Rate Of Nitric Oxide Release Research Articles

Structural heterogeneity in polymeric nitric oxide donor nanoblended coatings for controlled release behaviors.

Nitric oxide (NO) gas delivery has attracted extensive interest due to its endogenous therapeutic functions and potential biomedical applications for the treatment of various diseases. The important thing about NO delivery is the emission control due to the fast diffusion rate of gas molecules. To develop NO delivery platforms using macromolecules and to comprehend the chemical NO donor generation and release mechanisms, we studied branched polyethyleneimine/alginate (BPEI/ALG) nanoblended coatings fabricated by giving structural heterogeneity to the structure through a self-assembly process for the controlled release of gas molecules. NO release could be remarkably expected via the well-organized coating structures and explained by quantification of the NO donors. Taking advantage of these polymeric coatings, this process could be applied to the treatment of various diseases based on the biocompatibility of materials and the fine control of NO release rate and its amount.

Furoxan Nitric Oxide Donors Disperse Pseudomonas aeruginosa Biofilms, Accelerate Growth, and Repress Pyoverdine Production.

The use of nitric oxide (NO) as a signal for biofilm dispersal has been shown to increase the susceptibility of many biofilms to antibiotics, promoting their eradication. The delivery of NO to biofilms can be achieved by using NO donors with different kinetics and properties of NO release that can influence their efficacy as biofilm control agents. In this study, the kinetics of three furoxan derivatives were evaluated. The effects of these NO donors, which have an advantageous pharmacological profile of slower onset with an extended duration of action, on Pseudomonas aeruginosa growth, biofilm development, and dispersal were also characterized. Compound LL4254, which showed a fast rate of NO release, induced biofilm dispersal at approximately 200 μM. While LL4212 and LL4216 have a slower rate of NO release, both compounds could induce biofilm dispersal, under the same treatment conditions, when used at higher concentrations. In addition, LL4212 and LL4216 were found to promote P. aeruginosa growth in iron-limited minimal medium, leading to a faster rate of biofilm formation and glucose utilization, and ultimately resulted in early dispersal of biofilm cells through carbon starvation. High concentrations of LL4216 also repressed production of the siderophore pyoverdine by more than 50-fold, via both NOx-dependent and NOx-independent mechanisms. The effects on growth and pyoverdine levels exerted by the furoxans appeared to be mediated by NO-independent mechanisms, suggesting functional activities of furoxans in addition to their release of NO and nitrite. Overall, this study reveals that secondary effects of furoxans are important considerations for their use as NO-releasing dispersal agents and that these compounds could be potentially redesigned as pyoverdine inhibitors.

Study of Crystal Formation and Nitric Oxide (NO) Release Mechanism from S-Nitroso-N-acetylpenicillamine (SNAP)-Doped CarboSil Polymer Composites for Potential Antimicrobial Applications.

Stable and long-term nitric oxide (NO) releasing polymeric materials have many potential biomedical applications. Herein, we report the real-time observation of the crystallization process of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), within a thermoplastic silicone-polycarbonate-urethane biomedical polymer, CarboSil 20 80A. It is demonstrated that the NO release rate from this composite material is directly correlated with the surface area that the CarboSil polymer film is exposed to when in contact with aqueous solution. The decomposition of SNAP in solution (e.g. PBS, ethanol, THF, etc.) is a pseudo-first-order reaction proportional to the SNAP concentration. Further, catheters fabricated with this novel NO releasing composite material are shown to exhibit significant effects on preventing biofilm formation on catheter surface by Pseudomonas aeruginosa and Proteus mirabilis grown in CDC bioreactor over 14 days, with a 2 and 3 log-unit reduction in number of live bacteria on their surfaces, respectively. Therefore, the SNAP-CarboSil composite is a promising new material to develop antimicrobial catheters, as well as other biomedical devices.

Effect of chemical structure of S-nitrosothiols on nitric oxide release mediated by the copper sites of a metal organic framework based environment.

