• Journal of Plant Biotechnology
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• 제37권2호
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• pp.174-185
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• 2010

Full-length cDNAs are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. To elucidate the functions of a large population of Chinese cabbage (Brassica rapa) genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the full-length cDNA Over-eXpresser (FOX) gene hunting system. With oligo dT column it purify the each mRNA from the flower organs, leaf and stem tissue. And about 120,000 cDNAs from the library were transformed into $\lambda$-pFLCIII-F vector. Of which 115,000 cDNAs from the library were transformed into T-DNA binary vector, pBigs for transformation study. We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. Full-length Chinese cabbage cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Selfed seeds were harvested from transgenic Arabidopsis. We had selected 2,500 transgenic plants by hygromycin antibiotic tolerant test, and obtained a number of transgenic mutants. Each transgenic Arabidopsis was investigated in morphological changes, fertility and leaf colour. As a result, 285 possible morphological mutants were identified. Introduced cDNA was isolated by PCR amplification of the genomic DNA from the transgenic mutants. Sequencing result and BLAST analysis showed that most of the introduced cDNA were complete cDNAs and functional genes. Also, we examined the effect of Bromelain on enhancing resistance to soft rot in transgenic Chinese cabbage 'Osome'. The bromelain gene identified from FOX hunting system was transformed into Chinese cabbage using Agrobacterium methods. Transformants were screened by PCR, then RT-PCR and real time PCR were performed to analyze gene expression of cysteine protease in the T1 and T2 generations. The anti-bacterial activity of bromelain was tested in Chinese cabbages infected with soft rot bacteria. The results showed that the over-expressed bromelain gene from pineapple conferred enhanced resistance to soft rot in Chinese cabbage.

• 대한화장품학회지
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• 제39권3호
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• pp.177-186
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• 2013

피부 섬유아세포는 인간 피부의 주요 콜라겐 생산 세포이다. 노화가 진행되면, 섬유아세포에서의 콜라겐 생산이 감소되고, matrix metalloproteinase-1 (MMP-1)에 의해 시작되는 콜라겐 조각화가 증가된다. 즉 섬유아세포의 콜라겐 항상성의 불균형으로 인해 피부 collagenous, 세포외기질(ECM)의 구조와 기능이 변형되어, 피부노화가 촉진되는 것이다. Cysteine rich protein 61 (CCN1)는 CCN family의 일부이며, 인간피부의 섬유아세포에서 콜라겐 항상성을 조절하는 단백질이다. 노화된 인간 피부 섬유아세포에서의 CCN1 과 발현은 실질적으로 유형 I procollagen 생성을 감소시킴과 동시에 MMP-1의 발현을 증가시켜 섬유의 콜라겐 저하를 일으킨다. 그리고 노화된 섬유아세포는 노화 전 섬유아세포에 비해 증식률이 감소한다. 본 연구에서 만들어 사용한 복제 노화 피부 섬유아세포는 유형 I procollagen의 생성량이 감소하였고, MMP-1의 발현 수준이 증가하는 특징을 나타냈다. 또한 CCN1 단백질의 발현이 증가되고, 증식률이 감소하는 특징을 나타냈다. 가수분해 구멍갈파래 추출물은 노화 전 섬유아세포에서 새로운 콜라겐의 합성을 촉진하고 자외선에 의해 증가된 MP-1의 발현을 감소시켜 광노화를 개선하는 물질로 알려져 있다. 본 연구에서는 이러한 활성을 나타내는 가수분해 구멍갈파래 추출물을 사용하여, 복제 노화 피부 섬유아세포에서 가수분해 구멍갈파래 추출물에 의한 CN1 단백질의 발현 억제 여부를 조사하였으며, 이들 추출물은 배양된 복제 노화 피부 섬유아세포에서 유형 I procollagen의 생성을 증가시켰으며, MMP-1 발현을 억제시키는 것을 확인하였다. 또한, 콜라겐 항상성을 조절하는 단백질인 CN1 발현을 크게 감소시켰으며, 노화세포의 증식률을 증가시켰다. 이 결과는 복제 노화 섬유아세포가 in vitro 자연 노화모델로 화장품 원료 활성 연구에 사용될 수 있음을 말한다. 그리고 가수분해 구멍갈파래 추출물은 광노화 뿐 아니라 자연노화를 개선하는 피부미용제로 주름개선 기능성 화장품에 사용가능 하다는 것을 의미한다.

• Clinical and Experimental Pediatrics
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• 제45권7호
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• pp.902-905
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• 2002

선천성 신성 요붕증은 일반적으로 성염색체 열성 유전 양식을 취하여 보인자인 어머니로부터 아들에게 유전되고, 발생 빈도는 드문 것으로 알려져 있으며, 항이뇨호르몬의 혈장 농도가 정상이거나 상승되어 있으면서도 신세뇨관이 항이뇨호르몬에 대한 저항성 때문에 요농축능 장애가 발생하여 다음과 다뇨를 특징으로 하는 질환으로 이뇨제와 프로스타글란딘 합성억제제의 사용이 치료의 근간을 이루어 왔다. 본 증례에서 발견된 AVPR2 수용체 유전자의 돌연변이 부위는 선천성 신성 요붕증의 표현형을 보이는 것으로 외국에서는 보고된 바 있으나 국내에서는 최초로 확인된 것이며, 고용량의 항이뇨 호르몬과 이뇨제 치료에 반응을 보였다.

