Vertebrates originally possess four functional subtypes of G protein-coupled melatonin receptor.

Melatonin receptors (MTNRs) belonging to the G protein-coupled receptor family are considered to consist of three subtypes in vertebrates: MTNR1a, MTNR1b and MTNR1c. Additionally, MTNR1a-like genes have been identified in teleostean species as a fish-specific subtype of MTNR1a. However, similar molecules to this MTNR1a-like gene can be found in some reptiles upon searching the DNA database. We hypothesized that a vertebrate can essentially have four functional subtypes of MTNR as ohnologs. Thus, in the present study we examined the molecular phylogeny, expression patterns and pharmacological profile(s) using the teleost medaka (Oryzias latipes). The four conserved subtypes of MTNR (MTNR1a, MTNR1b, MTNR1c and MTNR1a-like) in vertebrates were classified based on synteny and phylogenetic analysis. The fourth MTNR, termed MTNR1a-like, could be classified as MTNR1d. It was observed by using RT-qPCR that expression patterns differed amongst these subtypes. Moreover, mtnr1a, mtnr1c and mtnr1a-like/mtnr1d expression was elevated during short days compared to long days in diencephalons. All the subtypes were activated by melatonin and transduced signals into the Gi pathway, to perform a cAMP-responsive reporter gene assay. It was shown that MTNR originally consisted of four subtypes: MTNR1a, MTNR1b, MTNR1c and MTNR1d. These subtypes were functional, at least in fish, although some organisms, including mammals, have lost one or two subtypes.

A fourth subtype of retinoic acid receptor-related orphan receptors is activated by oxidized all-trans retinoic acid in medaka (Oryzias latipes).

BACKGROUND: The three known subtypes of the retinoic acid receptor-related orphan receptor (ROR) have been implicated in the control of immunity, brain function, and circadian rhythm in mammals. Here, we demonstrate by phylogenetic analysis that there were originally four subtypes of RORs in vertebrates. One of the novel ror paralogs, rord1 (rorca in the Ensembl database), is conserved among teleosts, but absent in mammals. Using medaka (Oryzias latipes) as a model teleost, we evaluated the expression pattern of this gene, its transactivational properties for endogenic chemicals, and its ability to activate the promoters of putative target genes. RESULTS: In eyes, the transcript of rord1 was expressed at higher levels during the day than at night. Interestingly, cholesterol derivatives, which are well-known ligands for mammalian RORs, did not efficiently promote transcriptional activity via RORd1. Thus we sought to identify the ligands that regulate the transcriptional activity of RORd1 using a luciferase reporter cell-based screening system. Using this system, we identified two metabolites of all-trans retinoic acid (ATRA), 4OH-ATRA and 4-keto ATRA, as potential ligands of RORd1. Moreover, RORd1 activated the promoter of cyp26a1 in a 4OH-ATRA -dependent manner. CONCLUSIONS: A novel ror subtype, rord has two paralogs, rord1 and rord2, in teleost. Rord1 mRNA is highly abundant in the eyes of medaka during light periods, suggesting that rord1 expression is involved in the regulation of circadian rhythm. We identified two ATRA metabolites, 4OH-ATRA and 4 K-ATRA, as endogenous candidate ligands of RORd1. We also show that 4-oxygenated ATRA metabolites have the potential to activate cyp26a1, the metabolic enzyme of ATRA. Our results support the notion that RORd1 is involved in the metabolism of ATRA in medaka.

Chlorophyll-Based Organic-Inorganic Heterojunction Solar Cells.