The effect of chemical structure of different biologically compatible S-nitrosothiols on the solvation environment at catalytic copper sites in a metal organic framework (MOF) suspended in a solution of ethanol is probed using computational methods. The use of a copper based MOF as a storage vehicle and catalyst (copper sites of the MOF) in the controlled and sustained release of chemically stored nitric oxide (NO) from S-nitrosocysteine has been shown to occur both experimentally and computationally [J. Am. Chem. Soc., 2012, 134, 3330-3333; Phys. Chem. Chem. Phys., 2015, 17, 23403]. Previous studies on a copper based MOF, namely HKUST-1, concluded that modifications in the R-group of s-nitrosothiols and/or organic linkers of MOFs led to a method capable of modulating NO release. In order to test the hypothesis that larger R-groups slow down NO release, four different RSNOs (R = cysteine, N-acetylcysteine, N-acetyl-d,l-penicillamine or glutathione) of varying size were investigated, which in turn required the use of a larger copper based MOF. Due to its desirable copper centers and more extensive framework, MOF-143, an analog of HKUST-1 was chosen to further explore both the effect of different RSNOs as well as MOF environments on NO release. Condensed phase classical molecular dynamics simulations are utilized to study the effect of the complex MOF environment as well as the chemical structure and size of the RSNO on the species on the catalytic reaction. The results indicate that in addition to the size of the RSNO species and the organic linkers within the MOF, the reaction rates can be modulated by the molecular structure of the RSNO and furthermore combining different RSNO species can also be used to tune the rate of NO release.

Potential use and perspectives of nitric oxide donors in agriculture.

Nitric oxide (NO) has emerged in the last 30 years as a key molecule involved in many physiological processes in plants, animals and bacteria. Current research has shown that NO can be delivered via donor molecules. In such cases, the NO release rate is dependent on the chemical structure of the donor itself and on the chemical environment. Despite NO's powerful signaling effect in plants and animals, the application of NO donors in agriculture is currently not implemented and research remains mainly at the experimental level. Technological development in the field of NO donors is rapidly expanding in scope to include controlling seed germination, plant development, ripening and increasing shelf-life of produce. Potential applications in animal production have also been identified. This concise review focuses on the use of donors that have shown potential biotechnological applications in agriculture. Insights are provided into (i) the role of donors in plant production, (ii) the potential use of donors in animal production and (iii) future approaches to explore the use and applications of donors for the benefit of agriculture. © 2016 Society of Chemical Industry.

Nitric Oxide Releasing Coronary Stent: A New Approach Using Layer-by-Layer Coating and Liposomal Encapsulation.

The sustained or controlled release of nitric oxide (NO) can be the most promising approach for the suppression or prevention of restenosis and thrombosis caused by stent implantation. The aim of this study is to investigate the feasibility in the potential use of layer-by-layer (LBL) coating with a NO donor-containing liposomes to control the release rate of NO from a metallic stent. Microscopic observation and surface characterizations of LBL-modified stents demonstrate successful LBL coating with liposomes on a stent. Release profiles of NO show that the release rate is sustained up to 5 d. In vitro cell study demonstrates that NO release significantly enhances endothelial cell proliferation, whereas it markedly inhibits smooth muscle cell proliferation. Finally, in vivo study conducted with a porcine coronary injury model proves the therapeutic efficacy of the NO-releasing stents coated by liposomal LBL technique, supported by improved results in luminal healing, inflammation, and neointimal thickening except thrombo-resistant effect. As a result, all these results demonstrate that highly optimized release rate and therapeutic dose of NO can be achieved by LBL coating and liposomal encapsulation, followed by significantly efficacious outcome in vivo.

