Biomedical subjects

J A Kim

Publications and source records attributed to J A Kim.

At least 19 recordsLinked to original sources

PubMed2001

Cleavage of purified neuronal clathrin assembly protein (CALM) by caspase 3 and calpain.

The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.

Adaptor Proteins, Vesicular Transport↗

PubMed2001

Role of reactive oxygen species in apoptosis induced by N-ethylmaleimide in HepG2 human hepatoblastoma cells.

We have previously reported that N-ethylmaleimide induces apoptosis through activation of K(+), Cl(-)-cotransport in HepG2 human hepatoblastoma cells. In this study, we investigated the role for reactive oxygen species as a mediator of the apoptosis induced by N-ethylmaleimide. N-ethylmaleimide induced a significant elevation of intracellular level of reactive oxygen species. Treatment with antioxidants (N-acetyl cysteine, N,N'-diphenyl-p-phenylenediamine) which markedly suppressed generation of reactive oxygen species, significantly inhibited the N-ethylmaleimide-induced activation of K(+), Cl(-)-cotransport and apoptosis. Inhibitors of NADPH oxidase (diphenylene iodonium, apocynin, D-(+)-neopterine) also significantly blunted the generation of reactive oxygen species, activation of K(+), Cl(-)-cotransport and apoptosis induced by N-ethylmaleimide. These results suggest that reactive oxygen species generated through activation of NADPH oxidase may play a role in the N-ethylmaleimide-induced stimulation of K(+), Cl(-)-cotransport and apoptosis in HepG2 cells.

Apoptosis↗

PubMed2001

Two-photon absorption and nonlinear optical properties of octupolar molecules.

Two-photon absorption (TPA) cross sections of four representative series of octupolar molecules are theoretically investigated. The general structure--TPA-property relationship is described by using the effective four-state valence-bond three-charge-transfer model. As the charge-transfer character of the ground electronic state increases due to the strong donors or acceptors, (i) the transition dipole matrix elements between the ground and 2-fold degenerate excited states increase, (ii) the energy gap decreases, and consequently (iii) the TPA transition amplitude monotonically increases. Thus, the design strategy to maximize the TPA cross section of the octupolar molecule is established. On the basis of the four-state model, the first hyperpolarizability of the octupolar molecule is found to be linearly proportional to the TPA cross section. This theoretical relationship is confirmed by using the ab initio calculation results. The Hammett correlation analysis of the TPA cross section and first hyperpolarizability is also presented.

Journal Article↗

PubMed2001

Synthesis, topoisomerase I inhibition and antitumor cytotoxicity of 2,2':6',2"-, 2,2':6',3"- and 2,2':6',4"-terpyridine derivatives.

For the development of new anticancer agents, 2,2':6',2"-, 2,2':6',3"- and 2,2':6',4"-terpyridine derivatives were designed and evaluated for their topoisomerase I inhibitory activity and antitumor cytotoxicity. Structure-activity relationship studies indicated that 2,2':6',2"-terpyridine derivatives were highly cytotoxic toward several human tumor cell lines, whereas 2,2':6',3"- and 2,2':6',4"-terpyridine derivatives were potent topoisomerase I inhibitors.

Antineoplastic Agents↗

PubMed2001

RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes.

Extensive work on the maturation of lagging strands during the replication of simian virus 40 DNA suggests that the initiator RNA primers of Okazaki fragments are removed by the combined action of two nucleases, RNase HI and Fen1, before the Okazaki fragments join. Despite the well established in vitro roles of these two enzymes, genetic analyses in yeast revealed that null mutants of RNase HI and/or Fen1 are not lethal, suggesting that an additional enzymatic activity may be required for the removal of RNA. One such enzyme is the Saccharomyces cerevisiae Dna2 helicase/endonuclease, which is essential for cell viability and is well suited to removing RNA primers of Okazaki fragments. In addition, Dna2 interacts genetically and physically with several proteins involved in the elongation or maturation of Okazaki fragments. Here we show that the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA. The sequential action of these enzymes is governed by a single-stranded DNA-binding protein, replication protein-A (RPA). Our results demonstrate that the processing of Okazaki fragments in eukaryotes differs significantly from, and is more complicated than, that occurring in prokaryotes. We propose a novel biochemical mechanism for the maturation of eukaryotic Okazaki fragments.

