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Biomedical subjects

Y R Kim

Publications and source records attributed to Y R Kim.

At least 37 records · Page 2Linked to original sources
PubMed2000

Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA.

In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS (toxRS(Vc)) homologs in V. vulnificus. By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus (toxRS(Vp)), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb BglII-HindIII fragment and a 1.2-kb HindIII fragment containing two complete open reading frames and one partial open reading frame attributable to toxR(Vv), toxS(Vv), and htpG(Vv) were cloned. ToxR(Vv) shared 55.0 and 63.0% sequence homology with ToxR(Vc) and ToxR(Vp), respectively. ToxS(Vv) was 71.5 and 65.7% homologous to ToxS(Vc) and ToxS(Vp), respectively. The amino acid sequences of ToxRS(Vv) showed transmembrane and activity domains similar to those observed in ToxRS(Vc) and ToxRS(Vp). Western blot analysis proved the expression of ToxR(Vv) in V. vulnificus. ToxRS(Vv) enhanced, in an Escherichia coli background, the expression of the V. vulnificus hemolysin gene (vvhA) fivefold. ToxRS(Vv) also activated the ToxR(Vc)-regulated ctx promoter incorporated into an E. coli chromosome. A toxR(Vv) null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The toxR(Vv) mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxR(Vv) may regulate the virulence expression of V. vulnificus.

Amino Acid Sequence↗
PubMed2000

A rapid and accurate closed-tube immunoassay for platelets on an automated hematology analyzer.

Accurate and precise platelet counts are important for patients with severe thrombocytopenia or who are receiving chemotherapy. We developed a novel flow cytometric analysis of platelets that may be particularly valuable for assessing the necessity for platelet transfusions. This ImmunoPlt (CD61) assay is based in part on CD61 monoclonal antibody labeling and has been automated and implemented on the CELL-DYN 4000 hematology analyzer. It is well suited for thrombocytopenic specimens, since it reduces interference by nonplatelet particles. It takes less than 5 minutes from closed-tube aspiration to report. Data for more than 350 thrombocytopenic specimens demonstrate that the ImmunoPlt (CD61) assay is more accurate than the optical scatter or the impedance count for specimens with platelet counts between 1 and 60 x 10(3)/microL (1 and 60 x 10(9)/L). The ImmunoPlt (CD61) assay is more precise than the optical scatter or the impedance count for specimens with platelet counts between 1 and 50 x 10(3)/microL (1 and 50 x 10(9)/L).

Antibodies, Monoclonal↗
PubMed1999

Spectral characterization and chemical modification of FMN-containing ascorbyl free-radical reductase from Pleurotus ostreatus.

Ascorbyl free-radical reductase was purified 1143-fold with an overall yield of 9.9% from the cytosolic fraction of Pleurotus ostreatus. The native enzyme had a molecular mass of 127 kDa and SDS/PAGE revealed that the enzyme consists of two subunits, each with a molecular mass of 62 kDa. The enzyme utilized only NADH as an electron donor. The enzyme was highly specific for ascorbyl free radical as an electron acceptor and capable of catalysing the reduction of ferricyanide and 2,6-dichloroindophenol as artificial electron acceptors. The apparent K(m) values of the enzyme towards NADH and ascorbyl free radical were 35+/-0.22 and 2.1+/-0.03 microM, respectively. The catalytic mechanism of this enzyme is of Ping Pong type. The enzyme contained FMN as a prosthetic group and showed the characteristic absorption spectrum ascribed to the charge-transfer interaction of thiolate anion with FMN. The enzyme contained eight cysteine residues per monomer and was inactivated more rapidly by mercurials than by thiol-alkylating reagents. Kinetic analysis of the inactivation process revealed that the enzyme had 1 mol of thiol group/mol of subunit in the active site with a pK(a) of 6.9. The modification of the thiol group of the enzyme caused the loss of charge-transfer absorbance centred at 540 nm and blocked the electron-transfer process from NADH to FMN. The modification of lysine, arginine and histidine residues led to the loss of its activity. Unlike the active enzyme, the fluorescence quenching of NADH was not observed in the lysine-modified enzyme, which implies that lysine residues can participate in the interaction with NADH.

