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Biomedical subjects

D H Kim

Publications and source records attributed to D H Kim.

At least 19 recordsLinked to original sources
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

Solution structure of the antiapoptotic protein bcl-2.

The structures of two isoforms of Bcl-2 that differ by two amino acids have been determined by NMR spectroscopy. Because wild-type Bcl-2 behaved poorly in solution, the structures were determined by using Bcl-2/Bcl-x(L) chimeras in which part of the putative unstructured loop of Bcl-2 was replaced with a shortened loop from Bcl-x(L). These chimeric proteins have a low pI compared with the wild-type protein and are soluble. The structures of the two Bcl-2 isoforms consist of 6 alpha-helices with a hydrophobic groove on the surface similar to that observed for the homologous protein, Bcl-x(L). Comparison of the Bcl-2 structures to that of Bcl-x(L) shows that although the overall fold is the same, there are differences in the structural topology and electrostatic potential of the binding groove. Although the structures of the two isoforms of Bcl-2 are virtually identical, differences were observed in the ability of the proteins to bind to a 25-residue peptide from the proapoptotic Bad protein and a 16-residue peptide from the proapoptotic Bak protein. These results suggest that there are subtle differences in the hydrophobic binding groove in Bcl-2 that may translate into differences in antiapoptotic activity for the two isoforms.

Amino Acid Sequence↗
PubMed2001

Long-term preservation of rat pancreatic islets under physiological conditions.

In this study, we found that islet cells treated with polyphenol could be preserved for over 2 months under physiological conditions retaining their original function and maintaining their spherical shapes without any insulin secretion. When islets were treated at higher concentration than 250 microg ml(-1), these islets could retain their compact spherical shape over 65 days whereas non-treated islets were scattered ease to break within 2 weeks. The secretional capacity from treated islets in the initial stage is also lower than untreated islets. However, in the case of untreated islets, insulin release rapidly lowered with the progress in the culture time and secretion completely disappeared after 9 days. On the contrary, islets treated with polyphenol (250 microg ml(-1)) in RPMI culture medium showed significant enhancement of insulin secretion on 40th day. The secretional capacity of islets was greatly dependent on the treating concentration. Polyphenol treatment may be a useful method for preservation of mammalian islet cells. By changing the concentration of polyphenol, it is possible to control the preservation duration and insulin secretion of islets.

Animals↗
PubMed2001

Identification of a negative Cis-regulatory element and multiple DNA binding proteins that inhibit transcription of the transforming growth factor-beta type II receptor gene.

Expression of the transforming growth factor-beta type II receptor (TGF-beta RII) is highly regulated and is a critical determinant of the cellular response to TGF-beta. Previous analysis of the promoter region for the TGF-beta RII gene introduced the possible existence of a negative regulatory element (NRE) upstream adjacent to the core promoter region (Bae et al., 1995. J. Biol. Chem. 270, 29460-29468). We have confirmed the presence of a strong NRE located between base pairs -100 and -67 relative to the transcription start site. Utilizing DNA transfection techniques and a series of synthesized oligonucleotide promoter fragments, we have shown that this NRE is active in a variety of cell lines. Electrophoretic mobility shift assays have revealed the presence of multiple DNA binding proteins specifically interacting with the NRE. At least three distinct protein complexes are variably present depending on the specific cell line examined, and mutational analysis of the NRE has identified a ten-base pair recognition sequence which is shared by all three complexes. This palindromic sequence has not been previously reported and does not share homology with any known transcription factor consensus sequences. When inserted into an E4Delta heterologous promoter construct, the NRE binding sequence failed to inhibit either basal or activated transcription of the target gene, indicating that the NRE does not act as a general repressor but may specifically operate within the context of the TGF-beta RII core promoter.

Animals↗
PubMed2001

Differential regulation of protein tyrosine kinase on free radical production, granule enzyme release, and cytokine synthesis by activated murine peritoneal macrophages.

