Micron-sized domains in quasi single-component giant vesicles.

Giant unilamellar vesicles (GUVs), are a convenient tool to study membrane-bound processes using optical microscopy. An increasing number of studies highlights the potential of these model membranes when addressing questions in membrane biophysics and cell-biology. Among them, phase transitions and domain formation, dynamics and stability in raft-like mixtures are probably some of the most intensively investigated. In doing so, many research teams rely on standard protocols for GUV preparation and handling involving the use of sugar solutions. Here, we demonstrate that following such a standard approach can lead to the abnormal formation of micron-sized domains in GUVs grown from only a single phospholipid. The membrane heterogeneity is visualized by means of a small fraction (0.1 mol%) of a fluorescent lipid dye. For dipalmitoylphosphatidylcholine GUVs, different types of membrane heterogeneities were detected. First, the unexpected formation of micron-sized dye-depleted domains was observed upon cooling. These domains nucleated about 10 K above the lipid main phase transition temperature, TM. In addition, upon further cooling of the GUVs down to the immediate vicinity of TM, stripe-like dye-enriched structures around the domains are detected. The micron-sized domains in quasi single-component GUVs were observed also when using two other lipids. Whereas the stripe structures are related to the phase transition of the lipid, the dye-excluding domains seem to be caused by traces of impurities present in the glucose. Supplementing glucose solutions with nm-sized liposomes at millimolar lipid concentration suppresses the formation of the micron-sized domains, presumably by providing competitive binding of the impurities to the liposome membrane in excess. It is likely that such traces of impurities can significantly alter lipid phase diagrams and cause differences among reported ones.

Improvement of chlorophyll identification in foodstuffs by MALDI ToF/ToF mass spectrometry using 1,5-diaminonaphthalene electron transfer secondary reaction matrix.

Chlorophylls (Chls) are important pigments responsible for the characteristic green color of chloroplasts in algae and plants. In this study, 1,5-diaminonaphthalene (DAN) was introduced as an electron transfer secondary reaction matrix for the identification of intact chlorophylls and their derivatives, by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). DAN was proved to drastically outperform conventional matrices such as α-cyano-4-hydroxycinnnamic acid, dithranol, antracene, and even terthiophene, since loss of the metal ion and fragmentation of the phytol-ester linkage are negligible. Absence of significant fragmentation of radical cations of Chls a and b at m/z 892.529 and 906.513, respectively, makes MALDI MS capable of following natural degradation of intact porphyrin-based pigments whose initial steps are just represented by demetalation and dephytylation. Chl by-products, such as pyropheophytins, have been identified in dried tea leaves showing the potential of MALDI MS to follow chlorophyll biotransformation occurring in processed foodstuffs. Finally, preliminary results show the potential of MALDI MS to detect illegal vegetable oil re-greening practices.

Selenium speciation in tea by dispersive liquid-liquid microextraction coupled to high-performance liquid chromatography after derivatization with 2,3-diaminonaphthalene.

Selenium is an important element for human health, and it is present in many natural drinks and foods. Present study described a new method using dispersive liquid-liquid microextraction prior to high-performance liquid chromatography with a UV variable wavelength detector for the determination of the total selenium, Se(IV), Se(VI), and total organoselenium in tea samples. In the procedure, 2,3-diaminonaphthalene was used as the chelating reagent, 400 µL acetonitrile was used as the disperser solvent and 60 µL chlorobenzene was used as the extraction solvent. The complex of Se(IV) and 2,3-diaminonaphthalene in the final extracted phase was analyzed by high-performance liquid chromatography. The factors influencing the derivatization and microextraction were investigated. Under the optimal conditions, the limit of detection was 0.11 µg/L for Se(IV) and the linearity range was in the range of 0.5-40 µg/L. This method was successfully applied to the determination of selenium in four tea samples with spiked recoveries ranging from 91.3 to 100%.

FLiK: a direct-binding assay for the identification and kinetic characterization of stabilizers of inactive kinase conformations.

Despite the hundreds of kinase inhibitors currently in discovery and preclinical phases, the number of FDA-approved kinase inhibitors remains very low by comparison, a discrepancy which reflects the challenges which accompanies kinase inhibitor development. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibit kinase activity, but the highly conserved nature of the ATP-binding site often contributes to the poor inhibitor selectivity. To address this problem, we developed a high-throughput screening technology that can discriminate for inhibitors, which stabilize inactive kinase conformations by binding within allosteric pockets in the kinase domain. Here, we describe how to use the Fluorescence Labels in Kinases approach to measure the K(d) of ligands as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries in high throughput.

