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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 29, Issue 6 - Nov 1996
Volume 29, Issue 5 - Sep 1996
Volume 29, Issue 4 - Jul 1996
Volume 29, Issue 3 - May 1996
Volume 29, Issue 2 - Mar 1996
Volume 29, Issue 1 - Jan 1996
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Tyrosine Hydroxylase Activity and mRNA in Rat Locus Coeruleus and Adrenals Following Chronic Ethanol Treatment and Acute Cold Stress
Lee, Yong-Kyu ; Park, Dong-Ha ;
BMB Reports , volume 29, issue 5, 1996, Pages 393~397
Sprague-Dawley male rats (150 g) were chronically treated with 5 v/v % ethanol admixed with nutritionally complete liquid diet and fed ad libitum for 3 weeks. Controls were pair fed with the isocaloric sucrose liquid diet. One half of each group was exposed to cold stress at
either for 24 h (for determination of mRNA by in situ hybridization) or for 48 h (for determination of enzyme activity). Chronic ethanol treatment (ethanol) did not affect tyrosine hydroxylase (TH) mRNA level in locus coeruleus (LC) of brain and adrenal medulla (AM) compared to controls. Cold stress showed strong increase of TH mRNA level in LC and AM compared to controls. Pretreated ethanol reduced the increased TH mRNA level by cold stress in LC and AM. Ethanol did not affect TH activity in LC and adrenal glands (adrenals). Cold stress increased TH activity in LC but not in adrenals. Pretreated ethanol did not reduce the increased TH activity by cold stress in LC but this result was not shown in adrenals. It is suggested that ethanol does not affect the message level and enzyme protein level for TH in LC and AM in normal rat. It is also hypothesized that pretreated ethanol reduces the magnitude of acute cold stress response, that is induction of TH mRNA in LC and AM, and does not reduce the increased TH enzyme protein that is also acute cold stress response in LC.
Effects of Chilling Injury in the Light on Chlorophyll Fluorescence and D1 Protein Turnover in Cucumber and Pea Leaves
Eu, Young-Jae ; Ha, Suk-Bong ; Lee, Choon-Hwan ;
BMB Reports , volume 29, issue 5, 1996, Pages 398~404
Light-chilling effects were investigated in chilling-sensitive cucumber (Cucumis sativus L. cv. Ilmichungjang) and chilling-resistant pea (Pisum sativum L. cv. Giant) leaf discs in relation to possible damage in D1 protein. In both plants, dark-chilling did not cause any noticeable changes in (Fv)m/Fm and lincomycin did not affect the decrease in (Fv)m/Fm caused by light-chilling. This result suggests that the de novo synthesis of D1 protein did not occur actively during light-chilling. In pea light-chilled for 6 h. the decreased (Fv)m/Fm was partly recovered in the dark, and almost complete recovery was observed in the light. In cucumber light-chilled for 3 h. the reduced (Fv)m/Fm decreased further for the initial 2 h recovery process in the light regardless of the treatment of lincomycin and recovered very slowly. In both plant species, the treatment of lincomycin inhibited the recovery process in the light, but did not significantly inhibit the process in the dark. In cucumber leaves pulse-labeled with
, the labeled band intensities of isolated pigment-protein complexes were almost the same during the 6 h light-chilling, but significant decreases in band intensities were observed during the 3 h recovery period. This result suggests that the irreversibly damaged D1 protein was degraded during the recovery period. However, no noticeable changes were observed in the pea leaves during the 12 h chilling and 3 h recovery period. The polyacrylamide gel electrophoresis of the pigment-protein complexes showed that the principal lesion sites of light-chilling were different from those of room temperature photoinhibition.
Effects of Transforming Growth Factor Beta on Cytoskeleton Structure and Extracellular Matrix in Mv1Lu Mink Epithelial Cells
Choi, Eui-Yul ; Lee, Kyung-Mee ; Chung, So-Young ; Nham, Sang-Uk ; Yie, Se-Won ; Chun, Gie-Taek ; Kim, Pyeung-Hyun ;
BMB Reports , volume 29, issue 5, 1996, Pages 405~410
Previous studies have shown that transforming growth factor beta (
) is a potent regulator of cell growth and differentiation. To study the effects of
on cell morphology and cytoskeleton reorganization, we conducted a survey using Mv1Lu mink lung epithelial cells with antibodies to cytoskeletal proteins and an extracellular matrix protein. While the untreated cells showed a cuboidal shape of typical epithelia, the Mv1Lu cells displayed a drastic shape change in the presence of
. This alteration was most prominent when near-confluent cells were treated with
. Since the morphology alteration is known to be accompanied by the reorganization of cytoskeletal proteins in other cell types, we investigated the intracellular distribution of the three major cytoskeletal structures: microfilaments, microtubules, and intermediate filaments. In the microfilament system,
induced new stress fiber formation, which was caused primarily by the polymerization of cytoplasmic G-actin. However,
appeared not to induce any significant changes in microtubular structures and vimentin filaments as determined by indirect fluorescence microscopy. Finally we confirmed the rapid accumulation of fibronectin by immunoblot analysis and chased the protein locations by immunofluorescence microscopy.
