We examined the cold and heat denaturation of horse heart metmyoglobin (HHM) using an improved version of our recently published fluorescence ratio intrinsic basis states analysis (FRIBSTA). This protein is of considerable interest because its thermodynamic properties have been extensively studied, and it is one of several globular proteins used as models to investigate native protein stability. According to the published analyses, at pH values close to neutral in dilute electrolyte solution native sperm whale metmyoglobin has a stability maximum relative to its unfolded form in non-denaturing conditions of about +50kJ mole^<-1> at 〜35℃. At 0℃ the native protein is estimated to have a somewhat decreased stability of +35kJ mole^<-1>. In contrast, FRIBSTA indicates a detectable level of denaturation at 0℃ and pH 7, and considerably more at 0℃ and pH 4. Comparison with far UV circular dichroism (CD) spectra recorded at -2℃, 35℃, and in the range of heat denaturation reinforce the view that HHM has an intact secondary structure at -2℃ and pH 7. This would indicate that a significant proportion of myoglobin molecules are in the molten globule configuration at modest subambient temperatures. FRIBSTA derived values of the heat denaturation enthalpy of myoglobin at pH 4 and pH 7 are consistent with the literature data. When utilizing the published thermodynamic parameters of myoglobin's heat denaturation, the two state model gives estimates of the extent of cold denaturation fundamentally different from the low temperature FRIBSTA estimates of the thermodynamic stability of the protein. We briefly discuss the implications of these findings.

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We have shown that the 90-kDa heat shock protein (Hsp90) is able to bind partially unfolded firefly luciferase and maintain it in a refoldable state, and that the subsequent successive action of the 20S proteasome activator, PA28, Hsc70 and Hsp40 enables the refolding of the unfolded luciferase. It is most plausible that both the N- and C-terminal chaperone sites of Hsp90 participate in this protein refolding pathway.

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Upon exposure to low temperature, organisms perceive the change in temperature by certain mechanism(s) and regulate the expression of genes to acclimate to the new environment. In order to identify the low-temperature sensors from the cyanobacterium Synechocystis sp. PCC 6803, we inactivated each gene for 43 histidine kinases in the chromosome and found that in the mutant of histidine kinase 33, Hik33, the cold-inducibility of the desB gene for the ω3 acyl-lipid desaturase was clearly decreased. We also compared the gene expression in the cells of Hik33 mutant and cells of wild type after exposure to the low temperature by DNA microarray. The results indicated that Hik33 regulates the expression of some of the cold-inducible genes and cold-repressible genes. These results suggested that Synechocystis might have the second low-temperature sensor which regulates the expression of the genes which were not regulated by Hik33.

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Plant productivity is greatly affected by environmental stresses such as drought, salt loading and freezing. Recently, a major transcription system that controls gene expression in response to dehydration and low temperature has been identified. The system includes the DRE/CRT cis-acting element and its DNA-binding proteins, DREBs/CBFs, which have an AP2 domain. DREBs/CBFs contain two subclasses, DREB1/CBF and DREB2, which are induced by cold and dehydration, respectively, and control the expression of various genes involved in stress tolerance. Overexpression of the cDNA encoding DREB1/CBF in transgenic Arabidopsis activated the expression of many of these stress tolerance genes and resulted in improved tolerance to drought, saltloading and freezing. Overexpression of Arabidopsis DREB1/CBF genes in several kinds of crop plants induced expression of orthologs of Arabidopsis DREB-targeted genes and increased the freezing tolerance of transgenic plants. These observations suggest that the DRE/DREB regulon can be used to improve the tolerance of various kinds of agriculturally important crop plants to drought, high-salt, and freezing stresses by gene transfer.

