Biomedical subjects

J R Wands

Publications and source records attributed to J R Wands.

At least 91 records · Page 5Linked to original sources

PubMed1997

Comparison between cytomegalovirus promoter and elongation factor-1 alpha promoter-driven constructs in the establishment of cell lines expressing hepatitis C virus core protein.

The establishment of stable cell lines expressing the hepatitis C virus (HCV) core protein may be important for studies of HCV pathogenesis. Human and mouse cell lines were generated expressing the HCV core protein using expression vectors driven by either the cytomegalovirus (CMV) or elongation factor-1 alpha (EF-1 alpha) promoters. Following transient transfection, HCV core protein was expressed in all cell lines. However, stable human hepatocellular carcinoma (HCC) and murine myeloma cell lines expressing the HCV core protein were only established using constructs driven by the EF-1 alpha promoter. In contrast, stable expression of the hepatitis B virus (HBV) middle envelope protein (MHBs) was obtained successfully in these cell lines using an expression vector driven by the CMV promoter. Inhibitory activity of the first 69 amino acids of the HCV core protein on the CMV promoter was found by using chimeric MHBs/HCV core protein constructs. Growth of cloned cell lines expressing the HCV core protein was slower than that of nonexpressing cell lines. However, morphological changes and cell death were not observed in the stable cell lines expressing HCV core protein. These results indicate that the HCV core protein was not directly cytotoxic to HCC and myeloma cell lines but that specific promoter elements are required to establish stable expression of the nucleocapsid structural protein.

Animals↗

PubMed1997

Neuronal thread protein gene modulation with cerebral infarction.

Neuronal thread proteins (NTP) are a family of phosphoproteins expressed during neuritic sprouting. The 15 to 18 kD NTP cluster is associated with development and neuronal differentiation, whereas the 21 kD and 39 to 42 kD species are overexpressed in Alzheimer's disease, correlating with neurodegenerative sprouting and synaptic disconnection. Empirical observations suggested that NTP might also be modulated with central nervous system injury and stroke. In this study of both human and experimental (rat) focal cerebral infarcts, in situ hybridization and immunocytochemical staining revealed NTP gene expression up-regulated in perifocal neurons. These findings were confirmed by quantitative Northern and Western blot analyses. Moreover, Western blot analysis demonstrated selectively increased expression of the 15 to 18 kD NTP species during the acute, subacute, and healing phases of cerebral infarction in both humans and experimental animals, corresponding with the expected period of neuronal repair. These results suggest an additional role for the 15 to 18 kD NTP species in neuritic sprouting required for neuronal regeneration after injury in the mature central nervous system.

Adult↗

PubMed1997

Immunological approach to hepatocellular carcinoma.

A library of monoclonal antibodies (MoAbs) has been produced against a human hepatocellular carcinoma (HCC) cell line designated FOCUS in order to study the antigenic properties of transformed hepatocytes. Several monoclonal antibodies (MoAbs) were initially selected for study since they bound to antigens which were overexpressed in HCC tissues compared with the adjacent uninvolved normal liver counterpart; in addition, these MoAbs revealed low level antigen expression on other normal human tissues. Subsequently, HCC cell lines were metabolically labelled and the antigens further characterized by immunoprecipitation and Western blot analysis. If the MoAb recognized a primary linear epitope on a protein, cloning was performed using a lambda GT11 cDNA expression library prepared from the FOCUS HCC cell line. These studies characterized the HCC associated antigen(s) at the molecular level. This review illustrates the value of such an experimental approach to search for and identify HCC associated antigens and emphasizes the biological properties of novel proteins may be defined and characterized by these techniques. More important, our investigations have described unique proteins that may not only be important in the pathogenesis of HCC but also demonstrates how such antigen-antibody systems may be used to develop strategies for immunotargetting and gene therapy of HCC.

Animals↗

PubMed1997

Nucleic acid-based antiviral and gene therapy of chronic hepatitis B infection.

Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. There is a need to develop new antiviral approaches for the treatment of this disease. We have explored various nucleic acid-based strategies designed to inhibit HBV replication including: the use of antisense RNA and DNA constructs, DNA-based immunization techniques to stimulate broad-based cellular immune responses with particular emphasis on the generation of cytotoxic lymphocyte (CTL) activity to viral structural proteins, hammerhead ribozymes to cleave HBV pregenomic RNA in vitro and dominant negative HBV core mutant proteins as inhibitors of nucleocapsid formation within cells. In order to optimize these antiviral effects, various novel expression vectors have been developed to deliver such DNA constructs to cells. For example, adenoviral vectors carrying genes that encode for dominant negative proteins have been employed to transfect hepatocytes in vitro and in vivo. In addition, plasmid vectors have been produced to promote expression of HBV structural genes following injection into muscle cells as a means to stimulate the host's cellular and humoral immune response in the context of histocompatibility antigen (HLA) class I and II antigen presentation. These experimental approaches may have important implications for the generation of efficient antiviral effects during chronic HBV infection.

Antiviral Agents↗

PubMed1997

Posttranscriptional regulation of hepatitis B virus replication by the precore protein.

Hepadnaviruses encode two core-related open reading frames. One directs the synthesis of the p21 core protein, which subsequently becomes a structural component of the viral nucleocapsid. The other produces a p25 precore protein that is targeted by a signal peptide to a cell secretory pathway where N-terminal processing will create a p22 species. This molecule will be further modified at the C-terminal region to generate p17, and the truncated protein is secreted from the cell as hepatitis B e antigen (HBeAg). The function of the precore gene in the biology of hepadnaviruses is unknown. We found that ablation of the precore gene resulted in the generation of a hepatitis B virus (HBV) species with a high-replication-level phenotype. More important, expression in trans of physiologic levels of p25 restored viral replication to wild-type levels. Moreover, transient or stable overexpression of the precore gene resulted in striking inhibition of HBV replication. The molecular species responsible for this viral inhibitory effect was identified as the p22 nonsecreted HBeAg precursor protein. By sucrose gradient sedimentation analysis, we determined that expression of p22 leads to the formation of nucleocapsids similar to those made with wild-type p21 core protein. Immunoprecipitation experiments revealed that the p21 and p22 physically interact and form hybrid nucleocapsid structures devoid of pregenomic viral RNA. These experiments suggest that expression of the precore gene may be important in the regulation of HBV replication and describe a possible molecular mechanism(s) for this effect.

Chromobox Protein Homolog 5↗

PubMed1997

The small envelope protein is required for secretion of a naturally occurring hepatitis B virus mutant with pre-S1 deleted.

Naturally occurring deletions in the hepatitis B virus pre-S1 domain have been frequently found during persistent viral infection. In this study we have investigated the functional properties of a mutant viral genome that carries an in-frame deletion of 183 nucleotides in the pre-S1 region. This deletion removes the promoter of the small envelope gene. Transfection into human hepatocellular carcinoma cells of a replication-competent construct containing this deletion resulted in an increase of intermediate DNA replicative forms compared to those produced by wild-type hepatitis B virus. Northern blot analysis revealed that such cells lack the 2.1-kb transcripts encoding the small envelope protein and that hepatitis B surface antigen was absent as well. Furthermore, nucleocapsids containing the genome with pre-S1 deleted were not secreted, and the deleted large envelope protein was retained with the cytoplasm and exhibited a perinuclear pattern of distribution. However, coexpression with the small envelope protein was sufficient to restore virion secretion and to change the cellular distribution of the deleted large envelope protein. In addition, the creation of point mutations that prevent the synthesis of large or small envelope proteins also inhibited viral secretion and led to increased levels of hepatitis B virus intermediate replicative forms within the cell. These studies suggest that naturally occurring viral mutants with pre-S1 deletions involving the promoter region of the small envelope gene will generate a deleted large envelope protein that is retained in the endoplasmic reticulum, resulting in the accumulation of nucleocapsids containing viral DNA; transcomplementation with the wild-type small envelope protein will allow mutant virion secretion to occur.

Animals↗

PubMed1997

Genetic immunization with the free human chorionic gonadotropin beta subunit elicits cytotoxic T lymphocyte responses and protects against tumor formation in mice.

