Analytical method for determination of nitric oxide in zebrafish larvae: Toxicological and pharmacological applications

Zebrafish are currently used at various stages of the drug discovery process and can be a useful and cost-effective alternative to some mammalian models. Nitric oxide (NO) plays an important role in physiology of zebrafish. The availability of appropriate analytical techniques to quantify the NO is crucial for studying its role in physiological and pathological conditions. This work aimed at establishing a high-performance liquid chromatography method for determination of NO levels in zebrafish larvae. Attempts were also made to assess the normal levels of NO at the first days postfertilization and the possible changes under pathological conditions. The method validation was quantitatively evaluated in terms of sensitivity, specificity, precision, accuracy, linearity, and recovery. NO levels from zebrafish larvae at the first days postfertilization and larvae challenged to N(G)-nitro-L-arginine methyl ester, sodium nitroprusside, Escherichia coil lipopolysaccharide, and copper sulfate were analyzed. The samples were derivatized with 2,3-diaminonaphthalene, and fluorescence detection was used for the indirect determination of NO. The method showed a good performance for all validation parameters evaluated and was efficient to monitor changes in NO concentration under physiological and pathophysiological conditions. This method might represent a powerful tool to be applied in NO studies with zebrafish larvae. (C) 2011 Elsevier Inc. All rights reserved.

Blood transfusions increase circulating plasma free hemoglobin levels and plasma nitric oxide consumption: a prospective observational pilot study

Introduction: The increasing number of reports on the relation between transfusion of stored red blood cells (RBCs) and adverse patient outcome has sparked an intense debate on the benefits and risks of blood transfusions. Meanwhile, the pathophysiological mechanisms underlying this postulated relation remain unclear. The development of hemolysis during storage might contribute to this mechanism by release of free hemoglobin (fHb), a potent nitric oxide (NO) scavenger, which may impair vasodilation and microcirculatory perfusion after transfusion. The objective of this prospective observational pilot study was to establish whether RBC transfusion results in increased circulating fHb levels and plasma NO consumption. In addition, the relation between increased fHb values and circulating haptoglobin, its natural scavenger, was studied. Methods: Thirty patients electively received 1 stored packed RBC unit (n = 8) or 2 stored packed RBC units (n = 22). Blood samples were drawn to analyze plasma levels of fHb, haptoglobin, and NO consumption prior to transfusion, and 15, 30, 60 and 120 minutes and 24 hours after transfusion. Differences were compared using Pearson's chi-square test or Fisher's exact test for dichotomous variables, or an independent-sample t test or Mann-Whitney U test for continuous data. Continuous, multiple-timepoint data were analyzed using repeated one-way analysis of variance or the Kruskall-Wallis test. Correlations were analyzed using Spearman or Pearson correlation. Results: Storage duration correlated significantly with fHb concentrations and NO consumption within the storage medium (r = 0.51, P < 0.001 and r = 0.62, P = 0.002). fHb also significantly correlated with NO consumption directly (r = 0.61, P = 0.002). Transfusion of 2 RBC units significantly increased circulating fHb and NO consumption in the recipient (P < 0.001 and P < 0.05, respectively), in contrast to transfusion of 1 stored RBC unit. Storage duration of the blood products did not correlate with changes in fHb and NO consumption in the recipient. In contrast, pre-transfusion recipient plasma haptoglobin levels inversely influenced post-transfusion fHb concentrations. Conclusion: These data suggest that RBC transfusion can significantly increase post-transfusion plasma fHb levels and plasma NO consumption in the recipient. This finding may contribute to the potential pathophysiological mechanism underlying the much-discussed adverse relation between blood transfusions and patient outcome. This observation may be of particular importance for patients with substantial transfusion requirements.

