• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2005년도 국제학술심포지움 The 44th Annual Meeting of Korean Society for Life Science
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• pp.87-87
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• 2005

• 한국양식학회지
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• 제12권3호
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• pp.175-183
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• 1999

We have shown previously that the sequence of olive flounder (Paralichthys olivaceus) hsp70-related cDAN has a high similarity with those of cognate hsc70 of other species (Kim et al., 1999; J. Aquaculture, 12:91-100). In order to investigate whether this gene encodes the congate hsc70, we examined the expression of this gene in normal and heat-shocked conditions. By in vitro translation, this gene encoded a 70 kD protein which was constitutively experessed and was not induced by heat shock. This translated protein was recognized by anti-hsp/hsc70 antibody. Tests of heat-inducibility showed that this gene was constitutively expressed in normal conditions and its expression was not increased after heat shock. The expression levels of this gene were high in stomach, gill, intestine, kidney and brain, moderate in liver, and comparatively low in overy and heart. Furthermore, Northern blot analysis of transcript expression showed that the corresponding mRNA were detected throughout embryonic development in the absence of any heat shock. These results provided evidence that olive flounder hsp70-related cDNA encoded to cognate member of hsp70 family, hsc70.

• 한국동물학회지
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• 제33권1호
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• pp.35-44
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• 1990

생쥐 악하선으로부터 분리한 전 RNA를 poly(U)-sepharose chromatography와 sucrose linear density gradient centrifugation 방법으로 레닌 mRNA를 분리하여 in vitro translation과 immunoprecipitation에 의하여 레닌 mRNA를 확인하였다. 레닌 mRNA로부터 레닌 cDNA를 합성하여 EcoRI inker를 이용하여 pUC19에 삽입시켰고, Taq, polymerase를 이용한 PCR방법으로 합성한 레닌 cDNA는 pUC19의 Hinalll 부의에 삽입하여 재조합 plamid를 각각 작성하였다. 이것을 JM103에 형질전환시켜 레닌 유전자 발현을 유도하여 45,000의 분자량을 갖는 레닌을 얻었으며 이 레닌 단백은 토끼으 혈압을 850115mmHg에서 115-140mmHg로 증가시키는 생리 활성을 나타냈다. 토끼의 신장 DNA를 EMBL3 phage에 삽입시켜 genomic library를 작성한 후, 레닌 cDNA로부터 합성한 probe로 plaque hybridization시켜 genomic 유전자를 갖는 재조합 phage를 분리하였다.

• Journal of Plant Biotechnology
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• 제7권1호
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• pp.51-55
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• 2005

Many cloning vectors in which cDNAs can be inserted to the sense orientation have been developed. Uni-ZAP XR vector (Stratagene) should contain clones that are oriented to sense direction with respect to T3 RNA polymerase primer. Unexpectedly, large portions of cDNAs in Chinese cabbage cDNA library showed unusual insertions, antisense orientation and a hybrid of two different clones. Using two clones, 4H03 and 53-B10, derived from different cDNA libraries, we proposed and demonstrated the possibility of unusual-construct formation by in vitro translation and northern blot analysis. The 4H03 clone was inserted with inverse direction, and its transcript and translation product could be produced by T7 RNA polymerase, indicating that this clone is definitely inserted into inverse orientation. The 53-B10 that contains two independent genes was turned out to be a hybrid in which two genes are inserted to opposite direction each other. All unusual constructs might be due to the presence of small fragments of DNA, like adapter. However, the mechanism underlined the formation of unusual constructs is still remain to be solved.

• 미생물학회지
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• 제30권6호
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• pp.466-471
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• 1992