Effects of surfactin on membrane models displaying lipid phase separation.

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.

Application of 2,3-naphthalenediamine in labeling natural carbohydrates for capillary electrophoresis.

Neutral and acidic monosaccharide components in Ganoderma lucidum polysaccharide are readily labeled with 2,3-naphthalenediamine, and the resulting saccharide-naphthimidazole (NAIM) derivatives are quantified by capillary electrophoresis (CE) in borate buffer. Using sulfated-α-cyclodextrin as the chiral selector, enantiomers of monosaccharide-NAIMs are resolved on CE in phosphate buffer, allowing a simultaneous determination of the absolute configuration and sugar composition in the mucilage polysaccharide of a medicinal herb Dendrobium huoshanense. Together with the specific enzymatic reactions of various glycoside hydrolases on the NAIM derivatives of glycans, the structures of natural glycans can be deduced from the digestion products identified by CE analysis. Though heparin dissachrides could be successfully derived with the NAIM-labeling method, the heparin derivatives with the same degree of sulfation could not be separated by CE.

Predicting phospholipidosis: a fluorescence noncell based in vitro assay for the determination of drug-phospholipid complex formation in early drug discovery.

This paper describes for the first time, a high-throughput fluorescence noncell based assay to screen for the drug-phospholipid interaction, which correlates to phospholipidosis. Anionic amphiphilic phospholipids can form complexes in aqueous solution, and its critical micelle concentration (CMC) can be determined using the fluorescence probe N,N-dimethyl-6-propionyl-2-naphthylamine (Prodan). Upon interaction with drug candidates, this CMC may shift to a lower value due to the association between lipids and drug candidates, the stronger the interaction, the greater the shift. Metabolism of a drug can change the degree of phospholipidosis depending on the rate of metabolism and the nature of the metabolite(s). Our data from 45 drugs and metabolites of 10 drugs using this fluorescence approach demonstrate a good correlation with phospholipidosis as reported with human studies, in vivo testing, and cellular assays. This assay therefore offers a fast, reliable, and cost-effective screening tool for early prediction of the phospholipidosis-inducing potential of drug candidates.

Solid phase extraction--multisyringe flow injection system for the spectrophotometric determination of selenium with 2,3-diaminonaphthalene.

In the present work, a solid phase extraction (SPE) is hyphenated with an automatic MSFIA system to improve the selenite determination based on the reaction of selenite with aromatic o-diamines (such as 2,3-diaminonaphthalene (DAN)) to form the piazselenol complex. This reaction is greatly influenced by acid concentration, temperature, the time needed for colour development, and presence of foreign ions. For these reasons a thermostatic bath, glycine, and Na(2)-EDTA are used as heater, buffer, and masking agent, respectively. The principle of the determination is based on the sorption of the piazselenol onto a C(18) membrane disk, followed by its elution by acetonitrile. The piazselenol can then be detected by absorptiometry or fluorometry, both detection techniques being tested in our system. The best detection limit (1.7 microg L(-1)) and RSD (3.04%) are obtained by absorptiometry at 380 nm. Environmental samples were spiked and analyzed, with recoveries close to 100%.

Plant catechols and their S-glutathionyl conjugates as antinitrosating agents: expedient synthesis and remarkable potency of 5-S-glutathionylpiceatannol.

With a view to elucidating the structural requisites for effective antinitrosating properties in plant polyphenolics and their metabolites, we have undertaken a comparative investigation of the nitrite scavenging effects of representative catechol derivatives of dietary relevance in the 2,3-diaminonaphthalene (DAN) nitrosation and tyrosine nitration assays. Compounds tested included caffeic acid (1), chlorogenic acid (2), piceatannol (3), hydroxytyrosol (4), and the corresponding S-glutathionyl conjugates 5-8, which were prepared using either tyrosinase (5 and 6) or a novel, o-iodoxybenzoic acid (IBX)-based oxygenation/ conjugation methodology (7b and 8). In the DAN nitrosation assay at pH 4.0, the rank order of inhibitory activities was found to be 5-S-glutathionylpiceatannol (7b) > 3 > 1 > 2 > 2-S-glutathionylcaffeic acid (5) > 2-S-glutathionylchlorogenic acid (6) > 4 approximately 5-S-glutathionylhydroxytyrosol (8). Quite unexpectedly, in the tyrosine nitration assay in 0.5 M HCl, 2 was the most efficient inhibitor followed by 1 > 4 > 3 > 7b approximately 5 > 8 > 6. Under the assay conditions, the glutathionyl conjugates were usually consumed at faster rates than the parent catechols (decomposition rates: 3 > 1 > 4 > 2). The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay indicated that the most effective hydrogen donors were 4 > 7b > 1 approximately 3. Overall, these results indicated that catechol compounds and their glutathionyl conjugates may exhibit profoundly different inhibitory properties depending on the specific conditions of the assay, including especially pH, and that their antinitrosating properties do not correlate tout-court with their hydrogen donor capacity. The glutathionyl-piceatannol conjugate 7b was found to be one of the most potent inhibitors in the physiologically relevant DAN assay and may provide a new structural lead for the design of effective antinitrosating agents based on dietary polyphenolic compounds.

