Detection of γ-H2AX,a Biomarker for DNA Double-strand Breaks,in Urinary Bladders of N -Butyl- N -(4-Hydroxybutyl)-Nitrosamine-Treated Rats

To evaluate the potential role of DNA repair in bladder carcinogenesis, we performed an immunohistochemical analysis of expression of various DNA repair enzymes and γ-H2AX, a high-sensitivity marker of DNA double-strand breaks, in the urothelium of male F344 rats treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), a bladder-specific carcinogen. Our results clearly demonstrated that γ-H2AX aggregation was specifically generated in nuclei of bladder epithelial cells of BBN-treated rats, which was not found in untreated controls or mesenchymal cells. γ-H2AX-positive cells were detected not only in hyperplastic and neoplastic areas but also in the normal-like urothelium after BBN treatment. These data indicate that γ-H2AX has potential as a useful biomarker for early detection of genotoxicity in the rat urinary bladder. To the best of our knowledge, this is the first report demonstrating expression of γ-H2AX during bladder carcinogenesis.     

Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria

Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 10⁴ clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.     

Effect of trehalose supplementation in milk replacer on the incidence of diarrhea and fecal microbiota in preweaned calves

Trehalose, a nonreducing disaccharide consisting of d-glucose with α,α-1,1 linkage, was evaluated as a functional material to improve the gut environment in preweaned calves. In experiment 1, 173 calves were divided into two groups; the trehalose group was fed trehalose at 30 g/animal/d with milk replacer during the suckling period, and the control group was fed nonsupplemented milk replacer. Medication frequency was lower in the trehalose group (P < 0.05). In experiment 2, calves (n = 20) were divided into two groups (control group [n = 10] and trehalose group [n = 10]) based on their body weight and reared under the same feeding regimens as in experiment 1. Fresh feces were collected from individual animals at the beginning of the trial (average age 11 d), 3 wk after trehalose feeding (experimental day 22), and 1 d before weaning, and the fecal score was recorded daily. Fecal samples were analyzed for fermentation parameters and microbiota. The fecal score was significantly lower in the trehalose group than in the control group in the early stage (at an age of 14 to 18 d; P < 0.05) of the suckling period. Calves fed trehalose tended to have a higher proportion of fecal butyrate on day 22 than calves in the control group (P = 0.08). Population sizes of Clostridium spp. were significantly lower (P = 0.036), whereas those of Dialister spp. and Eubacterium spp. tended to be higher in the feces of calves in the trehalose group on day 22 (P = 0.060 and P = 0.083). These observations indicate that trehalose feeding modulated the gut environment and partially contributed to the reduction in medication frequency observed in experiment 1.     

Relationships between body size and secondary sexual characters,and sperm characters in male Dolly Varden char (Salvelinus malma)

In Salmonidae, subordinate males are exposed to higher risks of sperm competition than dominant males and thus are expected to improve the sperm characteristics (sperm concentrations, sperm velocity and sperm longevity). In this study, we investigated the relationships between body size and secondary sexual characters (breeding colour, hump height and snout length), and sperm characteristics of one‐year‐old (newly matured) Dolly Varden char. Small males displayed higher sperm concentrations than large males. Moreover, males with dull breeding colours, but not with lesser snout length and hump height, displayed an increased sperm velocity compared to males with bright colours, suggesting a trade‐off between sperm quantity and the investment in breeding colour. In addition, sperm longevity decreased as sperm swimming velocity increased. These findings indicate that small males with dull breeding colours improve the quantity and quality of their sperm to a great extent to enhance their chances of reproductive success.     

Loop-mediated isothermal amplification for the rapid, sensitive, and specific detection of the O9 group of Salmonella in chickens

In the present study, the loop-mediated isothermal amplification (LAMP) assay was developed to amplify the fragments of the O9 Salmonella-specific insertion element and evaluated in the laboratory for its potential use in a field situation, such as poultry farms. Among the bacteria tested, a positive reaction was observed only for 128 strains of 6 serovars of the O9 group Salmonella, such as Enteritidis (SE) and Pullorum. The detection limit of the LAMP assay was 10(3)CFU/ml, which was more sensitive than that of the polymerase chain reaction (PCR) assay with the same target gene (10(6)CFU/ml). The final results were obtained within 30 min for the LAMP assay, while the PCR assay needed a total of 120 min. When the LAMP assay was applied to the enrichment broth mixed with cecal dropping samples either spiked with SE in vitro or excreted by SE-inoculated hens, the results were comparable to those of the conventional plating method including 2 separate enrichments. In conclusion, the LAMP assay developed in the present study is an effective method for the specific detection of the O9 group Salmonella serovars, including SE.     

