Calcium oxalate precipitation by diffusion using laminar microfluidics: toward a biomimetic model of pathological microcalcifications.

The effect of mixing calcium and oxalate precursors by diffusion at miscible liquid interfaces on calcium oxalate crystalline phases, and in physiological conditions (concentrations and flow rates), is studied using a microfluidic channel. This channel has similar dimensions as the collection duct in human kidneys and serves as a biomimetic model in order to understand the formation of pathological microcalcifications.

Efficiency of the refocused 31P-29Si MAS-J-INEPT NMR experiment for the characterization of silicophosphate crystalline phases and amorphous gels.

One- and two-dimensional refocused MAS-J-INEPT NMR experiments in the solid state (through-bond polarization transfer) involving the highly abundant 31P spin and the rare 29Si spin are described for the crystalline silicophosphate phase Si5O(PO4)6 and complex mixtures of SiP2O7 polymorphs. The evaluation of the 2JP-O-Si coupling constants for all 29Si sites is obtained by the careful analysis of the INEPT build-up curves under fast MAS. The results are in agreement with the crystallographic data, taking into account the various J coupling paths. The efficiency of the experiment is demonstrated by its application to more complex systems such as silicophosphate amorphous gels (obtained by the sol-gel process).

Application of the MAS-J-HMQC experiment to a new pair of nuclei {29Si,31P}: Si5O(PO4)6 and SiP2O7 polymorphs.

We report the results of the two-dimensional MAS-J-HMQC experiment providing scalar correlations between 29Si and 31P nuclei in solid state NMR, and we give the first evaluation of the 2JSi-O-P coupling constants (approximately 15 Hz) for a crystalline silicophosphate phase Si5O(PO4)6. The experiment is applied to the characterization of complex mixtures of SiP2O7 phases, through editing of 31P spin pairs by the heteronuclear 2JP-O-Si interaction.

Liquid chromatography with multi-channel electrochemical detection for the determination of epigallocatechin gallate in rat plasma utilizing an automated blood sampling device.

A liquid chromatography method with multi-channel electrochemical detection was developed for the determination of epigallocatechin gallate (EGCG) in rat plasma. After administration of EGCG, blood samples were periodically collected by Culex (an automated blood sampling robot). EGCG was extracted from 50 microl of diluted blood (blood and saline at a ratio of 1:1) with ethyl acetate. Chromatographic separation was achieved within 10 min using a C8 (150x4.6 mm) 5 microm column with a mobile phase containing 20 mM sodium monochloroacetate, pH 2.8 and 12% acetonitrile at a flow-rate of 1.2 ml/min. A four-channel detector with glassy carbon electrodes was used with applied potentials of +700, 600, 500, 400 mV vs. Ag/AgCl. The limit of detection was 2 ng/ml at a signal-to-noise ratio of 3:1 and the limit of quantitation was 5 ng/ml. The calibration curve was linear over the range of 5-800 ng/ml. The intra- and inter-assay precisions were in the range of 1.3-4.5% and 2.2-4.4%, respectively. Using this method it was possible to determine plasma concentration following a single dose of EGCG to rats with good accuracy and precision. Thus the pharmacokinetic properties of EGCG in rats can be examined for intravenous, intraperitoneal and oral dosing.

In vivo microdialysis and reverse phase ion pair liquid chromatography/tandem mass spectrometry for the determination and identification of acetylcholine and related compounds in rat brain.

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. A microdialysis probe was surgically implanted into the striatum of the rats and Ringer's solution was used as the perfusion medium at a flow rate of 2 microL per minute. The samples were then analyzed off-line by LC/MS/MS experiments. The separation of ACh and choline (Ch) was carried out using reverse phase ion pair liquid chromatography with heptafluorobutyric acid as a volatile ion pairing reagent. Analytes were detected by electrospray ionization tandem mass spectrometry in the positive ion mode. The detection limit for ACh was 1.4 fmol on column, which is at least three times lower than previously reported. Three quaternary ammonium compounds in the rat brain microdialysate were also identified by tandem mass spectrometry experiments in which the unknown mass spectra were compared with standard reference compounds. These compounds were identified as carnitine, acetylcarnitine and (3-carboxypropyl)trimethylammonium. This is the first known report of the compound (3-carboxypropyl)trimethylammonium being found in rat brain.

Electrode materials for nitric oxide detection.

