Biomedical subjects

S B Hwang

Publications and source records attributed to S B Hwang.

At least 19 recordsLinked to original sources

PubMed2001

Hepatitis C virus core protein potentiates TNF-alpha-induced NF-kappaB activation through TRAF2-IKKbeta-dependent pathway.

Previous work has implicated that the core protein of hepatitis C virus (HCV) may play a modulatory effect on NF-kappaB activation induced by TNF-alpha. However, it is unclear how HCV core protein modulates TNF-alpha-induced NK-kappaB activation. Here we show that overexpression of HCV core protein potentiates NF-kappaB activation induced by TNF-alpha. Expression of dominant negative form of TRAF2 inhibits the synergistic effects of HCV core protein on NF-kappaB activation, suggesting that HCV core protein potentiates NF-kappaB activation through TRAF2. Moreover, we demonstrate that HCV core protein potentiates TRAF2-mediated NF-kappaB activation via IKKbeta. In addition, HCV core protein associates with TNF-R1-TRADD-TRAF2 signaling complex, resulting in synergistically activation of NF-kappaB induced by TNF-alpha. Thus, these observations indicate that HCV core protein may play an important role in the regulation of the cellular inflammatory and immune responses through NF-kappaB.

Antigens, CD↗

PubMed2001

Anti-inflammatory activities of LDP-392, a dual PAF receptor antagonist and 5-lipoxygenase inhibitor.

Leukotrienes (LTs) and platelet-activating factor (PAF) are important mediators of inflammation and allergy. LDP-392, a novel dual PAF receptor antagonist and 5-lipoxygenase (5-LO) inhibitor, has been identified. LDP-392 is 17.9-fold more potent than zileuton (5-LO inhibitor) in the RBL cytosolic 5-LO assay, and equally potent as MK 287 (PAF receptor antagonist) in the human platelet PAF receptor binding assay. The in vivo dual activities of LDP-392 were confirmed by measuring the inhibition of ex vivo LTB(4)production in rats and PAF-induced hemoconcentration in mice. Intravenous administration of LDP-392 demonstrated greater inhibition than zileuton, BN 50739 or MK 287 on arachidonic acid-induced ear edema and protected mice from LPS-induced lethality. Topical administration of LDP-392, in a dose-dependent manner, inhibited TPA-induced ear edema in mice and UVB-induced erythema in guinea-pigs. These data suggest that LDP-392, as a dual PAF receptor antagonist and 5-LO inhibitor, may be of greater clinical effectiveness.

Animals↗

PubMed2001

Hepatitis C virus core protein potentiates c-Jun N-terminal kinase activation through a signaling complex involving TRADD and TRAF2.

The hepatitis C virus (HCV) core protein is a multifunctional viral nucleocapsid protein. Previously, it has been demonstrated that the HCV core protein interacts with the cytoplasmic domain of tumor necrosis factor receptor 1 (TNFR1). Since the TNFR1 is engaged in stimulation of transcriptional factor NF-kappaB and AP-1 through activation of IkappaB kinase and c-Jun N-terminal kinase (JNK, or stress-activated protein kinase), respectively, we have examined whether the interaction between core protein and TNFR1 can modulate JNK. In this study, we demonstrate that the HCV core protein synergistically activates TNFalpha-induced JNK at a core concentration dependent manner in human embryonic kidney (HEK) 293 cells. HCV core-mediated synergism of JNK activation was also detected in stable cells expressing HCV core protein. Furthermore, we demonstrate that HCV core protein does not compete with TNF receptor-associated death domain (TRADD) for its interaction with the death domain of TNFR1. Our in vivo data show that HCV core and TRADD form a ternary complex with TNFR1. These findings suggest that the HCV core protein modulates TNFR1 signaling and may, thus, play a role in chronic infection of HCV patients.

Apoptosis↗

PubMed2000

Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.).

Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

5' Untranslated Regions↗

PubMed1999

Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein.

Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-ethylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP-33 and NS5A. Interestingly, hVAP-33 was also shown to interact with NS5B, the viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-33: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex.

