• Fisheries and Aquatic Sciences
• /
• 제18권2호
• /
• pp.137-142
• /
• 2015

Antifreeze proteins and glycoproteins [AF(G)Ps] constitute a group of proteins that lower the freezing but not the melting points of aqueous solutions, enabling polar and north-temperate fish to survive in ice-laden environments. However, little is known about antifreeze activity in temperate fish; such work would extend our knowledge on the functions and evolution of AF(G)Ps. In the present study, we screened for antifreeze activity in temperate fish caught off the coast of Jumunjin ($37.89^{\circ}N$), Gangneung, Korea. Thermal hysteresis (TH) and the ability to inhibit ice recrystallization (IR) in blood, liver, and muscle samples from nine fish were examined to assess antifreeze activity. As the East Sea off the coast of Jumunjin is ice-free year round, we thought it most unlikely that the fish would express antifreeze proteins. Surprisingly, the blood of Pleurogrammus azonus and three types of tissue from Gymnocanthus herzensteini, Zoarces gilli and Kareius bicoloratus exhibited measurable TH values together with the ability to trigger characteristic morphological changes in ice crystals. Blood samples from the three species also evidenced ice recrystallization (IR) inhibition. This implies that AF(G)Ps or other antifreeze-like substances are present in temperate fish even under nonfreezing conditions. These results contribute to our understanding of the functions and origins of antifreeze activity in fish.

• 한국작물학회지
• /
• 제48권5호
• /
• pp.407-412
• /
• 2003

In order to measure an antifreezing tolerance, antifreeze proteins accumulated upon cold acclimation in apoplast were analyzed. As Dongborilho were cold-acclimated for 3 to 74 days there was an abrupt increase in apoplastic proteins up to 30 days and then decrease to the similar levels. Among the known antifreeze proteins, CLP produced in E. coli. and TLP purified from apoplast were used to generate antisera that allow to measure and localize the proteins in leaves of barley. The CLP of 27.7 kDa and TLPs of 6, 26, 27, 30, and 31 kDa were increased in their amounts in apoplast as cold treatment being longer. There was a correlation among the amounts of those proteins accumulated in apoplast and freezing tolerance as shown in field and ion leakage tests for five cultivars. The deposit of CLP was localized in the marginal area and the area adjacent to leaf vescular bundle cells in an increasing manner according to duration of cold acclimation but no variation was observed in terms of it's distribution. Based on the close correlation between levels of antifreeze proteins and degrees of freezing tolerance, the immunological methods was to develop to estimate a freezing tolerance in barley.

• 한국작물학회지
• /
• 제43권3호
• /
• pp.172-178
• /
• 1998

Freezing-resistant plants can survive subzero temperatures by withstanding extracellular ice formation. During cold acclimation, their leaves accumulate antifreeze proteins (AFPs) that are secreted into the apoplast and have the ability to modify the normal growth of ice crystals. Three barley, two wheat and two rye cultivars were grown under two different temperature regimes (20/16$^{\circ}C$ and 5/2$^{\circ}C$, day/night). Apoplastic proteins from winter cereals were separated by SDS-PAGE and detected with antisera to AFPs from winter rye. Apoplastic proteins accumulated to much higher levels in cold-acclimated (CA) leaves compared with nonacclimated (NA) ones in winter cereals. After cold acclimation, the protein concentration of apoplastic extracts increased significantly from 0.088 $mgmL^{-1}$ to 0.448 $mgmL^{-1}$, with about 5-fold increment. Also, the apoplastic protein content per gram leaf fresh weight in CA leaves ranged from 31 $\mu\textrm{g}$ $(gFW)^{-1}$ to 120 $\mu\textrm{g}$ $(gFW)^{-1}$ with an averaged value of 77 $\mu\textrm{g}$ $(gFW)^{-1}$, and coefficients of variation of 54.9%. The CA leaves in Musketeer (a Canadian winter rye cultivar) showed the greatest AFPs and antifreeze activity followed by 'Geurumil' (a Korean winter wheat cultivar), and 'Dongbori l' (Korean facultative barley cultivar). The proteins secreted into the wheat leaf apoplast at CA condition were more numerous than those observed in winter rye, where two $\beta$-1,3-glucanase-like proteins (GLPs), two chitinase-like proteins (CLPs) and two thaumatin-like proteins (TLPs) accumulated during cold acclimation. The proteins in barley leaf apoplast at CA conditions were a little different from those in wheat leaves. The AFPs were various among and within species. More freezing-resistant cultivars had more clear and numerous bands than less freezing-resistant ones. The high determination coefficient ($R^2$ =91 %) between freezing resistance and AFPs per gram leaf fresh weight indicated that the amount of AFPs was highly related to freezing resistance in winter cereal crops.