The free beta subunit of human chorionic gonadotropin (hCG beta) is produced and secreted by human lung, bladder, and pancreatic tumors. We attempted to generate cytotoxic T lymphocytes (CTL) with activity against free hCG beta-producing tumors by genetic immunization using a construct containing a beta subunit expressing cDNA. To assess CTL activity in vivo, a cloned syngeneic SP2/O myeloma call line was established that constitutively expresses the free hCG beta protein. Inoculation of this cell line into BALB/c mice produced large tumors within 2 weeks. However, mice immunized with the free hCG beta expression construct demonstrated a marked reduction of tumor size and weight compared with animals immunized with mock DNA ("empty" plasmid). Indeed, 30% of immunized mice were tumor-free after 3 months and thus considered long-term survivors. Inhibition of tumor growth was strongly associated with the level of CTL activity present in CD8+ cells derived from the spleen. In addition, immunized mice developed high titer anti-hCG beta antibodies that neutralized the biologic effects of the intact hCG glycoprotein hormone on its cellular receptor as well. These results illustrate that substantial cellular and humoral immune responses to the free hCG beta subunit may be generated by DNA immunization. This study thus presents a potential approach to inhibiting growth of human tumor cells that produce and secrete the free hCG beta protein.

Animals↗

PubMed1996

A carboxy-terminal truncated insulin receptor substrate-1 dominant negative protein reverses the human hepatocellular carcinoma malignant phenotype.

Insulin receptor substrate-1 (IRS-1), a substrate of various receptor tyrosine kinases transmits mitogenic signals initiated by extracellular ligands. This protein is involved in normal hepatocyte growth and has been found to be overexpressed in human hepatocellular carcinoma. Expression of a carboxy-terminal truncated IRS-1 molecule containing the pleckstrin homology and phosphotyrosine-binding domains associates with the insulin receptor and prevents tyrosyl phosphorylation of endogenous IRS-1 and Shc proteins. Thus, subsequent activation of downstream signaling molecules induced by insulin and IGF-1 such as phosphatidylinositol-3 kinase and mitogen activated protein kinase is inhibited. The morphologic features of transformed human hepatocellular carcinoma cells change to a differentiated hepatocyte appearance and characteristics of the malignant phenotype as manifested by anchorage independent cell growth and tumor formation in nude mice are lost. These studies demonstrate that signal transduction pathways mediated through or by IRS-1 are important in hepatocyte and human hepatocellular carcinoma cell growth.

Animals↗

PubMed1996

Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma.

To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines. This antigen was also highly expressed in neoplastic epithelial cells of breast and colon carcinomas in contrast to its low level of expression in normal hepatocytes and in non-neoplastic epithelial cells. Among the normal adult tissues studied, high levels were observed only in proliferating trophoblastic cells of the placenta and in adrenal glands. A 636-bp partial cDNA, isolated from a gamma GT11 expression library generated with HepG2 human hepatoblastoma cells, and a complete cDNA, generated by reverse transcriptase-PCR, identified the antigen as the human form of aspartyl(asparaginyl)beta-hydroxylase. This enzyme catalyzes posttranslational hydroxylation of beta carbons of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins. Analyses of extracts prepared from several human tumor cell lines compared to their normal tissue counterparts indicate that the increase in hydroxylase, approximately 10-fold, is controlled at the level of transcription and the protein is expressed in an enzymatically active form. In similar analyses, comparing hepatocellular carcinomas to adjacent uninvolved liver from five patients, enzymatic activity was much higher in the tumor tissue from the four patients whose immunoblots revealed increased hydroxylase protein in the malignant tissue. EGF repeats in the extracellular domain of Notch or its homologs contain the consensus sequence for hydroxylation. Deletion mutants lacking this domain are gain-of-function mutants, suggesting that the domain modulates signal transduction by the cytoplasmic domain. While the function imparted by beta hydroxylation is unknown, our studies raise the possibility that beta hydroxylation is regulated in proteins like the mammalian Notch homologs, whose cytoplasmic domains have been shown to be oncogenic.

