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J R Wands

Publications and source records attributed to J R Wands.

At least 109 records · Page 6Linked to original sources
PubMed1996

Recent advances in the molecular biology of hepatitis B virus.

Hepatitis B virus (HBV) is an enveloped hepatotropic DNA virus. Acute and chronic HBV infection causes significant liver diseases such as acute hepatis, fulminant hepatitis and chronic active hepatitis that may lead to liver cirrhosis and the development of hepatocellular carcinoma. The use of molecular biological techniques has substantially improved our understanding of the HBV life cycle. In this review, we discuss recent advances that have contributed to a better understanding of HBV biology. Recent studies in the understanding of the life cycle of HBV such as viral entry, replication, transcriptional regulation, viral regulatory proteins, viral assembly and secretion, and nucleic acid based approaches to antiviral therapy will be emphasized. These advances in molecular biology and relationship to clinical disease will be instrumental in developing effective therapeutic approaches for the estimated 300 million individuals worldwide chronically infected with HBV.

Animals↗
PubMed1996

Characterization of a 120-Kilodalton pre-S-binding protein as a candidate duck hepatitis B virus receptor.

Infection by human and animal hepadnaviruses displays remarkable host and tissue tropism. The infection cycle probably initiates with binding of the pre-S domain of viral envelope protein to surface receptors present on the hepatocyte. Three types of neutralizing monoclonal antibodies against duck hepatitis B virus (DHBV) have their binding sites clustered within residues 83 to 107 of the pre-S protein, suggesting that this region may constitute a major receptor binding site. A 170- or 180-kDa duck protein (p170 or gp180) which binds DHBV particles through this part of the pre-S sequence has been identified recently. Although the p170 binding protein is host (duck) specific, its distribution is not restricted to DHBV-infectible tissues. Using the pre-S protein fused to glutathione S-transferase and immobilized on Sepharose beads, we have now identified an additional binding protein with a size of 120 kDa (p120). p120 expression is restricted to the liver, kidney, and pancreas, the three major organs of DHBV replication. While optimal p170 binding requires an intact pre-S protein, binding to p120 occurs much more efficiently with a few N- or C-terminally truncated forms. The p120 binding site was mapped to residues 98 to 102 of the pre-S region, which overlaps with a cluster of known virus-neutralizing epitopes. Site-directed mutagenesis revealed residues 100 to 102 (Phe-Arg-Arg) as the critical p120 contact site; nonconservative substitution in any of the three positions abolished p120 binding. Double mutations at positions 100 to 102 markedly reduced DHBV infectivity in cell culture. Short pre-S peptides covering the clustered neutralizing epitopes (also p170 and p120 binding sites) reduced DHBV infectivity in primary duck hepatocyte cultures. Thus, p120 represents a candidate component of the DHBV receptor complex.

Amino Acid Sequence↗
PubMed1996

Overexpression of human insulin receptor substrate 1 induces cellular transformation with activation of mitogen-activated protein kinases.

The receptor insulin substrate 1 protein (IRS-1) is a specific substrate for insulin receptor tyrosine kinase. Expression and tyrosyl phosphorylation of IRS-1 play an important role during normal hepatocyte growth, and the gene is overexpressed in hepatocellular carcinoma tissue. We determined if IRS-1 overexpression directly contributes to cellular transformation. The human IRS-1 gene was subcloned into a mammalian expression vector driven by the cytomegalovirus early promoter. NIH 3T3 cells transiently transfected with this vector subsequently developed transformed foci. Several stably transfected cell lines were established, and they grew efficiently under low-serum conditions and formed colonies when plated in soft agar. Cell lines overexpressing IRS-1 displayed increased tyrosyl phosphorylation of IRS-1 and association with Grb2 but not with the p85 subunit of phosphatidylinositol 3' kinase. Since Grb2 is a component of the son-of-sevenless-Ras pathway and upstream in the mitogen-activated protein kinase (MAPK) cascade, enzymatic activities of the major components of this cascade, such as MAPK kinase and MAPK were evaluated and found to be substantially increased in three independent cell lines with IRS-1 protein overexpression. Such cells, when injected into nude mice, were highly tumorigenic, and there may be a correlation between the degree of MAPK activation and tumor growth rate. This report describes the generation of a transformed phenotype by overexpression of a molecule without a catalytic domain far upstream in the signal transduction cascade and suggests that prolonged activation of MAPKs by this mechanism may be one of the molecular events related to hepatocellular transformation.