Determination of selenium in human serum by liquid chromatography/electron capture atmospheric pressure chemical ionization mass spectrometry after acid digestion and derivatization using 2,3-diaminonaphthalene.

Analysis of selenium in biological samples is very important and numerous analytical methods for the element have been developed. One of the most convenient and widely used methods for routine determination of serum selenium is a fluorometric method using 2,3-diaminonaphthalene (DAN); however, this method lacks specificity. We observed that 4,5-benzopiazselenol (BPS), a selenium derivative of DAN, is ionized with electron capture in an atmospheric pressure chemical ionization (APCI) interface, and subsequently established a method for determining total human serum selenium by means of liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. All pretreatment procedures were carried out in a single test tube to minimize selenium loss. The recovery of organic or inorganic selenium spiked to human serum was 97-103%. The detection limit of BPS was equivalent to 0.2 ng of selenium and the lower quantitative limit of serum selenium was 10 ng mL(-1). The coefficient of variation of standard concentrations in control serum samples was 4.5%. The purity of the observed peak obtained from serum samples was confirmed using the ion cluster technique.

Successive determinations of platinum(II) and selenium(IV) with 1,4-dibromo-2,3-diaminonaphthalene in aqueous micellar solutions.

Highly sensitive successive determinations for PtII and SeIV ions have been developed based upon reactions with 1,4-dibromo-2,3-diaminonaphthalene (Br2DAN), which forms a near-infrared (NIR) absorbing complex (epsilon = 1.2 x 10(5) l mol-1 cm-1 at 800 nm) and an emissive complex (ex. 386 nm, em. 604 nm) for PtII and SeIV ions, respectively, in acidic aqueous micellar solutions. In the presence of a cationic surfactant, cetyltrimethylammonium chloride, the detection limits for PtII and SeIV ions are 1.2 ng ml-1 (3 sigma) and 0.98 ng ml-1 (S/N = 3), respectively. Hydrobromic acid plays a key role to enhance the color development of the NIR-absorbing PtII complex. The influences of CuII and ZnII ions at the normal human serum levels are readily tolerated, and interference from FeIII ion at 35 mumol l-1 is circumvented by the addition of 50 mumol l-1 of polyaminocarboxylates, such as EDTA.

Quantitative evaluation of selenium contained in tea by high performance liquid chromatography.

For determination of selenium (Se) in biological materials, an improved method based on high performance liquid chromatographic determination of the fluorophore formed by reaction of selenite with 2,3-diaminonapththalene was developed. The concentration detection limits were 0.5 ng/g in dried materials and 0.03 ng/mL in fluid materials. In quadruplicate assays of 11 biological reference materials using the proposed method, measured Se concentrations were not significantly different from their certified values. Thus, the proposed method is reliable and suitable for the determination of trace levels of Se in foods. Using the proposed method, Se concentrations in various kinds of tea were determined to assess the contribution of tea to daily Se intake in the Japanese population. Se concentration in the leaves of general black, green and oolong tea obtained in local retail stores was 33 +/- 19 ng/g (n=440). The leaves of a particular Chinese green tea sold under the name "high Se tea" were found to contain 455 +/- 184 ng/g (n= 14) of Se. While the percentage of Se extractable by infusion was less than 5% for the general teas, that in the high Se tea was more than 20%. These results indicated that intake of tea does not contribute to daily Se intake in the Japanese population. However, since infusions from high Se tea contained over 5 ng/mL of Se, consumption of over 1 L/d of tea derived from such high Se teas may increase the daily Se intake by close to 10%.