Animals↗

PubMed1999

Cell cycle arrest mediated by hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only viral-encoded protein of the hepatitis delta virus (HDV). This protein has been extensively characterized with respect to its biochemical and functional properties. However, the molecular mechanism responsible for persistent HDV infection is not yet clear. Previously, we reported that overexpression of HDAg protects insect cells from baculovirus-induced cytolysis [Hwang, S.B. Park, K.-J. and Kim, Y.S. (1998) Biochem. Biophys. Res. Commun. 244, 652-658]. Here we report that HDAg mediates cell cycle arrest when overexpressed in recombinant baculovirus-infected insect cells. Flow cytometry analysis has shown that HDAg expression in Spodoptera frugiperda cells causes an accumulation of substantial amounts of polyploid DNA in the absence of cell division. This phenomenon may be partly responsible for the persistent infection of chronic HDV patients.

Animals↗

PubMed1998

Characterization of mRNA for hepatitis delta antigen: exclusion of the full-length antigenomic RNA as an mRNA.

Hepatitis delta virus (HDV) encodes a single protein, the hepatitis delta antigen (HDAg), which is thought to be translated from a 0. 8-kb RNA of antigenomic sense. This subgenomic RNA species is present in very small amounts in HDV-infected liver tissues and in cultured cells infected or transfected with HDV, and in some cases it cannot be detected at all. In contrast, HDAg protein is present in large amounts in all natural and experimental models of HDV infection. This study addresses whether other HDV RNA species, such as the antigenomic-sense, genome-size HDV RNA can also serve as the mRNA for HDAg synthesis. Taking advantage of the ability of herpes simplex virus (HSV) to degrade only polyadenylated mRNAs, we examined the effect of HSV coinfection on HDAg synthesis. It was shown that HSV infection did degrade the subgenomic 0.8-kb HDV mRNA but not HDV genome-length RNA. Under such conditions, HDAg synthesis was completely inhibited. Furthermore, the genome-length HDV RNA was found not to be associated with polysomes. Finally, in vitro translation studies demonstrated that HDAg could not be translated directly from the genome-length antigenomic-sense HDV RNA. These results suggest that only the subgenomic RNA species of HDV possesses properties characteristic of the mRNA for HDAg and that the genome-length RNA cannot be used for translating HDAg. In addition, we found that HDV RNA replication did not depend on de novo HDAg synthesis.

5' Untranslated Regions↗

PubMed1998

Hepatitis C virus core protein interacts with heterogeneous nuclear ribonucleoprotein K.

Hepatitis C virus (HCV) core protein, a component of viral nucleocapsid, has been shown to modulate cellular and viral promoter activities. To identify potential cellular targets for HCV core protein, a human liver cDNA library was screened for core-interacting proteins using the yeast two-hybrid system. Among the proteins identified was heterogeneous nuclear ribonucleoprotein K (hnRNP K), which has been demonstrated to be a transcriptional regulator. The interaction of HCV core protein with hnRNP K was confirmed by glutathione S-transferase fusion protein binding assay, protein-protein blotting assay, and coimmunoprecipitation in vitro and in vivo. Additionally, these two proteins were shown to be partially colocalized in the nucleus. The hnRNP K-binding site in HCV core protein was mapped to the region from amino acid residues 25-91, a hydrophilic area near the N terminus. The HCV core protein-binding domain was located within amino acid residues 250 to 392, which contain the three proline-rich domains, of hnRNP K. Furthermore, HCV core protein relieved the suppression effect of hnRNP K on the activity of the human thymidine kinase gene promoter. The specific binding of HCV core protein to hnRNP K suggests that multiple functions of hnRNP K may be disrupted by the core protein during HCV infection and thus explains, in part, the pathogenesis of HCV.

Amino Acid Sequence↗

PubMed1998

(+/-)-trans-2-[3-methoxy-4-(4-chlorophenylthioethoxy)-5-(N-methyl-N- hydroxyureidyl)methylphenyl]-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran (CMI-392), a potent dual 5-lipoxygenase inhibitor and platelet-activating factor receptor antagonist.

