Psychrophilic bacteria have acquired cold-resistance in order to protect themselves against freezing temperatures, which would otherwise be lethal. DnaK/DnaJ/GrpE systems are molecular chaperones which facilitate proper folding of newly synthesized proteins. Efficient folding processes are of great importance especially in a cold environment, such as the Arctic. In order to understand the protection mechanisms of psychrophilic bacteria against cold temperatures, we have explored a genome of KOPRI22215, tentatively identified as Psychromonas arctica, whose genome sequence has not yet been discovered. With an aim of searching for a coding gene of DnaK from KOPRI22215, we have applied a series of polymerase chain reactions (PCR) with homologous primers designed from other Psychromonas species and LA PCR in vitro cloning. 1917 bp complete coding sequence of dnaK from KOPRI22215 was identified including upstream promoter sites. Recombinant plasmids to overexpress PaDnaK along with EcDnaK (DnaK of E. coli) were then constructed in pAED4 vector and the pET-based system to induce PaDnaK expression by IPTG. Characterization assays of expressed PaDnaK were carried out by measuring survival rates upon 4 day incubation at 4 $^{\circ}C$: a refolding assay as molecular chaperone, and ATPase assay for functional activity. Taking account of all the data together, we conclude that PaDnaK was identified, successfully expressed, and found to be more efficient in providing cold-resistance for bacterial cells.
Proceedings of the Botanical Society of Korea Conference
/
2002.04a
/
pp.62-72
/
2002
This study has investigated the biosynthesis and function of the heavy metal binding peptides, the phytochelatins, in plants. PCs are synthesised enzymatically from glutathione by the enzyme PC synthase in the presence of heavy metal ions. Using Arabidopsis thaliana as a model organism cadmium-sensitive, phytochelatin-deficient mutants have been isolated and characterised in previous studies. The cadl mutants have wildtype levels of glutathione, are PC deficient and lack PC synthase activity. Thus, the CADl gene has been proposed to encode PC synthase. The CADl gene was isolated by a positional cloning strategy The gene was mapped and a candidate identified. Each of four cadl mutants had a single base pair change in the candidate gene and the cadmium-sensitive, cadl phenotype was complemented by the candidate gene. This demonstrated the CADl gene had been cloned. A homologous gene in the fission yeast, Schizosaccharomyces pombe was identified through database searches. A targeted-deletion mutation of this gene was constructed and the mutant, like cadl mutants of Arabidopsis, was cadmium-sensitive and PC-deficient. A comparison of the redicted amino acid sequences reveals a highly conserved N-terminal region Presumed to be the catalytic domain and a variable C-terminal region containing multiple Cys residues proposed to be involved in activation of the enzyme by metal ions. Similar genes were also identified in animal species. The Arabidopsis CADl/AtPCSl and S. pombe SpbPCS genes were expressed in E. coli and were shown to be sufficient for glutathione-dependent, heavy metal activate PC synthesis in vitro, thus demonstrating these genes encode PC synthase enzymes. Using RT-PCR, AtPCSl expression appeared to be independent of Cd exposure. However, at higher levels of Cd exposure a AtPCSl-CUS reporter gene construct appeared to be more highly expressed. Using the reporter gene construct, AtPCSl was expressed most tissues. Expression appeared to be greater in younger tissues and same higher levels of expression was observed in some regions, including carpels and the base of siliques. AtPCS2 was a functional gene encoding an active PC synthase. However, its Pattern of expression and the phenotype of a mutant (or antisense line) have not been determined. Assuming the gene is functional then it has clearly been maintained through evolution and must provide some selective advantage. This implies that, at least in some cells or tissue, it is likely to be the dominant PC synthase expressed. This remains to be determined
Journal of the Korean Applied Science and Technology
/
v.34
no.3
/
pp.613-622
/
2017
The object of this study was to determine the influence of dietary metabolic energy (ME) on ..... A total of 240 meat ducks Cherry valley (Anas platyrhynchos) were assigned into four treatment groups with a randomized block design for 42 days. The four treatments were: ME 2900 kcal/kg, ME 3000 kcal/kg, ME 3100 kcal/kg, and ME 3200 kcal/kg. There was no difference in liver tissue among the treatments. The duodenal villi and crypt depth length decreased by 10.58% in 2900 compared with ME 3000, but there was no difference between 3100 and 3200. Counts of caecal Latobacillus decreased by 9.47% in 2900 compared to ME 3000, but increased by 2.52 and 3.24% in 3100 and 3200, respectively. Total aerobic bacteria, E. coli and Coliform bacteria were increased by 2900 when compared to ME 3000, but there was no difference between 3100 and 3200. HSP $90-{\alpha}$ among the heat shock proteins (HSPs)-mRNA in the liver was reduced by 48.60% in 2900 compared to ME 3000, while 3100 and 3200 showed no difference or increased.
