Biomedical subjects

H J Kwon

Publications and source records attributed to H J Kwon.

At least 37 records · Page 2Linked to original sources

PubMed2001

Radicicol binding to Swo1/Hsp90 and inhibition of growth of specific temperature-sensitive cell cycle mutants of fission yeast.

A panel screening using cdc mutants of Schizosaccharomyces pombe identified radicicol as a potent growth inhibitor of certain mutants at the permissive temperature. The strains sensitive to radicicol were cdc7, cdc11, and cdc14, all of which are defective in early septum formation. Cytokinesis but not nuclear division of these mutants was inhibited by radicicol, but that of cells with the wild-type background was not. A biologically active derivative of radicicol with a biotin moiety at the C-11 position bound Swo1, an Hsp90 homologue in S. pombe. Increased Swo1 expression partially suppressed radicicol sensitivity of cdc14 and almost completely rescued morphological abnormalities in cdc14 and cdc7 cells induced by radicicol at the permissive temperature. On the other hand, the increased Swo1 expression did not restore septum formation at the nonpermissive temperature. These results suggest that Swo1, as a molecular chaperone, plays a role in stabilizing these temperature-sensitive proteins at the permissive temperature or in activating the cytokinesis signaling cascade.

Antifungal Agents↗

PubMed2001

Juxtaglomerular cell tumor of the kidney: a case report.

We report a case of renin-secreting juxtaglomerular cell tumor which developed in a hypertensive 47-yr-old Korean man. Presumptive clinical diagnosis was made before surgery based on the high level of plasma renin and the radiologic evidence of renal mass. Grossly, a round, bulging, well-encapsulated mass of 3 x 3 cm was located in the mid-portion of the right kidney. On microscopic examination, the tumor was composed of ovoid to polyhedral cells with bland nuclei, indistinct nucleoli and light eosinophilic cytoplasm. The immunostaining for renin showed strong positivity in the cytoplasm of tumor cells. The characteristic rhomboid shaped renin protogranules were observed in ultrastructural analysis.

Humans↗

PubMed2001

Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin.

Quercetin, a widely distributed bioflavonoid, has been shown to induce growth inhibition in certain cancer cell types. In the present study we have pursued the mechanism of growth inhibition in MCF-7 human breast cancer cells. Quercetin treatment resulted in the accumulation of cells specifically at G2/M phase of the cell cycle. Mitotic index measured by MPM2 staining clearly showed that cells were transiently accumulated in M phase, 24 h after treatment. The transient M phase accumulation was accompanied by a transient increase in the levels of cyclin B1 and Cdc2 kinase activity. However, 24 h or longer treatment caused a marked accumulation of cells in G2 instead of M phase. Levels of cyclin B1 and cyclin B1-associated Cdc2 kinase activity were also decreased. We also found that quercetin markedly increased Cdk-inhibitor p21CIP1/WAF1 protein level after treatment for 48 h or longer, and the induction of p21CIP1/WAF1 increased its association with Cdc2-cyclin B1 complex, however, up-regulation of p53 by quercetin was not observed. Quercetin also induced significant apoptosis in MCF-7 cells in addition to cell cycle arrest, and the induction of apoptosis was markedly blocked by antisense p21CIP1/WAF1 expression. The present data, therefore, demonstrate that a flavonoid quercetin induces growth inhibition in the human breast carcinoma cell line MCF-7 through at least two different mechanisms; by inhibiting cell cycle progression through transient M phase accumulation and subsequent G2 arrest, and by inducing apoptosis.

Antisense Elements (Genetics)↗

PubMed2000

SIMPL is a tumor necrosis factor-specific regulator of nuclear factor-kappaB activity.

