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Biomedical subjects

R J Stockert

Publications and source records attributed to R J Stockert.

At least 55 records · Page 3Linked to original sources
PubMed1989

Hepatocellular copper toxicity and its attenuation by zinc.

We studied the mechanisms by which excess copper exerts, and zinc mitigates, toxic effects on HepG2 cells. Survival and cell growth were reduced in media containing greater than 500 microM copper chloride for 48 h; LD50 was 750 microM. At 1,000 microM copper for 1 h, there was a general reduction of protein synthesis, and no recognizable changes in cellular ultrastructure. Incubation of cells with 200 microM zinc acetate before exposure to copper, raised the LD50 for confluent cells to 1,250 microM copper chloride, improved protein synthesis, and increased synthesis of a 10-kD protein, apparently metallothionein. The mitigation, by zinc, of copper's toxicity may in part be mediated through induction of this protein in the hepatocyte.

Adenosine Triphosphate↗
PubMed1988

Biotin-dependent expression of the asialoglycoprotein receptor in HepG2.

The asialoglycoprotein receptor (AsGR) is characteristic of fully differentiated hepatocytes. AsGR expression in confluent cultures of HepG2 cells grown in minimal essential medium (MEM) requires a 300-350-dalton dialyzable fraction of fetal bovine serum (FBS). Addition to dialyzed FBS (dFBS) of 10(-7) M biotin or biocytin (Mr 372) permitted full expression of AsGR by HepG2. Affinity chromatography of FBS on streptavidin-Sepharose abolished its ability to support AsGR production. The bound material, when released by heat denaturation and resolved by thin layer chromatography, yielded three cinnamaldehyde-positive components, of which the major detectable one migrates with authentic biocytin and reconstitutes dFBS. Sera from several species, which do not support AsGR production by HepG2, contain less than 10% biotin found in FBS as determined by direct enzyme-linked immunosorbent assay. These results indicate that biotin or a derivative is the low molecular weight serum factor of FBS required for expression of AsGR. Isolation of messenger RNA from HepG2 revealed no difference in AsGR transcripts when cells were grown in MEM-10% FBS or MEM-10% dFBS. Thus a biotin-dependent post-transcriptional event permits the ultimate expression of the AsGR by HepG2 cells.

Actins↗
PubMed1988

A membrane-bound lectin responsive to monocytic maturation in the promyelocytic leukemia cell line HL-60.

A novel mammalian lectin activity responsive to monocytic differentiation is described in the human promyelocytic leukemia cell line HL-60. Glycoprotein binding indicates that the lectin recognizes both N-acetylneuraminic acid and galactose-terminating biantennary oligosaccharide structures. Lectin activity is independent of calcium and appears to reside in a Mr 17,000 intracellular membrane protein. Induction of wild-type HL-60 cells into their macrophage-like counterparts by 1,25-dihydroxyvitamin D3 markedly enhances lectin activity. Induction of granulocytic differentiation by retinoic acid does not affect expression of the lectin. HL-60 sublines which are resistant to granulocytic differentiation by retinoic acid, dimethylsulfoxide, or 6-thioguanine are largely deficient in orosomucoid-binding activity. Induction of monocyte/macrophage differentiation of these sublines upregulates lectin activity to the level seen in induced wild-type cells.

Asialoglycoproteins↗
PubMed1988

Effect of monensin on receptor recycling during continuous endocytosis of asialoorosomucoid.

The binding of asialoglycoproteins to their liver cell receptor results in internalization of the ligand-receptor complex. These complexes rapidly appear in intracellular compartments termed endosomes whose acidification results in ligand-receptor dissociation. Ligand and receptor subsequently segregate: ligand is transported to lysosomes and is degraded while receptor recycles to the cell surface. The proton ionophore monensin prevents acidification of endosomes and reversibly inhibits this acid-dependent dissociation of ligand from receptor. The present study determined the effect of monensin treatment of short-term cultured rat hepatocytes on cell-surface-receptor content, determined both by their binding activity and immunologically, following continuous endocytosis of asialoorosomucoid. Inclusion of 5 microM monensin in the incubation medium reduced the number of immunologically detectable cell-surface receptors by 20% in the absence of ligand. During continuous endocytosis of asialoorosomucoid, inclusion of monensin resulted in a 30-40% reduction of cell-surface receptor detectable either by ligand binding or immunologically. These results suggest that the reduced liver-cell-surface content of receptor in monensin is due to intracellular trapping of ligand-receptor complexes. The reduction of surface receptor during monensin incubation in the absence of ligand suggests that "constitutive recycling" of plasma membrane components also requires intracellular acidification.

Animals↗
PubMed1988

Influence of cytosolic pH on receptor-mediated endocytosis of asialoorosomucoid.

Inhibitors of specific steps in the endocytosis of galactose-terminating glycoproteins (asialoglycoproteins) by cultured rat hepatocytes have been described (J. Cell Biol. 98: 375-381, 1984). In particular, substitution of K+ for Na+ in the culture medium results in reduced delivery of ligand to lysosomes; ligand-receptor internalization, dissociation, and segregation remain normal. We have now demonstrated by direct microelectrode measurement that incubation of hepatocytes in K+-substituted medium results in a reduction of intracellular pH by greater than or equal to 0.5 U. In addition, we have shown that reduced intracellular pH in these cells produced by either direct (CO2 diffusion) or indirect (K+ substitution) acidification inhibits ligand delivery to lysosomes. Return of internalized ligand-receptor complex to the cell surface (diacytosis) is also inhibited by these manipulations. These studies suggest that intracellular pH may modulate specific steps involving vesicle translocation and fusion in the receptor-mediated endocytosis of asialoglycoproteins. Similar effects of direct acidification of hepatocytes by CO2 diffusion and indirect acidification by K+ substitution for Na+, on diacytosis and ligand delivery to lysosomes, suggest that K+ substitution may influence these events by altering intracellular pH.

Animals↗
PubMed1987

Hodgkin's cell lectin: an ectosialyltransferase and lymphocyte agglutinant related to the hepatic asialoglycoprotein receptor.

The galactophilic lectin expressed on the surface of cultured Hodgkin's cells, recently described by this laboratory, has binding characteristics similar to those of the hepatic asialoglycoprotein receptor (HBP), and has been recognized as a Mr 55,000 (p55) membrane glycoprotein by a polyclonal antiserum to rat HBP. This study confirms the close structural relationship between the two lectins showing immunological cross-reactivity of monoclonal and polyclonal antibodies recognizing distinct epitopes on rat or human HBP. In support of the suggested dual nature of p55 as lectin and ectosialyltransferase, enzyme activity is inhibited by the monoclonal anti-HBP antibody, anti-HA 116. Cultured Hodgkin's cells, as purified HBP, agglutinate T-lymphocytes expressing hyposialylated membrane glycosyl determinants. This cell-cell interaction mediated by p55 results in the incorporation of sialic acid into lymphocyte surface asialo-glycans. 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.

Agglutinins↗
PubMed1987

Macrophages specifically regulate the concentration of their own growth factor in the circulation.

The physiological mechanism of clearance of the mononuclear phagocyte growth factor, colony-stimulating factor 1 (CSF-1), from the circulation of normal mice was investigated by following the fate of a trace amount of i.v. injected 125I-labeled CSF-1. Macrophages selectively cleared CSF-1 by CSF-1 receptor-mediated endocytosis and degraded the growth factor intracellularly. This manner of clearance provides a feedback control mechanism whereby the rate of macrophage production is determined by the number of mature macrophages.

Animals↗
PubMed1987

HL-T, a new cell line derived from HL-60 promyelocytic leukemia cell cultures expressing terminal transferase and secreting suppressor activity.

A cell line with immature blast cell morphology was isolated from HL-60 promyelocytic leukemia cell cultures and designated HL-T. This new cell type is biphenotypic, expressing terminal transferase (TdT) together with myelomonocytoid immunologic features. TdT enzymatic activity, undetectable in HL-60, was determined to be 140 to 180 units/10(8) HL-T cells by the dGTP-assay, approximately 20% of the activity found in lymphoblastoid cell lines. HL-T predominantly synthesize the known 58-kDa TdT-protein plus a minor 54/56-kDa doublet. The 58-kDa steady state form is nonglycosylated and is phosphorylated. Precursor antigens S3.13 and MY-10, absent on HL-60, are expressed by HL-T; however, the cells are negative for HLA-Dr. Southern blot analysis by hybridization with immunoglobulin heavy chain (JH) and T cell-receptor chain gene (T beta) probes shows JH to be in the germ-line configuration in both cell lines and the T beta gene to be in germ-line in HL-60 but to be rearranged in HL-T. Truncation of the gene encoding the granulocyte-macrophage-colony-stimulating factor (GM-CSF), as found in HL-60, is not observed in HL-T. HL-T are resistant to differentiation-induction by retinoic acid and 1,25-dihydroxyvitamin D3. Cytogenetically HL-T share with HL-60 a deletion of the short arm of chromosome 9 at breakpoint p13, an aberration frequently found in patients with T cell leukemia. In addition, HL-T display t(8;9)(p11;p24) and trisomy 20. Tetraploidy is observed in 80% of HL-T metaphases with aberrations identical to those in the diploid karyotype. Like HL-60, the new line shows some surface-antigenic-T cell characteristics. Despite an antigenic pattern most consistent with that of helper-inducer T cells (T4+, D44+/-, 4B4+, 2H4-, TQ1+/-), HL-T cells and their conditioned culture medium suppress antigen, mitogen, and mixed-leukocyte-culture-mediated lymphocyte proliferation.

