Chemical chaperoning action of glycerol on the antifreeze protein of rainbow smelt

Rainbow smelt (Osmerus mordax) accumulate high levels of glycerol and moderate levels of trimethylamine oxide (TMAO) that lower the colligative freezing point of the serum and thereby contribute to seasonal freeze resistance. In the current study, the possibility that one or both of these compounds might also have a chaperoning role at low temperatures in smelt was investigated by studying their effects on the smelt antifreeze protein (AFP). Activity of the AFP in the presence of physiological levels of TMAO and glycerol was observed by means of ice crystal morphology and measured as thermal hysteresis. Ice crystals in AFP solutions were not visibly modified by either compound and TMAO at 25 and 50 mM had no appreciable effect on hysteresis; however, glycerol at 250 and 500 mM increased hysteresis. An equiosmolar level of NaCl was not as effective as glycerol in enhancing hysteresis, suggesting that osmolarity had little or no role. Although cross-linking experiments showed dimerization of AFP to be unchanged in the presence of glycerol, circular dichroism and intrinsic fluorescence analyses revealed enhanced protein folding. As glycerol enhances the folding and consequent activity of smelt AFP, protein chaperoning appears to be an endogenous role of glycerol in this vertebrate species.     

Control of glycerol production by rainbow smelt (Osmerus mordax) to provide freeze resistance and allow foraging at low winter temperatures

The rainbow smelt (Osmerus mordax) is a small anadromous fish that actively feeds under the ice at temperatures as low as the freeze point of seawater. Freezing is avoided through the production of both non-colligative antifreeze protein (AFP) and glycerol that acts in a colligative manner. Glycerol is constantly lost across the gills and skin, thus glycerol production must continue on a sustained basis at low winter temperatures. AFP begins to accumulate in early fall while water temperatures are still high. Glycerol production is triggered when water temperatures decrease to about 5 degrees C. Glycerol levels rapidly increase with carbon flow from dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P) to glycerol. Glucose/glycogen serves as the initial carbon source for glycerol accumulation with amino acids contributing thereafter. The period of glycerol accumulation is associated with increases in GPDH mRNA and PEPCK mRNA followed by elevations in protein synthesis and enzyme activities. Plasma glycerol levels may reach in excess of 500 mM in winter. The high freeze resistance allows rainbow smelt to invade water of low temperature and forage for food. The lower the temperature, the higher the glycerol must be, and the higher the glycerol the greater the loss to the environment through diffusion. During the winter, rainbow smelt feed upon protein rich invertebrates with glycerol production being fueled in part by dietary amino acids via the gluconeogenic pathway. At winter temperatures, glycerol is quantitatively more important than AFP in providing freeze resistance of blood; however, the importance of AFPs to other tissues is yet to be assessed. Glycerol levels rapidly plummet in the spring when water temperature is still close to 0 degrees C. During this period, freeze resistance must be provided by AFP alone. Overall, the phenomenon of glycerol production by rainbow smelt reveals an elegant connection of biochemistry to ecology that allows this species to exploit an otherwise unavailable food resource.     

Seasonal freeze resistance of rainbow smelt (Osmerus mordax) is generated by differential expression of glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, and antifreeze protein genes

In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol-3-phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall-winter-spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression.     

Some characteristics of freezing avoidance in two osmerids, rainbow smelt and capelin

Winter-acclimatised rainbow smelt Osmerus mordax have elevated levels of glycerol and trimethylamine oxide (TMAO), and a protein antifreeze, all of which are lost in summer. No seasonal differences were found in glycerol-3-P-dehydrogenase, glycerol-3-phosphatase and glutamate dehydrogenase, which are involved in the synthesis of glycerol or its precursors. Although glycerol production requires NADH, no large seasonal differences were found in the activities of glucose-6-P-dehydrogenase and 6-phosphogluconate dehydrogenase (pentose phosphate pathway), malate dehydrogenase and aspartate aminotransferase (malate-aspartate shuttle) or in the NAD : NADH ratio. Thus, other regulatory mechanisms must be involved. Capelin Mallotus villosus collected from mid water in the Barents Sea in September (water temperatures +0·4 to −1·6°C) had only negligible amounts of glycerol, and no protein antifreeze, although serum TMAO levels (44 mm) were unusually high for a teleost. Capelin appear to avoid freezing largely by avoiding water layers with temperatures below their body freezing point, and by occasional supercooling.     