Adult↗

PubMed1996

Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein.

A tetracycline-regulated system was used to generate cell lines allowing tightly controlled expression of a hepatitis C virus (HCV) cDNA comprising the 5' noncoding, the core, and part of the E1 regions. Production of 21-kDa processed nucleocapsid protein could be regulated over a broad range by the concentration of tetracycline present in the culture medium. Induction ratios of over 1,000-fold were found using an HCV core-luciferase fusion construct. Core protein had an intracellular half-life of 9 hr and corresponded to the product of 173 amino-terminal amino acids of the HCV open reading frame. Sequential immunofluorescence microscopy revealed the presence of core antigen first in a predominantly perinuclear fine-reticular staining pattern and subsequently also in cytoplasmic granules and vesicles. By immunoelectron microscopy core protein was found on the endoplasmic reticulum membrane and on the surface of cytoplasmic lipid droplets. Growth rate analyses and colony formation efficiency assays showed no major cytotoxic effect of HCV core protein expression per se. HCV gene expression could be inhibited by an antisense oligonucleotide targeting a region immediately downstream of the translation initiation codon. These cell lines represent important tools to investigate structural and functional properties of HCV core protein and may be useful to evaluate gene therapeutic strategies against HCV in a cellular system.

Cell Division↗

PubMed1996

Insulin receptor substrate 1 overexpression in human hepatocellular carcinoma cells prevents transforming growth factor beta1-induced apoptosis.

Insulin-like growth factors initiate tyrosyl phosphorylation of the insulin receptor substrate I (IRS-I) protein and activate multiple signaling pathways essential for liver growth. This gene has been found to be up-regulated in human hepatocellular carcinomas (HCCs), and overexpression of IRS-1 in NIH 3T3 cells leads to malignant transformation with activation of the mitogen-activated protein kinase cascade. To explore another possible role of IRS-I in hepatocarcinogenesis, we examined the capability of transforming growth factor beta1 (TGF-beta1), a known negative regulator of hepatocyte growth, to induce programmed cell death in the context of IRS-I overexpression. Hep3B HCC cells were stably transfected with a retroviral vector containing the IRS-I gene. The overexpressed IRS-I protein was highly tyrosyl phosphorylated following insulin/insulin- like growth factor I stimulation and led to constitutive activation of downstream signal transduction molecules such as phosphatidylinositol-3 kinase and mitogen-activated protein kinase. Although parental Hep3B cells were sensitive to apoptosis, the Hep3B-IRS-I-transfected cells acquired resistance to TGF-beta1-induced programmed cell death. Our investigations suggest that IRS-I-mediated signals may act as survival factors and protect against TGF-beta1-induced apoptosis in HCC; this phenomenon may contribute to hepatic oncogenesis.

Apoptosis↗

PubMed1996

Neoplastic transformation induced by insulin receptor substrate-1 overexpression requires an interaction with both Grb2 and Syp signaling molecules.

The insulin receptor substrate-1 (IRS-1) is the major intracellular substrate of insulin and insulin-like growth factor-I (IGF-I) receptor tyrosine kinase activity, and this protein has been found to be overexpressed in human hepatocellular carcinomas. IRS-1 contains several src homology 2 (SH2) binding motifs that interact following tyrosyl phosphorylation with SH2-containing proteins, and this interaction may be essential for transmitting the growth signal from the cell surface to the nucleus. We have previously reported that overexpression of IRS-1 may induce neoplastic transformation of NIH 3T3 cells. This study examines the role of two SH2-containing molecules, namely the Grb2 adapter and Syp tyrosine phosphatase proteins as important components of the cellular transforming activity of IRS-1. Mutations of tyrosine 897 in the YVNI motif (Y897F) and of tyrosine 1180 in the YIDL motif (Y1180F) reduced the intracellular interaction of IRS-1 with Grb2 and Syp proteins, respectively. Furthermore, a single mutation at either Phe-897 or Phe-1180 substantially but not completely reduced IGF-I-dependent transforming activity of IRS-1, whereas creation of a double mutation of both tyrosine residues (Y897F/Y1180F) strikingly attenuated the transforming activity of IRS-1. Stable expression of the IRS-1 mutant constructs in NIH 3T3 cells was associated with a lower level of activation of the mitogen-activated protein kinase kinase (MAPKK)/MAPK cascade following IGF-I stimulation compared with cells stably transfected with the "wild-type" IRS-1 gene. These results suggest that IRS-1-induced cellular transformation requires an interaction with both Grb2 and Syp signal transduction molecules since neither interaction alone appears to be required, and this event subsequently leads to activation of the MAPKK/MAPK cascade.

