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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 32, Issue 6 - Nov 1999
Volume 32, Issue 5 - Sep 1999
Volume 32, Issue 4 - Jul 1999
Volume 32, Issue 3 - May 1999
Volume 32, Issue 2 - Mar 1999
Volume 32, Issue 1 - Jan 1999
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Structure and Function of the Developmental Signaling Molecule Hedgehog
Leahy, Daniel J. ;
BMB Reports , volume 32, issue 2, 1999, Pages 103~111
Hh proteins represent a new signaling paradigm in metazoan development. In species ranging from fruit flies to humans, Hh proteins mediate multiple processes vital to appropriate pattern formation in the developing embryo. Hh proteins undergo an autoprocessing event in which the full-length protein is cleaved into N-terminal and C-terminal domains (Hh-N and Hh-C, respectively), and a cholesterol moiety becomes covalently attached to Hh-N. All known signaling activities of Hh proteins are mediated by Hh-N while both the cleavage and cholesterol transfer reactions are mediated by Hh-C. The cholesterol attached to Hh-N is required to retrict the range of Hh signaling and may be involved in ensuring appropriate reception of the Hh signal in target tissues. Disruptions of Hh signaling pathways lead to severe developmental defects in newborns and cancers in adults. While studies of Hh proteins have yielded a wealth of new insight into the molecular mechanisms of metazoan development, many outstanding questions concerning Hh signaling mechanisms ensure that unraveling the secrets of this molecule will keep scientists well entertained for the foreseeable future.
Hypoxic Microenvironmental Control of Stress Protein and Erythropoietin Gene Expression
Beak, Sun-Hee ; Han, Mi-Young ; Lee, Seung-Hoon ; Choi, Eun-Mi ; Park, Young-Mee ;
BMB Reports , volume 32, issue 2, 1999, Pages 112~118
The presence of hypoxic cells in solid tumors has long been considered a problem in cancer treatment such as in radiation therapy or treatment with some anticancer drugs. It has been suggested that hypoxic cells are involved in the development of a more aggressive phenotype and contribute to metastasis. In this study, as an attempt to understand how tumor cells adapt to hypoxic stress, we investigated the regulation of the hypoxia-induced expression of proteins that control essential processes of tumor cell survival and angiogenesis. We first examined whether hypoxia induces stress protein gene expression of murine solid tumor RIF cells. We also examined hypoxia-induced changes in angiogenic gene expression in these cells. Finally, we investigated the association of the elevated levels of stress proteins with the regulation of hypoxia-induced angiogenic gene expression. Results demonstrated that hypoxia induced the expression of the erythropoietin (EPO) gene and at least two major members of stress proteins, heat shock protein 70 (HSP70) and 25 (HSP25) in RIF tumor cells. Evidence that the expression of EPO gene was greatly potentiated in TR cells suggested that the elevated levels of HSPs may play an important role in the regulation of the hypoxia-induced EPO gene expression. One of the RIF variant cell lines, TR, displays elevated levels of HSPs constitutively. Taken together, our results suggest that a hypoxic tumor microenvironment may promote the survival and malignant progression of the tumor cells by temporarily increasing the level of stress proteins and expressing angiogenic genes. We suspect that stress proteins may be associated with the increase of the angiogenic potential of tumor cells under hypoxia.
The SH3 Domain of Phospholipase C-
Associates with Shc
Kim, Myung-Jong ; Hwang, Jong-Ik ; Chang, Jong-Soo ; Ryu, Sung-Ho ; Suh, Pann-Ghill ;
BMB Reports , volume 32, issue 2, 1999, Pages 119~126
The SH3 domain of PLC-
has been known to induce DNA synthesis. However, little is known about the putative effector proteins that associate with the domain. In this report, we provide evidence that the SH3 domain of PLC-
associates with Shc, which has been implicated in the activation of p21Ras in response to many growth factors. The association between Shc and PLC-
is enhanced either by v-Src-induced transformation or EGF-stimulation in vivo and in vitro. Furthermore, from transient expression studies with COS-7 cells, we show that the SH3 domain of PLC-
is required for association with Shc in vivo, whereas tyrosyl phosphorylation of PLC-
is not. Taken together, we suggest that Shc might be involved in the PLC-
-mediated signaling pathway.
