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Biomedical subjects

S J Lo

Publications and source records attributed to S J Lo.

At least 37 records · Page 2Linked to original sources
PubMed1995

Deletion or alteration of hydrophobic amino acids at the first and the third transmembrane domains of hepatitis B surface antigen enhances its production in Escherichia coli.

To investigate the failure of high-level production of hepatitis B viral (HBV) surface antigen (HBsAg), including three authentic forms, large (L), middle (M) and major/small (S) HBsAg, in Escherichia coli, we employed the high-expression vector pGEX containing the glutathione S-transferase-encoding gene (GST) to study HBsAg production. Different fragments of HBV DNA containing the entire pre-S1/pre-S2/S region (for L protein), or partial pre-S1, pre-S2, pre-S1/pre-S2 and pre-S2/S region (for M protein), were fused downstream from the GST gene, in order to obtain five plasmids which encode GST-HBsAg fusion proteins. SDS-PAGE analyses revealed that cells containing plasmids with a full-length S region (pGLS and pGMS) produced undetectable GST-HBsAg fusion proteins, in contrast to those cells harboring plasmids without the S region (pGS1, pGS2 and pGS1S2), which synthesized fusion proteins in 3-10% of the total cellular protein. Using an immunoblot method to screen HBsAg production in cells which harbored plasmids derived from exonuclease BAL 31-digested pGLS, we obtained eight positive clones. Nucleotide sequence analyses of plasmids from the positive clones revealed that termination, deletion or frameshift occurred at the regions encoding either the first or the third transmembrane domain of the major HBsAg. Correlation between the production level of GST-HBsAg fusion proteins and their constituent and arrangement of amino acids (aa) at the last 20 aa among 15 clones suggested that the fusion protein ended with a longer stretch of or a higher ratio of hydrophobic aa had a lower production in E. coli.

Amino Acid Sequence↗
PubMed1995

Demonstration of the presence of protease-cutting site in the spacer of hepatitis B viral Pol protein.

Molecular genetic studies have revealed that the human hepatitis B viral (HBV) Pol protein, a polypeptide of about 94 kDa, contains four domains. These are the 5'-terminal protein, spacer, RNA reverse transcriptase/DNA polymerase, and RNase H, respectively, from the amino (N) to carboxy (C) terminus. No evidence indicates as yet the involvement of a specific protease in cleaving the Pol protein or the presence of protease-cutting sites in the Pol protein. An in vitro-translated Pol protein was shown to be cleaved by purified thrombin but not in the presence of its inhibitor, hirudin. Two thrombin-cutting sites, spanning 194 amino acids, were then deduced by thrombin digestion of Pol protein with various lengths of C-terminal deletion. These two putative cutting sites, one located in the spacer region and the other in the beginning of the polymerase region, were found to be conserved at similar positions in the Pol protein of all hepadnaviruses. By using a novel method called the LacZ localization assay (LLA), it was demonstrated that a tripartite fusion protein containing the nucleus localization sequence (NLS) of SV40 large T Ag, the putative thrombin cutting sequence (Ile-Arg-Ile-Pro-Arg320-Thr) of HBV Pol protein and the full length beta-galactosidase of E. coli, exhibited a lower percentage (approximately 53%) of targeting into the nucleus of transfected hepatoma cells when compared with a similar tripartite protein containing a single mutation (Arg320 residue into Trp320) of HBV Pol protein (approximately 78%) or with a bipartite protein of SV40 NLS-beta-galactosidase (approximately 90%). These results indicate that the putative thrombin-cutting site in the spacer region of HBV Pol protein could be cleaved by a cellular protease resulting in the separation of NLS sequence from the beta-galactosidase and rendering a lower frequency of X-gal staining in the nucleus.

Amino Acid Sequence↗
PubMed1995

Biocorrosion study of titanium-cobalt alloys.

The present work provides experimental results of corrosion behaviour in Hank's physiological solution and some other properties of in-house fabricated titanium-cobalt alloys with cobalt ranging from 25-30% in weight. X-ray diffraction (XRD) shows that, in water-quenched (WQ) alloys, beta-titanium is largely retained, whereas in furnace-cooled (FC) alloys, little beta-titanium is found. Hardness of the alloys increases with increasing cobalt content, ranging from 455 VHN for WQ Ti-25 wt% Co to 525 VHN for WQ Ti-30 wt% Co. Differential thermal analysis (DTA) indicates that melting temperatures of the alloys are lower than that of pure titanium by about 600 degrees C. Potentiodynamic polarization results show that all measured break-down potentials in Hank's solution at 37 degrees C are higher than 800 mV. The breakdown potential for the FC Ti-25 Wt% Co alloy is even as high as nearly 1200 mV.

