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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal DOI :
Korean Society for Biochemistry and Molecular Biology
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Volume & Issues
Volume 31, Issue 6 - Nov 1998
Volume 31, Issue 5 - Sep 1998
Volume 31, Issue 4 - Jul 1998
Volume 31, Issue 3 - May 1998
Volume 31, Issue 2 - Mar 1998
Volume 31, Issue 1 - Jan 1998
Selecting the target year
Generation of Anti-HLA-DR4 Specific Antibodies by Immunization of the Recombinantly Expressed Allelic Subtype-Specific Region of the
Park, Jung-Hyun ; Cho, Eun-Wie ; Lee, Yun-Jung ; Chung, Jin ; Hahm, Kyung-Soo ; Kim, Kil-Lyong ;
BMB Reports , volume 31, issue 2, 1998, Pages 111~116
HLA-DR4 is the dominant allele of MHC class II genes in Koreans. In particular, the
subtype has been reported to be almost exclusively expressed in Far East Asians, and has also been observed to be strongly associated with rheumatoid arthritis in Koreans and the Japanese. Identification of this specific allele has been mainly performed by PCR-based methods, which is often time consuming, costly, and involves tedious procedures such as the isolation of genomic DNA, PCR, and gel electrophoresis. To develop a more convenient tool for screening vast amounts of samples as well as to generate reagents which might also be used in other applications, in this study, antibodies were produced against this specific HLA subtype. By PCR, an allelespecific region covering the
was amplified and recombinantly expressed in E.coli. Immunization of Lewis rats with the purified protein yielded an allele specific antiserum. Western blot analysis showed the selective detection of the HLA-DR
. Using this antiserum, established cell lines and peripheral blood lymphocytes were analyzed on their HLA haplotype by fluorescence activated flow cytometry. These novel antibodies will provide a powerful tool in the detection and investigation of DR4 alleles.
Transient Increase of Lipocortin 1 in Nuclei of the Hippocampal Pyramidal Neurons in Rats Induced by Immobilization Stress
Park, Hyoung-Sup ; Jang, Yeon-Jin ; Kim, Dong-Hou ; Lee, Su-Ok ; Na, Doe-Sun ;
BMB Reports , volume 31, issue 2, 1998, Pages 117~122
Changes of lipocortin 1 (LC1) in the brain induced by immobilization stress were investigated in rats. Rats were immobilized for 0,1,2,3,4, and 5 h, and the brain slices were immunostained with anti-human LC1 antibodl (anti-LC1). Immunoreactivity of LCI (iLC1) was most prominent in neuronal cell bodies and processes of hippocampal CA regions and dentate gyrus. At rest without stress, most of the LC1 in the neuron located in the cytoplasm with the nuclei exhibiting relatively scarce immunoreactivity. Immobilization stress changed this intracellular distribution of LC1 by increasing nuclear LC1. The change was apparent in 1 h and reached the peak by 3 h. However, by 5 h of immobilization, the distribution pattern returned to that of the resting state. This transient nuclear translocation of LC1 was most prominent in
pyramidal neurons, and was not observed in areas other than the hippocampus. Adrenalectomy abolished this transient translocation of LC1. The roles of hippocampal LC1 as a mediator of glucocorticoid feedback signal and/or as an intracellar stress signaling protein could be suggested.
Expression and Secretion of Foreign Proteins in Yeast Using the ADH1 Promoter and 97 K Killer Toxin Signal Sequence
Hong, Seok-Jong ; Kang, Hyen-Sam ;
BMB Reports , volume 31, issue 2, 1998, Pages 123~129
of Bacillus subtilis, preS1+S2 region of hepatitis B virus large surface antigen, human
), and bovine growth hormone (bGH) were expressed in Saccharomyces cerevisiae and secreted into the medium. These proteins were expressed using the alcohol dehydrogenase I (ADH1) promoter of Saccharomyces cerevisiae and secreted by signal sequence of the 97 K killer toxin gene of doublestranded linear DNA plasmid (pGKL1) of S. cerevisiae. All these proteins underwent severe modifications; in particular, N-glycosylation in the case of
, and preS1+S2. Seventy four percent of the expressed
was secreted into the culture medium. Highly modified proteins were detected in the culture medium and in the cell. Expressed
, which has seven transmembrane domains, remained in the cell. The degrees of secretion and modification and the states of proteins in the culture medium and in the cell were quite different. These results indicated that the nature of the protein has a critical role in its secretion and modifications.