Biochemical Properties of NAD(P)H-Quinone Oxidoreductase from Saccharomyces cerevisiae
Kim, Kyung-Soon ; Suk, Hee-Won ;
BMB Reports , volume 32, issue 2, 1999, Pages 127~132
The NAD(P)H-quinone oxidoreductase (EC 1. 6. 99. 2) was purified from S. cerevisiae. The native molecular weight of the enzyme is approximately 111 kDa and is composed of five identical subunits with molecular weights of 22 kDa each. The optimum pH of the enzyme is pH 6.0 with 1,4-benzoquinone as a substrate. The apparent
for 1,4-benzoquinone and 1,4- naphthoquinone are 1.3 mM and
, respectively. Its activity is greatly inhibited by
ions, nitrofurantoin, dicumarol, and Cibacron blue 3GA. The purified NAD(P)H-quinone oxidoreductase was found capable of reducing aromatic nitroso compounds as well as a variety of quinones, and can utilize either NADH or NADPH as a source of reducing equivalents. The nitroso reductase activity of the purified NAD(P)H-quinone oxidoreductase is strongly inhibited by dicumarol.
A Second Thioltransferase from Chinese Cabbage: Purification and Characterization
Cho, Young-Wook ; Park, Eun-Hee ; Lim, Chang-Jin ;
BMB Reports , volume 32, issue 2, 1999, Pages 133~139
Thioltransferase, also known as glutaredoxin, was previously purified and characterized from Chinese cabbage (Brassica campestris ssp. napus var. pekinensis). However, in the process of gel filtration on Sephadex G-75, there were two activity peaks. In this study, a second thioltransferase (TTase CC-2) in the minor peak of the Sephadex G-75 elution profile was further purified using affinity chromatography on an S-hexylglutathione-agarose column by eluting with buffer solution containing 2.5 mM S-hexylglutathione. It showed a single band on SDS-PAGE indicating that TTase CC-2 is electrophoretically homogeneous. The molecular weight of TTase CC-2 was estimated to be about 22,000 daltons, and its isoelectric point was determined to be 6.73. Its size appears to be atypical and much larger than that of the first thioltransferase (TTase CC-1) from Chinese cabbage, and it can utilize 2-hydroxyethyl disulfide, S-sulfocysteine, and insulin as substrates. S-sulfocysteine was found to be a superior substrate for TTase CC-2. TTase CC-2 also displayed the reducing activity for non-disulfides such as dehydroascorbic acid. Its optimum pH was 8.5, which was consistent with that of TTase CC-1. TTase CC-2 activity was greatly activated by L-cysteine and reduced glutathione, and was found to be less heat-stable compared with TTase CC-1. Molecular and physiological differences between TTase CC-1 and TTase CC-2 remain to be elucidated. Chinese cabbage is the first plant which is known to contain two kinds of thioltransferases.
Comparison of TNF-Mediated Glucose Catabolism between the TNF-Sensitive and -Resistant Cell Lines
Kim, Yeon-Hyang ; Park, Bok-Ryun ; Cheong, Hee-Sun ; Kwon, Oh-Hwan ; Kim, Dae-Que ; Kim, Soung-Soo ;
BMB Reports , volume 32, issue 2, 1999, Pages 140~146
When murine fibrosarcoma L929 cells, a TNF-sensitive cell line, were treated with recombinant human tumor necrosis factor-
), the activities of glycolytic regulatory enzymes and lactate dehydrogenase increased up to 100-150% compared to the control L929 cells after TNF treatment. By using various metabolic inhibitors and activators, it was found that cAMP-dependent protein kinase is responsible for the increase of activities of the glycolytic enzymes. The activities of glycolytic regulatory enzymes and lactate dehydrogenase of TNF-resistant A549 cells, a human lung carcinoma cell line, did not increase significantly compared to TNF-sensitive L929 cells upon TNF treatment. In contrast, the pyruvate carboxylase activities of A549 cells, but not L929 cells, increased up to 30~40% after TNF treatment. The data suggest that pyruvate carboxylase activity may contribute to the compensation of energy loss mediated by TNF treatment in TNF-resistant A549 cells.