To understand the molecular structure and pathogenesis mechanism of Korean garlic viruses, we have isolated cDNA clones for garlic viruses. The partial nucleotide sequences of 24 cDNA clones were determined and those of five clones containing poly(A) tail were compared with sequences of other plant viruses. One of these clones, V9, has a primary structure similar to the carlavirus group, suggesting that the clone V9 derived from a part of garlic latent virus (GLV). Northern blot analysis with the clone V9 as a probe demonstrated that GLV genome is 8.5 knt long and has a poly(A) tail. The clone V9 encodes coat protein (CP) of 33 kDa and nucleic acid binding protein of 10 kDa in different reading frame. The hexanucleotide motif, 5'-ACCUAA, which is conserved in the 3' noncoding region arid was proposed to be a cis-acting element involved in the production of negative strand genomic RNA was noticed. Complementary sequence to the hexanucleotide motif, 5'-TTAGGT, is also found in the positive strand of V9 RNA. The putative CP gene was cloned into the pRSET-A expression vector and expressed in E. coli BL21. The expressed recombinant V9CP protein was purified by $Ni^{2+}$ NTA affinity chromatography. The anti-V9CP antibody recognizes 34 kDa polypeptide which could be CP of GLV in infected garlic leaf extract. Immunoblot and Northern blot analysis of various cultivars shows wide occurrence of GLV in Korean garlic plants.
So Jin Kim;Su Hyeong Heo;Min Gun Kim;Kyung Hwan Boo;Chang Sook Kim
Proceedings of the Plant Resources Society of Korea Conference
/
2022.09a
/
pp.112-112
/
2022
This study aims to confirm the possibility of using the invasive alien plants in Jeju as a functional biomaterial. To achieve this purpose, 70% ethanol extract and solvent fractions were prepared for five invasive alien plants (Hypochaeris radicata, Rumex acetosella, Humulus japonicus Siebold & Zucc., Solanum viarum, Lactuca scariolar) and their antioxidant, antibacterial anti-inflammatory and anti-obesity effects were investigated. The DPPH radical scavenging activity of ethanol extract from invasive alien plants was shown in the order of Rumex acetosella > Hypochaeris radicata > Humulus japonicus. Antimicrobial activity of ethanol extract against food poisoning bacteria (4 species) and oral cavity-induced microorganisms (6 species) was measured. As a result, the extract of Humulus japonicus showed high antibacterial effects against food poisoning bacteria (E. coli, V. parahaemolyticus) and oral microbes (L. casei, S. epidermidis, E. faecalis). In LPS-induced RAW 264.7 cells, the anti-inflammatory effect of ethanol extract from invasive alien plants was investigated. As a result, the NO production inhibition activity was highest in the Rumex acetosella and the Humulus japonicus Siebold & Zucc. ethanol extract, and the NO production inhibition activity was concentration-dependent. In addition, the Rumex acetosella and the Humulus japonicus Siebold & Zucc. ethanol extract showed a concentration-dependent inhibitory effect on cytokine (IL-6) production. These extracts also showed inhibitory activity of COX-2, an inflammatory protein. This suggests that NO production inhibition activity by the extract of invasive alien plants is the result of inhibition of iNOS and COX-2 expression. Currently, organic solvent fractions of crude extract are manufactured and the investigation of active ingredients is continuing along with evaluation of biological activity such as anti-inflammatory. These results are expected to be a major data for the study on the separation and utilization of active ingredients with antioxidant, antibacterial and anti-inflammatory effects using foreign plant crude extract and solvent fractions, and are highly likely to be applied to the development of functional food and cosmetics materials.