• Animal cells and systems
• /
• 제3권1호
• /
• pp.47-52
• /
• 1999

Change of proteins was confirmed during low temperature acclimation of overwintering larva, and some biochemical characteristics of the induced antifreeze protein-1 (AFP-1) were investigated in Protaetia brevitarsis. As the freezing point depression by the action of induced AFPs, a considerable thermal hysteresis was observed in the haemolymph and in partially purified proteins. AFP-1 was purified from the cold acclimation larvae by ammonium sulfate precipitation ion exchange chromatography, gel permeation chromatography, and electroelution. The purified AFP-1 was determined to be a glycoprotein (approximately 320 kDa, pl 5.8) composed of a single type of subunit (80 kDa). The high contents of hydrophilic amino acids (Asp, Glu, Lys, Asn, Gln, Arg, Ser, Thr) were also confirmed, showing similarity with antifreeze proteins from other insects.

• 식물조직배양학회지
• /
• 제27권4호
• /
• pp.339-343
• /
• 2000

When plants are exposed to subfreezing temperatures ice crystals are forming within extracelluar space in leaves. The growth of ice crystal is closely related to the degree of freezing injury. It was shown that an antifreeze protein binds to an ice nucleator through hydrogen bonds to prevent growth of ice crystal and also reduces freezing damage. The antifreeze proteins in plants are similar to PR proteins but only the PR proteins induced upon cold acclimation were shown to have dual functions in antifreezing as well as antifungal activities. Three of the genes encoded for CLP, GLP, and TLP were isolated from barley and Kentucky bluegrass based on amino acid sequence revealed after purification and low temperature-inducibility as shown in analysis of the protein. The deduced amino acid of the genes cloned showed a signal for secretion into extracellular space where the antifreezing activity sup-posed to work. The western analysis using the antisera raised against the antifreeze proteins showed a positive correlation between the amount of the protein and the level of freeze tolerance among different cultivars of barely. Besides it was revealed that TLP is responsible for a freeze tolerance induced by a treatment of trinexapac ethyl in Kentucky bluegrass. Analysis of an overwintering wild rice, Oryza rufipogon also showed that an acquisition of freeze tolerance relied on accumulation of the protein similar to CLP. The more direct evidence for the role of CLP in freeze tolerance was made with the analysis of the transgenic tobacco showing extracellular accumulation of CLP and enhanced freeze tolerance measured by amount of ion leakage and rate of photosynthetic electron transport upon freezing. These antifreeze proteins genes will be good candidates for transformation into crops such as lettuce and strawberry to develop into the new crops capable of freeze-storage and such as rose and grape to enhance a freeze tolerance for a safe survival during winter.

• Ocean and Polar Research
• /
• 제33권2호
• /
• pp.159-169
• /
• 2011

Antifreeze proteins (AFPs) have ice binding affinity, depress freezing temperature and inhibit ice recystallization which protect cellular membranes in polar organisms. Recent structural studies of antifreeze proteins have significantly expanded our understanding of the structure-function relationship and ice crystal growth inhibition. Although AFPs (Type I-IV AFP from fish, insect AFP and Plant AFP) have completely different fold and no sequence homology, they share a common feature of their surface area for ice binding property. The conserved ice-binding sites are relatively flat and hydrophobic. For example, Type I AFP has an amphipathic, single ${\alpha}$-helix and has regularly spaced Thr-Ala residues which make direct interaction with oxygen atoms of ice crystals. Unlike Type I AFP, Type II and III AFP are compact globular proteins that contain a flat ice-binding patch on the surface. Type II and Type III AFP show a remarkable structural similarity with the sugar binding lectin protein and C-terminal domain of sialic acid synthase, respectively. Type IV is assumed to form a four-helix bundle which has sequence similarity with apolipoprotein. The results of our modeling suggest an ice-binding induced structural change of Type IV AFP. Insect AFP has ${\beta}$-helical structure with a regular array of Thr-X-Thr motif. Threonine residues of each Thr-X-Thr motif fit well into the ice crystal lattice and provide a good surface-surface complementarity. This review focuses on the structural characteristics and details of the ice-binding mechanism of antifreeze proteins.

• Ocean and Polar Research
• /
• 제25권4호
• /
• pp.607-615
• /
• 2003

Many organisms are known to survive in icy environments. These include both over wintering terrestrial insects and plants as well the marine fish inhabiting high latitudes. The adaptation of these organisms is both a fascinating and important topic in biology. Marine teleosts in particular, can encounter ice-laden seawater that is approximately $1^{\circ}C$ colder than the colligative freezing point of their body fluids. These animals produce a unique group of proteins, the antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) that absorb the ice nuclei and prevent ice crystal growth. Presently, there are at least four different AFP types and one AFGP type that are isolated from a wide variety of fish. Despite their functional similarity, there is no apparent common protein homology or ice-binding motifs among these proteins, except that the surface-surface complementarity between the protein and ice are important for binding. The remarkable diversity of these proteins and their odd phylogenetic distribution would suggest that these proteins might have evolved recently in response to sea level glaciations just 1-2 million years ago in the northern hemisphere and 10-30 million years ago around Antarctica. Winter flounder, Pleuronectes americanus, has been used as a popular model to study the regulation of AFP gene expression. It has a built-in annual cycle of AFP expression controlled negatively by the growth hormone. The signal transduction pathways, transcription factors and promoter elements involved in this process have been studied in our laboratory and these studies will be presented.

