Hepatitis: Lee J

Feitelson MA, Lee J. · Department of Pathology, Anatomy and Cell Biology, Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA. · Cancer Lett. · Pubmed #17188425 No free full text.

Abstract: Chronic liver disease associated with long term hepatitis B virus (HBV) infection contributes importantly to the development of hepatocellular carcinoma (HCC). A salient feature of these chronic infections is the integration of subgenomic HBV DNA fragments into many different locations within the host DNA, suggesting that integration is random. Although this may promote genetic instability during liver regeneration which accompanies a bout of chronic liver disease, the actual role of integrated HBV DNA in hepatocarcinogenesis is uncertain. Importantly, most integration events retain the HBV open reading frame encoding the HBx antigen (HBxAg), which is the virus contribution to HCC. In addition, many integration events reported in the literature occur near or within fragile sites or other cancer associated regions of the human genome that are prone to instability in tumor development and progression. Genetic instability associated with integration potentially alters the expression of oncogenes, tumor suppressor genes, and microRNAs (miRNAs) that may contribute importantly to tumorigenesis. If so, then selected integration events may alter pathways that are rate limiting in hepatocarcinogenesis, thereby providing targets with diagnostic/prognostic potential and for therapeutic intervention.

Lee J, Yoo BC, Lee HS, Yoo HW, Yoo HH, Kang MJ, Kim DH. · Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, Chungryang, Seoul, Korea. · Ther Drug Monit. · Pubmed #16418707 No free full text.

Abstract: The purpose of this study was to develop an analytical method for the determination of 2'-fluoro-5-methyl-beta-l-arabinofuranosyl uracil triphosphate (L-FMAU-TP) in human peripheral blood mononuclear cells (PBMCs), and its application in the determination of cellular levels of L-FMAU-TP in PBMCs isolated from patients treated with 2'-fluoro-5-methyl-beta-l-arabinofuranosyl uracil (L-FMAU). An ion-pairing liquid chromatography (IPC) method, coupled with negative ion electrospray ionization tandem mass spectrometry (ESI-MS/MS), was developed for the accurate and repeatable detection of L-FMAU-TP, with a limit of detection of 1.6 pmol/10 cells. The calibration curve for L-FMAU-TP was linear over the concentration range 1.6 to 80 pmol/10(6) cells. The intra- and inter-day precision was lower than 11.2%, and the accuracy was between 97.1 and 106.9%. When applied to the determination of L-FMAU-TP in PBMCs isolated from HBV-infected patients undergoing L-FMAU treatment, the levels reached a steady state concentration 4 weeks after daily single oral administration of 20 mg L-FMAU, and these levels were maintained for up to 12 weeks, but then decreased 12 weeks after drug cessation. The terminal half-life of L-FMAU-TP in PBMCs after drug cessation was estimated to be 15.6 days.

Kim S, Lee J, Ryu WS. · Department of Biochemistry, Yonsei University, Seoul, Korea. · J Virol. · Pubmed #19515776 No free full text.

Abstract: Hepadnaviruses replicate via reverse transcription of an RNA template, the pregenomic RNA (pgRNA). Although hepadnaviral polymerase (Pol) and retroviral reverse transcriptase are distantly related, some of their features are distinct. In particular, Pol contains two additional N-terminal subdomains, the terminal protein and spacer subdomains. Since much of the spacer subdomain can be deleted without detrimental effects to hepatitis B virus (HBV) replication, this subdomain was previously thought to serve only as a spacer that links the terminal protein and reverse transcriptase subdomains. Unexpectedly, we found that the C terminus of the spacer subdomain is indispensable for the encapsidation of pgRNA. Alanine-scanning mutagenesis revealed that four conserved cysteine residues, three at the C terminus of the spacer subdomain and one at the N terminus of the reverse transcriptase subdomain, are critical for encapsidation. The inability of the mutant Pol proteins to incorporate into nucleocapsid particles, together with other evidence, argued that the four conserved cysteine residues are critical for RNA binding. One implication is that these four cysteine residues might form a putative zinc finger motif. Based on these findings, we speculate that the RNA binding activity of HBV Pol may be mediated by this newly identified putative zinc finger motif.