Nitric Oxide in the Exhaled Breath Condensate of Healthy Volunteers Collected With a Reusable Device

Background: The analysis of exhaled breath condensate (EBC) is a non-invasive technique that enables the determination of several volatile and nonvolatile substances produced in the respiratory tract, whose measurement may be useful for the diagnosis and monitoring of several respiratory diseases. Objective: The aim of this study was to produce a low-cost reusable device in order to sample exhaled breath condensate in healthy adult volunteers, and to determine the concentration of nitric oxide in the sample collected. Material and methods: The apparatus was made with a U-shaped tube of borosilicate glass. The tube was placed in a container with ice, and unidirectional respiratory valves were fitted to the distal end. Afterwards, nitric oxide was measured in the exhaled breath condensate (EBC) by chemiluminescence. Results: The total cost of the device was $120.20. EBC samples were obtained from 116 volunteers of both sexes, aged between 20 and 70. The mean volume of exhaled breath condensate collected during 10 minutes was 1.0 +/- 0.6 mL, and the mean level of nitric oxide was 12.99 +/- 14.38 mu M (median 8.72 mu M). There was no correlation between the nitric oxide levels in the exhaled breath condensate and age or gender. Conclusion: We demonstrate that it is possible to fabricate a low-cost, efficient, reusable device in order to collect and determine nitric oxide levels in EBC. We have identified no correlation between the nitric oxide levels present in the EBC obtained with this method with either age or sex. (C) 2011 SEPAR. Published by Elsevier Espana, S.L. All rights reserved.

Preparation, structural characterization and catalytic properties of Co/CeO2 catalysts for the steam reforming of ethanol and hydrogen production

In this paper, Co/CeO2 catalysts, with different cobalt contents were prepared by the polymeric precursor method and were evaluated for the steam reforming of ethanol. The catalysts were characterized by N-2 physisorption (BET method), X-ray diffraction (XRD), UV-visible diffuse reflectance, temperature programmed reduction analysis (TPR) and field emission scanning electron microscopy (FEG-SEM). It was observed that the catalytic behavior could be influenced by the experimental conditions and the nature of the catalyst employed. Physical-chemical characterizations revealed that the cobalt content of the catalyst influences the metal-support interaction which results in distinct catalyst performances. The catalyst with the highest cobalt content showed the best performance among the catalysts tested, exhibiting complete ethanol conversion, hydrogen selectivity close to 66% and good stability at a reaction temperature of 600 degrees C. (c) 2012 Elsevier B.V. All rights reserved.

An optimization study of PtSn/C catalysts applied to direct ethanol fuel cell: Effect of the preparation method on the electrocatalytic activity of the catalysts

The aim of this work was to perform a systematic study of the parameters that can influence the composition, morphology, and catalytic activity of PtSn/C nanoparticles and compare two different methods of nanocatalyst preparation, namely microwave-assisted heating (MW) and thermal decomposition of polymeric precursors (DPP). An investigation of the effects of the reducing and stabilizing agents on the catalytic activity and morphology of Pt75Sn25/C catalysts prepared by microwave-assisted heating was undertaken for optimization purposes. The effect of short-chain alcohols such as ethanol, ethylene glycol, and propylene glycol as reducing agents was evaluated, and the use of sodium acetate and citric acid as stabilizing agents for the MW procedure was examined. Catalysts obtained from propylene glycol displayed higher catalytic activity compared with catalysts prepared in ethylene glycol. Introduction of sodium acetate enhanced the catalytic activity, but this beneficial effect was observed until a critical acetate concentration was reached. Optimization of the MW synthesis allowed for the preparation of highly dispersed catalysts with average sizes lying between 2.0 and 5.0 nm. Comparison of the best catalyst prepared by MW with a catalyst of similar composition prepared by the polymeric precursors method showed that the catalytic activity of the material can be improved when a proper condition for catalyst preparation is achieved. (C) 2012 Elsevier B.V. All rights reserved.

Urea-Based Synthesis of Zinc Oxide Nanostructures at Low Temperature

The preparation of nanometer-sized structures of zinc oxide (ZnO) from zinc acetate and urea as raw materials was performed using conventional water bath heating and a microwave hydrothermal (MH) method in an aqueous solution. The oxide formation is controlled by decomposition of the added urea in the sealed autoclave. The influence of urea and the synthesis method on the final product formation are discussed. Broadband photoluminescence (PL) behavior in visible-range spectra was observed with a maximum peak centered in the green region which was attributed to different defects and the structural changes involved with ZnO crystals which were produced during the nucleation process.