HTLV-I 의 gag-pro 유전자 중첩영역내에서 -1 ribosomal frameshifting 이 일어나는 자리를 결정하기 위하여 gag-pro 중첩영역의 일부를 SP6 promoter 를 가진 백터내에 클로닝하였다. 그 결과 닭의 prelysozyme 에서 유래한 5개의 아미노산을 코드하는 합성유전자와 141 bp 로된 gag-pro 중첩영역의 뒤에 Straphylococcus aureus 의 protein A 유전자단편이 연결된 hybrid 유전자를 보유한 플라스미드를 제작하였다. 이 DNA 클론을 주형으로 SP6 RNA polymerase 의 작용에 의해 한종류의 mRNA 를 다량으로 합성하였다. Invitro 에서 합성된 mRNA 로 무세포계에서 단백질을 합성한 결과 21 kDal 의 단백질이 생성되었고 IgG-Sepharose 를 사용한 affinity chromatography 로 합성된 단백질을 순수하게 정제할 수 있었다. 본연구에서 설명한 in vitro 실험계는 Gag-Pro transframe 단백질의 신속한 정제 및 일차구조의 결정에 유익하게 사용될 것으로 보이며 이와 같은 실험의 결과 mRNA 에서 ribosomal frameshifting 이 일어나는 정확한 site 를 결정할 수 있을 뿐 같은 실험의 결과 mRNA 에서 ribosomal frameshifting 이 일어나는 정확한 site 를 결정할 수 있을 뿐 아니가 pro 유전자의 발현에 필요한 frameshift 를 유도하는 tRNA 의 동정도 가능하게 될 것이다.

• Applied Biological Chemistry
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• 제40권5호
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• pp.375-379
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• 1997

리보즘불활성화 단백질(Ribosome-inactivating protein, RIP)을 생성하는 식물을 탐색하여 그중 호박$(Cucurbita\;moschata\;D_{UCHESNE})$ 잎에서 ammonium sulfate 침전, DE 52-Cellulose, S-Sepharose, FPLC Superose 12 HR, FPLC Mono-S column chromatography에 의하여 ribosome-Inactivating 활성이 있는 단백질(PR 1, PRIP 2)을 분리하였다. 정제된 단백질의 분자량은 SDS-PAGE에서 약 31,000과 30,500인 염기성 단백질로서, 특히 PRIP 1은 열에도 안정하여 $50^{\circ}C$에서 30분간 처리한 경우에도 활성이 유지되었다. 이 단백질들의 ribosome-inactivating 활성을 in vitro translation system에서 측정한 결과 50% 활성저해농도 $(IC_{50})$는 PRIP 1은 0.82nM, PRIP 2은 0.79 nM이었다. PRIP 1과 PRIP 2의 N-말단부분의 아미노산 서열을 분석하여, 이미 밝혀진 리보즘불활성화 단백질들과 아미노산서열의 유사성을 분석해 본 결과, PRIP 1은 Luffa cylindrica에서 분리된 Luffin B 및 Trichosanthes kirilowii Maximowicz에서 분리된 Trichokirin과, PRE 2은 Momordia charantia에서 분리된 Momordin II 및 MAP 30과 유사성이 매우 높았다.

• Asian-Australasian Journal of Animal Sciences
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• 제29권8호
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• pp.1102-1110
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• 2016

The mammalian target of rapamycin (mTOR) regulates cellular processes such as cell growth, metabolism, transcription, translation, and autophagy. Rapamycin is a selective inhibitor of mTOR, and induces autophagy in various systems. Autophagy contributes to clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified-warmed mouse oocytes show acute increases in autophagy during warming, and suggested that it is a natural response to cold stress. In this follow-up study, we examined whether the modulation of autophagy influences survival, fertilization, and developmental rates of vitrified-warmed mouse oocytes. We used rapamycin to enhance autophagy in metaphase II (MII) oocytes before and after vitrification. The oocytes were then subjected to in vitro fertilization (IVF). The fertilization and developmental rates of vitrified-warmed oocytes after rapamycin treatment were significantly lower than those for control groups. Modulation of autophagy with rapamycin treatment shows that rapamycin-induced autophagy exerts a negative influence on fertilization and development of vitrified-warmed oocytes.

• Journal of Plant Biotechnology
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• 제40권3호
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• pp.169-177
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• 2013

