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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Korean Society for Biochemistry and Molecular Biology
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Volume & Issues
Volume 34, Issue 6 - Nov 2001
Volume 34, Issue 5 - Sep 2001
Volume 34, Issue 4 - Jul 2001
Volume 34, Issue 3 - May 2001
Volume 34, Issue 2 - Mar 2001
Volume 34, Issue 1 - Jan 2001
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Recognition of DNA Damage in Mammals
Lee, Suk-Hee ;
BMB Reports , volume 34, issue 6, 2001, Pages 489~495
DNA damage by UV and environmental agents are the major cause of genomic instability that needs to be repaired, otherwise it give rise to cancer. Accordingly, mammalian cells operate several DNA repair pathways that are not only responsible for identifying various types of DNA damage but also involved in removing DNA damage. In mammals, nucleotide excision repair (NER) machinery is responsible for most, if not all, of the bulky adducts caused by UV and chemical agents. Although most of the proteins involved in NER pathway have been identified, only recently have we begun to gain some insight into the mechanism by which proteins recognize damaged DNA. Binding of Xeroderma pigmentosum group C protein (XPC)-hHR23B complex to damaged DNA is the initial damage recognition step in NER, which leads to the recruitment of XPA and RPA to form a damage recognition complex. Formation of damage recognition complex not only stabilizes low affinity binding of XPA to the damaged DNA, but also induces structural distortion, both of which are likely necessary for the recruitment of TFIIH and two structure-specific endonucleases for dual incision.
Regulation of Chlorophyll-Protein Complex Formation and Assembly in Wheat Thylakoid Membrane
Guseinova, I.M. ; Suleimanov, S.Y. ; Aliev, J.A. ;
BMB Reports , volume 34, issue 6, 2001, Pages 496~501
Lincomycin, an inhibitor of plastid protein synthesis, was found to block the synthesis of apoprotein P700 with a molecular mass of 72 kDa and the assembly of the Chl a-protein of PS I. Synthesis of the polypeptides of 48, 43.5, and 32 kDa of the PS II complex is also suppressed. This process is accompanied by the disappearance of the PS Two reaction center Chl a at 683 nm, and of the PS One reaction center Chl a at 690, 696, and 705 nm on the fourth derivative of the absorption spectra at 77K. Lincomycin does not affect the synthesis of LHC subunits. It increases the content of the two main Chl forms of LHC at 648 nm (Chl b) and 676 nm (Chl a). The low-temperature fluorescence ratio F736/F685 is also increased. However, the effect of cycloheximide (an inhibitor of cytoplasmic protein synthesis) leads to the reduction of polypeptides of the light-harvesting Chl a/b-protein complex in the range of 29.5-22 kDa. Under these conditions, the relative amount of Chl b and the F736/ F685 fluorescence ratio decrease significantly. This is obviously the result of blocking the LHC I and LHC II synthesis. At the same time rifampicin and actinomycin D (inhibitors which block transcription in chloroplast and nuclear genome, respectively) inessentially affect the characteristics of these complexes.
A Rapid and Simple Method for Construction and Expression of a Synthetic Human Growth Hormone Gene in Escherichia coli
Roytrakul, Sittiruk ; Eurwilaichitr, Lily ; Suprasongsin, Chittiwat ; Panyim, Sakol ;
BMB Reports , volume 34, issue 6, 2001, Pages 502~508
A cDNA, encoding the human growth hormone (hGH), was synthesized based on the known 191 amino acid sequence. Its codon usage was optimized for a high level expression in Escherichia coli. Unique restriction sites were incorporated throughout the gene to facilitate mutagenesis in further studies. To minimize an initiation translation problem, a 624-bp cassette that contained a ribosome binding site and a start codon were fused to the hGH-coding sequence that was flanked between the EcoRI and HindIII sites. The whole fragment was synthesized by an overlapped extension of eight long synthetic oligonucleotides. The four-short duplexes of DNA, which were first formed by annealing and filling-in with a Klenow fragment, were assembled to form a complete hGH gene. The hGH was cloned and expressed successfully using a pET17b plasmid that contained the T7 promoter. Recombinant hGH yielded as much as 20% of the total cellular proteins. However, the majority of the protein was in the form of insoluble inclusion bodies. N-terminal amino acid sequencing also showed that the hGH produced in E. coli contained formyl-methionine. This study provides a useful model for synthesis of the gene of interest and production of recombinant proteins in E. coli.
