bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vesicles isolated from ATP‐depleted erythrocytes and out of thrombocyte‐rich plasma
Authors: Lutz Hans U. 
Published: May 2005

AbstractWhen human erythrocytes are depleted of endogenous ATP they release spectrin‐free vesicles as a light vesicle fraction [Lutz et al: J Cell Biol 73:548, 1977] and chains of rounded vesicles as well as flattened myelin forms in a heavy vesicle fraction. The heavy fraction retains some spectrin, and glycophorin is partially degraded. The release of both types of fragments is not dependent upon added Ca+2, and 50 μM EGTA does not prevent the vesicle release. Concomitant with vesicle release, a large fraction of the major protein components of the cell is found in disulfide‐bonded aggregates.A protocol is outlined to recover erythrocyte‐specific fragments from thrombocyte‐rich plasma. It allows detection of spectrin‐free vesicles in whole blood stored under blood bank conditions for 12 days. In freshly drawn blood no such vesicles are observed, but particles are obtained that are different from thrombocyte fragments and that show a prominent glycoprotein running slightly faster than glycophorin.
Spectrin binding and the control of membrane protein mobility
Authors: Goodman Steven R. , Branton Daniel 
Published: May 2005

AbstractTransmembrane proteins of the human erythrocyte show restricted in‐plane mobility. Many of the restrictions on mobility are attributable to the molecules of spectrin which are located on the protoplasmic surface of the erythrocyte membrane. These molecules are elongate, form end‐to‐end heterodimer associations, and bind selectively to protein (or proteins) accessible on inside‐out, but not right‐side out, membrane vesicles.
Isolation and partial characterization of human erythrocyte membrane NADH: (Acceptor) oxidoreductase
Authors: Wang Chi‐Sun , Alaupovic Petar 
Published: May 2005

AbstractThe NADH: (acceptor) oxidoreductase (EC 1.6.99.3) was isolated from human erythrocyte ghosts by a procedure including Triton X‐100 solubilization, affinity chromatography on an NAD+‐Sepharose 4B column, ammonium sulfate precipitation, and isoelectric focusing. This enzyme preparation was characterized by a single band on the urea‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and by a single precipitin line with its corresponding antiserum on double diffusion and immunoelectrophoresis. A 103‐fold purification indicates that the oxidoreductase represents approximately 1% of the ghost protein mass. The specific activity of the purified enzyme was 112 units/mg protein. The pH optimum was 6.8 and the isoelectric point, pI, was 6.6. The oxidoreductase has a specificity for NADH as a cofactor. The NADPH was ineffective as a reducing agent. The enzyme activity was strongly temperature‐dependent, displaying maximal activity between 35 and 40°C. The energy of activation was 4.9 kcal. The enzyme activity was inhibited by sulfhydryl reagents, anionic detergents, and divalent ions. The amino acid composition of the purified enzyme is characterized by the presence of all common amino acids including half‐cystine and tryptophan. The results of carbohydrate and lipid analyses indicated that the oxidoreductase is a glycolipoprotein with fucose, galactose, mannose, and glucosamine as the sugar components and cholesterol and sphingomyelin as the lipid constituents. The apparent subunit molecular weight estimated by urea‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the absence and presence of 2‐mercaptoethanol was 40,000. The antiserum completely inhibited the enzymic activity at the equivalence point. We suggest that the membrane‐bound NADH: (acceptor) oxidoreductase might be a transmembrane protein.
Mitogenic response to epidermal growth factor: Relationship to number, affinity, and down‐regulation of EGF receptors in three murine embyro cell lines
Authors: Aharonov Aharon , Passovoy Dennis S. , Herschman Harvey R. 
Published: May 2005

AbstractSwiss 3T3 and C3H‐M2 cells have a greater mitogenic response to epidermal growth factor (EGF) than do C3H‐10T1/2 cells. The latter cell line, however, has a number of EGF receptors per cell intermediate between the two cell lines that have a more vigorous response to EGF. Scatchard analysis of binding data indicate that all three cell lines have one class of EGF receptor, with indistinguishable affinity for the ligand. When exposed to 10‐nM EGF all three cell lines “down‐regulate” their EGF receptors with the same time course, and to the same precentage of initial receptors.
Studies of the diphtheria toxin receptor on chinese hamster cells
Authors: Draper Rockford K. , Chin Daniel , Stubbs Lisa , Simon Melvin I. 
Published: May 2005

