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A comprehensive assessment of indoor carbonyl compounds for the academic staff workers, and students was conducted on a university campus in Xiamen, China. A total of 15 representative environment categories, including 12 indoor workplaces and three residential units, were selected. The potential indoor pollution sources were identified based on the variability in the molar compositions and correlation analyses for the target carbonyls. Furnishing materials, cooking emissions, and electronic equipment, such as photocopiers, can generate various carbonyls in the workplace. Comparison studies were conducted in the clerical offices, demonstrating that off-gases from wooden furniture and lacquer coatings, environmental tobacco smoke (ETS), and the use of cleaning reagents elevated the indoor carbonyl levels. The measured concentrations of formaldehyde and acetaldehyde in most locations surpassed the exposure limit levels. The lifetime cancer hazard risk (R) associated with formaldehyde was above the concern risk level (1 x 10(-6)) in all of the workplaces. The results indicate that formaldehyde exposure is a valid occupational health and safety concern. Wooden furniture and refurbishing materials can pose serious health threats to occupants. The information in this study could act as a basis for future indoor air quality monitoring in Mainland China. Implications: A university campus represents a microscale city environment consisting of all the working, living, and commercial needs of staff and students. The scope of this investigation covers 21 hazardous carbonyl species based on samples collected from 15 categories of workplaces and residential building in a university campus in southern China. Findings of the study provide a comprehensive assessment of indoor air quality with regards to workers' health and safety. No similar study has been carried out in China.

Unlabelled: A pilot hazardous airborne carbonyls study was carried out in Hong Kong and the Mainland of China. Workplace air samples in 14 factories of various types of manufacturing and industrial operations were collected and analyzed for a panel of 21 carbonyl compounds. The factories can be classified into five general categories, including food processing, electroplating, textile dyeing, chemical manufacturer, and petroleum refinery. Formaldehyde was invariably the most abundant carbonyl compound among all the workplace air samples, accounting for 22.0-44.0% of the total measured amount of carbonyls on a molar basis. Acetone was also found to be an abundant carbonyl in workplace settings; among the selected industrial sectors, chemical manufacturers' workplaces had the highest percentage (an average of 42.6%) of acetone in the total amount of carbonyls measured in air. Benzaldehyde accounted for an average of 20.5% of the total amount of detected carbonyls in electroplating factories, but its contribution was minor in other industrial workplaces. Long-chain aliphatic carbonyls (C6-C10) accounted for a large portion (37.2%) of the total carbonyls in food-processing factories. Glyoxal and methylglyoxal existed at variable levels in the selected workplaces, ranging from 0.2% to 5.5%. The mixing ratio of formaldehyde ranged from 8.6 to 101.2 ppbv in the sampled workplaces. The observed amount of formaldehyde in two paint and wax manufacturers and food-processing factories exceeded the World Health Organization (WHO) air quality guideline of 81.8 ppbv. Carcinogenic risks of chronic exposure to formaldehyde and acetaldehyde by the workers were evaluated. The lifetime cancer hazard risks associated with formaldehyde exposure to male and female workers ranged from 2.01 x 10(-5) to 2.37 x 10(-4) and 2.68 x 10(-5) to 3.16 x 10(-4), respectively. Such elevated risk values suggest that the negative health impact of formaldehyde exposure represents a valid concern, and proper actions should be taken to protect workers from such risks., Implications: Many carbonyl species (e.g., formaldehyde, acetaldehyde, and acrolein) are air toxins and they pose public healt risks. The scope of this investigation covers 21 types of carbonyls based on samples collected from 14 different workplaces. Findings of the study will not only provide a comprehensive assessment of indoor air quality with regard to workers' healthy and safety, but also establish a theoretical foundation for future formulation of intervention strategies to reduce occupational carbonyl exposures. No similar study has been carried out either in Hong Kong or the Mainland of China.

Infection of human hepatitis B virus (HBV) causes acute hepatitis. Its persistent infection leads to a high risk of developing chronic hepatitis, cirrhosis and hepatocellular carcinoma. The levels of HBV 3.5 kb and 2.4/2.1 kb RNAs transcribed from a replicating HBV expression plasmid in human hepatoma HuH-7 cells are repressed by tumour necrosis factor alpha treatment or overexpressed p65 in a dose-dependent manner. The diminished expression of endogenous p65 by a p65-specific siRNA or IkappaB-alpha overexpression enhances the HBV gene expression. The protein bound to the Specificity protein 1 (Sp1) binding sites (nt 1733-1753) of HBV core promoter is reduced by either tumour necrosis factor alpha treatment or overexpressed p65. The N-terminal 43-amino-acid region of p65, which is required to interact with Sp1, is essential to repress the Sp1-mediated transactivation. The binding of Sp1 to Sp1 site and the Sp1-dependent reporter expression are inhibited by p65 in a dose-dependent manner. Furthermore, nuclear factor-kappa B-mediated repression of HBV gene expression is abolished by deletion of Sp1 sites of HBV gene promoter. Together, these results demonstrate that nuclear factor-kappa B represses the HBV gene expression through its interaction with Sp1 and repression of Sp1-mediated transcriptional activation.

Human hepatitis B virus (HBV) causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Here we report that HBV core protein interacts with a cellular SKIP (skeletal muscle and kidney enriched inositol phosphatase) protein, an endoplasmic reticulum-located phosphoinositide 5-phosphatase, both in vivo and in vitro. The minimal sequence required for interaction is the amino acid region from 116 to 149 for the core protein and the SKIP carboxyl homology (SKICH) domain for SKIP. When HBV replicates in HuH-7 cells, overexpressed SKIP localizes to nucleus in addition to ER and suppresses HBV gene expression and replication. SKIP loses its nuclear localization and suppressive effect during replication of a core-negative HBV mutant. HBV gene expression is enhanced significantly when endogenous SKIP expression is knocked down by a SKIP-specific siRNA. SKIP mutation analysis shows that its 5-phosphatase activity is not required for the suppressive effect and that the suppression domain maps to amino acids 199-226. These results demonstrate that SKIP is translocated from endoplasmic reticulum into nucleus through its interaction with core protein and suppresses HBV gene expression via a novel suppression domain.

