Biomedical subjects

H Kim

Publications and source records attributed to H Kim.

At least 361 records · Page 20Linked to original sources

PubMed1999

Assay of 2-naphthol in human urine by high-performance liquid chromatography.

This paper describes a novel liquid chromatographic method for the quantitation of 2-naphthol in human urine. Urine samples were extracted after enzymatic hydrolysis of glucuronides and sulfates; 2-naphthol was then separated using reversed-phase high-performance liquid chromatography. The corresponding detection limits were 0.04 ng/ml for the standard sample in acetonitrile and 0.13 ng/ml for urine samples. The level of urinary 2-naphthol in 100 Korean shipyard workers was analyzed using this new method. The level ranged from 0.21 ng/ml (0.26 micromol/mol creatinine) to 34.19 ng/ml (59.11 micromol/mol creatinine), and the mean+/-standard deviation was 5.08 ng/ml (6.60 micromol/mol creatinine)+/-5.75 ng/ml (9.22 micromol/mol creatinine). The mean+/-standard deviation of urinary 2-naphthol level of smokers, 7.03 ng/ml (8.49 micromol/mol creatinine)+/-6.16 ng/ml (10.23 micromol/mol creatinine), was significantly higher than that of non-smokers, 2.49 ng/ml (4.10 micromol/mol creatinine)+/-3.92 ng/ml (7.03 micromol/mol creatinine).

Acetonitriles↗

PubMed1999

Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning.

Lipase of Staphylococcus haemolyticus L62 was purified from culture supernatant and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for the hydrolysis of olive oil was 28 degrees C and pH 8.5, respectively. The enzyme was stable up to 50 degrees C in the presence of Ca(2+)and over the pH range 5-11. It had high hydrolytic activity against tributyrin, tripropionin, and trimyristin among various triglycerides. The gene encoding the lipase was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 2136 bp, which encodes a preproenzyme of 711 amino acids. The preproenzyme is composed of a signal peptide (60 aa), a pro-peptide (259 aa), and a mature enzyme (392 aa). The mature enzyme has 49-67% amino acid sequence homology with other staphylococcal lipases.

Amino Acid Sequence↗

PubMed1999

Neuroprotective effect of high glucose against NMDA, free radical, and oxygen-glucose deprivation through enhanced mitochondrial potentials.

Cultured cortical neurons maintained in 25 mM glucose underwent a widespread neuronal death after exposure to NMDA, AMPA, and kainate. Among these, NMDA toxicity was substantially reduced in neurons maintained in 100 mM glucose. NMDA-induced increase in [Ca(2+)](i) and reactive oxygen species was attenuated in neurons maintained in high glucose that revealed increased mitochondrial membrane and redox potentials as determined using rhodamine 123 and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. p-trifluoromethoxy-phenylhydrazone, KCN, and rotenone, the selective inhibitors of mitochondrial potential, abrogated neuroprotective effect of high glucose against NMDA. The neuroprotective action of high glucose was extended against oxygen or combined oxygen-glucose deprivation. The present study provides evidence that prolonged exposure of cortical cells to high glucose attenuates NMDA- and free radical-mediated neuronal death via enhanced mitochondrial function.

Animals↗

PubMed1999

Active site determination of yeast geranylgeranyl protein transferase type I expressed in Escherichia coli.

The ram2 and cal1 genes encode the alpha and beta subunits of yeast geranylgeranyl protein transferase type I (GGPT-I), respectively. Arginine 166 of the beta subunit was changed to isoleucine (betaR166I), histidine 216 to aspartic acid (betaH216D), and asparagine 282 to alanine (betaN282A) by sequential PCR using mutagenic primers. The mutants were expressed under the same conditions as the wild-type and were assayed for GGPT-I activity. Wild-type yeast GGPT-I, alphaH145D, alphaD140N, betaR166I, betaH216D and betaN282A mutant GGPT-Is were partially purified by ammonium sulfate fractionation followed by a Q-Sepharose column. Characterization studies were performed using the active fraction of the Q-Sepharose column. In the chemical modification reactions, the catalytic activity of purified enzyme decreased in proportion to the concentration of modifying reagents, such as phenylglyoxal and diethyl pyrocarbonate (DEPC). Geranylgeranyl pyrophosphate (GGPP) protected the enzyme activity from the modification with phenylglyoxal. The measurement of GGPP binding to wild-type and five mutant GGPT-Is was performed by a gel-filtration assay. The binding of GGPP to the betaR166I mutant was low and the Km value for GGPP in the betaR166I mutant increased about 29-fold. Therefore, the results suggest a role for this arginine residue that directly influences the GGPP binding. The activity of the DEPC-modified GGPT-I was inhibited by 80% at 5 mM DEPC. The differential absorption at 242 nm may suggest that at this concentration the modified histidine residues were 1.5 mol per GGPT-I. The protein substrate, glutathione S-transferase fused undecapeptide (GST-CAIL) protected the enzyme from inactivation by DEPC, and the Km value for GST-CAIL in the betaH216D mutant increased about 12-fold. The trypsin digestion of [14C]DEPC-modified enzyme yielded a single radioactive peptide. As a result of the sequence of this radioactive peptide, the histidine 216 residue was assumed to be an essential part of binding of peptide substrate.