Expression of a Carboxy-Terminal Deletion Mutant of Recombinant Tadpole H-Chain Ferritin in Escherichia coli
Lee, Mi-Young ; Kim, Young-Taek ; Kim, Kyung-Suk ;
BMB Reports , volume 29, issue 5, 1996, Pages 411~416
In order to study the role of the protein shell in both iron uptake and iron core formation of ferritin, we constructed a deletion mutant of the ferritin gene and expressed the mutant gene in Escherichia coli, This mutant was obtained by introducing an amber mutation at position Pro-157 and a deletion of the 19 amino acid residues at the carboxy-terminus of the recombinant tadpole H-chain ferritin. The deleted amino acids correspond to E-helix forming the hydrophobic channel in the protein. E. coli harboring the plasmid pTHP157, which contains the deleted gene, was grown at
in the presence of 0.1 mM IPTG, and the induced protein appeared to be partly soluble. Nondenaturing polyacrylamide gel electrophoresis showed that the expressed mutant H-chains coassemble into holoprotein, suggesting that E-helix is not necessary for assembly of the subunits as reported for human H-chain ferritin. Its ability in iron core formation was proven in an Fe staining gel, the result disagreeing with the observation that the hydrophobic channel is necessary for iron core formation in human H-chain ferritin.
Phosphotyrosine Protein Phosphatase Activity Is Inversely Related to Metastatic Ability in Rat Prostatic Tumor Cell Subclonal Lines
Lee, Han-Soo ;
BMB Reports , volume 29, issue 5, 1996, Pages 417~422
In clonal sublines with different metastatic ability derived from Dunning rat prostate tumor, phosphoamino acid levels of cellular proteins were determined. Cell lines with high metastatic ability exhibited 5-fold higher phosphotyrosine level than did cell lines with low metastatic ability, while the contents of phosphoserine and phosphothreonine were similar among cell lines examined, All cell lines showed similar activities of protein tyrosine kinases as well as overall protein kinases. Phosphotyrosine protein phosphatase (PTPP) activities of the cells with high metastatic ability were very low, compared to those of the cells with low metastatic ability, suggesting that the different phosphotyrosine levels among the cell lines were due to the difference in PTPP activities rather than protein tyrosine kinase activities. Cellular activities of prostatic acid phosphatase (PAcP), which has been reported to possess phosphotyrosine protein phosphatase activity, were shown to be inversely related to the phosphotyrosine levels and metastatic abilities of the prostate tumor cells, These results suggest that cellular PAcP activity, regulating phosphotyrosine levels of cellular proteins, is closely connected with the metastatic process in prostate tumor cells and can be utilized as a good biochemical marker for the diagnosis of metastasis of prostate tumor.
