Concentration Of Antifreeze Proteins Research Articles

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage.

Polycarboxylates enhance beetle antifreeze protein activity

Antifreeze proteins (AFPs) lower the noncolligative freezing point of water in the presence of ice below the ice melting point. The temperature difference between the melting point and the noncolligative freezing point is termed thermal hysteresis (TH). The magnitude of the TH depends on the specific activity and the concentration of AFP, and the concentration of enhancers in the solution. Known enhancers are certain low molecular mass molecules and proteins. Here, we investigated a series of polycarboxylates that enhance the TH activity of an AFP from the beetle Dendroides canadensis (DAFP) using differential scanning calorimetry (DSC). Triethylenetetramine- N, N, N′, N″, N′″, N′″-hexaacetate, the most efficient enhancer identified in this work, can increase the TH of DAFP by nearly 1.5 fold over than that of the published best enhancer, citrate. The Zn 2+ coordinated carboxylate results in loss of the enhancement ability of the carboxylate on antifreeze activity. There is not an additional increase in TH when a weaker enhancer is added to a stronger enhancer solution. These observations suggest that the more carboxylate groups per enhancer molecule the better the efficiency of the enhancer and that the freedom of motion of these molecules is necessary for them to serve as enhancers for AFP. The hydroxyl groups in the enhancer molecules can also positively affect their TH enhancement efficiency, though not as strongly as carboxylate groups. Mechanisms are discussed.

Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

Cold hardiness in relation to trace metal stress in the freeze-avoiding beetle Tenebrio molitor

The antifreeze proteins (AFPs) are a family of proteins characterised by their ability to inhibit the growth of ice. These proteins have evolved as a protection against lethal freezing in freeze avoiding species. Metal stress has been shown to reduce the cold hardening in invertebrates, but no study has investigated how this type of stress affects the production of AFPs. This study demonstrates that exposure to cadmium (Cd), copper (Cu) and zinc (Zn) reduces the normal developmental increase in AFP levels in Tenebrio molitor larvae reared under summer conditions. Exposure to winter conditions, however stimulated the production of AFPs in the metal exposed larvae, and raised the concentrations of AFPs to normal winter levels. The reduced level of AFPs in metal-stressed animals acclimated to summer conditions seems to arise from alterations in the normal gene expression of AFPs. The results indicate that metal exposure may cause freeze avoiding insects to become more susceptible to lethal freezing, as they enter the winter with lowered levels of AFPs. Such an effect cannot be revealed by ordinary toxicological tests, but may nevertheless be of considerable ecological importance.

Application of Antifreeze Protein for Food Preservation: Effect of Type III Antifreeze Protein for Preservation of Gel‐forming of Frozen and Chilled Actomyosin

ABSTRACTType III antifreeze protein (AFP) remarkably preserved Ca2+ ATPase activity of actomyosin during frozen and chilled storage. Under frozen conditions, AFP helped to retain the Ca2+ ATPase activity of actomyosin much higher than that of conventional cryoprotectants (sucrose‐sorbitol mixture). The Ca2+ ATPase activity increased with increasing AFP concentration and reached a maximum at 50g/L AFP. After 3‐d chilled storage, the Ca2+ ATPase activity of control and sucrose‐sorbitol samples had lost 80% and 50%, respectively, while the AFP samples remained unchanged. A Type III AFP mechanism based on freezing temperature depression (more unfrozen water) and inhibition of ice recrystallization that protects against the freezing of muscle proteins in chilled or frozen conditions is proposed.