3T3 Cells↗
PubMed1996

Profiles of neuronal thread protein expression in Alzheimer's disease.

Neuronal thread proteins (NTPs) comprise a family of molecules expressed in brain and primitive neuroectodermal tumor cell lines. In Alzheimer's disease (AD), increased CNS levels of the 21 kD NTP species are correlated with dementia. The present study characterizes the nature and distribution of NTP expression using recently generated brain-derived polyclonal and monoclonal antibodies (MoAbs) to recombinant AD7c-NTP protein. In AD, high levels of NTP immunoreactivity were detected in neuronal perikarya, neuropil fibers, and white matter fibers (axons). In addition, 4 of the 23 AD7c-NTP MoAbs labeled degenerating neurons (with or without neurofibrillary tangles), axonal spheroids, dystrophic neurites, or irregular, wavy threadlike neuropil fibers in AD. Increased neuronal AD7c-NTP immunoreactivity in AD colocalized with perikaryal accumulations of tau-1, phosphorylated neurofilament, and the ganglioside, A2B5. In addition, AD7c-NTP immunoreactivity was detected in early neuritic plaques along with beta-amyloid-containing fibrils, but not in mature plaques, nor was it colocalized in beta A4-immunoreactive fibrils. This study demonstrates the profiles of NTP overexpression in relation to paired helical filament-associated neurodegenerative lesions in AD.

Aged↗
PubMed1995

Ethanol inhibits insulin receptor substrate-1 tyrosine phosphorylation and insulin-stimulated neuronal thread protein gene expression.

Neuronal thread proteins (NTPs) are molecules that accumulate in the brains of patients with Alzheimer's disease, and may play a key role in both normal and neurodegenerative neuritic sprouting. In this investigation we determined whether NTP expression is up-regulated by insulin, an important neurotrophic factor that stimulates differentiation-associated neurite outgrowth, and studied the effects of ethanol, a known inhibitor of growth factor receptor tyrosine phosphorylation, on NTP expression and insulin-mediated signal transduction cascade in neuronal [primitive neuroectodermal tumour cell line 2; (PNET2)] cells. PNET2 cells were treated with 50 m-units/ml insulin in the presence or absence of 100 mM ethanol for 0.2-96 h, and cell proliferation and expression of NTP molecules were investigated by metabolic labelling, immunoprecipitation and immunohistochemical staining. Insulin stimulation resulted in an immediate increase in the levels of three (38, 18 and 15 kDa) of five NTP species (the others were of 26 and 21 kDa), followed by a decline in expression within 120 min; however, studies performed up to 96 h of culture demonstrated up-regulation by insulin of all five NTP species. Ethanol either abolished or severely muted the short- and long-term insulin-mediated upregulation of NTP expression, and substantially reduced insulin-mediated neuronal differentiation. The effects of ethanol on NTP gene expression were associated with impaired insulin-mediated tyrosine phosphorylation of both the insulin receptor beta subunit and the insulin receptor substrate-1 (IRS-1), resulting in decreased association of phosphatidylinositol 3-kinase with IRS-1. The findings suggest that ethanol may inhibit NTP expression associated with central nervous system neuronal differentiation by uncoupling the IRS-1-mediated insulin signal transduction pathway.

Calcium-Binding Proteins↗
PubMed1995

Insulin-induced differentiation and modulation of neuronal thread protein expression in primitive neuroectodermal tumor cells is linked to phosphorylation of insulin receptor substrate-1.

