Takashi Yagi

Vicariant speciation due to 1.55 Ma isolation of the Ryukyu islands, Japan, based on geological and GenBank data

The Ryukyu island arc, originally a continental margin arc, separated from the Chinese continent by the rifting of the Okinawa trough, a process which began at 1.55 million years ago (Ma) and continues to the present. In addition, the Ryukyu arc was simultaneously divided into the northern Amami–Okinawa and southern Yaeyama islands by the Kerama rift valley, and consequently formed two isolated island units. The Kuroshio warm current began to flow into the Okinawa trough from the Yonaguni Strait, and flow out through the Tsushima and Tokara straits also at 1.55 Ma, and these seaways effectively acted as barriers between the Ryukyu islands and Taiwan, China and Japan. Through this geological process, vicariant speciation generated Ryukyu endemic animal species. We support this hypothesis by drawing linearized maximum likelihood (ML) phylogenetic trees of the species in four endemic insect groups (peacock butterfly, Chinese windmill butterfly, golden‐ringed dragonfly, window firefly) using GenBank sequence data. We determined the precise branching ages for these phylogenetic trees, and show simultaneous speciation at 1.55 Ma for Amami–Okinawa and Yaeyama units. The Taiwan and Tsushima straits, barriers between Taiwan and China, and Japan and Korea, respectively, did not form sufficient barriers to migration during glacial low stands, and species were intermingled. A marine embayment may have posed as a migration barrier between northern and southern China in the Quaternary or a little earlier. From our study we also estimate the precise molecular evolution rate and justify the molecular clock.

Development of yeast reporter assay for screening specific ligands of retinoic acid and retinoid X receptor subtypes

INTRODUCTION Retinoic acids are essential for embryonic development, tissue organization, and homeostasis and act via retinoic acid receptors (RARs) that form heterodimers with retinoid X receptors (RXRs). Human RARs and RXRs include the three subtypes α, β, and γ, which have varying distributions and physiological functions among human tissues. Recent reports show that subtype-specific binding of several chemicals to RARs or RXRs may lead to endocrine disruption. To evaluate these ligand-like chemicals, convenient assay systems for each receptor subtype are required. METHODS We developed reporter assay yeasts to screen ligands for RXR subtype receptor homodimers. To screen RAR ligands, yeasts were engineered to express RAR subtypes with defective RXRα, which fails to bind to coactivators because of its shortened c-terminus. RESULTS These assay yeasts were validated using known RXR- and RAR-specific ligands and subtype-specific responses were clearly shown. Subtype-specific ligand activities of the suspected chemical RAR or RXR ligands o-t-butylphenol, triphenyltin chloride, tributyltin chloride, and 4-nonylphenol were determined. DISCUSSION The present assay yeasts may be valuable tools for subtype-specific assessments of unidentified environmental ligand chemicals and receptor-specific pharmaceuticals.

Genotoxicity of formaldehyde: molecular basis of DNA damage and mutation

Formaldehyde is commonly used in the chemical industry and is present in the environment, such as vehicle emissions, some building materials, food and tobacco smoke. It also occurs as a natural product in most organisms, the sources of which include a number of metabolic processes. It causes various acute and chronic adverse effects in humans if they inhale its fumes. Among the chronic effects on human health, we summarize data on genotoxicity and carcinogenicity in this review, and we particularly focus on the molecular mechanisms involved in the formaldehyde mutagenesis. Formaldehyde mainly induces N-hydroxymethyl mono-adducts on guanine, adenine and cytosine, and N-methylene crosslinks between adjacent purines in DNA. These crosslinks are types of DNA damage potentially fatal for cell survival if they are not removed by the nucleotide excision repair pathway. In the previous studies, we showed evidence that formaldehyde causes intra-strand crosslinks between purines in DNA using a unique method (Matsuda et al. Nucleic Acids Res. 26, 1769-1774,1998). Using shuttle vector plasmids, we also showed that formaldehyde as well as acetaldehyde induces tandem base substitutions, mainly at 5’-GG and 5’-GA sequences, which would arise from the intra-strand crosslinks. These mutation features are different from those of other aldehydes such as crotonaldehyde, acrolein, glyoxal and methylglyoxal. These findings provide molecular clues to improve our understanding of the genotoxicity and carcinogenicity of formaldehyde.

Construction of sensitive reporter assay yeasts for comprehensive detection of ligand activities of human corticosteroid receptors through inactivation of CWP and PDR genes

INTRODUCTION The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are members of the nuclear receptor superfamily and ligand-dependent transcription factors, whose major ligands are glucocorticoid and mineralocorticoid, so-called corticosteroids. The corticosteroids are a class of substances that include steroid hormones naturally produced in the adrenal cortex of vertebrates and analogues of these hormones that are synthesized in industry. They are involved in a wide range of physiological processes including stress and immune responses, and the regulation of carbohydrate metabolism, protein catabolism, sodium homeostasis, and inflammation. These substances are potential environmental contaminants because they are clinically consumed in large amounts worldwide. To develop a simple and sensitive bioassay to detect corticosteroids, we newly established reporter assay yeasts expressing human GR and MR. METHODS Ligand responses of the established assay yeasts were improved by forced expression of a human transcription coactivator SRC-1e. Further enhancement of the responses was achieved by inactivating the CWP and PDR genes that encode cell wall mannoproteins and plasma membrane efflux pumps, respectively, which may be attributable to an increased intracellular concentration of ligands. RESULTS These new assay yeasts were more responsive to both natural and synthetic agonist ligands than the conventional assay yeasts. They detected both agonistic and antagonistic activities of mifepristone, spironolactone, and eplerenone in a receptor-selective manner. They also detected ligand activities contained in oral pharmaceutical tablets and human urine. DISCUSSION This assay system will be a valuable tool to detect agonists as well as antagonists of corticosteroid receptors, in the fields of drug discovery and the assessment of environmental pollutants.