Engineering nitric oxide synthase chimeras to function as NO dioxygenases

Nitric oxide synthases (NOSs) catalyze a two-step oxidation of l-arginine to form nitric oxide (NO) and l-citrulline. NOS contains a N-terminal oxygenase domain (NOSoxy) that is the site of NO synthesis, and a C-terminal reductase domain (NOSred) that binds nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) and provides electrons to the NOSoxy heme during catalysis. The three NOS isoforms in mammals inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) share high structural similarity but differ in NO release rates and catalytic properties due to differences in enzyme kinetic parameters. These parameters must be balanced for NOS enzymes to release NO, rather than consume it in a competing, inherent NO dioxygenase reaction. To improve understanding, we drew on a global catalytic model and previous findings to design three NOS chimeras that may predominantly function as NO dioxygenases: iNOSoxy/nNOSred (Wild type (WT) chimera), V346I iNOSoxy/nNOSred (V346I chimera) and iNOSoxy/S1412D nNOSred (S1412D chimera). The WT and S1412D chimeras had higher NO release than the parent iNOS, while the V346I chimera exhibited much lower NO release, consistent with expectations. Measurements indicated that a greater NO dioxygenase activity was achieved, particularly in the V346I chimera, which dioxygenated an estimated two to four NO per NO that it released, while the other chimeras had nearly equivalent NO dioxygenase and NO release activities. Computer simulations of the global catalytic model using the measured kinetic parameters produced results that mimicked the measured outcomes, and this provided further insights on the catalytic behaviors of the chimeras and basis of their increased NO dioxygenase activities.

Nitric oxide releasing acridone carboxamide derivatives as reverters of doxorubicin resistance in MCF7/Dx cancer cells

A series of nitric oxide donating acridone derivatives are synthesized and evaluated for in vitro cytotoxic activity against different sensitive and resistant cancer cell lines MCF7/Wt, MCF7/Mr (BCRP overexpression) and MCF7/Dx (P-gp expression). The results showed that NO-donating acridones are potent against both the sensitive and resistant cells. Structure activity relationship indicate that the nitric oxide donating moiety connected through a butyl chain at N10 position as well as morpholino moiety linkage through an amide bridge on the acridone ring system at C-2 position, are required to exert a good cytotoxic effect. Further, good correlations were observed when cytotoxic properties were compared with in vitro nitric oxide release rate, nitric oxide donating group potentiated the cytotoxic effect of the acridone derivatives. Exogenous release of nitric oxide by NO donating acridones enhanced the accumulation of doxorubicin in MCF7/Dx cell lines when it was coadministered with doxorubicin, which inhibited the efflux process of doxorubicin. In summary, a nitric oxide donating group can potentiate the anti-MDR property of acridones.

Development of nitric oxide catalytic coatings by conjugating 3,3-disulfodipropionic acid and 3,3-diselenodipropionic acid for improving hemocompatibility.

Nitric oxide (NO), discovered as an endothelium-derived relaxing factor, has been found to have multiple intracellular effects in vascular diseases including vasorelaxation regulation, endothelial regeneration, inhibition of leukocyte chemotaxis, and platelet activation. In the work described here, the authors have developed a NO-catalytic bioactive coating for improving hemocompatibility. The authors first prepared a dopamine and hexamethylendiamine (PDAM/HD) amine-rich adherent copolymer coating to introduce amine groups onto 316L stainless steel, followed by covalently conjugating 3,3-disulfodipropionic acid (S-S) and 3,3-diselenodipropionic acid (Se-Se), which mimic glutathione peroxidase-like catalytic production of NO. S-S and Se-Se were immobilized on the PDAM/HD surface via carbodiimide coupling chemistry. X-ray photoelectron spectroscopy analysis revealed clear S2p and Se3d signals, confirming the immobilization of S-S and Se-Se on the PDAM/HD surface. The NO release behavior of different samples was investigated. In detail, two species of thionitrites (RSNO), S-nitrosoglutathione (GSNO, endogenous NO donors) and S-nitrosoacetylpenicillamine (SNAP) were chosen as NO donors to investigate the NO catalytic properties of S-S and Se-Se modified PDAM/HD surfaces. Not only Se-Se@PDAM/HD but also S-S@PDAM/HD coatings showed the ability to continuously catalyze RSNO to generate NO in the presence of proper thiol reducing agent. For the Se-Se@PDAM/HD coating, the NO release amount and rate were greater than S-S@PDAM/HD in both GSNO and SNAP conditions. The results showed that organosulfide species possesses NO catalytic ability as well as organoselenium species. The authors demonstrated that both S-S@PDAM/HD and Se-Se@PDAM/HD coatings exhibited outstanding inhibition effect on platelet adhesion, aggregation and activation via the cyclic guanylate monophosphate signal pathway. Thus these results suggested that NO catalytic coatings based on organoselenium and organosulfide species immobilization can help to improve hemocompatibility. NO-catalytic strategies possess huge potential applications in blood-contacting devices.