• 한국작물학회지
• /
• 제44권2호
• /
• pp.176-179
• /
• 1999

Trinexapac-ethyl[ 4-(cyclopropyl- $\alpha$ -hydroxy-methylene)-3,5-dioxocyclohexane carboxylic acid ethylester] is a growth-retardant for plants by inhibiting a key step in biosynthesis of GA. A treatment of trinexapacethyl generally induces a reduction in vegetative growth and also inhibits heading. In addition, the trinexapacethyl was known to enhance the freeze-tolerance in annual bluegrass, however, the mechanism is not known yet. One possible reason for the enhanced freeze-tolerance may be the antifreeze protein known to be accumulated in intercellular space of the leaf during cold acclimation. In order to see the possible in-duction of the synthesis of antifreeze proteins by trinexacpacethyl, the apoplastic proteins extracted from Kentucky bluegrass treated with trinexapacethyl were analyzed by SDS-PAGE and the presence of the antifreeze protein was observed. In addition, western analysis showed the identity of the protein induced by both a cold acclimation and a trinexapacethyl treatment. It appears that an enhanced freeze-tolerance of the turf grass by trinexapacethyl is due to the synthesis and/or accumulation of the antifreeze protein similar to the enhanced freeze tolerance induced by cold acclimation.

• 식물조직배양학회지
• /
• 제22권1호
• /
• pp.15-28
• /
• 1995

보리의 내동성기작에 관여할 것으로 예상되는 반결빙단백질을 분리하기 위해서 저온순화기간 중에 세포밖 공간에 축적되는 단백질을 분리 및 비교하였다. 42일간의 저온처리를 통해 70, 21, 16, 14 KDa의 단백질과 10 KDa 이하의 연속된 크기의 단백질들의 축적이 증가됨을 확인하였다. 이들 단백질들은 3일간의 저온처리에서도 어느 정도 축적되었으나, 42일간의 처리시 그 양에 있어서 더욱 증가됨을 볼 수 있었다. 위 방법으로 얻어진 단백질을 전체 잎조직의 단백질과 비교하여 세포밖 공간의 단백질 추출방법의 정확도를 검증하였다. 또한 호밀의 저온처리시 세포밖 공간에 축적하는 단백질과의 비교를 통해 구성 단백질의 크기에 있어서 차이를 확인하였으나, 호밀과 보리에서 공통적으로 10 KDa 이하의 범위에서 연속적인 크기의 단백질이 축적됨을 볼 수 있었다. 알려진 광어의 반결빙단백질은 크기가 3300에서 33,000 dalton에 이르는 점으로 미루어 보리와 호밀에서 공통적으로 발견되는 10 KDa 이하의 단백질이 반결빙단백질로 작용할 가능성은 매우 높다. Griffith등(1992)은 호밀의 세포밖 공간 단백질중 일부에서 반결빙활성도를 확인하였고 보리와 호밀간의 해당단백질의 전체적인 profile에서의 유사성을 미루어 보리로부터 얻어진 세포밖 공간의 단백질에서 반결빙단백질을 발견할 가능성은 매우 높다.

• Clinical and Experimental Reproductive Medicine
• /
• 제44권1호
• /
• pp.8-14
• /
• 2017

Objective: The aim of this study was to analyze the effect of supplementing vitrification and warming solutions with two types of antifreeze proteins (AFPs) and the combination thereof on the follicular integrity of vitrified-warmed mouse ovaries. Methods: Ovaries (n=154) were obtained from 5-week-old BDF1 female mice (n=77) and vitrified using ethylene glycol and dimethyl sulfoxide with the supplementation of 10 mg/mL of Flavobacterium frigoris ice-binding protein (FfIBP), 10 mg/mL of type III AFP, or the combination thereof. Ovarian sections were examined by light microscopy after hematoxylin and eosin staining, and follicular intactness was assessed as a whole and according to the type of follicle. Apoptosis within the follicles as a whole was detected by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. Results: The proportion of overall intact follicles was significantly higher in the type III AFP-supplemented group (60.5%) and the combination group (62.9%) than in the non-supplemented controls (43.8%, p<0.05 for each). The proportion of intact primordial follicles was significantly higher in the FfIBP-supplemented (90.0%), type III AFP-supplemented (92.3%), and combination (89.7%) groups than in the non-supplemented control group (46.2%, p<0.05 for each). The proportions of non-apoptotic follicles were similar across the four groups. Conclusion: Supplementation of the vitrification and warming solutions with FfIBP, type III AFP, or the combination thereof was equally beneficial for the preservation of primordial follicles in vitrified mouse ovaries.