Biocompatible Materials↗
PubMed1994

Biogenesis of the hepatitis B viral middle (M) surface protein in a human hepatoma cell line: demonstration of an alternative secretion pathway.

In the serum of hepatitis B virus (HBV)-infected patients, two different types of particles, a 42 nm virion and a 22 nm subviral particle, were identified. The envelope of both particles is composed of three proteins, the large (L), middle (M), and major/small (S) surface proteins but the ratio between these components varies in each. The M protein appears in a lesser amount than the S protein in both virion and subviral particles, although it is translated from the same subgenomic RNA, and this is due to its poor initiation context of translation. In addition, only the glycosylated form of M protein is secreted in contrast to both glycosylated and unglycosylated forms of L and S proteins that are secreted. To investigate the biogenesis of M protein, human hepatoma cells transfected with plasmids containing a mutated HBV DNA were used to produce a high amount of M protein. Electron microscopic observation revealed that despite a higher proportion of the M protein being found in the transfected cells, the secreted surface antigen particles possess similar size and density to 22 nm subviral particles. Detailed biochemical analyses showed the following. (1) The unglycosylated M protein was predominantly present in the microsomal fraction but not present in any other subcellular fractions. (2) The M protein formed 22-nm-like particles in the endoplasmic reticulum (ER) and was retained in the post-ER or pre-Golgi regions. (3) In addition to the complex glycosylated form of M protein, a high-mannose form of M protein could be secreted. (4) Normally, no unglycosylated M protein was secreted. However, glycosylation was not essential for M protein secretion since M protein deprived of glycosylation by tunicamycin treatment was detected in the medium. These findings suggest that (i) the M protein was probably translated and co-translocated into the ER and at least one site was glycosylated before leaving the ER resulting in no secretion of unglycosylated M protein, and (ii) the M protein had two secretion pathways, one through the conventional pathway and the other probably directly through the ER.

Autoradiography↗
PubMed1993

Rhodostomin, an RGD-containing peptide expressed from a synthetic gene in Escherichia coli, facilitates the attachment of human hepatoma cells.

Rhodostomin (Rho) from snake venom, a potent inhibitor of platelet aggregation, contains 68 amino acids having an RGD sequence and 12 cysteine residues. A chemically synthesized Rho gene was cloned and expressed in Escherichia coli. The expression of Rho gene fused with the glutathione S-transferase (GST) gene was about 10-30% of total cell proteins. The Rho-fusion protein could be recognized by antibodies raised against either a native Rho peptide or a synthetic peptide. The purified GST-Rho coated on culture plates facilitated the attachment of human hepatoma cells, which was inhibitable by co-incubation with a synthetic hexapeptide GRGDSP but not with a related peptide of GRGESP, suggesting that the E. coli-expressed Rho-fusion protein was properly folded and biologically functional.

Amino Acid Sequence↗
PubMed1993

Induction of cell expansion of goldfish melanocytoma cells (GMM-1) by epinephrine and dexamethasone requires external calcium.

Treatment of GMM-1 (a goldfish melanocytoma cell line) cells with epinephrine induced a rapid cell expansion (flattening of cells, extension and broadening of cellular processes) similar to the effect of dexamethasone reported previously (Shih et al., 1990). Studies on the possible involvement of secondary messengers in cell expansion indicated that (i) both 8-bromo-CAMP and forskolin caused cell shrinking (the opposite of cell expansion); (ii) TPA also caused cell shrinking; (iii) phospholipid derivatives, such as 1,2-dioctanoyl-sn-glycerol, lysophosphatidic acid, and arachidonic acid caused cell expansion; and (iv) EGTA (calcium chelator) and nifedipine (calcium channel blocker) inhibited the effect of epinephrine. Together with the previous findings, these observations indicate that epinephrine and dexamethasone may share a common pathway in triggering an external calcium influx to cause cell expansion. The results of the effects of epinephrine agonists and antagonists, together with those of other workers, also show that there are multiple isoforms of adrenoceptor in the goldfish.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HuH-7 cells.

