Intragenic recombination within the pun allele of the pink-eyed dilution locus in pre-melanocytes and primordial germ cells of embryonic mice treated with N-ethyl-N-nitrosourea.

The forward or reverse processes of intragenic recombination (IGR), which occur through the addition or deletion of duplicated homologous exons of the pun allele in Pun mice, was observed in vivo, after introducing an homozygous pun allele in a C57BL/6 background. We assessed the frequency of IGR upon N-ethyl-N-nitrosourea (ENU) treatment of pre-melanocytes (PMCs: somatic cells) and primordial germ cells (PGCs: germ cells) of embryonic mice at 10.5 days of development (E10.5). We simultaneously examined IGR and other mutations at the p locus of PMCs responsible for coat color in the offspring obtained by crossing pun/pun with pun/P mice. The frequencies of both spontaneous and ENU-induced IGR were markedly higher than that of the recessive mutation (RM) in PMCs obtained from crossing C57BL/6 and PW strains (Shibuya et al., 1982). ENU also induces IGR at a higher frequency in PGCs at E10.5, which was observed in the next generation. These results indicate that ENU, which preferentially induces gene mutations through base substitution, also induces IGR at a high frequency in the pun allele in both somatic and germ cells of embryonic mice at the E10.5 developmental stage.

Correlation between fundus autofluorescence and visual function in patients with cone-rod dystrophy.

This study aimed to investigate the relationship between autofluorescence (AF) signal measured with ultra-wide field imaging and visual functions in patients with cone-rod dystrophy (CORD). A retrospective chart review was performed for CORD patients. We performed the visual field test and fundus autofluorescence (FAF) measurement and visualized retinal structures with optical coherence tomography (OCT) on the same day. Using binarised FAF images, we identified a low FAF area ratio (LFAR: low FAF/30°). Relationships between age and logMAR visual acuity (VA), central retinal thickness (CRT), central choroidal thickness (CCT), mean deviation (MD) value, and LFAR were investigated. Thirty-seven eyes of 21 CORD patients (8 men and 13 women) were enrolled. The mean patient age was 49.8 years. LogMAR VA and MD were 0.52 ± 0.47 and - 17.91 ± 10.59 dB, respectively. There was a significant relationship between logMAR VA and MD (p = 0.001). LogMAR VA significantly correlated with CRT (p = 0.006) but not with other parameters. Conversely, univariate analysis suggested a significant relationship between MD and LFAR (p = 0.001). In the multivariate analysis, LFAR was significantly associated with MD (p = 0.002). In conclusion, it is useful to measure the low FAF area in patients with CORD. The AF measurement reflects the visual field deterioration but not VA in CORD.

Quantification of residual ellipsoid zone and its correlation with visual functions in patients with cone-rod dystrophy.

Cone-rod dystrophy (CORD) is one of the inherited retinal diseases that result in central visual field deterioration and decreased visual acuity (VA). In CORD patients, impaired photoreceptor cells are observed as the disruption of ellipsoid zone (EZ) on optical coherence tomography (OCT) images. In the present study, we calculated the index of residual EZ (rEZ) to quantify the function of photoreceptor cells and investigated the correlation between rEZ index and visual functions. Twenty-six eyes of 13 patients with clinical suspicion of CORD were examined. Visual field was tested with the Humphrey Visual Field Analyzer (HFA 10-2 program). We simultaneously obtained OCT images and calculated the area of decreased EZ intensity (EZa). Using the binarized OCT images, the percentage of the rEZ in a 3 × 3 mm area surrounding the macula was analyzed. To clarify interrator reproducibility, intraclass correlation coefficient (ICC) was calculated. Moreover, we investigated the association between OCT parameters and VA as well as the mean deviation (MD) value measured with HFA. The mean age of the patients was 48.5 ± 16.9 years. The mean central retinal thickness was 122.7 ± 73.2 µm. The mean EZa and rEZ were 22.2 ± 23.6 µm2 and 0.35 ± 0.31, respectively. The ICC of each rEZ index was 0.91 (95% CI: 0.89 < ICC < 0.93). Multivariate analysis indicated rEZ was significantly related to logMAR VA (p = 0.05) and rEZ and EZa were associated with the MD value (p = 0.014 and p = 0.009, linear mixed model). Furthermore, rEZ was also associated with photopic a- and b-wave amplitudes (p = 0.027 and p = 0.0024, respectively, linear mixed model). Taken together, the current results suggested the usefulness of rEZ quantification for predicting visual functions in CORD patients.

Short wavelength automated perimetry and standard automated perimetry in central serous chorioretinopathy.

