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D H Kim

Publications and source records attributed to D H Kim.

At least 73 records · Page 4Linked to original sources
PubMed2001

Cross-linking of MHC class II molecules with anti-MHC class II antibody or epitope peptide prevents resting B lymphocyte differentiation by inhibiting NF-kappaB-dependent gene expression.

To understand the mechanism(s) involved in anti-MHC class II antibody-mediated inhibition of B lymphocyte differentiation, we investigated the influence of anti-MHC class II antibody treatment on the gene expression of IL-6 in resting B lymphocytes, which had been known to be one of the most important cytokines involved in B cell physiology. The level of the IL-6 mRNA expression in the LPS-stimulated resting B cells was remarkably reduced by treatment of the corresponding anti-MHC class II antibodies. The inhibition was exerted in haplotype-specific and dose-dependent manners. Similarly, MHC class II-restricted epitope peptides, when applied as a dimer form, revealed haplotype-specific and dose-dependent inhibitory effects on the IL-6 gene expression by the LPS-stimulated B cells. In addition, treatment of the anti-MHC class II antibody and MHC class II-restricted epitope peptide inhibited, in haplotype-specific and dose-dependent manners, the activation of NF-kappaB, which had been known to be one of the critical transcription factors involved in the IL-6 gene expression. Interestingly, however, exogenous addition of the recombinant IL-6 did not reverse this inhibitory effect by the anti-MHC class II antibody. These results suggest that conjugation of the MHC class II molecules by the anti-MHC class II antibody inhibited B cell differentiation, possibly through the interruption of signaling pathways leading to the IL-6 gene expression via NF-kappaB activation in B lymphocytes.

Animals↗
PubMed2001

Characterization of the CDC10 product and the timing of events of the budding site of Saccharomyces cerevisiae.

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of septin filaments of unknown biochemical nature that lies under the inner surface of the plasma membrane in the neck that connects the mother cell to its bud. Mutants, defective in any of the four genes (CDC3, CDC10, CDC11, CDC12), lack these septin filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. The cloned CDC10 was fused to bacterial genes to generate antibodies specific for the CDC10 product was a constituent of the septin filaments. Cdc10p-specific antibodies for septin staining and actin-specific rhodamine-phalloidine were used to investigate the timing of the localization of septin and actin at the budding site using the immunofluorescence microscopic technique. In wild-type cells, the timing of the appearance and disappearance of these proteins was indistinguishable. In addition, the cdc10 mutant did not prevent actin localization at the budding site. The mutant that was blocked in the actin function also did not prevent the septin localization of the Cdc10p. This result may suggest an organizational independence between these proteins in the bud formation. Finally, the localization of septin and actin in the cdc24 mutant cell was examined. It was found that the CDC24 function was necessary for the organization of septin and actin at the budding site.

Actins↗
PubMed2001

The role of Tyr248 probed by mutant bovine carboxypeptidase A: insight into the catalytic mechanism of carboxypeptidase A.

We have investigated the function of Tyr248 using bovine wild-type CPA and its Y248F and Y248A mutants to find that the K(M) values were increased by 4.5-11-fold and the k(cat) values were reduced by 4.5-10.7-fold by the replacement of Tyr248 with Phe for the hydrolysis of hippuryl-L-Phe (HPA) and N-[3-(2-furyl)acryloyl]-Phe-Phe (FAPP), respectively. In the case of O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate (ClCPL), an ester substrate, the K(M) value was increased by 2.5-fold, and the k(cat) was reduced by 20-fold. The replacement of Tyr248 with Ala decreased the k(cat) values by about 18- and 237-fold for HPA and ClCPL, respectively, demonstrating that the aromatic ring of Tyr248 plays a critical role in the enzymic reaction. The increases of the K(M) values were only 6- and 5-fold for HPA and ClCPL, respectively. Thus, the present study indicates clearly that Tyr248 plays an important role not only in the binding of substrate but also in the enzymic hydrolysis. The kinetic results may be rationalized by the proposition that the phenolic hydroxyl of Tyr248 forms a hydrogen bond with the zinc-bound water molecule, causing further activation of the water molecule by reducing its pK(a) value. The pH dependency study of k(cat) values and the solvent isotope effects also support the proposition. A unified catalytic mechanism is proposed that can account for the different kinetic behavior observed in the CPA-catalyzed hydrolysis of peptide and ester substrates.

