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J R Wands

Publications and source records attributed to J R Wands.

At least 127 records · Page 7Linked to original sources
PubMed1994

Ethanol impairs insulin receptor substrate-1 mediated signal transduction during rat liver regeneration.

Chronic ethanol exposure inhibits the capacity of the liver to regenerate. Insulin is a potent hepatotrophic factor and it was determined if ethanol interferes with insulin receptor substrate (IRS-1)-protein mediated signal transduction during liver regeneration. Tyrosyl phosphorylation of IRS-1 was strikingly increased prior to the major wave of DNA synthesis in isocaloric pair-fed control rats; a blunted and delayed response was found in ethanol-fed rats. Enzymatic activity of phosphatidylinositol 3-kinase, a Src homology 2 (SH2) domain containing signal transduction molecule was enhanced by the association with tyrosyl phosphorylated IRS-1, whereas in ethanol-fed rats, this activity was greatly diminished and delayed. These results indicate that one potential molecular mechanism whereby ethanol inhibits hepatocyte DNA synthesis is through its action on the IRS-1-mediated signal transduction cascade.

Animals↗
PubMed1994

Presence of hepatitis B and C viral genomes in US blood donors as detected by polymerase chain reaction amplification.

Hepatitis C virus (HCV) represents a major cause of posttransfusion hepatitis worldwide. Posttransfusion hepatitis associated with hepatitis B virus (HBV) continues to occur. HBsAg-negative donor sera from the Rhode Island Blood Center between 1987 and 1988 were screened using more sensitive techniques to assess the prevalence of low level HBV infection. Group I consists of 866 healthy blood donors without HBV serologic markers, group II consists of 377 donors with ALT elevations (> 45 IU/L), group II consists of 148 donors positive for anti-HBc, and group IV consists of eight donors positive for both surrogate markers. A sensitive monoclonal immunoradiometric assay (M-IRMA) was employed for detection of HBsAg-associated epitopes (detection limit of 20 pg/ml) in serum. A subset of sera were analyzed for the presence of HBV DNA using the method of anti-HBs capture of HBV related virions in serum followed by polymerase chain reaction (PCR) amplification. Using these techniques, 0.8% and 1.7% of donors were positive for HBsAg and HBV DNA respectively in group I. In contrast, 0.9% and 9.5% in group II and 0.7% and 18.1% in group III were positive, respectively. There were eight donors with both ALT elevation and anti-HBc; and four (50%) of these were positive for HBV DNA. In the group with anti-HBc, the majority (80%) of donors with HBV DNA had either no or low (signal to noise ratio < 10) anti-HBs titer. Using anti-HCV testing and reverse transcription-PCR for detection of HCV genomes, we detected evidence of HCV infection in nine of the 49 donors with low level HBV DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Donors↗
PubMed1994

Properties of hepatitis B virus pre-S1 deletion mutants.

Deletion mutants of hepatitis B virus (HBV) pre-S proteins were detected in serum in 2 of 10 (20%) individuals with chronic hepatitis B infection following the initiation of interferon treatment. The size of these deletions was up to one-half of the entire pre-S1 region. In vivo, all HBV deletion mutants were found to coexist with a full-length "wild-type" viral genome. The functional properties of a HBV-deleted mutant were studied in detail and revealed a stop codon in the pre-S2 open reading frame in all 20 of the clones sequenced. Several of the deleted mutants produced low-level HBsAg in culture supernatants compared to wild-type virus due to a putative loss of transcription factor binding sites. Transfection experiments in human hepatoma cells (HuH-7) demonstrated that the polymerase gene function was not affected by the large pre-S1 deletions and mutant viral genomes were capable of replication. However, secretion of incapsidated mutant viral genomes was blocked in HuH-7 cells. Cotransfection studies with a plasmid expressing only the HBV pre-S1, pre-S2, and S proteins resulted in complete restoration of viral particle secretion. Our findings suggest that an in vivo trans-complementation phenomenon would have had to occur to permit secretion from the liver into serum of the nucleocapsids containing these deleted viral genomes.

