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G Acs

Publications and source records attributed to G Acs.

At least 73 records · Page 4Linked to original sources
PubMed1988

Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions.

Clonal cells derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete hepatitis B surface antigen particles, nucleocapsids, and virions (M. A. Sells, M.-L. Chen, and G. Acs, Proc. Natl. Acad. Sci. USA 84:1005-1009, 1987) which elicit acute hepatitis in chimpanzees (G. Acs, M. A. Sells, R. H. Purcell, P. Price, R. Engle, M. Shapiro, and H. Popper, Proc. Natl. Acad. Sci. USA 84:4641-4644, 1987). We report here the initial characterization of the viral nucleic acids produced in this culture system. Kinetic analyses of nuclear, cytoplasmic, and extracellular HBV DNAs were performed by Southern blotting with radiolabeled HBV strand-specific probes. The results from these analyses indicate that at the stationary cellular growth phase, there is a dramatic increase in the rate at which HBV DNA accumulates. Incomplete double- and single-stranded forms of the HBV genome were detected in the nuclear and cytoplasmic fractions as well as in the extracellular medium. In addition, the nuclear DNA apparently includes multiple complete copies of the HBV genome chromosomally integrated and full-length covalently closed circular HBV DNA. Multiple HBV-specific polyadenylated RNAs with lengths of 3.5, 2.5, and 2.1 kilobases were identified by Northern (RNA) blot analysis. S1 nuclease mapping and primer extension identified a single 3' end and multiple unique initiation sites corresponding to nucleotides just 5' to the pre-S1 region, as well as upstream and within the pre-S2 and precore regions. The nucleic acid profile obtained from these analyses is essentially a facsimile of that obtained by studying liver tissue from HBV-infected individuals.

Cell Transformation, Viral↗
PubMed1988

Identification of protein-binding sites in the hepatitis B virus enhancer and core promoter domains.

We have investigated the role of liver-specific trans-acting factor(s) in the regulation of hepatitis B virus (HBV) gene expression. A recorder plasmid (pEcoAluCAT; HBV nucleotides 1 through 1878) was constructed containing the HBV enhancer and the promoter region of the pregenomic RNA, which was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene. Upon transfecting this plasmid into various cell lines, the CAT gene was expressed only in cells of liver origin. Moreover, competition cotransfections with pEcoAluCAT and plasmids containing HBV enhancer sequences in human hepatoblastoma-derived HepG2 cells indicated the presence of titratable trans-acting factor(s) in these cells. Gel mobility shift assays using HBV enhancer and core promoter domains confirmed the existence of sequence-specific DNA-binding proteins in liver cell nuclear extract which bound to these regions. These binding sites encompass 17- and 12-nucleotide palindromes in the HBV enhancer and core promoter domains, respectively, when mapped by the methylation interference assay.

Base Sequence↗
PubMed1988

Morphologic, immunohistochemical, and ultrastructural studies of the production of hepatitis B virus in vitro.

Well differentiated human hepatoblastoma Hep G2 cells after transfection with cloned hepatitis B virus (HBV) genomes produce replicative HBV DNA intermediates, high levels of HBsAg, HBeAg and HBcAg as well as mature Dane particles. To analyze the replication cycle of HBV, we studied the expression of HBV antigens with monoclonal antibodies by immunomorphologic methods in the transfected cells at various time intervals after plating. HBcAg and HBeAg were detected in the cytoplasm and less frequently in the nuclei of transfected cells. The percentage of positive cells increased with time after plating and reached a plateau of about 50% positive cells at 10 days. HBsAg and the large and middle HBsAg polypeptides were observed in the cytoplasm of transfected cells and a maximum of 20 to 30% positive cells was reached during the 3rd week after plating. Examination of viable cells in suspension revealed HBcAg/HBeAg and HBsAg expression on the cell surface. Electron microscopy demonstrated characteristic core particles in the nuclei and cytoplasm and Dane particles in cytoplasmic vesicles and culture media of transfected cells. The HBV producing cells did not show any evidence of a cytopathic effect. These observations demonstrate significant similarities between the HBV DNA transfected cells and infected human hepatocytes which support active HBV replication in vivo. Taken together, the results suggest that the cultured cells may serve as a model to elucidate a number of unsolved problems of the molecular and cellular pathobiology of hepatitis B.