Deltorphin II analogues with 6-hydroxy-2-aminotetralin-2-carboxylic acid in position 1.

Two approaches to the design of very active and highly selective delta opioid peptides were used to obtain new deltorphin analogues with altered hydrophobic and stereoelectronic properties. Deltorphin II analogues were synthesized with the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 6-hydroxy-2-aminotetralin-2-carboxylic acid (Hat) instead of Tyr(1) in the message domain. In the radioreceptor-binding studies, in which type-specific tritiated opioid ligands were used, (R)- and (S)-Hat-deltorphins exhibited similar K(i) values, revealing high delta selectivity. The peptides displayed agonist properties in the in vitro bioassay, with IC(50) values in the subnanomolar range in the mouse vas deferens assay and in the micromolar or higher range in the guinea pig ileum assay, again demonstrating a high selectivity toward delta receptors. The agonist property of the new ligands was confirmed by means of [(35)S]GTPgammaS-binding experiments in membranes of the rat frontal cortex.

Disclosure of discrete sites for phospholipid and sterols at the protein-lipid interface in native acetylcholine receptor-rich membrane.

There is an increasing body of evidence to support the notion that the function of the nicotinic acetylcholine receptor (AChR) is influenced by its lipid microenvironment [see Barrantes, F. J. (1993) FASEB J. 7, 1460-1467]. We have recently made use of the so-called generalized polarization (GP) of the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) to learn about the physical state of the lipids in Torpedo marmorata AChR native membrane [Antollini, S. S., Soto, M. A., Bonini de Romanelli, I., Gutiérrez Merino, C., Sotomayor, P., and Barrantes, F. J. (1996) Biophys. J. 70, 1275-1284] and cells expressing endogenous or heterologous AChR [Zanello, L. P., Aztiria, E., Antollini, S., and Barrantes, F. J. (1996) Biophys. J. 70, 2155-2164]. In the present work, Laurdan GP was measured in T. marmorata native AChR membrane by direct excitation or under energy transfer conditions in the presence of exogenous lipids. GP was found to diminish in these two regions upon addition of oleic acid and dioleoylphosphatidylcholine and not to vary significantly upon addition of cholesterol hemisuccinate, indicating an increase in the polarity of the single, ordered-liquid lipid phase in the two former cases. Complementary information about the bulk lipid order was obtained from measurements of fluorescence anisotropy of DPH and two of its derivatives. The membrane order diminished in the presence of oleic acid and dioleoylphosphatidylcholine. The location of Laurdan was determined using the parallax method. Laurdan lies at approximately 10 A from the center of the bilayer, i.e., at depth of approximately 5 A from the lipid-water interface. Exogenous lipids modified the energy transfer efficiency from the intrinsic fluorescence to Laurdan. This strategy is introduced as a new analytic tool that discloses for the first time the occurrence of discrete and independent sites for phospholipids and sterols, respectively, both accessible to fatty acids, and presumably located at a shallow depth close to the phospholipid polar head region in the native AChR membrane.

Determination of selenium in infant formula and enteral formula by dry ash graphite furnace atomic absorption spectrometry with deuterium background correction.

A method was developed to determine selenium in infant formula using a graphite furnace equipped with deuterium background correction after dry ashing. The method circumvents the use of perchloric acid, 2,3-diaminonapthalene (DAN) and hydride generation without the use of Zeeman background correction. Twelve commercial infant and enteral formulas and corresponding spiked products (30-500 ng) were analyzed in triplicate for Se to evaluate this method. All test portions were digested on a hot plate after addition of magnesium nitrate-nitric acid. Following heating, digests were evaporated to dryness and placed in a 500 degrees C muffle furnace for 30 min to complete ashing. All Se was converted to Se+4 by dissolving the ash in HCl (5 + 1) and holding the solution for 20 min in a 60 degrees C water bath. Se+4 was subsequently reduced to Se(zero) with ascorbic acid and collected on a membrane filter. The membrane filters were digested in a small volume of nitric acid in a microwave oven. Following digestion, contents of the vessels were diluted and analyzed for Se by graphite furnace atomic absorption spectrometry. Selenium standards in starch or in unfortified formula containing trace levels of Se were carried through the entire process. The recovery range for Se was 85-127%, and analyzed reference materials fell within their certified range for Se. This method is as sensitive (detection limit 0.44 ng Se/g) as methods reported in the literature and may be applicable to other foods.