Cryopreserved cardiac myocytes are expected to be useful for the development of new drug against heart disease. However, the appropriate cryopreservation process for cardiac myocytes has not been established yet. Therefore, in this study, we evaluate the effectiveness of dimethyl sulfoxide (DMSO) on the cryopreservation of cardiac myocytes by three measurements: 1) the total viability (including cardiac myocytes) just after thawing with trypan blue staining, 2) the observation of morphology of cardiac myocytes for 7 days culture by real-time phase contrast microscopy and immunofluorescent staining, and 3) the variation of their beating rates for 7 days after thawing. The total viabilities of cryopreserved cells with 3v/v% to 20v/v% DMSO concentration were higher than those without DMSO. Thawed cells contracted regularly and spontaneously during culture, as did the non-cryopreserved cells. The immunofluorescence observation indicated that the thawed cardiac myocytes expressed α-actinin. Therefore, cardiac myocytes could be survived by the cryopreservation with DMSO concentrations between 3v/v% and 20v/v%, which had the normal contraction rates during culture for 7 days.

View full abstract

The objective of this study is to establish a cryopreservation protocol for shoot apices of Cardamine yezoensis. Cryopreservation was carried out using a vitrification method on shoot apices excised from in vitro cultures. Plant Vitrification Solution 2 (PVS2) was demonstrated to be an optimal vitrification solution for shoot apices in terms of its higher recovery after cryopreservation compared with PVS1 and PVS3. Smaller excised apices (1mm x 1mm square in size) showed higher regrowth after cryopreservation using PVS2 compared with larger apices (3mm x 3mm). The vitrification protocol leading to the optimal regrowth was as follows: Excised shoot tips were pretreated for 24hr at 25℃ on hormone-free basal medium with 0.4mol/L sucrose, then precultured in liquid basal medium supplemented with 0.4mol/L sucrose and 2.0mol/L glycerol (a loading solution) for 30min at 25℃. Precultured specimens were soaked in PVS2 for 60min at 0℃ in cryotubes before cryostorage for 1hr. We also examined the effects of various nutrient media on regrowth of cryopreserved apices. It was demonstrated that 4-times dilution of inorganic salts of Murashige and Skoog's medium (1/4MS) or Woody Plant medium (WPM) as basal medium resulted in higher regrowth percentages (both 66.7%) than other six media. We succeeded in improving regrowth after vitrification-based cryopreservation up to 96.7% by exchanging PVS2 twice during 60min of PVS2 loading.

View full abstract

Food oil-in-water (o/w) emulsions such as mayonnaise, some kind of dressing and milk are often destabilized during storage at low temperatures. The destabilization is considered to be induced by fat crystallization in a process of freeze-thawing. In this work, the relation between fat crystallization below freezing temperatures and oil separation was investigated for two types of o/w emulsions made from canola oil and soybean oil, using differential scanning calorimetry (DSC) and polarized optical microscopy. DSC measurements revealed the differences of crystallization temperature (T_c) between canola oil emulsion and soybean oil emulsion; that is, emulsified canola oil crystallized rapidly from -48℃. On the other hand emulsified soybean oil crystallized gradually in a wide range of subzero temperatures. Polarized optical microscopy showed a significant difference in the crystallization behavior in droplets. In the soybean oil droplets, fat crystals were located at oil-water interface while the fat crystals in the canola oil droplets were dispersed in the droplet. Freeze-thawing test showed that soybean oil emulsion exhibited higher stability than the canola oil. These results suggested that the crystals at the interface in the soybean oil droplets played a key role in its freeze-thaw stability.

View full abstract

Overwintering larvae of the rice stem borer, Chilo suppressalis obtains freezing tolerance to about -25℃ by the accumulation of glycerol in the haemolymph and replacing water with glycerol in the cell through the water channel during freezing. Moreover, when fat body tissue was frozen with an inhibitor of the water channel, the degree of freezing injury in overwintering larvae was significantly lower than that in non-diapausing larvae. In the present study the qualitative and quantitative changes of phospholipids in the last instar larvae of the rice stem borer were examined in summer and winter. Results showed that the total amount of their phospholipids did not change significantly between summer and winter and that the sum of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) comprised about 85% of their total phospholipids. In summer, the ratio of the PE to PC was almost one, while from autumn to mid winter it increased and reached three in February. The fatty acid compositions of PC hardly changed, and their percentage of unsaturated fatty acids did not exceed 50%. In contrast, the percentage of unsaturated fatty acids of PE in overwintering larvae increased up to 80% as ambient temperatures fell and oleic acid mainly contributed to the high percentage of unsaturation.

View full abstract

The raffinose family oligosaccharides (RFOs) are supposed to play a number of roles in plant development and stress tolerance. Galactinol synthase (GolS) catalyzing UDP-galactose and myo-inositol to form galactinol is a key enzyme in the raffinose synthesis pathway. Recently, it has been reported that the expression of enzymes related to the biosynthesis of galactinol and RFOs and their intracellular accumulation in plant cells are involved in acquired tolerance to various stresses. A rice GolS-homolog was found to be expressed in rice under drought stress, but it is unclear whether or not temperature stress changes the expression levels of rice GolS. By semi-quantitative RT-PCR, we analyzed the expression profiles of two GolS homologs in rice seedlings under chilling and salt stress. The predicted protein of OsGolS1 revealed a high similarity to those of OsGolS2 (78% in amino acid identity), AtGolS1 and AtGolS3 (72% and 67%, respectively). Chilling and salt stress induced mRNAs of OsGolS1 but not OsGolS2. It has been reported that an oxidative stress specific heat shock factor (HSFA2)is involved in the induction of AtGolS1 mRNA under heat stress via generation of reactive oxygen species (ROS). In this study, we examined whether HSFA2 functions in the induction of OsGolS1 in response to chilling stress. The expression level of rice HSFA2 homolog did not change under chilling stress. These results suggest that induction of OsGolS1 is involved in chilling stress response of rice and that expression of OsGolS1 at low temperature is regulated at downstream of OsMyb4 rather than DREB/CBF or HSFA2.

