Organisms are able to sense environmental information and must respond appropriately to live and proliferate. Information on environmental temperature is essential for organisms because temperature directly affects biochemical reactions and metabolism. Here, we review the molecular and physiological mechanisms responsible for cold tolerance and cold acclimation in a simple model animal, the nematode Caenorhabditis elegans. Recent molecular-biological studies and neural calcium-imaging analyses have revealed novel concepts of cold tolerance in this animal. Most significantly, a light-sensing neuron (ASJ) acts as temperature-sensing neuron that regulates the intestine and sperm in the cold-tolerance pathway. Sperm regulates cold tolerance through negative feedback onto this temperature-sensing neuron.

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In Arabidopsis, three transcription factors DRE-binding protein 1/C-repeat binding factors (DREB1/CBFs) function as master switches in cold stress-inducible gene expression. Because the expression of the DREB1 genes is rapidly induced in the cold stress responses, this induction seems to be the first step for the cold stressinducible transcriptional cascade followed by the expression of numerous genes that function in the cold stress response. We have revealed that the 65-bp fragment of the DREB1C promoter contains positive regulatory elements for the cold-inducible expression. By using yeast one-hybrid screens, one of Calmodulin-binding transcription activator (CAMTA) family proteins was identified as a positive regulatory factor that interacts with this fragment. The analyses using the multiple mutants of the CAMTA genes revealed that plants recognize cold stress as two different signaling pathways for inducing the expression of the DREB1 genes under cold stress conditions. In one pathway, CAMTA3 and CAMTA5 activate the expression of DREB1B and DREB1C by a rapid temperature decrease. In another one, the circadian clock components including CCA1/LHY induce the expression of DREB1A and DREB1C only in the daytime independent to the cooling rate. These two signaling pathways enable plants to efficiently acquire cold acclimation capacity or freezing tolerance.

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Group 3 late embryogenesis abundant (G3LEA) proteins have a protective function against desiccation stress. In our previous work, the model peptide, PvLEA-22, was developed based on one tandem repeat of the conserved 11-mer motif in G3LEA proteins. PvLEA-22 peptide showed an anti-aggregation activity for dried liposomes and other proteins in vitro, leading to the hypothesis that PvLEA-22 peptide could suppress aggregation of proteins and membrane structures in vivo and raise the survival rate after rehydration. To address whether PvLEA-22 peptide could mimic the function of G3LEA in vivo, we used the Pv11 cell line established from an anhydrobiotic chironomid, Polypedilum vanderplanki, investigated the beneficial effect of either extracellular or intracellular occurrence of PvLEA-22 peptide on the survival rate of the dried Pv11 cells after rehydration. Consequently, both experiments did not show any beneficial effect of PvLEA-22 peptide on the survival rate. Interestingly, the treatment of high concentration PvLEA- 22 peptide caused the cell death. It should be due to strong binding activity of the peptide to cell membrane, resulting in loss of membrane flexibility. Our results demonstrated that PvLEA-22 peptide did not have the same function as G3LEA proteins in Pv11 cells and treatment of high-concentration PvLEA-22 peptide spoiled desiccation tolerance instead.

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Many unicellular microalgae and cyanobacteria are successfully cryopreserved by snap-freezing in liquid nitrogen. However, high survival rates have not been obtained for most filamentous multicellular microalgae and cyanobacteria. This study was conducted to attain high survival rates of Spirulina (Arthrospira) platensis NIES-46 after snap-freezing in liquid nitrogen by establishing a simple and reliable cryopreservation method. To this end, we investigated the dependence of the survival rate on trichome morphology and recovery from damage during preculture. The survival rate was estimated in the presence of 5% v/v dimethyl sulfoxide. The rate was higher for S. platensis NIES-46 with loosely coiled trichomes than for those with tightly coiled trichomes, and was higher for mutant with straight trichomes than with coiled trichomes. Although trichomes were damaged by agitation during preculture, the survival rate after cryopreservation by snap-freezing was improved by stationary culture before freezing. Ultimately, a high survival rate of approximately 80% was obtained for S. platensis NIES-46 after cryopreservation by snap-freezing in liquid nitrogen by considering trichome morphology.

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It is known that group 3 late embryogenesis abundant (G3LEA) proteins have the ability to protect membrane and proteins from desiccation stress. G3LEA proteins commonly have characteristic 11-mer repeat motif. In previous studies using model peptides composed of two or four tandem repeat of the 11-mer motif, we suggested that the 11-mer motif region forms the core functional site of G3LEA proteins. However, the biological role of non-repeat sequence regions remains unclear. In a previous preliminary study, we examined the structural and functional properties of 22-mer model peptides for non-repeat regions of a G3LEA protein (PvLEA4) in an African sleeping chironomid. Here, we more extensively investigate the role of non-repeat regions using the same peptides.

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We report solvatochromic shift of the absorption spectrum and fluorescent red-edge effect (REE) of a betaine molecule, 2-(1-pyridinio)benzimidazolate (SBPa) in saccharide glasses. Interestingly, SBPa exhibits REE even in aqueous solution, where the microscopic environmental inhomogeneity is normally averaged over time by the solvent thermal fluctuation. We conclude that SBPa exists in an equilibrium state between betaine and protonated form in aqueous solution. Thus, frequency shift of absorption and fluorescence spectra of SBPa could be not only a measure of solvent polarity but also that of solvent acidity, and the data are reanalyzed by the new knowledge. Nevertheless, Stokes shift of SBPa in trehalose glass is the smallest compared to other saccharide glasses measured in this study, indicating significant suppression of molecular fluctuation by trehalose glass.

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We have studied the recovery of activity and secondary structure of bovine pancreatic ribonuclease A (RNase A) in aqueous ionic liquid (IL) solutions including methylammonium nitrate (MAN), ethylammonium nitrate (EAN), and propylammonium nitrate (PAN) after cryopreservation (77 K). We found that activity and secondary structure of RNase A in aqueous IL solutions were recovered. Remarkably, concentrated aqueous EAN solutions showed the highest recovery ability for RNase A among the studied IL concentrations. The present results indicate that concentrated aqueous EAN solutions may have potential as a novel cryopreservation/recovery solvent for wide variety of proteins. (Received

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The viability of cells after freezing and thawing depends on kinds of cells, kinds and concentration of chemical additives, cooling and warming rates, minimum temperature, cycles of repetition of freezing and thawing, etc. In cryosurgery, destruction of diseased biological tissues in vivo by freezing, tumor cells could remain in the area away from the freezing probe if the temperature drop there is not sufficient to destroy the cells. However, the destruction of the cells can be promoted by the repeat of freezing and thawing. In this study, double freezing and thawing was imposed on a cell-sample to investigate the morphological change and viability of cells after freezing and thawing and the relation between both the characteristics. The characteristics were also compared between the 1st and 2nd cycles of repetition of freezing and thawing. Furthermore, the correlation between the cell viabilities after the 1st and 2nd cycles of repetition of freezing and thawing was determined on the basis of a mathematical model with reaction kinetic formulation of cell death. The correlation predicted from the mathematical model was compared with the experiment to clear the effect of the double freezing and thawing.

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