The IL-1 receptor-associated kinase (IRAK/mPLK) is linked to the regulation of nuclear factor-kappaB (NF-kappaB)-dependent gene expression. Here we describe a novel binding partner of IRAK/mPLK that we term SIMPL (signaling molecule that associates with the mouse pelle-like kinase). Overexpression of SIMPL leads to the activation of NF-kappaB-dependent promoters, and inactivation of SIMPL inhibits IRAK/mPLK as well as tumor necrosis factor receptor type I-induced NF-kappaB activity. Dominant inhibitory alleles of IkappaB kinase (IKKalpha or IKKbeta) block the activation of NF-kappaB by IRAK/mPLK and SIMPL. Furthermore, SIMPL binds IRAK/mPLK and the IKKs in vitro and in vivo. In the presence of antisense mRNA to SIMPL, the physical association between IRAK/mPLK and IKKbeta but not IRAK/mPLK and IKKalpha is greatly diminished. Moreover, dominant-negative SIMPL blocks IKKalpha- or IKKbeta-induced NF-kappaB activity. These results lead us to propose a model in which SIMPL functions to regulate NF-kappaB activity by linking IRAK/mPLK to IKKbeta/alpha-containing complexes.

Amino Acid Sequence↗

PubMed2000

Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis.

A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His(30), His(274), His(291), His(313), and His(365)) and five acidic amino acid residues (Glu(253), Asp(255), Asp(262), Asp(275), and Asp(290)), which are fully conserved in the amino acid sequences of family I.3 lipases, by site-directed mutagenesis. We showed that the mutation of His(313) or Asp(255) to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His(313) or Asp(255) is not due to marked changes in the tertiary structure. We propose that His(313) and Asp(255), together with Ser(207), form a catalytic triad in PML.

Amino Acid Substitution↗

PubMed2000

Heat shock protein 90 mediates protein-protein interactions between human aminoacyl-tRNA synthetases.

Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo. Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments. This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS. Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS). Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90. EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors. hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex. In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex. Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases.

Adenosine Triphosphate↗

PubMed2000

Inhibition of NFkappaB by methyl chlorogenate from Eriobotrya japonica.

Methylchlorogenic acid (MC) is one of the main components in the leaves of Eriobotrya japonica. We previously reported that MC is the most potent antioxidant among several components of Eriobotrya japonica, and its antioxidant activity is stronger than that of chlorogenic acid. Antioxidants are expected to inhibit redox-sensitive NFkappaB activation since NFkappaB is readily influenced by cellular oxidative state. Based on these findings, in vivo experiments with MC were conducted to determine its ability to downregulate the NFkappaB activation in mouse liver. Results clearly showed that MC is a potent suppressor of BHP-induced NFkappaB activation. We observed a significant reduction by MC on BHP-induced translocation of p65 subunit of NFkappaB. This may be due to formation of p50/p65 heterodimer, which is mainly inducible NFkappaB. MC slightly blocked the BHP-induced IkappaB alpha degradation. There is a possibility of IkappaB alpha resynthesis via activated NFkappaB during a 5 h waiting period following BHP injection. The present results suggest that MC may inhibit NFkappaB activation, exhibiting its ability to downregulate the NFkappaB-dependent gene expression. Thus, it can be expected that MC may have potential for therapeutic intervention on various NFkappaB-dependent pathological conditions such as inflammatory or possibly mutagenic processes.

Active Transport, Cell Nucleus↗

PubMed2000

Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein. Implication for the E7-mediated immune evasion mechanism in cervical carcinogenesis.

In studying biological roles of interferon regulatory factor (IRF)-1 tumor suppressor in cervical carcinogenesis, we found that HPV E7 is functionally associated with IRF-1. Binding assays indicate a physical interaction between IRF-1 and HPV E7 in vivo and in vitro. The carboxyl-terminal transactivation domain of IRF-1 was required for the interaction. Transient co-expression of E7 significantly inhibits the IRF-1-mediated activation of IFN-beta promoter in NIH-3T3 cells. Co-transfection of E7 mutants reveals that the pRb-binding portion of E7 is necessary for the E7-mediated inactivation of IRF-1. It was next determined whether histone deacetylase (HDAC) is involved in the inactivation mechanism as recently suggested, where the carboxyl-terminal zinc finger domain of E7 associates with NURD complex containing HDAC. When trichostatin A, an inhibitor of HDAC, was treated, the repressing activity of E7 was released in a dose-dependent manner. Furthermore, the mutation of zinc finger abrogates such activity without effect on the interaction with IRF-1. These results suggest that HPV E7 interferes with the transactivation function of IRF-1 by recruiting HDAC to the promoter. The immune-promoting role of IRF-1 evokes the idea that our novel finding might be important for the elucidation of the E7-mediated immune evading mechanism that is frequently found in cervical cancer.

