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Biomedical subjects

W N Chen

Publications and source records attributed to W N Chen.

At least 19 recordsLinked to original sources
PubMed2001

Hepatitis B virus X protein in the proteasome of mammalian cells: defining the targeting domain.

The hepatitis B virus X (HBX) protein has been implicated in both hepatitis B virus-related pathogenesis and also in diverse cellular processes. The diversity of its activities may be mediated through its interaction with cellular organelles. However no clearly defined subcellular localization of HBX is available. We report here the localization of HBX in the proteasome complexes using green fluorescent protein tag. A new proteasome-targeting domain has also been defined in HBX by deletion study. Furthermore, a functional role of HBX in the cellular processes mediated by the proteasome complexes has been suggested by its cell cycle-independent localization in the proteasome. Further analysis of the functional role of HBX in the proteasome complexes should provide more information on the underlying mechanism of HBX ativities.

Animals↗
PubMed2000

Expression of integrated hepatitis B virus X variants in human hepatocellular carcinomas and its significance.

Hepatitis B virus X protein (HBX) has been implicated in the transactivation of diverse cellular genes and possibly also the pathogenesis of human hepatocellular carcinoma (HCC). We report the characterization of HBX variants from HBV-related human hepatocellular carcinoma (HCC). These HBX variants were integrated into the host chromosomes and also expressed in the HCC tissues. In addition, we report a novel in vitro HBX activity assay based on color changes that were indicative of the beta-galactosidase enzyme activity. Conducted in wheat germ lysates, the transactivating function of either wild type or mutant HBX protein was measured through their interaction with the Early Growth Response factor 1 (Egr-1) that controls the beta-galactosidase gene. Further analysis of these HBX deletion mutants using this assay may shed new insights on the significance of various mutations occurring in HCC-associated HBX.

Amino Acid Sequence↗
PubMed2000

Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy.

Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathological progressions during tumor formation and oxidation stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A inhibits cathepsin D activity with an IC50 of 10 nM. The nature of its binding to cathepsin D was further characterized using a fluorescence polarization measurement. Results showed that BODIPY FL-pepstatin A selectively binds to cathepsin D at pH 4.5. In fixed cells, BODIPY FL-pepstatin A stained lysosomes, where it co-localized with cathepsin D. This staining was depleted when cells were co-incubated with unlabeled pepstatin A in acidic buffer. In live cells, BODIPY FL-pepstatin A is internalized and transported to lysosomes. The staining in the lysosomes can be competed with unlabeled pepstatin A. These properties, along with the good photostability of the BODIPY FL fluorophore, make this probe a novel tool for the study of the secretion and trafficking of cathepsin D.

Animals↗
PubMed2000

Detection of hepatitis B virus surface antigen mutants in paraffin-embedded hepatocellular carcinoma tissues.

The antigenic "a" determinant of the human hepatitis B virus surface antigen (HBsAg) is highly conserved and involved in inducing neutralizing antibody. Mutations on this determinant have been associated with the hepatitis B virus (HBV) escape from vaccination but are also found in chronic HBV carriers, but their involvement in liver diseases including hepatocellular carcinoma (HCC) is unclear. To investigate the possible liver disease-associated role of HBsAg mutants, their incidence was analyzed in 11 paraffin embedded HCC tissues using an improved DNA extraction method. Mutations on the "a" determinant (Thr126Ser, Gly145Arg, a double mutant Thr126Ser/Gln129Asn, Met 133Leu and Thr140Ile) were identified in 5 samples while the wild type sequence was found in 2 others. Future characterization of these HCC-associated HBsAg mutants should provide new insights on their role in the pathogenesis of HCC.

Adult↗
PubMed2000

Mutations and deletions in core promoter and precore stop codon in relation to viral replication and liver damage in Singaporean hepatitis B virus carriers.

