and methamphetamine-induced locomotor stimulation and stereotyped behaviors in rats [S-7]. Despite these significant functional roles in the central nervous system, the presence of free D-amino acids in mammalian brain has not yet btien demonstrated, with the exception of the small amounts of free neutral amino acids in mice and human brains [8,9] and of free D-aspartate in adult rat and human brains (less than 3% of total aspartate) [l&12]. In the present study, we report the identification and the concentration of the free D-enantiomer of serine in adult rat brain using gas chromatographic (GC) analy- MATERIALS AND METHODSAll amino acids and trichloroacctic acid (TCA) were oblainrd rrom Nakardi Tcsquc (Japan). All solvcnls were HPLC grade and purchased from Kanto Chemicals (Japan). A pcntafluoropropionic anhydride (PFPA) was purchased from G.L.C. Scicncc (Japun). The column head pressure was I IO kPa. and the helium flow-mu on the column was 0.4 ml/min; the flow-rate (plus auxiliary gas) was 30 mllmin. The hydrogen flow-ralc and the air flow-rate al the dctcctor were 3.5 and I IO ml/min, rcspcctivcly. Peak arca was dclcrmined using a SIC chromalocorder I I intcgralor (Syslcm Inslrumcnu Co.). rWS umi/~v.si,sThe GC-MS analysis was pcrrormcd on a V.G. Masslab Trio-l mass spectrometer (Jasco, ImcrnaGonal Co., Japan), equipped with a Hcwlcu Packard 5890A gas chromalograph. The GC condilions wcrc the Same as for the GC analysis with the cxcplion oi a spiititisj. injection. The mass spcctromctcr con&ions wcrc: clcc~ron accclcraling voliugc, 70 cV; inicrracc tcmpcr?lurc, 170°C; photomul1iplicr 33

Microbiota have been shown to have a great influence on functions of intestinal epithelial cells (ECs). The role of indole as a quorum-sensing (QS) molecule mediating intercellular signals in bacteria has been well appreciated. However, it remains unknown whether indole has beneficial effects on maintaining intestinal barriers in vivo. In this study, we analyzed the effect of indole on ECs using a germ free (GF) mouse model. GF mice showed decreased expression of junctional complex molecules in colonic ECs. The feces of specific pathogen-free (SPF) mice contained a high amount of indole; however the amount was significantly decreased in the feces of GF mice by 27-fold. Oral administration of indole-containing capsules resulted in increased expression of both tight junction (TJ)- and adherens junction (AJ)-associated molecules in colonic ECs in GF mice. In accordance with the increased expression of these junctional complex molecules, GF mice given indole-containing capsules showed higher resistance to dextran sodium sulfate (DSS)-induced colitis. A similar protective effect of indole on DSS-induced epithelial damage was also observed in mice bred in SPF conditions. These findings highlight the beneficial role of indole in establishing an epithelial barrier in vivo.

Many priniciples of sequence-specific DNA recognition have been established over the past decade, largely from structural studies of protein-DNA and drug-DNA complexes. On the basis of these principles, it has been possible to design or select variants of known structural motifs, including zinc-fingers and minor groove-binding drugs, that bind desired sequences. Here we describe a strategy, based on transcriptional termination in bacteria, to identify specific RNA-binding peptides using the arginine-rich RNA-binding motif as a framework. Peptides were isolated from two combinatorial libraries that bind tightly and specifically to the Rev response element of HIV. It appears that alpha-helical peptides resembling Rev were selected from one library whereas new peptides that probably do not form helices were selected from the other, suggesting that the arginine-rich motif may be a particularly versatile framework for recognizing RNA structures.

No abstract

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.

Interindividual differences in hepatic metabolism, which are mainly due to genetic polymorphism in its gene, have a large influence on individual drug efficacy and adverse reaction. Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells have the potential to predict interindividual differences in drug metabolism capacity and drug response. However, it remains uncertain whether human iPSC-derived HLCs can reproduce the interindividual difference in hepatic metabolism and drug response. We found that cytochrome P450 (CYP) metabolism capacity and drug responsiveness of the primary human hepatocytes (PHH)-iPSHLCs were highly correlated with those of PHHs, suggesting that the PHH-iPS-HLCs retained donor-specific CYP metabolism capacity and drug responsiveness. We also demonstrated that the interindividual differences, which are due to the diversity of individual SNPs in the CYP gene, could also be reproduced in PHH-iPS-HLCs. We succeeded in establishing, to our knowledge, the first PHH-iPS-HLC panel that reflects the interindividual differences of hepatic drugmetabolizing capacity and drug responsiveness.human iPS cells | hepatocyte | CYP2D6 | personalized drug therapy | SNP D rug-induced liver injury (DILI) is a leading cause of the withdrawal of drugs from the market. Human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) are expected to be useful for the prediction of DILI in the early phase of drug development. Many groups, including our own, have reported that the human iPS-HLCs have the ability to metabolize drugs, and thus these cells could be used to detect the cytotoxicity of drugs that are known to cause DILI (1, 2). However, to accurately predict DILI, it will be necessary to establish a panel of human iPS-HLCs that better represents the genetic variation of the human population because there are large interindividual differences in the drug metabolism capacity and drug responsiveness of hepatocytes (3). However, it remains unclear whether the drug metabolism capacity and drug responsiveness of human iPS-HLCs could reflect those of donor parental primary human hepatocytes (PHHs). To address this issue, we generated the HLCs differentiated from human iPSCs which had been established from PHHs (PHH-iPS-HLCs). Then, we compared the drug metabolism capacity and drug responsiveness of PHH-iPS-HLCs with those of their parental PHHs, which are genetically identical to the PHH-iPS-HLCs.Interindividual differences of cytochrome P450 (CYP) metabolism capacity are closely related to genetic polymorphisms, especially single nucleotide polymorphisms (SNPs), in CYP genes (4). Among the various CYPs expressed in the liver, CYP2D6 is responsible for the metabolism of approximately a quarter of commercially used drugs and has the largest phenotypic variability, largely due to SNPs (5). It is known that certain alleles result in the poor metabolizer phenotype due to a decrease of CYP2D6 metabolism. Therefore, the appropriate dosage for drugs that are metabolized ...

No abstract

SummaryIncretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting β cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion.