709 results on '"Willingham M"'
Search Results
352. Apical membrane endocytosis via coated pits is stimulated by removal of antidiuretic hormone from isolated, perfused rabbit cortical collecting tubule.
- Author
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Strange K, Willingham MC, Handler JS, and Harris HW Jr
- Subjects
- Animals, Body Water metabolism, Cell Membrane Permeability drug effects, Female, Horseradish Peroxidase pharmacokinetics, Kidney Tubules, Collecting drug effects, Microscopy, Electron, Perfusion, Rabbits, Spectrophotometry, Coated Pits, Cell-Membrane physiology, Endocytosis, Endosomes physiology, Kidney Tubules physiology, Kidney Tubules, Collecting physiology, Vasopressins physiology
- Abstract
Antidiuretic hormone increases the water permeability of the cortical collecting tubule and causes the appearance of intramembrane particle aggregates in the apical plasma membrane of principal cells. Particle aggregates are located in apical membrane coated pits during stimulation of collecting ducts with ADH in situ. Removal of ADH causes a rapid decline in water permeability. We evaluated apical membrane retrieval associated with removal of ADH by studying the endocytosis of horseradish peroxidase (HRP) from an isotonic solution in the lumen. HRP uptake was quantified enzymatically and its intracellular distribution examined by electron microscopy. When tubules were perfused with HRP for 20 min in the absence of ADH, HRP uptake was 0.5 +/- 0.3 pg/min/micron tubule length (n = 6). The uptake of HRP in tubules exposed continuously to ADH during the 20-min HRP perfusion period was 1.3 +/- 0.8 pg/min/micron (n = 8). HRP uptake increased markedly to 3.2 +/- 1.1 pg/min/micron (n = 14), when the 20-min period of perfusion with HRP began immediately after removal of ADH from the peritubular bath. Endocytosis of HRP occurred in both principal and intercalated cells via apical membrane coated pits. We suggest that the rapid decline in cortical collecting duct water permeability which occurs following removal of ADH is mediated by retrieval of water permeable membrane via coated pits.
- Published
- 1988
- Full Text
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353. pH-dependent lysis of liposomes by adenovirus.
- Author
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Blumenthal R, Seth P, Willingham MC, and Pastan I
- Subjects
- Fluoresceins, Hydrogen-Ion Concentration, Indicators and Reagents, Kinetics, Microscopy, Electron, Adenoviruses, Human metabolism, Liposomes, Pulmonary Surfactants
- Abstract
Purified adenovirus induced a dose-dependent release of the water-soluble markers calcein and carboxyfluorescein from liposomes. Marker release was strongly dependent on pH, and at temperatures below 5 degrees C, the rate of release showed an optimum at a pH of about 6. This pH dependence parallels disruption of endocytic vesicles by adenovirus and the permeabilization that adenovirus induces on the cell surface. There did not seem to be a striking dependence on the lipid composition of the liposomes. Electron microscopy using a negative stain shows liposomes bound to adenovirus. In some cases, the liposomes were still intact, but many liposomes, which were attached to the vertices of the virus, appeared lysed. These data support the notion that adenovirus, which enters the host cell by receptor-mediated endocytosis, gains access to the cytoplasm by a subsequent pH-dependent disruption of the membrane of the endocytic vesicle.
- Published
- 1986
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354. Binding and internalization of alpha 2-microglobulin by cultured fibroblasts. Effects of monovalent ionophores.
- Author
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Dickson RB, Schlegel R, Willingham MC, and Pastan I
- Subjects
- Animals, Cadaverine analogs & derivatives, Cadaverine pharmacology, Cells, Cultured, Coated Pits, Cell-Membrane metabolism, Endocytosis, Fibroblasts metabolism, Humans, Low Density Lipoprotein Receptor-Related Protein-1, Mice, Microscopy, Electron, Monensin pharmacology, Protein Binding, Rats, Receptors, Immunologic drug effects, Rhodamines metabolism, Temperature, Ionophores pharmacology, alpha-Macroglobulins metabolism
- Abstract
Receptor-bound alpha 2-macroglobulin (alpha 2M) undergoes a two-step process in its internalization by cultured fibroblasts. First, the receptor- alpha 2M complexes concentrate in coated pits on the cell surface. Second, the alpha 2M is internalized into endocytic vesicles we have termed receptosomes. Using a variety of monovalent ionophores and inhibitors of ATP synthesis, the present report provides data that discriminates between these two steps. Appearance of alpha 2M-receptor complexes in coated pits occurs at 4 degrees C and is inhibited by primary amines as well as some other drugs and chemical reagents [1, 2]. Internalization of alpha 2M-receptor complexes into receptosomes is inhibited by monovalent ionophores that disrupt proton gradients (monensin, nigericin, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and 3,3',4',5-tetrachlorosalicyanilide), but not the Na+ specific ionophore antamanide or the K+ specific ionophore valinomycin. Using electron microscopy, the proton ionophores appear to interfere with the transfer of alpha 2M from coated pits to receptosomes. Prolonged incubation with monensin in the presence of alpha 2M also decreases the number of alpha 2M receptors on the cell surface, but this did not appear sufficient to account for the extensive inhibition of internalization. Monensin also inhibited the internalization of vesicular stomatitis virus and epidermal growth factor (EGF). Our data suggest that a proton gradient may be necessary for receptor-mediated endocytosis of alpha 2M and some other ligands.
- Published
- 1982
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355. The E5 oncoprotein of bovine papillomavirus is oriented asymmetrically in Golgi and plasma membranes.
- Author
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Burkhardt A, Willingham M, Gay C, Jeang KT, and Schlegel R
- Subjects
- Amino Acid Sequence, Animals, Cell Fractionation, Cell Membrane ultrastructure, Genetic Vectors, Golgi Apparatus ultrastructure, Immunohistochemistry, Insect Viruses genetics, Intracellular Membranes ultrastructure, Mice, Molecular Sequence Data, Molecular Weight, Protein Conformation, Bovine papillomavirus 1 ultrastructure, Membrane Proteins ultrastructure, Papillomaviridae ultrastructure
- Abstract
The E5 oncoprotein of BPV-1 is a 44 amino acid, hydrophobic polypeptide which is present in membrane fractions of transformed cell homogenates. To define the specific cellular membrane compartments with which E5 associates, immunofluorescence and immunoelectron microscopy were performed using an affinity-purified antibody specific for the E5 carboxyl-terminus. Two cell systems which overexpressed the E5 protein were analyzed: (1) Balb/c 3T3 mouse cells transformed by a BPV-1 cDNA expressing the E2-E5 ORFs, and (2) Sf9 insect cells infected with recombinant baculovirus expressing the E5 ORF. In both cell types, E5 protein was found in the membranes of the Golgi apparatus with its carboxyl-terminus oriented intraluminally. In the Sf9 cells, which synthesize much greater quantities of viral protein, E5 protein was observed additionally in plasma membranes with the carboxyl-terminus facing extracellularly. The concentration of E5 protein within the Golgi and plasma membranes suggests several potential mechanisms for inducing cellular transformation.
- Published
- 1989
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356. Endocytosis and exocytosis: current concepts of vesicle traffic in animal cells.
- Author
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Willingham MC and Pastan I
- Subjects
- Animals, Cell Membrane physiology, Clathrin physiology, Coated Pits, Cell-Membrane ultrastructure, Endoplasmic Reticulum physiology, Golgi Apparatus physiology, Hormones physiology, Humans, Kinetics, Lysosomes physiology, Organoids ultrastructure, Coated Pits, Cell-Membrane physiology, Endocytosis, Endosomes physiology, Exocytosis, Organoids physiology
- Abstract
Animal cells have specific pathways to transport macromolecules from their surrounding environment to their interior, and from internal compartments to the cell surface or other intracellular locations. Many of these movements appear to be receptor-dependent processes in which specific membrane receptors bind macromolecules, segregate them into discrete membrane-limited compartments, and move the molecules to new locations. Such processes include the clustering and internalization of receptor-bound ligands at the cell surface in clathrin-coated pits, the formation of endocytic vesicles (receptosomes) from coated pits, the movement of receptosomes by saltatory motion to the Golgi system, the concentration of materials in the coated pits of the Golgi system that are destined for delivery to lysosomes, and the directed traffic of materials destined for exocytosis out of the Golgi to the cell surface. This review describes some of the experiments which have led to our current understanding of the various organelles involved in this traffic and some of the biochemical mechanisms involved.
