Biomedical subjects

H J Kwon

Publications and source records attributed to H J Kwon.

At least 55 records · Page 3Linked to original sources

PubMed1999

Molecular basis for enhanced biosynthesis of clavulanic acid by a redox-cycling agent, phenazine methosulfate, in Streptomyces clavuligerus.

Phenazine methosulfate (PMS), a generator of superoxide, evoked the transcription of cas2 and cefF, ultimately resulting in the enhanced biosyntheses of clavulanic acid (CA) and cephamycin C (CMC) in Streptomyces clavuligerus. The transcriptional activation of cas2 and cefF was accompanied with that of ccaR, a regulatory gene for biosyntheses of CA and CMC. PMS or H2O2 in cell-free extract exerted a positive regulation on in vitro protein phosphorylation. The PMS-mediated activation of protein phosphorylation was significantly offset by butylated hydroxyanisole, a radical scavenger. Staurosporine, a protein kinase inhibitor, was shown to have a negative effect on PMS-promoted CA accumulation. Therefore, it is suggestive that PMS-activated transcription of cas2 and cefF is mediated by protein phosphorylation and the expression of a pathway-specific transcriptional activator as found in other streptomycetes. These experimental results present an example of the functional relationship between oxidative stimuli and secondary metabolite production in streptomycetes.

Anti-Bacterial Agents↗

PubMed1999

Germline mutations of the APC gene in Korean familial adenomatous polyposis patients.

We extensively analyzed genomic DNA and messenger RNA (mRNA) from 62 unrelated Korean patients with familial adenomatous polyposis (FAP) for identification of germline adenomatous polyposis coli (APC) gene mutations. We adopted both single-strand conformation polymorphism (SSCP) analysis and a method of analysis involving the reverse transcription-polymerase chain reaction (RT-PCR) followed by a protein truncation test (PTT). DNA sequencing confirmed all alterations represented by aberrant bands. Germline mutations were identified in 38 patients (61%). Nineteen of the detected mutations were presumed to be novel, thus emphasizing the heterogeneity of the mutational spectrum in Korean FAP patients. In the initial 48 patients, SSCP analysis was followed by PTT for those patients for whom no detectable mutations were found by SSCP. Using this combined approach, we identified germline APC gene mutations in 29 of the 48 FAP patients (60%), including 6 patients in whom SSCP analysis failed to distinguish the mutant allele. In the 14 later patients, we identified truncating mutations in 9 patients (64%) using PTT only. Our results confirm that the mutation detection rate with PTT was superior to that with SSCP, and suggest that PTT would be a more practical screening method to detect germline mutations of the APC gene in FAP patients.

Adenomatous Polyposis Coli↗

PubMed1999

Immune-stimulating properties of polysaccharides from Phellodendri cortex (Hwangbek).

Heteropolysaccarides were isolated from the Korean medicinal plant, Phellodendri cortex (Hwangbek), by hot water and alkali extractions. The extracted polysaccharides were fractionated into eight fractions and they are mainly composed of D-N-acetylglucosamine, D-galactose, D-mannose, and D-glucose. Among the polysaccharide fractions, Fr.-2 showed a potent B-lymphocyte-stimulating activity in a system using polyclonal antibody forming cells in C57BL/6XC3H mice at dosages of 2-10 mg. On the basis of their solubility in aqueous ethanol, four fractions of Fr.-2-1 to Fr.-2-4 were further obtained from the Fr.-2, and Fr.-2-3 was divided into Fr.-2-3-1, 2, 3, and 4 by DEAE cellulose chromatography. The main activity was found in Fr.-2-3-2, which contained 100% (w/w) of carbohydrates and further purified to Fr.-2-3-2-2 by gel filtration chromatography using TSK Gel HW50S. Fr.-2-3-2-2, having a molecular weight of about 230 kDa, showed the highest B-cell-stimulating activity and the half-maximal concentration for B-lymphocyte-stimulating activity was ca. 2.2 microg/ml.

Animals↗

PubMed1999

Nasal angiocentric lymphoma with hemophagocytic syndrome.

