The need for an alternative to red cells in transfusions has led to the creation of hemoglobin-based oxygen carriers (HBOCs). However, evaluations of all products tested in clinical trials have noted cardiovascular complications, raising questions about their safety that led to the abandonment of all those products. It has been considered that the adverse side effects come from the scavenging of the vasodilator – nitric oxide (NO) by the deoxyheme sites of the hemoglobin derivatives. Another observation is that HBOCs with lower oxygen affinity than red cells release oxygen prematurely in arterioles, triggering an unwanted homeostatic response. Since the need for such a product remains critical, it is important to understand the reactivity patterns that contribute to the observed complications. Various alterations of the protein have been attempted in order to reduce HBOC-induced vasoconstriction. Recent reports suggest that a safe and effective product should be pure, homogenous and have a high molecular weight along with appropriate oxygenation properties. While these properties are clearly important, vasodilatory features of hemoglobin through its nitrite reductase activity may also act as an in situ source of NO. It follows that HBOCs with an enhanced ability to produce NO from endogenous nitrite may serve to counteract vasoactivity associated with NO-scavenging by hemoglobin. Here we characterize the effects of different protein modifications on the nitrite reductase activity of hemoglobin. We produced a variety of HBOCs that include cross-linked tetramers, polyethylene glycol (PEG) conjugates and bis-tetramers of hemoglobin. We report that the rate of NO production strongly depends on the conformational state of the protein, with R-state stabilized proteins (PEG-Hbs), exhibiting the fastest rates. In particular, we found that PEGylated bis-tetramers of hemoglobin (BT-PEG) exhibit increased nitrite reductase activity while retaining cooperativity and stability. Animal studies of BT-PEG demonstrated that this material is benign: it did not cause significant increases in systemic blood pressure in mice, the major side effect associated with existing HBOCs. BT-PEG exhibits an enhanced nitrite reductase activity together with sample purity and homogeneity, molecular size and shape, and appropriate oxygenation properties, characteristics of a clinically useful HBOC.

In this study, Azotobacter vinelandii ATCC13705 (A. vinelandii), which is a free-living, nitrogen-fixing, gram-negative, and aerobic rod bacterium, was need to evaluate its ability to biodegrade tetracyanonickelate (TCN) under different conditions. Results show that A. vinelandii was able to biodegrade various concentrations of TCN (1, 10, and 20 mM) under aerobic conditions. Oxygen consumption and nitrogenase activity were investigated at 1 mM of TCN. Results indicate that the production of ammonia and methane was observed when TCN was consumed. Results suggest that nitrogenase was possibly involved in the enzymatic degradation of TCN. Moreover, higher degradation rate of TCN, higher nitrogenase activity, higher oxygen consumption, and higher specific growth rates were also observed at log growth period. Results suggest that the hypothesis of respiratory protection of nitrogenase is supported. Moreover, the addition of ammonia (1, 5, and 10 mM) would cause the decrease of TCN degradation rate (28%) during a 24-hr incubation period. Inhibition of TCN degradation (degradation rate¡G16% for 24 hrs) was observed when nitrite (5 and 10 mM) was added into the growth medium. Furthermore, the addition of 8% of glucose would significantly enhance the TCN degradation by the resting cells (degradation rate¡G43% for 8 hrs) . Results from this study provide us insight into the characteristics and mechanisms of TCN conversion by A. vinelandii.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 4, 2010). Includes bibliographical references (p. 120-131).