- Published
- 1984
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357. Image intensification techniques for detection of proteins in cultured cells.
- Author
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Willingham MC and Pastan IH
- Subjects
- Animals, Cells, Cultured, Culture Techniques methods, Microscopy, Fluorescence instrumentation, Microscopy, Fluorescence methods, Proteins analysis
- Abstract
We describe here the components and uses of two image intensifier systems. The SIT camera system is convenient, relatively inexpensive, readily adaptable to most microscopes, and reliable. It suffers from lack of resolution at high gain levels and the inability to extend its sensitivity by accumulating an image over time. The EMI system is expensive and bulky and requires special adaptation to the microscope and special image recording devices in its output. It has extraordinary sensitivity and resolution, however, and allows experiments to be carried out that are otherwise not possible. Other systems similar to those described here are also commercially available and, in general, have similar advantages and disadvantages. The choice of the proper type of system varies with the particular application. These systems amplify the amount of light in an available image within constraints of gain and resolution and produce a publishable record of what otherwise might not be able to be recorded. These systems cannot improve images that contain too much background nor improve the resolution inherent in the microscopic method employed.
- Published
- 1983
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358. Co-clustering and internalization of low-density lipoproteins and alpha 2-macroglobulin in human skin fibroblasts.
- Author
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Via DP, Willingham MC, Pastan I, Gotto AM Jr, and Smith LC
- Subjects
- Bacitracin pharmacology, Cadaverine analogs & derivatives, Cadaverine pharmacology, Cells, Cultured, Coated Pits, Cell-Membrane metabolism, Endosomes metabolism, Fibroblasts ultrastructure, Humans, Methylamines pharmacology, Skin, Temperature, Endocytosis drug effects, Fibroblasts metabolism, Lipoproteins, LDL metabolism, alpha-Macroglobulins metabolism
- Published
- 1982
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359. Filamin-actin interaction. Dissociation of binding from gelation by Ca2+-activated proteolysis.
- Author
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Davies PJ, Wallach D, Willingham MC, Pastan I, Yamaguchi M, and Robson RM
- Subjects
- Adenosine Triphosphatases metabolism, Animals, Chickens, Enzyme Activation, Gels, Gizzard, Avian, Myosin Subfragments, Peptide Fragments, Peptide Hydrolases, Protein Binding, Actins metabolism, Calcium pharmacology, Carrier Proteins metabolism, Contractile Proteins metabolism
- Abstract
Chicken gizzard filamin has been digested with purified Ca2+-activated protease. The subunits of (Mr = 250,000) of the protein are cleaved asymmetrically into two fragments, heavy merofilamin, Mr = 240,000, and light merofilamin, Mr = 9,500. Digestion is complete at substrate to enzyme ratios of 100:1 and requires Ca2+ concentrations in excess of 0.3 mM. Heavy merofilamin binds to F-actin as evidenced by cosedimentation with F-actin, by direct observation under the electron microscope, and by its ability to inhibit actin activation of heavy meromyosin ATPase. Heavy merofilamin does not form a gel when mixed with actin, except at very low concentrations of KCl. Thus, actin binding and gelation are separable activities of filamin. We speculate that Ca2+-stimulated proteolysis may play a role in the regulation of actin-filamin interactions.
- Published
- 1978
360. Reversible and irreversible inhibitors of clustering of alpha 2M in clathrin-coated pits on the surface of fibroblasts.
- Author
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Dickson RB, Schlegel R, Willingham MC, and Pastan IH
- Subjects
- Animals, Cell Membrane drug effects, Cells, Cultured, Cytoplasmic Granules drug effects, Cytoplasmic Granules metabolism, Fibroblasts drug effects, Low Density Lipoprotein Receptor-Related Protein-1, Mice, Rats, Receptors, Immunologic drug effects, Receptors, Immunologic metabolism, Temperature, Amines pharmacology, Cell Membrane metabolism, Enzyme Inhibitors pharmacology, Fibroblasts metabolism, alpha-Macroglobulins metabolism
- Published
- 1982
- Full Text
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361. Self-association of chicken gizzard filamin and heavy merofilamin.
- Author
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Davies PJ, Wallach D, Willingham M, Pastan I, and Lewis MS
- Subjects
- Animals, Chickens, Filamins, Gels, Mathematics, Microscopy, Electron, Models, Biological, Molecular Weight, Protein Binding, Temperature, Ultracentrifugation, Contractile Proteins metabolism, Gizzard, Avian analysis, Microfilament Proteins
- Abstract
Filamin is a high molecular weight (subunit Mr 250 000) actin-binding protein isolated from smooth muscle. The protein forms a gel when mixed with solutions of F-actin. A proteolytic fragment of filamin, heavy merofilamin (subunit Mr 240 000), generated by the action of Ca2+-activated protease binds to actin but does not produce gelation. We have studied the self-association properties of filamin and heavy merofilamin by direct examination in the electron microscope and by equilibrium sedimentation distribution studies in the ultracentrifuge. Filamin self-associates reversibly to form dimers; the free energy of dimerization is approximately 7 kcal/mol. Further association to form tetramer and multimer appears to be irreversible. Warming of filamin solutions accelerates aggregation. Heavy merofilamin does not appear to self-associate but is entirely monomeric. These studies suggest that filamin produces gelation of F-actin by binding to actin and then self-associating to cross-link actin filaments into a gel.
- Published
- 1980
- Full Text
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362. Immunocytochemical localization in normal and transformed human cells in tissue culture using a monoclonal antibody to the src protein of the Harvey strain of murine sarcoma virus.
- Author
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Willingham MC, Banks-Schlegel SP, and Pastan IH
- Subjects
- Animals, Antibodies, Monoclonal, Cell Line, Cell Transformation, Viral, Fluorescent Antibody Technique, Harvey murine sarcoma virus physiology, Humans, Mice, Oncogene Protein p21(ras), Viral Proteins immunology, Cell Membrane analysis, Cell Transformation, Neoplastic, Epithelium analysis, Neoplasms analysis, Viral Proteins analysis
- Abstract
Using a rat monoclonal antibody directed against the p21 src protein of the Harvey strain of Murine Sarcoma Virus (MSV), we have examined the reactivity of human cells in tissue culture using immunofluorescence and electron microscopy. Qualitative results indicated that untransformed mouse and human fibroblastic cells have undetectable amounts of p21; these levels were greatly increased after transformation with Harvey MSV. A group of human tumor cell lines adapted to tissue culture were examined and almost all of the epithelial tumor lines showed significant localization with this antibody. Notable exceptions were two melanoma cell lines which were negative for p21 by immunofluorescence. When normal human epithelial cells derived from esophageal or foreskin epithelium were examined, the antibody showed significant reactivity with subconfluent growing cells. After the normal cells were allowed to become quiescent, the reactivity with this antibody decreased. All of the localization seen by fluorescence was in a distribution consistent with the previously demonstrated location of p21 scr on the inner aspect of the plasma membrane. Electron microscope localization showed labeling for this antigen on the inner surface of the plasma membrane in both transformed mouse cells and in the human tumor cell lines MCF-7 and HTB-2 (RT4). These results suggest that the amounts of p21-like proteins detectable in human epithelial tumor cells do not necessarily reflect their malignant potential, but may be related to their epithelial nature. The loss of detectable localization at quiescence suggests that p21 levels decrease when these epithelial cells stop growing, and raises the possibility that an analog of p21 may be used by these human epithelial cells to regulate cell growth.
- Published
- 1983
- Full Text
- View/download PDF
363. Clathrin and coated vesicle proteins Immunological characterization.
- Author
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Keen JH, Willingham MC, and Pastan I
- Subjects
- Animals, Antigen-Antibody Complex, Cattle, Clathrin, Fibroblasts immunology, Fluorescent Antibody Technique, Immune Sera, Membrane Proteins immunology, Mice, Molecular Weight, Nerve Tissue Proteins immunology, Rabbits immunology, Tropomyosin isolation & purification, Brain Chemistry, Membrane Proteins analysis, Nerve Tissue Proteins analysis
- Abstract
Antisera to a purified extract of bovine brain coated vesicles have been prepared. The extract contained clathrin (greater than 90%) and polypeptides of 35,000 (less than 5%) and 38,000 (less than 5%) molecular weight. Antibodies to clathrin were affinity purified on a homogeneous clathrin-Sepharose column and demonstrated to be monospecific by sensitive immunoprecipitation and immunofluorescence experiments. These antibodies give a fluorescence pattern consistent with coated pit localization in cultured cells from diverse species indicating extensive cross-reaction, and, thus, conservation of the clathrin antigen. Antibodies to the lower molecular weight proteins (LMWP) present in the clathrin preparation were also affinity purified on a column containing these polypeptides but devoid of clathrin. These antibodies cross-reacted completely and immunoprecipitated only clathrin from 3T3 cell extracts. Although the molecular weights and isoelectric points of the LMWP presented are quite similar to tropomyosins, these immunological results and limited protease digestion data indicate that tropomyosin and the LMWP are not related. Rather, the latter may be breakdown products of clathrin.
