Yasuko Yanagida

Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization

The B-domain, which is one of IgG-binding domains of staphylococcal protein A, was repeated five times and a cysteine residue was introduced at its C-terminus by a genetic engineering technique. The resulting protein, designated B5C1, retained the same IgG-binding activity as native protein A. The B5C1 was assembled on a gold plate surface by utilizing a strong affinity between thiol of cysteine and a gold surface. IgG-binding activity of B5C1 on a gold surface was much higher than that of physically adsorbed B5, which lacks cysteine residue. Furthermore, antigen-binding activity of immobilized antibody molecules through the use of assembled B5C1 on a gold surface was about 4.3 times higher than that of physically adsorbed antibody molecules. Immobilization of highly oriented antibody molecules was realized with the engineered IgG-binding protein.

Stimuli-responsive hydrogel-silver nanoparticles composite for development of localized surface plasmon resonance-based optical biosensor.

In this paper, the development of a localized surface plasmon resonance (LSPR)-based optical enzyme biosensor using stimuli-responsive hydrogel-silver nanoparticles composite is described. This optical enzyme biosensor was constructed by immobilizing glucose oxidase (GOx) into the stimuli-responsive hydrogel. When a sample solution such as glucose was applied to the surface of this optical enzyme biosensor, the interparticle distances of the silver nanoparticles present in the stimuli-responsive hydrogel were increased, and thus the absorbance strength of the LSPR was decreased. Furthermore, hydrogen peroxide, which was produced by the enzymatic reaction, induced the degradation of highly clustered silver nanoparticles by the decomposition of hydrogen peroxide. Hence, a drastic LSPR absorbance change, which depends on the glucose concentrations, could be observed. On the basis of the abovementioned mechanism, the characterization of the LSPR-based optical enzyme biosensor was carried out. It was found that the LSPR-based optical enzyme biosensor could be used to specifically determine glucose concentrations. Furthermore, the detection limit of this biosensor was 10 pM. Therefore, this LSPR-based optical enzyme biosensor has the potential to be applied in cost-effective, highly simplified, and highly sensitive test kits for medical applications.

Electrically induced neurite outgrowth of PC12 cells on the electrode surface.

Morphological differentiation of PC12 cells cultured on an indium-tin oxide (ITO) electrode has been induced to grow neurites in the absence of nerve growth factor (NGF) by electrical stimulation. Rectangular pulse wave potentials were applied to the electrode at amplitudes of 200 mV and 400 mV with frequencies of 50Hz, 500Hz, and 1 kHz. The PC12 cells differentiated most prominently at 200 mV with 100Hz. No statistically significant differences were observed among the electrically induced neurite lengths. The electrically induced differentiation was completely inhibited by a blockade of calcium influx using LaCl3. This indicates that repeated potential shift in the vicinity of a cellular membrane may stimulate morphological response, probably through calcium ion channels.

Gene expression in the electrically stimulated differentiation of PC12 cells

Cell differentiation of PC12 cells was electrically induced to grow neurites in the absence of nerve growth factor (NGF) on the electrode surface, of which potential was modulated by a rectangular wave of potential. The electric stimulation induced the c-fos expression which is essential for cell differentiation. Non-specific calcium channel blocker, lanthanum ion, inhibited the electrically induced differentiation, while NGF-induced differentiation was not suppressed. An L-type calcium channel blocker, nifedipine, also inhibited the electrically induced calcium influx and c-fos expression. Moreover, a stretch-activated (SA) channel blocker, gadolinium ion, inhibited the electrically stimulated differentiation by blocking the calcium influx, but gave no prominent effects on the potassium ion-induced differentiation. Chelerythrine, a specific protein kinase C (PKC) inhibitor, almost inhibited the cell differentiation by the electric stimulation but not by the NGF treatment. These results indicate that the alternative potential may stimulate cell differentiation through a PKC cascade.

Two types of electrochemical nitric oxide (NO) sensing systems with heat-denatured Cyt C and radical scavenger PTIO

To develop an in situ NO sensing system for primarily biological and medical uses, two types of NO sensing materials, which may be coupled with an electrochemical reaction for signal transduction, have been investigated. Heat-denatured Cyt C and a radical scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetra methyl imidazoline-1-oxyl-3-oxide (C-PTIO) were found to be effective and were incorporated into electrochemical sensing systems. Heat-denatured Cyt C deposited on a 4-mercaptopyridine modified gold electrode responded to NO with an increase of cathodic current through electrochemical reduction of Cyt C (Fe3+), when the electrode potential was controlled at 0 mV vs Ag/AgCl. The dynamic range of the sensing system was 0.5-4 microM. The sensing system with C-PTIO exhibited an anodic output in response to NO at 0.7 V vs Ag/AgCl, and showed a wider dynamic range from 0.05 to 100 microM.