Zangwill KM, Eriksen E, Lee M, Lee J, Marcy SM, Friedland LR, Weston W, Howe B, Ward JI. · UCLA Center for Vaccine Research, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Los Angeles, CA, USA. · Pediatrics. · Pubmed #19047220 No free full text.

Abstract: BACKGROUND: Prelicensure studies of diphtheria, tetanus, acellular pertussis, hepatitis B, inactivated polio virus vaccine suggested that there were higher rates of fever after its administration than when its component antigens were given separately. METHODS: We conducted an open, controlled, cohort study to evaluate selected potential adverse events after receipt of diphtheria, tetanus, acellular pertussis, hepatitis B, inactivated poliovirus vaccine in the Southern California Kaiser Permanente Health Care Plan. From April 2003 through June 2005, we identified 61,004 infants who received >or=1 dose of vaccine (120000 total doses). This group was compared with a previous cohort of 58,251 age-, gender-, and medical center-matched infants (116,637 doses) who received diphtheria, tetanus, acellular pertussis vaccine and separate doses of hepatitis B and inactivated poliovirus vaccines from January 2002 through March 2003. We compared the incidence of seizures, medically attended events that were associated with fever, and other selected adverse outcomes. RESULTS: We identified 16 infants (8 with fever) who had a seizure in the diphtheria, tetanus, acellular pertussis, hepatitis B, inactivated poliovirus cohort and 15 infants (6 with fever) among control subjects in the 8-day period after receipt of any dose of vaccine. The incidence of all seizures or seizures associated with fever was not significantly different between cohorts. The incidence of medically attended events that were associated with fever in the 4-day period after any dose of vaccine was also similar in both cohorts. As well, no significant differences between the diphtheria, tetanus, acellular pertussis, hepatitis B, inactivated poliovirus and control cohorts, were noted in the incidence of allergic reactions within 48 hours of any dose of vaccine, outpatient visits within 21 days, hospitalizations within 21 days, or death within 1 year. CONCLUSIONS: We did not observe a statistically significant increase in any of several clinically important safety events after diphtheria, tetanus, acellular pertussis, hepatitis B, inactivated poliovirus vaccination compared with a historical cohort who received separate component vaccines.

Seo HS, Park JS, Han KY, Bae KD, Ahn SJ, Kang HA, Lee J. · Department of Chemical and Biological Engineering, Korea University, Anam-Dong 5-1, Sungbuk-Ku, Seoul 136-713, South Korea. · Vaccine. · Pubmed #18586361 No free full text.

Abstract: The biochemical and physical properties of hepatitis B virus (HBV) small surface antigen (S-HBVsAg) from Berna Biotech Korea Corp. were systematically analyzed and characterized. Through various electrophoresis and immunoblotting assay of S-HBVsAg and its proteolytic products, it was confirmed that the S-HBVsAg vaccine particles are present in the form of covalent multimers that are assembled via strong intermolecular disulfide bonds. The S-HBVsAg particles contain no N-glycosylation moiety but some O-glycosidically linked mannoses. Evidently from N-terminus sequencing of both monomers and dimers that are formed by complete and partial reduction, respectively, of the S-HBVsAg particles under reducing SDS-PAGE condition, it is evident that each polypeptide within S-HBVsAg particles has authentic sequence of N-terminus. Denaturation plot shows that the S-HBVsAg vaccine particles were extremely stable especially in the solution with high acidity. This stability property of S-HBVsAg vaccine particles could provide very useful information for the optimization of the downstream process of recombinant S-HBVsAg particles synthesized from yeast cultures.

Lee HJ, Lee J, Shin MK, Ryu WS. · Department of Biochemistry, Yonsei University, Seoul, 120-749 Korea. · Mol Cells. · Pubmed #18460902 No free full text.

Abstract: A hepadnaviruses replicates its DNA genome via reverse transcription of an RNA template (pregenomic RNA or pgRNA), which has a cap structure at the 5' end and a poly(A) tail at the 3' end. We have previously shown that the 5' cap is indispensable for encapsidation of the pgRNA. A speculative extension of the above finding is that the cap contributes to encapsidation via its interaction with the poly(A) tail, possibly involving eIF4E-eIF4G-PABP interaction. To test this hypothesis, poly(A)-less pgRNAs were generated via cleavage by a cis-acting hepatitis delta virus ribozyme sequence. We found that accumulation of the poly(A)-less pgRNA was markedly diminished, mostly likely due to its reduced stability. Importantly, however, the remaining poly(A)-less pgRNAs were nonetheless encapsidated and reverse transcribed normally when the reduced stability was taken account. Our finding clearly contradicts the notion that the poly(A) tail has any function in encapsidation and viral reverse transcription.