Cell Line↗
PubMed1986

Lectin activity as a marker for Hodgkin disease cells.

Treatment of cultured Hodgkin disease (HD) cells with neuraminidase results in decreased reactivity of monoclonal antibody VIM-D5 with its antigen, the X hapten, a fucosyl-N-acetyllactosamine. The other feature characteristic of HD cells is the expression of high levels of ectosialyltransferase activity. We present evidence for a cause-effect relationship between these two findings in that VIM-D5 antigenicity can be restored on neuraminidase-treated HD cells by modulating transferase activity. This can be interpreted in terms of a lectin activity of the ectosialyltransferase that binds the X hapten's desialylated galactosyl residues, thereby preventing antigen recognition by VIM-D5 antibody. This proposed mechanism is indistinguishable from the autoinhibition phenomenon described for another galactophilic binding protein, the hepatic binding protein (HBP), which binds its own terminal galactosyl residues following neuraminidase treatment. We establish a close relationship between the HD galactophilic binding site and HBP in that antiserum to HBP (i) inhibits the neuraminidase-induced loss of VIM-D5 antigenicity, (ii) blocks the binding of asialoglycoprotein to hepatocytes after being absorbed by and eluted from HD cells, and (iii) recognizes a single HD protein, which in its high level of expression is unique to HD cells. The presence of lectin activity in its classic sense on the surface of HD cells is confirmed by the erythrocyte-agglutinating ability of these cells. This lectin activity, which appears to be related to an ectosialyltransferase on the surface of HD cells, may serve as a marker for the abnormal cells characteristic of HD.

Animals↗
PubMed1986

Unique antigen of cultured Hodgkin's cells. A putative sialyltransferase.

Hodgkin's disease-derived giant cell lines (HD-cells) express high levels of ectosialyltransferase activity presumed to be a galactose-specific lectin recognizing the desialylated 3-fucosyl-N-acetyllactosamine structure (X-hapten). Both the anti-X-hapten monoclonal antibody VIM-D5 and a polyclonal antiserum to another galactose-lectin, the hepatic asialoglycoprotein receptor (HBP), recognize a 55,000-mol wt HD-cell protein (Paietta, E., R. J. Stockert, A. G. Morell, V. Diehl, and P. H. Weirnik. 1986. Proc. Natl. Acad. Sci. USA. 83:3451-3455.) That the expression of the 55,000-mol wt protein is restricted to HD-cells among X-hapten positive cells lines is confirmed in this study. The 55,000-mol wt protein is shown to be present on the cell surface and intracellularly, where an additional immunocrossreactive 150,000-mol wt protein is recognized. Extraction of the 55,000 mol wt protein from HD-cell lysates by affinity chromatography results in the loss of sialyltransferase activity. While evidence for a single protein possessing both the antigenic and the enzymatic activity is not direct, these results suggest that the ectosialyltransferase unique to HD-cells is a 55,000-mol wt membrane glycoprotein possessing the X-hapten oligosaccharide.

Antibodies, Neoplasm↗
PubMed1986

Selective expression of hepatocellular membrane proteins in mice homozygous for a lethal chromosomal deletion.

Previous studies of mice homozygous for one of several overlapping radiation-induced deletions in chromosome 7 revealed reduced expression of a number of hepatocyte proteins. These proteins include the plasma membrane receptors for insulin, epidermal growth factor, and glucagon, all of which are reduced in number by over 70% with no change in apparent affinity constants. It is not known whether all or only select liver cell surface proteins are so affected in newborn deletion homozygotes. In the present study, we investigated expression of two additional, functionally distinct intrinsic hepatocellular membrane binding proteins: the hepatic binding protein (HBP: the receptor for asialoglycoproteins), and the organic anion binding protein (OABP) which may play a role in organic anion transport. Immunoblot analysis showed the content of OABP and HBP in neonatal mutants to be identical to that in controls. As compared to adults, neonates showed levels of only about 8% of HBP and 75% of OABP binding proteins. Assays of HBP binding activities confirmed these results. The normal levels of these hepatocyte binding proteins in the deletion homozygotes suggest that the DNA sequences deleted in these mutants do not regulate all genes encoding such proteins but only a selected number of them.