Asymmetric Anion‐selective Membrane Electrode for Determining Nitric Oxide Release Rates from Ppolymeric Films/Electrochemical Devices

AbstractA novel potentiometric method for the determination of nitric oxide (NO) is presented. The assessment of NO levels is based on the rapid reaction of NO with oxyhemoglobin, during which nitrate is formed and then detected potentiometrically using a polymeric membrane anion‐selective electrode. The composition of the anion‐selective membrane is optimized to eliminate the response to high levels of oxyhemoglobin reagent used to react with NO to form NO3−. The practical utility of the method is demonstrated by determining the NO release rates from NO donor‐doped polymeric films, as well as from a new electrochemical NO generation system within the range of 10 to 55×10−10 mol ⋅ min−1. The values measured with the new assay system are shown to correlate well with a chemiluminescence reference method after accounting for the trapping efficiency of NO by the oxyhemoglobin solution.

Enhanced production of nitric oxide may contribute to the induction of detrusor underactivity in sucrose fed rats

Objective: In a previous study we showed that sucrose feeding creates cystometric properties resembling detrusor underactivity (DU) associated with enhanced production of nitric oxide (NO) in rats. Our present objective was to evaluate the possible role of P2X-purinergic and cholinergic receptors as molecular mechanisms underlying DU-like conditions associated with enhanced production of NO and altered contractile properties in rat bladder strips. Materials and methods: Female Sprague-Dawley rats had ad-libitum access to 5% sucrose in water for two to four weeks. Isometric detrusor contractions were characterized in response to electrical field stimulation, the cholinergic agonist carbachol, the P2X agonist alpha, beta-methylene-ATP or KCl-induced depolarization. ATP and NO release rates were determined from bladder strips after stimulation of cholinergic or P2X-type purinergic receptors.Results: Electrical field stimulations generated stronger contractile responses in the strips from sucrose fed animals, while KCl induced similar contractions. The release of ATP after cholinergic or purinergic stimulation was similar in control or sucrose fed bladder strips. Nonetheless, NO production was significantly higher when pharmacologically triggered with either cholinergic or purinergic stimulation in the bladder strips from rats drinking sucrose for four weeks. The contractile responses to carbachol or abMe-ATP were also significantly reduced in this group.Conclusions: Detrusor contractions in these underactive-like conditions are gradual, and may be generated by excessive NO production that is mediated by individual activation of cholinergic or purinergic receptors, with higher effects by the latest. Our results suggest that nitric oxide production and not impaired contractile mechanisms are involved in this rat model of detrusor underactivity.

Highly sensitive amperometric Pt–Nafion gas phase nitric oxide sensor: Performance and application in characterizing nitric oxide-releasing biomaterials

A highly sensitive amperometric gas-phase nitric oxide (NO) sensor based on a Pt working electrode chemically deposited on a Nafion film is described. The Pt electrode is chemically deposited on a Nafion 117 membrane by impregnating the film with Pt(NH3)42+ ions, which are then exposed to NaBH4 to precipitate conductive Pt metal. The sensor was characterized with a mass-flow controlled 1 ppm NO standard gas and has an electrochemical surface area of 34 ± 9 cm2, low limits of detection (4.3 ± 1.1 ppb) and a fast response time (<5s) toward changes in gas phase NO levels. Good correlation was found for measurements with the new amperometric Pt–Nafion sensor vs. chemiluminescence results for detecting the rates of NO release from Carbosil2080A polymer films doped with S-nitroso-N-acetylpenicillamine (R = 0.999, m = 0.999, n = 6) and for electrochemical reduction of nitrite to NO (R = 0.999, m = 0.938, n = 3) mediated by a copper(II)-tri(2-pyridylmethyl)amine complex.

Nitric oxide released from luminal s-nitroso-N-acetylcysteine increases gastric mucosal blood flow.

