Biomedical subjects

Eun Ju Kim

Publications and source records attributed to Eun Ju Kim.

At least 19 recordsLinked to original sources

PubMed2006

Caenorhabditis elegans ortholog of a diabetes susceptibility locus: oga-1 (O-GlcNAcase) knockout impacts O-GlcNAc cycling, metabolism, and dauer.

A dynamic cycle of O-linked N-acetylglucosamine (O-GlcNAc) addition and removal acts on nuclear pore proteins, transcription factors, and kinases to modulate cellular signaling cascades. Two highly conserved enzymes (O-GlcNAc transferase and O-GlcNAcase) catalyze the final steps in this nutrient-driven "hexosamine-signaling pathway." A single nucleotide polymorphism in the human O-GlcNAcase gene is linked to type 2 diabetes. Here, we show that Caenorhabditis elegans oga-1 encodes an active O-GlcNAcase. We also describe a knockout allele, oga-1(ok1207), that is viable and fertile yet accumulates O-GlcNAc on nuclear pores and other cellular proteins. Interfering with O-GlcNAc cycling with either oga-1(ok1207) or the O-GlcNAc transferase-null ogt-1(ok430) altered Ser- and Thr-phosphoprotein profiles and increased glycogen synthase kinase 3beta (GSK-3beta) levels. Both the oga-1(ok1207) and ogt-1(ok430) strains showed elevated stores of glycogen and trehalose, and decreased lipid storage. These striking metabolic changes prompted us to examine the insulin-like signaling pathway controlling nutrient storage, longevity, and dauer formation in the C. elegans O-GlcNAc cycling mutants. Indeed, we found that the oga-1(ok1207) knockout augmented dauer formation induced by a temperature sensitive insulin-like receptor (daf-2) mutant under conditions in which the ogt-1(ok430)-null diminished dauer formation. Our findings suggest that the enzymes of O-GlcNAc cycling "fine-tune" insulin-like signaling in response to nutrient flux. The knockout of O-GlcNAcase (oga-1) in C. elegans mimics many of the metabolic and signaling changes associated with human insulin resistance and provides a genetically amenable model of non-insulin-dependent diabetes.

Acetylglucosamine↗

PubMed2006

Enzymatic characterization of O-GlcNAcase isoforms using a fluorogenic GlcNAc substrate.

A highly sensitive fluorogenic hexosaminidase substrate, fluorescein di(N-acetyl-beta-D-glucosaminide) (FDGlcNAc), was prepared essentially as described previously [Chem. Pharm. Bull. 1993, 41, 314] with some modifications. The fluorescent analog is a substrate for a number of hexosaminidases but here we have focused on the cytoplasmic O-GlcNAcase isoforms. Kinetic analysis using purified O-GlcNAcase and its splice variant (v-O-GlcNAcase) expressed in Escherichia coli suggests that FDGlcNAc is a much more efficient substrate (Km = 84.9 microM) than the conventional substrate, para-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (pNP-beta-GlcNAc, Km = 1.1 mM) and a previously developed fluorogenic substrate, 4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside [MUGlcNAc, Km = 0.43 mM; J. Biol. Chem. 2005, 280, 25313] for O-GlcNAcase. The variant O-GlcNAcase, a protein lacking the C-terminal third of the full-length O-GlcNAcase, exhibited a Km of 2.1 mM with respect to FDGlcNAc. This shorter isoform was not previously thought to exhibit O-GlcNAcase activity based on in vitro studies with pNP-beta-GlcNAc. However, both O-GlcNAcase isoforms reduced O-GlcNAc protein levels extracted from HeLa and HT-29 cells in vitro, indicating that the splice variant is a bona fide O-GlcNAcase. Fluorescein di-N-acetyl-beta-D-galactosaminide (FDGalNAc) is not cleaved by these enzymes, consistent with previous findings that the O-GlcNAcase has substrate specificity toward O-GlcNAc but not O-GalNAc. The enzymatic activity of the shorter isoform of O-GlcNAcase was first detected by using highly sensitive fluorogenic FDGlcNAc substrate. The finding that O-GlcNAcase exists as two distinct isoforms has a number of important implications for the role of O-GlcNAcase in hexosamine signaling.

Acetylglucosaminidase↗

PubMed2006

An O-GlcNAcase-specific inhibitor and substrate engineered by the extension of the N-acetyl moiety.

