Bioaccumulation of copper and toxic effects on feeding, growth, fecundity and development of pond snail Lymnaea luteola L

We studied the bioaccumulation and the toxic effects of Cu on survival, number of eggs and eggmasses laying, embryo development, growth, and food consumption in an Indian pond snail, Lymnaea luteola L. exposed for 7 weeks. Copper caused loss of chemoreception, locomotion and inhibited food consumption significantly during 7 weeks of exposure. Food consumption in Cu exposed snails significantly decreased and at 56 and 100 μg L(-1), snail stopped feeding activity. Mean number of eggmasses or eggs significantly decreased in Cu concentrations during the 7 week study. The percentage hatching decreased in Cu concentrations but there was more than 95% hatched in control in 10-11 days after spawning. Egg development was completely inhibited at 100 μg L(-1), while abnormal embryonic development observed at 32 and 56 μg L(-1) of Cu. The Cu concentration in tissues increased in Cu treated snails and bioaccumulation factor ranged from 2.3 to 18.7. Snail growth at 5.6 and 10 μg L(-1) was reduced by 6.2% and 16.9%, respectively. The study revealed that snail embryos and adults could be used as in vivo test models for ecotoxicological studies. Findings of present study are helpful for advancing water quality guidelines for protecting aquatic biota.     

Increased in vivo glucose recovery via nitric oxide release

The in vivo glucose recovery of subcutaneously implanted nitric oxide (NO)-releasing microdialysis probes was evaluated in a rat model using saturated NO solutions to steadily release NO. Such methodology resulted in a constant NO flux of 162 pmol cm(-2) s(-1) from the probe membrane over 8 h of perfusion daily. The in vivo effects of enhanced localized NO were evaluated by monitoring glucose recovery over a 14 day period, with histological analysis thereafter. A difference in glucose recovery was observed starting at 7 days for probes releasing NO relative to controls. Histological analysis at 14 days revealed lessened inflammatory cell density at the probe surface and decreased capsule thickness. Collectively, the results suggest that intermittent sustained NO release from implant surfaces may improve glucose diffusion for subcutaneously implanted sensors by mitigating the foreign body reaction.     

Cell-based indicator to visualize picomolar dynamics of nitric oxide release from living cells

We report a novel cell-based indicator that is able to visualize picomolar dynamics of nitric oxide release from living cells. Cells from a pig kidney-derived cell line (PK15) endogenously express soluble guanylate cyclase (sGC), which is a receptor protein for the selective recognition of NO. Binding of NO by sGC causes the amplified generation of guanosine 3',5'-cyclic monophosphate (cGMP). To make the PK15 cells into NO indicators, the cells are transfected with a plasmid vector encoding a fluorescent indicator for cGMP and fluorescence resonance energy transfer is recorded at 480 +/- 15 and 535 +/- 12.5 nm upon excitation of the cells at 440 +/- 10 nm. The cell-based indicator exhibits exceptional sensitivity (detection limit of 20 pM), selectivity, reversibility, and reproducibility. The outstanding sensitivity of the present indicator has led us to uncover an oscillatory release of picomolar concentrations of NO from hippocampal neurons. We present evidence that Ca2+ oscillations in hippocampal neurons underlie the oscillatory NO release from the neurons during neurotransmission. We also have succeeded in visualizing the extent of diffusing NO from single vascular endothelial cells. The present cell-based indicator provides a powerful tool to uncover picomolar dynamics of NO that regulates a wide range of cell functions in biological systems.     

Measuring nitric oxide in single neurons by capillary electrophoresis with laser-induced fluorescence: use of ascorbate oxidase in diaminofluorescein measurements

