Hepcidin, a 25‐amino acid peptide encoded by the HAMP gene and produced mainly by hepatocytes and... more Hepcidin, a 25‐amino acid peptide encoded by the HAMP gene and produced mainly by hepatocytes and macrophages, is a mediator of innate immunity and the central iron‐regulatory hormone. Circulating hepcidin controls iron efflux by inducing degradation of the cellular iron exporter ferroportin. HCV infection is associated with hepatic iron overload and elevated serum iron, which correlate with poor antiviral responses. The HCV nonstructural NS5A protein is known to function in multiple aspects of the HCV life cycle, probably exerting its activity in concert with cellular factor(s). In this study, we attempted to delineate the effect of HCV NS5A on HAMP gene expression. We observed that transient transfection of hepatoma cell lines with HCV NS5A resulted in down‐regulation of HAMP promoter activity. A similar effect was evident after transduction of Huh7 cells with a recombinant baculovirus vector expressing NS5A protein. We proceeded to construct an NS5A‐expressing stable cell line, which also exhibited down‐regulation of HAMP gene promoter activity and significant reduction of HAMP mRNA and hepcidin protein levels. Concurrent expression of HCV core protein, a well‐characterized hepcidin inducer, revealed antagonism between those two proteins for hepcidin regulation. In attempting to identify the pathways involved in NS5A‐driven reduction of hepcidin levels, we ruled out any NS5A‐induced alterations in the expression of the well‐known hepcidin inducers SMAD4 and STAT3. Further analysis linked the abundance of intracellular zinc ions and the deregulation of the MTF‐1/MRE/hepcidin axis with the observed phenomenon. This effect could be associated with distinct phases in HCV life cycle.
Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically con... more Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically consists of the administration of expression vectors coding viral antigen sequences into the host's cells. Targeting of conserved viral epitopes by antibody fragments specific to activating cell surface co-receptor molecules on antigen-presenting cells could be an alternative approach for inducing protective immunity. It has been shown that FcγRI on human monocytes enhances antigen presentation in vivo. Various DNA constructs, encoding a Single-chain variable antibodies (scFv) from mouse anti-human FcγRI monoclonal antibody, coupled to a sequence encoding a T- and B-cell epitope-containing influenza A virus hemagglutinin inter-subunit peptide were inserted into the eukaryotic expression vector system pTriEx-3 Neo. The constructed chimeric DNA molecules were expressed by transfected Chinese hamster ovary cells and the ability of the engineered proteins to interact with FcγRI-expressing c...
Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to a... more Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to an extracellular ligand-binding domain of a single-chain antibody (scFv) have served as effective tools for redirecting cytotoxic T lymphocytes (CTL) against tumour cells. In this report, we constructed a chimeric scFv/zeta gene composed of the variable regions of an HER-2/neu-specific monoclonal antibody (MAb) joined to the TCR-zeta chain. The scFv(anti-HER-2/neu)/zeta chimeric gene was successfully expressed as a functional surface receptor in the MD.45 CTL hybridoma (MD.45-HER/zeta). More importantly, the scFv(anti-HER-2/neu)/zeta receptor was functionally active, since it triggered cytokine secretion by the MD.45-HER/zeta cells upon recognition of HER-2/neu-positive (+) tumour cell lines, or primary tumour cells from patients with HER-2/neu(+) cancers. The MD.45-HER/zeta-transduced cells also lysed HER-2/neu(+) target cells in vitro with high specificity. We tested the antitumour effi...
Porcine BM88 is a neuron-specific protein that enhances neuroblastoma cell differentiation in vit... more Porcine BM88 is a neuron-specific protein that enhances neuroblastoma cell differentiation in vitro and may be involved in neuronal differentiation in vivo. Here we report the identification, by Western blotting, of homologous proteins in human and mouse brain and the isolation of their respective cDNAs. Several human and mouse clones were identified in the EST database using porcine BM88 cDNA as a query. A human and a mouse EST clone were chosen for sequencing and were found both to predict a protein of 149 amino acids, with 79.9% reciprocal identity, and 76.4% and 70.7% identities to the porcine protein, respectively. This indicated that the clones corresponded to the human and mouse BM88 homologues. In vitro expression in a cell-free system as well as transient expression in COS7 cells yielded polypeptide products that were recognized by anti-BM88 antibodies and were identical in size to the native BM88 protein. Northern-blot analysis showed a wide distribution of the gene in hum...
