Chestnut goby, <Emphasis Type="Italic">Gymnogobius castaneus</Emphasis> (O’Shaughnessy 1875), as a suitable host of <Emphasis Type="Italic">Anemina arcaeformis</Emphasis> (Heude 1877) in floodplain water bodies,Hokkaido, Northern Japan

This study examined a host-parasite relationship between chestnut goby Gymnogobius castaneus (O’Shaughnessy 1875) and a unionid mussel Anemina arcaeformis (Heude 1877) in three floodplain water bodies of the Ishikari River. Field observations of gravidity between summer (July 2016) and spring (May 2017) revealed that female A. arcaeformis began incubating eggs in September, and all reached a fully ripe-glochidia-gravid stage by May with a decreasing trend in the proportion of gravid individuals. Two laboratory experiments, in which field-caught G. castaneus were artificially infected by glochidia of A. arcaeformis, showed that transformation rates were among the highest ever reported for A. arcaeformis (an average across all the surviving individuals: 47.9%) with a shorter development time in warmer environments. Transformation rates did not differ among sympatric or allopatric pairs, nor decrease when infected twice. Relatively high transformation rates were attributed to high glochidia attachments to gills and mouth cavity (approximately 30% of initially attached individuals) following the highest to fins, or a reduced immunity against infection due to colder environments. Overall, our study demonstrated that chestnut goby (G. castaneus) served as a suitable host of winter-breeding A. arcaeformis. Together with records of the expanded distribution of these two species in the past 30 years, it might be relatively recently that these two have colonized the study areas after extensive land changes, with A. arcaeformis having benefited from a successful host-parasite relationship.     

Enhancement of phase II enzyme activity by purpurin resulting in the suppression of MeIQx-DNA-adduct formation in mice

We previously demonstrated using a bacterial system that the antigenotoxic activity of the anthraquinone compounds purpurin and alizarin was due to the suppression of microsomal enzyme activity involved in the activation of mutagens. In the present study we determined the effect of purpurin and alizarin on (i) MeIQx-DNA-adduct formation in mouse tissues and (ii) the activity of phases I and II enzymes in liver fractions, the liver being the target tissue of MeIQx. The amount of MeIQx-DNA adduct formed was determined using 32P-postlabeling methods. Methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD) enzyme activities, which reflect CYP 1A activity, were measured as markers for phase I enzymes, and UDP-glucuronyltransferase (UGT) and glutathione S-transferase (GST) activities were determined as markers for phase II enzymes. Mice fed with a diet containing 0.5% purpurin for 3 days prior to MeIQx administration had 70% fewer MeIQx-DNA adducts in the lung and kidney, and fewer DNA adducts (insignificant, statistically) in the liver compared with mice fed a diet lacking purpurin. MROD and EROD activities in the liver of these mice increased six- and eight-fold, respectively, and were higher than those determined for the control mice within 1 day following commencement of purpurin treatment. These elevated activities were maintained during treatment and declined immediately following removal of purpurin from the diet. GST and UGT activities gradually increased 2.5- and 3-fold, respectively, following purpurin treatment, and were maintained at significantly high levels even after purpurin administration ceased. Alizarin did not significantly affect DNA-adduct formation and enzyme activity, except in the case of UGT. Taken together, our results show that purpurin reduced MeIQx-DNA-adduct formation by maintaining elevated phase II enzyme activities, thereby facilitating accelerated excretion of MeIQx.     

The Nuclear Orphan Receptor CAR-Retinoid X Receptor Heterodimer Activates the Phenobarbital-Responsive Enhancer Module of the CYP2B Gene

PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.     

Mouse steroid 15 alpha-hydroxylase gene family: identification of type II P-450(15)alpha as coumarin 7-hydroxylase

We identified type II P-450(15)alpha as mouse coumarin 7-hydroxylase (P-450coh). Unlike type I P-450(15)alpha, the other member within the mouse steroid 15 alpha-hydroxylase gene family, type II catalyzed little steroid 15 alpha-hydroxylase activity, yet structurally there were only 11 substitutions between type I and type II P-450(15)alphaS within their 494 amino acid residues (Lindberg et al., 1989), and the N-terminal sequence (21 residues) of P-450coh was identical with that of both P-450(15)alphaS. Induction by pyrazole of coumarin 7-hydroxylase activity correlated well with the increase of type II P-450(15)alpha mRNA in 129/J male and female mice. Pyrazole, on the other hand, was less in males or not effective in females in inducing the 15 alpha-hydroxylase activity and type I P-450(15)alpha mRNA. Expression of type I and II in COS-1 cells revealed that the latter catalyzed coumarin 7-hydroxylase activity at 10 to approximately 14 pmol min-1 (mg of cellular protein)-1. The former, on the other hand, had a high testosterone 15 alpha-hydroxylase but little coumarin 7-hydroxylase activity. It was concluded, therefore, that type II P-450(15)alpha is the mouse coumarin 7-hydroxylase. Identification of type II as the P-450 specific to coumarin 7-hydroxylase activity and characterization of its cDNA and gene, therefore, were significant advances toward understanding the basis of genetic regulation of this activity in mice (known as Coh locus).     

Analysis of 2-amino-N6-hydroxyadenine-induced mutagenesis in phage M13mp2

The mechanism of mutagenesis induced by 2-amino-N6-hydroxyadenine (AHA) and its deoxyriboside (AHAdR) was studied by determining the nucleotide sequences of phage M13mp2 mutant DNA samples. Mutations in the lac promoter-lacZ alpha region of the phage were induced by addition of this agent to culture media in which the phage was growing inside the host bacteria. The spectrum of spontaneous mutation was also investigated. The induced sequence changes were mostly base transitions (80% with AHA and 90% with AHAdR). A few single-base deletions and additions were detected, but they were ascribable to spontaneous mutations. These results are consistent with the incorporation type mechanism proposed by Janion (this issue). In the Ames Salmonella assay, both AHA and AHAdR showed strong mutagenicity in strain TA100 but no activity in TA98.     

Immunization protected well nourished mice but not undernourished ones from lung injury in Methicillin-resistant Staphylococcus aureus (MRSA) infection

Background  

Staphylococcus aureus methicillin-resistant (MRSA) has been frequently isolated from endotracheal and lung puncture aspirates in malnourished children with pneumonia. In this work we evaluated the susceptibility of undernourished BALB/c mice and its ability to mount a protective immunity against MRSA with emphasis on the lung involvement.     

Polo-like kinase 1 is required for localization of Posterior End Mark protein to the centrosome-attracting body and unequal cleavages in ascidian embryos

In ascidian embryos, the posterior-localized maternal factor Posterior End Mark (PEM) is responsible for patterning embryos along the anterior-posterior axis with regard to both cleavage pattern involving unequal cell divisions and gene expression. Although PEM plays important roles in embryogenesis, its mechanism of action is still unclear because PEM has no known functional domain. In the present study, we explored the candidate of PEM partner proteins in Halocynthia roretzi using yeast two-hybrid screening. We isolated a homologue of Polo-like kinase 1 (Plk1), a key regulator of cell division and highly conserved in eukaryotes, as the first potential binding partner of PEM. We biochemically confirmed that interaction occurred between the Plk1 and PEM proteins. Immunostaining showed that Plk1 protein concentrates in the centrosome-attracting body (CAB) at the posterior pole, where PEM protein is also localized. The CAB is a subcellular structure that plays an important role in generating the posterior cleavage pattern. Plk1 localization to the CAB was dependent on the cell cycle phases during unequal cleavage. Inhibition of Plk1 with specific drugs resulted in failure of the nucleus to migrate towards the posterior pole and formation of a microtubule bundle between the CAB and a centrosome, similarly to inhibition of PEM function, suggesting that both proteins are involved in the same process of unequal cleavages. This interrupted nuclear migration was rescued by overexpression of PEM. In Plk1-inhibited embryos, the localization of PEM protein to the CAB was impaired, indicating that Plk1 is required for appropriate localization of PEM.