Schwarz K, Garrett B, Lee J, Thompson D, Thiel T, Alter MJ, Strathdee S. · Johns Hopkins University School of Medicine, 600 N. Wolfe Street-Brady 320, Baltimore, MD 21287, USA. · J Urban Health. · Pubmed #18274908 links to  free full text

Abstract: Homeless youth are at increased risk for hepatitis B virus (HBV) infection and HBV vaccine coverage is poor in this group. The purpose of our study was to determine if a shelter-based HBV vaccine program in children and adolescents 2-18 years of age with a randomized controlled trial using a culturally appropriate HBV video could increase HBV vaccine coverage rates. Subjects were randomized to an 8 min HBV video or a control, smoking prevention video. Before exposure to the videos, HBV knowledge, and demographics were assessed in caregivers and adolescents. HBV vaccine no. 1 was offered to all subjects who did not produce a vaccine record; subsequently, an accurate HBV vaccine history was obtained from medical providers. Subjects were asked to return 1 and 3 months after visit 1, HBV vaccine was offered to all with incomplete coverage, and HBV knowledge was reassessed. There were 328 children and adolescents cared for by 170 caregivers enrolled in the study. One hundred and four had incomplete HBV vaccine coverage. Data are reported for all family units with at least one subject needing vaccine. There were 53 children and adolescents randomized to the HBV video vs. 51 to the smoking video. HBV knowledge scores of caregivers improved at Visit no. 2 vs. no. 1 in the HBV video group (p = 0.01) but not in the smoking group (p = 0.82). Similar results were observed for adolescents in the HBV video group (p = 0.05) but not in the smoking group (p = 0.40). Exposure to the HBV video vs the smoking video had a significant effect on return rates for vaccine at Visit no. 2 (59 vs. 31%; p = 0.05) but not at Visit no. 3 (47 vs. 18%, p = 0.06). The shelter-based vaccine program was very effective in increasing HBV coverage rates in the entire group of 328 children and adolescents enrolled in the study, from 68% coverage at baseline to 85% at the conclusion of the study. We conclude that shelter-based HBV vaccine programs can be highly effective in increasing vaccine coverage rates in older children and adolescents. A brief exposure to a culturally appropriate HBV video improves HBV knowledge and may improve return rates for vaccine.

Supekova L, Supek F, Lee J, Chen S, Gray N, Pezacki JP, Schlapbach A, Schultz PG. · Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. · J Biol Chem. · Pubmed #17951261 links to  free full text

Abstract: The propagation of the hepatitis C virus (HCV) is a complex process that requires both host and viral proteins. To facilitate identification of host cell factors that are required for HCV replication, we screened a panel of small interference RNAs that preferentially target human protein kinases using an HCV replicon expressing the firefly luciferase gene as a genetic reporter. Small interference RNAs specific for three human kinases, Csk, Jak1, and Vrk1, were identified that reproducibly reduce viral RNA and viral protein levels in HCV replicon-bearing cells. Treatment of replicon cells with a small molecule inhibitor of Csk also resulted in a significant reduction in HCV RNA and proteins, further supporting a role for Csk in HCV replication. The effects of siRNAs targeting eight kinases known to be negatively regulated by Csk were then examined; knock down of one of these kinases, Fyn, resulted in up-regulation of the HCV replicon, suggesting that Csk mediates its effect on HCV replication through Fyn. This conclusion was further corroborated by demonstration that replicon cells treated with Csk inhibitor contained lower levels of the phosphorylated form of Fyn than control cells.

Han HK, Han CY, Cheon EP, Lee J, Kang KW. · BK21 Project Team, College of Pharmacy, Chosun University, Gwangju 501-759, South Korea. · Biochem Biophys Res Commun. · Pubmed #17433259 No free full text.