OBJECTIVES: Hemophagocytic syndrome (HS) is a fatal complication of nasal angiocentric lymphoma (AL) and difficult to distinguish from malignant histiocyosis. Epstein-Barr virus (EBV)-associated HS is frequently observed in lymphoma of T-cell lineage and EBV is highly associated with nasal AL. Clinicopathologic features of 10 nasal ALs with HS were reviewed to determine the clinical significance and the pathogenetic association with EBV. METHODS: Ten patients of HS were identified from a retrospective analysis of 42 nasal ALs diagnosed from 1987 to 1996. Immunohistochemical study and in situ hybridization were performed on the paraffin-embedded tumor specimens obtained from 10 patients. Serologic study of EBV-Ab was performed in 3 available patients. RESULTS: Five patients had HS as initial manifestation, 3 at the time of relapse and 2 during the clinical remission of AL. Four patients were treated by combination chemotherapy (CHOP) and others had only supportive care. The median survival of all patients with HS was 4.1 months (range 2 days-36.5 months) and all had fatal outcome regardless of the treatment-modality. All cases were positive for UCHL1 (CD45RO) and EBV by EBER in situ hybridization. The data of serologic tests indicated the active EBV infection. CONCLUSIONS: HS is a fatal complication of nasal AL and has a high association with EBV. Reactivation of EBV may contribute to HS and further investigation of predictive factors and effective treatment of HS should be pursued in the future.

Adult↗

PubMed1998

Immunity to T cell receptor peptides in multiple sclerosis. III. Preferential immunogenicity of complementarity-determining region 2 peptides from disease-associated T cell receptor BV genes.

Vaccination with synthetic TCR peptides from the BV5S2 complementarity-determining region 2 (CDR2) can boost significantly the frequency of circulating CD4+ peptide-specific Th2 cells in multiple sclerosis (MS) patients, with an associated decrease in the frequency of myelin basic protein (MBP)-reactive Th1 cells and possible clinical benefit. To evaluate the immunogenicity of CDR2 vs other regions of the TCR, we vaccinated seven MS patients with overlapping BV5S2 peptides spanning amino acids 1-94. Six patients responded to at least one of three overlapping or substituted CDR2 peptides possessing a core epitope of residues 44-52, and one patient also responded to a CDR1 peptide. Of the CDR2 peptides, the substituted (Y49T)BV5S2-38-58 peptide was the most immunogenic but cross-reacted with the native sequence and had the strongest binding affinity for MS-associated HLA-DR2 alleles, suggesting that position 49 is an MHC rather than a TCR contact residue. Two MS patients who did not respond to BV5S2 peptides were immunized successfully with CDR2 peptides from different BV gene families overexpressed by their MBP-specific T cells. Taken together, these results suggest that a widely active vaccine for MS might well involve a limited set of slightly modified CDR2 peptides from BV genes involved in T cell recognition of MBP.

Adult↗

PubMed1998

Recognition of core-type DNA sites by lambda integrase.

Escherichia coli phage lambda integrase (Int) is a 40 kilodalton, 356 amino acid residue protein, which belongs to the lambda Int family of site-specific recombinases. The amino-terminal domain (residues 1 to 64) of Int binds to "arm-type" DNA sites, distant from the sites of DNA cleavage. The carboxy-terminal fragment, termed C65 (residues 65 to 356), binds "core-type" DNA sites and catalyzes cleavage and ligation at these sites. It has been further divided into two smaller domains, encompassing residues 65 to 169 and 170 to 356, respectively. The latter has been characterized and its crystal structure has been determined. Although this domain catalyzes the cleavage and rejoining of DNA strands it, unexpectedly, does not form electrophorectically stable complexes with core-type DNA. Here we have investigated the critical features of lambda Int binding to core-type DNA sites; especially, the role of the central 65 to 169 domain. To eliminate the complexities arising from lambda Int's heterobivalency we studied Int C65, which was shown to be as competent as Int, in binding to, and cleaving, core-type sites. Zero-length UV crosslinking was used to show that Ala125 and Ala126 make close contact with bases in the core-type DNA. Modification by pyridoxal 5'-phosphate was used to identify Lys103 at the protein-DNA interface. Since both of the identified loci are in the central domain, it was cloned and purified and found to bind to core-type DNA autonomously and specifically. The synergistic roles of the catalytic and the central, or core-binding (CB), domains in the interaction with core-type DNA are discussed for (Int and related DNA recombinases.

Bacteriophage lambda↗

PubMed1998

Depudecin induces morphological reversion of transformed fibroblasts via the inhibition of histone deacetylase.

