Characterization and partial purification of a novel mannosylphosphodolichol phosphodiesterase from chicken liver microsomes

Mannosylphosphodolichol phosphodiesterase, which catalyzes the release of mannose from mannosylphosphodolichol, was solubilized from chicken liver microsomes by treatment with the non-ionic detergent, Emulgen 909. The enzyme was partially purified using ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration on Sepharose 6B. The enzyme showed absolute requirement for sulfhydryl reducing agents. The enzyme activity was stimulated by the addition of CaCl2 and Emulgen 909 and exhibited a pH optimum around 5.3. The Km value for mannosylphosphodolichol was found to be 0.43 microM. The activity was competitively inhibited by dolichyl phosphate and dolichol and the Ki value for dolichyl phosphate was estimated to be 12.5 microM. The purified preparation had no activity toward N-acetylglucosaminyldiphosphodolichol, glucosylphosphodolichol, mannose 1-phosphate, or artificial substrates for mannosidases, glucosidases, acid phosphatase, and acid phosphodiesterase. A heat-stable factor which stabilizes the mannosylphosphodolichol phosphodiesterase was separated from the enzyme by DEAE-cellulose chromatography. It was precipitated by trichloroacetic acid and not extracted into lipid solvents. The separation resulted in the complete loss of the enzyme activity and the restoration of the activity was not observed when the factor was added back to the enzyme solution.     

Site-saturation mutagenesis for β-glucosidase 1 from Aspergillus aculeatus to accelerate the saccharification of alkaline-pretreated bagasse

Applied Microbiology and Biotechnology - Aspergillus aculeatus β-glucosidase 1 (AaBGL1) is one of the best cellobiose hydrolytic enzymes without transglycosylation products, among...     

Freezing of cell sheets using a 3D freezer produces high cell viability after thawing

In cell therapy, transplanting an appropriate number of cells to the target site is crucial. One way to achieve this is to transplant cell sheets. Transplantation of cell sheets has already been utilized for various diseases in clinical practice. However, reducing the cost of cell sheet utilization is essential so as to facilitate the spread of regenerative medicine. Several ways to reduce costs are available, one of which is the use of allogenic cells. Another alternative is the use of cell sheets, which necessitates the development of methods for freezing cell sheets. This is the first study to report the use of a 3D Freezer for freezing cells. 3D Freezers have been used in the field of food processing and technology for a long time. The 3D Freezer freezes objects using cold air at a uniform temperature from all directions. In this study, we analyzed the cooling speed of human fibroblast sheets in 11 cell preservation solutions using a 3D Freezer and a Program Freezer. The cooling speed was ?2 °C per min in the 3D Freezer. Supercooling in 10 cell preservation solutions was lower in the 3D Freezer than in the Program Freezer. Cell viability after freeze–thaw of the cell sheets using 3D Freezer was more than 70% in five cell preservation solutions. The levels of hepatocyte growth factor and transforming growth factor-β1 were the same not only in the fibroblast sheets frozen using the five cell preservation solutions but also in the non-frozen fibroblast sheets. These results suggest that the 3D Freezer can freeze implantable cell sheets immediately after thawing.     

Movement and assemblage of fish in an artificial wetland and canal in a paddy fields area,in eastern Japan

This study, carried out in a paddy fields area in eastern Japan, investigated the timing and patterns of fish movement and assemblage in an artificial wetland and canal. The number of Misgurnus anguillicaudatus and Lefua echigonia immigrating and emigrating between the wetland and the canal accounted for 80–90 % of all the sampled fish. M. anguillicaudatus, L. echigonia, Pseudorasbora parva and Rhynchocypris lagowskii were dominant in the wetland. Immigration of mature M. anguillicaudatus and L. echigonia was detected between late winter and spring. The standard length of the two loach species in the wetland was smaller than that in the canal. These results confirm that wetlands play a role in spawning and nursery for the two species of loaches. The standard length of P. parva in the wetland was smaller than in the canal. This suggests that the wetland was a more suitable spawning and nursery area for this fish species than the canal. L. echigonia used the wetland as a spawning and nursery area, but previous studies reported that the loach did not use paddy fields near the wetland. This could be because the paddy fields were irrigated between June and September and this period did not largely overlap with the fish spawning season. Therefore, we conclude that the conservation and restoration of wetlands, where water is present throughout the year, will contribute toward the preservation of the fish population in a paddy fields area.     