Expression, Purification, and Characterization of Prothrombin Kringle 2
Rhim, Tai-Youn ; Kim, Eun-kyung ; Park, Chan-Soo ; Kim, Soung-Soo ;
BMB Reports , volume 32, issue 2, 1999, Pages 147~153
Previously, we reported that the prothrombin kringle 2 (fragment 2), induced by LPS administration into rabbit, inhibited bFGF-stimulated BCE cell growth (Lee et al., 1998). In this study, we cloned and overexpressed the kringle 2 domain of rabbit and human prothrombin as a fusion protein with the pelB leader sequence in E. coli using the T7 promoter. The fusion protein was cleaved during translocation into the peri plasmic space, and cleaved recombinant protein was readily isolated from whole cell lysate by DEAE-Sepharose and Sephacryl S-200 gel filtration chromatography. Both the recombinant rabbit and human prothrombin kringle 2 showed very similar biochemical and functional characteristics to the rabbit prothrombin kringle 2 purified from rabbit serum, in terms of abnormal electrophoretic migration and endothelial cell growth inhibitory activity.
Characteristics of Cytosolic Calcium-Independent Phospholipase
Isolated from Rat Liver
Won, Jong-Hak ; Na, Doe-Sun ; Rhee, Hae-Jin ; Park, Young-Min ;
BMB Reports , volume 32, issue 2, 1999, Pages 154~160
A calcium-independent phospholipase
) was identified from the cytosolic fraction of rat liver cells. On gel filtration chromatography, the
activity was eluted as broad peaks of 150 to 500 kDa. The enzyme was maximally active at pH 7.5, retained 75% of its original activity after heating at
for 5 h, and was inhibited by
ions, but was not affected by
ions. The enzymatic activity was increased up to 150% by 1 to 4 mM DTT and was inhibited up to 25% by 0.1 to 1 mM PMSF. The
activity had preference for the head group of phospholipids, where phosphatidylethanolamine was preferred to phosphatidylcholine. The results suggest that the
may be a novel enzyme distinct from the previously reported
Oxidative DNA Damage in Rats with Diabetes Induced by Alloxan and Streptozotocin
Lee, Young-Jin ; Park, Young-Mee ; Choi, Eun-Mi ;
BMB Reports , volume 32, issue 2, 1999, Pages 161~167
The role of oxidative stress in the initiation and the complication of diabetes was examined by monitoring blood glucose increase and oxidative DNA damage in rats treated with alloxan or streptozotocin (STZ). Oxidative DNA damage was assessed by quantitating 8-oxo-2'-deoxyguanosine (
excreted in urine and the
accumulated in pancreas DNA. Both alloxan and STZ treatments resulted in an abrupt increase in blood glucose and significant increases in urinary and pancreatic
. Pretreatment of buthionine sulfoximine (BSO), a glutathione-depleting agent, slightly potentiated the increase of blood glucose and urinary
in the alloxan- and STZ-treated rats. Furthermore, the BSO pretreatment caused significant amplification of pancreatic
increase in the rats. On the other hand, pretreatment with 1,10- phenanthroline (o-phen), a chelator of divalent cations, showed different results between alloxan- and STZ-treated rats. The o-phen pretreatment completely blocked diabetes and the increase of
by alloxan treatment, while it potentiated the increase of blood glucose and
by STZ treatment. The results demonstrate that the causative effect of alloxan on diabetes may be the generation of reactive oxygen species through a Fenton type reaction, but that of STZ may not.
