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.     

Pathogenicity of border disease virus FNK2012-1 strain isolated from a pig in the natural host,sheep

A first isolation of border disease virus (BDV) in Japan was from a pig on a farm without keeping any ruminants. Our previous study showed that this BDV, termed the FNK2012-1 strain, replicated inefficiently in swine-derived cells compared with those of ruminant origin. Pigs inoculated with this virus showed neither clinical symptoms nor viremia. In this study, we evaluated the pathogenicity of the FNK2012-1 strain in sheep, its natural host. The inoculated sheep showed clinical symptoms and transient viremia. Seroconversion was observed in the inoculated sheep. These results suggest that the FNK2012-1 strain was introduced from sheep and has not yet adapted to swine. Therefore, surveillance of border disease in Japan is necessary among both the swine and ruminant populations.     

Abnormal clonalities of B-lymphocytes in bovine leukemia virus-infected cattle with persistent lymphocytosis

Peripheral B-lymphocyte clonality of 274 bovine leukemia virus-infected cattle with lymphocytosis was analyzed using clonality PCR based on sequences of the variable region of the bovine immunoglobulin H chain. None of the cattle showed monoclonal proliferation, while 10, 31, and 233 showed minor-clonal, oligoclonal, and polyclonal proliferation, respectively. A total of 163 cattle were analyzable the following year, and lymphocytosis was maintained in 157, indicating persistent lymphocytosis (PL). B-lymphocyte clonality of the 157 PL cattle was minor-clonal in 6 (3.8%), oligoclonal in 8 (5.1%), and polyclonal in 143 (91.1%). A higher rate of enzootic bovine leukosis (EBL) onset within a year was observed in PL cattle with minor-clonal (50.0% (3/6)) and oligoclonal (25.0% (2/8)) proliferation compared to those with polyclonal (5.6% (8/143)) proliferation. Minor-clonal and oligoclonal proliferation in PL cattle may be a prognosis factor for developing EBL.     

Inheritance of resistance to brown leaf spot disease in gentians

Brown leaf spot caused by Mycochaetophora gentianae is a serious disease on gentian in Japan. Previous studies revealed that Gentiana triflora cultivars were susceptible to M. gentianae and that G. scabra cultivars and their interspecific hybrid cultivars were resistant. We subsequently analyzed the mode of inheritance of resistance to M. gentianae in gentians using several populations derived from crosses between G. scabra and G. triflora. Parental G. scabra and G. triflora lines, F₁ and BC₁ plants were inoculated with a conidial suspension and assessed for susceptibility to M. gentianae. All tested F₁ plants were resistant to M. gentianae, indicating that the resistance of G. scabra was inherited dominantly. Segregation ratios in backcross progenies using G. scabra line OK fit the Mendelian ratio of 1:1, showing the involvement of a single dominant locus. These results indicate that the resistance to M. gentianae in G. scabra line OK is controlled by a single dominant allele designated as gentian brown leaf spot resistance (GBLS-1). Finding this allele should facilitate future breeding efforts to develop resistant gentian cultivars.     

Isolation of multiple drug-resistant enteric bacteria from feces of wild Western Lowland Gorilla (Gorilla gorilla gorilla) in Gabon

Prevalence of drug-resistant bacteria in wildlife can reveal the actual level of anthropological burden on the wildlife. In this study, we isolated two multiple drug-resistant strains, GG6-2 and GG6-1-1, from 27 fresh feces of wild western lowland gorillas in Moukalaba-Doudou National Park, Gabon. Isolates were identified as Achromobacter xylosoxidans and Providencia sp., respectively. Minimum inhibitory concentrations of the following 12 drugs—ampicillin (ABPC), cefazolin (CEZ), cefotaxime (CTX), streptomycin (SM), gentamicin (GM), kanamycin (KM), tetracycline (TC), nalidixic acid (NA), ciprofloxacin (CPFX), colistin (CL), chloramphenicol (CP) and trimethoprim (TMP)—were determined. Isolate GG6-2 was resistant to all antimicrobials tested and highly resistant to CTX, SM, TC, NA and TMP. Isolate GG6-1-1 was resistant to ABPC, CEZ, TC, CL, CP and TMP.     

A new ENU-induced mutant mouse with defective spermatogenesis caused by a nonsense mutation of the syntaxin 2/epimorphin (Stx2/Epim) gene

Repro34 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing male-specific infertility caused by defective spermatogenesis. In the present study, we investigated pathogenesis and molecular lesions in relation to spermatogenesis in the repro34/repro34 homozygous mouse. Histological examination of the testis showed that the seminiferous epithelium of the repro34/repro34 mouse contained spermatogonia and spermatocytes but no round and elongating spermatids. Instead of these haploid cells, multinucleated giant cells occupied the niche of the seminiferous tubules. Immunohistochemical staining for Hsc70t, an elongating spermatid specific protein, confirmed the absence of elongating spermatids. Furthermore, RT-PCR showed that there were significantly reduced expressions of the marker genes specifically expressed in the spermatid and that there was no difference in the expressions of the spermatocyte specific marker genes. These findings indicated interruption of the spermatogenesis during transition from the spermatocyte to spermatid in the repro34/repro34 mouse. The repro34 locus has been mapped on a 7.0-Mb region of mouse chromosome 5 containing the Syntaxin 2/Epimorphin (Stx2/Epim) gene, and targeted disruption of this gene has been reported to cause defective spermatogenesis. We therefore sequenced the entire coding region of the Stx2/Epim gene and found a nucleotide substitution that results in a nonsense mutation of this gene. The expression pattern of the Stx2/Epim gene during the first wave of spermatogenesis, increased expression at later stages of spermatogenesis, was in agreement with the affected phase of spermatogenesis in the adult repro34/repro34 testis. We therefore concluded that the male infertility of the repro34/repro34 mouse is caused by the interruption of spermatogenesis during transition from the spermatocyte to spermatid and that the nonsense mutation of the Stx2/Epim gene is responsible for the interruption of spermatogenesis.     

Differential behavior between acacia and Japanese larch woods in the formation and decomposition of hexenuronic acid during alkaline cooking

The effects of anthraquinone (AQ) and polysulfide (PS) on the hexenuronic acid (HexA) content of pulp during kraft cooking were studied using Acacia mearnsii (acacia) and Larix leptolepis (Japanese larch) sapwood. In contrast to the results of cooking Japanese larch at an H-factor of 1200, the HexA contents of acacia pulp with a kappa number of 20 at an H-factor of 291 did not differ greatly between the kraft, kraft-AQ, and PS-AQ cooking methods, although the hydroxide ion concentration in the acacia cooking liquor decreased on the addition of AQ or sulfur. To explain this difference, we studied the behavior of the formation and degradation of HexA during alkaline cooking of glucuronoxylan from cotton linter, which was cooked with 1.0 and 2.0 mol/l NaOH. The relationship between HexA content and H-factor during alkaline cooking of glucuronoxylan was clarified. The amount of HexA and its rate of decomposition were higher in the 2.0 mol/l solution than in the 1.0 mol/l solution. At a low H-factor similar to that for hardwood cooking, HexA content increased to a maximum level and then started to decrease at high hydroxide ion concentrations such as 2.0 mol/l, whereas it slowly decreased at low hydroxide concentrations such as 1.0 mol/l. At an H-factor of around 450, the HexA formation/degradation curve for 1.0 mol/l of hydroxide crossed the decomposition curve for 2.0 mol/l of hydroxide. Therefore, it was shown that at a low H-factor, a decrease in hydroxide ion concentration during acacia wood cooking had little effect on the HexA content of pulp.