Search PubMed⌕ Search

Biomedical subjects

R J Stockert

Publications and source records attributed to R J Stockert.

At least 37 records · Page 2Linked to original sources
PubMed1995

Conditional immortalization of Gunn rat hepatocytes: an ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer.

Viral vectors and protein carriers utilizing asialoglycoprotein receptor (ASGR)-mediated endocytosis are being developed to transfer genes for the correction of bilirubin-UDP-glucuronosyltransferase (bilirubin-UGT) deficiency. Ex vivo evaluation of these gene transfer vectors would be facilitated by a cell system that lacks bilirubin-UGT, but expresses differentiated liver functions, including ASGR. We immortalized primary Gunn rat hepatocytes by transduction with a recombinant Moloney murine leukemia virus expressing a thermolabile mutant SV40 large T antigen (tsA58). At 33 degrees C, the immortalized hepatocyte clones expressed SV40 large T antigen, synthesized DNA, and doubled in number every 2 to 3 days. At this temperature, differentiated hepatocyte markers, e.g., albumin, ASGR, and androsterone-UGT, were expressed at 5% to 10% of the levels found in primary hepatocytes maintained in culture for 24 hours. Glutathione-S-transferase Yp (GST-Yp), an oncofetal protein, was expressed in these cells at 33 degrees C, but was undetectable in primary hepatocytes. In contrast, when the cells were cultured at 39 degrees C or 37 degrees C, the large T antigen was degraded, DNA synthesis and cell growth stopped, and morphologic characteristics of differentiated hepatocytes were observed. The expression of albumin, ASGR, and androsterone-UGT, and their corresponding mRNAs, increased to 25% to 40% of the level in primary hepatocytes, whereas GST-Yp expression decreased. Functionality of ASGR was demonstrated by internalization of Texas red-labeled asialoorosomucoid, and binding and degradation of 125I-asialoorosomucoid. After liposome-mediated transfer of a plasmid containing the coding region of human bilirubin-UGT1, driven by the SV40 large T promoter, active human bilirubin-UGT1 was expressed in these cells. The immortalized cells were not tumorigenic after transplantation into severe combined immunodeficiency mice. These conditionally immortalized cells will be useful for ex vivo evaluation of bilirubin-UGT gene transfer vectors.

Animals↗
PubMed1994

Effects of cytokines on synthesis and function of the hepatic asialoglycoprotein receptor.

In this study we have investigated whether cytokines, critical mediators of the immune response, might have a direct effect on the expression and/or function of the human hepatic asialoglycoprotein receptor (ASGPR). Binding and uptake of asialoglycoproteins by the human hepatoma cell line, HepG2, and by freshly isolated rat hepatocytes were inhibited by 50% after 3-6 hours and completely abolished following a 24 hour exposure to tumor necrosis factor (TNF) alpha, interferon (INF) alpha or gamma, or interleukin-2 (IL-2). The loss of ASGPR binding activity mediated by IL-2 was reversible up to 4 hours of exposure and accompanied by the selective phosphorylation of the cell-surface receptor. Steady-state levels of total cellular ASGPR protein remained unchanged over the first 6 hours of IL-2 incubation but declined in a dose dependent manner thereafter. This down regulation of ASGPR expression was due to reduced synthesis as a result of reduced receptor transcript levels. No loss was detected, however, of cell surface-associated receptor protein even after 24 hours of IL-2 incubation, suggesting that cytokine induced phosphorylation constitutes a mechanism to regulate receptor activity.

Antibody Formation↗
PubMed1994

The asialoglycoprotein receptor mediates hepatic binding and uptake of natural hepatitis B virus particles derived from viraemic carriers.