Amino Acid Sequence↗
PubMed2001

Interaction of HRC (histidine-rich Ca(2+)-binding protein) and triadin in the lumen of sarcoplasmic reticulum.

HRC (histidine-rich Ca(2+) binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca(2+) with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca(2+)-sensitive. Our data suggest that HRC may play a role in the regulation of Ca(2+) release from the sarcoplasmic reticulum by interaction with triadin.

Amino Acid Sequence↗
PubMed2001

Transparent thin-film TiO2 photocatalysts with high activity.

A transparent, thin-film TiO2 layer prepared by sol-gel deposition is shown to be more active for photocatalytic oxidation (PCO) of acetaldehyde, acetic acid, and toluene than Degussa P25 thin films. The sol-gel TiO2 adsorbs 30-70% less organic, but the PCO activity per adsorbed molecule is 3.5-8.5 times higher on the sol-gel TiO2 than on Degussa P25. In addition, less-reactive intermediates do not appear to form as readily on the sol-gel catalyst as they do on Degussa P25, and thus the sol-gel catalyst deactivates slower during toluene PCO. Rates were measured in transient experiments for a monolayer of adsorbed organic, and transient experiments are shown to be an effective way to measure rates, selectivities, surface coverages, and formation of less-reactive intermediates in the same set of experiments.

Air Pollution↗
PubMed2001

Characterization of a novel allele of glucose oxidase from a Korean wild type strain of Aspergillus niger.

We have identified a novel allele of the glucose oxidase (GO, EC1.1.3.4) gene (GO) from a Korean wild type strain of Aspergillus niger, ACMO4, with an increased GO activity in culture filtrate. Southern blot analyses of GO from ACMO4 (GO-ACMO4) revealed that the gene was present as a single copy in the genome of A. niger. However, its sequence differs from that of GO from A. niger ATCC 9029 (GO-ATCC9029). GO-ACMO4 appears to be a functional gene based on the fact that it is enzymatically active when heterologously expressed in yeast. Sequence comparisons of the coding region of GO-ACMO4 revealed 16 nucleotide changes that resulted in four amino acid substitutions; T432D, G517D, G530S, and Q542R. The GO proteins from both ATCC 9029 and ACMO4 were heterologously expressed, purified, and compared biochemically. The two enzymes showed no difference in their apparent Km value for glucose (30 mM), but the Vmax of GO-ACMO4 (515.6 unit/mg) was 10% higher than that of GO-ATCC 9029, resulting in a 10% higher specific activity.

Alleles↗
PubMed2001

Hydrogen peroxide is a novel inducer of connective tissue growth factor.

Connective tissue growth factor (CTGF) has recently been described as a fibrogenic factor and is greatly induced by various extracellular stimuli, such as transforming growth factor-beta (TGF-beta), dexamethasone, and serotonin. CTGF induces collagen type I and fibronectin, and the deposition of such molecules leads to fibrotic disease in many tissues. Intracellular reactive oxygen species (ROS) are generated by extracellular stress conditions and are produced as by-products of cellular metabolism. Imbalanced cellular redox status is a potent pathogenic factor that leads to various degenerative diseases, including tissue fibrosis. Since CTGF is believed to play a crucial role in fibrotic disease formation in many tissues, we examined the role of ROS in CTGF gene expression in human lens epithelial cell line B3. The results showed that CTGF was induced by reactive oxygen species such as hydrogen peroxide and hydroxyl radicals. Next, we examined whether CTGF induction by ROS is via newly synthesized TGF-beta. The results showed that ROS directly induced CTGF mRNA not via the increased TGF-beta synthesis or activation. Next, we treated AG490, which is the well-known inhibitor of Janus kinase (JAK), with hydrogen peroxide. AG490 abrogated the CTGF induction by ROS in a dose-dependent manner. The results suggest that JAK-2/-3 seems to be involved in the enhanced CTGF mRNA expression by hydrogen peroxide. In this report, we present that hydrogen peroxide is a novel inducer of CTGF gene expression and that JAK-2/-3 activation seems to play a role in CTGF induction.