Nitric oxide (NO)-mediated vasodilation plays a key role in gastric mucosal defense, and NO-donor drugs may protect against diseases associated with gastric mucosal blood flow (GMBF) deficiencies. In this study, we used the ex vivo gastric chamber method and Laser Doppler Flowmetry to characterize the effects of luminal aqueous NO-donor drug S-nitroso-N-acetylcysteine (SNAC) solution administration compared to aqueous NaNO2 and NaNO3 solutions (pH 7.4) on GMBF in Sprague-Dawley rats. SNAC solutions (600 μM and 12 mM) led to a rapid threefold increase in GMBF, which was maintained during the incubation of the solutions with the gastric mucosa, while NaNO2 or NaNO3 solutions (12 mM) did not affect GMBF. SNAC solutions (600 μM and 12 mM) spontaneously released NO at 37 °C at a constant rate of 0.3 or 14 nmol·mL−1·min−1, respectively, while NaNO2 (12 mM) released NO at a rate of 0.06 nmol·mL−1·min−1 and NaNO3 (12 mM) did not release NO. These results suggest that the SNAC-induced GMBF increase is due to their higher rates of spontaneous NO release compared to equimolar NaNO2 solutions. Taken together, our data indicate that oral SNAC administration is a potential approach for gastric acid-peptic disorder prevention and treatment.

Nitric oxide release from polydimethylsiloxane-based polyurethanes.

Localized nitric oxide (NO) release from polymeric materials holds much promise for the prevention of coagulation often associated with implantable and extracorporeal blood-contacting devices. Films of polyurethane (PU) containing incorporated polyethyleneimine were thus exposed to NO gas to form diazeniumdiolates (NONOates) in situ. Donor incorporation and NO gas exposure did not affect the mechanical properties of the films. The NO release capacity increased with increasing polydimethylsiloxane (PDMS) content in the soft segment of the PU: total capacity could be more than doubled (P<0.05) from 0.093 ± 0.028 to 0.225 ± 0.004 mmol/g when the PDMS content was increased from 0 to 100%. Release kinetics were best approximated using a modified Korsemeyer-Peppas power law (R2=0.95-0.99). Despite the resultant rapid initial decrease in NO release rates, values above that observed for quiescent endothelial cells (0.83 pmol·cm−2·s−1) were maintained for extended periods of 5-10 days, while rates above that of a stimulated endothelium (2.7-6.8 pmol·cm−2·s−1) were achieved for the first 24 hours. This method of NONOate formation may be advantageous, as potential premature NO release by exposure of diazeniumdiolated donors during incorporation, processing and storage, can be avoided by in situ diazoniumdiolation closer to the time of implantation.

Optimized polymeric film-based nitric oxide delivery inhibits bacterial growth in a mouse burn wound model

Nitric oxide (NO) has many biological roles (e.g. antimicrobial agent, promoter of angiogenesis, prevention of platelet activation) that make NO releasing materials desirable for a variety of biomedical applications. Localized NO release can be achieved from biomedical grade polymers doped with diazeniumdiolated dibutylhexanediamine (DBHD/N2O2) and poly(lactic-co-glycolic acid) (PLGA). In this study, the optimization of this chemistry to create film/patches that can be used to decrease microbial infection at wound sites is examined. Two polyurethanes with different water uptakes (Tecoflex SG-80A (6.2±0.7wt.%) and Tecophilic SP-60D-20 (22.5±1.1wt.%)) were doped with 25wt.% DBHD/N2O2 and 10wt.% of PLGA with various hydrolysis rates. Films prepared with the polymer that has the higher water uptake (SP-60D-20) were found to have higher NO release and for a longer duration than the polyurethane with the lower water uptake (SG-80A). The more hydrophilic polymer enhances the hydrolysis rate of the PLGA additive, thereby providing a more acidic environment that increases the rate of NO release from the NO donor. The optimal NO releasing and control SG-80A patches were then applied to scald burn wounds that were infected with Acinetobacter baumannii. The NO released from these patches applied to the wounds is shown to significantly reduce the A. baumannii infection after 24h (∼4 log reduction). The NO release patches are also able to reduce the level of transforming growth factor-β in comparison to controls, which can enhance re-epithelialization, decrease scarring and reduce migration of bacteria. The combined DBHD/N2O2 and PLGA-doped polymer patches, which could be replaced periodically throughout the wound healing process, demonstrate the potential to reduce risk of bacterial infection and promote the overall wound healing process.

