Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii

In general, the enzymes of thermophilic organisms are more resistant to thermal denaturation than are those of mesophilic or psychrophilic organisms. Further, as is true for their mesophilic and psychrophilic counterparts, the activities of thermophilic enzymes are smaller at temperatures that are less than the optimal temperature. In an effort to characterize the properties that would improve its activity at temperatures less than the optimal, we subjected the thermostable Sulfolobus tokodaii (S. tokodaii) 3-isopropylmalate dehydrogenase to two rounds of random mutagenesis and selected for improved low-temperature activity using an in vivo recombinant Escherichia coli system. Five dehydrogenase mutants were purified and their catalytic properties and thermostabilities characterized. The mutations favorably affect the K(m) values for NAD (nicotinamide adenine dinucleotide) and/or the k(cat) values. The results of thermal stability measurements show that, although the mutations somewhat decrease the stability of the enzyme, the mutants are still very resistant to heat. The locations and properties of the mutations found for the S. tokodaii enzyme are compared with those found for the previously isolated low-temperature adapted mutants of the homologous Thermus thermophilus enzyme. However, there are few, if any, common properties that enhance the low-temperature activities of both enzymes; therefore, there may be many ways to improve the low-temperature catalytic activity of a thermostable enzyme.     

Allene as an alternative functional group for drug design: effect of C--C multiple bonds conjugated with quinazolines on the inhibition of EGFR tyrosine kinase

A series of allenic quinazolines were synthesized as receptor tyrosine kinase inhibitors by using a simple protocol for palladium-catalyzed allene transformation. Among the compounds synthesized, two allenic 4-anilinoquinazolines selectively suppressed epidermal growth factor receptor (EGFR) tyrosine kinase activity in vitro. According to immunoblot analysis, the allenic quinazolines inhibited the EGF-mediated phosphorylation of EGFR and its downstream kinases in A431 cells. Furthermore, one of these allenic quinazolines decreased the proliferation of A431 cells through the induction of cell-cycle arrest and apoptosis.     

Fabrication of Si-based planar type patch clamp biosensor using silicon on insulator substrate

The aim of this paper is to fabricate the planar type patch clamp ion-channel biosensor, which is suitable for the high throughput screening, using silicon-on-insulator (SOI) substrate. The micropore with 1.2 μm diameter is formed through the top Si layer and the SiO₂ box layer of the SOI substrate by focused ion beam (FIB). Then the substrate is assembled into the microfluidic circuit. The human embryonic kidney 293 (HEK-293) cell transfected with transient receptor potential vanilloid type 1 (TRPV1) is positioned on the micropore and the whole-cell configuration is formed by the suction. Capsaicin is added to the extracellular solution as a ligand molecule, and the channel current showing the desensitization unique to TRPV1 is measured successfully.     

Simultaneous purification and site-specific modification of pyrroline-carboxy-lysine proteins

Sticky residue: Pyrroline-carboxy-lysine (Pcl) can be readily incorporated into proteins expressed in E. coli and mammalian cells by using the pyrrolysyl tRNA/tRNA synthetase pair. Pcl can be used as a single amino acid purification tag and can be site-specifically modified with functional probes during the elution process.     

Application of citric acid as natural adhesive for wood

The development of natural adhesives derived from nonfossil resources is very important for the future. Besides, it is desirable to be safe adhesives without using harmful chemical substances. In this study, application of citric acid as a natural adhesive was investigated. Citric acid powder and bark powder obtained from Acacia mangium were used as raw materials. Citric acid powder was mixed with the bark powder, and the resulting powder mixture was poured into a metal mold. The mold was hot‐pressed at 180°C and 4 MPa for 10 min, and a bark molding was then obtained. The specific modulus of rupture and modulus of elasticity values of the molding containing 20 wt % citric acid were 18.1 MPa and 4.9 GPa, respectively. The molding did not decompose during a repeated boiling treatment. To clarify the effect of tannin on the adhesiveness of molding, bark was separated into tannin and residue. The molding was not obtained while using the tannin due to the marked fluidity, whereas it was obtained while using the residue, the same as while using the bark. It was considered that components other than tannin contributed to the adhesiveness. Based on the results of Fourier transform infrared spectra, the formation of ester linkages between carboxyl groups derived from citric acid and hydroxyl groups in the bark was confirmed. Accordingly, citric acid brought an adhesion by chemical bonding, and it could be used as a safe natural adhesive. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Acoustic phonon modulation and electron�Cphonon interaction in?semiconductor slabs and nanowires

