Expression and subcellular localization of GSE protein in germ cells and preimplantation embryos

We previously identified a novel gonad-specific expression gene (Gse) and investigated its expression during gametogenesis in the mouse testis and ovary. In this study, we generated a polyclonal antibody to GSE protein and determined the profiles of the protein's expression in germ cells and preimplantation embryos in detail using immunocytochemical and immunofluorescence staining. In a Western blot analysis, the anti-GSE antibody recognized long and short isoforms (approximately 27.6 kDa and 23.1 kDa) of the protein in the mouse testis and the long isoform in the ovary. In the mouse testis, GSE protein was expressed in spermatocytes I in the pachytene stage, round spermatids, and elongated spermatids. In the mouse ovary, the protein was located in the cytoplasm and nucleus of all oocytes regardless of the stage of the ovarian follicles. In preimplantation embryos from the pronuclear to blastocyst stage, however, GSE protein was mainly detected in the nuclei of cells. At the blastocyst stage, the protein was confirmed to have accumulated in the inner cell mass (ICM), whereas it had mostly disappeared from the trophectoderm (TE). These findings suggest that GSE protein may play a role in the establishment of nuclear totipotency and may be associated with early lineage specification.     

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts.     

A cytolethal distending toxin gene-based multiplex PCR assay for detection of Campylobacter spp. in stool specimens and comparison with culture method

In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248).     

Trapped neutrophil syndrome in a Border Collie dog: clinical, clinico-pathologic, and molecular findings

Trapped neutrophil syndrome (TNS) is an autosomal recessive inherited neutropenia known in Border Collies since the 1990's. Recently, the causative mutation has been identified in the canine VPS13B gene and a DNA-based diagnosis has now become available. The present paper describes clinical and clinico-pathologic findings in a Border Collie with TNS that was molecularly diagnosed for the first time in Japan. In a 10-week-old male Border Collie with microgenesis and symptoms related to recurrent infections, a hematological examination revealed severe leukopenia due to neutropenia, suggesting the dog to be affected by inherited neutropenic immunodeficiency. Direct DNA sequencing demonstrated that the dog was homozygous for the causative mutation of TNS and both its parents were heterozygous carriers. In addition, a simple and rapid polymerase chain reaction-based length polymorphism analysis coupled with microchip electrophoresis was developed for the genotyping of TNS. This assay could discriminate clearly all genotypes, suggesting that it was suitable for both individual diagnosis and large-scale surveys for prevention.     

Humeral Chondrosarcoma in a Hokkaido Brown Bear (Ursus arctos yesoensis)

Humeral chondrosarcoma was found in an 18-year-old male Hokkaido brown bear (Ursus arctos yesoensis). Necropsy revealed a large firm mass under the left superficial pectoral muscle at the axillary region. The mass involved the left shoulder joint and peripheral muscles, and connected to the head of the humerus with osteolysis. Histopathologically, the mass was composed of irregularly shaped myxomatous to cartilaginous tumor lobules. The tumor cell showed moderate nuclear atypia with a relatively high mitotic index, especially in the edges of the myxomatous lobules. The tumor cells were positively immunostained with vimentin and S-100 protein. Based on these findings, the tumors were diagnosed as chondrosarcoma. Metastases were found in the left axillary lymph node, lungs, liver and kidney.     

Plasma Profiles of Glucose, Insulin and Lipids in the Male WBN/Kob-Lepr(fa) Rat, a New Model of Type 2 Diabetes with Obesity

Plasma profiles of glucose, insulin and lipids were examined in the male WBN/Kob-Lepr(fa) (fa/fa) rat, a new model of type 2 diabetes (T2D), in comparison with age-matched original male WBN/Kob (lean) rats. The fa/fa rats developed hypertriglycemia, obesity and hyperglycemia from 5, 7, and 9 weeks of age, respectively. Plasma insulin levels in fa/fa rats were significantly higher than those in lean rats at 5 weeks of age, but after 11 weeks of age gradually declined to the levels in lean rats. HOMA-IR, a measure of insulin resistance status, showed that fa/fa rats had insulin resistance. The fa/fa rat has the potential to become an important animal model of T2D with obesity.     

