Impact of Nucleotide Mutations at the HNF3- and HNF4-Binding Sites in Enhancer 1 on Viral Replication in Patients with Chronic Hepatitis B Virus Infection

BACKGROUND/AIMS: The hepatitis B virus (HBV) genome contains binding sites for hepatocyte nuclear factors (HNF) 3 and 4 in the core domain of enhancer 1 (Enh1), and mutations in this domain have a strong impact on virus replication. We aimed to identify frequent base-mutation sites in the core domain of Enh1 and to examine the impact of these mutations on viral replication. METHODS: We studied virological characteristics and genetic sequences in 387 patients with chronic hepatitis B. We evaluated functional differences associated with specific mutations within the core domain of Enh1. RESULTS: Mutations in the core domain were found with significant frequency in C1126 (122/387 [31.5%], the binding site for HNF3) and in C1134 (106/387 [27.4%], the binding site for HNF4). A single mutation at nt 1126 (C1126) was identified in 17/123 (13.8%), and 105/123 (85.4%) had double mutations (C1126/1134). The level of HBV DNA (log10 copies/mL) was lower in single mutants (C1126, 5.81+/-1.25) than in wild (6.80+/-1.65) and double mutants (C1126/1134, 6.81+/-1.54). Similarly, the relative luciferase activity of C1126 and C1126/C1134 was 0.18 and 1.12 times that of the wild-type virus, respectively. CONCLUSIONS: Mutations in the HNF3 binding site inhibit viral replication, whereas mutations at the HNF4 binding site restore viral replication.

Comparison of Biological and Genetic Characteristics Between Sucrose-Fermenting and Sucrose-Nonfermenting Vibrio vulnificus Isolates

Twelve strains of V. vulnificus isolated from clinical specimens in 2002~2004 in Jeollado province were determined for their biologic groups, serotypes, presence of vvhA (hemolysin/cytolysin) gene, DNA sequence, and PFGE patterns of NotI-restricted genomic DNA. The following results were obtained. All 12 isolates were biogroup 1, and API 20E profiles were: 5146105 for 5 (41.7%) isolates, and 5148125 for 2 isolates with sucrose fermentation. Ten (83.3%) of the 12 isolates was V. vulnificus serotype O4A, and two sucrose-fermenting isolates belonged to serotype O2. Alleles of cytolysin-hemolysin gene were detected in all 12 isolates. The nucleotide sequences of vvhA genes from strains WKHC 212 and WKHC 221 showed 94~97% similarity compared with those from previously reported 7 strains, YJ016, CMCP6, L-180, CDC B3547, IF Vv10, CIP 75.4T and CNRVC 970121. PFGE of NotI-restricted genomic DNA from the 12 isolates showed approximately 48.5 to 873-kb fragments and they were clustered to five (A to E) patterns. Two sucrose-fermenting isolates belonged to pattern D with 95% similarity with each other. Two strains isolated from two different patients had two identical patterns C and D. It is concluded that sucrose-fermenting strains also exist among clinical isolates of V. vulnificus in Korea, and they can be identified by using API 20E system, and by detecting vvhA gene. DNA sequences and PFGE pattern of NotI-restricted genomic DNA suggested that the two sucrose-fermenting isolates belonged to an identical clone, and two strains each isolated from two different patients belonged to two identical clones.

Protective Effect of PKC Affecting Taxol-induced Cytotoxicity in MCF-7 Cells / 대한해부학회지

Paclitaxel (taxol) is known as effective drug inhibition of cell cycle encouraging activity in human ovarian and metastatic breast cancers and malignant melanoma. It is an antimicrotubule agent that is believed to mediate its antineoplastic effects by inducing mitotic arrest followed by apoptosis. The relation between phorbol 12 myristate 13 acetate (PMA), protein kinase C (PKC) activator, and taxol-induced apoptosis is not well understood until now. This study was performed to investigate the effects of PMA on the signal transduction pathways of taxol-induced apoptosis in MCF-7 human breast carcinoma cells. Taxol-induced apoptosis is attenuated by curcumine, JNK inhibitor, and pyrrolidine dithiocarbamate (PDTC), inhibitor of NFkB. Pretreatment with PKC activator (PMA) or protein kinase A (PKA) activators (forskolin and dibutyryl cAMP) inhibited taxol-induced apoptosis in MCF-7 cells. In addition, thapsigargin, a specific inhibitor of endoplasmic reticulum(ER) Ca(2+)-ATPase and CaCl2, also blocked the activation of caspases by taxol. From these results suggest that taxol-induced apoptosis may be mediated via JNK or NFkB pathway and PKC activation.