Solid-state chlorophyll solar cells (CSCs) employing a carboxylated chlorophyll derivative, methyl trans-32 -carboxypyropheophorbide a, as a light-harvesting dye sensitizer chlorophyll (DSC) deposited on mesoporous TiO2 , on which four zinc hydroxylated chlorophyll derivatives were spin-coated for hole transporter chlorophylls (HTCs), are described. Key parameters, including the effective carrier mobility of the HTC films, as determined by the space charge-limited current method, and the frontier molecular orbitals of these DSCs and HTCs, as estimated from cyclic voltammetry and electronic absorption spectra, suggest that both charge separation and carrier transport are favorable. The power conversion efficiencies (PCEs) of the present CSCs with fluorine-doped tin oxide (FTO)/TiO2 /DSC/HTCs/Ag were determined to follow the order of HTC-1>HTC-2>HTC-3>HTC-4, which coincided perfectly with the order of their hole mobilities. The maximum PCE achieved was 0.86 % with HTC-1. The photovoltaic devices studied herein with two types of chlorophyll derivatives as dye sensitizers and hole transporters provide a unique solution for the utilization of solar energy with a view to truly realizing "green energy".

Principal function of mineralocorticoid signaling suggested by constitutive knockout of the mineralocorticoid receptor in medaka fish.

As in osmoregulation, mineralocorticoid signaling is implicated in the control of brain-behavior actions. Nevertheless, the understanding of this role is limited, partly due to the mortality of mineralocorticoid receptor (MR)-knockout (KO) mice due to impaired Na+ reabsorption. In teleost fish, a distinct mineralocorticoid system has only been identified recently. Here, we generated a constitutive MR-KO medaka as the first adult-viable MR-KO animal, since MR expression is modest in osmoregulatory organs but high in the brain of adult medaka as for most teleosts. Hyper- and hypo-osmoregulation were normal in MR-KO medaka. When we studied the behavioral phenotypes based on the central MR localization, however, MR-KO medaka failed to track moving dots despite having an increase in acceleration of swimming. These findings reinforce previous results showing a minor role for mineralocorticoid signaling in fish osmoregulation, and provide the first convincing evidence that MR is required for normal locomotor activity in response to visual motion stimuli, but not for the recognition of these stimuli per se. We suggest that MR potentially integrates brain-behavioral and visual responses, which might be a conserved function of mineralocorticoid signaling through vertebrates. Importantly, this fish model allows for the possible identification of novel aspects of mineralocorticoid signaling.

High levels of plasma cortisol and impaired hypoosmoregulation in a mutant medaka deficient in P450c17I.

scl is a spontaneous medaka mutant deficient in P450c17I, which is required for production of sex steroids, but not of cortisol, the major role of which is osmoregulation in teleost fish. The scl mutant provides a new model to study the functions of these hormones. We first found that fish homozygous for this mutation have plasma cortisol constitutively at a high physiological level (1000 nM). Since we previously showed that this level reversed the seawater-type differentiation of the medaka gastrointestinal tract, hypoosmoregulation of the scl mutant was analyzed. Muscle water contents in freshwater were normal in scl homozygotes, but the contents were lower than those of the wild type (WT) after seawater transfer. There were no differences in gill mRNA levels of corticosteroid receptors or ion transporters between scl homozygotes and WT. In the intestine, expression of glucocorticoid receptors and Na(+)/K(+)/2Cl(-) cotransporter were induced in WT during seawater acclimation, but not in scl homozygotes. The high plasma cortisol may prevent hypoosmoregulation by inhibition of increased intestinal water absorption, essentially by the Na(+)/K(+)/2Cl(-) cotransporter, in seawater.

Stable reporter gene assay based on Gal4-vitamin D receptor ß fusion proteins in medaka (Oryzias latipes), and its transactivational properties.

The transactivational property of natural and synthetic chemicals via medaka vitamin D receptor ß subtype (VDRß) was investigated after the development of a stable cell line expressing a Gal4-VDRß fusion protein for reporter gene assay. Members of vitamin D class, including 1α, 25- dihydroxyvitamin D3 (1,25VD3) were specifically detected as agonists in our system. Although other steroids and chemicals used in the present estimation induced no agonistic response, 10 compounds displayed antagonistic or synergistic activity. Spironolactone, which is an antagonist of corticoid receptors in mammals, competitively inhibited the transactivity of 1,25VD3 by over 80% in a dose dependent manner. Mifepristone and cyproterone acetate were also detected as antagonists, but they significantly acted only at 10µ. Pregnenolone and raloxifene dose-dependently enhanced the activity of 1,25VD3 at EC50 to the maximum level. Diethylstilbestrol, 17α-ethynylestradiol, genistein, and stanozolol were also synergists, but their potency was low. Interestingly, dibutyltin dichloride, which is used as a stabilizer in the production of polyvinyl chloride plastics, produced greater response than maximum effect of 1,25VD3 although the concentration-response curve was not typically sigmoidal. In the present study, we successfully developed a stable reporter gene assay, which allows assessment of the vitamin D-like chemicals toward the medaka VDRß.