Thioredoxin in the Periplasmic Space of Escherichia coli as a Physiological Electron Donor to Periplasmic Thiol Peroxidase, p20
Cha, Mee-Kyung ; Kim, Il-Han ;
BMB Reports , volume 32, issue 2, 1999, Pages 168~172
We previously reported that a novel thiol peroxidase (p20) from Escherichia coli is a distinct periplasmic peroxidase that detoxifies hydroperoxides together with glutathione or thioredoxin. Until now, there was no experimental evidence for the presence of thioredoxin (Trx) in the periplasmic space. In an attempt to confirm the physiological function of p20 as a thiol peroxidase supported by Trx in the periplasmic space, we have purified a Trx activity from the periplasmic space of Escherichia coli and identified the Trx as the same protein as the cytoplasmic Trx. The presence of Trx in the periplasmic space of Escherichia coli suggests that p20 is a unique extracellular Trx-linked thiol peroxidase.
Effects of Agmatine on Polyamine Metabolism and the Growth of Prostate Tumor Cells
Choi, Yon-Sik ; Cho, Young-Dong ;
BMB Reports , volume 32, issue 2, 1999, Pages 173~180
The effects of agmatine on the enzymes responsible for the biosynthesis of polyamines, the resultant levels of polyamines, and their effect on the growth of DU145 human prostate tumor cells were investigated. When agmatine was added to the medium, ornithine decarboxylase (ODC, EC 220.127.116.11) activity was substantially reduced, but S-adenosylmethionine decarboxylase (SAMDC, EC 18.104.22.168) activity increased markedly. These changes in ODC and SAMDC activities were the result of an induction of ODC-antizyme and a decreased turnover rate of SAMDC in the presence of agmatine. Accordingly, there was a decrease in the intracellular levels of putrescine and spermidine but an increase in the intracellular level of spermine. Cell growth was markedly inhibited by agmatine treatment and this inhibition was not recovered by the addition of putrescine or spermidine. Our results suggest that agmatine alters the intracellular amounts of polyamine in the cells, closely related to the inhibition of cell growth.
Functional Expression and Characterization of C-terminal Mutant of 4-Aminobutyrate Aminotransferase
Sung, Bo-Kyung ; Cho, Jung-Jong ; Kim, Young-Tae ;
BMB Reports , volume 32, issue 2, 1999, Pages 181~188
4-Aminobutyrate aminotransferase plays an essential role in the 4-aminobutyric acid shunt, converting 4-aminobutyrate to succinic semialdehyde. Recombinant 4-aminobutyrate aminotransferases were overexpressed as their catalytically active forms in E. coli by coproduction with thioredoxin and their solubilities were also dramatically increased. In order to study the structural and functional aspects of the C-terminal domain of brain 4-aminobutyrate aminotransferase, we have constructed a C-terminal mutant of pig brain 4-aminobutyrate aminotransferase and analyzed the functional and structural roles of C-terminal amino acids residues on the enzyme. The deletion of five amino-acid residues from C-terminus did not interfere with the kinetic parameters and functional properties of the enzyme. Also, the deletion did not affect the dimeric structure of the protein aligned along the subunit interface at neutral pH. However, the deletion of the C-terminal region of the protein changed the stability of its dimeric structure at acidic pH. The dissociation of the enzyme acidic, facilitated by the deletion of five amino acids from C-terminus, abolished the catalytic activity.
Cloning and Characterization of the psbEF Gene Encoding Cytochrome b-559 of the Panax ginseng Photosystem II Reaction Center
Lee, Won-Kyu ; Park, Dae-Sung ; Tae, Gun-Sik ;
BMB Reports , volume 32, issue 2, 1999, Pages 189~195
From the Panax ginseng chloroplast, the psbE and psbF genes, encoding the
-subunits of cytochrome b-559 of the photosystem II reaction center, respectively, were cloned and characterized. The psbE and psbF genes were composed of 252 and 117 nucleotides, respectively. The deduced amino acid sequence of the
-subunits showed 95%, 93%, and 91% homology to monocots, dicots, and liverwort, respectively, whereas the
-subunits showed approximately 98% to 95% homology to the same species. Southern blot analysis revealed that a single copy of the psbEF gene exists in the chloroplast plastid. Northern blot analysis indicated that the psbE and psbF genes are cotranscribed as a polycistron.