Low temperature directly activates the initial glycerol antifreeze response in isolated rainbow smelt (Osmerus mordax) liver cells

Rainbow smelt (Osmerus mordax) accumulate high levels of glycerol in winter that serve as an antifreeze. Liver glycogen is a source of glycerol during the early stages of glycerol accumulation, whereas dietary glucose and amino acids are essential to maintain rates of glycerol synthesis. We presently report rates of glycerol and glucose production by isolated hepatocytes. Cells from fish held at 0.4 to -1.5 degrees C and incubated at 0.4 degrees C were metabolically quiescent with negligible rates of glycerol or glucose production. Hepatocytes isolated from fish maintained at 8 degrees C and incubated at 8 degrees C produced glucose but not glycerol. Glycerol production was activated in cells isolated from 8 degrees C fish and incubated at 0.4 degrees C without substrate or when glucose, aspartate, or pyruvate was available in the medium. Incubation at 0.4 degrees C without substrate resulted in similar molar rates of glucose and glycerol production in concert with glycogen mobilization. Glycogenolysis and glycerol production were associated with increases in total in vitro activities of glycogen phosphorylase and glycerol-3-phosphate dehydrogenase. Maximal in vitro activities of hexokinase and glucokinase were not influenced by temperature, but high activities of a low-K(m) hexokinase may serve to redirect glycogen-derived glucose to glycolysis as opposed to releasing it from the cells. Rates of glycerol production were not enhanced in cells from fish held at 8 degrees C and incubated at 0.4 degrees C with adrenergic or glucocorticoid stimulation. As such, low temperature alone is sufficient to activate the glycerol production mechanism and results in a shift from glucose to a mix of glucose and glycerol production.     

Responses of protein phosphatases and cAMP-dependent protein kinase in a freeze-avoiding insect, Epiblema scudderiana

Larvae of the goldenrod gall moth, Epiblema scudderiana, use the freeze avoidance strategy of winter cold hardiness and show multiple metabolic adaptations for subzero survival including accumulation of large amounts of glycerol as a colligative antifreeze. Induction and regulation of cold hardiness adaptations requires the intermediary action of signal transduction enzymes. Changes in the activities of several signaling enzymes including cAMP-dependent protein kinase (PKA), protein phosphatases 1 (PP1), 2A, 2C, and protein tyrosine phosphatases (PTPs) were monitored over the winter and during experimental exposures of larvae to subzero temperatures (-4 degrees C, a temperature that triggers rapid glycerol synthesis, or -20 degrees C, a common midwinter ambient temperature) or anoxia. A strong increase in the amount of active PP1 in the latter part of the winter may be responsible for shutting off glycogenolysis once glycerol levels are maximized. There appears to be a limited role for PKA in overwintering but PP2A and PP2C activities rose when larvae were exposed to -20 degrees C and PTP activities rose significantly over the winter months and also in response to laboratory subzero (-20 degrees C) and anoxia exposures. The strong responses by PTPs suggest that these may be involved in cell cycle and growth arrest during winter diapause.     

Accelerated hepatic glycerol synthesis in rainbow smelt (Osmerus mordax) is fuelled directly by glucose and alanine: a 1H and 13C nuclear magnetic resonance study