3T3 Cells↗

PubMed1996

Efficient gene transfer into mammalian cells with cholesteryl-spermidine.

The naturally occurring polyamine spermidine was covalently conjugated with cholesterol, resulting in a novel cationic compound that mediates efficient gene transfer into mammalian cells. Using reporter plasmids coding for firefly luciferase and beta-galactosidase, a simple procedure was developed allowing highly reproducible and efficient transient and stable transfection of HuH-7 cells. Transfection efficiency could be further increased when a fusogenic peptide derived from the influenza virus hemagglutinin HA2 aminoterminal sequence was included in the cholesteryl-spermidine-DNA complex. Cholesteryl-spermidine (Transfectall) represents a novel cationic compound for efficient transfection of cultured cells in vitro and has the potential to be used for gene transfer in vivo.

3T3 Cells↗

PubMed1996

Chromosomal assignment of human genomic NotI restriction fragments in a two-dimensional electrophoresis profile.

Using DNA from sorted human chromosomes and two-dimensional gel electrophoresis, we assigned 2295 NotI sites, 43% of the total, to specific chromosomes and designated the procedure CA-RLGS (chromosome-assigned restriction landmark genomic scanning). Although the NotI enzyme is sensitive to DNA methylation, our results suggested that the majority of the spots did not seem to be affected by this modification. The NotI sites were distributed at higher levels in chromosomes 17, 19, and 22, suggesting higher gene content in these chromosomes. Most spots were assigned to unique chromosomes, but some spots were found on two or more chromosomes. Quantitative analysis revealed the intensity of the DNA spots on the sex chromosomes to be haploid and that of the chromosome 21 spots in DNA from a male with Down syndrome to be trisomic, although there were exceptions. We report here the first-generation CA-RLGS map of the human genome.

Chromosome Aberrations↗

PubMed1996

Characterization of three novel monoclonal antibodies against hepatitis C virus core protein.

Three novel monoclonal antibodies (MAbs) were established against a recombinant hepatitis C virus (HCV) core protein derived from cloned genotype 1b HCV cDNA. MAbs C7-50 and C8-59 recognize a conserved linear epitope represented by amino acid residues 21 to 40 of the nucleocapsid protein. MAb C8-48 is directed against a strain-specific conformational epitope located within the first 82 amino acids. A sensitive two-site MAb-based immunoradiometric assay was established using antibodies directed against distinct epitopes on the nucleocapsid protein. Processed 21 kDa core protein was detected by immunoblotting in human hepatocellular carcinoma cell lines and primary adult rat hepatocytes transfected with a cytomegalovirus promoter-driven expression construct. Immunofluorescence microscopy studies revealed a granular and vesicular cytoplasmic staining pattern. MAb C7-50 was used successfully to detect HCV core antigen in chronically infected chimpanzee liver tissue. These MAbs represent important reagents for the study of HCV biology and for the development of immunodiagnostic assays.

Animals↗

PubMed1996

Expression and immune response to hepatitis C virus core DNA-based vaccine constructs.