Hepatitis B and C viruses (HBV and HCV, respectively) are associated with acute and chronic liver diseases and hepatocellular carcinoma. To elucidate the molecular status of superinfection with these two hepatitis viruses, we cotransfected the full-length or truncated version of HCV structural genes (core and envelope 1) together with the cloned HBV DNA into a human hepatoma cell line (HuH-7). Expression of HBV-specific major transcripts (3.5 and 2.1 kb), as well as HBV antigens (hepatitis B surface antigen and hepatitis B e and core antigens), was reduced about two- to fourfold by the presence of the HCV structural genes. In addition, the secretion of HBV viral particles, including the viral nucleocapsid and mature virion, was drastically suppressed about 20-fold. Analysis of the intracellular HBV core protein-associated nucleic acid indicated that the encapsidated HBV pregenomic RNA was similarly reduced about 14-fold. Deletion analysis of the HCV structural genes demonstrated that the core gene alone or the fragment containing the core protein's N-terminal 122 amino acid residues conferred the same level of suppressive activity as the full-length structural genes. By indirect immunofluorescence, we found that the core protein of HCV was located in the cytoplasm of transfected HuH-7 cells at day 3 posttransfection and was targeted to the nucleus at day 6. Thus, the kinetics of the suppressive effect exerted by HCV constructs matched the timing of core protein entrance into the nucleus. Our results substantiate the clinical finding that HBV markers are suppressed by superinfection with HCV and further imply that this inhibitory effect may occur in the processes of transcription and encapsidation of HBV pregenomic RNA and may be mediated by the core protein of HCV. The deduced amino acid sequence of the HCV core protein has revealed that it is a basic protein which contains a putative DNA-binding motif (SPRG), as well as triplicate nuclear localization signals and several putative protein kinase A and C recognition sites. These characteristics imply that the HCV core protein can also function as a gene-regulatory protein.

Amino Acid Sequence↗
PubMed1992

Evidence for involvement of a ribosomal leaky scanning mechanism in the translation of the hepatitis B virus pol gene from the viral pregenome RNA.

In retroviruses, the pol gene is expressed in the form of a gag-pol fusion protein by the mechanism of ribosomal frameshifting. In studies of the possible mechanism of hepadnaviral pol protein synthesis, recent results have ruled out core-pol fusion protein synthesis by ribosomal frameshifting. In this study, an in vitro transcription and translation coupling system was used to demonstrate that the HBV core and pol proteins could be synthesized independently using the pregenome RNA template. The result has led us to design experiments to distinguish between the involvement of a termination-reinitiation, internal initiation, or leaky scanning mechanism in the pol protein synthesis. In vitro experiments were then carried out to measure the amount of pol proteins being synthesized from (i) the preC mRNA, which contained an extra AUG and seven more nucleotides at the 5'-end in comparison with the pregenome RNA; (ii) the pregenome RNA in the presence of various amounts of antisense RNA annealing to the 5'-end of the pregenome RNA; and (iii) the pregenome RNA with an additional hairpin structure located upstream of the C gene. Results indicated that the synthesis of both core and pol proteins was concomitantly reduced in these three conditions, which suggested that leaky scanning is the most probable mechanism for pol protein synthesis in vitro. To further verify the mechanism in vivo, experiments were performed to assay the activity of DNA polymerase in virions, which were obtained from hepatoma cells transfected by plasmids containing either a wild-type sequence (5'-GGCATGG-3') or an optimal initiation context (5'-ACCATGG-3') of the C gene. Transfection results showed that the plasmid-containing mutations of the C gene significantly decreased the DNA polymerase activity in virions. This observation supports our hypothesis that the leaky scanning model is involved in the synthesis of pol protein.

Base Sequence↗
PubMed1992

Preferential ribosomal scanning is involved in the differential synthesis of the hepatitis B viral surface antigens from subgenomic transcripts.