Ribosome display for selection of active dihydrofolate reductase mutants using immobilized methotrexate on agarose beads

Ribosome display was applied to the selection of an enzyme. As a model, we selected and amplified the dihydrofolate reductase (DHFR) gene by ribosome display utilizing a wheat germ cell‐free protein synthesis system based on binding affinity to its substrate analog, methotrexate, immobilized on agarose beads. After three rounds of selection, the DHFR gene could be effectively selected and preferentially amplified from a small proportion in a mixture also containing competitive genes. Active enzymes were expressed and amplified and by sequence analysis, four mutants of DHFR were identified. These mutants showed as much activity as the wild‐type enzyme.

Osteogenesis Coordinated in C3H10T1/2 Cells by Adipogenesis-Dependent BMP-2 Expression System

A novel tissue engineering for bone formation has been proposed, to make osteoblast differentiation balanced by transfecting the mesenchymal stem cells with a gene encoding human bone morphogenetic protein-2 (hBMP-2) under the control of adipocyte specific lipoprotein lipase (LPL) promoter. Due to the promoter specificity, the initiation of BMP transcription is dependent on adipogenesis. For 14-day culture in the presence of ascorbic acid (asc) and beta-glycerophosphate (gly), nontransfected mouse embryonic fibroblast C3H10T1/2 (10T1/2) cells showed extensive accumulation of lipid droplets and adipocyte specific enzyme glycerol-3-phosphate dehydrogenase (G3PDH) mRNA expression, but exhibited neither BMP-2 expression, high alkaline phosphatase (ALP) activity which reflects osteoblast phenotype. On the other hand, transfected 10T1/2 cells showed hBMP-2 expression, high ALP activity and low level of G3PDH. mRNA expression accompanied with minimal lipid droplets. These results indicate that 10T1/2 cells are proved to be differentiated with maintaining coordinated balance of adipogenesis and osteogenesis, when they are transfected by the gene encoding hBMP-2 under the control of LPL promoter.

Electrically stimulated induction of hsp70 gene expression in mouse astroglia and fibroblast cells

Mouse astroglial cells, which were cultured on an electrode, were found responsive to an electric stimulation of sine wave potential in enhancing hsp70 mRNA resulting from an activation of hsp70 gene expression. On the basis of this finding, electrically responsive cells were established by transfecting mouse 3T3-L1 cells with a constructed plasmid encoding hsp70 promoter and the firefly luciferase gene. A stable cell line has been established through selection of heat-stimulated luciferase expression. A 1-h electric stimulation of the cells resulted in activation of luciferase expression, which was confirmed to produce an increase in light emission. The sequential pattern of the electrically stimulated expression of luciferase was found different from that of the heat stimulation. Furthermore, the promoter was activated depending on the potential and duration of the stimulation applied. Consequently, the electric stimulation has proven effective on activating hspP70 promoter. This cell line is feasible in expressing the gene of interest by electrical stimulation, which lead us to construct environment responsive cells in general.

Fabrication of core-shell structured nanoparticle layer substrate for excitation of localized surface plasmon resonance and its optical response for DNA in aqueous conditions

LSPR from nanostructured noble metals such as gold and silver offers great potential for biosensing applications. In this study, a core-shell structured nanoparticle layer substrate was fabricated and the localized surface plasmon resonance (LSPR) optical characteristics were investigated for DNA in aqueous conditions. Factors such as DNA length dependence, concentration dependence, and the monitoring of DNA aspects (ssDNA or dsDNA) were measured. Different lengths and concentrations of DNA solutions were introduced onto the surface of the substrate and the changes in the LSPR optical characteristics were measured. In addition, to monitor the changes in LSPR optical characteristics for different DNA aspects, a DNA solutions denatured by means of heat or alkali were introduced onto the surface, after which optical characterization of the core-shell structured nanoparticle substrate was carried out. With this core-shell structured nanoparticle layer for the excitation of LSPR, the dependence upon specific DNA conditions (length, concentration, and aspect) could be monitored. In particular, the core-shell structured nanoparticle layer substrate could detect DNA of length 100-5000 bp and 400-bp DNA at a concentration of 4.08 ng mL(-1) (1 x 10(7) DNA molecules mL(-1)). Furthermore, the changes in LSPR optical characteristics with DNA aspect could be monitored. Thus, LSPR-based optical detection using a core-shell structured nanoparticle layer substrate can be used to determine the kinetics of biomolecular interactions in a wide range of practical applications such as medicine, drug delivery, and food control.

Non-destructive monitoring of rpoS promoter activity as stress marker for evaluating cellular physiological status

To monitor the extent of cellular physiological stress, the activity of the rpoS promoter was evaluated as a marker of the stress pathway. A reporter plasmid was constructed by inserting the GFPuv gene under the rpoS promoter and used to transform Escherichia coli cells. The fluorescence of the GFPuv protein was measured in intact cells in a non-destructive manner. The physiological status of the cells could be conveniently monitored using the rpoS-GFPuv reporter gene with respect to the cellular growth phase and to elevated ethanol and NaCl concentrations as two examples of environmental stress factors. Comparison of the response of different E. coli strains demonstrated an essential role of the relA gene in the induction of the rpoS-GFPuv reporter gene.