Structural Characterization of a Flavonoid Compound Scavenging Superoxide Anion Radical Isolated from Capsella bursa-pastoris
Kweon, Mee-Hyang ; Kwak, Jae-Hyock ; Ra, Kyung-Soo ; Sung, Ha-Chin ; Yang, Han-Chul ;
BMB Reports , volume 29, issue 5, 1996, Pages 423~428
A superoxide anion radical scavenger isolated from Capsella bursa-pastoris was characterized by infrared (IR) spectroscopy, sugar analysis, ultraviolet (UV) spectroscopy,
nuclear magnetic resonance (NMR) spectroscopies, and fast atom bombardment (FAB) mass analysis. The compound was assumed to be a flavonoid-O-glycoside from IR spectrum and UV absorption maxima. When the sugar composition of the compound was examined by thin layer chromatography (TLC) and gas chromatography (GC) of the acid hydrolysate, only glucose was detected. According to the results of UV spectrotroscopy by using shift reagents, the compound was supposed to be luteolin (5,7,3',4'-tetrahydroxy flavone) or chrysoeriol (5,7,4'-trihydroxy-3'-methoxy flavone) with glucose. Based on
spectroscopies, the compound was deduced as 7,4'-dihydroxy-5,3'-dimethoxy-
-2"-o-glucosyl flavone. In FAB mass analysis the compound was finally characterized as 7,4'-dihydroxy-5,3'-dimethoxy-
-2"-o-glucosyl flavone (
Structure-Antagonistic Activity Relationships of an NK-2 Tachykinin Receptor Antagonist, L-659,877 and Its Analogues
Ha, Jong-Myung ; Shin, Song-Yub ; Hong, Hea-Nam ; Suh, Duk-Joon ; Jang, Tae-Sik ; Kang, Shin-Won ; Kuean, Sun-Jin ; Ha, Bae-Jin ;
BMB Reports , volume 29, issue 5, 1996, Pages 429~435
To investigate the structure-antagonistic relationship of the cyclohexapeptide L-659,877, a selective NK-2 tachykinin receptor antagonist, seven analogues were chemically synthesized by a solid phase method. The agonistic and antagonistic activities of the analogues were evaluated by contraction assay using the smooth muscle of guinea pig trachea (GPT) containing the NK-2 receptor. It was shown that the aromatic ring of Phe at position 3 and the sulfur group of Met at position 6 in L-659,877 were essential for binding to the NK-2 receptor. Decrease in antagonistic activity of L-659,877 caused by substituting Leu for Nle at position 5 indicates that the
methyl group and side chain length of Leu plays an important role in its antagonistic action. Although the activity was slightly lower than L-659,877, cyclo
(analogue 1) showed potential antagonistic activity for the NK-2 receptor. It was confirmed that the expansion of the ring in L-659,877 by substitution of
for Gly at position 4 stabilized its conformation monitored by CD spectra. The results suggest that analogue 1 can be used as a new leader compound to design a more powerful, selective, and stable NK-2 receptor antagonist.
Biochemical Characteristics of a Palmitoyl Acyl Carrier Protein Thioesterase Purified from Iris pseudoacorus
Kang, Han-Chul ; Hwang, Young-Soo ;
BMB Reports , volume 29, issue 5, 1996, Pages 436~441
The palmitoyl acyl carrier protein (ACP) specific thioesterase (EC 188.8.131.52) from Iris pseudoacorus was purified and characterized. The thioesterase which was very unstable in relatively high salt concentrations was eluted using a co-gradient of Triton X-100 and low concentration of KCl or Na-phosphate from Q-Sepharose, DEAE-Sepharose, and hydroxyapatite chromatography. SDS-PAGE analysis showed a single band with a molecular weight of 35,000. The native molecular weight of approximately 37,000 was estimated by Sephacryl S-200 chromatography, indicating that the enzyme is a monomer. The thioesterase activity was inhibited about 75% and 50% by N-ethylmaleimide (2 mM) and phenylmethylsulfonyl fluoride (2 mM). respectively. The N-ethylmaleimide-inactivation was protected by sodium palmitate but the inactivation with phenylmethylsulfonyl fluoride was not protected. Oxidation of thiols by 2 mM 5.5'-dithio-bis-(2-nitrobenzoic acid) resulted in 65% inactivation of the enzyme. These results suggest that a cysteinyl residue is essential to the catalytic reaction of the enzyme. The enzyme activity was increased by sodium citrate and also by
Rapid Detection of H-RAS Point Mutation Using Two-Step Polymerase Chain Reaction-Restriction Fragment Length Polymorphism
Park, Young-Suk ; Lee, Kyung-Ok ; Chai, Young-Gyu ;
BMB Reports , volume 29, issue 5, 1996, Pages 442~447
Mutations in codon 12, 13 and 61 of one of the three ras genes, H-ras, K-ras and N-ras, convert these genes into active oncogenes. The presence of H-ras gene mutations have important prognostic implications in various cancers. In this study, the H-ras gene mutations were investigated by two-step PCRRFLP in patients with bladder and stomach cancer. For the control experiments, T24 and SK2 cell lines were used. In a total of 36 bladder cancer patient cases, five (13.9%) mutations were found by this method. Of these, point 12 mutations were two (5.6%) cases and point 61 mutations were three (8.3%) cases. On the other hand, H-ras mutation was not found in 29 cases of stomach cancer. The results of the mutated H-ras gene confirmed by direct sequencing analysis were correlated well with PCR analysis. From the sensitivity test, the H-ras mutation was found to have about 0.2% of mutated DNA mingled in normal DNA. In conclusion, the H-ras mutation has a higher clinical Significance in bladder cancer than stomach cancer. Moreover the two-step PCR-RFLP method is sensitive, rapid and relatively simple for clinical work in detecting H-ras point mutations.
