Biomedical subjects

Hajime Watanabe

Publications and source records attributed to Hajime Watanabe.

At least 19 recordsLinked to original sources

PubMed2006

Ontogenic expression of estrogen receptor-alpha in female rat corneas.

PURPOSE: To investigate ontogenic expression of estrogen receptor (ER)-alpha in female rat corneas, as part of basic studies to elucidate the mechanism of estrogenic effects on the corneas. METHODS: The expression and localization of ERalpha were determined using quantitative reverse transcribed-polymerase chain reaction methodology and immunohistochemistry in the corneas of female rats on day 14 of gestation and postnatal days (PNDs) 0, 21, and 60. RESULTS: Quantitative analysis of ERalpha mRNA revealed that ERalpha gene expression increased approximately 4 times on PND 21 and about 10 times on PND 60, as compared with expression levels detected on PND 0. Immunohistochemical analysis revealed that expression of ERalpha protein was evident only in nuclei of the corneal epithelial cells from PND 21 onward. CONCLUSION: Ontogenic expression of ERalpha occurred in female rat corneas.

Animals↗

PubMed2006

Porphyrin accumulation in mitochondria is mediated by 2-oxoglutarate carrier.

Heme (Fe-protoporphyrin IX), an endogenous porphyrin derivative, is an essential molecule in living aerobic organisms and plays a role in a variety of physiological processes such as oxygen transport, respiration, and signal transduction. For the biosynthesis of heme or the mitochondrial heme proteins, heme or its biosynthetic precursor porphyrin must be transported into mitochondria from cytosol. The mechanism of porphyrin accumulation in the mitochondrial inner membrane is unclear. In the present study, we analyzed the mechanism of mitochondrial translocation of porphyrin derivatives. We showed that palladium meso-tetra(4-carboxyphenyl)porphyrin (PdTCPP), a phosphorescent porphyrin derivative, accumulated in the mitochondria of several cell lines. Using affinity latex beads, we showed that 2-oxoglutarate carrier (OGC), the mitochondrial transporter of 2-oxoglutarate, bound to PdTCPP, and in vitro PdTCPP inhibited 2-oxoglutarate uptake into mitochondria in a competitive manner (Ki = 15 microM). Interestingly, all types of porphyrin derivatives examined in this study competitively inhibited 2-oxoglutarate uptake into mitochondria, including protoporphyrin IX, coproporphyrin III, and hemin. Furthermore, mitochondrial accumulation of porphyrins was inhibited by 2-oxoglutarate or OGC inhibitor. These results suggested that porphyrin accumulation in mitochondria is mediated by OGC and that porphyrins are able to competitively inhibit 2-oxoglutarate uptake into mitochondria. This is the first report of a putative mechanism for accumulation of porphyrins in the mitochondrial inner membrane.

Amino Acid Sequence↗

PubMed2006

Endocrine-disrupting organotin compounds are potent inducers of adipogenesis in vertebrates.

Dietary and xenobiotic compounds can disrupt endocrine signaling, particularly of steroid receptors and sexual differentiation. Evidence is also mounting that implicates environmental agents in the growing epidemic of obesity. Despite a long-standing interest in such compounds, their identity has remained elusive. Here we show that the persistent and ubiquitous environmental contaminant, tributyltin chloride (TBT), induces the differentiation of adipocytes in vitro and increases adipose mass in vivo. TBT is a dual, nanomolar affinity ligand for both the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor gamma (PPARgamma). TBT promotes adipogenesis in the murine 3T3-L1 cell model and perturbs key regulators of adipogenesis and lipogenic pathways in vivo. Moreover, in utero exposure to TBT leads to strikingly elevated lipid accumulation in adipose depots, liver, and testis of neonate mice and results in increased epididymal adipose mass in adults. In the amphibian Xenopus laevis, ectopic adipocytes form in and around gonadal tissues after organotin, RXR, or PPARgamma ligand exposure. TBT represents, to our knowledge, the first example of an environmental endocrine disrupter that promotes adipogenesis through RXR and PPARgamma activation. Developmental or chronic lifetime exposure to organotins may therefore act as a chemical stressor for obesity and related disorders.

Adipogenesis↗

PubMed2006

TFII-I down-regulates a subset of estrogen-responsive genes through its interaction with an initiator element and estrogen receptor alpha.