Study of Hydroxyurea Induced Caspase Activities in Chronic Myelogenous Leukemic Cell Line, K562 cells / 대한임상병리학회지

BACKGROUND: Chronic myelogenous leukemia is a chronic myeloproliferative disorder characterized by leukocytosis with myeloid elements at all stages of differentiation, t(9;22)(q34;q11) and bcr/abl rearrangement. We studied hydroxyurea induced apoptotic changes such as externalization of phosphatidylserine, caspase activities on human chronic myelogenous leukemic cell line, K562 cells. METHODS: K562 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and treated hydroxyurea. Viability was examined by MTT assay. Apoptosis were examined by annexin V stain, caspase (such as caspase-, caspase-, caspase-, caspase-, and caspase-) activities, and DNA fragmentation. RESULTS: The viability of K562 cells were markedly decreased in a dose dependent manner of hydroxyurea. Phosphatidylserine externalization was detected by annexin V stain after 3 hours in hydroxyurea treated K562 cells and the value of lactate dehydrogenase was not significantly changed in their culture media. The upstream effector of caspase- was slightly increased and had influenced on caspase-. And downstream acting caspase protease of caspase- was markedly increased in a time dependent manner at hydroxyurea treated K562 cells. In addition, however the activities of caspase- and caspase- were not increased. We also found DNA fragmentation at hydroxyurea treated K562 cells between 48 hours and 72 hours on agarose gel electrophoresis. CONCLUSIONS: Hydroxyurea induces apoptotic change in K562 cells via externalization of phosphatidylserine, activations of caspase-, caspase-, caspase- proteases, and DNA fragmentation.

Bacterial Lipopolysaccharide - Induced Generation of Nitric Oxide is Mediated by ERK1 and JNK1 / SAPK in Primary Rat Neonatal Astrocytes / 대한면역학회지

No abstract available.

The Effects of Divalant Cation on the Idarubicin-Induced Apoptosis / 대한소아혈액종양학회지

PURPOSE: Zinc ion is critical for the functional and structural integrity of eukaryotic cells and participate in the regulation of apoptosis. In general, zinc inhibits a nuclear endonuclease, thereby causing inhibition of apoptosis. Recent studies have pointed to a role for a family of caspase proteases that act upstream of endonuclease. The widely used chemotherapeutic agents exert effects by inducing of apoptosis in sensitive tumor cells. In this study, we investigated the effects of zinc ion and other divalent cation on the idarubicin (IDA)-induced apoptosis of HL-60 cells. In addition, to determine whether Zn inhibits an event upstream of endonuclease activation, we analysed the activity of caspase-3, 9 and proteolytic cleavage of procaspase-3 and PARP [poly (ADP-ribose) polymerase]. METHODS: HL-60 cells were cultured in RPMI 1640 and treated with various doses and time periods of IDA with or without pretreatment of ZnCl2, CaCl2 and MgCl2. Cell viability was measured by trypan blue staining. For detection of apoptotic death, cells were stained with Hoechst dye and observed under fluorescence microscopy. The activities of caspase-3 and caspase-9 were measured by the proteolytic cleavages of Ac- DEVD-AMC and Ac-LEHD-AFC as flurogenic substrates, respectively. The proteolytic cleavages of procaspase-3 and PARP were analyzed by Western blotting using anti- caspase-3 and anti-PARP antibody, respectively. RESULTS: IDA induced the apoptotic death of HL-60 cells in a dose and time dependent manner, which was characterized by increasing chromatin condensation and DNA fragmentation. Pretreatment of HL-60 cells with ZnCl2 caused potent inhibition of IDA-induced apoptosis. Consistent with apoptotic death of HL-60 cells, IDA induced the catalytic activation of caspase-3 and caspase-9. After pretreatment of ZnCl2, the activation of caspase- 3 and the proteolysis of PARP induced by IDA were potently inhibited. But, after pretreatment of CaCl2 and MgCl2, there were no significant changes of IDA-induced apoptosis and proteases activity. CONCLUSION: Zinc ion suppressed the IDA-induced apoptosis via the inhibitions of caspase-9 and caspase-3. But calcium and magnesium ions didn't affect the IDA-induced apoptosis.

