Nitric oxide (NO) plays a role in regulating the rate of orthodontic tooth movement (OTM) in rat models; however, in humans this role remains less clear. In this study, samples of gingival crevicular fluid (GCF) were collected from each maxillary central incisor and first and second molar immediately before (T0), 1 hour after (T1), and 3-4 days after (T2) application of light orthodontic forces in thirteen male participants (ages 11-18 years) undergoing orthodontic therapy. NO levels were measured in each GCF sample, and significantly higher NO levels (p<0.05) were found at T1 at the buccal surfaces of the central incisors when compared to the posterior teeth. The results indicate a possible role for NO in OTM at the pressure sites of incisors at early time points. Further studies are required to determine whether NO levels in the PDL of human teeth are affected by the magnitude of an applied force.

The electronegativity of thiolate ligation in the hemeprotein nitric oxide synthase (NOS) proteins has been identified as an influence on autoinhibition in this enzyme. The mutation of a conserved tryptophan residue, which hydrogen bonds to the coordinating thiolate ligand and therefore influences its electronegativity, to either phenylalanine or tyrosine has had various effects including heme loss and dimer disruption in the inducible isoforms, while hyperactivity occurs in the neuronal isoforms. I have performed the analogous mutations in W180 of eNOSoxy, the endothelial isoform. UV/visible and resonance Raman spectroscopy have demonstrated that the mutants experienced increased basicity of the thiolate due to loss of the hydrogen bond between the mutated residue in the absence of the cofactor (6R)5,6,7,8-tetrahydrobiopterin (H4B). The mutants also displayed relative rates of NO2- production that were comparable to the nNOSoxy mutants, which is consistent with the nNOSoxy results. The presence of H4B alters porphyrin planarity, which enabled hydrogen bonding to occur in W180Y, thus restoring thiolate basicity to that of wild-type eNOSoxy. Reduced overall activities by the proteins suggest that H4B stabilizes the heme. The analogous W56 mutants of saNOS, a NOS oxygenase domain-like protein from Staphylococcus aureus (saNOS), have been previously characterized using resonance Raman spectroscopy. These mutants also exhibit increased thiolate electronegativity over wild-type. As the homodimers had already been investigated, saNOS was an ideal system in which to explore heterodimers. Heterodimers were generated through the co-expression of one wild-type and one mutated subunit, enabling the examination of each subunit individually through resonance Raman spectroscopy. The subunits of the resulting proteins were shown to have heme environments that resembled those of their corresponding homodimers. The activity of saNOS did not vary significantly for the various W56 mutants, suggesting that saNOS catalysis may be unaffected by thiolate electronegativity. / Thesis (Master, Chemistry) -- Queen's University, 2008-09-04 11:37:38.688

Blockade of the renin-angiotensin system lowers the rate of cardiovascular events in patients at risk for vascular disease and also improves endothelial function but the mechanism remains unclear. HUVECs were stimulated with Ang II (100 nM). Ang II produced a 2-fold increase in O2- production, which was measured by lucigenin-enhanced chemiluminescence. This increase was blocked by NAD(P)H oxidase inhibitor DPI, but not by eNOS inhibitor L-NAME. Ang II increased monocyte adhesion to ECs by 4.5-fold, and this increase was blocked by candesartan (AT1 receptor antagonist), DPI, L-NAME, wortmannin (PI3K inhibitor), dominant negative-AKT, and p22phox siRNA. Dominant active-AKT increased adhesion by 1.5-fold. Our findings indicate that the simultaneous activation by Ang II of eNOS and NAD(P)H oxidase leads to endothelial activation. This process can partially explain the therapeutic benefits of reducing the action of Ang II.