AbstractConcanavalin A, wheat germ agglutinin and the ovalbumin glycopeptide are all inhibitors of the cytotoxic effect of diphtheria toxin on Chinese hamster cells. Ovalbumin glycopeptide loses its inhibitory property after treatment with β‐N‐acetylglucosaminidase. This demonstrates the importance of the glycopeptide structure for the mechanism of inhibition. The glycopeptide may be a toxin cell‐surface receptor analogue.Diphtheria toxin‐resistant mutants were isolated in order to search for cells that might have an altered toxin receptor. One mutant was 10‐to 15‐fold more resistant to diphtheria toxin than wild‐type cells when protein synthesis was measured as a function of toxin concentration. However, when protein synthesis was measured as a function of time at a high toxin concentration, the time before onset of inhibition was identical in the mutant and wild‐type cells. We present evidence indicating that the resistance of this mutant can be accounted for by a decreased affinity of toxin for a cell‐surface receptor.
Erythrocyte membrane alterations in Huntington disease: Effects of γ‐aminobutyric acid
Authors: Butterfield D. A. , Braden M. L. , Markesbery W. R. 
Published: May 2005

AbstractThe interaction of the inhibitory neurotransmitter γ‐aminobutyric acid (GABA) with erythrocyte membranes from patients with Huntington disease and normal controls has been studied by electron spin resonance. GABA affects the physical state of erythrocyte membrane proteins in control and Huntington disease differently. In addition, after exposure of spin‐labeled Huntington disease erythrocyte membranes to 0.1 mM GABA, the relevant electron spin resonance parameters reflecting the physical state of membrane proteins are indistinguishable from those of untreated control membranes. These findings support the concept that this disease is associated with a generalized membrane defect.
Independence of normal phenotypic properties and cell surface receptor mobility in variant cell lines
Authors: Williams Robert E. , Linsley Peter S. , Rittenhouse Harry G. , Fox C. Fred , Boone Charles W. 
Published: May 2005

AbstractWe report the use of three classes of variants from the long‐established malignantly transformed LM cell line to demonstrate that the apparent mobility of cell surface receptors need not be dependent on the expression of the transformed phenotype in vitro.
Glycolipids as indicators of tumorigenesis
Authors: Morré D. J. , Kloppel T. M. , Merritt W. D. , Keenan T. W. 
Published: May 2005

AbstractHyperplastic liver nodules and hepatocellular carcinomas were induced in rats by oral administration of the carcinogen N‐2‐fluorenylacetamide. Neoplastic tissue was compared with control, fetal, neonatal, and precancerous liver tissues. The development of the tumors was slow, such that temporal changes in the biochemical and morphologic development of carcinogenesis could be identified. Ganglioside sialic acid levels were elevated in all but the most poorly differentiated tumors. Experiments to monitor individual enzymes suggested that the alterations in glycolipid composition were a direct effect of alterations in biosynthetic activities. The pattern during tumorigenesis was the inverse of that during normal development. Also, ganglioside patterns showed a progressive simplification from hyperplastic nodules to well‐differentiated hepatomas and through two grades of poorly differentiated hepatomas. An increase in the activity of the branchpoint enzyme of ganglioside biosynthesis preceded both a decrease in the branchpoint enzyme of the disialoganglioside pathway and a marked increase in the galactosyltransferase of GM1 formation. The results indicate that ganglioside deletions are the end result of a cascade of events in the tumorigenic transformation. The onset of ganglioside deletions but not of the cascade per se may correlate with the onset of malignancy.Glycolipid levels are elevated early in certain surrounding tissues especially in the blood. In rats bearing transplantable hepatomas, serum levels of lipidbound sialic acid were elevated 2.5‐fold. Similar results were obtained with sera of mice bearing transplantable mammary carcinomas and of cancer patients. These findings provide new emphasis for gangliosides in both cancer detection and as regulatory signals for growth and multiplication of cells.
Lipopolysaccharide effects on sensitive and resistant variant chinese hamster ovary cell lines
Authors: Brunson Kenneth W. , Nicolson Garth L. 
Published: May 2005