Enzyme Inhibitors↗
PubMed1999

Copper- and zinc-containing superoxide dismutase and its gene from Candida albicans.

Cytosolic copper- and zinc-containing superoxide dismutase was purified 136-fold with an overall yield of 2.5% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 39.4 kDa and the enzyme was composed of two identical subunits with a molecular mass of 19.6 kDa. The enzyme was stable in the range of pH 4.0-9.0 and up to 55 degrees C. The ultraviolet-visible absorption spectrum of the enzyme showed the absorption band of copper- and zinc-containing superoxide dismutase at 660 nm. The atomic absorption analysis revealed that the enzyme contained 0.87 g-atom of copper and 0.79 g-atom of zinc per mole of subunit. The N-terminal amino acid sequence alignments up to the 40th residue showed that copper- and zinc-containing superoxide dismutase from C. albicans has high similarity to other eukaryotic copper- and zinc-containing superoxide dismutases. The sod1 encoding copper- and zinc-containing superoxide dismutase has been cloned using a polymerase chain reaction fragment as a probe. Sequence analysis of the sod1 predicted a copper- and zinc-containing superoxide dismutase that contains 154 amino acids with a molecular mass of 16143 Da and displayed 79%, 69%, and 57% sequence identity to the homologues of Neurospora crassa, Saccharomyces cerevisiae, and bovine, respectively. The cloned sod1 contained an intron of 245 nucleotides, which was verified by reverse transcription-polymerase chain reaction.

Amino Acid Sequence↗
PubMed1999

Manganese-containing superoxide dismutase and its gene from Candida albicans.

Mitochondrial manganese-containing superoxide dismutase was purified around 112-fold with an overall yield of 1.1% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 106 kDa and the enzyme was composed of four identical subunits with a molecular mass of 26 kDa. The enzyme was not sensitive to either cyanide or hydrogen peroxide. The N-terminal amino acid sequence alignments (up to the 18th residue) showed that the enzyme has high similarity to the other eukaryotic manganese-containing superoxide dismutases. The gene sod2 encoding manganese-containing superoxide dismutase has been cloned using a product obtained from polymerase chain reaction. Sequence analysis of the sod2 predicted a manganese-containing superoxide dismutase that contains 234 amino acid residues with a molecular mass of 26173 Da, and displayed 57% sequence identity to the homologue of Saccharomyces cerevisiae. The deduced N-terminal 34 amino acid residues may serve as a signal peptide for mitochondrial translocation. Several regulatory elements such as stress responsive element and haem activator protein 2/3/4/5 complex binding sites were identified in the promoter region of sod2. Northern analysis with a probe derived from the cloned sod2 revealed a 0.94-kb band, which corresponds approximately to the expected size of mRNA deduced from sod2.

Amino Acid Sequence↗
PubMed1999

Dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha induced by guanidine hydrochloride.

The dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha (TNF-alpha) during denaturation at different guanidine hydrochloride (GdnHCl) concentrations (0-4.2 M) was investigated by steady-state fluorescence spectroscopy, potassium iodide (KI) fluorescence quenching, far-UV circular dichroism (CD), picosecond time-resolved fluorescence lifetime, and anisotropy decay measurements. We utilized the intrinsic fluorescence of Trp-28 and Trp-114 to characterize the conformational changes involved in the equilibrium unfolding pathway. The detailed unfolding pathway under equilibrium conditions was discussed with respect to motional dynamics and partially folded structures. At 0-0.9 M [GdnHCl], the rotational correlation times of 22-25 ns were obtained from fluorescence anisotropy decay measurements and assigned to those of trimeric states by hydrodynamic calculation. In this range, the solvent accessibility of Trp residues increased with increasing [GdnHCl], suggesting the slight expansion of the trimeric structure. At 1.2-2.1 M [GdnHCl], the enhanced solvent accessibility and the rotational degree of freedom of Trp residues were observed, implying the loosening of the internal structure. In this [GdnHCl] region, TNF-alpha was thought to be in soluble aggregates having distinct conformational characteristics from a native (N) or fully unfolded state (U). At 4.2 M [GdnHCl], TNF-alpha unfolded to a U-state. From these results, the equilibrium unfolding pathway of TNF-alpha, trimeric and all beta-sheet protein, could not be viewed from the simple two state model (N-->U).