Purification and Characterisation of a Burkholderia pseudomallei Protease Expressed in Recombinant E. coli
Ling, Jessmi M.L. ; Nathan, Sheila ; Hin, Lee Kok ; Mohamed, Rahmah ;
BMB Reports , volume 34, issue 6, 2001, Pages 509~516
A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and
, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.
Characterization of Protein Kinases Activated during Treatment of Cells with Okadaic Acid
Bogoyevitch, Marie A. ; Thien, Marilyn ; Ng, Dominic C.H. ;
BMB Reports , volume 34, issue 6, 2001, Pages 517~525
Six renaturable protein kinases that utilize the myelin basic protein (MBP) as a substrate were activated during prolonged exposure of cardiac myocytes to okadaic acid (OA). We characterized the substrate preference and activation of these kinases, with particular emphasis on 3 novel kinases-MBPK-55, MBPK-62 and MBPK-87. The transcription factors c-Jun, Elk, ATF2, and c-Fos that are used to assess mitogen-activated protein kinase activation were all poor substrates for these three kinases. MAPKAPK2 was also not phosphorylated. In contrast, Histone IIIS was phosphorylated by MBPK-55 and MBPK-62. These protein kinases were activated in cultured cardiac fibroblasts, H9c2 cardiac myoblasts, and Cos cells. High concentrations (0.5 to
) of OA were essential for the activation of the protein kinases in all of the cell types examined, whereas calyculin A [an inhibitor of protein phosphatase 1 (PP1) and PP2A], cyclosporin A (a PP2B inhibitor), and an inactive OA analog all failed to activate these kinases. The high dose of okadaic acid that is required for kinase activation was also required for phosphatase inhibition, as assessed by immunoblotting whole cell lysates with anti-phosphothreonine antibodies. A variety of chemical inhibitors, including PD98059 (MEK-specific), genistein (tyrosine kinase-specific) and Bisindolylmaleimide I (protein kinase C-specific), failed to inhibit the OA activation of these kinases. Thus, MBPK-55 and MBPK-62 are also Histone IIIS kinases that are widely expressed and specifically activated upon exposure to high OA concentrations.
Identification of the Interaction between Rat Translationally Controlled Tumor Protein/IgE-dependent Histamine Releasing Factor and Myosin Light Chain
Kim, Min-Jeong ; Jung, Jae-Hoon ; Choi, Eung-Chil ; Park, Hae-Young ; Lee, Kyung-Lim ;
BMB Reports , volume 34, issue 6, 2001, Pages 526~530
The translationally controlled tumor protein (TCTP), also known as the IgE-dependent histamine releasing factor (HRF), was used in the yeast two-hybrid system to screen the interacting molecules. We obtained the N-terminus truncated rat fast myosin alkai light chain from the rat skeletal muscle cDNA library in the screening. Since either TCTP/HRF or the myosin light chain is known to be associated with histamine secretion from RBL-2H3 cells, we investigated the possible interaction between rat TCTP/HRF and nonmuscle myosin light chain in these cells. We used affinity chromatography and coimmunoprecipitation. Our data suggests that HRF and the myosin light chain interact, which may play an important role in histamine release in RBL-2H3 cells.
Comparison of Three Substrates (Casein, Fibrin, and Gelatin) in Zymographic Gel
Choi, Nack-Shick ; Yoon, Kab-Seog ; Lee, Jin-Young ; Han, Kyoung-Yoen ; Kim, Seung-Ho ;
BMB Reports , volume 34, issue 6, 2001, Pages 531~536
Three zymographic techniques using casein, fibrin, and gelatin as substrates in SDS-PAGE were compared based on three aspects: (1) The proteolytic pattern of extracellular enzymes from the three bacterial strains, Bacillus sp. DJ-1, DJ-2, and DJ-3. (2) The enzymatic sensitivity of their activity on zymogram gels. (3) The stability of stained zymogram gels with Coomassie brilliant blue in the destaining solution. There was no significant difference on the pattern of extracellular enzymes from the three strains. The bands in the fibrin gel were clearer and more distinct from the extensive destaining process. It was also shown that the gelatin gel revealed the highest enzymatic sensitivity among the three gels, based on the densitometric analysis. In the casein gel, a trace that could be mistaken as a proteolytic band appeared around 40-50 kDa.