Carcinoma, Hepatocellular↗
PubMed1988

Regulation of interleukin-6 expression in cultured human blood monocytes and monocyte-derived macrophages.

A culture system that allows human blood monocytes to differentiate into macrophages in vitro was used to study B-cell stimulatory factor-2/interleukin-6 (interferon-beta 2/26 kd protein) expression in mononuclear phagocytes. Using B-cell stimulatory factor-2 (BSF-2) cDNA and a polyclonal, monospecific antibody directed against human BSF-2, we find that strong interleukin-6 (IL-6) expression is initiated in cultured monocytes on stimulation with endotoxin. Maximally induced monocytic BSF-2/IL-6 synthesis (1% to 2% of total proteins secreted by monocytes) is more than ten times stronger than in terminally differentiated macrophages (approximately 0.1% of total secretory proteins). BSF-2/IL-6 mRNA was detectable as early as one hour after stimulation with endotoxin, reaching maximum levels three hours after stimulus. Interleukin-1 (IL-1) was able to stimulate IL-6 synthesis in monocytes, but not in macrophages. Tumor necrosis factor, interferon-gamma and interleukin-2 (IL-2) had no effect on IL-6 synthesis in monocytes or macrophages. We found five molecular weight forms of BSF-2/IL-6 to be secreted by monocytes of 21.5 kd, 23.5 kd, 24 kd, 26 kd, and 28 kd apparent molecular weight. The 26 kd and 28 kd forms were found to represent N-glycosylated molecules, which were not detectable on treatment of the cells with the N-glycosylation inhibitor tunicamycin. The 21.5 kd, 23.5 kd, and 24 kd BSF-2/IL-6 forms were unaffected by tunicamycin treatment. We conclude from our data that cells of the mononuclear phagocyte lineage are one of the main sites of BSF-2/IL-6 (interferon-beta 2/26 kd protein/HSF) synthesis.

Cell Differentiation↗
PubMed1987

Hepatitis B virus produced by transfected Hep G2 cells causes hepatitis in chimpanzees.

We have reported that clonal cells derived from Hep G2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete spherical and filamentous forms of hepatitis B surface antigen (HBsAg), core particles, and virions into the culture medium. Here we describe the development of typical hepatitis in two chimpanzees following intravenous inoculation with the medium in which the transfected cells had grown. The liver biopsies from these animals showed characteristic lesions in parenchyma and portal tracts, more conspicuous at an earlier time in the chimpanzee that had received a greater number of virions. The amount of HBsAg in the serum of one infected chimpanzee increased with time after the initial inoculation and then decreased concomitantly with the appearance of antibodies against HBsAg and core antigens. HBsAg remained detectable in the other animal throughout the course of the experiment. The levels of hepatitis B "e" antigen in both animals peaked at week 5, signifying the acute phase of the infection. The activities of serum enzymes that are markers for necroinflammation also increased. The hepatitis HBsAg subtype of the virions isolated from the patient whose DNA was cloned and then used for transfection of the Hep G2 cells was the same as that found in the chimpanzees. Furthermore, the restriction enzyme analysis of the viral DNA isolated from the chimpanzees was identical to the cloned DNA. Thus, HBV DNA-transfected Hep G2 cells can support the replication of virions that, in turn, produce hepatitis in chimpanzees.

Animals↗
PubMed1987

Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.