The use of mouthwash is a common complement to oral care. However, the physiological implication of this use, besides of effects on oral hygiene, is poorly known. The research of the gut micro flora and its implications on the host is a very active area of research today. Many important connections between the gut micro flora and obesity and diabetes have been found. These billions of bacteria are part of the immune system, they produce essential vitamins and they make inaccessible polysaccharides more digestible to the host, just to mention a few of their symbiotic roles for the host. A less explored area is the micro flora in the oral cavity. On the back of the tongue, anaerobic bacteria can reduce dietary nitrate to nitrite which then further can be reduced to nitric oxide, NO. NO is important in several important biological functions, e.g. as a signal substance, vasoregulation, mucus production and antibacterial effects. Vegetables as beetroot and spinach are dietary sources with a high nitrate content. Also drinking water and processed meats can be of relevance. Nitrite is added to processed meat for the prevention of botulism but also adds taste and color.   Experiments on humans indicate that mitochondrial efficiency increases after nitrate load, manifested as a decreased oxygen demand during physical exercise. This can also be relevant under conditions where the mitochondrial function is impaired, such as in diabetes and cardiovascular diseases. First a pilot study was made to evaluate the nitrate reducing effect from the antibacterial mouthwash. The mouthwash proved very effective. The concentrations of nitrate and nitrite in saliva was analyzed by HPLC and saliva from the antibacterial treatment showed greatly reduced concentrations of nitrite and high concentrations of nitrate. Saliva from placebo mouthwash showed high concentrations of nitrite and low concentrations of nitrate as expected. To study the importance of oral bacteria on metabolism, we performed a randomized, cross-over double-blinded study with 19 healthy males between 22-43 years. During two separate three-day periods they used an antibacterial and placebo mouthwash, respectively. On the fourth day their basal metabolic rate (BMR) was measured with an indirect calorimetric system. Moreover, samples from saliva, urine and blood were collected but these results are not included in this thesis. An earlier, unpublished study has demonstrated that nitrate administration reduces the basal metabolic rate. Accordingly, our aim was to study potential effects on the basal metabolic rate following reduction of the number of oral bacteria by aid of antibacterial mouthwash. Our hypothesis was that the reduced availability of nitrite would decrease the availability of NO in the body and manifest as an increased basal metabolic rate. The results from indirect calorimetry measurements showed no significant difference between placebo and antibacterial mouthwash, but there may be confounding factors. Further study is needed to assess the potential effects on host metabolism by these bacteria.

Nitric oxide (NO) plays a role in regulating the rate of orthodontic tooth movement (OTM) in rat models; however, in humans this role remains less clear. In this study, samples of gingival crevicular fluid (GCF) were collected from each maxillary central incisor and first and second molar immediately before (T0), 1 hour after (T1), and 3-4 days after (T2) application of light orthodontic forces in thirteen male participants (ages 11-18 years) undergoing orthodontic therapy. NO levels were measured in each GCF sample, and significantly higher NO levels (p<0.05) were found at T1 at the buccal surfaces of the central incisors when compared to the posterior teeth. The results indicate a possible role for NO in OTM at the pressure sites of incisors at early time points. Further studies are required to determine whether NO levels in the PDL of human teeth are affected by the magnitude of an applied force.

The significant role that nitric oxide plays in human physiology is linked to the ability of NO to bind to iron forming mono-nitrosyl iron complexes. Protein-bound and low-molecular-weight dinitrosyl iron complexes (DNICs) are known to form in excess NO. Studies of such biological DNICs have relied on their paramagnetism and characteristic EPR signal of g value of 2.03. It has been suggested that DNICs act in vivo as NO storage (when protein-bound) and transfer agents (when released by, for example, free cysteine). Biological DNICs, mainly resulting from iron-sulfur cluster degradation, are difficult to extract and isolate, thereby preventing their full characterization. Thus, development of synthetic DNICs is a promising approach to model and better understand the formation and function of biological DNICs, the scope of donor ligands that might coexist with Fe(NO)2 units, the redox levels of bio-DNICs, and establish other spectroscopic techniques appropriate for characterization. A series of N-heterocyclic carbene (NHC) and imidazole (Imid) complexes has been characterized as mimics of histidine-containing DNICs. The pseudo-tetrahedral L2Fe(NO)2 complexes have NO stretching frequencies and redox potentials that suggest the NHCs are slightly better donors than Imids, however the two types of ligands have similar steric properties. Both the EPR-active, {Fe(NO)2}9 and the EPR-silent, {Fe(NO)2}10 states can be accessed and stabilized by the NHC. Nitric oxide transfer studies have shown that only the {Fe(NO)2}9 complexes are capable of transferring NO to a suitable NO trapping agent. Deprotonation of the distal nitrogen functionality in the imidazolate ligands of [(Imidazole)2Fe(NO)2]- leads to aggregation forming molecular squares of {Fe(NO)2}9 units bridged by the imidazolates. These interesting tetrameric complexes are examined by X-ray diffraction, EPR, and Mössbauer studies. The paramagnetic tetrameric complexes have multiple redox events observed by cyclic voltammetry. Mössbauer spectral data of the tetrameric complexes are compared with Mössbauer data obtained for a series of NHC-containing DNICs. Iron and cobalt-containing mononitrosyl N2S2 model complexes of the nitrile hydratase enzyme active site demonstrate sulfur-based reactivity resulting in the formation of polymetallic complexes. In all cases, shifts in the nitrosyl stretching frequencies demonstrate substantial transfer of electron density from the (NO)M(N2S2) moiety to the metal-acceptor site.

Previous research has shown that nitric oxide signalling in the lateral nucleus of the amygdala is required for the consolidation of Pavlovian conditioned fear. Given the evidence that nitric oxide can act as a retrograde signalling molecule in in vitro models of memory consolidation the question arises whether this is also occurring within behavioural memory models? Using auditory fear conditioning this research shows that nitric oxide does indeed act as retrograde signalling molecule in the fear system. Its synthesis in the lateral nucleus of the amygdala regulates conditioning induced expression of the immediate early gene early growth response gene 1 (EGR-1) in cells of the auditory thalamus that project to the lateral nucleus of the amygdala. The regulation of EGR-1 expression by the lateral nucleus of the amygdala was proven to be dependent on amygdala-based cellular excitation, nitric oxide synthesis and NR2B-NMDA receptor activation but not ERK/MAPK activity. Using an EGR-1 antisense oligonucleotide to prevent training induced EGR-1 expressions in the auditory thalamus it was shown that this gene upregulation is necessary for the consolidation of conditioned fear. Finally, inhibition of EGR-1 upregulation in the auditory thalamus was proven to impair conditioning induced increases in the presynaptic proteins synaptophysin, and synapsin II and II back in the lateral nucleus of the amygdala. Overall, the results of this dissertation have shown that nitric oxide acts as a retrograde messenger in a mammalian memory system by modulating gene expression in presynaptic cells. This modulation of gene expression serves to increase levels of presynaptic proteins back at the origin of nitric oxide synthesis. This supports the long standing doctrine that nitric oxide acts as a retrograde signalling molecule to coordinate presynaptic changes associated with memory formation.

Mitochondria are essential for promoting cell survival and growth through aerobic metabolism and energy production. Mitochondrial function is typically analyzed using mitochondria freshly isolated from tissues and cells because they yield tightly coupled mitochondria, whereas those from frozen tissue can consist of broken mitochondria and membrane fragments. A method, utilizing a well-characterized cryoprotectant such as dimethyl sulfoxide (DMSO), is described. Such mitochondria show preserved structure and function that presents us with a possible strategy to considerably expand the time-frame and the range of biochemical, molecular and metabolic studies that can be performed without the constraints of mitochondrial longevity ex vivo. Mitochondrial dysfunction is implicated in Alzheimer’s disease (AD) mainly through oxidative stress and altered metabolism. Mitochondria are isolated from post-mortem brain samples from selective regions of AD and control patients and, utilizing the cryopreservation strategy, analyzed for respiration and oxidative damage. While we did not observe increases in free radicals, we did observe decreased respiration and increases in oxidative damage markers in AD patients, suggesting a role for oxidative stress in mitochondrial dysfunction. While in the mitochondria, calcium (Ca2+) increases free radical generation by processes not completely understood. A new isoform of nitric oxide synthase (mtNOS) has been isolated and localized to mitochondria; though its existence and physiological role is debated. Nitric oxide synthase (NOS), when activated by Ca2+, produces nitric oxide (NO•) that can interact with ROS producing various reactive nitrogen species (RNS). These highly reactive radical species can damage DNA, proteins and lipids, ultimately resulting in cell death via apoptosis or necrosis. The current research is aimed at understanding the role of Ca2+ and NOS in oxidative stress leading to mitochondrial dysfunction. We observed a significant reduction in mitochondrial respiration with increasing doses of calcium. We also observed NOS enzyme activity and detected NOS protein in the purified mitochondrial fraction. Lastly, we were also able to show that Ca2+ increased the levels of free radicals and changes in oxidative damage markers. These results suggest the presence of NOS in mitochondria that could play a role in Ca2+ induced mitochondrial dysfunction and potentially leading to cell death as relevant to aging and neurodegenerative diseases.

Post-translational modification is an intracellular process that modifies the properties of proteins to extend the range of protein function without spending energy in de novo peptide synthesis. There are many post-translational modifications, for example, phosphorylation, ubiquitination, and S-nitrosylation. S-Nitrosylation is a post-translational modification which adds nitric oxide (NO) to sulfhydryl groups at cysteine residues to form S-nitrosothiol (SNO), and is required for plant immunity and fertility. Cellular NO changes between a pool of free NO and bound SNO. During pathogen infection, nitrosative stress in plants is mainly controlled by Snitrosothiolglutathione reductase (GSNOR) via the decomposition of GSNO. GSNOR is an alcohol dehydrogenase type 3 (ADH3) which has both GSNOR and formaldehyde dehydrogenase (FDH) activities. The roles of S-nitrosylation in mammals overlap with those in plants. This conservation led us to explore the relationship between S-nitrosylation, immune response, and fertility in Drosophila melanogaster as it might prove to be a good genetic model for further analysis of the role of S-nitrosylation in animals. I have identified fdh as the likely gsnor in D. melanogaster and have knocked this out using an overlapping deficiency technique in order to observe the effect on immunity and fertility. There are two main pathways in the Drosophila innate immune response, the Toll pathway for protecting against gram-positive bacteria and fungi, and the Imd pathway against gram-negative bacteria. I have investigated the effect of removing GSNOR on sensitivity to gramnegative bacteria (Escherichia coli and Erwinia carotovora) by septic and oral infection, and to fungi (Beauveria bassiana). Susceptibility to infection by the gram negative bacteria was similar to wild-type but susceptibility to B. bassiana was increased. This increase in susceptibility correlated with reduced anti-fungal antimicrobial peptide (AMP) production after B. bassiana infection. This suggests that GSNOR might be required for the normal activity of the Toll pathway or novel Toll-independent processes. We also observed that gsnor knockout impairs fertility and development of embryos.

