Identification of a c-kit exon 8 internal tandem duplication in a feline mast cell tumor case and its favorable response to the tyrosine kinase inhibitor imatinib mesylate

The gain-of-function mutations within c-kit, a protooncogene encoding KIT, induce constitutive ligand-independent kinase activation and are important for the pathogenesis of mast cell proliferative disease in humans as well as in dogs. Despite the clinical importance of feline mast cell tumors, no mutation has been shown within the c-kit gene in cats. In the present report, we analyzed the c-kit nucleotide sequence in the case of a cat that showed systemic mastocytosis and mastocytemia. Within the c-kit cDNA prepared from the malignant mast cells, we identified an 12-bp internal tandem duplication at the region corresponding to exon 8, resulting in a four amino acid insertion between residues Thr418 and His419 within the fifth immunoglobulin-like domain of KIT. The cat underwent therapy with the kinase inhibitor imatinib mesylate (Gleevec) at a dose of 10mg/kg. The tumor masses greatly responded and were undetectable after 5 weeks of treatment. Correspondingly, the number of mast cells in the peripheral blood was markedly reduced. It is, therefore, considered that the internal tandem duplication within the domain contributes to the neoplastic transformation of mast cells in the cat by increasing KIT phosphorylation.     

Molecular characterisation of equine group A rotavirus, Nasuno, isolated in Tochigi Prefecture, Japan

In this study, equine group A rotavirus (RV-A), Nasuno, isolated from foal diarrhoea in Tochigi Prefecture, Japan was characterised genetically by sequence analysis of the genome segments encoding VP4 and VP7. The nucleotide and deduced amino acid sequences revealed high homology with P[12] RV-As (94.0-99.3% and 94.9-99.4%) and G3 RV-As (86.9-99.5% and 91.1-99.4%). Nasuno was also classified into P[12] and G3 in the phylogenetic analysis of the nucleotide sequences of the genome segments encoding VP4 and VP7.     

Effect of forest structure on the spatial variation in soil respiration in a Bornean tropical rainforest

This study was undertaken to identify critical and practical factors explaining spatial variations in soil respiration and to estimate stand-scale soil respiration in an aseasonal tropical rainforest on Borneo Island. To this aim, we conducted soil respiration measurements at 25 points in a 40 m × 40 m subplot of a 4 ha study plot between 2002 and 2006, and examined the spatial variation in soil respiration averaged over the 4 years in relation to soil, root, and forest structural factors. In addition, we examined the spatial representativeness of soil respiration measured in the subplot using a specific scaling procedure. Consequently, we found significant positive correlation between the soil respiration and forest structural parameters such as the mean diameter at breast height (DBH), total basal area, and maximum DBH within 6 m of the measurement points. The most important factor was the mean DBH within 6 m of the measurement points, which had a significant linear relationship with soil respiration. Using the derived linear regression and an inventory dataset, we estimated the 4 ha plot-scale soil respiration. The 4 ha plot-scale estimation (6.0 μmol m⁻² s⁻¹) was nearly identical to the subplot-scale measurements (5.7 μmol m⁻² s⁻¹), which were roughly comparable to the nocturnal CO₂ fluxes calculated using the eddy covariance technique. In addition, we discuss characteristics of the stand-scale soil respiration at this site by comparing with those of other forests reported in previous literature in terms of the soil C balance. Soil respiration at our site was noticeably greater, relative to the incident litterfall amount, than soil respiration in other tropical and temperate forests probably owing to the larger total belowground C allocation by emergent trees. Overall, this study suggests the arrangement of emergent trees with larger DBH and their belowground C allocation could be primary factors controlling spatial variations in soil respiration in the tropical rainforest.     