A novel analogue of PUGNAc, a potent O-GlcNAcase inhibitor, was synthesized and analyzed as an inhibitor of O-GlcNAcase, hexosaminidase A, and hexosaminidase B. While PUGNAc does not demonstrate selective inhibition of these related enzymes, the extension of the acetyl moiety to the longer butyl chain provided a compound with depressed inhibition of O-GlcNAcase and no observed inhibition of either hexosaminidase A or hexosaminidase B. Further, we applied this knowledge of substrate recognition at the N-acetyl group to our recently reported fluorogenic substrate for monitoring O-GlcNAcase activity. Gratifyingly, this altered small molecule was demonstrated to be a potent substrate for O-GlcNAcase while possessing no activity at hexosaminidase A. This reagent provides, for the first time, a means for monitoring O-GlcNAcase activity independent of the related enzymes hexosaminidase A and hexosaminidase B.

Acetylglucosamine↗

PubMed2006

Critical roles of AMP-activated protein kinase in the carcinogenic metal-induced expression of VEGF and HIF-1 proteins in DU145 prostate carcinoma.

Epidemiological and experimental animal data indicate that exposure to both metals and metalloid species exacerbates the risk of human diseases, particularly cancers. Vascular endothelial growth factor (VEGF), which performs a primary function in both tumor progression and angiogenesis, is up-regulated due to exposure to an array of carcinogenic metals, but the mechanisms responsible for the metal activation remain somewhat poorly understood. Recently, we demonstrated that AMP-activated protein kinase (AMPK), which acts as an energy sensor, providing metabolic adaptation effects under ATP-deprived conditions, is critical for the expression of VEGF under oxygen- and glucose-deprived conditions. As carcinogenic metals are potent VEGF expression inducers, we hypothesized that AMPK would also play a crucial role in metal-induced VEGF expression. Here, we present evidence that carcinogenic metals such as arsenite, vanadate, and cobalt, induce AMPK activation and VEGF expression via several different mechanisms, and that AMPK is able to regulate the expression of VEGF mRNA in a hypoxia-inducible factor-1-dependent or -independent manner, depending on the metal applied. We also attempted to characterize the relevant signal transduction pathways in metal-induced VEGF expression and AMPK activation, as well as the role of reactive oxygen species within this context. Overall, our data suggest that AMPK is a critical regulatory component in metal-induced VEGF expression, which further implies its intrinsic involvement in metal-induced carcinogenesis.

AMP-Activated Protein Kinases↗

PubMed2006

Soochong virus: an antigenically and genetically distinct hantavirus isolated from Apodemus peninsulae in Korea.

Hantaan (HTN) virus, the etiologic agent of clinically severe hemorrhagic fever with renal syndrome (HFRS), was first isolated in 1976 from lung tissue of a striped-field mouse (Apodemus agrarius) captured in Songnae-ri, Gyeonggi Province, Republic of Korea. Found primarily in mountainous areas, the Korean field mouse (A. peninsulae) is the second-most dominant field rodent species found throughout Korea. A new hantavirus, designated Soochong (SOO), was isolated in Vero E6 cells from four A. peninsulae captured in August 1997 at Mt. Gyebang in Hongcheon-gun, Mt. Gachil, Inje-gun, Gangwon Province, and in September 1998 at Mt. Deogyu, Muju-gun, Jeollabuk Province. The entire S, M, and L genomic segments of SOO virus, amplified by RT-PCR from lung tissues of seropositive A. peninsulae and from virus-infected Vero E6 cells, diverged from HTN virus (strain 76-118) by 15.6%, 22.8%, and 21.7% at the nucleotide level and 3.5%, 9.5%, and 4.6% at the amino acid level, respectively. Phylogenetic analyses of the nucleotide and deduced amino acid sequences, using the maximum parsimony and neighbor-joining methods, indicated that SOO virus was distinct from A. agrarius-borne HTN virus. SOO virus shared a common ancestry with Amur virus from Far East Russia, as well as with H5 and B78 hantaviruses, previously isolated from HFRS patients in China. Cross-focus-reduction neutralizating antibody tests showed that SOO virus, which is the first hantavirus isolated in cell culture from A. peninsulae, could be classified as a new hantavirus serotype.