Base Sequence↗
PubMed1994

Polymerase chain reaction analysis of hepatitis B virus DNA in formalin-fixed, paraffin-embedded liver biopsies from alcoholics using a simplified and standardized amplification protocol.

Sixty-seven formalin-fixed and paraffin-embedded liver biopsies from HBsAg-negative alcoholics without previous blood transfusions or intravenous drug abuse were analyzed for the presence of low-level hepatitis B virus DNA by the polymerase chain reaction. To simplify and standardize the amplification procedure, aliquots of a complete polymerase chain reaction mix were prepared and frozen for storage; random samples were tested prior to analysis of clinical material. Freezing and storage of the aliquots did not affect the activity of Taq polymerase. One large batch of ready-to-use aliquots could thus be used as a standardized polymerase chain reaction kit for all experiments. The suitability of the extracted material for polymerase chain reaction analysis was tested in two ways. First, the absence of nonspecific polymerase chain reaction inhibitors was demonstrated in all samples by amplifying cloned hepatitis B virus DNA in the presence of extracted material. Second, the integrity of the extracted DNA was tested by amplifying a segment of the beta-globin gene. Twenty-three samples were beta-globin DNA positive and thus contained sufficient amounts of nondegraded DNA. These results emphasize the importance of testing both the absence of nonspecific inhibitors and DNA integrity in DNA samples extracted from fixed tissue. Among the 23 beta-globin positive samples, 12 had cirrhosis (52.1%). Two of these samples were hepatitis B virus DNA positive (8.7%); one of these cases had cirrhosis. Thus, even in the absence of common risk factors, the incidence of hepatitis B virus in this alcoholic population was increased compared to the general population.

Alcoholism↗
PubMed1994

Enhancement of HBsAg detection in serum of patients with chronic liver disease following removal of circulating immune complexes.

Patients with chronic liver disease and hepatocellular carcinoma may lack serological evidence of previous hepatitis B virus infection. The purpose of the present study was to test the hypothesis that circulating immune complexes may interfere with the detection of low levels of HBsAg in such patients. Sera from 190 patients were initially screened for the presence of circulating immune complexes. Patients belonged to three clinical categories: asymptomatic HBsAg carriers (50 patients), chronic liver disease (30 patients) and hepatocellular carcinoma (110 patients). Forty-one of the group of 190 patients (21%) were positive for circulating immune complexes. Sera from 21 patients were selected for further evaluation. The sera of 13 chronic liver disease or hepatocellular carcinoma patients (HBsAg negative, hepatitis B virus-DNA negative, with or without evidence of previous hepatitis B virus infection) and eight HBsAg positive carriers (four asymptomatic, three with chronic liver disease and one with hepatocellular carcinoma) were passed through a Clq affinity column (first column) to remove circulating immune complexes. Unbound material was then passed through a monoclonal IgG2a anti-HBs affinity column (second column). Unbound material (following both columns) contained free HBsAg, as determined by monocolonal radio-immunoassay, in eight patients in whom HBsAg had been undetectable in the original serum. Removal of circulating immune complexes from the serum of the three HBsAg positive patients with chronic liver disease also caused a significant increase in measurable circulating HBsAg compared with the original serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

The molecular pathogenesis of hepatocellular carcinoma.

Some of the multiple factors involved in the molecular pathogenesis of hepatocellular carcinoma have been elucidated in recent years but no clear picture of how and in what sequence these factors interact at the molecular level has emerged yet. Transformation of hepatocytes to the malignant phenotype may occur irrespective of the aetiological agent through a pathway of chronic liver injury, regeneration and cirrhosis. The activation of cellular oncogenes, the inactivation of tumour suppressor genes and overexpression of certain growth factors contribute to the development of HCC. There is increasing evidence that the hepatitis B virus may play a direct role in the molecular pathogenesis of HCC. Aflatoxins have been shown to induce specific mutations of the p53 tumour suppressor gene thus providing a clue to how an environmental factor may contribute to tumour development at the molecular level.