Neuronal thread proteins (NTPs) are a family of developmentally regulated molecules expressed in central nervous system (CNS) neurons and primitive neuroectodermal tumor (PNET) cell lines. NTP gene expression is modulated with DNA synthesis, neuritic sprouting, and neuronal differentiation. The present study explores the mechanism of insulin modulation of NTP gene expression during neuronal differentiation using PNET cell lines of CNS origin. PNET2 cells underwent neuronal differentiation with neurite outgrowth coupled with transient up-regulation of several species of NTP. In contrast, PNET1 cells failed to differentiate in response to insulin stimulation, although insulin receptors were more abundant than in PNET2 cells. Analysis of the insulin-mediated signal transduction pathway demonstrated that the lack of insulin responsiveness in PNET1 cells was primarily caused by impaired insulin-mediated tyrosyl phosphorylation of the insulin receptor substrate-1 (IRS-1). Correspondingly, the association between phosphatidyl-inositol 3 (PI3) kinase and phosphorylated IRS-1 was reduced in PNET1 compared with PNET2 cells. In contrast, the levels of IRS-1 protein were similar in PNET1 and PNET2 cells, and expression of the insulin receptor beta subunit (Ir beta) and insulin-mediated tyrosyl phosphorylation of the Ir beta were greater in PNET1 than PNET2 cells. The findings suggest that insulin effected neuronal differentiation and modulation of NTP gene expression in PNET cells utilizes a signal transduction cascade that requires tyrosyl phosphorylation of IRS-1.

Blotting, Western↗
PubMed1995

Inhibition of hepatic metastases of human colon cancer in nude mice by a chimeric SF-25 monoclonal antibody.

BACKGROUND/AIMS: Hepatic metastasis is one of the most serious complications of human colon cancer. A murine-human chimeric SF-25 monoclonal antibody was prepared, and this construct recognizes a cell surface antigen highly present in human colon adenocarcinoma. METHODS: This study determined if the chimeric SF-25 monoclonal antibody inhibits the outgrowth of hepatic metastases of human colon adenocarcinoma using an athymic nude mouse model. RESULTS: A single intravenous injection of chimeric SF-25 monoclonal antibody significantly inhibited the outgrowth of 5- and 7-day hepatic micrometastases (P = 0.0001 and 0.004, respectively, vs. untreated) and improved the survival of the animals. No detectable tumor was found in the liver when mice were treated by multiple injections of the antibody immediately after tumor cell grafting into the portal vein. In contrast, F(ab')2 fragments did not show antitumor effects, and the administration of natural killer cell or macrophage depleting agents (anti-asialo GM1 antibody and carrageenan, respectively) substantially inhibited the antitumor effects of chimeric SF-25 monoclonal antibody in vivo. CONCLUSIONS: Chimeric SF-25 monoclonal antibody inhibits growth of hepatic metastasis of human colon cancer, and cell-mediated host immune mechanisms seem to be important for its in vivo antitumor activity.

Adenocarcinoma↗
PubMed1995

Modulation of p36 gene expression in human neuronal cells.