- Published
- 1981
364. Kinetics of transit of transferrin and epidermal growth factor through clathrin-coated membranes.
- Author
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Hanover JA, Willingham MC, and Pastan I
- Subjects
- Biological Transport, Carcinoma, Cell Line, Coated Pits, Cell-Membrane ultrastructure, ErbB Receptors, Humans, Kinetics, Microscopy, Electron, Mouth Neoplasms, Receptors, Transferrin, Clathrin physiology, Coated Pits, Cell-Membrane metabolism, Endosomes metabolism, Epidermal Growth Factor metabolism, Receptors, Cell Surface metabolism, Transferrin metabolism
- Abstract
The kinetics of association of epidermal growth factor (EGF) and diferric-transferrin (TF) with clathrin-coated membranes of KB cells were examined using anti-clathrin antibody bound to Staphylococcus aureus. The ligands were bound to cells at 4 degrees C and after warming to 30 degrees C for 5 min, both ligands were found concentrated in a membrane fraction immunoadsorbed with anti-clathrin antibody. Fifteen minutes after entry both ligands moved to a non-clathrin-associated compartment. Twenty to 25 min after entry, EGF, but not TF, appeared in a second clathrin-associated compartment. In parallel morphologic experiments, EGF-horseradish peroxidase (EGF-HRP) and TF-HRP were both found at 6 min in coated pits associated with the plasma membrane. At 22 min EGF-HRP, but not TF-HRP, was localized in Golgi-associated coated pits. These studies provide biochemical and morphological evidence suggesting that coated membranes in the Golgi region are involved at some stage in the transfer of EGF to lysosomes. The method should be of general utility in the study of receptor-mediated endocytosis.
- Published
- 1984
- Full Text
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365. Transglutaminase is essential in receptor-mediated endocytosis of alpha 2-macroglobulin and polypeptide hormones.
- Author
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Davies PJ, Davies DR, Levitzki A, Maxfield FR, Milhaud P, Willingham MC, and Pastan IH
- Subjects
- Amines pharmacology, Animals, Cell Line, Contact Inhibition, Cricetinae, Membrane Proteins metabolism, Peptides, Rats, Receptors, Drug metabolism, gamma-Glutamyltransferase antagonists & inhibitors, Endocytosis drug effects, Hormones metabolism, alpha-Macroglobulins metabolism, gamma-Glutamyltransferase metabolism
- Abstract
The receptor-mediated endocytosis of alpha 2-macroglobulin can be inhibited by a diverse group of chemical compounds all of which share the property of being inhibitors of one form of cellular transglutaminase. The present results strongly suggest that protein cross-linking may be essential for receptor-mediated endocytosis of some protein and polypeptide hormones.
- Published
- 1980
- Full Text
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366. The role of intracellular pH in ligand internalization.
- Author
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Hwang J, Pouyssegur J, Willingham MC, and Pastan I
- Subjects
- Ammonium Chloride metabolism, Ammonium Chloride pharmacology, Animals, Cell Line, Cricetinae, Cricetulus, ErbB Receptors, Fibroblasts metabolism, Ion Channels metabolism, Receptors, Cell Surface analysis, Receptors, Cell Surface drug effects, Sodium-Potassium-Exchanging ATPase metabolism, Body Fluids metabolism, Epidermal Growth Factor metabolism, Hydrogen-Ion Concentration, Intracellular Fluid metabolism, Transferrin metabolism
- Abstract
Internalization of EGF and transferrin measured as the rate of uptake of 125I-labeled ligands was compared in the cell line CCL39 and a mutant derivative, PS-120, lacking the Na+/H+ antiport system. No significant alteration was detected between the two cell lines. In contrast, pretreatment of the mutant cells PS-120 with 20 mM NH4Cl for 30 min to decrease persistently intracellular pH resulted in an increase in 125I-EGF and 125I-transferrin uptake by 60% and 25%, respectively. However, similar NH4Cl pretreatment of the parental cell line, CCL-39, which only affected intracellular pH very transiently did not cause an increase of ligand uptake. The binding of 125I-EGF to CCL-39 and PS-120 cells with or without NH4Cl pretreatment showed that NH4Cl pretreatment did not affect EGF binding in either CCL-39 or PS-120 cells. Since cells regulate intracellular pH by ion transport systems, we also examined the role of Na+, K+-ATPase. Ouabain, an inhibitor of Na+, K+-ATPases, showed no effect on 125I-EGF uptake in either of the cell types with or without NH4Cl pretreatment. Taken together, these results suggest that the plasma membrane-bound Na+/H+ antiport, a major pHi-regulating system in vertebrates, indirectly plays a role in ligand internalization through regulation of intracellular pH.
- Published
- 1986
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367. Journey to the center of the cell: role of the receptosome.
- Author
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Pastan IH and Willingham MC
- Subjects
- Animals, Cell Compartmentation, Cells, Cultured, Clathrin, Cytoplasm physiology, Exocytosis, Ligands, Membrane Fluidity, Membrane Proteins metabolism, Mice, Microscopy, Fluorescence, alpha-Macroglobulins metabolism, gamma-Glutamyltransferase metabolism, Endocytosis, Fibroblasts physiology, Receptors, Drug physiology
- Abstract
Fibroblasts contain a specific internalization pathway that carries hormones as well as some proteins and viruses from the cell surface to the cell interior. Initially, the ligands bind to mobile receptors that are randomly distributed on the cell surface. Next the ligand-receptor complexes are trapped and concentrated in specialized regions of the membrane termed bristle-coated pits. From the pit a smooth-walled vesicle containing the ligand forms and carries the ligand to the cell interior. Because of its role in receptor-mediated endocytosis, this vesicle has been termed a "receptosome."
- Published
- 1981
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368. Cellular transformation and the 'morphologic phenotype' of transformed cells.
- Author
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Pastan I and Willingham M
- Subjects
- Animals, Cell Adhesion, Cell Division, Contact Inhibition, Cricetinae, Cytoskeleton pathology, Insulin physiology, Membrane Proteins metabolism, Mice, Microvilli pathology, Neoplasm Proteins metabolism, Phosphoproteins metabolism, Protein Kinases metabolism, Species Specificity, Viral Proteins physiology, Avian Sarcoma Viruses genetics, Cell Transformation, Neoplastic pathology, Cyclic AMP metabolism, Genes, Viral
- Abstract
Expression of the product of the transforming gene (src) of RNA tumour viruses promotes growth and usually alters the adhesion, appearance and surface properties of cultured fibroblasts. The latter group of properties termed the 'morphologic phenotype' of transformed cells is largely due to diminished cell-to-substratum adhesion. The role of cyclic AMP, cell surface protein (CSP), and other factors in producing the 'morphologic phenotype' are discussed. The effects of src expression bear a striking resemblance to the action of peptide hormones such as insulin on appropriate target cells.
- Published
- 1978
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369. Adenovirus-induced release of epidermal growth factor and pseudomonas toxin into the cytosol of KB cells during receptor-mediated endocytosis.
- Author
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FitzGerald DJ, Padmanabhan R, Pastan I, and Willingham MC
- Subjects
- Cell Line, Chemical Phenomena, Chemistry, Cytosol metabolism, Cytosol microbiology, Endosomes microbiology, Endosomes ultrastructure, Exotoxins toxicity, Humans, Microscopy, Electron, Pseudomonas aeruginosa Exotoxin A, ADP Ribose Transferases, Adenoviruses, Human physiology, Bacterial Toxins, Endocytosis, Endosomes physiology, Epidermal Growth Factor metabolism, Exotoxins metabolism, Virulence Factors
- Published
- 1983
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370. Electron microscopic immunocytochemical localization of intracellular antigens in cultured cells: the EGS and ferritin bridge procedures.
