Effect of 3-O-octanoyl-(+)-catechin on the responses of GABA(A) receptors and Na+/glucose cotransporters expressed in xenopus oocytes and on the oocyte membrane potential

Recently, 3-O-octanoyl-(+)-catechin (OC) was synthesized from (+)-catechin (C) by incorporation of an octanoyl chain into C in the light of (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), which are the major polyphenols found in green tea and have strong physiological activities. OC was found to inhibit the response of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA(A) receptors) and Na+/glucose cotransporters expressed in Xenopus oocytes in a noncompetitive manner more strongly than does C. OC also induced a nonspecific membrane current and decreased the membrane potential of the oocyte, and thus death of the oocyte occurred even at lower concentrations than that induced by C or EGCg. Although EGCg produced H2O2 in aqueous solution, OC did not. This newly synthesized catechin derivative OC possibly binds to the lipid membrane more strongly than does C, Ecg, or EGCg and as a result perturbs the membrane structure.     

Extended glucuronidation is another major path of cyanidin 3-O-beta-D-glucopyranoside metabolism in rats

We previously determined that five rather hydrophobic metabolites appeared in blood plasma after oral administration of cyanidin 3-O-beta-D-glucopyranoside, but a group of hydrophilic metabolites still remained unidentified. In the present study, 12 hydrophilic metabolites found were collected from urine and plasma samples by high-performance liquid chromatography (HPLC) and then analyzed by tandem MS spectrometry. From the MS spectra, four metabolites out of 12 were assigned as glucuronides of cyanidin 3-O-beta-D-glucopyranoside and six out of 12 were glucuronides of the primary metabolites of cyanidin 3-O-beta-D-glucopyranoside (O-methyl cyanidin 3-O-beta-D-glucopyranoside). Extended glucuronides of cyanidin 3-O-beta-D-glucopyranoside and O-methyl cyanidin 3-O-beta-D-glucopyranoside showed their maximum plasma concentrations at 15 and 60 min (or 30 min) after oral administration, respectively. Their maximum plasma concentrations ranged from 15 to 70 nM. From the profile of urinary-excreted anthocyanins after intravenous administration, it was deduced that extended glucuronides of cyanidin 3-O-beta-D-glucopyranoside and O-methyl cyanidin 3-O-beta-D-glucopyranoside were mainly produced in the liver rather than by intestinal flora. The area under the plasma concentration curve was 0.25 micromol min/L for extended glucuronides of cyanidin 3-O-beta-D-glucopyranoside and 0.14 micromol min/L for O-methyl cyanidin 3-O-beta-D-glucopyranoside, respectively, when evaluated as cyanidin 3-O-beta-D-glucopyranoside equivalent, indicating that extended glucuronidation is a critical pathway in cyanidin 3-O-beta-D-glucopyranoside metabolism in rats.     

Differentiation and elimination of uterine natural killer cells in delayed implantation and parturition mice

To clarify the roles of uterine natural killer (uNK) cells in implantation and parturition, differentiation and elimination of uNK cells in the pregnant uterus was examined using artificial delayed implantation (DI) and delayed parturition (DP) mice. To prepare DI mice, pregnant mice were ovariectomized on the third day of pregnancy (D3) and treated with 2 mg progesterone daily. The same amount of progesterone was administered on D15 or D17 of normal pregnant mice at 24 h intervals until sampling to prepare DP mice. The uNK cells contained PAS-positive granules on D8 in DI mice. The uNK cells in DI mice were smaller in size, and differentiation of these cells was delayed compared to those of the control mice. From D19 to D21 in DP mice, the metrial gland was well developed and uNK cells were present. The number of uNK cell granules decreased on D21, and there were no uNK cells in the normal pregnant mice. This result suggests that differentiation of uNK cells is not directly related to implantation, but elimination of these cells is closely involved in parturition.     

Histological study of granulated metrial gland (GMG) cells in beige (DA-bg/bg) rats

To clarify the roles of granulated metrial gland (GMG) cells for successful pregnancy in rats, GMG cells in beige rats (genotype: DA-bg/bg), whose NK cells show lysosomal dysfunction because of abnormalities in cytoplasmic granules, were examined in mid- and late-pregnancy by light and electron microscopies. The GMG cells of beige rats were significantly less in number than those of the two controls (genotypes: DA-bg/+ and DA-+/+) in mid- and late-pregnancy, and this accompanied a low reproductive performance in the beige rats. The size of intracellular granules in the GMG cells of the beige rats was larger than for the two controls on each corresponding day of pregnancy. These results suggest that the activity of rat GMG cells and peripheral NK cells might be influenced by the beige gene, which is involved in reproductive performance.     

Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil

Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.     

