bluuebunny/crossref_metadata_2025_split huggingface dataset:
Integral Membrane Glycoproteins Related to Cell–Substratum Adhesion in Mammalian Cells
Authors: Damsky Caroline H. , Knudsen Karen A. , Buck Clayton A. 
Published: December 2004

AbstractBroad spectrum antisera have been raised against surface membrane‐derived material from baby hamster kidney cells and mouse mammary tumor epithelial cells. These antisera disrupt cell–substratum adhesion in their respective cell types. Using an antibody neutralization (blocking) assay, adhesion‐related glycoproteins have been isolated from non‐ionic detergent extracts of each cell type. The purified material in each case consisted of a restricted population of glycoproteins of approximately 120,000–160,000 Mr. Purified material from each system blocked the disruption of adhesion induced by the heterologous antiserum on either cell type. The antisera were capable of disrupting cell–substratum adhesion of a large number of cell types and species sources. In addition, antibody blocking activity could be detected from partially purified extracts of several adult hamster cell types and a variety‐of cultured cell types. Thus, in addition to having similar substratum–associated glycoproteins (eg, fibronectin) and cytoskeleton‐associated proteins (eg, α‐actinin and vinculin) cells from different species and tissue sources appear to have a relatively conserved class of integral membrane glycoproteins involved in cell substratum–adhesion.
Growth of Human T Lymphocyte Colonies From Whole Blood: Culture Requirements and Applications
Authors: Knox S.J. , Wilson F.D. , Greenberg B.R. , Shifrine M. 
Published: December 2004

AbstractGrowth of human lymphocyte colonies from whole blood following stimulation with PHA, Con A, or PPD is described. Individual colony cells were identified as T lymphocytes on the basis of surface marker and enzyme cytochemical characterizations. Colony formation increased as a power function over a wide range of cell concentrations above a critical minimal concentration. The whole blood culture system eliminates possible selective effects of lymphocyte colony techniques utilizing gradient‐enriched lymphocyte fractions and more closely approximates the in vivo milieu. The whole blood colony method is more sensitive for the detection of low‐level radiation effects on lymphocytes than widely used tests that measure 3H‐thymidine incorporation. In preliminary studies, we used the whole blood method to determine the relative radiosensitivity of lymphocytes from humans with various hematopoietic disorders, and observed abnormalties in mitogen responsiveness and colony formation in some of the patient groups. This method has wide application for studies in cellular and clinical immunology.
Differential Reactivation of Zinc‐Mediated Metallothionein Induction in Ultraviolet‐Irradiated Normal and Repair‐Deficient Human Cells
Authors: Hildebrand C.E. , Strniste G.F. 
Published: December 2004

AbstractThe ubiquitous, low‐molecular‐weight, thiol‐rich, metal‐binding protein, metallothionein (MT), can be induced in cultured normal human fibroblasts (NF) and xeroderma pigmentosum (XP) cells by exposure to ZnCl2. Both NF and XP cells tolerate up to 200 μM ZnCl2 in the growth medium. Upon addition of ZnCl2 (200 μM) to monolayer cultures, both NF and XP cells showed similar kinetics for the induction of MT synthesis: Within 7 hours the MT synthesis rate rose from a low, marginally detectable rate to a maximal rate at least 50‐fold greater than the basal rate. The induction of MT synthesis in both cell types was inhibited by actinomycin D (5 μg/ml), indicating that the induction process is controlled at the level of transcription. Exposure of NF or XP cells to far ultraviolet light (UV) followed by induction with ZnCl2 resulted in a UV dose‐dependent decrease in the maximal rate of MT synthesis measured 8.5 hours postirradiation. The UV sensitivity of the MT induction was greater in XP cells than in NF cells. However, considerations of the differential repair capacities of NF and XP cells superimposed upon the kinetics of MT induction were invoked to explain the apparent differential UV sensitivity of MT induction. Liquid holding recovery experiments showed that NF cells possess the capacity to reactivate this inducible gene function rapidly while XP cells arc deficient in the reactivation capacity. These results are discussed in the context of both UV transcriptional mapping of this inducible gene function and development of techniques for measuring repair of transcription‐blocking lesions.
Carcinogen‐Mediated Amplification of Specific DNA Sequences
Authors: Lavi Sara 
Published: December 2004