As a putative mechanism of hepatitis B virus (HBV) uptake into hepatocytes the interaction between HBV and the hepatic, human-derived asialoglycoprotein receptor (ASGPR) was investigated. Sera from patients with different variations of hepatitis B surface antigen-(HBsAg) positive chronic hepatitis, HBV particles isolated from HBV carriers with high-titre viraemia and commercial HBsAg served as sources of HBV. ASGPR was affinity-purified from human liver. HBV that had bound to isolated ASGPR was either detected by radioimmunoassay using solid-phase bound ASGPR or enzyme immunoassay with biotin-ASGPR bound to immobilized HBV. Furthermore, binding and uptake of purified, 125I-labelled HBV particles into human hepatoma cell lines (HepG2 and HuH7), which constitutively express functional ASGPR molecules, were compared to that of ASGPR-negative COS cells. As a result HBV was found to bind to purified human ASGPR in two different assays. Circulating virus particles from sera with high titre viraemia showed the highest attachment activity to ASGPR. HBV binding to purified ASGPR was saturable and inhibitable by an excess of D-galactose-bearing ligands, by EDTA and anti-receptor immunoglobulin. Lysis of particles by adding detergent abolished immunodetectable HBV binding to purified ASGPR. Commercial HBsAg did not adhere to solid phase-immobilized ASGPR. Monoclonal anti-preS1 antibody (MA18/7) but not anti-preS2 antibody (Q19/10) inhibited virus attachment. Purified and radiolabelled HBV particles showed binding to HepG2 and HuH7 cells but to much lesser degree to COS cells. Cellular binding of HBV was significantly inhibited by blocking of ASGPR function. Both ASGPR ligands and rabbit anti-ASGPR immunoglobulin but not non-immune rabbit serum inhibited uptake of radiolabelled HBV particles into HepG2 cells or HuH7 cells, respectively. This study suggests that HBV virions may enter human hepatocytes via ASGPR molecules by attachment of viral preS1-related envelope binding sites to this receptor.

Acute Disease↗
PubMed1994

Pleiotropic effect of LEC mutation: a rodent model of Wilson's disease.

Metabolic studies with 67Cu were undertaken to identify the site of the cellular defect in copper metabolism in the Long-Evans Cinnamon (LEC) rat. The apparent rate of copper uptake by LEC primary hepatocytes was increased [maximal velocity (Vmax) = 259 pmol.min-1.mg protein-1] compared with controls (Vmax = 161 pmol.min-1.mg protein-1); however, Michaelis-Menten constant (Km) values were comparable (11.8 and 12.7 microM, LEC and control, respectively). Efflux of copper from LEC and control hepatocytes was similar from 0 to 15 min, but was reduced from 15 to 60 min in the former. Although hepatic copper contents were markedly elevated in LEC rats compared with controls (658 +/- 199 vs. 21.5 +/- 6.6 micrograms/g dry wt), biliary copper concentration was reduced in LEC rats compared with controls (0.187 vs. 1.39 +/- 0.66 microgram/ml). Subcellular fractionation of LEC liver homogenates revealed approximately 75% of copper to be present in cytosol, with gradients of copper concentration from cytosol to either lysosome or microsomal subcellular fractions. LEC rat bile and hepatic microsome and lysosome fractions contained smaller fractions of 67Cu administered intravenously as cupric acetate compared with control rats. However, recovery of 67Cu in bile and in lysosomal subcellular fractions were similar for LEC and controls following administration of 67Cu-labeled asialoceruloplasmin, which is targeted to lysosomes. This discordance suggests a possible defect in the entry of copper into lysosomes but normal delivery of lysosomal copper to bile. Based on these findings, we conclude that the mutation in LEC rats alters copper transport at more than one cellular site.

Animals↗
PubMed1994

Modulation of the asialoglycoprotein receptor in human hepatoma cells: effect of glucose.