By incorporating an N-hydroxyurea functionality onto diaryltetrahydrofurans, a novel series of compounds was investigated as dual 5-lipoxygenese (5-LO) inhibitor and platelet-activating factor (PAF) receptor antagonist. These dual functional compounds were evaluated in vitro for 5-LO inhibition in RBL cell extracts and human whole blood, and PAF receptor antagonism in a receptor binding assay. PAF-induced hemoconcentration and arachidonic acid- and TPA-induced ear edema in mice were used to determine in vivo activities. The structure-activity relationship analysis to define a preclinical lead is presented. (+/-)-trans-2-[3-methoxy-4-(4-chlorophenylthioethoxy)-5-(N-methyl- N-h ydroxyureidyl)methylphenyl]-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran (40, CMI-392) was selected for further study. In the arachidonic acid-induced mouse ear edema model, 40 was more potent than either zileuton (a 5-LO inhibitor) or BN 50739 (a PAF receptor antagonist), and it demonstrated the same inhibitory effect as a physical combination of the latter two agents. These results suggest that a single compound which both inhibits leukotriene synthesis and blocks PAF receptor binding may provide therapeutic advantages over single-acting agents. The clinical development of compound 40 is in progress.

Animals↗

PubMed1998

Overexpression of hepatitis delta antigen protects insect cells from baculovirus-induced cytolysis.

Hepatitis delta virus (HDV) is a human pathogen causing fulminant hepatitis and liver cirrhosis. HDV has a circular single-stranded RNA genome, which encodes only a single protein, hepatitis delta antigen (HDAg), from the antigenomic strand. Although the functional roles of HDAg have been extensively studied, the molecular mechanism of persistent infection and pathogenesis of HDV are not yet understood. Here we report that overexpressed HDAg protects cells from death in baculovirus-infected insect cells. Using both wild-type and recombinant baculovirus-infected insect cells, we have demonstrated that HDAg inhibited wild-type baculovirus-induced cytolysis and thus extended the survival of virus-infected insect cells. By deletion analysis, we show that N-terminal 25 amino acids are essential for this function. From these data, we suggest that HDAg may play a major role in persistent infection of HDV.

Animals↗

PubMed1998

Rac GTPase activity is essential for EGF-induced mitogenesis.

Rac, a member of the Rho family GTPases, has been implicated in the regulation of a wide range of biological processes including actin remodeling, cell transformation, G1 cell cycle progression, and gene expression. To determine whether Rac GTPase activity is required for epidermal growth factor-induced mitogenesis, Rat-2 stable cells expressing a dominant-negative Rac1 mutant, RacN17, were prepared. Exposure to EGF exhibited a significantly restricted growth response in Rat-2-RacN17 cells compared to Rat-2 parental cells, suggesting an essential role of Rac in EGF-induced mitogenesis. In contrast, addition of lysophosphatidic acid exerted the same level of growth in Rat-2 and Rat-2-RacN17 cells. To gain further evidence for the essential role of Rac in EGF-induced mitogenesis, we performed the microinjection experiment. EGF-induced DNA synthesis was significantly blocked by microinjection of recombinant RacN17 protein, and not control IgG. Our further study to analyze the downstream mediator of Rac in EGF-signaling to mitogenesis demonstrated that Rac-activated phospholipase A2 plays a critical role. Taken together, our results suggest that the "Rac and Rac-activated PLA2" cascade is one of the major mitogenic pathways induced by EGF.

Animals↗

PubMed1998

Hepatitis C virus core from two different genotypes has an oncogenic potential but is not sufficient for transforming primary rat embryo fibroblasts in cooperation with the H-ras oncogene.