AbstractA model experimental system based on SV40‐transformed Chinese hamster embryo cells and a highly sensitive in situ hybridization procedure was designed. Exposure of the cells to different categories of chemical and physical carcinogens resulted in the induction of SV40 DNA synthesis in the treated cells. Although the carcinogen‐mediated amplification of SV40 DNA sequences is regulated by the viral “A” gene, neither infectious virus nor complete viral DNA molecules were rescued from the treated cells. A heterogenous collection of DNA molecules containing SV40 sequences was generated following treatment with DMBA. Restriction enzyme analysis of the amplified DNA molecules in the Hirt supernatant revealed that not all sequences in the integrated SV40 inserts are present. The possibility that the amplification of SV40 sequences is a reflection of a general gene amplification phenomenon mediated by carcinogens is discussed.
Expression of Adhesion‐Related Membrane Components in Adherent Versus Nonadherent Hamster Melanoma Cells
Authors: Knudsen Karen A. , Damsky Caroline H. , Buck Clayton A. 
Published: December 2004

AbstractThe existence of integral membrane components that are involved in cell–substratum adhesion has been postulated. Using an immunochemical approach developed in this laboratory, we provide further evidence for the role in cell–substratum adhesion of integral membrane glycoproteins within a molecular weight region of 120,000–140,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of material enriched approximately 100‐fold in adhesion‐related components revealed the 120,000–140,000 Mr glycoproteins in an adherent hamster melanoma cell line. These glycoproteins are greatly reduced in a non‐adherent variant. Induction of adhesion in these cells by exposure to BudR is accompanied by re‐expression of the surface adhesion antigens.
Immunochemical Analysis of Discoidins I and II at the Cell Surface in Wild Type and Aggregation‐Defective Mutants of Dictyostelium discoideum
Authors: Armant D. Randall , Berger Edward A. 
Published: December 2004

AbstractThe endogenous lectins discoidins I and II are believed to be primary components of the morphogenetic cell cohesion system of D discoideum. We have developed two immunochemical methods to analyze the association of the discoidins with the cell surface. One method is a two‐stage specific antibody binding assay in which intact cells are incubated on ice with rabbit serum (either control serum or antidiscoidin I and II), washed, then incubated with 125I‐Protein A. Specific antibody binding is defined as the difference between percent radioactivity bound with antidiscoidin versus control serum during the first stage. Substantial specific binding was observed with developed A3 cells but not with vegetative cells, and nearly all of the activity could be removed by pread‐sorption of the antiserum with discoidin‐Sepharose. As a complementary method, quantitative immunoadsorption analysis was performed in which we tested the ability of intact cells to remove antibodies reactive with purified 125I‐discoidin I or II. Developed cells, but not vegetative cells, were capable of adsorbing antibodies reactive with discoidin I as well as those reactive with discoidin II. This represents the first demonstration that both lectins are present on the surface of cohesive cells.These procedures, coupled with other methods to analyze soluble discoidin in cell extracts, were used to study discoidin expression in wild type cells and in two newly isolated aggregation‐defective mutants. Strain EB‐32 fails to aggregate and displays little or no discoidin in cell extracts or at the cell surface. On the other hand, strain EB‐18 forms loose amorphous mounds, and expresses substantial quantities of the discoidins, both in cell extracts and at the cell surface. These mutants should prove valuable in studying the organization and regulation of discoidins I and II at the surface of aggregating cells.
Evidence for the Functional Association of Enzyme I and HPr of the Phosphoenolpyruvate‐Sugar Phosphotransferase System With the Membrane in Sealed Vesicles of Escherichia coli
Authors: Saier Milton H. , Cox David F. , Feucht Brigette U. , Novotny Michael J. 
Published: December 2004