At seawater temperatures below 1 degrees C, rainbow smelt (Osmerus mordax) accumulate plasma levels of glycerol up to 400 mM. Aspects of the synthesis of glycerol in liver and its regulation were previously investigated, but the pathways leading to glycerol synthesis remained unconfirmed. Here, we report nuclear magnetic resonance (NMR) studies which elucidate, in more detail, the fuel sources for rapid glycerol synthesis in rainbow smelt. Initial NMR analysis of liver homogenates from fish held at cold (-1 degrees C) temperatures and from fish transferred from 8 degrees C to -1 degrees C showed elevated glycerol, whereas those from fish held at 8 degrees C had far lower glycerol levels. These results confirm a temperature-responsive glycerol synthesis and show that NMR is a suitable approach to investigate the phenomenon. Further studies with fish held at low temperature and injected with labelled L-[2,3-(13)C(2)] alanine or D-[U-(13)C(6)]glucose revealed conversion of both alanine and glucose to glycerol. (13)C spectra showed satellites ((1)J(CC)=41.1 Hz) about the glycerol resonances indicating intact incorporation of a (13)C-(13)C unit in liver glycerol of fish injected with L-[2,3-(13)C(2)]alanine and a (13)C-(13)C-(13)C unit in liver glycerol of fish injected with D[U-(13)C(6)]glucose. Thus, glycerol can be efficiently produced directly from amino acid precursors by glyceroneogenesis, which is an abbreviated gluconeogenesis process leading to glycerol through dihydroxyacetone phosphate (DHAP). Glucose can also be metabolised to glycerol via an abbreviated form of glycolysis that similarly leads to glycerol through DHAP.     

Seasonal modulation of plasma antifreeze protein levels in Atlantic (Anarhichas lupus) and spotted wolffish (A. minor)

The Atlantic and spotted wolffish (Anarhichas lupus and A. minor, respectively) inhabit the cold waters of the northeast Atlantic Ocean. Although both species experience subzero water temperatures during winter, the Atlantic wolffish, which occupies shallower waters than the spotted wolffish, faces the greater threat of coming into contact with ice and freezing. This laboratory study was designed to determine whether these species differed in their abilities to resist freezing by examining the seasonal changes in blood plasma freezing points, antifreeze protein (AFP) activity and Na⁺ and Cl⁻ concentrations when exposed to seasonally cycling water temperatures and photoperiod. The plasma of both species showed distinct seasonal cycles in all parameters with the highest values occurring during the winter. However, of the two species, only the Atlantic wolffish produced sufficient AFP to protect the fish down to the freezing point of seawater (− 1.80 °C). The levels of AFP in the spotted wolffish were too low to impart any significant improvement in their resistance to freezing (approximately − 0.8 °C).When wolffish were maintained in warm water under a seasonally changing photoperiod, the amplitude of the seasonal cycle in AFP activity was greatly reduced, indicating that low water temperatures are necessary to maximize plasma AFP levels. However, despite being maintained in warm water, plasma levels of AFP activity began to increase over summer values at the same time of year as did the fish exposed to seasonally changing water temperatures. This suggests that photoperiod plays a major role in the timing of the annual AFP cycle.     

Structural and functional characterization of a C-type lectin-like antifreeze protein from rainbow smelt (Osmerus mordax).

Antifreeze proteins (AFPs) are produced by several cold-water fish species. They depress physiological freezing temperatures by inhibiting growth of ice crystals and, in so doing, permit the survival of these fish in seawater cooler than their normal freezing temperatures. The type II AFP from rainbow smelt (Osmerus mordax), which is a member of the C-type lectin superfamily, was characterized in terms of its Ca2+-binding quaternary structure and the role of its single N-linked oligosaccharide. The protein core of the smelt AFP, shown through sequence homology to be a C-type lectin carbohydrate-recognition domain, was found to be protease resistant. Smelt AFP was also shown to bind Ca2+, as determined by ruthenium red staining and a conformational change on Ca2+ binding detected by intrinsic fluorescence. The N-linked oligosaccharide was found to have no effect on protease resistance, dimerization, or antifreeze activity. Thus its role, if any, in the antifreeze function of this protein remains unknown. Smelt AFP was also shown to be a true intermolecular dimer composed of two separate subunits. This dimerization did not require the presence of N-linked oligosaccharide or bound Ca2+. Smelt AFP dimerization has implications for the effective solution concentration and measurement of its activity. This finding may also lead to new interpretation of the mechanism of ice-growth inhibition by this AFP.     

