Development of genotyping method for functionally relevant variants of cytochromes P450 in cynomolgus macaques.

In cynomolgus macaques (Macaca fascicularis), widely used in drug metabolism studies, CYP2C9, CYP2C76, CYP2D6, CYP3A4, and CYP3A5, important drug-metabolizing enzymes, are abundantly expressed in liver and metabolize cytochrome P450 substrates. CYP2C9 (c.334A>C), CYP2C76 (c.449TG>A), CYP2D6 (c.891A>G), CYP3A4 (IVS3 + 1G>del), and CYP3A5 (c.625A>T) substantially influence metabolic activity of enzymes, and thus are important variants in drug metabolism studies. In this study, a real-time PCR method was developed for genotyping these variants. The validity of the methods was verified by genotyping two wild type, two heterozygous, and two homozygous DNAs and was used to genotype 41 cynomolgus macaques (from Cambodia, Indonesia, the Philippines, or Vietnam) for the five variants, along with another important variant CYP2C19 (c.308C>T). The CYP2C9 and CYP2C19 variants were found only in Cambodian and Vietnamese animals, while the CYP2C76 and CYP2D6 variants were found only in Indonesian and Philippine animals. The CYP3A4 and CYP3A5 variants were not found in any of the animals analyzed. Mauritian animals, genotyped using next-generation sequencing data for comparison, possessed the CYP2C19 and CYP2D6 variants, but not the other variants. These results indicated differences in prevalence of these important variants among animal groups. Therefore, the genotyping tool developed is useful for drug metabolism studies using cynomolgus macaques.

Acotiamide improves stress-induced impaired gastric accommodation.

BACKGROUND: Gastric accommodation is a reflex reaction related to gastric reservoir function. Psychological stress, such as anxiety, inhibits gastric accommodation in humans. Acotiamide enhances the effect of acetylcholine in the enteric nervous system, enhances gastric contractility, and accelerates delayed gastric emptying. However, the effect of acotiamide on stress-induced impaired gastric accommodation remains unclear. Therefore, we examined the effect of acotiamide on gastric accommodation and stress-induced impaired gastric accommodation using a conscious guinea pig model. METHODS: A polyethylene bag was inserted through the distal region of the gastric body into the proximal stomach of 5-week-old male Hartley guinea pigs. Gastric accommodation was evaluated by measuring the intrabag pressure in the proximal stomach after oral administration of a liquid meal. In the stress model, animals were subjected to water-avoidance stress. Acotiamide (Z-338) or nizatidine was administered subcutaneously. Fecal output was determined as the number of fecal pellets. KEY RESULTS: Administration of the liquid meal significantly decreased intrabag pressure, indicating induction of gastric accommodation. Acotiamide treatment prolonged liquid meal-induced gastric accommodation and significantly increased the number of fecal pellets compared to controls. Water-avoidance stress significantly inhibited liquid meal-induced gastric accommodation. Pretreatment with acotiamide significantly improved stress-induced impaired gastric accommodation. The number of fecal pellets in the acotiamide group increased significantly compared to controls. Acotiamide, but not nizatidine, significantly decreased gastric emptying. CONCLUSIONS & INFERENCES: Acotiamide prolongs gastric accommodation and improves stress-induced impaired gastric accommodation, indicating a potential role for acotiamide in the treatment of functional dyspepsia through its effects on gastric accommodation reactions.

Molecular Evidence for Convergence and Parallelism in Evolution of Complex Brains of Cephalopod Molluscs: Insights from Visual Systems.

