Biomedical subjects

Eiichiro Fukusaki

Publications and source records attributed to Eiichiro Fukusaki.

8 recordsLinked to original sources

PubMed2007

Prediction of Japanese green tea ranking by gas chromatography/mass spectrometry-based hydrophilic metabolite fingerprinting.

An innovative technique for green tea's quality determination was developed by means of metabolomics. Gas-chromatography coupled with time-of-flight mass spectrometry and multivariate data analysis was employed to evaluate the quality of green tea. Alteration of green tea varieties and manufacturing processes effects a variation in green tea metabolites, which leads to a classification of the green tea's grade. Therefore, metabolic fingerprinting of green tea samples of different qualities was studied. A set of ranked green tea samples from a Japanese commercial tea contest was analyzed with the aim of creating a reliable quality-prediction model. Several multivariate algorithms were performed. Among those, the partial least-squares projections to latent structures (PLS) analysis with the spectral filtering technique, orthogonal signal correction (OCS), was found to be the most practical approach. In addition, metabolites that play an important role in green tea's grade classification were identified.

Algorithms↗

PubMed2006

Time-course metabolic profiling in Arabidopsis thaliana cell cultures after salt stress treatment.

Salt stress is one of the most important factors limiting plant cultivation. Many investigations of plant response to high salinity have been performed using conventional transcriptomics and/or proteomics approaches. However, transcriptomics and proteomics techniques are not all-encompassing methods that can achieve exclusive insights into the metabolite networks contributing to biochemical reactions. Hence, the functions of the complex stress response pathways are yet to be determined, especially at the metabolic level. A time-course metabolic profiling with Arabidopsis thaliana cell cultures after the imposition of salt stress is reported in this study. Analyses of primary metabolites, especially small polar metabolites such as amino acids, sugars, sugar alcohols, organic acids, and amines, was performed by GC/MS and LC/MS at 0.5, 1, 2, 4, 12, 24, 48, and 72 h after a salt-stress treatment with 100 mM NaCl being the final concentration. The mass chromatographic data were converted into matrix data sets, which were subjected to data mining processes, including principal component analysis (PCA) and batch-learning self-organizing mapping analysis (BL-SOM). The mining results suggest that the methylation cycle for the supply of methyl groups, the phenylpropanoid pathway for lignin production, and glycinebetaine biosynthesis are synergetically induced as a short-term response against salt-stress treatment. The results also suggest the the co-induction of glycolysis and sucrose metabolism as well as co-reduction of the methylation cycle as long-term responses to salt stress.

Amines↗

PubMed2006

Expression and purification of recombinant human annexin A2 in Pichia pastoris and utility of expression product for detecting annexin A2 antibody.

Annexin A2, a Ca2+-dependent phospholipid binding protein, is abundantly expressed in various human organs, which exists as either a membrane-associated, cytosolic or soluble form in serum. We constructed expression systems for recombinant human annexin A2 (rhA2) using Pichia pastoris. The systems are designed to secrete rhA2 as either the N- or C-terminally His6-tagged form to facilitate purification. Both types of rhA2 were overexpressed, but in the N-terminal-truncated form as revealed from the results of N-terminal amino acid sequencing and Western blotting. Therefore, further purification of N-terminally His6-tagged rhA2 was not feasible because of the removal of the N-terminal His6-tag sequence. C-terminally His6-tagged rhA2 was expressed as either a glycosylated or a nonglycosylated form, and the nonglycosylated form was purified using the combination of nickel-immobilized affinity, concanavalin A and cation exchanged column chromatographies. The solid-phase binding of rhA2 was examined by enzyme-linked immunosorbent assay (ELISA), which revealed the specific reactivity of rhA2 against an anti-annexin A2 monoclonal antibody. These results suggest that the expression system using P. pastoris is useful for the preparation of rhA2 that is applicable to the ELISA detection of the anti-annexin A2 antibody.

Amino Acid Sequence↗

PubMed2006

Pressure-assisted capillary electrophoresis mass spectrometry using combination of polarity reversion and electroosmotic flow for metabolomics anion analysis.

We have developed a method based on capillary electrophoresis (CE) coupled to mass spectrometry (MS) that is a powerful tool for metabolome analysis. In this paper, a simple method for the simultaneous analysis of anionic metabolites such as sugar phosphates, organic acids, nucleotides and CoA compounds based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS) is described. The feature of this method is that CE polarity, where the inlet of the capillary is at the anode and the outlet at the cathode, is inverted from conventional CE analysis for anions. Moreover, an ordinary fused silica capillary was chosen instead of a specific cationic polymer-coated capillary (SMILE (+) capillary). A robust and inexpensive analytical method was established from the above-mentioned CE polarity and noncoated fused silica capillary. A trimethylamine acetate electrolyte, pH 10.0, provides a high resolution between isomers. Electrolyte flow using an air pump after electrophoresis enables the comprehensive and simultaneous analyses of sugar phosphates, organic acids, nucleotides and a CoA compound that to date have been impossible to analyze. Our method can be used as a de facto standard for metabolome analysis.

