Establishment of a Conditional Transgenic System Using the 2A Peptide in the Female Mouse Germline

Transgenic mice are essential research tools in developmental biology studies. The 2A peptide allows multiple genes to be expressed simultaneously at comparable levels in somatic cells, but there are no reports of it being used successfully in germ cells. We constructed a Cre/loxP-based conditional vector containing the 2A peptide to significantly enhance the expression of a reporter and target gene from a constitutive promoter in oocytes. Mice with a transgene insertion containing the chicken β-actin promoter, floxed EGFP-polyA cassette, mCherry reporter, 2A peptide and target gene DNA methyltransferase 3A2 (Dnmt3a2) were crossed with TNAP- or Vasa-Cre mice to produce offspring, in which mCherry and DNMT3A2 proteins were highly expressed in oocytes upon Cre-mediated removal of EGFP-polyA. This novel transgenic mouse line based on the 2A expression system can serve as a useful tool for examining gene function during oogenesis.     

Concurrent infection with Trichinella spiralis and other helminths in pigs

The aim of this study was to investigate possible influence of different helmintosis in the development of Trichinella spiralis in experimental infected pigs. Forty-two Iberian pigs were allocated to six groups. Three groups were single inoculated with Ascaris suum, Metastrongylus apri or T. spiralis, respectively. Two groups were co-infected with T. spiralis and A. suum or T. spiralis and M. apri, respectively, while the last group included uninfected control pigs. Clinical signs were only observed in pigs with single or concurrent M. apri infections, with more severe respiratory symptoms in pigs with mixed M. apri infection. The number of A. suum and M. apri lung larvae, intestinal larvae of A. suum and adult M. apri were reduced in pigs with mixed Trichinella infections compared to pigs with single infections. In contrast, the number of liver white spots was higher in pigs with mixed infections. While T. spiralis muscular larval burdens were increased in pigs concomitantly infected with M. apri, they were reduced in pigs concomitantly infected with A. suum, compared to pigs receiving single infections with either of these helminths. Pigs with single or mixed A. suum infections showed higher eosinophil levels compared to the remaining groups. IgGt, IgG1, IgG2 and IgM against T. spiralis antigen could not be detected in pigs with single Ascaris or Metastrongylus infections, indicating that no cross-antibodies were produced. IgGt, IgG1 and IgM antibodies were detected earlier and generally at higher levels in mixed T. spiralis infections compared to single T. spiralis infections. The results suggest that T. spiralis had a low synergistic interaction with M. apri in concomitantly infected pigs, and an antagonistic interaction in concurrent infection with A. suum.     

Optimization and validation of a flow cytometric method for immunophenotyping peripheral blood lymphocytes from cynomolgus monkeys (Macaca fascicularis)

BACKGROUND: Guidelines published by the Food and Drug Administration and Center for Human Medicinal Products describe the need to assess immunotoxic effects in nonclinical studies that evaluate drug toxicity, including the use of immunophenotyping to measure immunotoxicity. We are not aware of previous studies, however, that have validated methods for immunophenotyping peripheral blood lymphocyte subsets in whole blood samples from cynomolgus monkeys. OBJECTIVE: The purpose of this study was to optimize and validate a flow cytometric assay for immunophenotyping lymphocytes in the peripheral blood of cynomolgus monkeys. METHODS: A series of prevalidation experiments were done to determine optimal reagents, volumes, timing, and other procedural details of the flow cytometric assay. Using the optimized method, we then determined precision, interindividual variation, laboratory-to-laboratory variability, and sample stability. Stabilized human blood was used as a positive control for staining, processing, and analysis. The percentage and number of pan-T cells (CD3+), T-helper cells (CD3+4+), T cytotoxic/suppressor cells (CD3+8+), natural killer cells (CD3-16+), and B-cells (CD3-20+) were determined in 146 male and 140 female, clinically healthy monkeys and reference intervals were calculated. RESULTS: By doing 4-color staining with a lyse-wash method, intra- and interassay precision were <5% for all lymphocyte subsets. Variability between technicians and laboratories was minimal (CVs<3%). Samples were stable for up to 24 hours after staining and fixing. CONCLUSIONS: The validated method is extremely robust and can be performed under good laboratory practice conditions to support nonclinical studies. Reference intervals for lymphocyte subsets were similar to those previously reported.     

