Hyperexpression of the gene for a Bacillus alpha-amylase in Bacillus subtilis cells: enzymatic properties and crystallization of the recombinant enzyme

We have constructed a new excretion vector, pHSP64, to develop a hyperexcretion system for Bacillus subtilis [Sumitomo et al., Biosci. Biotech. Biochem., 59, 2172-2175 (1995)]. The structural gene for a novel liquefying semi-alkaline alpha-amylase from the alkaliphilic Bacillus sp. KSM-1378 was amplified by PCR. It was cloned into a SalI-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B. subtilis. The transformed B. subtilis hyperproduced the alpha-amylase activity extracellularly, corresponding to approximately 1.0 g (5 x 10(6) units) per liter of an optimized liquid culture. The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield. No significant differences in physiochemical and catalytic properties were observed between the recombinant enzyme and the native enzyme produced by Bacillus sp. KSM-1378. The enzymatic properties of the recombinant enzyme were further examined with respect to the responses to various metal ions. The recombinant enzyme could easily be crystallized at room temperature within one day in a buffered solution of 10% (w/v) ammonium sulfate (pH 6.5).     

OCDMA over WDM PON-solution path to gigabit-symmetric FTTH

It will be revealed that a myth of deploying low bit-rate uplink fiber-to-the-home (FTTH) services while providing a high bit-rate downlink is wrong. Therefore, for the future broadband FTTH services, the focus should be on the capability to provide gigabit- or even multigigabits-per-second both in up- and downlinks, namely gigabit symmetric systems. Optical code-division multiple access (OCDMA) now deserves a revisit as a powerful alternative to time-division multiple access and wavelength-division multiple (WDM) access in FTTH systems. In this paper, the authors will first highlight the OCDMA systems. The system architecture and its operation principle, code design, optical en/decoding, using a long superstructured fiber Bragg grating (SSFBG) en/decoder, and its system performance will be described. Next, an OCDMA over WDM passive optical network (PON) as a solution for the gigabit-symmetric FTTH systems will be proposed. The system architecture and the WDM interchannel crosstalk will be studied. It will be shown that by taking advantage of reflection spectrum notches of the SSFBG en/decoder, the WDM interchannel crosstalk can be suppressed and can enable OCDMA over WDM PON to simultaneously provide multigigabit-per-second up- and downlinks to a large number of users.     

Strengthening mechanisms in nanostructured high-purity aluminium deformed to high strain and annealed

Samples of pure aluminium (99.99%) have been produced by accumulative roll-bonding to a large strain followed by a heat treatment, where a two-step annealing process has been used to produce samples with large variations in structural parameters such as boundary spacing, misorientation angle and dislocation density. These parameters have been quantified by a structural analysis applying transmission electron microscopy and electron backscatter diffraction, and the mechanical properties have been determined by tensile testing at room temperature. Strength–structure relationships have been analysed based on the operation of two strengthening mechanisms—grain boundary and dislocation strengthening—and good agreement with experiments has been found for the deformed sample. However, for samples where the density of dislocation sources has been reduced significantly by annealing, an additional strengthening mechanism, so-called dislocation source-limited hardening, may operate as a higher stress is required to activate alternative dislocation sources.     

A convenient synthesis of functionalized alkoxyamines as initiators for living free radical polymerization

Summary 2,2,6,6-Tetramethylpiperidine- 1-oxyl (TEMPO) was reacted with ethylbenzene (la), 1-bromo-4-ethylbenzene (lb), and 4-ethylphenyl acetate (lc), respectively, using tert-BuOOWCo(OAc)·4H₂O in acetonitrile at room temperature. The reactions produced the respective TEMPO-adducts (2a, 2b, and 2c) in the yields of 37, 44, and 45 %, which were based on TEMPO. Similarly, TEMPO was reacted with 4-ethylphenyl 2,3,6,2',3',4',6'-hepta-O-acetyl-β-D-cellobioside (1d) to afford the glycoconjugated TEMPO-adduct (2d) in 45 5% yield, which was based on 1d. These results indicated that the reaction has the potential to become an easy and also safe strategy, which provided various functionalized alkoxylamines. Received 20 November 2002/Revised Version: 5 December 2002/ Accepted: 7 December 2002 Correspondence to Naoya Sugimoto     

Demonstration of over 128-gb/s-capacity (12-User/spl times/10.71-gb/s/user) asynchronous OCDMA using FEC and AWG-based multiport optical encoder/decoders

For the first time, an arrayed-waveguide-grating (AWG)-based multiport optical encoder/decoder (E/D) and forward-error-correction (FEC) technique are applied in an optical code-division multiple-access (OCDMA) system. The AWG-based OCDMA E/D with high power contrast ratio between auto-/cross-correlation can significantly suppress the interference noises in an asynchronous OCDMA system without using ultralong optical codes and optical thresholder. A 12-user 10.71-Gb/s asynchronous OCDMA experiment has been successfully demonstrated to transmit ITU-T G.709 OTN frames including FEC.     