TFII-I was initially identified as the general transcription factor that binds to initiator (Inr) elements in vitro. Subsequent studies have shown that TFII-I activates transcription of various genes either through Inr elements or through other upstream elements in vivo. Since, however, most studies so far on TFII-I have been limited to over-expression and reporter gene assays, we reevaluated the role of TFII-I in vivo by using stable knockdown with siRNA and by examining the expression of endogenous genes. Contrary to the widely accepted view, here we show that TFII-I is not important for cell viability in general but rather inhibits the growth of MCF-7 human breast cancer cells. MCF-7 cells are known to proliferate in an estrogen-dependent manner. Through analysis of TFII-I's cell-type specific growth inhibitory effect, we show evidence that TFII-I down-regulates a subset of estrogen-responsive genes, only those containing Inr elements, by recruiting estrogen receptor (ER) alpha and corepressors to these promoters. Thus, this study has revealed an unexpected new role of TFII-I as a negative regulator of transcription and cell proliferation.

Cell Line, Tumor↗

PubMed2006

Chromatin immunoprecipitation-mediated target identification proved aquaporin 5 is regulated directly by estrogen in the uterus.

Estrogens play a central role in the reproduction of vertebrates and affect a variety of biological processes. The major target molecules of estrogens are nuclear estrogen receptors (ERs), which have been studied extensively at the molecular level. In contrast, our knowledge of the genes that are regulated directly by ERs remains limited, especially at the level of the whole organism rather than cultured cells. In order to identify genes that are regulated directly by ERs in vivo, we used estrogen treated mouse uterus and performed chromatin immunoprecipitation. Sequence analysis of a precipitated DNA fragment enabled alignment with the mouse genomic sequence and revealed that the promoter region of the gene encoding aquaporin 5 (AQP5) was precipitated with antibody against ER alpha. Quantitative PCR and DNA microarray analyses confirmed that AQP5 is activated soon after administration of estrogen. In addition, the promoter region of AQP5 contained a functional estrogen response element that was activated directly by estrogen. Although several AQP genes are expressed in the uterus, only direct activation of AQP5 could be detected following treatment with estrogen. This chromatin immunoprecipitation-mediated target identification may be applicable to the study of other transcription factor networks.

Animals↗

PubMed2006

Global gene expression in mouse vaginae exposed to diethylstilbestrol at different ages.

Estrogens regulate proliferation and differentiation of cells in target organs such as the female reproductive tract. In mature mice, estrogens stimulate cell proliferation, whereas ovariectomy results in atrophy of the female reproductive tract. In contrast, perinatal exposure to estrogens, including diethylstilbestrol (DES), induces persistent, ovary-independent vaginal stratification and cervico-vaginal tumors later in life. These effects are due to altered cell fate following DES exposure during a critical developmental period. The detailed mechanisms underlying the reversible and irreversible cell proliferation in vaginae induced by DES at different ages has not been clarified. Therefore, we examined differences in gene expression pattern using DNA microarray analysis in mouse vaginae 6 hrs after a single injection of 2 mug DES per gram of body weight, and proliferation of vaginal epithelial and stromal cells 24 hrs after the injection at postnatal days (PNDs) 0, 5, 20, and 70. After DES stimulation, vaginal epithelial and stromal cells showed cell proliferation at PNDs 20 and 70, and at PNDs 0 and 5, respectively. DNA microarray analysis exhibited 54 DES-induced genes and 9 DES-repressed genes in vaginae at PND 0, whereas more than 200 DES-induced genes were found in vaginae at PNDs 5 and 20, and 350 genes at PND 70. Clustering analysis of DES-induced genes in the vaginae at different ages revealed that genes induced by DES at PND 5 were closer to the adult type than that of PND 0. Genes related to keratinocyte differentiation, such as Gadd45alpha, p21, 14-3-3 sigma, small proline-rich protein 2f (Sprr2f), and Krupple-like factor 4 (Klf4), were induced by DES. The number of DES-induced genes during the critical period, PND 0, was smaller than those found after the critical period. These results give insight toward understanding the molecular mechanisms underlying the critical period in mouse vaginae.

Animals↗

PubMed2006

Application of ecotoxicogenomics for studying endocrine disruption in vertebrates and invertebrates.