Anisotropy↗
PubMed1999

Anti-allergic actions of the leaves of Castanea crenata and isolation of an active component responsible for the inhibition of mast cell degranulation.

The anti-allergic actions of the leaves of Castanea crenata (Fagaceae) were studied. The water extract demonstrated potent anti-allergic actions in in vivo and in vitro experiments. The oral or intraperitoneal administration of the extract (100 or 200 mg/kg) caused a significant inhibition of the 48 hr-PCA (up to 90%) and the vascular permeability induced by histamine or serotonin in rats (about 80%). The anaphylactic release of beta-hexosaminidase from RBL-2H3 cells was also significantly inhibited by the extract in a dose-dependent manner with an IC50 value of 230 microg/ml. The activity-guided fractionation of the extract, based on the determination of inhibitory effect upon the release of beta-hexosaminidase, led to the isolation of quercetin as an active principle responsible for the inhibition of degranulation.

Animals↗
PubMed1999

Panax ginseng blocks morphine-induced thymic apoptosis by lowering plasma corticosterone level.

The effects of Panax ginseng on morphine-induced immune suppression were studied. Morphine (20 mg/kg, SC, 4 days) decreased body weight increment rate and caused atrophy of thymus and spleen. These changes were partly reversed by concomitant administration of ginseng total saponin (GTS, 100 mg/kg, oral, 9 days). Morphine elevated the serum corticosterone level and caused the DNA fragmentation of thymocytes. These sequential events were completely blocked by a concomitant administration of GTS. Flow cytometry analysis showed that GTS specifically blocked morphine-induced apoptosis of thymocytes.

Animals↗
PubMed1999

Lyso-PAF analogues and lysophosphatidylcholines from the marine sponge Spirastrella abata as inhibitors of cholesterol biosynthesis.

A series of phospholipids, including previously undescribed compounds 4-7, were isolated by a bioactivity-guided fractionation from the marine sponge Spirastrella abata as inhibitors of cholesterol biosynthesis in human liver cells. These compounds were identified as lyso-PAF analogues (1-5) and lysophosphatidylcholines (6, 7) based on NMR and MS analyses. Compounds 1-7 specifically blocked the conversion of lanosterol into cholesterol in the Chang liver cell.

Animals↗
PubMed1998

Phosphorylation of rat insulin-like growth factor binding protein-1 does not affect its biological properties.

Insulin-like growth factors (IGFs) I and II stimulate growth and expression of specific genes through binding to cell membrane receptors. IGF binding proteins also bind IGF-I with higher affinity than the receptor. They are found in the circulation and tissues and can modulate IGF actions. Human IGFBP-1 is phosphorylated on serine residues, which increases its affinity for IGF-I. An acidic, presumably phosphorylated, form of human IGFBP-1 inhibits IGF-I-stimulated DNA synthesis in cultured cells, while a less acidic, unphosphorylated form potentiates this function. Phosphorylation of human IGFBP-3, however, does not affect its affinity for IGF-I. Previously we found that multiple forms of rat IGFBP-1 are obtained by anion-exchange chromatography, raising the possibility that it also is phosphorylated, which led us to examine its properties. Phosphopeptide analysis of 32P-labeled, immunoprecipitated rat IGFBP-1 synthesized by H-4-II-EC3 rat hepatoma cells indicated that it is phosphorylated on two sites that were deduced to be ser107 and ser132 in the central nonconserved domain. Dephosphorylation of purified phosphorylated rat IGFBP-1 did not affect its affinity for IGF-I or its specific binding activity, and the dephosphorylated form inhibited DNA synthesis in 3T3 cells. Incubation of cells labeled with radioactive proline in the presence of monensin and brefeldin A, which inhibit secretion at different sites, led to intracellular accumulation of the least phosphorylated form of rat IGFBP-1, but prevented further phosphorylation. The results suggested that phosphorylation occurs at two sites in cells, the cis-Golgi and the trans-Golgi network. In summary, these studies have shown that rat IGFBP-1 is phosphorylated on two sites by reactions that occur in different secretory organelles and that similar to human IGFBP-3, but unlike human IGFBP-1, phosphorylation does not affect its affinity for IGF-I.