Depudecin is a fungal metabolite that reverts the rounded phenotype of NIH 3T3 fibroblasts transformed with v-ras and v-src oncogenes to the flattened phenotype of the nontransformed parental cells. The mechanism of detransformation induced by this agent had not been determined. Here, we demonstrate that depudecin inhibits histone deacetylase (HDAC) activity effectively both in vivo and in vitro. Depudecin induces similar morphological reversion in v-ras transformed NIH 3T3 cells as do other naturally occurring HDAC inhibitors such as trichostatin A or trapoxin. It competitively inhibits the binding of [3H]trapoxin in vitro and the nuclear binding of a trapoxin-coumarin fluorophore in vivo, suggesting that depudecin shares a nuclear binding protein and site on that protein with trapoxin. Furthermore, depudecin induces hyperacetylation of histones in a dose-dependent manner and at concentrations comparable with that required for detransformation. An in vitro histone deacetylase assay, using purified recombinant HDAC1, reveals that depudecin inhibits 50% of the enzyme activity at a concentration of 4.7 microM. These results demonstrate that depudecin is a novel HDAC inhibitor and suggest that its ability to induce morphological reversion of transformed cells is the result of its HDAC inhibitory activity.

3T3 Cells↗

PubMed1998

Similarities and differences among 105 members of the Int family of site-specific recombinases.

Alignments of 105 site-specific recombinases belonging to the Int family of proteins identified extended areas of similarity and three types of structural differences. In addition to the previously recognized conservation of the tetrad R-H-R-Y, located in boxes I and II, several newly identified sequence patches include charged amino acids that are highly conserved and a specific pattern of buried residues contributing to the overall protein fold. With some notable exceptions, unconserved regions correspond to loops in the crystal structures of the catalytic domains of lambda Int (Int c170) and HP1 Int (HPC) and of the recombinases XerD and Cre. Two structured regions also harbor some pronounced differences. The first comprises beta-sheets 4 and 5, alpha-helix D and the adjacent loop connecting it to alpha-helix E: two Ints of phages infecting thermophilic bacteria are missing this region altogether; the crystal structures of HPC, XerD and Cre reveal a lack of beta-sheets 4 and 5; Cre displays two additional beta-sheets following alpha-helix D; five recombinases carry large insertions. The second involves the catalytic tyrosine and is seen in a comparison of the four crystal structures. The yeast recombinases can theoretically be fitted to the Int fold, but the overall differences, involving changes in spacing as well as in motif structure, are more substantial than seen in most other proteins. The phenotypes of mutations compiled from several proteins are correlated with the available structural information and structure-function relationships are discussed. In addition, a few prokaryotic and eukaryotic enzymes with partial homology with the Int family of recombinases may be distantly related, either through divergent or convergent evolution. These include a restriction enzyme and a subgroup of eukaryotic RNA helicases (D-E-A-D proteins).

Amino Acid Sequence↗

PubMed1998

Enhanced biosynthesis of clavulanic acid in Streptomyces clavuligerus due to oxidative challenge by redox-cycling agents.

Streptomyces clavuligerus produces a clinically important beta-lactamase inhibitor, clavulanic acid. When several of the selected redox-cycling agents were treated, an increase in clavulanate production was observed. The stimulatory effect was seen when the reaction was fed with menadione, plumbagin and phenazine methosulfate (PMS), whereas feeding with methyl viologen had a negative effect. PMS exerted the strongest effect, enhancing the accumulation of clavulanic acid by 150%. Induction of superoxide dismutase upon the addition of PMS suggested an involvement of superoxide in the enhancing process. The stimulatory effect of PMS was offset by the addition of butylated hydroxyanisole, further supporting the involvement of the active oxygen. The enhanced production of clavulanic acid correlated well with the increased total activity of clavaminic acid synthase, a key enzyme in its biosynthesis, and the transcription of cas2, its coding gene. The results suggested that active oxygen species could enhance the biosynthesis of secondary metabolites through the transcriptional activation of the biosynthetic gene.

Anti-Bacterial Agents↗

PubMed1998

Identification of an endonuclease secreted by human B lymphoblastic IM9 cells.

We have identified a Mg(2+)-dependent endonuclease from IM9 cell lysates and culture medium using DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay system. This particular endonuclease activity was not detectable in conventional DNA-SDS-polyacrylamide gel electrophoresis assay system which is similar to the method originally described by Rosenthal and Lacks (A.L. Rosenthal and S.A. Lacks, Anal. Biochem. 80 (1977) 76-90). Experimental results clearly demonstrated that the endonuclease activity was not derived from the fetal calf serum in which the cells were grown, but synthesized in the cell and secreted into the culture medium by IM9 cells. Biosynthesis and subsequent release of the endonuclease into the culture medium were significantly decreased by pretreatment of the cells with actinomycin D. Using supercoiled plasmid DNA as a substrate, the endonuclease activity was determined with the enzyme isolated from the cell culture medium by native-PAGE electroelution. The endonuclease, with Mg2+ alone, was able to catalyze the conversion of the plasmid into linear DNA followed by further degradation. This is the first report demonstrating that a distinct Mg(2+)-dependent endonuclease is secreted by a human immune cell line.