The 570-amino acid membrane form of IL-1RAcP (mIL-1RAcP) plays a pivotal role in the IL-1 signal transduction and response. We have identified another membrane form of IL-1RAcP with 687 amino acids (named as mIL-1RAcP687 hereon). Its except the last amino acid N-terminal 448 amino acid portion, containing three extracellular immunoglobulin domains, one transmembrane domain, and Box 1 and Box 2 of Toll/IL1 Receptor (TIR) domain, is identical to that of mIL-1RAcP. In contrast, the C-terminal 239 amino acid portion of mIL-1RAcP687, containing Box 3 of TIR domain, is unique. The mIL-1RAcP687 splice variant is derived from the first 11 exons except 9b, and a newly identified exon 13 of IL-1RAcP gene, while mIL-1RAcP is derived from the first 12 exons except 9b. Furthermore, mIL-1RAcP687 can associate with proteins involved in the upstream IL-1 signaling pathway such as IL-1RI, Tollip, and MyD88. It thus activates downstream signaling events to activate transcription factor NF-kappaB, and induce the expression of IL-1 responsive genes such as TNF-alpha and GM-CSF. These results demonstrate that like mIL-1RAcP, mIL-1RAcP687 functions in the IL-1 signal transduction and response. Identification of mIL-1RAcP687 adds further complexity to the regulation of IL-1 signaling and its subsequent response.

Protein-tyrosine phosphatase PTPN3 is a membrane-associated non-receptor protein-tyrosine phosphatase. PTPN3 contains a N-terminal FERM domain, a middle PDZ domain, and a C-terminal phosphatase domain. Upon co-expression of PTPN3, the level of human hepatitis B viral (HBV) RNAs, 3.5 kb, 2.4/2.1 kb, and 0.7 kb transcribed from a replicating HBV expression plasmid is significantly reduced in human hepatoma HuH-7 cells. When the expression of endogenous PTPN3 protein is diminished by specific small interfering RNA, the expression of HBV genes is enhanced, indicating that the endogenous PTPN3 indeed plays a suppressive role on HBV gene expression. PTPN3 can interact with HBV core protein. The interaction is mediated via the PDZ domain of PTPN3 and the carboxyl-terminal last four amino acids of core. Either deletion of PDZ domain of PTPN3 or substitution of PDZ ligand in core has no effect on PTPN3-mediated suppression. These results clearly show that the interaction of PTPN3 with core is not required for PTPN3 suppressive effect. Mutation of (359)serine and (835)serine of 14-3-3beta binding sites to alanine, which slightly reduces the interaction with 14-3-3beta, does not influence the PTPN3 effect. In contrast, mutation of the invariant (842)cysteine residue in phosphatase domain to serine, which makes the phosphatase activity inactive, does not change its subcellular localization and interaction with core or 14-3-3beta, but completely abolishes PTPN3-mediated suppression. Furthermore, deletion of FERM domain does not affect the phosphatase activity or interaction with 14-3-3beta, but changes the subcellular localization from cytoskeleton-membrane interface to cytoplasm and nucleus, abolishes binding to core, and diminishes the PTPN3 effect on HBV gene expression. Taken together, these results demonstrate that the phosphatase activity and FERM domain of PTPN3 are essential for its suppression of HBV gene expression.

Human hepatitis B virus (HBV) can cause acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV e antigen (HBeAg), a secreted protein and not required for viral replication, is thought to play an immunoregulatory role during viral infection. However, the functional involvement of HBeAg in host immune response has not been fully elucidated. We report in this study that HBeAg can bind to interleukin-1 receptor accessory protein (IL-1RAcP). Interleukin-1 (IL-1) plays an important role in inflammation and regulation of immune response, and membrane form of IL-1RAcP (mIL-1RAcP) is an essential component of trimeric IL-1/IL-1 receptor/mIL-1RAcP complex. We show that glutathione S-transferase- or polyhistidine-tagged recombinant HBeAg can interact with endogenous mIL-1RAcP in vitro. Purified (His)6-HBeAg added in the culture medium can interact with overexpressed FLAG-tagged mIL-1RAcP in vivo. Indirect immunofluorescence and confocal microscopy show that HBeAg colocalizes with mIL-1RAcP on the cell surface. Furthermore, HBeAg is able to induce the interaction of IL-1 receptor I (IL-1RI) with mIL-1RAcP and trigger the recruitment of adaptor protein myeloid differentiation factor 88 (MyD88) to the IL-1RI/mIL-1RAcP complex. Assembly and activation of IL-1RI/mIL-1RAcP signaling complex by HBeAg can activate downstream NF-kappaB pathway through IkappaB degradation, induce NF-kappaB-dependent luciferase expression, and induce the expression of IL-1-responsive genes. Silencing of IL-1RAcP by small interfering RNA dramatically abolishes HBeAg-mediated NF-kappaB activation. These results demonstrate that HBeAg can trigger host IL-1 response by binding to mIL-1RAcP. The interaction of HBeAg with mIL-1RAcP may play an important role in modulating host immune response in acute and chronic HBV infection.

A negative regulatory element (NRE) is located immediately upstream of the upstream regulatory sequence of core promoter and second enhancer of human hepatitis B virus (HBV). NRE represses the transcription activation function of the upstream regulatory sequence of core promoter and the second enhancer. In this study, we described the cloning and characterization of an NRE-binding protein (NREBP) through expression cloning. NREBP cDNA is 8266 nucleotides in size and encodes a protein of 2386 amino acids with a predicted molecular mass of 262 kDa. Three previously described cDNAs, DBP-5, SONB, and SONA, are partial sequence and/or alternatively spliced forms of NREBP. The genomic locus of the NREBP/SON gene is composed of 13 exons and 12 introns. The endogenous NREBP protein is localized in the nucleus of human hepatoma HuH-7 cells. Antibody against NREBP protein can specifically block the NRE binding activity present in fractionated nuclear extracts in gel shifting assays, indicating that NREBP is the endogenous nuclear protein that binds to NRE sequence. By polymerase chain reaction-assisted binding site selection assay, we determined that the consensus sequence for NREBP binding is GA(G/T)AN(C/G)(A/G)CC. Overexpression of NREBP enhances the repression of the HBV core promoter activity via NRE. Overexpression of NREBP can also repress the transcription of HBV genes and the production of HBV virions in a transient transfection system that mimics the viral infection in vivo.

Hepatitis B viral core protein is present in the nucleus and cytoplasm of infected hepatocytes. There is a strong correlation between the intrahepatic distribution of core protein and the viral replication state and disease activity in patients with chronic hepatitis. To understand the role of core protein in the pathogenesis of HBV, we used a yeast two-hybrid system to search for cellular proteins interacting with the carboxyl terminus of core protein, as this region is involved in a number of important functions in the viral replication cycle including RNA packaging and DNA synthesis. A cDNA encoding the extreme C-terminal region of human actin-binding protein, ABP-276/278, was identified. This interaction was further confirmed both in vitro and in vivo. In addition, the extreme C-terminal region of ABP-276/278 interacted with the nearly full-length HBV core protein. Since this region is present in both the core and the precore proteins, it is likely that both core and precore proteins of HBV can interact with the C-terminal region of ABP-276/278. The minimal region of ABP-276/278 which interacted with the HBV core protein was the C-terminal 199 amino acid residues which correspond to part of the 23rd repeat, the entire 24th repeat and the intervening hinge II region in ABPs. The potential functional outcome of ABP interaction in HBV replication and its contribution to the pathological changes seen in patients with chronic HBV infection are discussed.

