Haretsugu Hishigaki

Automated extraction of information on protein–protein interactions from the biological literature

MOTIVATION To understand biological process, we must clarify how proteins interact with each other. However, since information about protein-protein interactions still exists primarily in the scientific literature, it is not accessible in a computer-readable format. Efficient processing of large amounts of interactions therefore needs an intelligent information extraction method. Our aim is to develop an efficient method for extracting information on protein-protein interaction from scientific literature. RESULTS We present a method for extracting information on protein-protein interactions from the scientific literature. This method, which employs only a protein name dictionary, surface clues on word patterns and simple part-of-speech rules, achieved high recall and precision rates for yeast (recall = 86.8% and precision = 94.3%) and Escherichia coli (recall = 82.5% and precision = 93.5%). The result of extraction suggests that our method should be applicable to any species for which a protein name dictionary is constructed. AVAILABILITY The program is available on request from the authors.

A radiation hybrid map of the rat genome containing 5,255 markers

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

Cloning and Expression of a Novel NADP(H)-Dependent Daidzein Reductase, an Enzyme Involved in the Metabolism of Daidzein, from Equol-Producing Lactococcus Strain 20-92

ABSTRACT Equol is a metabolite produced from daidzein by enteric microflora, and it has attracted a great deal of attention because of its protective or ameliorative ability against several sex hormone-dependent diseases (e.g., menopausal disorder and lower bone density), which is more potent than that of other isoflavonoids. We purified a novel NADP(H)-dependent daidzein reductase (L-DZNR) from Lactococcus strain 20-92 (Lactococcus 20-92; S. Uchiyama, T. Ueno, and T. Suzuki, international patent WO2005/000042) that is involved in the metabolism of soy isoflavones and equol production and converts daidzein to dihydrodaidzein. Partial amino acid sequences were determined from purified L-DZNR, and the gene encoding L-DZNR was cloned. The nucleotide sequence of this gene consists of an open reading frame of 1,935 nucleotides, and the deduced amino acid sequence consists of 644 amino acids. L-DZNR contains two cofactor binding motifs and an 4Fe-4S cluster. It was further suggested that L-DZNR was an NAD(H)/NADP(H):flavin oxidoreductase belonging to the old yellow enzyme (OYE) family. Recombinant histidine-tagged L-DZNR was expressed in Escherichia coli. The recombinant protein converted daidzein to (S)-dihydrodaidzein with enantioselectivity. This is the first report of the isolation of an enzyme related to daidzein metabolism and equol production in enteric bacteria.

MUTATED G-PROTEIN-COUPLED RECEPTOR GPR10 IS RESPONSIBLE FOR THE HYPERPHAGIA/DYSLIPIDAEMIA/OBESITY LOCUS OF Dmo1 IN THE OLETF RAT

1. We have confirmed the Diabetes Mellitus OLETF type I (Dmo1) effect on hyperphagia, dyslipidaemia and obesity in the Otsuka Long‐Evans Tokushima Fatty (OLETF) strain. The critical interval was narrowed down to 570 kb between D1Got258 to p162CA1 by segregation analyses using congenic lines.

Identification of a Novel Dihydrodaidzein Racemase Essential for Biosynthesis of Equol from Daidzein in Lactococcus sp. Strain 20-92

ABSTRACT Equol is metabolized from daidzein, a soy isoflavone, by the gut microflora. In this study, we identified a novel dihydrodaidzein racemase (l-DDRC) that is involved in equol biosynthesis in a lactic acid bacterium, Lactococcus sp. strain 20-92, and confirmed that histidine-tagged recombinant l-DDRC (l-DDRC-His) was able to convert both the (R)- and (S)-enantiomers of dihydrodaidzein to the racemate. Moreover, we showed that recombinant l-DDRC-His was essential for in vitro equol production from daidzein by a recombinant enzyme mixture and that efficient in vitro equol production from daidzein was possible using at least four enzymes, including l-DDRC. We also proposed a model of the metabolic pathway from daidzein to equol in Lactococcus strain 20-92.