A lesson from polybutylene succinate plastisphere to the discovery of novel plastic degrading enzyme genes in marine vibrios

Polybutylene succinate (PBS) is an eco-friendly green plastic. However, PBS was shown as being non-biodegradable in marine environments, and up until now, only a limited number of PBS-degrading marine microbes have been discovered. We first set up in vitro PBS- and PBSA (polybutylene succinate adipate)-plastispheres to characterize novel PBS-degrading marine microbes. Microbial growth and oxygen consumption were observed in both PBS- and PBSA-plastispheres enriched with natural seawater collected from Usujiri, Hokkaido, Japan, and Vibrionaceae and Pseudoalteromonadaceae were significantly enriched on these films. Further gene identification indicated that vibrios belonging to the Gazogenes clade possess genes related to a PBS degrading enzyme (PBSase). The PBS degradation assay for six Gazogenes clade vibrios identified Vibrio ruber, Vibrio rhizosphaerae, and Vibrio spartinae as being capable of degrading PBS. We further identified the gene responsible for PBSase from the type strain of V. ruber, and the purified recombinant vibrio PBSase was found to have low-temperature adaptation and was active under high NaCl concentrations. We also provided docking models between the vibrio PBSase and PBS and PBSA units to show how vibrio PBSase interacts with each substrate compared to the Acidovorax PBSase. These results could contribute to a more sustainable society through further utilization of PBS in marine environments and plastic recycling.     

Distinct roles in phagocytosis of the early and late increases of cell surface calreticulin induced by oxaliplatin

Calreticulin (CRT), a chaperone typically located in the endoplasmic reticulum (ER), is known to translocate to the cell surface in response to anticancer drugs. Cell surface CRT (ecto-CRT) on apoptotic or pre-apoptotic cells serves as an “eat me” signal that can promote phagocytosis. In this study, we observed the biphasic (early transient and late sustained) increase of ecto-CRT on HT-29 cells after treatment with oxaliplatin (L-OHP). To investigate the role of ecto-CRT that accumulates in the early and late phases as “eat me” signals, we examined the phagocytosis of HT-29 cells by macrophage-like cells and dendritic cell (DC) -like cells prepared from THP-1 cells. The results indicated that the early ecto-CRT-expressed cells were phagocytosed by immature DC-like cells, and the late ecto-CRT-expressed cells were phagocytosed primarily by macrophage-like cells, while mature DC-like cells did not respond to the either class of ecto-CRT-expressed cells. Both types of phagocytotic events were inhibited by CRT Blocking Peptide, suggesting that such events depended on the ecto-CRT. Our results suggested that the early increase of ecto-CRT is related to phagocytosis as part of immunogenic cell death (ICD), while the late increase of ecto-CRT is related to the removal of apoptotic cells by macrophages.     

Legacy effects of canopy gaps on liana abundance 25 years later in a seasonal tropical evergreen forest in northeastern Thailand

Lianas require host trees to reach and stay in the forest canopy, but as seedlings and juveniles, they benefit from canopy gaps created by treefalls. Here, we evaluated the relative importance of these two aspects, that is, the availability of potential hosts vs. the legacy effect of past treefall gaps, on the local abundance of liana stems in a seasonal tropical evergreen forest in the Sakaerat Biosphere Reserve in northeastern Thailand. Within a 2.5-ha plot for forest dynamics monitoring, canopy height was measured in 1993 and 2018 at 5-m intervals to distinguish areas of mature (canopy height ≥ 20 m), building (10–20 m), and gap phases (< 10 m). In 2017–2018, we surveyed all liana stems ≥ 1 cm in diameter at breast height within 50 subplots (10 m × 10 m each) and recorded their diameter and the diameter of the host tree. Of a total of 445 liana individuals, 242 could be identified at least to the family level, while the others had clear morphological traits of climbing mechanisms. The number of liana stems was higher in areas that had been at the building/gap phase than those at the mature phase in 1993. When this 25-year-old legacy of past gap locations was considered, there was a positive association of local abundance between lianas and trees in areas at the mature phase in 2018. In conclusion, liana abundance reflected a long-term legacy of past treefall gaps more than 25 years earlier in this seasonal evergreen forest.