Molecular Characterization of the Region Encoding Integrative Functions from Enterococcal Bacteriophage
Kim, Min-Jung ; Lee, Jin-Young ; Kim, Young-Woo ; Sung, Ha-Chin ; Chang, Hyo-Ihl ;
BMB Reports , volume 29, issue 5, 1996, Pages 448~454
is a temperate phage which was identified as a prophage in the Enterococcus faecalis KBL703 chromosome. Phage
integrates into the host chromosome by site-specific recombination. The phage attachment site P (attP) was localized within the 0.65-kb XhoI-HindIII fragment and the nucleotide sequence of the region was determined. An open reading frame (mj1) which adjoined the phage attachment site encoded a deduced protein related to the site-specific recombinase family. The organization of this region was comparable to other site-specific recombination systems. The molecular weight of the expressed MJ1 in E. coli was in good agreement with the predicted 53,537 Da of the mj1 gene product. Elucidation of the phage-specific integration process in this study would provide useful genetic tools such as a chromosomal integration system.
Purification and Characterization of a Serine Proteinase from Acanthamoeba culbertsoni
Park, Ki-Won ; Song, Chul-Yong ;
BMB Reports , volume 29, issue 5, 1996, Pages 455~461
A serine proteinase was purified from Acanthamoeba culbertsoni by 41~80% ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was estimated to be 108.0 kDa by gel filtration chromatography and 54.0 kDa by SDS-PAGE. Therefore, the purified enzyme seemed to be a dimer. Isoelectric point was 4.5. The enzyme activity was highly inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate (OFP) and phenylmethyl sulfonylfluoride (PMSF). It had a narrow pH optimum of 6.5~7.5 with a maximum at pH 7.0. These data suggested that the purified enzyme was a neutral serine proteinase. Optimal temperature was
. It was stable for at least 16 h at
, but it was rapidly inactivated at
The activity of the purified enzyme was not influenced significantly by
. However, the enzyme activity was highly inhibited by
The enzyme degraded type I collagen and fibronectin, but not BSA, hemoglobin, lysozyme, immunoglobulin A or immunoglobulin G.
Overexpression of Nicotiana tabacum Acetolactate Synthase as an Inducible Fusion Protein in Escherichia coli: Production of a Polyclonal Antibody to Nicotiana tabacum Acetolactate Synthase
Chang, Soo-Ik ; Kang, Moon-Kyeong ; Kim, Hyun-Ju ; Choi, Jung-Do ; Namgoong, Sung-Keon ;
BMB Reports , volume 29, issue 5, 1996, Pages 462~467
Acetolactate synthase (ALS, EC 184.108.40.206) is the first common enzyme in the biosynthesis of leucine, isoleucine, and valine. It is the target enzyme for several classes of herbicides, including the sulfonylureas, the imidazolinones, the mazolopyrimidines, the pyrimidyl-oxy-benzoates, the pyrimidyl-thio-benzens, and the 4,6-dimethoxypyrimidines. An amino-terminal fragment of the sulfonylurea-resistant ALS gene (SurB) from Nicotiana tabaccum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS1 was used to transform Escherichia coli strain BL21, and the tobacco ALS was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). Polyclonal antibodies against the fusion product (GST-ALS) were produced, and the sensitivity of GST-ALS with the rabbit anti-GST-ALS IgG was up to 50 ng. This antibody was used for Western blot analysis of the partially purified ALS from barley shoots. The results suggest that the polyclonal antibody produced in this study can be used to detect plant ALS.
