Kinya Katayama

To clarify the relationship between irreversible inactivation and intracellular protein denaturation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles (CO2 MB) treatment, a storage test of S. pastorianus cells treated with CO2 MB was performed, and the effect on the intracellular protein was investigated. In the storage test, the S. pastorianus population, which decreased below the detection limit by CO2 MB treatment at a temperature of 45 and 50°C (MB45 and MB50), and thermal treatment at a temperature of 80°C (T80), remained undetectable during storage for 3 weeks at 25°C. However, 4.1 and 1.3-logs of the S. pastorianus populations, which survived after CO2 MB treatment at temperatures of 35 and 40°C (MB35 and MB40), increased gradually during storage for 3 weeks at 25°C. Insolubilization of intracellular proteins in S. pastorianus increased with increasing the temperature of CO2 MB treatment. Activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) identified as one of the insolubilized proteins increased at MB35 and MB40 than non-treatment but disappeared at MB45 and MB50, and T80. Therefore, it was revealed that S. pastorianus cells inactivated below the detection level by CO2 MB treatment did not regrow and that the denaturation of intracellular proteins of S. pastorianus was caused by CO2 MB and thermal treatments. Furthermore, it was suggested that denaturation of intracellular vital enzymes was an important factor for achieving irreversible inactivation of S. pastorianus by CO2 MB and thermal treatments.

Cancer consists of heterogeneous cells that contain a small population of cells that possess stem cell properties; these cells, referred to as cancer stem cells (CSCs) or tumor-initiating cells, are involved in tumor progression and metastasis. Using a sphere-forming assay, canine mammary CSCs were found to be similar to human breast CSCs. Metabolic reprogramming has been recognized as a hallmark of various cancers. However, the significance of cellular metabolism in CSCs remains unclear. The aim of this study was to define the metabolic characteristics of CSCs derived from canine mammary tumors and gain an understanding of the maintenance of stemness. We identified metabolite profiles of canine mammary adenocarcinoma cell lines using gas chromatography-mass spectrometry. Metabolites were extracted from both adherent and sphere-forming cells derived from three cell lines. Sphere-forming cells were separated from adherent cells using an orthogonal, partial least-squares discriminant analysis. Sphere-forming cells were found to contain high levels of the amino acids alanine, glycine and proline compared with adherent cells. They also had high levels of palmitoleate, palmitate and dihomo-gamma-linolenic acid compared with adherent cells. In a sphere-forming assay, palmitate increased the number of spheres for all cell lines. These results indicate that the sphere-forming cells derived from canine mammary adenocarcinoma cell lines have specific metabolic profiles that may be useful for the development of CSC-specific therapies targeting metabolic pathways and potential stemness biomarkers; these results also clarify the maintenance of stemness in canine mammary CSCs.

Among non-tuberculous mycobacteria (NTM), the Mycobacterium simiae complex is one of the largest groups, consisting of 18 species of slow-growing mycobacteria. In 2009, a case of NTM-associated infectious skin disease was reported in Shiga Prefecture, Japan. The patient presented with scattered nodules on the chest, back and extremities, and an M. simiae-like organism was isolated from skin biopsy specimens obtained from one of these lesions. Based on several assessments, including multiple-gene analyses, biochemical characterization and drug susceptibility testing, we concluded that this isolate represented a novel species of NTM, and proposed the name 'Mycobacterium shigaense'. Since 2009, five more cases of NTM-associated infectious disease in which there was a suspected involvement of 'M. shigaense' have been reported. Interestingly, four of these six cases occurred in Shiga Prefecture. Here we performed multiple-gene phylogenetic analyses, physiological and biochemical characterization tests, drug susceptibility tests, and profiling of proteins, fatty acids and mycolic acids of eight clinical isolates from the six suspected 'M. shigaense' cases. The results confirmed that all of the clinical isolates were 'M. shigaense', a slow-growing, scotochromogenic species. Here M. shigaense is validly proposed as a new member of the M. simiae complex, with the type strain being UN-152T (=JCM 32072T=DSM 46748T).