The purpose of this study was to investigate whether 3 days of dietary nitrate supplementation has positive effects on exercise performance and energy metabolism at rest and during strenuous knee exercise, and exercise recovery. The experimental protocol was a double blind, placebo-controlled, two-period, and within-subjects design. Fourteen healthy nonsmoking males (22.71 ± 0.72yr, 72.93 ± 2.35kg, and 47.67 ± 1.56 ml/kg/min VO2max) participated in the study. Two supplements (nitrate lozenge; NO-L or placebo; PLA) were orally administrated randomly at 48 and 24 hours before each trial day and again 40 minutes before each exercise trial. Total work, peak torque, and respiratory gases were automatically collected during repeated knee extensions/flexions (isokinetic concentric contractions with dominant leg; 4 sets of 28 repetitions at 180°/sec with 30 sec rest intervals) and recovery (6 contractions of 1 repetition maximum with 120 vii sec between contractions). For these results, peak torque, rate of fatigue, work efficiency, and rate of recovery were calculated. Blood specimens were collected at rest before and after the treatment, post exercise, and end of recovery to track the changes in blood glucose and lactate concentrations. There was a significant inverse correlation for total work during knee extension exercise and oxygen consumption (PLA: r = -.560 and NO-L: r = -.546; p < .01, respectively). During the exercise, RER was significantly higher for PLA compared with NO-L (PLA: 1.42 ± 0.02 vs. NO-L: 1.38 ± 0.02: p = .03). Work within each set and total work performed were higher for NO-L, but these differences were not significant. However, NO-L enhanced exercise efficiency by 3.3% when compared with PLA (PLA: 2497.5 ± 134.56 ft-lb/L/min vs. NO-L: 2578.7 ± 132.24 ftlb/L/min; p = .05). Extensor peak torque recovery rate slope was not significant, but meaningfully faster for NO-L (PLA: 2.39 ± 0.52% vs. NO-L: 3.06 ± 0.54%; p = .09) and significant interaction effect (p = .02) was found during recovery contractions, especially from contractions 2 to 3 (p = .03). It is also worth noting that differences in time to peak torque during knee extension exercise for NO-L and PLA approached significances (PLA: 0.213 ± 0.01 sec vs. NO-L: 0.200 ± 0.01 sec; p = .08). The results suggest that 3 days of dietary nitrate supplementation improves rate of exercise recovery and enhances work efficiency during vigorous resistance exercise. / text

Magistro darbe yra tiriama Kauno rajone, Mitkūnų kaime esančių 5 šachtinių šulinių vandens užterštumas azoto junginiais ir organinėmis medžiagomis. Darbo objektas – šachtinių šulinių vandens kokybės parametrai: nitratai (NO3ˉ ), nitritai (NO2ˉ ), permanganatinė oksidacija (ChDSMn), pH. Darbo tikslas – įvertinti ūkininkavimo įtaką geriamo vandens kokybei ir ryšio tarp sodybos dirbtinių vandens telkinių ir šachtinių šulinių vandens kokybės nustatymas.. Darbo metodai – mokslinės literatūros analizė; cheminės vandens kokybės parametrų kiekybiniai analizės metodai; statistinė analizė, panaudojant EXCEL ir STATISTCA programinius paketus. Darbo rezultatai :atlikus tyrimus nustatyta, kad vanduo užterštas nitratais. Šachtinių šulinių vandens kokybės kitimui būdinga sezoninė tarša. / Subject of research – shallows water quality indicators: nitrate (NO3ֿ), nitrite (NO2ֿ), organic matter (ChDSMn), water pH. Aim of research – to evaluate farming influence of drinking water quality and contact between homestead pond and shallows water quality. Methodology – analysis of scientific literature; chemical water quality analizes methods; statistical analysis using EXCEL, STATISTICA programs. Results of research – the results of scientific research showed, that water is polluted with nitrate. The tendency of water quality variations during seasons was observed.

Les ligands β-diiminates sont des plateformes intéressantes utilisées en chimie de coordination en particulier pour la catalyse homogène, leurs paramètres électroniques et géométriques facilement ajustables les rendant particulièrement attractifs. Toutefois, ces modifications sont limitées à l'introduction de bases de Lewis neutres (éthers, amines tertiaires) sur les substituants portés par les atomes d'azote du squelette β-diiminate. L'objectif principal de ce travail de thèse porte sur le dépassement de telles limitations avec la synthèse de nouveaux ligands β-diiminates fonctionnalisés par un ou deux groupements phénolates anioniques et l'étude de leur chimie de coordination avec des ions lanthanides et actinides. Un intérêt particulier a été apporté à l'élucidation du mécanisme de formation de ces ligands, qui a mis en lumière la sensibilité des fonctions imines vis-à-vis des fonctions phénols, à l'origine des limitations décrites ci-dessus. Deux nouveaux ligands N-aryloxy-β-diiminates ont été synthétisés à l'échelle de plusieurs grammes avec de très bons rendements. Ces ligands diffèrent par leur encombrement stérique, qui influe sur leur coordination aux ions lanthanides et actinides. La réactivité de trois des complexes N-aryloxy-β-diiminate a été explorée. Un complexe du Ce(III) possède des propriétés de réduction intéressantes, sa sphère de coordination permettant un abaissement significatif du potentiel d'oxydation du couple Ce(III)/Ce(IV). La présence d'une lacune de coordination dans un complexe du Th(IV) a été mise àprofit pour stabiliser différents adduits de bases de Lewis. Deux types de réarrangement du complexe de thorium ont été mis à jour, qui sont contrôlés par la minimisation de répulsions inter-ligands. La réactivité rédox d'un complexe N-aryloxy-β-diiminate de l'U(IV) a été exploitée pour préparer un exemple rare de complexe monooxo terminal d'uranium. Cette espèce possède une chimie rédox riche et sa réduction a permis la synthèse des analogues de l'U(V) et de l'U(IV), formant ainsi la première série de complexes monooxo terminaux d'uranium stables à trois degrés d'oxydation successifs. Cette série représente une opportunité unique d'étudier l'influence des électrons f sur le phénomène d'influence trans inverse (ITI) observés dans les complexes oxo de l'uranium. Des calculs DFT préliminaires ont été réalisés pour rationaliser les comportements expérimentaux.