Expression of inhibins, activins, insulin-like growth factor-I and steroidogenic enzymes in the equine placenta

In this study, the expression patterns of inhibins, activins, insulin-like growth factor-I (IGF-I) and steroidogenic enzymes in equine placentae recovered during the latter two-thirds of gestation were examined. Concentrations of inhibin A and inhibin pro-alphaC in endometrial and fetal placental tissue homogenates were very low during the period examined, whereas these tissues contained high concentrations of activin A. In both maternal endometrial and fetal placental tissues, activin A levels decreased as pregnancy progressed. Expression of inhibin alpha-subunit was not observed in the placenta using either immunohistochemistry or in situ hybridization. Inhibin/activin betaA-subunit and its mRNA were confined to maternal endometrial glands, whereas immunopositive betaB-subunit was not detected in either endometrial glands or microcotyledons. Cytochrome P450 side chain cleavage enzyme was detected by immunohistochemistry in both endometrial glands and microcotyledons, whereas cytochrome P450 17alpha-hydroxylase/lyase was absent in these tissues. Immunopositive signals for 3beta-hydroxysteroid dehydrogenase and cytochrome P450 aromatase were localized in microcotyledons but not in endometrial glands. Immunohistochemistry revealed that IGF-I was highly expressed in microcotyledons around Day 130, and decreased as pregnancy progressed. Changes in the expression of IGF-I were correlated with the number of PCNA positive cells in the placenta. The present study demonstrated the presence and localized the site of expression of activin, IGF-I and steroidogenic enzymes in equine placental tissues during the latter two-thirds of gestation; the results suggest that activin and IGF-I may be involved in the regulation of placental development.     

Induction of estrus by prostaglandin F2alpha administration in the vaginal vestibules of miniature pigs

Estrus induction is an important step in embryo production. It has been difficult to induce estrus in miniature pigs by intramascular (i.m.) injection of prostaglandin F2alpha (PGF2(2alpha)) in the early luteal stage of the estrous cycle. In the present study, we injected two different doses of PGF2(2alpha) i.m. and into the submucosa of the vaginal vestibule (i.ves.) of miniature pigs, and examined the effect of these treatments on estrus induction. Fifteen miniature pigs were divided into five experimental groups (control, saline injected i.m.; PGF2alpha treated, 1.0 or 1.5 mg of PGF2alpha injected twice i.m. or i.ves.), and the estrus length and concentrations of 17beta-estradiol (E2) and progesterone (P) in the blood were examined. Estrus length was significantly shortened by a large amount of PGF2alpha injected i.ves. In addition, the concentration of P in the blood significantly decreased after two injections of PGF2alpha (i.m. or i.ves.). These results suggest that in miniature pigs, administration of at least 3.0 mg of PGF2alpha is required for the induction of luteolysis and injection of PGF2alpha into the vaginal vestibule is a useful method of estrus induction.     

Phylogenetic analysis of AGAMOUS sequences reveals the origin of the diploid and tetraploid forms of self-pollinating wild buckwheat,Fagopyrum homotropicum Ohnishi

Fagopyrum homotropicum Ohnishi is a self-pollinating wild buckwheat species indigenous to eastern Tibet and the Yunnan and Sichuan Provinces of China. It is useful breeding material for shifting cultivated buckwheat (F. esculentum ssp. esculentum Moench) from out-crossing to self-pollinating. Despite its importance as a genetic resource in buckwheat breeding, the genetic variation of F. homotropicum is poorly understood. In this study, we investigated the genetic variation and phylogenetic relationships of the diploid and tetraploid forms of F. homotropicum based on the nucleotide sequences of a nuclear gene, AGAMOUS (AG). Neighbor-joining analysis revealed that representative individuals clustered into three large groups (Group I, II and III). Each group contained diploid and tetraploid forms of F. homotropicum. We identified tetraploid plants that had two diverged AG sequences; one belonging to Group I and the other belonging to Group II, or one belonging to Group II and the other belonging to Group III. These results suggest that the tetraploid form originated from at least two hybridization events between deeply differentiated diploids. The results also imply that the genetic diversity contributed by tetraploidization of differentiated diploids may have allowed the distribution range of F. homotropicum to expand to the northern areas of China.     

Antibody response in vaccinated pregnant mares to recent G3BP[12] and G14P[12] equine rotaviruses

Background

Both the G3P[12] and the G14P[12] type of equine group A rotavirus (RVA) have recently become predominant in many countries, including Japan. G3 types are classified further into G3A and G3B. The G3A viruses have been circulating in Europe, Australia, and Argentina, and the G3B viruses have been circulating in Japan. However, only an inactivated vaccine containing a single G3BP[12] strain is commercially available in Japan. To assess the efficacy of the current vaccine against recently circulating equine RVA strains, we examined antibody responses in pregnant mares to recent G3BP[12] and G14P[12] strains by virus neutralization test.

Findings

After vaccination in five pregnant mares, the geometric mean serum titers of virus-neutralizing antibody to recent G3BP[12] strains increased 5.3- to 7.0-fold and were similar to that against homologous vaccine strain. Moreover, antibody titers to recent G14P[12] strains were also increased 3.0- to 3.5-fold.

Conclusions

These results suggest that inoculation of mares with the current vaccine should provide foals with virus-neutralizing antibodies against not only the G3BP[12] but also the G14P[12] RVA strain via the colostrum.