Abstract: The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1alpha (HIF-1alpha) and induced the nuclear translocation of C/EBPbeta. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1alpha siRNA but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1alpha activation, and suggest HIF-1alpha for the therapeutic target of HBV-mediated chemoresistance.

Lee SH, Lee H, Park JS, Choi H, Han KY, Seo HS, Ahn KY, Han SS, Cho Y, Lee KH, Lee J. · Department of Chemical and Biological Engineering, Korea University, Seoul, South Korea. · FASEB J. · Pubmed #17283220 links to  free full text

Abstract: We report on the ultrasensitive protein nanoprobe system that specifically captures disease marker (autoantibodies of Type I diabetes in this case) with attomolar sensitivity. The system relies on supramolecular protein nanoparticles that bind a specific antibody [65 kDa glutamate decarboxylase (GAD65)-specific autoantibody, i.e., the early marker of Type I diabetes]. The ultrasensitive detection of early marker of Type I diabetes during the early phase of pancreatic beta-cell destruction is important because individuals at high risk of developing Type I diabetes can be identified several years before the clinical onset of the ailment. The bacterial expression of chimera genes encoding N-[human ferritin heavy chain (hFTN-H)]::[specific antigenic epitope]-C produces supramolecular nanoparticles with uniform diameters (10-15 nm), owing to self-assembly activity of hFTN-H. Each nanoparticle, formed by intermolecular self-assembly between the chimera protein molecules, is subjected to carrying a large number (presumably, 24) of epitopes with a homogeneous and stable conformation per autoantibody binding, thereby allowing substantial enhancement of sensitivity. The sensitivity was finally boosted to 3 attomolar concentration of the autoantibodies, 4-9 orders of magnitude more sensitive than conventional immunoassays. Also, this ultrasensitive protein nanoprobe successfully detected natural autoantibodies in the sera from Type I diabetic patients. The attomolar sensitivity was successfully reproduced on the detection of other antibodies, i.e., monoclonal antibodies against hepatitis B surface antigen. With the two antibody markers above, the feasibility of simultaneous and multiplexing-mode detection was also demonstrated.

Lee J, Kim JH, Kim K, Jin HM, Lee KB, Chung DJ, Kim N. · Research Institute of Medical Sciences, Chonnam National University Medical School, Gwangju, Korea. · Mol Cell Biochem. · Pubmed #16909305 No free full text.

Abstract: Hepatitis C combination therapy comprising ribavirin and interferon-alpha causes dramatic improvement with the sustained virological response; however, this treatment may result in the loss of bone mineral density. To investigate the effects of ribavirin on bone cells, we examined osteoblast differentiation as well as the formation of osteoclasts from their precursors. Ribavirin enhances osteoclast formation through osteoblasts by up-regulation of TRANCE/RANKL gene expression, whereas it has no significant effect on either osteoblast differentiation or on bone formation. Understanding ribavirin's underlying mechanism of action on bone cells will enable the improved management of bone loss in chronic hepatitis C patients.

Lee J, Wu CC, Lee KJ, Chuang TH, Katakura K, Liu YT, Chan M, Tawatao R, Chung M, Shen C, Cottam HB, Lai MM, Raz E, Carson DA. · Department of Medicine, University of California at San Diego, La Jolla, CA 92093-0663, USA. · Proc Natl Acad Sci U S A. · Pubmed #16446426 links to  free full text

Abstract: IFN-alpha is used to suppress the replication of hepatitis C virus (HCV) in chronically infected patients with partial success. Here we present evidence showing that a ligand of Toll-like receptor 7 (TLR7) can induce anti-HCV immunity not only by IFN induction, but also through an IFN-independent mechanism. Human hepatocyte line Huh-7 carrying an HCV replicon expressed TLR7, and activation of the receptor induced several antiviral genes including IFN regulatory factor-7. Inhibitors of the enzyme inosine monophosphate dehydrogenase augmented both IFN-dependent and -independent antiviral effect. Prolonged exposure of Huh-7 cells to a TLR7 ligand [SM360320 (9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine)], alone or in combination with an inosine monophosphate dehydrogenase inhibitor, reduced HCV levels dose dependently. Immunohistochemical analysis of livers shows that TLR7 is expressed in hepatocytes of normal or HCV-infected people. Because TLR7 agonists can impede HCV infection both via type I IFN and independently of IFN, they may be considered as an alternative treatment of chronic HCV infection, especially in IFN-alpha-resistant patients.