Overproduction, Purification, and Characterization of Heat Stable Aldolase from Methanococcus jannaschii, a Hyperthermophic Archaea
Choi, In-Geol ; Cho, Chun-Seok ; Cho, Yun-Je ; Yu, Yeon-Gyu ;
BMB Reports , volume 31, issue 2, 1998, Pages 130~134
An aldolase gene has been cloned from Methanococcus jannaschii. The coding region of the gene has been expressed in E. coli using a pET system to a level of 30% of total cellular proteins. The protein was purified to more than 95 % homogeneity by heat treatment and ion exchange chromatography. The protein performed an aldol condensation reaction with glyceraldehyde as substrate and dihydroxyacetone phosphate as a carboxyl donor. The protein was determined to be a type II aldolase which requires the
ion as a metal cofactor. This enzyme has a broad range of optimum pH (7-9) and temperature (
). It shows strong stability against heat, chemical denaturants, as well as a high percentage' of organic solvents. The half-life of this enzyme at
is more than 24 h and it maintains more than 90% of aldolase activity in the presence of 6 M urea, 50% acetonitrile, or 15% isopropyl alcohol.
Purification of YPTP1 with Immobilized Phosphonomethylphenylalanine-Containing Peptide as an Affinity Ligand
Han, Jun-Pil ; Kwon, Mi-Yun ; Cho, Hyeong-Jin ;
BMB Reports , volume 31, issue 2, 1998, Pages 135~138
A previous study on a yeast protein tyrosine phosphatase, YPTP1, using synthetic phosphotyrosine-containing peptides with various sequences as substrates revealed that DADEpYDA exhibits high affinity (
) toward the enzyme. A modified version of this peptide, GDADEpmFDA, immobilized on a resin, was used in this study as an affinity ligand for the purification of YPTP1. Phosphonomethyl-phenylalanine (pmF) was used as a nonhydrolyzable analog of the phosphotyrosine (pY) residue, with properties similar to pY. A protected form of pmF,
, was chemically synthesized and introduced during solid-phase peptide sythesis. YPTP1 was onrexpressed in an E. coli strain carrying a plasmid pT7-7-ptpl. Affinity chromatography of the crude lysate afforded PTPI (39 kDa) of about 50% purity.
Chemical Modification of Serratia marcescens Catabolic
Joo, Han-Seung ; Kim, Soung-Soo ;
BMB Reports , volume 31, issue 2, 1998, Pages 139~143
synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the tryptophane-specific reagent, N -bromosuccinimide, and the arginine-specific reagent, phenylglyoxal. The enzyme was inactivated slowly by the cysteine-specific reagent N-ethylmaleimide. The second-order rate constants for the inactivation by N-bromosuccinimide, phenylglyoxal. and N -ethylmaleimide were
, respectively. The reaction order with respect to N-bromosuccinimide, phenylglyoxal, and N-ethylmaleimide were 1.5,0.71, and 0.86, respectively. The inactivation of the catabolic aacetolactate synthase by these modifying reagents was protected by pyruvate. These results suggest that essential tryptophane, arginine, and cysteine residues are located at or near the active site of the catabolic
Purification and Characterization of Catalase-2 from Deinococcus radiophilus
Oh, Kyung-A ; Lee, Young-Nam ;
BMB Reports , volume 31, issue 2, 1998, Pages 144~148
A bifunctional catalase-peroxidase, designated catalase-2, of a UV resistant Deinococcus radiophilus was purified to electrophoretic homogeneity by both chromatographic and electrophoretic methods. Its molecular weight was 310 kDa and composed of a tetramer of 80 kDa subunits. The catalase-2 exerted its optimal activity at
and around pH 9. Its
was about 10 mM. It showed the typical ferric heme spectrum with maximum absorption at 403 nm which shifted to 419 nm in the presence of cyanide. The ratio of A40i' A2S0 was 0.48. Fifty percent inhibition of the enzyme activity was observed at
M of NaCN,
, respectively. The enzyme was thermostable and not sensitive to 3-amino-1,2,4-triazole. Treatment of the enzyme with ethanol-chloroform caused a partial loss (30%) of its activity. The catalase-2 was distinct from the Deinococcal bifunctional catalase-3 in a number of properties, particularly in its molecular structure and substrate affinity.
