Nucleophilic Addition Reaction with Dearomatization of Naphthalene Ring.

Various aromatic lactones have been synthesized and their regioselectivity (1,2-addition vs. 1,4- or 1,6-addition) investigated in reactions with organolithium species, particularly n-BuLi and sec-BuLi. The regioselectivity varied greatly depending on various factors, such as the bulkiness of both substrates and organolithium species, and types of solvent and cosolvent. In particular, 1,4-addition with dearomatization occurred preferentially using sec-BuLi as the nucleophile in tetrahydrofuran (THF) with hexamethylphosphoramide (HMPA) or N,N'-dimethylpropyleneurea (DMPU) as cosolvent. For sec-BuLi, the reaction was estimated to proceed through a single-electron transfer mechanism.

Nonlinear concentration gradients regulated by the width of channels for observation of half maximal inhibitory concentration (IC50) of transporter proteins.

This paper describes a simple microfluidic device that can generate nonlinear concentration gradients. We changed the "width" of channels that can drastically shorten the total microfluidic channel length and simplify the microfluidic network design rather than the "length" of channels. The logarithmic concentration gradients generated by the device were in good agreement with those obtained by simulation. Using this device, we evaluated a probable IC50 value of the ABC transporter proteins by the competitive transport assays at five different logarithmic concentrations. This probable IC50 value was in good agreement with an IC50 value (0.92 µM) obtained at the diluted concentrations of seven points.

Single-vesicle estimation of ATP-binding cassette transporters in microfluidic channels.

We have developed a method to analyze the substrate transport of ATP-binding cassette (ABC) transporters, which are associated with drug resistance in tumor cells. Our microfluidic method is well suited to the single-vesicle estimation of substrate transport and the rapid drug screening of ABC transporters. Using this method, we have demonstrated, for the first time, the analysis of substrate transport by a single transporter and performed drug-inhibition experiments in less than 3 h.

Rapid detection of a cocaine-binding aptamer using biological nanopores on a chip.

This paper describes a methodology for the rapid and highly selective detection of cocaine using a membrane protein channel combined with a DNA aptamer. The DNA aptamer recognizes the cocaine molecule with high selectivity. We successfully detected a low concentration of cocaine (300 ng/mL, the drug test cutoff limit) within 60 s using a biological nanopore embedded in a microchip.

Lipid-coated microdroplet array for in vitro protein synthesis.

Monitoring complex biological assays such as in vitro protein synthesis over long periods in micrometer-sized cavities of poly(dimethyl siloxane) (PDMS) microfluidic devices requires a strategy that solves the adsorption and absorption problems on PDMS surfaces. In this study, we developed a technique that instantaneously arrays aqueous microdroplets coated with a phospholipid membrane within a single microfluidic device. The simple lipid bilayer coating effectively inhibits the adsorption of proteins and DNA, whereas the encapsulation of the droplet reduces the area in contact with the PDMS surface, resulting in decreased absorption in part. Although the size becomes smaller during the first few hours, a lipid-coated microdroplet array demonstrated a temporal stability of more than 20 h and a size uniformity of CV 3% in the device. Furthermore, we succeeded in expressing a green fluorescent protein by confining an in vitro translation system in the microdroplets, which was confirmed by scanning the fluorescence spectrum of the droplets, demonstrating that the lipid coat secured the synthetic reaction from the adsorption problem.

Nonviral gene vector formation in monodispersed picolitre incubator for consistent gene delivery.

A novel picolitre incubator based microfluidic system for consistent nonviral gene carrier formulation is presented. A cationic lipid-based carrier is the most attractive nonviral solution for delivering plasmid DNA, shRNA, or drugs for pharmaceutical research and RNAi applications. The size of the cationic lipid and DNA complex (CL-DNA), or the lipoplex, is one of the important variations for consistency of gene transfection. CL-DNA size, in turn, may be controlled by factors such as the cationic lipid and DNA mixing order, mixing rate, and mixture incubation time. The Picolitre Microfluidic Reactor and Incubator (PMRI) system described here is able to control these parameters in order to create homogeneous CL-DNA. Compared with conventional CL-DNA preparation techniques involving hand-shaking or vortexing, the PMRI system demonstrates a greater ability to constantly and uniformly mix cationic lipids and DNA simultaneously. After mixing in the picolitre droplet reactors, the cationic lipid and DNA is incubated within the picolitre incubator to form CL-DNA. The PMRI generates a narrower size distribution band, while also turning the sample loading, mixing and incubation steps into an integrated process enabling the consistent formation of CL-DNA. The coefficient of variation (CV) of transfection efficiency is 0.05 and 0.30 for PMRI-based and conventional methods, respectively. In addition, this paper demonstrates that the gene transfection efficiency of lipoplex created in the PMRI is more reproducible.

A novel fluorescent probe that senses the physical state of lipid bilayers.