AbstractChinese hamster ovary (CHO · K1 · PRO) cell growth was inhibited by addition of a gram‐negative bacterial lipopolysaccharide (LPS) to the cell culture medium. Growth inhibition began after three or four days of incubation, was dose‐dependent up to a maximum at an LPS concentration of 500 μg/ml and was accompanied by cell shape changes and enhanced cytoplasmic vacuolization. Formation of bizarre CHO · K1 · PRO cell shapes and vacuole formation were most pronounced after seven days of incubation with LPS and could be observed by light and electron microscopy. An LPS‐resistant cell population was obtained by intermittent in vitro exposure to high levels of LPS; these variant cells or clones derived from them failed to display growth inhibition in the presence of LPS. A clone from the LPS‐resistant variant population showed altered cell properties compared to the parental cell line which included changes in cell morphology, adhesion, and endocytosis. Parental cells were markedly density‐inhibited, whereas the variant clone exhibited considerable growth after confluency. The LPS‐resistant variant cells showed a more elongated morphology than the parental line. No significant differences were observed between rates of detachment of parental and variant cells when sparse cultures of either line were removed from tissue culture dishes by ethylenediaminetetracetate (EDTA). However, at confluency approximately 100% of the variant cells versus 35% of the parental cells were removed by EDTA in one hour. Measurements of 125I‐ferritin uptake by parental and variant cells showed approximately twenty‐fold and twofold increases, respectively, in uptake induced by LPS when compared to untreated control cultures.
Binding of ricin A chain to rat liver ribosomes: Relationship to ribosome inactivation
Authors: Hedblom Mary L. , Cawley Daniel B. , Boguslawski Sophie , Houston L. L. 
Published: May 2005

AbstractRicin A chain was radioactively labeled using reductive alkylation, lactoperoxidase catalyzed iodination, and reaction with iodoacetamide or N‐ethylmaleimide (NEM). The inhibition of cell‐free rat liver protein synthesis by the modified A chains and the ribosome binding characteristics of each of the labeled derivatives was examined. [3H] NEM was found to quantitatively react with the A chain sulfhydryl group normally involved in a disulfide bond with the B chain in intact ricin. Labeling the protein with [3H] NEM had no effect on the in vitro inhibition of protein synthesis by the A chain. [3H] NEM‐labeled A chain binds to rat liver ribosomes in a manner which is dependent on the concentrations of NaCl and Mg2+. At optimal Mg2+ concentration (5.5 mM), A chain binding to ribosomes is saturable and fully reversible either by dilution of the reaction mixture or by addition of unlabeled A chain. At 5.5 mM Mg2+, A chain was found to bind to a single site on rat liver ribosomes with a dissociation constant of 6.2 X 10−8 M. [3H] NEM‐labeled A chain did not bind to isolated 40S ribosomal subunits and bound to 60S ribosomal subunits with a 1 : 1 molar stoichiometry and a dissociation constant of 2.2 X 10−7 M. The relationship between ribosome binding and A chain inhibition of eucaryotic protein synthesis is discussed.
Transmembrane action of thrombin initiates chick cell division
Authors: Carney Darrell H. , Cunningham Dennis D. 
Published: May 2005

AbstractThrombin immobilized on polystyrene beads initiates DNA synthesis and cell division in quiescent cultures of chick embryo (CE) cells in serum‐free medium. These thrombin beads also produce morphological changes in CE cells similar to those produced by soluble thrombin. The amount of acid‐precipitable material released from 125I‐thrombin‐beads into the culture medium was 60‐fold less than the amount of soluble thrombin required to produce an equivalent increase in cell number. Moreover, EM autoradiography of CE cells 10 h after 125I‐thrombin‐bead addition showed that there was no direct release and accumulation of radioactive material in the cytoplasm of these cells. These results demonstrate that thrombin action at the cell surface is sufficient to initiate proliferation of CE cells.
Resistance of the intact and reconstituted adipocyte hexose transport system to irreversible inhibition by sulfhydryl and amino reagents
Authors: Czech Michael P. , Pillion Dennis J. , Shanahan Michael F. 
Published: May 2005

