Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens)

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.     

A comparison of changes in stable isotope ratios in the epidermal mucus and muscle tissue of slow‐growing adult catfish

Although stable isotope analysis is a powerful tool for determining diet, migration patterns and the structure of food webs in aquatic systems, the slow response of isotopic ratios in the widely used muscle tissue often hampers this approach, particularly in slow‐growing or adult fishes. We conducted a diet‐switch experiment to compare the changes in the stable carbon isotope ratio (δ¹³C values) in the epidermal mucus and muscle tissue of five‐year‐old catfish (Silurus asotus). The isotope ratios in noninvasively sampled mucus changed more rapidly than those in the muscle tissue. As isotopic change in mucus was relatively rapid, this technique can be used over a finer timescale than traditional isotopic analyses using the muscle tissue. The isotopic change half‐life in our experimental condition was 200 days, which would not be short enough for some research purposes. Examining mucus along with the muscle would enable food habits of slow‐growing fishes to be determined over different timescales. However, the rate of isotopic change in the mucus was negatively affected by the size of fish and was slower than the previously reported rates in juvenile steelhead. These findings suggest that mucus turnover rates need to be determined prior to the field data interpretation.     

Development of a PCR test for the identification of Staphylococcus intermedius based on the 16S rDNA sequence

The development of a PCR assay based on the 16S ribosomal RNA gene (rDNA) sequence was carried out for the identification of Staphylococcus intermedius. Sixty-six strains of S. intermedius, 70 of Staphylococcus aureus and 2 of Staphylococcus hyicus were examined for the assay. The 16S rDNA, of which the PCR target fragment makes up 901 bp corresponding to the sequence data of the gene, was detected in all strains of S. intermedius, but it was not detected in any strains of either S. aureus or S. hyicus. These results suggest that the PCR allows a simple and precise identification of S. intermedius.     

断奶后多系统衰竭综合征病猪中存在沙门氏菌和猪繁殖与呼吸系统综合征病毒共感染

调查了曾经暴发沙门氏菌病的四个猪场的14头病猪,发现在这些猪的肿胀淋巴结中出现带有淋巴细胞排空的肉芽肿性炎症。应用免疫标记和PCR方法在病变部位检测到猪圆环病毒2(PCV2)抗原和PCV2DNA。此外,在这些病猪的肺脏中检测出猪繁殖与呼吸综合征病毒(PRRSV),分离到猪霍乱沙门氏菌。在9头沙门氏菌感染猪中,有5头为沙门氏菌、PMWS与PRRSV并发感染,其数量(55.6%)远远高于沙门氏菌与PMWS感染猪(22.2%)或沙门氏菌与PRRSV感染猪(22.2%)。     

Drought-induced root plasticity of two upland NERICA varieties under conditions with contrasting soil depth characteristics

To identify differences in root plasticity patterns of two upland New Rice for Africa (NERICA) varieties, NERICA 1 and 4, in response to drought under conditions with contrasting soil profile characteristics, soil moisture gradients were imposed using a sloping bed system with depths ranging 30–65 cm and a line-source sprinkler system with a uniformly shallow soil layer of 20 cm depth. Varietal differences in shoot and root growths were identified only under moderate drought conditions, 11–18% v/v soil moisture content. Further, under moderate drought soil conditions where roots could penetrate into the deep soil layer, deep root development was greater in NERICA 4 than in NERICA 1, which contributed to maintaining dry matter production. However, under soil conditions with underground impediment to deep root development, higher shoot dry weight was noted for NERICA 1 than for NERICA 4 at 11–18% v/v soil moisture content, which was attributed to increased lateral root development in the shallow soil layer in NERICA 1. Enhanced lateral root development in the 0–20-cm soil layer was identified in NERICA 1 even under soil conditions without an impediment to deep root development; however, this did not contribute to maintaining dry matter production in upland rice. Thus, we show different root developmental traits associated with drought avoidance in the two NERICA varieties, and that desirable root traits for upland rice cultivation vary depending on the target soil environment, such as the distribution of soil moisture and root penetration resistance.     

Comparison of dendritic cell-mediated immune responses among canine malignant cells

Dendritic cell (DC) vaccination is one of the most attractive immunotherapies for malignancies in dogs. To examine the differences in DC-mediated immune responses from different types of malignancies in dogs, we vaccinated dogs using autologous DCs pulsed with keyhole limpet hemocyanin (KLH) and cell lysate prepared from squamous cell carcinoma SCC2/88 (SCC-KLH-DC), histiocytic sarcoma CHS-5 (CHS-KLH-DC), or B cell leukemia GL-1 (GL-KLH-DC) in vitro. In vivo inductions of immune responses against these tumor cells were compared by the delayed-type hypersensitivity (DTH) skin test. The DTH response against SCC2/88 cells were observed in dogs vaccinated with autologous SCC-KLH-DC, while the response was undetectable against CHS-5 and GL-1 cells in dogs vaccinated with autologous CHS-KLH-DC and GL-KLH-DC. Skin biopsies taken from DTH challenge sites were then examined for immunohistochemistry, and recruitment of CD8 and CD4 T cells was detected at the site where SCC2/88 cells were inoculated in dogs vaccinated with SCC-KLH-DC. By contrast, neither CD8 nor CD4 T cell infiltration was found at the DTH challenge site in the dogs vaccinated with CHS-KLH-DC or GL-KLH-DC. These findings may reflect that the efficacy of immune induction by DC vaccination varies among tumor types and that immune responses could be inducible in squamous cell carcinoma. Our results encouraged further investigation of therapeutic vaccination for dogs with advanced squamous cell carcinoma in clinical trials.     

Control mechanism of the circadian clock for timing of cell division in vivo

Cell division in many mammalian tissues is associated with specific times of day, but just how the circadian clock controls this timing has not been clear. Here, we show in the regenerating liver (of mice) that the circadian clock controls the expression of cell cycle-related genes that in turn modulate the expression of active Cyclin B1-Cdc2 kinase, a key regulator of mitosis. Among these genes, expression of wee1 was directly regulated by the molecular components of the circadian clockwork. In contrast, the circadian clockwork oscillated independently of the cell cycle in single cells. Thus, the intracellular circadian clockwork can control the cell-division cycle directly and unidirectionally in proliferating cells.     

Spinal epidural empyema concurrent with sequestrum in a cat: a case report

A 5-month-old intact female mixed cat presented with repetitive paraplegia and drainage of pus from the back despite continuous antibiotic medication. Neurologic examination was consistent with below T3-L3 myelopathy. Computed tomography and magnetic resonance imaging revealed a contrast-enhanced mass in the L1-3 spinal canal, and bone fragments in the T13 and L1 spinal canal. Spinal epidural empyema was suspected, and hemilaminectomy was performed for T12-L2 on the right side and T11-12 on the left side. Bone fragments were diagnosed as sequestrum infected with Bacteroides sp. The cat recovered enough to ambulate next day. One month after surgery, there was no deficit in neurological function. This is the first report of spinal epidural empyema concurrent with sequestrum in a cat.