• Title/Summary/Keyword: Y2H library

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Purification and Characterization of 2,3-Dihydroxybiphenyl 1,2- Dioxygenase from Comamonas sp.

  • Lee Na Ri;Kwon Dae Young;Min Kyung Hee
    • Proceedings of the Microbiological Society of Korea Conference
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    • 2001.11a
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    • pp.16-25
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    • 2001
  • A genomic library of biphenyl-degrading strain Comamonas sp. SMN4 was constructed by using the cosmid vector pWE15 and introduced into Escherichia coli. Of 1,000 recombinant clones tested, two clones that expressed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity were found (named pNB 1 and pNB2). From pNB1 clone, subclone pNA210, demonstrated 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, is isolated. 2,3-Dihydroxybiphenyl 1,2-dioxygenase (23DBDO, BphC) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the Comamonas sp. SMN4 gene bphC, which encodes 23DBDO, was cloned into a plasmid pQE30. The His-tagged 23DBDO produced by a recombinant Escherichia coli, SG 13009 (pREP4)(pNPC), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 23DBDO construction was active. SDS-PAGE analysis of the purified active 23DBDO gave a single band of 32 kDa; this is in agreement with the size of the bphC coding region. The 23DBDO exhibited maximum activity at pH 9.0. The CD data for the pHs, showed that this enzyme had a typical a-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. This structure maintained up to pH 10.5. However, this high stable folding strucure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The result of CD spectra observed with pH effects on 23DBDO activity, suggested that charge transition by pH change have affected change of conformational structure for 23DBDO catalytic reaction. The $K_m$ for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7 $\mu$M, 24 $\mu$M, 50 mM and 625 $\mu$M.

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Yonsei Evolutionary Population Synthesis (YEPS) Model -III. Spectrophotometric Evolutions of Simple Stellar Population Models based on Empirical Spectra

  • Chung, Chul;Yoon, Suk-Jin;Lee, Young-Wook
    • The Bulletin of The Korean Astronomical Society
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    • v.38 no.2
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    • pp.38.1-38.1
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    • 2013
  • We present the Yonsei evolutionary population synthesis (YEPS) models based on the high-resolution empirical spectral energy distributions (SEDs). We have adopted the MILES library in the optical wavelength, and our new models based on the MILES library show good agreements with our previous models presented in the YEPS I. The effect of hot horizontal-branch (HB) stars on the integrated properties of simple stellar populations (SSPs) is again confirmed by our models based on empirical SEDs. In addition, we have extended our empirical models to the near-IR wavelength and predicted the strengths of the calcium II triplet (CaT) and the Paschen triplet (PaT) based on the INDO-US and the Cenarro library. We find that the effect of HB stars and the age of SSPs on the CaT is almost negligible. On the other hands, the PaT models are very sensitive to the existence of hot stars, e.g., HB stars and young turn-off stars, and show very similar results with Balmer lines. Interestingly, the CaT distribution of GCs in NGC 1407, which is at odds with the optical (B-I) color distribution, can be explained by the unique feature of the CaT-[Fe/H] relations that show almost the same equivalent widths in the metal-rich regime. We will also discuss the impact of the second-generation populations on the strength of the CaT.

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Screening of Growth- or Development-related Genes by Using Genomic Library with Inducible Promoter in Aspergillus nidulans

  • Lee Bang-Yong;Han Sang-Yong;Choi Han Gil;Kim Jee Hyun;Han Kap-Hoon;Han Dong-Min
    • Journal of Microbiology
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    • v.43 no.6
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    • pp.523-528
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    • 2005
  • Using the genomic library constructed at the downstream of the niiA promoter, which induces the over-expression of an inserted DNA fragment, we have attempted to screen the genes affecting growth or development by over-expression. The wild-type strain was transformed using the AMA-niiA(p) library and cultured on 1.2 M sorbitol media, in which asexual sporulation is induced, but sexual development is repressed. Over 100,000 strains transformed to $pyrG^+$ were analyzed with regard to any changes in phenotype. Consequently, seven strains were isolated for further analyses. These strains were designated NOT [niiA(p) over-expression transformants] stains. Four of the strains were of the inducible type, and the remaining strains were of the multi-copy suppression type. Two of the inducible-type strains, NOT 1 and NOT40, harbored genes which had been inserted in reverse direction, suggesting that the mutant phenotypes had been derived from an excess amount of anti-sense mRNA. Domain analyses of the deduced polypeptides from the DNA fragments rescued from the transformants revealed that NOT1, NOT40 and NOT6 harbored a LisH motif, a forkhead domain, and a $Zn(II)_2Cys_6$ binuclear zinc cluster, respectively.