Adenosine Triphosphatases↗

PubMed2001

Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease.

In order to gain insights into the structural basis of the multifunctional Dna2 enzyme involved in Okazaki fragment processing, we performed biochemical, biophysical and genetic studies to dissect the domain structure of Dna2. Proteolytic digestion of Dna2 using subtilisin produced a 127 kDa polypeptide that lacked the 45 kDa N-terminal region of Dna2. Further digestion generated two subtilisin-resistant core fragments of approximately equal size, 58 and 60 kDa. Surprisingly, digestion resulted in a significant (3- to 8-fold) increase in both ATPase and endonuclease activities compared to the intact enzyme. However, cells with a mutant DNA2 allele lacking the corresponding N-terminal region were severely impaired in growth, being unable to grow at 37 degrees C, indicating that the N-terminal region contains a domain critical for a cellular function(s) of Dna2. Analyses of the hydrodynamic properties of and in vivo complex formation by wild-type and/or mutant Dna2 lacking the N-terminal 45 kDa domain revealed that Dna2 is active as the monomer and thus the defect in the mutant Dna2 protein is not due to its inability to multimerize. In addition, we found that the N-terminal 45 kDa domain interacts physically with a central region located between the two catalytic domains. Our results suggest that the N-terminal 45 kDa domain of Dna2 plays a critical role in regulation of the enzymatic activities of Dna2 by serving as a site for intra- and intermolecular interactions essential for optimal function of Dna2 in Okazaki fragment processing. The possible mode of regulation of Dna2 is discussed based upon our recent finding that replication protein A interacts functionally and physically with Dna2 during Okazaki fragment processing.

Adenosine Triphosphatases↗

PubMed2001

Cell-free expression and functional reconstitution of CALM in clathrin assembly.

Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). In order to better understand a possible role of post-translational modification of CALM (clathrin assembly protein lymphoid myeloid), the homologue of AP180, in the assembly of CCVs, CALM was expressed in the cell-free reticulocyte translation system that is capable of carrying out post-translational modification. The apparent molecular weight of the expressed recombinant CALM was estimated as 105 kD. Alkaline phosphatase treatment of CALM resulted in a mobility shift on SDS-PAGE. We found that CALM was associated with the proteins harboring SH3 domain, promote assembly of clathrin triskelia into clathrin cage and bound to the preformed clathrin cage. CALM was also proteolyzed by caspase 3 and calpain but not by caspase 8. These results indicated that the post-translationally modified CALM, expressed in the eukaryotic cell-free reticulocyte translation system was able to mediate the assembly of clathrin and the coated-vesicle formation.

Adaptor Proteins, Vesicular Transport↗

PubMed2001

Association between insulin-like growth factor-2 and metastases after transcatheter arterial chemoembolization in patients with hepatocellular carcinoma: a prospective study.