Animals↗
PubMed1994

Diagnostic utility of quantitating neurofilament-immunoreactive Alzheimer's disease lesions.

The diagnosis of Alzheimer's disease (AD) neurodegeneration is based on histopathological detection of paired helical filament-associated lesions. Silver stains are routinely used but the results are fraught with intra- and interinstitutional variability. This study employed monoclonal antibodies to middle and high molecular weight neurofilament subunits in an immunohistochemical assay to assess the extent of paired helical filament-associated lesions in brains with AD, Down's syndrome plus AD lesions (AD+DN), Parkinson's disease dementia (PD), AD+PD, and normal aging changes. The densities of neurofilament-immunoreactive (NFI) cortical neurofibrillary tangles and plaques were significantly higher in AD and AD+DN than in PD and aged control brains (p < 0.001), and NFI neurofibrillary tangles and plaques were more abundant in AD and AD+DN compared with AD+PD and PD, yet all patients with AD, AD+PD, or PD died with end-stage dementia. In contrast, the densities of NFI dystrophic neurites (primarily dendrites) in cortical Layer 2 were similar among the AD, AD+DN, AD+PD, and PD groups, and all were significantly higher than control (p < 0.005). Stepwise multivariate regression analysis demonstrated significant correlations between AD diagnosis and high densities of NFI neurofibrillary tangles and plaques (p < 0.001) and between end-stage AD-type dementia and high densities of NFI dystrophic neurites (p < 0.001). This study demonstrates that the histopathological lesions correlated with AD dementia can be readily detected and quantified by immunostaining with monoclonal antibodies to phosphorylated and non-phosphorylated neurofilaments. Moreover, the findings suggest that NFI neurite pathology may be an important feature contributing to the clinically manifested AD-type dementia in individuals with Parkinson's disease.

Aged↗
PubMed1994

Influence of ethanol on insulin receptor substrate-1-mediated signal transduction during rat liver regeneration.

Chronic ethanol exposure inhibits the capacity of the liver to regenerate. Insulin is a potent hepatotrophic factor, and tyrosyl phosphorylation (TP) of the intracellular insulin receptor substrate-1 (IRS-1) protein plays a crucial role in hepatocyte growth. The present investigation determined whether ethanol interferes with IRS-1-mediated signal transduction during liver regeneration induced by partial hepatectomy (PH) using the chronic ethanol-fed rat model. Tyrosyl phosphorylation of IRS-1 was strikingly increased and two peaks of TP were observed at 2 and 12 hr prior to the major wave of DNA synthesis in isocaloric pair-fed control animals; a blunted and delayed response was found in the ethanol-fed group. Furthermore, association of IRS-1 with phosphatidylinositol 3-kinase (PtdIns 3-kinase), a Src homology 2 (SH(2)) domain containing signal transduction molecule, was enhanced at 2 and 12 hr after PH in controls. In ethanol-fed rats, a single peak of association at 4 hr was observed. More important, PtdIns 3-kinase enzymatic activity was strikingly enhanced by the association with tyrosyl phosphorylated IRS-1 at 2 and 12 hr after PH, whereas in ethanol-fed animals this activity was greatly diminished and delayed to 6 and 36 hr, respectively. The biological consequence of this ethanol effect on TP of IRS-1 was a reduction and delay of the hepatic regenerative response after PH. These results indicate that one potential molecular mechanism whereby ethanol inhibits hepatocyte DNA synthesis is through its action on the IRS-1-mediated signal transduction pathway.

