Total hepatocellular disposition profiling of rosuvastatin and pitavastatin in sandwich-cultured human hepatocytes.

This study describes the total disposition profiling of rosuvastatin (RSV) and pitavastatin (PTV) using a single systematic procedure called D-PREX (Disposition Profile Exploration) in sandwich-cultured human hepatocytes (SCHH). The biliary excretion fractions of both statins were clearly observed, which were significantly decreased dependent on the concentration of Ko143, an inhibitor for breast cancer resistance protein (BCRP). Ko143 also decreased the basolateral efflux fraction of RSV, whereas that of PTV was not significantly affected.

Novel multiple assessment of hepatocellular drug disposition in a single packaged procedure.

Unlabelled: Better prediction of drug disposition prior to the clinical trial is critical for the efficient development of new drugs. The purpose of this study is to develop a novel multiple assessment methodology of hepatocellular drug disposition from drug uptake to efflux including biliary and basolateral excretion, in a single packaged procedure. We started a sandwich culture using rat primary hepatocytes.

3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing.

Although it is expected that hepatocyte-like cells differentiated from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells will be utilized in drug toxicity testing, the actual applicability of hepatocyte-like cells in this context has not been well examined so far. To generate mature hepatocyte-like cells that would be applicable for drug toxicity testing, we established a hepatocyte differentiation method that employs not only stage-specific transient overexpression of hepatocyte-related transcription factors but also a three-dimensional spheroid culture system using a Nanopillar Plate. We succeeded in establishing protocol that could generate more matured hepatocyte-like cells than our previous protocol.

Identification and elimination of ion suppression in the quantitative analysis of sirolimus in human blood by LC/ESI-MS/MS.

Ion suppression can negatively affect the performance characteristics of LC/ESI-MS/MS based methods, and we wished to identify sources of ion suppression in an assay for quantitating sirolimus in human whole blood. We first compared the peak areas of sirolimus and ascomycin added to human blood samples treated with and without extraction using octadecyl silyl (ODS)-silica gel after protein precipitation, and we found that water-soluble compounds cause the ion suppression for both drugs. Post-column infusion studies indicated that compounds retained in the sample after ODS extraction and protein precipitation caused ion suppression.

The tyrosinase-encoding gene of Lentinula edodes, Letyr, is abundantly expressed in the gills of the fruit-body during post-harvest preservation.

The gill browning of Lentinula edodes fruit-bodies during preservation is thought to be due to melanin biosynthesis catalyzed by tyrosinase. We isolated a genomic DNA sequence and cDNA encoding a putative tyrosinase from the white rot basidiomycete Lentinula edodes (shiitake mushroom). The gene, named Letyr, consists of a 1,854-bp open reading frame interrupted by eight introns, and encodes a putative protein of 618 amino acid residues with an estimated molecular mass of 68 kDa.

Microarray techniques for more rapid protein quantification: use of single spot multiplex analysis and a vibration reaction unit.

Protein microarray technology is a powerful, popular tool for the high-throughput analysis of protein interactions. One important use for protein microarray technology is protein quantification by immunoassay, which was originally based on enzyme linked immunosorbent assay (ELISA) methods. Recently, new research and diagnostic applications have created a need for a rapid and easily applied high-throughput protein quantification method.

Vibratory reaction unit for the rapid analysis of proteins and glycochains.

A protein digestion system using immobilized enzymes for protein identification and glycochain analyses has been developed, and a vibration reaction unit for micro-scale sample convection on an enzyme-immobilized solid surface was constructed. BSA as a model substrate was digested by this unit, and was successfully identified by mass spectrometry (MS) analyses. Compared to the conventional liquid-phase digestion, the reaction unit increased the number of matched peptides from 9 to 26, protein score from 455 to 1247, and sequence coverage from 21% to 48%.

Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs).

Investigation of the relationship between protein-protein interaction and catalytic activity of a heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) by protein microarray.

Ec DOS, a heme-regulated phosphodiesterase from Escherichia coli, is composed of an N-terminal heme-bound PAS domain and a C-terminal phosphodiesterase domain. The heme redox state in the PAS domain regulates Ec DOS phosphodiesterase activity. Interestingly, the isolated heme-bound PAS fragment enhances phosphodiesterase activity of full-length Ec DOS.

Localization of actin filaments on mitotic apparatus in tobacco BY-2 cells.

Actin filaments are among the major components of the cytoskeleton, and participate in various cellular dynamic processes. However, conflicting results had been obtained on the localization of actin filaments on the mitotic apparatus and their participation in the process of chromosome segregation. We demonstrated by using rhodamine-phalloidin staining, the localization of actin filaments on the mitotic spindles of tobacco BY-2 cells when the cells were treated with cytochalasin D.

Protein microarray system for detecting protein-protein interactions using an anti-His-tag antibody and fluorescence scanning: effects of the heme redox state on protein-protein interactions of heme-regulated phosphodiesterase from Escherichia coli.

A highly sensitive microarray system for detecting protein-protein interactions has been developed. This method was successfully applied to analyze the interactions of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS). To immobilize (His)6-Tag fused Ec DOS, anti-(His)6-Tag monoclonal antibody (anti-(His)6-Tag mAb) was initially immobilized on the solid surface, and (His)6-Tag fused Ec DOS was fixed by antigen-antibody interactions.

Complete sequencing and characterization of 21,243 full-length human cDNAs.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding.

Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies.

A laccase (EC 1.10.3.