BACKGROUND: Hypoxia up-regulates insulin-like growth factor-2 (IGF-2) and thus stimulates the growth of hepatocellular carcinoma (HCC) cells. In the current study, the authors prospectively evaluated changes in plasma IGF-2 levels in HCC patients after transcatheter arterial chemoembolization (TACE), which usually results in hypoxic insult to liver tissue. The authors also examined the association between changes in plasma IGF-2 levels after TACE and HCC progression, especially in relation to metastasis. METHODS: Plasma IGF-2 levels were measured before and 4 weeks after TACE in 46 patients with HCC. Three months after TACE, the patients were evaluated for the occurrence of metastatic HCC. RESULTS: In 13 of the 46 patients, post-TACE plasma IGF-2 levels decreased by > 20% (Group 1) compared with their basal levels; in 18 patients, the IGF-2 levels changed within 20% (Group 2) and in 15 patients the IGF-2 levels increased by > 20% (Group 3). Plasma IGF-2 levels had a tendency to increase in patients with large-sized tumors, high serum alpha-fetoprotein (AFP) levels, and the heterogeneous uptake of iodized oil. Metastatic foci were found in 9 patients in Group 3 (60%), in contrast to only 3 patients in Group 2 (17%) and in none of the patients in Group 1 (P = 0.001). On multivariate analysis, higher Child-Pugh scores and increased plasma IGF-2 levels (Group 3) were found to be independent risk factors for metastasis. CONCLUSIONS: Increased plasma IGF-2 levels after TACE, which are common in patients with large-sized tumors and high serum AFP levels, appear to be associated with the occurrence of metastatic HCC after TACE.

Carcinoma, Hepatocellular↗

PubMed2001

Downregulated expression of integrin alpha6 by transforming growth factor-beta(1) on lens epithelial cells in vitro.

Integrins represent the main cell surface receptors that mediate cell-matrix and cell-cell interactions. They play critical roles in adhesion, migration, morphogenesis, and the differentiation of several cell types. Previous studies have demonstrated that members of the fibroblast growth factor (FGF)-2, transforming growth factor (TGF)-beta(1), and insulin growth factor (IGF)-1 play important roles in lens biology. In particularly, TGF-beta(1) appears to play a key role in extracellular matrix production, cell proliferation, and cell differentiation of lens epithelial cells. In this study we investigated the effects of FGF-2, TGF-beta(1), and IGF-1 on the modulation of integrin receptors using lens epithelial cell lines (HLE B-3 and alphaTN-4) and lens explants. We found that the expression of integrin alpha6 is downregulated by TGF-beta(1), but is not responsive to FGF-2 or IGF-1. The promoter activity of the integrin alpha6 gene decreased upon TGF-beta(1) treatment in a transient transfection assay, and flow cytometric analysis demonstrated the reduced expression of integrin alpha6 by TGF-beta(1), whereas significant changes were not observed in the level of integrin alpha6 after the addition of FGF-2. These findings suggest that the reduced expression of integrin alpha6 caused by TGF-beta(1) might play a role in the activation of the cell cycle genes required during the fiber differentiation of the lens.

Animals↗

PubMed2001

Involvement of K(+)-Cl(-)-cotransport in the apoptosis induced by N-ethylmaleimide in HepG2 human hepatoblastoma cells.

The role of K(+)-Cl(-)-cotransport in apoptosis in human cancer cells was investigated. N-Ethylmaleimide, a K(+)-Cl(-)-cotransport activator, induced apoptosis in a dose-dependent manner in HepG2 human hepatoblastoma cells. N-Ethylmaleimide induced Cl(-)-dependent K(+) efflux, indicating that K(+)-Cl(-)-cotransport is functionally present in HepG2 cells. Calyculin-A and genistein, inhibitors of K(+)-Cl(-)-cotransport, significantly prevented both K(+)-Cl(-)-cotransport activation and apoptosis induced by N-ethylmaleimide. These results demonstrate, for the first time, a novel role for K(+)-Cl(-)-cotransport in apoptosis in human hepatoma cells. These results further suggest that K(+)-Cl(-)-cotransport may be a valuable target for therapeutic interventions for human hepatoma.

Apoptosis↗

PubMed2001

Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study.