Animals↗
PubMed1993

The effect of ethanol and extracellular matrix on induction of p36 protein kinase substrate expression in rat hepatocytes.

p36 plays a direct role in DNA synthesis and is overexpressed in transformed cells. It is also an important component linking the cell membrane to intracellular cytoskeletal components. Experiments were performed in primary rat hepatocyte culture stimulated with epidermal growth factor (EGF) to determine if p36 expression was related to DNA synthesis or to the effect of the extracellular matrix on hepatocyte differentiation; ethanol was employed as an agent to inhibit hormone stimulated hepatocyte DNA synthesis. It was found that hepatocyte p36 expression was highly dependent on the type of extracellular matrix and the time in culture. There was no correlation of p36 expression with DNA synthesis and, therefore, p36 levels appeared more closely related to the differentiated phenotype, induced by the extracellular matrix interactions rather than cellular proliferation.

Animals↗
PubMed1993

Characterization of thread proteins expressed in neuroectodermal tumors.

Neuronal thread protein is a novel 21-kDa protein that accumulates in brains with Alzheimer's disease and exhibits developmentally associated changes in the level of expression. Recently, we discovered that primary human primitive neuroectodermal tumor (PNETs), malignant astrocytomas, and several human PNET and glioblastoma cell lines also express thread protein immunoreactivity. However, in addition to the 21-kDa species, there are approximately 17- and approximately 14-kDa thread protein-immunoreactive molecules expressed in both PNET and glioblastoma cell lines and a fourth approximately 8-kDa thread protein detected in glioblastoma cell lines. Metabolic labeling studies demonstrated that the 21-kDa thread proteins are phosphorylated, whereas the approximately 17-, approximately 14-, and approximately 8-kDa thread proteins are not. Glycosylated residues were not detected in either the PNET- or glioblastoma-derived thread proteins. Using a panel of monoclonal antibodies, we observed differences between PNET and glioblastoma cells suggesting that the thread proteins expressed in neuronal and glial cells are distinct. The levels of thread protein immunoreactivity in both PNET and glial cells were highest during the log phase of cell growth and lowest in serum-starved, nonproliferating cultures. The findings suggest that there are several distinct neuronal and glial derived thread proteins expressed in the central nervous system and that their levels of expression may be modulated with cell growth.

Antibodies, Monoclonal↗
PubMed1993

Expression and phosphorylation of insulin receptor substrate 1 during rat liver regeneration.

Insulin has been shown to be important for normal liver regeneration to occur. The mechanisms whereby insulin may exert its effects on hepatocyte growth, however, are still unknown. The rat and human insulin receptor substrate 1 (IRS-1) is a specific target molecule for the insulin receptor beta subunit kinase and will bind to signal transducing molecules containing Src homology 2 domains through its multiple tyrosyl phosphorylation (TP) sites. This investigation examined how IRS-1 may be involved in insulin action during hepatocyte growth induced by two-thirds partial hepatectomy. The TP of IRS-1 was strikingly enhanced prior to the major wave of hepatocyte DNA synthesis at 24 h; IRS-1 protein and mRNA expression increased in parallel to a lesser extent after partial hepatectomy. TP of insulin receptor beta subunit, which enhances its kinase activity toward IRS-1, was increased in association with TP of IRS-1. Finally, phosphatidylinositol 3-kinase, one of signal transducing molecules containing Src homology 2 domains, was associated with IRS-1 following TP in vivo. These observations suggest that IRS-1 protein may play an important role in transmitting the insulin signal to intracellular regulators involved in hepatocyte growth.

Animals↗
PubMed1993

Targeted transfection and expression of hepatitis B viral DNA in human hepatoma cells.