Alkyl and Aryl Transferases↗

PubMed1999

Mutation spectrum of 4-nitroquinoline N-oxide in the lacI transgenic Big Blue Rat2 cell line.

This paper describes the spectrum of mutations induced by 4-nitroquinoline N-oxide (4-NQO) in the lacI target gene of the transgenic Big Blue Rat2 cell line. There are only a few report for the mutational spectrum of 4-NQO in a mammalian system although its biological and genetic effects have been well studied. Big Blue Rat2 cells were treated with 0.03125, 0.0625 or 0.125 microg/ml of 4-NQO, the highest concentration giving 85% survival. Our results indicated that the mutant frequency (MF) induced by 4-NQO was dose-dependent with increases from three- to seven-fold. The DNA sequence analysis of lacI mutants from the control and 4-NQO treatment groups revealed an obvious difference in the spectra of mutations. In spontaneous mutants, transition (60%) mutations, especially G:C-->A:T transition (45%), were most frequent. However, the major type of base substitution after treatment of 4-NQO was transversions (68.8%), especially G:C-->T:A (43.8%), while only 25% of mutants were transitions. These results are consistent with those produced by 4-NQO in other systems and the transgenic assay system will be a powerful tool to postulate more accurately the mechanism of chemical carcinogenesis involved.

4-Nitroquinoline-1-oxide↗

PubMed1999

Molecular cloning, expression, and characterization of angiopoietin-related protein. angiopoietin-related protein induces endothelial cell sprouting.

Using degenerate polymerase chain reaction, we isolated a cDNA encoding a novel 493-amino acid protein from human and mouse adult heart cDNAs and have designated it angiopoietin-related protein-2 (ARP2). The NH(2)-terminal and COOH-terminal portions of ARP2 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in angiopoietins. ARP2 has two consensus glycosylation sites and a highly hydrophobic region at the NH(2) terminus that is typical of a secretory signal sequence. Recombinant ARP2 expressed in COS cells is secreted and glycosylated. In human adult tissues, ARP2 mRNA is most abundant in heart, small intestine, spleen, and stomach. In rat embryos, ARP2 mRNA is most abundant in the blood vessels and skeletal muscles. Endothelial and vascular smooth muscle cells also contain ARP2 mRNA. Recombinant ARP2 protein induces sprouting in vascular endothelial cells but does not bind to the Tie1 or Tie2 receptor. These results suggest that ARP2 may exert a function on endothelial cells through autocrine or paracrine action.

Amino Acid Sequence↗

PubMed1999

Expression of fas ligand in human hepatoma cell lines: role of hepatitis-B virus X (HBX) in induction of Fas ligand.

It has been postulated that tumor cells expressing Fas ligand (FasL) can evade immune surveillance by inducing apoptosis in T cells expressing Fas. In this study, we investigated FasL expression in 13 human hepatoma cell lines. Strong FasL expression was detected by reverse transcription-polymerase chain reaction or immunofluorescence in Hep G2.2.15, in which the hepatitis-B-virus (HBV) genome was transfected, and in SNU-354, which showed HBx transcripts. To determine the biological activity of FasL, Hep G2.2. 15 was co-cultured with MOLT-4, T-cell-leukemia cells. Hep G2.2.15 induced apoptosis in MOLT-4 and this was inhibited by the antagonistic anti-Fas antibody, ZB4. For further analysis of the role of HBx in the induction of FasL, PLC/PRF/5 cells were transfected transiently with the HBV genome, or HBx, or the frameshift mutant of HBx. In PLC/PRF/5 cells transfected with the HBV genome or HBx but not in cells transfected with the frameshift mutant of HBx, FasL expression was detected. Our data suggest that HBx plays a role in the induction of FasL in hepatoma cells and in the escape from immune surveillance.

