Mono-tailed glycolipids consisting of a sugar moiety and a single alkyl chain are effective stabilizer for maintaining protein activities during both freeze-thawing and freeze-drying processes. In order to understand the stabilizing mechanism, the behaviors of mono-tailed glycolipid under freezing of ice and desiccation have been investigated. In this review article, four topics in terms of mono-tailed glycolipids were summarized as following: The behaviors in (1) single component system, (2) water system with and without freezing and (3) electrolyte-water system with freezing and (4) the maintaining effect on enzyme activity of L-lactic dehydrogenase (LDH) during freeze-thawing and freeze-drying processes. In these topics, the glass formation of mono-tailed glycolipid have been mainly focused as a novel key functionality.

View full abstract

Mammalian hibernation is a strategy to survive during harsh winter with severe hypothermia and immobility state. It has attracted many researchers for a long time but still remains to be elucidated. Studies in obligate hibernators such as ground squirrels, chipmunk, and bears have revealed that they undergo systemic body remodeling in a season-dependent manner prior to hibernation. By contrast, a facultative hibernator, Syrian hamster, can hibernate in an environment-dependent manner; when they are exposed cold and short photoperiod condition for several months, they begin to hibernate. This inducible hibernation allows researchers to study mechanisms and significance of hibernation under a laboratory condition, whereas exact nature of systemic body remodeling for hibernation in Syrian hamsters remain unclear yet. Using histology and exhaustive gene expression analyses, we compared summer-like hamsters and winter-like hamsters and found that Syrian hamsters extensively remodel white adipose tissues during a pre-hibernation period. Particularly, simultaneous up-regulation of gene expression in both lipid catabolisms and lipid anabolisms takes place in winter-like hamsters, which is a unique property of Syrian hamsters, a“food-storing” hibernator who ingests food stored in the nest during hibernation season.

View full abstract

Most temperate fruit tree species enter dormancy in autumn and are released from dormancy after exposure of a certain amount of chilling. Recent reports suggested the involvement of some MADS-box genes in apple (Malus x domestica) dormancy regulation. In this review, gene expression patterns of apple MADS-box genes, named DORMANCY-ASSOCIATED MADS-box1 (MdDAM1), MdDAM2, and FLC-like in relation to dormancy progress and morphological changes of internal flower structure during autumn until bud break, were first summarized. Previous studies revealed that these three genes were up-regulated in apple flower buds during dormancy. MdDAM2 reached at maximum expression level before chilling requirement fulfillment time whereas MdDAM1 stayed at higher expression levels even after it. FLC-like were up-regulated by exposure to prolonged chilling. All these MADS-box genes were up-regulated in correlation with flower organ differentiation and development and then down-regulated at bud break stage. As for functional characterization, the reported transgenic studies revealed that Rosaceae DAMs may act as bud break repressors while FLC-like inhibited vegetative growth in Arabidopsis. Taken together, some apple homologs of MADS-box genes well-known as flowering regulators in Arabidopsis may contribute to dormancy regulation by acting as growth inhibitors and bud break repressors during dormancy and flower bud development.

View full abstract

Refrozen surimi loses gel forming ability while setting, for a variety of reasons. However, the setting gel formation rate of refrozen surimi has yet to be analyzed. While some studies have investigated the gel forming rate of frozen surimi, those studies focused solely on breaking strength and did not consider the concept of breaking strain. Therefore, we analyzed the rate of the setting gel forming ability using a novel analysis method that considered the concept of both breaking strength and strain. The breaking strength and breaking strain of the setting gels, which were prepared by frozen and/or refrozen surimi, were measured with a creep meter. The setting gel forming ability rate was calculated by determining the length of the line in the graph between breaking strength and gel stiffness (gel stiffness is the breaking strength divided by the breaking strain), and then, dividing the length by setting time. The setting gel forming rate was calculated using a novel analysis method, and was confirmed to be reasonable based on the analysis of SDS-PAGE patterns.