• 대한바이러스학회지
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• 제28권4호
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• pp.303-316
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• 1998

An attenuated classical swine fever virus (CSFV), Suri strain, is a variant derived from a vaccine virus, LOM strain. This study was performed to elucidate the molecular biologcal properties of CSFV Suri strain, and to obtain the basic data for molecular epidemiological approaches for the disease. The truncated form of gp55 gene without the C-terminal transmembrane domain, in size of 1,023bp, was amplified by RT-PCR and sequenced by dye terminator cyclic sequencing method, and inserted into BamHI site of pAcGP67B baculovirus vector, establishing a cloned pAcHEG plasmid. By the nucleotide sequences determined, 341 amino acid sequences were predicted. As compared the nucleotide and amino acid sequences of gp55 of Suri with the various CSFV, Suri strain showed the high homology over 99.1% with ALD and LOM strains, but comparably the lower homology with Alfort and Brescia. In comparison of amino acid sequence in variable domain of gp55 protein, the similar tendency of homology was observed. In hydrophobicity analysis, all of four CSFV strains revealed the analogous patterns of hydrophobicity. The numbers and locations of N-glycosylation site and cysteine residues in gp55 were analyzed, those of Suri strain being coincident with ALD and LOM strains. The results suggest that gp55 in Suri strain has the high similarity to those in ALD and LOM strains in terms of the nucleotide and amino acid sequences and the functional properties of gp55 protein.

• Journal of Dairy Science and Biotechnology
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• 제24권1호
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• pp.19-28
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• 2006

유아 분변으로부터 분리한 52 균주는 그람 양성균인 Listeria monocytogenes KCCM 40307$^T$에 대하여 항균활성이 있었고 그 중에서도 선별 균주 A24의 항균활성이 45% 이상으로 가장 높았다. Bifidobacterium longum A24의 생육 및 항균 물질 생산을 검토하였을 때 균체의 생육은 28시간 배양 시 최고에 도달하였고 항균 활성은 36시 간 배양 시 최고를 나타내었다. 선별균주 A24는 16S rRNA-based molecular typing 결과Bifidobacterium 속 균주임을 확인할 수 있었고 형태학적 ${\cdot}$ 생화학적인 방법으로 검토하여 보았을 때 Bifidobacterium longum으로 판단되었으며, 16s rDNA sequencing 결과 최종적으로 Bifidobacterium longum로 동정 되었으며 이것을 Bifidobacterium longum A24로 명명하였다. Bifidobacterium longum A24의 항균 활성 물질은 균체의 생육이 가장 좋은 28시간 배양에서가 아니라 그보다 늦은 36시간 배양에서 최고를 나타내었고 그 이후로는 활성이 감소하는 경향을 보였다. 이것은 Bifidobacterium longum A24이 생성하는 항균 활성 물질이 bacteriocin과 같은 2차 대사 산물임을 암시하는 결과로 해석된다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 Proceedings of 2000 KSAM International Symposium and Spring Meeting
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• pp.157-167
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• 2000

A thermostable chitosanase gene from the isolated strain, Bacillus sp. CK4, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30 kDa enzyme in size. The deduced amino acid sequence of the chitosanase from Bacillus sp. CK4 exhibits 76.6%, 15.3%, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. CK4 belongs to the Cluster III group with Bacillus subtilis. The size of the gene was similar to that of a mesophile, Bacillus subtilis showing a higher preference for codons ending in G or C. The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues were changed to E50D/Q, E62D/Q, and D66N/E by site-directed mutagenesis. The D66N/E mutants enzymes had remarkably decreased kinetic parameters such as $V_{max}$ and k$\sub$cat/, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three cysteine residues at position 49, 72, and 211. Titration of the Cys residues with DTNB showed that none of them were involved in disulfide bond. The C49S and C72S mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However the half-life of the C211S mutant enzyme was less than 60 min at 80$^{\circ}C$, while that of the wild type enzyme was about 90 min. Moreover, the residual activity of C211S was substantially decreased by 8 M urea, and fully lost catalytic activity by 40% ethanol. These results show that the substitution of Cys with Ser at position 211 seems to affect the conformational stability of the chitosanase.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.131-131
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• 2017