AbstractSeveral independent assay procedures were used to estimate the activities of the enzyme constituents of the phosphoenolpyruvate‐sugar phosphotransferase system (PTS) in osmotically shocked bacterial membrane vesicles. The soluble enzymes of the system were found to be in association with the membrane by several criteria. Phosphoenolpyruvate‐dependent sugar phosphorylation was catalyzed by this membrane‐bound enzyme system far more efficiently than by a mixture of the individual enzymes at corresponding concentrations. By contrast, the rates of the phosphoryl exchange reactions catalyzed by enzyme I and the enzyme II complexes were essentially the same for the associated and dissociated forms of the system. Functional association of the PTS‐enzyme complex was stabilized by Mg++ and phosphoenolpyruvate and could be destroyed by detergent treatment, sonication, or by passage of the vesicle preparation through a French pressure cell. These results lead to the possibility that in the intact bacterial cell the soluble enzymes of the phosphotransferase system exist, in part, as peripheral membrane constituents associated with the integral membrane enzyme II complexes.
Purification and Characterization of a Major Cell Surface Glycoprotein in Zajdela Ascites Hepatoma Cells Which Displays a Potent Concanavalin A Receptor Activity
Authors: Nato Farida , Bourrillon Roland 
Published: December 2004

AbstractA major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells.The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaIO4 followed by reduction with NaB3H4. Surface‐labeled glycoproteins were released by short incubations with TPCK‐trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II2, was then purified by chromatography on DEAE‐cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS‐polyacrylamide gels, isoelectric focusing, rechromatography on DEAE‐cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons.The location of GP II2on the cell surface was confirmed by the fact that it could be labeled metabolically with, D‐(3H) glucosamine and externally through the nonpenetrating periodate‐NaB3H4 system.GP II2could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II2could be an integral protein.Analysis of the carbohydrate composition of GP II2 revealed galactose, N‐acetylglucosamine, N‐acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O‐ and N‐linked to the polypeptide chain, most of them being O‐linked.Finally, GP II2has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.
Responses of Preneoplastic Epidermal Cells to Tumor Promoters and Growth Factors: Use of Promoter‐Resistant Variants for Mechanism Studies
Authors: Colburn Nancy H. , Wendel Edmund , Srinivas Leela 
Published: December 2004

AbstractThe JB6 mouse epidermal cell model system is being used to study the mechanism of promotion of transformation. Promotion of anchorage independence in JB6 cells occurs in response to second‐stage but not first‐stage promoters, and is inhibited by inhibitors of second‐stage not first‐stage promotion. A number of variants that are resistant to the phorbol diester TPA have been derived. Some are resistant to plateau density mitogenic stimulation by TPA; others are resistant to promotion of anchorage independence by TPA. Some of the mitogen‐resistant variants were promotable by TPA, thus ruling out a requirement for TPA mitogenesis in promotion of transformation in JB6 cells. TPA promotable clones were also sensitive to mezerein and EGF while the TPA nonpromotable variants were also resistant to mezerein and EGF, suggesting that sensitivity to promoters in these JB6 cells is determined at a level distal to receptor binding. Promotion sensitivity did not require available EGF receptors since two TPA promotable variants were EGF receptorless. The mitogenic response of JB6 cells to TPA may however be mediated by EGF since four of four mitogen‐resistant variants showed low to zero levels of EGF binding. Tumor promoting phorbol esters produce specific changes in cellular gangliosides. Certain of these changes occur in promotable but not nonpromotable variants of JB6 cells, suggesting that ganglioside changes may be involved in the process of promotion of transformation.
Differences in Metabolism of Chemical Carcinogens in Cultured Human Epithelial Tissues and Cells
Authors: Harris Curtis C. , Trump Benjamin F. , Grafstrom Roland , Autrup Herman 
Published: December 2004