3T3 Cells↗
PubMed1998

Simultaneous differentiation and quantitation of erythroblasts and white blood cells on a high throughput clinical haematology analyser.

After the neonatal period, the presence of nucleated red blood cells (NRBC) in peripheral blood is indicative of pathology. Despite the clinical utility of such measurements, automated NRBC counting has hitherto not been available on routine automated blood cell counting analysers. To address this, an automated method for the analysis of NRBC was developed and incorporated into the Abbott Cell Dyn 4000 (CD4000) haematology analyser. The system white blood cell (WBC) reagent was specifically formulated to preserve concomitantly white blood cell (WBC) morphology, rapidly lyse red blood cell and NRBC membranes, and subsequently stain NRBC nuclei with a nucleotide specific fluorochrome dye (Kim et al. 1996a). The fluorochrome itself does not permeabilize the membrane of intact viable white blood cells. The sample is processed by flow cytometry and the signals generated from an argon-ion laser light source are analysed. Axial light loss (AxLL), intermediate angle light scatter (IAS) and red fluorescence (FL3) are used to discriminate between particles of various types. By using these discriminators in a three-dimensional approach, NRBC from a discrete cluster which can easily be separated from leucocytes and enumerated as a distinct cell population during the optical WBC differential analysis. Consequently, accurate absolute WBC counts and differentials can be obtained even in the presence of NRBC. Background 'noise' (both fluorescent and non-fluorescent) from platelets. Howell-Jolly bodies, basophilic stippling, RNA from lysed reticulocytes, and DNA from leucocyte and megakaryocytic fragments are essentially eliminated (Kim et al. 1996b). While the membranes of intact and viable leucocytes remain impermeable to the passage of the fluorochrome stain, leucocytes with damaged membranes are permeable to the dye and generate FL3+ signals. Such cells, which are commonly seen as a consequence of sample ageing as well as in some distinctive pathologies, are identified by the algorithm (using their AxLL signal size) and are labelled as non-viable. Moreover, because non-viable leucocytes are retained in the WBC count and differential analyses, the CD4000 is further able to provide both numerical and graphical data regarding the relative frequency of viable and non-viable components. This additional information can serve as valuable 'decision-drivers' in the laboratory data review process.

Automation↗
PubMed1998

D-Erythroascorbic acid is an important antioxidant molecule in Saccharomyces cerevisiae.

D-Arabinono-1,4-lactone oxidase catalysing the final step of D-erythroascorbic acid biosynthesis was purified from the mitochondrial fraction of Saccharomyces cerevisiae. Based on the amino acid sequence analysis of the enzyme, an unknown open reading frame (ORF), YML086C, was identified as the ALO1 gene encoding the enzyme. The ORF of ALO1 encoded a polypeptide consisting of 526 amino acids with a calculated molecular mass of 59493Da. The deduced amino acid sequence of the enzyme shared 32% and 21% identity with that of L-gulono-1,4-lactone oxidase from rat and L-galactono-1,4-lactone dehydrogenase from cauliflower, respectively, and contained a putative transmembrane segment and a covalent FAD binding site. Blot hybridization analyses showed that a single copy of the gene was present in the yeast genome and that mRNA of the ALO1 gene was 1.8kb in size. In the alo1 mutants, D-erythroascorbic acid and the activity of D-arabinono-1,4-lactone oxidase could not be detected. The intracellular concentration of D-erythroascorbic acid and the enzyme activity increased up to 6.9-fold and 7.3-fold, respectively, in the transformant cells carrying ALO1 in multicopy plasmid. The alo1 mutants showed increased sensitivity towards oxidative stress, but overexpression of ALO1 made the cells more resistant to oxidative stress.