Cell membrane lipids and proteins are heterogeneously distributed in the membrane plane. In recent years, much attention has been paid to the heterogeneous distribution of the lipid components, particularly the formation of cholesterol-rich domains that are thought to be important in signaling processes. This has led to renewed interest in the phase diagrams of complex lipid mixtures, such as three-component mixtures containing phospholipids and cholesterol. We report here a novel fluorescent probe (NBD-R595) that is useful for exploring the phase behaviors of one-, two-, and three-component large unilamellar vesicles. In one-component fluid-phase membranes, the probe has the expected spectral characteristic of monomeric 7-nitrobenzo-2-oxa-1,3-diazol, with a fluorescence maximum of 540 nm when excited at 470 nm. But below the gel-to-liquid crystalline phase transition temperature, an additional emission peak appears at approximately 610 nm, because of Förster resonance energy transfer from NBD-R595 monomers to NBD-R595 Jelley aggregates of limited size formed by the association of 7-nitrobenzo-2-oxa-1,3-diazol moieties. This may be the first report of Förster resonance energy transfer from a single fluorophore in two different physical states. In a test of the probe, we found NBD-R595 to be remarkably sensitive to the molar composition of large unilamellar vesicles formed from cholesterol, distearoylphosphatidylcholine, and dioleoylphosphatidylcholine.

pH dependence of sphingosine aggregation.

Sphingosine and sphingosine 1-phosphate (S1P) are sphingolipid metabolites that act as signaling messengers to activate or inhibit multiple downstream targets to regulate cell growth, differentiation, and apoptosis. The amphiphilic nature of these compounds leads to aggregation above their critical micelle concentrations (CMCs), which may be important for understanding lysosomal glycosphingolipid storage disorders. We investigated the aggregation of sphingosine and S1P over a comprehensive, physiologically relevant range of pH values, ionic strengths, and lipid concentrations by means of dynamic light scattering, titration, and NMR spectroscopy. The results resolve discrepancies in literature reports of CMC and pK(a) values. At physiological pH, the nominal CMCs of sphingosine and S1P are 0.99 +/- 0.12 microM (pH 7.4) and 14.35 +/- 0.08 microM (pH 7.2), respectively. We find that pH strongly affects the aggregation behavior of sphingosine by changing the ionic and hydrogen-bonding states; the nominal critical aggregation concentrations of protonated and deprotonated sphingosine are 1.71 +/- 0.24 microM and 0.70 +/- 0.02 microM, respectively. NMR measurements revealed that the NH3+-NH2 transition of sphingosine occurs at pH 6.6, and that there is a structural shift in sphingosine aggregates caused by a transition in the predominant hydrogen-bonding network from intramolecular to intermolecular that occurs between pH 6.7 and 9.9.

Aggregation behavior of an ultra-pure lipopolysaccharide that stimulates TLR-4 receptors.

The innate immune systems of humans and other animals are activated by lipopolysaccharides (LPS), which are glucosamine-based phospholipids that form the outer leaflet of the outer membranes of Gram-negative bacteria. Activation involves interactions of LPS with the innate immunity-receptor comprised of toll-like receptor 4 in complex with so-called MD-2 protein and accessory proteins, such as CD14 and LPS binding protein. The Lipid Metabolites and Pathways Strategy (LIPID MAPS) Consortium has isolated in large amounts a nearly homogeneous LPS, Kdo(2)-Lipid A, and demonstrated that it activates macrophages via toll-like receptor 4. The active form of LPS, monomer or aggregate, is controversial. We have therefore examined the aggregation behavior and other physical properties of Kdo(2)-Lipid A. Differential scanning calorimetry of Kdo(2)-Lipid A suspensions revealed a gel-to-liquid crystalline phase transition at 36.4 degrees C (T(m)). The nominal critical aggregation concentration, determined by dynamic light scattering, was found to be 41.2 +/- 1.6 nM below the T(m) (25 degrees C), but only 8.1 +/- 0.3 nM above the T(m) (37 degrees C). The specific molecular volume of Kdo(2)-Lipid A, obtained by densitometry measurements was found to be 3159 +/- 71 A(3) at 25 degrees C, from which the number of molecules in each aggregate was estimated to be 5.8 x 10(5). The aggregation behavior of Kdo(2)-Lipid A in the presence of lipoprotein-deficient serum suggests that Re LPS monomers and multimers are the active units for the immune system in the CD14-dependent and -independent pathways, respectively.

The packing of lipid chains changes the character of bacteriorhodopsin reconstituted in a model membrane.

The packing of lipid chains in bicelles was 5-9% less than that in mixed micelles at temperatures between 298 and 318 K. This reduction of packing that accompanied the formation of bicelles changed the spectroscopic character of reconstituted bacteriorhodopsin, as indicated by static absorption measurements.