DNA-Binding Proteins↗

PubMed2000

Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G(i) protein-linked phospholipase C pathway.

We have previously shown that sphingosine 1-phosphate (S1P) stimulates motility of human umbilical vein endothelial cells (HUVECs) (O.-H. Lee et al., Biochem. Biophys. Res. Commun. 264, 743-750, 1999). To investigate the molecular mechanisms by which S1P stimulates HUVEC motility, we examined tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) which is important for cell migration. S1P induces a rapid increase in tyrosine phosphorylation of p125(FAK). Compared with other structurally related lipid metabolites such as sphingosine, C2-ceramide, and lysophosphatidic acid, S1P uniquely stimulated p125(FAK) tyrosine phosphorylation and migration of HUVECs. The effect of S1P on p125(FAK) tyrosine phosphorylation was markedly reduced by treatment with pertussis toxin or U73122, a phospholipase C (PLC) inhibitor. As a downstream signal of PLC, p125(FAK) tyrosine phosphorylation in response to S1P was totally blocked by depletion of the intracellular calcium pool. However, protein kinase C (PKC) inhibitor had no effect on the response to S1P. Finally, chemotaxis assays revealed that inhibition of PLC but not PKC significantly abrogated S1P-stimulated HUVEC migration. These results suggest that the G(i)-coupled receptor-mediated PLC-Ca(2+) signaling pathway may be importantly involved in S1P-stimulated focal adhesion formation and migration of endothelial cells.

Cell Adhesion Molecules↗

PubMed2000

Air pollution and daily mortality in seven major cities of Korea, 1991-1997.

The relationship between ambient air pollution and daily mortality in seven major cities of Korea for the period 1991-1997 was examined. These cities account for half of the Korean population (about 22 million). The observed concentrations of sulfur dioxide (SO(2), mean=23.3 ppb), ozone (O(3), mean=23.7 ppb), and total suspended particulates (TSP, mean=77.9 microg/m(3)) during the study period were at levels below Korea's current ambient air quality standards. Generalized additive models were applied to allow for the highly flexible fitting of seasonal and long-term time trends in air pollution as well as nonlinear associations with weather variables, such as air temperature and relative humidity. In city-specific analyses, an increase of 50 ppb of SO(2) corresponded to 1-12% more deaths, given constant weather conditions. The risk of all-cause mortality was estimated to increase by 0.5-4%, with an increase in the 2-day moving average of TSP levels equal to 100 microg/m(3). In multipollutant models with pooled data, we found that the estimated risk of death by SO(2) was notably unaffected by adding the other two pollutants (TSP and O(3)) to the model and was statistically significant in various regression models. The rate ratio (RR) for SO(2) remained elevated, indicating an excess mortality of 3% 50 ppb (RR=1.03; 95% CI, 1.01-1.05). TSP's effect on mortality maintained its significance with O(3), but not with SO(2). This implies that there may be collinearity problems where TSP and SO(2) are included in the same model or that TSP may function less than SO(2) as a surrogate for fine particles in the ambient air of Korea. In conclusion, increased mortality was associated with air pollution at SO(2) levels below the current recommendation for air quality. Further research is needed to clarify the relationship between SO(2) and fine particles in Korea.

Adolescent↗

PubMed2000

NT-4/5 exacerbates free radical-induced neuronal necrosis in vitro and in vivo.

Neurotrophins render neurons highly vulnerable to certain injuries. We examined the possibility that NT-4/5 would enhance free radical neurotoxicity in vivo as well as in vitro. Striatal neurons exposed to 10 microM Fe(2+) or 1 mM l-buthionine-[S, R]-sulfoximine (BSO) underwent mild degeneration within 24 h. With concurrent addition of 10-100 ng/ml NT-4/5, neuronal death following exposure to Fe(2+) or BSO was significantly increased and suppressed by addition of 100 microM trolox, an antioxidant. In the adult brain, the intrastriatal injections of 20 nmol Fe(2+) revealed features of neuronal necrosis such as swelling cell body and mitochondria, fenestration of plasma membrane prior to nuclear membrane, and scattering condensation of nuclear chromatin. Cotreatment with 1.8 microg NT-4/5 augmented the striatal damage 24 h following the injections of Fe(2+). This study implies that free radicals produce necrotic degeneration in vivo as well as in vitro that becomes more sensitive in the presence of neurotrophins.