The hepatoblastoma cell line Hep G2 was transfected with a plasmid carrying the gene that confers resistance to G418 and four 5'-3' tandem copies of the hepatitis B virus (HBV) genome positioned such that two dimers of the genomic DNA are 3'-3' with respect to one another. Cells of one clone that grew in the presence of G418 produce high levels of hepatitis B e antigen and of hepatitis B surface antigen. HBV DNA is carried by these cells as chromosomally integrated sequences and episomally as relaxed circular, covalently closed, and incomplete copies of the HBV genome. Viral DNA was detected also in conditioned growth medium at the buoyant densities characteristic for infectious Dane and immature core particles. Finally, HBV-specific components morphologically identical to the 22-nm spherical and filamentous hepatitis B surface antigen particles as well as 42-nm Dane particles were visualized by immunoelectron microscopic analysis. Therefore, we have demonstrated that the Hep G2 cell line can support the assembly and secretion not only of several of the replicative intermediates of HBV DNA but also of Dane-like particles. This in vitro system can now be used to study the life cycle of HBV and the reaction of immunocompetent cells with cells carrying HBV.

Carcinoma, Hepatocellular↗
PubMed1987

Murine cells carrying integrated tandem genomes of hepatitis B virus DNA transcribe RNAs from endogenous promoters on both viral strands and express middle and major viral envelope proteins.

Clone 4.10 cells were isolated as a methotrexate-resistant clone arising after cotransfection of mouse 3T3 cells with plasmid DNA containing a head-to-tail dimer of the hepatitis B virus (HBV) genome and DNA coding for methotrexate-resistant dihydrofolate reductase. The majority of methotrexate-resistant clones derived by this procedure have been found to contain multiple copies of the HBV genome, but the intact HBV dimer was rarely preserved. In contrast, 4.10 cells contained at least 40 copies of intact HBV dimer per cell. These cells produced large amounts of 22-nm hepatitis B surface antigen particles that included viral envelope proteins reactive with the pre-S2 region-specific antibody, indicating transcription and translation of the pre-S2 and S regions of the integrated viral genomes. The cells also synthesized viral e antigen, which was released into the culture medium. Characterization of polyadenylated viral RNAs transcribed from the long (minus) strand of the integrated HBV DNA demonstrated the presence of shorter-than-genome-length RNAs containing only X region sequences, shorter-than-genome-length RNAs containing both X and S region sequences, and longer-than-genome-length RNAs containing core, X, and S region sequences. Start sites for transcripts were mapped 5' to and within the pre-S region and 5' to and within the precore region at approximately the same sites as those utilized for HBV transcription during viral replication in infected livers. Polyadenylated RNA transcripts complementary to the short (plus) strand of HBV that initiated and terminated within the intact and integrated head-to-tail tandem viral genomes were also detected.

Animals↗
PubMed1987

Replicative intermediate of hepatitis B virus in transfected murine fibroblasts.

The NIH 3T3-derived cell line psi AM22b, which carries a defective Moloney murine leukemia virus, was transfected with a plasmid carrying the neo gene and two head-to-tail copies of the hepatitis B virus (HBV) genome positioned with opposing polarities. Both the two HBV dimers and the neo gene were located between two Moloney murine leukemia virus long terminal repeats. Poly(A)+ RNAs isolated from one clone that grew in the presence of G418 contained the two major classes of HBV-specific transcripts (3.5-kilobase pregenome and 2.1-kilobase mRNAs) in approximately equivalent amounts, which was reminiscent of the profiles of viral mRNAs from the livers of infected humans and chimpanzees.

Animals↗
PubMed1985

Formation of highly stable complexes between 5-azacytosine-substituted DNA and specific non-histone nuclear proteins. Implications for 5-azacytidine-mediated effects on DNA methylation and gene expression.