The hepatic receptor for asialoglycoproteins was found to be modulated by the glucose concentration in the medium of the human hepatoma cell line HepG2. The surface binding of asialoorosomucoid, a well-documented ligand for this receptor, increased from 20 ng/mg of cellular protein to about 40 ng/mg as the glucose concentration was increased from 10 to 50 mg/dl. The up-modulating effect of glucose was mimicked by pyruvate, a product of glucose metabolism, and abolished by both 2-deoxyglucose, an inhibitor of glucose metabolism, and by cycloheximide, an inhibitor of protein synthesis. Scatchard plot analysis indicated a rise in the number of binding sites and a twofold increase in binding affinity. In contrast, the binding of antibody remained unchanged with respect to alterations in glucose concentration, an indication that the actual number of receptors remained constant in face of an increased number of binding sites. Specificity of the glucose effect was shown by the binding of insulin and transferrin to their respective receptors, which was unaffected by the high glucose concentration that increased asialoorosomucoid binding. The repression of receptor binding seen with cells grown in biotin-deprived medium was reversed by increasing the glucose concentration of the medium. In this case, binding was restored to a level sixfold to sevenfold higher than that of the control cells grown in dialyzed serum. The stimulatory effect of glucose was shown to be independent of and significantly greater than that of cyclic GMP, a known regulator of receptor expression of biotin-deficient HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Asialoglycoprotein Receptor↗
PubMed1993

Regulation of the human asialoglycoprotein receptor by cAMP.

The mechanism of regulated expression of the asialoglycoprotein receptor (ASGR) by cAMP was investigated in the human hepatoblastoma cell line, HepG2. Incubation of HepG2 cells with the cell-permeant 8-bromo-cAMP or induction of intracellular cAMP with forskolin reduced receptor expression in confluent HepG2 cultures. Immunoblot analysis established that this reduction of receptor activity was due to a reduction of expression of both ASGR subunit polypeptides H1 and H2. Estimates of the steady-state levels of H1- and H2-related mRNA by Northern blot analysis indicated that reduced ASGR expression was a result of a decrease in gene transcript number. By a combination of run-on and mRNA turnover studies, it was suggested that this reduction of ASGR-related mRNA resulted from its destabilization induced by 8-bromo-cAMP. The effect of 8-bromo-cAMP appears not to be limited to ASGR expression as a rapid reduction in the albumin mRNA was also observed. In contrast, both the beta-actin and glyceraldehyde-3-phosphate dehydrogenase mRNA levels were elevated by exposure to 8-bromo-cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Caeruloplasmin biosynthesis by the human uterus.

The blue copper protein caeruloplasmin is synthesized mainly by hepatocytes. An alternative transcript for caeruloplasmin produced in certain extrahepatic tissues, CP-1, contains an additional 12 nucleotides encoding 4 amino acids not present in the hepatic transcript, CP-2 [Yang, Friedrichs, Cupples, Bonifacio, S Sanford, Horton & Bowman (1990) J. Biol. Chem. 265, 10780-10785]. We have demonstrated transcription of caeruloplasmin mRNA by a well-differentiated human uterine epithelial adenocarcinoma cell line, Ishikawa, and by human uterine endometrium and purified endometrial glands. Identical CP-2 nucleotide sequences were obtained for partial caeruloplasmin transcripts from human liver and Ishikawa cells, indicating that CP-2 transcripts are produced by uterine epithelial lining cells. The synthesis of caeruloplasmin protein was demonstrated for Ishikawa cells and another uterine adenocarcinoma cell line, ECC1. Peptide-mapping analysis indicated that caeruloplasmin secreted by Ishikawa cells was structurally identical with the protein synthesized by the human hepatoblastoma cell line HepG2. The secretion of a 135,000-M(r) caeruloplasmin by Ishikawa and ECC1 cells, comparable with that of the human hepatoblastoma cell line, HepG2, indicated similar processing of uterine and hepatic caeruloplasmin. Incorporation of 67Cu into caeruloplasmin was demonstrated for Ishikawa and ECC1 cells, suggesting that the human uterus produces a bioactive form of caeruloplasmin and possesses the necessary metal transporters and intracellular machinery for copper incorporation into this protein.

Base Sequence↗
PubMed1992

A role for microtubules in sorting endocytic vesicles in rat hepatocytes.