- Author
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Willingham MC
- Subjects
- Actins metabolism, Animals, Cell Membrane Permeability, Cell Transformation, Viral, Cytoplasm ultrastructure, Ferritins immunology, Fixatives, Mice, Tubulin metabolism, Viral Proteins metabolism, alpha-Macroglobulins metabolism, Cells, Cultured ultrastructure, Immunochemistry methods
- Abstract
A new primary fixative, ethyldimethylaminopropyl carbodi-imide-glutaraldehyde-Tris, has been combined with the use of saponin for membrane permeabilization to yield a procedure which preserves ultrastructural morphology, yet retains a cytoplasmic matrix permeable to globulin molecules. This allows pre-embedding localization of intracellular protein antigens in cultured cells by fluorescence or electron microscopy. A further combination of these methods with the 'ferritin bridge' techniqe has allowed discrete localization which is quantifiable. Together, these methods yield an overall technique which provides high quality ultrastructural morphological preservation and precise antigen localization. Examples of the localization of alpha 2-macroglobulin, actin, SV40 T-antigen, tubulin and p60src are demonstrated. Extension of these methods from cultured cells to intact tissue should be possible without major changes.
- Published
- 1980
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371. Internalization of alpha 2 macroglobulin in receptosomes. Studies with monovalent electron microscopic markers.
- Author
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Dickson RB, Nicolas JC, Willingham MC, and Pastan I
- Subjects
- Animals, Cell Line, Cell Membrane metabolism, Immunoenzyme Techniques, Low Density Lipoprotein Receptor-Related Protein-1, Mice, Microscopy, Electron, Organoids metabolism, Endocytosis, Receptors, Immunologic metabolism, alpha-Macroglobulins metabolism
- Published
- 1981
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372. Molecular biology of drug resistance.
- Author
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Fojo A, Cornwell M, Cardarelli C, Clark DP, Richert N, Shen DW, Ueda K, Willingham M, Gottesman MM, and Pastan I
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Gene Amplification, Glycoproteins analysis, Glycoproteins genetics, Humans, KB Cells drug effects, Neoplasms analysis, Neoplasms drug therapy, Phenotype, Drug Resistance genetics
- Published
- 1987
- Full Text
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373. A role for intracellular traffic of high-density lipoproteins in cholesterol metabolism.
- Author
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Willingham MC
- Subjects
- Endocytosis, Humans, Lipoproteins, LDL metabolism, Liver metabolism, Macrophages metabolism, Receptors, LDL metabolism, Cholesterol metabolism, Lipoproteins, HDL metabolism
- Published
- 1989
374. Serial section analysis of clathrin-coated pits in rat liver sinusoidal endothelial cells.
- Author
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Goldenthal KL, Pastan I, and Willingham MC
- Subjects
- Animals, Clathrin analysis, Endocytosis, Endothelium ultrastructure, Female, Methods, Rats, Rats, Inbred Strains, Coated Pits, Cell-Membrane ultrastructure, Endosomes ultrastructure, Liver ultrastructure
- Abstract
Electron microscopy and serial sections were used to examine the shape of clathrin-coated pits in sinusoidal endothelial cells of rat livers. Livers were perfused at 4 degrees C with either concanavalin A-horseradish peroxidase (conA-HRP), or HRP alone, followed by warm-up to 37 degrees C and fixation with glutaraldehyde. Alternatively, the livers were perfused with HRP at 37 degrees C, followed by fixation. All tissue was preserved using a membrane contrast enhancement technique (R-OTO) consisting of sequential osmium-ferrocyanide, thiocarbohydrazide, and osmium-ferrocyanide treatment. Peroxidase reaction product was used to identify structures participating in endocytosis. One hundred and ninety-three clathrin-coated structures were examined. Sixty-six were from livers perfused with conA-HRP at 4 degrees C, 63 were from livers perfused with only HRP at 4 degrees C, and 64 were from livers perfused with HRP at 37 degrees C. These coated structures were morphologically classified into three categories: (a) flat pits; (b) cup-shaped pits; (c) pits with a narrow neck. No isolated coated vesicles were found. In cells perfused at 4 degrees C followed by warming to 37 degrees C, the percentage of coated pits found connected to the cell surface by narrow necks was 31%, using conA-HRP, and 27% using HRP alone. In cells perfused continuously at 37 degrees C, the percentage of coated pits with narrow neck connections was 21% using HRP alone. These results suggest that the formation of coated pits connected to the surface by narrow necks is not an artifact of cell type, of experimental protocol or of incubation with a lectin.
- Published
- 1985
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375. Epidermal-growth-factor-dependent transformation by a human EGF receptor proto-oncogene.
- Author
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Velu TJ, Beguinot L, Vass WC, Willingham MC, Merlino GT, Pastan I, and Lowy DR
- Subjects
- Animals, Cell Transformation, Neoplastic chemically induced, Cells, Cultured, DNA, Recombinant, ErbB Receptors drug effects, Fibroblasts pathology, Genetic Vectors, Harvey murine sarcoma virus genetics, Humans, Male, Mice, Mice, Nude, Neoplasms, Experimental etiology, Proto-Oncogene Mas, Recombinant Proteins genetics, Cell Transformation, Neoplastic genetics, Epidermal Growth Factor pharmacology, ErbB Receptors genetics, Proto-Oncogenes
- Abstract
The epidermal growth factor (EGF) receptor gene EGFR has been placed in a retrovirus vector to examine the growth properties of cells that experimentally overproduce a full-length EGF receptor. NIH 3T3 cells transfected with the viral DNA or infected with the corresponding rescued retrovirus developed a fully transformed phenotype in vitro that required both functional EGFR expression and the presence of EGF in the growth medium. Cells expressing 4 x 10(5) EGF receptors formed tumors in nude mice, while control cells did not. Therefore, the EGFR retrovirus, which had a titer on NIH 3T3 cells that was greater than 10(7) focus-forming units per milliliter, can efficiently transfer and express this gene, and increased numbers of EGF receptors can contribute to the transformed phenotype.
- Published
- 1987
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376. Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells.
- Author
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Haigler HT, Maxfield FR, Willingham MC, and Pastan I
- Subjects
- Animals, Biological Transport drug effects, Cadaverine pharmacology, Cell Membrane metabolism, Cells, Cultured, Dansyl Compounds pharmacology, Kinetics, Mice, Mice, Inbred BALB C, Receptors, Cell Surface metabolism, Cadaverine analogs & derivatives, Diamines analogs & derivatives, Epidermal Growth Factor metabolism, Peptides metabolism
- Abstract
The binding and internalization of 125I-epidermal growth factor (125I-EGF) was studied in cultures of BALB 3T3 cells using a novel method that involved removal of cell-surface hormone by treatment with acetic acid under conditions that did not remove internalized hormone. In control cultures, 125I-EGF initially bound to its receptor on the plasma membrane and then was rapidly internalized. After 30 min, only 15% of the cell-bound hormone remained on the surface. In contrast, cultures treated with dansylcadaverine retained 82% of the cell-bound hormone on the cell surface. We propose that dansylcadaverine inhibits EGF internalization by preventing it from clustering in clathrin-coated pits.
- Published
- 1980
377. Inhibition of VSV binding and infectivity by phosphatidylserine: is phosphatidylserine a VSV-binding site?
- Author
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Schlegel R, Tralka TS, Willingham MC, and Pastan I
- Subjects
- Animals, Binding Sites, Cell Line, Chlorocebus aethiops, Liposomes metabolism, Phosphatidylcholines pharmacology, Phosphatidylserines pharmacology, Simplexvirus growth & development, Vesicular stomatitis Indiana virus drug effects, Vesicular stomatitis Indiana virus growth & development, Viral Plaque Assay, Phosphatidylserines metabolism, Receptors, Virus metabolism, Vesicular stomatitis Indiana virus metabolism
- Abstract
Recently we described a saturable, high-affinity binding site for vesicular stomatitis virus (VSV) on the surface of Vero cells that appears to mediate viral infectivity. To isolate this binding site, we have extracted Vero cells with the detergent, octyl-beta-D-glucopyranoside. The dialyzed detergent extract specifically inhibits the saturable, high-affinity binding of 35S-methionine-labeled VSV to Vero cells. The inhibitory activity is resistant to protease, neuraminidase and heating to 100 degrees C. It is soluble in chloroform-methanol and inactivated by phospholipase C, suggesting that it is a phospholipid. Of various purified lipids tested, only phosphatidylserine was capable of totally inhibiting the high-affinity binding of VSV. The half-maximal inhibitory concentration for phosphatidylserine was 1 microM. Phosphatidylserine also inhibited VSV plaque formation by 80%-90%; Herpes simplex virus plaque formation was unaffected. Centrifugation and electron microscopy studies have shown that phosphatidylserine-containing liposomes bind to VSV. The finding that phosphatidylserine directly binds to VSV and inhibits VSV attachment and infectivity suggests that plasma membrane phosphatidylserine could function as a binding site or portion of a binding site for VSV.