Incubation of 5-azacytosine-substituted DNA ([5-aza-C]DNA) with nuclear proteins leads to the formation of highly stable DNA . protein complexes which remain intact in the presence of 1 M NaCl and/or 0.6% Sarkosyl. The proteins involved in binding double-stranded [5-aza-C]DNA in these stable complexes comprise a specific subset of non-histone nuclear proteins that includes DNA methyltransferase. Complex formation does not require S-adenosylmethionine and does not involve covalent linkage of protein to DNA or modification of 5-azacytosine residues. Non-histone nuclear proteins do not form complexes with double-stranded unsubstituted DNA that are resistant to dissociation with NaCl and Sarkosyl but are capable of forming such complexes with single-stranded DNA regardless of whether it contains 5-azacytosine residues or not. However, it can be demonstrated 1) that single-stranded regions do not account for stable binding of proteins to native [5-aza-C]DNA and 2) that many nuclear proteins which form stable complexes with single-stranded DNA are incapable of forming such complexes with double-stranded [5-aza-C]DNA. Synthesis of [5-aza-C]DNA by cells growing in the presence of either 5-azacytidine or 5-aza-2'-deoxycytidine leads to rapid loss of extractable DNA methyltransferase (Creusot, F., Acs, G., and Christman, J.K. (1982) J. Biol. Chem. 257, 2041-2048). Analogous depletion of non-histone nuclear proteins capable of forming stable complexes with [5-aza-C]DNA in vitro is observed, suggesting that the same proteins can form highly stable complexes with [5-aza-C]DNA in vitro and in vivo. Formation of stable complexes between non-histone nuclear proteins and [5-aza-C]DNA could potentially affect not only the activity of DNA methyltransferase but the action of other regulatory proteins or enzymes that interact with DNA. Such interactions could explain effects of 5-azacytidine on gene expression that cannot be directly linked to loss of methyl groups from DNA.

Animals↗
PubMed1985

Long-term culture and passage of human fetal liver cells that synthesize albumin.

Long-term cultures of hepatocytes were established from livers of human fetuses obtained by abortion at 18 to 23 wk of gestation. Cells obtained by collagenase dissociation of liver were maintained in defined serum-free medium on a substratum of positively charged plastic. Under these conditions, the cells divide and form a confluent monolayer. After multiple passages over a period of 3 mo., the cells retained an epithelioid morphology and continued to synthesize and secrete albumin.

Albumins↗
PubMed1985

Enzyme-linked immunoassay of pre-S gene-coded sequences in hepatitis B vaccines.

Pre-S gene coded domains of the hepatitis B virus (HBV) envelope protein are highly immunogenic in experimental animals and humans. Their presence in HBV and hepatitis B surface antigen (HBsAg) particles leads to production of anti-pre-S-specific antibodies during the course of HBV infection. Since antibodies specific for pre-S domains are capable of preventing the attachment of HBV to hepatocytes and are virus neutralizing, it would seem desirable to produce HBV vaccines with a standardized level of pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after recovery from natural infection. However, a test with appropriate sensitivity for detecting pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after (ELISA) for detecting pre-S determinants in vaccines containing less than or equal to 20 micrograms of HBsAg. The components of this assay are (1) antibodies to a synthetic peptide pre-S (120-145) adsorbed to polystyrene beads, and (2) beta-lactamase-labelled antibodies purified from anti-HBV serum on the basis of their affinity for a pre-S (120-174) beta-galactosidase fusion protein produced in Escherichia coli. Results of an evaluation of the pre-S content of HBV vaccines from two different commercial sources are discussed.

Amino Acid Sequence↗
PubMed1985

Expression in Escherichia coli of a cloned DNA sequence encoding the pre-S2 region of hepatitis B virus.