Measurement of telomerase activity in bovine leukaemia virus infected cows

Telomerase adds new telomeric sequences to the end of chromosomal DNA in order to overcome the end-replication problem. The upregulation of telomerase activity in tumours has been reported in humans and some mammals and is considered to be a tumour marker; however, such activity has not been investigated in cows. Therefore, we investigated telomerase activity in bovine leukaemia, the most common tumour in cows and its relationship with the bovine leukaemia virus (BLV) infection, which is the major cause of leukaemia. Telomerase activity was detected in 25 of 29 bovine leukaemia tissue samples. In peripheral blood lymphocytes (PBL) from BLV-infected cases that did not develop the tumour, telomerase activity was detected in 11 of 71 cases (15.5%). When these cases were classified based on serological tests and the peripheral blood lymphocyte count, the telomerase activity was observed to be the highest in the seropositive, non-lymphoproliferative (PBL<8000 microl(-1)) cases (three of seven cases, 42.9%), and not observed in the lymphoproliferative cases (PBL<16,000 microl(-1)) except in one case. Although the precise pathogenesis of BLV-related diseases remains obscure, persistent lymphocytosis is considered as a pre-neoplastic state. In contrast, our results suggested that given the fact that telomerase activity indicates tumour development, the aleukaemic stage could be defined as the 'pre-neoplastic state'. In conclusion, similar to many tumours in humans, telomerase activity was detected in bovine leukaemia; further, this activity can be a potentially useful prediction marker for tumour development and/or a good therapeutic target.     

Salsolinol is present in the bovine posterior pituitary gland and stimulates the release of prolactin both in vivo and in vitro in ruminants

The aims of the present study were to determine whether salsolinol (SAL), a dopamine-related compound, is present in the bovine posterior pituitary (PP) gland, and to clarify the effect of SAL on the secretion of prolactin (PRL) in ruminants. SAL was detected in extract of bovine PP gland using high-pressure liquid chromatography with electrochemical detection (HPLC-EC). A single intravenous (i.v.) injection of SAL (5 and 10mg/kg body weight) significantly and dose-dependently stimulated the release of PRL in goats (P<0.05). Plasma PRL levels reached a peak 10min after the injection, then gradually returned to basal values in 60-80min. The PRL-releasing pattern was similar to that in response to sulpiride (a dopamine receptor antagonist). The intracerebroventricular (i.c.v.) injection of 1mg of SAL had no significant effect on the release of PRL in calves, however, 5mg significantly stimulated the release (P<0.05) with peak values reached 30-40min after the injection. Moreover, SAL significantly stimulated the release of PRL from cultured bovine anterior pituitary cells at doses of 10(-6) and 10(-5)M, compared to control cells (P<0.05). Taken together, our data clearly show that SAL is present in extract of the PP gland of ruminants, and has PRL-releasing activity both in vivo and in vitro. Therefore, this endogenous compound is a strong candidate for the factor having PRL-releasing activity that has been previously detected in extract of the bovine PP gland.     

Interaction of GH polymorphism with body weight and endocrine functions in Japanese black calves

We assessed the interaction of GH gene polymorphisms (AA, AB and BB genotypes) with body weight and measures of endocrine function in Japanese black calves at 10 months of age. The average body weight for the BB genotype (281+/-5 kg) was significantly lower (P=0.0017, ANOVA) than those for the AA (324+/-9 kg) and AB (317+/-7 kg) genotypes. Plasma concentrations of insulin and IGF-I were greater for the AA genotype than for the AB genotype, and AB and BB genotypes, respectively. There were significant differences in the triglyceride and cholesterol concentrations among the GH genotypes. The area under the basal GH concentration was significantly greater (P=0.0314) for the AA genotype than for the two other genotypes. The incremental area over the basal GH concentrations in response to intravenous GHRH injection (0.4 microg/kg BW) was significantly smaller (P=0.0005) for the BB genotype than for the two other genotypes. In addition, linear regression analysis between GH incremental area induced by GHRH and body weight demonstrated that there was a positive linear correlation (r=0.6496, P<0.002) for incremental areas less than 600 ng min/ml, but a negative correlation (r=0.6473, P<0.05) for incremental areas over 600 ng min/ml. These findings indicate that the GH genotypes of the animals could be associated with difference in the GH response in Japanese black cattle at 10 months of age. We also observed a relationship between genotype and animal performances, but other studies on more animals in different conditions must be realized to make a definite conclusion.     

Molecular characterization of an H1N2 swine influenza virus isolated in Miyazaki, Japan, in 2006

Swine influenza virus (SIV) was isolated from a farm in Miyazaki Prefecture in Japan in July 2006. An isolate was genetically subtyped as H1N2 and was designated A/swine/Miyazaki/1/2006 (H1N2). The nucleotide sequences of all eight viral RNA segments were determined, and then phylogenetic analysis was performed using the neighbor-joining method. All segments were shown to be closely related to those of Japanese SIV H1N2 isolates, which have been circulating since the 1980s. The results indicate the persistence of the SIV H1N2 subtype in the Japanese pig population for more than two decades and emphasize the importance of continuous surveillance for SIV.     

Preimplantation development of embryos in labrador retrievers

Preimplantation development of canine embryos is not well understood. To understand the timing of preattachment embryogenesis relative to the luteinizing hormone (LH) surge, early embryonic development was examined in Labrador Retrievers after artificial insemination. The embryos migrated from the oviduct to the uterus beginning on day 11 after the LH surge. This transport must be completed within 24 h. By day 13 after the LH surge, all of the embryos had moved and were localized in the uterus. The embryos developed to the morula stage within 11-13 days and to the blastocyst stage within 14 days after the LH surge, respectively. These findings add to the current understanding concerning the physiology of preimplantation development and should help further develop assisted reproductive techniques in canine species, such as cryopreservation and subsequent embryo transfer.