A phase I dose-escalation clinical trial of peritumoral injections of interleukin 12 (IL-12)-transduced autologous fibroblasts was performed in patients with disseminated cancer for whom effective treatment does not exist. The goals of this study were to assess the safety and toxicities as well as the efficacy, and ancillarily the immunomodulatory effects, of peritumoral IL-12 gene transfer. Primary dermal fibroblasts cultured from the patients were transduced with retroviral vector carrying human IL-12 genes (p35 and p40) as well as the neomycin phosphotransferase gene (TFG-hIL-12-Neo). Patients received four injections at intervals of 7 days. Nine patients were enrolled in this dose-escalation study, with secreted IL-12 doses ranging from 300 ng/24 hr for the first three patients to 1000, 3000, and 5000 ng/24 hr for two patients in each subsequent dosage level. Although a definite statement cannot be made, there appears to be perturbation of systemic immunity. Also, the locoregional effects mediated by tumor necrosis factor alpha (TNF-alpha) and CD8+ T cells were observed with tumor regression. Treatment-related adverse events were limited to mild to moderate pain at the injection site; clinically significant toxicities were not encountered. Transient but clear reductions of tumor sizes were observed at the injected sites in four of nine cases, and at noninjected distant sites in one melanoma patient. Hemorrhagic necrosis of tumors was observed in two melanoma patients. These data indicate that gene therapy by peritumoral injection of IL-12-producing autologous fibroblasts is feasible, and promising in patients with advanced cancer.

Adult↗

PubMed2001

Detection of circulating tumor cells in patients with gastrointestinal tract cancer using RT-PCR and its clinical implications.

To investigate the relationship between the presence of circulating tumor cells in different stages of gastrointestinal tract cancer and the subsequent relapse or distant metastasis, circulating levels of CEA mRNA was serially examined at an interval of 10.6+/-4.5 or 13.7+/-3.0 months in gastric or colorectal cancer patients, respectively. CEA mRNA was measured by means of RT-PCR amplification as an indicator for micrometastatic malignant cells. Seven of twenty-nine respectable gastric cancer patients (24.1%) [EGC: 2/9 (22.2%), AGC IIIa: 1/5 (20.0%), AGC IIIb: 4/15 (26.6%)] were positive for CEA mRNA on the initial test and 10 of 29 patients (34.4%) [EGC: 2/ 9 (22.2%), AGC IIIa: 1/5 (20.0%), AGC IIIb: 7/15 (46.7%)] were positive on a follow-up test. Only in AGC IIIb, the positive rate for CEA mRNA increased about twice and 6 of 7 positive cases (85.7%) relapsed within 2.6+/-2.4 months after the follow-up test. In colorectal cancer, 4 of 19 patients (21.1%) [B2: 1/6 (16.7%), C2: 3/13 (23.0%)] were positive on the initial test and 10 of 19 patients (52.6%) [B2: 4/6 (66.7%), C2: 6/13 (46.2%)] were positive on a follow-up test showing an increase in positive rates during a follow-up, however, no significant correlation between CEA mRNA positivity and subsequent relapse was demonstrated. These results suggest that an early tumor cell dissemination may occur in gastrointestinal tract cancer without subsequent relapse, however, the serial regular examination of CEA mRNA level may contribute to predicting a subsequent relapse in AGC IIIb in gastric cancer.

Adult↗

PubMed2001

Divergent effects of 4-1BB antibodies on antitumor immunity and on tumor-reactive T-cell generation.