The role of endogenous antifreeze protein enhancers in the hemolymph thermal hysteresis activity of the beetle Dendroides canadensis

Antifreeze proteins (AFPs) lower the freezing point of water by a non-colligative mechanism, but do not lower the melting point, therefore producing a difference between the freezing and melting points termed thermal hysteresis. Thermal hysteresis activity (THA) of AFPs from overwintering larvae of the beetle Dendroides canadensis is dependent upon AFP concentration and the presence of enhancers of THA which may be either other proteins or low molecular mass enhancers. The purpose of this study was to determine the relative contributions of endogenous enhancers in winter D. canadensis hemolymph. Winter hemolymph collected over four successive winters (1997–1998 to 2000–2001) was tested. The first three of these winters were the warmest on record in this area, while December of the final year was the coldest on record. Protein and low molecular mass enhancers raised hemolymph THA 60–97% and 35–55%, respectively, based on hemolymph with peak THA for each year collected over the four successive winters. However, the hemolymph AFPs were not maximally enhanced since addition of the potent enhancer citrate (at non-physiologically high levels) resulted in large increases in THA. 13NMR showed that glycerol was the only low molecular mass solute present in sufficiently high concentrations in the hemolymph to function as an enhancer. Maximum THA appears to be ∼8.5 °C.

The effects of antifreeze proteins on chilled and frozen meat

The effects of cryoprotectant proteins, trivially termed ‘antifreeze proteins’, from the Antarctic Cod and the Winter Flounder were assessed in meat during chilling and freezing. In light-microscopy studies, bovine muscle ( Sternomandibularis) samples were soaked in phosphate buffered saline with and without 0·1 mg/ml antifreeze protein. Samples were then held frozen (−20°C) or chilled (2°C) for 3 days. Samples were freeze-substituted, embedded in resin and sectioned. With antifreeze protein present, transverse sections of frozen samples had many small intracellular spaces, probably representing ice crystals. Frozen controls had much larger intracellular single spaces. Antifreeze protein had no effect on chilled samples. Similarly treated samples were examined by scanning electron microscopy using a cryostage attachment. Chilled ovine muscle samples ( Peroneus longus) were soaked for various periods (0–7 days) in 0·9% saline containing various concentrations of antifreeze proteins (0–1 mg/ml). Samples were then held frozen (−20°C) or chilled (2°C) for 5 or 7 days. With frozen samples, antifreeze proteins reduced the size of ice crystals, compared to the control. This effect depended upon the concentration used and the period of soaking before the samples were frozen, but was independent of source. Antifreeze proteins had no effect on chilled samples.

Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth.

Antifreeze proteins (AFPs) are extremely efficient at inhibiting ice recrystallization in frozen solutions. Knight and Duman [Knight, C. A. & Duman, J. G. (1986) Cryobiology 23, 256-263] have proposed that this may be an important function of the proteins in freeze-tolerant organisms. We have tested this proposal in vitro by characterizing the influence of AFP on the recovery of cryopreserved cells, which often can survive cooling and yet subsequently be damaged by ice crystal growth during warming. Relatively low concentrations (e.g., 5-150 micrograms/ml) of winter flounder (Pseudopleuronectes americanus) AFP enhance survival of red blood cells cryopreserved in hydroxyethyl starch solutions. This effect is most apparent in samples warmed at suboptimal rates, i.e., where ice recrystallization would be exaggerated. Cryomicroscopy demonstrates that AFP inhibits ice recrystallization in the extracellular regions during the latter stages of the warming cycle. AFP concentrations that enhance survival of red cells confer partial inhibition of recrystallization. Relatively high concentrations of AFP (e.g., 1.54 mg/ml) are much more effective at inhibiting extracellular recrystallization. However, extensive growth of ice around the cell, and concomitant cell damage, is noted. The mechanism for this AFP-induced ice growth is unknown. We propose that there is a delicate balance between AFP-induced enhancement of cell preservation and AFP-induced enhancement of cell preservation and AFP-induced enhancement of cell damage and that this balance hinges on the degrees of inhibition of ice recrystallization and of preferential growth of ice around the cells. We conclude that, under appropriate conditions, one of the proposed functions of AFPs in nature can be emulated, and perhaps have application, in cryopreservation of materials of biomedical interest.