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

• KSBB Journal
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• 제30권6호
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• pp.345-349
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• 2015

Antifreeze proteins (AFP) inhibit growth and recrystallization of ice, and permit organisms to survive in cold environments. The AFP from an Antarctic bacterium, Flavobacterium frigoris PS1, FfIBP (Flavobacterium frigoris icebinding protein), was produced in E. coli using a cold shock induction system. The culture temperature was shifted from $37^{\circ}C$ to $15^{\circ}C$ and a 20 L culture scale was used. The final weights of dried cell and FfIBP were estimated to be 126 g and 8.4 g, respectively. The thermal hysteresis (TH) activity ($1.53^{\circ}C$) of the produced FfIBP was 3.6-fold higher than that of the LeIBP (Leucosporidium ice-binding protein) produced in Picha. The current study demonstrates that large-scale production of FfIBP was successful and the result could be extended to further application studies using recombinant AFPs.

• 한국육종학회지
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• 제40권3호
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• pp.234-242
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• 2008

To identify low temperature-induced genes of wheat chromosome substitution line 5D, suppression subtractive hybridization (SSH) was performed with mRNAs from leaf samples that treated with low temperature ($4^{\circ}C$). A cDNA library was constructed using mRNA isolated from wheat chromosome substitution line 5D leaves treated with low temperature ($4^{\circ}C$). The nucleotide and deduced amino acid sequences of the putative gene products were compared. wfr-9 and wfr-32 showed identity over 90% related to vernalization gene. Other two genes, wfr-77 and wfr-83 which is related to freezing-resistant gene have also identity over 90%. This result suggest that those genes may be transcribed into antifreeze proteins which are accumulated within leaf apoplasts, when wheat chromosome substitution line 5D is acclimated during low temperature treatment.

• Journal of Microbiology and Biotechnology
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• 제30권11호
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• pp.1777-1784
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• 2020

To increase the availability of microalgae as producers of valuable compounds, it is necessary to develop novel systems for gene expression regulation. Among the diverse expression systems available in microalgae, none are designed to induce expression by low temperature. In this study, we explored a cold-inducible system using the antifreeze protein (AFP) promoter from a polar diatom, Chaetoceros neogracile. A vector containing the CnAFP promoter (pCnAFP) was generated to regulate nuclear gene expression, and reporter genes (Gaussia luciferase (GLuc) and mVenus fluorescent protein (mVenus)) were successfully expressed in the model microalga, Chlamydomonas reinhardtii. In particular, under the control of pCnAFP, the expression of these genes was increased at low temperature, unlike pAR1, a promoter that is widely used for gene expression in C. reinhardtii. Promoter truncation assays showed that cold inducibility was still present even when pCnAFP was shortened to 600 bp, indicating the presence of a low-temperature response element between -600 and -477 bp. Our results show the availability of new heterologous gene expression systems with cold-inducible promoters and the possibility to find novel low-temperature response factors in microalgae. Through further improvement, this cold-inducible promoter could be used to develop more efficient expression tools.

• 식물조직배양학회지
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• 제22권1호
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• pp.15-28
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• 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.

• 한국수정란이식학회지
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• 제31권4호
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• pp.355-365
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• 2016

The cryopreservation of sperm has become the subject of research for successful artificial insemination technologies. Antifreeze proteins (AFPs), one of the factors necessary for effective cryopreservation, are derived from certain Antarctic organisms. These proteins decrease the freezing point of water within these organisms to below the temperature of the surrounding seawater to protect the organism from cold shock. Accordingly, a recent study found that AFPs can increase the motility and viability of spermatozoa during cryopreservation. To evaluate this relationship, we performed cryopreservation of boar sperm with AFPs produced in the Arctic yeast Leucosporidium sp. AFP expression system at four concentrations (0, 0.01, 0.1, and $1{\mu}g/ml$) and evaluated motility using computer assisted sperm analysis. DNA damage to boar spermatozoa was measured by the comet assay, and sperm membrane integrity and acrosome integrity were evaluated by flow cytometry. The results showed that motility was positively affected by the addition of AFP at each concentration except $1{\mu}g/ml$ (p<0.001). Although cryopreservation with AFP decreased the viability of the boar sperm using, the tail DNA analyses showed that there was no significant difference between the control and the addition of 0.1 or $0.01{\mu}g/ml$ AFP. In addition, the percentage of live sperm with intact acrosomes showed the least significant difference between the control and $0.1{\mu}g/ml$ AFP (p<0.05), but increased with $1{\mu}g/ml$ AFP (p<0.001). Our results indicate that the addition of AFP during boar sperm cryopreservation can improve viability and acrosome integrity after thawing.