Amino Acid Sequence↗
PubMed2001

Light and brassinosteroid signals are integrated via a dark-induced small G protein in etiolated seedling growth.

Plant growth and development are regulated through coordinated interactions between light and phytohormones. Here, we demonstrate that a dark-induced small G protein, pea Pra2, regulates a variant cytochrome P450 that catalyzes C-2 hydroxylation in brassinosteroid biosynthesis. The cytochrome P450 is dark-induced and predominantly expressed in the rapidly elongating zone of etiolated pea epicotyls, where Pra2 is also most abundant. Transgenic plants with reduced Pra2 exhibit a dark-specific dwarfism, which is completely rescued by exogenous brassinolide. Overexpression of the cytochrome P450 results in enhanced hypocotyl growth even in the light, which phenocopies the etiolated hypocotyls. We therefore propose that Pra2 and its orthologs are molecular mediators for the cross-talk between light and brassinosteroids in the etiolation process in plants.

Amino Acid Sequence↗
PubMed2001

Synthesis of optically active 2-alkyl-3,4-iminobutanoic acids. Beta-amino acids containing an aziridine heterocycle.

All four stereoisomers of 2-alkyl-3,4-iminobutanoic acid, a novel class of beta-amino acids bearing a chemically versatile aziridine ring, were synthesized starting with aspartic acid. The synthetic strategy involves the introduction of an alkyl group at the beta-position of fully protected optically active aspartic acid followed by the construction of an aziridine ring making use of the alpha-carboxylate and alpha-amino groups. The alpha-carboxylate was reduced to the corresponding alcohol, which was then subjected to cyclization to form an aziridine ring with the N-protected amino group. Removal of the protection groups yielded the target compounds.

Journal Article↗
PubMed2001

Rebleeding leads to poor outcome in ultra-early craniotomy for intracerebral hemorrhage.

BACKGROUND: A modest benefit was previously demonstrated for hematoma evacuation within 12 hours of intracerebral hemorrhage onset. Perhaps surgery within 4 hours would further improve outcome. METHODS: Adult patients with spontaneous supratentorial intracerebral hemorrhage were prospectively enrolled. Craniotomy and clot evacuation were commenced within 4 hours of symptom onset in all cases. Mortality and functional outcome were assessed at 6 months. This group of patients was compared with patients treated within 12 hours of symptom onset using the same surgical and medical protocols. RESULTS: The study was stopped after a planned interim analysis of 11 patients in the 4-hour surgery arm. Median time to surgery was 180 minutes; median hematoma volume was 40 mL; median baseline NIH Stroke Scale score was 19 and Glasgow Coma Scale score was 12. Six-month mortality was 36% and median Barthel score was 75 in survivors. Postoperative rebleeding occurred in four patients, three of whom died. A relationship between postoperative rebleeding and mortality was apparent (p = 0.03). Rebleeding occurred in 40% of the patients treated within 4 hours, compared with 12% of the patients treated within 12 hours (p = 0.11). There was a clear correlation between improved outcome and smaller postsurgical hematoma volume (p = 0.04). CONCLUSIONS: Surgical hematoma evacuation within 4 hours of symptom onset is complicated by rebleeding, indicating difficulty with hemostasis. Maximum removal of blood remains a predictor of good outcome.

Adult↗
PubMed2001

Folding mechanism of ketosteroid isomerase from Comamonas testosteroni.