Box alpha is an essential element of both the upstream regulatory sequence of the core promoter and the second enhancer, which positively regulate the transcription of human hepatitis B virus (HBV) genes. In this paper, we describe the cloning and characterization of a box alpha binding protein, E4BP4. E4BP4 is a bZIP type of transcription factor. Overexpression of E4BP4 represses the stimulating activity of box alpha in the upstream regulatory sequence of the core promoter and the second enhancer in differentiated human hepatoma cell lines. E4BP4 can also suppress the transcription of HBV genes and the production of HBV virions in a transient-transfection system that mimics the viral infection in vivo. Expression of an E4BP4 antisense transcript can, instead, elevate the transcription of the core promoter. A low abundance of E4BP4 protein and mRNA in differentiated human hepatoma cell lines is detected, and E4BP4 is not a major component of box alpha binding proteins in untransfected differentiated human hepatoma cell lines. C/EBPalpha and C/EBPbeta, in contrast, are major components of the box alpha binding activity present in nuclear extracts. E4BP4 has a stronger binding affinity towards box alpha than the endogenous box alpha binding activity present in nuclear extracts. Structure and function analysis of E4BP4 reveals that DNA binding activity is sufficient to confer the negative regulatory function of E4BP4. These results indicate that binding site occlusion is the mechanism whereby E4BP4 suppresses transcription in HBV.

Core protein is the major component of the core particle (nucleocapsid) of human hepatitis B virus. Core particles and core proteins are involved in a number of important functions in the replication cycle of the virus, including RNA packaging, DNA synthesis, and recognition of viral envelope proteins. Core protein is a phosphoprotein with most, if not all, of the phosphorylation on C-terminal serine residues. In this study, we identified a serine kinase activity from the ribosome-associated protein fraction of cytoplasm that could specifically bind and phosphorylate the C-terminal portion of recombinant core protein. This kinase is referred to as core-associated kinase (CAK). CAK could be inhibited by the kinase inhibitors heparin and manganese ions but not by spermidine, DRB, H89, or H7, indicating that CAK is distinct from protein kinase A and protein kinase C. CAK could be partially purified by heparin-Sepharose CL-6B and phosphocellulose P11 columns. By using a far-Western assay, three specific proteins, of 46, 35, and 13 kDa, were shown to interact with the C-terminal part of the core protein. These three proteins were present only in the eluted fractions that contains the CAK activity. An in-gel kinase assay showed that a 46-kDa kinase in the same fraction could bind and phosphorylate the C-terminal part of the recombinant core protein. These results indicate that this 46-kDa kinase is most probably CAK. A similar 46-kDa kinase, which exhibits the same profile of sensitivity to kinase inhibitors as that of CAK, is present in both purified intracellular core particles and extracellular 42-nm virions, suggesting that CAK is a candidate for the core particle-associated kinase.

Polymerase of human hepatitis B virus is required for viral replication and pregenomic RNA encapsidation. Using recombinant GST fusion proteins, we show that the terminal protein domain of polymerase can interact specifically with a protein complex containing kinase activity and a tightly associated 35-kD protein (p35). This kinase is termed terminal-protein-associated kinase (TPAK). The phosphoamino acid analysis of phosphorylated p35 demonstrates that TPAK is a serine kinase. Analysis of deletion mutants shows that amino acids 1-95 of the terminal protein domain are required for the interaction with TPAK/p35 and phosphorylation of p35. TPAK/p35 are found predominantly in the cytoplasm. Furthermore, TPAK can be inhibited by heparin and manganese ions, but is resistant to spermidine, DRB, H89 or H7. These results indicate that TPAK is not protein kinase A or protein kinase C. Copyright 1997 S. Karger AG, Basel

The major surface promoter of human hepatitis B virus can produce three distinct groups of S transcripts. The initiation sites of these transcripts are in close proximity. Encompassing the ATG for the middle surface protein, the largest S transcript (+1) encodes the middle surface protein whereas the other two (+20 and +31) can only code for small surface protein. Sequence analysis does not reveal any TATA element. In this study, we employ deletion, linker scanning, and linker insertion analyses to study systematically the sequence requirements for the initiations of all three transcripts and their upstream regulatory sequences. Our study reveals that the sequence downstream of -16 is sufficient for precise initiation of all three groups of S transcripts. The 3' boundary of minimal promoter element is +15 for the +1 transcript, whereas it is +39 for both +20 and +31 transcripts. Furthermore, there are distinct sequence requirements for the initiations of three groups of S transcripts. The sequences from -17 to -10 and from -1 to +7 are required for the initiation of +1 transcript, the sequence from +16 to +39 is essential for the +20 transcript, and the sequences from -17 to -10 and from +24 to +39 are required for the + 31 transcript. Our results also suggest that the transcription initiations of major surface promoter may be mediated in part by initiators. The initiations of these three groups of S transcripts are under differential regulation. The region from -39 to -16 containing both negative and positive regulatory elements selectively regulates the transcription levels of the two major S transcripts. Most notably, mutation of the sequence from -17 to -10, which contains a Sp1 site, leads to an increase in the imprecise initiation at +1 site and depresses the initiation of +20 and, to a greater extent, +31 transcript. The relevance of differential regulation of major surface promoter to the varied production of different surface protein isoforms in viral life cycle is discussed.

The regulation of transcription of the hepatitis B virus core promoter is an important event in the viral life cycle. Two messages, precore and pregenomic RNAs, that are initiated 30 nucleotides apart are produced by the core promoter. Precore RNA encodes nucleocapsid protein and pregenomic RNA core and polymerase. The latter transcript also serves as a template for viral genome replication via reverse transcription. We have previously defined a basal core promoter, which contains four TA-rich sequences (TA1 through TA4) but no canonical TATA element, that can direct transcription of both messages. In this study, we demonstrated that a stretch of 15 nucleotides containing TA4 is sufficient to direct precise initiation of both precore and pregenomic transcripts. This sequence can function as both an initiator and a TATA element. Mutational analysis further revealed that sequences essential for either function are colocalized. The significance of this finding with respect to the basal transcription mechanism and regulation of viral gene expression is discussed.