Burkitt Lymphoma↗

PubMed1998

A Mg(2+)-dependent endonuclease is responsible for internucleosomal DNA fragmentation in human B lymphoblastic IM9 cells.

We have identified a Mg(2+)-dependent endonuclease activity from human B lymphoblastic IM9 cell lysates and nuclei using autodigestion method and DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay system. The level of the endonuclease activity in cell lysates was significantly decreased at certain stage by treatment of the cells with cycloheximide. However, the enzyme activity consistently remained for over 12 hours in the isolated nuclei of the apoptotic IM9 cells. The Mg(2+)-dependent endonuclease isolated from the nuclei by native-PAGE elution was able to catalyze the conversion of supercoiled plasmid DNA into linear form. This particular endonuclease activity was not detected in cycloheximide treated-U937 cells. Several lines of experimental evidence suggest that the Mg(2+)-dependent endonuclease localized in the nucleus may be responsible for the DNA fragmentation of apoptotic IM9 cells.

Apoptosis↗

PubMed1998

Multiple lentigines syndrome: a comparison of normal skin and lentiginous skin by electron microscopy and immunohistochemical staining.

A 26-year-old Korean man exhibited generalized lentigines, a slight prominence of the chin, short stature, and a familial history consistent with the autosomal dominant mode of inheritance. The multiple lentigines appeared at the age of 1 and spread over his entire body including the scalp, palms, and soles by the age of 4. There were several large dark pigmented spots on the trunk, but no other skin abnormalities were found. We performed histologic studies of the normal skin and the lentigo by both electron and light microscopy with immunohistochemical stains. Routine hematoxylin-eosin staining of the pigmented skin revealed heavily pigmented basal and spinous layers and prominent rete ridges. Fontana-Masson stain showed enormous black pigmentations on the basal and spinous layers of the pigmented macule. S-100 protein stain of the pigmented lesion demonstrated an increased number of melanocytes per unit skin area. Ultrastructural studies showed the presence of extensive aggregated melanosomes within the keratinocytes in the epidermis of lentigo, but not the presence of giant melanosomes in either normal skin or lentigines.

Adult↗

PubMed1998

Identification of radicicol as an inhibitor of in vivo Ras/Raf interaction with the yeast two-hybrid screening system.

Activation of cytoplasmic serine/threonine kinase Raf-1, an important effector of Ras, requires direct binding to Ras. The yeast two-hybrid screening system used for identification of inhibitors of Ras/Raf-1 interaction showed radicicol to be an inhibitor. Radicicol has been shown to induce morphological reversion of transformed cells. Immunoprecipitation with an anti-Ras antibody revealed that the in vivo Ras/Raf-1 binding in v-Ha-ras-transformed cells was also blocked by low concentrations of radicicol (0.1 approximately 1 microg/ml), while degradation of Raf-1 was induced at concentrations higher than 2 microg/ml. However, in vitro binding of glutathion S-transferase-fused Ras to a maltose binding protein-fused RIP3 containing the Ras-binding domain (RBD) of Raf-1 was not inhibited by radicicol. Similar two-hybrid assays with several truncated forms of Raf-1 showed that both the conserved serine/threonine-rich domain (CR2) and the C-terminal protein kinase domain (CR3) were required for the full inhibition by radicicol. These results suggest that radicicol interacts directly or indirectly with the region except with RBD of Raf-1, thereby inhibiting a conformational change of Raf-1 prerequisite for binding to Ras.

3T3 Cells↗

PubMed1997

Suppression of morphological transformation by radicicol is accompanied by enhanced gelsolin expression.

Radicicol, an inhibitor of Src-family protein-tyrosine kinases, causes morphological reversion of v-src- and v-Ha-ras-transformed fibroblasts and arrest of the cell cycle at both the G1 and the G2 phases. Radicicol was found to inhibit the growth of several other oncogene-transformed cell lines and human carcinoma cell lines and to revert their cell morphology to be flat. In the radicicol-treated flat cells, actin stress fiber bundles were reorganized. Since this effect of radicicol on these cell lines was inhibited by cycloheximide, de novo protein synthesis is required for the morphological reversion. Screening of cellular proteins enhanced in response to radicicol by two-dimensional gel electrophoresis suggested that the amount of gelsolin, an actin regulatory protein, was distinctly increased upon radicicol treatment. Western blot and Northern blot analyses showed that radicicol enhanced transcription of the gelsolin gene in human carcinoma cell lines, as a result of which the amount of gelsolin was increased several folds. Injection with an anti-gelsolin antibody into cells and successive treatment with radicicol resulted in approximately 80% reduction of the number of flat cells with stress fibers in comparison with controls treated with an irrelevant antibody. These results show that elevated expression of gelsolin is associated, at least in part, with the suppression of transformation and the restoration of actin stress fibers in human carcinoma cells by radicicol.

