• BMB Reports
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• 제30권2호
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• pp.95-100
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• 1997

Two kinds of cDNA encoding mouse $Gal{\beta}$1,3(4)GlcNAc ${\alpha}$2,3-sialyltransferase (mST3Gal III) and $Gal{\beta}$1,4(3)GlcNAc ${\alpha}$2,3-sialyltransferase (mST3Gal IV) were isolated from mouse brain cDNA library by means of a PCR-based approach. The cDNA sequences included an open reading frame coding for proteins of 374 and 333 amino acids, respectively, and the primary structure of these enzymes suggested a putative domain structure consisting of four regions, like that in other glycosyltransferases. The deduced amino acid sequences of mST3GaI III and IV showed a 98% and 89% identity with rat ST3GaI III and human ST3Gal IV, respectively. Northern analysis indicated that the expression of mST3Gal III mRNA was abundant in heart, liver and adult brain, while that of mST3GaI IV mRNA was detected in all tissues tested except for testis, but the level was the highest in liver. Soluble forms of mST3GaI III and IV transiently expressed in COS cells exhibited enzyme activity toward acceptor substrates containing the terminal either $Gal{\beta}$1,3GlcNAc or $Gal{\beta}$1,4GlcNAc sequences. The substrate preferences of both enzymes were stronger for tetrasaccharides than for disaccharides.

• Molecules and Cells
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• 제27권2호
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• pp.183-190
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• 2009

The plasma membrane-localized BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1) are a well-known receptor pair involved in brassinosteroids (BR) signaling in Arabidposis. The formation of a receptor complex in response to BRs and the subsequent activation of cytoplasmic domain kinase activity share mechanistic characteristics with animal receptor kinases. Here, we demonstrate that BRI1 and BAK1 are BR-dependently phosphorylated, and that phosphorylated forms of the two proteins persist for different lengths of time. Mutations of either protein abolished phosphorylation of the counterpart protein, implying transphosphorylation of the receptor kinases. To investigate the specific amino acids critical for formation of the receptor complex and activation of BAK1 kinase activity, we expressed several versions of BAK1 in yeast and plants. L32E and L46E substitutions resulted in a loss of binding of BAK1 to BRI1, and threonine T455 was essential for the kinase activity of BAK1 in yeast. Transgenic bri1 mutant plants overexpressing BAK1(L46E) displayed reduced apical dominance and seed development. In addition, transgenic wild type plants overexpressing BAK1(T455A) lost the phosphorylation activity normally exhibited in response to BL, leading to semi-dwarfism. These results suggest that BAK1 is a critical component regulating the duration of BR efficacy, even though it cannot directly bind BRs in plants.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1570-1576
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• 2006

An encephalomyocarditis virus (EMCV-CBNU) was isolated from an aborted swine fetus in October 2005. To investigate the genetic origin and virulence of the EMCV-CBNU strain, we determined the complete sequence of the virus and tested its virulence in mice. Genetic characterization revealed that the RNA genome was composed of 7,713 nucleotides with a single open reading frame (2,292 amino acids), coding 12 proteins. The EMCV-CBNU had the shortest poly(C) tract, consisting of 10 C's ($C_{10}$), compared with all the other EMCV strains reported in GenBank. Amino acid and phylogenetic analyses showed that EMCV-CBNU had the highest genetic identity with strain 2887A (99.7%), which was originally isolated from a fetus in a pig breeding farm that had a history of reproductive failure. Because rodents are the natural host of EMCV, we investigated the virulence of EMCV-CBNU in mice. Surprisingly, all mice inoculated with more than $1{\times}10^2\;TCID_{50}/0.1ml$ of EMCV-CBNU showed symptoms of hind limb paralysis and eventually died during 3 and 8 days postinoculation (DPI). Furthermore, when we inoculated the virus into pregnant mice, all dams and their fetuses died in 6 DPI. This is the first report on a full genomic analysis of swine EMCV in Korea, which exhibits high virulence in mice.