Enhancer II of human hepatitis B virus has dual functions in vivo. Located at nucleotides (nt) 1646 to 1741, it can stimulate the surface and X promoters from a downstream position. Moreover, the same sequence can also function as upstream regulatory element that activates the core promoter in a position- and orientation-dependent manner. In this study, we report the identification and characterization of a negative regulatory element (NRE) upstream of enhancer II (nt 1613 to 1636) which can repress both the enhancer and upstream stimulatory function of the enhancer II sequence in differentiated liver cells. This NRE has marginal inhibitory effect by itself but a strong repressive function in the presence of a functional enhancer II. Mutational analysis reveals that sequence from nt 1616 to 1621 is required for repression of enhancer activity by the NRE. Gel shift analysis reveals that this negative regulatory region can be recognized by a specific protein factor(s) present at the 0.4 M NaCl fraction of HepG2 nuclear extracts. The discovery of the NRE indicates that HBV gene transcription is controlled by combined effects of both positive and negative regulation. It also provides a unique system with which to study the mechanism of negative regulation of gene expression.

Hepatitis B virus is a hepatotropic virus. Its replication and gene expression are mainly restricted to hepatocytes in the infective process. The viral gene expression thus provides a unique system with which to study the control of tissue-specific gene expression. We have previously reported the identification and characterization of the second enhancer (enhancer II) of hepatitis B virus. In this report, we further demonstrate that the minimal functional constituents of the second enhancer, box alpha and box beta, display liver cell and differentiation state specificity. Moreover, box alpha exhibits the same liver cell and differentiation state specificity when functioning as an upstream regulator for the basal core promoter. Gel shift experiments reveal a unique box alpha-binding protein, protein a, which is present only in differentiated liver cells, where enhancer II is functional. The converse is true for another box alpha-binding protein, protein f, which is present only in poorly differentiated liver cells and nonliver cells. The simplest hypothesis that explains these results is that protein a activates and/or protein f suppresses the enhancer and upstream regulator functions. Although C/EBP is a candidate for a transcription factor that interacts with box alpha or box beta, none of the binding factors identified in the gel shift assays, including protein a and protein f, is likely to be C/EBP because they differ from C/EBP in heat lability and sequence preference.

To study the expression and localization of delta antigen during the replication cycle of hepatitis D virus, we cotransfected HuH-7 hepatoma cells with a hepatitis B virus expression plasmid and plasmids expressing the small or large delta antigen or the entire HDV genome. The transfected cells and culture medium were analyzed on double immunofluorescence staining for delta antigen and HBsAg, on Western blotting and on Northern-blot hybridization from 4 hr to 9 days after transfection. In cells transfected with the expression plasmid carrying the small delta antigen open reading frame, only the small delta antigen was expressed; it was localized in the nucleolus and was not released into the medium during the culture period. In cells transfected with the large delta antigen expression plasmid, the large delta antigen expressed was localized in the nucleolus at the initial stage; this was followed by relocalization in nucleoplasm. Finally, large delta antigen was released in HBsAg-enveloped particles within 1 day of transfection. In cells cotransfected with hepatitis B virus and hepatitis D virus expression plasmids, the small delta antigen was expressed 4 hr after transfection, whereas the large form was expressed 3 days after transfection. Expression of the large delta antigen coincided with the localization changes from nucleolar to mixed stage and, finally, to nucleoplasm and release of the hepatitis D virus particles. The large delta antigen appears to play a key role in relocalization of the delta antigen and packaging of the hepatitis D virus virions.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis B virus (HBV) infection, either acute or chronic, has been one of the leading health problems in the world. To understand the HBV life cycle and disease process, we set out to study the regulation of viral gene expression. In this paper, we report the characterization of the HBV core promoter: two 3.5-kb transcripts, precore and pregenomic, are made from it. The latter is itself a template for viral genome replication and also encodes viral proteins essential for both viral replication and virion assembly. We identify a short sequence (from nucleotides [nt] 1744 to 1851, referred to as the basic core promoter [BCP]) that is sufficient to direct correct initiation of both precore and pregenomic messages. In addition, the two appear to be regulated in a coordinate manner. Sequences upstream of the BCP (from nt 1636 to 1744, referred to as the core upstream regulatory sequence [CURS]), have a strong stimulating effect on the BCP. Addition of the CURS to the BCP leads to a dramatic increase in both the transcription of two 3.5-kb messages and the production of 42-nm virions from transiently transfected hepatoma cells. The CURS stimulates the BCP in a position- and orientation-dependent manner. Therefore, it is unlikely that the effect is mediated through enhancer II, which has been localized to the same sequence. Deletion analysis of the CURS suggests that it contains multiple regulatory elements that control the BCP in an interactive manner. In accord with this hypothesis, the CURS is found to be bound with many distinct protein factors in footprinting experiments. Among these elements, box alpha (from nt 1646 to 1668) and box gamma delta (from nt 1671 to 1703) are two regulatory elements which individually stimulate promoter activity more than 100-fold.

The second enhancer (enhancer II) of hepatitis B virus is functionally liver specific. Located within an open reading frame of the virus and immediately upstream of the initiation sites of viral major transcripts, enhancer II furnishes a unique model for use in investigating the structure and function of an enhancer. In this study, two functional constituents, a 23-bp box-alpha and a 12-bp box-beta, are identified as being both necessary and sufficient for enhancer II function. Examination of the box-alpha and box-beta sequences reveals a weak homology to the extended consensus for a C/EBP binding site. Gel shift and footprinting analyses indicate that multiple proteins bind to these sequences and thus are candidate transcription factors that mediate the enhancer function. One heat-resistant protein, protein a, and one heat-sensitive protein, protein b, bind to box-alpha. Protein a, which binds to box-alpha in a way indistinguishable from that seen with a recombinant C/EBP, appears not to be identical to C/EBP in that the binding of protein a requires a minimal sequence larger than the canonical C/EBP sites. Two box-beta-binding proteins, c and d, show greater affinity for the C/EBP consensus than for box-beta. However, both proteins c and d are relatively heat sensitive and display a distinct sequence preference from the recombinant C/EBP protein. Since the function of enhancer II is strictly dependent on a bipartite architecture, this system provides a unique model for studies of how the interactions of its binding proteins lead to the enhancer function.