Chemicals released into the environment potentially disrupt the endocrine system in wild animals and humans. Developing organisms are particularly sensitive to estrogenic chemicals. Exposure to estrogens or estrogenic chemicals during critical periods of development induces persistent changes in both reproductive and nonreproductive organs, including persistent molecular alterations. Estrogen-responsive genes and critical developmental windows of various animal species, therefore, need to be identified for investigators to understand the molecular basis of estrogenic activity during embryonic development. For investigators to understand molecular mechanisms of toxicity in various species, toxicogenomics/ecotoxicogenomics, defined as the integration of genomics (transcriptomics, proteomics, metabolomics) into toxicology and ecotoxicology, need to be established as powerful tools for research. As the initial step toward using genomics to examine endocrine-disrupting chemicals, estrogen receptors and other steroid hormone receptors have been cloned in various species, including reptiles, amphibians, and fish, and alterations in the expression of these genes in response to chemicals were investigated. We are identifying estrogen-responsive genes in mouse reproductive tracts using cDNA microarrays and trying to establish microarray systems in the American alligator, roach, medaka, and water fleas (Daphnia magna). It is too early to define common estrogen-responsive genes in various animal species; however, toxicogenomics and ectotoxicogenomics provide powerful tools to help us understand the molecular mechanism of chemical toxicities in various animal species.

Alligators and Crocodiles↗

PubMed2005

Effects of bisphenol A given neonatally on reproductive functions of male rats.

Male Sprague-Dawley rats (Crj:CD (IGS) were treated neonatally with bisphenol A (BPA) to evaluate effects on reproductive parameters. Animals were given BPA subcutaneously in corn oil to dosages of 0.002-97 mg/kg body weight, or 0.9 mg/kg 17beta-estradiol (E2) once a day from postnatal day (PND) 0 to PND 9. Preputial separation, copulatory rate, fertility rate, sperm analysis, serum testosterone levels, and gene expression in the testis were assessed. Males in the E2 group showed a decrease in testis weight and alterations of estrogen-mediated gene expression in the testis on PND 10, and by PND 150 incomplete preputial separation, decreases in the copulatory rate, testicular and accessory organ weights and number of sperm. In contrast, males in all BPA groups showed normal reproductive parameters. These results indicate that in male rats, BPA given during the neonatal period neither affected reproductive function nor evoked estrogen-mediated gene responses in the testis.

Animals↗

PubMed2005

Genetic differences in the production of male neonates in Daphnia magna exposed to juvenile hormone analogs.

We studied the susceptibility of three genetically different strains of the cyclical parthenogen Daphnia magna (Cladocera, Crustacea) in producing male neonates following exposure to juvenile hormone analogs. In experiment 1, NIES, Clone A, and Belgium A strains were exposed to the insect growth regulators (IGRs) fenoxycarb or epofenonane in a 21-day reproduction experiment. Fenoxycarb exposure decreased the total number of neonates and increased production of male neonates in a concentration-dependent manner in the NIES strain. The decrease in the total number of neonates was so great in Clone A following fenoxycarb exposure that male neonates were not observed, even at the highest concentration, where the total number of neonates was only 2% of the control. In the Belgium A strain, male neonates were observed at a rate of about 20% following exposure to the highest fenoxycarb concentration, but the total number observed was small. Epofenonane did not decrease reproduction in the NIES and Belgium A strains as dramatically as did fenoxycarb, but the neonatal sex ratio changed in a concentration-dependent manner. Although the ratio of males was as low as about 10%, induction of male neonates was also observed in Clone A following epofenonane exposure. In experiment 2, gravid females were exposed to high concentrations (5 or 10 microg/l) of fenoxycarb or pyriproxyfen for 12h. These treatments induced the production of male neonates in all strains, with a small decrease in the total number of neonates. Although induction of male neonates by juvenile hormones and their analogs was universal among genetically different strains, care is needed in interpreting the results of the 21-day reproduction tests, because decreased numbers of neonates at higher concentrations could obscure the presence of male neonates.

Animals↗

PubMed2005

Identification of genes whose expressions are enhanced or reduced in baker's yeast during fed-batch culture process using molasses medium by DNA microarray analysis.

Genes whose expression levels are enhanced or reduced during the cultivation process that uses cane molasses in baker's yeast production were identified in this study. The results showed that baker's yeast grown in molasses medium had higher fermentation ability and stress tolerance compared with baker's yeast grown in synthetic medium. Molasses apparently provided not only sugar as a carbon source but also provided functional components that enhanced or reduced expression of genes involved in fermentation ability and stress tolerance. To identify the genes whose expression is enhanced or reduced during cultivation in molasses medium, DNA microarray analysis was then used to compare the gene expression profile of cells grown in molasses with that of cells grown in synthetic medium. To simulate the commercial baker's yeast production process, cells were cultivated using a fed-batch culture system. In molasses medium, genes involved in the synthesis or uptake of vitamins (e.g., biotin, pyridoxine and thiamine) showed enhanced expression, suggesting that vitamin concentrations in molasses medium were lower than those in synthetic medium. Genes involved in formate dehydrogenase and maltose assimilation showed enhanced expression in molasses medium. In contrast, genes involved in iron utilization (e.g., siderophore, iron transporter and ferroxidase) showed enhanced expression in synthetic medium, suggesting that iron starvation occurred. The genes involved in the metabolism of amino acids also showed enhanced expression in synthetic medium. This identification of genes provides information that will help improve the baker's yeast production process.

