release of NO was reduced by about 21% (P $<$ 0.01). However, neutralization of IL-1$ beta$ and IL-6 did not have any effect on NO production by LPS-stimulated BMM. These results demonstrate that bovine casein peptides can costimulate naive macrophages with LPS for proinflammatory cytokine production and NO release and may play a role in host defense against pathogens.

This thesis studied the role of spinal nitric oxide (NO) in carrageenan-induced dermal hyperalgesia in the rat hindpaw by means of intrathecal (i.t.) administration of various NO synthase (NOS) inhibitors. A spinal role for NO in carrageenan-induced thermal hyperalgesia was confirmed by dose-dependently reducing the hyperalgesia with a non-selective NOS inhibitor. Next, it was determined that a relatively selective neuronal NOS (nNOS) inhibitor and two relatively selective inducible NOS (iNOS) inhibitors were able to reduce carrageenan-induced thermal hyperalgesia. Finally, early administration of either an nNOS or an iNOS inhibitor had no significant effect on carrageenan-induced thermal hyperalgesia. However, late administration of an iNOS, but not an nNOS inhibitor, significantly reduced the thermal hyperalgesia. We therefore suggest that iNOS contributes to only the late stages of carrageenan-induced thermal hyperalgesia, while nNOS likely plays a role throughout the entire time course of the injury.

Dissertation (PhD)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Thesis (Ph.D.)--Tufts University, 2003. / Adviser: Barry A. Trimmer. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 165-178). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (M.S.)--University of North Texas, Dec., 2007. / Title from title page display. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.