The envelope of the hepatitis B virus (HBV) is composed of three species of proteins, the large (L), middle (M), and major (S) surface proteins (HBsAgs), each of different molecular weights but sharing a common C-terminus. These three HBsAgs, encoded by two species of viral subgenomic transcripts (2.1 and 2.4 kb), which have a heterogenous 5'-terminus, appear in different amounts in both the 42-nm virions and the 22-nm subviral particles. To investigate the involvement of translational control in the differential expression of the L, M, and S proteins, we tested the translational capability of 2.1- and 2.4-kb transcripts in in vitro translation and of 2.1-kb transcripts in in vivo transfection experiments. Results of in vitro translation indicated that a large amount of the L protein and a very small amount of the M and S proteins were synthesized from the 2.4-kb mRNA. Translation of the 2.1-kb mRNA resulted in a 4:1 ratio of the S protein to the M protein. In contrast, translation of a similar 2.1-kb mRNA containing an optimal initiation context (5'-ACCATGG-3') of the pre-S2 region resulted in a reversed ratio, four times as much M protein as S protein. This result was also obtained by transfection of hepatoma cells with plasmid DNAs containing the mutated sequence (5'-ACCATGG-3') of the pre-S2 region. In considering these results, the production of a large amount of the L protein from the 2.4-kb mRNA and the determination of the level of the M protein by the context of translational initiation, we suggest that a preferential translational initiation is involved in the expression of differential amounts of the L, M, and S proteins.

Base Sequence↗
PubMed1990

Tropic effect of dexamethasone on goldfish melanocytoma cells: induction of calcium-dependent but protein synthesis-independent morphological changes.

Treatment of melanocytoma cells of goldfish origin with dexamethasone leads to rapid morphological changes, flattening of cell body and extension of dendrites. This effect is independent of protein synthesis but requires the presence of extracellular calcium, indicating that it is a "tropic effect" distinct from the typical "trophic effects" of steroid hormones that involve de novo protein synthesis.

Animals↗
PubMed1990

The hepatitis B virus X-C fusion protein is unlikely to be produced by the mechanism of ribosomal frameshifting.

A novel hepatitis B viral (HBV) protein of 35-40 kDa, characterized by antibodies and proposed as an X-C fusion protein, was previously described in core particles isolated from HBV-, WHV-, and GSHV-infected livers. The X and C genes are two adjacent genes in all mammalian hepadnaviruses but are not contiguous in WHV and GSHV. After examination of the X and preC/C junction sequences of 10 HBV, 4 WHV, and 1 GSHV, we found that the ORF of preC can be extended 7 more sense codons upstream so that X overlaps with the preC/C gene in all sequences. The number of overlapping base pairs (bp) is varied: 46 bp in HBV, 19 bp in WHV, and 10 bp in GSHV. In this region a conserved A-track was found to be followed by a pair of inverted repeats, suggesting that a ribosomal frameshift may occur for X-C fusion protein production. To assess this possibility, we have used an in vitro transcription and translation coupling system to identify X-C protein production. Two recombinant SP6 plasmids were used. One contained a full length of the X and preC/C gene of wild-type HBV-DNA and the other fused the X-preC/C gene by inserting a 10-bp HindIII linker at the junction of the X-preC/C region. No X-C fusion protein was detected from the wild-type plasmid. In contrast a large amount of X-C fusion protein was produced from the linker-inserted clone. It appears, therefore, that the X-C fusion protein is unlikely to be produced via the mechanism of ribosomal frameshifting.

Amino Acid Sequence↗
PubMed1990

Characterization of monoclonal antibody binding site to the polymorphic length of hepatitis B surface antigen synthesized in a cell-free system.

A combination of two modern biological techniques, hybridoma and recombinant DNA, was described to screen and characterize a monoclonal antibody binding site. Three monoclonal antibodies, M27, M9 and M01 recognizing to a specific domain of the hepatitis B virus surface antigen (HBsAg), were determined by their ability to immunoprecipitate the in vitro synthesized HBsAg in a polymorphic length. Results indicated that the M9 and M01 both recognize the S region of surface antigen while the M27 binds to the pre-S2 region of surface antigen.

Animals↗
PubMed1989

Analysis of the promoter region of the melanin locus from Streptomyces antibioticus.

Several approaches were used to study the transcriptional control region of the melanin-production locus (melC) of Streptomyces antibioticus. Filter-binding in combination with exonuclease III protection localized the 3' boundary of a Streptomyces RNA polymerase-binding site predominantly about 39 nucleotides (nt) upstream from the start codon of melC1, the first open reading frame in the melC locus. Deletion of nt 112-197 upstream from the melC1 start codon reduced melC expression to less than 10%, and deletion of nt 28-107 or 28-120 upstream from melC1 totally inactivated melC. High-resolution nuclease S1 mapping identified the in vitro transcriptional start point (tsp) at 33-34 nt upstream from the start codon of melC1. No sequence resembling the E. coli consensus promoter sequence was found in this region, and site-directed mutagenesis of such a sequence located 101-132 nt upstream from melC1 did not influence melC expression. These studies suggest that transcription of melC is principally from a single tsp and is positively regulated by a mechanism that involves sequences 87-163 nt upstream from the tsp.