4-1BB is an inducible receptor-like protein expressed rapidly by both CD4 and CD8 T-cells after activation. 4-1BB cross-linking, either by binding to 4-1BBL or by antibody ligation, delivers a costimulatory signal to enhance T-cell activation and proliferation. Previous studies have demonstrated that the administration of 4-1BB monoclonal antibodies (mAbs) induces antitumor immune responses. In the current study using several murine tumors, we examined the systemic effects of 4-1BB mAb on the growth of s.c., intracranial (i.c.), and pulmonary metastases. In addition, the effects of 4-1BB mAb on the generation of antitumor effector T cells were examined. Treatment of 3-day i.c. MCA 205 sarcoma and GL261 glioma with the antibody resulted in prolongation of survival and cure of disease in some mice, whereas only minimal therapeutic effects were observed in established s.c. and pulmonary tumors. No antitumor effects against the poorly immunogenic B16/D5 melanoma were observed. Interestingly, successful treatment of i.c. tumors induced concomitant regression of s.c. tumors. Experiments using severe combined immunodeficient mice and mice depleted of either CD4 or CD8 T cells demonstrated T-cell dependence of the antitumor effects. For generation of effector T cells in the tumor-draining lymph nodes (LNs), administration of 4-1BB mAb had adverse effects, despite the apparent hypertrophy of the LNs. During in vitro activation of tumor-draining LN T cells with anti-CD3 and interleukin 2, the 4-1BB mAb augmented proliferation, resulting in an increase in CD8 T cells. However, they were less therapeutic than not treated LN cells. In adoptive immunotherapy, the coadministration of 4-1BB mAb enhanced the therapeutic efficacy. These results thus demonstrate the limits and potential advantages of 4-1BB antibody interactions with antitumor T cells in vivo and in vitro and suggest that therapeutic interactions of the antibody may be used in a variety of immunotherapeutic approaches.

Animals↗

PubMed2001

Activation of Na(+), K(+), Cl(-)-cotransport mediates intracellular Ca(2+) increase and apoptosis induced by Pinacidil in HepG2 human hepatoblastoma cells.

The role of Na(+), K(+), Cl(-)-cotransport (NKCC) in apoptosis of HepG2 human hepatoblastoma cells was investigated. Pinacidil (Pin), an activator of ATP-sensitive K(+) (K(ATP)) channels, induced apoptosis in a dose- and time-dependent manner in HepG2 cells. Pin increased intracellular K(+) concentration ([K(+)](i)). Bumetanide and furosemide, NKCC inhibitors, significantly inhibited the Pin-induced increased [K(+)](i) and apoptosis, whereas K(ATP) inhibitors (glibenclamide and tolbutamide) had no effects. The Pin-induced [K(+)](i) increase was significantly prevented by reducing extracellular Cl(-) concentration, and Pin also increased intracellular Na(+) concentration ([Na(+)](i)), further indicating that these effects of Pin may be due to NKCC activation. In addition, Pin induced a rapid and sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which was completely prevented by the NKCC inhibitors. Treatment with EGTA or BAPTA/AM markedly inhibited the Pin-induced apoptosis. Inhibitors of Na(+), Ca(2+)-exchanger, bepridil, and benzamil significantly prevented both [Ca(2+)](i) increase and apoptosis induced by Pin. Taken together, these results suggest that Pin increases [Na(+)](i) through NKCC activation, which leads to stimulation of reverse-mode of Na(+), Ca(2+) exchanger, resulting in [Ca(2+)](i) increase, and in turn, apoptosis. These results further suggest that NKCC may be a good target for induction of apoptosis in human hepatoma cells.

Adenosine Triphosphate↗

PubMed2001

Role of pertussis toxin-sensitive G-proteins in intracellular Ca2+ release and apoptosis induced by inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in HepG2 human hepatoblastoma cells.

Previously, we have reported that inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by glibenclamide induced intracellular Ca2+ release from IP(3)-sensitive stores and apoptosis in HepG2 human hepatoblastoma cells (Kim JA, Kang YS, Lee SH, Lee EH, Yoo BH, Lee YS. 1999. Biochem Biophys Res Commun 261:682-688). In this study we investigated the upstream signals involved in the mechanism of these actions of glibenclamide. Treatment with glibenclamide initiated production of inositol 1,4,5-trisphosphate (IP(3)) in a dose- and time-dependent manner. The glibenclamide-induced formation of IP(3) was significantly inhibited by CFTR activators (levamisole and bromotetramisole). The intracellular Ca2+ release and apoptosis induced by glibenclamide were significantly suppressed by treatment with phospholipase C (PLC) inhibitors (U-73122 and manoalide) or by pretreatment with pertussis toxin (PTx). In addition, PTx-catalyzed ADP-ribosylation of GTP-binding proteins (G-proteins) was markedly enhanced by treatment with glibenclamide in a time-dependent manner. Taken together, these results suggest that PTx-sensitive G-proteins coupled to PLCbeta may mediate the intracellular Ca2+ release and apoptosis induced by inhibiting CFTR Cl- channels in HepG2 cells. These results further suggest that the PTx-sensitive G-proteins may be a valuable target for the therapeutic intervention of human hepatomas.