Nitric oxide oxidation signals were compared for uniform test electrodes of platinum, iridium, palladium, rhodium, ruthenium, gold, graphite, and a nickel-porphyrin on graphite in deaerated phosphate-buffered saline (pH 7.0) at 35 degrees C. All tested materials detected NO(*) amperometrically. Current densities (A/M/cm(2) +/- S.D.) were Ir (0.021 +/- 0.002), Rh (0.088 +/- 0.012), graphite (0.117 +/- 0.018), Pd (0.118 +/- 0.033), Au (0.149 +/- 0. 039), Pt (0.237 +/- 0.117), Ni (II)-tetra(3-methoxy-4-hydroxyphenyl) porphyrin on graphite (0.239 +/- 0.009), and Ru (0.680 +/- 0.058). NO(*) oxidation current on ruthenium was maximal at 675 mV (vs Ag/AgCl), nearly three times that on the next-best materials, platinum and Ni-porphyrin on graphite poised at 800 mV. The measured limit of detection for NO(*) on Ru was below 3 nM. Enhanced NO(*) oxidation current on ruthenium is apparently due to formation of nitrosyl- or chloronitrosyl-ruthenium complexes at the electrode surface. At fixed potentials above 675 mV, ruthenium exhibited an even larger NO(*) response, characterized by current flow opposite in polarity to an oxidation, which we hypothesize reflects suppression of the oxidative background current (presumably due to chloride oxidation or to the electrolysis of water) by a film consisting of nitrosyl- or chloronitrosyl-ruthenium complexes. The sensitive response of the ruthenium electrode to the direct oxidation of NO(*) may be useful in sensors for biomedical applications.

The yeast Saccharomyces cerevisiae does not sequester chloride but can express a functional mammalian chloride channel.

Chloride uptake into yeast was measured as a function of pH. A small amount of uptake was seen at pH values of 3.0 and 4.0; at pH 6.0 chloride uptake was substantially less than the uptake of phosphate and rubidium. Because chloride uptake is inefficient, we expressed the putative mammalian chloride channel, pI(Cln), in yeast and observed a chloride-selective current when total membrane protein was reconstituted into lipid bilayers. The current was inhibited by a specific chloride channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoic acid. These results suggest that yeast may serve as a means to characterize chloride channels from other organisms.

Water transport across yeast vacuolar and plasma membrane-targeted secretory vesicles occurs by passive diffusion.

To determine whether solute transport across yeast membranes was facilitated, we measured the water and solute permeations of vacuole-derived and late secretory vesicles in Saccharomyces cerevisiae; all permeations were consistent with passive diffusive flow. We also overexpressed Fps1p, the putative glycerol facilitator in S. cerevisiae, in secretory vesicles but observed no effect on water, glycerol, formamide, or urea permeations. However, spheroplasts prepared from the strain overexpressing Fps1p showed enhanced glycerol uptake, suggesting that Fps1p becomes active only upon insertion in the plasma membrane.

Reconstituted aquaporin 1 water channels transport CO2 across membranes.

Biological membranes provide selective barriers to a number of molecules and gases. However, the factors that affect permeability to gases remain unclear because of the difficulty of accurately measuring gas movements. To determine the roles of lipid composition and the aquaporin 1 (AQP1) water channel in altering CO2 flux across membranes, we developed a fluorometric assay to measure CO2 entry into vesicles. Maximal CO2 flux was approximately 1000-fold above control values with 0.5 mg/ml carbonic anhydrase. Unilamellar phospholipid vesicles of varying composition gave widely varying water permeabilities but similar CO2 permeabilities at 25 degreesC. When AQP1 purified from human red blood cells was reconstituted into proteoliposomes, however, it increased water and CO2 permeabilities markedly. Both increases were abolished with HgCl2, and the mercurial inhibition was reversible with beta-mercaptoethanol. We conclude that unlike water and small nonelectrolytes, CO2 permeation is not significantly altered by lipid bilayer composition or fluidity. AQP1 clearly serves to increase CO2 permeation, likely through the water pore; under certain circumstances, gas permeation through membranes is protein-mediated.

Reconstitution of water channel function of aquaporins 1 and 2 by expression in yeast secretory vesicles.

Aquaporins 1 (AQP1) and 2 (AQP2) were expressed in the yeast secretory mutant sec6-4. The mutant accumulates post-Golgi, plasma membrane-targeted vesicles and may be used to produce large quantities of membrane proteins. AQP1 or AQP2 were inducibly expressed in yeast and were localized within isolated sec6-4 vesicles by immunoblot analysis. Secretory vesicles containing AQP1 and AQP2 exhibited high water permeabilities and low activation energies for water flow, indicating expression of functional AQP1 and AQP2. AQP1 solubilized from secretory vesicles was successfully reconstituted into proteoliposomes, demonstrating the ability to use the yeast system to express aquaporins for reconstitution studies. The AQP2-containing secretory vesicles showed no increased permeability toward formamide, urea, glycerol, or protons compared with control vesicles, demonstrating that AQP2 is highly selective for water over these other substances. We conclude that the expression of aquaporins in yeast sec6 vesicles is a valid system to further study mammalian water channel function.