Mode of Action on EcoRI Restriction Endonuclease: EcoRI and EcoRI Variant N199H have Active Monomeric Forms
Kim, Jae-Jong ; Koh, Suk-Hoon ; Kim, Joong-Su ; Lee, Dae-Sil ;
BMB Reports , volume 31, issue 2, 1998, Pages 149~155
The N199H variant of the EcoRI endonuclease has about twice the catalytic activity of the wild-type. A comparison of their biochemical characteristics, using synthetic oligonucleotides 5'-dAAAACTTAAGAAAAAAAAAAA-3' (KA) and 5'-dTTTTTGAATTCTTTTTTTTTT-3' (KT), helps to define the cleavage reaction pathway of these enzymes. Both EcoRI and EcoRI variant N199H were found to cleave singlestranded KA or KT about three times faster than the double-stranded forms, although the KT oligonucleotide was more susceptible. Using the ssDNA substrate in kinetic analyses, lower
values were obtained for the N199H variant than for the wild-type at low (50 mM), as well as high (200 mM), sodium chloride concentrations. This difference between the endonucleases is attributed to a grealter accessibility for tbe substrate by the variant, and also a higher affinity for the DNA backbone. It also appears that the relative activities of the two enzymes, particularly at high ionic strength, are proportional to their populations in the monomeric enzyme form. That is, according to gel filtration data, half of the N199H molecules exist as monomers in 200 mM NaCl, whereas those of the wild-type are mainly dimeric. Consequently, the Asp199 residue of the EcoRI endonuclease may be implicated in the protein-protein interaction leading to dimerization, as well as in coupling to DNA substrates. In summary, it is proposed that active monomeric endonuclease molecules, derived from the dimeric enzyme, recognize and form a complex with a single stranded form of the DNA substrate, which then undergoes nucleophilic substitution and cleavage.
Primary Culture of Bovine Capillary Endothelial Cells for In Vitro Angiogenesis Assay
Lee, Tae-Hee ; Kim, Soung-Soo ;
BMB Reports , volume 31, issue 2, 1998, Pages 156~160
In this study, we cultured bovine capillary endothelial cells from adrenal cortex and compared these cells with capillary endothelial cells obtained from bovine adrenal medulla on morphological and cytokinetic properties. We demonstrated that bFGF and gelatin matrix were required for the growth of adrenal cortex-derived capillary endothelial cells over middle passage, but not for the growth of adrenal medulla-derived capillary endothelial cells. Also, we showed that the growth of adrenal cortex-derived capillary endothelial cells must be stimulated by bFGF and the gelatin matrix for the measurement of in vitro angiostatin activity. These data indicate that adrenal cortex-derived capillary endothelial cells over middle passage are more suitable than adrenal medulla-derived capillary endothelial cells for in vitro angiogenesis assay.
Comparative Studies of Protein Modification Mediated by Fenton-like Reactions of Iron, Hematin, and Hemoglobin: Generation of Different Reactive Oxidizing Species
Kim, Young-Myeong ; Kim, Sung-Soo ; Kang, Gu ; Yoo, Yeong-Min ; Kim, Ki-Mo ; Lee, Mi-Eun ; Han, Jeong-A ; Hong, Sun-Joo ;
BMB Reports , volume 31, issue 2, 1998, Pages 161~169
TThe reactive oxygen species oxidatively modify the biological macromolecules, including proteins, lipids, and nucleic acids. Iron- and heme-mediated Fenton-like reactions produce different pro-oxidants. However, these reactive products have not been clearly characterized. We examined the nature of the oxidizing species from the different iron sources by measuring oxidative protein modification and spectroscopic study. Hemoglobin (Hb) and methemoglobin (metHb) were oxidatively modified in
generating systems. Globin and bovine serum albumin (BSA) were also modified by iron, iron-EDTA, hematin, and Hb in an
generating system. In a
generating system, the iron- and iron-EDTA-mediated protein modifications were markedly reduced while the Hb-and hematin-mediated modifications were slightly increased. In the
generating system, the iron- and iron-EDTA-mediated protein modifications were strongly inhibited by superoxide dismutase (SOD) or catalase, but heme- and Hb-mediated protein modifications were inhibited only by catalase and slightly increased by SOD. Mannitol, 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), deoxyribose, and thiourea inhibited the iron-EDTA-mediated protein modification. Mannitol and DMPO, however, did not exhibit significant inhibition in the hematin-mediated modification. Desferrioxamine (DFO) inhibited protein modification mediated by iron, but cyanide and azide did not, while the hematin-mediated protein modification was inhibited by cyanide and azide, but not significantly by DFO. The protein-modified products by iron and heme were different. ESR and UV-visible spectroscopy detected the DMPO spin adduct of the hydroxyl radical and ferryl ion generated from iron-EDTA and metHb, respectively. These results led us to conclude that the main oxidizing species are hydroxyl radical in the iron-EDTA type and the ferry I ion in the hematin type, the latter being more effective for protein modification.