- Published
- 1983
- Full Text
- View/download PDF
378. A novel fibrillar structure in cultured cells detected by a monoclonal antibody.
- Author
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Willingham MC, Richert ND, and Rutherford AV
- Subjects
- Animals, Antigens immunology, Cattle, Cell Line, Chickens, Fluorescent Antibody Technique, Histocytochemistry, Humans, Immunoenzyme Techniques, Intermediate Filaments ultrastructure, Mice, Microscopy, Electron, Rats, Species Specificity, Thiocarbamates immunology, Tubulin analysis, Vimentin analysis, Antibodies, Monoclonal, Cytoplasm ultrastructure, Thiocarbamates analysis
- Abstract
Using a monoclonal antibody, we have detected an antigen present in a unique fibrillar structure in the cytoplasm of cultured cells by immunofluorescence. These structures have been identified by transmission electron microscopy and ultrastructural immunocytochemistry as large single paracrystalline arrays of individual filaments morphologically similar to intermediate filaments. The antibody detects these structures in fibroblastic and epithelioid cultured cell lines of mouse, rat, bovine, and human origin but not of avian origin. Only a small percentage of the cells in a culture contains these structures; each cell usually contains only one, although two or more have been observed in a single cell. The structures are elongated vermiform arrays of filaments in the cytoplasm (approximately 0.5 X 3 microns) which have a thread-like or toroidal appearance. Because of this shape, we have named the putative antigen recognized by this antibody "nematin." Double-label experiments showed that these structures had no relationship to tubulin or vimentin. Immunocytochemical localization in human tissues revealed a high concentration of a reactive antigen in the stratum granulosum of skin and in what probably are neuroglial cells in the central nervous system. This monoclonal antibody may detect a novel intermediate filament protein and/or a shared determinant of different intermediate filament proteins.
- Published
- 1987
- Full Text
- View/download PDF
379. Immunotoxins.
- Author
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Pastan I, Willingham MC, and FitzGerald DJ
- Subjects
- Animals, Antibodies, Monoclonal therapeutic use, Biological Availability, Bone Marrow Transplantation, Endocytosis, Humans, Immunotoxins metabolism, Immunotoxins pharmacology, Lymphocyte Depletion, Neoplasms drug therapy, Neoplasms therapy, Neoplasms, Experimental drug therapy, Immunotoxins therapeutic use
- Published
- 1986
- Full Text
- View/download PDF
380. Antipeptide antibodies recognize c-erbA and a related protein in human A431 carcinoma cells.
- Author
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Fukuda T, Willingham MC, and Cheng SY
- Subjects
- Antibodies immunology, Antibody Specificity, Cell Nucleus metabolism, Cyanogen Bromide, Cytosol analysis, Cytosol metabolism, Fluorescent Antibody Technique, Humans, Immunosorbent Techniques, Molecular Weight, Peptide Fragments immunology, Placenta analysis, Proto-Oncogene Proteins immunology, Proto-Oncogene Proteins metabolism, Receptors, Thyroid Hormone metabolism, Serine Endopeptidases metabolism, Triiodothyronine metabolism, Tumor Cells, Cultured, Peptide Fragments analysis, Protein Precursors analysis, Proto-Oncogene Proteins analysis
- Abstract
Peptides corresponding to the deduced amino acid residues 15-29 of the amino-terminal region and 445-456 of the carboxyl-terminal region of the human placental c-erbA protein (hc-erbA-beta) were synthesized and used to produce site-specific rabbit polyclonal antipeptide sera. Antibodies to the carboxyl-terminal peptide (C-91) and the amino-terminal peptide (N-98) specifically immunoprecipitated the hc-erbA-beta proteins synthesized in vitro. Furthermore, 68% and 48% of the T3-binding activity of the hc-erbA-beta protein were immunoabsorbed by antibodies C-91 and N-98, respectively. These results indicate that C-91 and N-98 recognized hc-erbA-beta proteins. These antibodies were used to study the subcellular distribution of hc-erbA-beta protein in human cultured cells. Nuclear extracts were prepared from human A431 carcinoma cells; C-91 immunoabsorbed 50% of the specific T3-binding activity in these extracts. These results provide structural evidence to confirming that hc-erbA-beta is the T3 nuclear receptor. Cells were metabolically labeled with [35S]methionine, and the cytosolic extracts were immunoprecipitated by C-91 or N-98. A protein with a mol wt of 58,000 (Cp58) was specifically immunoprecipitated by N-98 or C-91. Peptide mapping by V8 digestion and cyanogen bromide cleavage showed that the Cp58 molecules immunoprecipitated by N-98 or C-91 was identical. Indirect immunofluorescence using N-98 or C-91 indicated that Cp58 is present exclusively in the cytoplasm. Other human cultured cells, HepG2, MCF-7, IM-9, and KB, were also evaluated, and similar results were found. These results raised the possibility that a precursor of hc-erbA-beta may be present in the cytosol. The functional significance of the hc-erbA-beta-related cytosolic Cp58 remains to be established.
- Published
- 1988
- Full Text
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381. Quantitative determination of the lateral diffusion coefficients of the hormone-receptor complexes of insulin and epidermal growth factor on the plasma membrane of cultured fibroblasts.
- Author
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Schlessinger J, Shechter Y, Cuatrecasas P, Willingham MC, and Pastan I
- Subjects
- Azides pharmacology, Binding, Competitive, Cell Line, Cell Membrane metabolism, Fibroblasts metabolism, Fluorescent Dyes, Kinetics, Epidermal Growth Factor metabolism, Insulin metabolism, Peptides metabolism, Receptor, Insulin metabolism, Receptors, Cell Surface metabolism
- Abstract
Fluorescent derivatives of insulin and epidermal growth factor bound to 3T3 mouse fibroblasts are mobile on the cell surface, with similar diffusion coefficients, D approximately (3--5) x 10(-10) cm2/sec at 23 degrees C. Increasing the temperature to 37 degrees C results in rapid receptor immobilization. The immobilization is attributed to aggregation of hormone-receptor complexes, their internalization, or a combination of both processes.
- Published
- 1978
- Full Text
- View/download PDF
382. The cellular entry of EGF and transferrin: a problem in intracellular sorting.
- Author
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Pastan I, Hanover J, and Willingham M
- Subjects
- Cell Membrane metabolism, ErbB Receptors, Humans, KB Cells ultrastructure, Microscopy, Electron, Models, Biological, Organoids metabolism, Organoids ultrastructure, Receptors, Transferrin, Epidermal Growth Factor metabolism, Receptors, Cell Surface metabolism, Transferrin metabolism
- Published
- 1985
- Full Text
- View/download PDF
383. An immunoperoxidase study of senile cerebral amyloidosis with pathogenetic considerations.
- Author
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Powers JM, Schlaepfer WW, Willingham MC, and Hall BJ
- Subjects
- Amyloid immunology, Amyloidosis pathology, Amyloidosis physiopathology, Antibodies immunology, Arteries immunology, Cerebrovascular Circulation, Congo Red, Humans, Immunoenzyme Techniques, Nerve Tissue Proteins immunology, Neurofilament Proteins, Amyloidosis immunology, Brain Diseases immunology, Dementia immunology
- Abstract
Samples of human cerebral cortex were obtained from twelve autopsied patients with Alzheimer's disease or "normal" aging. Rabbit or goat anti-human antisera to the following plasma proteins: IgG, F(ab')2, Fc, kappa and lambda light chains, IgM, IgA, fibrinogen, albumin, C3, lysozyme, haptoglobin, macroglobulin, and microglobulin; antibodies to the following intracellular proteins: glial fibrillary acidic (GFA) protein, filamin, actin, non-muscle myosin, tubulin, cholinergic vesicle proteins, and neurofilament (NF) proteins were utilized in the immunoglobulin peroxidase bridge. Amyloid cores of classical or perivascular plaques and dyshoric angiopathy exhibited a strong reaction for intact IgG and for both of its light chains, moderate reactions for lysozyme, fibrinogen, albumin and IgA, and weak reactions for IgM, C3, Fc, F(ab')2, haptoglobin, macroglobulin and microglobulin. Antibodies to all three NF proteins, individually and pooled, stained dyshoric and plaque amyloid, while antibodies to other intracellular proteins did not. The coronae of classical plaques and many primitive plaques stained for GFA, but inconsistently for IgG, both light chains, lysozyme, actin, tubulin, and NF proteins. Affected vessels of three patients with Congophilic angiopathy were reactive for all plasma proteins (especially IgG, fibrinogen, and albumin) and for NF proteins. NF staining in Congophilic blood vessels, although variable, revealed a peripheral or adventitial distribution, whereas plasma proteins tended to be localized in the media of the vessel wall. The distributions of Congo red and NF positivity were often identical. Both NF and Congo red staining was sensitive to oxidation. Isolated NF proteins were Congophilic and capable of displaying apple-green birefringence. A hypothesis concerning the role of NF proteins in senile cerebral amyloid is presented.