The vectorial nature of hepatocyte receptor-mediated endocytosis (RME) and its susceptibility to cytoskeletal disruptors has suggested that a polarized network of microtubules plays a vital role in directed movement during sorting. Using as markers a well-known ligand, asialoorosomucoid, and its receptor, we have isolated endocytic vesicles that bind directly to and interact with stabilized endogenous hepatocyte microtubules at specific times during a synchronous, experimentally initiated, single wave of RME. Both ligand- and receptor-containing vesicles copelleted with microtubules in the absence of ATP but did not pellet under similar conditions when microtubules were not polymerized. When 5 mM ATP was added to preparations of microtubule-bound vesicles, ligand-containing vesicles were released into the supernatant, while receptor-containing vesicles remained immobilized on the microtubules. Release of ligand-containing vesicles from microtubules was prevented by monensin treatment during the endocytic wave. Several proteins, including the microtubule motor protein cytoplasmic dynein, were present in these preparations and were released from microtubule pellets by ATP addition concomitantly with ligand. These results suggest that receptor domains within the endosome can be immobilized by attachment to microtubules so that, following monensin-sensitive dissociation of ligand from receptor, ligand-containing vesicles can be pulled along microtubules away from the receptor domains by a motor molecule, such as cytoplasmic dynein, thereby delineating sorting.

Adenosine Triphosphate↗
PubMed1992

Alternatively spliced variants of the human hepatic asialoglycoprotein receptor, H2, differ in cellular trafficking and regulation of phosphorylation.

The less abundant subunit of the asialoglycoprotein receptor, H2, may be encoded by at least four variant transcripts as a result of alternative mRNA splicing. We had cloned two H2-cDNAs that differed predominantly by the presence (clone H2') or absence (clone L-H2) of two presumed exons; one of 57 nucleotides was near the 5' end of the sequence, and the other was within the transmembrane region consisting of 15 nucleotides. The relevance of these two segments to H2 processing was studied after expression in rat-6 fibroblasts of these two isolates and of artificial constructs containing either only the 57-nucleotide (transfectant-57) or the 15-nucleotide (transfectant-15) region. H2 proteins encoded by cDNAs containing the 15-nucleotide region were intracellularly retained and degraded independently of the presence of the 57-nucleotide sequence. Of proteins derived from clones lacking the 15-nucleotide region, only a fraction was processed through the trans-Golgi, as evidenced by sensitivity to O-glycanase and neuraminidase, and reached the cell surface. The presence of the 57-nucleotide sequence was necessary for protein phosphorylation. Phosphorylation of serine residue(s) was detected in the endoglycosidase H-sensitive and mature forms of H2 protein encoded by transfectant-57. Since the 57-nucleotide region does not encode for serine residues, it per se cannot be the site of phosphorylation but rather constitutes a regulatory element for post-translational modification.

Amino Acid Sequence↗
PubMed1992

Posttranscriptional regulation of the asialoglycoprotein receptor by cGMP.

The human asialoglycoprotein receptor expressed by the HepG2 cell line is composed of the two homologous polypeptides H1 and H2. Transblot analysis of HepG2 cell lysates indicated that the progressive loss in the steady-state level of asialoglycoprotein receptor (ASGR) when cells were maintained in medium supplemented with dialyzed fetal bovine serum was reversed by the addition of cell-permeant 8-bromo-cGMP. Estimates of the steady-state levels of H1- and H2-related mRNA by Northern blot analysis indicated that the reduction of ASGR was not the result of a concomitant reduction in gene transcript number. No difference in the translatability of the mRNAs derived from cells grown in medium supplemented with fetal bovine serum or its dialyzed counterpart was detected. Resolution of the mRNAs by sucrose gradient centrifugation suggests that cGMP-mediated posttranscriptional regulation of ASGR expression was due to a shift of both H1 and H2 mRNAs from the ribonucleoprotein fraction into a translationally active membrane-associated polysomal pool.