A DNA sequence encoding the entire pre-S2 region (amino acids 120-174; serotype ayw) of human hepatitis B virus envelope protein has been inserted into the lacZ gene of the plasmid pSKS105 yielding a recombinant, pWS3. Lac+ colonies of the Escherichia coli M182 delta (lacIOPZYA), isolated after transformation with pWS3, produced a pre-S2 peptide-beta-galactosidase fusion protein. This fusion protein, which comprised as much as 3% of the total bacterial protein, was purified to greater than 90% homogeneity by affinity chromatography on p-aminophenyl-beta-D-thiogalactoside-Sepharose. It is immunoprecipitable with rabbit antibodies to a synthetic peptide corresponding to amino acids 120-145 of the pre-S2 region of serotype adw [pre-S(120-145)] or with antibodies to hepatitis B virus. pre-S(120-145) completely blocked the binding of either antibody to the pre-S2 peptide-beta-galactosidase fusion protein. These results indicate that there are antigenic determinants on the fusion protein that are closely related to, if not identical to, determinants on synthetic pre-S(120-145) and on pre-S2 sequences of native hepatitis B virus particles. Thus, bacteria transformed with pWS3 can provide an abundant source of pre-S2-beta-galactosidase fusion protein, which may prove useful either as a diagnostic reagent possessing marker enzyme activity suitable for ELISA tests or as an immunogen with potential to contribute to active prophylaxis of hepatitis B.

Amino Acid Sequence↗
PubMed1985

Postextraction pain relief in children: a clinical trial of liquid analgesics.

Our objective was to evaluate the relative efficacies of four liquid analgesics in children, five to twelve years of age, following dental extractions. The analgesics, acetaminophen elixir (240 or 360 mg), acetaminophen with codeine elixir (240 mg and 24 mg, respectively), aluminum ibuprofen suspension (200 mg), and placebo liquid were administered at home, as a single dose, in a randomized double-blind study design. Of the 154 patients enrolled, 45 were evaluated, 39 patients never required medication, 12 were lost to follow-up, and 8 were excluded for other reasons. Aluminum ibuprofen provided significant relief in one-half hour compared with placebo. At one hour, both aluminum ibuprofen and acetaminophen with codeine provided significant relief compared with placebo. All three active agents were effective at two hours. The global rating of drug efficacy was statistically superior for aluminum ibuprofen. The majority of patients in all four groups were pain-free after four hours. No adverse reactions were reported during the study.

Acetaminophen↗
PubMed1983

Inhibition of development of characteristic indices of myeloid differentiation by a factor released from a tetraploid variant of the human promyelocytic cell line, HL-60.

HL-60TR, a tetraploid variant of the human promyeloid cell line HL-60, was obtained by culturing HL-60 cells for one week with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 400 pM. HL-60TR cells have lost the ability to develop several common markers of maturation in response to compounds that induce monocytoid or myelocytoid differentiation of HL-60 cells. In addition, they release a factor which inhibits induction of the same markers in HL-60 cells. Medium conditioned by HL-60TR cells also inhibits colony formation by normal mouse bone marrow cells. These properties have been maintained by HL-60TR cells through more than one year of constant subculture in the absence of TPA, a finding which suggests the possibility that TPA may promote tumor formation not only through direct effects on the phenotype of initiated cells but also through induction of continued production of factors that affect differentiation of normal stem cells.

Animals↗
PubMed1983

Characterization of RNA transcripts and virally coded proteins synthesized in mouse fibroblasts transfected with hepatitis B DNA: HBeAg synthesis in HBcAg-negative cells with active core-antigen genes.

A line of mouse cells expressing hepatitis B virus (HBV) surface and 'e' antigens identical in their physico-chemical properties to antigens from patients infected with HBV was isolated after transfection of 3T3 cells with cloned HBV DNA. The studies reported here indicate that the cells contain uninterrupted copies of the entire HBV genome which are unmethylated on CCGG sites and have no gross deletions or rearrangements. The entire core region is transcribed into polyadenylated RNAs large enough to serve as messengers for production of viral core antigen (HBcAg) yet no HBcAg can be detected. This suggests that the cells produce a primary translation product copied from the HBcAg messenger which either cannot assume the proper configuration for display of HBcAg determinants or is rapidly converted to HBeAg by proteolysis.

Animals↗