Analysis of the MVM P38 Promoter Distal DNA cis-Elements Responsible for Transactivation by Nonstructural Proteins
Kim, Yoo-Nha ; Ahn, Jeong-Keun ;
BMB Reports , volume 29, issue 5, 1996, Pages 468~473
The P38 promoter of minute virus of mice (MVM) is a very weak promoter which is strongly transactivated by viral nonstructural proteins. To analyze the upstream sequence of the P38 promoter which is responsible for the transactivation by nonstructural proteins in MVM, chloramphenicol acetyltransferase (CAT) reporter plasm ids containing a series of 5' deletion and internal deletion mutants of the P38 promoter were constructed. The wild type and mutant CAT constructs of P38 promoter were cotransfected into murine A92L fibroblast cells with a plasmid expressing viral nonstructural proteins by DEAE-dextran method. Each promoter activity was analyzed by CAT assay. As previously reported (Ahn et al., 1992), the proximal DNA cis-elements required for transactivation of the MVM P38 promoter are GC box and TATA box. However, the analysis of 5' deletion mutants showed that H-l tar like sequence (MVM TAR) which is located between -143 and -122 relative to the transcription initiation site is also required for transactivation of the P38 promoter by nonstructural proteins. Interestingly, even if the MVM TAR was removed by internal deletion, the level of the transactivation is still 70% of wild type level of transactivation. We also found that, in addition to the MVM TAR motif, there are two other motifs which are similar to the MVM TAR sequence. When these TAR like motifs were further deleted, the levels of transactivation were decreased further. Taken together, the MVM TAR sequence and TAR like motifs located upstream of P38 promoter are playing an important role for the transactivation of P38 promoter by nonstructural proteins in minute virus of mice.
Expression of CyI Cytoplasmic Actin Genes in Sea Urchin Development
Hahn, Jang-Hee ; Raff, Rudolf A. ;
BMB Reports , volume 29, issue 5, 1996, Pages 474~480
We present a study of evolutionary changes in expression of actin genes among closely related sea urchin species that exhibit different modes of early development. For this purpose, polyclonal antisera raised against peptides from the carboxyl terminus of the HeCyI cytoskeletal actin of Heliocidaris erythrogramma were used. H. erythrogramma is a direct developing sea urchin that proceeds from embryonic to adult stages without an intervening feeding larval stage. Expression patterns of the CyI actin isoform were compared with those of Heliocidaris tuberculata and to a related sea urchin Strongylocentrotus purpuratus, which both produce a feeding pluteus larval stage. The CyI actin of all three species is expressed in the same cell types. However, its expression patterns have been changed with reorganization of early cell lineage differentiation, which is apparent among the three species. Thus. evolutionary changes in CyI actin gene expression patterns are correlated with not only phylogenetic relationship, but developmental mode. The implication of this observation is that evolutionary changes in expression patterns of histospecific genes may underlie the emergence of novel developmental processes.
A Simple Method for Generation of Homologous Internal Standards for Competitive PCR
Choi, Eu-Na ; Hahn, Sung-Sik ; Choi, Kyung-Hee ; Na, Doe-Sun ;
BMB Reports , volume 29, issue 5, 1996, Pages 481~483
In competitive PCR, which is used to quantify target DNA an internal standard is needed. Here we present a simple method to construct homologous competitive standards. The method, which is based upon deletion of a portion of the target DNA, does not require any additional primers. This is the simplest method developed thus far to construct a competitive standard. The whole procedure. from construction of a competitive standard to quantitation by PCR, can be completed within a single day.
Expression of the Type IV Collagenase Genes and ras Oncogene in Various Human Tumor Cell Lines
Moon, A-Ree ; Park, Sang-Ho ; Lee, Sang-Hun ;
BMB Reports , volume 29, issue 5, 1996, Pages 484~487
The matrix metalloproteinases (MMPs) are members of a unique family of proteolytic enzymes that degrade components of the extracellular matrix. Significant evidence has accumulated to directly implicate members of the MMPs in tumor invasion and metastasis formation. To investigate the correlation between ras oncogene and MMP gene expression in various tumor cells, we detected mRNAs for the ras, MMP-2 and MMP-9 (72 kD and 92 kD type IV collagenases, respectively) genes in nine human tumor cell lines. The ras gene was expressed in seven cell lines; MMP-2 in three; MMP-9 in two cell lines tested. There was no direct correlation between the ras oncogene and MMP expression. A clear difference in the mRNA expression between MMP-2 and MMP-9 was observed among the cell lines. As an approach to study the effect of the ras oncogene on metastasis, we examined the expressions of MMP-2 and MMP-9 in HT1080 cells transfected with the v-H-ras gene. MMP-9 expression was Significantly enhanced in the ras-transfected HT1080 cells compared with the nontransfectants while ras transfection did not affect the expression of MMP-2. These results suggest the possible inducing effect of the ras oncogene on the metastasis by activation of the MMP-9 gene in HT1080.