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Artificial planar lipid bilayers are a powerful tool for the functional study of membrane proteins, yet they have not been widely used due to their low stability and reproducibility. This paper describes an accessible method to form a planar lipid bilayer, simply by contacting two monolayers assembled at the interface between water and organic solvent in a microfluidic chip. The membrane of an organic solvent containing phospholipids at the interface was confirmed to be a bilayer by the capacitance measurement and by measuring the ion channel signal from reconstituted antibiotic peptides. We present two different designs for bilayer formation. One equips two circular wells connected, in which the water/solvent/water interface was formed by simply injecting a water droplet into each well. Another equips the cross-shaped microfluidic channel. In the latter design, formation of the interface at the sectional area was controlled by external syringe pumps. Both methods are extremely simple and reproducible, especially in microdevices, and will lead to automation and multiple bilayer formation for the high-throughput screening of membrane transport in physiological and pharmaceutical studies.

A Moroccan family with autosomal recessive Stickler syndrome is clinically investigated for ear, nose and throat, ophthalmologic and radiological abnormalities. It was found that COL9A1, the fourth identified gene, could cause Stickler syndrome.

Using an expression cloning strategy, we have identified TFE3, a basic helix-loop-helix protein, as a transactivator of metabolic genes that are regulated through an E-box in their promoters. Adenovirus-mediated expression of TFE3 in hepatocytes in culture and in vivo strongly activated expression of IRS-2 and Akt and enhanced phosphorylation of insulin-signaling kinases such as Akt, glycogen synthase kinase 3[beta] and p70S6 kinase. TFE3 also induced hexokinase II (HK2) and insulin-induced gene 1 (INSIG1). These changes led to metabolic consequences, such as activation of glycogen and protein synthesis, but not lipogenesis, in liver. Collectively, plasma glucose levels were markedly reduced both in normal mice and in different mouse models of diabetes, including streptozotocin-treated, db/db and KK mice. Promoter analyses showed that IRS2, HK2 and INSIG1 are direct targets of TFE3. Activation of insulin signals in both insulin depletion and resistance suggests that TFE3 could be a therapeutic target for diabetes., Author(s): Yoshimi Nakagawa [1, 2]; Hitoshi Shimano (corresponding author) [1, 2]; Tomohiro Yoshikawa [1]; Tomohiro Ide [1]; Mariko Tamura [1]; Mika Furusawa [1]; Takashi Yamamoto [1]; Noriyuki Inoue [1]; Takashi [...]

An integrated system was developed that performs microfluidic transport, mixing, and sensing on a single chip. The operation principle for the microfluidic transport was based on electrowetting. A solution to be transported was confined in a space between a row of gold working electrodes and a protruding poly(dimethylsiloxane) (PDMS) structure. When a negative potential was applied to one of the gold working electrodes, it became hydrophilic, and the solution was transported through the flow channel. The solution could be transported in any desired direction in a network of flow channels by switching on necessary electrodes one by one. Furthermore, two solutions transported through two flow channels could be mixed using a mixing electrode based on the same principle. To demonstrate the applicability of a lab-on-a-chip, an air gap ammonia electrode was integrated by taking advantage of the open structure of the flow channel. Gaseous ammonia that was produced after pH adjustment and diffused through an air gap caused a pH change in the electrolyte layer, which was measured with an iridium oxide pH indicator electrode. The 90% response time was less than 1 min for the millimolar order of ammonia. The calibration curve was linear down to 10 [micro]M. The ammonia-sensing system was also applied to construct biosensing systems for urea and creatinine. A linear relationship was observed between the potential and the logarithm of the concentration of the analytes down to 50 [micro]M for both urea and creatinine. The developed microfluidic system can be a basic building block for future systems.

Cross-ring cleavage ions produced by in-source decay (ISD), as well as deprotonated molecular ions [M - H]-, are invariably observed in negative-ion linear-mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectra of neutral oligosaccharides with 9H-pyrido [3,4-blindole (norharman) as a matrix. The patterns of ISD ions depend on the oligosaccharide linkage type; thus, these ions are potentially useful in linkage analysis. In postsource decay (PSD) spectra from chlorinated molecular ions [[M + CI].sub.-], all PSD ions are observed in the deprotonated form, although no deprotonated molecular ions are detected. In oligosaccharides having an alditol at the reducing end, deprotonated molecular ions [[M - H].sup.-] are clearly seen in linear-mode mass spectra and survive in the PSD measurements. These results indicate that the deprotonation process drives ISD and PSD of oligosaccharides and that keto-enol tautomerization at the reducing terminal promotes ISD and PSD processes.