The hepatitis delta virus (HDV) is a defective virus with a coat composing of the surface antigen of its helper virus, hepatitis B virus (HBV). Replication of HDV in the absence of HBV has been shown in cell cultures by transient transfection of the HDV plasmid. However, the formation and release of HDV virions have not been observed. In this report, a human hepatoma cell line HuH-7 was transiently cotransfected with HDV and HBV plasmids. The production of monomeric and multimeric antigenomic RNAs of HDV in the transfected cells indicated replication of the HDV genome. The major 3.5- and 2.1-kb RNAs of HBV were also expressed. Virions of both HDV and HBV were released from the cotransfected cells, as shown by the detection of monomeric genomic HDV RNA and partially double-stranded HBV DNA in the culture medium. Thus, this is the first report that describes the assembly and the release of HDV viral particles in an in vitro cell culture. The HDV virions released possessed physicochemical properties identical to those of the HDV virions found in infected human serum. Furthermore, expression of both the 3.5- and 2.1-kb RNAs of HBV was shown to be dramatically decreased by the presence of HDV, indicating suppression of the expression of HBV genes by HDV. The amount of HBV virions released was similarly suppressed by HDV. Cotransfection of HBV with an expression plasmid of the HDV delta antigen remarkably reduced the levels of the 3.5- and 2.1-kb HBV RNAs, indicating that suppression of the expression of HBV RNAs by HDV occurs via the action of the delta antigen. This HBV- and HDV-cotransfected human hepatoma cell line should provide an excellent system for the study of the function of the delta antigen and the interaction between HDV and its helper, HBV.

Previous studies have identified an enhancer (enhancer I) at nucleotides (nt) 1074 to 1234 in the genome of the human hepatitis B virus (HBV), which locates immediately upstream from the X gene. By analysis of the expression of the chloramphenicol acetyltransferase gene driven by a heterologous simian virus 40 early promoter, we describe the identification of a second enhancer (enhancer II) at nt 1636 to 1741, which locates downstream of enhancer I and immediately upstream of the core gene. With various deletions at the 5' end of enhancer II, a positive regulatory element was identified at nt 1636 to 1690 (the II-A element), with the 5' boundary between nt 1636 and 1671. The II-A element alone did not have an enhancer function, but the enhancer activity was achieved by the concomitant presence of the sequence from nt 1704 to 1741 (the II-B element). The II-B element alone did not have enhancer activity. These results indicate that cooperation between the II-A and II-B elements is required to exhibit the enhancer activity of enhancer II. We also show that enhancer II stimulates the transcriptional activity of both the SPI and SPII promoters of the surface gene. Therefore, the SPI promoter activity is regulated by the proximal HNF-1 binding element and the distal enhancers I and II. These results indicate that multiple regulatory elements scattered over the whole viral genome are involved in the regulation of expression of each individual HBV gene and that the same regulatory element controls the expression of different HBV genes. The relative positions of these regulatory elements in the HBV genome suggest that they may control the expression of HBV genes in a coordinate and cooperative manner.

Twenty of 22 hepatitis delta virus (HDV)-superinfected chronic hepatitis B carriers who had detectable HDV RNA at the acute stage progressed to chronicity, while only 6 of 16 patients without HDV RNA did so (P less than .005). The poor outcome of patients with persistently positive or fluctuating HDV RNA has been indicated by the following findings: 24 of 38 patients suffered from prolonged hepatic inflammation complicated by three to eight episodes of exacerbations; among them, 5 developed cirrhosis and 2 died in a follow-up period of 4 years. For most patients, the replication of HBV was suppressed at acute stage; only 3 of the 38 cases had detectable HBV DNA in sera. While reactivation of HBV was found in another 8 patients in the follow-up period, for 5 it was in the presence of serum HDV RNA and 2 developed cirrhosis. Therefore, serial assays of serum HDV RNA and HBV DNA appeared to be of value in monitoring the clinical course and outcome of acute HDV superinfection and in the study of the long-term interactions between these two viruses.

Multivariate analysis of the risk factors for hepatitis D virus infection among 53 men with acute hepatitis D virus superinfection, 59 men with acute exacerbation of chronic hepatitis B and 54 asymptomatic male hepatitis B carriers revealed that sexual contact with prostitutes and use of nondisposable needles were the factors significantly associated with hepatitis D virus infection in Taiwan (odds ratio = 5.5, 95% confidence interval = 3.3 to 9.2; odds ratio = 2.4, 95% confidence interval = 1.3 to 4.4, respectively). The prevalence of antibody to hepatitis D virus antigen among hepatitis B virus carriers with histories of sexual exposure to prostitutes was 10.3% (14 of 136) and the prevalence of hepatitis D virus antigen among the female prostitutes who were hepatitis B virus carriers was 59% (24 of 51). Among those who admitted to sexual contact with prostitutes, testing for hepatitis D virus antigen revealed positive correlations with the frequency of such sexual contacts and with the frequency of history of venereal diseases. Among the female prostitutes, the prevalence of hepatitis D virus antigen had a positive correlation with the frequency of sexual contact and a negative correlation with age. Analysis of serum hepatitis D virus RNA among those with hepatitis D virus antigen revealed that 4 of the 9 with sexual exposure to prostitutes and 3 of the 24 prostitutes were positive. From these findings, we conclude that these two groups are not only at high risk for hepatitis D virus infection but also form a reservoir of this virus.

From January 1984-December 1987, 1783 patients received combination therapy of isoniazid, rifampin, and ethambutol for the control of tuberculosis. Forty-two developed symptomatic hepatitis during the period of treatment. Fifteen were hepatitis B virus carriers, and the remaining 27 were noncarriers. The peak serum transaminase and bilirubin levels were higher in carriers. Seven carriers died of fulminant or subacute hepatic failure, and only 1 noncarrier died. Eleven carriers had detectable serum hepatitis B virus deoxyribonucleic acid during the acute stage of hepatitis. The roles of isoniazid-rifampin combination therapy and hepatitis B virus in the adverse outcomes of carriers were discussed.

The human hepatoma Hep3B cells contain integrated hepatitis B viral genome and continually secret hepatitis B surface antigen (HBsAg). The production of HBsAg (but not alpha-fetoprotein) was suppressed by addition of low concentrations (0.1-1 nM) of insulin into serum-free medium. In addition, the suppression of HBsAg production by insulin was paralleled with the decrease in HBsAg mRNA abundance. Insulin also cause a rapid rate of disappearance of HBsAg mRNA (t 1/2, 2 h) in Hep3B cells. The Hep3B cells carry specific receptor with high affinity for insulin (Kd = 1.8 nM). The receptor showed an insulin-dependent protein tyrosine kinase activity. The half-maximal insulin concentration for the activation of the receptor kinase was about 5 nM. Only very high concentrations of insulin-like growth factor I and human proinsulin can compete for the insulin receptor binding and suppress HBsAg production, this suggests that insulin may act through its receptor binding to suppress HBsAg expression in human hepatoma Hep3B cells.