• International Journal of Industrial Entomology and Biomaterials
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• 제23권2호
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• pp.223-229
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• 2011

Cyclophilins are originally identified as cytosolic binding protein of the immunosuppressive drug cyclosporine A. They have an activity of peptidyl prolyl cis/trans-isomerases (PPIase), which may play important roles in protein folding, trafficking, assembly and cell signaling. In this study, we report the cloning and characterization of a Bombyx mori cyclophilin A (bCypA) cDNA. The full-length cDNA of bCypA consist of 947 nucleotides with a polyadenylation signal sequence AATAAA and contain an open reading frame of 498 nucleotides encoding a polypeptide of 166 amino acids. The deduced amino acid sequence of bCypA shares a central peptidyl prolyl cis/trans-isomerase and a cyclosporin-A-binding domain with other cyclophilin sequences. Relative quantification real-time (RT) PCR analysis shows that mRNA transcripts of bCypA are detected in all the investigated tissues and highest expression level in the skin of 3-day-old 5 instar larva. Also, bCypA had PPIase activity on the proline-containing peptides. Accordingly, we suggest that bCypA is a new member of the cyclophilin A (CyPA) family and will be useful for quality control of bioactivity recombinant proteins with proline-containing peptides.

• Journal of Microbiology
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• 제39권1호
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• pp.31-36
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• 2001

A cDNA clone encoding the ribosomal protein S20 has been isolated from the Schizosaccharomyces pombe cDNA library by colony hybridization. The insert contained in the original plasmid pYJ10 was transferred intro shuttle vector pRS316 generate plasmid pYJll. The dDNA insert of plasmid pYJll, contains 484 nucleotides and encodes a protein of 118 amino acids with a calculated mass of 13,544 daltons. The deduced amino acid sequence of S. pombe ribosomal protein S20 is very homologous with fruit fly, rat, and budding yeast counterparts. It is also homologous with Xenopus S22 ribosomal protein. S. pombe ribosomal protein S20 appears to be relatively hydruphobic except the C-terminal region. The 728 bp upstream region of the S20 gene was amplified from chromosomal DNA and transferred into the BamHI/EcoRI site of the promoterles $\beta$-galactosidase gene of the vector YEp357R, which resulted in fusion plasmid pYS20. The synthesis of $\beta$-galactosidase from the fusion plasmid appeared to be the highest in the mid-exponential phase. The S. pombe cells with the fusion plasmid grown at 35$\^{C}$ gave lower $\beta$-galactosidase activity than the cells grown at 30$\^{C}$. Computer analysis showed the consensus sequence CAGTCACA in the upstream regions of various ribosomal protein genes in S. pombe, which would be involved in the coordinated expression of small ribosomal proteins.

• Journal of Ginseng Research
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• 제47권5호
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• pp.662-671
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• 2023

Background: 20(S)-protopanaxadiol (PPD), a ginsenoside metabolite, has prominent benefits for the central nervous system, especially in improving learning and memory. However, its transcriptional targets in brain tissue remain unknown. Methods: In this study, we first used mass spectrometry-based drug affinity responsive target stability (DARTS) to identify the potential proteins of ginsenosides and intersected them with the transcription factor library. Second, the transcription factor PURA was confirmed as a target of PPD by biolayer interferometry (BLI) and molecular docking. Next, the effect of PPD on the transcriptional levels of target genes of PURA in brain tissues was determined by qRT-PCR. Finally, bioinformatics analysis was used to analyze the potential biological features of these target proteins. Results: The results showed three overlapping transcription factors between the proteomics of DARTS and transcription factor library. BLI analysis further showed that PPD had a higher direct interaction with PURA than parent ginsenosides. Subsequently, BLI kinetic analysis, molecular docking, and mutations in key amino acids of PURA indicated that PPD specifically bound to PURA. The results of qRT-PCR showed that PPD could increase the transcription levels of PURA target genes in brain. Finally, bioinformatics analysis showed that these target proteins were involved in learning and memory function. Conclusion: The above-mentioned findings indicate that PURA is a transcription target of PPD in brain, and PPD upregulate the transcription levels of target genes related to cognitive dysfunction by binding PURA, which could provide a chemical and biological basis for the study of treating cognitive impairment by targeting PURA.