Amino Acid Sequence↗
PubMed1997

Differential effects of ascorbate depletion and alpha,alpha'-dipyridyl treatment on the stability, but not on the secretion, of type IV collagen in differentiated F9 cells.

Ascorbic acid stimulates secretion of type I collagen because of its role in 4-hydroxyproline synthesis, but there is some controversy as to whether secretion of type IV collagen is similarly affected. This question was examined in differentiated F9 cells, which produce only type IV collagen, by labeling proteins with [14C]proline and measuring collagen synthesis and secretion. Hydroxylation of proline residues in collagen was inhibited to a greater extent in cells treated with the iron chelator alpha,alpha'-dipyridyl (97.7%) than in cells incubated without ascorbate (63.1%), but both conditions completely inhibited the rate of collagen secretion after 2-4 h, respectively. Neither treatment affected laminin secretion. Collagen synthesis was not stimulated by ascorbate even after treatment for 2 days. On SDS polyacrylamide gels, collagen produced by alpha,alpha'-dipyridyl-treated cells consisted mainly of a single band that migrated faster than either fully (+ ascorbate) or partially (- ascorbate) hydroxylated alpha 1(IV) or alpha 2(IV) chains. It did not contain interchain disulfide bonds or asn-linked glycosyl groups, and was completely digested by pepsin at 15 degrees C. These results suggested that it was a degraded product lacking the 7 S domain and that it could not form a triple helical structure. In contrast, the partially hydroxylated molecule contained interchain disulfide bonds and it was cleaved by pepsin to collagenous fragments similar in size to those obtained from the fully hydroxylated molecule, but at a faster rate. Kinetic experiments and monensin treatment suggested that completely unhydroxylated type IV collagen was degraded intracellularly in the endoplasmic reticulum or cis Golgi. These studies indicate that partial hydroxylation of type IV collagen confers sufficient helical structure to allow interchain disulfide bond formation and resistance to pepsin and intracellular degradation, but not sufficient for optimal secretion.

2,2'-Dipyridyl↗
PubMed1997

Evaluation of the Abbott Cell Dyn 4000 automated fluorescent reticulocyte measurements: comparison with manual, FACScan and Sysmex R1000 methods.

The Abbott Cell Dyn 4000 (CD4000) is the first haematology analyser in which fully-automated reticulocyte measurements can be routinely determined by fluorescence as part of the full blood count. This communication reports the first evaluation of this method which was undertaken by three independent reference laboratories in Belgium, Germany and Italy. A total of 695 different samples was entered into the study which was designed to compare CD4000 reticulocyte information (enumeration and qualitative maturational data) with results determined in parallel with the existing manual (supravital staining) reference procedure, and two semi-automated fluorescent assays (Becton Dickinson FACScan and Sysmex R1000 instruments). These studies revealed good agreement between the CD4000 and the manual procedure, with no inter-method bias. Comparison of CD4000 and FACScan reticulocyte measurements, however, indicated a distinct tendency for the FACScan to give higher reticulocyte estimates than the CD4000. Finally, the comparison of the CD4000 with the Sysmex R1000 showed excellent agreement in the range 0-6% reticulocytes, although there was some inter-method bias in the higher range (> 15%). Analysis of agreement levels between the methods using specific 'clinical decision points' confirmed the tendency for overestimation by the FACScan, in that 58% of the samples with a reticulocytopenia of < 0.5% as defined by the CD4000 gave FACScan results within the normal range (0.5-1.8%). In contrast, there was absolute agreement between the CD4000 and the Sysmex R1000 for all reticulocytopenias. Comparison (195 samples) of instrument fluorescent reticulocyte maturation profiles demonstrated an exponential relationship (r = 0.78) between CD4000 IRF and R1000 HFR (highly fluorescent reticulocyte fraction) values. The suggestion that the CD4000 IRF values includes some of the MFR as well the HFR reticulocyte fraction was confirmed as the correlation between the CD4000 IRF and the Sysmex R1000 MFR plus HFR percentages was linear (r = 0.82). This study confirms a high performance level for the CD4000 automated fluorescent reticulocyte method.

