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Biomedical subjects

Kenji Usui

Publications and source records attributed to Kenji Usui.

14 recordsLinked to original sources
PubMed2007

Interactions between peptides containing nucleobase amino acids and T7 phages displaying S. cerevisiae proteins.

The importance of high-throughput analyses of protein abundances and functions is interestingly increasing in genomic/proteomic studies. In such postgenome sequencing era, a protein-detecting chip, in which a large number of molecules specifically capturing target proteins (capturing agents) such as antibodies, recombinant proteins, and small molecules are arrayed onto solid, wet, or semi-wet substrates, enables comprehensive analysis of proteomes by a single experiment. However, whole proteomes are generally complicated for comprehensive analyses so that alternative approaches to subproteome analysis categorized by protein functions and binding properties (focused proteome) would be effective. Approaching the goal of development of designed peptide chip for protein analysis, diversity increases in peptide structures and validation of target proteins are needed. We herein describe design and synthesis of nucleobase amino acid (NBA)-containing peptides, selection of nucleic acid-related proteins derived from S. cerevisiae, and detection of interactions between NBA-containing peptides and T7 phages displaying proteins by both enzyme-linked immunosorbent assays (ELISA) and label-free anomalous reflection of gold (AR) measurements. Twenty-eight phage clones were obtained by the phage-display method and sequenced. Ten of 28 clones were expected to be nucleic acid-related proteins including initiation factor, TYB protein, ribosomal proteins, elongation factor, ATP synthase subunit, GTP-binding protein, and ribonuclease. Other phage clones encoded several classes of enzymes such as reductase, oxidase, aldolase, metalloprotease, and hexokinase. Both ELISA and AR measurements suggested that the methodology of in vitro selection for recognition of the NBA-containing peptide presented in this study was successfully established. Such a combination of NBA and phage display technologies would be potential to efficiently confirm valuable target proteins binding specifically to capturing agents, to be arrayed onto solid surfaces to develop the designed peptide chip.

Bacteriophage T7↗
PubMed2006

Screening of alpha-helical peptide ligands controlling a calcineurin-phosphatase activity.

In this paper, we describe an application of 202-membered fluorescently labeled peptide library designed to take an alpha-helix secondary structure. As a proof-of-concept experiment, a calmodulin (CaM)/calcineurin (Cn) pair was chosen to screen alpha-helical peptide ligands that tightly bind to CaM and also control enzymatic functions of Cn. Three peptides were successfully selected from the library by assaying Cn-phosphatase activities and peptide-CaM interactions (dual check process). The strategy using a designed peptide library shows real promise as a peptide-based high-throughput screening system.

Amino Acid Sequence↗
PubMed2006

The role of altered acetyl-CoA carboxylase in conferring resistance to fenoxaprop-P-ethyl in Chinese sprangletop (Leptochloa chinensis (L.) Nees).

From paddy field observations in 2002 and 2004, fenoxaprop-P-ethyl resistance in Chinese sprangletop (Leptochloa chinensis (L.) Nees) has been studied using information collected from 11 sites in the Saphan-Sung district of Bangkok, Thailand. The resistant Chinese sprangletop was found in nine rice fields, whereas the susceptible Chinese sprangletop was found in only two rice fields. In greenhouse experiments, both fenoxaprop-P-ethyl-resistant and susceptible Chinese sprangletop from the same location were investigated for 50% growth reduction based on phytotoxicity, plant height and fresh and dry weight. The resistant Chinese sprangletop showed apparent resistance at 14-21 days after herbicide application at a rate of 21.1-337.6 g AI ha(-1). The resistance index of resistant Chinese sprangletop was 10-25 times higher than that of the susceptible Chinese sprangletop. In addition, Chinese sprangletop did not exhibit multiple resistance to oxadiazon, propanil and quinclorac. According to acetyl-CoA carboxylase (ACCase) assays, the level of ACCase specific activity in the resistant Chinese sprangletop was significantly higher than that in the susceptible Chinese sprangletop. Similarly, the ACCase activity of the resistant Chinese sprangletop was 10 times less sensitive to fenoxaprop-P-ethyl than that of the susceptible Chinese sprangletop, based on the I50 values. The present study of the mechanism responsible for resistance in the biotypes investigated indicated that there was a close association between the concentration-response at the whole-plant level and ACCase sensitivity to fenoxaprop-P-ethyl, and resistance to fenoxaprop-P-ethyl was conferred by a modified ACCase at the target site, as suggested by higher specific activity and less sensitivity to the herbicide.

Acetyl-CoA Carboxylase↗
PubMed2006

Effects of five rice herbicides on the growth of two threatened aquatic ferns.