Asialoglycoprotein Receptor↗
PubMed1992

Differences in the abundance of variably spliced transcripts for the second asialoglycoprotein receptor polypeptide, H2, in normal and transformed human liver.

The human hepatic asialoglycoprotein receptor comprises two homologous polypeptides designated H1 and H2. Two distinct complementary DNA clones encoding these receptor subunits have been previously isolated from the human hepatoblastoma cell line HepG2. We discovered that multiple variants of H2 transcripts exist both in HepG2 cells and in the normal human liver that, at least in part, appear to be the result of alternative splicing events. We have found that (a) the complementary DNA clone for H2 previously isolated from HepG2 cells, characterized by a 57-nucleotide insertion within the 5' end of the complementary DNA that is absent from H1, represented only one third of H2-related sequences in an unamplified normal human liver complementary DNA library and less than 10% of H2 clones in HepG2 cells; (b) the predominant message for H2 expressed in the liver and HepG2 cells, designated L-H2, appeared to represent the fully processed product of the gene encoding both L-H2 and H2; and (c) a variant H2 transcript existed in HepG2 cells, designated H2', that contained a novel, 5' 88-bp nucleotide insertion. Poly(A+) RNA analysis of the normal liver and HepG2 cells by complementary RNA hybridization and ribonuclease protection corroborated the observations made during the screening of complementary DNA libraries regarding the abundance of the various messages. A striking incongruity was found between the levels of messenger RNA containing the H2-specific 57-nucleotide sequence and the levels of polypeptide expressed in the liver and HepG2 cells as recognized by antiserum specifically raised against this sequence.

Asialoglycoprotein Receptor↗
PubMed1990

Detection of multiple forms of human ceruloplasmin. A novel Mr 200,000 form.

Three polypeptides with apparent Mr = 200,000, 135,000, and 115,000, reacting with antibody to human ceruloplasmin (Cp), were consistently found in sera of normal adult and newborn subjects, patients with Wilson's disease, as well as in the oxidase-active fraction of purified human Cp, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentrations of the three Cp polypeptides were proportional to the total Cp oxidase activity measured in whole serum. Peptide mapping revealed that the three Cp polypeptides were closely related. Cross-linking of Cp135 resulted in dimers with electrophoretic mobility similar to that of Cp200. A common shift in electrophoretic mobility following N-glycanase treatment indicated that all three polypeptides were N-glycosylated, and that the apparent differences in molecular mass could not be related to the carbohydrate moiety. Immunoprecipitates of cell lysates of [35S]cysteine labeled HepG2 cells revealed the presence of two species of newly synthesized Cp polypeptides, Mr 200,000 and 135,000, which were secreted into the media. Secretion of Cp200 by the human liver appears to be physiologic and may be the result of posttranslational modification of Cp135.

Carcinoma, Hepatocellular↗
PubMed1990

Second messenger modulation of the asialoglycoprotein receptor.

Post-transcriptional regulation of the asialoglyco-protein receptor (ASGR) in the HepG2 cell line can be mediated by the presence of biotin in the culture medium. To determine if the induction by biotin of intracellular cGMP affects ASGR expression, HepG2 were grown in biotin-depleted medium with the cell-permeant 8-bromo-cGMP (8-Br-cGMP). Both cell-surface and total ASGR binding of iodinated asialoorosomucoid (125I-ASOR) was increased from 30 to 95% of control levels by the addition of increasing concentrations of 8-Br-cGMP. The rate of ASGR-mediated endocytosis of 125I-ASOR also increased with increasing concentrations of 8-Br-cGMP. Estimates of the steady state levels of ASGR by transblot analysis utilizing both antisera to affinity-purified ASGR and to isoform-specific antibodies prepared against synthetic peptides confirmed that the increase in 125I-ASOR binding was due to an increase in ASGR expression. Metabolic labeling of biotin-deprived HepG2 with [35S] cysteine and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitants revealed an increase of radiolabeled ASGR within 30 min of the addition of 8-Br-cGMP. Induction of cGMP by atrial natriuretic factor also increased the metabolic labeling of ASGR. ASGR expression in a second hepatocellular carcinoma cell line, HuH-7, responded in a similar fashion to the addition of 8-Br-cGMP. In contrast to 8-Br-cGMP, exposure to 8-bromo-cAMP results in a reduction of ASGR expression even in the presence of biotin-containing medium. The antagonistic roles of cGMP and cAMP suggest a balance between cyclic nucleotides is required for the maintenance of differentiated functions by the hepatocyte.