Effects of moderate electrical stimulation on reactive species production by primary rat skeletal muscle cells: Cross talk between superoxide and nitric oxide production

The effects of a moderate electrical stimulation on superoxide and nitric oxide production by primary cultured skeletal muscle cells were evaluated. The involvement of the main sites of these reactive species production and the relationship between superoxide and nitric oxide production were also examined. Production of superoxide was evaluated by cytochrome c reduction and dihydroethidium oxidation assays. Electrical stimulation increased superoxide production after 1?h incubation. A xanthine oxidase inhibitor caused a partial decrease of superoxide generation and a significant amount of mitochondria-derived superoxide was also observed. Nitric oxide production was assessed by nitrite measurement and by using 4,5-diaminofluorescein diacetate (DAF-2-DA) assay. Using both methods an increased production of nitric oxide was obtained after electrical stimulation, which was also able to induce an increase of iNOS content and NF-?B activation. The participation of superoxide in nitric oxide production was investigated by incubating cells with DAF-2-DA in the presence or absence of electrical stimulation, a superoxide generator system (xanthinexanthine oxidase), a mixture of NOS inhibitors and SOD-PEG. Our data show that the induction of muscle contraction by a moderate electrical stimulation protocol led to an increased nitric oxide production that can be controlled by superoxide generation. The cross talk between these reactive species likely plays a role in exercise-induced maintenance and adaptation by regulating muscular glucose metabolism, force of contraction, fatigue, and antioxidant systems activities. J. Cell. Physiol. 227: 25112518, 2012. (c) 2011 Wiley Periodicals, Inc.

Deconvolution of the EPR spectra of vanadium oxide nanotubes

In this work we report results of continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy of vanadium oxide nanotubes. The observed EPR spectra are composed of a weak well-resolved spectrum of isolated V4+ ions on top of an intense and broad structure-less line shape, attributed to spin-spin exchanged V4+ clusters. With the purpose to deconvolute the structured weak spectrum from the composed broad line, a new approach based on the Krylov basis diagonalization method (KBDM) is introduced. It is based on the discrimination between broad and sharp components with respect to a selectable threshold and can be executed with few adjustable parameters, without the need of a priori information on the shape and structure of the lines. This makes the method advantageous with respect to other procedures and suitable for fast and routine spectral analysis, which, in conjunction with simulation techniques based on the spin Hamiltonian parameters, can provide a full characterization of the EPR spectrum. Results demonstrate and characterize the coexistence of two V4+ species in the nanotubes and show good progress toward the goal of obtaining high fidelity deconvoluted spectra from complex signals with overlapping broader line shapes. (C) 2012 Elsevier Inc. All rights reserved.

Altered Neurotrophin, Neuropeptide, Cytokines and Nitric Oxide Levels in Autism

Introduction: Modifications in neurotrophins, neuropeptides, cytokines and nitric oxide (NO) levels in autism may represent different biological aspects of the disease. In the present study we investigate simultaneously all these variables as an attempt to clarify their interrelationships in autism. Methods: Plasma levels of vasoactive intestinal peptide (VIP), neurotrophin-3 (NT-3), cytokines and nitric oxide (NO) were determined in children with DSM-IV autistic disorder (n = 24) and in age- and gender-matched healthy controls (n = 24). VIP, NT-3, IFN-gamma and IL-1 beta levels were measured by ELISA, TNF-alpha, IL-10, IL-6, IL-4, IL-2 were evaluated by flow cytometry, and NO by Griess reaction. Results: Plasma levels of VIP, IFN-gamma and NO were significantly higher and NT-3 plasma levels were significantly lower in children with autism, compared to the healthy subjects. In children with autism there was a positive correlation between plasma levels of NO and IFN-gamma. Discussion: Our results indicate the presence of altered levels of neurotrophin and neuropeptide in infantile autism and provide additional evidence that higher levels of IFN-gamma may be associated with increased oxidative stress in autism.