Bovine heart mitochondria were treated with limited amounts of iodoacetamide, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, phenylglyoxal, tetranitromethane and 1-fluoro-2,4-dinitrobenzene, respectively. Examination of the respiration and proton extrusion characteristics of the chemically modified mitochondria suggests that sulfhydryl and imidazole groups are not directly involved in proton pumping, but that some of the labeled carboxyl, amino, guanidinium and phenolic groups may participate in an indirect proton-extrusion process. Cross-linking mitochondria with glutaraldehyde drastically decreases the efficiency of proton extrusion, whereas treatment of mitochondria with valeraldehyde under similar conditions did not affect the proton-pumping efficiency significantly. The latter observations show that conformational change in the inner mitochondrial membrane may play a crucial role in the active translocation of protons coupled to electron transport. Comparison of the reactivities of the essential amino and carboxyl groups in mitochondria in different oxidation states suggests that these two types of essential functional groups are more exposed to water in the oxidized state. An indirect mechanism for proton pumping based on protein conformational change driven by electron transport based on the results of the present chemical modification studies is suggested.

The HBV DNA sequence containing a primary part of pre-S2, the coding sequence of the major S protein and the enhancer sequence in pTWS1 was completely sequenced. A comparison with other previously published HBV sequences indicates that the nucleotide sequence homology in the pre-S2 region is 97%, 98% and 98% for adw, adr and ayw subtypes, respectively, and 76% among different subtypes. The nucleotide sequence homology in the major S protein region is 99% for each adw, adr and ayw subtype, and 90% among different subtypes. The important regulatory enhancer sequence (nucleotides 1036-1204) is identical in all subtypes.

Ninety-four patients, who were admitted with symptoms of liver disease and found to be positive for hepatitis B surface antigen and antibody to hepatitis B e antigen (anti-HBe), were examined for hepatitis B virus (HBV) DNA in serum and immunoglobulin antibody to hepatitis B core antigen and liver biopsies were stained for hepatic hepatitis B core antigen. Of 94 patients, 34 (36%) had evidence of HBV replication and 35 (37%) evidence of hepatitis D virus (HDV) superinfection. Most of the latter two groups of patients (greater than 70%) had evidence of chronic active hepatitis or active cirrhosis in their liver biopsies. The majority of these patients (greater than 80%) also had high levels of serum alanine aminotransferase (greater than 200 U/L) during the acute stage of their illness, and suffered from prolonged hepatic inflammation (greater than 1 year). Many of the patients (59%) also experienced frequent (1-6 episodes) relapsing exacerbations during a two-year follow-up period. Thus, persistent replication or reactivation of HBV and HDV superinfection were the two major causes of clinical exacerbations in anti-HBe-positive chronic HBV carriers in Taiwan, and also played an important role in the progression of their liver diseases and unfavorable outcomes.

In human hepatoma cell line Hep3B/T2, the human heat-shock-inducible gene hsp70 could be induced by insulin. The dose-dependent insulin effect correlates very well with the dissociation constant of the insulin receptor, indicating that the insulin effect is mediated by the insulin receptor. The expression of hsp70 gene was neither significantly induced by growth factors of epidermal and platelet-derived growth factors, nor by tumor promoter, calcium ionophore, cAMP, and glucocorticoids. These results indicate that the induction of expression of hsp70 gene by insulin is very specific and not cell cycle-related. Furthermore, the insulin-induced expression of hsp70 gene is transient, occurring specifically from 4 to 8 h after insulin addition.

The effect of inorganic phosphate, ADP, ATP, and their analogues on the rate of labeling of F1-ATPase by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and phenylglyoxal have been investigated. Analysis of the kinetic data indicate that the labeled functional groups of the essential tyrosine and arginine residues respectively are both located at the catalytic site of F1. The active phenolic group of tyrosine is located closer to the bound inorganic phosphate or the gamma-phosphate group than the alpha- and beta-phosphate groups of the bound ATP at the catalytic site, whereas the guanidinium group of arginine is located closer to the alpha- and beta-phosphate groups of the bound ATP than to its gamma-phosphate group or the bound inorganic phosphate. The kinetically deduced dissociation constants are 1.3 mM and 210 microM for the inorganic phosphate and aDP respectively bound to this catalytic site. Labeling the essential tyrosine residue by NBD-Cl has been found to facilitate subsequent labeling of the essential arginine residue by phenylglyoxal.

The transcriptional enhancer sequence has recently been demonstrated in the hepatitis B viral genome. This enhancer sequence has also been shown to increase the activity of HBcAg gene promoter. Using the chloramphenicol acetyltransferase gene expression system, we demonstrated that the intrinsic promoter activity of HBsAg gene promoter was stronger than that of HBcAg gene promoter in both human hepatoma cell lines, Hep3B and HuH-7. Furthermore, we showed that the HBV enhancer sequence not only stimulated the HBcAg gene promoter activity, but also stimulated the HBsAg gene promoter activity in both Hep3B and HuH-7 cells. The enhancer sequence increased the HBsAg gene promoter activity 20-fold in both Hep3B and HuH-7 cell lines, while the increase of the HBcAg gene promoter was 2- and 8-fold in Hep3B and HuH-7 cells, respectively.

Sera from 108 HBsAg carrier mothers at delivery and their respective offspring at birth and at 6 months of age were examined for hepatitis B virus DNA by the dot-blot hybridization technique. Hepatitis B virus DNA was detected in 83% of 88 carrier mothers who were HBeAg positive, and in 10% of 20 carrier mothers who were HBeAg negative. All five infants born to HBeAg-positive carrier mothers with hepatitis B virus DNA levels over 80 pg per 10 microliters of serum were infected by hepatitis B virus, in spite of receiving hepatitis B immunization. All 17 infants without hepatitis B immunization who were born to HBeAg and hepatitis B virus DNA-positive carrier mothers developed hepatitis B virus infection. Of 56 infants born to HBeAg and hepatitis B virus DNA-positive carrier mothers and who had received hepatitis B immunization, a higher hepatitis B virus infection rate was found in a group of infants whose sera hepatitis B virus DNA were positive (15/16, 93.8%) than in infants whose sera were negative (17/40, 42.5%) at birth (p less than 0.0005). These data suggest that the assay for hepatitis B virus DNA in sera of HBsAg carrier mothers at delivery or their infants at birth will predict the efficacy of hepatitis B immunization for prevention of maternal-infant hepatitis B virus transmission.

The expression of 20 known cellular proto-oncogenes in human well-differentiated hepatoma cell line Hep3B and poorly-differentiated hepatoma cell line HA22T/VGH was studied by Northern blot hybridization. Among the cellular proto-oncogenes examined, both cell lines express protein kinase genes including fps, mos and raf; PDGF B chain sis gene; GTP/GDP binding protein gene Ha-ras and nuclear protein genes including fos and myc. The expression of yes, abl, ros, src, erb-B, erb-A, fms, Ki-ras, myb, rel and bas genes was not detected in both cell lines.