As a family of novel fluorescent indicators for nitric oxide (NO), the diaminofluoresceins (DAFs) have allowed real-time measurement of neuronal NO, an important gaseous neurotransmitter. However, the measurement of NO by the most commonly used NO sensor, 4,5-diaminofluorescein (DAF-2), is altered by two processes: the interaction of DAF-2 with intracellular dehydroascorbic acid (DHA) and the impact of ascorbic acid (AA) on the levels of N2O3, the intermediate product of the oxidation of NO that reacts with DAF-2. Similar AA/DHA effects are observed with other DAF probes, including DAF-FM and DAR-4M. To overcome these limitations, we use a specific enzymatic reaction to eliminate the confounding effect of AA on DAF quantitation of NO and then use capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection to distinguish the various reaction products. First, the enzyme ascorbate oxidase (AO) is used to catalyze the oxidation of AA to DHA. Next, CE-LIF separates the fluorescent products of the reaction of DAF-2 with NO and DHA. Control experiments, including standard mixtures and single neurons with added NO donor, successfully demonstrate the utility of this approach. This protocol is further tested with homogenates of the mouth area from the sea slug Aplysia californica, previously shown to be NO-positive, and individual nitric oxide synthase-containing buccal neurons from the freshwater snail, Lymnaea stagnalis. In each case, significant amounts of NO are detected. This AO DAF methodology is specific, effective, simple, and allows NO to be measured in single cells without detectable interference from other compounds.     

Detection of nitric oxide and nitrogen dioxide with photoluminescent porous silicon

The visible photoluminescence of porous Si is quenched by nitric oxide and nitrogen dioxide to detection limits of 1.4 × 10(-)(3) and 5.3 × 10(-)(5) Torr, respectively (corresponding to 2 ppm and 70 ppb). At analyte partial pressures in the low milliTorr range, the photoluminescence quenching is partially reversible; recovery from nitrogen oxide exposure occurs on a time scale of minutes. For both NO and NO(2), the reversible photoluminescence quenching response fits a Stern-Volmer kinetic model. At higher partial pressures, quenching deviates from Stern-Volmer kinetics and some permanent loss of photoluminescence intensity occurs due to oxidation of the porous Si surface. Photoluminescence from porous Si is not quenched by nitrous oxide or carbon dioxide and only slightly quenched by carbon monoxide and oxygen.     

Detection of ATP-induced nitric oxide in a biomimetic circulatory vessel containing an immobilized endothelium

Conditions for the adhesion of bovine pulmonary artery endothelial cells (bPAECs) in microbore tubing of 250-microm i.d. are described. When immobilized to the lumen of microbore tubing, these cells represent a mimic of a circulatory vessel's endothelium. The microbore tubing is coated with 100 microg mL(-1) fibronectin in order to promote bPAEC adhesion to the lumen of the tubing. A series of micrographs of the cells inside of the tubing indicates that approximately 3.5 h is necessary for cell adhesion. In this study, adenosine triphosphate (ATP) is used to induce the release of nitric oxide from the endothelium mimic. The endothelium-derived NO is detected amperometrically at a parallel flow cell containing a glassy carbon working electrode modified with Nafion. Results indicate that detectable amounts of NO are only produced by the endothelium mimic when ATP is present in the buffer. The typical concentration of NO produced by the endothelium mimic upon the introduction of 100 microM ATP is approximately 0.80 microM. Based on the injection volume of ATP and the estimated number of cells on the tubing lumen, this value corresponds to approximately 1 amol of NO/cell. Moreover, shear stress alone does not provide the agonistic effect required for NO production in the submicromolar range.     

Control of nitric oxide, nitrous oxide, and ammonia emissions using microwave plasmas

The subject of this paper is mitigation of the undesirable side-effects of selective non-catalytic reduction (SNCR) and selective catalytic reduction (SCR): ammonia slip, residual NO(x), and N(2)O emissions. The use of microwave-plasma discharge within the flue gas was explored as a potential pollution-control method. The key issues addressed were: (1) N(2)O, NH(3), and NO removal efficiencies; and (2) sustaining a stable plasma at atmospheric, or close to atmospheric, pressure. In non-oxidizing atmospheres, removal efficiencies were always close to 100% for all species. In the presence of oxygen, however, appreciable amounts of nitric oxide and ammonia were formed. Methods leading to preventing these undesirable effects were examined. In a number of runs, stable plasma operation was attained at pressures close to atmospheric.     