Animals↗

PubMed2000

Differentiation of Salmonella enterica serotype gallinarum biotype pullorum from biotype gallinarum by analysis of phase 1 flagellin C gene (fliC).

Salmonella enterica serotype gallinarum biotype gallinarum and biotype pullorum are non-motile and pathogenic avian strains. Biotype gallinarum causes fowl typhoid and biotype pullorum is the cause of pullorum disease in chickens. The two biotypes could be differentiated based on biochemical characteristics. However, conventional culture and biochemical assays are time-consuming, laborious and need sterile laboratory practices. Although the two biotypes, gallinarum and pullorum are non-motile, they possess the phase 1 flagellin C gene. The variable regions of the flagellin C gene from 41 biotype pullorum and 52 biotype gallinarum were amplified by colony-PCR and analyzed by single strand conformational polymorphism (SSCP) method. Differences in SSCP electrophoretic patterns were confirmed by nucleotide sequencing. In addition, PCR-RFLP with Hinp1I was also successfully applied to differentiate the two biotypes. These results suggested that the variable regions of fliC could be used as a genetic marker to differentiate biotype gallinarum from biotype pullorum.

Animals↗

PubMed2000

Crystal structure of the Escherichia coli Rob transcription factor in complex with DNA.

The Escherichia coli Rob protein is a transcription factor belonging to the AraC/XylS protein family that regulates genes involved in resistance to antibiotics, organic solvents and heavy metals. The genes encoding these proteins are activated by the homologous proteins MarA and SoxS, although the level of activation can vary for the different transcription factors. Here we report a 2.7 A crystal structure of Rob in complex with the micF promoter that reveals an unusual mode of binding to DNA. The Rob-DNA complex differs from the previously reported structure of MarA bound to the mar promoter, in that only one of Rob's dual helix-turn-helix (HTH) motifs engages the major groove of the binding site. Biochemical studies show that sequence specific interactions involving only one of Rob's HTH motifs are sufficient for high affinity binding to DNA. The two different modes of DNA binding seen in crystal structures of Rob and MarA also match the distinctive patterns of DNA protection by AraC at several sites within the pBAD promoter. These and other findings suggest that gene activation by AraC/XylS transcription factors might involve two alternative modes of binding to DNA in different promoter contexts.

Amino Acid Sequence↗

PubMed2000

Characterization of AMC-HN-9, a cell line established from an undifferentiated carcinoma of the parotid gland: expression of alpha6beta4 with the absence of BP180 and 230.

We recently reported the development of a cell line, AMC-HN-9, established from an undifferentiated carcinoma (UDC) of the parotid gland. AMC-HN-9 consists mostly of spindle-shaped cells, has poor in vitro adhesiveness and an in vitro appearance that is different from that of other epithelial cell lines. To test the hypothesis that structural or functional abnormalities of the hemidesmosomes might contribute to the morphological appearance and biology of UDCs, we studied the expression of hemidesmosomal proteins in AMC-HN-9. Flow cytometry, indirect immunofluorescence, immunoprecipitation, reverse transcriptase-polymerase chain reaction, and cytogenetic analysis were used. AMC-HN-9 cells express the alpha6 and beta4 integrin subunits at nearly the same intensity as head and neck squamous cell carcinoma cell lines. However, AMC-HN-9 does not express BP180 and BP230, although there is no gross deletion of the loci of the BP180 and BP230 genes, suggesting that a more subtle mechanism has silenced these genes. In conclusion, the failure to express certain hemidesmosomal proteins is a likely explanation for the functional and morphologic characteristics of UDC cells both in vivo and in vitro.

Antibodies, Monoclonal↗

PubMed2000

Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts.

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful alternative to the more commonly used retrovirus- and adenovirus-based vectors for human gene therapy, efficient gene transfer and transgene expression by AAV vectors require that the following two obstacles be overcome. First, the target cell must express the receptor and the coreceptor for AAV infection, and second, the cell must allow for viral second-strand DNA synthesis. We now describe a third obstacle, impaired intracellular trafficking of AAV to the nucleus, which results in the lack of transgene expression in murine fibroblasts which do express the AAV receptor and the coreceptor and which are permissive for viral second-strand DNA synthesis. We document that AAV vectors bind efficiently and gain entry successfully into NIH 3T3 cells, but trafficking into the nucleus is significantly impaired in these cells. In contrast, viral trafficking to the nucleus in cells known to be efficiently transduced by AAV vectors is both rapid and efficient. The demonstration of yet another obstacle in AAV-mediated gene transfer has implications for the optimal use of these vectors in human gene therapy.