AbstractSensitivity of the adipocyte D‐glucose transport system in intact plasma membranes or following solubilization and reconstitution into phospholipid vesicles to several protein‐modifying reagents was investigated. When intact plasma membranes were incubated with N‐ethylmaleimide (20 mM) or fluorodinitrobenzene (4 mM), D‐glucose transport activity was virtually abolished. However, washing the membranes free of unreacted reagents restored transport activity, indicating that covalent interaction with the membranes did not mediate the transport inhibition. Reaction of [3H] N‐ethylmaleimide with plasma membranes under similar conditions resulted in extensive labeling of all protein fractions resolved on dodecyl sulfate gels. Similarly, addition of N‐ethyl‐maleimide to cholate‐solubilized membrane protein had no effect on transport activity in artifical phospholipid vesicles reconstituted under conditions where the membrane protein was free of unreacted N‐ethylmaleimide. Transport activity in plasma membranes was also inhibited by both reduced and oxidized dithiothreitol or glutathione (15 mM) in a readily reversible manner, consistent with a noncovalent mode of inhibition. Thus, the insulin‐responsive adipocyte D‐glucose transport system differs from the red cell hexose transport system in its remarkable insensitivity to modulation by covalent blockade of sulfhydryal or amino groups by the reagents studied.
Photolabile and paramagnetic reagents for the investigation of transmembrane signaling events
Authors: Bramhall John , Ishida Brian , Wisnieski Bernadine 
Published: May 2005

AbstractTo investigate the dynamics of membrane processes that may be integral components of specific transmembrane signaling events we have synthesized several novel paramagnetic probes and their photoreactive counterparts. The structure of these probes was designed to (1) restrict “flipping” across the membrane bilayer; (2) contain paramagnetic or photoreactive moieties that could be placed at specific depths within the bilayer; (3) provide information about membrane structure as well as dynamics of protein movement; and (4) in the case of the photoreactive probes, be of high specific radioactivity.The molecules described in this paper consist of amino acid, dipeptide, or carbohydrate groups attached to arylazide‐ or nitroxide‐bearing fatty acids. The synthesis and initial characterization of these membrane probes is described.
Redistribution of intramembrane particles of human erythrocytes induced by HVJ (Sendai virus): A prerequisite for the virus‐induced cell fusion
Authors: Asano Akira , Sekiguchi Kiyotoshi 
Published: May 2005

AbstractAggregation of intramembrane particles of human erythrocytes was found to be induced by HVJ (Sendai virus) under conditions which lead to cell fusion. Degree of polyerythrocyte formation was compared under a variety of conditions with extent of cluster formation observed with the same preparations. Both structural changes of the membranes, ie, fusion and clustering of the particles, behaved very similarly under widely different virus‐to‐cell ratios and over the time course of cell fusion. Furthermore, by inclusion of high concentrations of antispectrin antibodies within the ghosts, inhibition of clustering of intramembrane particles and hindrance of virus‐induced cell fusion were found to occur simultaneously. Antibodies by themselves did not induce aggregation of particles under isotonic conditions, whereas particle clustering could be induced under hypotonic conditions at antibody concentrations causing partial cross‐linking of spectrin molecules.In conclusion, clustering of intramembrane particles seems to be required for virus‐induced fusion of human erythrocytes.
Intracellular compartmentation and transport of metabolites
Authors: Davis Rowland H. , Bowman Barry J. , Weiss Richard L. 
Published: May 2005

