Shinya Ikeno

Discerning Data Analysis Methods to Clarify Agonistic/Antagonistic Actions on the Ion Flux Assay of Ligand-Gated Ionotropic Glutamate Receptor on Engineered Post-Synapse Model Cells

Cell-based experiments provide the efficacy of chemicals through the biological function. The authors have described post-synapse model cell-based assay based on qualified analysis for neural drug discoveries. However, in general, cell-based assays often include data fluctuation. This may be a barrier preventing the performance for various practical purposes. In this study, we tried discerning data analysis for clarify the chemical action to the ionotoropic glutamate receptor (GluR), whereby an ion-flux assay of post-synapse model cells is performed and are analyzed based on a chemometrics approach. The dynamic behavior of the GluR of post-synapse model cell was assayed with multivariate data analysis methods namely hierarchical cluster analysis (HCA) and principal component analysis (PCA). By using HCA, we can identify and remove the non-responding samples. By using PCA, the effect of chemicals on the dynamic behavior of ion flux through GluR can be recognized clearly; as either agonist or antagonist. As shown in the results, the GluR-based assay by post-synapse model cell with data analysis methods provide a sodium influx profile which is derived by an agonists or antagonists application. By employing the data analysis methods, PCA and HCA, it is possible to develop a smart cellular biosensing system for qualified analysis.

Gold Nanoparticles Functionalized with Peptides for Specific Affinity Aggregation Assays of Estrogen Receptors and Their Agonists

Nuclear receptors regulate the transcription of genes and various functions such as development, differentiation, homeostasis, and behavior by formation of complexes with ligand and co-activator. Recent findings have shown that agonists of a ligand may have a toxic effect on cellular/tissular function through improper activation of nuclear receptors. In this study, a simple assay system of hetero-complexes of three different molecules (estrogen receptor, ligand, and co-activator peptide) has been developed. This assay system employs functionalized gold nanoparticles (GNPs: 15 nm in diameter). The surfaces of the GNPs were modified by a 12- or 20-amino-acid peptide that contains the sequence of co-activator for activating nuclear receptor by an agonist ligand. Owing to the affinity of the peptide, the functionalized GNPs aggregate faster when the nuclear receptor and the agonist ligand are also present. The aggregation of GNPs can be identified by shifts in adsorption spectrum, which give information about the specificity of agonist ligands. Similarly, this spectrum shift can measure concentration of known agonist ligand. This simple agonist screening will be employed as high through-put analysis (HTA) in the discovery of drugs that act through nuclear receptors.

Boost protein expression through co-expression of LEA-like peptide in Escherichia coli.

The boost protein expression has been done successfully by simple co-expression with a late embryogenesis abundant (LEA)-like peptide in Escherichia coli. Frequently, overexpression of a recombinant protein fails to provide an adequate yield. In the study, we developed a simple and efficient system for overexpressing transgenic proteins in bacteria by co-expression with an LEA-like peptide. The design of this peptide was based on part of the primary structure of an LEA protein that is known hydrophilic protein to suppress aggregation of other protein molecules. In our system, the expression of the target protein was increased remarkably by co-expression with an LEA-like peptide consisting of only 11 amino acid residues. This could provide a practical method for producing recombinant proteins efficiently.

Molecular Commonality Sensing of Phosphoric Anhydride Substances Using an Ion-Sensitive Field-Effect Transistor Covered with an Artificial Enzyme Membrane

A new type of biosensor has been developed using an artificial enzyme membrane that recognizes and hydrolyzes phosphoric anhydride bonds selectively. The artificial enzyme (artificial P–P hydrolase) membrane was designed and synthesized by means of our original procedure, and mounted on a T2O5-gate ion-sensitive field-effect transistor (ISFET). H2PO4- ions are produced through the catalytic reaction in the artificial P–P hydrolase membrane, and accumulate in the membrane on the ISFET. The accumulated H2PO4- ions cause a shift in the static ISFET output as the sensor response. A key advantage of the artificial P–P hydrolase is its specific structure selectivity for the phosphoric anhydride bonds, which is one of the dominant and characteristic structures in biological energy substances. The molecular commonality detection of the phosphoric anhydride bonds is a novel tactic for biosurveillance and cellular viability assays.

Characterization of titanium dioxide nanoparticles modified with polyacrylic acid and H2O2 for use as a novel radiosensitizer

Abstract An induction of polyacrylic acid-modified titanium dioxide with hydrogen peroxide nanoparticles (PAA-TiO2/H2O2 NPs) to a tumor exerted a therapeutic enhancement of X-ray irradiation in our previous study. To understand the mechanism of the radiosensitizing effect of PAA-TiO2/H2O2 NPs, analytical observations that included DLS, FE-SEM, FT-IR, XAFS, and Raman spectrometry were performed. In addition, highly reactive oxygen species (hROS) which PAA-TiO2/H2O2 NPs produced with X-ray irradiation were quantified by using a chemiluminescence method and a EPR spin-trapping method. We found that PAA-TiO2/H2O2 NPs have almost the same characteristics as PAA-TiO2. Surprisingly, there were no significant differences in hROS generation. However, the existence of H2O2 was confirmed in PAA-TiO2/H2O2 NPs, because spontaneous hROS production was observed w/o X-ray irradiation. In addition, PAA-TiO2/H2O2 NPs had a curious characteristic whereby they absorbed H2O2 molecules and released them gradually into a liquid phase. Based on these results, the H2O2 was continuously released from PAA-TiO2/H2O2 NPs, and then released H2O2 assumed to be functioned indirectly as a radiosensitizing factor.

Construction and characterization of mutated LEA peptides in Escherichia coli to develop an efficient protein expression system

To develop an efficient protein expression system, we designed a late embryogenesis abundant (LEA) peptide by mutating the LEA peptide constructed in our previous study (LEA‐I). The peptide is based on the repeating units of an 11mer motif characteristic of LEA proteins from Polypedilum vanderplanki larvae. In the amino acid sequence of the 13mer LEA peptide, glycine at the 6th and 12th positions was replaced with other amino acids via point mutations. Glutamic acid, lysine, leucine, and asparagine in the LEA peptide at the 6th and 12th positions increased green fluorescence protein (GFP) expression. The GFP expression of the mutated LEA peptide was 1.5 to 2.0 times higher than that without LEA peptide. In contrast, the serine‐containing mutated LEA peptide has low GFP expression levels. We hypothesize that the position of amino acids and the nature of amino acid in LEA peptide are important for our coexpression system. These data suggest that the size, structure, and charge of amino acids in the LEA peptide improve the protection and expression of the target protein. The amino acid balance also plays an important role in the expression of the target protein.