3T3 Cells↗

PubMed1999

The homeodomain protein NK-3 recruits Groucho and a histone deacetylase complex to repress transcription.

Transcriptional repression by sequence-specific DNA binding factors is mediated by the recruitment of a corepressor complex to the promoter region. The NK-3 homeodomain protein is a transcriptional repressor that recruits the nuclear protein kinase, homeodomain interacting protein kinase 2 (HIPK2). Here we show that HIPK2 is a component of a corepressor complex containing Groucho and a histone deacetylase complex. Groucho, like HIPK2, acts as a corepressor for NK-3 and binds to NK-3 and HIPK2. Moreover, HIPK2 appears to regulate the corepressor activity of Groucho. Transcriptional repression by NK-3 and Groucho is relieved by the histone deacetylase inhibitor trichostatin A, and both NK-3 and Groucho directly interact with the histone deacetylase HDAC1 that is associated with mSin3A in vivo. Recruitment of the histone deacetylase complex by NK-3 decreases the acetylated histones that are associated with the target gene promoter. These results indicate that NK-3 represses transcription by recruiting a complex containing Groucho and a histone deacetylase complex that leads to histone modification on chromatin and suggest that HIPK2 may play a regulatory role in the corepressor complex formation.

Base Sequence↗

PubMed1999

Establishment and characterization of 12 human colorectal-carcinoma cell lines.

In this article, we describe the characteristics of 12 human colorectal-carcinoma cell lines established from 6 primary tumors and 6 metastatic sites of 11 Korean colorectal-carcinoma patients, including the morphology in vivo and in vitro and mutations of K-ras2, p15, p16, p53, APC, beta-catenin, hMLH1 and hMSH2 genes in vitro. No lines were contaminated with Mycoplasma or bacteria. All lines were proven to be unique by DNA-fingerprinting analysis. All lines expressed the surface carcino-embryonic antigen and secreted it into the supernatant fluid. The morphological correlation between the original tumors and cultured cells suggested that the original tumors showing mucinous adenocarcinoma correlated with floating aggregates in culture, and degree of desmoplasia in the original tumor correlated with attached growth in culture. Five of the cell lines showed mutations in the K-ras2 gene, and 6 of the cell lines showed mutations in the p53 gene. The p15 gene was deleted in 2 cell lines, and the p16 gene was hypermethylated in 3 cell lines. The mutation of mismatch-repair genes (hMLH1 and hMSH2) was found in 4 lines, the APC gene and beta-catenin gene were mutated in 9 and 2 lines respectively. These well-characterized colorectal-cancer cell lines should serve as useful tools for investigating the biological characteristics of colorectal cancer.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Regional difference of ROS generation, lipid peroxidation, and antioxidant enzyme activity in rat brain and their dietary modulation.

One of the potential causes of age-related neuronal damage can be reactive oxygen species (ROS), as the brain is particularly sensitive to oxidative damage. In the present study, we investigated the effects of aging and dietary restriction (DR) on ROS generation, lipid peroxidation, and antioxidant enzymes in cerebrum, hippocampus, and cerebellum of 6-, 12-, 18-, and 24-month-old rats. ROS generation significantly increased with age in cerebrum of ad libitum (AL) rats. However, no significant age-difference was observed in hippocampus and cerebellum. DR significantly decreased ROS generation in cerebrum and cerebellum at 24-months. On the other hand, the increased lipid peroxidation of AL rats during aging was significantly reduced by DR in all regions. Our results further showed that catalase activity decreased with age in cerebellum of AL rats, which was reversed by DR, although SOD activity had little change by aging and DR in all regions. In a similar way, glutathione (GSH) peroxidase activity increased with age in cerebrum of AL rats, while DR suppressed it at 24-months. These data further support the evidence that the vulnerability to oxidative stress in the brain is region-specific.

Aging↗