Ketosteroid isomerase (KSI) from Comamonas testosteroni is a homodimeric enzyme with 125 amino acids in each monomer catalyzing the allylic isomerization reaction at rates comparable to the diffusion limit. Kinetic analysis of KSI refolding has been carried out to understand its folding mechanism. The refolding process as monitored by fluorescence change revealed that the process consists of three steps with a unimolecular fast, a bimolecular intermediate, and most likely unimolecular slow phases. The fast refolding step might involve the formation of structured monomers with hydrophobic surfaces that seem to have a high binding capacity for the amphipathic dye 8-anilino-1-naphthalenesulfonate. During the refolding process, KSI also generated a state that can bind equilenin, a reaction intermediate analogue, at a very early stage. These observations suggest that the KSI folding might be driven by the formation of the apolar active-site cavity while exposing hydrophobic surfaces. Since the monomeric folding intermediate may contain more than 83% of the native secondary structures as revealed previously, it is nativelike taking on most of the properties of the native protein. Urea-dependence analysis of refolding revealed the existence of folding intermediates for both the intermediate and slow steps. These steps were accelerated by cyclophilin A, a prolyl isomerase, suggesting the involvement of a cis-trans isomerization as a rate-limiting step. Taken together, we suggest that KSI folds into a monomeric intermediate, which has nativelike secondary structure, an apolar active site, and exposed hydrophobic surface, followed by dimerization and prolyl isomerizations to complete the folding.

Aldose-Ketose Isomerases↗
PubMed2001

Determination of aloesin in rat plasma using a column-switching high-performance liquid chromatographic assay.

A column-switching high-performance liquid chromatography (HPLC) method for the determination of aloesin in rat plasma using column-switching and ultraviolet (UV) absorbance detection is described. Plasma was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The determination of aloesin was accurate and repeatable, with a limit of quantitation of 10 ng/ml in plasma. The standard calibration curve for aloesin was linear (r=0.998) over the concentration range of 10-1000 ng/ml in rat plasma. The intra- and inter-day assay variabilities of aloesin ranged from 1.0 to 4.7% and 1.1 to 8.8%, respectively. This highly sensitive and simple method has been successfully applied to a pharmacokinetic study after oral administration of aloesin to rats.

Administration, Oral↗
PubMed2001

p16(INK4a) and histology-specific methylation of CpG islands by exposure to tobacco smoke in non-small cell lung cancer.

The p16(INK4a) protein inhibits cyclin-dependent kinase 4, a key regulator of progression through the G(1) phase of the cell cycle. Methylation of CpG islands in the promoter region is an important avenue for inactivation of p16. The mechanism of methylation of the p16 promoter region, however, has not been elucidated. Recent reports investigating p16 methylation in non-small cell lung cancer (NSCLC) suggest that carcinogens in tobacco smoke induce the DNA methylation process. We investigated the association between methylation of the p16 promoter region and exposure to tobacco smoke in 185 primary NSCLCS: We also studied the relationship of p16 methylation with mutation of the K-ras and p53 genes, as well as with methylation at the DAP-kinase and p14(ARF) loci. Finally, we evaluated the prognostic significance of p16 methylation in NSCLC. The prevalence of p16 methylation was greater in squamous cell carcinoma (41%) compared with adenocarcinoma (22%; P = 0.03; Fisher's exact test). Methylation of p16 was significantly associated with pack-years smoked (P = 0.007; Wilcoxon rank sum test), duration of smoking (P = 0.0009; Wilcoxon rank sum test), and negatively with the time since quitting smoking (P = 0.03; Wilcoxon rank sum test). No methylation of the nearby p14(ARF) locus was detected, and methylation of the DAP-kinase locus was not associated with either p16 methylation or with exposure to tobacco smoke. In patients with stage 1 adenocarcinoma, p16 methylation was an independent risk factor predicting significantly shorter postsurgery survival (P = 0.03), controlling for the significant effects of other factors, including K-ras mutation. These findings suggest that methylation of CpG islands in tobacco-associated cancers occurs in a gene- and tissue-specific manner and is induced directly or indirectly by exposure to tobacco smoke in NSCLC.

Aged↗
PubMed2001

15N NMR relaxation studies of backbone dynamics in free and steroid-bound Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni.

