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Biomedical subjects

S J Lo

Publications and source records attributed to S J Lo.

At least 55 records · Page 3Linked to original sources
PubMed1988

Characteristics of the X gene of hepatitis B virus.

The 465-nucleotide sequence of the X gene from our cloned hepatitis B virus (HBV) DNA (subtype adw) was determined and compared to the same gene of 10 other published HBV sequences (3 of adw, 4 of adr, 2 of ayw, and 1 of ayr). We found (i) a total of 56 base differences among the 11 sequences (without counting the 27-base deletion in one adr) which resulted in 88% nucleotide homology, and (ii) 5 pairs of repeated sequence (3 direct repeats and 2 inverted repeats) that were highly conserved. Comparison of the protein amino acid sequences indicated that (i) there is 80% amino acid homology in total, and (ii) there are four highly conserved cysteine residues. In addition, the X gene of the adw subtype is more conserved than that of adr.

Amino Acid Sequence↗
PubMed1988

High-level production of hepatitis B viral X protein in Escherichia coli using gene II promoter of bacteriophage M13.

Region X is one of the four open reading frames (ORFs) of hepatitis B virus (HBV) and encodes a polypeptide of 154 amino acids (aa). A 584-bp BamHI-BglII fragment of the HBV DNA containing the major part of ORF X which encodes 145 aa was inserted into the BglII site within the gene II of bacteriophage M13. The insertion resulted in an in-phase gene II-X fused protein of 174 aa under the control of the gene II promoter. Cells harboring plasmids (pML alpha X.59 and pMLX.12d) derived from the above construct overproduced the 19-kDa fused protein in Escherichia coli at a level of 10%-20% of total cellular protein. The fused protein was recognized by the anti-X antibodies. This is the first demonstration of using gene II promoter of M13 to express a foreign gene efficiently.

Bacteriophage lambda↗
PubMed1988

Enhanced production of hepatitis B virus surface antigen in mouse C127 cell on a bovine papillomavirus-metallothionein vector.

We have constructed a recombinant plasmid pCPS12 containing the hepatitis B viral surface antigen (HBsAg) gene linked to the mouse metallothionein promoter on a BPV-pML2 vector. Two stable clones S12-8 and S12-2, obtained by transfection of the mouse C127 cells with pCPS12 propagated in dam+ dcm+ and dam- dcm- Escherichia coli respectively, exhibited different types of response to 5-azacytidine (5-aza-CR) and cadmium (Cd) induction. In S12-8, the productivity of HBsAg was enhanced by 5-aza-CR or 5-aza-CR plus Cd, but not by Cd alone. In S12-2, the expression of HBsAg was not affected by 5-aza-CR but was induced by Cd in the presence or absence of 5-aza-CR. This suggests that methylation may be important in controlling the HBsAg expression and the inducibility of Cd in the transfectants.

Animals↗
PubMed1987

Intra blood-cerebrospinal fluid-barrier detection of hepatitis B virus.

Hepatitis B, a major public health concern worldwide, is caused by hepatitis type B virus, a hepdnavirus that infects only human and certain nonhuman primates, and replicates strictly in hepatocytes. By using the techniques of slot and Southern blot DNA hybridization, and electron microscopy, the presence of HBV was identified in the cerebrospinal fluid of three affected individuals.

Blood-Brain Barrier↗
PubMed1987

Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core-related antigens (HBc/HBeAg) if well-differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH-7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH-7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus-associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS--PAGE after labeling with [35S]methionine. Antisera specific for X-gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV-related gene function and HBV replication.

Carcinoma, Hepatocellular↗
PubMed1987

Absence of cytokeratin in human hepatoma cell lines.

The immunofluorescence study revealed that both our established human hepatoma cell lines, HA22T/VGH and HA47T/VGH, were absent of cytokeratin. This observation was further confirmed by a western blot study. However, they as well as the other human hepatoma cells, Hep G2, Hep 3B, and SK-Hep-1 expressed vimentin.

Carcinoma, Hepatocellular↗
PubMed1986

Expression and secretion of hepatitis B viral surface antigen in E. coli.

Hepatitis B viral surface antigen (HBsAg) gene was subcloned into the BglII site of Bacillus licheniformis penicillinase (penP) gene of secretory vector pJP104. Expression and secretion of HBsAg protein was achieved by the E. coli CS412 carrying the plasmid pJPS2 in which the penP:HBsAg hybrid gene was under the control of two promoters, lipoprotein (lpp) and penP, spaced 450 bases apart. The secreted form of HBsAg encoded by the hybrid penP: HBsAg gene of plasmid pJPS2 was purified by immunoaffinity chromatography and found to be a 25 kilodalton protein.