- Published
- 1981
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384. Cyclic AMP and cell behavior in cultured cells.
- Author
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Willingham MC
- Subjects
- Adrenal Glands cytology, Agglutination drug effects, Biological Transport drug effects, Bucladesine pharmacology, Cell Adhesion drug effects, Cell Differentiation drug effects, Cell Division drug effects, Cell Membrane metabolism, Cell Movement drug effects, Cell Transformation, Neoplastic drug effects, Cells, Cultured cytology, Contact Inhibition, Cyclic AMP pharmacology, Enzyme Induction drug effects, Glycosaminoglycans biosynthesis, Models, Biological, Muscles cytology, Neurons cytology, Neurons drug effects, Skin cytology, Steroids biosynthesis, Cells, Cultured physiology, Cyclic AMP physiology
- Published
- 1976
- Full Text
- View/download PDF
385. Nuclear protein import: specificity for transport across the nuclear pore.
- Author
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Wolff B, Willingham MC, and Hanover JA
- Subjects
- Amino Acid Sequence, Animals, Biological Transport, Blotting, Western, Cell Line, Microinjections, Microscopy, Electron, Microscopy, Fluorescence, Molecular Sequence Data, Nuclear Envelope metabolism, Peptides metabolism, Phycoerythrin metabolism, Temperature, Time Factors, Cell Nucleus metabolism, Nuclear Proteins metabolism
- Abstract
Transport of proteins into the cell nucleus is thought to require specific localization sequences and may be mediated by nuclear pores. Following microinjection into fused cultured cells, nuclear protein import was directly monitored by fluorescence microscopy using B-phycoerythrin (PE; Mr 240,000) coupled to synthetic peptides corresponding to the simian virus 40 (SV-40) large T antigen nuclear localization signal. Peptides with a single amino acid replacement found in a cytoplasmic mutant of T antigen (cT) failed to promote uptake. Further studies with deletion peptides revealed the minimum sequence requirements for efficient nuclear import of PE conjugates to be similar to those previously defined genetically for large T antigen itself. No competitive inhibition of uptake was observed in cells expressing nuclear or cytoplasmic T antigen. Nuclear import was time- and temperature-dependent. The lectin wheat germ agglutinin (WGA) binds to glycoproteins bearing O-linked GlcNAc on the cytoplasmic face of the nuclear pore in vitro [J.A. Hanover et al. (1987) J. Biol. Chem. 262, 9887-9894] and in vivo. Microinjection of WGA into the cytoplasm of living cells did not alter the diffusion of dextran (Mr 10,000) into the nucleus, but blocked the uptake of PE conjugates. This inhibition was reversed when a competing saccharide was introduced into the cytoplasm.
- Published
- 1988
- Full Text
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386. Chemoimmunotoxin therapy against a human colon tumor (HT-29) xenografted into nude mice.
- Author
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Pearson JW, FitzGerald DJ, Willingham MC, Wiltrout RH, Pastan I, and Longo DL
- Subjects
- Animals, Antibodies, Monoclonal immunology, Antigens, Neoplasm immunology, Ascites, Cell Survival drug effects, Combined Modality Therapy, Cyclophosphamide therapeutic use, Drug Resistance, Humans, In Vitro Techniques, Mice, Mice, Nude, Neoplasm Transplantation, Pseudomonas aeruginosa, Tumor Cells, Cultured, Pseudomonas aeruginosa Exotoxin A, ADP Ribose Transferases, Bacterial Toxins, Colonic Neoplasms therapy, Exotoxins administration & dosage, Immunotoxins therapeutic use, Virulence Factors
- Abstract
The efficacy of intracavitary chemoimmunotoxin therapy for cancer treatment was evaluated using the human colon carcinoma (HT-29) which had been xenografted i.p. into nude mice. Mice bearing HT-29 were treated with an immunotoxin consisting of the monoclonal antibody OVB3 coupled to Pseudomonas exotoxin (OVB3-PE), with cyclophosphamide (Cy), or with both OVB3-PE plus Cy. Mice given injections i.p. of 3 x 10(6) HT-29 ascites cells developed a localized disease that presented as both malignant ascites and solid tumor confined to the peritoneal cavity. All mice died within 30 to 40 days. Mice that received either three or six injections of OVB3-PE at a dose of 0.5 micrograms every other day beginning 3 days post-tumor inoculation exhibited significantly increased median survival times (MSTs) (P = 0.002) of 62 and 68 days, respectively, as compared to a MST of 33 days for the controls. OVB3 alone or an irrelevant monoclonal antibody conjugated to PE exhibited no antitumor activity. The therapeutic effects of the immunotoxin could be blocked by giving a large amount of unconjugated OVB3 at the same time. Treatment of mice with Cy alone at the maximal tolerated dose (250 mg/kg) on Days 10 and 17 after tumor inoculation increased the MST from 33 days to 54 days. The maximum tolerated dose could be increased to 300 mg/kg per injection if the Cy treatment was preceded by 100 mg/kg of S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721), a sulfhydryl compound that selectively protects normal tissue against the toxicity of radiation and alkylating agents. Cy plus WR-2721 treatment on Days 10 and 17 increased the MST from 35 to 61 days (P = 0.002). Interestingly, groups of mice that received either two, four, or seven treatments of OVB3-PE following Cy plus WR-2721 therapy exhibited a further increase (P less than 0.002) in MSTs to 81, 87, and 96 days, respectively. Thus, the combination of cytoreductive chemotherapy with the OVB3-PE was significantly more effective for the intracavitary treatment of established HT-29 colon cancer xenografts than either chemotherapy or immunotoxin therapy alone.
- Published
- 1989
387. A high molecular weight phosphoprotein in cultured fibroblasts that associates with polymerized tubulin.
- Author
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Klein I, Willingham M, and Pastan I
- Subjects
- Cell Line, Cell Membrane analysis, Fibroblasts, Microsomes analysis, Molecular Weight, Phosphates metabolism, Phosphoproteins analysis, Subcellular Fractions analysis, Glycoproteins metabolism, Microtubules analysis, Phosphoproteins metabolism, Tubulin metabolism
- Published
- 1978
- Full Text
- View/download PDF
388. Prelysosomal divergence of transferrin and epidermal growth factor during receptor-mediated endocytosis.
- Author
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Dickson RB, Hanover JA, Willingham MC, and Pastan I
- Subjects
- Centrifugation, Density Gradient, Epithelium metabolism, ErbB Receptors, Humans, Microscopy, Fluorescence, Receptors, Cell Surface metabolism, Receptors, Transferrin, Endocytosis, Epidermal Growth Factor metabolism, Lysosomes metabolism, Transferrin metabolism
- Abstract
The routes followed by epidermal growth factor and transferrin during their endocytosis by human epithelial cells were compared in double-label studies by using density gradient centrifugation of cell homogenates and fluorescence microscopy with intact cells. Gradient centrifugation studies of cells incubated with radioactively labeled epidermal growth factor and transferrin indicated that both ligands initially were associated with a class of vesicles having a density of 1.037 g/mL and then were rapidly transferred to a membrane compartment having a slightly higher density (1.039 g/mL). Subsequently, the two ligands diverged. Epidermal growth factor ultimately was transferred to a membranous compartment containing lysosomal enzymes (density (1.08 g/mL) where it was degraded. Transferrin was released intact from the cells; very little was transferred to lysosomes. Using fluorescently labeled ligands, it was observed that after cells were warmed to 37 degrees C for 5 min, transferrin and epidermal growth factor gave coincident, punctate fluorescent patterns, strongly suggesting they were localized within the same endocytic vesicles. Subsequently, the epidermal growth factor signal was observed in lysosomes whereas the transferrin signal became weaker and diffuse and did not coincide with the punctate epidermal growth factor fluorescence. The time course of the divergence of the radioactive and fluorescent ligands coupled with the previous morphologic studies on the pathway of epidermal growth factor internalization [Willingham, M. C., & Pastan, I. (1982) J. Cell Biol. 94, 207-212] suggests that the sorting process is prelysosomal and possibly Golgi associated.
