Youn Uck Kim

Ca2+‐Mediated Activation of c‐Jun N‐Terminal Kinase and Nuclear Factor κB by NMDA in Cortical Cell Cultures

Abstract: We examined the possibility that c‐Jun N‐terminal kinase (JNK) and nuclear factor κB (NF‐κB) might be involved in intracellular signaling cascades that mediate NMDA‐initiated neuronal events. Exposure of cortical neurons to 100 µM NMDA induced activation of JNK within 1 min. Activity of JNK was further increased over the next 5 min and then declined by 30 min. Similarly, ionomycin, a selective Ca2+ ionophore, induced activation of JNK. The NMDA‐induced activation of JNK was abrogated in the absence of extracellular Ca2+, suggesting that Ca2+ entry is necessary and sufficient for the JNK activation. Immunohistochemistry with anti‐NF‐κB antibody demonstrated nuclear translocation of NF‐κB within 5 min following NMDA treatment. NMDA treatment also enhanced the DNA binding activity of nuclear NF‐κB in a Ca2+‐dependent manner. Treatment with 3 mM aspirin blocked the NMDA‐induced activation of JNK and NF‐κB. Neuronal death following a brief exposure to 100 µM NMDA was Ca2+ dependent and attenuated by addition of aspirin or sodium salicylate. The present study suggests that Ca2+ influx is required for NMDA‐induced activation of JNK and NF‐κB as well as NMDA neurotoxicity. This study also implies that aspirin may exert its neuroprotective action against NMDA through blocking the NMDA‐induced activation of NF‐κB and JNK.

Impact of the E540V Amino Acid Substitution in GyrB of Mycobacterium tuberculosis on Quinolone Resistance

ABSTRACT Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.

Androgen action on hepatic vitellogenin synthesis in the eel, Anguilla japonica is suppressed by an androgen receptor antagonist

Involvement of additional hormones other than estrogen in the control of vitellogenin (Vg) synthesis has been suggested in fish. However, no satisfactory explanation on the mechanism of the action of these hormones has been reported. In this study, we have exploited the possibility of androgen receptor mediation during the androgen action on the pathway of Vg synthesis. Hepatocytes were prepared from sexually immature Japanese eel Anguilla japonica and treated with estradiol-17beta (E2), 17alpha-methyltestosterone (MT), growth hormone, tamoxifen or flutamide, or in combination of these. Spent culture media were analysed by SDS-PAGE for Vg detection. Results from the chemical treatments demonstrated the necessity of E2 as the primary factor for Vg synthesis and requirement of additional hormones for the full expression of Vg. The effects of E2 and MT were effectively blocked by tamoxifen, an estrogen receptor antagonist and flutamide, an androgen receptor antagonist, respectively, indicating ER-mediated estrogen action and AR-mediated androgen action on Vg synthesis in this species.

In‐depth analysis of cysteine oxidation by the RBC proteome: Advantage of peroxiredoxin II knockout mice

Peroxiredoxin II (Prdx II, a typical 2‐Cys Prdx) has been originally isolated from erythrocytes, and its structure and peroxidase activity have been adequately studied. Mice lacking Prdx II proteins had heinz bodies in their peripheral blood, and morphologically abnormal cells were detected in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). In this study, a labeling experiment with the thiol‐modifying reagent biotinylated iodoacetamide (BIAM) in Prdx II−/− mice revealed that a variety of RBC proteins were highly oxidized. To identify oxidation‐sensitive proteins in Prdx II−/− mice, we performed RBC comparative proteome analysis in membrane and cytosolic fractions by nano‐UPLC‐MSE shotgun proteomics. We found oxidation‐sensitive 54 proteins from 61 peptides containing cysteine oxidation, and analyzed comparative expression pattern in healthy RBCs of Prdx II+/+ mice, healthy RBCs of Prdx II−/− mice, and abnormal RBCs of Prdx II−/− mice. These proteins belonged to cellular functions related with RBC lifespan maintain, such as cytoskeleton, stress‐induced proteins, metabolic enzymes, signal transduction, and transporters. Furthermore, protein networks among identified oxidation‐sensitive proteins were analyzed to associate with various diseases. Consequently, we expected that RBC proteome might provide clues to understand redox‐imbalanced diseases.

Molecular cloning and characterization of a single-chain variable fragment antibody specific for benzoylecgonine expressed in Escherichia coli

Benzoylecgonine is a major metabolite of cocaine. We generated hybridoma cells (C1303) producing anti-benzoylecgonine monoclonal antibody (mAb) with a single-chain variable fragment (scFv) and an antigen-binding domain from the C1303 cells. Genes encoding an scFv antibody and constant region (Fc) were amplified from a cDNA library of C1303 cells using PCR. The two frameworks built for scFv and scFv-Fc consisted of HL [(heavy chain variable region, VH) — linker — (light chain variable region, VL)] and HL-Fc, respectively. A 45 base-pair-long sequence encoding (Gly4-Ser)3 was used as the linker, and the mouse IgG1 constant region sequence (225 amino acids) was used as the Fc domain. These two types of recombinant Abs were determined to be 750 bp in length (which corresponds to a 30 kDa protein) in the HL and 1,432 bp in length (which corresponds to a 65 kDa protein) in the HL-Fc, respectively. The parental Ab and HL-Fc affinities against benzoylecgonine were measured by ELISA and found to be nearly equal to the Ab concentration. We were also able to measure HL affinity using an agarose diffusion assay (Ouchterlony test). The affinity of the recombinant single-chain antibody against benzoylecgonine was sufficiently comparable to that of the parent antibodies to be used for the immunodetection of specific drug compounds or the detoxification of drug abusers by immunotherapy.