Park K, Lee K, Lee J, Yeo S, Lee S, Cheon DS, Choi W, Ahn J, Kim S, Jee Y. · Division of Enteric and Hepatitis Viruses, Department of Virology, National Institute of Health, Korea Center for Disease Control and Prevention, Seoul, Korea. · J Med Virol. · Pubmed #16299722 No free full text.

Abstract: A variant of coxsackievirus A24 (CA24v) is one of the agents causing acute hemorrhagic conjunctivitis. There was an epidemic of acute hemorrhagic conjunctivitis caused by CA24v in Korea from 2002 to 2003. Seventy-one strains of CA24v were isolated from 159 conjunctival specimens (45%). Most of the patients were school children under the age of 20. The epidemic began in the first week of August in 2002, and spread extensively, with a peak in the third week of September. CA24v strains were also isolated from conjunctival specimens in 2003. Reverse transcription polymerase chain reaction (RT-PCR) was performed and sequencing of the 340 bp fragment of the VP1 region of the viruses. Sequencing data were multiple-aligned using CLUSTAL W (version 1.81). Phylogenetic trees were plotted using TreeView (version 1.6.6). Homologies ranged from 97.7%-100%, depending on geographical regions: from 99.4%-100% in 2002 and 98.4%-100% in 2003. A phylogenetic tree based on the nucleotide sequence homologies formed clusters depending on years rather than on geographical regions. Identities (98%-100%) were found among the Korean CA24v strains, and there was 85%-90% homology between these and the prototype strain.

Park JS, Seo HS, Yum JS, Moon HM, Lee J. · Department of Chemical and Biological Engineering, Korea University, Sungbuk-Ku, Seoul. · Biotechnol Bioeng. · Pubmed #16116655 No free full text.

Abstract: In Saccharomyces cerevisiae, we synthesized and secreted L-HBVsAg (named as pre-S(Met1 to Asn174)::S(Met175 to Ile400)) and three mutants, i.e., pre-S degree degree::S (Asn15Gln and Asn123Gln), pre-S degree degree::S degree (Asn15Gln, Asn123Gln, and Asn320Gln), and pre-S degree degree::S degree degree (Asn15Gln, Asn123Gln, Asn233Gln, and Asn320Gln). All of the secreted pre-S::S was N-glycosylated, i.e., hyper-mannosylated. In the secretion of pre-S degree degree::S and pre-S degree degree::S degree, besides the hyper-mannosylated form, another immunoreactive protein with much lower molecular mass was observed, which seems to be unglycosylated form of pre-S degree degree::S and pre-S degree degree::S degree. Only a part of the secreted pre-S degree degree::S or pre-S degree degree::S degree molecules was N-glycosylated, and the site for the partial N-glycosylation seems to be Asn233 in S-antigen region. Compared to the N-glycosylated pre-S degree degree::S and pre-S degree degree::S degree, pre-S degree degree::S degree degree (non-N-glycosylated mutant) was secreted with lower secretion efficiency but showed apparent immunoreactivity to anti-S antigen monoclonal Ab. Interestingly, unlike pre-S degree degree::S degree degree with authentic C-terminus, the recombinant pre-S degree degree::S degree degree with C-terminal myc or poly-histidine tag (pre-S degree degree::S degree degree::tag) was almost all aggregated into insoluble proteins in the intracellular region. Conclusively, the C-terminal sequence and glycosylation in S-antigen region seem to be of crucial importance in determining the secretion efficiency of L-HBVsAg in S. cerevisiae.