Identification of Ligands for Chicken Transient Receptor Potential Ankyrin 1 Channel and Chemosensory Perception of Herbal Compounds in Chickens

The pungency induced by spices and herbs plays an important role in food choice and appetite, and it is suggested that adding spices and herbs to feed as natural alternatives to antibiotics has beneficial effects in poultry farming. However, our knowledge of the chemosensory perception of herbal compounds in chickens is limited. Transient receptor potential ankyrin 1 (TRPA1) is involved in the sensory perception of various herbal compounds. Here, we performed calcium imaging and electrophysiological analyses using cells transiently expressing chicken TRPA1 (cTRPA1) and identified two novel cTRPA1 ligands—eugenol and thymol. In a behavioral assay, chickens responded to cTRPA1 ligands, including eugenol, thymol, cinnamaldehyde, carvacrol, and allyl isothiocyanate. These results provide evidence that chickens have a functional TRPA1 channel and chemosensory perception of various herbal compounds.     

Post-release movement and diel activity patterns of hatchery-reared and wild black-spot tuskfish <Emphasis Type="Italic">Choerodon schoenleinii</Emphasis> determined by ultrasonic telemetry

ABSTRACT:   Post-release movement and diel activity patterns of hatchery-reared and wild black-spot tuskfish were examined using ultrasonic telemetry. Five hatchery-reared and four wild fish were released in the sandy bottom of Urasoko Bay, Ishigaki Island, Okinawa, Japan, and monitored using automated monitoring receivers from November 2005 to February 2006. Both hatchery-reared and wild fish tended to stay near the release site for over two weeks, before leaving the release site. Both hatchery-reared and wild tuskfish showed diurnal rhythm intermittently; signals were recorded more frequently in the daytime and less frequently in the nighttime, suggesting that the fish of both origins were active during the day and inactive during the night. These findings indicate that the one-year-old hatchery-reared tuskfish have some consistent behavioral characteristics with those of the wild.     

Effect of water temperature and chlorophyll abundance on shell growth of the Japanese pearl oyster, Pinctada fucata martensii, in suspended culture at different depths and sites

To understand the relationships between shell growth and some environmental factors, we examined the relationships between water temperature or chlorophyll abundance and the shell growth of the Japanese pearl oyster, Pinctada fucata martensii, suspended at three different depths at two sites. Growth in height, length and thickness of the shells were limited by water temperature during winter (< 20 °C), whereas growth in thickness correlated with food abundance, measured as chlorophyll, during early summer (> 20 °C). These results suggest that the shell of P. fucata martensii could grow well at locations with greater abundance of food and adequate water temperatures (20–26 °C), resulting in a longer growing season.     

The palliative efficacy of modified Mohs paste for controlling canine and feline malignant skin wounds

In veterinary medicine, the management of malignant skin wounds is highly challenging. We conducted a study on seven case animals (four dogs and three cats) which presented with malignant skin wounds. All seven animals had signs and symptoms which were controlled following treatment with a modified Mohs paste. Upon obtaining informed consent from their owners, the animals requiring management of malignant wounds were enrolled in this study. The modified Mohs paste was prepared by mixing zinc chloride, zinc oxide starch powder, glycerin, and distilled water. The modified Mohs paste was topically applied to and left to remain on the malignant wounds for one hour, under controlled conditions. Once the paste was removed, the wounds were irrigated with a solution of sterile saline. At the first examination, the wounds of each animal were observed for signs of exudate, malodor, and bleeding. In every case, visible improvement was observed immediately after the modified Mohs paste treatment. Specifically, the size of the malignant wounds, and the number of times the dressing gauze required changing, significantly decreased (p < 0.05 and p < 0.01, respectively). The open malignant skin wounds caused by mammary gland tumors disappeared in two cases. The Mohs paste has been shown to be a viable option for the palliative treatment in canine and feline malignant skin wound management.     

Preliminary study on homing,site fidelity,and diel movement of black rockfish <Emphasis Type="Italic">Sebastes inermis</Emphasis> measured by acoustic telemetry

Several species of fish exhibit strong homing ability and distinct site fidelity to their original habitats and spawning sites. We have studied the homing ability, site fidelity, and diel movement patterns of three nocturnal black rockfish Sebastes inermis. The patterns were studied by radio-linked acoustic positioning system (VRAP) and fixed monitoring receivers (VR2) in Maizuru Bay, Japan from June to July 2004. All tagged fish were displaced by 80–120 m, but they returned to their respective captured sites at dusk, regardless of the time of release. The fish tagged with a depth sensor transmitter displayed active vertical movement just after the release, and subsequently swam near the bottom depth around the captured site. After homing, they displayed small home ranges [average 815 ± 265 m² (SD)] around a single rocky area with one or two core areas (average 64 ± 30 m²). One core area was utilized during the daylight and dark periods, whereas the other was used primarily during the dark period. The depth-tagged fish primarily used the depth of the rock crevice during the daylight, and it spent its dark period between the surface and the deeper bottom depth.