Short wavelength automated perimetry (SWAP) is known for detecting the early reduction of retinal sensitivity (RS) in glaucoma. It's application in retinal diseases have also been discussed previously. We investigated the difference in RS measured between standard white-on-white automated perimetry (WW) and blue-on-yellow SWAP in central serous chorioretinopathy (CSC). The overall RS (W-RS, S-RS) as well as the RS inside and outside of the serous retinal detachment (SRD) region were investigated in 26 eyes of 26 CSC patients using WW and SWAP. The central retinal thickness, central choroidal thickness, SRD area (SRDa), and SRD height at the fovea were measured using optic coherence tomography. RS inside the SRD region was lower than that of outside for both perimetries (both p < 0.001). The difference between RS inside and outside of the SRD region was greater in SWAP compared to WW (p < 0.001). Univariate analysis revealed significant correlations between SRDa and both W-RS and S-RS (both p < 0.001); moreover, multivariate analysis indicated that only S-RS was selected as the optimal model for SRDa. Our study demonstrated that SWAP was detected the decrease in RS more accurately than WW in CSC. These results may suggest the usefulness of SWAP for detecting change of retinal function in CSC.

Kojic acid -absence of tumor-initiating activity in rat liver, and of carcinogenic and photo-genotoxic potential in mouse skin.

Kojic acid (KA) has been widely used as a quasi-drug ingredient. Possible promotion activity of KA was suggested on livers of mouse and rat by findings obtained in genotoxicity and carcinogenicity studies performed thus far. Therefore, in order to examine safety as a quasi-drug ingredient, we investigated the presence of initiation activity in rat liver and the photo-genotoxicity and carcinogenicity in mouse skin. In medium-term carcinogenesis test in rats, 2.0% KA was orally given to F344/DuCrj rats for 4 weeks of the initiation period, followed by the combination of partial hepatectomy and treatment with a hepatocarcinogenesis promoter, phenobarbital (PB). As a result, glutathione S-transferase placental form (GST-P) positive foci of 0.2 mm or more in diameter in the KA group, which is usually used in determination of pre-cancerous lesions, did not increase significantly in both numbers and areas compared with those of the non-initiated controls. In the concurrent analysis, however, numbers of GST-P-positive foci of two cells or more and 0.1 mm or more in diameter increased slightly, and possible weak initiation activity of KA was equivocal. However, considering the known fact that KA exerts promotion activity in the liver of F344 rats by long-term dietary administration, it was suggested that the observed slight increase of the numbers of GST-P-positive foci in rat liver was the effect of promotion activity of KA rather than the initiation activity. In DNA adducts formation assay in a rat liver, no clear adducts derived from KA were detected in male F344/DuCrj rats administered 0.5% or 2% KA orally, and KA was considered not to form DNA adducts in rat liver. In the in vitro photo-reverse mutation assay with bacteria, KA exerted weak photo-mutagenicity. Furthermore, in chromosome aberration study in Chinese hamster lung cells (CHL/IU cells) with UV irradiation, KA induced chromosome aberration at high-dose (1.4 mg/mL) treatment with UV irradiation, but was negative without UV irradiation. However, in the in vivo photo-micronucleus study in mouse, in which 1.0 or 3.0% KA containing cream was applied twice to the back of the animals with a 24-hr interval, KA did not induce micronuclei in mouse epidermal cells. Based on these results, it is considered that the risk of KA to exert photo-carcinogenicity is quite low in the skin. In skin carcinogenesis bioassay for initiation-promotion potential, 3.0% KA cream formulation was applied to the back of the mouse for 1 week (once a day, total 7 times) and for 19 weeks (5 times a week, total 95 times) during the initiation and the promotion stages, respectively. No skin nodules were observed in any animal skins formed due to KA treatment given in either stage. Therefore, KA is considered not to possess initiation nor promotion activity of skin carcinogenesis. Furthermore, from the above findings, it is suggested that KA is virtually safe as a quasi-drug ingredient.

In vivo photochemical skin micronucleus test using a sunlight simulator: detection of 8-methoxypsoralen and benzo[a]pyrene in hairless mice.