Animals↗
PubMed1985

Immunological studies of an organic anion-binding protein isolated from rat liver cell plasma membrane.

The mechanism of organic anion uptake by hepatocytes has kinetics that suggest facilitated diffusion, and carrier-mediated membrane transport has been postulated. In previous studies, we purified a 55,000-mol wt organic anion-binding protein (OABP) by affinity chromatography on sulfobromophthalein (BSP)-Sepharose of deoxycholate solubilized liver cell plasma membrane preparations. Using specific goat and rabbit antibodies to OABP, we have now investigated the distribution of this protein in liver fractions and other tissues by an enzyme-linked immunosorbent assay and by the immunoblot (Western blot) procedure. These studies indicated that OABP is present in significant amounts in all tissues examined except for blood. Although OABP has not as yet been isolated from each of these tissues and characterized, OABP in heart retained the ability to bind organic anions, and was purified by affinity chromatography on BSP-sepharose. In liver, OABP was membrane bound and remained so after extraction with 0.9 M NaCl, which suggests that it is an intrinsic membrane protein. OABP did not have a ubiquitous subcellular distribution within the hepatocyte. Preparation of subfractions of liver cell plasma membrane revealed that OABP is present in the sinusoidal and absent from the canalicular membrane. Immunofluorescence studies performed in short-term cultured hepatocytes suggest that OABP is associated with the surface of these cells and does not have a significant intracellular distribution.

Animals↗
PubMed1983

Two galactose-specific receptors in the liver with different function.

In the rat liver both hepatocytes and macrophages have been shown to express on the surface lectins with similar binding specificity for galactose residues. Functionally the two lectins differ in the uptake of ligands. Whereas the hepatocytes ingest molecules and small particles (less than 10 nm), the macrophages take up particles only. Antisera raised against hepatic galactose-specific receptor failed to react with the macrophage lectin but blocked ligand binding to the hepatocyte only, indicating either a different antigenic structure or membrane localization of the two lectins.

Animals↗
PubMed1983

Growth-dependent expression of a cell surface glycoprotein.

The expression of the hepatocellular membrane receptor for desialylated galactose-termining glycoproteins was studied during different proliferative stages of a human hepatoma cell line. Rapidly growing cells exhibited a reduced endocytotic rate of desialylated orsomucoid as compared to non-growing cells. This reduction was shown to be the consequence of a lower concentration of active cell-surface associated receptor protein in the dividing cells.

Carcinoma, Hepatocellular↗
PubMed1983

Model for specific rescue of normal hepatocytes during methotrexate treatment of hepatic malignancy.

We utilized the presence of hepatic receptors for galactose-terminal glycoproteins to achieve specific rescue of differentiated hepatocytes from methotrexate toxicity. This was accomplished by administration of a conjugate formed by covalent coupling of the methotrexate antagonist, folinic acid (leucovorin), to galactose-terminating (desialylated) bovine fetuin. Two cultured hepatocyte-derived cell lines were chosen to test rescue from methotrexate toxicity: Hep G2 cells are capable of receptor-mediated endocytosis of galactose-terminating glycoproteins; PLC/PRF/5 cells are not. In the presence of methotrexate alone, both cells lines were reduced in number to 20% of control populations by day 5. Growth rates of both lines returned to normal when free folinic acid was added to the medium containing methotrexate. When asialofetuin-folinic acid conjugate was given to both cell lines in the presence of methotrexate, Hep G2 cells doubled their numbers by day 4 and reached control (without methotrexate) populations by day 8. However, PLC/PRF/5 cells failed to respond to administered asialofetuin-folinic acid conjugate under identical conditions, and the growth curve was the same as that for cells which received methotrexate alone. Conjugates of asialofetuin bound to folic acid (folate derivative requiring dihydrofolate reductase activity for conversion to an active metabolite) and fetuin (non-galactose-terminal glycoprotein) linked to folinic acid also were made for control studies. These conjugates failed to rescue either cell line.

Asialoglycoprotein Receptor↗
PubMed1983

Immunocytochemical localization of lysosomal beta-galactosidase in rat liver.

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta-galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta-galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.

Animals↗