BACKGROUND: The core promoter of hepatitis B virus (HBV) is crucial for the viral replication and mutations may lead to the establishment of chronic infection and development of liver diseases. We analysed this region in Singaporean HBV carriers and assessed their association with viral replication and liver damage. MATERIALS AND METHODS: Thirty-three Singaporean HBV carriers were selected. Serological markers for HBV infection and indicators for liver functions were analysed using commercial kits. Among these patients, 17 were chronic carriers, 10 had cirrhotic livers and 6 others had hepatocellular carcinoma (HCC). The region on the HBV genome covering the entire core promoter and core gene was amplified for each patient by polymerase chain reaction. The amplified DNA fragments were sequenced and analysed. RESULTS: The incidence of mutations in the core promoter or the precore gene product (stop codon at amino acid 28) was not significantly higher compared with the wild type sequences in patients with liver damage. Most mutations in either the core promoter or precore gene significantly reduced the viral replication, as indicated by HBV DNA levels. High levels of HBV DNA were found in three mutants with deletion in the same region, presumably the binding site of liver enriched factor, within the core promoter. CONCLUSION: Our findings revealed a different mutation pattern in the core promoter in Singaporean HBV carriers. While most mutations may not be directly associated with the development of liver diseases, deletions in the core promoter could contribute to enhanced viral replication and should be studied further.

Adult↗
PubMed2000

Intra-familial evidence of horizontal transmission of hepatitis B virus surface antigen mutant G145R.

OBJECTIVES: To provide intra-familial evidence on the horizontal transmission of hepatitis B virus surface antigen (HBsAg) mutant G145R. METHODS: Serum samples from family members of 10 vaccinated infants who carried this G145R mutant were collected. The presence of the mutant was analysed by polymerase chain reaction (PCR) and sequencing. RESULTS: The G145R mutant was identified in family members of three of the 10 infants. In family 1, the mutant found initially in child 1 was identified in another child and the father. In families 2 and 3, the G145R mutant detected previously in child 1 was detected in the father. Additional mutations in HBsAg were identified in at least two members in family 1 and 2, suggesting horizontal transmission of the mutant among them. The G145R mutant was found in samples with high levels of neutralizing antibody against HBV (anti-HBs). In addition, liver damage was seen in one G145R carrier infant. CONCLUSIONS: The G145R mutant could be transmitted horizontally among family members, and this could occur in the presence of high levels of anti-HBs. Improvement of detection system for the G145R and other HBsAg mutant will be needed for their effective control.

Disease Transmission, Infectious↗
PubMed2000

Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes.

We have developed fluo-4, a new fluorescent dye for quantifying cellular Ca2+ concentrations in the 100 nM to 1 microM range. Fluo-4 is similar in structure and spectral properties to the widely used fluorescent Ca(2+)-indicator dye, fluo-3, but it has certain advantages over fluo-3. Due to its greater absorption near 488 nm, fluo-4 offers substantially brighter fluorescence emission when used with excitation by argon-ion laser or other sources in conjunction with the standard fluorescein filter set. In vitro, fluo-4 exhibited high fluorescence emission, a high rate of cell permeation, and a large dynamic range for reporting [Ca2+] around a Kd(Ca2+) of 345 nM. We have also developed several Ca(2+)-indicators related to fluo-4 having lower affinities for Ca2+ that are useful in cellular studies requiring quantification of higher [Ca2+]. In a variety of physiological studies of live cells, fluo-4 labeled cells more brightly than did fluo-3, when challenged with procedures designed to elevate calcium levels. Fluo-4 is well suited for photometric and imaging applications that make use of confocal laser scanning microscopy, flow cytometry, or spectrofluorometry, or in fluorometric high-throughput microplate screening assays. Because of its higher fluorescence emission intensity, fluo-4 can be used at lower intracellular concentrations, making its use a less invasive practice.

3T3 Cells↗
PubMed1999

Mutation "hot spot" in HLA class I-restricted T cell epitope on hepatitis B surface antigen in chronic carriers and hepatocellular carcinoma.

