Identification and characterization of a novel thermo-stable endo-ß-N-acetylglucosaminidase from Rhizomucorpusillus.

Endo-ß-N-acetylglucosaminidase (ENGase) is an enzyme that hydrolyzes the chitobiose core of N-glycans and is widely used for glycan analysis on glycoproteins and preparation of precursors for glycosylated compounds. While most of the ENGases that can hydrolyze complex-type glycans are derived from eukaryotes, their production by heterologous expression using Escherichia coli is insufficient, making the production process expensive. From an industrial perspective, there is a need for a less expensive enzyme with higher activity and stability. In this study, we identified a novel ENGase gene from a thermophilic fungus, Rhizomucor pusillus, and named it Endo-Rp. Characterization of the recombinant Endo-Rp showed that the enzyme had maximum hydrolytic activity at 60 °C and hydrolyzed high-mannose-type and biantennary complex-type glycans, but not (2,4)-branched triantennary complex-type or fucosylated glycans. Endo-Rp also hydrolyzed N-glycans attached to RNase B and human transferrin. In summary, we consider Endo-Rp to be a valuable enzyme in various scientific and industrial applications.

Historical control data on prenatal developmental toxicity studies in rabbits.

Historical control data on rabbit prenatal developmental toxicity studies, performed between 1994-2010, were obtained from 20 laboratories, including 11 pharmaceutical and chemical companies and nine contract laboratories, in Japan. In this paper, data were incorporated from a laboratory if the information was based on 10 studies or more. Japanese White rabbits and New Zealand White rabbits were used for prenatal developmental toxicity studies. The data included maternal reproductive findings at terminal cesarean sections and fetal findings including spontaneous incidences of morphological alterations. No noticeable differences between strains or laboratories were observed in the maternal reproductive and fetal developmental data. The inter-laboratory variations in the incidences of fetal external, visceral, and skeletal alterations seem to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, and terminology of fetal alterations.

Effects of exposure in utero to bisphenol a on the expression of aryl hydrocarbon receptor, related factors, and xenobiotic metabolizing enzymes in murine embryos.

To evaluate the effects of bisphenol A (BPA), a candidate endocrine disruptor (ED), on embryonic development, we examined the mRNA expression levels of the aryl hydrocarbon receptor (AhR; which binds with many EDs and plays crucial roles in their metabolism) and related factors [aryl hydrocarbon receptor repressor (AhRR) and AhR nuclear translocator (Arnt)], xenobiotic metabolizing enzymes [XMEs; cytochrome P450 1A1 (CYP1A1) and UDP-glucuronosyltransferase, and the glutathione S-transferase Ya subunit (GST)], in murine embryos exposed in utero to BPA (0.02, 2, 200, and 20,000 microg/kg/day) and 17beta-estradiol (E2; 5 microg/kg/day, used as a positive control) at 6.5-13.5 or 6.5-17.5 days post coitum (dpc) using the quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) method. Protein levels of CYP1A1 and GST in embryonic livers were estimated by Western immunoblotting. Exposure in utero to BPA [0.02 (1/100 dose of environmental exposure), 2, 200, and 20,000 microg/kg/day] increased AhR mRNA expression in the cerebra, cerebella, and gonads (testes and ovaries) of male and female mid-and late-developmental stage (14.5- and 18.5-dpc, respectively) embryos. BPA dose-independently up-regulated the expression of AhRR and Arnt in mid- and late-stage embryos. BPA had no remarkable effect on the mRNA levels of XMEs in mid-stage embryos, but dose-dependently up-regulated the expression in late-stage embryos. Moreover, the protein levels of these enzymes in the livers of late-stage embryos were increased. The present findings revealed that exposure to BPA in utero disrupts the expression of AhR and related factors and of xenobiotic metabolizing enzymes, and that mid-stage embryos, in the organogenic stage, are sensitive to BPA.

Effects of in utero exposure to bisphenol A on mRNA expression of arylhydrocarbon and retinoid receptors in murine embryos.