Short Communication: Increased gene dosage augments antifreeze protein levels in transgenic Drosophila melanogaster

One of the principal environmental adaptations of certain fishes inhabiting polar and northern coastal waters is the synthesis of antifreeze proteins (AFPs). AFPs bind to and prevent the growth of nascent ice crystals, thus depressing the serum freezing point. The transgenic expression of AFP holds great promise for conferring freeze resistance to commercially important plant and animal species. Since fish at the greatest risk of freezing have multiple AFP gene copies in order to synthesize higher levels of this protein, we have evaluated this evolutionary strategy as a way to maximize AFP expression in a model transgenic host, the fruit fly Drosophila melanogaster. A construct in which AFP genes of the Atlantic wolffish are fused to the Drosophila yolk protein 1,2 promoter/enhancer region was transferred to flies through P-element mediated transformation. Several independent transgenic fly lines were used in genetic crosses to obtain multi-insert lines. Haemolymph freezing point depression (thermal hysteresis) was greater in homozygotes relative to heterozygotes for a given insert. Similarly, multi-insert lines consistently displayed greater haemolymph AFP activity than the single insert lines from which they were derived. The thermal hysteresis value obtained with a fly line harboring 8 AFP gene copies, 0.43 °C, represents the highest such value to date recorded in a transgenic host, and is even higher than the levels found in some AFP-producing fish.     

Insect antifreezes and ice-nucleating agents

Cold-tolerant, freeze-susceptible insects (those which die if frozen) survive subzero temperatures by proliferating antifreeze solutes which lower the freezing and supercooling points of their body fluids. These antifreezes are of two basic types. Lowmolecular-weight polyhydroxy alcohols and sugars depress the freezing point of water on a colligative basis, although at higher concentrations these solutes may deviate from linearity. Recent studies have shown that these solutes lower the supercooling point of aqueous solutions approximately two times more than they depress the freezing point. Consequently, if a freeze-susceptible insect accumulates sufficient glycerol to lower the freezing point by 5 °C, then the glycerol should depress the insect's supercooling point by 10 °C.Some cold-tolerant, freeze-susceptible insects produce proteins which produce a thermal hysteresis (a difference between the freezing and melting point) of several degrees in the body fluids. These thermal hysteresis proteins (THPs) are similar to the antifreeze proteins and glycoproteins of polar marine teleost fishes. The THPs lower the freezing, and presumably the supercooling, point by a noncolligative mechanism. Consequently, the insect can build up these antifreezes, and thereby gain protection from freezing, without the disruptive increases in osmotic pressure which accompany the accumulation of polyols or sugars. Therefore the THPs can be more easily accumulated and maintained during warm periods in anticipation of subzero temperatures. It is not surprising then that photoperiod, as well as temperature, is a critical environmental cue in the control of THP levels in insects.Some species of freeze-tolerant insects also produce THPs. This appears somewhat odd, since most freeze-tolerant insects produce ice nucleators which function to inhibit supercooling and it is therefore not clear why such an insect would produce antifreeze proteins. It is possible that the THPs have an alternate function in these species. However, it also appears that the THPs function as antifreezes during those periods of the year when these insects are not freeze tolerant (i.e., early autumn and spring) but when subzero temperatures could occur. In addition, at least one freeze-tolerant insect which produces THPs, Dendroides canadensis, typically loses freeze tolerance during midwinter thaws and then regains tolerance. The THPs could be important during those periods when Dendroides loses freeze tolerance by making the insect less susceptible to sudden temperature decreases.Comparatively little is known of the biochemistry of insect THPs. However, comparisons of those few insect THPs which have been purified with the THPs of fishes show some interesting differences. The insect THPs lack the large alanine component commonly found in the fish THPs. In addition, the insect THPs generally contain greater percentages of hydrophilic amino acids than do those of the fish. Perhaps the most interesting insect THPs are those from Tenebrio molitor which have an extremely large cysteine component (28% in one THP). Studies on the primary and higher-order structure of the insect THPs need to be carried out so that more critical comparisons with the fish THPs can be made. This may provide important insights into the mechanisms of freezing point and supercooling point depression exhibited by these molecules. In addition, comparative studies of the freezing and supercooling point depressing activities of the various THPs, in relation to their structures, should prove most interesting.It has become increasingly apparent over the last few years that most freeze-tolerant insects, unlike freeze-susceptible species, inhibit supercooling by accumulating ice-nucleating agents in their hemolymph. These nucleators function to ensure that ice formation occurs in the extracellular fluid at fairly high temperatures, thereby minimizing the possibility of formation of lethal intracellular ice. Little is known of the nature of the insect ice-nucleating agents. Those few which have been studied are heat sensitive and nondialyzable and are inactivated by proteolytic enzymes, thus indicating that they are proteinaceous. Studies on the structure-function relationships of these unique molecules should be done.     

