Analysis of genomic mutation and immunohistochemistry of platelet‐derived growth factor receptors in canine vascular tumours

We examined whether mutation of the platelet‐derived growth factor receptor protein tyrosine kinase (PDGFR)‐α and PDGFR‐β genes contributes to their overexpression in canine vascular tumours. Genomic sequences of trans‐ or juxtamembrane regions of PDGFR‐α and PDGFR‐β were analysed with immunohistochemical staining and polymerase chain reaction‐direct sequencing using DNA from paraffin‐embedded neoplastic tissues of 27 hemangiosarcomas (HSAs) and 20 hemangiomas (HAs). Immunohistochemically, 75% of the HA cases were positive for PDGFR‐α and almost most of the HA cases were negative for PDGFR‐β. Of the HSA cases, 55.6% were negative for PDGFR‐α and 63% were strongly positive for PDGFR‐β. Among the HA cases, 1 missense mutation was detected in PDGFR‐α exon 18 and 1 in PDGFR‐β exon 17. Two HSA cases had missense mutations in exon 14 and 1 in exon 17 of PDGFR‐β. Thus, genomic mutation of trans‐ or juxtamembrane regions of PDGFRs was not the main mechanism driving the activation of receptors in HSA and HA.     

Isolation and chemiluminescent properties of ferret (Mustela putorius furo) polymorphonuclear cells

Ferret polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation. Using a 50% Percoll solution (density=1.066), PMNs and PBMCs were successfully isolated after centrifugation; the purities of the PMNs and PBMCs were 94.2% and 95.6%, respectively. To evaluate the function of isolated ferret PMNs, we measured the superoxide generation with a MCLA-dependent chemiluminescence assay. The isolated ferret PMNs responded to phorbol 12-myristate 13-acetate (PMA) with kinetics similar to that of human PMNs. The ferret PMNs did not respond to N-formyl-Met-Leu-Phe (fMLF), unlike human PMNs, which rapidly responded. Thus, authors established a method for the rapid separation of highly purified populations of functional PMNs from the whole blood of ferrets.     

Phylogenetic characterization of rabies virus isolates from Carnivora in Brazil

The incidence of canine rabies has been widely reported in Brazil, and new rabies virus (RV) variants, genetically similar to canine RV, have recently been isolated from foxes. In order to derive the epidemiological characteristics of Brazilian Carnivora RV, Brazilian RVs isolated from dogs, cats, and foxes were genetically analyzed. Brazilian Carnivora RV isolates were divided into 2 main lineages. The predominant lineage was found in dogs and cats, which included the Argentinean and Bolivian Carnivora RV isolates, and was extensively distributed throughout Brazil and surrounding countries. The other lineage consisted of three sublineages containing Brazilian dog and fox RV isolates, with the dog sublineages located on an internal branch of 2 fox sublineages, suggesting that RV transmission events might have occurred between foxes and dogs in the past. These results suggest that contact between dogs and wildlife has the potential to generate new rabies variants and that it is important to control RV infection cycles in both dogs and wildlife to prevent spread of rabies infection.     

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.     

Identification of ommastrephid squid paralarvae collected in northern Hawaiian waters and phylogenetic implications for the family Ommastrephidae using mtDNA analysis

ABSTRACT:   A total of 110 adult individuals from four ommastrephid (family Ommastrephidae) squid species ( Ommastrephes bartramii, Sthenoteuthis oualaniensis, Eucleoteuthis luminosa, and Hyaloteuthis pelagica ) were used to obtain diagnostic DNA markers for species identification. Restriction fragment length polymorphism (RFLP) analysis of a partial segment (855 bp) of the mitochondrial DNA cytochrome oxidase I (COI) gene amplified by polymerase chain reaction (PCR) revealed that the restriction profiles of two endonucleases ( Alu  I and Tsp 509 I) were diagnostic for species identification. The restriction assay partially supplemented with nucleotide sequence analysis successfully assigned 69 damaged and morphologically equivocal ommastrephid paralarvae collected in northern Hawaiian waters, identifying 60 O. bartramii , eight S. oualaniensis , and one E. luminosa . The family Ommastrephidae appears to be monophyletic. Although the phylogenetic relationships among genera were not resolved well due to apparent homoplasy and large genetic divergence between species, COI sequence data without transitions provided support for subfamily level relationships.     

Studies on the green liver in cultured red sea bream fed low level and non-fish meal diets: Relationship between hepatic taurine and biliverdin levels

SUMMARY: The aim of this study was to investigate the cause of green liver in red sea bream fed substitute protein diets. Red sea bream Pagrus major was given either of the following diets for 28 weeks: (1) control diet (50% fish meal), (2) low level fish meal diet (15% fish meal), and (3) non-fish meal diet (0% fish meal). The green liver was observed in all groups tested, but the incidence was much higher in the experimental diet groups. The feeding of substitute protein diets reduced plasma triglyceride and cholesterol levels. However, there was no significant difference in plasma hepatic enzyme activities and plasma bile salts concentration among the treatments. Fish fed the substitute protein diets showed low hepatic taurine levels with an appearance of a biliverdin in the liver. Moreover, the proportion of ditaurobilirubin to total biliary bile pigments was significantly lower in fish fed the substitute protein diets. These data indicate that feeding of substitute protein diets did not induce any cholestatic hepatobiliary obstructions and that the low hepatic taurine level was one of the probable factors responsible for the occurrence of green liver in red sea bream fed substitute protein diets.