Choonmi Kim

Identification of the calcium binding sites in translationally controlled tumor protein

Translationally controlled tumor protein (TCTP), also known as lgE-dependent histamine-releasing factor, is a growth-related tumor protein. Although the primary sequence of rat TCTP does not reveal any recognizable Ca2+-binding motif, previous studies have demonstrated that rat TCTP consisting of 172 amino acids is a Ca2+-binding protein. However, the region of TCTP required for Ca2+ interaction has not been mapped to the molecule. Here, we reported that the Ca2+ binding region of TCTP, which was mapped by using a combination of deletion constructs of rat TCTP and45Ca2+-overlay assay, was confined to amino acid residues 81–112. This binding domain did not show any peculiar loop of calcium-binding motif such as CaLB domain and EF hand motif and it seems to be constituted of random coil regions neighboring the a helix. Thus, our data confirm that TCTP is a novel family of Ca2+-binding protein.

Immunological analysis of methamphetamine antibody and its use for the detection of methamphetamine by capillary electrophoresis with laser-induced fluorescence

An accurate, simple and rapid immunoassay is demonstrated for the detection of methamphetamine in urine by capillary electrophoresis (CE) with laser-induced fluorescence (LIF). An aminobutyl derivative of methamphetamine was conjugated with proteins, and used as an immunogen to produce antibodies for the assay. The methamphetamine derivative was also labeled with fluorescein isothiocyanate (FITC) to compete with free methamphetamine in the sample for the antibody binding site. Levels of free and antibody-bound FITC-labeled methamphetamine were monitored by performing CE-LIF using an untreated fused-silica column. This competitive immunoassay used antiserum instead of purified antibody or antibody fragment, yet was found to have good precision with a sensitivity of lower than 20 ng/ml. Various antibodies were also screened, and cross-reactivity of anti-MA antibody with methamphetamine analogues were also investigated. The results indicate that CE-LIF-based immunoassay is a powerful tool for the screening and characterization of antibody and may have possible applications in the detection of abused drugs in urine.

Cytotoxicity and DNA Topoisomerase Inhibitory Activity of Benz[f]indole-4,9-dione Analogs

A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC50<20.0 μg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC50=0.4 μg/ml)) compared to colon (IC50>20.0 μg/ml) and stomach (IC50>20.0 μg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.

The optimization of ELISA for methamphetamine determination: the effect of immunogen, tracer and antibody purification method on the sensitivity

To obtain more sensitive immunoassay for methamphetamine (MA) determination, the optimum condition of enzyme-linked immunosorbent assay (ELISA) was investigated in regard to immunogens, antibody purification methods and coating tracers. Activated MA, N-(4-aminobutyl)methamphetamine (4-ABMA), was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) and used as immunogen. The antibodies were purified by protein G chromatography or various immunoaffinity chromatography-linked MA-protein ligands, such as MA-BSA, MA-KLH or MA-ovalbumin (OVA). Each purified antibody was characterized by means of sensitivity and cross-reactivity using the three MA-protein coating tracers, MA-BSA, MA-KLH and MA-OVA. The best sensitivity of each antibody was acquired with the MA-OVA tracer although the tracer concentration and the antibody titer level at optimum condition were varied. The antibody with high titer level did not always yield good sensitivity. At optimum condition, immunoaffinity chromatography-purified antibodies were better for sensitivity and for specificity than protein G-purified antibodies. The cross-reactivity of the purified antibodies seemed to be affected by immunogen structure and showed somewhat different patterns according to the immunoaffinity ligand utilized. These data show that the antibody purification method as well as choice of coating tracer and immunogen is essential for the sensitivity and specificity of EIA; the optimum condition for assay should be discovered using various methods and combinations.