The protection of F1 ATPase by inorganic phosphate, ADP, ATP, and magnesium ion against inactivation by 1-fluoro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, respectively, has been investigated. Dissociation equilibrium constants and rate constants for the labeling reactions have been deduced from a quantitative treatment of the kinetic data. Comparison of these dissociation constants with each other and with the corresponding literature values indicates that the essential Tyr, Arg, Lys, and Glu or Asp residues are indeed located at the catalytic site of the enzyme. Examination of the rate constants for the labeling reactions in the presence of excess inorganic phosphate, ADP, ATP, or magnesium ion, respectively, suggests that the essential phenol and amino groups are located nearer to the bound inorganic phosphate or the gamma-phosphate group than to the alpha- or beta-phosphate group of the bound ATP, that the essential guanidinium group is located nearer to the alpha- or beta-phosphate group than to the gamma-phosphate group of the bound ATP or the bound inorganic phosphate, and that the essential carboxylate group is located slightly farther away but complexed with magnesium ion which it shares with the bound inorganic phosphate. A mechanism consistent with these topographical relationships is proposed for the catalytic hydrolysis and synthesis of ATP.

The level of c-myc transcript was examined in liver samples from seven hepatoma patients. Transcripts were detected in all the normal liver parts examined; in contrast, in two hepatoma parts, there was a dramatic reduction in c-myc transcripts. The restriction enzyme pattern of c-myc gene appeared the same among samples. The data suggest that c-myc gene expression might not be required for the maintenance of the tumor state in human liver carcinogenesis.

Two monoclonal antibodies, designated 4C4 and 4G1, were produced by immunization of BALB/c mice with a human esophageal carcinoma cell line, CE69T/VGH, followed by fusion of the spleen cells from an immunized mouse with myeloma cells NS-1. 4C4 showed strong binding activity to three human esophageal carcinoma cell lines and one human hepatoma cell line, but not to any other cell lines tested. 4G1 reacted with three human esophageal carcinoma cell lines and four other cell lines. By peroxidase-antiperoxidase staining, 4C4 and 4G1 detected antigens of the epithelial cells on 10 pairs of esophageal carcinoma and normal esophageal specimens. 4G1 recognized a CE69T/VGH antigen with a molecular weight of 180K. Since 4G1 also reacted with purified carcinoembryonic antigen (CEA) and immunoprecipitated 125I-CEA, 4G1 seems to be an antibody recognizing CEA produced by CE69T/VGH cells. Since 4C4 also bound to the epithelial cells of normal uterine, vaginal, breast and liver tissues, it seems to recognize an epithelial antigen, and can be used to characterize the antigen in the specialization or differentiation of epithelial cells.

The promoter sequence (from position -500 to -21) of the yeast actin gene was subcloned into the multiple cloning site of pUC12 to generate a new recombinant plasmid pYAP12. The actin gene promoter can therefore be readily excised from pYAP12 by several restriction enzymes and subsequently placed upstream of the desired protein coding sequence. Results of expression of the hepatitis B surface antigen controlled by the actin gene promoter of pYAP12 in yeast suggest it is a strong functional promoter. To our knowledge, this is the first demonstration of the potential application of the yeast actin gene promoter for expression of foreign proteins in yeast.

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.

Previous reports indicate that human hepatocytes do not express class I and class II MHC antigens. Our analyses on 10 human hepatocellular carcinoma (HCC) cell lines by immunofluorescence tests and RIA, demonstrate that all the human HCC cell lines tested express class I MHC antigens and among them, three poorly differentiated human HCC cell lines also express class II MHC antigens. Results of immunoprecipitation and/or Western blotting experiments indicate similarity in the chemical nature of both the class I and class II MHC antigens expressed by the human HCC cell lines and by a human B lymphoblastoid cell line Raji. Furthermore, a new variant form of class I antigen was detected in some of these HCC cell lines. Immunohistochemical studies of HCC tissues using the peroxidase-antiperoxidase staining method indicated that class I and class II antigens were detectable in 7 out of 11 and 3 out of 11 HCC tissues from patients, respectively. The availability of MHC class I antigen-positive cultured HCC cell lines, including the poorly differentiated lines that also express MHC class II antigen, has provided us with interesting models to study the relationship between expression of MHC antigen and transformation and differentiation of human hepatocytes. These studies will also allow us some insight into the role of MHC class I and class II antigen in the immunosensitivity and immunogenicity of HCC cells to the host-immune response.

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.

By transfections of hepatitis B virus (HBV) DNA into five human hepatoma cell lines with the characteristics of differentiated human hepatocytes, three human hepatoma cell lines possessing partial hepatocyte-associated markers, and one non-liver cell line, we demonstrated that the expression of hepatitis B surface and core genes preferentially occurred in hepatoma cell lines with differentiated hepatocyte-associated characteristics. With a heterologous CAT gene as a reporter, the transcriptional activity of the promoter region containing both the distal (SPI) and the proximal (SPII) promoters of hepatitis B surface gene was found to show a preference for differentiated hepatoma cell lines. The SPI promoter which produces a RNA transcript for the synthesis of the large surface protein shows a strong preference, at least 750-fold, for differentiated hepatoma cells, while the SPII promoter which produces RNA transcripts for the synthesis of the middle and major surface proteins shows a moderate preference, about 20- to 59-fold. Further study indicates that this 750-fold preference of the SPI transcriptional activity for differentiated hepatoma cell lines can be attributed to the regulatory sequences of both the SPI and the HBV enhancer regions. These results also imply the important role of the large surface protein of HBV on the hepatocyte-specific infectivity of this virus.