- Published
- 1983
- Full Text
- View/download PDF
389. Morphologic demonstration of clathrin-coated pits in frog and turkey erythrocytes.
- Author
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Willingham MC, Strader CD, Lefkowitz RJ, and Pastan I
- Subjects
- Animals, Clathrin blood, Coated Pits, Cell-Membrane analysis, Erythrocytes analysis, Fluorescent Antibody Technique, Microscopy, Electron, Clathrin analysis, Coated Pits, Cell-Membrane ultrastructure, Endosomes ultrastructure, Erythrocytes ultrastructure, Ranidae blood, Turkeys blood
- Abstract
We have examined nucleated erythrocytes of frog and turkey for the presence of clathrin-coated structures using electron microscopy and immunocytochemistry. By electron microscopy, coated pits were found on the plasma membrane of peripheral blood erythrocytes of both species. These structures had an appearance similar to coated pits seen in non-erythroid mammalian cells. Using immunofluorescence with anti-(bovine) clathrin antibody, erythrocytes of both species showed punctate membrane fluorescence similar to the pattern of coated pits seen in other cells. By both methods, frog erythrocytes showed considerable heterogeneity, such that only about 50% of the cells showed significant numbers of coated pits, usually fewer than 20-50 per cell. In contrast, the vast majority of turkey erythrocytes showed no detectable coated pits, but occasional cells (less than 10%) showed large numbers of coated structures. These results suggest that a functional endocytic system may be present in a subpopulation of these nucleated erythrocytes. These findings may be of significance in understanding the ligand-induced loss of some receptors from the surface of these cells, and may serve as an indication of morphologic differentiation.
- Published
- 1984
- Full Text
- View/download PDF
390. Enhancement of the activity of immunotoxins made with either ricin A chain or Pseudomonas exotoxin in human ovarian and epidermoid carcinoma cell lines.
- Author
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Pirker R, FitzGerald DJ, Willingham MC, and Pastan I
- Subjects
- Cadaverine analogs & derivatives, Cadaverine pharmacology, Calmodulin antagonists & inhibitors, Cell Line, Chlorpromazine pharmacology, Drug Synergism, Female, Humans, Trifluoperazine pharmacology, Verapamil pharmacology, Pseudomonas aeruginosa Exotoxin A, ADP Ribose Transferases, Bacterial Toxins, Carcinoma, Squamous Cell pathology, Exotoxins, Immunotoxins pharmacology, Ovarian Neoplasms pathology, Ricin, Virulence Factors
- Abstract
The present study evaluates whether the in vitro activity of immunotoxins can be enhanced by verapamil or by various antagonists of calmodulin (dansylcadaverine, trifluoperazine, chlorpromazine). The following immunotoxins made with either Pseudomonas exotoxin (PE), recombinant ricin A chain (rRTA), or ricin A chain (RTA) were used: HB21-PE and 454A12-rRTA that both recognize the human transferrin receptor; and 260F9-rRTA and 454C11-RTA that both react with human ovarian and breast cancer cells. The cytotoxicity of these immunotoxins was determined in human ovarian carcinoma cell lines and KB cells. Verapamil, that was demonstrated previously to enhance the cell-killing activity of PE immunotoxins, enhanced the activity of several ricin A chain immunotoxins, including 454A12-rRTA, 260F9-rRTA, and 454C11-RTA. Comparing 50% inhibitory dose values for inhibition of protein synthesis by 454A12-rRTA, enhancement ranged from 2- to greater than 25-fold, was dependent on the concentration of verapamil, and was greatest at short incubation times. In addition, the cytotoxicity of HB21-PE and of selected RTA immunotoxins was increased up to 30-fold by the addition of various calmodulin antagonists. The enhancing drugs did not decrease the specificity of the immunotoxins.
- Published
- 1988
391. Inhibitors of 125I-epidermal growth factor internalization.
- Author
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Haigler HT, Willingham MC, and Pastan I
- Subjects
- Animals, Azo Compounds pharmacology, Bacitracin pharmacology, Cells, Cultured, Chlorpromazine pharmacology, Methylamines pharmacology, Phenylglyoxal pharmacology, Quinacrine pharmacology, Rats, Acetophenones pharmacology, Endocytosis drug effects, Epidermal Growth Factor antagonists & inhibitors, Peptides antagonists & inhibitors, Phospholipases antagonists & inhibitors, Phospholipases A antagonists & inhibitors
- Published
- 1980
- Full Text
- View/download PDF
392. Expression of a multidrug resistance gene in human cancers.
- Author
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Goldstein LJ, Galski H, Fojo A, Willingham M, Lai SL, Gazdar A, Pirker R, Green A, Crist W, and Brodeur GM
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1, Drug Resistance genetics, Humans, Neoplasm Recurrence, Local, Membrane Glycoproteins genetics, Neoplasms genetics, RNA, Messenger analysis
- Abstract
Many cancers have been cured by chemotherapeutic agents. However, other cancers are intrinsically drug resistant, and some acquire resistance following chemotherapy. Cloning of the cDNA for the human MDR1 gene (also known as PGY1), which encodes the multidrug efflux protein P-glycoprotein, has made it possible to measure levels of MDR1 RNA in human cancers. We report the levels of MDR1 RNA in greater than 400 human cancers. MDR1 RNA levels were usually elevated in untreated, intrinsically drug-resistant tumors, including those derived from the colon, kidney, adrenal gland, liver, and pancreas, as well as in carcinoid tumors, chronic myelogenous leukemia in blast crisis, and cell lines of non-small cell carcinoma of the lung (NSCLC) with neuroendocrine properties. MDR1 RNA levels were occasionally elevated in other untreated cancers, including neuroblastoma, acute lymphocytic leukemia (ALL) in adults, acute nonlymphocytic leukemia (ANLL) in adults, and indolent non-Hodgkin's lymphoma. MDR1 RNA levels were also increased in some cancers at relapse after chemotherapy, including ALL, ANLL, breast cancer, neuroblastoma, pheochromocytoma, and nodular, poorly differentiated lymphoma. Many types of drug-sensitive and drug-resistant tumors, including NSCLC and melanoma, contained undetectable or low levels of MDR1 RNA. The consistent association of MDR1 expression with several intrinsically resistant cancers and the increased expression of the MDR1 gene in certain cancers with acquired drug resistance indicate that the MDR1 gene contributes to multidrug resistance in many human cancers. Thus, evaluation of MDR1 gene expression may prove to be a valuable tool in the identification of individuals whose cancers are resistant to specific agents. The information may be useful in designing or altering chemotherapeutic protocols in these patients.
- Published
- 1989
- Full Text
- View/download PDF
393. Characterization and tumorigenicity of a butyrate-adapted T24 bladder cancer cell line.
- Author
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Flatow U, Rabson AB, Hand PH, Willingham MC, and Rabson AS
- Subjects
- Acetylation, Adaptation, Physiological, Animals, Butyric Acid, Culture Media, Drug Resistance, Genes, ras, Histones metabolism, Humans, Mice, Neoplasm Transplantation, Ouabain pharmacology, Tumor Cells, Cultured, Urinary Bladder Neoplasms genetics, Butyrates pharmacology, Urinary Bladder Neoplasms pathology
- Abstract
We have adapted T24P, a tumorigenic subline of the T24 human bladder cancer cell line, to grow in 5 mM butyrate. In the presence of butyrate, the adapted cells (T24P/B) grow more slowly than the unadapted cells (T24P/C), have a lower saturation density, increased serum requirement for growth, loss of ability to form colonies when plated at low cell density, and decreased ouabain sensitivity. Morphologically, T24P/B cells in butyrate are large and flattened with increased cytoplasm. When T24P/B cells are grown without butyrate, the morphological changes, growth rate, plating efficiency, and ouabain sensitivity return to those of T24P/C. While the saturation density increases, it does not return to levels of T24P/C, and the size of colonies never reaches that of the T24P/C colonies. Both T24P/C and T24P/B are tumorigenic in nude mice, however, the T24P/B tumors differ grossly and microscopically from those produced by T24P/C in that they contain large cystic structures filled with clear fluid and lined by transitional cell epithelium with flattened surface layers. Although the transformed phenotype and tumorigenicity of T24P are modified by adaptation to growth in butyrate, no significant changes in ras oncogene RNA or protein expression were identified.