The present study examined the regulatory effect of tyrosine kinase inhibitors (genistein, tyrphostin, and 2,5-dihydroxycinnamate) on the free radical production, granule enzyme release, and synthesis of interleukin (IL)-8 and granulocyte macrophage-colony stimulating factor (GM-CSF) in murine peritoneal macrophages exposed to different stimulators [10 ng/mL of IL-1, 1 microgram/mL of lipopolysaccharide (LPS), and 1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP)]. Protein tyrosine kinase (PTK) inhibitors attenuated the stimulated superoxide, hydrogen peroxide, and nitric oxide production in macrophages stimulated with IL-1, LPS, or fMLP. N,N-Dimethylsphingosine (DMS) alone stimulated superoxide and hydrogen peroxide production by intact macrophages, but at 45 microM the stimulatory effect on superoxide production was not found. In contrast, DMS attenuated nitric oxide production by macrophages. High concentrations of DMS, tyrphostin, and 2,5-dihydroxycinnamate showed cytotoxic effects. PTK inhibitors did not exhibit a significant effect on granule enzyme release induced by IL-1, whereas they attenuated the effect of LPS and fMLP on degranulation. Genistein and tyrphostin decreased the production of IL-8 and GM-CSF in macrophages activated by IL-1, whereas 2,5-dihydroxycinnamate did not affect it. The results suggest that tyrosine kinases exposed to IL-1, LPS, and fMLP may exert different modulatory actions on macrophage responses. The IL-1-activated macrophage responses, particularly degranulation, appear to be differently regulated by tyrosine kinases compared with the responses activated by LPS and fMLP.

Animals↗
PubMed2001

Constituents and the antitumor principle of Allium victorialis var. platyphyllum.

To search for cytotoxic components from Allium victorialis, MTT assays on each extract and an isolated component, gitogenin 3-O-lycotetroside, were performed against cancer cell lines. Cytotoxicities of most extract were shown to be comparatively weak, though IC50 values of CHCl3 fraction was found to be <31.3-368.4 microg/ml. From the incubated methanol extract at 36 degrees C, eleven kinds of organosulfuric flavours were predictable by GC-MS performance. The most abundant peak was revealed to be 2-vinyl-4H-1,3-dithiin (1) by its mass spectrum. Further, this extract showed significant cytotoxicities toward cancer cell lies. Silica gel column chromatography of the n-butanol fraction led to the isolation of gitogenin 3-O-lycotetroside (3) along with astragalin (4) and kaempferol 3, 4'-di-O-beta-D-glucoside (5). This steroidal saponin exhibited significant cytotoxic activities (IC50, 6.51-36.5 microg/ml) over several cancer cell lines. When compound 3 was incubated for 24 h with human intestinal bacteria, a major metabolite was produced and then isolated by silica gel column chromatography. By examining parent- and prominent ion peak in FAB-MS spectrum of the metabolite, the structure was speculated not to be any of prosapogenins of 3, suggesting that spiroketal ring were labile to the bacterial reaction. These suggest that disulfides produced secondarily are the antitumor principles.

Allium↗
PubMed2001

Albumin release from biodegradable hydrogels composed of dextran and poly(ethylene glycol) macromer.

Biodegradable hydrogels based on glycidyl methacrylate dextran (GMD) and dimethacrylate poly(ethylene glycol) (DMP) were proposed for colon-specific drug delivery. GMD was synthesized by coupling of glycidyl methacrylate with dextran in the presence of 4-(N,N-dimethyl-amino)pyridine (DMAP) using dimethylsulfoxide as a solvent. Methacrylate-terminated poly (ethylene glycol) (PEG) macromer was prepared by the reaction of PEG with methacryloyl chloride. GMD/DMP hydrogels were prepared by radical polymerization of phosphate buffer solution (0.1M, pH 7.4) of GMD and DMP, using ammonium peroxydisulfate (APS) and UV as initiating system. The synthetic GMD, DMP, and GMD/DMP hydrogels were characterized by fourier transform infrared (FT-IR) spectroscopy. The FITC-albumin loaded hydrogels were prepared by adding FITC-albumin solution before UV irradiation. Swelling capacity of GMD/DMP hydrogels was controlled not only by molecular weight of dextran, but also by incorporation ratio of DMP Degradation of the hydrogels has been studied in vitro with dextranase. FITC-albumin release from the GMD/DMP hydrogels was affected by molecular weight of dextran and the presence of dextranase in the release medium.