Base Sequence↗
PubMed1989

Rearrangements of pterinosomes and cytoskeleton accompanying pigment dispersion in goldfish xanthophores.

The cytoskeleton of goldfish xanthophores contains an abundance of unique dense structures (400 nm in diameter) that are absent in goldfish nonpigment cells and are probably remnants of pterinosomes. No major difference in protein composition between xanthophores and nonpigment cells (without these structures) was found that could account for these structures. In xanthophores, these structures are foci of radiating filaments. The addition or withdrawal of ACTH causes a radical rearrangement of the xanthophore cytoskeleton accompanying redistribution of carotenoid droplets, namely, the virtual exclusion of these dense bodies with associated filaments from the space occupied by the carotenoid droplet aggregate vs. a relatively even cytoplasmic distribution of these structures when the carotenoid droplets are dispersed. These changes in cytoskeletal morphology are not accompanied by any major changes in the protein or phosphoprotein composition of the cytoskeleton.

Adrenocorticotropic Hormone↗
PubMed1989

Hepatitis B virus transcript produced by RNA splicing.

A new hepatitis B virus (HBV) transcript of about 2.2 kilobases was identified in HBV DNA-transfected human hepatoma cells. The 5' terminus of this viral RNA appears to map at one or more of the precore initiation sites, contains a deletion of 1,223 bases corresponding to the last codon of the core gene to the middle of the surface antigen gene, and terminates at the 3' polyadenylation site used by the other known HBV RNAs. The junction region of the deleted sequences showed the conserved splice donor and acceptor GT-AG sequences. Moreover, when a mutant HBV DNA in which the splice acceptor site was changed from AG to CG was transfected into human hepatoma cells, no 2.2-kilobase RNA was detected, further suggesting that this RNA represents a spliced transcript. The core gene, although an amino acid shorter, still encoded a functional viral core protein in complementation experiments. Sequence analysis of the cDNA of the 2.2-kilobase RNA suggests that this transcript can potentially encode a new protein that comprises the reverse transcriptase domain of HBV. However, genetic analysis using a transient DNA transfection system suggests that the gene product(s) of this transcript is not essential for viral replication. The function of this transcript remains to be studied.

Base Sequence↗
PubMed1989

Chicken mitochondrial cytochrome C oxidase subunit II: comparative analysis among the vertebrates.

The chicken cytochrome c oxidase subunit II (COII) was cloned and sequenced. A comparison of the deduced chicken COII sequence with 4 other vertebrate counterparts revealed 64-66% amino acid sequence homology and 68-70% nucleotide sequence homology. Four peptide segments each of nine amino acids long are highly conserved across the 5 species. A redox-center was formed by three of these highly conserved domains, which include two invariant Cys and two invariant His residues for copper ion coordination, three strictly conserved Glu or Asp residues for cytochrome c binding, and highly conserved aromatic acid residues for electron transfer.

Amino Acid Sequence↗
PubMed1988

Regulation of the distribution of carotenoid droplets in goldfish xanthophores and possible implication to secretory processes.

In goldfish xanthophores, the formation of pigment aggregate requires: 1) that a pigment organelle (carotenoid droplet) protein p57 be in the unphosphorylated state; 2) that self-association of pigment organelles occur in a microtubule-independent manner; and 3) that pigment organelles via p57 associate with microtubules. In the fully aggregated state, the pigment organelles are completely stationary. Pigment dispersion is initiated by activation of a cAMP-dependent protein kinase, which phosphorylates p57 and allows pigment dispersion via an active process dependent on F-actin and a cytosolic factor. This factor is not an ATPase, and its function is unknown. However, its abundance in different tissues parallels secretory activity of the tissues, suggesting a similarity between secretion and pigment dispersion in xanthophores. The identity of the motor for pigment dispersion is unclear. Experimental results show that pigment organelles isolated from cells with dispersed pigment have associated actin and ATPase activity comparable to myosin ATPase. This ATPase is probably an organelle protein of relative molecular mass approximately 72,000, and unlikely to be an ion pump. Isolated pigment organelles without associated actin have 5x lower ATPase activity. Whether this organelle ATPase is the motor for pigment dispersion is under investigation. The process of pigment aggregation is poorly understood, with conflicting results for and against the involvement of intermediate filaments.

Actin Cytoskeleton↗