Proceedings of the Korean Society of Applied Pharmacology
/
1997.04a
/
pp.95-95
/
1997
Among the various receptor molecules discovered so far the ${\beta}$2-adrenergic receptors have been regarded as excellent model systems for the so called 7 transmembrane helix receptor and have been the focus of extensive studies. For the analysis of receptor structure and function a monoclonal antibody plays a crucial role, thus providing useful tools for the study of receptor. However, because of the minute quantity of receptor molecules which could be obtained from natural sources, the generation of specific monoclonal antibody against receptor molecules from the purified receptors has been regarded as virtually impractical in consideration of cost and experimental times. The purpose of the present study was to generate and characterize a monoclonal antibody against human ${\beta}$2-adrenergic receptor. For the production of antibody, C-terminal regions of the human ${\beta}$2-adrenergic receptor was produced as a fusion protein with Glutathion S-transferase (GST) in E. Coli. The expression of the fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein was purified to an apparent homogeniety by affinity chromatography with Glutathion Sepharose CL-4B and used as an antigen for the immunization of BALB/C mice. The Production of monoclonal antibody was achieved by fusion of the immunized spleen cells and SP/2-0 myeloma cells. Positive hybridomas were screened by ELISA and were cloned by two consecutive rounds of limiting dilution. The monoclonal antibody produced in this study (mAb${\beta}$C02) was IgM type and purified by immunoaffinity chromatography using anti-mouse IgM agarose as an affinity matrix. MAb${\beta}$C02 showed strong and specific immunoreactivity against both the fusion protein and human ${\beta}$2-adrenergic receptor in ELISA and Western blot. The molecular weight of immunoreactive band was 64 kDa and exactly coincided with the previously reported molecular weight of ${\beta}$2-adrenergic recepters. The results of the present study suggest that mAb${\beta}$C02 may be used for the study of receptor function and regulation in normal or nonphysiological status.
Vaccination is the most effective way to prevent influenza virus infections. However, conventional vaccines based on hemagglutinin (HA) have to be annually updated because the HA of influenza viruses constantly mutates. In this study, we produced a 3M2e-3HA2-NP chimeric protein as a vaccine antigen candidate using an Escherichia coli expression system. The vaccination of chimeric protein (15 ㎍) conferred complete protection against A/Puerto Rico/8/1934 (H1N1; PR8) in mice. It strongly induced influenza virus-specific antibody responses, cytotoxic T lymphocyte activity, and antibody-dependent cellular cytotoxicity. To spare the dose and enhance the cross-reactivity of the chimeric, we used a complex of poly-γ-glutamic acid and alum (PGA/alum) as an adjuvant. PGA/alum-adjuvanted, low-dose chimeric protein (1 or 5 ㎍) exhibited higher cross-protective effects against influenza A viruses (PR8, CA04, and H3N2) compared with those of chimeric alone or alum-adjuvanted proteins in vaccinated mice. Moreover, the depletion of CD4+ T, CD8+ T, and NK cells reduced the survival rate and efficacy of the PGA/alum-adjuvanted chimeric protein. Collectively, the vaccination of PGA/alum-adjuvanted chimeric protein induced strong protection efficacy against homologous and heterologous influenza viruses in mice, which suggests that it may be a promising universal influenza vaccine candidate.