Identification of two novel reductases involved in equol biosynthesis in Lactococcus strain 20-92.

Lactococcus strain 20–92 is a bacterium that produces equol directly from daidzein under anaerobic conditions. In this study, we reveal that the transcription of the gene encoding daidzein reductase in Lactococcus strain 20–92 (L-DZNR), which is responsible for the first stage of the biosynthesis of equol from daidzein, is regulated by the presence of daidzein. We analyzed the sequence surrounding the L-DZNR gene and found six novel genes, termed orf-US4, orf-US3, orf-US2, orf-US1, orf-DS1 and orf-DS2. These genes were expressed in Escherichia coli, and the resulting gene products were assayed for dihydrodaidzein reductase (DHDR) and tetrahydrodaidzein reductase (THDR) activity. The results showed that orf-US2 and orf-US3 encoded DHDR and THDR, respectively. DHDR in Lactococcus strain 20–92 (L-DHDR) was similar to the 3-oxoacyl-acyl-carrier-protein reductases of several bacteria and belonged to the short chain dehydrogenase/reductase family. THDR in Lactococcus strain 20–92 (L-THDR) was similar to several putative fumarate reductase/succinate dehydrogenase flavoprotein domain proteins. L-DHDR required NAD(P)H for its activity, whereas L-THDR required neither NADPH nor NADH. Thus, we succeeded in identifying two novel enzymes that are related to the second and third stages of the biosynthetic pathway that converts daidzein to equol.

A whole-genome radiation hybrid panel and framework map of the rat genome

Linda C. McCarthy, * ** Marie-Therese Bihoreau,* Susanna L. Kiguwa,* Julie Browne, Takeshi K. Watanabe, Haretsugu Hishigaki, Atsushi Tsuji, Susanne Kiel, 2 Caleb Webber, Maria E. Davis, Catherine Knights, Angela Smith, Ricky Critcher, 1 Patrick Huxtall, 1 James R. Hudson, Jr., 4 Toshihide Ono, Hiroumi Hayashi, Toshihisa Takagi, Yusuke Nakamura, Akira Tanigami, 3 Peter N. Goodfellow, *** G. Mark Lathrop, 2 Michael R. James

Characterization of newly developed SSLP markers for the rat.

Abstract. We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27–36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F2 progeny of (OLETFxBN)F2 crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species.

Non-helical DNA Triplex Forms a Unique Aptamer Scaffold for High Affinity Recognition of Nerve Growth Factor.

Discerning the structural building blocks of macromolecules is essential for understanding their folding and function. For a new generation of modified nucleic acid ligands (called slow off-rate modified aptamers or SOMAmers), we previously observed essential functions of hydrophobic aromatic side chains in the context of well-known nucleic acid motifs. Here we report a 2.45-Å resolution crystal structure of a SOMAmer complexed with nerve growth factor that lacks any known nucleic acid motifs, instead adopting a configuration akin to a triangular prism. The SOMAmer utilizes extensive hydrophobic stacking interactions, non-canonical base pairing and irregular purine glycosidic bond angles to adopt a completely non-helical, compact S-shaped structure. Aromatic side chains contribute to folding by creating an unprecedented intercalating zipper-like motif and a prominent hydrophobic core. The structure provides compelling rationale for potent inhibitory activity of the SOMAmer and adds entirely novel motifs to the repertoire of structural elements uniquely available to SOMAmers.

Conservation of the rat X chromosome gene order in rodent species

We constructed the comparative cytogenetic maps of X chromosomes in three rodent species, Indian spiny mouse (Mus platythrix), Syrian hamster and Chinese hamster, using 26 mouse cDNA clones. Twenty-six, 22 and 22 out of the 26 genes, which were mapped to human, mouse and rat X chromosomes in our previous study, were newly localized to X chromosomes of Indian spiny mouse, and Syrian and Chinese hamsters, respectively. The order of the genes aligned on the long arm of human X chromosome was highly conserved in rat and the three rodent species except mouse. The present results suggest a possibility that the rat X chromosome retains the ancestral form of the rodent X chromosomes.