- Published
- 1989
- Full Text
- View/download PDF
394. Alpha 2-macroglobulin binding to cultured fibroblasts: identification by affinity chromatography of high-affinity binding sites.
- Author
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Hanover JA, Rudick JE, Willingham MC, and Pastan I
- Subjects
- Animals, Cattle, Cells, Cultured, Chromatography, Affinity, Chromatography, DEAE-Cellulose, Chromatography, Ion Exchange, Electrophoresis, Polyacrylamide Gel, Fibroblasts metabolism, Humans, Ligands, Low Density Lipoprotein Receptor-Related Protein-1, Receptors, Immunologic metabolism
- Abstract
Binding sites having the properties of high-affinity receptors for activated alpha 2-macroglobulin (alpha 2M) have been purified over 100-fold from membranes of spontaneously transformed NIH-3T3 cells (J. A. Hanover, S.-y. Cheng, M. C. Willingham, and I. H. Pastan [1983] J. Biol. Chem. 258, 370-377). To identify the molecular species involved in high-affinity binding, the solubilized receptor has been purified 500-fold by conventional procedures and further purified by affinity chromatography. After radioiodination of the 500-fold-purified preparation, the detergent-solubilized extract was applied to alpha 2M-Sepharose and an 85,000 +/- 5000 Mr species was selectively retained by the column. Binding of the 85,000 +/- 5000 Mr species to the affinity resin was inhibited by EDTA and by excess alpha 2M. Elution from the affinity column could be accomplished with bacitracin, a competitive inhibitor of alpha 2M binding, or with EDTA. Consistent with the previously reported characteristics of the high-affinity alpha 2M receptor, the 85,000 Mr species bound much more efficiently to methylamine-activated alpha 2M-Affigel than to alpha 2M-Affigel which had not been amine-activated. The present data suggest that a protein with a subunit Mr of 85,000 +/- 5000 may represent a component of the high-affinity alpha 2M receptor present on cultured fibroblasts.
- Published
- 1983
- Full Text
- View/download PDF
395. Monoclonal antibody 101 that precipitates the glycoprotein receptor for epidermal growth factor is directed against the Y antigen, not the H type 1 antigen.
- Author
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Le Pendu J, Fredman P, Richter ND, Magnani JL, Willingham MC, Pastan I, Oriol R, and Ginsburg V
- Subjects
- Animals, Carbohydrate Conformation, Carbohydrate Sequence, Carcinoma, Squamous Cell, Cell Line, Cell Membrane metabolism, Epidermal Growth Factor metabolism, Epitopes analysis, ErbB Receptors, Glycoproteins immunology, Humans, Hybridomas immunology, Mice, Receptors, Cell Surface metabolism, ABO Blood-Group System immunology, Antibodies, Monoclonal, H-Y Antigen immunology, Receptors, Cell Surface immunology
- Published
- 1985
- Full Text
- View/download PDF
396. Binding, surface mobility, internalization, and degradation of rhodamine-labeled alpha 2-macroglobulin.
- Author
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Maxfield FR, Willingham MC, Haigler HT, Dragsten P, and Pastan IH
- Subjects
- Animals, Binding Sites, Cells, Cultured, Fibroblasts metabolism, Humans, Mice, Protein Binding, Spectrometry, Fluorescence, Surface Properties, Rhodamines, Xanthenes, alpha-Macroglobulins metabolism
- Abstract
We have used quantitative fluorescence methods to examine the fate of rhodamine-labeled alpha 2-macroglobulin (R-alpha 2 M) after binding to cell-surface receptors on NRK and Swiss 3T3 cells. From measurements of fluorescence intensities in NRK cells fixed after incubation with R-alpha 2M, we found that uptake was saturable and that half-maximal uptake occurred at 130 nM R-alpha 2M. Fluorescence measurements on cell extracts of NRK and Swiss 3T3 cells also showed a half-maximal uptake of R-alpha 2M near 130 nM. We estimate that NRK cells can take up 10(6) molecules of R-alpha 2M per hour via receptor-mediated endocytosis. The mobility of alpha 2-macroglobulin receptors on the surface of Swiss 3T3 cells was measured by using fluorescence photobleaching recovery. The two-dimensional effective diffusion coefficient of R-alpha 2M receptors was approximately 8 X 10(-10) cm2 s-1, a value close to that previously obtained for insulin and epidermal growth factor receptors. Degradation of R-alpha 2M by the cells was followed by using the loss of fluorescence from the 185000-dalton band in sodium dodecyl sulfate--polyacrylamide gels. Rhodamine fluorescence was detected in the gels by using a microscope fluorescence spectrophotometer. NRK cells degraded alpha 2M to low molecular weight fragments with a t 1/2 of 15 min. Swiss 3T3 cells degraded about 75% of the alpha 2M with a t 1/2 of 1 h. The remaining 25% remained as the intact 185000-dalton peptide after 24 h. No significant accumulation of large breakdown products was observed in Swiss 3T3 or NRK cells.
- Published
- 1981
- Full Text
- View/download PDF
397. Involvement of Na+ and HCO-3 in receptor-mediated endocytosis of alpha 2-macroglobulin, epidermal growth factor, and vesicular stomatitis virus.
- Author
-
Dickson RB, Schlegel R, Willingham MC, and Pastan IH
- Subjects
- Animals, Cells, Cultured, Horseradish Peroxidase metabolism, Humans, Mice, Bicarbonates pharmacology, Endocytosis, Epidermal Growth Factor metabolism, Sodium pharmacology, Vesicular stomatitis Indiana virus physiology, alpha-Macroglobulins metabolism
- Abstract
alpha 2-Macroglobulin (alpha 2M), epidermal growth factor (EGF), and vesicular stomatitis virus (VSV) each enter cultured fibroblasts by receptor-mediated endocytosis. The present study defines some basic ionic requirements in the cell culture medium which are necessary for the maximal rate of endocytosis of these three ligands. Na+ and HCO-3 were both necessary for maximal endocytosis of 125I-alpha 2M, 125I-EGF, and 35S-VSV at 37 degrees C. The ion specificities for both the anion and cation requirements were established. The binding of 125I-alpha 2M to its cellular receptors at 4 degrees C was unaffected by the absence of Na+ and HCO-3 in the culture medium. In addition, the absence of Na+ and HCO-3 in the culture medium did not reduce cellular uptake of horseradish peroxidase by fluid phase endocytosis. Na+ and HCO-3 may be general requirements in receptor-mediated endocytosis.
- Published
- 1982
- Full Text
- View/download PDF
398. Receptor-mediated endocytosis of hormones in cultured cells.
- Author
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Pastan IH and Willingham MC
- Subjects
- Animals, Cells, Cultured, Humans, Kinetics, Receptors, Cell Surface metabolism, Endocytosis, Hormones metabolism, Receptors, Cell Surface physiology
- Published
- 1981
- Full Text
- View/download PDF
399. Isolation of receptosomes (endosomes) from human KB cells.
- Author
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Dickson RB, Hanover JA, Pastan I, and Willingham MC
- Subjects
- Endosomes metabolism, Enzymes metabolism, Epidermal Growth Factor metabolism, Humans, KB Cells, Lipid Metabolism, Neoplasms metabolism, Neoplasms ultrastructure, Cell Fractionation methods, Endosomes ultrastructure
- Published
- 1985
- Full Text
- View/download PDF
400. Ultrastructural immunocytochemical localization of clathrin in cultured fibroblasts.
- Author
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Willingham MC, Keen JH, and Pastan IH
- Subjects
- Animals, Antigen-Antibody Reactions, Cell Line, Clathrin, Fibroblasts, Mice, Microscopy, Electron, Organoids analysis, Cell Membrane analysis, Golgi Apparatus analysis, Intracellular Membranes analysis, Membrane Proteins analysis
- Published
- 1981
- Full Text
- View/download PDF
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