Nitrous oxide emissions from gasohol, ethanol and CNG light duty vehicles

A sample of 21 light duty vehicles powered by Otto cycle engines were tested on a chassis dynamometer to measure the exhaust emissions of nitrous oxide (N2O). The tests were performed at the Vehicle Emission Laboratory of CETESB (Environmental Company of the State of Sao Paulo) using the US-FTP-75 (Federal Test Procedure) driving cycle. The sample tested included passenger cars running on three types of fuels used in Brazil: gasohol, ethanol and CNG. The measurement of N2O was made using two methods: Non Dispersive InfraRed (NDIR) analyzer and Fourier Transform InfraRed spectroscopy (FTIR). Measurements of regulated pollutants were also made in order to establish correlations between N2O and NOx. The average N2O emission factors obtained by the NDIR method was 78 +/- 41 mg.km(-1) for vehicles running with gasohol, 73 +/- 45 mg.km(-1) for ethanol vehicles and 171 +/- 69 mg.km(-1) for CNG vehicles. Seventeen results using the FTIR method were also obtained. For gasohol vehicles the results showed a good agreement between the two methods, with an average emission factor of 68 +/- 41 mg.km(-1). The FTIR measurement results of N2O for ethanol and CNG vehicles were much lower than those obtained by the NDIR method. The emission factors were 17 +/- 10 mg.km(-1) and 33 +/- 17 mg.km(-1), respectively, possibly because of the interference of water vapor (present at a higher concentration in the exhaust gases of these vehicles) on measurements by the NDIR method.

The Effects of Palmitic Acid on Nitric Oxide Production by Rat Skeletal Muscle: Mechanism via Superoxide and iNOS Activation

Background: Increased plasma concentrations of free fatty acids (FFA) can lead to insulin resistance in skeletal muscle, impaired effects on mitochondrial function, including uncoupling of oxidative phosphorylation and decrease of endogenous antioxidant defenses. Nitric oxide (NO) is a highly diffusible gas that presents a half-life of 5-10 seconds and is involved in several physiological and pathological conditions. The effects of palmitic acid on nitric oxide (NO) production by rat skeletal muscle cells and the possible mechanism involved were investigated. Methods: Primary cultured rat skeletal muscle cells were treated with palmitic acid and NO production was assessed by nitrite measurement (Griess method) and 4,5-diaminofluorescein diacetate (DAF-2-DA) assay. Nuclear factor-kappa B (NF-kappa B) activation was evaluated by electrophoretic mobility shift assay and iNOS protein content by western blotting. Results: Palmitic acid treatment increased nitric oxide production. This effect was abolished by treatment with NOS inhibitors, L-nitro-arginine (LNA) and L-nitro-arginine methyl esther (L-NAME). NF-kappa B activation and iNOS content were increased due to palmitic acid treatment. The participation of superoxide on nitric oxide production was investigated by incubating the cells with DAF-2-DA in the presence or absence of palmitic acid, a superoxide generator system (X-XO), a mixture of NOS inhibitors and SOD-PEG (superoxide dismutase linked to polyethylene glycol). Palmitic acid and X-XO system increased NO production and this effect was abolished when cells were treated with NOS inhibitors and also with SOD-PEG. Conclusions: In summary, palmitic acid stimulates NO production in cultured skeletal muscle cells through production of superoxide, nuclear factor-kappa B activation and increase of iNOS protein content. Copyright (C) 2012 S. Karger AG, Basel

Analysis of kinematic, kinetic and electromyographic patterns during root canal preparation with rotary and manual instruments

Objective: This study assessed the muscular activity during root canal preparation through kinematics, kinetics, and electromyography (EMG). Material and Methods: The operators prepared one canal with RaCe rotary instruments and another with Flexofiles. The kinematics of the major joints was reconstructed using an optoelectronic system and electromyographic responses of the flexor carpi radial's, extensor carpi radialis, brachioradialis, biceps brachii, triceps brachii, middle deltoid, and upper trapezius were recorded. The joint torques of the shoulder, elbow and wrist were calculated using inverse dynamics. In the kinematic analysis, angular movements of the wrist and elbow were classified as low risk factors for work-related musculoskeletal disorders. With respect to the shoulder, the classification was medium-risk. Results: There was no significant difference revealed by the kinetic reports. The EMG results showed that for the middle deltoid and upper trapezius the rotary instrumentation elicited higher values. The flexor carpi radialis and extensor carpi radialis, as well as the brachioradialis showed a higher value with the manual method. Conclusion: The muscular recruitment for accomplishment of articular movements for root canal preparation with either the rotary or manual techniques is distinct. Nevertheless, the rotary instrument presented less difficulty in the generation of the joint torque in each articulation, thus, presenting a greater uniformity of joint torques.