Hann HW, Lee J, Bussard A, Liu C, Jin YR, Guha K, Clayton MM, Ardlie K, Pellini MJ, Feitelson MA. · Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania, USA. · Cancer Res. · Pubmed #15492253 links to  free full text

Abstract: Hepatitis B virus (HBV) carriers are at high risk for the development of hepatocellular carcinoma (HCC), but there are no reliable markers that will identify such high-risk carriers. The objective of this work is to identify serologic markers that may indicate the early presence of HCC. Since HBV-encoded X antigen (HBxAg) likely contributes to HCC by up- or down-regulation of host gene expression, X positive and negative HepG2 cells were made and subjected to cDNA subtraction. When specific ELISAs were constructed measuring differentially expressed antigens and corresponding antibodies, antibodies to several differentially expressed genes were detected. In cross-sectional and longitudinal studies, antibodies were predominantly present in patients with HBV-associated cirrhosis and HCC, but not in most carriers with hepatic inflammation alone or without active liver disease. Antibodies were also present in patients with hepatitis C virus (HCV)-related HCC, but rarely detected in sera from uninfected individuals, those with tumors other than HCC, or those with drug-induced hepatitis. Statistical analysis showed that HCC patients with four or more antibodies detectable before the appearance of HCC had decreased survival, suggesting that these markers may reflect stepwise hepatocarcinogenesis. Hence, these antibodies may serve as preneoplastic markers for HCC in HBV carriers with chronic liver disease, and may be identified by a simple blood test.

Lee J, Shin MK, Lee HJ, Yoon G, Ryu WS. · Department of Biochemistry, Yonsei University, Seoul 120-749, Korea. · J Virol. · Pubmed #15220419 links to  free full text

Abstract: Synthesis of the relaxed-circular (RC) DNA genomes of hepadnaviruses by reverse transcriptase involves two template switches during plus-strand DNA synthesis. These template switches require repeat sequences (so-called donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. To determine cis-acting elements apart from the donor and acceptor sites that are required for plus-strand RC DNA synthesis by hepatitis B virus (HBV), a series of mutants bearing a small deletion were made and analyzed for their impact on the viral genome synthesis. We found three novel cis-acting elements in the HBV genome: one element, located in the middle of the minus strand, is indispensable, whereas the other two elements, located near either end of the minus strand, contribute modestly to the plus-strand RC DNA synthesis. The data indicated that the first element facilitates plus-strand RNA primer translocation or subsequent elongation during plus-strand RC DNA synthesis, while the last two elements, although distantly located on the minus strand, act at multiple steps to promote plus-strand RC DNA synthesis. The necessity of multiple cis-acting elements on the minus-strand template reflects the complex nature of hepadnavirus reverse transcription.

Yoon SW, Park IY, Sohn BH, Lee J, Yeo WH, Lee YI. · Liver Cell Signal Transduction Laboratory, Bioscience Research Division, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon 305-600, Republic of Korea. · Biochem Biophys Res Commun. · Pubmed #15184062 No free full text.

Abstract: A new compound from Micromonospora sp. SA246, 9-hydroxycrisamicin-A (9-HCA-A), showed potential for activating hepatitis B virus (HBV) replication. To define the mechanism of 9-HCA-A, we used HepG2 2.2.15 cells which support HBV replication. 9-HCA-A activated HBV replication, increased episomal and integrated HBV DNA content, and increased secretions of HBV antigens (HBsAg and HBeAg) into culture medium. 9-HCA-A also activated HBV transcription in Hep2 2.2.15 cell line. To examine transcriptional control mechanisms, we analyzed the effect of 9-HCA-A on four different HBV promoters (Core, PreS1, PreS2, and X) in hepatoma cell line. 9-HCA-A responsive element was located at HBx promoter. By EMSA, we showed that 9-HCA-A activated the HBx promoter by detaching the 9-HCA-A responsive element binding protein (9H-REBP). Protein phosphatase (PP2A1) treatment detaches the 9H-REBP from the HBx promoter, similar to 9-HCA-A, while protein kinase A treatment does not detach the 9H-REBP from the HBx promoter. Our results showed that 9H- REBP functions as a repressor of HBV replication while 9-HCA-A activated protein phosphatase released the BP on the HBx promoter, thus activating HBV replication.