Persistent infection with hepatitis C virus (HCV) is associated with the development of liver cirrhosis and hepatocellular carcinoma. To examine the oncogenic potential of the HCV core gene product, primary rat embryo fibroblasts (REFs) were transfected with the core gene in the presence or absence of the H-ras oncogene. In contrast to a previous report (R. B. Ray, L. M. Lagging, K. Meyer, and R. Ray, J. Virol. 70:4438-4443, 1996), HCV core proteins from two different genotypes (type 1a and type 1b) were not found to transform REFs to tumorigenic phenotype in cooperation with the H-ras oncogene, although the core protein was successfully expressed 20 days after transfection. In addition, REFs transfected with E1A- but not core-expressing plasmid showed the phenotype of immortalized cells when selected with G418. The biological activity was confirmed by observing the transcription activation from two viral promoters, Rous sarcoma virus long terminal repeat and simian virus 40 promoter, which are known to be activated by the core protein from HCV-1 isolate. In contrast to the result with primary cells, the Rat-1 cell line, stably expressing HCV core protein, exhibited focus formation, anchorage-independent growth, and tumor formation in nude mice. HCV core protein was able to induce the transformation of Rat-1 cells with various efficiencies depending on the expression level of the core protein. These results indicate that HCV core protein has an oncogenic potential to transform the Rat-1 cell line but is not sufficient to either immortalize primary REFs by itself or transform primary cells in conjunction with the H-ras oncogene.

Animals↗

PubMed1998

TR4 orphan receptor crosstalks to chicken ovalbumin upstream protein-transcription factor and thyroid hormone receptor to induce the transcriptional activity of the human immunodeficiency virus type 1 long-terminal repeat.

Here we investigate the roles of human testicular orphan receptors, TR2 and TR4, on the gene regulation of the long-terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). In gel-retardation assays, a palindromic element at the 5'-end of HIV-LTR,5'-AGGGGTCAGATATCCACTGACCTTT-3',showed high affinity to TR2 and TR4 with an equilibrium dissociation constant (Kd) of 1.11 +/- 0.48 (n = 3) and 0.52 +/- 0.12 nM (n = 3), respectively. Interestingly, each half-site of the palindromic element is sufficient to compete with the binding of the labeled palindromic element to TR2 or TR4 with an equilibrium inhibition constant (ki) around 10 nM. However, the transiently expressed TR2 or TR4 in Chinese hamster ovary (CHO) cells or Japanese quail muscle myoblasts (QM7) cells showed no activity in regulating the transcriptional activity of the chloramphenicol acetyltransferase (CAT) reporter gene inserted downstream of the HIV-LTR promoter. Although both TR2 and TR4 showed no effect on CAT activity by itself, our data showed only the TR4 could crosstalk to the chicken ovalbumin upstream protein-transcription factor (COUP-TF1) and thyroid hormone receptor (TR alpha 1), and potentiated the transcriptional activity of HIV-LTR on the CAT reporter gene regulated by COUP-TF1 and TR alpha 1. These results indicate that TR4, but not TR2, may couple to other nuclear receptors in the upregulation of the HIV replication.

Animals↗

PubMed1997

Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization.

Hepatitis C virus NS5B protein is an RNA-dependent RNA polymerase. To investigate the properties and function of this protein, we have expressed the NS5B protein in insect and mammalian cells. NS5B was found to be present as fine speckles in the cytoplasm, particularly concentrated in the perinuclear region, suggesting its association with the nuclear membrane, the endoplasmic reticulum, or the Golgi complex. This conclusion was supported by the biochemical demonstration that NS5B was associated with the membranes in the cells. Furthermore, it was shown that NS5B protein is a phosphoprotein. These properties may be related to its function as an RNA polymerase.

Animals↗

PubMed1997

Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor.