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

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.

Nitrosamines have garnered increasing attention from researchers and policy makers in recent years due to potential human health implications associated with their unintentional formation in water and wastewater treatment facilities. This work addresses a crucial nitrosamine formation pathway concerning the catalysis of amine nitrosation by activated carbon materials whose use is widespread in municipal and industrial systems. Experimental results show that this catalysis is highly pH dependent, with maximum formation achieved near the pKa value for each of the secondary amines tested. This result suggests that the overall formation potential is governed by individual amine properties and their interactions with carbon surfaces, rather than solely nitrite speciation as previously reported. Formation of the most commonly studied nitrosamine, N-nitrosodimethylamine, was shown to be highly dependent on initial dimethylamine (DMA) solution concentration, with yields of approximately 0.11% of the spiked secondary amine at pH 7.5. Morpholine and dibutylamine, larger and bulkier secondary amines, formed their corresponding nitrosamines at higher yields than DMA (0.21% and 1.69%, respectively). Additionally, select tertiary amines were shown to be capable of undergoing nitrosation on the same order of magnitude as the secondary amines under neutral conditions in the presence of activated carbons. The magnitude of these results indicates that greater attention should be paid to this previously overlooked mechanism for nitrosamine formation.

Exhaled breath condensate (EBC) is a non-invasive method of investigating airway inflammation associated with nitric oxide (NO) and the metabolites nitrite/nitrates (NOx) in diseases such as chronic obstructive pulmonary disease (COPD), but some of the variables affecting the results are unknown. It was hypothesised that 1) EBC would be influenced by lung volumes and the type of EBC collection device; 2) fractional exhaled NO (FENO) and EBC NOx in COPD patients would be altered by smoking and glucocorticosteroids (GCS); 3) cigarette smoke could contribute to the EBC NOx concentration while it may also decrease FENO indirectly by converting airway NO to NOx. It was found that EBC volume was significantly correlated with both tidal volume and minute volume. Comparing four EBC collection devices demonstrated greater efficiency with the ECoScreen?? than siliconised glass tubes or RTube?? but it gave factitiously high NOx levels. Total EBC protein levels over a 10-minute collection were significantly higher using the ECoScreen?? than either glass or RTube?? devices. A cross-sectional study of 96 COPD patients and 80 age-matched control subjects demonstrated that FENO levels in COPD patients were significantly higher than normal subjects when comparing either the combined groups or appropriate two subgroups: ex-smokers and smokers. GCS treatment demonstrated no significant effect on either FENO levels or EBC NOx, but EBC NOx was elevated in smokers. In vitro, cigarette smoke extract (CSE) induced significantly higher NOx and asymmetric dimethylarginine (ADMA) levels in A549 cells when compared with control media. The anti-oxidant, NAC pre-treatment partially reversed the elevated NOx levels but not the ADMA levels. This thesis is the first to report FENO and EBC NOx in COPD patients in an appropriate sample size to be able to evaluate each subgroup, and the increased EBC NOx levels found in smokers in vivo was consistent with the elevated NOx level in response to CSE observed in vitro. These data indicate that smoking-related airway inflammation and activation of the NO pathway are complex with both an increase in ADMA, NO, NOx and may be regulated by oxidative stress rather than the nitric oxide synthase (NOS) pathway.