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Using the extremely freeze-tolerant woody shrub C. sericea as a model system, we investigated the association of xylem proteins to cold acclimation. We identified a predominant 24 kDa dehydrin-like protein that accumulated during cold acclimation, raised a polyclonal antibody and screened a cDNA expression library to search for related genes. In controlled environment conditions, we identified and deciphered the mode of regulation for the 24 kDa protein by manipulating abiotic factors and monitoring subsequent physiological and molecular effects. With immunolocalization techniques, we clarified the subcellular localization of 24 kDa-like proteins in an effort to provide insight into a potential biological role. To assess the proposed role of dehydrins to freeze induced desiccation (caused by extracellular ice formation), we characterized freezing behavior within 30 Cornus species into classifications of either supercooling or non-supercooling behaviors. Subsequent comparisons of total xylem protein blot analysis determined that 24 kDa-like proteins were not limited to species that display non-supercooling behavior, nor to those species that are extremely freeze-tolerant.

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Nostoc commune, a nitrogen-fixing filamentous cyanobacterium, produces copious amounts of the EPS (extracellular polysaccharide) around the cells and has remarkable desiccation tolerance. Recently, biochemical experiment revealed that the EPS can serve to stabilize cyanobacterial cells during desiccation and subsequent rehydration. The EPS also seems to act as the physical and/or chemical barrier to the surroundings. Despite marked features of EPS of this cyanobacteria, little is known about its chemical structure, due largely to structural complexity. In this paper, the chemical and biochemical aspects of this cyanobacterial EPS are discussed.

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Organisms often suffer from water stress or dehydration under drought, high salt or freezing conditions. They have evolved various physiological and biochemical mechanisms to survive these water stresses. The most unique and ultimate strategy against drought stress is cryptobiosis, which is the state of an organism that can tolerate extremely desiccation without ill effects and survive for an extended period. An African chironomid, Polypedilum vanderplanki, is the highest and largest multicellular animal with cryptobiotic ability. The cryptobiotic larva can tolerate not only exposure to extremely high (106℃) and low (-270℃) temperatures but also soak into pure ethanol. We found that this chironomid accumulated a large amount of trehalose (ca. 20% of the dry body weight) at the state of cryptobiosis, and cerebral regulation was not involved in the process for induction of cryptobiosis.

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Freeze-drying is a popular method to stabilize many therapeutic proteins that are not stable enough to meet the long-term stability required for pharmaceutics. In spite of the improved long-term stability, various stresses during the freeze-drying and subsequent storage often induce structural (e.g. denaturation, aggregation) and chemical (e.g., oxidation) changes of the proteins. This review describes how the various excipients (e.g., saccharides, polyols, polymers, surfactants, buffer components) stabilize proteins in each process. Effects of solute miscibility and crystallinity on the stabilizing effect were also discussed.

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Fibrin glue is a topical biological adhesive widely used in surgery for tissue adhesion and wound healing. The effect of fibrin glue imitates the final stages of coagulation. We prepared cryo-fibrin glue from autologous blood using the CryoSeal System. We chose the CryoSeal^<TM> System because the cryo-fibrin glue can be produced in a automated, rapid way. In fact, the entire process only required about 56 minutes. After separating fresh plasma (average volume: 199.2±1.8mL) from whole blood, cryoprecipitate was extracted. The concentration and volume of fibrinogen were 33.4±7.5mg/mL and 3.9±0.9mL, respectively (n=9), and the yields of fibronectin, plasminogen, and blood coagulation factor XIII were 10.9±1.4mg/mL, 177±51%, and 761±310%, respectively (n=9). The tensile strength of fibrin glue when mixed with thrombin was comparable to that of commercialy available fibrin glue. This system may be useful to prepare cryo-fibrin glue from autologous blood at the medical facilities.

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Freezing and drying behavior of polymer gels in a bead form, used as models of biological systems, has been studied as understanding of the behavior is of practical importance for a successful preservation of the systems. Attention was focused on the anomalous ice crystallization during rewarming observed with a dextran gel of a certain density of crosslink. Size of ice crystals formed or presence of glassy water in the frozen gel was found, as a result, to depend on the polymer network characterized especially by the crosslink density of the gels. Drying behavior is also influenced by the characteristics of the polymer network. In conclusion, water in polymer gels is obstructed in diffusion by the polymer network, and freezing and drying behavior in the gels becomes different from that in aqueous solutions. These results should be taken into consideration in the practice of biopreservation as most biological systems have an extensive polymer network.

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