Development of a method for detecting Coxiella burnetii in cheese samples

Coxiella burnetii is the causative agent of Q fever, and the main route of infection in humans is inhalation of contaminated aerosols. Although oral transmission by contaminated raw milk or dairy products is also a possible route of human infection, there have been few studies investigating the presence of C. burnetii in dairy products. We developed a new method of extracting DNA from cheese and detecting C. burnetii DNA in cheese samples with a nested PCR assay. The limit of detection was 6.0 × 10(2) C. burnetii particles per gram. We subsequently used this method to examine the presence of C. burnetii in cheese at commercial markets in Tokyo from June 2005 to December 2008. Twenty-eight of 147 cheese samples were found to be positive for C. burnetii DNA. However, when we assessed the viability of C. burnetii by inoculating mice with DNA-positive samples, all of the samples were found to be negative. Thus, the viability of C. burnetii appears to have been lost in these cheese samples.     

Effects of soil scraping on the reclamation of tsunami-damaged paddy soil

We investigated the possibility of tsunami-damaged paddy soil remediation in Japan using a scraping method. We collected undisturbed soil blocks to maintain the soil structure from paddy damaged by salt from the tsunami. Scraping treatments to remove the top 2 cm of the soil surface were conducted once, twice, and thrice after drying naturally in a greenhouse environment, as well as a noscraping control. Electrical conductivity (EC) of the soil dramatically decreased to < 1.36 dS m^?1 in the residual soils after scraping. The EC values of the residual soils were significantly lower after being scraped twice or thrice compared with a single scraping. Similarly, the mean NaCl removal rates were significantly higher with two (91%) or three scrapings (97.2%) compared with a single scraping (73.3%). The growth of rice plants was almost normal in the residual soils based on a visual score (approximately 3 on average) using a standard evaluation system at 4 weeks after transplantation. There were no significant differences in the grain yields from the residual soils with different scraping frequencies, and the EC values of all the residual soils were < 1.36 dS m^?1. This study clearly indicates that there were reductions in the EC and good plant growth performance in the residual soils after scraping, and there was also a high rate of NaCl removal. Therefore, scraping may be a useful method for the remediation of salt-damaged paddy soils when irrigation or culvert systems are affected severely by tsunamis and earthquakes.     

Fine mapping of quantitative trait loci affecting organ weights by mouse intersubspecific subcongenic strain analysis

The genetic architecture of organ weights is not well understood. In this study, we fine‐mapped quantitative trait loci (QTLs) affecting organ weights by characterizing six intersubspecific subcongenic mouse strains with overlapping and non‐overlapping genomic regions on chromosome 2 derived from wild Mus musculus castaneus. QTLs for heart, lung, spleen and kidney weights were revealed on a 6.38‐Mb genomic region between two microsatellite markers, D2Mit323 and D2Mit472. Effects of the castaneus alleles at the organ weight QTLs were all opposite in direction to a body weight QTL previously mapped to the same genomic region. In addition, new QTLs for lung and kidney weights were revealed on a different 3.57‐Mb region between D2Mit205 and D2Mit182. Their effects were dependent on that of another body weight QTL previously mapped to that genomic region. The organ weight QTLs revealed were all duplicated in independent analyses with F₂ intercross populations between subcongenic strains carrying these QTLs and their background strain. The findings suggested that organ weights are not exclusively regulated by genetic loci that commonly influence overall body weight and rather that there are loci contributing to the growth of specific organs only.     

Empirical Percentile Growth Curves with Z-scores Considering Seasonal Compensatory Growths for Japanese Thoroughbred Horses

Percentile growth curves are often used as a clinical indicator to evaluate variations of children’s growth status. In this study, we propose empirical percentile growth curves using Z-scores adapted for Japanese Thoroughbred horses, with considerations of the seasonal compensatory growth that is a typical characteristic of seasonal breeding animals. We previously developed new growth curve equations for Japanese Thoroughbreds adjusting for compensatory growth. Individual horses and residual effects were included as random effects in the growth curve equation model and their variance components were estimated. Based on the Z-scores of the estimated variance components, empirical percentile growth curves were constructed. A total of 5,594 and 5,680 body weight and age measurements of male and female Thoroughbreds, respectively, and 3,770 withers height and age measurements were used in the analyses. The developed empirical percentile growth curves using Z-scores are computationally feasible and useful for monitoring individual growth parameters of body weight and withers height of young Thoroughbred horses, especially during compensatory growth periods.