We have previously reported recombinant productions of bacteriocins using yeast expression plasmid pAUR123, which contains the alcohol dehydrogenase (ADH) promoter, in Saccharomyces cerevisiae cells and their antibacterial activities. In order to improve the antibacterial activities of bacteriocidal yeast cells, a strong glyceraldehyde phosphate dehydrogenase (GPD) promoter gene of S. cerevisiae was amplified and inserted upstream into bacteriocin genes such as the OR-7, Subpeptin JM4-A or JM4-B gene in the corresponding recombinant yeast plasmid. Yeast cells transformed by the recombinant plasmid containing the GPD promoter represented higher antibacterial activities against both Gram positive B. subtilis and Gram negative E. coli cells compared to those transformed by the corresponding recombinant plasmid containing the ADH promoter. Thus, yeast cells harboring the recombinant plasmid containing the GPD promoter constructed in this study could be applied in the food preservative or animal feed industries.
L-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the L-asparaginase gene (L-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino acid sequence of L-ASPG86 (L-ASPG86) was grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The L-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant L-asparaginase (r-L-ASPG86) showed optimum conditions at 37-40℃, pH 9. Moreover, r-L-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-L-ASPG86 was 687.1 units/mg under optimum conditions (37℃, pH 9, and 5 mM MnSO4).
The nuclear matrix was isolated from Misgumus mizolepis liver nuclei by low salt extraction and restriction enzyme treatment. The structure was digested with proteinase K. After centrifugation, matrix attachment regions (MARs) were obtained by RNase treatment and phenol-chloroform extraction. The result leads to the appearance of smeared bands in the range of about 0.3-15 kb. pURY19 vector was constructed by inserting 2.13 kb Eco47 III fragment of the yeast uracil 3 gene into the unique Ssp I site of pUC19 plasmid vector as a selection marker. This vector is unable to be maintained in Sacrharomyces cerevisiae by itself since it cannot replicate as an extrachromosomal element. Using this system, we attempted cloning the ARS (autonomously replicating sequence) from M. mizelepis to develop an efficient expression vector for the transgenic fish. pURY19N_{l-62}$ were constructed by inserting MARs in pURY19 plasmid vector and transformation of E. coli $DH5\alpha$. Replication origins (ARS) of M. mizolepis were isolated, which enabled the vector to replicate autonomously in S. cerevisiae. The cloned DNA fragments were sequenced by Sanger's dideoxy-chain termination method. All clones were AT-rich. $pURY19N_6$, one of the clones, expecially contained ARS consensus sequence, Topoisomerase II consensus, near A-box and T-box.
본 웹사이트에 게시된 이메일 주소가 전자우편 수집 프로그램이나
그 밖의 기술적 장치를 이용하여 무단으로 수집되는 것을 거부하며,
이를 위반시 정보통신망법에 의해 형사 처벌됨을 유념하시기 바랍니다.
[게시일 2004년 10월 1일]
이용약관
제 1 장 총칙
제 1 조 (목적)
이 이용약관은 KoreaScience 홈페이지(이하 “당 사이트”)에서 제공하는 인터넷 서비스(이하 '서비스')의 가입조건 및 이용에 관한 제반 사항과 기타 필요한 사항을 구체적으로 규정함을 목적으로 합니다.
제 2 조 (용어의 정의)
① "이용자"라 함은 당 사이트에 접속하여 이 약관에 따라 당 사이트가 제공하는 서비스를 받는 회원 및 비회원을
말합니다.
② "회원"이라 함은 서비스를 이용하기 위하여 당 사이트에 개인정보를 제공하여 아이디(ID)와 비밀번호를 부여
받은 자를 말합니다.
③ "회원 아이디(ID)"라 함은 회원의 식별 및 서비스 이용을 위하여 자신이 선정한 문자 및 숫자의 조합을
말합니다.
④ "비밀번호(패스워드)"라 함은 회원이 자신의 비밀보호를 위하여 선정한 문자 및 숫자의 조합을 말합니다.
제 3 조 (이용약관의 효력 및 변경)
① 이 약관은 당 사이트에 게시하거나 기타의 방법으로 회원에게 공지함으로써 효력이 발생합니다.