Optimization of ruthenium-containing,stabilized, nitric oxide reduction catalysts

To optimize the stabilized ruthenate catalysts for NO_x reduction in automotive exhaust treatment with respect to selectivity and stability, we studied dilution of ruthenium in the ruthenates by Ni³⁺ and Mn⁴⁺ ions in presynthesized specimens and the effect of stabilizer content in sequentially impregnated specimens. The replacement of Ru in BaRuO₃ by Mn⁴⁺ and in LaRuO₃ by Ni³⁺ increases the stability of the ruthenates with respect to volatilization in oxidizing environments. An increase in the resistance to volatilization is also observed in impregnated samples with the increase in the content of stabilizing component. These effects are substantial in shifting the onset of volatilization by several hundred degrees centigrade upwards on the temperature scale. However, all of these stabilization improvements result in decrease in the desired selectivity.     

Improving the thromboresistivity of chemical sensors via nitric oxide release: fabrication and in vivo evaluation of NO-releasing oxygen-sensing catheters

The development and in vivo analytical performance of a nitric oxide (NO)-releasing amperometric oxygen sensor with greatly enhanced thromboresistivity are reported. Gas permeable coatings formulated with cross-linked silicone rubber (SR) containing NO-generating compounds (diazeniumdiolates) are shown to release NO for extended periods of time (> 20 h) while reducing platelet adhesion and activation. Oxygen-sensing catheters prepared by dip-coating the NO-releasing films over the outer SR tubes of the implantable devices display similar analytical response properties in vitro (sensitivity, selectivity, response times) when compared to analogous sensors prepared without the NO release coatings. Superior analytical accuracy (relative to blood PO2 values measured in vitro) and greatly reduced thrombus formation on the outer surface of the sensors are observed in vivo (in canine model) with the NO release PO2 sensors compared to control sensors (without NO release) implanted simultaneously within the same animals. Based on these preliminary studies, the use of NO release polymers to fabricate catheter-style chemical sensors may be a potential solution to lingering biocompatibility and concomitant performance problems encountered when attempting to employ such devices for continuous intravascular measurements of blood gases and electrolytes.     

Confocal, two-photon laser-induced fluorescence technique for the detection of nitric oxide

We describe a confocal two-photon laser-induced fluorescence scheme for the detection of gaseous NO. Excitation from a simple YAG-pumped Coumarin 450 dye system near 452.6 nm was used to promote the two-photon NO(A (2)?(+), nu? = 0 ? X (2)?, nu? = 0) transition in the gamma(0, 0) band. Subsequent fluorescence detection in the range 200-300 nm permitted almost total rejection of elastic and geometric scatter of laser radiation for excellent signal/noise ratio characteristics. The goal of the research was to apply NO fluorescence to a relatively realistic limited optical access combustion environment. A confocal optical arrangement was demonstrated for single-point measurements of NO concentration in gas samples and in atmospheric-pressure flames. The technique is suitable for applications that offer only a single direction for optical access and when significant elastic scatter is present.     

Correlative analyses of nitric oxide generation rates and nitric oxide synthase levels in individual cells using a modular cell-retaining device

Nitric oxide (NO) is recognized as one of the major immune system agents involved in the pathogenesis and control of various diseases that may benefit from novel drug development, by exploiting NO signaling pathways and targets. This calls for detection of both intracellular levels of NO and expression of its synthesizing enzymes (NOS) in individual, intact, living cells. Such measurements are challenging, however, due to short half-life, low and fluctuating concentrations of NO, cellular heterogeneity, and inability to trace the same cells over time. The current study presents a device and methodology for correlative analysis of NO generation rates and NOS levels in the same individual cells, utilizing fluorescent imaging followed by immunohistochemistry (IHC). U937 promonocyte cell populations demonstrated significant heterogeneity in their baseline levels, in NO-generation kinetics, and in their response rates to stimuli. Individual cell analysis exposed cell subgroups which showed enhanced NO production upon stimulation, concomitantly with significant up-regulation of inducible NOS (iNOS) levels. Exogenous NO modulated the expression of iNOS in nondifferentiated cells within 1 h, in a dose-dependent manner, while treatment with lysophosphatidylcholine (LPC) enhanced the expression of iNOS, demonstrating a nondependence on NO production.     