Enzymatically amplified voltammetric sensor for microliter sample volumes of salicylate.

A new voltammetric sensing strategy for salicylate employing two enzymes and applicable to microliter sample volumes is demonstrated. The method involves the use of the enzyme salicylate hydroxylase to convert salicylate to catechol, which is oxidized at a carbon electrode. The product of this oxidation reaction, o-quinone, is then reduced by a second enzyme, glucose oxidase, to regenerate catechol. Reoxidation of catechol results in a signal that is amplified due to repeated cycling of catechol molecules between the oxidized and reduced states. This chemistry is implemented in two configurations. (i) A paper disk into which both enzymes have been absorbed is mounted on a coplanar three-electrode assembly for aqueous experiments. Determination of salicylate in a nonprescription dermatological product is demonstrated. (ii) A small solution volume confined directly on the coplanar electrodes is used for determination of salicylate in whole blood. The advantages of the use of two enzymes and of monitoring steady-state catalytic currents are discussed.

Mediated, anaerobic voltammetry of sulfite oxidase.

The anaerobic voltammetry of the Mo/Fe enzyme, sulfite oxidase (SO), is described for the mediators cytochrome c, [Ru(NH3)6]3+/2+, TMPD+/0, and [Co(bpy)3]3+/2+. Theory derived for steady-state voltammetric catalysis correctly predicts the observed concentration and scan-rate dependencies of the catalytic waves. The instances for which existing ECcat theories may be applied to two catalytic reactions coupled to an interfacial charge transfer are considered. The biomolecular rate constant for the reaction of [Co(bpy)3]3+ with reduced SO is calculated and determined to be approximately 5 X 10(4) L.mol-1.s-1. The appearance of catalytic prepeaks at low sulfite concentrations is noted and the shape of corresponding i/t curves from chronoamperometry is examined. The analytical implications of the novel time dependence of the catalytic current under these conditions are discussed.

T-cell receptor gene rearrangement and expression in human natural killer cells: natural killer activity is not dependent on the rearrangement and expression of T-cell receptor alpha, beta, or gamma genes.

To test the hypothesis that the T-cell receptor (Tcr) gamma gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16+ lymphocytosis were analyzed for rearrangements and expression of the human Tcr alpha, beta, and gamma genes. Two of the clones displayed distinct rearrangements of their Tcr beta and gamma genes and expressed mature Tcr alpha, beta, and gamma RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr gamma gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr alpha and beta gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.

Sensitivity of phospholipase C (Bacillus cereus) activity to phosphatidylcholine structural modifications.

The structural features of a phosphatidylcholine molecule important for binding to phospholipase C (Bacillus cereus) have been examined using kinetic analyses of a series of short-chain phosphatidylcholines and analogues. Lipids examined had varying chain lengths, methyl branched chains, phenyl alkanoate chains, and a single fatty acyl chain (lysophosphatidylcholines). A comparison of Vmax and Km for monomolecularly dispersed dibutyroyl-, dihexanoyl- and diheptanoylphosphatidylcholine indicates that the length of the fatty acyl chains must be at least six carbons for efficient binding of the phosphatidylcholine to the enzyme. Enzymatic rates of hydrolysis for pure short-chain phosphatidylcholine micelles of different chain lengths or detergent mixed micelles with comparable concentrations of short- and long-chain phosphatidylcholines show no dependence on substrate chain length greater than six carbons. Methyl branching of short-chain phosphatidylcholines only inhibits phospholipase C activity when the methyl group is adjacent to the carbonyl (e.g., di(2-methyl)hexanoylphosphatidylcholine). In a similar fashion, phosphatidylcholines with phenylalkanoate chains become poor substrates when the phenyl group is near the acyl linkage. As the phenyl group is moved from C-4 to C-2 a large increase in the micellar apparent Km is observed. Chain specificity (sn-1 and/or sn-2 ester linkages) for binding is not absolute, since phospholipase C will hydrolyze micellar short-chain lysophosphatidylcholines at rates one tenth of phosphatidylcholines. In contrast, substitution of ester linkages with ether moieties yields phosphatidylcholine analogues which are even poorer substrates and not good inhibitors of phospholipase C. These results suggest that the carbonyl group and its immediate environment are important for phospholipid interacting with this water-soluble lipolytic enzyme.