Shin MK, Lee J, Ryu WS. · Department of Biochemistry, Yonsei University, 134 Shinchondong, Seodaemungu, Seoul 120-749, Korea. · J Virol. · Pubmed #15163718 links to  free full text

Abstract: Hepadnaviruses replicate through reverse transcription of an RNA pregenome, resulting in a relaxed circular DNA genome. The first 3 or 4 nucleotides (nt) of minus-strand DNA are synthesized by the use of a bulge in a stem-loop structure near the 5' end of the pregenome as a template. This primer is then transferred to a complementary UUCA motif, termed an acceptor, within DR1* near the 3' end of the viral pregenome via 4-nt homology, and it resumes minus-strand DNA synthesis: this process is termed minus-strand transfer or primer translocation. Aside from the sequence identity of the donor and acceptor, little is known about the sequence elements contributing to minus-strand transfer. Here we report a novel cis-acting element, termed the beta5 region (28 nt in length), located 20 nt upstream of DR1*, that facilitates minus-strand DNA synthesis. The deletion or inversion of the sequence including the beta5 region diminished minus-strand DNA synthesis initiated at DR1*. Furthermore, the insertion of the beta5 region into its own position in a mutant in which the sequences including the beta5 region were replaced restored minus-strand DNA synthesis at DR1*. We speculate that the beta5 region facilitates minus-strand transfer, possibly by bringing the acceptor site in proximity to the donor site via base pairing or by interacting with protein factors involved in this process.

Lee J, Lee HJ, Shin MK, Ryu WS. · Yonsei University, Seoul, Korea. · Biotechniques. · Pubmed #15038153 No free full text.

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Boucek C, Krishnamurthy P, Marsh JW, Lee J. · University of Pittsburgh School of Medicine, Pennsylvania 15213, USA. · Anesthesiology. · Pubmed #14695743 links to  free full text

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Lee J, Park JS, Moon JY, Kim KY, Moon HM. · Department of Chemical and Biological Engineering, Korea University, Anam-Dong, Sungbuk-Ku, Seoul 136-701, Republic of Korea. · Biochem Biophys Res Commun. · Pubmed #12659834 No free full text.

Abstract: Three types of recombinant pre-S antigens (i.e., pre-S1S2) of hepatitis B virus (HBV) were synthesized in Saccharomyces cerevisiae and secreted into extracellular medium: wild type (pre-S1S2) and two mutant antigens, pre-S1 degrees S2 (Asn15Gln) and pre-S1 degrees S2 degrees (Asn15Gln and Asn123Gln). An N-terminus sequence (Ser5-Ala28) of human interleukin 1 beta (hIL-1 beta) was used as synthetic prosequence of recombinant HBV surface antigen (pre-S), secreted from S. cerevisiae. The expression cassette comprised the signal peptide of the killer toxin of Kluyveromyces lactis, the synthetic prosequence above, KEX2 dibasic endopeptidase cleavage site (-Lys-Arg-), and the surface antigen. The recombinant pre-S1S2 and pre-S1 degrees S2 were secreted in the hyper-mannosylated form, while the recombinant pre-S1 degrees S2 degrees was produced without N-glycosylation. It has been demonstrated that the two particular N-linked glycans at Asn15 and Asn123 interfered with the B-cell response to the HBV-derived pre-S1S2, resulting in low titers of pre-S1S2-neutralizing antibodies. This problem was overcome by eliminating both of the N-glycosylation signals. Despite enhanced immunogenicity, the recombinant pre-S1 degrees S2 degrees showed two major problems: (1) inefficient Kex2 cleavage process in the secretory pathway and (2) the severe proteolytic degradation by yeast proteases. The efficiency of Kex2 cleavage increased dramatically by removing N-glycosylation signal in the synthetic prosequence, but the proteolysis of pre-S1 degrees S2 degrees was somewhat inevitable. Further systematic approaches including modulation of degree of N-glycosylation or relocation of N-glycosylation sites in the recombinant pre-S1S2 may make it possible to achieve both enhanced immunogenicity and resistance towards proteolytic degradation of the secreted pre-S antigen.

