高野 貴士

Mesenchymal stem cells are expected to be a cell source for stem cell therapy of various diseases in veterinary medicine. However, donor-dependent cell heterogenicity has been a cause of inconsistent therapeutic efficiency. Therefore, we established immortalized cells from canine adipose tissue-derived mesenchymal stem cells (ADSCs) to minimize cellular heterogeneity by reducing the number of donors, evaluated their properties, and compared them to the primary cells with RNA-sequencing. Immortalized canine ADSCs were established by transduction with combinations of the R24C mutation of human cyclin-dependent kinase 4 (CDKR24C), canine cyclin D1, and canine TERT. The ADSCs transduced with CDK4R24C, cyclin D1, and TERT (ADSC-K4DT) or with CDK4R24C and cyclin D1 (ADSC-K4D) showed a dramatic increase in proliferation (population doubling level >100) without cellular senescence compared to the primary ADSCs. The cell surface markers, except for CD90 of the ADSC-K4DT and ADSC-K4D cells, were similar to those of the primary ADSCs. The ADSC-K4DT and ADSC-K4D cells maintained their trilineage differentiation capacity and chromosome condition, and did not have a tumorigenic development. The ability to inhibit lymphocyte proliferation by the ADSC-K4D cells was enhanced compared with the primary ADSCs and ADSC-K4DT cells. The pathway analysis based on RNA-sequencing revealed changes in the pathways mainly related to the cell cycle and telomerase. The ADSC-K4DT and ADSC-K4D cells had decreased CD90 expression, but there were no obvious defects associated with the decreased CD90 expression in this study. Our results suggest that ADSC-K4DT and ADSC-K4D cells are a potential novel cell source for mesenchymal stem cell therapy.

A Japanese resident bird, Phalacrocorax carbo hanedae (Japanese name: Kawa-u), was threatened with extinction due to deterioration of its habitat in the 1970s, but the population has since recovered thanks to environmental protection measures. This study analyzed the genetic diversity of 18 Kawa-u individuals living in the basins of the Abe and Warashina rivers in Shizuoka Prefecture, Japan. We obtained seven haplotypes of mitochondrial D-loop sequences and compared them with 49 European P. carbo D-loop haplotypes. We identified four new haplotypes but no clear genetic evidence distinguishing the Kawa-u as a distinct subspecies of P. carbo. Our results suggest the need for further surveillance of the P. carbo genetic lineage, regardless of the geographical distribution.

Some Listeria monocytogenes strains are persistent in food processing environments, where this pathogen may be subjected to various stresses. This study aimed to elucidate the response of persistent strains of L. monocytogenes to low pH and H2O2 exposure. Almost all of the persistent strains examined were highly susceptible to low pH, whereas H2O2 susceptibility was comparable to that of control strains. Two persistent strains isolated from the same sample, however, exhibited lower susceptibility to low pH. These findings suggest an acid-susceptible phenotype predominates in the habitat, indicating that environmental conditions contribute to the establishment of persistence. Representative strains exhibiting acid-susceptible and less acid-susceptible phenotypes were further investigated regarding acid response characteristics. Less acid-susceptible strains exhibited increased survival in acidified brain heart infusion (BHI) broth compared with acidified phosphate-buffered saline (PBS). These strains also exhibited increased survival in acidified PBS containing glucose and glutamate, which are involved in acid response mechanisms, compared with acidified PBS alone. However, neither acidified BHI broth nor exogenous glucose and glutamate increased survival of acid-susceptible strains. An adaptive acid tolerance response of the acid-susceptible phenotype was observed, but this was limited compared with that of the less acid-susceptible phenotype.

The level of Listeria monocytogenes contamination of domestic retail meat in Tokyo, Japan, was assessed by comparison of isolates from 2004 to 2007 with those isolated before 2003. The overall prevalence of L. monocytogenes among these samples significantly diminished over time (1998-2003, 28.0%; 2004-2007, 17.6%) reflecting a significant decrease in the frequency of contamination of beef. Serotype 1/2a was isolated most frequently, reflecting a change in the predominant serotype in pork from 1/2c to 1/2a. We performed a simple genetic subtyping method based on 3 genes, iap, sigB, and actA, as well as traditional multilocus sequence typing to classify the allele types (ATs). No extensive variation among sequence types was detected. However, increased genetic diversity among the ATs of the 3 genes in the 2004-2007 isolates was evident. We identified AT 26 of the iap gene, which was not previously reported in Japanese isolates, and 6 ATs of the sigB gene, including 4 with nonsense mutations not currently registered in L. monocytogenes DNA databases. sigB is an evolutionally conserved gene that plays a role in the stress response. Our results indicate that the sigB gene may be relatively unstable among L. monocytogenes strains circulating in Japan.