Evaluating in vivo photochemical genotoxicity (photogenotoxicity) or photochemical carcinogenicity (photocarcinogenicity) in the skin that is actually exposed to light is important for estimating the risk of human exposure to chemicals under sunlight. With regard to the skin micronucleus test, Nishikawa et al. developed a reliable technique that is simple and in which the negative control has a stable background. In the present study, we applied 8-methoxypsoralen (8-MOP) and benzo[a]pyrene (B[a]P) to the backs of hairless mice and subjected the mice to irradiation by a sunlight simulator in order to investigate whether this test can detect photogenotoxicity of these chemicals. In the treatment with 8-MOP [0.00075% and 0.0015% (w/v)], a significant increase was observed in the frequency of micronucleated cells only under light irradiation using the sunlight simulator. At a high chemical dose, the frequency of micronucleated cells increased from 48h after the treatment, peaked at 96h, and then decreased at 168h. Furthermore, at 96h with the high dose under light irradiation, we frequently observed cells with nuclear buds. In the treatment with B[a]P [first experiment: 0.025% and 0.05% (w/v); second experiment: 0.005%, 0.01%, and 0.02% (w/v)], a significant increase was observed in the frequency of micronucleated cells at skin-irritating doses [0.01%, 0.02%, 0.025%, and 0.05% (w/v)] at 72 or 96h after the treatment only under light irradiation using the sunlight simulator. In conclusion, photogenotoxicity of 8-MOP and B[a]P was detected in the in vivo photochemical skin micronucleus study.

Lewis acid-assisted nucleophilic substitution of fullerene epoxide.

[Structure: see text] A 1,4-bis(phenyl)-1,4-dihydro[60]fullerene resulting from an efficient nucleophilic substitution has been obtained by reaction of a fullerene epoxide, C60O, with nucleophilic aromatic compounds in the presence of boron trifluoride etherate as a Lewis acid.

Enhanced bioavailability of a new thiazolidine derivative FPFS-410, an antidiabetic and lipid-lowering drug, after oral administration of its hydroxypropyl-beta-cyclodextrin complex to bile duct-cannulated rats.

The effect of bile acids on bioavailability of FPFS-410 (2-(N-Cyanoimino)-5-{(E)-4-styrylbenzylidene}-4-oxothiazolidine) after oral administration of the drug and its 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) complex was investigated. The complexation with HP-beta-CyD increased the oral bioavailability of FPFS-410 in normal rats in a HP-beta-CyD concentration-dependent manner, compared with that of drug alone. In bile duct-cannulated rats, bile acid concentrations in pylic serum and biliary were decreased to 18% and 14% of sham-operated rats, respectively. After oral administration of the HP-beta-CyD complex, the plasma levels of FPFS-410 were lower in bile duct-cannulated rats than in sham-operated rats up to 1 h, however, this order reversed from 2 to 12 h. The plasma levels of M1, a dominant metabolite of FPFS-410 in rats, significantly decreased until 2 h after administration of the complex in bile duct-cannulated rats, compared with in sham-operated rats. Bioconversion of FPFS-410 to M1 and CYP3A2 expression in the liver was markedly lowered by bile duct-cannulation. Bile duct-cannulation did not, however, affect the serum levels of estradiol. These results suggest that bile acids have a pivotal role for bioavailability of FPFS-410 after oral administration of the FPFS-410 complex with HP-beta-CyD through CYP3A2 activity in liver of rats.

Improvement of solubility and oral bioavailability of 2-(N-cyanoimino)-5-[(E)-4-styrylbenzylidene]-4-oxothiazolidine (FPFS-410) with antidiabetic and lipid-lowering activities in dogs by 2-hydroxypropyl-beta-cyclodextrin.

2-(N-Cyanoimino)-5-[(E)-4-styrylbenzylidene]-4-oxothiazolidine (FPFS-410) is a newly synthesized thiazolidine derivative having not only antidiabetic but also lipid-lowering activities. However, this compound has an extremely low aqueous solubility (2.8 (+/-0.33) x 10(-8) M (0.0094+/-0.0011 microg/ml) in 1.0 M phosphate buffer (pH 7.0) at 25 degrees C). In this study, we investigated the effect of various hydrophilic cyclodextrins (CyDs) on the solubility of FPFS-410 to select a CyD suitable for formulations of the compound. Among various CyDs, 2-hydroxypropyl-beta-CyD (HP-beta-CyD) had the highest solubilizing ability to FPFS-410, e.g., the solubility of the compound was increased 200000-fold by the addition of 40 mM HP-beta-CyD, which was attributable to the formation of the 1 : 2 (guest : host) inclusion complexes. The interaction of HP-beta-CyD with FPFS-410 was studied using 1H-nuclear magnetic resonance (NMR) spectroscopies including ROESY spectroscopy and a molecular modeling calculation. These results suggested that HP-beta-CyD forms a 1:2 (guest : host) inclusion complex with FPFS-410 by including both the stilbene and thiazolidine moieties. FPFS-410/HP-beta-CyD solid complexes with various stoichiometries were prepared by the spray drying and cogrinding methods, and confirmed by powder X-ray diffractometry that these complexes are in an amorphous state. The dissolution of FPFS-410 in water was significantly accelerated by the complexation with HP-beta-CyD. In vivo studies revealed that HP-beta-CyD markedly increases the bioavailability of FPFS-410 after oral administration in dogs. The present results suggest that HP-beta-CyD is useful for improvement of the extremely low bioavailability of FPFS-410.