Comparison of two strains of avian infectious bronchitis virus for their interferon induction, viral growth and development of virus-neutralizing antibody in experimentally-infected chickens

Two field strains of avian infectious bronchitis virus were tested in specific pathogen-free chickens for interferon induction, growth and stimulation of virus-neutralizing antibody. Both strains induced interferon in lungs and trachea and neither strain induced it in kidneys. Strain Kagoshima-34, isolated from the kidneys of a chicken that had died of nephrosis/nephritis, induced interferon earlier than Strain Tottori-2, which was isolated from the trachea of a chicken with respiratory disease. Virus growth was generally more prolonged with Kagoshima-34, especially in the kidneys, but Tottori-2 persisted longer in the lungs.     

Suppression of heat-stable enterotoxin production by Yersinia spp. in milk

One of 16 raw milk isolates of Yersinia enterocolitica and Y. intermedia produced heat-stable enterotoxin (ST) in milk at 25 degrees C but not at 4 degrees C after 21 days of incubation. A catabolite repression of ST synthesis by the lactose-fermenting strain of Y. enterocolitica was observed when 4.6% lactose was added to trypticase soy broth. However, the lactose-fermenting strain was killed by acid produced by lactose fermentation in milk and did not produce ST in milk with the pH adjusted to neutrality. This study suggested that lactose and fat in milk are not the fundamental inhibitors of ST synthesis by Y. enterocolitica and that repression of ST synthesis may be related to other components.     

Induction of dendritic cell-mediated immune responses against canine malignant melanoma cells

To establish the basis for the use of dendritic cells (DC) in the treatment of canine melanoma, dogs were vaccinated using autologous DC pulsed with canine melanoma CMM2 cell lysate in the presence of keyhole limpet haemocyanin (KLH) in vitro (CMM2-KLH-DC), and the induction of immune responses against CMM2 cells in vivo was examined using the delayed-type hypersensitivity (DTH) skin test. The DTH responses against CMM2 cells and KLH were observed in dogs vaccinated with CMM2-KLH-DC, while the responses against KLH but not CMM2 cells were detected with DC pulsed with KLH alone (KLH-DC). Recruitment of CD8 and CD4 T cells was detected in the positively responding sites, suggested that vaccination with CMM2-KLH-DC efficiently elicits T cell-mediated immunity against CMM-2 cells in vivo. These findings demonstrate the potential utility of DC-based tumour vaccination in the treatment of canine malignant melanoma.     

Establishment of a BRAF V595E-mutant canine prostate cancer cell line and the antitumor effects of MEK inhibitors against canine prostate cancer

Canine prostate cancer (cPCa) is a malignant neoplasm with no effective therapy. The BRAF V595E mutation, corresponding to the human BRAF V600E mutation, is found frequently in cPCa. Activating BRAF mutations are recognized as oncogenic drivers, and blockade of MAPK/ERK phosphorylation may be an effective therapeutic target against BRAF-mutated tumours. The aim of this study was to establish a novel cPCa cell line and to clarify the antitumor effects of MEK inhibitors on cPCa in vitro and in vivo. We established the novel CHP-2 cPCa cell line that was derived from the prostatic tissue of a cPCa patient. Sequencing of the canine BRAF gene in two cPCa cell lines revealed the presence of the BRAF V595E mutation. MEK inhibitors (trametinib, cobimetinib and mirdametinib) strongly suppressed cell proliferation in vitro, and trametinib showed the highest efficacy against cPCa cells with minimal cytotoxicity to non-cancer COPK cells. Furthermore, we orally administered 0.3 or 1.0 mg/kg trametinib to CHP-2 xenografted mice and examined its antitumor effects in vivo. Trametinib reduced tumour volume, decreased phosphorylated ERK levels, and lowered Ki-67 expression in xenografts in a dose-dependent manner. Although no clear adverse events were observed with administration, trametinib-treated xenografts showed osteogenesis that was independent of dosage. Our results indicate that trametinib induces cell cycle arrest by inhibiting ERK activation, resulting in cPCa tumour regression in a dose-dependent manner. MEK inhibitors, in addition to BRAF inhibitors, may be a targeted agent option for cPCa with the BRAF V595E mutation.