Homology modeling and molecular docking study of translationally controlled tumor protein and artemisinin

Translationally controlled tumor protein (TCTP), also known as histamine releasing factor (HRF), is found abundantly in different eukaryotic cell types. The sequence homology of TCTP between different species is very high, belonging to the MSS4/DSS4 superfamily of proteins. TCTP is involved in both cell growth and human late allergy reaction, as well as having a calcium binding property; however, its primary biological functions remain to be clearly elucidated. In regard to many possible functions, the TCTP ofPlasmodium falciparum (Pf) is known to bind with an antimalarial agent, artemisinin, which is activated by heme. It is assumed that the endoperoxide-bridge of artemisinin is opened up by heme to form a free radical, which then eventually alkylates, probably to the Cys14 ofPfTCTP. Study of the docking of artemisinin with heme, and subsequently withPfTCTP, was carried out to verify the above hypothesis on the basis of structural interactions. The three dimensional (3D) structure ofPfTCTP was built by homology modeling, using the NMR structure of the TCTP ofSchizosaccharomyces pombe as a template. The quality of the model was examined based on its secondary structure and biological function, as well as with the use of structure evaluating programs. The interactions between artemisinin, heme andPfTCTP were then studied using the docking program, FlexiDock. The center of the peroxide bond of artemisinin and the Fe of heme were docked within a short distance of 2.6Å, implying the strong possibility of an interaction between the two molecules, as proposed. When the activated form of artemisinin was docked on thePfTCTP, the C4-radical of the drug faced towards the sulfur of Cys14 within a distance of 2.48Å, again suggesting the possibility of alkylation having occurred. These results confirm the proposed mechanism of the antimalarial effect of artemisinin, which will provide a reliable method for establishing the mechanism of its biological activity using a molecular modeling study.

Flexible docking of an acetoxyethoxymethyl derivative of thiosemicarbazone into three different species of dihydrofolate reductase

Dihydrofolate reductases (DHFR) of human,Candida albicans andE. coli were docked with their original ligands of X-ray crystal complex using QXP (Quick eXPIore), a docking program. Conditions to reproduce the crystal structures within the root mean square deviation (rmsd) of 2.00 Å were established. Applying these conditions, binding modes and species-specificities of a novel antibacterial compound, N4-(2-acetoxyethoxymethyl)-2-acetylpyridine thiosemicarbazone (AATSC), were studied. As the results, the docking program reproduced the crystal structures with average rmsd of six ligands as 0.91 A ranging from 0.49 to 1.45 ÅA. The interactions including the numbers of hydrogen bonds and hydrophobic interactions were the same as the crystal structures and superposition of the crystal and docked structures almost coincided with each other. For AATSC, the results demonstrated that it could bind to either the substrate or coenzyme sites of DHFR in all three species with different degrees of affinity. It confirms the experimentally determined kinetic behavior of uncompetitive inhibition against either the inhibitor or the coenzyme. The docked AATSC overlapped well with the original ligands and major interactions were consistent with the ones in the crystal complexes. The information generated from this work should be useful for future development of antibacterial and antifungal agents.

Thermotolerance inhibits various stress-induced apoptosis in NIH3T3 cells.

When NIH3T3 cells were exposed to mild heat and recovered at 37°C for various time intervals, they were thermotolerant and resistant to subsequent stresses including heat, oxidative stresses, and antitumor drug methotrexate which are apoptotic inducers. The induction kinetics of apoptosis by stresses were determined by DNA fragmentation and protein synthesis using [35S]methionine pulse labeling. We investigated the hypothesis that thermotolerant cells were resistant to apoptotic cell death compared to control cells when both cells were exposed to various stresses inducing apoptosis. The cellular changes in thermotolerant cells were examined to determine which components are involved in this resistance. At first, the degree of resistance correlates with the extent of heat shock protein synthesis which were varied depending on the heating times at 45°C and recovery times at 37°C after heat shock. Secondly, membrane permeability change was observed in thermotolerant cells. When cells prelabeled with [3H]thymidine were exposed to various amounts of heat and recovered at 37°C for 1/2 to 24 h, the permeability of cytosolic [3H]thymidine in thermotolerant cells was 4 fold higher than that in control cells. Thirdly, the protein synthesis rates in thermotolerant and control cells were measured after exposing the cells to the same extent of stress. It turned out that thermotolerant cells were less damaged to same amount of stress than control cells, although the recovery rates are very similar to each other. These results demonstrate that an increase of heat shock proteins and membrane changes in thermotolerant cells may protect the cells from the stresses and increase the resistance to apoptotic cell death, even though the exact mechanism should be further studied.