• Applied Biological Chemistry
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• 제11권
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• pp.1-27
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• 1969

우리나라에서 알려진 젓갈 30여종(餘種)에서 그중(中) 대표적(代表的)인 것으로 알려진 조기젓. 조개젓(고식(高食) 염성(鹽性)인 20%내외(內外))과 굴젓, 오징어젓(저식염성(低食鹽性)인 10%내외(內外))의 4종(種)을 시료(試料)로하여 일반성분(一般成分) 분석(分析), microflora, 주요발효미생물(主要醱酵微生物)의 동정(同定) 및 효소(酵素)의 특성(特性)을 조사(調査)하는 동시(同時)에 숙성(熟成)에 관여(關與)하는 미생물(微生物)의 효소작용(酵素作用)이 정미성(呈味性) 5’-mononucleotide 및 amino 산(酸)의 생성(生成)에 미치는 영향(影響)을 조사(調査)하여 다음과 같은 결과(結果)를 얻었다. (1) 4종(種) 젓갈에 대(對)한 microflara를 조사(調査)한 결과(結果)는 다음과 같다. a) 젓갈담금 $1{\sim}2$개월(個月)의것은 생균수(生菌數) total counts가 $10^7$이었으며 담금후(後) 6개월(個月)의것은 $10^4$이였다. b) 젓 갈 담금 $1{\sim}2$개월(個月)의것은 Micrococcus 속(屬)이 $10{\sim}20%$, Brevibacterium 속(屬)이 $10{\sim}20%$, Sarcina 속(屬)이 $0{\sim}30%$, Leuconostoc 속(屬) $20{\sim}30%$, Bacillus 속(屬)이 30%내외(內外) Pseudomonas 속(屬)이 $0{\sim}10%$ Flavobacterium 속(屬)이 $0{\sim}10%$ Yeast가 $0{\sim}20%$인 분포상(分布相)을 나타냈다. c) 젓갈숙성(熟成)에 관여(關與)하는 미생물(微生物)로서 초기이후(初期以後)에는 주(主)로 내염성(耐鹽性) Bacillus subtilis, Leuconostoc mesenteroides, Pediococcus halophilus, Sarcina litoralis 등(等)임을 동정(同定)하였고 이들의 효소활성(酵素活性)이 젓갈숙성(熟成)을 지배(支配)하는 요소(要素)임을 알 수 있었다. d) 숙성(熟成)에 관여(關與)하는 세균중(細菌中)에 Sarcina litoralis 8­14 및 8-16. 양균주(兩菌株)는 영양요구성(榮養要求性)이 현저(顯著)하여 전자(前者)는 purine, pyrimidine base 및 cystine 후자(後者)는 purine, pyrimidine base 및 glutamic acid가 공존(共存)하여야 생육(生育)이 가능(可能)하였다. (2) 젓갈원료(原料) 및 젓갈에서 분리(分離)한 관여미생물(關與微生物)에 대(對)하여 효소특성(酵素特性)을 검토(檢討)한 결과(結果)는 다음과 같다. a) 젓갈원료중(原料中)에 protease는 소량(少量) 존재(存在)하나 식염농도(食鹽濃度) 7%에서 protease 활성(活性)이 현저(顯著)하게 조해(阻害)를 받아 그 효소활성(酵素活性)의 $30{\sim}60%$나 감소(減少)하였다. b) 내염성세균(耐鹽性細菌)인 Bacillus subtilis 7-6, 11-1, 3-6 9-4 등(等) 4 균주(菌株)는 complete media에서 생성(生成)한 protease 활성(活性)은 식염농도(食鹽濃度) 7%에서 약간 조해(阻害)를 받아 활성(活性)이 $10{\sim}30%$가 감소(減少)하였고 Sarcina litoralis 8-14 및 8­16 양균주(兩菌株)는 동배지(同培地)에서 생성(生成)한 protease 활성(活性)은 식염농도(食鹽濃度) 7%에서 그 발효활성(醱酵活性)의 $10{\sim}20%$가 감소(減少)하였다. c) 젓갈 원료중(原料中)의 단백질(蛋白質)은 자가효소(自家酵素)에 의한것보다 주(主)로 젓갈숙성중(熟成中) 관여미생물(關與微生物)의 protease에 의(依)한 가수분해(加水分解)로 유리(遊離) amino 산(酸)이 생성(生成)됨을 알 수 있었다. d) 젓갈원료(原料) 및 젓갈의 RNA-depolymerase는 젓갈중(中) RNA를 nucleoside 및 유리인산(遊離燐酸)까지 분해(分解)하므로 정미성(呈味性) 5'-mononucleotide로서 축적(蓄積)되기 어렵다 e) 조개젓에서 분리(分離)한 Bacillus subtilis 3-6 