Culture Media↗

PubMed2005

HEXIM1 forms a transcriptionally abortive complex with glucocorticoid receptor without involving 7SK RNA and positive transcription elongation factor b.

The HEXIM1 protein has been shown to form a protein-RNA complex composed of 7SK small nuclear RNA and positive transcription elongation factor b (P-TEFb), which is composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1, and to inhibit the kinase activity of CDK9, thereby suppressing RNA polymerase II-dependent transcriptional elongation. Here, we biochemically demonstrate that HEXIM1 forms a distinct complex with glucocorticoid receptor (GR) without RNA, CDK9, or cyclin T1. HEXIM1, through its arginine-rich nuclear localization signal, directly associates with the ligand-binding domain of GR. Introduction of HEXIM1 short interfering RNA and adenovirus-mediated exogenous expression of HEXIM1 positively and negatively modulated glucocorticoid-responsive gene activation, respectively. In the nucleus, HEXIM1 was shown to localize in a distinct compartment from that of the p160 coactivator transcriptional intermediary factor 2. Overexpression of HEXIM1 decreased ligand-dependent association between GR and transcriptional intermediary factor 2. Antisense-mediated disruption of 7SK blunted the negative effect of HEXIM1 on arylhydrocarbon receptor-dependent transcription but not on GR-mediated one, indicating that a class of transcription factors are direct targets of HEXIM1. These results indicate that HEXIM1 has dual roles in transcriptional regulation: inhibition of transcriptional elongation dependent on 7SK RNA and positive transcription elongation factor b and interference with the sequence-specific transcription factor GR via a direct protein-protein interaction. Moreover, the fact that the central nuclear localization signal of HEXIM1 is essential for both of these actions may argue the crosstalk of these functions.

Animals↗

PubMed2005

Production of male neonates in Daphnia magna (Cladocera, Crustacea) exposed to juvenile hormones and their analogs.

We exposed the water flea Daphnia magna (Cladocera, Crustacea) to either juvenile hormone I (JH I), juvenile hormone II (JH II), or the juvenile hormone-mimicking insecticides kinoprene, hydroprene, epofenonane, or fenoxycarb. By 21-day reproduction tests, we investigated the effects on the number of neonates born per female and the offspring sex ratio. All six chemicals induced D. magna to produce male neonates; the male sex ratio of the offspring increased as the chemical concentration increased. EC50 values for production of male neonates were estimated as 400 (JH I), 410 (JH II), 190 (kinoprene), 2.9 (hydroprene), 64 (epofenonane), and 0.92 (fenoxycarb) microg/l. The number of neonates produced was reduced with all chemicals at the concentrations investigated. At the EC50 for male production, five of the six chemicals reduced the reproductive rate to less than 50%; the exception was epofenonane, which caused only a slight reduction in reproductive rate. These results were similar to those obtained for five juvenoids studied previously, one of which was studied here again. There are now 10 chemical substances--all juvenile hormones or their analogs-that are known to induce D. magna to produce male neonates. This suggests that juvenile hormone is involved in initiating male production followed by sexual reproduction in D. magna, and probably in most cladocerans that exhibit cyclic parthenogenesis.

Animals↗

PubMed2005

Effects of an androgenic growth promoter 17beta-trenbolone on masculinization of Mosquitofish (Gambusia affinis affinis).