Development of ELISA System for Screening of Specific Binding Inhibitors for Src Homology (SH)2 Domain and Phosphotyrosine Interactions
Lee, Sang-Seop ; Lee, Kyung-Im ; Yoo, Ji-Yun ; Jeong, Moon-Jin ; Park, Young-Mee ; Kwon, Byoung-Mog ; Bae, Yun-Soo ; Han, Mi-Young ;
BMB Reports , volume 34, issue 6, 2001, Pages 537~543
In the present study, an in vitro ELISA system to assess the interaction between Src homology (SH)2 domains and phosphotyrosine that contain peptides was established using purified GST-conjugated SH2 proteins and synthetic biotinylated phosphotyrosine that contain oligopeptides. The SH2 domains bound the relevant phosphopeptides that were immobilized in the streptavidin-coated microtiter plate in a highly specific and dose-dependent manner. The epidermal growth factor receptor (EGFR)-, T antigen (T Ag)-, and platelet-derived growth factor receptor (PDGFR)-derived phosphopeptides interacted with the growth factor receptor binding protein (Grb)2/SH2, Lck/SH2, and phosphatidyl inositol 3-kinase (PI3K) p85/SH2, respectively. No cross-reactions were observed. Competitive inhibition experiments showed that a short phosphopeptide of only four amino acids was long enough to determine the binding specificity. Optimal concentrations of the GST-SH2 fusion protein and phosphopeptide in this new ELISA system for screening the binding blockers were chosen at 2nM and 500nM, respectively. When two candidate compounds were tested in our ELISA system, they specifically inhibited the Lck/SH2 and/or p85/SH2 binding to the relevant phosphopeptides. Our results indicate that this ELISA system could be used as an easy screening method for the discovery of specific binding blockers of protein-protein interactions via SH2 domains.
Role of Oxidative Stress in the Radiation-Induced Lung Pathogenesis in Mice
Park, Eun-Mi ; Park, Ji-Sun ; Kim, Yun-Jeong ; Sung, Jae-Suk ; Hwamg, Tea-Sook ; Kim, Woo-Chul ; Han, Mi-Young ; Park, Young-Mee ;
BMB Reports , volume 34, issue 6, 2001, Pages 544~550
In pre-transplant total-body irradiation (TBI), the lung is a critical dose-limiting organ. Also, the possible role of oxidative stress was suggested in the development of TBI-induced lung damage. This study explores the association between TBI-induced oxidative stress and the induction of lung pathogenesis by investigating TBI-induced oxidative stress in the lungs of male C57BL/6 mice after a single dose of 10 Gy TBI. We showed significant increases of reactive oxygen species (ROS) formation and lipid peroxidation, and also a depletion and oxidation of glutathione after TBI. There is evidence that pretreatment with 1,10-phenanthroline (o-phen) significantly reduces oxidative stress in the lung. This indicates that the TBI-induced ROS generation involves a metal-catalyzed Fenton-type reaction. A pretreatment of buthionine sulfoximine (BSO) augmented the glutathione depletion and oxidation, but had no effect on the ROS formation and lipid peroxidation up to 6 h after TBI. Histopathological features that are consistent with pneumonitis were observed in the BSO pretreated-mice 1 week after irradiation. The results suggest that TBI-induced oxidative stress in the lung involves a generation of ROS through a Fenton-type reaction. Also, glutathione plays an important inhibitory role in the radiation-induced lung pathogenesis by participating in the self-amplifying cascade subsequent to the ROS generation by irradiation.