Functional and Physical Interaction between Human Lactate Dehydrogenase B and $Na^+/H^+$ Exchanger Isoform 1

  • Kim, Eun-Hee
    • Animal cells and systems
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    • v.13 no.3
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    • pp.283-288
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    • 2009
  • The ubiquitous plasma membrane $Na^+/H^+$ exchanger 1 (NHE1) is rapidly activated in response to various extracellular stimuli and maintains normal cytoplasmic pH. Yeast two-hybrid screening was used in order to identify proteins interacting with NHE1 using its cytoplasmic domain as a bait from HeLa cDNA library. One of the interacting cDNA clones was human Lactate dehydrogenase B (LDHB). In vitro translated LDHB was pulled down together with GST-NHE1.cd protein in the GST pull down assay, confirming the interaction in vitro. LDHB antibody immunoprecipitated endogenous LDHB together with NHE1 from H9c2 cells, validating cellular interaction between NHE1 and LDHB. Subsequent analysis revealed that the overexpression of LDHB increased intracellular PH, implying opening of the NHE1 transporter. Moreover, overexpression of LDHB activated caspase 3 and induced cell death, consistent with the expected phenotype of hyper-activation of NHE1. Collectively, our data indicate that LDHB modulates NHE1 activity via physical interaction.

A Structured System Analysis and System Specifications for Circulation Control in a University Libraries (구조적 분석 기법을 이용한 대출 업무의 분석과 설계)

  • 유재옥
    • Journal of the Korean Society for information Management
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    • v.9 no.2
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    • pp.118-153
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    • 1992
  • This study att.empts to conduct a system an;~lysis and dcsign for circt~lation control in a univcr~ity library. In this proccss, structured sysltcm analys15 tcrhn~clucs Iikr data flolv diagrnms. data dictiomiry, and entity - relationship analysis, arc. vmploycd to construct both concqitual and p h p c a l data models of current and new circulation systems. Thc whole design aims at an al~tomntctf c~r.culation system on thc [nlcro-compurcr --basis in a mid dle - sized library.

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Expression and purification of human mPGES-1 in E. coli and identification of inhibitory compounds from a drug-library

  • Kim, Woo-Il;Choi, Kyung-A;Do , Hyun-Soo;Yu, Yeon-Gyu
    • BMB Reports
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    • v.41 no.11
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    • pp.808-813
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    • 2008
  • Human microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane associated protein that catalyzes the conversion of prostaglandin $H_2$ ($PGH_2$) into prostaglandin $E_2$ ($PGE_2$). In this study, the expression of human mPGES-1 in E. coli was significantly enhanced by modifying the utility of specific codons and the recombinant mPGES-1 was efficiently purified to homogeneity. The $K_m$ and $V_{max}$ of the purified enzyme were determined and the trimeric state characterized by chemical cross-linking with glutaraldehyde. The purified mPGES-1 was used for the screening of a chemical library of bioactive or drug compounds to identify novel inhibitors, and oxacillin and dyphylline were identified as moderately inhibiting mPGES-1 with $I_{C50}$ values of 100 and 200 ${\mu}M$, respectively. As these compounds competitively inhibited the catalysis of $PGH_2$, their binding sites appeared to be located near the $PGH_2$ binding pocket.

A NOVEL APPROACH TO FIND OPTIMIZED NEUTRON ENERGY GROUP STRUCTURE IN MOX THERMAL LATTICES USING SWARM INTELLIGENCE

  • Akbari, M.;Khoshahval, F.;Minuchehr, A.;Zolfaghari, A.
    • Nuclear Engineering and Technology
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    • v.45 no.7
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    • pp.951-960
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    • 2013
  • Energy group structure has a significant effect on the results of multigroup transport calculations. It is known that $UO_2-PuO_2$ (MOX) is a recently developed fuel which consumes recycled plutonium. For such fuel which contains various resonant nuclides, the selection of energy group structure is more crucial comparing to the $UO_2$ fuels. In this paper, in order to improve the accuracy of the integral results in MOX thermal lattices calculated by WIMSD-5B code, a swarm intelligence method is employed to optimize the energy group structure of WIMS library. In this process, the NJOY code system is used to generate the 69 group cross sections of WIMS code for the specified energy structure. In addition, the multiplication factor and spectral indices are compared against the results of continuous energy MCNP-4C code for evaluating the energy group structure. Calculations performed in four different types of $H_2O$ moderated $UO_2-PuO_2$ (MOX) lattices show that the optimized energy structure obtains more accurate results in comparison with the WIMS original structure.