② 당 사이트는 이 약관을 개정할 경우에 적용일자 및 개정사유를 명시하여 현행 약관과 함께 당 사이트의
초기화면에 그 적용일자 7일 이전부터 적용일자 전일까지 공지합니다. 다만, 회원에게 불리하게 약관내용을
변경하는 경우에는 최소한 30일 이상의 사전 유예기간을 두고 공지합니다. 이 경우 당 사이트는 개정 전
내용과 개정 후 내용을 명확하게 비교하여 이용자가 알기 쉽도록 표시합니다.
제 4 조(약관 외 준칙)
① 이 약관은 당 사이트가 제공하는 서비스에 관한 이용안내와 함께 적용됩니다.
② 이 약관에 명시되지 아니한 사항은 관계법령의 규정이 적용됩니다.
제 2 장 이용계약의 체결
제 5 조 (이용계약의 성립 등)
① 이용계약은 이용고객이 당 사이트가 정한 약관에 「동의합니다」를 선택하고, 당 사이트가 정한
온라인신청양식을 작성하여 서비스 이용을 신청한 후, 당 사이트가 이를 승낙함으로써 성립합니다.
② 제1항의 승낙은 당 사이트가 제공하는 과학기술정보검색, 맞춤정보, 서지정보 등 다른 서비스의 이용승낙을
포함합니다.
제 6 조 (회원가입)
서비스를 이용하고자 하는 고객은 당 사이트에서 정한 회원가입양식에 개인정보를 기재하여 가입을 하여야 합니다.
제 7 조 (개인정보의 보호 및 사용)
당 사이트는 관계법령이 정하는 바에 따라 회원 등록정보를 포함한 회원의 개인정보를 보호하기 위해 노력합니다. 회원 개인정보의 보호 및 사용에 대해서는 관련법령 및 당 사이트의 개인정보 보호정책이 적용됩니다.
제 8 조 (이용 신청의 승낙과 제한)
① 당 사이트는 제6조의 규정에 의한 이용신청고객에 대하여 서비스 이용을 승낙합니다.
② 당 사이트는 아래사항에 해당하는 경우에 대해서 승낙하지 아니 합니다.
- 이용계약 신청서의 내용을 허위로 기재한 경우
- 기타 규정한 제반사항을 위반하며 신청하는 경우
제 9 조 (회원 ID 부여 및 변경 등)
① 당 사이트는 이용고객에 대하여 약관에 정하는 바에 따라 자신이 선정한 회원 ID를 부여합니다.
② 회원 ID는 원칙적으로 변경이 불가하며 부득이한 사유로 인하여 변경 하고자 하는 경우에는 해당 ID를
해지하고 재가입해야 합니다.
③ 기타 회원 개인정보 관리 및 변경 등에 관한 사항은 서비스별 안내에 정하는 바에 의합니다.
제 3 장 계약 당사자의 의무
제 10 조 (KISTI의 의무)
① 당 사이트는 이용고객이 희망한 서비스 제공 개시일에 특별한 사정이 없는 한 서비스를 이용할 수 있도록
하여야 합니다.
② 당 사이트는 개인정보 보호를 위해 보안시스템을 구축하며 개인정보 보호정책을 공시하고 준수합니다.
③ 당 사이트는 회원으로부터 제기되는 의견이나 불만이 정당하다고 객관적으로 인정될 경우에는 적절한 절차를
거쳐 즉시 처리하여야 합니다. 다만, 즉시 처리가 곤란한 경우는 회원에게 그 사유와 처리일정을 통보하여야
합니다.
제 11 조 (회원의 의무)
① 이용자는 회원가입 신청 또는 회원정보 변경 시 실명으로 모든 사항을 사실에 근거하여 작성하여야 하며,
허위 또는 타인의 정보를 등록할 경우 일체의 권리를 주장할 수 없습니다.
② 당 사이트가 관계법령 및 개인정보 보호정책에 의거하여 그 책임을 지는 경우를 제외하고 회원에게 부여된
ID의 비밀번호 관리소홀, 부정사용에 의하여 발생하는 모든 결과에 대한 책임은 회원에게 있습니다.