균주(菌株)가 생성(生成)한 효소(酵素)는 RNA를 분해(分解)하여 5’-mononucleotide로 축적(蓄積)하므로 이 균주(菌株)가 생성(生成)한 RNA 분해효소(分解酵素)는 5’-phosphodiesterase임을 밝혔다. f) Bacillus subtilis 3-6 strain의 효소생성배지중(酵素生成培地中)에 corn steep liquor 0.5% 농도(濃度)로 첨가(添加)한 구(區)와 조개젓 0.5% 농도(濃度)로 첨가(添加)한 구(區)에서 5’-phosphodiesterase 생성증가(生成增加)를 보았으므로 corn steep liquor 및 조개젓 성분(成分)이 각각(各各) 5’-phosphodiesterase 생성증가제(生成增加劑)가 됨을 밝혔으며 또한 이 효소(酵素)는 10%의 식염농도(食鹽濃度)에서 활성조해(活性阻害)를 받아 $10{\sim}30%$에 해당(該當)되는 활성(活性)이 감소(減少)되었고 식염농도(食鹽濃度) 20%에서 이 효소활성(酵素活性)의 $40{\sim}60%$가 감소(減少)함을 나타냈다. 그리고 이 균주(菌株)의 5’­phosphodiesterase에 의(依)하여 젓갈중(中) 5’-mononucleotides가 생성(生成)되는 것이라고 생각된다. (3) 젓갈의 유리(遊離) amino 산(酸)을 Autoanalyzer로 정량(定量)한 결과(結果)는 다음과 같다. a) 조개젓은 산성(酸性) amino 산(酸)인 glutamic acid, asp artic acid 함량(含量)이 다른 젓갈보다 $2{\sim}10$배(倍) 정도(程度)로 현저하게 많아서 조개젓의 구수한 맛이 발현(發現)되는 것이라고 생각(生覺)된다. b) 조기젓에는 basic amino acid인 arginine, histidine함량(含量)이 다른 젓갈보다 특이(特異)하게 많았다. c) 오징어젓은 함류황(含硫黃) amino산(酸)인 cystine함량(含量)이 다른 젓갈의 $17{\sim}130$배(倍), methionine함량(含量)이 $7{\sim}17$배정도(倍程度)로 현저하게 많았다. d) 굴젓에는 필수(必須) amino 산(酸)인 lysine, threonine iso leucine, leucine 함량(含量)이 다른 젓갈보다 현저하게 많을 뿐아니라 또한 감미성(甘味性) amino 산(酸)인 alanine 함량(含量)이 다른 젓갈들보다 4배(倍), glycine 함량(含量)이 $3{\sim}14$배정도(倍程度)로 함유(含有)하고 있어 이것이 굴젓 특유(特有)의 단맛을 발현(發現)하는 것이라 생각(生覺)된다. (4) 4종(種) 젓갈에 대(對)한 5'-mononucleotide를 ion exchange column chromatography로 측정(測定)한 결과(結果)는 다음과 같다. a) 조개젓에는 5'-adenylic acid, 3’-adenylic acid 가 많이 들어있고 5'-inosinic acid가 미량(微量)들어 있다. b) 굴젓에는 5'-adenylic acid와 3’-adenylic acid가 다른 mononucleotides보다 현저하게 많았다. c) 오징어젓에는 5'-adenylic 및 3'-adenylic acid만이 함유(含有)되어 있는데 이것은 패류(貝類)의 젓갈들과 같 이 무척추연체동물(無脊椎軟體動物)이며 이것들은 adenylic deaminase가 결여(缺如)되어 있어 mononucleotide중(中) adenylic acid의 함량(含量)이 높아 adenylic acid-type 임을 알았다. d) 조기젓 중(中)에는 5'-inosinic acid 함량(含量)이 다른젓갈보다 현저하게 많았으므로 이것은 다른 척추동물(脊椎動物)의 어육(魚肉)이나 수육(獸肉)과 같이 adenylic deaminase가 있어 5'-inosinic acid가 많은 inosinic acid-type임을 알았다. (5) 정미성(呈味性) amino 산(酸) 및 5'-mononucleotide의 조성(組成)과 함량(含量)으로 맛의 조화성(調和性)을 비교(比較)하여 보면 다음과 같다. a) 조기젓은 glutamic acid와 aspartic acid가 풍부(豊富)하고 소량(小量)의 5'-inosinic acid가 공존(共存)된 상태(狀態)이므로 정미성분조성(呈味成分組成)과 함량(含量)이 잘 조화(調和)되어 구수한 맛의 상승작용(相乘作用)이 적당(適當)하게 발현(發現)됨을 구명(究明)하였다. b) 조개젓의 맛은 정미성(呈味性) 5’-mononucleotides에서 유래(由來)되는것 보다는 현저하게 많이 들어있는 glutamic acid 및 aspartic acid에 기인됨을 알았다.