Detection of Cytosolic Phosphatidylethanolamine N -Methyltransferase in Rat Brain
Kim, Young-Jun ; Park, Heung-Soon ; Choi, Myung-Un ;
BMB Reports , volume 31, issue 2, 1998, Pages 170~176
Phosphatidylethanolamine N-methyltransferase (PEMT) is known to be a membrane-associated protein. However, cytosolic PEMT was detected when sufficient amounts of exogenous phospholipids were added in the incubation media. The methylation of phospholipids was measured by the incorporation of the
group from S-adenosylmethionine and the methylated phospholipids were analyzed by thinlayer chromatography. The essence of the assay condition for the cytosolic enzyme was the inclusion of 200
of each substrate, phosphatidylethanolamine (PE), phosphatidyl N-monomethylethanolamine (PME) and phosphatidyl N,N-dimethylethanolamine (PDE), in the reaction mixture of 100
. The subcellular fractionation of brain PEMT activities revealed that approximately 38.1 % for PME, 39.5% for PDE, and 22.4% for PC formation was present in the cytosolic fraction. The general properties of cytosolic PEMT were characterized and compared with those of neuronal nuclei PEMT.
In Vitro Selection of Hammerhead Ribozymes with Optimized Stems I and III
Sim, So-Yeong ; Kim, Se-Mi ; Kim, Ha-Dong ; Ahn, Jeong-Keun ; Lee, Young-Hoon ; Cho, Bong-Rae ; Park, In-Won ;
BMB Reports , volume 31, issue 2, 1998, Pages 177~182
A pool of cis-acting hammerhead ribozymes randomized in their substrate recognition sequences was constructed. A variety of active cis-acting ribozymes which had various structures of stems I and III was selected from the pool by in vitro selection. The selected ribozymes were cloned and sequenced. The relationship between the cleavage efficiency and base-pairing in stems I and III of the selected ribozymes was investigated. The ribozymes with the smaller difference in folding energies between the active conformation and the stable but inactive conformation showed a tendency to have the better cleavage efficiency. The optimum length of stem I was 5 or 6 bases while the longer stem III, in general, appeared to be required for efficient cleavage. The specificity of the ribozyme reaction is discussed in terms of the length of stems I and III.
2-Chloroethylethyl Sulfide Induces Apoptosis and Necrosis in Thymocytes
Hur, Gyeung-Haeng ; Kim, Yun-Bae ; Shin, Sung-Ho ;
BMB Reports , volume 31, issue 2, 1998, Pages 183~188
2-chloroethylethyl sulfide (CEES) is an alkylating agent that readily reacts with a wide variety of biological molecules causing metabolic abnormality. The mechanism of cell death during CEES injury is poorly understood. We have examined the effect of exposure of thymocytes with various concentrations of CEES to determine the pattern of cell death in thymocytes injury induced by CEES. In the present study, we show that two patterns of cell death occurred by either one of two mechanisms: apoptosis and necrosis. Exposure to low level of CEES (100
) for 5 h caused an induction of apoptosis on thymocytes, as identified by the following criteria: DNA fragmentation visualized by the characteristic "ladder" pattern was observed upon agarose gel electrophoresis and morphological features were revealed by microscopical observations. In contrast, exposure to high levels of CEES (500
) induce necrotic features such as cell lysis. Thus, depending on the concentrations, CEES can result in either apoptotic or necrotic cell damage. Our findings suggest that thymocytes which are not killed directly, but merely injured by low levels of CEES, are able to activate an internally-programmed cell death mechanism, whereas thymocytes receiving severe damages apparently can not.
