Conformational change detection in nonmetal proteins by direct electrochemical oxidation using diamond electrodes

In this report, we established a new electrochemical method for the detection of conformational changes in large, non-metalloproteins such as bovine serum albumin, using flow injection analysis coupled with hydrogen-terminated, boron-doped diamond electrodes. The oxidation current was used as a signal reporter in the monitoring of urea-induced BSA denaturation. In the denatured state at high urea concentrations, the electrochemical signal increased, and the amperometric responses for the oxidation potential at 1300 mV were consistent with the results of conventional methods of denaturation monitoring using fluorescence spectroscopy. The oxidation involved at least five redox-active species (cysteine, tryptophan, tyrosine, methionine, and disulfide bonds). Furthermore, the method also showed high sensitivity for quantitative analysis of protein. A linear dynamic in the concentration range 50-400 microg/mL (r(2) = 0.977) with a lower detection limit of 190 ng/mL was achieved for BSA. Direct electrochemical detection of conformation changes of proteins using BDD electrodes can be performed with advantages in terms of simplicity and sensitivity.     

Early Fixation of Cobalt-Chromium Based Alloy Surgical Implants to Bone Using a Tissue-engineering Approach

To establish the methods of demonstrating early fixation of metal implants to bone, one side of a Cobalt-Chromium (CoCr) based alloy implant surface was seeded with rabbit marrow mesenchymal cells and the other side was left unseeded. The mesenchymal cells were further cultured in the presence of ascorbic acid, β-glycerophosphate and dexamethasone, resulting in the appearance of osteoblasts and bone matrix on the implant surface. Thus, we succeeded in generating tissue-engineered bone on one side of the CoCr implant. The CoCr implants were then implanted in rabbit bone defects. Three weeks after the implantation, evaluations of mechanical test, undecalcified histological section and electron microscope analysis were performed. Histological and electron microscope images of the tissue engineered surface exhibited abundant new bone formation. However, newly formed bone tissue was difficult to detect on the side without cell seeding. In the mechanical test, the mean values of pull-out forces were 77.15 N and 44.94 N for the tissue-engineered and non-cell-seeded surfaces, respectively. These findings indicate early bone fixation of the tissue-engineered CoCr surface just three weeks after implantation.     

Cloning and in vitro and in vivo expression of plant glutathione S-transferase zeta class genes

The cDNAs encoding the zeta class of glutathione S-transferases (GSTs) (GSTZs), which are multifunctional enzymes, were cloned from Arabidopsis thaliana L. and three lines of Brassica napus L. by RT PCR, and named AtGSTZ for A. thaliana ecotype Columbia gll, and BnGSTZ-A, BnGSTZ-B and BnGSTZ-C for B. napus cv. Shan 2A, Shan 213 and Ken C1, respectively. Sequence analysis revealed that the sequences of Shan 2A and Shan 2B were identical, and Ken C1 was different only in 3.8% of the sequence. Comparison of these sequences with published sequences in GeneBank showed that the sequences of the Brassica species were unpublished. The cDNAs were then inserted into two vectors for in vivo expression in Escherichia coli and in vitro expression in a wheat-germ cell-free protein synthesis system. All GSTZs were well expressed both in vivo and in vitro as an enzymatically active form, showing that all of them had both dichloroacetic acid dechlorinating and maleylacetone isomerase activities.     

In vitro selection of proteins that undergo covalent labeling with small molecules by thiol-disulfide exchange by using ribosome display

There is a great deal of interest in proteins that can bind covalently to target molecules, as they allow unambiguous experiments by tight binding to molecules of interest. Here, we report the generation of proteins that undergo covalent labeling with small molecules through in vitro selection by using ribosome display. Selection was performed from a mutant library of the WW domain with a biotinylated peptide as its binding target, in which the biotin and the peptide are connected by a disulfide bond. After five rounds of selection, we identified mutants carrying a particular cysteine mutation. The binding target reacted specifically with the selected mutant, even in the presence of other proteins, and resulted in the generation of biotin- or peptide-labeled WW domains by thiol-disulfide exchange. When the mutant was fused to a protein of interest, the fusion protein was also labeled with biotin. Thus, the characteristics of the selected mutant should be suitable as a tag sequence that can be covalently labeled with small synthetic molecules. These results indicate that the rapid and efficient generation of such proteins is possible by ribosome display.     

Improvement in microstructure of SBT thin films

Abstract

Various attempts to improve the microstructure of SBT thin films were carried out. One was to employ ultra-thin BT film as a top layer on the conventional SBT thin film. After optimization of the BT top layer thickness, a very smooth SBT surface was successfully achieved. Particularly, the insulation break down field was improved to more than 1.0 MV/cm. Next we used UV exposure during the baking process. By optimizing the UV assisted process, surface morphology was successfully improved with fine grain microstructure. The break down field was improved to more than 1.0 MV/cm. Also, these two process combinations successfully lead to more reliable SBT thin films. The break down field was drastically improved to more than 1.2 MV/cm.     

Reduced Thermal Budget Process for SBT Thin Film in Planer Type Stack Cell FeRAMs

In order to reduce the thermal budget for SBT crystallization process in planer type stack cell FeRAMs, Rapid Thermal Anneal (RTA) based process for SBT thin film was investigated. Our new process is characterized by crystallization in RTA without any furnace annealing process, and includes a low temperature recovery annealing process (hereafter RTB (Reduced Thermal Budget) process). As a result of only 750C RTA for accumulated time of 60s without furnace annealing process, the sufficient ferroelectric properties were derived in comparison with that of conventional SBT thin film. In the RTB process, two approaches to improve the break down voltage were carried out. First, we used UV exposure during the baking process. By optimizing the UV assisted baking process, a high break down field due to smooth surface morphology was successfully obtained, resulting in break down field of more than 1.2 MV/cm. Secondly, ultra thin SBT films as a top few layer on the base SBT thin film were employed. After optimization of the ultra thin SBT layer thickness, a very smooth SBT surface was successfully achieved, resulting in improvement of the break down field of more than 1.1 MV/cm.     

Recent trend of the partial discharge measurement technique using the UHF electromagnetic wave detection method

The ultra high frequency (UHF) electromagnetic wave detection method has been widely studied and used in partial discharge (PD) measurement and as a diagnostic technique for insulation performance in gas‐insulated switchgears (GIS) and transformers. The UHF method has advantages such as high sensitivity, wide detection range and reduced external disturbances. On the other hand, there are still some issues to be solved in the UHF methods, such as a clear understanding of the propagation characteristics of electromagnetic waves arising from the structure of the equipment, optimization of antenna design, calibration of charge, etc. This article deals with the present status and future trend of the technology of this promising UHF method of PD measurement, together with recent activities and results from our laboratory. Copyright © 2007 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.