Variations in antifreeze activity and serum inorganic ions in the eelpout Zoarces viviparus: antifreeze activity in the embryonic state.

The eelpout Zoarces viviparus is a common inhabitant in the shallow waters along the Danish coastline. Specimens were caught in the brackish (12-16 per thousand) Roskilde fjord where water temperatures range from >20 degrees C during summer to subzero in winter. The serum melting points found in Z. viviparus varied between -0.76 (September) to -0.94 degrees C (January). Eighty to 97% of the serum melting points could be attributed to sodium, chloride and potassium. Hysteresis freezing points showed seasonal variation varying from -0.83 (September) to -2.08 degrees C (February). Serum antifreeze activity showed a seasonal variation with high levels (>1.2 degrees C) in winter and low levels (<0.1 degrees C) during summer and autumn. Antifreeze proteins are responsible for this antifreeze activity. Antifreeze activity was also found in Z. viviparus during their embryological development in the female ovary. Embryo thermal hysteresis reached the maximum level (approx. 0.6 degrees C) during December and maintained this level until parturition in January. Antifreeze activity seems unaffected by diminishing ice crystal fractions at ice fractions below 0.1 whereas ice fractions above 0.1 caused a decline in antifreeze activity.     

Comparison of liver enzymes in osmerid fishes: key differences between a glycerol accumulating species,rainbow smelt (Osmerus mordax), and a species that does not accumulate glycerol,capelin (Mallotus villosus)

Activities of enzymes associated with glycerol synthesis were compared in the liver of two osmerid fishes, the smelt (Osmerus mordax), which can accumulate high (400 mM) levels of glycerol and capelin (Mallotus villosus) that does not accumulate glycerol. Animals were sampled at approximately the same time of year and temperature thus negating potential seasonal effects. These species are closely related, reducing interpretative issues involving comparison between unrelated species. We found that key enzyme activities were elevated in the smelt relative to the non-glycerol accumulating capelin, namely enzymes involved with glycolysis (phosphofructose kinase-1 and aldolase), amino acid metabolism (aspartate aminotransferase and alanine aminotransferase), gluconeogenesis (phosphoenolpyruvate carboxykinase) and glycerol synthesis (glycerol-3-phosphate dehydrogenase). The enzyme profiles strongly support the hypothesis that smelt can synthesize glycerol by utilizing glycogen and amino acids as the carbon source and that they have increased capacity for metabolic flux through loci required for synthesis of the three carbon intermediate dihydroxyacetone phosphate and subsequently glycerol synthesis.     

Temperature effects on trimethylamine oxide accumulation and the relationship between plasma concentration and tissue levels in smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) were maintained in a long term acclimation study to elucidate temperature effects on the accumulation of trimethylamine oxide (TMAO) and to determine if the activity of trimethylamine oxidase (TMAoxi) plays a role in modulating the seasonally variable levels of TMAO. In the same experiment, the TMAO content was determined for several tissues at varying plasma TMAO concentrations. TMAO accumulation begins at 5-7 degrees C, well above that which might be normally associated with an antifreeze response. The plasma concentration reached a plateau of 20 mM as temperatures reached 0 degrees C. Plasma TMAO concentration drops to pre-accumulation levels, less than 5 mM, when fish are held at elevated temperature (8-11 degrees C) and increases when fish are chilled below ambient seawater temperatures. However, despite temperatures near or below 0 degrees C, plasma TMAO decreases after the winter season. Changes in TMAoxi activity do not correlate with TMAO levels, suggesting that the activity of this enzyme does not play a key role in regulating TMAO concentrations in smelt. For the first time in any teleost fish, tissue TMAO contents in liver, kidney, brain, and intestine were found to strongly correlate with plasma TMAO concentrations. For these tissues, the intracellular and extracellular concentration of TMAO appears to be approximately equal. Conversely, the heart and white muscle accumulate TMAO, and in the case of white muscle, intracellular concentration is maintained at a constant level of approximately 35 mmol/kg, despite fluctuating plasma concentrations over a range from 0 to over 25 mM.     