p36 is a calcium/lipid-binding phosphoprotein that is expressed at high levels in proliferating and transformed cells, and at low levels in terminally differentiated cells, such as CNS neurons. The calcium-dependent binding to membrane phospholipids, and its capacity to interact with intermediate filament proteins suggest that p36 may be involved in the transduction of extracellular signals. The present work examines p36 gene expression in the mature CNS, primary primitive neuroectodermal tumors (PNETs), and transformed PNET cell lines. p36 immunoreactivity was not observed in normal adult human brain, but low levels of the protein were detected by Western blot analysis. Following acute anoxic cerebral injury, the mean levels of p36 protein were elevated two-fold, and injured neurons exhibited increased p36 immunoreactivity. This phenomenon was likely to have been mediated by post-transcriptional mechanisms since there was no corresponding change in the level p36 mRNA. p36 immunoreactivity was detected in 8 of 9 primary PNETs, and in 3 of 3 neurofilament-expressing PNET cell lines. The levels of p36 protein in PNET cell lines were 5-fold higher than in adult human brain tissue. Although p36 gene expression was generally high in proliferating PNET cells, the levels of p36 mRNA and protein were not strictly correlated with DNA synthesis. Instead, p36 gene expression was modulated in both proliferating and non-proliferating PNET cell cultures by treatment with 50 mIU/ml of insulin, 100 mM ethanol, or 5 microM retinoic acid. The frequent discordances observed experimentally and in vivo between p36 mRNA and p36 protein expression suggest that the steady-state levels of p36 protein in neuronal cells may be regulated primarily by post-transcriptional mechanisms.

Adult↗
PubMed1995

Development of a two-site immuno-PCR assay for hepatitis B surface antigen.

Hepatitis B virus (HBV) gene transcription may occur at very low levels resulting in HBsAg concentrations in serum and liver below the limit of detection by currently available immunoassays. An assay has been developed that combines the specificity of two high affinity anti-HBs monoclonal antibodies (MAb) directed against distinct and separate determinants in the 'a' domain of HBsAg with the highly sensitive polymerase chain reaction (PCR) detection method. Following capture of HBsAg present in serum samples, the second anti-HBs MAb, which is biotinylated, is added. Binding of the second antibody allows the subsequent specific binding of streptavidin and a biotinylated linear DNA molecule derived from a bluetongue virus (BTV) gene. Presence of this DNA is then detected by PCR using BTV-specific primers. The PCR product is quantified by a liquid-phase oligonucleotide enzymatic assay, which further increases the sensitivity of the technique. The use of a two-site MAb capture and PCR detection system for HBsAg was shown to greatly enhance the specificity and sensitivity of the assay and detect as little as 0.5 pg of HBsAg in serum samples. It is suggested that the principles of this technique could be applied to measure other low level viral antigens in serum and biological samples.

Antibodies, Monoclonal↗
PubMed1995

Interferon gamma production by peripheral blood lymphocytes to hepatitis C virus core protein in chronic hepatitis C infection.

Evidence suggests that cellular immunity to hepatitis C virus (HCV) core protein may be important in the pathogenesis of viral infection. Therefore, interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) derived from patients with chronic HCV infection (genotype 1b) was examined. The cellular immune response was evaluated with a recombinant HCV core fusion protein derived from a patient with genotype 1b. To identify the immunodominant epitopes, IFN-gamma production in responders was also assessed with a panel of nine synthetic peptides that covered the entire core region. It was found that mononuclear cells from 24 (52%) of 46 patients with chronic liver disease responded to the core protein; asymptomatic HCV carriers demonstrated a lower response rate (14%, P < .05). More important, individuals who had received IFN-alpha treatment and went into clinical and virological remission had a higher response rate (75%, P < .05) compared with those with ongoing hepatitis whose treatment failed (31%). Of 25 patients whose mononuclear cells responded to HCV core protein, 18 had a significant response to one or more peptides; 12 patients reacted to a peptide mixture containing hydrophilic sequences. The core peptide amino acid sequence 141 to 160 was recognized in 9 patients. Interestingly, 7 of 8 patients bearing HLA DR 4 and w53 haplotypes recognized the peptide sequence 141 to 160. Thus, IFN-gamma production of the mononuclear cell response appeared to be HLA DR restricted, and the responding cells were identified as CD4+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Effect of ethanol on p36 protein kinase substrate and insulin receptor substrate 1 expression and tyrosyl phosphorylation in human hepatocellular carcinoma cells.