Amino Acid Sequence↗
PubMed1990

The rat hepatocyte plasma membrane organic anion binding protein is immunologically related to the mitochondrial F1 adenosine triphosphatase beta-subunit.

A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.

Affinity Labels↗
PubMed1989

Hodgkin's cell lectin, a lymphocyte adhesion molecule and mitogen.

We have previously shown a novel galactose/N-acetylgalactosamine specific lectin activity (Hodgkin's disease (HD) lectin) on the surface of cultured HD cells (lines L428, its variants, and line L540) to mediate lymphocyte adhesion. We here demonstrate that both surface membrane-bound and secreted HD lectin activities participate in the activation of agglutinated lymphocytes. Among known adhesion molecules expressed by the HD cells, only the intercellular adhesion molecule-1 (ICAM-1) contributed to this activation as an alternative PBL binding site. As yet we have not identified the cellular ligand(s) for the HD lectin on the lymphocyte surface. Pretreatment of lymphocytes with mAb to the accessory molecules CD2, CD3, CD4, CD8, CD11b, or CD11c did not interfere with their response to HD cells. mAb to CD11a (LFA-1), the alleged ligand of ICAM-1, inhibited the ICAM-1 but not the HD lectin-mediated lymphocyte stimulation. Although lymphocyte binding could proceed via either pathway, lymphocyte activation always depended upon factors secreted by the HD cells, one of which we identified as a soluble form of the HD lectin based on its shared properties with the membrane-bound form including immunologic cross-recognition and carbohydrate-binding specificity. Although HD cell-conditioned medium alone stimulated lymphocytes, HD cell plasma membranes could compensate for low concentrations of this medium. In addition, resting lymphocytes, normally unresponsive, were triggered into DNA synthesis by growth medium when cocultured with HD cell membranes. The unique functions of the surface-expressed HD lectin and its soluble counterpart as lymphocyte adhesion molecule and mitogen might be physiologically relevant to the severe immunodeficiencies occurring in patients with HD.

Antigens, CD↗
PubMed1989

A marker and putative pathoantigen of Hodgkin's cells.

A galactose-specific lectin, recently described by our laboratory, is immunologically demonstrable on the surface of neoplastic cells derived from patients with Hodgkin's disease. This Hodgkin's lectin is shown to be functionally and antigenically related to the galactose-N-acetylgalactosamine-specific lectin of the hepatocyte (HBP). Poly- and monoclonal antibodies against either the cytoplasmic tail or the cell-surface binding site of HBP recognize the Hodgkin's lectin as a 55 Kd protein. Expression of the 55 Kd antigen appears to be restricted to Hodgkin's disease involved tissues and cells of the monocyte/macrophage lineage. The putative identification of the Hodgkin's lectin as an ectosialyltransferase unique to Hodgkin's cells is supported by inhibition of enzymatic activity by anti-HBP antibodies. Cultured Hodgkin's cells, in analogy to purified HBP, agglutinate T-lymphocytes mediated by the Hodgkin's lectin. This cell-to-cell interaction results in the incorporation of sialic acid into lymphocyte surface asialoglycans as well as in the stimulation of lymphocyte proliferation. The function of the Hodgkin's lectin as lymphocyte agglutinant in vitro suggests its role as an immunomodulator contributing to the immunodeficiencies associated with Hodgkin's disease.

Antigens, Neoplasm↗