Previous studies have shown that the BM88 antigen, a novel neuron-specific molecule, promotes the... more Previous studies have shown that the BM88 antigen, a novel neuron-specific molecule, promotes the differentiation of mouse neuroblastoma (Neuro 2a) cells. In particular, stably transfected, with the BM88 cDNA, Neuro 2a cells overexpressing the BM88 antigen (Neuro2a-BM88 cells) are morphologically distinct from the nontransfected Neuro 2a cells; they exhibit enhanced process outgrowth and a slower rate of division. In this study we used Neuro2a and the morphologically differentiated Neuro 2a-BM88 cells to compare their responsiveness to growth factors. The growth factors we used were nerve growth factor (NGF), basic-fibroblast growth factor (b-FGF), and glial cell-line derived neurotrophic factor (GDNF). In addition, we used glial conditioned medium derived from either newborn mouse cerebral cortex (NBCC) or aged mouse cerebral hemispheres (MACH), as a source of normal glial factors. Because these cells express the cholinergic phenotype, we used choline acetyltransferase (ChAT) activ...
Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the ce... more Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the cerebral cortex in Alzheimer's disease (AD), but to date it has not been demonstrated which nicotinic receptor subunits contribute to this deficit. In the present study, autopsy tissue from the temporal cortex of 14 AD cases and 15 age-matched control subjects was compared using immunoblotting with antibodies against recombinant peptides specific for alpha3, alpha4, and alpha7 subunits, in conjunction with [3H]epibatidine binding. Antibodies to alpha3, alpha4, and alpha7 produced one major band on western blots at 59, 51, and 57 kDa, respectively. [3H]Epibatidine binding and alpha4-like immunoreactivity (using antibodies against the extracellular domain and cytoplasmic loop of the alpha4 subunit) were reduced in AD cases compared with control subjects (p < 0.02) and with a subgroup of control subjects (n = 9) who did not smoke prior to death (p < 0.05) for the former two paramete...
The nicotinic acetylcholine receptor (AChR) family is a member of the ligand-gated ion-channel ge... more The nicotinic acetylcholine receptor (AChR) family is a member of the ligand-gated ion-channel gene superfamily, which includes the y-aminobutyric acid (GABA) and glycine receptors (Barnard et al., 1987; Stroud et al., 1990). It contains receptors from the post-synaptic membranes of vertebrate neuromuscular junctions, from the electric organs of electric fish (Torpedo and Electrophorus) and from the central nervous systems of vertebrates and insects. These receptors have several structural and functional features in common, but also have many differences (Lucas and Bencherif, 1992). The muscle-type AChR (i.e. that of the neuromuscular junction and fish electric organs) is the best-known member of the AChR family. It is an integral pentameric membrane protein (Popot and Changeux, 1984) formed from four types of subunits in the stoichiometry @yS or OL$ES (Reynolds and Karlin, 1978; Numa, 1987). Three major factors have facilitated its extensive characterisation: (a) the large quantities that can be isolated from the electric organs of fish, (b) the presence of several snake venom neurotoxins, that bind specifically to the AChR and (c) the application of recombinant DNA techniques. In the present review, we will discuss only this type of AChR. Structural and functional studies of the muscle AChR are of great importance as the molecule acts as the autoantigen in the autoimmune disease myasthenia gravis (MG) and in its experimental animal model (Oosterhuis, 1984; Lindstrom et al., 1988; Penn et al., 1992). MG is characterized by weakness and fatiguability of the skeletal muscles. The disease occurs in about 1 in 20,000 people. As the autoantigen is a well characterized molecule, MG represents an excellent model for the study of the molecular interactions involved in a human autoimmune disease.