Animals↗

PubMed2006

[Comparison of influential variables for smoking temptation between adolescent and adult smokers].

PURPOSE: This study attempted to identify influential variables on smoking temptation between groups: adolescent smokers and adult smokers. METHOD: A survey was conducted with 376 adolescent smokers in 4 high schools and 451 adult smokers in community settings in South Korea. Univariate statistics and regression were used for data analysis. RESULT: The most powerful predictor of smoking temptation for adolescent smokers was nicotine dependency. On the other hand, the most powerful predictor of smoking temptation for adult smokers was self-efficacy for smoking abstinence. In the high smoking temptation group, depression and nicotine dependency were the predictors for smoking temptation for adolescent smokers and nicotine dependency and pros for smoking were the predictors for smoking temptation for adult smokers. In the low smoking temptation group, cons for smoking and process of change for smoking abstinence were the predictors on smoking temptation for adolescent smokers and self-efficacy for smoking abstention and pros for smoking were the predictors on smoking temptation for adult smokers. CONCLUSION: There were different influential variables on smoking temptation according to age groups and level of smoking temptation. Smoking-cessation interventions should be tailored to the level of smoking temptation of the individual smokers.

Adolescent↗

PubMed2005

Inhibition of O-GlcNAcase by PUGNAc is dependent upon the oxime stereochemistry.

The potent O-GlcNAcase inhibitor PUGNAc was synthesized and two isomers based on the E and Z stereochemistry of the oxime moiety were separated, defined, and tested for activity. Several lines of evidence were examined in an effort to define the correct stereochemical assignments of each form of PUGNAc. The ability of the Z stereoisomer to undergo the Beckmann rearrangement was ultimately the most definitive proof. It was determined via both in vitro and intact cell experiments that the Z form of PUGNAc was vastly more potent an inhibitor of O-GlcNAcase than the E form.

Acetylglucosamine↗

PubMed2005

The effect of sativan from Viola verecunda A. Gray on the expressions of matrix metalloproteinase-1 caused by ultraviolet irradiated cultured primary human skin fibroblasts.

Matrix metalloproteinases (MMPs) are a family of enzymes whose main function is degradation of the extracellular matrix. Matrix metalloproteinase-1 (MMP-1) degrades type I procollagen constituting the major structural component of the basement membrane and extra cellular membrane. The enzymatic activity is found to be elevated in photoaging. With the aim of finding novel MMP-1 inhibitors from natural products, 15 extracts of the Viola taxa, which are used as prescriptions for skin eruption treatment in traditional medicine, were screened for their inhibitory activities towards MMP-1. Among the 15 tested materials, the methanol extracts of Viola hondoensis W. BECKER et H. BOISS, Viola ibukiana MAKINO, Viola lactiflora NAKAI and Viola verecunda A. GRAY were potential inhibitory to MMP-1, and other Viola taxa showed a weak inhibitory effect at a concentration of 100 microg/ml. We investigated the effect of MMP-1 inhibitory of the solvent fractions of the same plants (Viola hondoensis, Viola ibukiana and Viola verecunda). Therefore, a strong inhibition was found in the ethylacetate fractions of Viola verecunda with inhibitory activity (>90%) at a concentration of 10 microg/ml. Here we investigated the effect of sativan isolated from the ethylacetate fractions of Viola verecunda on the expression of MMPs in UV-irradiated human skin fibroblasts in vitro. Sativan markedly reduced UV-induced MMP-1 expression at the protein levels in a dose-dependent manner. Our report is the first description for the ability of sativan to regulate UV-induced MMP-1 expression.

Cells, Cultured↗

PubMed2005

[Influential variables on intention and action to quit smoking between adolescent smokers and adult smokers-based on the transtheoretical model].

PURPOSE: This study identified and compared influential variables on intention and action to quit smoking between adolescent smokers and adult smokers. METHODS: For the selection of variables, the transtheoretical theory was used. A survey was conducted with 376 adolescent smokers in 4 high schools and 451 adult smokers in community settings in South Korea. Discriminant analysis was used for data analysis. RESULTS: The variables of adolescent smokers that predicted an intention to quit smoking were: smoking temptation, self re-evaluation, counter conditioning and stimulus control. The variables that predicted an action to quit smoking were: self-efficacy for smoking abstinence, pros for smoking, self reevaluation, and self liberation. The variables of adult smokers that predicted an intention to quit were: smoking temptation, pros for smoking, cons for smoking, self reevaluation. The variables that self liberation and predicted an action to quit smoking were: self efficacy for smoking abstinence, smoking temptation, and counter conditioning. CONCLUSIONS: Developing stage specific smoking intervention methods based on different ways of how individuals make a decision to quit smoking within their contexts needs to be done.