The outer envelope of the 42-nm virion of the human hepatitis B virus (HBV) is composed of the large, the middle, and the major surface proteins. Whereas the middle and the major surface proteins are transcribed from the SPII promoter of the pre-S/S gene, the large surface protein is transcribed from the SPI promoter located upstream of SPII. We have previously shown that transcription of SPI (comprising nucleotides [nt] -380 to +17) occurs preferentially in differentiated hepatoma cell lines (H.K. Chang and L.P. Ting, Virology 170:176-183, 1989). In this report, we further demonstrated that a sequence of 95 base pairs in the upstream region of SPI (nt -95 to +17) was necessary and sufficient for such preferential expression in differentiated hepatoma cells. By analysis of the expression of the chloramphenicol acetyltransferase gene in a series of mutants with deletions at the 5' end of SPI, we identified a positive transcriptional cis-acting element mapping at nt -95 to -72 which appears to play a key role in the regulation of the expression of the large surface protein. This region shared a high degree of sequence homology with regulatory sequences of several liver-specific genes from human, mouse, and rat, with a consensus sequence (G/A)GTTA(A/C)TNNT(C/T)NNC(A/C). We further identified a nuclear factor present in the nuclear extracts of differentiated human hepatoma cell lines which interacted specifically with this element of the SPI promoter. This nuclear factor was similar to the rat liver-specific factor HNF-1, since an oligonucleotide containing the recognition sequence of HNF-1 could efficiently compete for the human factor in a footprinting assay. The sequence at nt -93 to -68 which was bound by this factor in SPI was termed the HNF-1-binding element. Activation of the SPI promoter by human differentiated hepatocyte nuclear factor 1, described in this report, probably explains, first, the formation of the 42-nm virion specifically in liver but not in several other tissues despite the synthesis of the middle and the major surface proteins in those tissues, and second, why only differentiated hepatoma cell lines are able to produce 42-nm-like virion particles on transfection by HBV DNA.

Three enzyme makers, glucose-6-phosphatase, ATPase and gamma-glutamyl transpeptidase, have been used in studying carcinogenesis of hepatocellular carcinoma. They have been investigated in animal models and human hepatocellular carcinoma in vivo and in vitro. But the inconsistent levels of these three enzymes associated with this type of carcinoma raised the possibility that the carcinoma cells might have derived from the cells originating from different stages of differentiation. To evaluate this possibility, three human cell lines, Hep G2, Hep 3B, and HA 22T, all thought to be arrested in different stages of differentiation based on their biochemical and morphological characteristics, were used as models. The three enzyme markers glucose-6-phosphatase, ATPase and gamma-glutamyl transpeptidase were examined cytochemically and biochemically. Our results showed that there was no correlation between the ATPase levels and the stages of the cell line's differentiation. But both glucose-6-phosphatase and gamma-glutamyl-transpeptidase were higher in cells that were more differentiated.

Paired liver biopsy specimens and serum samples from 76 patients with chronic hepatitis B virus (HBV) infection were taken for staining of hepatitis B core antigen (HBcAg) by immunoperoxidase and testing of HBV-DNA by a spot hybridization technique, respectively. Thirty-two tissue specimens showed positive staining for HBcAg in their hepatocytes. The two patients with diffuse HBcAg expression in liver tissue also had high serum concentrations of HBV-DNA (greater than 10 pg/10 microL). Among 30 patients with focal HBcAg distribution, 28 patients (93.3%) had measurable levels of serum HBV-DNA and 17 patients (60.7%) had high levels of serum HBV-DNA. Of 44 patients without hepatic HBcAg expression, only 12 patients (27.3%) had detectable serum HBV-DNA, and most patients (93.1% [11/12]) had low concentrations (less than 10 pg/10 microL). Nineteen patients had superimposed hepatitis D virus infection, and, of these, three patients (15.8%) had detectable serum HBV-DNA in low concentrations, while one of the three patients had stainable HBcAg in his hepatocytes with focal distribution. Two of the three patients with hepatitis A virus superinfection who had focal HBcAg expression in their liver tissue had serum HBV-DNA levels that were high during the acute phase of hepatitis A virus infection, and in one patient his serum HBV-DNA levels further increased from 10 pg/10 microL to 40 pg/10 microL during the recovery phase. Thus, measurement of serum HBV-DNA levels in patients with chronic HBV infection correlated well with their hepatic HBcAg expression, and both represent the precise status of HBV replication.

Human hepatoma cell lines were studied for the expression of platelet-derived growth factor (PDGF), insulin-like growth factor-I (IGF-I) and their receptors at the mRNA level. Transcripts of PDGF were consistently detected in these cell lines. In addition, some cell lines also expressed PDGF receptor RNA. Moreover, RNA of IGF-I and its receptor were detected in every cell line examined. These results suggest that autocrine regulation may be an important mechanism for the maintenance of the transformed state of human hepatoma cells.

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.

The rates of inactivation of BF1 adenosinetriphosphatase (BF1-ATPase) from Escherichia coli by 7-chloro-4-nitro-2,1,3-benzoxadiazole, 1-fluoro-2,4-dinitrobenzene, 2,4,6-trinitrobenzenesulfonate, phenylglyoxal, and N,N'-dicyclohexylcarbodiimide have been measured in the presence and absence of various concentrations of inorganic phosphate, ADP, ATP, or magnesium ion. Dissociation equilibrium constants and rate constants for the labeling reactions have been deduced from a quantitative treatment of the kinetic data. The results suggest that the essential Tyr, Lys, Arg, and Glu or Asp residues are probably located at the catalytic site of BF1-ATPase and that in addition to steric interference, the effect of charge interaction should also be considered in interpreting the kinetic data on the protection of BF1-ATPase by substrate molecules against inactivation by the above labeling reagents. Examination of the relative values of the rate constants for the labeling reactions in the presence and absence of inorganic phosphate, ADP, ATP, or magnesium ion, respectively, and the effect of NBD label on the rates of labeling of the essential guanidinium, amino, and carboxyl groups suggest that the arrangement of these four functional groups at the catalytic site of BF1 may be similar to that previously proposed for MF1-ATPase from bovine heart; namely, the essential amino group and the unusually reactive phenol group are probably located near the bound inorganic phosphate or the gamma-phosphate group of the bound ATP, the essential guanidinium group is probably located nearer to the alpha- or beta-phosphate group than to the gamma-phosphate group of the bound ATP or the bound inorganic phosphate, and the essential carboxylate group is probably complexed with a magnesium ion which it shares with the bound inorganic phosphate.

The effects of somatostatin (SRIF), insulin, and triiodothyronine (T3) on the growth of human hepatoma cells were investigated on the well-differentiated human hepatoma cell line Hep3B. Results showed that both insulin and T3 can stimulate cell growth of serum starved Hep3B cells at physiological concentrations. SRIF alone showed little growth-promoting activity. When added concurrently with insulin, however, SRIF suppressed the insulin-induced cell proliferation in a dose-dependent manner. On the other hand, SRIF had no inhibitory effect on T3-induced cell proliferation. SRIF is labile in the medium, with a half-life of about 2 h during culture incubation. SRIF did not disturb the insulin binding to its surface receptors nor inhibit the insulin-dependent receptor kinase activity of Hep3B cells in vitro. These results suggest that postreceptor regulation may be involved. The selective suppression by SRIF of insulin-induced cell growth provides an unique approach to the study of insulin actions on proliferation of human hepatoma cells.