Thermodynamic nonequilibrium phase change behavior and thermal properties of biological solutions for cryobiology applications

Understanding the phase change behavior of biomaterials during freezing/thawing including their thermal properties at low temperatures is essential to design and improve cryobiology applications such as cryopreservation and cryosurgery. However, knowledge of phase change behavior and thermal properties of various biomaterials is still incomplete, especially at cryogenic temperatures (< or = -40 degrees C). Moreover, in these applications, chemicals are often added to improve their outcome, which can result in significant variation in the phase change behavior and thermal properties from those of the original biomaterials. These chemical additives include cryoprotective agents (CPAs), antifreeze protein (AFP), or cryosurgical adjuvants like sodium chloride (NaCl). In the present study, phase change behavior and thermal properties of saline solutions--either water-NaCl or phosphate buffered saline (PBS)--with various chemical additives were investigated. The chemical additives studied are glycerol and raffinose as CPAs, an AFP (Type III, molecular weight = 6500), and NaCl as a cryosurgical adjuvant. The phase change behavior was investigated using a differential scanning calorimeter (DSC) and a cryomicroscope. The specific and latent heat of these solutions were also measured with the DSC. The saline solutions have two distinct phase changes--water/ice and eutectic phase changes. During freezing, eutectic solidification of both water-NaCl and PBS are significantly supercooled below their thermodynamic equilibrium eutectic temperatures. However, their melting temperatures are close to thermodynamic equilibrium during thawing. These eutectic phase changes disappear when even a small amount (0.1 M glycerol) of CPA was added, but they are still observed after the addition of an AFP. The specific heats of these solutions are close to that of ice at very low temperatures (< or = -100 degrees C) regardless of the additives, but they increase between -100 degrees C and -30 degrees C with the addition of CPAs. The amount of latent heat, which is evaluated with sample weight, generally decreases with the addition of the additives, but can be normalized to approximately 300 J/g based on the weight of water which participates in the phase change. This illustrates that thermal properties, especially latent heat, of a biomaterial should be evaluated based on the understanding of its phase change behavior. The results of the present study are discussed in the context of the implications for cryobiology applications.     

The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate

Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.     

Role of membrane transport of water and glycerol in the freeze tolerance of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

Overwintering larvae of the rice stem borer, Chilo suppressalis accumulate glycerol and are freezing tolerant to about -25 degrees C. However, non-diapausing larvae cannot accumulate glycerol and are killed by freezing. We compared the extent of tissue damage, the effects of glycerol concentration, and the transport of glycerol and water in fat body tissues from these larvae at selected freezing temperatures. Tissues from overwintering larvae, but not non-diapausing larvae, survive when frozen at -20 degrees C with 0.25 M glycerol, but the protection afforded by glycerol is offset by the water-channel inhibitor mercuric chloride. Glycerol in higher concentration (0.75 M) affords some protection even to the fat body of non-diapausing larvae. Radiotracer assays of overwintering larvae show that water leaves the tissues during freezing while glycerol enters, and that mercuric chloride disrupts this process. Transport is also disrupted after lethal freezing at -35 degrees C. Therefore, membrane transport of water and glycerol is involved in the avoidance of freezing injury to fat body cells of the rice stem borer, apparently by mediating the replacement of water with glycerol in freezing-tolerant tissues.     

Solution Structures,Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.