Hepatitis C virus (HCV) is a major worldwide cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The development of vaccines against HCV have been complicated by the high variability of the envelope region, and it is likely that the cellular immune responses to viral structural proteins may be important for eradicating persistent viral infection. Recently, it was reported that the injection into muscle cells of plasmids encoding viral genes resulted in the generation of strong cellular immune responses. We constructed vectors that express the highly conserved HCV core gene. In this regard, the pHCV 2-2 construct contained the entire HCV core region and pHCV 4-2 contained both the 5' noncoding region and the core gene. Cellular expression of HCV core protein was assessed following transfection into human and murine cell lines, and higher intracellular levels of the 21-kd core protein were observed with pHCV 2-2. These HCV core DNA constructs were used to immunize BALB/c mice and produced low-level anti-HCV core humoral immune responses. To assess cytotoxic T-lymphocyte (CTL) activity generated in vivo, a cloned syngeneic SP2/O myeloma cell line constitutively expressing HCV core protein was established and inoculated into BALB/c mice to produce growth of plasmacytomas. Strong CTL activity was generated because the tumor size and weight in pHCV 2-2-immunized mice were remarkably reduced compared with mice injected with mock DNA. Spontaneous CTL activity was also exhibited by splenocytes in an in vitro cytotoxicity assay. These investigations demonstrate that plasmid constructs expressing HCV core protein generate strong CTL activity, as assessed both in vivo and in vitro, and are promising candidates as antiviral agents.

Animals↗

PubMed1996

Developmental patterns of neuronal thread protein gene expression in Down syndrome.

Neuronal thread proteins (NTP) are a group of immunologically related molecules expressed in brain and neuroectodermal tumor cell lines. NTP gene expression is up-regulated and NTP molecules accumulate in Alzheimer's disease (AD) brains, pathological states associated with regenerative neuritic sprouting, and during brain development. To investigate the role of NTP over-expression in AD, we examined NTP immunoreactivity in brains from differently aged individuals with Down syndrome, since patients with Down syndrome nearly always develop AD neuropathology and dementia. Using SMI monoclonal antibodies to neurofilament protein, we detected age-associated increases in neurofilament immunoreactive (SMI-positive) neurites in Layers I and II of the cerebral cortex beginning at 1 year of age, followed by SMI-positive neurofibrillary tangles beginning at age 5 years, and then SMI-positive plaques beginning in the third decade. Increased NTP immunoreactivity in Down syndrome brains began in the second decade, prior to establishment of widespread AD neurodegeneration (Down syndrome + AD), and at an age when low-level or absent NTP expression was observed in control brains. Analysis of SDS and Triton X-100-treated histological sections and tissue extracts demonstrated that a largely insoluble, denaturation-resistant form of NTP accumulates in both Down syndrome + AD and AD brains. The findings provide further evidence that abnormal NTP expression and accumulation in brain may be an early marker of AD neurodegeneration in Down syndrome.

Adolescent↗

PubMed1996

Modulation of neuronal thread protein expression with neuritic sprouting: relevance to Alzheimer's disease.

Widespread proliferation of dystrophic neurites in the cerebral cortex represents an important neuroanatomical correlate of dementia in Alzheimer's disease (AD). Increased CNS expression of the 21-kDa neuronal thread protein (NTP) species is also correlated with dementia in AD. Pilot in vitro experiments provided evidence that high-level NTP expression might be linked to neuritic growth. The present study examines retinoic acid (RA) modulation of NTP expression during neurite outgrowth and neuronal differentiation in SH-Sy5y neuroblastoma and PNET2 CNS-derived cells. In both cell lines, RA-induced neuronal differentiation resulted in increased synthesis, expression, and phosphorylation of several NTP species, with high steady-state levels and stepwise hyper-phosphorylation of 21-kDa NTP molecules. With neurite outgrowth, NTP molecules were translocated from the perikarya to long, slender, unbranched cell processes (axons) and growth cones. RA-mediated changes in NTP expression were independent of DNA synthesis. The findings suggest that high-level expression of 21-kDa, and closely related phosphorylated NTP molecules correlates with neuritic growth. Therefore, over-expression of 21-kDa NTP molecules in AD probably reflects the widespread cortical neuritic sprouting associated with dementia. In view of the rapid phosphorylation and cell process translocation of NTP that occurs during neurite outgrowth in vitro, the accumulation of NTP in AD cortical neuronal perikarya suggests a further problem related to post-translational processing and transport of NTP molecules in AD neurodegeneration.

Alzheimer Disease↗