AbstractThe metabolism of chemical carcinogens has been studied in cultured human bronchus, colon, duodenum, pancreatic duct, and esophagus. Metabolite patterns and carcinogen‐DNA adducts are generally qualitatively similar among animal species, individuals within a species, and tissues within an individual. However, wide quantitative differences are observed between individuals in out‐bred animal species, including humans. These interindividual differences in amounts of carcinogen‐DNA adducts and in activities of enzymes that are important in the metabolism of chemical carcinogens are similar in magnitude (10‐to 150‐fold) to those observed in pharmacogenetic studies of drug metabolism. The role of these differences as risk factors in human cancer is being investigated.
Changes in the Expression of Membrane Antigens During the Differentiation of Chicken Erythroblasts
Authors: Hayman Michael J. , Beug Hartmut , Savin Keith W. 
Published: December 2004

AbstractChicken erythroblasts can be transformed by the avian retrovirus, avian erythroblastosis virus (AEV). Earlier studies have shown that the mechanism of transformation appears to involve a “block” in differentiation, in that when erythroblasts are transformed by a temperature‐sensitive mutant of ts34 AEV and incubated at the nonpermissive temperature, the cells start to differentiate and produce hemoglobin. We have decided to use this system to isolate pure populations of chicken erythroblasts and raise monoclonal antibodies against their cell surface proteins. Three monoclonal antibodies were isolated and tested for their ability to bind to various hematopoietic cell types; two were shown to be erythroid‐specific, whereas the other antibody bound to proliferating cells but not to erythrocytes or granulocytes. Of the erythroid‐specific antibodies, one precipitated a 94,000 molecular weight protein, whereas the other precipitated a 11,000 molecular weight protein that was tentatively identified as hemoglobin. The use of this system and approach to identify and evaluate changes that occur during the differentiation is discussed.
Structural Characteristics of the Short‐Tail Fibers of T4 Bacteriophage
Authors: Zorzopulos Jorge , DeLong Sara , Chapman Virginia , Kozloff Lloyd M. 
Published: December 2004

AbstractThe characteristics of pure preparations of short‐tail fibers of bacteriophage T4 have been studied in the optical and electron microscope. Three main structures were observed: 1) spheres of 8.1 nm diameter; 2) fibers 43 nm long and 3.8 nm thick; and 3) fibers 54 nm long and 3.2 nm thick. Both types of fibers exhibited a regular beaded appearance. The 43‐nm fibers were the most abundant structure. During the process of purification of the short‐tail fibers, the formation of aggregates was observed each time the material containing the short‐tail fibers was dialyzed against saline solutions. These aggregates became increasingly fibrous (as observed in the optical microscope) as the material used was increasingly enriched in short‐tail fibers. Finally, most of the aggregates were of the fibrous type when they were formed from a purified preparation of short‐tail fibers. In the electron microscope, it was found that the filamentous aggregates were organized in well‐defined bundles. The amino acid composition of the highly purified short‐tail fibers was also determined. Among the known fibrous proteins, the ones that most resemble the amino acid composition of the short‐tail fibers are actin and fibrinogen. These observations are discussed in relation to the T4 short‐tail fiber structure and their localization on the hexagonal baseplate of the T4 tail structure.
The Role of Regulatory Components From Resident T Lymphocytes in Polyclonal B Cell Activation
Authors: Goodman Michael G. , Weigle William O. 
Published: December 2004