The effects of five rice herbicides bensulfuron methyl, mefenacet, quinoclamine, simetryn, and thiobencarb on the growth of two threatened aquatic ferns Azolla japonica and Salvinia natans were tested using 12-day exposure experiments at 0.1-100 nM which are expected to be present in drainages and rivers in Japan. As a reference species, Lenma minor was also used to examine the toxicity of bensulfuron methyl. Bensulfuron methyl had the most pronounced effect on the relative growth rate (RGR) of A. japonica, S. natans, and L. minor with an EC50 of 5.0, 0.54, and 10 nM, respectively. The other herbicides reduced the RGR of the aquatic ferns only at the highest concentration (100 nM) or not at all. S. natans showed the highest susceptibility to bensulfuron methyl among the three species, and the EC50 for this species was comparable to or below the maximum concentration (0.49-5.6 nM) that had been previously detected in 7 of 11 rivers in Japan. These results suggest that bensulfuron methyl runoff in drainages and rivers in Japan is expected to have adverse effects on the growth of threatened aquatic ferns in some cases, and that no or small effects occur for the other four herbicides tested.

Dose-Response Relationship, Drug↗
PubMed2005

A novel peptide microarray for protein detection and analysis utilizing a dry peptide array system.

A novel dry peptide microarray system has been constructed that affords a practical solution for protein detection and analysis. This system is an array preparation and assay procedure under dry conditions that uses designed peptides as non-immobilized capture agents for the detection of proteins. The system has several advantages that include its portability and ease-of-use, as well as the fact that vaporization of sample solutions need not be considered. In this study, various proteins have been characterized with an alpha-helical peptide mini-library. When proteins were added to the peptide library array, the fluorescent peptides showed different fluorescent intensities depending on their sequences. The patterns of these responses could be regarded as 'protein fingerprints' (PFPs), which are sufficient to establish the identities of the target proteins. Furthermore, statistical analysis of the resulting PFPs was performed using cluster analysis. The PFPs of the proteins were clustered successfully depending on their families and binding properties. Additionally, the target protein was characterized using a nanolitre system and could be detected down to 1.2 fmol. These studies imply that the dry peptide array system is a promising tool for detecting and analyzing target proteins. The dry peptide array will play a role in development of high-throughput protein-detecting nano/micro arrays for proteomics and ligand screening studies.

Amino Acid Sequence↗
PubMed2005

A PNA-DNA hybridization chip approach for the detection of beta-secretase activity.

Developed was the addressable chip technology based on the PNA-DNA complementary hybridization equipped with short seven-mer PNA-encoded peptides that can be a versatile scaffold to monitor on-chip immunoassays. We also developed and validated a methodology to perform beta-secretase enzyme assay with a highly sensitive fashion, resulting that a peptide substrate tethering dual fluorescent probes allowed us to detect beta-secretase activity 10 times more sensitively than assays in solution.

Amyloid Precursor Protein Secretases↗
PubMed2005

Protein-detecting microarrays: current accomplishments and requirements.

The sequencing of the human genome has been successfully completed and offers the chance of obtaining a large amount of valuable information for understanding complex cellular events simply and rapidly in a single experiment. Interestingly, in addressing these proteomic studies, the importance of protein-detecting microarray technology is increasing. In the coming few years, microarray technology will become a significantly promising and indispensable research/diagnostic tool from just a speculative technology. It is clear that the protein-detecting microarray is supported by three independent but strongly related technologies (surface chemistry, detection methods, and capture agents). Firstly, a variety of surface-modification methodologies are now widely available and offer site-specific immobilization of capture agents onto surfaces in such a way as to keep the native conformation and activity. Secondly, sensitive and parallel detection apparatuses are being developed to provide highly engineered microarray platforms for simultaneous data acquisition. Lastly, in the development of capture agents, antibodies are now probably the most prominent capture agents for analyzing protein abundances. Alternative scaffolds, such as phage-displayed antibody and protein fragments, which provide the advantage of increasing diversity of proteinic capture agents, however, are under development. An approach involving recombinant proteins fused with affinity tag(s) and coupled with a highly engineered surface chemistry will provide simple production protocols and specific orientations of capture agents on the microarray formats. Peptides and other small molecules can be employed in screening highly potent ligands as well as in measuring enzymatic activities. Protein-detecting microarrays supported by the three key technologies should contribute in accelerating diagnostic/biological research and drug discovery.

Animals↗
PubMed2005

A hydroponic rice seedling culture model system for investigating proteome of salt stress in rice leaf.