Anions↗

PubMed2005

Thermostable direct hemolysin of Vibrio parahaemolyticus is a bacterial reversible amyloid toxin.

Thermostable direct hemolysin (TDH), a major virulence factor of Vibrio parahaemolyticus, is detoxified by heating at approximately 60-70 degrees C but is reactivated by additional heating above 80 degrees C. This paradoxical phenomenon, known as the Arrhenius effect, has remained unexplained for approximately 100 years. We now demonstrate that the effect is related to structural changes in the protein that produce fibrils. The native TDH (TDHn) is transformed into nontoxic fibrils rich in beta-strands by incubation at 60 degrees C (TDHi). The TDHi fibrils are dissociated into unfolded states by further heating above 80 degrees C (TDHu). Rapid cooling of TDHu results in refolding of the protein into toxic TDHn, whereas the protein is trapped in the TDHi structure by slow cooling of TDHu. Transmission electron microscopy indicates the fibrillar structures of TDHi. The fibrils show both the property of the nucleation-dependent elongation and the increase in its thioflavin T fluorescence. Formation of beta-rich structures of TDH was also observed in the presence of lipid vesicles containing ganglioside G(T1b), a putative TDH receptor. Congo red was found to inhibit the hemolytic activity of TDH in a dose-dependent manner. These data reveal that the mechanism of the Arrhenius effect which is tightly related to the fibrillogenicity of TDH.

Amyloid↗

PubMed2005

Plant metabolomics: potential for practical operation.

In the postgenomic era, metabolomics is expected to be the newest useful omics science for functional genomics. However, in plant science, the present metabolomics technology cannot be considered a universal tool to perfectly elucidate perturbations imposed on sample plants although this is desired by plant physiologists. Despite it being an immature technology, metabolomics has already been used as a powerful tool for precise phenotyping, particularly for industrial application. Metabolomics is the best technology for the analysis of large mutant or transgenic libraries of model experimental plants, such as Arabidopsis, rice, etc. Here, we review the applications and technical problems of metabolomics. We also suggest the potential of metabolomics for plant post-genomic science.

Computational Biology↗

PubMedDate not supplied

In vivo 15N-enrichment of metabolites in suspension cultured cells and its application to metabolomics.

The incorporation of stable isotopes in suspension cultured cells is very simple and useful as a preliminary experimental method in the experimental scene of plant metabolomics to elucidate the metabolic profiles of mutants and transformants. Stable isotope methods would afford a dynamic explanation of turnover speed that would concern the metabolic flux. Utilization of suspension cultured cells allows genes to be easily induced or suppressed, culture conditions to be controlled, and samples to be easily prepared. Stable isotope tracing allows an index of metabolic flux to be obtained. Here we present an experiment feeding (15)N-labeled inorganic salts to Arabidopsis (cell line T87) and Coptis cultured cells. Results of a comparison of (15)N labeling ratios of amino acids derived from T87 cells cultured under light with those cultured in the dark corresponded to transcriptional expressions revealed by microarray experiments published previously, demonstrating the validity of this procedure. Furthermore, (15)N labeling ratios of Coptis cultured cells revealed arginine and lysine metabolism inhibition, which should result in inhibition of polyamine biosynthesis and cell division. This very simple experiment allowed us to uncover metabolic dynamic features of the plant cell. Therefore this method is very useful for forming working hypotheses and experimental design.

Amino Acids↗

PubMedDate not supplied

Metabolic fingerprinting and profiling of Arabidopsis thaliana leaf and its cultured cells T87 by GC/MS.

Cell suspension cultures are now recognized as important model materials for plant bioscience and biotechnology. Very few studies of metabolic comparisons between cell cultures and original plants have been reported, even though the biological identity of cultured cells with the normally grown plant is of great importance. In this study, a comparison of the metabolome for primary metabolites extracted from the leaves of Arabidopsis thaliana and cultured cells from an Arabidopsis suspension culture (cell line T87) was performed. The results suggest that although cell suspension cultures and Arabidopsis leaves showed similarities in the common primary metabolite profile, nonetheless, moderate differences in quantitative profile were revealed.

Amino Acids↗