Abstract 321: A Biomimetic Approach to Developing Antithrombotic Small-Caliber Prosthetic Vascular Grafts

Introduction: Prosthetic grafts smaller than 6 mm in diameter are linked with a high incidence of thrombosis and neointimal hyperplasia. We developed a novel graft material that provides controlled release of nitric oxide (NO), an antithrombotic agent that also promotes endothelialization within the lumen. Our very recent study focused on NO release kinetics from a similar composite that demonstrated controlled NO release for up to 200 d with a total release of 1.03 ± 0.09 μmol/mg. Methods: Grafts were fabricated using an electrospinning process that yields a micro-fibrous web composed of poly(ε-caprolactone) (PCL) . These fibers were doped with NO donor diethylenetriamine diazeniumdiolate (DETA/NO) and embedded within a polydimetheylsiloxane (PDMS) elastomer, which was found to regulate water transport and NO release rate. In vitro studies were tested in PBS at 37 C using a Griess assay to compute NO release. The shape memory properties inherent to this composite were used to form kink resistant acute angles through exposure to specific thermal cycles under strain. Results: NO release was proportional to DETA/NO doping and regulated by PDMS. Our present study revealed that the critical radius of curvature, below which kinks form, is a function of graft wall thickness and that a radii of curvature as small as 6 mm can be achieved using our unique process. The accompanying figure shows a 4 mm diameter graft with 0.8 mm wall thickness formed into a kink-free form with a radius of curvature of 6.7 mm. Conclusions: Incorporation of DETA/NO into a PCL/PDMS composite offers tunable long-term NO release and is a candidate for kink-resistant vascular grafts.

Accurate Nitric Oxide Measurements from Donors in Cell Media: Identification of Scavenging Agents

Nitric oxide (NO) is an essential messenger in human physiology, mediating cellular processes ranging from proliferation to apoptosis. The effects of NO are concentration dependent, and control over the instantaneous amount of NO available to cells is essential for determining the therapeutic NO dosages for various applications. As such, the development of NO therapeutic materials relies on accurate quantitative NO measurements that provide both total NO release from the NO donor as well as instantaneous NO concentrations. On the basis of the complexity of the cell media environment, inaccurate NO reporting often occurs for in vitro studies. These inaccuracies result from using inert media such as phosphate buffer saline (PBS), failing to account for the reactivity of media components. In this work, we describe a method for directly quantifying the instantaneous and total amounts of NO from commonly used NO donors in commercially available cell media routinely used for endothelial and neural cell lines. A riboflavin-tryptophan complex found in the media was identified as the major scavenger of NO in the cell media and likely reacts with NO via a radical-radical reaction. This finding significantly impacts the amount of available NO. The scavenging effects are concentration dependent on the riboflavin-tryptophan complex and the NO release rate from the NO donor. The results of this study provide insights on the exogenous amounts of NO that are present in cell media and may provide an explanation for differences in NO dosages between buffer experiments and in vitro and in vivo studies.

Analysis of the coexisting pathways for NO and N2O formation in Chernozem using the 15N-tracer SimKIM-Advanced model