Effects of radioprotective ginseng protein on UV induced sister chromatid exchanges

To elucidate the reaction mechanism of ginseng protein on its antiradiation activity, its effects were studied on sister chromatid exchanges (SCE) induced by UV irradiation in CHO-Kl cells. When cells were irradiated with 254 nm UV light at the dose of 0 to 80 erg/mm2, the frequencies of SCE were increased more than two fold. However, when radio-protective ginseng protein was added to the cells before and after UV irradiation, SCE frequencies were decreased significantly at all UV doses in both cases with no significant differences. As the amount of ginseng protein was varied from 100 to 500 μg/ml, with UV irradiation at 60 erg/mm2, SCE frequencies dropped sharply at the first two concentrations and then reached a sort of plateau in both cases of pre-and post-treatment. When the ginseng protein was treated alone without UV irradiation, there were no changes in SCE frequencies no matter when the protein was added. These results suggest that the ginseng protein could reduce DNA damages, which may play an important role in the reaction mechanism of radioprotective activity of the protein.

Preferred conformations of cyclic Ac-Cys-Pro-Xaa-Cys-NHMe peptides: a model for chain reversal and active site of disulfide oxidoreductase

The conformational study on cyclic Ac-Cys-Pro-Xaa-Cys-NHMe (Ac-CPXC-NHMe; X=Ala, Val, Leu, Aib, Gly, His, Phe, Tyr, Asn and Ser) peptides has been carried out using the Empirical Conformational Energy Program for Peptides, version 3 (ECEPP/3) force field and the hydration shell model in the unhydrated and hydrated states. This work has been undertaken to investigate structural implications of the CPXC sequence as the chain reversal for the initiation of protein folding and as the motif for active site of disulfide oxidoreductases. The backbone conformation DAAA is commonly the most feasible for cyclic CPXC peptides in the hydrated state, which has a type I beta-turn at the Pro-Xaa sequence. The proline residue and the hydrogen bond between backbones of two cystines as well as the formation of disulfide bond appear to play a role in stabilizing this preferred conformation of cyclic CPXC peptides. However, the distributions of backbone conformations and beta-turns may indicate that the cyclic CPXC peptide seems to exist as an ensemble of beta-turns and coiled conformations in aqueous solution. The intrinsic stability of the cyclic CPXC motif itself for the active conformation seems to play a role in determining electrochemical properties of disulfide oxidoreductases.

Effects of ginseng protein on relative survival and chromosome aberration of UV irradiated cells

A ginseng protein fraction which has been reported to have radiation protective effect was purified from Korean ginseng and its effects on relative survival and chromosome aberration were studied in UV irradiated CHO-K1 cells. When the protein fraction (100 μg/ml) was added to the cells before UV irradiation at 4 J/m2, the survival rates were increased to 53.8% from 40.6% in control. Addition of the protein (100 μg/ml) after UV irradiation at 4 and 8 J/m2 raised the rates to 85.4 and 24.0% from 79.2 and 11.5% in control, respectively. When the ginseng protein (800 μg/ml) was added to the cells exposed to UV light at 10, 20, 30 J/m2, the frequencies of chromosome aberration (CA) were reduced significantly to almost same level regardless of the UV dose increment and there was no significant difference between pre- and post-treatment. When the concentration of ginseng protein was increased from 200 to 800 μg/ml, at UV dose of 10, 20, 30 J/m2 each, the CA frequencies were decreased consistently as the dose of ginseng protein increased, at all UV doses tested. Similar effects were observed in both cases of pre- and post-treatment. The data suggest that the protein may reduce cell damage caused by UV light, especially damage to DNA molecule, or play a role in repair processes of damaged DNA, to increase cell survival and reduce chromosome aberrations.