Coleoid cephalopods show remarkable evolutionary convergence with vertebrates in their neural organization, including (1) eyes and visual system with optic lobes, (2) specialized parts of the brain controlling learning and memory, such as vertical lobes, and (3) unique vasculature supporting such complexity of the central nervous system. We performed deep sequencing of eye transcriptomes of pygmy squids (Idiosepius paradoxus) and chambered nautiluses (Nautilus pompilius) to decipher the molecular basis of convergent evolution in cephalopods. RNA-seq was complemented by in situ hybridization to localize the expression of selected genes. We found three types of genomic innovations in the evolution of complex brains: (1) recruitment of novel genes into morphogenetic pathways, (2) recombination of various coding and regulatory regions of different genes, often called "evolutionary tinkering" or "co-option", and (3) duplication and divergence of genes. Massive recruitment of novel genes occurred in the evolution of the "camera" eye from nautilus' "pinhole" eye. We also showed that the type-2 co-option of transcription factors played important roles in the evolution of the lens and visual neurons. In summary, the cephalopod convergent morphological evolution of the camera eyes was driven by a mosaic of all types of gene recruitments. In addition, our analysis revealed unexpected variations of squids' opsins, retinochromes, and arrestins, providing more detailed information, valuable for further research on intra-ocular and extra-ocular photoreception of the cephalopods.

Integration of cap analysis of gene expression and chromatin immunoprecipitation analysis on array reveals genome-wide androgen receptor signaling in prostate cancer cells.

The androgen receptor (AR) is a critical transcriptional factor that contributes to the development and the progression of prostate cancer (PCa) by regulating the transcription of various target genes. Genome-wide screening of androgen target genes provides useful information to understand a global view of AR-mediated gene network in PCa. In this study, we performed 5'-cap analysis of gene expression (CAGE) to determine androgen-regulated transcription start sites (TSSs) and chromatin immunoprecipitation (ChIP) on array (ChIP-chip) analysis to identify AR binding sites (ARBSs) and histone H3 acetylated (AcH3) sites in the human genome. CAGE determined 13 110 distinct, androgen-regulated TSSs (P<0.01), and ChIP-chip analysis identified 2872 androgen-dependent ARBSs (P<1e-5) and 25 945 AcH3 sites (P<1e-4). Both androgen-regulated coding genes and noncoding RNAs, including microRNAs (miRNAs) were determined as androgen target genes. Besides prototypic androgen-regulated TSSs in annotated gene promoter regions, there are many androgen-dependent TSSs that are widely distributed throughout the genome, including those in antisense (AS) direction of RefSeq genes. Several pairs of sense/antisense promoters were newly identified within single RefSeq gene regions. The integration of CAGE and ChIP-chip analyses successfully identified a cluster of androgen-inducible miRNAs, as exemplified by the miR-125b-2 cluster on chromosome 21. Notably, the number of androgen-upregulated genes was larger in LNCaP cells treated with R1881 for 24 h than for 6 h, and the percentage of androgen-upregulated genes accompanied with adjacent ARBSs was also much higher in cells treated with R1881 for 24 h than 6 h. On the basis of the Oncomine database, the majority of androgen-upregulated genes containing adjacent ARBSs and CAGE tag clusters in our study were previously confirmed as androgen target genes in PCa. The integrated high-throughput genome analyses of CAGE and ChIP-chip provide useful information for elucidating the AR-mediated transcriptional network that contributes to the development and progression of PCa.

Molecular characterization of the developmental gene in eyes: through data-mining on integrated transcriptome databases.

OBJECTIVES: Our aim was to utilize publicly available and proprietary sources to discover candidate genes important for ocular development. DESIGN AND METHODS: The collated information on our 5092 non-redundant clusters was grouped and functional annotation was conducted using gene ontology (FatiGO) for categorizing them with respect to molecular function. The web-based viewer technological platform (H-InvDB) was employed for transcription analyses of in-house high quality fetal eye Expressed Sequence Tags (ESTs). Eye-specific ESTs were also analyzed across species by using EMBEST. RESULTS: According to adult eye cDNA libraries, nucleic acid binding and cell structure/cytoskeletal protein genes were the most abundant among the ESTs of fetal eyes. Using cDNA assembly in H-InvDB, 20 (80%) of the 25 most commonly expressed genes in the human eye are also expressed in extraocular tissues. The crystalline gamma S gene is highly expressed in the eye, but not in other tissues. We used EMBEST to compare human fetal eye and octopus eye ESTs and the expression similarity was low (1.6%). This indicated that our fetal eye library contains genes necessary for the developmental process and biological function of the eye, which may not be expressed in the fully developed octopus eyes. The human fetal eye cDNA library also contained highly abundant eye tissue genes, including alphaA-crystallin, eukaryotic translation elongation factor 1 alpha 1 (EEF1A1), bestrophin (VMD2), cystatin C, and transforming growth factor, beta-induced (BIGH3). CONCLUSIONS: Our annotated EST set provides a valuable resource for gene discovery and functional genomic analysis. This display will help to appreciate the strengths and weaknesses of the different technological platforms, so that in future studies the maximum amount of beneficial information can be derived from the appropriate use of each method.