Quantification of vitellogenin in several developmental stages of medaka (Oryzias latipes) S-rR strain.

Concentrations of vitellogenin (VTG) in different developmental stages: embryo (on the day of fertilization); yolk-sac larva (on the day of hatching); 2, 4, 6 and 8 weeks posthatching; were determined in medaka (Oryzias latipes) S-rR strain. Both sexes were bred separately from 1 week after hatching and fed only Artemia nauplii to avoid contamination by xenoestrogen from females and feed. Whole bodies of ten test fish/group were homogenized individually and the supernatants were quantified. Quantification of VTG was performed by an enzyme-linked immunosorbent assay (ELISA). In most males, VTG levels were less than 1 microg/g body weight and no fluctuation was observed throughout the developmental stages. In 2-week-old females, five had no detectable VTG, and the others had 5.18+/-2.37 microg/g of VTG. The 4-week-old females had 16.3+/-12.0 microg/g VTG, and the concentrations increased with maturity to 5.54+/-3.09 mg/g in 6-week-old and 8.99+/-3.10 mg/g in 8-week-old specimens. These results show that the concentrations of VTG in males are routinely close to the detection limit independent of the developmental stages in an environment with low contamination by xenoestrogen derived from artificial feed and females. Females have detectable VTG levels even in the juvenile phase, and the level increases markedly with maturation.

Adhesion and colonization of enterohemorrhagic Escherichia coli O157:H7 in cecum of mice.

Infectious diseases due to enterohemorrhagic Escherichia coli (EHEC) are characterized by diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The adherence of EHEC on intestinal epithelial cells is a first step for developing these diseases. In the present study, we examined whether EHEC O157:H7 adhere to intestinal epithelial cells of mice and cause F-actin accumulation in the epithelial cells following the intragastric inoculation of the pathogen. Fecal shedding of the EHEC O157:H7 strain was observed in ICR mice up to 3 weeks. Fecal shedding periods of the type III secretion system-related gene (espA and sepL) deletion mutants were clearly shorter than that of the wild-type EHEC O157:H7 strain. The EHEC O157:H7 colonies were found on the epithelial surfaces of the ceca in association with F-actin accumulation beneath the attached bacteria.

A murine model of enterohemorrhagic Escherichia coli O157:H7 infection to assess immunopotentiating activity of drugs on mucosal immunity: effect of drugs.

An enterohemorrhagic Escherichia coli (EHEC) O157 oral infection murine model was established to examine the potentiating activity of drugs on mucosal immune responses. Groups of ICR mice inoculated intragastrically with 10(11) CFU/kg EHEC O157 showed chronic intestinal infection with the pathogen that persisted over 3 weeks and resulted in the synthesis of relatively high levels of antigen specific fecal IgA antibody. Intraperitoneal administration of 80 NU/kg Neurotropin, an immunopotentiator, augmented the antigen specific mucosal immune responses to EHEC O157. On the other hand, FK506 clearly suppressed the response. To further document the augmenting effect of Neurotropin on mucosal immune responses, mice were immunized intranasally with a mixture of ovalbumin and cholera toxin. Co-administration of 80 NU/kg Neurotropin significantly potentiated the synthesis of fecal IgA and serum IgG antibodies. These results suggest that Neurotropin has potential as a mucosal adjuvant to promote secretory IgA antibody production and that the mice model of oral infection with EHEC O157 is useful for immunopharmacological studies of bacterial infection-defensive mucosal immune responses.

Regulation of the expressions of HCO3- uptake and intracellular carbonic anhydrase in response to CO2 concentration in the marine diatom Phaeodactylum sp.

The marine diatom, Phaeodactylum tricornutum Bohlin, is probably one of the most extensively studied marine alga with respect to carbon acquisition and assimilation mechanisms. However, quantitative analyses of HCO3-utilization and the detailed process of acclimation of cells from high CO2 to limited CO2 are yet to be done extensively. Suitable molecular markers for this acclimation process are not established, either. Recently, it became clear that the rate of CO2 formation in artificial seawater is about eight times slower than that in freshwater, and thatP. tricornutum cells utilize HCO3- quite efficiently. Despite their great capacity to take up HCO3-, the signal controlling photosynthetic affinity for dissolved inorganic carbon has been shown to be CO2 in the medium. Furthermore, light seems to be required for this process. Internal carbonic anhydrase (CA) activity has been shown to be crucial for high-affinity photosynthesis in a number of algae, including marine diatoms. Internal ß-type CA, which has been isolated in one strain of P. tricornutum, was clearly shown to be a low-CO2 inducible enzyme. This review paper additionally includes data showing that this CA occurs generally in P. tricornutum species.