Though the Platycodon grandiflorum, has a broad range of pharmacologic properties, but the mechanisms underlying these effects remain unclear. In order to profile proteins from the nodal segment, callus, root and shoot, high throughput proteome approach was executed in the present study. Two-dimensional gels stained with CBB, a total of 84 differential expressed proteins were confirmed out of 839 protein spots using image analysis by Progenesis SameSpot software. Out of total differential expressed spots, 58 differential expressed protein spots (${\geq}2-fold$) were analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. Out of 58 differential expressed protein, 32 protein spots were up-regulated such as ribulose-1,5-bisphosphate carboxylase, endoplasmic oxidoreductin-1, heat stress transcription factor A3, RNA pseudourine synthase 4, cysteine proteinase, GntR family transcriptional regulator, E3 xyloglucan 6-xylosyltransferase, while 26 differential protein spots were down-regulated such as L-ascorbate oxidase precursor, late embryogenesis abundant protein D-34, putative SCO1 protein, oxygen-evolving enhancer protein 3. However, the frequency distribution of identified proteins using iProClass databases, and assignment by function based on gene ontology revealed that the identified proteins from the explants were mainly associated with the nucleic acid binding (17%), transferase activity (14%) and ion binding (12%). Taken together, the protein profile may provide insight clues for better understanding the characteristics of proteins and its metabolic activities in various explants of this essential medicinal plant P. grandiflorum.

• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2019-2029
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• 2016

Zinc finger proteins are among the most extensively applied metalloproteins in the field of biotechnology owing to their unique structural and functional aspects as transcriptional and translational regulators. The classical zinc fingers are the largest family of zinc proteins and they provide critical roles in physiological systems from prokaryotes to eukaryotes. Two cysteine and two histidine residues ($Cys_2His_2$) coordinate to the zinc ion for the structural functions to generate a ${\beta}{\beta}{\alpha}$ fold, and this secondary structure supports specific interactions with their binding partners, including DNA, RNA, lipids, proteins, and small molecules. In this account, the structural similarity and differences of well-known $Cys_2His_2$-type zinc fingers such as zinc interaction factor 268 (ZIF268), transcription factor IIIA (TFIIIA), GAGA, and Ros will be explained. These proteins perform their specific roles in species from archaea to eukaryotes and they show significant structural similarity; however, their aligned amino acids present low sequence homology. These zinc finger proteins have different numbers of domains for their structural roles to maintain biological progress through transcriptional regulations from exogenous stresses. The superimposed structures of these finger domains provide interesting details when these fingers are applied to specific gene binding and editing. The structural information in this study will aid in the selection of unique types of zinc finger applications in vivo and in vitro approaches, because biophysical backgrounds including complex structures and binding affinities aid in the protein design area.

• 한국작물학회지
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• 제60권1호
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• pp.97-106
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• 2015

Platycodon grandiflorum, commonly known as Doraji in Korea, has a wide range of pharmacologic properties, such as reducing adiposity and hyperlipidemia, and antiatherosclerotic effects. However, the mechanisms underlying these effects remain unclear. In order to profile proteins from the nodal segment, callus, root and shoot, high throughput proteome approach was executed in the present study. Two dimensional gels stained with CBB, a total of 84 differential expressed proteins were confirmed out of 839 protein spots using image analysis by Progenesis SameSpot software. Out of total differential expressed spots, 58 differential expressed protein spots (${\geq}$ 2-fold) were analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. Out of 58 differential expressed protein, 32 protein spots were up-regulated such as ribulose-1,5-bisphosphate carboxylase, endoplasmic oxidoreductin-1, heat stress transcription factor A3, RNA pseudourine synthase 4, cysteine proteinase, GntR family transcriptional regulator, E3 xyloglucan 6-xylosyltransferase, while 26 differential protein spots were down-regulated such as L-ascorbate oxidase precursor, late embryogenesis abundant protein D-34, putative SCO1 protein, oxygen-evolving enhancer protein 3. However, frequency distribution of identified proteins using iProClass databases, and assignment by function based on gene ontology revealed that the identified proteins from the explants were mainly associated with the nucleic acid binding (17%), transferase activity (14%) and ion binding (12%). In that way, the exclusive protein profile may provide insight clues for better understanding the characteristics of proteins and metabolic activity in various explants of the economically important medicinal plant Platycodon grandiflorum.

• Molecules and Cells
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• 제42권12호
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• pp.884-892
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• 2019

Piperlongumine (PL), a natural alkaloid compound isolated from long pepper (Piper longum), can selectively kill cancer cells, but not normal cells, by accumulation of reactive oxygen species (ROS). The objective of this study was to investigate functional roles of expression of SETDB1 and FosB during PL treatment in MCF7 breast cancer cells. PL downregulates SETDB1 expression, and decreased SETDB1 expression enhanced caspase 9 dependent-PARP cleavage during PL-induced cell death. PL treatment generated ROS. ROS inhibitor NAC (N-acetyl cysteine) recovered SETDB1 expression decreased by PL. Decreased SETDB1 expression induced transcriptional activity of FosB during PL treatment. PARP cleavage and positive annexin V level were increased during PL treatment with FosB overexpression whereas PARP cleavage and positive annexin V level were decreased during PL treatment with siFosB transfection, implying that FosB might be a pro-apoptotic protein for induction of cell death in PL-treated MCF7 breast cancer cells. PL induced cell death in A549 lung cancer cells, but molecular changes involved in the induction of these cell deaths might be different. These results suggest that SETDB1 mediated FosB expression may induce cell death in PL-treated MCF7 breast cancer cells.