③ 회원은 당 사이트 및 제 3자의 지적 재산권을 침해해서는 안 됩니다.
제 4 장 서비스의 이용
제 12 조 (서비스 이용 시간)
① 서비스 이용은 당 사이트의 업무상 또는 기술상 특별한 지장이 없는 한 연중무휴, 1일 24시간 운영을
원칙으로 합니다. 단, 당 사이트는 시스템 정기점검, 증설 및 교체를 위해 당 사이트가 정한 날이나 시간에
서비스를 일시 중단할 수 있으며, 예정되어 있는 작업으로 인한 서비스 일시중단은 당 사이트 홈페이지를
통해 사전에 공지합니다.
② 당 사이트는 서비스를 특정범위로 분할하여 각 범위별로 이용가능시간을 별도로 지정할 수 있습니다. 다만
이 경우 그 내용을 공지합니다.
제 13 조 (홈페이지 저작권)
① NDSL에서 제공하는 모든 저작물의 저작권은 원저작자에게 있으며, KISTI는 복제/배포/전송권을 확보하고
있습니다.
② NDSL에서 제공하는 콘텐츠를 상업적 및 기타 영리목적으로 복제/배포/전송할 경우 사전에 KISTI의 허락을
받아야 합니다.
③ NDSL에서 제공하는 콘텐츠를 보도, 비평, 교육, 연구 등을 위하여 정당한 범위 안에서 공정한 관행에
합치되게 인용할 수 있습니다.
④ NDSL에서 제공하는 콘텐츠를 무단 복제, 전송, 배포 기타 저작권법에 위반되는 방법으로 이용할 경우
저작권법 제136조에 따라 5년 이하의 징역 또는 5천만 원 이하의 벌금에 처해질 수 있습니다.
제 14 조 (유료서비스)
① 당 사이트 및 협력기관이 정한 유료서비스(원문복사 등)는 별도로 정해진 바에 따르며, 변경사항은 시행 전에
당 사이트 홈페이지를 통하여 회원에게 공지합니다.
② 유료서비스를 이용하려는 회원은 정해진 요금체계에 따라 요금을 납부해야 합니다.
제 5 장 계약 해지 및 이용 제한
제 15 조 (계약 해지)
회원이 이용계약을 해지하고자 하는 때에는 [가입해지] 메뉴를 이용해 직접 해지해야 합니다.
제 16 조 (서비스 이용제한)
① 당 사이트는 회원이 서비스 이용내용에 있어서 본 약관 제 11조 내용을 위반하거나, 다음 각 호에 해당하는
경우 서비스 이용을 제한할 수 있습니다.
- 2년 이상 서비스를 이용한 적이 없는 경우
- 기타 정상적인 서비스 운영에 방해가 될 경우
② 상기 이용제한 규정에 따라 서비스를 이용하는 회원에게 서비스 이용에 대하여 별도 공지 없이 서비스 이용의
일시정지, 이용계약 해지 할 수 있습니다.
제 17 조 (전자우편주소 수집 금지)
회원은 전자우편주소 추출기 등을 이용하여 전자우편주소를 수집 또는 제3자에게 제공할 수 없습니다.
제 6 장 손해배상 및 기타사항
제 18 조 (손해배상)
당 사이트는 무료로 제공되는 서비스와 관련하여 회원에게 어떠한 손해가 발생하더라도 당 사이트가 고의 또는 과실로 인한 손해발생을 제외하고는 이에 대하여 책임을 부담하지 아니합니다.
제 19 조 (관할 법원)
서비스 이용으로 발생한 분쟁에 대해 소송이 제기되는 경우 민사 소송법상의 관할 법원에 제기합니다.
[부 칙]
1. (시행일) 이 약관은 2016년 9월 5일부터 적용되며, 종전 약관은 본 약관으로 대체되며, 개정된 약관의 적용일 이전 가입자도 개정된 약관의 적용을 받습니다.