To evaluate the effects of bisphenol A (BPA), a candidate endocrine disruptor (ED), on embryonic development, we examined the mRNA expression levels of the arylhydrocarbon receptor (AhR), which binds with many EDs and plays crucial roles in xenobiotic metabolism, and of the retinoic acid receptor (RAR) alpha and retinoid X receptor (RXR) alpha, key factors in nuclear receptor-dependent retinoid signal transduction, in murine embryos exposed in utero to BPA (0.02, 2, 200, and 20,000 microg/kg/day) at 6.5-13.5 or 6.5-17.5 days post coitum (dpc), using the real-time reverse transcription-polymerase chain reaction (RT-PCR) method. Extremely low-dose BPA (0.02 microg/kg/day; 1/100 the dose of environmental exposure) remarkably increased AhR mRNA expression in the cerebra, cerebella, and gonads (testes and ovaries) of male and female 14.5- and 18.5-dpc-embryos. In utero exposure to BPA at 2, 200, and 20,000 microg/kg/day also increased levels of AhR mRNA. In gonads of 14.5-dpc-embryos, AhR mRNA levels were elevated and showed diphasic (U) dose-response curves following exposure to BPA, but inverted U dose-response curves were obtained for 18.5-dpc-embryos. Exposure to BPA increased expression levels of RARalpha and RXRalpha mRNAs in the cerebra, cerebella, and gonads of male and female 14.5- and 18.5-dpc-embryos. Extremely low-dose BPA (0.02 microg/kg/day) increased RARalpha mRNA expression in the cerebella of male and female 14.5- and 18.5-dpc-embryos and in the gonads of female 14.5-dpc-embryos, and significantly increased RXRalpha mRNA expression in the cerebra and cerebella of male and female 14.5-dpc-embryos. The present findings confirm that in utero exposure to an extremely low dose of BPA up-regulates the mRNA expression of AhR, RARalpha, and RXRalpha in murine embryos and disrupts the receptor-dependent signal transducing systems, and will contribute to the assessment of the toxic effects of BPA on xenobiotic metabolism and retinoid signals in embryogenesis.

Effects of in utero exposure to bisphenol A on expression of RARalpha and RXRalpha mRNAs in murine embryos.

Retinoic acid receptor (RAR) alpha and retinoid X receptor (RXR) alpha are key factors in a nuclear receptor-dependent signal. To evaluate the effects of bisphenol A (BPA), a candidate endocrine disruptor (ED), on embryonic development, we examined the mRNA levels of RARalpha and RXRalpha in murine embryos, exposed in utero to BPA (2 microg/kg/day) at 6.5-17.5 days post-coitum (dpc), by the real-time reverse transcription-polymerase chain reaction (RT-PCR) method. Higher levels of RARalpha mRNA in cerebra of male and female embryos of control groups were detected at 14.5 dpc. In utero BPA reduced the RARalpha mRNA expression. Higher levels of RXRalpha mRNA in cerebra of male and female embryos were seen at 12.5 dpc. The exposure decreased RXRalpha mRNA expression in male but not female embryos. No remarkable change in the RARalpha mRNA expression level was noted in cerebella of male or female embryos of the control group during embryonic development. Exposure to BPA increased expression levels of RARalpha mRNA in cerebella of male and female embryos at 12.5 dpc. Higher levels of RXRalpha mRNA in cerebella of male and female embryos were seen, but no remarkable changes were noted during embryonic development. BPA significantly decreased the expression levels of RXRalpha mRNA in cerebella of female embryos at 12.5, 14.5 and 18.5 dpc. RARalpha and RXRalpha mRNAs were expressed in gonads (testes and ovaries) of murine embryos from 12.5 to 18.5 dpc. In utero exposure to BPA decreased levels of RARalpha mRNA in testes of 14.5- and 18.5-dpc-embryos, levels of RXRalpha mRNA in testes of 14.5-dpc-embryos, and levels of RXRalpha mRNA in ovaries of 14.5-dpc-embryos. The present findings indicate that RARalpha and RXRalpha play crucial roles in organogenesis, and the growth and development of murine embryos, and will contribute to the assessment of the toxic effects of BPA on retinoid signals in embryogenesis.

Effects of oral exposure of bisphenol A on mRNA expression of nuclear receptors in murine placentae assessed by DNA microarray.

Bisphenol A (BPA), a candidate endocrine disruptor (ED), is considered to bind to estrogen receptors and to regulate expressions of estrogen responsive genes. It has also shown evidence of affecting the reproductive, immunological and nervous systems of mammalian embryos. However, the effects of BPA on placentae, a central organ of feto-maternal interlocution, are still unclear. To reveal the mechanisms of BPA effects on placentae in mammals, we compared the mRNA expression of 20 nuclear receptors between placentae of vehicle controls and those of orally BPA exposed pregnant mice by a DNA microarray technique. In murine placentae, mRNAs of 11 nuclear receptors were not detected. However, greater than 1.5 fold changes in mRNA expression of nine nuclear receptors between vehicle control and BPA treated mice were noted. Moreover, remarkable changes in mRNA expression of six non-nuclear receptor proteins were induced by BPA exposure. There were various differences in the effects of BPA on the expression of these mRNAs between the placentae with male embryos and those with female embryos. Such embryo-sex dependent differences are interesting and important pointers to understanding of the endocrine disrupting effect of BPA. The present data indicate that BPA affects the expression of nuclear receptor mRNAs in placentae and may disrupt the physiological functions of placentae.