Ethanol inhibits insulin (IN) and epidermal growth factor (EGF)-induced hepatocyte DNA synthesis. Growth factor receptor kinases, such as IN and EGF, phosphorylate insulin receptor substrate (IRS-1) and p36 protein kinase substrate, respectively, on tyrosine residues. IRS-1 and p36 are thought to be important intracellular signal transduction molecules involved in the regulation of cell growth. These investigations explored the effect of ethanol additions on the expression and tyrosyl phosphorylation (TP) of p36 and IRS-1 in a human hepatocellular carcinoma cell line (FOCUS) in relationship to cell proliferation induced by IN and serum growth factor stimulation. It was found that p36 was constitutively and highly expressed in serum-starved cells and protein, and mRNA levels did not change with cell proliferation induced by growth factors. However, exposure of FOCUS cells to ethanol additions substantially inhibited TP of p36. The early TP of IRS-1 induced by IN stimulation was also reduced by ethanol additions. Finally, there was a parallel decrease of FOCUS cell proliferation in ethanol-exposed cultures. These studies suggest that one possible mechanism of ethanol inhibitory effect on cell proliferation is through reduced TP of putative intracellular signal transduction molecules, such as p36 and IRS-1.

Annexin A2↗
PubMed1995

Interaction between duck hepatitis B virus and a 170-kilodalton cellular protein is mediated through a neutralizing epitope of the pre-S region and occurs during viral infection.

Identification of cell surface viral binding proteins is important for understanding viral attachment and internalization. We have fused the pre-S domain of the duck hepatitis B virus (DHBV) large envelope protein to glutathione S-transferase and demonstrated a 170-kDa binding protein (p170) in [35S]methionine-labeled duck hepatocyte lysates. This glycoprotein was found abundantly in all extrahepatic tissues infectible with DHBV and in some noninfectible tissues, though it is not secreted into the blood. The interaction of pre-S fusion protein with p170 was competitively inhibited by wild-type DHBV in a dose-dependent manner. In addition, infection of hepatocytes with DHBV blocked the binding of pre-S fusion protein to p170, which suggests a biological role for p170 during natural infection. The p170 binding site was mapped to a conserved sequence of 16 amino acid residues (positions 87 to 102) by using 24 pre-S deletion mutants; this binding domain coincides with a major virus-neutralizing antibody epitope. Furthermore, site-directed mutagenesis revealed that an arginine residue at position 97 is critical for p170 binding. p170 was purified by a combination of ion-exchange and affinity chromatographies, and four peptide sequences were obtained. Two peptides showed significant similarities to human and animal carboxypeptides H, M, and N. Taken together, these results raise the possibility that the p170 binding protein is important during the replication cycle of DHBV.

Amino Acid Sequence↗
PubMed1995

Specific targeting of adriamycin conjugates with monoclonal antibodies to hepatoma associated antigens to intrahepatic tumors in athymic mice.

Doxorubicin (adriamycin), once considered the treatment of choice for hepatocellular carcinoma (HCC), is known to cause cardiotoxicity and myelotoxicity. To reduce the systemic toxicity of adriamycin by direct delivery of the drug to the tumor site, we established a panel of monoclonal antibodies (MAbs) to hepatoma associated antigens that were conjugated to adriamycin by a dextran bridge. Initially, the efficacy of these conjugates in suppressing tumor growth was assessed using a model of subcutaneous HCC tumors injected in athymic mice. In the second stage of the study, we tested these conjugates in an experimental model in which human HCC was transplanted intrahepatically by intrasplenic injection, thus providing the tumor cells with growth factors and an adequate cellular matrix, similar to the natural microenvironment of HCC. Anti-tumoral therapy resulted in lower serum alpha-fetoprotein (AFP) levels in two of three experimental groups treated with different specific conjugates as compared with control mice treated with the individual components. Efficacy of targeting was enhanced using the intrahepatic model system for propagation of HCC and was demonstrated by fluorescence of adriamycin and MAb in tumor tissue and absence of this fluorescence in healthy liver tissue surrounding the tumor. Reduction of systemic toxicity was shown by the absence of adriamycin fluorescence in myocardial tissue in conjugate-treated mice, whereas in all other treatment groups, including mice treated with a mixture of adriamycin and specific MAb, there was strong myocardial fluorescence of adriamycin.