Acrylates↗
PubMed2001

N-(Hydroxyaminocarbonyl)phenylalanine: a novel class of inhibitor for carboxypeptidase A.

N-(Hydroxyaminocarbonyl)phenylalanine (1) was designed rationally as a new type of inhibitor for carboxypeptidase A (CPA). The designed inhibitor was readily prepared from phenylalnine benzyl ester in two steps and evaluated to find that rac-1 inhibits CPA in a competitive fashion with the Ki value of 2.09 microM. Surprisingly, inhibitor 1 having the D-configuration is more potent (Ki = 1.54 microM) than its antipode by about 3-fold. A possible explanation for the stereochemistry observed in the inhibition of CPA with 1 is presented.

Binding Sites↗
PubMed2001

A new inhibitor design strategy for carboxypeptidase A as exemplified by N-(2-chloroethyl)-N-methylphenylalanine.

N-(2-Chloroethyl)-N-methylphenylalanine was designed and synthesized in an optically active form as a novel class of mechanism-based inactivator for carboxypeptidase A (CPA). It was anticipated that the chloroethylamino moiety of the CPA bound inhibitor undergoes an intramolecular SN2 reaction to generate a chemically reactive species (an aziridinium ion) which is expectedly subjected to a nucleophilic attack by the carboxylate of Glu-270, leading to covalent modification of the carboxylate. The irreversible nature of the inhibition of CPA by the inhibitor was supported by the kinetic data: the enzyme lost its enzymic activity in a time-dependent manner in the presence of the inhibitor and the inactivated CPA failed to regain the activity upon dialysis. Interestingly, the (R)-isomer that belongs to the D-series was more potent than its enantiomer.

Animals↗
PubMed2001

Evidence for direct involvement of beta-catenin in phorbol ester-induced neurite outgrowth in GT1-1 hypothalamic neurones.

Gonadotropin-releasing hormone (GnRH) is a pivotal neuroendocrine regulator controlling reproductive functions. However, the scattered distribution of GnRH neurones in the mammalian brain has hindered studies on the development and differentiation of GnRH neurones. In the present study, we used the immortalized GnRH-producing GT1-1 cells to examine whether activation of protein kinase C (PKC) pathway with 12-O-tetradecanoyl-13-acetate (TPA) induces morphological and functional differentiation of GnRH neurones. TPA induced neurite outgrowth and inhibited proliferation of GT1-1 cells that were specifically antagonized by cotreatment of PKC inhibitor, calphostin C. The functional significance of TPA-induced differentiation of GT1-1 cells was manifested in part by the changes in the effects of gamma-aminobutyric acid (GABA) on intracellular Ca2+ levels. In untreated GT1-1 cells, activation of GABA-A receptor with 10 microM muscimol increased intracellular Ca2+ levels, whereas such stimulatory effects disappeared in GT1-1 cells bearing neurites. Accordingly, muscimol could not stimulate GnRH release in TPA-treated GT1-1 cells. To elucidate the molecular mechanism underlying TPA-induced neurite outgrowth, we performed differential display reverse transcription-polymerase chain reaction. Among several genes that are affected by TPA treatment, we found a significant induction of beta-catenin mRNA expression. Along with the rapid induction of beta-catenin protein levels, we observed that beta-catenin was reallocated from cell-cell adhesion sites to the growth cones within 3 h of TPA treatment. Transient transfection studies with green fluorescent protein as a reporter gene demonstrated that beta-catenin overexpression alone can promote neurite outgrowth in GT1-1 cells. Moreover, TPA was found to increase the transcription-activational roles of beta-catenin. Together, these data provide evidence that beta-catenin is involved in the TPA-induced functional differentiation of immortalized GnRH neurones.