Response to fish specific reproductive hormones and endocrine disrupting chemicals of a Sertoli cell line expressing endogenous receptors from an endemic cyprinid Gnathopogon caerulescens.

Fish Sertoli cells play a critical role in spermatogenesis by mediating androgen and progestogen signaling. Their hormonal response, however, considerably differ among species. Therefore it would be ideal to use Sertoli cells originated from the fish of interest to investigate the effects of hormones as well as endocrine disrupting chemicals (EDCs). The aim of this study was to investigate the responses to reproductive hormones and EDCs of a Sertoli cell line that we established from an endemic cyprinid Gnathopogon caerulescens. As the Sertoli cell line expressed endogenous androgen and progestogen receptors, we were able to detect hormone responses by transfecting only a reporter vector (pGL4.36) expressing luciferase under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter into the cell line. Unlike previous reporter gene assays using fish steroid hormone receptors expressed in mammalian cell lines, luciferase activities were induced by the fish specific androgen (11-ketotestosterone) and progestogen (17α,20ß-dihydroxy-4-pregnen-3-one), but not by testosterone and progesterone, at physiologically relevant concentrations. Furthermore, we found 4-nonylphenol (NP) but not bisphenol A showed strong anti-androgenic effects, implying that NP may have direct anti-androgenic effects on fish Sertoli cells in vivo. This is the first evidence, to the best of our knowledge, of anti-androgenic effects of NP in a fish Sertoli cell line. In addition, neither NP nor BPA showed anti-progestogenic effects. These results suggest that the Sertoli cell line established from the fish of interest can be a useful in vitro tool for investigating the mechanisms of reproductive hormones and EDCs in the specific fish.

Petroacetylene, a new polyacetylene from the marine sponge Petrosiasolida that inhibits blastulation of starfish embryos.

A new C30 linear polyacetylene compound designated petroacetylene (1) has been isolated from the marine sponge Petrosia solida Hoshino 1981, collected off the coast of Amami-Oshima, Kagoshima Prefecture, Japan. The structure was elucidated on the basis of spectroscopic data and chemical means. Petroacetylene (1) inhibited blastulation of starfish embryos at a concentration of 3.1 µg mL(- 1) or greater.

Bromotheoynic acid, a brominated acetylenic acid from the marine sponge Theonella swinhoei.

A new brominated C(17) acetylenic acid (1) designated as bromotheoynic acid has been isolated from the marine sponge Theonella swinhoei, collected off the coast of Tanegashima, Kagoshima Prefecture, Japan. The structure was determined on the basis of the analysis of its extensive 2D NMR spectroscopic data as well as HRMS. Bromotheoynic acid (1) inhibited maturation of starfish oocytes and cell division of fertilised starfish eggs. Bromotheoynic acid (1) also inhibited proliferation of human leukaemia U937 and HL60 cells, human lung cancer A549 and H1299 cells, and human embryonic kidney 293 (HEK293) cells.

Growth, energetics and the cortisol-hepatic glucocorticoid receptor axis of medaka (Oryzias latipes) in various salinities.