Hepcidin, a 25‐amino acid peptide encoded by the HAMP gene and produced mainly by hepatocytes and... more Hepcidin, a 25‐amino acid peptide encoded by the HAMP gene and produced mainly by hepatocytes and macrophages, is a mediator of innate immunity and the central iron‐regulatory hormone. Circulating hepcidin controls iron efflux by inducing degradation of the cellular iron exporter ferroportin. HCV infection is associated with hepatic iron overload and elevated serum iron, which correlate with poor antiviral responses. The HCV nonstructural NS5A protein is known to function in multiple aspects of the HCV life cycle, probably exerting its activity in concert with cellular factor(s). In this study, we attempted to delineate the effect of HCV NS5A on HAMP gene expression. We observed that transient transfection of hepatoma cell lines with HCV NS5A resulted in down‐regulation of HAMP promoter activity. A similar effect was evident after transduction of Huh7 cells with a recombinant baculovirus vector expressing NS5A protein. We proceeded to construct an NS5A‐expressing stable cell line, which also exhibited down‐regulation of HAMP gene promoter activity and significant reduction of HAMP mRNA and hepcidin protein levels. Concurrent expression of HCV core protein, a well‐characterized hepcidin inducer, revealed antagonism between those two proteins for hepcidin regulation. In attempting to identify the pathways involved in NS5A‐driven reduction of hepcidin levels, we ruled out any NS5A‐induced alterations in the expression of the well‐known hepcidin inducers SMAD4 and STAT3. Further analysis linked the abundance of intracellular zinc ions and the deregulation of the MTF‐1/MRE/hepcidin axis with the observed phenomenon. This effect could be associated with distinct phases in HCV life cycle.
Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically con... more Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically consists of the administration of expression vectors coding viral antigen sequences into the host's cells. Targeting of conserved viral epitopes by antibody fragments specific to activating cell surface co-receptor molecules on antigen-presenting cells could be an alternative approach for inducing protective immunity. It has been shown that FcγRI on human monocytes enhances antigen presentation in vivo. Various DNA constructs, encoding a Single-chain variable antibodies (scFv) from mouse anti-human FcγRI monoclonal antibody, coupled to a sequence encoding a T- and B-cell epitope-containing influenza A virus hemagglutinin inter-subunit peptide were inserted into the eukaryotic expression vector system pTriEx-3 Neo. The constructed chimeric DNA molecules were expressed by transfected Chinese hamster ovary cells and the ability of the engineered proteins to interact with FcγRI-expressing c...
Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to a... more Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to an extracellular ligand-binding domain of a single-chain antibody (scFv) have served as effective tools for redirecting cytotoxic T lymphocytes (CTL) against tumour cells. In this report, we constructed a chimeric scFv/zeta gene composed of the variable regions of an HER-2/neu-specific monoclonal antibody (MAb) joined to the TCR-zeta chain. The scFv(anti-HER-2/neu)/zeta chimeric gene was successfully expressed as a functional surface receptor in the MD.45 CTL hybridoma (MD.45-HER/zeta). More importantly, the scFv(anti-HER-2/neu)/zeta receptor was functionally active, since it triggered cytokine secretion by the MD.45-HER/zeta cells upon recognition of HER-2/neu-positive (+) tumour cell lines, or primary tumour cells from patients with HER-2/neu(+) cancers. The MD.45-HER/zeta-transduced cells also lysed HER-2/neu(+) target cells in vitro with high specificity. We tested the antitumour effi...
Porcine BM88 is a neuron-specific protein that enhances neuroblastoma cell differentiation in vit... more Porcine BM88 is a neuron-specific protein that enhances neuroblastoma cell differentiation in vitro and may be involved in neuronal differentiation in vivo. Here we report the identification, by Western blotting, of homologous proteins in human and mouse brain and the isolation of their respective cDNAs. Several human and mouse clones were identified in the EST database using porcine BM88 cDNA as a query. A human and a mouse EST clone were chosen for sequencing and were found both to predict a protein of 149 amino acids, with 79.9% reciprocal identity, and 76.4% and 70.7% identities to the porcine protein, respectively. This indicated that the clones corresponded to the human and mouse BM88 homologues. In vitro expression in a cell-free system as well as transient expression in COS7 cells yielded polypeptide products that were recognized by anti-BM88 antibodies and were identical in size to the native BM88 protein. Northern-blot analysis showed a wide distribution of the gene in hum...