Calcium↗
PubMed2001

Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells.

Very limited information is available on the role of phosphatidylinositol 3-phosphate (PI[3]P) in vesicle trafficking in plant cells. To investigate the role of PI(3)P during the vesicle trafficking in plant cells, we exploited the PI(3)P-specific binding property of the endosome binding domain (EBD) (amino acids 1257 to 1411) of human early endosome antigen 1, which is involved in endosome fusion. When expressed transiently in Arabidopsis protoplasts, a green fluorescent protein (GFP):EBD fusion protein exhibited PI(3)P-dependent localization to various compartments--such as the trans-Golgi network, the prevacuolar compartment, the tonoplasts, and the vesicles in the vacuolar lumen--that varied with time. The internalized GFP:EBD eventually disappeared from the lumen. Deletion experiments revealed that the PI(3)P-dependent localization required the Rab5 binding motif in addition to the zinc finger motif. Overexpression of GFP:EBD inhibited vacuolar trafficking of sporamin but not trafficking of H(+)-ATPase to the plasma membrane. On the basis of these results, we propose that the trafficking of GFP:EBD reflects that of PI(3)P and that PI(3)P synthesized at the trans-Golgi network is transported to the vacuole through the prevacuolar compartment for degradation in plant cells.

Arabidopsis↗
PubMed2001

Enhanced iron uptake of Saccharomyces cerevisiae by heterologous expression of a tadpole ferritin gene.

We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 microg per gram (dry cell weight) of the recombinant yeast but was 210 microg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.

Animals↗
PubMed2001

Metabolism of dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate (DDB) by human liver microsomes: characterization of metabolic pathways and of cytochrome P450 isoforms involved.

Metabolic fate of DDB and identification of P450 isozymes involved in the metabolism of DDB were investigated in human liver microsomes. DDB was rapidly metabolized to five different metabolites, and the structures of each metabolite were characterized based on UV, mass, and NMR spectral analyses. The major metabolic pathways of DDB in human liver microsomes were identified as O-demethylation of the carboxymethyl moiety (M4) and demethylenation of the methylenedioxyphenyl group (M2). The intramolecular lactonization between the hydroxyl group at the C6 and carboxymethyl group at the C2' of M2 resulted in the generation of M5, which was either hydrolyzed to its hydrolyzed derivative (M1) or further metabolized to the O-demethylated derivative (M3). The interconversion of M1, M2, and M5 took place nonenzymatically depending on the solvent condition. M5 was predominantly detected at the acidic condition, whereas M1 was preferentially detected at the basic environment. Cytochrome P450 (P450) isoform(s) involved in the metabolism of DDB was identified using several in vitro approaches. Chemical inhibition using isoform-selective P450 inhibitors, correlation of DDB metabolites formation with several isoform-specific P450 activities in a panel of liver microsomes, metabolism by microsomes derived from P450 cDNA-expressed B-lymphoblastoid cells, and immunoinhibition by isoform-specific anti-P450 antibodies collectively indicated that CYP1A2, CYP2C9, and CYP3A4 are responsible for the metabolism of DDB. O-Dealkylation of the carboxymethyl group was preferentially catalyzed by CYP1A2, whereas demethylenation of the methylenedioxyphenyl moiety was catalyzed by CYP3A4 and CYP2C9.

Antibodies↗
PubMed2000

Flow cytometric analysis and chromosome sorting of barley (hordeum vulgare L).

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 microM trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.

Analysis of Variance↗
PubMed2000

The asp-rich region at the carboxyl-terminus of calsequestrin binds to Ca(2+) and interacts with triadin.