Apoptosis↗

PubMed1999

Magnetic resonance image-based cerebellar volumetry in healthy Korean adults.

The effects of age and gender on cerebellar size have not been established yet. To understand these effects, the area of cerebellar vermis and the volume of cerebellum were measured using serial magnetic resonance images of 124 Korean adults free of neurologic symptoms and signs. Cerebellar volume of male was significantly larger than that of female, although the size of vermis did not show significant gender difference. Correlation analysis revealed that cerebellar volume was not affected by aging. Regressional analysis demonstrated that female vermis had a tendency to shrink after age of 50, whereas male vermis and total cerebellar volume in both sexes were not altered with aging. The different response of vermis with aging and maintenance of cerebellum volume need to be more explored.

Adult↗

PubMed1999

Arylpropane-1,3-diols in lignins from normal and CAD-deficient pines.

Significant quantities of arylopropane-1,3-diols have been identified in lignins isolated from a CAD-deficient pine mutant; smaller amounts are also present in lignins from normal pine. They arise from dihydroconiferyl alcohol via the action of peroxidases which are responsible for the radical generation steps of lignification. The structures in the complex lignin polymers are proven using 2D and 3D NMR of isolated lignin fractions.

Alcohol Oxidoreductases↗

PubMed1999

Beta-catenin mutations are specific for colorectal carcinomas with microsatellite instability but occur in endometrial carcinomas irrespective of mutator pathway.

Some colorectal tumors with wild-type adenomatous polyposis coli gene have activating mutations in beta-catenin (encoded by CTNNB1) that result in decreased phosphorylation by GSK-3beta and increased signaling through the Tcf/Lef transcription factors. To investigate the relationship between CTNNB1 mutations and underlying pathways of genomic instability, we examined 80 colorectal cancers stratified by the presence or absence of microsatellite instability (MSI). CTNNB1 mutations were identified in 13 (25%) of 53 cancers with high frequency MSI (MSI-H), including 12 point mutations at exon 3 phosphorylation sites (codons 41 and 45) and one deletion of the entire exon 3 degradation box. No CTNNB1 mutations were identified in 27 microsatellite stable or low frequency MSI (MSI-L) colorectal cancers (P < 0.01). In contrast, CTNNB1 mutations were identified in 3 of 9 (33%) MSI-H and 10 of 20 (50%) MSS/MSI-L endometrial carcinomas, suggesting a more generalized involvement in these tumors. Only six (46%) of the endometrial carcinoma CTNNB1 mutations occurred at residues directly phosphorylated by GSK-3beta, and only one of these was at either codon 41 or 45. All point mutations in MSI-H cancers were transitions, whereas 64% of those in MSS/MSI-L cancers were transversions (P < 0.01). The differences in the mutation profiles suggest that there may be molecular fingerprints of CTNNB1 mutations, determined by biological factors related to both tumor type and underlying pathways of genomic instability.

Adenocarcinoma↗

PubMed1999

A functional bone morphogenetic protein system in the ovary.

Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor beta superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions. In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought "luteinization inhibitor" in Graafian follicles during their growth and development.

Animals↗

PubMed1999

High-performance liquid chromatographic analysis of the anti-tumor agent SCH 66336 in cynomolgus monkey plasma and evaluation of its chiral inversion in animals.

SCH 66336 is a novel non-cytotoxic anti-tumor agent that is in phase I/II clinical trials for the treatment of solid tumors. This compound is a single enantiomer with one chiral center. Prior to evaluation of this drug candidate in man, it was necessary to evaluate its pharmacokinetics and possible chiral inversion in animals. Thus, high-performance liquid chromatographic (HPLC) methods have been developed for its determination in cynomolgus monkey plasma and for the evaluation of its chiral inversion in rats and cynomolgus monkeys. The achiral HPLC analysis involved extraction with 30% methylene chloride in hexane followed by separation on a CN column and quantitation by UV absorbance at 280 nm. The method was linear over a concentration range of 0.1 to 20 microg/ml in monkey plasma. The chiral HPLC analysis involved the use of a Chiralpak AD column set at 39 degrees C with a mobile phase of hexane-ethanol-diethylamine mixture and a UV detector set at 280 nm. Plasma samples were subjected to solid-phase extraction on a C2 cartridge prior to HPLC analysis. The method was linear over a concentration range of 0.25 to 10 microg/ml in rat and cynomolgus monkey plasma for both enantiomers. Both methods showed good linearity (r2>0.99), accuracy (bias< 13%) and precision (CV<12%). Chiral HPLC analysis indicated that SCH 66336 was not subjected to chiral inversion in rats and cynomolgus monkeys.