Genomic annotation of 15,809 ESTs identified from pooled early gestation human eyes.

To complement cDNA libraries from the human eye at early gestation and to discover candidate genes associated with early ocular development, we used freshly dissected human eyeballs from week 9-14 of gestation to construct the early human fetal eye cDNA library. A total of 15,809 clones were isolated and sequenced from the unamplified and unnormalized library. We screened 11,246 good-quality ESTs, leading to the identification of 5,534 nonredundant clusters. Among them, 4,010 (72%) genes matched in the human protein database (Ensembl). The remaining 28% (1,524) corresponded to potentially novel or previously unidentified ESTs. We used BLASTX to compare our EST data with eight organisms and found common expression of a high portion of genes: Caenorhabditis briggsae (26%), Caenorhabditis elegans (27%), Anopheles gambiae (37%), Drosophila melanogaster (32%), Danio rerio (42%), Fugu rubripes (49%), Rattus norvegicusvalitus (52%), and Mus musculus (59%). Nevertheless, 48% (2,680 of 5,534) of the genes expressed in the early developing eye were not shared with current NEIBank human eye cDNA data. In addition, eight known retinal disease genes existed in our ESTs. Among them, six (COL11A1, BBS5, PDE6B, OAT, VMD2, and PGK1) were conserved among the genomes of other organisms, indicating that our annotated EST set provides not only a valuable resource for gene discovery and functional genomic analysis but also for phylogenetic analysis. Our foremost early gestation human eye cDNA library could provide detailed comparisons across species to identify physiological functions of genes and to elucidate evolutionary mechanisms.

Proteomic signatures and aberrations of mouse embryonic stem cells containing a single human chromosome 21 in neuronal differentiation: an in vitro model of Down syndrome.

Neurodegeneration in fetal development of Down syndrome (DS) patients is proposed to result in apparent neuropathological abnormalities and to contribute to the phenotypic characteristics of mental retardation and premature development of Alzheimer disease. In order to identify the aberrant and specific genes involved in the early differentiation of DS neurons, we have utilized an in vitro neuronal differentiation system of mouse ES cells containing a single human chromosome 21 (TT2F/hChr21) with TT2F parental ES cells as a control. The paired protein extracts from TT2F and TT2F/hChr21 cells at several stages of neuronal differentiation were subjected to two-dimensional polyacrylamide gel electrophoresis protein separation followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to identify the proteins differentially expressed between TT2F and TT2F/hChr21 cells. We provide here a novel set of specific gene products altered in early differentiating DS neuronal cells, which differs from that identified in adult or fetal brain with DS. The aberrant protein expression in early differentiating neurons, due to the hChr21 gene dosage effects or chromosomal imbalance, may affect neuronal outgrowth, proliferation and differentiation, producing developmental abnormalities in neural patterning, which eventually leads to formation of a suboptimal functioning neuronal network in DS.

DDBJ in the stream of various biological data.

In the past year we at DDBJ (http://www.ddbj.nig. ac.jp) have made a steady increase in the number of data submissions with a 50.6% increment in the number of bases or 46.5% increment in the number of entries. Among them the genome data of man, ascidian and rice hold the top three. Our activity has extended to providing a tool that enables sequence retrieval using regular expressions, and to launching our SOAP server and web services to facilitate the acquisition of proper data and tools from a huge number of biological data resources on websites worldwide. We have also opened our public gene expression database, CIBEX.

Phylogenetic analysis of Vietnamese isolates of feline immunodeficiency virus: genetic diversity of subtype C.