The Effect of Protein Kinase C Pretreatment on Gliotoxin Induced Apoptosis in H9c2 Cells / 체질인류학회지

Aspergillus funigatus and other pathogenic fungi synthesize a toxic epidithi- odiopiperzine (ETP) metabolite called gliotoxin. Gliotoxin is an epidithiodiopiperzine compound which can both react with sulfhydryl groups and form hydrogen peroxide. The fungal toxin gliotoxin induces apoptotic cell death in a variety of cells. Apoptosis induced by gliotoxin need calcium but effect of calcium preconditioning is unknown by gliotoxin. We studied the effect of protein kinase C and calcium preconditioning on gliotoxin-induced apoptosis in H9c2 cell. PKC and calcium preconditiong inhibited DNA fragmentation by gliotoxin. From this above results suggest that gliotoxin induce apoptosis via caspase-3 activation, because caspase-3 inhibitor (DEVD-CHO) didn't induce apoptosis in gliotoxin treated H9c2 clls. Calcium and PKC preconditioning inhibit caspase-3 activation by gliotoxin. These data means that PKC preconditioning is related with caspase-3 regulate in gliotoxin-induced apoptosis.

Radiation-induced Apoptotic Signaling Pathway in HL - 60 Cells / 대한암학회지

PURPOSE: The mechanical insights of death of cancer cells by ionizing radiation are not yet clearly defined. Recent evidences have demonstrated that radiation therapy may induce cell death via activation of signaling pathway for apoptosis in target cells. This study was designed whether ionizing radiation may activate the signaling cascades of apoptosis including caspase family cystein proteases, mitogen-activated protein (MAP) kinases, and transcriptional activation factors in target cells eventually leading to death. MATERIALS AND METHODS: HL-60 cell line in the log phase was used in this study and the culture media was RPMI 1640. The irradiation was done using the linear accelarator and the radiation does was 10 Gy, 20 Gy, and 30 Gy, respectively. The cell viability was tested by MTT assay and apoptosis was identified by the DNA fragmentation assay. JNK1 (cJun N-terminal kinase) and ERK (extracellular-signal regulated protein kinase) activity was analyzed by the in vitro Ig complex kinase assay. NF- kB (Nuclear Factor- kB) and AP-1 (activator protein-1) activity was assayed by the electrophoretic mobility sbift assay. RESULTS: Ionizing radiation decreased the viability of HL-60 cells in a time and dose dependent manner. Ionizing radiation-induced cell death of HL-60 cells may be an apo- ptotic death which was evidenced as apoptotic characteristic ladder pattern fragmentation of DNA over 20 Gy at 4 hours. Ionizing radiation specifically induced the activation of CPP32-like cystein protease rather than ICE-like protease of HL-60 cells in a time and dose dependent manner. The activation of CPP32-like cystein protease was also evidenced by the digestion of poly (ADP-ribose) polymerase with 30 Gy ionizing irradiation at 2 hours. The activity of JNK1 was transiently increased up to 3.6 fold by 30 Gy ionizing radiation at 2 hours. Ionizing radiation also rapidly activated the transcriptional activation factors including AP-1 and NF- kB at 10 or 30 min. CONCLUSION: These data suggested that ionizing radiation-induced apoptosis was mediated by the activation of CPP32-like cystein protease, JNK1, and transcriptional activation factors

Signaling Pathway of the Bacterial Lipopolysaccharide-induced Generation of Nitric Oxide in Rat Primary Astorcytes / 신경정신의학