3T3 Cells↗

PubMed1997

Estrogen blocks neurotoxic effects of beta-amyloid (1-42) and induces neurite extension on B103 cells.

Clinical studies have shown that estrogen replacement therapy is associated with reduced risk of Alzheimer's disease (AD). We tested whether or not estrogen blocks neurotoxic effects of beta-amyloid (1-42) (A beta1-42) on cultured B103 cells. A beta1-42 (1 microM) induced typical necrotic cell death, as revealed by light and electron microscopic examinations. Co-administration of estrogen not only blocked A beta1-42 toxicity to a large degree, but also enhanced neurite extension. Pretreatment with estrogen was even more effective in blocking A beta1-42 toxicity. When added 18 h after the beginning of A beta1-42 treatment, estrogen was still effective in halting the progress of cell death and enhancing neurite extension. The protection against A beta1-42-induced neuronal death by estrogen was unlikely due to a blockade of lipid peroxidation injury, since estrogen completely failed to attenuate ferrous chloride-induced cell death. These results demonstrate that estrogen blocks A beta1-42-induced neurotoxicity and enhances neurite extension on B103 cells, both of which may well be underlying mechanisms of beneficial effects of estrogen in AD.

Amyloid beta-Peptides↗

PubMed1997

Flexibility in DNA recombination: structure of the lambda integrase catalytic core.

Lambda integrase is archetypic of site-specific recombinases that catalyze intermolecular DNA rearrangements without energetic input. DNA cleavage, strand exchange, and religation steps are linked by a covalent phosphotyrosine intermediate in which Tyr342 is attached to the 3'-phosphate of the DNA cut site. The 1.9 angstrom crystal structure of the integrase catalytic domain reveals a protein fold that is conserved in organisms ranging from archaebacteria to yeast and that suggests a model for interaction with target DNA. The attacking Tyr342 nucleophile is located on a flexible loop about 20 angstroms from a basic groove that contains all the other catalytically essential residues. This bipartite active site can account for several apparently paradoxical features of integrase family recombinases, including the capacity for both cis and trans cleavage of DNA.

Amino Acid Sequence↗

PubMed1996

Regulation of interleukin-4 on collagen gene expression by systemic sclerosis fibroblasts in culture.

This study was performed to evaluate the transcriptional regulation of interleukin-4 (IL-4) on collagen gene expression in systemic sclerosis (SSc) fibroblasts. The pro alpha 2(I) collagen promotor activity has been examined by transfection experiments and chloramphenicol acetyl transferase (CAT) assay. Maximal elevation of collagen synthesis was presented at a concentration of 5 ng/ml of IL-4. In the CAT assay, the percentage of acetylation was 7.0% +/- 2.0% in untreated controls and 12.5% +/- 3.5% in SSc fibroblasts at 5.0 ng/ml of IL-4. In normal skin (NS), 3.5% +/- 1.0%, and 10.2% +/- 2.5% were acetylated, respectively. The promoter activity was increased 1.8 +/- 0.7-fold and 2.9 +/- 1.3-fold in IL-4-treated SSc and NS, respectively, as compared to untreated groups. In Northern and dot-blot analysis, the level of types I and III collagen mRNA increased 1.1 +/- 0.3-fold and 1.3 +/- 0.3-fold, respectively, in IL-4-treated SSc fibroblasts compared to 3.0 +/- 0.4-fold and 3.0 +/- 0.3-fold, respectively, in NS fibroblasts. These data may indicate that IL-4 could be important in promoting biogenesis of collagen proteins by increased stability and transcription of the collagen mRNA. Also, transcriptional activation of collagen gene expression appears to have a less sensitive effect on SSc than on NS.

Adult↗

PubMed1996

Production of methyl 3-oxoartemisinate from methyl artemisinate by suspension cell culture of Mentha piperita.

Methyl artemisinate was fed to the suspension cell culture of Mentha piperita. The biotransformation product was isolated and identified as a novel compound, methyl 3-oxoartemisinate. The Mentha cells were apparently capable of extensively oxidizing at the allylic C-3 position, to give rise to an oxo group. The conversion of the fed methyl ester of the acid reached a maximum in 48 h with 5.5% conversion. The physicochemical data of the oxo compound are presented.

Acrylates↗