The acoustic phonon modulation (confinement) in semiconductor nanostructures and their interaction with electrons are reviewed. Special emphasis will be placed on free-standing and layered slabs, as well as nanowires. Analysis includes acoustic phonon dispersion relations, displacement wave functions, amplitudes, form factor, electron-phonon scattering rate, and electron mobility.     

Simple Short-Pulse CO<Subscript>2</Subscript> Laser Excited by Longitudinal Discharge without High-Voltage Switch

We have developed a longitudinally excited CO₂ laser without a high-voltage switch. The laser produces a short laser pulse similar to those from TEA and Q-switched CO₂ lasers. This system, which is the simplest short-pulse CO₂ laser yet constructed, includes a pulsed power supply, a high-speed step-up transformer, a storage capacitor, and a laser tube. At high pressure (4.2 kPa and above), a rapid discharge produces a short laser pulse with a sharp spike pulse. In mixed gas (CO₂: N₂: He = 1: 1: 2) at a pressure of 9.0 kPa, the laser pulse contains a spike pulse of 218 ns and has a pulse tail length of 16.7 μs.     

Re-evaluation of the phase relationship between plutonium and zirconium dioxides

The phase relationship between ZrO₂ and PuO₂ was examined in a low PuO₂ content region, from 3.1 to 11.2 mol% PuO₂, at temperatures between 1273 K and 1473 K, by high temperature X-ray diffractometry. The measurements were carried out in air. At 1273 K, the samples in this composition range consisted of two phases, monoclinic and cubic. Another phase, tetragonal, was observed at 1373 K. The low temperature monoclinic phase disappeared at 1473 K. It was confirmed that the monoclinic phase disappears around 1463 K; the disappearance temperature does not depend on the composition of the sample. It was, thus, inferred that there should be a eutectoid line in the phase diagram. Though the eutectoid point is not clear, the PuO₂ content at the point should be less than 3.1 mol%.     

Synthesis of cyclic poly(methyl methacrylate) by the intramolecular cyclization of α-amino, ω-carboxyl heterodifunctional poly(methyl methacrylate)

Summary α-Amino, ω-carboxyl heterodifunctional poly(methyl methacrylate) was prepared by a living anionic polymerization of methyl methacrylate using N,N'-diphenylethylenediamine monolithium amide and succinic anhydride as an initiator and terminator, respectively. Its intramolecular cyclization was carried out to obtain a well-defined cyclic poly(methyl methacrylate). Received: 27 June 2001/Accepted: 16 July 2001     

Fast Algorithms to Enumerate All Common Intervals of Two Permutations

Given two permutations of n elements, a pair of intervals of these permutations consisting of the same set of elements is called a common interval . Some genetic algorithms based on such common intervals have been proposed for sequencing problems and have exhibited good prospects. In this paper we propose three types of fast algorithms to enumerate all common intervals: (i) a simple O(n ² ) time algorithm (LHP), whose expected running time becomes O(n) for two randomly generated permutations, (ii) a practically fast O(n ² ) time algorithm (MNG) using the reverse Monge property, and (iii) an O(n+K) time algorithm (RC), where K is the number of common intervals. It will also be shown that the expected number of common intervals for two random permutations is O(1) . This result gives a reason for the phenomenon that the expected time complexity O(n) of the algorithm LHP is independent of K . Among the proposed algorithms, RC is most desirable from the theoretical point of view; however, it is quite complicated compared with LHP and MNG. Therefore, it is possible that RC is slower than the other two algorithms in some cases. For this reason, computational experiments for various types of problems with up to n=10 ⁶ are conducted. The results indicate that (i) LHP and MNG are much faster than RC for two randomly generated permutations, and (ii) MNG is rather slower than LHP for random inputs; however, there are cases in which LHP requires Ω(n ² ) time, but MNG runs in o(n ² ) time and is faster than both LHP and RC. Received December 21, 1996; revised June 2, 1998.