최근 급속도로 당뇨병 환자가 증가하면서 인슐린 시장이 크게 성장하고 있다. 또한 최근 식물체를 이용하여 의약용 단백질 생산이 경제적인 측면과 안정성 측면에서 매우 효과적임이 보고되고 있어 이를 이용한 분자농업이 주목을 받고 있다. 본 연구에서는 인슐린 단백질을 식물체에서 생산하기 위한 유전자 발현 construct를 설계하기 위한 실험으로서 식물발현용 preprominiinsulin construct를 제조하기 위한 단계적 실험을 수행하였다. 우선 proinsulin이 무세포 식물 전사/번역시스템에서 성공적으로 발현됨을 확인하였다. Prominiinsulin construct를 제조하여 대장균에서 발현시키는데 성공하였으며, 이를 트립신으로 절단한 후 인간 항인슐린 항체를 이용한 western 분석을 통하여 효과적으로 A-펩타이드와 B-펩타이드가 형성되며 이후 적절하게 접힘이 일어나고 hexamer로 조립됨을 확인하였다. 이후 식물체에서 재조합 인슐린 유전자가 발현되는지를 확인하기 위하여 RFP 결합 construct를 제조하여 담배의 현탁배양세포인 BY-2 세포에 형질전환시켜 RFP 결합 preprominiinsulin이 성공적으로 발현됨을 확인 하였다. 이러한 성공적인 연구 결과를 토대로 향후 이 construct는 RFP 단백질을 제거하여 35S 프로모터에 직접 유도되는 [N 말단 ${\rightarrow}$ tobacco E2 시그널 펩타이드 ${\rightarrow}$ B-펩타이드(1-29 AA) ${\rightarrow}$ AAK ${\rightarrow}$ A-펩타이드(1-21 AA) ${\rightarrow}$ RR ${\rightarrow}$ His6 ${\rightarrow}$ KDEL ${\rightarrow}$ C 말단] construct를 제조하여 담배 식물체에 형질전환시켜 분자농업을 통해 인간 인슐린 단백질을 생산하는데 활용하고자 한다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권5호
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• pp.2287-2290
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• 2014

Background: In the past few years, a considerable number of preclinical studies have been proposed metformin as a potential anticancer agent, but some of these studies suffer from a number of methodological limitations such as assessment of cytotoxicity in the presence of supraphysiological glucose concentrations or applying suprapharmacological levels of the drug. These objections have limited the translation of published preclinical data to the clinical setting. The present study aimed to investigate direct anticancer effects of metformin on different molecular subtypes of breast cancer with pharmacological concentrations and under normoglycemic conditions in vitro. Materials and Methods: Breast cancer cell lines from luminal A, luminal B, ErbB2 and triple-negative molecular subtypes were treated with a pharmacological concentration of metformin (2mM) at a glucose concentration of 5.5mM. Time-dependant cell viability was assessed by dye exclusion assay. MTTbased cytotoxicity assays were also performed with metformin alone or in combination with paclitaxel. Results: Metformin did not show any growth inhibitory effects or time-dependant cytotoxicity on breast cancer cell lines in the presence of normal glucose concentrations at the therapeutic plasma level. No augmentation of the antineoplastic properties of paclitaxel was apparent under the tested conditions. Conclusions: Metformin is probably unable to exert cytotoxic or cytostatic effects on breast cancer subtypes at pharmacological concentrations and normal plasma glucose levels. These results highlight the importance of establishing a higher steady-state plasma concentration of metformin in the clinical setting for assessment of anticancer effects in normoglycemic patients.

• The Journal of Advanced Prosthodontics
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• 제9권4호
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• pp.244-251
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• 2017

PURPOSE. The purpose of this study was to determine fracture resistance and failure modes of three-unit fixed dental prostheses (FDPs) made of lithium disilicate pressed on zirconia (LZ), monolithic lithium disilicate (ML), and monolithic zirconia (MZ). MATERIALS AND METHODS. Co-Cr alloy three-unit metal FDPs model with maxillary first premolar and first molar abutments was fabricated. Three different FDPs groups, LZ, ML, and MZ, were prepared (n = 5 per group). The three-unit FDPs designs were identical for all specimens and cemented with resin cement on the prepared metal model. The region of pontic in FDPs was given 50,000 times of cyclic preloading at 2 Hz via dental chewing simulator and received a static load until fracture with universal testing machine fixed at $10^{\circ}$. The fracture resistance and mode of failure were recorded. Statistical analyses were performed using the Kruskal-Wallis test and Mann-Whitney U test with Bonferroni's correction (${\alpha}=0.05/3=0.017$). RESULTS. A significant difference in fracture resistance was found between LZ ($4943.87{\pm}1243.70N$) and ML ($2872.61{\pm}658.78N$) groups, as well as between ML and MZ ($4948.02{\pm}974.51N$) groups (P<.05), but no significant difference was found between LZ and MZ groups (P>.05). With regard to fracture pattern, there were three cases of veneer chipping and two interfacial fractures in LZ group, and complete fracture was observed in all the specimens of ML and MZ groups. CONCLUSION. Compared to monolithic lithium disilicate FDPs, monolithic zirconia FDPs and lithium disilicate glass ceramics pressed on zirconia-based FDPs showed superior fracture resistance while they manifested comparable fracture resistances.