• Journal of Microbiology
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• 제45권2호
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• 미생물학회지
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• 제38권4호
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• pp.247-253
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• 2002

환경오염원으로서 폭약 2,4,6-trinitrotoluene (TNT)에 대한 TNT 분해세균 Stenotrophomonas sp. OK-5의 세포반응에 대하여 조사하였다. 아치사조건의 TNT농도와 노출시간에 따른 균주 OK-5의 생존율을 분석한 결과, 이 세균의 생존율은 스트레스 충격 단백질의 생성과 비례하였다. 총세포 지방산 조성분석에서 균주 OK-5는 tryp-ticase soy agar에서 자랄 때보다 TNT 배지에서 자랄 때 여러 가지 종류의 지방산이 생성되거나 사라지는 것이 밝혀졌다. 주사전자현미경하에서 TNT에 노출된 세포는 쭈글쭈글하고 불규칙적인 간상형으로 나타났다. Anti-DnaK와 anti-GroEL을 이용하여 SDS-PAGE와 Western blot을 통한 분석으로 균주 OK-5는 70 kDa DanK와 60 kDa GroEL을 포함하는 몇가지 스트레스충격단백질을 생성하는 것으로 밝혀졌다. TNT에 노출된 OK-5 배양에서 수용성 단백질 분획에 대하여 2-D PAGE를 실시하였으며, pH 3에서 pH 10의 범위에서 약 300여 개 spot들이 silver로 염색된 gel상에서 관찰되었다. 이들 가운데 TNT의 반응으로 현저하게 유도되고 발현된 10개의 spot들을 확인하였으며, 2개의 단백질, spot #1과 spot #10에 대한 내부아미노산 서열을 ESI-Q TOF로 분석한 결과, Xylella fastidiosa의 DnaK protein XF2340와 Mesorhizobium loti의 스트레스 유도단백질로 각각 밝혀졌다.

• 한국식품저장유통학회지
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• 제14권6호
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• pp.639-644
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• 2007

본 연구는 뜰보리수의 영양성분 및 생리활성 물질을 조사하여 식품학적 가치를 평가하고자 하는 연구의 일환으로 뜰보리수 잎의 영양성분을 분석하였다. 뜰보리수 잎의 수분, 탄수화물, 조단백, 조지방 및 조회분의 함량은 각각 71.6, 24.1, 1.4, 0.4 그리고 2.5%이었다. 뜰보리수 잎의 환원당과 수용성 단백질의 함량은 각각 460.0, 503.3 mg/100 g이었으며, 항산화성 기능을 가지고 있는 폴리페놀의 함량은 805.6mg/100 g이었다. 뜰보리수에 함유된 유리당은 fructose가 가장 많았으며, 그 외 arabinose, maltose, glucose 순이었으며, trehalose는 미량 검출되었다. 뜰보리수 잎은 malic acid가 주요 유기산이었으며, 이외 acetic acid, citric acid, lactic acid, succinic acid순으로 높았다. 뜰보리수 잎의 무기질 성분중 K의 함량이 840.5 mg/100 g으로 가장 높았고, Ca과 Ma도 많은 양 함유되어 있었다. 뜰보리수 잎의 구성아미노산 중 alanine의 함량이 112.0 mg/100 g으로 가장 높았으며, threonine, leucine, valine, phenylalanine 순으로 높았다. 그리고 유리아미노산은 glutamic acid가 57.3 mg/100 g으로 가장 많이 함유되어 있었으며, 그 다음으로는 arginine, leucine, glycine, phenylalanine 순이었다. 아미노산의 유도체는 11종이 검출되었으며, ornithine의 함량이 48.5 mg/100 g으로 가장 높게 분석되었다.