OBJECTIVES: Nitric oxide(NO)plays an important role in pathophysiology of stroke and various neurodegenerative diseases. This study is designed to elucidate the mechanisms by which signaling pathway of LPS-stimulated NO generation in rat prmary astrocytes may be mediated by MAP kinase cascades and transcriptional activation of NF-kB. METHOD: The generation of NO from primary rat neonatal astrocytes was measured by using Greese's reagents and Western blot analysis with including Erk, JNK1 and p38 was assessed by in vitro immunecomplex kinase assay. Activation of transcriptional activator, NF-kB, was determined by using electrophoretic mobility shift assay. RESULTS: Treatment of cultured rat primary neonatal astorcytes with LPS results in the generation of NO as well as increase in the expression of inducible nitric oxide synthase(iNOS) LPS-induced NO generation is inhibited by the addition of inhibitors of MEK and JNK1/SPAK, PD 98059 and curcumin. LPS also imcreases the phosphotransferase activity of Erk as well as JNK1 and increases the phosphorylation of p38. Inhibition of Ras results in decrease of LPS-inudced NO generation. cAMP decreases the LPS-induced NO generation via inhibition of JNK1. Furthermore LPS activates transcriptional activator, NF-kB which is inhibited by the addition of inhibitors of MEK and JNK. CONCLUSION: These data suggest that MAP kinases, especially Erk and JNK1, may mediate the signaling cascade of LPS-induced NO generation in rat primary astrocytes via activation of transcriptional factor, NF-kB.

Bacterial Endotoxin, Lipopolysaccaride, and Induced Activation of Stress-Activated Protein Kinase and Generation of Nitric Oxide

BACKGROUND: Previously, it has been suggested that lipopolysaccaride (LPS) stimulates the activation of the transcriptional factor activator protein (AP-1) which is in part regulated by activation of the c-Jun N-terminal kinase (JNK) / stress-activated protein kinase (SAPK) in the murine macrophage cell line RAW 264.7. METHODS: Consistent with this notion, we find that treatment of LPS on RAW 264.7 cells induces the generation of nitric oxide (NO) and results in the activation of JNK and treated with NO donors and NO inhibitors. RESULTS: NO donors including sodium nitroprusside (1 mM), GSNO (0.2 mM), or SNAP (0.5 mM) treatment of the macrophage cell line markedly induces the activation of JNK. However NGMMA (2 mM), a competitive inhibitor of NO, does not inhibit the activation of JNK induced by LPS. SIN-1, NO, and superoxide donor induce an activation of JNK that is slightly decreased by treatment with sodium dismutase whereas the activation of JNK is significantly augmented by adding sodium dismutase with catalase. C2 ceramide suppresses the generation of NO induced by LPS, but significantly increases the activity of JNK in vivo. LPS can induce the activation of JNK at 30 min after stimulation in RAW 264.7 cells. Exposure to SNP does not affect the enzymatic activity of JNK, immunoprecipitates, JNK, and c-Jun N-terminal proteins. CONCLUSIONS: These data suggest that even though NO is one of the major activators of JNK induced by LPS, there is, at least, an NO-independent JNK activation, signaling a pathway for LPS. Also, there may be an undefined NO-sensitive JNK-regulator (s) in vivo.

Roles of Nitric Oxide and Tumor Necrosis Factor in Liver Inloammation Induced by C . parvum and LPS / 대한면역학회지

No abstract available.

Gliotoxin induces the Apoptosis in HL-60 Cells / 대한면역학회지

Many fungi including Penicillium, Aspergillus, Gliocladium, and Thermoascus produce an epipolythiodioxopiperazine class of fungal metabolite, gliotoxin, which contirbutes the pathogenesis of fungal infection as an immunomodulator and cytotoxic agent. This study is designed to define the mechanism by which gliotoxin exerts the cytotoxic effect of gliotoxin on human promyelocytic leukemic cells, HL-60. Gliotoxin induces the apoptosis of HL-60 cells which is characterized by the ladder pattern fragmentation of DNA. Gliotoxin induces the activation of DEVD-specific cysteine protease in a time- and dose-dependent rnanner. It also increases the phosphotransferase activities of c-Jun N-terminal kinase1 (JNK1) and p38 in gliotoxin-treated HL-60 cells. Furthermore, gliotoxin decreases the activation of transcriptional activator, actiating protein (AP-1) and NF-kB. These results suggest that gliotoxin induces the apoptotic death of HL-60 cells via activation of DEVD- specific caspase as well as mitogen activated protein kinases (MAP kinases) including JNK1 and p38, and inhibition of transcriptional activators, AP-1 and NF-kB.