A pilot study for construction of a new cadmium-sensing yeast strain carrying a reporter plasmid with the JLP1 promoter

Cadmium contamination still occurs in some parts of the world, and its concentrations in the environment are monitored in most countries due to its adverse effects on human health. We herein established yeast (Saccharomyces cerevisiae) reporter assay strains carrying plasmids with the yeast JLP1, SEO1, and CUP1 promoters connected to the bacterial lacZ reporter gene. The strain carrying the high-copy number pESC-JLP1-lacZ reporter plasmid was more responsive to cadmium than strains with other reporter plasmids. This JLP1-lacZ reporter assay strain will be useful for monitoring cadmium contamination in environmental water and soil as a first screening tool preceding official instrumental analyses, because the assay is rapid, easy to handle, and has the ability to process a large number of samples at a low cost.

Genetic variations and phylogeography of the swallowtail butterfly Papilio machaon on the Japanese Islands: Genetic variations of Papilio machaon

The swallowtail butterfly Papilio machaon Linnaeus, 1758 is widely distributed in the Holarctic region, including all of the main islands of Japan, as well as Sakhalin, and on other smaller islands south to Yakushima Island. The Japanese population is situated at the margin of the Eurasian distribution range of this species. It is morphologically different from other populations and has been classified as the subspecies hippocrates C. & R. Felder, 1864. The population of the Japanese Islands is considered to be genetically distinct from the continental populations in relation to the geographical history of the Japanese Islands. Therefore, we examined a part of the ND5 gene sequence of the mitochondrial DNA for P. machaon individuals of various localities in Japan and some nearby countries, and found 68 haplotypes in 400 individuals from the Japanese Islands and Sakhalin. A DNA polymorphism analysis revealed that the genetic structure of the Hokkaido population was significantly different from that of the southern populations on the main Japanese islands. These results imply that P. machaon expanded its range from the Amur region of Russia southward through Sakhalin to the Japanese Islands, and that the Tsugaru Strait between Hokkaido and Honshu may have subsequently limited their gene flow as a geographical barrier.

Development of yeast reporter assays for the enhanced detection of environmental ligands of thyroid hormone receptors α and β from Xenopus tropicalis

Thyroid hormones (THs) are involved in the regulation of metabolic homeostasis during the development and differentiation of vertebrates, particularly amphibian metamorphosis, which is entirely controlled by internal TH levels. Some artificial chemicals have been shown to exhibit TH-disrupting activities. In order to detect TH disruptors for amphibians, we herein developed a reporter assay using yeast strains expressing the thyroid hormone receptors (TRs) α and β together with the transcriptional coactivator SRC-1, all of which were derived from the frog Xenopus tropicalis (XT). These yeast strains responded to endogenous THs (T2, T3, and T4) in a dose-dependent manner. They detected the TR ligand activities of some artificial chemicals suspected to exhibit TH-disrupting activities, as well as TR ligand activity in river water collected downstream of sewage plant discharges, which may have originated from human excrement. Moreover, the responses of XT TR strains to these endogenous and artificial ligands were stronger than those of yeast strains for human TRα and β assays, which had previously been established in our laboratory. These results indicate that the yeast reporter assay system for XT TRα and β is valuable for assessing TR ligand activities in environmental samples that may be particularly potent in amphibians.

New reporter gene assays for detecting natural and synthetic molting hormone agonists using yeasts expressing ecdysone receptors of various insects

Synthetic nonsteroidal ecdysone agonists, a class of insect growth regulators (IGRs), target the ecdysone receptor (EcR), which forms a heterodimer with ultraspiracle (USP) to transactivate ecdysone response genes. These compounds have high binding affinities to the EcR–USP complexes of certain insects and their toxicity is selective for certain taxonomic orders. In the present study, we developed reporter gene assay (RGA) systems to detect molting hormone (ecdysone) activity by introducing EcR–USP cDNA and a bacterial lacZ reporter gene into yeast. EcR and USP were derived from the insect species of three different taxonomic orders: Drosophila melanogaster (Diptera), Chilo suppressalis (Lepidoptera), and Leptinotarsa decemlineata (Coleoptera). Transcriptional coactivator taiman (Tai) cDNA cloned from D. melanogaster was also used in this RGA system. This yeast RGA system responded to various EcR ligands in a dose‐dependent and ecdysteroid‐specific manner. Furthermore, the insect order‐selective ligand activities of synthetic nonsteroidal ecdysone agonists were linearly related to their binding activities, which were measured against in vitro translated EcR–USP complexes. Our newly established yeast RGA is useful for screening new molting hormone agonists that work selectively on target insects.