AbstractThe intracellular locations of enzymes and metabolites were determined for ornithine metabolism in Neurospora. Pulse label experiments were used to measure the rates of intracellular translocations and the sizes of compartmented pools of metabolites in the mitochondrial, cytosolic and vesicular compartments. The results indicate that rapid equilibration occurs between these pools during growth in minimal medium, although the vast majority of the ornithine is confined to the vesicular compartment. Arginine, the biosynthetic end‐product of ornithine metabolism, regulates ornithine utilization through a combination of feedback inhibition, repression, and control of intracellular translocations. The last phenomenon plays a decisive role indicating that the regulation of intercompartmental translocations may be a common mechanism in rapid adaptation responses in eukaryotic cells.
Increase in the affinity of the uridine phosphorylation system for ATP after serum or insulin activation of 3T3 fibroblasts
Authors: Goldenberg Gerald J. , Stein Wilfred D. 
Published: May 2005

AbstractThe stimulation of uridine uptake, brought about by the addition of serum or insulin to quiescent 3T3 fibroblasts, is associated in the half‐saturation concentration of the uridine phosphorylating system for the substrate ATP, with relatively little change in the maximum uptake or in the affinity for uridine. In stimulated cells the Km towards ATP fell in the range 0.053–0.187 mM, while V max was 34 to 52 pmoles/106 cells/min. In quiescent cells these values were 2.89–4.22 mM and 74.5–126 pmoles/106 cells/min, respectively. No difference was found, however, between the Km's for ATP when phosphorylation of uridine was determined using cell‐free extracts prepared from either quiescent cells or from stimulated cells.
Steroid hormone receptor characterization of several histologic variants of a rat prostatic adenocarcinoma
Authors: Markland Francis S. , Chopp Richard T. , Cosgrove Malcolm D. , Howard Edwin B. 
Published: May 2005

AbstractSeveral histologic variants of the transplantable R‐3327 prostatic adenocarcinoma carried in male Copenhagen rats have been characterized and the histologic types have been correlated with steroid hormone receptor content. One type is clearly an adenocarcinoma; this tumor is hormonally responsive and contains substantial amounts of both androgen and estrogen receptors. In contrast, another histologic type, a fibrosarcoma, is hormonally nonresponsive and does not contain either receptor. A third histologic variant is classified as a carcinosarcoma and contains histological elements of both adenocarcinoma and fibrosarcoma and is also hormonally responsive. This tumor contains lower receptor levels than the adenocarcinomas but more than the fibrosarcomas. The androgen receptor appears to be identical in the different histologic forms of the tumor; the sedimentation coefficient is 7.8S and the dissociation constant for methyltrienolone is 4 X 10−9 M. Similarly, the estrogen receptor from the different histologic forms of the tumor has a sedimentation coefficient of 8.3S and the dissociation constant for estradiol is 7 X 10−10 M. These findings clearly distinguish the cytosol binding macromolecules from plasma binding proteins, and classify them as steroid hormone receptors. Further, rat serum was devoid of androgen and estrogen binding in the 8S region. Normal prostate tissue from Copenhagen rats contained low levels of an androgen receptor, but no estrogen receptor. It is possible that during growth and/or passage of the R‐3327 tumor, the hormonally responsive adenocarcinoma cells do not survive and there is a gradual emergence of the nonresponsive fibrosarcoma. If, as we suspect, the receptors are found in the epithelial cells and not the stromal cells, there clearly should be considerable variation of receptor content in the different intermediary histologic forms of the tumor.
Requirement for guanosine triphosphate in the activation of adenylate cyclase by cholera toxin
Authors: Enomoto Keiichi , Gill D. Michael 
Published: May 2005

AbstractThe activation of adenylate cyclase in lysed pigeon erythrocytes requires, among several cofactors, a nucleotide which may be ATP, GTP, or many other triphosphates. However, after removal of endogenous nucleotides by gel filtration or by adsorption onto charcoal the requirement can be met only by GTP, or an analog of GTP. The GTP is required during the activation of the cyclase by toxin even if GTP is also included during the subsequent adenylate cyclase assay, conducted without toxin. In the presence of GTP it is possible to assay for the cytosolic protein that is also required for the action of cholera toxin. By gel filtration, its apparent molecular weight is 15,000–20,000.
Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells
Authors: Maher P. , Molday R. S. 
Published: May 2005

AbstractConcanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either 125I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 107 binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy‐dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein‐tagged Con A and rhodamine‐tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.