Chun E, Lee J, Cheong HS, Lee KY. · Division of Virology and Immunology, Mogam Biotech. Institute, Koosung-myun, Yongin-city Kyonggi-do, South Korea. · J Immunol. · Pubmed #12538674 links to  free full text

Abstract: We have previously reported several CTL epitopes derived from the hepatitis B viral X Ag (HBx). In this study, we evaluated whether HBx-specific CTLs can be effectively used in adoptive cancer immunotherapy. To validate the possibility, four peptides containing a HLA-A2.1-restricted binding consensus motif were identified from the HBx protein and tested for their ability to activate CTL from PBMCs isolated from chronic carriers of HBV (n = 12). We selected two highly potent epitopes, HBx 52-60 (HLSLRGLFV) and HBx 115-123 (CLFKDWEEL), that are capable of inducing Ag-specific cytotoxic T cells in patient PBMCs. For adoptive immunotherapy using HBx-specific CTLs, we generated CTL clones restricted to the HBx 52-60 or HBx 115-123 peptide using a limiting dilution technique. LC-46, an HBx 52-60-specific clone, is CD62L(-)CD69(+)CD45RO(+)CD45RA(-)CD25(dim) and is stained by IFN-gamma (approximately 92%), IL-2 (30%), and TNF-alpha (56%), but not by IL-5, IL-10, IL-12, or TNF-beta, indicating that the cells are fully activated T cytotoxic 1-type cells. When LC-46 cells were adoptively transferred into xenografted nude mice bearing human hepatomas expressing HLA-A2.1 molecules and intracellular HBx proteins, the tumors were eradicated. Taken together, our data provide solid evidence for the feasibility of adoptive immunotherapy with HBx-sensitized CTLs in hepatitis disease, including hepatocellular carcinoma (HCC).

Lee J, Shim H, Park Y, Park S, Shin J, Yang W, Lee H, Park W, Chung Y, Lee S. · Central Research Laboratory, DongWha Pharm. Ind. Co. Ltd., Seoul, South Korea. · Bioorg Med Chem Lett. · Pubmed #12217361 No free full text.

Abstract: 2,5-Pyridinedicarboxylic acid derivatives were found to be the potent non-nucleoside inhibitors of hepatitis B virus (HBV) with IC(50) <or=0.01 microg/mL in a reverse transcriptase inhibitory effect. And they showed the low toxicity compared with the nucleoside analogues.

Ha-Lee YM, Lee J, Pyun H, Kim Y, Sohn J, Cho YJ, Kim Y. · Korea Nutrition Research Institute, Korea University, Seoul, Korea. · Arch Virol. · Pubmed #11315638 No free full text.

Abstract: The HBV in the sera of two chronic active hepatitis patients were analyzed for the promoter sequence heterogeneity. In most cases, the proportion of any particular clone in the total viral populations was less than 50%, showing high mutation rates. In contrast, promoter sequences of HBV from asymptomatic carriers revealed only a few point mutations with no deletions. HBV in chronic patient harbored variants with multiple mutations throughout promoters including 1762 (A-to-T), 1764 (G-to-A) double mutation in C promoter and deletions near CCAAT site in S promoter. Unlike other three promoter regions, C, pre-S1 and S, of HBV which revealed a high level of sequence heterogeneity, the X promoter region (from nt985 to 1430) showed little sequence heterogeneity within a patient. However, the predominant viral clones in two patients were quite different from each other. In addition to mutations in promoter regions, a deletion mutation in the translation start codon was also found in pre-S1 gene. The results in this report indicate that the mutation rates are not the same in all four promoters and that one of the strategies for maintaining persistent infection could be through mutations in viral promoters which then impair the balance of viral gene expressions.

Chung KM, Lee J, Kim JE, Song OK, Cho S, Lim J, Seedorf M, Hahm B, Jang SK. · Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, Korea. · J Virol. · Pubmed #10799599 links to  free full text

Abstract: It has been suggested that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays a role in the incapacitation of interferon by inactivation of RNA-dependent protein kinase PKR. In order to further investigate the role of NS5A, we tried to identify cellular proteins interacting with NS5A by using the yeast two-hybrid system. The karyopherin beta3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin beta3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin beta3 activity was investigated using a yeast cell line containing mutations in both PSE1 and KAP123, genes that are homologous to the human karyopherin beta3 gene. Human karyopherin beta3 complemented the loss of the PSE1 and KAP123 functions, supporting growth of the double mutant cells. However, expression of NS5A hampered the growth of the double mutant cells supplemented with human karyopherin beta3. On the other hand, expression of NS5A by itself had no effect on the growth of the double mutant expressing wild-type yeast PSE1. This indicates that NS5A may inhibit karyopherin beta3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.