Fluorescence Resonance Energy Transfer in Calf Thymus DNA from a Long-Lifetime Metal-Ligand Complex to Nile Blue
Kang, Jung-Sook ; Lakowicz, Josepb R. ;
BMB Reports , volume 34, issue 6, 2001, Pages 551~558
We extended the measurable time scale of DNA dynamics to submicrosecond using a long-lifetime metal-ligand complex,
(phen=1,10-phenanthroline, dppz=dipyrido[3,2-a:2',3'-c]phenazine) (RuPD), which displays a mean lifetime near 350 ns. We partially characterized the fluorescence resonance energy transfer (FRET) in calf thymus DNA from RuPD to nile blue (NB) using frequency-domain fluorometry with a high-intensity, blue light-emitting diode (LED) as the modulated light source. There was a significant overlap of the emission spectrum of the donor RuPD with the absorption spectrum of the acceptor NB. The F
rster distance (
) that was calculated from the spectral overlap was
. We observed dramatic decreases in the steady-state fluorescence intensities of RuPD when the NB concentration was increased. The intensity decays of RuPD were matched the closest by a triple exponential decay. The mean decay time of RuPD in the absence of the acceptor NB was 350.7 ns. In a concentration-dependent manner, RuPD showed rapid intensity decay times upon adding NB. The mean decay time decreased to 184.6 ns at
NB. The FRET efficiency values that are calculated from the mean decay times increased from 0.107 at
NB to 0.474 at
NB concentration. The use of FRET with a long-lifetime metal-ligand complex donor is expected to offer the opportunity to increase the information about the structure and dynamics of nucleic acids.
Cloning, Expression, and Characterization of Protein Carboxyl O-methyltransferase from Porcine Brain
Koh, Eun-Jin ; Shim, Ki-Shuk ; Kim, Hyun-Kyu ; Park, Ki-Moon ; Lee, Suk-Chan ; Kim, Jung-Dong ; Yoo, Sun-Dong ; Chi, Sang-Chul ; Hong, Sung-Youl ;
BMB Reports , volume 34, issue 6, 2001, Pages 559~565
Protein carboxyl O-methyltransferase (E.C.22.214.171.124) may play a role in the repair of aged protein that is spontaneously incorporated with isoaspartyl residues. The porcine brain carboxyl O-methyltransferase was cloned in the pET32 vector, and overexpressed in E.coh (BL21) that harbors pETPCMT, which encodes 227 amino acids, including tagging proteins at the N-terminus. The protein sequence of the cloned porcine brain PCMT (r-pbPCMT) shares a 98% identity with that of human erythrocyte PCMT and rat brain PCMT. It is 100% identical with that of bovine brain. The r-pbPCMT was purified using Ni-NTA affinity chromatography and digested by enterokinase in order to remove the protein tags. Then Superdex 75HR gel filtration chromatography was performed. The r-pbPCMT exhibited similar in vitro substrate specificities with the PCMT that was purified from porcine brain. The molecular weight of the enzyme was estimated to be 24.5 kDa on the SDS polyacrylamide gel electrophoresis. The
for S-adenosyl-L-methionine. S-adnosyl-L-homocysteine was a competitive type of inhibitor with the
. The enzyme has optimal activity at pH 6.0 and
. These results indicate that the expressed enzyme is functionally similar to the natural protein. It also suggests that it may be a suitable model to further understand the function of the mammalian enzyme.
Defective Interfering HIV-1 Pseudotypes Carrying Chimeric CD4 Protein
Park, Seung-Won ; Ye, Zhiping ; Schubert, Manfred ; Paik, Soon-Young ;
BMB Reports , volume 34, issue 6, 2001, Pages 566~572
Chimeric CD4 proteins were assembled. They contained the entire CD4 ectodomain that is linked to different membrane anchors. Membrane anchors consisted of either glucosyl phosphatidyl inositol (gpi), the transmembrane and cytoplasmic regions of HIV-1 Env protein, or the vesicular stomatitis virus G glycoprotein, respectively. The HIV-1 co-receptor CXCR4 and CD4 were independently inserted into viral envelopes. We compared the insertion of six different CD4/CXCR4 constructs into HIV-1 envelopes, as well as their functionality in targeting and specific infection of cells that constitutively express the HIV-1 Env protein. All of the six different HIV-1 (CD4/CXCR4) pseudotypes were able to transduce Env (+) cells at similar efficiency. In addition, stable transduction of the Env (+) recipient cells demonstrated that all chimeric proteins were functional as receptors for Env when inserted into HIV-1 envelopes. In fact, these results demonstrate for the first time a stable transduction by a targeted HIV-1 pseudotype virus.