Calsequestrin (CSQ) is a high capacity Ca(2+) binding protein in the junctional sarcoplasmic reticulum of striated muscles, and has been shown to regulate the ryanodine receptor (RyR) through triadin and junctin. In order to identify the functional roles of specific regions on CSQ, several CSQ deletion mutants were prepared by molecular cloning and Escherichia coli expression. 45Ca(2+) overlay assay using a native gel system revealed that the major Ca(2+) binding motif of CSQ resides in the asp-rich region (amino acids 354-367). In an in vitro binding assay using a glutathione-S-transferase affinity column, the interaction between CSQ and triadin was found to be Ca(2+)-dependent, and the site of interaction was confined to the asp-rich region of CSQ. Our results suggest that the asp-rich region of CSQ could participate in the RyR-mediated Ca(2+) release process by offering a direct binding site to luminal Ca(2+) as well as triadin.

Animals↗
PubMed2000

Increased plasma corticosterone, aggressiveness and brain monoamine changes induced by central injection of pertussis toxin.

The effects of intracerebroventricular (i.c.v.) injection of pertussis toxin, a specific inhibitor of G(i)/G(o) proteins, on plasma corticosterone levels, aggressiveness, and hypothalamic and hippocampal monoamines and their metabolites levels were examined in mice. Plasma corticosterone level was markedly increased at 3 h after pertussis toxin injection (0.03 and 0.2 microg/mouse), peaked at 6 h and was still increased for up to 6 days after injection. Mice injected with pertussis toxin (0.2 microg/mouse) did not show weight gain between day 0 and day 6 after injection. In addition, pertussis toxin (0.2 microg/mouse) induced a progressive increase in aggressiveness, i.e. a decrease in attack latency and an increase in number of attacks, on day 1 and 6 after injection. Brain monoamines and their metabolites levels were changed on day 1 and 6 after pertussis toxin injection (0.2 microg/mouse): in the hypothalamus, levels of dopamine and 3,4-dihydroxyphenylacetic acid were increased, norepinephrine level decreased, and 5-hydroxyindole acetic acid (5-HIAA) level was markedly increased, with no changes in 5-hydroxytryptamine (5-HT) level, whereas in the hippocampus, 5-HT level was significantly decreased, with no changes in 5-HIAA and catecholamines. These results suggest that signal transduction through G(i)/G(o) proteins in the brain is involved in the modulation of hypothalamo-pituitary-adrenal axis, aggressiveness, and monoamine levels in vivo.

Aggression↗
PubMed2000

Development of spontaneous hyperplastic skin lesions and chemically induced skin papillomas in transgenic mice expressing human papillomavirus type 16 E6/E7 genes.

Human papillomavirus type 16 (HPV16) has been known to be the major factor for the development of uterine cervical carcinomas. We have developed a line of transgenic mice that express the HPV16 E6 and E7 genes in certain organs using a fusion gene which consists of the tyrosinase promoter and E6/E7 of HPV16, and have chosen the tyrosinase minigene as a co-injected visual marker for the identification of transgenic mice. Our transgenic mice (1) expressed E6/E7 transgene mainly in skin and heart, and (2) showed skin and eye pigmentation profiles, and (3) raised incidence of hyperplastic skin lesions. We had performed two-stage skin carcinogenesis experiment to detect the susceptibility of skin papilloma development in our transgenic mice, using dimethylbenz-anthracene (DMBA) as a initiating agent and 12-O-tetradecanoyl-phorbol-13-acetate (TPA). After 1 week of DMBA treatment (25 microg dissolved in 0.2 ml acetone) and 15 consecutive weeks of TPA treatment (2.5 microg dissolved in 0.2 ml acetone) on the back of transgenic and non-transgenic control mice (Fv-1(b) strain mice which are Friend virus B-type susceptible (FVB)/N), papilloma incidence was increased in our transgenic mice approximately 2-fold higher than in control (in female mice, 69.2 vs. 30%, respectively). Thus our transgenic mice may be useful for the development of immunological or other therapies for HPV-associated cancers.

9,10-Dimethyl-1,2-benzanthracene↗