Endocrine disrupting chemicals can affect normal hormone dependent processes through numerous mechanisms, including ligand mimicky. 17beta-Trenbolone (TB), a pharmaceutical, androgenic, anabolic steroid, is a potent agonist of androgen receptors, and has been extensively used as a growth promoter for beef cattle in the US. The effects of TB on adult and newborn mosquitofish (Gambusia affinis affinis) were examined. Two forms of mosquitofish androgen receptor (AR), ARalpha and ARbeta, were cloned. The mRNA expression levels of ARalpha and ARbeta were transiently increased in the anal fin of adult females at day 3 following exposure to TB (1-10 microg/L) or methyltestosterone (MT) (0.1-10 microg/L), a pharmaceutical androgen used as a positive control. Gonopodium differentiation from the adult female anal fin was induced after 28 days of exposure to TB (1-10 microg/L) or MT (0.1-10 microg/L). Gonopodium differentiation also was induced in all mosquitofish fry exposed for 28 days to 0.3, 1 or 10 microg/L TB. Furthermore, spermatozoa were observed histologically in the testes of male fry exposed for 28 days to 1 or 10 microg/L TB; spermatozoa are normally observed only in the testes of mature males. Surprisingly, all female fry exposed for 28 days to 1 or 10 microg/L TB displayed the formation of an ovotestis, as spermatozoa were found in the ovary. Thus, TB, like MT, induced masculinization of the anal fin accompanied by a transient up-regulation of ARalpha and ARbeta in adult females. TB also induced differentiation of the anal fin into a gonopodium in fry of both sexes, stimulated precocious spermatogenesis in the testes of males and the formation of ovotestes in females.

Amino Acid Sequence↗

PubMed2005

Selective ligand purification using high-performance affinity beads.

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.

Animals↗

PubMed2005

Production of male neonates in four cladoceran species exposed to a juvenile hormone analog, fenoxycarb.

Previous studies have found that exposure of a cyclic parthenogen, the water flea Daphnia magna (Cladocera, Crustacea), to juvenile hormones and their analogs results in the production of neonates of male sex at concentration-dependent rates. We conducted reproduction experiments in four different species (Moina macrocopa, M. micrura, Ceriodaphnia dubia and C. reticulata) of cladoceran to test for the first time whether the occurrence of this phenomenon after exposure of the parent to such hormones is a generalized phenomenon. In the presence of a juvenile hormone analog, fenoxycarb, all four species produced male neonates and showed reduced rates of reproduction. The estimated median effective concentration (EC50) for the production of male neonates varied with species, ranging from 0.60 x 10(3) to 9.3 x 10(3) ng/l. Although there was a wide range of sensitivity to fenoxycarb, the production of male neonates in all four species demonstrates that this phenomenon is a common response to juvenile hormone analogs and further suggests that these hormones are capable of initiating sexual reproduction in cladocerans, most of which exhibit cyclic parthenogenesis.

Animals↗

PubMed2005

Analysis of expressed sequence tags of the water flea Daphnia magna.

To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.

Animals↗

PubMed2004

Effects of 17beta-estradiol, nonylphenol, and bisphenol-A on developing Xenopus laevis embryos.

Many chemicals released into the environment have the capacity to disrupt the normal development of aquatic animals. We investigated the influence of nonylphenol (NP), bisphenol-A (BPA), and 17beta-estradiol (E2) on developing Xenopus laevis embryos, as a model animal in the aquatic environment. Embryos were exposed to eight different concentrations of NP, BPA or E2 between 3 and 96 h post-fertilization (p.f.). Short body length, microcephaly, flexure, edema, and abnormal gut coiling were induced by 20 microM NP, BPA or 10 microM E2 by 96 h p.f. To clarify sensitive stages to these compounds, embryos were exposed to chemicals for 45 or 48 h starting at different developmental stages and experiments were terminated 96 h p.f. BPA and NP induced abnormalities in developing X. laevis, though the sensitive stages of embryos to these chemicals are different, BPA affecting earlier stages and NP affecting at later stages. To analyze the functional mechanisms of BPA and NP in induction of morphological changes, we adapted a DNA array technology and identified 6 X. laevis genes, XIRG, alpha skeletal tropomyosin, cyclin G1, HGF, troponin C2, and ribosomal protein L9. These findings may provide important clues to elucidate common mechanisms underlying teratogenic effects of these chemicals.

Animals↗

PubMed2004

Cloning of a cDNA encoding an ETR2-like protein (Os-ERL1) from deep water rice (Oryza sativa L.) and increase in its mRNA level by submergence, ethylene, and gibberellin treatments.

A cDNA from deep water rice treated with ethylene, encoding an ethylene receptor homologous to Arabidopsis thaliana ETR2 and EIN4, was isolated using differential display and RACE techniques. The cDNA (2880 bp), corresponding to the Os-ERL1 gene (Oryza sativa ETHYLENE RESPONSE 2 like 1; GenBank accession number AB107219), contained an open reading frame of 2289 bp coding for 763 amino acids. The protein Os-ERL1 has 50% and 52% similarity to Arabidopsis ETR2 and EIN4, respectively. The Os-ERL1 gene was up-regulated by flooding, and by treatment with ethylene and gibberellin. These results suggest that deep water rice responds to flooding by increasing the number of its ethylene receptors.

Amino Acid Sequence↗