Escherichia coli↗
PubMed1986

Characteristics of pre-S2 region of hepatitis B virus.

The nucleotide sequence of our cloned HBV DNA (subtype adw) has been determined. When the 165-nucleotide sequence of the pre-S2 region was compared with 7 other published sequences (subtypes adw, adr, ayw, and adyw), we found 38 nucleotide substitutions among different subtypes and 4 (adr) or 6 (adw and ayw) substitutions within the same subtype. Analysis of the predicted amino acid sequence from the known nucleotide sequences indicates that: there are 20 amino acid substitutions, the longest conserved amino acid sequence is located between amino acids 23 to 34, and the 54th amino acid is identical within the same subtype but varies in different subtypes.

Amino Acid Sequence↗
PubMed1985

Characterization of restriction endonuclease maps of hepatitis B viral DNAs.

The HBV DNA isolated from Dane particles of 9 patients' plasma was cloned into the EcoRI or BamHI site of the pUC8 plasmids. Two plasmids with full length HBV DNA and four plasmids containing the HBV surface antigen gene were obtained. Based on our cloned HBV DNA and a comparison with 7 complete sequences and 5 restriction endonuclease patterns of HBV DNA published by others, we can recognize common restriction sites shared by different subtypes (adw, adr, ayw, and adyw): (1) a HincII site in the S gene, (2) a BamHI site in the X region, and (3) two BglII sites in the C gene. In addition adw has specific sites for HincII, BamHI, and PstI in the pre-S region. A unique XhoI site is present in the pre-S region in all subtypes except for adw.

Bacterial Proteins↗
PubMed1985

Restriction endonuclease mapping of the hepatitis B viral genome isolated from Taiwan.

The Eco RI fragment of hepatitis B virus (HBV) DNA isolated from human blood plasma Dane particles were inserted into plasmid pUC8 Eco RI site and transformed into E. coli JM103 host. Two recombinants pTWL1 and pTWL2 were found to carry 3.2 kbp fragment and proved to have HBV genome by Southern hybridization method. The 1.4 kbp Bam HI fragment which carried the hepatitis B viral surface antigen (HBsAg) gene, obtained via Bam HI digestion of Dane particles DNA which was made fully double stranded by endogenous DNA polymerase reaction, was also inserted into plasmid pUC8 Bam HI site. Four recombinant clones, pTWS1, pTWS2, pTWS3, and pTWS4 were found. Only one of the clones pTWS1 carried the HBsAg gene in a correct orientation with respect to the lac promoter sequence. The physical mapping of HBV DNA was performed with several restriction endonucleases. Our results indicated that the HBV DNA insert contains unique XbaI and HpaI cleavage sites and lacks the cleavage sites for the HindIII, SmaI, KpnI, SalI, and SstI endonucleases. The locations of Bam HI, BglII, and HincII endonucleases cleavage sites within the cloned HBV DNA of the pTWL1 plasmid were similar to that HBV DNA of adw and adw2 subtypes.

Chromosome Mapping↗
PubMed1984

Biologic properties of three newly established human esophageal carcinoma cell lines.

Three epithelial cell lines, CE-48T/VGH, CE-69T/-VGH, and CE-81T/VGH, were established from human squamous cell carcinoma of the esophagus. The cells were polygonal with a high nucleus-to-cytoplasm ratio. Many cells were multinucleate. Electron microscopy revealed the presence of tonofilaments and desmosomes. Chromosome analysis showed that these 3 cell lines were heteroploids of human origin. When transplanted into BALB/c (nu/nu) mice, CE-69T/VGH and CE-81T/VGH produced tumors, the histology of which proved to be carcinomas. All 3 cell lines secreted carcinoembryonic antigen. However, the secretion patterns were different. These 3 cell lines may provide useful models for the study of human esophageal cancer.

Animals↗
PubMed1982

Isolation of xanthophores from the goldfish (Carassius auratus L.).

A method is described for the isolation of milligram quantities of viable, hormone-responsive xanthophores from goldfish scales. The preparations are typically 70 to 90% pure and are useful for biochemical analyses or for establishing primary cultures.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1980

Hormone-induced filopodium formation and movement of pigment, carotenoid droplets, into newly formed filopodia.