View full abstract

A technique for micropropagation and cryopreservation of an endangered plant Nymphoides indica from subcultured multiple shoot primordia is described. Excised tissues from leaves cultured on Murashige and Skoog medium (MS medium) supplemented with 3 % (w/v) sucrose, 10 μmol L-1 Thidiazuron (TDZ) and 0.1 μmol L-1 1-naphthaleneacetic acid (NAA) formed multiple shoot primordia. These shoot primordia were subcultured repeatedly at 3-week intervals. Subcultured shoot primordia were cryopreserved in liquid nitrogen. Cell clumps of multiple shoot primordia treated with 5% (v/v) dimethyl sulfoxide (DMSO) and 10% (w/v) glucose were cooled at a rate of 1°C/min to -40°C in a programmed freezer, then immersed in liquid nitrogen. Pre-culture of shoot primordia on a medium containing 5% (w/v) sucrose for 2 days enhanced survival rate of cryopreserved tissues. Plants with developed shoots and roots were obtained by transferring the survived shoot primordia to MS medium lacking growth regulators.

View full abstract

Cryoprotectant agents (CPA) that can suppress generating ice crystals during the freezing process are essential for cell cryopreservation. On the other hand, a CPA should ideally be avoided due to their cytotoxicity and potential side effects. We previously reported a novel cryopreservation method without a CPA by ultrarapid cooling. In the method, cells were ejected as tiny droplets by inkjet cell printing. The droplets were deposited on the liquid nitrogen-cooled substrate and were vitrified, that enables cell cryopreservation. While, these droplets can devitrify easily due to CPA-free and low thermal volume of the droplets. In this study, we fabricated an aluminum career and a silicon cover to inhibit devitrification of the droplets. The experimental results indicated that the glass substrate with the career and the cover was kept under the glass-transition temperature for a several seconds. The efficacy of the career and the cover was also evaluated on mouse fibroblast 3T3 cells. The cells transferred to the cryotube and stored for one week with the career and the cover had higher viability than the ones without them. These results indicate that the career and the cover were able to inhibit devitrification of the cells frozen by our method.

View full abstract

Vitrification based method combined with carboxylated poly-L-lysine (PLL) treatment was successfully developed using in vitro-grown chrysanthemum (Chrysanthemum morifolium) shoot tips. Shoots of chrysanthemum cultured on 1/2 MS medium were cold-hardened at 4oC for 30 days. Shoot tips (1.5 x 1.0 mm) were excised from shoot cultures and precultured at 4oC for 3 days on solidified 1/2 MS medium containing 0.3 M sucrose. Osmoprotection and PLL treatment were performed by immersing for 30 min at 25oC in loading solution with PLL. Pretreated shoot tips were placed in elliptical wells on an aluminium cryo-plate and embedded in calcium alginate gel. The shoot tips were dehydrated by plant vitrification solution 2 for 20 min. In this procedure, cooling was performed by transferring the cryo-plate in uncapped 2.0 mL cryotubes, and then directly plunging into liquid nitrogen. For rewarming, shoot tips attached to the cryo-plates were immersed in petri dish containing 1.0 M sucrose solution at 25oC. Regrowth rate of cryopreserved shoot tips using 5% and 10% PLL treatment were 83.3% and 85.0%, respectively. Although further investigation should be required, PLL might protect membrane from the freezing or osmotic damage. This protocol appears to be a promising technique for cryopreservation of chrysanthemum genetic resources.

View full abstract

Recently, discrimination of numerous odors with high sensitivity grows in demand. Olfactory system is the pattern recognition of the activation of olfactory receptors. We can mimic olfactory system by evaluating olfactory receptor’s response. Insect olfactory receptors are heterotetramers consisting of one olfactory receptor (OR) and the olfactory receptor co-receptor (Orco). They form a ligand-gated cation ion channel. Sf9 cells can grow at room temperature without CO2 gas and can produce high amount of proteins. Thus, Sf9 cells are commonly used to express olfactory receptors. One way to evaluate the response to odorants is live-cell calcium imaging. However, the live-cell imaging for Sf9 cells are difficult because their adherence capability onto a culture plate is low relatively. Here, we introduce the live-cell calcium imaging method in Sf9 cells by immobilization with low melting point agarose (LMPA). Or47a and Orco from Drosophila melanogaster were cloned and co-expressed in Sf9 cells with a calcium indicator protein GCaMP6f. We dropped an OR-expressing Sf9 cells solution to a plate and covered it with LMPA to immobilize. After addition of a ligand solution to immobilized Sf9 cells, we succeeded to observe the OR response in the same Sf9 cells without any toxicity by LMPA.

View full abstract