次号（Vol.70, No.2）掲載予定の原著論文
Water Sorption and Glass Transition Behaviors of Calcium Lactate
Takumi MOCHIZUKI and Kiyoshi KAWAI
The water sorption, water content dependence of the glass transition temperature (Tg ), and freeze-concentrated glass transition temperature (Tg ’) of calcium lactate (CaLt 2 ) were investigated, and the results were compared to those of calcium maltobionate (CaMb 2 ) and maltose. The water sorption behavior of amorphous CaLt 2 was analyzed by the Guggenheim-Anderson-de Boer model. The results showed that the monolayer water content of amorphous CaLt2 was higher than that of amorphous CaMb 2 and maltose. The Tg of amorphous CaLt 2 and the Tg ’ of CaLt 2 solution were investigated by differential scanning calorimetry. The results revealed that the anhydrous Tg of amorphous CaLt 2 was slightly lower than that of amorphous CaMb2 and was much higher than that of amorphous maltose. The Tg of amorphous CaLt 2 decreased with increasing water content due to water plasticizing. The Tg ’ of CaLt 2 solution was much higher than those of CaMb 2 and maltose solutions. CaLt 2 may form a reversible complex in dehydrated and freeze-concentrated states.
(Received Mar. 29, 2024; Accepted May. 09, 2024)
乾燥耐性細胞Pv11 におけるFlp/FRT 部位特異的組換えシステム
"Flp/FRT" Site-Specific Recombination System in the Anhydrobiotic Pv11 Cells
堀田真彦, 吉田祐貴, 黄川田隆洋
Masahiko HOTTA, Yuki YOSHIDA, Takahiro KIKAWADA
Loss of body water through freezing or desiccation causes most terrestrial organisms to suspend their animation and eventually die. However, a cultured cell line derived from the sleeping chironomid Polypedilum vanderplanki (Pv11 cells) is capable of anhydrobiosis, a latent state of life induced by desiccation. To date, molecular genetic analysis of Pv11 cells has identified several genes involved in anhydrobiosis, but the low efficiency of transfection into the cells has been an experimental limitation. In this study, to achieve highly efficient and selective gene insertion into or excision from the genome of Pv11 cells, we investigated the availability of recombinase-mediated cassette exchange (RMCE). We selected the Flp/FRT site-specific recombination system, which has been demonstrated to function in other insect cells, as a first target to test the functionality of the recombinase. Flow cytometry and sequence analysis showed that recombination occurs between two equi-oriented FRT sites in the cells transiently expressing Flp recombinase. Thus, we confirmed the Flp/FRT system is available in Pv11 cells.
(Received Apr. 11, 2024; Accepted May. 16, 2024)
Carnosine's Cryoprotective Effect on Lactate Dehydrogenase Compared with Carbohydrate Cryoprotectants
Zihao CHEN, Dan QIAO, Mario SHIBATA, Tomoaki HAGIWARA
The cryoprotective effect of carnosine on lactate dehydrogenase (LDH) was compared with that of typical carbohydrate cryoprotectants (sucrose, glucose, and trehalose). Carnosine significantly inhibited the freeze-induced inactivation of LDH, surpassing the other cryoprotectants. Differential scanning fluorimetry results indicated that the other four cryoprotectants stabilized the structure of LDH, whereas carnosine exhibited the least degree of stabilization. Using differential scanning calorimetry analysis, it was shown that the amount of freezable water was larger in the presence of carnosine than in the presence of sucrose. Dielectric relaxation measurements revealed little difference in the quality and quantity of free water between the sucrose and carnosine solutions. Based on these results, two hypothetical factors contributing to the cryoprotective effect of carnosine were proposed.
(Received May. 25, 2024; Accepted Aug. 08, 2024)
Cryopreservation of CHO-TRET1 cells using Pressure Liquid Cooling Vitrification method
Tsutomu UCHIDA, Yoshiharu SUZUKI, Kaito SASAKI, Takahiro KIKAWADA
The importance of vitrification in the mechanism of cell cryopreservation was examined using a physical vitrification (pressure liquid cooling vitrification) method. Trehalose was used as a cryoprotectant, and a freezing solution containing a suspension of trehalose-transporter-expressing Chinese hamster oocytes (CHOTRET1 cells) or non-expressing cells (CHO-vector cells) was pressurized at 0.05–0.3 GPa and frozen by liquid nitrogen under pressure. After decompression and storage at −196°C, the frozen samples were rapidly thawed and their viabilities were measured by flow cytometry. When frozen under 0.3 GPa, a pressure sufficient to induce vitrification for this solution, the viability of CHO-TRET1 cells was very low. However, at pressures less than 0.2 GPa, where normal ice was formed, the viabilities were at most 30% but exhibited positive pressure-dependence. The viabilities of CHO-vector cells frozen in the same freezing solution also showed similar positive pressure-dependence, which resulted in the large viability difference between the two kinds of cells under the 0.3 GPa condition. These results suggest that merely the vitrification itself does not make a major contribution to the cryopreservation mechanism, but that the suppression of crystal growth during thawing is more important.
(Received Aug. 18, 2024; Accepted Sep. 03, 2024)