AbstractResident T lymphocytes have been found to exert helper and suppressor regulatory influences with regard to polyclonal activation of murine splenic B lymphocytes elicited by lipopolysaccharide. In the normal adult spleen, only T cell helper influences are exercised over polyclonal B cell activation. This activity is a property of Lyt 1+2− T cells and does not appear to be subject to MHC restriction. Suppressive influence evidently is either latent or it exists at such a low level that its effects are difficult to detect. No regulatory activity can be recovered from the supernatants of T cells, cultured either with or without LPS. However, suppressor T cell function may be evoked by activating splenic T cells with Concanavalin A or by sonicating unstimulated splenic T cells in order to liberate a suppressive potential which is not expressed by these unstimulated cells when intact. The soluble fraction of resident splenic T cell sonicates exerts both helper and suppressor regulatory influences. The soluble helper activity is derived from Lyt 1+2− T cells, whereas suppressor activity is generated from Lyt 1−2+ T cells. The suppressive activity of T cell sonicates is not restricted by the MHC gene complex. Helper and suppressor activities contained in splenic T cell sonicates were separated by gel chromatography; the suppressive activity was found to elute with a molecular weight between 68,000 and 84,000 daltons, and the helper activity eluted with a molecular weight between 15,000 and 23,000 daltons. The data indicate that helper and suppressor activities are distinct molecular entities derived from distinct splenic T lymphocyte subpopulations. The possibility that these molecules are precursors to or components of antigen‐specific or nonspecific helper and suppressor factors described in the literature is discussed.
Occluding Junctions in MDCK Cells: Modulation of Transepithelial Permeability by the Cytoskeleton
Authors: Meza Isaura , Sabanero Myrna , Stefani Enrique , Cereijido Marcelino 
Published: December 2004

AbstractIn MDCK cell monolayers the opening and resealing of occluding junctions can be induced by removal and restoration of calcium to the external medium. The overall changes in permeability of the occluding junctions in the monolayer can be monitored by the drop and recovery of the total transepithelial electrical resistance. We have investigated the effects of cytochalasin B (CB) on this process. When CB is added to sealed monolayers there is a gradual drop in the electrical resistance across the monolayer. This drop is accompanied by a slow disorganization of the microfilament pattern of these cells, including a disturbance of a ring of cortical microfilaments that is normally associated with the junctions. Cells in open monolayers treated with CB will not reseal and have an altered filament distribution. These cells do not have a continuous cortical ring.We have used a voltage scanning technique that uses a microelectrode to measure the resistance at selected points along the junction which surrounds a single cell. In untreated, closed monolayers, the junction is heterogeneous with alternating points of high and low conductance. In closed monolayers treated with CB, although there are low conductance points, we have observed an increased frequency of high conductance points that correlates with the change in the overall conductance. The frequency of high conductance points along the junction and the overall conductance both increase with time of exposure to CB.In an effort to understand the molecular basis for the permeability changes induced by EGTA and CB, we have looked for differences in the protein components of the cell membranes of open, closed, and CB‐treated MDCK monolayers. This was done by radioiodinating the surface membrane proteins under control and experimental conditions that bring about permeability changes. No significant differences in the labeled protein patterns were found under these conditions. These results suggest that the permeability changes involve only a structural rearrangement of membrane components. In addition we have observed that about 36% of the surface label remains bound to the insoluble cytoskeletons obtained from cells in control and experimental conditions that alter the permeability of the tight junctions. The iodinated proteins attached to the CS include polypeptides with Mr of ≥ 120K daltons as well as peptides with Mr = 56K, 50K, 36K, and 18K daltons.
Inhibition by Glucocorticoids of Endocytosis in a Macrophage‐Like Cell Line
Authors: Miller Steven C. , Melnykovych George 
Published: December 2004

AbstractA macrophage‐like cell line (P388D1) has been used to demonstrate that glucocorticoids inhibit the fluid‐phase endocytosis of fluorescein‐labeled dextran (FITC‐dextran). Initial experiments demonstrated that the interaction of FITC‐dextran with cells had all the features of fluid‐phase uptake, ie, the amount taken up was proportional to the concentration in the medium, the uptake proceeded continuously with time and was blocked at 4°C. Dexamethasone (10−7M) had no effect on endocytosis until 11 hours after addition of the steroid, when it inhibited the uptake of FITC‐dextran by 35%. The amount of inhibition increased with longer exposure times to the hormone up to 50% after 22 hours. Although this effect on endocytosis was Observed prior to any effect on growth of the cells, endocytosis as well as cell proliferation were inhibited in a dose‐dependent fashion. A preliminary survey of selected steroids has established that the inhibition of endocytosis was restricted to steroids of the glucocorticoid class. The key experiments were also performed using horseradish peroxidase instead of FITC‐dextran with, essentially, identical results.