The protective effect of cilostazol on isolated rabbit femoral arteries under conditions of ischemia and reperfusion: the role of the nitric oxide pathway

OBJECTIVES: The clinical significance of ischemia/reperfusion of the lower extremities demands further investigation to enable the development of more effective therapeutic alternatives. This study investigated the changes in the vascular reactivity of the rabbit femoral artery and nitric oxide metabolites under partial ischemia/reperfusion conditions following cilostazol administration. METHODS: Ischemia was induced using infrarenal aortic clamping. The animals were randomly divided into seven groups: Control 90 minutes, Ischemia/Reperfusion 90/60 minutes, Control 120 minutes, Ischemia/Reperfusion 120/90 minutes, Cilostazol, Cilostazol before Ischemia/Reperfusion 120/90 minutes, and Ischemia 120 minutes/Cilostazol/Reperfusion 90 minutes. Dose-response curves for sodium nitroprusside, acetylcholine, and the calcium ionophore A23187 were obtained in isolated femoral arteries. The levels of nitrites and nitrates in the plasma and skeletal muscle were determined using chemiluminescence. RESULTS: Acetylcholine- and A23187-induced relaxation was reduced in the Ischemia/Reperfusion 120/90 group, and treatment with cilostazol partially prevented this ischemia/reperfusion-induced endothelium impairment. Only cilostazol treatment increased plasma levels of nitrites and nitrates. An elevation in the levels of nitrites and nitrates was observed in muscle tissues in the Ischemia/Reperfusion 120/90, Cilostazol/Ischemia/Reperfusion, and Ischemia/Cilostazol/Reperfusion groups. CONCLUSION: Hind limb ischemia/reperfusion yielded an impaired endothelium-dependent relaxation of the femoral artery. Furthermore, cilostazol administration prior to ischemia exerted a protective effect on endothelium-dependent vascular reactivity under ischemia/reperfusion conditions.

Cellulose micro/nanofibres from Eucalyptus kraft pulp: Preparation and properties

There is growing interest in cellulose nanofibres from renewable sources for several industrial applications. However, there is a lack of information about one of the most abundant cellulose pulps: bleached Eucalyptus kraft pulp. The objective of the present work was to obtain Eucalyptus cellulose micro/nanofibres by three different processes, namely: refining, sonication and acid hydrolysis of the cellulose pulp. The refining was limited by the low efficiency of isolated nanofibrils, while sonication was more effective for this purpose. However, the latter process occurred at the expense of considerable damage to the cellulose structure. The whiskers obtained by acid hydrolysis resulted in nanostructures with lower diameter and length, and high crystallinity. Increasing hydrolysis reaction time led to narrower and shorter whiskers, but increased the crystallinity index. The present work contributes to the different widespread methods used for the production of micro/nanofibres for different applications. (C) 2012 Elsevier Ltd. All rights reserved.

Preparation and Structural Characterization of Vulcanized Natural Rubber Nanocomposites Containing Nickel-Zinc Ferrite Nanopowders

Single-phase polycrystalline mixed nickel-zinc ferrites belonging to Ni0.5Zn0.5Fe2O4 were prepared on a nanometric scale (mean crystallite size equal to 14.7 nm) by chemical synthesis named the modified poliol method. Ferrite nanopowder was then incorporated into a natural rubber matrix producing nanocomposites. The samples were investigated by means of infrared spectroscopy, X-ray diffraction, scanning electron microscopy and magnetic measurements. The obtained results suggest that the base concentration of nickel-zinc ferrite nanoparticles inside the polymer matrix volume greatly influences the magnetic properties of nanoconnposites. A small quantity of nanoparticles, less than 10 phr, in the nanocomposite is sufficient to produce a small alteration in the semi-crystallinity of nanocomposites observed by X-ray diffraction analysis and it produces a flexible magnetic composite material with a saturation magnetization, a coercivity field and an initial magnetic permeability equal to 3.08 emu/g, 99.22 Oe and 9.42 X 10(-5) respectively.