Animals↗

PubMed1999

Interaction of diphtheria toxin T domain with molten globule-like proteins and its implications for translocation.

The transmembrane (T) domain of diphtheria toxin has a critical role in the low pH-induced translocation of the catalytic domain (A chain) of the toxin across membranes. Here it is shown that at low pH, addition of proteins in a partly unfolded, molten globule-like conformation converted the T domain from a shallow membrane-inserted form to its transmembrane form. Fluorescence energy transfer demonstrated that molten globule-like proteins bound to the T domain. Thus, the T domain recognizes proteins that are partly unfolded and may function in translocation of the A chain as a transmembrane chaperone.

Antibodies↗

PubMed1999

Compartmental organization of the olfactory bulb glomerulus.

Olfactory receptor cell (ORC) axons terminate in the olfactory bulb glomerular neuropil, where they synapse with dendrites of mitral, tufted, and periglomerular neurons. We investigated the organization of the glomerular neuropil by using antibodies to both single- and double-label constituents for analyses with confocal microscopy. Electron microscopy (EM) was employed to assess the distribution of synaptic appositions within the glomerulus. Adult Sprague-Dawley rats were processed for immunocytochemistry with olfactory marker protein (OMP), synaptophysin, synapsin 1, glial fibrillary acidic protein (GFAP), and/or microtubule-associated protein 2 (MAP2). Equivalent rats were processed for transmission EM. Double labeling for OMP and MAP2 revealed two distinctive subcompartments within glomeruli: an axonal compartment containing predominately primary afferent axons with individual dendritic inserts and a complementary dendritic compartment that excluded primary afferent axons. Areas not occupied by OMP or MAP2 immunoreactivity were either immunoreactive for GFAP, indicating a glial process, or were blood vessels. Synaptophysin and synapsin 1 also showed differential labeling within the glomerulus. Synaptophysin strongly colocalized with OMP, whereas synapsin 1 was associated most strongly with MAP2. Reconstructions of glomeruli from EM montages revealed interdigitating axonal and dendritic subcompartments. The axonal subcompartments were composed primarily of ORC processes with individual or small groups of dendrites interspersed. Dendritic subcompartments were composed predominately of dendritic processes. Primary afferent axodendritic and local-circuit dendrodendritic synapses segregated within the glomerulus into the axonal and dendritic subcompartments, respectively. The results support the hypothesis of subcompartmental organization within olfactory bulb glomeruli.

Animals↗

PubMed1999

Morphologic investigation of rolling mouse Nagoya (tg(rol)/tg(rol)) cerebellar Purkinje cells: an ataxic mutant, revisited.

Rolling mouse Nagoya (rolling: tg(rol)) is a neurologic mutant mouse exhibiting severe ataxia. Two alleles of the rolling mutation, tottering (tg) and leaner(tg(la)), have been identified as mutations in the voltage-dependent calcium channel alpha1A subunit. No specific light and electron microscopic findings have been reported for the rolling mouse cerebellum except a decreased number of granule cells, while altered Purkinje cell/parallel fiber synapses have been observed in tottering and leaner cerebella. Rolling mouse cerebella were analyzed using anti-calbindin-D immunohistochemistry and transmission electron microscopy to investigate Purkinje cell morphology and synaptic contacts between Purkinje cell dendritic spines and parallel fiber varicosities. Multiple Purkinje cell dendritic spines synapsing with single parallel fiber varicosities were frequently observed in rolling cerebella. The correlation between the presence of altered Purkinje cell synapses and ataxia in rolling mice warrants further investigation.

Alleles↗

PubMed1999

Overexpression of GLUT4 in mice causes up-regulation of UCP3 mRNA in skeletal muscle.