The nitrogen (N) cycle consists of a variety of microbial processes. These processes often occur simultaneously in soils, but respond differently to local environmental conditions due to process-specific biochemical restrictions (e.g. oxygen levels). Hence, soil nitrogen cycling (e.g. soil N gas production through nitrification and denitrification) is individually affected through these processes, resulting in the complex and highly dynamic behaviour of total soil N turnover. The development and application of methods that facilitate the quantification of individual contributions of coexisting processes is a fundamental prerequisite for (i) understanding the dynamics of soil N turnover and (ii) implementing these processes in ecosystem models. To explain the unexpected results of the triplet tracer experiment (TTE) of Russow et al. (Role of nitrite and nitric oxide in the processes of nitrification and denitrification in soil: results from 15N tracer experiments. Soil Biol Biochem. 2009;41:785–795) the existing SimKIM model was extended to the SimKIM-Advanced model through the addition of three separate nitrite subpools associated with ammonia oxidation, oxidation of organic nitrogen (Norg), and denitrification, respectively. For the TTE, individual treatments with 15N ammonium, 15N nitrate, and 15N nitrite were conducted under oxic, hypoxic, and anoxic conditions, respectively, to clarify the role of nitric oxide as a denitrification intermediate during N2O formation. Using a split nitrite pool, this analysis model explains the observed differences in the 15N enrichments in nitric oxide (NO) and nitrous oxide (N2O) which occurred in dependence on different oxygen concentrations. The change from oxic over hypoxic to anoxic conditions only marginally increased the NO and N2O release rates (1.3-fold). The analysis using the model revealed that, under oxic and hypoxic conditions, Norg-based N2O production was the dominant pathway, contributing to 90 and 50 % of the total soil N2O release. Under anoxic conditions, denitrification was the dominant process for soil N2O release. The relative contribution of Norg to the total soil NO release was small. Ammonia oxidation served as the major pathway of soil NO release under oxic and hypoxic conditions, while denitrification was dominant under anoxic conditions. The model parameters for soil with moderate soil organic matter (SOM) content were not scalable to an additional data set for soil with higher SOM content, indicating a strong influence of SOM content on microbial N turnover. Thus, parameter estimation had to be re-calculated for these conditions, highlighting the necessity of individual soil-dependent parameter estimations.

Antimicrobial efficacy and wound-healing property of a topical ointment containing nitric-oxide-loaded zeolites.

Topical delivery of nitric oxide (NO) through a wound dressing has the potential to reduce wound infections and improve healing of acute and chronic wounds. This study characterized the antibacterial efficacy of an ointment containing NO-loaded, zinc-exchanged zeolite A that releases NO upon contact with water. The release rate of NO from the ointment was measured using a chemiluminescence detection system. Minimum bactericidal concentration assays were performed using five common wound pathogens, including Gram-negative bacteria (Escherichia coli and Acinetobacter baumannii), Gram-positive bacteria (Staphylococcus epidermidis and meticillin-resistant Staphylococcus aureus) and a fungus (Candida albicans). The time dependence of antimicrobial activity was characterized by performing log-reduction assays at four time points after 1-8 h ointment exposure. The cytotoxicity of the ointment after 24 h was assessed using cultured 3T3 fibroblast cells. Minimum microbicidal concentrations (MMCs) for bacterial organisms (5×10(7) c.f.u.) ranged from 50 to 100 mg ointment (ml media)(-1); the MMC for C. albicans (5×10(4) c.f.u.) was 50 mg ointment (ml media)(-1). Five to eight log reductions in bacterial viability and three log reductions in fungal viability were observed after 8 h exposure to NO-zeolite ointment compared with untreated organisms. Fibroblasts remained viable after 24 h exposure to the same concentration of NO-zeolite ointment as was used in antimicrobial tests. In parallel studies, full-thickness cutaneous wounds on Zucker obese rats healed faster than wounds treated with a control ointment. These data indicate that ointment containing NO-loaded zeolites could potentially be used as a broad-spectrum antimicrobial wound-healing dressing.

NMR studies of the dynamics of nitrophorin 2 bound to nitric oxide.

The Rhodnius nitrophorins are β-barrel proteins of the lipocalin fold with a heme protruding from the open end of the barrel. They are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands, where NO is bound to iron. NO is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1-NP4. At pH 7.3, NP4 releases NO 17 times faster than NP2 does, as measured by stopped-flow kinetics. A number of crystal structures of the least abundant protein, NP4, are available. These structures have been used to propose that two loops between adjacent β-strands at the front opening of the protein, the A-B and G-H loops, determine the rate of NO release. To learn how the protein loops contribute to the release of NO for each of the nitrophorins, the dynamics of these proteins are being studied in our laboratory. In this work, the NP2-NO complex has been investigated by nuclear magnetic resonance relaxation measurements to probe the picosecond-to-nanosecond and microsecond-to-millisecond time scale motions at three pH values, 5.0, 6.5, and 7.3. It is found that at pH 5.0 and 6.5, the NP2-NO complex is rigid and only a few residues in the loop regions show dynamics, while at pH 7.3, somewhat more dynamics, particularly of the A-B loop, are observed. Comparison to other lipocalins shows that all are relatively rigid, and that the dynamics of lipocalins in general are much more subtle than those of mainly α-helical proteins.