Enhancement of a Liver Form of Cytosolic Phospholipase
Activity by Methylmercury
Huh, Don-Haeng ; Kang, Mi-Sun ; Sohn, Dong-Hun ; Na, Doe-Sun ; Kim, Dae-Kyong ;
BMB Reports , volume 31, issue 2, 1998, Pages 189~195
Methylmercury (MeHg), which is widely distributed in the environment, is well known for both its acute and chronic poisoning effects on the human health; however, the precise biochemical mechanisms by which this compound elicits its toxicity in a cellular level are still poorly understood. To examine whether MeHg-induced liver injury involves activation of Phospholipase
activity of control and MeHg-administrated livers was measured. MeHg stably enhanced a liver form of cytosolic
activity, which exhibited several biochemical properties similar to those of the 100 kDa
, except in its elution profile of a DEAE-5PW HPLC, and it migrated as a molecular weight of 80 kDa in Western blot analysis. This blotting analysis also indicated that the MeHg-induced enhancement of the activity could be due to the increase in the amount of the enzyme protein rather than a stable modification of the enzyme such as phosphorylation. Our data also showed the higher myeloperoxidase activity in MeHg-administrated liver than in the control, suggesting that this increase in the amounts of the 80 kDa
and its activity may be resulted from infiltration of neutrophils into the liver during a hepatic injury process such as MeHg-induced inflammation. Taken together, these data suggest that MeHg-induced liver injury may be mediated by activation of the 80 kDa form of liver cytosolic
Characterization of 27K Zein as a Transmembrane Protein
Lee, Dong-Hee ;
BMB Reports , volume 31, issue 2, 1998, Pages 196~200
Zeins, maize storage proteins, are retained in the endoplasmic reticulum (ER) during the subcellular targeting process without the ER retention signal. Circumstantial data indicate that the 27K zein is an ER transmembrane protein. The potential transmembrane domain may permit the 27K zein to remain in the ER. This study investigated the potential transmembrane feature by employing alkaline extraction, proteinase K digestion, and surface biotinylation on isolated intact protein bodies. These assays consistently support the possibility of the 27K zein as a transmembrane protein. The 27K zein polypeptide was shown to be associated with alkali-stripped membranes. The polypeptide was digested by proteinase K to a smaller fragment. According to surface biotinylation, the 27K zeins was labeled to the exclusion of other classes of zeins. This study, therefore, concludes that the 27K zein has an ER transmembrane domain, which may serve as an anchor for zeins' ER retention.
Proline Analogs, L-Azetidine-2-Carboxylic Acid and 3,4-Dehydro-L-Proline, Induce Stress Response in Drosophila Kc Cells
Moon, Sung-Joon ; Han, Ching-Tack ;
BMB Reports , volume 31, issue 2, 1998, Pages 201~208
Amino acid analogs, like other inducers of stress response, induce the synthesis of stress proteins in mammalian cells. In this study, Drosophila Kc cells, in which translation is tightly controlled during stress response, was treated with proline analogs, L-azetidine-2-carboxylic acid (AzC) and 3,4-dehydro-L-proline (dh-P). Kc cells exposed to AzC or dh-P induced the synthesis of several proteins which had the same molecular weights as known heat shock proteins. However, in Kc cells, normal protein synthesis still continued in the presence of amino acids analogs unlike in heat-shocked cells. For the induction of stress response, the incorporation of dh-P into the protein was not essential, but the incorporation of AzC was. The stress protein synthesis was regulated mainly at the transcriptional level by AzC, whereas it was regulated by dh-P at the transcription level and possibly posttranscription level. During recovery, the stress protein synthesis stopped sooner in analog-treated cells than in heat-shocked cells even though the accumulated amount of Hsp70 was much less in proline analogstreated cells. It could be concluded that the proline analogs, AzC and dh-P, induced stress response through a different mechanism from heat shock.