Adolescent↗

PubMed2004

Evidence that the fully assembled capsid of Leishmania RNA virus 1-4 possesses catalytically active endoribonuclease activity.

In this study, Leishmania RNA virus 1-4 (LRV1-4) particles purified from host Leishmania guyanensis promastigotes were examined for capsid endoribonuclease. Temperature optimum for the endoribonuclease activity was found to be at 37(O)C to 42(O)C and the activity was specifically inhibited by the aminoglycoside antibiotics, neomycin, kanamycin, and hygromycin and by 100 mM levels of NaCl or KCl. To determine the catalytic domain of the capsid endoribonuclease activity, three point-mutation at cysteine residues at C47S (P1), C128/ 133S (P2), and C194R (P3) were prepared and each gene was constructed into baculoviruses and expressed in Sf9 insect cells. LRV1-4 capsid N- terminus (N2 and N3) and C-terminus (C1 and C2) deletion mutants (Cadd et al., 1994) were also examined by in vitro RNA cleavage assay. The results showed that the capsid mutants; C1, C2, N3, P1, and P2 were capable of forming proper virus-like particles (VLPs) and they all possessed the specific endoribonuclease activity. However, two assembly-defective capsid mutants, N2 (N- terminus 24-amino acids deletion) and P3 mutants, did not retain the specific endoribonuclease activity. Taken together, the results suggest that at least 24 amino acids from the N-terminal region and C194 residue in LRV1-4 capsid protein are functionally important for LRV1-4 viral assembly and the capsid endoribonuclease activity may be dependent upon the properly assembled LRV1-4 virus particles.

Amino Acid Substitution↗

PubMed2004

Ectopic expression of the catalytic subunit of telomerase protects against brain injury resulting from ischemia and NMDA-induced neurotoxicity.

The catalytic subunit of telomerase reverse transcriptase (TERT) protects dividing cells from replicative senescence in vitro. Here, we show that expression of TERT mRNA is induced in the ipsilateral cortical neurons after occlusion of the middle cerebral artery in adult mice. Transgenic mice that overexpress TERT showed significant resistance to ischemic brain injury. Among excitotoxicity, oxidative stress, and apoptosis comprising of routes of ischemic neuronal death, NMDA receptor-mediated excitotoxicity was reduced in forebrain cell cultures overexpressing TERT. NMDA-induced accumulation of cytosolic free Ca2+ ([Ca2+]c) was reduced in forebrain neurons from TERT transgenic mice, which was attributable to the rapid flow of [Ca2+]c into the mitochondria from the cytosol without change in Ca2+ influx and efflux through the plasma membrane. The present study provides evidence that TERT is inducible in postmitotic neurons after ischemic brain injury and prevents NMDA neurotoxicity through shift of the cytosolic free Ca2+ into the mitochondria, and thus plays a protective role in ameliorating ischemic neuronal cell death.

Animals↗

PubMed2004

Analysis of bgl operon structure and characterization of beta-glucosidase from Pectobacterium carotovorum subsp. carotovorum LY34.

A putative bgl operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) was isolated. Sequence analysis of the 5,557 bp cloned DNA fragment (accession no. AY542524) showed three open reading frames (bglT, bglP, and bglB) predicted to encode 287, 633, and 468 amino acid proteins respectively. BglT and BglP ORFs show high similarity to that of the Pectobacterium chrysanthemi ArbG antiterminator and ArbF permease respectively. Also, the latter contains most residues important for phosphotransferase activity. The amino acid sequence of BglB showed high similarity to various beta-glucosidases and is a member of glycosyl hydrolase family 1. The purified BglB enzyme hydrolyzed salicin, arbutin, pNPG, and MUG. The molecular weight of the enzyme was estimated to be 53,000 Da by SDS-PAGE. The purified beta-glucosidase exhibited maximal activity at pH 7.0 and 40 degrees C, and its activity was enhanced in the presence of Mg(2+). Two glutamate residues (Glu(173) and Glu(362)) were found to be essential for enzyme activity.