Animals↗
PubMed1995

Specific targeting of human hepatocellular carcinoma cells by immunoliposomes in vitro.

The monoclonal antibody AF-20 was raised against the human hepatocellular carcinoma (HCC) cell line FOCUS and binds with high affinity to a rapidly internalized 180-kd homodimeric glycoprotein that is abundantly expressed on the surface of human HCC and other human cancer cell lines. Immunoliposomes were produced by covalently coupling AF-20 to liposomes containing carboxyfluorescein. Interaction of immunoliposomes with various HCC cell lines in vitro was quantitatively assessed by flow cytometry and qualitatively analyzed by fluorescence microscopy. Liposomes bearing an isotype-matched nonrelevant monoclonal antibody (MAb) and cell lines not expressing AF-20 antigen served as controls. AF-20-immunoliposomes specifically bound to HCC and other human cancer cell lines expressing the AF-20 antigen and were rapidly internalized at 37 degrees C. Interaction of AF-20-conjugated liposomes with these cell lines was between 5 and 200 times greater than that of unconjugated liposomes, whereas no difference was observed between control liposomes bearing a nonrelevant antibody and unconjugated liposomes. Specificity of liposome-target cell interaction was confirmed by competitive inhibition assays. Kinetic analysis showed rapid association of AF-20 immunoliposomes with target cells, with saturation conditions being reached after 60 minutes. We conclude that the MAb AF-20 directs highly efficient, specific, and rapid targeting of immunoliposomes to human HCC and other human cancer cell lines in vitro. This targeted liposomal delivery system represents a promising approach for the development of immunotargeted diagnosis and therapy strategies against HCC.

Antibodies, Monoclonal↗
PubMed1994

Characterization of hepatitis B virus core mutants that inhibit viral replication.

We have generated and functionally characterized dominant negative core protein variants of the hepadnaviruses to determine their effects on "wild type" viral replication. Plasmids expressing these constructs were introduced into hepatoma cell lines by transient transfection and effects on wild type woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) replication were evaluated by Southern blot analysis of purified viral core particles. WHV and HBV constructs expressing a truncated core protein fused in frame with the C-terminus of the small surface protein were found to inhibit viral replication by 90-95% due to disruption of the viral nucleocapsid assembly process and preventing encapsidation of pregenomic RNA. The antiviral effects were found to be specific for the targeted virus. These results demonstrate that mutants of hepadnaviral core protein may represent a novel class of antiviral agents.

DNA Replication↗
PubMed1994

Specific inhibition of hepatitis C virus expression by antisense oligodeoxynucleotides. In vitro model for selection of target sequence.

The effect of sense and antisense oligodeoxynucleotides (ODNs) on hepatitis C virus (HCV) gene expression was studied to determine the role of the highly conserved 5'-untranslated region in the life cycle of the virus. It was found that antisense ODNs complementary to nucleotides (nt) 38-65, 134-175, and 312-339 in the 5' noncoding region and 341-377 in the core open reading frame efficiently blocked HCV RNA translation. Overlapping ODNs that differed by only several nucleotides showed substantially different inhibition of HCV RNA translation. Fine sequence specificity testing at nt positions 351-377 revealed that ODNs as small as a 12-mer (nt 351-363) retained a high degree (80%) of inhibitory activity compared to ODNs of longer sequences. These results suggest that there are three highly specific domains in the 5' noncoding region and a sequence immediately downstream of the HCV core initiation codon that may be critical for translation of HCV RNA. This study also provides an experimental approach for the selection of target HCV RNA sequences susceptible to antisense effects, as well as for definition of functional regions of the genome necessary for viral replication.

Base Sequence↗