Mitochondrial uncoupling protein 3 (UCP3) is expressed in skeletal muscles. We have hypothesized that increased glucose flux in skeletal muscles may lead to increased UCP3 expression. Male transgenic mice harboring insulin-responsive glucose transporter (GLUT4) minigenes with differing lengths of 5'-flanking sequence (-3237, -2000, -1000 and -442 bp) express different levels of GLUT4 protein in various skeletal muscles. Expression of the GLUT4 transgenes caused an increase in UCP3 mRNA that paralleled the increase of GLUT4 protein in gastrocnemius muscle. The effects of increased intracellular GLUT4 level on the expression of UCP1, UCP2 and UCP3 were compared in several tissues of male 4 month-old mice harboring the -1000 GLUT4 minigene transgene. In the -1000 GLUT4 transgenic mice, expression of GLUT4 mRNA and protein in skeletal muscles, brown adipose tissue (BAT), and white adipose tissue (WAT) was increased by 1.4 to 4.0-fold. Compared with non-transgenic littermates, the -1000 GLUT4 mice exhibited about 4- and 1.8-fold increases of UCP3 mRNA in skeletal muscle and WAT, respectively, and a 38% decrease of UCP1 mRNA in BAT. The transgenic mice had a 16% increase in oxygen consumption and a 14% decrease in blood glucose and a 68% increase in blood lactate, but no change in FFA or beta-OHB levels. T3 and leptin concentrations were decreased in transgenic mice. Expression of UCP1 in BAT of the -442 GLUT4 mice, which did not overexpress GLUT4 in this tissue, was not altered. These findings indicate that overexpression of GLUT4 up-regulates UCP3 expression in skeletal muscle and down-regulates UCP1 expression in BAT, possibly by increasing the rate of glucose uptake into these tissues.

Adipose Tissue, Brown↗

PubMed1999

Deletion mapping on the short arm of chromosome 8 in hepatocellular carcinoma.

To define the commonly deleted region on chromosome 8p for further positional cloning of the putative tumor suppressor gene, we carried out allelic imbalance (AI) studies in 41 HCCs using a panel of 37 microsatellite markers. The overall AI on 8p was 87.8% (36 of 41). Among the 36 cases with AI, 13 cases showed AI in all of the loci, suggesting entire deletion on the short arm of chromosome 8, while the remaining 23 cases showed partial AI. Detailed deletion mapping identified two independent commonly deleted regions on chromosome 8p. These were as follows: (1) centered by the D8S1819 and D8S1706 loci between the D8S561 and D8S1825 loci, (2) centered by the D8S1733 locus between the D8S298 and D8S1739 loci. These results suggest that the two putative tumor suppressor genes may be present on chromosome 8p.

Carcinoma, Hepatocellular↗

PubMed1999

Up-regulation of liver uncoupling protein-2 mRNA by either fish oil feeding or fibrate administration in mice.

Fish oil feeding showed less obesity in rodents, relative to other dietary oils. N-3 fatty acids rich in fish oil and fibrate compounds are peroxisome proliferator-activated receptor alpha (PPARalpha) ligands that stimulate beta-oxidation of fatty acids in liver and are used for treatment of hypertriglycemic patients. Since UCP-2, a member of an uncoupling protein family, has been shown to express in hepatocytes, the effects of these agents on the expression of UCP2 mRNA were investigated. C57BL/6J mice were divided into three groups; the first group was given a high-carbohydrate diet, and the other two groups were given a high-fat diet (60% of total energy) as safflower oil or fish oil for 5 months. Safflower oil diet fed mice developed obesity, but those fed fish oil diet did not. Therefore, the effects of fish oil feeding on the expression of UCP1, UCP2 and UCP3 in liver, skeletal muscle (gastrocnemius), white adipose tissue (WAT) and brown adipose tissue (BAT) were assessed by Northern blotting. Compared with safflower oil feeding, fish oil feeding up-regulated liver UCP2, BAT UCP2 and skeletal muscle UCP3 mRNA, while down-regulated WAT UCP2 and BAT UCP3 mRNA. Among these alterations, 5-fold up-regulation of liver UCP2 mRNA, relative to carbohydrate feeding, was noteworthy. Fenofibrate administration (about 500 mg/kg BW/d) for 2 wks also induced liver UCP2 expression by 9-fold. These data indicated that fish oil feeding and fibrate administration each up-regulated UCP2 mRNA expression in liver possibly via PPARalpha and hence each has the potential of increasing energy expenditure for prevention of obesity.

Adipose Tissue↗