We examined growth of euryhaline Japanese medaka (Oryzias latipes) after transfer to freshwater or seawater from isotonic saline. Growth was unaffected by the different salinities for 1 week, but the body weight increase and BMI of fish kept in freshwater for 2-3 weeks were significantly higher than those in the isotonic controls. These results may reflect the usual habitat of this species. To assess the basis for the difference in growth, energetics and the hepatic stress axis were evaluated 1 week after the transfer. Unexpectedly, despite the higher growth rate, the rate of routine oxygen consumption was significantly higher in freshwater. Plasma cortisol levels in freshwater were significantly higher than those in seawater, and the mRNA levels of the glucocorticoid receptor (GR1) in the liver were significantly lower in freshwater and seawater, compared to that in isotonic saline. Branchial Na(+)/K(+)-ATPase activities were also reduced significantly in freshwater and seawater, compared to that in isotonic saline. The higher levels of hepatic GR1 expression and branchial Na(+)/K(+)-ATPase activity in isotonic salinity than those in freshwater and seawater for 1 week may account for the lower growth rate under the isotonic condition. After 3 weeks, however, the Na(+)/K(+)-ATPase activity in seawater was significantly higher than that in freshwater. No significant difference in growth rate between freshwater and seawater groups indicates that medaka is a good model for studies of hypo- and hyperosmotic adaptations, since osmoregulation is not strongly associated with size and growth.

Bioactive jatrophane diterpenes from Euphorbia guyoniana.

Chromatographic investigation of the methylenechloride/methanol extract of the aerial parts of Euphorbia guyoniana afforded two jatrophane diterpenes, designated guyonianins E and F, in addition to a known jatrophane diterpene. The structures of the compounds were determined by comprehensive NMR analyses, including DEPT, COSY, HMQC, HMBC, NOESY and HRMS. These compounds exhibited cytotoxicity against human embryonic kidney 293 (HEK293) cells with IC(50) values of 35-100 microM.

Molecular cloning and characterization of estrogen, androgen, and progesterone nuclear receptors from a freshwater turtle (Pseudemys nelsoni).

Steroid hormones are essential for the normal function of many organ systems in vertebrates. Reproductive activity in females and males, such as the differentiation, growth, and maintenance of the reproductive system, requires signaling by the sex steroids. Although extensively studied in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens, androgens, and progestins) action are poorly understood in reptiles. Here we evaluate hormone receptor ligand interactions in a freshwater turtle, the red-belly slider (Pseudemys nelsoni), after the isolation of cDNAs encoding an estrogen receptor alpha (ERalpha), an androgen receptor (AR), and a progesterone receptor (PR). The full-length red-belly slider turtle (t)ERalpha, tAR, and tPR cDNAs were obtained using 5' and 3' rapid amplification cDNA ends. The deduced amino acid sequences showed high identity to the chicken orthologs (tERalpha, 90%; tAR, 71%; tPR, 71%). Using transient transfection assays of mammalian cells, tERalpha protein displayed estrogen-dependent activation of transcription from an estrogen-responsive element-containing promoter. The other receptor proteins, tAR and tPR, also displayed androgen- or progestin-dependent activation of transcription from androgen- and progestin-responsive murine mammary tumor virus promoters. We further examined the transactivation of tERalpha, tAR and tPR by ligands using a modified GAL4-transactivation system. We found that the GAL4-transactivation system was not suitable for the measurement of tAR and tPR transactivations. This is the first report of the full coding regions of a reptilian AR and PR and the examination of their transactivation by steroid hormones.

Expression of DMY and DMRT1 in various tissues of the medaka (Oryzias latipes) [corrected].

Two DM-domain genes, DMY (sex-determining gene) and DMRT1, have been reported to be expressed in the testis of medaka. In this study, a specific RT-PCR assay was used to determine the expression patterns of DMY and DMRT1 in various tissues of medaka during different stages of development. The results show that the transcripts of both DMY and DMRT1 are present not only in testes but also in several other tissues from fry and adults of medaka.

A novel progestogen receptor subtype in the Japanese eel, Anguilla japonica.

A cDNA encoding a second type of a progestogen receptor (ePR2) was isolated from the same library as we had previously cloned a functional PR (ePR1) in eel testis. The amino acid sequence of the ePR2 shows low homology with ePR1 (34%), but both PRs showed progestogen-dependent transactivation in transfection experiment. Tissue distribution of ePR2 mRNA was clearly different from that of ePR1. Protein interaction between two PRs was demonstrated in vitro by a glutathione S-transferase pull-down assay. These results indicate that ePR2 is also a functional PR. This is the first isolation of two different functional PR molecules from a vertebrate.