A soluble DNA carrier system consisting of an asialoglycoprotein covalently linked to poly-L-lysine was used to bind DNA and deliver hepatitis B virus (HBV) DNA constructs to asialoglycoprotein receptor-positive human hepatoma cells. 4 d after transfection with surface or core gene expression constructs, HBsAg and HBeAg in the media were measured to be 16 ng/ml and 32 U/ml per 10(7) cells, respectively. Antigen production was completely inhibited by the addition of an excess of asialoorosomucoid. On the other hand, asialoglycoprotein receptor-negative human hepatoma cells, SK-Hep1, did not produce any viral antigens under identical conditions after incubation with HBV DNA complexed to a conjugate composed of asialoorosomucoid and poly-L-lysine. Using a complete HBV genome construct, HBsAg and HBeAg levels reached 16 ng/ml and 16 U/ml per 10(7) cells, respectively. Northern blots revealed characteristic HBV RNA transcripts including 3.5-, 2.4-, and 2.1-kb fragments. Intracellular and extracellular HBV DNA sequences including relaxed circular, linear and single stranded forms were detected by Southern blot hybridization. Finally, 42-nm Dane particles purified from the spent cultures medium were visualized by electron microscopy. This study demonstrates that a targetable DNA carrier system can transfect HBV DNA in vitro resulting in the production of complete HBV virions.

Carcinoma, Hepatocellular↗
PubMed1992

Cleavage of hepatitis B virus RNA by three ribozymes transcribed from a single DNA template.

Hepatitis B virus is a DNA virus which replicates asymmetrically through reverse transcription of an RNA intermediate (pregenomic RNA). As a first step toward an antiviral strategy, a single synthetic oligodeoxynucleotide coding for 3 ribozyme motifs directed against three adjacent sites within the pregenomic RNA was synthesized, cloned and transcribed in vitro. Experiments utilizing 5' and 3' end labeling of RNA demonstrated that the three ribozymes accurately cleaved hepatitis B virus substrate RNA. Cleavage efficiency was similar to that of single ribozyme constructs. These results demonstrate that hepatitis B virus RNA is susceptible to ribozyme induced cleavage and illustrate that three ribozymes were simultaneously active when transcribed from a single DNA template. The expression of multiple ribozyme motifs may offer an advantage if there is high viral target sequence variability.

Base Sequence↗
PubMed1992

The major histocompatibility complex class I antigen-binding protein p88 is the product of the calnexin gene.

A 90-kDa phosphoprotein (p90) of the endoplasmic reticulum was identified by a monoclonal antibody generated against human hepatoma cells. Pulse-chase experiments with [32P]phosphate and [35S]methionine demonstrated that p90 formed both stable and transient complexes with other cellular proteins, suggesting its role as a molecular chaperone. This protein associates with heavy chains of major histocompatibility complex class I proteins, suggesting that it is the human homolog of the recently described 88-kDa protein that transiently associates with murine class I molecules in the endoplasmic reticulum. The p90 protein also associates in B lymphocytes with membrane immunoglobulin mu heavy chains and may serve as a chaperone for many membrane-bound polypeptides. A partial human p90 cDNA was cloned from a lambda gt11 expression library and identified as the human homolog of calnexin, a major canine calcium-binding protein found to be associated with the signal-sequence receptor in endoplasmic reticulum membranes.

Amino Acid Sequence↗
PubMed1992

Cloning and increased expression of an insulin receptor substrate-1-like gene in human hepatocellular carcinoma.

Human insulin receptor substrate-1 (hIRS-1) cDNAs were cloned from a lambda GT11 expression library using a monoclonal antibody (MAb) produced against a human hepatocellular carcinoma (HCC) cell line (FOCUS). The predicted amino acid sequence derived from both a genomic DNA fragment and the cDNAs showed a 90.5% identity to the previously reported rat IRS-1 cDNA [Sun, X.P. (1991) Nature 352, 73-77]. Multiple potential phosphorylation sites, that suggest an intrinsic function of this molecule in response to insulin action, were highly conserved between the two species. A c.a. 180 kDa hIRS-1 protein was immunoprecipitated and found to be phosphorylated on tyrosine residue(s) following insulin stimulation of HuH-7 HCC cells. Northern blot analysis demonstrated a single c.a. 5 kb transcript in HCC cell lines and tissues. Higher levels of hIRS-1 gene transcripts were observed in HCC tumors compared to adjacent non-involved normal liver.

Amino Acid Sequence↗
PubMed1992

Increased levels of neuronal thread protein in cerebrospinal fluid of patients with Alzheimer's disease.