Construction of Complementary DNA Library and cDNA Cloning for Cy Strain of Odontoglossum Ringspot Virus Genomic RNA (오돈토글로썸 윤문 바이러스 Cy계통 게놈 RNA의 cDNA 구축 및 유전자 크로닝)

  • 류기현;박원목
    • Korean Journal Plant Pathology
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    • v.10 no.3
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    • pp.228-234
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    • 1994
  • Genomic RNA was extracted from Cy strain of odontoglossum ringspot tobamovirus (ORSV-Cy) isolated from infected leaves of tobacco cv. Samsun. Size of the genomic RNA was about 6.6 kb in length. The genomic RNA was fractionated using Sephadex G-50 column chromatography into 2 fractions. They were polyadenylated at their 3'-end using E. coli poly(A) polymerase. Polyadenylated viral RNA was recovered by oligo (dT) primer adapter containing NotI restriction site and Moloney murine leukemia virus SuperScript reverse transcriptase (RNase H-). Second-strand cDNA was synthesized by using E. coli DNA ligase, E. coli DNA polymerase I and E. coli RNase H. Recombinant plasmids containing cDNAs for ORSV-Cy RNA ranged from about 800 bp to 3,000 bp. Among the selected 238 recombinants, pORCY-124 clone was the largest one covering 3'-terminal half of the viral RNA. This clone contained two restriction sites for EcoRI and XbaI and one site for AccI, AvaI, BglII, BstXI, HindIII, PstI, and TthIII 1. respectively. The clone contained partial viral replicase, a full-length movement protein and a complete coat protein genes followed by a 3' untranslated region of 414 nucleotides based on restriction mapping and nucleotide sequencing analyses. Clones pORCY-028, -068, -072, -187 and -224 were overlapped with the pORCY-124. Clones pORCY-014 and -095 covered 5' half upstream from the middle region of the viral RNA, which was estimated based on restriction mapping and partial sequence analysis. Constructed cDNA library covered more than 90% of the viral genome.

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Comparative Analysis of Korean Universities' Journal Publication Research Performance Evaluation Standards (국내대학의 학술논문 연구업적평가기준 비교 분석)

  • Lee, Hye-Kyung;Yang, Kiduk
    • Journal of Korean Library and Information Science Society
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    • v.48 no.2
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    • pp.295-322
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    • 2017
  • As a first step in developing a more effective and robust approach to faculty research performance assessment, this study analyzed the existing faculty research assessment methods by comparing Korean universities' research evaluation standards, National Research Foundation of Korea's (KRF) standard for assessing research proposals, and various bibliometric measures. The study data included research evaluation standards of KRF along with 27 Korean universities with Library and Information Science (LIS) department, and the publication data of 183 LIS faculties in Korean universities from 2001 to 2015, which consisted of 3,863 papers 16,978 citations. After the comparative analysis of research evaluation standards, the study examined the rankings of institutions and faculty produced by applying the collected standards and bibliometric metrics to the publication data. The study showed that Korean university standards for research evaluation, which aim to consider both productivity and impact of faculty research, differ from similar bibliometric measures such as h-index, and the difference could significantly impact the assessment of faculty research performance.

Facile Synthesis of 3-Thioxo-3H-benzo[f]chromen-2-yl methanone and 3H-Benzo[f]chromene-3-one Under Solvent Free Condition

  • Singh, Okram Mukherjee;Devi, Nepram Sushuma;Devi, Laishram Ronibala;Lim, Ki-Bum;Yoon, Yong-Jin;Lee, Sang-Geyong
    • Bulletin of the Korean Chemical Society
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    • v.32 no.1
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    • pp.175-178
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    • 2011
  • A facile, convenient, efficient and high yielding synthesis of a combinatorial library of coumarins has been developed by the condensation of readily available $\beta$-oxodithioesters and S,S-acetal with 2-hydroxy-1-naphthaldehyde in the presence of catalytic amount of $CuCl_2$ under solvent free conditions.