Treatment of cultured goldfish xanthophores by hormone (ACTH) or c-AMP induces not only pigment dispersion, but subsequent outgrowth of processes, and pigment translocation into these processes. These latter effects are shown to proceed as follows: First the edge of the cytoplasmic lamellae takes on a scalloped contour with numerous protrusions. These presumably serve as nucleation centers where short microfilament bundles are assembled. Later, the microfilament bundles elongate ("grow"), often resulting in an extension of the protrusions to become filopodia while the proximal end of the microfilaments penetrates into the thicker portion of the cellular process which now houses the pigment, i.e., the carotenoid droplets. Carotenoid droplets appear to migrate along the microfilament bundles, or cytoplasmic channels associated with them, into the filopodia. Finally, some of the filopodia become broader, thicker and laden with carotenoid droplets and are then recognized by light microscopy as pigmented cellular processes. The microfilaments have been shown to be actin filaments by their thickness, the size of their subunits, and decoration by heavy meromyosin. Evidence is presented which suggests that the growth of these actin filaments may come about by recruitment from short F-actin strands found in random orientation in adjacent areas.

Adrenocorticotropic Hormone↗
PubMed1978

Ultrastructural demonstration of hormone-induced movement of carotenoid droplets and endoplasmic reticulum in xanthophores of the goldfish, Carassius auratus L.

The hormone-induced pigment dispersion in primary cultures of xanthophores of goldfish (Carassius auratus L.) has been shown to involve the dispersion of not only carotenoid droplets but also of smooth endoplasmic reticulum. The dispersion of these organelles is inhibited by cytochalasin B and is accompanied by thinning of the cell body, thickening of the processes, and also overall changes in cellular morphology (process extension) under certain conditions. Electron microscopic examination of heavy meromyosin treated glycerinated xanthophores in scales revealed the presence of actin filaments in these cells.

Adrenocorticotropic Hormone↗
PubMedDate not supplied

Receptor-mediated endocytosis as a selection force to enrich bacteria expressing rhodostomin on their surface.

Previously, we developed a TraT display system to express snake venom rhodostomin (RHO), a disintegrin, on the external surface of Escherichia coli [J Biomed Sci 6:64-70;1999]. To show a new potential use of the TraT display system, we employed a biotin labeling technique coupled with SDS-PAGE and flow cytometry analyses to further demonstrate and confirm the expression of TraT-RHO on the E. coli surface. We also showed that the expression of TraT-RHO on the cell surface not only facilitated the bacteria adhesion to BHK-21 cells but also induced bacterial internalization into BHK-21 cells. This feature allowed us to enrich the TraT-RHO expression bacteria about 10,000-fold starting with a mixture of TraT-RHO bacteria with beta-galactosidase-positive bacteria in a ratio of 10(2):10(7) through four cycles of BHK-21 cell endocytosis and replating of engulfed bacteria on agar plates. We therefore suggest that the TraT display system can be applied to select out bacteria expressing a specific peptide sequence from a large population of display library through the process of receptor-mediated endocytosis and reamplification cycles.

Animals↗
PubMedDate not supplied

Hepatitis B viral polymerase fusion proteins are biologically active and can interact with the hepatitis C virus core protein in vivo.

Hepadnaviruses and retroviruses are evolutionarily related families because they both require a process of reverse transcription for genome replication. However, hepadnaviruses produce polymerase (pol) and core proteins separately, while retroviruses synthesize a gag-pol fusion protein that is subsequently cleaved by a virally encoded protease to release a functional polymerase. To test whether an additional sequence at the N-terminus of pol in hepatitis B virus (HBV) interferes with its function, we created two plasmids expressing core-pol fusion proteins, core144-pol and core31-pol. Secreted particles obtained from HuH-7 cells, which were cotransfected with a core-pol fusion protein-expressing plasmid and a core-expressing plasmid, showed a positive signal of HBV DNA by the endogenous polymerase assay, indicating that the core-pol fusion proteins retain DNA priming, polymerization and RNase H activities. The fusion protein was detected in the cytoplasm of transfected cells and in secreted virions by immunoprecipitation. Furthermore, we found by immunofluorescence staining that the HBV core-pol fusion protein colocalized with the hepatitis C virus (HCV) core protein in cytoplasm and in lipid droplets. Immunoprecipitation studies showed that the anti-HCV core complex contained the HBV core-pol fusion protein while the anti-HBV pol complex contained the HCV core protein, which supports the hypothesis that the HCV core protein can form a complex with the HBV core-pol fusion protein.

Cytoplasm↗