An efficient magnetic force driven mixer with simple configuration is designed, fabricated, and tested. It is designed to facilitate the mixing of magnetic beads and biomolecules in a microchannel, where mixing is unavoidably inefficient due to its low Reynolds number. With appropriate temporal variations of the force field, chaotic mixing is achieved, hence the mixing becomes effective. The mixing device consists of embedded microconductors as a magnetic field source and a microchannel that guides the streams of working fluid. It is demonstrated that a pair of integrated micro conductors provides a local magnetic field strong enough to attract nearby magnetic beads. Mixing of magnetic beads is accomplished by applying a time-dependent control signal to a row of conductors, at the Reynolds number of as low as [10.sup.-2]. Two-dimensional numerical simulation has been performed to design the configuration of the channel and electrodes, which creates chaotic motion of beads. It is found that a simple two-dimensional serpentine channel geometry with the transverse electrodes is able to create the stretching and folding off material lines, which is a manifestation of chaos. The mixing pattern predicted by the simulation has been confirmed by both flow visualization and PTV (Particle Tracking Velocimetry) in the chaotic mixer fabricated, which should greatly increase the attachment of beads onto the target biomolecules. The optimum frequency of applied control signal is searched by evaluating the Lyapunov exponent in both numerical and experimental particle tracking. It is found that the range of optimum Strouhal number is 5 < St < 12, which is defined by the channel width and the mean velocity. [1200] Index Terms--Bio MEMS, cell sorting, chaotic mixing, magnetic beads, magnetic force, micro fluidic system.

An electrochemical protein chip was microfabricated. A thin-film three-electrode system, including an array of 36 platinum working electrodes, a set of thin-film Ag/AgCl electrodes, and platinum auxiliary electrodes, was integrated on a glass substrate. Capture antibodies were immobilized in a 4.5-nm-thick double layer of a hexamethyldisiloxane plasma-polymerized film. Because of their highly cross-linked network structure, the capture antibodies could be firmly immobilized. No nonspecific adsorption was observed during a series of procedures to detect target proteins, and electrochemical cross talk between neighboring sites was negligible. The sandwich immunoassay was conducted on a single chip using model proteins, ([alpha]-1-fetoprotein and [[beta].sub.2]-microglobulin. A distinct current increase following the oxidation of hydrogen peroxide produced by the enzymatic reaction of glucose oxidase was observed, which indicates that the capture proteins could actually bind the target proteins. Two kinds of protein were detected independently on multiple sites with respective capture antibodies.

A synthetase is a lipogenic enzyme controlled by SREBP-1 and energy status. Am J Physiol Endocrinol Metab 282: E222-E230, 2002; 10.1152/ajpendo.00189.2001.--DNA microarray analysis on upregulated genes in the livers from transgenic mice overexpressing nuclear sterol regulatory element-binding protein (SREBP)-1a, identified an espressed sequence tag (EST) encoding a part of murine cytosolic acetylcoenzyme A synthetase (ACAS). Northern blot analysis of the livers from transgenic mice demonstrated that this gene was highly induced by SREBP-1a, SREBP-1c, and SREBP-2. DNA sequencing of the 5' flanking region of the murine ACAS gene identified a sterol regulatory element with an adjacent Sp1 site. This region was shown to be responsible for SREBP binding and activation of the ACAS gene by gel shift and luciferase reporter gene assays. Hepatic and adipose tissue ACAS mRNA levels in normal mice were suppressed at fasting and markedly induced by refeeding, and this dietary regulation was nearly abolished in SREBP-1 knockout mice, suggesting that the nutritional regulation of the ACAS gene is controlled by SREBP-1. The ACAS gene was downregulated in streptozotocin-induced diabetic mice and was restored after insulin replacement, suggesting that diabetic status and insulin also regulate this gene. When acetate was administered, hepatic ACAS mRNA was negatively regulated. These data on dietary regulation and SREBP-1 control of ACAS gene expression demonstrate that ACAS is a novel hepatic lipogenic enzyme, providing further evidence that SREBP-1 and insulin control the supply of acetyl-CoA directly from cellular acetate for lipogenesis. However, its high conservation among different species and the wide range of its tissue distribution suggest that this enzyme might also play an important role in basic cellular energy metabolism. lipogenic enzyme; acetate; diabetes; insulin; transcription