Phylogenetic relationships of novel Vietnamese strains of feline immunodeficiency virus (FIV) were analysed. One Vietnamese strain was found to cluster with subtype D, which was previously known only in Japan, while the other seven strains were placed with members of subtype C. Calculation of the relative numbers of mutations resulting in amino acid and silent changes in FIV env subtypes suggested that subtype C isolates may be less structurally constrained (potentially more pathogenic) than subtype B.

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA.

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.

Functional conservation of the promoter regions of vertebrate tyrosinase genes.

Tyrosinase is the key enzyme for synthesizing melanin pigments, which primarily determine mammalian skin coloration. Considering the important roles of pigments in the evolution and the adaptation of vertebrates, phylogenetic changes in the coding and flanking regulatory sequences of the tyrosinase gene are particularly intriguing. We have now cloned cDNA encoding tyrosinase from Japanese quail and snapping turtle. These nonmammalian cDNA are highly homologous to those of the mouse and human tyrosinases, whereas the 5' flanking sequences are far less conserved except for a few short sequence motifs. Nevertheless, we demonstrate that the 5' flanking sequences from the quail or turtle tyrosinase genes are capable of directing the expression of a fused mouse tyrosinase cDNA when introduced into cultured mouse albino melanocytes. This experimental method, which reveals the functional conservation of regulatory sequences in one cell type (the melanocyte), may be utilized to evaluate phylogenetic differences in mechanisms controlling specific gene expression in many other types of cells. We also provide evidence that the 5' flanking sequences from these nonmammalian genes are functional in vivo by producing transgenic mice. Phylogenetic changes of vertebrate tyrosinase promoters and the possible involvement of conserved sequence motifs in melanocyte-specific expression of tyrosinase are discussed.

Searching for the prototypic eye genetic network: Sine oculis is essential for eye regeneration in planarians.

We have identified a sine oculis gene in the planarian Girardia tigrina (Platyhelminthes; Turbellaria; Tricladida). The planarian sine oculis gene (Gtso) encodes a protein with a sine oculis (Six) domain and a homeodomain that shares significant sequence similarity with so proteins assigned to the Six-2 gene family. Gtso is expressed as a single transcript in both regenerating and fully developed eyes. Whole-mount in situ hybridization studies show exclusive expression in photoreceptor cells. Loss of function of Gtso by RNA interference during planarian regeneration inhibits eye regeneration completely. Gtso is also essential for maintenance of the differentiated state of photoreceptor cells. These results, combined with the previously demonstrated expression of Pax-6 in planarian eyes, suggest that the same basic gene regulatory circuit required for eye development in Drosophila and mouse is used in the prototypic eye spots of platyhelminthes and, therefore, is truly conserved during evolution.

Evolutionary aspects of gobioid fishes based upon a phylogenetic analysis of mitochondrial cytochrome B genes.

The Gobioidei is a large suborder in the order Perciformes and consists of more than 2000 species belonging to about 270 genera. The vast number of species and their morphological specialization adapted to diverse habits and habitats makes the classification of the gobioid fishes very difficult.A comprehensive estimation of the evolutionary scenario of all gobioid fishes using only morphological information is difficult for two major reasons: first, in addition to wide ecological diversification, there is a trend towards specialization and degeneration of morphological characters among these species; second, an appropriate outgroup of gobioid fishes has not been recognized. Based upon nucleotide sequence comparisons of gobioid mitochondrial cytochrome b genes, we established the phylogenetic relationships of their differentiation into many groups of morphological and ecological diversity. The phylogenetic trees obtained show that most species examined have diverged from each other almost simultaneously or during an extremely short period of time.

Pigment cell-specific expression of the tyrosinase gene in ascidians has a different regulatory mechanism from vertebrates.