Neuronal thread protein is a recently characterized, approximately 20-kd protein that accumulates in brains with Alzheimer's disease (AD) lesions. This study examined whether concentrations of neuronal thread protein (NTP) were also increased in the cerebrospinal fluid (CSF) of individuals with probable (clinically diagnosed) and definite (histopathologically proved) AD. Using a highly sensitive three-site monoclonal antibody-based immunoradiometric assay, we measured NTP concentrations in CSF from 84 patients with probable AD and mild dementia (duration, 4.05 +/- 0.36 years), 45 with Parkinson's disease and minimal or no dementia (duration, 4.73 +/- 0.78 years), 73 with multiple sclerosis, and 73 nondemented control subjects. NTP concentrations were also measured in postmortem ventricular fluid and temporal lobe neocortex extracts from 31 subjects with histopathologically proved AD and 14 age-matched control subjects. The mean concentration of NTP in the CSF was higher in AD (4.15 +/- 0.25 ng/ml; 95% confidence interval [CI] limits, 3.65-4.65) than in Parkinson's disease (1.96 +/- 0.16 ng/ml; 95% CI, 1.65-2.27), multiple sclerosis (1.6 +/- 0.14 ng/ml; 95% CI limits, 1.33-1.88), or control subjects (1.27 +/- 0.06 ng/ml; 95% CI limits, 1.15-1.40) (p < 0.001). In addition, 70% of the patients with probable AD had concentrations of NTP in CSF that were higher than 2.5 ng/ml (> upper 99% CI limit in the control group), compared with 23% of Parkinson's disease patients, 11% of multiple sclerosis patients, and 4% of control subjects. The mean concentrations of NTP in the ventricular fluid and brain tissue from individuals with documented AD and end-stage dementia were threefold higher than the levels detected in the CSF from the remaining patients with probable AD and mild dementia. Moreover, of 9 patients with AD, postmortem brain and CSF manifested 5- to 50-fold higher levels of NTP compared with the CSF samples obtained an average of 6 years earlier. These findings demonstrate that NTP levels are elevated in the CSF of individuals with AD and that NTP levels in the CSF increase strikingly with progression of dementia and neuronal degeneration.

Adult↗
PubMed1992

Immunotargeting of daunomycin to localized and metastatic human colon adenocarcinoma in athymic mice.

A monoclonal antibody (designated SF25), which recognizes a protein antigen expressed on a large number of human colon carcinomas, was used for drug targeting. Daunomycin-antibody conjugates were prepared by two previously described procedures. In one, the drug was bound to the antibody through a spacer of small molecular mass (cis-aconitic acid), while in the other a dextran bridge served as the link between drug and antibody. High substitution rates of drug to antibody were obtained using the latter binding procedure. Both conjugates were tested in vitro against two human colon carcinoma cell lines, LS180 and KM-12. The efficacy of a daunomycin-dextran-SF25 antibody conjugate was tested against colon carcinoma LS180 tumors transplanted at different sites into athymic mice. The specific conjugate was significantly more inhibitory to a subcutaneous tumor growth than its components or their mixture. SF25 antibody alone showed antitumoral effects against all three forms of transplanted tumor tested, namely, local, metastatic or intrahepatic, whereas daunomycin, on its own, was effective only against the subcutaneous tumor. Binding of daunomycin to dextran partially improved its inhibitory activity against the metastatic tumor. The conjugate, daunomycin-dextran-SF25 antibody reduced the number of metastatic foci, increased the survival rate and delayed death. Yet against lymph node metastases it was not significantly better than a mixture of both constituents. However, results obtained with an intrahepatic tumor, a model that mimics the natural progression of the disease, resembled those described with the subcutaneous tumor. Daunomycin-dextran-SF25 antibody was significantly more effective than all components separately and than a mixture of drug and antibody, provided a highly drug-substituted conjugate was used.

Adenocarcinoma↗