• Journal of Plant Biotechnology
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• 제44권4호
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• pp.388-393
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• 2017

We report an Agrobacterium-mediated transformation procedure optimized for 'Kyoho' that is a major table grapevine cultivar in Korea, and its transgenic plants with antifreeze protein gene of carrot (DcAFP). The full length of DcAFP coding region in accordance with the previous report was isolated from young leaves of carrot and recombined into a plant transformation vector. Ethylene inhibitors such as silver nitrate and aminoethoxyvinylglycin (AVG) supplemented in a co-cultivation medium distinctly increased frequency of shoot regeneration when explants were sub-cultured in a selection medium: particularly ten-fold higher in treatment with 0.1 mg/L AVG than one without ethylene inhibitor. Among various antibiotics and their concentrations, the combination of 150 mg/L cefotaxime plus 150 mg/L $Clavamox^{TM}$ was selected for elimination of Agrobacterium cells in addition to minimization of adverse effect on shoot regeneration, while 50 mg/L kanamycin monosulfate effectively suppressed regeneration of non-transgenic shoots. Applying the elucidated culture condition, we finally obtained a total of 5 transgenic 'Kyoho' plantlets with DcAFP, of which integration with the grapevine genome and transcription was confirmed by nucleic acid analyses.

• Journal of Advanced Marine Engineering and Technology
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• 제26권2호
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• pp.240-247
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• 2002

Recently, a thermal energy storage system has been developed actively fur the purpose of saving energy and reducing the peak electrical demand. Especially, ice slurry is a promising working fluid for low temperature energy storage systems. A flow of ice crystals has a large cooling capacity as a result of the involvement of latent heat. However, there are still problems related to the recrystallization of ice crystals for realizing long term storage and long distance transportation. To find improvements fur this, a method for the creation of ice crystals resistant to recrystallization has been proposed and researched by the use of an antifreeze protein (AFP) solution etc. In the present study, it has been investigated the growth of ice crystal in several kinds of water solution added non-ionic surfactant. The results shows that size of ice crystal was smaller with increasing in added surfactant. And ice crystal was not increased with added surfactant.

• Journal of Microbiology and Biotechnology
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• 제25권12호
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• pp.1989-1996
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• 2015

Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1℃/min in a -80℃ freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

• Journal of Plant Biotechnology
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• 제30권4호
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• pp.315-321
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• 2003

고비사막 접경지역의 식물인 Artermisia adamsii의 내건성 및 내동성 증진을 위해 조직배양과 CLP및 Dhn5유전자의 형질전환을 수행하였다. 다양한 호르몬 농도 조건에서의 성장을 확인한 결과, 0.05mg/L의 NAA와 0.5mg/L의 BAP조건과 0.1mg/L의 NAA와 0.5mg/L의 BAP가 포함된 배지의 암조건 하에서 최적의 캘러스 생장을 확인하였으며 유전자 도입 및 유전자의 발현이 확인된 캘러스가 세포 수준에서도 내건성 및 내동성이 증진되었음을 확인하였다. 그러나 Atremisia속 다른 식물과 다르게 조직절편에서 직접 기관분화를 유도하거나 캘러스를 통한 식물체 재생에 어려움이 있어 식물체 수준에서 형질전환에 따른 환경스트레스 내성의 증진을 확인하지 못하고 있다. 앞으로 진행될 A. adamsii의 식물체 재생에 대한 연구를 통해 내동성 및 내건성이 증진된 식물체를 육성하여 고비사막 지역적응력을 조사할 예정이다.