Previous studies have shown that the BM88 antigen, a novel neuron-specific molecule, promotes the... more Previous studies have shown that the BM88 antigen, a novel neuron-specific molecule, promotes the differentiation of mouse neuroblastoma (Neuro 2a) cells. In particular, stably transfected, with the BM88 cDNA, Neuro 2a cells overexpressing the BM88 antigen (Neuro2a-BM88 cells) are morphologically distinct from the nontransfected Neuro 2a cells; they exhibit enhanced process outgrowth and a slower rate of division. In this study we used Neuro2a and the morphologically differentiated Neuro 2a-BM88 cells to compare their responsiveness to growth factors. The growth factors we used were nerve growth factor (NGF), basic-fibroblast growth factor (b-FGF), and glial cell-line derived neurotrophic factor (GDNF). In addition, we used glial conditioned medium derived from either newborn mouse cerebral cortex (NBCC) or aged mouse cerebral hemispheres (MACH), as a source of normal glial factors. Because these cells express the cholinergic phenotype, we used choline acetyltransferase (ChAT) activ...
Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the ce... more Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the cerebral cortex in Alzheimer's disease (AD), but to date it has not been demonstrated which nicotinic receptor subunits contribute to this deficit. In the present study, autopsy tissue from the temporal cortex of 14 AD cases and 15 age-matched control subjects was compared using immunoblotting with antibodies against recombinant peptides specific for alpha3, alpha4, and alpha7 subunits, in conjunction with [3H]epibatidine binding. Antibodies to alpha3, alpha4, and alpha7 produced one major band on western blots at 59, 51, and 57 kDa, respectively. [3H]Epibatidine binding and alpha4-like immunoreactivity (using antibodies against the extracellular domain and cytoplasmic loop of the alpha4 subunit) were reduced in AD cases compared with control subjects (p < 0.02) and with a subgroup of control subjects (n = 9) who did not smoke prior to death (p < 0.05) for the former two paramete...
The nicotinic acetylcholine receptor (AChR) family is a member of the ligand-gated ion-channel ge... more The nicotinic acetylcholine receptor (AChR) family is a member of the ligand-gated ion-channel gene superfamily, which includes the y-aminobutyric acid (GABA) and glycine receptors (Barnard et al., 1987; Stroud et al., 1990). It contains receptors from the post-synaptic membranes of vertebrate neuromuscular junctions, from the electric organs of electric fish (Torpedo and Electrophorus) and from the central nervous systems of vertebrates and insects. These receptors have several structural and functional features in common, but also have many differences (Lucas and Bencherif, 1992). The muscle-type AChR (i.e. that of the neuromuscular junction and fish electric organs) is the best-known member of the AChR family. It is an integral pentameric membrane protein (Popot and Changeux, 1984) formed from four types of subunits in the stoichiometry @yS or OL$ES (Reynolds and Karlin, 1978; Numa, 1987). Three major factors have facilitated its extensive characterisation: (a) the large quantities that can be isolated from the electric organs of fish, (b) the presence of several snake venom neurotoxins, that bind specifically to the AChR and (c) the application of recombinant DNA techniques. In the present review, we will discuss only this type of AChR. Structural and functional studies of the muscle AChR are of great importance as the molecule acts as the autoantigen in the autoimmune disease myasthenia gravis (MG) and in its experimental animal model (Oosterhuis, 1984; Lindstrom et al., 1988; Penn et al., 1992). MG is characterized by weakness and fatiguability of the skeletal muscles. The disease occurs in about 1 in 20,000 people. As the autoantigen is a well characterized molecule, MG represents an excellent model for the study of the molecular interactions involved in a human autoimmune disease.
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