Base Sequence↗

PubMed2003

Purification, characterization, and physiological response of a catalase-peroxidase in Mycobacterium sp. strain JC1 DSM 3803 grown on methanol.

A novel catalase-peroxidase (CP) from methanol-grown cells of Mycobacterium sp. strain JC1 was purified. The CP exhibited properties of both typical mycobacterial CPs (i.e. strict pH optimum, labile to heat treatment, capable of oxidizing NADH, and resistant to inhibition by 3-amino-1,2,4-triazole) and true catalases (i.e. stable against ethanol-chloroform treatment). The enzyme oxidized methanol and shared common antigenic groups with other mycobacteria. Isoniazid had almost no effect on the growth and expression of CP but inhibited the enzyme activity to some extent. Sodium nitroprusside arrested the growth but strongly stimulated the expression of CP with a concomitant increase in activity after the mid-exponential growth phase.

Amitrole↗

PubMed2003

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35.

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.

1,4-alpha-Glucan Branching Enzyme↗

PubMed2003

Cloning and characterization of a thermostable intracellular alpha-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8.

The gene encoding an intracellular alpha-amylase, AmyB (TM1650), from Thermotoga maritima MSB8, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli. The AmyB enzyme hydrolyzed alpha-1,4 starch linkage. The amyB gene is 1269 bp in length, encoding a protein of 422 amino acids (calculated molecular mass of 50187 Da). The molecular weight of the enzyme was estimated to be 50000 Da by SDS-PAGE after starch-nondenaturing-PAGE. The amino acid sequence of AmyB showed less than 12% identity to other amylases, but contained four regions that are highly conserved among alpha-amylases. The AmyB alpha-amylase exhibited maximal enzymatic activity at pH 7.0 and its optimum temperature for activity was 70 degrees C. Like the alpha-amylases of many other organisms, the thermostability of T. maritima MSB8 alpha-amylase, AmyB expressed in E. coli was enhanced in the presence of Ca(2+) (10 mM).

Amino Acid Sequence↗

PubMed2003

Insertional mutation in the intron 1 of Unc5h3 gene induces ataxic, lean and hyperactive phenotype in mice.

Mice carrying a mutation in the first intron of Unc5h3 were accidentally produced by transgenic insertion and characterized for their homozygous mutant phenotypes. Morphological and histological analysis revealed cerebellar and midbrain abnormalities, which are similar to the previously reported phenotypes of the Unc5h3 mutant. Behavioral analysis showed higher ambulatory activity and circling, and defects in habituation in a novel environment. Their body weights were 10-30% less than wildtype mice from 2-3 weeks of age to 22 months possibly due to reduced accumulation of adipose tissues. The transgenic insertion site was identified and mapped to the intron 1 of Unc5h3 gene with approximately 50 kb deletion of the intron sequence. This intronic mutation interfered with the mRNA expression of the Unc5h3 gene not in testes, but in many tissues including the brain, implying that this intronic region may play a role in regulating tissue-specific transcription of Unc5h3.

Animals↗

PubMed2002

Control of singlet oxygen-induced oxidative damage in Escherichia coli.

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. The oxyR gene product regulates the expression of the enzymes and proteins that are needed for cellular protection against oxidative stress. In this study, the role of oxyR in cellular defense against a singlet oxygen was investigated using Escherichia coli oxyR mutant strains. Upon exposure to methylene blue and visible light, which generates singlet oxygen, the oxyR overexpression mutant was much more resistant to singlet oxygen-mediated cellular damage when compared to the oxyR deletion mutant in regard to growth kinetics, viability and protein oxidation. Induction and inactivation of major antioxidant enzymes, such as superoxide dismutase and catalase, were observed after their exposure to a singlet oxygen generating system in both oxyR strains. However, the oxyR overexpression mutant maintained significantly higher activities of antioxidant enzymes than did the oxyR deletion mutant. These results suggest that the oxyR regulon plays an important protective role in singlet oxygen-mediated cellular damage, presumably through the protection of antioxidant enzymes.

Antioxidants↗

PubMed2002

Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen.

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe(3+)/O(2)) but not against an oxidation system without thiol. This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx). The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis. Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase. The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.

Cloning, Molecular↗