Air emissions from the combustion of fossil fuel, including carbon dioxide, sulfur dioxide, nitrogen dioxide and nitric oxide, have caused severe health and environmental problems. The post-combustion wet scrubbing has been employed for control of carbon dioxide and sulfur dioxide emissions. However, it is restricted by the sparingly water soluble nitric oxide, which accounts for 90-95% of nitrogen oxides. It is desirable and cost-effective to remove nitric oxide from flue gas by existing wet scrubbers for reduced capital costs and foot prints. In this research, absorption of nitric oxide from simulated flue gas using three different absorbents was first conducted in a bubble column system at room temperature and atmospheric pressure. Through performance comparison, ammoniacal cobalt(II) solutions were chosen as the optimum absorbent for nitric oxide absorption. Then the effects of fresh absorbent composition, pH value and temperature on nitric oxide absorption were investigated. Experimental results showed that the best initial NO removal efficiency of 96.45% was measured at the inlet flow rate of 500 mL·min-1; the room temperature of 292.2 K; the pH value of 10.50; and the concentrations of cobalt(II) solution, NO and O2 of 0.06 mol·L-1, 500 ppmv and 5.0%, respectively. For in-depth understanding of NO absorption into ammoniacal cobalt(II) complexes, equilibrium constants of reactions between nitric oxide and penta- and haxa-amminecobalt(II) solutions, respectively were determined using a bubble column reactor, in which the operation was performed continuously with respect to gas phase and batch-wise with respect to liquid phase. The experiments were conducted at temperatures from 298.2 to 310.2 K and pH from 9.06 to 9.37, all under atmospheric pressure. All experimental data fitted well to the following equations: K_NO^5=1.90×10^7 exp(3598.5/T) and K_NO^6=3.56×10^11 exp(1476.4/T), which give the enthalpy of reactions between NO and penta- and hexa-amminecobalt (II) nitrates as ∆H^5=-29.92 kJ·mol^(-1) and ∆H^6=-12.27 kJ·mol^(-1). In kinetic study, a number of experiments were conducted in a home-made double-stirred reactor at temperatures of 298.2 and 303.2 K and pH from 8.50 to 9.87 under atmospheric pressure. The reaction rate constants were calculated with the use of enhancement factor derived for gas absorption accompanied by parallel chemical reactions. The reaction between NO and pentaaminecobalt(II) was first order with respect to NO and pentaamminecobalt(II) ion, respectively. Similarly, the reaction between NO and hexaaminecobalt(II) was also first order with respect to NO and hexaamminecobalt(II) ion, respectively. The forward reaction rate constants of these two reactions were 6.43×10^6 and 1.00×10^7 L·mol-1·s-1, respectively at 298.2 K, and increased to 7.57×106 and 1.12×107 L∙mol-1∙s-1, respectively at 303.2 K. Furthermore, regeneration of used absorbent was attempted but fails. None of the additives tested herein including potassium iodide (KI), sodium persulphate (Na2S5O8) and activated carbon (AC) showed capability of regeneration at room temperature and atmospheric pressure. In addition, the effect of oxygen was investigated. With ammoniacal cobalt(II) compounds a positive effect of oxygen on NO absorption was observed. Calculated NO amount absorbed into the aqueous solution showed that with the oxygen the absorption reaction could be considered as irreversible. This fact was probably the reason for the failure of regeneration of the tested reagents. Last but not least, volumetric liquid-phase mass transfer coefficient, kLa, in some popular industrial absorbers including bubble column (BC), conventional stirred tank reactor (CSTR) and gas-inducing agitated tank (GIAT) were determined by modeling removal of oxygen from water. The experimental results could be well interpreted by mathematical models with 90% of deviations less than ±10 %.