By using an in vivo hydroponic rice seedling culture system, we investigated the physiological and biochemical responses of a model rice japonica cultivar Nipponbare to salt stress using proteomics and classical biochemical methods. Yoshida's nutrient solution (YS) was used to grow rice seedlings. YS-grown 18-day-old seedlings manifested highly stable and reproducible symptoms, prominently the wilting and browning of the 3rd leaf, reduced photosynthetic activity, inhibition in overall seedling growth, and failure to develop new (5th) leaf, when subjected to salt stress by transferring them to YS containing 130 mM NaCl for 4 days. As leaf response to salt stress is least investigated in rice by proteomics, we used the 3rd leaf as source material. A comparison of 2-DE protein profiles between the untreated control and salt-stressed 3rd leaves revealed 55 differentially expressed CBB-stained spots, where 47 spots were increased over the control. Of these changed spots, the identity of 33 protein spots (27 increased and 5 decreased) was determined by nESI-LC-MS/MS. Most of these identified proteins belonged to major metabolic processes like photosynthetic carbon dioxide assimilation and photorespiration, suggesting a good correlation between salt stress-responsive proteins and leaf morphology. Moreover, 2-DE immunoblot and enzymatic activity analyses of 3rd leaves revealed remarkable changes in the key marker enzymes associated with oxidative damage to salt stress: ascorbate peroxidase and lipid peroxidation were induced, and catalase was suppressed. These results demonstrate that hydroponic culture system is best suited for proteomics of salt stress in rice seedling.

Amino Acid Sequence↗
PubMed2004

First total synthesis of antimitotic compound, (+)-phomopsidin.

[reaction: see text] The first total synthesis of (+)-phomopsidin has been achieved via a diastereoselective transannular Diels-Alder (TADA) reaction. Key steps in the synthesis include diastereoselective ynone reduction with (-)-alpha-pinene and 9-BBN, macrocyclization by E-selective intramolecular Horner-Wadsworth-Emmons (HWE) reaction, as well as carbometalation under Wipf's conditions, followed by HWE reaction at low temperature to selectively construct the (E)-1-methylpropenyl and (1E,2E)-4-carboxy-1,3-butadienyl substituents.

Antineoplastic Agents↗
PubMed2004

Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns.

To realize a practical high-throughput protein-detection system, novel peptide arrays have been constructed using designed peptide libraries with loop, alpha-helix, or beta-strand structures. Here, we describe the overview of the reported designed peptide arrays with loop and alpha-helix structures and the new results of those with beta-strand structures. Initially, several model peptides known to interact with model structured proteins were selected to establish the present strategy for high-throughput detection of proteins. The fluorescent probes and suitable scaffolds of peptides were examined for the effective detection of proteins. The detection methods were established in solution and in an immobilized manner using the model systems. In the case of alpha-helix peptide, the response of a peptide with fluorescent resonance energy transfer between two probes at both termini was several times higher than that of a peptide with a single probe. In the cases of peptides with other structures, however, proteins were effectively detectable even by the fluorescent change of one probe. Furthermore, structurally focused libraries consisting of a total of ca. 250 different peptides based on the model peptides with secondary and/or tertiary structures were constructed with systematic replacement of residues. Using these libraries, various proteins were characterized effectively to give their own fluorescent "protein fingerprint" patterns. The resulting protein fingerprints correlated with the recognition properties of the proteins. These studies demonstrate that arrays with peptide libraries based on designed structures can be promising tools for detecting the target proteins. Designed synthetic peptides play roles as the capturing agents to be developed for practical protein chips.

Amino Acid Sequence↗
PubMed2004

Peptide arrays with designed alpha-helical structures for characterization of proteins from FRET fingerprint patterns.

A practical high-throughput protein detection system is described, based on synthetic peptide arrays consisting of designed alpha-helical peptides, detected by fluorescence resonance energy transfer (FRET). Initially a model alpha-helical peptide known to interact with a structured protein, calmodulin, was selected to establish the strategy for high-throughput detection. In comparison to peptides with a single probe, a much higher FRET response has been observed with two fluorescent probes (7-diethylaminocoumarin-3-carboxylic acid and 5(6)-carboxy-fluorescein) at both termini of the synthetic peptides. To establish a reproducible high-throughput detection system, peptides were also immobilized onto a solid surface for detection of the target proteins. A small library of 112 different peptides was constructed, based on a model of the alpha-helical peptide with systematic replacement of residues carrying specific charges and/or hydrophobicities. The library was used to effectively characterize various proteins, giving their own 'protein fingerprint' patterns. The resulting 'protein fingerprints' correlate with the recognition properties of the proteins. The present microarray with designed synthetic peptides as the capturing agents is promising for the development of protein detection chips.

Amino Acid Sequence↗