A continuous spectrophotometric assay for NADPH-cytochrome P450 reductase activity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 and also catalyzes the one-electron reduction of many drugs and foreign compounds. Various spectrophotometric assays have been performed to examine electron-accepting properties of CPR and its ability to reduce cytochrome b5, cytochrome c, and ferricyanide. In this report, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by CPR has been assessed as a method for monitoring CPR activity. The principle advantage of this substance is that the reduction of MTT can be assayed directly in the reaction medium by a continuous spectrophotometric method. The electrons released from NADPH by CPR were transferred to MTT. MTT reduction activity was then assessed spectrophotometrically by measuring the increase of A610. MTT reduction followed classical Michaelis-Menten kinetics (K(m)= 20 microM, k(cat)= 1,910 min(-1)). This method offers the advantages of a commercially available substrate and short analysis time by a simple measurement of enzymatic activity of CPR.     

Crystallization and preliminary X-ray crystallographic analysis of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin

The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to 2.8 A resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to 2.2 A have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or I212121) with unit-cell parameters of a = 63.7, b = 124.5, and c = 126.3 A. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter (VM) is 2.71 A3 Da-1 and solvent content is 54.6%.     

Efficient and accurate analysis of microRNA using a specific extension sequence

Molecular Biology Reports - We present here on an innovative assay for detecting miRNAs using a uniquely designed specific extension sequence that provides high efficiency and accuracy. This assay...     

Ordered Nanoscale Heterojunction Architecture for Enhanced Solution‐Based CuInGaS2 Thin Film Solar Cell Performance

Nanopatterned CuInGaS₂ (CIGS) thin films synthesized by a sol‐gel‐based solution method and a nanoimprint lithography technique to achieve simultaneous photonic and electrical enhancements in thin film solar cell applications are demonstrated. The interdigitated CIGS nanopatterns in adjacent CdS layer form an ordered nanoscale heterojunction of optical contrast to create a light trapping architecture. This architecture concomitantly leads to increased junction area between the p‐CIGS/n‐CdS interface, and thereby influences effective charge transport. The electron beam induced current and capacitance–voltage characterization further supports the large carrier collection area and small depletion region of the nanopatterned CIGS solar cell devices. This strategic geometry affords localization of incident light inside and between the nanopatterns, where created excitons are easily dissociated, and it leads to the enhanced current generation of absorbed light. Ultimately, this approach improves the efficiency of the nanopatterned CIGS solar cell by 55% compared to its planar counterpart, and offers the possibility of simultaneous management for absorption and charge transport through a nanopatterning process.     

Imobilization of β-glucosidase using the cellulose-binding domain of Bacillus subtilis endo-β-1,4-glucanase

A recombinant plasmid pβCBD was constructed for immobilization of Cellulomonas fimi β-glucosidase (Cbg) using the cellulose-binding domain (CBD) of Bacillus subtilis BSE 616 endo-β-1,4-glucanase (Beg). The Cbg-CBD _Beg fusion protein, 80 kDa, was expressed in Escherichia coli and immobilized to Avicel. Cellobiose was completely hydrolyzed with the immobilized fusion protein. The fusion protein bound to Avicel retained full activity during continuous operation for 24 h at 4°C. This revised version was published online in November 2006 with corrections to the Cover Date.     

Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2

Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C(6)-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A(2) (PLA(2)) inhibitors. Moreover, cytosolic PLA(2) GIVA (cPLA(2) GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells. Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A(2) GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA(2) are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA(2).     

A sol-gel-based microfluidics system enhances the efficiency of RNA aptamer selection

RNA and DNA aptamers that bind to target molecules with high specificity and affinity have been a focus of diagnostics and therapeutic research. These aptamers are obtained by SELEX often requiring many rounds of selection and amplification. Recently, we have shown the efficient binding and elution of RNA aptamers against target proteins using a microfluidic chip that incorporates 5 sol-gel binding droplets within which specific target proteins are imbedded. Here, we demonstrate that our microfluidic chip in a SELEX experiment greatly improved selection efficiency of RNA aptamers to TATA-binding protein, reducing the number of selection cycles needed to produce high affinity aptamers by about 80%. Many aptamers were identical or homologous to those isolated previously by conventional filter-binding SELEX. The microfluidic chip SELEX is readily scalable using a sol-gel microarray-based target multiplexing. Additionally, we show that sol-gel embedded protein arrays can be used as a high-throughput assay for quantifying binding affinities of aptamers.