Mutations in the human hepatitis B virus (HBV) genome contribute to its escape from host immune surveillance and result in persistent infection. We report the identification of frequent mutations encompassing residues 29 to 53 of the HBV surface antigen in chronic HBV carriers as well as in patients with hepatocellular carcinoma. The location of these mutations, not found in patients with acute hepatitis and vaccinated infants, coincides with a human leukocyte antigen class I-restricted cytotoxic T lymphocyte epitope. Significantly, mutations occur at a higher frequency (83%) compared with those identified on the immunogenic "a" determinant (25%) of the corresponding patients. Our findings therefore suggest the potential importance of this novel mutation "hot spot" in the establishment of chronic HBV infection.

Adolescent↗
PubMed1999

Human hepatitis B virus mutants: significance of molecular changes.

Human hepatitis B virus, the leading pathogen for hepatitis B, is a compact DNA virus with viral genes that largely overlap. An increasing number of mutations have emerged following human interventions such as vaccination and anti-viral therapy. While vaccine escape mutants are characterized by mutations on the antigenic hepatitis B surface antigen, those carrying mutations in other viral proteins are either resistant to anti-viral therapy or implicated in acute liver diseases. Molecular identification of these various mutants should shed new lights on the underlying mechanism of hepatitis B virus viral escape and resistance and provide helpful information on their effective eradication.

Antiviral Agents↗
PubMed1999

Detection of hepatitis B surface antigen mutants and their integration in human hepatocellular carcinoma.

Vaccine escape hepatitis B virus surface antigen (HBsAg) mutants are capable of independent replication and have been implicated in acute hepatitis. We now report the detection of these mutants with changes at various positions of the antigenic 'a' determinant in human hepatocellular carcinoma (HCC). Southern blot analysis indicated that the HBsAg mutant with the Glycine to Arginine change at position 145 was integrated in HCC, whereas those with a Threonine at position 133 instead of a Methionine were identified in the serum of aggressive HCC. Further studies on HBsAg mutants in HCC should provide new insights on their involvement in the hepatocarcinogenesis.

Arginine↗
PubMed1999

Hepatitis B virus variants with lamivudine-related mutations in the DNA polymerase and the 'a' epitope of the surface antigen are sensitive to ganciclovir.

Lamivudine is a new antiviral agent effective against hepatitis B viral (HBV) infections but can result in virus-drug resistance associated with mutations in the conserved 'YM552DD' motif of the HBV DNA polymerase. Due to their overlapping coding regions in the HBV genome, mutations in the DNA polymerase may result in substitutions in the hepatitis B surface antigen (HBsAg), albeit outside the antigenic 'a' epitope. Here we report the identification of a novel type of lamivudine-related mutations located in both the polymerase (YM552DD-->Y1552DD) and the 'a' epitope of HBsAg (Gly130-->Asp130). The same virus carried a HBsAg Gly145-->Arg145 mutation prior to therapy. Both the wild type HBV and lamivudine-related mutants with the Gly145-->Arg145 HBsAg mutation were suppressed following ganciclovir treatment, indicating a beneficial additive effect of both drugs against different forms of HBV mutants.

Adult↗
PubMed1998

Current aspects of hepatitis B surface antigen mutants in Singapore.

Mutations occurring on the major antigenic 'a' determinant of hepatitis B surface antigen (HBsAg) have been detected in immunized Singapore children, despite immunoprophylaxis with hepatitis B immune globulin and HBV vaccine. These vaccine-escape HBsAg mutants display a predominance of the Gly145-to-Arg145 mutation in the antigenic 'a' determinant. Our latest follow-up studies indicate the stability of this as well as other vaccine-escape HBsAg mutants over time. We have also identified HBsAg mutants in immunized children with amino acid substitutions outside the 'a' determinant. Transmission of various vaccine-escape HBsAg mutants have been shown in our epidemiological studies. Detection of HBsAg mutants, carrying amino acid changes at various position of the 'a' determinant in random population, points to their emergence in Singapore before the implementation of the vaccination programme. Of significant interest is the recent identification of vaccine-escape HBsAg mutants in hepatocellular carcinoma.

Adolescent↗