Evidence for the Ras-Independent Signaling Pathway Regulating Insulin-Induced DNA Synthesis
Jhun, Byung-H. ;
BMB Reports , volume 32, issue 2, 1999, Pages 196~202
The existence of the Ras-independent signal transduction pathway of insulin leading to DNA synthesis was investigated in Rat-1 fibroblasts overexpressing human insulin receptor (HIRc-B) using the single-cell microinjection technique. Microinjection of a dominant-negative mutant
protein into quiescent HIRc-B cells inhibited the DNA synthesis stimulated by insulin. Microinjection of oncogenic H-
) (0.1 mg/ml) induced DNA synthesis by 35%, whereas that of control-injected IgG was induced by 20%. When the marginal amount of oncogenic H-
protein was coinjected with a dominant-negative mutant of the H-Ras protein (
), DNA synthesis was 35% and 74% in the absence and presence of insulin, respectively. This full recovery of DNA synthesis by insulin suggests the existence of the Ras-independent pathway. The same recovery was observed in the cells coinjected with either H-
plus SH2 domain of the p85 subunit of PI3-kinase (
) or H-
plus interfering anti-Shc antibody. When co-injected with a dominant-negative H-
, the DNA synthesis induced by the Ras-independent pathway was blocked. These results indicate that the Ras-independent pathway of insulin leading to DNA synthesis exists, bypassing the p85 of PI3-kinase and Shc protein, and requires Rac1 protein.
Sulfhydryl-Related and Phenylpropanoid-Synthesizing Enzymes in Arabidopsis thaliana Leaves after Treatments with Hydrogen Peroxide, Heavy Metals, and Glyphosate
Park, Keum-Nam ; Sa, Jae-Hoon ; Lim, Chang-Jin ;
BMB Reports , volume 32, issue 2, 1999, Pages 203~209
Three-week grown Arabidopsis thaliana leaves were wounded by cutting whole leaves with a razor blade into pieces (about
) submerged in various solutions, and incubated in a growth chamber for 24 h. We measured and compared activities of several enzymes such as phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), thioredoxin, thioredoxin reductase, thioltransferase, glutathione reductase, and
-malate dehydrogenase. PAL activity was decreased in
-, and glyphosate-treated leaf slices, and could not be detected after treatment with
. TAL activity was found to be maximal in the
-treated leaf slices. Activity of thioredoxin, a small protein known as a cofactor of ribonucleotide reductase and a regulator of photosynthesis, was significantly increased in the
-treated leaf slices, while thioredoxin reductase activity was maximal in the
-treated leaf slices. Thioltransferase and glutathione reductase activities were significantly decreased in the
-treated leaf slices.
-malate dehydrogenase activity remained relatively constant after the chemical treatments. Our results strongly indicate that sulfhydryl-related and phenylpropanoid-synthesizing enzyme activities are affected by chemical treatments such as hydrogen peroxide, heavy metals, and glyphosate.
Regulation of the Expression of the Catabolic Acetolactate Synthase by Branched Chain Amino Acids in Serratia marcescens
Joo, Han-Seung ; Kim, Soung-Soo ;
BMB Reports , volume 32, issue 2, 1999, Pages 210~213
In Serratia marcescens, acetolactate produced by the catabolic acetolactate synthase (ALS) is converted into acetoin, its physiological role of which is to maintain intracellular pH homeostasis. In this study, the expression mode of catabolic ALS by aeration and branched-chain amino acids was examined by the ELISA method. The amount of catabolic ALS decreased approximately 93% under aerobic conditions. We also showed that the expression of catabolic ALS decreased approximately 34 % and 65 % in the presence of 2.5 mM and 10 mM leucine, respectively. The repression of catabolic ALS by leucine has not been reported previously. In contrast to leucine, catabolic ALS levels increased approximately 13% and 38% by treatment with 2.5 mM and 10 mM isoleucine, respectively, while valine alone did not have any significant effect on the synthesis of catabolic ALS. The amount of catabolic ALS was also reduced to approximately 32% and 45% in the presence of 10 mM Leu+Ile and Leu+Ile+Val, respectively. The regulatory mode of the Serratia catabolic ALS suggests that catabolic ALS may also have a role in supplying acetolactate as an intermediate of valine and leucine biosynthesis in addition to the maintenance of internal pH.