Modeling of nitrogen oxide release in coal combustion

A model for and results of calculating the chemical-thermodynamic equilibrium of compounds and elements contained in coal and in the air required for combustion are presented. They can be used to evaluate the possibility of decreasing the NO_x release in the process of coal combustion.     

Background corrections for laser-induced-fluorescence measurements of nitric oxide in lean, high-pressure, premixed methane flames

An experimental technique is presented that both minimizes and accounts for the interference background when laser-induced-fluorescence (LIF) measurements are made of NO in lean, high-pressure, premixed, CH(4)/O(2)/N(2) flames. Measurement interferences such as fluorescence and Raman scattering from secondary species become increasingly important for high-pressure LIF studies. O(2) fluorescence interferences are particularly troublesome in lean premixed flames. An excitation-detection scheme that minimizes the effects of these interferences is identified. A procedure that corrects the resulting LIF signal so as to account for any remaining interference signal is then developed. This correction is found to vary from less than 10% of the overall NO signal at atmospheric pressure to over 40% of the overall signal at 14.6 atm for LIF measurements of NO in a series of worst-case flames (phi = 0.6, dilution ratio 2.2). The correction technique is further demonstrated to be portable over a useful range of flame conditions at each pressure.     

The release of 5-fluorouracil from microspheres of poly(epsilon-caprolactone-co-ethylene oxide)

The purpose of this study was to evaluate the in vitro release of 5-fluorouracil from microspheres prepared using a novel triblock copolymer of ε-caprolactone and ethylene oxide as the encapsulating material. Microspheres of poly(ε-caprolactone-co-ethylene oxide) were prepared by employing the “hot-melt” method of microencapsulation. Microspheres were sized using sieve analysis and scanning electron microscopy (SEM). Release studies were performed using a custom-made rotating paddle dissolution apparatus. Copolymer microspheres, fabricated by the hot melt method were shown by electron microscopy to have smooth, nonporous surfaces. Drug-loaded microspheres were found to have a broad distribution of sizes, which was thought to be a consequence of the wide range of crystal sizes of the encapsulated unmilled drug. Nonlinear release kinetics were observed from microspheres in the size fraction 75-250 μm, with a pronounced “burst release” associated with the presence of drug at the surface of the microspheres. A specific delineation of the drug release mechanism was not possible due to rapid gelation, swelling, and subsequent dissolution of the microspheres that occurred on hydration. This work describes the preparation of microspheres that swell rapidly and coalesce together on hydration, accompanied by rapid drug release and copolymer dissolution over a 2-hr period.     

In vivo electrochemical detection of nitric oxide in tumor-bearing mice

Interest in elucidating the mechanisms of action of various classes of anticancer agents and exploring the pathways of the induced-nitric oxide (NO) release provides an impetus to conceive a better designed approach to locally detect NO in tumors, in vivo. We report here on the first use of an electrochemical sensor that allows the in vivo detection of NO in tumor-bearing mice. In a first step, we performed the electrochemical characterization of a stable electroactive probe, K4Fe(CN)6, directly injected into the liquid microenvironment especially created around the electrode in the tumor. Second, the ability of the inserted electrode system to detect the presence of NO itself in the tumoral tissue was achieved by using the chemically modified Pt/Ir electrode as NO sensor and two NO donor molecules: diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium 1,2-diolate (DEA-NONOate) and (Z)-1-[N-(2-aminopropyl)-N-(2-ammonio propyl)amino]diazen-1-ium 1,2-diolate (PAPA-NONOate). These two NO donor molecules allowed proving the electrochemical detection of (i) directly injected exogenous NO phosphate buffer solution into the tumor (decomposed DEA-NONOate) and (ii) biomimetically induced endogeneous release of NO in the tumoral tissue, upon injection of PAPA-NONOate into the tumor. This approach could be applied to the in vivo study of candidate anticancer drugs acting on the NO pathways.     