Adenosine Diphosphate Ribose↗

PubMed2001

Microscopic and macroscopic approaches of Cu(II) removal by FSM-16.

The removal of Cu(II) by a mesoporous material, FSM-16, was studied using electron paramagnetic resonance (EPR) spectroscopy and surface complexation modeling (SCM). Free copper ions, adsorbed and precipitated Cu(II) species were qualitatively identified by in situ EPR spectroscopy of Cu-FSM-16 suspensions at room temperature and at 77 K. In addition, the adsorbed species was identified as a Cu(II) species with an axial symmetry from an analysis of the EPR spectra of "dry" Cu-FSM-16 at 77 K. On the basis of the EPR results, the removal of Cu(II) as a function of pH under various experimental conditions was successfully simulated by assuming two removal mechanisms such as surface complexation and surface precipitation. In the acidic pH range (< pH 6), free copper ions were predominant, and surface complexed then surface precipitated species became dominant as the pH increased.

Adsorption↗

PubMed2001

The role of cholecystokinin in conditional compensatory responding and morphine tolerance in rats.

As elaborated in the conditioning analysis of tolerance, cues present at the time of drug administration become associated with the drug effect. A particularly salient cue that may become associated with the drug effect is the pharmacological drug-onset cue inherent to drug administration. Drug-associated cues contribute to tolerance by eliciting a conditional compensatory response that attenuates the drug effect. For example, the early drug effect, having been paired with the subsequent larger drug effect, may elicit the release of antiopioid peptides that counter opioid effects. The role of a putative antiopioid peptide, cholecystokinin-8 (CCK), in the associative mechanisms of opiate tolerance was evaluated. The results of these experiments suggest that a CCK2 receptor antagonist attenuates both the expression of opiate tolerance and the conditional compensatory response hypothesized to mediate such tolerance.

Animals↗

PubMed2001

Identification and subtyping of Trichophyton mentagrophytes by random amplified polymorphic DNA.

Trichophyton mentagrophytes is one of the most common pathogens for human and animal dermatophytoses and known as a complex species with variable morphology, and ecologic and genetic backgrounds. We performed random amplified polymorphic DNA (RAPD) analysis on 43 human and 18 animal isolates of T. mentagrophytes along with other 10 anamorphic species of dermatophytes and three teleomorphic species of T. mentagrophytes. Using RAPD analysis with primer 5'-ATGGATC(G,C)(G,C)C-3' (ATGS), all T. mentagrophytes strains produced identical band patterns with those of Arthroderma vanbreuseghemii, one of the teleomorphs of T. mentagrophytes, regardless of their phenotypes. Therefore, T. mentagrophytes could be identified by RAPD analysis with primer ATGS. Using RAPD analysis with primer 5'-ATGGATCGGC-3' (ATG) on T. mentagrophytes, human isolates yielded two distinct subgroups related by their colony morphologies at the time of primary isolation from patients. Three morphologic types--cottony, powdery and persicolor--revealed identical bands whereas the granular type lacked one minor band (0.74 kbp). Animal isolates of T. mentagrophytes produced five band patterns and some of them were identical with those of human isolates. With primer 5'-GAAGGCTCCC-3' (OPAO-15), animal isolates of T. mentagrophytes showed diverse band patterns in contrast to the uniform band pattern of human isolates. These results suggest that RAPD analysis may be a useful tool to identify and subtype T. mentagrophytes complex.

Animals↗