Autoanalysis↗
PubMed1997

Foodborne outbreaks of human toxoplasmosis.

Two outbreaks of acute toxoplasmosis involving 8 adult patients in Korea were linked to eating uncooked pork. In the first outbreak, 3 patients developed unilateral chorioretinitis within 3 months of eating a meal consisting of raw spleen and liver of a wild pig. In the second outbreak, 5 of 11 soldiers who ate a meal consisting of raw liver of a domestic pig developed lymphadenopathy. All 8 patients had high levels of IgG Toxoplasma gondii antibodies (> or = 1:1024) in the Sabin-Feldman dye test, modified agglutination test incorporating mercaptoethanol, and latex agglutination test. T. gondii IgM antibodies persisted in these patients for several months. Most patients had a favorable response to anti-T. gondii chemotherapy with pyrimethamine and sulfanomides.

Adult↗
PubMed1997

A small epidemiological survey for vivax malaria in Kimpo-gun, Kyonggi-do, Korea undertaken after detecting two consecutive cases.

On July and August 1997, two 15-year-old mates of a football team of Tongjin Middle School in northern Kyonggi-do, Korea were consecutively diagnosed as vivax malaria by peripheral blood smear. They had no histories of travelling abroad or drug abuse. They witnessed that other mates in the team were ill of fever in the same period. A small survey was therefore undertaken to determine whether vivax malaria was outbroken locally. A total of 57 students of the team living together in a dormitory was examined for history of fever, presence of splenomegaly, blood smear and anti-P. vivax antibody test by immunofluorescent antibody test (IFAT). Except for the above two patients, only one case revealed a marginal titer of IFAT. No other positive findings of vivax malaria were found. In the results of this local survey, no more cases of vivax malaria were revealed except the two sporadic cases.

Adolescent↗
PubMed1996

A heme-containing ascorbate oxidase from Pleurotus ostreatus.

A novel type of ascorbate oxidase was purified 420-fold from the cytosolic fraction of the mycelia of Pleurotus ostreatus with an overall yield of 13%. The molecular mass of the native enzyme determined by high performance gel permeation chromatography was 94 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme consists of two subunits with a molecular mass of 46 kDa. The N-terminal amino acid sequence of the enzyme was Asp-Val-Lys-Thr-Leu-Gln-Glu-His-Leu-Gln-Leu-Ala-Leu-Met-Val-. The enzyme was optimally active at pH 5.2, monitored at 37 degrees C. The enzyme had affinity toward L-ascorbic acid, D-ascorbic acid, L-erythroascorbic acid, and D-erythroascorbic acid. Under optimal conditions, the Km value of the enzyme toward L-ascorbic acid was 0.48 mm. The absorption spectra of the native enzyme exhibited a Soret maximum at 418 nm in its oxidized form and at 426 nm in its reduced form, and alpha and beta bands at 558 and 527 nm only in its reduced form, respectively. On the basis of spectral changes after treatment with cyanide and carbon monoxide, the enzyme is a hemoprotein, quite similar to b-type cytochrome, and contains 2 mol of heme per molecule. The reaction catalyzed by the enzyme was L-ascorbic acid + O2 --> dehydro-L-ascorbic acid + H2O2.

Amino Acid Sequence↗