Characterization of the Ryanodine Receptor and SERCA in Fetal, Neonatal, and Adult Rat Hearts
Ramesh, Venkat ; Kresch, Mitchell J. ; Park, Woo-Jin ; Kim, Do-Han ;
BMB Reports , volume 34, issue 6, 2001, Pages 573~577
The mammalian heart is known to undergo significant mechanical changes during fetal and neonatal development. The objective of this study was to define the ontogeny of the ryanodine receptor/
release channel and SERCA that play the major roles in excitation-contraction coupling. Whole ventricular homogenates of fetal (F) (19 and 22 days in gestation), postnatal (N) (1 and 7 days postnatal), and adult (A) (5 weeks postnatal) Sprague-Dawley rat hearts were used to study [
]ryanodine binding and oxalate-supported
uptake. For the ryanodine receptor, the major findings were: (1) The ryanodine receptor density, as determined by maximal [
]ryanodine binding (
), increased 3 fold between the F22 and A periods (
pmoles/mg protein, p<0.01), whereas there was no significant change during the F22 and N1 development phases (
). (2) Affinity of the ryanodine receptor to ryanodine did not significantly change, as suggested by the lack of change in the
during the development and maturation. For SERCA, changes started early with an increased rate of
uptake in the fetal periods (F19:
nmoles/g protein/min; p<0.05) and peaked by 7 days (N7) of the postnatal age (
). Thus, we conclude that the quantitative changes occur in the ryanodine receptor during myocardial development. Also, the maturation of the
uptake appears to start earlier than that of the
Cross-talk between STAT6 and Ras/MAPK Pathway for the IL-4-mediated T Cell Survival
So, Eui-Young ; Jang, Ji-Young ; Lee, Choong-Eun ;
BMB Reports , volume 34, issue 6, 2001, Pages 578~583
As a prototypic Thl vs Th2 cytokine, IFN-
and IL-4 activate distinct STAT proteins, STAT1 and STATE, respectively. In cytokine-producing Jurkat T cells, IL-4 is effectively rescued from cell death that is induced by dexamethasone, but IFN-
failed to do so. Since the Ras/MAPK pathway is known to play an important role in cytokine-induced cell survival, we investigated the mechanism of T cell survival through the analysis of functional cross-talk between Ras/MAPK and distinct STAT proteins that are activated by IL-4 and IFN-
. Although IL-4 and IFN-
each induced the activation of STATE and STATI. in Jurkat T cells, respectively, only IL-4 was capable of inducing MAPK. Along with tyrosine kinase inhibitors, MEK/MAPK inhibitors also caused a significant suppression of the IL-4-induced STATE activity. This suggests a positive regulation of STATE by MAPK during IL-4 signal transduction. Furthermore, transfection studies with dominant active (da) vs dominant negative (dn) Ras revealed that daRas, but not dnRas, selectively up-regulated the expression and activity of STATE with a concomitant increase in MAPK activity. These results, therefore, suggest that there is a functional cross-talk between the Ras/MAPK and Jak/STAT6 pathways, which may have a role in the IL-4-induced T cell survival.
Characterization of the Small Cryptic Plasmid, pGD2, of Klebsiellia sp. KCL-2.
Yoo, Ju-Soon ; Kim, Hae-Sun ; Chung, Soo-Yeol ; Lee, Young-Choon ; Cho, Young-Soo ; Choi, Yong-Lark ;
BMB Reports , volume 34, issue 6, 2001, Pages 584~589
One of the cryptic plasmids from the oil degrading bacterium Klebsiella sp. KCL-2, the small plasmid pGD2, has been identified and characterized. This plasmid has a size of 3.6 kb with unknown functions. We constructed the recombinant plasmid pMGD2. The nucleotide sequences of the plasmid were determined and two open reading frames were detected. ORF1 encodes a replication initiator protein (RepA), which has a high degree of homology with the protein of ColE2 plasmid. The product encoded by ORF2 showed a high similarity with the transposase protein of IS5. IS5 is 1195 by long and contains an inverted terminal repetition of 16 bp with one mismatch. Stem-loop structures in the 5'untranslated region of the repA suggest that a putative gene, incA, is located in a complementary strand to the leader region of the repA mRNA.