Hepatitis C virus (HCV) core protein is a multifunctional protein. We examined whether it can interact with cellular proteins, thus contributing to viral pathogenesis. Using the HCV core protein as a bait to screen a human liver cDNA library in a yeast two-hybrid screening system, we have isolated several positive clones encoding cellular proteins that interact with the HCV core protein. Interestingly, more than half of these clones encode the cytoplasmic domain of lymphotoxin-beta receptor (LT betaR), which is a member of the tumor necrosis factor receptor family. Their binding was confirmed by in vitro glutathione S-transferase fusion protein binding assay and protein-protein blotting assay to be direct and specific. The binding sites were mapped within a 58-amino-acid region of the cytoplasmic tail of LT betaR. The binding site in the HCV core protein was localized within amino acid residues 36 to 91 from the N terminus, corresponding to the hydrophilic region of the protein. In mammalian cells, the core protein was found to be associated with the membrane-bound LT betaR. Since the LT betaR is involved in germinal center formation and developmental regulation of peripheral lymphoid organs, lymph node development, and apoptotic signaling, the binding of HCV core protein to LT betaR suggests the possibility that this viral protein has an immunomodulating function and may explain the mechanism of viral persistence and pathogenesis of HCV.

Binding Sites↗

PubMed1996

Homotypic interaction and multimerization of hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein constitutes a viral nucleocapsid and may possess multiple functions. In this study, we demonstrated the homotypic interaction and multimerization of HCV core protein in vitro and in vivo. By using a yeast two-hybrid system, we showed that the amino-terminal hydrophilic portion (amino acids 1-115) of the core protein could interact with itself. Deletion analysis mapped the interacting domain within amino acid residues 36-91. The homotypic interaction of the core protein was also confirmed by in vitro protein-protein blotting assay using the recombinant HCV core proteins and by its binding to the glutathione S-transferase core fusion protein. The biological significance of the core protein self-interactions was demonstrated by the detection of multimeric forms of the core protein in mammalian cells. The domain responsible for multimerization was determined to be within the amino-terminal hydrophilic region (amino acids 1-115). Both the membrane-bound and the free core proteins exist in dimeric and multimeric forms, suggesting that multimerization of the HCV core protein occurred at an early stage of viral assembly and that the multimer forms may be involved in multiple functions of the core protein.

Animals↗

PubMed1995

A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus.

Mouse hepatitis virus (MHV), a murine coronavirus known to cause encephalitis and demyelination, uses murine homologues of carcinoembryonic antigens as receptors. However, the expression of these receptors is extremely low in the brain. By low-stringency screening of a mouse brain cDNA library, we have identified a member of the pregnancy-specific glycoprotein (PSG) subgroup of the carcinoembryonic antigen gene family. Unlike other PSG that are expressed in the placenta, it is expressed predominantly in the brain. Transfection of the cDNA into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to infection by some MHV strains, including A59, MHV-2, and MHV-3, but not JHM. Thus, this is a virus strain-specific receptor. The detection of multiple receptors for MHV suggests the flexibility of this virus in receptor utilization. The identification of this virus in receptor utilization. The identification of a PSG predominantly expressed in the brain also expands the potential functions of these molecules.

Amino Acid Sequence↗

PubMed1995

Differential subcellular localization of hepatitis C virus core gene products.

The expression of the core gene of two different hepatitis C virus (HCV) isolates was analyzed. In the presence of its downstream E1 envelope protein sequence, two major core protein products with molecular masses of 21 kDa (P21) and 19 kDa (P19) and a minor protein product with molecular mass of 16 kDa (P16) were detected. In the absence of its downstream E1 envelope protein sequence, P21 and P19 remained the major protein products expressed from the core gene of the HCV-RH isolate, whereas P16 became the major protein product of the core gene of the HCV-1 isolate. Analysis of the amino-terminal sequences of P21 and P16 expressed in Escherichia coli revealed that P21 and P16 were co-amino terminal. Deletion-mapping analysis indicated that P16 lacked the carboxy-terminal sequence of P21. Immunofluorescence analysis of the subcellular localization of different HCV core proteins indicated that P21 and P19 displayed a reticular and punctate staining pattern typical of endoplasmic reticulum-associated proteins, while P16 was localized to the nucleus. The distinct subcellular localization of P16 raises the possibility that P16 may have a biological function very different from those of P21 and P19.

Amino Acid Sequence↗