Triggered release from liposomes through magnetic actuation of iron oxide nanoparticle containing membranes

The ideal nanoscale drug delivery vehicle allows control over the released dose in space and time. We demonstrate that this can be achieved by stealth liposomes comprising self-assembled superparamagnetic iron oxide nanoparticles (NPs) individually stabilized with palmityl-nitroDOPA incorporated in the lipid membrane. Alternating magnetic fields were used to control timing and dose of repeatedly released cargo from such vesicles by locally heating the membrane, which changed its permeability without major effects on the environment.     

Numerical simulation of the crystallization front inversion in oxide single crystals grown from melt using the Czochralski method

The process of pulling a Czochralski grown oxide single crystal out from melt has been numerically simulated for the first time from the moment of seeding until attaining a stationary growth regime. It is established that the phase boundary inversion at the lateral (shouldering) growth stage is a rather complicated process accompanied by intense oscillations of the crystallization rate. Diagrams illustrating variations of the crystal shape during this process are presented.     

Imaging of Zn2+ release from pancreatic beta-cells at the level of single exocytotic events

Regulated secretion of Zn2+ from isolated pancreatic beta-cells was imaged using laser-scanning confocal microscopy. In the method, beta-cells were incubated in a solution containing the novel fluorescent Zn2+ indicator FluoZin-3. Zn2+ released from the cells reacted with the dye to form a fluorescent product, which was detected by the confocal microscope. The new dye is much brighter than Zinquin, previously used for this application, allowing detection limits of 10-40 nM and temporal resolution of 16 ms/image. The high temporal resolution allowed imaging of isolated fluorescent transients that occurred at the edge of the cells following stimulation with 20 mM glucose or 40 mM K+. Fluorescent transients took 16-50 ms to reach a peak from the initial rise and returned to baseline after 170 +/- 50 ms (n = 78 transients from 15 cells). It was concluded that the transients correspond to detection of exocytotic release of Zn2+. Analysis of the temporal and spatial dispersion of the transients indicates that the release of Zn2+ is not diffusion limited but is instead kinetically controlled in agreement with previous observations of insulin release detected by amperometry.     

Simultaneous electrochemical detection of nitric oxide and carbon monoxide generated from mouse kidney organ tissues

A planar-type amperometric dual microsensor for simultaneous detection of nitric oxide and carbon monoxide is presented. The sensor consists of a dual platinum microdisk-based working electrode (WE) and a Ag/AgCl counter/reference electrode covered with an expanded poly(tetrafluoroethylene) (Tetra-tex) gas-permeable membrane. The dual WE possesses two different platinized platinum disks (WE1 and WE2, 250 and 25 microm in diameter, respectively). The larger WE1 is further modified with electrochemical deposition of tin. Use of two sensing disks different in their size as well as in their surface modification produces apparently different sensitivity ratios of NO to CO at WE1 and at WE2 (approximately 2 and approximately 10, respectively) that are induced by favorable CO oxidation on the surface of tin versus platinum. Anodic currents independently measured at WE1 and at WE2 are successfully converted to the concentrations of NO and CO in the co-presence of these gases using the differentiated sensitivities at each electrode. The sensor is evaluated in terms of its analytical performance: respectable linear dynamic range (sub nM to microM); low detection limit (approximately 1 nM for NO and <5 nM for CO); selectivity (over nitrite up to approximately 1 mM); and sensitivity (sufficient for analyzing physiological levels of NO and CO). Using the NO/CO dual microsensor, real-time, simultaneous, direct, and quantitative measurements of NO and CO generated from living biological tissue (mouse, c57, kidney) surfaces, for the first time, are reported.     

Laser-induced-fluorescence detection of nitric oxide in high-pressure flames with A-X(0, 2) excitation

Laser-induced fluorescence techniques have been used successfully for quantitative two-dimensional measurements of nitric oxide. The commonly applied D-X(0, 1) or A-X(0, 0) schemes are restricted to atmospheric-pressure flames and engines driven with gaseous fuels because of strong attenuation of the exciting laser beam by combustion intermediates. The properties of a detection scheme for which excitation in the nitric oxide A-X(0, 2) band was used were investigated. We discuss the advantages of the A-X(0, 2) system (excited at 247.95 nm) based on measurements in laminar premixed methane/air flames at 1-40 bars.