The backbone dynamics of Delta(5)-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been studied in free enzyme and its complex with a steroid ligand, 19-nortestosterone hemisuccinate (19-NTHS), by (15)N relaxation measurements. The relaxation data were analyzed using the model-free formalism to extract the model-free parameters (S(2), tau(e), and R(ex)) and the overall rotational correlation time (tau(m)). The rotational correlation times were 19.23 +/- 0.08 and 17.08 +/- 0.07 ns with the diffusion anisotropies (D( parallel)/D( perpendicular)) of 1.26 +/- 0.03 and 1.25 +/- 0.03 for the free and steroid-bound KSI, respectively. The binding of 19-NTHS to free KSI causes a slight increase in the order parameters (S(2)) for a number of residues, which are located mainly in helix A1 and strand B4. However, the majority of the residues exhibit reduced order parameters upon ligand binding. In particular, strands B3, B5, and B6, which have most of the residues involved in the dimer interaction, have the reduced order parameters in the steroid-bound KSI, indicating the increased high-frequency (pico- to nanosecond) motions in the intersubunit region of this homodimeric enzyme. Our results differ from those of previous studies on the backbone dynamics of monomeric proteins, in which high-frequency internal motions are typically restricted upon ligand binding.

Binding Sites↗
PubMed2001

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells.

3T3 Cells↗
PubMed2001

Fungicidal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Candida albicans.

The antifungal activity and mechanism of a 23-mer peptide, PMAP-23, derived from pig myeloid was investigated. PMAP-23 displayed strong antifungal activity against yeast and mold. To investigate the antifungal mechanism of PMAP-23, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed. Candida albicans treated with PMAP-23 showed higher fluorescence intensity by propidium iodide(PI) staining, which was similar to that of Melittin than untreated cells. Confocal microscopy showed that the peptide was located in the plasma membrane. The action of peptides against fungal cell membranes was examined by treating prepared protoplasts of C. albicans with the peptide and lipid vesicle titration test. The result showed that the peptide prevented the regeneration of fungal cell walls and induced release of the fluorescent dye trapped in the artificial membrane vesicles, indicating that the peptide exerts its antifungal activity by acting on the plasma lipid membrane.

Amino Acid Sequence↗
PubMed2001

Promoter methylation of DAP-kinase: association with advanced stage in non-small cell lung cancer.

Death associated protein (DAP)-kinase is a 16 kDa calmodulin-dependent serine/threonine kinase that carries a death domain at its C-terminus. DAP-kinase functions as a positive mediator of apoptosis that is induced by interferon-gamma. Recent studies suggest that DAP-kinase is involved in tumor metastasis and that it can be inactivated by methylation of CpG islands in the promoter region of the gene in some human tumors. However, little is known about the factors that are associated with the occurrence of DAP-kinase promoter methylation. We investigated both the possible associations of tobacco carcinogen and asbestos exposure with DAP-kinase promoter methylation, and the demographic and clinical factors associated with DAP-kinase promoter methylation in non-small cell lung cancer (NSCLC). One hundred and eighty-five patients diagnosed with NSCLC undergoing surgical resection from June, 1992 through December, 1996 at Massachusetts General Hospital participated in this study. Methylation-Specific PCR (MSP), performed using fresh-frozen tissue, was used to determine the methylation status of the promoter region of the DAP-kinase gene. Forty-seven (25%) of 185 tumors showed DAP-kinase promoter methylation. There was a significant association between methylation and an advanced pathologic stage (P=0.003, Fisher's exact test). Methylation of the DAP-kinase promoter was also associated with an increase in tumor size (P=0.009, Fisher's exact test) and lymph node involvement (P=0.04). No association was found between promoter methylation of DAP-kinase and k-ras or p53 mutation. In addition there was no association with a history of exposure to tobacco or asbestos. Controlling for age, sex, and histology, the odds ratios describing the association of DAP-kinase hypermethylation with stage were 2.70 (1.13--6.45), 3.11 (1.37--7.08) and 7.77 (1.21--50.03) in stages II, III and IV, respectively. Stage I cases with DAP-kinase promoter methylation had worse overall survival, but with the small sample size and limited follow-up this did not reach statistical significance. Our findings suggest that methylation of the promoter region of the DAP-kinase gene is not associated with exposure to tobacco or asbestos. However, they strongly suggest that DAP-kinase may be important in the progression of non-small cell lung cancer from early to late stage disease.

Adult↗