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.     

Preparation and electrochemical properties of SiO2–non-graphitizable carbon composites as negative electrode materials for Li-ion batteries

SiO₂–non-graphitizable carbon composites were prepared by pyrolysis of a mixture of ethyl cellulose and nano-sized SiO₂. The composite electrode showed high reversibility in insertion and/or extraction reactions of Li ions at potentials below 1 V with little hysteresis after the 2nd cycle, whereas a large irreversible capacity was observed in the 1st cycle. This reversible capacity increased with increasing SiO₂ content above 5 wt%. Li ion transfer at the interface between a composite electrode and an electrolyte was studied by ac impedance spectroscopy. In the Nyquist plots, a semi-circle that was assigned to charge-transfer resistance (R _ct) because of Li ion transfer across the interface between the composite electrode and electrolyte appeared at potentials below 1 V. The values of R _ct decreased with increasing SiO₂ content. These results indicate that both a decrease in R _ct and an increase in reversible capacity can be achieved by use of SiO₂–non-graphitizable carbon composite electrodes; this would lead to Li-ion batteries with higher power and energy density.     

Living activity recognition using off-the-shelf sensors on mobile phones

In aging societies, such as that of Japan, there is growing awareness that robotic technology has the potential to help both physical and mental labor. To take an example of mental labor, the robotic technology can contribute as an interface to home electric appliances and a conversation partner with interactive communication. In this case, it is important to recognize the elderly user’s activities for not only watching-over services but also improving the quality of the conversation. We propose a low-throughput recognition method for in-home living activity recognition using only off-the-shelf sensors, namely an accelerometer and a microphone, which are commonly applied in mobile phones. The system can determine whether the user is walking, quiet, or performing a task by acceleration sensing, and then in the latter case, acoustic sensing can be used to classify the nature of the task that the user is performing. We conducted two experiments to confirm the feasibility of the proposed method. As a result of the first experiment, three movement conditions are classified with more than 95?% accuracy by acceleration sensing: walking, quiet, or performing a task. And it classified the nature of the task into brushing teeth, shaving, drying the hair with a hairdryer, flushing the toilet, vacuuming, washing the dishes, and ironing with 75.8?% accuracy by acoustic sensing and improved the accuracy to 85.9?% by training with only the subject’s own data. Moreover, the result of the second experiment shows that it is effective to adopt instance-based recognition which is an additional recognition scheme per each continuous task, according to the assumed application.     

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.     

Estimation of the Surface Energy of Polymer Solids

The methods to estimate the surface tension of polymer solids using contact angles have been reviewed in the first part. They are classified into the following three groups depending on the theories or the equations applied: (1) the methods using the Young's equation alone, (2) the methods using the combined equation of Young and Good-Girifalco, and (3) the methods using the equations of work of adhesion. Some notes and comments are given for each method and results are compared with each other. The two-liquids method for rather high energy surface is also introduced.

Next, some new possibilities to evaluate the surface tension of polymer solids are presented by our new contact angle theory in consideration of the friction between a liquid drop and a solid surface. The advancing and receding angles of contact (θ _a and θ _r ) are explained by the frictional tension γ_F and accordingly two kinds of the critical surface tension γ_C (γ_Ca and γ_Cr ) are given.

This work has shown that one of the recommendable ways to evaluate γ_S is either the maximum γ_LV cos θ_a or the maximum γ_C using the advancing contact angle θ_a alone, and another way is the arithmetic or the harmonic mean of the γ_Ca and γ_Cr . A depiction to determine the γ_C such as ln(1 + cos θ₀ ) vs. γ_LV with cos θ₀ = (cos θ₀ + cos θ_r )/2 has also been proposed.