Expression of c-Myc is related to host cell death following Salmonella typhimurium infection in macrophage

It has been known that ornithine decarboxylase (ODC) induced by the binding of c-Myc to odc gene is closely linked to cell death. Here, we investigated the relationship between their expressions and cell death in macrophage cells following treatment with Salmonella typhimurium or lipopolysaccharide (LPS). ODC expression was increased by bacteria or LPS and repressed by inhibitors against mitogen-activated protein kinases (MAPKs) in Toll-like receptor 4 (TLR4) signaling pathway. In contrast, c-Myc protein level was increased after treatment with bacteria, but not by treatment with LPS or heat-killed bacteria although both bacteria and LPS increased the levels of c-myc mRNA to a similar extent. c-Myc protein level is dependent upon bacterial invasion because treatment with cytochalasin D (CCD), inhibitors of endocytosis, decreased c-Myc protein level. The cell death induced by bacteria was significantly decreased after treatment of CCD or c-Myc inhibitor, indicating that cell death by S. typhimurium infection is related to c-Myc, but not ODC. Consistent with this conclusion, treatment with bacteria mutated to host invasion did not increase c-Myc protein level and cell death rate. Taken together, it is suggested that induction of c-Myc by live bacterial infection is directly related to host cell death.

Gene regulation function of the three specificity protein-1 (Sp1) within the human collectin placenta-1 proximal promoter

Scavenger receptors including collection-placenta 1 (CL-P1) are cell surface glycoproteins capable of binding to oxidized low density lipoprotein (oxLDL), which are expressed on endothelial cells, macrophages, and smooth muscle cells. We cloned and characterized a 750 base-pair fragment containing the 5′-flanking region of the human CL-P1 gene using a human genomic library. The fragment obtained from the translational start site, which contained three specificity protein-1 (Sp1) sites upstream of transcriptional start site and putative binding sites for granulocyte chemotactic factor and early growth response factor-1, was ligated to the pGL4.10-basic vector, and promoter activity was confirmed by transfection studies. Luciferase and electrophoretic gel motility shift assays revealed that three Sp1 binding sites showed specific DNA-protein binding. Deletion and mutation analyses showed that the region comprising the three Sp1 sites was required for CL-P1 proximal promoter activity in human umbilical vein endothelial cells, Hs683 cells, and SK-LMS cells. Furthermore, the third Sp1 (−28/−20) binding site was regulated through some factor, not the Sp1 protein, by hydrogen peroxide stimulation.

Involvement of androgens and growth hormone in the synthesis of vitellogenin in Japanese eel (Anguilla japonica)

Immature eels positively responded to estradiol-17β (E2) injection in terms of vitellogenin (Vg) synthesis but not to growth hormone (GH) or 17α-methyltestosterone (MT) injection. However, injection of MT or GH combined with E2 strongly increased Vg synthesis. In in vitro experiments, eel hepatocytes treated with E2, GH, or MT alone did not produce detectable amount of Vg, whereas the combination of E2 with GH or MT, or both greatly increased Vg synthesis in the hepatocytes.

Functional analysis of the receptor binding domain of SARS coronavirus S1 region and its monoclonal antibody

Severe acute respiratory syndrome (SARS) is caused by the SARS coronavirus (CoV). The spike protein of SARS-CoV consists of S1 and S2 domains, which are responsible for virus binding and fusion, respectively. The receptor-binding domain (RBD) positioned in S1 can specifically bind to angiotensin-converting enzyme 2 (ACE2) on target cells, and ACE2 regulates the balance between vasoconstrictors and vasodilators within the heart and kidneys. Here, a recombinant fusion protein containing 193-amino acid RBD (residues 318–510) and glutathione S-transferase were prepared for binding to target cells. Additionally, monoclonal RBD antibodies were prepared to confirm RBD binding to target cells through ACE2. We first confirmed that ACE2 was expressed in various mouse cells such as heart, lungs, spleen, liver, intestine, and kidneys using a commercial ACE2 polyclonal antibody. We also confirmed that the mouse fibroblast (NIH3T3) and human embryonic kidney cell lines (HEK293) expressed ACE2. We finally demonstrated that recombinant RBD bound to ACE2 on these cells using a cellular enzyme-linked immunosorbent assay and immunoassay. These results can be applied for future research to treat ACE2-related diseases and SARS.