Interaction of FcalphaR with gamma Subunit of FcgammaRIalpha to Modulate Cbl, Shc and Grb2 Adaptor Proteins / 대한면역학회지

No abstract available.

Effect of Nitric Oxide on the Viability of Bone Marrow - Derived Cultured Mast Cells / 대한면역학회지

It is well established that mast cell proliferation and maturation are regulated by two principle cytokines, IL-3 and the c-kit ligand stem cell factor (SCF). Previous reports have demonstrated that bone marrow-derived IL-3-dependent mast cells exhibit the characteristic apoptosis on removal of IL-3. To know how the number of mast cells is controlled, we observed the effects of nitric oxide (NO) on the murine bone marrow-derived cultured mast cells (BMCMC). Apoptosis was measured by the analysis of flow cytometric data and electrophoretic evidence of DNA fragmentation. Our data showed that sodiurn nitroprusside (SNP)-a NO releasing substance- induced apoptosis in BMCMC. Cell cycle analysis showed that the number of the G,/G, and S phase decreased markedly, while the percentage of cell in G,/M phase was increased. Also, SNP alone induced cell death, whereas SNP in combination with SCF markedly decreased cell death of BMCMC. SNP-induced apoptosis was partially inhibited by the treatment of BMCMC with SCF. Our results suggest that NO might have sorne role in the regulation of the number of mast cells.

Effects of Taxol on the Synthesis of Nitric Oxide in Murine Microglial Cells / 대한면역학회지

Taxol, an anticancer drug, blocks cell division by stabilizing microtubules. However, taxol has distinct cell-cycle-independent effects. For example, taxol and bacterial LPS induce strikingly similar responses in murine microglial cells. Here, we report that taxol, like LPS, provides a ""second"" signal for murine microglial cell activation to induce tumoricidal activity. Tumoricidal activity determined by MTT assay appeared that taxol or LPS alone weakly activated microglial cells to kill P815 mastocytoma cells, whereas combinations of taxol or LPS with IFN-r synergized to activate macrophages to lyse tumor cells in a dose dependent manner. Secretion of nitric oxide (NO) correlated with tumor cell killing, and the activated microglial cells failed to kill tumor cell targets in the presence of N'-monomethyl-L-arginine (N'MMA), a competitive inhibitor of NO synthase (NOS). Treatment of the cells with anti-TNF-a neutralizing antibodies clearly blocked taxol plus IFN-r induced tumoricidal activity as well as NO production. Collectively, the data illustrate the potential for taxol to activate microglial cell mediated-antitumor mechanisms in addition to its better characterized role as an anti-mitotic agent.

Flow Cytometric Analysis of T-cell Subsets in Brain Tumor Patients

The immunocompetence is important not only to kill the neoplastic cells but also to keep the neoplastic cells from growing further. T lymphocyte is plays the most important role in maintaining the tumor immunity efficiently. T lymphocyte has its specific functions depending in the subset of T lymphocytes. The author analyzed the T lymphocyte subsets in 31 brain tumor patients using anti-CD3, anti-CD4, anti-CD8 monoclonal antibodies and flow cytometry to determine the immunological status of brain tumor patients. All CD3, CD4 and CD8 subsets were reduced in both benign and malignant brain tumor patients but more signigicantly reduced in malignant tumor group. But in benign tumor group, the subtypes of T lymphocytes were not so different from those of normal healthy controls except the pituitary tumor patients, who showed the significant decrease in all the subtypes. In malignant tumor group, each subtype was signigicantly reduced and CD8 subtypes was markedly reduced in metastatic tumor patients, These analyses were considered to have the possibility to be contributable to planning the further immunotherapy and also the possibility to moniter the brain tumor patients clinically.