Tyrosinase is the key enzyme required for the synthesis of melanin pigments. Sequence comparison and functional analysis of the 5' upstream regions of vertebrate tyrosinase genes have revealed the importance of conserved E-box motifs in regulating their specific expression in pigment cells, optic cup-derived retinal pigment epithelium (RPE) and neural crest-derived melanocytes. In ascidians (more basal protochordates), two pigment cells that resemble vertebrate RPE cells are formed and specifically express the orthologous tyrosinase gene (HrTyr) in the cerebral vesicle located at the anterior end of the neural tube. To define regulatory sequences required for pigment cell-lineage-specific expression of HrTyr during embryogenesis, a series of mutations of the 5' upstream region of HrTyr were fused to the lacZ reporter gene and were microinjected into fertilized eggs. We found that the -152bp upstream of the translational start site is essential for expression in pigment cell precursors of tailbud-stage embryos. Further, additional positive and unique restriction elements were identified in the region up to -1.8kb. Surprisingly, in the -152bp minimal promoter or in other regions with regulatory activities, there are no E-box motifs or sequences correlating with other conserved elements regulating vertebrate tyrosinase promoters. The possibility that Pax proteins regulate HrTyr expression is also discussed.

Pax 6: mastering eye morphogenesis and eye evolution.

Pax 6 genes from various animal phyla are capable of inducing ectopic eye development, indicating that Pax 6 is a master control gene for eye morphogenesis. It is proposed that the various eye-types found in metazoa are derived from a common prototype, monophyletically, by a mechanism called intercalary evolution.

Structure and developmental expression of the ascidian TRP gene: insights into the evolution of pigment cell-specific gene expression.

The tyrosinase family in vertebrates consists of three related melanogenic enzymes: tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2. These proteins control melanin production in pigment cells and play a crucial role in determining vertebrate coloration. We have isolated a gene from the ascidian Halocynthia roretzi which encodes a tyrosinase-related protein (HrTRP) with 45-49% identity with vertebrate TRP-1 and TRP-2. The expression of the HrTRP gene in pigment lineage a8.25 cells starts at the early-mid gastrula stage, which coincides with the stage when these cells are determined as pigment precursor cells; therefore, it provides the earliest pigment lineage-specific marker, which enables us to trace the complete cell lineage leading to two pigment cells in the larval brain. In addition, the expression pattern of the HrTRP gene appears to share similar characteristics with the mouse TRP-2 gene although structurally the HrTRP gene is more closely related to mammalian TRP-1 genes. Based on these observations and on results from molecular phylogenetic and hybridization analyses, we suggest that triplication of the tyrosinase family occurred during the early radiation of chordates. Initially, duplication of an ancestral tyrosinase gene produced a single TRP gene before the urochordate and cephalochordate-vertebrate divergence, and a subsequent duplication of the ancestral TRP gene in the vertebrate lineage gave rise to two TRP genes before the emergence of teleost fishes. Evolution of the melanin synthetic pathway and possible phylogenetic relationships among chordate pigment cells that accommodate the metabolic process are discussed. Dev Dyn 1999;215:225-237.

Neural network in planarian revealed by an antibody against planarian synaptotagmin homologue.

In order to investigate the neural connection of planarian, it is imperative to produce an antibody that specifically stains axons. To identify axon-specific genes, we constructed a cDNA library from a single eye by using a single cell PCR method, in which visual neurons are major components, and sequenced one thousand independent clones. We succeeded in the identification of a planarian homologue of synaptotagmin, Djsyt, whose specific expression in neurons was confirmed by in situ hybridization. The antibody against DjSYT specifically stained axons although its mRNA is distributed in the cell bodies. By using anti-DjSYT, we succeeded in the visualization of neural connections in planarians by whole mount staining. The anti-DjSYT antibody will become a powerful tool to analyze the molecular mechanisms underlying neural network formation in planarian.

Mutation patterns for two flaviviruses: hepatitis C virus and GB virus C/hepatitis G virus.

We studied the mutation patterns of hepatitis C virus (HCV) and GB virus C/hepatitis G virus (HGV). Although the mutation patterns of the two viruses were similar to each other, they were quite different from that of HIV. In particular, the similarity of the patterns between HCV or HGV and human nuclear pseudogenes was statistically significant whereas there was no similarity between HIV and human nuclear pseudogenes. This finding suggests that the mutation patterns of HCV and HGV are similar to the patterns of spontaneous substitution mutations of human genes, implying that nucleotide analogues which are effective against HCV and HGV may have a side effect on the normal cells of humans.