Short-term memory circuit using hardware ring neural networks

A number of studies have recently been made on various neuron models and neural networks. This research is studied for applications to engineering problems and an understanding of the information processing functions of living organisms. We are studying an asynchronous neural network using a pulse-type hardware neuron model (P-HNM). Recently, we have been trying to construct a short-term memory circuit using hardware ring neural networks (RNN) with P-HNM. In this article, we discuss the construction of a short-term memory circuit using the hardware RNN, and conduct experiments that explain the characteristics of the network through circuit simulation using PSpice. As a result, we verify that the RNN which is proposed in this article can be used as the short-term memory circuit.This work was presented, in part, at the 9th International Symposium on Artificial Life and Robotics, Oita, Japan, January 28–30, 2004     

A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells,Based on One‐Step Cleavage of the Dabcyl Quencher

Sirtuins (SIRTs) are a family of NAD⁺‐dependent histone deacetylases. In mammals, dysfunction of SIRTs is associated with age‐related metabolic diseases and cancers, so SIRT modulators are considered attractive therapeutic targets. However, current screening methodologies are problematic, and no tools for imaging endogenous SIRT activity in living cells have been available until now. In this work we present a series of simple and highly sensitive new SIRT activity probes. Fluorescence of these probes is activated by SIRT‐mediated hydrolytic release of a 4‐(4‐dimethylaminophenylazo)benzoyl (Dabcyl)‐based FRET quencher moiety from the ?‐amino group of lysine in a nonapeptide derived from histone H3K9 and bearing a C‐terminal fluorophore. The probe SFP3 detected activities of SIRT1, ‐2, ‐3, and ‐6, which exhibit deacylase activities towards long‐chain fatty acyl groups. We then truncated the molecular structure of SFP3 in order to improve both its stability to peptidases and its membrane permeability, and developed probe KST‐F, which showed specificity for SIRT1 over SIRT2 and SIRT3. We show that KST‐F can visualize endogenous SIRT1 activity in living cells.     

Application of chitosan solutions gelled by melB tyrosinase to water‐resistant adhesives

An investigation was undertaken on the application of dilute chitosan solutions gelled by melB tyrosinase‐catalyzed reaction with 3,4‐dihydroxyphenethylamine (dopamine). The tyrosinase‐catalyzed reaction with dopamine conferred water‐resistant adhesive properties to the semi‐dilute chitosan solutions. The viscosity of the chitosan solutions highly increased by the tyrosinase‐catalyzed quinone conversion and the subsequent nonenzymatic reactions of o‐quinones with amino groups of the chitosan chains. The viscosity of chitosan solutions highly increased in shorter reaction times by addition of melB tyrosinase. Therefore, in this study, the gelation of a chitosan solution was carried out without poly(ethylene glycol) (PEG), which was added for the gelation of chitosan solutions using mushroom tyrosinase. The highly viscous, gel‐like modified chitosan materials were allowed to spread onto the surfaces of the glass slides, which were tightly lapped together and were held under water. Tensile shear adhesive strength of over 400 kPa was observed for the modified chitosan samples. An increase in either amino group concentration of the chitosan solutions or molecular mass of the chitosan samples used effectively led to an increase in adhesive strength of the glass slides. Adhesive strength obtained by chitosan materials gelled enzymatically was higher than that obtained by a chitosan gel prepared with glutaraldehyde as a chemical crosslinking agent. In addition, the use of melB tyrosinase led to a sharp increase in adhesive strength in shorter reaction times without other additives such as PEG. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Identification of the glucosamine kinase in the chitinolytic pathway of Thermococcus kodakarensis

1. Download high-res image (165KB)
2. Download full-size image

     

Improvement of Strength–Ductility Balance by the Simultaneous Increase in Ferrite and Martensite Strength in Dual-Phase Steels

Metallurgical and Materials Transactions A - This work investigates the effect of increasing both martensite phase and ferrite phase strength on tensile properties and fracture behavior of...