View full abstract

Model freeze-dried foods which consist of dextrin and glucose were prepared, and their glass transition temperature (T_g) was systematically investigated by using a differential scanning calorimetry. The T_g of the sample decreased readily with an increase in glucose content in the range of O to 20% (w/w). At the glucose content of 30% the T_g of the sample decreased drastically, and the temperature range of the glass transition was extended. The T_g of the sample decreased gradually with an increase in glucose content above the glucose content of 30%. Furthermore, the glass transition occurred in two steps. It is suggest that dextrin-glucose and glucose rich region showed glass transition individually.

View full abstract

A HEWL (Hen egg white lysozyme) orthorhombic crystal is grown by the two liquid interface method. The dynamics in a gigahertz range is measured over the wide temperature range in cryoprotective glycerol and propylene glycol aqueous solutions by using micro-Brillouin scattering. The elastic anomaly and structural relaxation are clearly observed. The structural relaxation time is assumed that it obeys Debye relaxation model. The temperature dependence of the relaxation time obeys the Arrhenius law at low temperatures. The correlation is found between the activation energy ΔE and the pre-exponential factor τ_0. The log τ_0 vs. ΔE plot indicates that the Mayer-Neldel rule holds.

View full abstract

The excited state (S_1) lifetime of a triphenylmethane (TPM) dye, malachite green (MG), the nonradiative decay of which is couple to the diffusive rotation of the phenyl ring, was monitored by means of femtosecond pump-probe spectroscopy in water-alcohol binary solvents. The dynamics observed in the ultrafast time range of ≤3.0ps was not very sensitive to the bulk solvent viscosity, indicating that the phenyl rotation is only influenced by the ambient local solvation structure.

View full abstract

Thermal analysis of liposome suspensions was performed to study their freezing behavior and effect of trehalose distributed across the membranes. Cooling scan of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) liposome suspensions showed exotherm peaks of bulk (heterogeneous ice nucleation, -15 to -25℃) and intraliposomal (homogeneous ice nucleation, approx. -45℃) solution freezing. Changing size of the intraliposomal ice formation peak indicated dehydration of liposomes after the bulk solution freezing. Liposomes of larger size and/or lower membrane fluidity showed slower rate of dehydration upon the bulk solution freezing. Addition of trehalose before and after the liposome preparation resulted in their different distribution across the membrane and varied freezing behaviors.

View full abstract

Thermal unfolding of ribonucrease A (RNase) in osmolyte (betaine, sarcosine, trehalose) solutions could be described well by the van't Hoff equation. All the osmolytes used stabilized RNase. Effect of osmolyte concentration on unfolding ratio of RNase at a constant temperature was described well by the reciprocal Wyman-Tanford plot based on water activity. Effect of cosolute on thermal stability of protein could be explained by the balance between the stabilization effect by hydration and the destabilization effect of cosolute binding to protein. By the integration of the reciprocal Wyman-Tanford equation, the stabilization free energy of protein in a solution as compared with that in pure water,Δ Δ G, could be obtained theoretically.

View full abstract

Exogenous application of a phytohormone abscisic acid (ABA) has been shown to induce both morphological changes leading to brood cell formation and stress tolerance in Physcomitrella patens protonema cells accompanied by the accumulation of low-molecular-weight soluble sugars and LEA-like boiling-soluble proteins. We used the transgenic D2-1 line expressing the catalytic domain of protein phosphatase 2C negatively regulating ABA signaling, and the ABA-less-sensitive AR7 mutant which has been isolated by ultraviolet mutagenesis, to compare their stress tolerance with that of wild type. Without ABA treatment, D2-1 and AR7 showed similar growth to that of wild type. However, the both lines were sensitive to freezing, desiccation and osmotic stresses with or without ABA treatment, whereas the ABA treatment enhanced tolerance in wild type to these abiotic stresses. These results indicate that ABA plays a major role in stress tolerance in P. patens.

View full abstract

According to our previous NMR study, trehalose forms an intermolecular complex with the cis-type double bond of diene compounds in aqueous solution. To elucidate the main driving force of such an intriguing complex formation, we calculated the potential of mean force (PMF) as a function of the distance (R) between the center of mass of trehalose and one double-bond of heptadiene. PMF exhibited the single minimum at a value of R=5-6Å, where the heptadiene molecule was located around the C3 (C3') methine groups and the exocyclic methylene groups of the trehalose, consistent with the results from the NMR study. Ab initio quantum chemical calculations indicated that the multiple interactions through weak hydrogen bonds such as CH/π and OH/π interactions play an important role in stabilizing the intermolecular complex. Furthermore, a significant increase in the activation energy was found for hydrogen abstraction reaction from the activated methylene group of heptadiene. On the basis of these results, we propose a novel mechanism for the antioxidant function of trehalose.

View full abstract