The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma

Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.     

Allele frequencies of the extension locus encoding the melanocortin-1 receptor in Japanese and Korean cattle

In order to estimate the influence of the Extension (E) locus in cattle coat color, the melanocortin‐1 receptor (MC1R) gene in Japanese Black, Japanese Brown and Korean (Hanwoo) cattle were sequenced. The sequences of the coding region revealed three alleles (E^D, E⁺ and e), which were previously reported. Polymerase chain reaction‐restriction fragment length polymorphism was performed to investigate the gene frequencies of the three breeds. Japanese Black was almost composed of E^D and E⁺ individuals, E^D = 0.481 and E⁺ = 0.514, and no homozygous e/e, therefore that is consistent with the hypothesis that E^D and E⁺ induce black pigment synthesis. Allele frequencies between Japanese Brown and Hanwoo were obviously different; however, recessive red e allele frequency was 0.038 for Japanese Brown and 0.948 for Hanwoo, even though both breeds have quite similar coat colors (ranging from yellowish brown to dark brown including a red coat color). This result suggested that other genes are also associated with a coat color of red and brown in cattle.     

Late seeding of soybean after rice transplanting period using variety "Yukihomare" in central area of Hokkaido

(Jpn. J. Soil Sci.Plant Nutr., 77, 293–298, 2006)
"Kitanokaori" is a new variety of wheat for bread use bred at the National Agricultural Research Center for Hokkaido Region. The grain protein content of wheat for bread use should be higher than 120 g kg−1. Much nitrogen application is necessary to obtain high grain protein content. Therefore, it is necessary to determine the optimum amount of nitrogen to obtain the required protein content and to prevent nitrogen from remaining in the soil. The nutrition diagnosis using leaf color was investigated to predict the need and the amount of top-dressing. Field experiments were conducted for four years with nitrogen treatments in Andosol, which has moderate nitrogen fertility, and in Histosol, which is a fertile soil. The leaf color was measured using a chlorophyll meter SPAD502 (CM value) at the middle part of the leaf, avoiding the center rib. The colors of the 10·15 uppermost second leaves were measured in one plot and averaged.
A close relation was found between leaf color at the full heading stage and grain protein content at harvest. Leaf color at the full heading stage is therefore a good index to control the protein content. Considering the effect of top-dressing at the full heading stage in each CM value, the diagnosis criterion was decided. When the CM value is over 52 at the full heading stage, more nitrogen application is not needed. When the CM value is 50·52, 30 kg N ha−1 of top-dressing at the full heading stage is needed, and when the CM value is 45·50, 60 kg N ha−1 of top-dressing is needed to obtain a grain protein content of more than 120 g kg−1.     

Exclusion of NEU1 and PPGB from candidate genes for a lysosomal storage disease in Japanese Black cattle

A case of lysosomal storage disease has been reported in a calf of Japanese Black cattle. Lysosomal storage diseases are hereditary diseases caused by deficiency of lysosomal hydrolases. The clinical and pathological features and accumulated substrates of the affected animal indicated a possibility of sialidosis or galactosialidosis caused by deficiency of neuraminidase (NEU1) or protective protein for β-galactosidase (PPGB). In the present study, we investigated nucleotide sequences of the genes encoding these two proteins to evaluate whether mutation of these genes is involved in this disease. We determined cattle genomic sequences of these two genes by using bovine EST sequences and the nucleotide sequences of all exons of these genes were compared between affected and normal animals. The results showed several nucleotide substitutions, but none of them was a functional mutation or specific to the affected animal. Furthermore, genotyping of the microsatellite markers in the vicinity of these two genes revealed no homozygosity of the chromosomal regions including these genes in the affected animal. These findings indicated that neither NEU1 nor PPGB gene is responsible for the lysosomal storage disease of Japanese Black cattle and therefore the disease is neither sialidosis nor galactosialidosis.     

Homozygosity of single nucleotide polymorphisms in the 3′ region of the canine estrogen receptor 1 gene is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas

Differences in the distribution of single nucleotide polymorphisms (SNPs) and haplotypes in the estrogen receptor α gene (ESR1) were examined in Miniature Dachshunds (n = 48), Chihuahuas (n = 20) and Toy Poodles (n = 18). Five DNA fragments located in the 40‐kb region at the 3′ end of ESR1 were amplified by polymerase chain reaction and were directly sequenced. We compared allele, genotype and estimated haplotype frequencies at each SNP in the 3′ end of ESR1 for these three breeds of small dog. The frequency of the major allele and the genotype frequency of the major allele homozygotes, were significantly higher in Toy Poodles for five SNPs (SNP #5, #14–17) than in Miniature Dachshunds, and significantly higher in Toy Poodles than Chihuahuas for three SNPs (SNP #15–17). A common haplotype block was identified in an approximately 20‐kb region encompassing four SNPs (SNPs # 14–17). The frequencies of the most abundant estimated haplotype (GTTG) and GTTG homozygotes were significantly higher in Toy Poodles than in the other two breeds. These results imply that homozygosity for the allele, genotype and haplotype distribution within the block at the 3′ end of ESR1 is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas.